Assessment of the subchronic oral toxicity of d-limonene in dogs

Several hydrocarbons, including d-limonene, have been shown to produce a male-rat-specific nephrotoxicity that is manifested acutely as exacerbation of hyaline droplet formation. In a study to assess the presence or absence of this response in a non-rodent species, the dog was selected as a relevant model because of an earlier report suggesting that d-limonene may be nephrotoxic in this species. Five male and five female adult beagle dogs per treatment group were gavaged twice daily over a 6-month period with tap-water (control) or d-limonene at 0.12 or 1.2 ml/kg body weight/day (100 or 1000 mg/kg body weight/day). The highest daily dose was determined in a pilot study to be close to the maximum tolerated dose for emesis (ED50 1.6 ml/kg body weight). The test compound was administered in divided doses to minimize the incidence of emesis. Feed consumption and body weight were unaffected by treatment. Linear regression analyses indicated a positive dose-related trend for absolute and relative female kidney weight and relative male kidney weight. There were no histopathological changes in the kidneys, evaluated by both haematoxylin and eosin and Mallory-Heidenhain staining, that could be associated with the organ-weight changes. Furthermore, there was no evidence of hyaline droplet accumulation nor of any other sign of hydrocarbon-induced nephropathy typical of those seen in male rats treated with d-limonene. Thus, dogs are refractory to the hyaline droplet nephropathy observed in male rats, thereby providing additional evidence that the male rat kidney is uniquely sensitive to hydrocarbons like d-limonene, and that this specific male rat nephropathic response may be inappropriate for interspecies extrapolation and human risk assessment.     

Review of kidney sections from a subchronic d-limonene oral dosing study conducted by the National Cancer Institute

d-Limonene administered by oral gavage at 150-2400 mg/kg/day in a subchronic (91-day) study conducted for the National Cancer Institute induced renal alterations in male rats at all dose levels, whereas kidneys of male mice, female rats and female mice were unaffected. The renal alterations were dose responsive, and were similar to changes observed as sequelae to oral or inhalation exposure to decalin, a model compound used in a volatile hydrocarbon toxicology programme. Decalin induces a nephrotoxic response unique to the male rat, but the primary response associated with decalin exposure--hyaline droplet formation within the cytoplasm of the epithelial cells of the proximal convoluted tubule--was not recognized in the kidneys of d-limonene-exposed male rats. A possible explanation for the absence of this primary response from kidneys of the d-limonene-treated male rats could be the 4-5-day interval between administration of the final dose and the killing of the animals.     

Morphogenesis of decalin-induced renal alterations in the male rat

Adult male Fischer 344 rats were killed after 5, 12, 19 or 31 days' 'occupational' (6 hr/day, 5 days/wk), 'semi-continuous' (22 hr/day, 5 days/wk) or 'continuous' (22 hr/day, 7 days/wk) exposure to 125 ppm decalin vapour. Control rats were exposed to filtered air. Kidney sections were evaluated to determine the nature and time-course of development of decalin-induced lesions. The development of renal lesions was characterized by a specific sequence of light microscopically evident alterations. The extent of the alterations was dependent on time and exposure regimen. Severe exacerbation of the spontaneous protein accumulation (hyaline droplets) routinely observed in the kidneys of control male rats was present in kidneys of all decalin-exposed animals at day 5, and was considered to be the primary morphological alteration associated with decalin exposure. The following sequelae of the hyaline droplet response were observed: the variable occurrence of light microscopically evident proximal convoluted tubule (PCT) epithelial cell degeneration/necrosis, presumably a reflection of cellular injury associated with excessive protein accumulation; the occurrence of granular casts at the junction of the inner and outer bands of the outer zone of the medulla secondary to PCT epithelial cell injury; chronic nephrosis, occurring secondary to tubular obstruction by granular casts. This triad of lesions (hyaline droplet accumulation, granular cast formation and chronic nephrosis) lends specificity to the decalin response and establishes a potential mechanistic relationship with other chemicals that induce these effects.     

