Pyrene Monooxygenase Activity Research Articles

Mixed-function oxidases in fish: their role in adaptation to pollution

Comparison of the average benzo[a]pyrene monooxygenase (BaPMO) activity in the livers of the natural populations of barbel (Barbus barbus L.), chub (Leuciscus cephalus L.) and nase (Chondrostoma nasus L.) in the rivers Sava, Krka and Kupa, with the load of pollution equivalents in these waters, demonstrated a highly significant correlation between these two factors (P < 0.001). Uncertainty in this parameter due to lack of knowledge either about the actual exposure of natural fish populations, about, avoidance reaction, about the intermittency in the nature of pollution or about the duration of exposure, could be eliminated simply by exposing caged fish that had no known history of contamination exposure to the selected environments in these rivers. Oneyear-old carp responded to such exposure in a time-dependent, highly correlated fashion in such a way that 99 % of the variation was explained by linear correlation between BaPMO activity and the site of exposure (r = 0.989). The correlation of BaPMO activity in such exposed fish to

Genetic variation in cytochrome P-450 and xenobiotic metabolism in Drosophila melanogaster

A marked genetic variation in the capacity to perform xenobiotic metabolism was observed in microsomal fractions from the seven Drosophila strains studied. A 1,5 to 2-fold variation was found in the content of cytochrome P-450 and in the NADPH-cytochrome c reductase activity. The two insecticide-resistant strains Hikone R and Oregon R differed markedly when compared to sensitive strains by having a 3 to 17-fold higher p-nitroanisole (PNA) demethylase activity and biphenyl-3-hydroxylase activity. SDS-polyacrylamide gel electrophoresis of the microsomes also revealed an increased content of a protein band with an apparent mol. wt of 54,000 in the resistant strains. The 4-hydroxylation of biphenyl was also 2–7-fold higher in the Oregon R strain, and the band with a mol. wt of 56,000 had a higher protein content compared to the other strains. The biphenyl-4-hydroxylation was several-fold lower in the strain Berlin K. 2-OH-biphenyl was formed only in trace amounts by all strains. 7-Ethoxycoumarin (EC) deethylase activity and benzo(a)pyrene (BP) monooxygenase activity was 2–6-fold lower in the Hikone R strain. An increased amount of the protein with a mol. wt of 58,000 was noted in the Canton S strain. No concomitant increase in any enzyme activity was observed. A genetic variation between the strains was observed after phenobarbital (PB) treatment in the content of cytochrome P-450 and in the various enzyme activities, varying from non-responsiveness to a 4- to 5-fold increase. Aroclor 1254 (PCB) was less efficient in enhancing the activities. It caused maximally a 3-fold increase, had often no effect and in some cases even decreased the metabolism. β-naphthoflavone (BNF) caused only marginal increases in the activities in most strains. The only significant effects were an increased formation of 3-OH-biphenyl in Berlin K and an enhanced NADPH-cytochrome c reductase activity in Lausanne-S. In conclusion, the variations observed in this study provide a basis for further studies on the genetic regulation of the cytochrome P-450 system in Drosophila. Furthermore, similarities in the regulation when compared to mammals indicate that studies on this genetically well characterized organism might contribute to the general understanding of the genetics of xenobiotic metabolism.

Activity of benzo[ a]pyrene monooxygenase in fish from the Sava river, Yugoslavia: Correlation with pollution

Benzo[ a]pyrene monooxygenase (B[ a]PMO) activity in non-migratory fish from a given river segment is highly correlated to the recent pollutional history of that part of the river. The enzyme activity level can serve as a relevant measure for the harmful pollutant potential in aquatic ecosystems. Caged experimental fish exposed for about 10 days in river segments show BaPMO activity changes with the same predictive validity as that of the natural population.

