Metabolism and binding of benzo[a]pyrene in randomly-proliferating,confluent and s-phase human skin fibroblasts

The metabolism of benzo[a]pyrene in randomly proliferating and confluent cultures of human skin fibroblast cells was compared with cell cultures in early S phase of the cell cycle after a G1 block. When each cell population was exposed to [G-³H]benzo[a]pyrene for 24 hours and the organic soluble metabolites in the extracellular medium and intracellular components were analyzed by HPLC, a quantitative increase in metabolism was observed in the confluent cell populations. The amount of organic soluble metabolites in the extracellular medium of the confluent dense cultures was 2.7 times the amount found in randomly proliferating cultures and 1.5 times that of the synchronized cultures. The trans-7,8- and 9,10 dihydrodiols and 3-hydroxy benzo[a]pyrene were the major metabolites formed. Small amounts of the sulphate conjugate, 9-hydroxy-benzo[a]pyrene and the tetrols were also detected. Cytoplasmic as well as nuclear extracts from the confluent cell cultures also contained higher amounts of metabolites compared to those from the randomly proliferating and S-phase cells. The levels of DNA modification by metabolically activated benzo[a]pyrene did not differ among the randomly proliferating, confluent and S-phase cells. However, the S-phase cells exhibited approximately 50-fold increase in the frequency of transformation compared to the randomly proliferating cells. Confluent cells were not transformed by benzo[a]pyrene. These data suggest that factors other than random modification of DNA by the carcinogen might have a significant role in the expression of a transformed phenotype and that metabolism and transformation are not directly related. Furthermore, confluent dense cultures with a heightened capability for metabolism of benzo[a]pyrene were more active in the detoxification of benzo[a]pyrene than in the production of the metabolites associated with cellular transformation.Abbreviations BaP benzo[a]pyrene - BaP-4,5-diol trans-4,5 dihydroxy-4,5-dihydrobenzo[a]pyrene - BaP-7,8-diol trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene - Bap-9,10-diol trans-9,10-dihydroxy-9,10 dihydrobenzo[a]pyrene - CM complete medium - HNF human neonatal foreskin - HPLC high pressure liquid chromatography - PAH polycyclic aromatic hydrocarbon - PDL population doubling - RP randomly proliferating     

Pyrene acts as a cocarcinogen with the carcinogens benzo [a] pyrene, β-propiolactone and radiation in the induction of malignant transformation in cultured mouse fibroblasts; soybean extract containing the Bowman-Birk inhibitor acts as an anticarcinogen

Pyrene was found to act as a cocarcinogen in the induction of transformation of cultured Balb/c3T3 cells by three different types of carcinogens: a direct acting chemical carcinogen, -propiolactone, a chemical carcinogen requiring metabolic activation, benzo[a]pyrene, and a physical carcinogen (⁶⁰Co) gamma radiation. Since pyrene enhanced transformation in vitro by approximately the same amount for all the carcinogens tested, these results suggest that the carcinogenic action of pyrene is not related to carcinogen metabolism or uptake in vitro. An extract of soybeans containing the Bowman-Birk protease inhibitor was shown to reduce transformation induced by -propiolactone, benzo[a]pyrene and -rays, both with and without the cocarcinogenic effect of pyrene, to background levels; the magnitude of the reduction in transformation by the protease inhibitor preparation was unrelated to the concentration of carcinogen. Neither the mechanism for the cocarcinogenic action of pyrene nor the anticarcinogenic effect of the soybean extract is known, but several hypotheses are discussed.Abbreviations BaP benzo[a]pyrene - BBI Bowman-Birk inhibitor - BPL -propiolactone     

Induction of anchorage-independent growth in human diploid fibroblasts by the cyclopenta-polycyclic aromatic hydrocarbon, benz[l]aceanthrylene

Cyclopenta-fused polycyclic aromatic hydrocarbons are a class of environmental PAH that have been recently identified. Many of these chemicals have been found to be more active than benzo[a]pyrene in tests for genetic toxicity using bacterial and rodent cells. Benz[l]aceanthrylene, a cyclopenta-polycyclic aromatic hydrocarbon related to benz[a]anthracene, and benzo[a]pyrene were compared for their activity to induce cytotoxicity and anchorage-independent growth with normal human diploid fibroblasts. Both benz[l]aceanthrylene and benzo[a]pyrene were relatively non-cytotoxic to normal human diploid fibroblasts. However, benz[l]aceanthrylene was twice as active compared to benzo[a]pyrene over the concentration range examined as an inducer of anchorage-independent growth. The ability of benz[l]aceanthrylene to induce anchorage-independent colony growth in normal human cells, in combination with its demonstrated ability as a mouse-skin tumorigen, suggests this PAH to be a potential multi-species carcinogen.     

