Adaptive responses of rat liver to the gestagen and anti-androgen cyproterone acetate and other inducers. I. Induction of drug-metabolizing enzymes

Treatment of female Wistar rats with cyproterone acetate (CPA) was shown to cause pronounced increases of hepatic microsomal monooxygenase activity towards the following substrates: ethylmorphine (EM), aminopyrine (AP), benzphetamin (BPA) and benzo[a]pyrene (BP). Minor increases were seen using p-nitroanisole (pNA) and aniline (AN). Monooxygenase activity reached maximal levels within 24 h. The effects were dose-dependent, the threshold dose being about 4 mg/kg, and were reversible within 6 days. The results of comparative studies with several ‘classical’ microsomal enzyme inducers, i.e. pregnenolone-(16α)-carbonitrile (PCN), phenobarbital (PB), α-hexachlorocyclohexane (α-HCH) and 3-methylcholanthrene (3-MC) suggest that CPA belongs to the PCN-type and α-HCH to the phenobarbital type of inducers. In male rats CPA induced only moderate increases of monooxygenase activities which can be explained by decreased testosterone secretion due to anti-gonadotropic effect of CPA.     

Differential localization of microsomal mixed function oxidase activity in periportal and perivenous hepatocytes isolated from rat liver

1. The activity per mg of microsomal protein of aminopyrine N-demethylase was higher in perivenous (PV) than in periportal (PP) hepatocytes of rat, but when it was expressed per cytochrome P-450 content the difference in the activity was not significant. 2. The activity of 7-ethoxycoumarin O-deethylase, when expressed per mg protein and per P-450 content, was significantly higher in PV than in PP cells. 3. The activities of dimethylnitrosamine(DMNA) N-demethylase and aniline p-hydroxylase were not significantly different between two subpopulations of isolated hepatocytes when either expressed per mg protein or per P-450 content.     

Effects of β-naphthoflavone on the cytochrome P450 system,and phase II enzymes in gilthead seabream (Sparus aurata)

The effect of β-naphthoflavone (β-NF) on several catalytic activities of cytochrome P450 (CYP) and phase II enzymes putatively controlled by [Ah]-receptor activation in the liver, heart and kidney of gilthead seabream, was investigated. In the liver, β-NF treatment [intraperitoneal injection (i.p.) 50 mg/kg] resulted in an increase of CYP content, immunoreactive CYP 1A and methoxyresorufin-O-demethylase (MEROD), pentoxyresorufin O-depentylase (PROD) and ethoxyresorufin-O-deethylase (EROD) activities. However, β-NF had no effect on any of the hepatic phase II enzymes examined (benzaldehyde dehydrogenase, propionaldehyde dehydrogenase, glutathione S-transferase, UDP-glucuronyl-transferase, DT-diaphorase). Single i.p. injection of 10 mg/kg β-NF showed a maximal induction of CYP 1A-like protein and EROD activity after 3–7 days. CYP 1A and EROD returned to control levels 18-days post-treatment. β-NF injection also caused a rapid increase of a single band size of mRNA recognized by a CYP 1A1 cDNA fragment from sea bass (Dicentrarchus labrax). Expression of mRNA preceded the increase of EROD activity and declined rapidly by 96 h. Dose–response experiments demonstrated that EROD was significantly enhanced in liver by a single injection of 0.3 mg/kg β-NF and was the most sensitive measurement for CYP 1A-like induction. β-NF treatments also increased the expression of CYP 1A-like protein, mRNA and EROD, but not MEROD and PROD activities in heart and kidney.     

Stability of microsomal mono-oxygenase during incubations for the liver microsomal assay with S9 fractions of mouse liver under various inductions

Aminopyrine-N-demethylase and p-nitroanisole-O-demethylase activities were determined in incubation mixtures for the liver microsomal assay at time zero and after 1 h of incubation in the conditions for the mutagenic assay. The experiments were performed with the S9 liver fraction of mice in the basal state and induced with sodium phenobarbital, β-naphthoflavone or both. Lipid peroxidation was also determined.

