Interactions between metallothionein inducers in rat liver and primary cultures of rat hepatocytes.

The interaction of Zn, stress and endotoxin on liver metallothionein (MT) regulation has been studied in the rat. Zn, stress and endotoxin increased liver MT levels significantly, by 12-, 5- and 8-fold, respectively. The previous administration of Zn to stress or endotoxin treatments increased MT levels by 35- and 42-fold, respectively, indicating a synergistic effect in both cases. In contrast, when liver MT was preinduced by stress, MT levels were further increased by endotoxin only in an additive manner. In another experiment where liver MT induction by stress was studied in control rats and in rats with preinduced MT by Zn, endotoxin or stress, it was found that Zn pretreated animals had higher MT-I mRNA levels than endotoxin- or stress-pretreated ones. No synergisms between dexamethasone, Zn, TNF and IFN were observed in primary culture of hepatocytes. These results suggest that the observed synergisms between Zn and other MT inducers in vivo in the liver is a consequence of increased Zn levels in the body and mobilization capacity, with concomitant MT synthesis.

Development of a competitive double antibody radioimmunoassay for rat metallothionein.

A competitive double antibody radioimmunoassay (RIA) for rat metallothionein (MT) has been developed that has a detection limit of 100 pg and a range of 100 to 100000 pg. The antibody was raised in rabbits against rat MT-2 but it crossreacts equally with MT-1 and MT-2. However, when the assay is done in the presence of 2-mercaptoethanol the antibody is more specific for MT-2. Zn- and Cd- saturated MTs have similar responses in the assay. Addition of Cu(II) to Zn-MT (more than 6 mol Cu/mol MT) in non-reducing conditions modifies the response of the antibody, probably because of Cu(II) oxidation and later MT polymerization. Standard curves developed in the presence of cytosols from brain cortex, hypothalamus or liver did not differ from the standard curve, indicating the absence of interfering substances in the assay. Furthermore, serial dilutions of those cytosols paralleled the response of the standard curve, indicating that the response of the antibody was specific. For comparison, MT levels in some brain areas measured with the present RIA were compared with those measured with an established RIA. In addition, the expected effect of dexamethasone and stress on liver MT levels was clearly identified by this RIA. The results suggest that the present RIA can be used for quantitation of metallothionein.