Repair of daughter strand gaps in nascent DNA from mouse epidermal cells treated with dihydrodiol epoxide derivatives of benzo[a]pyrene

Alkaline sucrose gradient analysis of [methyl-3H]thymidine-pulse-labeled DNA was used to study the effect of (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (benzo[a]pyrene-diol epoxide I), a potent mutagen and carcinogen, and (+/-)-7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (benzo[a]pyrene-diol epoxide II), a weaker mutagen and carcinogen, on the size of newly synthesized DNA in primary cultures of mouse epidermal cells. Both isomers caused a dose-dependent decrease in the size of newly synthesized DNA and in the rate of [methyl-3H]thymidine incorporation into DNA. When the pulse time was increased in the treated cells so that the amount of [methyl-3H]thymidine incorporation was equal to the control, newly synthesized DNA from exposed cells was still considerably smaller than DNA from control cells. The low molecular weight of the nascent DNA from treated cells was consistent with, but not indicative of, the presence of gaps in the nascent DNA from the treated cells. Evidence of gapped DNA synthesis was obtained by treatment of extracted DNA with a single-strand specific endonuclease from Neurospora crassa. The endonuclease treatment did not significantly alter the profile of [methyl-3H]thymidine prelabeled DNA from benzo[a]pyrene-diol epoxide-treated cultures but did introduce double-stand breaks in pulse-labeled DNA from treated cultures. The numbers of [14C]benzo[a]pyrene-diol epoxide I or [3H]benzo[a]pyrenediol epoxide II-DNA-bound adducts and daughter strand gaps were compared at several dose levels. Treatment with either isomer yielded one gap in the nascent DNA/DNA-bound adduct. Pulse-chase experiments showed that gaps in the nascent DNA were closed with time.     

Oxidation of Anthracene and Benzo[a]pyrene by Laccases from Trametes versicolor

The in vitro oxidation of the two polycyclic aromatic hydrocarbons anthracene and benzo[a]pyrene, which have ionization potentials of <=7.45 eV, is catalyzed by laccases from Trametes versicolor. Crude laccase preparations were able to oxidize both anthracene and the potent carcinogen benzo[a]pyrene. Oxidation of benzo[a]pyrene was enhanced by the addition of the cooxidant 2,2(prm1)-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS), while an increased anthracene oxidizing ability was observed in the presence of the low-molecular-weight culture fluid ultrafiltrate. Two purified laccase isozymes from T. versicolor were found to have similar oxidative activities towards anthracene and benzo[a]pyrene. Oxidation of anthracene by the purified isozymes was enhanced in the presence of ABTS, while ABTS was essential for the oxidation of benzo[a]pyrene. In all cases anthraquinone was identified as the major end product of anthracene oxidation. These findings indicate that laccases may have a role in the oxidation of polycyclic aromatic hydrocarbons by white rot fungi.     

Importance of the route of administration for genetic differences in benzo[a]pyrene-induced in utero toxicity and teratogenicity

