Binding of dimethylaminoazobenzene metabolites to DNA and proteins. I. In vitor studies on a microsomal dependent system

Incubating dimethylaminoazobenzene [aniline ring (U)¹⁴ C] (DAB), purified thymus DNA and rat liver microsomes led to a covalent binding of radioactivity to DNA and proteins, which implied the presence of bound DAB metabolites. This binding required an enzymatic activation. We studied some characteristics of the enzymatic process, i.e. NADPH requirement, effect of pH, effect of various concentrations of microsomes and DNA, induction by 3-methylcholanthrene. This in vitro system is discussed in view of its validity for studying interactions between the carcinogenic azodyes and the cellular constituents.     

Binding of metabolites of dietary 4-dimethyl-aminoazobenzene and 2-methyl-4-dimethylamino-azobenzene to rat liver DNA and protein of subcellular fractions

The binding of metabolites of two related azo dyes of different carcinogenic potency, the carcinogenic 4-dimethylaminoazobenzene (DAB) and the weakly carcinogenic 2-methyl-4-dimethylaminoazobenzene (2-Me-DAB), to rat liver DNA and to subcellular fraction protein was studied following chronic oral administration for 1 to 3 weeks. Different techniques for measuring the amount of DAB metabolites bound to protein were first compared, then the whole study was performed with labelled DAB and 2-Me-DAB (aniline ring-¹⁴C) of moderate specific activity. DAB metabolites were bound to liver DNA to a higher extent than those of 2-Me-DAB. In contrast, the binding of 2-Me-DAB metabolites was equal to or higher than that of DAB metabolites to protein. The amount of protein-bound metabolites was studied on the nucleo-mito-chondrial fraction, microsomes, supernatant, nuclei, chromatin, nucleoplasm, nucleolar fraction and nuclear membrane. Following the administration of both dye diets, the supernatant protein bound the highest level of metabolites. The time-course of binding of DAB metabolites to DNA and protein was different from that of 2-Me-DAB metabolites. These results show the possible involvement of carcinogen-DNA binding in the mechanism of carcinogenesis.     

Cytotoxicity,DNA cross-linking,and DNA single-strand breaks induced by cyclophosphamide in a rat leukemia in vivo

Summary A study of cyclophosphamide (CP)-induced DNA damage and repair occurring in vivo was conducted in the brown Norway rat myelocytic leukemia (BNML) model. DNA single-strand breaks (SSB), DNA-DNA interstrand cross-links (DIC), DNA-protein cross-links (DPC), and DNA double-strand breaks (DSB) were measured by alkaline and neutral elution. After i. p. injection of 50 mg/kg CP, DIC were detectable at 1 h and peaked at 8 h. DPC were detectable at 2 h and peaked at 6 h. Both DIC and DPC persisted at a relatively high level until 28 h. Dose-response curves for both DIC and DPC were determined at 4 h after CP injection over the dose range of 25–150 mg/kg. These doses ranged from the minimally effective dose to doses curative for rats bearing this leukemia (1- to 9-log kill of leukemia cells). No SSB or DSB was observed at 4 h after CP injection over the dose range of 15–250 mg/kg, but a low level of SSB was observed at 18–28 h after CP treatment. These data suggest that the cytotoxic effect of CP in vivo is mediated mostly by DIC and DPC. SSB appearing late after CP injection in vivo may be a reflection of repair of DIC and DPC and an indication of the optimal timing for administration of DNA-repair inhibitors. This observation is of interest since our earlier work demonstrated that hydroxyurea can potentiate the therapeutic benefit of CP in this model when it is given over the 4-day period immediately after CP treatment.Supported by NIH grant RO1 CA455329     

Metabolism of 7,8-dihydrobenzo(a)pyrene by rat liver microsomal enzymes and mutagenicity of metabolites

