Detection of 1,N6-etheno-2'-deoxyadenosine and 3,N4-etheno-2'-deoxycytidine occurring endogenously in DNA

1,N6-Etheno-2'-deoxyadenosine (epsilon dA) and 3,N4-etheno-2'-deoxycytidine (epsilon dC) are DNA adducts formed by a number of genotoxic chemicals, including vinyl chloride. They are also formed endogenously in tissue DNA, probably from a reactive metabolite of lipid peroxidation. Both the qualitative and quantitative detection of endogenous adducts is important in order to place adduct formation by chemicals such as vinyl chloride in the context of this natural background level. Methods with sufficient sensitivity are therefore being developed to measure the natural background of epsilon dA and epsilon dC adducts. We have developed a high-performance liquid chromatography (HPLC)-32P-postlabelling method to measure epsilon dA and epsilon dC at alkylation frequencies of 1 adduct in 10(7)-10(8) nucleotides in 10-microgram samples of DNA. In HPLC-32P-postlabelling analysis of liver DNA from control Wistar rats, epsilon dA and epsilon dC were determined at levels of 1 adduct in 8.1 x 10(7) and 1 adduct in 1.8 x 10(7) nucleotides, respectively. The levels of epsilon dA and epsilon dC measured in liver DNA of animals exposed orally to five daily doses of 50 mg/kg body weight vinyl chloride were found by this method to be 1 adduct in 2.9 x 10(7) and 1 adduct in 1.4 x 10(7) nucleotides, respectively. In contrast, in a direct labelling study, radiolabelled epsilon dA and epsilon dC were not detected in liver DNA of rats exposed for 6 h by nose-only inhalation to [1,2-14C]vinyl chloride at up to 45 ppm v/v. Immunochemical procedures are also being developed for recognizing etheno adducts. Thus, a monoclonal antibody raised to protein conjugates of epsilon dC showed high selectivity in the recognition of this DNA adduct. When the antibody was immobilized on a solid support and used in an immunoenrichment procedure to purify epsilon dC from a large excess of normal nucleotides, one epsilon dC adduct from about 10(8) normal nucleotides could be resolved. Coupling the immunoaffinity enrichment procedure with capillary zone electrophoresis permitted the detection of approximately one epsilon dC adduct in 3 x 10(6) nucleotides.     

A comparison of two ultrasensitive methods for measuring 1,N6-etheno-2'- deoxyadenosine and 3,N4-etheno-2'-deoxycytidine in cellular DNA

1,N⁶-Ethenodeoxyadenosine (edA) and 3,N⁴-ethenodeoxycytidine(edC) are two mutagenic adducts associated with exposure toethyl carbamate (urethane) and vinyl chloride. We have recentlydeveloped two ultrasensitive methods for determining the moleculardose of these adducts in cellular DNA. In both methods, purifiedDNA was first enzymatically digested to 2c-deoxyribonucleotide3c-monophosphates. Etheno-modified nucleotides were then separatedfrom normal nucleotides in one of two ways: either by reversephase, ion-pair HPLC coupled with 260 nm UV detection, or byimmunoaffinity chromatography using reusable microcolumns containingspecific monoclonal antibodies coupled to Protein A–Sepharose.Fractions enriched for the adducted nucleotides were labeledusing T4 polynucleotide kinase and [³²P]ATP, and individualnucleotides were subsequently resolved by two-dimensional TLC,visualized by autoradiography, and quantified by liquid scintillationcounting. When used to analyze the same sample of etheno-modifiedcalf thymus DNA, both assays produced similar results. However,when both methods were used to analyze rat liver DNA ‘spiked’with known amounts of etheno nucleotide standards, the immunoaffinity/³²PTLC procedure proved to be more sensitive and more reproduciblethan the HPLC/³²P TLC method: while the detection limit of theimmunoaffinity/³²P TLC technique was < 4 etheno adducts/10⁹parent deoxynucleotides, the HPLC/³²P TLC method often failedto detect adducts at concentrations <2/108. In other experiments,the immunoaffinity/³²P TLC method was used to demonstrate formationof edA and edC in cells treated with vinyl chloride monomer.Because of its exquisite sensitivity, the immunoaffinity/³²PTLC method promises to be extremely useful for measuring bothbackground and induced levels of etheno adducts, making it possibleto examine the role of these adducts in inducing mutations and/orcarcinogenesis.     

