Adduct Profiles Research Articles

Tumor multiplicity, DNA adducts and K-ras mutation pattern of 5-methylchrysene in strain A/J mouse lung.

This study was undertaken to evaluate the carcinogenic potential of 5-methylchrysene (5-MeC) in strain A/J mouse lung and to correlate the 5-MeC-DNA adduct profile in lung tissue with the mutation spectrum in the K-ras gene of lung tumors. Strain A/J mice received a single i.p. injection of 5-MeC at doses of 10, 50, 100 and 200 mg/kg and after 24, 48 and 72 h their lungs were collected for DNA adduct analysis. Eight months later, lungs from the remaining mice were harvested and the lung tumors counted and collected for subsequent mutational analysis of the K-ras gene. 5-MeC was found to be a potent lung carcinogen in strain A/J mice, inducing more than 100 tumors/mouse at a concentration of 200 mg/kg. Six 5-MeC-DNA adducts were observed; one adduct comigrated with the standard N2-deoxyguanosine adduct of 5-MeC-diol-epoxide I [1R,2S,3S-trihydroxy-4R-(N2-deoxy-guanosyl-3'-phosphate)- 1,2,3,4-tetrahydro-5-methyl-chrysene], derived from the bay-region diol-epoxide of 5-MeC. DNAs isolated from 5-MeC-induced lung tumors were evaluated for activating mutations in the K-ras gene by polymerase chain reaction-single strand conformation polymorphism and direct DNA sequencing analysis. Mutations were detected in 44 of 49 (90%) 5-MeC-induced tumors and the mutations were GGT-->TGT (50%), GGT-->GTT (23%) and GGT-->CGT (27%) in codon 12 of the gene. These results suggest that the N2-deoxyguanosine adduct of 5-MeC-diol-epoxide I may be one of the promutagenic adducts of 5-MeC in strain A/J mouse lung.

Differential recognition of stereochemically defined base adducts by antibodies against anti-benzo[a]pyrene diol-epoxide-modified DNA.

The configurational isomers of benzo[a]pyrene diol-epoxide exhibit a range of reactivity, adduct profiles, genotoxic, mutagenic and tumorigenic responses. Whilst the (+)-enantiomer of 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) is the most potent genotoxic species, studies dealing with many critical aspects of BPDE genotoxicity have predominantly been done with the racemic mixture of anti-BPDE. By utilizing highly sensitive non-competitive immunoassays, we have shown that both polyclonal and monoclonal antibodies developed against anti-BPDE-modified DNA exhibit a high degree of stereospecific adduct selectivity with 11mer oligodeoxynucleotides containing a single well-defined base adduct. The polyclonal antibody (PAb BP1) distinctly recognized the highly carcinogenic lesion (+)-anti-BPDE-N2-dG with a 40-fold preference over the (-)-anti-BPDE-N2-dG adduct. In contrast, the monoclonal antibody (MAb 5D2) bound avidly to (-)-anti-BPDE-N2-dG and exhibited very little affinity for the (+)-anti-BPDE-N2-dG adduct. The overall sensitivity of detection of polyclonal antibodies for adducts in (+/-)-anti-BPDE-modified DNA was about 90-fold higher than monoclonal antibodies. Neither antibody showed any detectable reactivity with (+)-or (-)-anti-BPDE-N6-dA and with unmodified DNA antigens. The distinct preference of antibodies for particular enantiomeric adducts was observed in both single and duplexed oligomeric conformations. The demonstrated differential interaction of antibodies with the established conformations of (+)- and (-)-enantiomer anti-BPDE-DNA adducts (de los Santos et al., Biochemistry, 31, 5245-5252, 1992), has significant implications for in vitro and in vivo adduct processing and risk assessment biomonitoring studies.

Metabolic activation of N-hydroxy-4-acetylaminobiphenyl by cultured human breast epithelial cell line MCF 10A.

