Aromatic Carcinogens Research Articles

Treatment of Human HeLa Cells with Black Raspberry Extracts Enhances the Removal of DNA Lesions by the Nucleotide Excision Repair Mechanism.

As demonstrated by us earlier and by other researchers, a diet containing freeze-dried black raspberries (BRB) inhibits DNA damage and carcinogenesis in animal models. We tested the hypothesis that the inhibition of DNA damage by BRB is due, in part, to the enhancement of DNA repair capacity evaluated in the human HeLa cell extract system, an established in vitro system for the assessment of cellular DNA repair activity. The pre-treatment of intact HeLa cells with BRB extracts (BRBE) enhances the nucleotide excision repair (NER) of a bulky deoxyguanosine adduct derived from the polycyclic aromatic carcinogen benzo[a]pyrene (BP-dG) by ~24%. The NER activity of an oxidatively-derived non-bulky DNA lesion, guanidinohydantoin (Gh), is also enhanced by ~24%, while its base excision repair activity is enhanced by only ~6%. Western Blot experiments indicate that the expression of selected, NER factors is also increased by BRBE treatment by ~73% (XPA), ~55% (XPB), while its effects on XPD was modest (<14%). These results demonstrate that BRBE significantly enhances the NER yields of a bulky and a non-bulky DNA lesion, and that this effect is correlated with an enhancement of expression of the critically important NER factor XPA and the helicase XPB, but not the helicase XPD.

Identification of cancer chemopreventive isothiocyanates as direct inhibitors of the arylamine N-acetyltransferase-dependent acetylation and bioactivation of aromatic amine carcinogens.

Aromatic amines (AAs) are chemicals of industrial, pharmacological and environmental relevance. Certain AAs, such as 4-aminobiphenyl (4-ABP), are human carcinogens that require enzymatic metabolic activation to reactive chemicals to form genotoxic DNA adducts. Arylamine N-acetyltransferases (NAT) are xenobiotic metabolizing enzymes (XME) that play a major role in this carcinogenic bioactivation process. Isothiocyanates (ITCs), including benzyl-ITC (BITC) and phenethyl-ITC (PEITC), are phytochemicals known to have chemopreventive activity against several aromatic carcinogens. In particular, ITCs have been shown to modify the bioactivation and subsequent mutagenicity of carcinogenic AA chemicals such as 4-ABP. However, the molecular and biochemical mechanisms by which these phytochemicals may modulate AA carcinogens bioactivation and AA-DNA damage remains poorly understood.This manuscript provides evidence indicating that ITCs can decrease the metabolic activation of carcinogenic AAs via the irreversible inhibition of NAT enzymes and subsequent alteration of the acetylation of AAs. We demonstrate that BITC and PEITC react with NAT1 and inhibit readily its acetyltransferase activity (ki = 200 M−1.s−1 and 66 M−1.s−1 for BITC and PEITC, respectively). Chemical labeling, docking approaches and substrate protection assays indicated that inhibition of the acetylation of AAs by NAT1 was due to the chemical modification of the enzyme active site cysteine. Moreover, analyses of AAs acetylation and DNA adducts in cells showed that BITC was able to modulate the endogenous acetylation and bioactivation of 4-ABP. In conclusion, we show that direct inhibition of NAT enzymes may be an important mechanism by which ITCs exert their chemopreventive activity towards AA chemicals.

Hydroxyl radical-dependent two-step chemiluminescence production by polyhalogenated quinones and H&amp;lt;sub&amp;gt;2&amp;lt;/sub&amp;gt;O&amp;lt;sub&amp;gt;2&amp;lt;/sub&amp;gt;

Tetrachloro-1,4-benzoquinone (TCBQ) is one of the major genotoxic and carcinogenic quinoid metabolites of the widely used wood preservative pentachlorophenol. We found previously that TCBQ and H2O2 could produce hydroxyl radicals (·OH) independent of transition metal ions. However, the underlying molecular mechanism is still unclear. Here we show that an unusual two-step chemiluminescence (CL) can be produced by TCBQ and H2O2. The two-step CL produced by TCBQ/H2O2 was found to be well correlated to and dependent on its two-step production of ·OH: markedly quenched by the classic ·OH scavengers, but enhanced further by classic Fenton reagent, which is known to produce extra ·OH. The initial reaction intermediate of TCBQ/H2O2 were found to be trichlorohydroxy-1,4-benzoquinone, and the major final products were identified as 2,5-dichloro-3,6-dihydroxy-1,4-benzoquonine, and the ring-opening 2,3-dichloromaleic acid and CO2 by complementary applications of multiple analytical methods such as HPLC-MS, GC-MS, ion chromatography (IC) and total organic carbon (TOC). Based on these data, we proposed that ·OH-dependent formation of quinone-dioxetane and electronically excited carbonyl species might be responsible for this unusual two-step CL production by TCBQ and H2O2. These findings may have potential applications in detecting and quantifying · OH and the ubiquitous polyhalogenated aromatic carcinogens, which may have broad biological and environmental implications for future research on these types of important species.

