David M. Janz

Developmental estrogenic exposure in zebrafish (Danio rerio): I. Effects on sex ratio and breeding success

In vivo studies of fishes exposed to xenoestrogens have reported vitellogenin (Vtg) induction, ovatestes, altered sex ratios, and impaired reproductive capacity. The objective of this study was to determine concentration dependent effects of a weak estrogen receptor agonist, 4-nonylphenol (NP) and a potent estrogen receptor agonist, 17alpha-ethinylestradiol (EE) on sex ratios, gonad morphology, Vtg induction and breeding success in zebrafish (Danio rerio). Fish were exposed from 2 to 60 days post-hatch (dph) to NP (10, 30, or 100 microg/l nominal), EE (1, 10, or 100 ng/l nominal), or solvent control (acetone; 0.2% v/v) in a static-renewal system with replacement every 48 h. At 60 dph, 20 fish from each treatment were euthanized for histological examination of gonads and Western blotting for Vtg using pooled heart homogenates. Remaining fish were reared in clean water until adulthood (120 dph) for breeding studies. Due to high mortality in the 100 ng/l EE group, insufficient fish were available for analyses. The percentage of males at 60 dph changed from 45% (9/20) in solvent controls to 0% at 10 ng/l EE and 10% at 100 microg/l NP. A concentration dependent increase in the number of fish with undeveloped gonads at 60 dph was observed in the EE exposure group. Two fish with ovatestes were observed at 100 microg/l NP, while one was observed at 30 microg/l NP. Western blotting showed induction of Vtg at 30 and 100 microg/l NP and 10 ng/l EE. The sex ratios of adults determined at 160 dph revealed no significant departure from 1 male:1 female, suggesting that exposure of zebrafish to estrogenic chemicals during sexual differentiation and early gametogenesis did not irreversibly alter phenotypic sex. The condition factor of adult fish and ovo-somatic index of adult female fish were also unaffected by developmental exposure to NP or EE. Despite this, breeding trials conducted in adult fish from 120 to 160 dph revealed significant reductions in the percent of viable eggs, hatchability, and swim-up success at 10 ng/l EE and 100 microg/l NP. Our results suggest that functional reproductive capacity (breeding success) may be more sensitive than gross morphological endpoints (condition, ovo-somatic index, sex ratio) in adult zebrafish exposed to xenoestrogens during sexual differentiation and early gametogenesis.

Developmental estrogenic exposure in zebrafish (Danio rerio): II. Histological evaluation of gametogenesis and organ toxicity.

Aquatic species can be exposed to endocrine disrupting chemicals (EDCs) in wastewater that often includes the weak estrogen, 4-nonylphenol (NP) and the potent estrogen, 17alpha-ethinylestradiol (EE). The goal of the present study was to determine concentration-dependent effects of developmental exposure to NP and EE on gametogenesis, as well as gonad, kidney and liver pathology using quantitative histological evaluation of hematoxylin/eosin-stained saggital sections of zebrafish (Danio rerio). The major finding of the present study was that exposure to NP (>/=100 microg/l nominal) and EE (>/=1 ng/l nominal) from 2 to 60 days post-hatch (dph) caused concentration-dependent suppression of gametogenesis in both male and female zebrafish. Severe kidney pathology was observed in 60 dph zebrafish, specifically glomerular dilation or degeneration, fibrosis, tubule enlargement and tubule necrosis, at a threshold of 10 ng/l EE. However, minor kidney histopathology indicated by increased pyknotic nuclei in kidney tubule and interstitial (hematopoietic) cells was detected at lower estrogenic exposures (>/=10 microg/l NP nominal) than delayed gametogenesis. Considering all histological parameters in the current study, the rank order of potency for pathological effects in 60 dph zebrafish was 10 ng/l EE>1 ng/l EE=100 microg/l NP>30 microg/l NP>10 microg/l NP10 (nominal concentrations). Zebrafish from the same cohort examined in the current study that had been placed in clean water from 60 to 300 dph had histologically normal testes and no kidney or liver histopathology. However, increased ovarian follicle atresia was detected at 300 dph in zebrafish exposed developmentally to 100 microg/l NP. Therefore, we conclude that functional rather than morphological changes may be more important for future evaluations of developmental exposure to estrogens in fish, and that negative effects in female rather than male gonads may contribute to prolonged breeding impairment.

