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.     

Comparative kinetic studies on aflatoxin B1- DNA binding and aflatoxin B1-glutathione conjugation with rat and hamster livers in vitro

Inhibition of microsome mediated aflatoxin B₁ (AFB₁) bindingto exogenous or endogenous DNA by cytosolic glutathione (GSH)S-transferases is well established from our earlier studies.Correlation between inhibition of AFB₁-DNA binding and AFB₁-GSHconjugation in vitro using rat and hamster liver subcellularfractions is elucidated in this report. Even though hamsterliver microsomes catalyzed AFB₁ binding to exogenous DNA threetimes as much as the rat, hamster cytosol inhibited AFB₁-DNAbinding catalyzed by either microsomes severalfold more thanthe rat cytosol. AFB₁ - DNA binding is found to be inverselyrelated to AFB₁-GSH conjugation at all AFB₁ concentrations (2–100µM)studied. Presence of either styrene oxide or 3,3,3-trichloropropeneoxide at 1 mM level diminished AFB₁-GSH formation in vitro confirmingsome competition by these epoxides with AFB₁-epoxide for cytosolicGSH S-transferases. In a reconstituted system with endogenousDNA, the ratio of AFB₁-GSH to AFB₁-DNA binding was found tobe 10–15 times higher with the hamster in comparison withthe rat indicating enhanced inactivation of the ultimate carcinogenicmetabolite in the hamster. These results are discussed in relationto AFB₁-DNA binding and AFB₁ hepatocardnogenicity in resistantand sensitive species.     

Structure-activity relationships of arylalkyl isothiocyanates for the inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone metabolism and the modulation of xenobiotic-metabolizing enzymes in rats and mice

Many arylalkyl isothiocyanates are potent inhibitors of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK)-induced lung tumorigenesis in rats and mice. In the mouse,4-phenylbutyl isothiocyanate (PBITC) and 6-phenylhexyl isothiocyanate(PHTTC) exhibited greater inhibition than benzyl isothiocyanate(BITC) and phenethyl isothiocyanate (PEITC). The present studywas conducted to investigate the structure-activity relationshipsof these four arylalkyl isothiocyanates for their inhibitionof NNK oxidation and effects on xenobiotic-metabolizing enzymesin rats and mice. A single dose (0.25 or 1.00 mmol/kg) of eachisothiocyanate was given to F344 rats 6 or 24 h before death.The rates of NNK oxidation were decreased in microsomes fromthe liver, lung and nasal mucosa of rats. Generally, PEITC wasmore potent than BITC but less potent than PBITC and PHlTC.The rates in rat liver microsomes were decreased at 6 h butrecovered or increased at 24 h; and the rates in rat lung microsomeswere markedly decreased at both 6 and 24 h; and the rates inrat nasal mucosa microsomes were also significantly decreased.The same treatment decreased the rat liver N-nitrosodimethyl-aminedemethylase activity dramatically and ethoxyresorufin O-dealkylaseand erythromycin N-demethylase activities moderately. However,the rat liver microsomal pentoxy-resorufin O-dealkylase activitywas decreased at 6 h but increased at 24 h, with PEITC showingthe most marked induction. The rat liver NAD(P)H: quinone oxidoreductaseactivity was increased 1.4- to 3.3-fold, with PEITC being mosteffective; and the glutathione S-transferase activity was increasedslightly. Similarly, at a single dose of 0.25 mmol/kg (5 µmol/mouse)24 h before death, PEITC, PBITC, PHlTC but not BITC, decreasedNNK oxidation in mouse lung microsomes by 40–85%, withPBITC and PHlTC showing greater inhibition. Furthermore, allfour isothiocyanates extensively inhibited NNK oxidation inrat lung and nasal mucosa microsomes as well as mouse lung microsomesin vitro, with PEITC (IC₅₀ of 120–300 nM) being more potentthan BITC (IC₅₀ of 500–1400 nM) but less potent than PBITCand PHITC (IC50 of 15–180 nM). PHITC was a very potentcompetitive inhibitor of NNK oxidation in mouse lung microsomeswith apparent K₁ values of 11–16 nM. These results indicatethat PBITC and PHITC are more potent inhibitors of NNK bioactivationin rats and mice than PEITC. In addition, these arylalkyl isothiocyanatescould be effective in protecting against the actions of a broadspectrum of carcinogenic or toxic compounds.     

