Xenobiotic Carcinogens Research Articles

Validating Disease Associations of Drug-Metabolizing Enzymes through Genome-Wide Association Study Data Analysis

Background and Objectives: Phase I and phase II drug-metabolizing enzymes are crucial for the metabolism and elimination of various endogenous and exogenous compounds, such as small-molecule hormones, drugs, and xenobiotic carcinogens. While in vitro and animal studies have suggested a link between genetic mutations in these enzymes and an increased risk of cancer, human in vivo studies have provided limited supportive evidence. Methods: Genome-wide association studies (GWASs) are a powerful tool for identifying genes associated with specific diseases by comparing two large groups of individuals. In the present study, we analyzed a GWAS database to identify key diseases genetically associated with drug-metabolizing enzymes, focusing on UDP-glucuronosyltransferases (UGTs). Results: Our analysis confirmed a strong association between the UGT1 gene and hyperbilirubinemia. Additionally, over ten studies reported a link between the UGT1 gene and increased low-density lipoprotein (LDL) cholesterol levels. UGT2B7 was found to be associated with testosterone levels, total cholesterol levels, and vitamin D levels. Conclusions: Despite the in vitro capability of UGT1 and UGT2 family enzymes to metabolize small-molecule carcinogens, the GWAS data did not indicate their genetic association with cancer, except for one study that linked UGT2B4 to ovarian cancer. Further investigations are necessary to fill the gap between in vitro, animal, and human in vivo data.

Conformation and Pairing Properties of an O6-Methyl-2'-deoxyguanosine-Directed Benzimidazole Nucleoside Analog in Duplex DNA.

O6-Methyl-2'-deoxyguanosine (O6-MeG) is one of the most common DNA lesions and arises as a consequence of both xenobiotic carcinogens and endogenous methylation by S-adenosylmethionine. O6-MeG frequently causes G-to-A mutations during DNA replication due to the misincorporation of dTTP and continued DNA synthesis. Efforts to detect DNA adducts such as O6-MeG, and to understand their impacts on DNA structure and function, have motivated the creation of nucleoside analogs with altered base moieties to afford a more favorable interaction with the adduct as compared to the unmodified nucleotide. Such analogs directed at O6-MeG include benzimidazolinone and benzimidazole nucleotides, as well as their extended π surface analogs naphthimidazolinone and napthimidazole derivatives. These analogs form a more stable pair with O6-MeG than with G, most likely due to a combination of H-bonding and stacking. While extending the π surface of the analogs enhances their performance as adduct-directed probes, the precise origins of the increased affinity between the synthetic analogs and O6-MeG remain unclear. To better understand relevant conformational and pairing properties, we used X-ray crystallography and analyzed the structures of the DNA duplexes with naphthimidazolinone inserted opposite G or O6-MeG. The structures reveal a complex interaction of the analog found either in an anti orientation and stacked inside the duplex, either above or below G or O6-MeG, or in a syn orientation and paired opposite G with formation of a single H-bond. The experimental structural data are consistent with the stabilizing effect of the synthetic analog observed in UV melting experiments and calculations and moreover reveal that the origin of these observations appears to be superior stacking between O6-MeG and the extended π system of the synthetic probe.

Interaction of Hepatitis B Virus X Protein with the Pregnane X Receptor Enhances the Synergistic Effects of Aflatoxin B1 and Hepatitis B Virus on Promoting Hepatocarcinogenesis

Background and AimsHepatitis B virus (HBV) infection has been found to increase hepatocellular sensitivity to carcinogenic xenobiotics, by unknown mechanisms, in the generation of hepatocellular carcinoma. The pregnane X receptor (PXR) is a key regulator of the body’s defense against xenobiotics, including xenobiotic carcinogens and clinical drugs. In this study, we aimed to investigate the molecular mechanisms of HBV X protein (HBx)-PXR signaling in the synergistic effects of chemical carcinogens in HBV-associated hepatocarcinogenesis.MethodsThe expression profile of PXR-cytochrome p450 3A4 (CYP3A4) signaling was determined by PCR, western blotting, and tissue microarray. Cell viability and aflatoxin B1 (AFB1) cytotoxicity were measured using the cell counting kit-8 assay. Target gene expression was evaluated using transient transfection and real time-PCR. The genotoxicity of AFB1 was assessed in newborn mice with a single dose of AFB1.ResultsHBx enhanced the hepatotoxicity of AFB1 by activating CYP3A4 and reducing glutathione S-transferase Mu 1 (GSTM1) in cell lines. Activation of PXR by pregnenolone 16α-carbonitrile increased AFB1-induced liver tumor incidence by up-regulating oncogenic KRAS to enhance interleukin (IL)-11:IL-11 receptor subunit alpha-1 (IL11RA-1)-mediated inflammation in an HBx transgenic model.ConclusionsOur finding regarding AFB1 toxicity enhancement by an HBx-PXR-CYP3A4/ GSTM1-KRAS-IL11:IL11RA signaling axis provides a rational explanation for the synergistic effects of chemical carcinogens in HBV infection-associated hepatocarcinogenesis.

