Interleukin-22 drives nitric oxide-dependent DNA damage and dysplasia in a murine model of colitis-associated cancer.

The risk of colon cancer is increased in patients with Crohn's disease and ulcerative colitis. Inflammation-induced DNA damage could be an important link between inflammation and cancer, although the pathways that link inflammation and DNA damage are incompletely defined. RAG2-deficient mice infected with Helicobacter hepaticus (Hh) develop colitis that progresses to lower bowel cancer. This process depends on nitric oxide (NO), a molecule with known mutagenic potential. We have previously hypothesized that production of NO by macrophages could be essential for Hh-driven carcinogenesis, however, whether Hh infection induces DNA damage in this model and whether this depends on NO has not been determined. Here we demonstrate that Hh infection of RAG2-deficient mice rapidly induces expression of iNOS and the development of DNA double-stranded breaks (DSBs) specifically in proliferating crypt epithelial cells. Generation of DSBs depended on iNOS activity, and further, induction of iNOS, the generation of DSBs, and the subsequent development of dysplasia were inhibited by depletion of the Hh-induced cytokine IL-22. These results demonstrate a strong association between Hh-induced DNA damage and the development of dysplasia, and further suggest that IL-22-dependent induction of iNOS within crypt epithelial cells rather than macrophages is a driving force in this process.

Chemistry meets biology in colitis-associated carcinogenesis.

The intestine comprises an exceptional venue for a dynamic and complex interplay of numerous chemical and biological processes. Here, multiple chemical and biological systems, including the intestinal tissue itself, its associated immune system, the gut microbiota, xenobiotics, and metabolites meet and interact to form a sophisticated and tightly regulated state of tissue homoeostasis. Disturbance of this homeostasis can cause inflammatory bowel disease (IBD)-a chronic disease of multifactorial etiology that is strongly associated with increased risk for cancer development. This review addresses recent developments in research into chemical and biological mechanisms underlying the etiology of inflammation-induced colon cancer. Beginning with a general overview of reactive chemical species generated during colonic inflammation, the mechanistic interplay between chemical and biological mediators of inflammation, the role of genetic toxicology, and microbial pathogenesis in disease development are discussed. When possible, we systematically compare evidence from studies utilizing human IBD patients with experimental investigations in mice. The comparison reveals that many strong pathological and mechanistic correlates exist between mouse models of colitis-associated cancer, and the clinically relevant situation in humans. We also summarize several emerging issues in the field, such as the carcinogenic potential of novel inflammation-related DNA adducts and genotoxic microbial factors, the systemic dimension of inflammation-induced genotoxicity, and the complex role of genome maintenance mechanisms during these processes. Taken together, current evidence points to the induction of genetic and epigenetic alterations by chemical and biological inflammatory stimuli ultimately leading to cancer formation.

Gut microbes define liver cancer risk in mice exposed to chemical and viral transgenic hepatocarcinogens.

BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) frequently results from synergism between chemical and infectious liver carcinogens. Worldwide, the highest incidence of HCC is in regions endemic for the foodborne contaminant aflatoxin B1 (AFB1) and hepatitis B virus (HBV) infection. Recently, gut microbes have been implicated in multisystemic diseases including obesity and diabetes. Here, the hypothesis that specific intestinal bacteria promote liver cancer was tested in chemical and viral transgenic mouse models. METHODS: Helicobacter-free C3H/HeN mice were inoculated with AFB1 and/or Helicobacter hepaticus. The incidence, multiplicity and surface area of liver tumours were quantitated at 40 weeks. Molecular pathways involved in tumourigenesis were analysed by microarray, quantitative real-time PCR, liquid chromatography/mass spectrometry, ELISA, western blot and immunohistochemistry. In a separate experiment, C57BL/6 FL-N/35 mice harbouring a full-length hepatitis C virus (HCV) transgene were crossed with C3H/HeN mice and cancer rates compared between offspring with and without H hepaticus. RESULTS: Intestinal colonisation by H hepaticus was sufficient to promote aflatoxin- and HCV transgene-induced HCC. Neither bacterial translocation to the liver nor induction of hepatitis was necessary. From its preferred niche in the intestinal mucus layer, H hepaticus activated nuclear factor-kappaB (NF-kappaB)-regulated networks associated with innate and T helper 1 (Th1)-type adaptive immunity both in the lower bowel and liver. Biomarkers indicative of tumour progression included hepatocyte turnover, Wnt/beta-catenin activation and oxidative injury with decreased phagocytic clearance of damaged cells. CONCLUSIONS: Enteric microbiota define HCC risk in mice exposed to carcinogenic chemicals or hepatitis virus transgenes. These results have implications for human liver cancer risk assessment and prevention.

