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.
Lehman-McKeeman
L D
LD
Miami Valley Laboratories, Procter & Gamble Company, Cincinnati, Ohio 45239-8707.
Rodriguez
P A
PA
Takigiku
R
R
Caudill
D
D
Fey
M L
ML
D000284:Administration, Oral; D000510:Alpha-Globulins / Q000378:metabolism*; D000818:Animals; D002250:Carbon Radioisotopes; D002851:Chromatography, High Pressure Liquid; D053138:Cyclohexenes; D003600:Cytosol / Q000378:metabolism; D005260:Female; D007668:Kidney / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D000077222:Limonene; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D012737:Sex Factors; D013729:Terpenes / Q000378:metabolism / Q000633:toxicity*
D000510:Alpha-Globulins; D002250:Carbon Radioisotopes; D053138:Cyclohexenes; D013729:Terpenes; C013803:alpha 2u globulin; D000077222:Limonene