Covalent binding of hexachlorobutadiene metabolites to renal and hepatic mitochondrial DNA

The covalent binding of [¹⁴C]hexachlorobutadiene (HCBD) metabolitesto DNA was investigated in mouse liver and kidney after applicationof a single oral dose of 30 mg/kg. A low level of binding of[¹⁴C]HCBD metabolites to nuclear DNA (nDNA) from kidney witha covalent binding index (CBI) of 27 was detectable, whereasnDNA from liver did not retain radioactivity significantly exceedingbackground activity (background CBI_nDNAna = 6 and CBI_mtDNA =60). Considerably higher binding of metabolites to mitochondrialDNA (mtDNA) from liver (CBI = 500) and kidney (CBI = 7500) couldbe demonstrated. Enzymatic hydrolysis followed by HPLC fractionationof mtDNA from mouse kidney revealed the presence of three radioactivecompounds which may represent DNA bases altered by HCBD metabolites.The observed binding of HCBD metabolites to DNA constituentsin vivo suggests that genotoxic mechanisms are operative ininitiating HCBD nephrocarcinogenesis.     

Covalent binding of diethylstilbestrol to microsomal protein in vitro correlates with the organotropism of its carcinogenicity

Diethylstilbestrol (DES) was incubated in vitro with liver andkidney microsomes from male and female hamsters and rats, andthe extent of non-extractable binding of radioactivity to microsomalprotein was determined. Binding to microsomes from male hamsterkidney, which is a target organ for DES carcinogenicity in vivo,was found to be 5-10 times higher than binding to microsomesfrom non-target tissues. Pretreatment with phenobarbital ledto a marked increase in binding of DES to kidney microsomesbut not to liver microsomes from female hamsters and male andfemale rats. The correlation of in vitro covalent binding withorgan susceptibility implies a role for metabolic activationof DES in the mechanism of its carcinogenicity.     

Covalent binding of estrogen metabolites to hamster liver microsomal proteins: inhibition by ascorbic acid and catechol-O-methyl transferase

We have studied the irreversible binding of [¹⁴C] to hamsterliver and kidney microsomes of castrated hamsters. The bindingof estradiol metabolites to kidney microsomes was {small tilde}25times lower than seen for liver microsomes, and was not affectedby increases in protein or substrate concentration. Our resultsindicate that this irreversible binding, covalent in nature,is dependent on the metabolism of estrogens to catechols sincethe absence of NADPH markedly reduces this binding. The irreversiblebinding was inhibited nearly 70% by addition of catechol-O-methyltransferase (COMT) and S-adenosylinethionine (SAM). Ascorbicacid also inhibited the binding to 85% in a dose-dependent manner.Utilizing a displacement assay to assess the relative covalentbinding of different stilbene and steroidal estrogens with homologousradiolabeled hormones, we found that only inden estrol B exhibitedgreater ability than diethylslilbestrol (DES) to displace [¹⁴C]from hepatic microsomal proteins. Except for hydroxyproplophenoneand ß-dienestrol, all of the stilbene estrogens studieddisplaced the radioactive DES bind ing from these liver proteinsto a greater extent than estradlol at comparable concentrations.A marked difference was observed in the ability of - and ß-dienestrolto displace [¹⁴C] DES. Using radioinert steroldal estrogensto displace [¹⁴C] we observed that both estriol and deoxoestronewere significantly less effective in displacing radlolabeledestradiol from liver microsomal proteins. Only ethinyl estradioland 2-hydroxyestradiol displaced >50% of the radio activehormone at 1-fold excess concentrations. Interestingly, 11ß-methoxyethinyl estradiol (Moxestrol) exhibited essentially thesame ability to bind liver microsomal proteins as radlol. Whenestrone and 2,4-dibromoestradiol were used as substrates together,we found the latter compound to be inactive as a substrate forestrogen hydroxylase (ESH) and additionally inhibited the metabolismof estrone to form the catechol metabolite. Data presented hereinsuggest that the chemically reactive estrogen metabolites responsiblefor covalent binding of both stilbene and steroidal estrogensare quinoids derived from catechols formed earlier in metabolism.Except for ethinyl estradiol which is a good substrate for liver,but not kidney microsomal ESH, the carcinogenicity data forthe hamster kidney with respect to these estrogens is consistentwith the covalent binding data presented.     

