Metabolic and redox barriers in the skin exposed to drugs and xenobiotics.

PubMed

Korkina, Liudmila

2016-01-01

Growing exposure of human skin to environmental and occupational hazards, to numerous skin care/beauty products, and to topical drugs led to a biomedical concern regarding sustainability of cutaneous chemical defence that is essential for protection against intoxication. Since skin is the largest extra-hepatic drug/xenobiotic metabolising organ where redox-dependent metabolic pathways prevail, in this review, publications on metabolic processes leading to redox imbalance (oxidative stress) and its autocrine/endocrine impact to cutaneous drug/xenobiotic metabolism were scrutinised. Chemical and photo-chemical skin barriers contain metabolic and redox compartments: their protective and homeostatic functions. The review will examine the striking similarity of adaptive responses to exogenous chemical/photo-chemical stressors and endogenous toxins in cutaneous metabolic and redox system; the role(s) of xenobiotics/drugs and phase II enzymes in the endogenous antioxidant defence and maintenance of redox balance; redox regulation of interactions between metabolic and inflammatory responses in skin cells; skin diseases sharing metabolic and redox problems (contact dermatitis, lupus erythematosus, and vitiligo) Due to exceptional the redox dependence of cutaneous metabolic pathways and interaction of redox active metabolites/exogenous antioxidants with drug/xenobiotic metabolism, metabolic tests of topical xenobiotics/drugs should be combined with appropriate redox analyses and performed on 3D human skin models.

Effects of Sublethal Exposure to a Glyphosate-Based Herbicide Formulation on Metabolic Activities of Different Xenobiotic-Metabolizing Enzymes in Rats.

PubMed

Larsen, Karen; Najle, Roberto; Lifschitz, Adrián; Maté, María L; Lanusse, Carlos; Virkel, Guillermo L

2014-07-01

The activities of different xenobiotic-metabolizing enzymes in liver subcellular fractions from Wistar rats exposed to a glyphosate (GLP)-based herbicide (Roundup full II) were evaluated in this work. Exposure to the herbicide triggered protective mechanisms against oxidative stress (increased glutathione peroxidase activity and total glutathione levels). Liver microsomes from both male and female rats exposed to the herbicide had lower (45%-54%, P < 0.01) hepatic cytochrome P450 (CYP) levels compared to their respective control animals. In female rats, the hepatic 7-ethoxycoumarin O-deethylase (a general CYP-dependent enzyme activity) was 57% higher (P < 0.05) in herbicide-exposed compared to control animals. Conversely, this enzyme activity was 58% lower (P < 0.05) in male rats receiving the herbicide. Lower (P < 0.05) 7-ethoxyresorufin O-deethlyase (EROD, CYP1A1/2 dependent) and oleandomycin triacetate (TAO) N-demethylase (CYP3A dependent) enzyme activities were observed in liver microsomes from exposed male rats. Conversely, in females receiving the herbicide, EROD increased (123%-168%, P < 0.05), whereas TAO N-demethylase did not change. A higher (158%-179%, P < 0.01) benzyloxyresorufin O-debenzylase (a CYP2B-dependent enzyme activity) activity was only observed in herbicide-exposed female rats. In herbicide-exposed rats, the hepatic S-oxidation of methimazole (flavin monooxygenase dependent) was 49% to 62% lower (P < 0.001), whereas the carbonyl reduction of menadione (a cytosolic carbonyl reductase-dependent activity) was higher (P < 0.05). Exposure to the herbicide had no effects on enzymatic activities dependent on carboxylesterases, glutathione transferases, and uridinediphospho-glucuronosyltransferases. This research demonstrated certain biochemical modifications after exposure to a GLP-based herbicide. Such modifications may affect the metabolic fate of different endobiotic and xenobiotic substances. The pharmacotoxicological significance of these

Modulation of Xenobiotic Metabolizing Enzyme and Transporter Gene Expression in Primary Cultures of Human Hepatocytes by ToxCast Chemicals

EPA Science Inventory

ToxCast chemicals were assessed for induction or suppression of xenobiotic metabolizing enzyme and transporter gene expression using primary human hepatocytes. The mRNA levels of 14 target and 2 control genes were measured: ABCB1, ABCB11, ABCG2, SLCO1B1, CYP1A1, CYP1A2, CYP2B6, C...

Phase I to II cross-induction of xenobiotic metabolizing enzymes: a feedforward control mechanism for potential hormetic responses.

PubMed

Zhang, Qiang; Pi, Jingbo; Woods, Courtney G; Andersen, Melvin E

2009-06-15

Hormetic responses to xenobiotic exposure likely occur as a result of overcompensation by the homeostatic control systems operating in biological organisms. However, the mechanisms underlying overcompensation that leads to hormesis are still unclear. A well-known homeostatic circuit in the cell is the gene induction network comprising phase I, II and III metabolizing enzymes, which are responsible for xenobiotic detoxification, and in many cases, bioactivation. By formulating a differential equation-based computational model, we investigated in this study whether hormesis can arise from the operation of this gene/enzyme network. The model consists of two feedback and one feedforward controls. With the phase I negative feedback control, xenobiotic X activates nuclear receptors to induce cytochrome P450 enzyme, which bioactivates X into a reactive metabolite X'. With the phase II negative feedback control, X' activates transcription factor Nrf2 to induce phase II enzymes such as glutathione S-transferase and glutamate cysteine ligase, etc., which participate in a set of reactions that lead to the metabolism of X' into a less toxic conjugate X''. The feedforward control involves phase I to II cross-induction, in which the parent chemical X can also induce phase II enzymes directly through the nuclear receptor and indirectly through transcriptionally upregulating Nrf2. As a result of the active feedforward control, a steady-state hormetic relationship readily arises between the concentrations of the reactive metabolite X' and the extracellular parent chemical X to which the cell is exposed. The shape of dose-response evolves over time from initially monotonically increasing to J-shaped at the final steady state-a temporal sequence consistent with adaptation-mediated hormesis. The magnitude of the hormetic response is enhanced by increases in the feedforward gain, but attenuated by increases in the bioactivation or phase II feedback loop gains. Our study suggests a

Activity of xenobiotic-metabolizing enzymes in the liver of rats with multi-vitamin deficiency.

PubMed

Tutelyan, Victor A; Kravchenko, Lidia V; Aksenov, Ilya V; Trusov, Nikita V; Guseva, Galina V; Kodentsova, Vera M; Vrzhesinskaya, Oksana A; Beketova, Nina A

2013-01-01

The purpose of the study was to determine how multi-vitamin deficiency affects xenobiotic-metabolizing enzyme (XME) activities in the rat liver. Vitamin levels and XME activities were studied in the livers of male Wistar rats who were fed for 4 weeks with semi-synthetic diets containing either adequate (100 % of recommended vitamin intake) levels of vitamins (control), or decreased vitamin levels (50 % or 20 % of recommended vitamin intake). The study results have shown that moderate vitamin deficiency (50 %) leads to a decrease of vitamin A levels only, and to a slight increase, as compared with the control, in the following enzyme activities: methoxyresorufin O-dealkylase (MROD) activity of CYP1 A2 - by 34 % (p < 0.05), UDP-glucuronosyl transferase - by 26 % (p < 0.05), and quinone reductase - by 55 % (p < 0.05). Profound vitamin deficiency (20 %) led to a decrease of vitamins A, E, B1, B2, and C, and enzyme activities in the liver: MROD - to 78 % of the control level (p < 0.05), 4-nitrophenol hydroxylase - to 74 % (p < 0.05), heme oxygenase-1 - to 83 % (p < 0.05), and quinone reductase - to 60 % (p < 0.05). At the same time, the UDP-glucuronosyl transferase activity and ethoxyresorufin O-dealkylase activity of CYP1A1, pentoxyresorufin O-dealkylase activity of CYP2B1/2 and 6β-testosterone hydroxylase, as well as the total activity of glutathione transferase did not differ from the control levels. The study has demonstrated that profound multi-vitamin deficiency is associated with a decrease in the expression of CYP1A2 and CYP3A1 mRNAs to 62 % and 79 %, respectively. These data indicated that a short-term but profound multi-vitamin deficiency in rats leads to a decrease in the activities and expression of the some XME that play an important role in detoxification of xenobiotics and metabolism of drugs and antioxidant protection.

Characterization of xenobiotic metabolizing enzymes of a reconstructed human epidermal model from adult hair follicles.

PubMed

Bacqueville, Daniel; Jacques, Carine; Duprat, Laure; Jamin, Emilien L; Guiraud, Beatrice; Perdu, Elisabeth; Bessou-Touya, Sandrine; Zalko, Daniel; Duplan, Hélène

2017-08-15

In this study, a comprehensive characterization of xenobiotic metabolizing enzymes (XMEs) based on gene expression and enzyme functionality was made in a reconstructed skin epidermal model derived from the outer root sheath (ORS) of hair follicles (ORS-RHE). The ORS-RHE model XME gene profile was consistent with native human skin. Cytochromes P450 (CYPs) consistently reported to be detected in native human skin were also present at the gene level in the ORS-RHE model. The highest Phase I XME gene expression levels were observed for alcohol/aldehyde dehydrogenases and (carboxyl) esterases. The model was responsive to the CYP inducers, 3-methylcholanthrene (3-MC) and β-naphthoflavone (βNF) after topical and systemic applications, evident at the gene and enzyme activity level. Phase II XME levels were generally higher than those of Phase I XMEs, the highest levels were GSTs and transferases, including NAT1. The presence of functional CYPs, UGTs and SULTs was confirmed by incubating the models with 7-ethoxycoumarin, testosterone, benzo(a)pyrene and 3-MC, all of which were rapidly metabolized within 24h after topical application. The extent of metabolism was dependent on saturable and non-saturable metabolism by the XMEs and on the residence time within the model. In conclusion, the ORS-RHE model expresses a number of Phase I and II XMEs, some of which may be induced by AhR ligands. Functional XME activities were also demonstrated using systemic or topical application routes, supporting their use in cutaneous metabolism studies. Such a reproducible model will be of interest when evaluating the cutaneous metabolism and potential toxicity of innovative dermo-cosmetic ingredients. Copyright © 2017 Elsevier Inc. All rights reserved.

Maternal drug abuse and human term placental xenobiotic and steroid metabolizing enzymes in vitro.

PubMed

Paakki, P; Stockmann, H; Kantola, M; Wagner, P; Lauper, U; Huch, R; Elovaara, E; Kirkinen, P; Pasanen, M

2000-02-01

We evaluated the impact of maternal drug abuse at term on human placental cytochrome P450 (CYP)-mediated (Phase I) xenobiotic and steroid-metabolizing activities [aromatase, 7-ethoxyresorufin O-deethylase (EROD), 7-ethoxycoumarin O-deethylase (ECOD), pyrene 1-hydroxylase (P1OH), and testosterone hydroxylase], and androstenedione-forming isomerase, NADPH quinone oxidoreductase (Phase II), UDP-glucuronosyltransferase (UGT), and glutathione S-transferase (GST) activities in vitro. Overall, the formation of androstenedione, P1OH, and testosterone hydroxylase was statistically significant between control and drug-abusing subjects; we observed no significant differences in any other of the phase I and II activities. In placentas from drug-abusing mothers, we found significant correlations between ECOD and P1OH activities (p < 0. 001), but not between ECOD and aromatase or P1OH and EROD activities; we also found significant correlations between blood cotinine and UGT activities (p < 0.01). In contrast, in controls (mothers who did not abuse drugs but did smoke cigarettes), the P1OH activity correlated with ECOD, EROD (p < 0.001), and testosterone hydroxylase (p < 0.001) activities. Our results (wider variation in ECOD activity among tissue from drug-abusing mothers and the significant correlation between P1OH and ECOD activities, but not with aromatase or EROD activities) indicate that maternal drug abuse results in an additive effect in enhancing placental xenobiotic metabolizing enzymes when the mother also smokes cigarettes; this may be due to enhancing a "silent" CYP form, or a new placental CYP form may be activated. The change in the steroid metabolism profile in vitro suggests that maternal drug abuse may alter normal hormonal homeostasis during pregnancy.

GENE EXPRESSION PROFILING OF XENOBIOTIC METABOLIZING ENZYMES (XMES) IN THE AGING MALE FISHER RAT

EPA Science Inventory

Detoxification and elimination of xenobiotics is a major function of the liver and is important in maintaining the metabolic homeostasis of the organism. The degree to which aging affects hepatic metabolism is not known. The expression of XMEs, in part, determines the fate of the...

Xenobiotic-metabolizing enzymes in Bacillus anthracis: molecular and functional analysis of a truncated arylamine N-acetyltransferase isozyme.

PubMed

Kubiak, Xavier; Duval, Romain; Pluvinage, Benjamin; Chaffotte, Alain F; Dupret, Jean-Marie; Rodrigues-Lima, Fernando

2017-07-01

The arylamine N-acetyltransferases (NATs) are xenobiotic-metabolizing enzymes that play an important role in the detoxification and/or bioactivation of arylamine drugs and xenobiotics. In bacteria, NATs may contribute to the resistance against antibiotics such as isoniazid or sulfamides through their acetylation, which makes this enzyme family a possible drug target. Bacillus anthracis, a bacterial species of clinical significance, expresses three NAT isozymes with distinct structural and enzymatic properties, including an inactive isozyme ((BACAN)NAT3). (BACAN)NAT3 features both a non-canonical Glu residue in its catalytic triad and a truncated C-terminus domain. However, the role these unusual characteristics play in the lack of activity of the (BACAN)NAT3 isozyme remains unclear. Protein engineering, recombinant expression, enzymatic analyses with aromatic amine substrates and phylogenetic analysis approaches were conducted. The deletion of guanine 580 (G580) in the nat3 gene was shown to be responsible for the expression of a truncated (BACAN)NAT3 isozyme. Artificial re-introduction of G580 in the nat3 gene led to a functional enzyme able to acetylate several arylamine drugs displaying structural characteristics comparable with its functional Bacillus cereus homologue ((BACCR)NAT3). Phylogenetic analysis of the nat3 gene in the B. cereus group further indicated that nat3 may constitute a pseudogene of the B. anthracis species. The existence of NATs with distinct properties and evolution in Bacillus species may account for their adaptation to their diverse chemical environments. A better understanding of these isozymes is of importance for their possible use as drug targets. This article is part of a themed section on Drug Metabolism and Antibiotic Resistance in Micro-organisms. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.14/issuetoc. © 2016 The British Pharmacological Society.

Pharmacogenetic profile of xenobiotic enzyme metabolism in survivors of the Spanish toxic oil syndrome.

PubMed Central

Ladona, M G; Izquierdo-Martinez, M; Posada de la Paz, M P; de la Torre, R; Ampurdanés, C; Segura, J; Sanz, E J

2001-01-01

In 1981, the Spanish toxic oil syndrome (TOS) affected more than 20,000 people, and over 300 deaths were registered. Assessment of genetic polymorphisms on xenobiotic metabolism would indicate the potential metabolic capacity of the victims at the time of the disaster. Thus, impaired metabolic pathways may have contributed to the clearance of the toxicant(s) leading to a low detoxification or accumulation of toxic metabolites contributing to the disease. We conducted a matched case-control study using 72 cases (54 females, 18 males) registered in the Official Census of Affected Patients maintained by the Spanish government. Controls were nonaffected siblings (n =72) living in the same household in 1981 and nonaffected nonrelatives (n = 70) living in the neighborhood at that time, with no ties to TOS. Genotype analyses were performed to assess the metabolic capacity of phase I [cytochrome P450 1A1 (CYP1A1), CYP2D6] and phase II [arylamine N-acetyltransferase-2 (NAT2), GSTM1 (glutathione S-transferase M1) and GSTT1] enzyme polymorphisms. The degree of association of the five metabolic pathways was estimated by calculating their odds ratios (ORs) using conditional logistic regression analysis. In the final model, cases compared with siblings (72 pairs) showed no differences either in CYP2D6 or CYP1A1 polymorphisms, or in conjugation enzyme polymorphisms, whereas cases compared with the unrelated controls (70 pairs) showed an increase in NAT2 defective alleles [OR = 6.96, 95% confidence interval (CI), 1.46-33.20] adjusted by age and sex. Glutathione transferase genetic polymorphisms (GSTM1, GSTT1) showed no association with cases compared with their siblings or unrelated controls. These findings suggest a possible role of impaired acetylation mediating susceptibility in TOS. PMID:11335185

Retinoid-xenobiotic interactions: the Ying and the Yang

PubMed Central

2015-01-01

The literature provides compelling evidence pointing to tight metabolic interactions between retinoids and xenobiotics. These are extensive and important for understanding xenobiotic actions in the body. Within the body, retinoids affect xenobiotic metabolism and actions and conversely, xenobiotics affect retinoid metabolism and actions. This article summarizes data that establish the importance of retinoid-dependent metabolic pathways for sustaining the body’s responses to xenobiotic exposure, including the roles of all-trans- and 9-cis-retinoic acid for protecting mammals from harmful xenobiotic effects and for ensuring xenobiotic elimination from the body. This review will also consider molecular mechanisms underlying xenobiotic toxicity focusing on how this may contribute to retinoid deficiency and disruption of normal retinoid homeostasis. Special attention is paid to xenobiotic molecular targets (nuclear receptors, regulatory proteins, enzymes, and transporters) which affect retinoid metabolism and signaling. PMID:26311625

Spatio-temporal Model of Xenobiotic Distribution and Metabolism in an in Silico Mouse Liver Lobule

NASA Astrophysics Data System (ADS)

Fu, Xiao; Sluka, James; Clendenon, Sherry; Glazier, James; Ryan, Jennifer; Dunn, Kenneth; Wang, Zemin; Klaunig, James

Our study aims to construct a structurally plausible in silico model of a mouse liver lobule to simulate the transport of xenobiotics and the production of their metabolites. We use a physiologically-based model to calculate blood-flow rates in a network of mouse liver sinusoids and simulate transport, uptake and biotransformation of xenobiotics within the in silico lobule. Using our base model, we then explore the effects of variations of compound-specific (diffusion, transport and metabolism) and compound-independent (temporal alteration of blood flow pattern) parameters, and examine their influence on the distribution of xenobiotics and metabolites. Our simulations show that the transport mechanism (diffusive and transporter-mediated) of xenobiotics and blood flow both impact the regional distribution of xenobiotics in a mouse hepatic lobule. Furthermore, differential expression of metabolic enzymes along each sinusoid's portal to central axis, together with differential cellular availability of xenobiotics, induce non-uniform production of metabolites. Thus, the heterogeneity of the biochemical and biophysical properties of xenobiotics, along with the complexity of blood flow, result in different exposures to xenobiotics for hepatocytes at different lobular locations. We acknowledge support from National Institute of Health GM 077138 and GM 111243.

GENE EXPRESSION PROFILING OF XENOBIOTIC METABOLIZING ENZYMES (XMES) THROUGH THE LIFE STAGES OF THE MALE C57BL/6 MOUSE

EPA Science Inventory

In the presence of foreign compounds, metabolic homeostasis of the organism is maintained by the liver's ability to detoxify and eliminate these xenobiotics. This is accomplished, in part, by the expression of XMEs, which metabolize xenobiotics and determine whether exposure will...

Molecular, cellular, and tissue impact of depleted uranium on xenobiotic-metabolizing enzymes.

PubMed

Gueguen, Yann; Rouas, Caroline; Monin, Audrey; Manens, Line; Stefani, Johanna; Delissen, Olivia; Grison, Stéphane; Dublineau, Isabelle

2014-02-01

Enzymes that metabolize xenobiotics (XME) are well recognized in experimental models as representative indicators of organ detoxification functions and of exposure to toxicants. As several in vivo studies have shown, uranium can alter XME in the rat liver or kidneys after either acute or chronic exposure. To determine how length or level of exposure affects these changes in XME, we continued our investigation of chronic rat exposure to depleted uranium (DU, uranyl nitrate). The first study examined the effect of duration (1-18 months) of chronic exposure to DU, the second evaluated dose dependence, from a level close to that found in the environment near mining sites (0.2 mg/L) to a supra-environmental dose (120 mg/L, 10 times the highest level naturally found in the environment), and the third was an in vitro assessment of whether DU exposure directly affects XME and, in particular, CYP3A. The experimental in vivo models used here demonstrated that CYP3A is the enzyme modified to the greatest extent: high gene expression changed after 6 and 9 months. The most substantial effects were observed in the liver of rats after 9 months of exposure to 120 mg/L of DU: CYP3A gene and protein expression and enzyme activity all decreased by more than 40 %. Nonetheless, no direct effect of DU by itself was observed after in vitro exposure of rat microsomal preparations, HepG2 cells, or human primary hepatocytes. Overall, these results probably indicate the occurrence of regulatory or adaptive mechanisms that could explain the indirect effect observed in vivo after chronic exposure.

Computational Prediction of Metabolism: Sites, Products, SAR, P450 Enzyme Dynamics, and Mechanisms

PubMed Central

2012-01-01

Metabolism of xenobiotics remains a central challenge for the discovery and development of drugs, cosmetics, nutritional supplements, and agrochemicals. Metabolic transformations are frequently related to the incidence of toxic effects that may result from the emergence of reactive species, the systemic accumulation of metabolites, or by induction of metabolic pathways. Experimental investigation of the metabolism of small organic molecules is particularly resource demanding; hence, computational methods are of considerable interest to complement experimental approaches. This review provides a broad overview of structure- and ligand-based computational methods for the prediction of xenobiotic metabolism. Current computational approaches to address xenobiotic metabolism are discussed from three major perspectives: (i) prediction of sites of metabolism (SOMs), (ii) elucidation of potential metabolites and their chemical structures, and (iii) prediction of direct and indirect effects of xenobiotics on metabolizing enzymes, where the focus is on the cytochrome P450 (CYP) superfamily of enzymes, the cardinal xenobiotics metabolizing enzymes. For each of these domains, a variety of approaches and their applications are systematically reviewed, including expert systems, data mining approaches, quantitative structure–activity relationships (QSARs), and machine learning-based methods, pharmacophore-based algorithms, shape-focused techniques, molecular interaction fields (MIFs), reactivity-focused techniques, protein–ligand docking, molecular dynamics (MD) simulations, and combinations of methods. Predictive metabolism is a developing area, and there is still enormous potential for improvement. However, it is clear that the combination of rapidly increasing amounts of available ligand- and structure-related experimental data (in particular, quantitative data) with novel and diverse simulation and modeling approaches is accelerating the development of effective tools for

Coordinated changes in xenobiotic metabolizing enzyme (XME) gene expression through the life stages of the male C57BL/6 mouse

EPA Science Inventory

Metabolic homeostasis of the organism is maintained by the liver's ability to detoxify and eliminate xenobiotics. This is accomplished, in part, by the expression of XMEs, which metabolize xenobiotics and determine whether exposure will result in toxicity. Some evidence indicates...

The Therapeutic Role of Xenobiotic Nuclear Receptors against Metabolic Syndrome.

PubMed

Pu, Shuqi; Wu, Xiaojie; Yang, Xiaoying; Zhang, Yunzhan; Dai, Yunkai; Zhang, Yueling; Wu, Xiaoting; Liu, Yan; Cui, Xiaona; Jin, Haiyong; Cao, Jianhong; Li, Ruliu; Cai, Jiazhong; Cao, Qizhi; Hu, Ling; Gao, Yong

2018-06-10

Xenobiotic nuclear receptors (XNRs) are nuclear receptors that characterized by coordinately regulating the expression of genes encoding drug-metabolizing enzymes and transporters to essentially eliminate and detoxify xenobiotics and endobiotics from the body, including the peroxisome proliferator-activated receptor (PPAR), the farnesoid X receptor (FXR), the liver X receptor (LXR), the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR). Heretofore, increasing evidences have suggested that these five XNRs are not only involved in the regulation of xeno-/endo-biotics detoxication but also the development of human diseases, such as cancer, obesity and diabetes. PPAR, FXR, LXR, PXR and CAR, as the receptors for numerous natural or synthetic compounds may be the most effective therapeutic targets in the treatment of metabolic diseases. In this review, we will focus on these five XNRs and their recently discovered functions in diabetes and its complications. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Acetaminophen induces xenobiotic-metabolizing enzymes in rat: Impact of a uranium chronic exposure.

PubMed

Rouas, Caroline; Souidi, Maâmar; Grandcolas, Line; Grison, Stephane; Baudelin, Cedric; Gourmelon, Patrick; Pallardy, Marc; Gueguen, Yann

2009-11-01

The extensive use of uranium in civilian and military applications increases the risk of human chronic exposure. Uranium is a slightly radioactive heavy metal with a predominantly chemical toxicity, especially in kidney but also in liver. Few studies have previously shown some effects of uranium on xenobiotic-metabolizing enzymes (XME) that might disturb drug pharmacokinetic. The aim of this study was to determine whether a chronic (9 months) non-nephrotoxic low dose exposure to depleted uranium (DU, 1mg/rat/day) could modify the liver XME, using a single non-hepatotoxic acetaminophen (APAP) treatment (50mg/kg). Most of XME analysed were induced by APAP treatment at the gene expression level but at the protein level only CYP3A2 was significantly increased 3h after APAP treatment in DU-exposed rats whereas it remained at a basal level in unexposed rats. In conclusion, these results showed that a chronic non-nephrotoxic DU exposure specially modify CYP3A2 after a single therapeutic APAP treatment. Copyright © 2009 Elsevier B.V. All rights reserved.

Interplay of drug metabolizing enzymes with cellular transporters.

PubMed

Böhmdorfer, Michaela; Maier-Salamon, Alexandra; Riha, Juliane; Brenner, Stefan; Höferl, Martina; Jäger, Walter

2014-11-01

Many endogenous and xenobiotic substances and their metabolites are substrates for drug metabolizing enzymes and cellular transporters. These proteins may not only contribute to bioavailability of molecules but also to uptake into organs and, consequently, to overall elimination. The coordinated action of uptake transporters, metabolizing enzymes, and efflux pumps, therefore, is a precondition for detoxification and elimination of drugs. As the understanding of the underlying mechanisms is important to predict alterations in drug disposal, adverse drug reactions and, finally, drug-drug interactions, this review illustrates the interplay between selected uptake/efflux transporters and phase I/II metabolizing enzymes.

Hepatoprotective effects of lycopene on liver enzymes involved in methionine and xenobiotic metabolism in hyperhomocysteinemic rats.

PubMed

Yefsah-Idres, Aicha; Benazzoug, Yasmina; Otman, Amel; Latour, Alizée; Middendorp, Sandrine; Janel, Nathalie

2016-06-15

Hyperhomocysteinemia, defined by an increased plasma homocysteine level, is commonly associated with chronic liver diseases. A link between the elevated homocysteine level and oxidative stress has been demonstrated. Indeed the pathogenesis of liver diseases in the case of hyperhomocysteinemia could be due to this production of oxidative stress. Many studies have demonstrated the antioxidative properties of lycopene, a carotenoid. Therefore, the present study was designed to induce hyperhomocysteinemia in male Wistar rats in order to analyze the effect of lycopene supplementation on homocysteine metabolism, on phase I and phase II xenobiotic-metabolizing enzyme activities, and on liver injury by histological examination and analysis of biochemical markers. We found that rats with a high methionine diet showed abnormal histological features, with an increase of serum homocysteine, alanine aminotransferase and aspartate aminotransferase levels, decreased hepatic cystathionine beta synthase and S-adenosyl-homocysteine hydrolase activities and an increased hepatic malondialdehyde level. We demonstrated the reversal effect of lycopene supplementation on hyperhomocysteinemia. Taken together, these findings provide additional clues on the hepatoprotective effects of lycopene.

[Human drug metabolizing enzymes. II. Conjugation enzymes].

PubMed

Vereczkey, L; Jemnitz, K; Gregus, Z

1998-09-01

In this review we focus on human conjugation enzymes (UDP-glucuronyltransferases, methyl-trasferases, N-acetyl-transferases, O-acetyl-transferases, Amidases/carboxyesterases, sulfotransferases, Glutation-S-transferases and the enzymes involved in the conjugation with amino acids) that participate in the metabolism of xenobiotics. Although conjugation reactions in most of the cases result in detoxication, more and more publications prove that the reactions catalysed by these enzymes very often lead to activated molecules that may attack macromolecules (proteins, RNAs, DNAs), resulting in toxicity (liver, neuro-, embryotoxicity, allergy, carcinogenecity). We have summarised the data available on these enzymes concerning their catalytic profile and specificity, inhibition, induction properties, their possible role in the generation of toxic compounds, their importance in clinical practice and drug development.

Differences in the expression of xenobiotic-metabolizing enzymes between islets derived from the ventral and dorsal anlage of the pancreas.

PubMed

Standop, Jens; Ulrich, Alexis B; Schneider, Matthias B; Büchler, Markus W; Pour, Parviz M

2002-01-01

Chronic pancreatitis and pancreatic cancer have been linked to the exposure of environmental chemicals (xenobiotics), which generally require metabolic activation to highly reactive toxic or carcinogenic intermediates. The primary enzyme system involved is made up of numerous cytochrome P450 mono-oxygenases (CYP). Glutathione S-transferases (GST) belong to the enzyme systems that catalyze the conjugation of the reactive intermediates produced by CYPs to less toxic or readily excretable metabolites. Because the majority of chronic pancreatitis and pancreatic cancers develop in the organ's head, we compared the expression of selected CYP and GST enzymes between the tissues deriving from the ventral anlage (head) and dorsal anlage (corpus, tail). A total of 20 normal pancreatic tissue specimen from organ donors and early autopsy cases were processed immunohistochemically by using antibodies to CYP 1A1, 1A2, 2B6, 2C8/9/19, 2D6, 2E1, 3A1, 3A2 and 3A4, GST-alpha, GST-mu and GST-pi, and the NADPH cytochrome P450 oxido-reductase (NA-OR), the specificity of which has been verified in our previous study by Western blot and RT-PCR analyses. In all pancreatic regions, most of the enzymes were expressed in islet cells. However, more islets in the head region expressed CYP 2B6, 2C8/9/19, 2E1 and the NA-OR, than those in the body and tail. Moreover, the expression of CYP 2B6 and 2E1 was restricted to the pancreatic polypeptide (PP) cells, and the concentration of CYP 3A1 and 3A4 was stronger in PP cells than in other islet cells. On the other hand, GST-mu and GST-pi were expressed primarily in islet cells of the body and tail. The greater content of xenobiotic-metabolizing and carcinogen-activating CYP enzymes and a lower expression of detoxifying GST enzymes in the head of the pancreas could be one reason for the greater susceptibility of this region for inflammatory and malignant diseases. Copyright 2002 S. Karger AG, Basel and IAP

Xenobiotic metabolizing enzyme activities in cells used for testing skin sensitization in vitro.

PubMed

Fabian, E; Vogel, D; Blatz, V; Ramirez, T; Kolle, S; Eltze, T; van Ravenzwaay, B; Oesch, F; Landsiedel, R

2013-09-01

For ethical and regulatory reasons, in vitro tests for scoring potential toxicities of cosmetics are essential. A test strategy for investigating potential skin sensitization using two human keratinocytic and two human dendritic cell lines has been developed (Mehling et al. Arch Toxicol 86:1273–1295, 2012). Since prohaptens may be metabolically activated in the skin, information on xenobiotic metabolizing enzyme (XME) activities in these cell lines is of high interest. In this study, XME activity assays, monitoring metabolite or cofactor, showed the following: all three passages of keratinocytic (KeratinoSens® and LuSens) and dendritic (U937 und THP-1) cells displayed N-acetyltransferase 1 (NAT1) activities (about 6–60 nmol/min/mg S9-protein for acetylation of para-aminobenzoic acid). This is relevant since reactive species of many cosmetics are metabolically controlled by cutaneous NAT1. Esterase activities of about 1–4 nmol fluorescein diacetate/min/mg S9-protein were observed in all passages of investigated keratinocytic and about 1 nmol fluorescein diacetate/min/mg S9-protein in dendritic cell lines. This is also of practical relevance since many esters and amides are detoxified and others activated by cutaneous esterases. In both keratinocytic cell lines, activities of aldehyde dehydrogenase (ALDH) were observed (5–17 nmol product/min/mg cytosolic protein). ALDH is relevant for the detoxication of reactive aldehydes. Activities of several other XME were below detection, namely the investigated cytochrome P450-dependent alkylresorufin O-dealkylases 7-ethylresorufin O-deethylase, 7-benzylresorufin O-debenzylase and 7-pentylresorufin O-depentylase (while NADPH cytochrome c reductase activities were much above the limit of quantification), the flavin-containing monooxygenase, the alcohol dehydrogenase as well as the UDP glucuronosyl transferase activities.

Xenobiotic metabolism capacities of human skin in comparison with a 3D-epidermis model and keratinocyte-based cell culture as in vitro alternatives for chemical testing: phase II enzymes.

PubMed

Götz, Christine; Pfeiffer, Roland; Tigges, Julia; Ruwiedel, Karsten; Hübenthal, Ulrike; Merk, Hans F; Krutmann, Jean; Edwards, Robert J; Abel, Josef; Pease, Camilla; Goebel, Carsten; Hewitt, Nicola; Fritsche, Ellen

2012-05-01

The 7th Amendment to the EU Cosmetics Directive prohibits the use of animals in cosmetic testing for certain endpoints, such as genotoxicity. Therefore, skin in vitro models have to replace chemical testing in vivo. However, the metabolic competence neither of human skin nor of alternative in vitro models has so far been fully characterized, although skin is the first-pass organ for accidentally or purposely (cosmetics and pharmaceuticals) applied chemicals. Thus, there is an urgent need to understand the xenobiotic-metabolizing capacities of human skin and to compare these activities to models developed to replace animal testing. We have measured the activity of the phase II enzymes glutathione S-transferase, UDP-glucuronosyltransferase and N-acetyltransferase in ex vivo human skin, the 3D epidermal model EpiDerm 200 (EPI-200), immortalized keratinocyte-based cell lines (HaCaT and NCTC 2544) and primary normal human epidermal keratinocytes. We show that all three phase II enzymes are present and highly active in skin as compared to phase I. Human skin, therefore, represents a more detoxifying than activating organ. This work systematically compares the activities of three important phase II enzymes in four different in vitro models directly to human skin. We conclude from our studies that 3D epidermal models, like the EPI-200 employed here, are superior over monolayer cultures in mimicking human skin xenobiotic metabolism and thus better suited for dermatotoxicity testing. © 2012 John Wiley & Sons A/S.

Xenobiotic metabolism in the fourth dimension: PARtners in time.

PubMed

Green, Carla B; Takahashi, Joseph S

2006-07-01

A significant portion of the transcriptome in mammals, including the PAR bZIP transcription factors DBP, HLF, and TEF, is under circadian clock control. In this issue of Cell Metabolism, Gachon and colleagues (Gachon et al., 2006) show that disruption of these three genes in mice alters gene expression patterns of many proteins involved in drug metabolism and in liver and kidney responses to xenobiotic agents. Triple mutant mice have severe physiological deficits, including increased hypersensitivity to xenobiotic agents and premature aging, highlighting the profound effect the circadian clock has on this important response system.

Monocrotophos Induces the Expression and Activity of Xenobiotic Metabolizing Enzymes in Pre-Sensitized Cultured Human Brain Cells

PubMed Central

Tripathi, Vinay K.; Kumar, Vivek; Singh, Abhishek K.; Kashyap, Mahendra P.; Jahan, Sadaf; Pandey, Ankita; Alam, Sarfaraz; Khan, Feroz; Khanna, Vinay K.; Yadav, Sanjay; Lohani, Mohtshim; Pant, Aditya B.

2014-01-01

The expression and metabolic profile of cytochrome P450s (CYPs) is largely missing in human brain due to non-availability of brain tissue. We attempted to address the issue by using human brain neuronal (SH-SY5Y) and glial (U373-MG) cells. The expression and activity of CYP1A1, 2B6 and 2E1 were carried out in the cells exposed to CYP inducers viz., 3-methylcholanthrene (3-MC), cyclophosphamide (CPA), ethanol and known neurotoxicant- monocrotophos (MCP), a widely used organophosphorous pesticide. Both the cells show significant induction in the expression and CYP-specific activity against classical inducers and MCP. The induction level of CYPs was comparatively lower in MCP exposed cells than cells exposed to classical inducers. Pre-exposure (12 h) of cells to classical inducers significantly added the MCP induced CYPs expression and activity. The findings were concurrent with protein ligand docking studies, which show a significant modulatory capacity of MCP by strong interaction with CYP regulators-CAR, PXR and AHR. Similarly, the known CYP inducers- 3-MC, CPA and ethanol have also shown significantly high docking scores with all the three studied CYP regulators. The expression of CYPs in neuronal and glial cells has suggested their possible association with the endogenous physiology of the brain. The findings also suggest the xenobiotic metabolizing capabilities of these cells against MCP, if received a pre-sensitization to trigger the xenobiotic metabolizing machinery. MCP induced CYP-specific activity in neuronal cells could help in explaining its effect on neurotransmission, as these CYPs are known to involve in the synthesis/transport of the neurotransmitters. The induction of CYPs in glial cells is also of significance as these cells are thought to be involved in protecting the neurons from environmental insults and safeguard them from toxicity. The data provide better understanding of the metabolizing capability of the human brain cells against xenobiotics

Xenobiotics and the Glucocorticoid Receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gulliver, Linda S M, E-mail: linda.gulliver@otago.

Glucocorticoid Receptor (GR) is present in virtually every human cell type. Representing a nuclear receptor superfamily, GR has several different isoforms essentially acting as ligand-dependent transcription factors, regulating glucocorticoid-responsive gene expression in both a positive and a negative manner. Although the natural ligand of the Glucocorticoid Receptor, glucocorticoids (GC) represent only some of the multiple ligands for GR. Xenobiotics, ubiquitous in the environment, bind to GR and are also capable of activating or repressing GR gene expression, thereby modulating GR cell and tissue-specific downstream effects in a multitude of ways that include responses to inflammatory, allergic, metabolic, neoplastic and autoimmunemore » processes. Many xenobiotics, if inadequately metabolized by xenobiotic metabolizing enzymes and not wholly eliminated, could have deleterious toxic effects with potentially lethal consequences. This review examines GR, the genomic and non-genomic actions of natural and synthetic GC and the body's handling of xenobiotic compounds, before reviewing what is presently known about GR's interactions with many of the more commonly encountered and some of the less well known GR-associated xenobiotics. GR promiscuity and crosstalk with other signaling pathways is discussed, alongside novel roles for GR that include mood disorder and addiction. A knowledge of GR interactions with xenobiotics is increasingly relevant when considering aging populations and the related prevalence of neoplastic disease, together with growing concerns around human exposure to mixtures of chemicals in the environment. Furthermore, escalating rates of obesity, Type 2 diabetes; autoimmune, allergy, addiction and mood disorder-related pathologies, require novel targeted interventions and GR appears a promising pharmacological candidate. - Highlights: • Biological impact of xenobiotics acting through Glucocorticoid Receptor. • Promiscuity of Glucocorticoid

High-throughput metagenomic analysis of petroleum-contaminated soil microbiome reveals the versatility in xenobiotic aromatics metabolism.

PubMed

Bao, Yun-Juan; Xu, Zixiang; Li, Yang; Yao, Zhi; Sun, Jibin; Song, Hui

2017-06-01

The soil with petroleum contamination is one of the most studied soil ecosystems due to its rich microorganisms for hydrocarbon degradation and broad applications in bioremediation. However, our understanding of the genomic properties and functional traits of the soil microbiome is limited. In this study, we used high-throughput metagenomic sequencing to comprehensively study the microbial community from petroleum-contaminated soils near Tianjin Dagang oilfield in eastern China. The analysis reveals that the soil metagenome is characterized by high level of community diversity and metabolic versatility. The metageome community is predominated by γ-Proteobacteria and α-Proteobacteria, which are key players for petroleum hydrocarbon degradation. The functional study demonstrates over-represented enzyme groups and pathways involved in degradation of a broad set of xenobiotic aromatic compounds, including toluene, xylene, chlorobenzoate, aminobenzoate, DDT, methylnaphthalene, and bisphenol. A composite metabolic network is proposed for the identified pathways, thus consolidating our identification of the pathways. The overall data demonstrated the great potential of the studied soil microbiome in the xenobiotic aromatics degradation. The results not only establish a rich reservoir for novel enzyme discovery but also provide putative applications in bioremediation. Copyright © 2016. Published by Elsevier B.V.

[Study of enzymes of xenobiotic metabolism in the evaluation of quality of protein-containing wheat germ flakes and wallpaper flour].

PubMed

Martinchuk, A N; E En Gyn; Safronova, A M; Peskova, E V

1991-01-01

Intake of wheat upholstery meal by growing rats was attended by a sharp decrease in the content and activity of xenobiotic metabolism enzymes in the hepatic microsomes, that was caused by the low biological value of the meal proteins. Hepatic microsomes of the rats that were fed with wheat germ flakes showed increased specific content of cytochromes P-450 and b5, but the total blood protein content per 100 g of body mass was lower than during casein consumption. No significant changes were detected in hydroxylation rate of benz(a)pyrene, aniline and ethylmorphine. During consumption of wheat germ flakes induction of UDP-glucuronide-transferase was detected in hepatic microsomes. Wheat germ flakes induced a 5-fold increase of Se-dependent glutathione peroxidase activity. Wheat germ flakes produced no significant effect on glutathione-S-aryltransferase and glutathione reductase activity.

Polymorphisms in xenobiotic metabolizing enzymes and diet influence colorectal adenoma risk.

PubMed

Northwood, Emma L; Elliott, Faye; Forman, David; Barrett, Jennifer H; Wilkie, Murray J V; Carey, Francis A; Steele, Robert J C; Wolf, Roland; Bishop, Timothy; Smith, Gillian

2010-05-01

We have earlier shown that diet and xenobiotic metabolizing enzyme genotypes influence colorectal cancer risk, and now investigate whether similar associations are seen in patients with premalignant colorectal adenomas (CRA), recruited during the pilot phase of the Scottish Bowel Screening Programme. Nineteen polymorphisms in 13 genes [cytochrome P450 (P450), glutathione S-transferase (GST), N-acetyl transferase, quinone reductase (NQ01) and microsomal epoxide hydrolase (EPHX1) genes] were genotyped using multiplex PCR or Taqman-based allelic discrimination assays and analyzed in conjunction with diet, assessed by food frequency questionnaire, in a case-control study [317 CRA cases (308 cases genotyped), 296 controls]. Findings significant at a nominal 5% level are reported. CRA risk was inversely associated with fruit (P=0.02, test for trend) and vegetable (P=0.001, test for trend) consumption. P450 CYP2C9*3 heterozygotes had reduced CRA risk compared with homozygotes for the reference allele [odds ratio (OR): 0.60; 95% confidence interval (CI): 0.36-0.99], whereas CYP2D6*4 homozygotes (OR: 2.72; 95% CI: 1.18-6.27) and GSTM1 'null' individuals (OR: 1.43; 95% CI: 1.04-1.98) were at increased risk. The protective effect of fruit consumption was confined to GSTP1 (Ala114Val) reference allele homozygotes (OR: 0.49; 95% CI: 0.34-0.71, P=0.03 for interaction). CRA risk was not associated with meat consumption, although a significant interaction between red meat consumption and EPHX1 (His139Arg) genotype was noted (P=0.02 for interaction). We report the novel associations between P450 genotype and CRA risk, and highlight the risk association with GSTM1 genotype, common to our CRA and cancer case-control series. In addition, we report a novel modifying influence of GSTP1 genotype on dietary chemoprevention. These novel findings require independent confirmation.

It’s War Out There: Fighting for life with xenobiotic degrading enzymes

USDA-ARS?s Scientific Manuscript database

It’s War Out There: Fighting for life with xenobiotic degrading enzymes Beta-lactamase enzymes are well studied because of their tremendous impact on medicine. Their prominent role is in resistance to beta-lactam (four membered lactam ring) antibiotics including the first and most famous fungally d...

Paternal nicotine exposure alters hepatic xenobiotic metabolism in offspring

PubMed Central

Vallaster, Markus P; Kukreja, Shweta; Bing, Xin Y; Ngolab, Jennifer; Zhao-Shea, Rubing; Gardner, Paul D; Tapper, Andrew R; Rando, Oliver J

2017-01-01

Paternal environmental conditions can influence phenotypes in future generations, but it is unclear whether offspring phenotypes represent specific responses to particular aspects of the paternal exposure history, or a generic response to paternal ‘quality of life’. Here, we establish a paternal effect model based on nicotine exposure in mice, enabling pharmacological interrogation of the specificity of the offspring response. Paternal exposure to nicotine prior to reproduction induced a broad protective response to multiple xenobiotics in male offspring. This effect manifested as increased survival following injection of toxic levels of either nicotine or cocaine, accompanied by hepatic upregulation of xenobiotic processing genes, and enhanced drug clearance. Surprisingly, this protective effect could also be induced by a nicotinic receptor antagonist, suggesting that xenobiotic exposure, rather than nicotinic receptor signaling, is responsible for programming offspring drug resistance. Thus, paternal drug exposure induces a protective phenotype in offspring by enhancing metabolic tolerance to xenobiotics. DOI: http://dx.doi.org/10.7554/eLife.24771.001 PMID:28196335

Xenobiotic-metabolizing enzymes in the skin of rat, mouse, pig, guinea pig, man, and in human skin models.

PubMed

Oesch, F; Fabian, E; Guth, K; Landsiedel, R

2014-12-01

The exposure of the skin to medical drugs, skin care products, cosmetics, and other chemicals renders information on xenobiotic-metabolizing enzymes (XME) in the skin highly interesting. Since the use of freshly excised human skin for experimental investigations meets with ethical and practical limitations, information on XME in models comes in the focus including non-human mammalian species and in vitro skin models. This review attempts to summarize the information available in the open scientific literature on XME in the skin of human, rat, mouse, guinea pig, and pig as well as human primary skin cells, human cell lines, and reconstructed human skin models. The most salient outcome is that much more research on cutaneous XME is needed for solid metabolism-dependent efficacy and safety predictions, and the cutaneous metabolism comparisons have to be viewed with caution. Keeping this fully in mind at least with respect to some cutaneous XME, some models may tentatively be considered to approximate reasonable closeness to human skin. For dermal absorption and for skin irritation among many contributing XME, esterase activity is of special importance, which in pig skin, some human cell lines, and reconstructed skin models appears reasonably close to human skin. With respect to genotoxicity and sensitization, activating XME are not yet judgeable, but reactive metabolite-reducing XME in primary human keratinocytes and several reconstructed human skin models appear reasonably close to human skin. For a more detailed delineation and discussion of the severe limitations see the "Overview and Conclusions" section in the end of this review.

Rb and p53 Liver Functions Are Essential for Xenobiotic Metabolism and Tumor Suppression

PubMed Central

Nantasanti, Sathidpak; Toussaint, Mathilda J. M.; Youssef, Sameh A.; Tooten, Peter C. J.; de Bruin, Alain

2016-01-01

The tumor suppressors Retinoblastoma (Rb) and p53 are frequently inactivated in liver diseases, such as hepatocellular carcinomas (HCC) or infections with Hepatitis B or C viruses. Here, we discovered a novel role for Rb and p53 in xenobiotic metabolism, which represent a key function of the liver for metabolizing therapeutic drugs or toxins. We demonstrate that Rb and p53 cooperate to metabolize the xenobiotic 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). DDC is metabolized mainly by cytochrome P450 (Cyp)3a enzymes resulting in inhibition of heme synthesis and accumulation of protoporphyrin, an intermediate of heme pathway. Protoporphyrin accumulation causes bile injury and ductular reaction. We show that loss of Rb and p53 resulted in reduced Cyp3a expression decreased accumulation of protoporphyrin and consequently less ductular reaction in livers of mice fed with DDC for 3 weeks. These findings provide strong evidence that synergistic functions of Rb and p53 are essential for metabolism of DDC. Because Rb and p53 functions are frequently disabled in liver diseases, our results suggest that liver patients might have altered ability to remove toxins or properly metabolize therapeutic drugs. Strikingly the reduced biliary injury towards the oxidative stress inducer DCC was accompanied by enhanced hepatocellular injury and formation of HCCs in Rb and p53 deficient livers. The increase in hepatocellular injury might be related to reduce protoporphyrin accumulation, because protoporphrin is well known for its anti-oxidative activity. Furthermore our results indicate that Rb and p53 not only function as tumor suppressors in response to carcinogenic injury, but also in response to non-carcinogenic injury such as DDC. PMID:26967735

Comparative liver accumulation of dioxin-like compounds in sheep and cattle: Possible role of AhR-mediated xenobiotic metabolizing enzymes.

PubMed

Girolami, F; Spalenza, V; Benedetto, A; Manzini, L; Badino, P; Abete, M C; Nebbia, C

2016-11-15

PCDDs, PCDFs, and PCBs are persistent organic pollutants (POPs) that accumulate in animal products and may pose serious health problems. Those able to bind the aryl hydrocarbon receptor (AhR), eliciting a plethora of toxic responses, are defined dioxin-like (DL) compounds, while the remainders are called non-DL (NDL). An EFSA opinion has highlighted the tendency of ovine liver to specifically accumulate DL-compounds to a greater extent than any other farmed ruminant species. To examine the possible role in such an accumulation of xenobiotic metabolizing enzymes (XME) involved in DL-compound biotransformation, liver samples were collected from ewes and cows reared in an area known for low dioxin contamination. A related paper reported that sheep livers had about 5-fold higher DL-compound concentrations than cattle livers, while the content of the six marker NDL-PCBs did not differ between species. Specimens from the same animals were subjected to gene expression analysis for AhR, AhR nuclear translocator (ARNT) and AhR-dependent oxidative and conjugative pathways; XME protein expression and activities were also investigated. Both AhR and ARNT mRNA levels were about 2-fold lower in ovine samples and the same occurred for CYP1A1 and CYP1A2, being approximately 3- and 9-fold less expressed in sheep compared to cattle, while CYP1B1 could be detectable in cattle only. The results of the immunoblotting and catalytic activity (most notably EROD) measurements of the CYP1A family enzymes were in line with the gene expression data. By contrast, phase II enzyme expression and activities in sheep were higher (UGT1A) or similar (GSTA1, NQO1) to those recorded in cattle. The overall low expression of CYP1 family enzymes in the sheep is in line with the observed liver accumulation of DL-compounds and is expected to affect the kinetics and the dynamics of other POPs such as many polycyclic aromatic hydrocarbons, as well as of toxins (e.g. aflatoxins) or drugs (e.g. benzimidazole

Altered carbohydrate, lipid, and xenobiotic metabolism by liver from rats flown on Cosmos 1887

NASA Technical Reports Server (NTRS)

Merrill, A. H. Jr; Hoel, M.; Wang, E.; Mullins, R. E.; Hargrove, J. L.; Jones, D. P.; Popova, I. A.; Merrill AH, J. r. (Principal Investigator)

1990-01-01

To determine the possible biochemical effects of prolonged weightlessness on liver function, samples of liver from rats that had flown aboard Cosmos 1887 were analyzed for protein, glycogen, and lipids as well as the activities of a number of key enzymes involved in metabolism of these compounds and xenobiotics. Among the parameters measured, the major differences were elevations in the glycogen content and hydroxymethylglutaryl-CoA (HMG-CoA) reductase activities for the rats flown on Cosmos 1887 and decreases in the amount of microsomal cytochrome P-450 and the activities of aniline hydroxylase and ethylmorphine N-demethylase, cytochrome P-450-dependent enzymes. These results support the earlier finding of differences in these parameters and suggest that altered hepatic function could be important during spaceflight and/or the postflight recovery period.

Knockout of cytochrome P450 3A yields new mouse models for understanding xenobiotic metabolism

PubMed Central

van Herwaarden, Antonius E.; Wagenaar, Els; van der Kruijssen, Cornelia M.M.; van Waterschoot, Robert A.B.; Smit, Johan W.; Song, Ji-Ying; van der Valk, Martin A.; van Tellingen, Olaf; van der Hoorn, José W.A.; Rosing, Hilde; Beijnen, Jos H.; Schinkel, Alfred H.

2007-01-01

Cytochrome P450 3A (CYP3A) enzymes constitute an important detoxification system that contributes to primary metabolism of more than half of all prescribed medications. To investigate the physiological and pharmacological roles of CYP3A, we generated Cyp3a-knockout (Cyp3a–/–) mice lacking all functional Cyp3a genes. Cyp3a–/– mice were viable, fertile, and without marked physiological abnormalities. However, these mice exhibited severely impaired detoxification capacity when exposed to the chemotherapeutic agent docetaxel, displaying higher exposure levels in response to both oral and intravenous administration. These mice also demonstrated increased sensitivity to docetaxel toxicity, suggesting a primary role for Cyp3a in xenobiotic detoxification. To determine the relative importance of intestinal versus hepatic Cyp3a in first-pass metabolism, we generated transgenic Cyp3a–/– mice expressing human CYP3A4 in either the intestine or the liver. Expression of CYP3A4 in the intestine dramatically decreased absorption of docetaxel into the bloodstream, while hepatic expression aided systemic docetaxel clearance. These results suggest that CYP3A expression determines impairment of drug absorption and efficient systemic clearance in a tissue-specific manner. The genetic models used in this study provide powerful tools to further study CYP3A-mediated xenobiotic metabolism, as well as interactions between CYP3A and other detoxification systems. PMID:17975676

Functioning of Microsomal Cytochrome P450s: Murburn Concept Explains the Metabolism of Xenobiotics in Hepatocytes.

PubMed

Manoj, Kelath Murali; Parashar, Abhinav; Gade, Sudeep K; Venkatachalam, Avanthika

2016-01-01

Using oxygen and NADPH, the redox enzymes cytochrome P450 (CYP) and its reductase (CPR) work in tandem to carry out the phase I metabolism of a vast majority of drugs and xenobiotics. As per the erstwhile understanding of the catalytic cycle, binding of the substrate to CYP's heme distal pocket allows CPR to pump electrons through a CPR-CYP complex. In turn, this trigger (a thermodynamic push of electrons) leads to the activation of oxygen at CYP's heme-center, to give Compound I, a two-electron deficient enzyme reactive intermediate. The formation of diffusible radicals and reactive oxygen species (DROS, hitherto considered an undesired facet of the system) was attributed to the heme-center. Recently, we had challenged these perceptions and proposed the murburn ("mured burning" or "mild unrestricted burning") concept to explain heme enzymes' catalytic mechanism, electron-transfer phenomena and the regulation of redox equivalents' consumption. Murburn concept incorporates a one-electron paradigm, advocating obligatory roles for DROS. The new understanding does not call for high-affinity substrate-binding at the heme distal pocket of the CYP (the first and the most crucial step of the erstwhile paradigm) or CYP-CPR protein-protein complexations (the operational backbone of the erstwhile cycle). Herein, the dynamics of reduced nicotinamide nucleotides' consumption, peroxide formation and depletion, product(s) formation, etc. was investigated with various controls, by altering reaction variables, environments and through the incorporation of diverse molecular probes. In several CYP systems, control reactions lacking the specific substrate showed comparable or higher peroxide in milieu, thereby discrediting the foundations of the erstwhile hypothesis. The profiles obtained by altering CYP:CPR ratios and the profound inhibitions observed upon the incorporation of catalytic amounts of horseradish peroxidase confirm the obligatory roles of DROS in milieu, ratifying

Cytotoxic effects and aromatase inhibition by xenobiotic endocrine disrupters alone and in combination.

PubMed

Benachour, Nora; Moslemi, Safa; Sipahutar, Herbert; Seralini, Gilles-Eric

2007-07-15

Xenobiotics may cause long-term adverse effects in humans, especially at the embryonic level, raising questions about their levels of exposure, combined effects, and crucial endpoints. We are interested in the possible interactions between xenobiotic endocrine disrupters, cellular viability and androgen metabolism. Accordingly, we tested aroclor 1254 (A1254), atrazine (AZ), o,p'-DDT, vinclozolin (VZ), p,p'-DDE, bisphenol A (BPA), chlordecone (CD), nonylphenol (NP), tributylin oxide (TBTO), and diethylstilbestrol (DES) for cellular toxicity against human embryonic 293 cells, and activity against cellular aromatase, but also on placental microsomes and on the purified equine enzyme. Cellular viability was affected in 24 h by all the xenobiotics with a threshold at 50 microM (except for TBTO and DES, 10 microM threshold), and aromatase was inhibited at non-toxic doses. In combination synergism was observed reducing the threshold values of toxicity to 4-10 microM, and aromatase activity by 50% in some cases. In placental microsomes the most active xenobiotics rapidly inhibited microsomal aromatase in a manner independent of NADPH metabolism. Prolonged exposures to low doses in cells generally amplified by 50 times aromatase inhibition. These xenobiotics may act by inhibition of the active site or by allosteric effects on the enzyme. Bioaccumulation is a feature of some xenobiotics, especially chlordecone, DDT and DDE, and low level chronic exposures can also affect cell signaling mechanisms. This new information about the mechanism of action of these xenobiotics will assist in improved molecular design with a view to providing safer compounds for use in the (human) environment.

Interference with xenobiotic metabolic activity by the commonly used vehicle solvents dimethylsulfoxide and methanol in zebrafish (Danio rerio) larvae but not Daphnia magna

PubMed Central

David, Rhiannon M.; Jones, Huw S.; Panter, Grace H.; Winter, Matthew J.; Hutchinson, Thomas H.; Kevin Chipman, J.

2012-01-01

Organic solvents, such as dimethylsulfoxide (DMSO) and methanol are widely used as vehicles to solubilise lipophilic test compounds in toxicity testing. However, the effects of such solvents upon innate detoxification processes in aquatic organisms are poorly understood. This study assessed the effect of solvent exposure upon cytochrome P450 (CYP)-mediated xenobiotic metabolism in Daphnia magna and zebrafish larvae (4 d post fertilisation). Adult D. magna were demonstrated to have a low, but detectable, metabolism of ethoxyresorufin in vivo and this activity was not modulated by pre-exposure to DMSO or methanol (24 h, up to 0.1% and 0.05% v/v, respectively). In contrast, the metabolism of ethoxyresorufin in zebrafish larvae was significantly reduced by both solvents (0.1% and 0.05% v/v, respectively) after 24 h of exposure. In zebrafish, these observed decreases in activity towards ethoxyresorufin were accompanied by decreased expression of a variety of genes coding for drug metabolising enzymes (corresponding to CYP1, CYP2, CYP3 and UDP-glucuronyl transferase [UGT] family enzymes), measured by quantitative PCR. Reduction of gene expression and CYP1 enzyme activities by methanol (0.05% v/v) in zebrafish larvae was partially reversed by co-exposure with Aroclor 1254 (100 μg L−1). Overall this study suggests that relatively low concentrations of organic solvents can impact upon the biotransformation of certain xenobiotics in zebrafish larvae, and that this warrants consideration when assessing compounds for metabolism and toxicity in this species. PMID:22472102

A QUANTITATIVE MODEL FOR XENOBIOTIC METABOLIZING ENZYME (XME) INDUCTION REGULATED BY THE PREGNANE X RECEPTOR (PXR)

EPA Science Inventory

The nuclear receptor, PXR, is an integral part of the regulation of hepatic metabolism. It has been shown to regulate specific CYPs (phase I drug-metabolizing enzymes) as well as certain phase II drug metabolism activities, including UDP-glucuronosyl transferase (UGT), sulfotran...

Xenobiotic metabolism capacities of human skin in comparison with a 3D epidermis model and keratinocyte-based cell culture as in vitro alternatives for chemical testing: activating enzymes (Phase I).

PubMed

Götz, Christine; Pfeiffer, Roland; Tigges, Julia; Blatz, Veronika; Jäckh, Christine; Freytag, Eva-Maria; Fabian, Eric; Landsiedel, Robert; Merk, Hans F; Krutmann, Jean; Edwards, Robert J; Pease, Camilla; Goebel, Carsten; Hewitt, Nicola; Fritsche, Ellen

2012-05-01

Skin is important for the absorption and metabolism of exposed chemicals such as cosmetics or pharmaceuticals. The Seventh Amendment to the EU Cosmetics Directive prohibits the use of animals for cosmetic testing for certain endpoints, such as genotoxicity; therefore, there is an urgent need to understand the xenobiotic metabolizing capacities of human skin and to compare these activities with reconstructed 3D skin models developed to replace animal testing. We have measured Phase I enzyme activities of cytochrome P450 (CYP) and cyclooxygenase (COX) in ex vivo human skin, the 3D skin model EpiDerm™ (EPI-200), immortalized keratinocyte-based cell lines and primary normal human epidermal keratinocytes. Our data demonstrate that basal CYP enzyme activities are very low in whole human skin and EPI-200 as well as keratinocytes. In addition, activities in monolayer cells differed from organotypic tissues after induction. COX activity was similar in skin, EPI-200 and NHEK cells, but was significantly lower in immortalized keratinocytes. Hence, the 3D model EPI-200 might represent a more suitable model for dermatotoxicological studies. Altogether, these data help to better understand skin metabolism and expand the knowledge of in vitro alternatives used for dermatotoxicity testing. © 2012 John Wiley & Sons A/S.

Effect of sprout extract from Tuscan black cabbage on xenobiotic-metabolizing and antioxidant enzymes in rat liver.

PubMed

Melega, Simone; Canistro, Donatella; Pagnotta, Eleonora; Iori, Renato; Sapone, Andrea; Paolini, Moreno

2013-02-18

In recent years, health protection by natural products has received considerable attention, and a multitude of nutraceuticals have been characterized and their use promoted. Dietary consumption of Cruciferous vegetables, rich in glucosinolates (GLs), and their myrosinase-mediated hydrolysis products isothiocyanates (ITCs), were associated with reductions in cancer risk. In this study, the chemo-preventive potential of sprout extract of Tuscan black cabbage (Brassica oleracea L. var. acephala subvar. Laciniata L.) (TBCSE), through modulation of the xenobiotic-metabolizing apparatus and antioxidant defenses, was investigated in Sprague-Dawley rat liver. TBCSE was administered either orally or intraperitoneally, at a dose of 15mg/kg b.w., daily for twenty-one consecutive days, in the absence or presence of exogenous myrosinase, β-thioglucoside glucohydrolase (MYR), to distinguish the effects of intact GLs and ITCs, in the context of the extract. A complex, mild modulation pattern of P450-related monooxygenases was observed, mainly regarding CYP content (up to 36% loss), NADPH cytochrome (P450) c-reductase (up to 26% loss), CYP1A1 (up to 23% loss), but no evident distinctions among the effects of the extracts containing GLs or ITCs, were noted. In contrast, significant inductions of phase-II enzymes (up to 107% for UDP-glucuronosyl-transferase, and up to 36% for glutathione S-transferase) were recorded only where the GLs to ITCs conversion had occurred. A boosting effect on catalase (up to 38%), NAD(P)H:quinone reductase (up to 70%), glutathione reductase and glutathione peroxidase (up to 10%) was also recorded, suggesting an indirect antioxidant capacity of the extracts. Overall, the general phase-I inhibition, together with the up-regulation of detoxifying phase-II and antioxidant enzymes, exerted by the TBCSE supplementation, seem to be in line with the classical chemopreventive theory, but whether the addition of exogenous MYR is relevant, still remains to be

The Chemically Inducible Plant Cytochrome P450 CYP76B1 Actively Metabolizes Phenylureas and Other Xenobiotics1

PubMed Central

Robineau, Tiburce; Batard, Yannick; Nedelkina, Svetlana; Cabello-Hurtado, Francisco; LeRet, Monique; Sorokine, Odile; Didierjean, Luc; Werck-Reichhart, Danièle

1998-01-01

Cytochrome P450s (P450s) constitute one of the major classes of enzymes that are responsible for detoxification of exogenous molecules both in animals and plants. On the basis of its inducibility by exogenous chemicals, we recently isolated a new plant P450, CYP76B1, from Jerusalem artichoke (Helianthus tuberosus) and showed that it was capable of dealkylating a model xenobiotic compound, 7-ethoxycoumarin. In the present paper we show that CYP76B1 is more strongly induced by foreign compounds than other P450s isolated from the same plant, and metabolizes with high efficiency a wide range of xenobiotics, including alkoxycoumarins, alkoxyresorufins, and several herbicides of the class of phenylureas. CYP76B1 catalyzes the double N-dealkylation of phenylureas with turnover rates comparable to those reported for physiological substrates and produces nonphytotoxic compounds. Potential uses for CYP76B1 thus include control of herbicide tolerance and selectivity, as well as soil and groundwater bioremediation. PMID:9808750

Characterization and profiling of hepatic cytochromes P450 and phase II xenobiotic-metabolizing enzymes in beluga whales (Delphinapterus leucas) from the St. Lawrence River Estuary and the Canadian Arctic.

PubMed

McKinney, Melissa A; Arukwe, Augustine; De Guise, Sylvain; Martineau, Daniel; Béland, Pierre; Dallaire, André; Lair, Stéphane; Lebeuf, Michel; Letcher, Robert J

2004-07-30

Cytochromes P450 (CYP, phase I) and conjugating (phase II) enzymes can be induced by and influence the toxicokinetics (metabolism) and toxicity of xenobiotic contaminants in exposed organisms. Beluga whale (Delphinapterus leucas) from the endangered St. Lawrence (SL) River Estuary population exhibit deleterious health effects and various severe pathologies that have been associated with contaminant exposure. In contrast, such effects (e.g. reproductive and immunological impairment) are generally less frequent in less exposed populations in the Canadian Arctic (CA). In the present study, opportunistic sampling resulted in the collection immediately after death of liver tissue from a single female neonate SL beluga (SL6) and male and female CA beluga (n=10) from the Arviat region of western Hudson Bay, in addition to sampling of stranded carcasses of male and female SL beluga (n=5) at least 12 h postmortem. We immunologically characterized cross-reactive proteins of hepatic microsomal CYP1A, CYP2B, CYP3A, CYP2E, epoxide hydrolase (EH) and uridine diphosphoglucuronosyl transferase (UDPGT) isozymes. Cross-reactive proteins were found in all SL and CA beluga using anti-rat CYP1A1, anti-rainbow trout CYP3A, anti-human CYP2E1, anti-rabbit EH and anti-human UDPGT1A1 polyclonal antibodies (Abs), whereas faintly cross-reactive CYP2B proteins were only found in SL6 and the CA samples using an anti-rabbit CYP2B1 Ab. In corresponding catalytic activity assessments, only SL6 and all CA beluga microsomal samples exhibited CYP1A-mediated 7-ethoxyresorufin O-deethylase (EROD) activity (51-260 pmol/mg/min), CYP3A-mediated activity (113-899 pmol/mg/min) based on the formation of 6beta-hydroxytestosterone using a testosterone hydroxylase assay, and UDPGT activity (830-4956 pmol/mg/min) based on 1-naphthylglucuronide formation. The marginal cross-reactivity with the anti-CYP2B1 Ab and lack of catalytically measurable hydroxytestosterone isomers associated with CYP2B-type activity in

Retrofit Strategies for Incorporating Xenobiotic Metabolism into High Throughput Screening Assays (EMGS)

EPA Science Inventory

The US EPA’s ToxCast program is designed to assess chemical perturbations of molecular and cellular endpoints using a variety of high-throughput screening (HTS) assays. However, existing HTS assays have limited or no xenobiotic metabolism which could lead to a mischaracterization...