7,8-Dihydrobenzo[a]pyrene (7,8-H2BaP) was metabolized by rat liver microsomes to form 7,8,9,10-tetrahydro-BaP trans-9,10-diol, 7,8,9,10-tetrahydro-BaP cis-9,10-diol, 7-hydroxy-7,8-H2BaP, 8-hydroxy-7,8-H2BaP, two phenolic products of 7,8-H2BaP [abbreviated as 7,8-H2BaP phenol 1 and phenol 2 according to their elution order on reversed-phase high-performance liquid chromatography (HPLC)], 4,5,7,8-tetrahydro-BaP trans-4,5-diol, BaP cis-7,8-dihydrodiol, BaP, and the metabolites known to be formed from the metabolism of BaP. Metabolites were isolated by reversed-phase and normal-phase HPLC and identified by ultraviolet-visible absorption and mass spectral analyses and by comparing their retention times with synthetic standards whenever available. The enantiomeric compositions of some mono-ol and diol metabolites were determined by chiral stationary phase HPLC. The optical purities of monool and diol metabolites formed were found to be dependent on the nature of cytochrome P-450 isozymes present in liver microsomes. Metabolites formed by liver microsomes from untreated, phenobarbital-treated, 3-methylcholanthrene-treated, and polychlorinated biphenyls (Aroclor 1254)-treated male Sprague-Dawley rats were quantified by using specifically tritium-labeled [10-3H]-7,8-H2BaP and liquid scintillation counting of fractions collected from reversed-phase HPLC. A portion (2-7% depending on the type of microsomes used) of the BaP found was formed nonenzymatically in microsomal metabolism of 7,8-H2BaP. The formations of other major metabolites were all cytochrome P-450 isozymes dependent since their formations were inhibited by carbon monoxide and were dependent on the presence of reduced nicotinamide adenine dinucleotide phosphate. Furthermore, the formations of tetrahydrodiols, monools, and phenols were not inhibited by the epoxide hydrolase inhibitor, 3,3,3-trichloropropylene 1,2-oxide. The relative mutagenic activities toward Salmonella typhimurium TA98 at 2 nmol of chemical per plate and 10 microliters of liver S9 fraction were: (+/-)BaP trans-7,8-dihydrodiol approximately equal to 7,8-H2BaP approximately equal to 7,8-H2BaP phenol 2 greater than (+/-)Bap cis-7,8-dihydrodiol greater than BaP approximately equal to 8-hydroxy-7,8-H2BaP greater than 7,8-H2BaP phenol 1 greater than 7-hydroxy-7,8-H2BaP. The results suggest that, in addition to the bay region 7,8,9,10-tetrahydro-BaP 9,10-epoxide, metabolic products formed by hydroxylations at the aliphatic and aromatic carbons of 7,8-H2BaP and their subsequent metabolism at the 9,10-double bond may also contribute to the carcinogenic activities of 7,8-H2BaP.     

Stereoselectivity of rat liver microsomal enzymes in the metabolism of 7-fluorobenz(a)anthracene and mutagenicity of metabolites

