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PubMed1999

Epimerization of benzo[a]pyrene-tetrols after acid hydrolysis, implications for determination of benzo[a]pyrene adducts in protein and DNA.

Determination of benzo[a]pyrene-DNA or protein adducts with high performance liquid chromatography (HPLC) after acid hydrolysis at high temperature (90 degrees C) enables four isomers of benzo[a]pyrene tetrahydrotetrol to be identified and quantitated. We have investigated the effect of acid treatment of benzo[a]pyrene-tetrahydrotetrol isomers using HPLC and nuclear magnetic resonance spectroscopy (NMR) analysis. By HPLC, we found reversible epimerization of (+/-)-benzo[a]pyrene-r-7,t-8,9, 10-tetrahydrotetrol to (+/-)-benzo[a]pyrene-r-7,t-8,9, c-10-tetrahydrotetrol and of (+/-)-benzo[a]pyrene-r-7,t-8,c-9, t-10-tetrahydrotetrol to (+/-)-benzo[a]pyrene-r-7,t-8,c-9, 10-tetrahydrotetrol, but no interconversion between the two isomer groups. After acid hydrolysis, we found an equilibrium of 87% (+/-)-benzo[a]pyrene-r-7,t-8,9,c-10-tetrahydrotetrol and 9% (+/-)-benzo[a]pyrene-r-7,t-8,9,10-tetrahydrotetrol and 68% (+/-)-benzo[a]pyrene-r-7,t-8,c-9,10-tetrahydrotetrol and 20% (+/-)-benzo[a]pyrene-r-7,t-8,c-9,t-10-tetrahydrotetrol. Minor amounts of two unknown compounds with similar chromatographic characteristics were also found. We have established a NMR method for determination of underivatized (+/-)-benzo[a]pyrene-r-7,t-8,9, c-10-tetrahydrotetrol and (+/-)-benzo[a]pyrene-r-7,t-8,9, 10-tetrahydrotetrol confirming the epimerization of (+/-)-benzo[a]pyrene-r-7,t-8,9,10-tetrahydrotetrol to (+/-)-benzo[a]pyrene-r-7,t-8,9,c-10- tetrahydrotetrol. (+/-)-Benzo[a]pyrene-r-7,t-8,9,10-tetrahydrotetrol was treated with aqueous hydrochloric acid in tetrahydro- furan-d8 to give (+/-)-benzo[a]pyrene-r-7,t-8,9,c-10-tetrahydrotetrol at 57 degrees C while observing the 1H NMR resonances at 500 MHz. Gradient-selected correlation spectroscopy (COSY), heteronuclear multiple quantum correlation (HMQC) and heteronuclear multiple bond correlation (HMBC) experiments were performed to confirm the assignments of the aliphatic hydrogens in the product (+/-)-benzo[a]pyrene-r-7,t-8,9, c-10-terahydrotetrol. Thus, when analyzing benzo[a]pyrene-DNA or protein adducts by cleaving the adducts with acid hydrolysis, the only ratio of biological significance is between (+/-)-benzo[a]pyrene-r-7,t-8,9,c-10-tetrahydrotetrol plus (+/-)-benzo[a]pyrene-r-7,t-8,9,10-tetrahydrotetrol and (+/-)-benzo[a]pyrene-r-7,t-8,c-9,10-tetrahydrotetrol plus (+/-)-benzo[a]pyrene-r-7,t-8,c-9,t-10-tetrahydrotetrol, due to interconversion (epimerization) at C-10.

Benzo(a)pyrene↗

PubMed1978

Comparative tumor initiating activity of 10-methylbenzo-[a]pyrene, 7,10-dimethylbenzo[a]pyrene and benzo[a]pyrene.

The tumor initiating activity on mouse skin of benzo[a]pyrene (BaP), 7,10-dimethylBaP, and 10-methylBaP was determined. Each compound was tested at initiating doses of 50 microgram and 100 microgram with promotion by application 3 times weekly of 2.5 microgram tetradecanoylphorbol acetate. BaP induced tumors in 40% (100 microgram) and 25% (50 microgram) of the animals. No tumors were observed in either group treated with 7,10-dimethylBaP. In the groups treated with 10-methyl-Bap, the incidence of tumor bearing animals was 20% at both doses. These results and the results of previous studies on other methylated BaP derivatives suggest that the mechanism of activation of these compounds is similar to that observed for the parent hydrocarbon and probably involves formation of an angular ring diol-epoxide or epoxide.

