Oxidation of benzo[a]pyrene by recombinant human cytochrome P450 enzymes

The oxidation of benzo[a]pyrene (B[a]P) was examined using reconstituted systems prepared with recombinant human cytochrome P450 (P450) enzymes 1A1, 1A2, 2C8, 2C10, 2E1, and 3A4 and with microsomes prepared from Saccharomyces cerevisiae expressing recombinant human P450s 2C8, 2C9, and 2C18. Products measured by HPLC included the 3- and 9-phenols, the 4,5-, 7,8-, and 9,10-dihydrodiols (detected in the presence of epoxide hydrolase), and products in the polar fraction eluting immediately after the void volume. The most active enzyme in all reactions was P450 1A1. P450 3A4 and P450 1A2 formed appreciable amounts of several of the products, including the 3-phenol. P450 2C enzymes and P450 2E1 formed relatively low amounts of all B[a]P products. Consideration of these patterns along with knowledge of levels of expression of the P450s in human tissues and previous results with microsomes leads to the conclusion that P450 1A1 should dominate the oxidation of B[a]P in tissues where it is present and inducible. In human liver the level of P450 1A1 is low and P450 3A4, P450 2C subfamily enzymes, and P450 1A2 probably all contribute. Of the human P450s considered here, P450 1A2 was the most active hepatic enzyme forming the 7,8-dihydrodiol. 7,8-Benzoflavone stimulated the oxidation of B[a]P by P450 3A4 and inhibited the oxidations catalyzed by P450 1A2. The extent of inhibition of P450 1A1 was less (than with P450 1A2), probably due to the rapid oxidation of 7,8-benzoflavone by P450 1A1. The major 7,8-benzoflavone product appears to be the 5,6-oxide.     

Interindividual variation in binding of benzo[a]pyrene to DNA in cultured human bronchi

The binding of benzo[a]pyrene to DNA in cultured human bronchus was measured in specimens from 37 patients. The binding values ranged from 2 to 151 picomoles of benzo[a]pyrene per milligram of DNA with an overall mean +/- standard error of 34.2 +/- 5.2. This 75-fold interindividual variation in the binding of benzo[a]pyrene to DNA is similar in magnitude to that found in pharmacogenetic studies of drug metabolism. Aryl hydrocarbon hydroxylase is also inducible by benz[a]anthracene in the bronchial mucosa.     

The mutagenicity of benzo[a]pyrene in mouse small intestine

OBJECTIVE: To determine the effects of controlled ovarian hyperstimulation (COH) on endometrial maturation. DESIGN: Prospective, before and after evaluation of midluteal endometrial biopsies in oocyte donor's spontaneous and subsequent COH cycles. SETTING: Tertiary academic medical center assisted reproductive technologies clinic. PATIENT(S): Nineteen oocyte donors. INTERVENTION(S): Exogenous gonadotropins, endometrial biopsies. MAIN OUTCOME MEASURE(S): Endometrial histology and an immunohistochemical marker of uterine receptivity, the alphavbeta3 vitronectin. RESULT(S): Glandular and stromal dyssynchrony was more common after COH in 16 (80%) of 20 cycles than 6 (30%) of 20 spontaneous cycles (P <.05). Glandular lag was more frequent in COH cycles and unaffected by progesterone administration. The beta3 subunit of the alphavbeta3 vitronectin receptor was present in 9 (45%) of 20 spontaneous and 2 (10%) of 20 COH cycles (P <.05). CONCLUSION(S): Exogenous gonadotropin use in healthy reproductive age women did not result in endometrial evidence of a luteal phase defect. A greater incidence of glandular-stromal dyssynchrony resulted from the use of exogenous gonadotropins. The presence of alphavbeta3 was noted in most endometrial specimens demonstrating in phase glandular maturation. We conclude that endometrial dyssynchrony that results from delayed glandular development most likely represents a normal histologic variant.     

DNA strand scission by benzo[a]pyrene diol epoxides

Syn-and anti-benzo[a]pyrene diol epoxides elicit a concentration-dependent nicking of superhelical Col E1 DNA in an in vitro reaction monitored by agarose gel electrophoresis and electron microscopy. This strand scission represents less than 1 percent of the DNA modification by diol epoxide. Kinetic analysis implicates the formation of unstable phosphotriesters, hydrolysis of which nick the DNA.     

