Hepatic Microsomal Mixed-function Oxidase Activities Research Articles

Induction and autoinduction properties of rifamycin derivatives: a review of animal and human studies.

Animal studies have demonstrated that the mouse and rabbit are far more responsive to the inductive properties of rifamycin derivatives than the rat and guinea pig. The rat hepatic cytochrome P450 system seems to be resistant to the action of rifampicin unless very high doses are used. Mouse hepatic microsomal mixed-function oxidase activity is markedly increased by repeated dosing with rifampicin, whereas administration of rifabutin may be ineffective. In humans, both rifampicin and rifabutin are extensively metabolized and induce their own metabolism. The induced metabolic pathways remain essentially unknown. Under autoinduction conditions, the elimination half-life of rifampicin decreases, whereas that of rifabutin is not altered. Although the effects of repeated administration of rifampicin and rifabutin on the various forms of cytochrome P450 in humans have not been extensively examined, there is convincing evidence that the P4503A subfamily is induced by either drug, whereas the P4501A subfamily and P4502D6 do not appear to be affected by rifampicin. Limited reliable information is available concerning the induction of human glucuronyltransferase activities by rifampicin and rifabutin which, however, do not seem to influence zidovudine glucuronide formation in healthy subjects.

Toxicity studies of a synthetic antioxidant, 2,2'-methylenebis (4-ethyl-6-tert-butylphenol) in rats. 2. Uncoupling effect on oxidative phosphorylation of liver mitochondria.

Effects of 2,2'-methylenebis (4-ethyl-6-tert-butylphenol) (MBEBP) on hepatic mitochondrial oxidative phosphorylation in vitro, and on hepatic peroxisomal enzymes activities and microsomal mixed-function oxidase activities were studied. 1. A low concentration of MBEBP, less than 50 microM, increased state 4 respiration and decreased state 3 respiration. However, a higher concentration of MBEBP, greater than 100 microM, acted as a respiratory inhibitor. Therefore, MBEBP was found to act as an uncoupler of oxidative phosphorylation in rat liver mitochondria. 2. MBEBP significantly decreased peroxisomal enzymes, cyanide-insensitive palmitoyl-CoA oxidizing activity and catalase activity in the livers of rats fed 0.2, 1.0 or 5.0% MBEBP for 4 weeks. 3. In microsomal enzyme assay, NADPH cytochrome c reductase activity was significantly increased, however, cytochrome P-450, cytochrome b5 levels, aminopyrine N-demethylase and benzo [a] pyrene hydroxylase activities were not significantly increased in the livers of rats fed 1.0 or 5.0% MBEBP for 4 weeks. The weight loss and the decrease of serum triglyceride level observed in the MBEBP-treated rats seemed to be caused by its uncoupling effects, which might also be the cause of the testicular damage induced by MBEBP.

Effect of Sizofilan, an Immunomodulator, on Hepatic Microsomal Mixed-Function Oxidase Activities in Rats

Mixed-function oxidase activities of hepatic microsomal preparations from rats were examined after intraperitoneal administration of sizofilan (SPG), an immunomodulator. Repeated doses of SPG (3 mg/kg/12 hr, 4 times) depressed the hepatic cytochrome P-450 content and the activities of aminopyrine N-demeth-ylase and aniline hydroxylase.

Effect of sizofilan, an immunomodulator, on hepatic microsomal mixed-function oxidase activities in rats.

Mixed-function oxidase activities of hepatic microsomal preparations from rats were examined after intraperitoneal administration of sizofilan (SPG), an immunomodulator. Repeated doses of SPG (3 mg/kg/12 hr, 4 times) depressed the hepatic cytochrome P-450 content and the activities of aminopyrine N-demethylase and aniline hydroxylase.

Effect of vitamin A on rat hepatic mixed-function oxidases, glutathione transferase activity and generations of oxygen radicals.

Rat hepatic microsomal mixed-function oxidase activities were not significantly affected by vitamin A deficiency. Similarly cytosolic glutathione S-transferase and glutathione reductase activities as well as total glutathione levels were unaffected by the vitamin A status. Induction of the mixed-function oxidases by 3-methylcholanthrene or phenobarbitone was independent of the vitamin A status. No significant differences in microsomal chemiluminescence, before and following challenge with tertiary butyl hydroperoxide, were evident between the vitamin-A-deficient animals and those maintained on vitamin-A-supplemented diets. The present findings indicate that the protective action of vitamin A against chemical carcinogens is unlikely to involve modulation of the enzyme systems responsible for their metabolism.

