Bioactivation Of Xenobiotics Research Articles

Strategies to Mitigate the Bioactivation of Aryl Amines.

The bioactivation of xenobiotics to yield reactive metabolites can lead to tolerability and toxicity concerns within a drug discovery program. Development of strategies for mitigating the metabolic liability of commonly encountered toxicophores, such as anilines, relies on an understanding of the relative tendency of these functionalities to undergo bioactivation. In this report, we present the first systematic study of the structure-activity relationships of the bioactivation of aryl amine fragments (molecular weight < 250 Da) using a glutathione (GSH) trapping assay in the presence of human liver microsomes and the reduced form of nicotinamide adenine dinucleotide phosphate. This study demonstrates that conversion of anilines to nitrogen-containing heteroarylamines results in a lower abundance of GSH conjugates in the order phenyl > pyrimidine ≈ pyridine > pyridazine. Introduction of electron-withdrawing functionality on the aromatic ring had a less pronounced effect on the extent of GSH conjugation. Examination of more drug-like compounds sourced from in-house drug discovery programs revealed similar trends in bioactivation between matched pairs containing (hetero)aryl amines. This study provides medicinal chemists with insights and qualitative guidance for the minimization of risks related to aryl amine metabolism.

Molecular characterization and functional analysis of glutathione S-transferase kappa 1 (GSTκ1) from the big belly seahorse (Hippocampus abdominalis): Elucidation of its involvement in innate immune responses

Glutathione S-transferases (GSTs) are essential enzymes for the bioactivation of xenobiotics through the conjugation of the thiol group of glutathione (GSH). In this study, a kappa class of GST was identified from the big belly seahorse (Hippocampus abdominalis) (HaGSTκ1) and its biochemical and functional properties were analyzed. HaGSTκ1 has 231 amino acids encoded by a 696 bp open reading frame (ORF). The protein has a predicted molecular mass of 26.04 kDa and theoretical isoelectric point (pI) of 8.28. It comprised a thioredoxin domain, disulfide bond formation protein A (DsbA) general fold, and Ser15 catalytic site as well as GSH-binding and polypeptide-binding sites. Phylogenetic analysis revealed that HaGSTκ1 is closely clustered with the kappa class of GSTs from teleost fishes. The recombinant (rHaGSTκ1) protein exhibited activity toward 1-chloro-2,4-dinitrobenzene (CDNB), 4-nitrobenzyl (4-NBC), and 4-nitrophenethyl bromide (4-NPB) but not 1,2-dichloro-4-nitrobenzene (DCNB). The optimum pH and temperature were 8 and 30 °C, respectively, for the catalysis of CDNB and the universal substrate of GSTs. The rHaGSTκ1 activity was efficiently inhibited in the presence of Cibacron blue (CB) as compared with hematin. Most prominent expression of HaGSTκ1 was observed in the liver and kidney among the fourteen different tissues of normal seahorse. After challenge with lipopolysaccharide (LPS), polyinosinic-polycytidylic (poly I:C), gram-negative Edwardsiella tarda, and gram-positive Streptococcus iniae, HaGSTκ1 expression was significantly modulated in the liver and blood tissues. Altogether, our study proposes the plausible important role of HaGSTκ1 in innate immunity and detoxification of harmful xenobiotics.

Coumarins and P450s, Studies Reported to-Date.

Cytochrome P450 enzymes (CYPs) are important phase I enzymes involved in the metabolism of endogenous and xenobiotic compounds mainly through mono-oxygenation reactions into more polar and easier to excrete species. In addition to their role in detoxification, they play important roles in the biosynthesis of endogenous compounds and the bioactivation of xenobiotics. Coumarins, phytochemicals abundant in food and commonly used in fragrances and cosmetics, have been shown to interact with P450 enzymes as substrates and/or inhibitors. In this review, these interactions and their significance in pharmacology and toxicology are discussed in detail.