Formation and repair of DNA adducts in vinyl chloride- and vinyl fluoride-induced carcinogenesis

Vinyl chloride is a known human and animal carcinogen that induces angiosarcomas of the liver. We review here studies on the formation and repair of DNA adducts associated with vinyl chloride and vinyl fluoride in exposed and control rodents and unexposed humans. These vinyl halides induce etheno (epsilon) adducts that are identical to those formed after lipid peroxidation. Of these adducts, N2,3-ethenoguanine (epsilon G) is present in greatest amounts in tissues of exposed animals. After exposure to vinyl chloride for four weeks, epsilon G levels attain steady-state concentrations, such that the amount of newly formed adducts equals the number of adducts that are lost each day. We report the first dosimetry of epsilon G in rats exposed to 0, 10, 100 or 1100 ppm vinyl chloride for five days or four weeks. The number of adducts increased in a supralinear manner. Exposure to 10 ppm vinyl chloride for five days caused a two- to threefold increase in epsilon G over that of the controls, while four weeks' exposure resulted in a fivefold increase. This was confirmed with [13C2]vinyl chloride and by measuring exogenous and endogenous adducts in the same animals. Exposure to 100 ppm vinyl chloride for four weeks caused a 25-fold increase in epsilon G levels over that found in control rats, while exposure to 1100 ppm resulted in a 42-fold increase. The amount of endogenous epsilon G was similar in liver DNA from rats and humans. A comparable response to exposure was seen in rats and mice exposed to 0, 25, 250 or 2500 ppm vinyl fluoride for 12 months. There was a very high correlation between epsilon G levels in rat and mouse liver at 12 months and the incidence of haemangiosarcoma at two years. We were able to demonstrate that the target cell population for angiosarcoma, the nonparenchymal cells, contained more epsilon G than hepatocytes, even though nonparenchymal cells are exposed by diffusion of vinyl halide metabolites formed in hepatocytes. The expression of N-methylpurine-DNA glycosylase mRNA was induced in rat liver after exposure to either 25 or 2500 ppm vinyl fluoride. When this induction was investigated in hepatocytes and nonparenchymal cells, it was found that the latter had only 20% of the N-methylpurine-DNA glycosylase mRNA of hepatocytes, and that only the hepatocytes had induction of this expression after exposure to vinyl fluoride. Thus, the target cells for vinyl halide carcinogenesis have much lower expression of this DNA repair enzyme, which has been associated with etheno adduct repair.     

Formation of N2,3-ethanoguanine in DNA after in vitro treatment with the therapeutic agent, N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea

HPLC analyses of the bases released by acid from N-(2-chloroethyl)-N-nitrosourea-treated DNA and N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea-treated DNA show the presence of a new guanine adduct, N2,3-ethanoguanine. This derivative can be synthesized at the monomer level by treating 2-hydroxyethylguanine with thionyl chloride. The product of this reaction, purified by HPLC, has been shown to have a mol. wt corresponding to ethanoguanine by mass spectrometry; NMR spectrometry also supports this structural assignment. The UV and fluorescence spectra are very similar to those of N2,3-ethenoguanine, providing evidence that the ethano bridge is attached between N2 and 3 positions. Proof that the derivative is N2,3-ethanoguanine comes from the fact that it can be converted to N2,3-ethenoguanine by dehydrogenation on a palladium catalyst. The discovery of this new derivative raises to four the number of tricylic derivatives that have been isolated from DNA treated with 2-haloethylnitrosoureas. The new adduct, N2,3-ethanoguanine, is closely related to an etheno adduct formed by chloroacetaldehyde, a metabolite of the human carcinogen vinyl chloride, and may have relevance to either the therapeutic or carcinogenic actions of the 2-haloethylnitrosoureas.     

