Total P450 Content Research Articles

Alkaloid metabolism by cytochrome P-450 enzymes in Drosophila melanogaster

The cytochrome P-450 family of enzymes is the primary means of foreign compound detoxification in virtually all organisms. Cytochrome P-450s have been strongly implicated in the metabolism of cactus alkaloids, and consequently, the observed patterns of host plant utilization by cactophilic species of Drosophila in the Sonoran Desert. The current study looked for evidence of alkaloid-metabolizing P-450 enzymes in a non-cactophilic species, D. melanogaster. The results of in vitro metabolism assays indicate the presence of a phenobarbital-inducible P-450 in adult D. melanogaster which is capable of metabolizing alkaloids. P-450 quantification data suggest that the enhanced level of metabolism is not the result of an overall increase in total P-450 content. Results from larval viability and adult longevity studies indicate that D. melanogaster's in vitro activity does not produce an enhanced in vivo tolerance of alkaloids.

5-epi-Aristolochene 3-hydroxylase from green pepper

5-epi-Aristolochene 3-hydroxylase, one of the key enzymes of the biosynthetic pathway leading to capsidiol, a major phytoalexin of green pepper ( Capsicum annuum), is localized in the microsomes of elicitor treated green pepper fruit. The enzyme was found to be a P-450-dependent enzyme with an estimated molecular mass of 59 kDa. The total microsomal P-450 content increased and the λ max of the CO-binding spectrum changed in response to elicitation. Immunoblot analysis of fruit microsomal proteins was performed with anti-tulip constitutive P-450 antibody during the elicitation period. A 48 kDa protein strongly immunoreacted with the P-450 antibody, and a decrease of this P-450-like 48 kDa protein was observed with time. These results suggest that elicitation induces the production of P-450 isoforms.

Effects of 5-fluorouracil treatment on rat liver microsomal enzymes.

1. The effects of the administration of the anticancer drug 5-fluorouracil on P450-mediated metabolizing capacity, and the activity of several related enzymes were examined by comparing two different dosage regimes, i.e. a single ip dose of 5-fluorouracil (120 mg/kg) at 1, 4 and 7 days prior to killing, and a repeated dose regimen of 5-fluorouracil (24 mg/kg/day for 5 consecutive days) with animals being killed at 1, 4 and 7 days after the last 5-fluorouracil dose. 2. Rat liver microsomes were assayed for total P450 content, reduced nicotinamide adenine dinucleotide phosphate-P450-reductase, aminopyrine demethylase, cyclophosphamide 4-hydroxylase, aniline hydroxylase, androstenedione 6 beta- and 16 alpha-hydroxylase and delta 4-3-oxo-steroid 5 alpha-oxidoreductase activity. 3. The animals that received 5-fluorouracil as a single, high dose did not show any alterations in any of the measured parameters at any of the times at which they were killed. 4. The animals that received chronic low doses of 5-fluorouracil, however, showed a statistically significant decrease in androstenedione 6 beta- and 16 alpha-hydroxylase and cyclophosphamide 4-hydroxylase activity and a statistically significant increase in delta 4-3-oxo-steroid 5 alpha-oxidoreductase activity 4 and 7 days after completion of dosing. 5. Immunoquantified P4502C11 and 3A content was also decreased at these time points. These alterations occurred concurrently with depleted plasma testosterone levels in the groups that received repeated low doses of 5-fluorouracil.

Modification of hepatic cytochrome P450 profile by cocaine-induced hepatotoxicity in DBA/2 mouse

