The effect of norharman on the metabolism of benzo[alpha]pyrene by rat-liver microsomes in vitro in relation to its enhancement of the mutagenicity of benzo[alpha]pyrene.

The effect of norharman on the metabolism of benzo[alpha]pyrene by rat-liver microsomes was studied. Separation of the metabolites into hydrophilic and hydrophobic fractions showed that norharman inhibited the conversion of hydrophobic metabolites to hydrophilic ones. Analysis of the hydrophobic metabolites by high-pressure liquid chromatography showed that norharman also inhibited the disappearance of benzo[alpha]pyrene itself. However, large amounts of hydrophobic metabolites, such as phenol, quinones and diols, were formed in the presence of norharman, and formation of the strong mutagen 7,8-dihydroxybenzo[alpha]pyrene was increased 10-fold by norharman. The increase in formation of this compound may be one of the chief reasons why norharman enhances the mutagenicity of benzo[alpha]pyrene on Salmonella typhimurium.     

Successive transformation of benzo[a]pyrene by laccase of Trametes versicolor and pyrene-degrading Mycobacterium strains

We previously hypothesized that polycyclic aromatic hydrocarbon (PAH)-degrading bacteria that produce laccase may enhance the degree of benzo[a]pyrene mineralization. However, whether the metabolites of benzo[a]pyrene oxidized by laccase can be further transformed by PAH degraders remains unknown. In this study, pyrene-degrading mycobacteria with diverse degradation properties were isolated and employed for investigating the subsequent transformation on the metabolites of benzo[a]pyrene oxidized by fungal laccase of Trametes versicolor. The results confirm the successive transformation of benzo[a]pyrene metabolites, 6-benzo[a]pyrenyl acetate, and quinones by Mycobacterium strains, and report the discovery of the involvement of a O-methylation mediated pathway in the process. In detail, the vast majority of metabolite 6-benzo[a]pyrenyl acetate was transformed into benzo[a]pyrene quinones or methoxybenzo[a]pyrene, via two distinct steps that were controlled by the catechol-O-methyltransferase mediated O-methylation, while quinones were reduced to dihydroxybenzo[a]pyrene and further transformed into dimethoxy derivatives.     

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.     

The metabolism of benzo[a]pyrene phenols by rat liver microsomal fractions

The oxidative metabolism of benzo[a]pryrene (B[a]P) phenols catalyzed by liver microsomes in vitro leads to multiple products. High-pressure liquid chromatography analysis of the organic-soluble products formed indicates that regardless of the animal pretreatment regime, 3-hydroxy-B[a]P is metabolized to the 3,6-quinone and to a hydroxylated derivative tentatively identified as 3,9-dihyroxy-B[a]P. However, the distribution of products obtained with 9-hydroxy-B[a]P varied with animal pretreatment. A maximum of three distinct metabolites was obtained when the 9-phenol was metabolized in vitro with microsomes from phenobarbital-pretreated rats and the tentative 3,9-dihydroxy derivative was a common metabolite for all pretreatment regimes. Physical characterization, including mass spectrometry, indicates that all three products have an extra oxygen atom incorporated into their molecular structure from molecular oxygen. Studies utilizing specific inhibitors of the cytochrome P-450-dependent monooxygenase clearly suggest that the formation of dihydroxy or phenol-oxide derivatives is catalyzed by the hemoprotein, cytochrome P-450. These metabolites of the benzo[a]pyrene phenols are most likely related to the putative phenol-oxides of benzo[a]pyrene which have been demonstrated to alkylate DNA and protein. Repetitive scan difference spectrophotometric analysis of incubation mixtures containing rat liver microsomes, 3- or 9-hydroxy-B[a]P, NADPH, and oxygen shows the conversion of the phenols into products which absorb in the region from 400 to 500 nm. During and after the steady state of the reaction, it can be seen that certain of the hydroxy compounds produced are in equilibrium with their respective quinone form and may be involved in an oxygen-coupled redox cycle.     