Formation of mutagenic metabolites from benzo( a)pyrene and 2-aminoanthracene by the S-9 fraction from the liver of the Northern pike ( Esox lucius): Inducibility with 3-methylcholanthrene and correlation with benzo[ a]pyrene monooxygenase activity

The mutagenicity of benzo[ a]pyrene and 2-aminoanthracene in the Ames test using the S-9 fraction from the liver of the Northern pike ( Esox lucius) was tested. S-9 fractions were prepared both from fish injected intraperitoneally with 3-methylcholanthrene and from control animals. In addition benzo[ a]pyrene monooxygenase activity was assayed in the same S-9 fractions used in the Ames test. S-9 fractions from the liver of the Northern pike were found to convert benzo[ a]pyrene and 2-aminoanthracene to mutagenic metabolites. The number of revertants obtained was increased 2–4-fold in the case of 2-aminoanthracene and 3–14-fold in the case of benzo[ a]pyrene by pretreatment of the pike with a single intraperitoneal injection of 3-methylcholanthrene. This injection also caused a 2–6-fold increase in the benzo[ a]pyrene monooxygenase activity of the S-9 fractions used. These increases in mutagenicity and activity occur mainly during the first 4–12 days after the injection, but further small increases are observed for as long as 60 days. A strong positive correlation was found between the benzo[ a]pyrene monooxygenase activity of the S-9 fractions used and their ability to give rise to revertants in the Ames test using 2-aminoanthracene or benzo[ a]pyrene. This indicates that the major determining factor in the production of reactive metabolites which attack DNA in this in vitro system is the activity of the phase I cytochrome P-450 system.

Effects of dietary aroclor 1254 and cyclopropene fatty acids on hepatic enzymes in rainbow trout.

Diets containing Aroclor 1254 (PCB); cyclopropene fatty acids (CPFA), and PCB plus CPFA were fed to rainbow trout (Salmo gairdneri) for 15 weeks to determine the effects on hepatic microsomal activities of ethoxyresorufin-O-deethylase, ethoxycoumarin-O-deethylase, and benzo(a)pyrene monooxygenase. Ethoxyresorufin-O-deethylase activity continued to increase to a level 77-fold higher than control at week 15. Ethoxycoumarin O-deethylase and benzo(a)pyrene monooxygenase activities increased to 7.1-fold and 47-fold over control at week 9, respectively. Cytochrome P-450 values remained approximately 2-fold above controls from week 5 through week 15. At weeks 1 and 3, cytochrome P-450 levels were not significantly different from control. Dietary CPFA significantly depressed ethoresorufin-O-deethylase and ethoxycoumarin-O-deethylase activities, but had no effect on benzo(a)pyrene monooxygenase activity. Ethoxyresorufin-O-deethylase, ethoxycoumarin-O-deethylase, nd benzo(a)pyrene monooxygenase activities in the combined PCB-CPFA-fed trout were significantly higher than in control- or CPFA-fed trout, and significantly lower than in PCB-fed trout. This is the first time dietary PCBs have been shown to induce the MFO system in rainbow trout. These results provide a possible explanation for the effects of dietary PCBs on the metabolism and expression of other chemical carcinogens.

Further studies on the increase in drug-metabolizing capacity adjacent to intrahepatic Morris hepatomas

Microsomal cytochrome P-450 content was higher in histologically non-tumorous liver adjacent to intrahepatically implanted Morris hepatomas 5123D or 7795 than in histologically normal liver far removed from each tumor. V max values for microsomal benzo[ a]pyrene monooxygenase activity and cyclophosphamide activation were also significantly higher in tumor-adjacent liver than in normal liver far removed from tumor. K m values of these reactions were unchanged. After intrahepatic implantation, inert spheres of several different materials produced no regional differences in hepatic microsomal cytochrome P-450 content. Both intrahepatic Morris hepatomas exhibited markedly reduced cytochrome P-450 content and benzo[ a]pyrene monooxygenase activity. Cyclophosphamide biotransformation could not be detected in microsomes from either Morris hepatoma. Similar recoveries from microsomes of far-removed and tumor-adjacent liver indicated that differences between these regions in drug-metabolizing activity could not be attributed to different stabilities or sedimenting properties of their microsomes. Although microsomal recovery was significantly less from hepatomas than from far-removed or tumor-adjacent liver, this loss of tumor microsomes accounted for only a small part of the reductions in cytochrome P-450-mediated monooxygenases observed within tumors. Compared to control rats. tumor-bearing rats exhibited no change in hepatic drug-metabolizing capacity measured in vivo by hexobarbital sleeping times and antipyrine elimination rates. Phenobarbital (PB) pretreatment of tumor-bearing rats induced cytochrome P-450 to different extents within far-removed liver, tumoradjacent liver, and both hepatomas. The same differential inducibility occurred with PB pretreatment for cyclophosphamide activation. After PB induction, differences in drug-metabolizing activity between far-removed and tumor-adjacent liver disappeared; though induced, these activities remained lower in the hepatomas than in other regions. These changes in drug-metabolizing activity in both basal and PB-induced states of various hepatic regions were related to changes in cellularity of tumor-adjacent tissue. Hepatocellular nuclei prepared from tumor-containing liver were separated into diploid and tetraploid classes by sucrose density gradient centrifugation. Compared to far-removed liver, tumoradjacent liver contained significantly more diploid nuclei and less tetraploid nuclei.

