R.C. Schnell

Direct enzymatic assay for reduced and oxidized glutathione

A modification of a specific assay for the reduced (GSH) and oxidized (GSSG) species of glutathione is presented and compared with the method of Ellman (1959). The present method has an enzymatic basis using GSH as the specific cosubstrate for glutathione S-transferase activity. The enzymatic method resulted in comparable, but consistently lower, values for GSH and GSSG than did the method of Ellman. The greatest differences between the two methods occurred when measuring renal GSH and GSSG, possibly due to the presence of mixed thiols in the kidney. This enzymatic method was more specific and provided an accurate and reproducible method of GSH and GSSG determination.

Circadian variations in glutathione-S-transferase and glutathione peroxidase activities in the mouse

Circadian variations in hepatic glutathione-S-transferase and glutathione peroxidase activities were found in male Swiss-Webster mice maintained under a 12/12 lighting schedule (L: 06.00-18.00 h) for 3 weeks prior to use. Hepatic glutathione-S-transferase activity was significantly elevated during the dark phase as compared to the light phase. The glutathione peroxidase activity, using two different substrates, was biphasic in nature. Using cumene hydroperoxide, the enzyme activity was significantly elevated at 18.00 and 06.00 h; nadirs occurred at 10.00, 14.00 and 22.00 h. Using hydrogen peroxide as the substrate, enzyme activity was greatest at 14.00 and 06.00 h; nadirs occurred at 10.00 and 22.00 h. Glutathione concentration also varied in a circadian manner with peak levels occurring between 06.00 and 10.00 h and the nadir occurring at 18.00 h. These studies have shown that glutathione levels and two enzymes utilizing glutathione as a cosubstrate vary in a similar circadian manner, with the highest values primarily occurring during the dark phase. Thus, these results may have considerable toxicological importance.

Protective effects of selenium on acetaminophen-induced hepatotoxicity in the rat

Experiments were undertaken to examine the ability of selenium to protect against acetaminophen-induced hepatotoxicity and to examine possible mechanisms for this protective effect. Pretreatment of male, Sprague-Dawley rats with sodium selenite (12.5 mumol Se/kg, ip) 24 hr prior to acetaminophen administration produced a significant protection against the hepatotoxic effects of acetaminophen as assessed by a decrease in the plasma appearance of alanine aminotransferase and aspartate aminotransferase activities following acetaminophen. This was accompanied by an increase in the hepatic glutathione levels in selenium-treated animals and an inhibition in the decrease in hepatic glutathione content observed in animals receiving hepatotoxic doses of acetaminophen. Selenium pretreatment decreased the in vivo covalent binding of acetaminophen metabolites to hepatic protein, but did not alter hepatic microsomal cytochrome P-450 content or NADPH cytochrome c reductase activity, suggesting that selenium does not significantly alter the metabolism of acetaminophen to reactive electrophilic metabolites by the cytochrome P-450-dependent mixed-function oxidase enzyme system. Selenium produced an increase in the activity of gamma-glutamylcysteine synthetase which may account for the increased glutathione availability in selenium-treated animals and increased the activities of glutathione S-transferase and glucose-6-phosphate dehydrogenase. Examination of the urinary metabolite profile in selenium-treated animals revealed that the urinary excretion of acetaminophen and its metabolites was significantly increased over a 72-hr period. The increase occurred in the AAP-glucuronide metabolite while parent AAP and AAP-sulfate were actually decreased in selenium-treated rats. No change in recovery was observed in the AAP-glutathione or AAP-mercapturate urinary metabolites. While the glutathione conjugating system is enhanced by selenium treatment, amelioration of acetaminophen toxicity is most likely the result of enhanced glucuronidation which effectively diverts the amount of acetaminophen to be converted by the cytochrome P-450 system to the toxic metabolite.