7-Fluorobenz(a)anthracene (7-FBA) was metabolized by rat liver microsomes predominantly to 4-hydroxy-7-FBA and 7-FBA trans-3,4-, 5,6-, 8,9-, and 10,11-dihydrodiols. Proton nuclear magnetic resonance spectral analyses indicated that the fluoro substituent causes 7-FBA trans-5,6- and 8,9-dihydrodiols to adopt preferentially quasidiaxial conformations (Chiu, P.-L., Fu, P. P., and Yang, S. K. Biochem. Biophys. Res. Commun., 106: 1405-1411, 1982). The major enantiomers of the quasidiaxial trans-5,6- and trans-8,9-dihydrodiols have been determined by the exciton chirality method to have R,R absolute stereochemistries. By comparing with the circular dichroism spectra of BA 3R,4R- and 10R,11R-dihydrodiols, the major enantiomers of the quasidiequatorial 7-FBA trans-3,4- and trans-10,11-dihydrodiols were also found to have R,R absolute configurations. All four 7-FBA trans-dihydrodiol metabolites obtained from incubations of 7-FBA with liver microsomes prepared from untreated and 3-methylcholanthrene-, phenobarbital-, and polychlorinated biphenyl-treated male Sprague-Dawley rats were enriched in R,R enantiomers, differing only in optical purities. Pretreatment of rats with phenobarbital, 3-methylcholanthrene, and polychlorinated biphenyls changed the rate of 7-FBA metabolism by 0.47-, 1.14-, and 1.70-fold, respectively. Pretreatment of rats with enzyme inducers also altered the quantitative distribution of metabolites formed. The relative mutagenic activities of metabolites toward Salmonella typhimurium TA 100 were: 7-FBA trans-3,4-dihydrodiol greater than 7-FBA trans-10,11-dihydrodiol greater than 7-methyl-BA approximately equal to 7-FBA greater than 7-FBA trans-8,9-dihydrodiol approximately equal to 7-methyl-BA trans-10,11-dihydrodiol greater than 7-FBA trans-5,6-dihydrodiol approximately equal to 4-hydroxy-7-FBA. The relatively high mutagenic activities of 7-FBA trans-3,4- and trans-10,11-dihydrodiols suggest that both 7-FBA trans-3,4-dihydrodiol 1,2-epoxide(s) and 7-FBA trans-10,11-dihydrodiol 8,9-epoxide(s) may be the major metabolites which contribute to the carcinogenic properties of 7-FBA.     

Binding of N-acetylbenzidine and N,N''-diacetylbenzidine to hepatic DNA of rat and hamster in vivo and in vitro

Benzidine, a potent hepatocarcinogen in rodents, is readilymetabolised to acetylated derivatives. In this study, the covalentbinding of [³H-acetyl]N-acetylbenzidine and [³H-acetyl]N,N'-diacetylbenzidineto liver DNA in rats and hamsters was investigated. Bindingto liver DNA of rats at 1 or 7 days after i.p. injection ofN-acetylbenzidine was 2-fold higher than that observed in theliver DNA of hamsters which had been similarly treated. Analysisof enzymically hydrolysed DNA from both species indicated thepresence of a single adduct which co-eluted with N-(deoxyguanosin-8-yl)-N'-acetylbenzidine. In vitro treatment of rat or hamster liverslices with N-acetylbenzidine also resulted in covalent bindingto hepatic DNA and the identical DNA adduct was detected atlevels comparable to that observed in vivo. When N,N'-diacetylbenzidinewas injected i.p. into rats, a comparatively low level of bindingto liver DNA was observed. Following enzymic hydrolysis, themajor DNA adduct detected by h.p.l.c. analysis was again N-(deoxyguanosin-8-yl)-N'-acetylbenzidineaccompanied by a small amount of N-(deoxyguanosin-8-yl)-N,N'-diacetylbenzidine.In vitro incubation of N,N'-diacetylbenzidine with rat liverslices resulted in DNA binding levels similar to that observedwith N-acetylbenzidine. In contrast to what was found in vivo,N-(deoxyguanosin-8-yl)-N,N'-diacetylbenzidine was the majoradduct detected in DNA from rat liver slices. These data suggestthat both N-hydroxy-N'-acetylbenzidine and N-hydroxy-N,N'-diacetylbenzidineare proximate carcinogenic species of benzidine, with N-hydroxy-N'-acetylbenzidinethe more important. The low level of N-(deoxyguanosin-8-yl)N,N'-diacetylbenzidineobserved in vivo may be due to its rapid repair. Alternatively,N-sulphonyloxy-N,N'-diacetylbenzidine, which would produce thisadduct on reaction with DNA, may be efficiently detoxified invivo.     