Mechanisms of Insertion of dCTP and dTTP Opposite the DNA Lesion O6-Methyl-2′-deoxyguanosine by Human DNA Polymerase η

O6-Methyl-2'-deoxyguanosine (O6-MeG) is a ubiquitous DNA lesion, formed not only by xenobiotic carcinogens but also by the endogenous methylating agent S-adenosylmethionine. It can introduce mutations during DNA replication, with different DNA polymerases displaying different ratios of correct or incorrect incorporation opposite this nucleoside. Of the "translesion" Y-family human DNA polymerases (hpols), hpol η is most efficient in incorporating equal numbers of correct and incorrect C and T bases. However, the mechanistic basis for this specific yet indiscriminate activity is not known. To explore this question, we report biochemical and structural analysis of the catalytic core of hpol η. Activity assays showed the truncated form displayed similar misincorporation properties as the full-length enzyme, incorporating C and T equally and extending from both. X-ray crystal structures of both dC and dT paired with O6-MeG were solved in both insertion and extension modes. The structures revealed a Watson-Crick-like pairing between O6-MeG and 2"-deoxythymidine-5"-[(α, β)-imido]triphosphate (approximating dT) at both the insertion and extension stages with formation of two H-bonds. Conversely, both the structures with O6- MeG opposite dCTP and dC display sheared configuration of base pairs but to different degrees, with formation of two bifurcated H-bonds and two single H-bonds in the structures trapped in the insertion and extension states, respectively. The structural data are consistent with the observed tendency of hpol η to insert both dC and dT opposite the O6-MeG lesion with similar efficiencies. Comparison of the hpol η active site configurations with either O6-MeG:dC or O6-MeG:dT bound compared with the corresponding situations in structures of complexes of Sulfolobus solfataricus Dpo4, a bypass pol that favors C relative to T by a factor of ∼4, helps rationalize the more error-prone synthesis opposite the lesion by hpol η.

Genetic variants of the CYP1B1 gene as predictors of biochemical recurrence after radical prostatectomy in localized prostate cancer patients.

Clinically localized prostate cancer is curative. Nevertheless many patients suffered from biochemical recurrence (BCR) after radical prostatectomy (RP). Mounting evidence suggest that estrogen and xenobiotic carcinogens play an essential role in progression of prostate cancervia oxidative estrogen metabolism. CYP1B1 is an enzyme involved in the hydroxylation of estrogens, a reaction of key relevance in estrogen metabolism. Given the role of CYP1B1 in the oxidative metabolism of endogenous/exogenous estrogen and compounds, CYP1B1 polymorphisms have the potential to modify its expression and subsequently lead to progression. We hypothesize that genetic variants of the CYP1B1 gene may influence clinical outcome in clinically localized prostate cancer patients. In this cohort study, we genotyped 9 tagging single nucleotide polymorphisms (SNPs) from the CYP1B1 gene in 312 patients treated with RP. For replication, these SNPs were genotyped in an independent cohort of 426 patients. The expression level of CYP1B1 in the adjacent normal prostate tissues was quantified by reverse transcription and real-time polymerase chain reaction. Kaplan-Meier analysis and Cox proportional hazard models were utilized to identify SNPs that correlated with BCR. CYP1B1 rs1056836 was significantly associated with BCR (hazard ratio [HR]: 0.69; 95% confidence interval [CI]: 0.40-0.89, P = 0.002) and relative CYP1B1 mRNA expression. Our findings suggest inherited genetic variation in the CYP1B1 gene may contribute to variable clinical outcomes for patients with clinically localized prostate cancer.