Pregnane xenobiotic receptor in cancer pathogenesis and therapeutic response

PubMed Central

Pondugula, Satyanarayana R.; Mani, Sridhar

2012-01-01

Pregnane xenobiotic receptor (PXR) is an orphan nuclear receptor that regulates the metabolism of endobiotics and xenobiotics. PXR is promiscuous and unique in that it is activated by a diverse group of xenochemicals, including therapeutic anticancer drugs and naturally-occurring endocrine disruptors. PXR has been predominantly studied to understand its regulatory role in xenobiotic clearance in liver and intestine via induction of drug metabolizing enzymes and drug transporters. PXR, however, is widely expressed and has functional implications in other normal and malignant tissues, including breast, prostate, ovary, endometrium and bone. The differential expression of PXR and its target genes in cancer tissues has been suggested to determine the prognosis of chemotherapeutic outcome. In addition, the emerging evidence points to the implications of PXR in regulating apoptotic and antiapoptotic as well as growth factor signaling that promote tumor proliferation and metastasis. In this review, we highlight the recent progress made in understanding the role of PXR in cancer, discuss the future directions to further understand the mechanistic role of PXR in cancer, and conclude with the need to identify novel selective PXR modulators. PMID:22939994

Current knowledge of detoxification mechanisms of xenobiotic in honey bees.

PubMed

Gong, Youhui; Diao, Qingyun

2017-01-01

The western honey bee Apis mellifera is the most important managed pollinator species in the world. Multiple factors have been implicated as potential causes or factors contributing to colony collapse disorder, including honey bee pathogens and nutritional deficiencies as well as exposure to pesticides. Honey bees' genome is characterized by a paucity of genes associated with detoxification, which makes them vulnerable to specific pesticides, especially to combinations of pesticides in real field environments. Many studies have investigated the mechanisms involved in detoxification of xenobiotics/pesticides in honey bees, from primal enzyme assays or toxicity bioassays to characterization of transcript gene expression and protein expression in response to xenobiotics/insecticides by using a global transcriptomic or proteomic approach, and even to functional characterizations. The global transcriptomic and proteomic approach allowed us to learn that detoxification mechanisms in honey bees involve multiple genes and pathways along with changes in energy metabolism and cellular stress response. P450 genes, is highly implicated in the direct detoxification of xenobiotics/insecticides in honey bees and their expression can be regulated by honey/pollen constitutes, resulting in the tolerance of honey bees to other xenobiotics or insecticides. P450s is also a key detoxification enzyme that mediate synergism interaction between acaricides/insecticides and fungicides through inhibition P450 activity by fungicides or competition for detoxification enzymes between acaricides. With the wide use of insecticides in agriculture, understanding the detoxification mechanism of insecticides in honey bees and how honeybees fight with the xenobiotis or insecticides to survive in the changing environment will finally benefit honeybees' management.

Systems approaches evaluating the perturbation of xenobiotic metabolism in response to cigarette smoke exposure in nasal and bronchial tissues.

PubMed

Iskandar, Anita R; Martin, Florian; Talikka, Marja; Schlage, Walter K; Kostadinova, Radina; Mathis, Carole; Hoeng, Julia; Peitsch, Manuel C

2013-01-01

Capturing the effects of exposure in a specific target organ is a major challenge in risk assessment. Exposure to cigarette smoke (CS) implicates the field of tissue injury in the lung as well as nasal and airway epithelia. Xenobiotic metabolism in particular becomes an attractive tool for chemical risk assessment because of its responsiveness against toxic compounds, including those present in CS. This study describes an efficient integration from transcriptomic data to quantitative measures, which reflect the responses against xenobiotics that are captured in a biological network model. We show here that our novel systems approach can quantify the perturbation in the network model of xenobiotic metabolism. We further show that this approach efficiently compares the perturbation upon CS exposure in bronchial and nasal epithelial cells in vivo samples obtained from smokers. Our observation suggests the xenobiotic responses in the bronchial and nasal epithelial cells of smokers were similar to those observed in their respective organotypic models exposed to CS. Furthermore, the results suggest that nasal tissue is a reliable surrogate to measure xenobiotic responses in bronchial tissue.

Systems Approaches Evaluating the Perturbation of Xenobiotic Metabolism in Response to Cigarette Smoke Exposure in Nasal and Bronchial Tissues

PubMed Central

Iskandar, Anita R.; Martin, Florian; Talikka, Marja; Schlage, Walter K.; Mathis, Carole; Hoeng, Julia; Peitsch, Manuel C.

2013-01-01

Capturing the effects of exposure in a specific target organ is a major challenge in risk assessment. Exposure to cigarette smoke (CS) implicates the field of tissue injury in the lung as well as nasal and airway epithelia. Xenobiotic metabolism in particular becomes an attractive tool for chemical risk assessment because of its responsiveness against toxic compounds, including those present in CS. This study describes an efficient integration from transcriptomic data to quantitative measures, which reflect the responses against xenobiotics that are captured in a biological network model. We show here that our novel systems approach can quantify the perturbation in the network model of xenobiotic metabolism. We further show that this approach efficiently compares the perturbation upon CS exposure in bronchial and nasal epithelial cells in vivo samples obtained from smokers. Our observation suggests the xenobiotic responses in the bronchial and nasal epithelial cells of smokers were similar to those observed in their respective organotypic models exposed to CS. Furthermore, the results suggest that nasal tissue is a reliable surrogate to measure xenobiotic responses in bronchial tissue. PMID:24224167

Human extrahepatic cytochromes P450: function in xenobiotic metabolism and tissue-selective chemical toxicity in the respiratory and gastrointestinal tracts.

PubMed

Ding, Xinxin; Kaminsky, Laurence S

2003-01-01

Cytochrome P450 (CYP) enzymes in extrahepatic tissues often play a dominant role in target tissue metabolic activation of xenobiotic compounds. They may also determine drug efficacy and influence the tissue burden of foreign chemicals or bioavailability of therapeutic agents. This review focuses on xenobiotic-metabolizing CYPs of the human respiratory and gastrointestinal tracts, including the lung, trachea, nasal respiratory and olfactory mucosa, esophagus, stomach, small intestine, and colon. Many CYPs are expressed in one or more of these organs, including CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2F1, CYP2J2, CYP2S1, CYP3A4, CYP3A5, and CYP4B1. Of particular interest are the preferential expression of certain CYPs in the respiratory tract and the regional differences in CYP expression profile in different parts of the gastrointestinal tract. Current research activities on the characterization of CYP expression, function, and regulation in these tissues, as well as future research needs, are discussed.

Metagenomic analysis of the pinewood nematode microbiome reveals a symbiotic relationship critical for xenobiotics degradation

PubMed Central

Cheng, Xin-Yue; Tian, Xue-Liang; Wang, Yun-Sheng; Lin, Ren-Miao; Mao, Zhen-Chuan; Chen, Nansheng; Xie, Bing-Yan

2013-01-01

Our recent research revealed that pinewood nematode (PWN) possesses few genes encoding enzymes for degrading α-pinene, which is the main compound in pine resin. In this study, we examined the role of PWN microbiome in xenobiotics detoxification by metagenomic and bacteria culture analyses. Functional annotation of metagenomes illustrated that benzoate degradation and its related metabolisms may provide the main metabolic pathways for xenobiotics detoxification in the microbiome, which is obviously different from that in PWN that uses cytochrome P450 metabolism as the main pathway for detoxification. The metabolic pathway of degrading α-pinene is complete in microbiome, but incomplete in PWN genome. Experimental analysis demonstrated that most of tested cultivable bacteria can not only survive the stress of 0.4% α-pinene, but also utilize α-pinene as carbon source for their growth. Our results indicate that PWN and its microbiome have established a potentially mutualistic symbiotic relationship with complementary pathways in detoxification metabolism. PMID:23694939

Survey of Human Oxidoreductases and Cytochrome P450 Enzymes Involved in the Metabolism of Xenobiotic and Natural Chemicals

PubMed Central

2015-01-01

Analyzing the literature resources used in our previous reports, we calculated the fractions of the oxidoreductase enzymes FMO (microsomal flavin-containing monooxygenase), AKR (aldo-keto reductase), MAO (monoamine oxidase), and cytochrome P450 participating in metabolic reactions. The calculations show that the fractions of P450s involved in the metabolism of all chemicals (general chemicals, natural, and physiological compounds, and drugs) are rather consistent in the findings that >90% of enzymatic reactions are catalyzed by P450s. Regarding drug metabolism, three-fourths of the human P450 reactions can be accounted for by a set of five P450s: 1A2, 2C9, 2C19, 2D6, and 3A4, and the largest fraction of the P450 reactions is catalyzed by P450 3A enzymes. P450 3A4 participation in metabolic reactions of drugs varied from 13% for general chemicals to 27% for drugs. PMID:25485457

Foreign compounds and intermediary metabolism: sulfoxidation bridges the divide.

PubMed

Mitchell, S C; Steventon, G B

2009-03-01

It is widely appreciated that as a xenobiotic travels through an organism and interacts with the biochemical machinery of a living system, it most probably will undergo a number of metabolic alterations usually leading to a cluster of differing chemical species. Indeed, the modern 'metabonomic' approach, where earlier studied drug metabolism profiles have been reassessed, has indicated that there are normally many more previously unrecognised minor metabolites, and when all such biotransformation products are considered, then their total number is legion. It is now being recognised also that the same metabolic alteration of a substrate, especially a xenobiotic substrate, may be catalysed by more than one enzyme and that the previously sacrosanct notion of an enzyme's 'substrate specificity' may well be inverted to read a substrate's 'enzyme preference'. The following brief article attempts to highlight another aspect where our general acceptance of the 'status quo' needs to be reconsidered. The conventionally acknowledged division between the collection of enzymes that undertake intermediary metabolism and the group of enzymes responsible for xenobiotic metabolism may be becoming blurred. It may well be a prudent time to reassess the current dichotomous view. Overcoming inertia, with a realignment of ideas or alteration of perception, may permit new concepts to emerge leading to a more profound understanding and hopefully eventual benefits for mankind.

Xenobiotica-metabolizing enzymes in the skin of rat, mouse, pig, guinea pig, man, and in human skin models.

PubMed

Oesch, F; Fabian, E; Landsiedel, Robert

2018-06-18

Studies on the metabolic fate of medical drugs, skin care products, cosmetics and other chemicals intentionally or accidently applied to the human skin have become increasingly important in order to ascertain pharmacological effectiveness and to avoid toxicities. The use of freshly excised human skin for experimental investigations meets with ethical and practical limitations. Hence information on xenobiotic-metabolizing enzymes (XME) in the experimental systems available for pertinent studies compared with native human skin has become crucial. This review collects available information of which-taken with great caution because of the still very limited data-the most salient points are: in the skin of all animal species and skin-derived in vitro systems considered in this review cytochrome P450 (CYP)-dependent monooxygenase activities (largely responsible for initiating xenobiotica metabolism in the organ which provides most of the xenobiotica metabolism of the mammalian organism, the liver) are very low to undetectable. Quite likely other oxidative enzymes [e.g. flavin monooxygenase, COX (cooxidation by prostaglandin synthase)] will turn out to be much more important for the oxidative xenobiotic metabolism in the skin. Moreover, conjugating enzyme activities such as glutathione transferases and glucuronosyltransferases are much higher than the oxidative CYP activities. Since these conjugating enzymes are predominantly detoxifying, the skin appears to be predominantly protected against CYP-generated reactive metabolites. The following recommendations for the use of experimental animal species or human skin in vitro models may tentatively be derived from the information available to date: for dermal absorption and for skin irritation esterase activity is of special importance which in pig skin, some human cell lines and reconstructed skin models appears reasonably close to native human skin. With respect to genotoxicity and sensitization reactive

Polymorphisms in xenobiotic metabolizing genes, intakes of heterocyclic amines and red meat, and postmenopausal breast cancer

PubMed Central

Lee, Hae-Jeung; Wu, Kana; Cox, David G.; Hunter, David; Hankinson, Susan E.; Willett, Walter C.; Sinha, Rashmi; Cho, Eunyoung

2013-01-01

Heterocyclic amines (HCAs) are mutagenic compounds generated when meats are cooked at high temperature and for long duration. The findings from previous studies on the relation between HCAs and breast cancer are inconsistent, possibly due to genetic variations in the enzymes metabolizing HCAs. To evaluate whether the associations of intakes of estimated HCAs, meat-derived mutagenicity (MDM), and red meat with risk of postmenopausal breast cancer were modified by N-acetyltransferase 2 (NAT2) acetylator genotype or cytochrome P450 1A2 -164 A/C (CYP1A2) polymorphism, we conducted a nested case-control study with 579 cases and 981 controls within a prospective cohort, the Nurses’ Health Study (NHS). HCAs and MDM intakes were derived using a cooking method questionnaire administered in 1996. NAT2 acetylator genotype, the CYP1A2 polymorphism, and intakes of HCAs, MDM, and red meat were not associated with risk of postmenopausal breast cancer. There was also no interaction between NAT2 acetylator genotype or CYP1A2 polymorphism and HCAs and MDM and red meat intake in relation to breast cancer. These results do not support the hypothesis that genetic polymorphisms of xenobiotic enzymes involved in the metabolism of HCAs may modify the associations between intakes of red meat or meat-related mutagens and breast cancer risk. PMID:24099317

Transgenic plants for enhanced biodegradation and phytoremediation of organic xenobiotics.

PubMed

Abhilash, P C; Jamil, Sarah; Singh, Nandita

2009-01-01

, PCBs etc. Another approach to enhancing phytoremediation ability is the construction of plants that secrete chemical degrading enzymes into the rhizosphere. Recent studies revealed that accelerated ethylene production in response to stress induced by contaminants is known to inhibit root growth and is considered as major limitation in improving phytoremediation efficiency. However, this can be overcome by the selective expression of bacterial ACC deaminase (which regulates ethylene levels in plants) in plants together with multiple genes for the different phases of xenobiotic degradation. This review examines the recent developments in use of transgenic-plants for the enhanced metabolism, degradation and phytoremediation of organic xenobiotics and its future directions.

Synthetic metabolism: metabolic engineering meets enzyme design.

PubMed

Erb, Tobias J; Jones, Patrik R; Bar-Even, Arren

2017-04-01

Metabolic engineering aims at modifying the endogenous metabolic network of an organism to harness it for a useful biotechnological task, for example, production of a value-added compound. Several levels of metabolic engineering can be defined and are the topic of this review. Basic 'copy, paste and fine-tuning' approaches are limited to the structure of naturally existing pathways. 'Mix and match' approaches freely recombine the repertoire of existing enzymes to create synthetic metabolic networks that are able to outcompete naturally evolved pathways or redirect flux toward non-natural products. The space of possible metabolic solution can be further increased through approaches including 'new enzyme reactions', which are engineered on the basis of known enzyme mechanisms. Finally, by considering completely 'novel enzyme chemistries' with de novo enzyme design, the limits of nature can be breached to derive the most advanced form of synthetic pathways. We discuss the challenges and promises associated with these different metabolic engineering approaches and illuminate how enzyme engineering is expected to take a prime role in synthetic metabolic engineering for biotechnology, chemical industry and agriculture of the future. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Xenobiotic metabolism in human skin and 3D human skin reconstructs: a review.

PubMed

Gibbs, Sue; van de Sandt, Johannes J M; Merk, Hans F; Lockley, David J; Pendlington, Ruth U; Pease, Camilla K

2007-12-01

In this review, we discuss and compare studies of xenobiotic metabolism in both human skin and 3D human skin reconstructs. In comparison to the liver, the skin is a less studied organ in terms of characterising metabolic capability. While the skin forms the major protective barrier to environmental chemical exposure, it is also a potential target organ for adverse health effects. Occupational, accidental or intended-use exposure to toxic chemicals could result in acute or delayed injury to the skin (e.g. inflammation, allergy, cancer). Skin metabolism may play a role in the manifestation or amelioration of adverse effects via the topical route. Today, we have robust testing strategies to assess the potential for local skin toxicity of chemical exposure. Such methods (e.g. the local lymph node assay for assessing skin sensitisation; skin painting carcinogenicity studies) incorporate skin metabolism implicitly in the in vivo model system used. In light of recent European legislation (i.e. 7(th) Amendment to the Cosmetics Directive and Registration Evaluation and Authorisation of existing Chemicals (REACH)), non-animal approaches will be required to reduce and replace animal experiments for chemical risk assessment. It is expected that new models and approaches will need to account for skin metabolism explicitly, as the mechanisms of adverse effects in the skin are deconvoluted. 3D skin models have been proposed as a tool to use in new in vitro alternative approaches. In order to be able to use 3D skin models in this context, we need to understand their metabolic competency in relation to xenobiotic biotransformation and whether functional activity is representative of that seen in human skin.

Humanized mouse lines and their application for prediction of human drug metabolism and toxicological risk assessment

PubMed Central

Cheung, Connie; Gonzalez, Frank J

2008-01-01

Cytochrome P450s (P450s) are important enzymes involved in the metabolism of xenobiotics, particularly clinically used drugs, and are also responsible for metabolic activation of chemical carcinogens and toxins. Many xenobiotics can activate nuclear receptors that in turn induce the expression of genes encoding xenobiotic metabolizing enzymes and drug transporters. Marked species differences in the expression and regulation of cytochromes P450 and xenobiotic nuclear receptors exist. Thus obtaining reliable rodent models to accurately reflect human drug and carcinogen metabolism is severely limited. Humanized transgenic mice were developed in an effort to create more reliable in vivo systems to study and predict human responses to xenobiotics. Human P450s or human xenobiotic-activated nuclear receptors were introduced directly or replaced the corresponding mouse gene, thus creating “humanized” transgenic mice. Mice expressing human CYP1A1/CYP1A2, CYP2E1, CYP2D6, CYP3A4, CY3A7, PXR, PPARα were generated and characterized. These humanized mouse models offers a broad utility in the evaluation and prediction of toxicological risk that may aid in the development of safer drugs. PMID:18682571

Xenobiotic/medium chain fatty acid: CoA ligase - a critical review on its role in fatty acid metabolism and the detoxification of benzoic acid and aspirin.

PubMed

van der Sluis, Rencia; Erasmus, Elardus

2016-10-01

Activation of fatty acids by the acyl-CoA synthetases (ACSs) is the vital first step in fatty acid metabolism. The enzymatic and physiological characterization of the human xenobiotic/medium chain fatty acid: CoA ligases (ACSMs) has been severely neglected even though xenobiotics, such as benzoate and salicylate, are detoxified through this pathway. This review will focus on the nomenclature and substrate specificity of the human ACSM ligases; the biochemical and enzymatic characterization of ACSM1 and ACSM2B; the high sequence homology of the ACSM2 genes (ACSM2A and ACSM2B) as well as what is currently known regarding disease association studies. Several discrepancies exist in the current literature that should be taken note of. For example, the single nucleotide polymorphisms (SNPs) reported to be associated with aspirin metabolism and multiple risk factors of metabolic syndrome are incorrect. Kinetic data on the substrate specificity of the human ACSM ligases are non-existent and currently no data exist on the influence of SNPs on the enzyme activity of these ligases. One of the biggest obstacles currently in the field is that glycine conjugation is continuously studied as a one-step process, which means that key regulatory factors of the two individual steps remain unknown.

A Compilation of In Vitro Rate and Affinity Values for Xenobiotic Biotransformation in Fish, Measured Under Physiological Conditions

EPA Science Inventory

This manuscript presents a summary of in vitro rate and affinity data for xenobiotic biotransformation enzymes in fish...One potential use of this data summary is to support in vitro to in vivo metabolism extrapolations which can be used as inputs to chemical kinetic models for f...

Bisphenol A-Associated Alterations in the Expression and Epigenetic Regulation of Genes Encoding Xenobiotic Metabolizing Enzymes in Human Fetal Liver

PubMed Central

Nahar, Muna S.; Kim, Jung H.; Sartor, Maureen A.; Dolinoy, Dana C.

2014-01-01

Alterations in xenobiotic metabolizing enzyme (XME) expression across the life course, along with genetic, nutritional, and environmental regulation, can influence how organisms respond to toxic insults. In this study, we investigated the hypothesis that in utero exposure to the endocrine active compound, bisphenol A (BPA), influences expression and epigenetic regulation of phase I and II XME genes during development. Using healthy 1st to 2nd trimester human fetal liver specimens quantified for internal BPA levels, we examined XME gene expression using PCR Array (n =8) and RNA-sequencing (n =12) platforms. Of the greater than 160 XME genes assayed, 2 phase I and 12 phase II genes exhibited significantly reduced expression with higher BPA levels, including isoforms from the carboxylesterase, catechol O-methyltransferase, glutathione S-transferase, sulfotransferase, and UDP-glucuronosyltransferase families. When the promoters of these candidate genes were evaluated in silico, putative binding sites for the E-twenty-six (ETS) and activator protein1 (AP1) related transcription factor families were identified and unique to 97% of all candidate transcripts. Interestingly, many ETS binding sites contain cytosine-guanine dinucleotides (CpGs) within their consensus sequences. Thus, quantitative analysis of CpG methylation of three candidate genes was conducted across n =50 samples. Higher BPA levels were associated with increased site-specific methylation at COMT (P <0.005) and increased average methylation at SULT2A1 (P <0.020) promoters. While toxicological studies have traditionally focused on high-dose effects and hormonal receptor mediated regulation, our findings suggest the importance of low-dose effects and nonclassical mechanisms of endocrine disruption during development. PMID:24214726

Systemic responses to inhaled ozone in mice: cachexia and down-regulation of liver xenobiotic metabolizing genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Last, Jerold A.; Gohil, Kishorchandra; Mathrani, Vivek C.

2005-10-15

Rats or mice acutely exposed to high concentrations of ozone show an immediate and significant weight loss, even when allowed free access to food and water. The mechanisms underlying this systemic response to ozone have not been previously elucidated. We have applied the technique of global gene expression analysis to the livers of C57BL mice acutely exposed to ozone. Mice lost up to 14% of their original body weight, with a 42% decrease in total food consumption. We previously had found significant up-regulation of genes encoding proliferative enzymes, proteins related to acute phase reactions and cytoskeletal functions, and other biomarkersmore » of a cachexia-like inflammatory state in lungs of mice exposed to ozone. These results are consistent with a general up-regulation of different gene families responsive to NF-{kappa}B in the lungs of the exposed mice. In the present study, we observed significant down-regulation of different families of mRNAs in the livers of the exposed mice, including genes related to lipid and fatty acid metabolism, and to carbohydrate metabolism in this tissue, consistent with a systemic cachexic response. Several interferon-dependent genes were down-regulated in the liver, suggesting a possible role for interferon as a signaling molecule between lung and liver. In addition, transcription of several mRNAs encoding enzymes of xenobiotic metabolism in the livers of mice exposed to ozone was decreased, suggesting cytokine-mediated suppression of cytochrome P450 expression. This finding may explain a previously controversial report from other investigators more than 20 years ago of prolongation of pentobarbital sleeping time in mice exposed to ozone.« less

Systemic responses to inhaled ozone in mice: cachexia and down-regulation of liver xenobiotic metabolizing genes.

PubMed

Last, Jerold A; Gohil, Kishorchandra; Mathrani, Vivek C; Kenyon, Nicholas J

2005-10-15

Rats or mice acutely exposed to high concentrations of ozone show an immediate and significant weight loss, even when allowed free access to food and water. The mechanisms underlying this systemic response to ozone have not been previously elucidated. We have applied the technique of global gene expression analysis to the livers of C57BL mice acutely exposed to ozone. Mice lost up to 14% of their original body weight, with a 42% decrease in total food consumption. We previously had found significant up-regulation of genes encoding proliferative enzymes, proteins related to acute phase reactions and cytoskeletal functions, and other biomarkers of a cachexia-like inflammatory state in lungs of mice exposed to ozone. These results are consistent with a general up-regulation of different gene families responsive to NF-kappaB in the lungs of the exposed mice. In the present study, we observed significant down-regulation of different families of mRNAs in the livers of the exposed mice, including genes related to lipid and fatty acid metabolism, and to carbohydrate metabolism in this tissue, consistent with a systemic cachexic response. Several interferon-dependent genes were down-regulated in the liver, suggesting a possible role for interferon as a signaling molecule between lung and liver. In addition, transcription of several mRNAs encoding enzymes of xenobiotic metabolism in the livers of mice exposed to ozone was decreased, suggesting cytokine-mediated suppression of cytochrome P450 expression. This finding may explain a previously controversial report from other investigators more than 20 years ago of prolongation of pentobarbital sleeping time in mice exposed to ozone.

Impact of environmental exposures on ovarian function and role of xenobiotic metabolism during ovotoxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bhattacharya, Poulomi; Keating, Aileen F., E-mail: akeating@iastate.edu

2012-06-15

The mammalian ovary is a heterogeneous organ and contains oocyte-containing follicles at varying stages of development. The most immature follicular stage, the primordial follicle, comprises the ovarian reserve and is a finite number, defined at the time of birth. Depletion of all follicles within the ovary leads to reproductive senescence, known as menopause. A number of chemical classes can destroy follicles, thus hastening entry into the menopausal state. The ovarian response to chemical exposure can determine the extent of ovotoxicity that occurs. Enzymes capable of bioactivating as well as detoxifying xenobiotics are expressed in the ovary and their impact onmore » ovotoxicity has been partially characterized for trichloroethylene, 7,12-dimethylbenz[a]anthracene, and 4-vinylcyclohexene. This review will discuss those studies, as well as illustrate where knowledge gaps remain for chemicals that have also been established as ovotoxicants. -- Highlights: ► Summary of ovotoxicant action during ovotoxicity. ► Discussion of impact of biotransformation on chemical toxicity. ► Identification of knowledge gaps in chemical metabolism.« less

Sex- and age-dependent gene expression in human liver: An implication for drug-metabolizing enzymes.

PubMed

Uno, Yasuhiro; Takata, Ryo; Kito, Go; Yamazaki, Hiroshi; Nakagawa, Kazuko; Nakamura, Yusuke; Kamataki, Tetsuya; Katagiri, Toyomasa

2017-02-01

Sex and age differences in hepatic expression of drug-metabolizing enzyme genes could cause variations in drug metabolism, but has not been fully elucidated, especially in Asian population. In this study, the global expression of human hepatic genes was analyzed by microarrays in 40 Japanese subjects (27 males and 13 females). Thirty-five sex-biased genes were identified (P < 0.005). Whereas, 60 age-biased genes in two age groups, <60 years and ≥70 years (P < 0.001), were identified in males. By Gene Ontology analysis, the sex-biased genes were related to protein catabolism and modification, while the age-biased genes were related to transcription regulation and cell death. Quantitative polymerase chain reaction confirmed the female-biased expression of drug-metabolizing enzyme genes BChE, CYP4X1, and SULT1E1 (≥1.5-fold, P < 0.05). Further analysis of drug-metabolizing enzyme genes indicated that expression of CYP2A6 and CYP3A4 in females in the ≥70 age group was less than in the <60 age group (≥1.5-fold, P < 0.05), and this trend was also observed for PXR expression in males (≥1.5-fold, P < 0.05). The results presented provide important insights into hepatic physiology and function, especially drug metabolism, with respect to sex and age. Copyright © 2016 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

Pigs in Toxicology: Breed Differences in Metabolism and Background Findings.

PubMed

Helke, Kristi L; Nelson, Keith N; Sargeant, Aaron M; Jacob, Binod; McKeag, Sean; Haruna, Julius; Vemireddi, Vimala; Greeley, Melanie; Brocksmith, Derek; Navratil, Nicole; Stricker-Krongrad, Alain; Hollinger, Charlotte

2016-06-01

Both a rodent and a nonrodent species are required for evaluation in nonclinical safety studies conducted to support human clinical trials. Historically, dogs and nonhuman primates have been the nonrodent species of choice. Swine, especially the miniature swine or minipigs, are increasingly being used in preclinical safety as an alternate nonrodent species. The pig is an appropriate option for these toxicology studies based on metabolic pathways utilized in xenobiotic biotransformation. Both similarities and differences exist in phase I and phase II biotransformation pathways between humans and pigs. There are numerous breeds of pigs, yet only a few of these breeds are characterized with regard to both xenobiotic-metabolizing enzymes and background pathology findings. Some specific differences in these enzymes based on breed and sex are known. Although swine have been used extensively in biomedical research, there is also a paucity of information in the current literature detailing the incidence of background lesions and differences between commonly used breeds. Here, the xenobiotic-metabolizing enzymes are compared between humans and pigs, and minipig background pathology changes are reviewed with emphasis on breed differences. © The Author(s) 2016.

Xenobiotic metabolizing gene variants, pesticide use, and risk of prostate cancer

PubMed Central

Koutros, Stella; Andreotti, Gabriella; Berndt, Sonja I.; Barry, Kathryn Hughes; Lubin, Jay H.; Hoppin, Jane A.; Kamel, Freya; Sandler, Dale P.; Burdette, Laurie A.; Yuenger, Jeffrey; Yeager, Meredith; Alavanja, Michael C.R.; Beane Freeman, Laura E.

2011-01-01

Background To explore associations with prostate cancer and farming, it is important to investigate the relationship between pesticide use and single nucleotide polymorphisms (SNPs) in xenobiotic metabolic enzyme (XME) genes. Objectives We evaluated pesticide-SNP interactions between 45 pesticides and 1,913 XME SNPs with respect to prostate cancer among 776 cases and 1,444 controls in the Agricultural Health Study. Methods We used unconditional logistic regression to estimate odds ratios (ORs) and 95% confidence intervals (CIs). Multiplicative SNP-pesticide interactions were calculated using a likelihood ratio test. Results A positive monotonic interaction was observed between petroleum oil/petroleum distillate use and rs1883633 in the oxidative stress gene glutamate-cysteine ligase (GCLC) (p-interaction=1.0×10−4); men carrying at least one variant allele (minor allele) experienced an increased prostate cancer risk (OR=3.7, 95% CI: 1.9–7.3). Among men carrying the variant allele for thioredoxin reductase 2 (TXNRD2) rs4485648, microsomal epoxide hyrdolase 1 (EPHX1) rs17309872, or myeloperoxidase (MPO) rs11079344, increased prostate cancer risk was observed with high compared to no petroleum oil/petroleum distillate (OR=1.9, 95% CI: 1.1–3.2, p-interaction=0.01), (OR=2.1, 95% CI: 1.1–4.0, p-interaction=0.01), or terbufos (OR=3.0, 95% CI: 1.5–6.0 p-interaction=2.0×10−3) use, respectively. No interactions were deemed noteworthy at the false discovery rate = 0.20 level; the number of observed interactions in XMEs was comparable to the number expected by chance alone. Conclusions We observed several pesticide-SNP interactions in oxidative stress and phase I/phase II enzyme genes and risk of prostate cancer. Additional work is needed to explain the joint contribution of genetic variation in XMEs, pesticide use, and prostate cancer risk. PMID:21716162

Biological definition of multiple chemical sensitivity from redox state and cytokine profiling and not from polymorphisms of xenobiotic-metabolizing enzymes

DOE Office of Scientific and Technical Information (OSTI.GOV)

De Luca, Chiara; Scordo, Maria G.; Cesareo, Eleonora

Background: Multiple chemical sensitivity (MCS) is a poorly clinically and biologically defined environment-associated syndrome. Although dysfunctions of phase I/phase II metabolizing enzymes and redox imbalance have been hypothesized, corresponding genetic and metabolic parameters in MCS have not been systematically examined. Objectives: We sought for genetic, immunological, and metabolic markers in MCS. Methods: We genotyped patients with diagnosis of MCS, suspected MCS and Italian healthy controls for allelic variants of cytochrome P450 isoforms (CYP2C9, CYP2C19, CYP2D6, and CYP3A5), UDP-glucuronosyl transferase (UGT1A1), and glutathione S-transferases (GSTP1, GSTM1, and GSTT1). Erythrocyte membrane fatty acids, antioxidant (catalase, superoxide dismutase (SOD)) and glutathione metabolizing (GST,more » glutathione peroxidase (Gpx)) enzymes, whole blood chemiluminescence, total antioxidant capacity, levels of nitrites/nitrates, glutathione, HNE-protein adducts, and a wide spectrum of cytokines in the plasma were determined. Results: Allele and genotype frequencies of CYPs, UGT, GSTM, GSTT, and GSTP were similar in the Italian MCS patients and in the control populations. The activities of erythrocyte catalase and GST were lower, whereas Gpx was higher than normal. Both reduced and oxidised glutathione were decreased, whereas nitrites/nitrates were increased in the MCS groups. The MCS fatty acid profile was shifted to saturated compartment and IFNgamma, IL-8, IL-10, MCP-1, PDGFbb, and VEGF were increased. Conclusions: Altered redox and cytokine patterns suggest inhibition of expression/activity of metabolizing and antioxidant enzymes in MCS. Metabolic parameters indicating accelerated lipid oxidation, increased nitric oxide production and glutathione depletion in combination with increased plasma inflammatory cytokines should be considered in biological definition and diagnosis of MCS.« less

Effects of Brown Rice and White Rice on Expression of Xenobiotic Metabolism Genes in Type 2 Diabetic Rats

PubMed Central

Imam, Mustapha Umar; Ismail, Maznah

2012-01-01

Xenobiotics constantly influence biological systems through several means of interaction. These interactions are disturbed in type 2 diabetes, with implications for disease outcome. We aimed to study the implications of such disturbances on type 2 diabetes and rice consumption, the results of which could affect management of the disease in developing countries. In a type 2 diabetic rat model induced through a combination of high fat diet and low dose streptozotocin injection, up-regulation of xenobiotic metabolism genes in the diabetic untreated group was observed. Xenobiotic metabolism genes were upregulated more in the white rice (WR) group than the diabetic untreated group while the brown rice (BR) group showed significantly lower expression values, though not as effective as metformin, which gave values closer to the normal non-diabetic group. The fold changes in expression in the WR group compared to the BR group for Cyp2D4, Cyp3A1, Cyp4A1, Cyp2B1, Cyp2E1, Cyp2C11, UGT2B1, ALDH1A1 and Cyp2C6 were 2.6, 2, 1.5, 4, 2.8, 1.5, 1.8, 3 and 5, respectively. Our results suggest that WR may upregulate these genes in type 2 diabetes more than BR, potentially causing faster drug metabolism, less drug efficacy and more toxicity. These results may have profound implications for rice eating populations, constituting half the world’s population. PMID:22942722

The in vivo effects of adenine-induced chronic kidney disease on some renal and hepatic function and CYP450 metabolizing enzymes.

PubMed

Al Za'abi, M; Shalaby, A; Manoj, P; Ali, B H

2017-05-04

Adenine-induced model of chronic kidney disease (CKD) is a widely used model especially in studies testing novel nephroprotective agents. We investigated the effects of adenine-induced CKD in rats on the activities of some xenobiotic metabolizing enzymes in liver and kidneys, and on some in vivo indicators of drug metabolism (viz pentobarbitone sleeping time, and plasma concentration of theophylline 90 min post administration). CKD was induced by orally feeding adenine (0.25 % w/w) for 35 days. Adenine induced all the characteristics of CKD, which was confirmed by biochemical and histological findings. Glutathione concentration and activities of some enzymes involved in its metabolism were reduced in kidneys and livers of rats with CKD. Renal CYP450 1A1 activity was significantly inhibited by adenine, but other measured isoenzymes (1A2, 3A4 and 2E1) were not significantly affected. Adenine significantly prolonged pentobarbitone-sleeping time and increased plasma theophylline concentration 90 min post administration. Adenine also induced a moderate degree of hepatic damages as indicated histologically and by significant elevations in some plasma enzymes. The results suggest that adenine-induced CKD is associated with significant in vivo inhibitory activities on some drug-metabolizing enzymes, with most of the effect on the kidneys rather than the liver.

Expression profiles of phases 1 and 2 metabolizing enzymes in human skin and the reconstructed skin models Episkin and full thickness model from Episkin.

PubMed

Luu-The, Van; Duche, Daniel; Ferraris, Corinne; Meunier, Jean-Roch; Leclaire, Jacques; Labrie, Fernand

2009-09-01

Episkin and full thickness model from Episkin (FTM) are human skin models obtained from in vitro growth of keratinocytes into the five typical layers of the epidermis. FTM is a full thickness reconstructed skin model that also contains fibroblasts seeded in a collagen matrix. To assess whether enzymes involved in chemical detoxification are expressed in Episkin and FTM and how their levels compare with the human epidermis, dermis and total skin. Quantification of the mRNA expression levels of phases 1 and 2 metabolizing enzymes in cultured Episkin and FTM and human epidermis, dermis and total skin using Realtime PCR. The data show that the expression profiles of 61 phases 1 and 2 metabolizing enzymes in Episkin, FTM and epidermis are generally similar, with some exceptions. Cytochrome P450-dependent enzymes and flavin monooxygenases are expressed at low levels, while phase 2 metabolizing enzymes are expressed at much higher levels, especially, glutathione-S-transferase P1 (GSTP1) catechol-O-methyl transferase (COMT), steroid sulfotransferase (SULT2B1b), and N-acetyl transferase (NAT5). The present study also identifies the presence of many enzymes involved in cholesterol, arachidonic acid, leukotriene, prostaglandin, eicosatrienoic acids, and vitamin D3 metabolisms. The present data strongly suggest that Episkin and FTM represent reliable and valuable in vitro human skin models for studying the function of phases 1 and 2 metabolizing enzymes in xenobiotic metabolisms. They could be used to replace invasive methods or laboratory animals for skin experiments.

Alginate Immobilization of Metabolic Enzymes (AIME) for High ...

EPA Pesticide Factsheets

Alginate Immobilization of Metabolic Enzymes (AIME) for High-Throughput Screening Assays DE DeGroot, RS Thomas, and SO SimmonsNational Center for Computational Toxicology, US EPA, Research Triangle Park, NC USAThe EPA’s ToxCast program utilizes a wide variety of high-throughput screening (HTS) assays to assess chemical perturbations of molecular and cellular endpoints. A key criticism of using HTS assays for toxicity assessment is the lack of xenobiotic metabolism (XM) which precludes both metabolic detoxification as well as bioactivation of chemicals tested in vitro thereby mischaracterizing the potential risk posed by these chemicals. To address this deficiency, we have developed an extracellular platform to retrofit existing HTS assays with XM activity. This platform utilizes the S9 fraction of liver homogenate encapsulated in an alginate gel network which reduces the cytotoxicity caused by direct addition of S9 to cells in culture. Alginate microspheres containing encapsulated human liver S9 were cross-linked to solid supports extending from a 96-well plate lid and were assayed using a pro-luciferin substrate specific for CYP3A4 (IPA). We demonstrate that S9 was successfully encapsulated and remained enzymatically active post-encapsulation with 5-10X the CYP3A4 activity as compared to 1 µg solubilized human liver S9. Ketoconazole, a known inhibitor of human CYP3A4, inhibited CYP3A4 activity in a concentration-dependent manner (IC50: 0.27 µM) and inhibiti

Structural insights into xenobiotic and inhibitor binding to human aldehyde oxidase.

PubMed

Coelho, Catarina; Foti, Alessandro; Hartmann, Tobias; Santos-Silva, Teresa; Leimkühler, Silke; Romão, Maria João

2015-10-01

Aldehyde oxidase (AOX) is a xanthine oxidase (XO)-related enzyme with emerging importance due to its role in the metabolism of drugs and xenobiotics. We report the first crystal structures of human AOX1, substrate free (2.6-Å resolution) and in complex with the substrate phthalazine and the inhibitor thioridazine (2.7-Å resolution). Analysis of the protein active site combined with steady-state kinetic studies highlight the unique features, including binding and substrate orientation at the active site, that characterize human AOX1 as an important drug-metabolizing enzyme. Structural analysis of the complex with the noncompetitive inhibitor thioridazine revealed a new, unexpected and fully occupied inhibitor-binding site that is structurally conserved among mammalian AOXs and XO. The new structural insights into the catalytic and inhibition mechanisms of human AOX that we now report will be of great value for the rational analysis of clinical drug interactions involving inhibition of AOX1 and for the prediction and design of AOX-stable putative drugs.

Dietary lipids differentially modulate the initiation of experimental breast carcinogenesis through their influence on hepatic xenobiotic metabolism and DNA damage in the mammary gland.

PubMed

Manzanares, Miguel Ángel; de Miguel, Cristina; Ruiz de Villa, M Carme; Santella, Regina M; Escrich, Eduard; Solanas, Montserrat

2017-05-01

Breast cancer is the most common malignancy among women worldwide. In addition to reproductive factors, environmental factors such as nutrition and xenobiotic exposure have a role in the etiology of this malignancy. A stimulating and a potentially protective effect on experimental breast cancer has been previously described for high corn oil and high extra-virgin olive oil diets, respectively. This work investigates the effect of these lipids on the metabolism of 7,12-dimethylbenz(a)anthracene (DMBA), a polycyclic aromatic hydrocarbon that can initiate carcinogenesis and its consequences in an experimental rat breast cancer model. The PUFA n-6-enriched diet increased expression of Phase I enzymes prior to DMBA administration and raised the activity of CYP1s in the hours immediately after induction, while reducing the activity of Phase II enzymes, mainly NQO1. The levels of reactive metabolites measured in plasma by GC-MS and DMBA-DNA adducts in the mammary gland of the animals fed the high corn oil diet were also higher than in the other groups. On the other hand, the high extra-virgin olive oil diet and the control low-fat diet exhibited better coordinated Phase I and Phase II activity, with a lower production of reactive metabolites and less DNA damage in the mammary gland. The concordance between these effects and the different efficacy of the carcinogenesis process due to the dietary treatment suggest that lipids may differently modify mammary gland susceptibility or resistance to cancer initiation over the exposure to environmental carcinogens. Dietary lipids influence the initiation of DMBA-induced mammary cancer through the modulation of liver xenobiotic metabolism, formation of reactive metabolites and subsequent DNA damage in the target tissue. Copyright © 2017 Elsevier Inc. All rights reserved.

Nature's inordinate fondness for metabolic enzymes: why metabolic enzyme loci are so frequently targets of selection.

PubMed

Marden, James H

2013-12-01

Metabolic enzyme loci were some of the first genes accessible for molecular evolution and ecology research. New technologies now make the whole genome, transcriptome or proteome readily accessible, allowing unbiased scans for loci exhibiting significant differences in allele frequency or expression level and associated with phenotypes and/or responses to natural selection. With surprising frequency and in many cases in proportions greater than chance relative to other genes, glycolysis and TCA cycle enzyme loci appear among the genes with significant associations in these studies. Hence, there is an ongoing need to understand the basis for fitness effects of metabolic enzyme polymorphisms. Allele-specific effects on the binding affinity and catalytic rate of individual enzymes are well known, but often of uncertain significance because metabolic control theory and in vivo studies indicate that many individual metabolic enzymes do not affect pathway flux rate. I review research, so far little used in evolutionary biology, showing that metabolic enzyme substrates affect signalling pathways that regulate cell and organismal biology, and that these enzymes have moonlighting functions. To date there is little knowledge of how alleles in natural populations affect these phenotypes. I discuss an example in which alleles of a TCA enzyme locus associate with differences in a signalling pathway and development, organismal performance, and ecological dynamics. Ultimately, understanding how metabolic enzyme polymorphisms map to phenotypes and fitness remains a compelling and ongoing need for gaining robust knowledge of ecological and evolutionary processes. © 2013 John Wiley & Sons Ltd.

Natural variations in xenobiotic-metabolizing enzymes: developing tools for coral monitoring

NASA Astrophysics Data System (ADS)

Rougée, L. R. A.; Richmond, R. H.; Collier, A. C.

2014-06-01

The continued deterioration of coral reefs worldwide demonstrates the need to develop diagnostic tools for corals that go beyond general ecological monitoring and can identify specific stressors at sublethal levels. Cellular diagnostics present an approach to defining indicators (biomarkers) that have the potential to reflect the impact of stress at the cellular level, allowing for the detection of intracellular changes in corals prior to outright mortality. Detoxification enzymes, which may be readily induced or inhibited by environmental stressors, present such a set of indicators. However, in order to apply these diagnostic tools for the detection of stress, a detailed understanding of their normal, homeostatic levels within healthy corals must first be established. Herein, we present molecular and biochemical evidence for the expression and activity of major Phase I detoxification enzymes cytochrome P450 (CYP450), CYP2E1, and CYP450 reductase, as well as the Phase II enzymes UDP, glucuronosyltransferase (UGT), β-glucuronidase, glutathione- S-transferase (GST), and arylsulfatase C (ASC) in the coral Pocillopora damicornis. Additionally, we characterized enzyme expression and activity variations over a reproductive cycle within a coral's life history to determine natural endogenous changes devoid of stress exposure. Significant changes in enzyme activity over the coral's natural lunar reproductive cycle were observed for CYP2E1 and CYP450 reductase as well as UGT and GST, while β-glucuronidase and ASC did not fluctuate significantly. The data represent a baseline description of `health' for the expression and activity of these enzymes that can be used toward understanding the impact of environmental stressors on corals. Such knowledge can be applied to address causes of coral reef ecosystem decline and to monitor effectiveness of mitigation strategies. Achieving a better understanding of cause-and-effect relationships between putative stressors and biological

An enhanced in vivo stable isotope labeling by amino acids in cell culture (SILAC) model for quantification of drug metabolism enzymes.

PubMed

MacLeod, A Kenneth; Fallon, Padraic G; Sharp, Sheila; Henderson, Colin J; Wolf, C Roland; Huang, Jeffrey T-J

2015-03-01

Many of the enzymes involved in xenobiotic metabolism are maintained at a low basal level and are only synthesized in response to activation of upstream sensor/effector proteins. This induction can have implications in a variety of contexts, particularly during the study of the pharmacokinetics, pharmacodynamics, and drug-drug interaction profile of a candidate therapeutic compound. Previously, we combined in vivo SILAC material with a targeted high resolution single ion monitoring (tHR/SIM) LC-MS/MS approach for quantification of 197 peptide pairs, representing 51 drug metabolism enzymes (DME), in mouse liver. However, as important enzymes (for example, cytochromes P450 (Cyp) of the 1a and 2b subfamilies) are maintained at low or undetectable levels in the liver of unstimulated metabolically labeled mice, quantification of these proteins was unreliable. In the present study, we induced DME expression in labeled mice through synchronous ligand-mediated activation of multiple upstream nuclear receptors, thereby enhancing signals for proteins including Cyps 1a, 2a, 2b, 2c, and 3a. With this enhancement, 115 unique, lysine-containing, Cyp-derived peptides were detected in the liver of a single animal, as opposed to 56 in a pooled sample from three uninduced animals. A total of 386 peptide pairs were quantified by tHR/SIM, representing 68 Phase I, 30 Phase II, and eight control proteins. This method was employed to quantify changes in DME expression in the hepatic cytochrome P450 reductase null (HRN) mouse. We observed compensatory induction of several enzymes, including Cyps 2b10, 2c29, 2c37, 2c54, 2c55, 2e1, 3a11, and 3a13, carboxylesterase (Ces) 2a, and glutathione S-transferases (Gst) m2 and m3, along with down-regulation of hydroxysteroid dehydrogenases (Hsd) 11b1 and 17b6. Using DME-enhanced in vivo SILAC material with tHR/SIM, therefore, permits the robust analysis of multiple DME of importance to xenobiotic metabolism, with improved utility for the study of

Studies on the role of six enzymes in the metabolism of kinetin in mustard aphid, Lipaphis erysimi (Kalt.).

PubMed

Rup, Pushpinder J; Sohal, S K; Kaur, H

2006-07-01

The activity of catalase, glutathione peroxidase, superoxide dismutase, O-demethylase, ATPase and succinate dehydrogenase, belonging to two main classes of detoxification enzymes (i.e. hydrolases and oxido-reductases), mostly involved in metabolism and degradation of xenobiotics in insects, were assessed under the influence of kinetin, a plant growth regulator (PGR). The nymphs (48-52 hr old) of Lipaphis erysimi (Kalt.) were permitted to feed on radish plant, Raphanus sativus L. treated with kinetin (400 ppm) for 13, 25 and 37 hr. It was found that the activity of catalase, glutathione peroxidase and superoxide dismutase increased significantly when compared with the control of the same age group, which indicated that these enzymes might be playing a significant role in the metabolism of kinetin in this insect. The activity of O-demethylase showed an increase up to 25 hr of the treatment but it decreased under prolonged treatment whereas the activity of succinate dehydrogenase fluctuated insignificantly. ATPase showed a decrease in the activity with the treatment suggesting kinetin's interference in synthesis of ATPase.

Addressing species diversity in biotransformation: variability in expressed transcripts of hepatic biotransformation enzymes among fishes

EPA Science Inventory

There is increasing evidence that diverse xenobiotic metabolizing enzymes exist among fishes, potentially resulting in different chemical sensitivities and accumulation, but this has never been systematically evaluated. One concern is that model test species such as rainbow trou...

INTERINDIVIDUAL VARIANCE OF CYTOCHROME P450 FORMS IN HUMAN HEPATIC MICROSOMES: CORRELATION OF INDIVIDUAL FORMS WITH XENOBIOTIC METABOLISM AND IMPLICATIONS IN RISK ASSESSMENT

EPA Science Inventory

Differences in biotransformation activities may alter the bioavailability or efficacy of drugs, provide protection from certain xenobiotic and environmental agents, or increase toxicity of others. Cytochrome P450 (CYP450) enzymes are responsible for the majority of oxidation reac...

Biotransformation enzymes in the rodent nasal mucosa: the value of a histochemical approach.

PubMed Central

Bogdanffy, M S

1990-01-01

An increasing number of chemicals have been identified as being toxic to the nasal mucosa of rats. While many chemicals exert their effects only after inhalation exposure, others are toxic following systemic administration, suggesting that factors other than direct deposition on the nasal mucosa may be important in mechanisms of nasal toxicity. The mucosal lining of the nasal cavity consists of a heterogeneous population of ciliated and nonciliated cells, secretory cells, sensory cells, and glandular and other cell types. For chemicals that are metabolized in the nasal mucosa, the balance between metabolic activation and detoxication within a cell type may be a key factor in determining whether that cell type will be a target for toxicity. Recent research in the area of xenobiotic metabolism in nasal mucosa has demonstrated the presence of many enzymes previously described in other tissues. In particular, carboxylesterase, aldehyde dehydrogenase, cytochromes P-450, epoxide hydrolase, and glutathione S-transferases have been localized by histochemical techniques. The distribution of these enzymes appears to be cell-type-specific and the presence of the enzyme may predispose particular cell types to enhanced susceptibility or resistance to chemical-induced injury. This paper reviews the distribution of these enzymes within the nasal mucosa in the context of their contribution to xenobiotic metabolism. The localization of the enzymes by histochemical techniques has provided important information on the potential mechanism of action of esters, aldehydes, and cytochrome P-450 substrates known to injure the nasal mucosa. Images PLATE 1. PLATE 2. PLATE 3. PMID:2200661

Cytochrome P450 peroxidase/peroxygenase mediated xenobiotic metabolic activation and cytotoxicity in isolated hepatocytes.

PubMed

Anari, M R; Khan, S; Liu, Z C; O'Brien, P J

1995-12-01

Cytochrome P450 (P450) can utilize organic hydroperoxides and peracids to support hydroxylation and dealkylation of various P450 substrates. However, the biological significance of this P450 peroxygenase/peroxidase activity in the bioactivation of xenobiotics in intact cells has not been demonstrated. We have shown that tert-butyl hydroperoxide (tBHP) markedly enhances 3-20-fold the cytotoxicity of various aromatic hydrocarbons and their phenolic metabolites. The tBHP-enhanced hepatocyte cytotoxicity of 4-nitroanisole (4-NA) and 4-hydroxyanisole (4-HA) was also accompanied by an increase in the hepatocyte O-demethylation of 4-NA and 4-HA up to 7.5- and 21-fold, respectively. Hepatocyte GSH conjugation by 4-HA was also markedly increased by tBHP. An LC/MS analysis of the GSH conjugates identified hydroquinone-GSH and 4-methoxy-catechol:GSH conjugates as the predominant adducts. Pretreatment of hepatocytes with P450 inhibitors, e.g., phenylimidazole, prevented tBHP-enhanced 4-HA metabolism, GSH depletion, and cytotoxicity. In conclusion, hydroperoxides can therefore be used by intact cells to support the bioactivation of xenobiotics through the P450 peroxidase/peroxygenase system.

In Silico Prediction of Cytochrome P450-Mediated Biotransformations of Xenobiotics: A Case Study of Epoxidation.

PubMed

Zhang, Jing; Ji, Li; Liu, Weiping

2015-08-17

Predicting the biotransformation of xenobiotics is important in toxicology; however, as more compounds are synthesized than can be investigated experimentally, powerful computational methods are urgently needed to prescreen potentially useful candidates. Cytochrome P450 enzymes (P450s) are the major enzymes involved in xenobiotic metabolism, and many substances are bioactivated by P450s to form active compounds. An example is the conversion of olefinic substrates to epoxides, which are intermediates in the metabolic activation of many known or suspected carcinogens. We have calculated the activation energies for epoxidation by the active species of P450 enzymes (an iron-oxo porphyrin cation radical oxidant, compound I) for a diverse set of 36 olefinic substrates with state-of-the-art density functional theory (DFT) methods. Activation energies can be estimated by the computationally less demanding method of calculating the ionization potentials of the substrates, which provides a useful and simple predictive model based on the reaction mechanism; however, the preclassification of these diverse substrates into weakly polar and strongly polar groups is a prerequisite for the construction of specific predictive models with good predictability for P450 epoxidation. This approach has been supported by both internal and external validations. Furthermore, the relation between the activation energies for the regioselective epoxidation and hydroxylation reactions of P450s and experimental data has been investigated. The results show that the computational method used in this work, single-point energy calculations with the B3LYP functional including zero-point energy and solvation and dispersion corrections based on B3LYP-optimized geometries, performs well in reproducing the experimental trends of the epoxidation and hydroxylation reactions.

Computational Functional Analysis of Lipid Metabolic Enzymes.

PubMed

Bagnato, Carolina; Have, Arjen Ten; Prados, María B; Beligni, María V

2017-01-01

The computational analysis of enzymes that participate in lipid metabolism has both common and unique challenges when compared to the whole protein universe. Some of the hurdles that interfere with the functional annotation of lipid metabolic enzymes that are common to other pathways include the definition of proper starting datasets, the construction of reliable multiple sequence alignments, the definition of appropriate evolutionary models, and the reconstruction of phylogenetic trees with high statistical support, particularly for large datasets. Most enzymes that take part in lipid metabolism belong to complex superfamilies with many members that are not involved in lipid metabolism. In addition, some enzymes that do not have sequence similarity catalyze similar or even identical reactions. Some of the challenges that, albeit not unique, are more specific to lipid metabolism refer to the high compartmentalization of the routes, the catalysis in hydrophobic environments and, related to this, the function near or in biological membranes.In this work, we provide guidelines intended to assist in the proper functional annotation of lipid metabolic enzymes, based on previous experiences related to the phospholipase D superfamily and the annotation of the triglyceride synthesis pathway in algae. We describe a pipeline that starts with the definition of an initial set of sequences to be used in similarity-based searches and ends in the reconstruction of phylogenies. We also mention the main issues that have to be taken into consideration when using tools to analyze subcellular localization, hydrophobicity patterns, or presence of transmembrane domains in lipid metabolic enzymes.

Passive rGE or developmental gene-environment cascade? An investigation of the role of xenobiotic metabolism genes in the association between smoke exposure during pregnancy and child birth weight

PubMed Central

Marceau, Kristine; Palmer, Rohan H.C.; Neiderhiser, Jenae M.; Smith, Taylor F.; McGeary, John E.; Knopik, Valerie S.

2016-01-01

There is considerable evidence that smoke exposure during pregnancy (SDP) environmentally influences birth weight after controlling for genetic influences and maternal characteristics. However, maternal smoking during pregnancy – the behavior that leads to smoke exposure during pregnancy – is also genetically-influenced, indicating the potential role of passive gene-environment correlation. An alternative to passive gene-SDP correlation is a cascading effect whereby maternal and child genetic influences are causally linked to prenatal exposures, which then have an ‘environmental’ effect on the development of the child’s biology and behavior. We describe and demonstrate a conceptual framework for disentangling passive rGE from this cascading GE effect using a systems-based polygenic scoring approach comprised of genes shown to be important in the xenobiotic (substances foreign to the body) metabolism pathway. Data were drawn from 5,044 families from the Avon Longitudinal Study of Parents and Children with information on maternal SDP, birth weight, and genetic polymorphisms in the xenobiotic pathway. Within a k-fold cross-validation approach (k=5), we created weighted maternal and child polygenic scores using 18 polymorphisms from 10 genes that have been implicated in the xenobiotic metabolism pathway. Mothers and children shared variation in xenobiotic metabolism genes. Amongst mothers who smoked during pregnancy, neither maternal nor child xenobiotic metabolism polygenic scores were associated with a higher likelihood of smoke exposure during pregnancy, or the severity of smoke exposure during pregnancy (and therefore, neither proposed mechanism was supported), or with child birth weight. SDP was consistently associated with lower child birth weight controlling for the polygenic scores, maternal educational attainment, social class, psychiatric problems, and age. Limitations of the study design and the potential of the framework using other designs are

Transcriptional regulation of xenobiotic detoxification in Drosophila

PubMed Central

Misra, Jyoti R.; Horner, Michael A.; Lam, Geanette; Thummel, Carl S.

2011-01-01

Living organisms, from bacteria to humans, display a coordinated transcriptional response to xenobiotic exposure, inducing enzymes and transporters that facilitate detoxification. Several transcription factors have been identified in vertebrates that contribute to this regulatory response. In contrast, little is known about this pathway in insects. Here we show that the Drosophila Nrf2 (NF-E2-related factor 2) ortholog CncC (cap ‘n’ collar isoform-C) is a central regulator of xenobiotic detoxification responses. A binding site for CncC and its heterodimer partner Maf (muscle aponeurosis fibromatosis) is sufficient and necessary for robust transcriptional responses to three xenobiotic compounds: phenobarbital (PB), chlorpromazine, and caffeine. Genetic manipulations that alter the levels of CncC or its negative regulator, Keap1 (Kelch-like ECH-associated protein 1), lead to predictable changes in xenobiotic-inducible gene expression. Transcriptional profiling studies reveal that more than half of the genes regulated by PB are also controlled by CncC. Consistent with these effects on detoxification gene expression, activation of the CncC/Keap1 pathway in Drosophila is sufficient to confer resistance to the lethal effects of the pesticide malathion. These studies establish a molecular mechanism for the regulation of xenobiotic detoxification in Drosophila and have implications for controlling insect populations and the spread of insect-borne human diseases. PMID:21896655

Metabolic enzymes dysregulation in heart failure: the prospective therapy.

PubMed

Parihar, Priyanka; Parihar, Mordhwaj Singh

2017-01-01

The heart failure accounts for the highest mortality rate all over the world. The development of preventive therapeutic approaches is still in their infancy. Owing to the extremely high energy demand of the heart, the bioenergetics pathways need to respond efficiently based on substrate availability. The metabolic regulation of such heart bioenergetics is mediated by various rate limiting enzymes involved in energy metabolism. Although all the pertinent mechanisms are not clearly understood, the progressive decline in the activity of metabolic enzymes leading to diminished ATP production is known to cause progression of the heart failure. Therefore, metabolic therapy that can maintain the appropriate activities of metabolic enzymes can be a promising approach for the prevention and treatment of the heart failure. The flavonoids that constitute various human dietary ingredients also effectively offer a variety of health benefits. The flavonoids target a variety of metabolic enzymes and facilitate effective management of the equilibrium between production and utilization of energy in the heart. This review discusses the broad impact of metabolic enzymes in the heart functions and explains how the dysregulated enzyme activity causes the heart failure. In addition, the prospects of targeting dysregulated metabolic enzymes by developing flavonoid-based metabolic approaches are discussed.

[Interaction between CYP450 enzymes and metabolism of traditional Chinese medicine as well as enzyme activity assay].

PubMed

Lu, Tu-lin; Su, Lian-lin; Ji, De; Gu, Wei; Mao, Chun-qin

2015-09-01

Drugs are exogenous compounds for human bodies, and will be metabolized by many enzymes after administration. CYP450 enzyme, as a major metabolic enzyme, is an important phase I drug metabolizing enzyme. In human bodies, about 75% of drug metabolism is conducted by CYP450 enzymes, and CYP450 enzymes is the key factor for drug interactions between traditional Chinese medicine( TCM) -TCM, TCM-medicine and other drug combination. In order to make clear the interaction between metabolic enzymes and TCM metabolism, we generally chose the enzymatic activity as an evaluation index. That is to say, the enhancement or reduction of CYP450 enzyme activity was used to infer the inducing or inhibitory effect of active ingredients and extracts of traditional Chinese medicine on enzymes. At present, the common method for measuring metabolic enzyme activity is Cocktail probe drugs, and it is the key to select the suitable probe substrates. This is of great significance for study drug's absorption, distribution, metabolism and excretion (ADME) process in organisms. The study focuses on the interaction between TCMs, active ingredients, herbal extracts, cocktail probe substrates as well as CYP450 enzymes, in order to guide future studies.

21 CFR 862.3360 - Drug metabolizing enzyme genotyping system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Drug metabolizing enzyme genotyping system. 862... Test Systems § 862.3360 Drug metabolizing enzyme genotyping system. (a) Identification. A drug metabolizing enzyme genotyping system is a device intended for use in testing deoxyribonucleic acid (DNA...

21 CFR 862.3360 - Drug metabolizing enzyme genotyping system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Drug metabolizing enzyme genotyping system. 862... Test Systems § 862.3360 Drug metabolizing enzyme genotyping system. (a) Identification. A drug metabolizing enzyme genotyping system is a device intended for use in testing deoxyribonucleic acid (DNA...

21 CFR 862.3360 - Drug metabolizing enzyme genotyping system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Drug metabolizing enzyme genotyping system. 862... Test Systems § 862.3360 Drug metabolizing enzyme genotyping system. (a) Identification. A drug metabolizing enzyme genotyping system is a device intended for use in testing deoxyribonucleic acid (DNA...

21 CFR 862.3360 - Drug metabolizing enzyme genotyping system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Drug metabolizing enzyme genotyping system. 862... Test Systems § 862.3360 Drug metabolizing enzyme genotyping system. (a) Identification. A drug metabolizing enzyme genotyping system is a device intended for use in testing deoxyribonucleic acid (DNA...

21 CFR 862.3360 - Drug metabolizing enzyme genotyping system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Drug metabolizing enzyme genotyping system. 862... Test Systems § 862.3360 Drug metabolizing enzyme genotyping system. (a) Identification. A drug metabolizing enzyme genotyping system is a device intended for use in testing deoxyribonucleic acid (DNA...

Genetic variation in genes for the xenobiotic-metabolizing enzymes CYP1A1, EPHX1, GSTM1, GSTT1 and GSTP1 and susceptibility to colorectal cancer in Lynch syndrome

PubMed Central

Pande, Mala; Amos, Christopher I.; Osterwisch, Daniel R.; Chen, Jinyun; Lynch, Patrick M.; Broaddus, Russell; Frazier, Marsha L.

2011-01-01

Individuals with Lynch syndrome are predisposed to cancer due to an inherited DNA mismatch repair gene mutation. However, there is significant variability observed in disease expression, likely due to the influence of other environmental, lifestyle, or genetic factors. Polymorphisms in genes encoding xenobiotic-metabolizing enzymes may modify cancer risk by influencing the metabolism and clearance of potential carcinogens from the body. In this retrospective analysis, we examined key candidate gene polymorphisms in CYP1A1, EPHX1, GSTT1, GSTM1, and GSTP1 as modifiers of age at onset of colorectal cancer among 257 individuals with Lynch syndrome. We found that subjects heterozygous for CYP1A1 I462V (c.1384A>G) developed colorectal cancer 4 years earlier than those with the homozygous wild-type genotype (median ages 39 and 43 years, respectively; log-rank test P = 0.018). Furthermore, being heterozygous for the CYP1A1 polymorphisms, I462V and Msp1 (g.6235T>C), was associated with an increased risk for developing colorectal cancer [adjusted hazard ratio for AG relative to AA = 1.78, 95% CI = 1.16–2.74, P = 0.008; and hazard ratio for TC relative to TT = 1.53, 95% CI = 1.06–2.22, P = 0.02]. Since homozygous variants for both CYP1A1 polymorphisms were rare, risk estimates were imprecise. None of the other gene polymorphisms examined were associated with an earlier onset age for colorectal cancer. Our results suggest that the I462V and Msp1 polymorphisms in CYP1A1 may be an additional susceptibility factor for disease expression in Lynch syndrome since they modify the age of colorectal cancer onset by up to 4 years. PMID:18768509

Rapid birth-and-death evolution of the xenobiotic metabolizing NAT gene family in vertebrates with evidence of adaptive selection

PubMed Central

2013-01-01

Background The arylamine N-acetyltransferases (NATs) are a unique family of enzymes widely distributed in nature that play a crucial role in the detoxification of aromatic amine xenobiotics. Considering the temporal changes in the levels and toxicity of environmentally available chemicals, the metabolic function of NATs is likely to be under adaptive evolution to broaden or change substrate specificity over time, making NATs a promising subject for evolutionary analyses. In this study, we trace the molecular evolutionary history of the NAT gene family during the last ~450 million years of vertebrate evolution and define the likely role of gene duplication, gene conversion and positive selection in the evolutionary dynamics of this family. Results A phylogenetic analysis of 77 NAT sequences from 38 vertebrate species retrieved from public genomic databases shows that NATs are phylogenetically unstable genes, characterized by frequent gene duplications and losses even among closely related species, and that concerted evolution only played a minor role in the patterns of sequence divergence. Local signals of positive selection are detected in several lineages, probably reflecting response to changes in xenobiotic exposure. We then put a special emphasis on the study of the last ~85 million years of primate NAT evolution by determining the NAT homologous sequences in 13 additional primate species. Our phylogenetic analysis supports the view that the three human NAT genes emerged from a first duplication event in the common ancestor of Simiiformes, yielding NAT1 and an ancestral NAT gene which in turn, duplicated in the common ancestor of Catarrhini, giving rise to NAT2 and the NATP pseudogene. Our analysis suggests a main role of purifying selection in NAT1 protein evolution, whereas NAT2 was predicted to mostly evolve under positive selection to change its amino acid sequence over time. These findings are consistent with a differential role of the two human isoenzymes

Do heavy metals and metalloids influence the detoxification of organic xenobiotics in plants?

