Metabolism of the carcinogen N-hydroxy-N-2-fluorenylacetamide by rat peritoneal neutrophils

The in vitro metabolism of a locally carcinogenic N-hydroxy-N-2-fluorenylacetamide(N-OH-2-FAA) by rat peritoneal polymorphonuclear leukocytes(PMNL), chiefly neutrophils, elicited with intraperitoneal injectionsof proteose peptone, was examined. At 10⁶ PMNL/ml in media containinghalide (X^–), 0.14 M Cl^– ± 0.1 mM Br^–(without Ca⁺⁺ and Mg⁺⁺), addition of 10 nM phorbol myristateacetate (PMA) resulted in generation of superoxide anion andH₂O₂. Subsequent cetyltrimethylammonium Cl^– (Cetac) additionat 0.002% effected myeloperoxidase (MPO) activity release. PMNLtreated with PMA and/or Cetac did not metabolize N-OH-2-FAA(30 µM). However, 1–2 pulses of H₂O₂ (50 µM)after Cetac addition resulted in oxidation of N-OH-2-FAA toN-acetoxy-2-FAA (<0.5 µM) and 2-nitrosofluorene (2-NOF)(1–2 µM). In the presence of Br^– 2-NOF wasincreased (3–5 µM). The results are consistent withoxidation of N-OH-2-FAA by MPO/H₂O₂ and MPO/H₂O₂/X^– viatwo pathways: one electron oxidation leading to N-acetoxy-2-FAAand 2-NOF, and X^–-dependent oxidation to 2-NOF. N-Acetoxy-2-FAA(10 µM) incubated with PMNL under similar conditions wasconverted non-enzymatically to 4-OH-2-FAA (5 µM) and enzymaticallyto N-OH-2-FAA (3 µM). In the presence of H₂O₂, smalleramounts of these products were formed. Formation of N-OH-2-FAAwas prevented by paraoxon (0.1 mM) suggesting O-deacetylaseactivity. However, accountability for N-acetoxy-2-FAA decreasedwith time, presumably because of binding to cellular macromolecules.With H₂O₂ addition, 2-NOF (10 µM) was converted to 0.5or 0.25 µM 2-nitrofluorene by active PMNL or heat-inactivatedcell lysates, respectively. Low recoveries of 2-NOF were alsoattributed to binding. The results suggest that PMNL may beinvolved in activation of the carcinogenic N-arylhydroxamicacids in vivo.     

Recombinant rat and hamster N-acetyltransferases-1 and -2: relative rates of N-acetylation of arylamines and N,O-acyltransfer with arylhydroxamic acids

Genes for the 290 amino acid, 33–34 kDa cytosolic acetyltransferases(NAT1^* and NAT2^*) from rat and hamster were cloned and expressedin Escherichia coli. Active clones were selected by a simplevisual test for their ability to decolorize 4-aminoazobenzenein bacterial medium by acetylation. These recombinant acetyltransferaseswere analyzed for: (i) N-acetyltransferase, which was assayedby the rate of acetyl coenzyme A-dependent N-acetylation of2-aminofluorene (2-AF) or 4-aminoazobenzene (AAB); (ii) arylhydroxamicacid acyltransferase, assayed by N, O-acyltransfer with N-hydroxy-N-acetyl-2-aminofluorene.Both NAT2s showed first order increases in N-acetylation rateswith increasing 2-AF or AAB concentrations between 5 and 100µM, with apparent K_m values of 22–32 and 62–138µM respectively. Although under the same conditions theN-acetylation rates for the two NAT1s declined by >50%, below5 µM 2-AF or AAB, the NAT rate data fit Michaelis-MentenKinetics, and the apparent K_m values were 0.2–0.9 µM.For N, O-acetyltransferase, the apparent K_m values of the NAT1swere 6 µM, while the K_m values of the NAT2s were 20- to70-fold higher. SDS-PAGE/Western blot analysis of the recombinantacetyltransferases gave apparent relative molecular weights(MW_r) of 31 kDa for both NAT1s and rat NAT2 and 29 kDa for hamsterNAT2. Comparable MW_r values were observed for native hamsterliver NAT1 and NAT2 and for rat NAT1 under the same conditions.Although we did not detect NAT2-like activity in rat liver cytosolpreviously, the present data show that the rat NAT2^* gene doescode for a functional acetyltransferase, with properties similarto those of hamster liver NAT2. The data also indicate thatat low substrate concentrations, NAT1 would apparently playthe predominant role in vivo in N-acetylation and N, O-acyltransferof aromatic amine derivatives, including their metabolic activationto DNA-reactive agents.     

