Acute intramuscular injection of oils or the oleic acid component protects mice against paraquat lethality.

Although selenium or vitamin E deficiencies or changing from cereal-based to purified diets augments paraquat toxicity, the action of other dietary components in normal animals fed nutritionally adequate diets is not clear. Upon injection of mice with antiinflammatory agents, a protective action of the corn oil vehicle against paraquat lethalities was noted. This preventive action of a large parenteral administration of unknown components in oils served as the basis for this study. Intramuscular injection of various vegetable oils protected similarly, indicating that in mixtures, the degree of lipid saturation did not seem to be an important factor. Injection of the monounsaturated fatty acid oleic acid decreased oral paraquat lethalities in mice, but linoleic, gamma-linoleic or linolenic acids were not protective in either male or female mice. Measurement of paraquat concentrations in various tissues at various times after administration indicated no effect of corn oil on paraquat distribution. Although the exact mechanism of the complex nature of oil protection against paraquat toxicity in mice is still unknown, this study provides evidence for in vivo oxidant protection by a monounsaturated fatty acid.

Mercaptans decrease selenium-dependent glutathione peroxidase activity in the chick.

A variety of mercaptans, especially the beta-mercaptocarboxylic acids, inhibited the selenium (Se)-dependent glutathione peroxidase (SeGSHpx) activity of chick liver postmitochondrial supernatant or cytosol and of purified bovine erythrocyte SeGSHpx. The effects of mercaptans and a glutathione analogue on Se utilization were determined by subcutaneous injection of test compounds into vitamin E-deficient chicks fed diets containing 0.1 ppm Se (as Na2SeO3) at times when greater than 50% of vitamin E- and Se-deficient chicks showed the vitamin E-, Se-deficiency disease exudative diathesis (ED). D-(-)-Penicillamine hydrochloride (the positive control model compound), sodium beta-mercaptopyruvate, t-butyl mercaptan and S-methylglutathione (nonmercaptan glutathione analogue) decreased SeGSHpx activity in chick liver postmitchondrial supernatants within 24 h of injection and increased the incidence of ED within 4 d. Other mercaptans tested did not increase ED incidence or affect liver, kidney or plasma SeGSHpx activities. Although mercaptosuccinic acid, N-(2-mercaptopropionyl)glycine and sodium thioglycolate each strongly inhibited SeGSHpx in vitro, each also significantly increased chick mortality in the dosage range tested; therefore, their effects on SeGSHpx in vivo could not be evaluated. It appears that the beta-mercaptocarboxylic acids, mercaptans with a high degree of steric hinderance in close proximity to the thiol group and a close structural analogue of glutathione are capable of altering selenium status in chicks.

Synthetic seleno-organic compound with glutathione peroxidase-like activity in the chick.

The glutathione peroxidase activity catalyzed by the seleno-organic anti-inflammatory drug Ebselen (registered under the trademark of the Natterman Corp. Cologne, FRG) [PZ51, 2-phenyl-1,2-benzisoselenazol-3(2H)on], as measured by NADPH oxidation, was inhibited in vitro by the selenium-dependent glutathione peroxidase (SeGSHpx) inhibitors aurothioglucose and D-(-)penicillamine HCl. Vitamin E- and selenium-deficient chicks were given 0, 80 or 320 ppm PZ51 in diets devoid of vitamin E and supplemented with low levels of sodium selenite (0.04 ppm selenium added to the basal diet containing ca. 0.015 ppm selenium) when a small number of chicks (ca. 13%) had exudative diathesis (ED). By 24 hr, the high PZ51 dose (320 ppm) delayed the onset of ED compared to untreated controls. Similarly, vitamin E-deficient chicks fed diets containing 0, 80, 160, 320, 640 or 1280 ppm PZ51 and supplemented with 0.04 ppm selenium showed ED in inverse proportion to log PZ51 dose. Plasma and liver post-mitochondrial supernatant samples from these chicks also exhibited log-linear relationships between dietary PZ51 level and selenium content or SeGSHpx-like activity. The amount of SeGSHpx-like activity for chicks given PZ51 above that determined for untreated chicks was extractable into ethanol, indicating that those PZ51-associated increases were not due to protein-bound selenium or SeGSHpx. This suggests that selenium from PZ51 was not available to support synthesis of SeGSHpx. Dietary PZ51 (1280 ppm) or selenium (0.1 ppm) alone or in combination decreased the acute lethalities of nitrofurantoin or paraquat in vitamin E-adequate chicks. The results indicate that SeGSHpx-like activity in selenium-deficient chicks is increased by oral administration of PZ51, which appears to mimic the true enzyme by affording protection against clinical signs of selenium deficiency (i.e. ED) and pro-oxidant drug lethality.

Selenium-dependent glutathione peroxidase inhibitors increase toxicity of prooxidant compounds in chicks.