Previous studies in our laboratory have shown that a hepatotoxic dose of cocaine increases coumarin 7-hydroxylase activity in male DBA/2 mouse liver. In the present study, the dose- and time-dependent responses of the hepatic CYP2A4/5 complex to cocaine-induced liver damage were studied. Cocaine increased CYP2A4/5 levels in a dose-dependent manner. The maximal increases in coumarin 7-hydroxylase activity (4-fold), microsomal CYP2A4/5 content (3-fold) and steady-state mRNA levels (10-fold) were observed at 24 h after administration of a single dose of 60 mg/kg cocaine coinciding with morphologically detectable diffuse liver damage, while the total P450 content was not changed. 3 and 5 days after the daily administration of cocaine severe, mainly pericentral (zone III of Rappaport), liver damage was apparent in parallel with a clear decline in CYP2A4/5 mRNA, protein content and coumarin 7-hydroxylase activity. After 5 days of treatment, CYP2A5 still remained at a very low level but an induction in CYP2B10 protein and related pentoxyresorufin O-dealkylase activity was observed. No marked changes in microsomal CYP2Cx and CYP1A1/2 contents or associated activities were observed. Dimethylnitrosamine N-demethylase activity, a marker for CYP2E1, decreased in parallel with increased cocaine dose and time and the severity of liver damage. Our results demonstrate that (i) administration of cocaine causes a clear but transient increase in the expression of Cyp2a-4/5 gene complex prior to overt liver damage and (ii) microsomal CYP2B10 and related 7-pentoxyresorufin O-dealkylase activity is markedly increased in animals treated for 5 days with cocaine concomitantly with the decrease in other monooxygenases indicating an association of coumarin 7-hydroxylase activity with liver injury and different roles for coumarin 7-hydroxylase and 7-pentoxyresorufin O-dealkylase activities in cocaine hepatotoxicity.

Toxicity of bromodichloromethane in female rats and mice after repeated oral dosing

The carcinogenic water disinfection byproduct, bromodichloromethane (BDCM), produces renal and hepatic toxicity in rodents in acute and subchronic studies. In the present investigation, female rats and mice ( n=6) were dosed daily for 5 consecutive days with BDCM (dissolved in an aqueous, 10% Emulphor solution) by gavage. Rats received 75, 150 and 300 mg BDCM/kg body weight/day and mice received 75 and 150 mg BDCM/kg body weight/day. Two rats in the 300 mg/kg/day treatment group died on day 5. On day 6, the animals were sacrificed and serum samples were taken for analysis of indicators of hepatic and renal toxicity. Livers and kidneys were excised and samples taken for histopathological evaluation. Portions of the livers were also utilized to produce microsomes for analysis of cytochrome P450 enzyme activities and total P450 content. Total hepatic cytochrome P450 was decreased in rats dosed with 150 and 300 mg BDCM/kg body weight/day, but was not significantly affected in BDCM-treated mice. Serum lactate (LDH) and sorbitol (SDH) dehydrogenase, aspartate aminotransferase (AST), creatinine and blood urea nitrogen were increased above those of controls in rats dosed with 300 mg BDCM/kg/day. These data suggested that hepatic and renal damage had occurred in this treatment group. This was confirmed by histopathologically analyses which revealed that lesions occurred in both hepatic and renal tissues from rats dosed with 150 and 300 mg BDCM/kg/day. The hepatic lesions were centrilobular and primarily consisted of vacuolar degeneration. The hepatotoxicity indicators alanine aminotransferase (ALT) and SDH were increased in mice dosed with 150 mg BDCM/kg/day. However, no histopathological lesions were observed in these animals. This study shows that BDCM is both hepatotoxic and nephrotoxic to female rats after repeated dosing, but is only weakly hepatotoxic to female mice at the administered doses. Also, reduced activities of hepatic cytochrome P450 were observed in rats, but not mice. These species differences in toxicity and xenobiotic metabolizing enzyme inhibition caused by BDCM suggest that an understanding of the mechanism of toxicity of this compound will be critical when extrapolating rodent toxicity data to humans for this environmental pollutant.

Induction of cytochrome P450 isozymes in rat renal microsomes by cyclosporin A

To examine the effects of cyclosporin A (CsA) on renal cytochrome P450 forms, CsA was administered to rats, and the renal levels of P450 were determined by immunoblotting. CsA treatment for 17 days increased total renal P450 content by 40% with a concomitant elevation the ω- and (ω-1)-hydroxylation activities of lauric acid. Arachidonic acid ω-hydroxylation activity was also induced 2-fold by treatment with CsA for 17 days. Among the P450 forms, CYP4A2 was induced significantly, whereas CYP2C23, CYP4A1 and CYP4A8 were unaffected. These changes were accompanied by slight but significant increases in blood urea nitrogen and systolic blood pressure. These data suggest that CsA increased arachidonic acid ω-hydroxylation activity by the induction of CYP4A2. The specific induction of CYP4A2 may be related to CsA-induced nephrotoxicity and elevated blood pressure, because ω-hydroxyarachidonic acid is a potent vasoconstrictor.

