7-ethoxycoumarin O-deethylase Activity Research Articles

Inhibition of P-450 by aucubin: is the biological activity of aucubin due to its glutaraldehyde-like aglycone?

The inhibition of ethoxy coumarin O-deethylase (ECOD) activity by aucubin and its aglycone was examined in a microsomal system and in freshly isolated hepatocytes. Aucubin was found to be inactive but the aglycone was found to be a potent time-dependent inhibitor of ECOD activity in both systems. The close structural similarity between the aglycone of aucubin and glutaraldehyde suggests a similiar mechanism of enzyme inhibition through protein cross-linking by Schiff reactions. The similarity between the 2 compounds was demonstrated through their closely similar binding spectra to bovine serum albumin. The biological activities reported for the aglycone are suggested to be due to this similarity to glutaraldehyde.

β-Naphthoflavone-inducible cytochrome P4501A1 activity in liver microsomes of the marine safi fish ( Siganus canaliculatus)

The cytochrome P450-dependent metabolism of benzo( a)pyrene and other xenobiotics has been investigated in liver microsomes prepared from a local marine safi fish, Siganus canaliculatus. The safi fish was found to have a well-developed microsomal monooxygenase system consisting of cytochrome P450, cytochrome b 5 and NADPH-cytochrome c reductase. The fish microsomal enzyme system was able to metabolize benzo( a)pyrene, 7-ethoxycoumarin and 7-ethoxyresorufin. Male fish were found to exhibit a higher monooxygenase activity than female fish. Treatment of fish with β-naphthoflavone was found to induce (2- to 4-fold) the activities of aryl hydrocarbon hydroxylase, ethoxycoumarin O-deethylase and ethoxyresorufin O-deethylase. HPLC analysis of the metabolites produced by incubation of benzo( a)pyrene with the liver microsomal preparation showed a predominant formation of 3-OH and 9-OH benzo( a)pyrene. There was an increased formation of benzo( a)pyrene 7,8-diol and benzo( a)pyrene 7,8,9,10-tetrol in liver microsomes prepared from β-naphthoflavone-treated fish. Western immunoblot analysis of liver microsomes from β -naphthoflavone-treated fish using an antibody to rat liver cytochrome P4501A1 (CYP1A1) suggested the presence of an inducible cytochrome P450 enzyme that was comparable with that of rat liver enzyme. Our results suggest that liver microsomes from the safi fish have multiple forms of cytochrome P450 with a specific β-naphthoflavone-inducible CYP1A1 homologous protein that can metabolize a variety of substrates.

Octopus pallidus cytochrome P-450: characterisation and induction studies with β-naphthoflavone and Aroclor 1254

Levels of cytochrome P-450 and related enzyme activities in Octopus pallidus were investigated for the first time to assess their potential for use as biomarkers of xenobiotic exposure. Spectral analysis revealed cytochrome P-450 in the digestive gland microsomes. Enzyme assays 7-ethoxyresorufin O-deethylase and 7-ethoxycoumarin O-deethylase were optimised with respect to temperature, pH and incubation time and NADPH cytochrome c reductase assays were performed. Corn oil was demonstrated to have an inhibitory effect on octopus cytochrome P-450 system. The study with Aroclor 1254 suspended in glycerol (100 mg/kg) revealed an elevation of 7-ethoxycoumarin O-deethylase activity, which would indicate cytochrome P-450 was induced.

Effect of fluvastatin, an inhibitor of 3-hydroxy-3-methyl glutaryl CoA reductase, on drug-metabolizing enzymes in rats

Fluvastatin (FV), a new cholesterol-lowering agent, has been studied for its effects on hepatic microsomal drug-metabolizing enzymes in male rats. FV was orally administered in dosages of 1, 5, and 30 mg/kg/day for 7 consecutive days. 3-Hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase activities were markedly decreased at all dose levels. The amount of microsomal protein and the contents of cytochromes P450 and b5 did not change. No induction of aniline hydroxylase, aminopyrine N-demethylase, testosterone hydroxylases (15 alpha-, 7 alpha-, 6 beta-, 16 alpha-, and 16 beta-), and UDP-glucuronosyltransferase were found. On the other hand, 7-ethoxycoumarin o-deethylase activity was slightly increased and lauric acid omega-1-hydroxylase activity tended to be decreased after treatment with FV.

