Heterocyclic aromatic amine-DNA-adducts in bacteria and mammalian cells detected by 32P-postlabeling analysis

The formation of DNA adducts by the fried meat mutagen and carcinogen2-amino-3-methylimidazo[4,5-f]quinoline (IQ) was studied bymeans of ³²P-postlabeling of DNA digests and four-directionalt.l.c. Three major and five minor adducts were detected in assaysof DNA digests obtained from Salmonella typhimurium TA98 cellsafter treatment with IQ and rat liver postmitochondrial supernatant(S9). A qualitatively identical adduct pattern was obtainedwith nitro-IQ (3-methyl-2-nitrounidazo[4,5-f]quinoline), a newanalogue of IQ with a nitro instead of the amino group. Thesetwo compounds, therefore, form the same ultimate metabolite.The same adduct pattern was also found after TA98/1,8-DNP⁶ (acetyltransferase-deficient)cells were treated with nitro-IQ; this is probably due to aresidual acetyltransferase activity in this strain. Upon treatmentof TA98 cells with 1 mM IQ for 3 h one adduct was detected in4.7 x 10⁵ total bases; a considerably higher adduct frequency,one in 4.2 x 10³, was induced by nitro-IQ (70 µM, 30 min).The IQ isomer 2-amino-1-methyliniidazo[4,5-f]quinoline (isoIQ)and its nitro-analogue nitro-isoIQ (1-methyl-2-nitroimidazo[4,5-f]-quinoline)also produced identical adducts. Their common adduct patternwas very similar to the IQ adduct pattern but was located ina position different from that of the IQ adduct pattern. DNAfrom Syrian hamster embryo (SHE) cells treated with IQ and S9exhibited adducts apparently identical with those of SalmonellaDNA.     

Effect of methyl substitution on mutagenicity of 2-amino-3-methylimidazo[4,5-f]quinoline isolated from broiled sardine

2-Amino-3-methylimldazo [4,5-f]quinoline [I] is a potent mutagenisolated from broiled sun-dried sardine. [I] and its seven ofderivatives, (two isomers, one demethylated derivative and fourmethyl-substituted derivatives), were tested for mutagenicityon Salmonella typhimurium TA98 and TA100 in the presence ofS9 mix. 2-Amino-1,4-dlmethylimldazo [4,5-f]quinoline was thestrongest mutagen of these 8 compounds on TA98, giving 159,000revertants/nmol (750,000 revertants/µg). The demethylatedderivative, 2-aminoimidazo [4,5-f]quinoline, had very weak mutagenicity,inducing only 55 revertants/nmol (200 revertants/µg).Compounds having a methyl group at position N-1 or N-3 of 2-aminoimidazo[4,5-f]quinoline were strong mutagens. The 1,5-dimethyl-derivativewas more mutagenic than 3,5-dimethyl-derivative. Introductionof a methyl group at position 4 and position 5 enhanced andreduced the mutagenicity, respectively. All the compounds testedwere more mutagenic to TA98 than to TA100, but their relativeorders of mutgenlcity with TA98 and TA100 were the same.     

Synthesis, purification and mutagenicity of 2-hydroxyamino-3-methylimidazolo[4,5-f] quinoline

Synthesis of 2-hydroxyamino-3-methylimidazolo [4,5-f]quinoline(N-hydroxy-IQ), a reactive metabolite of 2-amino-3-methylimidazolo[4,5-f]quinoline(IQ), was achieved by a modification of an earlier method. N-Hydroxy-IQwas purified by a two-step procedure involving C₁₈ Sep-Packand semi-preparative h.p.l.c. Additional h.p.l.c. methods weredeveloped to monitor the synthesis of N-hydroxy-IQ, and to measureIQ and other IQ derivatives on the same h.p.l.c. profile. Thestructure of N-hydroxy-IQ was confirmed by mass spectral analysisfollowing derivatization to azoxy-IQ, phenyl-azoxy-IQ and acetoxy-acetamido-IQ,and by chemical reactivity studies. Mutagenicity studies withthe nitroreductase-deficient strain of Salmonella TA98 showedthat N-hydroxy-IQ is directly mutagenic, having a specific activityof 2 x 10⁴ revertants/nmol. The data confirm that N-hydroxy-IQis a mutagenic metabolite of IQ and further implicate the hydroxylaminein the carcinogenkity of IQ.     