DNA adduct formation by tamoxifen with rat and human liver microsomal activation systems

Using microsomal preparations from rat and human liver, we investigatedthe activation of the anti-estrogen compound tamoxifen (TMX)to form DNA adducts. Pretreatment of rats with phenobarbitalincreased DNA adduct formation by microsomal activation of TMX3- to 6-fold, depending on the cofactors used. When reducednicotinamide-adenine dinucleotide phosphate (NADPH) was usedas a cofactor in human and rat microsomal activation systems,the relative DNA adduct levels were 2.9 and 5.2 x 10^–8respectively and 1-3 TMX-DNA adducts were detected by ²³P-postlabeling;DNA adduct 1 was the same in both microsomal systems. When cumenehydroperoxide (CuOOH) was used as a cofactor, activation ofTMX produced four major DNA adducts and several minor DNA adductsin both rat and human liver microsomes; the relative adductlevels were 11.1 and 23.1 xlO^–8 respectively. TMX-DNAadducts 1, 4, 5 and 6 were similar in both human and rat microsomalsystems with CuOOH as the cofactor. The TMX-DNA adducts formedwith NADPH as the cofactor were clearly different from thoseformed with CuOOH as the cofactor, which implies that the metabolitesleading to the individual DNA adducts were different. Additionof a P450 inhibitor, either n-octylamine or     

In vivo binding of N-nitrosopyrrolidine and N-nitrosohexamethyleneimine to non-purine sites on rat liver DNA

Liver DNA and RNA were isolated from rats treated with the liver carcinogens N-nitrosopyrrolidine (NPYR) and N-nitrosohexamethyleneimine (NHX). After hydrolysis in 70% perchloric acid (100°C, 1.0 h), 70% of the radioactivity in both the DNA and RNA hydrolysates chromatographed as a single peak. The material from both hydrolysates had comparable R_f values on cation exchange and Sephadex G-10 chromatography. Subsequent experiments indicated this material was volatile.After depurination (0.1 M HCl) of the DNA from NPYR- and NHX-treated rats, Sephadex G-10 chromatography separated only a single radio-active peak which co-eluted with the apurinic acid at the void volume. The material which comprised this peak was not volatile. After dialysis of the same 0.1 M HCl hydrolysate from NHX-treated rats, 98% of the radio-activity remained attached to the apurinic acid. These 2 cyclic nitrosamines appear to produce alkylating species that: (10 are capable of extensive, if not exclusive, phosphotriester formation; or (2) have 2 active sites that cross-link to keep purines attached to apurinic acid after 0.1 M HCl hydrolysis.     

Mutagenicity of cyclopenta-fused isomers of benz(a)anthracene in bacterial and rodent cells and identification of the major rat liver microsomal metabolites

