Alpha 2u-globulin nephropathy and carcinogenicity following exposure to decalin (decahydronaphthalene) in F344/N rats.

Decalin (decahydronaphthalene) is a widely used industrial solvent known to cause male rat-specific alpha2u-globulin nephropathy. In this project, 13-week and two-year inhalation studies of decalin were conducted consecutively in both sexes of F344/N rats. The key objectives were to (1) characterize the 13-week toxicity of decalin in rats, with an emphasis on nephropathy in males; (2) compare the kidney concentrations of decalin, 2-decalone, and alpha2u-globulin in males over 2 to 13 weeks of decalin exposure; and (3) correlate male rat nephropathy observed in the 13-week study with renal carcinogenicity in the two-year study. F344 rats (M/F) were exposed via whole-body inhalation to 0, 25, 50, 100, 200, or 400 ppm decalin for 13 weeks. Urine was collected at weeks 2 and 6 for creatinine and decalol analyses and at week 12 for clinical urinalysis. Right kidneys were collected from male rats at weeks 2 and 6 and from both sexes at week 13, homogenates were prepared using the whole kidney, and these homogenates were analyzed for alpha2u-globulin, decalin, and 2-decalone. Left kidneys were evaluated for histopathology and cell proliferation utilizing a proliferating cell nuclear antigen technique and counting proximal renal tubular epithelial cells to determine cell labeling indices. Necropsies and histopathologic evaluations were performed at week 13. Decalin exposure caused increases in kidney weight, urinalysis parameters (protein, AST, LDH), kidney alpha2u-globulin concentration, and proximal convoluted renal tubular cell proliferation in males. These changes were accompanied by microscopic lesions (accumulation of hyaline droplets in cortical tubules, regeneration of proximal tubular epithelium, and granular casts in medullary tubules) clearly linked to alpha2u-globulin nephropathy. Both decalin and 2-decalone were related to increased alpha2u-globulin in male kidneys. Kidney concentrations of decalin, 2-decalone, and alpha2u-globulin in exposed females were negligible, while females excreted greater amounts of decalol metabolites in urine than males at weeks 2 and 6. There were no exposure-related microscopic lesions in females. For chronic exposure, F344 rats were exposed via whole-body inhalation to 0, 25, 50 (males only), 100, or 400 ppm decalin for two years. Chronic exposure induced a spectrum of nonneoplastic and neoplastic lesions in the renal cortex of males, ranging from regenerative lesions of chronic nephropathy to tubular carcinomas. Incidences of renal tubular adenoma, tubular carcinoma, combined tubular adenomas and carcinomas, cortical tubular hyperplasia, hyaline droplet accumulation, hyperplasia of pelvic epithelium, and mineralization in renal papilla were increased in exposed males compared to controls. There was a clear increase in the mean severity of chronic nephropathy in decalin-exposed males. It was concluded that the carcinogenic effect on the renal cortical epithelium of male rats exposed to decalin was related to increased turnover of this epithelium, resulting from the cytotoxic effects of alpha2u-globulin accumulation in the renal cortical tubular cell cytoplasm.     

Metabolism of nephrotoxic cis- and trans-decalin in Fischer-344 rats

The cis- and trans-decalin stereoisomers have found many uses as solvents and fuel components. The metabolism of the decalin isomers in male and female Fischer-344 rats and the effects of the decalins on renal damage were evaluated. Only male rats had kidney damage. Metabolism of cis-decalin yielded cis,trans-1-decalol and cis,cis-2-decalol in the urine of both sexes, with the male also producing cis,cis-1-decalol. The urinary metabolites of trans-decalin included trans,cis-2-decalol in both male and female rats and trans,trans-1-decalol in males. Extracts of kidney homogenates from male rats, but not from females, dosed with cis- and trans-decalin yielded cis-2-decalone and trans-2-decalone, respectively. A single male rat treated with trans-decalin produced no 2-decalone in the kidney extract and also showed no renal damage.     

NTP toxicology and carcinogenesis studies of decalin (CAS No. 91-17-8) in F344/N rats and B6C3F(1) mice and a toxicology study of decalin in male NBR rats (inhalation studies)