Linked organs-on-chips for probing xenobiotic bioactivation, transport and toxicity

Linked organs-on-chips for probing xenobiotic bioactivation, transport and toxicity

Crypt Organoid Culture as an in Vitro Model in Drug Metabolism and Cytotoxicity Studies.

The gastrointestinal tract is enriched with xenobiotic processing proteins that play important roles in xenobiotic bioactivation, metabolism, and detoxification. The application of genetically modified mouse models has been instrumental in characterizing the function of xenobiotic processing genes (XPG) and their proteins in drug metabolism. Here, we report the utilization of three-dimensional crypt organoid cultures from these animal models to study intestinal drug metabolism and toxicity. With the successful culturing of crypt organoids, we profiled the abundance of Phase I and Phase II XPG expression, drug transporter gene expression, and xenobiotic nuclear receptor (XNR) gene expression. Functions of XNRs were examined by treating crypt cells with XNR prototypical agonists. Real-time quantitative polymerase chain reaction demonstrated that the representative downstream target genes were induced. These findings were validated from cultures developed from XNR-null mice. In crypt cultures isolated from Pxr-/- mice, pregnenolone 16α-carbonitrile failed to induce Cyp3a11 gene expression; similarly, WY14643 failed to induce Cyp4a10 in the Pparα-/- crypts. Crypt cultures from control (Ugt1F/F ) and intestinal epithelial cell (IEC) specific Ugt1 null mice (Ugt1ΔIEC ) were treated with camptothecin-11, an anticancer prodrug with severe intestinal toxicity that originates from insufficient UGT1A1-dependent glucuronidation of its active metabolite SN-38. In the absence of Ugt1 gene expression, Ugt1ΔIEC crypt cultures exhibit very limited production of SN-38 glucuronide, concordant with increased apoptosis in comparison with Ugt1F/F crypt cultures. This study suggests crypt organoid cultures as an effective in vitro model for studying intestinal drug metabolism and toxicity.

Crypt Organoids Culture as an In Vitro Model in Studying Regulation of Xenobiotic Metabolism and drug‐induced Cytotoxicity

The gastrointestinal (GI) tract is the largest absorption organ in the body and is also enriched with many of the Xenobiotic Processing Genes (XPGs), which play an important role in xenobiotic bioactivation, metabolism, and detoxification. The application of genetically modified mouse models to study drug metabolism and toxicity has proven beneficial over the past decade, and with the ability to culture primary cells from these animal experiments can now be developed in vitro to examine mechanistic properties. Intestinal stem cells (ISCs), located in the crypt zone at the bottom of the villi, produce and differentiate into intestinal epithelial cells (IECs) as a supply for continuous intestinal renewal. Methods have been established in long‐term culture conditions in which one single Lgr5+ stem cell embedded in matrix gel can independently generate villus‐like epithelial domains with the presence of all differentiated IECs. Here, we report the application of 3D crypt organoid cultures as a tool for the study of intestinal drug metabolism and toxicity evaluation. Crypt cells were isolated from mouse intestinal tissue, suspended into matrigels, and cultured in the advanced DMEM/F12 medium supplemented with growth factors. Following continuous budding, crypts were further expanded to create organoids and the function of genes and enzymes involved in drug metabolism were evaluated. Crypt cultures were treated with vehicle (control) or different prototypical agonists of the xenobiotic nuclear receptors (XNR), including the AhR agonist TCDD, PXR agonist PCN, CAR agonist TCPOBOP, PPARa agonist Wy14643, and LXRa agonist T0901317. RT‐QPCR results demonstrated that in response to the different XNR agonists, the representative downstream target genes, including Cyp1a1 (by TCDD), Cyp3a11 (by PCN), Cyp2b10 (by TCPOBOP), Cyp4a10 (by Wy14643), and Abca1 (by T0901317) were largely induced. To further validate receptor‐specific gene induction by their corresponding ligands, we validated these findings using XNR‐null mice. In crypts isolated from Pxr−/−, PCN failed to induce Cyp3a11 gene expression; similarly, TCPOBOP and Wy14643 failed to induce Cyp2b10 or Cyp4a10 in the crypt organoids isolated from Car−/− and Ppara−/− mice, respectively. Camptothecin (CPT)‐11 (Irinotecan), an anticancer drug that generates substantial intestinal toxicity, is bioactivated by carboxylesterases to form the active topoisomerase 1 inhibitor SN‐38. Previous studies in our laboratory have shown that the deletion of intestinal UGT1A1 greatly sensitizes mice toward irinotecan induced intestinal toxicity induced by SN‐38. Bioactivation of irinotecan and cytotoxicity of SN‐38 can also be evaluated in novel crypt organoid cultures. This study suggests that crypt organoid cultures can be exploited as an effective in vitro model for studying the impact of xenobiotics on intestinal XPGs, drug metabolism, and toxicity.Support or Funding InformationSupported by ES010337 and ES024818.

