Increase In Cytochrome P-450 Content Research Articles

Detoxification and antioxidant function of liver of the marsh frog (Ranaridibunda) intoxicated with oil from Kenkiyak oilfield

Detoxification and antioxidant functions of liver of marsh frog (Ranaridibunda) exposed to water-soluble fraction of oil from Kenkiyak oilfieldin concentrations of 0.05, 0.5 and 1% during 30 days were studied. Dosedependent increase of cytochrome P450 content (1.5-2 times) in comparison to intact animals (control), indicating the activation of detoxificationprocesses in liver of intoxicated frogs was observed. It was shown that thegrowth of monooxigenases was accompained by enchancement of lipidperoxidation processes: the content of MDA in liver of intoxicated marshfrogs also rose dose-dependently 1.5-2 times(Р≤0,05)compared with control. Herewith, the content of reduced glutathione, one of the key substances of antioxidant and detoxification systems of the body, decreased1.3-2 times (Р≤0,05) as a result of almost the same induction ofglutathioneS-transferase (1.3-2.1 times, Р≤0,05). It was found that activation of lipidperoxidation in liver of intoxicated marsh frogs was caused by supressionofactivity of catalase (1.2-1.5 times) and superoxide dismutase (1.1-1.4times).On the basis of obtained results a conclusion about activation of detoxification functions and inhibition of antioxidant defence in liver of frogs,leading to strengthening of oxidative stress in studied animalsas a result ofoil intoxication, was made. Accumulation of lipid peroxides in liver led todisruption of adaptive reactions of intoxicated animals and developmentof destructive and necrobioticchanges of hepatocytes.Key words: marsh frog, liver, oil, cytochrome P450, malondialdehyde,reduced glutathione, glutathione-S-transferase, catalase, superoxide dismutase.

The effects of 3-methylcholanthrene on lymphocyte proliferation in the common carp ( Cyprinus carpio L.)

The sensitivity of lymphocyte proliferation as bioindicator of pollution stress was evaluated in the common carp ( Cyrinus carpio L.). The time course response of peripheral blood leukocyte proliferation in response or not to mitogens was measured from 1 to 7 days after peritoneal injection of 3-methylcholantrene (3-MC), and compared to the time course response of a highly sensitive biomarker, induction of cytochrome P450. 3-Methylcholanthrene (40 mg kg −1) inhibited both B- and T-lymphocyte proliferation in response to lipopolysaccharide (LPS) and concanavalin A (Con A). Studies with α-naphtofiavone, suggest the lack of metabolic processes. 3-Methylcholanthrene alone strongly stimulated resting peripheral blood leukocytes (PBLs) proliferation. This effect was not transient. The induction of lymphocyte proliferation paralleled the increase in cytochrome P450 content in the liver. The specificity of polycyclic aromatic hydrocarbon (PAH)-induced lymphocyte proliferation suggests that this immune activity may be an early marker of exposure to PAHs in aquatic environments. The capacity of 3-MC to induce rapid lymphocyte proliferation may be related to PAH-induced rapid clonal expansion in mammals. These results strongly suggested that the underlying mechanism might be the same in both models. More studies are needed in fish to explain this phenomenon and may be helpful in understanding the occurrence of neoplastic epizootics in fish associated with PAH exposition.

Effects of amitraz on cytochrome P450-dependent monooxygenases and estrogenic activity in MCF-7 human breast cancer cells and immature female rats

This study investigated the ability of amitraz, a formamidine insecticide, to induce cytochrome P450-dependent monooxygenases and to disrupt estrogenic activity in human breast cancer MCF-7 cells and immature female rats. In MCF-7 cells, treatment with 10 μM amitraz for 24 h increased 7-ethoxyresorufin O-deethylase activity in cell homogenate. Treatment of MCF-7 cells with 1 and 10 μM amitraz for 3 h replaced previously bound [ 3H]17β-estradiol (E 2) from estrogen receptors. Treatment with 0.1 and 1 μM amitraz for 2 days inhibited [ 3H]thymidine incorporation into the DNA of MCF-7 cells while the inhibition was blocked in cells co-treated with 1 nM E 2 and amitraz. In immature female rats, treatment with 50 mg/kg amitraz intraperitoneally for 3 days increased cytochrome P450 content, 7-ethoxyresorufin, methoxyresorufin and pentoxyresorufin O-dealkylases, and benzo[ a]pyrene hydroxylase activities in liver microsomes. The results of immunoblot analysis revealed that amitraz induced liver microsomal CYP1A1/2, 2B1/2B2, and 3A proteins. Treatment with 10 and 25 mg/kg amitraz for 3 days dose-dependently decreased uterine weight and peroxidase activity in immature female rats while the decreases were blocked in rats co-treated with 10 μg/kg E 2 and 10 or 25 mg/kg amitraz. These in vitro and in vivo findings suggest that amitraz induces multiple forms of P450 and exerts weak antiestrogenic activity.