The experiments were repeated with female mice and also in the presence of styrene, as an example of a xenobiotic substance. The activity of pNAD was much more stable than that of APD in all the conditions tested. The pattern of stability, however, was similar for the two activities: more stable than controls with S9 fractions from β-NF-induced mice, less stable than controls in PB-induced mice, intermediate between controls and PB-induced mice in those with combined induction by PB + βNF. Styrene 50 mM in the incubation mixtures led to a marked inactivation of enzymic activity, similar in all cases and reaching about 90% in 1 h. S9 fractions from female mice gave enzymes slightly more stable in almost all cases. Lipid peroxidation was appreciably more elevated in basal than in induced animals.

It was concluded that, for a mutagenesis test on an unknown xenobiotic, S9 fractions from mice following PB and β-NF induction are to be preferred from the point of view of activation.     

NADPH-generating system: Influence on microsomal mono-oxygenase stability during incubation for the liver-microsomal assay with rat and mouse S9 fractions

Activity levels of 7-ethoxycoumarin O-deethylase (ED), aminopyrine N-demethylase (APD), p-nitroanisoleO-demethylase (p-NAD) and glucose-6-phosphate dehydrogenase (G-6-PDH) were determined in incubation mixtures for the liver-microsomal assay (LMA) at time 0 and after 1 and 2 h incubation under conditions for mutagenic assay. The experiments were performed with S9 liver fractions from mice (induced with Na-phenobarbital and β-naphthoflavone) and rats (induced with Aroclor 1254) with and without G-6-PDH in the incubation mixtures.

In the absence of G-6-PDH the activities were significantly lower at time 0 in the mouse. The pattern of stability, however, was similar for the activities, with an increase of stability after 1 and 2 h of pre-incubation (an exception for p-NAD).

Only ED activity showed a similar behaviour in the rat. No differences were present for APD and p-NAD activities at time 0 in the rat, but the enzyme stabilities were significantly decreased after 2 h of incubation (about 15% and 10% for APD and p-NAD respectively) in the absence of G-6-PDH.

At time 0, the amounts of G-6-PDH differed between mouse and rat fractions; however, during the incubations for LMA they decreased by about 57% and 53% for the two species, respectively. In addition to the above biochemical results, the presence of exogenous G-6-PDH in the incubations for the mutagenic assay, significantly increased the mitotic gene conversion and mitotic crossing-over of dimethylnitrosamine (DMN) and AR2MNFN (a nitroimidazo[2,1-b]thiazole) in the D₇ strain of Saccharomyces cerevisiae.     

Expression analysis of the mixed function oxidase system in rat brain by the polymerase chain reaction

Metabolism of therapeutic drugs in the body by the mixed function oxidase system is an important consideration in the analysis of a drug's effectiveness. P450-dependent metabolism within the brain of a neuro-specific drug may affect the drug's course of action. To determine whether cytochrome P450 was expressed in brain, RNA was isolated from the whole brains of rats treated with a variety of known hepatic P450 inducers, including amitriptyline, imipramine, isosafrole, phenobarbital, and -naphthoflavone. The RNA was analyzed for the presence of P450 isozymes by the PCR technique. Differential expression of P450IA1, P450IIB1, P450IIB2, P450IID, and P450IIE1 was detected in the brain samples, depending on the treatment. Cytochrome P450 reductase expression was also detected in the brain samples, giving strong evidence that the brain contains a competent mixed function oxidase system under all conditions studied. (Mol Cell Biochem120: 171–179, 1993)Thesis student of the Graduate School of Biomedical Sciences, the University of Texas Health Science Center at Houston     

Induction by 9-hydroxyellipticine of aryl hydrocarbon hydroxylase in perinatal rat liver and in primary fetal hepatocytes in culture

N-nitrosodiethanolamine is converted to N-(2-hydroxyethyl)-N-(formylmethyl)nitrosamine (EFMN) and N-(2-hydroxyethyl)-N-carboxymethyl) nitrosamine (ECMN) by rat S9 liver preparation as a result of beta-oxidation. The beta-oxidized metabolites were isolated and identified by gas chromatography-mass spectrometry (GC-MS) by comparison with authentic standards. An original gas chromatographic method with thermal energy detection was set up to measure both metabolites quantitatively. Under the experimental conditions described, when NAD+ was used as cofactor, about 1% of N-nitrosodiethanolamine (NDELA) was converted to EFMN and about half of the latter product was in turn converted to ECMN. The beta-nitrosamino aldehyde seems to transfer the nitroso moiety to other amino-compounds, even at physiological pH. The significance of the metabolic formation of EFMN in relation to the carcinogenicity of NDELA is discussed.     

