Max G. Parkki

Organ specific induction of drug metabolizing enzymes by 2,3,7,8-tetrachlorodibenzo-p-dioxin in the rat

Abstract The effect of 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD) was studied on the activities of arylhydrocarbon hydroxylase, ethoxycoumarin deethylase, cytochrome c reductase, epoxide hydratase, UDP glucuronosyltransferase, and glutathione S -transferase in the liver, kidney, lung, small intestinal mucosa, and testis of male Wistar rats. There was a severalfold increase in the activity of monooxygenase in the liver, kidney, and lung, whereas virtually no effect could be detected in the intestine or testes. The proportion of 3- and 9-hydroxylation of the total hydroxylation of benzo( a )pyrene decreased in the liver, but increased in the kidney. TCDD had no significant effect on epoxide hydratase or glutathione S -transferase activities in any tissues. UDP glucuronosyltransferase exhibited a sevenfold increase in the liver, less than twofold in the kidney, and none in other tissues. Treatment of the microsomes with digitonin, trypsin, or phospholipase A did not reveal additional induction UDP glucurnosyltransferase, although all were able to increase measurable enzymatic activity in control and TCDD-treated animals. TCDD seems to be different from phenobarbital and polycyclic hydrocarbons as an effector of not only monooxygenase but also epoxide hydratase, UDP glucuronosyltransferase, and glutathione S -transferase.

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.

Long‐term effects of single and combined doses of DDT and PCB on drug‐metabolizing enzymes in rat liver

Two common environmental pollutants, DDT and a polychlorinated biphenyl (PCB) mixture (Clophen A-50), were administered ip to rats in discrete single doses (160 and 100 mg/kg, respectively) and in combination. All the enzyme activities studied were enhanced by DDT and PCB. the overall drug hydroxylation reactions, and their components, achieved maximal induction in 1 wk. The cytochrome P-450 content of microsomes was increased nearly fourfold and NADPH-cytochrome c reductase activity was enhanced twofold by both compounds. p-Nitroanisole O-demethylase was increased sevenfold by PCB and fourfold by DDT, aryl hydrocarbon hydroxylase threefold by PCB and 1.7-fold by DDT. After 2 wk the activities began to decline. Distinct increases in enzyme activities were still detectable 1 month after a single dose. Epoxide hydratase and UDPglucuronosyltransferase activities were also enhanced in 1 wk (epoxide hydratase 2.5-fold by both compounds, UDPglucuronosyltransferase tenfold by PCB in trypsin-activated microsomes but only threefold by DDT). The disappearance of induction in epoxide hydratase was slower than in the monooxygenases, and UDPglucuronosyltransferase still showed a trend toward increased activity 4 wk after the administration. The DDT-enhanced UDPglucuronosyltransferase activity slightly returned toward the control level. Glutathione S-transferase differed from the microsomal enzymes in that it was already elevated 1 day after the administration of both DDT and PCB. Its activity was only doubled, but the increased activity remained at almost the same level through the whole 1 month period.

Inhibition of drug metabolizing enzymes by heavy metals in vitro

Abstract Cadmium iodide was a potent inhibitor of benzo(a)pyrene hydroxylation, ethoxycoumarin deethylation, epoxide hydration, and glutathione conjugation, but had no effect on cytochrome c reduction. Mercuric acetate inhibited cytochrome c reduction, benzo(a)pyrene hydroxylation, ethoxycoumarin deethylation, and glutathione conjugation, but was without effect on epoxide hydration. Nickel, cobalt, chromium and zinc salts had no effect on epoxide hydration or glutathione conjugation. Lead salts did not affect either monooxygenation, cytochrome c reduction, epoxide hydration, or glutathione conjugation.

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.

Enhancement of microsomal monooxygenase, epoxide hydrase and udpglucuronyltransferase by aldrin, dieldrin and isosafrole administrations in rat liver

The effects of intraperitoneal administration of aldrin (10 mg/kg), dieldrin (10 mg/kg) and isosafrole (50 mg/kg) were investigated on the activities of drug biotransformation enzymes in rat liver. All the compounds studied were found to enhance the activities of microsomal monooxygenase (e.g. p-nitroanisole O-demethylase, aryl hydrocarbon hydroxylase), epoxide hydrase (styrene oxide as substrate) and UDPglucuronyltransferase (p-nitrophenol as aglycone). Dieldrin, the epoxidized derivative aldrin, the eposidized derivative of aldrin, was a more potent inducer than the parent compound itself. NADPH CYTOCHROME C REDUCTASE ACTIVITY WAS EHANCED 2.5-FOLD, P-NITROANISOLE O-demethylase 7-fold, benzpyrene hydroxylase 2-fold, and epoxide hydrase 5-fold after treating rats with dieldrin for 6 days. The increase in activity of the microsomal UDPglucuronyltransferase could be only detected after an in vitro digitonin treatment of microsomal membranes, the enhancement being about 1.5-fold after administering dieldrin for 6 days. The administration of isosafrole to rats increased especially p-nitroanisole O-demethylase activity in liver microsomes (10-fold in 3 days). NADPH cytochrome c reducatase activkty was increased 2-fold, cytochrome P-450 content 1.2-fold, benzpyrene hydrozylase activity 2.5-fold and epoxide hydrase activity 1.2-fold after treatment of rats for 3 days. UDPglucuronyltransferase activity increased 2.2-fold by treating rats for 6 days with isosafrole. This increase was, however, only to be seen in in vitro digitonin-activated microsomes due to the latency of UDPglucuronyltransferase.

Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the hepatic metabolism of testosterone in the rat.

A dose of 20 micrograms/kg of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was given to adult male rats intragastrically and the metabolism of [4-14C]-testosterone by liver homogenate was examined 1 week later. The overall catabolism of testosterone was significantly reduced by the TCDD treatment. Dihydroxysteroids (intermediate reduced metabolites) were increased, while the production of polar non-conjugated metabolites (end products) was diminished.

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.