Jukka Marniemi

Radiochemical assay of glutathione S-epoxide transferase and its enhancement by phenobarbital in rat liver in vivo

Abstract A rapid and sensitive assay of glutathione S -epoxide transferase was developed using tritium-labeled styrene oxide as the epoxide substrate. The method is based on the separation of unreacted styrene oxide from the glutathione conjugate formed after the incubation by the extraction with light petroleum. The radioactivity of the conjugate remaining in the aqueous phase can be determined subsequently by liquid scintillation. Using the present radiochemical method we found, after the intraperitoneal administration of phenobarbital (80 mg/kg) in vivo , a significant enhancement (about 56%) in the conjugation of styrene oxide with glutathione catalyzed by rat liver soluble fraction. No enhancement in the enzyme activity could be detected, on the other hand, after administration with styrene (1 g/kg) or 20-methylcholanthrene (20 mg/kg).

Action of styrene and its metabolites styrene oxide and styrene glycol on activities of xenobiotic biotransformation enzymes in rat liver in vivo

Abstract The effects of the administration of styrene and its metabolites, styrene oxide and styrene glycol, on drug metabolizing enzymes of rat liver were studied. Styrene (three or six daily doses of 500 g/kg) doubled the activities of microsomal p -nitroanisole O -demethylase, epoxide hydrase (styrene oxide as substrate), and UDP glucuronosyltransferase ( p -nitrophenol) when measured in microsomes pretreated in vitro with digitonin or trypsin. On the other hand, glycine conjugation, the cytochrome P-450 content, and activities of NADPH cytochrome c reductase and benzpyrene hydroxylase, were less affected by styrene. Styrene oxide was more toxic to rats than styrene or styrene glycol and it also caused (one dose of 375 mg/kg) a significant decrease in the activities of benzpyrene hydroxylase and p -nitroanisole O -demethylase and in the cytochrome P-450 content. Epoxide hydratase NADPH cytochrome c reductase, and UDP glucuronosyltransferase were more resistant towards styrene oxide. Styrene glycol did not significantly alter the activities of drug metabolizing enzymes. The current data show that styrene causes an increase in the activities of some enzymes involved in its own metabolism.

COVALENT BINDING OF STYRENE OXIDE TO RAT LIVER MACROMOLECULES IN VIVO AND IN VITRO

ABSTRACT Styrene oxide was found to bind covalently to homogenate, microsomes and protein and nucleic fractions of the rat liver after intraperitoneal administration in vivo . Styrene oxide also bound covalently in vitro when incubated with different liver fractions, and reduced glutathione decreased this binding. The free glutathione content of the liver was decreased by styrene oxide in vivo .

Effects of aliphatic chlorohydrocarbons on drug-metabolizing enzymes in rat liver in vivo.

1. Various polychlorinated hydrocarbons were administered intragastrically to rats to examine their effects on the biotransformation capacity of the liver. Due to high toxicity, 1,1,2,2-tetrachloroethane and pentachloroethane were given at a dose level equivalent to one quarter of that of CCl4 and the other chlorohydrocarbons (i.e. 2-6 mmol/kg). 2. Carbon tetrachloride at 10-3 mmol/kg was the most active in decreasing cytochrome P-450 content and the overall drug hydroxylation activities in rat liver. 1,1,2,2-Tetrachloroethane was the next most active in decreasing the hepatic drug oxidizing enzymic activities. 3. Expoxide hydratase activity in rat liver declined significantly after CCl4, 1,1,2,2-tetrachloroethane and pentachloroethane administrations. 4. UDP-Glucuronosyltransferase was affected to a lesser extent than the microsomal mono-oxygenase or epoxide hydratase by chlorohydrocarbon treatment.

Different effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on glucuronide conjugation of various aglycones. Studies in Wistar and Gunn rats

Abstract 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) (20 μg/kg) was administered to homozygous Gunn and Wistar as well as heterozygous rats, and the activity of UDP-glucuronosyltransferase determined in the liver and kidney toward o-aminophenol, p-nitrophenol, 4-methylumbelliferone, and bilirubin. Diethylnitrosamine was used to activate the enzyme in vitro. TCDD did not enhance UDP-glucuronosyltransferase (UDPGT) activity in the Gunn rat, either in the liver or in the kidney. In the Wistar rat, the UDPGT activity toward methylumbelliferone was enhanced eightfold in the liver and twofold in the kidney. TCDD did not affect UDPGT activity toward bilirubin. Diethylnitrosamine activated o-aminophenol and p-nitrophenol glucuronidation in vitro in the liver only. No activation in vitro due to diethylnitrosamine treatment could be seen in the liver of the TCDD-treated Wistar rat.

Radiochemical assay of UDP glucuronyltransferase (p-nitrophenol).