The metabolism of drugs and carcinogens in isolated subcellular fractions of Drosophila melanogaster. II. Enzyme induction and metabolism of benzo[ a]pyrene

The effect of various pretreatments on the activities of several drug metabolizing enzymes was investigated in microsomes and postmicrosomal supernatant fractions isolated from whole body homogenates of Drosophila melanogaster larvae of different strains. Pretreatments of larvae with either phenobarbital (PB), β-naphthoflavone (BNF) or a mixture of polychlorinated biphenyls (Aroclor 1254, PCB) for 24 h increased microsomal benzo[ a]pyrene (BP) monooxygenase activity 2- to 6-fold in all strains as compared to untreated larvae. A simultaneous increase in the contents of cytochrome P-450 occurred after pretreatment with PB and PCB. Comparison of the turnover rates of BP per molecule of cytochrome P-450 indicated that BP was a poor substrate for control cytochrome P-450 whereas BNF induced a most active hemoprotein for this metabolism. Marked differences in the qualitative pattern of BP metabolites were obtained between microsomes isolated from BNF-treated larvae or rat liver microsomes. 3-Hydroxy-BP (3-OH-BP) was the dominating metabolite with both preparations, while the BP dihydrodiols were formed in minor quantities in Drosophila as compared to rat liver. Metyrapone and SKF 525-A inhibited BP metabolism in microsomes isolated from untreated and BNF treated larvae of all strains. In contrast, α-naphthoflavone (ANF) stimulated the BP monooxygenase activity of microsomes isolated from untreated larvae approx. 3-fold but only slightly influenced the activity of microsomes from BNF treated larvae indicating that the latter species of cytochrome P-450 was less sensitive to ANF. In all strains, PCB and PB treatments approximately doubled microsomal epoxide hydrolase activity and increased cytosolic glutathione- S-transferase activity 25–60%, significant only in strain Berlin K after PB treatment. The activities of epoxide hydrolase and glutathione- S-transferase in control larvae were comparable in the different strains, whereas the content of cytochrome P-450 and BP monooxygenase activity was higher in the Hikone R strain. Variability in the induction response to the various pretreatment was observed among the three strains.

The effect of long term treatment with disulfiram on content of cytochrome P-450 and on benzo(a)pyrene monooxygenase activity in microsomes isolated from the rat small intestinal mucosa

Disulfiram (DS) was administered perorally once daily to rats for 30 days to investigate the effects on cytochrome P-450 content and benzo(a)pyrene (BP) monooxygenase activity in microsomes isolated from the small intestinal mucosa. 50 mg or 100 mg DS/kg body weight caused a dose-related increase in BP monooxygenase activity, whereas the content of cytochrome P-450 was increased at the higher dose only. Similar absorption characteristics of cytochrome P-450 and turnover rates for BP on the basis of cytochrome P-450 was observed among the different microsomal preparations. The addition of DS or diethyldithiocarbamate (DDTC) to incubates of intestinal microsomes inhibited BP monooxygenase activity. Microsomes isolated from DS-treated rats were however less sensitive to in vitro inhibition by DS.