Animals↗

PubMed1998

Metabolic activation of racemic and enantiomeric trans-8, 9-dihydroxy-8,9-dihydrodibenzo[a,l]pyrene (dibenzo[def,p]chrysene) to dibenzo[a,l]pyrene-bis-dihydrodiols by induced rat liver microsomes and a recombinant human P450 1A1 system: the role of the K-region-derived metabolic intermediates in the formation of dibenzo[a,l]pyrene-DNA adducts.

Metabolic activation studies of dibenzo[a,l]pyrene (DB[a,l]P) (dibenzo[def,p]chrysene), an extremely potent environmental carcinogen, have been focused on metabolism at the fjord region, a region associated with high mutagenic and carcinogenic activities of the corresponding fjord-region DB[a,l]P-11,12-diol-13,14-epoxides. DB[a,l]P is metabolized by beta-naphthoflavone (BNF)- and 3-methylcholanthrene-induced rat liver microsomes and a recombinant human P450 1A1 system to two major dihydrodiols, the K-region dihydrodiol, DB[a,l]P-8,9-dihydrodiol (DB[a,l]P-8,9-diol), and the fjord-region dihydrodiol, DB[a,l]P-11,12-dihydrodiol. We have investigated the further metabolic activation of DB[a,l]P-8,9-diol by BNF-induced rat liver microsomes and a recombinant human P450 1A1 system with epoxide hydrolase to DB[a,l]P-bis-diols and to DNA adducts. (+/-)-trans-DB[a,l]P-8,9-diol was synthesized and resolved into its enantiomers. Racemic trans-DB[a,l]P-8,9-diol was metabolized by BNF-induced rat liver microsomes to six metabolites: two diastereomers of trans,trans-DB[a,l]P-8,9:11,12-bis-diol, two diastereomers of trans,cis-DB[a,l]P-8,9:11,12-bis-diol, and two diastereomers of trans-DB[a,l]P-8,9:13,14-bis-diol as characterized by NMR, MS, and UV spectroscopy. Metabolic studies using both enantiomeric (-)- and (+)-trans-DB[a,l]P-8,9-diol further demonstrated that each diastereomer of trans,trans-DB[a,l]P-8,9:11, 12-bis-diol and trans-DB[a,l]P-8,9:13,14-bis-diol was comprised of two enantiomers. Similarly, incubations of enantiomeric or racemic trans-DB[a,l]P-8,9-diol with a recombinant human P450 1A1 system and epoxide hydrolase also gave the same two enantiomeric mixtures of diastereomers of trans,trans-DB[a,l]P-8,9:11,12-bis-diol and the same two enantiomeric mixtures of diastereomers of trans-DB[a,l]P-8, 9:13,14-bis-diol. This suggested that the microsomal oxidations of (-)- and (+)-trans-DB[a,l]P-8,9-diol were stereospecific. The stereospecific formation of enantiomers of trans-DB[a,l]P-8,9-diol from DB[a,l]P was examined using both BNF-induced rat liver microsomes and a recombinant human P450 1A1 system with epoxide hydrolase. Stereospecificity was observed as both metabolic systems favored the formation of (-)-trans-DB[a,l]P-8,9-diol by 8-9-fold. DNA adduct studies were undertaken using TLC/HPLC 32P-postlabeling techniques. In the presence of a recombinant human P450 1A1 system with epoxide hydrolase, DB[a,l]P gave two groups of calf thymus DNA adducts. The group of later-eluting adducts were identified as arising from syn- and anti-DB[a,l]P-11,12-diol-13,14-epoxides, while the more polar early-eluting adducts were derived, in part, from the further activation of trans-DB[a,l]P-8,9-diol. Our data indicate that, in P450 1A1-mediated microsomal incubations, DB[a,l]P is metabolized to trans-DB[a,l]P-8,9-diol which is further metabolized to DB[a,l]P-bis-diols. trans-DB[a,l]P-8,9-diol is metabolically activated to intermediates that can bind to DNA and give DNA adducts similar to those observed with DB[a,l]P. These results indicate that DB[a,l]P can be metabolically activated by both fjord-region and K-region pathways.

Animals↗

PubMed1999

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.

Animals↗

PubMed1976

Metabolism of benzo(a)pyrene and benzo (a)pyrene derivatives to mutagenic products by highly purified hepatic microsomal enzymes.

