193
Toxicology Letters, 53 (1990) 193-195 Elsevier TOXLET 02415
Comparative disposition of d-limonene in rats and mice: relevance to male-rat-specific nephrotoxicity
Lois D. Lehman-McKeeman, Douglas Caudill, Ray Takigiku, Robert E. Schneider and Judith A. Young Human and Environmental Safety Division, Miami
Valley Laboratories, Procter and Gamble Company,
Cincinnati, OH (U.S.A.)
Key words: d-Limonene; Hyaline droplet nephropathy; a2wGlobulin; Mouse urinary protein
It is well established that d-limonene is a male-rat-specific nephrotoxin and nephrocarcinogen. The toxicity of this monoterpene results from the interaction of a metabolite, cis-d-limonene-1,2-oxide, with the male-rat-specific protein, a2u-globulin [l]. This epoxide is a stable inte~ediate which binds reversibly to a2u-globulin and disrupts the lysosomal degradation of the protein, thereby causing its accumulation in renal cortical lysosomes [2]. It is generally believed that the male rat specificity of d-limonene-induced nephrotoxicity is determined by the presence of a2u-globulin exclusively in male rats. However, a family of proteins which is structurally similar to a2u-globulin has been identified [3], raising some concern over the male rat specificity of this syndrome. The protein which is most similar to a2u-globulin is mouse urinary protein (MUP)[4]. This protein is essentially the mouse equivalent of a2u-globulin, as it is the major urinary protein excreted by adult male mice. To determine the abundance of these proteins, a2u-globulin and MUP were quantitated in adult male Fischer 344 rats and male and female B6C3Fl mice with a reverse-phase HPLC method. Male rats excreted 12.24 & 0.60 (n=24) mg of cr2u-globulin/d. Male mice excreted 14.88 Ilt 0.99 (n = 24), whereas female mice excreted 2.06 + 0.25 (n = 24) mg of MUP/d. If normalized to body weight, a male rat (250 g) excretes approximately 50 mg a2uglobulin/kg/d, whereas a male mouse (25 g) excretes approximately 600 mg MUP/kg/ d. However, despite the abundance of a homologous protein, d-limonene is neither nephrotoxic nor nephrocarcinogenic in mice [S]. This difference in the toxicity of d-
for correspondence: Lois D. Lehman-McKeeman, Ph.D., Miami Valley Laboratories, Procter & Gamble Company, P.O. Box 398707, Cincinnati, OH 45239-8707, U.S.A.
Address
0378~274/~/$3..50 @ 1990 Elsevier Science Publishers B.V. (Biom~ica~ Division)
194
limonene between species may result from differences in: (1) the biotransformation of d-limonene; (2) the disposition of the terpene; or (3) the renal protein binding of d-limonene. To determine whether both rats and mice oxidize d-limonene to cis-d-limonene1,2-oxide, liver microsomes, isolated from untreated male Fischer 344 rats and B6C3Fl mice, were incubated with d-limonene, and the formation of the 1,Zoxide was quantitated by gas chromatographic analysis. The results indicated that cis-dlimonene-I ,2-oxide was a minor metabolite formed by both species, representing less than 1% of the total d-limonene metabolized. There were, however, quantitative and qualitative species differences noted in these experiments. Mouse liver microsomes formed approximately 2-times more epoxide than rats and, whereas rat liver microsomes catalyzed formation of only the c-is-isomer of the oxide, mice formed both cisand tvans-isomers of the epoxide. Species differences in renal disposition and protein binding of d-limonene were also determined. Previous experiments have shown that, in rats, d-limonene exhibits sexdependent renal disposition, as the concentration of d-limonene equivalents are approximately 2.5-times higher in males than in females [l]. At 24 h after oral dosing, the amount of d-limonene equivalents in male rat kidney represented approximately 0.1% of the administered dose, and of this total, approximately 407 (or 0.04% of the total dose) bound reversibly to ~2u-globulin. Mice were dosed orally with 500 mg d-limonenelkg (2.75 mCi [14C]d-limonene/kg) to evaluate the renal disposition of the terpene. In mice, no sex differences in the renal disposition of d-limonene were noted. Twenty-four hours after dosing, the