Investigations on cell proliferation and enzyme induction in male rat kidney and female mouse liver caused by tetrahydrofuran.

To elucidate possible mechanism(s) of carcinogenic action of tetrahydrofuran (THF) that had been demonstrated in previous inhalation studies, groups of male F344 rats and female B6C3F(1) mice were exposed to dynamic atmospheric concentrations of 0, 600, 1800, or 5400 mg/m(3) for 6 h per day, either for 5 consecutive days or for a period of 4 weeks (5 days per week). The reversibility of treatment-related changes was investigated in rats and mice exposed for 5 days and sacrificed 21 days after the last exposure. Female B6C3F(1) mice exposed to 5400 mg/m(3) showed significantly increased cytochrome P450 content, increased ethoxyresorufin-O-deethylase and pentoxyresorufin-O-depentylase activities, increased cell proliferation (5-bromo-2'-deoxyuridine-method) and an increased mitotic index in liver zones 2 (midzonal region) and 3 (central vein region). The changes were found to be reversible after a 3-week treatment-free period (cell proliferation examined, only). Male F344 rats showed dose-related alpha2u-globulin (alpha2u) accumulation in the renal cortex after 5 or 20 exposures, and there were no signs of reversal after a 3-week treatment-free period. After 20 exposures at 5400 mg/m(3), the alpha2u accumulation was found to be associated with increased cell proliferation in "hot spots" of the renal cortex and increased apoptosis. Increased cell proliferation was also detected after 20 exposures at 1800 mg/m(3). There were no effects at 600 mg/m(3). It is concluded that THF enhances tumor formation in male rat kidney and female mouse liver via induction of cell proliferation. These features present essential elements that should be taken into account for the carcinogenic risk assessment of THF.

Chronic treatment with the endocannabinoid anandamide increases cytochrome P450 metabolizing system in the rat

The aim of this work was to investigate the effects of single and repeated administration of the endogenous cannabinoid anandamide (20 mg/kg i.p.) on cytochrome P450-mediated biotransformation in the rat. In liver microsomes from chronically treated rats, an increase in cytochrome P450 content and in the activity and immunoreactivity of cytochrome P450 reductase was detected. Immunoblot analysis of the hepatic microsomal proteins revealed an increase in the relative level of cytochrome P450 2B1/2 and 3A2. The activity of monooxygenase enzymes linked to specific cytochrome P450 isoforms was significantly enhanced. This increase in the content and activity of the cytochrome P450 system was also seen in liver microsomes from acutely treated rats; however, these increases were smaller than those seen after prolonged treatment. After acute treatment, the brain cytochrome P450 and b5 content was increased, whereas after chronic treatment, only that of b5 was enhanced. Cytochrome P450 reductase activity and its relative abundance were increased only in the brains of chronically treated rats. The present findings demonstrate that anandamide administration increased the metabolic activity of the cytochrome P450 system in rat liver and brain.

Pretreatment of rats with the inducing agents phenobarbitone and 3-methylcholanthrene ameliorates the toxicity of chromium (VI) in hepatocytes

