Marc H. Davies

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 rhythm in acetaminophen toxicity: Role of nonprotein sulfhydryls

A circadian rhythm in the toxicity of acetaminophen administered at different times of the day was exhibited in male mice. Peak lethality (70% dead) occurred at 1800 hr and nadir lethality (10% dead) occurred at 1000 hr. Hepatic nonprotein sulfhydryl (NPS) concentrations also varied in a circadian manner and were found to exhibit an inverse relationship to the acetaminophen lethality rhythm, with the highest hepatic concentrations occurring at 1000 hr and the lowest concentrations occurring at 1800 hr. At 1800 hr when acetaminophen lethality was greatest and NPS levels the lowest, more reactive acetaminophen metabolites were found covalently bound to hepatic macromolecules as compared to those bound at 1000 hr when NPS levels were highest and acetaminophen lethality was lowest. Hepatic cytochrome P-450 levels measured at these two daily time periods did not differ. The plasma half-life of acetaminophen was significantly decreased at the 1000 hr time period as compared to the 1800 hr time period. These findings suggest that the normal daily changes in NPS levels may influence the lethality of acetaminophen.

Factors Influencing Circadian Rhythms in Acetaminophen Lethality

Experiments were conducted to examine the effects of changes in lighting schedules and food consumption on circadian rhythms in acetaminophen lethality and hepatic glutathione levels in male mice. Under a normal lighting schedule (light: 06.00-18.00 h), male mice exhibited a circadian rhythm in acetaminophen lethality (peak: 18.00 h; nadir: 06.00, 10.00 h) and an inverse rhythm in hepatic glutathione concentrations (peak: 06.00, 10.00 h; nadir: 18.00 h). Under a reversed lighting schedule (light: 18.00-06.00 h) the glutathione rhythm was reversed and the rhythm in acetaminophen lethality was altered showing greater sensitivity to the drug. Under continuous light, there was a shift in the acetaminophen lethality and the hepatic glutathione rhythms. Under continuous dark, both rhythms were abolished. Under a normal lighting regimen, hepatic glutathione levels were closely correlated with food consumption; i.e., both were increased during the dark phase and decreased during the light phase. Fasting the mice for 12 h abolished the rhythms in acetaminophen lethality and hepatic glutathione levels; moreover, the lethality was increased and the hepatic glutathione levels were decreased. These experiments show that both lighting schedules and feeding can alter the circadian rhythms in acetaminophen lethality and hepatic glutathione levels in male mice.

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.

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.

Sex differences in the effects of retinoids on carcinogenesis by N-nitrosobis(2-oxopropyl)amine in Syrian hamsters

Syrian hamsters were given in a single dose of N-nitrosobis(2-oxopropyl)-amine (BOP) (40 mg/kg, s.c.) and 1 week later were fed 1 of 4 retinoid types (13-cis-retinoic acid (13-cis-RA), N-ethylretinamide (ERA), 2-hydroxyethylretinamide (OH-ERA), or 4-hydroxyphenylretinamide (PRA)) each at 3 levels (0.05, 0.1, 0.2 mM/kg diet). The pancreatic carcinoma incidence was not influenced significantly by feeding retinoids. The pancreatic adenoma incidence, however, was reduced by feeding each of the retinoids to female hamsters, with the reduction varying with the retinoid fed (13-cis-RA greater than ERA and OH-ERA greater than PRA). In male hamsters increased numbers of pancreatic adenomas were observed after feeding OH-ERA and PRA. Tumors induced in other tissues were reduced by retinoids in females, but not in males. Females fed 13-cis-RA and ERA had a lower incidence of gall bladder polyps, and feeding OH-ERA reduced the liver tumor incidence. Food consumption and serum alkaline phosphatase ans aspartate amino transferase activities were not influenced by BOP or retinoid type or level. Body and pancreas weight were influenced by retinoid level, but the effects were not consistently dose-related.