Paradoxical effect of Sudan III on the in vivo and in vitro genotoxicity elicited by 7,12-dimethylbenz(a)anthracene.

Effect of the induction of drug metabolizing enzymes by Sudan III on the in vivo and in vitro genotoxicity elicited by 7,12-dimethyl-benz(a)anthracene (DMBA) was investigated. A significant suppression of DMBA-induced micronucleated reticulocytes was observed in C57BL/6 mice treated with Sudan III intraperitoneally for 3 or 5 days before injection of the DMBA. However, the preincubation of DMBA with hepatic microsomes from Sudan III-treated rats caused a marked increase in the in vitro mutagenicity in the Ames assay, paradoxically. Sudan III was found to induce CYP 1A1, 7-ethoxycoumarin O-deethylase activity as well as both UDP-glucuronyl transferase and glutathione S-transferase activities. The increase of mutagenicity of DMBA observed in the Ames assay using hepatic microsomes from Sudan III-treated rats was inhibited by the addition of uridine 5'-diphosphoglucuronic acid or reduced glutathione with cytosol. Mutagenic metabolites of DMBA formed by CYP1A1 appeared to be effectively detoxified by these phase II enzymes. The results of this study suggest that Sudan III-induced prevention of in vivo mutagenesis is due to the induction of both CYP 1A1 and detoxifying phase II enzymes. The induced CYP1A1 may accelerate formation of active metabolic intermediates, but phase II enzymes are also induced and detoxify these intermediates to inactive metabolites. This would reduce residence time of the carcinogen in the body and the time of exposure to active metabolites for target organs.

Polybrominated biphenyl induction of cytochrome P450 mixed function oxidase activity in primary rat and human hepatocytes

Polybrominated biphenyl (PBB) is an industrial chemical and environmental contaminant with known incidence of significant human exposure. PBB has been studied in laboratory animals and found to have significant toxicological effects as well as being a potent inducer of hepatic cytochrome P450 mixed function oxidase (MFO) activity. As part of our program to compare the response of laboratory animals and humans to industrial and environmental toxicants, we studied the effect of a major component of commercial PBB mixtures, 2,2′,4,4′,5,5′-hexabromobiphenyl (HBB), on MFO induction in primary cultures of human and rat hepatocytes. MFO induction was evaluated by measuring the deethylation of 7-ethoxycoumarin by intact hepatocytes. Rat hepatocytes were found to be highly susceptible to HBB induction of 7-ethoxycoumarin O-deethylase (ECOD) activity, with significant induction observed at the lowest concentration tested of 10 −8 M. Human hepatocytes were found to have a higher threshold for HBB induction of ECOD activity than rat hepatocytes. The lowest concentration of HBB required for ECOD induction observed for human hepatocytes was 10- to 1000-fold higher (10 −7, 10 −6, 10 −5 and 10 −5 M for the four human samples) than that found in rat hepatocytes. Future mechanistic investigation of this observed difference in sensitivity towards PBB between rat and human hepatocytes may aid the extrapolation of human health risk from toxicological data obtained from laboratory animals.

Induction of hepatic microsomal drug-metabolizing enzymes by methylsulphonyl metabolites of polychlorinated biphenyl congeners in rats

The effect of methylsulphonyl (MeSO 2) metabolites of 2,3′,4′,5-tetrachlorobiphenyl (tetraCB) (IU-70), 2,2′,3′,4′,5-pentachlorobiphenyl (pentaCB) (IU-87), 2,2′,4′,5,5′ -pentaCB (IU-101) and 2,2′,3′,4′,5,5′-hexachlorobiphenyl (hexaCB) (IU-141), on the hepatic microsomal drug-metabolizing enzyme system was investigated in rats. The administration of 3-MeSO 2-2,3′,4′,5-tetraCB (10 μmol/kg), 3-MeSO 2-2,2′,3′,4′,5-pentaCB (0.5 μmol/kg), 3-MeSO 2-2,2′,4′,5,5′-pentaCB (0.5 μmol/kg) and 3-MeSO 2-2,2′,3′,4′,5,5′-hexaCB (2 μmol/kg) to rats significantly increased the contents of cytochromes P-450 and b 5 and the activities of aminopyrine N-demethylase, 7-ethoxycoumarin O-deethylase and benzo[ a]pyrene hydroxylase. From these results, it is suggested that the 3-MeSO 2 derivatives studied are possibly potent phenobarbital-like inducers of microsomal drug-metabolizing enzymes. On the other hand, 4-MeSO 2-2,3′,4′,5-tetraCB, 4-MeSO 2-2,2′,3′,4′,5-pentaCB, 4-MeSO 2-2,2′, 4′,5,5′-pentaCB and 4-MeSO 2-2,2′,3′,4′,5,5′-hexaCB had almost no effect on both cytochrome contents and these enzyme activities. After 96 h, following administration of 2,3′,4′,5-tetraCB, 2,2′,3′,4′,5-pentaCB, 2,2′,4′,5,5′-pentaCB and 2,2′,3′,4′,5,5′-hexaCB

