Formation of DAN-binding products from isolated benzo[a]pyrene metabolites in rat liver nuclei

Liver nuclei from 3-methylcholanthrene-treated rats in the presence of NADPH metabolized 3- and 9-hydroxybenzo[a]pyrene and 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene to products that bound to DNA. Maximal binding was obtained with the dihydrodiol which was approximately 3-fold that with 9-hydroxybenzo[a]pyrene, and 60-fold that with 3-hydroxybenzo[a]pyrene, as substrates. Both 4,5-dihydro-4,5-dihydroxybenzo[a]pyrene and 9,10-dihydro-9,10-dihydroxybenzo[a]pyrene were also extensively metabolized by the nuclear fraction but did not give rise to DNA-binding products.The available evidence suggests that the DNA binding species derived from 9-hydroxy-benzo[a]pyrene is 9-hydroxy-benzo[a]pyrene-4,5-oxide and from 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene, as previously observed in different systems, 7,8-dihydro-7,8-dihydroxy-benzo[a]pyrene-9,10-oxide.     

Dual-label high-performance liquid chromatographic assay for femtomole levels of benzo[a]pyrene metabolites

A dual-label HPLC assay to measure femtomole quantities of ethyl acetate-extractable [3H]benzo[a]pyrene metabolites was developed. 14C-labeled metabolites of benzo[a]pyrene formed by rat liver 9000g supernatant were used as both internal standards and chromatographic markers. The percentage deviation between assays was determined to be between 11 and 13% for 9,10-dihydro-9,10-dihydroxybenzo[a]pyrene, 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene, benzo[a]pyrene-3,6-quinone, benzo[a]pyrene-1,6-quinone, and 9-hydroxybenzo[a]pyrene, 22% for 4,5-dihydro-4,5-dihydroxybenzo[a]pyrene, and less than 5% for 3-hydroxybenzo[a]pyrene. The detection limit of this assay was between 3 and 10 fmol per metabolite. The application of this technique to the metabolism of [3H]benzo[a]pyrene by microsomes of hamster and human oral cavity tissue is described.     

Fluorescence study of DNA-complexes formed after metabolic activation of benzo(a)pyrene derivatives

Benzo(a)pyrene derivatives (1-, 2-, 3-, 7-, and 9-hydroxy-benzo(a)pyrene and trans-9,10-dihydro-9,10-dihydroxy-, -4,5-dihydro-4,5-dihydroxy-, and -7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene) were metabolized by liver microsomes isolated from 3-methylcholanthrene-treated rats in the presence of calf thymus DNA. The isolated DNA was then assayed by fluorescence for bound metabolic products. Only 2-hydroxy-benzo(a)pyrene, 9-hydroxy-benzo(a)pyrene and trans-7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene yielded detectable amounts of DNA-bound products. In contrast to the product(s) from 9-hydroxy-benzo(a)pyrene, the metabolites of 2-hydroxy-benzo(a)pyrene and trans-7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene, both strong carcinogens, had similar excitation spectra and gave considerably increased fluorescence intensities when the DNA was denatured. These data indicate structural similarities in the DNA complexes formed after metabolic activation of 2-hydroxy-benzo(a)pyrene and trans-7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene.     

Chemical rearrangement of phenol-epoxide metabolites of polycyclic aromatic hydrocarbons to quinone-methides

Evidence of the involvement of triol-epoxide and phenol-epoxide metabolites in the metabolic activation of polycyclic hydrocarbons is accumulating. It is proposed that the phenolic OH-groups present in such epoxides will activate the epoxide moieties and permit their rearrangement to quinone-methides. These quinone-methides are highly reactive, potentially-isolable chemical entities with strong alkylating activity. In one resonance form they are resonance-stabilized carbonium ions. Only epoxides that also possess phenolic OH-groups in certain positions will form quinone-methides: these appear to include 9-hydroxybenzo [a] pyrene 4,5-oxide and the triol-epoxides 9-hydroxy-trans-1,2-dihydro-1,2-dihydroxychrysene 3,4-oxide and 2-hydroxy-trans-9,10-dihydro-9,10-dihydroxybenzo[a]pyrene 7,8-oxide.     

