Role of etheno DNA adducts in carcinogenesis induced by vinyl chloride in rats

Vinyl chloride, a hepatocarcinogen in humans and rodents, can form promutagenic etheno bases in DNA after metabolic activation. The formation of 1,N6-ethenoadenine (epsilon A) and 3,N4-ethenocytosine (epsilon C) was measured in adult Sprague-Dawley rats by immunoaffinity purification and 32P-postlabelling. A highly variable background was found in all tissues from untreated animals: the mean molar ratios of epsilon A:A and epsilon C:C in DNA ranged from 0.043 x 10(-8) to 31.2 x 10(-8) and from 0.062 x 10(-8) to 20.4 x 10(-8), respectively. After exposure to 500 ppm vinyl chloride by inhalation (4 h/day, 5 days/week for 8 weeks), increased levels of epsilon A were found in the liver, lung, circulating lymphocytes and testis, the mean (+/- SD) of induced levels (treated-control values) being (4.1 +/- 1.5) x 10(-8) for these tissues. No increase in the epsilon A:A ratio was observed in kidney, brain or spleen. The levels of epsilon C increased in all the tissues examined except the brain. The mean value of the induced epsilon C:C ratios was (7.8 +/- 1.2) x 10(-8) for the liver, kidney, lymphocytes and spleen, and these ratios were higher in the lung (28 x 10(-8)) and testis (19 x 10(-8)). The results suggest a variable repair capacity for epsilon A or epsilon C in different tissues. The results are discussed in relation to published studies on the accumulation and persistence of etheno bases in the liver during and after exposure to vinyl chloride and on mutation spectra in the ras and p53 genes in liver tumours induced by vinyl chloride. In addition, we show that the linear relationship established for monofunctional alkylating agents between their carcinogenic potency in rodents and their covalent binding index for promutagenic bases in hepatic DNA holds for vinyl chloride. It is concluded that etheno bases are critical lesions in hepatocarcinogenesis induced by vinyl chloride. For a better understanding of the mechanism of action of this compound, further work is needed on the role of DNA repair pathways and of endogenous lipid peroxidation products in the formation and persistence of etheno bases in vivo.     

Formation and repair of cisplatin-induced adducts to DNA in cultured normal and repair-deficient human fibroblasts

The formation and repair of cisplatin [cis-PtCl2(NH3)2] adducts in the DNA of cultured normal and repair-deficient human fibroblasts are presented in relation to cell survival after cisplatin treatment. Directly after treatment with cisplatin, in normal (MB), Fanconi's anemia (FA), and xeroderma pigmentosum (XP) fibroblasts four platinated products are found. The major adduct is cisplatin bound to two neighboring guanines, Pt-GG (62-75%). A less abundant product is cisplatin bound to an AG sequence (Pt-AG). Binding to two guanines separated by one or more bases or to two guanines in opposite DNA strands (together measured as G-Pt-G) and cisplatin bound monofunctionally to guanine (Pt-G) are also found in small amounts. The distribution of the four products is similar to that found previously, in in vitro systems as well as in living cells. Directly after cisplatin treatment, the removal of cisplatin-DNA adducts is fast in normal and FA fibroblasts, whereas in XP fibroblasts adduct removal proceeds slowly throughout the repair period studied. Both FA and XP fibroblasts are extremely sensitive to cisplatin with regard to cell killing. For FA fibroblasts this sensitivity may be attributed to the fact that in these cells initially more DNA-adducts are formed than in normal fibroblasts, and/or to their known deficiency in the repair of DNA interstrand cross-links. For XP fibroblasts this sensitivity may be caused by their deficiency in the fast repair process, known as excision repair.     

