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Carps, both sexes, 3 years old, weighing about 1 kg, and tenches of both sexes, 6 years old, weight about 250 g, were caught from a Thuringian lake without industrial pollution in November 1995 (fish without food uptake, water temperature at about 10 degrees C) and kept for 2 weeks in basins with clean water and addition of 0, 0.1, 1.0 or 10.0 mg/l phenobarbital-Na (PB). The concentration of PB was controlled during and at the end of the exposure period. The animals were fed pellets, but no food uptake was observed. After 24-48 h in fresh water the fish were sacrificed and the following hepatic parameters were immediately determined biochemically: monooxygenase functions: cytochrome P450 (P450) content, ethylmorphine N-demethylation (EN), ethoxycoumarin O-deethylation (ECOD), ethoxyresorufin O-deethylation (EROD), 7-benzyloxy-4-methyl-coumarin O-debenzylation (BCDB); oxidase function indicators: microsomal Fe2+/NADPH dependent hydrogen peroxide formation (H2O2), microsomal Fe2+/NADPH dependent luminol and lucigenin amplified chemiluminescence (LMCL, LCCL), microsomal Fe2+/NADPH dependent lipid peroxide formation (LPO); oxidative state: lipid peroxidation products (TBARS) and GSH and GSSG. Additionally, the expression of three P450 isoforms, 1A1, 2B and 3A, was assessed immunohistochemically in tissue samples from brain, gill, heart, spleen, liver, gut and ovary of both fish species and in kidney of tenches. PB did not influence body or liver weights, but increased liver P450 concentration in both species by 50-100%, though not significantly. Carp: PB increased both EN and EROD significantly, but not ECOD and BCDB; H2O2 and TBARS were enhanced significantly. LPO, LMCL and LCCL were not significantly influenced. Tench: PB increased all monooxygenase reactions (EN, ECOD, BCDB and EROD), though only significantly ECOD; H2O2 was elevated only after treatment with 0.1 mg/l PB, whereas LPO was decreased (!) after treatment by all three concentrations, though significantly only after 1.0 mg/l PB. LMCL was depressed (not significantly), but LCCL increased 5fold. TBARS were significantly enhanced. P450 1A1 subtype expression was concentration dependently elevated by PB in gill and liver of both fish and in the heart and kidney of tenches, P450 2B and 3A isoforms expression was induced in brain, gill, heart, liver and gut of both fish and in the kidney of tenches. In summary, the increased activities of the monooxygenase reactions tested and the elevated expression of all three P450 isoforms investigated in certain tissues indicate an induction of the P450 families 1, 2 and 3 by PB in fish.

In the present study the effects of beta-naphthoflavone (BNF), phenobarbital (PB) and dexamethasone (DEX) on cytochrome P450 (P450) dependent monooxygenase functions were investigated in intrasplenic liver cell explants in comparison to adult liver. Fetal liver tissue suspensions were transplanted into the spleens of 60-90 days old adult male syngenic Fisher 344 inbred rats. 2, 4 or 6 months after surgery, transplant recipients and age matched controls were orally treated with BNF (1x50 mg/kg body weight (b.wt.)), PB (1x50 mg/kg b.wt.), DEX (for 3 days 4 mg/kg b.wt. per day), or the respective solvents (dimethylsulfoxide or 0.9% NaCl). The animals were sacrificed 24 (BNF, DEX) or 48 (PB) hours after the last treatment. P450 mediated monooxygenase functions were measured in spleen and liver 9000 g supernatants by three model reactions for different P450 subtypes: ethoxyresorufin O-deethylation (EROD; 1A), ethoxycoumarin O-deethylation (ECOD; 1A, 2A, 2B), and ethylmorphine N-demethylation (END; 3A). Spleen weights were significantly higher in transplanted rats, compared to controls, at all three time points after surgery. Induction with PB or DEX, and in some cases also with BNF, lead to a significant increase in liver weights of transplant recipients and control rats independent of the time after transplantation. In contrast, there was no influence on spleen weights due to BNF or PB. At all time points after surgery, with DEX a marked decrease in body weights, weights of adrenal glands and of lymphatic organs like thymus glands and spleens was observed, with the weights of the transplant containing spleens being still higher in comparison to control organs. Spleens of control animals displayed nearly no P450 mediated monooxygenase functions neither without nor with induction. After transplantation, however, significant EROD and ECOD, but hardly any END activities were seen in the host organs at all three time points after surgery. In transplant containing spleens EROD and ECOD were significantly increased after BNF or PB treatment at all three time points after surgery, and ECOD after DEX administration, but at 4 and 6 months after transplantation only. END was only induced after DEX treatment at 6 months after transplantation. With the livers of both transplant recipients and control rats EROD and ECOD were increased after BNF induction and EROD, ECOD, and END after PB treatment at all three time points after transplantation. After DEX administration END was significantly enhanced only at 2 and 4 months after transplantation, ECOD was decreased at 2 and 4 months, and EROD was diminished at all three time points after surgery. Transplantation of fetal liver tissue suspensions into the spleens did not influence monooxygenase functions and their inducibility within the respective livers of the animals. These results demonstrate that transplanted liver cells originating from syngenic fetal liver tissue suspensions display P450 dependent monooxygenase functions which are, simi lar to normal adult liver, inducible by BNF, PB and DEX. Both monooxygenase functions and their inducibility within the transplant containing spleens display quantitative and qualitative developmental changes.