Elevated levels of ornithine decarboxylase and polyamines in the kidneys of estradiol-treated male hamsters

The effects of 17 ß-estradiol (E₂) on the levels ofornithlne decarboxytase (ODC), S-adenosylmethionine decarboxylase(SAMDC), and of the polyamines putrescine, spermidine and sperminein the kidneys of castrated male hamsters were determined. Thei.p. injection of E₂ into male hamsters led to renal ODC levelsthree times above the control levels 6–12 h after treatment.Similarly, the renal ODC levels in hamsters treated with chronicdoses of E₂ for 60–180 days were 13–1.9 times thecorresponding enzyme levels in control, sham-operated animals.With a series of estrogen analogues, there was a direct correlationbetween the rise in renal ODC in vitro and the binding to renalestradiol receptor sites in vitro. The hamster kidney levelsof the polyamines putrescine, spermidine and spermine all declinedduring the 180-day experimental period. A single i.p. injectionof E led to a 70% increase in renal SAMDC activity 12 h aftertreatment. However, the administration of E for 180–270days was without effect on the normal age-dependent declinein SAMDC levels noted in kidneys. These results indicate that,like other carcinogens and promoters, E₂ increases the levelsof ODC and of polyamines in its target tissue and that the risein ODC is mediated by a specific estradiol-binding protein.     

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.     

The activation of aflatoxin B1 in liver slices and in bacterial mutagenicity assays using livers from different species including man

The activation of aflatoxin B₁ (AFB₁) has been compared in twoin vitro systems: (1) binding to DNA in liver slices incubatedwith [¹⁴C] or [³H]AFB₁; (2) standard bacterial mutation systemsusing 9000 x g supernatant (S-9) fractions from uninduced liversfor activation. Several factors which modify aflatoxin carcinogenesiswere investigated, namely species, sex, phenobarbitone pretreatmentand aflatoxin G₁ (AFG₁) compared with AFB₁. The results fromDNA binding in liver slices showed the following trends: rat> hamster > mouse, control > phenobarbitone-pretreatedrat and AFB₁ > AFG₁ which correlated directly with trendsin carcinoenicity. An exception to this trend was the similarlevel of binding found in male and female rat livers, the latterbeing less susceptible to AFB₁ carcinogenesis. This result suggeststhat sex differences in AFB₁ carcinogenicity may be due to differencesin repair of lesions or during the promotion phase of carcinogenesis.The levels of binding of [³H]AFB₁ to DNA slices from fresh humanliver biopsies showed considerable variation between the sixsamples. Values ranged from 0.7–8.5 ng AFB₁/mg DNA, whichare in between values observed in the hamster and mouse. Mutagenicitydata did not correlate with carcinogenicity in relation to speciesdifferences (hamster > rat > mouse) nor phenobarbitonepretreatment. Supplementation of the top agar mixture with glutathioneand/or pre-incubation of S-9, AFB₁ and cofactors did not improvethis correlation. Nevertheless, it is expected that differencesbetween these two systems are due to limitations of the metabolizingsystem in mutagenicity tests, rather than either DNA bindingor bacterial mutation being the more valid end point.     

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.     

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.     

Metabolism of the cis and trans isomers of N-nitroso-2,6-dimethylmorpholine and their deuterated analogs by liver microsomes of rat and hamster

