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The combination of immunotherapy and molecular targeted therapy exhibits promising therapeutic efficacy in hepatocellular carcinoma (HCC), but the underlying mechanism is still unclear. Here, phosphoglycerate mutase 1 (PGAM1) is identified as a novel immunometabolic target by using a bioinformatic algorithm based on multiple HCC datasets. PGAM1 is highly expressed in HCC and associated with a poor prognosis and a poor response to immunotherapy. In vitro and in vivo experiments indicate that targeting PGAM1 inhibited HCC cell growth and promoted the infiltration of CD8+ T-cells due to decreased enzymatic activity. Mechanistically, inhibition of PGAM1 promotes HCC cell ferroptosis by downregulating Lipocalin (LCN2) by inducing energy stress and ROS-dependent AKT inhibition, which can also downregulate Programmed death 1-ligand 1 (PD-L1). Moreover, an allosteric PGAM1 inhibitor (KH3) exhibits good antitumor effects in patient-derived xenograft (PDX) models and enhanced the efficacy of anti-PD-1 immunotherapy in subcutaneous and orthotopic HCC models. Taken together, the findings demonstrate that PGAM1 inhibition exerts an antitumor effect by promoting ferroptosis and CD8+ T-cell infiltration and can synergize with anti-PD-1 immunotherapy in HCC. Targeting PGAM1 can be a promising new strategy of "killing two birds with one stone" for HCC treatment. [ABSTRACT FROM AUTHOR]

Naphthalene (NA) is a ubiquitous pollutant to which humans are widely exposed. 1,2-Dihydro-1,2-dihydroxynaphthalene (NA-dihydrodiol) is a major metabolite of NA generated by microsomal epoxide hydrolase (mEH). To investigate the role of the NA-dihydrodiol and subsequent metabolites (i.e. 1,2-naphthoquinone) in cytotoxicity, we exposed both male and female wild type (WT) and mEH null mice (KO) to NA by inhalation (5, 10, 20 ppm for 4h). NA-dihydrodiol was ablated in the KO mice. High-resolution histopathology was used to study site-specific cytotoxicity, and formation of naphthalene metabolites was measured by HPLC in microdissected airways. Swollen and vacuolated airway epithelial cells were observed in the intra- and extrapulmonary airways of all mice at and below the current OSHA standard (10 ppm). Female mice may be more susceptible to this acute cytotoxicity. In the extrapulmonary airways, WT mice were more susceptible to damage than KO mice, indicating that the metabolites associated with mEH-mediated metabolism could be partially responsible for cytotoxicity at this site. The level of cytotoxicity in the mEH KO mice at all airway levels suggests that non-mEH metabolites are contributing to NA cellular damage in the lung. Our results indicate that the apparent contribution of mEH-dependent metabolites to toxicity differs by location in the lung. These studies suggest that metabolites generated through the mEH pathway may be of minor importance in distal airway toxicity and subsequent carcinogenesis from NA exposure.

Arylamine N-acetyltransferase 1 (NAT1) plays a pivotal role in the metabolism of carcinogens and is a drug target for cancer prevention and/or treatment. A protein–ligand virtual screening of 2 million chemicals was ranked for predicted binding affinity towards the inhibition of human NAT1. Sixty of the five hundred top-ranked compounds were tested experimentally for inhibition of recombinant human NAT1 and N-acetyltransferase 2 (NAT2). The most promising compound 9,10-dihydro-9,10-dioxo-1,2-anthracenediyl diethyl ester (compound 10) was found to be a potent and selective NAT1 inhibitor with an in vitro IC50 of 0.75 µM. Two structural analogs of this compound were selective but less potent for inhibition of NAT1 whereas a third structural analog 1,2-dihydroxyanthraquinone (a compound 10 hydrolysis product also known as Alizarin) showed comparable potency and efficacy for human NAT1 inhibition. Compound 10 inhibited N-acetylation of the arylamine carcinogen 4-aminobiphenyl (ABP) both in vitro and in DNA repair-deficient Chinese hamster ovary (CHO) cells in situ stably expressing human NAT1 and CYP1A1. Compound 10 and Alizarin effectively inhibited NAT1 in cryopreserved human hepatocytes whereas inhibition of NAT2 was not observed. Compound 10 caused concentration-dependent reductions in DNA adduct formation and DNA double-strand breaks following metabolism of aromatic amine carcinogens beta-naphthylamine and/or ABP in CHO cells. Compound 10 inhibited proliferation and invasion in human breast cancer cells and showed selectivity towards tumorigenic versus non-tumorigenic cells. In conclusion, our study identifies potent, selective, and efficacious inhibitors of human NAT1. Alizarin's ability to inhibit NAT1 could reduce breast cancer metastasis particularly to bone. [ABSTRACT FROM AUTHOR]

