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)     

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.     

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.     

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.     

Alpha 2u-globulin is the only member of the lipocalin protein superfamily that binds to hyaline droplet inducing agents.

The rate-limiting step in chemically induced, male rat-specific hyaline droplet nephropathy is the reversible binding of a xenobiotic to alpha 2u-globulin. In this study, equilibrium saturation binding experiments were conducted to evaluate the in vitro binding of d-limonene-1,2-oxide (dLO) and 2,4,4-trimethyl-2-pentanol (TMP-OH) to alpha 2u-globulin and members of the alpha 2u-globulin protein superfamily. Both dLO and TMP-OH bound to alpha 2u-globulin, with Scatchard analysis yielding dissociation constants of 5.6 and 6.4 x 10(-7) M, respectively. The Bmax for binding (nmol bound/mg protein) was 50.7 and 61.1 for dLO and TMP-OH, respectively, yielding a molar ratio of approximately 1 for both ligands. The ability of dLO and TMP-OH to bind to human-derived alpha 1-acid glycoprotein, rat-derived retinol-binding protein, human protein-1, and bovine beta-lactoglobulin was also studied. These superfamily proteins are generally abundant in plasma, are freely filtered across the glomerulus, and can bind a wide range of ligands. However, neither dLO nor TMP-OH bound to any of the superfamily proteins. In contrast, under identical experimental conditions, alpha 1-acid glycoprotein did bind progesterone (Kd = 10(-6) M), whereas both beta-lactoglobulin and retinol-binding protein bound retinol (Kd = 10(-8) M for both proteins). These results indicate that, under conditions where alpha 2u-globulin superfamily proteins bind to established ligands, the proteins do not interact with hyaline droplet inducing agents. Thus, the interaction between male rat-specific nephrotoxicants and alpha 2u-globulin is unique to this protein. More importantly, these results provide direct evidence that the presence of the alpha 2u-globulin superfamily proteins does not predispose humans to develop hyaline droplet nephropathy and renal cancer from this class of chemicals.     

14C-labeled pulegone and metabolites binding to alpha2u-globulin in kidneys of male F-344 rats

Pulegone is a major constituent of pennyroyal oil and a minor component of peppermint oil. Pulegone is biotransformed to menthofuran and menthones (diastereomeric menthone and isomenthone) in pennyroyal and peppermint as well as in rodents. Pulegone and menthofuran are hepatotoxic to rodents, and menthones are less toxic. The metabolism and disposition of pulegone and menthofuran were previously studied in rodents, and higher concentrations of pulegone- and menthofuran-derived radioactivity were observed in male than female rat kidney. One explanation is the association of pulegone and metabolites with a male rat-specific protein, alpha2u-globulin. To test this hypothesis, male and female rats were dosed orally with 14C-labeled pulegone (80 mg/kg, 120 microCi/kg) or menthofuran (60 mg/kg, 120 microCi/kg) or menthones (80 mg/kg, 120 microCi/kg) in corn oil, and the kidney cytosol was prepared 24 h after dosing. An equilibrium dialysis experiment showed that in all three studies the radioactivity was associated with kidney cytosol proteins of male but not female rats. The chemicals present in the male rat kidney cytosol after dialysis were extracted with dichloromethane and characterized by high-performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC-MS). All parent compounds were detected, and the metabolites characterized included piperitone from pulegone or menthones treatment, menthones and possibly 8-hydroxymenthones from pulegone treatment, and mintlactones (diastereomeric mintlactone and isomintlactone) and 7a-hydroxymintlactone from menthofuran treatment. Analysis of the male rat kidney cytosol by a gel filtration column demonstrated that the retention was due to reversible binding of these chemicals with the male rat-specific protein alpha2u-globulin. However, binding of pulegone and/or metabolites to alpha2u-globulin did not produce accumulation of this protein in the kidney.     

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.     

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

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

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.     

