Use of the photo-micronucleus assay in Chinese hamster V79 cells to study photochemical genotoxicity

2-Methoxyaniline (o-anisidine) is a urinary bladder carcinogen in both mice and rats. Since the urinary bladder contains substantial peroxidase activity, we investigated the metabolism of this carcinogen by prostaglandin H synthase (PHS), a prominent enzyme in the urinary bladder, and lactoperoxidase as model mammalian peroxidases. Horseradish peroxidase (HRP)-mediated oxidation of o-anisidine was also determined and compared with the reactions catalyzed by mammalian peroxidases. All three peroxidases oxidized o-anisidine via a radical mechanism. Using HPLC combined with electrospray tandem mass spectrometry, we determined that peroxidases oxidized o-anisidine to a diimine metabolite, which subsequently hydrolyzed to form a quinone imine. Two additional metabolites were identified as a dimer linked by an azo bond and another metabolite consisting of three methoxybenzene rings, which exact structure has not been identified as yet. Using [¹⁴C]-labeled o-anisidine, we observed substantial peroxidase-dependent covalent binding of o-anisidine to DNA, tRNA and polydeoxynucleotides [poly(dX)]. The ³²P-postlabeling assay (a standard procedure and enrichment of adducts by digestion with nuclease P1 or by extraction into 1-butanol prior to ³²P-labeling) was employed as the second method to detect and quantitate binding of o-anisidine to DNA. Using these versions of the ³²P-postlabeling technique we did not observe any DNA adducts derived from o-anisidine. The o-anisidine-DNA adducts became detectable only when DNA modified by o-anisidine was digested using three times higher concentrations of micrococcal nuclease and spleen phosphodiesterase (MN/SPD). We found deoxyguanosine to be the target for o-anisidine binding in DNA using poly(dX) and deoxyguanosine 3′-monophosphate (dGp). A diimine metabolite of o-anisidine is the reactive species forming adducts in dGp. The results strongly indicate that peroxidases play an important role in o-anisidine metabolism to reactive species, which might be responsible for its genotoxicity, and its carcinogenicity to the urinary bladder in rodents. The limitation of the ³²P-postlabeling technique to analyze DNA adducts derived from o-anisidine as a means to estimate its genotoxicity is discussed.     

Evidence for reductive activation of carcinogenic aristolochic acids by prostaglandin H synthase -- (32)P-postlabeling analysis of DNA adduct formation

Aristolochic acid (AA), a naturally occurring nephrotoxin and carcinogen, is implicated in an unique type of renal fibrosis, designated Chinese herbs nephropathy (CHN), which can develop to urothelial cancer. Understanding which enzymes are involved in AA activation and/or detoxication is important in the assessment of an individual susceptibility to this natural carcinogen. We examined the ability of prostaglandin H synthase (PHS) to activate AA to metabolites forming DNA adducts with the nuclease P1 and 1-butanol extraction enrichment procedure of the (32)P-postlabeling assay. PHS is a prominent enzyme in the kidney and urothelial tissues. Ram seminal vesicle (RSV) microsomes, which contain high levels of PHS, generated AA-DNA adduct patterns reproducing those found in renal tissues in CHN patients. 7-(Deoxyadenosin-N(6)-yl)aristolactam I, 7-(deoxyguanosin-N(2)-yl)aristolactam I and 7-(deoxyadenosin-N(6)-yl)aristolactam II were identified as AA-DNA adducts formed by AAI. Two adducts, 7-(deoxyguanosin-N(2)-yl)aristolactam II and 7-(deoxyadenosin-N(6)-yl)aristolactam II, were generated from AAII. According to the structures of the DNA adducts identified, nitroreduction is the crucial pathway in the metabolic activation of AA. The identity of PHS as the activating enzyme in RSV microsomes was proven with different cofactors and inhibitors. Only indomethacin, a selective inhibitor of PHS, significantly decreased the amount of adducts formed by RSV microsomes. The inhibitor of NADPH:CYP reductase (alpha-lipoic acid) and some selective inhibitors of cytochromes P450 (CYP) were not effective. Likewise, only cofactors of PHS, arachidonic acid and hydrogen peroxide, supported the DNA adduct formation of AAI and AAII, while NADPH and NADH were ineffective. These results demonstrate a key role of PHS in the activation pathway of AAI and AAII in the RSV microsomal system and were corroborated with the purified enzyme, namely ovine PHS-1. The results presented here are the first report demonstrating a reductive activation of nitroaromatic compounds by PHS-1.     