A highly purified and reconstituted hepatic microsomal monooxygenase system, completely free of epoxide hydrase and consisting of cytochrome P-448 from 3-methylcholanthrene-treated rats, NADPH-cytochrome c reductase, phosphatidylcholine, and NADPH, metabolizes benzo (a)pyrene to products highly mutagenic in strains TA 98 and TA 1538 of Salmonella typhimurium. The formation of mutagenic metabolites is completely dependent on the presence of benzo (a)pyrene, NADPH, NADPH-cytochrome c reductase, and cytochrome P-448 and is partially dependent on phosphatidylcholine. Mutation frequency in both strains is linearly related to amount of cytochrome P-448 and to time of incubation. Highly purified cytochrome P-450 from phenobarbital-treated rats is relatively poor in catalyzing the formation of mutagenic metabolites from benzo (a)pyrene. Addition of 7.5 to 75 units of highly purified epoxide hydrase to the cytochrome P-448-dependent monooxygenase system decreases the number of mutations by approximately 50% and30% in strains TA 1538 and TA 98, respectively. Additional amounts of epoxide hydrase (300 units) fail to further suppress mutations, indicating that at least some, but probably not all, of the mutagenic metabolites of benzo (a)pyrene are arene oxides. In the absence of a monooxygenase system, mutations induced by benzo (a)pyrene 4,5-oxide are readily quenched by epoxide hydrase, whereas mutations induced by a diol epoxide metabolite of benzo (a)pyrene [(+/-)-7 beta, 8alpha-dihydroxy-9beta, 10beta-epoxy-7,8,9,10-tetrahydrobenzo (a)pyrene] are not. Several known and potential phenolic and dihydrodiol metabolites of benzo (a)pyrene are metabolized to products mutagenic in the Salmonella. The number of mutations induced per nmol of hemoprotein is approximately 3- to 4-fold higher when trans-7,8-dihydroxy-7,8-dihydrobenzo (a)pyrene replaces benzo (a)pyrene as a substrate for the cytochrome P-448-dependent monooxygenase system. Little or no mutagenic activity is observed with trans-dihydrodiols at positions 4,5, 9,10, or 11,12 of the hydrocarbon, either in the absence or presence of the active monooxygenase system. Of the 12 possible isomeric monophenols of benzo (a)-pyrene, only 6- and 12-hydroxybenzo (a)pyrene are moderately active bacterial mutagens; 1-, 2-, 3-, 6-, 9-, and 12-hydroxybenzo (a)pyrene are premutagens (i.e. metabolized to mutagenic products); and 4-, 5-, 7-, 8-, 10-, and 11-hydroxybenzo (a)pyrene have little or no mutagenic activity with or without further oxidative metabolism. Benzo (a)pyrene 7,8-oxide, a carcinogen on mouse skin, is weakly mutagenic but can be further metabolized to a highly active bacterial mutagen(s), presumably diol epoxide(s), by a combination of epoxide hydrase and the cytochrome P-448 monooxygenase system. This is the first example of a direct role of epoxide hydrase in the metabolic activation of a chemical to a toxic product.

Animals↗

PubMed2005

The influence of single and multiple applications of pyrene on the evolution of pyrene catabolism in soil.

The influence of pyrene added in a single application (0, 50, 100 and 200 mg kg(-1)) was investigated in multiple applications (1 x 50, 2 x 50 and 4 x 50 mg kg(-1)) on the evolution of catabolic activity in a pristine pasture soil. The microbial community's ability to degrade pyrene was assessed at 0, 4, 8 and 12 weeks by the mineralization of added 14C-pyrene. Significant mineralization (>5%) of added 14C-pyrene only occurred after 4 weeks soil-pyrene contact time in most of the pyrene-amended soils. Pyrene-amended soils showed statistically significantly shorter (P<0.05) lag times compared to the control soil after 8 and 12 weeks soil-pyrene contact time. Further, the rates of degradation increased in the presence of pyrene, peaking at 8 weeks. In terms of the overall extents of pyrene mineralization, there were statistically significant increases (P<0.05) between 4 and 8 weeks, with little difference between 8 and 12 weeks, with the general trend that an increase in pyrene concentration resulted in higher levels of mineralization. Increasing the concentration and number of pyrene additions can have a significant impact on the adaptation of the soil microflora to degrade pyrene over time.

Adaptation, Physiological↗

PubMed1987

Topical treatment of mice with benzo[a]pyrene or parenteral administration of benzo[a]pyrene diol epoxide-DNA to rats results in faecal excretion of a putative benzo[a]pyrene diol epoxide-deoxyguanosine adduct.