Assessment of ionic liquids' toxicity through the inhibition of acylase I activity on a microflow system

Acylase I (ACY I) plays a role in the detoxication and bioactivation of xenobiotics as well in other physiological functions. In this context, an automated ACY I assay for the evaluation of ionic liquids' (ILs) toxicity was developed. The assay was implemented in a sequential injection analysis (SIA) system and was applied to eight commercially available ILs. The SIA methodology was based on the deacetylation of N-acetyl-l-methionine with production of l-methionine, which was determined using fluorescamine. ACY I inhibition in the presence of ILs was monitored by the decrease of fluorescence intensity. The obtained results confirmed the influence of ILs' structural elements on its toxicity and revealed that pyridinium and phosphonium cations, longer alkyl side chains and tetrafluoroborate anion displayed higher toxic effect on enzyme activity.The developed methodology proved to be robust and exhibited good repeatability (RSD < 1.3%, n = 10), leading also to a reduction of reagents consumption and effluents production. Thus, it is expected that the proposed assay can be used as a novel tool for ILs' toxicity screening.

The Role of CYP2E1 in the Drug Metabolism or Bioactivation in the Brain.

Organisms have metabolic pathways that are responsible for removing toxic agents. We always associate the liver as the major organ responsible for detoxification of the body; however this process occurs in many tissues. In the same way, as in the liver, the brain expresses metabolic pathways associated with the elimination of xenobiotics. Besides the detoxifying role of CYP2E1 for compounds such as electrophilic agents, reactive oxygen species, free radical products, and the bioactivation of xenobiotics, CYP2E1 is also related in several diseases and pathophysiological conditions. In this review, we describe the presence of phase I monooxygenase CYP2E1 in regions of the brain. We also explore the conditions where protein, mRNA, and the activity of CYP2E1 are induced. Finally, we describe the relation of CYP2E1 in brain disorders, including the behavioral relations for alcohol consumption via CYP2E1 metabolism.

Managing the Liabilities Arising from Structural Alerts: A Safe Philosophy for Medicinal Chemists

Bioactivation of xenobiotics can, in certain circumstances, result in the formation of reactive electrophilic species. These reactive metabolites may covalently modify proteins and macromolecules and it has been suggested that protein modification is a key initial step in provoking idiosyncratic adverse drug reactions. Understanding these bioactivation pathways is critical in order to rationally design drug candidates with a lower propensity to form reactive intermediates. Herein, we provide an overview of the importance of Structural Alerts and bioactivation pathways and describe the creation of an in-house database as a tool aimed at informing medicinal chemists about these potential liabilities.

Profiling the Reactive Metabolites of Xenobiotics Using Metabolomic Technologies

A predominant pathway of xenobiotic-induced toxicity is initiated by bioactivation. Characterizing reactive intermediates will provide information on the structure of reactive species, thereby defining a potential bioactivation mechanism. Because most reactive metabolites are not stable, it is difficult to detect them directly. Reactive metabolites can form adducts with trapping reagents, such as glutathione, which makes the reactive metabolites detectable. However, it is challenging to "fish" these adducts out from a complex biological matrix, especially for adducts generated via uncommon metabolic pathways. In this regard, we developed a novel approach based upon metabolomic technologies to screen trapped reactive metabolites. The bioactivation of pulegone, acetaminophen, and clozapine were reexamined by using this metabolomic approach. In all these cases, a large number of trapped reactive metabolites were readily identified. These data indicate that this metabolomic approach is an efficient tool to profile xenobiotic bioactivation.

