Rat Hepatic Microsomal Preparations Research Articles

Sex-dependent toxicity of a novel acyl-CoA:cholesterol acyltransferase inhibitor, YIC-C8-434, in relation to sex-specific forms of cytochrome P450 in rats.

YIC-C8-434 is a novel inhibitor of acyl coenzyme A:cholesterol acyltransferase (ACAT). To clarify the toxicity of YIC-C8-434, the compound was given orally to Sprague-Dawley rats for 28 days at 0, 4, 20, 100, or 500 mg/kg/day. The toxicity of the drug differed significantly between male and female rats. In female rats treated at 500 mg/kg, many symptoms including moribund condition, suppression of weight gain and food consumption, abnormal blood chemistry, and decreases in organ weights (thymus, ovaries, and uterus) were observed. In male rats by contrast, no significant toxicity was observed at any dose. After a single administration of YIC-C8-434 at 500 mg/kg, female rats had a higher blood concentration of the compound than male rats. Little elimination of YIC-C8-434 was observed in female rats on analysis of drug-elimination kinetics. Furthermore, the metabolism of YIC-C8-434 was analyzed using rat hepatic microsomal preparations from both sexes. Consistent with the observations in vivo, hepatic microsomes from male rats better metabolized YIC-C8-434 than those from females. In addition, the metabolism of YIC-C8-434 by hepatic microsomes from male rats was blocked by SKF525A, a P450 inhibitor. Inhibition experiments using anti-rat CYP1A1, CYP1A2, CYP2B1, CYP2C11, CYP2E1, CYP3A2, and CYP4A1 antisera indicated that CYP3A2 played the predominant role in the metabolism of YIC-C8-434 in rats. Since there is less CYP3A2 in the liver of female than male rats, the involvement of CYP3A2 in YIC-C8-434 metabolism has implications for the sex-related metabolic activity and toxicity of YIC-C8-434.

The in vitro hepatic microsomal metabolism of N-benzyladamantanamine in rats.

The metabolism of N-benzyladamantanamine (NBAD) was studied in vitro using rat hepatic microsomal preparations. The substrate and proposed metabolites were synthesized and characterized using spectroscopic techniques and separated using a reverse phase HPLC system. NBAD was incubated with rat microsomal preparations, extracted into DCM in the presence of NaCl and evaporated under a stream of nitrogen. The results from HPLC studies showed that NBAD produced the corresponding nitrone and hydroxylamine. This experiment also revealed that dealkylation occurred. No metabolites were observed which corresponded to authentic amide or oxaziridine. The reactions required a microsomal enzyme source and NADPH as a cofactor. The results indicate that the nitrone observed as a metabolite of NBAD is not an intermediate leading to the formation of an oxaziridine and hence an amide, under careful experimental conditions excluding light.

The in vitro hepatic microsomal metabolism of 3,5-dimethyl-4-(phenylazo)-(1H)-pyrazole in rats.

The in vitro hepatic microsomal metabolism of 3,5-dimethyl-4-(phenylazo)-(1H)-pyrazole (DMPAP) was studied using washed rat hepatic microsomal preparations fortified with NADPH. The substrate, DMPAP, and its potential metabolites, i.e. the corresponding reduction product, 3,5-dimethyl-4-amino-(1H)-pyrazole (DMAP), and the oxidation product, 3,5-dimethyl-4-(phenylazoxy)-(1H)-pyrazole (DMAPO), were synthesized and their structures elucidated by use of their spectral characteristics. DMPAP and its potential metabolites were then separated using a reverse phase HPLC system consisting of a C18 column and a mobile phase of acetonitrile:water (50:50) at a flow rate of 1 ml/min with UV detection at 254 nm. DMPAP was incubated with rat microsomal preparations, extracted into DCM in the presence of NaCl, and finally evaporated under a stream of nitrogen. The results from HPLC studies showed that DMPAP was metabolised to the corresponding reduction and oxidation products in the presence of NADPH.

The substrate specificity of the rat hepatic cytosolic arylamine oxidase catalyzing the bioactivation of aromatic amines

The ability of the cytosolic arylamine oxidase to convert structurally diverse aromatic and heterocyclic amines to mutagens in the Ames test was investigated using hepatic cytosol from Aroclor 1254-treated rats as the activation system. Using this system, only amino compounds containing at least three fused aromatic rings elicited a strong mutagenic effect; heterocyclic amines failed to exhibit mutagenicity. In contrast, when Aroclor 1254-induced rat hepatic microsomal preparations served as the activation system, monocyclic, bicyclic as well as larger amino compounds induced a clear mutagenic response; moreover, heterocyclic amines were potent mutagens. Nitrosamines displayed mutagenicity in the presence of only the microsomal activation system. In the presence of the cytosol, the mutagenic response of aromatic amines was much lower in the bacterial strain TA98-1,8-DNP 6, which is deficient in O-acetyltransferase activity, compared to the normal TA98 strain. This finding implies that the cytosolic activation of aromatic amines involves N-hydroxylation.