The administration of [3H]BPDE-DNA, whether by i.p. or i.v. injection, to male Wistar rats resulted in the majority of the radioactivity being recovered in the faeces. Excretion was rapid: within 24 h post-injection, 45% of the applied dose was recovered in the faeces. H.p.l.c. analysis of radioactive material extracted from the faeces by methanol showed that it contained a single component which co-chromatographed with [3H]BPDE-dGuo and which was not affected by treatment with alkaline phosphatase, aryl sulphatase or beta-glucuronidase. To determine if this phenomenon occurs after topical application of BP to a target tissue, such as mouse skin, animals were treated with [3H]BP and their faeces collected. After an extensive extraction procedure involving differential solubility in organic solvents, Sephadex LH-20 chromatography and h.p.l.c., a product was isolated from mice faeces which had characteristics consistent with a [3H]BPDE-dGuo adduct. These findings are discussed in relation to detection of BPDE adducts in human populations.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1999

Metabolism of benzo[a]pyrene to trans-7,8-dihydroxy-7, 8-dihydrobenzo[a]pyrene by recombinant human cytochrome P450 1B1 and purified liver epoxide hydrolase.

Recombinant human enzymes expressed in membranes obtained from Escherichia coli transformed with cytochrome P450 (P450) and NADPH-P450 reductase cDNAs were used to identify the human P450 enzymes that are most active in catalyzing the oxidative transformation of benzo[a]pyrene in vitro. Activation of benzo[a]pyrene to genotoxic products that cause induction of umu gene expression in Salmonella typhimurium NM2009 by P450 1A1 and P450 1B1 enzymes was found to be enhanced by inclusion of purified epoxide hydrolase (isolated from rat or human livers) with the reaction mixture. High-performance liquid chromatographic analysis showed that P450 1B1 catalyzed benzo[a]pyrene to trans-7, 8-dihydroxy-7,8-dihydrobenzo[a]pyrene at level of approximately 3 nmol min(-)(1) nmol of P450(-)(1) only when epoxide hydrolase was present and P450 1A1 (with the hydrolase) was able to catalyze benzo[a]pyrene at one-tenth of the activity catalyzed by P450 1B1. Kinetic analysis showed that ratio of V(max) to K(m) for the formation of trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene in this assay system was 3.2-fold higher in CYP1B1 than in CYP1A1. Other human P450s (including P450s 1A2, 2E1, and 3A4) were found to have very low or undetectable activities toward the formation of trans-7, 8-dihydroxy-7,8-dihydrobenzo[a]pyrene. A reconstituted system containing purified P450 1B1, rabbit liver NADPH-P450 reductase, and human liver epoxide hydrolase was found to catalyze benzo[a]pyrene to trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene at a rate of 0.86 nmol min(-)(1) nmol of P450(-)(1); the activities were found to be largely dependent on the presence of sodium cholate in the system. These results suggest that P450 1B1 is a principal enzyme in catalyzing the oxidation of benzo[a]pyrene to trans-7,8-dihydroxy-7, 8-dihydrobenzo[a]pyrene and that the catalytic functions of P450 1B1 may determine the susceptibilities of individuals to benzo[a]pyrene carcinogenesis.

Animals↗

PubMed1996

Lateral organization of pyrene-labeled lipids in bilayers as determined from the deviation from equilibrium between pyrene monomers and excimers.

In lipid bilayers, pyrene and pyrene-labeled lipids form excimers in a concentration-dependent manner. The aromatic amine N, N-diethylaniline (DEA), which has a high membrane-to-medium partition coefficient, quenches the monomers only, and therefore it is expected that under conditions in which the monomers are in equilibrium with the excimers due to the mass law, the Stern-Volmer coefficient (Ksv) for monomers (M), defined as KM, should be identical to that of the excimer (E), defined as KE, and KE/KM = 1. 0. This is indeed the case for pyrene and pyrene valerate in egg phosphatidylcholine small unilamellar vesicles. However, for pyrene decanoate and pyrene dodecanoate in these vesicles, and for N-[12-(1-pyrenyl)dodecanoyl]sphingosylphosphocholine in a matrix of either N-stearoyl sphingosylphosphocholine or 1-palmitoyl-2-oleoyl phosphatidylcholine, KE < KM. This can be explained either by the existence of (a) two subpopulations of excimers, one in fast equilibrium with the monomers and the other, related to ground-state protoaggregates of pyrene lipids; (b) two monomer subpopulations where part of M cannot be quenched by DEA; or (c) two monomer subpopulations, both quenched by DEA, but only one of which produces excimers. The good agreement between the photophysical processes determined by steady state and time-resolved measurements supports the third explanation for the bilayers containing pyrene phospholipids. It also suggests that the main factors determining the immiscibility of pyrene lipids in phospholipid bilayers are the temperature, the difference in the gel-to-liquid-crystalline phase transition temperature (deltaTm) between the matrix and the pyrene lipid, and the structural differences between the matrix lipid and the pyrene-labeled lipid. These results indicate that the KE/KM ratio can serve as a very sensitive tool to quantify isothermal microscopic immiscibility in membranes. This novel approach has the following advantages: applicability to fluid phase immiscibility, requirement of a relatively low mol fraction of pyrene lipids, and conceivably, applicability to biological membranes.