Metabolic activation of benzo[a]pyrene in human skin maintained in short-term organ culture

The metabolic activation of benzo[a]pyrene (BP) was examined in six samples of human skin after topical application of the hydrocarbon to the skin in short-term organ culture. The results show that all of the samples were capable of metabolizing BP to water-soluble products and to ether-soluble products that included the 4,5-, 7,8- and 9,10-dihydrodiols and a product which had chromatographic properties identical with those of authentic trans-11,12-dihydro-11,12-dihydroxybenzo[a]pyrene (BP-11,12-diol). The major BP-deoxyribonucleoside adduct detected in each skin sample appeared to be formed from the reaction of r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BP-7,8-diol 9,10-oxide) with deoxyguanosine residues in DNA.     

Effects of defined synthetic phospholipids on glycosylation processes. Modifications of membrane-bound N-acetylglucosaminyl-transferase and solubilized sialyl-transferase activities

In cultures of hamster embryo cells, benzo[a]pyrene (B[a]P) is metabolized primarily in the bay region. In contrast, little or no bay region metabolism of the noncarcinogenic isomer benzo[e]pyrene (B[e]P) could be detected during 12–96-h incubations of hamster embryo cells with 4 μM [³H]B[e]P. The upper limit to 9,10-dihydro-9,10-dihydroxy-B[e]P formation is about 0.2% of the ethyl acetate-soluble metabolites ( <0.1% of the total metabolites). The major identified metabolites of B[e]P were 4,5-dihydro-4,5-dihydroxy B[e]P and the glucuronide conjugates of 3-OH-B[e]P and 4,5-dihydro-4,5-dihydroxy B[e]P. Simultaneous treatment of cells with either B[a]P or 7,8-benzoflavone (BF) did not induce bay region metabolism of [³H]B[e]P.     

The formation of dihydrodiols from benzo[alpha]pyrene by oxidation with an ascorbic acid/ferrous sulphate/EDTA system.

In the oxidation of benzo[alpha]pyrene in an abscorbic acid-ferrous sulphate-EDTA system, four dihydrodiols were detected. Three, trans-4,5-dihydro-4,5-dihydroxybenzo[alpha]pyrene, trans-7,8-dihydro-7,8-dihydroxybenzo[alpha]pyrene and trans-9,10-dihydro-9,10-dihydroxybenzo[alpha]pyrene were identified by their UV spectra and by direct comparisons of their chromatographic properties, using HPLC, with those of the authentic compounds. The fourth compound appeared to be trans-11,12-dihydro-11,12-dihydroxybenzo[alpha]pyrene since its ultraviolet spectrum was identical to that of the cis-dihydrodiol. Time-course experiments showed that the maximum amounts of products were obtained after 8 h of oxidation. A re-examination of the dihydrodiols formed from benzo[alpha]pyrene by rat-liver microsomal fractions failed to show the formation of the trans-11,12-dihydrodiol.     

Unusual patterns of benzo[a]pyrene metabolites and DNA-benzo[a]pyrene adducts produced by human placental microsomes in vitro

Human placental microsomes were incubated with [³H]benzo[a]pyrene (BP) and Salmon sperm DNA and the resulting metabolite-nucleoside complexes resolved by Sephadex LH-20 chromatography. The metabolite pattern was analyzed by high-pressure liquid chromatography (HPLC). The incubates were also co-chromatographed with extracts obtained from incubates with rat liver microsomes and [¹⁴C]BP. Phenols, quinones and 7,8-dihydrodiol were detected in the placental incubates. Both 9,10- and 4,5-dihydrodiols were very low as compared with control rat liver samples. Placental microsomes catalyzed the binding of BP metabolites to DNA in vitro, giving rise to two main complexes which co-chromatographed with rat liver-produced peaks attributable to 7,8-diol-9,10-epoxide and 7,8-oxide and/or quinones when metabolized further. The nucleoside metabolite peaks attributable to 4,5-oxide and 9-phenol-4,5-oxide were lacking when compared with the binding pattern catalyzed by rat liver. Both the total binding and specific metabolite-nucleoside adducts in the placenta correlated with fluorometrically measured aryl hydrocarbon hydroxylase (AHH) activity and with the amount of dihydrodiol formed. The results demonstrate that both the metabolite pattern and the nucleoside-metabolite complexes formed by the placental microsomes in vitro differed greatly from those produced by rat liver microsomes. These studies also suggest that it is not possible to predict specific patterns of DNA binding from AHH measurements or even from BP metabolite patterns, especially when comparing different tissues and species.     