To exert cytotoxicity chromium VI (Cr(VI)) has to be reduced inside cells. This is achieved through both enzymatic and non-enzymatic mechanisms. Enzymatic mechanisms include DT-diaphorase, cytochrome P450, and NADPH cytochrome c reductase, and non-enzymatic mechanisms involve reduced glutathione (GSH) and ascorbic acid. The extent of cytotoxicity of Cr(VI) may thus be influenced by the availability of non-enzymatic reductants, and by the activities of the reductase enzymes. In the present paper we have investigated the effect of pretreatment with the inducing agents, phenobarbitone (PB) and 3-methylcholanthrene (3-MC), on the response of rat hepatocytes to Cr(VI). Pretreatment with PB increased the activity of NADPH cytochrome c reductase, and 3-MC increased DT-diaphorase activity in hepatocytes. Both inducers increased cytochrome P450 content, while neither influenced intracellular GSH content or the activity of glutathione reductase. Pretreatment with either PB or 3-MC resulted in amelioration of Cr(VI) toxicity both in terms of hepatocyte viability, and to a greater extent, in terms of Cr(VI) induced GSH loss. We propose that the inducing agents increase the amount of enzymatic reduction of Cr(VI) relative to non-enzymatic reduction. Thus, less GSH is used in the reduction of Cr(VI), and intracellular GSH does not fall as rapidly as in cells from control animals therefore cell integrity is better maintained. Exposure to environmental inducing agents in vivo may also alter the response of human tissues to Cr(VI).

Effects of β-naphthoflavone on the cytochrome P450 system, and phase II enzymes in gilthead seabream ( Sparus aurata)

The effect of β-naphthoflavone (β-NF) on several catalytic activities of cytochrome P450 (CYP) and phase II enzymes putatively controlled by [Ah]-receptor activation in the liver, heart and kidney of gilthead seabream, was investigated. In the liver, β-NF treatment [intraperitoneal injection (i.p.) 50 mg/kg] resulted in an increase of CYP content, immunoreactive CYP 1A and methoxyresorufin- O-demethylase (MEROD), pentoxyresorufin O-depentylase (PROD) and ethoxyresorufin- O-deethylase (EROD) activities. However, β-NF had no effect on any of the hepatic phase II enzymes examined (benzaldehyde dehydrogenase, propionaldehyde dehydrogenase, glutathione S-transferase, UDP-glucuronyl-transferase, DT-diaphorase). Single i.p. injection of 10 mg/kg β-NF showed a maximal induction of CYP 1A-like protein and EROD activity after 3–7 days. CYP 1A and EROD returned to control levels 18-days post-treatment. β-NF injection also caused a rapid increase of a single band size of mRNA recognized by a CYP 1A1 cDNA fragment from sea bass ( Dicentrarchus labrax). Expression of mRNA preceded the increase of EROD activity and declined rapidly by 96 h. Dose–response experiments demonstrated that EROD was significantly enhanced in liver by a single injection of 0.3 mg/kg β-NF and was the most sensitive measurement for CYP 1A-like induction. β-NF treatments also increased the expression of CYP 1A-like protein, mRNA and EROD, but not MEROD and PROD activities in heart and kidney.

Methyl jasmonate induces lauric acid omega-hydroxylase activity and accumulation of CYP94A1 transcripts but does not affect epoxide hydrolase activities in vicia sativa seedlings

Treatment of etiolated Vicia sativa seedlings by the plant hormone methyl jasmonate (MetJA) led to an increase of cytochrome P450 content. Seedlings that were treated for 48 h in a 1 mM solution of MetJA stimulated omega-hydroxylation of 12:0 (lauric acid) 14-fold compared with the control (153 versus 11 pmol min-1 mg-1 protein, respectively). Induction was dose dependent. The increase of activity (2.7-fold) was already detectable after 3 h of treatment. Activity increased as a function of time and reached a steady level after 24 h. Northern-blot analysis revealed that the transcripts coding for CYP94A1, a fatty acid omega-hydroxylase, had already accumulated after 1 h of exposure to MetJA and was maximal between 3 and 6 h. Under the same conditions, a study of the enzymatic hydrolysis of 9,10-epoxystearic acid showed that both microsomal and soluble epoxide hydrolase activities were not affected by MetJA treatment.

Cytochrome P450 monooxygenases and insecticide resistance in insects.

Cytochrome P450 monooxygenases are involved in many cases of resistance of insects to insecticides. Resistance has long been associated with an increase in monooxygenase activities and with an increase in cytochrome P450 content. However, this increase does not always account for all of the resistance. In Drosophila melanogaster, we have shown that the overproduction of cytochrome P450 can be lost by the fly without a corresponding complete loss of resistance. These results prompted the sequencing of a cytochrome P450 candidate for resistance in resistant and susceptible flies. Several mutations leading to amino-acid substitutions have been detected in the P450 gene CYP6A2 of a resistant strain. The location of these mutations in a model of the 3D structure of the CYP6A2 protein suggested that some of them may be important for enzyme activity of this molecule. This has been verified by heterologous expression of wild-type and mutated cDNA in Escherichia coli. When other resistance mechanisms are considered, relatively few genetic mutations are involved in insecticide resistance, and this has led to an optimistic view of the management of resistance. Our observations compel us to survey in more detail the genetic diversity of cytochrome P450 genes and alleles involved in resistance.

