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The effects of dietary corn oil on the metabolism and activation of benzo[a]pyrene by the benzo[a]pyrene metabolizing enzymes of the mouse.

Male ICR Swiss mice, weighing 16-20 g, were fed ad libitum either a fat-free diet or a diet containing 10% corn oil. After three weeks on these diets, the rates of benzo[a]pyrene (B[a]P) metabolite formation and metabolism to products which covalently bind with macromolecules were compared using hepatic nuclei and microsomal preparations. The maximum activity of B[a]P hydroxylase in microsomes from untreated animals was increased 50% by feeding the corn oil diet, however, B[a]P hydroxylase in microsomes from 3-methylcholanthrene (3-MC)-treated mice was unaffected by diet. In animals treated with phenobarbital, B[a]P metabolism and B[a]P-DNA adduct formation were greater in microsomes from corn oil fed mice compared to those fed the fat-free diet. At a B[a]P concentration of 96 microM, microsomes from corn oil fed untreated mice produced 26% more extractable metabolites and covalent binding to exogenous DNA was increased 46%. At lower substrate concentrations (0.94-15.0 microM B[a]P), B[a]P-DNA and B[a]P-protein binding were 300-400% greater when incubated with microsomes from corn oil fed mice than when incubated with microsomes from mice fed fat-free diet. The apparent Vmax's determined for the formation of each extractable metabolite were increased 1.5-3.0 times by the corn oil diet. Hepatic nuclear B[a]P hydroxylase and nuclear activation of B[a]P to products which covalently bind to DNA in both non-induced and 3-MC-pretreated animals fed the corn oil diet were greater than that observed in animals fed the fat-free diet. B[a]P hydroxylase activities in the lungs of these animals were unaltered by diet.

Animals↗

Instability of (+/-)-7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (syn-BaPDE)-DNA adducts formed in benzo[a]pyrene-treated Wistar rat embryo cell cultures.

One of the peaks present in HPLC profiles of [3H]benzo[a]-pyrene (BaP)-deoxyribonucleosides prepared by enzymatic degradation of [3H]BaP-DNA isolated from Wistar rat embryo cell cultures exposed to [G-3H]BaP was found to be r-7,c-9,c-10,t-8-tetrahydroxy-7,8,9,10-tetrahydroBaP, a BaP-DNA adduct decomposition product (Pruess-Schwartz, D. and Baird, W.M., Cancer Res., 46, 545-552, 1986). To investigate the stability of the hydrocarbon-deoxyribonucleoside linkages in intact BaP-modified DNA, DNA was isolated from Wistar rat embryo cells that had been exposed to [G-3H]BaP and incubated in darkness at 37 degrees C at a range of pH values from 5 to 11 for 72 h or for 1-150 h at pH 7. The rate of breakdown of [3H]BaP-DNA adducts (0.25%/h) was linear over 150 h. The amounts of the two major BaP-DNA adduct decomposition products, I and II (present in a ratio of 1:3), increased with length of time of incubation. Formation of I was not affected by pH, whereas, formation of II was highest at acidic and neutral pH. Analysis of the decomposition products by immobilized boronate chromatography and reverse-phase HPLC demonstrated that both I and II contained cis-vicinal hydroxyl groups and decomposition product II cochromatographed with r-7,c-9, c-10,t-8-tetrahydroxy-7,8,9,10-tetrahydroBaP, a (+/-)-7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydroBaP (syn-BaPDE)-derived tetraol. At neutral pH [3H](+/-)-syn-BaPDE-modified calf thymus DNA formed a decomposition product identical to II. Analysis of the BaP-DNA adducts that remained covalently bound to the DNA after the above incubations demonstrated that the amounts of both major syn-BaPDE-deoxyguanosine adducts decreased with length of time of incubation. Thus, syn-BaPDE-deoxyribonucleoside adducts formed in the DNA of [3H]BaP-treated Wistar rat embryo cells are unstable and breakdown spontaneously in the absence of light to yield syn-BaPDE-tetraol decomposition products.

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

Hydroxylated benzo[a]pyrene metabolites are responsible for in vitro estrogen receptor-mediated gene expression induced by benzo[a]pyrene, but do not elicit uterotrophic effects in vivo.