The relationship between the levels of DNA-hydrocarbon adducts and the formation of sister-chromatid exchanges in Chinese hamster ovary cells treated with derivatives of polycyclic aromatic hydrocarbons

The frequencies of the induction of sister-chromatid exchanges and the levels of deoxyribonucleoside-hydrocarbon adducts formed in Chinese hamster ovary cells that had been treated with either dihydrodiols or a diol-epoxide derived from polycyclic aromatic hydrocarbons were determined. Up to 6-fold increases in the incidence of these exchanges were observed when the cells were treated either with the dihydrodiols, trans-3,4-dihydro-3,4-dihydroxy-7-methylbenz[a]anthracene,trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene or the diol-epoxide, (±)-r-7, t-8dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a] pyrene but when the cells were transferred to media free of these compounds, there were rapid reductions in the frequency of these exchanges. When the exchanges were induced by the diol-epoxide, the decreases in frequency were paralleled by decreases in the levels of deoxyribonucleoside-diol-epoxide adducts that were present in hydrolysates of DNA isolated from the cells. There thus appears to be a close relationship between the frequency of sister-chromatid exchanges and the levels of deoxyribonucleoside-diol-epoxide adduct formation.     

Magnesium ions affect the quantitative but not the qualitative microsome mediated binding of benzo[alpha]-pyrene to DNA

Five distinct hydrocarbon-deoxyribonucleoside adducts are separated by high pressure liquid chromatography after reaction of benzo[alpha]pyrene with calf thymus DNA in the presence of liver microsomes from 3-methylcholanthrene treated rats. The two major adducts co-chromatography with deoxyribonucleoside adducts obtained after hydrolysis of calf thymus DNA previously reacted with liver microsomal metabolically activated 9-hydroxy-benzo[alpha]pyrene or trans-7,8-dihydro-7,8-dihydroxybenzo[alpha]pyrene. High magnesium ion concentrations in the microsomal incubations cause a significant decrease in the covalent binding of the hydrocarbon to DNA but do not affect the qualitative distribution of the individual benzo[alpha]pyrene-deoxyribonucleoside adducts.     

Effect of purified epoxide hydrolase on metabolic activa-tion and binding of benzo(a)pyrene to exogenous DNA. Shift of the activation pathway

The effect of purified epoxide hydrolase (E.C. 3.3.2.3) on the binding of benzo(a)pyrene metabolites 9-hydroxybenzo(a)pyrene and 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene to DNA catalyzed by cytochrome P 448 from liver microsomes of methylcholanthrene pretreated rats has been investigated. The total binding and the major binding species derived from 9-hydroxybenzo(a)pyrene were strongly inhibited by the presence of purified epoxide hydrolase and the species derived from 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene was slightly increased. By modifying the balance between cytochrome P 448 and epoxide hydrolase it is possible to shift quantitatively the binding of these two main reactive intermediates to DNA.     

Induction of sister-chromatid exchanges in Chinese hamster ovary cells treated in vitro with non-k-region dihydrodiols of 7-methylbenz[a]anthracene and benzo[a]pyrene

Studies were carried out on the incidence of sister-chromatid exchanges induced in Chinese hamster ovary cells by in vitro treatment with the polycyclic aromatic hydrocarbons 7-methylbenz[a]anthracene and benzo[a]pyrene and with related K-region and non-K-region dihydrodiols. Appreciable increases in the incidence of sister-chromatid exchanges were apparent in cells treated with non-K-region dihydrodiols: the most active compounds were 3,4-dihydro-3,4-dihydroxy-7-methylbenz[a]anthracene and 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene and the effects were dose-dependent. The parent hydrocarbons and the related K-region dihydrodiols induced some sister-chromatid exchanges but they were considerably less active than these two non-K-region diols. The results suggest that this system may usefully be applied to studies aimed at determining which dihydrodiols are important in the metabolic activation of the carcinogenic polycyclic hydrocarbons. These and other results also infer that Chinese hamster ovary cells possess some intrinsic ability to metabolize such compounds in the absence of exogenous activation systems.     