Induction of rat hepatic mixed function oxidases by aromatic amines and its relationship to their bioactivation to mutagens

Hepatic microsomal mixed-function oxidase activities were determined in rats pretreated with the aromatic amines 2-aminoanthracene, 2-naphthylamine or 4-aminobiphenyl. All three amines stimulated the O-deethylation of ethoxyresorufin (cytochromes P-448) but none had any effect on the p-hydroxylation of analine. 2-Aminoanthracene and 4-aminobiphenyl also stimulated the NADPH-dependent reduction of cytochrome c and 2-naphthylamine inhibited the N-demethylation of benzphetamine. Hepatic preparations from animals pretreated with 2-aminoanthracene were more efficient in converting this carcinogen to mutagens while in contrast pretreatment with Aroclor 1254 caused a marked decrease in mutagenicity. 4-Aminobiphenyl also enhanced its own activation but Aroclor-pretreated preparations were the most effective. The latter preparations were also more efficient than controls in activating 2-naphthylamine to mutagens. It is concluded that 4-aminobiphenyl and 2-aminoanthracene enhance their own activation at least partly, by inducing the synthesis of cytochromes P-448.

Induction of renal and hepatic mixed function oxiases in the hamster and guinea pig

A marked species differences exists in the induction of renal and hepatic mixed function oxidase (MFO) activity between rats and rabbits. However, little is known about MFO induction in these organs from other laboratory animals. Male Golden Syrian hamsters and male Hartley guinea pigs were administered phenobarbital (PB) or β-napthoflavone (BNF) at 70 and 40 mg/ kg, respectively, as daily i.p. injections for 4 days. Polybrominated biphenyl (PBB) (Firemaster BP-6) was given as a single i.p. injection (50 mg/kg). Hamster hepatic microsomal ethoxyresorufin- O-deethylase (EROD) and benzphetamine- N-demethylase (BPND) were selectively induced by BMF and PB, respectively. PBB administration induced both hamster hepatic EROD and BPND. In contrast, hepatic microsomal MFO activity from the guinea pig was inducible by PB, PBB and BMF. Renal microsomal MFO activity in both species was inducible by BNF and PBB as arylhydrocarbon hydroxylase and EROD were induced approximately 10-fold. On the other hand, hamster BPND was induced by PB whereas guinea pig MFO activity was unaffected. Total renal cytochrome P-450 content was not affected by any of these inducers in either species. These data demonstrate selective patterns of induction in both hamster and guinea pig liver and kidney suggesting the involvementof multiple forms of cytochrome P-450.

Hepatic microsomal mixed-function oxidase activity in ethanol-treated hamsters and its consequences on the bioactivation of aromatic amines to mutagens

Male golden Syrian hamsters were maintained on ethanol-containing liquid diets for 4 weeks, corresponding to an average daily intake of 17 g/kg body wt. The p-hydroxylation of aniline was markedly enhanced by this treatment while minimal effects were seen in benzphetamine N-demethylase and ethoxyresorufin O-deethylase activities; there was no change in the microsomal levels of cytochromes P-450. Hepatic microsomal preparations from the ethanol-treated hamsters were more efficient than controls fed isocaloric diets in converting 2-aminofluorene, 4-aminobiphenyl, benzidine and 2-acetylaminofluorene into mutagens in the Salmonella mutagenicity test. The same treatment had no effect on the metabolic activation of 2-naphthylamine and even inhibited the mutagenicity of 2-aminoanthracene. No increase was seen in the activation of the two polycyclic aromatic hydrocarbons, benzo[a]pyrene and 3-methylcholanthrene to mutagens and an inhibitory effect was seen with the former. The ethanol-induced increase in the mutagenicity of 2-aminofluorene was inhibited by 2-butanol but not by the hydroxyl radical scavenger dimethylsulphoxide. It is concluded that chronic ethanol ingestion modulates the bioactivation of aromatic amines and amides to mutagens, the effect being substrate dependent. This effect of ethanol may be catalysed by unique form(s) of cytochrome P-450 whose synthesis is induced by such treatment.

Effects of oral contraceptive steroids (norethisterone/mestranol) on the activities of hepatic drug-metabolizing enzymes in iron-deficient anemic rats.

Either oral contraceptive steroid (norethisterone/mestranol; N/M) treatment or iron-deficiency (Fe(-] anemia alone caused an increase in NADPH cytochrome c reductase and in three hepatic microsomal mixed-function oxidase activities in female rats. When N/M treatment and the Fe(-) diet are combined, no further change in hepatic enzyme activity is seen compared with that with either treatment alone.

