Mechanism of formation and 32P-postlabeling of DNA adducts derived from peroxidative activation of carcinogenic non-aminoazo dye 1-phenylazo-2-hydroxynaphthalene (Sudan I)

Horseradish peroxidase in the presence of hydrogen peroxide mediates the activation of carcinogenic 1-phenylazo-2-hydroxynaphthalene (Sudan I) to DNA-bound products in vitro. The peroxidase activating system is greater than 10 times more effective with respect to DNA modification by Sudan I than the microsomal enzymes containing cytochrome P450. The DNA-binding reaction of the Sudan I metabolite(s) formed by the peroxidase system is dependent on Sudan I and H2O2 concentration and pH. Reactive intermediate(s) or product(s) of the Sudan I oxidation by peroxidase with a short half-life are responsible for the DNA modification. DNA modified by peroxidase-activated Sudan I becomes colored and has an absorption maximum at approximately 480 nm. The modification of DNA by Sudan I metabolites(s) formed by the peroxidase system is inhibited by some compounds of physiological importance (ascorbate, glutathione, Mg2+ ions) and by radical trapping agents (nitrosobenzene, methyl viologen). 32P-Postlabeling assay of the DNA modified by Sudan I activated by the peroxidase system indicates that the covalent DNA adduct formation is the principal type of the DNA modification. Four major and several minor adducts of deoxyribonucleotide 3',5'-bisphosphate from DNA with Sudan I metabolite(s) were detected by the classical Randerath 32P-postlabelling assay as well as by the nuclease P1 version of the same method.     

Formation and 32P-postlabeling of DNA and tRNA adducts derived from peroxidative activation of carcinogenic azo dye N,N-dimethyl-4-aminoazobenzene.

Peroxidase in the presence of hydrogen peroxide catalyzes in vitro the activation of carcinogenic N,N-dimethyl-4-aminoazobenzene (DAB) to DNA-, tRNA- and homopolydeoxyribonucleotide-bound products. tRNA is the most susceptible to modification by the activated DAB. Binding of DAB products to macromolecules is inhibited by methyl viologen, nitrosobenzene, ascorbate, glutathione, NADH and MgCl2. The mechanism of these inhibitions was studied. The nuclease P1 version of the 32P-postlabeling assay was employed for detection and quantitation of some major DNA or tRNA adducts formed with DAB activated by a peroxidase system. tRNA modified by activated DAB shows a significantly increased acceptance for L-methionine.     

ras gene activation in rat tumors induced by benzidine congeners and derived dyes

Dimethoxybenzidine (DMO) and dimethylbenzidine (DM) are used to synthesize dyes such as C.I. Direct Blue 15 and C.I. Acid Red 114, respectively. These commercially used dyes are metabolically degraded to DMO or DM in the intestinal tract of rodents and subsequently DMO and DM are absorbed into the blood stream. Animals were exposed to DMO, DM, or the dyes in the drinking water. Tumors obtained from control and chemical-treated animals were examined for the presence of activated oncogenes by the NIH 3T3 DNA transfection assay. Activated oncogenes were detected in less than 3% (1/38) of the tumors from control animals whereas 68% (34/50) of the tumors from chemical-treated animals contained detectable oncogenes. Activated oncogenes were detected in both malignant (25/36) and benign (9/14) tumors from the chemically treated animals but only in one of 13 malignant tumors from the control animals. The presence of oncogenes in the chemically induced benign tumors suggests that oncogene activation was an early event in those tumors. Southern blot analysis of transfectant DNA showed that the transforming properties of the chemically induced rat tumor DNAs were due to the transfer of an activated H-ras (31/34) or N-ras (3/34) gene. One spontaneous rat tumor DNA was found to contain an activated H-ras gene. Oligonucleotide hybridization analysis indicated that the H-ras oncogenes from chemical-associated tumors contained mutations at codons 12, 13, or 61 whereas the spontaneously activated H-ras gene contained a point mutation at codon 61. These data suggest that activation of cellular ras genes by point mutation is an important step in the induction of tumors, at least in rats, by this class of benzidine-derived dyes. Moreover, in light of common histogenesis of the normal counterparts of many of the chemically induced neoplasms and histological evidence of varied tissue differentiation in some basal cell neoplasms, it is possible that most or all of the chemically induced neoplasms were derived from a common epidermal progenitor stem cell population.     