Prostaglandin H synthase-dependent mutagenic activation of heterocyclic aromatic amines of the IQ-type

Microsomes from ram seminal vesides known as a rich source ofprostaglandin H synthase (PHS) activate the food mutagen IQ(2-amino-3-methylimidazo[4,5-f]quinoline) to (a) product(s)mutagenic in Salmonella typhimurium TA98. The activation isdependent on the PHS cofactor arachidonic acid and is stronglyinhibited by the PHS inhibitor indomethacin. In this system,the mutagenic potency of IQ is 22 and 110 times higher thanthat of 2-aminofluorene and benzidine, respectively. The highmutagenic potency of IQ observed previously with mono-oxygenaseactivation is thus extended to the PHS system. The mutagenicactivity produced by PHS increases for 4 h; this contrasts withthe relatively short lifetime of the activity produced by mono-oxygenaseand suggests that different agents are involved in the two processes.The PHS-mediated mutagenic activity of IQ is strongly dependenton the bacterial O-acetyltransferase which is defective in strainTA98/1,8-DNP₆. Further, the responses of the strains TA1978and TA1538 indicate that the mutagenic activity is dependenton lack of the bacterial DNA excision repair and independentof the plasmid pkM101 coded error-prone DNA repair system. Structuralanalogs of IQ without a methyl group on the imidazole ring andwith a naphthalene instead of the quinoline ring show greatlydiminished PHS-mediated mutagenic activity. The strain responsepattern and structure-activity relationships are similar tothose found with mono-oxygenase activation of IQ and thus indicatea basic similarity of the IQ activation via PHS with that viamono-oxygenase. It is hypothesized that PHS may activate carcinogenicheterocyclic aromatic amines in vivo.     

Effects of {alpha}-deuterium substitution on the mutagenicity of 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK)1

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a carcinogenictobacco specific nitrosamine, can be converted to electrophilicdiazohydroxide intermediates by metabolic hydroxylalion of eitherthe methylene carbon (carbon 4) or the methyl carbon attachedto the nitrosamine group. To investigate the relative importanceof these two processes in NNK mutagenesis, we synthesized 4,4-dideutero-4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone([4,4,-D₂]NNK)and 4-(trideuteromethylnitrosamino)-1-(3-pyridyl)-1-butanone([CD₃] NNK), and evaluated their mutagenic activities in Salmonellatyphimurium tester strains. In the presence of Aroclor inducedrat liver 9000 g supernatant, NNK and [4,4-D₂]NNK had comparablemutagenic activities towards S. typhimurium TA 1535 and TA 100,but [CD₃]NNK was inactive in both strains. These results suggestthat hydroxylation of the methyl group of NNK is more importantthan hydroxylation of carbon 4 in its activation to a mutagen.To test the inherent mutagenicity of 4-oxo-4-(3-pyridyl)butyldiazohydroxideand methyldiazohydroxide which would be formed by methyl hydroxylationor carbon 4 hydroxylation, respectively, we compared the mutagenicities,without activation, of the corresponding model compounds, 4-(carbethoxynitrosamino)-1-(3-pyridyl)-1-butanoneand carbethoxynitrosaminomethane (methylnitrosourethane). Bothcompounds were highly mutagenic toward S. typhimurium TA 1535and TA 100, but at doses of 4 x 10^–3 to 4 x 10^–4µmol/plate,only 4-(carbethoxynitrosamino)-1-(3-pyridyl)-1-butanone wasmutagenic. These results are consistent with those obtainedwith the deuterium substituted compounds and indicate the importanceof 4-oxo-4-(3-pyridyl)butylation of DNA in NNK mutagenesis.     

Binding of 2-amino-3-methylimidazo[4,5-f]quinoline to hemoglobin and albumin in vivo in the rat. Identification of an adduct suitable for dosimetry