C57BL/6N (Ahb/Ahb) mice have a high-affinity Ah receptor in tissues, whereas AKR/J and DBA/2N (Ahd/Ahd) mice have a poor-affinity Ah receptor. The cytochrome P1-450 induction response (enhanced benzo[a]pyrene metabolism) occurs much more readily in Ahb/Ahb and Ahb/Ahd than in Ahd/Ahd mice, at any given dose of the inducer benzo[a]pyrene. Embryos from the AKR/J X (C57BL/6N)(AKR/J)F1 and the reciprocal backcross were studied during benzo[a]pyrene feeding of the pregnant females. Oral benzo[a]pyrene (120 mg/kg/day) given to pregnant Ahd/Ahd mice between gestational day 2 and 10 produces more intrauterine toxicity and malformations in Ahd/Ahd than Ahb/Ahd embryos. This striking allelic difference is not seen in pregnant Ahb/Ahd mice receiving oral benzo[a]pyrene. Pharmacokinetics studies with [3H]benzo[a]pyrene in the diet and high-performance liquid chromatographic analysis of benzo[a]pyrene metabolism in vitro by the maternal intestine, liver, and ovary and the embryos of control and oral benzo[a]pyrene-treated pregnant females are consistent with "first-pass elimination" kinetics and differences in benzo[a]pyrene metabolism by the embryos and/or placentas versus maternal tissues. In the pregnant Ahd/Ahd mouse receiving oral benzo[a]pyrene, little induction of benzo[a]pyrene metabolism occurs in her intestine and liver; this leads to much larger amounts of benzo[a]pyrene reaching her embryos, and genetic differences in toxicity and teratogenesis are manifest. In the pregnant Ahb/Ahd mouse receiving oral benzo[a]pyrene, benzo[a]pyrene metabolism is greatly enhanced in her intestine and liver; this leads to less benzo[a]pyrene reaching her embryos, much less intrauterine toxicity and malformations, and no genetic differences are manifest. More toxic metabolites (especially benzo[a]pyrene 1,6- and 3,6-quinones) are shown to occur in Ahd/Ahd embryos than in Ahb/Ahd embryos. In additional studies, no prenatal or neonatal "imprinting" effect in C57BL/6N mice by 2,3,7,8-tetrachlorodibenzo-p-dioxin or Aroclor 1254 on benzo[a]pyrene metabolism later in life was detectable. These genetic differences in intrauterine toxicity and teratogenicity induced by oral benzo[a]pyrene are just opposite those induced by intraperitoneal benzo[a]pyrene [Shum et al., '79; Hoshino et al., '81). The data in the present report emphasize the importance of the route of administration when the teratogen induces its own metabolism.     

Specificity in the activation and inhibition by flavonoids of benzo[a]pyrene hydroxylation by cytochrome P-450 isozymes from rabbit liver microsomes

The effect of flavone and 7,8-benzoflavone on the metabolism of benzo[a]pyrene to fluorescent phenols by five cytochrome P-450 isozymes obtained from rabbit liver microsomes was determined. Benzo[a]pyrene metabolism was stimulated more than 5-fold by the addition of 600 microM flavone to a reconstituted monooxygenase system consisting of NADPH, cytochrome P-450 reductase, dilauroylphosphatidylcholine, and cytochrome P-450LM3c or cytochrome P-450LM4. In contrast, an inhibitory effect of flavone on benzo[a]pyrene metabolism was observed when cytochrome P-450LM2, cytochrome P-450LM3b, or cytochrome P-450LM6 was used in the reconstituted system. 7,8-Benzoflavone (50-100 microM) stimulated benzo[a]pyrene metabolism by the reconstituted monooxygenase system about 10-fold when cytochrome P-450LM3c was used, but benzo[a]pyrene hydroxylation was strongly inhibited when 7,8-benzoflavone was added to the cytochrome P-450LM6-dependent system. Smaller effects of 7,8-benzoflavone were observed on the metabolism of benzo[a]pyrene by the cytochrome P-450LM2-, cytochrome P-450LM3b-, and cytochrome P-450LM4-dependent monooxygenase systems. These results demonstrate that the activating and inhibiting effects of flavone and 7,8-benzoflavone on benzo[a]pyrene metabolism depend on the type of cytochrome P-450 used in the reconstituted monooxygenase system.     

Formation of radical cations in a model for the metabolism of aromatic hydrocarbons

To test the hypothesis that electrophilic radical cations are the major ultimate electrophilic and carcinogenic forms of benz[a]anthracene (BA), dibenz[a,h]anthracene (DBA), and benzo[a]pyrene (BP), we have focused on a chemical model of metabolism which parallels and duplicates known or potential metabolites of some polycyclic hydrocarbons formed in cells. Studies of this model system show that radical cations are hardly formed, if at all, in the case of BA or DBA but are definitely formed in the cases of the carcinogen BP as well as the non-carcinogenic hydrocarbons, pyrene and perylene. We conclude that the carcinogenicities of BA, DBA, BP, pyrene, and perylene are independent of one-electron oxidation to radical cation intermediates.     

