Evaluation of Benzo[c]Chrysene Dihydrodiols in the Morphological Cell Transformation of Mouse Embryo Fibroblast C3H10T1/2CL8 Cells

The morphological cell transforming activities of three dihydrodiols of benzo[c]chrysene (B[c]C), trans?B[c]C-7,8-diol, trans?B[c]C-9,10-diol, and trans?B[c]C-1,2-diol were compared to those of B[c]C in order to study the possible routes of metabolic activation in transformable C3H10T1/2 mouse embryo fibroblasts. B[c]C-treated C3H10T1/2 cells exhibited a concentration-related increase in morphologically transformed foci over a concentration range of 0–3 μg/ml. At 3 μg/ml, B[c]C induced 1.23 Type II & III foci/dish, with 73% of the dishes exhibiting Type II or Type III foci, and a survival of 87%. trans?B[c]C-7,8-diol produced concentration-related responses over a range of 0–5 μg/ml. At 3 μg/ml, trans?B[c]C-7,8-diol produced 1.13 Type II & III foci/dish with 72% of the dishes exhibiting foci, and a survival of 76%. trans?B[c]C-9,10-diol was inactive as a morphological cell transforming agent over a concentration range of 0–3 μg/ml. trans?B[c]C-1,2-diol was also inactive as a morphological cell transforming agent over a concentration range of 0–3 μg/ml. These results suggest that a K-region dihydrodiol of B[c]C, trans?B[cC-7,8-diol, may play a role in the ability of B[c]C to morphologically transform C3H10T1/2 cells.     

Species-Dependent Metabolism of Benzo[c]Chrysene Mediated by c-DNA-Expressed Human,Rodent and Fish Cytochrome P450 Enzymes

The metabolism of benzo[c]chrysene (B[c]Ch) with various cytochrome P450 (CYP) enzymes including rat 1A1, 1A2, 2B1 and 2E1, human 1A1, 1A2, 2A6, 1B1, 3A4 and 2E1, mouse 1B1, and scup fish 1A1 expressed in Chinese hamster V79 cells has been investigated to clarify the role of individual enzymes in the regioselective oxidation of B[c]Ch and the species dependency. In six cell lines expressing individual CYP enzymes from four different species B[c]Ch was metabolized to several isomeric phenols and trans?dihydrodiols. However, cell lines expressing human 3A4, 2A6 and 2E1 or rat 1A2, 2B1 and 2E1 were metabolically in-competent towards B[c]Ch. Among the trans?dihydrodiols the 9,10-isomer could be detected in cells expressing human, rat and fish CYP 1A1 and to a minor extent in cells with human 1A2, but not in cells expressing human and mouse CYP 1B1. The latter two cell lines produced high amounts of the bay region 3,4-dihydrodiol, whereas the K-region 7,8-dihydrodiol was a minor metabolite. Oxidation of B[c]Ch to the 1,2-dihydrodiol could not be catalyzed by any of the CYP enzymes investigated except fish 1A1. Our results suggest that metabolic activation of B[c]Ch is initiated predominantly by CYP 1A1 to result selectively in the formation of fjord region 9,10-dihydrodiol 11,12-epoxides regardless of the species involved. The activation of B[c]Ch appears to be limited by a low regioselectivity for the 9,10-oxidation.     

Relationship Between Inhaled PAH and Urinary Excretion of Phenanthrene,Pyrene and Benzo[a]pyrene Metabolites in Coke Plant Workers

