Hepatic Microsomal Monooxygenase Research Articles

Reponses of the Hepatic Microsomal Cytochrome P450 Monooxygenase System in Rock Bream Oplegnathus fasciatus Exposed to Tributyltin (TBT)

Abstract The study was conducted to investigate the responses of the hepatic microsomal cytochrome P450 monooxygenase system in the rock bream Oplegnathus fasciatus after chronic exposure to 0, 1, 2, 4, and 8 μg/L tributyltin (TBT) concentrations for 4 weeks. Hepatic cytochrome 450 content and ethoxyresorufin O -deethylation (EROD) activity were found to significantly increase in fish treated with the higher concentration of TBT (≥4 μg/L); however, no significant changes were observed in penthoxyresorufin O -deethylation (PROD) activity in all treated groups compared to the control group. These findings suggest that exposure to a low TBT concentration (≥4 μg/L) has the potential to induce cytochrome 450 content and EROD enzyme activity in hepatic tissue in the rock bream. Key words: Oplegnathus fasciatus , Tributyltin, Cytochrome 450, EROD, PROD Introduction Many aquatic ecosystems are faced with spatially or tem-porally alarmingly high levels of xenobiotic chemicals (Brack et al., 2002; Diez et al., 2002). Tributyltin (TBT) is a typi-cal synthetic pollutant, which was selected for investigation in this study. TBT is an organotin compound used primarily as a biocide in antifouling paints for ships, boats, and fishing nets. The use of TBT as an antifouling agent in paint has been banned for small boats and fishing nets in most countries as it has been shown to be toxic to aquatic life and is an endo-crine disrupting chemical that generates severe reproductive effects in aquatic animals (Horiguchi et al., 1994). Reproduc-tive impairment in gastropods has been reported at low TBT concentrations (Gibbs et al., 1988). TBT has been shown to induce imposex and intersex in brosobranchs (Oehlmann et al., 1998), and impaired reproductive function in fish has also been described (Mc Allister and Kime, 2003).Some studies have reported toxic effects of TBT such as morphological and functional alterations of teleost gills in aquatic media (Byrne et al., 1989; Schwaiger et al., 1992; Tsu -da et al., 1992; Wang and Huang, 1998). TBT has also been shown to markedly inhibit fish hepatic CYP and in particu-lar the isozyme cytochrome P4501A1 (CYP1A), both in vivo and vitro (Fent and Bucheli, 1994; Morcillo and Porte, 1997). The CYP1A isozyme plays a key role in the biotransformation of xenobiotic compounds, leading to either their detoxifica-tion or bioactivation, while other CYP forms are important

Sex-related differences in rat liver microsomal enzymes and their induction by doxapram.

The effects of doxapram on the hepatic microsomal mono-oxygenase system of male and female rats were investigated. Male and female rats were administered doxapram (10-120 mg kg-1 day-1, i.p.) for 4 days. In female rats, administration of doxapram (20, 40, 60, 80, 100 and 120 mg kg-1) elevated the parameters in a dose-dependent manner while doxapram (100 and 120 mg kg-1) elevated the levels of cytochrome P450 and hexobarbitone hydroxylase in male rats. Doxapram (40 mg kg-1) caused induction of hepatic drug metabolism typified by an increase of hepatic microsomal cytochrome P450 content and activities of hexobarbitone hydroxylase, benzphetamine N-demethylase and ethylmorphine N-demethylase in female rats, but no change in male rats. These findings were supported by the results of SDS/polyacrylamide-gel electrophoresis. However, 7-ethoxycoumarin O-de-ethylase and arylhydrocarbon hydroxylase activities were significantly increased in male rats. NADPH-cytochrome c reductase and NADH-cytochrome c reductase activities, and cytochrome b5 content were unaffected in rats of both sexes. The sex-dependent cytochrome P450 species may be selectively sensitive to the action of doxapram.

Inhibition of hepatic microsomal monooxygenase activity by cinchocaine: mechanistic studies and effects of ionization.