Dual-label high-performance liquid chromatographic assay for femtomole levels of benzo[a]pyrene metabolites

A dual-label HPLC assay to measure femtomole quantities of ethyl acetate-extractable [3H]benzo[a]pyrene metabolites was developed. 14C-labeled metabolites of benzo[a]pyrene formed by rat liver 9000g supernatant were used as both internal standards and chromatographic markers. The percentage deviation between assays was determined to be between 11 and 13% for 9,10-dihydro-9,10-dihydroxybenzo[a]pyrene, 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene, benzo[a]pyrene-3,6-quinone, benzo[a]pyrene-1,6-quinone, and 9-hydroxybenzo[a]pyrene, 22% for 4,5-dihydro-4,5-dihydroxybenzo[a]pyrene, and less than 5% for 3-hydroxybenzo[a]pyrene. The detection limit of this assay was between 3 and 10 fmol per metabolite. The application of this technique to the metabolism of [3H]benzo[a]pyrene by microsomes of hamster and human oral cavity tissue is described.     

Formation of sulfate and glucoside conjugates of benzo[e]pyrene by Cunninghamella elegans

 Benzo[e]pyrene is a pentacyclic aromatic hydrocarbon, which, unlike its structural isomer benzo[a]pyrene, is not a potent carcinogen or mutagen. The metabolism of benzo[e]pyrene was studied using the filamentous fungus Cunninghamella elegans ATCC 36112. C. elegans metabolized 65% of the [9, 10, 11, 12-³H]benzo[e]pyrene and unlabeled benzo[e]pyrene added to Sabouraud dextrose broth cultures after 120 h of incubation. Three major metabolites of benzo[e]pyrene were separated by reversed-phase high-performance liquid chromatography. These metabolites were identified by ¹H and ¹³C NMR, UV-visible, and mass spectral analyses as 3-benzo[e]pyrenylsulfate, 10-hydroxy-3-benzo[e]pyrenyl sulfate, and benzo[e]pyrene 3-O-β-glucopyranoside. Received: 7 September 1995/Received revision: 14 November 1995/Accepted: 11 December 1995     

The in vitro metabolism of benzo[a]pyrene by polychlorinated and polybrominated biphenyl induced rat hepatic microsomal monooxygenases

The metabolism of benzo[a]pyrene by halogenated biphenyl-induced rat hepatic microsomal monooxygenases was determined using a high pressure liquid chromatographic assay system. Incubation of benzo[a]pyrene with microsomes from rats pretreated with phenobarbitone or phenobarbitone-type inducers (2,2',4,4',5,5'-hexachlorobiphenyl, 2,2',4,4',6,6'-hexachlorobiphenyl, 2,2',5,5'-tetrachlorobiphenyl, 2,2',4,4',5,5'-hexabromobiphenyl, and 2,2',5,5'-tetrabromobiphenyl) resulted in increased overall metabolism of the hydrocarbon (less than fourfold) into phenolic, quinone, and diol metabolites, with the most striking increase observed in the formation of 4,5-dihydro-4,5-dihydroxybenzo[a]pyrene. In contrast, the metabolism of benzo[a]pyrene by microsomes from rats induced with 3-methylcholanthrene or 3,3',4,4'-tetrachlorobiphenyl resulted in a greater than 10-fold increase in overall benzo[a]pyrene metabolism, with the largest increases observed in the formation of the trans-7,8- and -9,10-dihydrodiol metabolites of benzo[a]pyrene. However, in comparison to control and phenobarbitone-induced microsomes, the oxidative conversion of benzo[a]pyrene by microsomes induced with 3-methylcholanthrene and 3,3',4,4'-tetrachlorobiphenyl into the 6,12-quinone was substantially inhibited. Previous reports have shown that the commercial halogenated biphenyl mixtures, fireMaster BP-6, and Aroclor 1254 are mixed-type inducers and that microsomes from rats pretreated with these mixtures markedly enhance the overall metabolism of benzo[a]pyrene. Not surprisingly, the metabolism of benzo[a]pyrene by microsomes from rats pretreated with the mixed-type inducers, 2,3,3',4,4'-penta-,2,3,3',4,4',5-hexa-, and 2',3,3',4,4',5-hexa- chlorobiphenyl was also increased and the metabolic profile was similar to that observed with fireMaster BP-6 and Aroclor 1254 induced microsomes.     

