Lois D. Lehman-McKeeman

d-Limonene-induced male rat specific nephrotoxicity: evaluation of the association between d-limonene and α2u-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.

Lysosomal degradation of α2u-globulin and α2u-globulin-xenobiotic conjugates☆

Abstract A diverse group of chemicals cause a male rat-specific nephrotoxicity in which α2u-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 α2u-globulin and that to which d -limonene, d -limonene-1,2-oxide, isophorone, 1,4-dichlorobenzene, and 2,5-dichlorophenol were bound was studied. α2u-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 α2u-globulin degradation to 28 ± 8 and 17 ± 5% of control, respectively, whereas addition of both inhibitors decreased α2u-globulin degradation to 8 ± 1% of control values. These results indicate that both classes of endopeptidases are important in the degradation of α2u-globulin. Under the incubation conditions used, 30% of native α2u-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 α2u-globulin did not alter the degradation of the protein, whereas binding of d -limonene-1,2-oxide, 2,5-dichlorophenol, and isophorone decreased α2u-globulin degradation by 33%. These results indicate that not all chemicals which have been shown to bind in vivo to α2u-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 α2u-globulin, suggesting that a decrease in lysosomal degradation is involved in the accumulation of this protein in male rat kidney lysosomes.

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)

Improved high-performance liquid chromatographic procedure for the separation and quantification of hydroxytestosterone metabolites

A reproducible, sensitive high-performance liquid chromatography (HPLC) method has been developed to quantify hydroxytestosterone metabolites. The method features a simple one-step linear gradient of methanol in water (10%-60% methanol) for separation of the testosterone metabolites on a Supelcosil LC-18 column; metabolites are detected at 247 nm. This method provides a distinct advantage over previously developed assays in that the solvent gradient does not contribute to baseline changes throughout the chromatographic run. In this way, the flat baseline markedly improves robustness of the assay and simplifies peak integration and quantification. At the same time, resolution of 15 different steroid metabolites catalyzed by the cytochrome P-450 enzymes are readily separated in rat or mouse liver microsomes. An internal standard, cortexolone, was selected for use based on its structural and spectral similarity to the hydroxytestosterone metabolites, and quantification is based on the molar response for the testosterone/cortexolone peak area ratio. The limit of detection (LOD) is 1 pmol on-column with a limit of quantitation (LOQ) of 4 pmol on-column. The intraday repeatability is approximately 3%. This simplified procedure is straightforward and should greatly facilitate the routine use of the testosterone hydroxylation assay to measure cytochrome P-450 isozyme activity.

α2u-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.

The role of metallothionein induction and altered zinc status in maternally mediated developmental toxicity: Comparison of the effects of urethane and styrene in rats

We hypothesize that maternal metallothionein (MT) induction by toxic dosages of chemicals may contribute to or cause developmental toxicity by a chain of events leading to a transient but developmentally adverse decrease in Zn availability to the embryo. This hypothesis was tested by evaluating hepatic MT induction, maternal and embryonic Zn status, and developmental toxicity after exposure to urethane, a developmental toxicant, or styrene, which is not a developmental toxicant. Pregnant Sprague-Dawley rats were given 0 or 1 g/kg urethane ip, or 0 or 300 mg/kg styrene in corn oil po, on Gestation Day 11 (sperm positive = Gestation Day 0). These were maternally toxic dosages. As both treatments decreased food consumption, separate pair-fed control groups were also evaluated for effects on MT and Zn status and development. In addition, Gestation Day 11 rat embryos were exposed to urethane in vitro in order to determine whether urethane has the potential to be directly embryotoxic. Urethane treatment induced hepatic MT 14-fold over control; styrene treatment induced MT 2.5-fold. The MT induction by styrene could be attributed to decreased food intake, as a similar level of induction was observed in a pair-fed untreated control group. However, the level of MT induction by urethane was much greater than that produced by decreased food intake alone. Hepatic Zn concentration, particularly in the cytosol, was increased in the presence of increased hepatic MT concentration. Plasma Zn concentration was significantly decreased (approximately 30%) by urethane treatment, but not by styrene or food restriction (pair-feeding). Distribution of 65Zn to the liver of urethane-treated dams was significantly greater (by 30%), while distribution to embryonic tissues was significantly lower (by at least 50%) than in pair-fed or ad lib.-fed controls. Styrene treatment had no effect on 65Zn distribution. Urethane was developmentally toxic, causing an 18% decrease in fetal weight and a significant delay in skeletal ossification, but was not toxic to rat embryos in vitro. Styrene was not developmentally toxic. The changes observed after urethane treatment, namely substantial hepatic MT induction and altered maternal and embryonic Zn status, along with the lack of direct embryotoxicity of urethane in vitro, support the hypothesis that these maternal effects contribute to developmental toxicity. The lack of similar changes in styrene-intoxicated dams provides one explanation for its low developmental toxicity at maternally toxic dosages.

