Benzo[a]pyrene Hydroxylase Research Articles

Interaction of tributyltin with hepatic cytochrome P450 and uridine diphosphate-glucuronosyl transferase systems of fish: in vitro studies.

Hepatic microsomes of red mullet (Mullus barbatus) and flounder (Platichthys flesus) were preincubated in the presence of a concentration range of the antifouling agent tributyltin (TBT) chloride, and the interactions of TBT with cytochrome P450 and uridine diphosphate-glucuronyl transferase systems were investigated. The enzyme systems were examined in terms of cytochrome P4501A (CYP1A)-catalyzed 7-ethoxyresorufin O-deethylase (EROD) activity and benzo[a]pyrene (BaP) metabolism and in terms of glucuronidation of testosterone and 17beta-estradiol, respectively. Ethoxyresorufin O-deethylase and BaP hydroxylase (BPH) activities of both fish species were progressively inhibited by increasing concentrations of TBT, and the effects were more pronounced for EROD than for BPH (maximal inhibition at 100 microM TBT for EROD and 250-500 microM TBT for BPH). Hydroxylated metabolites of BaP (3-hydroxy-, 7,8-dihydrodiol, and 9,10-dihydrodiol), representing 95% of the total metabolites formed, were reduced up to 75% in the presence of 100 to 500 microM TBT, whereas the formation of other metabolites was less affected. This may alter BaP toxicity and carcinogenicity. Overall, the results were consistent with a specific inhibitory effect of TBT on CYP1A in the two fish species. Additionally, the conjugation of testosterone was significantly inhibited (20%) at low TBT doses (5 microM), with no effect on the glucuronidation of estradiol.

The role of algae ( Isochrysis galbana) enrichment on the bioaccumulation of benzo[a]pyrene and its effects on the blue mussel Mytilus edulis

The role of algal concentration in the transfer of organic contaminants in a food chain has been studied using the ubiquitous model polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) as the contaminant, Isochrysis galbana as the phytoplankton food source, and the common mussel ( Mytilus edulis) as the primary consumer. The effect of algal concentration on BaP uptake by M. edulis was determined by feeding M. edulis daily with I. galbana which had previously been kept in the presence of BaP for 24 h. Four combinations of concentrations of algae and BaP were used to give final exposure concentrations of 30,000 or 150,000 algal cells ml −1 in combination with either 2 or 50 μg BaP l −1. BaP concentrations were determined fluorometrically in rest tissues (excluding digestive glands) and digestive gland microsomal fractions of M. edulis after 1, 7 and 15 days exposure, and also in isolated algae. Potentially toxic effects of BaP on M. edulis were examined in terms of blood cell lysosomal membrane damage (neutral red dye retention assay) and induction of digestive gland microsomal mixed-function oxygenase (MFO) parameters [BaP hydroxylase (BPH) and NADPH-cytochrome c (P450) reductase activities]. BaP bioaccumulation in rest tissues (and to a lesser extent in digestive gland microsomes) of M. edulis increased with both increasing BaP and algal exposure concentrations, and over time, producing maximal bioconcentration factors in rest tissues after 15 days exposure to 150,000 algal cells ml −1 and 50 μg BaP l −1 of 250,000. The five-fold higher concentration of algae increased BaP bioaccumulation by a factor of approximately 2 for 50 μg BaP l −1 at day 15. Blood cell neutral red dye retention time decreased linearly with increasing log 10 tissue BaP body burden, indicating an increased biological impact on M. edulis with increasing BaP exposure possibly due to a direct effect of BaP on blood cell lysosomal membrane integrity. An increase was seen in NADPH-cytochrome c reductase activity, and indicated in BPH activity, with 1 but not 7 or 15 days exposure to BaP, indicating a transient response of the digestive gland microsomal MFO system to BaP exposure.

