Characterization of thymidine adducts formed by acrolein and 2-bromoacrolein.

Thymidine was permitted to react with the known mutagens acrolein and 2-bromoacrolein under physiological conditions. The products of these reactions were separated by HPLC and characterized by UV, FAB/MS, electrospray MS, 1H NMR and chemical transformation. The reaction with acrolein gave one major product, N3-(3'-oxopropyl)thymidine, which is unstable in aqueous solution and was reduced with sodium borohydride to the corresponding alcohol. Reaction with 2-bromoacrolein yielded the unstable intermediate, N3-(2'-bromo-3'-oxopropyl)thymidine, and two stable products, the diastereomers of N3-(2'-hydroxy-3'-oxopropyl)thymidine, which are slowly transformed to N3-(2'-oxo-3'-hydroxypropyl)thymidine. Reactions with both mutagens proceed most rapidly at pH 9.2, less rapidly at pH 7.4, and no products are found at pH 4.2. Stable adducts found in the reaction of 2-bromoacrolein were also identified in reactions with single-strand oligodeoxynucleotides using a sensitive, selected ion monitoring GC/MS procedure.     

Identification of the major adducts formed by reaction of 5-methylchrysene anti-dihydrodiol-epoxides with DNA in vitro

5-Methylchrysene is metabolically converted to the bay-region dihydrodiol-epoxides, trans-1,2-dihydroxy-anti-3,4-epoxy-1,2,3,4-tetrahydro-5-methylchrysene (DE-I), in which the methyl group and the epoxide ring are in the same bay region, and trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydro-5-methylchrysene (DE-II). Previous studies have indicated that DE-I is more important in 5-methylchrysene carcinogenesis than is DE-II. Both DE-I and DE-II were individually reacted with calf thymus DNA in vitro. The DNA was enzymatically hydrolyzed to deoxyribonucleosides, and the modified deoxyribonucleosides were separated by chromatography on Sephadex LH-20 and analyzed by high-performance liquid chromatography. One major adduct and seven minor adducts were formed from each dihydrodiol-epoxide. The major adduct was, in each case, characterized by its pH-dependent partition coefficient, stability to base, mass spectrum, ultraviolet spectrum, and nuclear magnetic resonance spectrum as a deoxyguanosine derivative resulting from addition of the exocyclic amino group of deoxyguanosine to the benzylic carbon of the epoxide ring of the dihydrodiol-epoxide. The results of this study show that the major DNA adducts formed from 5-methylchrysene via DE-I and DE-II are structurally similar.     

In vitro reactions of butadiene monoxide with single- and double-stranded DNA: characterization and quantitation of several purine and pyrimidine adducts

We have previously shown that butadiene monoxide (BM), the primary metabolite of 1,3-butadiene, reacted with nucleosides to form alkylation products that exhibited different rates of formation and different stabilities under in vitro physiological conditions. In the present study, BM was reacted with single-stranded (ss) and double-stranded (ds) calf thymus DNA and the alkylation products were characterized after enzymatic hydrolysis of the DNA. The primary products were regioisomeric N-7-guanine adducts. N-3-(2-hydroxy-3-buten-1-yl)adenine and N-3-(1-hydroxy-3-buten-2-yl)adenine, which were depurinated from the DNA more rapidly than the N-7-guanine adducts, were also formed. In addition, N6-(2-hydroxy-3-buten-1-yl)deoxyadenosine and N6-(1-hydroxy-3-buten-2-yl)deoxyadenosine were detected and evidence was obtained that these adducts were formed by Dimroth rearrangement of the corresponding N-1-deoxyadenosine adducts, not while in the DNA, but following the release of the N-1-alkylated nucleosides by enzymatic hydrolysis. N-3-(2-hydroxy-3-buten-1-yl)deoxyuridine adducts, which were apparently formed subsequent to deamination reactions of the corresponding deoxycytidine adducts, were also detected and were stable in the DNA. Adduct formation was linearly dependent upon BM concentration (10-1000 mM), with adduct ratios being similar at the various BM concentrations. At a high BM concentration (750 mM), the adducts were formed in a linear fashion for up to 8 h in both ssDNA and dsDNA. However, the rates of formation of the N-3-deoxyuridine and N6-deoxyadenosine adducts increased 10- to 20-fold in ssDNA versus dsDNA, whereas the N-7-guanine adducts increased only slightly, presumably due to differences in hydrogen bonding in ssDNA versus dsDNA. These results may contribute to a better understanding of the molecular mechanisms of mutagenesis and carcinogenesis of both BM and its parent compound, 1,3-butadiene.     