Effects of the induction of hepatic microsomal metabolism on the toxicity of cyclophosphamide.

Cyclophosphamide (CP) administration to rats in a single i.p. dose (200 mg kg-1), while producing urinary bladder toxicity and 30-40% depression of the hepatic microsomal mixed function oxidase (MFO), failed to produce any depression of MFO activities in extrahepatic tissues such as lung, kidney and intestine. Phenobarbital pretreatment of the rats, which is known to enhance hepatic microsomal activation of CP, protected against CP-induced urinary bladder toxicity and the depression of hepatic MFO activities. This protection appears to be, at least in part, related to phenobarbital induction of hepatic cytochrome P-450 isozyme(s) that metabolizes CP to a new metabolite tentatively identified as didechlorodihydroxycyclophosphamide.

Effect of carbon tetrachloride--alcohol-induced liver fibrosis on microsomal mixed-function oxidases and the cytosolic glutathione-conjugating system in rat liver.

1. Subchronic treatment of male and female rats with CCl4 (0.2 ml/kg orally twice weekly) and drinking water containing 5% ethanol for four weeks led to a 20 to 40-fold increase in serum sorbitol dehydrogenase activity and to an augmentation of the liver triglyceride and hydroxyproline contents, indicating steatosis and fibrosis, respectively. Liver fibrosis was less pronounced in females than in male rats. 2. As a consequence of these alterations the hepatic microsomal mixed-function oxidase activity as measured by aminopyrine demethylation was decreased with concomitant loss of cytochrome P-450 in both sexes. Aniline hydroxylation as well as the activity of the NADPH-cytochrome c reductase showed no significant alterations. 3. While the hepatic glutathione content remained unchanged, the cytosolic glutathione S-transferase activities towards both an aryl and an epoxide substrate were markedly decreased following the development of liver fibrosis both in male and female rats.

Toxicologic studies with swine fed corn grown on municipal sewage sludge-amended soil.

Over a 3-yr period 336 MT/ha of dried sewage sludge, from a municipal waste treatment plant, was applied in liquid form to land subsequently used to grow corn. The sludge contained 115 mg Cd, 4,200 mg Zn and 538 mg Ni/kg dry matter and comprised the solids remaining after treatment of the waste waters of approximately 260 industries as well as domestic users. Corn grain harvested from the plot amended with sewage sludge (SA corn) or corn grown on a plot without sludge addition (control corn) was fed to a total of 56 pigs to determine the effect on growth performance and parameters indicative of toxicity. Each treatment consisted of seven pens of four pigs each with an average initial weight of 17.6 kg. The growth trial was terminated when pigs weighed approximately 90 kg. No differences were observed between treatment groups for average daily gain, feed: gain ratio or daily feed intake. Higher (P less than .01) concentrations of Cd, Ni and Zn were found in SA corn compared with the control corn. This resulted in higher (P less than .01) concentrations of Cd in kidney and liver and Ni in kidney of pigs fed SA corn as compared with pigs fed control corn. No significant differences were observed in Cd or Ni concentrations in muscle or in hepatic microsomal mixed-function oxidase (MFO) activity and liver:body weight ratios, which is indicative of the absence or low levels of organic toxicants in SA corn. In addition, no significant treatment effects were observed when corn, feces and urine samples were evaluated for the presence of mutagenic substances. Histopathologic analysis of various tissues for lesions demonstrated that pigs fed SA corn were not adversely affected.

The induction of hepatic microsomal mixed-function oxidase activities in the mouse by mirex, 3,4,5,3′,4′,5′-hexachlorobiphenyl, and equimolar dosages of both

At low oral dosages (15 μmole/kg) neither mirex nor 3,4,5,3′,4′,5′-hexachlorobiphenyl was found to significantly elevate hepatic microsomal cytochrome(s) P-450 in the mouse, 7 days post-treatment. The associated drug metabolic activities, rmaminopyrine- N-demethylase, p-nitroanisole- O-demethylase, and aniline hydroxylase, were also not significantly elevated above control levels. However, aminopyrine- N-demethylase and p-nitroanisole- O-demethylase activities were significantly elevated by a 1:1 molar combination of the two compounds (“Both”), administered at a total molar concentration (15 μmole/kg) equal to that of either of the two constituents. Concomitant administration of the molar sum of Mirex and the hexachlorobiphenyl significantly elevated all three metabolic activities and cytochrome(s) P-450 concentration. The relatively greater effect of “Both” in elevating these activities may be due to the simultaneous elevation of cytochromes P-450 and P-448.