Nitric oxide and TNF-alpha trigger colonic inflammation and carcinogenesis in Helicobacter hepaticus-infected, Rag2-deficient mice.

Recombinase-activating gene-2-deficient (Rag2(-/-)) mice lacking functional lymphocytes provide a useful model of chronic inflammatory bowel disease-emulating events in human colon cancer. Infection of Rag2(-/-) mice with Helicobacter hepaticus led to accumulation of macrophages and neutrophils in the colon, a process temporally related to up-regulation of tissue inducible nitric oxide synthase (iNOS) expression at the site of infection and increased nitric oxide (NO) production, as evidenced by urinary excretion of nitrate. Progressive development of increasingly severe inflammation, hyperplasia, dysplasia, and cancer accompanied these changes. Concurrent administration of an iNOS inhibitor prevented NO production and abrogated epithelial pathology and inhibited the onset of cancer. The presence of Gr-1(+) neutrophils and elevated tumor necrosis factor-alpha (TNF-alpha) expression in colon were required for increased iNOS expression and cancer, whereas interleukin-10 (IL-10) down-regulated TNF-alpha and iNOS expression and suppressed cancer. Anti-inflammatory CD4(+) regulatory lymphocytes also down-regulated iNOS and reduced cancer formation. Collectively, these results confirm essential roles for inflammation, increased TNF-alpha expression, and elevated NO production in colon carcinogenesis.

Nitric oxide enhances aggrecan degradation by aggrecanase in response to TNF-alpha but not IL-1beta treatment at a post-transcriptional level in bovine cartilage explants.

OBJECTIVE: The objective of this study was to determine the role of nitric oxide (NO) in tumor necrosis factor alpha (TNF-alpha)-induced matrix damage, compared to interleukin 1 beta (IL-1beta), in bovine cartilage explant cultures. METHODS: Cartilage explants were subjected to treatment with TNF-alpha (100ng/ml), IL-1beta (10 ng/ml) and to the nitric oxide synthase inhibitor, N-methyl-arginine (L-NMA; 1.25 mM) for 26, 50 or 120 h (5 days). The collected medium was analyzed for sulfated glycosaminoglycan (sGAG), nitrate and nitrite, matrix metalloproteinase (MMP) activity by zymography, and aggrecan degradation by immunoblotting of aggrecan-G1 and aggrecan-G1-NITEGE fragments. RNA was extracted from the 26 and 50 h treated explants for real time quantitative PCR analyses. RESULTS: TNF-alpha and IL-1beta treatment caused a 3-5 fold increase in sGAG release with an increase in aggrecanase-specific aggrecan breakdown and an increase in nitrate and nitrite production. L-NMA treatment inhibited almost 50% of the sGAG release caused by TNF-alpha treatment, with concomitant decrease in the aggrecanase-specific-NITEGE neo-epitope of aggrecan released into the medium. No L-NMA effect was identified with IL-1beta. TNF-alpha and IL-1beta both increased a disintegrin and matrix metalloproteinase with thrombospondin motif (ADAMTS)4 and ADAMTS5 transcription with no effect by L-NMA, suggesting that NO regulates aggrecanase activity at a post-transcriptional level in response to TNF-alpha. TNF-alpha and IL-1beta both caused an increase in protease transcription (MMP-3, MMP-13, ADAMTS4 and ADAMTS5) and in pro-inflammatory enzymes, inducible nitric oxide synthase and cyclooxygenase (COX)-2, as well as a decrease in matrix protein transcription, including collagen II, aggrecan, fibromodulin and link protein (IL-1beta only), and an increase in MMP-3 and MMP-9 secretion. L-NMA had no effect on gene transcription or MMP secretion. CONCLUSION: NO regulates aggrecanase activity at a post-transcriptional level in response to TNF-alpha treatment while having no effect on IL-1beta treated cartilage explants.

A microscale in vitro physiological model of the liver: predictive screens for drug metabolism and enzyme induction.

In vitro models of the liver using isolated primary hepatocytes have been used as screens for measuring the metabolism, toxicity and efficacy of xenobiotics, for studying hepatocyte proliferation, and as bioartificial liver support systems. Yet, primary isolated hepatocytes rapidly lose liver specific functions when maintained under standard in vitro cell culture conditions. Many modifications to conventional culture methods have been developed to foster retention of hepatocyte function. Still, not all of the important functions -- especially the biotransformation functions of the liver -- can as yet be replicated at desired levels, prompting continued development of new culture systems. In the first part of this article, we review primary hepatocyte in vitro systems used in metabolism and enzyme induction studies. We then describe a scalable microreactor system that fosters development of 3D-perfused micro-tissue units and show that primary rat cells cultured in this system are substantially closer to native liver compared to cells cultured by other in vitro methods, as assessed by a broad spectrum of gene expression, protein expression and biochemical activity metrics. These results provide a foundation for extension of this culture model to other applications in drug discovery -- as a model to study drug-drug interactions, as a model for the assessment of acute and chronic liver toxicity arising from exposure to drugs or environmental agents; and as a disease model for the study of viral hepatitis infection and cancer metastasis.

