Α2u-globulin Nephropathy Research Articles

A 13-week subchronic toxicity study of linalool oxide in Crl:CD(SD) rats.

Linalool oxide is frequently used as a flavoring agent, however, data on its toxicity is limited. In this study, we performed a 13-week subchronic toxicity study of linalool oxide (furanoid) in male and female Crl:CD(SD) rats. Doses of 0, 80, 250, and 800 mg/kg body weight (bw) per day were orally administered by gavage, using corn oil as the vehicle. Abnormal gait in both sexes and decreased locomotor activity in males were observed in the 800 mg/kg group. Reduced body weight gain was noted in both sexes at 800 mg/kg and at 250 mg/kg in males. In the 800 mg/kg group, serum biochemistry showed increased γ-glutamyl transpeptidase and decreased glucose in both sexes, increased total protein in males, and increased total cholesterol and phospholipids in females, suggesting that linalool oxide may have adverse effects on the liver. Increased relative and/or absolute liver weights, centrilobular hepatocellular hypertrophy in both sexes, and periportal microvesicular fatty changes in females were observed in the 800 mg/kg group. Increased relative liver weights and decreased serum glucose levels were observed in the 250 mg/kg male and female groups, respectively. Increased serum magnesium levels and relative kidney weights were observed in both sexes in the 800 mg/kg group, suggesting possible adverse effects of linalool oxide. Although histopathology showed accumulation of hyaline droplets in the male kidneys, immunohistochemistry revealed α2u-globulin nephropathy, which was not considered toxicologically significant. These results indicate that the no-observed-adverse-effect level of linalool oxide was 80 mg/kg bw/day for both sexes.

Nephrotoxic Potential of the Three Dichlorobenzene Isomers in Isolated Kidney Cells from Male Fischer 344 Rats

Dichlorobenzenes (DCBs) are commonly used as chemical intermediates in the manufacture of a wide range of agricultural, industrial and pharmaceutical products. Previous studies have shown that 1,4-DCB can induce nephrotoxicity in male rats characterized as α2u-globulin nephropathy. Initially, 1,4-DCB binds to α2u-globulin at extra-renal sites. The 1,4-DCB-protein complex then accumulates in kidney and causes protein droplet formation, ultimately leading to cell death and/or eventually tumor formation. The purpose of this study was to explore the direct cytotoxic effects of the three DCB isomers in isolated kidney cells (IKCs) from male Fischer 344 rats. IKCs (~4 million/ml, 3 ml) were incubated with a DCB (0.25-1.0 mM) or dimethyl sulfoxide (DMSO, vehicle) for 30 or 60 min with shaking at 37oC under a 95% oxygen/5% carbon dioxide atmosphere. Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) release. In separate experiments, IKCs were pretreated with an antioxidant (ascorbate, 1.0 mM; N-acetyl-L-cysteine, 2.0 mM; glutathione, 1.0 mM) prior to the addition of 1,4-DCB (0.5 mM) and followed by a 60 min co-incubation. 1,4-DCB increased LDH release at all times and concentrations tested. 1,3-DCB increased LDH release at 0.5 mM or greater at both time points, while 1,2-DCB only increased LDH release at 0.5 mM or greater at 60 min. Thus, the order of decreasing nephrotoxic potential was 1,4-DCB > 1,3- DCB > 1,2-DCB. Pretreatment with any of the antioxidants tested attenuated 1,4-DCB nephrotoxicity. These results indicate that DCBs can induce nephrotoxicity in vitro without the presence of extra-renal α2u-globulin, 1,4-DCB is the most potent nephrotoxicant among the three DCB isomers in IKCs, and free radicals play a role in DCB nephrotoxicity.

A 13-week subchronic toxicity study of 2-(l-menthoxy)ethanol in F344 rats.

