INDUCTION OF CYTOCHROME P-448 BY 3-METHYLCHOLANTHRENE IN THE RAT DURING INHIBITION OF PROTEIN SYNTHESIS IN VIVO

Administration of cycloheximide in vivo during induction of rats with 3-methylcholanthrene prevents the increase in total cytochrome P-450 content usually seen under the influence of the inducer. The population of cytochromes P-450 in the livers of these animals is, however, similar to that in the completely induced animals. Microsomal aryl hydrocarbon hydroxylase activity and biphenyl-2-hydroxylation are enhanced severalfold and biphenyl-4-hydroxylation is enhanced twofold. Monooxygenase activity shows the same pattern of preferential inhibition as in microsomes from animals which had received the inducer only. The affinity of the reduced cytochromes for the ligand metyrapone is considerab…

Enzymic control of irreversible binding of metabolically activated benzo(a)pyrene in perfused rat liver by monooxygenase activity.

Addition of [3H]-benzo(a)pyrene to the perfusion medium of isolated rat livers results in irreversible binding of radioactivity to DNA, RNA and protein. Binding to DNA accounted for about 0.1% of the total radioactivity which was bound in livers from animals treated with oil or saline and was increased by a factor of 3–5 after pretreatment of the animals with β-naphthoflavone or with phenobarbital. When the inhibitiors of monooygenase activity, α-naphthoflavone or metyrapone, were present in the perfusion medium, irreversible binding was reduced in livers from both β-naphthoflavone- and phenobarbital-pretreated animals, irrespective of the inhibitor used.

Synthetic antioxidants: biochemical actions and interference with radiation, toxic compounds, chemical mutagens and chemical carcinogens.

Abstract Biological actions of 4 commonly used synthetic antioxidants — butylated hydroxyanisole, butylated hydroxytoluene, ethoxquin and propul gallate — on the molecular, cellular and organ level are compiled. Such actions may be divided into modulation of growth, macromolecule synthesis and differentiation, modulation of immune response, interference with oxygen activation and miscellaneous. Moreover, an overview of beneficial and adverse interactions of these antioxidants with exogenous noxae is given. Beneficial interactions include radioprotection, protection against acute toxicity of chemicals, antimutagenic activity and antitumorigenic action. Possible mechanisms of the antitumorige…

Elevation of hepatic epoxide hydratase activity by ethoxyquin is due to increased synthesis of the enzyme.

Abstract Feeding of the antioxidant ethoxyquin to rats leads to an increase of epoxide hydratase activity in liver microsomes. The apparent half life of the increase is 3–4 days. Elevation of epoxide hydratase activity is also obtained by intraperitoneal treatment of mice with ethoxyquin. This elevation is prevented by concomitant treatment with cycloheximide. When radiolabelled leucine is incorporated into microsomal protein by liver cell fractions from either ethoxyquin-fed or untreated rats, gel electrophoresis reveals that ethoxyquin feeding increases incorporation into epoxide hydratase. These results suggest that the elevation of epoxide hydratase activity by ethoxyquin is due to incr…

Differential inhibition of biphenyl hydroxylation in perfused rat liver

A differential inhibition of biphenyl hydroxylation by alpha-naphthoflavone and metyrapone was observed in isolated perfused rat liver. alpha-Naphthoflavone inhibited 2- and 4-hydroxylation in livers from beta-naphthoflavone-pretreated animals but had no effect on both reactions in livers from phenobarbital-pretreated animals. Metyrapone inhibited 2- and 4-hydroxylation in phenobarbital-stimulated livers, but only insignificant inhibition of 2-hydroxylation and a slight enhancement of 4-hydroxylation by metyrapone was observed in beta-naphthoflavone-stimulated livers. Conjugation of 2-hydroxybiphenyl and 4-hydroxybiphenyl by isolated perfused livers was also studied. 4-Hydroxybiphenyl prefe…

ENHANCEMENT OF MICROSOMAL TYPE II SUBSTRATE OXIDATION BY HEMOGLOBIN

Effect of nitrite on microsomal cytochrome P-450.