Induction, suppression and inhibition of multiple hepatic cytochrome P450 isozymes in the male rat and bobwhite quail ( Colinus virginianus) by ergosterol biosynthesis inhibiting fungicides (EBIFs)

Ergosterol biosynthesis inhibiting fungicides (EBIFs) have complex effects on the hepatic microsomal monooxygenase systems of vertebrate species, having been described as mixed inducers and inhibitors of cytochrome P450. In the current study, we examined the effects of two EBIFs in clinical use, clotrimazole and ketoconazole, and two agricultural EBIFs, propiconazole and vinclozolin, on hepatic monooxygenase activities and P450 apoprotein expression in the male Sprague-Dawley rat and the male bobwhite quail. EBIFs produced Type II binding spectra with hepatic microsomes from both species and were effective inhibitors of methoxyresorufin O-demethylase, an activity selective for P450 isozymes in gene family 1. However, the EBIFs varied widely in their effectiveness as inducers of P450 isozymes in gene families 1, 2, 3 and 4, both within the same species and between species. In the rat, clotrimazole was the most effective inducer, increasing expression of CYP 3A isozymes over 450-fold, CYP 2B1/2 30-fold and CYP 1A1/2 12-fold and suppressing expression of CYP 2C11 nearly 70%. By contrast, in the quail, clotrimazole was the least effective inducer. In quail, vinclozolin and propiconazole elevated total P450 content 10- and 7-hold, respectively. The induction response also appeared to be mixed, but in this case consisted of a 5-fold induction of P450s in gene family 1A, a 3-fold induction of P450s in gene family 3A and 4A, and induction of protein(s) from gene family 2, cross-reactive with antisera against rat CYP 2C11 and CYP 2A1. A protein that was cross-reactive with antibodies raised against rat CYP 2B1 was decreased with EBIF treatment. In conclusion, EBIFs have complex patterns of induction, suppression and inhibition of cytochrome P450 isozymes in both mammals and birds, which vary according to both the fungicide and the species.

Temporal aspects of induction of hepatic cytochrome P450 1A and conjugating enzymes in the viviparous blenny ( Zoarces viviparus) treated with petroleum hydrocarbons

The heavy gas oil fraction of distilled North Sea crude oil was intraperitoneally injected into the marine teleost viviparous blenny ( Zoarces viviparus). The effects were compared with those of the model inducer, β-naphthoflavone (β-NF), on the hepatic cytochrome P450 1A (P450 1A or CYP 1A) system and on conjugating enzymes in a time course study over 14 days. The injection of heavy gas oil resulted in temporal responses in P450 1A protein content and P450 1A catalytic activity similar to those obtained with β-NF, although differences existed in terms of their effects on CYP1A1 mRNA levels and on the total P450 content. In contrast to β-NF, heavy gas oil had no effect on UDP-glucuronosyltransferase, glutathione S-transferase and glutathione reductase activities. GC-MS analyses of pooled bile, the major route of excretion of organic hydrocarbons, of viviparous blenny treated with heavy gas oil revealed that besides naphthalene the bile also contained polycyclic aromatic hydrocarbons (PA11s) such as phenanthrenes, anthracene, pyrenes, fluoranthene, benzo(a)anthracene and chrysene. None of these compounds were detected in bile from control fish, with the only exception of 3-methylphenanthrene. This study indicates the usefulness of the P450 1A system in the viviparous blenny as a sensitive biomarker in monitoring studies in petroleum oil contaminated areas in Northern Europe.