Detection and identification of mutagens by the adducts formed upon reaction with guanosine derivatives

Mutagens present in crude samples such as heated glucose can be detected or identified by means of the adducts formed upon reaction with a fluorescent guanosine derivative (FG) or isopropylideneguanosine (IPG). After the reaction of IPG with heated glucose, two adducts were isolated by high-performance liquid chromatography. One of the adducts was identified as the cyclic adduct formed between IPG and glyoxal. Mesoxaldialdehyde, which is structurally related to glyoxal, also produced a cyclic IPG-adduct and showed mutagenic activity in Salmonella typhimurium strain TA100. The other adduct isolated from the reaction mixture of IPG and heated glucose was 8-hydroxy-IPG. Various reagents which generate oxygen radicals were effective in the hydroxylation of guanosine derivatives at the C-8 position. These reagents also cause hydroxylation of guanine residues in DNA.     

Characterization of 4,4'-methylenebis(2-chloroaniline)--DNA adducts formed in vivo and in vitro.

4,4'-Methylenebis(2-chloroaniline) (MOCA) is a genotoxic and carcinogenic industrial chemical to which there is considerable potential human exposure. Since metabolic activation and formation of DNA adducts are believed to be important for the induction of these effects, DNA was treated in vitro with radiolabeled N-hydroxy-MOCA, the presumed proximate carcinogenic metabolite formed in vivo. Two major radioactive peaks were observed after HPLC separation of enzymatic hydrolysates. The two products were analyzed by MS and characterized as N-(deoxyadenosine-8-yl)-4-amino-3-chlorobenzyl alcohol and N-(deoxyadenosin-8-yl)-4-amino-3-chlorotoluene. The same adducts were also the major adducts formed in DNA of tissues from rats treated with radiolabeled MOCA. They were eliminated from rat liver with non-linear kinetics, in agreement with observations made for other carcinogens. The selective reaction of N-hydroxy-MOCA with DNA-adenine and the formation of single arylamine ring adducts suggest a substitution mechanism involving an intermediate with strong SN1 character, aided by the negative inductive effect of the ortho-chlorine. Due to tautomer formation, the initial adduct may be inherently unstable and undergo cleavage at the 1'-carbon-methylene bond to yield the observed adducts.     

32P-Postlabelling of DNA adducts formed by allyl glycidyl ether in vitro and in vivo

32P-Postlabelling analysis of allyl glycidyl ether-treated DNA after adduct enrichment on anion-exchange cartridges revealed two major and one minor DNA adducts. The major adducts were shown to originate from alkylation at N-7-guanine and N-1-adenine, respectively, while the minor adduct was at N-3-cytosine. In addition, rearrangement products of the 1-adenine and 3-cytosine adducts to N6-adenine and 3-uracil were indicated. The relative amounts of adenine, cytosine and uracil products appeared to be dependent upon conditions (in particular pH) during sample processing and analysis. When nuclease P1 was used for adduct enrichment the adenine, cytosine and uracil adducts, but not the 7-guanine adduct, were detected. The labelling efficiency of the 7- guanine adduct standard was 40-45%. Total recovery of this adduct from allyl glycidyl ether-modified DNA was 9-12%. The labelling efficiency of the 1-adenine adduct standard was 78-82%. Total recovery of this adduct from DNA was approximately 20% when using anion-exchange chromatography for adduct enrichment and 30-34% when using nuclease P1. Preliminary analysis of DNA from mice treated with allyl glycidyl ether indicated 57 times higher level of the 7-guanine adduct, per unit dose, in skin DNA (120 per 10(8) normal nucleotides) after topical application when compared to liver DNA after i.p. administration. The 1- adenine adduct could not be quantified in liver DNA (due to an interfering background product present in untreated animals) and the level of the 3-cytosine adduct was below the detection limit of the method. After topical application the level of the 1 adenine adduct in skin DNA was approximately 30 per 10(8), using either column or nuclease P1 enrichment. The 3-cytosine adduct was detected in skin, but was not quantified.      