DNA strand breaks, cytochrome P-450-dependent monooxygenase system activity and levels of chlorinated biphenyl congeners in the pyloric caeca of the seastar (Asterias rubens) from the North Sea

Seastars (Asterias rubens L.) were collecte d at sampling locations in different areas along transects radiating into the southern North Sea, representing areas impacted by contaminants to different degrees. Strand breakage in DNA isolated from tissue of the pyloric caeca was measured by the alkaline unwinding assay, modified to allow for the isolation of highly intact DNA. The interpretation of the results is based on the incidence of DNA strand breaks (expressed as the double:total DNA ratio or F value, indicating the degree of double-strandedness). The cytochrome P-450 concentration and the benzo [a] pyrene (BaP) hydroxylase activity were measured in microsomal fractions of the pyloric caeca. The chlorinated biphenyl (CB) congeners in tissue samples from the digestive gland were determined by temperature-programmed gas-chromatography, with a CPSil8 capillary column as the stationary phase, hydrogen as the carrier gas and 63Ni-electron capture detection. Areas where seastars showed different DNA integrity could be described. The highest integrity (0.75 < F < 0.85) was found in the off-shore reference sites of the Dogger Bank and Southern Bight. The mean F value in seastars from most sampling locations varied between 55 and 75%. The lowest DNA integrity (0.35 < F < 0.55) was found in specimens obtained from sampling locations near the river Rhine delta, along the Dutch coast and at two expected uncontaminated offshore areas. The BaP hydroxylase activity was relatively high near the mouth of the rivers Rhine and Scheldt, but also at a supposedly clean site near Dogger Bank. The concentration of CB congeners in the pyloric caeca of seastars decreased along transects radiating into the southern North Sea from the coastal areas of the Netherlands; the highest concentrations were in the nearby coastal areas and the lowest were in the open sea sampling locations. The data indicate that there might be a relationship between pollution from the Rivers Rhine, Meuse and Scheldt and the incidence of DNA strand breaks and/or BaP hydroxylase activity

Benzo[a]pyrene hydroxylase and glutathione s‐transferase activities as biomarkers in Lymnaea palustris (mollusca, gastropoda) exposed to atrazine and hexachlorobenzene in freshwater mesocosms

AbstractFreshwater pond mesocosms were used to validate xenobiotic‐metabolizing enzymes as biomarkers of contamination by atrazine and hexachlorobenzene (HCB) in a basommatophoran gastropod, Lymnaea palustris (Müller). Over long‐term (21‐d) exposure to 5, 25, and 125 μg/L atrazine and to 0.5, 1.25, and 5 μg/L HCB, the uptake and internal concentration of both pesticides were followed, and the activities of benzo[a]pyrene hydroxylase (BaPH) and glutathione S‐transferases (GSTs) of pesticide‐exposed snails were compared with those of control animals maintained in untreated mesocosms. Internally recovered HCB concentrations were much higher than internal atrazine concentrations, but the uptake of atrazine was faster than that of HCB. Although it affected the integrity of microsomal membranes, HCB had no relevant effects on BaPH and GST activities at concentrations which affected growth and fecundity, thus confirming the low inducibility of mollusc xenobiotic‐metabolizing enzymes by chlorinated compounds. In contrast, atrazine markedly inhibited BaPH and both postmitochondrial and cytosolic GSTs at the same concentrations, which had no effects on growth or reproduction. Enzyme inhibition was negatively correlated with the maximal internal amount of atrazine and positively correlated with the bioconcentration factor, suggesting that effects on xenobiotic‐metabolizing enzymes may affect pharmacokinetics of atrazine within the snail body. Correlation between the bioconcentration factor and enzyme inhibition may serve as a descriptor of the physiological status of animals and can also be used to indirectly estimate the pesticide concentration in the environment. Laboratory data were considered for the interpretation of results obtained in the mesocosms. In the biomarker context, BaPH and GST activities are proposed, along with other biochemical markers already identified in atrazine‐ and HCB‐exposed L. palustris, as elements of a multiparametric approach of the ecotoxicological effects of pesticides on freshwater ecosystems.