The quinoline-based local anaesthetic cinchocaine (dibucaine) was found to be a mixed-type inhibitor of microsomal aminopyrine N-demethylase and 7-ethoxycoumarin O-deethylase activities from control and phenobarbitone-induced rat liver in-vitro. Cinchocaine also elicited a characteristic type I optical difference spectrum in oxidized liver microsomes (Ks = 24 microM; delta Amax = 3.4 X 10(-3) absorbance units (nmol cytochrome P450)-1) but did not appear to bind to the reduced form of the cytochrome. Additional studies indicated that cinchocaine competitively inhibited the type I spectral binding of substrate (aminopyrine) to ferric cytochrome P450. Studies of monooxygenase inhibition by cinchocaine over a relatively narrow pH range (6.5-8.5) indicated that, as might be expected, the un-ionized form of the drug is associated with inhibitory potency superior to that of the ionized form. Thus 40% inhibition of aminopyrine N-demethylase activity was observed with 100 microM cinchocaine at pH 8.0 and 8.5 (24% and 50% un-ionized drug, respectively), whereas only 16% inhibition was observed at pH 6.5 (1% un-ionized drug). These findings suggest that the inhibitory action of cinchocaine is mediated exclusively via an interaction with ferric cytochrome P450 and that the extent of ionization is a determinant of mixed function oxidase inhibition.

Comprehensive Spectroscopic, Steady State, and Transient Kinetic Studies of a Representative Siderophore-associated Flavin Monooxygenase

Many siderophores used for the uptake and intracellular storage of essential iron contain hydroxamate chelating groups. Their biosyntheses are typically initiated by hydroxylation of the primary amine side chains of l-ornithine or l-lysine. This reaction is catalyzed by members of a widespread family of FAD-dependent monooxygenases. Here the kinetic mechanism for a representative family member has been extensively characterized by steady state and transient kinetic methods, using heterologously expressed N(5)-l-ornithine monooxygenase from the pathogenic fungus Aspergillus fumigatus. Spectroscopic data and kinetic analyses suggest a model in which a molecule of hydroxylatable substrate serves as an activator for the reaction of the reduced flavin and O(2). The rate acceleration is only ∼5-fold, a mild effect of substrate on formation of the C4a-hydroperoxide that does not influence the overall rate of turnover. The effect is also observed with the bacterial ornithine monooxygenase PvdA. The C4a-hydroperoxide is stabilized in the absence of hydroxylatable substrate by the presence of bound NADP(+) (t(½) = 33 min, 25 °C, pH 8). NADP(+) therefore is a likely regulator of O(2) and substrate reactivity in the siderophore-associated monooxygenases. Aside from the activating effect of the hydroxylatable substrate, the siderophore-associated monooxygenases share a kinetic mechanism with the hepatic microsomal flavin monooxygenases and bacterial Baeyer-Villiger monooxygenases, with which they share only moderate sequence homology and from which they are distinguished by their acute substrate specificity. The remarkable specificity of the N(5)-l-ornithine monooxygenase-catalyzed reaction suggests added means of reaction control beyond those documented in related well characterized flavoenzymes.

DOSE- AND TIME-DEPENDENT EFFECTS OF 2,3,7,8-TETRABROMODIBENZO-P-DIOXIN ON RAT LIVER

Dose- and time-dependent effects of 2,3,7,8-tetrabromodibenzo-p-dioxin (TBDD) on the liver were examined by single administration of TBDD by gavage to male and female rats. Fifteen Wistar rats of each sex per group received 0, 10, 30, 100 or 300 microg TBDD/kg body weight. Rats surviving to scheduled necropsy on Day 2, 7 or 36 after the TBDD administration were examined for hepatic histopathology, activities of hepatic microsomal enzymes and serum levels of lipids, total cholesterol and transaminases and hepatic concentrations of TBDD. Tigroid basophilic cytoplasm and hepatocellular hypertrophy were observed at 10 microg/kg on Day 2 or 7 through 36, whereas degenerative and aggressive lesions such as necrosis, fibrosis, multinucleated hepatocytes and disarrangement of hepatocytes occurred later at higher dose levels. Persistently increased activities of hepatic aryl hydrocarbon hydroxylase (AHH), ethoxycoumarin O-deethylase (ECOD) and ethoxyresorufin O-deethylase (EROD), increased serum levels of total cholesterol and phospholipid and increased relative liver weight were observed in all groups dosed 10 mug/kg and above, suggesting that hepatic microsomal monooxygenases and basophilic cytoplasm of hepatocytes were early and sensitive indicators among those TBDD-induced effects. A dose-dependent increase in liver concentrations of TBDD on Day 2 was followed by logarithmic decreases in TBDD concentrations against the days elapsed after the TBDD administration. An elimination half-life (t(1/2)) of TBDD from the liver was estimated to range from 12 to 16 days. It was suggested that females were more susceptible to TBDD than males, and that acute hepatotoxicity of TBDD was as potent as that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