Characterization of DNA adducts at the bay region of dibenz[a,h]anthracene formed in vitro

Bay region diolepoxide—DNA adducts of dibenz[a,h]anthracene(DBA) formed in vitro were identified and their absolute stereochemistrywas assigned. After activation of [5,12-¹⁴C]DBA with liver microsomesobtained from Aroclor 1254 treated male Sprague—Dawleyrats in the presence of calf thymus DNA for 1 h, the amountof DNA adducts was found to be 9.9 ± 2.4 pmol/mg DNA,calculated on the basis of the portion of radioactivity elutedfrom the HPLC reversed-phase column with a water/acetonitrilegradient. Bay region diolepoxide—DNA adducts represented27.5% of radioactivity associated with DNA adducts. The absoluteconfiguration of the various adducts was determined from thereaction of the (+)- and (–)-3,4-dihydrodiol after metabolicactivation and the reaction of the anti- andsyn-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrodibenz[a,h]anthracenewith DNA or with the individual deoxyribonucleotides. The mainbay region adduct was identified as a deoxyguanosine adductof (anti)-3S,4R-dihydroxy-1R,2S-epoxy-1,2,3,4-tetrahydrodibenz[a,h]anthracene, a metabolite of (–)-3,4-dihydroxy-3,4-dihydrodi-benz[a,h]anthracene.Anti bay region diolepoxide-deoxyguanosine adducts of DBA contributedto 17.7% and syn diolepoxide-derived deoxyguanosine adductsto 5.8% of adduct-associated radioactivity. The amount of bayregion deoxyadenosine adducts was calculated to be 4%. For sixof probably eight different deoxyadenosine adducts absolutestereochemistry could be assigned. ₃₂P-Postlabelling experimentsrevealed a binding of 23 ± 6 pmol/mg DNA for (–)-3,4-dihydrodioland of 1.5 ± 0.4 pmol/mg DNA for (+)-3,4-dihydrodiolof DBA.     

Characterization of phosphodiester adducts produced by the reaction of cyanoethylene oxide with nucleotides

Cyanoethylene oxide (CEO), a putative toxic and carcinogenicmetabolite of acrylonitrile, is a direct-acting mutagen. Thefocus of this study was to elucidate potential adducts responsiblefor the mutagenic effect of CEO by characterizing products fromthe reaction of CEO with nucleotides. The reaction of CEO withthe 5'-monophosphates of deoxy-guanosine, deoxyadenosine, deoxycytidineor deoxythymidine resulted in the formation of at least oneadduct for each nucleotide. Using two-dimensional NMR spectroscopyand fast atom bombardment mass spectrometry, CEO-nucleotideadducts (     

Identification of the major metabolites and DNA adducts formed from 2-nitropyrene in vitro

In contrast to 1-nitropyrene (1-NP), which is the most abundant nitropolycyclic aromatic hydrocarbon in numerous environmental sources, 2-nitropyrene (2-NP) has been detected only in the ambient air and not in direct emissions. Thus, 2-NP can be used as an indicator for monitoring human exposure to nitropolynuclear aromatic hydrocarbons in ambient air. Therefore, it is essential to determine the possible metabolic pathways of 2-NP. The metabolism of 2-NP by rat liver 9000 g supernatant was investigated. Under aerobic conditions, ring oxidation to 6-hydroxy-2-nitropyrene and nitroreduction to 2-aminopyrene (2-AP) were observed. When incubations were carried out in an atmosphere of nitrogen, 2-AP was the only metabolite detected. These results are consistent with those observed with 1-NP. In vitro metabolic activation of 2-NP to DNA adducts catalyzed by xanthine oxidase was also examined. Two adducts were characterized as N-(deoxyguanosin-8-yl)-2-aminopyrene and N-(deoxyadenosin-8-yl)-2-aminopyrene. The presence of deoxyadenosine adduct, which is derived from the nitroreduction pathway, may contribute to the powerful direct-acting mutagenicity of 2-NP.     