Liver microsomes from male Syrian golden hamsters and SpragueDawley rats metabolize the cis and trans isomers of N-nitroso-2,6-dimethylmorpholine(NNDM) to N-nitroso-(2-hydroxypropyl)(2-oxopropyl)amine (HPOP)as the major product detectable by h.p.l.c. The rates of totalmetabolism are similar for both the cis and trans isomers; butthe cis isomer of NNDM yields >70% of the total product asHPOP while the trans isomer yields HPOP only as a minor product(20–30%) in both hamster and rat. The inability to identifyother products could be attributed to -hydroxylation which leadsto fragmentation of NNDM and loss of tritium label to water.In order to investigate the possibility of the participationof an -hydroxylation reaction, the metabolism of NNDM fullydeuterated at either the 3 and 5 (-d₄) or the 2 and 6 (ß-d₂)positions was examined and compared to the metabolism of theundeuterated compound (d0). Although the rates of metabolismof all of the cis and trans derivatives of NNDM were similar(^Vmax = 2.13 nmol/min/mg hamster microsomal protein) as determinedfrom measurements of substrate disappearance, the yields ofHPOP were different. Maximum HPOP yields were observed withcis -(d₄) NNDM (93.9% of the total), followed by cis d₀ NNDM(72.3%), trans -(d₄) NNDM (60.1%), trans d₀ NNDM (30.2%), cisß-(d₂) NNDM (19.5%) and trans ß-(d₂) NNDM(8.5%). These results suggest that a-hydroxylation is an alternativeto ß-hydroxylation. Since the carcinogenic potencyof the various deuterium derivatives of NNDM for the Syriangolden hamster parallels their ability to yield HPOP, ß-hydroxylationis closely related to pancreatic carcinogenesis in the hamster.Rat liver microsomal fractions showed the same patterns of HPOPformation to total metabolite yields as hamster liver microsomeswith both the cis and trans isomers. However, rates of NNDMmetabolism and HPOP formation were 7 times faster with hamsterthan with rat liver microsomes. Such a difference may be relatedto the failure of the cis isomer to induce pancreatic cancerin rats.     

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.     

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.     

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.     

Catechol-induced alterations in metabolic activation and binding of enantiomeric and racemic 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrenes to DNA in mouse skin

Catechol (1,2-dihydroxybenzene) is a potent co-carcinogen withbenzo[a]pyrene (BaP) and with (?)-7,8-dihydroxy-7, 8-dihydrobenzo[a]pyrene(BaP-7, 8-diol) in mouse skin. The effects of catechol on themetabolic activation of (+)- and (–)- [³H]BaP-7,8-diolsand on epidermal DNA adduct formation of racemic and enantiomeric[³H]BaP-7, 8-diols were examined by applying the tritlated diolsto mouse skin. The major metabolite of the (+)- [³H]BaP-7, 8-diolswas the hydrolysis product of (–)- [³H]-7,8ß-diolsepoxy-9ß,10ß-epoxy-7,8,9, 10-tetrahydroheiizo[a]pyrene (anti-BPDE).This suggests that a peroxyl radical-mediated pathway Is predominantlyresponsible for the epoxidation of this diol. Formation of (–)-anti[³H]BPDEfrom (+)-[³H]BaP-7,8-diol was greater than that of (+)-anti-BPDEfrom (–)-[³H]BaP-7,8-diol Co-application of catechol with[³H]BaP-7,8-diols inhibited epoxidation of the (+) enantiomerto a greater extent than that of the (–) enantiomer. Catecholdecreased the total DNA-binding and the formation of the majoradduct with (+)-[³H]BaP-7, 8-diols metabolites but catecholhad no significant effect on the binding and formation of (+)-anti-[³H]BPDE-deoxyguanosine the major DNA adduct derived from (–)-[³H]BaP-7,8-diolsCo-administration of catechol with (?)-[³H]BaP-7,8-diols increasedthe ratio of (–)- to (+)-[³H]BaP-7, 8-diols major DNAadducts in mouse skin suggesting that catechol selectively inhibitscertain pathways of metabolic activation of (? )-[³H]BaP-7,8-diols Thus, catechol modifies the tumorigenic activity of(?)- BaP-7 ,8.-diol either by alteration of the relative proportionof various hydrocarbon:DNA adducts or by a totally differentas yet unexplored mechanism.     