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)     

Decalin-induced nephrotoxicity: light and electron microscopic examination of the effects of oral dosing on the development of kidney lesions in the rat

Male and female Fischer 344 rats were given decalin by oral gavage for 5 or 12 consecutive days in order to determine whether oral dosing would result in light microscopically evident renal effects that were comparable to those that have been observed after inhalation exposure. Decalin (in corn oil vehicle) was administered at doses of 0, 0.1, 0.5, 1.0 or 2.0 g/kg body weight to male rats, and 0, 1.0, 1.5, 1.75 or 2.0 g/kg to female rats. Biopsies of the kidneys of selected control and high-dose male rats were taken for examination by electron microscopy. Sections of kidneys from all control and treated rats were examined by light microscopy. The kidneys of all male control rats contained minimal levels of hyaline droplets within the cytoplasm of proximal convoluted tubule (PCT) epithelial cells. Decalin-induced alterations in the kidneys of male rats included an exacerbation of the hyaline droplet/globule levels found in controls and the formation of granular casts in the outer zone of the renal medulla. The exacerbated formation of hyaline droplets was characterized light microscopically by a marked dose-related increase in the number and size of individual droplets/globules and ultrastructurally by a marked increase in the size range of, and the presence of crystalline inclusions in, the PCT epithelial cell phagolysosomal populations. No other ultrastructural alterations occurred that differentiated treated male rats from control males. The formation of granular casts was dose and time related, occurring in 60% of male rats given 0.5 g decalin/kg for 12 days and in 100% of those given 1.0 g decalin/kg for 12 days. Light microscopy revealed no differences between the kidneys of control and decalin-treated female rats, and no hyaline droplets or granular casts were observed in the kidneys of any female rat killed after 5 or 12 days. These results were in agreement with those of inhalation studies and provide additional evidence that the formation of hyaline droplets in response to exposure to volatile hydrocarbons may be unique to the male rat.     

Development of a short-term model of decalin inhalation nephrotoxicity in the male rat

Fischer 344 male rats and C57BL/6 male mice were exposed 'continuously' (22 hr/day, 7 days/wk) for 20, 28 or 35 days to a model compound, decalin, at 0, 25, 62.5 or 125 ppm. Fischer 344 female rats were exposed 'continuously' to decalin at 0 or 125 ppm for 28 days. No histopathological changes were observed in selected organs of female rats or male mice exposed to up to 125 ppm decalin for 28 or 35 days, respectively. However, kidney lesions were observed in all three test groups of male rats after 20, 28 and 35 days' exposure. The nephrotoxicity was characterized by the formation of hyaline droplets in the cytoplasm of proximal convoluted tubule epithelial cells, by the presence of granular casts at the outer zone of the medulla, and by chronic nephrosis. These changes were time and dose dependent and were identical to the renal toxicity that has been reported to occur in male rats following 90 days of continuous exposure to decalin by inhalation. No histopathological effects were observed in the heart, liver, lung or nasal turbinates of male rats. Our results indicate a sex and species specificity for the kidney toxicity. This leads to questions with regard to the appropriateness of using the male rat to assess the potential inhalation toxicity of volatile hydrocarbons. By producing nephrotoxicity in less than 90 days, decalin may now be used to examine, in a well-defined manner, the effect on nephrotoxicity of variables such as dose, exposure regimen, sex, species, and route of exposure. Data from these studies can be used to ascertain whether or not the male rat is an appropriate test animal for predicting potential human nephrotoxic responses to volatile chemicals such as perfumes and perfume raw materials.     