Intrinsic Chemiluminescence Generation during Advanced Oxidation of Persistent Halogenated Aromatic Carcinogens.

The ubiquitous distribution coupled with their carcinogenicity has raised public concerns on the potential risks to both human health and the ecosystem posed by the halogenated aromatic compounds (XAr). Recently, advanced oxidation processes (AOPs) have been increasingly favored as an "environmentally-green" technology for the remediation of such recalcitrant and highly toxic XAr. Here, we show that AOPs-mediated degradation of the priority pollutant pentachlorophenol and all other XAr produces an intrinsic chemiluminescence that directly depends on the generation of the extremely reactive hydroxyl radicals. We propose that the hydroxyl radical-dependent formation of quinoid intermediates and electronically excited carbonyl species is responsible for this unusual chemiluminescence production. A rapid, sensitive, simple, and effective chemiluminescence method was developed to quantify trace amounts of XAr and monitor their real-time degradation kinetics. These findings may have broad biological and environmental implications for future research on this important class of halogenated persistent organic pollutants.

Induction of Non-Cytochrome Mediated Enzymes- Xanthine Oxidase and Glutathione-S-Transferase By 3-Methylcholanthrene in Kidney Tissues of Male Albino Mice

3-Methycholanthrene, a potent polycyclic aromatic carcinogen, induces the activities of Phase I and Phase II drug metabolism enzymes in the body. Increase in the activities of Cytochrome P-450, a most important Phase I enzyme, by 3-Methylcholanthrene in liver and kidney tissues are well established. However the effect of this aromatic carcinogen on non-cytochrome mediated drug metabolizing enzymes-renal xanthine oxidase and Glutathione-s-transferase in renal tissues is still not clear. In the present investigation it is found that intraperitonal administration of 0.25 ml of 3-Methylcholanthrene significantly elevates the activities of xanthine oxidase (p< 0.05) and Glutathione-s-transferase (p<0.05) activities in the kidney tissues. From the present study it can be concluded that like liver tissues, kidney tissues are highly modulated by the administration of xenobiotics and carcinogens. This finding can be of significant therapeutic, toxicological and pharmacological importance.

Oxidative DNA Adducts Detected in Vitro from Redox Activity of Cigarette Smoke Constituents

Cigarette smoke contains a variety of carcinogens, cocarcinogens, mutagens, and tumor promoters. In addition to polycyclic aromatic carcinogens and tobacco-specific nitrosamines, cigarette smoke also contains an abundance of catechols, aldehydes, and other constituents, which are DNA damaging directly or indirectly; therefore, they can also contribute to cigarette smoke-mediated carcinogenicity. In this study, we investigated the potential of cigarette smoke constituents to induce oxidative damage to DNA through their capacity to redox cycle. When DNA (300 μg/mL) was incubated with cigarette smoke condensate (0.2 mg of tobacco particulate matter/mL) and CuCl(2) as a catalyst (50-100 μM), a variety of oxidative DNA adducts were detected by (32)P-postlabeling/TLC. Of the total adduct burden (2114 ± 419 adducts/10(6) nucleotides), over 40% of all adducts were attributed to the benchmark oxidative DNA lesion, 8-oxodeoxyguanosine (8-oxodG). Adducts were formed dose dependently. Essentially, similar adduct profiles were obtained when cigarette smoke condensate was substituted with ortho- and para-dihydroxybenzenes. Vehicle treatment with Cu(2+) or CSC alone did not induce any significant amount of oxidative DNA damage. Furthermore, coincubation of cigarette smoke condensate and ortho-dihydroxybenzene with DNA resulted in a higher amount of oxidative DNA adducts than obtained with the individual entity, suggesting that adducts presumably originated from catechols or catechol-like compounds in cigarette smoke condensate. Adducts resulting from both cigarette smoke condensate and pure dihydroxybenzenes were chromatographically identical to adducts formed by reaction of DNA with H(2)O(2), which is known to produce 8-oxodG, and many other oxidative DNA adducts. When the cigarette smoke condensate-DNA reaction was performed in the presence of ellagic acid, a known antioxidant, the adduct formation was inhibited dose dependently, further suggesting that adducts originated from oxidative pathway. Our data thus provide evidence of the capacity of catechols or catechol-like constituents in cigarette smoke to produce oxidative DNA damage, which may contribute to the tumor-promoting activity of cigarette smoke.