Further characterization of the DNA adducts formed by electrophilic esters of the hepatocarcinogens 1'-hydroxysafrole and 1'-hydroxyestragole in vitro and in mouse liver in vivo, including new adducts at C-8 and N-7 of guanine residues

The identities of the adducts formed on reaction of the model electrophilic and carcinogenic esters 1'-acetoxysafrole or 1'-acetoxyestragole with deoxyguanosine in vitro and those formed in vivo in the hepatic DNA of 12-day-old male C57BL/6 X C3H/He F1 (hereafter called B6C3F1) mice treated with 1'-hydroxysafrole or 1'-acetoxysafrole were investigated further with more discriminating high-performance liquid chromatography systems than previously used. The adducts formed from the reactions of 1'-acetoxysafrole or 1'-acetoxyestragole are strictly analogous and are distinguished by the prefixes S and E, respectively. Five adducts, including S(E)-II identified by Phillips et al. (Cancer Res., 41: 176-186, 2664-2671, 1981) as N2-(trans-isosafrol-3'-yl)deoxyguanosine and the analogous isoestragole derivative, have been characterized from the reactions with each ester. Adducts S-I and E-I, tentatively identified by Phillips et al. as N2-(safrol-1'-yl)- or N2-(estragol-1'-yl)deoxyguanosine, were each resolved into a pair of diastereomers. The proposed structures for each diastereomer were confirmed by nuclear magnetic resonance and circular dichroism spectroscopy. Two new adducts, i.e., S(E)-V and S(E)-VI, were isolated from each reaction mixture. On the basis of their pKas, their loss of 3H from [8-3H]deoxyguanosine, their retention of 3H from [1',2'-3H]deoxyguanosine, and their nuclear magnetic resonance spectra, Adducts S-V and E-V were characterized as 8-(trans-isosafrol-3'-yl)- and 8-(trans-isoestragol-3'-yl)deoxyguanosine, respectively. Adducts S-VI and E-VI were characterized in a similar manner as 7-(trans-isosafrol-3'-yl)- and 7-(trans-isoestragol-3'-yl)guanine, respectively. Adducts S-III and E-III, minor components described in the earlier studies, were not observed in the present work. High-performance liquid chromatography of hydrolysates of the hepatic DNA of male 12-day-old B6C3F1 mice killed 9 h after a single dose (0.1 mumol/g body weight) of [2',3'-3H]-1'-hydroxysafrole showed that Adducts S-Ia, S-Ib, S-II, S-IV (identified by Phillips et al. as N6-(trans-isosafrol-3'-yl)deoxyadenosine), S-V, and S-VI were present at average levels of 3.5, 7.0, 24.4, 2.9, 1.2, and 3.6 pmol/mg DNA, respectively. Similar levels of these adducts were found in the hepatic DNA after administration of the same dose of [2',3'-3H]-1'-acetoxysafrole under identical conditions.     

32P-HPLC suitable for characterization of DNA adducts formed in vitro by polycyclic aromatic hydrocarbons and derivatives

Analysis of DNA adducts demands both high sensitivity and goodresolution. A high-performance liquid chromato-graphy methodfor ³²P-postlabeled DNA adducts (³²P-HPLC) was used to investigateDNA adduct formation from 38 polycyclic hydrocarbons and biphenylsin vitro. The ³²P-HPLC method proved to be useful for separation,detection and characterization of DNA adducts from most of thesubstances. The in vitro method used to form the DNA adducts,with calf thymus DNA, nucleotide 3'-phosphates and metabolicactivation through S-9 liver homo-genate, gave poor quantitativereproducibility. However, the results showed that the ³²P-HPLCmethod was suitable for characterizing DNA adducts from manysubstances. From 35 of the tested substances 365 DNA and nucleotide3'-phospate adducts were detected and characterized concerningretention times. Of the adducts, 171 were detected in DNA and39 of them from five substances were characterized concerningtarget nucleotides. The retention time library built can beused in future analyses of DNA with complex patterns of DNAadducts.     