Blood protein binding by the food-borne carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline(IQ) was investigated using male Sprague-Dawley rats. Amongthe many blood proteins modified in rats dosed intragastricallywith [³H(G)]IQ, hemoglobin and albumin were modified in a dosedependent fashion. Albumin bound 3 – 5 times more IQ thanhemoglobin at doses from 2 to 150 µmol. IQ-modified serumalbumin was enzymatically digested using Pronase and analyzedby h.p.l.c. Many peptide fragments containing radioactivitywere detected but the low level of protein modification (0.01– 0.04% of dose) prevented spectroscopic analyses of theseadducts. An in vitro system containing hepatic microsomes metabolizedIQ to a reactive species which could bind to serum albumin.One major adduct was formed at the cysteine³⁴ residue usingthis activation system and was identical to an adduct isolatedfrom in vivo-modified albumin. Chemical and spectroscopic analysesof the Pronase fragment proved the adduct was a tripeptide containingN²-cysteinesulfinyl-IQ. A chemically identical adduct was formedin vitro when N-hydroxy-IQ was incubated with serum albumin.As much as 10% of the IQ bound to serum albumin in vivo waspresent as this sulfur-linked adduct based on h.p.l.c. analysisof the Pronase digest fragments and on the acid-labile activitywhich could be recovered as IQ.     

Mutagenic activation of IQ, PhIP, and MeIQx by hepatic microsomes from rat, monkey and man: low mutagenic activation of MeIQx in cynomolgus monkeys in vitro reflects low DNA adduct levels in vivo

Cooked meat, poultry and fish contain a number of mutagenicand carcinogenic heterocyclic amines, including 2-amino-3-methylimidazo[4,5-f]quinoline(IQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx)and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP).In the present study we examined the capacity of hepatic microsomesfrom Fischer 344 rats, cynomolgus monkeys and humans to metabolicallyactivate IQ, MeIQx and PhIP in vitro using the Ames Salmonellamutagenicity assay. The mutagenic activation of IQ was similaramong the three species; however, there were significant differencesamong the species in the activation of PhIP and MeIQx. Livermicrosomes from humans showed the greatest capacity to activatePhIP and MeIQx, followed by rats, and then monkeys. The largestdifferences between the species were observed when MeIQx wasused as the mutagen. MeIQx–DNA adducts formed in vivowere then compared among rats and monkeys given MeIQx by gavage(20 mg/kg/day, 10 doses). ³²P-Postlabeling analysis, carriedout under intensification conditions, was used to examine MeIQx–DNAadducts in the liver, kidney, heart, colon and white blood cells.MeIQx–DNA adducts were highest in all tissues examinedfrom male rats, followed by female rats, and much lower in monkeys.In the liver, the total MeIQx–DNA adduct levels of monkeyswere {small tilde}19 and {small tilde}10 times lower than inmale and female rats respectively. In extrahepatic tissues,the differences in MeIQx–DNA adduct levels between monkeysand rats were even greater. The results suggest that the lowlevel of MeIQx–DNA adducts found in vivo in cynomolgusmonkeys reflects a low capacity to activate MeIQx via the hepaticcytochrome P450 monooxygenase system.     

Synthesis of 2-azido-3-methylimidazo[4,5-f]quinoline and photolytic generation of a highly reactive and mutagenic IQ derivative

Azido-IQ (2-azido-3-methylimidazo[4,5-fquinoline), a novel analogof the food mutagen and carcinogen IQ (2-amino-3-methylimidazo[4,5-f]quinoline)was synthesized and characterized. Both thermolysis and photolysisof this azide yield a short-lived nitrene which can react asan electrophile either directly or via protonation to a nitreniumion. Reaction of the nitrene/nitrenium ion with water producesN-hydroxy-IQ; reactions with nucleotides and with DNA (in vitroand in cells) produce adducts efficiently. Correspondingly,high frequencies of mutations are induced in Salmonella typhimuriumby photolyzed azido-IQ. Comparative mutation assays with theS.typhimurium strains TA98 and the hydroxyl-amine-resistantTA98/1,8-DNP₆ provide evidence for a novel mechanism of mutation,direct reaction of the nitrene or nitrene-derived nitreniumion with DNA without involvement of the hydroxylamine. The photolysisof arylazides promises to be a very convenient and generallyapplicable non-enzymatic procedure for the cell-free or intracellulargeneration of short-lived and highly reactive electrophilicspecies which are assumed to be identical with the metabolicallyformed ultimate mutagens/carcinogens of arylamines and nitroarenes.     