PubMed

Schröder, Peter; Lyubenova, Lyudmila; Huber, Christian

2009-11-01

Mixed pollution with trace elements and organic industrial compounds is characteristic for many spill areas and dumping sites. The danger for the environment and human health from such sites is large, and sustainable remediation strategies are urgently needed. Phytoremediation seems to be a cheap and environmentally sound option for the removal of unwanted compounds, and the hyperaccumulation of trace elements and toxic metals is seemingly independent from the metabolism of organic xenobiotics. However, stress reactions, ROS formation and depletion of antioxidants will also cause alterations in xenobiotic detoxification. Here, we investigate the capability of plants to detoxify chlorophenols via glutathione conjugation in a mixed pollution situation. Typha latifolia and Phragmites australis plants for the present study were grown under greenhouse conditions in experimental ponds. A Picea abies L. suspension culture was grown in a growth chamber. Cadmium sulphate, sodium arsenate and lead chloride in concentrations from 10 to 500 microM were administered to plants. Enzymes of interest for the present study were: glutathione transferase (GST), glutathione reductase, ascorbate peroxidase and peroxidase. Measurements were performed according to published methods. GST spectrophotometric assays included the model substrates CDNB, DCNB, NBC, NBoC and the herbicide Fluorodifen. Heavy metals lead to visible stress symptoms in higher plants. Besides one long-term experiment of 72 days duration, the present study shows time and concentration-dependent plant alterations already after 24 and 72 h Cd incubation. P. abies spruce cell cultures react to CdSO(4) and Na(2)HAsO(4) with an oxidative burst, similar to that observed after pathogen attack or elicitor treatment. Cd application resulted in a reduction in GSH and GSSG contents. When a heavy metal mixture containing Na(2)HAsO(4), CdSO(4) and PbCl(2) was applied to cultures, both GSH and GSSG levels declined. Incubation with

Short-term calorie restriction feminizes the mRNA profiles of drug metabolizing enzymes and transporters in livers of mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fu, Zidong Donna; Klaassen, Curtis D., E-mail: cklaasse@kumc.edu

2014-01-01

Calorie restriction (CR) is one of the most effective anti-aging interventions in mammals. A modern theory suggests that aging results from a decline in detoxification capabilities and thus accumulation of damaged macromolecules. The present study aimed to determine how short-term CR alters mRNA profiles of genes that encode metabolism and detoxification machinery in the liver. Male C57BL/6 mice were fed CR (0, 15, 30, or 40%) diets for one month, followed by mRNA quantification of 98 xenobiotic processing genes (XPGs) in the liver, including 7 uptake transporters, 39 phase-I enzymes, 37 phase-II enzymes, 10 efflux transporters, and 5 transcription factors.more » In general, 15% CR did not alter mRNAs of most XPGs, whereas 30 and 40% CR altered over half of the XPGs (32 increased and 29 decreased). CR up-regulated some phase-I enzymes (fold increase), such as Cyp4a14 (12), Por (2.3), Nqo1 (1.4), Fmo2 (5.4), and Fmo3 (346), and numerous number of phase-II enzymes, such as Sult1a1 (1.2), Sult1d1 (2.0), Sult1e1 (33), Sult3a1 (2.2), Gsta4 (1.3), Gstm2 (1.3), Gstm3 (1.7), and Mgst3 (2.2). CR feminized the mRNA profiles of 32 XPGs in livers of male mice. For instance, CR decreased the male-predominantly expressed Oatp1a1 (97%) and increased the female-predominantly expressed Oatp1a4 (11). In conclusion, short-term CR alters the mRNA levels of over half of the 98 XPGs quantified in livers of male mice, and over half of these alterations appear to be due to feminization of the liver. - Highlights: • Utilized a graded CR model in male mice • The mRNA profiles of xenobiotic processing genes (XPGs) in liver were investigated. • CR up-regulates many phase-II enzymes. • CR tends to feminize the mRNA profiles of XPGs.« less

Scaling of muscle metabolic enzymes: an historical perspective.

PubMed

Moyes, Christopher D; Genge, Christine E

2010-07-01

In this paper, we take an historical approach to reviewing research into the patterns of metabolic enzymes in muscle in relation to body size, focusing on mitochondrial enzymes. One of the first studies on allometric scaling of muscle enzymes was published in an early issue of this journal (George and Talesara, 1961 Comp. Biochem. Physiol. 3: 267-273). These researchers studied a number of locally available birds and a bat, measuring the activity of the mitochondrial enzyme succinate dehydrogenase in relation to body mass and muscle structure. Though the phenomenon of allometric scaling of metabolism was well recognized even 50 years earlier, this study was one of the first to explore the enzymatic underpinnings of the metabolic patterns in different animals. In this review, we begin by considering the George and Talesara study in the context of this early era in metabolic biochemistry and comparative physiology. We review subsequent studies in the last 50 years that continued the comparative analysis of enzyme patterns in relation to body size in diverse experimental models. This body of work identified a recurrent (though not ubiquitous) reciprocal relationship between oxidative and glycolytic enzymes. In the last 10 years, studies have focused on identifying the molecular mechanisms that determine the muscle metabolic enzyme phenotype. Copyright 2010 Elsevier Inc. All rights reserved.

Metabolic Enzymes Enjoying New Partnerships as RNA-Binding Proteins.

PubMed

Castello, Alfredo; Hentze, Matthias W; Preiss, Thomas

2015-12-01

In the past century, few areas of biology advanced as much as our understanding of the pathways of intermediary metabolism. Initially considered unimportant in terms of gene regulation, crucial cellular fate changes, cell differentiation, or malignant transformation are now known to involve 'metabolic remodeling' with profound changes in the expression of many metabolic enzyme genes. This review focuses on the recent identification of RNA-binding activity of numerous metabolic enzymes. We discuss possible roles of this unexpected second activity in feedback gene regulation ('moonlighting') and/or in the control of enzymatic function. We also consider how metabolism-driven post-translational modifications could regulate enzyme-RNA interactions. Thus, RNA emerges as a new partner of metabolic enzymes with far-reaching possible consequences to be unraveled in the future. Copyright © 2015 Elsevier Ltd. All rights reserved.

Two horizontally transferred xenobiotic resistance gene clusters associated with detoxification of benzoxazolinones by Fusarium species

USDA-ARS?s Scientific Manuscript database

Microbes encounter a broad spectrum of chemical compounds in their diverse environments. These xenobiotics may negatively impact growth or cause death. To counter such adverse effects, many microbes possess metabolic strategies to detoxify and biotransform xenobiotics. Fusarium verticillioides is a ...

Chemical Screening for Bioactivated Electrophilic Metabolites Using Alginate Immobilization of Metabolic Enzymes (AIME) (SOT)

EPA Science Inventory

The US EPA's ToxCast program is designed to assess chemical perturbations of molecular and cellular endpoints using a variety of high-throughput screening (HTS) assays. However, existing HTS assays have limited or no xenobiotic metabolism which could lead to a mischaracterization...

Comparative metabolism as a key driver of wildlife species sensitivity to human and veterinary pharmaceuticals

PubMed Central

Hutchinson, Thomas H.; Madden, Judith C.; Naidoo, Vinny; Walker, Colin H.

2014-01-01

Human and veterinary drug development addresses absorption, distribution, metabolism, elimination and toxicology (ADMET) of the Active Pharmaceutical Ingredient (API) in the target species. Metabolism is an important factor in controlling circulating plasma and target tissue API concentrations and in generating metabolites which are more easily eliminated in bile, faeces and urine. The essential purpose of xenobiotic metabolism is to convert lipid-soluble, non-polar and non-excretable chemicals into water soluble, polar molecules that are readily excreted. Xenobiotic metabolism is classified into Phase I enzymatic reactions (which add or expose reactive functional groups on xenobiotic molecules), Phase II reactions (resulting in xenobiotic conjugation with large water-soluble, polar molecules) and Phase III cellular efflux transport processes. The human–fish plasma model provides a useful approach to understanding the pharmacokinetics of APIs (e.g. diclofenac, ibuprofen and propranolol) in freshwater fish, where gill and liver metabolism of APIs have been shown to be of importance. By contrast, wildlife species with low metabolic competency may exhibit zero-order metabolic (pharmacokinetic) profiles and thus high API toxicity, as in the case of diclofenac and the dramatic decline of vulture populations across the Indian subcontinent. A similar threat looms for African Cape Griffon vultures exposed to ketoprofen and meloxicam, recent studies indicating toxicity relates to zero-order metabolism (suggesting P450 Phase I enzyme system or Phase II glucuronidation deficiencies). While all aspects of ADMET are important in toxicity evaluations, these observations demonstrate the importance of methods for predicting API comparative metabolism as a central part of environmental risk assessment. PMID:25405970

Contributions of Human Enzymes in Carcinogen Metabolism

PubMed Central

Rendic, Slobodan; Guengerich, F. Peter

2012-01-01

Considerable support exists for roles of metabolism in modulating the carcinogenic properties of chemicals. In particular, many of these compounds are procarcinogens that require activation to electrophilic forms to exert genotoxic effects. We systematically analyzed the existing literature on metabolism of carcinogens by human enzymes, which has been developed largely in the past 25 years. The metabolism and especially bioactivation of carcinogens are dominated by cytochrome P450 enzymes (66% of bioactivations). Within this group, six P450s—1A1, 1A2, 1B1, 2A6, 2E1, and 3A4—accounted for 77% of the P450 activation reactions. The roles of these P450s can be compared with those estimated for drug metabolism and should be considered in issues involving enzyme induction, chemoprevention, molecular epidemiology, inter-individual variations, and risk assessment. PMID:22531028

Regulation of hepatic energy metabolism by the nuclear receptor PXR.

PubMed

Hakkola, Jukka; Rysä, Jaana; Hukkanen, Janne

2016-09-01

The pregnane X receptor (PXR) is a nuclear receptor that is traditionally thought to be specialized for sensing xenobiotic exposure. In concurrence with this feature PXR was originally identified to regulate drug-metabolizing enzymes and transporters. During the last ten years it has become clear that PXR harbors broader functions. Evidence obtained both in experimental animals and humans indicate that ligand-activated PXR regulates hepatic glucose and lipid metabolism and affects whole body metabolic homeostasis. Currently, the consequences of PXR activation on overall metabolic health are not yet fully understood and varying results on the effect of PXR activation or knockout on metabolic disorders and weight gain have been published in mouse models. Rifampicin and St. John's wort, the prototypical human PXR agonists, impair glucose tolerance in healthy volunteers. Chronic exposure to PXR agonists could potentially represent a risk factor for diabetes and metabolic syndrome. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie. Copyright © 2016 Elsevier B.V. All rights reserved.

The biochemistry and molecular biology of xenobiotic polymer degradation by microorganisms.

PubMed

Kawai, Fusako

2010-01-01

Research on microbial degradation of xenobiotic polymers has been underway for more than 40 years. It has exploited a new field not only in applied microbiology but also in environmental microbiology, and has greatly contributed to polymer science by initiating the design of biodegradable polymers. Owing to the development of analytical tools and technology, molecular biological and biochemical advances have made it possible to prospect for degrading microorganisms in the environment and to determine the mechanisms involved in biodegradation when xenobiotic polymers are introduced into the environment and are exposed to microbial attack. In this review, the molecular biological and biochemical aspects of the microbial degradation of xenobiotic polymers are summarized, and possible applications of potent microorganisms, enzymes, and genes in environmental biotechnology are suggested.

Advances in drug metabolism and pharmacogenetics research in Australia.

PubMed

Mackenzie, Peter I; Somogyi, Andrew A; Miners, John O

2017-02-01

Metabolism facilitates the elimination, detoxification and excretion in urine or bile (as biotransformation products) of a myriad of structurally diverse drugs and other chemicals. The metabolism of drugs, non-drug xenobiotics and many endogenous compounds is catalyzed by families of drug metabolizing enzymes (DMEs). These include the hemoprotein-containing cytochromes P450, which function predominantly as monooxygenases, and conjugation enzymes that transfer a sugar, sulfate, acetate or glutathione moiety to substrates containing a suitable acceptor functional group. Drug and chemical metabolism, especially the enzymes that catalyse these reactions, has been the research focus of several groups in Australia for over four decades. In this review, we highlight the role of recent and current drug metabolism research in Australia, including elucidation of the structure and function of enzymes from the various DME families, factors that modulate enzyme activity in humans (e.g. drug-drug interactions, gene expression and genetic polymorphism) and the application of in vitro approaches for the prediction of drug metabolism parameters in humans, along with the broader pharmacological/clinical pharmacological and toxicological significance of drug metabolism and DMEs and their relevance to drug discovery and development, and to clinical practice. Copyright © 2016 Elsevier Ltd. All rights reserved.

Back to the future: transgenerational transmission of xenobiotic-induced epigenetic remodeling

PubMed Central

Jiménez-Chillarón, Josep C; Nijland, Mark J; Ascensão, António A; Sardão, Vilma A; Magalhães, José; Hitchler, Michael J; Domann, Frederick E; Oliveira, Paulo J

2015-01-01

Epigenetics, or regulation of gene expression independent of DNA sequence, is the missing link between genotype and phenotype. Epigenetic memory, mediated by histone and DNA modifications, is controlled by a set of specialized enzymes, metabolite availability, and signaling pathways. A mostly unstudied subject is how sub-toxic exposure to several xenobiotics during specific developmental stages can alter the epigenome and contribute to the development of disease phenotypes later in life. Furthermore, it has been shown that exposure to low-dose xenobiotics can also result in further epigenetic remodeling in the germ line and contribute to increase disease risk in the next generation (multigenerational and transgenerational effects). We here offer a perspective on current but still incomplete knowledge of xenobiotic-induced epigenetic alterations, and their possible transgenerational transmission. We also propose several molecular mechanisms by which the epigenetic landscape may be altered by environmental xenobiotics and hypothesize how diet and physical activity may counteract epigenetic alterations. PMID:25774863

Substrate specificity of xenobiotic metabolizing esterases in the liver of two catfish species

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jaiswal, R.G.; Huang, T.L.; Obih, P.O.

1994-12-31

The preliminary studies were conducted on the characterization of substrate specificity in the liver microsomes and cytosol of two catfish species, Ictalurus punctatus and Ictalurus natalie. A series of five esters of p-nitrophenol were used as calorimetric substrates to assay the carboxylesterases. The substrate specificity of liver microsomal and cytosolic carboxylesterases were remarkably different from each other. The valerate ester of p-nitrophenol was most rapidly hydrolyzed by the microsomal carboxylesterases, whereas the prioponate ester was the best substrate for cytosolic carboxylesterases. The Ictalurus natalie catfish species were obtained from the Devil Swamp site of the Mississippi River Basin which ismore » known to be heavily contaminated with toxic and hazardous industrial wastes. These results will be discussed in relation to the responses of xenobiotic metabolizing esterases to environmental pollutants and their possible use as biomarkers.« less

Non-metabolic functions of glycolytic enzymes in tumorigenesis.

PubMed

Yu, X; Li, S

2017-05-11

Cancer cells reprogram their metabolism to meet the requirement for survival and rapid growth. One hallmark of cancer metabolism is elevated aerobic glycolysis and reduced oxidative phosphorylation. Emerging evidence showed that most glycolytic enzymes are deregulated in cancer cells and play important roles in tumorigenesis. Recent studies revealed that all essential glycolytic enzymes can be translocated into nucleus where they participate in tumor progression independent of their canonical metabolic roles. These noncanonical functions include anti-apoptosis, regulation of epigenetic modifications, modulation of transcription factors and co-factors, extracellular cytokine, protein kinase activity and mTORC1 signaling pathway, suggesting that these multifaceted glycolytic enzymes not only function in canonical metabolism but also directly link metabolism to epigenetic and transcription programs implicated in tumorigenesis. These findings underscore our understanding about how tumor cells adapt to nutrient and fuel availability in the environment and most importantly, provide insights into development of cancer therapy.

From 20th century metabolic wall charts to 21st century systems biology: database of mammalian metabolic enzymes

PubMed Central

Corcoran, Callan C.; Grady, Cameron R.; Pisitkun, Trairak; Parulekar, Jaya

2017-01-01

The organization of the mammalian genome into gene subsets corresponding to specific functional classes has provided key tools for systems biology research. Here, we have created a web-accessible resource called the Mammalian Metabolic Enzyme Database (https://hpcwebapps.cit.nih.gov/ESBL/Database/MetabolicEnzymes/MetabolicEnzymeDatabase.html) keyed to the biochemical reactions represented on iconic metabolic pathway wall charts created in the previous century. Overall, we have mapped 1,647 genes to these pathways, representing ~7 percent of the protein-coding genome. To illustrate the use of the database, we apply it to the area of kidney physiology. In so doing, we have created an additional database (Database of Metabolic Enzymes in Kidney Tubule Segments: https://hpcwebapps.cit.nih.gov/ESBL/Database/MetabolicEnzymes/), mapping mRNA abundance measurements (mined from RNA-Seq studies) for all metabolic enzymes to each of 14 renal tubule segments. We carry out bioinformatics analysis of the enzyme expression pattern among renal tubule segments and mine various data sources to identify vasopressin-regulated metabolic enzymes in the renal collecting duct. PMID:27974320

Enzyme clustering accelerates processing of intermediates through metabolic channeling

PubMed Central

Castellana, Michele; Wilson, Maxwell Z.; Xu, Yifan; Joshi, Preeti; Cristea, Ileana M.; Rabinowitz, Joshua D.; Gitai, Zemer; Wingreen, Ned S.

2015-01-01

We present a quantitative model to demonstrate that coclustering multiple enzymes into compact agglomerates accelerates the processing of intermediates, yielding the same efficiency benefits as direct channeling, a well-known mechanism in which enzymes are funneled between enzyme active sites through a physical tunnel. The model predicts the separation and size of coclusters that maximize metabolic efficiency, and this prediction is in agreement with previously reported spacings between coclusters in mammalian cells. For direct validation, we study a metabolic branch point in Escherichia coli and experimentally confirm the model prediction that enzyme agglomerates can accelerate the processing of a shared intermediate by one branch, and thus regulate steady-state flux division. Our studies establish a quantitative framework to understand coclustering-mediated metabolic channeling and its application to both efficiency improvement and metabolic regulation. PMID:25262299

DrugMetZ DB: an anthology of human drug metabolizing Chytochrome P450 enzymes.

PubMed

Antony, Tresa Remya Thomas; Nagarajan, Shanthi

2006-11-14

Understandings the basics of Cytochrome P450 (P450 or CYP) will help to discern drug metabolism. CYP, a super-family of heme-thiolate proteins, are found in almost all living organisms and is involved in the biotransformation of a diverse range of xenobiotics, therapeutic drugs and toxins. Here, we describe DrugMetZ DB, a database for CYP metabolizing drugs. The DB is implemented in MySQL, PHP and HTML. www.bicpu.edu.in/DrugMetZDB/

Drug Metabolism by the Host and Gut Microbiota: A Partnership or Rivalry?

PubMed Central

2015-01-01

The importance of the gut microbiome in determining not only overall health, but also in the metabolism of drugs and xenobiotics, is rapidly emerging. It is becoming increasingly clear that the gut microbiota can act in concert with the host cells to maintain intestinal homeostasis, cometabolize drugs and xenobiotics, and alter the expression levels of drug-metabolizing enzymes and transporters and the expression and activity levels of nuclear receptors. In this myriad of activities, the impact of the microbiota may be beneficial or detrimental to the host. Given that the interplay between the gut microbiota and host cells is likely subject to high interindividual variability, this work has tremendous implications for our ability to predict accurately a particular drug’s pharmacokinetics and a given patient population’s response to drugs. In this issue of Drug Metabolism and Disposition, a series of articles is presented that illustrate the progress and challenges that lie ahead as we unravel the intricacies associated with drug and xenobiotic metabolism by the gut microbiota. These articles highlight the underlying mechanisms that are involved and the use of in vivo and in vitro approaches that are currently available for elucidating the role of the gut microbiota in drug and xenobiotic metabolism. These articles also shed light on exciting new avenues of research that may be pursued as we consider the role of the gut microbiota as an endocrine organ, a component of the brain-gut axis, and whether the gut microbiota is an appropriate and amenable target for new drugs. PMID:26261284

Drug Metabolism by the Host and Gut Microbiota: A Partnership or Rivalry?

PubMed

Swanson, Hollie I

2015-10-01

The importance of the gut microbiome in determining not only overall health, but also in the metabolism of drugs and xenobiotics, is rapidly emerging. It is becoming increasingly clear that the gut microbiota can act in concert with the host cells to maintain intestinal homeostasis, cometabolize drugs and xenobiotics, and alter the expression levels of drug-metabolizing enzymes and transporters and the expression and activity levels of nuclear receptors. In this myriad of activities, the impact of the microbiota may be beneficial or detrimental to the host. Given that the interplay between the gut microbiota and host cells is likely subject to high interindividual variability, this work has tremendous implications for our ability to predict accurately a particular drug's pharmacokinetics and a given patient population's response to drugs. In this issue of Drug Metabolism and Disposition, a series of articles is presented that illustrate the progress and challenges that lie ahead as we unravel the intricacies associated with drug and xenobiotic metabolism by the gut microbiota. These articles highlight the underlying mechanisms that are involved and the use of in vivo and in vitro approaches that are currently available for elucidating the role of the gut microbiota in drug and xenobiotic metabolism. These articles also shed light on exciting new avenues of research that may be pursued as we consider the role of the gut microbiota as an endocrine organ, a component of the brain-gut axis, and whether the gut microbiota is an appropriate and amenable target for new drugs. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Non-Alcoholic Fatty Liver Disease (NAFLD) - Pathogenesis, Classification, and Effect on Drug Metabolizing Enzymes and Transporters

PubMed Central

Cobbina, Enoch; Akhlaghi, Fatemeh

2017-01-01

Non-alcoholic fatty liver disease (NAFLD) is a spectrum of liver disorders. It is defined by the presence of steatosis in more than 5 % of hepatocytes with little or no alcohol consumption. Insulin resistance, the metabolic syndrome or type 2 diabetes and genetic variants of PNPLA3 or TM6SF2 seem to play a role in the pathogenesis of NAFLD. The pathological progression of NAFLD follows tentatively a ‘three-hit’ process namely steatosis, lipotoxicity and inflammation. The presence of steatosis, oxidative stress and inflammatory mediators like TNF-α and IL-6 have been implicated in the alterations of nuclear factors such as CAR, PXR, PPAR-α in NAFLD. These factors may results in altered expression and activity of drug metabolizing enzymes (DMEs) or transporters. Existing evidence suggests that the effect of NAFLD on CYP3A4, CYP2E1 and MRP3 are more consistent across rodent and human studies. CYP3A4 activity is down-regulated in NASH whereas the activity of CYP2E1 and the efflux transporter MRP3 are up-regulated. However, it is not clear how the majority of CYPs, UGTs, SULTs and transporters are influenced by NAFLD either in vivo or in vitro. The alterations associated with NAFLD could be a potential source of drug variability in patients and could have serious implications for the safety and efficacy of xenobiotics. In this review, we summarize the effects of NAFLD on the regulation, expression and activity of major drug metabolizing enzymes and transporters. We also discuss the potential mechanisms underlying these alterations. PMID:28303724

COMMENTS ON "EFFECT OF PRENATAL EXPOSURE OF DELTAMETHRIN ON THE ONTOGENY OF XENOBIOTIC METABOLIZING CYTOCHROME P450S IN THE BRAIN AND LIVER OF OFFSPRINGS.

EPA Science Inventory

Comments on: Effect of prenatal exposure of deltamethrin on the ontogeny of xenobiotic metabolizing cytochrome P450s in the brain and liver of offsprings [Johri et al. Toxicol Appl Pharmacol. 214:279-289, 2006]

Johri and colleagues recently reported that maternal exposur...

The evolution of new enzyme function: lessons from xenobiotic metabolizing bacteria versus insecticide-resistant insects

PubMed Central

Russell, Robyn J; Scott, Colin; Jackson, Colin J; Pandey, Rinku; Pandey, Gunjan; Taylor, Matthew C; Coppin, Christopher W; Liu, Jian-Wei; Oakeshott, John G

2011-01-01

Here, we compare the evolutionary routes by which bacteria and insects have evolved enzymatic processes for the degradation of four classes of synthetic chemical insecticide. For insects, the selective advantage of such degradative activities is survival on exposure to the insecticide, whereas for the bacteria the advantage is simply a matter of access to additional sources of nutrients. Nevertheless, bacteria have evolved highly efficient enzymes from a wide variety of enzyme families, whereas insects have relied upon generalist esterase-, cytochrome P450- and glutathione-S-transferase-dependent detoxification systems. Moreover, the mutant insect enzymes are less efficient kinetically and less diverged in sequence from their putative ancestors than their bacterial counterparts. This presumably reflects several advantages that bacteria have over insects in the acquisition of new enzymatic functions, such as a broad biochemical repertoire from which new functions can be evolved, large population sizes, high effective mutation rates, very short generation times and access to genetic diversity through horizontal gene transfer. Both the insect and bacterial systems support recent theory proposing that new biochemical functions often evolve from ‘promiscuous’ activities in existing enzymes, with subsequent mutations then enhancing those activities. Study of the insect enzymes will help in resistance management, while the bacterial enzymes are potential bioremediants of insecticide residues in a range of contaminated environments. PMID:25567970

From 20th century metabolic wall charts to 21st century systems biology: database of mammalian metabolic enzymes.

PubMed

Corcoran, Callan C; Grady, Cameron R; Pisitkun, Trairak; Parulekar, Jaya; Knepper, Mark A

2017-03-01

The organization of the mammalian genome into gene subsets corresponding to specific functional classes has provided key tools for systems biology research. Here, we have created a web-accessible resource called the Mammalian Metabolic Enzyme Database ( https://hpcwebapps.cit.nih.gov/ESBL/Database/MetabolicEnzymes/MetabolicEnzymeDatabase.html) keyed to the biochemical reactions represented on iconic metabolic pathway wall charts created in the previous century. Overall, we have mapped 1,647 genes to these pathways, representing ~7 percent of the protein-coding genome. To illustrate the use of the database, we apply it to the area of kidney physiology. In so doing, we have created an additional database ( Database of Metabolic Enzymes in Kidney Tubule Segments: https://hpcwebapps.cit.nih.gov/ESBL/Database/MetabolicEnzymes/), mapping mRNA abundance measurements (mined from RNA-Seq studies) for all metabolic enzymes to each of 14 renal tubule segments. We carry out bioinformatics analysis of the enzyme expression pattern among renal tubule segments and mine various data sources to identify vasopressin-regulated metabolic enzymes in the renal collecting duct. Copyright © 2017 the American Physiological Society.

[THE SYSTEM OF XENOBIOTICS BIOTRANSFORMATION OF HELMINTHS. RESEMBLANCE AND DIFFERENSES FROM SIMILAR HOST SYSTEMS (REWEW)].

PubMed

Smirnov, L P; Borvinskaya, E V; Suhovskaya, I V

2016-01-01

The three phases system xenobiotic biotransformation in cells as prokaryotes as eukaryotes was formed during the process of evolution. Clear and managed function of all three links of this system guarantee the survival of living organisms at alteration of chemical component of environment. Oxidation, reduction or hydrolysis of xenobiotics realize in phase I by insertion or opening reactive and hydrophilic groups in structure of drug molecule. In phase II xenobiotics or their metabolites from phase I conjugate with endogenic compounds, main of there are glutathione, glucuronic acid, amino acids and sulphates. Active transport of substrata, metabolites and conjugates through cell lipid membranes special transport proteins carry out (phase III). The system of xenobiotics biotransformation of helminths has essential differences from the same of vertebrate hosts. In particular, parasites do not reveal the activity of prime oxidases of phase I, such as CYP or FMO, in spite of the genes of these enzymes in DNA. As this phenomenon displays mainly in adult helminths, living in guts of vertebrates, then the hypothesis was formulated that this effect is related with adaptation to conditions of strong deficiency of oxygen, arise in a process of evolution (Kotze et al., 2006). Literature data testify the existence in helminths of unique forms of enzymes of phase II, the investigation of which present doubtless interest in relation with possible role in adaptation to parasitic mode of life. Notwithstanding that many of helminths GST in greater or lesser degree similar with enzymes of M, P, S and О classes of other organisms, nevertheless they have essential structural differences as compared with enzymes of hosts that makes perspective the search of specific anthelminthics vaccines. Transport of xenobiotics is now considered phase III of biotransformation. It was shown that proteins of this phase (ATP binding cassette transporters (ABC ) of parasites) play a key role in efflux

Cadmium effects on oxidative metabolism in a marine seagrass: Posidonia oceanica

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hamoutene, D.; Romeo, M.; Gnassia, M.

1996-02-01

Posidonia oceanica, a seagrass in contact with sediment, is an interesting organism for environmental biomonitoring. In fact, up to now, scientists have studied some pollutant effects on growth rate leaf length, or productivity of this species. In recent years, the topic of metabolism of xenobiotics in plants has arisen. Many of the environmental chemicals are biotransformed by plants in a manner similar to those in mammalian systems. Studies of the influence of pollutants on biotransformation enzymes in posidonia oceanica are of great interest in pollution monitoring programs especially in the Mediterranean sea. In a previous study, we characterized some enzymaticmore » systems involved in the metabolism of xenobiotics in Posidonia oceanica. This study was designed to determine the effects of cadmium on certain biochemical functions in this species.« less

Research Resource: A Reference Transcriptome for Constitutive Androstane Receptor and Pregnane X Receptor Xenobiotic Signaling

PubMed Central

Ochsner, Scott A.; Tsimelzon, Anna; Dong, Jianrong; Coarfa, Cristian

2016-01-01

The pregnane X receptor (PXR) (PXR/NR1I3) and constitutive androstane receptor (CAR) (CAR/NR1I2) members of the nuclear receptor (NR) superfamily of ligand-regulated transcription factors are well-characterized mediators of xenobiotic and endocrine-disrupting chemical signaling. The Nuclear Receptor Signaling Atlas maintains a growing library of transcriptomic datasets involving perturbations of NR signaling pathways, many of which involve perturbations relevant to PXR and CAR xenobiotic signaling. Here, we generated a reference transcriptome based on the frequency of differential expression of genes across 159 experiments compiled from 22 datasets involving perturbations of CAR and PXR signaling pathways. In addition to the anticipated overrepresentation in the reference transcriptome of genes encoding components of the xenobiotic stress response, the ranking of genes involved in carbohydrate metabolism and gonadotropin action sheds mechanistic light on the suspected role of xenobiotics in metabolic syndrome and reproductive disorders. Gene Set Enrichment Analysis showed that although acetaminophen, chlorpromazine, and phenobarbital impacted many similar gene sets, differences in direction of regulation were evident in a variety of processes. Strikingly, gene sets representing genes linked to Parkinson's, Huntington's, and Alzheimer's diseases were enriched in all 3 transcriptomes. The reference xenobiotic transcriptome will be supplemented with additional future datasets to provide the community with a continually updated reference transcriptomic dataset for CAR- and PXR-mediated xenobiotic signaling. Our study demonstrates how aggregating and annotating transcriptomic datasets, and making them available for routine data mining, facilitates research into the mechanisms by which xenobiotics and endocrine-disrupting chemicals subvert conventional NR signaling modalities. PMID:27409825

Research Resource: A Reference Transcriptome for Constitutive Androstane Receptor and Pregnane X Receptor Xenobiotic Signaling.

PubMed

Ochsner, Scott A; Tsimelzon, Anna; Dong, Jianrong; Coarfa, Cristian; McKenna, Neil J

2016-08-01

The pregnane X receptor (PXR) (PXR/NR1I3) and constitutive androstane receptor (CAR) (CAR/NR1I2) members of the nuclear receptor (NR) superfamily of ligand-regulated transcription factors are well-characterized mediators of xenobiotic and endocrine-disrupting chemical signaling. The Nuclear Receptor Signaling Atlas maintains a growing library of transcriptomic datasets involving perturbations of NR signaling pathways, many of which involve perturbations relevant to PXR and CAR xenobiotic signaling. Here, we generated a reference transcriptome based on the frequency of differential expression of genes across 159 experiments compiled from 22 datasets involving perturbations of CAR and PXR signaling pathways. In addition to the anticipated overrepresentation in the reference transcriptome of genes encoding components of the xenobiotic stress response, the ranking of genes involved in carbohydrate metabolism and gonadotropin action sheds mechanistic light on the suspected role of xenobiotics in metabolic syndrome and reproductive disorders. Gene Set Enrichment Analysis showed that although acetaminophen, chlorpromazine, and phenobarbital impacted many similar gene sets, differences in direction of regulation were evident in a variety of processes. Strikingly, gene sets representing genes linked to Parkinson's, Huntington's, and Alzheimer's diseases were enriched in all 3 transcriptomes. The reference xenobiotic transcriptome will be supplemented with additional future datasets to provide the community with a continually updated reference transcriptomic dataset for CAR- and PXR-mediated xenobiotic signaling. Our study demonstrates how aggregating and annotating transcriptomic datasets, and making them available for routine data mining, facilitates research into the mechanisms by which xenobiotics and endocrine-disrupting chemicals subvert conventional NR signaling modalities.

Regulation of yeast central metabolism by enzyme phosphorylation

PubMed Central

Oliveira, Ana Paula; Ludwig, Christina; Picotti, Paola; Kogadeeva, Maria; Aebersold, Ruedi; Sauer, Uwe

2012-01-01

As a frequent post-translational modification, protein phosphorylation regulates many cellular processes. Although several hundred phosphorylation sites have been mapped to metabolic enzymes in Saccharomyces cerevisiae, functionality was demonstrated for few of them. Here, we describe a novel approach to identify in vivo functionality of enzyme phosphorylation by combining flux analysis with proteomics and phosphoproteomics. Focusing on the network of 204 enzymes that constitute the yeast central carbon and amino-acid metabolism, we combined protein and phosphoprotein levels to identify 35 enzymes that change their degree of phosphorylation during growth under five conditions. Correlations between previously determined intracellular fluxes and phosphoprotein abundances provided first functional evidence for five novel phosphoregulated enzymes in this network, adding to nine known phosphoenzymes. For the pyruvate dehydrogenase complex E1 α subunit Pda1 and the newly identified phosphoregulated glycerol-3-phosphate dehydrogenase Gpd1 and phosphofructose-1-kinase complex β subunit Pfk2, we then validated functionality of specific phosphosites through absolute peptide quantification by targeted mass spectrometry, metabolomics and physiological flux analysis in mutants with genetically removed phosphosites. These results demonstrate the role of phosphorylation in controlling the metabolic flux realised by these three enzymes. PMID:23149688

Identifying metabolic enzymes with multiple types of association evidence

PubMed Central

Kharchenko, Peter; Chen, Lifeng; Freund, Yoav; Vitkup, Dennis; Church, George M

2006-01-01

Background Existing large-scale metabolic models of sequenced organisms commonly include enzymatic functions which can not be attributed to any gene in that organism. Existing computational strategies for identifying such missing genes rely primarily on sequence homology to known enzyme-encoding genes. Results We present a novel method for identifying genes encoding for a specific metabolic function based on a local structure of metabolic network and multiple types of functional association evidence, including clustering of genes on the chromosome, similarity of phylogenetic profiles, gene expression, protein fusion events and others. Using E. coli and S. cerevisiae metabolic networks, we illustrate predictive ability of each individual type of association evidence and show that significantly better predictions can be obtained based on the combination of all data. In this way our method is able to predict 60% of enzyme-encoding genes of E. coli metabolism within the top 10 (out of 3551) candidates for their enzymatic function, and as a top candidate within 43% of the cases. Conclusion We illustrate that a combination of genome context and other functional association evidence is effective in predicting genes encoding metabolic enzymes. Our approach does not rely on direct sequence homology to known enzyme-encoding genes, and can be used in conjunction with traditional homology-based metabolic reconstruction methods. The method can also be used to target orphan metabolic activities. PMID:16571130

Free Radical Mechanisms of Xenobiotic Mammalian Cytotoxicities

DTIC Science & Technology

1991-06-30

injury process was mediated through biotransformation of the halocarbons to a free radical intermediate, similar to what happens in the liver . However...peroxidation) of antioxidant agents - is not limited to the liver , but also occurs in vascular cells as well. Unlike the liver , where most of the injury is...frequent mechanism of xenobiotic liver toxicity is biotransformation by cytochrome P,5o-enzymes to toxic free radical intermediates. The primary objective

Transposable elements are enriched within or in close proximity to xenobiotic-metabolizing cytochrome P450 genes

PubMed Central

Chen, Song; Li, Xianchun

2007-01-01

Background Transposons, i.e. transposable elements (TEs), are the major internal spontaneous mutation agents for the variability of eukaryotic genomes. To address the general issue of whether transposons mediate genomic changes in environment-adaptation genes, we scanned two alleles per each of the six xenobiotic-metabolizing Helicoverpa zea cytochrome P450 loci, including CYP6B8, CYP6B27, CYP321A1, CYP321A2, CYP9A12v3 and CYP9A14, for the presence of transposon insertions by genome walking and sequence analysis. We also scanned thirteen Drosophila melanogaster P450s genes for TE insertions by in silico mapping and literature search. Results Twelve novel transposons, including LINEs (long interspersed nuclear elements), SINEs (short interspersed nuclear elements), MITEs (miniature inverted-repeat transposable elements), one full-length transib-like transposon, and one full-length Tcl-like DNA transpson, are identified from the alleles of the six H. zea P450 genes. The twelve transposons are inserted into the 5'flanking region, 3'flanking region, exon, or intron of the six environment-adaptation P450 genes. In D. melanogaster, seven out of the eight Drosophila P450s (CYP4E2, CYP6A2, CYP6A8, CYP6A9, CYP6G1, CYP6W1, CYP12A4, CYP12D1) implicated in insecticide resistance are associated with a variety of transposons. By contrast, all the five Drosophila P450s (CYP302A1, CYP306A1, CYP307A1, CYP314A1 and CYP315A1) involved in ecdysone biosynthesis and developmental regulation are free of TE insertions. Conclusion These results indicate that TEs are selectively retained within or in close proximity to xenobiotic-metabolizing P450 genes. PMID:17381843

Phosphorylation of Isoflavones by Bacillus subtilis BCRC 80517 May Represent Xenobiotic Metabolism.

PubMed

Hsu, Chen; Wu, Bo-Yuan; Chang, Yu-Chuan; Chang, Chi-Fon; Chiou, Tai-Ying; Su, Nan-Wei

2018-01-10

The soy isoflavones daidzein (DAI) and genistein (GEN) have beneficial effects on human health. However, their oral bioavailability is hampered by their low aqueous solubility. Our previous study revealed two water-soluble phosphorylated conjugates of isoflavones, daidzein 7-O-phosphate and genistein 7-O-phosphate, generated via biotransformation by Bacillus subtilis BCRC80517 cultivated with isoflavones. In this study, two novel derivatives of isoflavones, daidzein 4'-O-phosphate and genistein 4'-O-phosphate, were identified by HPLC-ESI-MS/MS and 1 H, 13 C, and 31 P NMR, and their biotransformation roadmaps were proposed. Primarily, isoflavone glucosides were deglycosylated and then phosphorylated predominantly into 7-O-phosphate conjugates with traces of 4'-O-phosphate conjugates. Inevitably, trace quantities of glucosides were converted into 6″-O-succinyl glucosides. GEN was more efficiently phosphorylated than DAI. Nevertheless, the presence of GEN prolonged the time until the exponential phase of cell growth, whereas the other isoflavones showed little effect on cell growth. Our findings provide new insights into the novel microbial phosphorylation of isoflavones involved in xenobiotic metabolism.

Mechanistic insights into the regulation of metabolic enzymes by acetylation

PubMed Central

2012-01-01

The activity of metabolic enzymes is controlled by three principle levels: the amount of enzyme, the catalytic activity, and the accessibility of substrates. Reversible lysine acetylation is emerging as a major regulatory mechanism in metabolism that is involved in all three levels of controlling metabolic enzymes and is altered frequently in human diseases. Acetylation rivals other common posttranslational modifications in cell regulation not only in the number of substrates it modifies, but also the variety of regulatory mechanisms it facilitates. PMID:22826120

Altered drug metabolism during pregnancy: Hormonal regulation of drug-metabolizing enzymes

PubMed Central

Jeong, Hyunyoung

2013-01-01

Importance of the field Medication use during pregnancy is prevalent, but pharmacokinetic information of most drugs used during pregnancy is lacking in spite of known effects of pregnancy on drug disposition. Accurate pharmacokinetic information is essential for optimal drug therapy in mother and fetus. Thus, understanding how pregnancy influences drug disposition is important for better prediction of pharmacokinetic changes of drugs in pregnant women. Areas covered in this review Pregnancy is known to affect hepatic drug metabolism, but the underlying mechanisms remain unknown. Physiological changes accompanying pregnancy are likely responsible for the reported alteration in drug metabolism during pregnancy. These include elevated concentrations of various hormones such as estrogen, progesterone, placental growth hormones and prolactin. This review covers how these hormones influence expression of drug-metabolizing enzymes, thus potentially responsible for altered drug metabolism during pregnancy. What the reader will gain The reader will gain a greater understanding of the altered drug metabolism in pregnant women and the regulatory effects of pregnancy hormones on expression of drug-metabolizing enzymes. Take home message In-depth studies in hormonal regulatory mechanisms as well as confirmatory studies in pregnant women are warranted for systematic understanding and prediction of the changes in hepatic drug metabolism during pregnancy. PMID:20367533

[Carbohydrate metabolism in acute poisoning with xenobiotics].

PubMed

Pach, Dorota; Szurkowska, Magdalena; Szafraniec, Krystyna; Targosz, Dorota; Sułek, Monika; Kamenczak, Aleksandra; Huszno, Bohdan

2007-01-01

The aim of the study was to evaluate carbohydrate metabolism in patients hospitalised because of acute intoxication with xenobiotics. An analysis of 3628 patients (1553 females and 2075 males; age: 40.6 +/- 15.9 y) hospitalized at the Ward of Toxicology and Environmental Diseases because of acute poisoning in 2004-2006 was done. The patients with diabetes mellitus diagnosed prior to hospitalisation were excluded from the analysis. The blood ethanol concentration was measured, medication drugs and/or psychoactive substance screening test were performed in all patients on admission. Fasting glucose level on admission and control level on second or third day of hospitalisation were determined. Risk ratio of hyperglycaemia according to toxic agent was assessed using multiple regression model considering age, gender, and the patient education. In 18.2% (398 males and 254 females) of the patients the blood glucose level on admission was > or = 7.8 mmol/l; in u 3.6% (78 males and 50 females) > or = 11.1 mmol/l. In 24 (0.6%) of the patients glycaemia on admission was < or = 3.5 mmol/l. Control fasting glucose level of > or = 7.0 mmol/l was determined in 115 males and 76 female patients. 42% elevation in risk of hyperglycaemia was noted in acute carbon monoxide poisoning (OR = 1.42; 95% PU: 1.11-1.82). In ethanol intoxicated patients 12% drop in risk of hyperglycaemia was noted (OR = 0.88; 95% PU: 0.72-1.07). Benzodiazepine poisoning diminished risk of hyperglycaemia in 36% (OR=0.64; 95%PU: 0.48-0.84). Risk of hyperglycaemia in poisoning by medicines co-ingested with ethanol was always lower compared to poisoning with the single agent. A higher risk of hyperglycaemia was related to acute carbon monoxide poisoning whereas lower risk of hyperglycaemia was attributed to benzodiazepines and alcohol. A frequency of hypoglycaemia in the group of poisoned patients was much more lower compared to hyperglycaemia.

Metabolic enzymes: key modulators of functionality in cancer stem-like cells.

PubMed

Dong, Bo-Wen; Qin, Guang-Ming; Luo, Yan; Mao, Jian-Shan

2017-02-21

Cancer Stem-like Cells (CSCs) are a subpopulation of cancer cells with self-renewal capacity and are important for the initiation, progression and recurrence of cancer diseases. The metabolic profile of CSCs is consistent with their stem-like properties. Studies have indicated that enzymes, the main regulators of cellular metabolism, dictate functionalities of CSCs in both catalysis-dependent and catalysis-independent manners. This paper reviews diverse studies of metabolic enzymes, and describes the effects of these enzymes on metabolic adaptation, gene transcription and signal transduction, in CSCs.

Nuclear localization of metabolic enzymes in immunity and metastasis.

PubMed

He, Yuchen; Gao, Menghui; Cao, Yiqu; Tang, Haosheng; Liu, Shuang; Tao, Yongguang

2017-12-01

Metabolism is essential to all living organisms that provide cells with energy, regulators, building blocks, enzyme cofactors and signaling molecules, and is in tune with nutritional conditions and the function of cells to make the appropriate developmental decisions or maintain homeostasis. As a fundamental biological process, metabolism state affects the production of multiple metabolites and the activation of various enzymes that participate in regulating gene expression, cell apoptosis, cancer progression and immunoreactions. Previous studies generally focus on the function played by the metabolic enzymes in the cytoplasm and mitochondrion. In this review, we conclude the role of them in the nucleus and their implications for cancer progression, immunity and metastasis. Copyright © 2017 Elsevier B.V. All rights reserved.

Differential expression of glucose-metabolizing enzymes in multiple sclerosis lesions.

PubMed

Nijland, Philip G; Molenaar, Remco J; van der Pol, Susanne M A; van der Valk, Paul; van Noorden, Cornelis J F; de Vries, Helga E; van Horssen, Jack

2015-12-04

Demyelinated axons in multiple sclerosis (MS) lesions have an increased energy demand in order to maintain conduction. However, oxidative stress-induced mitochondrial dysfunction likely alters glucose metabolism and consequently impairs neuronal function in MS. Imaging and pathological studies indicate that glucose metabolism is altered in MS, although the underlying mechanisms and its role in neurodegeneration remain elusive. We investigated expression patterns of key enzymes involved in glycolysis, tricarboxylic acid (TCA) cycle and lactate metabolism in well-characterized MS tissue to establish which regulators of glucose metabolism are involved in MS and to identify underlying mechanisms. Expression levels of glycolytic enzymes were increased in active and inactive MS lesions, whereas expression levels of enzymes involved in the TCA cycle were upregulated in active MS lesions, but not in inactive MS lesions. We observed reduced expression and production capacity of mitochondrial α-ketoglutarate dehydrogenase (αKGDH) in demyelinated axons, which correlated with signs of axonal dysfunction. In inactive lesions, increased expression of lactate-producing enzymes was observed in astrocytes, whereas lactate-catabolising enzymes were mainly detected in axons. Our results demonstrate that the expression of various enzymes involved in glucose metabolism is increased in both astrocytes and axons in active MS lesions. In inactive MS lesions, we provide evidence that astrocytes undergo a glycolytic shift resulting in enhanced astrocyte-axon lactate shuttling, which may be pivotal for the survival of demyelinated axons. In conclusion, we show that key enzymes involved in energy metabolism are differentially expressed in active and inactive MS lesions. Our findings imply that, in addition to reduced oxidative phosphorylation activity, other bioenergetic pathways are affected as well, which may contribute to ongoing axonal degeneration in MS.

Tyrosine metabolic enzymes from insects and mammals: a comparative perspective.

PubMed

Vavricka, Christopher John; Han, Qian; Mehere, Prajwalini; Ding, Haizhen; Christensen, Bruce M; Li, Jianyong

2014-02-01

Differences in the metabolism of tyrosine between insects and mammals present an interesting example of molecular evolution. Both insects and mammals possess fine-tuned systems of enzymes to meet their specific demands for tyrosine metabolites; however, more homologous enzymes involved in tyrosine metabolism have emerged in many insect species. Without knowledge of modern genomics, one might suppose that mammals, which are generally more complex than insects and require tyrosine as a precursor for important catecholamine neurotransmitters and for melanin, should possess more enzymes to control tyrosine metabolism. Therefore, the question of why insects actually possess more tyrosine metabolic enzymes is quite interesting. It has long been known that insects rely heavily on tyrosine metabolism for cuticle hardening and for innate immune responses, and these evolutionary constraints are likely the key answers to this question. In terms of melanogenesis, mammals also possess a high level of regulation; yet mammalian systems possess more mechanisms for detoxification whereas insects accelerate pathways like melanogenesis and therefore must bear increased oxidative pressure. Our research group has had the opportunity to characterize the structure and function of many key proteins involved in tyrosine metabolism from both insects and mammals. In this mini review we will give a brief overview of our research on tyrosine metabolic enzymes in the scope of an evolutionary perspective of mammals in comparison to insects. © 2013 Institute of Zoology, Chinese Academy of Sciences.

Metabolic enzymes: key modulators of functionality in cancer stem-like cells

PubMed Central

Dong, Bo-Wen; Qin, Guang-Ming; Luo, Yan; Mao, Jian-Shan

2017-01-01

Cancer Stem-like Cells (CSCs) are a subpopulation of cancer cells with self-renewal capacity and are important for the initiation, progression and recurrence of cancer diseases. The metabolic profile of CSCs is consistent with their stem-like properties. Studies have indicated that enzymes, the main regulators of cellular metabolism, dictate functionalities of CSCs in both catalysis-dependent and catalysis-independent manners. This paper reviews diverse studies of metabolic enzymes, and describes the effects of these enzymes on metabolic adaptation, gene transcription and signal transduction, in CSCs. PMID:28009990

Dynamic Reorganization of Metabolic Enzymes into Intracellular Bodies

PubMed Central

O’Connell, Jeremy D.; Zhao, Alice; Ellington, Andrew D.; Marcotte, Edward M.

2013-01-01

Both focused and large-scale cell biological and biochemical studies have revealed that hundreds of metabolic enzymes across diverse organisms form large intracellular bodies. These proteinaceous bodies range in form from fibers and intracellular foci—such as those formed by enzymes of nitrogen and carbon utilization and of nucleotide biosynthesis—to high-density packings inside bacterial microcompartments and eukaryotic microbodies. Although many enzymes clearly form functional mega-assemblies, it is not yet clear for many recently discovered cases whether they represent functional entities, storage bodies, or aggregates. In this article, we survey intracellular protein bodies formed by metabolic enzymes, asking when and why such bodies form and what their formation implies for the functionality—and dysfunctionality—of the enzymes that comprise them. The panoply of intracellular protein bodies also raises interesting questions regarding their evolution and maintenance within cells. We speculate on models for how such structures form in the first place and why they may be inevitable. PMID:23057741

Genome-Wide Prediction of Metabolic Enzymes, Pathways, and Gene Clusters in Plants

DOE PAGES

Schläpfer, Pascal; Zhang, Peifen; Wang, Chuan; ...

2017-04-01

Plant metabolism underpins many traits of ecological and agronomic importance. Plants produce numerous compounds to cope with their environments but the biosynthetic pathways for most of these compounds have not yet been elucidated. To engineer and improve metabolic traits, we will need comprehensive and accurate knowledge of the organization and regulation of plant metabolism at the genome scale. Here, we present a computational pipeline to identify metabolic enzymes, pathways, and gene clusters from a sequenced genome. Using this pipeline, we generated metabolic pathway databases for 22 species and identified metabolic gene clusters from 18 species. This unified resource can bemore » used to conduct a wide array of comparative studies of plant metabolism. Using the resource, we discovered a widespread occurrence of metabolic gene clusters in plants: 11,969 clusters from 18 species. The prevalence of metabolic gene clusters offers an intriguing possibility of an untapped source for uncovering new metabolite biosynthesis pathways. For example, more than 1,700 clusters contain enzymes that could generate a specialized metabolite scaffold (signature enzymes) and enzymes that modify the scaffold (tailoring enzymes). In four species with sufficient gene expression data, we identified 43 highly coexpressed clusters that contain signature and tailoring enzymes, of which eight were characterized previously to be functional pathways. Finally, we identified patterns of genome organization that implicate local gene duplication and, to a lesser extent, single gene transposition as having played roles in the evolution of plant metabolic gene clusters.« less

Genome-Wide Prediction of Metabolic Enzymes, Pathways, and Gene Clusters in Plants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schläpfer, Pascal; Zhang, Peifen; Wang, Chuan

Plant metabolism underpins many traits of ecological and agronomic importance. Plants produce numerous compounds to cope with their environments but the biosynthetic pathways for most of these compounds have not yet been elucidated. To engineer and improve metabolic traits, we will need comprehensive and accurate knowledge of the organization and regulation of plant metabolism at the genome scale. Here, we present a computational pipeline to identify metabolic enzymes, pathways, and gene clusters from a sequenced genome. Using this pipeline, we generated metabolic pathway databases for 22 species and identified metabolic gene clusters from 18 species. This unified resource can bemore » used to conduct a wide array of comparative studies of plant metabolism. Using the resource, we discovered a widespread occurrence of metabolic gene clusters in plants: 11,969 clusters from 18 species. The prevalence of metabolic gene clusters offers an intriguing possibility of an untapped source for uncovering new metabolite biosynthesis pathways. For example, more than 1,700 clusters contain enzymes that could generate a specialized metabolite scaffold (signature enzymes) and enzymes that modify the scaffold (tailoring enzymes). In four species with sufficient gene expression data, we identified 43 highly coexpressed clusters that contain signature and tailoring enzymes, of which eight were characterized previously to be functional pathways. Finally, we identified patterns of genome organization that implicate local gene duplication and, to a lesser extent, single gene transposition as having played roles in the evolution of plant metabolic gene clusters.« less

Iron Sulfur and Molybdenum Cofactor Enzymes Regulate the Drosophila Life Cycle by Controlling Cell Metabolism.

PubMed

Marelja, Zvonimir; Leimkühler, Silke; Missirlis, Fanis

2018-01-01

Iron sulfur (Fe-S) clusters and the molybdenum cofactor (Moco) are present at enzyme sites, where the active metal facilitates electron transfer. Such enzyme systems are soluble in the mitochondrial matrix, cytosol and nucleus, or embedded in the inner mitochondrial membrane, but virtually absent from the cell secretory pathway. They are of ancient evolutionary origin supporting respiration, DNA replication, transcription, translation, the biosynthesis of steroids, heme, catabolism of purines, hydroxylation of xenobiotics, and cellular sulfur metabolism. Here, Fe-S cluster and Moco biosynthesis in Drosophila melanogaster is reviewed and the multiple biochemical and physiological functions of known Fe-S and Moco enzymes are described. We show that RNA interference of Mocs3 disrupts Moco biosynthesis and the circadian clock. Fe-S-dependent mitochondrial respiration is discussed in the context of germ line and somatic development, stem cell differentiation and aging. The subcellular compartmentalization of the Fe-S and Moco assembly machinery components and their connections to iron sensing mechanisms and intermediary metabolism are emphasized. A biochemically active Fe-S core complex of heterologously expressed fly Nfs1, Isd11, IscU, and human frataxin is presented. Based on the recent demonstration that copper displaces the Fe-S cluster of yeast and human ferredoxin, an explanation for why high dietary copper leads to cytoplasmic iron deficiency in flies is proposed. Another proposal that exosomes contribute to the transport of xanthine dehydrogenase from peripheral tissues to the eye pigment cells is put forward, where the Vps16a subunit of the HOPS complex may have a specialized role in concentrating this enzyme within pigment granules. Finally, we formulate a hypothesis that (i) mitochondrial superoxide mobilizes iron from the Fe-S clusters in aconitase and succinate dehydrogenase; (ii) increased iron transiently displaces manganese on superoxide dismutase, which

Iron Sulfur and Molybdenum Cofactor Enzymes Regulate the Drosophila Life Cycle by Controlling Cell Metabolism

PubMed Central

Marelja, Zvonimir; Leimkühler, Silke; Missirlis, Fanis

2018-01-01

Iron sulfur (Fe-S) clusters and the molybdenum cofactor (Moco) are present at enzyme sites, where the active metal facilitates electron transfer. Such enzyme systems are soluble in the mitochondrial matrix, cytosol and nucleus, or embedded in the inner mitochondrial membrane, but virtually absent from the cell secretory pathway. They are of ancient evolutionary origin supporting respiration, DNA replication, transcription, translation, the biosynthesis of steroids, heme, catabolism of purines, hydroxylation of xenobiotics, and cellular sulfur metabolism. Here, Fe-S cluster and Moco biosynthesis in Drosophila melanogaster is reviewed and the multiple biochemical and physiological functions of known Fe-S and Moco enzymes are described. We show that RNA interference of Mocs3 disrupts Moco biosynthesis and the circadian clock. Fe-S-dependent mitochondrial respiration is discussed in the context of germ line and somatic development, stem cell differentiation and aging. The subcellular compartmentalization of the Fe-S and Moco assembly machinery components and their connections to iron sensing mechanisms and intermediary metabolism are emphasized. A biochemically active Fe-S core complex of heterologously expressed fly Nfs1, Isd11, IscU, and human frataxin is presented. Based on the recent demonstration that copper displaces the Fe-S cluster of yeast and human ferredoxin, an explanation for why high dietary copper leads to cytoplasmic iron deficiency in flies is proposed. Another proposal that exosomes contribute to the transport of xanthine dehydrogenase from peripheral tissues to the eye pigment cells is put forward, where the Vps16a subunit of the HOPS complex may have a specialized role in concentrating this enzyme within pigment granules. Finally, we formulate a hypothesis that (i) mitochondrial superoxide mobilizes iron from the Fe-S clusters in aconitase and succinate dehydrogenase; (ii) increased iron transiently displaces manganese on superoxide dismutase, which

Molecular insights into the association of obesity with breast cancer risk: relevance to xenobiotic metabolism and CpG island methylation of tumor suppressor genes.

PubMed

Naushad, Shaik Mohammad; Hussain, Tajamul; Al-Attas, Omar S; Prayaga, Aruna; Digumarti, Raghunadha Rao; Gottumukkala, Suryanarayana Raju; Kutala, Vijay Kumar

2014-07-01

Obesity, genetic polymorphisms of xenobiotic metabolic pathway, hypermethylation of tumor suppressor genes, and hypomethylation of proapoptotic genes are known to be independent risk factors for breast cancer. The objective of this study is to evaluate the combined effect of these environmental, genetic, and epigenetic risk factors on the susceptibility to breast cancer. PCR-RFLP and multiplex PCR were used for the genetic analysis of six variants of xenobiotic metabolic pathway. Methylation-specific PCR was used for the epigenetic analysis of four genetic loci. Multifactor dimensionality reduction analysis revealed a significant interaction between the body mass index (BMI) and catechol-O-methyl transferase H108L variant alone or in combination with cytochrome P450 (CYP) 1A1m1 variant. Women with "Luminal A" breast cancer phenotype had higher BMI compared to other phenotypes and healthy controls. There was no association between the BMI and tumor grade. The post-menopausal obese women exhibited lower glutathione levels. BMI showed a positive association with the methylation of extracellular superoxide dismutase (r = 0.21, p < 0.05), Ras-association (RalGDS/AF-6) domain family member 1 (RASSF1A) (r = 0.31, p < 0.001), and breast cancer type 1 susceptibility protein (r = 0.19, p < 0.05); and inverse association with methylation of BNIP3 (r = -0.48, p < 0.0001). To conclude based on these results, obesity increases the breast cancer susceptibility by two possible mechanisms: (i) by interacting with xenobiotic genetic polymorphisms in inducing increased oxidative DNA damage and (ii) by altering the methylome of several tumor suppressor genes.

Establishing a herbicide-metabolizing enzyme library in Beckmannia syzigachne to identify genes associated with metabolic resistance.

PubMed

Pan, Lang; Gao, Haitao; Xia, Wenwen; Zhang, Teng; Dong, Liyao

2016-03-01

Non-target site resistance (NTSR) to herbicides is an increasing concern for weed control. Metabolic herbicide resistance is an important mechanism for NTSR. However, little is known about metabolic resistance at the genetic level. In this study, we have identified three fenoxaprop-P-ethyl-resistant American sloughgrass (Beckmannia syzigachne Steud.) populations, in which the molecular basis for NTSR remains unclear. To reveal the mechanisms of metabolic resistance, the genes likely to be involved in herbicide metabolism (e.g. for cytochrome P450s, esterases, hydrolases, oxidases, peroxidases, glutathione S-transferases, glycosyltransferases, and transporter proteins) were isolated using transcriptome sequencing, in combination with RT-PCR (reverse transcription-PCR) and RACE (rapid amplification of cDNA ends). Consequently, we established a herbicide-metabolizing enzyme library containing at least 332 genes, and each of these genes was cloned and the sequence and the expression level compared between the fenoxaprop-P-ethyl-resistant and susceptible populations. Fifteen metabolic enzyme genes were found to be possibly involved in fenoxaprop-P-ethyl resistance. In addition, we found five metabolizing enzyme genes that have a different gene sequence in plants of susceptible versus resistant B. syzigachne populations. These genes may be major candidates for herbicide metabolic resistance. This established metabolic enzyme library represents an important step forward towards a better understanding of herbicide metabolism and metabolic resistance in this and possibly other closely related weed species. This new information may help to understand weed metabolic resistance and to develop novel strategies of weed management. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Genome-Wide Prediction of Metabolic Enzymes, Pathways, and Gene Clusters in Plants.

PubMed

Schläpfer, Pascal; Zhang, Peifen; Wang, Chuan; Kim, Taehyong; Banf, Michael; Chae, Lee; Dreher, Kate; Chavali, Arvind K; Nilo-Poyanco, Ricardo; Bernard, Thomas; Kahn, Daniel; Rhee, Seung Y

2017-04-01

Plant metabolism underpins many traits of ecological and agronomic importance. Plants produce numerous compounds to cope with their environments but the biosynthetic pathways for most of these compounds have not yet been elucidated. To engineer and improve metabolic traits, we need comprehensive and accurate knowledge of the organization and regulation of plant metabolism at the genome scale. Here, we present a computational pipeline to identify metabolic enzymes, pathways, and gene clusters from a sequenced genome. Using this pipeline, we generated metabolic pathway databases for 22 species and identified metabolic gene clusters from 18 species. This unified resource can be used to conduct a wide array of comparative studies of plant metabolism. Using the resource, we discovered a widespread occurrence of metabolic gene clusters in plants: 11,969 clusters from 18 species. The prevalence of metabolic gene clusters offers an intriguing possibility of an untapped source for uncovering new metabolite biosynthesis pathways. For example, more than 1,700 clusters contain enzymes that could generate a specialized metabolite scaffold (signature enzymes) and enzymes that modify the scaffold (tailoring enzymes). In four species with sufficient gene expression data, we identified 43 highly coexpressed clusters that contain signature and tailoring enzymes, of which eight were characterized previously to be functional pathways. Finally, we identified patterns of genome organization that implicate local gene duplication and, to a lesser extent, single gene transposition as having played roles in the evolution of plant metabolic gene clusters. © 2017 American Society of Plant Biologists. All Rights Reserved.

Colonization-Induced Host-Gut Microbial Metabolic Interaction

PubMed Central

Claus, Sandrine P.; Ellero, Sandrine L.; Berger, Bernard; Krause, Lutz; Bruttin, Anne; Molina, Jérôme; Paris, Alain; Want, Elizabeth J.; de Waziers, Isabelle; Cloarec, Olivier; Richards, Selena E.; Wang, Yulan; Dumas, Marc-Emmanuel; Ross, Alastair; Rezzi, Serge; Kochhar, Sunil; Van Bladeren, Peter; Lindon, John C.; Holmes, Elaine; Nicholson, Jeremy K.

2011-01-01

The gut microbiota enhances the host’s metabolic capacity for processing nutrients and drugs and modulate the activities of multiple pathways in a variety of organ systems. We have probed the systemic metabolic adaptation to gut colonization for 20 days following exposure of axenic mice (n = 35) to a typical environmental microbial background using high-resolution 1H nuclear magnetic resonance (NMR) spectroscopy to analyze urine, plasma, liver, kidney, and colon (5 time points) metabolic profiles. Acquisition of the gut microbiota was associated with rapid increase in body weight (4%) over the first 5 days of colonization with parallel changes in multiple pathways in all compartments analyzed. The colonization process stimulated glycogenesis in the liver prior to triggering increases in hepatic triglyceride synthesis. These changes were associated with modifications of hepatic Cyp8b1 expression and the subsequent alteration of bile acid metabolites, including taurocholate and tauromuricholate, which are essential regulators of lipid absorption. Expression and activity of major drug-metabolizing enzymes (Cyp3a11 and Cyp2c29) were also significantly stimulated. Remarkably, statistical modeling of the interactions between hepatic metabolic profiles and microbial composition analyzed by 16S rRNA gene pyrosequencing revealed strong associations of the Coriobacteriaceae family with both the hepatic triglyceride, glucose, and glycogen levels and the metabolism of xenobiotics. These data demonstrate the importance of microbial activity in metabolic phenotype development, indicating that microbiota manipulation is a useful tool for beneficially modulating xenobiotic metabolism and pharmacokinetics in personalized health care. PMID:21363910

Enzyme Recruitment and Its Role in Metabolic Expansion

PubMed Central

2015-01-01

Although more than 109 years have passed since the existence of the last universal common ancestor, proteins have yet to reach the limits of divergence. As a result, metabolic complexity is ever expanding. Identifying and understanding the mechanisms that drive and limit the divergence of protein sequence space impact not only evolutionary biologists investigating molecular evolution but also synthetic biologists seeking to design useful catalysts and engineer novel metabolic pathways. Investigations over the past 50 years indicate that the recruitment of enzymes for new functions is a key event in the acquisition of new metabolic capacity. In this review, we outline the genetic mechanisms that enable recruitment and summarize the present state of knowledge regarding the functional characteristics of extant catalysts that facilitate recruitment. We also highlight recent examples of enzyme recruitment, both from the historical record provided by phylogenetics and from enzyme evolution experiments. We conclude with a look to the future, which promises fruitful consequences from the convergence of molecular evolutionary theory, laboratory-directed evolution, and synthetic biology. PMID:24483367

Differential Regulation of CYP3A4 and CYP3A5 and Its Implication in Drug Discovery

PubMed Central

Lolodi, Ogheneochukome; Wang, Yue-Ming; Wright, William C.; Chen, Taosheng

2017-01-01

Cancer cells use several mechanisms to resist the cytotoxic effects of drugs, resulting in tumor progression and invasion. One such mechanism capitalizes on the body’s natural defense against xenobiotics by increasing the rate of xenobiotic efflux and metabolic inactivation. Xenobiotic metabolism typically involves conversion of parent molecules to more soluble and easily excreted derivatives in reactions catalyzed by Phase I and Phase II drug metabolizing enzymes. Recent reports indicate that components of the xenobiotic response system are upregulated in some diseases, including many cancers. Such components include the pregnane X receptor (PXR) and the cytochrome P450 (CYP) 3A4 and 3A5 enzymes. The CYP3A enzymes are a subset of the numerous enzymes that are transcriptionally activated following the interaction of PXR and many ligands. Intense research is ongoing to understand the functional ramifications of aberrant expression of these components in diseased states with the goal of designing novel drugs that can selectively target them. PMID:28558634

Transcriptional profiling of mouse and human livers at different life stages

EPA Science Inventory

In the presence offoreign compounds,metabolichomeostasis oftheorganismismaintained by the liver's ability to detoxify and eliminate these xenobiotics. This is accomplished, in part, by the expression ofxenobiotic metabolizing enzymes (XMEs), which metabolize xenobiotics and det...

Tracing the Repertoire of Promiscuous Enzymes along the Metabolic Pathways in Archaeal Organisms.

PubMed

Martínez-Núñez, Mario Alberto; Rodríguez-Escamilla, Zuemy; Rodríguez-Vázquez, Katya; Pérez-Rueda, Ernesto

2017-07-13

The metabolic pathways that carry out the biochemical transformations sustaining life depend on the efficiency of their associated enzymes. In recent years, it has become clear that promiscuous enzymes have played an important role in the function and evolution of metabolism. In this work we analyze the repertoire of promiscuous enzymes in 89 non-redundant genomes of the Archaea cellular domain. Promiscuous enzymes are defined as those proteins with two or more different Enzyme Commission (E.C.) numbers, according the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. From this analysis, it was found that the fraction of promiscuous enzymes is lower in Archaea than in Bacteria. A greater diversity of superfamily domains is associated with promiscuous enzymes compared to specialized enzymes, both in Archaea and Bacteria, and there is an enrichment of substrate promiscuity rather than catalytic promiscuity in the archaeal enzymes. Finally, the presence of promiscuous enzymes in the metabolic pathways was found to be heterogeneously distributed at the domain level and in the phyla that make up the Archaea. These analyses increase our understanding of promiscuous enzymes and provide additional clues to the evolution of metabolism in Archaea.

