Ulla Koivusaari

Seasonal variation of hepatic biotransformation in female and male rainbow trout (Salmo gairdneri)

Abstract 1. 1. Seasonal and sex variations in the cytochrome P450 content and monooxygenase and glucuronidation activities were observed in the liver microsomes of rainbow trout cultivated in North-European waters. 2. 2. During the summer the specific monooxygenase activities lowered but the total calculated capacity remained unchanged (measured at 18°C) or increased (measured at environmental temperatures) due to liver growth. 3. 3. During water cooling in autumn the specific monooxygenase activity as well as the total capacity showed an ideal temperature compensation and remained unchanged if measured at environmental temperatures but increased considerably if measured at 18°C. 4. 4. Before the spawning time cytochrome P450 and the monooxygenase activities lowered in both sexes but more in females revealing a temporary sex difference which disappeared after spawning. 5. 5. The specific UDP glucuronosyltransferase activity lowered considerably during the cooling of environmental temperature, and thereafter an increase in both sexes was observed towards the spawning time (significantly higher in females) and coming summer.

Extrahepatic xenobiotic metabolism in North-European freshwater fish

Abstract 1. 1. Cytochrome P450 and monooxygenase activity and glucuronidation were measured in the gills, intestine, heart and kidney as well as in the liver of vendace, perch and roach and rainbow trout for comparison. 2. 2. Cytochrome P450 content and the specific monooxygenase activities were always highest in the liver. In the rainbow trout and vendace the renal 7-ethoxycoumarin-O-deethylase and in the roach the renal 3,4-benzpyrene hydroxylase activity was about half of that in liver. The activities in gills were relatively high in roach and vendace. In rainbow trout, perch and roach the intestinal activities were easily detectable. 3. 3. In all fish high UDPglucuronosyltransferase activities were found in the liver and intestine. In vendace and roach both the gills and the kidney showed, however, even higher specific activities. 4. 4. The rainbow trout showed higher hepatic and intestinal biotransformation activities than the other species.

Metabolism of foreign compounds in freshwater crayfish (Astacus astacus L.) tissues

Abstract Freshwater crayfish, Astacus astacus L. were found to have considerable amounts of cytochrome P-450 in the hepatopancreas microsomes. The content was almost as high as in rat liver microsomes. The low (7-ethoxycoumarin O-deethylase and ethylmorphine demethylase) or absent (3,4-benzpyrene hydroxylase and 7-ethoxyresorufin deethylase) monooxygenase activities recorded did not, however, correlate with the high amount of cytochrome P-450. The binding of type I and II substrates to the enzyme was poorer in crayfish hepatopancreas than in rat liver microsomes. There was also less NADPH than NADH cytochrome c reductase activity in crayfish hepatopancreas microsomes. NADH supported faster demethylation of ethylmorphine than NADPH. Studies with hepatopancreas subfractions added to rat liver microsomes showed that hepatopancreas cytosol contained heat labile monooxygenase inhibitor(s). Only traces of 7-ethoxycoumarin O-deethylase were found in total homogenates of extrahepatopancreatic tissues like gills, intestine and green glands. From conjugation activities considerable levels of glutathione S-transferase were found in all the crayfish tissues studied. UDP-glucuronosyltransferase activity was very low in microsomal and cytosol fractions of hepatopancreas.

Metabolism of xenobiotics by vendace (Coregonus albula)

Abstract 1. 1. The xenobiotic biotransformation ability of vendace (Coregonus albula) was studied. 2. 2. Cytochrome P-450, monooxygenase and UDPglucuronosyltransferase activities were detected in vendace liver, gills, kidneys, intestine and heart. 3. 3. The monooxygenase activity seems to be very sensitive to temperature and it is already inactivated at temperatures above 20°C. 4. 4. Monooxygenase activities in vendace tissues were lower than in the liver of cultivated rainbow trout (Salmo gairdnerii) or rat (Rattus norvegicus). UDPglucuronosyltransferase activities in vendace liver were as high as in the liver of rainbow trout and higher than in rat (measured at 18°C). The UDPglucuronosyltransferase activity in vendace gills was much higher than in the liver of all the species studied.