Comparison of supercritical fluid extraction and conventional liquid-solid extraction for the determination of benzo[a]pyrene in water-soluble smoke

In patients undergoing bone marrow transplantation cryptococcosis is rarely encountered. We report a fatal case of Cryptococcus meningitis in a 12-year-old girl with acute lymphoblastic leukemia (ALL) in second remission who had a transplant from a human leukocyte antigen (HLA)-identical unrelated bone marrow donor. The conditioning regimen was thiotepa, cyclophosphamide, and total body irradiation (TBI); graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporin A, methotrexate, and antilymphocyte globulin (ALG). The patient experienced stage III GVHD responsive to high-dose corticosteroids. On day +54 a thrombotic microangiopathy occurred. On day +64 neurological status worsened; a brain computed tomographic (CT) scan showed hyperdense lesions suggesting fungal infection. Detection of cryptococcal antigen by latex agglutination was positive but India ink stain and culture were negative. Despite treatment with amphotericin B, 5-flucytosine, and granulocyte-macrophage colony-stimulating factor, the patient died 13 days after the diagnosis.     

Tissue distribution of DNA adducts in rats treated by intramammillary injection with dibenzo[a,l]pyrene, 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene

Recombinant human bone morphogenetic protein (rhBMP-2) was examined for its in vitro effects on biochemical markers representing osteoblast phenotype. Primary cultures of fetal rat calvarial osteoblasts were used in this study. The results indicated that rhBMP-2 stimulated alkaline phosphatase activity, parathyroid hormone (PTH)-induced cyclic AMP production, and collagen biosynthesis in a dose-dependent manner in confluent cultures. The percent collagen synthesis also increased in a dose-dependent manner. Alkaline phosphatase activity was stimulated in a time-dependent manner by rhBMP-2 that reached its maximum 5 days after initiation. Cycloheximide (2 micrograms/ml) inhibited rhBMP-2-stimulated alkaline phosphatase indicating de novo protein synthesis of the enzyme. Transforming growth factor-beta 1 (TGF-beta 1)-induced inhibition of alkaline phosphatase activity observed in confluent primary cultures was completely abolished by rhBMP-2 at a concentration that was 43 times greater than the TGF-beta 1 concentration. Also, rhBMP-2 produced a small stimulation of alkaline phosphatase activity in cells grown in the absence of ascorbic acid; however, the effect was greatly enhanced in cells cultivated in the presence of ascorbic acid (50 micrograms/ml). In view of the potentiating effect of ascorbic acid on rhBMP-2-induced stimulation of alkaline phosphatase, we speculate that ascorbic acid could amplify the osteoinductive effects of rhBMP-2 and thereby augment the efficacy of the BMP when used as bone repair material in vivo. rhBMP-2 (4.3-86 ng/ml) did not exhibit mitogenic effects on cultured osteoblasts. These data suggest that rhBMP-2 has the ability to induce expression of various markers associated with the osteoblast phenotype in primary cultures of fetal rat calvarial osteoblasts. In addition, we speculate that TGF-beta 1 may play a regulatory role in BMP-induced bone formation and ascorbic acid may potentiate the effects of rhBMP-2 in vivo.     

Hemoglobin adducts of benzo[a]pyrene diolepoxide in newspaper vendors: association with traffic exhaust

Benzo[a]pyrene diol epoxide adducts with hemoglobin (Hb) were measured to detect human exposure to environmental benzo[a]pyrene from traffic exhaust. Benzo[a]pyrene tetrahydrotetrols (BPTs) released from Hb after acid hydrolysis were quantitated by gas chromatography-mass spectrometry after immunoaffinity chromatography. Fifty three newspaper vendors were enrolled. The median adduct concentration was 0.3 fmol BPTs/mg Hb in high density traffic-exposed vendors and < or = 0.1 fmol BPTs/mg Hb in those exposed to low density traffic; the difference was not significant (P = 0.09). Among non-smokers, adducts were detectable in 60% of high exposure subjects (median 0.3 fmol BPTs/mg Hb) and in 28% of those with low exposure (median < or = 0.1 fmol/mg Hb). This difference was significant (P = 0.02). In low exposure smokers the median of adducts was 0.26 fmol BPTs/mg Hb, while in low exposure non-smokers it was < or = 0.1 fmol BPTs/mg Hb (P = 0.08, not significant). Adduct concentration was no different for low and high density traffic-exposed smokers (P = 0.82). The data indicate a significant difference in adduct concentration related to traffic exhaust exposure among non-smokers.     