Effects of harman and norharman on the mutagenicity and binding to DNA of benzo[a]pyrene metabolites in vitro and on aryl hydrocarbon hydroxylase induction in cell culture

Harman and norharman, two β-carboline derivatives known to exist in certain foods and to be formed during pyrolysis of tobacco and meat, were tested for mutagenic activity in the presence of benzo[a]pyrene, mouse liver enzymes, and Salmonella typhimurium TA98 in vitro. Both harman and norharman inhibit benzo[a]pyrene mutagenicity, benzo[a]pyrene metabolism (as measured by aryl hydrocarbon hydroxylase activity), and the binding of all benzo[a]pyrene metabolites to DNA in vitro. Moreover, harman and norharman are quite toxic to cultures of hepatoma-derived H-4-II-E and Hepa-1 established cell lines and therefore were found to be very weak inducers of aryl hydrocarbon hydroxylase activity.     

Metabolite repression inhibits degradation of benzo[a]pyrene and dibenz[a,h]anthracene by Stenotrophomonas maltophilia VUN 10,003

Large inocula of Stenotrophomonas maltophilia VUN 10,003 were used to investigate bacterial degradation of benzo[a]pyrene and dibenz[a,h]anthracene. Although strain VUN 10,003 was capable of degrading 10–15 mg l⁻¹ of the five-ring compounds in the presence of pyrene after 63 days, further addition of pyrene after degradation of the five-ring polycyclic aromatic hydrocarbons (PAHs) ceased did not stimulate significant decreases in the concentration of benzo[a]pyrene or dibenz[a,h]anthracene. However, pyrene was degraded to undetectable levels 21 days after its addition. The amount of benzo[a]pyrene and dibenz[a,h]anthracene degraded by strain VUN 10,003 was not affected by the initial concentration of the compounds when tested at 25–100 mg l⁻¹, by the accumulation of by-products from pyrene catabolism or a loss of ability by the cells to catabolise benzo[a]pyrene or dibenz[a,h]anthracene. Metabolite or by-product repression was suspected to be responsible for the inhibition: By-products from the degradation of the five-ring compounds inhibited their further degradation. Journal of Industrial Microbiology & Biotechnology (2002) 28, 88–96 DOI: 10.1038/sj/jim/7000216 Received 30 January 2001/ Accepted in revised form 10 October 2001     

Formation of sulfate and glucoside conjugates of benzo[e]pyrene by Cunninghamella elegans

 Benzo[e]pyrene is a pentacyclic aromatic hydrocarbon, which, unlike its structural isomer benzo[a]pyrene, is not a potent carcinogen or mutagen. The metabolism of benzo[e]pyrene was studied using the filamentous fungus Cunninghamella elegans ATCC 36112. C. elegans metabolized 65% of the [9, 10, 11, 12-³H]benzo[e]pyrene and unlabeled benzo[e]pyrene added to Sabouraud dextrose broth cultures after 120 h of incubation. Three major metabolites of benzo[e]pyrene were separated by reversed-phase high-performance liquid chromatography. These metabolites were identified by ¹H and ¹³C NMR, UV-visible, and mass spectral analyses as 3-benzo[e]pyrenylsulfate, 10-hydroxy-3-benzo[e]pyrenyl sulfate, and benzo[e]pyrene 3-O-β-glucopyranoside. Received: 7 September 1995/Received revision: 14 November 1995/Accepted: 11 December 1995     

Formation and removal of benzo[a]pyrene metabolites-DNA adducts in cultured normal human fibroblasts using a microsome-activating system

The rate of removal of DNA adducts of several benzo[a]pyrene from nuclear DNA was compared by introducing a microsome-activating system in human fibroblast cells. Conlfuent human fibroblasts were exposed to benzo[a]pyrene in the presence of a microsomal activating system and DNA adducts were formed in the nuclear DNA. The adducts present in DNA were determined after 1 h of incubation and 48 h later. There was no difference in the rate of removal between 7S- and 7R-N²-[10- (7β,8α,9α-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene)yl]deoxyguanosine, 7R-N²-[10(7β,8α,9β,trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene)yl]deoxyguanosine and the covalent adduct of 9-hydroxybenzo[a]pyrene-4,5-epoxide to guanosine. This finding does not agree with the idea that metabolites forming ‘persistent DNA adducts’ are always responsible for the carcinogenicity of their parent compound.     