Depletion of O6-methylguanine--DNA methyltransferase and potentiation of 1,3-bis(2-chloroethyl)-1-nitrosourea antitumor activity by O6-benzylguanine in vitro

The overcoming effect of O⁶-benzylguanine on O⁶-methylguanine-DNAmethyltransferase (MGMT)-mediated 1,3-bis(2-chloroethyl)-1-nitrosourea(BCNU) resistance in vitro was evaluated. Depletion of MGMTactivity in Mer⁺ HeLa S3 cells by O⁶-benzylguanine was dose-dependentand a complete loss of MGMT activity was achieved at a concentrationof 0.5 µM. The cytotoxic potential of BCNU on MGMT proficientHeLa S3 (1.10 pmol/mg of protein), SMMC-7721 (0.72 pmol/mg ofprotein) and Cc801 (0.39 pmol/mg of protein) was greatly enhancedwhen cells were exposed to 10 µM O⁶-benzylguanine for1 h, but there was a lack of potentiation of BCNU sensitivityin Mer^– HeLa MR cells due to its nearly undetectable levelof MGMT. There existed a correlation between the extent of enhancementand the amount of MGMT activity. The intensity of enhancementexpressed as dose modifying factor = IC₅₀ (BCNU alone)/IC₅₀(10 µM O⁶-benzylguanine + BCNU) was 4.56, 3.89, 3.67 and0.97 in HeLa S3, SMMC-7721, Cc801 and HeLa MR cells respectively.The results further demonstrated that O⁶-benzylguanine may havepotential utility as an adjuvant in combination chemotherapywith chloroethylnitrosourea agents.     

Inhibition of aflatoxin B1 mutagenesis in Salmonella typhimurium and DNA damage in cultured rat and human tracheobronchial tissues by ellagic acid

Ellagic acid (EA), a plant phenol found in various fruits andnuts, was examined for its ability to inhibit aflatoxin B₁ (AFB₁)mutagenesis in strain TA 100 of Salmonella typhimurium. In thepresence of rat liver S-9 microsomal preparation, EA (1.5 µg/plate)inhibited the number of mutations induced by AFB, (0.5 µg/plate)by 50%. EA at a dose of 1000 µg/plate inhibited the mutationfrequency by >90%. EA was also tested for its ability toinhibit the DNA binding and adduct formation of AFB₁ in culturedexplants of rat trachea and human tracheobronchus. Explantswere incubated in medium containing EA at concentrations of10, 50 and 100 µM for 16 h foUowed by the addition of1 µM [₃H]AFB₁ and EA for 24 h. DNA was isolated by phenolextraction and hydroxylapatite chromatography. EA caused a dose-dependentinhibition in the covalent binding of AFB₁ to the DNA of boththe rat trachea (9—57% inhibition) and human tracheobronchus(24—79% inhibition). After acid hydrolysis of the isolatedDNA, the AFB₁—DNA adducts were separated by h.p.l.c. Intissues from both species, the major AFB₁—DNA adductswere AFB₁-N⁷-Gua [8,9-dihydro-8-(N⁷-guanyl)-9-hydroxyAFB₁] andAFB₁-N⁷-FaPyr (major) [8,9-dihydro-8- (2, 6-diamino-4-oxo-3,4-dihydro-pyrimid-5-ylformamido)-9-hydroxyAFB₁], and the formation of these adductswas reduced by 28—76% in the presence of EA. These dataindicate that EA has the potential to act as a naturally occurringinhibitor of AFB₁-related respiratory damage in rats and inhumans.     