Metabolism and nucleic acid binding of the mammary gland carcinogen N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) was investigated using the human mammary epithelial cell line MCF 10A. Chromatographic analysis of the ethyl acetate extract of the media from cultured MCF 10A after 24 h exposure to N-OH-AABP revealed the formation of two metabolites, 4-aminobiphenyl (ABP) and 4-acetylaminobiphenyl (AABP). Incubation of [3H]N-OH-AABP with calf thymus DNA in the presence of the cytosols or microsomes revealed a binding of 0.21 and 2.36 nmol/mg DNA/mg protein respectively. In contrast to cytosol-mediated binding, the microsome-mediated binding of [3H]N-OH-AABP to DNA was inhibited by paraoxon. Furthermore, exogenous addition of non-labelled N-hydroxy-4-aminobiphenyl (N-OH-ABP) to the incubation mixture blocked the binding of [3H]N-OH-AABP to DNA, suggesting that the metabolic activation process involves inter-molecular transacetylation. Cytosols from MCF 10A also catalyzed acetyl coenzyme A (AcCoA)-dependent binding of [3H]N-OH-ABP to DNA; the amount of binding was 0.51 nmol/mg DNA/mg protein. HPLC of the DNA hydrolysate obtained after incubation of [3H]N-OH-AABP and [3H]N-OH-ABP with the MCF 10A microsomes and cytosols showed N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-ABP) as the primary adduct, based on the mobility of the radioactive peak in comparison with the synthetic standard. 32P-postlabeling of adducted DNA obtained on incubation with N-OH-ABP or N-OH-AABP showed similar adduct profiles, with the major adduct corresponding with the bisphospho derivative of dG-ABP and a minor adduct corresponding with N-(deoxyadenosin-8-yl)-4-aminobiphenyl (dA-ABP). Additionally, the cellular DNA isolated from MCF 10A following exposure to N-OH-AABP also revealed a major spot corresponding with the dG-ABP derivative. These results suggest that the mammary gland carcinogen N-OH-AABP is activated to reactive electrophilic species in the target human mammary tissues by acetyl transferase(s) enzyme systems.

Identification and quantitation of benzo[a]pyrene-DNA adducts formed in mouse skin.

The DNA adducts of benzo[a]pyrene (BP) formed in vitro were previously identified and quantitated. In this paper, we report the identification and quantitation of the depurination adducts of BP, 8-(benzo[a]pyren-6-yl)guanine (BP-6-C8Gua), BP-6-N7Gua, and BP-6-N7Ade, formed in mouse skin by one-electron oxidation, as well as the major stable adduct formed via the diolepoxide pathway, BP diolepoxide bound at C-10 to the 2-amino of dG (BPDE-10-N2dG). Identification of the depurination adducts was achieved by HPLC and fluorescence line narrowing spectroscopy. The depurination adducts, BP-6-C8Gua (34%), BP-6-N7Gua (10%), and BP-6-N7Ade (30%), constituted 74% of the adducts found in mouse skin 4 h after treatment with BP. The stable adduct BPDE-10-N2dG accounted for 22% of the adducts. Treatment of the skin with BP-7,8-dihydrodiol or BP diolepoxide yielded almost exclusively the stable adduct BPDE-10-N2dG. When BP or BP-7,8-dihydrodiol was bound to RNA or denatured DNA in reactions catalyzed by rat liver microsomes, no depurination adducts were detected. The profiles of stable adducts were similar both qualitatively and quantitatively with native or denatured DNA. With activation of BP by horseradish peroxidase, the profiles of stable adducts differed with native and denatured DNA. The total amount of adducts with denatured DNA was only 25% of the amount detected with native DNA. No depurination adducts were detected with denatured DNA or RNA in the peroxidase system.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitation of 2‐amino‐3‐methylimidazo[4,5‐f]quinoline and 2‐amino‐3,8‐dimethylimidazo[4,5‐f]quinoxaline DNA adducts in specific sequences using alkali or uvrABC excinuclease

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MelQx) are carcinogens found in cooked meats that form DNA adducts upon metabolic activation. Purified DNA from Chinese hamster ovary (CHO) cells was reacted in vitro with the active metabolites N-acetoxy-IQ or N-acetoxy-MelQx, and the adduct levels in the 5' dihydrofolate reductase (DHFR) gene and downstream region were quantitated by Southern hybridization. Adducted and restricted DNA was treated with Escherichia coli uvrABC excinuclease or alkali (0.1 N NaOH, 37 degrees C, 60 min) to incise DNA at IQ and MelQx adduct sites. The DNA was then denatured with formamide, electrophoresed on a neutral agarose gel, transferred to a support membrane, and hybridized with sequence-specific DNA probes. Both uvrABC and alkali reduced the intensity of Southern hybridization in proportion to the number of IQ or MelQx adducts in DNA, indicating that these adducts are substrates for uvrABC and that they form alkali-labile lesions in DNA. IQ and MelQx adduct levels were the same in the 5' DHFR gene and in the downstream region. Southern hybridization analysis of pBR322 containing known levels of IQ or MelQx adducts showed that the efficiency of cutting IQ or MelQx adducts by uvrABC excinuclease and alkali was approximately 30% and 15%, respectively. 32P-postlabeling studies examining adduct level in bulk DNA further showed that the adduct profiles were identical in pBR322, CHO DNA, and cultured CHO cells exposed to the reactive metabolites of IQ or MelQx. The results indicate that IQ and MelQx adducts can be quantitated in specific genomic sequences and that this method should be directly applicable to studies of gene-specific repair of these adducts in cultured cells.