Kinetics↗

PubMed1986

Inhibitory effect of 3-hydroxybenzo(a)pyrene on the mutagenicity and tumorigenicity of (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene.

The 12 isomeric phenols of benzo(a)pyrene were tested for their ability to inhibit the mutagenic activity of (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene [B(a)P 7,8-diol-9,10-epoxide-2], an ultimate mutagenic and carcinogenic metabolite of benzo(a)pyrene. 3-Hydroxybenzo(a)pyrene [3-HO-B(a)P], a major metabolite of benzo(a)pyrene, was the most potent antagonist tested. Approximately 3 nmol of 3-HO-B(a)P, 14 nmol of 10-HO-B(a)P, and 5-8 nmol of 1-, 2-, 4-, 5-, 6-, 7-, 8-, 9-, 11-, and 12-HO-B(a)P inhibited the mutagenic activity of 0.05 nmol of B(a)P 7,8-diol-9,10-epoxide-2 by 50% in Salmonella typhimurium strain TA 100. The importance of the phenolic group for antimutagenic activity was indicated by the lack of antimutagenic activity of benzo(a)pyrene itself. 3-HO-B(a)P also inhibited the mutagenic activity resulting from the metabolic activation of benzo(a)pyrene and (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene by rat liver microsomes. This inhibition may have resulted from an effect of 3-HO-B(a)P on the metabolic activation of these carcinogens and/or from a direct effect on the action of B(a)P 7,8-diol-9,10-epoxide-2. In a mammalian cell culture system utilizing Chinese hamster V79 cells, 3-HO-B(a)P (8 microM) inhibited the mutagenicity of B(a)P 7,8-diol-9,10-epoxide-2 (0.2 microM) by 50%. Although 3-HO-B(a)P was a potent inhibitor of the mutagenic activity of bay-region diol epoxides of benzo(a)pyrene, dibenzo(a,h)pyrene, and dibenzo(a,i)pyrene in S. typhimurium strain TA 100, higher concentrations of 3-HO-B(a)P were needed to inhibit the mutagenicity of the chemically less reactive benzo(a)pyrene 4,5-oxide and the bay-region diol epoxides of benz(a)anthracene, chrysene, and benzo(c)phenanthrene. Both 3-HO-B(a)P and 10-HO-B(a)P accelerated the disappearance of B(a)P 7,8-diol-9,10-epoxide-2 from 1:9 dioxane-water solutions at pH 7 and 25 degrees C. 3-HO-B(a)P, the most effective antimutagen of the B(a)P phenols tested, was much more reactive with the diol epoxide than 10-HO-B(a)P, the least effective antimutagen. The rate constant for the reaction of 3-HO-B(a)P with the diol epoxide exhibited a nonlinear (greater than first-order) dependence on the concentration of the phenol. Evidence was obtained for covalent adduct formation between the diol epoxide and each of the two phenols.(ABSTRACT TRUNCATED AT 400 WORDS)

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1985

Benzo[a]pyrene dione-benzo[a]pyrene diol oxidation-reduction couples; involvement in DNA damage, cellular toxicity, and carcinogenesis.