In vitro metabolic N- and C-oxidation of phenanthridine.

The in vitro microsomal metabolism of phenanthridine has been studied to establish as to whether phenanthridine produces the corresponding N-oxide and lactam as metabolites and the mechanism involved. We now report our preliminary findings using rat hepatic microsomal preparations (control and induced with phenobarbitone) fortified with NADPH. The potential metabolite, phenanthridine-N-oxide, was prepared by m-CPBA oxidation of substrate; the lactam was commercially available. The substrate and metabolites were extracted and analysed by HPLC and TLC. Five metabolites, i.e. the corresponding N-oxide, lactam and three other products, were detected. Both N-oxide and lactam metabolites showed identical chromatographic behaviour and UV spectrum--using a multi-array UV detector linked to a HPLC system--as the authentic compounds. The uncharacterised metabolites are proposed to be phenolic because of their chromatographic behaviour and response to detection reagents. The amount of N-oxide and lactam formed was significantly increased when phenobarbitone induced rat microsomes were used as enzyme source. The results indicate that these latter metabolites are probably formed by a phenobarbitone inducible CYP450 isozyme. It may be that the lactam was produced via the N-oxide and experiments are under way to investigate the proposed pathway.

Effect of acute and chronic butylated hydroxyanisole administration on in vivo glucuronidation of N-nitrosobutyl(4-hydroxybutyl)amine in rats

The urinary metabolic pattern of N-nitrosobutyl(4-hydroxybutyl)amine (NB4HBA) administered ip at a dose of 5 mg/kg body weight was studied in animals either pretreated with butylated hydroxyanisole (BHA) as a single oral dose of 50 or 250 mg/kg, or fed a diet containing 0.1 or 0.5% BHA. The 24-hr urinary excretion of NB4HBA, its glucuronic acid-conjugate (NB4HBA-G) and N-nitrosobutyl(3-carboxypropyl)amine (NB3CPA) in control rats were 0.12, 0.75 and 30% of the administered dose, respectively, and were not changed after a single oral dose of 50 mg BHA/kg. NB4HBA-G was significantly reduced in the urine of rats given 250 mg BHA/kg. In vitro assays carried out using rat-hepatic microsomal preparations as the source of the enzyme UDP-glucuronyl transferases (GT) and NB4HBA as the substrate, suggest that a competition between NB4HBA and BHA for the same enzyme may be the cause of the decreased NB4HBA-G excretion observed in vivo. A fourfold increase in NB4HBA-G urinary excretion was observed after chronic 0.5% BHA feeding; moreover, the glucuronic acid-conjugate of NB3CPA (NB3CPA-G), which was not detected in the controls or after acute BHA treatment, appeared in the urine of rats given dietary BHA for 3 wk, accounting for about 10% of the administered NB4HBA. In vitro experiments indicate that the increased glucuronides excretion may be the result of an elevated hepatic GT activity.

Regioselective progesterone hydroxylation catalyzed by eleven rat hepatic cytochrome P-450 isozymes

Quantitative high-pressure liquid chromatographic assays were developed that separate progesterone and 17 authentic monohydroxylated derivatives. The assays were utilized to investigate the hydroxylation of progesterone by 11 purified rat hepatic cytochrome P-450 isozymes and 8 different rat hepatic microsomal preparations. In a reconstituted system, progesterone was most efficiently metabolized by cytochrome P-450h followed by P-450g and P-450b. Seven different monohydroxylated progesterone metabolites were identified. 16 alpha-Hydroxyprogesterone, formed by 8 of the 11 isozymes, was the only detectable metabolite formed by cytochromes P-450b and P-450e. 2 alpha-Hydroxyprogesterone was formed almost exclusively by cytochrome P-450h, and 6 alpha-hydroxyprogesterone and 7 alpha-hydroxyprogesterone were only formed by P-450a. 6 beta-hydroxylation of progesterone was catalyzed by four isozymes with cytochrome P-450g being the most efficient, and 15 alpha-hydroxyprogesterone was formed as a minor metabolite by cytochromes P-450g, P-450h, and P-450i. None of the isozymes catalyzed 17 alpha-hydroxylation of progesterone, and only cytochrome P-450k had detectable 21-hydroxylase activity. 16 alpha-Hydroxylation catalyzed by cytochrome P-450b was inhibited in the presence of dilauroylphosphatidylcholine (1.6-80 microM), while this phospholipid either stimulated (up to 3-fold) or had no effect on the metabolism of progesterone by the other purified isozymes. Results of microsomal metabolism in conjunction with antibody inhibition experiments indicated that cytochromes P-450a and P-450h were the sole 7 alpha- and 2 alpha-hydroxylases, respectively, and that P-450k or an immunochemically related isozyme contributed greater than 80% of the 21-hydroxylase activity observed in microsomes from phenobarbital-induced rats.

