Development of an immunohistochemical stain to detect heavy metal contamination in biological materials.

摘要

A low molecular weight cadmium-associated protein was isolated from rainbow trout liver. Molecular weight was determined to be 14,000 D based on sodium dodecyl sulphate polyacrylamide gel electrophoresis. Isoelectric points were 5.0 and 5.9. Amino acid analysis after purification by carbonyldiimidazole affinity chromatography showed the protein contained 4 mol% cysteine, 3.5 mol% phenylalanine, 2.2 mol% tyrosine and 1.8 mol% histidine. This protein was present in both cadmium injected and control fish as determined by Western blotting using polyclonal and monoclonal antibodies against the protein. A monoclonal antibody specific for metallothionein (MAb) (1E9) did not react against the protein in Western blots.; Immunohistochemical analysis of tissue sections from metal-loaded fish demonstrated no reactivity with either polyclonal or monoclonal antibodies against the cadmium-associated protein. However, immunostaining using polyclonal and monoclonal antibodies against rabbit metallothionein was positive in both copper-loaded and control rat sections of kidney, liver and small intestine. In copper-loaded rats, intense staining was present within hepatocytes in periportal regions, renal proximal convoluted tubular epithelial cells, and Paneth cells of the small intestine. Staining was both cytoplasmic and nuclear with variability in staining intensity between cells in copper-loaded rats. Staining followed a similar distribution but was much less intense in control rats. Intensity of immunostaining correlated well with hepatic metal content determined by atomic absorption spectrophotometry (AAS). As hepatic copper content increased so did the intensity of immunostaining. Rubeanic acid staining was present only in copper-loaded rats and appeared as small, discrete intracellular granules within renal proximal convoluted tubular epithelial cells and hepatocytes in periportal areas. (Abstract shortened by UMI.)