California Sea Grant College Program

Apoptosis, or programmed cell death, may be induced in fishes following exposure to a diversity of toxicants. While some apoptosis may prevent cancerous cell proliferation, the inappropriate stimulation of apoptosis by toxicants may lead to developmental and reproductive abnormalities. Little is known about the applicability of apoptosis as a biomarker in wild-caught fishes; however, the development of apoptosis as a biomarker is essential because it may be used as an early warning indicator for severe toxicant effects. Here, 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, salinity-tolerant goby found in tidal marshes. In the laboratory, topsmelt (Atherinops affinis) was used because it is an EPA­ toxicity test organism that inhabits the same marshes as mudsuckers, and Cadmium (Cd) was chosen as a model contaminant because its toxicity has long been studied in fishes.

First, I determined whether TUNEL and caspase activity assays were reliable methods for measuring apoptosis as a biomarker response in estuarine fish from California tidal marshes varying in contamination levels. I measured increased apoptotic DNA fragmentation (TUNEL positive cells), and DEVDase activity (caspase-3 like protease activity) in hepatocytes of topsmelt exposed to non-cytotoxic Cd concentrations in the laboratory, indicating that TUNEL and caspase activity assays were sensitive apoptosis methods. In the field, apoptotic DNA fragmentation and DEVDase activity levels were significantly higher in the liver oflongjaw mudsuckers from Stege Marsh (ST) relative to mudsuckers from the reference marsh. Moreover, average concentrations of many sediment contaminants including metals, legacy organic chemicals, and pesticides were highest at ST, and apoptotic DNA fragmentation correlated with numerous sediment contaminants, suggesting that apoptosis was a reliable biomarker. I then examined how increases in apoptosis and altered physiological responses were related to growth impairment of Cd-exposed larval topsmelt. Apoptotic DNA fragmentation and metallothionein-like protein levels were elevated, and Ca content of fish diminished at Cd concentrations that also 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 more detailed investigation of Cd-impaired growth of topsmelt because growth measurements (i.e. final body size) used in standard toxicity tests with topsmelt were insensitive endpoints of toxicity. Otolith growth rate analysis was a useful method because it allowed for the detection of small differences in growth rates among treatments, even when differences in somatic growth were not observed.

This study demonstrates that 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. The use of apoptosis as an early warning indicator of ecologically relevant effects of toxicant exposure needs further exploration.