Characterization of hepatic microsomal cytochrome P-450 from rats treated with methylsulphonyl metabolites of polychlorinated biphenyl congeners

The inducing potency of 3-methylsulphonyl(MeSO 2)-2,2′,4′,5,5′-pentachlorobiphenyl (pentaCB), which was one of the major MeSO 2 metabolites of polychlorinated biphenyls (PCBs) present in seal blubber[1], on the hepatic drug metabolizing enzyme activities was examined in comparison with that of the parent compound and phenobarbital (PB). The inducing fashion of the above enzymes and changes in the contents of PB-inducible P-450 forms by 2,3′,4′,5-tetrachlorobiphenyl (tetraCB) (IU-70), 2,2′,3′,4′,5-pentaCB (IU-87), 2,2′,4′,5,5′-pentaCB (IU-101) and 2,2′,3′,4′,5,5′-hexachlorobiphenyl (hexaCB) (IU-141), and their MeSO 2 metabolites were investigated in rats. Administration at various doses (0.2-1.0 μmol/kg) of 3-MeSO 2-2,2′,4′,5,5′-pentaCB produced nearly dose-related increases in the hepatic concentration of this methyl sulphone, in the contents of cytochromes P-450 and b 5, and in activities of aminopyrine N-demethylase, 7-ethoxycoumarin O-deethylase and benzo[ a]pyrene hydroxylase of liver microsomes. Major PB-inducible forms, CYP2B1, CYP2B2, CYP3A2 and CYP2C6 were induced with four PCBs (342 μmol/kg) and their 3-MeSO 2 metabolites (0.5–10 μmol/kg), indicating that 3-MeSO 2 metabolites were strong PB-type inducers of hepatic drug-metabolizing enzymes. 3-MeSO 2-2,2′,4′,5,5′-pentaCB was an especially strong inducer. On the other hand, four PB-inducible forms of cytochrome P-450 were not induced with the 4-MeSO 2 isomers. The relation between liver concentrations of the corresponding 3-MeSO 2 derivatives and induction of four PB-inducible forms of cytochrome P-450 after administration of four PCBs and their 3-MeSO 2 derivatives further confirmed that the 3-MeSO 2 metabolites played an important role in the induction which parent PCB congeners caused on the hepatic drug-metabolizing enzyme system.

The Effect of Insulin-like Growth Factor-1 on Protein Metabolism and Hepatic Response to Endotoxemia in Parenterally Fed Rats