Functional alterations of the endoplasmic reticulum and the detoxification systems during diethyl-nitrosamine carcinogenesis in rat liver

The mixed-function oxidase system shows a number of variations in the liver of diethyl-nitrosamine (DEN) treated rats. These include a decrease of the cytochrome P450 content and of the aminopyrine demethylase activity both in the hyperplastic nodules and in the hepatoma. Processes of detoxification, such as the glutathione system, show some modifications. These alterations are in accordance with the decrease of glutathione peroxidase and the increase of gamma-glutamyltranspeptidase during diethyl-nitrosamine carcinogenesis.     

Change in the mixed function oxidase system of liver microsomes in rats treated with 3'-methyl-4-dimethylaminoazobenzene.

The mixed function oxidase systems of plasma membranes, the Golgi apparatus, and smooth and rough endoplasmic reticula of the livers of rats fed on a standard diet containing 0.06% (w/w) 3'-methyl-4-dimethylaminoazobenzene were investigated. The components and activities of the mixed function oxidase systems of the smooth endoplasmic reticulum and Golgi apparatus were especially reduced by the carcinogen. The activities for hydroxylation of anilines and demethylation of p-nitroanisole and the contents of cytochromes P-450 and b5 of the submicrosomal fractions of the rats decreased considerably more than the activities of NADH- and NADPH-ferricyanide reductases. More than 90% of the ferri-cytochrome P-450 content of the 3'-methyl-4-dimethylaminoazobenzene induced microsomes at 20 K was in the low spin form.     

The effect of nicotinamide on the activity of the glutathione- glutathione reductase system in subcellular fractions of regenerating rat liver

Experiments on white rats have shown that growth rates of the glutathione reductase activity and reduced glutathione concentration in the cytoplasmic fraction of the generating liver tissue and especially in the mitochondrial one are more pronounced with an increase of the nicotinamide dose from 50 mg/kg to 150 mg/kg, than after administration of nicotinamide in a dose of 300 mg/kg. Higher doses of nicotinamide (500 mg/kg) produce less pronounced changes in these parameters.     

Induction by butylated hydroxytoluene of rat liver gamma-glutamyl transpeptidase activity in comparison to expression in carcinogen-induced altered lesions

Butylated hydroxytoluene (BHT) at concentrations of 300-6000 ppm in the diet caused a dose-dependent increase in gamma-glutamyl transpeptidase (GGT) activity in normal F344 male rat liver at 18 weeks. However, the activities of glutathione S-transferases (GSTs) of rat liver cytosol were enhanced only at concentrations of 3000 or 6000 ppm BHT. Histochemically, the enhanced GGT activity was localized to hepatocytes surrounding the portal areas. Autoradiographic measurements of DNA synthesis showed that dietary BHT did not increase the level of cell proliferation and the GGT-positive hepatocytes did not exhibit different rates of DNA synthesis from those of GGT-negative cells. Feeding of the liver carcinogen N-2-fluorenylacetamide (FAA) induced foci and nodules of GGT-positive altered cells which exhibited higher rates of DNA synthesis than those of surrounding GGT-negative hepatocytes. Following iron loading, the periportal GGT-positive hepatocytes produced by BHT accumulated cellular iron, whereas the cells in FAA-induced lesions excluded iron. These results suggest that dietary BHT induces GGT activity in periportal hepatocytes without proliferation of the cells and that induction does not represent fetal expression or a preneoplastic alteration.     