Use of an orthogonal design method to study two-stage chemical carcinogenesis in BALB/3T3 cells

An orthogonal design method was used to study two-stage chemicalcarcinogenesis in BALB/3T3 cells. Four factors were studied:(0 different concentrations of the initiator N-methyl-N-nitro-N'-nitrosoguanidine(MNNG); (ii) different concentrations of the promoter 12-O-tetradecanoyl-phorbol-13-acetate(TPA); (iii) different concentrations of fetal calf serum (FCS)and (iv) different times at which TPA was added after cell initiation.From the results of three experiments designed by Table L₉(3⁴),L₈(2⁷) and L₁₆(4⁵), 0.3µg/ml MNNG was the highest possibleinitiating concentration and 0.25 µg/ml TPA was the minimumeffective concentration for promoting activity. There is synergybetween MNNG and TPA, the optimum combination in sequentialtreatment being 0.3 µg/ml MNNG and 0.25 µg/ml TPA.The 10% FCS standard concentration was the optimal one; however,below 5% few foci appeared. The time at which TPA was addedhad little effect on cloning efficiency and transformation frequency.So the use of this orthogonal design in cell culture has manyadvantages: several factors can be tested simultaneously; itis easy to find the optimal protocol conditions and the dose-responserelationship is stable, which enables the reproducibility tobe improved. In addition, the different tables proposed mayhelp to reveal unexpected problems.     

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.     

Biotransformation of aflatoxin B1 in human lung

In addition to being a potent hepatocarcinogen, aflatoxin B₁(AFB₁) is a pulmonary carcinogen in experimental animals, andepidemiological studies have shown an association between AFB₁exposure and lung cancer in humans. This study investigatedAFB₁ bioactivation and detoxification in human lung tissue obtainedfrom patients under-going clinically indicated lobectomy. [³H]AFB₁was bioactivated to a DNA binding metabolite by human wholelung cytosols in a time-, protein concentration-, and AFB₁ concentration-dependentmanner. Cytosolic activation of [³H]AFB₁ correlated with lipoxygenase(LOX) activity and was inhibited by the LOX inhibitor nordihydroguaiareticacid (NDGA; 100 µM), indicating that LOXs were largelyresponsible for the observed cytosolic activation of AFB₁. Inwhole lung microsomes, low levels of indomethacin inhibitableprostaglandin H synthase (PHS)-mediated [³H]AFB₁-DNA bindingand cytochrome P-450 (P450)-mediated [³H]AFB₁-DNA binding wereobserved. Cytosolic glutathione S-transferase (GST)-catalyzeddetoxification of AFB₁–8,9-epoxide, produced by rabbitliver microsomes, was minimal at 1 and 10 µM [³H]AFB₁.With 100 µM [³H]AFB₁, [³H]AFB₁–8, 9-epoxide conjugationwith reduced glutathione was 0.34 ± 0.26 pmol/mg/h (n= 10). In intact, isolated human lung cells, [³H]AFB₁ bindingto cellular DNA was higher in cell fractions enriched in macrophagesthan in either type II cell-enriched fractions or fractionscontaining unseparated cell types. Indomethacin produced a 63–100%decrease in [³H]AFB₁-DNA binding in macrophages from five ofseven patients, while NDGA inhibited [³H]AFB₁ -DNA adduct formationby 19, 40 and 56% in macrophages from three of seven patients.In alveolar type O cells, NDGA decreased [³H]AFB₁-DNA bindingby 30–100% in cells from three patients and indomethacinhad little effect. SKF525A, an isozyme non-selective P450 inhibitor,enhanced [³H]AFB₁ binding to cellular DNA in unseparated cells,macrophages, and type II cells, suggesting that P450-mediatedbioactivation of AFB₁ is not a major pathway by which AFB₁–8,9-epoxideis formed in human lung cells. Overall, these studies suggestthat P450 has a minor role in the bioactivation of AFB₁ in humanlung. Rather, LOXs and PHS appear to be important bioactivationenzymes. Co-oxidative bioactivation of AFB₁, in combinationwith the low conjugating activity displayed by human lung cytosolicGSTs, likely contributes to human pulmonary susceptibility toAFB₁.     