To determine the influence of insulin-like growth factor-1 (IGF-1) on nitrogen loss and hepatic response to critical illness, 34 male Sprague-Dawley rats (190-230 g) were randomized to receive parenteral nutrition (PN) only (Ctrl), PN plus continuous infusion of Escherichia coli 026:B6 lipopolysaccharide at 6 mg/kg/day (LPS), or PN plus LPS plus rhIGF-1 (IGF-1) at 3 Mg/ kg/day for 48 hr. Prior to randomization, all animals underwent iv cannulation and 30 hr of adaptation to PN. All animals received isocaloric and isonitrogenous PN (glucose 170 kcal/kg/day and nitrogen 1.1 g/kg/ day) and were kept NPO except for water ad libitum. [ 15N]glycine was infused in all animals for determination of liver fractional synthetic rate. Cumulative nitrogen balance during endotoxemia was significantly different from each other (+72 ± 42, -217 ± 131, -114 ± 137 mg/kg/48 hr for the Ctrl, LPS, and IGF-1 groups, respectively; ANOVA, P < 0.001). Endotoxin significantly increased the urinary 3-methylhistidine/creatinine ratio (0.24 ± 0.05, 0.55 ± 0.12, 0.48 ± 0. 17 for the Ctrl, LPS, and IGF-1 groups, respectively; ANOVA, P < 0.001); however, IGF-1 did not significantly reduce the ratio. Endotoxin induced a significant increase in liver fractional synthetic rate (29 ± 8, 56 ± 18, 64 ± 12%/day for the Ctrl, LPS, and IGF-1 groups, respectively; ANOVA, P < 0.01) and depressed hepatic cytochrome P450 concentration (0.54 ± 0.19, 0.22 ± 0.07, 0.19 ± 0.07 nmol/mg protein, respectively; ANOVA, P < 0.05) and ethoxycoumarin O-deethylase (ECOD) activity (103 ± 73, 29 ± 13, 17 ± 11, pmol/mg/ min, respectively; ANOVA, P < 0.01); however, rhIGF-1 did not significantly alter these hepatic variables during endotoxin infusion. Recombinant human insulin-like growth factor-1 significantly improved nitrogen balance without compromising hepatic response as measured by liver fractional synthetic rate, cytochrome P450 concentration, and ECOD activity in endotoxemic parenterally fed rats.

Mechanisms of interleukin-2-induced depression of hepatic cytochrome P-450 in mice

Interleukin-2 (15 μg/mouse, i.p. twice daily for 4 days and once on the 5th day) significantly lowered cytochrome P-450 and heme content and increased heme oxygenase mRNA accumulation; the activities of 7-ethoxycoumarin O-deethylase, ethoxy- and pentoxyphenoxazone O-dealkylases were decreased. The activity of the type O form of hepatic xanthine oxidase increased, but there was no increase in lipid peroxide, expressed in terms of microsomal malondialdehyde. In vivo inactivation of xanthine oxidase activity by feeding mice with tungstate did not substantially change the degree of interleukin-2-induced cytochrome P-450 depression, suggesting that the two processes are not causally linked. Induction of tolerance to endotoxin by a 4-day pretreatment with lipopolysaccharide resulted in 50% protection against this depression despite inhibition of the interleukin-2 induced formation of tumor necrosis factor. This suggests that the release of tumor necrosis factor per se does not fully account for the depression of cytochrome P-450. Dexamethasone, already used in patients to reduce toxicity of interleukin-2 therapy, provided full protection against the cytochrome P-450 depression.

Validation of biomarkers of marine pollution exposure in sand flathead using Aroclor 1254

The Australian marine fish, sand flathead ( Platycephalus bassensis), were exposed to the polychlorinated biphenyl (PCB) mixture Aroclor 1254 by intraperitoneal injections of 10, 100. 200 and 400 mg/kg in corn oil. Cytochrome P450 content, ethoxycoumarin O-deethylase (ECOD), ethoxyresorufin O-deethylase (EROD) and uridine diphospho-glucuronosyltransferase (UDP-GT) activities were measured to quantify liver detoxication enzyme response. The type and amount of metabolised PCBs in the bile were quantified by gas chromatography/mass spectrometry. EROD activity increased in a dose-dependent manner. At the 400 mg/kg dose, EROD activity was 2.64 ± 0.34 nmol/min per g liver (0.07 ± 0.01 nmol/min per mg protein), approximately 5 times control levels ( P < 0.05). UDP-GT activity at 400 mg/kg was 63.99 ± 8.69 U/g liver (1.53 ± 0.29 U/mg protein), an increase of 75% above control levels. Protein content of the liver increased 1.5-fold to 41.25 ± 2.52 mg/g liver and cytochrome P450 content increased 1.8-fold to 6.82 ± 0.91 nmol/g liver. No differences with treatment were seen in ECOD activity. Metabolised PCBs excreted in the bile were hydroxylated and conjugated with glucuronic acid or sulphate, and the increases in production of biliary metabolites paralleled the increases in total liver EROD activity. These results indicate that EROD and UDP-GT activities and biliary metabolites are sensitive biomarkers of exposure and that sand flathead represents an appropriate fish for use in further biomonitoring work.