Decalin is used as an industrial solvent for naphthalene, fats, resins, oils, and waxes. It is also used as a substitute for turpentine in lacquers, paints, and varnishes; as a solvent and stabilizer for shoe polishes and floor waxes; and as a constituent of motor fuels and lubricants. Other applications include use as a paint thinner and remover, a patent fuel in stoves, a high-density fuel in submarine-launched cruise missile systems, and in stain removal and cleaning machinery. Decalin was nominated for study by the National Cancer Institute because of its chemical structure, its potential for consumer exposure, and a lack of adequate testing of the chemical. Male and female F344/N rats and B6C3F(1) mice were exposed to decalin (greater than 99% pure) by inhalation for 2 weeks, 3 months, or 2 years. Groups of male NBR rats were exposed to decalin for 2 weeks. Male NBR rats do not produce alpha2u-globulin; the NBR rats were included to study the relationship of alpha2u-globulin and renal lesion induction. Genetic toxicology studies were conducted in Salmonella typhimurium and mouse peripheral blood erythrocytes. 2-WEEK STUDIES IN RATS: Groups of five male and five female F344/N rats and five male NBR rats were exposed to 0, 25, 50, 100, 200, or 400 ppm decalin vapor 6 hours per day, 5 days per week for 16 days. All rats survived to the end of the study, and mean body weights of exposed groups were similar to those of the chamber controls. Renal toxicity studies were performed in male F344/N and NBR rats. The numbers of labeled cells and the labeling indices in the left kidney of 200 and 400 ppm F344/N male rats were significantly greater than those in the chamber controls. The alpha2u-globulin/soluble protein ratios were significantly increased in all exposed groups of F344/N rats. Liver weights of male F344/N and NBR rats exposed to 100 ppm or greater were significantly increased, as were those of all exposed groups of females. Kidney weights of male F344/N rats exposed to 50 ppm or greater were significantly increased. Exposure-related hyaline droplet accumulation, degeneration and regeneration of renal cortical tubules, and granular casts occurred in the kidney of exposed F344/N male rats. 2-WEEK STUDIES IN MICE: Groups of five male and five female B6C3F(1) mice were exposed to 0, 25, 50, 100, 200, or 400 ppm decalin vapor 6 hours per day, 5 days per week for 17 days. All mice survived to the end of the study, and mean body weights of exposed groups were similar to those of the chamber control groups. Liver weights of 200 and 400 ppm males and females and 100 ppm females were significantly increased. 3-MONTH STUDY IN RATS: Groups of 25 male and 20 female F344/N rats were exposed to 0, 25, 50, 100, 200, or 400 ppm decalin vapor 6 hours per day, 5 days per week for 2 (five male renal toxicity rats), 6 (10 male and 10 female clinical pathology rats), or 14 (10 core study rats) weeks. All rats survived to the end of the study, and mean body weights of exposed groups were similar to those of the chamber control groups. Urinalysis results indicated that decalin exposure caused increases in urine glucose and protein concentrations and enzyme activities that were consistent with the renal lesions observed microscopically. Renal toxicity studies were performed on rats sacrificed at 2 and 6 weeks and at the end of the study. In kidney tissue examined for cell proliferation, the numbers of PCNA-labeled cells and labeling indices were generally significantly greater than those of the chamber controls in exposed groups of rats at all three time points. Concentrations of alpha2u-globulin in the kidney as well as the alpha2u-globulin/soluble protein ratios were significantly increased at week 2 in all exposed groups and in the 200 and 400 ppm groups at week 6 and at the end of the study. Absolute and/or relative kidney and liver weights of male rats exposed to 50 ppm or greater were increased. Incidences of renal tubule regeneration and granular casts in the medulla of the kidney in exposed male rats were increased, and the severities of hyaline droplets generally increased with increasing exposure concentration. 3-MONTH STUDY IN MICE: Groups of 10 male and 10 female B6C3F(1) mice were exposed to 0, 25, 50, 100, 200, or 400 ppm decalin vapor 6 hours per day, 5 days per week for 14 weeks. All mice survived to the end of the study, and mean body weights of exposed groups were similar to those of the chamber control groups. Liver weights of 200 and 400 ppm males and females were significantly increased. There was a significant exposure concentration-related decrease in the absolute spermatid head count and a significant decrease in absolute head count of the 400 ppm group compared to the chamber controls. Incidences of centrilobular cytomegaly of the liver were increased in exposed male mice. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female F344/N rats were exposed to 0, 25, 50 (male rats only), 100, or 400 ppm (female rats only) decalin vapor 6 hours per day, 5 days per week for 105 weeks. A group of 20 male rats was exposed to 400 ppm. Survival of exposed groups was similar to that of the chamber control groups. Mean body weights of 400 ppm males were slightly less than those of the chamber controls during the second year of the study. Incidences of renal tubule adenoma and adenoma or carcinoma (combined) and of benign or malignant pheochromocytoma (combined) of the adrenal medulla in 100 and 400 ppm males were significantly increased. There was a significant association between nephropathy severity and adrenal pheochromocytoma incidence. Nonneoplastic lesions related to decalin exposure occurred in the kidney of male rats. 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female B6C3F(1) mice were exposed to 0, 25, 100, or 400 ppm decalin vapor 6 hours per day, 5 days per week for 105 weeks. Survival of exposed mice was similar to that of the chamber controls. Mean body weights of exposed groups were generally similar to those of the chamber control groups throughout the study. Increased incidences of hepatocellular neoplasms occurred in 25 and 400 ppm female mice, and the incidences of centrilobular hypertrophy, necrosis, syncytial alteration, and erythrophagocytosis of the liver in 400 ppm males were significantly increased. The incidences of uterine stromal polyp and stromal polyp or stromal sarcoma (combined) occurred with positive trends in female mice. PHARMACOKINETIC MODEL: The rate of metabolism of decalin was the same for males and females in rats and mice. Also in rats and mice, decalin metabolism was saturated at less than 400 ppm. Increased labeling indices in male rats were likely due to changes related to alpha2u-globulin. GENETIC TOXICOLOGY: Decalin was not mutagenic in S. typhimurium strains TA97, TA98, TA100, or TA1535, with or without induced hamster or rat liver S9 enzymes. A small but significant increase in the frequency of micronucleated normochromatic erythrocytes was noted in male mice exposed to decalin for 3 months; however, no induction of micronuclei was observed in female mice. CONCLUSIONS: Under the conditions of these studies, there was clear evidence of carcinogenic activity of decalin in male F344/N rats based on increased incidences of renal tubule neoplasms. The increased incidences of benign or malignant pheochromocytoma (combined) of the adrenal medulla in male rats were also considered to be exposure related. There was no evidence of carcinogenic activity of decalin in female F344/N rats exposed to 25, 100, or 400 ppm. There was no evidence of carcinogenic activity of decalin in male B6C3F(1) mice exposed to 25, 100, or 400 ppm. There was equivocal evidence of carcinogenic activity of decalin in female B6C3F(1) mice based on marginally increased incidences of hepatocellular and uterine neoplasms. Exposure of male rats to decalin resulted in nonneoplastic lesions of the kidney characteristic of alpha2u-globulin accumulation. Nonneoplastic lesions of the liver were observed in male mice exposed to decalin.     