Activated neutrophils induce prolonged DNA damage in neighboring cells

We have measured the capacity of highly-purified, paraffin oil-elicitedneutrophils to induce DNA single-strand breaks in a newly establishedplasmacytoma cell line, RIMPC 2304, which was induced by a retroviruscontaining the c-myc and V-Ha-ras oncogenes. This cell lineeffectively repairs DNA damage induced by -irradiation. DNAdamage induced by neutrophils was correlated with the oxidativeburst of the neutrophils. The levels of superoxide anion, H₂O₂and HOCl produced after stimulation of the neutrophils (6 x10⁵/cm³) with the tumor promoter phorbol myristate acetate (100nM) were 33.8 µM, 12.8µM and 1.7 µM respectivelyin 15 mm, and 98 µM, 20 µM and 8.7 µM respectivelyin 90 mm.The results of alkaline elution experiments revealedthat when the same concentration of neutrophils was co-incubatedfor15 min in serum-free medium with an equal number of radioactivelylabeled RIMPC 2304 cells, the latter incurred a level of damagethat approximated that caused by 300 rad equivalents of -irradiationor by a 1-min treatment with 20 µM H₂O₂ at 37C. Damagefrom neutrophils was coincident with the oxidative burst; itwas induced rapidly (within 5 min) but remained high for morethan 90 min. The level of damage achieved was dependent uponthe ratio of neutrophils: target cells and was clearly detectableat ratios as low as 0.25:1. Induction of single-strand breakswas completely inhibited by catalase and partially inhibitedby superoxide dismutase, mannitol, and reduced glutathione butnot by Na azide. Addition of the non-steroidal anti-inflammatorydrug indomethacin either enhanced (at 50 µM) or had noeffect (at 2 µM) on the damage detected. Finally, repairof strand breaks induced by neutrophils was significantly slower(half time 10 min) than that observed for repair of similarlevels of damage induced by H₂O₂ or -irradlatlon (half-times3 min, each). The results indicate that neutrophils cause prolongedDNA damage in neighboring cells. Moreover, they indicate thatalthough H₂O₂ produced in the oxidative burst is an essentialmediator of the damage observed, additional reactive oxygenintermediates including the superoxide anion are also implicated.The data are discussed in relation to the possible role of neutrophilsin chronic inflammation and in pristane-induced plasmacytomaformation in mice.     

Sulfite-dependent mutagenicity of benzo[a]pyrene derivatives

Benzo[a]pyrene (BP) and sulfur dioxide (SO₂) are ubiquitousair pollutants and are also components of tobacco smoke. AlthoughSO₂ itself is not carcinogenic, concurrent administration withBP results in enhancement of respiratory tract tumorigenesis.In biological systems, SO₂ exists as its hydrated form, sulfite(SO₃^2–). Sulfite readily undergoes autoxidation, generatingpotent oxidant species. When 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene(BP-7,8-diol) is included in sulfite autoxidation mixtures itis converted to more polar products, most notably 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrenes(BP tetraols). This implies the intermediacy of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetra-hydro-benzo[a]pyrenes (BPDE). We report herethe sulfite-dependent conversion of BP-7,8-diol to forms highlymutagenic to Salmonella typhimurium strain TA 98. This activationis observed at BP-7,8-diol concentrations of from 2 to 40 µMand at sulfite concentrations of from 0.5 to 10 mM. In the presenceof 10 µM BP-7,8-diol, half-maximal activation is observedat 1.6 mM sulfite. Sulfite itself is neither toxic nor mutagenicto the bacteria under these conditions. The time course of theactivation of BP-7,8-diol and its sensitivity to inhibitionby antioxidants indicate a requirement for sulfite autoxidation.These data further support the sulfite-dependent epoxidationof BP-7,8-diol. Not only does sulfite convert this promutagento its active mutagenic form, sulfite also enhances the mutagenicactivity of BP diolepoxides toward the tester strain. The reversionfrequency in response to 0.1–0.5 µM anti-EPDE isincreased by up to 33% in the presence of 1 mM sulfite, andby up to 270% with 10 mM sulfite. The mechanism of this enhancementof anti-BPDE activity is not known, but could be related toinhibition of the glutathione-S-transferase system which hasbeen previously reported for sulfite. These results are discussedin regard to the noted cocarcinogenicity of sulfur dioxide forBP.     

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.     