Regio- and stereospecificity of homogeneous 3 alpha-hydroxysteroid-dihydrodiol dehydrogenase for trans-dihydrodiol metabolites of polycyclic aromatic hydrocarbons

The homogeneous 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) of rat liver cytosol is indistinguishable from dihydrodiol dehydrogenase (trans-1,2-dihydrobenzene-1,2-diol dehydrogenase EC 1.3.1.20), Penning, T. M., Mukharji, I., Barrows, S., and Talalay, P. (1984) Biochem. J. 222, 601-611). Examination of the substrate specificity of the purified dehydrogenase for trans-dihydrodiol metabolites of polycyclic aromatic hydrocarbons indicates that the enzyme will catalyze the NAD(P)-dependent oxidation of trans-dihydrodiols of benzene, naphthalene, phenanthrene, chrysene, 5-methylchrysene, and benzo[a]pyrene under physiological conditions. Comparison of the utilization ratios Vmax/Km indicates that benzenedihydrodiol and the trans-1,2- and trans-7,8-dihydrodiols of 5-methylchrysene were most efficiently oxidized by the purified dehydrogenase, followed by the trans-7,8-dihydrodiol of benzo[a]pyrene and the trans-1,2-dihydrodiols of phenanthrene, chrysene, and naphthalene. The purified enzyme appears to display rigid regio-selectivity, since it will readily oxidize non-K-region trans-dihydrodiols but will not oxidize the K-region trans-dihydrodiols of phenanthrene and benzo[a]pyrene. The stereochemical course of enzymatic dehydrogenation was investigated by circular dichroism spectrometry. For the trans-1,2-dihydrodiols of benzene, naphthalene, phenanthrene, chrysene, and 5-methylchrysene, the dehydrogenase preferentially oxidized the (+)-[S,S]-isomer. Apparent inversion of this stereochemical preference occurred with the trans-7,8-dihydrodiol of 5-methylchrysene, as the (-)-enantiomer was preferentially oxidized. No change in the sign of the Cotton Effect was observed following oxidation of the racemic trans-7,8-dihydrodiol of benzo[a]pyrene, suggesting that both stereoisomers of this compound were substrates. Large-scale incubation of the [3H]-(+/-)-trans-7,8-dihydrodiol of benzo[a]pyrene with the purified dehydrogenase resulted in greater than 90% utilization of this potent proximate carcinogen, suggesting that the enzyme utilizes both the (-)-[R,R] and the (+)-[S,S]-stereoisomers, which confirms the circular dichroism result. These data show that dihydrodiol dehydrogenase displays the appropriate regio- and stereospecificity to catalyze the oxidation of both the major and minor non-K-region trans-dihydrodiols that arise from the microsomal metabolism of benzo[a]pyrene in vivo.     