Three isomeric quinone metabolites of the environmental carcinogen benzo[a]pyrene undergo reversible, univalent oxidation-reduction cycles involving the corresponding benzo[a]pyrene diols and intermediate semiquinone radicals. Under anaerobic conditions, benzo[a]pyrene 1,6-dione, benzo[a]pyrene 3,6-dione, and benzo[a]pyrene 6,12-dione are readily reduced by mild biological agents such as NADH and glutathione. The benzo[a]pyrene diols, in turn, are very rapidly autooxidized to diones when exposed to air. Substantial amounts of hydrogen peroxide are produced during these autooxidations. The benzo[a]pyrene diol/benzo[a]pyrene dione interconversions proceed by one-electron steps; the corresponding semiquinone radicals were detected as intermediates when the reactions were carried out at high pH. Benzo[a]pyrene diones are electron-acceptor substrates for NADH dehydrogenase. Catalytic amounts of these metabolites, together with this respiratory enzyme, function as cyclic oxidation-reduction couples to link NADH and molecular oxygen in the continuous production of hydrogen peroxide. Benzo[a]pyrene diones induce strand scissions when incubated with T7 DNA. The damage is modified by conditions that indicate that reduced oxygen species propagate the reactions responsible for strand scission. Benzo[a]pyrene diones are cytotoxic at low concentrations to cultured hamster cells. The cytotoxic effect can be substantially reduced by depletion of oxygen from the growth medium and the atmosphere in which the cells are incubated. The results support the hypothesis that the biological activity of benzo[a]pyrene diones is due to the regenerative oxidation-reduction cycles involving quinone and hydroquinone forms; activated oxygen species and semiquinone radicals formed during these cycles are most likely responsible for the observed cytotoxic action. The role of activated oxygen species in carcinogenesis is discussed.

Animals↗

PubMed1998

Kinetics of tissue distribution and elimination of pyrene and 1-hydroxypyrene following intravenous administration of [14C]pyrene in rats.

The tissue distribution and elimination of pyrene and 1-hydroxypyrene (1-OHP) were evaluated in male Sprague-Dawley rats (210-240 g) following an intravenous injection of 50 micromol/kg of [14C]pyrene. Blood and tissues were removed and urine and feces were collected at 1, 2, 4, 8, 16, and 24 h postdosing. [14C]Pyrene equivalents were measured by liquid scintillation counting, and beta-glucuronidase/arylsulfatase-treated blood, tissues, and excreta were analyzed for pyrene and 1-OHP by HPLC/fluorescence. At 1 h, the largest fraction of the dose was found in adipose tissue, essentially as pyrene, and its elimination followed first-order monophasic kinetics with a half-life (t(1/2)) of 4.9 h. In blood, liver, kidney, lung, muscle, and gastrointestinal (GI) tract, kinetics of [14C]pyrene equivalents were biphasic and average t(1/2) values for the terminal elimination phase (8 to 24 h) ranged between 6.2 and 8.7 h. Elimination of pyrene in blood and these tissues except the GI tract followed first-order biphasic kinetics with average t(1/2) values of the terminal phase ranging between 3.6 and 5.4 h. In the GI tract, a monophasic elimination kinetics of pyrene was observed with mean t(1/2) value of 3.1 h. Kinetics of 1-OHP in blood and liver showed a monophasic elimination with mean t(1/2) values of 6.7 and 6.2 h, respectively. Kinetics of 1-OHP in the other tissues were biphasic with average t(1/2) values of the terminal elimination phase ranging between 5.2 and 6.2 h. At 24 h, on average, 81.7% of the dose was recovered in the urine (57.2%), feces (18.3%), and GI tract (6.2%) as [14C]pyrene equivalents with 2.7 and 1.9% of dose excreted as total 1-OHP in urine and feces, respectively. At all time points, 1-OHP in urine represented a constant fraction of total 14C in urine and feces. These results indicate that (i) [14C]pyrene was rapidly distributed, metabolized, and eliminated from the body, and (ii) although 1-OHP represents a small percentage of total pyrene eliminated from the body, it remains a reliable indicator of systemic exposure to, and overall elimination of the 14C associated with, this polycyclic aromatic hydrocarbon.

Animals↗

PubMed1997

Characterization of DNA adducts in Chinese hamster ovary cells treated with mutagenic doses of 1- and 3-nitrosobenzo[a]pyrene and the trans-7,8-diol-anti-9,10-epoxides of 1- and 3-nitrobenzo[a]pyrene.