DNA binding and adduct formation of aflatoxin B1 in cultured human and animal tracheobronchial and bladder tissues

DNA binding and adduct formation of aflatoxin B₁ (AFB₁) wasstudied in cultured bladder and tracheobronchial explants fromhuman, monkey, dog, hamster and rat. Explants were exposed to[³H]AFB₁ (1 µM final concentration) in PFMR-4 medium (pH7.4) without serum for 24 h, after which epithetial cell DNAwas isolated by hydroxylapatite chromatography. Binding (µmolAFB₁/mol deoxyribo-nucledetide, mean ± SD) was higherin tracheobronchial tissues (human, 2.2 ± 2.4; rat, 5.7± 2.4; dog, 10.6 ± 6.6; hamster 134.6 ±44.6) than in bladder tissues (human, 1.5 ± 2.3; monkey,2.5 ± 1.1; rat, 3.8 ± 1.1; dog, 5.2 ± 2.3;hamster, 26.2 ± 13.3). These binding levels were notcorrelated with the relative susceptibilities of these speciesto AFB₁ hepatocarcinogenesis, in that the hamster and the dogare insensitive, but exhibited the highest binding, while thesusceptible species, the rat and the monkey, had lower binding.After acid hydrolysis of the isolated DNA, the [³H]AFB₁-DNAadducts were separated by h.p.l.c. In all cases, almost allof the [³H]AFB1-DNA represented addition of AFB₁ to the N⁷ atomof guanine, the major adduct (40–79% of the total) being8, 9-dihydro-8-(N⁵-formyl-2', 5', 6' -triamino-4' -oxo-N⁵-pyrimidyl)-9-hydroxyAFB₁,with minor amounts (7–28%) of 8,9-dihydro-8-(N⁷-guanyl)-9-hydroxyAFB₁.In some cases small amounts (0–8%) of unknown, polar adductscould be detected. It is concluded that, qualitatively, AFB₁-DNAadduct formation by human and animal bladder and tracheobronchilalexplants is similar to that found in other in vitro and in vivoextrahepatic and hepatic systems, but that in vitro bindingdata of AFB₁ to extrahepatic animal tissues can probably notbe used to predict the susceptibility of the human to AFB₁-relatedcardnogenesis in these tissues.     

Inhibition of trans-dihydrodiol oxidation by the non-steroidal anti-inflammatory drugs

The homogeneous dihydrodiol dehydrogenase of rat liver cytosol(EC 1.3.1.20 [EC] ) reduces the mutagenicity of benzo[a]-pyrene inthe Ames test, suggesting that the enzyme may detoxify the trans-dihydrodiolproximate carcinogens formed from this compound. This reportdirectly demonstrates for the first time that the purified dehydrogenasecatalyzes the NADP-dependent oxidation of the potent proximatecarcinogen [l, 3–³H](±)-trans-7, 8-dihydroxy-7,8-dihydrobenzo-[ajpyrene at physiological pH. An initial velocityof 1.8 nmol [3H]trans-dibydrodiol oxidized/min/mg protein wasobserved at a substrate concentration of 20 µM. This potentialdetoxification reaction was potently inhibited by the non-steroidalanti-inflammatory drug indomethacin, yielding an IC50 valueof 10 µM. At higher drug concentrations (30 /M), the inhibitionpersisted for many hours. In an extension of this work, benzenedihydrodiol(trans-l, 2-dihydroxy-3, 5-cyclo-bexadiene) and naphthalenedihydrodiol(trans-l,2-dihydroxy-1, 2-dihydronaphthalene) were synthesizedas models of the scarce proximate carcinogen trans-7, 8-dihydroxy-7,8-dihydro-benzo[a]pyrene and examined as substrates. The K_mfor ben-zenedihydrodiol was 1.74 mM and the V_max was 530 nmolsubstrate oxidized/min/mg protein, while the K_m for naphthalenedihydrodiolwas 10.71 mM and the V_max was 268 nmol oxidized/min/mg protein;the former compound was oxidized to catechol. Eight differentnon-steroidal anti-inflammatory drugs were found to inhibitthe oxidation of these model trans-dihydrodiols, yielding IC50values comparable to or lower than peak plasma concentrationsobserved in man. These results suggest that therapeuticallyrelevant concentrations of the non-steroidal anti-inflammatorydrugs may inhibit the oxidation of trans-dihydrodiol proximatecarcinogens by this route.     

