Occupational Exposure to Polycyclic Aromatic Hydrocarbons and Elevated Cancer Incidence in Firefighters.

Cancer incidence appears to be higher amongst firefighters compared to the general population. Given that many cancers have an environmental component, their occupational exposure to products of carbon combustion such as polycyclic aromatic hydrocarbons (PAHs) is of concern. This is the first UK study identifying firefighters exposure to PAH carcinogens. Wipe samples were collected from skin (jaw, neck, hands), personal protective equipment of firefighters, and work environment (offices, fire stations and engines) in two UK Fire and Rescue Service Stations. Levels of 16 US Environmental Protection Agency (EPA) PAHs were quantified together with more potent carcinogens: 7,12-dimethylbenzo[a]anthracene, and 3-methylcholanthrene (3-MCA) (12 months post-initial testing). Cancer slope factors, used to estimate cancer risk, indicate a markedly elevated risk. PAH carcinogens including benzo[a]pyrene (B[a]P), 3-MCA, and 7,12-dimethylbenz[a]anthracene PAHs were determined on body surfaces (e.g., hands, throat), on PPE including helmets and clothing, and on work surfaces. The main exposure route would appear to be via skin absorption. These results suggest an urgent need to monitor exposures to firefighters in their occupational setting and conduct long-term follow-up regarding their health status.

Multi-walled carbon nanotube-impregnated agarose film microextraction of polycyclic aromatic hydrocarbons in green tea beverage.

A new microextraction procedure termed multi-walled carbon nanotube-impregnated agarose film microextraction (MWCNT-AFME) has been developed. The method utilized multi-walled carbon nanotubes (MWCNTs) immobilized in agarose film to serve as adsorbent in solid phase microextraction (SPME). The film was prepared by mixing the MWCNTs in agarose solution and drying the mixture in oven. Extraction of selected polycyclic aromatic hydrocarbons was performed by inserting a needle through circular MWCNT-impregnated agarose films (5 mm diameter) and the assembly was dipped into an agitated sample solution prior to micro high performance liquid chromatography-ultraviolet analysis. Back extraction was then performed using ultrasonication of the films in 100 µL of solvent. The film was discarded after single use, thus avoiding any analyte carry-over effect. Due to the mesoporous nature of the agarose film, the MWCNTs were immobilized easily within the film and thus allowing for close contact between adsorbent and analytes. Under the optimized extraction conditions, the technique achieved trace LODs in the range of 0.1 to 50 ng L(-1) for the targeted analytes, namely fluoranthene, phenanthrene and benzo[a]pyrene. The method was successfully applied to the analysis of spiked green tea beverage samples with good relative recoveries in the range of 91.1 to 107.2%. The results supported the feasibility of agarose to serve as adsorbent holder in SPME which then minimizes the consumption of chemicals and disposal cost of organic wastes.

Antifungal activities of metabolites produced by a termite-associated Streptomyces canus BYB02.

Two main antifungal metabolites resistomycin and tetracenomycin D were isolated and purified from a termite-associated Streptomyces canus BYB02 by column chromatography. The structures of isolated compounds were determined on the basis of extensive spectroscopic analysis. Resistomycin possessed strong activities against mycelial growth of Valsa mali (IC(50) = 1.1 µg/mL) and Magnaporthe grisea (IC(50) = 3.8 µg/mL), which were comparable to those of referenced cycloheximide, with IC(50) values of 2.3 and 0.3 µg/mL, respectively. A further spore germination test showed that resistomycin exhibited potent reduction in spore germination for M. grisea , with an IC(50) value of 5.55 µg/mL. Finally, the in vivo antifungal activity experiment showed that resistomycin possessed significant preventive efficacy against rice blast, which was more potent than that of referenced carbendazim, with control efficacies of 66.8 and 58.7%, respectively. The present results suggest that resistomycin has potential to be used as a fungicide.

Sensitive immunosensor for benzo[a]pyrene detection based on dual amplification strategy of PAMAM dendrimer and amino-modified methylene blue/SiO2 core-shell nanoparticles.