Changes in the activity of the alternative oxidase in Orobanche seeds during conditioning and their possible physiological function

The appearance of the activity of the cyanide insensitive, alternative oxidase (AOX), pathway of oxygen uptake was followed in seeds of Orobanche aegyptiaca during conditioning. The pathway becomes operative during conditioning, up to day three as determined by inhibition of oxygen uptake of the seeds by propyl gallate. At the same time an increasing percentage of oxygen uptake is insensitive to cyanide and an increased oxygen uptake, responsive to propyl gallate, is induced by brief salicylic acid treatment of seeds. By day six of conditioning, these responses decrease and the AOX pathway could not be detected in germinating seeds, after treatment with a germination stimulant. These results were confirmed by following the reaction of extracts of fractions enriched with mitochondria from the conditioned seeds, using a specific antibody against AOX. Treatment of the seeds with inhibitors of AOX during conditioning significantly inhibited their subsequent germination. Addition of hydrogen peroxide after 4 and 7 days of conditioning resulted in reduced germination. In addition treatment of seed with propyl or octyl gallate during conditioning reduced the infection of tomato plants by Orobanche seeds and the development of tubercles of the parasite on the host roots. These results together indicate that the operation of AOX during conditioning has a significant function on the subsequent germination behaviour and pathogenicity of the root parasite. Some potential practical applications of these findings are discussed.     

Enhancement of nuclear Ca2+-ATPase activity in regenerating rat liver: involvement of nuclear DNA increase

The alteration of calcium content, Ca²⁺-ATPase activity, DNA content and DNA fragmentation in the nuclei of regenerating rat liver was investigated. Liver was surgically removed about 70% of that of sham-operated rats. the reduced liver weight by partial hepatectomy was completely restored at 3 days after the surgery. Regenerating liver significantly increased Ca²⁺-ATPase activity and DNA content in the nuclei between 1 and 5 days after hepatectomy. The nuclear calcium content was clearly increased from 2 days after hepatectomy. The increase of Ca²⁺-ATPase activity in regenerating liver was clearly inhibited by the presence of trifluoperazine (10 M), staurosporine (2.5 M) and dibucaine (10 M), which are inhibitors of calmodulin and protein kinase, in the enzyme reaction mixture. However, the nuclear enzyme activity in normal rat liver was not significantly altered by these inhibitors. Meanwhile, the increase of nuclear DNA content in regenerating liver was completely blocked by the administration of trifluoperazine (2.5 mg/100 g body weight), suggesting an involvement of calmodulin. Now, the nuclear DNA fragmentation was significantly decreased in regenerating liver, suggesting that this decrease is partly contributed to the increase in nuclear DNA content. The present study clearly demonstrates that regenerating liver enhances nuclear Ca²⁺-ATPase activity and induces a corresponding elevation of nuclear calcium content. This Ca²⁺-signaling system may be involved in the regulation of nuclear DNA functions in regenerating rat liver.     

Inhibitory effect of calcium-binding protein regucalcin on protein kinase activity in the nuclei of regenerating rat liver

The effect of Ca²⁺-binding protein regucalcin on protein kinase activity in the nuclei of normal and regenerating rat livers was investigated. Protein kinase activity in the nuclei isolated from normal rat liver was significantly increased by addition of Ca²⁺ (500 μM) and calmodulin (10 μg/ml) in the enzyme reaction mixture. Nuclear protein kinase activity was significantly decreased in the presence of EGTA (1.0 mM), trifluoperazine (TFP; 20 μM), dibucaine (10⁻⁴ M), or staurosporine (10⁻⁷ M), indicating that Ca²⁺-dependent protein kinases are present in the nuclei. Protein kinase activity was significantly elevated in the liver nuclei obtained at 6 to 48 h after a partial hepatectomy. Hepatectomy-increased nuclear protein kinase activity was significantly decreased in the presence of EGTA (1.0 mM), TFP (20 μM), or staurosporine (10⁻⁷ M) in the enzyme reaction mixture. The presence of regucalcin (0.1–0.5 μM) caused a significant decrease in protein kinase activity in the nuclei obtained from normal and regenerating rat livers. Meanwhile, the nuclear protein kinase activity from normal and regenerating livers was significantly elevated in the presence of anti-regucalcin monoclonal antibody (50–200 ng/ml). The present study suggests that regucalcin plays a role in the regulation of protein kinase activity in the nuclei of proliferative liver cells. J. Cell. Biochem. 71:569–576, 1998. © 1998 Wiley-Liss, Inc.