Quantifying genotoxicity and non-genotoxicity

Since the ability to induce genotoxicity is often equated with the potential for initiating the carcinogenic process, a method for quantitating genotoxicity would provide a useful measure for this potential. It is demonstrated herein that CPBS, the Carcinogenicity Prediction and Battery Selection method, provides a useful quantitative measure of genotoxicity as well as allowing for the detailed evaluation of the performance of batteries of short-term tests in order to select those predictive of carcinogenic potential.     

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.     

Rhodanobacter sp. strain BPC1 in a benzo[a]pyrene-mineralizing bacterial consortium

A bacterial consortium which rapidly mineralizes benzo[a]pyrene when it is grown on a high-boiling-point diesel fuel distillate (HBD) was recovered from soil and maintained for approximately 3 years. Previous studies have shown that mobilization of benzo[a]pyrene into the supernatant liquid precedes mineralization of this compound (R. Kanaly, R. Bartha, K. Watanabe, and S. Harayama, Appl. Environ. Microbiol. 66:4205-4211, 2000). In the present study, we found that sterilized supernatant liquid filtrate (SSLF) obtained from the growing consortium stimulated mineralization of benzo[a]pyrene when it was readministered to a consortium inoculum without HBD. Following this observation, eight bacterial strains were isolated from the consortium, and SSLF of each of them was assayed for the ability to stimulate benzo[a]pyrene mineralization by the original consortium. The SSLF obtained from one strain, designated BPC1, most vigorously stimulated benzo[a]pyrene mineralization by the original consortium; its effect was more than twofold greater than the effect of the SSLF obtained from the original consortium. A 16S rRNA gene sequence analysis and biochemical tests identified strain BPC1 as a member of the genus Rhodanobacter, whose type strain, Rhodanobacter lindaniclasticus RP5557, which was isolated for its ability to grow on the pesticide lindane, is not extant. Strain BPC1 could not grow on lindane, benzo[a]pyrene, simple hydrocarbons, and HBD in pure culture. In contrast, a competitive PCR assay indicated that strain BPC1 grew in the consortium fed only HBD and benzo[a]pyrene. This growth of BPC1 was concomitant with growth of the total bacterial consortium and preceded the initiation of benzo[a]pyrene mineralization. These results suggest that strain BPC1 has a specialized niche in the benzo[a]pyrene-mineralizing consortium; namely, it grows on metabolites produced by fellow members and contributes to benzo[a]pyrene mineralization by increasing the bioavailability of this compound.     

Mutagenicity of some methylated benzo[a]pyrene derivatives

The mutagenicity of benzo[a]pyrene (BP) and a number of methylated derivatives towards Salmonella typhimurium has been tested. The most mutagenic derivative tested was 6-methylbenzo[a]pyrene which produced about twice the number of revertants as did BP, 11-Methylbenzo[a]pyrene was slightly more mutagenic than BP. All the other compounds tested (7-, 8-, 9- and 10-methylbenzo[a]pyrene and 7,8- and 7,10-dimethylbenzo[a]pyrene) were significantly less active than benzo[a]pyrene. With the exception of 6-methylbenzo[a]pyrene, these results closely parallel the known carcinogenicity of the methylated benzo[a]pyrenes, and support the view that metabolic activation of BP may involve the 7-10 positions which are blocked in the methylated compounds.     

HPLC analysis of benzo[a]pyrene-albumin adducts in benzo[a]pyrene exposed rats. Detection of cis-tetrols arising from hydrolysis of adducts of anti- and syn-BPDE-III with proteins