Indole-3-carbinol protects against covalent binding of benzo[a]pyrene and N-nitrosodimethylamine metabolites to mouse liver macromolecules

Benzo[a]pyrene (BaP) and N-nitrosodimethylamine (NDMA) are carcinogens and indirect acting mutagens. A naturally occurring dietary indole, indole-3-carbinol (I-3-C), has been shown to decrease the incidence of aryl hydrocarbon induced neoplasia in experimental animals. We examined the relationship between the ability of I-3-C to alter the rate of carcinogen oxidation and its ability to decrease the rate of covalent binding of carcinogen metabolites to DNA and protein. We found that I-3-C inhibited the covalent binding of NDMA oxidation products to DNA in vitro in proportion to its ability to inhibit carcinogen metabolism. Pretreatment of mice by gavage with I-3-C resulted in no change in the rate of aryl hydrocarbon hydroxylase or NDMA demethylase in hepatic post-mitochondrial supernatant. However, this pretreatment resulted in a 60-90% decrease in the ability of carcinogen oxidative metabolites to bind covalently to DNA or protein in vitro. Similarly, in in vivo experiments, gavage with I-3-C, followed by gavage with BaP or NDMA, resulted in a 63-85% decrease in covalent binding to macromolecules, with no concomitant change in carcinogen metabolism. The results suggest that the in vivo administration of I-3-C may confer protection for hepatic macromolecules against covalent binding of the metabolites of these two indirect acting mutagens.     

Oxidative DNA damage induced by a metabolite of carcinogenic o-anisidine: enhancement of DNA damage and alteration in its sequence specificity by superoxide dismutase

The mechanism of DNA damage by a metabolite of the carcinogen o-anisidine in the presence of metals was investigated by the DNA sequencing technique using 32P-labeled human DNA fragments. The o-anisidine metabolite, o-aminophenol, caused DNA damage in the presence of Cu(II). The DNA damage was inhibited by catalase and bathocuproine, suggesting the involvement of H2O2 and Cu(I). The formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine by o-aminophenol increased in the presence of Cu(II). We conclude that Cu(II)-mediated oxidative DNA damage by this o-anisidine metabolite seems to be relevant for the expression of the carcinogenicity of o-anisidine. o-Aminophenol plus Cu(II) caused preferential DNA damage at the 5'-site guanine of GG and GGG sequences. When CuZn-SOD or Mn-SOD was added, the DNA damage was enhanced and its predominant cleavage sites were changed into thymine and cytosine residues. We consider that SOD may increase the frequency of mutations due to DNA damage induced by o-aminophenol and thus increase its carcinogenic potential.     

Carcinogenic pollutants o-nitroanisole and o-anisidine are substrates and inducers of cytochromes P450

2-Methoxyaniline (o-anisidine) and 2-methoxynitrobenzene (o-nitroanisole) are important pollutants and potent carcinogens for rodents. o-Anisidine is oxidized by microsomes of rats and rabbits to N-(2-methoxyphenyl)hydroxylamine that is also formed as the reduction metabolite of o-nitroanisole. o-Anisidine is a promiscuity substrate of rat and rabbit cytochrome P450 (CYP) enzymes, because CYPs of 1A, 2B, 2E and 3A subfamilies oxidize o-anisidine. Using purified CYP enzymes, reconstituted with NADPH: CYP reductase, rabbit CYP2E1 was the most efficient enzyme oxidizing o-anisidine, but the ability of CYP1A1, 1A2, 2B2, 2B4 and 3A6 to participate in o-anisidine oxidation was also proved. Utilizing Western blotting and consecutive immunoquantification employing chicken polyclonal anti bodies raised against various CYPs, the effect of o-anisidine and o-nitroanisole on the expression of the CYP enzymes was investigated. The expression of CYP1A1/2 was found to be strongly induced in rats treated with either compounds. In addition, 7-ethoxyresorufin O-deethylation, a marker activity for both CYP1A1 and 1A2, was significantly increased in rats treated with either carcinogen. The data demonstrate the participation of different rat and rabbit CYP enzymes in o-anisidine oxidation and indicate that both experimental animal species might serve as suitable models to mimic the o-anisidine oxidation in human. Furthermore, by induction of rat hepatic and renal CYP1A1/2, both o-nitroanisole and o-anisidine influence their carcinogenic effects, modifying their detoxification and/or activation pathways.     