amount of d-limonene in kidney was much less than that observed in rats, accounting for 0.023 and 0,015$%of the administered dose for male and female mice, respectively. EquiIibrium dialysis ex~riments demonstrated that there was essentially no binding of d-limonene equivalents to kidney proteins in male or female mice. Additionally, no interaction between d-limonene equivalents and MUP in urine was observed. These experiments suggest that although mice do make cis-d-limonene-1,2-oxide, MUP is sufficiently different from a2u-globulin that the epoxide does not bind to it in vivo. Furthermore, these results provide evidence that the interaction between dlimonene and a2u-globulin is very specific, thereby casting doubt on the likelihood that hyaline droplet nephropathy could occur with other members of the a2u-globulin superfamily, including those present in humans. REFERENCES
1 Lehman-McKeeman, L.D., Rodriguez, P.A., Takigiku, R., Caudill, D. and Fey, M.L. (1989) d-limonene-induced male rat-specific nephrotoxicity: evaluation of the association between d-hmonene and a2uglobulin. Toxicol. Appl. Pharmacol. 99,25&259. 2 Lehman-McK~man, L.D., Rivera-Torres, M.I. and Caudill, D. (1990) Lysosomal degradation of a2uglobulin and a2u-globulin-xenobioti~ conjugates. Toxicol. Appl. Pharmacof. 103,53%548.
195 3 Snyder, S.H., Sklar, P.B. and Pewter, J. (1988) Molecular mechanisms of olfaction. J. Biol. Chem. 263, 13971-13974. 4 Ali, S. and Clark, A.J. (1988) Characterization of the gene encoding ovine beta-lactoglobulin. Similarity to the genes for retinol binding protein and other secretory proteins. J. Mol. Biol. 199,415426. 5 Jameson, C.W., Eustis, S.L. and Hong, L.H. (1990) Toxicology and carcinogenicity studies of d-limonene in male and female F344 rats and B6C3Fl mice. Toxicologist 10, 142 (Abstract).
Toxicology Letters, 53 (1990) 193-195 Elsevier TOXLET 02415
Comparative disposition of d-limonene in rats and mice: relevance to male-rat-specific nephrotoxicity
Lois D. Lehman-McKeeman, Douglas Caudill, Ray Takigiku, Robert E. Schneider and Judith A. Young Human and Environmental Safety Division, Miami
Valley Laboratories, Procter and Gamble Company,
Cincinnati, OH (U.S.A.)
Key words: d-Limonene; Hyaline droplet nephropathy; a2wGlobulin; Mouse urinary protein
It is well established that d-limonene is a male-rat-specific nephrotoxin and nephrocarcinogen. The toxicity of this monoterpene results from the interaction of a metabolite, cis-d-limonene-1,2-oxide, with the male-rat-specific protein, a2u-globulin [l]. This epoxide is a stable inte~ediate which binds reversibly to a2u-globulin and disrupts the lysosomal degradation of the protein, thereby causing its accumulation in renal cortical lysosomes [2]. It is generally believed that the male rat specificity of d-limonene-induced nephrotoxicity is determined by the presence of a2u-globulin exclusively in male rats. However, a family of proteins which is structurally similar to a2u-globulin has been identified [3], raising some concern over the male rat specificity of this syndrome. The protein which is most similar to a2u-globulin is mouse urinary protein (MUP)[4]. This protein is essentially the mouse equivalent of a2u-globulin, as it is the major urinary protein excreted by adult male mice. To determine the abundance of these proteins, a2u-globulin and MUP were quantitated in adult male Fischer 344 rats and male and female B6C3Fl mice with a reverse-phase HPLC method. Male rats excreted 12.24 & 0.60 (n=24) mg of cr2u-globulin/d. Male mice excreted 14.88 Ilt 0.99 (n = 24), whereas female mice excreted 2.06 + 0.25 (n = 24) mg of MUP/d. If normalized to body weight, a male rat (250 g) excretes approximately 50 mg a2uglobulin/kg/d, whereas a male mouse (25 g) excretes approximately 600 mg MUP/kg/ d. However, despite the abundance of a homologous protein, d-limonene is neither nephrotoxic nor nephrocarcinogenic in mice [S]. This difference in the toxicity of d-
for correspondence: Lois D. Lehman-McKeeman, Ph.D., Miami Valley Laboratories, Procter & Gamble Company, P.O. Box 398707, Cincinnati, OH 45239-8707, U.S.A.