Communicated by A.A. Maradudin on 15 August 2005

Antioxidants inhibit the propagation of free radical reactions. Because of their widespread use as preservatives in processed foods, their biochemical effects have been investigated. Early feeding studies indicated that butylated hydroxytoluene (BHT) increased liver weight, induced proliferation of smooth endoplasmic reticulum, and elevated several hepatic microsomal mono-oxygenase activities typical of phase 1 metabolism. In addition to phase1, antioxidants also induce phase2 xenobiotic-metabolizing enzymes. Phase2 enzymes detoxify activated electrophilic metabolites of xenobiotics via conjugation of endogenous substrates such as glutathione (GSH). Antioxidants act to directly terminate the propagation of free radical reactions, and increase the activity of enzymes those readily metabolize and aid in the elimination of potential cytotoxic chemicals. Dietary administration of antioxidants induced phase 2 xenobiotic-metabolizing enzymes. Induction of phase 2 enzymes by antioxidants was found in many organs and tissues, such as liver, lung, kidney, small intestine, colon, and spleen, thereby affording protection at many anatomical sites .This chapter presents evidence for several newly identified antioxidant-inducible genes, describes the proposed mechanisms of antioxidant signal transduction leading to enhanced expression of these enzymes, and complements the information presented in related reviews concerning the mechanisms and consequences of induction of phase 2 xenobiotic-metabolizing enzymes by antioxidants. The chapter discusses the genes induced by antioxidants—cytochrome P450s, Glutathione, S-transferases, NAD(P)H: quinone reductase, UDP-glucuronosyltransferases, microsomal epoxide hydrolase, aflatoxin BI-aldehyde reductase, dihydrodiol dehydrogenases, aldehyde dehydrogenases, enzymes of glutathione and reduced nicotinamide metabolism, and other proteins and enzymes. The chapter delves into the mechanisms of gene induction by antioxidants—the antioxidant-response element (ARE), proteins binding and signal transduction through the ARE—and the consequences of antioxidant gene induction. The regulation of antioxidant-inducible genes helps understand the signals leading to cellular transformation and carcinogenesis. Antioxidants can be used to decipher the encrypted signal transduction pathways that prevent cell transformation and carcinogenesis.

Effects of Light and Dark Beer on Hepatic Cytochrome P-450 Expression in Male Rats Receiving Alcoholic Beverages as Part of Total Enteral Nutrition

Alcoholic beverages contain many congeners in addition to ethanol. Therefore, consumption of alcoholic beverages may have considerably different effects on expression of hepatic microsomal monooxygenases than the relatively selective induction of cytochrome P-450 (CYP) 2E1 observed after consumption of pure ethanol. : In the current study, we compared the effects of two beers: lager (a light roasted beer) and stout (a dark roasted beer) with those of an equivalent amount of pure ethanol on hepatic CYP expression. Beer or pure ethanol was part of a complete total enteral nutrition diet that was infused intragastrically into male Sprague Dawley rats for 21 days. At the end of the infusion period, rats were euthanized, and liver and intestinal microsomes were prepared. Expression and activity of CYP1A1/2, CYP2B1, CYP2E1, CYP3A, and CYP4A were assessed by Western immunoblotting and by using CYP enzyme-specific substrates, respectively. mRNA and protein levels of CYP4A1 were elevated only in stout-treated animals. However, lauric acid 12-hydroxylase activity (a CYP4A-specific activity) was reduced (p < or = 0.05) in microsomes from lager- and stout-fed rats. After oxidation with potassium ferricyanide, this activity was significantly increased in microsomes from stout-fed animals. The relative expression of CYP2E1 and CYP2B1 and the activities toward p-nitrophenol, pentoxyresorufin, or benzyloxyresorufin did not differ between beers or compared with pure ethanol or controls. However, the mean expression of CYP1A2, CYP3A, and CYP4A apoproteins was greater in liver microsomes from stout-infused rats than in those from lager-infused rats, ethanol-infused rats, and diet controls (p < or = 0.05). In addition, although no significant differences were observed in ethoxyresorufin O-dealkylase (EROD), methoxyresorufin O-dealkylase (MROD), midazolam, or testosterone hydroxylase activities between groups, stout-infused rats had greater hepatic microsomal erythromycin N-demethylase activity than other groups (p < or = 0.05). Stout contains components other than ethanol that interact in a complex fashion with the monooxygenase system.

