Embryo Microinjection of Selenomethionine Reduces Hatchability and Modifies Oxidant Responsive Gene Expression in Zebrafish.

In previous studies we demonstrated that exposure to selenomethionine (SeMet) causes developmental toxicities in zebrafish (Danio rerio). The objectives of this study were to establish a dose-response relationship for developmental toxicities in zebrafish after embryo microinjection of Se (8, 16 or 32 µg/g dry mass of eggs) in the form of SeMet, and to investigate potential underlying mechanism(s) of SeMet-induced developmental toxicities. A dose-dependent increase in frequencies of mortality and total deformities, and reduced hatchability were observed in zebrafish exposed to excess Se via embryo microinjection. The egg Se concentration causing 20% mortality was then used to investigate transcript abundance of proteins involved in antioxidant protection and methylation. Excess Se exposure modified gene expression of oxidant-responsive transcription factors (nuclear factor erythroid 2-related factor nrf2a and nrf2b), and enzymes involved in cellular methylation (methionine adenosyltransferase mat1a and mat2ab) in zebrafish larvae. Notably, excess Se exposure up-regulated transcript abundance of aryl hydrocarbon receptor 2 (ahr2), a signalling pathway involved in the toxicity of dioxin-related compounds. Our findings suggest that oxidative stress or modification of methylation, or a combination of these mechanisms, might be responsible for Se-induced developmental toxicities in fishes.

Mercury and cortisol in Western Hudson Bay polar bear hair.

Non-invasive methods of assessing animal health and life history are becoming increasingly popular in wildlife research; hair samples from polar bears (Ursus maritimus), are being used to study an ever broader range of anthropogenic and endocrine compounds. A number of contaminants are known to disrupt endocrine function in polar bears. However, the relationship between mercury and cortisol remains unknown, although mercury is an endocrine disruptor in other species. Here, we examine the relationship between concentrations of cortisol and total mercury (THg) analyzed in guard hair from 378 polar bears (184 females, 194 males) sampled in Western Hudson Bay, 2004-2012. The difference in mean cortisol concentration between female (0.8 ± 0.6 pg/mg) and male (0.7 ± 0.5 pg/mg) polar bears bordered on significance (p = 0.054). However, mean mercury concentration was significantly greater (p = 0.009) in females (4.7 ± 1.4 µg/g) than males (4.3 ± 1.2 µg/g). Hair cortisol in males was significantly influenced by mercury, age, and fatness, as well as interactions between mercury and year, mercury and fatness, and year and fatness (all: p < 0.03) (multiple regression analysis, whole model: r(2) = 0.14, F(7,185) = 4.43, p = 0.0001). Fatness was the only significant variable in the multiple regression analysis for females (r(2) = 0.06, F(1,182) = 13.0, p = 0.0004). In conclusion, a significant, but complex, relationship was found between mercury and cortisol concentrations in hair from male, but not female, polar bears.

Glucocorticosteroid concentrations in feces and hair of captive caribou and reindeer following adrenocorticotropic hormone challenge.

Climate change and industrial development are contributing to synchronous declines in Rangifer populations across the Arctic. Chronic stress has been implicated as a proximate factor associated with decline in free-ranging populations, but its role in Rangifer is unspecified. Analysis of glucocorticosteroid (GC) concentration in feces, and more recently in hair, is a non-invasive method for monitoring stress in wildlife. Adrenocorticotropic hormone (ACTH) released from the pituitary gland stimulates GC release from the adrenals and can be administered to reflect adrenal activation. In this study, we assessed concentrations of GC metabolites in feces and cortisol in hair of Alaskan caribou (Rangifer tarandus granti) and reindeer (R. t. tarandus) following ACTH treatment. We predicted that ACTH challenge would increase concentrations of fecal GCs, but not hair cortisol because steroid deposited into the hair shaft occurs over an extended period of time (months) and is likely insensitive to acute adrenal stimulation. Adult caribou (n=10; mean age, 6.5 years old) exhibited a peak increase in fecal GCs 8h following a 2 IU/kg dose of ACTH compared to pre-injection concentrations. In contrast, sub-adult reindeer (n=10, 0.8 years old) elicited a diminished response to the same dose. Quadrupling the dose (8 IU/kg) prolonged the fecal GC response in female reindeer, but male reindeer were unresponsive. Hair cortisol was unaffected by a single ACTH challenge. Further investigation is required to ascertain whether subspecific differences in adrenal sensitivity are attributed to age or sex differences, or historical selective pressures from semi-domestication and/or sedentary life cycle in reindeer.