Immunohistochemical detection of 1,N(6)-ethenodeoxyadenosine, a promutagenic DNA adduct, in liver of rats exposed to vinyl chloride or an iron overload

Etheno adducts in DNA bases are formed from exogenous agents such as vinyl chloride and urethane, but also via endogenous lipid peroxidation products like trans-4-hydroxy-2-nonenal. An immunohistochemical method was developed to localize the promutagenic 1,N(6)-ethenodeoxyadenosine DNA adduct in liver of rats exposed to vinyl chloride or an iron overload with or without carbon tetrachloride. Six monoclonal antibodies, previously produced through collaborative efforts, were screened for their optimal adduct recognition and low background formation. The antibody generated by clone EM-A-4 was found to be most suitable. Semi-quantitative image analysis of relative pixel intensity showed approximately 1.5 times higher adduct levels (P < 0.05) in the livers of rats treated with vinyl chloride or an iron overload when compared with untreated controls. Significantly elevated adduct levels persisted in vinyl chloride-treated rat liver 14 days after cessation of exposure, suggesting that this adduct is not rapidly eliminated from rat liver DNA. Using the new immunohistochemical method it is possible to visualize this promutagenic etheno-DNA adduct that may play a role in oxidative stress and lipid peroxidation-induced DNA damage in carcinogenesis.     

Development of techniques to monitor for exposure to vinyl chloride: monoclonal antibodies to ethenoadenosine and ethenocytidine

Monoclonal antibodies which specifically recognize etheno-adenosineand ethenocytidine, two of the adducts resulting from exposureto vinyl chloride, have been developed. The sensitivity andspecificity of these antibodies have been determined by enzyme-linkedimmunosorbent assay (ELISA). The antibody to ethenoadenosine(1G4) reacts with both the ribose (50% inhibition at 600 fmol)and deoxyribose (50% inhibition at 980 fmol) form of the adduct.The antibody to ethenocytidine (6F5) also reacts with both theribose (50% inhibition at 800 fmol) and deoxyribose (50% inhibitionat 1000 fmol) form of the adduct. Neither antibody cross-reactswith non-modified DNA or the normal nucleotides. A more sensitivefluorescence ELISA was developed for antibody 1G4 with 50% inhibitionat 212 fmol of ethenoadenosine and for antibody 6F5 with 50%inhibition at 192 fmol ethenocytidine. These antibodies havebeen used to determine the level of etheno derivatives in DNAmodified in vitro with chloroacet-aldehyde and in the DNA andRNA of cells treated in culture.     

Detection of 1,N6-ethenoadenine in rat urine after chloroethylene oxide exposure

The four etheno adducts of vinyl chloride formed in DNA, 1,N6- ethenoadenine (epsilonA), 3,N4-ethenocytosine, 1,N2-ethenoguanine and N2,3-ethenoguanine were previously reported to be released from DNA by a family of enzymes in the base-excision repair pathway (Dosanjh et al., Proc. Natl Acad. Sci. USA, 91, 1024-1028, 1994; Hang et al., Carcinogenesis, 17, 155-157, 1996; Hang et al., Proc. Natl Acad. Sci. USA, 94, 12869-12874, 1997). Adducts excised from DNA by glycosylases are usually excreted in urine and have been reported to be potential biomarkers of DNA damage in exposed individuals. In this study, we report the detection of epsilonA in the urine of rats exposed to chloroethylene oxide (CEO) using immunoaffinity columns made with specific monoclonal antibodies for enrichment, followed by quantitation by HPLC with fluorescence detection. Chemical analysis of urine samples revealed the presence of a compound chromatographically identical to authentic epsilonA standard. This compound was confirmed by mass spectral analysis. EpsilonA was present in urine of control and CEO- treated rats, with the latter having up to 50-fold greater amounts. The cumulative excretion of epsilonA reached a plateau between 24 and 48 h post-exposure. While it is clear that CEO treatment results in increased excretion of epsilonA, the exact source of the adduct is unknown. When rats were administered epsilonA i.v., approximately 10% of the administered dose was excreted in urine. This research demonstrates that urinary excretion of epsilonA may be a potential biomarker for in vivo alkylation of DNA and nucleotide pools.      