O-Aminoazotoluene does induce the enzymes of its own mutagenic activation in mouse liver

The objective of this study was to investigate the CYP1A1 and CYP1A2 mRNAs and enzyme activities in mouse liver during induction with o-aminoazotoluene (OAT) as well as the capability of the hepatic S9-fraction from OAT-treated mice to induce its own activation to mutagens in the Ames test using S. typhymurium strain TA98. The data obtained indicate that when used at appropriate doses, OAT is a PAH-type inducer of mouse hepatic microsomal monooxygenases, which activity is not less than that of the known inducer 3,4-benzo[α]pyrene. In the absence of S9-fraction enzymes no OAT-mediated mutagenicity was observed in the Ames test. In the presence of the S9-fraction from OAT-pretreated mice, OAT induced as high revertant numbers, as it did in the presence of the S9 fraction from the liver of Aroclor 1254-treated mice. Thus, OAT does induce the enzymes of its own mutagenic activation in mouse liver.

Effects of the mosquito larvicide GB-1111 on red-winged blackbird embryos

Golden Bear Oil (GB-1111; legal trade name for GB-1313) is a petroleum distillate that is used in the United States and other countries as a larvicide for mosquito suppression. As part of a multi-species evaluation of the potential effects of GB-1111 on birds, red-winged blackbird eggs were collected, artificially incubated, and treated with one of five amounts of GB-1111 varying from 0 to 10 times the expected exposure from a spray application of the maximum recommended amount ( X=47 l/ha, 5 gal/ac). The application of 10 X caused a significant reduction in hatching success. A dose-related reduction of hepatic microsomal monooxygenase activity (EROD) was detected. Among body weights, skeletal measurements, and age at death, only crownrump length was different among experimental groups. Overall, the potential hazard to embryos of a representative wetland passerine appears minimal until the application rate exceeds 3 X.

Hepatotoxicity and aging: endogenous antioxidant systems in hepatocytes from 2-, 6-, 12-, 18- and 30-month-old rats following a necrogenic dose of thioacetamide

The influence of aging on the mechanisms of liver injury and regeneration was studied in a model of hepatotoxicity induced in 2-, 6-, 12-, 18- and 30-month-old rats by a sublethal dose of thioacetamide (500 mg/kg body weight), a soft nucleophilic and hepatotoxic compound metabolized by the hepatic microsomal FAD monooxygenase system. Samples—blood and hepatocytes—were obtained at 0, 12, 24, 48, 72 and 96 h following thioacetamide intoxication. Parameters of liver injury in serum (NADPH-isocitrate dehydrogenase (ICDH) activity) indicate that the severity of injury was significantly higher in the adult groups (6 and 12 months old) when compared either with the youngest (2 months old) or oldest (18 and 30 months old) groups. Parameters related to biotransformation, such as microsomal FAD monooxygenase, followed mainly the same pattern of age-dependent changes as those observed for injury. The profile of glutathione-S-transferase activity showed an initial induction parallel to liver injury and opposite to the levels of reduced glutathione and protein -SH groups. Enzyme activities and gene expression of the systems involved in the cell endogenous antioxidant defense, such as Mn- and Cu,Zn-superoxide dismutases (SOD), catalase and glutathione peroxidase (GPX) showed significant age-dependent changes that can be summarized as follows: an increase in all enzyme activities and gene expression and a decreased ability to restore the initial activities following 96 h of thioacetamide. We conclude, first, that the gene expression and activity of the enzymes involved in the intracellular antioxidant defense system increased with aging, which can be considered a consequence of the enhanced oxidative state of the cell (decreased in GSH level); and second, that the lower and delayed response in the aged groups significantly influenced the restoration towards normal of GSH and the antioxidant enzyme activities.

The effect of hepatic microsomal cytochrome P450 monooxygenases on monensin–sulfadimidine interactions in broilers