Induction of atypical squamous metaplasia with benzo[a]pyrene in cultured hamster tracheas

Areas of hyperplasia were produced in hamster tracheal epithelium maintained in vitro by exposure to a suspension of benzo[a]pyrene (BP) in gelatin. Typical and atypical epiodermoid metaplasia were seen by 2 weeks. In atypical areas, cell nuclei were enlarged with prominent nucleoli, the cytoplasm contained dense bundles of tonofilaments and the cells were joined by numerous desmosomes. The peak response to the carcinogen was reached 4 weeks after the application of BP and consisted of extensive atypical epidermoid metaplasia. Tracheas treated with gelatin alone maintained a columnar epithelium for 6 weeks of culture. The characteristics of the metaplastic changes in vitro are essentially identical to those described after exposure of the hamster tracheobronchial epithelium to benzo[a]pyrene-ferric oxide in vivo.     

Tumour-initiating activities on mouse skin of dihydrodiols derived from benzo[a]pyrene

Three dihydrodiols that are metabolites of benzo[a]pyrene and benzo[a]-pyrene itself have been tested in a comparative experiment for their activities as initiators of tumours in mouse skin. A single application (25 mug) of 4,5-dihydro-4,5-dihydroxybenzo[a]pyrene, of 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene, of 9,10-dihydro-9,10-dihydroxybenzo[a]pyrene, or of benzo[a]pyrene was made to the shaved dorsal skin of adult female CDI mice; this was followed 2 weeks later by multiple thrice-or twice-weekly applications (1 mug) of 12-O-tetradecanoyl-phorbol-13-acetate as promoting agent. A control group of 30 mice received the promoting agent alone. The experiments were terminated 52 weeks after initiation. At this stage, all the groups contained mice bearing skin papillomas, some of which had progressed to malignancy. Quantitatively the results show that the 7,8-dihydrodiol is almost as active an initiator of mouse skin tumours as benzo[a]pyrene itself; the 4,5- and 9,10-dihydrodiols were significantly less active. The significance of these results is discussed in relation to the hypothesis that diol-epoxides are important in the metabolic activation of polycyclic hydrocarbons like benzo[a]pyrene.     

Antispermatogenic effects of ethyl methanesulfonate and benzo[a]pyrene in PD4 Lakeview hamsters

Mammalian sperm seems to provide an excellent cell type for monitoring mutagenic and other toxicological damage to the germinal tissue. Studies with mice indicated that most agents known for their mutagenic activity in vivo produced marked elevations in sperm abnormalities. To determine whether this response is typical of other species, groups of inbred Lakeview hamsters were exposed to ethyl methane-sulfonate (EMS) and benzo[a]pyrene (BP) in five daily subacute ip doses ranging from 5 to 125 mg/kg and 2 to 50 mg/kg, respectively. Percentage of abnormal sperm, testis weight, and body weight were monitored at wk 1, 4, and 10 after treatment. EMS exposures increased the frequency of sperm abnormalities and reduced sperm numbers and testis weights. Body weights were also affected. BP exposures did not induce sperm abnormalities; however, there were marked reductions in sperm number and testis weight. These findings are in agreement with results of EMS studies in the mouse; however, BP exposure did induce sperm abnormalities in the mouse.     

Anti-benzo[a]pyrene diolepoxide--DNA adduct levels in peripheral mononuclear cells from coke oven workers and the enhancing effect of smoking

The level of (+/-)-r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) bound to DNA of lymphocytes plus monocytes in 39 coke oven workers exposed to polycyclic aromatic hydrocarbons (PAH) and 39 non-exposed persons (controls) were investigated, each of the groups consisting of smokers and non-smokers. The adduct level was measured by an improved HPLC/fluorescence method (Rojas, M., Alexandrov, K., van Schooten, F. J., Hillebrand, M., Kriek, E. and Bartsch, H., Carcinogenesis, 15, 557-560, 1994) through the release of the corresponding benzo[a]pyrene (B[a]P) tetrols. The anti-BPDE-DNA adduct was detected in 51% of coke oven workers exposed to PAH and in 18% of the non-exposed (control) subjects. The mean level of anti-BPDE-DNA adducts/10(8) nucleotides in coke oven workers (15.7 +/- 37.8) was approximately 8 times higher than in non-exposed subjects (2.0 +/- 8.7). The interindividual variation of adduct levels was approximately 100-fold in coke oven workers and approximately 50-fold in controls respectively. Smokers in the exposed group had 3.5 times more DNA adducts than non-smokers. With the exception of one non-smoker with very high adduct levels (52.8 adducts/10(8)), the control subjects showed the presence of barely detectable adducts in only 16% of the samples examined. The increased in vivo formation in some smokers and high variability of anti-BPDE-DNA adducts in coke oven workers suggests variations in genetically controlled activation/inactivation reactions of PAH metabolism.     