Abstract

By means of personal air samplers the exposure to polycyclic aromatic hydrocarbons (PAH) of four coke employees working at different locations was measured during 4 running days. Simultaneously the 24 hrs urines were collected. A simple, well reproducible method for the determination of 9,10-dihydroxy-9,10-dihydrobenzo[a]pyrene, 1-; 2-; 3-; 4- and 9-hydroxyphenanthrenes, 1,2-; 3,4-; and 9,10-dihydroxydihydrophenanthrenes as well as 1-hydroxypyrene and 1,2-dihydroxy-1,2-dihydropyrene was used. As expected, workers on the battery topside are most exposed and, accordingly, excrete by far the highest amount of PAH metabolites. A good correlation between the PAH inhaled during 8 hrs and the metabolites excreted in the 24 hrs urine is observed. 20% to 30% of the inhaled phenanthrene is excreted as dihydrodiols, but only 0.5% of the inhaled benzo[a]pyrene forms 9,10-dihydrodiol in the urine. The individual invariance of the metabolite profile of the isomeric phenols and dihydrodiols indicates a genetically caused enzyme pattern of oxygenases, which can or cannot be induced by exogenous factors. More investigations are necessary to clarify whether this metabolic profile may be suitable as a marker for carcinogenic risk.     

Comparison of the Morphological Transforming Activities of Fjord-Region PAHs with Dibenzo[a,e]Pyrene and Benzo[a]Pyrene

The morphological transforming activities in mouse embryo C3H10T1/2CL8 (C3H10T1/2) cells were examined for six PAHs: benzo[c]chrysene (B[c]C); benzo[g]chrysene (B[g]C); benzo[c]phenanthrene (B[c]P); dibenzo[a, l]pyrene (DB[a, l]P); dibenzo[a,e]pyrene (DB[a,e]P) and benzo[a]pyrene (B[a]P). C3H10T1/2 cells treated with B[c]P or B[g]C at concentrations of 0–3 μg/ml did not produce any transformed Type II or III foci after 24 hr of exposure. Concurrent cytotoxicity was observed. Under the same conditions, B[a]P and B[c]C were active, with B[c]C approximately one-half the activity of B[a]P. However, after a 48-hr treatment, B[c]P and B[g]C gave significant activity measured as both foci/dish or the number of dishes exhibiting foci. After a 24-hr treatment, comparison of B[a]P with two dibenzopyrenes, DB[a, l]P and DB[a,e]P, gave activities in the order: DB[a, l]P > B[a]P > DB[a,e]P. After 48 hr of treatment, both B[a]P and DB[a,e]P had similar activities.     

Microscale Solid-Phase Extraction and HPLC Separation of Purine Deoxyribonucleoside Monophosphate Adducts of Benzo[a]pyrene Diol Epoxide

Benzo[a]pyrene, a polycyclic aromatic hydrocarbon, is a potent mutagen and genotoxic compound. Activation of benzo[a]pyrene by oxidative metabolism in cells leads to the formation of highly reactive (±) anti benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxides, which bind covalently to DNA bases, in particular to the nucleophilic N² atom of guanine and the N⁶ atom of adenine. Here we have investigated the recovery of eight different enantiomers of purine deoxyribonucleoside monophosphate benzo[a]pyrene diol epoxide adducts after a microscale solid-phase extraction (SPE). Microscale SPE was carried out using a pipette tip with a bed of C₁₈ chromatograph packing material. We also tested the ability of microscale SPE to isolate adducts derived from the in vitro reaction of calf thymus DNA with (±) anti benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide. The recoveries of benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide monophosphate adduct standards were good, varying between 65% and 92%, except for the trans BPDE-dGMP adduct derived from the (?) enantiomer of benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide, which presumably became decomposed to tetrols during the purification process. After using a neutral condition during the SPE treatment, the decomposition to tetrols of the trans BPDE-dGMP adduct could be avoided and the recovery increased to 79%.     

Tumorigenicity of Fjord Region Diol Epoxides of Polycyclic Aromatic Hydrocarbons

(±)-anti-Benzo[c]phenanthrene-3,4-diol-1,2-epoxide (BcPDE), (±)-anti-benzo[g]-chrysene-11,12-diol-13,14-epoxide (BgCDE), and (±)-anti-dibenzo[a,l]pyrene-11,12-diol-13,14-epoxide (DB[a,l]PDE) were synthesized for use in biological assays. To compare mammary carcinogenicity, BcPDE and (±)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE) were tested by direct application to the mammary glands of female CD rats at a total dose of 12.2 μmol per animal. 6-Nitrochrysene (6-NC) at the same dose was used as a positive control. BcPDE was found to be a potent mammary tumorigen and carcinogen, inducing significantly more fibroadenomas and adenocarcinomas than the other two compounds. In a second bioassay, BcPDE, BgCDE, and DB[a,l]PDE at a total dose of 1.2 μmol were compared. All three diol epoxides were potent mammary carcinogens and based on the collective results to date, the relative carcinogenic activities of the diol epoxides can be approximated as DB[a,l]PDE>BcPDE>BgCDE>BPDE.

