P450-dependent Enzyme Activities Research Articles

In vitro accumulation of thyroid hormones by cultured rat hepatocytes and the biliary excretion of iodothyronines in rats treated with a novel histamine H 2-receptor antagonist

The administration of SK&F 93479, a novel histamine H 2 antagonist, to Wistar rats has been shown to produce thyroid lesions associated with an increased clearance of plasma thyroxine (T 4) and elevated plasma TSH concentrations. To determine if these changes were mediated via an increase in the hepatic clearance of thyroid hormones, the biliary clearance and hepatic accumulation of iodothyronines were measured in rats and cultured hepatocytes treated with SK&F 93479. Both the in vitro and in vivo results showed that treatment with SK&F 93479 caused an increased hepatic accumulation (approx. 200% of control) and biliary excretion (2–3-fold control values) of T 4 but had little or no effect on T 3 uptake. The in vitro studies showed that the treatment related increase in hepatic thyroxine accumulation was temperature and therefore probably energy dependent, while inhibition of cytochrome P-450 dependent enzyme activity did not alter the accumulation of T 4 suggesting that the parent compound and not some oxidative metabolite was responsible for the hepatic effects. Chromatographic analysis of bile from SK&F 93479 and phenobarbitone-treated rats showed that in the latter animals 81% of the T 4 was present as the glucuronide conjugate (consistent with enzyme induction) whereas in the SK&F 93479-treated rats only 25% was present as the conjugate with 75% being in the unconjugated form. These studies show that SK&F 93479 increases the hepatic accumulation and biliary clearance of T 4 by a novel mechanism not associated with liver microsomal enzyme induction.

Effects of tin-porphyrins on developmental changes in hepatic cytochrome P450 content, selected P450-dependent drug-metabolizing enzyme activities and brain glutathione levels in the newborn rat.

Sn-mesoporphyrin is considerably more effective than Sn-protoporphyrin in inhibiting bilirubin production in vivo, in the experimental animal. In this study the effects of Sn-mesoporphyrin, administered in doses ranging from 1 to 20 mumol/kg b.w., on the developmental patterns of hepatic cytochrome P450 content and cytochrome P450-dependent drug metabolism in rat neonates were examined at various time points during the 5-week period immediately after birth. No detrimental alterations in cytochrome P450 content or in cytochrome P450-dependent drug metabolism were observed. In addition no deleterious effects were noted on total glutathione content in brain of Sn-mesoporphyrin-treated animals. After single doses of Sn-protoporphyrin of 20, 50 or 100 mumol/kg b.w. were administered at birth, transient decreases in hepatic cytochrome P450 content (days 1 and 2), and ethylmorphine demethylase (days 2 and 5) and 7-ethoxycoumarin deethylase (days 1, 2 and 5) activities were observed in the period immediately after birth. However no sustained alterations in the developmental patterns of these enzymes were observed even at the highest dose (100 mumol/kg b.w.) of Sn-protoporphyrin administered. These findings indicate that in the doses utilized in this study both metalloporphyrins have no long-term effects on cytochrome P450-dependent drug metabolism. Furthermore, in doses up to 20 mumol/kg b.w., neither compound produced any short-term effects on hepatic cytochrome P450 content or functional activity in newborn rats.

The biologic and toxic effect of 2,3,7,8-tetrachlorodibenzo- p-dioxin in chickens

The administration of 2,3,7,8-TCDD to 2-week-old White Leghorn cockerels produced a dose-dependent induction of hepatic microsomal ethoxyresorufin O-deethylase (EROD) and benzo[α]pyrene (B[α]P) hydroxylase activities with induction EC 50 values of 778 and 302 ng/kg, respectively. In addition, 2,3,7,8-TCDD also induced 4-dimethylaminoantipyrine (DMAP) N-demethylase (EC 50 = 561 ng/kg), this result contrasting with studies reported for other animal species in which 2,3,7,8-TCDD either does not induce or inhibits this cytochrome P-450 dependent monooxygenase enzyme activity. However, the reduced cytochrome P-450 : CO binding difference spectral absorption maxima for the 2,3,7,8-TCDD induced microsomes was observed at 448 nm which was similar to that reported for most animals which have been investigated. Electrophoresis of control and 2,3,7,8-TCDD induced microsomal proteins using SDS polyacrylamide slab gels showed intensification of 3 protein staining bands at M r 53 000, 56 000 and 58 000. Incubation of the 2,3,7,8-TCDD-induced microsomes with SKF-525A and α-naphthoflavone showed that both compounds inhibited DMAP N-demethylase, EROD and AHH and that α-naphthoflavone was the more potent inhibitor of all 3 microsomal monooxygenases. 2,3,7,8-TCDD treatment caused significant involution of the Bursa of Fabricius when administered at a dose level of 10 μg/kg for 3 days and this result confirmed the extreme sensitivity of the immature White Leghorn cockerel to the biologic and toxic effects elicited by 2,3,7,8-TCDD. However, in contrast to other sensitive species, no high affinity cytosolic receptor protein for [ 3H]2,3,7,8-TCDD could be detected in the liver of chick embryos or 2-week-old birds.

