Vitellogenin (vtg) was isolated from greenback flounder (Rhombosolea tapirina), rainbow trout, (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar) plasma. The molecular weight of each native molecule, as determined by gel filtration, was 540, 383 and 557 kD respectively. With SDS-PAGE under reducing conditions, rainbow trout vtg dissociated into 1 major subunit (~154 kD), and Atlantic salmon and greenback flounder vtg dissociated into 3 major subunits (~ 159, 117, 86 kD and 155, 104, 79 kD respectively). Polyclonal antisera, produced against only the highest molecular weight subunit from each species following excision of these bands from reducing gels, were reactive with all subunits in Western blots.These antisera were found to be highly species-specific as determined by competition ELISA.No cross-reactivity was noted, even between the two salmonid species. These data suggest that excision of bands from gels is a simple procedure for the preparation of highly specific antisera, and that cross-species assays employing heterologous antisera are unlikely to be possible.

This study investigates oxidative stress and bioindicators of reproductive function in wild white sucker (Catostomus commersoni) collected from environments receiving pulp and paper mill effluent discharges in northern Ontario. Samples were collected over an eight-year period adjacent to three pulp and paper mills using a variety of processing and bleaching techniques. Fish collected downstream of pulp and paper mills within the Moose River basin exhibited elevated hepatic and gonadal 2-thiobarbituric acid reactive substances (TBARS), the presence of which is indicative of oxidative stress in these tissues. Within the Jackfish Bay system, exposure to pulp and paper mill effluent did not elevate hepatic or gonadal TBARS. Hepatic cytochrome P4501A activity (CYP1A) and fatty acyl-CoA oxidase (FAO) activities were frequently increased in livers of Moose River basin fish exposed to pulp and paper mill effluent, while lower activities of both enzymes were found within fish from the Jackfish Bay system. This suggests that oxidative stress may be related to CYP1A and FAO activities. Within the Moose River system, increases in measures of oxidative stress (TBARS, FAO) were generally coincident with decreased levels of 17 beta-estradiol; however, testosterone was often lower in Jackfish Bay system fish without any commensurate changes in oxidative stress. The suite of reproductive and oxidative stress parameters measured in this study varied between seasons and mills suggesting responses to effluent are dynamic and effects are complicated by different receiving environments. The relationship between gonad size, gonadal oxidative stress, and circulating plasma steroids remains unclear.

The accumulation of ligands for the aryl hydrocarbon receptor (AhR) and fish sex steroid receptors was investigated using two separate controlled fish exposures to final effluent from a bleached sulfite/groundwood mill in New Brunswick, Canada. In the first experiment, hepatic tissue extracts from exposed fish were fractionated according to lipophilicity. Fractions with different octanol-water (Kow) partition coefficients were tested for the presence of bioavailable chemicals that function as ligands for the AhR in H4IIE cells, rainbow trout hepatic estrogen receptors (ER), goldfish testicular androgen receptors (AR), and goldfish sex steroid binding protein (SSBP). Fish accumulated ligands for each receptor after 4-d exposure to effluent. Single fractions contained ligands for the AhR and the ER, while multiple fractions competed for the AR and SSBP. Fish also accumulated ligands for the AhR and SSBP from Saint John River dilution water, indicating upstream sources of bioactive substances. Semipermeable-membrane devices deployed concurrently with fish accumulated ligands from effluent for all receptors except the ER. In the second experiment, accumulated ligands were evaluated after exposure of fish to effluent for two different durations and following a depuration period. Hepatic mixed function oxygenase activity and whole-liver hormonal activity, measured as binding to SSBP, returned to background following 6 d depuration and were reduced but still significant after 12-d exposure to effluent. Whole-liver extract affinities for the AR were maintained after extended exposure and depuration, indicating the potential for AR ligands to bioaccumulate. The accumulation of AhR ligands and ligands for sex steroid receptors provides a mechanistic linkage to effects on growth, development, and performance of fish exposed to effluent from this and other mills.

A study was initiated in the spring of 2001 to determine if hormonally active chemicals are bioavailable to white sucker (Catostomus commersoni) residing in the Wabigoon River, downstream of the bleached kraft pulp mill and municipal sewage outfall located in Dryden, Ontario. Females collected during spawning migrations in May 2001 at a near-field exposure site exhibited increased condition factor, liver size and fecundity relative to those collected from a reference site. Liver samples within each site were pooled, soxhlet extracted and fractionated by gel permeation chromatography and HPLC according to octanol/water partition coefficient (Kow). Fractions were then tested to determine if fish accumulated ligands which bind to the goldfish testicular androgen receptor (AR) and sex steroid binding protein (SSBP). The levels of hormonally active substances were low in males whereas females had accumulated significant amounts of several chemicals capable of binding to the hormone receptors. The levels of ligands detected in pulp mill effluent samples were substantially reduced after secondary treatment. Fractionation of final effluent extracts showed only ligands for the AR in the hydrophobic region (log Kow >5), which corresponded to the activities detected in fish tissues from both sexes. It can be concluded that ligands for fish sex steroid receptors are present in final effluent after treatment at the pulp mill and chemicals with the same Kow are bioavailable to fish under spring freshet conditions. Further work at this site is necessary to determine the contribution of pulp mill and municipal sewage effluents to the physiological performance of fish in the Wabigoon River and to their burden of accumulated compounds.