Metabolism and covalent binding of benzo[alpha]pyrene in human peripheral lung

Short-term organ cultures of peripheral lung from lung cancer patients metabolise benzo[alpha]pyrene to ethylacetate-soluble metabolites, which covalently bind to tissue macromolecules. The nature and quantities of metabolites formed and the extent of covalent binding are dependent upon the time of incubation, the substrate concentration and interindividual variability in the metabolic activity of the lung. Individuals whose lungs rapidly metabolise the carcinogen exhibit more extensive further metabolism of primary metabolites and higher levels of covalent binding. Certain striking differences in the relative retention in the tissue or release into the extra-cellular medium of different metabolites have been found as illustrated by the observation that the ratio of 7,8-dihydro-7,8-dihydroxybenzo[alpha]-pyrene to 9,10-dihydro-9,10-dihydroxybenzo[alpha]pyrene was always significantly higher in the tissue than in the extracellular medium.     

Epoxy derivatives of aromatic polycyclic hydrocarbons. The preparation and metabolism of epoxides related to benzo[a]pyrene and to 7,8- and 9,10-dihydrobenzo[a]pyrene

Products that appeared to be mainly benzo[a]pyrene 7,8-oxide and benzo[a]pyrene 9,10-oxide were synthesized and their chemical and biochemical properties were investigated. The oxides were unstable and readily rearranged to phenols. They were converted by rat liver homogenates and microsomal preparations into phenols and dihydrodiols, but glutathione conjugates were not formed in appreciable amounts. The dihydrodiols formed from benzo[a]pyrene 7,8- and 9,10-oxide by rat liver microsomal preparations were identical in their chromatographic and spectrographic properties with dihydrodiols formed when benzo[a]pyrene was metabolized by rat liver homogenates. 9,10-Dihydrobenzo[a]pyrene 7,8-oxide and 7,8-dihydrobenzo[a]pyrene 9,10-oxide were also synthesized. They were converted by rat liver homogenates and microsomal preparations into the related cis- and trans-dihydroxy compounds. Glutathione conjugates were formed from the oxides by rat liver homogenates. Both 7,8- and 9,10-dihydrobenzo[a]pyrene were metabolized by rat liver homogenates to mainly the trans-isomers of the related dihydroxy compounds. In experiments with boiled homogenates, the benzo[a]pyrene oxides were converted into phenols, whereas the dihydrobenzo[a]pyrene oxides yielded small amounts of the related dihydroxy compounds.     

Oxidation of benzo[a]pyrene by the filamentous fungus Cunninghamella elegans.

Cunninghamella elegans oxidized benzo[a]pyrene to several metabolic products. Compounds that were isolated and identified were: trans-9,10-dihydroxy-9,10-dihydrobenzo[a]pyrene, trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, benzo[a]pyrene 1,6-quinone, benzo[a]pyrene 3,6-quinone, 9-hydroxybenz[a]pyrene, and 3-hydroxybenzo[a]pyrene. In addition, an unidentified dihydroxybenzo[a]pyrene metabolite was also formed. Experiments with [14C]benzo[a]pyrene showed that over a 96-h period, 18.4% of the hydrocarbon was converted to metabolic products. Most of the metabolites were sulfate conjugates as demonstrated by the formation of benzo[a]pyrene quinones and phenols after treatment with aryl sulfatase. Glucuronide and sulfate conjugates were also detected as water-soluble metabolites. The results show that benzo[a]pyrene is metabolized by a filamentous fungus in a manner that is remarkably similar to that observed in higher organisms.     