UDP glucuronyltransferas¢ (UDPglucuronate glucuronyltransfera~, EC 2.4. I. 17) catalyzing the glucuron:de formation of foreign compounds Is fimdy bound to the membranes of endoplasmtc rctlculum. The kinetics of the UDP glucuronyltransferase has therefore been studied with only crude preparations. These kinetic studies have been hampered by tl,e lack of an accurate and sensitive method for determining enzyme activity, p-Nitrophenol is very often used as an aglycone, because its spectrophotometric quantttation ts rapid and simple [ 1 ]. it has also been widely used in recent detailed kinetic studies of ttus enzyme [2-8] . This method has, howe,,ecertain disadvantages, smce the consumption of Ul¢ substrat¢ is measured and mot the formation of the product, whsch is preferable in kinetic studies. The absorption spectrum of p-mtrophenyl glucuronide and free a~lycone have different absorption maxima (glucur,~, ide at 312 and agl:,-cone at 400 nm, respectively), and this has been utilized in the direct rn.,2~.?~,tmment of IIDP #tte~srgn~,!tr,anqfera~ activity [9]. This method is, however, rather insensl. tire. In kinetic studies low substrate concentrations should also be us¢¢l due to their physiological relevance. With the old spectt~photometric methods mentioned above this has been very difficult. In the present paper we describe a radiochvmtcal method for UDP glucuronyltransferas¢ determination with IncRaae d accuracy and sensitivity, t4C-Labe!ed pnittophenol, which is now commercially available, wa~,used itSa ~UCu/onyl ac~ept0r mbstrat¢,

Drug Conjugation in Gunn Rats: Reduced UDP-Glucuronosyl Transferase and UDP-Glucosyl Transferase Activities with Increased Glycine-N-Acyltransferase Activity

Glucuronyl, glucosyl, glutathione, glycine conjugations and epoxide hydration were studied in livers of Wistar and Gunn rats. The activities of bilirubin UDP-glucuronosyl transferase and UDP-glucosyl transferase were reduced in microsomes of homozygous and heterozygous Gunn rats compared to Wistar rats (over 90 and 55-65%, respectively). Conjugation of 4-methylumbelliferone by UDP-glucuronosyl transferase was also reduced (30-35%) in Gunn rats. Treatment of microsomes in vitro with membrane perturbating agents increased the measurable activities but did not change the activity relationships between Wistar and Gunn rats. Microsomal epoxide hydrase and soluble glutathione-S-epoxide transferase activities occurred in Gunn rats at the Wistar level, but mitochondrial glycine-N-acyltransferase activity was elevated in homozygous and heterozygous Gunn rats (66 and 47%, respectively). The reciprocal velocity plots of UDP-glucuronosyl transferase activity were similarly concave in Gunn and Wistar rats.

Inhibition of glucuronide synthesis by physiological metabolites in liver slices

The synthesis of glucuronides @-D-glucopyranosiduronic acids) is catalyzed by rather poorly characterized UDPglucuronyltransferase(s?) (UDPglucuronate glucuronyltransferase (acceptor unspecific), (EC 2.4.1. 17). No specific inhibitor is known, which could be used to block the glucuronide synthesis in living cells. The glucuronide synthesis is insensitive to the various metabolites of the glucuronic acid pathway [l] . The synthesis of the glucuronyl donor, UDPglucuronic acid, by UDPglucose dehydrogenase (UDPglucase: NAD oxidoreductase, EC 1.1.1.22) can, however, be inhibited effectively at least in vitro, by UDPgalactose [2] and UDPxylose [3]. Furthermore, the level of UDPglucose, which is a precursor of UDPglucuronic acid, can be lowered in the liver by perfusion with a Dgalactosamine containing medium [4]. In this study data are presented, which indicate that UDPgalactose, Dgalactose, UDPxylose and Dgalactosamine can be used to block glucuronide synthesis from phenolic aglycone in liver slices. D-galactose, UDPxylose and D-galactosamine have no effect on the oxygen uptake of the slices. None of these compounds inhibited the glucuronide synthesis in isolated microsomal membranes.

Bilirubin UDP-glucosyl- and UDP-glucuronosyltransferase of rat liver. A comparative study of the effects of membrane perturbants in vitro and of chrysene administration in vivo

Abstract The effect of several membrane perturbants (digitonin, cetylpyridinium chloride (CPC), trypsin and phospholipase C) on bilirubin-conjugating UDP-glucosyl and UDPglucuronosyltransferase of rat liver microsomes was studied. All the compounds appeared to activate (maximally 6–11-fold) UDP-glucuronosyl transferase. Digitonin, CPC and trypsin had the same kind of activating effect on UDPglucosyltransferase (maximally 3–4-fold). The action of phospholipase C on these enzymes was found to be different. UDPglucosyltransferase was more sensitive toward this agent than UDPglucuronosyltransferase. The intraperitoneal administration of a polycyclic hydrocarbon, chrysene, to rats was shown to enhance the UDPglucuronosyltransferase activity of liver native microsomes about 1.5-fold. In the perturbant-treated microsomes this increment could not be found. The activity of UDPglucosyltransferase was not affected by the same chrysene treatment either in native or activated microsomes. The present data suggest that there is a difference in the phospholipid environment of bilirubin UDPglucosyI and UDPglu curonosyltransferases. Furthermore support for the hypothesis that two different enzymes are involved in the synthesis of glucoside and glucuronide conjugates of bilirubin is given.

Studies on the action of d-galactose and d-galactosamine on the glucuronide synthesis in the rat tissues

Abstract 1. 1. d -Galactose was found to inhibit the o -aminophenyl glucuronide synthesis in the rat liver and kidney slices, but no effect was obterved in the slices of jejunum. 2. 2. d -Galactosamine inhibited the glucuronide synthesis only in the liver. 3. 3. The sensitivity of glucuronide synthesis in the liver depends on the rapid activation of d -galactose and d -galactosamine to UDP compounds. 4. 4. UDP-Glucuronic acid and, to a lesser degree, UDP-glucose reversed the inhibition caused by d -galactose and d -galactosamine in the liver slices.