Vince P. Palace

Collapse of a fish population after exposure to a synthetic estrogen

Municipal wastewaters are a complex mixture containing estrogens and estrogen mimics that are known to affect the reproductive health of wild fishes. Male fishes downstream of some wastewater outfalls produce vitellogenin (VTG) (a protein normally synthesized by females during oocyte maturation) and early-stage eggs in their testes, and this feminization has been attributed to the presence of estrogenic substances such as natural estrogens [estrone or 17β-estradiol (E2)], the synthetic estrogen used in birth-control pills [17α-ethynylestradiol (EE2)], or weaker estrogen mimics such as nonylphenol in the water. Despite widespread evidence that male fishes are being feminized, it is not known whether these low-level, chronic exposures adversely impact the sustainability of wild populations. We conducted a 7-year, whole-lake experiment at the Experimental Lakes Area (ELA) in northwestern Ontario, Canada, and showed that chronic exposure of fathead minnow (Pimephales promelas) to low concentrations (5–6 ng·L−1) of the potent 17α-ethynylestradiol led to feminization of males through the production of vitellogenin mRNA and protein, impacts on gonadal development as evidenced by intersex in males and altered oogenesis in females, and, ultimately, a near extinction of this species from the lake. Our observations demonstrate that the concentrations of estrogens and their mimics observed in freshwaters can impact the sustainability of wild fish populations.

The role of oxidative stress in the genesis of heart disease

Although researchers in radiation and cancer biology have known about the existence of free radicals and their potential role in pathobiology for several decades, cardiac biologists only began to take notice of these noxious species in the 1970s. Exponential growth of free radical research occurred after the discovery of superoxide dismutase in 1969. This antioxidant enzyme is responsible for the dismutation of superoxide radical--a free radical chain initiator. A fine balance between free radicals and a variety of endogenous antioxidants is believed to exist. Any disturbance in this equilibrium in favour of free radicals causes an increase in oxidative stress and initiates subcellular changes leading to cardiomyopathy and heart failure. Our knowledge about the role of free radicals in the pathogenesis of cardiac dysfunction is fast approaching the point where newer therapies employing antioxidants are in sight.

Hexabromocyclododecane: Current Understanding of Chemistry, Environmental Fate and Toxicology and Implications for Global Management

Hexabromocyclododecane (HBCD) is a globally produced brominated flame retardant (BFR) used primarily as an additive FR in polystyrene and textile products and has been the subject of intensified research, monitoring and regulatory interest over the past decade. HBCD is currently being evaluated under the Stockholm Convention on Persistent Organic Pollutants. HBCD is hydrophobic (i.e., has low water solubility) and thus partitions to organic phases in the aquatic environment (e.g., lipids, suspended solids). It is ubiquitous in the global environment with monitoring data generally exhibiting the expected relationship between proximity to known sources and levels; however, temporal trends are not consistent. Estimated degradation half-lives, together with data in abiotic compartments and long-range transport potential indicate HBCD may be sufficiently persistent and distributed to be of global concern. The detection of HBCD in biota in the Arctic and in source regions and available bioaccumulation data also support the case for regulatory scrutiny. Toxicity testing has detected reproductive, developmental and behavioral effects in animals where exposures are sufficient. Recent toxicological advances include a better mechanistic understanding of how HBCD can interfere with the hypothalamic-pituitary-thyroid axis, affect normal development, and impact the central nervous system; however, levels in biota in remote locations are below known effects thresholds. For many regulatory criteria, there are substantial uncertainties that reduce confidence in evaluations and thereby confound management decision-making based on currently available information.

Dietary accumulation of hexabromocyclododecane diastereoisomers in juvenile rainbow trout (Oncorhynchus mykiss) I: bioaccumulation parameters and evidence of bioisomerization.

Juvenile rainbow trout (Oncorhynchus mykiss) were exposed to three diastereoisomers (alpha, beta, gamma) of hexabromocyclododecane (C12H18Br6) via their diet for 56 d followed by 112 d of untreated food to examine bioaccumulation parameters and test the hypothesis of in vivo bioisomerization. Four groups of 70 fish were used in the study. Three groups were exposed to food fortified with known concentrations of an individual diastereoisomer, while a fourth group were fed unfortified food. Bioaccumulation of the gamma-diastereoisomer was linear during the uptake phase, while the alpha- and beta-diastereoisomers were found to increase exponentially with respective doubling times of 8.2 and 17.1 d. Both the beta- and the gamma-diastereoisomers followed a first-order depuration kinetics with calculated half-lives of 157 +/- 71 and 144 +/- 60 d (+/-1 x standard error), respectively. The biomagnification factor (BMF) for the alpha-diastereoisomer (BMF = 9.2) was two times greater than the beta-diastereoisomer (BMF = 4.3); the large BMF for the beta-diastereoisomer is consistent with this diastereoisomer dominating higher-trophic-level organisms. Although the BMF of the beta-diastereoisomer suggests that it will biomagnify, it is rarely detected in environmental samples because it is present in small quantities in commercial mixtures. Results from these studies also provide evidence of bioisomerization of the beta- and gamma-diastereoisomers. Most importantly, the alpha-diastereoisomer that was recalcitrant to bioisomerization by juvenile rainbow trout in this study and known to be the dominant diastereosiomer in fish was bioformed from both the beta- and the gamma-diastereoisomers. To our knowledge, this is the first report of bioisomerization of a halogenated organic pollutant in biota.