A novel electrochemical immunosensor for sensitive detection of benzo[a]pyrene (BaP) is constructed using poly(amido amine) (PAMAM) dendrimer and functionalized methylene blue/SiO2 core-shell nanoparticle (MB/SiO2) loaded with horseradish peroxidase (HRP) and HRP-secondary antibody (HRP-Ab2). Greatly enhanced sensitivity for BaP analysis is based on a dual signal amplification strategy. Firstly, the gold electrode (GE) was amino-functioned by electropolymerization of a novel compound, 2-amino-5,2':5'2''-terthiophene, followed by the modification of G 2.0 PAMAM dendrimer to amplify functional groups on the substrate and thus enhance the immobilization capacity of BaP antigen (BaP-Ag). Secondly, amino-functionalized MB/SiO2 was used to load HRP and HRP-Ab2, and the resulting nanostructure (HRP-MB/SiO2-Ab2) was applied as the detection label for the immunosensor. The proposed immunosensor exhibited a relatively wide linear response between 0.01 and 2.0 ng/mL with a detection limit of 6 pg/mL. This amplification strategy shows excellent promise for environmental monitoring of some pollutants and a potential application in the immunosensor.

Benzo[a]pyrene degradation using simultaneously combined chemical oxidation, biotreatment with Fusarium solani and cyclodextrins.

The interest of simultaneously combining chemical (Fenton's reaction) and biological treatments for the degradation of a high molecular weight polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) has been studied in laboratory tests. An optimal concentration of 1.5x10(-3) M H(2)O(2) as Fenton's reagent was firstly determined as being compatible with the growth of Fusarium solani, the Deuteromycete fungus used in the biodegradation process. For the enhancement of BaP solubilisation, cyclodextrins were also used in the performed tests. The best degradation performance was achieved through the use of 5x10(-3) M hydroxypropyl-beta-cyclodextrin (HPBCD) in comparison with randomly methylated-beta-cyclodextrin (RAMEB). When Fenton's treatment was combined with biodegradation, a beneficial effect on BaP degradation (25%) was obtained in comparison with biodegradation alone (8%) or with chemical oxidation alone (16%) in the presence of HPBCD for 12 days of incubation.

Method development and validation for optimized separation of benzo[a]pyrene-quinone isomers using liquid chromatography-mass spectrometry and chemometric response surface methodology.

The successful separation of three benzo[a]pyrene-quinone isomers, two of which were previously unresolved, using liquid chromatography-mass spectrometry (LC-MS) and response surface methodology is presented. Initial efforts centered on chromatographic separation of benzo[a]pyrene-1,6/3,6-quinone peaks evaluated for both resolution and retention time. The mergence of the two parameters was accomplished using the Derringer's desirability function with subsequent optimization by a Box-Behnken response surface design. By implementing the optimal flow rate, column temperature and eluent composition predicted by the validated model, enhanced resolution of the two isomers was achieved in less than 20 min. Calibrations were performed to quantify these isomers and the limits of detection were determined. Optimal model conditions were then used to identify three independent benzo[a]pyrene-quinone isomers produced in the irradiation of a benzo[a]pyrene standard solvated in oxygen-saturated methanol/methylene chloride. This work is not only highly significant to the field of environmental chemistry, but instructive for investigators struggling with the co-elusion of isomeric compounds in their work.

Spectral differentiation and immunoaffinity capillary electrophoresis separation of enantiomeric benzo(a)pyrene diol epoxide-derived DNA adducts.

Antibody cross-reactivity makes separation and differentiation of enantiomeric analytes one of the most challenging problems in immunoanalytical research, particularly for the analysis of structurally related biological molecules [such as benzo( a)pyrene (BP) metabolites and BP-derived DNA adducts]. It has recently been shown that the interaction of enantiomers of BP tetrols (BPT) with a promiscuous anti-polycyclic aromatic hydrocarbon ( anti-PAH) monoclonal antibody (mAb) allowed for separation of all four enantiomeric isomers using immunoaffinity capillary electrophoresis [ Grubor, N. M. , Armstrong, D. W. , and Jankowiak, R. ( 2006) Electrophoresis 27, 1078 ] and unambiguous spectral resolution using fluorescence line narrowing spectroscopy (FLNS) [ Grubor, N. M. , Liu, Y. , Han, X. , Armstrong, D.W. , and Jankowiak, R. ( 2006) J. Am.Chem. Soc. 128, 6409 ]. Here, we expand the use of the above two methodologies to the group of biologically important molecules that are products of BP diol epoxide (BPDE)-induced DNA damage. Four diastereomeric anti-BPDE-derived deoxyguanosine (dG) adducts, that is, (+)- and (-)- anti-trans-BPDE- N (2)-dG and (+)- and (-)- anti-cis-BPDE- N (2)-dG, were electrophoretically separated and spectroscopically differentiated using 8E11 mAb raised against BP-DNA conjugates. In fluorescence line narrowing spectroscopy (FLNS) experiments, complexes of BPDE-dG adducts with mAb revealed differences in fluorescence origin band positions, bandwidths, and vibrational patterns for all four BPDE- N (2)-dG adducts. Narrow fluorescence origin bands observed for (-)- trans-BPDE-dG (70 cm (-1)) and (+)- trans-BPDE- N (2)-dG (80 cm (-1)) suggest spatial constraint within the mAb binding pocket. Broader origin bands observed for cis type adducts ( approximately 120 cm (-1)) in 8E11 mAb suggest different binding geometries and/or conformational changes, as also indicated by changes in vibrational frequencies observed for the (+)- anti-cis and (-)- anti-cis adducts complexed with mAb. FLNS revealed that binding conformations and interactions within the mAb binding pocket are different for each adduct, enabling unambiguous positive identification. The methodologies described in this manuscript could also be used for analysis of DNA adducts following enzymatic hydrolysis of BPDE-adducted DNA to free nucleosides.