Four-week repeated inhalation study of HCFC 225ca and HCFC 225cb in the common marmoset

Four male and three female marmosets in each group were exposed to air only, 1000 ppm of HCFC 225ca or 5000 ppm of HCFC 225cb, for 6 h per day for 28 consecutive days. HCFC 225ca caused a slight reduction in body weight. HCPC 225cb occasionally caused somnolence during exposure and vomiting on the first day of exposure. Clinical chemistry findings included a mild reduction of triglyceride, cholesterol and phospholipid levels and increased GOT level m the HCFC 225ca exposure group. HCFC 225cb also caused a reduction of triglyceride levels in some animals. HCFC 225ca caused a slight increase of hepatic carnitine palmitoyltransferase (CPT) activity while HCFC 225cb slightly increased cyanide-insensitive palmitoyl CoA β-oxidation (FAOS) activity. In the HCFC 225cb exposure group, an increase in cytochrome P-450 content was also observed. HCFC 225ca caused a fatty change in the hepatic cells. Increased incidence of lipid droplets in the hepatic cells and myelin-like bodies in hepatic cells, Kupffer's cells and hepatic blood vessels were observed electron microscopically in the HCFC 225ca exposure group. A proliferation of smooth endoplasmic reticulum was observed in the HCFC 225cb exposure group. Decreased peroxisome volume density in the HCFC 225ca group, and increased volume density m the HCFC 225cb exposed females were seen. However, organ weight measurement and histopathological examination did not reveal hepatomegaly or hypertrophy with either substance. Although slight changes were noticed in peroxisome volume density and in some of the peroxisomal enzyme; activities, the changes related to peroxisome proliferation with HCFC 225ca and 225cb were minimal in marmosets compared to those seen in rats. Histopathological examination and hormonal analysis did not reveal any abnormalities in the pancreas or testes.

Effect of nifedipine, verapamil, diltiazem and trifluoperazine on acetaminophen toxicity in mice

The hepatotoxicity of acetaminophen overdose depends on the metabolic activation to a toxic reactive metabolite by the hepatic mixed function oxidases. There is evidence that an increase in cytosolic Ca2+ is involved in acetaminophen hepatotoxicity. The effects of the Ca2+-antagonists nifedipine (NF), verapamil (V), diltiazem (DL) and of the calmodulin antagonist trifluoperazine (TFP) on the activity of some drug-metabolizing enzyme systems, lipid peroxidation and acute acetaminophen toxicity were studied in male albino mice. No changes in the drug-metabolizing enzyme activities studied and in the cytochrome P-450 and b5 contents were observed 1 h after oral administration of V (20 mg/kg). DL (70 mg/kg) and TFP (3 mg/kg). NF (50 mg/kg) increased cytochrome P-450 content, NADPH-cytochrome c reductase and ethylmorphine-N-demethylase activities. DL and TFP significantly decreased lipid peroxidation. NF, V, DL and TFP administered 1 h before acetaminophen (700 mg/kg orally) increased the mean survival time of animals. A large increase of serum aspartate aminotransferase(AST), and liver weight and depletion of liver reduced glutathione (GSH) occurred in animals receiving toxic acetaminophen dose. NF, V and DL prevented and TFP decreased the acetaminophen-induced hepatic damage measured both by plasma AST and by liver weight. NF, V, DL and TFP changed neither the hepatic GSH level nor the GSH depletion provoked by the toxic dose of acetaminophen. This suggests that V, DL and TFP do not influence the amount of the acetaminophen toxic metabolite formed in the liver. The possible mechanism of the protective effect of NF, V, DL and TFP on the acetaminophen-induced toxicity is discussed.