Genetic alterations and DNA repair in human carcinogenesis

A causal association between genetic alterations and cancer is supported by extensive experimental and epidemiological data. Mutational inactivation of tumor suppressor genes and activation of oncogenes are associated with the development of a wide range of cancers. The link between mutagenesis and carcinogenesis is particularly evident for cancers induced by chemical exposures, which, in some cases, lead to characteristic patterns of mutations. These "genotoxic," direct-acting carcinogens form covalent adducts with DNA, which cause mutations during DNA replication. The link between mutagenesis and carcinogenesis is also supported by the observation that DNA repair defects are associated with an increased cancer risk. Normally, DNA repair mechanisms serve to suppress mutagenesis by correcting DNA damage before it can lead to heritable mutations. It has been postulated that mutagenesis plays a role in both the initiation phase and the progression phase of carcinogenesis, and that an essential step in the carcinogenic process is the development of a mutator state in which the normal cellular processes that suppress mutagenesis become compromised. Given the link between mutations and cancer, attempts have been made to use the mutational profile of cancer cells as an indicator of the causative agent. While this may be a valid approach in some cases, it is complicated by the role of endogenous processes in promoting mutagenesis. In addition, many important carcinogenic agents may enhance mutagenesis indirectly through suppression of DNA repair functions or stimulation of inappropriate cell proliferation. Epigenetic phenomena may also suppress gene expression without causing overt changes in DNA sequence.     

Metabolic activation of vinyl chloride, formation of nucleic acid adducts and relevance to carcinogenesis

Comparative experiments with vinyl chloride and 2,2'-dichlorodiethyl ether confirm that chloroacetaldehyde, which is formed during metabolism of both compounds, binds covalently to proteins, but not to nucleic acids. The putative nucleic-acid-binding agent, chloroethylene oxide, is formed from vinyl chloride, not from 2,2'-dichlorodiethyl ether. The degree of potential carcinogenicity of epoxides formed from substituted ethylenes is considered to be determined by factors of stability/reactivity and by the rate of breakdown of epoxide and the rate of its formation. Hence, pharmacokinetic data are of considerable importance in assessing the comparative carcinogenic potencies of ethylene derivatives.     

Mutagenic and promutagenic properties of DNA adducts formed by vinyl chloride metabolites

Published results and work from this laboratory permit the characterization of the possible promutagenic lesions induced by chloroethylene oxide (CEO) and chloroacetaldehyde (CAA), both known as bifunctional alkylating metabolites of vinyl chloride (VC). The mutagenic effectiveness of CEO and CAA in Escherichia coli, when compared to their nucleophilic selectivity, suggests that the critical target site in DNA bases is not an oxygen atom, and/or that the reaction mechanism of CEO and CAA is different from a simple alkylation. CEO-mutagenicity in E. coli is recA-independent, and CEO preferentially induces GC----AT transitions; accordingly, the mutagenicity of CEO in bacteria may result mainly from a miscoding guanosine or cytosine adduct. Two observations argue against the role of 1,N6-ethenoadenine (epsilon A) and 3,N4-ethenocytosine (epsilon C) in VC-induced mutagenesis/carcinogenesis: i) the lack of detection in double-stranded DNA in vivo and in vitro; ii) the inconsistency between mutational specificity of CEO and miscoding properties of epsilon A and epsilon C. The lack of miscoding properties of 7-(2-oxoethyl)guanine (oxet-G), the major in-vivo VC-DNA adduct, suggests a minor miscoding base adduct. Several lines of evidence point to N4-(2-chlorovinyl)cytosine as one possible putative promutagenic lesion produced by VC, but this compound has yet to be identified in DNA.     

DNA adducts of heterocyclic amine food mutagens: implications for mutagenesis and carcinogenesis

The heterocyclic amines (HCAs) are a family of mutagenic/carcinogenic compounds produced during the pyrolysis of creatine, amino acids and proteins. The major subclass of HCAs found in the human diet comprise the aminoimidazoazaarenes (AIAs) 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). All, except DiMeIQx, have been shown to be carcinogenic in animals. These compounds are present in cooked muscle meats at the p.p.b. level. Since the discovery of the HCAs in the late 1970s, many studies have examined the DNA adducts of these compounds. This review compiles the literature on AIA-DNA adducts including their identification and characterization, pathways of formation, mutagenesis in vitro and in vivo, and their association with carcinogenesis in animal models. It is now known that metabolic activation leading to the formation of DNA adducts is critical for mutagenicity and carcinogenicity of these compounds. All of the AIAs studied adduct to the guanine base, the major adduct being formed at the C8 position. Two AIAs, IQ and MeIQx, also form minor adducts at the N2 position of guanine. A growing body of literature has reported on the mutation spectra induced by AIA-guanine adducts. Studies of animal tumors induced by AIAs have begun to relate AIA-DNA adduct-induced mutagenic events with the mutations found in critical genes associated with oncogenesis. Several studies have demonstrated the feasibility of chemoprevention of AIA tumorigenesis. Only a few studies have reported on the detection of AIA-DNA adducts in human tissues; difficulties persist in the routine detection of AIA-DNA adducts in humans for the purpose of biomonitoring of exposure to AIAs. The AIAs are nevertheless regarded as possible human carcinogens, and future research on AIA-DNA adducts is likely to help address the role of AIAs in human cancer.     

