Secondary metabolites of benzo[a]pyrene: 3-hydroxy-trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene, a biliary metabolite of 3-hydroxybenzo[a]pyrene in the rat

Rats administered 3-hydroxybenzo[a]pyrene (50 mg/kg, i.p.),excrete via the bile metabolites which, after treatment withß-glucuronidase and aryl sulphatase, yield, in additionto 3-hydroxybenzo[a]pyrene, 3-hydroxy-trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene(3-OH-BP-7, 8-diol) and a minor, highly labile, metabolite tentativelyidentified as 3,5-dihydroxybenzo[a]pyrene. These novel metabolitesare readily isolated in a pure state via preparative layer chromatography.The structure of the 3-OH-BP-7, 8-diol was revealed by its u.v.,proton magnetic resonance and mass spectral properties. Itshydroxyl functions are in a predominantly quasi-diequatorialconformation.     

Characterization of neutral metabolites of benzo[a]pyrene in urine from germfree rats

[¹⁴C] Benzo[a]pyrene (BP) was administered to male germfreerats. Urinary metabolites, constituting 9% of the administeredradioactivity, were fractionated by lipophilic ion exchangechromatography. More than 80% of the urinary metabolites wereconjugated, while neutral metabolites constituted 13–18%.The latter group was characterized by reversed-phase HPLC, ultravioletspectrometry (UV) and gas chromatography/mass spectrometry (GC/MS).Relative quantities of BP metabolites were estimated from thedistribution of radioactivity upon HPLC fractionation. Somecoeluted peaks were further quantitated from the total ion currentchromatograms obtained in the GC/MS analysis. Two 7,8,9,10-tetrolswhich might be produced from the ultimate carcinogen r-7-t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydro-BP(anti-7,8-diol-9,10-epoxide) were detected at trace levels andindicated to be the r-7-t-8,9-c-10-(7,10/8,9) and r-7-t-8,9,10-(7/8,9,10-)isomers. A trans-11,12-dihydrodiol was characterized as a majormetabolite, while a trans-7,8-dihydrodiol was present at tracelevels. Three additional quantitatively important metaboliteswere identified as isomeric trihydroxy-BPs. Two metabolitescoeluting with BP quinones on HPLC were detected with relativelyhigh abundance and tentatively identified as carboxylic methylester derivatives of BP quinones. Three qulnones were detectedwith 1,6-, 3,6- and 6,12-substitutions. The 6,12- and 11,12-dihydroxy-BPswere also found at trace levels. A group of quinone-like metaboliteswere tentatively identified as trioxo-BPs. No monohydroxy-BPwas detected in the neutral fraction of the urine extract. Thetime course of excretion was also studied and found to differbetween individual metabolites.     

Inactivation of DNA-binding metabolites of benzo[a]pyrene and benzo[a]pyrene-7,8-dihydrodiol by glutathione and glutathione S-transferases

The binding to DNA of reactive metabolites of trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene(BP-7, 8-diol) was studied following the incubation of tritiatedbenzo[a]pyrene (BP) and BP-7, 8-diol with nuclei from liversof 3-methyl-cholanthrene-treated rats. Binding was inhibitedto a small extent by glutathione (GSH) alone and to a much greaterextent by GSH and cytosol or purified GSH-transferases B andE. In this respect GSH-transferases A and C were also active,but less so. Inhibition of binding of BP-7,8-diol metabolitesto DNA mediated by GSH-transferases was associated with theformation of GSH conjugates. The extent of inhibition of bindingwas similar in incubations of nuclei alone, nuclei and rat livermicrosomes, and calf thymus DNA and rat liver microsomes. Thisindicates that reactive metabolites of BP-7, 8-diol, formedeither by nuclei or microsomes, are readily accessible to solubleGSH-transferases. GSH and cytosol were also active in inhibitingDNA-binding of reactive metabolites from 9-hydroxybenzo[a]pyrene(9-OH-BP). Thus, in the rat hepatocyte GSH and GSH-transferasesmay be important in protecting DNA from electrophilic attackby reactive BP-7, 8-diol and 9-OH-BP species.     