Quantitation of protein-benzo[a]pyrene adducts represent a more sensitive analysis method than quantitation of benzo[a]pyrene-DNA adducts. By accurate analysis of benzo[a]pyrene-protein adducts several different molecular adduct forms can be studied. Male Wistar rats were injected i.p. with benzo[a]pyrene, and serum albumin was isolated and subjected to acid hydrolysis at 90 degrees C for 3 h. The hydrolysate was analyzed by HPLC with fluorescence detection. The HPLC profiles obtained after albumin hydrolysis from benzo[a]pyrene exposed animals were compared to similar HPLC profiles from in vitro adducted bovine serum albumin (BSA) and direct hydrolysis of both r-10,t-9-dihydrodiol-c-7,8-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (syn-BPDE-III) and r-10,t-9-t-dihydrodiol-t-7,8-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE-III). After acid hydrolysis of albumin from benzo[a]pyrene exposed rats, 6 fluorescent peaks were separated. Four of the peaks were isomers of benzo[a]pyrene-tetrahydrotetrols, (+/-)-benzo[a]pyrene-r-7,t-8,9,10-tetrahydrotetrol, (+/-)-benzo[a]pyrene-r-7,t-8,9,c-10-tetrahydrotetrol, (+/-)-benzo[a]pyrene-r-7,t-8,c-9,t-10-tetrahydrotetrol and (+/-)-benzo[a]pyrene-r-7,t-8,c-9,10-tetrahydrotetrol. In addition we found two fluorescent peaks, named X1 and X2 with retention times similar to the benzo[a]pyrene-tetrols. The unknown fluorescent peaks reacted similar to the four known tetrols in both dose response experiments and time course experiments. Fluorescent material with retention times equal to X1 and X2 were found after acid hydrolysis of syn-BPDE-III and anti-BPDE-III in acid and in hydrolysates from BSA treated in vitro with syn-BPDE-III and anti-BPDE-III. The ratio X1/X2 was relatively constant indicating epimerization equilibrium between these to species. Synchronous fluorescence analysis of fractions containing X1 or X2 from both in vivo and in vitro experiments showed fluorescence spectra characteristic of benzo[a]pyrene tetrols using a wavelength difference of 34 nm.     

Importance of the route of administration for genetic differences in benzo[a]pyrene-induced in utero toxicity and teratogenicity

C57BL/6N (Ahb/Ahb) mice have a high-affinity Ah receptor in tissues, whereas AKR/J and DBA/2N (Ahd/Ahd) mice have a poor-affinity Ah receptor. The cytochrome P1-450 induction response (enhanced benzo[a]pyrene metabolism) occurs much more readily in Ahb/Ahb and Ahb/Ahd than in Ahd/Ahd mice, at any given dose of the inducer benzo[a]pyrene. Embryos from the AKR/J X (C57BL/6N)(AKR/J)F1 and the reciprocal backcross were studied during benzo[a]pyrene feeding of the pregnant females. Oral benzo[a]pyrene (120 mg/kg/day) given to pregnant Ahd/Ahd mice between gestational day 2 and 10 produces more intrauterine toxicity and malformations in Ahd/Ahd than Ahb/Ahd embryos. This striking allelic difference is not seen in pregnant Ahb/Ahd mice receiving oral benzo[a]pyrene. Pharmacokinetics studies with [3H]benzo[a]pyrene in the diet and high-performance liquid chromatographic analysis of benzo[a]pyrene metabolism in vitro by the maternal intestine, liver, and ovary and the embryos of control and oral benzo[a]pyrene-treated pregnant females are consistent with "first-pass elimination" kinetics and differences in benzo[a]pyrene metabolism by the embryos and/or placentas versus maternal tissues. In the pregnant Ahd/Ahd mouse receiving oral benzo[a]pyrene, little induction of benzo[a]pyrene metabolism occurs in her intestine and liver; this leads to much larger amounts of benzo[a]pyrene reaching her embryos, and genetic differences in toxicity and teratogenesis are manifest. In the pregnant Ahb/Ahd mouse receiving oral benzo[a]pyrene, benzo[a]pyrene metabolism is greatly enhanced in her intestine and liver; this leads to less benzo[a]pyrene reaching her embryos, much less intrauterine toxicity and malformations, and no genetic differences are manifest. More toxic metabolites (especially benzo[a]pyrene 1,6- and 3,6-quinones) are shown to occur in Ahd/Ahd embryos than in Ahb/Ahd embryos. In additional studies, no prenatal or neonatal "imprinting" effect in C57BL/6N mice by 2,3,7,8-tetrachlorodibenzo-p-dioxin or Aroclor 1254 on benzo[a]pyrene metabolism later in life was detectable. These genetic differences in intrauterine toxicity and teratogenicity induced by oral benzo[a]pyrene are just opposite those induced by intraperitoneal benzo[a]pyrene [Shum et al., '79; Hoshino et al., '81). The data in the present report emphasize the importance of the route of administration when the teratogen induces its own metabolism.     