To determine tumorigenic potencies in newborn mice, BPDE was compared with BgCDE, DB[a,l]PDE, and BcPDE at a total dose of 25 nmol per mouse, administered on days 1, 7, and 15 of life. BgCDE and BcPDE had similar potency in inducing lung tumors. BgCDE induced more liver tumors in male mice than BcPDE. Both compounds were more tumorigenic than BPDE. DB[a,l]PDE was highly toxic at 25 nmol and all animals died within one week after the first dose. DB[a,l]PDE was also tested at total intended doses of 5 nmol and 1 nmol. Due to high toxicity, only the first dose of the intended 5 nmol was given. This dose as well as the 1 nmol total dose caused significant incidence and multiplicity of lung tumors.

These results support the hypothesis that sterically hindered fjord region diol epoxides are potent mammary carcinogens in CD rats and strong lung tumorigens in newborn mice.     

Effect of Structure and Sequence on Mutations Induced by Diol Epoxide-DNA Adducts in an E. Coli−M13 Vector System

The effects of two DNA sequence contexts on the mutagenic response to dG and dA adducts derived from the optically active 7,8-diol 9,10-epoxides of benzo[a]pyrene (BaP DEs) and 3,4-diol 1,2-epoxides of benzo[c]phenanthrene (BcPh DEs) were examined. On replication of the adduct-containing, single-stranded vector M13mp 7L2 in E. coli SMH77, frequencies of substitution mutations ranging from 0.05% to 68% were observed. In general, the highest mutational frequencies were observed for BaP DE-dA adducts at the central position in a ～TAG～ sequence. The BaP DE adducts showed no relationship between stereochemistry and mutagenic response. In contrast, mutagenicity of the BcPh DE-dA adducts in the ～TAG～ sequence depended strongly on the configuration at C-1, the site of attachment of the hydrocarbon to the purine. In this sequence, BcPh DE-dA adducts with 1R configuration were 16-600 times less mutagenic than those with 1S configuration.     

Substrate Enantioselective and Product Stereoselective Metabolism of Racemic 2-Hydroxy-3-methylcholanthrene by Rat Liver Microsomes

Racemic 2-hydroxy-3-methylcholanthrene (rac-2-OH-3MC) is a potent carcinogen and 2S-OH-3MC is the most abundant metabolite formed in the metabolism of 3-methylcholanthrene (3MC) by rat liver microsomes. Eighteen identifiable metabolites were formed in the metabolism of rac-2-OH-3MC by liver microsomes prepared from phenobarbital-treated rats. Metabolites were separated by sequential use of reversed-phase and normal-phase high performance liquid chromatography (HPLC) and characterized by ultraviolet-visible absorption, mass, and circular dichroism spectral, and chiral stationary phase (CSP) HPLC analyses. Identified metabolites were: 2-OH-3MC 7,8-dihydrodiol (enriched in 7R,8R enantiomer), 2-OH-3MC 9,10-dihydrodiol (2S,9R,10R:2R,9S,10S ≈ 27:73 and 2R,9R,10R:2S,9S,10S ≈ 81:19), 2-OH-3MC 11,12-dihydrodiol (2R,11R,12R:2S,11S,12S ≈ 5:95 and an essentially optically pure 2S,11R,12R enantiomer), 3MC trans-1,2-diol (1S,2S:1R,2R ≈ 68:32), 3MC cis-1,2-diol (1S,2R:1R,2S ≈ 18:82), 2-OH-3-hydroxymethyl-cholanthrene (2-OH-3-OHMC, 2R:2S ≈ 19:81), 2-OH-3-OHMC 9,10-dihydrodiol (9R,10R:9S,10S ≈ 62:38), 3MC-2-one 9,10-dihydrodiol (9R,10R:9S,10S ≈ 7:3), 3MC cis-1,2-diol:trans-9,10-dihydrodiol, 8-hydroxy-2-OH-3-OHMC, 9-hydroxy-2-OH-3MC, 10-hydroxy-2-OH-3MC, and 3MC-2-one. The enantiomer ratios of some metabolites formed were determined by circular dichroism spectra and/or CSP-HPLC. Six 9,10-dihydrodiols formed in the metabolism of 2-OH-3MC may be further converted to potentially reactive 9,10-diol-7,8-epoxides and may contribute to the overall carcinogenicity exhibited by 3MC.     

