Endocrine disruption and altered gonadal development in white perch (Morone americana) from the lower Great Lakes region.

High prevalences of gonadal intersex have been observed in wild fish populations in areas affected by domestic and industrial effluents. For this study, fish were collected in 1998 from the Cootes Paradise region of Hamilton Harbour in western Lake Ontario, Canada, to determine whether gonadal abnormalities, including intersex, were present in young of the year (YOY) fish. No gonadal abnormalities were observed in goldfish (Carassius auratus), common carp (Cyprinus carpio), gizzard shad (Dorosoma cepedianum), brown bullhead (Ictalurus ameiurus), pumpkinseed (Lepomis gibbosus), and bluegill (Lepomis macrochirus). However, intersex gonads were observed in 8 of 16 male white perch (Morone americana) examined in this survey. Subsequently, in 1999 and 2000 white perch estimated to be YOY to approximately 2 years of age were collected from Cootes Paradise and from two other sites in the lower Great Lakes region. Gonadal intersex was observed in male white perch collected from the Bay of Quinte (22-44%) and Lake St. Clair (45%), although the prevalence and the extent of the intersex condition were lower relative to the 83% prevalence in white perch collected in Cootes Paradise. Intersex was not observed in hatchery-reared white perch or in white perch collected from an uncontaminated reference site (i.e., Deal Lake) in the United States. An analysis of plasma collected in the spring of 2002 from male adult white perch in Cootes Paradise revealed high concentrations of vitellogenin, ranging from 49 to 1,711 microg/mL. These observations indicate that male white perch are exposed to estrogenic endocrine-disrupting substances that may be responsible for the induction of gonadal intersex.

Application of Alamar blue/5-carboxyfluorescein diacetate acetoxymethyl ester as a noninvasive cell viability assay in primary hepatocytes from rainbow trout.

We have adopted the application of two fluorescent indicator dyes to studying the viability of monolayers of primary rainbow trout hepatocytes. The two fluorescent dyes--Alamar blue, which indicates metabolic activity of a cell, and 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM), which is an indirect measure of cell membrane integrity-are noninvasive and can be monitored conveniently directly in multiwell plates. According to these dyes, L-15 culture medium supported hepatocyte viability over 96 h more stably than did M199. The two dyes proved to be capable of detecting a concentration-dependent toxic insult to hepatocytes caused by the model compound, pentachlorophenol. In contrast, a lack of impact on cell viability was indicated for up to 10(-5) M 17beta-estradiol, and that observation was supported by the induction of vitellogenin (VTG) mRNA/protein as indicator of hepatocyte-differentiated function. Application of the Alamar blue/CFDA-AM for 30 min did not alter gene expression either specifically as reflected by VTG or generally as reflected by a random selection of gene sequences that were amplified by differential display reverse transcription PCR (dd-rt-PCR). Thus, the assay represents a resource-efficient way of integrating measures of cell viability and gene expression that should aid in the interpretation of in vitro results. The assay can be applied repeatedly to the same set of cells and can be performed just prior to analysis of gene expression.