Mechanism of peroxidative activation of the bladder carcinogen 2-amino-4-(5-nitro-2-furyl)-thiazole (ANFT): comparison with benzidine

The mechanism of activation of the bladder carcinogen 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT) was investigated by comparison with benzidine. In comparison with benzidine, ANFT has a higher electrochemical potential (approximately 700 mV) and is less effective as a reducing co-substrate for either prostaglandin H synthase (PHS) or horseradish peroxidase. Activation was monitored by measuring binding to protein (BSA) and DNA. ANFT binding to protein was reduced by indomethacin, a fatty acid cyclooxygenase inhibitor; phenol and aminopyrine, competitive reducing co-substrates; ascorbic acid, an antioxidant; and glutathione, thioether conjugate formation. These results are consistent with those previously reported for benzidine and demonstrate a peroxide co-substrate requirement, interaction of peroxidase with amine, formation of reactive intermediates and inactivation of reactive intermediates. 5,5-Dimethyl-1-pyrroline N-oxide (DMPO), a radical trap, also reduced ANFT binding to protein. Similar results were observed whether activation by PHS or horseradish peroxidase was investigated. Peroxidative activation of ANFT and benzidine to bind DNA was inhibited by these test agents in a manner similar to that observed with protein except that DMPO did not reduce binding. In addition, 2-methyl-2-nitrosopropane and methyl viologen, which are radical traps, and methionine and p-nitrobenzyl-pyridine, which are strong nucleophiles, did not reduce ANFT or benzidine binding to DNA. These agents also did not prevent binding of benzidinediimine, the two-electron product of benzidine oxidation, to polydeoxyguanosine. Glutathione inhibited diimine binding by forming a conjugate. Results demonstrate that activation of ANFT to bind protein and DNA is similar to benzidine. Peroxidative activation of benzidine occurs by both one- and two-electron oxidation. A similar mechanism would explain ANFT binding to protein (one electron) and DNA (two electron).     

Immunochemical quantitation of DNA adducts derived from the human bladder carcinogen 4-aminobiphenyl

To assess target-tissue exposure to the human urinary bladder carcinogen 4-aminobiphenyl (ABP), we have developed a sensitive immunochemical method for measuring the major arylamine-DNA adduct formed, N-(guan-8-yl)-ABP (Gua-C8-ABP). High-affinity polyclonal antisera from rabbits immunized with N-(guanosin-8-yl)-ABP coupled to keyhole limpet hemocyanin were characterized and shown to have high specificity for antigenic determinants on the purine and biphenyl rings of Gua-C8-ABP and minimal cross-reactivity with ABP, deoxyguanosine, or hydrolyzed DNA. Assay standards containing ABP-modified DNA were prepared by reacting [3H]N-hydroxy-ABP with calf thymus DNA. DNA samples were hydrolyzed with trifluoroacetic acid and dried under vacuum, and the residues were dissolved in dimethyl sulfoxide under argon. Using a streptavidin-biotin amplified enzyme-linked immunosorbent assay, DNA hydrolysates competed at 25 micrograms DNA/microtiter well for a limiting amount of anti-keyhole limpet hemocyanin-(Gua-C8-ABP) in the presence of excess solid-phase bovine serum albumin-(Gua-C8-ABP) coating antigen. The limit of sensitivity for this assay using 25 micrograms DNA was 2 adducts/10(8) nucleotides. Gua-C8-ABP adducts in liver and bladder epithelial DNAs were readily quantified after p.o. administration of 5 mg/kg ABP to dogs. This methodology is capable of detecting adducts at levels of biological significance and should be applicable to human target-tissue dosimetry.     

Peroxidative activation of o-phenylhydroquinone leads to the formation of DNA adducts in HL-60 cells.