Chinese medicinal herbs modulate mutagenesis, DNA binding and metabolism of benzo[a]pyrene 7,8-dihydrodiol and benzo[a]pyrene 7,8-dihydrodiol-9,10-epoxide.

Oldenlandia diffusa(OD) and Scutellaria barbata (SB) have been used in traditional Chinese medicine for treating liver, lung and rectal tumors. In this study, the effects of aqueous extracts of these two herbs on benzo[a]pyrene 7,8-dihydrodiol. (BaP 7,8-DHD) and benzo[a]pyrene 7,8-dihydrodiol-9,10-epoxide (BPDE)-induced mutagenesis using Salmonella typhimurium TA100 as the bacterial tester strain and rat liver 9000 x g supernatant (S9) as the metabolic activation system were assessed. We also determined the effects of these two herbs on BaP 7,8-DHD and BPDE binding to calf thymus DNA. Organosoluble metabolites of BaP 7,8-DHD and water-soluble conjugates of BaP 7,8-DHD and BPDE were analyzed by high-performance liquid chromatography (HPLC) and alumina column liquid chromatography. Mutagenesis assays revealed that these two herbs produced a significant concentration-dependent inhibition of histidine-independent (His+) revertants induced by BaP 7,8-DHD and BPDE. OD and SB also inhibited BPDE-induced mutagenesis in a concentration-dependent manner in the absence of S9. SB had a greater inhibitory effect than OD. SB significantly inhibited BaP 7,8-DHD and BPDE binding to DNA while OD significantly enhanced DNA binding of both compounds. OD and SB inhibited the formation of organosoluble metabolites of BaP 7,8-DHD and decreased the formation of water-soluble conjugates of BaP 7,8-DHD and BPDE. However, the fraction of the total radioactivity in the water-soluble conjugates present as sulfate and glutathione was increased by OD and SB. Glucuronide fraction was decreased. The results of this study affirm our previous work suggesting that these two Chinese medicinal herbs possess antimutagenic properties and further suggest that they act as blocking agents through a scavenging mechanism.     

Metabolic activation to a mutagen of 3-hydroxy-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, a secondary metabolite of benzo[a]pyrene

3-Hydroxy-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (3-OH-BP-7,8-diol)wag isolated from arylsulfatase/ß-glucuronidase-treatedbile of rats to which 3-hydroxybenzo[a]pyrene (3-OH-BP) hasbeen administered. This triol was investigated for mutagenicityin Salmonella typhimurium (reversion to histidine prototrophyof strains TA 97, TA 98, TA 100 and TA 1537) and in V79 Chinesehamster cells (acquisition of resistance to 6-thioguanine).When no exogenous metabolizing system was added the triol wasinactive, while 3-OH-BP showed weak mutagenic effects with allfour bacterial strains. In the presence of NADPH-fortified postmitochondrialsupernatant fraction (S9 mix) of liver homogenate from Aroclor1254-treated rats, the mutagenicity of 3-OH-BP was potentiated,and the triol was activated to a mutagen(s). In the presenceof S9 mix, the triol was 5—18 times more mutagenic than3-OH-BP in strains TA 97, TA 100 and TA 1537, but both compoundsshowed similar mutagenic potencies with strain TA 98. Thesestrain differences strongly suggest that the mutagenicity of3-OH-BP in the S9 mix-mediated test was not exclusively dueto metabolites of 3-OH-BP-7, 8-diol. Trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene(BP-7,8-diol), like the triol, showed mutagenic effects onlyin the presence of S9 mix. Strain TA 1537 was reverted by thetriol but not by the diol. In the other bacterial strains thediol was more mutagenic than the triol, the difference in potencybeing largest in strain TA 100 (2.5-to 10-fold, depending onthe experimental conditions). In V79 cells, the diol was a potentmutagen, while the triol showed only very weak mutagenic effects.However the triol was more cytotoxic than the diol. High cytotoxicityof the triol was observed even in the absence of S9 mix. Theresults of the present study demonstrate that metabolites of3-OH-BP-7, 8-diol) are biologically-active derivatives of benzo[a]pyrene.Comparison of the mutagenic effectiveness in different bacterialstrains also reveals that metabolites of 3-OH-BP-7, 8-diol andof BP-7, 8-diol substantially differ in the kind of geneticalterations they evoke.     