Alterations in hepatic mixed-function oxidase activity of rainbow trout after acute treatment with polybrominated biphenyl isomers and FireMaster BP-6.

The effects of FireMaster BP-6 and several pure isomers on hepatic microsomal mixed-function oxidase (MFO) activity in rainbow trout have been investigated. After one parenteral injection of these agents at 150 mg/kg and sacrifice 5 d later, there was no elevation of hepatic cytochrome P-450. The noncoplanar isomers 2,4,5,2',4',5'-hexa and 2,3,4,5,2',4',5'-heptabromobiphenyl did not increase any MFO activity generally associated with an increase in cytochrome P-450 levels, including the dealkylations of ethoxycoumarin and benzphetamine. The coplanar isomer 3,4,5,3',4',5'-hexabromobiphenyl produced an increase in O-deethylation of both ethoxycoumarin and ethoxyresorufin, suggesting enhancement of cytochrome P-448 associated activity, but produced no increase in benzphetamine N-dealkylation. The "mixed inducer" 2,4,5,3',4',5'-hexabromobiphenyl, produced a small but insignificant elevation of cytochrome P-448 associated MFO activities and no increase in cytochrome P-450 associated activities. At both 150 and 500 mg/kg, the commercial mixture of polybrominated biphenyls FireMaster BP-6 produced marked elevations of both ethoxycoumarin and ethoxy-resorufin dealkylations but had no effect on demethylation of benzphetamine. When microsomes from rainbow trout treated with FireMaster BP-6 were examined electrophoretically, A coomassie Blue-staining band at 57,000 daltons was intensified, as seen after treatment of fish with the cytochrome P-448 inducer beta-naphthoflavone. It is concluded that coplanar polybrominated isomers produce an induction of hepatic cytochrome P-448 associated activity, while noncoplanar isomers are ineffective as inducers of cytochrome P-450 related MFO activities in rainbow trout.

Exposure of newborn rats to pharmacologically active compounds may permanently alter carcinogen metabolism.

Administration of phenobarbital to mother rats during early lactation causes long-term, perhaps permanent, alteration of hepatic microsomal mixed-function oxidase activity and aflatoxin B1 adduct formation in the adult male offspring. These findings suggest that perinatal exposure to pharmacologically active compounds may be a determinant of cancer risk.

Polycyclic aromatic hydrocarbon induction of cytochrome P-450-dependent mixed-function oxidases in marine fish

Administration of 3-methylcholanthrene (3-MC) and related chemicals (1,2,3,4-dibenzanthracene, 7,12-dimethylbenzanthracene, and 5,6-benzoflavone) by ip or im injection to five species of marine fish resulted in increases in their hepatic microsomal mixed-function oxidase activities with benzo( a)pyrene, 7-ethoxycoumarin, and 7-ethoxyresorufin as substrates. Cytochrome P-450 content was increased in some treated fish. Sheepshead injected im with 3-MC showed the most marked induction of cytochrome P-450 content. In sheepshead, the Soret maximum of the CO-reduced difference spectrum changed from 450 nm in control to 448 or 449 nm in 3-MC-treated fish. The time course of 3-MC induction of benzo( a)pyrene hydroxylase activity in sheepshead changed with season (water temperature) and was not the same as the time course of induction of cytochrome P-450 content or 7-ethoxyresorufin O-deethylase activity. In summer (water temperature 26°C), benzo( a)pyrene hydroxylase activities in hepatic microsomes from 3-MC-treated sheepshead were maximum at 3 days and were back to control levels at 14 days after the dose, whereas in winter (water temperature 14°C) maximum activities were observed 8 days after dosing, but elevated activities were still observed 28 days after one ip dose. Epoxide hydrase and glutathione S-transferase activities were not induced by polycyclic aromatic hydrocarbons or 5,6-benzoflavone in the marine fish studied, at the doses tested.