Unprecedented hydroxyl radical-dependent two-step chemiluminescence production by polyhalogenated quinoid carcinogens and H 2 O 2

Most chemiluminescence (CL) reactions usually generate only one-step CL, which is rarely dependent on the highly reactive and biologically/environmentally important hydroxyl radicals ((•)OH). Here, we show that an unprecedented two-step CL can be produced by the carcinogenic tetrachloro-1,4-benzoquinone (also known as p-chloranil) and H(2)O(2), which was found to be well-correlated to and directly dependent on its two-step metal-independent production of (•)OH. We proposed that (•)OH-dependent formation of quinone-dioxetane and electronically excited carbonyl species might be responsible for this unusual two-step CL production by tetrachloro-1,4-benzoquinone/H(2)O(2). This is a unique report of a previously undefined two-step CL-producing system that is dependent on intrinsically formed (•)OH. These findings may have potential applications in detecting and quantifying (•)OH and the ubiquitous polyhalogenated aromatic carcinogens, which may have broad biological and environmental implications for future research on these types of important species.

Abstract 4423: Transgenic liver expression of human arylamine N-acetyltransferase NAT2 restores sensitivity of male Nat1/2 null mice to 4-aminobiphenyl-induced liver tumorigenesis

Abstract We recently observed that male Nat1/2(-/-) mice, which are deficient in the arylamine N-acetyltransferases Nat1 and Nat2, have a significantly lower incidence and multiplicity of liver tumors than wild-type C57BL/6 mice after their neonatal exposure to the aromatic amine carcinogen 4-aminobiphenyl (ABP). Female mice showed low or no liver tumors from ABP exposure regardless of Nat1/2 status. The goal of the present study was to determine the effect of liver-selective transgenic expression of human NAT2 on ABP-induced liver tumorigenicity in male and female Nat1/2(-/-) mice. We generated a hNAT2tgNat1/2(-/-) strain of mice that express human NAT2 selectively in liver on a Nat1/2 null background, exposed male and female mice to either DMSO vehicle, a low dose (600 nmoles) or a high dose (1200 nmoles) of ABP during the second postnatal week, and assessed mice for tumor growth at one year of age. Male hNAT2tgNat1/2(-/-) mice had liver tumor incidences of 29% (low dose) and 61% (high dose), which more closely resembled those of C57BL/6 mice (60% and 69%, respectively) than those of Nat1/2(-/-) mice (0% and 35%, respectively). This suggests that human NAT2 is capable of substantially restoring the sensitivity of mice to ABP-induced liver tumorigenicity that is lost by the absence of mouse Nat1 and Nat2. On the other hand, female hNAT2tgNat1/2(-/-) mice were still as resistant to liver tumors as C57BL/6 or Nat1/2(-/-) mice. No tumors were observed in any other organs. These results suggest that the human and mouse NAT enzymes play similar roles in enhancing liver tumor growth, and that the mechanism of female protection against ABP-induced tumor growth is unrelated to NAT status. However, neither the sex nor the NAT status of the mice appear to be correlated with the ability of ABP to produce acute DNA-damaging or mutation-inducing effects in liver. This suggests that the role of the human and mouse NAT enzymes in promoting liver tumor growth in mice may be due to a mechanism that is independent of their ability to influence the process of ABP metabolic activation and subsequent DNA damage leading to tumor initiation. 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 4423. doi:1538-7445.AM2012-4423

ChemInform Abstract: The Synthesis of C8‐Aryl Purines, Nucleosides, and Phosphoramidites

AbstractReview: 79 refs.

