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 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.     

Single administration toxicokinetic studies of decalin (decahydronaphthalene) in rats and mice.

Decalin (decahydronaphthalene) is an industrial solvent known to cause alpha2u-globulin nephropathy in male rats. Studies were conducted using decalin (mixture of cis and trans isomers) to (1) characterize systemic elimination of decalin in rats and mice and (2) evaluate disposition of decalin, its metabolites, and kidney alpha2u-globulin in young and old rats of both sexes following a single 6-h whole-body inhalation exposure at up to 400 ppm decalin. Additionally, a separate group of young male F344/N rats were administered either cis- or trans-decalin iv at doses up to 20 mg/kg to assess disposition of each isomer, its metabolites, and kidney alpha2u-globulin. Decalin was eliminated from blood in a dose-dependent manner, regardless of sex, age, or species. C0 and AUC infinity increased supra-proportionally with exposure concentration. Mice were more efficient in eliminating decalin than rats at lower exposure concentrations, but nonlinear elimination kinetics were more noticeable at 400 ppm. Sex differences in blood decalin elimination were observed in rats; females had a consistently higher AUC infinity at all exposure concentrations. There was a dose-dependent increase in kidney decalin, decalone, and alpha2u-globulin in male rats exposed to decalin. Kidney alpha2u-globulin and decalone concentrations in old male rats were substantially lower than those in young males, but were similar to those observed in all (young and old) females. Compared to old males and all females, young male rats had significantly lower urinary decalol concentrations, but higher kidney decalin, decalone, and alpha2u-globulin concentrations. Administration of decalin to male rats as either the cis or trans isomer revealed that more cis -decalone is produced per unit dose as compared to trans-decalone, and that more trans-decalin accumulated in the kidney (as alpha2u-globulin-ligand complexes) compared to cis-decalin. These patterns of isomer-specific metabolism were also reflected in the cis/trans ratios of decalin in blood, as well as urinary decalol metabolites. The ratio of alpha2u-globulin to the total amount of decalin plus decalone measured in the male rat kidney was approximately 1.0. Therefore, alpha2u-globulin was a key factor in the accumulation of decalin and decalone in kidneys of young male rats, decalin and decalone were practically absent in all females and in old males.     

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.     

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.     

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.     

2,2,4-Trimethylpentane-induced nephrotoxicity. II. The reversible binding of a TMP metabolite to a renal protein fraction containing alpha 2u-globulin

Trimethylpentane (TMP) produces nephrotoxicity in male but not in female rats. The toxicity is characterized by an increase in protein droplets in proximal convoluted tubular cells and an increase in the renal concentration of the male-rat-specific protein alpha 2u-globulin. Subcellular fractionation of the kidneys from male rats 24 hr after [3H]TMP administration showed that about 60% of the radiolabeled material was localized in the 116,000g supernatant. Column chromatography of this supernatant resolved the radioactivity into two components; one, which contained about 26% of the radiolabel, coeluted with alpha 2u-globulin and cross-reacted with an antibody specific for alpha 2u-globulin. The remaining component eluted in the low-molecular-weight range (less than 1000 Da) and was assumed to be TMP metabolites. Radiolabel from [3H]TMP in male rat urine also resolved into two components with about 0.1% of the radiolabel in urine coeluting with the alpha 2u-globulin-containing fraction. Radiolabel from TMP in male rat liver 116,000g supernatant and plasma and in female rat kidney 116,000g supernatant eluted as a single component in the low-molecular-weight range. Dialysis (1000-Da cutoff) of male kidney 116,000g supernatant led to a loss of the low-molecular-weight components, but nondialyzable radiolabel (about 20%) still coeluted with the alpha 2u-globulin after gel chromatography. Dialysis against 0.1% sodium dodecyl sulfate led to a loss of both the low- and high-molecular-weight radioactive material. These results suggested that the high-molecular-weight radioactive material was formed by the reversible binding of a radioactive component of TMP to a male-rat-specific protein. Gas chromatography-mass spectrometry of an ethyl acetate extract of the alpha 2u-globulin-containing fractions of TMP-treated male rat kidney 116,000g supernatant identified 2,4,4-trimethyl-2-pentanol as the only bound metabolite to alpha 2u-globulin. These studies provide the first evidence for a reversible binding between a metabolite of TMP and a male-rat-specific protein in the kidney and thus provide important insight delineating a potential mechanism of hydrocarbon-induced hyaline-droplet nephropathy.     