Exposure to aristolochic acid (AA) is associated with human nephropathy and urothelial cancer. The tumour suppressor TP53 is a critical gene in carcinogenesis and frequently mutated in AA-induced urothelial tumours. We investigated the impact of p53 on AAI-induced nephrotoxicity and DNA damage in vivo by treating Trp53(+/+), Trp53(+/−) and Trp53(−/−) mice with 3.5 mg/kg body weight (bw) AAI daily for 2 or 6 days. Renal histopathology showed a gradient of intensity in proximal tubular injury from Trp53(+/+) to Trp53(−/−) mice, especially after 6 days. The observed renal injury was supported by nuclear magnetic resonance (NMR)-based metabonomic measurements, where a consistent Trp53 genotype-dependent trend was observed for urinary metabolites that indicate aminoaciduria (i.e. alanine), lactic aciduria (i.e. lactate) and glycosuria (i.e. glucose). However, Trp53 genotype had no impact on AAI-DNA adduct levels, as measured by 32P-postlabelling, in either target (kidney and bladder) or non-target (liver) tissues, indicating that the underlying mechanisms of p53-related AAI-induced nephrotoxicity cannot be explained by differences in AAI genotoxicity. Performing gas chromatography–mass spectrometry (GC–MS) on kidney tissues showed metabolic pathways affected by AAI treatment, but again Trp53 status did not clearly impact on such metabolic profiles. We also cultured primary mouse embryonic fibroblasts (MEFs) derived from Trp53(+/+), Trp53(+/−) and Trp53(−/−) mice and exposed them to AAI in vitro (50 µM for up to 48 h). We found that Trp53 genotype impacted on the expression of NAD(P)H:quinone oxidoreductase (Nqo1), a key enzyme involved in AAI bioactivation. Nqo1 induction was highest in Trp53(+/+) MEFs and lowest in Trp53(−/−) MEFs; and it correlated with AAI-DNA adduct formation, with lowest adduct levels being observed in AAI-exposed Trp53(−/−) MEFs. Overall, our results clearly demonstrate that p53 status impacts on AAI-induced renal injury, but the underlying mechanism(s) involved remain to be further explored. Despite the impact of p53 on AAI bioactivation and DNA damage in vitro, such effects were not observed in vivo. [ABSTRACT FROM AUTHOR]

The combination of immunotherapy and molecular targeted therapy exhibits promising therapeutic efficacy in hepatocellular carcinoma (HCC), but the underlying mechanism is still unclear. Here, phosphoglycerate mutase 1 (PGAM1) is identified as a novel immunometabolic target by using a bioinformatic algorithm based on multiple HCC datasets. PGAM1 is highly expressed in HCC and associated with a poor prognosis and a poor response to immunotherapy. In vitro and in vivo experiments indicate that targeting PGAM1 inhibited HCC cell growth and promoted the infiltration of CD8 + T-cells due to decreased enzymatic activity. Mechanistically, inhibition of PGAM1 promotes HCC cell ferroptosis by downregulating Lipocalin (LCN2) by inducing energy stress and ROS-dependent AKT inhibition, which can also downregulate Programmed death 1-ligand 1 (PD-L1). Moreover, an allosteric PGAM1 inhibitor (KH3) exhibits good antitumor effects in patient-derived xenograft (PDX) models and enhanced the efficacy of anti-PD-1 immunotherapy in subcutaneous and orthotopic HCC models. Taken together, the findings demonstrate that PGAM1 inhibition exerts an antitumor effect by promoting ferroptosis and CD8 + T-cell infiltration and can synergize with anti-PD-1 immunotherapy in HCC. Targeting PGAM1 can be a promising new strategy of "killing two birds with one stone" for HCC treatment., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Exposure to aristolochic acid (AA) causes aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN). Conflicting results have been found for the role of human sulfotransferase 1A1 (SULT1A1) contributing to the metabolic activation of aristolochic acid I (AAI) in vitro. We evaluated the role of human SULT1A1 in AA bioactivation in vivo after treatment of transgenic mice carrying a functional human SULT1A1- SULT1A2 gene cluster (i.e. hSULT1A1/2 mice) and Sult1a1(−/−) mice with AAI and aristolochic acid II (AAII). Both compounds formed characteristic DNA adducts in the intact mouse and in cytosolic incubations in vitro. However, we did not find differences in AAI-/AAII-DNA adduct levels between hSULT1A1/2 and wild-type (WT) mice in all tissues analysed including kidney and liver despite strong enhancement of sulfotransferase activity in both kidney and liver of hSULT1A1/2 mice relative to WT, kidney and liver being major organs involved in AA metabolism. In contrast, DNA adduct formation was strongly increased in hSULT1A1/2 mice compared to WT after treatment with 3-nitrobenzanthrone (3-NBA), another carcinogenic aromatic nitro compound where human SULT1A1/2 is known to contribute to genotoxicity. We found no differences in AAI-/AAII-DNA adduct formation in Sult1a1(−/−) and WT mice in vivo. Using renal and hepatic cytosolic fractions of hSULT1A1/2, Sult1a1(−/−) and WT mice, we investigated AAI-DNA adduct formation in vitro but failed to find a contribution of human SULT1A1/2 or murine Sult1a1 to AAI bioactivation. Our results indicate that sulfo-conjugation catalysed by human SULT1A1 does not play a role in the activation pathways of AAI and AAII in vivo, but is important in 3-NBA bioactivation. [ABSTRACT FROM AUTHOR]

Non-small cell lung cancer (NSCLC) is the most common form of lung cancer. Genetic polymorphisms in tumour suppressor genes and genes encoding xenobiotic metabolising enzymes alter the activity of their corresponding enzymes and are important individual susceptibility factors for NSCLC. Because of the lack of information in literature, the aim of our study was to investigate the role of the tumour suppressor gene TP53 (Arg72Pro) and the xenobiotic metabolising CYP2E1*5B gene polymorphisms on the risk of NSCLC development. The study population consisted of 172 patients and 172 controls (156 men and 16 women in each group). Genetic polymorphisms were determined with real-time polymerase chain reaction (PCR) and PCR restriction fragment length polymorphism (PCR-RFLP). Multivariate analysis showed a significant association with NSCLC for the combination between the TP53 codon72 Arg/Pro and the Pro/Pro genotypes (OR 2.21, 95% CI 1.39-3.51; p=0.001). We also analysed whether combinations of these gene variants with GSTM1, GSTT1, GSTP1 exon 5 (Ile105Val), and GSTP1 exon 6 (Ala114Val) gene polymorphisms were associated with the NSCLC risk. A significant increase in the risk was observed for the following combinations: TP53 codon72 variant with GSTM1 null (OR 2.22, 95% CI 1.23-4.04; p=0.009), GSTT1 null (OR 2.98, 95% CI 1.49-5.94; p=0.002), and GSTP1 (Ala114Val) variant genotypes (OR 3.38, 95% CI 1.54-7.41; p=0.002). Further studies with larger samples are needed to verify these findings. [ABSTRACT FROM AUTHOR]