Structural characterization by mass spectrometry of hemoglobin adducts formed after in vitro exposure to methyl bromide

A mass spectrometric procedure is described for the structuralstudy of the adducts formed in human hemoglobin by in vitroexposure of erythrocytes to the alkylating agent methyl bromideusing different protein to reagent ratios. Peptide mapping byHPLC and tandem mass spectrometry allowed location of methylatedamino acids within the protein sequence. A prominent reactivityof several nucleo-philic side chains in human hemoglobin summitswas observed, which was modulated by the concentration of thealkylating agent Cysteine residues, the main reactive sites,were fully methylated in hemoglobin exposed to a 10-fold excessof methyl bromide, differently from other residues, includinghistidines, showing a heterogeneous pattern of methylation thatwas largely directed by their environment. No evidence of methylationwas found at the heme proximal histidines ß92 and     

Comparison of the DNA adducts formed by tamoxifen and 4-hydroxytamoxifen in vivo

Tamoxifen is a liver carcinogen in rats and has been associated with an increased risk of endometrial cancer in women. Recent reports of DNA adducts in leukocyte and endometrial samples from women treated with tamoxifen suggest that it may be genotoxic to humans. One of the proposed pathways for the metabolic activation of tamoxifen involves oxidation to 4-hydroxytamoxifen, which may be further oxidized to an electrophilic quinone methide. In the present study, we compared the extent of DNA adduct formation in female Sprague-Dawley rats treated by gavage with seven daily doses of 54 micromol/kg tamoxifen or 4-hydroxytamoxifen and killed 24 h after the last dose. Liver weights and microsomal rates of ethoxyresorufin O-deethylation, 4-dimethylaminopyrine N-demethylation and p-nitrophenol oxidation were not altered by tamoxifen or 4-hydroxytamoxifen treatment. Uterine weights were decreased significantly and uterine peroxidase activity was decreased marginally in treated as compared with control rats. DNA adducts were assayed by 32P-post-labeling in combination with HPLC. Two major DNA adducts were detected in liver DNA from rats administered tamoxifen. These adducts had retention times comparable with those obtained from in vitro reactions of alpha-acetoxytamoxifen and 4-hydroxytamoxifen quinone methide with DNA. Hepatic DNA adduct levels in rats administered 4-hydroxytamoxifen did not differ from those observed in control rats. Likewise, adduct levels in uterus DNA from rats treated with tamoxifen or 4-hydroxytamoxifen were not different from those detected in control rats. These data suggest that a metabolic pathway involving 4-hydroxytamoxifen is not a major pathway in the activation of tamoxifen to a DNA-binding derivative in Sprague-Dawley rats.     

Structural characterization of the major adducts formed by reaction of 4,5-epoxy-4,5-dihydro-1-nitropyrene with DNA.

The only available marker of DNA adducts formed from 1-nitropyrene (1-NP) and DNA, N-(deoxyguanosin-8-yl)-1-aminopyrene, is derived from the nitroreduction pathway. Our studies, as well as those of others, have indicated that multiple DNA adducts are formed from 1-NP in vivo and in vitro. Thus the need for additional DNA adduct markers was apparent. Therefore, it was our goal to characterize the DNA adducts formed from 4,5-epoxy-4,5-dihydro-1-nitropyrene, a metabolite of 1-NP. The epoxide was incubated with calf thymus DNA (pH 5.4). The DNA was enzymatically hydrolyzed to deoxyribonucleosides which were analyzed by reverse phase HPLC. Three major peaks were obtained in yields less than 5%. The structural assignment of these adducts was made by comparison of their proton nuclear magnetic resonance spectra with those of cis- and trans-4,5-dihydro-4.5-dihydroxy-1-nitropyrene, and by long range coupling constants, decoupling experiments, D2O exchange, partitions and acid hydrolysis. Two adducts result from trans and one from cis addition of the N2-exocyclic amino group of deoxyguanosine to the C5-benzylic carbon of the epoxide ring. This is the first report that describes the structure of the DNA adducts formed with a ring-oxidized metabolite of 1-NP. On the basis of this finding we suggest that K-region oxides of 1-NP may be responsible for the formation of the putative 1-NP-DNA adducts in vivo.     