Molecular analysis of DNA adducts and hprt mutations produced by 6-nitrosochrysene in Chinese hamster ovary cells.

We have characterized the mutational spectrum of 6-nitrosochrysene in the hprt gene of Chinese hamster ovary (CHO-K1) cells and also examined the adducts formed by this compound in CHO-K1 cells by quantitative 32P-postlabeling analysis. Seventy percent of the identified mutations were simple basepair substitutions, and they occurred more often at A:T (14/17) than at G:C. Furthermore, 13 of the basepair substitutions at A:T had the mutated dA, the probable adducted residue, on the non-transcribed DNA strand. The preference for mutation at A:T contrasted sharply with the distribution of adducts formed by 6-nitrosochrysene: 80% of the identified adducts were with dG, while only 20% were probably formed through binding with dA. Analyses conducted with excision-repair-defective CHO-UV5 cells revealed both a preference for basepair substitution at A:T and an adduct profile that were similar to those found for repair-proficient CHO-K1 cells. However, basepair substitutions from CHO-UV5 cell mutants had the mutated dAs distributed randomly between the non-transcribed and transcribed DNA strands. The mutational spectra found for solvent control CHO-K1 and CHO-UV5 cells differed from those of the 6-nitrosochrysene-treated cultures. These findings suggest that 6-nitrosochrysene-induced mutations are targeted to DNA damage, but that 6-nitrosochrysene-derived dA adducts are much more effective at producing mutations than 6-nitrosochrysene-derived dG adducts. The extreme strand bias for mutated dAs in the CHO-K1 mutational spectrum appears to result from preferential removal of 6-nitrosochrysene-induced DNA lesions from the transcribed DNA strand.

Comparative 32P-postlabeling analysis of benzo[a]pyrene--DNA adducts formed in vitro upon activation of benzo[a]pyrene by human, rabbit and rodent liver microsomes.

An interspecies comparison was made of the DNA-adducts formed in vitro upon incubation of rat liver DNA (RL-DNA) with benzo[a]pyrene (BP) in the presence of liver microsomes. Incubations were carried out with RL-DNA, BP (100 microM) and liver microsomes from hamsters, mice, rabbits, rats, 3-methylcholanthrene (3MC) pretreated rats and from humans. To analyse the adduct profiles, the 32P-postlabeling technique with the nuclease P1-enhancement procedure was used. The total amount of adduct formed varied greatly with the species; also the number of adduct spots detected was different, ranging from 1 to 5. In all incubations the BP-N2-deoxyguanosine adduct was formed. Relative to the total adduct level, the level of this adduct varied from 26% with rat, 54% with hamster, 56% with 3MC-pretreated rat, 58% with mouse and 75% with rabbit, to 100% with human liver microsomes. In human liver microsomes both the total amount of cytochrome P-450 per mg microsomal protein and the ethoxyresorufin O-deethylation (EROD) activity were low compared to that in animal liver microsomes. In microsomes from 3MC-pretreated rats the EROD activity was strongly induced. There was no correlation between EROD activity in non-induced microsomes and total adduct level. To compare BP-DNA adduct formation in human white blood cells (WBC) with that in RL-DNA, WBC were incubated with BP and 3MC-pretreated rat microsomes. The adduct profile in WBC-DNA differed from that observed after incubation of RL-DNA: the BP-N2-deoxyguanosine adduct in WBC-DNA accounted for 97% of the total adduct level. It is concluded that the 32P-postlabeling method is a suitable technique to investigate both qualitative and quantitative differences in BP-DNA adduct formation between species. Furthermore, the incubation of microsomes from the liver (or other sources) with a genotoxic agent and isolated DNA or cells can be a useful approach to study the formation and stability of reactive intermediates that are able to bind to DNA, also with respect to differences between species or tissue.

