Analysis of metallothionein brain gene expression in relation to ethanol preference in mice using cosegregation and gene knockouts.

BACKGROUND: Metallothioneins (MTs) are ubiquitously expressed intracellular proteins that bind heavy metals and are involved in cytoprotection against several types of stress agents including chemicals, hormones, and oxidants. We have previously reported 1 isoform, MT-II, as a possible candidate gene for ethanol (EtOH) preference (EP) determination in mice. METHODS: Semiquantitative RT-PCR was used to determine brain mRNA levels of MT-I and MT-III in 4 inbred mouse strains with variable EP. Following this, cosegregation of MT-II brain expression with EP was analyzed in F2 mice from 2 intercrosses (C57BL/6J x BALB/cJ and C57BL/6J x DBA/2J). Studies on MT-I/MT-II knockout (KO) mice were also undertaken to further explore this relationship. RESULTS: Our results suggest that MT-I is responsive to EtOH, with no evidence of basal-level differences between strains. Conversely, MT-III shows no EtOH response, yet indicates a possible strain-specific feature with C57BL/6J having the lowest levels of brain MT-III. Metallothionein-II expression cosegregates with EP in F2 mice from a C57BL/6J (preferring) and DBA/2J (avoiding) intercross. Although F2 mice from a cross with C57BL/6J and BALB/cJ (avoiding) strains follow a similar pattern, the results are not statistically significant. Metallothionein-I/MT-II knockout (MT-KO) mice appear to have smaller litter sizes as well as higher weight compared with controls (129S1/SvImJ) and also show a slight increase in EP. CONCLUSIONS: Metallothionein-II remains the primary candidate of the mouse MT gene family for involvement in EP. Its effect on EP appears to be dependent on the genetic background. Such conclusions are based on results from C57BL/6J, BALB/cJ, DBA/2J, and 129 inbred mouse strains. Evidence also points to shared neural pathways involved in weight gain and obesity. The complex interactions between MT-II, EP, and weight gain/obesity remain to be studied.

Strain-specific brain metallothionein II (MT-II) gene expression, its ethanol responsiveness, and association with ethanol preference in mice.

BACKGROUND: Metallothioneins (MTs) are ubiquitously expressed intracellular proteins that bind heavy metals such as zinc, copper, and cadmium. Although their specific function has yet to be discovered, they are known to regulate the metabolism of these metals as well as respond to cellular stress agents, particularly oxidants. METHODS: Brain RNA from experimental (8 g/kg 25% ethanol injection) and control (saline injection) mice from four strains (A/J, BALB/cJ, C57BL/6J, DBA/2J) that are known to differ with respect to ethanol preference was used in differential displays. This report includes molecular results on one gene (MT-II) identified. RESULTS: Our results on differential displays suggest that a proportion of genes are differentially expressed across pair-wise strain comparisons. We identified MT-II as a strain-specific and ethanol-responsive gene. The level of MT-II messenger RNA (mRNA) in control mice of A/J, BALB/cJ, C57BL/6J, and DBA/2J strains was variable (0.50, 0.51, 0.90, and 0.14 times G3PDH expression, respectively). The degree of up-regulation in experimental mice was also somewhat variable among strains, ranging from 2.5 to 3.2 times expression over the matched controls. Experiments indicate that the promoter and genomic organization of the MT-II gene is identical in sequence for all four strains, and methylation studies revealed that the MT-II promoter region is unmethylated in the brains of these mice. Interestingly, MT-II expression in control mice demonstrated a positive correlation with the ethanol preference phenotype. CONCLUSION: An increase in MT-II mRNA levels after injection of ethanol is attributed to the antioxidant properties of MT-II. The differential mRNA levels of this gene among four strains are not accounted for by the genomic organization, DNA sequence, or methylation status of this gene. Furthermore, the observed correlation between MT-II mRNA levels and ethanol preference raises an interesting hypothesis about the possible role of MT-II in ethanol effects and preference in mice.