Mutagenicity of nitrosated cimetidines

Dinitrosocimetidine and mononitrosocimetidine were tested ina series of short term assay systems. Both compounds were mutagenicin the absence of rat liver microsomes. The dinitrosocimetidineproduced higher mutagenic or clastogenic effects at concentrationsthat were 50 to 500 times lower than the concentrations at whichthe mononitrosocimetidine produces its maximum effects. Themost sensitive short term assay system was the Chinese hamsterovary cell culture system. Dinitrosocimetidine caused sisterchromatid exchanges at a concentration of 10^–8 M and chromosomeaberrations at 10^–7 M in this system. Dinitrosocimetidinehad moderate activity in the bacterial short term assay systems.In the Ames test, strain TA 100 was the most sensitive. Thecompound was of lower activity in the E. coli WP-2 and the E.colirec_– systems.     

The phorbol ester TPA markedly enhances the binding of calcium to the regulatory domain of protein kinase C {beta}1 in the presence of phosphatidylserine

Activation of protein kinase enzyme activity by Ca²⁺ and diacylglycerolor phorbol esters is a feature of certain isoforms of proteinkinase C (PKC). Although the binding sites of phorbol esteron the regulatory domain of PKC have been extensively studied,little is known about the actual mechanism of Ca²⁺ binding andhow this leads to enzyme activation. We previously reportedthat high affinity binding of ⁴⁵Ca²⁺ to the regulatory domainof PKCß1, expressed as a GST fusion protein in Escherichiacoli, is dependent on the presence of phosphatidylserine (PS)or 12O-tetradecanoylphorbol-13-acetate (TPA). In the presentstudy we have used this system to further analyze Ca²⁺ binding.Using various deletions, we found that different domains inthe regulatory domain of PKCß1 are involved in TPA-inducedCa²⁺ binding, depending on whether or not PS was also presentin the binding assay. In addition, Ca²⁺ binding in the presenceof TPA alone displayed very different kinetics than Ca²⁺ bindingin the presence of TPA and PS. Scatchard analysis indicatedthat in the presence of TPA, the K_d value for Ca²⁺ binding was51.9 µM. However, in the presence of both TPA and PS,the K_d value dropped to 0.23 µM. These results providedirect evidence that TPA activates certain isoforms of PKC byenhancing PS-dependent Ca²⁺ binding, thus decreasing the K_dvalue for Ca²⁺ binding to a physiological level.     

The enzymatic conjugation of glutathione with bay-region diol-epoxides of benzo[a]pyrene, benz[a]anthracene and chrysene

The kinetics of the enzymatic conjugation of glutathione (GSH)with the anti-diastereoisomers of trans-7, 8-dihydroxy-9, 10-epoxy-7,8, 9, 10-tetrahydrobenzo[a]pyrene (BPDE), trans-3, 4-dihydroxy-1,2-epoxy-1, 2, 3, 4-tetrahyd-robenz[a] anthracene (BADE) andtrans-1, 2-dihydroxy-3, 4-epoxy-1, 2, 3, 4-tetrahydrochrysene(CDE) catalyzed by transferase 4-4 from rat liver have beencompared. When the concentration of these diol-epoxides wasvaried (using 2mM GSH) the apparent V_max values were 560, 2100and 1500 nmol/ing/min for (?)-anti-BPDE, (?)-anti-BADE and (?)-anti-CDE,respectively, with corresponding apparent K_m values of 11, 125and 105 µM. The catalytic efficiency of transferase 4–4in the GSH conjugation of (?)-anti-BADE and (?)-anti-CDE isthus approximately one-third of (?)-anti-BPDE (0.014 and 0.012s^-1 µM^-1 respectively versus 0.042 s^-1µM^-1). Similarnon-linear Lineweaver-Burk plots were obtained with each diol-epoxidewhen the concentration of GSH was varied, and two apparent.K_m values of 0.02–0.04 and 0.4–0.9 mM GSH were estimated.The GSH-conjugates formed with the individual enantiomers ofthe recemic substrates used were resolved by h.p.l.c. The dataindicate that with each diol-epoxide transferase 4–4 ishighly selective(95%) towards the biologically most active (+)-enantiomer.     