Journal of Veterinary Pharmacology and TherapeuticsVolume 24, Issue 1 p. 73-76 The effect of hepatic microsomal cytochrome P450 monooxygenases on monensin–sulfadimidine interactions in broilers E. Ershov, E. Ershov Kimron Veterinary Institute, P.O. Box 12, Bet Dagan 50250, IsraelSearch for more papers by this authorM. Bellaiche, M. Bellaiche Kimron Veterinary Institute, P.O. Box 12, Bet Dagan 50250, IsraelSearch for more papers by this authorV. Hanji, V. Hanji Kimron Veterinary Institute, P.O. Box 12, Bet Dagan 50250, IsraelSearch for more papers by this authorS. Soback, S. Soback Kimron Veterinary Institute, P.O. Box 12, Bet Dagan 50250, IsraelSearch for more papers by this authorM. Gips, M. Gips Kimron Veterinary Institute, P.O. Box 12, Bet Dagan 50250, IsraelSearch for more papers by this authorY. Weisman, Y. Weisman Kimron Veterinary Institute, P.O. Box 12, Bet Dagan 50250, IsraelSearch for more papers by this authorA. Shlosberg, A. Shlosberg Kimron Veterinary Institute, P.O. Box 12, Bet Dagan 50250, IsraelSearch for more papers by this author E. Ershov, E. Ershov Kimron Veterinary Institute, P.O. Box 12, Bet Dagan 50250, IsraelSearch for more papers by this authorM. Bellaiche, M. Bellaiche Kimron Veterinary Institute, P.O. Box 12, Bet Dagan 50250, IsraelSearch for more papers by this authorV. Hanji, V. Hanji Kimron Veterinary Institute, P.O. Box 12, Bet Dagan 50250, IsraelSearch for more papers by this authorS. Soback, S. Soback Kimron Veterinary Institute, P.O. Box 12, Bet Dagan 50250, IsraelSearch for more papers by this authorM. Gips, M. Gips Kimron Veterinary Institute, P.O. Box 12, Bet Dagan 50250, IsraelSearch for more papers by this authorY. Weisman, Y. Weisman Kimron Veterinary Institute, P.O. Box 12, Bet Dagan 50250, IsraelSearch for more papers by this authorA. Shlosberg, A. Shlosberg Kimron Veterinary Institute, P.O. Box 12, Bet Dagan 50250, IsraelSearch for more papers by this author First published: 07 July 2008 https://doi.org/10.1046/j.1365-2885.2001.00299.xCitations: 6 SHLOSBERG Kimron Veterinary Institute, P.O. Box 12, Bet Dagan 50250, Israel Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Citing Literature Volume24, Issue1February 2001Pages 73-76 RelatedInformation

Toxicity of Anacostia River, Washington, DC, USA, sediment fed to mute swans (Cygnus olor)

AbstractSediment ingestion is sometimes the principal route by which waterfowl are exposed to environmental contaminants, and at severely contaminated sites waterfowl have been killed by ingesting sediment. Mute swans (Cygnus olor) were fed a diet for 6 weeks with a high but environmentally realistic concentration (24%) of sediment from the moderately polluted Anacostia River in the District of Columbia, USA, to estimate the sediment's toxicity. Control swans were fed the same diet without the sediment. Five organochlorine compounds were detected in the treated diets, but none of 22 organochlorine compounds included in the analyses was detected in livers of the treated swans. The concentrations of 24 polynuclear aromatic hydrocarbons measured in the treated diet were as high as 0.80 mg/kg, and they were thought to have been responsible for the observed induction of hepatic microsomal monooxygenase activity in livers. A concentration of 85 mg/kg of lead in the diet was enough to decrease red blood cell ALAD activity but was not high enough to cause more serious effects of lead poisoning. The dietary concentrations of Al, Fe, V, and Ba were high compared to the concentrations of these elements known to be toxic in laboratory feeding studies. However, the lack of accumulation in the livers of the treated swans suggested that these elements were not readily available from the ingested sediment. We did not study all potential toxic effects, but, on the basis of those that we did consider, we concluded that the treated swans were basically healthy after a chronic exposure to the sediment.

Potentiation of thioacetamide hepatotoxicity by phenobarbital pretreatment in rats. Inducibility of FAD monooxygenase system and age effect