Time-related binding of the hepatocarcinogen carbon tetrachloride to hepatic chromatin proteins in vitro

The in vitro covalent binding of ¹⁴C-labelled carbon tetrachloride[¹⁴C]CCl₄ to histones and non-histone chromosomal proteins (NHCP)under microsome-mediated aerobic conditions was determined.Whole chromatin was prepared from purified nuclei isolated fromlivers of B6C3F1 hybrid mice and incubated with 2.5, 5.0 and10.0 µmol [¹⁴C]CCl₄, in the presence of microsomes isolatedfrom the same tissue, at 4 mg protein, and an NADPH-regeneratingsystem at 37°C for varying incubation times. Binding of[¹⁴C]CCl₄ to histones and NHCP was also determined in the presenceof 5 mM L-cysteine. The results show that the activated intermediateof CCl₄ bound more to histones than to NHCP in a dose- and time-dependentmanner, and that 5 mM L-cysteine inhibited the binding of theactivated intermediate of CCl₄ to histones by 59%, without affectingthe binding to NHCP. These data suggest different extents ofalkylation or acylation between histones and NHCP by metabolicallyactivated CCl₄ under aerobic in vitro conditions, and differentialinhibition of CCl₄-alkylation-acylation by cysteine. This suggestiondoes not exclude other possible mechanisms of action.     

Covalent binding of styrene to DNA in rat and mouse

Covalent binding of (7-³H)styrene (S) to DNA in vivo was measuredand evaluated in a quantitative manner in order to investigatewhether DNA adduct formation could form a mechanistic basisfor tumor induction in a carcinogenicity bioassay. [7-³H]S wasadministered by inhalation in a closed chamber to male and femaleCD rats and B6C3F1 mice. After 4.5–6 h (rats) and 6–9h (pools of four mice), S doses of 23–39 and 85–110mg/kg respectively had been metabolized. DNA was purified toconstant specific radioactivity which was measurable in allsamples. DNA was enzymatically degraded to the 3'-nucleotideswhich were separated by HPLC for the detection of radiolabelednucleotide-S adducts. The fractions with the normal nucleotidescontained most of the radioactivity. In mouse liver DNA, a minutebut significant level of adduct radioactivity was also detected.In the units of the Covalent Binding Index CBI = (µmoladduct/mol DNA nucleotide)/(mmol chemical/kg body wt), valuesof 0.05–0.09 and 0.07–0.18 were calculated for malesand females respectively. In the rat, no DNA adducts were detectablein the liver at a limit of detection of 0.1 CBI units. Two ofthe four lung samples of the female rats showed adduct-relatedradioactivity corresponding to 0.07 CBI units. The CBI valuesare compatible with styrene 7,8-oxide as the reactive intermediate.The data are compared with CBI values and carcinogenic potenciesof established genotoxic carcinogens. It is concluded that theDNA-binding potency of S is so low that significant tumor inductionin a standard bioassay for carcinogenicity is unlikely to bedue to DNA adduct formation alone. Consequences for a humanrisk estimation are discussed.     

Covalent binding of genotoxic agents to proteins and nucleic acids

To a large extent, initiators of current cancer incidence are unknown. This is due partly to lack of proper variables and low statistical power of epidemiological studies and to difficulties of risk estimation from experimental data. Considering these facts, as well as long latent times of genotoxic effects, monitoring systems aiming at risk prevention should: (1) respond soon after onset of exposure; (2) have sufficiently high power; (3) identify causative agents; and (4) permit risk quantitation. The determination of in-vivo adducts to DNA (the target in most genotoxic effects) and, especially, to blood proteins, fulfils these criteria, since the demonstration of protein adducts is a relevant measure of formation of the corresponding DNA adducts.     

Mechanism of DNA binding by the oesophageal carcinogen N-nitroso-N-methylaniline.