High-Fat Diets Alter the Modulatory Effects of Xenobiotics on Cytochrome P450 Activities

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sadler, Natalie C.; Webb-Robertson, Bobbie-Jo M.; Clauss, Therese R.

Cytochrome P450 monooxygenases (P450) are key to the metabolism of myriad endogenous chemicals and xenobiotics, including the majority of therapeutic drugs. Dysregulated P450 activities can lead to altered drug metabolism and toxicity, oxidative stress, and inflammation; all physiological states frequently charged as the impetus for various chronic pathologies. We characterized the impact of common xenobiotic exposures, specifically high-fat diet and active or passive cigarette smoke, on the functional capacity of hepatic and pulmonary P450s. We employed an activity-based protein profiling approach to characterize the identity and activity level of measured individual P450 isoforms. Our results confirm expectations of significant alterationsmore » in pulmonary P450s due to cigarette smoke, but now reveal the repressive impact of high-fat diet-induced obesity on many hepatic P450s activities, and the dynamic alterations due to concomitant diet and smoke exposures on liver and lung P450 activities impacting drug metabolism and pathways of inflammation.« less

Decreased detoxification genes and genome size make the human body louse an efficient model to study xenobiotic metabolism

PubMed Central

Lee, Si Hyeock; Kang, Jae Soon; Min, Jee Sun; Yoon, Kyong Sup; Strycharz, Joseph P.; Johnson, Reed; Mittapalli, Omprakash; Margam, Venu M.; Sun, Weilin; Li, Hong-Mei; Xie, Jun; Wu, Jing; Kirkness, Ewen F.; Berenbaum, May R.; Pittendrigh, Barry R.; Clark, J. Marshall

2010-01-01

The human body louse, Pediculus humanus humanus, has one of the smallest insect genomes, containing ~10,775 annotated genes (Kirkness et al. 2010). Annotation of detoxification [cytochrome P450 monooxygenase (P450), glutathione-S-transferase (GST), esterase (Est), and ATP-binding cassette transporter (ABC transporter)] genes revealed that they are dramatically reduced in P. h. humanus compared to other insects except for Apis mellifera. There are 37 P450, 13 GST and 17 Est genes present in P. h. humanus, approximately half of that found in Drosophila melanogaster and Anopheles gambiae. The number of putatively functional ABC transporter genes in P. h. humanus and A. mellifera are the same (36) but both have fewer than An. gambiae (44) or D. melanogaster (65). The reduction of detoxification genes in P. h. humanus may be due to their simple life history, where they do not encounter a wide variety of xenobiotics. Neuronal component genes are highly conserved across different insect species as expected due to their critical function. Although reduced in number, P. h. humanus still retains at least a minimum repertoire of genes known to confer metabolic or toxicokinetic resistance to xenobiotics (e.g., Cyp3 clade P450s, Delta GSTs, B clade Ests and B/C subfamily ABC transporters), suggestive of its high potential for resistance development. PMID:20561088

Decreased detoxification genes and genome size make the human body louse an efficient model to study xenobiotic metabolism.

PubMed

Lee, S H; Kang, J S; Min, J S; Yoon, K S; Strycharz, J P; Johnson, R; Mittapalli, O; Margam, V M; Sun, W; Li, H-M; Xie, J; Wu, J; Kirkness, E F; Berenbaum, M R; Pittendrigh, B R; Clark, J M

2010-10-01

The human body louse, Pediculus humanus humanus, has one of the smallest insect genomes, containing ∼10 775 annotated genes. Annotation of detoxification [cytochrome P450 monooxygenase (P450), glutathione-S-transferase (GST), esterase (Est) and ATP-binding cassette transporter (ABC transporter)] genes revealed that they are dramatically reduced in P. h. humanus compared to other insects except for Apis mellifera. There are 37 P450, 13 GST and 17 Est genes present in P. h. humanus, approximately half the number found in Drosophila melanogaster and Anopheles gambiae. The number of putatively functional ABC transporter genes in P. h. humanus and Ap. mellifera are the same (36) but both have fewer than An. gambiae (44) or Dr. melanogaster (65). The reduction of detoxification genes in P. h. humanus may be a result of this louse's simple life history, in which it does not encounter a wide variety of xenobiotics. Neuronal component genes are highly conserved across different insect species as expected because of their critical function. Although reduced in number, P. h. humanus still retains at least a minimum repertoire of genes known to confer metabolic or toxicokinetic resistance to xenobiotics (eg Cyp3 clade P450s, Delta GSTs, B clade Ests and B/C subfamily ABC transporters), suggestive of its high potential for resistance development. © 2010 The Authors. Insect Molecular Biology © 2010 The Royal Entomological Society.

Phase 0 and phase III transport in various organs: combined concept of phases in xenobiotic transport and metabolism.

PubMed

Döring, Barbara; Petzinger, Ernst

2014-08-01

The historical phasing concept of drug metabolism and elimination was introduced to comprise the two phases of metabolism: phase I metabolism for oxidations, reductions and hydrolyses, and phase II metabolism for synthesis. With this concept, biological membrane barriers obstructing the accessibility of metabolism sites in the cells for drugs were not considered. The concept of two phases was extended to a concept of four phases when drug transporters were detected that guided drugs and drug metabolites in and out of the cells. In particular, water soluble or charged drugs are virtually not able to overcome the phospholipid membrane barrier. Drug transporters belong to two main clusters of transporter families: the solute carrier (SLC) families and the ATP binding cassette (ABC) carriers. The ABC transporters comprise seven families with about 20 carriers involved in drug transport. All of them operate as pumps at the expense of ATP splitting. Embedded in the former phase concept, the term "phase III" was introduced by Ishikawa in 1992 for drug export by ABC efflux pumps. SLC comprise 52 families, from which many carriers are drug uptake transporters. Later on, this uptake process was referred to as the "phase 0 transport" of drugs. Transporters for xenobiotics in man and animal are most expressed in liver, but they are also present in extra-hepatic tissues such as in the kidney, the adrenal gland and lung. This review deals with the function of drug carriers in various organs and their impact on drug metabolism and elimination.

Cryptic indole hydroxylation by a non-canonical terpenoid cyclase parallels bacterial xenobiotic detoxification

NASA Astrophysics Data System (ADS)

Kugel, Susann; Baunach, Martin; Baer, Philipp; Ishida-Ito, Mie; Sundaram, Srividhya; Xu, Zhongli; Groll, Michael; Hertweck, Christian

2017-06-01

Terpenoid natural products comprise a wide range of molecular architectures that typically result from C-C bond formations catalysed by classical type I/II terpene cyclases. However, the molecular diversity of biologically active terpenoids is substantially increased by fully unrelated, non-canonical terpenoid cyclases. Their evolutionary origin has remained enigmatic. Here we report the in vitro reconstitution of an unusual flavin-dependent bacterial indoloterpenoid cyclase, XiaF, together with a designated flavoenzyme-reductase (XiaP) that mediates a key step in xiamycin biosynthesis. The crystal structure of XiaF with bound FADH2 (at 2.4 Å resolution) and phylogenetic analyses reveal that XiaF is, surprisingly, most closely related to xenobiotic-degrading enzymes. Biotransformation assays show that XiaF is a designated indole hydroxylase that can be used for the production of indigo and indirubin. We unveil a cryptic hydroxylation step that sets the basis for terpenoid cyclization and suggest that the cyclase has evolved from xenobiotics detoxification enzymes.

Interactions between Urinary 4-tert-Octylphenol Levels and Metabolism Enzyme Gene Variants on Idiopathic Male Infertility

PubMed Central

Xu, Bin; Tang, Rong; Chen, Xiaojiao; Du, Guizhen; Lu, Chuncheng; Meeker, John D.; Zhou, Zuomin; Xia, Yankai; Wang, Xinru

2013-01-01

Octylphenol (OP) and Trichlorophenol (TCP) act as endocrine disruptors and have effects on male reproductive function. We studied the interactions between 4-tert-Octylphenol (4-t-OP), 4-n- Octylphenol (4-n-OP), 2,3,4-Trichlorophenol (2,3,4-TCP), 2,4,5-Trichlorophenol (2,4,5-TCP) urinary exposure levels and polymorphisms in selected xenobiotic metabolism enzyme genes among 589 idiopathic male infertile patients and 396 controls in a Han-Chinese population. Ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to measure alkylphenols and chlorophenols in urine. Polymorphisms were genotyped using the SNPstream platform and the Taqman method. Among four phenols that were detected, we found that only exposure to 4-t-OP increased the risk of male infertility (P trend = 1.70×10−7). The strongest interaction was between 4-t-OP and rs4918758 in CYP2C9 (P inter = 6.05×10−7). It presented a significant monotonic increase in risk estimates for male infertility with increasing 4-t-OP exposure levels among men with TC/CC genotype (low level compared with non-exposed, odds ratio (OR) = 2.26, 95% confidence intervals (CI) = 1.06, 4.83; high level compared with non-exposed, OR = 9.22, 95% CI = 2.78, 30.59), but no associations observed among men with TT genotype. We also found interactions between 4-t-OP and rs4986894 in CYP2C19, and between rs1048943 in CYP1A1, on male infertile risk (P inter = 8.09×10−7, P inter = 3.73×10−4, respectively).We observed notable interactions between 4-t-OP exposure and metabolism enzyme gene polymorphisms on idiopathic infertility in Han-Chinese men. PMID:23555028

Detoxification function of the Arabidopsis sulphotransferase AtSOT12 by sulphonation of xenobiotics.

PubMed

Chen, Jinhua; Gao, Liqiong; Baek, Dongwon; Liu, Chunlin; Ruan, Ying; Shi, Huazhong

2015-08-01

Cytosolic sulphotransferases have been implicated in inactivation of endogenous steroid hormones and detoxification of xenobiotics in human and animals. Yet, the function of plant sulphotransferases in xenobiotic sulphonation and detoxification has not been reported. In this study, we show that the Arabidopsis sulphotransferase AtSOT12 could sulphonate the bacterial-produced toxin cycloheximide. Loss-of-function mutant sot12 exhibited hypersensitive phenotype to cycloheximide, and expression of AtSOT12 protein in yeast cells conferred resistance to this toxic compound. AtSOT12 exhibited broad specificity and could sulphonate a variety of xenobiotics including phenolic and polycyclic compounds. Enzyme kinetics analysis indicated that AtSOT12 has different selectivity for simple phenolics with different side chains, and the position of the side chain in the simple phenolic compounds affects substrate binding affinity and catalytic efficiency. We proposed that the broad specificity and induced production of AtSOT12 may have rendered this enzyme to not only modify endogenous molecules such as salicylic acid as we previously reported, but also sulphonate pathogen-produced toxic small molecules to protect them from infection. Sulphonation of small molecules in plants may constitute a rapid way to inactivate or change the physiochemical properties of biologically active molecules that could have profound effects on plant growth, development and defence. © 2015 John Wiley & Sons Ltd.

Expression of Enzymes that Metabolize Medications

NASA Technical Reports Server (NTRS)

Wotring, Virginia E.; Peters, C. P.

2012-01-01

Most pharmaceuticals are metabolized by the liver. Clinically-used medication doses are given with normal liver function in mind. A drug overdose can result if the liver is damaged and removing pharmaceuticals from the circulation at a rate slower than normal. Alternatively, if liver function is elevated and removing drugs from the system more quickly than usual, it would be as if too little drug had been given for effective treatment. Because of the importance of the liver in drug metabolism we want to understand the effects of spaceflight on the enzymes of the liver.

A single amino acid controls the functional switch of human constitutive androstane receptor (CAR) 1 to the xenobiotic-sensitive splicing variant CAR3.

PubMed

Chen, Tao; Tompkins, Leslie M; Li, Linhao; Li, Haishan; Kim, Gregory; Zheng, Yuxin; Wang, Hongbing

2010-01-01

The constitutive androstane receptor (CAR) is constitutively activated in immortalized cell lines independent of xenobiotic stimuli. This feature of CAR has limited its use as a sensor for xenobiotic-induced expression of drug-metabolizing enzymes. Recent reports, however, reveal that a splicing variant of human CAR (hCAR3), which contains an insertion of five amino acids (APYLT), exhibits low basal but xenobiotic-inducible activities in cell-based reporter assays. Nonetheless, the underlying mechanisms of this functional shift are not well understood. We have now generated chimeric constructs containing various residues of the five amino acids of hCAR3 and examined their response to typical hCAR activators. Our results showed that the retention of alanine (hCAR1+A) alone is sufficient to confer the constitutively activated hCAR1 to the xenobiotic-sensitive hCAR3. It is noteworthy that hCAR1+A was significantly activated by a series of known hCAR activators, and displayed activation superior to that of hCAR3. Moreover, intracellular localization assays revealed that hCAR1+A exhibits nuclear accumulation upon 6-(4-chlorophenyl) imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl) oxime (CITCO) treatment in COS1 cells, which differs from the spontaneous nuclear distribution of hCAR1 and the nontranslocatable hCAR3. Mammalian two-hybrid and glutathione S-transferase pull-down assays further demonstrated that hCAR1+A interacts with the coactivator SRC-1 and GRIP-1 at low level before activation, while at significantly enhanced level in the presence of CITCO. Thus, the alanine residue in the insertion of hCAR3 seems in charge of the xenobiotic response of hCAR3 through direct and indirect mechanisms. Activation of hCAR1+A may represent a sensitive avenue for the identification of hCAR activators.

A Single Amino Acid Controls the Functional Switch of Human Constitutive Androstane Receptor (CAR) 1 to the Xenobiotic-Sensitive Splicing Variant CAR3

PubMed Central

Chen, Tao; Tompkins, Leslie M.; Li, Linhao; Li, Haishan; Kim, Gregory; Zheng, Yuxin

2010-01-01

The constitutive androstane receptor (CAR) is constitutively activated in immortalized cell lines independent of xenobiotic stimuli. This feature of CAR has limited its use as a sensor for xenobiotic-induced expression of drug-metabolizing enzymes. Recent reports, however, reveal that a splicing variant of human CAR (hCAR3), which contains an insertion of five amino acids (APYLT), exhibits low basal but xenobiotic-inducible activities in cell-based reporter assays. Nonetheless, the underlying mechanisms of this functional shift are not well understood. We have now generated chimeric constructs containing various residues of the five amino acids of hCAR3 and examined their response to typical hCAR activators. Our results showed that the retention of alanine (hCAR1+A) alone is sufficient to confer the constitutively activated hCAR1 to the xenobiotic-sensitive hCAR3. It is noteworthy that hCAR1+A was significantly activated by a series of known hCAR activators, and displayed activation superior to that of hCAR3. Moreover, intracellular localization assays revealed that hCAR1+A exhibits nuclear accumulation upon 6-(4-chlorophenyl) imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl) oxime (CITCO) treatment in COS1 cells, which differs from the spontaneous nuclear distribution of hCAR1 and the nontranslocatable hCAR3. Mammalian two-hybrid and glutathione S-transferase pull-down assays further demonstrated that hCAR1+A interacts with the coactivator SRC-1 and GRIP-1 at low level before activation, while at significantly enhanced level in the presence of CITCO. Thus, the alanine residue in the insertion of hCAR3 seems in charge of the xenobiotic response of hCAR3 through direct and indirect mechanisms. Activation of hCAR1+A may represent a sensitive avenue for the identification of hCAR activators. PMID:19820207

Preliminary Evaluation of Three-Dimensional Primary Human Hepatocyte Culture System for Assay of Drug-Metabolizing Enzyme-Inducing Potential.

PubMed

Arakawa, Hiroshi; Kamioka, Hiroki; Jomura, Tomoko; Koyama, Satoshi; Idota, Yoko; Yano, Kentaro; Kojima, Hajime; Ogihara, Takuo

2017-01-01

Drug-induced liver injury (DILI) is a common reason for withdrawal of candidate drugs from clinical trials, or of approved drugs from the market. DILI may be induced not only by intact parental drugs, but also by metabolites or intermediates, and therefore should be evaluated in the enzyme-induced state. Here, we present a protocol for assay of drug-metabolizing enzyme-inducing potential using three-dimensional (3D) primary cultures of human hepatocytes (hepatocyte spheroids). Hepatocyte spheroids could be used up to 21 d after seeding (pre-culture for 7 d and exposure to inducer for up to 14 d), based on preliminary evaluation of basal activities of CYP subtypes and mRNA expression of the corresponding transcription factor and xenobiotic receptors (aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR) and pregnane X receptor (PXR)). After 2 d exposure of hepatocyte spheroids to omeprazole, phenobarbital and rifampicin (typical inducers of CYP1A2, 2B6 and 3A4, respectively), CYP1A2, 2B6 and 3A4 mRNA expression levels were significantly increased. The mRNA induction of CYP2B6 remained reasonably stable between days 2 and 14 of exposure to inducers, while induction of both CYP1A2 and 3A4 continued to increase up to day 14. These enzyme activities were all significantly increased compared with the control until day 14. Our findings indicate that our 3D hepatocyte spheroids system would be especially suitable for long-term testing of enzyme activity induction by drugs, either to predict or to verify clinical events.

Genome-Wide Prediction of Metabolic Enzymes, Pathways, and Gene Clusters in Plants1[OPEN

PubMed Central

Zhang, Peifen; Kim, Taehyong; Banf, Michael; Chavali, Arvind K.; Nilo-Poyanco, Ricardo; Bernard, Thomas

2017-01-01

Plant metabolism underpins many traits of ecological and agronomic importance. Plants produce numerous compounds to cope with their environments but the biosynthetic pathways for most of these compounds have not yet been elucidated. To engineer and improve metabolic traits, we need comprehensive and accurate knowledge of the organization and regulation of plant metabolism at the genome scale. Here, we present a computational pipeline to identify metabolic enzymes, pathways, and gene clusters from a sequenced genome. Using this pipeline, we generated metabolic pathway databases for 22 species and identified metabolic gene clusters from 18 species. This unified resource can be used to conduct a wide array of comparative studies of plant metabolism. Using the resource, we discovered a widespread occurrence of metabolic gene clusters in plants: 11,969 clusters from 18 species. The prevalence of metabolic gene clusters offers an intriguing possibility of an untapped source for uncovering new metabolite biosynthesis pathways. For example, more than 1,700 clusters contain enzymes that could generate a specialized metabolite scaffold (signature enzymes) and enzymes that modify the scaffold (tailoring enzymes). In four species with sufficient gene expression data, we identified 43 highly coexpressed clusters that contain signature and tailoring enzymes, of which eight were characterized previously to be functional pathways. Finally, we identified patterns of genome organization that implicate local gene duplication and, to a lesser extent, single gene transposition as having played roles in the evolution of plant metabolic gene clusters. PMID:28228535

Identification of parallel and divergent optimization solutions for homologous metabolic enzymes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Standaert, Robert F.; Giannone, Richard J.; Michener, Joshua K.

Here, metabolic pathway assembly typically involves the expression of enzymes from multiple organisms in a single heterologous host. Ensuring that each enzyme functions effectively can be challenging, since many potential factors can disrupt proper pathway flux. Here, we compared the performance of two enzyme homologs in a pathway engineered to allow Escherichia coli to grow on 4-hydroxybenzoate (4-HB), a byproduct of lignocellulosic biomass deconstruction. Single chromosomal copies of the 4-HB 3-monooxygenase genes pobA and praI, from Pseudomonas putida KT2440 and Paenibacillus sp. JJ-1B, respectively, were introduced into a strain able to metabolize protocatechuate (PCA), the oxidation product of 4-HB. Neithermore » enzyme initially supported consistent growth on 4-HB. Experimental evolution was used to identify mutations that improved pathway activity. For both enzymes, silent mRNA mutations were identified that increased enzyme expression. With pobA, duplication of the genes for PCA metabolism allowed growth on 4-HB. However, with praI, growth required a mutation in the 4-HB/PCA transporter pcaK that increased intracellular concentrations of 4-HB, suggesting that flux through PraI was limiting. These findings demonstrate the value of directed evolution strategies to rapidly identify and overcome diverse factors limiting enzyme activity.« less

Identification of parallel and divergent optimization solutions for homologous metabolic enzymes

DOE PAGES

Standaert, Robert F.; Giannone, Richard J.; Michener, Joshua K.

2018-04-18

Here, metabolic pathway assembly typically involves the expression of enzymes from multiple organisms in a single heterologous host. Ensuring that each enzyme functions effectively can be challenging, since many potential factors can disrupt proper pathway flux. Here, we compared the performance of two enzyme homologs in a pathway engineered to allow Escherichia coli to grow on 4-hydroxybenzoate (4-HB), a byproduct of lignocellulosic biomass deconstruction. Single chromosomal copies of the 4-HB 3-monooxygenase genes pobA and praI, from Pseudomonas putida KT2440 and Paenibacillus sp. JJ-1B, respectively, were introduced into a strain able to metabolize protocatechuate (PCA), the oxidation product of 4-HB. Neithermore » enzyme initially supported consistent growth on 4-HB. Experimental evolution was used to identify mutations that improved pathway activity. For both enzymes, silent mRNA mutations were identified that increased enzyme expression. With pobA, duplication of the genes for PCA metabolism allowed growth on 4-HB. However, with praI, growth required a mutation in the 4-HB/PCA transporter pcaK that increased intracellular concentrations of 4-HB, suggesting that flux through PraI was limiting. These findings demonstrate the value of directed evolution strategies to rapidly identify and overcome diverse factors limiting enzyme activity.« less

Identification of parallel and divergent optimization solutions for homologous metabolic enzymes.

PubMed

Standaert, Robert F; Giannone, Richard J; Michener, Joshua K

2018-06-01

Metabolic pathway assembly typically involves the expression of enzymes from multiple organisms in a single heterologous host. Ensuring that each enzyme functions effectively can be challenging, since many potential factors can disrupt proper pathway flux. Here, we compared the performance of two enzyme homologs in a pathway engineered to allow Escherichia coli to grow on 4-hydroxybenzoate (4-HB), a byproduct of lignocellulosic biomass deconstruction. Single chromosomal copies of the 4-HB 3-monooxygenase genes pobA and praI , from Pseudomonas putida KT2440 and Paenibacillus sp. JJ-1B, respectively, were introduced into a strain able to metabolize protocatechuate (PCA), the oxidation product of 4-HB. Neither enzyme initially supported consistent growth on 4-HB. Experimental evolution was used to identify mutations that improved pathway activity. For both enzymes, silent mRNA mutations were identified that increased enzyme expression. With pobA , duplication of the genes for PCA metabolism allowed growth on 4-HB. However, with praI , growth required a mutation in the 4-HB/PCA transporter pcaK that increased intracellular concentrations of 4-HB, suggesting that flux through PraI was limiting. These findings demonstrate the value of directed evolution strategies to rapidly identify and overcome diverse factors limiting enzyme activity.

Development of gold-immobilized P450 platform for exploring the effect of oligomer formation on P450-mediated metabolism for in vitro to in vivo drug metabolism predictions

NASA Astrophysics Data System (ADS)

Kabulski, Jarod L.

The cytochrome P450 (P450) enzyme family is responsible for the biotransformation of a wide range of endogenous and xenobiotic compounds, as well as being the major metabolic enzyme in first pass drug metabolism. In vivo drug metabolism for P450 enzymes is predicted using in vitro data obtained from a reconstituted expressed P450 system, but these systems have not always been proven to accurately represent in vivo enzyme kinetics, due to interactions caused by oligomer formation. These in vitro systems use soluble P450 enzymes prone to oligomer formation and studies have shown that increased states of protein aggregation directly affect the P450 enzyme kinetics. We have developed an immobilized enzyme system that isolates the enzyme and can be used to elucidate the effect of P450 aggregation on metabolism kinetics. The long term goal of my research is to develop a tool that will help improve the assessment of pharmaceuticals by better predicting in vivo kinetics in an in vitro system. The central hypothesis of this research is that P450-mediated kinetics measured in vitro is dependent on oligomer formation and that the accurate prediction of in vivo P450-mediated kinetics requires elucidation of the effect of oligomer formation. The rationale is that the development of a P450 bound to a Au platform can be used to control the aggregation of enzymes and bonding to Au may also permit replacement of the natural redox partners with an electrode capable of supplying a constant flow of electrons. This dissertation explains the details of the enzyme attachment, monitoring substrate binding, and metabolism using physiological and electrochemical methods, determination of enzyme kinetics, and the development of an immobilized-P450 enzyme bioreactor. This work provides alternative approaches to studying P450-mediated kinetics, a platform for controlling enzyme aggregation, electrochemically-driven P450 metabolism, and for investigating the effect of protein

Targeting xenobiotic receptors PXR and CAR in human diseases

PubMed Central

Banerjee, Monimoy; Robbins, Delira; Chen, Taosheng

2014-01-01

Nuclear receptors such as the pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are xenobiotic receptors regulating not only drug metabolism and disposition but also various human diseases such as cancer, diabetes, inflammatory disease, metabolic disease and liver diseases, suggesting that PXR and CAR are promising targets for drug discovery. Consequently, there is an urgent need to discover and develop small molecules that target these PXR- and/or CAR-mediated human-disease-related pathways for relevant therapeutic applications. This review proposes approaches to target PXR and CAR, either individually or simultaneously, in the context of various human diseases, taking into consideration the structural differences between PXR and CAR. PMID:25463033

The expression of xenobiotic-metabolizing enzymes in human prostate and in prostate epithelial cells (PECs) derived from primary cultures.

PubMed

Al-Buheissi, S Z; Cole, K J; Hewer, A; Kumar, V; Bryan, R L; Hudson, D L; Patel, H R; Nathan, S; Miller, R A; Phillips, D H

2006-06-01

Dietary heterocyclic amines (HCAs) are carcinogenic in rodent prostate requiring activation by enzymes such as cytochrome P450 (CYP) and N-acetyltransferase (NAT). We investigated by Western blotting and immunohistochemistry the expression of CYP1A1, CYP1A2, and NAT1 in human prostate and in prostate epithelial cells (PECs) derived from primary cultures and tested their ability to activate the dietary carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and its N-hydroxy metabolite (N-OH-IQ) to DNA-damaging moieties. Western blotting identified CYP1A1, CYP1A2, and NAT1. Immunohistochemistry localized NAT1 to the cytoplasm of PECs. Inter-individual variation was observed in the expression levels of CYP1A1, 1A2, and NAT1 (11, 75, and 35-fold, respectively). PECs expressed CYP1A1 and NAT1 but not CYP1A2. When incubated with IQ or N-OH-IQ, PECs formed DNA adducts indicating their ability to metabolically activate these compounds. Prostate cells possess the capacity to activate dietary carcinogens. PECs may provide a useful model system to study their role in prostate carcinogenesis.

Molecular Identification, Enzyme Assay, and Metabolic Profiling of Trichoderma spp.

PubMed

Bae, Soo-Jung; Park, Young-Hwan; Bae, Hyeun-Jong; Jeon, Junhyun; Bae, Hanhong

2017-06-28

The goal of this study was to identify and characterize selected Trichoderma isolates by metabolic profiling and enzyme assay for evaluation of their potential as biocontrol agents against plant pathogens. Trichoderma isolates were obtained from the Rural Development Administration Genebank Information Center (Wanju, Republic of Korea). Eleven Trichoderma isolates were re-identified using ribosomal DNA internal transcribed spacer (ITS) regions. ITS sequence results showed new identification of Trichoderma isolates. In addition, metabolic profiling of the ethyl acetate extracts of the liquid cultures of five Trichoderma isolates that showed the best anti- Phytophthora activities was conducted using gas chromatography-mass spectrometry. Metabolic profiling revealed that Trichoderma isolates shared common metabolites with well-known antifungal activities. Enzyme assays indicated strong cell walldegrading enzyme activities of Trichoderma isolates. Overall, our results indicated that the selected Trichoderma isolates have great potential for use as biocontrol agents against plant pathogens.

Reciprocal regulation of p53 and malic enzymes modulates metabolism and senescence.

PubMed

Jiang, Peng; Du, Wenjing; Mancuso, Anthony; Wellen, Kathryn E; Yang, Xiaolu

2013-01-31

Cellular senescence both protects multicellular organisms from cancer and contributes to their ageing. The pre-eminent tumour suppressor p53 has an important role in the induction and maintenance of senescence, but how it carries out this function remains poorly understood. In addition, although increasing evidence supports the idea that metabolic changes underlie many cell-fate decisions and p53-mediated tumour suppression, few connections between metabolic enzymes and senescence have been established. Here we describe a new mechanism by which p53 links these functions. We show that p53 represses the expression of the tricarboxylic-acid-cycle-associated malic enzymes ME1 and ME2 in human and mouse cells. Both malic enzymes are important for NADPH production, lipogenesis and glutamine metabolism, but ME2 has a more profound effect. Through the inhibition of malic enzymes, p53 regulates cell metabolism and proliferation. Downregulation of ME1 and ME2 reciprocally activates p53 through distinct MDM2- and AMP-activated protein kinase-mediated mechanisms in a feed-forward manner, bolstering this pathway and enhancing p53 activation. Downregulation of ME1 and ME2 also modulates the outcome of p53 activation, leading to strong induction of senescence, but not apoptosis, whereas enforced expression of either malic enzyme suppresses senescence. Our findings define physiological functions of malic enzymes, demonstrate a positive-feedback mechanism that sustains p53 activation, and reveal a connection between metabolism and senescence mediated by p53.

Development of radiometric assays for quantification of enzyme activities of the key enzymes of thyroid hormones metabolism.

PubMed

Pavelka, S

2014-01-01

We newly elaborated and adapted several radiometric enzyme assays for the determination of activities of the key enzymes engaged in the biosynthesis (thyroid peroxidase, TPO) and metabolic transformations (conjugating enzymes and iodothyronine deiodinases, IDs) of thyroid hormones (THs) in the thyroid gland and in peripheral tissues, especially in white adipose tissue (WAT). We also elaborated novel, reliable radiometric methods for extremely sensitive determination of enzyme activities of IDs of types 1, 2 and 3 in microsomal fractions of different rat and human tissues, as well as in homogenates of cultured mammalian cells. The use of optimized TLC separation of radioactive products from the unconsumed substrates and film-less autoradiography of radiochromatograms, taking advantage of storage phosphor screens, enabled us to determine IDs enzyme activities as low as 10(-18) katals. In studies of the interaction of fluoxetine (Fluox) with the metabolism of THs, we applied adapted radiometric enzyme assays for iodothyronine sulfotransferases (ST) and uridine 5'-diphospho-glucuronyltransferase (UDP-GT). Fluox is the most frequently used representative of a new group of non-tricyclic antidepressant drugs--selective serotonin re-uptake inhibitors. We used the elaborated assays for quantification the effects of Fluox and for the assessment of the degree of potential induction of rat liver ST and/or UDP-GT enzyme activities by Fluox alone or in combination with T(3). Furthermore, we studied possible changes in IDs activities in murine adipose tissue under the conditions that promoted either tissue hypertrophy (obesogenic treatment) or involution (caloric restriction), and in response to leptin, using our newly developed radiometric enzyme assays for IDs. Our results suggest that deiodinase D1 has a functional role in WAT, with D1 possibly being involved in the control of adipose tissue metabolism and/or accumulation of the tissue. Significant positive correlation between

A Kinetic Modelling of Enzyme Inhibitions in the Central Metabolism of Yeast Cells

NASA Astrophysics Data System (ADS)

Kasbawati; Kalondeng, A.; Aris, N.; Erawaty, N.; Azis, M. I.

2018-03-01

Metabolic regulation plays an important role in the metabolic engineering of a cellular process. It is conducted to improve the productivity of a microbial process by identifying the important regulatory nodes of a metabolic pathway such as fermentation pathway. Regulation of enzymes involved in a particular pathway can be held to improve the productivity of the system. In the central metabolism of yeast cell, some enzymes are known as regulating enzymes that can be inhibited to increase the production of ethanol. In this research we study the kinetic modelling of the enzymes in the central pathway of yeast metabolism by taking into consideration the enzyme inhibition effects to the ethanol production. The existence of positive steady state solution and the stability of the system are also analysed to study the property and dynamical behaviour of the system. One stable steady state of the system is produced if some conditions are fulfilled. The conditions concern to the restriction of the maximum reactions of the enzymes in the pyruvate and acetaldehyde branch points. There exists a certain time of fermentation reaction at which a maximum and a minimum ethanol productions are attained after regulating the system. Optimal ethanol concentration is also produced for a certain initial concentration of inhibitor.

Xenobiotics: Chapter 15

USGS Publications Warehouse

Bridges, Christine M.; Semlitsch, Raymond D.; Lannoo, Michael

2005-01-01

While a number of compounds have been reported as toxic to amphibians, until recently, there have been conspicuously few ecotoxicological studies concerning amphibians. Studies are now focusing on the effects of xenobiotics on amphibians, an interest likely stimulated by widespread reports of amphibian declines. It has been speculated that chemical contamination may be partially to blame for some documented amphibian declines, by disrupting growth, reproduction, and behavior. However, evidence that xenobiotics are directly to blame for population declines is sparse because environmental concentrations are typically not great enough to generate direct mortality. Although the effects of environmental contaminants on the amphibian immune system are currently unknown, it is possible that exposure to stressors such as organic pollutants (which enter ecosystems in the form of pesticides) may depress immune system function, thus allowing greater susceptibility to fungal infections. This chapter discusses toxicity testing for xenobiotics and presents the results of a study that has focused on the subtle effects of sublethal concentrations of the chemical carbaryl on tadpoles.

Interactions among infections, nutrients and xenobiotics.

PubMed

Ilbäck, Nils-Gunnar; Friman, Göran

2007-01-01

During recent years there have been several incidents in which symptoms of disease have been linked to consumption of food contaminated by chemical substances (e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD). Furthermore, outbreaks of infections in food-producing animals have attracted major attention regarding the safety of consumers, e.g., Bovine Spongiform Encephalitis (BSE) and influenza in chicken. As shown for several xenobiotics in an increasing number of experimental studies, even low-dose xenobiotic exposure may impair immune function over time, as well as microorganism virulence, resulting in more severe infectious diseases and associated complications. Moreover, during ongoing infection, xenobiotic uptake and distribution are often changed resulting in increased toxic insult to the host. The interactions among infectious agents, nutrients, and xenobiotics have thus become a developing concern and new avenue of research in food toxicology as well as in food-borne diseases. From a health perspective, in the risk assessment of xenobiotics in our food and environment, synergistic effects among microorganisms, nutrients, and xenobiotics will have to be considered. Otherwise, such effects may gradually change the disease panorama in society.

Polychlorinated Biphenyl-Xenobiotic Nuclear Receptor Interactions Regulate Energy Metabolism, Behavior, and Inflammation in Non-alcoholic-Steatohepatitis.

PubMed

Wahlang, Banrida; Prough, Russell A; Falkner, K Cameron; Hardesty, Josiah E; Song, Ming; Clair, Heather B; Clark, Barbara J; States, J Christopher; Arteel, Gavin E; Cave, Matthew C

2016-02-01

Polychlorinated biphenyls (PCBs) are environmental pollutants associated with non-alcoholic-steatohepatitis (NASH), diabetes, and obesity. We previously demonstrated that the PCB mixture, Aroclor 1260, induced steatohepatitis and activated nuclear receptors in a diet-induced obesity mouse model. This study aims to evaluate PCB interactions with the pregnane-xenobiotic receptor (Pxr: Nr1i2) and constitutive androstane receptor (Car: Nr1i3) in NASH. Wild type C57Bl/6 (WT), Pxr(-/-) and Car(-/-) mice were fed the high fat diet (42% milk fat) and exposed to a single dose of Aroclor 1260 (20 mg/kg) in this 12-week study. Metabolic phenotyping and analysis of serum, liver, and adipose was performed. Steatohepatitis was pathologically similar in all Aroclor-exposed groups, while Pxr(-/-) mice displayed higher basal pro-inflammatory cytokine levels. Pxr repressed Car expression as evident by increased basal Car/Cyp2b10 expression in Pxr(-/-) mice. Both Pxr(-/-) and Car(-/-) mice showed decreased basal respiratory exchange rate (RER) consistent with preferential lipid metabolism. Aroclor increased RER and carbohydrate metabolism, associated with increased light cycle activity in both knockouts, and decreased food consumption in the Car(-/-) mice. Aroclor exposure improved insulin sensitivity in WT mice but not glucose tolerance. The Aroclor-exposed, Pxr(-/-) mice displayed increased gluconeogenic gene expression. Lipid-oxidative gene expression was higher in WT and Pxr(-/-) mice although RER was not changed, suggesting PCB-mediated mitochondrial dysfunction. Therefore, Pxr and Car regulated inflammation, behavior, and energy metabolism in PCB-mediated NASH. Future studies should address the 'off-target' effects of PCBs in steatohepatitis. Published by Oxford University Press on behalf of the Society of Toxicology 2015. This work is written by US Government employees and is in the public domain in the US.

Maintenance of drug metabolism and transport functions in human precision-cut liver slices during prolonged incubation for 5 days.

PubMed

Starokozhko, Viktoriia; Vatakuti, Suresh; Schievink, Bauke; Merema, Marjolijn T; Asplund, Annika; Synnergren, Jane; Aspegren, Anders; Groothuis, Geny M M

2017-05-01

Human precision-cut liver slices (hPCLS) are a valuable ex vivo model that can be used in acute toxicity studies. However, a rapid decline in metabolic enzyme activity limits their use in studies that require a prolonged xenobiotic exposure. The aim of the study was to extend the viability and function of hPCLS to 5 days of incubation. hPCLS were incubated in two media developed for long-term culture of hepatocytes, RegeneMed ® , and Cellartis ® , and in the standard medium WME. Maintenance of phase I and II metabolism was studied both on gene expression as well as functional level using a mixture of CYP isoform-specific substrates. Albumin synthesis, morphological integrity, and glycogen storage was assessed, and gene expression was studied by transcriptomic analysis using microarrays with a focus on genes involved in drug metabolism, transport and toxicity. The data show that hPCLS retain their viability and functionality during 5 days of incubation in Cellartis ® medium. Albumin synthesis as well as the activity and gene expression of phase I and II metabolic enzymes did not decline during 120-h incubation in Cellartis ® medium, with CYP2C9 activity as the only exception. Glycogen storage and morphological integrity were maintained. Moreover, gene expression changes in hPCLS during incubation were limited and mostly related to cytoskeleton remodeling, fibrosis, and moderate oxidative stress. The expression of genes involved in drug transport, which is an important factor in determining the intracellular xenobiotic exposure, was also unchanged. Therefore, we conclude that hPCLS cultured in Cellartis ® medium are a valuable human ex vivo model for toxicological and pharmacological studies that require prolonged xenobiotic exposure.

Nontargeted analysis of the urine nonpolar sulfateome: a pathway to the nonpolar xenobiotic exposome

PubMed Central

Yao, Yuanyuan; Wang, Poguang; Shao, Gang; Anzalota Del Toro, Liza V.; Codero, Jose; Giese, Roger W.

2016-01-01

RATIONALE Testing the urine nonpolar sulfateome can enable discovery of xenobiotics that are most likely to be bioactive. This is based on the fact that nonpolar xenobiotics are more likely to enter cells where they tend to undergo metabolism, in part, to sulfates that are then largely excreted into the urine. METHODS The following sequence of steps, with conditions that achieve high reproducibility, was applied to large human urine samples: (1) competitive nonpolar extraction with a porous extraction paddle; (2) weak anion exchange extraction with strong organic washing; and (3) UHPLC/negative ion-MALDI-TOF/TOF-MS with recording of ions with S/N ≥ 20 that yielded M-1-80 (loss of SO3) or m/z 97 (HSO4−) upon fragmentation. RESULTS From a collection of urine samples from six pregnant women, the masses of 1129 putative sulfates were measured. Three lists of candidate compounds (preliminary hits) from these masses were formed by searching METLIN, especially via MATLAB, yielding putative xenobiotic contaminants (35 compounds), steroids (122), and flavonoids (1582). CONCLUSION A new way to reveal some of the nonpolar xenobiotic exposome has been developed that applies to urine samples. The value of the method is to suggest xenobiotics for subsequent targeted analysis in the population of people under study, in order to relate the environment to health and disease. PMID:27557133

Proteolytic regulation of metabolic enzymes by E3 ubiquitin ligase complexes: lessons from yeast.

PubMed

Nakatsukasa, Kunio; Okumura, Fumihiko; Kamura, Takumi

2015-01-01

Eukaryotic organisms use diverse mechanisms to control metabolic rates in response to changes in the internal and/or external environment. Fine metabolic control is a highly responsive, energy-saving process that is mediated by allosteric inhibition/activation and/or reversible modification of preexisting metabolic enzymes. In contrast, coarse metabolic control is a relatively long-term and expensive process that involves modulating the level of metabolic enzymes. Coarse metabolic control can be achieved through the degradation of metabolic enzymes by the ubiquitin-proteasome system (UPS), in which substrates are specifically ubiquitinated by an E3 ubiquitin ligase and targeted for proteasomal degradation. Here, we review select multi-protein E3 ligase complexes that directly regulate metabolic enzymes in Saccharomyces cerevisiae. The first part of the review focuses on the endoplasmic reticulum (ER) membrane-associated Hrd1 and Doa10 E3 ligase complexes. In addition to their primary roles in the ER-associated degradation pathway that eliminates misfolded proteins, recent quantitative proteomic analyses identified native substrates of Hrd1 and Doa10 in the sterol synthesis pathway. The second part focuses on the SCF (Skp1-Cul1-F-box protein) complex, an abundant prototypical multi-protein E3 ligase complex. While the best-known roles of the SCF complex are in the regulation of the cell cycle and transcription, accumulating evidence indicates that the SCF complex also modulates carbon metabolism pathways. The increasing number of metabolic enzymes whose stability is directly regulated by the UPS underscores the importance of the proteolytic regulation of metabolic processes for the acclimation of cells to environmental changes.

Molecular cloning of a family of xenobiotic-inducible drosophilid cytochrome P450s: Evidence for involvement in host-plant allelochemical resistance

PubMed Central

Danielson, Phillip B.; MacIntyre, Ross J.; Fogleman, James C.

1997-01-01

Cytochrome P450s constitute a superfamily of genes encoding mostly microsomal hemoproteins that play a dominant role in the metabolism of a wide variety of both endogenous and foreign compounds. In insects, xenobiotic metabolism (i.e., metabolism of insecticides and toxic natural plant compounds) is known to involve members of the CYP6 family of cytochrome P450s. Use of a 3′ RACE (rapid amplification of cDNA ends) strategy with a degenerate primer based on the conserved cytochrome P450 heme-binding decapeptide loop resulted in the amplification of four cDNA sequences representing another family of cytochrome P450 genes (CYP28) from two species of isoquinoline alkaloid-resistant Drosophila and the cosmopolitan species Drosophila hydei. The CYP28 family forms a monophyletic clade with strong regional homologies to the vertebrate CYP3 family and the insect CYP6 family (both of which are involved in xenobiotic metabolism) and to the insect CYP9 family (of unknown function). Induction of mRNA levels for three of the CYP28 cytochrome P450s by toxic host-plant allelochemicals (up to 11.5-fold) and phenobarbital (up to 49-fold) corroborates previous in vitro metabolism studies and suggests a potentially important role for the CYP28 family in determining patterns of insect–host-plant relationships through xenobiotic detoxification. PMID:9380713

Something Old, Something New: Conserved Enzymes and the Evolution of Novelty in Plant Specialized Metabolism1

PubMed Central

Moghe, Gaurav D.; Last, Robert L.

2015-01-01

Plants produce hundreds of thousands of small molecules known as specialized metabolites, many of which are of economic and ecological importance. This remarkable variety is a consequence of the diversity and rapid evolution of specialized metabolic pathways. These novel biosynthetic pathways originate via gene duplication or by functional divergence of existing genes, and they subsequently evolve through selection and/or drift. Studies over the past two decades revealed that diverse specialized metabolic pathways have resulted from the incorporation of primary metabolic enzymes. We discuss examples of enzyme recruitment from primary metabolism and the variety of paths taken by duplicated primary metabolic enzymes toward integration into specialized metabolism. These examples provide insight into processes by which plant specialized metabolic pathways evolve and suggest approaches to discover enzymes of previously uncharacterized metabolic networks. PMID:26276843

Experiment K-6-21. Effect of microgravity on 1) metabolic enzymes of type 1 and type 2 muscle fibers and on 2) metabolic enzymes, neutransmitter amino acids, and neurotransmitter associated enzymes in motor and somatosensory cerebral cortex. Part 1: Metabolic enzymes of individual muscle fibers; part 2: metabolic enzymes of hippocampus and spinal cord

NASA Technical Reports Server (NTRS)

Lowry, O.; Mcdougal, D., Jr.; Nemeth, Patti M.; Maggie, M.-Y. Chi; Pusateri, M.; Carter, J.; Manchester, J.; Norris, Beverly; Krasnov, I.

1990-01-01

The individual fibers of any individual muscle vary greatly in enzyme composition, a fact which is obscured when enzyme levels of a whole muscle are measured. The purpose of this study was therefore to assess the changes due to weightless on the enzyme patterns composed by the individual fibers within the flight muscles. In spite of the limitation in numbers of muscles examined, it is apparent that: (1) that the size of individual fibers (i.e., their dry weight) was reduced about a third, (2) that this loss in dry mass was accompanied by changes in the eight enzymes studied, and (3) that these changes were different for the two muscles, and different for the two enzyme groups. In the soleus muscle the absolute amounts of the three enzymes of oxidative metabolism decreased about in proportion to the dry weight loss, so that their concentration in the atrophic fibers was almost unchanged. In contrast, there was little loss among the four enzymes of glycogenolysis - glycolysis so that their concentrations were substantially increased in the atrophic fibers. In the TA muscle, these seven enzymes were affected in just the opposite direction. There appeared to be no absolute loss among the oxidative enzymes, whereas the glycogenolytic enzymes were reduced by nearly half, so that the concentrations of the first metabolic group were increased within the atrophic fibers and the concentrations of the second group were only marginally decreased. The behavior of hexokinase was exceptional in that it did not decrease in absolute terms in either type of muscle and probably increased as much as 50 percent in soleus. Thus, their was a large increase in concentration of this enzyme in the atrophied fibers of both muscles. Another clear-cut finding was the large increase in the range of activities of the glycolytic enzymes among individual fibers of TA muscles. This was due to the emergence of TA fibers with activities for enzymes of this group extending down to levels as low as

Modeling antibiotic and cytotoxic effects of the dimeric isoquinoline IQ-143 on metabolism and its regulation in Staphylococcus aureus, Staphylococcus epidermidis and human cells

PubMed Central

2011-01-01

Background Xenobiotics represent an environmental stress and as such are a source for antibiotics, including the isoquinoline (IQ) compound IQ-143. Here, we demonstrate the utility of complementary analysis of both host and pathogen datasets in assessing bacterial adaptation to IQ-143, a synthetic analog of the novel type N,C-coupled naphthyl-isoquinoline alkaloid ancisheynine. Results Metabolite measurements, gene expression data and functional assays were combined with metabolic modeling to assess the effects of IQ-143 on Staphylococcus aureus, Staphylococcus epidermidis and human cell lines, as a potential paradigm for novel antibiotics. Genome annotation and PCR validation identified novel enzymes in the primary metabolism of staphylococci. Gene expression response analysis and metabolic modeling demonstrated the adaptation of enzymes to IQ-143, including those not affected by significant gene expression changes. At lower concentrations, IQ-143 was bacteriostatic, and at higher concentrations bactericidal, while the analysis suggested that the mode of action was a direct interference in nucleotide and energy metabolism. Experiments in human cell lines supported the conclusions from pathway modeling and found that IQ-143 had low cytotoxicity. Conclusions The data suggest that IQ-143 is a promising lead compound for antibiotic therapy against staphylococci. The combination of gene expression and metabolite analyses with in silico modeling of metabolite pathways allowed us to study metabolic adaptations in detail and can be used for the evaluation of metabolic effects of other xenobiotics. PMID:21418624

Application of a hierarchical enzyme classification method reveals the role of gut microbiome in human metabolism

PubMed Central

2015-01-01

Background Enzymes are known as the molecular machines that drive the metabolism of an organism; hence identification of the full enzyme complement of an organism is essential to build the metabolic blueprint of that species as well as to understand the interplay of multiple species in an ecosystem. Experimental characterization of the enzymatic reactions of all enzymes in a genome is a tedious and expensive task. The problem is more pronounced in the metagenomic samples where even the species are not adequately cultured or characterized. Enzymes encoded by the gut microbiota play an essential role in the host metabolism; thus, warranting the need to accurately identify and annotate the full enzyme complements of species in the genomic and metagenomic projects. To fulfill this need, we develop and apply a method called ECemble, an ensemble approach to identify enzymes and enzyme classes and study the human gut metabolic pathways. Results ECemble method uses an ensemble of machine-learning methods to accurately model and predict enzymes from protein sequences and also identifies the enzyme classes and subclasses at the finest resolution. A tenfold cross-validation result shows accuracy between 97 and 99% at different levels in the hierarchy of enzyme classification, which is superior to comparable methods. We applied ECemble to predict the entire complements of enzymes from ten sequenced proteomes including the human proteome. We also applied this method to predict enzymes encoded by the human gut microbiome from gut metagenomic samples, and to study the role played by the microbe-derived enzymes in the human metabolism. After mapping the known and predicted enzymes to canonical human pathways, we identified 48 pathways that have at least one bacteria-encoded enzyme, which demonstrates the complementary role of gut microbiome in human gut metabolism. These pathways are primarily involved in metabolizing dietary nutrients such as carbohydrates, amino acids, lipids

Application of a hierarchical enzyme classification method reveals the role of gut microbiome in human metabolism.

PubMed

Mohammed, Akram; Guda, Chittibabu

2015-01-01

Enzymes are known as the molecular machines that drive the metabolism of an organism; hence identification of the full enzyme complement of an organism is essential to build the metabolic blueprint of that species as well as to understand the interplay of multiple species in an ecosystem. Experimental characterization of the enzymatic reactions of all enzymes in a genome is a tedious and expensive task. The problem is more pronounced in the metagenomic samples where even the species are not adequately cultured or characterized. Enzymes encoded by the gut microbiota play an essential role in the host metabolism; thus, warranting the need to accurately identify and annotate the full enzyme complements of species in the genomic and metagenomic projects. To fulfill this need, we develop and apply a method called ECemble, an ensemble approach to identify enzymes and enzyme classes and study the human gut metabolic pathways. ECemble method uses an ensemble of machine-learning methods to accurately model and predict enzymes from protein sequences and also identifies the enzyme classes and subclasses at the finest resolution. A tenfold cross-validation result shows accuracy between 97 and 99% at different levels in the hierarchy of enzyme classification, which is superior to comparable methods. We applied ECemble to predict the entire complements of enzymes from ten sequenced proteomes including the human proteome. We also applied this method to predict enzymes encoded by the human gut microbiome from gut metagenomic samples, and to study the role played by the microbe-derived enzymes in the human metabolism. After mapping the known and predicted enzymes to canonical human pathways, we identified 48 pathways that have at least one bacteria-encoded enzyme, which demonstrates the complementary role of gut microbiome in human gut metabolism. These pathways are primarily involved in metabolizing dietary nutrients such as carbohydrates, amino acids, lipids, cofactors and

Multifunctional enzymes from reduced genomes - model proteins for simple primordial metabolism?

PubMed

Seelig, Burckhard

2017-08-01

Billions of years of evolution have yielded today's complex metabolic networks driven by efficient and highly specialized enzymes. In contrast, the metabolism of the earliest cellular life forms was likely much simpler with only a few enzymes of comparatively low activity. It has been speculated that these early enzymes had low specificities and in turn were able to perform multiple functions. In this issue of Molecular Microbiology, Ferla et al. describe examples of enzymes that catalyze chemically distinct reactions while using the same active site. Most importantly, the authors demonstrated that the comparatively weak activities of these multifunctional enzymes are each physiologically relevant. These findings contrast with simply promiscuous enzyme activities, which have been described numerous times but are not physiologically relevant. Ferla et al. elegantly combined initial bioinformatics searches for enzyme candidates with sound kinetic measurements, evolutionary considerations and even structural discussions. The phenomenon of multifunctionality appears to be a mechanism for bacteria with reduced genomes to compensate for their lack of certain enzymes. In the broader context of evolution, these organisms could be considered living model systems to study features of long-extinct early cellular life. © 2017 John Wiley & Sons Ltd.

Simultaneous prediction of enzyme orthologs from chemical transformation patterns for de novo metabolic pathway reconstruction

PubMed Central

Tabei, Yasuo; Yamanishi, Yoshihiro; Kotera, Masaaki

2016-01-01

Motivation: Metabolic pathways are an important class of molecular networks consisting of compounds, enzymes and their interactions. The understanding of global metabolic pathways is extremely important for various applications in ecology and pharmacology. However, large parts of metabolic pathways remain unknown, and most organism-specific pathways contain many missing enzymes. Results: In this study we propose a novel method to predict the enzyme orthologs that catalyze the putative reactions to facilitate the de novo reconstruction of metabolic pathways from metabolome-scale compound sets. The algorithm detects the chemical transformation patterns of substrate–product pairs using chemical graph alignments, and constructs a set of enzyme-specific classifiers to simultaneously predict all the enzyme orthologs that could catalyze the putative reactions of the substrate–product pairs in the joint learning framework. The originality of the method lies in its ability to make predictions for thousands of enzyme orthologs simultaneously, as well as its extraction of enzyme-specific chemical transformation patterns of substrate–product pairs. We demonstrate the usefulness of the proposed method by applying it to some ten thousands of metabolic compounds, and analyze the extracted chemical transformation patterns that provide insights into the characteristics and specificities of enzymes. The proposed method will open the door to both primary (central) and secondary metabolism in genomics research, increasing research productivity to tackle a wide variety of environmental and public health matters. Availability and Implementation: Contact: maskot@bio.titech.ac.jp PMID:27307627

Sensor potency of the moonlighting enzyme-decorated cytoskeleton: the cytoskeleton as a metabolic sensor

PubMed Central

2013-01-01

Background There is extensive evidence for the interaction of metabolic enzymes with the eukaryotic cytoskeleton. The significance of these interactions is far from clear. Presentation of the hypothesis In the cytoskeletal integrative sensor hypothesis presented here, the cytoskeleton senses and integrates the general metabolic activity of the cell. This activity depends on the binding to the cytoskeleton of enzymes and, depending on the nature of the enzyme, this binding may occur if the enzyme is either active or inactive but not both. This enzyme-binding is further proposed to stabilize microtubules and microfilaments and to alter rates of GTP and ATP hydrolysis and their levels. Testing the hypothesis Evidence consistent with the cytoskeletal integrative sensor hypothesis is presented in the case of glycolysis. Several testable predictions are made. There should be a relationship between post-translational modifications of tubulin and of actin and their interaction with metabolic enzymes. Different conditions of cytoskeletal dynamics and enzyme-cytoskeleton binding should reveal significant differences in local and perhaps global levels and ratios of ATP and GTP. The different functions of moonlighting enzymes should depend on cytoskeletal binding. Implications of the hypothesis The physical and chemical effects arising from metabolic sensing by the cytoskeleton would have major consequences on cell shape, dynamics and cell cycle progression. The hypothesis provides a framework that helps the significance of the enzyme-decorated cytoskeleton be determined. PMID:23398642

Non-alcoholic fatty liver disease (NAFLD) - pathogenesis, classification, and effect on drug metabolizing enzymes and transporters.

PubMed

Cobbina, Enoch; Akhlaghi, Fatemeh

2017-05-01

Non-alcoholic fatty liver disease (NAFLD) is a spectrum of liver disorders. It is defined by the presence of steatosis in more than 5% of hepatocytes with little or no alcohol consumption. Insulin resistance, the metabolic syndrome or type 2 diabetes and genetic variants of PNPLA3 or TM6SF2 seem to play a role in the pathogenesis of NAFLD. The pathological progression of NAFLD follows tentatively a "three-hit" process namely steatosis, lipotoxicity and inflammation. The presence of steatosis, oxidative stress and inflammatory mediators like TNF-α and IL-6 has been implicated in the alterations of nuclear factors such as CAR, PXR, PPAR-α in NAFLD. These factors may result in altered expression and activity of drug metabolizing enzymes (DMEs) or transporters. Existing evidence suggests that the effect of NAFLD on CYP3A4, CYP2E1 and MRP3 is more consistent across rodent and human studies. CYP3A4 activity is down-regulated in NASH whereas the activity of CYP2E1 and the efflux transporter MRP3 is up-regulated. However, it is not clear how the majority of CYPs, UGTs, SULTs and transporters are influenced by NAFLD either in vivo or in vitro. The alterations associated with NAFLD could be a potential source of drug variability in patients and could have serious implications for the safety and efficacy of xenobiotics. In this review, we summarize the effects of NAFLD on the regulation, expression and activity of major DMEs and transporters. We also discuss the potential mechanisms underlying these alterations.

Altered drug metabolism during pregnancy: hormonal regulation of drug-metabolizing enzymes.

PubMed

Jeong, Hyunyoung

2010-06-01

Medication use during pregnancy is prevalent, but pharmacokinetic information of most drugs used during pregnancy is lacking in spite of known effects of pregnancy on drug disposition. Accurate pharmacokinetic information is essential for optimal drug therapy in mother and fetus. Thus, understanding how pregnancy influences drug disposition is important for better prediction of pharmacokinetic changes of drugs in pregnant women. Pregnancy is known to affect hepatic drug metabolism, but the underlying mechanisms remain unknown. Physiological changes accompanying pregnancy are probably responsible for the reported alteration in drug metabolism during pregnancy. These include elevated concentrations of various hormones such as estrogen, progesterone, placental growth hormones and prolactin. This review covers how these hormones influence expression of drug-metabolizing enzymes (DMEs), thus potentially responsible for altered drug metabolism during pregnancy. The reader will gain a greater understanding of the altered drug metabolism in pregnant women and the regulatory effects of pregnancy hormones on expression of DMEs. In-depth studies in hormonal regulatory mechanisms as well as confirmatory studies in pregnant women are warranted for systematic understanding and prediction of the changes in hepatic drug metabolism during pregnancy.

Characterization of the human cytochrome P450 enzymes involved in the metabolism of dihydrocodeine

PubMed Central

Kirkwood, L. C.; Nation, R. L.; Somogyi, A. A.

1997-01-01

Aims Using human liver microsomes from donors of the CYP2D6 poor and extensive metabolizer genotypes, the role of individual cytochromes P-450 in the oxidative metabolism of dihydrocodeine was investigated. Methods The kinetics of formation of N- and O-demethylated metabolites, nordihydrocodeine and dihydromorphine, were determined using microsomes from six extensive and one poor metabolizer and the effects of chemical inhibitors selective for individual P-450 enzymes of the 1A, 2A, 2C, 2D, 2E and 3A families and of LKM1 (anti-CYP2D6) antibodies were studied. Results Nordihydrocodeine was the major metabolite in both poor and extensive metabolizers. Kinetic constants for N-demethylation derived from the single enzyme Michaelis-Menten model did not differ between the two groups. Troleandomycin and erythromycin selectively inhibited N-demethylation in both extensive and poor metabolizers. The CYP3A inducer, α-naphthoflavone, increased N-demethylation rates. The kinetics of formation of dihydromorphine in both groups were best described by a single enzyme Michaelis-Menten model although inhibition studies in extensive metabolizers suggested involvement of two enzymes with similar Km values. The kinetic constants for O-demethylation were significantly different in extensive and poor metabolizers. The extensive metabolizers had a mean intrinsic clearance to dihydromorphine more than ten times greater than the poor metabolizer. The CYP2D6 chemical inhibitors, quinidine and quinine, and LKM1 antibodies inhibited O-demethylation in extensive metabolizers; no effect was observed in microsomes from a poor metabolizer. Conclusions CYP2D6 is the major enzyme mediating O-demethylation of dihydrocodeine to dihydromorphine. In contrast, nordihydrocodeine formation is predominantly catalysed by CYP3A. PMID:9431830

Metabolic Diseases Downregulate the Majority of Histone Modification Enzymes, Making a Few Upregulated Enzymes Novel Therapeutic Targets--"Sand Out and Gold Stays".

PubMed

Shao, Ying; Chernaya, Valeria; Johnson, Candice; Yang, William Y; Cueto, Ramon; Sha, Xiaojin; Zhang, Yi; Qin, Xuebin; Sun, Jianxin; Choi, Eric T; Wang, Hong; Yang, Xiao-feng

2016-02-01

To determine whether the expression of histone modification enzymes is regulated in physiological and pathological conditions, we took an experimental database mining approach pioneered in our labs to determine a panoramic expression profile of 164 enzymes in 19 human and 17 murine tissues. We have made the following significant findings: (1) Histone enzymes are differentially expressed in cardiovascular, immune, and other tissues; (2) our new pyramid model showed that heart and T cells are among a few tissues in which histone acetylation/deacetylation, and histone methylation/demethylation are in the highest varieties; and (3) histone enzymes are more downregulated than upregulated in metabolic diseases and regulatory T cell (Treg) polarization/ differentiation, but not in tumors. These results have demonstrated a new working model of "Sand out and Gold stays," where more downregulation than upregulation of histone enzymes in metabolic diseases makes a few upregulated enzymes the potential novel therapeutic targets in metabolic diseases and Treg activity.

Short-term calorie restriction feminizes the mRNA profiles of drug metabolizing enzymes and transporters in livers of mice.

PubMed

Fu, Zidong Donna; Klaassen, Curtis D

2014-01-01

Calorie restriction (CR) is one of the most effective anti-aging interventions in mammals. A modern theory suggests that aging results from a decline in detoxification capabilities and thus accumulation of damaged macromolecules. The present study aimed to determine how short-term CR alters mRNA profiles of genes that encode metabolism and detoxification machinery in the liver. Male C57BL/6 mice were fed CR (0, 15, 30, or 40%) diets for one month, followed by mRNA quantification of 98 xenobiotic processing genes (XPGs) in the liver, including 7 uptake transporters, 39 phase-I enzymes, 37 phase-II enzymes, 10 efflux transporters, and 5 transcription factors. In general, 15% CR did not alter mRNAs of most XPGs, whereas 30 and 40% CR altered over half of the XPGs (32 increased and 29 decreased). CR up-regulated some phase-I enzymes (fold increase), such as Cyp4a14 (12), Por (2.3), Nqo1 (1.4), Fmo2 (5.4), and Fmo3 (346), and numerous number of phase-II enzymes, such as Sult1a1 (1.2), Sult1d1 (2.0), Sult1e1 (33), Sult3a1 (2.2), Gsta4 (1.3), Gstm2 (1.3), Gstm3 (1.7), and Mgst3 (2.2). CR feminized the mRNA profiles of 32 XPGs in livers of male mice. For instance, CR decreased the male-predominantly expressed Oatp1a1 (97%) and increased the female-predominantly expressed Oatp1a4 (11). In conclusion, short-term CR alters the mRNA levels of over half of the 98 XPGs quantified in livers of male mice, and over half of these alterations appear to be due to feminization of the liver. Copyright © 2013 Elsevier Inc. All rights reserved.

Differences in glycogen, lipids, and enzymes in livers from rats flown on Cosmos 2044

NASA Technical Reports Server (NTRS)

Merrill, Alfred H., Jr.; Wang, Elaine; Laroque, Regina; Mullins, Richard E.; Morgan, Edward T.; Hargrove, James L.; Bonkovsky, Herbert L.; Popova, Irina A.

1992-01-01

Livers from rats flown aboard Cosmos 2044 were analyzed for protein, carbohydrate (glycogen), and lipids as well as the activities of a number of key enzymes involved in metabolism of these compounds and xenobiotics. The major differences between the flight group and the synchronous control were elevations in microsomal protein, liver glycogen content, tyrosine aminotransferase, and tryptophan oxygenase and reductions in sphingolipids and the rate-limiting enzyme of heme biosynthesis delta-aminolevulinic acid synthase. These results provide further evidence that spaceflight has pronounced and diverse effects on liver function; however, some of the results with samples from Cosmos 2044 differed notably from those from previous spaceflights. This may be due to conditions of spaceflight and/or the postflight recovery period for Cosmos 2044.

A study of inter-individual variability in the Phase II metabolism of xenobiotics in human skin.

PubMed

Spriggs, Sandrine; Cubberley, Richard; Loadman, Paul; Sheffield, David; Wierzbicki, Antonia

2018-08-01

Understanding skin metabolism is key to improve in vitro to in vivo extrapolations used to inform risk assessments of topically applied products. However, published literature is scarce and usually covers a limited and non-representative number of donors. We developed a protocol to handle and store ex vivo skin samples post-surgery and prepare skin S9 fractions to measure the metabolic activity of Phase II enzymes. Preincubation of an excess of cofactors at 37 °C for fifteen minutes in the S9 before introduction of the testing probe, greatly increased the stability of the enzymes. Using this standardised assay, the rates of sulphation (SULT) and glucuronidation (UGT) of 7-hydroxycoumarin, methylation (COMT) of dopamine and N-acetylation (NAT) of procainamide were measured in the ng/mg protein/h (converted to ng/cm 2 /h) range in eighty-seven individuals. Glutathione conjugation (GST) of 1-chloro-2,4-dinitrobenzene was assessed in a smaller pool of fifty donors; the metabolic rate was much faster and measured over six minutes using a different methodology to express rates in μg/mg protein/min (converted to μg/cm 2 /min). A comprehensive statistical analysis of these results was carried out, separating donors by age, gender and metabolic rate measured. Copyright © 2018 Elsevier B.V. All rights reserved.

Pentachlorophenol: Uptake/elimination kinetics and metabolism in an aquatic plant, Eichhornia crassipes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Roy, S.; Haenninen, O.

1994-05-01

Eichhornia crassipes [(Mart) Solms], an aquatic plant widely used for the treatment of wastewaters, was used to study uptake/elimination kinetics and metabolism of pentachlorophenol (PCP). PCP is a well-known industrial by-product and a major pollutant of the aquatic environment. The initial phase of PCP uptake by the plant was rapid and reached a nearly steady state between 24 and 48 h of exposure to PCP. The major by-products of PCP metabolisms in Eichhornia crassipes were identified as ortho- and para- substituted chlorohydroxyphenols (chlorocatechols and -hydroquiones), -anisoles, and -veratroles. Partially dechlorinated products of PCP were also detected. A major portion ofmore » the absorbed PCP and metabolites was found in bound/conjugated form. The response of the enzyme systems involved in the xenobiotic metabolism and antioxidative system were also studied following PCP exposure. A significant increase was observed in the activity of glutathione S-transferase (GST), a major conjugating enzyme, and in the activities of superoxide dismutase and ascorbate peroxidase of PCP exposed plants. Such responses of plant enzymes may be implemented as useful markers of aquatic pollution. The result related to the uptake and metabolism of PCP obtained from the present study suggests a crucial role of aquatic plants in determining the fate of environmental chemicals.« less

Mathematical Modeling of Cellular Metabolism.

PubMed

Berndt, Nikolaus; Holzhütter, Hermann-Georg

Cellular metabolism basically consists of the conversion of chemical compounds taken up from the extracellular environment into energy (conserved in energy-rich bonds of organic phosphates) and a wide array of organic molecules serving as catalysts (enzymes), information carriers (nucleic acids), and building blocks for cellular structures such as membranes or ribosomes. Metabolic modeling aims at the construction of mathematical representations of the cellular metabolism that can be used to calculate the concentration of cellular molecules and the rates of their mutual chemical interconversion in response to varying external conditions as, for example, hormonal stimuli or supply of essential nutrients. Based on such calculations, it is possible to quantify complex cellular functions as cellular growth, detoxification of drugs and xenobiotic compounds or synthesis of exported molecules. Depending on the specific questions to metabolism addressed, the methodological expertise of the researcher, and available experimental information, different conceptual frameworks have been established, allowing the usage of computational methods to condense experimental information from various layers of organization into (self-) consistent models. Here, we briefly outline the main conceptual frameworks that are currently exploited in metabolism research.