Stereoselective aspects of the metabolic activation of benzo[a]pyrene by human skin in vitro

Benzo[a]pyrene (BP) is activated within tissues in both a regio- and a stereoselective manner and, since human skin is sensitive to tumour induction by polycyclic aromatic hydrocarbons (PAH), the steroselective metabolism of BP in this tissue has been investigated. Samples of skin from eleven individuals were treated with [3H]BP in short-term organ culture. Two samples were also treated with mixtures of [14C](+)- and (-)-trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene (BP-7,8-dihydrodiol) in varying proportions. Following application of [3H] BP, more 7,8-dihydrodiol was recovered from the skin itself than from the culture fluid in ten cases; no 7.8-dihydrodiol was detected in extracts from the eleventh. The 7,8-dihydrodiol metabolite was extracted predominantly (range 74-greater than 99%) as the (-)-enantiomer in nine of these ten patients, although proportionately more (+)-enantiomer was recovered from the culture fluid than from the skin in each case. The relative proportions of [3H]BP tetrols derived from syn- and anti-7,8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydroxybenzo[a]pyrene (BPDE) detected in these extracts was more variable. When skin samples were treated with [14C]BP-7,8-dihydrodiol, more anti- than syn-BPDE-derived tetrols were extracted, irrespective of the optical purity of the dihydrodiol applied. These findings provide evidence for interindividual variations in the stereoselective metabolism of BP, which may be of some importance in determining individual susceptibility to PAH-induced skin carcinogenesis.     

Mutagenicity of benzylic acetates, sulfates and bromides of polycyclic aromatic hydrocarbons

Studies were performed to determine the direct mutagenicity of the acetates and some bromides and sulfates of hydroxymethyl polycyclic aromatic hydrocarbons in S. typhimurium strains TA98 and TA100. Benzylic acetates, bromides and sulfates were synthesized and characterized. The compounds tested were benzyl alcohol, 5-hydroxymethylchrysene, 1-hydroxymethylpyrene, 6-hydroxymethylbenzo[a]pyrene, 6-(2-hydroxyethyl)benzo[a]pyrene, 6-hydroxymethylanthanthrene, 9-hydroxymethylanthracene, 9-hydroxymethyl-10-methylanthracene, 7-hydroxymethylbenz[a]anthracene, 7-(2-hydroxyethyl)benz[a]anthracene, 12-hydroxymethylbenz[a]anthracene, 7-hydroxymethyl-12-methylbenz[a]anthracene, 12-hydroxymethyl-7-methylbenz[a]anthracene, 1-hydroxy-3-methylcholanthrene, 2-hydroxy-3-methylcholanthrene, 3-hydroxy-3, 4-dihydrocyclopental[cd]pyrene and 4-hydroxy-3, 4-dihydrocyclopental[cd]pyrene. The benzylic sulfate esters of 6-hydroxymethylbenzo[a]pyrene and 7-hydroxymethylbenz[a]anthracene were the most mutagenic compounds, whereas the aliphatic sulfate ester of 7-hydroxyethylbenz[a]anthracene did not cause an increase in mutations above background. All meso-anthracenic benzylic acetate esters were mutagenic in both strains with various degrees of activity, whereas the corresponding non-benzylic esters were inactive, as expected. Of the non-meso-benzylic acetate esters, only the 3-acetoxy-3, 4-dihydrocyclopenta[cd]pyrene was mutagenic. In the benzylic bromide series, only the eight mesoanthracenic were mutagenic, whereas benzyl bromide and 5-bromomethylchrysene were inactive. The aliphatic bromides, 6-(2-bromoethyl)benzo[a]pyrene and 7-(2-bromoethyl)benz[a]anthracene did not display significant activity. The potencies of the acetate esters more accurately reflect the mutagenicity because the rate of solvolysis did not compete with the reactivity of the esters with bacterial DNA. In the case of benzylic sulfates and bromides, the rate of solvolysis was very rapid and could have diminished the level of mutagenicity, depending on the assay conditions. These results demonstrate that meso-anthracenic benzylic acetates, sulfates and bromides are mutagenic, whereas benzylic acetate esters attached to other carbon atoms are inactive.     

Origins of stereospecificity in DNA damage by anti-benzo[a]pyrene diol-epoxides. A molecular modelling study

A general computational procedure for the modelling of intercalated DNA-ligand complexes has been developed, and is used here to model intercalated complexes of the (+)-anti and (-)-anti enantiomers of benzo[a]pyrene diol-epoxide (BPDE) with cytosine-3',5'-guanosine double-stranded DNA sequences (dCpG). Results are presented indicating differences between the behaviours of the two enantiomers which have implications for the understanding of the stereospecificity of DNA strand breakage by benzo[a]pyrene diol-epoxides.     