D-Isomer of gly-tyr-pro-cys-pro-his-pro peptide: A novel and sensitive in vitro trapping agent to detect reactive metabolites by electrospray mass spectrometry

This paper describes a d-peptide isomer-based trapping assay using an LC/MS ion-trap spectrometer with an electrospray ionization (ESI) source as the analytical tool to study bioactivation of xenobiotics. Reactive metabolites were generated from parent compounds in in vitro incubations with different sources of CYP enzymes. A short d-isomer of gly-tyr-pro-cys-pro-his-pro proved to be a sensitive trapping agent and resistant to proteases. This method was tested with 16 probe substances. Acetaminophen, 1-chloro 2,4-dinitrobenzene, clozapine, diclofenac, imipramine, menthofuran, propranolol, pulegone and ticlopidine all formed d-peptide adducts, which were analogous to the GSH adducts previously described in the literature. New adducts were identified with clopidogrel (–Cl + peptide), nicotine (–CH 3+H + peptide), nimesulide (+peptide) and tolcapone (+peptide), i.e., no GSH adducts of those drugs have been described in the literature. No adducts were identified with ciprofloxacin, ketoconazole and verapamil. In the literature no GSH adducts have been described with ciprofloxacin and verapamil. d-Peptide-based trapping proved to be a reliable and reproducible method to identify bioactivated intermediates. d-Peptide is a new and convenient protein trapping agent for use in early phase screening of bioactivation of new chemical entities and evaluation of toxic properties of chemicals.

Rapid estimation of activation enthalpies for cytochrome‐P450‐mediated hydroxylations

Cytochrome P450 (CYP) enzymes play a critical role in detoxication and bioactivation of xenobiotics; thus, the ability to predict the biotransformation rates and regioselectivity of CYP enzymes toward substrates is an important goal in toxicology and pharmacology. Here, we present the use of the semiempirical quantum chemistry method SAM1 to rapidly estimate relative activation enthalpies (ΔH(‡)) for the hydroxylation of aliphatic carbon centers of various substrates. The ΔH(‡) were determined via a reaction path calculation, in the reverse direction (RRP), using the iron-hydroxo-porphine intermediate and the substrate radical. The SAM1 ΔH(‡) were calculated via unrestricted Hartree-Fock (UHF) and configuration interaction (CI) formalisms for both the doublet and quartet spin states. The SAM1 RRP ΔH(‡), after subtracting a correction factor, were compared with density functional theory (DFT) B3LYP activation energies for two sets of substrates and showed R(2) ranging from 0.69 to 0.89, and mean absolute differences ranging from 1.2 ± 1.0 to 1.7 ± 1.5 kcal/mol. SAM1 UHF and CI RRP calculation times were, on average, more than 200 times faster than those for the corresponding forward reaction path DFT calculations. Certain key transition-state (TS) geometry measurements, such as the forming O···H bond length, showed good correlation with the DFT values. These results suggest that the SAM1 RRP approach can be used to rapidly estimate the DFT activation energy and some key TS geometry measurements and can potentially be applied to estimate substrate hydroxylation rates and regioselectivity by CYP enzymes.

