Doranne Borsay Horowitz

Vitellogenin-induced pathology in male summer flounder (Paralichthys dentatus)

Male summer flounder (Paralichthys dentatus) were given two injections (initially and 2 weeks later) of 17beta-estradiol (E2) totaling 0.2 (2 x 0.1), 2.0 (2 x 1.0) or 20.0 (2 x 10.0) mg E2/kg body weight. Blood and tissue samples were collected 4, 6 and 8 weeks after the initial injection in the (2 x 0.1) mg/kg treatment, 4, 6, 8, and 15 weeks after the first injection in the (2 x 1.0) mg/kg treatment and at 4 weeks only in the (2 x 10.0) mg/kg treatment. Five of the 12 fish injected twice with 10.0 mg/kg were moribund before the first sampling period. Circulating levels of vitellogenin (VTG) in the blood of all E2-injected fish from all treatments were comparable with those concentrations found in the blood of wild male carp (Cyprinus carpio) and walleye (Stezostedion vitreum) previously collected near a sewage treatment plant (0.1-10.0 mg VTG/ml plasma). Excessive hyalin material accumulated in the livers, kidneys and testes of the treated fish. A portion of that material was identified as VTG by immunohistochemistry. The accumulation of VTG, and possibly other estrogen-inducible proteins, resulted in hepatocyte hypertrophy, disruption of spermatogenesis, and obstruction or rupture of renal glomeruli.

Modulation of aromatase activity as a mode of action for endocrine disrupting chemicals in a marine fish

The steroidogenic enzyme aromatase catalyzes the conversion of androgens to estrogens and therefore plays a central role in reproduction. In contrast to most vertebrates, teleost fish have two distinct forms of aromatase. Because brain aromatase activity in fish is up to 1000 times that in mammals, fish may be especially susceptible to negative effects from environmental endocrine-disrupting chemicals (EDCs) that impact aromatase activity. In this study, the effects of estradiol (E2), ethynylestradiol (EE2), octylphenol (OP), and androstatrienedione (ATD) on reproduction and aromatase activity in brains and gonads from the marine fish cunner (Tautogolabrus adspersus) was investigated. The purpose of the study was to explore the relationship between changes in aromatase activity and reproductive output in a marine fish, as well as compare aromatase activity to two commonly used indicators of EDC exposure, plasma vitellogenin (VTG) and gonadosomatic index (GSI). Results with E2, EE2, and ATD indicate that aromatase activity in cunner brain and ovary are affected differently by exposure to these EDCs. In the case of E2 and EE2, male brain aromatase activity was signficantly increased by these treatments, female brain aromatase activity was unaffected, and ovarian aromatase activity was significantly decreased. Treatment with the aromatase inhibitor ATD resulted in significantly decreased aromatase activity in male and female brain, but had no significant impact on ovarian aromatase activity. Regardless of test chemical, a decrease or an increase in male brain aromatase activity relative to controls was associated with decreased egg production in cunner and was also correlated with significant changes in GSI in both sexes. E2 and EE2 significantly elevated plasma VTG in males and females, while ATD had no significant effect. Treatment of cunner with OP had no significant effect on any measured endpoint. Overall, results with these exposures indicate EDCs that impact aromatase activity also affect reproductive output in spawning cunner.

Demonstration of Aspergillus sp. in Tissues of the Common Loon, Gavier immer: Incidence, Progression, and Severity

Abstract By studying a large group of loons affected by an oil spill, much can be learned about the toxic effects of petroleum hydrocarbons in exposed birds, their ability to handle these environmental stressors, and their ability to combat natural pathogens. On January 19, 1996 the North Cape oil barge ran aground off the coast of Rhode Island, releasing 800,000 gallons of #2 fuel oil into the marine environment. Several hundred sea birds overwintering in near shore waters were exposed to the chemical stressors found in this highly refined oil. One hundred and fourteen oiled birds were captured alive, and the most abundant of these birds was the common loon, Gavia immer. As a result of oil toxicity, which caused stress and weakened the immune system in the loons, all efforts of treatment and rehabilitation were unsuccessful. Gross necropsies and histopathological examinations were performed on the loons lost in captivity. At necropsy, lesions of various sizes were observed covering many organs in the most severely affected birds. Histochemical techniques revealed dramatic growths of Aspergillus sp., showing the morphology, mode of reproduction, and spread of this fungus. Over time, the Aspergillus infection became more prevalent, correlating to the increased physiological stress By using histopathology to trace the incidence, progression, and severity of the fungal infections throughout organ systems, we could assess the health impacts. (The J Histotechnol 24:101, 2001) Submitted: January 4, 2001; Accepted with revisions: January 25, 2001

An Injectable, Slow-Release Implantation Method for Exposing Fish to Chemicals over a Period of Weeks

Abstract A slow-release, injectable implant method was developed for administering test chemicals to cunners Tautogolabrus adspersus. The implant is composed of a matrix of a test chemical homogenized in a mixture of Ethocel (Dow Chemical) and coconut oil. The effectiveness of a subcutaneous implant of this matrix in vivo was determined by tracing plasma concentrations of three separate chemicals (estradiol, ethynylestradiol, and atrazine) over time in treated male cunners. Release from the implant was determined based on the percentage of the implanted concentration of test chemical (plus metabolites) that was detected in fish plasma over a 1–2-week period after implantation. Circulating estrogen concentrations measured in plasma from two different cunners that received the estradiol implant were almost identical, indicating that there is a reasonably even distribution of test chemical within the Ethocel–coconut oil preparation and that individual variability may be minimal for release of test chemical f...

Approaches for predicting effects of unintended environmental exposure to an endocrine active pharmaceutical, tamoxifen

Tamoxifen is an endocrine‐active pharmaceutical (EAP) that is used world‐wide. Because tamoxifen is a ubiquitous pharmaceutical and interacts with estrogen receptors, a case study was conducted with this compound to (1) determine effects on reproductive endpoints in a nontarget species (i.e., a fish), (2) compare biologically‐active metabolites across species, (3) assess whether in vitro assays predict in vivo results, and (4) investigate metabolomic profiles in tamoxifen‐treated fish to better understand the biological mechanisms of tamoxifen toxicity. In reproductive assays, tamoxifen exposure caused a significant reduction in egg production and significantly increased ovarian aromatase activity in spawning adult cunner fish (Tautogolabrus adspersus). In plasma from tamoxifen‐exposed cunner, the predominant metabolite was 4‐hydroxytamoxifen, while in rats it was N‐desmethyltamoxifen. Because 4‐hydroxytamoxifen is a more biologically active metabolite than N‐desmethyltamoxifen, this difference could result in a different level of risk for the two species. The results of in vitro assays with fish hepatic microsomes to assess tamoxifen metabolism did not match in vivo results, indicating probable differences in excretion of tamoxifen metabolites in fish compared with rats. For the first time, a complete in vitro characterization of the metabolism of tamoxifen using fish microsomes is presented. Furthermore, a metabolomic investigation of cunner gonad extracts demonstrates that tamoxifen alters the biochemical profile in this nontarget species. Understanding the consequence of tamoxifen exposure in nontarget species, and assessing the discrepancies between sex‐ and species‐mediated endpoints, is a step toward understanding how to accurately assess the risks posed by EAPs, such as tamoxifen, in the aquatic environment.