O‐Dealkylation of Phenacetin in the Olfactory Rosette in Rainbow Trout (Salmo Gairdneri)

Olfactory rosettes from trout (Salmo gairdneri) were found to metabolise (14C-ethyl)-phenacetin to 14CO2 in vitro. Based on wet weight, the rate of metabolism was lower than that in liver and gills. Based on protein content, the rate of metabolism was about equal to that in liver and gills. Addition of the cytochrome P-450 inhibitors metyrapone, 9-hydroxyellipticine and piperonyl butoxide significantly decreased the formation of 14CO2 from (14C-ethyl)-phenacetin in olfactory rosettes. Microautoradiography of rosettes incubated with (14C-ethyl)-phenacetin showed the presence of non-extractable metabolites in the sensory and indifferent epithelium. Addition of metyrapone decreased the binding of radioactivity in the epithelia considerably. When olfactory rosettes were incubated with (14C-dimethylamine)-aminopyrine, no formation of 14CO2 was observed. The results are concluded to indicate that cytochrome P-450 dependent enzyme activity is present in the epithelia of the trout olfactory rosette.

Neonatal exposure to toluene: Effects on the development of liver microsomal cytochrome P-450 and serum hormone levels in the rat

Lactating Sprague-Dawley rats and their pups were exposed on postnatal days 1–7, 6 h/day, to 80,500 and 1000 ppm toluene, respectively, by inhalation. Exposure to 80 ppm toluene decreased the liver microsomal AHH activity and the rate of 7α- and 6β-hydroxylation of androstenedione in 8-day-old-pups. On the other hand, neonatal exposure to 500 or 1000 ppm toluene resulted in a significant increase in AHH and 7-ethoxyresorufin O-deethylase activities and in the formation of 16-oxygenated metabolites of androstenedione in 8-day-old animals. Exposure to toluene increased the cytochrome P-450 content at all 3 dose levels in male but not in female pups. Twenty-one days after neonatal exposure no such effects were seen in young animals of either sex. In 56-day-old male rats, however, neonatal exposure to 80 ppm toluene resulted in a decreased rat of 6β-hydroxylation of androstenedione and a reduced AHH activity. No such effects were seen in female rats of the same age. Neonatal exposure to toluene affected the body and liver weights in 8-day-old pups of both sexes but had no effect on these parameters in 21-day-old animals of either sex. Exposure to 80 ppm toluene during the neonatal period gave a significantly increased body weight of 56-day-old male but not of female rats of the same age although this treatment increased liver weight in both sexes at this age. Serum testosterone levels were decreased in 21-day-old male rats following neonatal exposure to 80 or 500 ppm toluene and in 56-day-old male rats exposed neonatally to 1000 ppm toluene. In conclusion, exposure to toluene during the first week of life caused significant changes in various liver microsomal cytochrome P-450 dependent enzyme activities in 8-day-old pups, whereas the long-term effects on liver metabolism of the adult animal were small.

Comparison of kidney, lung and liver coumarin 7-hydroxylases in phenobarbital pretreated DBA/2J and C57BL/6J mice

In this paper we describe catalytic and immunological properties of coumarin 7-hydroxylase, a cytochrome P-450 dependent enzyme activity, in the liver, kidney and lung of C57BL/67 and DBA/2J mice. Coumarin 7-hydroxylase activity was higher in D2 than in B6 mice in all three organs. For both strains of mice, liver had the highest enzyme activity when expressed per milligram of microsomal protein. However, when expressed per nmole cytochrome P-450 there was no difference in the enzyme activity between the tissues. Inhibition of microsomal coumarin 7-hydroxylase by antibodies previously developed in our laboratory against a cytochrome P-450 fraction from D2 and B6 mouse liver, associated with coumarin 7-hydroxylase, occurred as follows. In D2 mice both antibodies caused approximately 50% inhibition of the enzyme activity of all three organs. In B6 mice, however, the only organ where considerable inhibition took place was the liver, and only when antibody against B6 cytochrome P-450 was used. Ouchterlony immunodiffusion analysis revealed a 100% crossreactivity between the two strains of mice when similar organs were compared. The 100% crossreactivity was also found between the liver and lung in both strains of mice. However, only a 50% crossreactivity was found between kidney and liver or kidney and lung in B6 and between the kidney and lung in D2. The data demonstrate interorgan and interstrain differences in the immunological and catalytical properties of cytochrome(s) P-450 catalysing coumarin 7-hydroxylation.

