DNA-reactive Metabolites Research Articles

P53 Mutagenesis by benzo[a]pyrene derived radical cations.

Benzo[a]pyrene (B[a]P), a major human carcinogen in combustion products such as cigarette smoke and diesel exhaust, is metabolically activated into DNA-reactive metabolites via three different enzymatic pathways. The pathways are the anti-(+)-benzo[a]pyrene 7,8-diol 9,10-epoxide pathway (P450/epoxide hydrolase catalyzed) (B[a]PDE), the benzo[a]pyrene o-quinone pathway (aldo ketose reductase (AKR) catalyzed) and the B[a]P radical cation pathway (P450 peroxidase catalyzed). We used a yeast p53 mutagenesis system to assess mutagenesis by B[a]P radical cations. Because radical cations are short-lived, they were generated in situ by reacting B[a]P with cumene hydroperoxide (CuOOH) and horse radish peroxidase (HRP) and then monitoring the generation of the more stable downstream products, B[a]P-1,6-dione and B[a]P-3,6-dione. On the basis of B[a]P-1,6 and 3,6-dione formation, approximately 4 μM of radical cation was generated. In the mutagenesis assays, the radical cations produced in situ showed a dose-dependent increase in mutagenicity from 0.25 μM to 10 μM B[a]P with no significant increase seen with further escalation to 50 μM B[a]P. However, mutagenesis was 200-fold less than with the AKR pathway derived B[a]P, 7-8-dione. Mutant p53 plasmids, which yield red colonies, were recovered from the yeast to study the pattern and spectrum of mutations. The mutation pattern observed was G to T (31%) > G to C (29%) > G to A (14%). The frequency of codons mutated by the B[a]P radical cations was essentially random and not enriched at known cancer hotspots. The quinone products of radical cations, B[a]P-1,6-dione and B[a]P-3,6-dione were more mutagenic than the radical cation reactions, but still less mutagenic than AKR derived B[a]P-7,8-dione. We conclude that B[a]P radical cations and their quinone products are weakly mutagenic in this yeast-based system compared to redox cycling PAH o-quinones.

Abstract 4473: Urinary biomarkers of exposure to 1,3-Butadiene and its bioactivation to DNA-reactive metabolites

Abstract 1,3-butadiene (BD) is one of the most potent and abundant carcinogens present in cigarette smoke. BD is metabolically activated to 3,4-epoxy-1-butene (EB), 1,2,3,4-diepoxy butane (DEB), hydroxymethylvinylketone (HMVK), and 3,4-epoxy-1,2-butanediol (EBD), which can react with DNA nucleobases and proteins to form promutagenic adducts. Alternatively, these electrophilic metabolites can be detoxified via hydrolysis or conjugation with glutathione. The glutathione conjugates of these metabolites are ultimately excreted in urine as 1-hydroxy 2-(N-acetylcysteinyl)-3-butene (MHBMA), 1,4-bis-(N-acetylcysteinyl)butane-2,3-diol (bis-BDMA), 1,2-dihydroxy-4-(N-acetylcysteinyl)-butane (DHBMA), and 1,2,3-trihydroxy-4-(N-acetylcysteinyl)-butane (THBMA), respectively. Since epoxide metabolites of BD are thought to be responsible for its adverse health effects, it is important to develop specific biomarkers of their formation in vivo to be used in molecular epidemiology studies. While MHBMA, DHBMA, THBMA have been previously detected in smoker's urine, bis-BDMA has not been reported. In the present study, novel stable isotope dilution HPLC-ESI-MS/MS methods for accurate and precise quantification of MHBMA, DHBMA, THBMA, and bis-BDMA in urine have been developed. Dose dependent formation of all BD-mercapturic acids including bis-BDMA was observed in urine samples from male and female F344 rats exposed to 0, 62.5 and 200 ppm of BD for 2 weeks by inhalation. The relative amounts of MHBMA, DHBMA, THBMA, and bis-BDMA in rat urine were 20:50:50:1. Furthermore, MHBMA, DHBMA, and THBMA were detected in urine samples from known smokers and non-smokers, with significantly higher concentrations observed in smokers. The mean levels of these BD-mercapturic acids in smokers were 3, 340, and 27 ng/mg creatinine, respectively. In contrast, bis-BDMA could not be detected in human urine samples, probably a result of the inefficient DEB formation in humans. These methods are now being applied to a multi-ethnic cohort study to identify any differences in metabolism that contribute to ethnic and inter-individual differences in lung cancer incidence. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4473. doi:1538-7445.AM2012-4473