The oxidation of isomeric amino and acetamidobiphenyls by rat hepatic microsomal preparations.

The metabolism of isomeric amino and acetamidobiphenyls was studied using rat liver microsomal preparations. The aromatic amines were hydroxylated ortho or para to the amino group, whereas the aromatic amides were mainly oxidized in the para position. The carcinogenic amines 3- and 4- aminobiphenyl were converted to hydroxylamines and nitroso compounds. The non-carcinogenic amine 2-aminobiphenyl was resistant to enzymic nitrogen oxidation. The results support the concept that oxidation of aromatic amino groups is a prerequisite for carcinogenic and mutagenic activity.

Lack of hepatic microsomal metabolism of deoxynivalenol and its metabolite, DOM-1

Rat hepatic microsomal preparations were used to study the metabolism of deoxynivalenol (DON) and its metabolite 3α,7α,15-trihydroxytrichothec-9,12-dien-8-one (DOM-1). The N-demethylation of ethylmorphine was monitored to assess the viability of the mixed-function oxidase. DON was incubated with microsomes and an NADPH-generating system. Samples were removed from the incubation system and analysed for DON using an HPLC equipped with a UV detector. After incubation for 30 min, there was no evidence of disappearance of DON or of the presence of new metabolites; neither was microsomal NADPH oxidation altered by the addition of DON. Rat and pig hepatic microsomal preparations were used to assess DON glucuronidation, using p-nitrophenol disappearance to check the viability of the microsomal glucuronidating system. When DON was incubated with microsomes and 14C-labelled uridine 5′-diphosphoglucuronic acid, no radioactivity was detected in the TLC zone where the glucuronide was expected. Three rats and one pig were dosed orally with 2 mg DON/kg and samples of their urine and faeces were extracted and incubated with β-glucuronidase or with buffer only. No differences in DON or DOM-1 concentrations were detected between samples incubated with or without β-glucuronidase. These results suggest that DON was neither bioactivated to a more toxic product nor oxidized to a less toxic compound by the rat hepatic mixed-function oxidase system. Likewise, DOM-1 was not reactivated or metabolized by this system. Neither DON nor DOM-1 glucuronides were formed either in in vitro liver systems or in vivo.

Rat hepatic cholesterol 7α-hydroxylase: Biochemical properties and comparison to constitutive and xenobiotic-inducible cytochrome P-450 enzymes

Cytochrome P-450 Ch7 α (cholesterol 7α-hydroxylase) catalyzes the first and rate-limiting step in the conversion of cholesterol to bile acids in mammalian liver. The properties of this cytochrome P-450 ( P-450) form were studied in rat hepatic microsomal preparations in comparison to those of several well characterized constitutive and xenobiotic-inducible rat hepatic P-450s. Administration of the bile acid-sequestering resin cholestyramine [4% ( w w ) in the diet] to male or female rats maintained on a reverse light cycle led to a 10- to 15-fold induction of P-450 Ch7 α activity relative to untreated, standard light cycle controls. By contrast, the levels of four hepatic steroid hormone hydroxylating P-450 enzymes, designated 2a, 2c, 3, and PB-4 [Waxman, D. J. (1984) J. Biol. Chem. 259, 15481–15490] , were not significantly affected by cholestyramine treatment. Antibody inhibition experiments established that P-450 Ch7 α is immunochemically distinct from nine other rat hepatic P-450s, including P-450 3, a highly regio- and stereoselective steroid hormone 7α-hydroxylase. P-450 Ch7 α was shown to be selectively inactivated by micromolar concentrations of the disulfide-containing reagents disulfiram (Antabuse ®) and 2,2′-dithiopyridine. This inactivation was readily reversed upon incubation with 2-mercaptoethylamine, suggesting the presence of a highly reactive thiol group at the active site of P-450 Ch7 α . These findings demonstrate that P-450 Ch7 α corresponds to a unique P-450 enzyme exhibiting inductive, biochemical, immunochemical, and regulatory properties distinct from those of nine well-characterized rat hepatic P-450 forms.