Effects of acetylsalicylic acid on parameters related to peroxisome proliferation in mouse liver

Male C57 BL/6 mice were exposed to 1.0% (w/w) acetylsalicylic acid (ASA) in their diet for 10 days and effects related to peroxisome proliferation were subsequently examined. A 2.2-fold increase in mitochondrial protein content was obtained. The activities of the peroxisomal enzymes, lauroyl-CoA oxidase, palmitoyl-CoA oxidation and catalase, were enhanced 4.5-, 4.0- and 2.1-fold, respectively. There was a dramatic increase (9.1-fold) in microsomal cytochrome P450 IVA-catalysed activity, a 1.6-fold induction of total microsomal P450 content and a 2-fold induction of microsomal cytochrome P450 reductase activity (measured as NADPH-cytochrome c reductase). Catalase activity in the cytosol was induced 5.2-fold and DT-diaphorase activity was increased 3.5- and 3.2-fold in the cytosol and mitochondria, respectively. There was a significant increase in the susceptibility of microsomes to lipid peroxidation. Smaller increases in superoxide dismutase, glutathione transferase and glutathione peroxidase activities were also observed. The possible relevance of these effects to the pharmacology of ASA is discussed.

Toxicological and ecological implications of biotransformation enzymes in the tropical teleost Chaetodon capistratus

Hepatic cytochromes P450 (phase I monooxygenases) and glutathione transferases (phase II conjugating enzymes) were investigated in Chaetodon capistratus (Linnaeus) collected in Florida and Belize in June and December 1991, respectively. These biotransformation enzymes play major roles in the detoxification of xenobiotics by converting lipophilic chemicals to more hydrophilic, readily excretable metabolites. Content of total microsomal P450 (0.501 to 0.821 nmol mg-1 microsomal protein) and rates of NADPH-cytochrome c (P450) reductase (270.7 to 330.2 nmol min-1 mg-1 microsomal protein) and glutathione transferase (2.81 to 3.12 μg min-1 mg-1 cytosolic protein) in these fish were greater than in most untreated fish species, i.e., fish that have not been exposed to PAHs (polycyclic aromatic hydrocarbons) or PCBs (polycyclic biphenyls). Ethoxyresorufin O-deethylase (EROD) rates (0.029 to 0.171 nmol min-1 mg-1) were also comparable to those in most untreated marine fish. Immunoblot analysis with monoclonal antibody (MAb) 1-12-3 to scup P450E (the EROD catalyst and a teleost representative of the PAH-inducible CYP1A gene subfamily) showed slight amounts of cross-reacting protein in C. capistratus liver microsomes. Hepatic CYP1A content and EROD activity did not differ significantly between fish collected in Florida and Belize, suggesting that the two sites differed little in contamination by CYP1A inducers. Immunochemical analyses with polyclonal antibodies to scup P450B (a teleost representative of the CYP2B subfamily) and human CYP3A4 cross-reacted strongly with C. capistratus hepatic proteins. The CYP2B and CYP3A subfamilies in mammals are believed to have partially evolved in response to toxic dietary allelochemicals. C. capistratus regularly feeds on terpenoid-rich gorgonian corals, suggesting that biotransformation enzymes may be involved in the metabolism of dietary allelochemicals as well as anthropogenic xenobiotics in this species.

Piperine effects on the expression of P4502E1, P4502B and P4501A in rat.