Role of biotransformation in the alterations of chloroform hepatotoxicity produced by Kepone and mirex

A previous report demonstrated that pretreatment of mice with Kepone resulted in marked potentiation of CHCl 3-induced hepatotoxicity whereas pretreatment with a structural analog, mirex, had no effect on the liver injury produced by a challenging dose of CHCl 3. To determine some of the possible mechanisms for this difference in potentiating ability, various parameters were studied. The hepatic content of mirex and Kepone was determined 42 hr after the oral administration of each agent to male Swiss-Webster mice. The concentration of mirex within the liver increased in a dose-related fashion. Following a single dose (50 mg/kg) of either mirex or Kepone, the hepatic content of each agent was approximately equal. Kepone (50 mg/kg, po) had no effect on mouse hepatic glutathione content at 18 hr. The relationship between the effects of mirex and Kepone on microsomal mixed function oxidase (MFO) activity and the in vivo and in vitro irreversible binding of 14CHCl 3 reactive metabolite(s) to hepatic constituents was assessed at 18 hr. Mirex pretreatment (10, 50, 250 mg/kg) resulted in a more profound effect on hepatic MFO activity than did pretreatment with Kepone (50 mg/kg). However, mirex pretreatment (50 mg/kg) did not alter either the in vivo or in vitro irreversible binding of 14CHCl 3-derived radioactivity to mouse hepatic constituents. In contrast, pretreatment of mice with Kepone (50 mg/kg) resulted in profound increases in both the in vivo and in vitro irreversible binding of 14CHCl 3 metabolites. Thus it appears that the disparate potentiating abilities of mirex and Kepone are related to a difference in the capacity of these agents to increase the activity of the CHCl 3 bioactivation system of the liver.

The inhibition and potentiation of procarbazine on hepatic mixed-function oxidases in phenobarbital tolerant and nontolerant mice.

The effect of procarbazine on mixed-function oxidases was investigated in naive and pentobarbital tolerant mice. In mice receiving procarbazine, 200 mg/kg, i.p. 1 hr earlier, metabolisms of pentobarbital, aniline and ethylmorphine in vitro and cytochrome P-450 content of hepatic microsomes were significantly decreased. The drug binding of either aniline or pentobarbital to cytochrome P-450 was also decreased. However, procarbazine failed to exert this effect after the enzymes had been induced by continuous administration of pentobarbital. Interestingly, procarbazine enhanced the barbiturate induced hepatic microsomal mixed-function oxidase activities when it was administered before the implantation of pentobarbital pellet. Both cytochrome P-450 and cytochrome b5 content after pentobarbital pellet implantation were further increased by pretreatment with procarbazine. This finding was further substantiated by the increase in pentobarbital and aniline binding to cytochrome P-450. The present studies may provide another model for studying the nature of hepatic mixed-function oxidase induction process.

Hepatic microsomal mixed-function oxidase activities in several marine species common to coastal Florida

1. NADPH-dependent hepatic microsomal mixed-function oxidase activities were measured in a number of marine vertebrate and invertebrate species common to the Florida Atlantic coast, using benzo(a)pyrene, 7-ethoxycoumarin, benzphetamine and aniline as substrates. Optimal assay conditions were established using sheepshead hepatic microsomes. 2. Mixed-function oxidase activity was easily detected in hepatic microsomes from teleost and elasmobranch fish, but was low or undetectable in hepatopancreas microsomes from crustaceans. However, the cytochrome P-450 content of hepatopancreas microsomes often exceeded that found in microsomes from teleost or elasmobranch liver. 3. Mixed-function oxidase activity in microsomes prepared from extrahepatic organs was usually much lower than was found in hepatic microsomes. However, for two teleost species (sheepshead and black drum), benzphetamine N-demethylase activity of gill microsomes approached that found in hepatic microsomes. 4. The temperature optimum for in vitro mixed-function oxidase activity was higher in the Florida fish tested than has been reported for cold-water acclimated marine or freshwater fish.

Effect of hexachlorophene, tribromsalan, trichlorcarban and cloflucarban on hexobarbital sleeping time and hepatic drug-metabolizing enzyme activity in vitro in the rat

Oral administration of the antibactenals hexachlorophene, tribromsalan and trichlorcarban at doses of 50, 10 and 40 mg/kg, respectively, caused significant increases in hexobarbital sleeping time in male Wistar rats, while an oral dose of 40 mg/kg of cloflucarban gave only a slight increase. The four halogenated antibacterials inhibited hepatic microsomal aminopyrine N-demethylase and aniline hydroxylase activities in vitro but had no effect on hepatic microsomal NADPH-cytochrome c reductase activity. The addition of each of the antibacterials to hepatic microsomes produced a type I difference spectrum. The spectral binding constants for interaction of hexachlorophene, tribromsalan, trichlorcarban and cloflucarban with hepatic microsomal cytochrome P450 were 33, 62, 66 and 40 μM respectively. The per cent of in vitro inhibition of hepatic microsomal mixed-function oxidase activity by these agents increased with increasing incubation times. The addition of bovine serum albumin to the incubation mixtures partially reversed the inhibition of microsomal drug metabolism caused by the halogenated antibacterial agents in vitro.