Metabolic Toxicity Screening Using Electrochemiluminescence Arrays Coupled with Enzyme-DNA Biocolloid Reactors and Liquid Chromatography–Mass Spectrometry

New chemicals or drugs must be guaranteed safe before they can be marketed. Despite widespread use of bioassay panels for toxicity prediction, products that are toxic to a subset of the population often are not identified until clinical trials. This article reviews new array methodologies based on enzyme/DNA films that form and identify DNA-reactive metabolites that are indicators of potentially genotoxic species. This molecularly based methodology is designed in a rapid screening array that utilizes electrochemiluminescence (ECL) to detect metabolite-DNA reactions, as well as biocolloid reactors that provide the DNA adducts and metabolites for liquid chromatography-mass spectrometry (LC-MS) analysis. ECL arrays provide rapid toxicity screening, and the biocolloid reactor LC-MS approach provides a valuable follow-up on structure, identification, and formation rates of DNA adducts for toxicity hits from the ECL array screening. Specific examples using this strategy are discussed. Integration of high-throughput versions of these toxicity-screening methods with existing drug toxicity bioassays should allow for better human toxicity prediction as well as more informed decision making regarding new chemical and drug candidates.

Microfluidic Electrochemical Array for Detection of Reactive Metabolites Formed by Cytochrome P450 Enzymes

A novel, simple, rapid microfluidic array using bioelectronically driven cytochrome P450 enzyme catalysis for reactive metabolite screening is reported for the first time. The device incorporates an eight-electrode screen-printed carbon array coated with thin films of DNA, [Ru(bpy)(2)(PVP)(10)](ClO(4)) {RuPVP}, and rat liver microsomes (RLM) as enzyme sources. Catalysis features electron donation to cyt P450 reductase in the RLMs and subsequent cyt P450 reduction while flowing an oxygenated substrate solution past sensor electrodes. Metabolites react with DNA in the film if they are able, and damaged DNA is detected by catalytic square wave voltammetry (SWV) utilizing the RuPVP polymer. The microfluidic device was tested for a set of common pollutants known to form DNA-reactive metabolites. Logarithmic turnover rates based on SWV responses gave excellent correlation with the rodent liver TD(50) toxicity metric, supporting the utility of the device for toxicity screening. The microfluidic array gave much better S/N and reproducibility than single-electrode sensors based on similar principles.

Evaluation of Electrochemiluminescent Metabolic Toxicity Screening Arrays Using a Multiple Compound Set

Arrays for screening metabolite-generated toxicity utilizing spots containing DNA, enzyme, and electroluminescent (ECL) polymer ([Ru(bpy)(2)PVP(10)](2+)) were extended to include a fully representative set of metabolic enzymes from human and rat liver microsomes, human and rat liver cytosol, and mouse liver S9 fractions. Array use involves two steps: (1) enzyme activation of the test chemical and metabolite reaction with DNA, and then, (2) capture of ECL resulting from DNA damage using a charge coupled device (CCD) camera. Plots of ECL increase vs enzyme reaction time monitor relative rates of DNA damage and were converted into turnover rates for enzymic production of DNA-reactive metabolites. ECL turnover rates were defined by R, the initial slope of ECL increase versus enzyme reaction time normalized for amounts of enzyme and test chemical. R-values were used to establish correlations for 11 toxic compounds with the standard toxicity metrics rodent liver TD(50) and lethal dose (LD(50)), Ames tests, and Comet assays for in vitro DNA damage. Results support the value of the ECL genotoxicity arrays together with toxicity bioassays for early screening of new chemicals and drug candidates.

Regulation of Benzo[a]pyrene-Mediated DNA- and Glutathione-Adduct Formation by 2,3,7,8-Tetrachlorodibenzo-p-dioxin in Human Lung Cells