1. Treatment of rat with piperine (PIP) (1.4 mmol/kg, 3 days ip injections) resulted in an approximate two-fold increase in total liver microsomal P450 content relative to that in uninduced animals. 2. 4-Nitrophenol and aniline hyroxylase activities in the hepatic microsomes prepared from rat treated with PIP decreased by 30 and 28% respectively as compared with control. Immunoblot analyses also revealed decreased P4502E1 levels in hepatic microsomes from PIP-treated animals. 3. In contrast with P4502E1 suppression, hepatic 2B1 and 2B2 levels were significantly increased in PIP-induced animals, as evidence by both metabolic activity and immunoblot analysis of the liver microsomal fractions. The rate of hexobarbital hydroxylase activity in microsomes from PIP-treated animals was markedly elevated and was inhibited by approximately 62% in the presence of monoclonal anti-P4502B IgG. Immunoblot analyses demonstrated that P4502B1 and 2B2 levels in hepatic microsomes from PIP-treated animals were comparable with those from phenobarbital-treated animals. 4. 7-Ethoxycoumarin deethylase activity was elevated approximately two-fold in PIP-induced animals and was 17% of that derived from 3-methylcholanthrene-induced animals. 7-ethoxycoumarin deethylase activity in PIP-induced hepatic microsomes was inhibited 63% in the presence of monoclonal anti-P4501A antibody. Immunoblot analysis confirmed the increase in P4501A levels by PIP, which was 15% of that in hepatic microsomes from 3-methylcholanthrene-induced animals. 5. PIP treatment failed to affect microsomal epoxide hydrolase (mEH) and glutathione S-transferases (GST) expression, as indicated by immunoblot analyses using polyclonal antibodies toward mEH and GST subunits Ya, Yb1, Yb2 and Yc. 6. These results demonstrate that PIP treatment suppressed P4502E1 expression and enhanced 2B and 1A expression, whereas this agent failed to affect hepatic mEH and GST expression.

Inhibition of hepatic microsomal monooxygenase system by organotins in vitro in freshwater fish

The interaction in vitro of organotins tributyltin (TBT) and triphenyltin (TPT) with the hepatic microsomal monooxygenase systems in the freshwater fish, rainbow trout ( Oncorhynchus mykiss), European eel ( Anguilla anguilla), and bullhead ( Cottus gobio) was studied. Hepatic microsomes were incubated in vitro with TBT and TPT and various components analyzed. Ethoxyresorufin O-deethylase (EROD) activity was strongly inhibited by TBT and TPT in a concentration-dependent manner in all fish. Rainbow trout microsomes were more sensitive than were eel or bullhead microsomes. Total inhibition of EROD activity occurred both at 0.5 mM TBT and TPT in rainbow trout, whereas I mM decreased EROD activity to 15% (TBT) or 3% (TPT) in eel, and 35% (TBT) or 18% (TPT) in bullhead, respectively. As this effect may be caused by inhibition of different components of the microsomal electron transport system, different enzymes were studied separately. In all fish, both organotins led to a time- and concentration-dependent decrease in spectral total microsomal P450 content, and formation of cytochrome P420. TPT led to a greater inactivation of P450 enzyme than TBT, and induced a 50% loss in all fish at 0.08 mM TPT, whereas in case of TBT a 50% loss occurred at 0.18 mM in rainbow trout, 0.30 mM in bullhead, and 0.83 mM in eel. Cytochrome b 5 content was not affected, but both organotins led to an almost selective inhibition of either NADH or NADPH cytochrome c reductase activity in trout and eel, or of both in bullhead. Whereas TBT inhibited NADH cytochrome c reductase, TPT acted strongly on NADPH cytochrome e reductase. The study demonstrates species-related significant and selective effects of TBT and TPT on different components of the microsomal monooxygenase system in freshwater fish. This leads to inactivation of native enzymes and inhibition of enzyme activities.

Induction of cytochrome P450 1A in fish treated with 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin or chemically contaminated sediment