N-Nitroso-N-methylaniline (NMA) is a strong oesophageal carcinogen in rats but exhibits few overt genotoxic effects. Previous work from our laboratory established that NMA is readily metabolized by cytochrome P450-catalysed N-demethylation to produce the benzenediazonium ion (BDI), a relatively stable but reactive electrophilic agent. We have also shown that BDI reacts with DNA to form an acid-labile adduct. We have now shown that BDI, generated chemically or by the metabolism of NMA in vitro, reacts with DNA to form a triazene coupling product at the N6-position of adenine residues. This adduct has also been shown to be produced in the liver DNA of rats treated with NMA. Procedures for the isolation of DNA and for analysis of the adduct are presented.     

Formation of urothelial and hepatic DNA adducts from the carcinogen 2-naphthylamine

The carcinogen, 2-naphthylamine (2-NA), induces tumor formationin the urinary bladder but not the liver of several species,including humans and dogs. Since its proximate carcinogenicmetabolite, N-hydroxy-2-NA, was known to react directly withDNA in vitro to give specific carcinogen-base adducts, we investigatedthe in vivo formation and persistence of (2-NA) - DNA adductsin the bladder and liver and attempted to determine whetheror not these lesions correlated with tissue susceptibility.Male beagle dogs were administered [³H]2-NA and sacrificed after2 or 7 days. The DNA was isolated from the liver and urotheliumand hydrolyzed enzymatically. The (2-NA)-deoxyribonucleosideadducts, which were quantitated by high pressure liquid chromatographicanalysis, were the same as those found in vitro, namely 1-(deoxyguanosin-N²-yl)-2-NA,1-(deoxyadenosin-N⁶-yl)-2-NA, and an imidazole ring-opened derivativeof N-(deoxyguanosin-8-yl)-2-NA. The major difference detectedbetween target and non-target tissues was in the total levelof binding to DNA, which was 4-fold higher in the urotheliumat 2 days and 8-fold higher at 7 days after 2-NA dosing. Analysisof specific adducts suggested that this difference may be dueto the relative persistence of the C-8-guanine adduct in theurothelium as compared to the liver. Similar experiments withthe non-carcinogen, 1-naphthylamine, failed to reveal bindingin urothelial DNA and indicated a 20-fold lower binding levelin hepatic DNA. Evidence for binding of 2-NA to glycogen isalso presented and problems associated with measuring totalradioactivity in glycogen-contaminated DNA fractions are discussed.The data obtained in this study, though from a necessarily limitednumber of animals, are consistent with the hypothesis that theformation and persistence of DNA-carcinogen adducts may be importantin the initiation of the neoplastic process.     

Comparison of the in vitro and in vivo hepatic metabolism of the carcinogen 1-nitropyrene

[4, 5, 9, 10–³H]1-nitropyrene was incubated with NADH-or NADPH-fortified rat liver microsomes under an argon atmosphere.Residual substrate and metabolites were extracted with ethylacetate and analyzed by high pressure liquid chromatography.Both reduced and oxidized products were formed: namely, 1-aminopyrene,trans-4, 5-dihydroxy-4, 5-dihydro-1-nitropyrene, and 3-, 6-and 8-hydroxy-1-nitropyrene. When incubations were conductedwith rat liver cytosol, only the reduced products 1-nitrosopyreneand 1-aminopyrene were detected. In parallel experiments, [4,5, 9, 10–³H]1-nitropyrene was administered to rats byintravenous injection or gavage and the bile was collected.After 4 h, approximately one-third of the intravenously-administeredcompound appeared in the bile as O-glucuronides of 3-, 6- and8-hydroxy-1-nitropyrene, the O-glucuronide of trans-4, 5-dihydroxy-4,5-dihydro-1-nitropyrene, and unidentified glutathione conjugates.The same metabolites were found in rats treated with 1-nitropyreneby gavage; however, only 10% of the dose appeared in the bilewithin 12 h. These studies indicate that both nitroreductionand ring oxidation are involved in the hepatic metabolism of1-nitropyrene. The importance of these pathways in the etiologyof 1-nitropyrene tumorigenesis is discussed.     

An investigation into the binding of the carcinogen 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one to DNA in vitro.