Hepatic and Adrenal Toxicity of a Novel Lipid Regulator in Beagle Dogs

Hepatic and Adrenal Toxicity of a Novel Lipid Regulator in Beagle Dogs. Wolfgang, G. H. I., Robertson. D. G., Welty, D. F., and Metz, A. L. (1995). Fundam. Appl. Toxicol. 26, 272-281. PD 138142-15 is a substituted urea hypolipidemic and potential anti-atherosclerotic agent. To determine the toxicity of PD 138142-15, beagle dogs were given oral doses of 1, 10, 30, and 100 mg/kg daily for 13 weeks. Two animals at 100 mg/kg were euthanized during Week 5 due to poor condition. Clinical findings included decreased serum albumin at ⩾30 mg/kg, and increased ALP (up to 30-fold) and 5′-nucleotidase activities (up to 9-fold) at doses ⩾10 mg/kg. ALT and AST activities were elevated only at 100 mg/kg. There was a two- to threefold increase in cytochrome P450 content of hepatic microsomes from all treated animals and increases in liver weights at 10 mg/kg and above. Hepatic changes included hepatocellular hypertrophy and increased cytoplasmic eosinophilia at ⩾10 mg/kg; single cell necrosis of hepatocytes was noted in moribund animals. ACTH-stimulated cortisol levels were decreased at 30 and 100 mg/kg. Adrenal cholesterol esters were decreased at 10 mg/kg and above, while total adrenal cholesterol was decreased at ⩾30 mg/kg. These changes correlated with adrenal cortical zonal atrophy, principally of the zona fasciculata and zona reticularis, present at 30 and 100 mg/kg. Plasma concentrations of PD 138142-15 increased with increasing dose; plasma levels were significantly lower during Week 12 than those on Day 1, possibly due to autoinduction. Overt hepatotoxicity occurred at 100 mg/kg, whereas hepatic changes at 10 and 30 mg/kg were consistent with cytochrome P450 induction. The hepatic lesions were reversible within 4 weeks, while adrenal lesions were still evident after 4 weeks without treatment.

Effects of dexamethasone on steroidogenesis in Leydig cells from rats of different ages.

The effects of 0.1 microM dexamethasone on cytochrome P450 content, 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity, and basal and LH-induced testosterone production of Leydig cells from rats 3, 5, 7 and 10 weeks old were examined. The cytochrome P450 content of Leydig cells from rats 3 weeks old was increased by treatment with dexamethasone for 22 h, while 3 beta-HSD activity was decreased. The cytochrome P450 content of Leydig cells from rats 5 weeks old was increased after 3 and 22 h of culture, while 3 beta-HSD activity was decreased after 22 and 44 h of treatment. The cytochrome P450 content of rats 7 weeks old was increased after 3 h of culture, while 3 beta-HSD activity was decreased after 22 and 44 h of culture. Leydig cells from rats 10 weeks old showed increased cytochrome P450 content upon dexamethasone treatment after 3 h. The activity of 3 beta-HSD was decreased after 44 h of treatment. In Leydig cells from rats 3 and 5 weeks old, dexamethasone decreased basal testosterone production after 22 h of treatment, but not after 44 h, and did not affect LH-induced testosterone production. Leydig cells from rats 7 weeks old showed decreased basal and LH-induced testosterone production, when treated with dexamethasone for 22 and 44 h. Basal testosterone production was unaffected by dexamethasone in rats 10 weeks old, while LH-induced testosterone production was decreased after 44 h of treatment. The effect of dexamethasone on testosterone secretion changed during development, as a transient, early effect on basal testosterone secretion was observed in Leydig cells from prepubertal and pubertal rats. These data suggest that dexamethasone affects Leydig cells differently, depending on the age of the rat, the older rats being more sensitive than the younger rats.