The estrogenic activities of benzo[a]pyrene (B[a]P) and 10 metabolites (1, 3-, 7-, and 9-hydroxy-B[a]P; 4,5-, 7,8-, and 9,10-dihydrodihydroxy-B[a]P; and 1,6-, 3,6-, and 6,12-B[a]P-dione) were investigated. In vitro, B[a]P did not displace tritiated 17beta-estradiol ([3H]E2) from either a bacterially expressed fusion protein consisting of glutathione-S:-transferase linked to the D, E, and F domains of human ERalpha (GST-hERalphadef), or from full-length human ERbeta (hERbeta) at concentrations as high as 60 microM. However, 10 microM B[a]P demonstrated partial agonist activity in human Gal4-ERalphadef and mouse Gal4-ERbetadef reporter gene assays in transiently transfected MCF-7 cells, relative to 10 nM E2. 1-, 3-, 7-, and 9-hydroxy-B[a]P were found to bind to both receptor isoforms, each showing a higher affinity for the beta isoform. At 10 microM the four monohydroxylated metabolites were able to induce Gal4-hERalphadef- and Gal4-mERbetadef-mediated reporter gene expression to levels 20-100% of that caused by 10 nM E2, suggesting that these metabolites, and not the parent compound, induced reporter gene expression following B[a]P treatment of transiently transfected MCF-7 cells. In addition, the effect of B[a]P on two estrogen-inducible end points, uterine weight and lactoferrin mRNA levels, was determined in ovariectomized DBA/2 and C57BL/6 mice. Neither orally administered B[a]P at doses as high as 10 mg/kg body weight nor subcutaneously injected 3- or 9-hydroxy-B[a]P at doses as high as 20 mg/kg induced effects on uterine wet weight or uterine lactoferrin mRNA levels in either strain. These data suggest that B[a]P metabolites that are estrogenic at high concentrations in vitro do not induce estrogenic effects in the mouse uterus.

Animals↗

Contribution of human cytochrome P450 to benzo[a]pyrene and benzo[a]pyrene-7,8-dihydrodiol metabolism, as predicted from heterologous expression in yeast.

The metabolism of benzo[a]pyrene (B[a]P) and its proximate mutagen B[a]P-7,8-dihydrodiol (7,8-diol) was investigated in the presence of human microsomal epoxide hydrolase and P450 1A1, 1A2, 2C8, 2C9, 2C18, 2C19, 2D6 and 3A4 expressed in the yeast Saccharomyces cerevisiae. P450 1A1 had the highest turnover numbers for the formation of all B[a]P metabolites, including phenols and dihydrodiols. P450 1A2, 2C8, 2C9, 2C18, 2C19 and 3A4, which are well represented in the liver, gave rise to the formation of appreciable amounts of 3-hydroxy-B[a]P and of some dihydrodiols from B[a]P. When 7,8-diol was used as substrate, P450 1A1 also exhibited the highest turnover numbers for the formation of tetrols, the hydrolysis products of the diolepoxides, whereas P450 1A2, 2C8, 2C19 and 3A4 showed moderate activities. In order to test the validity of the yeast system, the contribution of each P450 isoform to B[a]P and 7,8-diol metabolism was evaluated as the product of the turnover numbers of recombinant P450s by specific contents of each P450 in human liver microsomes. Calculated formation rates for each B[a]P and 7,8-diol metabolite globally matched experimental values. There is evidence that P450 3A4 and 2C9 play a major role in the formation of 3-hydroxy-B[a]P from B[a]P. Accumulation of the proximate mutagen 7,8-diol was predicted to be mainly driven by P450 1A2, 2C9 and 2C19, while formation of the genotoxic diolepoxides from 7,8-diol appeared to be dependent on P450 1A2 and 3A4 in the liver.

Benzo(a)pyrene↗

A bioactive metabolite of benzo[a]pyrene, benzo[a]pyrene-7,8-dione, selectively alters microsomal Ca2+ transport and ryanodine receptor function.