Using 32P-postlabeling we studied DNA adduct formation in HL-60 cells treated with the o-phenylphenol metabolites o-phenylhydroquinone (o-PHQ) and o-phenylbenzoquinone (o-PBQ). Treatment with 25-500 microM o-PHQ for 8 h produced one principal and three minor adducts with a relative distribution of 80, 10, 6 and 4%. The relative adduct levels from these treatments were 0.26-2.31 adducts/10(7) nucleotides. Treatment with 25-250 microM o-PBQ for 2 h resulted in a similar level of DNA modification and adduct distribution. Reaction of purified calf thymus DNA with o-PBQ produced one DNA adduct, which did not correspond to the major adduct produced in HL-60 cells. These results show that o-PHQ and o-PBQ can form DNA adducts. Peroxidase activation of o-phenylphenol may therefore play a role in the carcinogenic effect of this compound.     

Glyoxal-guanine DNA adducts: detection, stability and formation in vivo from nitrosamines

The glyoxal-deoxyguanosine adduct (gG) is formed from alpha-nitrosamino aldehydes and dG in vitro and in vivo from nitrosamines carrying the 2-hydroxyethyl side-chain as well as from N-nitrosomorpholine. The structures of all of the diastereomeric forms of both the cis and trans isomers of the adduct have been investigated by ab initio calculations and with nuclear magnetic resonance spectroscopy at 500 MHz. The preferred orientation of the OH groups is trans, but at equilibrium a small amount of the cis isomer was observed. The pH-independent equilibrium constant for the hydrolysis of the gG adduct is K = 1.36 x 10(-4) mol/L, and its rate of formation at pH 7.3 is k = 5.3 min-1 mol-1. In acid (pH 2), the hydrolysis of the nucleosidic linkage is nearly twice as rapid as the hydrolysis of gG to glyoxal and dG. We used a gG analogue to explore a number of reductive methods for derivatization of the adduct, but all of the processes either gave low yields or product mixtures which rendered them impractical for derivatizing the adduct in DNA. A 32P-postlabelling method for detection of the pH-sensitive gG adduct has been developed, which permitted detection of the adduct in the liver DNA of male Wistar rats after administration of selected nitrosamines. The levels of adducts found were: N-nitrosodiethanolamine > 2-hydroxyethylmethynitrosamine > N-nitrosomorpholine > 2-hydroxyethyethylnitrosamine. In separate experiments, N-nitrosodiethanolamine gave greater adduct levels than its metabolite 2-hydroxy-N-nitrosomorpholine. Mechanistic pathways for the generation of gG adducts in vitro and in vivo are discussed.     

Hemoglobin adducts in workers exposed to benzidine and azo dyes

Benzidine (Bz) is a known human carcinogen. Several azo dyes have been synthesized with Bz. Bz can be metabolically released from azo dyes. In a group of Indian workers producing Bz and azo dyes the presence of hemoglobin (Hb) adducts was investigated. The following Hb adducts were identified and quantified by GC-MS: Bz, N-acetylbenzidine (AcBz), 4-aminobiphenyl (4ABP), aniline. 4ABP and aniline were quantitatively the major adducts. In the exposed workers (n = 33) all correlations between 4ABP, Bz and AcBz were r = 0.89 (P < 0.01) or greater. The group of workers exposed to Bz (Bz workers, n = 15) had 10-17-fold higher adduct levels than the workers exposed to dyes (dye workers, n = 18). 4ABP can be metabolically released from Bz and azo dyes. Aniline can be metabolically released from azo dyes. Therefore, the presence of 4ABP and aniline as Hb adducts is a consequence of exposure to the parent compounds or to the exposure of Bz and azo dyes and a consequent metabolical release of the arylamine moiety. The mean adduct ratios of 4ABP/(AcBz + Bz) varied up to 4-fold across all seven factories. Therefore, it is possible that 4ABP may have derived from general contamination in the work environment or endogenous metabolism, or a combination of the two. Since 4ABP is also a known human carcinogen, tumors observed in workers exposed to Bz or Bz dyes might be caused by both compounds. Further, these results suggest that understanding the role that genetic variants in NAT1 and NAT2 play in modifying the impact of Bz on bladder cancer risk may be complicated, as N-acetylation detoxifies 4ABP and activates Bz.     