Degradation of benzo[a]pyrene by Mycobacterium vanbaalenii PYR-1

Metabolism of the environmental pollutant benzo[a]pyrene in the bacterium Mycobacterium vanbaalenii PYR-1 was examined. This organism initially oxidized benzo[a]pyrene with dioxygenases and monooxygenases at C-4,5, C-9,10, and C-11,12. The metabolites were separated by reversed-phase high-performance liquid chromatography (HPLC) and characterized by UV-visible, mass, nuclear magnetic resonance, and circular dichroism spectral analyses. The major intermediates of benzo[a]pyrene metabolism that had accumulated in the culture media after 96 h of incubation were cis-4,5-dihydro-4,5-dihydroxybenzo[a]pyrene (benzo[a]pyrene cis-4,5-dihydrodiol), cis-11,12-dihydro-11,12-dihydroxybenzo[a]pyrene (benzo[a]pyrene cis-11,12-dihydrodiol), trans-11,12-dihydro-11,12-dihydroxybenzo[a]pyrene (benzo[a]pyrene trans-11,12-dihydrodiol), 10-oxabenzo[def]chrysen-9-one, and hydroxymethoxy and dimethoxy derivatives of benzo[a]pyrene. The ortho-ring fission products 4-formylchrysene-5-carboxylic acid and 4,5-chrysene-dicarboxylic acid and a monocarboxylated chrysene product were formed when replacement culture experiments were conducted with benzo[a]pyrene cis-4,5-dihydrodiol. Chiral stationary-phase HPLC analysis of the dihydrodiols indicated that benzo[a]pyrene cis-4,5-dihydrodiol had 30% 4S,5R and 70% 4R,5S absolute stereochemistry. Benzo[a]pyrene cis-11,12-dihydrodiol adopted an 11S,12R conformation with 100% optical purity. The enantiomeric composition of benzo[a]pyrene trans-11,12-dihydrodiol was an equal mixture of 11S,12S and 11R,12R molecules. The results of this study, in conjunction with those of previously reported studies, extend the pathways proposed for the bacterial metabolism of benzo[a]pyrene. Our study also provides evidence of the stereo- and regioselectivity of the oxygenases that catalyze the metabolism of benzo[a]pyrene in M. vanbaalenii PYR-1.     

Carcinogenicity and metabolic profiles of 6-substituted benzo[a]pyrene derivatives on mouse skin

The ability was tested of appropriate substituents of benzo[a]pyrene (BP) at C-6 to decrease or suppress the carcinogenic activity for these BP derivatives relative to the parent compound. 8-week-old female Swiss mice in 9 groups of 30 were treated on the back with 0.2 mumol of compound in acetone 4 times weekly for 20 weeks. The following compounds were administered: BP, 6-methylbenzo[a]pyrene (BP-6-CH3), 6-hydroxymethylbenzo[a]pyrene (BP-6-CH2OH), benzo[a]pyrene-6-carboxaldehyde (BP-6-CHO), benzo[a]pyrene-6-carboxylic acid, 6-methoxybenzo[a]pyrene, 6-acetoxybenzo[a]pyrene, 6-bromobenzo[a]pyrene, and 6-iodobenzo[a]pyrene. Two additional groups received BP or BP-6-CH3 twice weekly for 20 weeks at a total dose 25% of that above. In addition, the metabolism of selected 6-substituted BP derivatives was studied, using mouse skin homogenates in vitro and mouse skin in vivo. Only four compounds were carcinogenic; the order of potency was BP greater than BP-6-CH3 greater than BP-6-CH2OH and BP-6-CHO. The difference in carcinogenicity between BP-6-CH2OH and BP-6-CHO could not be assessed by this experiment. In a further tumorigenesis experiment the carcinogenicity of BP-6-CH2OH was compared to that of BP-6 CHO, BP-6-CH3 and 6-hydroxymethylbenzo[a]pyrere sulfate ester (BP-6-CH2OSO3Na) on mouse skin. 9-week-old female Swiss mice in groups of 28 were treated at three dose levels with 0.8, 0.2 and 0.05 mumol of compounds in dioxane--dimethyl sulfoxide (75 : 25) twice weekly for 40 weeks. After 40 experimental weeks BP-6-CH2OSO3Na proved to be a more potent carcinogen than BP-6-CH2OH, which, in turn was more active than BP-6-CHO. The greater carcinogenicity of BP-6-CH3 relative to BP-6-CH2OH and BP-6-CHO is confirmed, suggesting that BP-6-CH2OH is not a proximate carcinogenic metabolite for BP-6-CH3. Since BP-6-CHO is a weaker carcinogen than BP-6-CH2OH and is efficiently reduced metabolically to BP-6-CH2OH, the latter compound may be a common proximal carcinogenic metabolite. The stronger potency of BP-6-CH2OSO3Na, compared to its alcohol, suggests that an ester of BP-6-CH2OH might be the ultimate alkylating compound reacting with cellular nucleophiles.     