Inhibitory effect of dietary 4-ipomeanol on DNA adduct formation by the food mutagen 2-amino-3-methylimidazo [4,5-f]quinoline (IQ)in male CDF1 mice

The food mutagen 2-amino-3-methlimidazo[4,5-f]quinoline (IQ)is carcinogenic in the CDF₁ mouse liver, lungs and stomach.IQ Is activated to its ultimate carcinogenic form by N-hydroxylation,catalyzed principally by hepatic microsomal cytochrome P450IA2,and further esterification, resulting in the formation of N-(deoxyguanosin-8-yl)-IQand other adducts. The furanoterpenold 4-ipomeanol (IPO) isa naturally occurring pneumotoxin which exerts its specifictoxicity in Clara cells of the lung after activation by microsomalcytochrome P450. Because IPO is activated in the liver by acytochrome P450IA2 enzyme, we evaluated IPO as a possible chemopreventiveagent by assessing its ability to inhibit IQ-DNA adduct formationin the CDF₁ mouse. Mice were put on an AIN-76A diet with orwithout 0.075% IPO from day 0 to 54. IQ (0.01%) was added tothe diets from day 22 to 41 and animals were killed (four animals/timepoint) on days 42, 44, 46, 48, 50 and 54. Blood (for white bloodcell isolation), liver, lungs, stomach, small intestine, cecun,colon, kidneys, spleen and heart were collected for analysisof IQ-DNA adducts by .³²P-post-labeling. During the 12 day periodafter cessation of IQ exposure (days 42–54) IQ-DNA adductformation was significantly inhibited in the liver (33.6–46.4%),lungs (29.9–58.6%), stomach (33.2–51.5%) and whiteblood cells (24.5–63.7%), but not in the other organs.Except in the colon, adduct removal from organs during days42–54 was relatively slow (36.0–81.9% of day 42levels remaining on day 54, 9.4–16.7% in the colon), butthe presence of IPO in the diet did not influence the rate ofadduct removal. Measurement of hepatic microsomal ethoxyresorufindeethylase, an activity specific for cytochrome P450IA isozymes,showed that the enzyme could be inhibited (14.1–68.1%)by IPO (0.05–10.0 mM) in vitro. It is concluded that IPOinhibits IQ-DNA adduct formation in target organs of the CDF₁mouse and that IPO may act by inhibiting N-hydroxylation ofIQ. It is therefore possible that IPO may be a candidate chemopreventiveagent against IQ-induced carcinogenesis.     

Cytotoxicity, DNA adduct formation and DNA repair induced by 2-hydroxyamino-3-methylimidazo[4,5-f]quinoline and 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine in cultured human mammary epithelial cells

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine(PhIP) are heterocyclic amines (HAs) found in cooked meats.Both compounds are mammary gland carcinogens in rats. The initiationof carcinogenesis by the HAs is believed to be associated withtheir DNA adduct formation that occurs after metabolic activationof the parent amines via cytochrome P-450-mediated N-hydroxylationand esterification. To assess the capacity of the human mammaryepithelium to metabolically activate the HAs, we used the ³²P-postlabelingmethod to measure the levels of DNA adducts in a culture ofhuman mammary epithelial cells exposed to IQ, PhIP or theirN-hydroxylamine metabolites. Whereas 50 µM parent aminesdid not form detectable levels of DNA adducts in cultured humanmammary epithelial cells after 24 h incubations, concentrationsas low as 1 µM N-hydroxylamines produced detectable levelsof adducts after 2 h incubations. N-Hydroxy-PhIP formed higheradduct levels than N-hydroxy-IQ at all concentrations tested.For example, following a 2 h incubation at 50 µM, adductlevels (per 10⁷ nucleotides) were 674 and 16 for N-hydroxy-PhIPand N-hydroxy-IQ, respectively. At similar initial adduct levels(10–11/10⁷ nucleotides), 60–80% of IQ- and PhIP—DNAadducts were removed after 24 h, indicating that the mammaryepithelial cell culture showed efficient repair of HA adducts.Whereas neither IQ nor PhIP was cytotoxic, both N-hydroxy-IQand N-hydroxy-PhIP were cytotoxic as assessed by a dose-dependentinhibition of colony formation. After exposure to 0.1, 1, 10or 50 µM N-hydroxy-PhIP (or N-hydroxy-IQ), colony formationwas 103 (94), 84 (74), 37 (29) and 3 (2)% of the control values,respectively. Only N-hydroxy-PhIP (at 10 and 50 µM), however,was cytotoxic as assessed by the MTT cell survival assay (whichmeasures the capacity of mitochondria to metabolize a tetrazoliumsalt). The ability of the N-hydroxylamines to form DNA adductsand to be cytotoxic in a culture of human mammary epithelialcells may implicate these metabolites as proximate carcinogenicforms of IQ and PhIP in the human mammary gland. However, whetherthere are inter-individual differences in N-hydroxylamine metabolism,adduct formation and repair in human mammary epithelial cellsrequires further study. The results from this study support the usefulness of culturedhuman mammary epithelial cells for studies on the genotoxicityand metabolism of the N-hydroxylamines of HA food mutagens.     