alpha 2u-Globulin nephropathy, renal cell proliferation, and dosimetry of inhaled tert-butyl alcohol in male and female F-344 rats.

tert-Butyl alcohol (TBA) has been shown to cause kidney tumors in male rats following chronic administration in drinking water. The objective of the present study was to determine whether TBA induces alpha 2u-globulin (alpha 2u) nephropathy (alpha 2u-N) and enhanced renal cell proliferation in male, but not female, F-344 rats, and whether the dosimetry of TBA to the kidney is gender specific. Male and female F-344 rats were exposed to 0, 250, 450, or 1750 ppm TBA vapors 6 h/day for 10 consecutive days to assess alpha 2u-nephropathy and renal cell proliferation and for 1 and 8 days to evaluate the dosimetry of TBA following a single and repeated exposure scenario. Protein droplet accumulation was observed in kidneys of male rats exposed to 1750 ppm TBA, with alpha 2u-globulin immunoreactivity present in these protein droplets. A statistically significant increase in alpha 2u concentration in the kidney, as measured by an enzyme-linked immunosorbent assay, was observed in male rats exposed to 1750 ppm TBA with a exposure-related increase in renal cell proliferation. Renal alpha 2u concentration was positively correlated with cell proliferation in male rat kidney. No histological lesions or increased renal cell proliferation was observed in female rats exposed to TBA compared to controls. The TBA kidney:blood ratio was higher at all concentrations and time points in male rats compared with female rats, which suggests that TBA is retained longer in male rat kidney compared with female rat kidney. Together these data suggest that TBA causes alpha 2u-N in male rats, which is responsible for the male rat-specific increase in renal cell proliferation.     

Characterization of spontaneous and decalin-induced hyaline droplets in kidneys of adult male rats

Studies were conducted to gain additional information about the spontaneous and decalin-exacerbated formation of hyaline droplets within the cytoplasm of proximal convoluted tubule (PCT) epithelial cells of the adult male rat. Renal cortical tissue protein patterns determined through two-dimensional gel electrophoresis revealed four species of a low-molecular-weight protein (18,000-20,000 daltons). Treatment groups differed only with respect to this protein, the relative concentrations of which paralleled the numbers of hyaline droplets in mature treated and untreated male rats. The increase in the numbers of hyaline droplets and protein accumulation were dose related. Neither this protein or hyaline droplets were detected in the renal cortical tissues of untreated or decalin-exposed adult female or immature male control rats. However this protein, and hyaline droplet formation, could be induced in the kidneys of adult, ovariectomized female rats by repeated testosterone injections. This protein was then demonstrated to be immunologically identical to alpha 2u-globulin, a protein synthesized by hepatic parenchymal cells. Alpha 2u-globulin protein has also been shown to be the major urinary component responsible for the proteinuria routinely observed in normal control adult male rats. PCT epithelial cell reabsorption and lysosomal accumulation of alpha 2u-globulin, reflected morphologically as hyaline droplets, occurs spontaneously only in the mature male rat. Decalin, a model compound, exacerbates this accumulation as a specific integral step in the pathogenesis of the nephropathy induced in male rats by volatile hydrocarbons. Hence, since men and women lack this specific PCT cell peculiarity, they would not be expected to respond to decalin exposure in a manner similar to the male rat.     

