Hepatic Mixed Function Oxidase System Research Articles

Perspectives on selenium anticarcinogenicity†

Although the earlier literature regarding selenium stressed its toxicity, especially its role in carcinogenesis, there is an increasingly held view that selenium is an essential trace element with antioxidant, anticarcinogenic (e.g., against colon, liver, and skin cancer in animals) and antimutagenic properties. A variety of malfunctions, heart diseases, and increased cancer risk have been connected with selenium deficiency. The role of selenium as a cofactor of the enzyme glutathione peroxidase as well as in the hepatic mixed function oxidase system is also recognized. The focus of this presentation is to review the role of selenium in the inhibition of both the initiation and promotion phases of chemical carcinogenesis in animals and to assess human epidemiological studies that suggest a link between selenium and the incidence of certain types of human cancer.

Free radicals and environmental toxins

Some chemicals that contaminate our environment exert their toxic effects by virtue of their ability to form free radicals. In the absence of sufficient quenching reactions, these reactive radicals can attack biomolecules, resulting in their oxidative degradation. Biological membranes which contain polyunsaturated fatty acids are most susceptible to oxidative degradation (lipid peroxidation), although oxidation of DNA may have more severe biological consequences. Free radicals species can be generated by at least two mechanisms in vivo. The first, of which carbon tetrachloride (CCl4) is the classic example, is the biotransformation of the chemical to a free radical species. Metabolism of CCl4 to the trichloromethyl radical by the hepatic mixed-function oxidase system results in the initiation of lipid peroxidation, protein-lipid cross linkages, and trichloromethyl adducts with DNA, protein, and lipid. The second mechanism for forming free radicals involves their reduction to less stable free radical intermediates which are oxidized by molecular oxygen to give superoxide (O2-.). In the presence of transition metals, such as iron, O2-. can be converted to other oxygen radical species, such as the hydroxyl radical (.OH), an extremely powerful oxidant capable of cleaving DNA, oxidizing protein, and initiating lipid peroxidation. Under many conditions, lipid peroxidation appears not to be initiated by .OH, but rather by an iron-oxygen complex. Regardless of the identity of the initiating species, transition metals are required for most of the deleterious reactions of oxygen. Superoxide and certain organic radicals have been found to release iron from ferritin.

Characterization of mixed‐function oxidase systems of the nestling herring gull and its implications for bioeffects monitoring

AbstractThe hepatic mixed‐function oxidase system was characterized in embryonic and nestling herring gulls (Larus argentatus). The activity of aminopyrine N‐demethylase decreased significantly with the age of the nestlings. No consistent changes in 7‐ethyoxyresorufin O‐deethylase, benzo[a]pyrene 3‐hydroxylase or cytochrome P450 levels with age were found. Levels of organochlorines were determined in individual livers for the 21‐d‐old nestlings. Correlations among organochlorine residue levels were good, and the activities of aminopyrine N‐demethylase and benzo[a]pyrene 3‐hydroxylase correlated with each other and with cytochrome P450 levels. No correlations were found with 7‐ethoxyresorufin O‐deethylase activity, and none of the enzyme levels correlated with the organochlorine residue levels. However, cytochrome P450 levels correlated with both hexachlorobenzene and oxychlordane levels. The implications for the use of the mixed‐function oxidase system for environmental monitoring of exposure to organochlorines and polynuclear aromatic hydrocarbons are discussed.

Investigation of the hepatic mixed‐function oxidase system in herring gull embryos in relation to environmental contaminants

AbstractWe measured hepatic enzyme activity in 25‐d embryos from eight colonies in the Great Lakes and one relatively uncontaminated colony in the Bay of Fundy. This was done to further investigate the use of the mixed‐function oxidase system of herring gull (Larus argentatus) embryos for bioeffects monitoring. The changes in ethoxyresorufin o‐dealkylation (ERoD) and the rate of aminopyrene‐N‐demethylation (APDM) across the Great Lakes were small. However, the rate of aniline hydroxylation (AnH) was significantly depressed in seven of eight colonies when compared with control colony. This suppression was greatest in embryos from the three colonies in Lake Ontario, where mirex levels were highest. Organochlorines were measured in egg homogenates from each colony. There was a significant negative correlation of the rate of APDM with both polychlorinated biphenyl and hexachlorobenzene levels and also of the rate of AnH with mirex levels. In conjunction with the above, an egg injection experiment using three doses each of hexachlorobenzene and mirex was conducted to elucidate these compounds' effects on the enzymes measured. The effects on body weight, liver weight and enzyme activity were small compared with the mortality incurred in the eggs injected with hexachlorobenzene. The embryonic LD50 for hexachlorobenzene was calculated as 4.3 ppm. Mortality in the controls injected with 1,4‐dioxane was 20%, and that of eggs injected with mirex was similar to that of controls.