Metabolic Activation of Dibenzo[a,l]Pyrene by Cytochrome P450 Enzymes to Stable DNA Adducts Occurs Exclusively Through the Formation of the (−)-trans−(11R, 12R)-Diol

At low doses, the carcinogenic hexacyclic hydrocarbon dibenzo[a,l]pyrene (DB[a,l]P) forms DNA adducts in human MCF-7 cells exclusively through formation of the (–)-anti?(11R, 12S)-diol (13S, 14R)-epoxide (DB[a,l]PDE) via its metabolic precursor, the (–)-(11R, 12R)-diol. The same result was obtained by exposure of V79 cells stably expressing human cytochrome P450 (P450) 1B1. Although several other metabolites such as the 7-phenol and a 11, 12-diol phenol are formed, no other DNA adducts are detectable after exposure to 0.1 μM DB[a,l]P. Exposure to 1 μM DB[a,l]P leads to the formation of low levels of (+)-syn?DB[a,l]P-DE-DNA adducts through intermediate generation of the (+)-(11S, 12S)-diol. In contrast, treatment of P450 1A1-expressing V79 cells results also in the formation of several polar DNA adducts. Incubation of the trans?8,9-diol and trans?11, 12-enantiomers of DB[a,l]P demonstrated that all polar DNA adducts detected in 1A1-expressing cells resulted from intermediate formation of the (–)-trans?11, 12-diol. Although the K-region trans?8,9-diol is metabolically converted to several diol phenols and bis?diols, this compound does not contribute to the strong DNA binding or cytotoxicity observed after exposure of P450 1A1- and 1B1-expressing cells to the parent hydrocarbon.     

Selective Recognition of Substituted Chrysene-Diol Epoxide-2-DNA Adducts by Antiserum prepared Against DNA Adducts of Benzo[c]Phenanthrene-Diol Epoxide-2

Polyclonal antiserum prepared against DNA that was modified with racemic benzo[c]phenanthrene-3,4-diol-1,2-epoxide-2 (B[c]PhDE-2; benzylic hydroxyl and epoxide oxygen trans) was characterized for specificity of antigen recognition. Previous studies have demonstrated that the antisera stereoselectively recognized B[c]PhDE-2-DNA and failed to recognize DNA modified with racemic benzo[c]phenanthrene-3,4-diol-1,2-epoxide-1 (B[c]PhDE-1-DNA, benzylic hydroxyl and epoxide oxygen cis), benzo[a]pyrene-7,8-diol-9, 10-epoxide-2-DNA (B[a]PDE-2-DNA) and 7,12-dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide-1-DNA (DMBADE-1-DNA). DNA samples modified by diol-epoxide-2 diastereomers of several hydrocarbons were tested in competitive ELISA assays utilizing B[c]PhDE-2-DNA (270 fmol adducts per well). DNA modified with racemic diol-epoxide-2 of various substituted chrysenes (including chrysene, benzo[g]chrysene (B[g]C), 6-methylchrysene (6-MeC), and 5-methychrysene (5-MeC), gave 50% inhibition of antisera binding at significantly higher concentrations (5 to 7-fold) than the parent B[c]PhDE-2-DNA or 5,6-diMeCDE-DNA. DNA modified with 5,7-dimethylchryseneDE-2 (5,7-diMeCDE-2) and dibenzo[a,l]pyreneDE-2 (DB[a,l] PDE-2) required 20 and > 100-fold greater levels of adducts to give 50% inhibition. Results with B[c]Pb, 5,6-diMeC, chrysene, 6-MeC and 5-MeC diol epoxide-2-DNA     

anti-Benzo[a]pyrene-7,8-diol-9,10-epoxide Treatment Increases Levels of the Proteins p53 and p21WAF1 in the Human Mammary Carcinoma Cell Line MCF-7

