Microsomal Protein Levels Research Articles

Xenobiotic imprinting of the hepatic monooxygenase system: Effects of neonatal phenobarbital administration

The ability of phenobarbital (PB) to neonatally “imprint” or “program” the hepatic microsomal cytochrome P-450-dependent monooxygenase system (MOS) was investigated. Phenobarbital (30 mg/kg) was administered subcutaneously to neonatal rats of both sexes on days 1–5 postpartum. various hepatic MOS activities were measured at 6, 22, 50 and 140 days of age. Six-day-old animals of both sexes displayed the increased hepatic microsomal protein levels and enzyme activities expected from the action of phenobarbital as a transitory MOS inducer. Most of these increases dissipated by 22 and 50 days of age. However, at 140 days of age rats of both sexes that had received neonatal phenobarbital showed increased levels of cytochrome P-450, as well as both P-450 and cytochrome c reductase, ethoxycoumarin O-deethylation, glucuronyl transferase activity, in vitro covalent binding of benzo[ a]pyrene to DNA and in vivo covalent binding of aflatoxin B 1 to hepatic macromolecular fractions. Neonatal phenobarbital administration can alter the metabolic profile of rats in adulthood, apparently by a mechanism different from that responsible for either transitory PB induction or testosterone imprinting.

No effect of prolonged fluoride exposure on cytochrome P-450 and associated monooxygenases or on the level of polyamines in the rat.

When exposing rats to drinking water containing 100 p.p.m. fluoride for 8 weeks, no effect could be detected in biochemical parameters of the liver, such as the concentrations of the polyamines putrescine, spermidine and spermine; the levels of microsomal protein and cytochrome P-450; or the activities of two associated monooxygenases, aryl hydrocarbon hydroxylase and ethylmorphine N-demethylase. Neither was there any increase in plasma glutamic-oxalacetic transaminase indicative of liver damage.

Inhalation toxicity studies of hydrogen cyanide (HCN) in spbague-dawley rats

Groundwater contamination with arsenic is a major global health concern. The organophosphorus insecticide malathion has gained significance as an environmental pollutant due to its widespread use in agriculture, grain storage, ectoparasite control and public health management. The deleterious effects produced by arsenic or malathion alone are documented, but very little is known about the consequences of their coexposure. The aim of the current study was to examine the effects of repeated simultaneous exposure to arsenic and malathion on drug-biotransforming enzymes in the liver of broiler chickens. One-month-old broiler chickens were exposed daily to arsenic (50 ppm)-supplemented drinking water, malathion (500 ppm)-mixed diet or in a similar fashion coexposed to these agents for 28 days. At the term, changes in body weight, organ weights, and levels of hepatic cytochrome P450 (CYP), cytochrome b5, microsomal and cytosolic proteins; aminopyrine N-demethylase (ANDM), aniline P-hydroxylase (APH), glutathione S-transferase (GST) and uridine diphosphate glucuronosyltransferase (UGT) were assessed. Arsenic, malathion or their coexposure decreased the body weight gain and liver weight. Brain weight (relative) was increased with arsenic or malathion, but not with the coexposure. Treatment with arsenic decreased the CYP and cytochrome b5 contents by 39 and 36%, than with malathion by 54 and 22% and the coexposure by 45 and 28%, respectively. The ANDM activity was decreased with arsenic (44%), malathion (23%) and the coexposure (32%). Arsenic (23%) and the coexposure (37%), but not malathion (14%), reduced the APH activity. The activities of hepatic microsomal and cytosolic GST were increased with all the three treatments [Arsenic (microsomal: 88% cytosolic: 113%), malathion (microsomal: 137%, cytosolic: 94%) and coexposure (microsomal: 140%, cytosolic: 148%)]. These treatments did not significantly affect the hepatic UGT activity, but reduced the hepatic microsomal (arsenic: 28%, malathion: 34% and coexposure: 43%) and cytosolic (17–19%) protein contents. The effects of coexposure on the activities of various phase I and phase II drug-biotransforming enzymes were almost similar to that of arsenic or malathion. This study provides evidence that repeated coexposure to arsenic and malathion may influence the extent of drug metabolism in chickens.