Susceptibility of Human Lymphocytes to 2-Nitrofluorene-Induced Gene Mutation is Strongly Dependent on Metabolic Genotypes

Genetic polymorphisms in drug metabolism contribute largely to adverse drug reactions in susceptible individuals due to excess production of reactive metabolites and active oxygen species. We conducted an in vitro study that clearly demonstrates that functional impact of common polymorphisms in the metabolic genes, such as N-acetyltransferase 2 (NAT2), can be easily reflected in mutant induction in the gene coding for hypoxanthineguanine phosphoribosyl transferase (HPRT) in T-lymphocytes isolated from human peripheral blood. NAT2 is involved in the metabolic activation of 2-nitrofluorene (2-NF) to the known aromatic amide carcinogen N-acetyl- 2-aminofluorene. Subsequent deactivation through glutathione conjugation involves glutathione S-transferase M1 (GSTM1). We obtained a clear dose-related increase in the HPRT mutant frequency after treating mitogen-stimulated lymphocytes isolated from a normal blood donor with 2-NF (up to 5 fold at 400 μg/mL, 24h exposure), while no HPRT mutant induction was observed using cells from another blood donor. The susceptible cells turned out to have the NAT2 rapid and GSTM1 null genotype combination (capable of activation, with insufficient deactivation), while the resistant cells had the NAT2 slow and GSTM1 positive combination. Although the contribution of GSTM1 genotype is less clear, our finding suggests that functional polymorphisms in key metabolic genes do affect induction of gene mutations at the HPRT locus and at least the NAT2 genotype plays a critical role in determining the susceptibility of human cells to genotoxicity of 2-NF. Further, human peripheral T-lymphocytes and the in vitro HPRT gene mutation assay can be utilized to study the functional impact of common genetic polymorphisms in drug metabolism and to identify risk genotypes susceptible for drug toxicity and somatic mutations.

Abstract 1850: Benzyl isothiocyanate causes proteasome-mediated degradation of aryl hydrocarbon receptor in Hepa1c1c7 cells

Abstract The biotransformation and detoxification of polycyclic aromatic carcinogens is dependent upon the activity of CYP1A1 and NQO1. Transcriptional activation of these enzymes is regulated by the aryl hydrocarbon receptor (AhR) and Nuclear Factor-Erythroid 2-Related Factor. These transcription factors reside in the cytoplasm and when activated, shed their respective chaperones and translocate into the nucleus where they interact with nuclear proteins involved with transcription of genes possessing xenobiotic (XRE) and antioxidant (ARE) response elements. We have previously reported that the phytochemical benzyl isothiocyanate (BITC) induces ligand-independent nuclear translocation of the AhR in Hepa1c1c7 cells and also induces AhR heterodimerization with ARNT. This suggests that BITC might bind the AhR and affect enzyme activities through transcriptional regulation of XRE-responsive genes. We also have observed that BITC causes rapid degradation of the AhR both alone and in combination with known receptor ligands. In the current study, we investigated whether BITC causes proteasomal degradation of the AhR and whether this degradation occurs through increased ubiqitination of the receptor. This is important because it provides a potential mechanism by which BITC affects AhR turnover and consequently its regulation of phase I and II enzymes. Hepa1 cells were treated overnight with DMSO (control), 10 µM BITC, 10 µM β-napthoflavone (BNF) or a combination of BITC and BNF both at 10 µM. AhR was immunoprecipitated and detected using western blot. AhR was degraded in samples treated with BITC alone compared to control. Additionally, BITC enhanced degradation of AhR in samples treated with both BITC and BNF compared to BNF alone. Western blots re-probed with anti-ubiquitin showed increased ubiquitination of proteins in BITC and BNF-treated samples compared to control. In additional experiments, cells were treated for 16 hours with DMSO, 10 µM BITC, 10 µM BNF or a combination of BNF (10 µM) and increasing concentrations of BITC (1-10 µM). Sub-cellular fractionation of samples treated with a combination of BITC and BNF showed a dose-dependent increase in the amount of insoluble AhR compared to either BITC or BNF alone. AhR degradation was blocked in cells pre-treated with the proteasome inhibitor MG132 but not in cells pre-treated with broad spectrum protease inhibitors or PMSF. This suggests that degradation is specific to the proteasome rather than other proteases such as serine proteases and calpains. Collectively these results show that BITC degrades the AhR and this is mediated by ubiquitin and the proteasome. AhR degradation might affect transcription of XRE responsive genes and therefore carcinogen activation. These results also provide a potential mechanism for BITC as a cancer chemopreventive agent. (Support: NIH MBRS/GM028248; RCMI RR033034) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1850. doi:10.1158/1538-7445.AM2011-1850