Bladder freeze ulceration and sodium saccharin feeding in the rat: Examination for urinary nitrosamines, mutagens and bacteria, and effects on hepatic microsomal enzymes

We previously demonstrated that long-term feeding of sodium saccharin, a non-mutagen, induced bladder carcinomas when administered to F344 male rats with regenerative hyperplasia of the urothelium induced by the freeze-ulceration technique, even without prior chemical initiation (Cohen et al. Cancer Res. 1982, 42, 65). In the present study, we examined the urine of rats subjected to freeze ulceration of the bladder and then fed sodium saccharin at 5% in the diet to evaluate the possibility of a mutagen being generated as a result of ulceration and/or saccharin feeding. Urine was collected into a syringe by aspiration from the urinary bladder after ligating the urethra for 2 hr at intervals from day 0 to day 14 after ulceration. After ulceration and/or sodium saccharin feeding, the urine showed no bacterial contamination, no mutagenic activity in the standard Ames assay, no production of nitrosamines, and no nitrosating environment. In addition, no significant changes in activities of liver microsomal enzymes (i.e. cytochrome P-450, NADPH-cytochrome c reductase, aniline hydroxylase, or ethylmorphine N-demethylase) were observed in rats fed sodium saccharin for 1, 5 or 14 days. Thus, freeze ulceration, and the consequent regenerative hyperplasia of the epithelium, compared with sodium saccharin feeding do not involve the administration of an exogenous mutagenic substance or the generation of a detectable mutagen in the urine.

Toxic Effects of Dietary Selenium in the Syrian Hamster

Selenium (Se) toxicity and utilization was evaluated in hamsters fed casein- and torula yeast-based diets. 4-week-old hamsters received semipurified diets for 21 days. In experiment I diets were supplemented with either 0.25, 10, 20, 40 or 80 ppm Se as sodium selenite (SS) and in experiment II diets were supplemented with 0.1, 5.0 or 10.0 ppm as SS or selenomethionine (SM). Blood and tissue Se concentrations and glutathione peroxidase (GSH-Px) activity were measured at the termination of the feeding period. In both studies growth rate was depressed and food consumption decreased in hamsters given diets supplemented with 10 ppm or greater SS. Mortality associated with Se toxicity occurred only in females fed the 80 ppm Se-supplemented diet. Whole blood and tissue Se concentrations rose with increasing dietary Se and occurred up to the 80 ppm Se level in blood. Liver, kidney and lung Se concentrations were higher in hamsters fed SM than for those fed SS. Plasma GSH-Px activity was not significantly affected by increasing dietary Se levels, and hamsters fed dietary Se levels above 10 ppm did not have increased erythrocyte GSH-Px activity associated with increased blood Se concentrations. Liver GSH-Px activity was higher in SM-fed hamsters. The results suggest that dietary Se, fed as SS, becomes toxic for Syrian hamsters at levels of 10 ppm and above.

Effect of sodium selenite upon bromobenzene toxicity in rats: I. Hepatotoxicity

The effects of sodium selenite on bromobenzene hepatotoxicity were examined in male rats. Rats pretreated with sodium selenite (12.5 or 30 mumol/kg, ip) 72 hr prior to injection of bromobenzene (7.5 mmol/kg, ip) showed a marked reduction in bromobenzene-induced liver injury as evidenced by decreased plasma alanine and aspartate transaminase values, sorbitol dehydrogenase activity, and reduced histologic damage. Administration of bromobenzene did not affect the selenium content of blood or liver. At 72 hr after treatment with selenite, hepatic reduced (GSH) and oxidized (GSSG) glutathione values or GSH synthetic and degradation enzyme activities were not altered. However, from 3 to 12 hr following bromobenzene administration, hepatic GSH and cysteine amounts declined less rapidly in selenite-treated rats compared to control. Thus, acute selenite treatment ameliorated bromobenzene hepatotoxicity in a manner suggesting facilitation of hepatic GSH production by selenite for use in bromobenzene detoxication.

Effect of acute and repeated selenium treatment on hepatic monooxygenase enzyme activity in male rats

The effect of sodium selenite administered acutely or repeatedly on the biochemical components of the hepatic microsomal monooxygenase enzyme system was examined in male rats. 72 h following acute administration of selenium (2.4 mg Se/kg, i.p.), there was a significant decrease in ethylmorphine-N-demethylase activity and cytochrome P-450 levels but no change in aniline hydroxylase or NADPH cytochrome c reductase activity. Following repeated administration of selenite in the drinking water (1, 2, or 4 ppm Se) for 30 days, there was no alteration in any of the parameters measured. Following the in vitro additions of selenite to microsomes obtained from untreated rats, ethylmorphine-N-demethylase and aniline hydroxylase activities were inhibited at selenium concentrations of 10(-4) M or greater, but the inhibition achieved was less than 50%. No alterations in cytochrome P-450 levels were observed. These results indicate that selenium is a rather weak, indirect, and substrate-specific inhibitor of the hepatic monooxygenase enzyme system.