Characterization of tert-butyl alcohol binding to alpha2u-globulin in F-344 rats.

tert-Butyl alcohol (TBA) is widely used in the manufacturing of certain perfumes, cosmetics, drugs, paint removers, methyl tert-butyl ether (MTBE), and industrial solvents. In both rodents and humans, TBA is a major metabolite of MTBE, an oxygenated fuel additive. Chronic TBA exposure causes protein droplet nephropathy, alpha2u-globulin (alpha2u) accumulation, renal cell proliferation, and with chronic exposure, renal tumors in male, but not female, rats. These effects suggest an alpha2u-mediated mechanism for renal tumors. The objective of the present study was to determine whether TBA or its metabolites bind to alpha2u. Mature male and female F-344 rats were administered a single gavage dose of 500 mg/kg TBA, 500 mg/kg (14)C-TBA, or corn oil. TBA equivalents/gram or ml of tissue in the male rat kidney, liver, and blood were higher than the levels measured in female rat tissue 12 h after (14)C-TBA administration. Gel filtration and anion-exchange chromatography demonstrated that (14)C-TBA-derived radioactivity co-eluted with alpha2u from male kidney cytosol. Protein dialysis studies demonstrated that the interaction between (14)C-TBA-derived radioactivity and alpha2u was reversible. Incubations of the low-molecular-weight protein fraction (LMWPF) isolated from (14)C-TBA-treated male rat kidneys with d-limonene oxide (a chemical with a high affinity to alpha2u) demonstrated that (14)C-TBA-derived radioactivity was displaced. Gas chromatography-mass spectrometry analysis confirmed that TBA was present in this LMWPF fraction. These results demonstrate that TBA interacts with alpha2u, which explains the accumulation of alpha2u in the male rat kidney following TBA exposure.     

Biotransformation and male rat-specific renal toxicity of diethyl ethyl- and dimethyl methylphosphonate.

Dimethyl methylphosphonate (DMMP) is a widely used chemical. Diethyl ethylphosphonate (DEEP) has been proposed as a replacement for DMMP in several applications. A long-term carcinogenesis study with DMMP in rats and mice showed a significant increase in the incidence of kidney tumors after 2 years of exposure in male but not in female rats and both sexes of mice. DMMP is not genotoxic. Due to these findings, a role of alpha(2u)-globulin accumulation in organ-specific tumorigenicity may be possible. alpha(2u)-Globulin is a low-molecular-weight protein synthesized in male rats under androgen control. Several male rat specific renal carcinogens have been shown to bind to alpha(2u)-globulin and to impair the renal degradation of this protein. This impairment results in alpha(2u)-globulin accumulation in the kidney, lysosomal overload, cell death, cell proliferation, and finally, renal tumor induction. To further characterize the toxicology of DMMP and DEEP, we investigated the biotransformation of these compounds and their ability to induce alpha(2u)-globulin accumulation in kidney. Biotransformation of both DMMP and DEEP were studied in male and female rats after single oral doses of 50 and 100 mg/kg. 31P-NMR and GC/MS showed that unchanged DMMP was excreted with urine; methyl phosphonate was identified as the only metabolite in urine. Unchanged DEEP was also excreted with urine; in addition, ethyl ethylphosphonate and ethylphosphonate were urinary metabolites. The majority of the applied dose of both compounds was recovered in urine within 24 h indicating rapid absorption and excretion. No sex-differences in rates of formation or excretion of metabolites were seen. To investigate alpha(2u)-globulin accumulation in the kidney after DMMP and DEEP, male and female Fischer-344 rats were administered DMMP or DEEP daily for five consecutive days by gavage. DMMP doses were 500- and 1,000-mg/kg body weight (bw); due to marked toxicity, daily DEEP dose of 50 and 100 mg/kg had to be used. Control rats received corn oil only and positive controls received five doses of 500-mg/kg bw trimethylpentane (TMP). Relative kidney weights were increased in male rats dosed with DMMP, DEEP, and TMP. alpha(2u)-Globulin in kidney cytosol was separated and quantified by capillary electrophoresis and by SDS-PAGE and Western blotting. In DMMP-, DEEP-, and TMP-treated rats, dose-dependent increases in the alpha(2u)-globulin content were observed by both methods in male, but not female rats. The increase of alpha(2u)-globulin accumulation was accompanied by the formation of protein droplets in the proximal tubules of male rats. These data demonstrate that the sex specific increase in kidney tumors by DMMP in male rats may be due to alpha(2u)-globulin accumulation and that similar toxic effects are to be expected from DEEP.     

Behavior of alpha 2u-globulin accumulating in kidneys of male rats treated with d-limonene: kidney-type alpha 2u-globulin in the urine as a marker of d-limonene nephropathy.

Effects of d-limonene on alpha 2u-globulin in the kidneys, urine and serum were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis. Treatment of male rats with d-limonene by gavage for 14 consecutive days (300 mg/kg/day) caused accumulation of hyaline droplets in renal proximal tubule cells, and a marked intensification of a protein band corresponding to the kidney-type alpha 2u-globulin, with a molecular weight calculated to be approximately 16 kDa. However, no significant changes in the serum alpha 2u-globulin (native-type) band, of approximately 19 kDa, were observed between treated rats and controls, suggesting that circulating alpha 2u-globulin levels were not affected by the d-limonene administration. While the molecular weight of the major alpha 2u-globulin in the urine from control rats was the same as that in the serum (native-type), marked increase in the protein band corresponding to kidney-type-alpha 2u-globulin was observed in the urine from treated rats. The results were indicative of elimination of alpha 2u-globulin from the kidney to urine, the appearance of kidney-type-alpha 2u-globulin in urine implying disruption or exfoliation of proximal tubule cells. Therefore, it is suggested that the presence of the alpha 2u-globulin (kidney-type) in the urine might be used as an indicator of chemically induced alpha 2u-globulin nephropathy.     

