Induction of metallothionein mRNA and protein in primary murine neuron cultures

Metallothionein (MT)-I and -II are present in most if not all cells of the body and are readily inducible both in vivo and in vitro. MT-III is present only in brain, primarily in neurons, and is seemingly not inducible. Because it is difficult to determine whether inducing agents reach neurons (in vivo) in sufficient concentrations, it was of interest to directly test the inducibility of MT-III in vitro. A further objective was to examine the inducibility of MT-I and -II so that comparisons could be made to responses in glial cells. Cultures were established from fetal (Days 14-17) CF-1 mice. Cells were treated after they achieved a mature phenotype (around 6 days) with various concentrations of the following MT inducers: dexamethasone (Dex), cadmium (Cd), zinc (Zn), or inorganic mercury (Hg). MT protein was quantified by the Cd-hemoglobin assay at 24, 48, 72, 96, and 120 hr. All inducers produced significant increases in MT protein between 24 and 96 hr. MT protein increased in a dose-dependent manner, and maximum (up to 6-fold over controls) increases were seen at 3, 30, 100, and 1000 microM for Cd, Hg, Zn, and Dex, respectively. The expression of MT-I, -II, and -III mRNA was examined by dot-blot analysis 6 hr following the addition of inducers at concentrations known to maximally stimulate the induction of MT protein. Zinc and Cd produced 2.5 to 3.5-fold increases in MT-I and -II mRNA, whereas Dex and Hg produced 1.5- to 2.5-fold increases. However, all inducers assayed decreased MT-III mRNA about 30-60%. The present results indicate that MT-I and -II are inducible in neurons as they are in astrocytes, but the basal level and induced level of MT protein is about one-third in neurons as in astrocytes. Unlike MT-I and -II, MT-III is apparently not inducible under the present experimental conditions.