Alterations in Hepatic Microsomal Drug Metabolism and Cytochrome P-450 Proteins in Spontaneously Hypertensive Rats

Experiments were conducted to determine if substrate-specific changes in microsomal metabolism and liver proteins occurred in young (12-13 weeks) spontaneously hypertensive rats (SHR) fed ad libitum compared to age-matched normotensive Wistar Kyoto (WKY) control rats. The hepatic microsomal protein content in SHR rats was significantly increased compared to WKY rats while cytosolic and total liver protein levels did not differ between the two groups. Liver microsomal ethylmorphine-N-demethylase activity was substantially enhanced in SHR rats with only slight increases in cytochrome P-450 content and aniline hydroxylase activity compared to WKY rats. The substrate-specific increases in the microsomal drug metabolism in SHR rats were accompanied by an increase in the prominence of a protein with molecular weight 55,000 in the cytochrome P-450 region. These preliminary observations may be clinically relevant in that alterations in hepatic drug metabolism may be associated with endogenous biochemical processes underlying the hypertensive state.

Influence of cadmium on calcium absorption from the rat intestine

Influence of Cadmium on Calcium Absorption from the Rat Intestine. Yuhas, E. M., Miya, T. S., and Schnell, R. C. (1978). Toxicol. Appl. Pharmacol. 43 , 23–31. The effect of cadmium on gastrointestinal calcium absorption in male rats was examined using an in situ single-pass intestinal perfusion technique. The addition of cadmium, (10 −5 , 10 −3 , or 10 −2 m ) to the perfusion medium caused a decrease in net calcium absorption. At Cd concentrations greater than 10 −5 m , a net negative calcium balance occurred. The decrease in net calcium absorption resulted from both a decrease in the lumen-to-plasma flux of calcium (10 −5 to 10 −2 m Cd) and an increase in the plasma-to-lumen flux to calcium (10 −4 to 10 −2 m Cd). Intestinal calcium absorption was unaltered 72 hr following a single acute injection of cadmium acetate (2 mg/kg, ip). In another experiment, rats were given cadmium (0, 1, 10 or 100 ppm) in the drinking water for 13, weeks, and then in situ intestinal calcium absorption was determined. At 1 or 10 ppm of Cd, there was no effect on net calcium absorption or lumen-to-plasma or plasmato-lumen fluxes of calcium. At 100 ppm of Cd, there was an increase in the lumen-to-plasma-to-lumen fluxes of calcium. At 100 ppm of Cd, there was an increase in the lumen-to-plasma flux of calcium since there was no effect on the plasma-to-lumen flux of calcium. While cadmium added to the perfusate may exert a direct inhibitory effect on intestinal calcium absorption, the treatment in vivo of rats with cadmium, either acutely or chronically, does not decrease intestinal calcium absorption.

Cadmium-induced potentiation of hexobarbital sleep time in rats.

Nachweis, dass Cadmium bei Ratten die Hexobarbital-Schlafdauer erhöht. Eine wirksame Reaktion war bereits bei einer Cadmium-Dosis von 840 μg/kg beobachtet, und die Schlafdauer wurde bereits nach einer einzigen Dosis vom 1. bis zum 10. Tage erhöht, wobei zwischen dem 2. und 5. Tage die Maximalwirkung beobachtet wurde.

Oltipraz-induced amelioration of acetaminophen hepatotoxicity in hamsters: I. Lack of dependence on glutathione

These studies were designed to test the hypothesis that oltipraz (OTP) provided protection against AAP intoxication in a sensitive species, the hamster; and further, to show that the sparing effect was related to the marked increase in hepatic reduced glutathione (GSH) levels. Dose-response and time-course experiments demonstrated that maximal increases in liver GSH occurred at 48 hr after an oltipraz dose of approximately 2.0 mmol/kg (po). Accompanying greater GSH levels were increased glutathione disulfide (GSSG) levels. Decreased indices of the oxidation state of glutathione and of hepatic pyridine nucleotides indicated a greater share of glutathione existed as GSH and that increased reducing equivalents were present, respectively. Additionally, glutathione disulfide reductase activity was greater in OTP-treated groups. Glutathione S-transferase activities were only marginally increased. OTP treatment did not elicit observable hepatotoxicity, whereas AAP (2.6 mmol/kg, ip) resulted in a reproducible model of liver damage. OTP-treated groups were protected from AAP-induced toxicity, as shown by decreased plasma appearance of liver enzymes and unremarkable histopathology. However, the degree of liver GSH depletion by AAP was fourfold greater in non-OTP treated groups compared to those which had received the dithiolthione. To test the importance of increased hepatic GSH, the biosynthesis of glutathione was interrupted. Buthionine sulfoximine (BSO) treatment decreased hepatic GSH, the biosynthesis of glutathione was interrupted. Buthionine sulfoximine (BSO) treatment decreased hepatic GSH content to 50% of control in hamsters which either had or had not received OTP. The groups receiving BSO and AAP incurred 83% lethality, while no lethality, unremarkable liver histopathology, and plasma enzyme levels consistent with control were found in the group receiving OTP, BSO, and AAP. Treatment with BSO only had no influence on hepatotoxicity parameters. These results indicate that the increased GSH levels in the OTP-treated hamster are coincidental to the sparing effect of OTP and are not central to the protection scheme in AAP-induced hepatotoxicity.

