Stuart D. Sleight

Lipid peroxidation: A possible mechanism of cephaloridine-induced nephrotoxicity

Cephaloridine produces renal cortical injury, but the precise mechanism responsible for this nephrotoxicity remains unclear. Recently cephaloridine has been shown to deplete reduced glutathione (GSH) concentration selectively in renal cortex. Cephaloridine nephrotoxicity can be potentiated by diethyl maleate (a GSH depletor), but no glutathione conjugate can be detected. Thus, it was of interest to investigate further the mechanism of depletion of renal cortical GSH by cephaloridine. In the present study, cephaloridine markedly decreased GSH in rat and rabbit renal cortex while concomitantly increasing oxidized glutathione (GSSG). Furthermore, cephaloridine increased lipid peroxidation specifically in renal cortical cells. Conjugated diene formation (an index of lipid peroxidation) was increased in renal cortex but not in the liver shortly following administration of cephaloridine. Removal of selenium and/or vitamin E from the diet, which should enhance lipid peroxidation, potentiated cephaloridine nephrotoxicity and enhanced cephaloridine-induced morphological damage in the kidney. These findings are consistent with a major role of lipid peroxidation in the etiology of cephaloridine nephrotoxicity.

Mechanism of chloroform nephrotoxicity. I. Time course of chloroform toxicity in male and female mice.

Chloroform (CHCl3) nephrotoxicity in male mice could be detected as early as 2 hr after CHCl3 administration (250 microliter/kg, sc) as decreased ability of renal cortical slices to accumulate p-aminohippurate (PAH) and tetraethylammonium (TEA). The decrease was preceded and paralleled by a reduction of renal cortical nonprotein sulfhydryl (NPSH) concentration, an index of tissue reduced glutathione concentration. Histologic alterations were not observed until NPSH concentrations and PAH and TEA accumulation had reached the nadir, 5 hr after CHCl3 administration. Female mice exhibited no evidence of nephrotoxicity to CHCl3 even when the dose was increased to 1000 microliter/kg or when pretreated with diethyl maleate to reduce renal cortical NPSH concentrations prior to CHCl3 injection. The extent of hepatotoxicity was similar in male and female mice and decreases of hepatic NPSH concentrations also were detected by 1.5 hr after CHCl3 administration. The rapid response of the kidney to CHCl3 toxicity in male mice and the similarity of liver toxicity in both sexes suggests that nephrotoxicity occurs independently of hepatotoxicity. Furthermore, the ability to detect these early changes in vivo following CHCl3 administration may permit the development of an in vitro model to evaluate the mechanism of CHCl3 nephrotoxicity.

Induction of liver microsomal drug-metabolizing enzymes by 2,2′,4,4′,5,5′-hexabromobiphenyl

The major component of Firemaster, a mixture of polybrominated biphenyls (PBBs), is 2,2′,4,4′,5,5′-hexabromobiphenyl (HBB). HBB was isolated to greater than 99.9% purity, and its effects on tissue structure and liver microsomal drug-metabolizing enzymes were examined. Male rats were injected ip with 90 mg of HBB/kg and sacrified 1 to 14 days later. Ten tissues were examined microscopically; only liver revealed HBB-dependent lesions. Hepatic cells were swollen and vacuolated, predominantly in the midzonal region. Liver to body weight ratios were increased by a third, and microsomal protein was nearly tripled. NADPH-cytochrome P-450 reductase was induced, and cytochrome P-450 was increased more than 2.5-fold, with the spectral maximum remaining at 450 nm. Aminopyrine demethylation was doubled by HBB, while PBBs and phenobarbital (Pb) both tripled this activity. Induction of benzo[a]pyrene hydroxylation was similar to that caused by Pb, but the level was fivefold smaller than that obtained by 3-methylcholanthrene (MC) induction. PBBs induced this activity to half of the MC level. HBB induced epoxide hydratase more than threefold. While PBBs tripled UDP-glucuronyltransferase activity, HBB was a poor inducer. SDS-polyacrylamide gel electrophoresis of HBB-induced microsomes revealed heme and protein profiles similar to those of microsomes induced by Pb, but markedly different from those of control microsomes. While PBBs are a mixed-type inducer of microsomal drug-metabolizing enzymes, these results show that HBB is strictly a Pb-type inducer.

