Dibenz[a,j]acridine metabolism: identification of in vitro products formed by liver microsomes from 3-methylcholanthrene-pretreated rats

The metabolism of the carcinogenic pentacyclic azaaromatic compound,dibenz[a,j]acridine, has been examined in liver microsomal incubationsusing preparations from 3-methylcholanthrene-pretreated Wistarrats. Using authentic synthetic standards, u.v. spectroscopyand mass spectrometry, the following were proved to be metabolites:trans-5, 6-dihydro-5, 6-dihydroxydibenz[a,j]acridine, trans-3,4-dihydro-3, 4-dihydroxydibenz[a,j]acridine, dibenz[a,j]acridine-5,6-oxide, 3-hydroxydibenz[a,j]acridine and 4-hydroxydibenz[a,j]acridine.The 3, 4-dihydrodiol appeared to be the major metabolite. Thesecondary metabolites were also examined and evidence is presentedfor the additional formation of dibenz[a,j]acridine-5, 6, 8,9-dioxide,tetrols, diol epoxides and phenolic dihydrodiols.     

On the metabolic activation of benz[a]acridine and benz[c]acridine by rat liver and lung microsomes

The metabolism of benz[a]- and benz[c]acridine by liver and lung microsomes from untreated, phenobarbital (PB)-treated and benzo[k]fluoranthene (BkF)-treated rats has been studied by gas chromatography/mass spectrometry (GC/MS). Epoxidation and hydrolysis of the epoxides to dihydrodiols were found to be the predominant pathways for all substrates. N-Oxidation is likely to occur in the case of benz[c]acridine. However, no unequivocal evidence could be obtained for the formation of the ultimate carcinogens — the t-3,4-dihydrodiol-1,2-epoxides — in case of both benz[a]- and benz[c]acridine. K-Region oxidation was induced by phenobarbital, whereas the formation of non-K-region metabolites increased after BkF treatment in the case of benz[c]acridine.     

Metabolism of dibenz[a,h]acridine by rat liver microsomes

As part of a project to assess the effect of heterocyclic nitrogenin modifying the metabolism and mutagenicity of polycyclic aromatichydrocabons, we investigated the metabolism of dibenz[a,h]acridine(DB[a,h]AC) by liver microsomes prepared from male Sprague-Dawleyrats. During a 6-min incubation 21, 14, 0.7 or 0.2 nmol DB[a,h]ACper mg protein were metabolized by microsomes from rats pre-treatedwith DB[a,h]AC, 3-methylcholanthrene (3-MC), phenobarbital (PB)or corn oil, respectively. In each case the predominant metaboliteswere the dihydrodiols with bay-region double bonds, namely,DB[a,h]AC-3,4-dihydrodiol and DB[a,h]AC-10,11-dihydrodiol, eachof which accounted for 21–23% of the total metabolismdetermined during a 7-min incubation with microsomes from 3-MC-treatedrats. Other metabolites produced by these microsomes includedDB[a,h]AC-l,2-dihydrodiol ({small tilde}5% of total metabolites);two K-region oxides [DB[a,h]AC-12,13- and 5,6-oxides (estimatedto represent 5% and 2% of total metabolites, respectively)];several unidentified polar metabolites (10–15%) and severalunidentified metabolites which co-eluted with 3-hydroxy-DB[a,h]AC(20%). DB[a,h]AC-8,9-dihydrodiol was not detected (<2%).The metabolite profiles produced by microsomes prepared fromrats pretreated with DB[a,h]AC, PB or corn oil were very similarto the profile produced by 3-MC-induced microsomes. We concludethat: (i) the potentially mutagenic benzo-ring dihydrodiolswith bay-region double bonds are the predominant metaboliteof DB[a,h]AC; (ii) the heterocyclic nitrogen atom has littleeffect in modifying the relative extents of formation of thesetwo benzo-ring dihydrodiols with bay-region double bonds; (iii)metabolism at the K-region is only a minor pathway for DB[a,h]AC,as is also true for the carbon analogue dibenz[a,h]anthracene;and (iv) induction by a 3-MC-type inducer (e.g. DB[a,h]AC) isrequired for substantial metabolism to occur.     