Annual Differences in Daily Variations in Hepatic Drug Metabolizing Enzyme Activity and Plasma Hormone Levels in the Rat

Daily variations in hepatic drug metabolism and in plasma corticosterone and insulin levels were examined at monthly intervals over the duration of 1 year. Annual differences were observed in the basal levels of all parameters. Consistent daily variations were observed only in plasma corticosterone levels, whereas daily variations in each of the other parameters occurred inconsistently over the year with significant variations being found in ethylmorphine metabolism and insulin levels only five times, and in aniline metabolism, six times. These data show that daily variations in the parameters examined do not occur in a regular manner.Daily variations in hepatic drug metabolism and in plasma corticosterone and insulin levels were examined at monthly intervals over the duration of 1 year. Annual differences were observed in the basal levels of all parameters. Consistent daily variations were observed only in plasma corticosterone levels, whereas daily variations in each of the other parameters occurred inconsistently over the year with significant variations being found in ethylmorphine metabolism and insulin levels only five times, and in aniline metabolism, six times. These data show that daily variations in the parameters examined do not occur in a regular manner.

Oltipraz-induced amelioration of acetaminophen hepatotoxicity in hamsters. II. Competitive shunt in metabolism via glucuronidation.

Previously, we demonstrated that oltipraz [OTP: 5-(2-pyrazinyl)-4-methyl-1,2-dithiol-3-thione] prevented the hepatotoxicity of acetaminophen (AAP) in hamsters and that the observed protection was not related to increases in hepatic reduced glutathione (GSH) levels. These experiments were designed to elucidate the mechanism of OTP-induced protection with respect to an apparent non-GSH-dependent system. Marked differences in the relative amounts of hepatic GSH content depleted by AAP in control vs OTP-treated hamsters occurred. Urinary recoveries of AAP and metabolites indicated that more AAP-glucuronide was formed at the expense of other major metabolites (AAP-GSH, -N-acetylcysteine, and -sulfate) in OTP-treated hamsters, while plasma toxicokinetic modeling suggested a greater rate of AAP systemic clearance. An increased apparent formation rate constant for AAP glucuronidation (135%), in concert with significantly lower apparent formation rate constants for those metabolites which reflect the production of the reactive intermediate from AAP (glutathione and N-acetylcysteine), provide the rationale for this shift of metabolism. The biochemical basis for metabolic shunting is significantly elevated hepatic UDP-glucuronic acid content, an increased calculated UDP-glucuronic acid synthetic rate, and an increased liver microsomal UDP-glucuronyl transferase activity in OTP-treated animals. These changes in AAP conjugation were concomitant with decreased fractional clearance of AAP via bioactivation and less in vivo AAP covalent binding. These data support the hypothesis that OTP provides a protecting effect from AAP hepatotoxicity due to an augmented and predisposing glucuronidation capacity.

Sex-related differences in selenium-induced alterations in drug action in the rat.

Three days after pretreatment of rats of both sexes with sodium selenite (2.4 mg Se/kg, ip) the duration of pentobarbital-induced hypnosis was potentiated in males but not females. Similar treatment with selenium led to a significant inhibition of ethylmorphine demethylase activity by hepatic microsomal enzymes obtained from male but not female rats. Selenium treatment did not alter the activity of aniline hydroxylase in animals of either sex. These data show that there is a sex-related difference in the ability of selenium to alter drug response and hepatic drug metabolism, which is substrate-specific, in the rat.