Aryl hydrocarbon hydroxylase in rat brain microsomes

Trp-P-1 (3-amino-1,4-dimethyl-5H-pyrido(4,3-b)indole) is known to be metabolized by cytochrome P4501A (P4501A) to reactive intermediates which may bind irreversibly to tissue macromolecules. The irreversible binding of [3H]Trp-P-1 in the brain of NMRI-mice was studied by microautoradiography. There was a selective irreversible binding of radioactivity in endothelial cells following an i.v. or i.p. injection of [3H]Trp-P-1 (100 μg/kg or 1.5 mg/kg) in mice treated with the P4501A-inducing agent β-naphthoflavone (BNF). The binding of radioactivity was highest in capillary loops of the choroid plexus, less marked in large cerebral veins and in arachnoidal veins whereas no binding was observed in cerebral capillaries, arteries, neurons or in other brain cells. In endothelial cells of vehicle-treated control mice injected with [3H]Trp-P-1 no binding of radioactivity was observed. At incubation of brain slices with [3H]Trp-P-1, there was a marked irreversible binding of radioactivity in endothelial cells in the choroid plexus of BNF-treated mice but not in vehicle-treated control mice. The P4501A inhibitor ellipticine abolished the BNF-induced endothelial binding of [3H]Trp-P-1-derived radioactivity in vivo and in vitro. The marked binding of Trp-P-1-derived radioactivity in endothelial cells of the choroid plexus of BNF-treated mice demonstrate that a BNF-responsive enzyme activity, possibly P4501A, may be induced at this site and suggests that a P450-dependent enzyme activity be part of the blood-cerebrospinal fluid barrier regulating the transendothelial passage of compounds.

The effects of buthionine sulphoximine (BSO) on glutathione depletion and xenobiotic biotransformation

Buthionine sulphoximine (BSO) is an inhibitor of γ-glutamylcysteine synthetase (γ-GCS) and consequently lowers tissue glutathione (GSH) concentrations. In fed male C3H mice, liver and kidney GSH levels were depleted by BSO in a dose dependent manner with maximum effect (35% of initial levels) occurring with doses between 0.8 and 1.6 g/kg, i.p. At these doses maximum effects on γ-GCS and GSH were observed 2–4 hr after BSO administration; initial γ-GCS activity and GSH content were restored approximately 16 hr post BSO. BSO, either in vivo or in vitro, had no effect on hepatic microsomal cytochrome P-450 levels, a range of cytochrome P-450 dependent enzyme activities or p-nitrophenol glucuronyl transferase activity. Similarly, BSO had no effect on phenol sulphotransferase and two GSH-transferase activities in the 105, 000 g supernatant fraction. BSO had no effect on the duration of hexobarbitone induced narcosis in mice. Consistent with specific inhibition of GSH synthesis, BSO pretreatment of mice decreased the proportion of a 50 mg/kg dose of paracetamol excreted in the urine as GSH-derived conjugates but did not affect paracetamol clearance through the glucuronidation or sulphation pathways. Since BSO does not affect cytochrome P-450 or conjugating enzyme activity, its use as a specific depletor of tissue GSH in the investigation of mechanisms of xenobiotic-induced toxicities is preferable to the standard GSH-depleting agents as these have other enzymic effects.

1-Ethynylpyrene, a suicide inhibitor of cytochrome P-450 dependent benzo[a]pyrene hydroxylase activity in liver microsomes.