The tumor suppressor p53 is involved in the recognition of DNA damage induced by radiation and chemicals. The effect of polycyclic hydrocarbons on p53 was investigated by treating MCF-7 cells with anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE), an ultimate carcinogenic metabolite of benzo[a]pyrene. Western blotting of lysates with antibody mAb1801 showed that B[a]PDE doses of 0.1 to 0.5 μM caused detectable increases in p53 protein. In cells treated with 0.3 μM B[a]PDE, p53 protein levels increased by 2 hours after treatment, reached a maximum between 8 and 24 hours and returned to control value by 120 hours after treatment. Levels of p21^WAF1 protein increased by 8 hours after treatment, reached a maximum by 48 hours and returned to control value by 168 hours after treatment. B[a]PDE-DNA adducts were quantitated by [γ-³³P]ATP postlabeling and separation on reverse-phase HPLC. Adduct levels dropped rapidly between 2 and 24 hrs after treatment (48 to 15 pmol/mg DNA), and subsequently decreased more slowly (12 pmol/mg DNA at 48 hrs to 3 at 168 hrs). By 96 hours after treatment, p53 protein levels were comparable to those from untreated controls, although a low level of B[a]PDE-DNA adducts remained. Cells treated with B[a]PDE also incorporated significantly less [³H]thymidine into DNA than did untreated cells for at least 24 hours after treatment. These results provide preliminary evidence that B[a]PDE-induced DNA damage is recognized by a p53-dependent signal transduction pathway and suggest that cell cycle arrest occurs during the repair of the majority of the B[a]PDE-DNA adducts.     

Correlation of the Extent of Fjord-Region Oxidation with DNA Binding and Mutagenicity of the Enantiomeric 11,12-Dihydrodiols of Dibenzo[a,l]pyrene

In vitro studies on the hepatic biotransformation of the enantiomeric trans-11,12-dihydrodiols of dibenzo[a,l]pyrene (DB[a,l]P) using microsomal fractions of animals pretreated with Aroclor 1254 revealed that the formation of fjord-region dihydrodiol epoxides strongly depends on the absolute configuration of the substrate. Both the (-)-11R,12R- and the (+)-11S,12S-enantiomer are converted diastereoselectively to the (-)- and (+)-anti-dihydrodiol epoxide, respectively, by either rat or mouse liver microsomes. Fjord-region oxidation occurs to greatest extent on incubation of the (-)-11R,12R-dihydrodiol with preparations from rats. This finding is in line with the differences seen for the two enantiomers on the total DNA binding under identical activation conditions as well as on the mutagenic activity in Chinese hamster V79 cells using the postmitochondrial hepatic fraction of Aroclor 1254-treated rats as metabolizing system.     

Gold(I)‐Catalyzed Cascade for Synthesis of Pyrrolo[1,2‐a:2′,1′‐c]‐/Pyrido[2,1‐c]pyrrolo[1,2‐a]quinoxalinones

An efficient and convenient method was developed for the one‐pot construction of the complex polycyclic heterocycles pyrrolo[1,2‐a:2′,1′‐c]‐/pyrido[2,1‐c]pyrrolo[1,2‐a]quinoxalinones from two simple starting materials via a gold(I)‐catalyzed domino reaction. This strategy presents an atom economical and environmentally friendly transformation, in which two new C N bonds and one new C C bond are formed in a one‐pot reaction process.     