A comparative study of the biochemical properties of human and mouse recombinant O6-methylguanine-DNA methyltransferases

The O⁶-methylguanine-DNA methyltransferase (MGMT) repairs mutagenicand carcinogenic O⁶-alkylguanine in DNA by accepting stoichiometricallythe alkyl group from the base. Although the mouse MGMT is largerthan the human protein because of an additional tetrapeptidesequence, these proteins are 70% homologous. Recombinant MGMTsof the human, the mouse and a mouse mutant with the tetrapeptidedeleted were purified to homogeneity from Escherichia coli.The N-terminal amino acid sequences of these proteins are identicalto those predicted from the nucleotide sequences, and theirmolecular masses deter mined by SDS-PAGE agreed with the predictedvalues. However, the observed isoelectric points of 9.3, 9.2and 9.3, for the human, mouse and mutant mouse proteins respectivelywere significantly different from the values, 8.09, 7.47 and7.49 calculated from the amino acid composition. The extinctioncoefficients E_1%^{280 nm} of human, mouse and mutant mouse proteinwere calculated from amino acid composition to be 18.2, 11.1and 11.3 respectively. These values agree fairly well with calculatedvalues. Human and wild-type mouse MGMTs react with the alkylatedbase in a synthetic DNA substrate poly(dC, dG, m⁶dG) with comparablesecond-order rate constants of 2.2x10⁸ and 3.7x10⁸ 1/M/min at37°C respectively and were inactivated by O⁶-benzylguanineat similar rates. The initial reaction rate (K_in) and rate ofinactivation (k_inact) constants for reaction with the base werecalculated to be 1.8x10^–4 M and 1.4x10^–3/s for thehuman protein, 2.3x10^–4 M and 1.1x10^–3/s for thewild-type mouse protein, and 2.1x10^–4 and 1.4x10^–3/sfor the mutant mouse protein respectively. The MGMTs were inactivatedto the extent of 55—65% after heating at 50°C in 20mMTris-HCI, pH 8.0, 1 mM EDTA, 1 mM DTT and 10% glycerol. However,in the presence of DNA (200 µg/ml), only 25—35%of the protein was inactivated. Both DNA and RNA inhibited allthree enzymes in a concentration-dependent fashion, althoughDNA was a better inhibitor than RNA. High salt (0.2 M NaCl)inhibited human MGMT by 80%, while the wild-type and the mutantmouse MGMTs were inhibited by 55%. The human protein had higheraffinity for binding to duplex DNAs than the mouse proteins.     

Induction of sister chromatid exchanges in cultured adult rat hepatocytes by directly and indirectly acting mutagens/carcinogens

Primary cultures of adult rat hepatocytes were tested for theirsuitability to assess sister chromatid exchange (SCE)-inducingDNA damage produced by both directly and indirectly acting mutagens/carcinogens.Compared to other genotoxicity assay systems which utilize themetabolizing activity of liver microsomes, this system is atleast 1–2 orders of magnitude more sensitive. The approximatedrug concentrations leading to a doubling of control SCE levelswere 2.5x10^–4 M for cyclophosphamide, 4.5x10^–5 Mfor dimethylnitrosamine, 2.5x10^–6 M for N-methyl-N-nitro-N-nitrosoguanidine,2x10^–10 M for aflatoxin B₁ (AFB₁) and 30 mJ for u.v. Themost potent inducer of SCE proved to be AFB₁, leading to a significantlyelevated level of exchanges at a concentration of 10^–12M. The increased background SCE levels observed (0.75 SCE/chromosome)appears to reflect the sensitivity of hepatocytes to SCE-inducingDNA damage resulting from the dietary intake of mutagenic/carcinogeniccompounds. In view of the high sensitivity and versatility ofthis genotoxicity assay system, it will be of use for the detectionof the low levels of mutagenic/carcinogenic compounds foundin the environment.     