Evaluation of a methodology for the use of preparations from rat small intestine in the Salmonella/microsome assay

A methodology is evaluated for the use in the Ames assay of a microsomal metabolising system derived from villous tip cells of rat small intestine. The procedure involved high frequency vibration of everted gut segments followed by gentle lysis and homogenisation. This technique, which has previously been shown to result routinely in high levels of cytochrome P450 and linked enzymes, has now been investigated for its ability to yield preparations capable of activating several promutagens in the Salmonella/plate incorporation test. The data obtained have been compared with results observed with standard rat liver metabolising fractions. In the presence of intestinal microsomes, 2-aminoanthracene, 2-aminofluorene, 2-acetylaminofluorene, aflatoxin B 1, benzo[ a]pyrene and cyclophosphamide all caused dose-related increases in revertants, the maximum yields of which were lower than those detected with liver microsomes or S9 mix. These and other differences in dose-responses have been discussed in relation to the levels of microsomal protein and cytochrome P450 plated and with respect to the activities of relevant enzymes in the tissue extracts.

Prochloraz, a potent inducer of the microsomal cytochrome P-450 system

Prochloraz ( N-propyl- N-[2-(2,4,6-trichlorophenoxy)ethyl]-imidazole-1-carboxamide), a recently developed agricultural fungicide, is a potent inducer of microsomal enzymes. Rats fed 7 days with a prochloraz-contaminated diet (2500 ppm) showed an increase in hepatic cytochrome P-450, cytochrome b 5, and microsomal protein level; aniline hydroxylase, 7-ethoxycoumarin dealkylase, 7-ethoxyresorufin dealkylase, NADPH-cytochrome c reductase, and epoxide hydrolase were significantly induced. At a lower dose (100 ppm), only an increase in cytochrome P-450 and 7-ethoxyresorufin dealkylase was noticed. As shown with aniline hydroxylase and 7-ethoxycoumarin dealkylase, prochloraz is also a potent inhibitor of drug-metabolizing enzymes. The interaction of prochloraz with hepatic microsomal fraction from rat liver was also studied. Prochloraz binds to oxidized cytochrome P-450 to produce a type II spectral change; the compound also binds to reduced cytochrome P-450. The binding of some ligands (7-ethoxycoumarin, n-octylamine, aniline, and imidazole) to oxidized cytochrome P-450 was determined after induction by prochloraz. Japanese quails ( Coturnix coturnix) fed 7 days with a prochloraz-contaminated diet (2000 ppm) showed a dramatic increase in liver weight (2.5-fold) and both hepatic and duodenal cytochrome P-450 (9- and 12-fold, respectively).

Liver microsomal membrane lipid composition in marasmic-kwashiorkor

The microsomal membrane cholesterol and phospholipid content and phospholipid composition of marasmic kwashiorkor rats have been compared with those of normal rats. A Significant increase in the cholesterol/phospholipid ratio, as well as in the sphingomyelin/phosphatidyl-choline ratio was observed in the marasmic-kwashiorkor rat. These effects would tend to decrease the fluidity of the phospholipid bilayer of the endoplasmic reticulum membrane and may thus affect drug metabolism. It is well known that a change in the quality or quantity of dietary protein causes an alteration in the rates of metabolism of many xenobiotics by the mammalian liver (1–3). These metabolic alteration have been attributed mainly to changes in the levels of microsomal membrane proteins themselves, especially that of cytochrome P-450 (4–6). However, a recent report by Suzuki et al. (7) indicates that the more subtle features of drug metabolism such as interactions between NADPH-cytochrome P-450 reductase, cytochrome P-450, cytochrome b and other specific drug metabolzing enzymes in the membrane of the endoplasmic reticulum might well be affected by the fluidity of the phospholipid bilayer. There is still a high incidence of protein-energy malnutrition (PEM) diseases such as kwashiorkor in many part of the world (8). The membrane lipid composition from microsomes of marasmic-kwashiorkor rats have therefore been investigated with a view to finding out if there are any changes in these components due to protein deficiency which could contribute to the decreased metabolism of xenobiotics in this condition.

Aniline and hexobarbital hydroxylases from rat lung and kidney: neither sex dependent nor inducible with phenobarbital.

The basal activities of aniline hydroxylase (AH), hexobarbital hydroxylase (HH), and ethylmorphine N-demethylase (ED) were measured in the 9000 X g supernatant of kidneys and lungs from male and female rats. No ED activity was detected in any tissue although all tissues N-demethylated three other substrates. The activities of AH and HH were not sex dependent in either kidney or lung. Similarly, pulmonary and renal microsomal protein concentrations were independent of sex. In addition, cytochrome P-450 levels in the kidney were the same in males and females (pulmonary P-450 was not measured). The pulmonary hydroxylases were more active than the renal enzymes in both sexes. In males, phenobarbital (ip, 50 mg X kg-1 X day-1 for 3 days) failed to induce AH or HH in either kidney or lung; it did not increase the weight or microsomal protein levels of these organs and it also failed to increase renal P-450. Thus, the basal activities of AH and HH in lungs and kidneys are not different in male and female rats and are not increased by phenobarbital.