The metabolism of dibenz[a,j]acridine in the isolated perfused lung

The metabolism of the carcinogenic N-heterocyclic aromatic, dibenz[a,j]acridine (DB[a,j]A), was investigated in an isolated perfused rabbit lung preparation. The rate of metabolism of DB[a,j]A was less than the rate of metabolism of 7H-dibenzo[c,g]carbazole (DB[c,g]C) in the untreated and corn oil-pretreated animals. A significantly increased rate of metabolism was observed for DB[a,j]A in benzo[a]pyrene(B[a]P)-pretreated animals. This resulted in marked increases in conjugation and distribution of conjugates and total metabolites in blood and lung. Two major metabolites characterized spectroscopically were assigned as the 3,4-dihydrodiol and a phenol of DB[a,j]A. The results indicate that in the lung DB[a,j]A is metabolized in a manner similar to that of B[a]P.     

Stereoselective metabolism of dibenz[a,h]acridine to bay-region diol epoxides by rat liver microsomes

The carcinogen dibenz[a,h]acridine (DB[a,h]ACR) is metabolizedpredominantly to trans-3,4-dihydroxy-3,4-dihydrodibenz[a,h]acridine(DB[a,h]ACR-3,4-diol) and the proximate carcinogen trans-10,11-dihydroxy-10,11-dihydrodibenz[a,h]acridine(DB[a,h]ACR-10,11-diol) [Steward et al. (1987) Carcinogenesis,8, 1043–1050]. In the present investigation, the stereoselectivityof rat liver enzymes in metabolism of DB[a,h]ACR to its 3,4-dioland 10,11-diol and of DB[a,h]ACR-10,11-diol enantiomers to theirbay-region diol epoxides has been examined with liver microsomesfrom control and 3-methylcholanthrene-treated rats. Both microsomalpreparations produced the major metabolites DB[a,h]ACR-3,4-dioland DB[a,h]ACR-10,11-diol containtaining predominantly R,R-enantiomerswith 38–54% optical purity. Metabolism of (–)-(10R,11R)-and (+)-(10S,11S)-enantiomers of DB[a,h]ACR-10,11-diol by livermicrosomes from control rats produced predominantly bay-regiondiol epoxides (46–59% of total metabolites), whereas verylittle bay-region diol epoxides (14–17% of total metabolites)were produced by liver microsomes from 3-methylcholanthrene-treatedrats. The bay-region diol epoxides produced in these studiesconsisted of predominantly DB[a,h]ACR-10,11-trans-diol epoxidediastereomer in which the benzylic hydroxyl group and epoxideoxygen are trans. However, (–)-DB[a,h]ACR-10R,11R-diol,a major metabolite of DB[a,h]ACR, was metabolized by liver microsomesfrom 3-methylcholanthrene-treated rats to (+)-[8R,9S,10S,11R]-DB[a,h]ACR-10,11-trans-diolepoxide, a diastereomer which displayed high mutagenic activityin V79 cells, in an amount which was 6.5-fold greater than thatof the corresponding cis-diol epoxide diastereomer. The relativeamounts of trans-diol epoxide versus cis-diol epoxide in themixture of bay-region diol epoxides produced from DB[a,h]ACR-10R,11R-dioland DB[a,h]ACR-10S,11R-diol with liver microsomes from controlrats and from DB[a,h]ACR-10S,11S-diol with liver microsomesfrom 3-methylcholanthrene-treated rats were 1.7, 2.1 and 2.3respectively.     