32P-postlabeling analysis of IQ, MeIQx and PhIP adducts formed in vitro in DNA and polynucleotides and found in vivo in hepatic DNA from IQ-, MeIQx- and PhIP-treated monkeys.

The 32P-postlabeling method was used to examine the adducts in DNA, polynucleotides, and mononucleotides reacted in vitro with the N-hydroxy and N-acetoxy derivatives of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3, 8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) or 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Adduct profiles were compared to those found in vivo in liver of cynomolgus monkeys fed IQ, MeIQx or PhIP. The N-acetoxy derivatives of IQ, MeIQx and PhIP (generated in situ from the corresponding N-hydroxylamine in the presence of acetic anhydride) each formed three principal adducts in DNA. Adduct 1 of IQ, MeIQx and PhIP was chromatographically identical to the 32P-labeled bis(phosphate) derivative of N-(deoxyguanosin-8-yl)-IQ, N-(deoxyguanosin-8-yl)-MeIQx, and N-(deoxyguanosin-8-yl)-PhIP respectively, and this adduct comprised approximately 65% of total adduct levels found in DNA in vitro. The C8-guanine adduct and the two minor adducts were also found in poly(dG-dC). poly(dG-dC), suggesting that the two minor adducts of IQ, MeIQx and PhIP are also formed on the guanine base. The N-acetoxy derivatives of IQ, MeIQx, and to a much lesser extent PhIP, also formed adducts with adenine-containing polynucleotides including poly(dA), poly(dA).poly(dT) and poly(dA-dT).poly(dA-dT), but these adenine adducts were chromatographically different from those found in DNA. The three guanine adducts of N-acetoxy-IQ, -MeIQx and -PhIP found in vitro in DNA and in guanine-containing polynucleotides were also found in the liver of monkeys fed IQ, MeIQx or PhIP respectively, indicating that metabolic activation via N-hydroxylation and esterification occurred in vivo in monkeys. With each compound, the C8-guanine adduct was the predominant adduct found in vivo. The results indicate similarities among IQ, MeIQx and PhIP in the DNA adducts formed in vitro and in vivo and substantiate the use of the 32P-postlabeling method for comparative adduct studies.

Detection of the major DNA adducts of benzo[j]fluoranthene in mouse skin: nonclassical dihydrodiol epoxides.

Studies have demonstrated that the metabolic activation of benzo[j]fluoranthene (B[j]F) to a genotoxic agent proceeds through the formation of either the trans-4,5-dihydrodiol (B[j]F-4,5-diol) or the trans-9,10-dihydrodiol (B[j]F-9,10-diol) metabolite of B[j]F. Using 32P-postlabeling analysis, the profiles of DNA adducts formed in vivo in mouse skin from B[j]F-4,5-diol and B[j]F-9,10-diol were obtained to establish the contribution of each of these dihydrodiols to the formation of B[j]F-DNA adducts in vivo. B[j]F, B[j]F-4,5-diol and B[j]F-9,10-diol were applied to the shaved backs of mice (100 micrograms/mouse), and DNA adducts were isolated and separated using multidimensional TLC and reverse-phase HPLC. The greatest level of adducts was observed with B[j]F-4,5-diol, which resulted in the formation of 383 pmol of DNA adducts/mg of DNA. This level of DNA modification was more than 2 orders of magnitude greater than that observed with B[j]F or B[j]F-9,10-diol. The major adducts detected from the application of B[j]F-4,5-diol to mouse skin in vivo had chromatographic properties similar to those of the major adducts detected with B[j]F. In contrast, the major DNA adducts detected with B[j]F-9,10-diol had chromatographic properties distinctly different than the adducts formed from either B[j]F or B[j]F-4,5-diol. DNA adducts formed from the syn and anti isomers of the 4,5-dihydrodiol 6,6a-epoxide and the 9,10-dihydrodiol 11,12-epoxide of B[j]F were also evaluated. Each dihydrodiol epoxide derivative was reacted with calf thymus DNA in vitro and applied to mouse skin in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Covalent modification of DNA by daunorubicin.