After metabolic activation the carcinogen 15,16-dihydro-11-[3H]methylcyclopenta[a]phenanthren-17-one binds to DNA in vitro, and this binding is prevented by 7,8-benzoflavone. Radioactivity cannot be removed from the DNA with organic solvents or by chromatography on Sephadex G-50, even after heat denaturation of the DNA. Enzymatic hydrolysis yields radioactive fractions, which elute from a column of Sephadex LH-20 immediately after the natural nucleosides. At least two species of reactive metabolites are involved in this bending, those with a half-life of a few hr and others with greater stability. After extraction from the aqueous incubation mixture, they could be detected in discrete polar fractions from separations of the complex metabolite mixture by high-pressure liquid chromatography. Their ability to bind to DNA decreased with time at ambient temperature, and they were rapidly deactivated by acid. 7,8-Benzolflavone acted by suppressing the formation of polar metabolites derived from enzymatic oxidation of the aromatic double bonds. The inhibitor had no effect on the enzymes hydroxylating saturated carbon; hence it is unlikely that metabolism of the methyl group is important in conversion of this carcinogen to its proximate form, although the presence of the 11-methyl group is essential for carcinogenic activity in this series.     

Preferential binding of the carcinogen benzo[a]pyrene to proteins of the nuclear matrix

Rat liver nuclei or hepatocytes were incubated with the procarcinogen benzo[a]pyrene (B[a]P) and its ultimate carcinogen, anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE). When nuclei were fractionated by mild micrococcal nuclease digestion into different chromatin regions to determine the distribution of covalent binding to proteins, there was a much higher level of B[a]P bound to proteins of the non-released fraction than to those of released mono- and oligonucleosomes. When non-released material was further fractionated with 2 M NaCl, the highest level of B[a]P binding was found in the proteins of the salt-insoluble fraction. Electrophoretic analysis of [3H]B[a]P-modified nuclear proteins revealed radioactive species migrating in the regions of histones H1 and H3, high mobility group (HMG) proteins 1 and 2, and various high mol. wt non-histone proteins. The non-released fraction contained prominent B[a]P-modified species migrating in the position of the lamins, major components of the nuclear matrix. To confirm B[a]P modification of nuclear matrix proteins, following exposure to B[a]P or BPDE, nuclei were fractionated by a different procedure into an active chromatin fraction, a bulk chromatin fraction, a high-salt-extracted chromatin fraction and a nuclear matrix fraction. Proteins of the nuclear matrix bound consistently more B[a]P metabolites than those of bulk chromatin. This was true following exposure to B[a]P or both low and high concentrations of BPDE, in contrast to previous data on damage to nuclear matrix DNA. Proteins of active chromatin bound more carcinogen than bulk chromatin proteins at low concentrations of BPDE, but less than bulk chromatin at higher concentrations, in parallel with previous data on DNA damage in active chromatin. The potential significance of B[a]P binding to nuclear matrix proteins is discussed.     

Covalent binding of isomeric benzo[a]pyrene diol-epoxides to DNA

We have compared the abilities of two diol-epoxide derivativesof benzo[a]pyrene (B[a]P) to bind covalently to DNA in a simplein vitro system. Purified DNA in aqueous solution was allowedto react with (±)-7, 8ß-dihydroxy-9ß,10ß-oxy-7,8,9,10-tetrahydroB[a]P (BPDE) or with (±)-9,10ß-dihydroxy-7,8-oxy-7,8,9,10-tetrahydroB[a]P (reverseBPDE) to completion. After repurification of the DNA, bindingwas detected by fluorescence spectroscopy or by absorbance spectroscopy.Both BPDE and reverse BPDE but not their hydrolysis productsexhibited binding which increased linearly with increasing diolepoxide concentration. When DNA modified by reverse BPDE wasenzymatically hydrolysed, two major fluorescent deoxyribonucleoside-adductswere detected by reverse phase h.p.l.c. These were separablefrom the major adduct obtained from BPDE-modified DNA and fromthe major products obtained by hydrolysis of reverse BPDE inthe absence of DNA. Absorbance and fluorescence spectroscopyof modified native DNA suggested that the pyrene nucleus ofreverse BPDE but not of BPDE was intercalated in the DNA doublehelix. This suggestion was supported by fluorescence-quenchingstudies. In the presence of increasing DNA concentrations, covalentbinding of both diol epoxides increased towards an apparentmaximum. Double reciprocal analysis of the data indicated amaximum binding level of 5% of the total dose for BPDE and 4%for reverse BPDE. This suggests that for both diol epoxidesthe ratio of the rate constants for covalent binding and forDNA-enhanced hydrolysis are nearly the same. Covalent bindingof reverse BPDE to DNA was effectively blocked by low concentrationsof Mg²⁺, suggesting that formation of a non-covalent intercalationcomplex may be a prerequisite for covalent reaction.     