Influence of estrogen levels on anticholinergic activity of tricyclic antidepressives

1. 1. Anticholinergic activity of a number of tricyclic antidepressives and nomifensine was demonstrated and their order of affinity for the cholinergic receptors of rat jejunum was determined. 2. 2. The influence of etninyl estradiol and a conjugated estrogen product, Premarin (R) on the binding of several tricyclic antidepressives to the hepatic mixed function oxidase system was investigated. 3. 3. The influence of these steroids on the metabolism of the antidepressives was evaluated and ethinyl estradiol was shown to have a marked influence on the metabolism of the antidepressives studied, while conjugated estrogens were shown to have little effect.

The effect of amiodarone on theophylline pharmacokinetics in the rat.

Amiodarone is an investigational antiarrhythmic agent which has been implicated in reducing the activity of the hepatic mixed-function oxidase system. To evaluate this effect further, two groups of six male Sprague-Dawley rats each received theophylline (6 mg/kg, iv) preceded by either normal saline or amiodarone HC1 (100 mg/kg, iv). Blood samples were obtained serially for a period of 6 hr and the sera were assayed for theophylline by high-pressure liquid chromatography (HPLC). In rats pretreated with amiodarone, a significant 45% reduction in the mean (± SD) systemic clearance [0.057 (0.010) vs 0.031 (0.004) liter/hr/kg, P < 0.001] and a greater than 100% increase in the mean elimination half-life [2.03 (0.46) vs 4.29 (0.71) hr, P < 0.001] of theophylline were observed. These data demonstrate an acute inhibitory effect of amiodarone on the hepatic microsomal enzyme system.

Alterations in Tissue Trace Element and Ascorbic Acid Metabolism in Phenytoin-Fed Rats and Mice

Phenytoin was fed to rats and mice in their diet for 6 wk. The dosage was about 80 mg/kg. In rats, phenytoin treatment had no effect on tissue and body weights and had a minimal effect on the hepatic mixed-function oxidase (MFO, EC 1.14.14.1) system. Plasma ascorbic acid levels were higher in the phenytoin group than in the controls, but tissue levels and the rate of ascorbic acid synthesis were similar in the two groups. Also, copper concentration in liver and kidney was significantly higher in phenytoin-treated rats than in controls. Iron, zinc and manganese levels were unchanged in comparison to control values in liver, kidney, heart and brain. In contrast to the results with rats, phenytoin treatment in mice resulted in a lower body weight and a clear induction in the hepatic MFO system compared to that in controls. Phenytoin treatment resulted in higher liver ascorbic acid tissue levels than in controls. Liver copper and kidney zinc were lower and liver and kidney calcium and bone iron were higher in phenytoin-fed mice than in controls. This study shows that for both species phenytoin feeding affected ascorbic acid and tissue trace element metabolism. The clinical significance of these findings with regard to the nutritional status of the human patient undergoing treatment with phenytoin needs to be considered.

Comparison of the hepatic mixed function oxidase system of young, adult, and old non- human primates (Macaca nemestrina)

The influence of age on the mixed function oxidase system from a non-human primate was studied. Microsomes were isolated from the livers of female Macaca nemestrina ranging from 2 to 21 years of age. No significant age-related change was observed in either the cytochrome P-450 content or the NADPH cytochrome c reductase activity. In addition, the ability of the microsomes to metabolize benzo[ a]pyrene did not change significantly with age. These observations contradict studies with liver tissue from laboratory rodents in which an age-related decline in the mixed function oxidase system is generally observed. The lipid composition of the liver microsomes was studied also. Both the cholesterol and total phospholipid content of the liver microsomes increased significantly with age; however, the ratio of cholesterol to phospholipid remained constant. The percentage of individual phospholipids in the microsomes changed only slightly with age. These results provide new information on the effect of age on the mixed function oxidase system and indicate that one must be cautious in extrapolating from studies with liver tissue from laboratory rodents to primates.