Acute and subchronic nephrotoxicity of d-limonene in Fischer 344 rats

In the studies described here, we have examined the sex-specific sensitivity of rat kidney to d-limonene. At 24 hr after an acute dose of 200 mg d-limonene/kg body weight administered to adult male and female Fischer 344 rats by oral gavage, an increase in the incidence and severity of hyaline droplets was observed in the kidneys of males only. This histological change was accompanied by a treatment-related increase in alpha 2u-globulin in males only and a greater accumulation of radioactivity in renal cortex of the male rat compared with that in the females dosed with [14C]d-limonene. In a separate subchronic study, groups of 5-wk-old male rats were administered d-limonene in a corn oil vehicle at 0 (control), 2, 5, 10, 30, or 75 mg/kg body weight by single daily gavage (5 days/wk) for 13 wk. Rats from selected dose groups received interim necropsies from days 8-29, while all groups were necropsied at the end of the study. Linear regression analyses indicated a dose-related trend in the increased relative weights of the kidney and liver at 30 and 75 mg d-limonene/kg body weight. Histological examination of kidney tissue confirmed that d-limonene induced changes characterized by hyaline droplets, granular casts at the corticomedullary junction and multiple cortical changes collectively classified as chronic nephrosis. The no-observable-effect level for these effects was 5 mg d-limonene/kg body weight. At the earliest necropsy, 8 days after the start of the treatment, it was evident that d-limonene exacerbated the hyaline droplets at the 10 mg/kg body weight dose. It is concluded that treatment with d-limonene caused an increase in the formation of hyaline droplets in male rats only, that this increase was associated with an accumulation of alpha 2u-globulin, that d-limonene (or its metabolite) accumulated significantly in male rat kidney compared with that in females and that subchronic dosing produced a triad of morphological changes in the male rat kidney. These observations suggest that d-limonene caused nephrotoxicity specific to the male rat and that this toxicity may not be predictive of a similar response in humans.     

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.     

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.     

Biochemical basis for mouse resistance to hyaline droplet nephropathy: lack of relevance of the alpha 2u-globulin protein superfamily in this male rat-specific syndrome.

It is well-established that binding of a chemical to alpha 2u-globulin is the rate-limiting step in the development of male rat-specific hyaline droplet nephropathy. Mice synthesize mouse urinary protein (MUP), a protein which is very similar to alpha 2u-globulin, but this protein does not render the mouse sensitive to a similar renal toxicity. Therefore, the purpose of the present study was to determine the biochemical basis for mouse resistance to hyaline droplet nephropathy. Male Fischer 344 rats and B6C3F1 mice excreted 12.24 +/- 0.60 and 14.88 +/- 0.99 mg of alpha 2u-globulin and MUP daily, indicating that quantitative differences in protein excretion were not involved in the species specificity of the nephropathy. With d-limonene as a model hyaline droplet inducing agent, both rat and mouse liver microsomes oxidized the terpene to its 1,2-epoxide (the metabolite that binds reversibly to alpha 2u-globulin in vivo), demonstrating that metabolic differences do not determine the mouse resistance to this lesion. In spite of the formation of the epoxide intermediate, no binding of [14C]d-limonene equivalents to mouse kidney proteins was observed. In contrast, about 40% of the d-limonene equivalents in male rat kidney was reversibly bound to renal proteins. The renal reabsorption of alpha 2u-globulin and MUP was markedly different, as rats reabsorbed about 60% of the total filtered load of alpha 2u-globulin, but MUP was not reabsorbed by the mouse kidney. Given the absence of MUP in mouse kidney, in vitro equilibrium saturation binding studies were also conducted to determine whether MUP could bind the epoxide metabolite. alpha 2u-Globulin bound [14C]d-limonene-1,2-oxide with an apparent Kd of 4 x 10(-7) M. However, under identical experimental conditions, MUP failed to bind the epoxide. These data indicate that two major biochemical differences between alpha 2u-globulin and MUP contribute to mouse resistance to hyaline droplet nephropathy. Under both in vivo and in vitro conditions, MUP does not bind d-limonene-1,2-oxide, the rate-limiting step in the development of the nephropathy. However, even if MUP did bind the epoxide, the fact that it is not reabsorbed into the mouse kidney precludes its involvement in a syndrome involving renal protein overload. Finally, the absence of an interaction between d-limonene, a model hyaline droplet inducer, and the protein most similar to alpha 2u-globulin suggests that no other protein in the alpha 2u-globulin superfamily is likely to cause hyaline droplet nephropathy in other species.     