Assay conditions and basal activity of CYP1A-dependent mixed function oxidase in parr and smolt of atlantic salmon ( Salmo salar)

Liver microsomes were prepared from parr and smolt of Atlantic salmon ( Salmo salar), and optimal assay conditions (pH, temperature, incubation time) were established to measure basal activities, and kinetic constants (Km, Vmax) of three CYP1A-dependent mixed function oxygenases—benzo-a-pyrene hydroxylase (BAPH), 7-ethoxyresorufin-O-deethylase (EROD), and ethoxycoumarin-O-deethylase (ECOD), cytochromes P-450 and b5 content, and NADH- and NADPH-cytochrome c reductase activities. Using the determined assay conditions, we measured basal MFO activities in parr and smolt over a one-year period. Mean basal activities were as follows: EROD 0.033–0.104 nmol min −1 mg −1, BAPH 0.022–0.223 nmol min −1 mg −1, cyt-P450 0.063–0.353 nmol mg −1, NADH-cyt c reductase 45.25–71.99 nmol min −1 mg −1, NADPH-cyt c reductase 34.01–46.27 nmol min −1 mg −1, cyt-b5 0.079–0.587 nmol mg −1. There was a consistent development-related shift in basal EROD and BAPH activities in Atlantic salmon juveniles as they proceeded through smoltification.

Interannual Mixed Function Oxidase (MFO) Activity in Winter Flounder (Pleuronectes americanus) from a Coal Tar Contaminated Estuary

7-Ethoxyresorufin-O-deethylase (EROD), benzo[a]pyrene hydroxylase (BaPH), and cytochromes P-450 (cyt-P450) and b5(cyt-b5) varied annually in winter flounder (Pleuronectes americanus) collected in August of 1987, 1988, and 1989 from a coal tar contaminated estuary (Sydney, Nova Scotia, Canada). For August 1989, with fish available from all estuary areas, these indices correlated strongly with a spatial (along estuary) gradient in PAH in bottom sediments (7.19 ± 6.59–191 ± 184 μg g dry weight−1). Mean EROD activities in flounder near the coal tar source were up to seven times those in other estuary areas and paralleled sediment PAH loadings; however, standard deviations were high. Correlations for all MFO indices and sediment PAH were obtained in female flounder (P &lt; 0.01: EROD, cyt-b5, cyt-P450; P &lt; 0.02: BaPH). For male flounder the trend was similar, but only cyt-P450 correlated with sediment PAH (P &lt; 0.017). BaPH activity was highest near the coal tar source but was more variable and less sensitive to pollutant levels than EROD activity. Somatic indices in fish from Sydney estuary and St. George's Bay were similar. Winter flounder are vulnerable to PAH-induced MFO activities from coal tar contaminated sediments, but MFO induction does not occur equally in all fish; single-season or single-year data must be interpreted with caution.

Time-course and dose-response of the apparent induction of the cytochrome P450 monooxygenase system of pyloric caeca musomes of the female sea star Asterias rubens L. by benzo[a]pyrene and polychlorinated biphenyls

A study was made on the time-course of putative monooxygenase (MO) induction and the dose-response relationships for the effects of 3,3′,4,4′,5-pentachlorobiphenyl (IUPAC congener 126), 2,3′,4,4′,5-pentachlorobiphenyl (congener 118), 2,2′,4,4′,5,5′-hexachlorobiphenyl (congener 153) and benzo[a]pyrene (BaP) on MO-system activities in female sea stars Asterias rubens L. Sea stars received a single injection of PCB or BaP at different concentrations. Cytochrome P450 concentration and activities of NADPH-cytochrome c(P450) reductase, BaP hydroxylase (BPH) and prenenolone hydroxylase were determined in musomal fractions of the pyloric caeca. Apparent induction of BPH activity by PCB was observed only with CB-126 (3-methylcholanthrene (3-MC)-type inducer) at 10 μmol/kg. BPH activity was not affected by CB-153 (phenobarbital-type inducer) or CB-118 (mixed-type inducer). BaP (3-MC-type inducer) elicited a clear dose-dependent BPH induction in the range 10–160 μmol/kg. The maximum observed increase in BPH activity was about 350%. In time-course studies with CB-126 and BaP, highest BPH activities were found 3–4 days after injection. HPLC analysis of musomal BaP metabolites revealed that the elevated metabolism was shifted towards phenol production. PCB or BaP had no effect on the level of cytochrome P450 or the NADPH-cytochrome c reductase activity. All three PCBs and BaP inhibited pregnenolone hydroxylase activity, but no dose-response relationships were found. Overall the results suggest that the echinoderm MO system is inducible in response to exposure to cytochrome P450 1A-type inducers. However, the mechanism of induction remains unclear.