Metabolism of benzo[a]pyrene and benzo[a]pyrene-7,8-dihydrodiol in human mammary epithelial cells: feedback inhibition by 7-hydroxybenzo[a]pyrene

The metabolism of benzo[a]pyrene (B[a]P) and (-)-transbenzo[a]pyrene-7,8-dihydrodiol (B[a]P-diol) was compared in human mammary epithelial cells (HMEC) grown in serum-free medium, MCDB-170. Conversion of B[a]P-diol to the carcinogen (+)-benzo[a]pyrene-7,8-dihydroxy-9,10-epoxide (BPDE), as measured by analysis of their tetraol hydrolysis products, occurred much more efficiently in cultures incubated with [3H]B[a]P-diol than in cultures incubated with [3H]B[a]P. In cultures pretreated with unlabeled B[a]P (24 h, 400 nM), the conversion of [3H]-B[a]P-diol to [3H]tetraols is inhibited 49%, while the conversion of [3H]B[a]P to [3H]B[a]P-diol- is not affected. These observations led to the identification of a major B[a]P-derived metabolite as 7-hydroxybenzo[a]pyrene (B[a]P-7-ol), which was found to be an extremely potent and selective inhibitor of the conversion of B[a]P-diol to BPDE, with a KI estimated at 3-12 nM. Thus B[a]P activation in HMEC appears to be significantly limited by a feedback inhibition pathway induced by B[a]P-7-ol. The potency and selectivity of the B[a]P-7-ol-induced inhibition suggests that the diol to diolepoxide conversion is affected by a selective oxygenase in HMEC, rather than a non-enzymatic, peroxy radical-induced mechanism. B[a]P-7-ol should prove to be a valuable tool in the study of B[a]P carcinogenesis.     

Inhibition of liver microsome-mediated mutagenesis, metabolism and DNA-binding of benzo[a]pyrene and benzo[a]pyrene 7,8-dihydrodiol in the rat following glucose administration

Aroclor 1254-induced rat liver microsomes prepared from control and glucose-treated rats (30% glucose in drinking water 48 h prior to sacrifice) were used in studies of benzo[a]pyrene (BaP) and BaP 7,8-dihydrodiol (BaP 7,8-DHD)-induced mutagenesis in Salmonella typhimurium TA100. Microsome-dependent metabolism and metabolite binding of BaP and BaP 7,8-DHD to calf thymus DNA was also investigated. BaP-induced mutagenesis in TA100 was inhibited 27% and BaP 7,8-DHD-induced mutagenesis was inhibited 55% by microsomes from glucose-treated rats. [3H]BaP and [3H]BaP 7,8-DHD metabolite binding to DNA was inhibited 17% and 20%, respectively. High performance liquid chromatographic (hplc) analysis of enzyme-hydrolyzed DNA yielded 7R and 7S-diol epoxide-1 deoxyguanosine (BPDE-1:dG) adducts and BPDE-2:dG adducts of [3H]BaP and [3H]BaP 7,8-DHD. These adducts were inhibited 38% and 50%, respectively, by microsomes from glucose-treated rats. Hplc analysis of organosoluble metabolites of [3H]BaP and [3H]BaP 7,8-DHD showed an inhibition of metabolism of 28% and 50%, respectively, by microsomes from glucose-treated rats. The inhibition of metabolism correlated with the effect of glucose treatment on inhibition of BaP and BaP 7,8-DHD-induced mutagenesis and adduct formation. These results suggest that the mechanism by which glucose produces its effects on mutagenesis, DNA-binding and adduct formation is by an inhibition of microsome-mediated metabolism of BaP and BaP 7,8-DHD.     