Daunorubicin, a clinically useful antitumor agent, induces mammary adenocarcinoma in Sprague-Dawley rats. As part of an investigation of the mechanism of tumor induction by daunorubicin, the formation of daunorubicin-DNA adducts has been investigated by 32P-postlabeling assay. Rat-liver DNA incubated with either 0.05 or 0.1 mM daunorubicin, rat-liver microsomes, and 5 mM reduced nicotinamide adenine dinucleotide phosphate (NADPH) for 1 h contained covalent DNA adducts in addition to the endogenous adduct profile present in control DNA. With 1.5 mM cumene hydroperoxide serving as a cofactor, higher levels of these two adducts and two additional adducts were formed, all of which most likely were daunorubicin-DNA adducts. This latter treatment also resulted in an intensification of three endogenous DNA modifications over levels occurring in control DNA. Covalent DNA alterations in vivo were studied in rats treated with 20 mg/kg daunorubicin for 2 days and 200 mg/kg on the 3rd day. Daunorubicin-DNA adducts as observed in vitro could not be detected in DNA of liver or mammary epithelial cells. The levels of endogenous modifications in drug-treated rats were increased by 200% in mammary DNA and by 50% in hepatic DNA as compared with controls. It was concluded from these experiments that daunorubicin may be metabolically activated to a reactive metabolite that binds covalently to DNA. These daunorubicin-DNA adducts may not play a role in tumor induction because they were not detectable in vivo. However, the increase in levels of endogenous DNA modifications induced by daunorubicin both in vitro and in vivo is consistent with a role of this class of DNA modification in the carcinogenic process.

Formation of DNA adducts following laboratory exposure of the mussel, Mytilus edulis, to xenobiotics

32P-Postlabelling was used to determine the formation of bulky hydrophobic DNA adducts following exposure of mussels ( Mytilus edulis) to the aromatic amide, 2-acetylaminofluorene (2AAF). Mussels were exposed to 2AAF either in their seawater (5 ppm) or by injection into the posterior adductor muscle (50 mg/kg total wt.). A major band of radioactivity close to the chromatogram origin was located following analysis of digestive gland DNA from treated mussels which was not present in controls. Identical adduct profiles were obtained irrespective of the route of exposure. This indicates that M. edulis can metabolize 2AAF to genotoxic products in vivo. The formation of 8-hydroxydeoxyguanosine (8OHdG) was used as a marker of oxidative DNA damage. No significant increase in 8OHdG was observed following in vivo exposure of mussels to two redox cycling compounds, menadione (100 ppb) and nitrofurantoin (100 ppb). Since treatment with these compounds has previously been shown to cause increased oxyradical production, the results may reflect limited access of reactive oxygen species to cellular DNA.

HPLC separation of 32P-postlabelled DNA adducts formed from dibenz[ a, h]anthracene in skin

Mouse skin and human skin have been treated in vivo or in short- term organ culture with dibenz[ a,h]anthracene (DB[ a,h]A), the related 3,4- or 5,6-diols or the anti- or syn-3,4-diol 1,2-oxides. DNA hydrolysates have been 32P-postlabelled and the adducts present examined by HPLC using a phenyl-modified reverse phase column and, for comparison, by PEI-cellulose TLC and autoradiography. The adducts formed when the diol-epoxides were reacted with salmon sperm DNA were also examined. The results show that in mouse skin treated in vivo, the major adducts formed from DB[ a,h]A and the 3,4-diol were the same and that two of them were more polar than those formed in skin or in DNA that had been treated with the related anti-or syn-diol epoxides. Human skin treated with DB[a,h]A in culture yielded an adduct profile that was qualitatively similar to the profiles obtained with mouse skin.

DNA damage induced by cigarette smoke condensate in vitro as assayed by 32P-postlabeling. Comparison with cigarette smoke-associated DNA adduct profiles in vivo