Assessment of the covalent binding potential of 2,2,4-trimethylpentane to rat alpha 2u-globulin

Subchronic exposure of male rats to the nephrotoxin 2,2,4-trimethylpentane (TMP) causes an accumulation of protein droplets in the epithelial cells of the renal cortex. Experimental evidence suggests that these droplets contain alpha 2u-globulin, a low-molecular-weight protein found specifically in the urine of male rats. It has been proposed that aldehyde metabolites of TMP form Schiff base adducts with the lysine groups of alpha 2u-globulin and thereby inhibit renal lysosomal processing of the protein. Accordingly, the ability of TMP and its metabolites to covalently bind to alpha 2u-globulin was examined. As a model, a [14C] formaldehyde-alpha 2u-globulin Schiff base was formed. This protein adduct was stabilized by reduction with cyanoborohydride and could be identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein analysis by SDS-PAGE demonstrated that hepatocytes isolated from male Fischer-344 rats produced significant quantities of alpha 2u-globulin in culture, whereas hepatocytes from female rats did not. A 15-hr exposure of metabolically competent, primary cultures of male rat hepatocytes to [14C] TMP (0.1 and 0.5%, v/v), followed by reduction with cyanoborohydride, dialysis, and analysis with SDS-PAGE, revealed no evidence of radiolabeled alpha 2u-globulin. When [14C]TMP was administered to an adult male Fischer-344 rat (300 mg/kg, ig) 22, 16, and 10 hr before sacrifice, 16% of the administered radioactivity was eliminated in the urine as TMP metabolites. Analysis as above showed no TMP-derived radioactivity in fractions containing alpha 2u-globulin from liver, blood, kidney cortex, or urine. The absence of a detectable covalent interaction between TMP and alpha 2u-globulin following in vitro or in vivo exposure suggests that a TMP-alpha 2u-globulin adduct is not responsible for the excessive formation of protein droplets in the renal cortex of exposed male rats.     

d-Limonene-induced male rat-specific nephrotoxicity: evaluation of the association between d-limonene and alpha 2u-globulin

d-Limonene is a naturally occurring monoterpene, which when dosed orally, causes a male rat-specific nephrotoxicity manifested acutely as the exacerbation of protein droplets in proximal tubule cells. Experiments were conducted to examine the retention of [14C]d-limonene in male and female rat kidney, to determine whether d-limonene or one or more of its metabolites associates with the male rat-specific protein, alpha 2u-globulin, and if so, to identify the bound material. The results indicated that, 24 hr after oral administration of 3 mmol d-limonene/kg, the renal concentration of d-limonene equivalents was approximately 2.5 times higher in male rats than in female rats. Equilibrium dialysis in the presence or absence of sodium dodecyl sulfate indicated that approximately 40% of the d-limonene equivalents in male rat kidney associated with proteins in a reversible manner, whereas no significant association was observed between d-limonene equivalents and female rat kidney proteins. Association between d-limonene and male rat kidney proteins was characterized by high-performance gel filtration and reverse-phase chromatography. Gel filtration HPLC indicated that d-limonene in male rat kidney is associated with a protein fraction having a molecular weight of approximately 20,000. Separation of alpha 2u-globulin from other kidney proteins by reverse-phase HPLC indicated that d-limonene associated with a protein present only in male rat kidney which was definitively identified as alpha 2u-globulin by amino acid sequencing. The major metabolite associated with alpha 2u-globulin was d-limonene-1,2-oxide. Parent d-limonene was also identified as a minor component in the alpha 2u-globulin fraction. Thus, d-limonene, and more specifically d-limonene-1,2-oxide, associates with alpha 2u-globulin in a reversible manner in male rat kidney. This interaction may be responsible for excessive accumulation of alpha 2u-globulin in kidneys of male rats exposed to d-limonene.     

Comparison of short-term renal effects due to oral administration of decalin or d-limonene in young adult male Fischer-344 rats

Groups of young adult male Fischer-344 rats given the vehicle (corn oil) or either decalin or d-limonene at dose levels of 75, 150 or 300 mg/kg body weight by a single daily gavage on 5 days/wk were killed on study days 6 or 27, approximately 24 hr after the fifth or 20th dose, to determine whether the specific time- and dose-related triad of renal alterations characterizing decalin-associated nephrotoxicity in the adult male rat also occurs in response to d-limonene. Dose-related hyaline droplet formation associated with renal accumulation of a specific protein alpha 2u-globulin) is considered the primary response in the morphogenesis of decalin-induced nephrotoxicity in the male rat and was present to a maximal degree in all decalin- and d-limonene-treated groups by day 6. Alterations considered to be sequelae of the hyaline droplet response, including granular casts in the outer zone of the medulla and multiple cortical changes collectively classified as chronic nephrosis, were present in the kidneys of both decalin- and d-limonene-treated rats killed on day 27. These findings demonstrate a uniformity of primary and secondary renal responses to the two chemicals, strongly suggesting that the morphogenesis of d-limonene-associated nephrotoxicity in the adult male rat is consistent with that of decalin. The response of the male rat kidney to decalin treatment has been shown to be uniquely different, by virtue of anatomical, physiological and biochemical peculiarities involving the proximal convoluted tubule, from that in female rats and higher mammalian species.     