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)     

Mutagenicity of C24H14 PAH in human cells expressing CYP1A1

Relatively little is known about the mutagenicity of C24H14 PAH, a diverse group of five- and six-ring PAH, some of which are present at trace levels in the environment. To better understand the mutagenicity of this class of compounds, 11 C24H14 PAH, including benzo[a]perylene, benzo[b]perylene, dibenzo[a,e]fluoranthene, dibenzo[a,f]fluoranthene, dibenzo[j,l]fluoranthene, dibenzo[a,h]pyrene, dibenzo[a,i]pyrene, dibenzo[e,l]pyrene, naphtho[1,2-b]fluoranthene, naphtho[2,3-a]pyrene, and naphtho[2,3-e]pyrene, were tested in a mutagenicity assay based on human h1A1v2 cells. h1A1v2 cells are a line of human B-lymphoblastoid cells that have been engineered to express cytochrome P4501A1 (CYP1A1), an enzyme capable of metabolizing promutagenic PAH. Mutagenicity was measured at the thymidine kinase (tk) locus following a 72-h exposure period. Our results show that nine of the compounds were mutagenic. Benzo[a]perylene, dibenzo[a,e]fluoranthene, dibenzo[a,i]pyrene, and naphtho[2,3-a]pyrene were the most potent mutagens, having minimum mutagenic concentrations (MMC) (i.e., the dose at which the induced response was twice that of the negative controls) in the 1-5 ng/ml range. Benzo[b]perylene, dibenzo[a,h]pyrene, dibenzo[a,f]fluoranthene, and naphtho[2,3-e]pyrene were somewhat less potent mutagens, having MMC in the 10-30 ng/ml range. Dibenzo[e,l]pyrene, which had an MMC of 280 ng/ml, was the least potent mutagen. Dibenzo[j,l]fluoranthene and naphtho[1,2-b]fluoranthene were not mutagenic at the doses tested (1-3000 ng/ml). The most mutagenic compounds were also quite toxic. At the highest doses tested, benzo[a]perylene, dibenzo[a,e]fluoranthene, dibenzo[a,i]pyrene, dibenzo[a,h]pyrene, and dibenzo[a,f]fluoranthene induced > 60% killing, and naphtho[2,3-a]pyrene and naphtho[2,3-e]pyrene induced > 50% killing. Benzo[b]perylene, dibenzo[e,l]pyrene, dibenzo[j,l]fluoranthene, and naphtho[1,2-b]fluoranthene induced < 50% killing at the highest doses tested. Comparing these results to a previous study in which nine other C24H14 PAH were tested for mutagenicity in this same assay, it was found that dibenzo[a]pyrene isomers were generally more mutagenic than the other groups of C24H14 PAH tested. These observations are discussed with emphasis given to identifying C24H14 PAH that may be important environmental mutagens.     

Rapid mineralization of benzo[a]pyrene by a microbial consortium growing on diesel fuel

A microbial consortium which rapidly mineralized the environmentally persistent pollutant benzo[a]pyrene was recovered from soil. The consortium cometabolically converted [7-(14)C]benzo[a]pyrene to (14)CO(2) when it was grown on diesel fuel, and the extent of benzo[a]pyrene mineralization was dependent on both diesel fuel and benzo[a]pyrene concentrations. Addition of diesel fuel at concentrations ranging from 0.007 to 0.2% (wt/vol) stimulated the mineralization of 10 mg of benzo[a]pyrene per liter 33 to 65% during a 2-week incubation period. When the benzo[a]pyrene concentration was 10 to 100 mg liter(-1) and the diesel fuel concentration was 0.1% (wt/vol), an inoculum containing 1 mg of cell protein per liter (small inoculum) resulted in mineralization of up to 17.2 mg of benzo[a]pyrene per liter in 16 days. This corresponded to 35% of the added radiolabel when the concentration of benzo[a]pyrene was 50 mg liter(-1). A radiocarbon mass balance analysis recovered 25% of the added benzo[a]pyrene solubilized in the culture suspension prior to mineralization. Populations growing on diesel fuel most likely promoted emulsification of benzo[a]pyrene through the production of surface-active compounds. The consortium was also analyzed by PCR-denaturing gradient gel electrophoresis of 16S rRNA gene fragments, and 12 dominant bands, representing different sequence types, were detected during a 19-day incubation period. The onset of benzo[a]pyrene mineralization was compared to changes in the consortium community structure and was found to correlate with the emergence of at least four sequence types. DNA from 10 sequence types were successfully purified and sequenced, and that data revealed that eight of the consortium members were related to the class Proteobacteria but that the consortium also included members which were related to the genera Mycobacterium and Sphingobacterium.     