2-(l-Menthoxy)ethanol has been frequently employed as a flavoring agent; however, data regarding 2-(l-menthoxy)ethanol toxicity remain limited. We performed a 13-week subchronic toxicity study of 2-(l-menthoxy)ethanol in male and female F344 rats, with doses of 0, 15, 60, or 250 mg/kg body weight (BW)/day orally administered by gavage using corn oil as the vehicle. No significant toxicological changes in general condition, body weight, or food intake were observed in any groups. The hematological assessment showed decreased hemoglobin, hematocrit, mean corpuscular volume, and mean corpuscular hemoglobin and increased platelet count in the male 250 mg/kg group. Serum biochemistry revealed elevated total cholesterol in the 250 mg/kg group of male and female rats, reduced triglyceride in the female 250 mg/kg group, and increased total protein in the male 250 mg/kg group, indicating effects on lipid metabolism and protein synthesis. For organ weights, absolute and relative weights of the liver and adrenal glands were increased in the 250 mg/kg group of both sexes and the male 250 mg/kg group, respectively. Histopathological analysis showed chronic nephropathy in the male 15 mg/kg or higher groups, with increased absolute and relative kidney weights, as well as elevated serum creatinine, in the male 60 and 250 mg/kg groups. However, eosinophilic granules containing α2u-globulin were identified in proximal tubules, suggesting α2u-globulin nephropathy specific to male rats and without toxicological significance. These results indicated that the no-observed-adverse-effect level of 2-(l-menthoxy)ethanol was 60 mg/kg BW/day for both sexes.

Dietary administration of β-caryophyllene and its epoxide to Sprague-Dawley rats for 90 days

Two independent 90-day GLP-compliant studies were conducted in Sprague-Dawley rats with β-caryophyllene or β-caryophyllene epoxide, two common flavoring and fragrance materials. Dietary concentrations of β-caryophyllene were 3500; 7000; and 21,000 ppm for males and 3500; 14,000; and 56,000 ppm for females. Dietary concentrations of β-caryophyllene epoxide were 1750; 10,500; and 21,000 ppm. There were no deaths or clinical toxicity attributable to either substance administration. Statistically significant, dose-dependent reductions in body weight, body weight gain, food consumption, and food efficiency at the highest dietary concentrations of β-caryophyllene, but not of β-caryophyllene epoxide, were attributed to palatability issues. Neither β-caryophyllene nor β-caryophyllene epoxide influenced estrus cyclicity or sperm parameters. Macroscopic and microscopic findings were primarily related to changes in the kidneys of male rats, consistent with α2u-globulin nephropathy, and in the liver of male and female rats, including hepatocyte hypertrophy at the middle and high intake levels. These changes correlated with increased absolute and relative organ weights. Since the kidney findings were a species- and sex-specific effect, the NOAEL in each study was based on hepatocyte hypertrophy at the two highest dietary concentrations and were determined to be 222 mg/kg bw/day for β-caryophyllene and 109 mg/kg bw/day for β-caryophyllene epoxide.

Drug-induced Obstructive and Retrograde Nephropathy Associated with α2u-globulin in Male Rats.

The chemically induced accumulation of α2u-globulin protein in male rats causes specific renal lesions and subsequent nephropathy. Herein, we report additional parallel findings in the kidney of male rats consistent with obstructive and retrograde nephropathy. Kidney and urinary bladder samples were evaluated from Wistar rats treated with RG7129 for 2 week and 8 week and from an 8-week mechanistic study using females, intact and castrated males. Histopathological findings were present in intact males in all studies, including hyaline droplet accumulation and granular casts consistent with α2u-globulin nephropathy. In addition, tubular degeneration and regeneration, tubular changes extending from papilla to cortex, tubular dilation, and interstitial and luminal inflammation were observed consistent with retrograde and obstructive nephropathy. Renal and urinary lesions and their severity increased in a time- and dose-dependent manner. Urinalysis findings, including increases in leukocytes, protein, and in kidney biomarkers, kidney injury molecule 1 and clusterin, were present only in intact males. No treatment-related changes were observed in female rats or in castrated males. These results indicate that RG7129 induces α2u-globulin nephropathy, associated with retrograde and obstructive nephropathy secondary to precipitation in intact male rats only, constituting a species- and sex-specific syndrome that is not expected to occur in humans or other species.

Two types of deposits, hyaline droplets and eosinophilic bodies, associated with α2u-globulin accumulation in the rat kidney.