Resistoflavine, cytotoxic compound from a marine actinomycete, Streptomyces chibaensis AUBN1/7.

In our systematic screening programme for marine actinomycetes, a bioactive Streptomycete was isolated from marine sediment samples of Bay of Bengal, India. The taxonomic studies indicated that the isolate belongs to Streptomyces chibaensis and it was designated as S. chibaensis AUBN1/7. The isolate yielded a cytotoxic compound. It was obtained by solvent extraction followed by the chromatographic purification. Based on the spectral data of the pure compound, it was identified as quinone-related antibiotic, resistoflavine (1). It showed a potent cytotoxic activity against cell lines viz. HMO2 (Gastric adenocarcinoma) and HePG2 (Hepatic carcinoma) in vitro and also exhibited weak antibacterial activities against Gram-positive and Gram-negative bacteria.

[Cytotoxic compounds from the marine actinobacterium].

We isolated a bioactive streptomycete from marine sediment samples collected at Bay of Bengal, India, during our systematic study of marine actinobacteria. The taxonomic studies indicated that the isolate is related to Strepomyces corchorusii. However, it differed in certain aspects, and, hence, was designated as S. corchorusii AUBN(1)/7. A solvent extraction followed by a chromatographic purification helped obtain from the isolate two cytotoxic compounds, which were identified as resistomycin, a quinone-related antibiotic, and tetracenomycin D, an anthraquinone antibiotic, on the basis of spectral data of pure compounds. They demonstrated in vitro a potent cytotoxic activity against cell lines HMO2 (gastric adenocarcinoma) and HepG2 (hepatic carcinoma) and also exhibited weak antibacterial activities against Gram-positive and Gram-negative bacteria. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2006, vol. 32, no. 3; see also http://www.maik.ru.

1-Hydroxy-1-norresistomycin and resistoflavin methyl ether: new antibiotics from marine-derived streptomycetes.

Cultivation of the marine-derived streptomycete isolate B8005 delivered three known antibiotics, resistomycin (1), resistoflavin (3a) and tetracenomycin (4), and a further member of the rare resistomycin class, the weakly antibiotically active 1-hydroxy-1-norresistomycin (2). From a related marine strain B4842, 1 and resistoflavin methyl ether (3b) have been isolated. The formation of 2 is of interest from a biosynthetic point of view.

Extraction and purification of depurinated benzo[a]pyrene-adducted DNA bases from human urine by immunoaffinity chromatography coupled with HPLC and analysis by LC/quadrupole ion-trap MS.