Evaluation of the in vivo interaction of methyl tert-butyl ether with alpha2u-globulin in male F-344 rats.

Methyl tert-butyl ether (MTBE), a fuel additive blended into unleaded gasoline, decreases emissions of selected air pollutants. Exposure to MTBE causes a low incidence of renal tumors in male, but not female, F-344 rats. A number of chemicals that cause male rat-specific renal tumors also cause a syndrome unique to male rats referred to as alpha2u-globulin (alpha2u) nephropathy (alpha2u-N). Previous investigations have demonstrated that MTBE exposure induces a mild accumulation of alpha2u in male F-344 rats. The objective of the present study was to determine if MTBE, or a metabolite of MTBE, interacts with alpha2u in male rats administered MTBE orally. Eleven-week-old male and female F-344 rats were administered 750 mg [14C]MTBE/kg body wt or an equivalent volume of 10% emulphor orally for 4 consecutive days. Although [14C]MTBE-treated male rats exhibited a statistically significant increase in renal alpha2u concentration, the total radioactivity recovered was similar in kidney samples from [14C]MTBE-treated male and female rats. Further analysis of kidney cytosol prepared from [14C]MTBE-treated rats revealed that a slightly greater percentage of radioactivity coeluted on a G-25 gel filtration column with the total protein fraction from male rats than from female rats. Gel filtration (Sephadex G-75 column) and anion exchange chromatography, however, did not demonstrate any coelution of MTBE-derived radioactivity with the low-molecular-weight protein fraction or alpha2u fraction, respectively, in kidney cytosol prepared from [14C]MTBE-treated male or female rats. Further experimentation using a sealed vial equilibration system demonstrated that d-limonene oxide, a chemical with a high affinity for alpha2u, displaced MTBE in male, but not female, rat kidney samples following administration of MTBE. These findings provide indirect evidence that MTBE interacts with a male-specific protein such as alpha2u in male F-344 rats. Since the pathogenesis of alpha2u-N is dependent on the formation of a reversibly bound chemical-alpha2u complex, demonstration of an in vivo interaction of MTBE or one of its metabolites with alpha2u supports the alpha2u mechanism as a cause of MTBE-induced protein droplet nephropathy in male rats.     

Inhalation toxicology and carcinogenesis studies of propylene glycol mono-t-butyl ether in rats and mice

Propylene glycol mono-t-butyl ether (PGMBE) is used as a solvent in a variety of commercial applications. Male and female F344/N rats and B6C3F(1) mice were exposed to PGMBE by whole-body inhalation for 2 or 14 weeks (0, 75, 150, 300, 600, or 1200 ppm) or 2 years (0, 75, 300, or 1200 ppm); male NBR rats were exposed for 2 weeks. The kidney and the liver were targets of PGMBE toxicity in rats. Renal lesions suggestive of alpha(2u)-globulin nephropathy were observed in male F344/N, in the 2 and 14-week studies, no kidney lesions were seen in NBR rats. In the 2-year study, male rats displayed exposure-related nonneoplastic lesions in the kidney, and may have shown marginal increases in tubular neoplasms. In the liver, the incidences of hepatocellular adenomas occurred with a positive trend in male rats, and may have been related to PGMBE exposure. In mice of both sexes, the major target of PGMBE toxicity was the liver. In the 2-week study, liver weights and in the 14-week study, liver weights and the incidences of centrilobular hypertrophy were increased. In the 2-year study, the incidences of exposure-related hepatocellular adenoma, adenoma or carcinoma combined, and hepatoblastoma occurred with a positive trend, and were significantly increased in 1200 ppm groups. In summary, exposure to PGMBE resulted in nonneoplastic lesions of the kidney characteristic of alpha(2u)-globulin nephropathy, and may have increased renal tubular neoplasms in male rats. Exposure to PGMBE also produced increases in hepatic tumors in male and female mice.     