Heterocyclic aromatic amines (HAA) are formed in cooked meat, poultry and fish but also arise in tobacco smoke and exhaust gases. HAA are potential human carcinogens, which require metabolic activation to exert their genotoxicity. Human tissues can bioactivate HAA to produce reactive intermediates that bind to DNA. HAA DNA adduct formation occurs in human hepatocytes; however, the potential of HAA to form DNA adducts has not been investigated in human T lymphocytes. In this study, we investigated the ability of human T lymphocytes activated with PMA/Ionomycin or CD3/CD28 to express functional CYP1 activity and bioactivate three major HAA: 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5- f]quinoxaline (MeIQx), and 2-amino-9 H-pyrido[2,3- b]indole (AαC) to form DNA adducts. Adducts were measured by ultraperformance liquid chromatography-electrospray ionization/multistage scan mass spectrometry. The highest level of DNA adducts occurred for AαC (16 adducts per 109 nucleotides), followed by PhIP (9 adducts per 109 nucleotides). In contrast, DNA adducts formed from MeIQx and the structurally related aromatic amine 4-aminobiphenyl, a known human carcinogen, were below the limit of detection (< 3 adducts per 109 nucleotides). Moreover, we demonstrate that AαC is a potent inducer of CYP1A1 and CYP1B1 activity through a transcriptional mechanism involving the AhR pathway. Overall, our results highlight the capacity of activated human T lymphocytes to more efficiently bioactivate AαC to form DNA adducts than other prominent HAA or 4-ABP. Environ. Mol. Mutagen. 57:656-667, 2016. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Neutrophils infiltrate tissues during inflammation, and when activated, they release β-glucuronidase. Since inflammation is associated with carcinogenesis, we investigated how extracellular β-glucuronidase changed the in vitro cellular response to the chemical carcinogen benzo(a)pyrene (B[a]P). For this we exposed human liver (HepG2) and lung (A549) cells to B[a]P in the presence or absence of β-glucuronidase. β-Glucuronidase reduced B[a]P-induced expression of CYP1A1 and CYP1B1 at 6 h after exposure, which did not depend on β-glucuronidase activity, because the inhibitor d-saccharic acid 1,4-lactone monohydrate did not antagonize the effect of β-glucuronidase. On the other hand, the inhibitory effect of β-glucuronidase on CYP expression was dependent on signalling via the insulin-like growth factor receptor (IGF2R, a known receptor for β-glucuronidase), because co-incubation with the IGF2R inhibitor mannose-6-phosphate completely abolished the effect of β-glucuronidase. Extracellular β-glucuronidase also reduced the formation of several B[a]P metabolites and B[a]P-DNA adducts. Interestingly, at 24 h of exposure, β-glucuronidase significantly enhanced CYP expression, probably because β-glucuronidase de-glucuronidated B[a]P metabolites, which continued to trigger the aryl hydrocarbon receptor (Ah receptor) and induced expression of CYP1A1 (in both cell lines) and CYP1B1 (in A549 only). Consequently, significantly higher concentrations of B[a]P metabolites and DNA adducts were found in β-glucuronidase-treated cells at 24 h. DNA adduct levels peaked at 48 h in cells that were exposed to B[a]P and treated with β-glucuronidase. Overall, these data show that β-glucuronidase alters the cellular response to B[a]P and ultimately enhances B[a]P-induced DNA adduct levels. [ABSTRACT FROM AUTHOR]

The tumour suppressor p53 is one of the most important cancer genes. Previous findings have shown that p53 expression can influence DNA adduct formation of the environmental carcinogen benzo[ a]pyrene (BaP) in human cells, indicating a role for p53 in the cytochrome P450 (CYP) 1A1-mediated biotransformation of BaP in vitro. We investigated the potential role of p53 in xenobiotic metabolism in vivo by treating Trp53(+ /+ ), Trp53(+ /- ) and Trp53(− /− ) mice with BaP. BaP-DNA adduct levels, as measured by P-postlabelling analysis, were significantly higher in liver and kidney of Trp53(− /− ) mice than of Trp53(+ /+ ) mice. Complementarily, significantly higher amounts of BaP metabolites were also formed ex vivo in hepatic microsomes from BaP-pretreated Trp53(− /− ) mice. Bypass of the need for metabolic activation by treating mice with BaP-7,8-dihydrodiol-9,10-epoxide resulted in similar adduct levels in liver and kidney in all mouse lines, confirming that the influence of p53 is on the biotransformation of the parent compound. Higher BaP-DNA adduct levels in the livers of Trp53(− /− ) mice correlated with higher CYP1A protein levels and increased CYP1A enzyme activity in these animals. Our study demonstrates a role for p53 in the metabolism of BaP in vivo, confirming previous in vitro results on a novel role for p53 in CYP1A1-mediated BaP metabolism. However, our results also suggest that the mechanisms involved in the altered expression and activity of the CYP1A1 enzyme by p53 in vitro and in vivo are different. [ABSTRACT FROM AUTHOR]