Cytotoxicity and genotoxicity of ({+/-})-benzo[a]pyrene-trans-7,8-dihydrodiol in CYP1A1-expressing human fibroblasts quantitatively correlate with CYP1A1 expression level

Cytochrome P450 1A1 (CYP1A1) activity is associated with increasedsusceptibility to lung cancer induced by polycyclic aromatichydrocarbons such as benzo[a]pyrene (BP). In non-hepatic humantissues, CYP1A1 is the principal enzyme responsible for themetabolic activation of the proximate BP mutagenic metabolite,(–)-benzo[a]pyrene-trans-7,8-dihydrodiol, to (+)-anti-benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide,the ultimate BP mutagen. We have genetically engineered bothDNA repair-deficient (xeroderma pigmentosum group A) and DNArepair-proficient human skin fibroblasts to express human CYP1A1under control of the inducible mouse metallothionein-I promoter.CYP1A1 activity was induced by CdSO₄ and monitored by followingthe O-deethylation of ethoxy fluorescein ethyl ester or of 7-ethoxyresorufin.Induced CYP1A1 activities were similar in both cell lines andwere dependent on CdSO₄ concentration and induction time. MaximalCYP1A1 activities were obtained in 4–6 h with 5–7µM CdSO₄. BPD-induced cytotoxicity and hypoxanthine phosphoribosyltransferase mutagenicity were both quantitatively correlatedwith the level of CYP1A1 activity and were greater in DNA repair-deficientcells than in DNA repair-proficient cells. The results suggestthat modestly induced CYP1A1 activity is a risk factor in polycyclicaromatic hydrocarbon-induced carcinogenesis.     

Production of monoclonal antibodies to guanine imidazole ring-opened aflatoxin B1DNA, the persistent DNA adduct in vivo

Monoclonal antibodies were produced following immunisation ofmice with guanine imidazole ring-opened aflatoxin B₁ DNA (iroAFB₁ DNA), coupled electrostatically to methylated keyhole limpethaemocyanin. Three monoclonal hybridoma lines producing antibodiesspecific for iro AFB₁ DNA were grown as ascites tumours andsuitable dilutions of the ascitic fluid (1:8000–1:50 000)used in a competitive enzyme linked immunosorbent assay (ELISA)to measure reactivity of the antibodies to a variety of aflatoxinand nucleic acid-related compounds. These antibodies recogniseAFB₁ bound to DNA at levels 10⁴–10⁵ times lower concentrationthan unmodified calf thymus DNA or 8,9-dihydro-8,9-dihydroxy-aflatoxinB₁; and show 2–5 times the affinity to iro AFB₁ DNA comparedto AFB₁ DNA. The concentration of AFB₁ in iro AFB₁ DNA producing50% inhibition in a competitive ELISA was 1.8 x 10^–7 molar.Using the most sensitive hybridoma line, levels of 1 adductin 300 000 nudeotides would be detectable, which is the levelof binding found in the rat and hamster in vivo. These monoclonalantibodies should therefore prove useful in detecting theselesions in animal and human tissue samples exposed to aflatoxins.     

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.     

Survey of cytotoxicities and antimutagenic and antitumor initiating activities of Cu(II)(3,5-diisopropylsalicylate)2 and its analogs in a keratinocyte-mediated mutation assay and the murine skin multistage carcinogenesis model

A keratinocyte-mediated mutagenesis assay, and the murine skinmultistage carcinogenesis tumor model were used to survey thechemopreventive properties of Cu(II)(3,5-diiso-propylsalicylate)₂[CuDIPS]and its analogs. Supplementation of cocultures of newborn SENCARkeratinocytes and Chinese hamster lung fibroblasts (V79 cells)with CuDIPS, 3,5-diisopropylsalicylate (DIPS), and CuSO₄ resultedin dose-dependent killings of V79 cells (LD₅₀ of 34, 75, 960µM, respectively), and inhibitions of benzo [a]pyrene(BP) and 7,12-dimethylbenz[a]anthracene (DMBA) mutagenesis (ED₅₀of 13, 95, 80 µM, and 40, 125, 110µM, respectively).Analyses of dose-response curvessuggest (i) CuDIPS preferentiallyin hibits BP mutagenesis; (ii) the antimutagemc activity ofCuDIPS towards DMBA and the cytotoxicity of the copper complexare derived from the DIPS component of the chelate; (iii) theantimutagenic activity of CuDIPS towards BP requires both copperand DIPS; and (iv) DIPS and CuDIPS induced cytotoxicity is requiredfor inhibition of mutagenesis. Inhibition of mutagenesis byCuDIPS was not mediated by modulation of promutagen metabolismbecause antimutagenic concentrations of the chelate had no significanteffects on DMBA- and BP-dependent cytotoxicities. Topical pretreatmentof SENCAR mice with CuDIPS (100–4000 nmol) 15 mm priorto initiation with DMBA or BP resulted in small (38% maximum)non-dose-responsive reductions of papillomas/mouse following20 weeks of promotion.     

