Metallothionein induction in fetal rat brain and neonatal primary astrocyte cultures by in utero exposure to elemental mercury vapor (Hg⁰)

Brain metallothionein (MT) protein and mRNA levels were determined in the fetal rat following in utero (gestational days 7-21) exposure to elemental mercury vapor (Hg⁰; 300 μg Hg/m³; 4 h/day). Total RNA was probed on northern blots with [ α- ³²P]dCTP-labeled synthetic cDNA probes specific for rat MT isoform mRNAs. The probes for MT-I and MT-II mRNA hybridized to a single band of approximately 550 and 450 nucleotides, respectively. Expression of whole brain MT-I mRNA in full-term fetal rats (day 21) was significantly increased (P < 0.03) by in utero exposure to Hg⁰ compared to nonexposed controls. This corresponded to a 14-fold increase (P < 0.001) in fetal brain Hg concentration after in utero Hg⁰ exposure. In addition, astrocytes from both control and in utero Hg⁰-exposed fetuses were isolated, and neonatal primary astrocyte cultures were established and maintained in vitro for up to 3 weeks without additional experimental intervention. Astrocyte monolayers derived from in utero Hg⁰-exposed fetuses consistently expressed increased abundance of MT-I mRNA transcripts after 1, 2, and 3 weeks in culture (P < 0.03, P < 0.01, and P < 0.03, respectively) compared with controls. The abundance of astrocyte MT-II mRNA was unchanged at 1 and 2 weeks in culture, but was significantly increased at 3 weeks in cultures derived from brains of Hg⁰-exposed fetuses (P < 0.04). Consistent with the increase in MT mRNA, an increase in astrocytic levels of MT proteins was noted by western blot analysis and MT-immunoreactivity. These studies suggest that in utero exposure to Hg⁰ induces brain MT gene expression, and that MT mRNAs and their respective proteins are useful quantitative biochemical markers of intrauterine exposure to Hg⁰, a potentially cytotoxic challenge to astrocytes in the developing brain. It is concluded that induction of MT by fetal/neonatal astrocytes represents an attempt by these glial cells to protect against Hg cytotoxicity in maintaining cerebral homeostasis.