In this paper, we describe implementation and testing of an immunoaffinity (IA) column for rapid and selective extraction of 7-(benzo[a]pyren-6-yl)adenine (BP-6-N7Ade) and 7-(benzo[a]pyren-6-yl)guanine (BP-6-N7Gua) from urine, where BP is benzo[a]pyrene. The BP radical cation is a carcinogenic metabolite that reacts with double-stranded DNA, producing depurinated BP-adducted DNA bases excreted in urine. The expected modified nucleobases are BP-6-N7Gua, BP-6-N7Ade, and 8-(benzo[a]pyren-6-yl)guanine (BP-6-C8Gua), and they may serve as important biomarkers for DNA damage by PAHs. IA extracts of urine from a cigarette smoker and a nonsmoker contained less than 5% of contaminants present in Sep-Pak extracts and, unlike the latter, were suitable for analytical HPLC. IA extraction achieved 75-95% recovery of BP-6-N7Gua (10 fmol/mL) and BP-6-N7Ade (1 fmol/mL) added to urine samples. Tandem mass spectrometry of IA/HPLC fractions of urine from two coal smoke-exposed women at high risk for lung cancer demonstrated the presence of 20 and 50 fmol BP-6-N7Gua per mL of urine. Unexposed controls were negative. With proposed modifications, the IA-based protocol can achieve a detection limit of 0.1 fmol/mL urine, which is sufficient for routine quantification of BP-adducted bases in urine of cigarette smokers. This procedure may allow screening of persons at risk for lung cancer associated with exposure to PAH in cigarette and other forms of smoke.

Preparation, isolation, and characterization of Dibenzo[a,l]pyrene diol epoxide-deoxyribonucleoside monophosphate adducts by HPLC and fluorescence line-narrowing spectroscopy.

Dibenzo[a,l]pyrene (DB[a,l]P) is the most potent carcinogenic polycyclic aromatic hydrocarbon that has been identified in the environment. Earlier studies in our laboratory indicated that more than 80% of the DB[a,l]P-DNA adducts formed in vitro were depurinating adducts and that most of the stable adducts were formed from diol epoxide intermediates. To complete the profile of both stable and depurinating adducts of DB[a,l]P, we have synthesized standard adducts by reacting 3'-dAMP or 3'-dGMP with either (+/-)-anti- or (+/-)-syn-dibenzo[a,l]pyrene 11,12-dihydrodiol 13, 14-epoxide (DB[a,l]PDE). The adducts were separated by HPLC with an ion-pair column and were identified by fluorescence line-narrowing spectroscopy (FLNS). A total of six pairs of stereoisomers along with another stable DB[a,l]PDE-DNA adduct were successfully isolated and identified. Pairs of (+/-)-trans and (+/-)-cis isomers were expected to be formed from the reaction of anti-DB[a,l]PDE with either dAMP or dGMP. While we were able to identify two pairs of stereoisomeric (+/-)-syn-DB[a,l]PDE-dAMP (cis and trans) and two pairs of stereoisomeric (+/-)-anti-DB[a,l]PDE-dAMP (cis and trans) adducts, identification of all the stereoisomers of dGMP adducts proved to be impossible. A pair of (+/-)-syn-trans-DB[a,l]PDE-dGMP adducts, a pair of (+/-)-anti-cis-DB[a,l]PDE-dGMP adducts, and one syn-cis-DB[a,l]PDE-dGMP adduct were conclusively identified by FLNS. These standard adducts will be used to identify the stable DNA adducts formed by DB[a,l]P and DB[a,l]PDE in vitro and in vivo.

3-Alkanoyl-5-hydroxymethyl tetronic acid homologues and resistomycin: new inhibitors of HIV-1 protease. I. Fermentation, isolation and biological activity.

In the course of a screening program for HIV-1 protease inhibiting activity, six new homologues of 3-alkanoyl-5-hydroxymethyl tetronic acids (1 approximately 6) and the known natural product resistomycin (7) were isolated from cultures of the Actinomycete strain DSM 7357. The substituted tetronic acids belong to a recently described structural class of secondary metabolites. The HIV-1 activity of resistomycin (7) has not been reported before.

Microsomal metabolism of 4-(N,N-diacetylamino)benzo[a]pyrene: a potent mutagenic arylamide derived from a carcinogenic polycyclic aromatic hydrocarbon.

4-(N,N-Diacetylamino)benzo[a]pyrene, a potent mutagen, is derived from a carcinogenic polycyclic aromatic hydrocarbon, benzo[a]pyrene. Metabolism of this compound by rat liver microsomes was studied. Metabolites were separated by reversed-phase high-performance liquid chromatography and were identified by analysis of their UV-vis absorption, mass, and proton nuclear magnetic resonance spectral data. Seven metabolites were identified, namely, the 9-phenol, 1,9-hydroquinone, and trans-9,10-dihydrodiol of 4-(N,N-diacetylamino)benzo[a]pyrene, 4-(N-acetylamino)benzo[a]pyrene, and the 5-phenol, 5,9-hydroquinone, and trans-9,10-dihydrodiol of 4-(N-acetylamino)benzo[a]pyrene. Comparison of these results with those of metabolism of benzo[a]pyrene indicates that the N,N-diacetylamino substitutent at the 4-carbon of benzo[a]pyrene inhibits metabolism at the peri position (3-carbon) and positions (6-, 7-, and 8-carbons) remote from the substituent. The results also indicate that while 4-(N,N-diacetylamino)-benzo[a]pyrene is a substrate of the rat liver microsomal deacetylase, the formed 4-(N-acetylamino)benzo[a]pyrene apparently is not a substrate.