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.     

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.     

Etheno adducts in spleen DNA of SJL mice stimulated to overproduce nitric oxide

In order to investigate specific DNA damage caused by nitric oxide (NO) induced lipid peroxidation, levels of promutagenic etheno adducts 1,N6- ethenodeoxyadenosine (epsilondA) and 3,N4-ethenodeoxycytidine (epsilondC) were measured in spleen DNA of SJL mice induced to produce high levels of NO by injection of RcsX (pre-B-cell lymphoma) cells. epsilondA and epsilondC levels were quantified by an ultrasensitive immunoaffinity-32P-post-labeling method. Spleen DNA of control mice (n = 5) had background levels of 9.2+/-5.4 epsilondA adducts per 10(9) dA and 13.1+/-5.7 epsilondC adducts per 10(9) dC. In RcsX cell-injected mice (n = 7), levels of these adducts were elevated approximately 6- fold, i.e. 53.9+/-39.4 epsilondA per 10(9) dA and 83.5+/-57.8 epsilondC per 10(9) dC (P < 0.05). Mice injected with RcsX cells and also treated with NG-methyl-L-arginine (NMA), an inhibitor of inducible nitric oxide synthase (n = 6), had significantly reduced levels (P < 0.05) of both epsilondA and epsilondC (13.5+/-5.7 epsilondA per 10(9) dA and 28.2+/- 15.7 epsilondC per 10(9) dC). These findings constitute the first available evidence of formation of etheno adducts associated with NO overproduction in vivo. The adducts were presumably formed from lipid peroxidation products such as trans-4-hydroxy-2-nonenal (HNE), generated via oxidation of lipids by peroxynitrite. The results suggest that etheno-DNA adducts, among other types of damage, may contribute to the etiology of cancers associated with chronic infection/inflammation in which NO is overproduced.      

Development of monoclonal antibodies recognizing 7-(2-hydroxyethyl)guanine and imidazole ring-opened 7-(2-hydroxyethyl)guanine

7-(2-Hydroxyethyl)guanine (7HEG) is of biological interest becauseit is formed in vivo by reaction of DNA with ethylene oxide(EO). Furthermore, the major DNA adduct of vinyl chloride, 7-(2-oxyethyl)guanine,can be converted to this adduct by reduction. Two monoclonalantibodies (9E2, 4A5) recognizing 7HEG have been developed fromBALB/c mice immunized with the adduct coupled to keyhole limpethemocyanin. In addition, another antibody (8E10) was developedagainst the imidazole ring-opened form of the adduct (ro-7HEG).ELISAs were used to determine the sensitivity and specificityof these antibodies. With antibody 9E2, 50% inhibition of antibodybinding in the competitive ELISA was at 54 pmol of the modifiedbase 7HEG/well and 67 pmol 7HEGR/well, while with antibody 4A5,the values were 3.6 pmol 7HEG/well and 6.7 pmol 7HEGR/well.Antibody 8E10 gave 50% inhibition at 48 pmol ro-7HEGR/well.Neither antibody 9E2 nor 8E10 cross-reacted with unmodifiedDNA or with the normal nudeosides at the highest concentrationtested. However, antibody 4A5 had a low affinity for deoxyguanosine(50% inhibition at 31 000 pmol). Sensitivity of adduct measurementcan be increased 3- to 10-fold using an ELISA with fluorescenceendpoint detection. These antibodies have been used to determinethe level of adducts in DNA modified in vitro with [³H]- or[¹⁴C]EO. Because of the cross-reactivity of the most sensitiveantibody, 4A5, with deoxyguanosine, a combined HPLC/immunoassaymethod was developed to quantitate 7HEG in DNA. The limit ofsensitivity of this method is dependent upon the amount of DNAavailable for analysis. Using 30 fmol as the lowest detectableamount (20% inhibition) in the fluorescent ELISA with antibody4A5 and 100 µg of DNA assayed per well, adduct levelsof 1/10⁷ nucleotide can be determined. This method was appliedto DNA adduct detection in EO-treated myeloma cells and wholeblood. Antibody 8E10 was also used in immunohistochemical studiesto visualize ring-opened adducts in cells treated with EO followedby high pH. These antibodies will be used for the detectionand quantitation of adducts in human samples.     