Induction of Cytochrome P-450 isoenzymes in cultured monkey hepatocytes

The effect of phenobarbital (PB), β-naphthoflavone (β-NF) and rifampicin (Rif) on the drug-metabolizing activity of cultured squirrel monkey hepatocytes was examined. The drug metabolizing activity (e.g. alkoxycoumarin dealkylase or steroid hydroxylase) gradually decreased during the culture period with 40–70% activity remaining at 72 hr. When 0.5 mM PB was added to the culture, the activities of 7-methoxycoumarin O-demethylase (MCOD) and 7-ethoxycoumarin O-deethylase (ECOD) increased to 6–7 fold higher level than those of control at 72 hr. Testosterone 6β-hydroxylase (6β-OH-T) and testosterone 16β-hydroxylase (16β-OH-T) activities were approx. 3-fold higher than those of the control. Addition of β-NF significantly increased the activities of 7-ethoxyresorufin O-deethylase (EROD) and ECOD. Though statistically insignificant, Rif slightly increased 6β-OH-T activity. Western blot analysis indicated PB induced production of the CYP 2B and 3A subfamilies, while β-NF and Rif induced that of the CYP 1A and the CYP 3A subfamily, respectively.

Effects of streptolysin O, picibanil (OK 432) and interferon alpha 2A on cytochrome P-450-dependent monooxygenases and arylamine N-acetyltransferase in rat liver.

Streptolysin O, a thiol-activated exotoxin from group A beta-haemolytic streptococci, caused a dose-dependent depression of aniline hydroxylase, aminopyrine N-demethylase and ethylmorphine N-demethylase activities when added into the hepatic microsomal mixtures from male rats at concentrations 0.02-0.4 HU/mL in vitro. The activities of 7-ethoxycoumarin O-deethylase, 7-ethylresorufin O-deethylase and 7-pentylresorufin O-depentylase were not altered with the used concentrations of the toxin. Specific antibody against haemolytic action of streptolysin O added to incubation mixtures in vitro was not able to protect streptolysin-sensitive monooxygenases from the inhibition. The addition of streptolysin O (0.01-0.8 HU/mL) into the cytosol-containing medium did not significantly influence the activity of procainamide N-acetyltransferase. Immunomodulators picibanil (OK 432) and human recombinant interferon alpha 2A which are known to suppress oxidative metabolism in vivo in humans and animals, were without effect either on the cytochrome P-450-dependent monooxygenases or on the N-acetyltransferase activity when administered in vitro at the doses real in their clinical application (0.001-0.1 KE/mL of picibanil and 10-500 U/mL of alpha-interferon).

Induction of rat hepatic P4501A1 by organic extracts from airborne particulate matter in Santiago, Chile

1. Organic extracts from particulate matter collected in downtown Santiago, Chile, in 1990 were administered to female Wistar rats. 2. Extracts shifted the maximal absorption wavelength P450 spectra of the reduced-CO complex of hepatic microsomal P450 from 450 to 448 nm, and enhanced the total content of P450. In addition, substantial increases in aryl hydrocarbon hydroxylase, ethoxyresorufin O-deethylase and ethoxycoumarin O-deethylase activities were observed, whereas aminopyrine N-demethylase activity was not affected by treatment. 3. Western blotting using polyclonal antibodies against P4501A isozymes showed the appearance of a distinct and intense P4501A1 band in microsomes from rat pretreated with air particle extracts, and was not observed in microsomes from control rat. On the other hand, the intensity of the P4501A2 isoenzyme was apparently not affected. 4. These findings suggest that the organic extracts from airborne particulate matter modify the composition of rat liver P450 isozymes by inducing those isoforms responsible for the activation of precarcinogenic to carcinogenic agents.

Interaction of tetrachloroethylene with rat hepatic microsomal P450-dependent monooxygenases.