Caffeine enhancement of the initiation of DNA replication in benzo[a]pyrene diol epoxide damaged cells

We have used a newly developed pH stepwise alkaline elution method to show that caffeine enhances DNA initiation (DNA replication in sub-replicon size nascent strands) in (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene (BPDEI) damaged mouse primary epidermal cells. Caffeine alone caused a dose-dependent increase in DNA initiation without an effect on DNA elongation (joining of replicon-sized nascent DNA). BPDEI alone inhibited DNA elongation as shown by a relative increase in sub-replicon size nascent DNA. When BPDEI treated cells were incubated with caffeine, there was a dose-dependent increase in sub-replicon size nascent DNA without a significant effect on the proportion of joined replicons. Therefore, caffeine can enhance DNA initiation in mammalian cells damaged with a reactive form of the carcinogen benzo[a]pyrene and this may account for the biological interaction between caffeine and the ultimate carcinogenic form of benzo[a]pyrene.     

Active transport of benzo[a]pyrene in apical membrane vesicles from normal human intestinal epithelium

Transport of the carcinogen benzo[a]pyrene in apical membrane vesicles (AMV) from normal human intestine, was investigated. Benzo[a]pyrene transport was found in AMV throughout the small intestine, but was greatest in colon. Evidence suggesting involvement of P-Glycoprotein (P-Gp), included (1) comparable transport of P-Gp substrate doxorubicin, (2) transport stimulation by ATP and (3) transport suppression by the P-Gp inhibitor, verapamil.     

Metabolism of benzo(a)pyrene,dimethylbenzanthracene and aflatoxin B1 by camel liver microsomes

The ability of camel liver microsomes to metabolise a range of common environmental carcinogens including benzo(a)pyrene, dimethylbenzanthracene and aflatoxin B1 has been investigated. The camel liver has shown the ability to metabolise benzo(a)pyrene, dimethylbenzanthracene and aflatoxin B1 to a number of metabolites. The major metabolites of benzo(a)pyrene produced by camel liver enzymes were identified as its mono-hydroxy derivatives and suggest that the metabolic detoxification pathways of carcinogen metabolism are predominant in this species. Benzo(a)pyrene metabolising activity in camel liver required NADPH and was inhibited by CO and alpha-naphthoflavone suggesting the involvement of cytochrome P450 in the metabolism of this carcinogen by camel liver. The cytochrome P450-dependent metabolism of carcinogen and other specific substrates such as ethoxyresorufin and ethoxycoumarin, by camel liver enzymes, was about 50% higher than that of rat liver enzymes. The cytochrome P450-dependent metabolism of a variety of carcinogenic and other substrates by camel liver demonstrated that there are multiple forms of cytochrome P450 enzymes involved in the metabolism of a wide array of xenobiotics and pollutants.     

Cellular uptake and intracellular localization of benzo(a)pyrene by digital fluorescence imaging microscopy

Uptake of benzo(a)pyrene by living cultured cells has been visualized in real time using digital fluorescence-imaging microscopy. Benzo(a)pyrene was noncovalently associated with lipoproteins, as a physiologic mode of presentation of the carcinogen to cells. When incubated with either human fibroblasts or murine P388D1 macrophages, benzo(a)pyrene uptake occurred in the absence of endocytosis, with a halftime of approximately 2 min, irrespective of the identity of the delivery vehicles, which were high density lipoproteins, low density lipoproteins, very low density lipoproteins, and 1-palmitoyl-2-oleoylphosphatidylcholine single-walled vesicles. Thus, cellular uptake of benzo(a)pyrene from these hydrophobic donors occurs by spontaneous transfer through the aqueous phase. Moreover, the rate constant for uptake, the extent of uptake, and the intracellular localization of benzo(a)pyrene were identical for both living and fixed cells. Similar rate constants for benzo(a)pyrene efflux from cells to extracellular lipoproteins suggests the involvement of the plasma membrane in the rate-limiting step. The intracellular location of benzo(a)pyrene at equilibrium was coincident with a fluorescent cholesterol analog, N-(7-nitrobenz-2-oxa-1,3-diazole)-23,24-dinor-5-cholen-22-amine-3 beta-ol. Benzo(a)pyrene did not accumulate in acidic compartments, based on acridine orange fluorescence, or in mitochondria, based on rhodamine-123 fluorescence. When the intracellular lipid volume of isolated mouse peritoneal macrophages was increased by prior incubation of these cells with either acetylated low density lipoproteins or with very low density lipoproteins from a hypertriglyceridemic individual, cellular accumulation of benzo(a)pyrene increased proportionately with increased [1-14C]oleate incorporation into cellular triglycerides and cholesteryl esters. Thus, benzo(a)pyrene uptake by cells is a simple partitioning phenomenon, controlled by the relative lipid volumes of extracellular donor lipoproteins and of cells, and does not involve lipoprotein endocytosis as an obligatory step.     