High excretion of etheno adducts in liver fluke-infected patients: protection by praziquantel against DNA damage

Chronic infection by Opisthorchis viverrini (OV) is a strong risk factor for developing cholangiocarcinoma (CCA). To clarify the involvement of oxidative stress and lipid peroxidation (LPO)-derived DNA damage, the excretion of LPO-derived etheno DNA adducts was measured in urine samples collected from healthy volunteers and OV-infected Thai subjects. 1,N(6)-etheno-2'-deoxyadenosine (epsilondA) and 3,N(4)-etheno-2'-deoxycytidine (epsilondC) levels were quantified by immunoprecipitation/high-performance liquid chromatography/fluorescence detection and (32)P-postlabeling TLC. Excreted etheno adduct levels were related to indicators of inflammatory conditions [malondialdehyde (MDA) and nitrate/nitrite levels in urine and plasma alkaline phosphatase (ALP) activity]. Mean epsilondA and epsilondC levels were 3 to 4 times higher in urine of OV-infected patients; MDA, nitrate/nitrite, and ALP were also increased up to 2-fold. MDA and ALP were positively related to epsilondA excretion. Two months after a single dose of the antiparasitic drug Praziquantel, epsilondA and epsilondC concentrations in urine of OV-infected subjects were decreased; MDA, nitrate/nitrite, and ALP were concomitantly lowered. We conclude that chronic OV infection through oxidative/nitrative stress leads to increased urinary excretion of the etheno-bridged deoxyribonucleosides, reflecting high LPO-derived DNA damage in vivo. These promutagenic DNA etheno adducts in bile duct epithelial cells may increase the risk of OV-infected patients to later develop CCA. Urinary epsilondA and epsilondC levels should be explored (a) as noninvasive risk markers for developing opisthorchiasis-related CCA and (b) as promising biomarkers to assess the efficacy of preventive and therapeutic interventions.     

Mammalian enzymatic repair of etheno and para-benzoquinone exocyclic adducts derived from the carcinogens vinyl chloride and benzene

Two human carcinogens that have been extensively studied are vinyl chloride and benzene. The active metabolites used in this study are chloroacetaldehyde (CAA) and para-benzoquinone (pBQ). Each forms exocyclic adducts between the N1 and N6 of A, the N3 and N4 of C and the N1 and N2 of G. Only CAA has been found to form the N2,3 adduct of G. CAA and pBQ adducts differ structurally in size and in the number of added rings, pBQ adding two rings to the base, while etheno bases have a single five-membered ring. The mechanism of repair of these two types of adducts by human enzymes has been studied in our laboratory with defined oligodeoxynucleotides and a site-specific adduct. The etheno derivatives are repaired by DNA glycosylase activity; two mammalian glycosylases are responsible: alkylpurine-DNA-N-glycosylase (APNG) and mismatch-specific thymine-DNA glycosylase. The former repairs 1,N6-ethenoA (epsilon A) as rapidly as the original substrate, 3-methyladenine, while the latter repairs 3,N4-ethenoC (epsilon C) more efficiently than the G/T mismatch. Our finding that there are separate enzymes for epsilon A and epsilon C has been confirmed by the use of tissue extracts from an APNG knockout mouse. As pBQ is much less efficient than CAA in modifying bases, the biochemical studies required total synthesis of the nucleosides. Furthermore, the pBQ adduct-containing oligomers are cleaved, to various extents by a different class of enzyme: human and bacterial N-5'-alkylpurine (AP) endonucleases. The enzyme incises such oligomers 5' to the adduct and generates 3'-hydroxyl and 5'-phosphoryl termini but leaves the modified base on the 5'-terminus of the 3' cleavage fragment ('dangling base'). Using active-site mutants of the human AP endonuclease, we found that the active site for the primary substrate, abasic (AP) site, is the same as that for the bulky pBQ adducts. There appears to be no clear rationale for the widely differing recognition and repair mechanisms for these exocyclic adducts, as shown for the repair of the same types of modification on different bases (e.g. epsilon A and epsilon C) and for completely unrelated lesions (e.g. AP site and pBQ adducts). Another important variable that affects the rate and extent of repair is the effect of neighbouring bases. In the case of epsilon A, this sequence-dependent repair correlates with the extent of double-strandedness of the substrate, as demonstrated by thermal stability studies.     