Polycyclic aromatic hydrocarbons are environmental pollutants known to be carcinogenic and immunotoxic. In intact cell assays, benzo[a]pyrene (B[a]P) disrupts Ca(2+) homeostasis in both immune and nonimmune cells, but the molecular mechanism is undefined. In this study, B[a]P and five metabolites are examined for their ability to alter Ca(2+) transport across microsomal membranes. Using a well-defined model system, junctional SR vesicles from skeletal muscle, we show that a single o-quinone metabolite of B[a]P, B[a]P-7,8-dione, can account for altered Ca(2+) transport across microsomal membranes. B[a]P-7,8-dione induces net Ca(2+) release from actively loaded vesicles in a dose-, time-, and Ca(2+)-dependent manner. In the presence of 5 microM extravesicular Ca(2+), B[a]P-7,8-dione exhibited threshold and EC(50) values of 0.4 and 2 microM, respectively, and a maximal release rate of 2 micromol of Ca(2+) min(-1) mg(-1). The mechanism by which B[a]P-7,8-dione enhanced Ca(2+) efflux was further investigated by measuring macroscopic fluxes and single RyR1 channels reconstituted in bilayer lipid membranes and direct measurements of SERCA catalytic activity. B[a]P-7,8-dione (< or = 20 microM) had no measurable effect on initial rates of Ca(2+) accumulation in the presence of ruthenium red to block ryanodine receptor (RyR1), nor did it alter Ca(2+)-dependent (thapsigargin-sensitive) ATPase activity. B[a]P-7,8-dione selectively altered the function of RyR1 in a time-dependent diphasic manner, first activating then inhibiting channel activity. Considering that RyR1 and its two alternate isoforms are broadly expressed in mammalian cells and their important role in Ca(2+)-signaling, the present results reveal a mechanism by which metabolic bioactivation of B[a]P may mediate RyR dysfunction of pathophysiological significance.

Animals↗

Urinary excretion of benzo[a]pyrene metabolites following intravenous, oral, and cutaneous benzo[a]pyrene administration.

The effect of the administration route, dose, and sampling time on the total urinary excretion of four major benzo[a]pyrene (BaP) metabolites, 3-hydroxyBaP (3-OHBaP), 9-hydroxyBaP 9-hydroxyBaP (9-OHBaP), trans-4,5-dihydrodiolBaP (4,5-diolBaP), and trans-9,10-dihydrodiolBaP (9,10-diolBaP), was studied in male Sprague-Dawley rats exposed to a single intravenous, oral, and cutaneous dose of 2, 6, 20, and 60 mumol BaP/kg. Urine samples were collected at 24-h intervals following treatment. Over the 0-72 h period and for a given dose, amounts of BaP metabolites were 3-OHBaP > 4,5-diolBaP > > 9-OHBaP following intravenous and oral dosing, and 3-OHBaP > > 9-OHBaP > or = 4,5-diolBaP after cutaneous treatment. 9,10-diolBaP was barely detected. On the other hand, amounts of 3-OHBaP and 4,5-diolBaP excreted in urine over the 0-72 h period and for a given dose appeared in the following order: intravenous approximately oral > or = cutaneous. Amounts of 9-OHBaP excreted varied as follows: oral > or = cutaneous > intravenous. For all routes of administration, excretion of 4,5-diolBaP was almost complete over the 0-24 h period in contrast with 3-OHBaP and 9-OHBaP. Peak excretion of 3-OHBaP and 9-OHBaP was reached in the 0-24 h period following intravenous and oral treatment and in the 24-48 h period following cutaneous application. Overall, for a given administration route and dose, there were variations in the time profiles between metabolites. In general, there was nonetheless a good correlation between the BaP dose and urinary excretion of 3-OHBaP, 9-OHBaP, and 4,5-diolBaP. Furthermore, total urinary excretion of a specific metabolite, its time profile, and the relative proportion of the metabolites studied depended on the administration route. Data also suggest that a measure of the concentration ratio of the different metabolites could reflect the time and main route of exposure.

Administration, Cutaneous↗

Suppression of the vitro humoral immune response of mouse splenocytes by benzo(a)pyrene metabolites and inhibition of benzo(a)pyrene-induced immunosuppression by alpha-naphthoflavone.