A benzo[a]pyrene -7,8-dihydrodiol-9,10-epoxide is the major metabolite involved in the binding of benzo[a]pyrene to DNA in isolated viable rat hepatocytes

Benzo[a]pyrene is metabolised by isolated viable hepatocytes from both untreated and 3-methylcholanthrene pretreated rats to reactive metabolites which covalently bind to DNA. The DNA from the hepatocytes was isolated, purified and enzymically hydrolysed to deoxyribonucleosides. The hydrocarbon-deoxyribonucleoside products after initial separation, on small columns of Sephadex LH-20, from unhydrolysed DNA, oligonucleotides and free bases, were resolved by high pressure liquid chromatography (HPLC). The qualitative nature of the adducts found in both control and pretreated cells was virtually identical; however pretreatment with 3-methylcholanthrene resulted in a quantitatively higher level of binding. The major hydrocarbon-deoxyribonucleoside adduct, found in hepatocytes co-chromatographed with that obtained following reaction of the diol-epoxide, (±)7α,8β-dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene with DNA. Small amounts of other adducts were also present including a more polar product which co-chromatographed with the major hydrocarbon-deoxyribonucleoside adduct formed following microsomal activation of 9-hydroxybenzo[a]pyrene and subsequent binding to DNA. In contrast to the results with hepatocytes, when microsomes were used to metabolically activate benzo[a]pyrene, the major DNA bound-product co-chromatographed with the more polar adduct formed upon further metabolism of 9-hydroxybenzo[a]pyrene. These results illustrate that great caution must be exercised in the extrapolation of results obtained from short-term mutagenesis test systems, utilising microsomes, to in vivo carcinogenicity studies.     

In vivo benzo[a]pyrene diol epoxide-induced alkali-labile sites are not apurinic sites

We have used endonuclease IV from Escherichia coli as a probe for apurinic sites in the DNA of HeLa cells following treatment with an activated diol epoxide derivative of benzo[a]pyrene. DNA strand breaks and alkali-labile sites were observed that were repaired following exposure to the carcinogenic alkylating agent. The alkali-labile sites were not substrates for the apurinic site-specific endonuclease IV. We conclude that the alkali-labile sites formed in vivo by benzo[a]pyrene derivatives are not apurinic sites and probably arise as a consequence of rearrangement of the abundant N²-guanine adducts. This finding questions the involvement of apurinic sites in the mutagenic activity of benzo[a]pyrene.     

Effects of the hepatic S9 fraction from aroclor-1254-treated rats on the mutagenicity of benzo[a]pyrene and 2-aminoanthracene in the salmonella/microsome assay

The mutagenicity of benzo[a]pyrene and 2-aminoanthracene for Salmonella typhimurium TA98 in the plate-incorporation test was studied using liver S9 from untreated and Aroclor-1254-treated rats. The induction of liver S9 protein, arylhydrocarbon hydroxylase (AHH), and cytochrome P448/450 was followed with time. There was no change in protein concentrations with induction; AHH and cytochrome levels were increased at 1, 3, 5 and 7 days post Aroclor treatment. Benzo[a]pyrene mutagenicity was enhanced with Aroclor treatment while 2-aminoanthracene mutagenicity was depressed. The benzo[a]pyrene mutagenicity showed a positive correlation with the levels of AHH and cytochrome on the plate; 2-aminoanthracene showed a negative correlation with activity in induced samples.     

Time course of metabolism of benzo[e]pyrene by hamster embryo cells and the effect of chemical modifiers

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.     

Metabolism of benzo[a]pyrene VI. Stereoselective metabolism of benzo[a]pyrene and benzo[a]pyrene 7,8-dihydrodiol to diol epoxides