Investigation of Xenobiotics Metabolism, Genotoxicity, and Carcinogenicity Using Cyp2e1-/- Mice

Cytochromes P450 (CYPs) comprise a number of enzyme subfamilies responsible for the oxidative metabolism of a wide range of therapeutic agents, environmental toxicants, mutagens, and carcinogens. In particular, cytochrome P450 2E1 (CYP2E1) is implicated in the oxidative bioactivation of a variety of small hydrophobic chemicals including a number of epoxide-forming drugs and environmentally important toxicants including urethane, acrylamide, acrylonitrile, benzene, vinyl chloride, styrene, 1-bromopropane, trichloroethylene, dichloroethylene, acetaminophen, and butadiene. Until recently, chemical modulators (inducers and inhibitors) were used in order to characterize the enzymatic basis of xenobiotic metabolism and the relationships between CYP-mediated bioactivation and chemical-induced toxicity/carcinogenicity. With the advent of genetically engineered knockout mice, the ability to evaluate the roles of specific CYPs in the metabolism of xenobiotics has become more attainable. The main focus of the current review is to present studies that characterized the enzymatic, metabolic, and molecular mechanisms of toxicity, genotoxicity, and carcinogenicity of various xenobiotics using Cyp2e1-/- mice. Data presented in this review demonstrated that the most comprehensive studies using Cyp2e1-/- mice, encompassing the entire paradigm of metabolism to toxicity, genotoxicity, and carcinogenicity were possible when a substrate was primarily metabolized via CYP2E1 (e.g. urethane and acrylamide). In contrast, when multiple CYP enzymes were prevalent in the oxidation of a particular substrate (e.g.: trichloroethylene, methacrylonitrile, crotononitrile), investigating the relationships between oxidative metabolism and biological activity became more complicated and required the use of chemical modulators. In conclusion, the current review showed that Cyp2e1-/- mice are a valuable animal model for the investigation of the metabolic and molecular basis of toxicity, genotoxicity, and carcinogenicity of xenobiotics.

Effects of the ADH3, CYP2E1, and GSTP1 genetic polymorphisms on their expressions in Caucasian lung tissue

Individual differences in lung cancer susceptibility should be considered for effective lung cancer prevention. We investigated the CYP2E1, ADH3, and GSTP1 genetic polymorphisms that biotransform xenobiotic carcinogens, and variations of their enzyme activity in Caucasian lung tissues ( N=28), and found a variant distribution in pulmonary ADH and CYP2E1 activity. The ADH3* 1/* 1 subjects ( N=8) showed significantly higher ADH activity than ADH3* 2/* 2 ( N=3) subjects ( P<0.01). On the other hand, we found a 5-fold variation in the pulmonary CYP2E1 activity using a sensitive HLPC/EC based technique. A subject with the CYP2E1- c/ t allele showed 2-fold higher CYP2E1 activity than subjects with the c/ c allele ( N=14). GSTP1 expression comprised 83% of the total pulmonary GSTs. However, neither the GSTP1 polymorphism, nor other lifestyle factors, such as age, gender, smoking status, were found to be associated with pulmonary GST expression. In conclusion, subjects with the ADH3* 1 allele showed higher ADH activity and acetaldehyde–DNA adducts in lung than other subjects; thus, the ADH3* 1 allele could be considered a risk factor for lung cancer.

Using cytochrome P-450 gene knock-out mice to study chemical metabolism, toxicity, and carcinogenicity.

Cytochrome P-450 (CYP) enzymes are heme-containing proteins that carry out oxidative metabolism of a wide range of structurally diverse exogenous chemicals and therapeutic agents as well as endogenous compounds. For some of these xenobiotics, oxidative metabolism results in the formation of toxic, mutagenic, or carcinogenic metabolites. In the past, the role of CYP enzymes in metabolism and chemical-induced toxicity was studied indirectly through use of specific antibodies or inducers and inhibitors of these enzymes. Progress in molecular biology and the ability to bioengineer animal models that do not express CYP1A2, CYP1A1, CYP1B1, CYP2E1, or both CYP1A2 and CYP2E1 isozymes has allowed for direct investigations of the in vivo role of these enzymes in the metabolism, toxicity, and carcinogenicity of xenobiotics. This article reviews research conducted to date that utilizes these genetically bioengineered mice in metabolism, toxicity, or carcinogenicity studies of chemicals. Some studies showed a positive correlation between in vivo results and in vitro predictions for the role of a specific CYP in chemical-induced effects, whereas other studies did not support in vitro predictions. Work reviewed herein demonstrates the importance of using animal models for investigating the role of specific CYP enzymes in metabolism and chemical-induced toxicity or carcinogenicity rather than relying solely on in vitro techniques. Eventually, studies of this nature will facilitate a more accurate assessment of human risks with regard to chemicals by helping us to understand the relationships between chemical metabolism, carcinogenicity, and polymorphisms in CYP enzymes.