The mutagenicity of dibenz[a,j]acridine, some metabolites and other derivatives in bacteria and mammalian cells

Dibenz[a,j]acridine (DBAJAC) was studied because of its close structural relationship with a number of important carcinogenic polycyclic and azaaromatic hydrocarbons. It was of particular relevance to examine the mutagenicity of known or proposed 'bay-region' metabolites, which may be proximate or ultimate carcinogenic derivatives of DBAJAC. Trans-1,2-, 3,4- and 5,6-dihydrodiols, the 4- and 6-phenols, the 5,6-oxide and N-oxide derivatives, and anti- and syn-3,4-diol 1,2-epoxides of DBAJAC were examined for their mutagenicity in Salmonella typhimurium TA98 and TA100 and in V79 Chinese hamster lung cells. Of all the compounds studied which require metabolic activation, the 3,4-dihydrodiol was the most active in both TA100 and in V79 cells. The activity of the 3,4-dihydrodiol enantiomers was also tested in strain TA100 where no difference was observed from that of the racemic mixture. In V79 cells only the 3R,4R-dihydrodiol was active, the activity being about three times that of the racemic material. Salmonella strains TA98 and TA100 also differed in their sensitivity towards DBAJAC dihydrodiols, the 1,2-isomer being of greatest activity in TA98. The most mutagenic compounds in both mammalian and bacterial cells were the 'bay-region' diol epoxides of DBAJAC which did not require metabolic activation by S9 mix. The anti-DBAJAC 3,4-diol 1,2-epoxide was more mutagenic than the syn form in V79, TA98 and TA100 cells. Overall these results suggest that the in vivo biological activity of DBAJAC metabolites is likely to reflect previous findings with other similar polycyclic aromatic hydrocarbons.     

Metabolism of 6-methylbenz[a]anthracene by rat liver microsomes and mutagenicity of metabolites

6-Methylbenz[a]anthracene (6-MBA) is metabolized by rat liver microsomes to form 3-hydroxy-6-MBA, 4-hydroxy-6-MBA, 5-hydroxy-6-MBA, 6-MBA trans-3,4-, 5,6-, 8,9-, and 10,11-dihydrodiols, and 4-hydroxy-6-MBA trans-10,11-dihydrodiol as the identifiable metabolites. 6-Hydroxymethylbenz[a]anthracene and its phenolic and dihydrodiol metabolites are also formed. The unique metabolites identified in 6-MBA metabolism are 6-MBA trans-5,6-dihydrodiol and 4-hydroxy-6-MBA trans-10,11-dihydrodiol. Metabolites were isolated by reversed-phase and normal-phase high-performance liquid chromatographies and identified by UV-visible absorption, mass, and proton nuclear magnetic resonance spectral analyses. Metabolites formed by low and high concentrations of liver microsomal enzymes from untreated, phenobarbital-treated, and 3-methylcholanthrene-treated male Sprague-Dawley rats were quantified by using [3H]6-MBA, with the tritium labeled at the methyl carbon, and liquid scintillation counting of fractions collected from reversed-phase high-performance liquid chromatography. Metabolic formations of 6-hydroxymethylbenz[a]anthracene, 6-MBA trans-dihydrodiols, and 4-hydroxy-6-MBA trans-10,11-dihydrodiol are highly dependent on the contents of cytochrome P-450 isozymes present in liver microsomes. The relative mutagenic activities of metabolites toward Salmonella typhimurium TA100 are: 6-MBA trans-3,4-dihydrodiol greater than 6-MBA trans-8,9-dihydrodiol greater than 6-MBA greater than 6-MBA trans-10,11-dihydrodiol greater than 4-hydroxy-6-MBA congruent to 4-hydroxy-6-MBA trans-10,11-dihydrodiol. The relatively high mutagenic activities of 6-MBA trans-3,4-dihydrodiol and 6-MBA trans-8,9-dihydrodiol suggest that both 6-MBA trans-3,4-dihydrodiol 1,2-epoxide(s) and 6-MBA trans-8,9-dihydrodiol 10,11-epoxide(s) may be the major metabolites which contribute to the carcinogenic properties of 6-MBA.     