Cigarette smoke induces a multitude of bulky/aromatic DNA adducts in vivo as revealed by 32P-postlabeling assay. The formation of such adducts is thought to involve metabolic activation of aromatic chemicals especially polycyclic aromatic hydrocarbons (PAHs) present in tumor-initiating cigarette tar fractions, via cytochrome P450-associated monooxygenases. Because radicals are present in both the gas and particulate (tar) phase of cigarette smoke and in aqueous extracts of cigarette smoke condensate (CSC), we addressed the question as to whether cytochrome P450-independent, possibly free radical-mediated reactions may contribute, also, to formation of cigarette smoke-associated bulky DNA adducts. Rat-lung DNA was incubated with aqueous extracts of CSC in the absence of microsomes under various conditions and analyzed by 32P-postlabeling. Radioactively labeled bulky reaction products were found to accumulate in a time- and CSC concentration-dependent manner. The resulting chromatographic profiles resembled cigarette smoke-associated DNA-adduct patterns observed in vivo. Pretreatment of aqueous CSC extract with radical scavengers/reducing agents ascorbic acid, glutathione) diminished adduct formation in a concentration-dependent manner. Adduct formation in vitro may involve oxygen-free radicals, which are known to be present in aqueous CSC extracts and could (i) attack DNA directly to produce bulky adducts, (ii) induce radical sites on DNA covalently binding CSC components, or (iii) convert CSC components to DNA-reactive electrophiles. In addition, DNA may react with direct-acting mutagens in CSC. Adduct fractions derived from in vitro and in vivo experiments showed similar chromatographic behavior, suggesting that metabolic activation as well as processes not involving metabolism lead to formation of smoking-induced bulky DNA adducts in vivo.

Storage phosphor imaging technique for detection and quantitation of DNA adducts measured by the 32P-postlabeling assay.

The 32P-postlabeling method has found wide application as a sensitive technique for detecting the presence of a broad range of bulky aromatic compounds covalently bound to DNA. In this method, the modified DNA is enzymatically degraded to 3'-mononucleotides and labeled with [32P]-phosphate at the 5'-position using [gamma-32P]ATP and T4 polynucleotide kinase. The 32P-labeled DNA digest is then chromatographed in two dimensions on polyethyleneimine - cellulose thin-layer plates. Screen-enhanced autoradiography is used to locate the presence of the radiolabeled adducts on the chromatogram, and the radioactive areas are generally excised and quantitated by liquid scintillation spectrometry. However, on a chromatogram with multiple adducts, it can be difficult to quantitative partially resolved adducts and evaluated background radioactivity levels. We have evaluated the use of storage phosphor imaging techniques to quantitate and map the radioactivity on chromatograms generated by the 32P-postlabeling method. The results showed that storage phosphor imaging was approximately 10 times more sensitive than screen-enhanced autoradiography at -80 degrees C for the detection of 32P, exhibits a greater linear range of response, has a resolution that compares favorably to film and has a lower background than does liquid scintillation spectrometry. Further, the generation of a digitized record of the distribution and intensity of radioactivity allows for computer-assisted assessment of adduct profiles and can facilitate quantitation of individual adducts and radioactive zones comprised of multiple overlapping adducts in complex chromatograms. Additionally, the permanent record created by the imaging technology permits facile retrospective analysis of samples, whereas with autoradiography and liquid scintillation spectrometry reanalysis of a replicate sample is required.

Microsome-mediated transacetylation and binding of N-hydroxy-4-aminobiphenyl to nucleic acids by hepatic and bladder tissues from dog.

Microsome-mediated metabolism of [3H]4-aminobiphenyl (ABP) and binding of [3H]N-hydroxy-4-aminobiphenyl (N-OH-ABP) to nucleic acids by dog hepatic and bladder microsomes were investigated. HPLC analysis of the ethyl acetate extracts of hepatic microsomal incubates of [3H]ABP in the presence of 4-acetylaminobiphenyl (AABP), N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP), or acetyl coenzyme A (AcCoA) as acetyl donors showed the formation of [3H]AABP, suggesting that microsomes catalyze N-acetylation of ABP involving transacetylation. Dog hepatic microsomes also catalyzed the binding of [3H]N-OH-ABP to RNA in the presence of AABP, N-OH-AABP or AcCoA, and the binding was blocked by paraoxon, an inhibitor of microsomal deacetylases. Binding of [3H]N-OH-ABP to DNA was catalyzed also by dog hepatic microsomes, and the extent of binding was 266, 156 and 135 pmol/mg DNA for AABP, N-OH-AABP and AcCoA as acetyl donors respectively. HPLC analyses of the DNA hydrolysates showed that the major adduct formed was N-(deoxyguanosine-8-yl)-4-aminobiphenyl, based on mobility of the adduct in comparison with the synthetic standard. The acetyl adduct N-(deoxyguanosine-8-yl)-4-acetylaminobiphenyl was not detected in the DNA hydrolysates. Adduct profiles obtained from 32P-postlabeling of DNA samples from the microsome-mediated binding of [3H]N-OH-ABP showed similarities to the profile obtained previously from the chemical interaction of N-OH-ABP with DNA under acidic conditions, suggesting that the microsome-mediated binding of N-OH-ABP may proceed via formation of aryl nitrenium ions as the ultimate electrophilic species. Microsomes from dog bladder also catalyzed the binding of [3H]N-OH-ABP to RNA and DNA in the presence of AABP, N-OH-AABP or AcCoA as acetyl donors, though the levels of binding were less than those observed with hepatic microsomes. The prevalence of these acetyl transferases in the target organs for ABP and AABP carcinogenesis raises the possibility that metabolic activation of the proximate metabolite N-OH-ABP could occur directly in these tissues and these reactions could play a critical role in the initiation of cancers.