1. We have studied the effects of tetrachloroethylene (PCE) on the kinetics of the P450-dependent monooxygenases in rat liver microsomes. 2. 7-Pentoxyresorufin O-depentylase (PROD) and 7-benzyloxyresorufin O-debenzylase (BROD) activities in phenobarbital (PB)-treated rat liver microsomes were substantially inhibited by PCE. The inhibition profiles were non-competitive for both enzyme activities; Ki's from Eadie-Hofsee plots were 0.16 and 0.29 mM for PROD and BROD respectively. In contrast, the enzyme activities in untreated, beta-naphthoflavone (BNF)-, isoniazid (ISN)- and pregnenolone-16 alpha-carbonitrile (PCN)-induced microsomes were not affected by PCE. 3. 7-Ethoxycoumarin O-deethylase (ECOD) activity in PB-induced microsomes was competitively inhibited by PCE, with a Ki that was lower than those of other microsomes. 4. PCE inhibited 7-ethoxyresorufin O-deethylase (EROD) activities in some microsomes slightly. The Ki for PCE was the lowest in untreated, followed by ISN-treated microsomes. 5. No effect of PCE upon aniline 4-hydroxylase (AN4H) and testosterone 6 beta-hydroxylase (TS6BH) activities was evident in any microsomal preparation. 6. These results indicate that PCE inhibits PB-inducible, P450-dependent monooxygenases in vitro non-competitively or competitively, and that the P450 enzymes of the P4502B subfamily may contribute to PCE toxicity.

Effect of swimming exercise and ethanol on rat liver P450-dependent monooxygenases

The interactive effects of 6 wk of repeated swimming exercise and chronic ethanol consumption (36% of total calories) on the hepatic cytochrome P450-dependent monooxygenase system were studied utilizing four groups of male rats in a 2 x 2 factorial design. The sedentary-control (S/C), sedentary-ethanol (S/E), and swim-control (SW/C) groups received the same amount of food that the swim-ethanol (SW/E) group consumed. The swimming groups were trained to swim for 2 h.d-1, 5 d.wk-1. Significant main effects due to ethanol (P < 0.002) and exercise (P < 0.02) were observed for the enhanced cytochrome P450 content and cytochrome P450 reductase activity, respectively. In addition, significant main effects for ethanol (P < 0.001), exercise (P < 0.0001), and significant interaction effects (P < 0.005) on aniline p-hydroxylase activity and significant main effects for ethanol (P < 0.01), exercise (P < 0.01), and interaction effects (P < 0.04) on 7-ethoxycoumarin o-deethylase activity were observed. Because the SW/C treatment had no effect on any of the measured cytochrome P450 activities and the SW/E treatment enhanced P450 activities much more than the S/E treatment, the main effects observed for exercise are accounted for by the alterations produced by combining swimming with the ethanol treatment. Based on these results, repeated exercise combined with ethanol consumption produces a synergistic increase in ethanol-inducible cytochrome P450-dependent activities.

Effect of environmental pollutants on hepatocellular function in rats: 3-methylcholanthrene and Aroclor-1254.

Environmental pollutants, Aroclor-1254 (PCB) and 3-methylcholanthrene (MC), were employed in this study to investigate some aspects of the induction of hepatic drug metabolism in rats. PCB and MC treatments increased 7-ethoxyresorufin and 7-ethoxycoumarin O-deethylase activities related to cytochrome P-448. Cytochrome P-450 reductase activity was increased by PCB while no effect was observed by MC treatment. Pretreatment with PCB resulted in approximately 50% increase in the phospholipid content of the microsomes whereas MC caused no change. Liver microsomal cholesterol content was decreased while triglycerides were increased by PCB. The ratio between saturated and unsaturated fatty acids (saturation index) decreased in the total microsomes and phospholipids with PCB treatment, whereas MC did not alter the ratio, except that the major effect of MC was observed in the acyl derivatives of microsomal phosphatidylethanolamine. It is proposed that the uniaxial rotation and mobility of hemoproteins may be restricted by an increase in the saturation index of the membrane, while a decreased index may facilitate contact with reductases for electron transfer by enhanced membrane fluidity. The decreased saturation index after treatment with MC may play a role in carcinogenicity by triggering induction of free radicals.

Effects of prolonged administration of perfluorooctanoic acid on hepatic activities of enzymes which detoxify peroxide and xenobiotic in the rat