Excretion of the N(2)-glucuronide conjugate of 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine in urine and its relationship to CYP1A2 and NAT2 activity levels in humans.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a mutagenic and carcinogenic heterocyclic aromatic amine formed in meat products during cooking. The genotoxity of PhIP requires an initial cytochrome P450-mediated N-oxidation followed by N-O-esterification catalyzed generally by N-acetyltransferases and sulfotransferases. This study examined the urinary excretion of N(2)-(beta-1-glucos-iduronyl)-2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine-the major human urinary N-oxidation metabolite of PhIP-and determined its relationship to individual activity levels of cytochrome P4501A2 (CYP1A2) and N-acetyltransferase (NAT2). The subjects (33 males and 33 females) in the dietary study were phenotyped for their CYP1A2 and NAT2 activity prior to consumption of meat-based diet, and urine collections were obtained 0-12 and 12-24 h after ingestion of the meal. Acidic hydrolysis of N(2)-(beta-1-glucosiduronyl)-2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine and its d(3)-analog to form their respective deaminated products 2-hydroxy-1-methyl-6-phenylimidazo[4,5-b]pyridine (2-OH-PhIP) was used in the assay. The products after derivatization were analyzed by capillary gas chromatography-negative ion chemical ionization mass spectrometry with selective ion monitoring. The amount of N(2)-(beta-1-glucosiduronyl)-2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine measured as the acid hydrolysis product 2-OH-PhIP in the 0-12 h urine was 20.2 +/- 8.0% (mean +/- SD) of the ingested dose; the median was 18.8% and the range varied from 5.4 to 39.6% within the group. In a subset (n = 18) of samples from individual urine collected from the 12-24 h period, an average value of 4.4 +/- 2.5% (+/- SD) of the dose was recovered. The excretion of N(2)-(beta-1-glucosiduronyl)-2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine in the 0-12 h urine was significantly related to the quantity of PhIP ingested for all subjects (r = 0.52, P <0.0001). Linear regression analysis of the relationship between the excretion level of N(2)-(beta-1-glucosiduronyl)-2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine, adjusted for meat intake and CYP1A2 activity in the combined group of males and females showed a low association (r = 0.25, P = 0.05). There was no association between the amount of N(2)-(beta-1-glucosiduronyl)-2-hydroxyamino-1-methyl-6-phenylimid-azo[4,5-b]pyridine in urine and NAT2 activity levels of the subjects nor with the age of the subjects. N(2)-(beta-1-glucosi-duronyl)-2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine comprised a significant proportion of the ingested dose in some individuals; however, considerable variation was found within the group. The results indicate that interindividual differences in the rates of N-oxidation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, as well as phase II glucuronidation reactions regulate the formation of this metabolite in humans.

Locating nucleobase lesions within DNA sequences by MALDI-TOF mass spectral analysis of exonuclease ladders.

The location of carcinogen-modified nucleobases (DNA adducts) within DNA sequences is a critical factor affecting their promutagenic properties and persistence in DNA. We now report the use of controlled exonuclease digestion followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to directly map modified nucleobases within DNA. The DNA sequence is determined by mass spectral analysis of the DNA ladders produced by sequential removal of nucleotides with either 5'-->3' or 3'-->5' exonuclease. Individual mononucleotides are identified from the mass differences between adjacent peaks corresponding to singly charged ions of the products of enzymatic cleavage. Chemically modified nucleotides are detected and identified by their molecular weight. The resolution and mass accuracy of this approach are sufficient to identify nucleobase modifications differing in mass by as little as 2 Da. No a priori information on the DNA sequence or adduct type is required. We demonstrate the general applicability of this method by sequencing synthetic oligonucleotides containing a range of nucleobase modifications: O(6)-methylguanine, peroxynitrite-induced oxidative lesions (oxaluric acid, oxazolone, cyanuric acid), and the N(2)-guanine adduct of (+,-)-7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydribenzo[a]pyrene. Sequence information is also obtained for DNA oligodeoxynucleotides containing O(6)-pyridyloxobutylguanine, despite the ability of this lesion to block 3'-phosphodiesterase.

Oxidation of 2,6-dimethylaniline by recombinant human cytochrome P450s and human liver microsomes.