Degradation of benzo[a]pyrene by Mycobacterium vanbaalenii PYR-1

Metabolism of the environmental pollutant benzo[a]pyrene in the bacterium Mycobacterium vanbaalenii PYR-1 was examined. This organism initially oxidized benzo[a]pyrene with dioxygenases and monooxygenases at C-4,5, C-9,10, and C-11,12. The metabolites were separated by reversed-phase high-performance liquid chromatography (HPLC) and characterized by UV-visible, mass, nuclear magnetic resonance, and circular dichroism spectral analyses. The major intermediates of benzo[a]pyrene metabolism that had accumulated in the culture media after 96 h of incubation were cis-4,5-dihydro-4,5-dihydroxybenzo[a]pyrene (benzo[a]pyrene cis-4,5-dihydrodiol), cis-11,12-dihydro-11,12-dihydroxybenzo[a]pyrene (benzo[a]pyrene cis-11,12-dihydrodiol), trans-11,12-dihydro-11,12-dihydroxybenzo[a]pyrene (benzo[a]pyrene trans-11,12-dihydrodiol), 10-oxabenzo[def]chrysen-9-one, and hydroxymethoxy and dimethoxy derivatives of benzo[a]pyrene. The ortho-ring fission products 4-formylchrysene-5-carboxylic acid and 4,5-chrysene-dicarboxylic acid and a monocarboxylated chrysene product were formed when replacement culture experiments were conducted with benzo[a]pyrene cis-4,5-dihydrodiol. Chiral stationary-phase HPLC analysis of the dihydrodiols indicated that benzo[a]pyrene cis-4,5-dihydrodiol had 30% 4S,5R and 70% 4R,5S absolute stereochemistry. Benzo[a]pyrene cis-11,12-dihydrodiol adopted an 11S,12R conformation with 100% optical purity. The enantiomeric composition of benzo[a]pyrene trans-11,12-dihydrodiol was an equal mixture of 11S,12S and 11R,12R molecules. The results of this study, in conjunction with those of previously reported studies, extend the pathways proposed for the bacterial metabolism of benzo[a]pyrene. Our study also provides evidence of the stereo- and regioselectivity of the oxygenases that catalyze the metabolism of benzo[a]pyrene in M. vanbaalenii PYR-1.     

Metabolism and binding of benzo[a]pyrene in randomly-proliferating,confluent and s-phase human skin fibroblasts

The metabolism of benzo[a]pyrene in randomly proliferating and confluent cultures of human skin fibroblast cells was compared with cell cultures in early S phase of the cell cycle after a G1 block. When each cell population was exposed to [G-³H]benzo[a]pyrene for 24 hours and the organic soluble metabolites in the extracellular medium and intracellular components were analyzed by HPLC, a quantitative increase in metabolism was observed in the confluent cell populations. The amount of organic soluble metabolites in the extracellular medium of the confluent dense cultures was 2.7 times the amount found in randomly proliferating cultures and 1.5 times that of the synchronized cultures. The trans-7,8- and 9,10 dihydrodiols and 3-hydroxy benzo[a]pyrene were the major metabolites formed. Small amounts of the sulphate conjugate, 9-hydroxy-benzo[a]pyrene and the tetrols were also detected. Cytoplasmic as well as nuclear extracts from the confluent cell cultures also contained higher amounts of metabolites compared to those from the randomly proliferating and S-phase cells. The levels of DNA modification by metabolically activated benzo[a]pyrene did not differ among the randomly proliferating, confluent and S-phase cells. However, the S-phase cells exhibited approximately 50-fold increase in the frequency of transformation compared to the randomly proliferating cells. Confluent cells were not transformed by benzo[a]pyrene. These data suggest that factors other than random modification of DNA by the carcinogen might have a significant role in the expression of a transformed phenotype and that metabolism and transformation are not directly related. Furthermore, confluent dense cultures with a heightened capability for metabolism of benzo[a]pyrene were more active in the detoxification of benzo[a]pyrene than in the production of the metabolites associated with cellular transformation.Abbreviations BaP benzo[a]pyrene - BaP-4,5-diol trans-4,5 dihydroxy-4,5-dihydrobenzo[a]pyrene - BaP-7,8-diol trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene - Bap-9,10-diol trans-9,10-dihydroxy-9,10 dihydrobenzo[a]pyrene - CM complete medium - HNF human neonatal foreskin - HPLC high pressure liquid chromatography - PAH polycyclic aromatic hydrocarbon - PDL population doubling - RP randomly proliferating     

The DNA binding of benzo[alpha]pyrene metabolites catalysed by rat lung microsomes in vitro and in isolated perfused rat lung.