Detection and identification of benzo[a]pyrene-DNA adducts by [35S]phosphorothioate labeling and HPLC

Two generally applicable [35S]phosphorothioate postlabeling procedures for the HPLC analysis of polycyclic aromatic hydrocarbon (PAH)-DNA adducts have been developed based upon [32P]phosphate postlabeling assays described by Gupta and Randerath et al. In one procedure, benzo[a]pyrene (B[a]P)-modified DNA was digested to nucleoside 3'-phosphates by micrococcal nuclease and spleen phosphodiesterase and the adducted nucleotides were extracted with 1-butanol. The adducted nucleoside-3'-phosphates were 5'-thiophosphorylated by T4 polynucleotide kinase (T4PNK) and adenosine 5'-O-(3-[35S]thiotriphosphate) to yield [35S]B[a]P-nucleoside-5'-phosphorothioate-3'-phosphate adducts. Although thiophosphorylation of B[a]P-DNA adducts was slower than the corresponding phosphorylation reaction, similar recoveries of the postlabeled adducts were achieved with longer incubation times and higher concentrations of T4PNK. A major advantage of this procedure over the 32P-postlabeling procedure is that the resistance of phosphorothioates to degradation by phosphatases allows selective removal of the unlabeled 3'-phosphate from the [35S]B[a]P-nucleoside-5'-phosphorothioate-3'-phosphate adducts by brief treatment with alkaline phosphatase. [35S]B[a]P-nucleoside-5'-phosphorothioate adducts were also prepared using a nuclease P1/prostatic acid phosphatase DNA degradation method. For anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-modified DNA, overall adduct recoveries were substantially higher with the nuclease P1/prostatic acid phosphatase method (48-51%) than with the micrococcal nuclease/spleen phosphodiesterase/alkaline phosphatase method (22-29%). There were no significant differences in the HPLC profiles of the [35S]phosphorothioate-postlabeled adducts obtained from these two procedures. HPLC analysis of B[a]P-DNA adducts formed in B[a]P-treated hamster embryo cell cultures demonstrated the formation of two major adducts, (+)syn-BPDE-deoxyguanosine-5'-phosphorothioate and (+)anti-BPDE-deoxyguanosine-5'-phosphorothioate, along with other minor adducts. Based upon an overall adduct recovery of 20% and 0.5 mol as the detection limit of this 35S-postlabeling/HPLC assay, the sensitivity of this assay is 1 adduct/10(8) nucleotides for a 60 micrograms DNA sample. This method offers the advantages of using 35S which has a longer half-life and lower radioactive decay energy than 32P and the ability to prepare PAH-DNA adducts at the monophosphorothioate level which greatly facilitates separation of individual 35S-postlabeled PAH-DNA adducts by HPLC.     

Genetic effects of exocyclic DNA adducts in vivo: heritable genetic damage in comparison with loss of heterozygosity in somatic cells