Differential metabolism of benzo[a]pyrene and benzo[a]pyrene-7,8-dihydrodiol by human CYP1A1 variants

Cytochrome P450 1A1 (CYP1A1) plays a key role in the metabolism of carcinogens, such as benzo[a]pyrene (B[a]P) and metabolites to ultimate carcinogens. Three human allelic variants, namely wild-type (CYP1A1.1), CYP1A1.2 (I462V) and CYP1A1.4 (T461N), were coexpressed by coinfection of baculovirus-infected insect cells with human NADPH-P450 reductase. These recombinant enzymes (in microsomal membranes) were used to analyze whether CYP1A1 polymorphisms affect catalytic activities towards B[a]P and B[a]P-7,8-dihydrodiol. The complete spectrum of phase I metabolites, including the tetrahydrotetrols resulting from hydrolysis of the ultimate carcinogen, B[a]P-7,8-dihydrodiol-9,10-epoxide, was examined by HPLC. Wild-type enzyme showed the highest total metabolism of B[a]P, CYP1A1.2 was approximately 50%, and CYP1A1.4 approximately 70%. Km values for all metabolites with CYP1A1.2 were generally significantly lower than with wild-type enzyme (e.g. B[a]P-7,8-diol formation: 13.8 microM for wild-type, 3.5 microM for CYP1A1.2 and 7.7 microM for CYP1A1.4). Addition of epoxide hydrolase markedly increases the relative diol-to-phenol activities by all three variants. However, CYP1A1.4 exhibits the greatest efficiency to produce diol species. Each variant produced the diol epoxides from B[a]P-7,8-dihydrodiol. CYP1A1.1 exhibited with 10.4 pmol/min/pmol CYP1A1 the greatest total rate for 7,8-diol metabolites followed by CYP1A1.2 (7.2 pmol/min/pmol CYP1A1) and CYP1A1.4 (5.5 pmol/min/pmol CYP1A1). All enzyme variants produced about three times more diol epoxide 2-derived metabolites than diol epoxide 1-derived ones, whereby both rare allelic variants exhibited statistically significantly increased formation of diol epoxide 2. This study showed that the three CYP1A1 variants had different enzyme kinetics properties to produce both the diol metabolites from B[a]P and the ultimate mutagenic species diol epoxide 2 from B[a]P-7,8-dihydrodiol, which must be considered in the evaluation of individual susceptibility to cancer.     

Pulmonary carcinogenicity of 3,9- and 3,7-dinitrofluoranthene, 3-nitrofluoranthene and benzo[a]pyrene in F344 rats

3,9- and 3,7-Dinitrofluoranthene (3,9- and 3,7-DNF), 3-nitrofluoranthene (3-NF) and benzo[a]pyrene (B[a]P) were tested for pulmonary carcinogenicity by intrapulmonary implantation of the compounds into rat lung. These chemicals were given in various doses as suspensions in beeswax-trycaprylin and the animals were observed for 100 weeks. The control group received no drugs. The incidences of lung tumors were 19/21 (90.5%), 7/10 (70%) and 1/10 (10%) in rats treated with 200, 100 and 50 micrograms of 3,9-DNF, 4/9 (44.4%), 3/10 (30%) and 0/10 (0%) in rats treated with 200, 100 and 50 micrograms of B[a]P, 12/22 (54.5%) in rats treated with 200 micrograms of 3,7-DNF and 1/20 (5%) in rats treated with 1000 micrograms of 3-NF respectively. No lung tumors were found in control rats. The incidence of lung tumors induced by 3,9-DNF was twice as high as that induced by B[a]P, when the equivalent dose levels of the two compounds were compared. Histologically, most of the tumors induced by 3,9- and 3,7-DNF and B[a]P were squamous cell carcinomas.     

Peroxidase-mediated glutathione conjugation of benzo[a]pyrene-7,8-dihydrodiol is enhanced by benzo[a]pyrene phenols in vitro.