Metal Complexes as DNA Intercalators

DNA has a strong affinity for many heterocyclic aromatic dyes, such as acridine and its derivatives. Lerman in 1961 first proposed intercalation as the source of this affinity, and this mode of DNA binding has since attracted considerable research scrutiny. Organic intercalators can inhibit nucleic acid synthesis in vivo, and they are now common anticancer drugs in clinical therapy. The covalent attachment of organic intercalators to transition metal coordination complexes, yielding metallointercalators, can lead to novel DNA interactions that influence biological activity. Metal complexes with σ-bonded aromatic side arms can act as dual-function complexes: they bind to DNA both by metal coordination and through intercalation of the attached aromatic ligand. These aromatic side arms introduce new modes of DNA binding, involving mutual interactions of functional groups held in close proximity. The biological activity of both cis- and trans-diamine Pt(II) complexes is dramatically enhanced by the addition of σ-bonded intercalators. We have explored a new class of organometallic "piano-stool" Ru(II) and Os(II) arene anticancer complexes of the type [(η(6)-arene)Ru/Os(XY)Cl](+). Here XY is, for example, ethylenediamine (en), and the arene ligand can take many forms, including tetrahydroanthracene, biphenyl, or p-cymene. Arene-nucleobase stacking interactions can have a significant influence on both the kinetics and thermodynamics of DNA binding. In particular, the cytotoxic activity, conformational distortions, recognition by DNA-binding proteins, and repair mechanisms are dependent on the arene. A major difficulty in developing anticancer drugs is cross-resistance, a phenomenon whereby a cell that is resistant to one drug is also resistant to another drug in the same class. These new complexes are non-cross-resistant with cisplatin towards cancer cells: they constitute a new class of anticancer agents, with a mechanism of action that differs from the anticancer drug cisplatin and its analogs. The Ru-arene complexes with dual functions are more potent towards cancer cells than their nonintercalating analogs. In this Account, we focus on recent studies of dual-function organometallic Ru(II)- and Os(II)-arene complexes and the methods used to detect arene-DNA intercalation. We relate these interactions to the mechanism of anticancer activity and to structure-activity relationships. The interactions between these complexes and DNA show close similarities to those of covalent polycyclic aromatic carcinogens, especially to N7-alkylating intercalation compounds. However, Ru-arene complexes exhibit some new features. Classical intercalation and base extrusion next to the metallated base is observed for {(η(6)-biphenyl)Ru(ethylenediamine)}(2+) adducts of a 14-mer duplex, while penetrating arene intercalation occurs for adducts of the nonaromatic bulky intercalator {(η(6)-tetrahydroanthracene)Ru(ethylenediamine)}(2+) with a 6-mer duplex. The introduction of dual-function Ru-arene complexes introduces new mechanisms of antitumor activity, novel mechanisms for attack on DNA, and new concepts for developing structure- activity relationships. We hope this discussion will stimulate thoughtful and focused research on the design of anticancer chemotherapeutic agents using these unique approaches.

The Synthesis of C8-Aryl Purines, Nucleosides and Phosphoramidites

C8-Aryl purines, their nucleosides, and phosphoramidites has been synthetic targets for more than 60 years. Interest in these compounds stems from their utility as fluorescent markers, they have therapeutic uses, are biomarkers, biomolecular probes, supramolecular building blocks, and for conformational studies. Until recently, the selective arylation of the C8-position of purines has been a challenging task. Several approaches have been explored including building them up from a pyrimidine or selective C8-modification of an unsubstituted purine. Neither of these approaches has proven to have broad scope. The discovery that C8-aryl purine nucleosides can be made via the Suzuki cross-coupling reaction has allowed a diverse array of analogues to be prepared and, in turn, the corresponding phosphoramidites. The latter is particularly significant as C8-aryl purine adducts are a major mutation observed from aromatic carcinogens and ready access to C8-aryl phosphoramidites will facilitate the synthesis and study of C8-aryl purine biomarkers and modified oligonucleotides.