Preferential binding of the carcinogen benzo[a]pyrene to DNA in active chromatin and the nuclear matrix

Rat liver nuclei or hepatocytes were incubated with the proximatecarcinogen, benzo[a]pyrene (BP) and its ultimate carcinogen,anti-benzo[a]pyrene-7, 8-dol-9,10-epoxide (BPDE. Following carcinogenexposure, nuclei were fractionated by micrococcal nuclease digestionand stepwise extraction to yield an active chromatin fractionenriched in transcribed versus non-transcribed genes, a bulkchromatin fraction, a high-salt-extracted chromatin fractionand a nuclear matrix fraction containing elevated concentrationsof transcribed and non-transcribed genes. BP binds more readilyto DNA of active chromatin and nuclear matrix than to bulk chromatin.Since low concentrations of BPDE also selectively damage activechromatin and matrix DNA, selectivity is not due to the sub-nuclearlocation of enzymes which activate BP to BPDE. Higher BPDE concentrationscause more uniform DNA damage. Selective carcinogen attack mayresult from an accessible DNA conformation in active chromatinand matrix or from partitioning of carcinogen in the nuclearmembrane.     

Covalent binding of polycyclic aromatic hydrocarbon components of coal tar to DNA in mouse skin

Treatment of mouse skin with coal tar is known to initiate tumourformation, with the carcinogenic activity associated mainlywith polycyclic aromatic hydrocarbons (PAHs). A sample of pharmaceuticalcoal tar was analysed by gas chromatography and 19 major PAHswere identified. ³²P-postlabelling analysis was used to characterizethose PAHs that are responsible for the DNA binding of coaltar and, by inference, its biological activity. PAHs were groupedaccording to their reported carcinogenic activities and appliedas mixtures to mouse skin. Group A contained all of the 19 PAHs,group B seven PAHs for which there is sufficient evidence forcarcinogenicity and group C 12 PAHs with only limited or inadequateevidence of carcinogenicity in experimental animals. ³²P-LabelledDNA adducts formed by coal tar were resolved on TLC into a patternof three discrete spots (2, 4 and 6) and four areas of diffuseradioactivity (1, 3, 5 and 7). By comparison of the patternof adducts formed by coal tar with those formed by the syntheticmixtures it appeared that PAHs in group B formed coal tar–DNAadduct spots 4 and 6, and that adduct spot 2 was formed by PAHsin group C. Attempts to identify those PAHs responsible forthe formation of coal tar–DNA adducts 4 and 6 were madeby comparing the chromatographic mobilities of ³²P-labelledcoal tar-derived DNA adducts formed in mouse skin, using TLCand HPLC, with those formed by PAHs in group B. As benzo[ghi]perylene(B[ghi]P), a component of group C, has been demonstrated toexhibit significant DNA binding ability previously, the chromatographicmobility of coal tar–DNA adduct spot 2 was compared tothat of the major DNA adducts formed by B[ghi]P in vivo andin vitro. It appeared that coal tar adduct spot 2 was the majoradduct formed by B[ghi]P in vitro and that benzo[a]pyrene, benzo[b]fluoranthene,benzo [j]fluoranthene and benzo[k]fluoranthene contributed tothe formation of adduct spot 6. None of the PAHs examined appearedto be responsible for the formation of adduct spot 4.     