Effect of sex hormone status on chloroform nephrotoxicity and renal mixed function oxidases in mice

In mice, only males are susceptible to chloroform (CHCl3) nephrotoxicity and the susceptibility appears to be related to renal mixed function oxidase activity. There were sex-related differences of renal cytochrome P-450 and b5 concentrations and of ethoxycoumarin O-deethylase activity in mouse kidneys; in all cases activity was higher in males. Castration of male mice eliminated susceptibility to CHCl3 nephrotoxicity and reduced renal mixed function oxidases to concentrations observed in female mice. Treatment of male and female mice with testosterone increased the susceptibility to CHCl3 nephrotoxicity and increased renal mixed function oxidases to similar activities in both sexes. Previous data have suggested that CHCl3 is metabolized in situ by the kidney, possibly by a mechanism similar to that occurring in the liver. The data from this investigation are consistent with the concept that CHCl3 is metabolized by a cytochrome P-450-dependent mechanism in the kidney.

Effects of 2,2'-dibromobiphenyl and 2,2',3,4,4',5,5'-heptabromobiphenyl on liver microsomal drug metabolizing enzymes.

Polybrominated biphenyls (PBBs) cause a mixed-type induction of liver microsomal drug-metabolizing enzymes. We have shown that 2,2′4,4′,5,5′-hexabromobiphenyl (HBB 6 ), the major component (56%) of PBBs (Firemaster), is strictly a phenobarbital-type inducer, and are examining the effects of other purified PBB components on these hepatic enzymes Male rats were given one 90 mg/kg ip injection of either 2,2′-dibromobiphenyl (DBB) or of 2,2′,3,4,4′,5,5′-heptabromobiphenyl (HBB 7 ), a major (27%) congener, and were sacrificed 1–22 days later. Eleven tissues were examined microscopically: Only HBB 7 caused lesions which were confined to hepatocyte swelling and vacuolation. DBB had little if any effect on any parameter examined. In contrast, HBB 7 increased liver weights and strongly induced microsomal protein, NADPH-cytochrome P -450 reductase, cytochrome P -450, aminopyrine demethylation, and epoxide hydratase. These effects were apparent within several days after treatment, and were still pronounced at Day 22. HBB 7 caused only small increases in benzo[ α ]pyrene hydroxylation and p -nitrophenol-UDP-glucuronyltransferase and failed to shift the cytochrome P -450 spectral maximum from 450 nm. These results, and the results of sodium dodecylsulfatepolyacrylamide gel electrophoresis, indicate that HBB 7 , like HBB 6 , is strictly a phenobarbital-type inducer of hepatic microsomal drug-metabolizing enzymes, and that all brominated biphenyls are not inducers. Seventeen percent by weight of Firemaster remains uncharacterized; one or more of these components is responsible for the 3-methylcholanthrene-like aspects of the mixed-type induction caused by the PBB mixture.

Effects of aminoglycosides on glomerular permeability, tubular reabsorption, and intracellular catabolism of the cationic low-molecular-weight protein lysozyme.

Gentamicin and other aminoglycoside antibiotics in high doses may produce proteinuria and other signs of nephrotoxicity. Proteinuria may result from general renal damage or may reflect alterations in specific steps in the renal handling of proteins. To distinguish between these two possibilities, experiments were designed to quantify the effects of nephrotoxic doses of several aminoglycosides on the renal handling of proteins in the isolated perfused rat kidney with the cationic low-molecular-weight protein lysozyme as a representative protein. Each aminoglycoside was administered ip to male Wistar rats (30 mg/kg/day) for 7 days. Lysozyme and 125I-lysozyme were added to the perfusate to achieve a lysozyme perfusate concentration of about 100 mg/liter. Clearances of inulin and lysozyme, release of tyrosine and trichloroacetic acid-soluble radioactive metabolites into the perfusate, and the glomerular sieving coefficient of lysozyme were determined. Scanning and transmission electron microscopy indicated that gentamicin and tobramycin decreased the number and diameter of the endothelial fenestrae of the glomerular capillaries. Concurrently, gentamicin and tobramycin decreased the glomerular sieving coefficient of lysozyme from 0.8 to 0.6 and 0.5, respectively. Netilmicin did not affect the percentage reabsorption of lysozyme whereas gentamicin and tobramycin decreased lysozyme reabsorption from 71.7 to 35.4 and 34.4% of the filtered load, respectively. Lysozyme degradation, estimated by the release of tyrosine into the perfusate during a 150-min perfusion period, was decreased from a control value of 12 mumol/liter to 4.43 and 4.65 mumol/liter in kidneys from rats treated with gentamicin and tobramycin, respectively. This study demonstrates that polycationic aminoglycosides may affect several processes involved in renal handling of lysozyme including glomerular permeability, tubular reabsorption, and intracellular proteolytic degradation.