Dietary selenomethionine exposure in adult zebrafish alters swimming performance, energetics and the physiological stress response.

Selenomethionine (Se-Met) is the major form of organoselenium present in food. Early life stages of oviparous vertebrate species, especially fish, are highly susceptible to dietary selenium (Se) exposure; however less is known concerning effects in adults. The present study was designed to investigate behavioral and physiological consequences of dietary Se-Met exposure to adult zebrafish (Danio rerio). Adult fish were fed either control food (1.3µg Se/g, dry weight or dw) or food spiked with varying measured concentrations of Se (3.7, 9.6 and 26.6 µg Se/g, dw) in the form of Se-Met for 60 days at 5% body weight/day ration, and an additional 30-40 days with equal ration (2.5%) of control or Se-Met spiked foods and clean chironomids. At the end of the exposure period, critical swimming speed (Ucrit), oxygen consumption (MO(2)), cost of transport (COT), tail beat amplitude, tail beat frequency, and whole body cortisol, triglyceride and glycogen levels were determined. Significantly reduced Ucrit was observed in fish fed 3.7, 9.6 and 26.6 µg Se/g when compared to control fish. Although MO(2) of fish fed >3 µg Se/g was consistently greater than control fish, those values were not statistically significant. There was no difference in COT among different treatment groups. Tail beat amplitudes of fish fed >3 µg Se/g were lower than control fish, however tail beat frequencies were not altered. Fish fed 3.7, 9.6 and 26.6 µg Se/g had greater whole body triglycerides and glycogen levels than control fish. Fish fed the highest concentration of Se (26.6 µg Se/g) had elevated levels of whole body cortisol compared to control fish. Our results suggest that environmentally relevant dietary Se-Met exposure can alter both behavioral and physiological responses in adult fish, and such consequences could threaten fitness of adult fish in Se impacted aquatic ecosystems.

Selenium accumulation in aquatic biota downstream of a uranium mining and milling operation.

Uranium mining and milling operations have the potential to release trace elements such as arsenic, molybdenum, nickel, selenium and uranium and ions (e.g., sulfate, ammonium) into the receiving aquatic ecosystem. The major implication of elevated environmental selenium is its propensity to accumulate in the aquatic food chain, potentially impairing fish reproduction. The objective of this study was to investigate the accumulation of selenium in the major compartments of aquatic ecosystems (lakes) upstream and downstream of a uranium mine in northern Saskatchewan, Canada. Selenium concentrations in aquatic biota were elevated in the exposure lake although water and sediment concentrations were low (0.43 microg/L and 0.54 microg/g dry weight, respectively). Biomagnification of selenium resulted in approximately 1.5 to 6 fold increase in the selenium concentration between plankton, invertebrates and fish. However, no biomagnification was observed between forage and predatory fish. Although some aquatic biota (e.g., forage fish) exceeded the lower limit of the proposed 3 to 11 microg/g (dry weight) dietary toxicity threshold for fish, no adverse effects of selenium could be identified in this aquatic system. Continued environmental monitoring is recommended to avoid potential selenium impacts.

Accumulation of selenium in aquatic systems downstream of a uranium mining operation in northern Saskatchewan, Canada.

The objective of this study was to investigate the accumulation of selenium in lakes downstream of a uranium mine operation in northern Saskatchewan, Canada. Selenium concentrations in sediment and biota were elevated in exposure areas even though water concentrations were low (<5 microg/L). The pattern (from smallest to largest) of selenium accumulation was: periphyton

Tissue-specific HSP70 levels and reproductive physiological responses in fishes inhabiting a metal-contaminated creek.

