Metallothionein (MT) -I and MT-II expression are induced and cause zinc sequestration in the liver after brain injury

Abstract

Experiments with transgenic over-expressing, and null mutant mice have determined that metallothionein-I and -II (MT-I/II) are protective after brain injury. MT-I/II is primarily a zinc-binding protein and it is not known how it provides neuroprotection to the injured brain or where MT-I/II acts to have its effects. MT-I/II is often expressed in the liver under stressful conditions but to date, measurement of MT-I/II expression after brain injury has focused primarily on the injured brain itself. In the present study we measured MT-I/II expression in the liver of mice after cryolesion brain injury by quantitative reverse-transcriptase PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) with the UC1MT antibody. Displacement curves constructed using MT-I/II knockout (MT-I/II(-/-)) mouse tissues were used to validate the ELISA. Hepatic MT-I and MT-II mRNA levels were significantly increased within 24 hours of brain injury but hepatic MT-I/II protein levels were not significantly increased until 3 days post injury (DPI) and were maximal at the end of the experimental period, 7 DPI. Hepatic zinc content was measured by atomic absorption spectroscopy and was found to decrease at 1 and 3 DPI but returned to normal by 7DPI. Zinc in the livers of MT-I/II(-/-) mice did not show a return to normal at 7 DPI which suggests that after brain injury, MT-I/II is responsible for sequestering elevated levels of zinc to the liver.

Conclusion: MT-I/II is up-regulated in the liver after brain injury and modulates the amount of zinc that is sequestered to the liver.

Figure 1. Standard curve generated by the UC1MT competitive ELISA with 4-parameter logistic curve fitted.

Each point shown is the average of 4 quadruplicate standards.

Figure 2. Cross-reactivity of the UC1MT antibody for MT-III was tested by direct ELISA.

Comparison of the standard curves for MT-IIA (blue lines) and MT-III (red lines) demonstrate UC1MT has very little if any cross-reactivity for MT-III. Data are expressed as the mean of triplicate measurements (error bars = SEM).

Figure 3. Displacement curves for MT-IIA in MT-I/II^−/− mouse brain homogenate (A) and MT-I/II^−/− mouse liver homogenate (B).

Displacement curves constructed in solutions with protein content of 0.01 mg/ml and 0.1 mg/ml are parallel to the standard curve constructed in PBS. Therefore, no matrix effects were observed at these concentrations, in these tissues (n = 3, error bars = SEM).

Figure 4. Expression of MT-I and MT-II mRNA in the liver of wild type and MT-I/II^−/− mice after brain injury was quantified by RT-PCR.

(A) MT-I mRNA expression showed its greatest increase at 1 DPI and 3 DPI in wild type mice. (B) MT-II mRNA was increased at 1 DPI in wild type mice but was at peak levels at 3 DPI. MT-I/II^−/− mice were unable to increase MT-I and MT-II mRNA levels to the same extent as wild type mice. Groups that share lower case letters are not significantly different from each other (for both graphs; n = 6–7, error bars = SEM).

Figure 5. Liver MT-I/II protein levels after cryolesion injury to the brain were assayed by UC1MT ELISA in wild type mice.

Hepatic MT-I/II protein levels were not increased until 3 DPI and showed a further increase at 7 DPI. Groups that share lower case letters are not significantly different from each other (n = 7, error bars = SEM). signalling mechanism is involved.

Figure 6. Corticosterone concentrations in plasma after cryolesion injury to the brain were assayed by RIA in wild type and MT-I/II^−/− mice (A).

No significant differences were found between the mouse strains. There was a significant increase in plasma corticosterone after cryolesion injury and sham surgery to a similar extent. Sham surgery does not induce a significant change in hepatic MT-I or MT-II mRNA expression (B). Time points that share letters are not significantly different (n = 5, error bars = SEM).

Figure 7. Liver zinc content was measured by atomic absorption spectroscopy.

Liver Zinc content was decreased in both wild type and MT-I/II^−/− mice at 1 and 3 DPI. 1-way ANOVA revealed significantly higher hepatic zinc in wild type than MT-I/II^−/− mice at 7 DPI. Groups that share lower case letters are not significantly different from each other (n = 5–6, error bars = SEM).