The ability of phenobarbital to induce the expression and activity of microsomal drug monooxygenases in the liver presents one of the most important issues in the field of chemical interactions and in the toxicity of xenobiotics. The model of rat liver injury induced by a single dose of thioacetamide (500 mg/kg intraperitoneally) was used to study the effect of phenobarbital (80 mg/kg/day intraperitoneally) for 5 days prior to thioacetamide. Serum parameters of liver injury such as aspartate aminotransferase activity, gamma-glutamyl transferase activity and the total bilirubin levels, as well as the activities of hepatic FAD and cytochrome P450 microsomal monooxygenases, were assayed in 2- and 12-month-old rats. Samples of blood and liver were obtained from controls (injected at 0 h with 0.5 ml of 0.9% NaCl) and at 12, 24, 48, 72 and 96 h of thioacetamide intoxication either to non-treated or phenobarbital pretreated rats. Potentiation of thioacetamide hepatotoxicity by phenobarbital pretreatment was demonstrated at morphological level, and by significant increases in the activities of serum aspartate aminotransferase and gamma-glutamyl transferase, and in the levels of total bilirubin. The extent of potentiation of thioacetamide-induced liver injury by phenobarbital pretreatment was similar in both age groups. Microsomal FAD monooxygenase activity, the enzyme responsible for thioacetamide biotransformation, was significantly enhanced (twofold) by phenobarbital pretreatment, and also underwent a further increase following thioacetamide, preceding the peak of necrosis. Cytochrome P-450 monooxygenases were induced by phenobarbital pretreatment more than sixfold, and sharply decreased when phenobarbital was withdrawn and thioacetamide administered, showing at 48 h intoxication values close to basal. Phenobarbital pretreatment potentiated thioacetamide necrogenicity, and this potentiation was parallel to the induction of the microsomal FAD monooxygenase system, both by phenobarbital and by thioacetamide itself. The extent of thioacetamide-induced liver injury was significantly higher in 12-month-old rats, but the effect of phenobarbital pretreatment was similar in both age groups.

Characterizations of mouse hepatic microsomal monooxygenase catalyzing 11β-hydroxylation of osaterone acetate

Osaterone acetate (17α-acetoxy-6-chloro-2-oxa-4,6-pregnadiene-3,20-dione, OA) is a new steroidal antiandrogen. There is a marked species difference in the metabolism of OA in that 11β-hydroxylated metabolites are found in the plasma, feces, and urine of mice after oral administration of OA, but there is very little metabolism in rats and humans. OA reduces the adrenal gland weight in mice, but not in rats, and this effect in mice might be explained by the species difference in 11β-hydroxylation activity. The objectives of this study were to elucidate the enzyme(s) involved in this particular oxidation and to explain the species difference observed. Mouse hepatic microsomes oxidize OA to 11β-OH OA, and this oxidation requires NADPH as a cofactor. The use of various competitive and allosteric inhibitors of cytochrome P450 and flavin-containing monooxygenase (i.e. CO, N-octylamine, and methimazole) showed that the oxidation of OA was catalyzed by cytochrome P450. In microsomes from mice pretreated with phenobarbital (a CYP2B-selective inducer), 3-methylcholanthrene (a CYP1A-selective inducer), pregnenolone-16α-carbonitrile (a CYP3A-selective inducer), and EtOH (a CYP2E-selective inducer), an increase in the rates of oxidation was seen only in microsomes from EtOH-treated animals. However, metyrapone, a selective inhibitor for enzymes of the cytochrome P45011B and P4502B family, inhibited mouse hepatic microsomal 11β-hydroxylation by < 30%. The results obtained showed that the production of 11β-OH OA may be catalyzed by a novel cytochrome P450 in mouse liver.

Effect of enzyme imprinting of liver microsomal monooxygenases upon lifespan of rats

Effect of the enzyme imprinting by phenobarbital upon alterations of hepatic microsomal monooxygenase activities and lifespan of Wistar rats has been studied. Phenobarbital-sodium (3.5 mg/100 g body weight per day, i.p.) was injected during 1–3 days after birth. This resulted in the enzyme imprinting of the liver microsomal monooxygenases, however, this effect being observed in female but not male rats. In the phenobarbital treated female rats of different age the duration of sleeping time was significantly lower than that in control animals, whereas it did not differ substantially in male rats. The cytochrome P-450 content increased by 34.5% in phenobarbital treated female rats in the age of 12 months in comparison with control animals. A mean lifespan of experimental female rats increased by 17.5% compared to the level of control animals and did not change in male rats. The analysis of survival of animals in Gompertz equation coordinates showed that enzyme imprinting by phenobarbital caused changes in the mortality patterns at different stages of ontogenesis in experimental female but not male rats. An inverse correlation was found between the duration of pentobarbital sleeping time and lifespan of female and male rats.