Alpha2u-globulin is an adult male rat-specific protein that accumulates spontaneously or inductively in the renal proximal tubular epithelium and forms microscopically observable deposits, which are generally referred to as “hyaline droplets,” whereas a specific type of deposits is referred to as “eosinophilic bodies” by Japanese toxicologic pathologists. We compared hyaline droplets and eosinophilic bodies using special stains including immunostaining for α2u-globulin and lysosome-associated membrane protein in spontaneously occurring and d-limonene-induced cases. Eosinophilic bodies appeared simultaneously and increased in parallel with the hyaline droplets in the induced case. In both of the spontaneous and induced cases, hyaline droplets and eosinophilic bodies were associated with α2u-globulin and lysosomes, although there were differences in the forms and staining properties that probably reflected the purity or density of α2u-globulin. According to the results, it is not necessary for eosinophilic bodies to be strictly distinguished from hyaline droplets, and it is reasonable to identify eosinophilic bodies as hyaline droplets in α2u-globulin nephropathy in routine toxicity studies, as they have been recognized to be a sequence of changes associated with accumulation of α2u-globulin.

Combined repeated-dose and reproductive/developmental toxicity screening test of 1-tert-butoxy-4-chlorobenzene in rats

We have carried out animal toxicity tests of chemicals for a chemical safety program implemented by the Ministry of Economy, Trade, and Industry of Japan. Here, we tested 1-tert-butoxy-4-chlorobenzene in a combined repeat-dose and developmental and reproductive toxicity test. The test chemical was administered daily by gavage to 9-week-old Crl:CD (SD) rats at doses of 0, 20, 100, and 500 mg/kg/d. Males were treated for 42 d beginning 14 d before mating. Females were treated from 14 d before mating to day 4 of lactation. Decreased spontaneous locomotion, decreased respiratory rate, and incomplete eyelid opening were observed at 500 mg/kg/d (both sexes), but resolved within 30 min of administration, suggesting central nervous system depression. No notable changes were observed in body weight, food consumption, functional battery tests, or blood test. Increased liver weight with centrilobular or diffuse hepatocyte hypertrophy was observed at 100 and 500 mg/kg/d (both sexes). There were no biochemical or histopathological changes related to hepatotoxicity. Increased kidney weight with basophilic tubules, tubule dilatation, and increased hyaline droplets were observed in males dosed at 100 and 500 mg/kg/d. Immunohistochemical staining indicated α2u-globulin nephropathy, a male rat-specific toxicity. Although kidney weight was also increased in females dosed at 500 mg/kg/d, it was not considered to be an adverse effect because there were no histopathological changes. Pup weights on postnatal day 0 were decreased at 500 mg/kg/d and still decreased on postnatal day 4. Our data indicated the no-observed-adverse-effect-level for repeated-dose and reproductive/developmental toxicity for 1-tert-butoxy-4-chlorobenzene was 100 mg/kg/d.

A Mechanism for the induction of renal tumours in male Fischer 344 rats by short-chain chlorinated paraffins

Short-chain chlorinated paraffins (SCCPs) cause kidney tumours in male rats, but not in female rats or mice of either sex. Male rat-specific tumours also occur in rats dosed with a range of compounds including 1,4- dichlorobenzene (DCB) and d-limonene (DL). These compounds bind to a male rat-specific hepatic protein, alpha-2-urinary globulin (α2u), and form degradationresistant complexes in the kidney. The resulting accumulation of α2u causes cell death and sustained regenerative cell proliferation, which in turn leads to the formation of renal tumours. To investigate whether the SCCP, Chlorowax 500C (C500C), causes tumours via the accumulation of α2u male rats were orally dosed with either C500C (625 mg/kg of body weight), DCB (300 mg/kg of body weight), or DL (150 mg/kg of body weight) for 28 consecutive days. An increase in renal α2u and cell proliferation was observed in DCB- and DL-treated rats but not in C500C-treated rats. C500C caused peroxisome proliferation and a down-regulation of α2u synthesis in male rat liver. This down-regulation occurred at the transcriptional level. Since less α2u was produced in C500C-treated rats, there was less available for accumulation in the kidney hence a typical α2u nephropathy did not appear. However, the administration of a radiolabelled SCCP, [14C]polychlorotridecane (PCTD), to male rats demonstrated its binding to renal α2u. Thus, it is possible that SCCPs bind to α2u and cause a slow accumulation of the protein in the kidney followed by delayed onset of α2u nephropathy. As a consequence of these findings in the current experiments, while evidence exists implicating α2u-globulin in the molecular mechanism of action of the C500C, the classic profile of a α2u-globulin nephropathy seen with other chemicals such as DCB and DL was not reproduced during this experimental protocol.