Quantitative and qualitative aspects of the binding of N-hydroxy-2-acetylaminofluorene to hepatic chromatin fractions

The binding of a chemical carcinogen to components of hepatic chromatin in male rats was examined. After a single injection of N-[3H]hydroxy-2-acetylaminofluorene ([3H]OH-AAF) covalent binding to chromatin RNA, protein, and DNA occurs. The amount of carcinogen bound to RNA was approximately 5 times greater than to DNA, and 10 times that of the protein. However, loss of carcinogen from RNA with time was rapid, whereas a persistent binding to DNA equal to 15% of the initial values was observed. To localize the initial and persistent DNA-bound carcinogen, the genome was fractionated using two different chromatin fractionation procedures. The procedures used yielded 3 chromatin fractions based on physical characteristics, degree of association with nascent RNA and in vitro template capacity. Based on those parameters, these chromatin fractions have been tentatively classified as template expressed euchromatin, a repressed heterochromatin, and a highly condensed pelleted heterochromatin. With both the glycerol gradient chromatin fractionation procedure and the selective MgCl2 chromatin precipitation procedure, the initial (2 h) binding of carcinogen was greatest on the euchromatin DNA. Loss of carcinogen from the DNA, however, was also significantly faster from the euchromatin when compared to the heterochromatin and the pelleted heterochromatin. By 10 days after a single injection of the carcinogen, the largest amount of bound fluorene residues was located on the pelleted heterochromatin DNA, an apparently repressed portion of the genome, while less than 5% of the initial values were found on either the eu- or heterochromatin. When the rats were fed a 2-acetylaminofluorene-containing diet, loss of carcinogen from the pelleted heterochromatin DNA was enhanced, while loss from the euchromatin DNA was reduced. The covalent nature of the carcinogen modification of DNA was confirmed by thin-layer chromatography (TLC). These studies also demonstrated 2 separate carcinogen-purine base adducts which were identified as N-(guanin-8-yl)-N-AF and 3-(guanin-N2-yl)-N-AAF based on either co-chromatography with an authentic standard or on published Rf-values, respectively. The pelleted heterochromatin DNA had a significantly greater proportion of the 3-guanine-N2 adduct when compared to DNA from either the eu- or heterochromatin.     

Carcinogen-nucleic acid interactions: equilibrium binding studies of aflatoxins B1 and B2 with DNA and the oligodeoxynucleotide d(ATGCAT)2