Differences in alpha 2u-globulins increased in male rat kidneys following treatment with several alpha 2u-globulin accumulating agents: cystein protease(s) play(s) an important role in production of kidney-type-alpha 2u-globulin.

Effects of alpha 2u-globulin accumulating agents on alpha 2u-globulins in rat kidneys were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis. Treatment of male animals with decalin (150 mg/kg), 2,2,4-trimethylpentane (50 mg/kg), isophorone (150 mg/kg), d-limonene (150 mg/kg) or 1,4-dichlorobenzene (150 mg/kg) by gavage for 14 consecutive days in each case resulted in a marked intensification of a protein band corresponding to kidney-type-alpha 2u-globulin, with a molecular mass calculated to be approximately 16 kDa. However, intraperitoneal treatment with leupeptin and E-64 (two times 0.07 mmol/kg, for each), well known cystein protease inhibitors, while only slightly increasing this kidney-type-alpha 2u-globulin band, caused the intensification of a approximately 19-kDa molecular mass protein band which was revealed to be a native-type-alpha 2u-globulin by SDS-PAGE and immunoblotting. These results indicated that at least two types of alpha 2u-globulin can be increased in male rat kidney by chemical treatment. Moreover, cystein protease(s) appear(s) to play an important role in the degradation of alpha 2u-globulin and particularly in the conversion of native-type-alpha 2u-globulin to kidney-type-alpha 2u-globulin in rat kidneys.     

Hyaline droplet nephropathy resulting from exposure to 3,5,5-trimethylhexanoyloxybenzene sulfonate.

Acute oral dosing of 3,5,5-trimethylhexanoyloxybenzene sulfonate (THBS) to adult male and female rats causes a male rat-specific nephrotoxicity manifested as exacerbation of hyaline droplet formation. This chemical is structurally distinct from the volatile hydrocarbons known to cause male rat-specific kidney lesions. Therefore, to classify THBS as a hyaline droplet-inducing agent, experiments were conducted to determine whether [14C]THBS equivalents bound to alpha 2 mu-globulin and caused the protein to accumulate in male rat kidney cortex. Two-dimensional gel electrophoretic separation of male rat kidney proteins indicated that alpha 2u-globulin levels in kidney increased 24 hr after a single oral dose of THBS (500 mg/kg). Furthermore, a sex-dependent retention THBS was noted as there was approximately 10 times more THBS equivalents in male rat kidney than in female rat kidney. Equilibrium dialysis experiments indicated that 40% of THBS equivalents bound reversibly to male rat kidney proteins, whereas no interaction between THBS and female rat kidney proteins was detected. Specific binding of THBS to alpha 2mu-globulin was determined by anion-exchange HPLC after which metabolites in the alpha 2u-globulin fraction were identified by gas chromatography with parallel radioactivity-mass spectrometry and mass spectrometry-matrix isolation Fourier-transform infrared analysis. Four metabolites of THBS were found in this protein fraction, and the major component (approximately 70%) was identified as the cis gamma-lactone of 3,5,5-trimethylhexanoic acid. Experiments were also conducted in mice to determine whether THBS bound to any mouse kidney proteins, particularly mouse urinary protein. The results indicated that there was no interaction between THBS and mouse urinary protein, a protein which shares significant homology with alpha 2u-globulin. These results indicate that THBS treatment exacerbates hyaline droplet formation in male rat kidneys by binding to alpha 2mu-globulin, thereby causing the protein to accumulate in the renal cortex. The interaction between THBS and alpha 2mu-globulin appears to be unique to this male rat-specific protein as THBS does not interact with a very similar protein found in mice.     

Rapid postexposure decay of alpha 2u-globulin and hyaline droplets in the kidneys of gasoline-treated male rats

Unleaded gasoline induces nephropathy, characterized by rapid accumulation of hyaline (protein resorption) droplets in epithelial cells of the renal proximal convoluted tubules, only in male rats. The hepatic synthesis of the male rat-specific protein alpha 2u-globulin, a constituent of renal hyaline droplets, is unaltered by gasoline treatment (Olson et al., 1987). Renal alpha 2u-globulin content increased to 210% of control within 18 h of a single oral dose of gasoline (2.0 ml/kg); maximal levels (320% of control) were attained following gasoline administration for 3 d. Increases in renal alpha 2u-globulin caused by gasoline were accompanied by concurrent proliferation of hyaline droplets. However, within 3 d of terminating gasoline administration renal alpha 2u-globulin content decreased to the same level as that in unexposed rats, although renal hyaline droplet number returned to pretreatment levels somewhat more slowly. The conjoint effect of postexposure recovery and estradiol (an inhibitor of hepatic alpha 2u-globulin synthesis) administration was also determined in male rats. On postexposure d 3, 6, and 9, estradiol treatment (1 mg/kg, sc, 4 d, starting on d 9 of gasoline treatment) decreased renal alpha 2u-globulin content to 75%, 59%, and 48%, respectively, of that in rats allowed to recover from gasoline with no hormone treatment. Hepatic alpha 2u-globulin content in estradiol-treated rats was decreased by 74%, 97%, and 96% at the same intervals. Estradiol treatment during recovery from gasoline also appeared to increase the removal of accumulated hyaline droplets from the renal cortex. Thus, accumulation of alpha 2u-globulin-containing hyaline droplets after subacute exposure of male rats to gasoline is rapidly reversible, dependent on continuous exposure to gasoline and maintenance of the normal rate of hepatic alpha 2u-globulin synthesis. These results emphasize the dynamic state of renal cortical hyaline droplets and suggest strongly that gasoline hydrocarbons cause hyaline droplet accumulation by prolonging the half-time of degradation of alpha 2u-globulin.     