Address
0378~274/~/$3..50 @ 1990 Elsevier Science Publishers B.V. (Biom~ica~ Division)
194
limonene between species may result from differences in: (1) the biotransformation of d-limonene; (2) the disposition of the terpene; or (3) the renal protein binding of d-limonene. To determine whether both rats and mice oxidize d-limonene to cis-d-limonene1,2-oxide, liver microsomes, isolated from untreated male Fischer 344 rats and B6C3Fl mice, were incubated with d-limonene, and the formation of the 1,Zoxide was quantitated by gas chromatographic analysis. The results indicated that cis-dlimonene-I ,2-oxide was a minor metabolite formed by both species, representing less than 1% of the total d-limonene metabolized. There were, however, quantitative and qualitative species differences noted in these experiments. Mouse liver microsomes formed approximately 2-times more epoxide than rats and, whereas rat liver microsomes catalyzed formation of only the c-is-isomer of the oxide, mice formed both cisand tvans-isomers of the epoxide. Species differences in renal disposition and protein binding of d-limonene were also determined. Previous experiments have shown that, in rats, d-limonene exhibits sexdependent renal disposition, as the concentration of d-limonene equivalents are approximately 2.5-times higher in males than in females [l]. At 24 h after oral dosing, the amount of d-limonene equivalents in male rat kidney represented approximately 0.1% of the administered dose, and of this total, approximately 407 (or 0.04% of the total dose) bound reversibly to ~2u-globulin. Mice were dosed orally with 500 mg d-limonenelkg (2.75 mCi [14C]d-limonene/kg) to evaluate the renal disposition of the terpene. In mice, no sex differences in the renal disposition of d-limonene were noted. Twenty-four hours after dosing, the amount of d-limonene in kidney was much less than that observed in rats, accounting for 0.023 and 0,015$%of the administered dose for male and female mice, respectively. EquiIibrium dialysis ex~riments demonstrated that there was essentially no binding of d-limonene equivalents to kidney proteins in male or female mice. Additionally, no interaction between d-limonene equivalents and MUP in urine was observed. These experiments suggest that although mice do make cis-d-limonene-1,2-oxide, MUP is sufficiently different from a2u-globulin that the epoxide does not bind to it in vivo. Furthermore, these results provide evidence that the interaction between dlimonene and a2u-globulin is very specific, thereby casting doubt on the likelihood that hyaline droplet nephropathy could occur with other members of the a2u-globulin superfamily, including those present in humans. REFERENCES
1 Lehman-McKeeman, L.D., Rodriguez, P.A., Takigiku, R., Caudill, D. and Fey, M.L. (1989) d-limonene-induced male rat-specific nephrotoxicity: evaluation of the association between d-hmonene and a2uglobulin. Toxicol. Appl. Pharmacol. 99,25&259. 2 Lehman-McK~man, L.D., Rivera-Torres, M.I. and Caudill, D. (1990) Lysosomal degradation of a2uglobulin and a2u-globulin-xenobioti~ conjugates. Toxicol. Appl. Pharmacof. 103,53%548.
195 3 Snyder, S.H., Sklar, P.B. and Pewter, J. (1988) Molecular mechanisms of olfaction. J. Biol. Chem. 263, 13971-13974. 4 Ali, S. and Clark, A.J. (1988) Characterization of the gene encoding ovine beta-lactoglobulin. Similarity to the genes for retinol binding protein and other secretory proteins. J. Mol. Biol. 199,415426. 5 Jameson, C.W., Eustis, S.L. and Hong, L.H. (1990) Toxicology and carcinogenicity studies of d-limonene in male and female F344 rats and B6C3Fl mice. Toxicologist 10, 142 (Abstract).