The microsomal metabolites and mutagenic activity of four cyclopenta-fused benz(a)anthracenes, benz(j)aceanthrylene [B(j)A], benz(e)aceanthrylene [B(e)A], benz(l)aceanthrylene [B(l)A], and benz(k)acephenanthrylene [B(k)A], have been studied. Aroclor 1254-induced rat liver microsomes metabolized B(j)A to B(j)A-1,2-dihydrodiol, B(j)A-9,10-dihydrodiol, B(j)A-11,12-dihydrodiol, and 10-hydroxy-B(j)A; B(e)A-1,2-dihydrodiol, B(e)A-3,4-dihydrodiol, and B(e)A-5,6-dihydrodiol; B(l)A to B(l)A-1,2-dihydrodiol, B(l)A-4,5-dihydrodiol, and B(l)A-7,8-dihydrodiol; and B(k)A to B(k)A-4,5-dihydrodiol and B(k)A-8,9-dihydrodiol. With each polycyclic aromatic hydrocarbon, metabolism occurred on the cyclopenta ring. All four isomers were active as gene mutagens in Salmonella typhimurium and in Chinese hamster V79 cells. In the S. typhimurium mutation studies, using Aroclor 1254-induced rat liver S9, B(j)A, B(e)A, and B(l)A required significantly less microsomal protein for maximal mutation response than B(k)A and B(a)P, suggesting a one-step activation mechanism, presumably on the cyclopenta-fused ring. B(j)A, B(e)A, and B(l)A were significantly more mutagenic than B(k)A and B(a)P in S. typhimurium. In the Aroclor 1254-induced rat liver S9-mediated V79 mutagenesis system, all four isomers were active, with B(l)A the most active. When Syrian hamster embryo cells were used as the metabolic activation component for V79 cells, only B(l)A produced a significant response and was equivalent in activity to B(a)P. A helical configuration for B(l)A is inferred from the identification of two trans-B(l)A-1,2-dihydrodiols, syn and anti, which have been synthesized, separated, and characterized. The metabolically formed dihydrodiol is anti-trans-B(l)A-1,2-dihydrodiol, and experimental evidence suggests that the metabolically formed B(l)A-1,2-oxide is the anti-isomer. Synthetic B(l)A-1,2-oxide was found to be a direct-acting mutagen in S. typhimurium and Chinese hamster V79 cells and is estimated to account for up to 40% of the mutagenic activity of the parent hydrocarbon. Therefore, certain cyclopenta-ring fusions on benz(a)anthracene appear to markedly increase its genotoxic and carcinogenic activities.     

Nafenopin-induced rat liver peroxisome proliferation reduces DNA methylation by N-nitrosodimethylamine in vivo

The hypolipidaemic drug nafenopin (NAF) has been shown to enhancethe hepatocarcinogenic effect of N-nitrosodimethylamine (NDMA)and N-nitrosodiethylamine in rats. We have investigated whetherthe NAF-induced peroxisome proliferation in hepatocytes interfereswith NDMA's metabolism and interaction with DNA. Adult maleWistar rats received a single i.p. injection of [¹⁴C]NDMA (2mg/kg) and were killed 4 h later. DNA was isolated from liverand kidney, hydrolysed in 0.1 N HCI and analysed by Sephasorbchromatography. In rats pre-treated with NAF (0.2% in the dietover a period of 3 weeks), the concentration of N7-methylguaninein hepatic DNA (µmol/mol guanine) was 46% below controlvalues. This is probably due to the greater amount of targetDNA, as NAF caused a marked hepatomegaly with a 50% increasein total liver DNA content. Concentrations of N7-methylguaninein kidney DNA were twice as high in NAF-pre-treated animalswhen compared to control rats. This is unlikely to result froma shift in the metabolism of NDMA from liver to other rat tissuessince the time course and extent of the conversion of [¹⁴C]NDMAto ¹⁴CO₂ and ¹⁴C-labelled urinary metabolites were identicalin NAF-treated and control animals. There was no indicationthat NAF inhibits the activity of the hepatic O⁶-alkylguanine-DNAalkyltransferase.     

Dose-dependent persistence of alkylation-induced single stranded regions in rat liver DNA in vivo

Single stranded regions in DNA, presumed to be indicative of DNA repair, may be readily detected in rat liver DNA following injections of nonnecrotizing single doses of methyl methanesulfonate, dimethylnitrosamine and diethylnitrosamine. The present study concerns persistence of such structural defects in vivo, as determined by benzoylated DEAE-cellulose chromatography, in relation to the dose of alkylating agent. For all these agents, the period during which structural damage may be detected is markedly dependent upon dose: prolonged persistence only occurs after the highest dose. The findings, in relation to other data, implicate reaction processes involving the final stages of repair as being critical to prolongation of this type of genomic damage in vivo.     