Renal biomarker changes associated with hyaline droplet nephropathy in rats are time and potentially compound dependent

Alpha 2u-globulin mediated hyaline droplet nephropathy (HDN) is a male rat specific lesion induced when a compound or metabolite binds to alpha 2u-globulin. The objective of this study was to investigate if the newer and more sensitive renal biomarkers would be altered with HDN as well as be able to distinguish between HDN and oxidative stress-induced kidney injury. Rats were dosed orally for 7 days to determine (1) if HDN (induced by 2-propanol or d-limonene) altered the newer renal biomarkers and not BUN or creatinine, (2) if renal biomarkers could distinguish between HDN and oxidative stress-induced kidney injury (induced by potassium bromate), (3) sensitivity of HDN-induced renal biomarker changes relative to d-limonene dose, and (4) reversibility of HDN and renal biomarkers, using vehicle or 300 mg/kg/day d-limonene with 7 days of dosing and necropsies scheduled over the period of Days 8–85. HDN-induced renal biomarker changes in male rats were potentially compound specific: (1) 2-propanol induced mild HDN without increased renal biomarkers, (2) potassium bromate induced moderate HDN with increased clusterin, and (3) d-limonene induced marked HDN with increased αGST, μGST and albumin. Administration of potassium bromate did not result in oxidative stress-induced kidney injury, based on histopathology and renal biomarkers creatinine and BUN. The compound d-limonene induced a dose dependent increase in HDN severity and renal biomarker changes without altering BUN, creatinine or NAG: (1) minimal induction of HDN and no altered biomarkers at 10 mg/kg/day, (2) mild induction of HDN with increased αGST and μGST at 50 mg/kg/day and (3) marked induction of HDN with increased αGST, μGST and albumin at 300 mg/kg/day. HDN induced by d-limonene was reversible, but with a variable renal biomarker pattern over time: Day 8 there was increased αGST, μGST and albumin; on Day 15 increased clusterin, albumin and Kim-1. In summary, HDN altered the newer and more sensitive renal biomarkers in a time and possibly compound dependent manner.     

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.     

Nephrotoxicity of hexachloro-1:3-butadiene in the rat: The effect of age,sex, and strain

Adult male rats are less susceptible to hexachloro-1:3-butadiene-induced nephrotoxicity than adult female and young male rats. A single dose of 50 mg/kg hexachloro-1:3-butadiene (HCBD) ip in adult Alderley Park (Wistar-derived) females produced marked renal tubular necrosis and an increased plasma urea by 24 hr. Young male rats were also more susceptible to HCBD-induced nephrotoxicity; a dose of 25 mg/kg produced marked tubular necrosis and an increased plasma urea in 21-day-old rats, while a dose of 200 mg/kg was required to produce a similar response in adult males. p-Aminohippurate accumulation by thin slices of renal cortex from rats treated 24 hr previously with HCBD was reduced in adult but not in young male rats. HCBD was more toxic to young male rats (21 and 29 days old, LD50 57 and 96 mg/kg. respectively) than to adult males (7 weeks old, LD50 360 mg/kg). HCBD administration produced a more marked nephrotoxicity in 21-day-old rats treated with phenobarbital in their drinking water for 7 days than in rats of the same age not treated with phenobarbital. Associated with the increased susceptibility of female rats, renal nonprotein sulfhydryl content (NP-SH) was decreased in female but not in male rats 4 hr after HCBD administration. This decrease suggests conjugation of HCBD by the female rat kidney. The sex and age differences observed in nephrotoxicity due to HCBD are probably related to differences in hepatic and renal enzymes responsible for the detoxification and/or activation of HCBD. Fischer 344 rats were slightly more susceptible and Long Evans rats slightly less susceptible than the Alderley Park strain to HCBD-induced nephrotoxicity, although the differences were not as marked as those seen with age and sex.     

Accumulation of alpha 2u-globulin in the renal proximal tubules of male rats exposed to unleaded gasoline