29 DECREASE IN CYTOCHROME P-450 DEPENDENT 3-N-DEMETHYLATION OF CAFFEINE MEASURED BY THE CAFFEINE 13CO2 BREATH TEST (CET) FOLLOWING GROWTH HORMONE THERAPY IN GROWTH HORMONE (GH) DEFICIENT CHILDREN

Significant changes in hepatic drug clearance occur in the transition from childhood to adulthood. These changes probably parallel the functional capacity of the hepatic cytochrome P-450 dependent mixed function oxidase (MFO) system. GH decreases hepatic MFO activity in animals and may act similarly in humans. Caffeine labelled with stable 13C has been shown to be a substrate for the MFO system. Five GH deficient children underwent CBT, with collections of expired air for 13CO2 enrichment analysis following ingestion of 13C-caffeine (3 mg/Kg) before, and 4-5 weeks following GH 0.1 u/Kg S.C. t.i.w. All children showed a decrease in % 13CO2 dose exhaled over 2 hours following GH administration: Pre 9.1±1.5, Post 7.3±2.9 (p < .05, paired t-test) The CBT is a safe effective technique for the measurement of the hepatic MFO system. N-demethylation of caffeine is decreased by GH therapy in GH deficient children. The capacity for N-demethylation is highest in prepubertal children. This may partially correlate with changing patterns of GH release.

Activities of liver mixed function oxidase system in rats fed trans fat.

Rats were fed diets containing either camellia oil or partially hydrogenated corn oil as a source of cis or trans octadecenoate, respectively, in the presence of adequate linoleic acid. After 35 days of feeding the diets, activities of several hepatic drug metabolizing enzymes as well as the content of hepatic microsomal cytochrome P-450 were determined. Geometrical difference in the dietary fat did not affect the amount of microsomal protein nor the content of cytochrome P-450. Also, activities of NADPH cytochrome C reductase, aminopyrine N-demethylase and biphenyl hydroxylase were approximately the same between two groups of rats. Aniline hydroxylase was slightly elevated in the rats fed trans fat. It was concluded that the difference in the geometry of dietary fatty acids had little effect in modulating the hepatic mixed function oxidase system.

Hepatocarcinogenicity of Benzo[&lt;italic&gt;a&lt;/italic&gt;]pyrene to Rainbow Trout by Dietary Exposure and Intraperitoneal Injection&lt;xref ref-type="fn" rid="FN1"&gt;2&lt;/xref&gt;&lt;xref ref-type="fn" rid="FN2"&gt;3&lt;/xref&gt;

The influence of benzo[a]pyrene [(BP) CAS: 50-32-8] on the induction of certain enzymes within the hepatic mixed-function oxidase (MFO) system and its potential carcinogenicity were examined in rainbow trout (Salmo gairdneri). Nine-week feeding trials were performed with 500 and 1,000 ppm BP to determine trout tolerance to BP. Levels of MFO enzymes, including ethoxyresorufin-O-deethylase (EROD), ethoxycoumarin-O-deethylase (ECOD), benzo[a]pyrene monooxygenase (BPMO), and cytochrome P450 were measured during this time. An 18-month feeding trial of a 1,000-ppm BP dose was initiated in duplicate groups of 100 fingerling rainbow trout. Samples of trout were killed at 6, 12, and 18 months for gross and histologic examination of the internal organs for neoplasms. A group of fifty 10-month-old rainbow trout were given 12 monthly ip injections of 1 mg BP in 0.4 ml propylene glycol (PG), and comparable controls were given PG injections only. The trout were held for an additional 6 months, killed at age 28 months, and examined as in the dietary study. Mean MFO enzyme levels of EROD, ECOD, BPMO, and cytochrome P450 were significantly (P less than .001) elevated, showing dose- and time-response relationships when compared to MFO enzyme levels in control fish. Twelve months after BP exposure was initiated, 15% of the BP-fed fish had histologically confirmed neoplasms of the liver. After 18 months the incidence increased to 25%. No evidence of neoplasia was observed in control fish. BP injected ip resulted in a 50% incidence of hepatocellular neoplasms and in a fibrosarcoma of the liver and papillary adenomas of the swim bladder in 1 fish. These results indicate that BP is a potent inducer of selected hepatic MFO enzymes and establish, for the first time, the hepatocarcinogenicity of BP in an aquatic species.