A framework and case studies for evaluation of enzyme ontogeny in children's health risk evaluation.

PubMed

Ginsberg, Gary; Vulimiri, Suryanarayana V; Lin, Yu-Sheng; Kancherla, Jayaram; Foos, Brenda; Sonawane, Babasaheb

2017-01-01

Knowledge of the ontogeny of Phase I and Phase II metabolizing enzymes may be used to inform children's vulnerability based upon likely differences in internal dose from xenobiotic exposure. This might provide a qualitative assessment of toxicokinetic (TK) variability and uncertainty pertinent to early lifestages and help scope a more quantitative physiologically based toxicokinetic (PBTK) assessment. Although much is known regarding the ontogeny of metabolizing systems, this is not commonly utilized in scoping and problem formulation stage of human health risk evaluation. A framework is proposed for introducing this information into problem formulation which combines data on enzyme ontogeny and chemical-specific TK to explore potential child/adult differences in internal dose and whether such metabolic differences may be important factors in risk evaluation. The framework is illustrated with five case study chemicals, including some which are data rich and provide proof of concept, while others are data poor. Case studies for toluene and chlorpyrifos indicate potentially important child/adult TK differences while scoping for acetaminophen suggests enzyme ontogeny is unlikely to increase early-life risks. Scoping for trichloroethylene and aromatic amines indicates numerous ways that enzyme ontogeny may affect internal dose which necessitates further evaluation. PBTK modeling is a critical and feasible next step to further evaluate child-adult differences in internal dose for a number of these chemicals.

Discovery and characterization of a prevalent human gut bacterial enzyme sufficient for the inactivation of a family of plant toxins

PubMed Central

Koppel, Nitzan; Bisanz, Jordan E; Pandelia, Maria-Eirini

2018-01-01

Although the human gut microbiome plays a prominent role in xenobiotic transformation, most of the genes and enzymes responsible for this metabolism are unknown. Recently, we linked the two-gene ‘cardiac glycoside reductase’ (cgr) operon encoded by the gut Actinobacterium Eggerthella lenta to inactivation of the cardiac medication and plant natural product digoxin. Here, we compared the genomes of 25 E. lenta strains and close relatives, revealing an expanded 8-gene cgr-associated gene cluster present in all digoxin metabolizers and absent in non-metabolizers. Using heterologous expression and in vitro biochemical characterization, we discovered that a single flavin- and [4Fe-4S] cluster-dependent reductase, Cgr2, is sufficient for digoxin inactivation. Unexpectedly, Cgr2 displayed strict specificity for digoxin and other cardenolides. Quantification of cgr2 in gut microbiomes revealed that this gene is widespread and conserved in the human population. Together, these results demonstrate that human-associated gut bacteria maintain specialized enzymes that protect against ingested plant toxins. PMID:29761785

Developmental roles of tyrosine metabolism enzymes in the blood-sucking insect Rhodnius prolixus

PubMed Central

Oliveira, Pedro L.

2017-01-01

The phenylalanine/tyrosine degradation pathway is frequently described as a catabolic pathway that funnels aromatic amino acids into citric acid cycle intermediates. Previously, we demonstrated that the accumulation of tyrosine generated during the hydrolysis of blood meal proteins in Rhodnius prolixus is potentially toxic, a harmful outcome that is prevented by the action of the first two enzymes in the tyrosine degradation pathway. In this work, we further evaluated the relevance of all other enzymes involved in phenylalanine/tyrosine metabolism in the physiology of this insect. The knockdown of most of these enzymes produced a wide spectrum of distinct phenotypes associated with reproduction, development and nymph survival, demonstrating a highly pleiotropic role of tyrosine metabolism. The phenotypes obtained for two of these enzymes, homogentisate dioxygenase and fumarylacetoacetase, have never before been described in any arthropod. To our knowledge, this report is the first comprehensive gene-silencing analysis of an amino acid metabolism pathway in insects. Amino acid metabolism is exceptionally important in haematophagous arthropods due to their particular feeding behaviour. PMID:28469016

Developmental roles of tyrosine metabolism enzymes in the blood-sucking insect Rhodnius prolixus.

PubMed

Sterkel, Marcos; Oliveira, Pedro L

2017-05-17

The phenylalanine/tyrosine degradation pathway is frequently described as a catabolic pathway that funnels aromatic amino acids into citric acid cycle intermediates. Previously, we demonstrated that the accumulation of tyrosine generated during the hydrolysis of blood meal proteins in Rhodnius prolixus is potentially toxic, a harmful outcome that is prevented by the action of the first two enzymes in the tyrosine degradation pathway. In this work, we further evaluated the relevance of all other enzymes involved in phenylalanine/tyrosine metabolism in the physiology of this insect. The knockdown of most of these enzymes produced a wide spectrum of distinct phenotypes associated with reproduction, development and nymph survival, demonstrating a highly pleiotropic role of tyrosine metabolism. The phenotypes obtained for two of these enzymes, homogentisate dioxygenase and fumarylacetoacetase, have never before been described in any arthropod. To our knowledge, this report is the first comprehensive gene-silencing analysis of an amino acid metabolism pathway in insects. Amino acid metabolism is exceptionally important in haematophagous arthropods due to their particular feeding behaviour. © 2017 The Author(s).

Understanding Substrate Selectivity of Human UDP-glucuronosyltransferases through QSAR modeling and analysis of homologous enzymes

PubMed Central

Dong, Dong; Ako, Roland; Hu, Ming; Wu, Baojian

2015-01-01

The UDP-glucuronosyltransferase (UGT) enzyme catalyzes the glucuronidation reaction which is a major metabolic and detoxification pathway in humans. Understanding the mechanisms for substrate recognition by UGT assumes great importance in an attempt to predict its contribution to xenobiotic/drug disposition in vivo. Spurred on by this interest, 2D/3D-quantitative structure activity relationships (QSAR) and pharmacophore models have been established in the absence of a complete mammalian UGT crystal structure. This review discusses the recent progress in modeling human UGT substrates including those with multiple sites of glucuronidation. A better understanding of UGT active site contributing to substrate selectivity (and regioselectivity) from the homologous enzymes (i.e., plant and bacterial UGTs, all belong to family 1 of glycosyltransferase (GT1)) is also highlighted, as these enzymes share a common catalytic mechanism and/or overlapping substrate selectivity. PMID:22385482

Monobromobimane occupies a distinct xenobiotic substrate site in glutathione S-transferase pi.

PubMed

Ralat, Luis A; Colman, Roberta F

2003-11-01

Monobromobimane (mBBr), functions as a substrate of porcine glutathione S-transferase pi (GST pi): The enzyme catalyzes the reaction of mBBr with glutathione. S-(Hydroxyethyl)bimane, a nonreactive analog of monobromobimane, acts as a competitive inhibitor with respect to mBBr as substrate but does not affect the reaction of GST pi with another substrate, 1-chloro-2,4-dinitrobenzene (CDNB). In the absence of glutathione, monobromobimane inactivates GST pi at pH 7.0 and 25 degrees C as assayed using mBBr as substrate, with a lesser effect on the enzyme's use of CDNB as substrate. These results indicate that the sites occupied by CDNB and mBBr are not identical. Inactivation is proportional to the incorporation of 2 moles of bimane/mole of subunit. Modification of GST pi with mBBr does not interfere with its binding of 8-anilino-1-naphthalene sulfonate, indicating that this hydrophobic site is not the target of monobromobimane. S-Methylglutathione and S-(hydroxyethyl)bimane each yield partial protection against inactivation and decrease reagent incorporation, while glutathionyl-bimane protects completely against inactivation. Peptide analysis after trypsin digestion indicates that mBBr modifies Cys45 and Cys99 equally. Modification of Cys45 is reduced in the presence of S-methylglutathione, indicating that this residue is at or near the glutathione binding region. In contrast, modification of Cys99 is reduced in the presence of S-(hydroxyethyl)bimane, suggesting that this residue is at or near the mBBr xenobiotic substrate binding site. Modification of Cys99 can best be understood by reaction with monobromobimane while it is bound to its xenobiotic substrate site in an alternate orientation. These results support the concept that glutathione S-transferase accomplishes its ability to react with a diversity of substrates in part by harboring distinct xenobiotic substrate sites.

Elucidation of metabolic pathways from enzyme classification data.

PubMed

McDonald, Andrew G; Tipton, Keith F

2014-01-01

The IUBMB Enzyme List is widely used by other databases as a source for avoiding ambiguity in the recognition of enzymes as catalytic entities. However, it was not designed for metabolic pathway tracing, which has become increasingly important in systems biology. A Reactions Database has been created from the material in the Enzyme List to allow reactions to be searched by substrate/product, and pathways to be traced from any selected starting/seed substrate. An extensive synonym glossary allows searches by many of the alternative names, including accepted abbreviations, by which a chemical compound may be known. This database was necessary for the development of the application Reaction Explorer ( http://www.reaction-explorer.org ), which was written in Real Studio ( http://www.realsoftware.com/realstudio/ ) to search the Reactions Database and draw metabolic pathways from reactions selected by the user. Having input the name of the starting compound (the "seed"), the user is presented with a list of all reactions containing that compound and then selects the product of interest as the next point on the ensuing graph. The pathway diagram is then generated as the process iterates. A contextual menu is provided, which allows the user: (1) to remove a compound from the graph, along with all associated links; (2) to search the reactions database again for additional reactions involving the compound; (3) to search for the compound within the Enzyme List.

Expression and Regulation of Drug Transporters and Metabolizing Enzymes in the Human Gastrointestinal Tract.

PubMed

Drozdzik, M; Oswald, S

2016-01-01

Orally administered drugs must pass through the intestinal wall and then through the liver before reaching systemic circulation. During this process drugs are subjected to different processes that may determine the therapeutic value. The intestinal barrier with active drug metabolizing enzymes and drug transporters in enterocytes plays an important role in the determination of drug bioavailability. Accumulating information demonstrates variable distribution of drug metabolizing enzymes and transporters along the human gastrointestinal tract (GI), that creates specific barrier characteristics in different segments of the GI. In this review, expression of drug metabolizing enzymes and transporters in the healthy and diseased human GI as well as their regulatory aspects: genetic, miRNA, DNA methylation are outlined. The knowledge of unique interplay between drug metabolizing enzymes and transporters in specific segments of the GI tract allows more precise definition of drug release sites within the GI in order to assure more complete bioavailability and prediction of drug interactions.

New glycyl radical enzymes catalysing key metabolic steps in anaerobic bacteria.

PubMed

Selmer, Thorsten; Pierik, Antonio J; Heider, Johann

2005-10-01

During the last decade, an increasing number of new enzymes containing glycyl radicals in their active sites have been identified and biochemically characterised. These include benzylsuccinate synthase (Bss), 4-hydroxyphenylacetate decarboxylase (Hpd) and the coenzyme B12-independent glycerol dehydratase (Gdh). These are involved in metabolic pathways as different as anaerobic toluene metabolism, fermentative production of p-cresol and glycerol fermentation. Some features of these newly discovered enzymes are described and compared with those of the previously known glycyl radical enzymes pyruvate formate-lyase (Pfl) and anaerobic ribonucleotide reductase (Nrd). Among the new enzymes, Bss and Hpd share the presence of small subunits, the function of which in the catalytic mechanisms is still enigmatic, and both enzymes contain metal centres in addition to the glycyl radical prosthetic group. The activating enzymes of the novel systems also deviate from the standard type, containing at least one additional Fe-S cluster. Finally, the available whole-genome sequences of an increasing number of strictly or facultative anaerobic bacteria revealed the presence of many more hitherto unknown glycyl radical enzyme (GRE) systems. Recent studies suggest that the particular types of these enzymes represent the ends of different evolutionary lines, which emerged early in evolution and diversified to yield remarkably versatile biocatalysts for chemical reactions that are otherwise difficult to perform in anoxic environments.

Targeted proteome analysis of single-gene deletion strains of Saccharomyces cerevisiae lacking enzymes in the central carbon metabolism.

PubMed

Matsuda, Fumio; Kinoshita, Syohei; Nishino, Shunsuke; Tomita, Atsumi; Shimizu, Hiroshi

2017-01-01

Central carbon metabolism is controlled by modulating the protein abundance profiles of enzymes that maintain the essential systems in living organisms. In this study, metabolic adaptation mechanisms in the model organism Saccharomyces cerevisiae were investigated by direct determination of enzyme abundance levels in 30 wild type and mutant strains. We performed a targeted proteome analysis using S. cerevisiae strains that lack genes encoding the enzymes responsible for central carbon metabolism. Our analysis revealed that at least 30% of the observed variations in enzyme abundance levels could be explained by global regulatory mechanisms. A enzyme-enzyme co-abundance analysis revealed that the abundances of enzyme proteins involved in the trehalose metabolism and glycolysis changed in a coordinated manner under the control of the transcription factors for global regulation. The remaining variations were derived from local mechanisms such as a mutant-specific increase in the abundances of remote enzymes. The proteome data also suggested that, although the functional compensation of the deficient enzyme was attained by using more resources for protein biosynthesis, available resources for the biosynthesis of the enzymes responsible for central metabolism were not abundant in S. cerevisiae cells. These results showed that global and local regulation of enzyme abundance levels shape central carbon metabolism in S. cerevisiae by using a limited resource for protein biosynthesis.

Enzymes and Inhibitors in Neonicotinoid Insecticide Metabolism

PubMed Central

Shi, Xueyan; Dick, Ryan A.; Ford, Kevin A.; Casida, John E.

2009-01-01

Neonicotinoid insecticide metabolism involves considerable substrate specificity and regioselectivity of the relevant CYP450, aldehyde oxidase, and phase II enzymes. Human CYP450 recombinant enzymes carry out the following conversions: CYP3A4, 2C19 and 2B6 for thiamethoxam (TMX) to clothianidin (CLO); 3A4, 2C19 and 2A6 for CLO to desmethyl-CLO; 2C19 for TMX to desmethyl-TMX. Human liver aldehyde oxidase reduces the nitro substituent of CLO to nitroso much more rapidly than that of TMX. Imidacloprid (IMI), CLO and several of their metabolites do not give detectable N-glucuronides but 5-hydroxy-IMI, 4,5-diol-IMI and 4-hydroxy-thiacloprid are converted to O-glucuronides in vitro with mouse liver microsomes and UDP-glucuronic acid or in vivo in mice. Mouse liver cytosol with S-adenosylmethionine converts desmethyl-CLO to CLO but not desmethyl-TMX to TMX. Two organophosphorus CYP450 inhibitors partially block IMI, thiacloprid and CLO metabolism in vivo in mice, elevating the brain and liver levels of the parent compounds while reducing amounts of the hydroxylated metabolites. PMID:19391582

Truffles contain endocannabinoid metabolic enzymes and anandamide.

PubMed

Pacioni, Giovanni; Rapino, Cinzia; Zarivi, Osvaldo; Falconi, Anastasia; Leonardi, Marco; Battista, Natalia; Colafarina, Sabrina; Sergi, Manuel; Bonfigli, Antonella; Miranda, Michele; Barsacchi, Daniela; Maccarrone, Mauro

2015-02-01

Truffles are the fruiting body of fungi, members of the Ascomycota phylum endowed with major gastronomic and commercial value. The development and maturation of their reproductive structure are dependent on melanin synthesis. Since anandamide, a prominent member of the endocannabinoid system (ECS), is responsible for melanin synthesis in normal human epidermal melanocytes, we thought that ECS might be present also in truffles. Here, we show the expression, at the transcriptional and translational levels, of most ECS components in the black truffle Tuber melanosporum Vittad. at maturation stage VI. Indeed, by means of molecular biology and immunochemical techniques, we found that truffles contain the major metabolic enzymes of the ECS, while they do not express the most relevant endocannabinoid-binding receptors. In addition, we measured anandamide content in truffles, at different maturation stages (from III to VI), through liquid chromatography-mass spectrometric analysis, whereas the other relevant endocannabinoid 2-arachidonoylglycerol was below the detection limit. Overall, our unprecedented results suggest that anandamide and ECS metabolic enzymes have evolved earlier than endocannabinoid-binding receptors, and that anandamide might be an ancient attractant to truffle eaters, that are well-equipped with endocannabinoid-binding receptors. Copyright © 2014 Elsevier Ltd. All rights reserved.

The roles of carboxylesterase and CYP isozymes on the in vitro metabolism of T-2 toxin.

PubMed

Lin, Ni-Ni; Chen, Jia; Xu, Bin; Wei, Xia; Guo, Lei; Xie, Jian-Wei

2015-01-01

T-2 toxin poses a great threat to human health because it has the highest toxicity of the currently known trichothecene mycotoxins. To understand the in vivo toxicity and transformation mechanism of T-2 toxin, we investigated the role of one kind of principal phase I drug-metabolizing enzymes (cytochrome P450 [CYP450] enzymes) on the metabolism of T-2 toxin, which are crucial to the metabolism of endogenous substances and xenobiotics. We also investigated carboxylesterase, which also plays an important role in the metabolism of toxic substances. A chemical inhibition method and a recombinant method were employed to investigate the metabolism of the T-2 toxin by the CYP450 enzymes, and a chemical inhibition method was used to study carboxylesterase metabolism. Samples incubated with human liver microsomes were analyzed by high performance liquid chromatography-triple quadrupole mass spectrometry (HPLC- QqQ MS) after a simple pretreatment. In the presence of a carboxylesterase inhibitor, only 20 % T-2 toxin was metabolized. When CYP enzyme inhibitors and a carboxylesterase inhibitor were both present, only 3 % of the T-2 toxin was metabolized. The contributions of the CYP450 enzyme family to T-2 toxin metabolism followed the descending order CYP3A4, CYP2E1, CYP1A2, CYP2B6 or CYP2D6 or CYP2C19. Carboxylesterase and CYP450 enzymes are of great importance in T-2 toxin metabolism, in which carboxylesterase is predominant and CYP450 has a subordinate role. CYP3A4 is the principal member of the CYP450 enzyme family responsible for T-2 toxin metabolism. The primary metabolite produced by carboxylesterase is HT-2, and the main metabolite produced by CYP 3A4 is 3'-OH T-2. The different metabolites show different toxicities. Our results will provide useful data concerning the toxic mechanism, the safety evaluation, and the health risk assessment of T-2 toxin.

Metabolic enzyme cost explains variable trade-offs between microbial growth rate and yield

PubMed Central

Ferris, Michael; Bruggeman, Frank J.

2018-01-01

Microbes may maximize the number of daughter cells per time or per amount of nutrients consumed. These two strategies correspond, respectively, to the use of enzyme-efficient or substrate-efficient metabolic pathways. In reality, fast growth is often associated with wasteful, yield-inefficient metabolism, and a general thermodynamic trade-off between growth rate and biomass yield has been proposed to explain this. We studied growth rate/yield trade-offs by using a novel modeling framework, Enzyme-Flux Cost Minimization (EFCM) and by assuming that the growth rate depends directly on the enzyme investment per rate of biomass production. In a comprehensive mathematical model of core metabolism in E. coli, we screened all elementary flux modes leading to cell synthesis, characterized them by the growth rates and yields they provide, and studied the shape of the resulting rate/yield Pareto front. By varying the model parameters, we found that the rate/yield trade-off is not universal, but depends on metabolic kinetics and environmental conditions. A prominent trade-off emerges under oxygen-limited growth, where yield-inefficient pathways support a 2-to-3 times higher growth rate than yield-efficient pathways. EFCM can be widely used to predict optimal metabolic states and growth rates under varying nutrient levels, perturbations of enzyme parameters, and single or multiple gene knockouts. PMID:29451895

Metabolism of deltamethrin and cis- and trans-permethrin by human expressed cytochrome P450 and carboxylesterase enzymes.

PubMed

Hedges, Laura; Brown, Susan; MacLeod, A Kenneth; Vardy, Audrey; Doyle, Edward; Song, Gina; Moreau, Marjory; Yoon, Miyoung; Osimitz, Thomas G; Lake, Brian G

2018-06-04

The metabolism of the pyrethroids deltamethrin (DLM), cis-permethrin (CPM) and trans-permethrin (TPM) was studied in human expressed cytochrome P450 (CYP) and carboxylesterase (CES) enzymes. DLM, CPM and TPM were metabolised by human CYP2B6 and CYP2C19, with the highest apparent intrinsic clearance (CL int ) values for pyrethroid metabolism being observed with CYP2C19. Other CYP enzymes contributing to the metabolism of one or more of the three pyrethroids were CYP1A2, CYP2C8, CYP2C9*1, CYP2D6*1, CYP3A4 and CYP3A5. None of the pyrethroids were metabolised by CYP2A6, CYP2E1, CYP3A7 or CYP4A11. DLM, CPM and TPM were metabolised by both human CES1 and CES2 enzymes. Apparent CL int values for pyrethroid metabolism by CYP and CES enzymes were scaled to per gram of adult human liver using abundance values for microsomal CYP enzymes and for CES enzymes in liver microsomes and cytosol. TPM had the highest and CPM the lowest apparent CL int values for total metabolism (CYP and CES enzymes) per gram of adult human liver. Due to their higher abundance, all three pyrethroids were extensively metabolised by CES enzymes in adult human liver, with CYP enzymes only accounting for 2%, 10% and 1% of total metabolism for DLM, CPM and TPM, respectively.

Radiation Exposure Alters Expression of Metabolic Enzyme Genes in Mice

NASA Technical Reports Server (NTRS)

Wotring, V. E.; Mangala, L. S.; Zhang, Y.; Wu, H.

2011-01-01

Most administered pharmaceuticals are metabolized by the liver. The health of the liver, especially the rate of its metabolic enzymes, determines the concentration of circulating drugs as well as the duration of their efficacy. Most pharmaceuticals are metabolized by the liver, and clinically-used medication doses are given with normal liver function in mind. A drug overdose can result in the case of a liver that is damaged and removing pharmaceuticals from the circulation at a rate slower than normal. Alternatively, if liver function is elevated and removing drugs from the system more quickly than usual, it would be as if too little drug had been given for effective treatment. Because of the importance of the liver in drug metabolism, we want to understand the effects of spaceflight on the enzymes of the liver and exposure to cosmic radiation is one aspect of spaceflight that can be modeled in ground experiments. Additionally, it has been previous noted that pre-exposure to small radiation doses seems to confer protection against later and larger radiation doses. This protective power of pre-exposure has been called a priming effect or radioadaptation. This study is an effort to examine the drug metabolizing effects of radioadaptation mechanisms that may be triggered by early exposure to low radiation doses.

Metabolic Diseases Downregulate the Majority of Histone Modification Enzymes, Making a Few Upregulated Enzymes Novel Therapeutic Targets – “Sand out and Gold Stays”

PubMed Central

Shao, Ying; Chernaya, Valeria; Johnson, Candice; Yang, William Y.; Cueto, Ramon; Sha, Xiaojin; Zhang, Yi; Qin, Xuebin; Sun, Jianxin; Choi, Eric T.; Wang, Hong; Yang, Xiao-feng

2016-01-01

To determine whether the expression of histone modification enzymes is regulated in physiological and pathological conditions, we took an experimental database mining approach pioneered in our labs to determine a panoramic expression profile of 164 enzymes in 19 human and 17 murine tissues. We have made the following significant findings: 1) Histone enzymes are differentially expressed in cardiovascular, immune and other tissues; 2) Our new pyramid model showed that heart and T cells are among a few tissues in which histone acetylation/deacetylation, histone methylation/demethylation are in the highest varieties; and 3) Histone enzymes are more downregulated than upregulated in metabolic diseases and Treg polarization/differentiation, but not in tumors. These results have demonstrated a new working model of “sand out and gold stays,” where more downregulation than upregulation of histone enzymes in metabolic diseases makes a few upregulated enzymes the potential novel therapeutic targets in metabolic diseases and Treg activity. PMID:26746407

Beyond triglyceride synthesis: the dynamic functional roles of MGAT and DGAT enzymes in energy metabolism.

PubMed

Shi, Yuguang; Cheng, Dong

2009-07-01

Monoacyglycerol acyltransferases (MGATs) and diacylglycerol acyltransferases (DGATs) catalyze two consecutive steps of enzyme reactions in the synthesis of triacylglycerols (TAGs). The metabolic complexity of TAG synthesis is reflected by the presence of multiple isoforms of MGAT and DGAT enzymes that differ in catalytic properties, subcellular localization, tissue distribution, and physiological functions. MGAT and DGAT enzymes play fundamental roles in the metabolism of monoacylglycerol (MAG), diacylglycerol (DAG), and triacylglycerol (TAG) that are involved in many aspects of physiological functions, such as intestinal fat absorption, lipoprotein assembly, adipose tissue formation, signal transduction, satiety, and lactation. The recent progress in the phenotypic characterization of mice deficient in MGAT and DGAT enzymes and the development of chemical inhibitors have revealed important roles of these enzymes in the regulation of energy homeostasis and insulin sensitivity. Consequently, selective inhibition of MGAT or DGAT enzymes by synthetic compounds may provide novel treatment for obesity and its related metabolic complications.

Beyond triglyceride synthesis: the dynamic functional roles of MGAT and DGAT enzymes in energy metabolism

PubMed Central

Shi, Yuguang; Cheng, Dong

2009-01-01

Monoacyglycerol acyltransferases (MGATs) and diacylglycerol acyltransferases (DGATs) catalyze two consecutive steps of enzyme reactions in the synthesis of triacylglycerols (TAGs). The metabolic complexity of TAG synthesis is reflected by the presence of multiple isoforms of MGAT and DGAT enzymes that differ in catalytic properties, subcellular localization, tissue distribution, and physiological functions. MGAT and DGAT enzymes play fundamental roles in the metabolism of monoacylglycerol (MAG), diacylglycerol (DAG), and triacylglycerol (TAG) that are involved in many aspects of physiological functions, such as intestinal fat absorption, lipoprotein assembly, adipose tissue formation, signal transduction, satiety, and lactation. The recent progress in the phenotypic characterization of mice deficient in MGAT and DGAT enzymes and the development of chemical inhibitors have revealed important roles of these enzymes in the regulation of energy homeostasis and insulin sensitivity. Consequently, selective inhibition of MGAT or DGAT enzymes by synthetic compounds may provide novel treatment for obesity and its related metabolic complications. PMID:19116371

The microbial pharmacists within us: a metagenomic view of xenobiotic metabolism

PubMed Central

Spanogiannopoulos, Peter; Bess, Elizabeth N.; Carmody, Rachel N.; Turnbaugh, Peter J.

2016-01-01

Although the significance of human genetic polymorphisms in therapeutic outcomes is well established, the importance of our “second genome” (the microbiome) has been largely overlooked. In this Review, we highlight recent studies that shed light on the mechanisms linking the human gut microbiome to the efficacy and toxicity of xenobiotics, including drugs, dietary compounds and environmental toxins. Continued progress in this area could enable more precise tools for predicting patient responses and the development of a next generation of therapeutics based on or targeted at the gut microbiome. Indeed, the admirable goal of precision medicine may require us to first understand the microbial pharmacists within. PMID:26972811

Multiple resistance to carcinogens and xenobiotics: P-glycoproteins as universal detoxifiers.

PubMed

Efferth, Thomas; Volm, Manfred

2017-07-01

The detoxification of toxic substances is of general relevance in all biological systems. The plethora of exogenous xenobiotic compounds and endogenous toxic metabolic products explains the evolutionary pressure of all organisms to develop molecular mechanisms to detoxify and excrete harmful substances from the body. P-glycoprotein and other members of the ATP-binding cassette (ABC) transporter family extrude innumerous chemical compounds out of cells. Their specific expression in diverse biological contexts cause different phenotypes: (1) multidrug resistance (MDR) and thus failure of cancer chemotherapy, (2) avoidance of accumulation of carcinogens and prevention of carcinogenesis in healthy tissues, (3) absorption, distribution, metabolization and excretion (ADME) of pharmacological drugs in human patients, (4) protection from environmental toxins in aquatic organisms (multi-xenobiotic resistance, MXR). Hence ABC-transporters may have opposing effects for organismic health reaching from harmful in MDR of tumors to beneficial for maintenance of health in MXR. While their inhibition by specific inhibitors may improve treatment success in oncology and avoid carcinogenesis, blocking of ABC-transporter-driven efflux by environmental pollutants leads to ecotoxicological consequences in marine biotopes. Poisoned seafood may enter the food-chain and cause intoxications in human beings. As exemplified with ABC-transporters, joining forces in interdisciplinary research may, therefore, be a wise strategy to fight problems in human medicine and environmental sciences.

The metabolism of aflatoxin B1 by hepatocytes isolated from rats following the in vivo administration of some xenobiotics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Metcalfe, S.A.; Neal, G.E.

Isolated rat hepatocytes, an intact cellular system capable of performing phase I and phase II metabolism, have been used to investigate metabolism of aflatoxin B1. These cells were found to metabolise (/sup 14/C)aflatoxin B1 to aflatoxins M1 and Q1, and to radiolabelled polar material, presumably conjugates, as analysed by h.p.l.c., t.l.c. and radioactive determination. In vivo administration of the mixed function oxidase inducers, phenobarbitone and 3-methylcholanthrene, resulted in enhanced hepatocyte phase I (microsomal) metabolism of aflatoxin B1. In contrast to metabolism of AFB1 by in vitro subcellular systems increased production of polar material (conjugated metabolites) derived from (/sup 14/C)aflatoxin B1more » was also detected in hepatocytes isolated from these pretreated animals. Formation of aflatoxin Q1 by isolated hepatocytes appeared to be mediated by cytochrome P450-linked enzymes whereas cytochrome P448-linked enzymes were apparently involved in aflatoxin M1 production. Chronic feeding of aflatoxin B1 to rats enhanced hepatocyte production of conjugated material only and did not elevate cellular cytochrome P450 levels, thus suggesting that aflatoxin B1 is not an inducer of its own primary metabolism.« less

Single-Walled Carbon Nanotubes Inhibit the Cytochrome P450 Enzyme, CYP3A4

NASA Astrophysics Data System (ADS)

El-Sayed, Ramy; Bhattacharya, Kunal; Gu, Zonglin; Yang, Zaixing; Weber, Jeffrey K.; Li, Hu; Leifer, Klaus; Zhao, Yichen; Toprak, Muhammet S.; Zhou, Ruhong; Fadeel, Bengt

2016-02-01

We report a detailed computational and experimental study of the interaction of single-walled carbon nanotubes (SWCNTs) with the drug-metabolizing cytochrome P450 enzyme, CYP3A4. Dose-dependent inhibition of CYP3A4-mediated conversion of the model compound, testosterone, to its major metabolite, 6β-hydroxy testosterone was noted. Evidence for a direct interaction between SWCNTs and CYP3A4 was also provided. The inhibition of enzyme activity was alleviated when SWCNTs were pre-coated with bovine serum albumin. Furthermore, covalent functionalization of SWCNTs with polyethylene glycol (PEG) chains mitigated the inhibition of CYP3A4 enzymatic activity. Molecular dynamics simulations suggested that inhibition of the catalytic activity of CYP3A4 is mainly due to blocking of the exit channel for substrates/products through a complex binding mechanism. This work suggests that SWCNTs could interfere with metabolism of drugs and other xenobiotics and provides a molecular mechanism for this toxicity. Our study also suggests means to reduce this toxicity, eg., by surface modification.

Single-Walled Carbon Nanotubes Inhibit the Cytochrome P450 Enzyme, CYP3A4

PubMed Central

El-Sayed, Ramy; Bhattacharya, Kunal; Gu, Zonglin; Yang, Zaixing; Weber, Jeffrey K.; Li, Hu; Leifer, Klaus; Zhao, Yichen; Toprak, Muhammet S.; Zhou, Ruhong; Fadeel, Bengt

2016-01-01

We report a detailed computational and experimental study of the interaction of single-walled carbon nanotubes (SWCNTs) with the drug-metabolizing cytochrome P450 enzyme, CYP3A4. Dose-dependent inhibition of CYP3A4-mediated conversion of the model compound, testosterone, to its major metabolite, 6β-hydroxy testosterone was noted. Evidence for a direct interaction between SWCNTs and CYP3A4 was also provided. The inhibition of enzyme activity was alleviated when SWCNTs were pre-coated with bovine serum albumin. Furthermore, covalent functionalization of SWCNTs with polyethylene glycol (PEG) chains mitigated the inhibition of CYP3A4 enzymatic activity. Molecular dynamics simulations suggested that inhibition of the catalytic activity of CYP3A4 is mainly due to blocking of the exit channel for substrates/products through a complex binding mechanism. This work suggests that SWCNTs could interfere with metabolism of drugs and other xenobiotics and provides a molecular mechanism for this toxicity. Our study also suggests means to reduce this toxicity, eg., by surface modification. PMID:26899743

Nuclear Receptors in Drug Metabolism, Drug Response and Drug Interactions

PubMed Central

Prakash, Chandra; Zuniga, Baltazar; Song, Chung Seog; Jiang, Shoulei; Cropper, Jodie; Park, Sulgi; Chatterjee, Bandana

2016-01-01

Orally delivered small-molecule therapeutics are metabolized in the liver and intestine by phase I and phase II drug-metabolizing enzymes (DMEs), and transport proteins coordinate drug influx (phase 0) and drug/drug-metabolite efflux (phase III). Genes involved in drug metabolism and disposition are induced by xenobiotic-activated nuclear receptors (NRs), i.e. PXR (pregnane X receptor) and CAR (constitutive androstane receptor), and by the 1α, 25-dihydroxy vitamin D3-activated vitamin D receptor (VDR), due to transactivation of xenobiotic-response elements (XREs) present in phase 0-III genes. Additional NRs, like HNF4-α, FXR, LXR-α play important roles in drug metabolism in certain settings, such as in relation to cholesterol and bile acid metabolism. The phase I enzymes CYP3A4/A5, CYP2D6, CYP2B6, CYP2C9, CYP2C19, CYP1A2, CYP2C8, CYP2A6, CYP2J2, and CYP2E1 metabolize >90% of all prescription drugs, and phase II conjugation of hydrophilic functional groups (with/without phase I modification) facilitates drug clearance. The conjugation step is mediated by broad-specificity transferases like UGTs, SULTs, GSTs. This review delves into our current understanding of PXR/CAR/VDR-mediated regulation of DME and transporter expression, as well as effects of single nucleotide polymorphism (SNP) and epigenome (specified by promoter methylation, histone modification, microRNAs, long non coding RNAs) on the expression of PXR/CAR/VDR and phase 0-III mediators, and their impacts on variable drug response. Therapeutic agents that target epigenetic regulation and the molecular basis and consequences (overdosing, underdosing, or beneficial outcome) of drug-drug/drug-food/drug-herb interactions are also discussed. Precision medicine requires understanding of a drug’s impact on DME and transporter activity and their NR-regulated expression in order to achieve optimal drug efficacy without adverse drug reactions. In future drug screening, new tools such as humanized mouse models and

Astrocyte-neuron crosstalk regulates the expression and subcellular localization of carbohydrate metabolism enzymes.

PubMed

Mamczur, Piotr; Borsuk, Borys; Paszko, Jadwiga; Sas, Zuzanna; Mozrzymas, Jerzy; Wiśniewski, Jacek R; Gizak, Agnieszka; Rakus, Dariusz

2015-02-01

Astrocytes releasing glucose- and/or glycogen-derived lactate and glutamine play a crucial role in shaping neuronal function and plasticity. Little is known, however, how metabolic functions of astrocytes, e.g., their ability to degrade glucosyl units, are affected by the presence of neurons. To address this issue we carried out experiments which demonstrated that co-culturing of rat hippocampal astrocytes with neurons significantly elevates the level of mRNA and protein for crucial enzymes of glycolysis (phosphofructokinase, aldolase, and pyruvate kinase), glycogen metabolism (glycogen synthase and glycogen phosphorylase), and glutamine synthetase in astrocytes. Simultaneously, the decrease of the capability of neurons to metabolize glucose and glutamine is observed. We provide evidence that neurons alter the expression of astrocytic enzymes by secretion of as yet unknown molecule(s) into the extracellular fluid. Moreover, our data demonstrate that almost all studied enzymes may localize in astrocytic nuclei and this localization is affected by the co-culturing with neurons which also reduces proliferative activity of astrocytes. Our results provide the first experimental evidence that the astrocyte-neuron crosstalk substantially affects the expression of basal metabolic enzymes in the both types of cells and influences their subcellular localization in astrocytes. © 2014 Wiley Periodicals, Inc.

Spatial localization of the first and last enzymes effectively connects active metabolic pathways in bacteria.

PubMed

Meyer, Pablo; Cecchi, Guillermo; Stolovitzky, Gustavo

2014-12-14

Although much is understood about the enzymatic cascades that underlie cellular biosynthesis, comparatively little is known about the rules that determine their cellular organization. We performed a detailed analysis of the localization of E.coli GFP-tagged enzymes for cells growing exponentially. We found that out of 857 globular enzymes, at least 219 have a discrete punctuate localization in the cytoplasm and catalyze the first or the last reaction in 60% of biosynthetic pathways. A graph-theoretic analysis of E.coli's metabolic network shows that localized enzymes, in contrast to non-localized ones, form a tree-like hierarchical structure, have a higher within-group connectivity, and are traversed by a higher number of feed-forward and feedback loops than their non-localized counterparts. A Gene Ontology analysis of these enzymes reveals an enrichment of terms related to essential metabolic functions in growing cells. Given that these findings suggest a distinct metabolic role for localization, we studied the dynamics of cellular localization of the cell wall synthesizing enzymes in B. subtilis and found that enzymes localize during exponential growth but not during stationary growth. We conclude that active biochemical pathways inside the cytoplasm are organized spatially following a rule where their first or their last enzymes localize to effectively connect the different active pathways and thus could reflect the activity state of the cell's metabolic network.

Development of in vivo biotransformation enzyme assays for ecotoxicity screening: In vivo measurement of phases I and II enzyme activities in freshwater planarians.

PubMed

Li, Mei-Hui

2016-08-01

The development of a high-throughput tool is required for screening of environmental pollutants and assessing their impacts on aquatic animals. Freshwater planarians can be used in rapid and sensitive toxicity bioassays. Planarians are known for their remarkable regeneration ability but much less known for their metabolic and xenobiotic biotransformation abilities. In this study, the activities of different phase I and II enzymes were determined in vivo by directly measuring fluorescent enzyme substrate disappearance or fluorescent enzyme metabolite production in planarian culture media. For phase I enzyme activity, O-deethylation activities with alkoxyresorufin could not be detected in planarian culture media. By contrast, O-deethylation activities with alkoxycoumarin were detected in planarian culture media. Increases in 7-ethoxycoumarin O-deethylase (ECOD) activities was only observed in planarians exposed to 1μM, but not 10μM, β-naphthoflavone for 24h. ECOD activity was inhibited in planarians exposed to 10 and 100μM rifampicin or carbamazepine for 24h. For phase II enzyme activity, DT-diaphorase, arylsulfatases, uridine 5'-diphospho (UDP)-glucuronosyltransferase or catechol-O-methyltransferase activity was determined in culture media containing planarians. The results of this study indicate that freshwater planarians are a promising model organism to monitor exposure to environmental pollutants or assess their impacts through the in vivo measurement of phase I and II enzyme activities. Copyright © 2016. Published by Elsevier Inc.

Biotransformation of anthelmintics and the activity of drug-metabolizing enzymes in the tapeworm Moniezia expansa.

PubMed

Prchal, Lukáš; Bártíková, Hana; Bečanová, Aneta; Jirásko, Robert; Vokřál, Ivan; Stuchlíková, Lucie; Skálová, Lenka; Kubíček, Vladimír; Lamka, Jiří; Trejtnar, František; Szotáková, Barbora

2015-04-01

The sheep tapeworm Moniezia expansa is very common parasite, which affects ruminants such as sheep, goats as well as other species. The benzimidazole anthelmintics albendazole (ABZ), flubendazole (FLU) and mebendazole (MBZ) are often used to treat the infection. The drug-metabolizing enzymes of helminths may alter the potency of anthelmintic treatment. The aim of our study was to assess the activity of the main drug-metabolizing enzymes and evaluate the metabolism of selected anthelmintics (ABZ, MBZ and FLU) in M. expansa. Activities of biotransformation enzymes were determined in subcellular fractions. Metabolites of the anthelmintics were detected and identified using high performance liquid chromatography/ultra-violet/VIS/fluorescence or ultra-high performance liquid chromatography/mass spectrometry. Reduction of MBZ, FLU and oxidation of ABZ were proved as well as activities of various metabolizing enzymes. Despite the fact that the conjugation enzymes glutathione S-transferase, UDP-glucuronosyl transferase and UDP-glucosyl transferase were active in vitro, no conjugated metabolites of anthelmintics were identified either ex vivo or in vitro. The obtained results indicate that sheep tapeworm is able to deactivate the administered anthelmintics, and thus protects itself against their action.

Characterization of the cytochrome P450 enzymes and enzyme kinetic parameters for metabolism of BVT.2938 using different in vitro systems.

PubMed

Baranczewski, Pawel; Edlund, Per Olof; Postlind, Hans

2006-03-18

An important step in the drug development process is identification of enzymes responsible for metabolism of drug candidates and determination of enzyme kinetic parameters. These data are used to increase understanding of the pharmacokinetics and possible metabolic-based drug interactions of drug candidates. The aim of the present study was to characterize the cytochrome P450 enzymes and enzyme kinetic parameters for metabolism of BVT.2938 [1-(3-{2-[(2-ethoxy-3-pyridinyl)oxy]ethoxy}-2-pyrazinyl)-2(R)-methylpiperazine], a potent and selective 5HT2c-receptor agonist. The enzyme kinetic parameters were determined for formation of three main metabolites of BVT.2938 using human liver microsomes and expressed cytochrome P450 (CYP) isoforms. The major metabolite was formed by hydroxylation of the pyridine ring (CL(int)=27 microl/mgmin), and was catalysed by both CYP2D6*1 and CYP1A1, with K(m) values corresponding to 1.4 and 2.7 microM, respectively. The results from enzyme kinetic studies were confirmed by incubation of BVT.2938 in the presence of the chemical inhibitor of CYP2D6*1, quinidine. Quinidine inhibited the formation of the major metabolite by approximately 90%. Additionally, studies with recombinant expressed CYP isoforms from rat indicated that formation of the major metabolite of BVT.2938 was catalysed by CYP2D2. This result was further confirmed by experiments with liver slices from different rat strains, where the formation of the metabolite correlated with phenotype of CYP2D2 isoform (Sprague-Dawley male, extensive; Dark Agouti male, intermediate; Dark Agouti female, poor metabolizer). The present study showed that the major metabolite of BVT.2938 is formed by hydroxylation of the pyridine ring and catalysed by CYP2D6*1. CYP1A1 is also involved in this reaction and its role in extra-hepatic metabolism of BVT.2938 might be significant.

EnzDP: improved enzyme annotation for metabolic network reconstruction based on domain composition profiles.

PubMed

Nguyen, Nam-Ninh; Srihari, Sriganesh; Leong, Hon Wai; Chong, Ket-Fah

2015-10-01

Determining the entire complement of enzymes and their enzymatic functions is a fundamental step for reconstructing the metabolic network of cells. High quality enzyme annotation helps in enhancing metabolic networks reconstructed from the genome, especially by reducing gaps and increasing the enzyme coverage. Currently, structure-based and network-based approaches can only cover a limited number of enzyme families, and the accuracy of homology-based approaches can be further improved. Bottom-up homology-based approach improves the coverage by rebuilding Hidden Markov Model (HMM) profiles for all known enzymes. However, its clustering procedure relies firmly on BLAST similarity score, ignoring protein domains/patterns, and is sensitive to changes in cut-off thresholds. Here, we use functional domain architecture to score the association between domain families and enzyme families (Domain-Enzyme Association Scoring, DEAS). The DEAS score is used to calculate the similarity between proteins, which is then used in clustering procedure, instead of using sequence similarity score. We improve the enzyme annotation protocol using a stringent classification procedure, and by choosing optimal threshold settings and checking for active sites. Our analysis shows that our stringent protocol EnzDP can cover up to 90% of enzyme families available in Swiss-Prot. It achieves a high accuracy of 94.5% based on five-fold cross-validation. EnzDP outperforms existing methods across several testing scenarios. Thus, EnzDP serves as a reliable automated tool for enzyme annotation and metabolic network reconstruction. Available at: www.comp.nus.edu.sg/~nguyennn/EnzDP .

Inhalation exposure of rats to asphalt fumes generated at paving temperatures alters pulmonary xenobiotic metabolism pathways without lung injury.

PubMed Central

Ma, Jane Y C; Rengasamy, Apavoo; Frazer, Dave; Barger, Mark W; Hubbs, Ann F; Battelli, Lori; Tomblyn, Seith; Stone, Samuel; Castranova, Vince

2003-01-01

Asphalt fumes are complex mixtures of various organic compounds, including polycyclic aromatic hydrocarbons (PAHs). PAHs require bioactivation by the cytochrome P-450 monooxygenase system to exert toxic/carcinogenic effects. The present study was carried out to characterize the acute pulmonary inflammatory responses and the alterations of pulmonary xenobiotic pathways in rats exposed to asphalt fumes by inhalation. Rats were exposed at various doses and time periods to air or to asphalt fumes generated at paving temperatures. To assess the acute damage and inflammatory responses, differential cell counts, acellular lactate dehydrogenase (LDH) activity, and protein content of bronchoalveolar lavage fluid were determined. Alveolar macrophage (AM) function was assessed by monitoring generation of chemiluminescence and production of tumor necrosis factor-alpha and interleukin-1. Alteration of pulmonary xenobiotic pathways was determined by monitoring the protein levels and activities of P-450 isozymes (CYP1A1 and CYP2B1), glutathioneS-transferase (GST), and NADPH:quinone oxidoreductase (QR). The results show that acute asphalt fume exposure did not cause neutrophil infiltration, alter LDH activity or protein content, or affect AM function, suggesting that short-term asphalt fume exposure did not induce acute lung damage or inflammation. However, acute asphalt fume exposure significantly increased the activity and protein level of CYP1A1 whereas it markedly reduced the activity and protein level of CYP2B1 in the lung. The induction of CYP1A1 was localized in nonciliated bronchiolar epithelial (Clara) cells, alveolar septa, and endothelial cells by immunofluorescence microscopy. Cytosolic QR activity was significantly elevated after asphalt fume exposure, whereas GST activity was not affected by the exposure. This induction of CYP1A1 and QR with the concomitant down-regulation of CYP2B1 after asphalt fume exposure could alter PAH metabolism and may lead to potential

Inhibitors of steroidal cytochrome p450 enzymes as targets for drug development.

PubMed

Baston, Eckhard; Leroux, Frédéric R

2007-01-01

Cytochrome P450's are enzymes which catalyze a large number of biological reactions, for example hydroxylation, N-, O-, S- dealkylation, epoxidation or desamination. Their substrates include fatty acids, steroids or prostaglandins. In addition, a high number of various xenobiotics are metabolized by these enzymes. The enzyme 17alpha-hydroxylase-C17,20-lyase (P450(17), CYP 17, androgen synthase), a cytochrome P450 monooxygenase, is the key enzyme for androgen biosynthesis. It catalyzes the last step of the androgen biosynthesis in the testes and adrenal glands and produces androstenedione and dehydroepiandrosterone from progesterone and pregnenolone. The microsomal enzyme aromatase (CYP19) transforms these androgens to estrone and estradiol. Estrogens stimulate tumor growth in hormone dependent breast cancer. In addition, about 80 percent of prostate cancers are androgen dependent. Selective inhibitors of these enzymes are thus important alternatives to treatment options like antiandrogens or antiestrogens. The present article deals with recent patents (focus on publications from 2000 - 2006) concerning P450 inhibitor design where steroidal substrates are involved. In this context a special focus is provided for CYP17 and CYP19. Mechanisms of action will also be discussed. Inhibitors of CYP11B2 (aldosterone synthase) will also be dealt with.

Dual Function of the Cytochrome P450 CYP76 Family from Arabidopsis thaliana in the Metabolism of Monoterpenols and Phenylurea Herbicides1[W][OPEN

PubMed Central

Höfer, René; Boachon, Benoît; Renault, Hugues; Gavira, Carole; Miesch, Laurence; Iglesias, Juliana; Ginglinger, Jean-François; Allouche, Lionel; Miesch, Michel; Grec, Sebastien; Larbat, Romain; Werck-Reichhart, Danièle

2014-01-01

Comparative genomics analysis unravels lineage-specific bursts of gene duplications related to the emergence of specialized pathways. The CYP76C subfamily of cytochrome P450 enzymes is specific to Brassicaceae. Two of its members were recently associated with monoterpenol metabolism. This prompted us to investigate the CYP76C subfamily genetic and functional diversification. Our study revealed high rates of CYP76C gene duplication and loss in Brassicaceae, suggesting the association of the CYP76C subfamily with species-specific adaptive functions. Gene differential expression and enzyme functional specialization in Arabidopsis thaliana, including metabolism of different monoterpenols and formation of different products, support this hypothesis. In addition to linalool metabolism, CYP76C1, CYP76C2, and CYP76C4 metabolized herbicides belonging to the class of phenylurea. Their ectopic expression in the whole plant conferred herbicide tolerance. CYP76Cs from A. thaliana. thus provide a first example of promiscuous cytochrome P450 enzymes endowing effective metabolism of both natural and xenobiotic compounds. Our data also suggest that the CYP76C gene family provides a suitable genetic background for a quick evolution of herbicide resistance. PMID:25082892

Modulation of xenobiotic biotransformation system and hormonal responses in Atlantic salmon (Salmo salar) after exposure to tributyltin (TBT).

PubMed

Mortensen, Anne Skjetne; Arukwe, Augustine

2007-04-01

Multiple biological effects of tributyltin (TBT) on juvenile salmon have been investigated. Fish were exposed for 7 days to waterborne TBT at nominal concentrations of 50 and 250 microg/L dissolved in dimethyl sulfoxide (DMSO). Hepatic samples were analyzed for gene expression patterns in the hormonal and xenobiotic biotransformation pathways using validated real-time PCR method. Immunochemical and several cytochrome P450 (CYP)-mediated enzyme activity (ethoxyresorufin: EROD, benzyloxyresorufin: BROD, methoxyresorufin: MROD and pentoxyresorufin: PROD) assays were analyzed. Our data show that TBT produced concentration-specific decrease of estrogen receptor-alpha (ERalpha), vitellogenin (Vtg), zona radiata protein (Zr-protein) and increase of estrogen receptor-beta (ERbeta) and androgen receptor-beta (ARbeta) in the hormonal pathway. In the xenobiotic biotransformation pathway, TBT produced apparent increase and decrease at respective low and high concentration, on aryl hydrocarbon receptor-alpha (AhRalpha), AhR nuclear translocator (ARNT) and AhR repressor (AhRR) mRNA. The expression of CYP1A1 and GST showed a TBT concentration-dependent decrease. The AhRbeta, CYP3A and uridine diphosphoglucuronosyl transferase (UGT) mRNA expressions were significantly induced after exposure to TBT. Immunochemical analysis of CYP3A and CYP1A1 protein levels confirmed the TBT effects observed at the transcriptional levels. The effect of TBT on the biotransformation enzyme gene expressions partially co-related but did not directly parallel enzyme activity levels for EROD, BROD, MROD and PROD. In general, these findings confirm previous reports on the endocrine effects of TBT, in addition to effects on hepatic CYP1A isoenzyme at the transcriptional level that transcends to protein and enzymatic levels. The induced expression patterns of CYP3A and UGT mRNA after TBT exposure, suggest the involvement of CYP3A and UGT in TBT metabolism in fish. The effect of TBT on CYP3A is proposed to

Xenobiotic-Metabolizing Enzyme and Transporter Gene Expression in Primary Cultures of Human Hepatocytes Modulated by Toxcast Chemicals

EPA Science Inventory

Primary human hepatocyte cultures are useful in vitro model systems of human liver because when cultured under appropriate conditions the hepatocytes retain liver-like functionality such as metabolism, transport, and cell signaling. This model system was used to characterize the ...

Phase I and phase II reductive metabolism simulation of nitro aromatic xenobiotics with electrochemistry coupled with high resolution mass spectrometry.

PubMed

Bussy, Ugo; Chung-Davidson, Yu-Wen; Li, Ke; Li, Weiming

2014-11-01

Electrochemistry combined with (liquid chromatography) high resolution mass spectrometry was used to simulate the general reductive metabolism of three biologically important nitro aromatic molecules: 3-trifluoromethyl-4-nitrophenol (TFM), niclosamide, and nilutamide. TFM is a pesticide used in the Laurential Great Lakes while niclosamide and nilutamide are used in cancer therapy. At first, a flow-through electrochemical cell was directly connected to a high resolution mass spectrometer to evaluate the ability of electrochemistry to produce the main reduction metabolites of nitro aromatic, nitroso, hydroxylamine, and amine functional groups. Electrochemical experiments were then carried out at a constant potential of -2.5 V before analysis of the reduction products by LC-HRMS, which confirmed the presence of the nitroso, hydroxylamine, and amine species as well as dimers. Dimer identification illustrates the reactivity of the nitroso species with amine and hydroxylamine species. To investigate xenobiotic metabolism, the reactivity of nitroso species to biomolecules was also examined. Binding of the nitroso metabolite to glutathione was demonstrated by the observation of adducts by LC-ESI(+)-HRMS and the characteristics of their MSMS fragmentation. In conclusion, electrochemistry produces the main reductive metabolites of nitro aromatics and supports the observation of nitroso reactivity through dimer or glutathione adduct formation.

Acute biotoxic effect of styrene on rat liver. Correlation with enzyme-mediated mutagenicity of benzpyrene and acrylonitrile.

PubMed

Roberfroid, M; Poncelet, F; Lambotte-Vandepaer, M; Duverger-Van Bogaert, M; de Meester, C; Mercier, M

1978-01-01

Styrene is commonly used in western Europe for the manufacture of plastics suitable for packaging foodstuffs. This report demonstrates that, injected intraperitoneally at a dose as low as 10 mg/kg, styrene modifies the catalytic properties of aryl hydrocarbon hydroxylase by reducing its KM value. A similar effect is reported for two potent chemical carcinogens, 3-methylcholanthrene and benzo(a)pyrene. Ethylbenzene and benzo(e)pyrene and phenobarbital do not produce the same effect. Pretreatments of the rats with chemicals which modify aryl hydrocarbon hydroxylase also increase the capacity of the liver enzymes to activate benzopyrene to a mutagenic intermediate in vitro, as measured by the Ames test for mutagenicity. Exposure to both styrene and the other modifiers of the xenobiotic-metabolizing enzymes could thus influence the carcinogenic and toxic effects of chemicals which are activated by these enzymes. This hypothesis needs further investigation.

EFFECTS OF X-IRRADIATION ON THE HEXOBARBITAL METABOLIZING ENZYME SYSTEM OF RAT LIVER MICROSOMES.

DTIC Science & Technology

RADIATION EFFECTS , *ENZYME INHIBITORS, *HYPNOTICS AND SEDATIVES, ENZYMES, BIOSYNTHESIS, METABOLISM, DETOXIFICATION, BARBITURATES, OXIDATION...MICROSOMES, LIVER, REGENERATION(ENGINEERING), EXCISION, SUBLETHAL DOSAGE, TOXICITY , HYPNOSIS, SLEEP, HEAD(ANATOMY), MALES, FEMALES, RATS.

Identification of a CYP3A form (CYP3A126) in fathead minnow (Pimephales promelas) and characterisation of putative CYP3A enzyme activity.

PubMed

Christen, Verena; Caminada, Daniel; Arand, Michael; Fent, Karl

2010-01-01

Cytochrome P450-dependent monooxygenases (CYPs) are involved in the metabolic defence against xenobiotics. Human CYP3A enzymes metabolise about 50% of all pharmaceuticals in use today. Induction of CYPs and associated xenobiotic metabolism occurs also in fish and may serve as a useful tool for biomonitoring of environmental contamination. In this study we report on the cloning of a CYP3A family gene from fathead minnows (Pimephales promelas), which has been designated as CYP3A126 by the P450 nomenclature committee (GenBank no. EU332792). The cDNA was isolated, identified and characterised by extended inverse polymerase chain reaction (PCR), an alternative to the commonly used method of rapid amplification of cDNA ends. In a fathead minnow cell line we identified a full-length cDNA sequence (1,863 base pairs (bp)) consisting of a 1,536 bp open reading frame encoding a 512 amino acid protein. Genomic analysis of the identified CYP3A isoenzyme revealed a DNA sequence consisting of 13 exons and 12 introns. CYP3A126 is also expressed in fathead minnow liver as demonstrated by reverse transcription PCR. Exposure of fathead minnow (FHM) cells with the CYP3A inducer rifampicin leads to dose-dependent increase in putative CYP3A enzyme activity. In contrast, inhibitory effects of diazepam treatment were observed on putative CYP3A enzyme activity and additionally on CYP3A126 mRNA expression. This indicates that CYP3A is active in FHM cells and that CYP3A126 is at least in part responsible for this CYP3A activity. Further investigations will show whether CYP3A126 is involved in the metabolism of environmental chemicals.

Enzymes and Metabolites in Carbohydrate Metabolism of Desiccation Tolerant Plants

PubMed Central

Zhang, Qingwei; Song, Xiaomin; Bartels, Dorothea

2016-01-01

Resurrection plants can tolerate extreme water loss. Substantial sugar accumulation is a phenomenon in resurrection plants during dehydration. Sugars have been identified as one important factor contributing to desiccation tolerance. Phylogenetic diversity of resurrection plants reflects the diversity of sugar metabolism in response to dehydration. Sugars, which accumulate during dehydration, have been shown to protect macromolecules and membranes and to scavenge reactive oxygen species. This review focuses on the performance of enzymes participating in sugar metabolism during dehydration stress. The relation between sugar metabolism and other biochemical activities is discussed and open questions as well as potential experimental approaches are proposed. PMID:28248249

Disposition of xenobiotic chemicals and metabolites in marine organisms.

PubMed Central

Varanasi, U; Stein, J E

1991-01-01

Studies with several bottom fish species from urban waterways show that of the identified xenobiotic chemicals in bottom sediments, polycylic aromatic hydrocarbons (PAHs) are the most strongly associated with the prevalence of liver lesions, including neoplasms. Accordingly, there is concern about the transfer of contaminants, such as PAHs, from aquatic species to humans. Because PAHs exert their toxicity only after being biotransformed, increasing attention has been focused on the ability of aquatic organisms to metabolize these chemicals. Overall, the results of both laboratory and field studies show that generally low levels (nanograms per gram wet weight) of a few low molecular weight PAHs may be present in edible tissue of fish from contaminated areas and that high molecular weight PAHs, such as the carcinogen benzo(a)pyrene, will rarely be detected because of extensive metabolism. Additionally, the results from a few studies suggest that even though interactions between xenobiotics can affect both biochemical and physiological systems to alter the disposition of PAHs in fish, these interactions do not markedly change the relative proportions of metabolites to parent PAH in tissues. Thus, these studies clearly demonstrate that to obtain some insight into the questions of whether there is any risk to human health from consuming fish and crustaceans from urban areas, techniques must be developed that measure metabolites of carcinogens, such as PAHs, in edible tissue. Initial attempts may focus on semiquantitative methods that permit rapid assessment of the level of metabolites in edible tissues of fish and crustaceans from many urban areas.(ABSTRACT TRUNCATED AT 250 WORDS) Images FIGURE 4. FIGURE 4. FIGURE 4. PMID:2050086

Heme-containing enzymes and inhibitors for tryptophan metabolism.

PubMed

Yan, Daojing; Lin, Ying-Wu; Tan, Xiangshi

2017-09-20

Iron-containing enzymes such as heme enzymes play crucial roles in biological systems. Three distinct heme-containing dioxygenase enzymes, tryptophan 2,3-dioxygenase (TDO), indoleamine 2,3-dioxygenase 1 (IDO1) and indoleamine 2,3-dioxygenase 2 (IDO2) catalyze the initial and rate-limiting step of l-tryptophan catabolism through the kynurenine pathway in mammals. Overexpression of these enzymes causes depletion of tryptophan and the accumulation of metabolic products, which contributes to tumor immune tolerance and immune dysregulation in a variety of disease pathologies. In the past few decades, IDO1 has garnered the most attention as a therapeutic target with great potential in cancer immunotherapy. Many potential inhibitors of IDO1 have been designed, synthesized and evaluated, among which indoximod (d-1-MT), INCB024360, GDC-0919 (formerly NLG-919), and an IDO1 peptide-based vaccine have advanced to the clinical trial stage. However, recently, the roles of TDO and IDO2 have been elucidated in immune suppression. In this review, the current drug discovery landscape for targeting TDO, IDO1 and IDO2 is highlighted, with particular attention to the recent use of drugs in clinical trials. Moreover, the crystal structures of these enzymes, in complex with inhibitors, and the mechanisms of Trp catabolism in the first step, are summarized to provide information for facilitating the discovery of new enzyme inhibitors.

Endogenous and xenobiotic metabolic stability of primary human hepatocytes in long-term 3D spheroid cultures revealed by a combination of targeted and untargeted metabolomics

PubMed Central

Vorrink, Sabine U.; Ullah, Shahid; Schmidt, Staffan; Nandania, Jatin; Velagapudi, Vidya; Beck, Olof; Ingelman-Sundberg, Magnus; Lauschke, Volker M.

2017-01-01

Adverse reactions or lack of response to medications are important concerns for drug development programs. However, faithful predictions of drug metabolism and toxicity are difficult because animal models show only limited translatability to humans. Furthermore, current in vitro systems, such as hepatic cell lines or primary human hepatocyte (PHH) 2-dimensional (2D) monolayer cultures, can be used only for acute toxicity tests because of their immature phenotypes and inherent instability. Therefore, the migration to novel phenotypically stable models is of prime importance for the pharmaceutical industry. Novel 3-dimensional (3D) culture systems have been shown to accurately mimic in vivo hepatic phenotypes on transcriptomic and proteomic level, but information about their metabolic stability is lacking. Using a combination of targeted and untargeted high-resolution mass spectrometry, we found that PHHs in 3D spheroid cultures remained metabolically stable for multiple weeks, whereas metabolic patterns of PHHs from the same donors cultured as conventional 2D monolayers rapidly deteriorated. Furthermore, pharmacokinetic differences between donors were maintained in 3D spheroid cultures, enabling studies of interindividual variability in drug metabolism and toxicity. We conclude that the 3D spheroid system is metabolically stable and constitutes a suitable model for in vitro studies of long-term drug metabolism and pharmacokinetics.—Vorrink, S. U., Ullah, S., Schmid, S., Nandania, J., Velagapudi, V., Beck, O., Ingelman-Sundberg, M., Lauschke, V. M. Endogenous and xenobiotic metabolic stability of primary human hepatocytes in long-term 3D spheroid cultures revealed by a combination of targeted and untargeted metabolomics. PMID:28264975

Endogenous and xenobiotic metabolic stability of primary human hepatocytes in long-term 3D spheroid cultures revealed by a combination of targeted and untargeted metabolomics.

PubMed

Vorrink, Sabine U; Ullah, Shahid; Schmidt, Staffan; Nandania, Jatin; Velagapudi, Vidya; Beck, Olof; Ingelman-Sundberg, Magnus; Lauschke, Volker M

2017-06-01

Adverse reactions or lack of response to medications are important concerns for drug development programs. However, faithful predictions of drug metabolism and toxicity are difficult because animal models show only limited translatability to humans. Furthermore, current in vitro systems, such as hepatic cell lines or primary human hepatocyte (PHH) 2-dimensional (2D) monolayer cultures, can be used only for acute toxicity tests because of their immature phenotypes and inherent instability. Therefore, the migration to novel phenotypically stable models is of prime importance for the pharmaceutical industry. Novel 3-dimensional (3D) culture systems have been shown to accurately mimic in vivo hepatic phenotypes on transcriptomic and proteomic level, but information about their metabolic stability is lacking. Using a combination of targeted and untargeted high-resolution mass spectrometry, we found that PHHs in 3D spheroid cultures remained metabolically stable for multiple weeks, whereas metabolic patterns of PHHs from the same donors cultured as conventional 2D monolayers rapidly deteriorated. Furthermore, pharmacokinetic differences between donors were maintained in 3D spheroid cultures, enabling studies of interindividual variability in drug metabolism and toxicity. We conclude that the 3D spheroid system is metabolically stable and constitutes a suitable model for in vitro studies of long-term drug metabolism and pharmacokinetics.-Vorrink, S. U., Ullah, S., Schmid, S., Nandania, J., Velagapudi, V., Beck, O., Ingelman-Sundberg, M., Lauschke, V. M. Endogenous and xenobiotic metabolic stability of primary human hepatocytes in long-term 3D spheroid cultures revealed by a combination of targeted and untargeted metabolomics. © The Author(s).

Metabolic enzyme expression highlights a key role for MTHFD2 and the mitochondrial folate pathway in cancer

NASA Astrophysics Data System (ADS)

Nilsson, Roland; Jain, Mohit; Madhusudhan, Nikhil; Sheppard, Nina Gustafsson; Strittmatter, Laura; Kampf, Caroline; Huang, Jenny; Asplund, Anna; Mootha, Vamsi K.

2014-01-01

Metabolic remodeling is now widely regarded as a hallmark of cancer, but it is not clear whether individual metabolic strategies are frequently exploited by many tumours. Here we compare messenger RNA profiles of 1,454 metabolic enzymes across 1,981 tumours spanning 19 cancer types to identify enzymes that are consistently differentially expressed. Our meta-analysis recovers established targets of some of the most widely used chemotherapeutics, including dihydrofolate reductase, thymidylate synthase and ribonucleotide reductase, while also spotlighting new enzymes, such as the mitochondrial proline biosynthetic enzyme PYCR1. The highest scoring pathway is mitochondrial one-carbon metabolism and is centred on MTHFD2. MTHFD2 RNA and protein are markedly elevated in many cancers and correlated with poor survival in breast cancer. MTHFD2 is expressed in the developing embryo, but is absent in most healthy adult tissues, even those that are proliferating. Our study highlights the importance of mitochondrial compartmentalization of one-carbon metabolism in cancer and raises important therapeutic hypotheses.

SIMULATING METABOLISM TO ENHANCE EFFECTS MODELING

EPA Science Inventory

A major uncertainty that has long been recognized in evaluating chemical toxicity is accounting for metabolic activation of chemicals resulting in increased toxicity. The proposed research will develop a capability for forecasting the metabolism of xenobiotic chemicals of EPA int...