Abstract B77: Translational effects of alternative NAT1 mRNA isoforms

Abstract B77 N-acetyltransferase 1 (NAT1) catalyzes the N-acetylation of many aromatic amine carcinogens such as 2-aminofluorene and 4-aminobiphenyl. Following N-oxidation by cytochrome P450’s, NAT1 catalyzes the O-acetylation of the N-hydroxy metabolites of many aromatic and heterocyclic amine carcinogens. NAT1 genetic polymorphisms have been associated with differential risks to various cancers related to aromatic and heterocyclic amine carcinogens, but the findings have been inconsistent. Individual susceptibility to these carcinogenic compounds could be modified by genetic polymorphism or by other factors causing differences in NAT1 expression. Our laboratory and others have reported that NAT1 is transcribed from a major promoter, NATb, and an alternative promoter, NATa, resulting in mRNAs with distinct 5’-UTR regions. The 117 nucleotide NATb 5’-UTR contains two noncoding exons, while the 371 nucleotide NATa 5’UTR contains four noncoding exons. The two 5’UTRs share a 79 nucleotide exon immediately upstream of the open reading frame (ORF). We hypothesized that mRNAs including either the NATa or NATb 5’-UTR may be associated with differences in protein expression and that SNPs included in variant NAT1 alleles may modify this effect. We therefore investigated effects on protein expression due to interaction of the different 5’-UTRs with the ORF and region 3’ to the ORF of the reference NAT1*4 allele, and two variant alleles: NAT1*10 and NAT1*11 that are associated with modified risk to arylamine-induced cancers. pcDNA5/FRT plasmid constructs were prepared for transient transfection of full length human mRNAs including either the NATa or NATb 5’-UTR, the ORF, and 885 nucleotides of the region 3’ to the ORF. For each allele of interest, two constructs were made, one including the NATa 5’-UTR and one including the NATb 5’-UTR. Following transient transfection into Chinese hamster ovary cells, NAT1-catalyzed N-acetylation of p-aminobenzoic was measured by HPLC and NAT1 protein expression was measured by Western blot. A difference of approximately 10 fold (p&amp;lt;0.05) in both NAT1 catalytic activity and protein was observed between the NATa and NATb mRNA forms but there was no evidence for allele specific 5’-UTR interactions. These results suggest that differences in NAT1 expression are modified by factors other than polymorphisms in the ORF. Further experiments to examine the possible roles of mRNA production, mRNA stability or differences in mRNA translation efficiency on NAT1 expression are needed. Partially supported by USPHS grants CA034627, ES014559, ES011564, and ES014443. Citation Information: Cancer Prev Res 2008;1(7 Suppl):B77.

Fluorescence line-narrowing spectroscopy for probing purposes in bioanalytical and environmental chemistry

Fluorescence line-narrowing spectroscopy (FLNS) is a cryogenic technique that can be used to obtain high-resolution fluorescence spectra. The technique has mainly been used for fingerprint identification. However, this review focuses on the trend to use FLNS for probing purposes: obtaining information on interactions with the local environment or determining conformations.We briefly discuss the basics of FLNS, solute-matrix interactions and important instrumental aspects. We describe examples from various bioanalytical fields: DNA adducts of the aromatic carcinogen benzo[a]pyrene (BP); binding of BP metabolites to monoclonal antibodies or the estrogen receptor; FLNS studies of porphyrin-containing proteins; and, photosynthetic systems.As an illustration from the field of environmental chemistry, we discuss the use of luminescence line-narrowing spectroscopy – a special mode of FLNS – to study the complexation of lanthanides to humic substances.

Inhibition by acetaminophen of neoplastic initiation elicited in rat liver by the DNA-reactive hepatocarcinogen N-acetyl-2-aminofluorene

Acetaminophen, a monocyclic phenolic compound and analgesic, when fed at 8900 p.p.m. in the diet, was reported to inhibit the hepatocarcinogenicity in rats of the aromatic amine proximate carcinogen N-hydroxy-N-acetyl-2-aminofluorene. To elucidate the mechanism(s) of this anticarcinogenicity, the present study examined whether acetaminophen at lower doses has the ability to inhibit the initiating effects in the rat liver of the precursor hepatocarcinogen N-acetyl-2-aminofluorene. Male F344 rats were allocated to six groups, which were maintained under reverse light cycle conditions to assure acetaminophen ingestion at the time of N-acetyl-2-aminofluorene administration during the dark phase, which was imposed from 07.00 to 19.00 h. Group 1 served as vehicle control (0.5% carboxymethylcellulose) for N-acetyl-2-aminofluorene, which was administered intragastrically 3 days per week at 2.6 mg/kg for 8 weeks (group 4) to achieve initiation. Acetaminophen was given in the diet either alone at 2400 or 4800 p.p.m. for 9 weeks (groups 2 and 3), or with N-acetyl-2-aminofluorene (groups 5 and 6), starting 1 week before N-acetyl-2-aminofluorene administration. Acetaminophen blood levels were about 1 and 4 microg/ml at the two dietary concentrations. N-acetyl-2-aminofluorene induced hepatocellular preneoplastic lesions measured as hepatocellular altered foci expressing glutathione S-transferase-P, reflecting initiation. Induced foci were reduced with administration of both concentrations of acetaminophen. Acetaminophen by itself produced no DNA adducts nor did it alter the high formation of N-acetyl-2-aminofluorene-DNA adducts, about 200 in 10 nucleotides, measured by nucleotide postlabeling. Acetaminophen did not affect background liver cell proliferation, but significantly reduced N-acetyl-2-aminofluorene-induced increased proliferation measured by proliferating cell nuclear antigen immunostaining. Thus, acetaminophen effectively protected hepatocytes from the initiating effects of N-acetyl-2-aminofluorene, possibly through a cytoprotective effect resulting from slowing the rate of induced cell turnover.