We reported previously that glutathione (GSH) is oxidized by peroxidases to a thiyl radical that can react with a number of chemicals, including the penultimate carcinogenic metabolite benzo[a]pyrene-7,8-dihydrodiol (7,8-B[a]PD), to give GSH conjugates. Here, we report that phenolic metabolites of benzo[a]pyrene (B[a]P) enhance the peroxidase-mediated formation of glutathione conjugates of 7,8-B[a]PD. The GSH conjugation of 7,8-B[a]PD in a horseradish peroxidase/peroxide system was increased over control values as follows: 9-OH-B[a]P by 4-fold, 7-OH-B[a]P by 3-fold, 1-OH-B[a]P by 2-fold. In contrast 3-OH-B[a]P was ineffective. A phenolic derivative of another polycyclic aromatic hydrocarbon (PAH), benz[a]anthracene, also enhanced GSH conjugation of 7,8-B[a]PD. The enhancement was dependent upon the presence of the phenol, horseradish peroxidase and peroxide. The phenolic compounds, including 3-OH-B[a]P, were also efficient reducing cofactors for the peroxidase. With the exception of 3-OH-B[a]P, the phenolic metabolites of PAH enhanced peroxidase-mediated formation of thiyl radical as detected by electron spin resonance spectrometry. Since both phenols and dihydrodiols are metabolites of B[a]P catalyzed by the cytochromes P450 system, enhancement of peroxidase-dependent 7,8-B[a]PD-GSH conjugation by phenols suggests a possible interaction between peroxidases and cytochromes P450 systems. This interaction may contribute to the detoxication of the penultimate carcinogenic PAH-dihydrodiols and other chemicals.     

Metabolism of benzo[a]pyrene and benzo[a]pyrene-7,8-diol by human cytochrome P450 1B1

Benzo[a]pyrene (B[a]P), a ubiquitous environmental, tobacco and dietary carcinogen, has been implicated in human cancer etiology. The role of human cytochrome P450 1B1 in the metabolism of B[a]P is poorly understood. Using microsomal preparations of human P450 1A1, 1A2 and 1B1 expressed in baculovirus-infected insect cells, as well as human and rat P450 1B1 expressed in yeast, we have determined the metabolism of B[a]P, with and without the addition of exogenous epoxide hydrolase, and B[a]P-7,8-dihydrodiol (7,8-diol), each substrate at a concentration of 10 microM. HPLC analysis detected eight major metabolites of B[a]P and four metabolites of the 7,8-diol. The results of these studies indicate that cytochrome P450 1B1 carries out metabolism of B[a]P along the pathway to the postulated ultimate carcinogen, the diol epoxide 2, at rates much higher than P450 1A2 but less than P450 1A1. The rates of formation of the 7,8-diol metabolite in incubations with epoxide hydrolase are 0.17 and 0.38 nmol/min/nmol P450 for human P450 1B1 and 1A1, respectively, and undetectable for 1A2. The rates of total tetrol metabolite formation from the 7,8-diol, which are indicative of diol epoxide formation, are 0.60, 0.43 and 2.58 nmol/min/nmol P450 for 1B1, 1A2 and 1A1 respectively. In agreement with other reports of rat P450 1B1 activity, our data show this rat enzyme to be very active for B[a]P and 7,8-diol, with rates higher than human P450 1B1. In addition to the established role of P450 1A1 in B[a]P metabolism, P450 1B1 may significantly contribute to B[a]P and 7,8-diol metabolism and carcinogenesis in rodent tumor models and in humans.      