Differential effects of disulfiram and diethyldithiocarbamate on small intestinal and liver microsomal benzo[ a]pyrene metabolism

Microsomes isolated from rat small intestinal mucosa and liver were used to study the effects of disulfiram and diethyldithiocarbamate on benzo[ a]pyrene monooxygenase activity. This activity was decreased in the intestinal microsomes to 25 per cent of control 24 hr after a single oral dose of disulfiram. In contrast, daily administration of disulfiram for 5 days produced a dose related increase of benzo[ a]pyrene monooxygenase activity, above control level. The elevated activities were accompanied by a concomitant increase in the concentration of cytochrome P-450. This benzo[ a]pyrene monooxygenase activity was further stimulated by addition of α-naphthoflavone to the incubation medium. Furthermore, the absorption maximum of this cytochrome was at 450 nm in the CO bound reduced difference spectrum. These observations indicate that the disulfiram induced cytochrome P-450 was of the control type. Daily pretreatment with diethyldithiocarbamate impaired both intestinal and liver microsomes at benzo[ a]pyrene monooxygenase activities. Pretreatment with a single dose of 3-methylcholanthrene resulted in a more than 10-fold increase of intestinal benzo[ a]pyrene monooxygenase activity after 24 hr. Administration of disulfiram 24 hr before treatment appeared to potentiate the 3-methylcholanthrene induced increase of intestinal benzo[ a]pyrene monooxygenase activity. In vitro addition of disulfiram and diethyldithiocarbamate to incubates of intestinal or liver microsomes inhibited benzo[ a]pyrene metabolism to various extents; the liver being more sensitive. Disulfiram was approximately 50-fold more potent as an inhibitor than diethyldithiocarbamate. The in vitro inhibition of intestinal benzo[ a]pyrene monooxygenase activity obtained with disulfiram appeared to be caused both by direct interaction with the monooxygenase system and through NADPH dependent metabolic activation of disulfiram, while the inhibition of diethyldithiocarbamate may be a result of the latter process only.

Species and organ specificity of the trans-stilbene oxide induced effects on epoxide hydratase and benzo(a)pyrene monooxygenase activity in rodents

It was investigated, whether the selective induction of epoxide hydratase by trans-stilbene oxide (TSO) represents a general phenomenon or is confined to the liver of male rats where it was discovered. Therefore the effect of treatment with TSO on epoxide hydratase and benzo(a)pyrene mono-oxygenase activities were investigated in other organs (kidney, lung, skin, testis), other species (mice, hamsters) and also in female rats. In female rat livers the effect of TSO on the measured enzyme activities was very similar to that found in the male rat liver, i.e. a large induction of epoxide hydratase activity to 300–400 per cent of controls without affecting the benzo(a)pyrene monooxygenase activity. The potency of TSO to induce liver epoxide hydratase activity expressed as per cent of controls was 350:180:140 in rat, mouse and hamster, respectively. Selective induction of epoxide hydratase was found in rat and hamster liver, but not in the mouse liver, where benzo(a)pyrene monooxygenase activity was induced to about the same extent as the epoxide hydratase activity. The only extrahepatie organ in which an increased epoxide hydratase activity was found after TSO treatment was the rat kidney. Subcutaneous and topical treatment with TSO for 12 and 10 days respectively did not induce rat skin epoxide hydratase activity, instead a decrease of the enzyme activity to about 70 per cent ofthat found in control animals was found. Thus, TSO which was demonstrated to be a selective inducer of epoxide hydratase in rat liver can be utilized so far only in a limited number of carcinogenicity test systems, since it failed to induce the skin epoxide hydratase activity, which would have been an excellent tool to study directly the role of epoxide hydratase in the mechanism of skin tumor formation caused by polycyclic hydrocarbons. Interestingly, the epoxide hydratase of the hamster, investigated for the first time in this study, proved quite different from that of rat and mouse in that it hydrated styrene oxide remarkably faster than benzo(a)pyrene 4,5-oxide. This was true for all organs investigated. Also, the organ distribution of epoxide hydratase proved to be very different from that in rat and mouse. In the mouse the activity (with benzo(a)pyrene 4,5-oxide as substrate) was amongst all organs investigated highest in the testis (2.5 fold as compared to liver) but in the hamster the activity was more than 100 fold lower in testis as compared to liver. On the other hand, the activity in kidney was about 50 fold higher in hamster as compared to mouse.