Several investigators have demonstrated that the humoral immune response of mice and splenocyte cultures was suppressed with benzo(a)pyrene [B(a)P] exposure. The mechanism of the B(a)P immunosuppression, however, has not been established. Since reactive metabolites of B(a)P, rather than the parent compound, have been shown to mediate the carcinogenic effects of B(a)P, it was hypothesized that the immunosuppression produced by B(a)P may also be mediated by its reactive metabolites. The objective of this investigation was to examine the role of B(a)P metabolism in the B(a)P-induced suppression of the in vitro humoral immune response. This was addressed by first determining if various B(a)P metabolites are capable of inhibiting the generation of antibody-forming cells (AFC) of splenocyte cultures. Addition of B(a)P or B(a)P-7,8-diol (2 X 10(-5) M) to splenocyte cultures produced a similar dose-dependent suppression of the in vitro T-dependent AFC response to sheep red blood cells. In contrast, decreases in the AFC response and cell viability of cultures exposed to the 4,5-diol or 9,10-diol were only observed at 2 X 10(-5) M. Exposure of cultures to 3-hydroxy-B(a)P resulted in a significant decrease in the AFC response at 2 X 10(-6) and 2 X 10(-5) M. Slight decreases in the AFC response were observed with the addition of B(a)P-4,5-epoxide or B(a)P-6,12-dione at 2 X 10(-6) M, whereas a dramatic decrease in the AFC response, as well as a 45% decrease in cell viability, was obtained at 2 X 10(-5) M. The second objective was to examine the effects of the cytochrome P-450 inhibitor, alpha-naphthoflavone (ANF), on the B(a)P- and B(a)P-7,8-diol-induced suppression of the in vitro AFC response. Exposure of splenocyte cultures to 2 X 10(-5) M ANF did not affect the AFC response. Coincubation of splenocytes with ANF was observed to attenuate the suppressive effects of B(a)P and B(a)P-7,8-diol. This concentration of ANF was observed to inhibit the metabolism of [3H]B(a)P by splenocyte cultures to water soluble metabolites. Moreover, B(a)P metabolism by splenic microsomal preparations of untreated mice was inhibited by ANF. These findings suggest that the B(a)P-induced suppression of the in vitro AFC response is mediated by B(a)P metabolites generated by cytochrome P-450 present within splenocytes.

Animals↗

Benzo(a)pyrene:DNA adduct formation in early-passage Wistar rat embryo cell cultures: evidence for multiple pathways of activation of benzo(a)pyrene.

The benzo(a)pyrene (BaP):DNA adducts formed in cells are present at very low levels and are usually identified by reverse-phase high-performance liquid chromatography of tritium labeled BaP:deoxyribonucleoside adducts with known standards. To improve the identification of the BaP:DNA adducts formed, acid hydrolysis techniques were used to convert the BaP:deoxyribonucleoside adducts formed in Wistar rat embryo cell cultures to BaP:purine adducts and BaP:tetraols. Early passage Wistar rat embryo cell cultures were exposed to [3H]BaP. The BaP:deoxyribonucleoside adducts were isolated by immobilized boronate chromatography and reverse-phase high-performance liquid chromatography. Three adducts (MS1, MS2, MS3) bound to the immobilized boronate column indicating that they contained cis-vicinal hydroxyl groups, a configuration which would result from reaction of 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydroBaP (anti-BaPDE) with DNA. MS2 resulted from reaction of (+)-anti-BaPDE with deoxyguanosine (dGuo), for it cochromatographed with a [14C]-(+)-anti-BaPDE:dGuo marker at the deoxyribonucleoside level and after hydrolysis of MS2 and the marker to 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydroBaP (BaPDE):guanine and BaPDE:tetraol. MS1, an adduct that eluted in the same region as a (-)-anti-BaPDE:dGuo marker, was not formed by reaction of anti-BaPDE with DNA. Exposure of (-)-anti-BaPDE:dGuo to 0.1 N HCl for 24 h at 37 degrees C resulted in cleavage of the glycosidic bond to give an enantiomer that cochromatographed with the (+)-anti-BaPDE:dGuo hydrolysis product. Hydrolysis of MS1 under the same conditions yielded a product that eluted earlier than the hydrolysis product of anti-BaPDE:dGuo. Hydrolysis of MS1 at 80 degrees C under conditions which produce tetraols from BaPDE:deoxyribonucleoside adducts resulted in the formation of a product which did not elute with either 7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydro-BaP (syn-BaPDE) or anti-BaPDE tetraols. MS1 was also not present in cells exposed to BaP-7,8-diol or 3-hydroxyBaP. These results demonstrated that MS1 is formed by a different mechanism of activation than a simple bay-region diol-epoxide. MS3 and its hydrolysis products had chromatographic properties identical to those of r-7,c-9,c-10,t-8-tetrahydroxy-7,8,9,10-tetrahydroBaP, a tetraol formed from syn-BaPDE. MS3 appears to result from spontaneous breakdown of a syn-BaPDE:DNA adduct to give a tetraol that contains cis-vicinal hydroxyls in the 9 and 10 positions and therefore binds to an immobilized boronate column.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Potential genoprotective role for UDP-glucuronosyltransferases in chemical carcinogenesis: initiation of micronuclei by benzo(a)pyrene and benzo(e)pyrene in UDP-glucuronosyltransferase-deficient cultured rat skin fibroblasts.