DNA adducts formed by ring-oxidation of the carcinogen 2-naphthylamine with prostaglandin H synthase in vitro and in the dog urothelium in vivo

The presence of relatively high levels of prostaglandin H synthase(PHS) in the dog urinary bladder and its ability to mediatethe activation of carcinogenic arylamines to DNA-bound productsin vitro suggests the involvement of this enzyme in arylamine-inducedbladder carcinogenesis. Since the PHS-dependent metabolism of2-naphthylamine (2-NA) had been shown to yield both ring- andN-oxidation products in vitro, we compared the reactivity of³H-labeled N-hydroxy-2-naphthylamine (N-OH-2-NA), 2-nitrosonaphthalene,and 2-amino-1-naphthol (2-AN) toward DNA and protein. In thePHS-incubation system, all three derivatives bound at high levelsto protein, but only N-OH-2-NA and 2-AN bound appreciably toDNA. Though ring-oxidation has usually been considered a detoxificationpathway, the covalent binding of [³H]2-AN to DNA was found tooccur readily under aerobic conditions and was enhanced at acidicpH. At pH 5 in air, the reactivity of [³H]2-AN with nucleicacids and protein was in the order: serum albumin > tRNA> poly G > poly C > DNA > poly A > rRNA >poly U. Enzymatic hydrolysis of DNA reacted with [³H]2-AN andsubsequent analysis by h.p.l.c. indicated the presence of severalcarcinogen-nucleoside adducts. The major product was characterizedas N⁴-(deoxyguanosin-N²-yl)-2-amino-1,4-naphthoquinoneimine;and two minor products were tentatively identified as N⁴-(deoxyadenosin-N⁶-yl)-2-amino-1,4-naphthoquinoneimineand a deoxyguanosin-N²-yl adduct of a naphthoquinoneimine dimer.These adducts accounted for 60% of the total DNA binding obtainedby incubation of [³H]2-NA with PHS in vitro and for 20% of the[³H]2-NA bound to dog urothelial DNA in vivo. The remainingadducts were identical to those previously reported as productsof the reaction of N-OH-2-NA with DNA. These results suggestthat a minor proportion of the DNA adducts found in vivo maybe formed by PHS-activation of 2-NA in the target tissue. Furthermore,the reactivity of 2-AN with cellular nucleophiles, presumablythrough formation of 2-imino-1-naphthoquinone or a protonated4-naphthocarbenium ion, indicates that ring-oxidation productsof arylamines and of other carcinogenic aryl compounds shouldbe evaluated as proximate carcinogenic metabolites.     

Excretion kinetics of the DNA adducts of cis-diamminedichloroplatinum(II) formed in vitro in rat urine

The main adduct of cis-diamminedichloroplatinum(ll) (cis-Pt)with DNA, cis-[Pt(NH₃)₂(dGpdG)], was administered i.p. to rats.Urine was collected daily for 4 days. The adduct was purifiedby a weak cation exchanger and quantitated by HPLC with UV detection.The recovery of the adduct was the chemical instability of cis-[Pt(NH₃₂(dGpdG)]in urine as shown in an in vitro incubation. Adjusted for thisinstability the recovery in urine was > 70% of the dose.When cis-Pt-DNA (the molar ratio of cis-Pt to nucleotide = 1:50) was administered i.p. to rats only 1.25 ± 0.23% ofplatinum was excreted in urine in the form of cis-[Pt(NH₃)₂(dGpdG)]and cis-[Pt(NH₃)₂(dApdG)] during the first 4 days. If the removalof the cis-Pt-DNA adducts from human tissues is to be followed,their possible slow excretion and chemical instability in urineneeds to be considered.     