Metabolism of l-nitrobenzo[a]pyrene by rat liver microsomes to potent mutagenic metabolites

1-, 3- and 6-Nitrobenzo[a]pyrene (nitro-BaP), which are prototypesof nitro polycyclic aromatic hydrocarbons (nitro-PAHs) derivedfrom a carcinogenic parent PAH, benzo[a]-pyrene, are environmentalcontaminants and potent bacterial mutagens. In this study, theaerobic and hypoxic metabolism of 1-nitro-BaP by rat liver microsomeswas studied. Aerobic metabolism of 1-nitro-BaP yielded 1-nitro-BaPtrans-7, 8- and 9, 10-dihydrodtol, while metabolism under hypoxicconditions yielded 1-amino-BaP. The metabolites formed fromaerobic metabolism of 1-nitro-BaP and 1-nitro-BaP trans-9, 10-dihy-drodiolby liver microsomes of untreated rats and rats pretreated with3-methylcholanthrene and phenobarbital were quantified. Comparisonof these results with those obtained with BaP and BaP trans-9,10-dihydrodiol indicates that nitro substitution at the 1-positionof BaP markedly affects the regioselectivity of the P-450-containingenzymes. 1-Nitro-BaP and the three metabolites were potent mutagensin Salmonella typhimurium TA98, both in the absence and in thepresence of an exogenous metabolic activation system (S9). Thedirect and S9-mediated mutagenkities of 1-nitro-BaP and thetwo dihydrodiols were decreased in the nitroreductase-deficientstrain TA98NR, while TA98/1, 8-DNP₆, an 0-acetylase-defidentstrain, was less sensitive to the two dihydrodiols, both withand without S9, and 1-nitro-BaP with S9. 1-Amino-BaP was equallymutagenic in all three tester strains. These observations indicatethat: (i) the metabolism of 1-nitro-BaP involves several pathwaysleading to mutagenic activation; (ii) the major activation pathwaysof 1-nitro-BaP involve nitroreduction; (iii) nitroreductionfollowed by O-acetylation is the major activation pathway of1-nitro-BaP trans-7, S- and 9, 10-dihydrodiol; and (iv) 1-amino-BaPis a potent direct-acting mutagen.     

Effect of dimethylnitrosamine concentration on its demethylation by liver microsomes from control and 3-methylcholanthrene pretreated rats, hamsters and guinea pigs

The effect of dimethylnitrosamine concentration on its demethylation by liver microsomes from control and 3-methylcholanthrene pretreated (100 mg/kg body wt. 24 h before sacrifice) rats, hamsters and guinea pigs was investigated. At low substrate concentration (2 mM), liver microsomes from pretreated rats and hamsters showed 30-50% lower demethylation activity than their respective controls. No such difference was found in the guinea pig. At high substrate concentration (100 mM), all 3 pretreated species showed 50-100% higher enzyme activity than their respective controls. Enzyme activities among the 3 species showed the following order of activity: hamster greater than guinea pig greater than rat.     

Stereoselective metabolism of 7-bromobenz [a]anthracene by rat liver microsomes: absolute configurations of trans-dihydrodiol metabolites