1. Addition of nitrite to anaerobic rat liver microsomes leads to the appearance of a difference spectrum similar to the spectrum of the ferrous cytochrome P-450-NO complex. A Soret band is found at 444 nm in phenobarbital-stimulated microsomes but at 442 nm in 3-methylcholanthrene-stimulated microsomes. An alpha-band is located at 583 nm in both types of microsome. 2. The initial nitrite-induced difference spectrum is converted into a spectrum lacking a Soret band but with a prominent absorbance minimum at 417 nm. This is more rapid in microsomes from phenobarbital-treated animals where it is completed in 8 min than in microsomes from 3-methylcholanthrene-treated animals. A similar spectru…

Antioxidants and carcinogen metabolism

Abstract Supplementation of diet with synthetic antioxidants has been shown to protect animals against a broad spectrum of chemical carcinogens. Some interest has been expressed in the use of these food additives as possible prophylactic agents. However, such considerations appear premature in view of the fact that the protective mechanism has not yet been elucidated. The present communication reviews the effect of antioxidants on carcinogen-metabolizing enzymes, one of the major candidates in the interpretation of the protective antioxidant action.

Significance of Induction Phenomena

A number of foreign compounds induce the proliferation of the hepatic smooth endoplasmatic reticulum and thereby increase the activity of monooxygenases that metabolize drugs and other foreign compound. With reference to the safety of food additives some antioxidants have been examined by various authors for their inducing capacity, in doses well above those ingested with treated food and above the stipulated accepted daily intake (ADI). Thus feeding of rats with the very high dose of 500 mg/kg body weight of butylated hydroxtoluene (BHT) resulted in an increase in its own oxidative metabolism. Also in monkeys BHT produces an inductive increase of microsomal enzyme activity, cytochrome P 45…

Induction of rat hepatic epoxide hydratase by dietary antioxidants

Abstract Supplementation of rat diet with butylated hydroxytoluene (BHT), butylated hydroxyanisole, or ethoxyquin resulted in increased liver epoxide hydratase activity. The increase was obvious at 0.1% and amounted to 200–400% at 0.5%. Increased activity was accompanied by increased proportion of the epoxide hydratase band in SDS polyacrylamide gels, indicating induction of the enzyme. Ethoxycoumarin deethylase activity and cytochrome b5 concentrations were moderately elevated while cytochrome P-450 concentrations and aryl hydrocarbon hydroxylase activity remained at control levels. Preferential inhibition of monooxygenase activity by metyrapone and not 7,8-benzoflavone, as well as increas…

Enhancement of Nitro Reduction in Rat Liver Microsomes by Haemin and Haemoproteins

1. Reductive metabolism of p-nitrobenzoic acid and neoprontosil in rat liver microsomes was studied in the presence of haemin, haemoglobin and myoglobin. 2. Microsomal nitro reduction is enhanced 4-fold in the presence of haemoglobin, whereas azo reduction is not affected. 3. Microsomal nitro reduction is enhanced to a similar extent by haemoglobin, haemin and boiled haemoglobin, whereas myoglobin is about half as active. 4. Maximal enhancement of microsomal nitro reductase activity by haemoglobin is achieved at high substrate concentration (6 mM) and low microsomal protein concentration (0.5--1.0 mg/ml). 5. Control microsomal nitro reduction as well as the haemoglobin-enhanced microsomal n…

Ethoxyquin as an inducer and inhibitor of phenobarbital-type cytochrome P-450 in rat liver microsomes.