Enhancement and inhibition of benzo[a]pyrene-induced SOS function in E. coli by synthetic antioxidants

8 antioxidants were tested in the SOS chromotest for induction of SOS function and for modulation of benzo[a]pyrene-induced SOS function. None of the antioxidants leads to increased beta-galactosidase activity by itself. Butylated hydroxytoluene at concentrations between 10(-5) M and 3 X 10(-4) M enhances benzo[a]pyrene-induced SOS function at benzo[a]pyrene concentrations between 10(-6) M and 3 X 10(-5) M. Butylated hydroxyanisole, ethoxyquin, propyl gallate and octyl gallate also slightly enhance benzo[a]pyrene-induced SOS function at concentrations up to 3 X 10(-4) M though to a lesser degree than butylated hydroxytoluene. Dodecyl gallate, vitamin C and alpha-tocopherol do not increase benzo[a]pyrene action. In concentrations exceeding 3 X 10(-4) M all synthetic antioxidants tested but not vitamin C and alpha-tocopherol decrease beta-galactosidase activity both in the absence and, more extensively, in the presence of benzo[a]pyrene. Preliminary data suggest that the apparent suppression of benzo[a]pyrene-induced SOS function is not due to an effect on the formation of benzo[a]pyrene metabolites by the metabolizing system used.     

Oxidation of polycyclic aromatic hydrocarbons by the bacterial laccase CueO from E. coli

Laccases produced by white rot fungi are capable of rapidly oxidizing benzo[a]pyrene. We hypothesize that the polycyclic aromatic hydrocarbon (PAH)-degrading bacteria producing laccase can enhance the degree of benzo[a]pyrene mineralization. However, fungal laccases are glycoproteins which cannot be glycosylated in bacteria, and there is no evidence to show that bacterial laccases can oxidize benzo[a]pyrene. In this study, the in vitro oxidation of PAHs by crude preparations of the bacterial laccase, CueO, from Escherichia coli was investigated. The results revealed that the crude CueO catalyzed the oxidation of anthracene and benzo[a]pyrene in the same way as the fungal laccase from Trametes versicolor, but showed specific characteristics such as thermostability and copper dependence. In the presence of 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid), high amounts of anthracene and benzo[a]pyrene, 80% and 97%, respectively, were transformed under optimal conditions of 60°C, pH 5, and 5 mmol l(-1) CuCl(2) after a 24-h incubation period. Other PAHs including fluorene, acenaphthylene, phenanthrene, and benzo[a]anthracene were also oxidized by the crude CueO. These findings indicated the potential application of prokaryotic laccases in enhancing the mineralization of benzo[a]pyrene by PAH-degrading bacteria.     

Simple luminescence method for estimation of benzo[a]pyrene in a complex mixture of polycyclic aromatic hydrocarbons without a pre‐separation procedure

Benzo[a]pyrene causes cancer at cellular level and is widely present in the environment. Conventional spectroscopic methods for analysis of this compound need a pre‐separation procedure due to severe spectral overlap from other polycyclic aromatic hydrocarbons. We report a simple method that avoids spectral overlap of benzo[a]pyrene from other impurities or polycyclic aromatic hydrocarbons (PAHs), thus it can easily identify benzo[a]pyrene in a complex PAH mixture. The method could easily identify benzo[a]pyrene in an 18‐component PAH mixture. Calibration plots in methanol solution and in micellar media show a good linearity (R > 0.9997) in the benzo[a]pyrene concentration range generally found in the environment. The method gives a detection limit of 1.52 × 10^?9 mol/L in CTAB micellar medium and 2.55 × 10^?9 mol/L in methanol solution. The proposed method is selective, sensitive and fast. The fluorescence response of benzo[a]pyrene is found to be a potential candidate to sense the critical micellar concentration (CMC) of CTAB micelles. Copyright © 2003 John Wiley & Sons, Ltd.     