153. XENOBIOTICS; INFLUENCE ON OVARIAN FOLLICULAR DEVELOPMENT

The mammalian female reproductive lifespan is largely defined by a finite pool of ovarian follicles established around the time of birth. It is now understood that certain synthetic chemical compounds, known as xenobiotics, can cause premature ovarian senescence through the destruction of small ovarian follicles. Although the ovotoxic effects of these chemicals are well documented, the exact molecular mechanisms behind their action are only just becoming understood. Recent evidence suggests that bioactivation of xenobiotics by Phase I detoxifying enzymes may lead to the generation of free oxygen radicals (ROS), which we suspect may perturb intracellular signalling pathways in primordial follicles. In this study we attempted to identify ovarian follicle signalling pathways activated by xenobiotic exposure using ovotoxic agents which target immature follicles. Neonatal ovaries obtained from 3/4-day old Swiss mice were exposed to either 4-Vinylcyclohexene (25µM), Methoxychlor (25µM) or Menadione (5µM) for 96hrs using our in vitro culture system. Total RNA was then collected and analysed using Affymetrix Mouse Genome 430 2.0 Arrays. Bioinformatic analysis identified between ~500–1000 genes with a two-fold significant difference in gene expression (p&lt;0.05) for each xenobiotic compared to the control. Differentially expressed genes were analysed for pathways and molecular functions using Ingenuity Pathways Analysis (Ingenuity Systems). In agreement with the current literature, many of the genes belonged to toxic response pathways, such as; Xenobiotic metabolism (10); p53 (15) and Apoptosis (11) signalling. However, the vast majority of the differentially expressed genes belonged to canonical pathways implicated in follicular development, such as PI3K/AKT (18), Wnt/ b -catenin (21), and JAK/Stat (8) signalling. Further qPCR analysis has confirmed a substantial increase in the transcription factor Sox4 and cell cycle inhibitor Cdkn2a in 4-Vinylcyclohexene and Menadione treated ovaries respectively. These results suggest that xenobiotics which target primordial follicles may exert part of their ovotoxic effects by perturbing signalling pathways involved in follicular activation and development.

Non-P450 aldehyde oxidizing enzymes: the aldehyde dehydrogenase superfamily

Background: Aldehydes are highly reactive molecules. While several non-P450 enzyme systems participate in their metabolism, one of the most important is the aldehyde dehydrogenase (ALDH) superfamily, composed of NAD(P)+-dependent enzymes that catalyze aldehyde oxidation. Objective: This article presents a review of what is currently known about each member of the human ALDH superfamily including the pathophysiological significance of these enzymes. Methods: Relevant literature involving all members of the human ALDH family was extensively reviewed, with the primary focus on recent and novel findings. Conclusion: To date, 19 ALDH genes have been identified in the human genome and mutations in these genes and subsequent inborn errors in aldehyde metabolism are the molecular basis of several diseases, including Sjögren-Larsson syndrome, type II hyperprolinemia, γ-hydroxybutyric aciduria and pyridoxine-dependent seizures. ALDH enzymes also play important roles in embryogenesis and development, neurotransmission, oxidative stress and cancer. Finally, ALDH enzymes display multiple catalytic and non-catalytic functions including ester hydrolysis, antioxidant properties, xenobiotic bioactivation and UV light absorption.

Is Serum Gamma-Glutamyltransferase a Biomarker of Xenobiotics, Which Are Conjugated by Glutathione?

A meta-analysis of 10 prospective cohort studies,1 including our own,2 reported that serum γ-glutamyltransferase (GGT) predicted incident vascular events. This meta-analysis posed one important, but unsolved question: why does serum GGT predict incident vascular events? In addition to vascular events, serum GGT predicts various important clinical outcomes including type 2 diabetes, metabolic syndrome, and renal problems.3–5 Serum GGT has traditionally been used as a marker of alcohol consumption; however, alcohol consumption cannot explain the disease relationships with serum GGT because these associations have been observed among nondrinkers as well as drinkers. The other prevailing interpretations are that serum GGT predicts clinical outcomes as a marker of nonalcoholic fatty liver disease (NAFLD) or oxidative stress.6–8 Although both of these explanations have some merit, each also has problems. Although not clearly stated in many of the reports,1–5 most associations of serum GGT with clinical outcomes were dose-response relations within the normal range of serum GGT; we submit that this aspect of the association is critical to interpretation. First, even though NAFLD may partially explain the associations among subjects with a relatively high serum GGT activity and NAFLD can present even among subjects with low normal serum GGT, NAFLD is unlikely to fully explain the graded associations with clinical outcomes which were observed among subjects with serum GGT in the low part of its normal range, less than 20 or 30 U/L. However, it is worthwhile to note that serum GGT may be a predictor of the future risk of NAFLD. In a prospective study by our group,9 serum GGT within its normal range strongly predicted the future risk of chronic elevation of serum alanine aminotransferase (ALT), but serum ALT within its normal range did not predict the future risk of chronic elevation of serum GGT. The …