UDP-glucuronosyltransferases (UGTs) are cytoprotective and may also be genoprotective. Since over 10% of the population have hereditary deficiencies in UGTs, this family of enzymes could constitute an important determinant of susceptibility to chemical carcinogenesis, teratogenesis, and neurodegeneration. Fibroblasts contain Phase I and II drug-metabolizing enzymes, including UGTs, and undergo mitosis, rendering them susceptible to xenobiotic genotoxicity associated with micronucleus formation, which is thought to reflect carcinogenic initiation. Accordingly, skin fibroblasts may provide an accessible model for elucidating genoprotective mechanisms in both animals and humans and for characterizing the potential role of UGTs as determinants of individual toxicological susceptibility. To test this hypothesis, the carcinogen/teratogen benzo(a)pyrene [B(a)P], or its noncarcinogenic B(e)P isomer, was incubated with cultured skin fibroblasts obtained from male RHA-J/J rats. These rats have a hereditary homozygous deficiency in bilirubin UGT and demonstrate reduced xenobiotic glucuronidation, enhanced cytochrome P-450-catalyzed bioactivation, covalent binding, and toxicity of acetaminophen and B(a)P. Control fibroblasts were cultured from UGT-normal congenic homozygous male RHA-(+/+) rats and male Wistar rats. The cells were incubated with 10 microM B(a)P or B(e)P either for assessment of micronucleus formation or for quantifying the bioactivation and covalent binding of B(a)P and the glucuronidation of its hydroxylated metabolites. Compared to control fibroblasts incubated only with buffer, micronucleus formation was not enhanced by either DMSO vehicle or B(e)P. In contrast, B(a)P significantly enhanced micronucleus formation in all cells, and UGT-deficient cells (RHA-J/J) had a > 2-fold higher B(a)P-initiated micronucleus formation compared to UGT-normal cells (RHA-(+/+)) (P < 0.05). Glucuronidation of total B(a)P metabolites was 10% lower in RHA-J/J UGT-deficient fibroblasts, and the covalent binding of B(a)P to protein, reflective of an electrophilic reactive intermediate and DNA-alkylating agent, was up to 3-fold higher in RHA-J/J UGT-deficient fibroblasts or fibroblast homogenates compared to UGT-normal controls (P < 0.05). In fibroblast homogenates, addition of the UGT cosubstrate UDP-glucuronic acid reduced B(a)P covalent binding, corroborating the cytoprotective importance of UGTs. There was a highly significant correlation between decreasing glucuronidation of B(a)P metabolites and increasing bioactivation and covalent binding of B(a)P (r = -0.889; P = 0.018) in fibroblasts from RHA-J/J and RHA-(+/+) rat strains, indicating an important genoprotective role for UGT. These results provide the first evidence that hereditary UGT deficiencies may enhance susceptibility to chemical carcinogenesis and suggest that skin fibroblasts may provide a useful and highly sensitive model for human risk assessment.