DNA adduct formation of the food carcinogen 2-amino-3-methylimidazo[4, 5-f]quinoline (IQ) in liver, kidney and colo-rectum of rats

DNA adducts of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)have been measured in the liver, kidney, and colorectum of maleFischer-344 rats given a single oral dose of IQ (20 mg/kg).The pattern and distribution of DNA adducts examined by ³²P-postlabelingwas similar in all tissues. N-(Deoxyguanosin-8-yl)-2-amino-3-methylimidazo-[4,5-f]quinoline(dG-C8-IQ) was the principal adduct identified and it accountedfor     

DNA adduct formation and assessment of aberrant crypt foci in vivo in the rat colon mucosa after treatment with N-methyl-N-nitrosourea

N-Nitroso-compound DNA adduct formation in vivo and occurrenceof aberrant crypt foci (ACF) were studied in the rat colon mucosaafter a single, local treatment with a carcinogen, N-methyl-N-nitrosourea(MNU), using a simple surgical approach. A segment of F344 ratcolon was ligated to make a pouch and injected with MNU solution.For the study of DNA adduct formation, the solution contained50 µCi of [³H]MNU. The results demonstrated that similarranges of carcinogen dose, i.e. 0.15x10^–2 –1.5x10^–2MMNU, could induce both DNA adduct formation and appearanceof ACF in the rat colon with both parameters showing a nearbylinear dose dependence. HPLC analysis revealed the DNA adductsto include both 7-methylguanine (7-mGua) and O⁶-methylguanine(O⁶-mGua) with the 7-mGua/O⁶-mGua ratio being 8.2–11.3:1in the system used. Assessment of ACF development from 4 to16 weeks after MNU treatment at a dose of 7.5x10^–2 M showedthe numbers to increase up to the 8th week, followed by a decreaseat weeks 12 and 16, when 40% of the ACF counted at the peaktime point were still present. The percentage of large ACF (     

Induction of oxygen-dependent lethal damage by monochromatic UVB (313 nm) radiation: strand breakage, repair and cell death

The action of 313 nm radiation in cellular inactivation (biologicalmeasurements) and induction and repair of DNA strand breaks(physical measurements) were studied in a repair proficientstrain and three repair deficient strains (polA, recA, uvrA)of Escherichia coli K-12. Although the induction of breaks waslinear in purified T₄ DNA (6.3 x 10^–4 breaks/2.5 x 10⁹daltons/Jm^–2) and the polA strain (4 x 10^–4 breaks/2.5x 10⁹ daltons/Jm^–2), simultaneous induction and repairof breaks were observed in the uvrA, recA and repair proficientstrains at doses <5 x 10⁴ Jm^–2. The final rates ofinduction in these strains were 1 x 10^–4, 7.5 x 10^–5and 7.5 x 10^–5 breaks/2.5 x 10⁹ daltons/Jm^–2, respectively.A highly efficient polA-dependent repair occurring at 0°Cin minimal buffer and a second slower type of repair occurringat 31°C in the polA strain were detected. Oxygen dependenceof cellular inactivation was observed for the polA and repairproficient strains irradiated at 313 nm thus providing biologicalevidence for an oxygen-dependent lesion involved in lethalityin the short wavelength range of the solar u.v. The lower hypoxicbreak induction rates of the pol4 (1.6 x 10^–4 breaks/2.5x 10⁹ daltons/Jm^–2) and the repair proficient (3.6 x 10^–5breaks/2.5 x 10⁹ daltons/Jm^–2) strains, indicate oxygen-enhancedDNA breakage by 313 nm radiation.     

Microsomal metabolism of 1-nitrobenzo[e] to a highly mutagenic K-region dihydrodiol