Formation of etheno adducts and their effects on DNA polymerases

Etheno (epsilon) and related DNA adducts are formed from the reaction of certain bifunctional electrophiles with DNA. Our interest has been focused on oxiranes substituted with leaving groups, e.g. 2-chlorooxirane, the epoxide derived from the carcinogen vinyl chloride. The chemical mechanisms of the formation of the major etheno products derived from adenine, cytosine and guanine have been elucidated by nuclear magnetic resonance analysis and 13C-labelled precursors. The amounts of all major etheno adducts have been quantified in DNA treated with 2-chlorooxirane by coupled high-performance liquid chromatography of nucleoside and base products. 1,N2-epsilon-Gua, its formally hydrated but stable hemiaminal HO-ethanoGua (5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine) and 1,N2-ethanoGua have all been inserted at a single site in oligonucleotides. All three of these bases block polymerases, cause misincorporations and produce some mutations in bacteria. The patterns of blockage and substitution vary among polymerases. In nucleotide excision repair-deficient Escherichia coli, 1,N2-epsilon-Gua yielded a calculated 16% mutation frequency (base-pair substitutions) when the results were corrected for strand usage. 1,N2-epsilon-Gua was also examined in Chinese hamster ovary cells with a stable integration system; the mutants are more complex than observed in bacteria and include rearrangements, deletions and base-pair substitutions other than at the adduct site.     

DNA polymerases in replication and repair of DNA during carcinogenesis induced by feeding N-acetylaminofluorene

To study the roles of DNA polymerases and ß duringreplication and repair of damaged DNA, use was made of the factthat during chronic treatment with carcinogens, replicationand repair do not necessarily follow the same time sequence.Early cell damage and restorative hyperplasia cause a transientwave of DNA synthesis, while repair replication might be expectedto continue throughout the period of treatment with the carcinogen.N-acetylaminoflluorene (AAF) was fed in the diet for periodsof up to 35 weeks, and at intervals during the feeding periodmeasurements were made of DNA synthesis in vivo, and off DNApolymerases a and ß as assayed in vitro after fractionation.The activity of polyararise increased and decreased with thetransient early wave of DNA synthesis. Polymerase ßshowed an initial rapid increase in activity which peaked beforethe increase in DNA synthesis, and then decreased. The decreasein activity may be due to the fact that, although AAF continuesto be fed in the diet, the foci and nodules which develop nolonger metabolise the carcinogen to a form which damages DNA.Thus replication occurs in the nodules while DNA damage andrepair occur in the surrounding non-neoplastic liver. With therapid growth of nodules there is overall an increase in neoplastictissue, a relative decrease in nonneoplastic tissue, and thusa relative decrease in DNA damage, repair, and induction ofpolymerase ß. Histo-logical examination showed thatby 35 weeks the conversion to neopflasia was virtually complete.These results support the concept that polymerase functionsin de novo replication of DNA, and is induced during cell replication,while polymerase it functions in repair replication, and increasesin activity during chronic damage to DNA. Whether it is inducedby treatment with carcinogens depends on the duration of treatment,and on other processes (e.g. metabolism of the carcinogen) whichtake place during the development of malignancy.     

