Development of methods to assess metallothionein expression in lake trout (Salvelinus namaycush) during a reproductive cycle and the effects of cadmium and ethynylestradiol

Metallothioneins (MT) are low molecular weight proteins rich in cysteine whose physiological role is thought to include the regulation of essential metals Cu and Zn and protection against heavy metal toxicity and oxidative stress damage. MT has been isolated from a wide variety of terrestrial and aquatic organisms and, because its production increases markedly in the tissues of organisms exposed to certain metals, has been proposed for use as a bioindicator. Specifically, MT is a good indicator of environmental exposure to group IB and IIB metals for fish.; However, before MT can be routinely applied as a bioindicator, the influences of other biotic and abiotic factors and methodological considerations must be addressed. In particular, it has been suggested that MT can vary widely with the reproductive stages of fish as a secondary consequence of intracellular metal mobilization.; Four methods for measuring MT were developed and applied to tissues from lake trout (Salvelinus namaycush). High performance liquid chromatography (HPLC) and western blot analysis were used to measure MT protein concentrations while quantification of mRNA for MT-I and MT-II was performed with Northern blot analysis and real-time PCR. These techniques were used to examine MT expression and protein production throughout a reproductive cycle in lake trout as well as to examine the effects of chronic exposure to Cd in a field setting or the potent estrogen mimic, ethynylestradiol (EE2) in the field and laboratory exposures.; Based on the idea that MT changes through the reproductive cycle of fish and that MT changes when the fish are exposed to Cd and EE2, two hypothesis were established. (a) Concentrations of the metal binding protein metallothionein will vary in female lake trout throughout the year as a function of reproductive stage. (b) Normal seasonal variations in MT concentrations will be disrupted in lake trout chronically exposed to Cd or to 17alpha-Ethynylestradiol.; MT protein and mRNA were elevated during the fall, at the time of spawning, relative to winter, spring and summer. Induction was greater for the MT-I isoform than MT-II. The elevated levels of MT-I during the fall are likely a secondary result of the release of Zn from organelles after the process of vitellogenesis. Higher concentrations of free Zn would enhance production of MT protein and bind and retain the metal away from sites of toxic action.; It has been proposed that contaminants found in complex effluents have the potential to disrupt normal MT regulation. For example, environmentally relevant concentrations of the synthetic estrogen, ethynylestradiol (EE2) can induce production of vitellogenin outside the normal reproductive window. This could affect Zn mobilization and MT expression. However, exposures at environmentally relevant EE2 concentrations were not sufficient to alter seasonal expression patterns of MT protein or mRNA in lake trout. Only when EE2 concentrations exceeded 40 ng/L, was a decrease in liver MT protein observed. The heavy metal Cd, also found in industrial effluents, can induce MT expression. Chronic exposure therefore has the potential to disrupt normal MT regulation.; Lake trout exposed to elevated waterborne concentrations of Cd in an experimentally treated lake were found to exhibit an increase in MT protein. However, no differences were observed in mRNA content of fish from the treated lake. It has been suggested that mRNA may be elevated soon after exposure, but that in chronic exposures elevated protein with no increase mRNA production can occur. This dynamic should be considered when applying MT analysis to fish from a specific location for biomonitoring purposes.; The techniques that have been developed for measuring MT and its isoforms will allow for more widespread application of MT as a bioindicator. Differences in the levels of expression of the two isoforms indicate their different roles in regulating essential metals during the reprodu...