The acute lethality of paraquat (1, 1'-dimethyl-4,4'-bipyridinium dichloride; also methyl viologen) for chicks was reduced in a dose-dependent manner by adding to a selenium-deficient torula-yeast-based diet low concentrations (0.02-0.04 ppm) of selenium (Se) as either Na2SeO3, selenomethionine or a high Se yeast without significantly increasing plasma Se-dependent glutathione peroxidase (Se GSH-Px) activity. Similarly, chicks orally dosed with 100 mg nitrofurantoin [N-(5-nitro-2 furfurylidine)-1-aminohydantoin] per kilogram had highest mortalities in the Se-deficient (unsupplemented) group; lowest mortalities occurred in chicks supplemented with 0.2 ppm Se; chicks supplemented with 0.02 ppm Se survived at rates not statistically different from chicks either unsupplemented or supplemented with 0.2 ppm Se. The activities of SeGSH-px in various vital organs were significantly elevated by supplementation of 0.2 ppm Se to Se-deficient chicks; but only kidney SeGSH-Px increased with 0.02 ppm Se. Additionally, no histopathology was observed in the vital organs of moribund chicks 5 or 24 h following nitrofurantoin administration at any dietary level of Se tested. Exposure of chicks to oxygen enhanced the toxicity of nitrofurantoin, but the protective effect of dietary Se was still evident. Two inhibitors of SeGSH-Px, D(-)-penicillamine X HCl and aurothioglucose, were found to increase the lethalities of both nitrofurantoin and paraquat. Aurothioglucose was most effective when administered simultaneously with the prooxidant compounds; penicillamine increased toxicities only when administered at least 24 h before paraquat or nitrofurantoin (it decreased nitrofurantoin lethality and did not significantly alter paraquat toxicity if given simultaneously). These data support an hypothesis that the protection offered by dietary Se against the acute toxicities of the prooxidant compounds paraquat and nitrofurantoin may be provided by SeGSH-Px in the chick.

Drug-induced changes in selenium-dependent glutathione peroxidase activity in the chick.

The ability of aurothioglucose and D(-)-penicillamine hydrochloride to inhibit selenium-dependent glutathione peroxidase (SeGSH-Px) in vitro and to increase exudative diathesis in vitamin E-deficient chickens was studied. Aurothioglucose and penicillamine competitively inhibited SeGSH-Px in inverse proportion to the concentration of hydrogen peroxide and reduced glutathione, respectively, in chick liver postmitochondrial supernatant assay preparations. Neither drug inhibited glutathione reductase or superoxide dismutase at the concentrations tested; however, both inhibited catalase in a semilogarithmic fashion. This was true for both the purified bovine enzyme and chick liver homogenate. Aurothioglucose and penicillamine injected subcutaneously at the back of the neck increased exudative diathesis in vitamin E-deficient chickens fed 0.1 ppm Se, and effectively overcame the protective effect of selenium 72 h after injection in chicks fed vitamin E-free, low selenium diets supplemented with 0.0-0.1 ppm Se. Assays of plasma and of liver, lung and kidney postmitochondrial supernatants indicated that all observed reductions in SeGSH-Px activity preceded increases in exudative diathesis. Plasma and liver SeGSH-Px activities were lower at early times (6-24 h) after treatment with high doses of either drug. Lung SeGSH-Px activities were only lower in chicks receiving 240 mg penicillamine/kg 6 h after treatment; kidney SeGSH-Px activities were only lower in chicks treated with the highest dose of aurothioglucose 48 h after treatment. Brain SeGSH-Px activities were unaffected by drug treatment and the heart had higher SeGSH-Px activities only at 6 h after treatment with the highest dose of either drug compared to saline controls. Catalase activities in liver homogenates were only significantly altered by penicillamine; the highest dose caused the activity to be higher than that in saline-treated chicks. The cause of the lower SeGSH-Px activities could be either lower enzyme concentrations in tissues of the drug-treated groups and/or direct inhibition. Whatever the mechanism, it is concluded that exudative diathesis can be used to determine which drugs reduce SeGSH-Px activity in the chick.

Separation of hepatic N-demethylase-inducing and opioid dependence-producing doses of levo-alpha-acetylmethadol in the pregnant rat.

A 2.0 mg per kg oral dose of 1-alpha-acetylmethadol (LAAM) administered daily to female rats prior to mating and throughout pregnancy increased ethylmorphine N-demethylase activity in liver microsomes of the dams measured 24 h after parturition. This dose of LAAM decreased maternal weight gain during gestation and increased postnatal mortality. However, 0.05 mg LAAM per kg was sufficient to produce dependence in the dams without affecting hepatic drug metabolism, gestational weight gain or neonatal mortality. The data indicate that it is not necessary to use doses of LAAM which can affect drug metabolizing enzymes in dams and increase pup mortality to maintain opioid-type physical dependence.

Regulation of monooxygenation during development of chick embryo liver: role of cytochrome P-450 isozymes and NADPH.

The endoplasmic reticulum of the liver cell, supplied with molecular oxygen and NADPH, can monooxygenate a variety of lipid soluble compounds including steroids and many drugs. The essential enzymatic steps are carried out by cytochrome P-450 and NADPH cytochrome P-450 reductase. There are several molecular forms (isozymes) of cytochrome P-450 in liver, and it has been shown that their relative concentrations change during development and in response to phenobarbital. Because substrate specificities of the isozymes differ to some extent, changes in the isozyme pattern should alter the pattern of substrate utilization if the isozymes are controlling the rates of monooxygenation. We found that the rates of testosterone hydroxylation and the oxidative N-demethylations of benzphetamine, codeine, and ethylmorphine were equal in endoplasmic reticulum isolated from chick embryo liver on day 11 of egg incubation, and this equality was unaffected by age or phenobarbital. Thus, monooxygenation rates do not appear to be controlled by the isozymes of cytochrome P-450. A factor that might affect the pattern of steroid and drug utilization by endoplasmic reticulum may be the availability of reducing equivalents. Lowering the concentration of NADPH to one half-optimum values in our reaction mixtures (0.5 to 0.25 mM) diminished the monooxygenation rate of benzphetamine by 8%, of testosterone by 40%, and of codeine and ethylmorphine by 60%. These differential effects were independent of age and phenobarbital. Thus, the pattern of substrate utilization by the monooxygenase system in vivo is probably altered by changes in the tissue concentration of NADPH.