The correlation between DNA adducts and chromosomal aberrations in the target organ of benzidine exposed, partially-henatectomized mice

An experimental system was developed to test the associationbetween benzidine—DNA adduct levels and chromosome aberrationsin the target organ, the liver, of mice. A 2/3 partial hepatectomywas performed (0 h), then the animals were treated with benzidine(0, 7.8, 19.5, 38.2 or 97.8 mg/kg, i.p.) and an agar-coated50 mg 5-bromodeoxyuridine tablet was implanted subcutaneously(58 h). Colcemid was given at 4 mg/kg i.p. (70 h), and the animalswere sacrificed 2 h later. The liver from each animal was divided,with portions allocated for cytogenetics and DNA adduct analysis.DNA adducts were analyzed with the ³²P-postlabeling technique.DNA adduct and chromosomal aberration data were available ona total of 43 animals. Benzidine was shown to be a potent clastogenin liver, the target organ, as opposed to its reported weakactivity in the bone marrow. A linear dose response was demonstratedfor benzidine—DNA adducts found in the liver. The correlationbetween adduct levels and aberrations in individual animalswas 0.43 (P < 0.05). However, most of the residual variancewas due to four outlying cases. When these cases were removedfrom the data set and the analysis repeated, the linear correlationcoefficient increased to 0.74. When the data were analyzed bydose groups, the correlation was 0.91. These data support thehypothesis that carcinogen—DNA adducts are responsiblefor the induction of chromosomal aberrations, and perhaps othergenotoxic events, induding neoplasia.     

In vitro formation of DNA adducts with bile acids

The in vitro experiment was carried out following ³²P-postlabelinganalysis to determine the DNA-reactive bile acids present inthe human body. The unconjugated and conjugated forms of cholic(CA), chenodeoxycholic (CDCA), deoxycholic (DCA) and lithocholicacid (LCA) were added to calf thymus DNA followed by 1 h ofincubation at 37°C. After the incubation the mixture wasanalyzed by the nuclease P1 modification of ³²P-postlabeling.Among the 12 bile acids tested, our results showed that unconjugatedCDCA and LCA and the glycine and taurine conjugates of LCA (g-LCA,t-LCA) were able to bind covalently with naked DNA in vitrowithout intervention of any catalyst. It was also shown thatbile acid alone did not give any spot on TLC. These bindingreactions depended on the bile acid concentration in a linearmanner. The data on the extent of binding at a concentrationof 0.1 mg/ml showed values of 28.5 (t-LCA), 23.7 (g-LCA), 3.47(LCA) and 1.32 (CDCA) adducts per 10⁸ nucleotides. These reactivebile acids were also incubated with COLO 205 human colon carcinomacells and Hep G2 human hepatocellular carcinoma cells for 24h, but no specific DNA adduct was formed. Further, when LCAor CDCA was administered into male Fischer 344 rats by gavageat a dose of 10 mg/rat every 8 h for 3 days, no specific DNAadduct was detected in their liver or colon. Covalent DNA adductsare believed to cause alteration of the primary structure ofgenes, which is potentially linked to carcinogenesis. Thoughour preliminary data failed to detect any bile acid-relatedDNA adducts in the cultured cells or in rats, the results mayprovide a basis for assuming some of these bile acids to bepotential initiators of colon cancer.     

Ram seminal vesicle microsome-catalyzed activation of benzidine and related compounds: dissociation of mutagenesis from peroxidase-catalyzed formation of DNA-reactive material