The induction of omega and beta-oxidation of fatty acids and effect on alpha 2u globulin content in the liver and kidney of rats administered 2,2,4-trimethylpentane

The effect of daily administration of 12 mmol/kg 2,2,4-trimethylpentane for 10 d on hepatic and renal microsomal mono-oxygenase activity, peroxisomal beta-oxidation and the concentration of alpha 2u-globulin has been examined in male and female rats. 2,2,4-Trimethylpentane produces liver and, to a lesser extent, kidney enlargement. This is associated with the selective induction of cytochrome P-450-mediated omega-oxidation and peroxisomal beta-oxidation of fatty acids and proliferation of peroxisomes. Male rats show a more marked response than female rats. 2,2,4-Trimethylpentane produces an increase in alpha 2u-globulin in the kidney of male rats. The relevance of selective induction of omega- and beta-oxidation of fatty acids and accumulation of alpha 2u-globulin to renal tubular necrosis in male rats requires further study.     

2,2,4-Trimethylpentane-induced nephrotoxicity. I. Metabolic disposition of TMP in male and female Fischer 344 rats

2,2,4-Trimethylpentane (TMP), a component of unleaded gasoline, causes nephrotoxicity in male, but not in female, rats. In the present study, male and female Fischer 344 rats were treated with a single oral dose of [14C]TMP (4.4 mmol/kg; 2 microCi/mmol). Radiolabeled material in kidney, liver, and plasma was determined at 4, 8, 12, 24, and 48 hr after dosing. Maximum concentration of TMP-derived radioactivity in kidney, liver, and plasma of male rats was found after 12 hr (1252, 1000, and 403 nmol eq/g, respectively), whereas those measured in females were found after 8 hr (577, 1163, and 317 nmol eq/g, respectively). A selective retention of the TMP-derived radiolabel in the kidneys of male rats was noted when peak tissue concentration was expressed as a percentage of administered dose. Kidney concentrations of TMP-derived radiolabel increased in a nonlinear, but dose-dependent, manner; the kidney to plasma ratio was greater at low doses than at higher doses. Increased retention of radiolabel material in the kidney was associated with a significant increase in renal concentration of the male-rat-specific protein, alpha 2u-globulin, 24 and 48 hr after TMP administration. Total radioactivity collected in urine 48 hr after TMP administration was similar in males and females (32 and 31% of dose). Identification and quantitation of the urinary metabolites of TMP showed that both male and female rats metabolize TMP via the same pathway and at a similar rate. Female rats, however, excreted more conjugates of 2,4,4-trimethyl-2-pentanol in urine than males. 2,4,4-Trimethyl-2-pentanol was the major metabolite present in the male rat kidney, but was absent in the female rat kidney. The renal retention of 2,4,4-trimethyl-2-pentanol appears to account for the delayed clearance observed in the disposition of [14C]TMP-derived radiolabel. Based on the concomitant accumulations in renal alpha 2u-globulin concentration and renal 2,4,4-trimethyl-2-pentanol concentration, an association is speculated between these two components. The male-rat-specific accumulation of 2,4,4-trimethyl-2-pentanol may therefore reflect the accumulation of a "metabolite-alpha 2u-globulin" complex. This may be relevant to the male-rat-specific nephrotoxicity produced by TMP.     

Leupeptin-mediated alteration of renal phagolysosomes: similarity to hyaline droplet nephropathy of male rats exposed to unleaded gasoline

alpha 2u-Globulin, a protein of hepatic origin found in the urine of male rats, is accumulated in the kidney cortex during exposure to unleaded gasoline and has been implicated in the development of fuel hydrocarbon-induced nephropathy and renal neoplasia. The principal morphological feature of gasoline-induced nephropathy is accumulation of hyaline droplets (enlarged secondary lysosomes or phagolysosomes) in epithelial cells of the proximal convoluted tubule S1 and S2 segments. Inhibition of cathepsin B (a major lysosomal peptidase) by treatment of male rats with leupeptin causes rapid accumulation of phagolysosomes and alpha 2u-globulin in the kidney very similar to gasoline exposure. Further, the renal cortical subcellular distribution of alpha 2u-globulin, determined with an electron microscopic immunochemical method, is almost totally confined to phagolysosomes following administration of either gasoline or leupeptin. These results, taken together, indicate that the mechanism of nephrotoxicity of gasoline involves inhibition of renal phagolysosomal proteolysis.     