Methyl isobutyl ketone (MIBK) induction of α2u-globulin nephropathy in male, but not female rats

Male F-344 rats were administered corn oil (vehicle control), d-limonene (positive control, 300 mg/kg), or MIBK (1000 mg/kg) and female F-344 rats corn oil (vehicle control) or MIBK for 10 consecutive days by oral gavage. Approximately 24 h after the final dose the kidneys were excised and the left kidney prepared and evaluated for histological changes including protein (hyaline) droplet accumulation, immunohistochemical staining for α2u-globulin (α2u), and proliferating cell nuclear antigen (PCNA) to quantitate renal cell proliferation. The right kidney was prepared for quantitation of total protein and α2u using an ELISA. MIBK elicited an increase in protein droplets, accumulation of α2u, and renal cell proliferation in male, but not female rats, responses characteristic of α2u-mediated nephropathy. MIBK produced identical histopathological changes in the male rat kidney when compared to d-limonene, an acknowledged inducer of α2u-nephropathy except that the grade of severity tended to be slightly lower with MIBK. MIBK did not induce any effects in female rats. Therefore, renal histopathology, along with the other measures of α2u accumulation, provides additional weight of evidence to support the inclusion of MIBK in the category of chemicals exerting renal effects through a α2u-nephropathy-mediated mode-of-action.

Development of α2u-Globulin Nephropathy and Adrenal Medullary Pheochromocytomas in Male Rats Following Exposure to Stoddard Solvent IIC

Stoddard solvent IIC is widely used as a solvent in paints and varnishes, and for dry cleaning and other grease removal applications. Because concern exists regarding the long-term effects of occupational exposure in industrial settings, the toxicity and carcinogenicity of Stoddard solvent IIC were evaluated in male and female F344/N rats and B6C3F1 mice. Rats and mice were exposed to 0, 138, 275, 550, 1100, or 2200 mg/m3 Stoddard solvent IIC by whole-body inhalation for 3 mo, and to 0, 138 (male rats), 550, 1100, or 2200 (female rats and male and female mice) mg/m3 for 2 yr. The kidney, liver, and adrenal medulla were targets of Stoddard solvent IIC toxicity in rats. After 3 mo of exposure, male rats developed lesions characteristic of α2u-globulin nephropathy. Male and female rats displayed increased liver weights and/or clinical pathology changes suggestive of hepatic injury, although no accompanying histopathologic changes were observed. After 2 yr, increased incidences of adrenal medullary pheochromocytomas provided some evidence of carcinogenicity in male rats. Renal tubule adenomas were slightly increased in male rats after 2 yr, and may have been related to exposure. In mice, there was no chemical-related toxicity after 3 mo, with the exception of increased liver weights in male mice exposed to 2200 mg/m3. After 2 yr, the incidences of hepatocellular adenomas were increased in female mice exposed to 2200 mg/m3; however, these increases were marginal and associated with increases in body weight. There was no evidence of Stoddard solvent IIC carcinogenicity in female rats or male mice. In summary, inhalation exposures of Stoddard solvent IIC resulted in renal toxicity and adrenal medullary pheochromocytomas in male rats. The liver also appeared to be a site of toxicity in male and female rats and mice.

P-Nitrobenzoic acid alpha2u nephropathy in 13-week studies is not associated with renal carcinogenesis in 2-year feed studies.

The objective of this study was to characterize the renal toxicity and carcinogenicity of p-nitrobenzoic acid in F344 rats. Dose levels in 13-week and 2-year studies ranged from 630-10,000 ppm and 1,250-5,000 ppm, respectively. At 13 weeks, renal lesions included minimal to mild hyaline droplet accumulation in male rats and karyomegaly in male and female rats. At 2 years, renal lesions included proximal tubule epithelial cell hyperplasia in male rats and oncocytic hyperplasia in high-dose male and female rats, and a decreased severity of nephropathy in males and females. The hvaline droplets in renal tubular epithelial cells of male rats at 13 weeks were morphologically similar to those described in alpha2u-globulin nephropathy. Using immunohistochemical methods, alpha2u-globulin accumulation was associated with the hyaline droplets. In addition, at 13 weeks, cell proliferation as detected by PCNA immunohistochemistry was significantly increased in males exposed to 5,000 and 10,000 ppm when compared to controls. Cytotoxicity associated with alpha2U-globulin nephropathy such as single-cell necrosis of the P2 segment epithelium or accumulation of granular casts in the outer medulla did not occur in the 13-week study. In addition, chronic treatment related nephrotoxic lesions attributed to accumulation of alpha2u-globulin such as linear foci of mineralization within the renal papilla, hyperplasia of the renal pelvis urothelium and kidney tumors were not observed. Although there was histologic evidence of alpha2u-globulin accumulation in male rats at 13 weeks, the minimal severity of nephropathy suggests that the degree of cytotoxicity was below the threshold, which would contribute to the development of renal tumors at 2 years.