Interference of benzo[a]pyrene with cytochrome P-450-mediated steroid metabolism in pyloric caeca microsomes of the sea star Asterias rubens L.

1. 1. The effects of benzo[a]pyrene (BaP) on the metabolism of progesterone and pregnenolone, and the effects of steroids on BaP metabolism were examined in pyloric caeca microsomes of female Asterias rubens. 2. 2. The patterns of metabolism of progesterone and pregnenolone in microsomes were similar to those found in previous studies for homogenates and tissue incubations of pyloric caeca. 3. 3. BaP reduced the rate of hydroxylation of progesterone and pregnenolone, but had no effect on metabolite formation by non-cytochrome P-450-catalysed reactions. 4. 4. Microsomal BaP hydroxylase activity was reduced by the presence of progesterone, but pregnenolone and testosterone had no such effect. 5. 5. The reductions in steroid or BaP metabolism were progressive with increasing ratios of the concentration of the interfering compound to that of the assay substrate and were maximally 50% or less at ratios of × 100. 6. 6. It is concluded that isoenzymic forms of cytochrome P-450 are present, with preferences towards either steroid or BaP metabolism. The implications of the results for the in vivo situation are discussed.

In vivo and in vitro metabolism of benzo(a)pyrene by the larva of the newt, Peurodeles waltl

1. The larva of the amphibian species, Pleurodeles waltl was shown to metabolize benzo( a)pyrene (BaP) in vivo into a variety of oxidized products. 2. In vitro, BaP hydroxylase (AHH) activity was found in hepatic microsomes and postmitochondrial fractions from both larvae and adults of the pleurodele. 3. The clastogenic effect of BaP (formation of micronuclei in the erythrocytes) was shown to be related to the presence of BaP quinones in the tissues of the newt.

Induction of unscheduled DNA synthesis in primary cultures of rat, mouse, hamster, monkey, and human hepatocytes

Variation in hepatic metabolism between species may be an important factor in the differences observed in chemical carcinogenesis. We examined 6 chemicals representative of 4 chemical classes in the in vitro hepatocyte DNA repair assay using cells isolated from the Fischer-344 rat, B6C3F1 mouse, Syrian golden hamster, cynomolgus monkey and from human liver. Hepatocytes were isolated by in situ or biopsy liver perfusion and incubated with [ 3H]-thymidine and the test chemical. Unscheduled DNA synthesis (UDS) was measured as net grains/nucleus (NG) by quantitative autoradiography. Qualitative and quantitative differences in UDS responses were observed for every chemical. Liver cultures isolated from the rat, mouse, hamster, human, and monkey and treated with aflatoxin B 1 or dimethylnitrosamine all yielded dose-related increases in NG. Human, rat, and hamster hepatocyte cultures yielded positive responses following exposure to the aromatic amines 2-acetylaminofluorene, 4-aminobiphenyl, and benzidine, whereas cultures isolated from the monkey and mouse yielded < 0 NG. Treatment with benzo[ a]pyrene (BAP) produced strong positive responses in monkey and human hepatocyte cultures, weak positive responses in hamster cultures, and equivocal or negative responses in rat and mouse hepatocyte cultures. Hepatocyte function was assessed by measurement of DNA content, glutathione content, BAP hydroxylase activity, p-nitroanisole- O-demethylase activity, p-nitrophenol conjugation, and urea synthesis rates. The functional capabilities of isolated hamster, monkey, and human hepatocyte cultures do not appear to correlate with UDS responses observed for any compound; however, they indicate that the cultures were metabolically competent at the time of chemical exposure. These studies suggest that rat hepatocytes are a suitable model for human hepatocytes, whereas mouse and male monkey hepatocytes may be insensitive to aromatic amines.

Control of glucose-6-phosphate dehydrogenase deficiency on the formation of mutagenic and carcinogenic metabolites derived from benzo(a)pyrene.