Environmental carcinogens, such as polycyclic aromatic hydrocarbons (PAHs), require metabolic activation to DNA-reactive metabolites in order to exert their tumorigenic effects. Benzo[a]pyrene (B[a]P), a prototypic PAH, is metabolized by cytochrome P450 (P450) 1A1/1B1 and epoxide hydrolase to (−)-B[a]P-7,8-dihydro-7,8-diol (B[a]P-7,8-dihydrodiol). B[a]P-7,8-dihydrodiol then undergoes further P4501A1/1B1-mediated metabolism to the ultimate carcinogen, (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro-B[a]P (B[a]PDE), which forms DNA-adducts primarily with 2′-deoxyguanosine (dGuo) to form (+)-anti-trans-B[a]PDE-N2-dGuo (B[a]PDE-dGuo) in DNA. Pretreatment of cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is known to induce P4501A1/1B1 mRNA expression through the aryl hydrocarbon receptor (AhR) pathway. This causes increased B[a]PDE-dGuo formation in liver cells. In contrast, TCDD induction of H358 lung cells surprisingly caused a decrease in (−)-B[a]P-7,8-dihydrodiol-mediated (+)-B[a]PDE-dGuo adduct formation when compared with the non-TCDD-induced cells. Furthermore, treatment of the TCDD-induced cells with (±)-B[a]PDE also resulted in decreased (+)-B[a]PDE-dGuo adduct formation when compared with the non-TCDD-induced cells. These data suggested that it was a detoxification pathway that had been up-regulated rather than an activation pathway that had been down-regulated. LC-MS was used to analyze B[a]PDE-dGuo and B[a]PDE-GSH-adducts in H358 lung and HepG2 liver cells. There was a significant increase in the (−)-B[a]PDE-GSH-adduct with high enantiomeric excess after treatment of the TCDD-induced H358 cells with (±)-B[a]PDE when compared with the noninduced cells. This could explain why increased activation of (−)-B[a]P-7,8-dihydrodiol through TCDD up-regulation of P4501A1/1B1 did not lead to increased (+)-B[a]PDE-dGuo adducts in the H358 lung cells. In addition, TCDD did not induce B[a]PDE-GSH-adduct formation in HepG2 liver cells. (±)-B[a]PDE-GSH-adducts were formed at much lower levels in both TCDD-induced and noninduced HepG2 cells when compared with (−)-B[a]PDE-GSH-adducts in the H358 lung cells. Therefore, our study has revealed that there is a subtle balance between activation and detoxification of B[a]P in lung-derived compared with liver-derived cells and that this determines how much DNA damage occurs.

Proximal Events in 7,12-Dimethylbenz[a]anthracene-Induced, Stromal Cell-Dependent Bone Marrow B Cell Apoptosis: Stromal Cell–B Cell Communication and Apoptosis Signaling

Intercellular communication is an essential process in stimulating lymphocyte development and in activating and shaping an immune response. B cell development requires cell-to-cell contact with and cytokine production by bone marrow stromal cells. However, this intimate relationship also may be responsible for the transfer of death-inducing molecules to the B cells. 7,12-Dimethylbenz[a]anthracene (DMBA), a prototypical polycyclic aromatic hydrocarbon, activates caspase-3 in pro/pre-B cells in a bone marrow stromal cell-dependent manner, resulting in apoptosis. These studies were designed to examine the hypothesis that an intrinsic apoptotic pathway is activated by DMBA and that the ultimate death signal is a DMBA metabolite generated by the stromal cells and transferred to the B cells. Although a loss of mitochondrial membrane potential did not occur in the DMBA/stromal cell-induced pathway, cytochrome c release was stimulated in B cells. Caspase-9 was activated, and formation of the apoptosome was required to support apoptosis, as demonstrated by the suppression of death in Apaf-1(fog) mutant pro-B cells. Investigation of signaling upstream of the mitochondria demonstrated an essential role for p53. Furthermore, DMBA-3,4-dihydrodiol-1,2-epoxide, a DNA-reactive metabolite of DMBA, was sufficient to upregulate p53, induce caspase-9 cleavage, and initiate B cell apoptosis in the absence of stromal cells, suggesting that production of this metabolite by the stromal cells and transfer to the B cells are proximal events in triggering apoptosis. Indeed, we provide evidence that metabolite transfer from bone marrow stromal cells occurs through membrane exchange, which may represent a novel communication mechanism between developing B cells and stromal cells.