AbstractMirror carp were exposed to Rotterdam Harbor sediment, highly contaminated with polychlorodibenzo‐p‐dioxins (PCDDs), polychlorodibenzofurans (PCDFs), and polychlorobiphenyls (PCBs) (0.5 μg 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin [TCDD] equivalents per kilogram dry weight). In two additional separate experiments rainbow trout (Oncorhynchus mykiss) and mirror carp (Cyprinus carpio) received a single intraperitoneal injection of approximately 0.01, 0.03, 0.06, 0.3, 0.6, or 3.0 μg TCDD per kilogram body weight. In all three experiments induction of hepatic P450 1A was measured with immunochemical and enzymatic methods. The polyclonal antibodies anticod (Gadus morhua), anti‐perch (Perca fluviatilis), and anti‐rainbow trout P450 1A all cross‐reacted with the P450 1A orthologue of the carp and rainbow trout. In most cases high correlations were found between 7‐ethoxyresorufin O‐deethylation (EROD) activity and cytochrome P450 1A protein contents, the latter measured by the enzyme‐linked immunosorbent assay (ELISA) and protein blot methods. However, the correlations between EROD activity and P450 1A protein levels were higher within the separate sampling periods (i.e., 3, 6, and 12 weeks after dosage) than with the total data set, especially in the dose‐effect study with the rainbow trout. This was probably caused by a difference in time‐dependent relationships between P450 1A protein content and EROD enzyme activity: 12 weeks after dosage the P450 1A protein was still increased, although EROD activity had returned to background level. In addition, there were higher correlations of the EROD activity and P450 1A protein content with total P450 content in rainbow trout and carp treated with a single dose of TCDD, than with total P450 content in carp exposed to contaminated sediment. In our study, the ELISA method appeared to be more useful than the protein blot technique, because the ELISA is faster and has higher reproducibility. In addition, in all our experiments EROD activity showed a higher induction than the P450 1A protein, indicating a higher sensitivity of the EROD assay. Our results strongly indicated that determination of the P450 1A protein content and EROD activity provides complementary information. Thus we recommended the use of both the ELISA and the EROD activity assay in order to understand the nature of P450 1A induction.

The Chronic Administration of Nicotine Induces Cytochrome P450 in Rat Brain

The objective of these studies was to determine whether chronic administration of nicotine altered the cytochrome P450 (P450) monooxygenase system in rat brain. Male Sprague-Dawley rats received injections of nicotine bitartrate (1.76 mg/kg, s.c., twice daily for 10 days), and total cytochrome P450 content, the activity of NADPH-cytochrome c reductase, and the activities and relative abundance of P4502B1 and P4502B2 (P4502B1/2) were determined in microsomal fractions from rat brain. The content of P450 increased significantly (p < 0.02) in all brain regions examined from nicotine-injected rats; the largest increase (208% of control) was in frontal cortex and the smallest increase (122% of control) in cerebellum. The activity of NADPH-cytochrome c reductase was unaltered by nicotine administration. Benzyloxyresorufin O-dealkylase (BROD) and pentoxyresorufin O-dealkylase (PROD) activities, mediated by P4502B1/2, increased significantly (p < 0.02) following nicotine administration; the largest increase (213-227% of control) was in frontal cortex. Western blots of microsomal proteins indicated that the increase in enzymatic activity was associated with an increase in content of P4502B1/2 immunoreactive proteins. In contrast to brain, total P450 content, activities of NADPH-cytochrome c reductase, BROD, and PROD, and levels of P4502B1/2 immunoreactive proteins in liver were unaffected by chronic nicotine administration. Results indicate that chronic nicotine administration regulates the expression of P4502B1/2 in brain and that at the dose schedule used this effect occurs without a demonstrable effect on the hepatic P450 monooxygenase system.

The Effect of PCB on the Induction of Cytochrome P450 Enzyme, Detected by Monoclonal Antibody against the P450IA1 Gene Products, in Barn Owl and Marsh Turtle Hepatic Microsomes

Induction of P450 species belonging to the P450IA1 gene family was demonstrated in liver microsomes of the barn owl Tytoalba and the marsh turtle Mauremyscaspicarivulata, after treatment with Aroclor 1254, using immunoblotting technique with monoclonal antibody (Mab) specific to P450IA1 (anti scup P450E, 1-12-6 Mab). P450IA1 could hardly be detected in barn owls collected from their natural habitat. Significant induction (p&amp;lt;0.05), of up to three times, in the content of the barn owls hepatic microsomal P450IA1 was recorded after intraperitoneal injection of Aroclor 1254, although the proportion of P450IA1 from total P450 content, did not exceed 5 percent. The content of P450IA1 in liver microsomes of marsh turtles collected from their natural habitat was relatively high, and values up to 225 pmol/mg protein were recorded. Treatment with Aroclor 1254 increased the mean content of P450IA1 in the turtle livers up to 5 times, and the proportion of P450IA1 from total hepatic P450 was about 20 percent.