Developmental effects of bioaccumulated selenium in eggs and larvae of two salmonid species

Elevated concentrations of Se have been detected in cold, flowing water habitats near uranium and coal mines in Canada. Fish from these systems have concentrations of Se in their tissues that exceed toxic effect thresholds that have been established for warm-water fishes. However, the applicability of toxic effect thresholds and guidelines to cold water, lotic habitats is a matter of contention in the literature since most cases of Se toxicosis have been documented in standing, warm-water systems. To examine the possibility of impaired reproduction in wild rainbow trout (Oncorhynchus mykiss) and brook trout (Salvelinusfontinalis) near coal mining activity in the northeastern slopes region of Alberta, Canada, spawn from both species were collected from exposure and reference sites. Gametes were fertilized in the laboratory, reared to the swim-up stage, and examined for deformities. A significant relationship was observed for rainbow trout between the amount of Se in eggs and the incidence of developmental abnormalities, specifically craniofacial defects, skeletal deformities, and edema. These associations approximate exponential functions with probabilities that 15% of the population would be affected occurring between 8.8 and 10.5 microg Se per gram of wet egg weight, based on probit analysis. These relationships are similar to those described for centrarchids inhabiting a seleniferous warm-water lake. No such relationships were established for brook trout.

IMMUNOTOXICITY OF THE COMMERCIAL POLYBROMINATED DIPHENYL ETHER MIXTURE DE-71 IN RANCH MINK (MUSTELA VISON)

Polybrominated diphenyl ethers (PBDEs) are persistent, bioaccumulative, organohalogen compounds that are increasing exponentially in the Great Lakes (Canada/USA) biota. The present study was undertaken to examine the immunological effects of a commercial PBDE mixture in ranch mink (Mustela vison). Twenty-week-old mink (n = 10 mink/group) were exposed to 0, 1, 5, or 10 ppm of DE-71 through their diet for eight weeks. The phytohemagglutinin-induced cutaneous reaction, and antibodies specific to keyhole limpet hemocyanin conjugated to dinitrophenol were measured. Liver microsomal ethoxyresorufin-O-deethylase (EROD) activity also was measured. Organs were weighed and spleens were examined histologically. No differences were found in the PHA-induced skin response in exposed mink; mink in the two highest treatments exhibited significant increases in antibody production over control mink. Systemic toxicity was apparent; significant body weight reductions were found in mink exposed to 5 and 10 ppm of DE-71. Exposed mink had significantly larger relative spleen, adrenal, and liver masses than control mink. Spleens of mink exposed to 10 ppm of DE-71 had significantly increased germinal center development and incidence of B-cell hyperplasia. The activity of EROD was induced in all treated mink relative to controls and was positively associated with the liver somatic index. Hematocrit in mink from the two highest exposure groups was significantly lower than control mink. Percentage neutrophils increased and percentage lymphocytes decreased significantly in mink from the higher two dosage groups. Our findings have direct relevance to wild mink in the Great Lakes ecosystem, because mink are top predators of the aquatic food web, providing evidence for the vulnerability of this species to the effects of environmental PBDE mixtures.

Biochemical and histopathological effects in pearl dace (Margariscus margarita) chronically exposed to a synthetic estrogen in a whole lake experiment.

Potential effects of exposure to the synthetic estrogen 17alpha-ethynylestradiol (EE2) were examined in several species of fish from a lake experimentally treated with environmentally relevant concentrations of the contaminant. Ethynylestradiol was added to Lake 260, a small Precambrian shield lake at the Experimental Lakes Area in northwestern Ontario, Canada, from May to October of 2001, 2002, and 2003. Mean concentrations of EE2 in epilimnetic waters ranged between 4.5 and 8.1 ng/L during the three years, with overall means of 6.1 (+/- 2.8), 5.0 (+/- 1.8), and 4.8 (+/- 1.0) ng/L for the three years, respectively. Male and female pearl dace (Margariscus margarita) captured after EE2 additions began contained up to 4,000-fold higher concentrations of the egg yolk precursor vitellogenin than fish captured from the same lake before the EE2 additions or when compared to fish from reference lakes. Edema in the ovaries, inhibited development of testicular tissue, intersex, and histopathological kidney lesions were all evident in fish exposed to EE2. Some indications that EE2 exposure affected in vitro steroidogenic capacity of the ovaries and the testes existed, although results were not always consistent between years. Pearl dace abundance was similar in the lake treated with EE2 and the reference lake. A trend exists toward a reduced overall population of pearl dace from the treated and reference lakes, as do indications that young-of-the-year size classes are less abundant in the EE2-treated lake. Biochemical and histopathological impacts observed in fish exposed to EE2 in this study have not yet been linked to clear population level impacts in pearl dace. Monitoring of these populations is ongoing.