Increase of liver microsomal benzo(a)pyrene monooxygenase activity by subsequent glucuronidation.

Benzo(a)pyrene monooxygenase activity in rat and guinea pig liver microsomes is markedly stimulated when UDP-glucuronic acid is present in the assay. This effect is further enhanced by addition of UDP-N-acetylglucosamine, an allosteric activator of UDP-glucuronyltransferase. The results suggest a coupling between microsomal monooxygenase and UDP-glucuronyltransferase which may assist elimination of toxic compounds.

Epoxide hydratase and benzo(A)pyrene monooxygenase activities in liver, kidney and lung after treatment of rats with epoxides of widely varying structures

The effect of treatment with various epoxides on epoxide hydratase and benzo(a)pyrene monooxygenase activities in rat liver, kidney and lung was tested with the aim possibly to find a selective inducer of epoxide hydratase. In a first series of epoxides, good substrates of epoxide hydratase and relatively small molecules did not lead to an increase in epoxide hydratase activity in any organ tested. Treatment with the poor substrates dieldrin and trans-stilbene oxide(TSO), however, increased epoxide hydratase activity in the liver and with TSO also in the kidney (3-fold). In contrast to all other epoxide hydratase inducers so far discovered, TSO did not affect the benzo(a)pyrene monooxygenase activities, even after doses leading to maximal induction of epoxide hydratase ( ~ 350 per cent of controls). In an attempt to find an even more potent selective inducer of epoxide hydratase several trans-stilbene oxide derivatives were synthesized. All modifications of the TSO molecule led to compounds which were less effective inducers of liver epoxide hydratase than the parent compound and also either increased or drastically decreased the benzo(a)pyrene monooxygenase activities. With TSO, 4-methoxy-TSO, 4-chloro-TSO and 4-nitro-TSO the tests were extended to three other parameters of the monooxygenase system, the cytochrome P450 content, the NADPH-cytochrome c reductase and the aminopyrine N-demethylase activities. All these derivatives but not TSO itself affected part of the monooxygenase system. Thus, with respect to five measured monooxygenase parameters TSO was found to be a selective inducer of epoxide hydratase in rat liver. An influence of TSO on the pattern of the various cytochrome P450 forms not leading to observable changes in monooxygenase activity towards two substrates known to be preferential substrates of different cytochrome P450 forms (aminopyrine and benzo(a)pyrene) is unlikely but cannot be excluded.

Benzo(a)pyrene monooxygenase induction in marine fish-molecular response to oil pollution

Induction of benzo (a) pyrene monooxygenase (BPMO) activity occurred in Blennius pavo, a species with a restricted territorial range, in response to exposure to a Diesel 2 oil. A response delay of 14 days was found at a concentration of 170 ppb and of 3 days when the water was saturated with Diesel 2 oil. When induced fish were transferred to clean water, elevated BPMO activity was maintained at a high level for at least a month. A benthic protochordate, Microcosmos sulcatus, showed no increase in BPMO activity when exposed to these concentrations even after 30 days of exposure. Field observations revealed a great variation in the BPMO activity from B. pavo caught at different sites. Fish from contaminated sites had significantly elevated levels of BPMO activity. Sardine schools caught at different sites had different, low levels of BPMO activities. However, specimens from the same school had closely similar levels of enzyme activity. An oil pollution incident (New Year 1977 oil spill in Northern Adriatic Sea) caused an increase in the BPMO activity in the livers of Blenniideae, reaching a peak on the 23rd day (representing an 8.5-fold increase in the background level), followed by a decrease in activity until a new background level, 3 times that of the original background level, was reached on the 45th day. This new background level is constant (through May-5 months after the incident). Measurement of BPMO activity in the livers of nonmigrant fish could serve as a useful biochemical parameter for monitoring and evaluation of acute or long-term oil pollution at a given site.