Male and female rats were fed a diet containing 0.01% (w/w) perfluorooctanoic acid (PFOA) for 2 or 26 weeks, and the effects on enzymes that participate in the metabolism of peroxides and xenobiotics in liver were studied. Elevated activity of peroxisomal β-oxidation persisted throughout the treatment of male rats with PFOA for 26 weeks. The activity of glutathione (GSH) peroxidase towards hydrogen peroxide was depressed significantly by the prolonged administration. The long-term treatment of male rats with PFOA decreased the activity of GSH peroxidase towards cumene hydroperoxide and increased the activity of microsomal NADPH-dependent lipid peroxidation. The activities of GSH reductase and hepatic content of GSH remained unchanged. There was no difference in the content of conjugated dienes in microsomal lipid between male rats exposed to PFOA for 26 weeks and age-matched control. The activities of GSH S-transferase towards 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene were depressed by the short-term administration of PFOA to male rats, and this inhibition became pronounced during the prolonged treatment. Microsomal cytochrome P450 was induced by the short-term treatment of male rats with PFOA, and elevated levels persisted throughout the treatment for 26 weeks. Upon the administration of PFOA to male rats for 2 weeks, the activity of 7-ethoxycoumarin O-deethylase was increased markedly, whereas the activities of either aniline p-hydroxylase or aminopyrine N-demethylase were unchanged. Although an age-dependent decrease was observed in the activity of 7-ethoxycoumarin O-deethylase, the activity in male rats treated with PFOA for 26 weeks was higher than that of age-matched control, to the same extent as was observed with the short-term treatment. The prolonged administration of PFOA to male rats caused a significant increase in the activity of both aniline p-hydroxylase and aminopyrine N-demethylase. Little changes were found in the same parameters tested in female rats even after the prolonged administration of PFOA.

Cytochrome P450 monooxygenase and glutathione S-transferase activity of two Australian termites: Mastotermes darwiniensis and Coptotermes acinaciformis

The major detoxication enzymes have een characterized in preparations of two economically important termite species. Mastotermes darwiniensis microsomes contained a similar quantity of total cytochrome P450 as Coptotermes acinaciformis but the activities of aldrin epoxidase (AE), 7-ethoxyresorufin O-deethylase (EROD) and 7-ethoxycoumarin O-deethylase (ECOD) were 4.5, 5.8 and 17 times higher, respectively. Compared with other insects, AE activity in these two termite species is low, EROD activity moderate and ECOD activities are high, especially in M. darwiniensis. The cytosolic GST activity of C. acinaciformis toward chloro-3,5-dinitrobenzene (CDNB) was 3.13 μmol min −1mg −1, > 3 fold higher activity than M. darwiniensis cytosol. Apparent K m values of 1.87 mM for CDNB and 0.84 mM for glutathione were determined in C. acinaciformis cytosol. 7,8-Benzoflavone dramatically reduced EROD activity in microsomes of both termite species but had significantly lower inhibitory effect on ECOD activity. The addition of SKF 525A to termite microsomes inhibited both EROD and ECOD activities. Multiple isoenzymes of cytochrome P450 in termites are indicated by these findings. The distinctly different substrate specificities and λ max of the CO difference spectra from M. darwiniensis and C. acibnaciformis microsomes may arise from differences in the cytochrome P450 isoenzymes between the two species.

Selectivity of isoprenoid-containing imidazole antifungal compounds for sterol 14-demethylase P450 (P450 14 dm) and 7-ethoxycoumarin O-deethylase P450 of rat liver microsomes

The imidazole antifungal compound AFK-108 (1-[2-(2,4-dichlorophenyl)-2-((2 E)-3,7-dimethylocta-2,6-dienyloxy)ethyl]-1 H-imidazole) has been shown to be a potent inhibitor for yeast lanosterol 14α-demethylase (P450 14 dm ), interacting specifically with the sterol side-chain recognition part of the substrate site through its geranyl moiety. AFK-108 acted as a potent inhibitor for rat liver P450 14 dm , while its farnesyl (AFK-110) and prenyl (AFK-122) homologues were weak inhibitors. This indicates that AFK-108 interacts with rat liver P450 14 dm in the same manner as with the yeast enzyme. However, the difference between the potency of AFK-108 and the homologues was greater in rat P450 14 dm than in the yeast enzyme. AFK-108 and its homologues partially inhibited 7-ethoxycoumarin O-deethylase activity of rat liver microsomes. The order of potency was AFK-122 > AFK-108 > AFK-110, indicating that some steric hindrance of the isoprenoid moiety might affect their potency. The inhibitory effect of AFK-108 for P450 14 dm was considerably higher than for 7-ethoxycoumarin O-deethylase P450, while the inhibition of AFK-110 and AFK-122 on these enzymes was of the same order of magnitude. These results suggest that azole compounds interacting with the side-chain recognition site of P450 14 dm may be good candidates as antifungal agents selective for fungal P450 14 dm .