Effect of ketoconazole on hepatic oxidative drug metabolism.

Several clinical reports have suggested (but not demonstrated) that ketoconazole, a broad-spectrum antifungal drug, may inhibit hepatic oxidative drug metabolism in man. We recently demonstrated that ketoconazole inhibits caffeine and aminopyrine oxidation in the rat; we now study the influence of ketoconazole on theophylline and chlordiazepoxide kinetics in man. These studies were performed before and after varying doses of ketoconazole within the currently accepted therapeutic range. Ketoconazole had no effect on theophylline clearance, whereas the drug impaired chlordiazepoxide clearance from plasma. After a single dose of ketoconazole, there was a 20% decrease in clearance and a 26% decrease in volume of distribution without evidence of inhibition of drug metabolism. These changes apparently were not related to ketoconazole dose. After repetitive dosing with ketoconazole, chlordiazepoxide clearance decreased by 38% and was associated with reduced concentrations of its first oxidative metabolite, N-desmethylchlordiazepoxide. We conclude that ketoconazole inhibits at least one subset of the hepatic mixed-function oxidase system, but is not as general an inhibitor of hepatic oxidative drug metabolism as cimetidine appears to be. For some coadministered drugs, ketoconazole may also have an effect on other kinetic parameters such as volume of distribution. Therefore, we caution that clinically important drug interactions may occur with the concurrent use of ketoconazole.

Turnover of hepatic hemoproteins in rainbow trout

The hepatic mixed function oxidase system in the fish differs from that in mammals in its responses to the two classic mammalian inducers. The cytochrome P-448-type inducers (polycyclic aromatic hydrocarbons) stimulate monooxygenase activity, but phenobarbital, a P-450-type inducer, does not. 1 We have compared the effects of phenobarbital (PB) and polychlorinated biphenyls (PCB) on the turnover of hepatic microsomal hemoproteins in trout (PCB's are P-448- and P-450-type inducers in mammals, which in fish induce only cytochrome P-448). We show here that neither PCB nor PB treatment changed the turnover rate. However, both the rates of synthesis and degradation were much slower than in the rat.

Effect of long-term administration of delta 9-tetrahydrocannabinol on hepatic mixed-function oxidase systems in the rat.

Chronic oral treatment of young female Fischer rats with 25 mg/kg delta 9-tetrahydrocannabinol (THC) per day inhibited aminopyrine demethylation and significantly increased benzo[a]pyrene oxidation by the liver. THC treatment also elevated serum corticosterone levels and produced a significant loss of body weight. The weight loss was not due to vehicle or food intake (pair-feeding). Pair-feeding did, however, produce a stimulation of both mixed function oxidase pathways as well as a marked elevation in serum corticosterone levels. The results indicate that THC has a differential effect on mixed function oxidase pathways in the liver that is not directly related to food intake or corticosterone levels.

Studies on the Hepatic Microsomal Mixed Function Oxidase System with Higenamine on the Rat

Studies on the Hepatic Microsomal Mixed Function Oxidase System with Higenamine on the Rat

A correlation of induced multifunction oxidase activity to bond strengths in partially chlorinated hydrocarbons