Exfoliated Cells in the Urine Reflect Transient and Sustained Elevation of Cell Proliferation in Rat .ALPHA.2u-Globulin Nephropathy.

2, 2, 4-Trimethylpentane (TMP) was administered orally to male Sprague-Dawley rats for 26 weeks to induce α2u-globulin nephropathy and histopathological examinations of the kidney as well as urinary sediment morphology and 5-bromo-2'-deoxyuridine (BrdU) incorporation were performed. The number of exfoliated renal epithelial cells in the urine increased with the TMP treatment until week 4, when a peak of approximately 90-fold as compared with the matched control values was observed. Significantly elevated levels then persisted throughout the study. Deposits of α2u-globulin and BrdU labeling in the kidney also reached maxima at week 4. A correlation among the amount of α2u-globulin deposits, the BrdU labeling indices, and the numbers of exfoliated cells in the urine was noted at each time point. The results thus suggest that damaged cells become detached following excessive accumulation of α2u-globulin and this stimulates regenerative cell proliferation in the kidney. Quantitative examination of exfoliated cells in the urine may thus provide a reliable estimate of cell proliferation induced by α2u-globulin nephropathy during rat toxicology studies.

Comparison of the Hepatic and Renal Effects of 1,4-Dichlorobenzene in the Rat and Mouse

The effects of 1,4-dichlorobenzene (DCB) have been compared in male F344 rats given 0 (corn oil control), 25, 75, 150, and 300 mg/kg DCB and male B6C3F1 mice given 0 (corn oil control), 300, and 600 mg/kg DCB by daily oral gavage five days per week for 1, 4, and 13 weeks. The two highest rat and both mouse dose levels were the same as those employed in a NTP bioassay, where DCB produced kidney tumors in male rats and liver tumors in mice. DCB produced significant dose-related increases in relative liver weight in both the rat and the mouse which was associated with, respectively, mild and marked centrilobular hypertrophy. Administration of DCB also produced a sustained induction of microsomal cytochrome P450 content and 7-pentoxyresorufin O-depentylase activity in both species. Western immunoblotting studies demonstrated that DCB induced CYP2B isoenzyme(s) in both rat and mouse liver microsomes. Replicative DNA synthesis was studied by implanting osmotic pumps containing 5-bromo-2'-deoxyuridine in study Weeks 0-1, 3-4, and 12-13. In the rat hepatocyte labeling index values were only increased in animals given 300 mg/kg DCB for 1 week, whereas hepatocyte labeling index values were significantly increased in mice given 300 and 600 mg/kg DCB for 1 and 4 weeks. DCB treatment produced significant increases in rat renal P1/P2 proximal tubule cell labeling index values at all time points, whereas little effect was observed in mouse kidney. The observed species difference in DCB-induced liver tumor formation may reflect the greater sensitivity of the mouse to tumor promotion by a CYP2B inducer. For the kidney, the present data provides further evidence that while DCB-induced alpha2U-globulin nephropathy is associated with a sustained stimulation of cell replication in male rat renal proximal tubule cells, this effect is not observed in the male mouse.

Methyltert-Butyl Ether Causes α2u-Globulin Nephropathy and Enhanced Renal Cell Proliferation in Male Fischer-344 Rats