It has been observed that human lymphocytes (HL) and fibroblasts, isolated in vitro from donors carrying the Mediterranean variant of glucose-6-phosphate dehydrogenase (G6PD), show a great decrease in this enzymatic activity, the hexose monophosphate shunt, and the NADPH/NADP+ ratio. This effect is associated with a decreased sensitivity of G6PD-deficient cells to the benzo(a)pyrene (BaP) cytotoxic effect and to a decreased in vitro transformation of BaP-treated fibroblasts. Further, benzo(a)anthracene (BaA)-induced BaP hydroxylase activity is lower in G6PD-deficient cells, when measured in the presence of endogenous NADPH. It has been hypothesized that the NADPH level could be rate-limiting for the NADPH-dependent steps of BaP metabolic activation. To test this hypothesis, the formation of BaP metabolites was studied in normal and G6PD-deficient HL incubated with the carcinogen. HPLC profiles of organic-soluble metabolites revealed that both types of HL produced all the following known BaP metabolites: 9,10-, 4,5- and 7,8-dihydrodiols, quinones, 9- and 3-hydroxy and two peaks of more polar metabolites. There was a great decrease of the various metabolites in the deficient HL. A decrease of total water-soluble BaP metabolites also occurred. HL formed mutagenic metabolites for S. typhimurium TA100 (his-) when incubated with the rat liver S9 fraction. When intact HL substituted S9 fraction, a significant reversed mutation occurred only with normal HL. This could indicate that the NADPH pool is inadequate in G6PD-deficient HL for active BaP metabolism. Accordingly, deficient HL formed lower amounts of BaP:DNA adducts than control during incubation with BaP.

Nondetectable concentrations of human placental ah receptors are associated with potent induction of microsomal benzo[ a]pyrene hydroxylase in individuals exposed to polychlorinated biphenyls, quaterphenyls, and dibenzofurans

Human exposure to polychlorinated biphenyls, quaterphenyls, and dibenzofurans occurred in an accidental food poisoning episode in Taiwan in 1979. Our objective was to evaluate the role of Ah receptors in the production of biochemical effects detected in placentas from exposed subjects. Placentas were obtained from pregnant women who were identified from a registry of exposed individuals. Benzo[ a]pyrene (BaP) hydroxylase was quantified in placental microsomes and Ah receptor was investigated in placental cytosolic and nuclear fractions by a variety of receptor-binding techniques using 2,3,7,8-[1,6- 3H]tetrachlorodibenzo- p-dioxin ([ 3H]TCDD) as the ligand. Microsomal BaP hydroxylase was dramatically elevated in placentas of exposed subjects compared to those of nonexposed, nonsmoking control subjects. However, concentrations of displaceable [ 3H]TCDD binding in placental preparations from exposed or control subjects were uniformally low; approximately 1.0 fmol/mg cytosolic protein. Further evaluation of displaceable TCDD binding by sucrose-gradient sedimentation and hydroxylapatite column chromatography revealed that binding properties were different than those for the Ah receptor. These data suggest that extremely low concentrations of Ah receptors in human placenta may be sufficient to markedly elevate microsomal BaP hydroxylation. Alternatively, induction in human placenta may be mediated by a mechanism not requiring Ah receptor.

Interaction of benzo(a)pyrene and cadmium on GSH-S-transferase and benzo(a)pyrene hydroxylase in the black sea bass Centropristis striata.

The effect of benzo(a)pyrene (BaP) and cadmium (Cd) upon the hepatic enzymes, BaP hydroxylase and GSH-S-transferase, was investigated in black sea bass. Intraperitoneal (ip) injections of BaP produced a significant increase in BaP hydroxylase activity in the microsomal fraction and GSH-S-transferase activity in the cytosol. Administration of Cd alone had no effect on either enzyme. However, when both Cd and BaP were injected (ip) in the fish, Cd had an inhibitory effect on the activity of these hepatic enzymes. Pretreatment of fish with a low dose of Cd (0.42 mg/kg) prior to injection of BaP and Cd produced a 30% increase in GSH-S-transferase activity and a 50% reduction in BaP hydroxylase activity when compared to fish injected with BaP alone following Cd pretreatment. Thus, a low dose of Cd was effective in producing a tolerance to a subsequent Cd challenge for the enzyme GSH-S-transferase but not for BaP hydroxylase. In contrast, fish pretreated with a high dose of Cd (2.5 mg/kg) prior to BaP treatment alone or with Cd displayed no difference in either enzyme activity. The implications from this study on the exposure of black sea bass to two xenobiotics, Cd and BaP, indicate that BaP hydroxylase may not serve as a good monitor for hydrocarbon pollution in the presence of Cd.