Naphthalene (NA) is a ubiquitous pollutant to which humans are widely exposed. 1,2-Dihydro-1,2-dihydroxynaphthalene (NA-dihydrodiol) is a major metabolite of NA generated by microsomal epoxide hydrolase (mEH). To investigate the role of the NA-dihydrodiol and subsequent metabolites (i.e. 1,2-naphthoquinone) in cytotoxicity, we exposed both male and female wild type (WT) and mEH null mice (KO) to NA by inhalation (5, 10, 20 ppm for 4 h). NA-dihydrodiol was ablated in the KO mice. High-resolution histopathology was used to study site-specific cytotoxicity, and formation of naphthalene metabolites was measured by HPLC in microdissected airways. Swollen and vacuolated airway epithelial cells were observed in the intra- and extrapulmonary airways of all mice at and below the current OSHA standard (10 ppm). Female mice may be more susceptible to this acute cytotoxicity. In the extrapulmonary airways, WT mice were more susceptible to damage than KO mice, indicating that the metabolites associated with mEH-mediated metabolism could be partially responsible for cytotoxicity at this site. The level of cytotoxicity in the mEH KO mice at all airway levels suggests that non-mEH metabolites are contributing to NA cellular damage in the lung. Our results indicate that the apparent contribution of mEH-dependent metabolites to toxicity differs by location in the lung. These studies suggest that metabolites generated through the mEH pathway may be of minor importance in distal airway toxicity and subsequent carcinogenesis from NA exposure. [ABSTRACT FROM AUTHOR]

The expression of the tumor suppressor p53 can influence the bioactivation of, and DNA damage induced by, the environmental carcinogen benzo[a]pyrene, indicating a role for p53 in its cytochrome P450 (CYP)-mediated biotransformation. The carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), which is formed during the cooking of food, is also metabolically activated by CYP enzymes, particularly CYP1A2. We investigated the potential role of p53 in PhIP metabolism in vivo by treating Trp53(+/+), Trp53(+/-) and Trp53(-/-) mice with a single oral dose of 50 mg/kg body weight PhIP. N-(Deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP-C8-dG) levels in DNA, measured by liquid chromatography-tandem mass spectrometry, were significantly lower in liver, colon, forestomach and glandular stomach of Trp53(-/-) mice compared to Trp53(+/+) mice. Lower PhIP-DNA adduct levels in the livers of Trp53(-/-) mice correlated with lower Cyp1a2 enzyme activity (measured by methoxyresorufin-O-demethylase activity) in these animals. Interestingly, PhIP-DNA adduct levels were significantly higher in kidney and bladder of Trp53(-/-) mice compared to Trp53(+/+) mice, which was accompanied by higher sulfotransferase (Sult) 1a1 protein levels and increased Sult1a1 enzyme activity (measured by 2-naphthylsulfate formation from 2-naphthol) in kidneys of these animals. Our study demonstrates a role for p53 in the metabolism of PhIP in vivo, extending previous results on a novel role for p53 in xenobiotic metabolism. Our results also indicate that the impact of p53 on PhIP biotransformation is tissue-dependent and that in addition to Cyp1a enzymes, Sult1a1 can contribute to PhIP-DNA adduct formation. [ABSTRACT FROM AUTHOR]

The tumour suppressor gene TP53 is mutated in more than 50 % of human tumours, making it one of the most important cancer genes. We have investigated the role of TP53 in cytochrome P450 (CYP)-mediated metabolic activation of three polycyclic aromatic hydrocarbons (PAHs) in a panel of isogenic colorectal HCT116 cells with differing TP53 status. Cells that were TP53(+/+), TP53(+/− ), TP53(−/−), TP53(R248W/+) or TP53(R248W/−) were treated with benzo[ a]pyrene (BaP), dibenz[ a,h]anthracene and dibenzo[ a,l]pyrene, and the formation of DNA adducts was measured by P-postlabelling analysis. Each PAH formed significantly higher DNA adduct levels in TP53(+/+) cells than in the other cell lines. There were also significantly lower levels of PAH metabolites in the culture media of these other cell lines. Bypass of the need for metabolic activation by treating cells with the corresponding reactive PAH-diol-epoxide metabolites resulted in similar adduct levels in all cell lines, which confirms that the influence of p53 is on the metabolism of the parent PAHs. Western blotting showed that CYP1A1 protein expression was induced to much greater extent in TP53(+/+) cells than in the other cell lines. CYP1A1 is inducible via the aryl hydrocarbon receptor (AHR), but we did not find that expression of AHR was dependent on p53; rather, we found that BaP-induced CYP1A1 expression was regulated through p53 binding to a p53 response element in the CYP1A1 promoter region, thereby enhancing its transcription. This study demonstrates a new pathway for CYP1A1 induction by environmental PAHs and reveals an emerging role for p53 in xenobiotic metabolism. [ABSTRACT FROM AUTHOR]

Pulmonary inflammation can contribute to the development of lung cancer in humans. We investigated whether pulmonary inflammation alters the genotoxicity of polycyclic aromatic hydrocarbons (PAHs) in the lungs of mice and what mechanisms are involved. To model nonallergic acute inflammation, mice were exposed intranasally to lipopolysaccharide (LPS; 20 µg/mouse) and then instilled intratracheally with benzo[a]pyrene (BaP; 0.5 mg/mouse). BaP-DNA adduct levels, measured by 32P-postlabeling analysis, were approximately 3-fold higher in the lungs of LPS/BaP-treated mice than in mice treated with BaP alone. Pulmonary Cyp1a1 enzyme activity was decreased in LPS/BaP-treated mice relative to BaP-treated mice suggesting that pulmonary inflammation impacted on BaP-induced Cyp1a1 activity in the lung. Our results showed that Cyp1a1 appears to be important for BaP detoxification in vivo and that the decrease of pulmonary Cyp1a1 activity in LPS/BaP-treated mice results in a decrease of pulmonary BaP detoxification, thereby enhancing BaP genotoxicity (ie, DNA adduct formation) in the lung. Because less BaP was detoxified by Cyp1a1 in the lungs of LPS/BaP-treated mice, more BaP circulated via the blood to extrapulmonary tissues relative to mice treated with BaP only. Indeed, we observed higher BaP-DNA adduct levels in livers of LPS/BaP-treated mice compared with BaP-treated mice. Our results indicate that pulmonary inflammation could be a critical determinant in the induction of genotoxicity in the lung by PAHs like BaP. Cyp1a1 appears to be involved in both BaP bioactivation and detoxification although the contribution of other enzymes to BaP-DNA adduct formation in lung and liver under inflammatory conditions remains to be explored. [ABSTRACT FROM AUTHOR]