In the present study, the effect of beta-naphthoflavone (BNF), phenobarbital (PB) and dexamethasone (DEX) on the expression of three cytochrome P450 (P450) isoforms, 1A1, 2B1 and 3A2, and on glycogen storage was investigated in intrasplenic liver cell explants in comparison to adult liver. Fetal liver tissue suspensions were transplanted into the spleens of adult male syngenic Fisher inbred rats. Four months after surgery, transplant recipients and age matched controls were orally treated with BNF (1 x 50 mg/kg body weight (b.wt.)), PB (1 x 50 mg/kg b.wt.), DEX (for 3 days 4 mg/kg b.wt. per day), or the respective solvents (dimethylsulfoxide or 0.9% NaCl). The animals were sacrificed 24 (BNF, DEX) or 48 (PB) hours after the last treatment. The livers of both solvent treated transplant recipients and control rats displayed only in few liver lobules a slight P450 1A1, but in all lobules a strong P450 2B1 and 3A2 expression, which was all mainly located in the hepatocytes around the central veins (zone III, according to Rappaport). After BNF administration a P450 1A1 expression was induced in the hepatocytes of the peripheral regions of the liver lobules (zone I, according to Rappaport), whereas the staining of the hepatocytes around the central veins disappeared. Also the staining for P450 2B1 in the hepatocytes of zone III became slightly more pronounced. Following PB treatment the P450 1A1 expression in the hepatocytes of the central regions (zone III), as seen in few lobules after solvent treatment only, was reduced, whereas the staining for P450 2B1 and 3A2 was more pronounced in the hepatocytes of the intermedial and central regions of the liver lobules (zone II and III). DEX treatment diminished P450 1A1 and 2B1 expression within the livers of both transplant recipients and control rats. In contrast, the staining for P450 3A2 was enhanced in all regions of the liver lobules. Transplantation of fetal liver tissue suspensions into the spleens did not influence the inducibility of P450 isoforms expression within the respective livers of the animals. Spleens of control rats displayed no P450 isoforms expression without as well as with induction. In the explant containing spleens, however, similar to normal liver, the transplanted hepatocytes displayed nearly no P450 1A1, but a strong P450 2B1 and 3A2 expression. After BNF treatment a staining for P450 1A1 was induced and also the P450 2B1 expression was slightly more pronounced. PB treatment caused an increase in the staining for P450 2B1 and 3A2 and DEX administration for P450 3A2 within the transplanted hepatocytes. Additionally, after DEX treatment some bile ducts of the explants displayed a slight staining for P450 1A1, 2B1 and 3A2. All hepatocytes within the livers of both solvent treated transplant recipients and control rats displayed a slightly PAS-positive cytoplasma and, in most cases, homogeneously distributed, fine-grained, strongly PAS-stained granules indicating glycogen storage. No regional variance in the glycogen content of the hepatocytes was seen within the liver lobules, but there was a marked difference between the individual hepatocytes of the same lobular region in the extent of glycogen accumulation. The hepatocytes within the explants displayed the same type of glycogen storage as did the adult liver cells. BNF treatment did not display any effect on the glycogen accumulation in livers and intrasplenic liver cell explants. After PB administration, only in livers, but not in the transplants, the glycogen content in the hepatocytes around the central veins was slightly reduced. DEX treatment lead to an excessive storage of fat within the hepatocytes of both livers and spleens. Thus, the glycogen was displaced, leading to a "spoke-wheel" like pattern of glycogen storage. Additionally, within the hepatocytes of both livers and liver cell explants a higher amount of glycogen seemed to be stored and the granules appeared to be more coarse-grained. (ABSTRACT

The NADPH-dependent lipid peroxidation is low in liver microsomes of newborns and increases to day 30 of life. Thereafter nearly no changes in lipid peroxidation occur. The relatively high rate of lipid peroxidation in fetal liver microsomes is of interest. The well known inducers of the monooxygenase system phenobarbital and beta-naphthoflavone change lipid peroxidation only in liver microsomes of newborns. Lipid peroxidation is connected with the formation of reactive oxygen species and organic radicals characterized by the chemiluminescence with the amplifiers luminol and lucigenin. Chemiluminescence increases with increasing age depending on the amplifier. Treatment of the animals with phenobarbital causes the most pronounced increase in chemiluminescence of the 10-day-old animals, independent of the amplifier used. beta-naphthoflavone leads to an increase in chemiluminescence in microsomes of all age groups investigated except the luminol chemiluminescence