2,6-Dimethylaniline (2,6-DMA) is classified as a rodent nasal cavity carcinogen and a possible human carcinogen. The major metabolite of 2,6-DMA in rats and dogs is 4-amino-3,5-dimethylphenol (DMAP) but oxidization of the amino group to produce metabolites such as N-(2,6-dimethylphenyl)hydroxylamine (DMHA) is also indicated by the occurrence of hemoglobin adducts of 2,6-DMA in human and rats. Previous studies have shown a large interindividual variability in human 2,6-DMA hemoglobin adduct levels. In the present study, 2,6-DMA oxidation in vitro by human liver microsomes and recombinant human P450 enzymes was investigated to assess whether the hemoglobin adduct variability could be attributed to metabolic differences. At micromolar concentrations, the only product detectable (UV) was DMAP, while at 10 nM, DMHA was a substantial product. 2E1 and 2A6 were identified as the major P450s in human liver microsomes responsible for the production of DMAP by using P450-specific chemical inhibitors and mouse monoclonal antibodies that selectively inhibit human P450 2E1 and 2A6. 2A6 was identified as the major P450 responsible for the N-hydroxylation. Native P450 2E1 and human liver microsomes catalyzed the rearrangement of DMHA to DMAP independent of NADPH. Consistent with a mechanism involving oxygen rebound to the heme iron center, labeled oxygen was not incorporated into DMAP from either 18O2 gas or H2 18O in this rearrangement. Results presented here suggest much of the observed interindividual variability of 2,6-DMA hemoglobin adduct levels could be due to differences in the relative amounts of hepatic 2E1 and 2A6.

Site-selective nitration of tyrosine in human serum albumin by peroxynitrite.

Peroxynitrite, which is formed in biological systems by the reaction of nitric oxide with superoxide anion, is a highly reactive molecule that can lead to cell injury or cell death. Reactions of peroxynitrite under physiological conditions include nitration of tyrosine-containing proteins or peptides, and we have been investigating the behavior of human serum albumin following exposure to peroxynitrite. Peroxynitrite, at relative concentrations ranging from 0.2 to 50 with respect to protein, was added to human serum albumin in buffer at pH 7.2. The resulting mixtures were dialyzed to remove small molecules, dried under vacuum, and then digested with trypsin. The digests were analyzed by high performance liquid chromatography with UV detection at 230 and 354 nm, the latter wavelength being selective for nitrotyrosine. At the higher relative concentrations of peroxynitrite, the 354-nm chromatograms contained a large number of peaks, including at least nine with molecular weights corresponding to nitration of nominal tryptic peptides. Following treatment with the lower relative concentrations of peroxynitrite, however, the 354-nm chromatograms were dominated by only two nitrated peptides; these were identified by comparison of LC retention times and collision-induced decomposition mass spectra as nitro-Y(411)TK(413) and nitro-Y(138)LYEIAR(144). Each of these tyrosines resides in a known reactive site within the protein, i.e., subdomains IIIA and IB, respectively.

Gender- and smoking-related bladder cancer risk.

BACKGROUND: There is growing evidence that, when smoking habits are comparable, women incur a higher risk of lung cancer than men. Because smokers are also at risk for bladder cancer, we investigated possible sex differences in the susceptibility to bladder cancer among smokers. METHODS: A population-based, case--control study was conducted in Los Angeles, CA, involving 1514 case patients with bladder cancer and 1514 individually matched population control subjects. Information on tobacco use was collected through in-person interviews. Peripheral blood was collected from study participants to measure 3- and 4-aminobiphenyl (ABP)-hemoglobin adducts, a marker of arylamine exposure. Data were analyzed to determine whether the risk of bladder cancer differs between male and female smokers and whether female smokers exhibit higher levels of ABP-hemoglobin adducts than male smokers with comparable smoking habits. All statistical tests were two-sided. RESULTS: Cigarette smokers had a statistically significant 2.5-fold higher risk (95% confidence interval = 2.1 to 3.0) of bladder cancer than never smokers. Use of filtered versus nonfiltered cigarettes, low-tar versus higher tar cigarettes, or the pattern of inhalation did not modify the risk. The risk of bladder cancer in women who smoked was statistically significantly higher than that in men who smoked comparable numbers of cigarettes (P =.016 for sex-lifetime smoking interaction). Consistent with the sex difference in smoking-related bladder cancer risk, the slopes of the linear regression lines of the 3- and 4-ABP--hemoglobin adducts by cigarettes per day were statistically significantly steeper in women than in men (P values for sex differences <.001 and.006, respectively). CONCLUSION: The risk of bladder cancer may be higher in women than in men who smoked comparable amounts of cigarettes.