(±)-7β,8α-Dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-1) and (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-2) are highly mutagenic diol epoxide diastereomers that are formed during metabolism of the carcinogen (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene. Remarkable stereoselectivity has been observed on metabolism of the optically pure (+)- and (?)-enantiomers of the dihydrodiol which are obtained by separation of the diastereomeric diesters with (?)-α-methoxy-α-trifluoromethylphenylacetic acid. The high stereoselectivity in the formation of diol epoxide-1 relative to diol epoxide-2 was observed with liver microsomes from 3-methylcholanthrene-treated rats and with a purified cytochrome P-448-containing monoxygenase system where the (?)-enantiomer produced a diol epoxide-2 to diol epoxide-1 ratio of 6 : 1 and the (+)-enantiomer produced a ratio of 1 : 22. Microsomes from control and phenobarbital-treated rats were less stereospecific in the metabolism of enantiomers of BP 7,8-dihydrodiol. The ratio of diol epoxide-2 to diol epoxide-1 formed from the (?)- and (+)-enantiomers with microsomes from control rats was 2 : 1 and 1 : 6, respectively. Both enantiomers of BP 7,8-dihydrodiol were also metabolized to a phenolic derivative, tentatively identified as 6,7,8-trihydroxy-7,8-dihydrobenzo[a]pyrene, which accounted for ～30% of the total metabolites formed by microsomes from control and phenobarbital-pretreated rats whereas this metabolite represents ～5% of the total metabolites with microsomes from 3-methylcholanthrene-treated rats. With benzo[a]pyrene as substrate, liver microsomes produced the 4,5-, 7,8- and 9,10-dihydrodiol with high optical purity (>85%), and diol epoxides were also formed. Most of the optical activity in the BP 7,8-dihydrodiol was due to metabolism by the monoxygenase system rather than by epoxide hydrase, since hydration of (±)-benzo[a]pyrene 7,8-oxide by liver microsomes produced dihydrodiol which was only 8% optically pure. Thus, the stereospecificity of both the monoxygenase system and, to a lesser extent, epoxide hydrase plays important roles in the metabolic activation of benzo[a]pyrene to carcinogens and mutagens.     

Liver microsomal electron transport systems. Properties of a reconstituted, NADH-mediated benzo[a]pyrene hydroxylation system.

An enzyme system from rat liver microsomes which catalyzes the NADH-mediated hydroxylation of benzo[a]pyrene has been reconstituted. The essential microsomal components of this NADH-dependent pathway were NADH-cytochrome b₅ reductase, cytochrome b₅, cytochrome P-448 and, phosphatidyl choline. Highly purified NADPH-cytochrome c reductase containing small amounts of deoxycholate stimulated this NADH-mediated pathway supported by 0.2 mm NADH whereas boiled reductase had little effect. Part of this stimulation could be attributed to hydroxylation of benzo[a]pyrene via a second pathway; i.e., NADPH-cytochrome c reductase in combination with cytochrome P-448 and phosphatidylcholine also supported a low rate of NADH-dependent hydroxylation. The mechanism of the remaining stimulation is not known. However, the effect of NADPH-cytochrome c reductase on the reconstituted cytochrome b₅-dependent pathway was not unique; high concentrations of deoxycholate also stimulated this pathway, perhaps by facilitating the transfer of electrons from NADH-cytochrome b₅ reductase to cytochrome b₅. The addition of NADPH-cytochrome c reductase to the cytochrome b₅-dependent reconstituted system also affected the apparent K_m of NADH for benzo[a]pyrene hydroxylation. In the absence of NADPH-cytochrome c reductase, the apparent K_m of NADH was 1.3 μm while in its presence a low (1.3 μm) and a high (1700 μm) K_m were observed, consistent with the affinities of the two flavoproteins for NADH. Our results also indicate that the relative contribution of the pathway due to NADPH-cytochrome c reductase in combination with phosphatidyl choline and cytochrome P-448 to the overall rate of NADH-supported benzo[a]pyrene hydroxylation in microsomes would be greatly dependent on the concentration of NADH chosen. The rate of benzo[a]pyrene hydroxylation by these reconstituted components was almost 10-fold greater with 10 mm NADH than with 0.2 mm NADH, a result consistent with the reduction of NADPH-cytochrome c reductase by high concentrations of NADH.     

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.     

A modified method for the assay of benzo(a)pyrene hydroxylase

An assay is described for the activity of benzo[a]pyrene hydroxylase. The method is based on the direct fluorimetric measurement of the metabolites of benzo[α]pyrene in alkaline medium after adding Triton X 100 to the testing sample.     