Acute pathologic effects of 3,3′,4,4′,5,5′-hexabromobiphenyl in rats: Comparison of its effects with Firemaster BP-6 and 2,2′,4,4′,5,5′-hexabromobiphenyl☆☆☆

Abstract Male Sprague-Dawley rats were fed diets containing 0, 0.1, 1, 10, or 100 ppm of 3,3′,4,4′,5,5′-hexabromobiphenyl (HBB), 2,2′,4,4′,5,5′-HBB, or Firemaster (FM) BP-6, a commercial mixture of polybrominated biphenyls, for 9 days. Although 3,3′,4,4′,5,5′-HBB is not in FM BP-6, it was used because it is a 3-methylcholanthrene (MC)-type inducer of hepatic drug-metabolizing enzymes. Nearly one-half of FM BP-6 is comprised of 2,2′,4,4′,5,5′-HBB and this congener is strictly a phenobarbital (PB)-type inducer. FM BP-6 has both MC- and PB-type induction capability. Feed consumption and body and organ weights were recorded and histologic and ultrastructural changes were evaluated. Significant effects on feed intake and body weight occurred only at 10 or 100 ppm of 3,3′,4,4′,5,5′-HBB. Therefore, two of six rats given 100 ppm of 3,3′,4,4′,5,5′-HBB were continued on the diet until death occurred at 20 days. Liver weights were increased by each of the three chemicals at 10 and 100 ppm. Hepatocytes were diffusely enlarged and contained lipid vacuoles. The degree of vacuolation was dose related, most prominent in the centrolobular to midzonal area, and most severe in rats given 3,3′,4,4′,5,5′-HBB. Thymic and splenic weights were decreased at 10 and 100 ppm of 3,3′,4,4′,5,5′-HBB and lymphocytic depletion was severe in the thymus. Ultrastructural hepatic lesions were seen with all three chemicals. For 2,2′,4,4′,5,5′-HBB and FM BP-6 at 10 and 100 ppm the changes consisted mainly of increased smooth endoplasmic reticulum and lipid vacuolation. Additional changes seen with 3,3′,4,4′,5,5′-HBB included disorganization of rough endoplasmic reticulum, myelin body formation, and bile ductule hyperplasia. Results indicated that 3,3′,4,4′,5,5′-HBB causes more severe pathologic effects than either FM BP-6 or 2,2′,4,4′,5,5′-HBB.

Acute and subchronic methylmercury toxicosis in the rat

Abstract A series of experiments was performed to ascertain the acute and subchronic effects of methylmercury dicyandiamide (MMD) in the rat. The 7 day LD50 of 1.335 mg/100 g of MMD was used for the acute study. Weekly sublethal doses of 1.0 mg/100 g were used in the subchronic study. In the acute experiment clinical signs were nonspecific and could not be attributed directly to methylmercury toxicosis. Deaths occurred within 72–78 hr after inoculation. In the subchronic study some rats bumped into objects and had difficulty in walking, indicative of a nervous system disorder. Emaciation and/or loss of weight were constant findings in rats that died in both experiments. Neuronal degeneration occurred in all levels of the cerebrum, hippocampus and cervical spinal cord in rats from the acute and subchronic experiments. Fibrinoid necrosis of the capillary walls, a heretofore unreported lesion in the rat, was also seen. The neural and vascular lesions apparently were related, suggesting that the vascular lesions were responsible for the neuronal changes. Demyelination was observed in the sciatic nerve of rats killed during the first week of the acute experiment and in rats examined throughout the subchronic experiment. Atrophy of the granular cells of the cerebellum and hepatocytes was present only in rats in the subchronic study. A correlation between the concentration of mercury in the tissues and the presence and severity of lesions could not be made.