The role of alpha2u-globulin in ochratoxin A induced renal toxicity and tumors in F344 rats

The mycotoxin ochratoxin A (OTA) was shown to be a potent kidney carcinogen in rats demonstrating a marked sex difference in the response. Compared to female rats, male rats had a 10-fold higher incidence of kidney carcinomas. The objective of this study was to investigate whether this sex difference in tumor response is due to an exacerbation of effect resulting from the interaction of the male rat specific urinary protein alpha2u-globulin (alpha2u) with OTA. Male and female rats were treated by oral gavage with OTA (1 mg/kg per day), D-limonene (dL; 1650 mg/kg per day) as a positive control or corn oil for 7 consecutive days. OTA induced severe renal lesions predominantly in the P3 region of the proximal tubules. The lesions consisted of necrotic cells and cell exfoliations. No hyaline droplets were found in the P2 segment following OTA treatment, whereas dL induced the expected accumulation of droplets. The results suggest that OTA induced kidney lesions are in all characteristic points different from the known alpha2u-nephropathy induced by dL. Based on these experiments the male rat specific protein alpha2u does not seem to be involved in the mechanism(s) leading to the high tumor incidence observed in OTA exposed male rats.     

Hepatic alpha(2u)-globulin mRNA levels and diethylstilbestrol-associated testicular atrophy in rats

Biosynthesis of alpha(2u)-Globulin (alpha(2u)-g) is under multihormonal regulation. In this study, we investigated histopathologic changes in the testis and hepatic alpha(2u)-g messenger ribonucleic acid (mRNA) levels in male rats after administration of the potent estrogen diethylstilbestrol (DES) at 0. 01, 0.1, or 1 mg/kg/day by gavage for 14 days. DES treatment decreased hepatic alpha(2u)-g mRNA levels in a dose-dependent manner accompanied by atrophic histopathologic changes in the testis. In addition, alpha(2u)-g mRNA levels were lowest in animals with the most marked testicular changes. Hepatic alpha(2u)-g mRNA may be a useful biomarker for the evaluation of endocrine disruption in male rats.     

Investigations on cell proliferation and enzyme induction in male rat kidney and female mouse liver caused by tetrahydrofuran.

To elucidate possible mechanism(s) of carcinogenic action of tetrahydrofuran (THF) that had been demonstrated in previous inhalation studies, groups of male F344 rats and female B6C3F(1) mice were exposed to dynamic atmospheric concentrations of 0, 600, 1800, or 5400 mg/m(3) for 6 h per day, either for 5 consecutive days or for a period of 4 weeks (5 days per week). The reversibility of treatment-related changes was investigated in rats and mice exposed for 5 days and sacrificed 21 days after the last exposure. Female B6C3F(1) mice exposed to 5400 mg/m(3) showed significantly increased cytochrome P450 content, increased ethoxyresorufin-O-deethylase and pentoxyresorufin-O-depentylase activities, increased cell proliferation (5-bromo-2'-deoxyuridine-method) and an increased mitotic index in liver zones 2 (midzonal region) and 3 (central vein region). The changes were found to be reversible after a 3-week treatment-free period (cell proliferation examined, only). Male F344 rats showed dose-related alpha2u-globulin (alpha2u) accumulation in the renal cortex after 5 or 20 exposures, and there were no signs of reversal after a 3-week treatment-free period. After 20 exposures at 5400 mg/m(3), the alpha2u accumulation was found to be associated with increased cell proliferation in "hot spots" of the renal cortex and increased apoptosis. Increased cell proliferation was also detected after 20 exposures at 1800 mg/m(3). There were no effects at 600 mg/m(3). It is concluded that THF enhances tumor formation in male rat kidney and female mouse liver via induction of cell proliferation. These features present essential elements that should be taken into account for the carcinogenic risk assessment of THF.     