Ram seminal vesicle (RSV) microsomal preparations activate benzidineand other arylamines to mutagenic species in a modified Amesassay. We have examined the mechanism of this activation processin more detail. The mutagenic effect was neither arachidonicacid-dependent nor indomethacin in hibitable. The mutagenicspecies was stable for at least 30 min in experiments in whichaddition of bacteria was delayed. Acetylbenzidine was a muchmore potent mutagen than benzidine in this system. Substitutionof the acetylase-deficient tester strain TA98/1,8-DNP₆ for strainTA98 markedly reduced the mutagenicity of acetylbenzidine andcompletely eliminated the mutagenicity of benzidine. Benzidineanalogues 3,3'-dimethoxybenzidine (o-dianisidine), o-tolidineand 3,3',- 5,5'-tetramethylbenzidine were not mutagenic in theRSV activation system. RSV-dependent activation of all radiolabeledcongeners examined resulted in covalent binding to calfthymusDNA. The rank order of binding was: 3,3'-dichloro-benzidine> henzidine > o-dianisidine > acetylbenzidine >tetramethylbenzidine. This binding required active enzyme andarachidonic acid or hydrogen peroxide. The reactive specieswas short-lived: delayed addition of DNA reduced the level ofbinding nearly to zero. Binding was inhibitable by indomethacin,but this inhibition was incomplete in the cases of dichlorobenzidineand acetylbenizidine. We conclude that the extracellular generationof peroxidase-catalyzed oxidation products does not explainthe RSV microsome-dependent mutagenicity observed with thesecompounds.     

Urinary mutagenicity as a biomarker in workers exposed to benzidine: correlation with urinary metabolites and urothelial DNA adducts

Urinary mutagenicity has been used in occupational and epidemiological studies for over two decades as a cost-effective, general biomarker of exposure to genotoxic agents. However, few studies have compared urinary mutagenicity to additional biomarkers determined among low- and high-exposed groups. To address this issue, we evaluated the relationship between urinary mutagenicity and other types of biomarkers in a cross-sectional study involving 15 workers exposed to the urinary bladder carcinogen benzidine (BZ, high exposure), 15 workers exposed to BZ-dyes (low exposure), and 13 unexposed controls in Ahmedabad, India. Urinary organics were extracted by C18/methanol and evaluated for mutagenicity in the presence of S9 in the Salmonella strain YG1024, which is a frameshift strain that overproduces acetyltransferase. The results were compared to biomarker data reported recently from the same urine samples (Rothman et al., Proc. Natl Acad. Sci. USA, 93, 5084- 5089, 1996) that included a metabolite biomarker (the sum of the urinary levels of BZ + N-acetylbenzidine + N,N'-diacetylbenzidine) and a DNA adduct biomarker [a presumptive N-(3'-phosphodeoxyguanosin-8-yl)- N'-acetylbenzidine (C8dG-ABZ) DNA adduct in exfoliated urothelial cells]. The mean +/- SE urinary mutagenicity (revertants/micromol of creatinine) of the low-exposure (BZ-dye) workers was 8.2 +/- 2.4, which was significantly different from the mean of the controls (2.8 +/- 0.7, P = 0.04) as was that of the mean of the high-exposure (BZ) workers (123.2 +/- 26.1, P < 0.0001). Urinary mutagenicity showed strong, positive correlations with urinary metabolites (r = 0.88, P < 0.0001) and the level of the presumptive C8dG-ABZ urothelial DNA adduct (r = 0.59, P = 0.0006). A strong association was found between tobacco use (bidi smoking) and urinary mutagenicity among the controls (r = 0.68, P = 0.01) but not among the exposed workers (r = 0.18, P = 0.11). This study confirms the ability of a biomarker such as urinary mutagenicity to detect low-dose exposures, identify additional genotoxic exposures among the controls, and correlate strongly with urinary metabolites and DNA adducts in the target tissue (urinary bladder epithelia) in humans.      

Metabolism of 1-nitropyrene and formation of DNA adducts in Salmonella typhimurium

1-Nitropyrene is slowly reduced by intact cells of Salmonellatyphimurium to yield 1-aminopyrene and N-acetyl-1-aminopyreneplus six unidentified minor products. When the bacteria areexposed to tritiated 1-nitropyrene, increasing amounts of radioactivitybecome bound to DNA as the nitropyrene is metabolized. Enzymatichydrolysis of the labelled DNA yields low molecular weight labelledcompounds which probably represent nucleoside adducts formedby the reaction of nitropyrene metabolites with DNA. Resultswith appropriate mutant strains indicate that bacterial nitroreductasesare involved in activating nitropyrene to a reactive intermediatethat binds to DNA and that nitropyrene adducts in DNA are subjectto excision repair.     