Genotoxic effects of estrogens

Estrogens are associated with several cancers in humans and are known to induce tumors in rodents. In this review a mechanism of carcinogenesis by estrogens is discussed which features the following key enents: (1) Steroid estrogens are metabolized by estrogen 2- and 4-hydroxylases to catecholestrogens. Target organs of estrogen-induced carcinogenesis, hamster kidney or mouse uterus, contain high levels of estrogen 4-hydroxylase activity. Since the methylation of 4-hydroxyestradiol by catechol- O-methyltransferase is inhibited by 2-hydroxyestradiol, it is proposed that a build up of 4-hydroxyestrogens precedes estrogen-induced cancer. (2) The catecholestrogen or diethylstilbestrol (DES) are oxidized to semiquinones and quinones by the peroxidatic activity of cytochrome P-450. The quinons are proposed to be (the reactive intermediates of estrogen metabolism. (3) The quinones may be reduced to catecholestrogens and DES and redox cycling may ensue. Redox cycling of estrogens has been shown to generate free reaicals which may react to form the organic hydroperoxides needed as cofactors for oxidation to quinones. (4) The quinone metabolites of catechol estrogens and of DES bind covalently to DNA in vitor whereas DNA binding in vivo has only been examined for DES. When DES is administered to hamsters, the resulting DES-DNA adduct profile in liver, kidney, or other organs closely matches that of DES quinone-DNA adducts in vitor. In vitor, DES-DNA adducts are chemically unstable and are generated in incubations with organic hydroperoxide as cofactor. It is proposed that the instability of adducts and the lower sensitivity of previous assay methods contributed to the reported failures to detect adducts. Steroid estrogen-DNA adducts in vivo are currently under investigation. (5) Tumors are postulated to arise in cells rapidly proliferating due to the growth stimulus provided by the estrogenic activity of the primary estrogen or of hormonally potent metabolites such as 4-hydroxyestradiol. The covalent modification of DNA in these cells is temporary because of the chemical instability of adducts and will result in altered genetic messages in daughter cells, whereas in non-proliferating cells there may be no lasting genetic damage. The sequence of events described above is a plausible mechanism for tumor initiation by estrogens and is partially substantiated by experimental evidence obtained in vitro and/or in vivo.

Role of metabolism on the DNA binding of MeIQx in mice and bacteria

We report the effects of several inducers of P450 metabolizing enzymes on DNA adduct formation by 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in C57BL/6 mice. We also examined the role of N:O-acetylation and the nitrenium ion in the genotoxicity of MeIQx, since these have been implicated in the activation of other aminoimidazoazaarenes (AIA) to DNA reactive species. Mice were given phenobarbital (PB), Aroclor 1254, beta-naphthoflavone (BNF) or corn oil, i.p., followed 3-5 days later with oral administration of MeIQx. Induction of Aroclor and BNF produced DNA with 8-fold more adducts than either the corn oil-alone or PB-treated animals. Both corn oil-alone and PB-treated animals were similar. Four major adducts were found in all cases with no differences among inducers as judged by co-chromatography. Azido-MeIQx induced calf-thymus-DNA adducts produced identical adduct profiles to those seen for the mouse DNA. Similar adduct profiles were obtained from Salmonella TA98, and the nitroreductase deficient strains (TA98NR and TA98/1,8-DNP6) exposed to MeIQx in the presence of Aroclor-induced-mouse-liver S9. Adduct frequencies in TA98/1,8-DNP6 were significantly lower than in TA98 and TA98NR. The data described in this report demonstrate that induction quantitatively increases adduct numbers but does not affect the types of DNA damage. These data also suggest that the same DNA reactive intermediates are formed in vivo as in vitro and support the hypothesis that the metabolism of MeIQx involves the P450I family of isozymes, N:O-acetyltransferases and possibly a nitrenium ion. The application of radioanalytic scanners for quantitation of 32P-postlabelling adduct maps is described.