Association of CYP1A1*2A polymorphism with male infertility in Indian population

Background The CYP1A1 gene is a polymorphic gene and encodes for the CYP1A1 enzyme that catalyzes the bioactivation of polycyclic aromatic hydrocarbons (PAHs). PAHs are ubiquitous pollutants in the natural environment, which are capable of forming DNA adducts once being activated to generate DNA reactive metabolites. DNA adducts in sperm cells could be considered as a sign of severe DNA damage, which played an important role in meiotic division during spermatogenesis and could be associated with infertility. Lipophilic compounds undergo metabolic activation by phase I enzymes, which introduce a reactive center into the molecule, followed by phase II conjugation reaction resulting in a water soluble product. Methods We genotyped CYP1A1*2A, using polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) assay in a hospital based case–control study including 206 infertile men and 230 healthy fertile (control) subjects. Results Analysis showed that CYP1A1*2A CC genotype is associated with increased risk of male infertility (OR = 6.08, 95% CI = 1.91–25.27), while TC genotype showed a non-significantly increased risk of male infertility (OR = 1.35 95% CI = 0.89–2.05). Further, when the variant genotypes were combined (CYP1A1*2A TC + CC) assuming a co-dominant allele effect, TC plus CC genotypes were also found to be significant with increased risk of male infertility (OR = 1.57 95% CI = 1.05–2.35 p = 0.02). Allele frequencies are calculated for each genotype of CYP1A1*2A and the differences for allele frequencies between the infertile and fertile men are determined using Fisher's exact test. T and C allele frequencies in infertile men are 71% and 29% as against 80% and 20% in fertile men. The differences for allele frequencies are found to be statistically significant ( p = 0.002). The results showed a drastic decrease in the sperm count and motility and increase in dead sperms in CC genotype when compared to other genotypes in infertile men. Conclusion Based on Indian study we conclude that CC genotype of CYP1A1 is associated in the pathogenesis of male infertility.

CYP1A2* 1C, CYP2E1* 5B, and GSTM1 polymorphisms are predictors of risk and poor outcome in head and neck squamous cell carcinoma patients

Head and neck squamous cell carcinoma (HNSCC) is associated with environmental factors, especially tobacco and alcohol consumption. Most of the carcinogens present in tobacco smoke are converted into DNA-reactive metabolites by cytochrome P450 (CYPs) enzymes and detoxification of these substances is performed by glutathione S-transferases (GSTs). It has been suggested that genetic alterations, such as polymorphisms, play an important role in tumorigenesis and HNSCC progression. The aim of this study was to investigate CYP1A1, CYP1A2, CYP2E1, GSTM1, and GSTT1 polymorphisms as risk factors in HNSCC and their association with clinicopathologic data. The patients comprised 153 individuals with HNSCC (cases) and 145 with no current or previous diagnosis of cancer (controls). Genotyping of the single nucleotide polymorphisms (SNPs) of the CYP1A1, CYP1A2, and CYP2E1 genes was performed by PCR-RFLP and the GSTM1 and GSTT1 copy number polymorphisms (CNPs) were analyzed by PCR-multiplex. As expected, a significant difference was detected for tobacco and alcohol consumption between cases and controls (P<0.001). It was observed that the CYP1A2*1D (OR=16.24) variant and GSTM1 null alleles (OR=0.02) confer increased risk of HNSCC development (P<0.001). In addition, head and neck cancer alcohol consumers were more frequently associated with the CYP2E1*5B variant allele than control alcohol users (P<0.0001, OR=190.6). The CYP1A2*1C polymorphism was associated with tumor recurrence (log-rank test, P=0.0161). The CYP2E1*5B and GSTM1 null alleles were significantly associated with advanced clinical stages (T3+T4; P=0.022 and P=0.028, respectively). Overall, the findings suggested that the genetic polymorphisms studied are predictors of risk and are also associated with tumor recurrence, since they are important for determining the parameters associated with tumor progression and poor outcomes in HNSCC.

Assessment of the Medicines Lidocaine, Prilocaine, and Their Metabolites, 2,6-Dimethylaniline and 2-Methylaniline, for DNA Adduct Formation in Rat Tissues

The local anesthetics lidocaine (lido) and prilocaine (prilo) are metabolized to their constituent aromatic amines 2,6-dimethylaniline (DMA, 2,6-xylidine) and 2-methylaniline (MA, o-toluidine), respectively, which are both tumorigenic in rats. The capacity of lido and prilo to form DNA adducts was assessed in major target tissues for aromatic amines in male F344 rats in comparison to equimolar doses of DMA and MA using the (32)P-postlabeling assay. Direct reaction of putative DNA-reactive metabolites N-hydroxy-DMA and N-hydroxy-MA with isolated DNA yielded reference adducts. Rats were dosed by p.o. gavage with 0.5 mmol/kg b.wt. of each test substance or the vehicle either once or daily for 7 days. After repeat administrations of either prilo or lido, DNA adducts were detected in the liver and nasal mucosa. Urinary bladder DNA adducts were detected only in lido and DMA repeat dosed rats. Groups dosed with DMA or MA showed adducts in both single- and multiple-dose groups, except for the single-dose DMA liver and urinary bladder samples, which were below the level of detection. No DNA adducts were detected in any of the white blood cell samples under either dosing regimen. The lido- and prilo-DNA adducts detected were chromatographically indistinguishable from those formed either in DMA- or MA-dosed rats, respectively, or by chemical reaction of the corresponding N-hydroxy derivatives with DNA. Thus, lido and prilo can generate DNA adducts in rats via their aromatic amine metabolites, although at lower levels than equal molar quantities of their amine metabolites.