Equilibrium binding is believed to play an important role in directing the subsequent covalent attachment of many carcinogens to DNA. We have utilized UV spectroscopy to examine the non-covalent interactions of aflatoxin B1 and B2 with calf thymus DNA, poly(dAdT):poly(dAdT), and poly(dGdC):poly(dGdC), and have utilized NMR spectroscopy to examine non-covalent interactions of aflatoxin B2 with the oligodeoxynucleotide d(ATGCAT)2. UV-VIS binding isotherms suggest a greater binding affinity for calf thymus DNA and poly(dAdT):poly(dAdT) than for poly(dGdC):poly(dGdC). Scatchard analysis of aflatoxin B1 binding to calf thymus DNA in 0.1 M NaCl buffer indicates that binding of the carcinogen at levels of bound aflatoxin less than 1 carcinogen per 200 base pairs occurs with positive cooperativity. The cooperative binding effect is dependent on the ionic strength of the medium; when the NaCl concentration is reduced to 0.01 M, positive cooperativity is observed at carcinogen levels less than 1 carcinogen per 500 base pairs. The Scatchard data may be fit using a "two-site" binding model [L.S. Rosenberg, M.J. Carvlin, and T.R. Krugh, Biochemistry 25, 1002-1008 (1986)]. This model assumes two independent sets of binding sites on the DNA lattice, one a high affinity site which binds the carcinogen with positive cooperativity, the second consisting of lower affinity binding sites to which non-specific binding occurs. NMR analysis of aflatoxin B2 binding to d(ATGCAT)2 indicates that the aflatoxin B2/oligodeoxynucleotide complex is in fast exchange on the NMR time scale. Upfield chemical shifts of 0.1-0.5 ppm are observed for the aflatoxin B2 4-OCH3, H5, and H6a protons. Much smaller chemical shift changes (less than or equal to 0.06 ppm) are observed for the oligodeoxynucleotide protons. The greatest effect for the oligodeoxynucleotide protons is observed for the adenine H2 protons, located in the minor groove. Nonselective T1 experiments demonstrate a 15-25% decrease in the relaxation time for the adenine H2 protons when aflatoxin B2 is added to the solution. This result suggests that aflatoxin B2 protons in the bound state may be in close proximity to these protons, providing a source of dipolar relaxation. Further experiments are in progress to probe the nature of the aflatoxin B1 and B2 complexes with polymeric DNA and oligodeoxynucleotides, and to establish the relationship between the non-covalent DNA-carcinogen complexes observed in these experiments, and covalent aflatoxin B1-guanine N7 DNA adducts.     

The importance of intercalation in the covalent binding of benzo(a)pyrene diol epoxide to DNA

The primary mode of non-covalent interaction of the strong carcinogen, benzo(a)pyrene diol epoxide, with DNA is through intercalation. It has variously been suggested that intercalative complexes may be prerequisite for either covalent binding or DNA-catalysed hydrolysis of the epoxide or both. Geacintov [Geacintov, N. E. (1986). Carcinogenesis 7, 589.] has recently argued that intercalation is important in covalent binding and presented theoretical constructs consistent with this proposal. A more general theoretical model is presented here which includes the possibilities that either catalysis of hydrolysis or covalent binding of benzo(a)pyrene diol epoxide DNA can occur (a) in an intercalation complex, or (b) without formation of a detectable, physically bound complex. It is shown that a variety of possible mechanisms formulated under this general theory lead to equations for overall reaction rates and covalent binding fractions which are all of the same form with respect to DNA concentration dependence. A consequence of this is that experimental studies of the dependence of hydrolysis rates and covalent binding fractions on DNA concentration do not distinguish between the various possible mechanisms. These findings are discussed in relation to the interactions of benzo(a)pyrene diol epoxide with chromatin in cells.     

Interaction of benzo[alpha]pyrene diol-epoxide with nuclei and isolated chromatin.

Chicken erythrocyte chromatin and nuclei were labeled with benzo[alpha]-pyrene (B[alpha]P) diol-epoxide (anti) and digested with micrococcal nuclease to mono- and dinucleosomes. Analysis of the distribution of the carcinogen showed that the internucleosomal region bound 3-4 times more carcinogen per unit DNA than did nucleosomes. The enhanced binding of the 'ultimate' carcinogen to the internucleosomal region was similar when isolated chromatin or nuclei were used for in vitro labeling. Furthermore, isolation of the histone core proteins, H2A, H2B, H3 and H4, revealed that only 15% of the carcinogen was associated with the histones and that the majority of the carcinogen was bound to chromosomal DNA. Fluorography of purified nucleosomal histones showed that the covalent association of the carcinogen was mainly with histones H3 and H2B.     