Alpha 2U-globulin: measurement in rat kidney following administration of 2,2,4-trimethylpentane

Hyaline droplet formation was stimulated markedly in the kidneys of post-puberty male rats 24-48 h after a single oral dose of 12/24 mmol/kg 2,2,4-trimethylpentane [TMP]. Renal hyaline droplet formation could not be detected in female rats or in pre-puberty male rats following similar doses of TMP. A dose-dependent increase in the renal concentration of the androgen-dependent low molecular weight protein, alpha 2U-globulin was observed in post-puberty male rats 24 h after a single oral dose of TMP, over the range 0.3-12.0 mmol/kg. After administration of a single dose of 12 mmol/kg TMP to male rats, the renal concentration of alpha 2U-globulin rose steadily up to a peak after 48 h and then returned slowly to near normal after 7 days. Renal alpha 2U-globulin could not be detected in female rats and in pre-puberty male rats. An immunocytochemical assay was developed to examine the distribution of alpha 2U-globulin within the kidney. alpha 2U-Globulin was localised primarily in the S2 segment of renal proximal tubules in untreated male rats. Rats which received a single dose of 12 mmol TMP/kg showed not only a greater staining intensity, due to the presence of a higher concentration of alpha 2U-globulin, but also staining in adjacent segments of the renal cortex. Several urinary biochemical indicators of nephrotoxicity were measured daily in male rats for up to 72 h following a single dose of 12 mmol TMP/kg. Renal proximal tubular function was unimpaired by TMP treatment. On the basis of studies in untreated and TMP-treated rats, a strong association has been found between the presence of renal hyaline droplets and the occurrence of renal alpha 2U-globulin. The findings in the present study provide an explanation for the occurrence of renal hyaline droplets only in adult male rats, but do not, as yet, establish the toxicological significance of increases in renal hyaline droplet formation.     

The induction of ω and β-oxidation of fatty acids and effect on α2u globulin content in the liver and kidney of rats administered 2, 2, 4-trimethylpentane

1. 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 β-oxidation and the concentration of α_2u-globulin has been examined in male and female rats.

2. 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 ω-oxidation and peroxisomal β-oxidation of fatty acids and proliferation of peroxisomes. Male rats show a more marked response than female rats.

3. 2, 2, 4-Trimethylpentane produces an increase in α_2u-globulin in the kidney of male rats.

4. The relevance of selective induction of ω- and β-oxidation of fatty acids and accumulation of α_2u-globulin to renal tubular necrosis in male rats requires further study.     

Male rat specific nephrotoxicity resulting from subchronic administration of hexachlorobenzene.

Male rats are more sensitive to the nephrocarcinogenic effect of hexachlorobenzene (HCB) than are female rats. The purpose of this study was to shed light on this phenomenon by investigating mechanisms of subchronic nephrotoxicity of HCB. Groups of rats were administered HCB in corn oil (po) at 100 mg/kg, 5 days per week for 15 days or at 50 mg/kg, 5 days per week for 50 days. Urine was collected on Days 1, 8, and 15 for the 15-day treatment and on Day 50 for the 50-day treatment. Glucosuria, proteinuria, and enzymuria (gamma-glutamyl transpeptidase) were measured to assess renal function. Twenty-four hours after the last HCB treatment, the animals were killed and kidneys were removed for histopathological evaluation. Urine analyses showed no indication of renal dysfunction in treated animals compared to controls during the 15-day treatment. However, histology of male rat kidneys revealed degenerative and regenerative cellular foci accompanied by an increased accumulation of protein droplets in epithelial cells of the proximal tubules. The same histological observations were also made in male rats after a 50-day HCB treatment but this time they were accompanied by renal function alterations. In female rats, no such renal functional or histological alterations were observed. The histopathological observations in male rats correspond well with the protein droplet nephropathy; the latter is characteristic of the accumulation in kidney cells of alpha 2u-globulin probably caused by the reversible binding of a chemical to alpha 2u that renders the protein indigestible to kidney proteases. alpha 2u-Globulin was measured in the cytosol of male rats and was found to be increased 11-fold compared to controls. Also, HCB was found to be bound reversibly to alpha 2u. These results suggest that HCB induces a male rat specific nephropathy that could explain the higher incidence of kidney tumors in male rats compared to female rats.     