DNA adducts formed by the comutagens harman and norharman in various tissues of mice

Covalent modifications of DNA in various tissues of mice with harman or norharman were analyzed by 32P-postlabeling assay. Administration of 0.1% harman to mice in their diet for 4 weeks resulted in DNA adducts in the liver and kidney. No specific DNA adduct was detected in other tissues, such as the glandular stomach, large intestine and brain. Similar treatment of mice with norharman resulted in DNA adducts in the kidney, glandular stomach and large intestine, but not in the liver or brain. These results suggests the in vivo genotoxicities of harman and norharman.     

32P-Postlabelling method for the detection of 7-alkylguanine adducts formed by the reaction of different 1,2-alkyl epoxides with DNA

A ³²P-postlabelling method is reported for the detection of7-alkylguanines, the major adducts formed by the reaction of1,2-alkylepoxides with DNA. Calf thymus DNA was reacted in vitrowith different epoxides (ethylene oxide through octylene oxide)and digested with micrococcal nuclease and spleen phosphodiesteraseto 3'-nucleotides. The adduct enrichment was carried out byan ion-exchange method and adducts were labelled with [     

Characterization and properties of the DNA adducts formed from N-methyl-4-aminoazobenzene in rats during a carcinogenic treatment regimen

Chronic oral administration of the carcinogenic aminoazo dyeN-methyl-4-aminoazobenzene (MAB) to rats is known to resultin the induction of liver tumors. In order to assess the roleof carcinogen-DNA adduct formation in MAB hepatocarcinogenesis,male rats were fed 0.06% [3'-³H]MAB in the diet for 1, 3 or5 weeks. Groups were sacrificed at 0, 24 and 72 h after dosing,and DNA was isolated from the liver and from two non-targettissues, the kidney and spleen. Upon enzymatic hydrolysis ofthe DNA, [³H]aminoazo dye-nucleoside adduct levels in thesetissues were determined by h.p.l.c. Rats concurrently administeredunlabeled MAB for 5 weeks and continued on a control diet for9 months developed hepatocellular carcinomas (16/30 animals).No tumors were observed in 21 rats given only control diets.After chronic administration of [³H]MAB, three major MAB-DNAadducts were found in vivo: N-(deoxyguanosin-8-yl)-MAB (C8-dG-MAB),3-(deoxyguanosin-N²-yl)-MAB (N²-dG-MAB) and 3-(deoxyadenosin-N⁶-yl)-MAB(N⁶-dA-MAB). In addition, several minor products were identifiedas: (i) an (8,9)-purine ring-opened derivative of C8-dG-MABthat may represent an intermediate in DNA repair; (ii) N-guanosin-8-yl-MABwhich is present due to trace RNA contamination; (iii) cis isomersof C8-dG-MAB and N-guanosin-8-yl-MAB, formed by photo-illuminationduring analyses; and (iv) N-(guanin-8-yl)-MAB, a deribosylatedproduct resulting from thermal depurination of C8-dG-MAB. Inaddition, N-(deoxyguanosin-8-yl)-4-aminoazobenzene (C8-dG-AB),a major adduct previously detected in mouse liver after a singledose of 4-aminoazobenzene, was found in rat liver but appearedto be present in significant amounts only after chronic treatmentwith MAB. This product co-chromatographed with N⁶-dA-MAB butcould be removed by selective decomposition in 0.1 N NaOH. Forall tissues examined N²-dG-MAB and C8-dG-MAB were the majoradducts observed with each accounting for 40-50% of the totalcarcinogen bound to DNA in rats that were sacrificed immediatelyafter MAB feeding for 1, 3 or 5 weeks. The levels of total MAB-DNAadducts in the liver were 2–10 times greater than in thekidney or spleen and appeared to increase 2- to 3-fold overthe dosing period. However, by 24–72 h after cessationof MAB treatment, hepatic C8-dG-MAB showed a rapid decline tolevels similar to that found in non-target tissues. The minoradducts, N⁶-dA-MAB and C8-dG-AB, exhibited similar behaviorand never accounted for > 5–10% of the total DNA binding.In contrast, hepatic N²-dG-MAB was a persistent lesion throughoutthe treatment regimen; at 72 h after dosing, it accounted for60–90% of the hepatic DNA adducts and was the only adductwhose levels correlated with target tissue specificity aftera complete hepatocarcinogenic dose of MAB.