Animals↗

Monoclonal anti-idiotypic antibodies as probes for common idiotopes shared by anti-"benzo(a)pyrene-like" IgA of cancer patients and rabbit anti-conjugated benzo(a)pyrene antibodies.

Anti-"benzo(a)pyrene [B(a)P]-like" IgA [referred as idiotypic antibodies (Abl)] from cancer patients' sera were found to react with conjugated B(a)P and a monoclonal anti-anti-conjugated B(a)P, internal image of conjugated B(a)P called AIB1 (referred to as Ab2 beta). These IgA were used to raise mouse monoclonal anti-idiotypic antibodies (Ab2). A monoclonal Ab2 called AIK1 was characterized as the internal image of a "B(a)P-like" structure. As shown by competitive experiments, AIK1 inhibited the reaction between Ab1 from a rabbit anti-conjugated B(a)P serum- and its relevant internal image, AIB1. Furthermore, AIB1 inhibited the reaction between AIK1 and anti-"B(a)P-like" IgA from cancer patients' sera. These observations confirmed the cross-reactivity between idiotypic determinants of human anti-"B(a)P-like" IgA and rabbit anti-conjugated B(a)P antibodies (Ab). This result was reinforced by a correlation between the anti-"B(a)P-like" IgA levels found in cancer patients' sera using indirect ELISA method with conjugated B(a)P, AIB1 and AIK1 coated on well-plates.

Animals↗

Tumor-initiating activity in mouse skin and carcinogenicity in rat mammary gland of dibenzo[a]pyrenes: the very potent environmental carcinogen dibenzo[a, l]pyrene.

Comparative studies of tumor-initiating activity in mouse skin and carcinogenicity in rat mammary gland were conducted with several dibenzo[a]-pyrenes (DBPs). SENCAR mice were initiated with DB[a, e]P, DB[a, h]P, DB[a, i]P, DB[a, l]P and anthanthrene, and promoted with tetradecanoyl-phorbol acetate. The same compounds were tested by intramammillary injection in female Sprague-Dawley rats. Anthanthrene was inactive in both mouse skin and rat mammary gland. DB[a, e]P was a very weak tumor-initiator in mouse skin and was inactive in rat mammary gland. DB[a, h]P induced twice as many papillomas in mouse skin as DB[a, i]P, although both compounds exhibited similar tumor latencies and percentages of tumor-bearing mice. These two compounds induced similar numbers of mammary tumors, but treatment of the rats with DB[a, i]P resulted in a significantly larger number of adenocarcinomas. DB[a, l]P was toxic to both the mice and rats. Treatment of mouse skin with this compound led to an erythema, which delayed the beginning of promotion until the 3rd week after initiation. Despite this delay, papillomas began appearing 5 weeks after initiation with DB[a, l]P and the number of tumors increased rapidly. The compound was so toxic in the rats that half of the animals died in the first 9 weeks and the remaining animals were sacrificed after 15 weeks. Nonetheless, DB[a, l]P was the strongest carcinogen tested, inducing seven tumors per rat within 10 weeks. These results demonstrate that DB[a, l]P, which is present in tobacco smoke, is an extremely potent carcinogenic aromatic hydrocarbon. Furthermore, some of these compounds can serve as useful models for elucidating their mechanisms of activation.

Adenocarcinoma↗

Metabolism of benzo[A]pyrene in hamster embryo cells. Effect of the concentration of benzo[A]pyrene on its metabolism.

The metabolism of benzo[a]pyrene (BP) by hamster embryo cells was studied. The production of water-soluble metabolites, shown to be conjugates with glucuronic acid, depended on BP concentration. With increased BP concentration the amount of glucuronic acid conjugates increased, but the proportion of conjugates in BP or its metabolites present in the medium decreased. The metabolites extracted with ethylacetate were trans-7,8-dihydrodiol-BP (7,8-dihydrodiol) and trans-9,10-dihydrodiol-BP (9,10-dihydrodiol), but large peaks of phenolic metabolites were found by high pressure liquid chromatography (HPLC) after digesting the medium with beta-glucuronidase. Therefore, BP is metabolized to oxygenated forms, and of these, most of the phenolic metabolites and parts of the dihydrodiols are conjugated with glucuronic acid. The proportions of dihydrodiols to phenols, estimated by HPLC after beta-glucuronidase digestion, decreased when the BP concentration was decreased. The results suggest that dihydrodiols are less readily glucuronidated than phenols and so may be metabolized further to metabolites other than glucuronic acid conjugates.