Increased Hyaline Droplet Formation in Male Rats Exposed to Decalin Is Dependent on the Presence of {alpha}2u-Globulin

Increased Hyaline Droplet Formation in Male Rats Exposed toDecalin Is Dependent on the Presence of _2u-Globulin. RIDDER,G. M., VON BARGEN, E. C., ALDEN, C. L., AND PARKER, R. D. (1990).Fundam. Appl. Toxicol. 15, 732–743. A peculiar decalin-inducedmale rat nephropathy associated with the altered renal handlingof filtered protein appears limited to the accumulation of theprotein, _2u-globulin. Several strains of male rats that produce_2u-globulin (Fischer-344, Sprague-Dawley, Buffalo, and NorwayBrown) demonstrate spontaneous renal cortical hyaline dropletswhich are exacerbated after exposure to decalin. In all cases,a close correlation exists between hyaline droplet formationobserved histologically and _2u-globulin accumulation measuredbiochemically. In stark contrast, the NCI-Black-Reiter strain,which does not produce measurable quantities of _2u-globulin,neither forms hyaline droplets nor accumulates any filteredprotein in its kidney cortex either spontaneously or after exposureto decalin. Also, female rats injected ip with male rat _2u-globulinexhibit increased hyaline droplet formation and _2u-globulinaccumulation when treated with decalin. These data provide evidencethat the presence of _2u-globulin is key in understanding whythis nephropathy appears unique to the male rat.     

Semi-quantitative immunohistochemical analysis of male rat-specific alpha2u-globulin accumulation for chemical toxicity evaluation

We purified male rat urinary alpha(2u)-globulin, prepared the antibody in rabbits, and improved an immunohistochemical detection method using this antibody for male rat-specific alpha(2u)-globulin accumulation appearing as hyaline droplets in the kidneys. Our prepared antibody reacted specifically with alpha(2u)-globulin in both immunohistochemical and Western blotting analyses, furthermore, and the graded immuno-reactivities on the slide were well associated with computational image analyzing results. Using this method, we retrospectively analyzed the renal sections from the toxicity studies of 12 nephrotoxic chemicals, which had already been conducted under the Japanese Existing Chemicals Survey Program. We demonstrated that the hyaline droplets induced by treatment with 10 chemicals (1,4-dibromobenzene, dicyclopentadiene, 3,4-dimethylaniline, 1,4-dicyanobenzene, tetrahydrothiophene-1,1-dioxide, 1,3-dicyanobenzene, acenaphthene, 3,4-dichloro-1-butene, 3a,4,7,7a-tetrahydro-1H-indene and 3,5,5-trimethylhexan-1-ol) were directly associated with alpha(2u)-globulin accumulation. This immunohistochemical method is convenient for applying, even retrospectively, paraffin sections from general toxicity studies and could be useful for qualifying male rat-specific hyaline droplets consisting of alpha(2u)-globulin and renal risk in humans.     

Characteristics of chemical binding to alpha 2u-globulin in vitro--evaluating structure-activity relationships.