Methyltert-butyl ether (MTBE), a fuel additive blended into unleaded gasoline to decrease carbon monoxide emissions, induces renal tumors in male, but not female, rats exposed by inhalation to ≥3000 ppm MTBE. A number of chemicals that induce male rat-specific renal tumors also cause a syndrome unique to male rats referred to as α2u-globulin nephropathy (α2u-N). The objective of the present study was to determine if MTBE induces an α2u-N and renal cell proliferation in male F-344 rats. Male and female F-344 rats were exposed to MTBE vapors of 0, 413, 1516, or 3013 ppm for 6 hr/day for 10 consecutive days. Significant proximal tubule necrosis and protein droplet accumulation were observed in kidneys from male rats exposed to 1516 and 3013 ppm MTBE. Significantly greater labeling indices were observed in all groups of MTBE-exposed male rats. α2u-Globulin immunoreactivity was present in and confined to protein droplets in male rat kidney. A mild dose-related increase in α2u concentration in the kidney, as measured by an enzyme-linked immunosorbent assay, was observed in male rats exposed to MTBE, with a statistically significant increase in α2u concentration in male rats exposed to 3013 ppm MTBE. There was a strong positive correlation (r= 0.994) with exposure concentration between cell proliferation and α2u concentration in male rat kidney. No significant differences were observed in female rats for any of these responses. Further analysis of kidney cytosol failed to demonstrate the accumulation of any protein besides α2u in MTBE-exposed male rat kidney. These findings demonstrate that MTBE causes a mild induction of α2u-N and enhanced renal cell proliferation in male, but not female, F-344 rats, suggesting a role for α2u-N in renal tumorigenesis.

Development of a Mechanism-Based Dosimetry Model for 2,4,4-Trimethyl-2-pentanol-Induced α2u-Globulin Nephropathy in Male Fischer 344 Rats

Development of a Mechanism-Based Dosimetry Model for 2,4,4-Trimethyl-2-pentanol-Induced α2u-Globulin Nephropathy in Male Fischer 344 Rats. Borghoff, S. J., Gargas, M. L., Andersen, M. E., and Conolly, R. B. (1995). Fundam. Appl. Toxicol. 25, 124-137. A mechanism-based dosimetry model was developed to describe 2,4,4-trimethyl-2-pentanol (TMP-2-OH) dosimetry and renal α2u-globulin (α2u) nephropathy in the male Fischer 344 rat. Experimental data were collected to estimate the chemical-specific parameters (metabolic constants, tissue solubility, and oral absorption rate) necessary to describe TMP-2-OH dosimetry in male rats. The concentrations of α2u and TMP-2-OH were measured in male rats up to 64 hr after a single oral dose of TMP-2-OH (6, 60, or 600 mg/kg). The model predicted the time course behavior of TMP-2-OH and α2u in the kidney, but overestimated their renal concentrations by two or threefold. Simulations of renal α2u concentration were sensitive to changes in TMP-2-OH-α2u-binding affinity and degradation rate of the TMP-2-OH-protein complex. In contrast, simulation of the concentration of TMP-2-OH in the kidney was most sensitive to the amount of protein present. Oral absorption of TMP-2-OH was dose dependent. The model predicted that α2u and TMP-2-OH concentration in the kidney is sensitive to changes in the rate of TMP-2-OH absorbed after oral administration. This model permitted a more rigorous evaluation than has previously been possible of the combination of protein characteristics and chemical dosimetry required for the accumulation of α2u in the kidney of male rats. The behavior of the model is consistent with the qualitative aspects of the α2u hypothesis. However, further characterization of α2u distribution and renal hydrolysis will be required in order to fully characterize the hypothesis at the quantitative level.

Comparative Hyaline Droplet Nephropathy in Male F344/NCr Rats Induced by Sodium Barbital and Diethylacetylurea, a Breakdown Product of Sodium Barbital

Hyaline droplet nephropathy in male rats due to α2u-globulin accumulation in proximal tubules is caused by chemicals from several chemical classes. We have previously shown that the well-known sedative/hypnotic barbiturate, sodium barbital, and its breakdown product, diethylacetylurea, are renal toxins and renal tumor promoters. To determine comparative induction of hyaline droplets in renal tubules by sodium barbital and diethylacetylurea, male F344/NCr rats, 6 weeks of age, were given diets containing 0, 170, 341, 500, or 1000 ppm of diethylacetylurea or containing 500, 1000, or 4000 ppm of sodium barbital for periods of 2 or 10 weeks. Rats were terminated at 2 or 10 weeks and the histology of the kidney was evaluated using light microscopy with hematoxylin and eosin staining and staining by the Heidenhain method. Quantitative analysis showed dose responses for the degree of droplet accumulation in the P2 and P3 segments of the proximal tubules. Diethylacetylurea was more potent. Immunohistochemistry and ultrastructural evaluation revealed the nature of the droplets. Western blotting confirmed the presence of α2u-globulin. Renal tubular necrosis, regeneration, and increased levels of cell proliferation using proliferating cell nuclear antigen immunohistochemistry were also found. Female rats similarly exposed to each chemical did not show tubule droplet accumulations nor renal lesions. We confirm for the first time that these two chemicals can be added to the enlarging list of nephrotoxic chemicals inducing α2u-globulin nephropathy and possessing tumor promoting and renal carcinogenic properties.