The model Ah-receptor agonist {beta}-naphthoflavone inhibits aflatoxin B1--DNA binding in vivo in rainbow trout at dietary levels that do not induce CYP1A enzymes

ß-Naphthoflavone (BNF), a well-known Ah-receptor agonist,has been believed to inhibit aflatoxin B₁(AFB₁) carcinogenesisin rats and rainbow trout primarily through induction of thecytochrome P450 1A (CYP1A) enzyme subfamily and consequent diversionof AFB₁ to the less carcinogenic phase I metabolite aflatoxinM₁(AFM₁). This study investigates the dose responsive effectsof dietary BNF treatment on CYP1A induction, AFM₁ formation,AFB₁–8, 9-epoxide formation and AFB₁-DNA binding in thetrout model. Pre-feeding diet containing 10–200 p.p.m.BNF after AFB₁ i.p. injection provided dose-dependent inductionof CYP1A-dependent ethoxyresorufin-O-deethylase (EROD) activityand inhibition of in vivo AFB₁-DNA binding. However, most ofthe observable inhibition of DNA adduction (45% inhibition)had occurred at 10 p.p.m. BNF without detectable EROD induction;higher doses of BNF up to 200 p.p.m. induced EROD >6-foldbut provided only another 15% inhibition of DNA adduction invivo. When in vitro AFB₁-DNA binding was assessed using livermicrosomes from trout fed 10–100 p.p.m. BNF, induced microsomalEROD activity correlated moderately with reduction of in vitroAFB₁-DNA binding activity. However, BNF treatment in a low doserange (0.2–10 p.p.m.) also strongly inhibited in vivohepatic AFB₁-DNA binding (69% inhibition at 5 p.p.m. BNF inthis experiment), in a dose-dependent manner, in the completeabsence of detectable EROD induction. The microsomes from 5p.p.m. BNF-treated trout had no more EROD activity than controlmicrosomes, and no less capacity for catalyzing AFB₁-DNA bindingin vitro than control microsomes. Thus, the potent inhibitionof hepatic AFB₁-DNA binding in vivo by 5 p.p.m. BNF was a resultof neither CYP1A enzyme induction nor irreversibly reduced catalyticcapacity for AFB₁–8, 9-epoxide formation. Direct analysisof AFB₁ metabolites formed in vitro by liver microsomes fromtrout fed 10, 100 and 500 p.p.m. BNF showed that low dietaryBNF (10 p.p.m.) neither induced microsomal CYP1A-mediated AFM₁formation nor altered AFB₁–8, 9-epoxide formation comparedto the control. By comparison, 100 and 500 p.p.m. BNF pretreatmentsignificantly elevated microsome-catalyzed AFM₁ formation invitro (P0.001), and this increase was highly correlated withincreased EROD activity (r² = 0.999, P< 0.001). Upon in vitroaddition, BNF was found to be a potent inhibitor of microsome-mediatedAFB₁–8,9-exo-epoxide formation (IC₅₀=2.6±0.1µM)and AFB₁-DNA binding (inhibition constant K₁= 3.03±0.25µM).These findings indicate that CYP1A enzyme induction can contributemodestly to BNF protection against AFB₁ in this species bothin vivo and in vitro at higher BNF doses, but does not do soat lower doses. Instead, enzyme inhibition by BNF against AFB₁8,9-epoxidation appears to be the predominant protective mechanismat higher BNF doses, and the sole protective mechanism at lowdoses, in the rainbow trout. These findings demonstrate thatmechanisms of chemoprevention can change with anticarcinogendose, and caution that even potent induction of phase I or phaseII activities does not assure that pathway to be a predominantprotective mechanism in vivo.     

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.