The 70-kDa stress protein family (HSP70) plays important roles in a variety of physiological processes, including protein chaperoning, protection against apoptosis, steroidogenesis, and general cellular stress responses in vertebrate organisms, and has also been proposed as a biochemical marker of environmental stress, such as toxicant exposure. The objectives of this study were to determine HSP70 protein expression in head kidney, liver, gill, and ovarian tissues and to examine reproductive physiological responses in female fishes exposed chronically to sublethal metal concentrations. Female black bullhead (Ameiurus melas) and bluegill sunfish (Lepomis macrochirus) were collected from Tar Creek, Oklahoma (flowing through the Tri-State mining district) and from a nearby reference creek (Lytle Creek) during spring (prespawning; 26.5 +/- 0.95 degrees C water temperature) and winter (ovarian recrudescence; 4.8 +/- 0.80 degrees C water temperature). Aqueous (dissolved and suspended) concentrations of Cd and Zn and liver concentrations of Cd and Zn in both fish species were significantly greater at Tar Creek compared to Lytle Creek. HSP70 expression was consistently elevated in the head kidney of both fish species collected at Tar Creek in comparison to fish collected from the reference creek. In contrast, no consistent differences were observed in HSP70 expression in liver, gill, or ovarian tissues between sites. Significant seasonal differences were observed in expression of HSP70 in gill tissue of both species, in ovarian and liver tissue of bluegill sunfish and in head kidney of black bullhead. Serum testosterone concentration was significantly reduced in sunfish collected from Tar Creek during winter. Gonadosomatic and hepatosomatic indices were significantly lower in black bullhead collected from Tar Creek during spring, and condition factors were lower in black bullhead collected from Tar Creek during both spring and winter. There was no significant difference in the extent of ovarian follicular cell apoptosis in either species collected during spring. In conclusion, we observed significant tissue specific differences and seasonal variation in expression of HSP70, as well as alterations in circulating testosterone levels in female fish chronically exposed to metals.

Effect of beta-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.

Elevated ovarian and thymic cell apoptosis in wild cotton rats inhabiting petrochemical-contaminated terrestrial ecosystems.

The objective of this study was to determine the rates of apoptotic cell death in ovary and thymus collected from wild female cotton rats (Sigmodon hispidus) inhabiting five petrochemical-contaminated and five ecologically matched reference sites in Oklahoma. Overall comparison of reference and contaminated sites, using individual sites as replicates, revealed a significantly increased rate of ovarian cell apoptosis in cotton rats inhabiting contaminated sites. In comparison to rats from reference sites, the number of uterine scars was lower in rats collected from the contaminated sites. There were no significant differences in the percentage of atretic follicles among animals collected from reference and contaminated sites. The rate of thymocyte apoptosis was elevated at one of five contaminated sites, although the overall rate of thymocyte apoptosis was not significantly different when comparing all sites. To our knowledge, this is the first study documenting elevated rates of ovarian and thymic cell apoptosis in wild mammals exposed chronically to environmental toxicants.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces hepatic cytochrome P450-dependent arachidonic acid epoxygenation in diverse avian orders: regioisomer selectivity and immunochemical comparison of the TCDD-induced P450s to CYP1A4 and 1A5.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) markedly induced cytochrome P450 (CYP)-dependent arachidonic acid metabolism in liver microsomes from hatchlings of four avian species belonging to four different orders: chick, pigeon, cormorant, and great blue heron, increasing formation of arachidonic acid epoxides (EETs), monohydroxyeicosatetraenoic acids (HETEs), omega-1, and omega-2 OH arachidonic acid products by fivefold or more. Microsomes from TCDD-induced hatchling chicks had the highest activity and the least restricted EET regioselectivity. omega-OH arachidonic acid, the principal constitutive metabolite in chick and pigeon liver microsomes and a major product for cormorant and great blue heron was not induced by TCDD. Constitutive EET formation in avian liver microsomes was very low except in cormorant microsomes where 8,9-EET was generated almost exclusively. Western blots of liver microsomes using polyclonal antisera to chick embryo-derived CYP1A4 and 1A5 recognized two TCDD-induced bands in each of the species. The chick bands had the same molecular weights as CYP1A4 and 1A5 (55 and 55.5 kDa, respectively) but those of the other species differed. Immunopurified antiserum monospecific for CYP1A5 recognized a band in microsomes from all of the avian species, and monospecific antiserum for CYP1A4 recognized a band in microsomes from chick, pigeon, and great blue heron. AntiCYP1A4 and 1A5 IgG immunoinhibited TCDD-induced mixed function oxidase activity completely in chick and chick embryo microsomes and only partially in the other avian microsomes. The results demonstrate that (1) TCDD causes much greater induction of CYP-dependent arachidonic acid metabolism, and of arachidonic acid epoxygenation in particular, in avian than in mammalian species; (2) TCDD induces two CYP1A-related enzymes in birds as in mammals; (3) CYP1A enzymes in the birds other than chicks are not identical to CYP1A4 and 1A5 but share some enzymatic and immunochemical characteristics with them; (4) constitutive omega-OH arachidonic acid in all of the avian species and 8,9-EET in cormorant are formed by CYP enzymes unrelated to CYP1A; and (5) two distinct characteristics of avian CYP1A enzymes are the acquisition by avian CYP1A4-related P450 of unique epitope(s) and by CYP1A5-related P450 of unusual catalytic effectiveness for arachidonic acid epoxygenation.