Risk for K-ras gene mutations in smoking-induced lung cancer is associated with cytochrome P4501A1 and glutathione S-transferase μ1 polymorphisms

Polycyclic aromatic hydrocarbons (PAHs) and other tobacco-related carcinogens are oxidized by phase I enzymes into reactive metabolites that are then detoxified by phase II enzymes. These findings suggested that polymorphisms in genes controlling metabolism of carcinogens underlie individual variations in cancer susceptibility. Moreover, it is unclear whether there is a relation between genetically determined individual susceptibilities and target gene mutations in lung carcinogenesis. We examined K-ras mutations in relation to polymorphisms in the cytochrome P4501A1 (CYP1A1) and glutathione S-transferase micro1 (GSTM1) genes in 246 patients with lung adenocarcinoma and 167 patients with lung squamous cell carcinoma. K-ras mutations were found in 33 of 413 (8.0%) tumors, and all K-ras gene mutations were found in habitual smokers, 110 non-smokers were excluded from final analysis. Among smokers with lung adenocarcinoma, K-ras mutations occurred with greater frequency in patients with the GSTM1(-) genotype than in those with the GSTM1(+) genotype. Patients with a combination of the CYP1A1 m1/m2 and GSTM1(-) genotypes showed an increased probability of having mutated K-ras genes (OR, 6.00; p=0.031; 95% CI, 1.18-30.62) in comparison to those with the CYP1A1 m1/m1 and GSTM1(+) genotypes. The impact of combined genotypes of the CYP1A1 Ile/Val polymorphism and GSTM1 on mutation of K-ras was also analyzed, and a higher risk of having a mutated K-ras gene was found for both the CYP1A1 Ile/Ile and GSTM1(-) (OR, 6.32; p=0.021; 95% CI, 1.33-30.19) and CYP1A1 Ile/Val and GSTM1(-) (OR, 6.09; p=0.042; 95% CI, 1.07-34.72) genotype combinations in patients with adenocarcinoma. There was no significant association for squamous cell carcinoma. In conclusion, these findings suggest that K-ras mutations in smokers with lung adenocarcinoma may be due in part to accumulation of carcinogens, which is not adequately detoxified in individuals with certain CYP1A1 genotypes and the GSTM1(-) genotype.

Prostate expression of N-acetyltransferase 1 (NAT1) and 2 (NAT2) in rapid and slow acetylator congenic Syrian hamster.

Arylamine carcinogens induce prostate tumours in rodent models and may contribute to the aetiology of human prostate cancers. N-acetylation and O-acetylation, catalysed by N-acetyltransferase 1 (NAT1) and 2 (NAT2), activate and/or deactivate arylamines to electrophilic intermediates that bind DNA and initiate tumours in target organs. NAT1 and NAT2 are both subject to genetic polymorphism in humans, and molecular epidemiological investigations suggest that NAT1 and/or NAT2 acetylator genotype modifies risk for prostate cancers. A Syrian hamster model congenic at the NAT2 locus was used to investigate the role of acetylator genotype in N- and O-acetylation of aromatic and heterocyclic amine carcinogens in the liver and prostate. A gene dose-response (NAT2*15/*15>NAT2*15/*16A>NAT2*16A/*16A) relationship was observed in liver and prostate cytosol towards the N-acetylation of p-aminobenzoic acid, 2-aminofluorene, beta-napthylamine, 4-aminobiphenyl, and 3,2'-dimethyl-4-aminobiphenyl. NAT1 and NAT2 were separated and partially purified from liver and prostate cytosol. NAT1 and NAT2 in liver and prostate catalysed -acetylation of the arylamines above and O-acetylation of N-hydroxy derivatives of 2-aminofluorene, 4-aminobiphenyl and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. Rates were higher in rapid versus slow acetylators when catalysed by NAT2 but not when catalysed by NAT1. Partially purified prostate NAT2 exhibited higher apparent K(m) and V(max) than NAT1. Prostate NAT1 mRNA levels were higher than NAT2 and neither NAT1 nor NAT2 mRNA level differed with NAT2 acetylator genotype. The results provide mechanistic support for a role of NAT1 and/or NAT2 acetylator polymorphism(s) in human prostate cancer risk related to aromatic and/or heterocyclic amine carcinogens.