Comparative dose-response study on the pulmonary carcinogenicity of 1, 6-dinitropyrene and benzo[a]pyrene in F344 rats

The dose dependencies of the lung carcinogenicity of 1, 6-dinitropyrene(1, 6-DNP) and benzo[a]pyrene (BaP) were examined by directinjections of these compounds into rat lungs. A total of 276male F344 rats were divided into 10 groups and given variousdoses of 1, 6-DNP or BaP, or no drug (control group). Both chemicalswere injected into the lung, as suspensions in beeswax-tricaprylinand the animals were then observed for 104 weeks. The incidencesof lung cancer were 0/39 (0%), 4/30 (13%), 13/31 (42%), 22/26(85%) and 6/9 (67%) in groups treated with 0.003, 0.01, 0.03,0.1 and 0.15 mg of 1, 6-DNP respectively, and 1/29 (3%), 7/30(23%) 22/29 (76%) and 9/13 (69%) in those treated with 0.03,0.1, 0.3 and 1.0 mg of BaP respectively. No lung cancer wasfound in control rats. Thus the incidences of lung cancer inducedby 1, 6-DNP and BaP showed significant dose dependence. At equaldoses, the incidence of lung cancer was much higher with 1,6-DNP than with BaP, and the induction of cancer by 1, 6-DNPwas higher even at one-third the dose of BaP. Histologically,most tumours induced by 1, 6-DNP were undifferentiated neoplasms,whereas most of those induced by BaP were well-differentiatedsquamous cell carcinomas.     

Binding of benzo[a]pyrene metabolites in the rat intestinal lumen by magnetic polyethyleneimine microcapsules following an intragastric dose of [14C]benzo[a]pyrene

Semi-permeable magnetic polyethyleneimine (PEI) microcapsuleshave been developed to trap carcinogens and their metabolitesin vivo and their time-dependent binding of a model carcinogen,[¹⁴C]benzo[a]pyrene ([¹⁴C]BaP), is studied within the intestinallumen. Overall, 0.5% of an intragastrk BaP dose was bound bythese microcapsules recovered from faeces with specific bindingof metabolites (nmol/10⁶ recovered microcapsules) being similarin the 0–24-h and 24–48-h periods, but 10-foldlower in the 48–72-h period. Successive extractions ofmicrocapsules with ammoniacal methanol, 2.5 N HCI, methanoland dimethylsulfoxide released 60% of bound radiolabel andthe unextracted radiolabel was presumed to have been bound covalently.By contrast, > 90% of bound radiolabel was extractable fromthe faeces of the treated animals and from microcapsules treatedin vitro with [¹⁴C]7,8-dihydroxy-9,10-epoxytetrahydrobenzo[a]-pyrene(BaPDE), indicating that the in vivo microcapsule- bound metaboliteswere not derived either from adsorbed faecal material or from[¹⁴C]BaPDE formed in situ. A time-dependent appearance of BaP3,6-dione was found. Also the qualitative and quantitative patternsof metabolites trapped by microcapsules, as assayed by h.p.l.c,were consistent only with a unique set of BaP metabolites beingbound within the intestinal lumen. Hence these carcinogen-bindingmicrocapsules can be used to investigate the in situ formationof carcinogen metabolites within the intestinal tract.     

Sodium nitrite-stimulated metabolic activation of benzo[a]pyrene 7,8-dihydrodiol in human polymorphonuclear leukocytes

Sodium nitrite was shown to enhance the metabolism of trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol) to 7/8,9,10- and 7,10/8,9-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (tetraols) in phorbol myristate acetate (PMA)-stimulated polymorphonuclear leukocytes (PMNs). The production of these tetraols implicates the intermediate formation of the corresponding trans-7,8-dihydroxy-9,10-epoxy-7,8-9,10-tetrahydrobenzo[a]pyrene (anti-BPDE). A 2- to 3-fold increase in the tetraol yield was observed in the presence of nitrite in excess of 1 mM. Sodium azide, an inhibitor of myeloperoxidase and catalase, reduced the nitrite-stimulated metabolism of BP-7,8-diol in PMA-activated leukocytes. Diphenylene iodonium sulphate, a NADPH-oxidase inhibitor, lowered the production of tetraols in PMA-stimulated leukocytes both in the absence and presence of nitrite. Additionally, nitrite markedly enhanced the covalent binding of metabolites derived from [3H](-)-BP-7,8-diol to leukocyte proteins as well as to DNA present extracellularly. The nitrite-stimulated covalent binding to both proteins and DNA was inhibited by the presence of sodium azide. The mechanism underlying the effect of nitrite on the metabolism of BP-7,8-diol to reactive intermediates in PMA-activated human polymorphonuclear leukocytes is not known. However, the results are compatible with a peroxidase-dependent mechanism although other possible pathways may contribute to the enhanced rate of metabolism.     