The environmental contaminants 1- and 3-nitrobenzo[a]pyrene (1- and 3-nitro-BaP) are mutagens in Chinese hamster ovary (CHO) cells with exogenous metabolic activation. Previous studies demonstrated the potent direct-acting mutagenicity of the oxidized metabolites, trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydro-1-nitrobenzo[a] pyrene (1-NBaPDE) and trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9, 10-tetrahydro-3-nitrobenzo[a]pyrene (3-NBaPDE), and the partially nitroreduced metabolites, 1- and 3-nitrosobenzo[a]pyrene (1- and 3-NO-BaP). In this study, we have identified the major adduct formed by incubation of calf thymus DNA with 1-NBaPDE and used this standard in conjunction with other adduct standards to characterize the 32P-postlabeled DNA adducts produced by 1- and 3-nitro-BaP metabolites in CHO cultures. The major adduct from 1-NBaPDE exposure was 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-1- nitrobenzo[a]pyrene; from 3-NBaPDE, 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-3- nitrobenzo[a]pyrene; from 1-NO-BaP, 6-(deoxyguanosin-N2-yl)-1-aminobenzo[a]pyrene; and from 3-NO-BaP, 6-(deoxyguanosin-N2-yl)-3-aminobenzo[a]pyrene. For comparison, the adducts formed by trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and the related nitroreduced derivative 6-nitrosobenzo[a]pyrene were also examined. The nitrobenzo[a]pyrene DNA adducts described in this study are proposed to be involved in the mutagenicity of 1- and 3-nitro-BaP upon either oxidative or reductive metabolism.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMedDate not supplied

Nitroreduction of 1- and 3-nitro-7,8,9,10-tetrahydrobenzo[a]pyrene and 1-nitrobenzo[a]pyrene resulting in formation of N2-deoxyguanosinyl adducts through long-range migration.

We recently reported that the reaction of N-hydroxy-3-aminobenzo[a] pyrene with calf thymus DNA produced 6-(deoxyguanosin-N2-yl)-3-aminobenzo[a]pyrene as the predominant adduct. The deoxyguanosinyl group of this adduct resides at the C6 position, which is remote from the reaction site, the nitrenium ion. It is significant to determine if formation of this type of DNA adduct is general and whether or not adduct formation is due to an increase in the stabilization of the nitrenium ion by increasing aromaticity. Thus, reduction of 1-nitro-7,8,9,10-tetrahydrobenzo[a]pyrene, 3-nitro-7,8,9,10-tetrahydrobenzo[a]pyrene, and 1-nitrobenzo[a]pyrene, both chemically and enzymatically, followed by reaction with calf thymus DNA was investigated. DNA was isolated and enzymatically digested, and the resulting modified nucleosides were separated by HPLC. Upon spectral analyses by mass and proton nuclear magnetic resonance spectroscopy, 6-(deoxyguanosin-N2-yl)-1-amino-7,8,9,10-tetrahydrobenzo[a] pyrene, 6-(deoxyguanosin-N2-yl)-3-amino-7,8,9,10-tetrahydrobenzo[a]pyrene, and 6-(deoxyguanosin-N2-yl)-1-aminobenzo[a]pyrene were identified, respectively. The same DNA adducts were formed from xanthine oxidase-mediated reductive metabolism of 1-nitro-7,8,9,10-tetrahydrobenzo[a]pyrene, 3-nitro-7,8,9,10-tetrahydrobenzo[a]pyrene, and 1-nitrobenzo[a]pyrene in the presence of calf thymus DNA. Thee results indicate that formation of N2-deoxyguanosinyl adducts of this type is common and that increasing the aromaticity by increasing the number of aromatic rings is not a decisive factor in directing their formation.

Animals↗

PubMedDate not supplied

Lipid peroxidation-coupled co-oxygenation of benzo(a)pyrene and benzo(a)pyrene-7,8-dihydrodiol in human term placental microsomes.

The link between lipid peroxidation and benzo(a)pyrene activation was studied in microsomes isolated from human term placenta. Lipid peroxidation was initiated in the presence of NADPH by partially chelated iron. Covalently bound and free metabolites of benzo(a)pyrene or benzo(a)pyrene-7,8-diol were quantitated by radiometry and/or HPLC. Peroxidative conditions increased the amounts of benzo(a)pyrene-trans-anti-tetrol produced from benzo(a)pyrene-7,8-diol, benzo(a)pyrene-diones from benzo(a)pyrene, and protein bound metabolites from both. A reactive oxo-iron complex is proposed as an ultimate species initiating hydrogen abstraction and lipid peroxidation. It is suggested that partially chelated iron catalyzes co-oxygenation of benzo(a)pyrene and benzo(a)pyrene-7,8-diol by peroxyl radical in placental microsomes. This peroxidative reaction may be crucial for bioactivation of benzo(a)pyrene in human term placenta.

Benzo(a)pyrene↗

PubMed2000

In vivo laser-induced fluorescence detection of pyrene in nematodes and determination of pyrene binding constants for humic substances by fluorescence quenching and bioconcentration experiments.

