Total Microsomal P450 Content Research Articles

Effects of fumonisin B1 present in Fusarium moniliforme culture material on drug metabolising enzyme activities in ducks

The effects of fumonisin B1 (0, 5, 15 and 45 mg/kg/day), obtained from culture material of Fusarium moniliforme, on drug metabolising enzyme activities were investigated in four groups of five growing ducks by daily oral administration over 12 days. No lethality or sign of toxicosis occurred. The liver and kidney weights were increased, whereas microsomal and cytosolic tissue fractions were unaffected. Although the total microsomal P450 content was unaffected, benzphetamine, ethylmorphine, erythromycin N-demethylases and ethoxyresorufin O-deethylase activities were together increased (respectively by 114, 242, 57 and 27% with 5 mg/kg/day and 1024, 969, 200 and 147% with 45 mg/kg/day). By contrast, aminopyrine and nitrosodimethylamine N-demethylases, methoxyresorufin and pentoxyresorufin O-dealkylases, and UDP-glucuronyltransferase activities were only increased by using 45 mg/kg/day, whereas glutathione S-transferases activities remained unaffected.

Cytochrome P450-dependent renal arachidonic acid metabolism in desoxycorticosterone acetate-salt hypertensive mice.

Cytochrome P450 (P450)-dependent arachidonic acid metabolites may act as mediators in the regulation of vascular tone and renal function. We studied arachidonic acid hydroxylase activities in renal microsomes from normotensive NMRI mice, desoxycorticosterone acetate (DOCA)-salt hypertensive mice, and DOCA-salt mice treated with either lovastatin or bezafibrate, both of which improve hemodynamics in this model. Control renal microsomes had arachidonic acid hydroxylase activities of 175+/-12 pmol. min(-1). mg(-1). The metabolites formed were 20- and 19-hydroxyarachidonic acid, representing approximately 80% and approximately 20% of the total hydroxylation. Treatment with DOCA-salt resulted in significantly decreased hydroxylase activities (to 84+/-4 pmol. min(-1). mg(-1)) of the total microsomal P450 content and a decrease in immunodetectable Cyp4a proteins. Lovastatin had no effect on these variables, whereas bezafibrate increased arachidonic acid hydroxylase activities to 163+/-12 pmol. min(-1). mg(-1). In situ hybridization with probes for Cyp4a-10, 12, and 14 revealed that Cyp4a-14 was the P450 isoform most strongly induced by bezafibrate. The expression was concentrated in the cortical medullary junction and was localized predominantly in the proximal tubules. In conclusion, these results suggest that the capacity to produce 20-hydroxyarachidonic acid is impaired in the kidneys of DOCA-salt hypertensive mice. Furthermore, bezafibrate may ameliorate hemodynamics in this model by restoring P450-dependent arachidonic acid hydroxylase activities. Lovastatin, on the other hand, exerts its effects via P450-independent mechanisms.

Nitric oxide mediates hepatic cytochrome P450 dysfunction induced by endotoxin.

Animals subjected to immunostimulatory conditions (sepsis) exhibit decreased total cytochrome P450 content and decreased P450-dependent drug metabolism. Cytochrome P450 function is of clinical significance because it mediates the metabolism of some opioid and hypnotic drugs. The authors tested the hypothesis that reduced P450 function and decreased drug metabolism in sepsis are mediated by endotoxin-enhanced synthesis of nitric oxide. Hepatic microsomes were prepared from male Sprague-Dawley rats in nontreated rats, rats pretreated with phenobarbital and rats receiving aminoguanidine or NG-L-monomethyl-arginine alone. Nitric oxide synthesis was augmented for 12 h with a single injection of bacterial lipopolysaccharides. Nitric oxide synthase was inhibited with aminoguanidine or N(G)-L-monomethyl-arginine during the 12 h of endotoxemia in some animals. Plasma nitrite and nitrate concentrations were measured in vivo, and total microsomal P450 content, and metabolism of ethylmorphine and midazolam in vitro. Administration of endotoxin increased plasma nitrite and nitrate concentrations, decreased total cytochrome P450 content, and decreased metabolism of ethylmorphine and midazolam. Inhibition of nitric oxide formation by aminoguanidine or N(G)-L-monomethyl-arginine partially prevented the endotoxin-induced effects in the nontreated and phenobarbital-treated groups. Aminoguanidine or N(G)-L-monomethyl-arginine alone did not have an effect on either total cytochrome P450 content or P450-dependent drug metabolism. Plasma nitrite and nitrate concentrations correlated significantly negatively with P450 content (nontreated r = -0.88, phenobarbital r = -0.91), concentrations of formed formaldehyde (nontreated r = -0.87, phenobarbital r = -0.95), and concentrations of midazolam metabolites (4-OH midazolam nontreated r = -0.88, phenobarbital r = -0.93, and 1'-OH midazolam nontreated r = -0.88, phenobarbital r = -0.97). Altered hepatic microsomal ethylmorphine and midazolam metabolism during sepsis is mediated in large part by nitric oxide.

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 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.

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.

Inhibition and induction of cytochrome P450 2B1 in rat liver by promazine and chlorpromazine

Phenothiazine tranquilizers have been associated with pharmacokinetic drug interactions in man. In this study the in vivo and in vitro effects of the clinically important phenothiazines promazine (PZ) and chlorpromazine (CPZ) on drug oxidations catalysed by specific cytochrome P450 (P450) enzymes were investigated in the rat. In vitro, the two drugs were relatively ineffective inhibitors of constitutive P450 activities, but were inhibitory toward the principal phenobarbital-inducible P450 2B1 and, to a lesser extent, P450 1A1. Administration of PZ and CPZ to male rats did not markedly influence the total microsomal P450 content of the liver. However, the quantitatively important male-specific P450 2C11 was down-regulated by CPZ and concomitant induction of P450 2B1 and associated 7-pentylresorufin O-depentylase activity were noted. A small increase in the activity of microsomal 7-ethylresorufin O-deethylase was also observed following administration of both drugs to rats, suggesting induction of P4501A1/2. Considered together, it is apparent that the two phenothiazines are preferential inhibitors and inducers of P450 2B1 in rat liver. Drug interactions in humans involving phenothiazines may reflect a combined effect of induction and inhibition processes as well as down-regulation of other P450s, such as that produced by CPZ on P450 2C11.

