• Investigation into a role for metallothionein in cadmium-induced immunotoxicity and immunocompetence of hemocytes from the eastern oyster, Crassostrea virginica

      Butler, Rondi Ann; Roesijadi, Guritno (2000)
      Metallothioneins (MT) are a class of low molecular weight, sulfhydryl-rich, metal binding proteins that function in metal tolerance, essential metal metabolism and radical scavenging. Mammalian research suggests MT plays a role in immune function, including the activities of macrophages/monocytes. MT is also found in the macrophage-like cells, hemocytes, of the bivalve, Crassostrea virginica. Hemocytes are known to accumulate high levels of several metals including cadmium (Cd). We hypothesize that MT expression influences hemocyte immune function: modulation of MT expression alters hemocyte resistance to Cd toxicity and alters cellular immune response to invading microorganisms. In vitro exposure of hemocytes to sublethal Cd concentrations had marked effects on MT expression, as measured by a quantitative RT-PCR assay, inducing MT in a dose-dependent manner to a maximum, above which greater Cd exposure inhibited induction. Cadmium also suppressed reactive oxygen species (ROS) levels and lysosomal stability. Conference of protection by oyster MT against Cd-induced toxicity was demonstrated in hemocytes through disruption of MT expression by phosphorothioate oligodeoxynucleotides antisense to a portion of the MT mRNA. Results from down regulation by antisense also suggest a role for MT in ROS generation by hemocytes, consistent with observations from higher organisms. The mechanism by which MT is involved in macrophage and hemocyte ROS generation remains to be elucidated. Biological significance of the Cd and antisense induced changes in immune activities were assessed in a functional assay for microbial killing. Immunocompetence was tested towards two different microbes: the non-pathogenic (to oysters) marine bacterium Bacillus megaterium and the oyster protozoan parasite, Perkinsus marinus. Despite the treatment induced suppression of ROS and decreased stability of the lysosomal compartment, no significant changes were observed in hemocyte ability to destroy either of the test microorganisms. Thus, MT expression does not appear to be critical for hemocytes to mount an effective immune response against at least certain invading microorganisms.
    • A molecular approach to the study of metallothionein induction in oysters

      Unger, Michael Eugene; Roesijadi, Guritno (1995)
      The objective of this study was to provide basic information about molluscan metallothionein (MT) induction at the molecular level in order to increase our understanding of the response of molluscs to metal exposure and metal tolerance. This required the development of tools and methods to analyze MT mRNA in oysters and examination of basic exposure parameters. This work will provide significant basic structural information for use in comparative studies and evaluate a possible mechanism of short-term tolerance in an aquatic invertebrate. The cDNA for oyster MT was isolated by cloning the product of a reverse transcription-polymerase chain reaction amplification. The cDNA was characteristic of MT sequences and confirmed the previous classification of Crassostrea virginica MT as a class I MT. The cDNA was used to develop methods to quantify MT mRNA in oyster gill tissue. Ribonuclease protection-based assays were necessary to quantify MT mRNA in oysters not exposed to metals. These methods were applied to examine the time- and exposure-concentration dependence of oyster gill MT mRNA during Cd exposure and the effect of sublethal Cd challenge on gill MT mRNA accumulation in oysters preexposed to a range of low Cd concentrations. Initial treatments of control (0.0003 {dollar}\mu{dollar}M) and 0.0044, 0.044, and 0.44 {dollar}\mu{dollar}M Cd exposure for 21 d were followed by challenge with 0.44 {dollar}\mu{dollar}M Cd. MT mRNA accumulation during preexposure exhibited concentration- and time-dependence, increasing at all preexposure concentrations. During the challenge, MT mRNA increased in all samples and the concentration-dependence of MT mRNA in relation to preexposure concentrations was maintained. MT mRNA concentrations during the challenge were significantly increased in oysters preexposed to the two highest Cd concentrations in comparison with the controls. Since MT mRNA levels are correlated with the rate of MT translation in previous studies, these data implicate new MT synthesis in acquired tolerance to Cd toxicity. The results of this study complement ongoing research at the protein level and will be part of a foundation upon which the effects of metal pollution on natural populations of organisms can be evaluated.