Quantification of (7S,8R)-dihydroxy-(9R,10S)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene adducts in human serum albumin by laser-induced fluorescence: implications for the in vivo metabolism of benzo[a]pyrene.

The ubiquitous environmental carcinogen benzo[a]pyrene (BaP) is metabolized in vivo in humans to its ultimate carcinogenic form of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). Mouse skin tumorigenicity studies indicate that the (7R,8S,9S,10R) enantiomer of BPDE, (7R,8S)-dihydroxy-(9S,10R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(7R,8S,9S,10R)-BPDE], is a potent tumor initiator, whereas the (7S,8R,9R,10S) enantiomer of BPDE, (7S,8R)-dihydroxy-(9R,10S)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(7S,8R,9R,10S)-BPDE], may act as a tumor promoter. In vitro experiments have shown that human liver microsomes are capable of metabolizing BaP to both the (7R,8S,9S,10R) and (7S,8R,9R,10S) enantiomers of BPDE. However, the metabolism of BaP to (7S,8R,9R,10S)-BPDE has not been demonstrated in humans in vivo. The adducts formed between human serum albumin (HSA) and the (7S,8R,9R,10R) and (7R,8S,9S,10R) enantiomers of BPDE have been described previously. (7S,8R,9R,10S)-BPDE forms a stable adduct at histidine146 of HSA, whereas (7R,8S,9R,10R)-BPDE forms a relatively unstable ester adduct at aspartate187 or glutamate188 of HSA. Using high-performance liquid chromatography with laser-induced fluorescence (LIF) detector, we quantified the level of (7S,8R,9R,10S)-BPDE adducts at histidine146 in HSA isolated from 63 healthy males who were population control subjects for an ongoing case-control study of bladder cancer. By design, roughly half of the participants were lifelong nonsmokers (n = 35), whereas the remaining 28 participants were current smokers of varying intensities. HP-BPDE adducts were detected in 60 of the 63 samples (95%) by HPLC-LIF. Adduct levels ranged from undetectable (<0.04 fmol/mg HSA) to 0.77 fmol/mg HSA. The samples had a mean and median (7S,8R,9R,10S)-BPDE-HSA adduct level of 0.22 and 0.16 fmol of adduct/mg albumin, respectively. Mean adduct levels did not differ between smokers and nonsmokers (P = 0.72). Occupational exposure to polycyclic aromatic hydrocarbons was unrelated to adduct level (P = 0.62). Intake frequencies of two food items showed statistically significant associations with adduct levels. Consumption of sweet potatoes was negatively related to adduct level (P = 0.029), whereas intake of grapefruit juice was positively related to adduct level (P = 0.045). None of the three indices of residential ambient air pollution under study showed a statistically significant association with adduct levels.

N-acetyltransferase 2 phenotype but not NAT1*10 genotype affects aminobiphenyl-hemoglobin adduct levels.

Aminobiphenyls (ABPs) in tobacco have been implicated in bladder cancer etiology in smokers. N-Acetylation of ABPs in the liver, predominantly by the N-acetyltransferase 2 (NAT2) isozyme, represents a detoxification pathway, whereas O-acetylation of N-hydroxy-ABPs in the bladder, predominantly by the N-acetyltransferase 1 (NAT1) isozyme, represents a bioactivation pathway. We and others have demonstrated that NAT2 phenotype affects 3- and 4-ABP-hemoglobin adduct levels (higher levels in slow acetylators), which are considered valid biomarkers of the internal dose of ABP to the bladder. We have also shown that NAT1 genotype (NAT1*10 allele) is associated with increased DNA adduct levels in urothelial tissue and higher risk of bladder cancer among smokers. It is not known whether NAT1*10 genotype influences ABP-hemoglobin adduct levels. Therefore, we assessed 403 primarily non-Hispanic white residents of Los Angeles County for their NAT2 acetylator phenotype, NAT1*10 acetylator genotype, and 3- and 4-ABP-hemoglobin adduct levels. Eighty-two subjects were current tobacco smokers of varying intensities. Tobacco smokers had significantly higher mean 3- and 4-ABP-hemoglobin adduct levels relative to nonsmokers. The levels increased with increased amounts smoked per day (two-sided, P < 0.0001 in all cases). With adjustment for NAT1 genotype and race, the smoking-adjusted geometric mean level of 3-ABP-hemoglobin adducts in NAT2 slow acetylators was 47% higher than that in NAT2 rapid acetylators (P = 0.01). The comparable value for 4-ABP-hemoglobin adducts was 17% (P = 0.02). In contrast, no association between NAT1*10 genotype and 3- or 4 ABP-hemoglobin adduct levels was observed after adjustment for NAT2 phenotype, smoking, and race. The present study suggests that the impact of the NAT1*10 genotype on 3- and 4-ABP-hemoglobin adducts is noninformative on the possible association between NAT1 activity and bladder cancer risk.