Mobilization of Antioxidant Vitamin Pools and Hemodynamic Function After Myocardial Infarction

BACKGROUND Although most previous studies have attempted to correlate plasma concentrations of vitamins with specific cardiovascular end points, metabolic considerations suggest that changes in myocardial tissue and storage organs may be better indicators of myocardial oxidative stress. METHODS AND RESULTS Rats fed commercial chow or a diet enriched with vitamin E for 2 weeks were subjected to either a surgical myocardial infarction (MI) or a sham procedure. Rats were hemodynamically assessed 16 weeks after surgery, and their heart, liver, kidney, and plasma were analyzed for antioxidant vitamins E (tocopherol) and A (retinol and total retinyl esters). At 16 weeks, MI rats on a control diet showed depressed peak systolic and elevated diastolic pressures in both right and left ventricles compared with their sham controls. Plasma concentrations of vitamins E and A in MI rats were not different from sham controls fed the same diet. However, concentrations of vitamin E in left ventricle and liver and of vitamin A in liver (retinol) and kidney (retinyl esters) were decreased in rats with MI compared with the sham controls. Vitamin E supplementation improved hemodynamic function in rats with MI and increased plasma, myocardial, liver, and kidney concentrations of vitamin E. The vitamin E diet also prevented the loss of total retinyl esters from the kidney but not of retinol from the liver in MI rats. CONCLUSIONS Dietary supplements of vitamin E can sustain better cardiac function subsequent to MI. Antioxidant vitamin levels in the myocardium or in storage organs and not in plasma may be better indicators of myocardial oxidative stress.

Waterborne ethynylestradiol induces vitellogenin and alters metallothionein expression in lake trout (Salvelinus namaycush)

Estrogenic contaminants isolated from waters receiving sewage treatment plant effluents are known to induce the egg yolk precursor vitellogenin (VTG) in male fish. Levels of the metal binding protein metallothionein (MT) have also been shown to be affected by estrogens in fish. It has been postulated that MT declines in estrogen exposed fish to facilitate transfer of the essential metal Zn to cellular components required for VTG synthesis. To examine the changes in MT and VTG concentrations in fish exposed to an estrogen contaminant, lake trout (Salvelinus namaycush) were exposed to waterborne ethynylestradiol at 0, 4, 40 or 400 ng/l(-1) for 21 days. Blood and tissues were collected after 21 days of exposure to measure circulating levels of VTG as well as MT concentrations in liver and kidney. VTG increased in male and female fish from all three exposure groups compared to control fish. MT in liver significantly decreased in males and females compared to the controls, in the two highest exposures. MT in kidney was significantly higher in both sexes of fish exposed to the two highest concentrations of ethynylestradiol. These data are supportive of a relationship between estrogen exposure and the regulation of MT. Further studies to examine the specific links between estrogen exposure, VTG induction and regulation of essential metals like Zn are required.

Oxidative stress in Lake Sturgeon (Acipenser fulvescens) orally exposed to 2,3,7,8-tetrachlorodibenzofuran

Juvenile lake sturgeon were orally dosed with gelatin containing nominal concentrations of 0, 0.16 or 1.6 ng [3H]-2,3,7,8-tetrachlorodibenzofuran (TCDF) kg−1 fish weight. Liver, kidney and blood were collected 10 and 27 days after exposure. Phase I mixed-function oxidase (MFO) and Phase II (glucuronyltransferase) enzyme activities were determined in liver. Concentrations of non-enzymatic (tocopherol, retinoids) and enzymatic (superoxide dismutase, catalase, glutathione peroxidase) antioxidant parameters were also quantified in liver and kidney. TCDF concentrations were elevated in liver and kidney of both dose groups at 10 and 27 days. Lower tissue concentrations of TCDF at 27 days, compared with 10 day exposures, as well as the presence of polar metabolites in bile, may indicate rapid metabolism and clearance of the contaminant. MFO enzyme activity, measured as ethoxyresorufin-O-deethylase (EROD), was induced in liver with activity at the two sample periods reflecting the TCDF concentrations. Greater concentrations of hepatic lipid peroxides in both dose groups, indicate that oxidative stress was produced by contaminant exposure and/or induced metabolism enzyme activity.