It has long been known that bladder cancer (BC) incidence is approximately four-fold higher in men than in women in the United States, and a similar disparity also exists in other countries. The reason for this phenomenon is not known, which impedes progress in BC prevention. However, BC incidence is also significantly higher in male animals than in their female counterparts after treatment with aromatic amines, which are principal human bladder carcinogens. These animal studies and related studies in the context of available human data provide significant insight into what may drive the excessive BC risk in men, which is the focus of this article. The carcinogenicity and biotransformation of bladder carcinogens as well as the impact of sex hormones on these processes are discussed, highlighting the novel concept that the gender disparity in BC risk may result primarily from the interplay of androgen, estrogen, and liver, with the liver functioning via its metabolic enzymes as the main decider of bladder exposure to carcinogens in the urine and the male and female hormones exerting opposing effects on carcinogenesis in the bladder and likely also on liver enzymes handling bladder carcinogens. The findings may facilitate further investigation into the mechanism of gender disparity in BC risk and may also have important implications for BC prevention. [ABSTRACT FROM AUTHOR]

Abstract: 1,3-Butadiene is metabolized mainly by cytochrome P450 2E1 to several epoxides that are considered toxic and carcinogenic. The first step of BD metabolism is oxidation to 1,2-epoxy-3-butene (EB), a reactive metabolite. It has been shown that P450s can be inactivated by covalent binding of reactive metabolites to protein or heme. Molecular dosimetry studies have clearly shown that BD metabolism follows a supralinear dose response, suggestive of saturation of metabolic activation. In this study, potential binding sites of EB in human P450 2E1 were identified and modeled to test whether EB covalently binds to residues important for enzyme activity. Commercially available human P450 2E1 was reacted with EB, digested with trypsin and the resulting peptides were analyzed by Matrix-Assisted Laser Desorption/Ionization tandem Time-of-Flight mass spectrometry (MALDI-MS). The identity of EB modified peptides was confirmed by Matrix-Assisted Laser Desorption/Ionization tandem mass spectrometry (MALDI-MS/MS) sequencing. It was shown that EB binds to four histidine and two tyrosine residues. All modification sites were assigned by at least two adjacent and a minimum of eight peptide specific fragments. Protein modeling revealed that two of these covalent modifications (His109, His370) are clearly associated with the active site, and that their Cα atoms are located less than 9Å from a known inhibitor binding site. In addition, the side chain of His370 is within 4Å of the heme group and its modification is expected to influence the orientation of the heme. The Cα atom of Tyr71 is within 14Å of the potential inhibitor binding site and within 7Å of the flap undergoing conformational change upon ligand binding, potentially placing Tyr71 near the substrate as it enters and leaves the active site. The data support the hypothesis that EB can inactivate P450 2E1 by covalent modifications and thus add an additional regulatory mechanism for BD metabolism. [Copyright &y& Elsevier]

Abstract: Cancer in children is a major concern in many countries. An important question is whether these childhood cancers are caused by something, or are just tragic random events. Causation of at least some children''s cancers is suggested by direct and indirect evidence, including epidemiological data, and animal studies that predict early life sensitivity of humans to carcinogenic effects. Candidate risk factors include genotoxic agents (chemicals and radiation), but also diet/nutrition, and infectious agents/immune responses. With regard to likelihood of risks posed by genotoxicants, there are pros and cons. The biological properties of fetuses and infants are consistent with sensitivity to preneoplastic genotoxic damage. Recent studies of genetic polymorphisms in carcinogen-metabolizing enzymes confirm a role for chemicals. On the other hand, in numerous epidemiological studies, associations between childhood cancers and exposure to genotoxicants, including tobacco smoke, have been weak and hard to reproduce. Possibly, sensitive genetic or ontogenetic subpopulations, and/or co-exposure situations need to be discovered to allow identification of susceptible individuals and their risk factors. Among the critical knowledge gaps needing to be bridged to aid in this effort include detailed tissue and cellular ontogeny of carcinogen metabolism and DNA repair enzymes, and associations of polymorphisms in DNA repair enzymes with childhood cancers. Perinatal bioassays in animals of specific environmental candidates, for example, benzene, could help guide epidemiology. Genetically engineered animal models could be useful for identification of chemical effects on specific genes. Investigations of interactions between factors may be key to understanding risk. Finally, fathers and newborn infants should receive more attention as especially sensitive targets. [Copyright &y& Elsevier]

Abstract: Studies of the impact of phase 1 enzyme polymorphisms on genetic damage have yielded mixed results. We studied how genetic damage would be altered when specific genes were ablated under low dose conditions. Methods: Knockouts (KO) were generated from c57bl6/J mice with mutations in Cyp1a2 or Ahr receptor that eliminated gene product function. Animals were treated topically with either 4-aminobiphenyl (4ABP) 10mg/kg, benzo(a)pyrene (BaP) 33.3mg/kg or dibenzo(c,g)carbazole (DBC) 8mg/kg, and sacrificed after 24h. DNA from livers, skin and/or urinary bladders were isolated and 32P-postlabelled. Results: Cyp1a2−/− mice did not differ in 4ABP DNA adduct levels in either urinary bladder or liver compared to wildtype. There was a sex difference in the organ affected. Cyp1a2 knockout reduced skin BAP adduct levels 50% and AHR knockout reduced skin BAP adduct levels by 90%. There was no impact of either knockout on the levels of DBC-DNA adducts in any tissue. Conclusions: Ablation of specific metabolizing enzymes had compound- and tissue-specific effects in mice. Phenotypic variability in single CYP enzymes may have minor impact in humans at low doses, but variation in the ability to induce the family of CYPs may have a greater impact. [Copyright &y& Elsevier]