The pattern of p53 mutations caused by PAH o-quinones is driven by 8-oxo-dGuo formation while the spectrum of mutations is determined by biological selection for dominance.

PAHs (polycyclic aromatic hydrocarbons) are suspect lung cancer carcinogens that must be metabolically converted into DNA-reactive metabolites. P4501A1/P4501B1 plus epoxide hydrolase activate PAH to (+/-)- anti-benzo[ a]pyrene diol epoxide ((+/-)- anti-BPDE), which causes bulky DNA adducts. Alternatively, aldo-keto reductases (AKRs) convert intermediate PAH trans-dihydrodiols to o-quinones, which cause DNA damage by generating reactive oxygen species (ROS). In lung cancer, the types or pattern of mutations in p53 are predominantly G to T transversions. The locations of these mutations form a distinct spectrum characterized by single point mutations in a number of hotspots located in the DNA binding domain. One route to the G to T transversions is via oxidative DNA damage. An RP-HPLC-ECD assay was used to detect the formation of 8-oxo-dGuo in p53 cDNA exposed to representative quinones, BP-7,8-dione, BA-3,4-dione, and DMBA-3,4-dione under redox cycling conditions. Concurrently, a yeast reporter system was used to detect mutations in the same cDNA samples. Nanomolar concentrations of PAH o-quinones generated 8-oxo-dGuo (detected by HPLC-ECD) in a concentration dependent manner that correlated in a linear fashion with mutagenic frequency. By contrast, micromolar concentrations of (+/-)- anti-BPDE generated (+)- trans- anti-BPDE-N (2)-dGuo adducts (detected by stable-isotope dilution LC/MS methodology) in p53 cDNA that correlated in a linear fashion with mutagenic frequency, but no 8-oxo-dGuo was detected. Previous studies found that mutations observed with PAH o-quinones were predominately G to T transversions and those observed with (+/-)- anti-BPDE were predominately G to C transversions. However, mutations at guanine bases observed with either PAH-treatment occurred randomly throughout the DNA-binding domain of p53. Here, we find that when the mutants were screened for dominance, the dominant mutations clustered at or near hotspots primarily at the protein-DNA interface, whereas the recessive mutations are scattered throughout the DNA binding domain without resembling the spectra observed in cancer. These observations, if extended to mammalian cells, suggest that mutagenesis can drive the pattern of mutations but that biological selection for dominant mutations drives the spectrum of mutations observed in p53 in lung cancer.

Investigation of the genotoxicity of dibenzo[c,p]chrysene in human carcinoma MCF-7 cells in culture.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants that have been linked to certain human cancers. The fjord region PAH dibenzo[a,l]pyrene exhibits the highest levels of carcinogenic activity of all PAH as yet tested in rodent tumor models. Another hexacyclic aromatic hydrocarbon, dibenzo[c,p]chrysene (DBC), is a unique PAH that possesses one bay region and two fjord regions within the same molecule. Due to its structure, which is a merger of the fjord region PAHs benzo[c]phenanthrene, benzo[c]chrysene, and benzo[g]chrysene, DBC is of considerable research interest. In order to investigate the pathway of regioselective metabolism we have studied the cytotoxicity, metabolic activation and DNA adduct formation of DBC in human mammary carcinoma MCF-7 cells in culture. The cytotoxicity assay indicated undisturbed cell proliferation even at concentrations as high as 4.5 microM (1.5 micro g/ml) DBC. Concurrently, DNA adducts were detected in MCF-7 cells treated with DBC only in low amounts (0.6 pmol adducts/mg DNA). On the contrary, exposure to anti-DBC-1,2-diol-3,4-epoxide and anti-DBC-11,12-diol-13,14-epoxide, two putatively genotoxic metabolites of DBC, resulted in high levels of DNA adducts (33 and 51 pmol adducts/mg DNA, respectively). Although DBC was not efficiently transformed into DNA-reactive metabolites in MCF-7 cells in culture, the results from our study indicate that the two fjord region diol-epoxide derivatives of DBC may serve as ultimate genotoxic metabolites once they are enzymatically generated under certain circumstances in vitro or in vivo.