Interaction Analyses of Binary Mixtures of Carcinogenic PAHs Using Morphological Cell Transformation of C3H10T1/2CL8 Mouse Embryo Fibroblasts in Culture

Studies of defined mixtures of carcinogenic polycyclic aromatic hydrocarbons (PAH) have identified three major categories of interactions: antagonism; synergism; and additivity depending on the biological model, tissue, route of exposure, and specific PAH. To understand the bases of these interactions we studied binary mixtures of benzo[a]pyrene (B[a]P) and dibenz[a,h]anthracene (DBA) in transformable C3H10T1/2C18 (C3H10T1/2) mouse embryo fibroblast cells in culture. C3H10T1/2 cells treated with binary mixtures of B[a]P and DBA gave less than additive morphological cell transformation based on response additivity. These results were consistent with those reported in mice and rats on the antagonistic effects of B[a]P and DBA on tumorigenesis. ³²P-Postlabeling DNA adduct studies revealed that DBA reduced B[a]P-DNA adduct levels by 47% with no effect on DBA-DNA adduct levels. This suggests that one mechanism for the inhibition of morphological cell transformation of binary mixtures of B[a]P and DBA is due to alterations in the metabolic activation of B[a]P.     

Benzo[a]pyrene-Induced Genotoxicity in Rats Is Affected by Co-Exposure to Sudan I by Altering the Expression of Biotransformation Enzymes

The environmental pollutant benzo[a]pyrene (BaP) is a human carcinogen that reacts with DNA after metabolic activation catalysed by cytochromes P450 (CYP) 1A1 and 1B1 together with microsomal epoxide hydrolase. The azo dye Sudan I is a potent inducer of CYP1A1/2. Here, Wistar rats were either treated with single doses of BaP (150 mg/kg bw) or Sudan I (50 mg/kg bw) alone or with both compounds in combination to explore BaP-derived DNA adduct formation in vivo. Using ³²P-postlabelling, DNA adducts generated by BaP-7,8-dihydrodiol-9,10-epoxide were found in livers of rats treated with BaP alone or co-exposed to Sudan I. During co-exposure to Sudan I prior to BaP treatment, BaP-DNA adduct levels increased 2.1-fold in comparison to BaP treatment alone. Similarly, hepatic microsomes isolated from rats exposed to Sudan I prior to BaP treatment were also the most effective in generating DNA adducts in vitro with the activated metabolites BaP-7,8-dihydrodiol or BaP-9-ol as intermediates. DNA adduct formation correlated with changes in the expression and/or enzyme activities of CYP1A1, 1A2 and 1B1 in hepatic microsomes. Thus, BaP genotoxicity in rats in vivo appears to be related to the enhanced expression and/or activity of hepatic CYP1A1/2 and 1B1 caused by exposure of rats to the studied compounds. Our results indicate that the industrially employed azo dye Sudan I potentiates the genotoxicity of the human carcinogen BaP, and exposure to both substances at the same time seems to be hazardous to humans.     

Detection of Reactive Quinones in the Metabolism of Polycyclic Aromatic Hydrocarbons by the Formation of their Glutathione Conjugates

The biotransformation of polycyclic aromatic hydrocarbons to quinones by rat liver microsomes was investigated. The employment of an electrochemical detector allowed the specific detection of quinones separated by reverse phase HPLC with higher sensitivity as compared to UV detection. Microsomal incubations of benzo[a]pyrene (BP) resulted in the formation of 1,6-, 3,6- and 6,12-quinones, of naphthalene in the detection of naphthalene-1,4-quinone, whereas ortho-quinones could only be detected in trace amounts. Additional protein binding studies showed that only 9–22% of synthetic ortho-quinones could be recovered from microsomal incubations. In order to scavenge possible reactive quinone metabolites with glutathione (GSH) and to identify these metabolites, GSH-conjugates of naphthalene-1,2-quinone, naphthalene-1,4-quinone, chrysene-1,2-quinone, BP-7,8-quinone and BP-9,10-quinone were synthesized and spectroscopically characterized. After incubations of 1-naphthol or naphthalene with rat liver microsomes the GSH conjugate of naphthalene-1,2-quinone could be identified by cochromatography with the authentic reference compound.     