Co-oxidation-mediated xenobiotic activation and cytotoxicity by 12-lipoxygenase in intact platelets

Peroxidase-mediated co-oxidation process has been suggested as a major alternative pathway for xenobiotic bioactivation other than hepatic cytochrome P450 monooxygenase system. Despite the wealth of reports on the possible involvement of co-oxidation in bioactivation of various compounds, clear manifestation of co-oxidation-mediated xenobiotic bioactivation in intact cell system without extra sources of peroxidase system has been difficult to demonstrate, mainly due to the natural scarcity of peroxidase activities in fresh intact cells. In the present study, arachidonic acid (AA) dependent bioactivation of α-naphthol, a representative phenolic compound, was demonstrated as shown by covalent binding increases in α-naphthol concentration dependent manner in freshly prepared intact platelets, where two AA dependent representative peroxidases, 12-lipoxygenase (12-LOX) and prostaglandin H synthase (PHS) are abundantly expressed. Inhibitors of 12-LOX attenuated the covalent binding of α-naphthol while inhibitors of PHS were not effective, indicating the predominant role of 12-LOX in AA-initiated co-oxidation in intact platelets. In addition, free radical scavengers and thiol donors prevented effectively the bioactivation of α-naphthol, suggesting the involvement of naphthoxy radical or naphthoxy-derived radical generation. Notably, the co-oxidation process resulted in enhanced cytotoxicities of α-naphthol against platelets indeed, as observed by cellular membrane disturbance and mitochondrial membrane potential decrease. With these results, we believe that an important in vitro evidence of peroxidase-mediated xenobiotic activation was provided for understanding the toxicological implication of peroxidase-mediated co-oxidation in the xenobiotic bioactivation.

Cytochrome P450 3A4 regiospecific oxygenation of raloxifene: Evidence for the formation of the di‐quinone methide

While the bioactivation of xenobiotics to toxic intermediates through cytochrome P450 (CYP)‐catalyzed oxygenation reactions is a well recognized process, the production of reactive electrophilic intermediates by P450s through dehydrogenation reactions is not well characterized. Raloxifene is a second generation selective estrogen receptor modulator used for the treatment and prevention of osteoporosis in postmenopausal women. Previous studies have shown CYP3A4‐mediated metabolism of raloxifene produces two hydroxylated metabolites and several glutathione (GSH) adducts. The hydroxylated metabolites and one of the GSH adducts could theoretically be derived from the same dehydrogenated intermediate. It is postulated that raloxifene is dehydrogenated to a reactive di‐quinone methide. To evaluate the formation of the di‐quinone methide, raloxifene was incubated with CYP3A4 in the presence of H218O or 18O2. LC/MS analysis showed that 3′‐hydroxyraloxifene incorporated oxygen only from 18O2, while the source of the oxygen for the 7‐hydroxyraloxifene was inconclusive. These results demonstrate CYP3A4 selectively oxygenates raloxifene from 18O2 at the 3′‐position, but not at the 7‐position. Furthermore, the lack of CYP3A4‐mediated oxygenation at the 7‐position suggests that the di‐quinone methide is hydrated by active‐site H2O, but labeled H218O could not serve as the source of hydration. Supported by NIH grant # GM074249 form the National Institute of General Medical Sciences.