Increased cellular apoptosis after chronic aqueous exposure to nonylphenol and quercetin in adult medaka (Oryzias latipes)

Increasing evidence suggests that sublethal effects of natural or xenobiotic chemicals in the environment may be mediated via the stimulation of apoptosis. To investigate whether apoptosis can be induced in fish by weakly estrogenic and androgenic chemicals, adult male Japanese medaka (Oryzias latipes) were exposed to 100 ppb of the estrogenic alkylphenol, 4-nonylphenol, and adult female medaka were exposed to 100 ppb of the aromatase-inhibiting bioflavonoid, quercetin, for 6 weeks. Exposure to nonylphenol and quercetin had no significant effect on the length, weight or condition factors compared to solvent (acetone) controls in male or female medaka. Apoptosis was evaluated in blinded histological sections of whole medaka using terminal dideoxynucleotidyl transferase dUTP nick end labeling (TUNEL) that labels nuclei of cells containing apoptotic (fragmented) DNA. There was a six-fold greater extent of apoptosis in spermatocytes, Sertoli cells and Leydig-homologue cells, but not in spermatids of testes from nonylphenol-exposed male medaka compared to testes of solvent controls. No significant differences in the extent of apoptosis were detected in intestine, liver or kidney from the same male fish. Quercetin-treated female medaka had a significantly increased number of atretic ovarian follicles, but no significant differences in the extent of apoptosis in intestine, liver or kidney. These results suggest that nonylphenol caused testicular degeneration via increased testicular cell apoptosis, while quercetin may be ovotoxic via increased follicular atresia.

Effect of β-naphthoflavone and dimethylbenz[a]anthracene on apoptosis and HSP70 expression in juvenile channel catfish (Ictalurus punctatus) ovary

Complex environmental mixtures such as pulp mill effluents and crude oil have been shown to increase ovarian cell apoptosis and affect heat shock protein (HSP) expression in fish. We hypothesize that polyaromatic hydrocarbons (PAH) mediate these effects. To test this hypothesis, we exposed juvenile channel catfish (Ictalurus punctatus) acutely to the aryl hydrocarbon receptor (AhR) agonists, beta-naphthoflavone (BNF; 75 mg/kg) or the model PAH, dimethylbenz[a]anthracene (DMBA; 50 mg/kg) via intraperitoneal injection. Apoptotic DNA fragmentation and HSP70 expression were determined in ovary and liver. Hepatic cytochrome P450 1A (CYP1A) was significantly induced, confirming that BNF and DMBA had distributed to internal organs and stimulated AhR. At 96 h post-injection, BNF and DMBA significantly increased apoptosis and decreased HSP70 expression in juvenile catfish ovaries. Although primary oocytes underwent the greatest rates of apoptosis compared to early or late vitellogenic follicles in all treatment groups, the cell type undergoing increased rates of apoptosis after BNF or DMBA exposure was not clear using terminal deoxynucleotidyl transferase (TdT)-mediated deoxyUTP nick end labeling (TUNEL). There was a significant negative relationship between expression of HSP70 and apoptosis in juvenile channel catfish ovaries. This differed from liver of these fish which did not exhibit increased apoptosis and instead increased hepatic HSP70 expression at 96 h post-injection. However, DMBA had no effect on apoptosis or HSP70 levels in more reproductively mature juvenile fish that were housed at a lower water temperature. This may be due to a developmental or temperature-dependent component to these responses. We propose that the decrease in ovarian HSP70 expression in response to BNF and DMBA may be causally related to the increase in ovarian cell apoptosis. Further experiments using a full time course, dose-response and methods to confirm that AhR is a direct mediator of these effects are required.