Lipid composition of liver microsomes from rats treated with acetone

The metabolism of certain compounds by rat hepatic microsomal preparations obtained from acetone-treated rats is greater than that measured in microsomes obtained from control rats. Since the lipid composition of membranes can significantly influence metabolism catalyzed by membrane-bound enzymes, studies were conducted in order to determine whether acetone-induced alterations in microsomal metabolism are associated with changes in the lipid composition of these membranes. Phospholipid and cholesterol concentrations were measured in extracts of rat liver microsomes obtained from rats treated with acetone, sodium phenobarbital, or vehicle. Acetone treatment did not alter the phospholipid and cholesterol content of the microsomes, whereas sodium phenobarbital (positive control) caused a significant decrease in cholesterol content/mg microsomal protein. These data indicate that acetone-induced alterations in microsomal metabolism may not be attributed to changes in the phospholipid and cholesterol content of the microsomes.

Comparison of the mutagenicities of 4-aminobiphenyl and benzidine in the Salmonella/microsome, Salmonella/hepatocyte and host-mediated assays

The applicability of suspensions of rat hepatocytes as the metabolic factor in a bacterial mutagenicity test was studied. Therefore, mutagenicity of 4-aminobiphenyl and benzidine towards the Salmonella typhimurium strains TA1538 and TA100 in the presence of rat-hepatic microsomal preparations or intact liver cells was measured. The mutagenic potency of these compounds was also established in the intrasanguineous host-mediated assay. Comparison of the test results showed that the mutagenic activity of 4-aminobiphenyl and benzidine after metabolism by intact rat hepatocytes resembled the mutagenic activity of these compounds after biotransformation in vivo more than after activation with a rat-liver S9 fraction.

Binding of acetaldehyde to rat liver microsomes: Enhancement after chronic alcohol consumption

Summary Rat hepatic microsomal preparations were assessed for their capacity to bind exogenous 14C-acetaldehyde and 14C-acetaldehyde formed endogenously from 14C-ethanol in the microsomal ethanol-oxidizing system (MEOS). Exogenous acetaldehyde was found to bind to rat liver microsomes. Blocking of the free amino groups and thiol groups with site specific reagents (pyridoxal 5′-phosphate and p-hydroxymercuribenzoate) reduced the binding of acetaldehyde. Acetaldehyde formed endogenously from ethanol also bound to hepatic microsomes. This was increased after chronic alcohol consumption, in association with enhanced MEOS activity. The binding of acetaldehyde produced by MEOS was significantly greater than that of an equivalent amount of exogenous acetaldehyde. Thus, acetaldehyde produced in situ may exert local toxic effects on the endoplasmic reticulum.

Unusual substrate specificity in the oxidative dearylation of paraoxon analogs by mouse hepatic microsomal enzymes

In previous studies with various dialkyl paraoxons, it was shown that the methyl, ethyl and i-propyl analog were dearylated by hepatic microsomes almost exclusively through an NADPH-independent A-esterase, while dearylation of n-propyl paraoxon was NADPH-dependent. The present investigation was initiated to study the basis for this specificity. Release of p-nitrophenol was measured spectrophotometrically after incubation of a series of dialkyl-substituted paraoxon analogs with rat hepatic microsomal preparations. Oxidative dearylation was shown to have the characteristics of a typical mixed-function oxygenase (MFO) reaction. Methyl, ethyl, n-butyl and n-amyl paraoxons were not readily metabolized by the MFO system, though considerable dearylation of n-propyl paraoxon was observed. When the two alkyl substituents were not identical, only the compounds with an n-propyl group were metabolized in this manner. Dearylation was also observed with analogs containing certain branched chains or modified ethyl groups (e.g. 2-chloroethyl and 2-methoxyethyl). The unusual specificity for this MFO reaction was associated with the presence of a 3-carbon length chain or its steric equivalent. The reaction mechanism is most likely different from that for the analogous MFO-catalyzed dearylation of parathion analogs, as p-nitrophenol production from the paraoxons is not accompanied by release of the corresponding dialkyl phosphate.