Identification of the 11,12-dihydro-11,12-dihydroxybenzo(a)pyrene as a major metabolite produced by the green alga, Selenastrum capricornutum.

Benzo(a)pyrene metabolites were isolated after incubation of [14C]-benzo(a)pyrene with the green alga, Selenastrum capricornutum. A significant amount of radioactivity chromatographed in the dihydrodiol region which did not coelute with any of the previously identified dihydrodiol metabolites isolated from this system. Following characterization by mass spectrometry, fluorescence spectroscopy, and high pressure liquid chromatography, this metabolite was identified as the cis-11,12-dihydro-11,12-dihydroxybenzo(a)pyrene. This metabolite has not been identified previously as a metabolite formed in a plant system.

Characterization of mutagenic glucuronide formation from benzo(a)pyrene in the nonrecirculating perfused rat liver.

Excretion of mutagenic metabolites of benzo(a)pyrene into bile from livers of corn oil- or 3-methylcholanthrene-treated Sprague-Dawley rats perfused with a nonrecirculating perfusion system was quantitated. Mutagenic benzo(a)pyrene metabolites were detected using Salmonella typhimurium (strain TA 98) grown in the presence of limiting amounts of histidine. Microsomes were not included in the bacterial assay since metabolic activation was carried out by the perfused liver. Mutagenic activity was detected only if beta-glucuronidase was added to the assay mixture or if bile was treated with acid to hydrolyze glucuronides prior to assay. When livers were perfused with 20 microM benzo(a)pyrene, stable, mutagenic glucuronides were exported from corn oil-treated livers at maximal rates of 149 +/- 24 (S.E.) revertants/g/hr and at rates of 225 +/- 22 revertants/g/hr in livers from 3-methylcholanthrene-treated rats. Chromatography of bile by high-performance liquid chromatography demonstrated that two peak areas contained phenolic glucuronides which were hydrolyzed by beta-glucuronidase. These two peaks, one which cochromatographed with authentic 3-benzo(a)pyrenyl-beta-D-glucuronide, accounted for all of the mutagenic activity in bile from livers perfused with benzo(a)pyrene. A good correlation (r = 0.86) between rates of mutagen production and rates of formation of phenolic glucuronides was observed under a variety of experimental conditions. The mutagenic activity observed with pure 3-benzo(a)pyrenyl-beta-D-glucuronide exposed to beta-glucuronidase was 4 revertants/nmol. When the rate of mutagen production was divided by the rate of production of 3-benzo(a)pyrenyl-beta-D-glucuronide by the perfused liver, a value of 4 revertants/nmol was also obtained. Therefore, it is concluded that mutagens exported in bile from livers perfused with benzo(a)pyrene can be accounted for predominantly by hydrolysis products of phenolic glucuronides.

Analysis of benzo(a)pyrene:DNA adducts formed in cells in culture by immobilized boronate chromatography.

A chromatographic procedure using boronic acid residues linked to a cellulose support [(N-(N'-[m-(dihydroxyboryl)-phenyl]succinamyl)amino]ethyl cellulose), used by Sawicki et al. (Cancer Res., 43: 3212-3218, 1983) for analysis of 7,12-dimethylbenz(a)anthracene:DNA adducts, was modified to allow the analysis of benzo(a)pyrene (BaP):DNA adducts formed in cells in culture. Adducts resulting from reaction of 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (anti-BaPDE) contain cis-vicinal hydroxyl groups that complex with the boronic acid residues; adducts resulting from 7 beta, 8 alpha-dihydroxy-9 beta, 10 beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (syn-BaPDE) do not. A mixture of [3H]-syn-BaPDE:deoxyguanosine (dGuo) adduct and [14C]-anti-BaPDE:dGuo adduct was completely resolved on a column of boronate:cellulose. Early-passage cultures of Sencar mouse, Syrian hamster, and Wistar rat embryo cells and a culture of a human hepatoma cell line (Hep G2) were exposed to [3H]BaP, and the BaP:DNA adducts were resolved by boronate chromatography and high-performance liquid chromatography. The Hep G2 cells and mouse embryo cells contained two major adducts, a (+)-anti-BaPDE:dGuo adduct and a syn-BaPDE:dGuo adduct. Boronate chromatography permitted the resolution of an additional minor syn-BaPDE:deoxyribonucleoside adduct in the mouse embryo cells. The hamster and rat embryo cells contained a number of major BaP-DNA adducts that were resolved by boronate chromatography followed by high-performance liquid chromatography. The rat embryo cells contained three syn-BaPDE:deoxyribonucleoside adducts and approximately equal amounts of two adducts tentatively identified as dGuo adducts of the (+) and (-) enantiomers of anti-BaPDE. The boronate chromatography-high-performance liquid chromatography procedure improves the separation of the BaP:DNA adducts formed in biological systems and facilitates the identification of the BaP metabolite(s) responsible for the formation of these adducts.