Ultraviolet irradiation of monkey cells enhances the repair of DNA adducts in alpha DNA

Excision repair of bulky adducts in alpha DNA of African greenmonkey cells has previously been shown to be deficient relativeto that in the overall genome. We have found that u.v. irradiationof these cells results in the enhanced removal of both aflatoxinB₁ (AFB₁) and acetylaminofluorene (AAF) adducts from the alphaDNA sequences without affecting repair in the bulk of the DNA.The degree of enhanced removal of AFB₁ is dependent upon theu.v. dose and the time interval between irradiation and AFB₁treatment. The u.v. enhancement is not inhibited by cydoheximide.Exposure of the cells to dimethylsulfate or gamma-rays doesnot affect AFB₁ adduct repair. The formation and removal ofN-acetoxy-2-acetylaminofluorene (NA-AAF) adducts from alphaand bulk DNA was studied in detail. A higher initial level ofthe acetytated C8 adduct of guanine was found in alpha DNA thanin bulk DNA. Although both the acetylated and deacetylated C8adducts were removed from the two DNA species, the level ofrepair was significantly greater in the bulk DNA. Irradiationof cells with U.V. prior to treatment with NA-AAF enhanced theremoval of both adducts from alpha DNA with little or no effecton repair in bulk DNA. We condude that the presence of u.v.photoproducts or some intermediate in their processing altersthe chromatin structure of alpha DNA thereby rendering bulkyadducts accessible to repair enzymes. In addition, the differentialformation and repair of AAF adducts in alpha DNA compared withthat in the bulk of the genome supports the hypothesis of analtered chromatin structure for alpha domains.     

Effect of DNA repair gene polymorphisms on BPDE-DNA adducts in human lymphocytes

To determine whether variations in DNA repair genes are related to host DNA damage, we investigated the association between polymorphism in the XPD gene (codon 199, 312, 751) and the XRCC1 gene (codon 194, 399) and the presence of benzo(a)pyrene diolepoxide adducts to lymphocyte DNA (BPDE-DNA) in a group of male patients with incident lung cancer, all current smokers. BPDE-DNA adducts were analyzed by high-resolution gas chromatography-negative ion chemical ionization-mass spectrometry. XPD and XRCC1 genotypes were identified by PCR-RFLP. XRCC1 and XPD genotypes did not affect the levels and proportion of detectable BPDE-DNA adducts. The patients were also genotyped for the GSTM1 polymorphism, given its role in the detoxification of BPDE. Individuals with the GSTM1 deletion had significantly higher levels of BPDE-DNA adducts when they were XPD-Asp312Asp+Lys751Lys than carriers of at least one variant allele. No such association was found with the XRCC1 genotypes. Because of the small study population (n = 60), further statistical analysis of possible gene-gene and gene-environment would not be informative. This is the first study analysing the specific BPDE-DNA adduct in vivo with regard to polymorphic repair genes (XPD, XRCC1) and xenobiotic metabolizing gene (GSTM1). Our results raise the possibility that the XPD-Asp312Asp+Lys751Lys genotype may increase BPDE-DNA damage; this effect might be evident in individuals who are especially likely to have accumulated damage, probably because of lower detoxification capacity and high environmental exposure.     

Formation of DNA adducts by the carcinogen N-hydroxy-2-naphthylamine

The probable ultimate urinary bladder carcinogen, N-hydroxy-2-naphthylamine (N-OH-2-NA), reacted with nucleic acids and proteins under mildly acidic conditions (pH 5) to form covalently bound derivatives. The extent of reaction was in the order: Polyguanylic acid greater than DNA approximately protein greater than rRNA greater than tRNA greater than polyadenylic acid approximately polyuridylic acid greater than polycytidylic acid. At pH 7, appreciable reaction occurred only with protein. Enzymatic hydrolyses of the DNA, which contained 1.5 naphthyl residues/1,000 nucleotides, yielded 3 nucleoside-arylamine adducts. From chemical, UV, nuclear magnetic resonance, and mass spectrometric analyses, the adducts were identified as 1-(deoxyguanosin-N2-yl)-2-NA, 1-(deoxyadenosin-N6-yl)-2-NA, and a purine ring-opened derivative of N-(deoxyguanosin-8-yl)-2-NA, tentatively identified as 1-[5-(2-6-diamino-4-oxopyrimidinyl-N6-deoxyriboside)]-2-(2-naphthyl)urea. Preliminary experiments with a dog given [3H]2-NA suggested the presence of these adducts in vivo. The properties of adducts derived from N-OH-1-NA and N-OH-2-NA and their possible roles in the initiation of carcinogenesis are discussed.     