Rat liver microsomes metabolized the weak carcinogen 7-bromobenz[a](7-Br-BA) to form predominantly trans-3,4-, 5,6-, 8,9-, and10,11-dihydrodiols. The dihydrodiol metabolites were isolatedby reversed-phase h.p.l.c. for structural and conformationalanalyses. N.m.r. spectral analysis indicated that the 3,4- and10,11-dihydrodiols preferentially adopted quasidiequatorialconformations whereas the 5,6- and 8,9-dihydrodiols (which havea peri bromo substituent) preferred quasidiaxial conformations.Comparison of c.d. spectra with those of quasidiequatorial benz[a]anthracene(BA) 3R,4R-dihydrodiol and 10R,11R-dihydrodiol indicated thatthe major enantiomeric 7-Br-BA- 3,4- and 10,11-dihydrodiol metaboliteshave R,R absolute stereochemistry. The absolute conformationof 7-Br-BA-5,6-and -8,9-dihydrodiol metabolites could not bededuced by comparing their c.d. spectra with those of BA 5R,6R-and 8R,9R-dihydrodiols. Catalytic hydrogenolysis converted theenzymalically formed 7-Br-BA trans-5,6-dihydrodiol to BA trans-5,6-dihydrodiolwhich had a c.d. spectrum identical to that of BA 5R,6R-dihydrodiol.Catalytic hydrogenolysis and hydrogenation converted the enzymaticallyformed 7-Br-BA trans-8,9-dihydrodiol to a BA 8,9,10,11-tetrahydro-trans-8,9-diol which had a retention time identical to that of BA 8,9,10,11-tetrahydro-8R,9R-diolon a chiral h.p.l.c. column. These results indicate that themajor enantiomers of trans-dihydrodiol metabolites formed inrat liver microsomal metabolism of 7-Br-BA all have R,R absolutestereochemistries.     

Comparative carcinogenic potencies of 7H-dibenzo[c,g]carbazole, dibenz[a,j]acridine and benzo[a]pyrene in mouse skin

The relative carcinogenic potencies of three combustion products of fossil fuels, 7H-dibenzo[c,g]carbazole (7H-DB[c,g]C), dibenz[a,j]acridine (DB[a,j]A) and benzo[a] pyrene (B[a]P) were compared using complete carcinogenicity C3H mouse skin bioassays. Both 7H-DB[c,g]C and B[a]P produced tumors in 48 of 50 mice with latency periods of 36.6 and 32.4 weeks, respectively. DB[a,j]A produced tumors in 25 of 50 mice with a latency period of 80 weeks. 7H-DB[c,g]C was found to be as potent a carcinogen as B[a]P when applied to mouse skin. These results have important implications in the determination of relative carcinogenic potencies of complex mixtures.     

Identification of tumorigenic metabolites of benzo[j]fluoranthene formed in vivo in mouse skin

The metabolism of benzo[j]fluoranthene (BjF) in vivo in mouse skin was investigated. trans-4,5-Dihydro-4,5-dihydroxybenzo[j]fluoranthene (BjF-4,5-diol) and trans-9,10-dihydro-9,10-dihydroxybenzo[j]fluoranthene (BjF-9,10-diol) have been identified as major metabolites. In addition, 4- and 10-hydroxybenzo[j]fluoranthene and benzo[j]fluoranthen-4,5-dione have been tentatively identified among the metabolites formed in vivo in mouse skin. The enantiomeric purity of the metabolic dihydrodiols of BjF as formed in vivo in mouse skin was determined. The major enantiomer of BjF-4,5-diol was present in 57-62% enantiomeric excess while that of BjF-9,10-diol was present in 66-71% enantiomeric excess. In each case the later-eluting enantiomer on chiral stationary-phase high performance liquid chromatography predominated. The tumor-initiating activity of trans-2,3-dihydro-2,3-dihydroxybenzo[j]fluoranthene (BjF-2,3-diol), BjF-4,5-diol, BjF-9,10-diol, and BjF was evaluated on the skin of female CD-1 mice. As a total initiation dose of 3 mumol/mouse BjF-4,5-diol resulted in a 100% incidence of tumor-bearing mice with 5.0 tumors/mouse. In comparison, BjF-9,10-diol elicited a 60% incidence of tumor-bearing mice with 1.7 tumors/mouse, while BjF-2,3-diol was inactive. At the same dose, BjF gave rise to a 90% incidence of tumor-bearing mice with 7.8 tumors/mouse. At a 1-mumol dose, BjF-4,5-diol induced a 78% incidence of tumor-bearing mice with 4.3 tumors/mouse while BjF gave rise to a 70% tumor incidence with 3.4 tumors/mouse while BjF gave rise to a 70% tumor incidence with 3.4 tumors/mouse. These studies indicate that while BjF-9,10-diol could contribute to the overall tumorigenic activity of BjF in mouse skin, BjF-4,5-diol is a more potent tumor initiator in the target tissue.     