Nitric oxide-induced mutations in the HPRT gene of human lymphoblastoid TK6 cells and in Salmonella typhimurium.

Characterization of mutations induced by NO in different experimental systems will facilitate elucidation of mechanisms underlying its genotoxicity. The mutagenic specificity of NO in human cells is of particular interest in view of its potential role in inflammation-associated carcinogenesis. We compared mutagenesis in human lymphoblastoid TK6 cells and in Salmonella typhimurium induced by exposure to NO delivered into the medium at rates approximating its production by activated macrophages. Exposure of TK6 cells continuously for 60 min decreased viability by 88%, and survivors exhibited a sixfold increase in mutant fraction in the hprt gene. Independent mutants were isolated and mutations characterized by RT-PCR and DNA sequencing. Among a total of 68 mutants analyzed, RT-PCR products were obtained in 41 (60%), and cDNA sequencing revealed that 26 (63%) of them contained mutations located in the hprt coding region. Base substitutions were present in 18 mutants, 12 occurring at A:T base pairs. Seven mutants contained deletions of 1-27 bp and one a 13-bp insertion; the 15 remaining RT-PCR products contained whole-exon deletions, 14 involving single exons. Six tester strains of S. typhimurium, each containing one of the six possible point mutations in the target codon of a gene in the histidine biosynthetic pathway, were similarly treated with NO and induction of mutation was detected by reversion to histidine auxotrophy. Significant increases were observed in frequencies of each of the six possible base mutations, with the highest occurring in G:C --> A:T transitions. The pattern of NO-induced hprt mutations in TK6 cells was similar to a recently published spectrum in spontaneous mutants, suggesting that reactive species derived from NO may contribute to spontaneous mutagenesis of the endogenous hprt gene in human cells.

Peroxynitrite-induced DNA damage in the supF gene: correlation with the mutational spectrum.

Tissue inflammation and chronic infection lead to the overproduction of nitric oxide and superoxide. These two species rapidly combine to yield peroxynitrite (ONOO(-)), a powerful oxidizing and nitrating agent that is thought be involved in both cell death and an increased cancer risk observed for inflamed tissues. ONOO(-) has been shown to induce single-strand breaks and base damage in DNA and is mutagenic in the supF gene, inducing primarily G to T transversions clustered at the 5' end of the gene. The mutagenicity of ONOO(-) is believed to result from chemical modifications at guanine nucleobases leading to miscoding DNA lesions. In the present work, we applied a combination of molecular and analytical techniques in an attempt to identify biologically important DNA modifications induced by ONOO(-). pUC19 plasmid treated with ONOO(-) contained single-strand breaks resulting from direct sugar damage at the DNA backbone, as well as abasic sites and nucleobase modifications repaired by Fpg glycosylase. The presence of carbon dioxide in the reaction mixture shifted the ONOO(-) reactivity towards reactions at nucleobases, while suppressing the oxidation of deoxyribose. To further study the chemistry of the ONOO(-) interactions with DNA, synthetic oligonucleotides representing the mutation-prone region of the supF gene were treated with ONOO(-), and the products were analyzed by liquid chromatography-negative ion electrospray ionization mass spectrometry (LC-ESI(-) MS) and tandem mass spectrometry. 8-Nitroguanine (8-nitro-G) was formed in ONOO(-)-treated oligonucleotides in a dose-dependent manner with a maximum at a ratio of [ONOO(-)]: [DNA]=10 and a decline at higher ONOO(-) concentrations, suggesting further reactions of 8-nitro-G with ONOO(-). 8-Nitro-G was spontaneously released from oligonucleotides (t(1/2)=1 h at 37 degrees C) and, when present in DNA, was not recognized by Fpg glycosylase. To obtain more detailed information on ONOO(-)-induced DNA damage, a restriction fragment from the pSP189 plasmid containing the supF gene (135 base pairs) was [32P]-end-labeled and treated with ONOO(-). PAGE analysis of the products revealed sequence-specific lesions at guanine nucleobases, including the sites of mutational "hotspots." These lesions were repaired by Fpg glycosylase and cleaved by hot piperidine treatment, but they were resistant to depurination at 90 degrees C. Since 8-nitro-G is subject to spontaneous depurination, and 8-oxo-guanine is not efficiently cleaved by piperidine, these results suggest that alternative DNA lesion(s) contribute to ONOO(-) mutagenicity. Further investigation of the identities of DNA modifications responsible for the adverse biological effects of ONOO(-) is underway in our laboratory.