A comparison of basal and induced hepatic microsomal cytochrome P450 monooxygenase activities in the cynomolgus monkey (Macaca fascicularis) and man

1. The specific activities of hepatic microsomal cortisol 6β-hydroxylase, coumarin 7-hydroxylase, S-mephenytoin 4'-hydroxylase and phenoxazone hydroxylase and the O-dealkylations of seven homologous alkoxyresorufins were < 3-fold different between the untreated (UT) cynomolgus monkey and man. 2. Heptoxy- and octoxyresorufin O-dealkylase, S-mephenytoin N-demethylase and dextromethorphan O-demethylase specific activities were > 6-fold higher, whereas tolbutamide hydroxylase was almost 5-fold lower in the UT monkey than in man. 3. Phenobarbitone induced (2-6-fold) coumarin 7-hydroxylase, cortisol 6β-hydroxylase, S-mephenytoin N-demethylase, phenoxazone hydroxylase and benzyloxyresorufin O-dealkylase activities, but not the O-dealkylations of pentoxyresorufin or other alkoxyresorufins, in monkey. 4. Rifampicin induced (2-3-fold) cortisol 6β-hydroxylase, S-mephenytoin 4′-hydroxylase, S-mephenytoin N-demethylase and tolbutamide hydroxylase activities, the O-dealkylations of methoxy-, ethoxy- and propoxyresorufin and CYP2C- and CYP3A- immunorelated proteins in monkey. 5. Dextromethorphan O-demethylase was significantly reduced by both phenobarbitone and rifampicin treatment in monkey. 6. β-Naphthoflavone induced (8-39-fold) the O-dealkylations of several alkoxyresorufins, the greatest effect being on propoxyresorufin, but had no effect on the other activities measured in monkey. 7. Constitutive hepatic microsomal CYP2D6-immunorelated proteins were expressed at apparently much higher levels in monkey than in man.

Lack of developmental and reproductive toxicity of 2,3,3',4, 4'-pentachlorobiphenyl (PCB 105) in ring-necked pheasants.

Mono-ortho PCBs are global contaminants of wildlife with the potential to produce toxicity by an aryl hydrocarbon receptor (AhR)-mediated mechanism. To determine the potency of 2,3,3',4, 4'-pentachlorobiphenyl (PCB 105) for producing reproductive and developmental toxicity, adult ring-necked pheasant hens (Phasianus colchicus) were orally dosed with 0, 0.06, 0.6, or 6 mg PCB 105/kg hen/week for 10 weeks to achieve cumulative doses of 0, 0.6, 6, or 60 mg PCB 105/kg hen after which hens were bred with untreated roosters once per week for 8 weeks. Except at week 6 of the egg-laying period when cumulative egg production in the 6 mg PCB 105/kg hen group was greater than controls, fertilized egg production was not significantly different between treatment groups. Embryo mortality and chick mortality were not significantly different between treatment groups. Total body and heart weights of all chicks 1 day posthatch (dph) were not different between groups, however, liver weights of chicks from the 60 mg/kg treatment group were greater than controls at 1 dph. The first chick to hatch from each hen was reared to 21 dph and among these birds, the total body, liver, and heart weights were not different between groups. There were no dose-related malformations of the beak or limbs, and no signs of subcutaneous edema, ascites, or pericardial edema in chicks at 1 or 21 dph. Hepatic microsomal monooxygenase activities [ethoxyresorufin-O-dealkylase (EROD), benzyloxyresorufin-O-dealkylase (BROD), and methyloxyresorufin-O-dealkylase (MROD)] were significantly elevated in chicks at 1 dph from hens given a cumulative PCB 105 dose of 6 mg/kg and in chicks at 21 dph from hens given a cumulative PCB dose of 60 mg/kg. These results indicate that a cumulative PCB 105 dose up to 60 mg/kg hen does not decrease the production of fertilized eggs or increase embryo or chick mortality in ring-necked pheasants, but does increase chick hepatic monooxygenase activity.

Hepatic P450 monooxygenase response in rainbow trout (Oncorhynchus mykiss (Walbaum)) administered aquaculture antibiotics

The effects of the 4-quinolones, oxolinic acid and flumequine on hepatic microsomal cytochrome P450 monooxygenases in rainbow trout were examined during this study. Following antibiotic administration in the diet for 10 days at a representative commercial medicated feed concentration, fish were killed for assay at various periods up to 12 days following a return to normal diet. Ethoxyresorufin O-deethylase (EROD) and benzyloxyresorufin O-dealkylase (BROD) activities were significantly elevated as a result of antibiotic treatment. The effects of oxolinic acid were delayed and longer-lasting compared with flumequine. The effects of flumequine were detectable 4 days earlier than those of oxolinic acid and lasted for less than ten days after treatment cessation. In contrast, oxolinic acid effects were apparent even on day 12 of the recovery period. The results of O-dealkylation, isoform-selective inhibition, and immunoblotting showed that the effects of both oxolinic acid and flumequine were related to P4501A subfamily. P450-binding spectra and experiments in vitro suggested that both antibiotics are weak dose-independent inhibitors of P450 activity, with flumequine exhibiting slightly higher binding affinity and inhibitory activity.