Cytochrome P450E (P450IA) induction and inhibition in winter flounder by 3,3′,4,4′‐tetrachlorobiphenyl: Comparison of response in fish from georges bank and narragansett bay

AbstractInduction of liver microsomal cytochrome P450 by 3,3′,4,4′‐tetrachlorobiphenyl (TCB) was evaluated in winter flounder from two different sites, one offshore (Georges Bank) and one coastal (Narrow River, Narraganseti, Rhode Island). Immunoblot analysis of liver microsomes with monoclonal antibody 1–12–3 to scup P450E (P450IA1) revealed P450IA protein content of 0.01 nmol/mg in Georges Bank fish that were not treated with TCB. By comparison, untreated Narrow River fish had an 80‐fold greater content of immunodetected P450IA protein, indicating a strong environmental induction in these fish. In Georges Bank fish the total (spectrophotometrically measured) microsomal P450 content and the content of P450IA protein were induced progressively by intraperitoneal doses of TCB ranging from 0.1 to 10.0 mg/kg. Ethoxyresorufin‐O‐deethylase (EROD) specific activity (activity per mg protein) was also progressively induced, but the catalytic efficiency or turnover number (i.e., activity/nmol P450IA) was less in fish given the greater doses of TCB. In Narrow River fish TCB treatment resulted in no significant change (at P ≤ 0.05) in total microsomal P450 content or in P450IA protein content, although they tended to be less (total P450) or greater (P450IA) in fish given the greater doses. The EROD activity per mg protein was less in Narrow River fish given greater TCB doses than it was in control fish. EROD activities per nmol of P4501A protein in control Narrow River fish were less than those in any treatment group of Georges Bank fish and tended to diminish even further with TCB treatment. The results show that 3,3′,4,4′‐TCB induces P450IA in winter flounder and that TCB acts in vivo to inhibit the activity of P450IA enzyme by mechanisms not yet known. The lower catalytic efficiency of P450IA enzyme in Narrow River fish than in Georges Bank fish indicates that P450IA inhibition, whether caused by TCB or some other agent, does occur in the environment. The data also indicate that prior condition, including existing environmental induction, can strongly influence the responses of P450IA protein to additional chlorobiphenyl exposure.

Identification of P-450 ALC in microsomes from alcohol dehydrogenase-deficient deermice: Contribution to ethanol elimination in vivo

Isozyme 3a of rabbit hepatic cytochrome P-450, also termed P-450 ALC, was previously isolated and characterized and was shown to be induced 3- to 5-fold by exposure to ethanol. In the present study, antibody against rabbit P-450 ALC was used to identify a homologous protein in alcohol dehydrogenase-negative (ADH −) and -positive (ADH +) deermice, Peromyscus maniculatus. The antibody reacts with a single protein having an apparent molecular weight of 52,000 on immunoblots of hepatic microsomes from untreated and ethanol-treated deermice from both strains. The level of the homologous protein was about 2-fold greater in microsomes from naive ADH − than from naive ADH + animals. Ethanol treatment induced the protein about 3-fold in the ADH + strain and about 4-fold in the ADH − strain. The antibody to rabbit P-450 ALC inhibited the microsomal metabolism of ethanol and aniline. The homologous protein, termed deermouse P-450 ALC, catalyzed from 70 to 80% of the oxidation of ethanol and about 90% of the hydroxylation of aniline by microsomes from both strains after ethanol treatment. The antibody-inhibited portion of the microsomal activities, which are attributable to the P-450 ALC homolog, increased about 3-fold upon ethanol treatment in the ADH + strain and about 4-fold in the ADH − strain, in excellent agreement with the results from immunoblots. The total microsomal P-450 content and the rate of ethanol oxidation were induced 1.4-fold and 2.2-fold, respectively, by ethanol in the ADH + strain and 1.9-fold and 3.3-fold, respectively, in the ADH − strain. Thus, the total microsomal P-450 content and ethanol oxidation underestimate the induction of the P-450 ALC homolog in both strains. A comparison of the rates of microsomal ethanol oxidation in vitro with rates of ethanol elimination in vivo indicates that deermouse P-450 ALC could account optimally for 3 and 8% of total ethanol elimination in naive ADH + and ADH − strains, respectively. After chronic ethanol treatment, P-450 ALC could account maximally for 8% of the total ethanol elimination in the ADH + strain and 22% in the ADH − strain. Further, cytochrome P-450 ALC appears to be responsible for about one-half of the increase in the rate of ethanol elimination in vivo after chronic treatment with ethanol. These results indicate that the contribution of P-450 ALC to ethanol oxidation in the deermouse is relatively small. Desferrioxamine had no effect on rates of ethanol uptake by perfused livers from ADH-negative deermice, indicating that ethanol oxidation by a hydroxyl radical-mediated mechanism was not involved in ethanol metabolism in this mutant. Peroxisomal β-oxidation capacity was increased 40% over control values by ethanol treatment, consistent with the hypothesis that the increase in ethanol elimination in the ADH-negative deermouse is mediated predominantly via catalase-H 2O 2.