Metabolism of 7-methylbenz[c]acridine: comparison of rat liver and lung microsomal preparations and identification of some minor metabolites

The metabolism of the carcinogenic polycydic aza-aromatic compound,7-methylbenz[c]acridine, has been studied in lung and livermicrosomal preparations obtained from control and induced rats.Minor metabolites not previously identified included, trans-10,11-dihydro-10,11-dihydroxy-7-methyl-benz[c]acridine,trans-1,2-dihydro-1,2-dihydroxy-7-methyl-benz[c]acridine, 7-methylbenz[c]acridine-5,6-oxideand 7-hydroxymethylbenz[c]acridine-5,6-oxide. Metabolite profilesfrom liver microsomes showed 7-hydroxymethylbenz[c]-acridine,trans-8,9-diydro-8,9-dihydroxy-7-methylbenz[c]-acridine, 7-methylbenz[c]acridine-5,6-oxideand phenols to be major products. Metabolite distributions obtainedwith lung microsomes were very similar although activities weremuch lower than those of liver microsomes prepared from thesame animals.     

Identification of the metabolites of benzo[f]quinoline and benzo[h]-quinoline formed by rat liver homogenate

Benzo[f]quinoline and benzo[h]quinoline are widespread environmentalpollutants which have been found to be mutagenic. The metabolismof benzo[f]quinoline and benzo[h]quinoline was investigatedusing a liver homogenate from Aroclor-pretreated rats. The metabolitesof benzof[f]-quinoline which were identified were 7,8-dihydroxy-7,8-di-hydrobenzo[f]quinoline,9,10-dihydroxy-9,10-dihydrobenzo-[f]quinoline, 7-hydroxybenzo[f]quinoline,and benzof[f]-quinoline-N-oxide. Metabolism studies on benzo[f]quinolineperformed in the presence of the epoxide hydratase inhibitor,3,3,3-trichloropropylene oxide, demonstrated that the formation of both of these dihydrodiols can be inhibited. The major metabolites of benzo[h]quinoline were identified as 5,6-di-hydroxy-5,6-dihydrobenzo[h]quinolineand 7,8-dihydroxy-7,8-dihydrobenzo[h]quinoline. Benzo[h]quinoIine-N-oxidewas not detected as a metabolite. In the presence of an epoxide hydratase inhibitor, the major metabolites of benzof[h]-quinolinewere 5,6-epoxybenzo[h]quinoline and 7-hydroxy-benzo[h]quinoline.The difference in the metabolism to N-oxides observed betweenbenzo[h]quinoline and benzof[f]-quinoline is consistent withprevious observations in which sterically hindered aromaticring nitrogen compounds such as benzo[h]quinoline are more resistantto N-oxide formation. The nitrogen atom of these aza-areneswith its lone pair of electrons has a significant influenceon sites at which dihydrodiols are formed. The data suggestthat the aromatic ring nitrogen of these azaphenanthrenes hasan effect similar to that of a methyl substituent in directingtheir metabolic oxidation.     

Comparative 32P-postlabeling analysis of benzo[a]pyrene--DNA adducts formed in vitro upon activation of benzo[a]pyrene by human, rabbit and rodent liver microsomes