Reactions of benzo]pa]pyrene diol-epoxides with DNA and nucleosomes in aqueous solutions

The rate of solvolysis of benzo[$\text{a}$]pyrene diol-epoxide in aqueous solutions can be followed by fluorescence spectroscopy. When DNA was present the rat of breakdown of benzo[$\text{a}$]pyrene diol-epoxide was substantially enhanced, while at the same time fluorescence intensity was decreased. This decrease, however, was due to noncovalently bound tetraols and does not seem to be a function of the covalent adducts formed. Nucleosomal core particles, reacted under identical conditions, showed very little quenching of the pyrene-like chromophore. When increasing amounts of cysteine were present the covalent binding could be prevented in both free DNA and nucleosomal DNA. Analysis of the distribution of the carcinogen to nucleosomal DNA showed that the covalently bound carcinogen was located at or within 10 bases of the 5′-OH region of the nucleosomal DNA.     

Nucleic acid binding and mutagenicity of active metabolites of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is a potent mutagen and carcinogen present in heated foodstuffs. The covalent binding of MeIQx to calf thymus DNA and calf liver RNA with microsomal activation was demonstrated. A major metabolite which exerts a direct mutagenic effect on S. typhimurium TA98 was found by HPLC analysis after incubation of MeIQx with rat liver microsomal fraction. The metabolite was identified as 2-hydroxyamino-3,8-dimethylimidazo[4,5-f]quinoxaline (N-OH-MeIQx). Synthetic N-OH-MeIQx was found to bind non-enzymatically to DNA and RNA at neutral pH even at 0 degrees C. Addition of acetic anhydride increased the binding of N-OH-MeIQx to DNA 10 times. These results suggest that MeIQx is metabolized to N-OH-MeIQx by microsomal cytochrome P-450 and further activated to an acetylated form that binds efficiently to nucleic acids in rat liver. Preferential modification of polyguanylic acid suggests that guanine residues of DNA are mainly modified with MeIQx. Synthetic N-OH-MeIQx exerted direct mutagenic activity on S. typhimurium TA98 inducing 150,000 rev/micrograms. Pentachlorophenol (PCP) caused a dose-dependent inhibition of this mutagenic effect, but 2,6-dichloro-4-nitrophenol (DCNP) did not. Thus the acetyltransferase of S. typhimurium seems to be important for the high mutagenicity of MeIQx after its microsomal activation.     

A comparison of cell-collecting methods for the Comet assay in urinary bladders of rats

Conducting the single-cell gel electrophoresis (Comet) assay in the urinary bladders of rodents is technically problematic because the bladder is small and thin, which makes it difficult to collect its mucosal cells by scraping. We performed the Comet assay using a simple mincing method in which tissues are minced with scissors. We then compared data obtained with this method with data obtained using the scraping method. Sprague-Dawley rats of both sexes were orally given twice the known carcinogens N-methyl-N-nitrosourea (MNU), ethyl methanesulfonate (EMS), or o-anisidine (OA). Three hours after the second administration, the bladder of each rat was divided into two parts and each part was processed by either the mincing or the scraping method. Both mincing and scraping methods detected DNA damage in MNU-, EMS-, but not OA-treated rats, and thus the mincing method had a sufficient capability to detect DNA damaging agents. The morphological analysis of the prepared cell suspensions revealed that more than 80% of the cells collected by the mincing method were from the epithelium. Because the mincing method requires only one-half of a bladder, the other half remains intact and can be used for histopathological examination. We conclude that the mincing method is easier and more appropriate for the Comet assay in urinary bladder tissue than the scraping method.     

Cooperative activation of the ATR checkpoint kinase by TopBP1 and damaged DNA

TopBP1, acting in concert with DNA containing bulky base lesions, stimulates ATR kinase activity under physiologically relevant reaction conditions. Here, we analyze the roles of the three components in ATR activation: DNA, base damage and TopBP1. We show that base adducts caused by a potent carcinogen, benzo[a]pyrene diol epoxide (BPDE), constitute a strong signal for TopBP1-dependent ATR kinase activity on Chk1 and p53. We find that the C-terminus of TopBP1 binds preferentially to damaged DNA and is sufficient to mediate damaged DNA-dependent ATR activation in a manner similar to full-length TopBP1. Significantly, we find that stimulation of ATR by BPDE-damaged DNA exhibits strong dependence on the length of DNA, with essentially no stimulation with fragments of 0.2 kb and reaching maximum stimulation with 2 kb fragments. Moreover, TopBP1 shows preferential binding to longer DNA fragments and, in contrast to previous biochemical studies, TopBP1 binding is completely independent of DNA ends. We find that TopBP1 binds to circular and linear DNAs with comparable affinities and that these DNA forms elicit the same level of TopBP1-dependent ATR activation. Taken together, these findings suggest a cooperative activation mechanism for the ATR checkpoint kinase by TopBP1 and damaged DNA.     