Rhodanese Functions as Sulfur Supplier for Key Enzymes in Sulfur Energy Metabolism

PubMed Central

Aussignargues, Clément; Giuliani, Marie-Cécile; Infossi, Pascale; Lojou, Elisabeth; Guiral, Marianne; Giudici-Orticoni, Marie-Thérèse; Ilbert, Marianne

2012-01-01

How microorganisms obtain energy is a challenging topic, and there have been numerous studies on the mechanisms involved. Here, we focus on the energy substrate traffic in the hyperthermophilic bacterium Aquifex aeolicus. This bacterium can use insoluble sulfur as an energy substrate and has an intricate sulfur energy metabolism involving several sulfur-reducing and -oxidizing supercomplexes and enzymes. We demonstrate that the cytoplasmic rhodanese SbdP participates in this sulfur energy metabolism. Rhodaneses are a widespread family of proteins known to transfer sulfur atoms. We show that SbdP has also some unusual characteristics compared with other rhodaneses; it can load a long sulfur chain, and it can interact with more than one partner. Its partners (sulfur reductase and sulfur oxygenase reductase) are key enzymes of the sulfur energy metabolism of A. aeolicus and share the capacity to use long sulfur chains as substrate. We demonstrate a positive effect of SbdP, once loaded with sulfur chains, on sulfur reductase activity, most likely by optimizing substrate uptake. Taken together, these results lead us to propose a physiological role for SbdP as a carrier and sulfur chain donor to these key enzymes, therefore enabling channeling of sulfur substrate in the cell as well as greater efficiency of the sulfur energy metabolism of A. aeolicus. PMID:22496367

Radiation Exposure Alters Expression of Metabolic Enzyme Genes In Mice

NASA Technical Reports Server (NTRS)

Wotring, Virginia E.; Mangala, L. S.; Zhang, Y.; Wu, H.

2010-01-01

Most pharmaceuticals are metabolized by the liver. The health of the liver, especially the rate of its metabolic enzymes, determines the concentration of circulating drugs as well as the duration of their efficacy. Because of the importance of the liver in drug metabolism it is important to understand the effects of spaceflight on the enzymes of the liver. Exposure to cosmic radiation is one aspect of spaceflight that can be modeled in ground experiments. This study is an effort to examine the effects of adaptive mechanisms that may be triggered by early exposure to low radiation doses. Using procedures approved by the JSC Animal Care & Use Committee, C57 male mice were exposed to Cs-137 in groups: controls, low dose (50 mGy), high dose (6Gy) and a fourth group that received both radiation doses separated by 24 hours. Animals were anesthetized and sacrificed 4 hours after their last radiation exposure. Livers were removed immediately and flash-frozen in liquid nitrogen. Tissue was homogenized, RNA extracted and purified (Absolutely RNA, Agilent). Quality of RNA samples was evaluated (Agilent Bioanalyzer 2100). Complementary DNA was prepared from high-quality RNA samples, and used to run RT-qPCR screening arrays for DNA Repair and Drug Metabolism (SuperArray, SABiosciences/Qiagen; BioRad Cfx96 qPCR System). Of 91 drug metabolism genes examined, expression of 7 was altered by at least one treatment condition. Genes that had elevated expression include those that metabolize promethazine and steroids (4-8-fold), many that reduce oxidation products, and one that reduces heavy metal exposure (greater than 200-fold). Of the 91 DNA repair and general metabolism genes examined, expression of 14 was altered by at least one treatment condition. These gene expression changes are likely homeostatic and could lead to development of new radioprotective countermeasures.

Extent of cutaneous metabolism during percutaneous absorption of xenobiotics.

PubMed

Bronaugh, R L; Stewart, R F; Storm, J E

1989-07-01

In vitro percutaneous absorption studies generally do not determine whether biotransformation occurs during passage of a substance through the skin. Since it has recently been demonstrated that several chemicals are metabolized during skin permeation, we investigated the metabolism of five additional compounds (14C-labeled) after application to fuzzy rat skin: caffeine, p,p'-DDT, butylated hydroxytoluene (BHT), salicylic acid, and acetyl ethyl tetramethyltetralin (AETT). The viability of skin was maintained with a tissue culture medium. Radioactivity of each substrate and any metabolites in skin and receptor fluid was measured so that the absorption and metabolism of water-insoluble compounds would be accurately determined. Percutaneous absorption ranged from a low of 13% of the applied dose for BHT to a high of 49% for DDT. BHT was metabolized in skin to 4-hydroxy-BHT and an unknown metabolite. Of the absorbed radioisotope, 6.6% was isolated in biotransformed products found mainly in the receptor fluid. AETT was also metabolized during absorption, with 1.9% of the absorbed radioisotope found in two unknown peaks. Caffeine, DDT, and salicylic acid were not metabolized during skin permeation. Skin and liver microsomal metabolism was measured for all compounds except DDT. Metabolism in skin was observed only for the compounds also biotransformed in the diffusion cell; BHT and AETT were metabolized at 113 and 2.5 pmol/min/mg protein, respectively. In this study, as in others, skin metabolism was substantially less than the corresponding metabolism in liver. Therefore, a low rate of liver metabolism such as that found for caffeine, salicylic acid, and DDT might often be predictive of the absence of measurable metabolism during skin permeation. It seems likely that for many compounds, the biotransformations in skin will be small in terms of the percentage of absorbed material that is metabolized. Nevertheless, with potent compounds, even small quantities of a metabolite

Expression of Enzymes that Metabolize Medications

NASA Technical Reports Server (NTRS)

Wotring, V. E.; Peters, C. P.

2011-01-01

INTRODUCTION: Increased exposure to radiation is one physiological stressor associated with spaceflight and it is feasible to conduct ground experiments using known radiation exposures. The health of the liver, especially the activity rate of its metabolic enzymes, determines the concentration of circulating drugs as well as the duration of their efficacy. While radiation is known to alter normal physiological function, how radiation affects liver metabolism of administered medications is unclear. Crew health could be affected if the actions of medications used in spaceflight deviated from expectations formed during terrestrial medication use. This study is an effort to identify liver metabolic enzymes whose expression is altered by spaceflight or by radiation exposures that mimic features of the spaceflight environment. METHODS: Using procedures approved by the Animal Care and Use Committee, mice were exposed to either 137Cs (controls, 50 mGy, 6Gy, or 50 mGy + 6Gy separated by 24 hours) or 13 days of spaceflight on STS 135. Animals were anesthetized and sacrificed at several time points (4 hours, 24 hours or 7 days) after their last radiation exposure, or within 6 hours of return to Earth for the STS 135 animals. Livers were removed immediately and flash-frozen in liquid nitrogen. Tissue was homogenized, RNA extracted, purified and quality-tested. Complementary DNA was prepared from high-quality RNA samples, and used in RT-qPCR experiments to determine relative expression of a wide variety of genes involved in general metabolism and drug metabolism. RESULTS: Results of the ground radiation exposure experiments indicated 65 genes of the 190 tested were significantly affected by at least one of the radiation doses. Many of the affected genes are involved in the metabolism of drugs with hydrophobic or steroid-like structures, maintenance of redox homeostasis and repair of DNA damage. Most affected genes returned to near control expression levels by 7 days post

Comparative and integrative metabolomics reveal that S-nitrosation inhibits physiologically relevant metabolic enzymes.

PubMed

Bruegger, Joel J; Smith, Brian C; Wynia-Smith, Sarah L; Marletta, Michael A

2018-04-27

Cysteine S -nitrosation is a reversible post-translational modification mediated by nitric oxide ( • NO)-derived agents. S -Nitrosation participates in cellular signaling and is associated with several diseases such as cancer, cardiovascular diseases, and neuronal disorders. Despite the physiological importance of this nonclassical • NO-signaling pathway, little is understood about how much S -nitrosation affects protein function. Moreover, identifying physiologically relevant targets of S -nitrosation is difficult because of the dynamics of transnitrosation and a limited understanding of the physiological mechanisms leading to selective protein S -nitrosation. To identify proteins whose activities are modulated by S -nitrosation, we performed a metabolomics study comparing WT and endothelial nitric-oxide synthase knockout mice. We integrated our results with those of a previous proteomics study that identified physiologically relevant S -nitrosated cysteines, and we found that the activity of at least 21 metabolic enzymes might be regulated by S -nitrosation. We cloned, expressed, and purified four of these enzymes and observed that S -nitrosation inhibits the metabolic enzymes 6-phosphogluconate dehydrogenase, Δ1-pyrroline-5-carboxylate dehydrogenase, catechol- O -methyltransferase, and d-3-phosphoglycerate dehydrogenase. Furthermore, using site-directed mutagenesis, we identified the predominant cysteine residue influencing the observed activity changes in each enzyme. In summary, using an integrated metabolomics approach, we have identified several physiologically relevant S -nitrosation targets, including metabolic enzymes, which are inhibited by this modification, and we have found the cysteines modified by S -nitrosation in each enzyme. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

The Aldo-Keto Reductase Superfamily and its Role in Drug Metabolism and Detoxification

PubMed Central

Barski, Oleg A.; Tipparaju, Srinivas M.; Bhatnagar, Aruni

2008-01-01

The Aldo-Keto Reductase (AKR) superfamily comprises of several enzymes that catalyze redox transformations involved in biosynthesis, intermediary metabolism and detoxification. Substrates of the family include glucose, steroids, glycosylation end products, lipid peroxidation products, and environmental pollutants. These proteins adopt a (β/α)8 barrel structural motif interrupted by a number of extraneous loops and helixes that vary between proteins and bring structural identity to individual families. The human AKR family differs from the rodent families. Due to their broad substrate specificity, AKRs play an important role in the Phase II detoxification of a large number of pharmaceuticals, drugs, and xenobiotics. PMID:18949601

Regional variation in muscle metabolic enzymes in individual American shad (Alosa sapidissima)

USGS Publications Warehouse

Leonard, J.B.K.

1999-01-01

Evaluation of the activity of metabolic enzymes is often used to asses metabolic capacity at the tissue level, but the amount of regional variability within a tissue in an individual fish of a given species is frequently unknown. The activities of four enzymes (citrate synthase (CS), phosphofructokinase, lactate dehydrogenase (LDH), and ??-hydroxyacyl coenzyme A dehydrogenase (HOAD) were assayed in red and white muscle at 10 sites along the body of adult American shad (Alosa sapidissima). Red and white muscle HOAD and white muscle CS and LDH varied significantly, generally increasing posteriorly. Maximal variation occurs in red muscle HOAD (~450%) and white muscle LDH (~60%) activity. Differences between the sexes also vary with sampling location. This study suggests that the variability in enzyme activity may be linked to functional differences in the muscle at different locations, and also provides guidelines for sample collection in this species.

Influence of multidrug resistance and drug transport proteins on chemotherapy drug metabolism.

PubMed

Joyce, Helena; McCann, Andrew; Clynes, Martin; Larkin, Annemarie

2015-05-01

Chemotherapy involving the use of anticancer drugs remains an important strategy in the overall management of patients with metastatic cancer. Acquisition of multidrug resistance remains a major impediment to successful chemotherapy. Drug transporters in cell membranes and intracellular drug metabolizing enzymes contribute to the resistance phenotype and determine the pharmacokinetics of anticancer drugs in the body. ATP-binding cassette (ABC) transporters mediate the transport of endogenous metabolites and xenobiotics including cytotoxic drugs out of cells. Solute carrier (SLC) transporters mediate the influx of cytotoxic drugs into cells. This review focuses on the substrate interaction of these transporters, on their biology and what role they play together with drug metabolizing enzymes in eliminating therapeutic drugs from cells. The majority of anticancer drugs are substrates for the ABC transporter and SLC transporter families. Together, these proteins have the ability to control the influx and the efflux of structurally unrelated chemotherapeutic drugs, thereby modulating the intracellular drug concentration. These interactions have important clinical implications for chemotherapy because ultimately they determine therapeutic efficacy, disease progression/relapse and the success or failure of patient treatment.

Tropinone reductases, enzymes at the branch point of tropane alkaloid metabolism.

PubMed

Dräger, Birgit

2006-02-01

Two stereospecific oxidoreductases constitute a branch point in tropane alkaloid metabolism. Products of tropane metabolism are the alkaloids hyoscyamine, scopolamine, cocaine, and polyhydroxylated nortropane alkaloids, the calystegines. Both tropinone reductases reduce the precursor tropinone to yield either tropine or pseudotropine. In Solanaceae, tropine is incorporated into hyoscyamine and scopolamine; pseudotropine is the first specific metabolite on the way to the calystegines. Isolation, cloning and heterologous expression of both tropinone reductases enabled kinetic characterisation, protein crystallisation, and structure elucidation. Stereospecificity of reduction is achieved by binding tropinone in the respective enzyme active centre in opposite orientation. Immunolocalisation of both enzyme proteins in cultured roots revealed a tissue-specific protein accumulation. Metabolite flux through both arms of the tropane alkaloid pathway appears to be regulated by the activity of both enzymes and by their access to the precursor tropinone. Both tropinone reductases are NADPH-dependent short-chain dehydrogenases with amino acid sequence similarity of more than 50% suggesting their descent from a common ancestor. Putative tropinone reductase sequences annotated in plant genomes other that Solanaceae await functional characterisation.

Xenobiotic Compounds Degradation by Heterologous Expression of a Trametes sanguineus Laccase in Trichoderma atroviride

PubMed Central

Balcázar-López, Edgar; Méndez-Lorenzo, Luz Helena; Batista-García, Ramón Alberto; Esquivel-Naranjo, Ulises; Ayala, Marcela; Kumar, Vaidyanathan Vinoth; Savary, Olivier; Cabana, Hubert; Herrera-Estrella, Alfredo; Folch-Mallol, Jorge Luis

2016-01-01

Fungal laccases are enzymes that have been studied because of their ability to decolorize and detoxify effluents; they are also used in paper bleaching, synthesis of polymers, bioremediation, etc. In this work we were able to express a laccase from Trametes (Pycnoporus) sanguineus in the filamentous fungus Trichoderma atroviride. For this purpose, a transformation vector was designed to integrate the gene of interest in an intergenic locus near the blu17 terminator region. Although monosporic selection was still necessary, stable integration at the desired locus was achieved. The native signal peptide from T. sanguineus laccase was successful to secrete the recombinant protein into the culture medium. The purified, heterologously expressed laccase maintained similar properties to those observed in the native enzyme (Km and kcat and kcat/km values for ABTS, thermostability, substrate range, pH optimum, etc). To determine the bioremediation potential of this modified strain, the laccase-overexpressing Trichoderma strain was used to remove xenobiotic compounds. Phenolic compounds present in industrial wastewater and bisphenol A (an endocrine disruptor) from the culture medium were more efficiently removed by this modified strain than with the wild type. In addition, the heterologously expressed laccase was able to decolorize different dyes as well as remove benzo[α]pyrene and phenanthrene in vitro, showing its potential for xenobiotic compound degradation. PMID:26849129

Thiamin diphosphate-dependent enzymes: from enzymology to metabolic regulation, drug design and disease models.

PubMed

Bunik, Victoria I; Tylicki, Adam; Lukashev, Nikolay V

2013-12-01

Bringing a knowledge of enzymology into research in vivo and in situ is of great importance in understanding systems biology and metabolic regulation. The central metabolic significance of thiamin (vitamin B1 ) and its diphosphorylated derivative (thiamin diphosphate; ThDP), and the fundamental differences in the ThDP-dependent enzymes of metabolic networks in mammals versus plants, fungi and bacteria, or in health versus disease, suggest that these enzymes are promising targets for biotechnological and medical applications. Here, the in vivo action of known regulators of ThDP-dependent enzymes, such as synthetic structural analogs of the enzyme substrates and thiamin, is analyzed in light of the enzymological data accumulated during half a century of research. Mimicking the enzyme-specific catalytic intermediates, the phosphonate analogs of 2-oxo acids selectively inhibit particular ThDP-dependent enzymes. Because of their selectivity, use of these compounds in cellular and animal models of ThDP-dependent enzyme malfunctions improves the validity of the model and its predictive power when compared with the nonselective and enzymatically less characterized oxythiamin and pyrithiamin. In vitro studies of the interaction of thiamin analogs and their biological derivatives with potential in vivo targets are necessary to identify and attenuate the analog selectivity. For both the substrate and thiamin synthetic analogs, in vitro reactivities with potential targets are highly relevant in vivo. However, effective concentrations in vivo are often higher than in vitro studies would suggest. The significance of specific inihibition of the ThDP-dependent enzymes for the development of herbicides, antibiotics, anticancer and neuroprotective strategies is discussed. © 2013 FEBS.

Applications of mass spectrometry in drug metabolism: 50 years of progress.

PubMed

Wen, Bo; Zhu, Mingshe

2015-02-01

Mass spectrometry plays a pivotal role in drug metabolism studies, which are an integral part of drug discovery and development nowadays. Metabolite identification has become critical to understanding the metabolic fate of drug candidates and to aid lead optimization with improved metabolic stability, toxicology and efficacy profiles. Ever since the introduction of atmospheric ionization techniques in the early 1990s, liquid chromatography coupled with mass spectrometry (LC/MS) has secured a central role as the predominant analytical platform for metabolite identification as LC and MS technologies continually advanced. In this review, we discuss the evolution of both MS technology and its applications over the past 50 years to meet the increasing demand of drug metabolism studies. These advances include ionization sources, mass analyzers, a wide range of MS acquisition strategies and data mining tools that have substantially accelerated the metabolite identification process and changed the overall drug metabolism landscape. Exemplary applications for characterization and identification of both small-molecule xenobiotics and biological macromolecules are described. In addition, this review discusses novel MS technologies and applications, including xenobiotic metabolomics that hold additional promise for advancing drug metabolism research, and offers thoughts on remaining challenges in studying the metabolism and disposition of drugs and other xenobiotics.

Lycopene, resveratrol, vitamin C and FeSO4 increase damage produced by pro-oxidant carcinogen 4-nitroquinoline-1-oxide in Drosophila melanogaster: Xenobiotic metabolism implications.

PubMed

Dueñas-García, I E; Heres-Pulido, M E; Arellano-Llamas, M R; De la Cruz-Núñez, J; Cisneros-Carrillo, V; Palacios-López, C S; Acosta-Anaya, L; Santos-Cruz, L F; Castañeda-Partida, L; Durán-Díaz, A

2017-05-01

4-nitroquinoline-1-oxide (4-NQO) is a pro-oxidant carcinogen bioactivated by xenobiotic metabolism (XM). We investigated if antioxidants lycopene [0.45, 0.9, 1.8 μM], resveratrol [11, 43, 172 μM], and vitamin C [5.6 mM] added or not with FeSO 4 [0.06 mM], modulate the genotoxicity of 4-NQO [2 mM] with the Drosophila wing spot test standard (ST) and high bioactivation (HB) crosses, with inducible and high levels of cytochromes P450, respectively. The genotoxicity of 4-NQO was higher when dissolved in an ethanol - acetone mixture. The antioxidants did not protect against 4-NQO in any of both crosses. In the ST cross, resveratrol [11 μM], vitamin C and FeSO 4 resulted in genotoxicity; the three antioxidants and FeSO 4 increased the damage of 4-NQO. In the HB cross, none of the antioxidants, neither FeSO 4 , were genotoxic. Only resveratrol [172 μM] + 4-NQO increased the genotoxic activity in both crosses. We concluded that the effects of the antioxidants, FeSO 4 and the modulation of 4-NQO were the result of the difference of Cyp450s levels, between the ST and HB crosses. We propose that the basal levels of the XM's enzymes in the ST cross interacted with a putative pro-oxidant activity of the compounds added to the pro-oxidant effects of 4-NQO. Copyright © 2017. Published by Elsevier Ltd.

Disposition of xenobiotic chemicals and metabolites in marine organisms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Varanasi, U.; Stein, J.E.

1991-01-01

Studies with several bottom fish species from urban waterways show that of the identified xenobiotic chemicals in bottom sediments, polycyclic aromatic hydrocarbons (PAHs) are the most strongly associated with the prevalence of liver lesions, including neoplasms. Accordingly, there is concern about the transfer of contaminants, such as PAHs, from aquatic species to humans. Because PAHs exert their toxicity only after being biotransformed, increasing attention has been focused on the ability of aquatic organisms to metabolize these chemicals. Overall, the results of both laboratory and field studies show that generally low levels of a few low molecular weight PAHs may bemore » present in edible tissue of fish from contaminated areas and that high molecular weight PAHs, such as the carcinogen benzo(a)pyrene, will rarely be detected because of extensive metabolism. Additionally, the results from a few studies suggest that even though interactions between xenobiotics can affect both biochemical and physiological systems to alter the disposition of PAHs in fish, these interactions do not markedly change the relative proportions of metabolites to parent PAH in tissues. Thus, these studies clearly demonstrate that to obtain some insight into the questions of whether there is any risk to human health from consuming fish and crustaceans from urban areas, techniques must be developed that measure metabolites of carcinogens, such as PAHs, in edible tissue. Initial attempts may focus on semiquantitative methods that permit rapid assessment of the level of metabolites in edible tissues of fish and crustaceans from many urban areas. Based on information from such screening studies, further refinement in methodology leading to identification of specific compounds may be needed because certain metabolites may not be as toxic or carcinogenic as others.« less

Xanthine Oxidoreductase in Drug Metabolism: Beyond a Role as a Detoxifying Enzyme.

PubMed

Battelli, Maria Giulia; Polito, Letizia; Bortolotti, Massimo; Bolognesi, Andrea

2016-01-01

The enzyme xanthine oxidoreductase (XOR) catalyzes the last two steps of purine catabolism in the highest uricotelic primates. XOR is an enzyme with dehydrogenase activity that, in mammals, may be converted into oxidase activity under a variety of pathophysiologic conditions. XOR activity is highly regulated at the transcriptional and post-translational levels and may generate reactive oxygen and nitrogen species, which trigger different consequences, ranging from cytotoxicity to inflammation. The low specificity for substrates allows XOR to metabolize a number of endogenous metabolites and a variety of exogenous compounds, including drugs. The present review focuses on the role of XOR as a drug-metabolizing enzyme, specifically for drugs with anticancer, antimicrobial, antiviral, immunosuppressive or vasodilator activities, as well as drugs acting on metabolism or inducing XOR expression. XOR has an activating role that is essential to the pharmacological action of quinone drugs, cyadox, antiviral nucleoside analogues, allopurinol, nitrate and nitrite. XOR activity has a degradation function toward thiopurine nucleotides, pyrazinoic acid, methylxanthines and tolbutamide, whose half-life may be prolonged by the use of XOR inhibitors. In conclusion, to avoid potential drug interaction risks, such as a toxic excess of drug bioavailability or a loss of drug efficacy, caution is suggested in the use of XOR inhibitors, as in the case of hyperuricemic patients affected by gout or tumor lysis syndrome, when it is necessary to simultaneously administer therapeutic substances that are activated or degraded by the drug-metabolizing activity of XOR.

Oxidation and adduct formation of xenobiotics in a microfluidic electrochemical cell with boron doped diamond electrodes and an integrated passive gradient rotation mixer.

PubMed

van den Brink, Floris T G; Wigger, Tina; Ma, Liwei; Odijk, Mathieu; Olthuis, Wouter; Karst, Uwe; van den Berg, Albert

2016-10-05

Reactive xenobiotic metabolites and their adduct formation with biomolecules such as proteins are important to study as they can be detrimental to human health. Here, we present a microfluidic electrochemical cell with integrated micromixer to study phase I and phase II metabolism as well as protein adduct formation of xenobiotics in a purely instrumental approach. The newly developed microfluidic device enables both the generation of reactive metabolites through electrochemical oxidation and subsequent adduct formation with biomolecules in a chemical microreactor. This allows us to study the detoxification of reactive species with glutathione and to predict potential toxicity of xenobiotics as a result of protein modification. Efficient mixing in microfluidic systems is a slow process due to the typically laminar flow conditions in shallow channels. Therefore, a passive gradient rotation micromixer has been designed that is capable of mixing liquids efficiently in a 790 pL volume within tens of milliseconds. The mixing principle relies on turning the concentration gradient that is initially established by bringing together two streams of liquid, to take advantage of the short diffusion distances in the shallow microchannels of thin-layer flow cells. The mixer is located immediately downstream of the working electrode of an electrochemical cell with integrated boron doped diamond electrodes. In conjunction with mass spectrometry, the two microreactors integrated in a single device provide a powerful tool to study the metabolism and toxicity of xenobiotics, which was demonstrated by the investigation of the model compound 1-hydroxypyrene.

Effects of a Model Inducer, Phenobarbital, on Thyroid Hormone Glucuronidation in Rat Hepatocytes

EPA Science Inventory

In vivo, hepatic enzyme inducers such as phenobarbital (PB) decrease circulating thyroid hormone (TH) concentrations. This decrease in circulating TH occurs in part through extrathyroidal mechanisms. Specifically, through the induction of hepatic xenobiotic metabolizing enzymes...

DrugBank 4.0: shedding new light on drug metabolism

PubMed Central

Law, Vivian; Knox, Craig; Djoumbou, Yannick; Jewison, Tim; Guo, An Chi; Liu, Yifeng; Maciejewski, Adam; Arndt, David; Wilson, Michael; Neveu, Vanessa; Tang, Alexandra; Gabriel, Geraldine; Ly, Carol; Adamjee, Sakina; Dame, Zerihun T.; Han, Beomsoo; Zhou, You; Wishart, David S.

2014-01-01

DrugBank (http://www.drugbank.ca) is a comprehensive online database containing extensive biochemical and pharmacological information about drugs, their mechanisms and their targets. Since it was first described in 2006, DrugBank has rapidly evolved, both in response to user requests and in response to changing trends in drug research and development. Previous versions of DrugBank have been widely used to facilitate drug and in silico drug target discovery. The latest update, DrugBank 4.0, has been further expanded to contain data on drug metabolism, absorption, distribution, metabolism, excretion and toxicity (ADMET) and other kinds of quantitative structure activity relationships (QSAR) information. These enhancements are intended to facilitate research in xenobiotic metabolism (both prediction and characterization), pharmacokinetics, pharmacodynamics and drug design/discovery. For this release, >1200 drug metabolites (including their structures, names, activity, abundance and other detailed data) have been added along with >1300 drug metabolism reactions (including metabolizing enzymes and reaction types) and dozens of drug metabolism pathways. Another 30 predicted or measured ADMET parameters have been added to each DrugCard, bringing the average number of quantitative ADMET values for Food and Drug Administration-approved drugs close to 40. Referential nuclear magnetic resonance and MS spectra have been added for almost 400 drugs as well as spectral and mass matching tools to facilitate compound identification. This expanded collection of drug information is complemented by a number of new or improved search tools, including one that provides a simple analyses of drug–target, –enzyme and –transporter associations to provide insight on drug–drug interactions. PMID:24203711

DrugBank 4.0: shedding new light on drug metabolism.

PubMed

Law, Vivian; Knox, Craig; Djoumbou, Yannick; Jewison, Tim; Guo, An Chi; Liu, Yifeng; Maciejewski, Adam; Arndt, David; Wilson, Michael; Neveu, Vanessa; Tang, Alexandra; Gabriel, Geraldine; Ly, Carol; Adamjee, Sakina; Dame, Zerihun T; Han, Beomsoo; Zhou, You; Wishart, David S

2014-01-01

DrugBank (http://www.drugbank.ca) is a comprehensive online database containing extensive biochemical and pharmacological information about drugs, their mechanisms and their targets. Since it was first described in 2006, DrugBank has rapidly evolved, both in response to user requests and in response to changing trends in drug research and development. Previous versions of DrugBank have been widely used to facilitate drug and in silico drug target discovery. The latest update, DrugBank 4.0, has been further expanded to contain data on drug metabolism, absorption, distribution, metabolism, excretion and toxicity (ADMET) and other kinds of quantitative structure activity relationships (QSAR) information. These enhancements are intended to facilitate research in xenobiotic metabolism (both prediction and characterization), pharmacokinetics, pharmacodynamics and drug design/discovery. For this release, >1200 drug metabolites (including their structures, names, activity, abundance and other detailed data) have been added along with >1300 drug metabolism reactions (including metabolizing enzymes and reaction types) and dozens of drug metabolism pathways. Another 30 predicted or measured ADMET parameters have been added to each DrugCard, bringing the average number of quantitative ADMET values for Food and Drug Administration-approved drugs close to 40. Referential nuclear magnetic resonance and MS spectra have been added for almost 400 drugs as well as spectral and mass matching tools to facilitate compound identification. This expanded collection of drug information is complemented by a number of new or improved search tools, including one that provides a simple analyses of drug-target, -enzyme and -transporter associations to provide insight on drug-drug interactions.

The BRG1 chromatin remodeling enzyme links cancer cell metabolism and proliferation

PubMed Central

Wu, Qiong; Madany, Pasil; Dobson, Jason R.; Schnabl, Jake M.; Sharma, Soni; Smith, Tara C.; van Wijnen, Andre J.; Stein, Janet L.; Lian, Jane B.; Stein, Gary S.; Muthuswami, Rohini; Imbalzano, Anthony N.; Nickerson, Jeffrey A.

2016-01-01

Cancer cells reprogram cellular metabolism to meet the demands of growth. Identification of the regulatory machinery that regulates cancer-specific metabolic changes may open new avenues for anti-cancer therapeutics. The epigenetic regulator BRG1 is a catalytic ATPase for some mammalian SWI/SNF chromatin remodeling enzymes. BRG1 is a well-characterized tumor suppressor in some human cancers, but is frequently overexpressed without mutation in other cancers, including breast cancer. Here we demonstrate that BRG1 upregulates de novo lipogenesis and that this is crucial for cancer cell proliferation. Knockdown of BRG1 attenuates lipid synthesis by impairing the transcription of enzymes catalyzing fatty acid and lipid synthesis. Remarkably, exogenous addition of palmitate, the key intermediate in fatty acid synthesis, rescued the cancer cell proliferation defect caused by BRG1 knockdown. Our work suggests that targeting BRG1 to reduce lipid metabolism and, thereby, to reduce proliferation, has promise for epigenetic therapy in triple negative breast cancer. PMID:27223259

Some enzymes of carbohydrate metabolism in Mesocestoides corti and Heterakis spumosa.

PubMed

Dubinský, P; Ruscinová, B; Hetmanski, S L; Arme, C; Turceková, L; Rybos, M

1991-09-01

The activities of selected enzymes of carbohydrate metabolism were measured in tetrathyridia of Mesocestoides corti and in adult females and males of Heterakis spumosa. When the species were compared, only lactate dehydrogenase and phosphoenolpyruvate carboxykinase activities were considerably higher in M. corti. Activities of other enzymes were higher in H. spumosa, with malate dehydrogenase activity being considerably so. In H. spumosa, enzyme activity was higher, and succinate dehydrogenase markedly so in males, when compared with females. Tetrathyridia aged 170 and 210 days show relatively stable malate and lactate dehydrogenase activities, and mice of ICR and BALB/c strains are suitable for the maintenance of tetrathyridia.

Highlighting the Need for Systems-Level Experimental Characterization of Plant Metabolic Enzymes.

PubMed

Engqvist, Martin K M

2016-01-01

The biology of living organisms is determined by the action and interaction of a large number of individual gene products, each with specific functions. Discovering and annotating the function of gene products is key to our understanding of these organisms. Controlled experiments and bioinformatic predictions both contribute to functional gene annotation. For most species it is difficult to gain an overview of what portion of gene annotations are based on experiments and what portion represent predictions. Here, I survey the current state of experimental knowledge of enzymes and metabolism in Arabidopsis thaliana as well as eleven economically important crops and forestry trees - with a particular focus on reactions involving organic acids in central metabolism. I illustrate the limited availability of experimental data for functional annotation of enzymes in most of these species. Many enzymes involved in metabolism of citrate, malate, fumarate, lactate, and glycolate in crops and forestry trees have not been characterized. Furthermore, enzymes involved in key biosynthetic pathways which shape important traits in crops and forestry trees have not been characterized. I argue for the development of novel high-throughput platforms with which limited functional characterization of gene products can be performed quickly and relatively cheaply. I refer to this approach as systems-level experimental characterization. The data collected from such platforms would form a layer intermediate between bioinformatic gene function predictions and in-depth experimental studies of these functions. Such a data layer would greatly aid in the pursuit of understanding a multiplicity of biological processes in living organisms.

Effects of phenobarbital on thyroid hormone contabolism in rat hepatocytes

EPA Science Inventory

Hepatic enzyme inducers such as phenobarbital (PB) decrease circulating thyroid hormone (TH) concentrations in rodents. PB induction of hepatic xenobiotic metabolizing enzymes increases thyroid hormones catabolism and biliary elimination. This study examines the catabolism and cl...

Metabolic enzyme microarray coupled with miniaturized cell-culture array technology for high-throughput toxicity screening.

PubMed

Lee, Moo-Yeal; Dordick, Jonathan S; Clark, Douglas S

2010-01-01

Due to poor drug candidate safety profiles that are often identified late in the drug development process, the clinical progression of new chemical entities to pharmaceuticals remains hindered, thus resulting in the high cost of drug discovery. To accelerate the identification of safer drug candidates and improve the clinical progression of drug candidates to pharmaceuticals, it is important to develop high-throughput tools that can provide early-stage predictive toxicology data. In particular, in vitro cell-based systems that can accurately mimic the human in vivo response and predict the impact of drug candidates on human toxicology are needed to accelerate the assessment of drug candidate toxicity and human metabolism earlier in the drug development process. The in vitro techniques that provide a high degree of human toxicity prediction will be perhaps more important in cosmetic and chemical industries in Europe, as animal toxicity testing is being phased out entirely in the immediate future.We have developed a metabolic enzyme microarray (the Metabolizing Enzyme Toxicology Assay Chip, or MetaChip) and a miniaturized three-dimensional (3D) cell-culture array (the Data Analysis Toxicology Assay Chip, or DataChip) for high-throughput toxicity screening of target compounds and their metabolic enzyme-generated products. The human or rat MetaChip contains an array of encapsulated metabolic enzymes that is designed to emulate the metabolic reactions in the human or rat liver. The human or rat DataChip contains an array of 3D human or rat cells encapsulated in alginate gels for cell-based toxicity screening. By combining the DataChip with the complementary MetaChip, in vitro toxicity results are obtained that correlate well with in vivo rat data.

Pharmacogenetics of drug-metabolizing enzymes in US Hispanics

PubMed Central

Duconge, Jorge; Cadilla, Carmen L.; Ruaño, Gualberto

2015-01-01

Although the Hispanic population is continuously growing in the United States, they are underrepresented in pharmacogenetic studies. This review addresses the need for compiling available pharmacogenetic data in US Hispanics, discussing the prevalence of clinically relevant polymorphisms in pharmacogenes encoding for drug-metabolizing enzymes. CYP3A5*3 (0.245–0.867) showed the largest frequency in a US Hispanic population. A higher prevalence of CYP2C9*3, CYP2C19*4, and UGT2B7 IVS1+985 A>Gwas observed in US Hispanic vs. non-Hispanic populations. We found interethnic and intraethnic variability in frequencies of genetic polymorphisms for metabolizing enzymes, which highlights the need to define the ancestries of participants in pharmacogenetic studies. New approaches should be integrated in experimental designs to gain knowledge about the clinical relevance of the unique combination of genetic variants occurring in this admixed population. Ethnic subgroups in the US Hispanic population may harbor variants that might be part of multiple causative loci or in linkage-disequilibrium with functional variants. Pharmacogenetic studies in Hispanics should not be limited to ascertain commonly studied polymorphisms that were originally identified in their parental populations. The success of the Personalized Medicine paradigm will depend on recognizing genetic diversity between and within US Hispanics and the uniqueness of their genetic backgrounds. PMID:25431893

Metabolism of clofibric acid in zebrafish embryos (Danio rerio) as determined by liquid chromatography-high resolution-mass spectrometry.

PubMed

Brox, Stephan; Seiwert, Bettina; Haase, Nora; Küster, Eberhard; Reemtsma, Thorsten

2016-01-01

The zebrafish embryo (ZFE) is increasingly used in ecotoxicology research but detailed knowledge of its metabolic potential is still limited. This study focuses on the xenobiotic metabolism of ZFE at different life-stages using the pharmaceutical compound clofibric acid as study compound. Liquid chromatography with quadrupole-time-of-flight mass spectrometry (LC-QToF-MS) is used to detect and to identify the transformation products (TPs). In screening experiments, a total of 18 TPs was detected and structure proposals were elaborated for 17 TPs, formed by phase I and phase II metabolism. Biotransformation of clofibric acid by the ZFE involves conjugation with sulfate or glucuronic acid, and, reported here for the first time, with carnitine, taurine, and aminomethanesulfonic acid. Further yet unknown cyclization products were identified using non-target screening that may represent a new detoxification pathway. Sulfate containing TPs occurred already after 3h of exposure (7hpf), and from 48h of exposure (52hpf) onwards, all TPs were detected. The detection of these TPs indicates the activity of phase I and phase II enzymes already at early life-stages. Additionally, the excretion of one TP into the exposure medium was observed. The results of this study outline the high metabolic potential of the ZFE with respect to the transformation of xenobiotics. Similarities but also differences to other test systems were observed. Biotransformation of test chemicals in toxicity testing with ZFE may therefore need further consideration. Copyright © 2016 Elsevier Inc. All rights reserved.

Etiological classification of depression based on the enzymes of tryptophan metabolism.

PubMed

Fukuda, Katsuhiko

2014-12-24

Viewed in terms of input and output, the mechanisms of depression are still akin to a black box. However, there must be main pivots for diverse types of depression. From recent therapeutic observations, both the serotonin (5-HT) and kynurenine pathways of tryptophan metabolism may be of particular importance to improved understanding of depression. Here, I propose an etiological classification of depression, based on key peripheral and central enzymes of tryptophan metabolism. Endogenous depression is caused by a larger genetic component than reactive depression. Besides enterochromaffin and mast cells, tryptophan hydroxylase 1 (TPH1), primarily expressed in the gastrointestinal tract, is also found in 5-hydroxytryptophan-producing cells (5-HTP cells) in normal intestinal enterocytes, which are thought to essentially shunt 5-HT production in 5-HT-producing cells. Genetic studies have reported an association between TPH1 and depression, or the responsiveness of depression to antidepressive medication. Therefore, it is possible that hypofunctional 5-HTP cells (reflecting TPH1 dysfunction) in the periphery lead to deficient brain 5-HT levels. Additionally,it has been reported that higher TPH2 expression in depressed suicides may reflect a homeostatic response to deficient 5-HT levels. Subsequently, endogenous depression may be caused by TPH1 dysfunction combined with compensatory TPH2 activation. Reactive depression results from life stresses and involves the hypothalamic-pituitary-adrenal axis, with resulting cortisol production inducing tryptophan 2,3-dioxygenase (TDO) activation. In secondary depression, caused by inflammation, infection, or oxidative stress, indoleamine 2,3-dioxygenase (IDO) is activated. In both reactive and secondary depression, the balance between 3-hydroxykynurenine (3-HK) and kynurenic acid may shift towards 3-HK production via kynurenine-3-monooxygenase (KMO) activation. By shifting the equilibrium position of key enzymes of tryptophan

The Effects of Pharmaceutical Excipients on Gastrointestinal Tract Metabolic Enzymes and Transporters-an Update.

PubMed

Zhang, Wenpeng; Li, Yanyan; Zou, Peng; Wu, Man; Zhang, Zhenqing; Zhang, Tao

2016-07-01

Accumulating evidence from the last decade has shown that many pharmaceutical excipients are not pharmacologically inert but instead have effects on metabolic enzymes and/or drug transporters. Hence, the absorption, distribution, metabolism, and elimination (ADME) of active pharmaceutical ingredients (APIs) may be altered due to the modulation of their metabolism and transport by excipients. The impact of excipients is a potential concern for Biopharmaceutics Classification System (BCS)-based biowaivers, particularly as the BCS-based biowaivers have been extended to class 3 drugs in certain dosage forms. The presence of different excipients or varying amounts of excipients between formulations may result in bio-inequivalence. The excipient impact may lead to significant variations in clinical outcomes as well. The aim of this paper is to review the recent findings of excipient effects on gastrointestinal (GI) absorption, focusing on their interactions with the metabolic enzymes and transporters in the GI tract. A wide range of commonly used excipients such as binders, diluents, fillers, solvents, and surfactants are discussed here. We summarized the reported effects of those excipients on GI tract phase I and phase II enzymes, uptake and efflux transporters, and relevant clinical significance. This information can enhance our understanding of excipient influence on drug absorption and is useful in designing pharmacokinetic studies and evaluating the resultant data.

Metabolic Mapping: Quantitative Enzyme Cytochemistry and Histochemistry to Determine the Activity of Dehydrogenases in Cells and Tissues.

PubMed

Molenaar, Remco J; Khurshed, Mohammed; Hira, Vashendriya V V; Van Noorden, Cornelis J F

2018-05-26

Altered cellular metabolism is a hallmark of many diseases, including cancer, cardiovascular diseases and infection. The metabolic motor units of cells are enzymes and their activity is heavily regulated at many levels, including the transcriptional, mRNA stability, translational, post-translational and functional level. This complex regulation means that conventional quantitative or imaging assays, such as quantitative mRNA experiments, Western Blots and immunohistochemistry, yield incomplete information regarding the ultimate activity of enzymes, their function and/or their subcellular localization. Quantitative enzyme cytochemistry and histochemistry (i.e., metabolic mapping) show in-depth information on in situ enzymatic activity and its kinetics, function and subcellular localization in an almost true-to-nature situation. We describe a protocol to detect the activity of dehydrogenases, which are enzymes that perform redox reactions to reduce cofactors such as NAD(P) + and FAD. Cells and tissue sections are incubated in a medium that is specific for the enzymatic activity of one dehydrogenase. Subsequently, the dehydrogenase that is the subject of investigation performs its enzymatic activity in its subcellular site. In a chemical reaction with the reaction medium, this ultimately generates blue-colored formazan at the site of the dehydrogenase's activity. The formazan's absorbance is therefore a direct measure of the dehydrogenase's activity and can be quantified using monochromatic light microscopy and image analysis. The quantitative aspect of this protocol enables researchers to draw statistical conclusions from these assays. Besides observational studies, this technique can be used for inhibition studies of specific enzymes. In this context, studies benefit from the true-to-nature advantages of metabolic mapping, giving in situ results that may be physiologically more relevant than in vitro enzyme inhibition studies. In all, metabolic mapping is an

mRNA levels of enzymes and receptors implicated in arachidonic acid metabolism in gliomas.

PubMed

De Armas, Rafael; Durand, Karine; Guillaudeau, Angélique; Weinbreck, Nicolas; Robert, Sandrine; Moreau, Jean-Jacques; Caire, François; Acosta, Gisela; Pebet, Matias; Chaunavel, Alain; Marin, Benoît; Labrousse, François; Denizot, Yves

2010-07-01

Gliomas are tumors of the central nervous system derived from glial cells. They show cellular heterogeneity and lack specific diagnostic markers. Although a possible role for the eicosanoid cascade has been suggested in glioma tumorigenesis, the relationship between enzymes and receptors implicated in arachidonic acid metabolism, with histological tumor type has not yet been determined. Quantitative real-time reverse transcription-polymerase chain reaction was performed to measure and compare transcript levels of enzymes and receptors implicated in both lipoxygenase and cyclooxygenase pathways between oligodendrogliomas, astrocytomas, glioblastomas and mixed oligoastrocytomas. Arachidonic acid metabolism-related enzymes and receptor transcripts (i) were underexpressed in classical oligodendrogliomas compared to astrocytomas and/or glioblastomas, (ii) differed between astrocytomas and glioblastomas and (iii) had an intermediate expression in mixed oligoastrocytomas. mRNA levels of enzymes and receptors implicated both in lipoxygenase and cyclooxygenase pathways differed significantly in gliomas according to the histological type. Copyright 2010 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Xenobiotic metabolism in the fish hepatic cell lines Hepa-E1 and RTH-149, and the gill cell lines RTgill-W1 and G1B: Biomarkers of CYP450 activity and oxidative stress.

PubMed

Franco, Marco E; Sutherland, Grace E; Lavado, Ramon

2018-04-01

The use of fish cell cultures has proven to be an effective tool in the study of environmental and aquatic toxicology. Valuable information can be obtained from comparisons between cell lines from different species and organs. In the present study, specific chemicals were used and biomarkers (e.g. 7-Ethoxyresorufin-O-deethylase (EROD) activity and reactive oxygen species (ROS)) were measured to assess the metabolic capabilities and cytotoxicity of the fish hepatic cell lines Hepa-E1 and RTH-149, and the fish gill cell lines RTgill-W1 and G1B. These cell lines were exposed to β-naphthoflavone (BNF) and benzo[a]pyrene (BaP), the pharmaceutical tamoxifen (TMX), and the organic peroxide tert-butylhydroperoxide (tBHP). Cytotoxicity in gill cell lines was significantly higher than in hepatic cells, with BNF and TMX being the most toxic compounds. CYP1-like associated activity, measured through EROD activity, was only detected in hepatic cells; Hepa-E1 cells showed the highest activity after exposure to both BNF and BaP. Significantly higher levels of CYP3A-like activity were also observed in Hepa-E1 cells exposed to TMX, while gill cell lines presented the lowest levels. Measurements of ROS and antioxidant enzymes indicated that peroxide levels were higher in gill cell lines in general. However, levels of superoxide were significantly higher in RTH-149 cells, where no distinctive increase of superoxide-related antioxidants was observed. The present study demonstrates the importance of selecting adequate cell lines in measuring specific metabolic parameters and provides strong evidence for the fish hepatocarcinoma Hepa-E1 cells to be an excellent alternative in assessing metabolism of xenobiotics, and in expanding the applicability of fish cell lines for in vitro studies. Copyright © 2018 Elsevier Inc. All rights reserved.

Neurotoxicity Linked to Dysfunctional Metal Ion Homeostasis and Xenobiotic Metal Exposure: Redox Signaling and Oxidative Stress.

PubMed

Garza-Lombó, Carla; Posadas, Yanahi; Quintanar, Liliana; Gonsebatt, María E; Franco, Rodrigo

2018-06-20

Essential metals such as copper, iron, manganese, and zinc play a role as cofactors in the activity of a wide range of processes involved in cellular homeostasis and survival, as well as during organ and tissue development. Throughout our life span, humans are also exposed to xenobiotic metals from natural and anthropogenic sources, including aluminum, arsenic, cadmium, lead, and mercury. It is well recognized that alterations in the homeostasis of essential metals and an increased environmental/occupational exposure to xenobiotic metals are linked to several neurological disorders, including neurodegeneration and neurodevelopmental alterations. Recent Advances: The redox activity of essential metals is key for neuronal homeostasis and brain function. Alterations in redox homeostasis and signaling are central to the pathological consequences of dysfunctional metal ion homeostasis and increased exposure to xenobiotic metals. Both redox-active and redox-inactive metals trigger oxidative stress and damage in the central nervous system, and the exact mechanisms involved are starting to become delineated. In this review, we aim to appraise the role of essential metals in determining the redox balance in the brain and the mechanisms by which alterations in the homeostasis of essential metals and exposure to xenobiotic metals disturb the cellular redox balance and signaling. We focus on recent literature regarding their transport, metabolism, and mechanisms of toxicity in neural systems. Delineating the specific mechanisms by which metals alter redox homeostasis is key to understand the pathological processes that convey chronic neuronal dysfunction in neurodegenerative and neurodevelopmental disorders. Antioxid. Redox Signal. 28, 1669-1703.

Functional Analogy in Human Metabolism: Enzymes with Different Biological Roles or Functional Redundancy?

PubMed Central

Piergiorge, Rafael Mina; de Miranda, Antonio Basílio; Catanho, Marcos

2017-01-01

Abstract Since enzymes catalyze almost all chemical reactions that occur in living organisms, it is crucial that genes encoding such activities are correctly identified and functionally characterized. Several studies suggest that the fraction of enzymatic activities in which multiple events of independent origin have taken place during evolution is substantial. However, this topic is still poorly explored, and a comprehensive investigation of the occurrence, distribution, and implications of these events has not been done so far. Fundamental questions, such as how analogous enzymes originate, why so many events of independent origin have apparently occurred during evolution, and what are the reasons for the coexistence in the same organism of distinct enzymatic forms catalyzing the same reaction, remain unanswered. Also, several isofunctional enzymes are still not recognized as nonhomologous, even with substantial evidence indicating different evolutionary histories. In this work, we begin to investigate the biological significance of the cooccurrence of nonhomologous isofunctional enzymes in human metabolism, characterizing functional analogous enzymes identified in metabolic pathways annotated in the human genome. Our hypothesis is that the coexistence of multiple enzymatic forms might not be interpreted as functional redundancy. Instead, these enzymatic forms may be implicated in distinct (and probably relevant) biological roles. PMID:28854631

Life-history evolution and the microevolution of intermediary metabolism: activities of lipid-metabolizing enzymes in life-history morphs of a wing-dimorphic cricket.

PubMed

Zera, Anthony J; Zhao, Zhangwu

2003-03-01

Although a considerable amount of information is available on the ecology, genetics, and physiology of life-history traits, much more limited data are available on the biochemical and genetic correlates of life-history variation within species. Specific activities of five enzymes of lipid biosynthesis and two enzymes of amino acid catabolism were compared among lines selected for flight-capable (LW[f]) versus flightless (SW) morphs of the cricket Gryllus firmus. These morphs, which exist in natural populations, differ genetically in ovarian growth (100-400% higher in SW) and aspects of flight capability including the size of wings and flight muscles, and the concentration of triglyceride flight fuel (40% greater in LW[f]). Consistently higher activity of each enzyme in LW(f) versus SW-selected lines, and strong co-segregation between morph and enzyme activity, demonstrated genetically based co-variance between wing morph and enzyme activity. Developmental profiles of enzyme activities strongly paralleled profiles of triglyceride accumulation during adulthood and previous measures of in vivo lipid biosynthesis. These data strongly imply that genetically based elevation in activities of lipogenic enzymes, and enzymes controlling the conversion of amino acids into lipids, is an important cause underlying the elevated accumulation of triglyceride in the LW(f) morph, a key biochemical component of the trade-off between elevated early fecundity and flight capability. Global changes in lipid and amino-acid metabolism appear to have resulted from microevolutionary alteration of regulators of metabolism. Finally, strong genotype x environment (diet) interactions were observed for most enzyme activities. Future progress in understanding the functional causes of life-history evolution requires a more detailed synthesis of the fields of life-history evolution and metabolic biochemistry. Wing polymorphism is a powerful experimental model in such integrative studies.

Effect of diet composition and mixture of selected food additives on the erythrocytic system and iron metabolism in peripheral blood of male rats.

PubMed

Sadowska, Joanna; Kuchlewska, Magdalena

2011-01-01

Metabolic processes of food additives which are "exogenous xenobiotics" are catalysed, primarily, by enzymes located in microsomes of hepatocytes affiliated to P-450 cytochrome superfamily, containing iron. The aim of the study was to investigate the effect of diet composition and selected food additives on the erythrocyte system and iron metabolism in peripheral blood of male rats. The experiment was carried out on 30 male rats sorted into three equinumerous groups. For drinking animals received pure, settled tap water, animals from group III were receiving additionally an aqueous solution of sodium (nitrate), potassium nitrite, benzoic acid, sorbic acid and monosodium glutamate. Ascertained a significant effect of changes in diet composition on the increase in hematocrit marker value and the count of red blood cells in blood of animals examined. Used food additives diminished hemoglobin concentration, hematocrit value and red blood cell count, diminishing also iron concentration in serum, the total iron binding capacity and transferrin saturation with iron. Analysis of the results allowed ascertain adverse changes in values of the erythrocytic system markers, occurring under the influence of the applied mixture of food additives. Used food additives change the iron metabolism, most likely from the necessity of applied xenobiotics biotransformation by heme-containing monoxygenases of P-450 cytochrome.

Metabolic cold adaptation in fishes occurs at the level of whole animal, mitochondria and enzyme

PubMed Central

White, Craig R.; Alton, Lesley A.; Frappell, Peter B.

2012-01-01

Metabolic cold adaptation (MCA), the hypothesis that species from cold climates have relatively higher metabolic rates than those from warm climates, was first proposed nearly 100 years ago and remains one of the most controversial hypotheses in physiological ecology. In the present study, we test the MCA hypothesis in fishes at the level of whole animal, mitochondria and enzyme. In support of the MCA hypothesis, we find that when normalized to a common temperature, species with ranges that extend to high latitude (cooler climates) have high aerobic enzyme (citrate synthase) activity, high rates of mitochondrial respiration and high standard metabolic rates. Metabolic compensation for the global temperature gradient is not complete however, so when measured at their habitat temperature species from high latitude have lower absolute rates of metabolism than species from low latitudes. Evolutionary adaptation and thermal plasticity are therefore insufficient to completely overcome the acute thermodynamic effects of temperature, at least in fishes. PMID:22158960

Metabolic cold adaptation in fishes occurs at the level of whole animal, mitochondria and enzyme.

PubMed

White, Craig R; Alton, Lesley A; Frappell, Peter B

2012-05-07

Metabolic cold adaptation (MCA), the hypothesis that species from cold climates have relatively higher metabolic rates than those from warm climates, was first proposed nearly 100 years ago and remains one of the most controversial hypotheses in physiological ecology. In the present study, we test the MCA hypothesis in fishes at the level of whole animal, mitochondria and enzyme. In support of the MCA hypothesis, we find that when normalized to a common temperature, species with ranges that extend to high latitude (cooler climates) have high aerobic enzyme (citrate synthase) activity, high rates of mitochondrial respiration and high standard metabolic rates. Metabolic compensation for the global temperature gradient is not complete however, so when measured at their habitat temperature species from high latitude have lower absolute rates of metabolism than species from low latitudes. Evolutionary adaptation and thermal plasticity are therefore insufficient to completely overcome the acute thermodynamic effects of temperature, at least in fishes.

Comparison of protein expression between human livers and the hepatic cell lines HepG2, Hep3B, and Huh7 using SWATH and MRM-HR proteomics: Focusing on drug-metabolizing enzymes.

PubMed

Shi, Jian; Wang, Xinwen; Lyu, Lingyun; Jiang, Hui; Zhu, Hao-Jie

2018-04-01

Human hepatic cell lines are widely used as an in vitro model for the study of drug metabolism and liver toxicity. However, the validity of this model is still a subject of debate because the expressions of various proteins in the cell lines, including drug-metabolizing enzymes (DMEs), can differ significantly from those in human livers. In the present study, we first conducted an untargeted proteomics analysis of the microsomes of the cell lines HepG2, Hep3B, and Huh7, and compared them to human livers using a sequential window acquisition of all theoretical mass spectra (SWATH) method. Furthermore, high-resolution multiple reaction monitoring (MRM-HR), a targeted proteomic approach, was utilized to compare the expressions of pre-selected DMEs between human livers and the cell lines. In general, the SWATH quantifications were in good agreement with the MRM-HR analysis. Over 3000 protein groups were quantified in the cells and human livers, and the proteome profiles of human livers significantly differed from the cell lines. Among the 101 DMEs quantified with MRM-HR, most were expressed at substantially lower levels in the cell lines. Thus, appropriate caution must be exercised when using these cell lines for the study of hepatic drug metabolism and toxicity. Copyright © 2018 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

The importance of sourcing enzymes from non-conventional fungi for metabolic engineering and biomass breakdown.

PubMed

Seppälä, Susanna; Wilken, St Elmo; Knop, Doriv; Solomon, Kevin V; O'Malley, Michelle A

2017-11-01

A wealth of fungal enzymes has been identified from nature, which continue to drive strain engineering and bioprocessing for a range of industries. However, while a number of clades have been investigated, the vast majority of the fungal kingdom remains unexplored for industrial applications. Here, we discuss selected classes of fungal enzymes that are currently in biotechnological use, and explore more basal, non-conventional fungi and their underexploited biomass-degrading mechanisms as promising agents in the transition towards a bio-based society. Of special interest are anaerobic fungi like the Neocallimastigomycota, which were recently found to harbor the largest diversity of biomass-degrading enzymes among the fungal kingdom. Enzymes sourced from these basal fungi have been used to metabolically engineer substrate utilization in yeast, and may offer new paths to lignin breakdown and tunneled biocatalysis. We also contrast classic enzymology approaches with emerging 'omics'-based tools to decipher function within novel fungal isolates and identify new promising enzymes. Recent developments in genome editing are expected to accelerate discovery and metabolic engineering within these systems, yet are still limited by a lack of high-resolution genomes, gene regulatory regions, and even appropriate culture conditions. Finally, we present new opportunities to harness the biomass-degrading potential of undercharacterized fungi via heterologous expression and engineered microbial consortia. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Xenobiotic Transporter Expression along the Male Genital Tract1

PubMed Central

Klein, David M.; Wright, Stephen H.; Cherrington, Nathan J.

2015-01-01

The male genital tract plays an important role in protecting sperm by forming a distinct compartment separate from the body which limits exposure to potentially toxic substrates. Transporters along this tract can influence the distribution of xenobiotics into the male genital tract through efflux back into the blood or facilitating the accumulation of toxicants. The aim of this study was to quantitatively determine the constitutive mRNA expression of 30 xenobiotic transporters in caput and cauda regions of the epididymis, vas deferens, prostate, and seminal vesicles from adult Sprague-Dawley rats. The epididymis was found to express at least moderate levels of 18 transporters, vas deferens 15, seminal vesicles 23, and prostate 18. Constitutive expression of these xenobiotic transporters in the male genital tract may provide insight into the xenobiotics that can potentially be transported into these tissues and may provide the molecular mechanism for site specific toxicity of select agents. PMID:24814985

Increments and duplication events of enzymes and transcription factors influence metabolic and regulatory diversity in prokaryotes.

PubMed

Martínez-Núñez, Mario Alberto; Poot-Hernandez, Augusto Cesar; Rodríguez-Vázquez, Katya; Perez-Rueda, Ernesto

2013-01-01

In this work, the content of enzymes and DNA-binding transcription factors (TFs) in 794 non-redundant prokaryotic genomes was evaluated. The identification of enzymes was based on annotations deposited in the KEGG database as well as in databases of functional domains (COG and PFAM) and structural domains (Superfamily). For identifications of the TFs, hidden Markov profiles were constructed based on well-known transcriptional regulatory families. From these analyses, we obtained diverse and interesting results, such as the negative rate of incremental changes in the number of detected enzymes with respect to the genome size. On the contrary, for TFs the rate incremented as the complexity of genome increased. This inverse related performance shapes the diversity of metabolic and regulatory networks and impacts the availability of enzymes and TFs. Furthermore, the intersection of the derivatives between enzymes and TFs was identified at 9,659 genes, after this point, the regulatory complexity grows faster than metabolic complexity. In addition, TFs have a low number of duplications, in contrast to the apparent high number of duplications associated with enzymes. Despite the greater number of duplicated enzymes versus TFs, the increment by which duplicates appear is higher in TFs. A lower proportion of enzymes among archaeal genomes (22%) than in the bacterial ones (27%) was also found. This low proportion might be compensated by the interconnection between the metabolic pathways in Archaea. A similar proportion was also found for the archaeal TFs, for which the formation of regulatory complexes has been proposed. Finally, an enrichment of multifunctional enzymes in Bacteria, as a mechanism of ecological adaptation, was detected.

Increments and Duplication Events of Enzymes and Transcription Factors Influence Metabolic and Regulatory Diversity in Prokaryotes

PubMed Central

Martínez-Núñez, Mario Alberto; Poot-Hernandez, Augusto Cesar; Rodríguez-Vázquez, Katya; Perez-Rueda, Ernesto

2013-01-01

In this work, the content of enzymes and DNA-binding transcription factors (TFs) in 794 non-redundant prokaryotic genomes was evaluated. The identification of enzymes was based on annotations deposited in the KEGG database as well as in databases of functional domains (COG and PFAM) and structural domains (Superfamily). For identifications of the TFs, hidden Markov profiles were constructed based on well-known transcriptional regulatory families. From these analyses, we obtained diverse and interesting results, such as the negative rate of incremental changes in the number of detected enzymes with respect to the genome size. On the contrary, for TFs the rate incremented as the complexity of genome increased. This inverse related performance shapes the diversity of metabolic and regulatory networks and impacts the availability of enzymes and TFs. Furthermore, the intersection of the derivatives between enzymes and TFs was identified at 9,659 genes, after this point, the regulatory complexity grows faster than metabolic complexity. In addition, TFs have a low number of duplications, in contrast to the apparent high number of duplications associated with enzymes. Despite the greater number of duplicated enzymes versus TFs, the increment by which duplicates appear is higher in TFs. A lower proportion of enzymes among archaeal genomes (22%) than in the bacterial ones (27%) was also found. This low proportion might be compensated by the interconnection between the metabolic pathways in Archaea. A similar proportion was also found for the archaeal TFs, for which the formation of regulatory complexes has been proposed. Finally, an enrichment of multifunctional enzymes in Bacteria, as a mechanism of ecological adaptation, was detected. PMID:23922780

Cross-induction of detoxification genes by environmental xenobiotics and insecticides in the mosquito Aedes aegypti: impact on larval tolerance to chemical insecticides.

PubMed

Poupardin, Rodolphe; Reynaud, Stéphane; Strode, Clare; Ranson, Hilary; Vontas, John; David, Jean-Philippe

2008-05-01

The effect of exposure of Aedes aegypti larvae to sub-lethal doses of the pyrethroid insecticide permethrin, the organophosphate temephos, the herbicide atrazine, the polycyclic aromatic hydrocarbon fluoranthene and the heavy metal copper on their subsequent tolerance to insecticides, detoxification enzyme activities and expression of detoxification genes was investigated. Bioassays revealed a moderate increase in larval tolerance to permethrin following exposure to fluoranthene and copper while larval tolerance to temephos increased moderately after exposure to atrazine, copper and permethrin. Cytochrome P450 monooxygenases activities were induced in larvae exposed to permethrin, fluoranthene and copper while glutathione S-transferase activities were induced after exposure to fluoranthene and repressed after exposure to copper. Microarray screening of the expression patterns of all detoxification genes following exposure to each xenobiotic with the Aedes Detox Chip identified multiple genes induced by xenobiotics and insecticides. Further expression studies using real-time quantitative PCR confirmed the induction of multiple CYP genes and one carboxylesterase gene by insecticides and xenobiotics. Overall, this study reveals the potential of xenobiotics found in polluted mosquito breeding sites to affect their tolerance to insecticides, possibly through the cross-induction of particular detoxification genes. Molecular mechanisms involved and impact on mosquito control strategies are discussed.

In-vitro synthesis of drug metabolites and their screening/characterization using liquid chromatography-mass spectrometry (LC-MS)

USDA-ARS?s Scientific Manuscript database

Drug metabolism is a biochemical process by which drugs and xenobiotics are chemically modified to metabolites, primarily by liver enzymes. Metabolites may sometimes affect cellular therapeutic or toxicological processes, therefore knowledge of metabolic processes is essential for understanding drug...

Molecular docking studies of 3-bromopyruvate and its derivatives to metabolic regulatory enzymes: Implication in designing of novel anticancer therapeutic strategies

PubMed Central

Yadav, Saveg; Pandey, Shrish Kumar; Singh, Vinay Kumar; Goel, Yugal; Kumar, Ajay

2017-01-01

Altered metabolism is an emerging hallmark of cancer, as malignant cells display a mammoth up-regulation of enzymes responsible for steering their bioenergetic and biosynthetic machinery. Thus, the recent anticancer therapeutic strategies focus on the targeting of metabolic enzymes, which has led to the identification of specific metabolic inhibitors. One of such inhibitors is 3-bromopyruvate (3-BP), with broad spectrum of anticancer activity due to its ability to inhibit multiple metabolic enzymes. However, the molecular characterization of its binding to the wide spectrum of target enzymes remains largely elusive. Therefore, in the present study we undertook in silico investigations to decipher the molecular nature of the docking of 3-BP with key target enzymes of glycolysis and TCA cycle by PatchDock and YASARA docking tools. Additionally, derivatives of 3-BP, dibromopyruvate (DBPA) and propionic acid (PA), with reported biological activity, were also investigated for docking to important target metabolic enzymes of 3-BP, in order to predict their therapeutic efficacy versus that of 3-BP. A comparison of the docking scores with respect to 3-BP indicated that both of these derivatives display a better binding strength to metabolic enzymes. Further, analysis of the drug likeness of 3-BP, DBPA and PA by Lipinski filter, admetSAR and FAF Drug3 indicated that all of these agents showed desirable drug-like criteria. The outcome of this investigation sheds light on the molecular characteristics of the binding of 3-BP and its derivatives with metabolic enzymes and thus may significantly contribute in designing and optimizing therapeutic strategies against cancer by using these agents. PMID:28463978

Molecular docking studies of 3-bromopyruvate and its derivatives to metabolic regulatory enzymes: Implication in designing of novel anticancer therapeutic strategies.

PubMed

Yadav, Saveg; Pandey, Shrish Kumar; Singh, Vinay Kumar; Goel, Yugal; Kumar, Ajay; Singh, Sukh Mahendra

2017-01-01

Altered metabolism is an emerging hallmark of cancer, as malignant cells display a mammoth up-regulation of enzymes responsible for steering their bioenergetic and biosynthetic machinery. Thus, the recent anticancer therapeutic strategies focus on the targeting of metabolic enzymes, which has led to the identification of specific metabolic inhibitors. One of such inhibitors is 3-bromopyruvate (3-BP), with broad spectrum of anticancer activity due to its ability to inhibit multiple metabolic enzymes. However, the molecular characterization of its binding to the wide spectrum of target enzymes remains largely elusive. Therefore, in the present study we undertook in silico investigations to decipher the molecular nature of the docking of 3-BP with key target enzymes of glycolysis and TCA cycle by PatchDock and YASARA docking tools. Additionally, derivatives of 3-BP, dibromopyruvate (DBPA) and propionic acid (PA), with reported biological activity, were also investigated for docking to important target metabolic enzymes of 3-BP, in order to predict their therapeutic efficacy versus that of 3-BP. A comparison of the docking scores with respect to 3-BP indicated that both of these derivatives display a better binding strength to metabolic enzymes. Further, analysis of the drug likeness of 3-BP, DBPA and PA by Lipinski filter, admetSAR and FAF Drug3 indicated that all of these agents showed desirable drug-like criteria. The outcome of this investigation sheds light on the molecular characteristics of the binding of 3-BP and its derivatives with metabolic enzymes and thus may significantly contribute in designing and optimizing therapeutic strategies against cancer by using these agents.

An MRM-based workflow for absolute quantitation of lysine-acetylated metabolic enzymes in mouse liver.

PubMed

Xu, Leilei; Wang, Fang; Xu, Ying; Wang, Yi; Zhang, Cuiping; Qin, Xue; Yu, Hongxiu; Yang, Pengyuan

2015-12-07

As a key post-translational modification mechanism, protein acetylation plays critical roles in regulating and/or coordinating cell metabolism. Acetylation is a prevalent modification process in enzymes. Protein acetylation modification occurs in sub-stoichiometric amounts; therefore extracting biologically meaningful information from these acetylation sites requires an adaptable, sensitive, specific, and robust method for their quantification. In this work, we combine immunoassays and multiple reaction monitoring-mass spectrometry (MRM-MS) technology to develop an absolute quantification for acetylation modification. With this hybrid method, we quantified the acetylation level of metabolic enzymes, which could demonstrate the regulatory mechanisms of the studied enzymes. The development of this quantitative workflow is a pivotal step for advancing our knowledge and understanding of the regulatory effects of protein acetylation in physiology and pathophysiology.

Interplay of Drug-Metabolizing Enzymes and Transporters in Drug Absorption and Disposition.