Changes in hepatic cytochrome P450 enzymes by cis- and trans-1,2-dichloroethylenes in rat

1. We examined the effect of cis- and trans-1,2-dichloroethylen es (DCEs) on hepatic microsomalcytochrome P450 (P450) enzymes in the male and female rat. Rats were treated intraperitoneal ly with 7 5 mmol kg cis- or trans-1,2-DCE daily for 4 days. 2. Among the hepatic microsomal P450-dependent monooxygenase activities tested, testosterone 2α-hydroxylase (T2AH) activity in the male rat, which is associated with CYP2C11, was decreased by both cis- and trans-1,2-DCE isomers. The levels to control activities were 53 and 63 % respectively. Furthermore, immunoblottingshowed that both isomers significantly reduced the CYP2C11 6 protein level in liver microsomes from the male. The levels of testosterone 6β-hydroxylase (T6BH) activity and CYP3A2 1 protein in the male rat were reduced by cis-1,2-DCE but not trans-1,2-DCE. 3. cis-1,2-DCE decreased ethoxycoumarin O-deethylase (ECOD), benzyloxyresorufin O-debenzylase (BROD), chlorzoxazone 6-hydroxylase (CZ6H) and testosterone 7α-hydroxylase (T7AH) activities in the male rat by 29, 28, 34 and 27 % respectively. On the other hand, trans-1,2-DCE significantly increased 7-ethoxyresorufin O-deethylase (EROD) and 7-methoxyresorufin O-demethylase (MROD) activities in the male rat 1.4- and 1.9-fold respectively. Immunoblottingshowed that cis-1,2-DCE reduced CYP1A1 2 and CYP2B1/2 protein levels, whereas trans-1,2-DCE increased CYP1A1/2 and CYP2B1/2 protein levels in the male rat. 4. The activities of other P450-dependent monooxygenases, namely benzphetamine N-demethylase (BZND), aminopyrine N-demethylase (APND), erythromycin N-demethylase (EMND) and lauric acid ω-hydroxylase (LAOH), in the male rat were hardly affected by either 1,2-DCE isomer. In the female rat there were no apparent changes in P450-dependent monooxygenase activities upon treatment with the 1,2-DCE isomers. 5. These results suggest that 1,2-DCEs mainly affect male-specific P450 isoforms in the rat liver and that these changes may relate to the toxicity of 1,2-DCEs.

Quantitation and kinetic properties of hepatic microsomal and recombinant flavin-containing monooxygenases 3 and 5 from humans

Variable amounts of flavin-containing monooxygenase isoforms 3 and 5 (FMO3 and FMO5) are present in microsomal preparations from adult, male, human liver. Quantitation with monospecific antibodies and recombinant isoforms as standards showed levels of FMO3 and of FMO5 that ranged from 12.5 to 117 and 3.5 to 34 pmol/mg microsomal protein, respectively. The concentration of FMO3 was greater than that of FMO5 in all samples, but the ratio of FMO3 to FMO5 varied from 2:1 to 10:1. Human hepatic microsomal samples also showed variable activities for the S-oxidation of methimazole. This activity was associated totally with FMO3; no participation of FMO5 was apparent. This conclusion was supported by several lines of evidence: first, the catalytic efficiency of FMO3 with methimazole was found to be ∼5000 times greater than that of FMO5; second, the rate of metabolism showed a direct, quantitative relationship with FMO3 content; third, the plot of the relationship between metabolism and FMO3 content extrapolated close to the origin. A second reaction, the N-oxidation of ranitidine, exhibited a much higher K m with recombinant FMO3 than did methimazole (2 mM vs. 35 μM). However, a direct relationship between this reaction and FMO3 content in human hepatic microsomal preparations was also apparent. This result shows that even with a high K m substrate, FMO3-catalyzed metabolism can account for the majority of the product formation with some drugs. Our findings demonstrate that the contribution of FMO isoforms to human hepatic drug metabolism can be assessed quantitatively on the basis of the characteristics of the enzymes expressed in Escherichia coli