Bacterial mutagenicity of two cyclopentafused isomers of benzpyrene

Two novel cyclopentafused polycyclic aromatic hydrocarbons, naphtho(1,2,3-mno)acephenanthrylene (cyclopenta benzo[e]pyrene) and naphtho(2,1,8-hij)acephenanthrylene (cyclopenta(ij)benzo[a]pyrene) were evaluated for mutagenic activity in the Ames Salmonella typhimurium plate incorporation assay. Both compounds required S9 metabolic activation, and showed optimal activity at low S9 concentrations (below 0.6 mg/plate). Both compounds induced frameshift and base-pair substitution mutations, being active in strains TA98, TA100, TA1537, TA1538 and TA104, but not in strain TA1535. Cyclopenta(ij)benzo[a]pyrene was more active than cyclopentabenzo[e]pyrene, and both were more potent than their parent ring systems, benzo[a]pyrene and benzo[e]pyrene, respectively. Cyclopenta(ij)benzo[a]pyrene was more active in strain TA104 than in TA100 or TA98 (250-470, 340 and 80-100 rev/nmole) as was benzo[a]pyrene (120, 70 and 40 rev/nmole respectively); cyclopentabenzo[e]pyrene was more active in TA100 than TA104 or TA98 (70 versus 50 and 40 rev/nmole), and benzo[e]pyrene showed a similar pattern (4, 3.5 and 0.6 rev/nmole). The relative potencies of the four compounds are in accord with predictions based on perturbational molecular orbital calculations. The peak of activity at low S9 concentrations is consistent with epoxidation at the cyclopentafused ring being the major route of metabolic activation for both these cyclopentafused compounds.     

Mutagenic activity of biliary metabolites of 6-hydroxymethylbenzo[a]pyrene

The biliary excretion of the carcinogen 6-hydroxy-methylbenzo[a]pyrene was investigated in rats after i.p. administration. Mutagenicity of the parent compound and its biliary metabolites was tested in Ames Salmonella/microsome mutagenicity assay. Approximately 40% of the dose administered (0.25-0.5 mg/kg) to the rats was excreted in the bile within 6 h. 6-Hydroxymethylbenzo[a]pyrene was excreted primarily as water-soluble metabolites, including glucuronide and sulfate conjugates. Negligible quantities of unchanged 6-hydroxymethylbenzo[a]pyrene were excreted in the bile. In the presence of Aroclor-induced S9, 6-hydroxymethylbenzo[a]pyrene was a potent mutagen. The mutagenicity of bile from rats treated with 6-hydroxymethylbenzo[a]pyrene was variable in the absence of an activation system. However, the same bile samples were mutagenic in the presence of beta-glucuronidase and/or S9. These results indicate that biliary metabolites of 6-hydroxymethylbenzo[a]pyrene can be metabolically activated to mutagenic species.     

Analysis of metabolism of carcinogenic polycyclic hydrocarbons by position-sensing proportional counting of thin-layer chromatograms.

A procedure which combines thin-layer chromatography with position-sensing proportional counting has been developed for analyzing the metabolism of carcinogenic polycyclic aromatic hydrocarbons. The profiles of the metabolites of [³H]benzo(a)pyrene and 7,12-[12-¹⁴C]dimethylbenz(a)anthracene produced in cell culture were comparable when obtained by this procedure and by standard methods. However, position-sensing proportional counting allows simultaneous counting of all components of a sample within 10–20 min, and thereby permits the analysis of many hydrocarbon samples in a short time. In addition, the procedure eliminates the necessity of cutting or scraping carcinogen-containing thin-layer chromatograms.     

Degradation of fluoranthene, pyrene, benz[a]anthracene and dibenz[a,h]anthracene by Burkholderia cepacia

Microbiological analysis of soils from a polycyclic aromatic hydrocarbon (PAH)-contaminated site resulted in the enrichment of five microbial communities capable of utilizing pyrene as a sole carbon and energy source. Communities 4 and 5 rapidly degraded a number of different PAH compounds. Three pure cultures were isolated from community 5 using a spray plate method with pyrene as the sole carbon source. The cultures were identified as strains of Burkholderia ( Pseudomonas ) cepacia on the basis of biochemical and growth tests. The pure cultures (VUN 10 001, VUN 10 002 and VUN 10 003) were capable of degrading fluorene, phenanthrene and pyrene (100 mg l⁻¹) to undetectable levels within 7–10 d in standard serum bottle cultures. Pyrene degradation was observed at concentrations up to 1000 mg l⁻¹. The three isolates were also able to degrade other PAHs including fluoranthene, benz[ a ]anthracene and dibenz[ a , h ]anthracene as sole carbon and energy sources. Stimulation of dibenz[ a , h ]anthracene and benzo[ a ]pyrene degradation was achieved by the addition of small quantities of phenanthrene to cultures containing these compounds. Substrate utilization tests revealed that these micro-organisms could also grow on n -alkanes, chlorinated- and nitro-aromatic compounds.     

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.