PubMed

Shi, Shaojun; Li, Yunqiao

2014-01-01

In recent years, the functional interplay between drug-metabolizing enzymes (DMEs) and drug transporters (DTs) in drug absorption and disposition, as well as the complex drug interactions (DIs), has become an intriguing contention, which has also been termed the "transport-metabolism interplay". The current mechanistic understanding for this interplay is first discussed. In the present article, studies investigating the interplay between cytochrome P450 enzymes (CYPs) and efflux transporters have been systematically reviewed in vitro, in situ, in silico, in animals and humans, followed by CYPs-uptake transporters, CYPs-uptake transporters-efflux transporters, and phase II metabolic enzymes-transporters interplay studies. Although several cellular, isolated organ and whole animal studies, in conjunction with simulation and modelling, have addressed the issue that DMEs and DTs can work cooperatively to affect the bioavailability of shared substrate drugs, convincing evidences in human studies are still lacking. Furthermore, the functional interplay between DMEs and DTs will be highly substrate- and dose- dependent. Additionally, we review recent studies to evaluate the influence of genetic variations in the interplay between DMEs and DTs, which might be helpful for the prediction of pharmacokinetics (PK) and possible DIs in human more correctly. There is strong evidence of coordinately regulated DEMs and DTs gene expression and protein activity (e.g. nuclear receptors). Taken together, further investigations and analysis are urgently needed to explore the functional interplay of DMEs and DTs and to delineate the underlying mechanisms.

Liver enzymes, the metabolic syndrome, and incident diabetes: the Mexico City diabetes study.

PubMed

Nannipieri, Monica; Gonzales, Clicerio; Baldi, Simona; Posadas, Rosalinda; Williams, Ken; Haffner, Steven M; Stern, Michael P; Ferrannini, Ele

2005-07-01

To test the hypothesis that enzymes conventionally associated with liver dysfunction (aspartate aminotransferase, alanine aminotransferase, gamma-glutamyltransferase [GGT], and alkaline phosphatase) may predict diabetes. From a population-based diabetes survey, we selected 1,441 men and women in whom serum enzyme levels were < or =3 SDs of the mean population value, alcohol intake was <250 g/week, and hepatitis B and C virus testing was negative. At follow-up (7 years), 94 subjects developed diabetes and 93 impaired glucose tolerance (IGT). At baseline, all four enzymes were related to most of the features of the metabolic syndrome. After controlling for sex, age, adiposity/fat distribution, alcohol intake, serum lipids, and blood pressure, higher alanine aminotransferase and GGT values were significantly (P < 0.01) associated with both IGT and diabetes, whereas alkaline phosphatase was associated with diabetes only (P = 0.0004) and aspartate aminotransferase with IGT only (P = 0.0001). Raised GGT alone was associated with all the features of the metabolic syndrome. Raised GGT was a significant predictor of either IGT or diabetes (odds ratio 1.62 [95% CI 1.08-2.42] top quartile vs. lower quartiles, P < 0.02) after controlling for sex, age, adiposity/fat distribution, alcohol consumption, fasting plasma insulin and proinsulin levels, and 2-h postglucose plasma glucose concentrations. Although mild elevations in liver enzymes are associated with features of the metabolic syndrome, only raised GGT is an independent predictor of deterioration of glucose tolerance to IGT or diabetes. As GGT signals oxidative stress, the association with diabetes may reflect both hepatic steatosis and enhanced oxidative stress.

Complete Proteomic-Based Enzyme Reaction and Inhibition Kinetics Reveal How Monolignol Biosynthetic Enzyme Families Affect Metabolic Flux and Lignin in Populus trichocarpa[W

PubMed Central

Wang, Jack P.; Naik, Punith P.; Chen, Hsi-Chuan; Shi, Rui; Lin, Chien-Yuan; Liu, Jie; Shuford, Christopher M.; Li, Quanzi; Sun, Ying-Hsuan; Tunlaya-Anukit, Sermsawat; Williams, Cranos M.; Muddiman, David C.; Ducoste, Joel J.; Sederoff, Ronald R.; Chiang, Vincent L.

2014-01-01

We established a predictive kinetic metabolic-flux model for the 21 enzymes and 24 metabolites of the monolignol biosynthetic pathway using Populus trichocarpa secondary differentiating xylem. To establish this model, a comprehensive study was performed to obtain the reaction and inhibition kinetic parameters of all 21 enzymes based on functional recombinant proteins. A total of 104 Michaelis-Menten kinetic parameters and 85 inhibition kinetic parameters were derived from these enzymes. Through mass spectrometry, we obtained the absolute quantities of all 21 pathway enzymes in the secondary differentiating xylem. This extensive experimental data set, generated from a single tissue specialized in wood formation, was used to construct the predictive kinetic metabolic-flux model to provide a comprehensive mathematical description of the monolignol biosynthetic pathway. The model was validated using experimental data from transgenic P. trichocarpa plants. The model predicts how pathway enzymes affect lignin content and composition, explains a long-standing paradox regarding the regulation of monolignol subunit ratios in lignin, and reveals novel mechanisms involved in the regulation of lignin biosynthesis. This model provides an explanation of the effects of genetic and transgenic perturbations of the monolignol biosynthetic pathway in flowering plants. PMID:24619611

The enzymes of biotin dependent CO2 metabolism: What structures reveal about their reaction mechanisms

PubMed Central

Waldrop, Grover L; Holden, Hazel M; Maurice, Martin St

2012-01-01

Biotin is the major cofactor involved in carbon dioxide metabolism. Indeed, biotin-dependent enzymes are ubiquitous in nature and are involved in a myriad of metabolic processes including fatty acid synthesis and gluconeogenesis. The cofactor, itself, is composed of a ureido ring, a tetrahydrothiophene ring, and a valeric acid side chain. It is the ureido ring that functions as the CO2 carrier. A complete understanding of biotin-dependent enzymes is critically important for translational research in light of the fact that some of these enzymes serve as targets for anti-obesity agents, antibiotics, and herbicides. Prior to 1990, however, there was a dearth of information regarding the molecular architectures of biotin-dependent enzymes. In recent years there has been an explosion in the number of three-dimensional structures reported for these proteins. Here we review our current understanding of the structures and functions of biotin-dependent enzymes. In addition, we provide a critical analysis of what these structures have and have not revealed about biotin-dependent catalysis. PMID:22969052

The MetaCyc database of metabolic pathways and enzymes and the BioCyc collection of pathway/genome databases

PubMed Central

Caspi, Ron; Altman, Tomer; Dale, Joseph M.; Dreher, Kate; Fulcher, Carol A.; Gilham, Fred; Kaipa, Pallavi; Karthikeyan, Athikkattuvalasu S.; Kothari, Anamika; Krummenacker, Markus; Latendresse, Mario; Mueller, Lukas A.; Paley, Suzanne; Popescu, Liviu; Pujar, Anuradha; Shearer, Alexander G.; Zhang, Peifen; Karp, Peter D.

2010-01-01

The MetaCyc database (MetaCyc.org) is a comprehensive and freely accessible resource for metabolic pathways and enzymes from all domains of life. The pathways in MetaCyc are experimentally determined, small-molecule metabolic pathways and are curated from the primary scientific literature. With more than 1400 pathways, MetaCyc is the largest collection of metabolic pathways currently available. Pathways reactions are linked to one or more well-characterized enzymes, and both pathways and enzymes are annotated with reviews, evidence codes, and literature citations. BioCyc (BioCyc.org) is a collection of more than 500 organism-specific Pathway/Genome Databases (PGDBs). Each BioCyc PGDB contains the full genome and predicted metabolic network of one organism. The network, which is predicted by the Pathway Tools software using MetaCyc as a reference, consists of metabolites, enzymes, reactions and metabolic pathways. BioCyc PGDBs also contain additional features, such as predicted operons, transport systems, and pathway hole-fillers. The BioCyc Web site offers several tools for the analysis of the PGDBs, including Omics Viewers that enable visualization of omics datasets on two different genome-scale diagrams and tools for comparative analysis. The BioCyc PGDBs generated by SRI are offered for adoption by any party interested in curation of metabolic, regulatory, and genome-related information about an organism. PMID:19850718

Two Horizontally Transferred Xenobiotic Resistance Gene Clusters Associated with Detoxification of Benzoxazolinones by Fusarium Species

PubMed Central

Glenn, Anthony E.; Davis, C. Britton; Gao, Minglu; Gold, Scott E.; Mitchell, Trevor R.; Proctor, Robert H.; Stewart, Jane E.; Snook, Maurice E.

2016-01-01

Microbes encounter a broad spectrum of antimicrobial compounds in their environments and often possess metabolic strategies to detoxify such xenobiotics. We have previously shown that Fusarium verticillioides, a fungal pathogen of maize known for its production of fumonisin mycotoxins, possesses two unlinked loci, FDB1 and FDB2, necessary for detoxification of antimicrobial compounds produced by maize, including the γ-lactam 2-benzoxazolinone (BOA). In support of these earlier studies, microarray analysis of F. verticillioides exposed to BOA identified the induction of multiple genes at FDB1 and FDB2, indicating the loci consist of gene clusters. One of the FDB1 cluster genes encoded a protein having domain homology to the metallo-β-lactamase (MBL) superfamily. Deletion of this gene (MBL1) rendered F. verticillioides incapable of metabolizing BOA and thus unable to grow on BOA-amended media. Deletion of other FDB1 cluster genes, in particular AMD1 and DLH1, did not affect BOA degradation. Phylogenetic analyses and topology testing of the FDB1 and FDB2 cluster genes suggested two horizontal transfer events among fungi, one being transfer of FDB1 from Fusarium to Colletotrichum, and the second being transfer of the FDB2 cluster from Fusarium to Aspergillus. Together, the results suggest that plant-derived xenobiotics have exerted evolutionary pressure on these fungi, leading to horizontal transfer of genes that enhance fitness or virulence. PMID:26808652

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thornton-Manning, J.R.; Hotchkiss, J.A.; Ding, Xinxin

The nasal mucosa, the first tissue of contact for inhaled xenobiotics, possesses substantial enobiotic-metabolizing capacti. Enzymes of the nasal cavity may metabolize xenobiotics to innocuous, more water-soluble compounds that are eliminated from the body, or they may bioactivate them to toxic metabolites. These toxic metabolites may find to cellular macromolecules in the nasal cavity or be transported to other parts of the body where they may react. Nasal carcinogenesis in rodents often results from bioactivation of xenobiotics. The increased incidences of nasal tumors associated with certain occupations suggest that xenobiotic bioactivation may be important in human nasal cancer etiology, asmore » well. The increasing popularity of the nose as a route of drug administration makes information concerning nasal drug metabolism and disposition vital to accomplish therapeutic goals. For these reasons, the study of xenobiotic-met abolizing capacity of the nasal cavity is an important area of health-related research. In the present study, we have confirmed the presence of CYP2A6 mRNA in human respiratory mucosa.« less

Potential role of liver enzymes levels as predictor markers of glucose metabolism disorders in Tunisian population.

PubMed

Bouhajja, Houda; Abdelhedi, Rania; Amouri, Ali; Hadj Kacem, Faten; Marrakchi, Rim; Safi, Wajdi; Mrabet, Houcem; Chtourou, Lassaad; Charfi, Nadia; Fourati, Mouna; Bensassi, Salwa; Jamoussi, Kamel; Abid, Mohamed; Ayadi, Hammadi; Feki, Mouna Mnif; Elleuch, Noura Bougacha

2018-03-10

The relationship between liver enzymes and type 2 diabetes (T2D) risk is inconclusive. We aimed to evaluate the association between liver markers and risk of carbohydrate metabolism disorders and their discriminatory power for T2D prediction. This cross-sectional study enrolled 216 participants classified as normoglycemic, prediabetes, newly-diagnosed diabetes and diagnosed diabetes. All participants underwent anthropometric and biochemical measurements. The relationship between hepatic enzymes and glucose metabolism markers was evaluated by ANCOVA analyses. The associations between liver enzymes and incident carbohydrate metabolism disorders were analyzed through logistic regression and their discriminatory capacity for T2D by receiver operating characteristic (ROC) analysis. High alkaline phosphatase (AP), alanine aminotransferase (ALT), γ-glutamyltransferase (γGT) and aspartate aminotrasferase (AST) levels were independently related to decreased insulin sensitivity. Interestingly, higher AP level was significantly associated with increased risk of prediabetes (p=0.017), newly-diagnosed diabetes (p=0.004) and T2D (p=0.007). Elevated γGT level was an independent risk factor for T2D (p=0.032) and undiagnosed-T2D (p=0.010) in prediabetic and normoglycemic subjects, respectively. In ROC analysis, AP was a powerful predictor of incident diabetes and significantly improved T2D prediction. Liver enzymes within normal range, specifically AP levels, are associated with increased risk of carbohydrate metabolism disorders and significantly improved T2D prediction.

Towards an Understanding of the Function of the Phytochelatin Synthase of Schistosoma mansoni

PubMed Central

Rigouin, Coraline; Nylin, Elyse; Cogswell, Alexis A.; Schaumlöffel, Dirk; Dobritzsch, Dirk; Williams, David L.

2013-01-01

Phytochelatin synthase (PCS) is a protease-like enzyme that catalyzes the production of metal chelating peptides, the phytochelatins, from glutathione (GSH). In plants, algae, and fungi phytochelatin production is important for metal tolerance and detoxification. PCS proteins also function in xenobiotic metabolism by processing GSH S-conjugates. The aim of the present study is to elucidate the role of PCS in the parasitic worm Schistosoma mansoni. Recombinant S. mansoni PCS proteins expressed in bacteria could both synthesize phytochelatins and hydrolyze various GSH S-conjugates. We found that both the N-truncated protein and the N- and C-terminal truncated form of the enzyme (corresponding to only the catalytic domain) work through a thiol-dependant and, notably, metal-independent mechanism for both transpeptidase (phytochelatin synthesis) and peptidase (hydrolysis of GSH S-conjugates) activities. PCS transcript abundance was increased by metals and xenobiotics in cultured adult worms. In addition, these treatments were found to increase transcript abundance of other enzymes involved in GSH metabolism. Highest levels of PCS transcripts were identified in the esophageal gland of adult worms. Taken together, these results suggest that S. mansoni PCS participates in both metal homoeostasis and xenobiotic metabolism rather than metal detoxification as previously suggested and that the enzyme may be part of a global stress response in the worm. Because humans do not have PCS, this enzyme is of particular interest as a drug target for schistosomiasis. PMID:23383357

Characterisation of the cytochrome P450 enzymes involved in the in vitro metabolism of granisetron.

PubMed Central

Bloomer, J C; Baldwin, S J; Smith, G J; Ayrton, A D; Clarke, S E; Chenery, R J

1994-01-01

1. The metabolism of granisetron was investigated in human liver microsomes to identify the specific forms of cytochrome P450 responsible. 2. 7-hydroxy and 9'-desmethyl granisetron were identified as the major products of metabolism following incubation of granisetron with human liver microsomes. At low, clinically relevant, concentrations of granisetron the 7-hydroxy metabolite predominated. Rates of granisetron 7-hydroxylation varied over 100-fold in the human livers investigated. 3. Enzyme kinetics demonstrated the involvement of at least two enzymes contributing to the 7-hydroxylation of granisetron, one of which was a high affinity component with a Km of 4 microM. A single, low affinity, enzyme was responsible for the 9'-desmethylation of granisetron. 4. Granisetron caused no inhibition of any of the cytochrome P450 activities investigated (CYP1A2, CYP2A6, CYP2B6, CYP2C9/8, CYP2C19, CYP2D6, CYP2E1 and CYP3A), at concentrations up to 250 microM. 5. Studies using chemical inhibitors selective for individual P450 enzymes indicated the involvement of cytochrome P450 3A (CYP3A), both pathways of granisetron metabolism being very sensitive to ketoconazole inhibition. Correlation data were consistent with the role of CYP3A3/4 in granisetron 9'-desmethylation but indicated that a different enzyme was involved in the 7-hydroxylation. PMID:7888294

Axonal and dendritic localization of mRNAs for glycogen-metabolizing enzymes in cultured rodent neurons

PubMed Central

2014-01-01

Background Localization of mRNAs encoding cytoskeletal or signaling proteins to neuronal processes is known to contribute to axon growth, synaptic differentiation and plasticity. In addition, a still increasing spectrum of mRNAs has been demonstrated to be localized under different conditions and developing stages thus reflecting a highly regulated mechanism and a role of mRNA localization in a broad range of cellular processes. Results Applying fluorescence in-situ-hybridization with specific riboprobes on cultured neurons and nervous tissue sections, we investigated whether the mRNAs for two metabolic enzymes, namely glycogen synthase (GS) and glycogen phosphorylase (GP), the key enzymes of glycogen metabolism, may also be targeted to neuronal processes. If it were so, this might contribute to clarify the so far enigmatic role of neuronal glycogen. We found that the mRNAs for both enzymes are localized to axonal and dendritic processes in cultured lumbar spinal motoneurons, but not in cultured trigeminal neurons. In cultured cortical neurons which do not store glycogen but nevertheless express glycogen synthase, the GS mRNA is also subject to axonal and dendritic localization. In spinal motoneurons and trigeminal neurons in situ, however, the mRNAs could only be demonstrated in the neuronal somata but not in the nerves. Conclusions We could demonstrate that the mRNAs for major enzymes of neural energy metabolism can be localized to neuronal processes. The heterogeneous pattern of mRNA localization in different culture types and developmental stages stresses that mRNA localization is a versatile mechanism for the fine-tuning of cellular events. Our findings suggest that mRNA localization for enzymes of glycogen metabolism could allow adaptation to spatial and temporal energy demands in neuronal events like growth, repair and synaptic transmission. PMID:24898526

Axonal and dendritic localization of mRNAs for glycogen-metabolizing enzymes in cultured rodent neurons.

PubMed

Pfeiffer-Guglielmi, Brigitte; Dombert, Benjamin; Jablonka, Sibylle; Hausherr, Vanessa; van Thriel, Christoph; Schöbel, Nicole; Jansen, Ralf-Peter

2014-06-04

Localization of mRNAs encoding cytoskeletal or signaling proteins to neuronal processes is known to contribute to axon growth, synaptic differentiation and plasticity. In addition, a still increasing spectrum of mRNAs has been demonstrated to be localized under different conditions and developing stages thus reflecting a highly regulated mechanism and a role of mRNA localization in a broad range of cellular processes. Applying fluorescence in-situ-hybridization with specific riboprobes on cultured neurons and nervous tissue sections, we investigated whether the mRNAs for two metabolic enzymes, namely glycogen synthase (GS) and glycogen phosphorylase (GP), the key enzymes of glycogen metabolism, may also be targeted to neuronal processes. If it were so, this might contribute to clarify the so far enigmatic role of neuronal glycogen. We found that the mRNAs for both enzymes are localized to axonal and dendritic processes in cultured lumbar spinal motoneurons, but not in cultured trigeminal neurons. In cultured cortical neurons which do not store glycogen but nevertheless express glycogen synthase, the GS mRNA is also subject to axonal and dendritic localization. In spinal motoneurons and trigeminal neurons in situ, however, the mRNAs could only be demonstrated in the neuronal somata but not in the nerves. We could demonstrate that the mRNAs for major enzymes of neural energy metabolism can be localized to neuronal processes. The heterogeneous pattern of mRNA localization in different culture types and developmental stages stresses that mRNA localization is a versatile mechanism for the fine-tuning of cellular events. Our findings suggest that mRNA localization for enzymes of glycogen metabolism could allow adaptation to spatial and temporal energy demands in neuronal events like growth, repair and synaptic transmission.

Xenobiotics and loss of cell adhesion drive distinct transcriptional outcomes by aryl hydrocarbon receptor signaling.

PubMed

Hao, Nan; Lee, Kian Leong; Furness, Sebastian G B; Bosdotter, Cecilia; Poellinger, Lorenz; Whitelaw, Murray L

2012-12-01

The aryl hydrocarbon receptor (AhR) is a signal-regulated transcription factor, which is canonically activated by the direct binding of xenobiotics. In addition, switching cells from adherent to suspension culture also activates the AhR, representing a nonxenobiotic, physiological activation of AhR signaling. Here, we show that the AhR is recruited to target gene enhancers in both ligand [isopropyl-2-(1,3-dithietane-2-ylidene)-2-[N-(4-methylthiazol-2-yl)carbamoyl]acetate (YH439)]-treated and suspension cells, suggesting a common mechanism of target gene induction between these two routes of AhR activation. However, gene expression profiles critically differ between xenobiotic- and suspension-activated AhR signaling. Por and Cldnd1 were regulated predominantly by ligand treatments, whereas, in contrast, ApoER2 and Ganc were regulated predominantly by the suspension condition. Classic xenobiotic-metabolizing AhR targets such as Cyp1a1, Cyp1b1, and Nqo1 were regulated by both ligand and suspension conditions. Temporal expression patterns of AhR target genes were also found to vary, with examples of transient activation, transient repression, or sustained alterations in expression. Furthermore, sequence analysis coupled with chromatin immunoprecipitation assays and reporter gene analysis identified a functional xenobiotic response element (XRE) in the intron 1 of the mouse Tiparp gene, which was also bound by hypoxia-inducible factor-1α during hypoxia and features a concatemer of four XRE cores (GCGTG). Our data suggest that this XRE concatemer site concurrently regulates the expression of both the Tiparp gene and its cis antisense noncoding RNA after ligand- or suspension-induced AhR activation. This work provides novel insights into how AhR signaling drives different transcriptional programs via the ligand versus suspension modes of activation.

Monobromobimane occupies a distinct xenobiotic substrate site in glutathione S-transferase π

PubMed Central

Ralat, Luis A.; Colman, Roberta F.

2003-01-01

Monobromobimane (mBBr), functions as a substrate of porcine glutathione S-transferase π (GST π): The enzyme catalyzes the reaction of mBBr with glutathione. S-(Hydroxyethyl)bimane, a nonreactive analog of monobromobimane, acts as a competitive inhibitor with respect to mBBr as substrate but does not affect the reaction of GST π with another substrate, 1-chloro-2,4-dinitrobenzene (CDNB). In the absence of glutathione, monobromobimane inactivates GST π at pH 7.0 and 25°C as assayed using mBBr as substrate, with a lesser effect on the enzyme’s use of CDNB as substrate. These results indicate that the sites occupied by CDNB and mBBr are not identical. Inactivation is proportional to the incorporation of 2 moles of bimane/mole of subunit. Modification of GST π with mBBr does not interfere with its binding of 8-anilino-1-naphthalene sulfonate, indicating that this hydrophobic site is not the target of monobromobimane. S-Methylglutathione and S-(hydroxyethyl)bimane each yield partial protection against inactivation and decrease reagent incorporation, while glutathionyl-bimane protects completely against inactivation. Peptide analysis after trypsin digestion indicates that mBBr modifies Cys45 and Cys99 equally. Modification of Cys45 is reduced in the presence of S-methylglutathione, indicating that this residue is at or near the glutathione binding region. In contrast, modification of Cys99 is reduced in the presence of S-(hydroxyethyl)bimane, suggesting that this residue is at or near the mBBr xenobiotic substrate binding site. Modification of Cys99 can best be understood by reaction with monobromobimane while it is bound to its xenobiotic substrate site in an alternate orientation. These results support the concept that glutathione S-transferase accomplishes its ability to react with a diversity of substrates in part by harboring distinct xenobiotic substrate sites. PMID:14573868

Glutamate and GABA-metabolizing enzymes in post-mortem cerebellum in Alzheimer's disease: phosphate-activated glutaminase and glutamic acid decarboxylase.

PubMed

Burbaeva, G Sh; Boksha, I S; Tereshkina, E B; Savushkina, O K; Prokhorova, T A; Vorobyeva, E A

2014-10-01

Enzymes of glutamate and GABA metabolism in postmortem cerebellum from patients with Alzheimer's disease (AD) have not been comprehensively studied. The present work reports results of original comparative study on levels of phosphate-activated glutaminase (PAG) and glutamic acid decarboxylase isoenzymes (GAD65/67) in autopsied cerebellum samples from AD patients and matched controls (13 cases in each group) as well as summarizes published evidence for altered levels of PAG and GAD65/67 in AD brain. Altered (decreased) levels of these enzymes and changes in links between amounts of these enzymes and other glutamate-metabolizing enzymes (such as glutamate dehydrogenase and glutamine synthetase-like protein) in AD cerebella suggest significantly impaired glutamate and GABA metabolism in this brain region, which was previously regarded as not substantially involved in AD pathogenesis.

Expression level, cellular compartment and metabolic network position all influence the average selective constraint on mammalian enzymes

PubMed Central

2011-01-01

Background A gene's position in regulatory, protein interaction or metabolic networks can be predictive of the strength of purifying selection acting on it, but these relationships are neither universal nor invariably strong. Following work in bacteria, fungi and invertebrate animals, we explore the relationship between selective constraint and metabolic function in mammals. Results We measure the association between selective constraint, estimated by the ratio of nonsynonymous (Ka) to synonymous (Ks) substitutions, and several, primarily metabolic, measures of gene function. We find significant differences between the selective constraints acting on enzyme-coding genes from different cellular compartments, with the nucleus showing higher constraint than genes from either the cytoplasm or the mitochondria. Among metabolic genes, the centrality of an enzyme in the metabolic network is significantly correlated with Ka/Ks. In contrast to yeasts, gene expression magnitude does not appear to be the primary predictor of selective constraint in these organisms. Conclusions Our results imply that the relationship between selective constraint and enzyme centrality is complex: the strength of selective constraint acting on mammalian genes is quite variable and does not appear to exclusively follow patterns seen in other organisms. PMID:21470417

Bioactive terpenoids and flavonoids from ginkgo biloba extract induce the expression of hepatic drug-metabolizing enzymes through Pregnane X receptor, Constitutive androstane receptor, and Aryl hydrocarbon receptor-mediated pathways

PubMed Central

Li, Linhao; Stanton, Joseph D; Tolson, Antonia H; Luo, Yuan; Wang, Hongbing

2008-01-01

Purpose The objective of the current study is to investigate the hypothesis that bioactive terpenoids and flavonoids of Ginkgo biloba extract (GBE) induce human hepatic drug metabolizing enzymes (DMEs) and transporters through the selective activation of pregnane X receptor (PXR), constitutive androstane receptor (CAR), and aryl hydrocarbon receptor (AhR). Methods Human primary hepatocyte (HPH), and HepG2 cells are used as in vitro models for enzyme induction and nuclear receptor activation studies. A combination of real-time RT-PCR, transient transfection, and cell-based reporter assays were employed. Results In human primary hepatocytes, real-time PCR analysis showed induction of CYP2B6, CYP3A4, UGT1A1, MDR1, and MRP2 by EGb 761, ginkgolide A (GA) and ginkgolide B (GB), but not by bilobalide (BB) or the flavonoids (quercetin, kaempferol and tamarixetin) of GBE. Cell-based reporter assays in HepG2 revealed that GA and GB are potent activators of PXR; quercetin and kaempferol activate PXR, CAR, and AhR, whereas BB exerts no effects on these xenobiotic receptors. Notably, the flavonoids induced the expression of UGT1A1 and CYP1A2 in HepG2 cells but not in HPH. Conclusion Our results indicate that terpenoids and flavonoids of GBE exhibit differential induction of DMEs through the selective activation of PXR, CAR, and AhR. PMID:19034627

[Important application of intestinal transporters and metabolism enzymes on gastrointestinal disposal of active ingredients of Chinese materia medica].

PubMed

Bi, Xiaolin; Du, Qiu; Di, Liuqing

2010-02-01

Oral drug bioavailability depends on gastrointestinal absorption, intestinal transporters and metabolism enzymes are the important factors in drug gastrointestinal absorption and they can also be induced or inhibited by the active ingredients of Chinese materia medica. This article presents important application of intestinal transporters and metabolism enzymes on gastrointestinal disposal of the active ingredients of Chinese materia medica, and points out the importance of research on transport and metabolism of the active ingredients of Chinese materia medica in Chinese extract and Chinese medicinal formulae.

Contributions of Human Cytochrome P450 Enzymes to Glyburide Metabolism*

PubMed Central

Zhou, Lin; Naraharisetti, Suresh B.; Liu, Li; Wang, Honggang; Lin, Yvonne S.; Isoherranen, Nina; Unadkat, Jashvant D.; Hebert, Mary F.; Mao, Qingcheng

2011-01-01

Glyburide (GLB) is a widely used oral sulfonylurea for the treatment of gestational diabetes. Therapeutic use of GLB is often complicated by a substantial inter-individual variability in the pharmacokinetics and pharmacodynamics of the drug in human populations, which might be caused by inter-individual variations in factors such as GLB metabolism. Therefore, there has been a continued interest in identifying human cytochrome P450 (CYP) isoforms that play a major role in the metabolism of GLB. However, contrasting data are available in the present literature in this regard. In the present study, we systematically investigated the contributions of various human CYP isoforms (CYP3A4, CYP3A5, CYP2C8, CYP2C9, and CYP2C19) to in vitro metabolism of GLB. GLB depletion and metabolite formation in human liver microsomes were most significantly inhibited by the CYP3A inhibitor ketoconazole compared with the inhibitors of other CYP isoforms. Furthermore, multiple correlation analysis between GLB depletion and individual CYP activities was performed, demonstrating a significant correlation between GLB depletion and the CYP3A probe activity in 16 individual human liver microsomal preparations, but not between GLB depletion and the CYP2C19, CYP2C8, or CYP2C9 probe activity. By using recombinant supersomes overexpressing individual human CYP isoforms, we found that GLB could be depleted by all the enzymes tested; however, the intrinsic clearance (Vmax/Km) of CYP3A4 for GLB depletion was 4 – 17 times greater than that of other CYP isoforms. These results confirm that human CYP3A4 is the major enzyme invovled in the in vitro metabolism of GLB. PMID:20437462

Environmental Factors and Bioremediation of Xenobiotics Using White Rot Fungi

PubMed Central

Fragoeiro, Silvia; Bastos, Catarina

2010-01-01

This review provides background information on the importance of bioremediation approaches. It describes the roles of fungi, specifically white rot fungi, and their extracellular enzymes, laccases, ligninases, and peroxidises, in the degradation of xenobiotic compounds such as single and mixtures of pesticides. We discuss the importance of abiotic factors such as water potential, temperature, and pH stress when considering an environmental screening approach, and examples are provided of the differential effect of white rot fungi on the degradation of single and mixtures of pesticides using fungi such as Trametes versicolor and Phanerochaete chrysosporium. We also explore the formulation and delivery of fungal bioremedial inoculants to terrestrial ecosystems as well as the use of spent mushroom compost as an approach. Future areas for research and potential exploitation of new techniques are also considered. PMID:23956663

Improved Xenobiotic Metabolism and Reduced Susceptibility to Cancer in Gluten-Sensitive Macaques upon Introduction of a Gluten-Free Diet

PubMed Central

Sestak, Karol; Conroy, Lauren; Aye, Pyone P.; Mehra, Smriti; Doxiadis, Gaby G.; Kaushal, Deepak

2011-01-01

Background A non-human primate (NHP) model of gluten sensitivity was employed to study the gene perturbations associated with dietary gluten changes in small intestinal tissues from gluten-sensitive rhesus macaques (Macaca mulatta). Methodology Stages of remission and relapse were accomplished in gluten-sensitive animals by administration of gluten-free (GFD) and gluten-containing (GD) diets, as described previously. Pin-head-sized biopsies, obtained non-invasively by pediatric endoscope from duodenum while on GFD or GD, were used for preparation of total RNA and gene profiling, using the commercial Rhesus Macaque Microarray (Agilent Technologies),targeting expression of over 20,000 genes. Principal Findings When compared with normal healthy control, gluten-sensitive macaques showed differential gene expressions induced by GD. While observed gene perturbations were classified into one of 12 overlapping categories - cancer, metabolism, digestive tract function, immune response, cell growth, signal transduction, autoimmunity, detoxification of xenobiotics, apoptosis, actin-collagen deposition, neuronal and unknown function - this study focused on cancer-related gene networks such as cytochrome P450 family (detoxification function) and actin-collagen-matrix metalloproteinases (MMP) genes. Conclusions/Significance A loss of detoxification function paralleled with necessity to metabolize carcinogens was revealed in gluten-sensitive animals while on GD. An increase in cancer-promoting factors and a simultaneous decrease in cancer-preventing factors associated with altered expression of actin-collagen-MMP gene network were noted. In addition, gluten-sensitive macaques showed reduced number of differentially expressed genes including the cancer-associated ones upon withdrawal of dietary gluten. Taken together, these findings indicate potentially expanded utility of gluten-sensitive rhesus macaques in cancer research. PMID:21533263

A Liver-Centric Multiscale Modeling Framework for Xenobiotics.

PubMed

Sluka, James P; Fu, Xiao; Swat, Maciej; Belmonte, Julio M; Cosmanescu, Alin; Clendenon, Sherry G; Wambaugh, John F; Glazier, James A

2016-01-01

We describe a multi-scale, liver-centric in silico modeling framework for acetaminophen pharmacology and metabolism. We focus on a computational model to characterize whole body uptake and clearance, liver transport and phase I and phase II metabolism. We do this by incorporating sub-models that span three scales; Physiologically Based Pharmacokinetic (PBPK) modeling of acetaminophen uptake and distribution at the whole body level, cell and blood flow modeling at the tissue/organ level and metabolism at the sub-cellular level. We have used standard modeling modalities at each of the three scales. In particular, we have used the Systems Biology Markup Language (SBML) to create both the whole-body and sub-cellular scales. Our modeling approach allows us to run the individual sub-models separately and allows us to easily exchange models at a particular scale without the need to extensively rework the sub-models at other scales. In addition, the use of SBML greatly facilitates the inclusion of biological annotations directly in the model code. The model was calibrated using human in vivo data for acetaminophen and its sulfate and glucuronate metabolites. We then carried out extensive parameter sensitivity studies including the pairwise interaction of parameters. We also simulated population variation of exposure and sensitivity to acetaminophen. Our modeling framework can be extended to the prediction of liver toxicity following acetaminophen overdose, or used as a general purpose pharmacokinetic model for xenobiotics.

A Liver-Centric Multiscale Modeling Framework for Xenobiotics

PubMed Central

Swat, Maciej; Cosmanescu, Alin; Clendenon, Sherry G.; Wambaugh, John F.; Glazier, James A.

2016-01-01

We describe a multi-scale, liver-centric in silico modeling framework for acetaminophen pharmacology and metabolism. We focus on a computational model to characterize whole body uptake and clearance, liver transport and phase I and phase II metabolism. We do this by incorporating sub-models that span three scales; Physiologically Based Pharmacokinetic (PBPK) modeling of acetaminophen uptake and distribution at the whole body level, cell and blood flow modeling at the tissue/organ level and metabolism at the sub-cellular level. We have used standard modeling modalities at each of the three scales. In particular, we have used the Systems Biology Markup Language (SBML) to create both the whole-body and sub-cellular scales. Our modeling approach allows us to run the individual sub-models separately and allows us to easily exchange models at a particular scale without the need to extensively rework the sub-models at other scales. In addition, the use of SBML greatly facilitates the inclusion of biological annotations directly in the model code. The model was calibrated using human in vivo data for acetaminophen and its sulfate and glucuronate metabolites. We then carried out extensive parameter sensitivity studies including the pairwise interaction of parameters. We also simulated population variation of exposure and sensitivity to acetaminophen. Our modeling framework can be extended to the prediction of liver toxicity following acetaminophen overdose, or used as a general purpose pharmacokinetic model for xenobiotics. PMID:27636091

Impact of expression of EMP enzymes on glucose metabolism in Zymomonas mobilis.

PubMed

Chen, Rachel Ruizhen; Agrawal, Manoj; Mao, Zichao

2013-06-01

Zymomonas mobilis is the only known microorganism that utilizes the Entner-Doudoroff (ED) pathway anaerobically. In this work, we investigated whether the overexpression of a phosphofructokinase (PFK), the only missing Embden-Meyerhof-Parnas (EMP) pathway enzyme, could establish the pathway in this organism. Introduction of a pyrophosphate-dependent PFK, along with co-expression of homologous fructose-1,6-bisphosphate aldolase and triosephosphate isomerase, did not result in an EMP flux to any appreciable level. However, the metabolism of glucose was impacted significantly. Eight percent of glucose was metabolized to form a new metabolite, dihydroxyacetone. Reducing flux through the ED pathway by as much as 40 % through antisense of a key enzyme, ED aldolase, did not result in a fully functional EMP pathway, suggesting that the ED pathway, especially the lower arm, downstream from glyceraldehyde-3-phosphate, is very rigid, possibly due to redox balance.

Geraniol Pharmacokinetics, Bioavailability and Its Multiple Effects on the Liver Antioxidant and Xenobiotic-Metabolizing Enzymes.

PubMed

Pavan, Barbara; Dalpiaz, Alessandro; Marani, Luca; Beggiato, Sarah; Ferraro, Luca; Canistro, Donatella; Paolini, Moreno; Vivarelli, Fabio; Valerii, Maria C; Comparone, Antonietta; De Fazio, Luigia; Spisni, Enzo

2018-01-01

Geraniol is a natural monoterpene showing anti-inflammatory, antioxidant, neuroprotective and anticancer effects. No pharmacokinetic and bioavailability data on geraniol are currently available. We therefore performed a systematic study to identify the permeation properties of geraniol across intestinal cells, and its pharmacokinetics and bioavailability after intravenous and oral administration to rats. In addition, we systematically investigated the potential hepatotoxic effects of high doses of geraniol on hepatic phase I, phase II and antioxidant enzymatic activities and undertook a hematochemical analysis on mice. Permeation studies performed via HPLC evidenced geraniol permeability coefficients across an in vitro model of the human intestinal wall for apical to basolateral and basolateral to apical transport of 13.10 ± 2.3 × 10 -3 and 2.1 ± 0.1⋅× 10 -3 cm/min, respectively. After intravenous administration of geraniol to rats (50 mg/kg), its concentration in whole blood (detected via HPLC) decreased following an apparent pseudo-first order kinetics with a half-life of 12.5 ± 1.5 min. The absolute bioavailability values of oral formulations (50 mg/kg) of emulsified geraniol or fiber-adsorbed geraniol were 92 and 16%, respectively. Following emulsified oral administration, geraniol amounts in the cerebrospinal fluid of rats ranged between 0.72 ± 0.08 μg/mL and 2.6 ± 0.2 μg/mL within 60 min. Mice treated with 120 mg/kg of geraniol for 4 weeks showed increased anti-oxidative defenses with no signs of liver toxicity. CYP450 enzyme activities appeared only slightly affected by the high dosage of geraniol.

Assessing in silico the recruitment and functional spectrum of bacterial enzymes from secondary metabolism.

PubMed

Veprinskiy, Valery; Heizinger, Leonhard; Plach, Maximilian G; Merkl, Rainer

2017-01-26

Microbes, plants, and fungi synthesize an enormous number of metabolites exhibiting rich chemical diversity. For a high-level classification, metabolism is subdivided into primary (PM) and secondary (SM) metabolism. SM products are often not essential for survival of the organism and it is generally assumed that SM enzymes stem from PM homologs. We wanted to assess evolutionary relationships and function of bona fide bacterial PM and SM enzymes. Thus, we analyzed the content of 1010 biosynthetic gene clusters (BGCs) from the MIBiG dataset; the encoded bacterial enzymes served as representatives of SM. The content of 15 bacterial genomes known not to harbor BGCs served as a representation of PM. Enzymes were categorized on their EC number and for these enzyme functions, frequencies were determined. The comparison of PM/SM frequencies indicates a certain preference for hydrolases (EC class 3) and ligases (EC class 6) in PM and of oxidoreductases (EC class 1) and lyases (EC class 4) in SM. Based on BLAST searches, we determined pairs of PM/SM homologs and their functional diversity. Oxidoreductases, transferases (EC class 2), lyases and isomerases (EC class 5) form a tightly interlinked network indicating that many protein folds can accommodate different functions in PM and SM. In contrast, the functional diversity of hydrolases and especially ligases is significantly limited in PM and SM. For the most direct comparison of PM/SM homologs, we restricted for each BGC the search to the content of the genome it comes from. For each homologous hit, the contribution of the genomic neighborhood to metabolic pathways was summarized in BGC-specific html-pages that are interlinked with KEGG; this dataset can be downloaded from https://www.bioinf.ur.de . Only few reaction chemistries are overrepresented in bacterial SM and at least 55% of the enzymatic functions present in BGCs possess PM homologs. Many SM enzymes arose in PM and Nature utilized the evolvability of enzymes

Prediction of metabolism-induced hepatotoxicity on three-dimensional hepatic cell culture and enzyme microarrays.

PubMed

Yu, Kyeong-Nam; Nadanaciva, Sashi; Rana, Payal; Lee, Dong Woo; Ku, Bosung; Roth, Alexander D; Dordick, Jonathan S; Will, Yvonne; Lee, Moo-Yeal

2018-03-01

Human liver contains various oxidative and conjugative enzymes that can convert nontoxic parent compounds to toxic metabolites or, conversely, toxic parent compounds to nontoxic metabolites. Unlike primary hepatocytes, which contain myriad drug-metabolizing enzymes (DMEs), but are difficult to culture and maintain physiological levels of DMEs, immortalized hepatic cell lines used in predictive toxicity assays are easy to culture, but lack the ability to metabolize compounds. To address this limitation and predict metabolism-induced hepatotoxicity in high-throughput, we developed an advanced miniaturized three-dimensional (3D) cell culture array (DataChip 2.0) and an advanced metabolizing enzyme microarray (MetaChip 2.0). The DataChip is a functionalized micropillar chip that supports the Hep3B human hepatoma cell line in a 3D microarray format. The MetaChip is a microwell chip containing immobilized DMEs found in the human liver. As a proof of concept for generating compound metabolites in situ on the chip and rapidly assessing their toxicity, 22 model compounds were dispensed into the MetaChip and sandwiched with the DataChip. The IC 50 values obtained from the chip platform were correlated with rat LD 50 values, human C max values, and drug-induced liver injury categories to predict adverse drug reactions in vivo. As a result, the platform had 100% sensitivity, 86% specificity, and 93% overall predictivity at optimum cutoffs of IC 50 and C max values. Therefore, the DataChip/MetaChip platform could be used as a high-throughput, early stage, microscale alternative to conventional in vitro multi-well plate platforms and provide a rapid and inexpensive assessment of metabolism-induced toxicity at early phases of drug development.

Metabolism of myclobutanil and triadimefon by human and rat cytochrome P450 enzymes and liver microsomes.

PubMed

Barton, H A; Tang, J; Sey, Y M; Stanko, J P; Murrell, R N; Rockett, J C; Dix, D J

2006-09-01

Metabolism of two triazole-containing antifungal azoles was studied using expressed human and rat cytochrome P450s (CYP) and liver microsomes. Substrate depletion methods were used due to the complex array of metabolites produced from myclobutanil and triadimefon. Myclobutanil was metabolized more rapidly than triadimefon, which is consistent with metabolism of the n-butyl side-chain in the former and the t-butyl group in the latter compound. Human and rat CYP2C and CYP3A enzymes were the most active. Metabolism was similar in microsomes prepared from livers of control and low-dose rats. High-dose (115 mg kg-1 day-1 of triadimefon or 150 mg kg-1 day-1 of myclobutanil) rats showed increased liver weight, induction of total CYP, and increased metabolism of the two triazoles, though the apparent Km appeared unchanged relative to the control. These data identify CYP enzymes important for the metabolization of these two triazoles. Estimated hepatic clearances suggest that CYP induction may have limited impact in vivo.

Visualizing and quantifying difference in cytoplasmic and nuclear metabolism in the hepatobiliary system in vivo

NASA Astrophysics Data System (ADS)

Lin, Chih-Ju; Kang, Ning; Lee, Jian-Ye; Lee, Hsuan-Shu; Dong, Chen-Yuan

2015-01-01

The liver is a major organ responsible for performing xenobiotic metabolism. In this process, xenobiotic is uptaken and processed in hepatocytes and subsequently excreted into the bile canaliculi. However, the intracellular heterogeneity in such metabolic processes is not known. We use the molecular probe 6-carboxyfluorescein diacetate (6-CFDA) to investigate xenobiotic metabolism in hepatocytes with intravital multiphoton fluorescence microscopy. 6-CFDA is processed by intracellular esterase to fluorescent 6-CF, which can be imaged and quantified. We found that compared to the nucleus, cytoplasmic 6-CF fluorescence intensity reached a maximum earlier (cytoplasm: 11.3±4.4 min nucleus: 14.7±4.9 min) following 6-CFDA injection. We also found a slight difference in the rate of 6-CFDA metabolism as the rates of 6-CF decay at rates of 1.43±0.75 and 1.27±0.72 photons/min for the cytoplasm and nucleus, respectively. These results indicate that molecular transport to the nucleus is additionally hindered and can affect drug transport there.

A Txnrd1-dependent metabolic switch alters hepatic lipogenesis, glycogen storage, and detoxification

PubMed Central

Iverson, Sonya V.; Eriksson, Sofi; Xu, Jianqiang; Prigge, Justin R.; Talago, Emily A.; Meade, Tesia A.; Meade, Erin S.; Capecchi, Mario R.; Arnér, Elias S.J.; Schmidt, Edward E.

2013-01-01

Besides helping to maintain a reducing intracellular environment, the thioredoxin (Trx) system impacts bioenergetics and drug-metabolism. We show that hepatocyte-specific disruption of Txnrd1, encoding Trx reductase-1 (TrxR1), causes a metabolic switch in which lipogenic genes are repressed and periportal hepatocytes become engorged with glycogen. These livers also overexpress machinery for biosynthesis of glutathione and conversion of glycogen into UDP-glucuronate; they stockpile glutathione-S-transferases and UDP-glucuronyl-transferases; and they overexpress xenobiotic exporters. This realigned metabolic profile suggested that the mutant hepatocytes might be preconditioned to more effectively detoxify certain xenobiotic challenges. Hepatocytes convert the pro-toxin acetaminophen (APAP, paracetamol) into cytotoxic N-acetyl-p-benzoquinone imine (NAPQI). APAP defenses include glucuronidation of APAP or glutathionylation of NAPQI, allowing removal by xenobiotic exporters. We found that NAPQI directly inactivates TrxR1, yet Txnrd1-null livers were resistant to APAP-induced hepatotoxicity. Txnrd1-null livers did not have more effective gene expression responses to APAP challenge; however their constitutive metabolic state supported more robust GSH biosynthesis-, glutathionylation-, and glucuronidation-systems. Following APAP challenge, this effectively sustained the GSH system and attenuated damage. PMID:23743293

Influence of bacterial N-acyl-homoserine lactones on growth parameters, pigments, antioxidative capacities and the xenobiotic phase II detoxification enzymes in barley and yam bean.

PubMed

Götz-Rösch, Christine; Sieper, Tina; Fekete, Agnes; Schmitt-Kopplin, Philippe; Hartmann, Anton; Schröder, Peter

2015-01-01

Bacteria are able to communicate with each other and sense their environment in a population density dependent mechanism known as quorum sensing (QS). N-acyl-homoserine lactones (AHLs) are the QS signaling compounds of Gram-negative bacteria which are frequent colonizers of rhizospheres. While cross-kingdom signaling and AHL-dependent gene expression in plants has been confirmed, the responses of enzyme activities in the eukaryotic host upon AHLs are unknown. Since AHL are thought to be used as so-called plant boosters or strengthening agents, which might change their resistance toward radiation and/or xenobiotic stress, we have examined the plants' pigment status and their antioxidative and detoxifying capacities upon AHL treatment. Because the yield of a crop plant should not be negatively influenced, we have also checked for growth and root parameters. We investigated the influence of three different AHLs, namely N-hexanoyl- (C6-HSL), N-octanoyl- (C8-HSL), and N-decanoyl- homoserine lactone (C10-HSL) on two agricultural crop plants. The AHL-effects on Hordeum vulgare (L.) as an example of a monocotyledonous crop and on the tropical leguminous crop plant Pachyrhizus erosus (L.) were compared. While plant growth and pigment contents in both plants showed only small responses to the applied AHLs, AHL treatment triggered tissue- and compound-specific changes in the activity of important detoxification enzymes. The activity of dehydroascorbate reductase in barley shoots after C10-HSL treatment for instance increased up to 384% of control plant levels, whereas superoxide dismutase activity in barley roots was decreased down to 23% of control levels upon C6-HSL treatment. Other detoxification enzymes reacted similarly within this range, with interesting clusters of positive or negative answers toward AHL treatment. In general the changes on the enzyme level were more severe in barley than in yam bean which might be due to the different abilities of the plants to

Influence of bacterial N-acyl-homoserine lactones on growth parameters, pigments, antioxidative capacities and the xenobiotic phase II detoxification enzymes in barley and yam bean

PubMed Central

Götz-Rösch, Christine; Sieper, Tina; Fekete, Agnes; Schmitt-Kopplin, Philippe; Hartmann, Anton; Schröder, Peter

2015-01-01

Bacteria are able to communicate with each other and sense their environment in a population density dependent mechanism known as quorum sensing (QS). N-acyl-homoserine lactones (AHLs) are the QS signaling compounds of Gram-negative bacteria which are frequent colonizers of rhizospheres. While cross-kingdom signaling and AHL-dependent gene expression in plants has been confirmed, the responses of enzyme activities in the eukaryotic host upon AHLs are unknown. Since AHL are thought to be used as so-called plant boosters or strengthening agents, which might change their resistance toward radiation and/or xenobiotic stress, we have examined the plants’ pigment status and their antioxidative and detoxifying capacities upon AHL treatment. Because the yield of a crop plant should not be negatively influenced, we have also checked for growth and root parameters. We investigated the influence of three different AHLs, namely N-hexanoyl- (C6-HSL), N-octanoyl- (C8-HSL), and N-decanoyl- homoserine lactone (C10-HSL) on two agricultural crop plants. The AHL-effects on Hordeum vulgare (L.) as an example of a monocotyledonous crop and on the tropical leguminous crop plant Pachyrhizus erosus (L.) were compared. While plant growth and pigment contents in both plants showed only small responses to the applied AHLs, AHL treatment triggered tissue- and compound-specific changes in the activity of important detoxification enzymes. The activity of dehydroascorbate reductase in barley shoots after C10-HSL treatment for instance increased up to 384% of control plant levels, whereas superoxide dismutase activity in barley roots was decreased down to 23% of control levels upon C6-HSL treatment. Other detoxification enzymes reacted similarly within this range, with interesting clusters of positive or negative answers toward AHL treatment. In general the changes on the enzyme level were more severe in barley than in yam bean which might be due to the different abilities of the plants to

Alginate Immobilization of Metabolic Enzymes (AIME) for High-Throughput Screening Assays (SOT)

EPA Science Inventory

Alginate Immobilization of Metabolic Enzymes (AIME) for High-Throughput Screening Assays DE DeGroot, RS Thomas, and SO SimmonsNational Center for Computational Toxicology, US EPA, Research Triangle Park, NC USAThe EPA’s ToxCast program utilizes a wide variety of high-throughput s...

Cytochrome P450 Monooxygenases for Fatty Acids and Xenobiotics in Marine Macroalgae1

PubMed Central

Pflugmacher, Stephan; Sandermann, Heinrich

1998-01-01

The metabolism of xenobiotics has mainly been investigated in higher plant species. We studied them in various marine macroalgae of the phyla Chlorophyta, Chromophyta, and Rhodophyta. Microsomes contained high oxidative activities for known cytochrome (Cyt) P450 substrates (fatty acids, cinnamic acid, 3- and 4-chlorobiphenyl, 2,3-dichlorobiphenyl, and isoproturon; up to 54 pkat/mg protein). The presence of Cyt P450 (approximately 50 pmol/mg protein) in microsomes of the three algal families was demonstrated by CO-difference absorption spectra. Intact algal tissue converted 3-chlorobiphenyl to the same monohydroxy-metabolite formed in vitro. This conversion was 5-fold stimulated upon addition of phenobarbital, and was abolished by the known P450 inhibitor, 1-aminobenzotriazole. It is concluded that marine macroalgae contain active species of Cyt P450 and could act as a metabolic sink for marine pollutants. PMID:9576781

Bile acid metabolism and signaling in cholestasis, inflammation and cancer

PubMed Central

Apte, Udayan

2015-01-01

Bile acids are synthesized from cholesterol in the liver. Some cytochrome P450 (CYP) enzymes play key roles in bile acid synthesis. Bile acids are physiological detergent molecules, so are highly cytotoxic. They undergo enterohepatic circulation and play important roles in generating bile flow and facilitating biliary secretion of endogenous metabolites and xenobiotics and intestinal absorption of dietary fats and lipid soluble vitamins. Bile acid synthesis, transport and pool size are therefore tightly regulated under physiological conditions. In cholestasis, impaired bile flow leads to accumulation of bile acids in the liver, causing hepatocyte and biliary injury and inflammation. Chronic cholestasis is associated with fibrosis, cirrhosis and eventually liver failure. Chronic cholestasis also increases the risk of developing hepatocellular or cholangiocellular carcinomas. Extensive research in the last two decades has shown that bile acids act as signaling molecules that regulate various cellular processes. The bile acid-activated nuclear receptors are ligand-activated transcriptional factors that play critical roles in the regulation of bile acid, drug and xenobiotic metabolism. In cholestasis, these bile acid-activated receptors regulate a network of genes involved in bile acid synthesis, conjugation, transport and metabolism to alleviate bile acid-induced inflammation and injury. Additionally, bile acids are known to regulate cell growth and proliferation, and altered bile acid levels in diseased conditions have been implicated in liver injury/regeneration and tumorigenesis. We will cover the mechanisms that regulate bile acid homeostasis and detoxification during cholestasis, and the roles of bile acids in the initiation and regulation of hepatic inflammation, regeneration and carcinogenesis. PMID:26233910

Geraniol Pharmacokinetics, Bioavailability and Its Multiple Effects on the Liver Antioxidant and Xenobiotic-Metabolizing Enzymes

PubMed Central

Pavan, Barbara; Dalpiaz, Alessandro; Marani, Luca; Beggiato, Sarah; Ferraro, Luca; Canistro, Donatella; Paolini, Moreno; Vivarelli, Fabio; Valerii, Maria C.; Comparone, Antonietta; De Fazio, Luigia; Spisni, Enzo

2018-01-01

Geraniol is a natural monoterpene showing anti-inflammatory, antioxidant, neuroprotective and anticancer effects. No pharmacokinetic and bioavailability data on geraniol are currently available. We therefore performed a systematic study to identify the permeation properties of geraniol across intestinal cells, and its pharmacokinetics and bioavailability after intravenous and oral administration to rats. In addition, we systematically investigated the potential hepatotoxic effects of high doses of geraniol on hepatic phase I, phase II and antioxidant enzymatic activities and undertook a hematochemical analysis on mice. Permeation studies performed via HPLC evidenced geraniol permeability coefficients across an in vitro model of the human intestinal wall for apical to basolateral and basolateral to apical transport of 13.10 ± 2.3 × 10-3 and 2.1 ± 0.1⋅× 10-3 cm/min, respectively. After intravenous administration of geraniol to rats (50 mg/kg), its concentration in whole blood (detected via HPLC) decreased following an apparent pseudo-first order kinetics with a half-life of 12.5 ± 1.5 min. The absolute bioavailability values of oral formulations (50 mg/kg) of emulsified geraniol or fiber-adsorbed geraniol were 92 and 16%, respectively. Following emulsified oral administration, geraniol amounts in the cerebrospinal fluid of rats ranged between 0.72 ± 0.08 μg/mL and 2.6 ± 0.2 μg/mL within 60 min. Mice treated with 120 mg/kg of geraniol for 4 weeks showed increased anti-oxidative defenses with no signs of liver toxicity. CYP450 enzyme activities appeared only slightly affected by the high dosage of geraniol. PMID:29422862

Pharmacogenetics of drug-metabolizing enzymes: implications for a safer and more effective drug therapy

PubMed Central

Ingelman-Sundberg, Magnus; Rodriguez-Antona, Cristina

2005-01-01

The majority of phase I- and phase II-dependent drug metabolism is carried out by polymorphic enzymes which can cause abolished, quantitatively or qualitatively decreased or enhanced drug metabolism. Several examples exist where subjects carrying certain alleles do not benefit from drug therapy due to ultrarapid metabolism caused by multiple genes or by induction of gene expression or, alternatively, suffer from adverse effects of the drug treatment due to the presence of defective alleles. It is likely that future predictive genotyping for such enzymes might benefit 15–25% of drug treatments, and thereby allow prevention of adverse drug reactions and causalities, and thus improve the health of a significant fraction of the patients. However, it will take time before this will be a reality within the clinic. We describe some important aspects in the field with emphasis on cytochrome P450 and discuss also polymorphic aspects of foetal expression of CYP3A5 and CYP3A7. PMID:16096104

Metabolic enzyme activities of abyssal and hadal fishes: pressure effects and a re-evaluation of depth-related changes

NASA Astrophysics Data System (ADS)

Gerringer, M. E.; Drazen, J. C.; Yancey, P. H.

2017-07-01

Metabolic enzyme activities of muscle tissue have been useful and widely-applied indicators of whole animal metabolic capacity, particularly in inaccessible systems such as the deep sea. Previous studies have been conducted at atmospheric pressure, regardless of organism habitat depth. However, maximum reaction rates of some of these enzymes are pressure dependent, complicating the use of metabolic enzyme activities as proxies of metabolic rates. Here, we show pressure-related rate changes in lactate and malate dehydrogenase (LDH, MDH) and pyruvate kinase (PK) in six fish species (2 hadal, 2 abyssal, 2 shallow). LDH maximal reaction rates decreased with pressure for the two shallow species, but, in contrast to previous findings, it increased for the four deep species, suggesting evolutionary changes in LDH reaction volumes. MDH maximal reaction rates increased with pressure in all species (up to 51±10% at 60 MPa), including the tide pool snailfish, Liparis florae (activity increase at 60 MPa 44±9%), suggesting an inherent negative volume change of the reaction. PK was inhibited by pressure in all species tested, including the hadal liparids (up to 34±3% at 60 MPa), suggesting a positive volume change during the reaction. The addition of 400 mM TMAO counteracted this inhibition at both 0.5 and 2.0 mM ADP concentrations for the hadal liparid, Notoliparis kermadecensis. We revisit depth-related trends in metabolic enzyme activities according to these pressure-related rate changes and new data from seven abyssal and hadal species from the Kermadec and Mariana trenches. Results show that, with abyssal and hadal species, pressure-related rate changes are another variable to be considered in the use of enzyme activities as proxies for metabolic rate, in addition to factors such as temperature and body mass. Intraspecific increases in tricarboxylic acid cycle enzymes with depth of capture, independent of body mass, in two hadal snailfishes suggest improved nutritional

Differences in response of glucuronide and glutathione conjugating enzymes to aflatoxin B/sub 1/ and N-acetylaminofluorene in underfed rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rajpurohit, R.; Krishnaswamy, K.

Changes in the hepatic drug/xenobiotic-metabolizing enzymes in underfed rats exposed to aflatoxin B/sub 1/ and N-acetylaminofluorene were investigated. Neither carcinogen, fed at the level of 10 ..mu..g and 0.667 mg per 100 g body weight, respectively, over a period of 3 wk, had any significant influence on cytochrome P-450 and aryl hydrocarbon hydroxylase in the undernourished rats. Significantly low activities of UDP-glucuronyltransferase and glutathione S-transferase were observed in food-restricted animals fed on aflatoxin B/sub 1/. N-acetylaminofluorene, on the other hand stimulated both the enzyme activities in the underfed group, to as much observed in the respective well-fed treated group. UDP-Glucuronyltransferasemore » and glutathione S-transferase in undernutrition seem to respond differently to aflatoxin B/sub 1/ and N-acetylaminofluorene. Further studies are needed to assess the possible consequences of such alterations.« less

Virtual Screening as a Strategy for the Identification of Xenobiotics Disrupting Corticosteroid Action

PubMed Central

Praxmarer, Lukas; Chantong, Boonrat; Cereghetti, Diego; Winiger, Rahel; Schuster, Daniela; Odermatt, Alex

2012-01-01

Background Impaired corticosteroid action caused by genetic and environmental influence, including exposure to hazardous xenobiotics, contributes to the development and progression of metabolic diseases, cardiovascular complications and immune disorders. Novel strategies are thus needed for identifying xenobiotics that interfere with corticosteroid homeostasis. 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) and mineralocorticoid receptors (MR) are major regulators of corticosteroid action. 11β-HSD2 converts the active glucocorticoid cortisol to the inactive cortisone and protects MR from activation by glucocorticoids. 11β-HSD2 has also an essential role in the placenta to protect the fetus from high maternal cortisol concentrations. Methods and Principal Findings We employed a previously constructed 3D-structural library of chemicals with proven and suspected endocrine disrupting effects for virtual screening using a chemical feature-based 11β-HSD pharmacophore. We tested several in silico predicted chemicals in a 11β-HSD2 bioassay. The identified antibiotic lasalocid and the silane-coupling agent AB110873 were found to concentration-dependently inhibit 11β-HSD2. Moreover, the silane AB110873 was shown to activate MR and stimulate mitochondrial ROS generation and the production of the proinflammatory cytokine interleukin-6 (IL-6). Finally, we constructed a MR pharmacophore, which successfully identified the silane AB110873. Conclusions Screening of virtual chemical structure libraries can facilitate the identification of xenobiotics inhibiting 11β-HSD2 and/or activating MR. Lasalocid and AB110873 belong to new classes of 11β-HSD2 inhibitors. The silane AB110873 represents to the best of our knowledge the first industrial chemical shown to activate MR. Furthermore, the MR pharmacophore can now be used for future screening purposes. PMID:23056542

Characterization of Sugar Contents and Sucrose Metabolizing Enzymes in Developing Leaves of Hevea brasiliensis

PubMed Central

Zhu, Jinheng; Qi, Jiyan; Fang, Yongjun; Xiao, Xiaohu; Li, Jiuhui; Lan, Jixian; Tang, Chaorong

2018-01-01

Sucrose-metabolizing enzymes in plant leaves have hitherto been investigated mainly in temperate plants, and rarely conducted in tandem with gene expression and sugar analysis. Here, we investigated the sugar content, gene expression, and the activity of sucrose-metabolizing enzymes in the leaves of Hevea brasiliensis, a tropical tree widely cultivated for natural rubber. Sucrose, fructose and glucose were the major sugars detected in Hevea leaves at four developmental stages (I to IV), with starch and quebrachitol as minor saccharides. Fructose and glucose contents increased until stage III, but decreased strongly at stage IV (mature leaves). On the other hand, sucrose increased continuously throughout leaf development. Activities of all sucrose-cleaving enzymes decreased markedly at maturation, consistent with transcript decline for most of their encoding genes. Activity of sucrose phosphate synthase (SPS) was low in spite of its high transcript levels at maturation. Hence, the high sucrose content in mature leaves was not due to increased sucrose-synthesizing activity, but more to the decline in sucrose cleavage. Gene expression and activities of sucrose-metabolizing enzymes in Hevea leaves showed striking differences compared with other plants. Unlike in most other species where vacuolar invertase predominates in sucrose cleavage in developing leaves, cytoplasmic invertase and sucrose synthase (cleavage direction) also featured prominently in Hevea. Whereas SPS is normally responsible for sucrose synthesis in plant leaves, sucrose synthase (synthesis direction) was comparable or higher than that of SPS in Hevea leaves. Mature Hevea leaves had an unusually high sucrose:starch ratio of about 11, the highest reported to date in plants. PMID:29449852

Expanding the Halohydrin Dehalogenase Enzyme Family: Identification of Novel Enzymes by Database Mining.

PubMed

Schallmey, Marcus; Koopmeiners, Julia; Wells, Elizabeth; Wardenga, Rainer; Schallmey, Anett

2014-12-01

Halohydrin dehalogenases are very rare enzymes that are naturally involved in the mineralization of halogenated xenobiotics. Due to their catalytic potential and promiscuity, many biocatalytic reactions have been described that have led to several interesting and industrially important applications. Nevertheless, only a few of these enzymes have been made available through recombinant techniques; hence, it is of general interest to expand the repertoire of these enzymes so as to enable novel biocatalytic applications. After the identification of specific sequence motifs, 37 novel enzyme sequences were readily identified in public sequence databases. All enzymes that could be heterologously expressed also catalyzed typical halohydrin dehalogenase reactions. Phylogenetic inference for enzymes of the halohydrin dehalogenase enzyme family confirmed that all enzymes form a distinct monophyletic clade within the short-chain dehydrogenase/reductase superfamily. In addition, the majority of novel enzymes are substantially different from previously known phylogenetic subtypes. Consequently, four additional phylogenetic subtypes were defined, greatly expanding the halohydrin dehalogenase enzyme family. We show that the enormous wealth of environmental and genome sequences present in public databases can be tapped for in silico identification of very rare but biotechnologically important biocatalysts. Our findings help to readily identify halohydrin dehalogenases in ever-growing sequence databases and, as a consequence, make even more members of this interesting enzyme family available to the scientific and industrial community. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Carboxylesterases: General detoxifying enzymes

PubMed Central

Hatfield, M. Jason; Umans, Robyn A.; Hyatt, Janice L.; Edwards, Carol C; Wierdl, Monika; Tsurkan, Lyudmila; Taylor, Michael R.; Potter, Philip M.

2016-01-01

Carboxylesterases (CE) are members of the esterase family of enzymes, and as their name suggests, they are responsible for the hydrolysis of carboxylesters into the corresponding alcohol and carboxylic acid. To date, no endogenous CE substrates have been identified and as such, these proteins are thought to act as a mechanism to detoxify ester-containing xenobiotics. As a consequence, they are expressed in tissues that might be exposed to such agents (lung and gut epithelia, liver, kidney, etc.). CEs demonstrate very broad substrate specificities and can hydrolyze compounds as diverse as cocaine, oseltamivir (Tamiflu), permethrin and irinotecan. In addition, these enzymes are irreversibly inhibited by organophosphates such as Sarin and Tabun. In this overview, we will compare and contrast the two human enzymes that have been characterized, and evaluate the biology of the interaction of these proteins with organophosphates (principally nerve agents). PMID:26892220

Assembly and multiple gene expression of thermophilic enzymes in Escherichia coli for in vitro metabolic engineering.

PubMed

Ninh, Pham Huynh; Honda, Kohsuke; Sakai, Takaaki; Okano, Kenji; Ohtake, Hisao

2015-01-01

In vitro reconstitution of an artificial metabolic pathway is an emerging approach for the biocatalytic production of industrial chemicals. However, several enzymes have to be separately prepared (and purified) for the construction of an in vitro metabolic pathway, thereby limiting the practical applicability of this approach. In this study, genes encoding the nine thermophilic enzymes involved in a non-ATP-forming chimeric glycolytic pathway were assembled in an artificial operon and co-expressed in a single recombinant Escherichia coli strain. Gene expression levels of the thermophilic enzymes were controlled by their sequential order in the artificial operon. The specific activities of the recombinant enzymes in the cell-free extract of the multiple-gene-expression E. coli were 5.0-1,370 times higher than those in an enzyme cocktail prepared from a mixture of single-gene-expression strains, in each of which a single one of the nine thermophilic enzymes was overproduced. Heat treatment of a crude extract of the multiple-gene-expression cells led to the denaturation of indigenous proteins and one-step preparation of an in vitro synthetic pathway comprising only a limited number of thermotolerant enzymes. Coupling this in vitro pathway with other thermophilic enzymes including the H2 O-forming NADH oxidase or the malate/lactate dehydrogenase facilitated one-pot conversion of glucose to pyruvate or lactate, respectively. © 2014 Wiley Periodicals, Inc.

P450 AND METABOLISM IN TOXICOLOGY

EPA Science Inventory

The cytochromes P450 catalyze the initial phase of detoxification of many environmental chemicals, xenobiotic, drugs and the secondary metabolic product of plants. Plant secondary chemicals can be highly toxic, and they evolved in a coevolving plant - animal warfare - the plants ...