Partial Characterization of Microsomes Isolated from Mung Bean Cotyledons

Microsomes isolated from excised cotyledons of 3-day-old, dark-grown, mung bean ( Vigna radiata, L., cv Berken) seedlings metabolized two endogenous substrates (cinnamic acid and lauric acid), three organophosphate insecticides (diazinon, isazofos, and methidathion), three acetamide herbicides (metolachlor, CGA-24704, and alachlor), and bentazon. Cinnamic acid was aryl hydroxylated forming p-coumaric acid. Lauric acid was primarily hydroxylated at the terminal carbon (ω-hydroxylation). The three α-chloroacetamides were O-demethylated. With all three organophosphate insecticides, the phosphorothionate sulfur was oxidized to the corresponding oxon and the phosphoroester oxygen was cleaved in both diazinon and isazofos. Bentazon was aryl hydroxylated forming the 6-hydroxy derivative. The concentration of cytochrome P450 in the microsomal preparations was marginally enhanced by pretreatment of the seed with naphthalic anhydride (NA), but was markedly increased by subirrigation of NA-treated seed with ethanol and was additionally increased with the combination of NA, clofibrate, and ethanol. The extent of metabolism of only lauric acid paralleled the increases in cytochrome P450 content. The various seed/seedling treatments, however, did approximately double the rate of metabolism of the three organophosphates, the three chloroacetamides, and bentazon. Metabolism required a reduced pyridine nucleotide and was affected by several cytochrome P450 monooxygenase inhibitors (carbon monoxide, tetcyclacis, piperonyl butoxide, 1-aminobenzotriazole, and SKF-525A). The inhibitors differentially affected metabolism of the substrates. Microsomal oxidations from both untreated and inducer-treated tissue responded similarly to the inhibitors. The differential inhibitory responses suggest that metabolism may involve several monooxygenase isoforms.

Liver enzyme activities after multiple administration of nifedipine in mice.

The effect of multiple nifedipine administration on hexobarbital sleeping time, liver monooxygenase and synthetase activities, lipid peroxidation and microsomal membrane fluidity were studied in male albino mice. The drug was administered orally at a dose of 25 mg/kg daily for 14 and 21 days. Nifedipine caused enzyme induction, demonstrated by shortened hexobarbital sleeping time, enhanced ethylmorphine N-demethylase, aniline 4-hydroxylase, ethoxycoumarine O-deethylase, UDP-glucuronyl transferase, glutathione S-transferase and NADPH-cytochrome c reductase activities and increased content of cytochrome P450 and cytochrome b5. This effect persisted until the 7th day after the last dose of nifedipine. There were no changes in lipid peroxidation and fluidity of the microsomal membranes after 14-day nifedipine administration. The increased cytochrome P450 content and drug metabolizing enzyme activities could be not associated with changes in these liver microsomal membrane properties.

Purification and properties of cytochrome P-450 ( P-450 lun) catalyzing steroid 11β-hydroxylation in Curvularia lunata

Addition of 11-deoxycortisol to the culture medium of Curvularia lunata induced the increase of cytochrome P-450 content and steroid 11β-hydroxylase activity. The enzyme in cell-free extract produces cortisol from 11-deoxycortisol in the presence of NADPH and O 2. The enzyme was partially stabilized by glycerol, 11-deoxycortisol, GSH and PMSF. The hydroxylation activity was strongly inhibited by carbon monooxid and sulfhydryl reagents. Cytochrome P-450 located on the microsomal fraction was solubilized with Triton X-100 and sodium cholate and purified to apparent homogeneity by column chromatography. The purified cytochrome P-450 ( P-450 lun) has a molecular mass of 60 kDa and exhibits the absorption maxium at 392 nm in the spectrum of oxidized form in the presence of 11-deoxycortisol. The reduced CO difference spectrum has a maximal peak at 448 nm. 11β-Hydroxylation of 11-deoxycortisol was reconstituted by cytochrome P-450 lun, C. lunata NADPH-cytochrome P-450 reductase and DLPC in the presence of NADPH and O 2 with a turnover number of 207 nmol/min per nmol of cytochrome P-450. The reductase and DLPC could be partially replaced with the enzyme purified from yeast or pig testis microsome and lipids purified from C. lunata, respectively. P-450 lun catalyzes bifunctionally 11β- and 14α-hydroxylations of 11-deoxycortisol. Deoxycorticosterone, progesterone, androstenedione and testosterone are hydroxylated in the similar manner.

Enhanced in vitro metabolism of testosterone by microsomes from insecticide-resistant Spodoptera littoralis larvae

In vitro testosterone hydroxylation was compared between susceptible and insecticide-resistant Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae) larvae. The susceptible larvae were topically applied with a non-lethal dose of lindane (γ-HCH) and the effects on microsomal P450 were studied within the 48 h following treatment. In vitro testosterone hydroxylation by phenobarbital-induced rat liver microsomes was used as a biological standard to investigate the functional role of P450 isozymes in S. littoralis. Depending on either the strain or the treatment, various amound of identical hydroxylated derivatives of testosterone were formed. Lindane increased cytochrome P450 content in susceptible S. littoralis but isoforms specific for testosterone hydroxylation were unaffected or even inhibited. In contrast, most of testosterone hydroxylase activities were enhanced in the resistant strain, uggesting either that not only xenobiotic-specific cytochromes P450 were overproduced, or that overproduced cytochrome P450 forms were not only specific for xenobiotics.