N-oxidative metabolism of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in humans: excretion of the N2-glucuronide conjugate of 2-hydroxyamino-MeIQx in urine.

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), a major heterocyclic aromatic amine (HAA) formed in cooked meats, is metabolically transformed to mutagenic/carcinogenic intermediates. Cytochrome P4501A2 (CYP1A2)-mediated N-hydroxylation followed by phase II O-esterification by N-acetyltransferase (NAT2) are generally regarded as activation processes in which MeIQx and other HAAs are converted to genotoxic species. In this study, we determined the relationship between the activities of these two enzymes and the urinary excretion level of the N2-glucuronide conjugate of 2-hydroxyamino-MeIQx--N2-(beta-1-glucosiduronyl)-2-hydroxyam ino-3,8-dimethylimidazo[4,5-f]quinoxaline (N-OH-MeIQx-N2-glucuronide)--among healthy subjects fed a uniform diet containing high-temperature cooked meat. The individuals (n = 66) in the study ate meat containing known amounts of MeIQx, and urine was collected from 0 to 12 h after the meal. After addition of the deuterium-labeled internal standard to urine, N-OH-MeIQx-N2-glucuronide was isolated using solid-phase extraction and immunoaffinity separation. The isolated conjugate was converted to the deaminated product 2-hydroxy-3,8-dimethylimidazo[4,5-f]quinoxaline (2-OH-MeIQx) by heating with acetic acid. 2-OH-MeIQx and its deuterated analogue were derivatized to form the corresponding 3,5-bis(trifluoromethyl)benzyl ether derivatives and analyzed by capillary gas chromatography-negative ion chemical ionization mass spectrometry using selected ion monitoring procedures. The subjects in the study excreted an average of 9.4 +/- 3.0% (+/-SD) of an ingested dose of MeIQx as N-OH-MeIQx-N2-glucuronide in urine; the range varied from 2.2 to 17.1%. A significant correlation was found between the level of N-OH-MeIQx-N2-glucuronide in urine and the amount of MeIQx ingested (r(s) = 0.44; P = 0.0002). The excretion level of N-OH-MeIQx-N2-glucuronide in urine was not associated with the enzyme activities of NAT2 or CYP1A2. This is expected with the latter enzyme because the metabolism of MeIQx is first order and very rapid at the amounts ingested. The amount of N-OH-MeIQx-N2-glucuronide in urine was not correlated with the age or sex of the individuals. Our results indicate that biotransformation of MeIQx via CYP1A2 oxidation to form the N-hydroxylamine followed by N2-glucuronidation is a general pathway of MeIQx metabolism in humans; the variability in the excreted levels of N-OH-MeIQx-N2-glucuronide is probably due to interindividual differences in UDP-glucuronosyltransferase activity and/or excretion pathways.

Biomonitoring of heterocyclic aromatic amine metabolites in human urine.

Human exposure to heterocyclic aromatic amines such as MeIQx (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline) may be monitored by measuring the levels of the heterocyclic aromatic amine in urine. In order to investigate the contribution of N-oxidation to the metabolism of MeIQx in vivo, we developed a biomonitoring procedure for the analysis and quantification of the N2-glucuronide conjugate of 2-hydroxyamino-3,8-dimethylimidazo[4,5-f]quinoxaline in human urine. Subjects (n = 66) in the dietary study ingested a uniform diet of cooked meat containing known amounts of MeIQx, and urine was collected after consumption of the test meal. A method based on solid-phase extraction and immunoaffinity separation was used to isolate N2-(beta-1-glucosiduronyl)-2-hydroxyamino-3,8-dimethylimidazo++ +[4,5-f]quinoxaline and its stable isotope-labeled internal standard from urine. The isolated conjugate was converted to the deaminated product 2-hydroxy-3,8-dimethylimidazo[4,5-f]quinoxaline by treatment with acetic acid under moderate heating. 2-Hydroxy-3,8-dimethylimidazo[4,5-f]quinoxaline and the [2H3]methyl analog were derivatized to form the corresponding 3,5-bis(trifluoromethyl)benzyl ether derivatives and quantified by capillary gas chromatography-negative ion chemical ionization mass spectrometry employing selected ion monitoring procedures. The amounts of N2-(beta-1-glucosiduronyl)-2-hydroxyamino-3,8-dimethylimidazo++ +[4,5-f]quinoxaline recovered in urine collected 0-12 h after the test meal accounted for 2.2-17.1% of the ingested dose, with a median value of 9.5%. The variability in the proportion of the dose excreted among the subjects may be reflective of several factors, including interindividual variation in the enzymic activity of CYP1A2 and/or conjugation reactions of the N-hydroxylamine metabolite with N-glucuronosyltransferase(s).