Oxidation of N-butyl-N-(3-formylpropyl)nitrosamine to N-butyl-N-(3-carboxypropyl)nitrosamine in rat liver and inhibition by disulfiram

The metabolism of N-butyl-N-(3-formylpropyl)nitrosamine, a presumptiveintermediate metabolite of the urinary bladder carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine,by rat liver has been examined. N-Butyl-N-(3-formylpropyl)nitrosaminewas metabolized by an NADH-dependent reduction to N-butyl-N-(4-hydroxybutyl)nitrosamineand by an NAD⁺-dependent oxidation to N-butyl-N-(3-carboxy-propyl)nitrosamine.The reduction of N-butyl-N-(3-formyl-propyl)nitrosamine wasinhibited by pyrazole. The oxidation of N-butyl-N-(3-formylpropyl)nitrosaminewas studied further. The rate of oxidation in total rat liverwas 3µmol/min/g liver or 21 nmol/min/mg protein and wassimilar to that found for the oxidation of propionaldehyde,a model substrate for isozymes of rat liver aldehyde dehydro-genase.The rate of oxidation of N-butyl-N-(3-formyl-propyl)nitrosamineby isozymes in rat liver cytosol was 2–2.5 times thatfound for propionaldehyde. The apparent K_m for the NAD⁺-dependentoxidation of N-butyl-N-(3-formyl-propyl)nitrosamine was 20–30µM, which is considerably lower than values reported forknown substrates of aldehyde dehydrogenase. The NAD⁺ -dependentoxidation of N-butyl-N-(3-formylpropyl)nitrosamine was inhibited40–50% by 50 µM disulfiram, 60–70% by 100µM disulfiram, and 50% by 0.4 mM sodium arsenite. Thesestudies show that N-butyl-N-(3-formylpropyl)nitrosamine is veryrapidly oxidized to N-butyl-N-(3-carboxypropyl)nitrosamine inrat liver by aldehyde dehydrogenase and the results may helpto explain why the 3-formylpropyl intermediate has not beendirectly identified as a metabolite of N-butyl-N-(4-hydroxybutyl)nitrosaminein urine or in isolated hepatocytes.     

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.     

Assessment of chemically-induced DNA repair in rat tracheal epithelial cells

An assay for measuring chemically-induced DNA repair in primarycultures of rat tracheal epithelial (TE) cells has been developedand characterized. Chemical exposure may be either in vitroor in vivo. Epithelial cells were removed from the trachea byprotease digestion, allowed to attach to collagen-coated glassslides, and incubated with [³H]-thymidine. DNA repair was assessedas unscheduled DNA synthesis by quantitative autoradiography.The direct acting genotoxicants methyl methanesulfonate (100µM) and N-methyl-N'-nitro-N-nitrosoguanidine (10 µM)yielded a positive response in vitro. 1, 6-Dinitropyrene (DNP)(0.05 µM) and dimethylnitrosamine (DMN) (1 mM) were alsopositive in vitro demonstrating that TE cells have the capacityto metabolically activate these compounds. 2-Acetylaminofluorene(AAF), aflatoxin B₁ (AFB₁), and benzo[a]pyrene (BP) were allnegative in vitro, suggesting organ specific patterns of metabolicactivation. DMN, which has been shown to induce DNA repair inTE cells following exposure by inhalation, was negative whenadministered by gavage. 1, 6-DNP, BP and AAF did not induceDNA repair or alter the fraction of cells in S-phase when administeredby gavage. Formaldehyde did not induce DNA repair or increasethe fraction of cells in S-phase in TE cells following eitherin vivo exposure by inhalation (0.47, 2, 5.9 or 14.8 p.p.m.for 1, 3 or 5 days) or exposure of the cultured cells in vitro(100 µM). This assay provides the means to assess thegenotoxic potential of environmental chemicals in the epithelialcells of the respiratory system.     