Etheno adducts are promutagenic lesions which generate point mutations, deletions, homologous recombination and gross structural DNA aberrations. High ratios of chromosome loss to forward mutations characterize vinyl bromide, vinyl chloride, ethyl carbamate, vinyl carbamate and its epoxide as effective clastogens in postmeiotic germ cells of Drosophila melanogaster. Of the mutants induced by vinyl carbamate at the vermilion gene, 68% were intra-locus or multi-locus deletions. In view of the far-reaching concordance between mutation spectra in mice and Drosophila observed in specific-locus tests with genotoxic agents, etheno bases are expected to generate mainly deletions in male mammals in the postmeiotic germ-cell stages. Twenty-two of 23 base substitutions induced in the vermilion gene after treatment of postmeiotic stages with vinyl carbamate or vinyl bromide fall into four categories of mutations expected from etheno bases: GC-->AT, AT-->GC, GC-->TA and AT-->TA base-pair changes. These types of point mutations occurred in mutated proto-oncogenes of tumours induced in rodents by vinyl chloride, ethyl carbamate or their metabolites. Of interest is the ability of vinyl carbamate to produce persistent lesions in otherwise highly repair-active premeiotic cells of Drosophila, leading to mutations of yet unknown nature. Etheno bases are also potent pro-clastogenic lesions in somatic cells in vivo. Strongly positive responses were reported for ethyl carbamate and vinyl carbamate in assays for micronucleus formation in mouse bone marrow and in the Drosophila white+/white eye mosaic test. Loss of heterozygosity in somatic cells of Drosophila was due primarily to ring-X chromosome loss, followed by homologous mitotic recombination. Particularly striking is the near failure of ethyl carbamate and vinyl carbamate to generate significant frequencies of intrachromosomal recombination. The overall genetic activity profiles of etheno adduct-forming chemicals in mice and in Drosophila support the hypothesis that vinyl carbamate is the proximate mutagen of ethyl carbamate, and vinyl carbamate epoxide is the ultimate electrophilic mutagen and carcinogen.     

Vinyl chloride-specific mutations in humans and animals

Vinyl chloride is a potent hepatocarcinogen which reacts with DNA to generate etheno bases. In order to determine whether mutational patterns in target genes in vivo are characteristic of vinyl chloride and could be explained by the mutagenic properties of the etheno bases, human and rat liver tumours associated with exposure to vinyl chloride were analysed for point mutations in the ras and p53 genes. In this paper, we review these data and report our latest results on animal tumours. Two alterations were found which could be attributed to a direct effect of vinyl chloride: a GC-->AT transition which leads to a GGC-->GAC mutation at codon 13 of the Ki-ras gene in human liver angiosarcomas, and lesions at AT base pairs, mostly AT-->TA transversions, which lead to mutations in the p53 gene in human and rat angiosarcomas and to a CAA-->CTA mutation at codon 61 of the Ha-ras gene in rat hepatocellular carcinomas.     

Fluorescence detection of benzo[a]pyrene--DNA adducts in human lung.

Improved techniques are described for the specific identification of benzo[a]pyrene-diolepoxide (BPDE)--DNA adducts in human tissues. Immunoaffinity chromatography, synchronous fluorescence spectroscopy and second-derivative synchronous fluorescence spectroscopy have previously been used to detect BPDE-DNA adducts in human placenta. Here we report how these methods, together with HPLC and the generation of complete fluorescence excitation--emission matrices, have been used to identify unequivocally BPDE-DNA adducts in samples of human lung. BPDE nucleotide adducts were isolated with immunoaffinity chromatography columns bearing antibodies raised against the (+/-)anti-7,8-diol-9,10-epoxide-deoxyguanosine adduct of benzo[a]pyrene. These adducts were hydrolyzed to tetrahydrotetrols and the hydrolysis products subjected to HPLC. The major product isolated by HPLC, benzo[a]-pyrene-7,10/8,9-tetrahydrotetrol, was determined by fluorescence spectroscopy. Using this method, levels of BPDE-DNA adducts in the range of 1-40 in 10(8) nucleotides were measured in 6 out of 25 samples, with a lower detection limit of one adduct in 10(8) nucleotides. The data may also indicate that adduct levels show regional variation in different parts of the same lung.     