The preparation of 1-ethynylpyrene (EP) is described. Incubation of EP with liver microsomes in the presence of NADPH yields fluorescent products, but the production of these products ceases after 15 min. Addition of fresh microsomes restores the original rate of EP metabolism. The metabolism of EP is initially more rapid in microsomes from 5,6-benzoflavone- (BF) pretreated rats than in microsomes from phenobarbital- (PB) pretreated rats or from untreated, control animals. EP strongly inhibits the hydroxylation of benzo[a]pyrene (BP) by liver microsomes; after 20 min in the presence of EP, BP metabolism nearly ceases. Addition of fresh microsomes restores the original rate of BP hydroxylation. EP more effectively inhibits the oxidation of BP in liver microsomes from rats pretreated with BF than from rats pretreated with PB or from untreated, control animals. The inhibition of BP hydroxylation activity due to EP is dependent upon NADPH and is apparently irreversible. Kinetic analyses demonstrate that the observed inhibition of BP hydroxylation is due to loss of the enzymatic activity by a process that is first order in EP and that reaches a limiting value at infinite EP concentrations. One such first-order process, with a t 1/2 of 3.5 min and a Ks for EP of 40 microM, is observed in microsomes from BF-pretreated rats. Two such first-order processes, one with t 1/2 of 6.9 min and Ks of 46 microM and one with t 1/2 of 12.7 min and Ks of 33 microM, are observed in microsomes from PB-pretreated rats. It is proposed that a self-catalyzed inhibition (suicide inhibition) of the cytochrome P-450 dependent BP hydroxylation occurs in the presence of EP. Incubation with EP under conditions that result in loss of about 90% of the BP hydroxylase activity in microsomes from BF-pretreated rats and about 66% of the activity in microsomes from PB-pretreated rats causes the loss of only 6 and 12% of the cytochrome P-450, respectively. It is concluded that loss of P-450 content is an insensitive measure of the effect of this inhibitor upon this cytochrome P-450 dependent enzyme activity. The selectivity of the loss of P-450 due to the incubation of the different microsomal preparations with EP is also observed to be different than the selectivity for loss of BP hydroxylase activity. It is proposed that the suicide inhibition of cytochrome P-450 dependent enzymes by alkynes need not involve heme alkylation and a resulting loss of P-450 content.(ABSTRACT TRUNCATED AT 400 WORDS)

Induction of cytochrome P-450 dependent reactions in the rat ventral prostate by β-naphthoflavone and 2,3,7,8-tetrachlorodibenzo- p-dioxin

The purpose of this study was to characterize drug metabolism in the rat ventral prostate in untreated, β-naphthoflavone (BNF) or 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) treated animals. Intraperitoneally administered [ 3H] benzo[a] pyrene (BP) was recovered in about equal quantities in rat ventral prostate, lungs and testes. The relatively high uptake of [ 3H]BP in the ventral prostate might possibly be related to the occurence of a macromolecule in the gland binding TCDD and 3-methylcholanthrene (MC) with a relatively high affinity ( K d 1.5 and 5.0 μM, respectively). BNF, BP and MC competed with the TCDD binding site of the macromolecule. The prostatic cytochrome P-450 dependent microsomal enzyme activities aryl hydrocarbon hydroxylase (AHH) and 7-ethoxyresorufin O-deethylase are very low in untreated animals (about 1 pmol/min/mg of microsomal protein). BNF- or TCDD treatment of the animals induced these activities about 500-fold, whereas aminopyrine N-demethylase was enhanced to a smaller degree. Maximal enzyme activities of AHH and 7-ethoxyresorufin O-deethylase were obtained 24 h after a single intraperitoneal injection of BNF and the activities decayed relatively rapidly after this peak. The magnitude of enzyme induction was much higher in the prostate than in the liver. Mutagenic BP metabolites were formed in vitro by induced prostatic microsomes as revealed by HPLC analysis. The activity of NADPH-cytochrome c reductase was 35 pmol/min/mg and was not changed by BNF- or TCDD treatment.

Stimulation of microsomal drug oxidation in liver and kidney of rats treated with the oncolytic agent cis-dichlorodiammineplatinum-II.

In vitro drug metabolism from liver and kidney of rats was studied for up to 12 days following a single intraperitoneal injection of the antineoplastic drug cisplatinum. At 3,5, and 8 days posttreatment, hepatic cytochrome P-450 dependent enzyme activity was increased 20-45%, but levels of cytochromes P-450 and b5 were unaltered. Hepatic in vitro stimulated thiobarbituric acid reactive (TBAR) substances were increased at all times, with a maximum of 20 times normal on day 5. Renal aniline hydroxylase activity was elevated 25-95% throughout the first 8 days. Renal in vitro stimulated TBAR substances were not significantly altered. In vitro addition of cisPt to control microsomes resulted in no consistent, dose-related changes in substrate metabolism.