Apoptotic and anti-proliferative effects of fumonisin B1 in human keratinocytes, fibroblasts, esophageal epithelial cells and hepatoma cells

Fumonisin B₁ is associated with various animal and human carcinomasand toxicoses, including leukoencelphalomalacia, hepatocarcinoma,pulmonary edema and esophageal carcinoma. We have examined thecellular effects of fumonisin B₁ in vitro using cellular modelsystems relevant to potential human target tissues. Althoughfumonisin B₁ has been described as a mitogen in Swiss 3T3 cellsbased on stimulation of [³H]thymidine incorporation, in thecurrent work it was found that fumonisin B₁ inhibited incorporationof [³H]thymidine by cultured neonatal human keratinocytes andHepG2 human hepatocarcinoma cells at 10^–7 and 10^–4M respectively. Fumonisin B₁ also inhibited clonal expansionof normal human keratinocytes and HET-1A human esophageal epithelialcells at 10^–5 M and growth in mass culture of normal humanfibroblasts at 10^–7 M. The clonogenicity of normal humankeratinocytes decreased to 45.5% of controls afterexposure to10^–4 M fumonisin B₁ for 2 days. However, no differencesin the cell cycle distribution of cultured keratinocytes wasnoted after exposure to 10^–5 M fumonisin B₁ for 40 h.Theviability of normal human keratinocytes and HET-1A cellsdecreased as a result of fumonisin B₁ treatment, as determinedby a fluorescein diacetate/propidium iodide flow cytometriccell viability assay. Fumonisin B₁-treated keratinocytes releasednucleosomal DNA fragments into the medium 2–3 days afterexposure to 10^–4 M fumonisin B₁ and increased DNA strandbreaks were detected in attached keratinocytes exposed to 0–10^–4M fumonisin B₁ using a terminal deoxy-nucleotidyl transferase-basedimmunochemical assay system. Furthermore, fumonisin B₁-treatedkeratinocytes and HET-1A cells developed morphological featuresconsistent with apoptosis, as determined by phase contrast microscopy,fluorescent microscopy of acridine orange stained cells andelectron microscopy. These results are consistent with the occurrenceof fumonisin B₁-mediated apoptosis in vitro.     

In vitro binding of aflatoxin B1 and 2-acetylaminofluorene to rat, mouse and human hepatocyte DNA: the relationship of DNA binding to carcinogenicity

DNA binding levels were determined and compared in culturedhepatocytes from male and female rats as well as other animalspecies following exposure to aflatoxin B₁ (AFB₁) or 2-acetylaminofluorene(2-AAF). When human, rat (both male and female) and mouse hepatocytesin primary culture were exposed to 2.0 ? 10^–7 M [³H]AFB₁(sp. act. 2.63 µCi/nmol) for 24 h, male rat hepatocyteshad the highest degree of [³H]AFB₁-DNA binding (203 pmol/mgDNA) and human hepatocytes contained the next highest bindinglevel (42 pmol/mg DNA). Hepatocytes from female rats contained38 pmol/mg DNA while cultured mouse hepatocytes contained only1.4 pmol/mg DNA. When the same dose of [³H]AFB₁ was administeredto the cultured male rat hepatocytes at 24 h, 48 h, 72 h and1 week after seeding, and incubated for 24 h, the DNA bindinglevels were 189, 175, 76, 75 pmol/mg DNA respectively. In parallelexperiments to the cultured male rat hepatocytes above, theAFB₁-DNA binding levels in the cultured female hepatocytes were42, 41, 37 and 34 pmol/mg DNA respectively. Human, male andfemale rat hepatocytes in primary culture were exposed to 5.2? 10^–5 M 2-acetyl-amino [9–¹⁴C]fluorene (sp. act.0.0094 µCi/nmol) for 24 h. It was determined that malerat hepatocytes contained the highest amount of radioactivelylabeled 2-AAF bound to their DNA (1.57 nmol/mg DNA), femalerat hepatocytes contained 0.62 nmol/mg DNA and human hepatocytescontained 0.29 nmol/mg DNA. Results from our in vitro hepatocyteculture system correlate well with in vivo animal studies dealingwith species and sex differences in DNA binding and carcinogenicsusceptability. This indicates that hepatocytes in vitro maintainmany of the biological properties necessary for carcinogen responsesimilar to liver cells in vivo. In addition, comparison of genotoxiceffect in cultured hepatocytes from animals as well as humansmay be useful in evaluating carcinogenic potential of xenobioticsin human liver.