Hepatic drug metabolism during ethanol ingestion in riboflavin deficient rats

Riboflavin deficiency was induced by feeding rats a riboflavin-deficient diet for 1 month. In order to find out if there are any combined effects of ethanol and riboflavin deficiency on drug metabolism, a group of riboflavin-deficient rats were also given ethanol in their drinking water. At the end of the feeding period, hepatic drug-metabolizing enzyme activities were determined. The hepatic phospholipid and protein contents were the same in rats receiving a standard diet and in those on a riboflavin-deficient diet. However, ethanol ingestion in both groups enhanced significantly the phospholipid content. Ethanol ingestion also markedly enhanced the hepatic cytochrome P-450 concentration in rats fed either a standard or riboflavin-deficient diet. Ethoxycoumarin O-deethylase activity was significantly lower in riboflavin-deficient rat livers than in those of the controls. In both groups ethanol ingestion nearly doubled the activities. Aryl hydrocarbon hydroxylase activity was also significantly decreased during riboflavin deficiency. However, ethanol administration did not change the activities of this enzyme. UDP glucuronosyltransferase activity was slightly lower in riboflavin-deficient rat livers than in those fed a standard diet. No significant decrease was found in the epoxide hydrase activity in the riboflavin-deficient rats. However, the riboflavin-deficient rats had enhanced activity after the ethanol ingestion.

Catalytic and immunological comparison of coumarin 7-hydroxylation in different species

Coumarin 7-hydroxylation activities were determined in the liver microsomes of eight different species: pig, mouse (three strains), rat, hamster, guinea-pig, rabbit, rainbow trout and crayfish. A high coumarin 7-hydroxylase activity was found in the microsomes of D2 mouse, rabbit, hamster and pig, an intermediate activity in case of B6 and AKR mice, rat and guinea-pig and a very low activity in rainbow trout and crayfish. In Ouchterlony immunodiffusion analysis our antibody against coumarin 7-hydroxylase specific cytochrome P-450 from D2 mice was able to form a weak precipitin line only with rat and hamster microsomes in addition to the three strains of mice (D2, B6, AKR) where the band was very sharp. Strongest inhibition by the antibody (50% or more) of microsomal coumarin 7-hydroxylase was found in the case of the three strains of mice and rabbit. In the case of the other species the inhibition was very weak or did not exist.

Polysubstrate monooxygenase activity and sex hormones in pre- and postspawning rainbow trout (Salmo gairdneri)

Abstract The hepatic microsomal cytochrome P -450 content and polysubstrate monooxygenase (PSMO) activities as well as plasma levels of sex hormones in gonadally mature rainbow trout ( Salmo gairdneri ) were studied. The plasma level of testosterone in prespawning female fish exceeded that of male fish. In both sexes, the testosterone level was at its highest in March and decreased considerably before the spawning time. The 17 β-estradiol content in female fish showed similar changes. The hepatic cytochrome P -450 content was at its lowest in prespawning fish in March, and significantly lower in females than in males. The sex difference in the cytochrome P -450 content disappeared after spawning. The absorption maximum of the reduced cytochrome-CO-complex varied from 449–450 nm without any correlation to the sex of the fish or the cytochrome P -450 content. In PSMO activities (benzo[ a ]pyrene, 7-ethoxycoumarin, aminopyrine and ethylmorphine as substrates), a significant sex difference could be observed during the prespawning period. In all cases the activity was lower in females than in males. The 7-ethoxy-resorufin deethylase activity did not, however, show any sex difference. Benzo[ a ]pyrene hydroxylase and 7-ethoxyresorufin deethylase activities in both sexes increased gradually during the pre- and postspawning times, being at their highest level in June. However, aminopyrine demethylase activity increased considerably during the prespawning period, and then decreased during and after spawning. The results indicate that changes in monooxygenase activities are dependent on the substrate used and the state of spawning, refering to the presence of multiple forms of cytochrome P -450 in the rainbow trout liver. Prior to spawning the rainbow trout is probably most sensitive to direct acting chemicals and most resistant to activation requiring compounds due to the much lowered levels of cytochrome P -450 and several monooxygenase activities.