Aerobic metabolism of 1-nitrobenzo [e](1-nitro-BeP) by rat livermicrosomes produced 1-nitro-BeP trans-4,5-dihydrodiol, 6-hydroxy-1-nitro-BeP,and 8-hydroxy-1-nitro-BeP. When 3,3,3-trichioropropylene 1,2-oxidewas incorporated into the metabolism, 1-nitro-BeP 4,5-oxidewas the predominant metabolite, and 1-nitro-BeP trans-4,5-dlhydrodiolwas not detected. All of the metabolites were purified by bothreversed- and normal-phase HPLC and characterized by analysisof their mass and 500 MHz proton NMR spectral data. 1-Nitro-BePwas not metabolized under hypoxic conditions. 1-Nitro-BeP andits four metabolites were assayed in Salmonella typhimuriumtester strains TA98, TA98NR and TA98/1,8-DNP₆, both in the presenceand absence of S9 activation. As predicted, 1-nitro-BeP wasa weak mutagen without S9 (2 revertants/µg in TA98); theaddition of S9 resulted in {small tilde}18, 17 and 4 revertants/µin TA98, TA98NR and TA98/1,8-DNP₆ respectively. The two phenolicmetabolites were mutagenic both in the presence and absenceof S9, producing moderate responses (19–84 revertants/µg)In addition, while the 1-nitro-BeP 4,5-oxide was only weaklymutagenic in TA98 (6–14 revertants/µg), 1-nitro-BePtrans 4,5-dihydrodiol was unexpectedly potent ({small tilde}300revertants/µg both with and without S9). These resultsindicate that microsomal epoxidation of 1-nitro-BeP followedby epoxide hydrolase-catalyzed hydrolysis of the resulting epoxideto the 1-nitro-BeP trans-4,5-dihydrodiol results in the mostpotent mutagenic derivatives. The weak mutagenicity of 1-nitro-BeP4,5-oxide demonstrates that not all epoxides of nitrated polycyclic aromatic hydrocarbons (PAHs) are more mutagenic thanthe corresponding parent nitro-PAHs. Also, the lower S9-mediatedmutagenicity of 1-nitro-BeP in TA98/1,8-DNP₆ compared with TA98indicates that the mutagenicity of 1-nitro-BeP is dependentupon nitroreduction and transesterification. Finally, we previouslyhypothesized that nitrated PAHs with their nitro substituentsperpendicular or nearly perpendicular to the aromatic ringsare very weak or non-direct-acting mutagens in Salmonella typhimuriumtester strains. The results reported in this communication demonstratethat ring-oxidized derivatives of nitro-PAHs do not always followthis structure— mutagenicity correlation.     

SHORT COMMUNICATION: Metabolic activation and DNA binding of food mutagens and other environmental carcinogens in human mammary epithelial cells

Cultures of human mammary epithelial cells were treated withone of seven heterocyclic amine food mutagens [2-amino-3-methylimidazo[4,5-f)quinoline (IQ), 2-amino-3, 4-dimethylimidazo[4, 5-f)quinoline(MelQ), 2-amino-3, 8-di-methylimidazo[4, 5-f]quinoxaline (MeIQx),2-amino-3, 4, 8-trimethylimidazo[4, 5-f]quinoxaline (4, 8-DiMelQx)2-amino-3, 7, 8-trimethylimidazo[4, 5-f]quinoline (7, 8-DiMelQx),2-amino-3, 4, 7, 8-tetramethylimidazo[4, 5-f]quinoxaline (4,7, 8-TriMelQx) or 2-amino-1-methy1–6-phenylimidazo[4,5-b] pyridine (PhlP)], four nitropyrenes (1-nitropyrene (1-NP),1, 3-dinitropyrene (1, 3-DNP), 1, 6-dinitropyrene (1, 6-DNP)or 1, 8-dinitropyrene (1, 8-DNP) or the Polycyclic aromatichydrocarbon dibenzo[a, l]pyrene (DB[a, l]P). DNA isolated fromthe cultures was analysed by ³²P-post-labelling and in eachcase the presence of carcinogen-DNA adducts was detected. Thepatterns and numbers of adducts obtained when human mammarycell DNA digests were separated on polyethyleneimine-celluloseTLC were found to closely resemble those previously demonstratedto be present in the DNA of tissues from rodents and other primatestreated with the same agents. Up to six DNA adducts were detectedin human breast cells treated with IQ and MelQ. Fewer adducts(1–3) were detected following treatment with MelQx orits methylated derivatives, whilst PhIP gave rise to at leastfour distinct adduct spots. Five adduct spots were detectedin breast cells treated with DB[a, l]P or with 1-NP, but feweradduct spots were formed by 1, 3-, 1, 6- and 1, 8-DNP. Thesedata demonstrate the ability of human breast epithelial cellsto activate to DNA binding species a range of carcinogenic compoundsknown to be present in the human diet or to which humans areknown to be exposed environmentally.     