An interspecies comparison was made of the DNA-adducts formedin vitro upon incubation of rat liver DNA (RL-DNA) with benzo[a]pyrene(BP) in the presence of liver microsomes. Incubations were carriedout with RL-DNA, BP (100 µM) and liver microsomes fromhamsters, mice, rabbits, rats, 3-methylcholanthrene (3MC) pretreatedrats and from humans. To analyse the adduct profiles, the ³²P-postlabelingtechnique with the nuclease P1-enhancement procedure was used.The total amount of adduct formed varied greatly with the species;also the number of adduct spots detected was different, rangingfrom 1 to 5. In all incubations the BP-N²-deoxyguanosine adductwas formed. Relative to the total adduct level, the level ofthis adduct varied from 26% with rat, 54% with hamster, 56%with 3MC-pretreated rat, 58% with mouse and 75% with rabbit,to 100% with human liver microsomes. In human liver microsomesboth the total amount of cytochrome P-450 per mg microsomalprotein and the ethoxyresorufin O-deethylation (EROD) activitywere low compared to that in animal liver microsomes. In microsomesfrom 3MC-pretreated rats the EROD activity was strongly induced.There was no correlation between EROD activity in non-inducedmicrosomes and total adduct level. To compare BP—DNA adductformation in human white blood cells (WBC) with that in RL-DNA,WBC were incubated with BP and 3MC-pretreated rat microsomes.The adduct profile in WBC-DNA differed from that observed afterincubation of RL-DNA: the BP-N²-deoxyguanosine adduct in WBC-DNAaccounted for 97% of the total adduct level. It is concludedthat the ³²P-postlabeling method is a suitable technique toinvestigate both qualitative and quantitative differences inBP — DNA adduct formation between species. Furthermore,the incubation of microsomes from the liver (or other sources)with a genotoxic agent and isolated DNA or cells can be a usefulapproach to study the formation and stability of reactive intermediatesthat are able to bind to DNA, also with respect to differencesbetween species or tissue.     

Time course of oxidative benz[a]anthracene metabolism by liver microsomes of normal and PCB-treated rats

The time course for the oxidative metabolism of benz-[a)anthraceneby liver microsomes of normal, 3,3',4,4' -tetrachlorobiphenyl-(TCBP)and polychlorinated biphenyl-(PCB) treated rats has been investigated.These are shown not to be linear in all cases. In normal microsomesthe 10, 11-dihydrodiol is the main metabolite, followed by the5,6- and 8,9-dihydrodiols. Secondary metabolism, i.e. formationof dihydrodiol epoxides, is observed only after 5 min. In contrast,TCBP microsomes produce predominantly the 5,6-dihy-drodiol followedby the 8,9-dihydrodiol, whereas the formation of the 10, 11-dihydrodiolis suppressed. Metabolism deriving from oxidation of the 5,6-positionis increased 15–20 fold; again secondary metabolites occurbetween the 5th and 10th min of incubation. Gas chromatographyand mass spectra data suggest the formation of the ultimatecarcinogen, 3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenz[a]anthracene,as concluded from detection of its rearrangement product, the2,3,4-triol. In PCB-treated rats secondary metabolism is observedwithin 2.5 min. 5,6-Oxidation is increased 27 fold, 8,9-oxidation10 fold, but 10,11-oxidation is completely suppressed. The above-mentionedultimate carcinogen is also formed. Moreover, a series of tetrolsis detected. Optimum incubation times dependent on the problemunder study are discussed.     

Metabolism of benzo[a]pyrene by brain microsomes of fetal and adult rats and mice. Induction by 5,6 benzoflavone, comparison with liver and lung microsomal activities

Using brain, lung and liver microsomes as the enzyme sourcein in vitro assays, benzo[a]pyrene (B[a]P) metabolism was studiedin fetuses and dams of mice (C57B1/6) and rats (WAG). Separationand quantitation of B[a]P metabolites were performed by h.p.l.c.Microsomal preparations were tested for cytochrome P-450 dependentO-dealkylatlon of 7-ethoxycoumarin and epoxide hydrolase activities.Another parameter measured included the conjugation of 1-chloro-2,4dinitrobenzene to glutathione by cytosolic glutathione-S-transferaseactivity. The induction of B[a]P metabolism was studied aftertreatment of animals with 5,6-benzoflavone (BF). Mixed functionoxygenase, epoxide hydrolase and glutathione-S-transferase activiteswere transpiacentally inducible after dams were treated withBF. Metabolic activation of B[a]P by fetal brain microsomeswas lower in both species than that by fetal lung and livermicrosomes, but it was higher in fetuses than in adults. Allmetabolites of B[a]P increased after BF treatment; the productionof 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene (7,8-dihydrodiolB[a]P) was higher in brain microsomes from BF-treated rats thanthat in mice. In stimulated rats, the formation of 7,8-dihydrodiolB[a]P by fetal brain microsomes was higher than that by fetallung microsomes, whereas in mice, the opposite was observed.These data suggest that initiation could occur in utero, andpartially explain the species-specific differences in susceptibilityto transplacental tumorigenesis by polycyclic aromatic hydrocarbonsby differences in biotransformation in the target organ.     