Molecular and genetic damage from environmental tobacco smoke in young children.

To assess the risks of early life exposure to environmental tobacco smoke (ETS), we tested whether four biomarkers in peripheral blood were associated with home ETS exposure in Hispanic and African-American children. The biomarkers included cotinine (a metabolite of nicotine) and three indicators of molecular and genetic damage from mutagens/carcinogens, protein adducts formed by the carcinogens 4-aminobiphenyl (4-ABP) and polycyclic aromatic hydrocarbons (PAHs), and sister chromatid exchanges (SCEs). We also explored possible ethnic differences in biomarkers. The study cohort comprised 109 Hispanic and African-American preschool children (1-6 years of age). Plasma cotinine was analyzed by gas chromatography, 4-ABP-hemoglobin adducts by gas chromatography-mass spectroscopy, PAH-albumin adducts by ELISA, and SCEs by cytogenetic techniques. Data on the amount of smoking by mothers (average 10.5 cigarettes per day) and other household members and regular visitors (average 6.5 cigarettes per day) were obtained by interview-administered questionnaires. Cotinine, 4-ABP-hemoglobin adducts, and PAH-albumin were significantly higher (P < 0.05) in the ETS-exposed children compared with the unexposed. SCEs were marginally higher (P = 0.076). African-American children had higher levels of cotinine (P = 0.059) and PAH-albumin (P = 0.02) than Hispanic children, after controlling for exposure to ETS. These results indicate molecular and genetic damage in minority children with

Urinary excretion of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in White, African-American, and Asian-American men in Los Angeles County.

Meats, such as beef, pork, poultry, and fish, cooked at high temperatures produce heterocyclic aromatic amines, which have been implicated indirectly as etiological agents involved in colorectal and other cancers in humans. This study examined the urinary excretion of a mutagenic/carcinogenic heterocyclic aromatic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), among 45 African-American, 42 Asian-American (Chinese or Japanese), and 42 non-Hispanic white male residents of Los Angeles who consumed an unrestricted diet. Total PhIP (free and conjugated) was isolated from overnight urine collections, purified by immunoaffinity chromatography, and then quantified by high-pressure liquid chromatography combined with electrospray ionization mass spectrometry. Geometric mean levels of PhIP in Asian-Americans and African-Americans were approximately 2.8-fold higher than in whites. The urinary excretion levels of PhIP were not associated with intake frequencies of any cooked meat based on a self-administered dietary questionnaire, in contrast to our earlier finding (Ji et al., Cancer Epidemiol. Biomark. Prev., 3: 407-411, 1994) of a positive and statistically significant association between bacon intake and the urinary level of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) among this same group of study subjects. Although there is a statistically significant association between urinary levels of PhIP and MeIQx (2-sided P = 0.001), 10 subjects (8%) displayed extreme discordance between urinary PhIP and MeIQx levels. Several factors, including variable contents of heterocyclic aromatic amines in food, enzymic and interindividual metabolic differences, and analytical methodology determine the degree of concordance between the urinary excretion levels of PhIP and MeIQx. Accordingly, urinary excretion levels of a single heterocyclic aromatic amine can only serve as an approximate measure of another in estimating exposure to these compounds in humans consuming unrestricted diets.

DNA adduct formation by secondary metabolites of cyclopenta[cd]pyrene in vitro.

In this study, calf thymus DNA was reacted in vitro with cyclopenta[cd]pyrene 3,4-epoxide (CPPE) or its metabolites, 3,4-dihydroCPP-3,4-diol (CPP-3,4-diol) and 4-hydroxy-3,4-dihydroCPP (4-OH-DCPP), activated with sulfotransferase. The adducts formed were analyzed by HPLC with fluorescence detection following enzymatic digestion of DNA to deoxynucleosides. We have shown previously that the major CPPE-reacted DNA adducts are cis-3-(deoxyguanosin-N2-yl)-4-hydroxy-3,4-dihydroCPP. Sulfotransferase activation of trans-CPP-3,4-diol yielded two adducts that were identical to the products resulting from the reaction of CPPE with DNA, while cis-CPP-3,4-diol gave very low covalent binding. Two adducts formed by sulfotransferase activation of 4-OH-DCPP were identical to the products of the reaction of synthetic 4-NaO3S-O-DCPP or sulfotransferase-activated 4-OH-DCPP with deoxyguanosine. These results indicate that guanine is the predominant site of CPP adduct formation in DNA, and that the 4-hydroxy-3-dGuo adducts can arise by reaction of DNA with either CPPE or sulfotransferase-activated trans-CPP-3,4-diol.