Differential response to adrenocorticotropic hormone analogs of bovine adrenal plasma membranes and cells.

The ability of three analogs of ACTH1-24 ([Gln5, Phe9] ACTH1-24, [Gln5, Ala9[Acth1-24, and [Gln5, Lys8, Phe9[ ACTH1-24) embodying tryptophan substitutions to activate the adenylate cyclase system of a bovine adrenal plasma membrane preparation was compared to the effect of the analogs on adenosine 3':5'-monophosphate (cyclic AMP) accumulation and steroidogenesis in viable bovine adrenocortical cells. The results were not comparable. Whereas the analogs antagonized the ACTH1-24-activated membrane cyclase they stimulated cyclic AMP accumulation as well as steroid production of the cells. None of the analogs inhibited steroidogenesis of ACTH1-24-stimulated cells, but two of them, at very high dose levels, inhibited cyclic AMP production. The ability of the analogs to stimulate steroidogenesis of the adrenal cells half-maximally decreased in the order tryptophan greater than phenylalanine greater than alanine, indicating that the aromaticity of the indole ring of tryptophan is necessary for maximal interaction between hormone and receptor. Both the absolute and relative steroidogenic potencies were the same for several analogs when assayed with rat adrenal cells. Although only a small fraction of the cell's potential to produce cyclic AMP was necessary to induce maximum steroid production, the relative activities of a series of analogs were the same for steroidogenesis as for cyclic AMP accumulation. Furthermore, the concentration of cyclic AMP necessary for full steroidogenesis was practically identical for a series of peptides that differed widely in potency. These findings support the postulate that cyclic AMP accumulation and steroidogenesis in adrenocortical cells are coupled processes. The differential behavior of bovine adrenal plasma membranes and bovine adrenocortical cells toward ACTH analogs indicates that structure-function studies using cyclase assays may not reflect events that take place in the intact adrenal or in cell preparations derived therefrom.     

Selective oxidation of mitochondrial glutathione in cultured rat adrenal cells and its relation to polycyclic aromatic hydrocarbon-induced cytotoxicity

Primary cultures of rat adrenal cells, as well as rat adrenals in vivo, are sensitive to the potent carcinogen 7,12-dimethylbenz[a]anthracene and its liver metabolite 7-hydroxymethyl-12-methylbenz[a]anthracene, whereas unmethylated polycyclic aromatic hydrocarbons like benzo[a]pyrene or benzo[a]anthracene are ineffective. The adrenocorticolytic potencies of the hydrocarbons are affected by adrenocorticotrophic hormone and various steroids, cytochrome P450 inhibitors, and antioxidants. In the present investigation digitonin was used to fractionate cultured rat adrenal cells. It was found that the mitochondria and cytosol of the cells contained 3-5 nmol/10(6) cells (approximately 15%) and 20-30 nmol/10(6) cells (approximately 85%) of the total soluble cellular glutathione equivalents, respectively. After exposing the cells to 7-hydroxymethyl-12-methylbenz[a]anthracene in the culture medium, a time- and concentration-dependent selective oxidation of mitochondrial glutathione was observed, whereas the effect on the cytosolic glutathione was negligible. Under the same conditions, 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene were unable to alter the redox levels of the subcellular pools of glutathione. Omission of adrenocorticotrophic hormone lowered the oxidation of mitochondrial glutathione induced by 7-hydroxymethyl-12-methylbenz[a]anthracene about twofold. The results suggest that rat adrenal cells contain two separate pools of glutathione, one cytosolic and one mitochondrial, of which the latter is selectively influenced by 7-hydroxymethyl-12-methylbenz[a]anthracene. Moreover, it is concluded that rat adrenal cells offer a unique model system for general studies of the effects of a selective oxidation of mitochondrial glutathione on various cell functions. These effects may constitute early changes in cytotoxicity, preceding, e.g., membrane damage and loss of cytosolic components.