Treated municipal sewage discharge affects multiple levels of biological organization in fish.

The objectives of this study were to examine cellular-, organ-, and organism-level responses in longear sunfish (Lepomis megalotis) and fish community structure in a stream in which treated municipal sewage effluent is discharged and in a nearby reference stream with little surrounding land use. A modified version of the U.S.E.P.A. Rapid Bioassessment Protocol V, which combines a habitat assessment with Karrs index of biotic integrity, was used on 400-m reaches of each stream. The study site had a higher proportion of tolerant species and omnivores and a lower proportion of top predators, suggesting alterations in the fish community and a slight level of water quality impairment. Significant increases in condition factor, hepatosomatic index, serum testosterone, and plasma vitellogenin concentrations were observed in male sunfish collected from the study stream in comparison to fish collected from the reference stream. There were no differences between sites in hepatic expression of the 70-kDa stress protein (HSP70). In conclusion, effects were observed at cellular, organ, organism, and community levels of biological organization in fishes exposed to treated municipal sewage effluent.

Expression of HSP70 and CYP1A protein in ovary and liver of juvenile rainbow trout exposed to β-naphthoflavone

Cytochrome P4501A (CYP1A) and the 70-kDa stress protein (HSP70) were determined using Western blotting in the ovary and liver of juvenile female rainbow trout (Oncorhynchus mykiss) exposed for 4 days to beta-naphthoflavone (betaNF) following a single intraperitoneal injection. Ovarian CYP1A protein was observed in both control and betaNF-exposed fish, indicating constitutive and inducible expression of CYP1A in immature trout ovaries. CYP1A protein levels determined using densitometry were 14- and 46-fold greater in betaNF-exposed trout compared to controls in the liver and ovary, respectively. Hepatic microsomal ethoxyresorufin O-deethylase (EROD) activity, a specific catalytic marker of CYP1A, was also induced 38-fold above controls following betaNF exposure. Hepatic HSP70 protein expression was significantly higher in whole cell homogenates, but not in cytosolic fractions, collected from betaNF-exposed fish in comparison to control fish. There was no difference in ovarian HSP70 levels determined in whole cell homogenates between control and betaNF-exposed fish. The observation that unlike liver, ovarian HSP70 expression remained unchanged following induction of CYP1A protein may be related to the sensitivity of the teleost ovary to environmental toxicants that act as aryl hydrocarbon receptor agonists.

Reproductive and thyroid hormone profiles in captive Western fence lizards (Sceloporus occidentalis) after a period of brumation.

Seasonal fluctuation in serum concentrations of sex steroid (testosterone [T] and 17beta-estradiol [E(2)]) and thyroid (triiodothyronine [T(3)] and thyroxine [T(4)]) hormones was determined in captive Western fence lizards (Sceloporus occidentalis). Samples were collected from male and female breeding pairs weekly for a 4-month period after their emergence from artificial brumation. Circulating levels of E(2) corresponded with the expected vitellogenic and ovulatory cycles in females, and surprisingly, E(2) in males followed a similar pattern, indicating a possible role in breeding behavior. Serum T was elevated in male lizards for the first 6 weeks after emergence from brumation, possibly related to an increase in the onset of active spermatogenesis. Thyroid hormones showed little cyclical activity throughout the breeding period, with the exception of small increases of T(3) at weeks 8 and 16, possibly implying an active role of this hormone with ovulation in females. Overall, these baseline hormone data are not only useful in developing this animal as a laboratory reptile model for assessment of endocrine-mediated toxicity, but also of value for understanding herpetological endocrinology and for application in the conservation of threatened species. Zoo Biol 27:36-48, 2008. (c) 2007 Wiley-Liss, Inc.