Evaluation of potential modes of action of inhaled ethylbenzene in rats and mice.

Potential factors underlying the tumorigenic activity of ethylbenzene (EB) were examined in F344 rats and B6C3F1 mice inhaling 750 ppm EB vapor 6 h/day, 5 days/week, for one or four weeks. Target tissues (kidneys of rats and livers and lungs of mice) were evaluated for changes in organ weights, mixed function oxygenases (MFO), glucuronosyl transferase activities, S-phase DNA synthesis, apoptosis, alpha2u-globulin deposition, and histopathology. In male rats, kidney weight increases were accompanied by focal increases in hyaline droplets, alpha2u-globulin, degeneration, and S-phase synthesis in proximal tubules. In female rats, only decreased S-phase synthesis and MFO activities occurred. In mice, increased liver weights were accompanied by hepatocellular hypertrophy, mitotic figures, S-phase synthesis, and enzyme activities. S-phase synthesis rates in terminal bronchiolar epithelium were elevated and accompanied by loss of MFO activity. Exposure to a nontumorigenic level of 75 ppm for one week caused few changes. These data, considered with the general lack of EB genotoxicity, suggest a mode of tumorigenesis dependent upon increased cell proliferation and altered population dynamics in male rat kidney and mouse liver and lungs. A similar response in the kidneys of female rats appears to require a longer exposure period than was employed.     

The human relevance of the renal tumor-inducing potential of d-limonene in male rats: implications for risk assessment

The monoterpene d-limonene is a naturally occurring chemical which is the major component in oil of orange. Currently, d-limonene is widely used as a flavor and fragrance and is listed to be generally recognized as safe (GRAS) in food by the Food and Drug Administration (21 CFR 182.60 in the Code of Federal Regulations). Recently, however, d-limonene has been shown to cause a male rat-specific kidney toxicity referred to as hyaline droplet nephropathy. Furthermore, chronic exposure to d-limonene causes a significant incidence of renal tubular tumors exclusively in male rats. Although d-limonene is not carcinogenic in female rats or male and female mice given much higher dosages, the male rat-specific nephrocarcinogenicity of d-limonene may raise some concern regarding the safety of d-limonene for human consumption. A considerable body of scientific data has indicated that the renal toxicity of d-limonene results from the accumulation of a protein, alpha 2u-globulin, in male rat kidney proximal tuble lysosomes. This protein is synthesized exclusively by adult male rats. Other species, including humans, synthesize proteins that share significant homology with alpha 2u-globulin. However, none of these proteins, including the mouse equivalent of alpha 2u-globulin, can produce this toxicity, indicating a unique specificity for alpha 2u-globulin. With chronic exposure to d-limonene, the hyaline droplet nephropathy progresses and the kidney shows tubular cell necrosis, granular cast formation at the corticomedullary junction, and compensatory cell proliferation. Both d-limonene and cis-d-limonene-1,2-oxide (the major metabolite involved in this toxicity) are negative in in vitro mutagenicity screens. Therefore, the toxicity-related renal cell proliferation is believed to be integrally involved in the carcinogenicity of d-limonene as persistent elevations in renal cell proliferation may increase fixation of spontaneously altered DNA or serve to promote spontaneously initiated cells. The scientific data base demonstrates that the tumorigenic activity of d-limonene in male rats is not relevant to humans. The three major lines of evidence supporting the human safety of d-limonene are (1) the male rat specificity of the nephrotoxicity and carcinogenicity; (2) the pivotal role that alpha 2u-globulin plays in the toxicity, as evidenced by the complete lack of toxicity in other species despite the presence of structurally similar proteins; and (3) the lack of genotoxicity of both d-limonene and d-limonene-1,2-oxide, supporting the concept of a nongenotoxic mechanism, namely, sustained renal cell proliferation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Toxic effects of l-aspartic acid at high dose levels on kidneys and salivary glands in Fischer 344 rats detected in a 90-day feeding study

A subchronic oral toxicity study of l-aspartic acid (l-Asp) was conducted with groups of 10 male and 10 female Fischer 344 rats fed a powder diet containing 0%, 0.05%, 1.25%, 2.5% and 5.0% concentrations for 90 days. Serum biochemistry showed treatment-related decreases of blood urea nitrogen, creatinine and uric acid levels in both sexes. In addition, incidences of urinary ketone and protein were significantly increased in treated both sexes, while relative kidney weight was significantly increased in the 5.0% male rat, and regenerative renal tubules with tubular dilation were histopathologically observed in male rats of the 2.5% or greater groups. The observed renal injury was confirmed not to be due to accumulation of alpha2u-globulin. Acinar cell hypertrophy of salivary glands was histopathologically evident in male and female rats of the 2.5% or greater groups. The present results indicate that l-Asp causes toxic effects on kidneys and possibly salivary glands at high dose levels in male and female Fischer 344 rats. Such toxic effects were observed only in animals given 2.5% and/or higher doses of l-Asp. In conclusion, the no-observed-adverse-effect-level (NOAEL) for l-Asp is 1.25% (696.6 mg/kg body weight/day for males and 715.2 mg/kg body weight/day for females) under the present experimental conditions.