The spectral changes due to the binding of hexobarbital and aniline to cytochrome P-450 of rat liver microsomes were investigated in 10-day- to 15-month-old rats. The Ks values for both substances and consequently the affinity for cytochrome P-450 do not change during ageing. Phenobarbital treatment does not alter the affinity of hexobarbital, but enhances the Ks value for aniline. The maximal spectral changes (delta A max) due to aniline are nearly equal in all age groups whereas delta A max due to hexobarbital is very small in young rats and increases considerably during ageing. The age-dependence of the hexobarbital-induced delta A max is similar to the development of drug-metabolizing reactions. delta A max due to hexobarbital and aniline is enhanced by phenobarbital treatment of the rats. The addition of aniline to microsomes enhances the Ks value and diminishes delta A max for hexobarbital.

Bile flow and biliary excretion of free cholesterol were estimated after single and/or repeated i.p. administration of phenobarbital (60 mg/kg b. w.), chlorpromazine (40 mg/kg b.w.), progesterone (50 mg/kg b.w.), and phenylbutazone (100 mg/kg b.w.) in male Wistar rats. The drugs enhanced the biliary cholesterol excretion after both single and repeated administration, whereas only phenobarbital and phenylbutazone pretreatment increased the bile flow. Bile acid excretion was concomitantly enhanced only after phenylbutazone pretreatment. These results suggest that cholesterol output in rats may be widely bile acid independent and can be influenced by different drugs.

A single intraperitoneal administration of 3-methylcholanthrene (3MC) or beta-naphthoflavone (BNF) increased aryl hydrocarbon hydroxylase (AHH) and 7-ethoxyresorufin 0-deethylase (EROD) activities. Following a second administration of both inducers 3-4 weeks later, the extent of induction was considerably greater. Such a "booster"-phenomenon was also observed with different cytochrome P-450 (P-450)-dependent N-demethylation reactions after repeated phenobarbital (PB) administrations. Again the P-450 content was higher than after a single PB administration.

Maturation and phenobarbital inducibility of hepatic uptake of the organic anions indocyanine green (ICG) and eosine were studied in rats of different ages. Bile duct ligation reduced the ICG and eosine liver concentration in young rats. Therefore, bile duct ligation cannot be recommended for uptake investigations in rats of different ages. The longest elimination half life time (12.4 min) was found in 10-day-old rats within the first 15 min after i.v. administration of 38 mumol ICG/kg. In 20-day-old rats the ICG half life was significantly shorter than in adult 110-day-old rats (5.8 versus 9.6 min). Phenobarbital pretreatment did not alter the ICG elimination half lives in 10-, 20- and 110-day-old rats. The shortest elimination half life time of eosine was found in 60-day-old rats (33.0 min versus 80.3 min in 10-day-old rats and 62.4 min in 240-day-old ones). Phenobarbital pretreatment did not influence eosine serum concentrations. Our results indicate for ICG that uptake comes to maturity earlier than biliary excretion. Differences between ICG and eosine in maturation of the two transport steps support the assumption of more than one carrier for exogenous organic anions. Dye uptake was not altered after phenobarbital and, consequently, cannot be responsible for enhanced biliary dye excretion in differently aged rats.

Bile flow and excretion of monohydroxy-, dihydroxy- and trihydroxy-bile acids (MBA, DBA and TBA) were estimated after acute and subacute phenobarbital and chlorpromazine pretreatment in 60-day-old male Wistar rats. Bile was collected in bile-fistula rats in three 1-hour periods. MBA were not detected. Neither single nor repeated ip. administration of different amounts of saline before bile sampling nor oral water supply within the bile collection period influenced the bile flow and excretion of DBA and TBA. Phenobarbital administration (60 mg/kg b.w. ip.) 2 hrs before bile sampling did not influence bile flow and bile acid (BA) excretion. After 3 days pretreatment with phenobarbital (3 X 60 mg/kg b.w. ip.) the bile flow was somewhat increased, BA-excretion was unchanged and the relation TBA/DBA diminished. Chlorpromazine administration (40 mg/kg b.w. ip.) 1,5 hrs before bile sampling decreased bile flow and BA excretion within the first collection period, whereas bile flow and BA excretion increased in the thrid collection period. No signs of cholestasis were observed after chlorpromazine pretreatment once a day for 3 days. Bile flow and BA excretion were increased and the relation TBA/DBA was unchanged.

The activity of NADPH-cytochrome P-450 reductase in liver microsomes of 10- to 60-day-old rats was determined. Neither the half life time of cytochrome P-450 reduction nor the absolute amount of cytochrome P-450 reduced per time unit depend on age. Phenobarbital pretreatment enhances the reduction rate in all age groups. The addition of hexobarbital or ethylmorphine to microsomal suspension accelerates the reduction of cytochrome P-450 in some age groups only. Age differences corresponding to developmental changes in drug-metabolizing activities are not detectable. The NADPH-cytochrome P-450 reductase seems to be not responsible for the age dependence of drug metabolism.