Effect of hepatocarcinogens on epoxide hydrolase and other xenobiotic metabolizing enzymes.

Hepatocarcinogens cause marked biochemical changes in the liver at short intervals after administration. The studies described were designed to investigate the effects of hepatocarcinogens and hepatotoxicants on the microsomal mixed function oxidase system. DT-diaphorase and epoxide hydrolase. Following 5 day p.o. treatment of male F-344 rats with aflatoxin B1 (AFB), 2-acetylaminofluorene (AAF), technical grade dinitrotoluene (DNT), or 2,4-diaminotoluene, microsomal cytochrome P450 dependent enzyme activities were depressed while epoxide hydrolase activity was markedly elevated (3-8 times control). Diethylnitrosamine (DEN) given at 5 mg/kg/day and DL-ethionine at 1000 mg/kg/day failed to increase epoxide hydrolase. 3-Methylcholanthrene, methylnitrosourea, carbon tetrachloride, bromobenzene and vinyl chloride all failed to increase epoxide hydrolase activity. Using 3 daily i.p. injections, dose-response relationships for increases in epoxide hydrolase were generated for the hepatocarcinogens. With the exception of p-dimethylaminoazobenzene (DAB) and DEN, the carcinogens studied produced log-linear dose response curves for increase in epoxide hydrolase. Both DEN and DAB caused increases in epoxide hydrolase but classical sigmoidal dose-response curves were not obtained. The order of potency for increasing epoxide hydrolase was AFB greater than AAF greater than 2,6-dinitrotoluene greater than 3'-methyl-N,N-dimethyl-4-aminoazobenzene greater than DNT greater than 2, 4-dinitrotoluene. The slopes of the linear portions of the log dose-response curves were not statistically different from the slope of the dose-response curve obtained with AAF suggesting that structurally diverse carcinogens elicit increases in epoxide hydrolase by a common mechanism.

Actions of danazol in vivo on cytochrome P-450 and steroidogenic enzymes in rat testis and liver microsomal preparations

The mechanism of action of danazol has not been established and the drug may act at multiple loci. Effects suggesting inhibition of pituitary gonadotropin release have been described while some in vitro studies have demonstrated competitive inhibition of steroidogenic enzymes. In addition, destruction of cytochrome P-450 by the acetylenic moiety of danazol is a possible mechanism. Following 14 days of danazol treatment (10 mg/kg/day), the specific content of rat testis microsomal cytochrome P-450 and the serum testosterone were decreased, in spite of no significant change in serum luteinizing hormone. Furthermore, simultaneous administration of human chorionic gonadotropin with danazol still resulted in a decrease in cytochrome. The activities of two testicular microsomal steroid-converting enzymes (17 alpha-hydroxylase and 17,20-lyase) were not altered when based on cytochrome P-450 content but were markedly depressed when based on microsomal protein. In liver, danazol showed a similar but less marked dimunition of the cytochrome. Surprisingly, steroid 17 alpha-hydroxylase activity in liver was significantly increased while other cytochrome P-450 dependent enzyme activities were not altered.

Determination of plasma trimethadione and its metabolite carbon tetrachloride-intoxicated rat liver: a useful tool for estimation of hepatic drug metabolizing capacity.

1. The relationship between the plasma concentration ratio of 5,5-dimethyl-2,4-oxazolidinedione (DMO) to trimethadione (TMO) and cytochrome P-450 dependent drug-metabolizing enzyme activities after administration of TMO to rats pretreated with different dose levels of carbon tetrachloride (CCl4) was investigated. 2. Pretreatment of rats with different dose levels of CCl4 resulted in a prolongation of TMO half-life, and increase of the area under the curve (AUC), and a decrease of clearance, but the apparent volume of distribution (Vd) was not significantly decreased. 3. Depending on the extent of liver-injury by CCl4, there was a good correlation between the ratio of DMO to TMO in plasma and hepatic cytochrome P-450 dependent drug -metabolizing enzyme activities such as cytochrome P-450 content (r=0.796 at 1 h, r=0849 at 2 h), aminopyrine N-demethylase activity (r=0.877 t 1 h, r=0905 at 2 h), TMO N-demethylase activity (r=0.876 at 1 h,r=0.928 at 2 h) and aniline hydroxylase activity (r=0.900 at 1 h, r=0.936 at 2 h). 4. These results, together with the previous findings, indicate determination of the plasma levels of TMO and DMO might be a useful tool as an index of drug metabolizing capacity of rat livers.