Depression of cytochrome P-450-dependent drug biotransformation by adriamycin

The cytochrome P-450-dependent mixed function oxidase system was depressed following the administration of adriamycin. Cytochrome P-450, aminopyrine N-demethylase, and benzo( a)pyrene hydroxylase were significantly decreased in hepatic microsomes prepared from rats treated with a single dose of adriamycin (10 mg/kg subcutaneously) 4 days previously. Total microsomal protein levels and cytochrome b 5 levels remained unchanged. The administration of cysteamine 1 hr prior to adriamycin prevented the loss of the cytochrome P-450 and the decrease in drug biotransformation. The administration of diethylmaleate had no effect on the ability of adriamycin to decrease this enzyme system. The loss of drug biotransformation and the protection offered by cysteamine is correlated with the lipid peroxidation activity in hepatic microsomes. This suggests that the loss of cytochrome P-450 and related drug biotransformation is related to the generation of free radicals and subsequent lipid peroxidation in the liver.

Effects of dietary lead acetate on hepatic detoxication enzyme activity.

Lead-containing compounds usually inhibit enzymic and metabolic processes. This inhibition is presumed to be the mechanism of intoxication by these compounds. Inhibition of detoxication activities of liver microsomal enzymes could be particularly detrimental because the toxicity of many different substances would be increased. Exposure of experimental animals to lead compounds in several studies has been associated with depressed activity of hepatic microsomal enzymes, reduced levels of hepatic cytochrome P-450, reduced levels of hepatic microsomal protein, and prolonged hexobarbital sleep times. The present report contains observations that under certain experimental conditions there is stimulated hepatic meicrosomal enzyme activity in rats fed lead acetate.

Acute inhibition of oxidative drug metabolism by propoxyphene (Darvon)

Propoxyphene is a potent inhibitor of the hepatic, microsomal mixed-function oxidases, acting in a manner similar to the prototype inhibitor of drug metabolism, SKF 525-A (β-diethylaminoethyl-2, 2-diphenylvalerate), which it resembles chemically. Propoxyphene ( K i = 4.6 ± 0.9 × 10 −5 M) and SKF 525-A ( K i = 4.0 ± 1.1 × 10 −6 M) inhibited competitively the activity of aminopyrine N-demethylase in washed microsomes from mouse liver. Both drugs were considerably weaker, noncompetitive inhibitors of the hydroxylation of aniline by microsomes. They manifested typical type I binding spectra to cytochrome P-450, propoxyphene binding approximately 15 per cent as avidly as SKF 525-A ( K s = 6.9 ± 0.5 × 10 −5 M for propoxyphene; 9.1 ± 1.8 × 10 −6 M for SKF 525-A). When propoxyphene and SKF 525-A were injected i.p. in equimolar (0.26 m-mole/kg) doses, both depressed the N-demethylation of aminopyrine and the hydroxylation of aniline by the 10, 000 g supernatant fraction of mouse liver removed 0.5 hr post-administration. Propoxyphene was 30–40 per cent as potent as SKF 525-A as an inhibitor of these activities. When potassium ferricyanide (50 μM) was added to suspensions of hepatic microsomes from animals pretreated with propoxyphene or SKF 525-A, the complexes formed between the drugs and cytochrome P-450 were destroyed. This same equimolar dose of propoxyphene and SKF 525-A prolonged hexobarbital sleeping time and zoxazolamine paralysis time, propoxyphene again being 30–40 per cent as potent as SKF 525-A in these tests. Chronically, propoxyphene increased the rate of its own metabolism as well as that of aminopyrine and aniline, and levels of cytochrome P-450 and microsomal protein. Propoxyphene thus seems to act in a manner very similar to that of SKF 525-A, acting as a potent inhibitor of microsomal drug metabolism when given acutely and as an inducer when given chronically.