Abstract: Sanguinarine''s use in human clinical applications is currently controversial. While some studies have demonstrated sanguinarine''s anti-inflammatory and anti-oxidant properties, other investigations reported sanguinarine''s procarcinogenic effects. Like the tobacco-associated carcinogen, benzo(a)pyrene (B(a)P), sanguinarine is a polycyclic aromatic hydrocarbon (PAH). PAH exposure activates the aryl hydrocarbon transcription activating factor (AhR), resulting in nuclear translocation, binding to the aryl hydrocarbon nuclear translocator (ARNT), which thereby increases expression of a pool of carcinogen metabolizing enzymes. The goal of this study was to investigate whether sanguinarine activates this PAH-associated signaling cascade in human oral cells and tissues. Our results demonstrate that sanguinarine: (i) results in formation of the AhR–ARNT complex, (ii) induces AhR-associated gene expression, (iii) inhibits cytochrome P450 1A1 (CYP 1A1) microsomal oxidative activity and (iv) pretreatment upregulates CYP 1A1 function. Collectively, these data provide evidence that sanguinarine activates PAH-associated signaling and metabolic pathways. Notably, previous studies have demonstrated that mammalian hepatic microsomes metabolize sanguinarine to a mutagenic epoxide. Persons who respond to sanguinarine exposure with induction of primarily Phase I relative to Phase II enzymes are, therefore, at risk for sanguinarine bioactivation and its potential mutagenic effects. [Copyright &y& Elsevier]

People are continuously exposed exogenously to varying amounts of chemicals that have been shown to have carcinogenic or mutagenic properties in experimental systems. Exposure can occur exogenously when these agents are present in food, air or water, and also endogenously when they are products of metabolism or pathophysiologic states such as inflammation. It has been estimated that exposure to environmental chemical carcinogens may contribute significantly to the causation of a sizable fraction, perhaps a majority, of human cancers, when exposures are related to “life-style” factors such as diet, tobacco use, etc. This chapter summarizes several aspects of environmental chemical carcinogenesis that have been extensively studied and illustrates the power of mechanistic investigation combined with molecular epidemiologic approaches in establishing causative linkages between environmental exposures and increased cancer risks.A causative relationship between exposure to aflatoxin, a strongly carcinogenic mold-produced contaminant of dietary staples in Asia and Africa, and elevated risk for primary liver cancer has been demonstrated through the application of well-validated biomarkers in molecular epidemiology. These studies have also identified a striking synergistic interaction between aflatoxin and hepatitis B virus infection in elevating liver cancer risk. Use of tobacco products provides a clear example of cancer causation by a life-style factor involving carcinogen exposure. Tobacco carcinogens and their DNA adducts are central to cancer induction by tobacco products, and the contribution of specific tobacco carcinogens (e.g. PAH and NNK) to tobacco-induced lung cancer, can be evaluated by a weight of evidence approach. Factors considered include presence in tobacco products, carcinogenicity in laboratory animals, human uptake, metabolism and adduct formation, possible role in causing molecular changes in oncogenes or suppressor genes, and other relevant data. This approach can be applied to evaluation of other environmental carcinogens, and the evaluations would be markedly facilitated by prospective epidemiologic studies incorporating phenotypic carcinogen-specific biomarkers.Heterocyclic amines represent an important class of carcinogens in foods. They are mutagens and carcinogens at numerous organ sites in experimental animals, are produced when meats are heated above 180 °C for long periods. Four of these compounds can consistently be identified in well-done meat products from the North American diet, and although a causal linkage has not been established, a majority of epidemiology studies have linked consumption of well-done meat products to cancer of the colon, breast and stomach. Studies employing molecular biomarkers suggest that individuals may differ in their susceptibility to these carcinogens, and genetic polymorphisms may contribute to this variability. Heterocyclic amines, like most other chemical carcinogens, are not carcinogenic per se but must be metabolized by a family of cytochrome P450 enzymes to chemically reactive electrophiles prior to reacting with DNA to initiate a carcinogenic response. These same cytochrome P450 enzymes—as well as enzymes that act on the metabolic products of the cytochromes P450 (e.g. glucuronyl transferase, glutathione S-transferase and others)—also metabolize chemicals by inactivation pathways, and the relative amounts of activation and detoxification will determine whether a chemical is carcinogenic. Because both genetic and environmental factors influence the levels of enzymes that metabolically activate and detoxify chemicals, they can also influence carcinogenic risk.Many of the phenotypes of cancer cells can be the result of mutations, i.e., changes in the nucleotide sequence of DNA that accumulate as tumors progress. These can arise as a result of DNA damage or by the incorporation of non-complementary nucleotides during DNA synthetic processes. Based upon the disparity between the infrequency of spontaneous mutations and the large numbers of mutations reported in human tumors, it has been postulated that cancers must exhibit a mutator phenotype, which would represent an early event in cancer progression. A mutator phenotype could be generated by mutations in genes that normally function to guarantee genetic stability. These mutations presumably arise via DNA damage by environmental or endogenous agents, but it remains to be determined whether the acquisition of a mutator phenotype is a necessary event during tumor progression. [Copyright &y& Elsevier]