Saturated branched-chain aliphatic hydrocarbons, found in motor fuels, induce nephrotoxicity in male rats. Treatment of male rats with unleaded gasoline (0.04-2.0 ml/kg body wt, po) for 9 days increased markedly the number and size of hyaline (protein resorption) droplets in epithelial cells of the renal proximal convoluted tubules (PCT) and enhanced cellular exfoliation at high dose levels. No other treatment-related pathological effects were observed in the glomeruli, distal tubules, or medulla. The renal content of alpha 2u-globulin, a major urinary protein of male rats, was increased maximally by about 4.4-fold after gasoline administration (1.0 ml/kg, po, 9 days); no further increase was observed at higher doses. Immunoperoxidase staining of kidney tissue sections for alpha 2u-globulin revealed large accumulations of antigen localized in many of the PCT epithelial cells which contained hyaline droplets. The hepatic content of alpha 2u-globulin and its mRNA were not altered by gasoline administration. These data show, for the first time, that alpha 2u-globulin is accumulated in the kidneys of gasoline-intoxicated male rats and sequestered specifically in some of the hyaline droplets characteristic of gasoline-induced nephropathy. A hydrocarbon-induced defect in the renal lysosomal degradation of low-molecular-weight urinary proteins, rather than increased synthesis of these proteins, appears to cause hyaline droplet accumulation.     

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.     

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.     

Induction of light hydrocarbon nephropathy by p-dichlorobenzene

In order to clarify the etiology of a dose-related increase in the incidence of tubular cell adenocarcinomas of the kidney in male rats, the nephrotoxicity of p-dichlorobenzene (p-DCB) was investigated in a subchronic study. Groups of ten male and ten female Fischer 344 rats were dosed by gavage with 0 (controls), 75, 150, 300 or 600 mg p-DCB/kg/day in corn oil. Half of the animals were sacrificed after 4 weeks and the remainder after 13 weeks. Increased urinary LDH and epithelial cell excretion and exacerbation of hyaline droplet accumulation in the cytoplasm of renal cortical cells were observed in male rats over the entire dose range investigated. Tubular single cell necrosis, dilated tubules with granular cast formation in the outer zone of the medulla, were evident in male rats after 4 and 13 weeks of treatment with doses of 150–600 mg/kg/day. In female rats there was no indication of a nephrotoxic action of p-DCB. The effects on the kidney, both in their morphological characteristics and the fact that they occur exclusively in male animals, correspond to the light hydrocarbon nephropathy observed as a result of short-term treatment with a number of aliphatic and cyclic hydrocarbons. The development of cortical renal tumors seems to be associated with this kind of kidney damage which is unique to male rats. The literature on this subject generally regards these renal effects as not predictive for man.     

Lysosomal degradation of alpha 2u-globulin and alpha 2u-globulin-xenobiotic conjugates.

A diverse group of chemicals cause a male rat-specific nephrotoxicity in which alpha 2u-globulin accumulates in renal lysosomes. It has been suggested that these chemicals bind to the protein and decrease its degradation by lysosomal proteinases. To test this hypothesis, the lysosomal degradation of native alpha 2u-globulin and that to which d-limonene, d-limonene-1,2-oxide, isophorone, 1,4-dichlorobenzene, and 2,5-dichlorophenol were bound was studied. alpha 2u-Globulin was purified from male rat urine, and male rat renal cortical lysosomes, isolated by differential centrifugation, served as the proteolytic enzyme source. Pepstatin, an inhibitor of aspartic acid proteinases, and leupeptin, an inhibitor of cysteine proteinases, reduced alpha 2u-globulin degradation to 28 +/- 8 and 17 +/- 5% of control, respectively, whereas addition of both inhibitors decreased alpha 2u-globulin degradation to 8 +/- 1% of control values. These results indicate that both classes of endopeptidases are important in the degradation of alpha 2u-globulin. Under the incubation conditions used, 30% of native alpha 2u-globulin was degraded in a 4-hr period. Conjugates of the protein were made for in vitro binding experiments. Binding of d-limonene and 1,4-dichlorobenzene to alpha 2u-globulin did not alter the degradation of the protein, whereas binding of d-limonene-1,2-oxide, 2.5-dichlorophenol, and isophorone decreased alpha 2u-globulin degradation by 33%. These results indicate that not all chemicals which have been shown to bind in vivo to alpha 2u-globulin alter the in vitro lysosomal degradation of the protein. However, in all cases, one metabolite of each hyaline droplet inducer did alter degradation of alpha 2u-globulin, suggesting that a decrease in lysosomal degradation is involved in the accumulation of this protein in male rat kidney lysosomes.     

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.     

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.