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 theradioactivity being recovered in the faeces. Excretion was rapid:within 24 h post-injection, 45% of the applied dose was recoveredin the faeces. H.p.l.c. analysis of radioactive material extractedfrom the faeces by methanol showed that it contained a singlecomponent which co-chromatographed with [³H]BPDE-dGuo and whichwas not affected by treatment with alkaline phosphatase, arylsulphatase or ß-glucuronidase. To determine if thisphenomenon occurs after topical application of BP to a targettissue, such as mouse skin, animals were treated with [³H]BPand their faeces collected. After an extensive extraction procedureinvolving differential solubility in organic solvents, SephadexLH-20 chromatography and h.p.l.c, a product was isolated frommice faeces which had characteristics consistent with a [³H]BPDE-dGuoadduct. These findings are discussed in relation to detectionof BPDE adducts in human populations.     

Synthesis of a novel fluorinated benzo[a]pyrene: 4,5-difluorobenzo[a]pyrene

The synthesis of 4,5-difluorobenzo[a]pyrene, as a fluorinated probe to investigate the involvement of the K-region in the further metabolic activation of benzo[a]pyrene metabolites, is described. Benzo[a]pyrene-4,5-dione obtained from 2,3-dichloro-5,6-dicyano-1,4-benzoquinone oxidation of cis-4,5-dihydro-4,5-dihydroxybenzo[a]pyrene was fluorinated with dimethylaminosulfur trifluoride to give 4H,5H,4,4,5,5,-tetra-fluorobenzo[a]pyrene. Defluorination using lithium aluminum hydride in tetrahydrofuran gave 4,5,-difluorobenzo[a]pyrene.     

Transport of DNA-adducting metabolites in mouse serum following benzo [a]pyrene administration

Metabolic activation of benzo[]pyrene (BaP) by cellular enzymesis required for DNA adduct formation. In vivo DNA adducts mightalso arise from BaP metabolites supplied via the systemic circulation,rather than from in situ activation. We determined whether electrophilicmetabolites could be detected in mouse serum 4 h after BaP dosing(i.p.) by trapping metabolites with salmon sperm DNA (ssDNA),followed by ³²P-postlabeling analysis for DNA adducts. In vitrostudies demonstrated that mouse serum sequesters BaP-7,8-diol-9,10-epoxide(BPDE) and protects it from hydrolysis. BPDE was rapidly transferredfrom serum to ssDNA or splenocytes, with adduct levels in ssDNA4- to 7-fold greater than in splenocytes. After BaP administration,mouse serum produced two adduct spots when incubated with ssDNA.The major adduct (spot 3) co-chromatographed with a BPDE adductstandard, while the minor adduct (spot 2) was unrelated to BPDE.A BPDE standard curve in control serum was developed to quantitateBPDE levels in dosed serum. These levels ranged from 13.1 to19.1 nM. Tissue DNA contained three adduct spots: spots 2 and3 appeared identical to the respective adducts arising fromdosed serum. BPDE-DNA adducts in tisues were highest in liver,lung and spleen, with kidney and stomach levels significantlylower. Levels of adduct 2 did not correlate with levels of adduct3, especially in spleen where the adduct 2/adduct 3 ratio wasvery low. In vitro studies in which splenocytes were presentedwith both adducting metabolites suggested that splenocytes preferentiallyform adduct 3. These results indicate that two of the threeBaP electrophilic metabolites responsible for cellular DNA damageare present in mouse serum. The levels of BPDE in serum maybe sufficient to account for a substantial portion of the tissueload of BPDE-DNA adducts.