Crude liver membrane fractions as substrate preserve liver-specific functions in long-term, serum-free rat hepatocyte cultures

Over time, rat hepatocytes cultured on collagen lose the capacity to express liver-specific functions. The influence on this degradation process of an alternative substratum--crude membrane fractions prepared from the liver of the same rat strain--was investigated. Freshly isolated rat hepatocytes were cultured in serum-free Williams E medium supplemented with aprotinin, selenium, dexamethasone, and insulin in flasks coated with a mixture of rat liver crude membrane fractions:collagen type I (100:1). The cells adhered firmly, exhibiting minimal spreading and remaining grouped in columns or in cell islands, and retained their liver-specific functions for more than 1 wk. Hepatocytes secreted substantially higher amounts of albumin than cells cultured on collagen-coated dishes, and on Days 1 and 9 in culture the total P-450 content was 72 and 40%, respectively, of that of freshly isolated cells. On Day 6, the 7-ethoxyresorufin-O-deethylase and the aldrin epoxidase activities were still more than 50% that of freshly isolated hepatocytes. Exposure to phenobarbital on Days 3 to 6 increased the total cytochrome P-450 content twofold; exposure to 3-methylcholanthrene increased the activity of the corresponding cytochrome P-450 isoforms to 20 times that observed in untreated cultures and 6 times that observed in freshly isolated cells. Thus, given the ease with which they are prepared, the use of crude membrane fractions combined with culture medium supplemented with aprotinin and selenium can facilitate the preparation of reproducible cultures suitable for long-term in vitro pharmacotoxicologic studies using rat hepatocytes.

High expression of cytochrome P450 2a‐5 (coumarin 7‐hydroxylase) in mouse hepatomas

A high level of Cyp2a-5 was found in spontaneous and transplanted mouse hepatomas compared with normal liver. Increased expression of Cyp2a-5 was associated with an increase in coumarin 7-hydroxylation, a marker activity of Cyp2a-5, and the corresponding mRNA, suggesting that regulation of Cyp2a-5 in hepatomas is pretranslational. In contrast, the total P450 content and arylhydrocarbon hydroxylase and amidopyrene demethylase activities decreased. Pyrazole, a strong inducer of Cyp2a-5 in normal mouse livers, also increases this isozyme in hepatomas. A parallel increase in the corresponding mRNA suggests that pyrazole, like the formation of hepatomas, affects the regulation of Cyp2a-5 pretranslationally.

Effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin or 2,2′,4,4′,5,5′-hexachlorobiphenyl on vitamin K-dependent blood coagulation in female germfree WAG/Rij-rats

Newborn infants are susceptible to bleeding disorders caused by a vitamin K deficiency, so called ‘haemorrhagic disease of the newborn’ (HDN). These bleedings often occur in infants after medication of the mother with antiepileptics, such as phenobarbital or phenytoin. It has been suggested that an increase in the late type of HDN in exclusively breast-fed infants might be related to the presence of cytochrome P450-inducing polychlorinated biphenyls (PCBs), dibenzo- p-dioxins (PCDDs) and dibenzo-furans (PCDFs) in human milk. In order to study this possible mechanistic relationship 5-week-old, germ-free, female WAG/Rij-rats were exposed to a single oral dose of either 1 μg 2,3,7,8-tetrachlorodibenzo- p-dioxin/kg body weight (TCDD) or 30 mg 2,2′,4,4′,5,5′ hexachlorobiphenyl/kg body weight (HxCB), representing cytochrome P-450 1A (3-methylcholanthrene type) and 2B (phenobarbital type) inducers. During the experiment blood coagulation time from each rat was measured. Also, hepatic 7-ethoxy-(EROD) and 7-pentoxyresorufin O-dealkylating (PROD) activities and total cytochrome P450 content were measured. Blood coagulation time (Thrombotest®) in the HxCB-treated rats was significantly prolonged and positively correlated to PROD activity and total P450 content. No clear effect of TCDD on coagulation time could be observed under these experimental conditions. These results suggest involvement of P450 2B isoenzymes in vitamin K metabolism.