Altered Zn status by α-hederin in the pregnant rat and its relationship to adverse developmental outcome

The hypothesis that an acute-phase reaction in the pregnant animal causes a systemic redistribution of Zn, resulting in a transient but developmentally adverse Zn deficiency in the embryo, was tested by treating pregnant rats during organogenesis with alpha-hederin, an agent reported to induce substantial metallothionein (MT) synthesis in rat liver, and determining hepatic MT concentration, hepatic and plasma Zn concentration, and systemic distribution of a pulse of 65Zn after treatment. Developmental toxicity was assessed by evaluating morphologic development in term fetuses. A single dose of alpha-hederin, injected sc at dosages of 3 to 300 mumol/kg, caused an acute phase response, indicated by decreased Fe and Zn, and increased Cu, alpha 1-acid glycoprotein, and ceruloplasmin concentration in plasma, along with a dosage-related increase in maternal hepatic MT concentration. The maximum induction of MT was 11 to 15-fold greater than control and occurred at dosages of 30 mumol/kg and higher, and MT concentration reached its peak 12 to 24 h after treatment. Zn concentration in liver and liver cytosol increased along with MT, reaching a maximum level at dosages of 30 mumol/kg and higher. Plasma Zn concentration decreased after alpha-hederin treatment to a level approximately 75% of control at a dosage of 30 mumol/kg and 50% of control at 300 mumol/kg. Therefore, hepatic MT induction was associated with most, but not all, of the decrease in plasma Zn concentration. Zn distribution was evaluated by giving an oral pulse of 65Zn 8 h after treatment with 0, 30, or 300 mumol/kg alpha-hederin on gestation day 11, and measuring 65Zn levels 18 h after treatment. The fraction of 65Zn distributed to the liver of treated rats (either dosage) was twice that of control, but distribution of 65Zn to other maternal tissues was decreased. 65Zn accumulation by conceptuses was significantly decreased, attributable to decreased accumulation in decidua, but not in visceral yolk sacs or embryos; however, at this stage of development the decidua accounts for a greater quantity of Zn than either of the other products of conception and may serve as the Zn-storing tissue for the conceptus. Both 30 and 300 mumol/kg increased resorption incidence, and 300 mumol/kg also decreased fetal weight and increased the incidence of abnormal fetuses. Serum collected from rats two hours after alpha-hederin treatment (i.e., before the onset of MT synthesis) supported rat embryo development in vitro, whereas serum collected 18 h after treatment did not. Adding Zn to this serum restored normal embryonic development.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochemical basis for mouse resistance to hyaline droplet nephropathy: Lack of relevance of the α2u-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.

Immunohistochemical analysis of Clara cell secretory protein expression in a transgenic model of mouse lung carcinogenesis.

Immunohistochemical methods have been widely used to determine the histogenesis of spontaneous and chemically-induced mouse lung tumors. Typically, antigens for either alveolar Type II cells or bronchiolar epithelial Clara cells are studied. In the present work, the morphological and immunohistochemical phenotype of a transgenic mouse designed to develop lung tumors arising from Clara cells was evaluated. In this model, Clara cell-specific transformation is accomplished by directed expression of the SV40 large T antigen (TAg) under the mouse Clara cell secretory protein (CC10) promoter. In heterozygous mice, early lesions at 1 month of age consisted of hyperplastic bronchiolar epithelial cells. These progressed to adenoma by 2 months as proliferating epithelium extended into adjacent alveolar spaces. By 4 months, a large portion of the lung parenchyma was composed of tumor masses. Expression of constitutive CC10 was diminished in transgenic animals at all time points. Only the occasional cell or segment of the bronchiolar epithelium stained positively for CC10 by immunohistochemistry, and all tumors were found to be uniformly negative for staining. These results were corroborated by Western blotting, where CC10 was readily detectable in whole lung homogenate from nontransgenic animals, but not detected in lung from transgenic animals at any time point. Tumors were also examined for expression of surfactant apoprotein C (SPC), an alveolar Type II cell-specific marker, and found to be uniformly negative for staining. These results indicate that, in this transgenic model, expression of CC10, which is widely used to determine whether lung tumors arise from Clara cells, was reduced and subsequently lost during Clara cell tumor progression.

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.