Base-sequence dependence of covalent binding of benzo[a]pyrene diol epoxide to guanine in oligodeoxyribonucleotides

We report a solitary nodular form of primary cutaneous amyloidosis due to locally infiltrating plasma cells. An 81-year-old Japanese women presented with a scarlet, dome-shaped 1.5-cm nodule with an irregular surface. Histology showed thick deposits of eosinophilic, oval, and homogeneous bodies in the dermis with mild infiltrates of mononuclear cells. The homogeneous bodies stained positively with periodic acid-Schiff, Congo red, and Yanagihara's Dylon stain, and immunohistochemically with anti-human lambda light-chain antibody. Methylgreen pyronine staining revealed that approximately half of the cellular infiltrates around the vessels were plasma cells. Electron microscopy demonstrated the homogeneous bodies to be amyloid masses, a part of which were in the cytoplasm of the plasma cells. Laboratory examination showed a slight elevation of IgG but no obvious findings suspicious for systemic amyloidosis or gammopathy.     

Inhibitory effects of naturally occurring coumarins on the metabolic activation of benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene in cultured mouse keratinocytes

Several naturally occurring coumarins to which humans are routinely exposed have been previously found to be potent inhibitors and inactivators of cytochrome P450 (P450) 1A1-mediated monooxygenase in both murine hepatic microsomes and in a reconstituted system using purified human P450 1A1 [Cai et al. (1993) Chem. Res. Toxicol., 6, 872-879 and Cai et al. (1996) Chem. Res. Toxicol., 9, 729-736]. In the present study, several of these coumarins were investigated for their inhibitory effects on the metabolism and metabolic activation of benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA) in cultured mouse keratinocytes. Initial analysis of B[a]P metabolism in cultured keratinocytes showed that imperatorin, isoimperatorin, coriandrin, and bergamottin, at concentrations of 2 nM equal with B[a]P, reduced the formation of water-soluble metabolites of B[a]P by 33% to 57%. Bergamottin and coriandrin were the most potent inhibitors of the compounds examined. HPLC analysis of organic solvent-soluble metabolites of B[a]P indicated that all the coumarins tested significantly reduced the formation of individual B[a]P metabolites (including phenols, diols and tetraols). However, the greatest effect was on the formation of B[a]P tetraols. Additional experiments determined the ability of selected coumarins to block covalent binding of B[a]P and DMBA to DNA in keratinocytes. Bergamottin preferentially inhibited the binding of B[a]P to DNA by 56%, while coriandrin preferentially inhibited the binding of DMBA to DNA by 48%. Notably, analysis of individual DNA adducts formed from B[a]P and DMBA indicated that both bergamottin and coriandrin specifically inhibited the formation of anti diol-epoxide DNA adducts derived from both hydrocarbons. The preferential inhibitory effect of bergamottin and coriandrin on the formation of anti diol-epoxide adducts derived from DMBA was further confirmed by separation of anti- and syn-diol-epoxide-DNA adducts using immobilized boronate chromatography. The current study demonstrates that certain naturally occurring coumarins inhibited metabolic activation of B[a]P and DMBA in cultured mouse keratinocytes and specifically inhibited the formation of DNA adducts derived from the anti diol-epoxide diastereomers from either hydrocarbon. The current data also suggest that certain naturally occurring coumarins may possess anticarcinogenic activity toward polycyclic aromatic hydrocarbons.     

Gas chromatographic-mass spectrometric determination of benzo[a]pyrene and chrysene diol epoxide globin adducts in humans

