Metallothionein Induction in Neonatal Rat Primary Astrocyte Cultures Protects Against Methylmercury Cytotoxicity

Abstract

Metallothionein (MT) protein and mRNA levels were monitored following exposure of rat neonatal primary astrocyte cultures to methylmercury (MeHg). MT-I and MT-II mRNAs were probed on northern blots with an [α-³²P]dCTP-labeled synthetic cDNA probe specific for rat MT mRNA. MT-I and MT-II mRNAs were detected in untreated cells, suggesting constitutive MT expression in these cells. The probes hybridize to a single mRNA with a size appropriate for MT, ∼550 and 350 bp for MT-I and MT-II, respectively. Expression of MT-I and MT-II mRNA in astrocyte monolayers exposed to 2 × 10⁻⁶M MeHg for 6 h was increased over MT-I and MT-II mRNA levels in controls. Western blot analysis revealed a time-dependent increase in MT protein synthesis through 96 h of exposure to MeHg. Consistent with the constitutive expression of MTs at both the mRNA level and the protein level, we have also demonstrated a time-dependent increase in MT immunoreactivity in astrocytes exposed to MeHg. The cytotoxic effects of MeHg were measured by the rate of astrocytic d-[³H]aspartate uptake. Preexposure of astrocytes to CdCl₂, a potent inducer of MTs, completely reversed the inhibitory effect of MeHg on d-[³H]aspartate uptake that occurs in MeHg-treated astrocytes with constitutive MT levels. Associated with CdCl₂ treatment was a time-dependent increase in astrocytic MT levels. In summary, astrocytes constitutively express MTs; treatment with MeHg increases astrocytic MT expression, and increased MT levels (by means of CdCl₂ pretreatment) attenuate MeHg-induced toxicity. Increased MT expression may represent a generalized response to heavy metal exposure, thus protecting astrocytes and perhaps also, indirectly, juxtaposed neurons from the neurotoxic effects of heavy metals.