Animals↗

Benzo[a]pyrene uptake into rat liver microsomes: effects of adsorption of benzo[a]pyrene to asbestos and non-fibrous mineral particulates.

The fluorescence yield of benzo[a]pyrene (BP) increases dramatically upon its transfer from the surface of particulates to rat liver microsomes. Adsorption of BP to Canadian chrysotile, anthophyllite, hematite and silica results in greatly enhanced uptake rates into microsomes when compared to uptake from a microcrystalline dispersion of BP. The fibrous minerals chrysotile and anthophyllite were more effective than silica and hematite in enhancing BP uptake. Simple mixtures of BP microcrystals and particles did not display enhanced transport, indicating that adsorption of BP to the particulate surface is necessary for enhanced microsomal uptake. BP was not released into microsomes from carbon black. We suggest that particulate-enhanced availability of BP may be of significance in the co-carcinogenesis between particulates and polynuclear aromatic hydrocarbons. However, other mechanisms are also possible, and are not excluded by our experiments. The fluorescence methodology, described in this paper provides a novel and convenient means to quantify microsomal uptake of BP and thereby investigate further the mechanisms of cocarcinogenesis.

Adsorption↗

Unusual patterns of benzo[a]pyrene metabolites and DNA-benzo[a]pyrene adducts produced by human placental microsomes in vitro.

Human placental microsomes were incubated with [3H]benzo[a]pyrene (BP) and Salmon sperm DNA and the resulting metabolite-nucleoside complexes resolved by Sephadex LH-20 chromatography. The metabolite pattern was analyzed by high-pressure liquid chromatography (HPLC). The incubates were also co-chromatographed with extracts obtained from incubates with rat liver microsomes and [14C] BP. Phenols, quinones and 7,8-dihydrodiol were detected in the placental incubates. Both 9,10- and 4,5-dihydrodiols were very low as compared with control rat liver samples. Placental microsomes catalyzed the binding of BP metabolites to DNA in vitro, giving rise to two main complexes which co-chromatographed with rat liver-produced peaks attributable to 7,8-diol-9,10-epoxide and 7,8-oxide and/or quinones when metabolized further. The nucleoside metabolite peaks attributable to 4,5-oxide and 9-phenol-4,5-oxide were lacking when compared with the binding pattern catalyzed by rat liver. Both the total binding and specific metabolite-nucleoside adducts in the placenta correlated with fluorometrically measured aryl hydrocarbon hydroxylase (AHH) activity and with the amount of dihydrodiol formed. The results demonstrate that both the metabolite pattern and the nucleoside-metabolite complexes formed by the placental microsomes in vitro differed greatly from thos produced by rat liver microsomes. These sstudies also suggest that it is not possible to predict specific patterns of DNA binding from AHH measurements or even from BP metabolite patterns, especially when comparing different tissues and species.

Aryl Hydrocarbon Hydroxylases↗

Mutagenicity to mammalian cells in culture by (+) and (-) trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrenes and the hydrolysis and reduction products of two stereoisomeric benzo(a)pyrene 7,8-diol-9,10-epoxides.

The mutagenicity for mammalian cells of benzo(a)pyrene (BP) and 9 of its derivatives was tested by resistance to ouabain in Chinese hamster V78 cells. The derivatives included the (-) and (+) enantiomers of trans-7,8-diol; the racemic (+/-)trans-7,8-diol; two triols, (7/8,9)-triol and (7,9/8)-triol; and four tetrols, (7,10/8,9)-tetrol, (7/8,9,10)-tetrol, (7,9/8,10-triol and (7,9,10/8)-tetrol. Since V78 cells do not metabolize polycyclic hydrocarbons, mutagenesis was tested both in the presence and in the absence of Golden hamster cells capable of metabolizing polycyclic hydrocarbons. Neither BP nor any of its 9 tested derivatives showed mutagenicity for V78 cells in the absence of normal Golden hamster cells. However, in the presence of these cells, BP and the optically active and racemic trans-7,8-diols exhibited a mutagenic response that was dose-dependent. All other derivatives were inactive. The most active mutagenic hydrocarbon was (-) trans-7,8-diol, and activity decreased in the order (+/-)trans-7,8-diol, (+) trans-7,8-diol and BP.