An alternative hypothesis on the role of chemically induced protein droplet (α2u-globulin) nephropathy in renal carcinogenesis

Based on associations between the accumulation of protein droplets containing α2u-globulin in proximal tubular epithelial cells and increased incidences of renal tubular neoplasms in male rats, it has been suggested that the carcinogenicity of chemicals that cause α2u-globulin nephropathy is unique to animals that synthesize this protein. Chemicals that caused α2u-globulin nephropathy and renal carcinogenicity in male rats have not been shown to produce renal tumors in animals that lack the capability for hepatic α2u-globulin systhesis, including female rats, male NBR rats, or mice of either sex. Because humans do not synthesize α2u-globulin it has been suggested that chemicals which cause renal toxicity associated with α2u-globulin accumulation do not pose an increased cancer risk to humans. In this review on the association between α2u-globulin nephropathy and renal carcinogenesis, it is apparent that (a) there are data inconsistent with the hypothesis linking these occurrences, (b) alternative mechanisms of renal toxicity and carcinogenicity are plausible, (c) data on quantitative dose-response correspondences between the various stages of α2u-globulin nephropathy and renal carcinogenicity are limited, and (d) a greater understanding of the molecular changes occurring during renal carcinogenesis is needed before assuming that the current hypothesis is correct. Future research aimed at resolving issues raised in this paper should help determine whether or not the association between α2u-globulin nephropathy and renal carcinogenesis represents a cause-and-effect relationship.

A comparison of European High Test gasoline and PS-6 unleaded gasoline in their abilities to induce α2 u-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, α 2 u -globulin (α2u) 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, α2u 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 α2u 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.

The metabolism of 2-methylheptane in male Fischer 344 rats

The urinary metabolites of 2-methylheptane (2-MH) in male Fischer 344 rats, administered the hydrocarbon by gavage, included 2-methyl-1,2-heptanediol, 2-methyl-1,5-heptanediol, 2-methyl-2,5-heptanediol, 2-methylheptanoic acid and 2-methyl-5-hydroxy-1-heptanoic acid. Metabolism strongly favored the formation of diols. The metabolites were identified using gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). Histopathologic examination of the kidneys revealed minimal hyaline droplet formation (α2u-globulin nephropathy) in the proximal tubule area.

Comparison of Hyaline Droplets in Rats With Chronic Progressive Nephropathy And Chemical-Induced .ALPHA.2u-Globulin Nephropathy.

Hyaline droplets in kidney tubules of rats with chronic progressive nephropathy were histopathologically compared with those found in 2, 2, 4-trimethylpentane (TMP) induced α2u-globulin nephropathy. Repeated oral administration of TMP at the dosage of 50mg/kg/day for four weeks caused development of hyaline droplets which were all immunohistochemically positive for α2u-globulin. In control rats (13 to 52 weeks-old), two types of hyaline droplets were observed: one small round and multiple type being deeply eosinophilic and refractile (hyaline droplet type); and the other being slightly eosinophilic, large, and generally single (eosinophilic body type). Both were positive for anti-α2u-globulin immunohistochemical staining. Fifty-two and 109 weeks-old male rats with chronic progressive nephropathy showed “hyaline droplet degeneration” of tubules with hyaline cast formation in the lumina. Immunohistochemical examination revealed such “hyaline droplet degeneration” to be positive for albumin, but negative for α2u-globulin. Under the electron microscope, round, amorphous, and moderately electron-dense phagolysosomes were observed in the tubules of “hyaline droplet degeneration” cases, clearly different from the polyangular and crystalline-like electron-dense phagolysosomes of normal α2u-globulin reabsorption droplets in young rats or TMP induced α2u-globulin accumulation droplets. The present results suggest that the etiology of chronic progressive nephropathy may be somewhat different from that of α2u-globulin induced nephropathy in terms of the causative protein.