Responses of channel catfish to xenobiotics: Induction and partial characterization of a mixed function oxygenase

The effects of the environmental pollutant benzo(a)pyrene (BP) were assessed on the hepatic mixed function oxygenase system (MFO) in the channel catfish,Ictalurus punctatus. Initially, the optimum conditions were established for measuring hepatic MFO in this species of fish. Parameters investigated were divalent metal ion requirement,in vitro incubation temperature for optimum activity, pH for optimum activity and the time course for enzyme induction. A single dose of BP (100Μg/kg body weight) given intraperitoneally to catfish kept in the laboratory at 19†C induced the hepatic MFO benzo(a)pyrene hydroxylase (BaPH) with maximum activity occurring 96 hr after the injection of BP. Cytochrome P450 assays were carried out at 24 hr and 48 hr after injection of BP. Hepatic cytochrome P450 and BaPH activity increased from 24 to 48 hr after the BP injection. In a temperature acclimation experiment, fish were maintained at 15†, 19†, and 26†C; assays for BaPH were conducted 24, 48, and 96 hr after an inoculation with BP. Induction occurred equally in all BP-injected fish by 24 hr; by 48 hr, induction was greatest in the fish kept at 26†C, least in the 15†C group. The fish kept at 19†C were intermediate. However, by 96 hr, induction had occurred to the same degree in all three experimental groups. BaPH activity was always greater in the BP-injected groups than in their respective control groups. Phenobarbital (75 mg/kg body weight) failed to cause an increase in hepatic dealkylating activities or cytochrome P450 concentration.

Induction of mixed function oxidases by petroleum in the American eel, Anguilla rostrata.

American eels, Anguilla rostrata, were exposed to crude oil by ingestion of a 10, 100, or 500 microliters/kg fish dose per day for five days. Depuration was followed for an additional twelve days. All oil doses caused an induction of hepatic microsomal enzymes, maximally by three days of exposure. Benzo(a)pyrene hydroxylase (BaPH) showed a dose related response, with greater induction at 100 microliters/kg than at the other doses. The highest dose was hepatotoxic. Cytochrome P-450 induction was dose independent, and remained induced maximally for the entire experimental period, in contrast to BaPH which declined in activity. Reaction optimum for BaPH was at pH 7.5 and 27 degrees C. A study of tissue distribution showed the liver to account for nearly all BaPH activity. A significant increase in the protein content of the hepatic postmitochondrial fraction of oil-exposed fish was also observed.

Enhanced pulmonary and intestinal activation of procarcinogens and mutagens after chronic ethanol consumption in the rat.

Recent epidemiological surveys have indicated that alcoholics exhibit increased incidences of a variety of cancers. We have investigated, as a possible contributing factor to carcinogenesis in this population, the effect of chronic ethanol consumption on metabolic activation of procarcinogens by microsomes isolated from lungs and small intestine. These tissues are major sites through which procarcinogens enter the body and are also potential sites of procarcinogen metabolism. Rat litter-mates were pair-fed nutritionally adequate liquid diets which contained either ethanol as 36% of total energy or an equivalent energy content of carbohydrates in place of ethanol. Chronic ethanol consumption produced significant increases in pulmonary microsomal cytochrome P-450 and microsomal ethanol oxidation. The ethanol diet also enhanced the capacity of pulmonary microsomes to activate compounds present in tobacco pyrolyzates to mutagens detectable in the Ames Salmonella auxotroph reversion assay. The ethanol diet did not alter the capacity of pulmonary microsomes to hydroxylate benzo(a)pyrene (BaP) or to activate BaP to a mutagen. In contrast, microsomes from the upper small intestine of ethanol-fed rats did exhibit both higher levels of BaP hydroxylase activity and enhanced activation of BaP to a mutagen. The ethanol feeding also enhanced the capacity of the intestinal microsomes to activate to mutagens both tryptophan pyrolyzate and 2-aminofluorene but did not influence the metabolic activation of these promutagens by pulmonary microsomes. Chronic ethanol consumption thus influences carcinogen metabolism in the intestine and lung in a manner which varies with respect to both carcinogen and tissue.