CHARACTERIZATION OF THE IN VITRO METABOLISM OF SELECTIVE ANDROGEN RECEPTOR MODULATOR USING HUMAN, RAT, AND DOG LIVER ENZYME PREPARATIONS

PubMed Central

Gao, Wenqing; Wu, Zengru; Bohl, Casey E.; Yang, Jun; Miller, Duane D.; Dalton, James T.

2007-01-01

Compound S4 [S-3-(4-acetylamino-phenoxy)-2-hydroxy-2-methyl-N-(4-nitro-3-trifluoromethyl-phenyl)-propionamide] is a novel nonsteroidal selective androgen receptor modulator that demonstrates tissue-selective androgenic and anabolic effects. The purpose of this in vitro study was to identify the phase I metabolites, potential species differences in metabolism, and the cytochromes P450 (P450s) involved in the phase I metabolism of S4 using 14C-S4, recombinant P450s, and other liver enzyme preparations from human, rat, and dog. The major phase I metabolism pathways of S4 in humans were identified as deacetylation of the B-ring acetamide group, hydrolysis of the amide bond, reduction of the A-ring nitro group, and oxidation of the aromatic rings, with deacetylation being the predominant pathway observed with most of the enzyme preparations tested. Among the major human P450 enzymes tested, CYP3A4 appeared to be one of the major phase I enzymes that could be responsible for the phase I metabolism of S4 [Km = 16.1 μM, Vmax = 1.6 pmol/(pmol · min)] in humans and mainly catalyzed the deacetylation, hydrolysis, and oxidation of S4. In humans, the cytosolic enzymes mainly catalyzed the hydrolysis reaction, whereas the microsomal enzymes primarily catalyzed the deacetylation reactions. Similar phase I metabolic profiles were observed in rats and dogs as well, except that the amide bond hydrolysis seemed to occur more rapidly in rats. In summary, these results showed that the major phase I reaction of S4 in human, rat, and dog is acetamide group deacetylation. PMID:16272404

Transformation of RDX and other energetic compounds by xenobiotic reductases XenA and XenB

PubMed Central

McClay, Kevin; Hawari, Jalal; Paquet, Louise; Malone, Thomas E.; Fox, Brian G.; Steffan, Robert J.

2017-01-01

The transformation of explosives, including hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), by xenobiotic reductases XenA and XenB (and the bacterial strains harboring these enzymes) under both aerobic and anaerobic conditions was assessed. Under anaerobic conditions, Pseudomonas fluorescens I-C (XenB) degraded RDX faster than Pseudomonas putida II-B (XenA), and transformation occurred when the cells were supplied with sources of both carbon (succinate) and nitrogen (NH4+), but not when only carbon was supplied. Transformation was always faster under anaerobic conditions compared to aerobic conditions, with both enzymes exhibiting a O2 concentration-dependent inhibition of RDX transformation. The primary degradation pathway for RDX was conversion to methylenedinitramine and then to formaldehyde, but a minor pathway that produced 4-nitro-2,4-diazabutanal (NDAB) also appeared to be active during transformation by whole cells of P. putida II-B and purified XenA. Both XenA and XenB also degraded the related nitramine explosives octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine and 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane. Purified XenB was found to have a broader substrate range than XenA, degrading more of the explosive compounds examined in this study. The results show that these two xenobiotic reductases (and their respective bacterial strains) have the capacity to transform RDX as well as a wide variety of explosive compounds, especially under low oxygen concentrations. PMID:19455327

Insights into drug metabolism by cytochromes P450 from modelling studies of CYP2D6-drug interactions

PubMed Central

Maréchal, J-D; Kemp, C A; Roberts, G C K; Paine, M J I; Wolf, C R; Sutcliffe, M J

2008-01-01

The cytochromes P450 (CYPs) comprise a vast superfamily of enzymes found in virtually all life forms. In mammals, xenobiotic metabolizing CYPs provide crucial protection from the effects of exposure to a wide variety of chemicals, including environmental toxins and therapeutic drugs. Ideally, the information on the possible metabolism by CYPs required during drug development would be obtained from crystal structures of all the CYPs of interest. For some years only crystal structures of distantly related bacterial CYPs were available and homology modelling techniques were used to bridge the gap and produce structural models of human CYPs, and thereby obtain useful functional information. A significant step forward in the reliability of these models came seven years ago with the first crystal structure of a mammalian CYP, rabbit CYP2C5, followed by the structures of six human enzymes, CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6 and CYP3A4, and a second rabbit enzyme, CYP2B4. In this review we describe as a case study the evolution of a CYP2D6 model, leading to the validation of the model as an in silico tool for predicting binding and metabolism. This work has led directly to the successful design of CYP2D6 mutants with novel activity—including creating a testosterone hydroxylase, converting quinidine from inhibitor to substrate, creating a diclofenac hydroxylase and creating a dextromethorphan O-demethylase. Our modelling-derived hypothesis-driven integrated interdisciplinary studies have given key insight into the molecular determinants of CYP2D6 and other important drug metabolizing enzymes. PMID:18026129

Experiment K-7-21: Effect of Microgravity on 1: Metabolic Enzymes of Type 1 and Type 2 Muscle Fibers, and on 2: Metabolic Enzymes, Neurotransmitter Amino Acids, and Neurotransmitter Associated Enzymes in Selected Regions of the Central Nervous System. Part 2; The Distribution of Selected Enzymes and Amino Acids in the Hippocampal Formation

NASA Technical Reports Server (NTRS)

Lowry, O. H.; Krasnov, I.; Ilyina-Kakueva, E. I.; Nemeth, P. M.; McDougal, D. B., Jr.; Choksi, R.; Carter, J. G.; Chi, M. M. Y.; Manchester, J. K.; Pusateri, M. E.

1994-01-01

Six key metabolic enzymes plus glutaminase and glutamate decarboxylase, as well as glutamate, aspartate and GABA, were measured in 11 regions of the hippocampal formation of synchronous, flight and tail suspension rats. Major differences were observed in the normal distribution patterns of each enzyme and amino acid, but no substantive effects of either microgravity or tail suspension on these patterns were clearly demonstrated.

Ecologically Appropriate Xenobiotics Induce Cytochrome P450s in Apis mellifera

PubMed Central

Johnson, Reed M.; Mao, Wenfu; Pollock, Henry S.; Niu, Guodong; Schuler, Mary A.; Berenbaum, May R.

2012-01-01

Background Honey bees are exposed to phytochemicals through the nectar, pollen and propolis consumed to sustain the colony. They may also encounter mycotoxins produced by Aspergillus fungi infesting pollen in beebread. Moreover, bees are exposed to agricultural pesticides, particularly in-hive acaricides used against the parasite Varroa destructor. They cope with these and other xenobiotics primarily through enzymatic detoxificative processes, but the regulation of detoxificative enzymes in honey bees remains largely unexplored. Methodology/Principal Findings We used several approaches to ascertain effects of dietary toxins on bee susceptibility to synthetic and natural xenobiotics, including the acaricide tau-fluvalinate, the agricultural pesticide imidacloprid, and the naturally occurring mycotoxin aflatoxin. We administered potential inducers of cytochrome P450 enzymes, the principal biochemical system for Phase 1 detoxification in insects, to investigate how detoxification is regulated. The drug phenobarbital induces P450s in many insects, yet feeding bees with phenobarbital had no effect on the toxicity of tau-fluvalinate, a pesticide known to be detoxified by bee P450s. Similarly, no P450 induction, as measured by tau-fluvalinate tolerance, occurred in bees fed xanthotoxin, salicylic acid, or indole-3-carbinol, all of which induce P450s in other insects. Only quercetin, a common pollen and honey constituent, reduced tau-fluvalinate toxicity. In microarray comparisons no change in detoxificative gene expression was detected in phenobarbital-treated bees. However, northern blot analyses of guts of bees fed extracts of honey, pollen and propolis showed elevated expression of three CYP6AS P450 genes. Diet did not influence tau-fluvalinate or imidacloprid toxicity in bioassays; however, aflatoxin toxicity was higher in bees consuming sucrose or high-fructose corn syrup than in bees consuming honey. Conclusions/Significance These results suggest that regulation of

The role of arginine and arginine-metabolizing enzymes during Giardia – host cell interactions in vitro

PubMed Central

2013-01-01

Background Arginine is a conditionally essential amino acid important in growing individuals and under non-homeostatic conditions/disease. Many pathogens interfere with arginine-utilization in host cells, especially nitric oxide (NO) production, by changing the expression of host enzymes involved in arginine metabolism. Here we used human intestinal epithelial cells (IEC) and three different isolates of the protozoan parasite Giardia intestinalis to investigate the role of arginine and arginine-metabolizing enzymes during intestinal protozoan infections. Results RNA expression analyses of major arginine-metabolizing enzymes revealed the arginine-utilizing pathways in human IECs (differentiated Caco-2 cells) grown in vitro. Most genes were constant or down-regulated (e.g. arginase 1 and 2) upon interaction with Giardia, whereas inducible NO synthase (iNOS) and ornithine decarboxylase (ODC) were up-regulated within 6 h of infection. Giardia was shown to suppress cytokine-induced iNOS expression, thus the parasite has both iNOS inducing and suppressive activities. Giardial arginine consumption suppresses NO production and the NO-degrading parasite protein flavohemoglobin is up-regulated in response to host NO. In addition, the secreted, arginine-consuming giardial enzyme arginine deiminase (GiADI) actively reduces T-cell proliferation in vitro. Interestingly, the effects on NO production and T cell proliferation could be reversed by addition of external arginine or citrulline. Conclusions Giardia affects the host’s arginine metabolism on many different levels. Many of the effects can be reversed by addition of arginine or citrulline, which could be a beneficial supplement in oral rehydration therapy. PMID:24228819

The effect of polymorphic metabolism enzymes on serum phenytoin level.

PubMed

Ozkaynakci, Aydan; Gulcebi, Medine Idrizoglu; Ergeç, Deniz; Ulucan, Korkut; Uzan, Mustafa; Ozkara, Cigdem; Guney, Ilter; Onat, Filiz Yilmaz

2015-03-01

Phenytoin has a widespread use in epilepsy treatment and is mainly metabolized by hepatic cytochrome P450 enzymes (CYP). We have investigated CYP2C9*2, CYP2C9*3, CYP2C19*2 and CYP2C19*3 allelic variants in a Turkish population of patients on phenytoin therapy. Patients on phenytoin therapy (n = 102) for the prevention of epileptic seizures were included. Polymorphic alleles were analyzed by restriction fragment length polymorphism method. Serum concentrations of phenytoin were measured by fluorescence polarization immune assay method. The most frequent genotype was detected for CYP2C9 wild-type alleles (78.43 %), whereas CYP2C19*2/*2 (5.88 %) was the least frequent genotype group. According to the classification made with both enzyme polymorphisms, CYP2C9*1/*1-CYP2C19*1/*1 (G1: 41.17 %) genotype group was the most frequent whereas CYP2C9*1/*2-CYP2C19*1/*3 (G7: 0.98 %) was the least frequent one. The highest mean phenytoin level (27.95 ± 1.85 µg/ml) was detected in the G8 genotype group (CYP2C9*1/*3-CYP2C19*2/*3) and the G1 genotype group showed the lowest mean phenytoin level (7.43 ± 0.73 µg/ml). The mean serum concentration of phenytoin of the polymorphic patients with epilepsy was higher than that for the wild-type alleles both in the monotherapy and polytherapy patients. These results show the importance of the genetic polymorphism analysis of the main metabolizing enzyme groups of phenytoin for the dose adjustment.

Enhanced hepatic and kidney cytochrome p-450 activities in nandrolone decanoate treated albino mice.

PubMed

Acharjee, B K; Mahanta, R

2009-04-01

Anabolic androgenic steroids are the xenobiotic substrates that are metabolized in the body by the protective enzyme systems. Mixed function oxygenase enzymes include a group of enzymes which play an essential role in the metabolism of a broad range of xenobiotics including endogenous and exogenous substrates. Cytochrome P-450, a member of mixed function oxygenase enzymes, plays an important role in oxidative metabolism of drugs and xenobiotics entering human body. Various anabolic steroids are found either to increase or decrease the activity of cytochrome P-450. However, effect of nandrolone decanoate, most commonly abused anabolic steroid, on cytochrome P-450 activity is still fragmentary. In the present study, albino mice were administered intramuscular 2.5 mg of nandrolone decanoate injection at 15 days interval. Cytochrome P-450 activity is determined by following the method of Omura and Sato (1964) in liver and kidney tissues of both normal and experimental groups upto 90 days. Investigation shows a significant (p <0.01) increase of cytochrome P-450 (nmol/mg) activity in liver tissue as compared to that of kidney tissues. A tissue specific and dose specific increase of cytochrome P-450 activity is observed. Mean cytochrome P-450 is found highest in liver tissue on 45(th) day whereas the activity in kidney tissue is noticed on 90(th) day of treatment. From the above observation, nandrolone decanoate can be suggested as a potent inducer of cytochrome P-450 activity like other anabolic steroids.

The RNA world and the origin of metabolic enzymes

PubMed Central

Ralser, Markus

2014-01-01

An RNA world has been placed centre stage for explaining the origin of life. Indeed, RNA is the most plausible molecule able to form both a (self)-replicator and to inherit information, necessities for initiating genetics. However, in parallel with self-replication, the proto-organism had to obtain the ability to catalyse supply of its chemical constituents, including the ribonucleotide metabolites required to replicate RNA. Although the possibility of an RNA-catalysed metabolic network has been considered, it is to be questioned whether RNA molecules, at least on their own, possess the required catalytic capacities. An alternative scenario for the origin of metabolism involves chemical reactions that are based on environmental catalysts. Recently, we described a non-enzymatic glycolysis and pentose phosphate pathway-like reactions catalysed by metal ions [mainly Fe(II)] and phosphate, simple inorganic molecules abundantly found in Archaean sediments. While the RNA world can serve to explain the origin of genetics, the origin of the metabolic network might thus date back to constraints of environmental chemistry. Interestingly, considering a metal-catalysed origin of metabolism gives rise to an attractive hypothesis about how the first enzymes could have formed: simple RNA or (poly)peptide molecules could have bound the metal ions, and thus increased their solubility, concentration and accessibility. In a second step, this would have allowed substrate specificity to evolve. PMID:25109990

A comparative study on the metabolism of Epimedium koreanum Nakai-prenylated flavonoids in rats by an intestinal enzyme (lactase phlorizin hydrolase) and intestinal flora.

PubMed

Zhou, Jing; Chen, Yan; Wang, Ying; Gao, Xia; Qu, Ding; Liu, Congyan

2013-12-24

The aim of this study was to compare the significance of the intestinal hydrolysis of prenylated flavonoids in Herba Epimedii by an intestinal enzyme and flora. Flavonoids were incubated at 37 °C with rat intestinal enzyme and intestinal flora. HPLC-UV was used to calculate the metabolic rates of the parent drug in the incubation and LC/MS/MS was used to determine the chemical structures of metabolites generated by different flavonoid glycosides. Rates of flavonoid metabolism by rat intestinal enzyme were quicker than those of intestinal flora. The sequence of intestinal flora metabolic rates was icariin>epimedin B>epimedin A>epimedin C>baohuoside I, whereas the order of intestinal enzyme metabolic rates was icariin>epimedin A>epimedin C>epimedin B>baohuoside I. Meanwhile, the LC/MS/MS graphs showed that icariin produced three products, epimedin A/B/C had four and baohuoside I yielded one product in incubations of both intestinal enzyme and flora, which were more than the results of HPLC-UV due to the fact LC/MS/MS has lower detectability and higher sensitivity. Moreover, the outcomes indicated that the rate of metabolization of flavonoids by intestinal enzyme were faster than those of intestinal flora, which was consistent with the HPLC-UV results. In conclusion, the metabolic pathways of the same components by intestinal flora and enzyme were the same. What's more, an intestinal enzyme such as lactase phlorizin hydrolase exhibited a more significant metabolic role in prenylated flavonoids of Herba Epimedi compared with intestinal flora.

Cadmium, cobalt and lead cause stress response, cell cycle deregulation and increased steroid as well as xenobiotic metabolism in primary normal human bronchial epithelial cells which is coordinated by at least nine transcription factors.

PubMed

Glahn, Felix; Schmidt-Heck, Wolfgang; Zellmer, Sebastian; Guthke, Reinhard; Wiese, Jan; Golka, Klaus; Hergenröder, Roland; Degen, Gisela H; Lehmann, Thomas; Hermes, Matthias; Schormann, Wiebke; Brulport, Marc; Bauer, Alexander; Bedawy, Essam; Gebhardt, Rolf; Hengstler, Jan G; Foth, Heidi

2008-08-01

Workers occupationally exposed to cadmium, cobalt and lead have been reported to have increased levels of DNA damage. To analyze whether in vivo relevant concentrations of heavy metals cause systematic alterations in RNA expression patterns, we performed a gene array study using primary normal human bronchial epithelial cells. Cells were incubated with 15 microg/l Cd(II), 25 microg/l Co(II) and 550 microg/l Pb(II) either with individual substances or in combination. Differentially expressed genes were filtered out and used to identify enriched GO categories as well as KEGG pathways and to identify transcription factors whose binding sites are enriched in a given set of promoters. Interestingly, combined exposure to Cd(II), Co(II) and Pb(II) caused a coordinated response of at least seven stress response-related transcription factors, namely Oct-1, HIC1, TGIF, CREB, ATF4, SRF and YY1. A stress response was further corroborated by up regulation of genes involved in glutathione metabolism. A second major response to heavy metal exposure was deregulation of the cell cycle as evidenced by down regulation of the transcription factors ELK-1 and the Ets transcription factor GABP, as well as deregulation of genes involved in purine and pyrimidine metabolism. A third and surprising response was up regulation of genes involved in steroid metabolism, whereby promoter analysis identified up regulation of SRY that is known to play a role in sex determination. A forth response was up regulation of xenobiotic metabolising enzymes, particularly of dihydrodiol dehydrogenases 1 and 2 (AKR1C1, AKR1C2). Incubations with individual heavy metals showed that the response of AKR1C1 and AKR1C2 was predominantly caused by lead. In conclusion, we have shown that in vivo relevant concentrations of Cd(II), Co(II) and Pb(II) cause a complex and coordinated response in normal human bronchial epithelial cells. This study gives an overview of the most responsive genes.

Applicability of the Rayleigh equation for enantioselective metabolism of chiral xenobiotics by microsomes, hepatocytes and in-vivo retention in rabbit tissues

PubMed Central

Jammer, Shifra; Gelman, Faina; Lev, Ovadia

2016-01-01

In this study we propose a new approach for analyzing the enantioselective biodegradation of some antidepressant drugs mediated by human and rat liver microsomes by using the Rayleigh equation to describe the enantiomeric enrichment−conversion dependencies. Analysis of reported degradation data of additional six pesticides, an alpha blocker and a flame retardant by microsomes or hepatocytes in vitro reaffirmed the universality of the approach. In all the in vitro studied cases that involved enantioselective degradation, a Rayleigh dependence of the enantiomeric enrichment was observed. Published data regarding in vivo retention of myclobutanil in liver, kidney, muscle and brain tissues of rabbits following injection of the racemate were remodeled showing prevalence of the Rayleigh law for the chiral enrichment of the fungicide in the various tissues. This approach will revolutionize data organization in metabolic pathway research of target xenobiotics by either liver microsomes, hepatocytes or their organ-specific in vivo retention. The fact that the enantiomeric enrichment as a function of the conversion can be described by a single quantifier, will pave the road for the use of structure activity predictors of the enantiomeric enrichment and for mechanistic discrimination based on parametric dependence of the quantifier. PMID:27021918

Chimeric mice with humanized liver: Application in drug metabolism and pharmacokinetics studies for drug discovery.

PubMed

Naritomi, Yoichi; Sanoh, Seigo; Ohta, Shigeru

2018-02-01

Predicting human drug metabolism and pharmacokinetics (PK) is key to drug discovery. In particular, it is important to predict human PK, metabolite profiles and drug-drug interactions (DDIs). Various methods have been used for such predictions, including in vitro metabolic studies using human biological samples, such as hepatic microsomes and hepatocytes, and in vivo studies using experimental animals. However, prediction studies using these methods are often inconclusive due to discrepancies between in vitro and in vivo results, and interspecies differences in drug metabolism. Further, the prediction methods have changed from qualitative to quantitative to solve these issues. Chimeric mice with humanized liver have been developed, in which mouse liver cells are mostly replaced with human hepatocytes. Since human drug metabolizing enzymes are expressed in the liver of these mice, they are regarded as suitable models for mimicking the drug metabolism and PK observed in humans; therefore, these mice are useful for predicting human drug metabolism and PK. In this review, we discuss the current state, issues, and future directions of predicting human drug metabolism and PK using chimeric mice with humanized liver in drug discovery. Copyright © 2017 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

Design and Performance of a Xenobiotic Metabolism Database Manager for Building Metabolic Pathway Databases

EPA Science Inventory

A major challenge for scientists and regulators is accounting for the metabolic activation of chemicals that may lead to increased toxicity. Reliable forecasting of chemical metabolism is a critical factor in estimating a chemical’s toxic potential. Research is underway to develo...

Pregnane X receptor- and CYP3A4-humanized mouse models and their applications

PubMed Central

Cheng, Jie; Ma, Xiaochao; Gonzalez, Frank J

2011-01-01

Pregnane X receptor (PXR) is a pivotal nuclear receptor modulating xenobiotic metabolism primarily through its regulation of CYP3A4, the most important enzyme involved in drug metabolism in humans. Due to the marked species differences in ligand recognition by PXR, PXR-humanized (hPXR) mice, and mice expressing human PXR and CYP3A4 (Tg3A4/hPXR) were established. hPXR and Tg3A4/hPXR mice are valuable models for investigating the role of PXR in xenobiotic metabolism and toxicity, in lipid, bile acid and steroid hormone homeostasis, and in the control of inflammation. PMID:21091656

DEVELOPMENT OF A CLASS-SELECTIVE ENZYME IMMUNOASSAY FOR URINARY PHENOLIC GLUCURONIDES. (R825433)

EPA Science Inventory

Class-selective immunoassays for the measurement of glucuronides in human urine can aid evaluation of human exposure to complex mixtures of xenobiotics. Therefore, an enzyme immunoassay (EIA) for the group-selective detection of phenolic Membrane Phospholipid Augments Cytochrome P4501a Enzymatic Activity by Modulating Structural Conformation during Detoxification of Xenobiotics

PubMed Central

Ghosh, Manik C.; Ray, Arun K.

2013-01-01

Cytochrome P450 is a superfamily of membrane-bound hemoprotein that gets involved with the degradation of xenobiotics and internal metabolites. Accumulated body of evidence indicates that phospholipids play a crucial role in determining the enzymatic activity of cytochrome P450 in the microenvironment by modulating its structure during detoxification; however, the structure-function relationship of cytochrome P4501A, a family of enzymes responsible for degrading lipophilic aromatic hydrocarbons, is still not well defined. Inducibility of cytochrome P4501A in cultured catfish hepatocytes in response to carbofuran, a widely used pesticide around the world, was studied earlier in our laboratory. In this present investigation, we observed that treating catfish with carbofuran augmented total phospholipid in the liver. We examined the role of phospholipid on the of cytochrome P4501A-marker enzyme which is known as ethoxyresorufin-O-deethylase (EROD) in the context of structure and function. We purified the carbofuran-induced cytochrome P4501A protein from catfish liver. Subsequently, we examined the enzymatic activity of purified P4501A protein in the presence of phospholipid, and studied how the structure of purified protein was influenced in the phospholipid environment. Membrane phospholipid appeared to accelerate the enzymatic activity of EROD by changing its structural conformation and thus controlling the detoxification of xenobiotics. Our study revealed the missing link of how the cytochrome P450 restores its enzymatic activity by changing its structural conformation in the phospholipid microenvironment. PMID:23469105

Membrane phospholipid augments cytochrome P4501a enzymatic activity by modulating structural conformation during detoxification of xenobiotics.

PubMed

Ghosh, Manik C; Ray, Arun K

2013-01-01

Cytochrome P450 is a superfamily of membrane-bound hemoprotein that gets involved with the degradation of xenobiotics and internal metabolites. Accumulated body of evidence indicates that phospholipids play a crucial role in determining the enzymatic activity of cytochrome P450 in the microenvironment by modulating its structure during detoxification; however, the structure-function relationship of cytochrome P4501A, a family of enzymes responsible for degrading lipophilic aromatic hydrocarbons, is still not well defined. Inducibility of cytochrome P4501A in cultured catfish hepatocytes in response to carbofuran, a widely used pesticide around the world, was studied earlier in our laboratory. In this present investigation, we observed that treating catfish with carbofuran augmented total phospholipid in the liver. We examined the role of phospholipid on the of cytochrome P4501A-marker enzyme which is known as ethoxyresorufin-O-deethylase (EROD) in the context of structure and function. We purified the carbofuran-induced cytochrome P4501A protein from catfish liver. Subsequently, we examined the enzymatic activity of purified P4501A protein in the presence of phospholipid, and studied how the structure of purified protein was influenced in the phospholipid environment. Membrane phospholipid appeared to accelerate the enzymatic activity of EROD by changing its structural conformation and thus controlling the detoxification of xenobiotics. Our study revealed the missing link of how the cytochrome P450 restores its enzymatic activity by changing its structural conformation in the phospholipid microenvironment.

Predicting the Metabolic Sites by Flavin-Containing Monooxygenase on Drug Molecules Using SVM Classification on Computed Quantum Mechanics and Circular Fingerprints Molecular Descriptors

PubMed Central

Fu, Chien-wei; Lin, Thy-Hou

2017-01-01

As an important enzyme in Phase I drug metabolism, the flavin-containing monooxygenase (FMO) also metabolizes some xenobiotics with soft nucleophiles. The site of metabolism (SOM) on a molecule is the site where the metabolic reaction is exerted by an enzyme. Accurate prediction of SOMs on drug molecules will assist the search for drug leads during the optimization process. Here, some quantum mechanics features such as the condensed Fukui function and attributes from circular fingerprints (called Molprint2D) are computed and classified using the support vector machine (SVM) for predicting some potential SOMs on a series of drugs that can be metabolized by FMO enzymes. The condensed Fukui function fA− representing the nucleophilicity of central atom A and the attributes from circular fingerprints accounting the influence of neighbors on the central atom. The total number of FMO substrates and non-substrates collected in the study is 85 and they are equally divided into the training and test sets with each carrying roughly the same number of potential SOMs. However, only N-oxidation and S-oxidation features were considered in the prediction since the available C-oxidation data was scarce. In the training process, the LibSVM package of WEKA package and the option of 10-fold cross validation are employed. The prediction performance on the test set evaluated by accuracy, Matthews correlation coefficient and area under ROC curve computed are 0.829, 0.659, and 0.877 respectively. This work reveals that the SVM model built can accurately predict the potential SOMs for drug molecules that are metabolizable by the FMO enzymes. PMID:28072829

In vitro metabolism of a novel JNK inhibitor tanzisertib: interspecies differences in oxido-reduction and characterization of enzymes involved in metabolism.

PubMed

Atsriku, Christian; Hoffmann, Matthew; Moghaddam, Mehran; Kumar, Gondi; Surapaneni, Sekhar

2015-01-01

1. In vitro metabolism of Tanzisertib [(1S,4R)-4-(9-((S)tetrahydrofuran-3-yl)-8-(2,4,6-trifluorophenylamino)-9H-purin-2-ylamino) cyclohexanol], a potent, selective c-Jun amino-terminal kinase (JNK) inhibitor, was investigated in mouse, rat, rabbit, dog, monkey and human hepatocytes over 4 h. The extent of metabolism of [(14)C]tanzisertib was variable, with <10% metabolized in dog and human, <20% metabolized in rabbit and monkey and >75% metabolized in rat and mouse. Primary metabolic pathways in human and dog hepatocytes, were direct glucuronidation and oxidation of cyclohexanol to a keto metabolite, which was subsequently reduced to parent or cis-isomer, followed by glucuronidation. Rat and mouse produced oxidative metabolites and cis-isomer, including direct glucuronides and sulfates of tanzisertib and cis-isomer. 2. Enzymology of oxido-reductive pathways revealed that human aldo-keto reductases AKR1C1, 1C2, 1C3 and 1C4 were responsible for oxido-reduction of tanzisertib, CC-418424 and keto tanzisertib. Characterizations of enzyme kinetics revealed that AKR1C4 had a high affinity for reduction of keto tanzisertib to tanzisertib compared to other isoforms. These results demonstrate unique stereoselectivity of the reductive properties documented by human AKR1C enzymes for the same substrate. 3. Characterization of UGT isoenzymes in glucuronidation of tanzisertib and CC-418424 revealed that, tanzisertib glucuronide was catalyzed by: UGT1A1, 1A4, 1A10 and 2B4, while CC-418424 glucuronidation was catalyzed by UGT2B4 and 2B7.

Regulation of sucrose metabolism in higher plants: localization and regulation of activity of key enzymes

NASA Technical Reports Server (NTRS)

Winter, H.; Huber, S. C.; Brown, C. S. (Principal Investigator)

2000-01-01

Sucrose (Suc) plays a central role in plant growth and development. It is a major end product of photosynthesis and functions as a primary transport sugar and in some cases as a direct or indirect regulator of gene expression. Research during the last 2 decades has identified the pathways involved and which enzymes contribute to the control of flux. Availability of metabolites for Suc synthesis and 'demand' for products of sucrose degradation are important factors, but this review specifically focuses on the biosynthetic enzyme sucrose-phosphate synthase (SPS), and the degradative enzymes, sucrose synthase (SuSy), and the invertases. Recent progress has included the cloning of genes encoding these enzymes and the elucidation of posttranslational regulatory mechanisms. Protein phosphorylation is emerging as an important mechanism controlling SPS activity in response to various environmental and endogenous signals. In terms of Suc degradation, invertase-catalyzed hydrolysis generally has been associated with cell expansion, whereas SuSy-catalyzed metabolism has been linked with biosynthetic processes (e.g., cell wall or storage products). Recent results indicate that SuSy may be localized in multiple cellular compartments: (1) as a soluble enzyme in the cytosol (as traditionally assumed); (2) associated with the plasma membrane; and (3) associated with the actin cytoskeleton. Phosphorylation of SuSy has been shown to occur and may be one of the factors controlling localization of the enzyme. The purpose of this review is to summarize some of the recent developments relating to regulation of activity and localization of key enzymes involved in sucrose metabolism in plants.

Molecular Basis of Microbial One-Carbon Metabolism 2008 Gordon Research Conference (July 20-25, 2008)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stephen W. Ragsdale

2009-08-12

One-carbon (C-1) compounds play a central role in microbial metabolism. C-1 compounds include methane, carbon monoxide, CO2, and methanol as well as coenzyme-bound one-carbon compounds (methyl-B12, CH3-H4folate, etc). Such compounds are of broad global importance because several C-1 compounds (e.g., CH4) are important energy sources, some (e.g., CO2 and CH4) are potent greenhouse gases, and others (e.g., CH2Cl2) are xenobiotics. They are central in pathways of energy metabolism and carbon fixation by microbes and many are of industrial interest. Research on the pathways of one-carbon metabolism has added greatly to our understanding of evolution, structural biology, enzyme mechanisms, gene regulation,more » ecology, and applied biology. The 2008 meeting will include recent important findings in the following areas: (a) genomics, metagenomics, and proteomic studies that have expanded our understanding of autotrophy and C-1 metabolism and the evolution of these pathways; (b) redox regulation of carbon cycles and the interrelationship between the carbon cycle and other biogeochemical cycles (sulfur, nitrogen, oxygen); (c) novel pathways for carbon assimilation; (d) biotechnology related to C-1 metabolism; (e) novel enzyme mechanisms including channeling of C-1 intermediates during metabolism; and (f) the relationship between metal homeostasis and the global carbon cycle. The conference has a diverse and gender-balanced slate of speakers and session leaders. The wide variety of disciplines brought to the study of C-1 metabolism make the field an excellent one in which to train young researchers.« less

Identification of the Human SULT Enzymes Involved in the Metabolism of Rotigotine.

PubMed

Jia, Chaojun; Luo, Lijun; Kurogi, Katsuhisa; Yu, Juming; Zhou, Chunyang; Liu, Ming-Cheh

2016-06-01

Sulfation has been reported to be a major pathway for the metabolism and inactivation of rotigotine in vivo. The current study aimed to identify the human cytosolic sulfotransferase (SULT) enzyme(s) capable of mediating the sulfation of rotigotine. Of the 13 known human SULTs examined, 6 of them (SULT1A1, 1A2, 1A3, 1B1, 1C4, 1E1) displayed significant sulfating activities toward rotigotine. pH dependence and kinetic parameters of the sulfation of rotigotine by relevant human SULTs were determined. Of the 6 human organ samples tested, small intestine and liver cytosols displayed considerably higher rotigotine-sulfating activity than did brain, lung, and kidney. Moreover, sulfation of rotigotine was shown to occur in HepG2 human hepatoma cells and Caco-2 human colon adenocarcinoma cells under metabolic conditions. Collectively, the results obtained provided a molecular basis underlying the previous finding of the excretion of sulfated rotigotine by patients undergoing treatment with rotigotine. © 2015, The American College of Clinical Pharmacology.

SIMULATING METABOLISM OF XENOBIOTIC CHEMICALS AS A PREDICTOR OF TOXICITY

EPA Science Inventory

EPA is faced with long lists of chemicals that need to be assessed for hazard. A major gap in evaluating chemical risk is accounting for metabolic activation resulting in increased toxicity. The goals of this project are to develop a capability to forecast the metabolism of xenob...

Developmental changes in drug-metabolizing enzyme expression during metamorphosis of Xenopus tropicalis.

PubMed

Mori, Junpei; Sanoh, Seigo; Kashiwagi, Keiko; Hanada, Hideki; Shigeta, Mitsuki; Suzuki, Ken-Ichi T; Yamamoto, Takashi; Kotake, Yaichiro; Sugihara, Kazumi; Kitamura, Shigeyuki; Kashiwagi, Akihiko; Ohta, Shigeru

2017-01-01

A large number of chemicals are routinely detected in aquatic environments, and these chemicals may adversely affect aquatic organisms. Accurate risk assessment requires understanding drug-metabolizing systems in aquatic organisms because metabolism of these chemicals is a critical determinant of chemical bioaccumulation and related toxicity. In this study, we evaluated mRNA expression levels of nuclear receptors and drug-metabolizing enzymes as well as cytochrome P450 (CYP) activities in pro-metamorphic tadpoles, froglets, and adult frogs to determine how drug-metabolizing systems are altered at different life stages. We found that drug-metabolizing systems in tadpoles were entirely immature, and therefore, tadpoles appeared to be more susceptible to chemicals compared with metamorphosed frogs. On the other hand, cyp1a mRNA expression and CYP1A-like activity were higher in tadpoles. We found that thyroid hormone (TH), which increases during metamorphosis, induced CYP1A-like activity. Because endogenous TH concentration is significantly increased during metamorphosis, endogenous TH would induce CYP1A-like activity in tadpoles.

Hepatotoxicity of Herbal Supplements Mediated by Modulation of Cytochrome P450

PubMed Central

Chen, Taosheng

2017-01-01

Herbal supplements are a significant source of drug-drug interactions (DDIs), herb-drug interactions, and hepatotoxicity. Cytochrome P450 (CYP450) enzymes metabolize a large number of FDA-approved pharmaceuticals and herbal supplements. This metabolism of pharmaceuticals and supplements can be augmented by concomitant use of either pharmaceuticals or supplements. The xenobiotic receptors constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) can respond to xenobiotics by increasing the expression of a large number of genes that are involved in the metabolism of xenobiotics, including CYP450s. Conversely, but not exclusively, many xenobiotics can inhibit the activity of CYP450s. Induction of the expression or inhibition of the activity of CYP450s can result in DDIs and toxicity. Currently, the United States (US) Food and Drug Administration does not require the investigation of the interactions of herbal supplements and CYP450s. This review provides a summary of herbal supplements that inhibit CYP450s, induce the expression of CYP450s, and/or whose toxicity is mediated by CYP450s. PMID:29117101

Reversal of high fat diet-induced obesity through modulating lipid metabolic enzymes and inflammatory markers expressions in rats.

PubMed

A, Kalaivani; Uddandrao, V V Sathibabu; Parim, Brahmanaidu; Ganapathy, Saravanan; P R, Nivedha; Kancharla, Sushma Chandulee; P, Rameshreddy; K, Swapna; Sasikumar, Vadivukkarasi

2018-03-19

In this study, we evaluated the ameliorative potential of Cucurbita maxima seeds oil (CSO (100 mg/kg body weight)) supplementation to high fat diet (HFD)-induced obese rats for 30 days on the changes in body weight, markers of lipid metabolism such as LDL, HDL, triglycerides, total cholesterol, adiponectin, leptin, amylase, and lipase. We also investigated the effects of CSO on the changes of lipid metabolic enzymes such as fatty-acid synthase, acetyl CoA carboxylase, carnitine palmitoyl transferase-1, HMG CoA reductase, and inflammatory markers (TNF-α and IL-6). Administration of CSO revealed significant diminution in body weight gain, altered the activity, expressions of lipid marker enzymes and inflammatory markers. It demonstrated that CSO had considerably altered these parameters when evaluated with HFD control rats. In conclusion, this study suggested that CSO might ameliorate the HFD-induced obesity by altering the enzymes and mRNA expressions important to lipid metabolism.

Human cytochrome-P450 enzymes metabolize N-(2-methoxyphenyl)hydroxylamine, a metabolite of the carcinogens o-anisidine and o-nitroanisole, thereby dictating its genotoxicity.

PubMed

Naiman, Karel; Martínková, Markéta; Schmeiser, Heinz H; Frei, Eva; Stiborová, Marie

2011-12-24

N-(2-Methoxyphenyl)hydroxylamine is a component in the human metabolism of two industrial and environmental pollutants and bladder carcinogens, viz. 2-methoxyaniline (o-anisidine) and 2-methoxynitrobenzene (o-nitroanisole), and it is responsible for their genotoxicity. Besides its capability to form three deoxyguanosine adducts in DNA, N-(2-methoxyphenyl)-hydroxylamine is also further metabolized by hepatic microsomal enzymes. To investigate its metabolism by human hepatic microsomes and to identify the major microsomal enzymes involved in this process are the aims of this study. N-(2-Methoxyphenyl)hydroxylamine is metabolized by human hepatic microsomes predominantly to o-anisidine, one of the parent carcinogens from which N-(2-methoxyphenyl)hydroxylamine is formed, while o-aminophenol and two N-(2-methoxyphenyl)hydroxylamine metabolites, whose exact structures have not been identified as yet, are minor products. Selective inhibitors of microsomal CYPs, NADPH:CYP reductase and NADH:cytochrome-b(5) reductase were used to characterize human liver microsomal enzymes reducing N-(2-methoxyphenyl)hydroxylamine to o-anisidine. Based on these studies, we attribute the main activity for this metabolic step in human liver to CYP3A4, 2E1 and 2C (more than 90%). The enzymes CYP2D6 and 2A6 also partake in this N-(2-methoxyphenyl)hydroxylamine metabolism in human liver, but only to ∼6%. Among the human recombinant CYP enzymes tested in this study, human CYP2E1, followed by CYP3A4, 1A2, 2B6 and 2D6, were the most efficient enzymes metabolizing N-(2-methoxyphenyl)hydroxylamine to o-anisidine. The results found in this study indicate that genotoxicity of N-(2-methoxyphenyl)hydroxylamine is dictated by its spontaneous decomposition to nitrenium/carbenium ions generating DNA adducts, and by its susceptibility to metabolism by CYP enzymes. Copyright © 2011 Elsevier B.V. All rights reserved.

Pyruvate decarboxylase and alcohol dehydrogenase overexpression in Escherichia coli resulted in high ethanol production and rewired metabolic enzyme networks.

PubMed

Yang, Mingfeng; Li, Xuefeng; Bu, Chunya; Wang, Hui; Shi, Guanglu; Yang, Xiushan; Hu, Yong; Wang, Xiaoqin

2014-11-01

Pyruvate decarboxylase and alcohol dehydrogenase are efficient enzymes for ethanol production in Zymomonas mobilis. These two enzymes were over-expressed in Escherichia coli, a promising candidate for industrial ethanol production, resulting in high ethanol production in the engineered E. coli. To investigate the intracellular changes to the enzyme overexpression for homoethanol production, 2-DE and LC-MS/MS were performed. More than 1,000 protein spots were reproducibly detected in the gel by image analysis. Compared to the wild-type, 99 protein spots showed significant changes in abundance in the recombinant E. coli, in which 46 were down-regulated and 53 were up-regulated. Most proteins related to tricarboxylic acid cycle, glycerol metabolism and other energy metabolism were up-regulated, whereas proteins involved in glycolysis and glyoxylate pathway were down-regulated, indicating the rewired metabolism in the engineered E. coli. As glycolysis is the main pathway for ethanol production, and it was inhibited significantly in engineered E. coli, further efforts should be directed at minimizing the repression of glycolysis to optimize metabolism network for higher yields of ethanol production.

The effect of chlorpyrifos-oxon and other xenobiotics on the human cytochrome P450-dependent metabolism of naphthalene and deet.

PubMed

Cho, Taehyeon M; Rose, Randy L; Hodgson, Ernest

2007-01-01

Chlorpyrifos-oxon (CPO), a metabolite of chlorpyrifos, is a potent inhibitor of acetylcholinesterase and, although the neurotoxicological impact of this organophosphorus compound has been broadly studied both in vitro and in vivo, there are few studies of metabolic interactions of CPO with other xenobiotics. CPO significantly activated the production of 1-naphthol (5-fold), 2-naphthol (10-fold), trans-1,2-dihydro-1,2-naphthalenediol (1.5-fold), and 1,4-naphthoquinone from naphthalene by human liver microsomes (HLM). It was further demonstrated that the production of naphthalene metabolites by CYP2C8, 2C9*(1), 2C19, 2D6*(1), 3A4, 3A5, and 3A7 was activated by CPO, while the production of naphthalene metabolites by CYP1A1, 1A2, 1B1, and 2B6 was inhibited by CPO. CPO inhibited CYP1A2 production of naphthalene metabolites, while activating their production by CYP3A4. Similarly, CPO inhibited the production of N,N-diethyl-m-hydroxymethylbenzamide (BALC) from DEET by human liver microsomes, but activated the production of N-ethyl-m-toluamide (ET) from this substrate. CYP2B6, the most efficient isoform for BALC production, was inhibited by CPO, while CYP3A4, the most efficient isoform for ET production, was activated by CPO. CPO inhibited CYP2B6 production of both BALC and ET from DEET, but activated CYP3A4 production of ET, while inhibiting CYP3A4 BALC production. CPO appears to facilitate the binding of naphthalene to CYP3A4. This metabolic activation is independent of cytochrome b5, suggesting that activation of CYP3A4 by CPO is associated with a conformational change of the isoform rather than facilitating electron transfer.

Identification of enzymes responsible for extracellular alginate depolymerization and alginate metabolism in Vibrio algivorus.

PubMed

Doi, Hidetaka; Tokura, Yuriko; Mori, Yukiko; Mori, Kenichi; Asakura, Yoko; Usuda, Yoshihiro; Fukuda, Hiroo; Chinen, Akito

2017-02-01

Alginate is a marine non-food-competing polysaccharide that has potential applications in biorefinery. Owing to its large size (molecular weight >300,000 Da), alginate cannot pass through the bacterial cell membrane. Therefore, bacteria that utilize alginate are presumed to have an enzyme that degrades extracellular alginate. Recently, Vibrio algivorus sp. SA2 T was identified as a novel alginate-decomposing and alginate-utilizing species. However, little is known about the mechanism of alginate degradation and metabolism in this species. To address this issue, we screened the V. algivorus genomic DNA library for genes encoding polysaccharide-decomposing enzymes using a novel double-layer plate screening method and identified alyB as a candidate. Most identified alginate-decomposing enzymes (i.e., alginate lyases) must be concentrated and purified before extracellular alginate depolymerization. AlyB of V. algivorus heterologously expressed in Escherichia coli depolymerized extracellular alginate without requiring concentration or purification. We found seven homologues in the V. algivorus genome (alyB, alyD, oalA, oalB, oalC, dehR, and toaA) that are thought to encode enzymes responsible for alginate transport and metabolism. Introducing these genes into E. coli enabled the cells to assimilate soluble alginate depolymerized by V. algivorus AlyB as the sole carbon source. The alginate was bioconverted into L-lysine (43.3 mg/l) in E. coli strain AJIK01. These findings demonstrate a simple and novel screening method for identifying polysaccharide-degrading enzymes in bacteria and provide a simple alginate biocatalyst and fermentation system with potential applications in industrial biorefinery.

Distinct patterns of dysregulated expression of enzymes involved in androgen synthesis and metabolism in metastatic prostate cancer tumors

PubMed Central

Mitsiades, Nicholas; Sung, Clifford C.; Schultz, Nikolaus; Danila, Daniel C.; He, Bin; Eedunuri, Vijay Kumar; Fleisher, Martin; Sander, Chris; Sawyers, Charles L.; Scher, Howard I.

2012-01-01

Androgen receptor (AR) signaling persists in castration-resistant prostate carcinomas (CRPCs), due to several mechanisms that include increased AR expression and intratumoral androgen metabolism. We investigated the mechanisms underlying aberrant expression of transcripts involved in androgen metabolism in CRPC. We compared gene expression profiles and DNA copy number alteration (CNA) data from 29 normal prostate tissue samples, 127 primary prostate carcinomas (PCas) and 19 metastatic PCas. Steroidogenic enzyme transcripts were evaluated by qRT-PCR in PCa cell lines and circulating tumor cells (CTCs) from CRPC patients. Metastatic PCas expressed higher transcript levels for AR and several steroidogenic enzymes, including SRD5A1, SRD5A3, and AKR1C3, while expression of SRD5A2, CYP3A4, CYP3A5 and CYP3A7 was decreased. This aberrant expression was rarely associated with CNAs. Instead, our data suggest distinct patterns of coordinated aberrant enzyme expression. Inhibition of AR activity by itself stimulated AKR1C3 expression. The aberrant expression of the steroidogenic enzyme transcripts were detected in CTCs from CRPC patients. In conclusion, our findings identify substantial interpatient heterogeneity and distinct patterns of dysregulated expression of enzymes involved in intratumoral androgen metabolism in PCa. These steroidogenic enzymes represent targets for complete suppression of systemic and intratumoral androgen levels, an objective that is supported by the clinical efficacy of the CYP17 inhibitor abiraterone. A comprehensive AR axis targeting approach via simultaneous, frontline enzymatic blockade and/or transcriptional repression of several steroidogenic enzymes, in combination with GnRH analogs and potent anti-androgens, would represent a powerful future strategy for PCa management. PMID:22971343

Cysteine S-conjugate β-lyases: Important roles in the metabolism of naturally occurring sulfur and selenium-containing compounds, xenobiotics and anticancer agents

PubMed Central

Cooper, Arthur J. L.; Krasnikov, Boris F.; Niatsetskaya, Zoya V.; Pinto, John T.; Callery, Patrick S.; Villar, Maria T.; Artigues, Antonio; Bruschi, Sam A.

2010-01-01

Summary Cysteine S-conjugate β-lyases are pyridoxal 5′-phosphate-containing enzymes that catalyze β-elimination reactions with cysteine S-conjugates that possess a good leaving group in the β-position. The end products are aminoacrylate and a sulfur-containing fragment. The aminoacrylate tautomerizes and hydrolyzes to pyruvate and ammonia. The mammalian cysteine S-conjugate β-lyases thus far identified are enzymes involved in amino acid metabolism that catalyze β-lyase reactions as non-physiological side reactions. Most are aminotransferases. In some cases the lyase is inactivated by reaction products. The cysteine S-conjugate β-lyases are of much interest to toxicologists because they play an important key role in the bioactivation (toxication) of halogenated alkenes, some of which are produced on an industrial scale and are environmental contaminants. The cysteine S-conjugate β-lyases have been reviewed in this journal previously [Cooper and Pinto, 2006]. Here we focus on more recent findings regarding: 1) the identification of enzymes associated with high-Mr cysteine S-conjugate β-lyases in the cytosolic and mitochondrial fractions of rat liver and kidney; 2) the mechanism of syncatalytic inactivation of rat liver mitochondrial aspartate aminotransferase by the nephrotoxic β-lyase substrate S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (the cysteine S-conjugate of tetrafluoroethylene); 3) toxicant channeling of reactive fragments from the active site of mitochondrial aspartate aminotransferase to susceptible proteins in the mitochondria; 4) the involvement of cysteine S-conjugate β-lyases in the metabolism/bioactivation of drugs and natural products; and 5) the role of cysteine S-conjugate β-lyases in the metabolism of selenocysteine Se-conjugates. This review emphasizes the fact that the cysteine S-conjugate β-lyases are biologically more important than hitherto appreciated. PMID:20306345

Identification of human drug-metabolizing enzymes involved in the metabolism of SNI-2011.

PubMed

Washio, T; Arisawa, H; Kohsaka, K; Yasuda, H

2001-11-01

In vitro studies were conducted to identify human drug-metabolizing enzymes involved in the metabolism of SNI-2011 ((+/-)-cis-2-methylspiro [1,3-oxathiolane-5,3'-quinuclidine] monohydrochloride hemihydrate, cevimeline hydrochloride hydrate). When 14C-SNI-2011 was incubated with human liver microsomes, SNI-2011 trans-sulfoxide and cis-sulfoxide were detected as major metabolites. These oxidations required NADPH, and were markedly inhibited by SKF-525A, indicating that cytochrome P450 (CYP) was involved. In a chemical inhibition study, metabolism of SNI-2011 in liver microsomes was inhibited (35-65%) by CYP3A4 inhibitors (ketoconazole and troleandomycin) and CYP2D6 inhibitors (quinidine and chlorpromazine). Furthermore, using microsomes containing cDNA-expressed CYPs, it was found that high rates of sulfoxidation activities were observed with CYP2D6 and CYP3A4. On the other hand, when 14C-SNI-2011 was incubated with human kidney microsomes, SNI-2011 N-oxide was identified as a major metabolite. This N-oxidation required NADPH, and was completely inhibited by thiourea, indicating that flavin-containing monooxygenase (FMO) was involved. In addition, microsomes containing cDNA-expressed FMO1, a major isoform in human kidney, mainly catalyzed N-oxidation of SNI-2011, but microsomes containing FMO3, a major isoform in adult human liver, did not. These results suggest that SNI-2011 is mainly catalyzed to sulfoxides and N-oxide by CYP2D6/3A4 in liver and FMOI in kidney, respectively.

Characterization of human DHRS4: an inducible short-chain dehydrogenase/reductase enzyme with 3beta-hydroxysteroid dehydrogenase activity.

PubMed

Matsunaga, Toshiyuki; Endo, Satoshi; Maeda, Satoshi; Ishikura, Shuhei; Tajima, Kazuo; Tanaka, Nobutada; Nakamura, Kazuo T; Imamura, Yorishige; Hara, Akira

2008-09-15

Human DHRS4 is a peroxisomal member of the short-chain dehydrogenase/reductase superfamily, but its enzymatic properties, except for displaying NADP(H)-dependent retinol dehydrogenase/reductase activity, are unknown. We show that the human enzyme, a tetramer composed of 27kDa subunits, is inactivated at low temperature without dissociation into subunits. The cold inactivation was prevented by a mutation of Thr177 with the corresponding residue, Asn, in cold-stable pig DHRS4, where this residue is hydrogen-bonded to Asn165 in a substrate-binding loop of other subunit. Human DHRS4 reduced various aromatic ketones and alpha-dicarbonyl compounds including cytotoxic 9,10-phenanthrenequinone. The overexpression of the peroxisomal enzyme in cultured cells did not increase the cytotoxicity of 9,10-phenanthrenequinone. While its activity towards all-trans-retinal was low, human DHRS4 efficiently reduced 3-keto-C(19)/C(21)-steroids into 3beta-hydroxysteroids. The stereospecific conversion to 3beta-hydroxysteroids was observed in endothelial cells transfected with vectors expressing the enzyme. The mRNA for the enzyme was ubiquitously expressed in human tissues and several cancer cells, and the enzyme in HepG2 cells was induced by peroxisome-proliferator-activated receptor alpha ligands. The results suggest a novel mechanism of cold inactivation and role of the inducible human DHRS4 in 3beta-hydroxysteroid synthesis and xenobiotic carbonyl metabolism.

Nuclear Receptor Involvement in PPAA-Induced Metabolic Changes.

EPA Science Inventory

It has been proposed that certain xenobiotics commonly identified in biomonitoring studies may play a role in the incidence of obesity and metabolic syndrome in the United States and other countries. The list of potential "environmental obesogens" includes endocrine disrupting co...

Adapting capillary gel electrophoresis as a sensitive, high-throughput method to accelerate characterization of nucleic acid metabolic enzymes

PubMed Central

Greenough, Lucia; Schermerhorn, Kelly M.; Mazzola, Laurie; Bybee, Joanna; Rivizzigno, Danielle; Cantin, Elizabeth; Slatko, Barton E.; Gardner, Andrew F.

2016-01-01

Detailed biochemical characterization of nucleic acid enzymes is fundamental to understanding nucleic acid metabolism, genome replication and repair. We report the development of a rapid, high-throughput fluorescence capillary gel electrophoresis method as an alternative to traditional polyacrylamide gel electrophoresis to characterize nucleic acid metabolic enzymes. The principles of assay design described here can be applied to nearly any enzyme system that acts on a fluorescently labeled oligonucleotide substrate. Herein, we describe several assays using this core capillary gel electrophoresis methodology to accelerate study of nucleic acid enzymes. First, assays were designed to examine DNA polymerase activities including nucleotide incorporation kinetics, strand displacement synthesis and 3′-5′ exonuclease activity. Next, DNA repair activities of DNA ligase, flap endonuclease and RNase H2 were monitored. In addition, a multicolor assay that uses four different fluorescently labeled substrates in a single reaction was implemented to characterize GAN nuclease specificity. Finally, a dual-color fluorescence assay to monitor coupled enzyme reactions during Okazaki fragment maturation is described. These assays serve as a template to guide further technical development for enzyme characterization or nucleoside and non-nucleoside inhibitor screening in a high-throughput manner. PMID:26365239

Comparative effects of dimethylsulfoxide on metabolism and toxicity of carbon tetrachloride and dichloromethane.

PubMed

Kim, Sun J; Jung, Young S; Yoon, Mi Y; Kim, Young C

2007-01-01

The effects of dimethylsulfoxide (DMSO) on the metabolism and toxicity of chlorinated methanes were examined. Male mice were treated with DMSO (1, 2.5 or 5 ml kg(-1), i.p.) prior to challenge with dichloromethane (CH(2)Cl(2)) or carbon tetrachloride (CCl(4)). Blood carboxyhemoglobin elevation resulting from metabolic conversion of CH(2)Cl(2) to carbon monoxide was inhibited dose-dependently by DMSO pretreatment. The elevation of serum aspartate aminotransferase, alanine aminotransferase and sorbitol dehydrogenase activities induced by CCl(4) (0.1 mmol kg(-1)) was not changed in mice pretreated with DMSO at 1 ml kg(-1), but depressed significantly at a greater dose of DMSO. However, DMSO failed to alter the hepatotoxicity of CCl(4) injected at a dose of 0.2 mmol kg(-1). DMSO induced the microsomal p-nitrophenol hydroxylase and p-nitroanisole O-demethylase activities as early as 2 h following the treatment. Microsomal disposition of CH(2)Cl(2) and CCl(4) was measured using a vial equilibration technique. The disappearance of CH(2)Cl(2) was inhibited competitively by addition of DMSO. But DMSO did not affect the metabolic degradation of CCl(4). The results indicate that DMSO has multiple effects on metabolism and toxicity of xenobiotics. DMSO induces the hepatic metabolizing activity mediated by CYP2E1, but the presence of this solvent in the enzyme site may inhibit directly the enzymatic interaction with a substrate. The toxicological significance of DMSO-induced effects on such an interaction may be variable depending on the properties of each substrate. The invulnerability of CCl(4) metabolism to the effects of DMSO appears to be related to its high affinity for the lipophilic CYP enzyme site. Copyright 2006 John Wiley & Sons, Ltd.

The metabolic enzyme fructose-1,6-bisphosphate aldolase acts as a transcriptional regulator in pathogenic Francisella.

PubMed

Ziveri, Jason; Tros, Fabiola; Guerrera, Ida Chiara; Chhuon, Cerina; Audry, Mathilde; Dupuis, Marion; Barel, Monique; Korniotis, Sarantis; Fillatreau, Simon; Gales, Lara; Cahoreau, Edern; Charbit, Alain

2017-10-11

The enzyme fructose-bisphosphate aldolase occupies a central position in glycolysis and gluconeogenesis pathways. Beyond its housekeeping role in metabolism, fructose-bisphosphate aldolase has been involved in additional functions and is considered as a potential target for drug development against pathogenic bacteria. Here, we address the role of fructose-bisphosphate aldolase in the bacterial pathogen Francisella novicida. We demonstrate that fructose-bisphosphate aldolase is important for bacterial multiplication in macrophages in the presence of gluconeogenic substrates. In addition, we unravel a direct role of this metabolic enzyme in transcription regulation of genes katG and rpoA, encoding catalase and an RNA polymerase subunit, respectively. We propose a model in which fructose-bisphosphate aldolase participates in the control of host redox homeostasis and the inflammatory immune response.The enzyme fructose-bisphosphate aldolase (FBA) plays central roles in glycolysis and gluconeogenesis. Here, Ziveri et al. show that FBA of the pathogen Francisella novicida acts, in addition, as a transcriptional regulator and is important for bacterial multiplication in macrophages.

Purification and characterization of aspartate N-acetyltransferase: A critical enzyme in brain metabolism.

PubMed

Wang, Qinzhe; Zhao, Mojun; Parungao, Gwenn G; Viola, Ronald E

2016-03-01

Canavan disease (CD) is a neurological disorder caused by an interruption in the metabolism of N-acetylaspartate (NAA). Numerous mutations have been found in the enzyme that hydrolyzes NAA, and the catalytic activity of aspartoacylase is significantly impaired in CD patients. Recent studies have also supported an important role in CD for the enzyme that catalyzes the synthesis of NAA in the brain. However, previous attempts to study this enzyme had not succeeded in obtaining a soluble, stable and active form of this membrane-associated protein. We have now utilized fusion constructs with solubilizing protein partners to obtain an active and soluble form of aspartate N-acetyltransferase. Characterization of the properties of this enzyme has set the stage for the development of selective inhibitors that can lower the elevated levels of NAA that are observed in CD patients and potentially serve as a new treatment therapy. Copyright © 2015 Elsevier Inc. All rights reserved.

Isoquercetin ameliorates hyperglycemia and regulates key enzymes of glucose metabolism via insulin signaling pathway in streptozotocin-induced diabetic rats.

PubMed

Jayachandran, Muthukumaran; Zhang, Tongze; Ganesan, Kumar; Xu, Baojun; Chung, Stephen Sum Man

2018-06-15

Among the foremost common flavonoids within the human diet, quercetin glycosides possess neuroprotective, cardioprotective, anti-oxidative, chemopreventive, and anti-allergic properties. Isoquercetin is one such promising candidate with anti-diabetic potential. However, complete studies of its molecular action on insulin signaling pathway and carbohydrate metabolizing enzymes remain unclear. Hence, we have designed this study to accumulate the experimental evidence in support of anti-diabetic effects of isoquercetin. Male albino Wistar rats were divided into seven groups. Rats (Groups 3-7) were administered a single intraperitoneal injection of streptozotocin (STZ; 40 mg/kg b.w) to induce diabetes mellitus. As an extension, STZ rats received isoquercetin at three different doses (20, 40 and 80 mg/kg b.w), and Group 7 rats received glibenclamide (standard drug) (600 μg/kg b.w). The results showed that STZ exaggerated blood sugar, decreased insulin, altered metabolizing enzymes, and impaired the mRNA expression of insulin signaling genes and carbohydrate metabolizing enzyme genes. Supplementation with isoquercetin significantly normalized blood sugar levels, insulin and regulated the mRNA expression of insulin signaling genes and carbohydrate metabolizing enzyme genes. The results achieved with isoquercetin are similar to that of standard drug glibenclamide. The findings suggest isoquercetin could be a possible therapeutic agent for treating diabetes mellitus in the near future. Copyright © 2018 Elsevier B.V. All rights reserved.

Interplay between adenylate metabolizing enzymes and amp-activated protein kinase.

PubMed

Camici, Marcella; Allegrini, Simone; Tozzi, Maria Grazia

2018-05-18

Purine nucleotides are involved in a variety of cellular functions, such as energy storage and transfer, and signalling, in addition to being the precursors of nucleic acids and cofactors of many biochemical reactions. They can be generated through two separate pathways, the de novo biosynthesis pathway and the salvage pathway. De novo purine biosynthesis leads to the formation of IMP, from which the adenylate and guanylate pools are generated by two additional steps. The salvage pathways utilize hypoxanthine, guanine and adenine to generate the corresponding mononucleotides. Despite several decades of research on the subject, new and surprising findings on purine metabolism are constantly being reported, and some aspects still need to be elucidated. Recently, purine biosynthesis has been linked to the metabolic pathways regulated by AMP-activated protein kinase (AMPK). AMPK is the master regulator of cellular energy homeostasis, and its activity depends on the AMP:ATP ratio. The cellular energy status and AMPK activation are connected by AMP, an allosteric activator of AMPK. Hence, an indirect strategy to affect AMPK activity would be to target the pathways that generate AMP in the cell. Herein, we report an up-to-date review of the interplay between AMPK and adenylate metabolizing enzymes. Some aspects of inborn errors of purine metabolism are also discussed. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Remarkable Reproducibility of Enzyme Activity Profiles in Tomato Fruits Grown under Contrasting Environments Provides a Roadmap for Studies of Fruit Metabolism1[W][OPEN

PubMed Central

Biais, Benoît; Bénard, Camille; Beauvoit, Bertrand; Colombié, Sophie; Prodhomme, Duyên; Ménard, Guillaume; Bernillon, Stéphane; Gehl, Bernadette; Gautier, Hélène; Ballias, Patricia; Mazat, Jean-Pierre; Sweetlove, Lee; Génard, Michel; Gibon, Yves

2014-01-01

To assess the influence of the environment on fruit metabolism, tomato (Solanum lycopersicum ‘Moneymaker’) plants were grown under contrasting conditions (optimal for commercial, water limited, or shaded production) and locations. Samples were harvested at nine stages of development, and 36 enzyme activities of central metabolism were measured as well as protein, starch, and major metabolites, such as hexoses, sucrose, organic acids, and amino acids. The most remarkable result was the high reproducibility of enzyme activities throughout development, irrespective of conditions or location. Hierarchical clustering of enzyme activities also revealed tight relationships between metabolic pathways and phases of development. Thus, cell division was characterized by high activities of fructokinase, glucokinase, pyruvate kinase, and tricarboxylic acid cycle enzymes, indicating ATP production as a priority, whereas cell expansion was characterized by enzymes involved in the lower part of glycolysis, suggesting a metabolic reprogramming to anaplerosis. As expected, enzymes involved in the accumulation of sugars, citrate, and glutamate were strongly increased during ripening. However, a group of enzymes involved in ATP production, which is probably fueled by starch degradation, was also increased. Metabolites levels seemed more sensitive than enzymes to the environment, although such differences tended to decrease at ripening. The integration of enzyme and metabolite data obtained under contrasting growth conditions using principal component analysis suggests that, with the exceptions of alanine amino transferase and glutamate and malate dehydrogenase and malate, there are no links between single enzyme activities and metabolite time courses or levels. PMID:24474652

Gene expression analysis of a critical enzyme in intermediary metabolism in oyster pathogen Perkinsus marinus .

NASA Astrophysics Data System (ADS)

Noell, K.

2016-02-01

A key regulatory component in the Krebs cycle pathway is the mitochondrial aconitase enzyme which has been posited to balance energy needs and oxidative growth total storage via citrate utilization. The presence of a cytosolic aconitase (cAcon) activity which serves as a competitor for citrate substrate has been recognized for years. cAcon is a dual function protein with mutually exclusive roles as a post transcriptional regulator of animal cell iron metabolism or as the cytosolic isoform of the iron sulfur enzyme aconitase. We are interested in establishing the role of this orthologue in Perkinsus marnius metabolism through demonstrating its function as aconitase, by looking at gene expression under certain environmental conditions. P. marinus is a close evolutionary relative of the dinoflagellates and is the causative agent of Dermo disease, which has significantly impacted oyster populations along the eastern seaboard. An understanding of intermediary metabolism will yield important insights into how c-aconitase may be involved in stress response systems such as oxidative tension and metabolite deficiency, which could be used to help aquaculturists alleviate the severe impact of "dermo" on the on the oyster population. This study will present data regarding our preliminary analysis of the gene aconitase and its role in intermediary metabolism.

Simultaneous quantification of the abundance of several cytochrome P450 and uridine 5'-diphospho-glucuronosyltransferase enzymes in human liver microsomes using multiplexed targeted proteomics.

PubMed

Achour, Brahim; Russell, Matthew R; Barber, Jill; Rostami-Hodjegan, Amin

2014-04-01

Cytochrome P450 (P450) and uridine 5'-diphospho-glucuronosyltransferase (UGT) enzymes mediate a major proportion of phase I and phase II metabolism of xenobiotics. In vitro-in vivo extrapolation (IVIVE) of hepatic clearance in conjunction with physiologically-based pharmacokinetics (PBPK) has become common practice in drug development. However, prediction of xenobiotic kinetics in virtual populations requires knowledge of both enzyme abundances and the extent to which these correlate. A multiplexed quantification concatemer (QconCAT) strategy was used in this study to quantify the expression of several P450 and UGT enzymes simultaneously and to establish correlations between various enzyme abundances in 24 individual liver samples (ages 27-66, 14 male). Abundances were comparable to previously reported values, including CYP2C9 (40.0 ± 26.0 pmol mg(-1)), CYP2D6 (11.9 ± 13.2 pmol mg(-1)), CYP3A4 (68.1 ± 52.3 pmol mg(-1)), UGT1A1 (33.6 ± 34.0 pmol mg(-1)), and UGT2B7 (82.9 ± 36.1 pmol mg(-1)), expressed as mean ± S.D. Previous reports of correlations in expression of various P450 (CYP3A4/CYP3A5*1/*3, CYP2C8/CYP2C9, and CYP3A4/CYP2B6) were confirmed. New correlations were demonstrated between UGTs [including UGT1A6/UGT1A9 (r(s) = 0.82, P < 0.0001) and UGT2B4/UGT2B15 (r(s) = 0.71, P < 0.0001)]. Expression of some P450 and UGT enzymes were shown to be correlated [including CYP1A2/UGT2B4 (r(s) = 0.67, P = 0.0002)]. The expression of CYP3A5 in individuals with *1/*3 genotype (n = 11) was higher than those with *3/*3 genotype (n = 10) (P < 0.0001). No significant effect of gender or history of smoking or alcohol use on enzyme expression was observed; however, expression of several enzymes declined with age. The correlation matrix produced for the first time by this study can be used to generate more realistic virtual populations with respect to abundance of various enzymes.