Bayo, M; Serra, MA; Clerici, LA
Mercury compounds are environmental pollutants long recognised as causing detrimental effects in the nervous system, inorganic mercury (Hg2J and, particularly, methylmercury (MeHg) are neurotoxic (Chang, 1990). Exposure to the metal causes cell death by the activation of apoptosis, either with Hg2+ (Rossi et al., 1997) or with MeHg (Nagashima et al., 1996). Environmental mercury contamination has increased very much in developed countries, thus increasing the risk of human exposure. The use of in vitro systems allows the characterisation of the mode of action and molecular effects of toxic metals, thus giving further and/or complementary insight in their neurotoxic mechanism to classical in vivo studies. A general aim of these studies is also to develop biomarkers of exposure/effect of these metals (Manzo et al., 1996). On the other hand, the possibility of exploiting the heat shock response system as biomarker of stress caused by many physical and chemical agents has been previously recognised (Gonzalez et al., 1989). Recently, it has also been suggested that heatshock protein 70 (hsp70) overproduction can protect neurons against several types of injury (Sharp et al., 1999).
The developing central nervous system of fetuses and neonates and, particularly, the cerebellum is one of the main targets of mercury toxicity and a selective damage is produced in these cells (Choi, 1989). In particular, cerebellar granule cells (CGCs), which are characterized as glutamatergic neurons (i.e. they synthesize, store and release glutamate (Glu) and aspartate (Asp) upon stimulation), are sensitive to methyl mercury (Clarkson, 1987, Fonnum and Lock, 2000). Moreover, interference of Hg"+ with the glutamatergic pathway has been suggested as a potential mechanism underlying mercury neurotoxicity (Chang, 1990). Primary cultures of cerebellum neurons (cerebellar granule cells, CGCs) are thus a suitable model for in vitro neurotoxicity studies with mercury.
We have already shown that primary cultures ofhepatocytes, embryonic fibroblasts, as well as kidney, lung and bone marrow cells, derived from our transgenic mouse model, in which a human growth hormone (hGH) gene has been put under the control of the heat shock protein 70 (hsp70) gene promoter, are quite sensitive to heavy metal insult, including MeHg (Sacco et al., 1997). Analogously, mouse NIH-3T3 fibroblasts transfected with this chimeric gene have been also shown to respond to toxic metals, including Hg2+ (Fischbach et al., 1993). The detection of the reporter gene product (hGH) in the extracellular medium by specific immunoassays constituted a quite practical advantage.
The aim of the present work was to investigate the usefulness of primary CGCs derived from our transgenic mouse model to study neurotoxic effects of inorganic (Hg2+) and organic (MeHg) mercury. A comprehensive strategy was proposed integrating cytotoxicity assays (MTI test) with the evaluation of the stress response (induction of hsp70). The possible use of the induction of hsp70 as biomarker of mercury-induced neurotoxicity is discussed.