Effects of temperature acclimation on the expression of hepatic cytochrome P4501A mRNA and protein in the fish Fundulus heteroclitus

Previous studies showed that hydrocarbon induction of hepatic microsomal monooxygenase activity is attenuated in the teleost fish Fundulus heteroclitus acclimated to low temperature. The basis of that attenuation, and the effects of temperature on monooxygenase activity, were examined by analyzing liver cytochrome P4501A (CYP1 A) mRNA, protein, and catalytic activity in control and β-naphthoflavone (BNF)-treated F. heteroclitus acclimated to 6 or 16 °C. There were no temperature-related differences in total P450 content, NADPH-cytochrome c (P450) reductase activity, ethoxyresorufin O-deethylase (EROD) activity, or immunoquantified CYP1A content in hepatic microsomes of untreated fish. Fish acclimated to 16 °C and given a single intraperitoneal injection of BNF exhibited a rapid rise and fall in CYP1A mRNA content and an induction of EROD activity and CYP1A protein that was undiminished over 7 days. Similarly treated fish acclimated at 6 °C showed an increase in CYP1A mRNA content greater than that in 16 °C fish, but with no significant increase in EROD activity or CYP1A content over 7 days. Examined over a longer term, microsomal EROD activity was significantly induced by BNF in fish at both temperatures; activity peaked at 5–7 days in 16 °C fish, while in 6 °C fish the activity continued to rise slowly over 25 days. However, the greatest activity reached in 6 °C fish (0.68 nmol/min/ mg) was less than half that seen in the warmer animals (1.46 nmol/min/mg). Immunodetectable CYP1A content showed the same trend as EROD activity, and the turnover number (nmol product formed/min/nmol CYP1A) for EROD activity was about the same in all groups, indicating that concentration of the catalyst alone could account for the different patterns of microsomal activity. CYP1A mRNA content was again induced to a similar degree by BNF in both the 6 and the 16 °C fish; the apparent half-life of the mRNA was substantially longer in cold-acclimated than in warm-acclimated BNF-treated fish. Comparing the levels of CYP1A mRNA and protein at the two acclimation temperatures following BNF treatment indicates that translational activity, rather than transcriptional activity, is the sensitive point in the effect of temperature on CYP1A induction in these fish.

Differences in hepatic microsomal cytochrome P-450 isoenzyme induction by pyrazole, chronic ethanol, 3-methylcholanthrene, and phenobarbital in high alcohol sensitivity (HAS) and low alcohol sensitivity (LAS) rats.

High and low alcohol sensitivity (HAS and LAS) rats have been selected for their differences in ethanol-induced sleep time. Liver monooxygenase activities were studied in HAS and LAS rats before and after treatments with known inducers such as chronic ethanol, pyrazole, 3-methylcholanthrene (3-MC) and phenobarbital (PB) to determine whether the selection procedure also selected for differences in the cytochrome P-450 (P-450) inducibility. This previously has been shown with long sleep (LS) and short sleep (SS) mice, which were selected using a similar criterion. 3-MC and PB, in conjunction with chronic ethanol treatment, were used in order to evaluate the interactions of ethanol with these inducers. Prior to treatment, total P-450 content was slightly lower in LAS than in HAS rats. However, both lines displayed the same microsomal monooxygenase activities related to different P-450 isozymes. This was demonstrated by ethoxyresorufin deethylation (EROD) for cytochrome P-450 1A1 (CYP1A1), acetanilide hydroxylation (ACET) for CYP1A2, pentoxyresorufin dealkylation (PROD) for CYP2B, 1-butanol oxidation (BUTAN) and N-nitrosodimethylamine demethylation (NDMA) for CYP2E1. After the different treatments, HAS rats did not differ from LAS rats in their CYP2E1 inducibility. However, pyrazole, PB and 3-MC treatment led to differences in CYP1A and CYP2B monooxygenase activities between the two lines. The enhancement of PROD by pyrazole treatment was less prominent in LAS (1.7-fold of the control value) than in HAS rats (3.8-fold).(ABSTRACT TRUNCATED AT 250 WORDS)