CARCINOGENSIS: Formation of DNA adducts by the co-mutagen norharman with aromatic amines

Norharman, widely distributed in our environment, is alone notmutagenic to Salmonella typhimurium TA98 and TA100 either withor without S9 mix, but becomes mutagenic to S. Typhimurium TA98with S9 mix when non-mutagenic aromatic amines like anilineor o- or m-toluidine are added. Thus norharman has been calleda ‘co-mutagen’. In the present study we examinedwhether or not DNA adducts are formed in DNA of S.typhimuriumTA98 by treatment with norharman and aromatic amines using ³²P-post-labelinganalysis under modified adduct intensification conditions. Whena sample of norharman (8 mg) and aniline (4 mg) was incubatedwith 4 ml of overnight culture of S.typhimurium TA98 in thepresence of 20 ml S9 mix for 6 h at 37°C, three adduct spotswere detected at a total relative adduct labeling (RAL) of 10.8± 2.27/10⁸ nucleotides. Under the same conditions, amixture of norharman (8 mg) and o-toluidine (4 mg) yielded threeadduct spots at a RAL of 3.74 ± 1.71/10⁸ nucleotides.With a combination of norharman and m-toluidine, a single adductspot was seen at a RAL of 0.04 ± 0.01/10⁸ nucleotides.In contrast, norharman with p-toluidine did not produce adductspots. Furthermore, neither norharman nor the aromatic aminesthemselves gave any evidence of adducts. Thus DNA adduct formationby norharman with aromatic amines correlates with the co-mutagenicaction of norharman in S.typhimurium TA98.     

Mutagenicity of butadiene and its epoxide metabolites: I. Mutagenic potential of 1,2-epoxybutene, 1,2,3,4-diepoxybutane and 3,4-epoxy-1,2-butanediol in cultured human lymphoblasts

The mutagenic potential of the epoxide metabolites of butadiene(BD) was measured at the tk and hprt loci in TK6 human lymphoblastoidcells. TK6 cells were exposed for 24 h to 0–400 µM1,2-epoxybutene (EB), 0–800 µM 3,4-epoxy-1,2-butanediol(EBD), or 0–6 µM 1,2,3,4-diepoxybutane (DEB). Treatedcells were allowed to grow for several days and then seededin medium containing either 6-thioguanine or trifluorothymidineto select for hprt^– or tk^–/– mutants, respectively.All three metaboiltes were mutagenic at both loci, with DEBexhibiting activity at concentrations approximately 100-foldlower than EB or EBD. At the hprt locus, an induced mutationfrequency of 5 x 10^–6 (approximately twice backgroundhprt^– frequency) was produced by treatment with 3.5 µMDEB, 150 µM EB and 450 µM EBD. At the tk locus,a similar increase in mutation frequency (total tk^–/–frequency) was produced by treatment with 1.0 µM DEB,100 µM EB and 350 µM EBD. Each epoxide tested wascapable of inducing slow growth tk^–/– mutants. Thismutant phenotype, as shown previously by others, results fromlarge alterations in the tk region which completely remove theactive tk allele. In addition, Southern blot analysis revealedthat approximately half of DEB-induced hprt^– mutants displayedloss of wild-type hprt restriction fragments. No statisticallysignificant increase in the fraction of hprt deletions amongEB mutants was observed. The ability of DEB to induce deletionsmay be related to its ability to form DNA-DNA and DNA-proteincross-links.     

32P-Post-labeling detection of DNA adducts in mononuclear cells of workers occupationally exposed to styrene

³²-Post-labeling was used to analyze for the presence of DNAadducts in 47 workers exposed to styrene in a boat manufacturingfacility. Individual airborne exposures measured several timesover the course of 1 year ranged from 1to 235 mg/m³ with a meanvalue of 65.6 mg/m³. Two adducts were detected in the DNA ofmononuclear cells of these workers. The following levels ofadducts were detected: adduct 1, range 0.6–102x10^–8(mean 15.8x10^–8); adduct 2, range 0.1–70.9x10^–8(mean 14.2x10^–8). Significant linear relationships werefound between styrene exposure and both DNA adducts (adduct2, r = 0.330, P = 0.012; adduct 1, r = 0.244, P = 0.049). Co-chromatographyexperiments identified DNA adduct 1 in the exposed samplesasN²-(2-hydroxy-1-phenylethyl)-2'-deoxyguanosine-3', 5'-bisphosphate.DNA adduct 2 remains unidentified. No significant linear relationshipswere observed between the level of DNA adducts and sister chromatidexchanges, possibly because of the poor precision of the ³²-post-labelingassay (the estimated coefficients of variation for adducts 1and 2 were 2.54 and 1.96, respectively). These results demonstratethat occupational exposure to styrene results in the formationof DNA adducts in human mononuclear cells.