An isolated perfused model for the study of colonic metabolism and transport

The murine colonic perfusion model allows for the examination of absorption, metabolism, and portal transfer by the colon under physiologic conditions. This model was characterized by the use of four radiolabeled compounds: estradiol and Vitamin D 3, both physiologically active circulating steroid compounds, and benzo[ a]pyrene and N-acetylaminofluorene, xenobiotic carcinogens of the aromatic hydrocarbon and aromatic amide classes, respectively. Hemodynamic parameters and oxygen consumption of the preparation were stable throughout perfusion. Estradiol and N-acetylaminofluorene entered the portal vein at a rate of 2% of the lumenal dose per hour. Benzo[ a]pyrene crossed at 0.4% of the lumenal dose per hour. The rate of transfer of Vitamin D 3 was negligible. Analysis of the lumenal label revealed only substrate. In all experiments <0.02% of the applied substrate remained in the tissue compartment. Analysis of the vascular perfusate demonstrated evidence for sulfates of estradiol and N-acetylaminofluorene. Three conjugate classes were found associated with benzo[ a]pyrene, constituting 42% of the portal label. Hydrolysis data suggests the presence of double conjugates of benzo[ a]pyrene involving glutathione. In the case of aromatic hydrocarbons, conjugation, particularly thioether formation, implies hydroxylation and epoxide formation. For sulfation an N-acetylaminofluorene ring or N-hydroxylation is required. The latter process could allow for the delivery of highly carcinogenic NO sulfates to the liver.

Acetylation phenotype and skin complexion

The relationship of acetylation phenotype to skin complexion was studied in 155 healthy Caucasians. Individuals received 500 mg sulphadimidine at 11.00 p.m.; urine was collected eight hours later. The percentage of acetylated sulphadimidine in urine was measured with high performance liquid chromatography. There was a slight but insignificant preponderance of slow acetylators in the dark skin types. It is concluded that slow acetylation phenotype is not correlated with light skin complexion. Therefore, it is unlikely that acetylation of xenobiotic carcinogens plays a dominant role in melanoma risk.

Sex-dependent activities of quinone reductases in rabbits indicate higher risk of bladder cancer in the male

The distribution of NADPH-dependent quinone reductase and NADPH-cytochrome P-450 reductase activities was determined in the urinary bladders of male and female rabbits. In urinary bladder transitional epithelium (UBTE) and in urinary bladder non-transitional tissue (UBNT) microsomal quinone reductases demonstrated significant (P less than 0.05) sex-dependent differences in the case of both dicoumarol-insensitive (male greater than female) and dicoumarol-sensitive or DT-diaphorase (female greater than male) activities. Microsomal NADPH-cytochrome P-450 reductase activities in UBTE and in UBNT were found to be similar in male and female rabbits. The activities of microsomal and cytosolic quinone reductases and the activity of microsomal NADPH-cytochrome P-450 reductase in UBNT were much lower than those in UBTE. NADPH-cytochrome P-450 reductase and similar flavo-enzymes activate quinones via one-electron reduction into semiquinone free radicals, which then react with molecular oxygen, forming superoxide anions. DT-diaphorase acts as a detoxifying enzyme by converting many quinones via a unique two-electron reduction into less reactive hydroquinones, enabling their excretion as water-soluble conjugates. Since UBTE contains substantial activities of prostaglandin H synthase (PHS) and NADPH-cytochrome P-450 reductase, unlike UBNT, the toxicity and carcinogenicity of xenobiotics which are either quinones or form quinones in situ through the mediation of PHS would be high in UBTE. The risk of carcinogenicity of quinones in UBTE would be higher in male rabbits than in female rabbits due to sex-dependent differences in the relative proportions of the one-electron reduction pathway, represented by NADPH-cytochrome P-450 reductase, and the two-electron reduction pathway, represented by DT-diaphorase (female greater than male).