Monocrotaline pyrrol is cytotoxic and alters the patterns of GFAP expression on astrocyte primary cultures

Dehydromonoctotaline (DHMC) is the main monocrotaline active cytochrome P450's metabolite, and has already been assessed in the CNS of experimentally intoxicated rats. DHMC effects were here investigated toward rat astroglial primary cultures regarding cytotoxicity, morphological changes and regulation of GFAP expression. Cells, grown in DMEM supplemented medium, were treated with 0.1-500 mu M DHMC, during 24- and 72-h. According to MTT and LDH tests, DHMC was toxic to astrocytes after 24-h exposure at 1 mu M and induced membrane damages at 500 mu M. Rosenfeld dying showed hypertrophic astrocytes after 72-h exposure to 0.1-1 mu M DHMC. GFAP immunocytochemistry and western immunoblot revealed an increase of GFAP labelling and expression, suggesting an astrogliotic reaction to low concentrations of DHMC. At higher concentrations (10-500 mu M), astrocytes shrank their bodies and retracted their processes, presenting a more polygonal phenotype and a weaker expression on GFAP labelling Nuclear chromatin staining by Hoechst-33258 dye, revealed condensed and fragmented chromatin in an important proportion (+/- 30%) of the astrocytes exposed to 100-500 mu M DHMC, suggesting signs of apoptosis. Our results confirm a cytotoxic and dose-dependant effect of DHMC on cultures of rat cortical astrocytes, leading to apoptortic figures. These effects might be related to the neurological damages and clinical signs observed in animals intoxicated by Crotalaria. (C) 2008 Elsevier Ltd. All rights reserved.