Benzopyrenes↗

Benzo[alpha]pyrene antibody inhibition of benzo[alpha]pyrene-induced mutageneis.

An antibody to benzo[alpha]pyrene (BP) was prepared. The isolated antibody showed a specificity for BP and a low reactivity with another carcinogenic hydrocarbon, 7,12-dimethylbenz[alpha]anthracene (DMBA). The BP-antibody inhibited the in vitro cytotoxic and mutagenic activity of BP in both a rat embryo fibroblast- and a rat lung cell-mediated mutagenesis system. A possible correlation of these in vitro findings to the in vivo carcinogenesis situation is discussed.

9,10-Dimethyl-1,2-benzanthracene↗

A novel method for the isolation and identification of stable DNA adducts formed by Dibenzo[a,l]pyrene and Dibenzo[a,l]pyrene 11, 12-dihydrodiol 13,14-epoxides in vitro.

Our laboratory previously reported the identification and quantification of depurinating DNA adducts of dibenzo[a,l]pyrene (DB[a,l]P) in vitro, which comprise about 84% of all the DNA adducts that are formed [Li, K.-M., et al. (1995) Biochemistry 34, 8043-8049]. To determine a complete adduct profile and identify both stable and depurinating DNA adducts, we have developed a relatively simple, nonradioactive method for the identification of stable DNA adducts by combining enzymatic digestion, HPLC, and fluorescence line-narrowing spectroscopy (FLNS) techniques. Calf thymus DNA, bound to either (+/-)-anti- or (+/-)-syn-DB[a,l]PDE or rat liver microsome-activated DB[a,l]P, was first digested to 3'-mononucleotides with micrococcal nuclease and spleen phosphodiesterase. The adducts were then separated by HPLC with an ion-pair column and identified by FLNS by using the spectra of standards for comparison. In reactions with (+/-)-anti-DB[a,l]PDE, three adducts, an anti-cis-DB[a,l]PDE-dGMP, an anti-trans-DB[a, l]PDE-dAMP, and an anti-cis-DB[a,l]PDE-dAMP, were identified by HPLC and FLNS. In reactions with (+/-)-syn-DB[a,l]PDE, a pair of syn-trans-DB[a,l]PDE-dGMP adducts as well as a syn-cis-DB[a, l]PDE-dGMP, a syn-cis-DB[a,l]PDE-dAMP, and a pair of syn-trans-DB[a, l]PDE-dAMP adducts were identified. From the digest of microsome-activated DB[a,l]P-bound DNA, a syn-trans-DB[a,l]PDE-dGMP, an anti-cis-DB[a,l]PDE-dGMP, a syn-trans-DB[a,l]PDE-dAMP, and a syn-cis-DB[a,l]PDE-dAMP adduct were identified. An anti-cis-DB[a, l]PDE-dAMP adduct was identified only by (32)P-postlabeling. A total of five of the stable adducts formed by DB[a,l]P and nine of the stable adducts formed by DB[a,l]PDE in vitro have been identified. These adducts were also correlated to adduct spots in the (32)P-postlabeling method by cochromatography with standards. Approximately 93% of the stable adducts formed in reactions with (+/-)-anti-DB[a,l]PDE, 90% of adducts with (+/-)-syn-DB[a,l]PDE, and 85% of adducts formed with microsome-activated DB[a,l]P have been identified as Gua or Ade adducts. Equal amounts of stable Gua and Ade adducts were observed in the microsome-catalyzed binding of DB[a, l]P to calf thymus DNA, while 1.4 times more Gua adducts than Ade adducts were obtained in reactions with (+/-)-anti- or (+/-)-syn-DB[a,l]PDE.

Animals↗