Using an integrated approach to evaluate apoptosis as a biomarker response in estuarine fishes

I used an integrated approach to evaluate apoptosis as a biomarker response in estuarine fishes. The longjaw mudsucker (Gillichthys mirabilis ) was used in field investigations because it is a bottom-dwelling goby found in diverse tidal marshes. In the laboratory, topsmelt ( Atherinops affinis) was used because it is an EPA-toxicity test organism that also inhabits tidal marshes, and cadmium (Cd) was chosen as a model environmental contaminant. First, I determined whether TUNEL and caspase activity assays were reliable methods for measuring apoptosis as a biomarker response in fish from California tidal marshes varying in contamination. Apoptotic DNA fragmentation (TUNEL positive cells) and DEVDase activity (caspase-3-like protease activity) were elevated in topsmelt hepatocytes exposed to non-cytotoxic Cd concentrations in the laboratory. Apoptotic DNA fragmentation and DEVDase activity also were higher in the liver of longjaw mudsuckers from Stege Marsh (ST) relative to fish from the reference marsh. Average concentrations of many sediment contaminants were highest at ST, and apoptotic DNA fragmentation correlated with numerous sediment contaminants. I then examined how apoptosis and altered physiological responses were related to growth of Cd-exposed larval topsmelt. Apoptotic DNA fragmentation and metallothionein-like protein levels were elevated, and Ca content diminished at Cd concentrations that impaired topsmelt growth. Oxygen consumption rates were correlated with growth impairment, and likely increased as a compensatory response to Cd exposure. These results indicate that less energy may have been allocated for growth because of an increased metabolic demand due to apoptosis, metallothionein synthesis, and changes in ion regulation. As part of this study, I applied otolith growth rate analysis to a study of Cd-impaired growth of topsmelt because growth measurements (i.e. final body size) used in standard toxicity tests were insensitive endpoints. Otolith growth rate analysis allowed for the detection of small differences in growth rates, even when differences in somatic growth were not observed. In sum, apoptosis was a sensitive biomarker response because (i) liver apoptosis in wild-caught longjaw mudsuckers was correlated with environmental contamination, (ii) tissue- and concentration-dependent differences in apoptosis were found in Cd-exposed topsmelt, and (iii) apoptosis was associated with impaired growth of Cd-exposed fish.