Molecular dosimetry of DNA adducts in Chinese hamster ovary cells and C3H10T1/2 mouse embryo fibroblast cells treated with benzo(a)pyrene

Chromatographic profiles of DNA adducts formed following treatment of Chinese hamster ovary (CHO) cells or C3H10T1/2 mouse embryo fibroblast cells with benzo[a]pyrene (BP), in the presence or absence of rat liver S9 fraction, have been obtained by both direct labelling ([3H]BP) and 32P-post-labelling methods. The principal adduct formed in both cell lines was (+)-N2-(7R,8S,9R-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene- 10S-yl) 2'-deoxyguanosine (detected as its 3',5'-bisphosphate by 32P-post-labelling). In both cell lines significantly higher levels of adducts were observed in the presence of S9 fraction. The results of these studies provide useful reference data on the genotoxic metabolism of BP in these widely used cell culture systems and allow comparisons with data obtained in other experimental mammalian systems. Valid comparisons of metabolism in experimental systems used as extrapolative models for man are particularly important where multiple bioactivation pathways are possible, such as in the case of polycyclic aromatic hydrocarbons.

32P-Postlabelling analysis of DNA adducts of benzo[ a]pyrene formed in complex metabolic activation systems in vitro

The influence of cytochrome P-450-linked monooxygenase, epoxide hydrolase, UDP-glucuronyltransferase and glutathione S-transferases on the metabolic activation of benzo[a]pyrene (BP) was studied by incubating BP with preparations of rat-liver microsomal and cytosolic fractions in the presence of exogenous DNA. 32P-Postlabelling analysis of the DNA revealed the presence of covalently bound adducts formed by BP, which were visualised as radioactive spots on autoradiographs of thin-layer chromatograms. The effects on the adduct profile of adding different combinations of 1,2-epoxy-3,3,3-trichloropropane (TCPO), UDP-glucoronic acid (UDPGA) and glutathione (GSH) to the incubation mixture were determined. As many as 14 different DNA adducts were resolved and quantitated on the chromatograms, the numbers and quantities of which varied within a large range depending on the incubation conditions. The influence of the enzyme inhibitor and cofactors on the adduct patterns reflects the complex effects of simultaneous enzyme interactions on the metabolic activation of BP.

Characterization of DNA adducts derived from (.+-.)-trans-3,4-dihydroxy-anti-1,2-epoxy-1,2,3,4-tetrahydrodibenz[a,j]anthracene and (.+-.)-7-methyl-trans-3,4-dihydroxy-anti-1,2-epoxy-1,2,3,4-tetrahydrodibenz[a,j]anthracene

Structural characterizations of the DNA adducts derived from reaction of the racemic bay region anti-diol epoxides of dibenz[a,j]anthracene and 7-methyldibenz[a,j]anthracene with calf thymus DNA are presented. Quantities of adducts necessary for spectroscopic characterization were obtained from reactions of the respective diol epoxides with individual deoxyribonucleotides. Both hydrocarbon diol epoxides showed similar adduct profiles upon reaction with calf thymus DNA in vitro which were composed mainly of three deoxyguanosine and four deoxyadenosine adducts. No significant modification of pyrimidine bases in DNA was detected with either of the diol epoxides. Approximately 3 times more deoxyguanosine than deoxyadenosine residues in the DNA were found to be modified by both diol epoxides. The DNA reactions showed very similar stereo- and enantioselectivities with both diol epoxides. The stereochemistries of addition of the purine bases to the diol epoxides were determined from analysis of the NMR spectra of individual adducts. The predominant adducts formed were products of trans addition of the exocyclic amino group of purines to the diol epoxides. The enantiomeric nature of the various adducts was determined from reaction of the individual deoxyribonucleotides with the pure (+)-anti-diol epoxide of dibenz[a,j]anthracene. The major deoxyguanosine and deoxyadenosine adducts from reactions with DNA were found to arise from the (+)-enantiomer of both hydrocarbon diol epoxides. The high reactivities of both diol epoxides (24-38%) with DNA in solution are consistent with the high tumor-initiating activity exhibited by the diol epoxide of dibenz[a,j]anthracene relative to the parent hydrocarbon.