The efficacy of a newly developed gas chromatography-negative ion chemical ionization-mass spectrometry-selected ion monitoring (GC-NICI-MS-SIM) assay for measuring globin adducts of benzo[a]pyrene (B[a]P) and chrysene diol epoxides in human was evaluated. In this pilot study, smokers and nonsmokers were selected as exposed and nonexposed groups. Using [2H12]r-7,t-8,9,c-10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyren e ([2H12]trans,anti-B[a]P-tetraol) as an internal standard, B[a]P-tetraols released from globin after hydrolysis and derivatization were quantified by GC-NICI-MS-SIM. Levels of trans-1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydrochrysene (chrysene-DE)-globin adducts were estimated by assuming that the recovery and the MS response of the perdeuterated B[a]P-tetraol internal standard reflected the recovery and MS response of chrysene tetraols. The assay was found to be reproducible and sensitive enough to detect both analytes in all samples. The mean levels of B[a]P-tetraols released from the corresponding benzo[a]pyrene diol epoxide (BPDE) globin adducts in smokers were significantly higher than those in nonsmokers, i.e., 2.6 +/- 0.6 SE fmol/mg globin (ranging from 1.2 to 7.8 fmol/mg globin) in smokers and 0.97 +/- 0.05 SE fmol/mg globin (ranging from 0.7 to 1.3 fmol/mg globin) in nonsmokers (P < 0.01). Interestingly, estimated levels of chrysene-DE-globin adducts in the same subjects were about two orders of magnitude higher than those of the globin adducts of BPDE. The mean of the chrysene-DE adducts in smokers was estimated to be 310 +/- 30 SE fmol/mg globin (ranging from 190 to 460 fmol/mg globin) and that in nonsmokers was 250 +/- 25 SE fmol/mg globin (ranging from 110 to 380 fmol/mg). Although the estimated mean of chrysene-DE adducts with globin in smokers appeared to be about 25% higher than in nonsmokers, the difference was not significant (P = 0.06). The results of this study demonstrate the feasibility of the GC-NICI-MS-SIM method for measurement of BPDE globin adducts in humans.     

Enrichment of benzo[a]pyrene in smoked food products and determination by high-performance liquid chromatography-fluorescence detection

We developed a procedure for trace enrichment of benzo[a]pyrene (BP) in extracts of smoked food products, and an HPLC-fluorescence detection (FL) method for determination of BP in the enriched extracts. The procedure consists in extraction/sonication of the lyophilized product in hexane, clean-up of the hexane extract by passage through a Sep-Pak Silica Plus cartridge and, subsequently, by partitioning between hexane and dimethyl sulphoxide, and concentration of the BP using a Sep-Pak C18 Plus cartridge. HPLC-FL and quantification limits were 0.049 microgram/l in acetonitrile (< 0.0067 microgram/kg of smoked food) and 0.089 microgram/l in acetonitrile (< 0.012 microgram/kg), respectively. Recovery (94.1%) and RSD (< 8.65%) were satisfactory. When applied to 15 types of sausage, mean BP content was 0.022 microgram/kg, and all but two samples (both treated with wood smoke) had BP contents below the 0.03 microgram/kg limit imposed in EU legislation for smoking-flavour agents.     

Elution of benzo[alpha]pyrene from carbon particles in the respiratory tract of mice

The effect of carrier particle size on the rate of dissociation of benzo[alpha]pyrene (BaP) from carrier particles deposited in the respiratory tract of mice was studied. BaP-coated carbon particles (in two size ranges, 0.5-1.0 and 15-30 mum) plus 103Ru-tagged carbon tracer particles were intratracheally instilled in mice. The clearance of carbon particles and the simultaneous rate of elimination of BaP from the respiratory tract was measured. BaP adsorbed to 15- to 30-muM carbon particles was eliminated from the lung at essentially the same rate as the carbon particles were cleared. In contrast, BaP adsorbed to 0.5- to 1.0-muM carbon particles was eliminated from the lung approximately 4 times faster than the carbon particles were cleared. The persistence of carcinogens and their rates of elution from carrier particles are discussed in relation to the pathogenesis of lung cancer in animals treated with carcinogen-carrier particle preparations.     

Mass spectrometric sequencing of site-specific carcinogen-modified oligodeoxyribonucleotides containing bulky benzo[a]pyrene diol epoxide-deoxyguanosyl adducts

Site-specific carcinogen-modified oligonucleotides are often used in site-directed mutagenesis and other biological and biochemical studies of structure-function relationships. Postsynthetic analysis and confirmation of the sites of carcinogen binding in such oligonucleotides is an important step in the characterization of these site-specific carcinogen-DNA adducts. It is shown here that negative ion mode electrospray tandem mass spectrometry methods and collision-induced dissociation offer a rapid and convenient approach for the sequencing of products derived from the reaction of the carcinogenic and mutagenic metabolite of benzo[a]pyrene, the diol epoxide r7,t8-dihydroxy-t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE), with the 11-mer oligonucleotide d(CATGCGGCCTAC). The site of reaction of anti-BPDE with either one of the three dG residues in this oligonucleotide can be accurately established by comparing the mass/charge ratios of the observed collision-induced dissociation fragments with calculated values.