Formation of phenolic and dihydrodiol metabolites of benzo(a)pyrene in freshly isolated human hair follicles occurs independently.

Freshly isolated hair follicles of 20 adult non-smoking volunteers were assayed for formation of phenolic and dihydrodiol metabolites of benzo(a)pyrene (BP). In each of a total of 14 experiments two volunteers were assayed simultaneously, and the ratios of both phenolic and dihydrodiol metabolites of BP between the two individuals were determined. It was obvious that the mean interindividual variation in formation of phenolic metabolites was greater than the variation in formation of dihydrodiol metabolites. No correlation existed between the amount of both types of metabolites formed. These observations indicate that for detection of differences in carcinogen metabolism to assess individual susceptibility to the carcinogenic action of polycyclic aromatic hydrocarbons (PAH), measurements of phenolic and dihydrodiol metabolites of BP are not interchangeable.

Stereoselectivity of the epoxidation of 7,8-dihydrobenzo[a]pyrene by prostaglandin H synthase and cytochrome P-450 determined by the identification of polyguanylic acid adducts.

The stereoselectivity of the oxidation of 7,8-dihydrobenzo[a]pyrene (H2BP) to 9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (H4BP-epoxide) by prostaglandin H (PGH) synthase and cytochrome P-450 has been studied using microsomal preparations from ram seminal vesicles and rat liver. Incubations were performed in the presence of polyguanylic acid and the adducts formed between H4BP-epoxide and guanosine were isolated following the recovery and hydrolysis of the poly(G). When (+/-)-H4BP-epoxide was reacted with poly(G), four diastereomeric adducts were formed by the cis and trans addition of the exocyclic amino group of guanine to the benzylic carbon of the epoxide enantiomers. Each diastereomer was identified by a combination of ultraviolet, nuclear magnetic resonance, circular dichroism, and mass spectroscopy. Under comparable conditions, ram seminal vesicle microsomes in the presence of arachidonic acid triggered the binding of H2BP to poly(G) to a greater extent than rat liver microsomes from untreated and phenobarbital- and methylcholanthrene pretreated animals in the presence of NADPH. Quantitation of the (-)-cis- and (+)-cis-guanosine adducts revealed the degree of stereoselectivity of epoxidation. The ratio of (-)/(+) adducts was 54:46 for PGH synthase and 89:11 (control), 62:38 (phenobarbital), and 69:31 (methylcholanthrene) for cytochrome P-450-catalyzed reactions. PGH synthase catalyzed the epoxidation of H2BP with little or no stereoselectivity in contrast to cytochrome P-450. The utility of the poly(G) binding technique for the elucidation of the stereoselective generation of chiral electrophiles is discussed along with the mechanistic implications of the results.

A chemiluminescence assay specific for the microsomal metabolite, benzo[a]pyrene-7,8-dihydrodiol.

It is possible to assay for trans-7,8-dihydroxy 7,8-dihydrobenzo[a]-pyrene (BP-7,8-dihydrodiol) in complex metabolite mixtures produced during microsomal metabolism of benzo[a]pyrene (BP) because only the BP-7,8-dihydrodiol metabolite will produce significant chemiluminescence (CL) in the NaOCl-H2O2 singlet oxygen-generating system. The limiting CL sensitivity is 30 pmol in a 1-ml CL reaction mixture. CL assays for BP-7,8-dihydrodiol in microsomal reaction solutions gave concentrations identical with those determined by calibrated high-performance liquid chromatography.