Chapter 13 – Endocrine System

Publisher Summary This chapter provides an overview of teleost endocrine physiology in terms of both the diverse and conserved nature of hormones in fishes. Techniques available for measuring hormones in body fluids are presented and approximate circulating levels in representative species are discussed. Despite adaptations to a diverse range of aquatic habitats, pituitary endocrine physiology and morphology are remarkably similar in all fishes. As in higher vertebrates the pituitary or hypophysis of fishes consists of two distinct tissues capable of producing hormones—the neurohypophysis and adenohypophysis. The neurosecretory nuclei of the hypothalamus produce neurohormones control pituitary hormone secretion, thus constituting the hypothalamo-hypophysial axis. The ovarian and testicular functions are controlled by the pituitary gonadotropins and also by multiple hormones and growth factors that act in an endocrine, autocrine, or paracrine manner. In fishes thyroid hormones are important in the control of development, growth, metabolism and osmoregulation, and may often act in concert with cortisol and growth hormone. Thyroid hormones play a key role in the posthatching metamorphosis of many teleosts. Further, there is close association between tissues secreting corticosteroids and catecholamines in most fishes. The actions of corticosteroids and catecholamines are similar in all vertebrates and generally include increased oxygen uptake, mobilization of energy stores, and reallocation of energy away from growth and reproduction, and immunosuppression.

Ecotoxicological risks associated with land treatment of petrochemical wastes. III. Immune function and hematology of cotton rats.

Landfarming is a widely used method of treating petrochemical waste through microbial biodegradation. The effects of residual petrochemical contamination on wildlife, especially terrestrial mammals, are poorly understood. The effects of contaminants on the immune system and hematology of cotton rats (Sigmodon hispidus) living on five abandoned petrochemical landfarms (units 1-5) in Oklahoma were studied. Cotton rats were sampled seasonally (summer and winter) from each landfarm and from five ecologically matched reference sites for 2 yr (1998-2000) and returned to the laboratory for immunological and hematological assays. Overall analysis indicated that rats inhabiting landfarms exhibited decreased relative spleen size compared to rats collected from reference sites, with the landfarm at unit 1 showing the greatest reduction. Cotton rats collected from landfarms also had increased hemoglobin, hematocrit, and platelet levels and decreased blood leukocytes during summer. During winter, an increase in the number of popliteal node white blood cells was observed from rats collected on landfarms. No marked difference was detected for lymphocyte proliferation in response to concanavalin A, pokeweed, or interleukin-2. Lymphokine-activated killer cell lytic ability showed a seasonal pattern, but no treatment differences. No differences between landfarm and reference sites were detected in the hypersensitivity reaction of rats given an intradermal injection of phytohemagluttinin (PHA-P). Comparisons within individual sites indicated that two sites (units 1 and 3) had the greatest effects on immune function and hematology of cotton rats. The results of this study suggest that residual petrochemical waste affects the immune system and hematology of cotton rats living on abandoned landfarms during summer and is complicated by variation in the contaminants found on individual petroleum sites.

Chapter 25 – Endocrine System

Publisher Summary There are four major categories of messenger molecules: amines, steroids, peptides and lipids. This chapter focuses on the steroids and peptides hormones. Growth hormone-releasing hormone (GHRH) is a peptide hormone of 45 amino acid residues in teleost and chondrostean species that belongs to the secretin-glucagon family of hormones. The target of GHRH is anterior pituitary cells and it has been shown to cause GH release. However, there appears to be species-specificity in the ability of GHRH to stimulate release of GH. The sex steroid hormones are organic fused ring structures that are derived from cholesterol. The main sex steroids in fish are 17β-estradiol and testosterone, but both 17α,20β-dihydroxy-4-pregnen-3-one and 11-ketotestosterone may play specific yet key roles in fish. Control of sex steroid hormone production occurs mainly by regulation of key enzymes in the biosynthetic pathway. Further, there are thyroid hormones, which are iodinated derivatives of the amino acid, tyrosine. In fish, the first type is thyroxine (T4), whereas the second type is its metabolite. Thyroid stimulating hormone (TSH) stimulates T4 release from thyroid follicular cells in all vertebrates. Additionally, there are two peptide hormone vital to the physiology—insulin and insulin-like growth factor.