On the enzyme-inducing action of calcium antagonists.

The effects of three calcium antagonists, nifedipine (NF), verapamil (V) and diltiazem (DL), on rat liver monooxygenases were studied. The drugs were administered in oral doses of 50, 40 and 30 mg/kg daily for 3 weeks in male Wistar rats. NF and V shortened the hexobarbital (HB) sleeping time and increased benzphetamin-N-demethylase (BND), ethylmorphine-N-demethylase (EMND), aniline hydroxylase (AH), ethoxycoumarine-O-deethylase (ECOD), ethoxyresorufin-O-deethylase (EROD) and NADPH-cytochrome c reductase activities and the content of cytochrome P-450 and microsomal heme, but did not change the content of cytochrome b5. The data suggest that these calcium antagonists exert an enzyme-inducing effect on the hepatic monooxygenases. DL significantly increased only the EROD and NADPH-cytochrome c reductase activities and shortened HB sleeping time to a lesser extent, suggesting a weaker enzyme-inducing effect as compared to NF and V. The three drugs increased the delta-aminolevulinic acid (ALA) synthetase activity and decreased heme oxygenase (HO) activity. The increased cytochrome P-450 content is probably due to the increased synthesis and the decreased breakdown of this hemoprotein.

The effects of aroclor 1254 and petrochemical pollutants on cytochrome P450 from the digestive gland microsomes of four species of mediterranean molluscs

1. Clear and significant increase in cytochrome P450 content, was recorded for the Mediterranean bivalve Donax trunculus and the gastropod Avicularia gibbosula after accidental pollution of their habitat by oil spill. 2. The significant increase in cytochrome P450 content in Donax trunculus from polluted sites or after treatment with Aroclor 1254, was not accompanied by an increase, but rather a drastic decrease, in 7-ethoxyresorufin O-deethylase (EROD) catalytic activity. 3. Immunoblotting, using monoclonal antibody (1-12-3) against scup cytochrome P450E, failed to reveal the existence of a hemoprotein of the P450IA1 gene family, in Donax trunculus or Patella caerulea collected from polluted sites or treated with Aroclor 1254.

Cytochrome P-450-Dependent Hydroxylation of Lauric Acid at the Subterminal Position and Oxidation of Unsaturated Analogs in Wheat Microsomes

Microsomes from etiolated wheat (Triticum aestivum L. cv Etoile de Choisy) shoots catalyzed the reduced nicotinamide adenine dinucleotide phosphate-dependent hydroxylation of lauric acid predominantly at the subterminal or (omega-1) position (65%). Minor amounts of 10-hydroxy- (31%) and 9-hydroxylaurate (4%) were also formed. The reaction was catalyzed by cytochrome P-450, since enzyme activity was strongly inhibited by tetcyclacis, carbon monoxide, and antibodies against NADPH-cytochrome c (P-450)-reductase. The apparent K(m) for lauric acid was estimated to be 8.5 +/- 2.0 mum. Seed treatment with the safener naphthalic acid anhydride or treatment of seedlings with phenobarbital increased cytochrome P-450 content and lauric acid hydroxylase (LAH) activity of the microsomes. A combination of both treatments further stimulated LAH activity. A series of radiolabeled unsaturated lauric acid analogs (8-, 9-, 10-, and 11-dodecenoic acids) was used to explore the regioselectivity and catalytic capabilities of induced wheat microsomes. It has been found that wheat microsomes catalyzed the reduced nicotinamide adenine dinucleotide phosphate-dependent epoxidation of sp2 carbons concurrently with hydroxylation at saturated positions. The regioselectivity of oxidation of the unsaturated substrates and that of lauric acid were similar. Preincubation of wheat microsomes with reduced nicotinamide adenine dinucleotide phosphate and 11-dodecenoic acid resulted in a partial loss of LAH activity.