Structural and biotransformational membrane changes in the liver and intestine during chronic ethanol administration

The binding of a fluorescent probe 1-anilinonaphthalene-8-sulphonic acid (1,8-ANS) to liver microsomal membranes was markedly increased after chronic ethanol administration while the binding of a non-ionised probe phenylnaphthylamine (PNA) was not altered. The increase in 1,8-ANS binding is in accordance with the simultaneous increase of the ethoxycoumarin O-de-ethylase activity and cytochrome P-450 concentration. Also the intestinal ethyoxycoumarin O-de-ethylase activity and cytochrome P-450 concentration were increased. No changes in the aryl hydrocarbon hydroxylase or UDP-glucuronosyltransferase activities were found. The chronic ehtanol administration increased the phospholipid amount in the liver microsomes and altered the fatty acid composition of microsomal phospholipids by decreasing the amount of oleic acid and increasing linoleic acid proportion. The data suggest that chronic ethanol administration may effect the biotransformation enzyme activities by changing the structural properties of the membranes as well as increasing the cytochrome P-450 concentration.

Effect of administration route of 3-methylcholanthrene on the inducibility of intestinal drug-metabolizing enzymes.

The inducibility of the mucosal drug-metabolizing enzymes of rat small intestine was studied by administering 3-methylcholanthrene either intragastrically or intraperitoneally. The aryl hydrocarbon hydroxylase activity was 4.1 times higher after intragastric than after intraperitoneal administration of methylcholanthrene. The ethoxycoumarin-O-diethylase activity was 11 times and UDP-glucuronosyltransferase (with p-nitrophenol as substrate) activity was 3 times higher after intragastric administration than after intraperitoneal administration. The epoxide hydratase activity was, on the other hand, 38% lower after intragastric administration of 3-methylcholanthrene than after intraperitoneal administration. The results suggest that compounds entering the body intragastrically, e.e. in the diet, might have profound enzyme-specific effects on the intestinal metabolic rates of drugs.

Microsomal drug metabolism and the interaction of three fluorescent probes with microsomes at different temperatures

Abstract The activity of drug metabolizing enzymes, both those involved in hydroxylation and in glucuronidation, in rat liver microsomes were studied at different temperatures from 10°C to 38°C. Alterations in the enzyme activities in microsomes were compared with the binding of three fluorescent probes, 1,8-ANS (1-anilinonaphthalene-8-sulphonic acid), N-phenyl-1-naphthylamin and DL-12-(9-anthroyl) stearic acid. A non-linear change in the activity of hydroxylation reactions (but not in that of the glucuronidation reactions) was found at 20– 22°C. The increase in incubation temperature from 10°C to 38°C decreased the quantum yield and increased the affinity of fluorescent probes from microsomes. N-Phenyl-1-naphthylamin had the highest affinity for microsomes and DL-12-(9-anthroyl) stearic acid the lowest. A sudden increase in the affinity of 1,8-ANS molecules for microsomes was found at 16–20°C. No similar increase of affinity could be detected for other probes used. Of the three probes, 1,8-ANS was the most important inhibitor of microsomal drug metabolism. All three probes inhibited more effectively the hydroxylation reactions than glucuronidation reactions. It is concluded that the binding sites of 1,8-ANS in microsomes are more important for drug metabolism than those of N-phenyl-1-naphthylamin or DL-12-(9-anthroyl) stearic acid. The mechanism by which the probe molecules inhibit microsomal drug metabolism is discussed.