The bioconcentration of pyrene by bacterivorous thread worms (nematodes) of the species Caenorhabditis elegans was studied with laser-induced fluorescence (LIF) spectroscopy, fluorescence imaging and a radiotracer method. The vibronic band intensities of the LIF spectra indicated that the microenvironment of pyrene in the nematodes was similar to a low-polarity solvent, and thus provided direct evidence that pyrene was accumulated in lipid-rich areas inside the nematodes. The concentration of pyrene in the nematodes was estimated from the monomer/excimer fluorescence intensity ratio. Results from this method were in fair agreement with results using 14C labeled pyrene for measuring pyrene bioconcentration. Preliminary results indicated that LIF measurements of pyrene may be possible even in single nematodes. Fluorescence microscopic observations revealed that pyrene was not adsorbed on the outside of the organisms, but was strongly concentrated in restricted areas inside the worms. In the second part of the study, the effects of six different humic substances (HS) on the bioconcentration of pyrene were investigated and sorption coefficients (KDOC) calculated from reductions in bioconcentration (KDOC(biol)) were compared with sorption coefficients measured with a fluorescence quenching technique (KDOC(flu)). The results of these two different experimental methods agreed well (with KDOC(biol) being slightly lower than KDOC(flu), indicating that the fraction of pyrene that was determined as freely dissolved by the fluorescence quenching method was comparable to the bioavailable fraction.

Adsorption↗

PubMed2006

Comparison of microbial pyrene and benzo[a]pyrene mineralization in liquid medium, soil slurry, and soil.

The microbial degradation of 14C-pyrene and 14C-benzo[a]pyrene by a bacterial mixed culture was studied within a mixture of the PAHs phenanthrene, anthracene, pyrene, fluoranthene, and benzo[a]pyrene as sole carbon source in the different culture systems: (i) liquid medium, (ii) soil slurry (surface and grinding influence), and (iii) soil. The fate of these two labeled compounds was followed in these systems with an emphasis on mineralization to carbon dioxide, extractability, and adsorption to humic materials and formation of unextractable residual. Mineralization showed the most obvious differences: soil slurries achieved the best results both concerning the extent of mineralization and the time required. The highest extent of pyrene mineralization (54% within 21 days) was observed in soil slurries; in liquid media, pyrene mineralization was slower, but reached approximately the same extent (54% in 150 days); in soils, mineralization reached only 36% of added pyrene after 160 days. Benzo[a]pyrene was mineralized in a mixture of PAHs in soil slurries to an extent of 34% within 70 days, whereas mineralization in liquid medium and soil occurred in the range of 5% (70 days). Mineralization of benzo[a]pyrene in sand slurries was lower compared to soil slurries (19% in sand slurries vs. 32% in soil slurries within 50 days).

Adsorption↗

PubMed1991

Indigenous and enhanced mineralization of pyrene, benzo[a]pyrene, and carbazole in soils.

We studied the mineralization of pyrene, carbazole, and benzo[a]pyrene in soils obtained from three abandoned coal gasification plants in southern Illinois. The soils had different histories of past exposure to hydrocarbon contamination and different amounts of total organic carbon, microbial biomass, and microbial activity. Mineralization was measured by using serum bottle radiorespirometry. The levels of indigenous mineralization of 14C-labeled compounds ranged from 10 to 48% for pyrene, from undetectable to 46% for carbazole, and from undetectable to 25% for benzo[a]pyrene following long-term (greater than 180-day) incubations. Pyrene and carbazole were degraded with short or no lag periods in all soils, but benzo[a]pyrene mineralization occurred after a 28-day lag period. Mineralization was not dependent on high levels of microbial biomass and activity in the soils. Bacterial cultures that were capable of degrading pyrene and carbazole were isolated by enrichment, grown in pure culture, and reintroduced into soils. Reintroduction of a pyrene-degrading bacterium enhanced mineralization to a level of 55% within 2 days, compared with a level of 1% for the indigenous population. The carbazole degrader enhanced mineralization to a level of 45% after 7 days in a soil that showed little indigenous carbazole mineralization. The pyrene and carbazole degraders which we isolated were identified as a Mycobacterium sp. and a Xanthamonas sp., respectively. Our results indicated that mineralization of aromatic hydrocarbons can be significantly enhanced by reintroducing isolated polycyclic aromatic hydrocarbon-degrading bacteria.

Bacteria↗