Mutagenicity of stereochemical configurations of 1,2-epoxybutene and 1,2:3,4-diepoxybutane in human lymphblastoid cells

The carcinogenicity of 1,3-butadiene (BD) is related to its bioactivation to several DNA-reactive metabolites; accumulating evidence suggests that the stereochemistry of these BD intermediates may play a significant role in the mutagenic and carcinogenic actions of the parent compound. The objective of this study was to evaluate the cytotoxicity and mutagenicity of stereochemical forms of 1,2-epoxybutene (EB) and 1,2:3,4-diepoxybutane (DEB), two genotoxic BD metabolites, in a human lymphoblastoid cell line, TK6. Cytotoxicity was measured by comparing cloning efficiencies in chemical-exposed cells versus those in control cells. The hypoxanthine-guanine phosphoribosyltransferase (HPRT) and thymidine kinase (TK) mutant frequencies (MFs) were measured using a cell cloning assay. HPRT mutants collected from cells exposed to the three forms of DEB were analyzed by PCR to characterize large genetic alterations. All the three stereoisomers of DEB caused increased HPRT and TK MFs compared to the concurrent control samples. There were no significant differences in cytotoxicity or mutagenicity among the three isomers of DEB in TK6 cells. Molecular analysis of HPRT mutants revealed similar distributions of types of mutations among the three isomers of DEB. There were also no statistically significant differences in mutagenic efficiencies between the two isomers of EB in TK6 cells. These results were consistent with the in vivo findings that there was little difference in the mutagenic efficiencies of racemic-DEB versus meso-DEB in rodents. Thus, in terms of mutagenic efficiency, stereochemical configurations of EB and DEB are not likely to play a significant role in the mutagenicity and carcinogenicity of BD.

Coffee consumption induces GSTP in plasma and protects lymphocytes against (±)-anti-benzo[ a]pyrene-7,8-dihydrodiol-9,10-epoxide induced DNA-damage: Results of controlled human intervention trials

A number of animal studies indicate that coffee protects against chemical induction of cancer; also human studies suggest that coffee consumption is inversely related with the incidence of different forms of cancer. The protective effects were attributed to induction of glutathione- S-transferases (GSTs) and aim of the present human study was to find out if coffee causes induction of GSTs and protects against DNA-damage caused by (±)-anti-B[ a]P-7,8-dihydrodiol-9,10-epoxide (BPDE), the DNA-reactive metabolite of benzo( a)pyrene. Ten participants consumed 1 L unfiltered coffee/d over 5 days. Before and after the intervention, saliva and blood were collected and the overall GST activity was measured with 1-chloro-2,4-dinitrobenzene (CDNB). Additionally, GSTP and GSTA were determined in plasma with immunoassays. In blood, only weak ( p = 0.042) induction of GST (CDNB) was found. Furthermore, pronounced (three-fold) induction of GSTP was observed in blood, whereas GSTA was not altered. No correlations were seen between induction of GST (CDNB) and GSTP activities and the GSTP1 genotypes of the participants. Also clinical parameters (creatinine, alanine, aminotransferase, aspartate aminotransferase, alkaline phosphatase), which are markers for organ damage, were monitored. None of them was altered by coffee, but serum cholesterol levels were slightly (not significantly) enhanced. In a second trial ( n = 7), GSTP induction by unfiltered and paper filtered coffees, differing in cafestol and kahweol contents, were compared. The participants consumed 1 L coffee/d over 3 days. Again significant (three-fold) induction of GSTP was observed. The effects seen with the two coffees were identical, indicating that the diterpenoid concentrations are not responsible for the effects. In a further trial ( n = 7), the effect of coffee (unfiltered, 1 L/d, 5 days) on BPDE induced DNA-migration was studied in comet assays. A 45% reduction effect was observed. Our findings show that coffee induces GSTP in humans and indicate that consumption may lead to protection towards polycyclic aromatic hydrocarbons.