Carcinogenic purine N-oxide ester modifies covalently all common bases in polynucleotides

The carcinogen 1-methyl-3-hydroxyxanthine after esterification binds covalently to polynucleotides, RNA and DNA. All four ribopolynucleotides and poly(dT) are targets. Depending on reaction conditions, covalent binding is greatest to poly(A) followed by poly(U), poly(dT), poly(G), poly(C), RNA and DNA. Maximal covalent modification of DNA is one moiety per 360 nucleotides. All modified polynucleotides, RNA and DNA, except poly guanylic acid have been enzymatically digested and the major adducts characterized as nucleosides.     

Catalysis of carcinogen-detoxification by DNA: comparison of enantiomeric diol epoxides

The interactions of the (+)- and (-)-enantiomers of 7r,8t-dihydroxy-9t,10t-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I) with purified DNA have been studied in vitro. These compounds are formed by cellular metabolism of the potent environmental carcinogen benzo[a]pyrene, and the (+)-enantiomer is thought to be the ultimate carcinogenic metabolite. Non-covalent, intercalative binding was measured spectrophotometrically, hydrolysis was measured spectrofluorometrically and covalent binding was detected by liquid scintillation counting. No significant differences were found in the association constants for intercalative binding or in the ability of DNA to catalyse the hydrolysis of the two enantiomers. Covalent DNA binding was 4.5-fold higher for the (+)-enantiomer. When DNA was pretreated with a molar equivalent of the (-)-enantiomer, its subsequent ability to enhance the rate of BPDE-I hydrolysis and to bind covalently to (+)-BPDE-I was unimpaired. This suggests that the participation of the DNA in the hydrolysis reaction does not alter the DNA and therefore that the rate-enhancement is true catalysis.     

DNA-binding studies with 6BT and 5I: implications for DNA-binding/carcinogenicity and DNA-binding/mutagenicity correlations

The divergent activities of a reported carcinogen/noncarcinogen pair of monoazo dyes related to the hepatocarcinogen Butter Yellow (DAB) are currently under investigation in our laboratories. As part of these studies we have determined (a) target organ distribution after oral dosing to rats and (b) covalent binding of 14C-labelled compound to DNA. In DNA-binding studies, 3 rat liver-metabolising systems were employed: in vivo (whole liver), isolated intact hepatocytes, and liver subcellular fractions. Distribution studies revealed that comparable levels of both compounds were detected in the liver at similar times after dosing, and these in vivo tissue concentrations were used for in vitro DNA-binding studies. At this 'in vivo equivalent dose', the carcinogen was consistently bound to DNA more effectively, and the difference (ratio of DNA binding) between the 2 compounds was far greater in vivo. In subsequent studies, covalent DNA binding to bacterial (Salmonella) DNA was assessed at the in vivo equivalent dose. In contrast to the afore-mentioned findings in mammalian systems, the carcinogen was bound less effectively to DNA, and gave fewer revertant counts/plate when the 2 compounds were bound to an equivalent extent. These data are discussed in view of their implications for DNA-binding/carcinogenicity correlations, and with respect to the relationship between DNA binding and mutagenicity in the Salmonella assay.     

Binding mode of chemically activated semiquinone free radicals from quinone anticancer agents to DNA

Chemical reduction of the highly active quinone-containing antitumor drugs, adriamycin and daunorubicin formed the same partially reduced free radical previously reported [9] by microsomal activation. In vitro incubation of the chemically activated free radical intermediates with DNA resulted in covalent binding of these drugs to DNA. The adriamycin semiquinone radical has a greater affinity for DNA and covalent complexes up to one adriamycin per 12 nucleotides were obtained. The daunorubicin semiquinone radical, on the other hand, showed a lesser binding affinity and gave rise to complexes in which one drug molecule was covalently bound per 135 nucleotides. The stronger covalent binding of adriamycin to DNA may account for more severe DNA damage induced by this drug.     