Metabolic activation of the potent carcinogen dibenzo[a, l]pyrene by human recombinant cytochromes P450, lung and liver microsomes

The metabolic activation of dibenzo[a, l]pyrene (DB[a, l]P),recently considered the most potent carcinogen among all polycyclicaromatic hydrocarbons, to the 11, 12-dihydrodiol, a precursorof the ultimate carcinogens, the 11, 12-diol-13, 14-epoxides,was investigated using eleven human recombinant cytochrome P450s,as well as human lung and liver microsomes. Of all human P450s,1A1 was the most active in the metabolism of DB[a, l]P (310pmol/min, nmol P450) and had 5–23-fold higher catalyticactivity than other P450s examined. The order of activity inthe formation of the 11, 12-dihydrodiol was as follows: 1A1(116 pmol/min, nmol P450) > 2C9 (29) > 1A2 (22) > 2B6(18) > 3A4 (16) > others (     

Metabolism of benzo[f]quinoline by rat liver microsomes

The metabolism of [1,3-¹⁴C]benzo[f]quinoline (BfQ) by livermicrosomes from control, 3-methylcholanthrene (3-MC)-pretreatedand phenobarbital (PB)-pretreated rats has been investigatedin order to gain insights into the effect of mixed functionoxidase inducers on the types and levels of specific metabolitesas formed in vitro. The rates of metabolism of BfQ by livermicrosomes from control, 3-MC- and PB-pretreated rats were 0.5,3.6 and 2.4 nmol/min/mg of respectively. The most predominantmetabolite of BfQ detected with liver microsomes from 3-MC-pretreatedrats was BfQ-7,8-dihydrodiol, a precursor of the bay-regiondiol epoxide, constituting 41% of the total ethyl acetate-extractablemetabolites. Other metabolites obtained along with their relativeproportions were as follows: BfQ-N-oxide, 23% 7-hydroxyBfQ,15%; 9-hydroxyBfQ, 9%; and BfQ-9,10-dihydrodiol, 6%. BfQ-5,6-dihydrodiol, a K-region dihydrodiol, was a trace metaboliterepresenting {small tilde}1.0% of the total metabolism. Livermicrosomes from PB-pretreated rats oxidized BfQ primarily toBfQ-N-oxide and 9-hydroxyBfQ, which constituted 41% and 20%of the total ethyl acetate-extractable metabolites of BfQ. Therelative proportions of BfQ-9,10-dihydrodiol, BfQ-7,8-dihydrodioland 7-hydroxy-BfQ formed were 12%, 3% and 13% respectively,while the figure for BfQ-5,6-dihydrodiol was 0.5%. The profileof metabolites formed by liver microsomes from control ratswas similar to that generated by microsomes from PB-pretreatedrats. While benzo-ring metabolites represented a major partof the metabolism of BfQ by liver microsomes from either 3-MC-or PB-pretreated rats, these two types of microsomes exhibiteda positional selectivity in the oxidation of BfQ, the formerprimarily attacking the 7,8-position of BfQ while the latterpreferentially oxidizing the 9,10-position. The preponderanceof the potentially mutagenic BfQ-7,8-dihydrodiol amongst themetabolites generated by liver microsomes from 3-MC-pretreatedrats suggests a possible role for cytochrome P-450c, the majorform of rat hepatic cytochrome P-450 induced by 3-MC, in themetabolic activation of BfQ.