Suiphotransferase-mediated covalent binding of the carcinogen 7,12-dihydroxymethylbenz to calf thymus DNA and its inhibition by glutatbione transferase

The carcinogen, 7,12-dihydroxymethylbenz[a]anthracene (DHBA),bound covalently through its 7-methylene carbon to calf thymusDNA via DHBA 7-sulphate, a regiospecifical-ly formed, reactivemetabolite, when incubated with rat liver cytosol in the presenceof 3'-phosphoadenosine 5'-phospho-sulphate (PAPS). The hydroxysteroidsulphotransferase in hibitor, dehydroepiandrosterone sulphate,strongly retarded the covalent binding of DHBA to DNA as wellas the DHBA 7-sulphate formation from DHBA in the biologicalsystem, while pentachlorophenol and dichloronitrophenol showedlittle effect on these reactions. DHBA 7-sulphate was a goodsub-strate for rat liver cytosolic glutathione (GSH) transferases,so that the PAPS-dependent covalent binding of DHBA to DNA couldbe markedly retarded in the presence of GSH with concomitantformation of a significant amount of the stable conjugate, S-(12-hydroxymethylbenz[a]anthracen-7-yl)meth-ylgiutathione.From DNA, incubated with DHBA in the pres ence of the hepaticcytosol and PAPS as well as with DHBA 7-sulphate alone, twopurine base adducts were isolated after hydrolysis. The purinebase adducts accounted for 70% of the total covalent bindingof the carcinogen or its 7-sulphate to the nucleic acid andwere identified with synthetic speci mens as N⁶(12-hydroxymethlbenz[a]anthracen-7-yl)-methylguanine.The ratio of the adenine to guanine adducts was 1:2.5 with DNA,incubated with DHBA in the presence of hepatic cytosol and PAPSas well as with various concen-trations of DHBA 7-sulphate.     

Induction of hepatic CYP1A in channel catfish increases binding of 2- aminoanthracene to DNA in vitro and in vivo

Data are presented from in vitro and in vivo studies that indicate cytochrome P4501A (CYP1A) in channel catfish (Ictalurus punctatus) hepatic tissue activates 2-amino-anthracene (AA) to a reactive metabolite that binds to DNA. Channel catfish were injected i.p. with vehicle or 10 mg/kg beta-naphthoflavone (betaNF) on two consecutive days. Two days after the final injection of vehicle or betaNF, vehicle or [3H]AA was injected i.p. at 10 mg/kg, creating four different treatments: vehicle only, betaNF only, [3H]AA only, and betaNF/[3H]AA. Hepatic tissue was examined for CYP1A-associated ethoxyresorufin-O-de- ethylase (EROD) activity, and for DNA adducts at 1, 2, 4 and 7 days following administration of vehicle or [3H]AA. Hepatic EROD activity in betaNF-treated fish was 17-fold higher at day 0 and remained significantly greater than untreated animals for the 7-day experiment. Hepatic DNA adducts, as measured by tritium-associated DNA, ranged from 4.8 to 8.6 pmol/mg DNA in vehicle-pretreated fish injected with [3H]AA, but ranged from 12.6 to 22.7 pmol/mg DNA in betaNF-pretreated fish injected with [3H]AA. Thus, pretreatment with betaNF significantly increased binding of [3H]AA to hepatic DNA in vivo at all four times. Analysis by 32P-post-labeling and thin layer chromatography of hepatic DNA from channel catfish treated with AA revealed two major and several minor spots, which are indicative of DNA adduct formation. Hepatic microsomes from betaNF-pretreated fish were more effective at catalysing the binding of [3H]AA to DNA in vitro than were microsomes from non-treated fish. In addition, binding was decreased by the CYP1A inhibitor 3,3',4,4'-tetrachlorobiphenyl. Collectively, these data demonstrate that CYP1A is involved in the activation of AA in channel catfish.      

Brain microsomal enzyme mediated covalent binding of benzo[a]pyrene to DNA

The covalent binding of benzo[a]pyrene (BP) to calf thymus DNA by brain microsomes isolated from control and 3-methylcholanthrene (3-MC) treated rats was investigated. The influence of incubation time, pH, and concentrations of protein, BP and NADPH on covalent binding was investigated to obtain optimum conditions for the in vitro binding of [3H]BP to DNA. Treatment of rats to 3-MC resulted in a 1.53-fold increase in the brain microsomal mediated covalent binding of [3H]BP to DNA. Inhibitors of monooxygenase enzyme activity such as alpha-naphthoflavone, metyrapone, 1-benzylimidazole and ellagic acid significantly inhibited the binding of [3H]BP to DNA from control and 3-MC stimulated brain microsomes. Our results indicate that inhibitors and inducers of monooxygenases may modulate brain enzyme-mediated binding of polycyclic aromatic hydrocarbons (PAHs) to DNA.