Abstract The effect of ethoxyquin in vivo and in vitro on drug metabolism in rat liver microsomes was studied. In feeding experiments, a threshold dose of induction was found at 0.05% ethoxyquin for 14 days. At 0.5% ethoxyquin, relative liver weight, cytochrome P-450 content, cytochrome b5 content, ethylmorphine demethylation, and ethoxycoumarin deethylation were increased by a factor of 1.5 to 2. Aryl hydrocarbon hydroxylase activity was, however, not induced but even decreased by 0.5% ethoxyquin in food. Induction of epoxide hydratase was marked, amounting to 400% of control after 0.5% ethoxyquin. The induced enzyme was similar to the phenobarbital-inducible cytochrome P-450 in its CO spe…

SEX DIFFERENCES IN THE PATTERN OF CYTOCHROMES P-450 IN RAT LIVER MICROSOMES

ABSTRACT A number of sex differences in the spectral and enzymic properties of rat liver microsomes have been observed which may reflect differences in the population of hepatic cytochromes P 450 of male and female rats: 1. a blue shift in the spectrum of the reduced P 450-CO complex in females as compared to males, 2. lower ΔA max values in the binding of metyrapone to reduced microsomes in females as compared to males, 3. a higher proportion of 2-hydroxylation in the metabolism of biphenyl in females as compared to males, 4. preferential inhibition of ethoxycoumarin deethylation, benzpyrene hydroxylation and biphenyl-4-hydroxylation by α-naphthoflavone in females but by metyrapone in male…

Binding of benzo(a)pyrene metabolites to cellular DNA in perfused rat lungs

The influence of pretreatment with monooxygenase inducers on total irreversible binding of metabolically activated [3H]-benzo(a)pyrene to cellular DNA and the formation of benzo(a)pyrene metabolite-deoxyribonucleoside adducts after cytochrome P-448 induction was studied in perfused rat lungs. Pretreatment with the cytochrome P-448 inducer beta-naphthoflavone increasing binding by a factor of 23. In lungs of induced animals, 0.45 pmoles of benzo(a)pyrene equivalents were bound per mg DNA. Binding to RNA and to protein was also considerably induced by beta-naphthoflavone. Phenobarbital treatment did not significantly increase binding to cellular macromolecules of rat lung. Analysis of hydroly…

Effect of dietary antioxidants on benzo[a]pyrene metabolism in rat liver microsomes

Feeding of rats with 1% ethoxyquin (EQ) and butylated hydroxytoluene (BHT) but not butylated hydroxyanisole (BHA) increases the formation rate of benzo[a]pyrene (BP)-4,5-dihydrodiol from BP in hepatic microsomes. The production of other BP-dihydrodiols and of BP phenols is decreased after treatment with EQ, BHT and BHA. EQ and BHT are more effective than BHA in inducing epoxide hydrolase (EH) activity towards styrene oxide as the substrate.

Ethoxyquin feeding to rats increases liver microsome-catalyzed formation of benzo(a)pyrene diol epoxide--DNA adduct.

Abstract The ability of rat liver microsomes to catalyze the formation of benzo(a)pyrene 7,8-diol-9,10-epoxide — DNA nucleoside adduct was increased threefold by feeding 0.5% ethoxyquin to the animals. Microsomal epoxide hydratase activity was enhanced i parallel by a factor of 3 while aryl hydrocarbon hydroxylase activity was not induced. Liver microsomes from rat pretreated with 3-methylcholanthrene produced an increased proportion of diol epoxide — DNA adduct when ethoxyquin had been fed to the animals. The main chromatographic peak formed by microsomes from 3-methylcholanthrene treated rats which contains DNA adducts of secondary benzo(a)pyrene phenol metabolites is reduced when the ani…

Similar level of metabolic activation of benzo(a)pyrene in perfused rat lung and liver and protection of lung by liver in a combined perfusion system

Abstract Irreversible binding of metabolically activated benzo(a)pyrene to DNA, RNA and protein proceeds by a different time course in perfused liver and lung of 5,6-benzoflavone-treated rats. Peak binding in liver is obtained after 15 min while binding in lung continuously increases over 120 min. Total irreversible binding per mg DNA or RNA is in the same order of magnitude in both organs. While binding in lung is lower at 15 min it exceeds binding in liver at 120 min. Binding per mg protein is higher in lung than in liver over the whole perfusion period. Introduction of a liver into the lung perfusion circuit decreases binding in lung. This protection effect is more pronounced when the li…