Multiple DNA adducts in lymphocytes of smokers and nonsmokers determined by 32P-postlabeling analysis

Identification of DNA adducts in peripheral lymphocytes could serve as a means of monitoring human exposure to potential genotoxic agents. In this study, DNA from peripheral lymphocytes of smokers and nonsmokers was examined for adducts by the P1 nuclease 32P-postlabeling technique. Thin layer chromatography (TLC) maps from both groups revealed multiple DNA adducts which ranged from no adducts for one individual to six adducts for a different individual. The total DNA adduct concentrations were approximately one adduct in 10(8)-10(10) normal nucleotides. Comparison of the adduct TLC profiles revealed individual variation in both pattern and level of DNA adducts. The type and amount of adduct was not influenced by smoking history and remained unchanged in four out of six subjects who were resampled after a 1 month interval. The capacity of lymphocytes to form BaP-derived DNA adducts after a 72 h incubation with 10(-6) M [3H]BaP was measured by both high-performance liquid chromatography (HPLC) and 32P-postlabeling analysis. The in vitro adduct values detected by [3H]nucleoside concentrations on HPLC ranged from 1 to 7 fmol adduct per micrograms DNA (3.3-23.3 adducts per 10(7) nucleotides). The [3H]nucleoside values were consistent with values obtained by 32P-postlabeling of the same sample (correlation coefficient of 0.88). No relationship was apparent between the capacity of lymphocytes to form a [3H]BaP-derived adduct in vitro and the concentration of any adduct, or total adducts present in untreated lymphocytes. These results suggest that multiple DNA adducts are present in lymphocytes from nonsmokers as well as smokers, although the profile and extent of these adducts can vary among individuals. The relationship of the lymphocyte DNA adducts detected in this study to human cancer susceptibility remains to be determined.     

Ethanol‐mediated carcinogenesis in the human esophagus implicates CYP2E1 induction and the generation of carcinogenic DNA‐lesions

Chronic alcohol consumption is a major risk factor for esophageal cancer. Various mechanisms may mediate carcinogenesis including the genotoxic effect of acetaldehyde and oxidative stress. Ethanol exerts its carcinogenic effect in the liver among others via the induction of cytochrome P450 2E1 (CYP2E1) and the generation of carcinogenic etheno‐DNA adducts. Here we investigated if such effects can also be observed in the human esophagus. We studied nontumorous esophageal biopsies of 37 patients with upper aerodigestive tract cancer and alcohol consumption of 102.3 ± 131.4 g/day (range: 15–600 g) as well as 16 controls without tumors (12 teetotalers and 4 subjects with a maximum of 25 g ethanol/day). CYP2E1, etheno‐DNA adducts and Ki67 as a marker for cell proliferation were determined immunohistologically. Chronic alcohol ingestion resulted in a significant induction of CYP2E1 (p = 0.015) which correlated with the amount of alcohol consumed (r = 0.6, p < 0.001). Furthermore, a significant correlation between CYP2E1 and the generation of the carcinogenic exocyclic etheno‐DNA adducts 1,N⁶‐ethenodeoxyadenosine (r = 0.93, p < 0.001) and 3,N⁴‐ethenodeoxycytidine (r = 0.92, p < 0.001) was observed. Etheno‐DNA adducts also correlated significantly with cell proliferation (p < 0.01), which was especially enhanced in patients who both drank and smoked (p < 0.001). Nonsmokers and nondrinkers had the lowest rate of cell proliferation, CYP2E1 expression and DNA lesions. Our data demonstrate for the first time an induction of CYP2E1 in the esophageal mucosa by ethanol in a dose dependent manner in man and may explain, at least in part, the generation of carcinogenic DNA lesions in this target organ.     

32P-postlabeling analysis of binding of the cyclophosphamide metabolite, acrolein, to DNA

The cyclophosphamide metabolite, acrolein, was reacted with 2'-deoxyguanosine-3'-monophosphate, and two adducts were detected by high performance liquid chromatography and 32P-postlabeling assay. These adducts were resistant to dephosphorylation by nuclease P1 and could be isolated and detected from calf thymus DNA that had been reacted in vitro with acrolein. A combination of HPLC purification and enzymatic digestion of normal nucleotides by nuclease P1 allowed for the detection of these adducts in hepatic DNA from mice treated with cyclophosphamide. The level of the two adducts in the hepatic DNA, as determined by 32P-postlabeling, was one adduct per 2.7-4.1 x 10(7) normal nucleotides.