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Toxicologic Pathology, Vol. 29, No. 6, 607-616 (2001)
DOI: 10.1080/019262301753385933

Protein Kinase Activity Is Central to Rat Germ Cell Apoptosis Induced by Methoxyacetic Acid

Toshimasa Jindo

Reproductive Toxicology Group, National Toxicology Program, NIEHS, Research Triangle Park, North Carolina, Drug Safety Research Laboratory, Daiichi Pharmaceuticals, Tokyo, Japan

Robert N. Wine

Reproductive Toxicology Group, National Toxicology Program, NIEHS, Research Triangle Park, North Carolina, wine{at}niehs.nih.gov

Ling-Hong Li

Reproductive Toxicology Group, National Toxicology Program, NIEHS, Research Triangle Park, North Carolina, Reproductive and Cancer Health Assessment/Office of Environmental Health Hazard Assessment, Sacramento, California 95812

Robert E. Chapin

Reproductive Toxicology Group, National Toxicology Program, NIEHS, Research Triangle Park, North Carolina, Stine-Haskell Research Center, DuPont Pharmaceuticals Company, Newark, Delaware 19711-3507

Methoxyacetic acid (MAA) is a major metabolite of ethylene glycol monomethyl ether (EGME). Previous investigations of the testicular lesion induced by EGME have found that dividing meiotic cells are the most sensitive, although several stages of spermatocytes are also vulnerable. Preliminary data from this lab suggested the involvement of protein kinase activity in the development of this lesion, a hypothesis explored in the present studies. We used cultured seminiferous tubules (STs) from juvenile rats (25-day-old), exposed in vitro to MAA and several inhibitors of protein kinases. Nineteen h following a 5-h exposure to 5 mM MAA (the plasma level in vivo after a toxic dose of EGME), apoptotic spermatocytes were seen in early- and late-stage STs. Cell death was prevented by cotreatment with broad-spectrum inhibitors of protein kinases such as H-7, H-8, K-252a, W-7, and genistein. In corroboration, immunocytochemistry with antibodies to various kinases (PKCµ, {zeta}, and {gamma} , AKAP220, CaMKII, MLCK, and Src) showed increased staining around dying spermatocytes following EGME treatment in vivo. 2D-PAGE, autoradiography, and nanospray mass spectrometry was used to separate and identify proteins whose phosphorylation status was most greatly changed following exposure to MAA. One protein was identified by sequence analysis as being glucose-regulated protein 94 (grp94). Western blotting and immunocytochemistry confi rmed this finding. The data we present implicate kinase activities in the pathogenesi s of this lesion and suggest the involvement of Sertoli cells.

Key Words: Apoptosis • EGME • MAA • protein kinase inhibitors • 2D PAGE • seminiferous tubule culture • rats.

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