Carcinogen-Nucleic Acid Interactions: Equilibrium Binding Studies of Aflatoxins B1 and B2 with DNA and the Oligodeoxynucleotide d(ATGCAT)2

Abstract

Equilibrium binding is believed to play an important role in directing the subsequent covalent attachment of many carcinogens to DNA. We have utilized UV spectroscopy to examine the non-covalent interactions of aflatoxin B₁ and B₂ with calf thymus DNA, poly(dAdT):poly(dAdT), and poly(dGdC):poly(dGdC), and have utilized NMR spectroscopy to examine non-covalent interactions of aflatoxin B₂ with the oligodeoxynucleotide d(ATGCAT)₂. UV-VIS binding isotherms suggest a greater binding affinity for calf thymus DNA and poly(dAdT):poly(dAdT) than for poly(dGdC):poly(dGdC). Scatchard analysis of aflatoxin B₁ binding to calf thymus DNA in 0.1 M NaCl buffer indicates that binding of the carcinogen at levels of bound aflatoxin ? 1 carcinogen per 200 base pairs occurs with positive cooperativity. The cooperative binding effect is dependent on the ionic strength of the medium; when the NaCl concentration is reduced to 0.01 M, positive cooperativity is observed at carcinogen levels ? 1 carcinogen per 500 base pairs. The Scatchard data may be fit using a “two-site” binding model [L.S. Rosenberg, M J. Carvlin, and T.R. Krugh, Biochemistry 25, 1002–1008 (1986)]. This model assumes two independent sets of binding sites on the DNA lattice, one a high affinity site which binds the carcinogen with positive cooperativity, the second consisting of lower affinity binding sites to which non-specific binding occurs. NMR analysis of aflatoxin B₂ binding to d(ATGCAT)₂ indicates that the aflatoxin B₂/oligodeoxynucleotide complex is in fast exchange on the NMR time scale. Upfield chemical shifts of 0.1–0.5 ppm are observed for the aflatoxin B₂ 4-OCH₃, H5, and H6a protons. Much smaller chemical shift changes ? 0.06 ppm) are observed for the oligodeoxynucleotide protons. The greatest effect for the oligodeoxynucleotide protons is observed for the adenine H2 protons, located in the minor groove. Nonselective T₁ experiments demonstrate a 15–25 % decrease in the relaxation time for the adenine H2 protons when aflatoxin B₂ is added to the solution. This result suggests that aflatoxin B₂ protons in the bound state may be in close proximity to these protons, providing a source of dipolar relaxation. Further experiments are in progress to probe the nature of the aflatoxin B₁ and B₂ complexes with polymeric DNA and oligodeoxynucleotides, and to establish the relationship between the non-covalent DNA-carcinogen complexes observed in these experiments, and covalent aflatoxin B₁,-guanine N7 DNA adducts.     

Dichlorobenzidine-DNA binding catalyzed by peroxidative activation in Salmonella typhimurium

Peroxidative oxidation of dichlorobenzidine in vitro results in covalent binding to exogenous DNA. In a modified Ames assay, mutagenicity is observed in S. typhimurium strain TA98 following the incubation of dichlorobenzidine, bacteria, and hydrogen peroxide. In this paper, we demonstrate that [14C]dichlorobenzidine becomes covalently bound to S. typhimurium macromolecules, including DNA, when exogenous hydrogen peroxide is supplied. We compared the levels of binding in a pair of otherwise isogenic strains with wild-type (oxyR+) versus constitutive (oxyR1) expression of the hydrogen peroxide stress-induced regulon. Binding was approximately twofold higher in TA4124 (oxyR1) than in TA4123 (oxyR+). Bacterial hydroperoxidases may catalyze the activation of dichlorobenzidine to mutagenic and DNA binding species in this system.