270 EFFECT OF ENDOTOXIN ON RAT HEPATIC DRUG METABOLISM DURING DEVELOPMENT

The frequency of gram negative infections and endotoxemia in the perinatal period prompted an investigation of the effects of endotoxin (E. coli. 026B6) on hepatic drug metabolism. Chronic dosing of endotoxin (0.2 mg/kg/d × 7 days) to lactating mothers significantly stimulated hepatic microsomal cytochrome P-450 (118%) and aminopyrine demethylase activity (114%). No alteration in neonatal enzyme activities was observed. Body and liver weight ratios or microsomal protein level of mothers and neonates were not altered by chronic administration of endotoxin to mothers during the experimental period. The acute i.p. administration of of endotoxin to mothers (1.4 mg/kg on the 7th day after parturition)significantly decreased the activity of aminopyrine demethylase (48%) and content of cytochrome P-450 (25%). When neonates themselves were injected (i.p.) with endotoxin (1.0 mg/kg) at 7, 16 and 27 days of age, a significant reduction in levels of mixed function oxidase enzymes was observed. Cytochrome P-450 contents were reduced by 57, 32 and 24% and aminopyrine demethylation decreased by 78, 48 and 34% in 7, 16 and 27 day old animals resptively. These observations indicate that the ability of mothers and neonates to metabolize drugs is significantly decreased upon acute exposure to endotoxin and this demands careful evaluation of drug disposition in gram negative sepsis during perinatal period. Supported by NIH Grant HD 10063.

Mixed function oxidase enzymes in trout ( Salvelinus fontinalis) liver: Absence of induction following feeding of p, p′-DDT or p, p′-DDE

1. 1. The characteristics of aldrin epoxidase, 7-ethoxycoumarin O-de-ethylase and Cytochrome P-450 (components of a mixed function oxidase system) in trout ( Salvelinus fontinalis) liver are described. 2. 2. Feeding of p, p′-DDT or p, p′-DDE to trout over a 4 week period to produce tissue concentrations of around 20 and 60 ppm wet wt, respectively, did not induce the activities of any of the enzymes listed above, of aniline hydroxylase, nor of microsomal protein levels above those occurring in trout not exposed to these compounds.

Effect of hexachlorobenzene on microsomal enzyme systems

Male rats were fed for 10 days on a diet containing 333 ppm hexachlorobenzene. Increased microsomal protein levels were noted compared to control rats. On a per g liver basis, the levels of aniline hydroxylase, biphenyl 4-hydroxylase, biphenyl 2-hydroxylase, 4-nitroanisole O-demethylase, esterase, cytochrome P-450 and cytochrome b 5 all increased compared with the control values. On a per mg microsomal protein basis, biphenyl 2-hydroxylase, 4-nitroanisole O-demethylase and cytochrome P-450 levels increased several-fold compared with the control values. It is suggested that, by inducing the 2-hydroxylation reaction, hexachlorobenzene might cause preferential ortho-hydroxylation, as do some carcinogenic polycyclic hydrocarbons, and that in some circumstances this could lead to the formation of carcinogens.

Activite enzymatique, au niveau des microsomes hepatiques du rat, apres administration d'acides phenoliques

Enzyme activity at the level of the hepatic microsomes has been evaluated after treatment of female rats with gallic acid, ethyl gallate, syringic acid, veratric acid, vanillic acid or 3,4,5-trimethoxybenzoic acid. These substances, administered ip in a dose of 150 mg/kg/day at 24-hr intervals for 4 days had no effect on microsomal-enzyme activity. In contrast, tannic acid given in a dose of 25 mg/kg/day for 4 days caused a significant inhibition of aniline hydroxylation and amidopyrine N-demethylation but had no significant effect on p-nitroanisole O-demethylation or the concentration of cytochrome P-450. At the same time, pentobarbitone sleeping was prolonged in vivo in the treated rats. These results indicate a change at the sub-cellular level, under the conditions of the experiment, and this was supported by a reduction in the level of microsomal protein. However, after oral administration of tannic acid in a dose of 500 mg/kg/day for 4 days, there was no change in microsomal-enzyme activity.

Microsomal drug-metabolizing enzyme activity inlivers of bats

1. 1. Hepatic drug-metabolizing enzyme activities in two species of eastern American bats ( Myotis lucifugus and Plecotus rafinesquii) wwere found to be qualitatively similar to, but quantitatively distinct from, most other mammals. 2. 2. Ethylmorphine N-demethylase and NADPH cytochrome c reductase activities in bats were only about 20–25 per cent those in other mammals while microsomal protein and cytochrome P-450 levels were only slightly less in bats. 3. 3. Ultrastructurally, liver cells of bats showed abundant mitochondria, many of which showed distinct configurational differences. Endoplasmic reticulum in bat liver cells was very sparse relative to most mammals. 4. 4. Electron micrographs of microsomal suspensions from bats demonstrated a relative lack of smooth membranes and the microsomes from M. lucifugus exhibited an uncommon non-versiculated appearance.