Formation of DHP-derived DNA adducts from metabolic activation of the prototype heliotridine-type pyrrolizidine alkaloid, lasiocarpine

Pyrrolizidine alkaloids (PAs) are probably the most common poisonous plants affecting livestock, wildlife, and humans. The PAs that have been found to be tumorigenic in experimental animals belong to the retronecine-, heliotridine-, and otonecine-type PAs. Our recent mechanistic studies indicated that riddelliine, a tumorigenic retronecine-type PA, induced tumors via a genotoxic mechanism mediated by the formation of 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-derived DNA adducts. The same adducts were formed from clivorine, a tumorigenic otonecine-type PA from metabolism of clivorine by rat liver microsomes in the presence of calf thymus DNA. In this study, we report that metabolism of lasiocarpine, the prototype heliotridine PA, by F344 rat liver microsomes resulted in the formation of DHP. When incubated in the presence of calf thymus DNA, the same DHP-derived DNA adducts were formed. These results suggest that these DHP-derived DNA adducts are potential biomarkers of exposure and tumorigenicity for all types of PAs.     

Microsomal and peroxidase activation of 4-hydroxy-tamoxifen to form DNA adducts: comparison with DNA adducts formed in Sprague-Dawley rats treated with tamoxifen

Using rat liver microsomal preparations and peroxidase enzymes,we have investigated the formation of DNA adducts by the antiestrogencompound tamoxifen (TAM) and its metabolite 4-hydroxy-tamoxifen(4-OH-TAM). When reduced nicotinamide-adenine dinucleotide phosphate(NADPH) was used as a cofactor in microsomal activation of either4-OH-TAM or TAM, one DNA adduct and relative DNA adduct levelsof 4.6 and 3.1x10^–8, respectively were detected by 32P-postlabeling.The DNA adduct produced by microsomal activation of 4-OH-TAMand TAM was the same. With cumene hydroperoxide (CuOOH) as thecofactor for the microsomal activation of either 4-OH-TAM orTAM, three to six DNA adducts were produced; the relative adductlevels were 8.0 and 20.6x10^–8, respectively. Comparisonof the DNA adduct patterns produced by 4-OH-TAM and TAM showedthat they were distinct However one of the DNA adducts (a) producedby microsomal activation of 4-OH-TAM using CuOOH was the sameas adduct a produced by microsomal activation of 4-OH-TAM withNADPH. Activation of 4-OH-TAM with horseradish peroxidase resultedin the formation of a single DNA adduct and a relative adductlevel of 20.7x10^–8. Rechromatography analysis of thisDNA adduct showed that it was identical to that produced bymicrosomal activation of 4-OH-TAM with NADPH and one of theadducts produced using CuOOH as the cofactor. Ten DNA adductsand a relative adduct level of 15.3x10^–8 were detectedin the liver of female Sprague-Dawley rats treated daily with20 mg/kg of TAM for 7 days. The DNA adduct pattern in the liverof the treated animals was similar to that produced by microsomalactivation of TAM using CuOOH as the co-factor. The principalDNA adduct (no. 6) formed in the livers of rats treated withTAM was the same as the principal DNA adduct formed followingmicrosomal activation of TAM using CuOOH as a cofactor. TheDNA adduct formed following microsomal activation of eitherTAM or 4-OH-TAM using NADPH was also present as one of the adducts(1^*) formed in vivo following TAM treatment These studies demonstratethat 4-OH-TAM can be activated to form DNA adducts and thatit contributes to the formation of DNA adducts in the liverof rats treated with TAM.     