When [³H]benzo[a]pyrene is incubated in vitro together with DNA, NADPH and rat lung microsomes, covalent binding of benzo[a]pyrene (BP) metabolites to DNA occurs. These metabolite-nucleoside complexes can be resolved into several distinct peaks by elution of a Sephadex LH-20 column with a water-methanol gradient. 3-Methylcholanthrene (MC) pretreatment of animals induces the total covalent binding in vitro several-fold and increases the amounts of at least five metabolite-nucleoside complexes associated with the 7,8-diol-9,10-epoxidcs, the 7,8-oxide or quinones oxygenated further, the 4,5-oxide and phenols oxygenated further. These increases correspond well with the increases in the production of both non-K-region and K-region metabolites of BP by lung microsomes, as determined by highpressure liquid chromatography (HPLC). On the other hand, when [³H]BP is metabolized in isolated perfused rat lung, only the peak representing the 7,8-diol-9,10-epoxide bound to nucleoside(s) is readily detectable and then only in lungs from MC-treated animals. The extent of binding of BP metabolites to lung DNA is very low, about 0.0004% of the total dose applied to the perfusion medium; more than 60% of this can be accounted for by the binding of the 7,8-diol-9,10-epoxides to nucleoside(s). It is suggested that the further metabolism leading to metabolites not available to covalent binding, (e.g. conjugation) of primary BP metabolites in the intact tissue is responsible for the differences in the metabolite-nucleoside patterns observed in vivo, as compared with microsomal metabolism in vitro.     

Neonatal modulation of adult rat hepatic microsomal benzo[a]pyrene hydroxylase activities by Aroclor 1254 or phenobarbital

The constitutive and Aroclor 1254-induced activities of hepatic microsomal benzo[a]pyrene hydroxylases in male and female rats were determined in animals from ages 11 to 120 days. In 11-day-old noninduced male rats, benzo[a]pyrenediones and 9-hydroxybenzo[a]pyrene were the major microsomal metabolites; in 21-day-old males benzo[a]pyrene-diones and benzo[a]pyrene-9,10-dihydrodiol were predominant. In 60- and 120-day-old animals 3-hydroxybenzo[a]pyrene was the major microsomal metabolite. A similar trend was observed for the development of benzo[a]pyrene hydroxylase activities in female rats. With the exception of 4,5-dihydrodiol formation, the highest induction of individual and total benzo[a]pyrene hydroxylase activities by Aroclor 1254 was observed in the 21-day-old immature male rats, in which there was a 330- and 4.5-fold increase in the formation of 3-hydroxybenzo[a]pyrene and quinone metabolites, respectively. The induction of benzo[a]pyrene total metabolite formation by Aroclor 1254 in female rats from 11 to 120 days of age was relatively constant (i.e., 13.3- to 10.1-fold induction); however, the relative induction of the individual benzo[a]pyrene hydroxylases was highly variable. In a second set of experiments, male and female rats were neonatally exposed to phenobarbital (600 mumol/kg) or Aroclor 1254 (100 mumol/kg), and the effects of these xenobiotics on neonatal imprinting of hepatic microsomal benzo[a]pyrene hydroxylase activities were determined in the 120-day-old animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

High mutagenicity and toxicity of a diol epoxide derived from benzo[a]pyrene

(±)-7β,8α-Dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BP 7,8-diol-9,10-epoxide) is a suspected metabolite of benzo[a]pyrene that is highly mutagenic and toxic in several strains of $\text{Salmonella}\text{typhimurium}$ and in cultured Chinese hamster V79 cells. BP 7,8-diol-9,10-epoxide was approximately 5, 10 and 40 times more mutagenic than benzo[a]pyrene 4,5-oxide (BP 4,5-oxide) in strains TA 98 and TA 100 of $\text{S.}\text{typhimurium}$ and in V79 cells, respectively. Both compounds were equally mutagenic to strain TA 1538 and non-mutagenic to strain TA 1535 of $\text{S.}\text{typhimurium}$. The diol epoxide was toxic to the four bacterial strains at 0.5–2.0 nmole/plate, whereas BP 4,5-oxide was nontoxic at these concentrations. In V79 cells, the diol epoxide was about 60-fold more cytotoxic than BP 4,5-oxide.