CYP2S1 is a recently discovered member of the cytochrome P450 (CYP) gene superfamily. Interestingly, even though the DNA sequence identifies it as the sole member of the new CYP2S family, CYP2S1 exhibits many features typical to CYP1 family members, e.g. dioxin-inducibility mediated by the aryl hydrocarbon receptor (AHR) and the aryl hydrocarbon receptor nuclear translocator (ARNT). In addition, CYP2S1 metabolises some aromatic hydrocarbons as well as cellular substances. These characteristics, together with a wide extrahepatic tissue distribution, suggest that CYP2S1 may have an important role in both exogenous and endogenous metabolism. This is the first study characterising CYP2S1 alleles and naming them with the recommended CYP allele nomenclature. We used denaturing gradient gel electrophoresis (DGGE) and direct sequencing to investigate genetic variation of CYP2S1 in 100 male Finnish Caucasians. Those exons in which variation was found were examined in subsequent 100 subjects. The coding region of all of the nine exons, as well as a 449 bp fragment of the proximal promoter region, was analysed. This systematic investigation revealed eight single nucleotide polymorphisms (SNPs), which comprise nine different variant alleles (haplotypes), in addition to the wild-type allele. Seven of the SNPs occurred in the protein-coding areas and one in the proximal 3′ untranslated region (3′UTR). Two of these sequence variations (10347C > T and 13106C > T) result in non-conservative amino acid substitutions, i.e. Arg380Cys and Pro466Leu, respectively. The respective allelic variants, CYP2S1*2 ([10347C > T]) and CYP2S1*3 (13106C > T; 13255A > G]), occurred in our study population at frequencies of 0.50 and 3.75%, respectively. The most common of the variant alleles was CYP2S1*1H (23.8%), harbouring a 13255A > G substitution located in the 3′UTR. [Copyright &y& Elsevier]

The contribution of polymorphisms in carcinogen metabolizing genes to overall cancer rates may vary widely between groups with differing allele frequencies and with varying levels of carcinogenic exposure. Their effects are modified by interactions with each other and with other genes, particularly those involved in DNA repair. Studies on the combined effects of particular polymorphisms on colorectal and other cancers, and also on intermediate markers such as DNA adduct formation, are discussed. Such susceptibility genes are of considerable scientific interest, but do not confer high enough risks to be clinically relevant.Oncogene (2004) 23, 6477-6483. doi:10.1038/sj.onc.1207897 [ABSTRACT FROM AUTHOR]

Almost all the polycyclic aromatic hydrocarbons (PAHs) require metabolic activation to exert their carcinogenic activity. Environmental carcinogen [3H] benzo[a]pyrene (BP) is carcinogenic only after its metabolic transformation to a reactive intermediate, which can then bind to cellular macromolecules. Inhibition of dimethylbenz anthracene–(DMBA–) DNA binding generally accompanied inhibition of tumor initiation as most inhibitors of initiation interfere with the metabolic activation of the initiator. The importance of carcinogen–DNA interaction and the enzymes involved in the metabolism of carcinogenic polycyclic hydrocarbons has led to a search for inhibitors that would be useful in modifying the cancer-causing effects of the PAHs. We tested the effect of calcium glucarate (Cag), a naturally occurring nontoxic compound, on carcinogen metabolism and DNA interaction. Cag inhibited [3H] BP binding to both calf thymus DNA in vitro and to epidermal DNA in vivo. Application of Cag to mouse skin caused a dose-dependent inhibition of [3H] BP binding to epidermal DNA. To establish the relevance of the in vivo results to the in vitro situation, we followed the in vitro effect of Cag on [3H] BP binding to calf thymus DNA and observed that Cag inhibited the [3H] BP binding to calf thymus DNA in the presence of microsomes prepared from animals treated with DMBA. We also studied related events like DNA synthesis and carcinogen metabolism. For assessing the DNA synthesis, thymidine kinase was used as marker. Cag caused a dose-dependent inhibition of DMBA-induced thymidine kinase activity. At the same time, Cag caused a marked inhibition of DMBA-induced aryl hydrocarbon hydroxylase (AHH) activity, an enzyme responsible for the metabolism of PAHs like BP, both in vivo and in vitro. Our study indicates that Cag exerted its antitumor effect possibly by inhibiting the carcinogen-DNA binding, which appears to be due to reduced DNA synthesis and AHH activity. [ABSTRACT FROM AUTHOR]

Although both non-starch polysaccharides (NSP) and resistant starches (RS) are included in current definitions of dietary fibre, our previous work has suggested fundamental differences in the way in which these two classes of material affect the disposition and absorption of a dietary carcinogen. The present studies explore whether different effects on carcinogen metabolism could play a role in the contrasting patterns seen previously. Groups of female Wistar rats were pre-fed for 4 weeks one of five types of defined diet (AIN-76). The control diet contained 35% maize starch and no dietary fibre. The RS-containing diets had all the maize starch substituted with either Hi-maize™ or potato starch. In the NSP-containing diets, 10% of the maize starch was substituted with dietary fibre in the form of either lignified plant cell walls (wheat straw) or soluble dietary fibre (apple pectin). Pre-fed rats were gavaged with the food carcinogen, [2-14C] 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and plasma and urinary metabolites characterized using HPLC at various time intervals after administration. After 4 h gavage, plasma from rats on both RS-containing diets contained significantly higher levels of intact IQ and lower levels of the major metabolites, IQ-5-O-glucuronide and IQ-5- sulfate, as compared with plasma from the negative control group at this time. In contrast, plasma from animals on the NSP-containing wheat straw diet (and to a lesser extent the apple pectin diet) showed significantly lower levels of intact IQ, and significantly higher levels of the two major metabolites, as compared with those from the control rats. These different metabolite profiles were also reflected in different urinary excretion profiles. Urine from rats pre-fed RS-containing diets revealed significantly slower metabolite excretion as compared with urine from rats that had been given the NSP-containing diets. Western blotting methodologies also profiled differences between the effects of these two types of dietary fibre in the expression of xenobiotic metabolizing enzymes. We conclude that changes in activity and expression of xenobiotic metabolising enzymes could play a role in the contrasting effects of these two types of dietary fibre on carcinogen uptake and disposition. [Copyright &y& Elsevier]