Human Papilloma Virus, Cellular Genetics and Susceptibility to Cervical Cancer

KEYWORDS Human papillmoa virus; cervical cancer; genetic susceptibility; polymorphisms ABSTRACT Research in relation to the etiology of cervical cancer has made substantial progress in the last two decades both in scientific and operational terms. In many countries, HPV is the most common sexually transmitted infection (STI) and cervical cancer remains the second most common cancer among women worldwide. Although high risk HPV infection has been identified as the primary etiological agent for cervical cancer, various co-factors such as tobacco derived carcinogens, inhalation of air cotaminated through the combustion of coal and kitchen smoke have also been reported to be associated with cervical cancer. Studies on HPV 16 E6 and E7 gene variations provide evidence for the association of specific E6 gene variants with the risk of cervical cancer. While E6 variants may be important in contributing to increased severity of cervical cancer, polymorphisms of other host cellular proteins, such as p53 and p73, may also play an as yet undefined role in modulating the E6-mediated carcinogenic process. Tobacco smoking and chewing has also been found to be associated with increased risk of cervical malignancy. Major classes of carcinogens present in tobacco and tobacco smoke are converted into DNA-reactive metabolites by cytochrome P450 (CYP)-related enzymes, several of which display genetic polymorphism. Individual susceptibility to cancer is likely to be modified by the genotype for enzymes involved in the activation or detoxification of carcinogens in tobacco and repair of DNA damage. Polymorphisms in the carcinogenmetabolizing enzymes are thought to play a role in cancer susceptibility in humans. Associations of polymorphisms with cancer risk will be especially important in cases where there are known exposures to chemical carcinogens such as with tobacco smoking, high intake of food mutagens and industrial exposures. HPV infection, through the modulation of cellular xenobiotic metabolizing enzymes, may play a role in the ability of cells to handle environmental carcinogens.

Coffee diterpenes prevent the genotoxic effects of 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) and N-nitrosodimethylamine in a human derived liver cell line (HepG2)

Aim of the present experiments was to study the genotoxic effects of coffee diterpenoids, namely cafestol palmitate and a mix of cafestol and kahweol (C + K) in human derived hepatoma (HepG2) cells. Furthermore, we investigated the potential protective properties of these substances towards carcinogens contained in the human diet, namely N-nitrosodimethylamine (NDMA) and 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP). C + K and cafestol palmitate were tested over a broad dose range in micronucleus (MN) assays and no indication for genotoxic effects was seen. In combination experiments with PhIP (300 μM), pronounced inhibition (≈1.7-fold) of MN formation was observed with C + K and cafestol palmitate at dose levels ⩾0.9 and 1.7 μg/ml, respectively. Enzyme measurements indicate that the protection is due to inhibition of sulfotransferase, an enzyme involved in the activation of the amine, and/or to induction of UDP-glucuronosyltransferase which detoxifies the DNA-reactive metabolites of PhIP. Furthermore, a significant increase of glutathione- S-transferase was seen, whereas the activities of cytochrome P-450 1A1 and N-acetyltransferase 1 were not significantly altered. Also in combination experiments with C + K and NDMA, strong protective effects (50% reduction of genotoxicity) were seen at low dose levels (⩾0.3 μg/ml). Since inhibition of MN was also observed when C + K were added after incubation with NDMA, it is likely that the chemoprotective effects are due to induction of DNA repair enzymes. Comparison of data on the effects of C + K on the cholesterol metabolism, which was investigated in earlier in vivo studies, with the present findings suggests that DNA-protective effects take place at exposure levels which are substantially lower than those which cause hypercholesterolemia.

DNA adducts in rats and mice following exposure to [4- [formula omitted]]-1,2-epoxy-3-butene and to [2,3- [formula omitted]]-1,3-butadiene

1,3-Butadiene (BD) is a major industrial chemical and a rodent carcinogen, with mice being much more susceptible than rats. Oxidative metabolism of BD, leading to the DNA-reactive epoxides 1,2-epoxy-3-butene (BMO), 1,2-epoxy-3,4-butanediol (EBD) and 1,2:3,4-diepoxybutane (DEB), is greater in mice than rats. In the present study the DNA adduct profiles in liver and lungs of rats and mice were determined following exposure to BMO and to BD since these profiles may provide qualitative and quantitative information on the DNA-reactive metabolites in target tissues. Adducts detected in vivo were identified by comparison with the products formed from the reaction of the individual epoxides with 2′-deoxyguanosine (dG). In rats and mice exposed to [4- 14 C ]-BMO (1–50 mg/kg, i.p.), DNA adduct profiles were similar in liver and lung with N 7-(2-hydroxy-3-butenyl)guanine ( G1) and N 7-(1-(hydroxymethyl)-2-propenyl)guanine ( G2) as major adducts and N 7-2,3,4-trihydroxybutylguanine ( G4) as minor adduct. In rats and mice exposed to 200 ppm [2,3- 14 C ]-BD by nose-only inhalation for 6 h, G4 was the major adduct in liver, lung and testes while G1 and G2 were only minor adducts. Another N 7-trihydroxybutylguanine adduct ( G3), which could not unambiguously be identified but is either another isomer of N 7-2,3,4-trihydroxybutylguanine or, more likely, N 7-(1-hydroxymethyl-2,3-dihydroxypropyl)guanine, was present at low concentrations in liver and lung DNA of mice, but absent in rats. The evidence indicates that the major DNA adduct formed in liver, lung and testes following in vivo exposure to BD is G4, which is formed from EBD, and not from DEB.