alpha 2u-Globulin (alpha 2u) has been shown to accumulate in the kidneys of male rats treated with 2,2,4-trimethylpentane (TMP). 2,4,4-Trimethyl-2-pentanol (TMP-2-OH), a metabolite of TMP, is found reversibly bound to alpha 2u isolated from the kidneys of these treated rats. The objectives of the following study were to characterize the ability of [3H]TMP-2-OH to bind to alpha 2u in vitro and to determine whether other compounds that cause this protein to accumulate have the same binding characteristics. Although compounds that have been shown to cause the accumulation of alpha 2u in male rat kidneys compete in vitro with [3H]TMP-2-OH for binding to alpha 2u, they do so to varying degrees. The binding affinity (Kd) of the [3H]TMP-2-OH-alpha 2u complex was calculated to be on the order of 10(-7) M. The inhibition constant values (Ki) determined for d-limonene, 1,4-dichlorobenzene, and 2,5-dichlorophenol were all in the range 10(-4) M, whereas the Ki values for isophorone, 2,4,4- or 2,2,4-trimethyl-1-pentanol, and d-limonene oxide were determined to be in the range 10(-6) and 10(-7) M, respectively. TMP and 2,4,4- and 2,2,4-trimethylpentanoic acid did not compete for binding. This suggests that other factors, besides binding, are involved in the accumulation of alpha 2u. In this study the ability of a chemical to bind to alpha 2u was used as a measure of biological activity to assess structure-activity relationships among the chemicals tested and known to cause the accumulation of alpha 2u. The results so far suggest that binding is dependent on both hydrophobic interactions and hydrogen bonding.     

A comparison of European High Test gasoline and PS-6 unleaded gasoline in their abilities to induce alpha 2u-globulin nephropathy and renal cell proliferation.

Male Fischer-344 rats were administered European High Test gasoline (EHT) (50-500 mg/kg), PS-6 unleaded gasoline (UG) (16-500 mg/kg) or 2,2,4-trimethylpentane (TMP) (0.95-30 mg/kg) by gavage for ten consecutive days. To measure cell replication, rats were exposed to [3H]thymidine continuously over the last 7 days of the exposure period. Twenty-four hours after the final dose, protein droplet (PD) accumulation, alpha 2u-globulin (alpha 2u) concentration and the nuclear labeling index (LI), as a measure of cell replication, were measured in the kidneys of control and treated rats. Dose-related increases in PD, alpha 2u and cell replication were detected in the kidneys of rats treated with either gasoline mixture or TMP. The accumulation of PD and the increase in alpha 2u was greater in the kidneys of UG- and TMP-treated rats than in the kidneys of rats treated with EHT. These differences were attributed to the higher composition of branched hydrocarbons in UG, which have been shown to be the biologically active components for these endpoints. The extent of renal cell proliferation was similar in both EHT-, UG- and TMP-treated rats. This suggests that other components besides the branched hydrocarbons are responsible for the increased renal cell replication in EHT-treated rats.     

Silymarin protects against liver damage in BALB/c mice exposed to fumonisin B1 despite increasing accumulation of free sphingoid bases.

Fumonisin B(1) (FB(1)) is a mycotoxin produced by Fusarium verticillioides found on corn and corn-based foods. It causes equine leukoencephalomalacia, porcine pulmonary edema, and liver and kidney damage in most animal species. Fumonisin B(1) perturbs sphingolipid metabolism by inhibiting ceramide synthase activity, leading to the production of cell signaling factors including tumor necrosis factor alpha (TNF-alpha). The signal pathways of TNF-alpha are important factors in the pathogenesis of FB(1) hepatotoxicity. In the present study, female BALB/c mice were treated daily with 750 mg/kg silymarin by gavage and 2.25 mg/kg FB(1) subcutaneously for 3 days. Then, 1 day after the last FB(1) injection, the mice were euthanized and blood and tissues were sampled for analyses. Silymarin significantly diminished FB(1)-induced elevation of plasma alanine aminotransferase and aspartate aminotransferase activities and the number of apoptotic hepatocytes, while it augmented hepatocyte proliferation indicated by an increase in proliferating cells. Silymarin dramatically potentiated FB(1)-induced accumulation of free sphinganine and sphingosine in both liver and kidney. Silymarin itself slightly increased expression of hepatic TNF-alpha; however, it prevented the FB(1)-induced increases in TNF-alpha, TNF receptor 1, TNF receptor-associated apoptosis-inducing ligand, lymphotoxin beta, and interferon gamma. The induction of transforming growth factor beta1 expression in liver following FB(1) treatment was not affected by silymarin. These findings suggest that silymarin protected against FB(1) liver damage by inhibiting biological functions of free sphingoid bases and increasing cellular regeneration.