In utero–initiated cancer: The role of reactive oxygen species

It is becoming more evident that not only can drugs and environmental chemicals interfere with normal fetal development by causing structural malformations, such as limb defects, but that xenobiotic exposure during development can also cause biochemical and functional abnormalities that may ultimately lead to cancer later on in life. Fetal toxicity may be partly mediated by the embryonic bioactivation of xenobiotics to free radical intermediates that can lead to oxidative stress and potentially lead, in some cases, to carcinogenesis. Using a number of examples, this review will focus on the role of reactive oxygen species (ROS) in the mechanisms pertaining to in utero initiated cancers.

An in vitro approach to detect metabolite toxicity due to CYP3A4-dependent bioactivation of xenobiotics

Many adverse drug reactions are caused by the cytochrome P450 (CYP) dependent activation of drugs into reactive metabolites. In order to reduce attrition due to metabolism-mediated toxicity and to improve safety of drug candidates, we developed two in vitro cell-based assays by combining an activating system (human CYP3A4) with target cells (HepG2 cells): in the first method we incubated microsomes containing cDNA-expressed CYP3A4 together with HepG2 cells; in the second approach HepG2 cells were transiently transfected with CYP3A4. In both assay systems, CYP3A4 catalyzed metabolism was found to be comparable to the high levels reported in hepatocytes. Both assay systems were used to study ten CYP3A4 substrates known for their potential to form metabolites that exhibit higher toxicity than the parent compounds. Several endpoints of toxicity were evaluated, and the measurement of MTT reduction and intracellular ATP levels were selected to assess cell viability. Results demonstrated that both assay systems are capable to metabolize the test compounds leading to increased toxicity, compared to their respective control systems. The co-incubation with the CYP3A4 inhibitor ketoconazole confirmed that the formation of reactive metabolites was CYP3A4 dependent. To further validate the functionality of the two assay systems, they were also used as a “detoxification system” using selected compounds that can be metabolized by CYP3A4 to metabolites less toxic than their parent compounds. These results show that both assay systems can be used to screen for metabolic activation, or de-activation, which may be useful as a rapid and relatively inexpensive in vitro assay for the prediction of CYP3A4 metabolism-mediated toxicity.

Genetic Polymorphisms of GSTT1, GSTM1, GSTP1, MnSOD, and Catalase in Nonhereditary Chronic Pancreatitis: Evidence of Xenobiotic Stress and Impaired Antioxidant Capacity

Epidemiological studies have demonstrated a variety of potential environmental factors that may alter susceptibility to chronic pancreatitis (CP) through oxidative/xenobiotic stress; however, a direct causal and mechanistic role has not been established. We aimed (1) to determine the prevalence of functional genetic polymorphisms in the antioxidant enzymes, glutathione S-transferase GSTM-1, GSTP-1, and GSTT-1, manganese superoxide dismutase, and catalase in CP and (2) to reveal evidence of oxidative stress in patients with CP by measuring whole-blood glutathione redox status. In total, 122 patients with CP (75 alcohol-induced [A1CP], 33 idiopathic [ICP], and 13 hereditary) and 245 age- and sex-matched controls were recruited. The prevalence of the functional GSTT-1 genotype (GSTT-1*A) was significantly higher in CP (88.5%) compared to healthy controls (76%; chi2 = 7.26, P = 0.007). Stratification to disease etiology demonstrated that the GSTT-1*A genotype was also significantly more prevalent among patients with ICP (94%; P = 0.02; 95% CI, 0.04-9.16) but not in those with A1CP. In 22 patients with stable CP, the whole-blood glutathione concentration (median [IQR]: 72 micromol/L [21-181 micromol/L]) and the glutathione redox ratio (GSH/GSSG) (median [IQR]: 9 (3-77]) were significantly reduced compared to those in 20 healthy volunteers (median [IQR]: 815 micromol/L [679-1148 micromol/L], P < 0.001, and 96 [52-347], P = 0.005, respectively). We conclude that the GSTT-1 functional genotype is associated with ICP. Evidence of altered glutathione redox status suggests that this disease modification may be a consequence of oxidative stress or the bioactivation of xenobiotics.