Enhanced hepatic microsomal activity by pretreatment of rats with acetone or isopropanol.

SummaryRat liver microsomes isolated from rats pretreated orally with acetone or isopropanol possessed an enhanced capacity to bind 14CCl4 and 14CHCl3 covalently and to demethylate DMN. The N-demethylation of ethylmorphine was unchanged by this pretreatment. Since there was no increase in the level of microsomal protein, and microsomal cytochrome P-450 or the activity of NADPH-cytochrome c reductase, the mechanism of this enhanced metabolism remains unclear.The authors acknowledge the excellent technical assistance of E. M. Boykins, M. A. Williams and W. Saul.

Vitamin A status and hepatic drug metabolism in the rat.

The effect of vitamin A status on hepatic drug metabolism was studied in rats. Animals were fed diets with and without vitamin A for 20 and 25 days. Weight gains of control and deficient animals were not significantly different, whereas liver vitamin A levels had decreased to less than 10% of control animals after 20 days and were essentially zero after eating the deficient diet for 25 days. Aniline metabolism in vitro and aminopyrine metabolism in vitro and in vivo were significantly lower in male weanling rats fed a vitamin A deficient diet for 20 days. No alteration in in vitro p-nitrobenzoic acid metabolism was noted after 25 days on the test. Vitamin A deficiency did not alter microsomal protein levels or cytochrome c reductase activity but deficient animals did have a lower microsomal cytochrome P-450 content. Hepatic enzyme activities and cytochrome P-450 levels were restored to values approaching those found in control animals by feeding vitamin A deficient rats the vitamin A containing diet for 21 days. Liver vitamin A levels were markedly increased after re-feeding studies but were still significantly lower than control animals.

Stimulation of the levels of cytochrome P-450 and 17 -hydroxylase in chick testis microsomes by pituitary hormones.

Chicks, one—day old, were injected with LH, FSH, and LH + FSH for two days and the levels of microsomal protein, cytochrome P— 450, and 17α-hydroxylase were measured in the testis. Since LH and FSH act on different cells in the testis, the levels of cytochrome P-450 and 17α—hydroxylase are reported both per mg microsomal protein and per chick. Between a dose range of 3–100 μg of LH, the levels of cytochrome P-450, 17α—hydroxylase, and microsomal protein were found to increase as a linear function of the log dose of LH. LH at the 100 μg dose level was found to increase, per mg microsomal protein, the levels of cytochrome P-450 and 17α—hydroxylase by 200% and 80%, respectively. On a per chick basis, these increases were 450% and 250%, respectively. FSH at the 100 μg dose level was found to decrease, per mg microsomal protein, the levels of cytochrome P-450 and 17α—hydroxylase by 27% and 48%, respectively. On the per chick basis, increases in the level of 61% and 30% were found after correction for a 1% cont...

Role of dietary magnesium in the metabolism of drugs by NADPH-dependent rat liver microsomal enzymes

Metabolism in vitro and in vivo of aniline and aminopyrine metabolism in vitro were significantly reduced in magnesium-deficient rats. Magnesium depletion did not alter the rate of metabolism in vitro of p-nitrobenzoic acid or pentobarbital, nor the duration of action of pentobarbital. Microsomal protein and RNA levels were maintained during magnesium depletion, but a significant decrease in the cytochrome P-450 content of the microsomes was noted in magnesium-deficient rats at approximately the same time as the lower rate of drug metabolism was noted. Hepatic enzyme activities and P-450 levels were restored to normal by feeding magnesium-deficient rats a magnesium-containing diet for 8 days. The results indicate that the impairment of hepatic drug metabolism in magnesium deficiency results from a decrease in the specific activity of the microsomal enzymes or from an alteration in the cytochrome P-450 levels.

Hepatic drug metabolism in zinc-deficient rats

The rates of metabolism in vitro of pentobarbital, aminopyrine and p-nitrobenzoic acid were significantly reduced and the duration of action of pentobarbital in vivo was prolonged in zinc-deficient rats. Zinc deficiency did not result in an alteration in the rate of aromatic ring hydroxylaiion in vitro, with either aniline or zoxazolamine as substrate. Microsomal protein and RNA levels were maintained during zinc depletion, but a significant decrease in the cytochrome P-450 content of the microsomes was noted in zinc-deficient rats at approximately the same time as the lower rate in metabolism of pentobarbital was found. Biochemical lesions and gross signs of zinc deficiency were completely ameliorated by refeeding zinc-deficient rats a zinc-containing diet for 14 days.