H2AX phosphorylation in A549 cells induced by the bulky and stable DNA adducts of benzo[a]pyrene and dibenzo[a,l]pyrene diol epoxides

Early events in the cellular response to DNA damage, such as double strand breaks, rely on lesion recognition and activation of proteins involved in maintenance of genomic stability. One important component of this process is the phosphorylation of the histone variant H2AX. To investigate factors explaining the variation in carcinogenic potency between different categories of polycyclic aromatic hydrocarbons (PAHs), we have studied the phosphorylation of H2AX (H2AXγ). A549 cells were exposed to benzo[a]pyrene diol epoxide [(+)-anti-BPDE] (a bay-region PAH) and dibenzo[a,l]pyrene diol epoxide [(−)-anti-DBPDE] (a fjord-region PAH) and H2AXγ was studied using immunocytochemistry and Western blot. Hydrogen peroxide (H₂O₂) was used to induce oxidative DNA damage and strand breaks. As showed with single cell gel electrophoresis, neither of the diol epoxides resulted in DNA strand breaks relative to H₂O₂. Visualisation of H2AXγ formation demonstrated that the proportion of cells exhibiting H2AXγ staining at 1 h differed between BPDE, 40% followed by a decline, and DBPDE, <10% followed by an increase. With H₂O₂ treatment, almost all cells demonstrated H2AXγ at 1 h. Western blot analysis of the H2AXγ formation also showed concentration and time-dependent response patterns. The kinetics of H2AXγ formation correlated with the previously observed kinetics of elimination of BPDE and DBPDE adducts. Thus, the extent of H2AXγ formation and persistence was related to both the number of adducts and their structural features.     

Activation of benzo[a]pyrene and 2-aminoanthracene to bacteria mutagens by mussel digestive gland postmitochondrial fraction

The ability of the mussel postmitochondrial fraction (S9) to activate benzo[a]pyrene (BaP) and 2-aminoanthracene (2AA) to mutagenic metabolites towards Salmonella typhimurium strain TA98 was tested. The mechanisms involved in this activation were investigated and mussel cytochrome P-450-dependent monooxygenases and its NADPH cytochrome c reductase were found to contribute to the activation of BaP. This activation was improved by treating the mussel with 4,5,4′,5′-tetrachlorobiphenyl (TCB) (a 3-methylcholanthrene-type inducer of cytochrome P-450-dependent monooxygenase in marine fish) and was inhibited by α-naphthoflavone (ANF), a cytochrome P-450 inhibitor. However, both BaP activation and cytchrome P-450-related metabolic activities are much weaker in mussels than in vertebrates. Mussel S9 activates aromatic amines more effectively than BaP. Pretreatment of mussels with TCB or addition of ANF in the incubation medium has no effect on 2AA activation. As suggested by Kurelec (1985), aromatic amine metabolism may be supported by a flavoprotein mixed-function amine oxidase which is NADPH-dependent.     

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.     

Effect of various substituents in the 6-position on the relative carcinogenic activity of a series of benzo[a]pyrene derivatives

To test the hypothesis that polycyclic aromatic hydrocarbons capable of being converted to a reactive ester of the mesohydroxymethyl metabolite would be carcinogenic, a series of 6-substituted derivatives of benzo[a]pyrene (B[a]P) were tested for carcinogenicity in Sprague-Dawley rats by subcutaneous injection of the compound in sesame oil on alternate days for 30 doses. At the 0.2-μmol dose level B[a]P, 6-acetoxymethyl(6-AcOCH₂)B[a]P, 6-hydroxymethyl(6-HOCH₂)B[a]P, 6-methyl(6-CH₃)B[a]P and 6-benzoyloxymethyl(6-BzOCH₂)B[a]P were nearly equipotent, 6-formyl(6-OCH)-and 6-chloromethyl(6-ClCH₂)B[a]P were less active, and 6-methoxymethyl (6-MeOCH₂)B[a]P was inactive. At lower doses the order of potency was estimated to be: 6-AcOCH₂- = 6-HOCH₂- = or > B[a]P > 6-CH₂- > 6-BzOCH₂- > 6-ClCH₂- > 6-OCH- > 6-BrCH₂B[a]P. Incubation of these compounds in the presence of cofactors or cofactors plus a microsomal preparation of rat subcutis indicated that enzymic activation was necessary for metabolism to highly polar products and for conversion of 6-AcOCH₂-, 6-BzOCH₂- and 6-OCHB[a]P to 6-HOCH₂B[a]P. The halomethyl compounds were converted to 6-HOCH₂B[a]P in the absence of enzyme by hydrolysis. 6-MeOCH₂B[a]P was unchanged in this system. These observations are consistent with the foregoing hypothesis with regard to derivatives of B[a]P and demonstrate that compounds of this series that are capable of conversion to the 6-HOCH₂-derivatives are carcinogenic.