A cyclophosphamide/DNA phosphoester adduct formed in vitro and in vivo

The antitumor activity of cyclophosphamide is thought to be due to the alkylating activity of phosphoramide mustard, a metabolite of cyclophosphamide. Reaction of 2'-deoxyguanosine 3'-monophosphate and phosphoramide mustard resulted in the formation of several adducts that could be detected by high performance liquid chromatography (HPLC). One of these adducts, isolated and purified by HPLC, could be detected by 32P postlabeling. This product was identified by UV, nuclear magnetic resonance, and mass spectrometry and by acid, base, and enzymatic hydrolysis to be 2'-deoxyguanosine 3'-monophosphate 2-(2-hydroxyethyl)aminoethyl ester. A combination of HPLC fractionation of digested DNA and 32P postlabeling was used to detect this adduct in calf thymus DNA incubated in vitro with metabolically activated cyclophosphamide and in DNA from the liver of mice treated with cyclophosphamide. In these DNA samples the adduct occurred at a level of 1/10(5) and 1/3 x 10(7) nucleotides, respectively.     

Rat liver microsome-mediated binding of benzo(a)pyrene metabolites to DNA.

Individual metabolites of benzo (a) pyrene were isolated from rat liver microsomal incubation of the parent hydrocarbon, and subsequently bound to DNA in separate incubations with microsomes. Of the metabolites examined, by far the greatest binding resulted with 7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene (BP-7,8-diol); the binding od the benzo (a) pyrene phenols (BP-OH) was about 50% that of the 7,8-diol. Analysis of the hydrocarbon-deoxyribonucleoside derivatives obtained by enzymic degradation of these DNA samples revealed that the binding of BP-7,8-diol was accounted for mainly by a single product identical to one of the product identical to one of the products obtained from DNA with bound benzo(a)pyrene. Furthermore, the microsome-induced binding of BP-OH to DNA yielded mainly a single product identical in chromatographic behaviour to the major product derived from benzo(a)pyrene when bound to DNA during incubation with microsomes.     

Effect of butylated hydroxyanisole on the metabolism of benzo[a]-pyrene and the binding of metabolites to DNA, in vitro and in vivo, in the forestomach, lung, and liver of mice

The effects of butyldted hydroxyanisole (BHA) administrationon the amounts of benzo[a]pyrene (BP) metabolite-DNA adductsformed in vivo in the forestomach of A/HeJ mice were investigated48 h after oral administration of BP. BP was administered tomice in amounts known to result in BPInduced neoplasia in certaintissues. Analysis of deoxyribonudeosides by h.p.l.c. showedthat several BP metabolite-DNA adducts were formed in this tissue.The major identified adduct was the (±)-7ß,8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDEI) deoxyguanosine adduct. Addition of BHA to the diet inhibitedBPDE I-DNA adduct formation in the forestomach. The inhibitionof BPDE I-DNA adduct formation by BHA occurred under the sameexperimental conditions as does inhibition of tumor formationby this compound. These results in forestomach and previousresults in lung and forestomach demonstrated that inhibitionof the formation of the BPDE I-DNA adduct in the target tissueis a possible mechanism by which BHA inhibits BP-induced neoplasia.BP metabolism and DNA binding were also studied under in vitroconditions using microsomes prepared from forestomach, lung,and liver of A/HeJ mice. The amount of BPDE-DNA adduct formedin vitro is either equal to or lower than the amount of BP phenol-oxide-DNAadduct formed. BPDE I-DNA adduct formation was not significantlydifferent in incubations containing microsomes prepared fromBHA-treated or untreated mice. These results suggest that alterationsof the microsomal monooxygenases induced by BHA feeding arenot sufficient to account for the observed decreases in BPDE-DNAadduct formation in vivo. The monooxygenases were apparentlyaltered by BHA feeding as indicated by the substantial changesin the metabolic profile of BP and the decrease in the formationof the BP phenol-oxide-DNA adducts in the forestomach. The exclusionof glutathione transferases from the in vitro incubations couldaccount for the lack of effect of BHA treatment on BPDE-DNAadduct formation. BHA enhancement of ghitathione transferaseactivity has been postulated to play a role in the anticarcinogenicaction of BHA.