LACK OF DNA BINDING IN THE RAT NASAL MUCOSA AND OTHER TISSUES OF THE NASAL TOXICANTS ROFLUMILAST, A PHOSPHODIESTERASE 4 INHIBITOR, AND A METABOLITE, 4-AMINO-3,5-DICHLOROPYRIDINE, IN CONTRAST TO THE NASAL CARCINOGEN 2,6-DIMETHYLANILINE*

The phosphodiesterase 4 inhibitor Roflumilast (B9302-107) (RF) and its metabolite 4-amino-3,5-dichloropyridine (ADCP) produced nasal toxicity in preclinical safety studies with rats. The purpose of this study was to assess the possible formation of DNA adducts, by RF and ADCP, in the nasal mucosa, liver and testes of male rats using the 32P-postlabeling assay. For comparison, rats were exposed to the DNA-reactive carcinogens 2,6-dimethylaniline (DMA), also known as 2,6-xylidine, a nasal carcinogen, and the aromatic amine carcinogens 4,4′-methylene-bis(2-chloroaniline) (MOCA), which yields monocyclic DNA adducts, and 2-acetylaminofluorene (2-AAF). In the case of RF, possible sources of DNA adducts include the parent molecule and its ADCP moiety by enzymatic N-hydroxylation and sulfation, reactions typical of carcinogenic aromatic amines. 4-Acetoxylamino-3,5-dichloropyridine (N-acetoxy-ADCP), a chemically activated derivative of ADCP, was prepared and used to modify DNA which was then used to establish the chromatographic conditions with which to reliably detect whether or not such adducts were formed metabolically from RF and ADCP. Similarly, a standard N-hydroxy-DMA was prepared, but the corresponding N-acetoxy derivative was unstable and decomposed during synthesis. Both N-hydroxy-DMA and N-acetoxy-ADCP were mutagenic in the Salmonella typhimurium Ames assay using strain TA100 without an exogenous bioactivation system, with the former being more potent. N-hydroxy-ADCP was essentially inactive in this assay. For the 32P-postlabeling assay, male Wistar rats were exposed to the test substances and carrier control compounds by intragastric instillation at the selected dose levels for 7 days. Subsequently, the nasal mucosa, liver, and testes of the rats exposed to the test or control compounds were extirpated, the DNA extracted and the samples postlabeled. The patterns of adducts formed with the test compounds were compared to those formed in N-acetoxy-ADCP- and N-hydroxy-DMA-adducted DNA, which were assayed by both nuclease P1 and butanol enhancement methods. Based upon the similarity of results from the two enhancement methods, only the former was used for the in vivo studies. No evidence was obtained for the formation of DNA adducts from RF or its metabolites, specifically ADCP, under the conditions of these assays despite the ability to detect adducts from DNA modified chemically with N-acetoxy-ADCP and DNA adducts from the other compounds in their target organs. In the absence of a pattern of compound-related spots, we conclude that RF does not form DNA adducts having the potential to initiate neoplasia in these three tissues.

Ethanol Enhances the Activation of 1-Hydroxymethylpyrene to DNA Adduct-Forming Species in the Rat

1-Methylpyrene, a hepatocarcinogen, is metabolized via 1-hydroxymethylpyrene (HMP) to a reactive sulfuric acid ester (SMP). SMP forms DNA adducts and, alternatively, is conjugated with glutathione, leading to the excretion of methylpyrenyl mercapturic acid (MPMA). However, 1-pyrenylcarboxylic acid and its derivatives constitute nearly 80% of the excreted metabolites after administration of HMP to rats. Thus, side-chain oxidation, probably catalyzed by alcohol and aldehyde dehydrogenases, competes with the activation to SMP. We therefore studied the bioactivation of HMP in rats treated with 4-methylpyrazole (an inhibitor of alcohol dehydrogenases), disulfiram (an inhibitor of aldehyde dehydrogenases), or ethanol (a substrate of alcohol dehydrogenases). These metabolic modulators enhanced the levels of hepatic DNA adducts 28-, 3.8-, and 15-fold, respectively. Exceptionally high levels of adducts, up to ∼1 per 10 4 nucleotides, were observed. The metabolic modulators also raised the excretion of MPMA, a possible biomarker for the formation of ultimate carcinogen. Thus, interaction with other chemicals may dramatically affect the toxicological effects of some polycyclic aromatic carcinogens. In particular, the effect of ethanol may be relevant for some human exposures to polycyclic aromatic compounds.