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

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

Involvement of reversible binding to alpha 2u-globulin in 1,4-dichlorobenzene-induced nephrotoxicity

Similarly to unleaded gasoline, 1,4-dichlorobenzene (1,4-DCB) administered for 2 years caused a dose-related increase in the incidence of renal tumors in male but not in female rats or in either sex of mice. Unleaded gasoline and 2,2,4-trimethylpentane (TMP), a component of unleaded gasoline, increased protein droplet formation and cell proliferation in male but not in female rat kidneys. These protein droplets contained, alpha 2u-globulin, a male rat-specific low-molecular-weight protein and 2,4,4-trimethyl-2-pentanol, a metabolite of TMP that was reversibly bound to this protein. Studies were undertaken to determine if 1,4-DCB produced similar effects; 1,2-DCB was used for comparison since it did not produce renal carcinogenesis in male rats. Gel filtration chromatography of a 116,000g supernatant prepared from kidneys of 1,4-[14C]DCB-treated rats showed that radiolabel coeluted with alpha 2u-globulin as one sharp peak as opposed to a multipeak pattern observed for 1,2-[14C]DCB; the maximal quantity of radiolabel for 1,4-DCB was twice that for 1,2-DCB. Equilibrium dialysis of kidney cytosol in the presence or absence of sodium dodecyl sulfate demonstrated that the radiolabel was reversibly bound to alpha 2u-globulin; the amount for 1,4-[14C]DCB-treated rats was almost twice as much as that for 1,2-[14C]DCB-treated rats. 1,2-DCB was also shown to be covalently bound to renal alpha 2u-globulin, and covalently bound to liver and plasma high-molecular-weight proteins. 1,4-DCB and, to a minor extent, 2,5-dichlorophenol, the major metabolite of 1,4-DCB, were reversibly bound to renal alpha 2u-globulin from 1,4-DCB-treated rats. 1,4-DCB increased protein droplet formation in male but not in female rat kidneys, whereas equimolar doses of 1,2-DCB showed no effect in either sex. Renal cell proliferation, measured by [3H]thymidine incorporation into renal DNA, was increased after 1,4-DCB but not after 1,2-DCB treatment. Nephrotoxicity and biochemical alterations induced by 1,4-DCB resemble those of unleaded gasoline and suggest that a similar mechanism is involved in the induction of alpha 2u-globulin nephropathy in male rats.     

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.     

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.     

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

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

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.     

Effects of a thirteen-week inhalation exposure to ethyl tertiary butyl ether on fischer-344 rats and CD-1 mice.

The 1990 Clean Air Act Amendments require that oxygenates be added to automotive fuels to reduce emissions of carbon monoxide and hydrocarbons. One potential oxygenate is the aliphatic ether ethyl tertiary butyl ether (ETBE). Our objective was to provide data on the potential toxic effects of ETBE. Male and female Fisher 344 rats and CD-1 mice were exposed to 0 (control), 500, 1750, or 5000 ppm of ETBE for 6 h/day and 5 days/wk over a 13-week period. ETBE exposure had no effect on mortality and body weight with the exception of an increase in body weights of the female rats in the 5000-ppm group. No major changes in clinical pathology parameters were noted for either rats or mice exposed to ETBE for 6 (rats only) or 13 weeks. Liver weights increased with increasing ETBE-exposure concentration for both sexes of rats and mice. Increases in kidney, adrenal, and heart (females only) weights were noted in rats. Degenerative changes in testicular seminiferous tubules were observed in male rats exposed to 1750 and 5000 ppm but were not seen in mice. This testicular lesion has not been reported previously for aliphatic ethers. Increases in the incidence of regenerative foci, rates of renal cell proliferation, and alpha2u-globulin containing protein droplets were noted in the kidneys of all treated male rats. These lesions are associated with the male rat-specific syndrome of alpha2u-globulin nephropathy. Increases in the incidence of centrilobular hepatocyte hypertrophy and rates of hepatocyte cell proliferation were seen in the livers of male and female mice in the 5000-ppm group, consistent with a mitogenic response to ETBE. These two target organs for ETBE toxicity, mouse liver and male rat kidney, have also been reported for methyl tertiary butyl ether and unleaded gasoline.     

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.     

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

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

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.     

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

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