Suppression of apoptosis by gonadotropin, 17beta-estradiol, and epidermal growth factor in rainbow trout preovulatory ovarian follicles.

In this study we present the first evidence for the occurrence of apoptotic cell death in ovarian follicles from teleost fish. Preovulatory ovarian follicles from mature hatchery-raised rainbow trout (Oncorhynchus mykiss) were collected and either immediately frozen in liquid nitrogen or incubated in serum-free medium at 18 degrees for 24 hr. The extent of ovarian apoptotic DNA fragmentation was determined using 3'-end labeling of DNA with [32P]dideoxy-ATP, size fractionation by agarose gel electrophoresis, and quantification of low-molecular-weight (<15 kb) DNA using autoradiography and liquid scintillation counting. The extent of apoptotic DNA fragmentation was eightfold greater in immediately frozen preovulatory follicles than in previtellogenic ovarian follicles collected from immature rainbow trout (P < 0.05), suggesting differences in the degree of apoptosis at different stages of follicular development. In preovulatory trout follicles, the extent of apoptotic DNA fragmentation was fivefold greater in follicles incubated for 24 hr. Treatment of incubated preovulatory follicles with either partially purified salmon gonadotropin SG-G100 (1 microg/ml) or epidermal growth factor (EGF; 100 ng/ml) suppressed apoptotic DNA fragmentation by 31 and 41%, respectively, in comparison to untreated incubated follicles (P < 0.01). Treatment of incubated follicles with 17beta-estradiol (1-100 ng/ml) caused a concentration-dependent suppression of apoptotic DNA fragmentation (P < 0.05). These results suggest that apoptosis is involved in teleost ovarian development and that several of the hormonal factors acting as follicle survival factors in mammalian and avian ovaries may play a similar role in teleost ovarian follicles.

Elevated ovarian follicular apoptosis and heat shock protein-70 expression in white sucker exposed to bleached kraft pulp mill effluent.

Exposure of feral fish populations to bleached kraft pulp mill effluent (BKME) results in a variety of negative impacts on reproductive fitness including reduced ovarian development, reduced egg size, decreased fecundity with age, delayed sexual maturation, and alterations in reproductive endocrine homeostasis at multiple sites along the pituitary-gonadal axis. The present study provides evidence of elevated apoptotic DNA fragmentation and increased expression of the 70-kDa heat shock protein (HSP70) in ovarian follicular cells from prespawning white sucker (Catostomus commersoni) exposed to BKME. Apoptosis is the molecular mechanism responsible for ovarian follicular atresia which is involved in various stages of vertebrate ovarian development such as follicular recruitment, growth, differentiation, and regression. In mammals, induction of HSP70 is associated with inhibition of hormone-sensitive steroidogenesis and mediation of luteal regression. The 3'-end labeling of isolated ovarian follicular cell DNA revealed a 10-fold increase in the extent of apoptosis in BKME-exposed white sucker in comparison to follicles collected from a nearby reference site. Western blotting for ovarian follicular HSP70 levels showed increased expression of this protein in fish exposed to BKME. The elevated ovarian cell apoptosis and increased HSP70 expression in BKME-exposed fish were associated with reduced ovary size, decreased plasma testosterone, and increased plasma 17 beta-estradiol concentrations, but not induction of hepatic ethoxyresorufin O-deethylase activity. It is not known whether increased ovarian HSP70 expression in BKME-exposed fish is related to elevated apoptosis or represents a general response to environmental stress. Since apoptosis is regulated by several hormonal factors and conserved gene products, these data suggest that certain components of BKME increase ovarian cell apoptosis in fish via stimulation of cell death signaling. However, it is unclear whether BKME stimulates ovarian cell apoptosis directly or if this response occurs secondarily as a result of altered reproductive endocrine homeostasis.

In ovo 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure in three avian species. 1. Effects on thyroid hormones and growth during the perinatal period.