Comparative Metabolism,Bioavailability, and Toxicokinetics of Benzo[ a ]pyrene in Rats After Acute Oral,Inhalation, and Intravenous Administration

The metabolic fate of high doses of B a P is not fully established. To fill this important data need, a comprehensive metabolism, bioavailability, and toxicokinetic study has been undertaken to track the fate of B a P subsequent to single acute exposures. Doses of 100 mg/kg body weight, 0.1 mg/m 3 (equivalent to 19 mg/kg oral dose), and 4.5 w g/kg B a P were administered to 8-week-old male F-344 rats via oral, inhalation (nose only), and intravenous routes, respectively. Rats were sacrificed at 0, 0.5, 1, 2, 4, 6, 24, 48, and 72 hr postexposure. Blood, liver, lung, brain, reproductive tissues, urine, and feces samples were analyzed for parent B a P and metabolites by HPLC with fluorescence detection. Most of the administered B a P was metabolized 4, 6, and 72 hr postexposure for inhalation, intravenous, and oral routes, respectively. The following metabolites were detected: 4,5-dihydrodiol, 7,8-dihydrodiol, 9,10-dihydrodiol, 3,6-dione, 3-hydroxy, and 9-hydroxy B a P (organic fraction), glucuronides, sulfates, and glutathione conjugates (aqueous fraction). Toxicokinetic data revealed a high mean residence time, and low clearance values for B a P metabolites in lung, liver, and brain relative to plasma. Findings of this study establish the relationship between bioavailability and the acute toxic effects of B a P observed in our laboratory at these high doses.     

SYNTHESIS OF DIHYDRODIOLS OF PHENANTHRO[3,4-b]THIOPHENE AND PHENANTHRO[4,3-b]THIOPHENE

A number of sulfur analogs of polynuclear aromatic hydrocarbons (thia-PAHs) have been identified in cigarette smoke condensate. Phenanthro[3,4-b]thiophene (P[3,4-b]T) and phenanthro[4,3-b]thiophene (P[4,3-b]T) are sulfur analogs of benzo[c]phenanthrene, which is known to be metabolized to one of the most tumorigenic fjord region diol epoxides tested thus far. Although fjord region diol epoxides of P[3,4-b]T and P[4,3-b]T are expected to be potent mutagens and tumorigens, these two thia-PAHs differ greatly in their mutagenic potencies. In contrast to P[3,4-b]T which is as mutagenic as benzo[a]pyrene, its isoster P[4,3-b]T is a nonmutagenic compound. In order to understand the basis underlying the difference in the mutagenic potency of P[3,4-b]T and P[4,3-b]T, we require these thia-PAHs and their dihydrodiol derivatives for investigating their metabolism and mutagenic/carcinogenic activity. In these studies, we have investigated the Suzuki cross-coupling reaction for an abbreviated synthesis of P[3,4-b]T, P[4,3-b]T, and their dihydrodiol derivatives from easily available reagents.     

New Rapid HPLC Method for Separation and Determination of Benzo[A]Pyrene Hydroxyderivatives

A new rapid, one-step semi-quantitative HPLC-MS method applicable for the separation and characterization of the group of total 10 BaP hydroxyderivatives (1-hydroxy benzo[a]pyrene, 2-hydroxybenzo[a]pyrene, 3-hydroxybenzo[a]pyrene, 4-hydroxybenzo [a]pyrene, 7-hydroxybenzo[a]pyrene, 8-hydroxybenzo[a]pyrene, 9-hydroxybenzo[a]py rene, 10-hydroxybenzo[a]pyrene, 12-hydroxybenzo[a]pyrene and benzo[a]pyrene-9,10-dihydrodiol) was developed, involving electrospray ionization technique (ESI) in positive mode. Its qualitative and quantitative characteristics were selected by the analysis of the model OH-BaP mixture consisted of compounds mentioned above. The model system offered the linear response in the whole concentration range used (50–1000 ng.mL^?1) as well as the resolution sufficient for semi-quantitative purposes (R_S > 0.5). Subsequently, the suitability of the developed method was tested on BaP decomposition products to be formed during photooxidation at λ = 365 nm. These analyses confirmed the applicability of the proposed method, succeeding in the identification of 5 BaP hydroxyderivatives formed upon the BaP photooxidation experiment.