Beer contains a variety of phenolic compounds. During the brewing process, some of these compounds are removed by polyvinylpolypyrrolidone (PVPP) to prevent haze formation. We have analyzed the phytochemical composition of a PVPP residue as well as of unstabilized beer and isolated a total of 51 compounds. Eight structures were identified as novel, i.e., 2-(4′-hydroxyphenyl)-3,5-dihydroxybenzoic acid ( 6), 2′-(4″-hydroxyphenyl)isoferulic acid ester ( 12), 1,2,5,7-tetrahydroxyanthraquinone ( 23) and 4,7-dihydroxy-5-(2′,4′,6′-trihydroxyphenyl)-indan-1,2-dione ( 24) from the PVPP residue, and catechin-7-O-β-(6″-O-nicotinoyl)-β-D-glucopyranoside ( 41), ent-epigallo-catechin-(4α to8, 2α toO to7)catechin ( 44), ent-epigallocatechin (4α to6, 2α toO to7)catechin ( 45) and 2,3- cis-3,4- trans-2-[2,3- trans-3,3′,4′,5,7-pentahydroxyflavan-8-yl]-4-(3,4-dihydroxyphenyl)3,5,7-trihydroxybenzopyran ( 46) from the unstabilized beer. Most of the compounds were tested for potential cancer chemopreventive activities in in vitro test systems detecting a modulation of carcinogen metabolism (inhibition of phase 1 cytochrome P450 1A (Cyp1A) activity, induction of NAD(P)H:quinone oxidoreductase (QR) activity) and anti-inflammatory mechanisms (inhibition of lipopolysaccharide (LPS)-mediated induction of inducible nitric oxide synthase (iNOS), inhibition of cyclooxygenase 1 (Cox-1) activity). 1,2,5,7-Tetrahydroxyanthraquinone ( 23) and xanthohumol ( 25), a prenylated chalcone derived from hop, were identified as the most potent compounds and were additionally tested for inhibition of chemically-induced preneoplastic lesions in an ex vivo mouse mammary gland organ culture model (MMOC). Importantly, both agents inhibited lesion formation with halfmaximal inhibitory concentrations (IC50) of 0.1 and 0.02 μM, respectively. Our results demonstrate that beer is an interesting source of potential cancer chemopreventive agents and should be further investigated with this respect. [ABSTRACT FROM AUTHOR]

Resveratrol (trans-3,4′,5-trihydroxystilbene) is a natural antioxidant found in plants, such as grapes, that studies suggest has cancer chemopreventive activity. We investigated the effects of resveratrol on DNA binding via esterification reactions with 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP) – a metabolite of a mammary gland carcinogen present in cooked meats. Treatment of primary cultures of human mammary epithelial cells with 50 μM resveratrol led to a decrease in PhIP-DNA adducts ranging from 31 to 69%. Using substrate-specific assays and mammary gland tissue cytosols, resveratrol inhibited PhIP-DNA adduct formation by O-acetyltransferase and sulfotransferase catalysis. Cytosols from tumor tissue and breast reduction tissue were similarly affected. Resveratrol also suppressed O-acetyltransferase and sulfotransferase activities from the breast cancer cell lines MCF-7 and ZR-75-1. It was also observed that resveratrol stimulated ATP-dependent cytosolic activation of N-OH-PhIP in all human samples but not in mouse liver samples. In addition to resveratol''s other preventive effects, the present data suggest that O-acetyltransferases and sulfotransferases may represent anti-oncogenic targets for resveratrol. [Copyright &y& Elsevier]

Objective: Altered metabolites are important for the tumourigenicity of hepatocellular carcinoma (HCC). We performed integrative metabolomics analysis of the metabolites changes in portal venous blood and in comparison with the metabolites changes in liver tissues and stool samples of HCC patients and healthy liver donors., Design: Serum (portal and central vein), liver tissue (HCC tumour and adjacent non-tumour, normal liver) and stool samples were collected from 102 subjects (52 HCC patients and 50 healthy controls) in the discovery cohort; and 100 subjects (50 HCC patients and 50 healthy controls) in an independent validation cohort. Untargeted metabolomic profiling was performed using high-performance liquid chromatography-mass spectrometry. The function of candidate metabolites was validated in hepatocyte cell lines., Results: Detailed metabolomic evaluation showed distinct clusters of metabolites in serum, liver tissue and stool samples from patients with HCC and control individuals (p<0.001). HCC patients had significantly higher levels of portal vein serum and HCC tissue metabolites of DL-3-phenyllactic acid, L-tryptophan, glycocholic acid and 1-methylnicotinamide than healthy controls, which were associated with impaired liver function and poor survival. On the other hand, HCC patients had lower levels of linoleic acid and phenol in portal vein and stool samples than healthy controls. Linoleic acid and phenol significantly inhibited HCC proliferation, inferring their anti-HCC function as protective metabolites., Conclusions: The integrative metabolome analysis of serum, tissue and stool metabolites revealed unreported metabolic alterations in HCC patients. In portal vein, we identified elevated and depleted metabolites signifying that they might play a role in HCC development., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The process of chemical carcinogenesis is a complex multistage process initiated by DNA damage in growth control genes. Carcinogens enter the body from a variety of sources, but most require metabolic activation before they can damage DNA. There are multiple protective processes that include detoxification and conjugation, DNA repair and programmed cell death. Most of these functions exhibit wide interindividual variation in the population and thus are thought to affect cancer risk. The role of gene-environment interactions is being explored, and current data indicate that genetic susceptibilities can modify carcinogen exposures from the diet and tobacco smoking, although much more data exist for the latter. This review addresses the relationships of human carcinogenesis to these interindividual differences of phase I, phase II and DNA repair enzymes. [ABSTRACT FROM AUTHOR]

A variety of approaches are used to study carcinogenesis. Recent advances in techniques for culture of human tissues and cells have provided additional experimental systems of study the process of carcinogenesis and the genetics of cancer. [ABSTRACT FROM AUTHOR]