Site-specific adducts derived from the binding of anti-5-methylchrysene diol epoxide enantiomers to DNA: Synthesis and characteristics

The direct synthesis and characterization of site-specific adductsderived from the binding of ( + )-1R, 2S-dihydroxy-3S, 4R-epoxide-l,2,3,4-tetrahydro-5-methylchryseneand the ( – )-1S, 2R, 3R, 4S-enantiomer [( + )- and (– )-5-MeCDE, respectively], to the N²-guanine residuesin the oligonucleo-tide d(CCATCGCTACC) are described. The spectroscopiccharacteristics of the 5-MeCDE-modified oligonucleotides arediscussed, and it is shown that their CD characteristics canbe used to distinguish between the trans-addition products ofthe binding of the ( + )- and ( – )-enantiomers of 5-MeCDE(C4 position). The 11-mer duplexes with the normal complementarystrands are destabilized by the site-specific, covalently bound5-MeCDE residues: the melting points, T_m, are 5-10° lowerthan in the case of the unmodified duplex. Stereoselective exonucleaseenzyme digestion patterns of the single-stranded ( + )- and( – )-trans-5-MeCDE-modified oligonucleotides (Mao etal., 1993, Biochemistry, 32, 11785-11793) were used to probethe orientations of the covalently bound 5-MeCDE residues relativeto the modified guanine and the 5'-3' strand polarity, the aromaticresidues are positioned either on the 5'-side [( + )-5-MeCDE],or the 3-side [( – )-5-MeCDE adduct] of the modified guanineresidues. The electrophoretic mobilities of the ( + )-5-MeCDE-modified11-mer duplexes in native polyacrylamide gels are slower thanthose of unmodified and modified duplexes containing the stereoisomeric( – )-5-MeCDE-N²-dG lesions. This indicates that the lesionsderived from the tumorigenic ( + )-5-MeCDE induce greater degreesof bending or local flexibility than the non-tumor-igenic (– )-5- MeCDE enantiomer. These differences in the orientationaland structural characteristics are similar to those observedwith analogous DNA adducts derived from the tumorigenic ( +)-7R, 8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo[]pyreneand the non-tumor-igenic 7S,8R,9R,10S-enantiomer, respectively.The adducts derived from BPDE and 5-MeCDE enantiomers thus displaysimilar characteristics that depend primarily on the PAH diolepoxide enantiomer stereochemistry. This direct synthesis approachcan be used to generate milligram quantities of site-specific5-MeCDE-modified oligonucleotides that are suitable for NMRstudies (Cosman et al., 1995, Biochemistry, 34, 6247-6260).     

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.     

Peroxidase activation of tamoxifen and toremifene resulting in DNA damage and covalently bound protein adducts

When [¹⁴C]tamoxifen was incubated with horseradish peroxidaseand H₂O2, two major metabolites, separated and identified byHPLC, were N-desmethyltamoxifen and tamoxifen N-oxide. Toremifeneincubated in a similar system yielded N-desmethyltoremifeneand toremifene N-oxide. No 4-hydroxylated metabolites were detectedwith either drug. When calf thymus DNA was included in peroxidaseincubation mixtures, DNA damage, as assessed by ³²P-postlabelling,could also be detected. The extent of damage caused by tamoxifenand toremifene was similar. The major adducts formed followingincubation of DNA with tamoxifen had similar R_f values to twoof the ³²P-postlabelled adducts seen following dosing of ratswith tamoxifen. Peroxidase was able to activate both drugs toderivatives which covalently bound to bovine serum albumin.The pH optimum for covalent binding and N-demethylation wasnear to pH 6.0. Results from liquid chromatography-electrospraysecondary ion mass spectrometry suggest that tamoxifen and toremifeneare metabolized by peroxidase to putative reactive epoxide intermediatesresponsible for the genotoxic effects. It is proposed that peroxidaseoxidizes tamoxifen to a carbon-centred free radical which reactswith oxygen to form peroxy radicals capable of inserting anoxygen atom into tamoxifen. Lactoperoxidase and prostaglandinsynthase are also able to catalyse tamoxifen N-demethylationand binding to protein. These data show that peroxidase canactivate both tamoxifen and toremifene to an intermediate(s)that can damage DNA and covalently react with protein. Sinceit is known that women treated with tamoxifen can develop endometrialtumours, it may be relevant to determine whether activationof tamoxifen by peroxidases may contribute to its carcinogenicaction at extrahepatic sites.