Polymorphisms in Drug-metabolizing Genes and Risk of Head and Neck Squamous Cell Carcinoma

One kind of defense mechanism against development of cancer involves activities of a series of enzymes that metabolize and excrete potentially toxic compounds and repair subtle mistakes in DNA. Head and neck squamous cell carcinoma (HNSCC) is causally associated with tobacco use alone and also in combination with alcohol consumption. Most of the carcinogens present in tobacco and tobacco smoke are converted into DNA-reactive metabolites by cytochrome P450 enzymes and detoxification of these metabolites are performed by glutathione S-transferases and N- acetyltransferases in humans. Several of these genes display polymorphisms that could modulate enzymatic activities like activation and detoxification of carcino- gens. Molecular epidemiological studies, to assess the risk associated with metabolic polymorphisms and HNSCC, have shown that overall effect of common polymorphisms is moderate in terms of penetrance and relative risk. However, it may have a great population impact to assess the risk of cancer caused by tobacco habit. In this review, we have discussed the reported prevalences of these common polymorphisms and risk of HNSCC causally related to tobacco habit in different populations. Although a large number of studies with small sample sizes have been conducted, the results have been inconsistent and therefore inconclusive. So, future studies should be conducted with sufficiently large sample sizes to detect the moderate or even small risks in a population.

Mutations induced by (-)-anti-11R,12S-dihydrodiol 13S,14R-epoxide of dibenzo[a,l]pyrene in the coding region of the hypoxanthine phosphoribosyltransferase (Hprt) gene in Chinese hamster V79 cells.

The polycyclic aromatic hydrocarbon dibenzo[a,l]pyrene (DB[a,l]P) is an exceptionally potent carcinogen. Its direct DNA-reactive metabolite, the fjord region (-)-anti-11R,12S-dihydrodiol 13S,14R-epoxide [(-)-anti-DB[a,l]PDE], was used to investigate induction of mutations in the coding region of the hypoxanthine phosphoribosyltransferase (Hprt) gene in Chinese hamster V79 cells. Cells exposed to 1-10 nM (-)-anti-DB[a,l]PDE exhibited a close dose-responsive increase in the frequency of mutant clones resistant to 6-thioguanine. RNA was isolated from mutant clones and cDNAs were prepared by reverse transcription. The coding region of the cDNA of the Hprt gene was amplified by polymerase chain reaction and sequenced. Analysis of the DNA base sequence changes induced by (-)-anti-DB[a,l]PDE indicated that base substitutions were the most prevalent mutations, followed by exon deletions. Among the groups of V79 cells treated with low concentrations of (-)-anti-DB[a,l]PDE, most displayed high selectivity for both A:T-->T:A transversions and A:T-->G:C transitions, while cells exposed to a higher dose (10 nM) formed predominantly G:C-->T:A transversions. Also, the number of base substitutions per mutant clone increased with dose. In general, the mutation profiles induced by (-)-anti-DB[a,l]PDE exhibited a wide spectrum; in addition to base substitutions, deletions, insertions, frameshift mutations, as well as tandem mutations were detected. Analysis of the DNA adduct levels induced by (-)-anti-DB[a,l]PDE revealed that a concentration-dependent increase in the level of adduct formation preceded the concentration-dependent increase in mutational events in these cells and that an increasing proportion of DNA adducts at deoxyadenosine were formed with dose. The results of this study demonstrate a correspondence between the concentration and types of DNA adducts and the frequency and types of mutations induced by (-)-anti-DB[a,l]PDE in V79 cells.

The Liverbeads® as a tool for the comet assay

Liverbeads®, cryopreserved hepatocytes entrapped within an alginate matrix, were examined for their relevance in the comet assay. It was estimated by their capacity to activate the indirectly acting mutagens, cyclophosphamide (CP), benzo[a]pyrene (BP), dimethylbenzanthracene (DMBA) and 2-acetylaminofluorene (2-AAF), into DNA reactive metabolites. The comet assay performed in alkaline condition is a sensitive method for detecting strand breaks at the level of individual cells and allows use of quiescent cells. Experimental conditions as treatment time, cell density, beads dissociation and viability were investigated. Significant statistical positive results assessed by the tail extent moment (TEM) were observed with both human and rat Liverbeads® after 12h duration incubation compared to metabolic non-competent cells, HeLa S3. Due to the maintenance of specific functions assessed by the observed capacity to metabolize xenobiotics, Liverbeads® represent a suitable tool system, easy to handle, for the detection of promutagens using the comet assay.