Thyroid hormones are important in the perinatal growth and development of avian species, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds have been shown to cause alterations in these hormones in laboratory animals. Since the decreased reproductive success in certain fish-eating bird populations exposed to TCDD and related compounds is characterized by high embryo and hatchling mortality, we examined the effects of in ovo TCDD exposure on plasma thyroid hormone concentrations (total T3, total T4) and body and skeletal growth during the perinatal period in the domestic chicken (Gallus gallus), domestic pigeon (Columba livia), and great blue heron (Ardea herodias). Hepatic ethoxyresorufin O-deethylase (EROD) activity was also determined as an enzymatic marker of cytochrome P450IA induction by TCDD. [3H]TCDD was injected into the air cell of chicken eggs (21-day incubation period) on Embryonic Day 4.5 (0.1 microgram/kg egg), pigeon eggs (18-day incubation period) on Embryonic Day 3.5 (1 microgram/kg egg) and Embryonic Day 14 (3 microgram/kg egg), and heron eggs (28-day incubation period) at approximately the midpoint of incubation (2 microgram/kg egg). Chickens were euthanized on Embryonic Days 17 and 19, day of hatch (Embryonic Day 21), and Days 2 and 4 after hatch. Pigeons and herons were euthanized either at hatch (Embryonic Days 18 and 28, respectively), or fed an uncontaminated diet for 7 days prior to sacrifice. Although hepatic EROD activity was induced 13- to 43-fold above controls in chickens, there was no effect of TCDD exposure on hatchability, body growth, subcutaneous edema, or plasma thyroid hormone levels. In pigeons exposed to TCDD on Embryonic Day 3.5, EROD was induced 6- to 15-fold, hatchability was decreased, liver to body weight ratio was elevated, and body and skeletal growth were decreased (p < 0.01); however, there was no effect of TCDD exposure on plasma thyroid hormone levels. Similarly, in pigeons exposed to TCDD on Embryonic Day 14, EROD was induced 10- to 14-fold, liver to body weight ratio was elevated, and body and skeletal growth were decreased (p < 0.01), but there was no effect of TCDD treatment on plasma thyroid hormone levels. In herons, hepatic EROD activity was induced 2- to 3-fold above control birds, similar to EROD activities measured in heron hatchlings exposed to environmental levels of TCDD and related chemicals in the Strait of Georgia, British Columbia. However, this level of TCDD exposure had no effect on plasma thyroid hormone levels or body growth in herons. Collectively, these results suggest that perinatal plasma thyroid hormone levels cannot be used as relatively noninvasive biomarkers of TCDD exposure during embryonic development in chickens, pigeons, and great blue herons.

In ovo 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure in three avian species. 2. Effects on estrogen receptor and plasma sex steroid hormones during the perinatal period.

As opposed to mammals, the heterogametic sex in birds is female, and sexual differentiation of the central nervous system away from the intrinsic male pattern is dependent on ovarian estrogen secretions during the perinatal period. The contamination of aquatic systems with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds has been suggested to be responsible for decreased reproductive success in certain wild fish-eating bird populations. Since TCDD has been shown to alter estrogenic status in laboratory animals, we determined the effects of in ovo TCDD exposure on hepatic estrogen receptor (ER) concentrations and affinities, and plasma estradiol concentrations during the perinatal period in the domestic chicken (Gallus gallus), domestic pigeon (Columba livia), and great blue heron (Ardea herodias). Plasma testosterone levels were also determined in herons as an indication of androgenic status. [3H]TCDD was injected into the air cell of chicken eggs on Embryonic Day 4.5 (0.1 microgram/kg egg), pigeon eggs on Embryonic Day 3.5 (1 microgram/kg egg) and Embryonic Day 14 (3 micrograms/kg egg), and heron eggs at approximately Embryonic Day 13 (2 micrograms/kg egg). Chickens were euthanized on Embryonic Days 17 and 19, hatch, and Days 2 and 4 after hatch. Pigeons and herons were either euthanized at hatch or fed an uncontaminated diet for 7 days prior to termination. Between 5 and 10% of the injected [3H]TCDD dose was measured in the liver of hatchlings. There was no effect of in ovo TCDD exposure on hepatic ER levels or plasma estradiol concentrations in female chickens and pigeons exposed early in incubation. In female pigeons exposed during the latter third part of incubation to a TCDD dose that would cause high embryo lethality if injected early in incubation, hepatic ER concentrations were elevated (p < 0.001) and plasma estradiol concentrations were decreased (p < 0.01) at hatch. There was no effect of TCDD exposure on plasma estradiol levels in male pigeons. In herons, TCDD exposure had no effect on hepatic ER levels or plasma estradiol and testosterone concentrations at either time point. We conclude that in chicken, pigeon, and great blue heron hatchlings exposed early in incubation to low doses of TCDD, hepatic ER levels and plasma estradiol concentrations are not biomarkers of toxicity.