Four xenobiotics, benzene, 1,2,4-trichlorobenzene, cyclohexane and γ-1,2,3,4,5,6-hexachlorocyclohexane were orally administered at 1 7 LD 50 dose levels to Sprague Dawley rats. Seventy-two hours after dosing, the specific activity of the hepatic MFO system of each animal was determined in vitro by measuring NADPH utilization when each of the four xenobiotics was added as the substrate. For each of the four dose types, the specific activity in vitro showed no dependence on substrate stereo-chemistry, but was inversely related to the C-H bond strength of the substrate. For each substrate, the specific activities of each of the four types of MFO preparations varied directly with the C-H bond strength of the inducer. This implies that the specific activity of the induced MFO enzyme system is adjusted to the C-H bond breaking requirement which leads to the hydroxylation of the xenobiotic. This hypothesis was further examined following in vivo exposure of rats to six polychlorinated derivatives of dibenzo-p-dioxin (PCDD). The specific activity of each MFO extract was determined by use of all other PCDD's as substrates. The dioxins showed a consistent ranking for both activity as substrate and as inducer but, unlike the monocyclic hydrocarbons, the two rankings were not a simple inverse of each other. By combining a series of in vivo and in vitro tests, a structure-activity-relationship can be developed to relate the mixed function oxidase (MFO) response to the physico-chemical properties of a xenobiotic. Following in vivo exposure to one of a series of hydrocarbons or their chlorinated

Temperature compensation in the hepatic mixed-function oxidase system of bluegill

1. Bluegill ( Lepomis macrochirus R.) were acclimated to 12, 22 or 32°C for 5 or 14 days. 2. Liver weight to body weight ratio and the rate of metabolism of benzo[ a]pyrene by liver microsomes varied inversely with the acclimation temperature of the fish. 3. Concentration of microsomal cytochrome P-450, as determined by CO-difference binding spectra, was not significantly affected by acclimation temperature. 4. There were no qualitative or quantitative differences in the electrophoretic patterns of proteins with molecular weights similar to those reported for cytochrome P-450. 5. There were no shifts in the temperature optima of the microsomal benzo[ a]pyrene hydroxylase activity.

Induction of hepatic mixed function oxidase system by endosulfan in rats.

Induction of hepatic mixed function oxidase system by endosulfan in rats.

Effect of manganese on the hepatic microsomal mixed function oxidase enzyme system in the rat

Experiments were conducted to examine the effect of manganese on the hepatic mixed function oxidase system in the rat. Acute treatment with manganese chloride (1–10 mg Mn/kg, ip) produced a significant prolongation of hexobarbital hypnosis in male rats on Days 2 and 3 following metal administration. The threshold dose of manganese to produce this alteration in response was 5 mg Mn/kg and the altered response returned to control values by Day 5. The prolonged hexobarbital hypnosis resulted from Mn inhibition of the hepatic microsomal mixed function oxidase system, the activity of which was assessed using aniline (23%), ethylmorphine (26%), and hexobarbital (27%) as substrates. Manganese treatment also produced significantly reduced levels of cytochrome P-450 (23%) and b 5 (21%), but the substrate-induced spectral binding of all three substrates was not altered significantly by Mn when expressed as Δ A per nanomole of cytochrome P-450. The activity of NADPH cytochrome c reductase was also significantly decreased (25%) by Mn treatment. Following the in vitro addition of Mn in concentrations ranging from 1 × 10 −6 to 1 × 10 −3 m Mn to microsomes derived from naive rats, there was no decrease in the metabolism of aniline or hexobarbital or cytochrome P-450 levels. Significant inhibition in ethylmorphine metabolism was observed with Mn concentrations of 1 × 10 −4 m and greater. These experiments indicate that acute Mn treatment can alter drug response as the result of decreased hepatic biotransformation which occurs by an indirect mechanism.

Effect of ampicillin on hepatic microsomal mixed-function oxidase system in male mice

The effect of ampicillin [( d)-α-aminobenzyl penicillin] administration on the hepatic mixed-function oxidase (MFO) system was studied in male mice. Ampicillin (100 mg/kg, i.p., 3 days) decreased the levels of cytochrome P-450, aminopyrine N-demethylase, acetanilide hydroxylase and cytochrome c-reductase activity significantly. In carbon tetrachloride (CCl 4)-pretreated mice, ampicillin increased acetanilide hydroxylation compared with CCl 4 treatment alone; however, all other parameters of the MFO system remained unchanged. Ampicillin exhibited type II binding with microsomes (trough at 388 nm, peak at 430 nm). Thus ampicillin acts as an inhibitor of the MFO system.