Formation of cigarette smoke-induced DNA adducts in the rat lung and nasal mucosa

The formation of DNA adducts in the nasal, lung, and liver tissues of rats exposed daily to fresh smoke from a University of Kentucky reference cigarette (2R1) for up to 40 weeks was examined. The amount of smoke total particulate matter (TPM) inhaled and the blood carboxyhemoglobin (COHb) values averaged 5-5.5 mg smoke TPM/day/rat and 5.5%, respectively. The pulmonary AHH activity measured at the termination of each experiment showed an average increase of about two- to threefold in the smoke-exposed groups. These observations suggested that animals effectively inhaled both gaseous and particulate phase constituents of cigarette smoke. DNAs from nasal, lung, and liver tissue were extracted and analyzed by an improved 32P-postlabeling procedure. The results showed that the mainstream cigarette smoke induced a spectrum of at least four new DNA adducts in the nasal mucosa of the exposed rats and the magnitude of these adducts increased with the duration of exposure. In the lung tissue, the smoke exposure induced an accumulation of one DNA adduct, which upon cessation of exposure for 19 weeks was reduced by about 75%. Smoke-related adducts were not detected in the liver, a nontarget tissue. Selective chromatography and butanol extractability suggested that the nasal and lung DNA adducts are aromatic and/or hydrophobic in nature and that the smoke-related lung DNA-adduct may contain polar group(s). These data demonstrate the DNA-damaging potential of long term fresh cigarette smoke exposure and suggest the ability of the tissue to partially recover from such damage following cessation of the exposure.     

Formation of DNA adducts in mouse skin treated with metabolites of chrysene

Analysis by 32P-postlabelling of DNA isolated from mouse skin that had been treated in vivo with the polycyclic hydrocarbon chrysene revealed the presence of 7 adducts. All 7 adducts were also present in DNA from mice treated with trans-1,2-dihydro-1,2-dihydroxychrysene (chrysene-1,2-diol), and one of them, adduct 2, was formed from the triol derivative 9-hydroxy-trans-1,2- dihydro-1,2-dihydroxychrysene (9-hydroxychrysene-1,2-diol) and from 3-hydroxychrysene. Adducts were not detected in DNA from mice treated with trans-3,4-dihydro-3,4-dihydroxychrysene (chrysine-3,4-diol) or with 1-, 2-, 4-, 5- or 6-hydroxychrysene. In vitro modification of DNA by the anti-isomer of the bay-region diol-epoxide yielded adducts 3-7, while the corresponding triol-epoxide yielded adducts 2. It is concluded that chrysene activation in mouse skin proceeds principally via the bay-region diol-epoxide and to a lesser extent via the related bay-region triol-epoxide.     

Etheno adducts formed in DNA of vinyl chloride-exposed rats are highly persistent in liver.

Preweanling rats were exposed to 600 p.p.m. (4h/day) of the human carcinogen vinyl chloride for 5 days to determine the molecular dosimetry of DNA adducts in liver, lung and kidney. 7-(2'-Oxoethyl)guanine (7OEG) was the major DNA adduct detected, representing approximately 98% of all adducts. N2,3-Ethenoguanine (epsilon G) and 3,N4-etheno-2'-deoxycytidine (epsilon dC) were present at approximately 1% of the 7OEG concentration, while 1,N6-etheno-2'-deoxyadenosine was present in even lower concentrations. Liver had 3- to 8-fold higher amounts of the DNA adducts than lung and kidney. The persistence of all four adducts was determined at 3, 7 and 14 days post-exposure. Whereas 7OEG had a t 1/2 of -62 h, all three etheno adducts were highly persistent. After accounting for dilution due to growth-related cell proliferation, epsilon G had a t 1/2 of approximately 30 days, while epsilon dC and epsilon dA were not repaired. These data suggest that these cyclic adducts are poorly recognized by liver DNA repair enzymes and have the potential for accumulation upon chronic exposure. This, coupled with the known miscoding properties of the ethenobases, provides a strong rational for examining their role in vinyl chloride-induced cancer and their utility as biomarkers of exposure.