Liver microsomal enzyme induction and toxicity studies with 2,4,5,3',4'-pentabromobiphenyl.

Abstract A relatively minor component of Firemaster, 2,4,5,3′,4′-pentabromobiphenyl (PenBB), was purified and compared to the effects of Firemaster and 3,4,5,3′,4′,5′-hexabromobiphenyl (HBB). Male rats were given 90 mg/kg, ip, of PenBB or Firemaster and killed 2 weeks later. Blood samples were clinically analyzed and various tissues were examined for pathological effects. Induction of hepatic microsomal drug metabolizing enzymes and effects on the immune system were also investigated. No hematologic changes could be detected 2 weeks after either treatment. PenBB caused a significant loss in body weight, and the spleen weight to body weight and thymus weight to body weight ratios were slightly decreased by this treatment. Liver weight to body weight ratios were increased by both treatments. Ultrastructural changes in hepatocytes included proliferation of the smooth endoplasmic reticulum and the appearance of lipid droplets and concentric membrane whorls. The splenic T helper and B cells were substantially impaired by Firemaster and particularly by PenBB. There was a marked increase in liver microsomal cytochrome P-450 content and its CO difference spectrum absorption maximum was shifted to 448.5 nm. The microsomal ethyl isocyanide difference spectrum 455 430 -nm ratio was also increased. Benzo[ a ]pyrene hydroxylase, p -nitrophenol UDP-glucuronyltransferase, and epoxide hydrolase activities were markedly increased. Less pronounced induction of NADPH-cytochrome P-450 reductase, and aminopyrine- N -demethylase activities occurred. These results, in addition to the results of SDS-polyacrylamide gel electrophoresis suggest that the mono- ortho -brominated congener is predominantly a 3-methylcholanthrene (MC)-like inducer and is toxic. In another experiment when enzyme induction by PenBB (30 mg/kg) was compared to that caused by 30 or 2 mg/kg of HBB it was evident that the non- ortho -substituted congener is the more potent MC-like inducer. Therefore, we conclude that the single ortho halogen of PenBB may weaken but not abolish its toxicity and MC-like enzyme induction effects.

Vitamin A status, polybrominated biphenyl (PBB) Toxicosis, and common bile duct hyperplasia in rats☆☆☆

The interaction between dietary concentrations of vitamin A and PBB was evaluated in two experiments. In the first experiment, male weanling rats were used in a 2 X 3 factorial experiment. Concentrations of PBB were 0, 10, or 100 mg/kg of diet and diets were either vitamin A-deficient or were supplemented with 3000 IU vitamin A palmitate/kg of diet. In the second experiment the design was similar except that two vitamin A-supplemented diets were used, one containing 3,000 IU/vitamin A palmitate/kg of diet and the other 30,000 IU. Diets contained either 0 or 100 mg of PBB/kg. Clinical signs of vitamin A deficiency and mortality occurred early in rats fed vitamin A-deficient diets containing 100 mg of PBB/kg. Vitamin A supplementation provided partial protection against decreased weight gain associated with PBB. Decreases in thymic weight associated with PBB toxicosis were prevented by supplementation with vitamin A. Massive enlargement of the common bile duct occurred in rats fed a vitamin A-deficient diet containing 100 mg of PBB/kg. Histologically, this lesion consisted of extensive hyperplasia. A significant decrease in retinol concentrations in the sera was recorded in rats fed vitamin A-deficient diets containing 100 mg of PBB/kg. Interaction between vitamin A deficiency and PBB toxicosis affected vitamin A metabolism as manifested by the appearance of appreciable amounts of retinyl acetate in the liver vitamin A profile. These results suggest an interaction between PBB toxicity and vitamin A and emphasize the importance of nutritional factors such as vitamin A in assessment of PBB toxicosis.