Jon A. Doering

Tissue specificity of aryl hydrocarbon receptor (AhR) mediated responses and relative sensitivity of white sturgeon (Acipenser transmontanus) to an AhR agonist

Sturgeons are endangered in some parts of the world. Due to their benthic nature and longevity sturgeon are at greater risk of exposure to bioaccumulative contaminants such as dioxin-like compounds that are associated with sediments. Despite their endangered status, little research has been conducted to characterize the relative responsiveness of sturgeon to dioxin-like compounds. In an attempt to study the biological effects and possible associated risks of exposure to dioxin-like compounds in sturgeon, the molecular and biochemical responses of white sturgeon (Acipenser transmontanus) to a model aryl hydrocarbon receptor (AhR) agonist, β-naphthoflavone (βNF) were investigated. White sturgeon were injected intraperitoneally with one of three doses of βNF (0, 50, or 500mg/kg, bw). Rainbow trout (Oncorhynchus mykiss) were used as a reference species since their responses have been well characterized in the past. Three days following injection with βNF, fish were euthanized and livers, gills, and intestines collected for biochemical and molecular analyses. White sturgeon exposed to βNF had significantly greater ethoxyresorufin O-deethylase (EROD) activity in liver (up to 37-fold), gill (up to 41-fold), and intestine (up to 36-fold) than did unexposed controls. Rainbow trout injected with βNF exhibited EROD activity that was significantly greater in liver (88-fold), than that of controls, but was undetectable in gills or intestine. Abundance of CYP1A transcript displayed a comparable pattern of tissue-specific induction with intestine (up to 189-fold), gills (up to 53-fold), and liver (up to 21-fold). Methoxyresorufin O-deethylase (MROD) and pentoxyresorufin O-deethylase (PROD) activities were undetectable in unexposed white sturgeon tissues while exposed tissues displayed MROD activity that was only moderately greater than the activity that could be detected. Differential inducibility among liver, gill, and intestine following exposure to an AhR agonist is likely associated with tissue-specific regulation of the AhR signalling pathway. Liver and gill of white sturgeon had significantly greater AhR transcript abundance than did the intestine, however following exposure to βNF, significantly greater induction in AhR transcript abundance was detected in intestine (up to 35-fold) compared to liver (up to 5-fold) or gills (up to 11-fold). It was shown that white sturgeon are responsive to AhR agonists in the liver, gill, and intestine and could be among the more sensitive fish species with regard to inducibility of CYP1A.

Predicting the sensitivity of fishes to dioxin-like compounds: possible role of the aryl hydrocarbon receptor (AhR) ligand binding domain

Dioxin-like compounds are chronically toxic to most vertebrates. However, dramatic differences in sensitivity to these chemicals exist both within and among vertebrate classes. A recent study found that in birds, critical amino acid residues in the aryl hydrocarbon receptor (AhR) ligand binding domain are predictive of sensitivity to dioxin-like compounds in a range of species. It is currently unclear whether similar predictive relationships exist for fishes, a group of animals at risk of exposure to dioxin-like compounds. Effects of dioxin-like compounds are mediated through the AhR in fishes and birds. However, AhR dynamics are more complex among fishes. Fishes possess AhRs that can be grouped within at least three distinct clades (AhR1, AhR2, AhR3) with each clade possibly containing multiple isoforms. AhR2 has been shown to be the active form in most teleosts, with AhR1 not binding dioxin-like compounds. The role of AhR3 in dioxin-like toxicity has not been established to date and this clade is only known to be expressed in some cartilaginous fishes. Furthermore, multiple mechanisms of sensitivity to dioxin-like compounds that are not relevant in birds could exist among fishes. Although, at this time, deficiencies exist for the development of such a predictive relationship for application to fishes, successfully establishing such relationships would offer a substantial improvement in assessment of risks of dioxin-like compounds for this class of vertebrates. Elucidation of such relationships would provide a mechanistic foundation for extrapolation among species to allow the identification of the most sensitive fishes, with the ultimate goal of the prediction of risk posed to endangered species that are not easily studied.

EFFECTS OF SUBCHRONIC EXPOSURE OF EARLY LIFE STAGES OF WHITE STURGEON (ACIPENSER TRANSMONTANUS) TO COPPER, CADMIUM, AND ZINC

Populations of sturgeon (Acipenseridae) are declining in many places in the world because of several potential factors, including overharvesting, habitat alteration, and pollution. In North America, populations of the white sturgeon (Acipenser transmontanus) have been experiencing poor annual recruitment in major river systems for more than three decades. Metal pollution has been hypothesized as a potential contributing factor to the poor recruitment in some of the water bodies. In general, little is known about the toxicity of metals such as Cu, Cd, and Zn to white sturgeon and their potential influence on survival of embryos and juveniles. The present study was conducted to establish baseline toxicity data for the subchronic exposure of early life stages of white sturgeon to Cu, Cd, and Zn that can be used in metal-related risk assessments. Embryos, larvae, and fry were exposed to increasing concentrations of dissolved Cu, Cd, or Zn for 66 d using laboratory-based flow-through exposure systems. Hatching success was greater than 79% for all controls, and no significant differences were observed among treatment groups or between treatments and controls. Chronic lethal concentrations at which 20% mortality occurred (LC20s) for Cd (1.5 µg/L), Cu (5.5 µg/L), and Zn (112 µg/L) obtained for white sturgeon in the present study were comparable to those of sensitive salmonid species. Based on LC20 values for 19 or 58 d posthatch white sturgeon, the United States national ambient water quality criteria and the Canadian water quality guidelines for the protection of aquatic life that have been established for Cd, Cu, and Zn protect white sturgeon early life stages.

Identification and expression of aryl hydrocarbon receptors (AhR1 and AhR2) provide insight in an evolutionary context regarding sensitivity of white sturgeon (Acipenser transmontanus) to dioxin-like compounds

Sturgeons are ancient fishes, which are endangered in many parts of the world. Due to their benthic nature and longevity, sturgeon are at great risk of exposure to bioaccumulative contaminants such as dioxin-like compounds (DLCs). Despite their endangered status, little research has been conducted to characterize the relative sensitivity of sturgeons to DLCs. Proper assessment of risk of DLCs posed to these fishes therefore, requires a better understanding of this sensitivity and the factors that are driving it. Adverse effects associated with exposure to DLCs are mediated by the aryl hydrocarbon receptor (AhR). This study identified and characterized two distinct AhRs, AhR1 and AhR2, in white sturgeon (Acipenser transmontanus) for the first time as a first step in studying the relative sensitivities of sturgeons to DLCs. Furthermore, tissue-specific expression of both AhRs under basal conditions and in response to exposure to the model DLC, β-naphthoflavone (βNF), was determined. The sequence of amino acids of AhR1 of white sturgeon had greater similarity to AhRs of tetrapods, including amphibians, birds, and mammals, than to AhR1s of other fishes. The sequence of amino acids in the ligand binding domain of the AhR1 had greater than 80% similarity to AhRs known to bind DLCs and was less similar to AhRs not known to bind DLCs. AhR2 of white sturgeon had greatest similarity to AhR2 of other fishes. Profiles of expression of AhR1 and AhR2 in white sturgeon were distinct from those known in other fishes and appear more similar to profiles observed in birds. Expressions of both AhR1 and AhR2 of white sturgeon were greatest in liver and heart, which are target organs for DLCs. Furthermore, abundances of transcripts of AhR1 and AhR2 in all tissues from white sturgeon were greater than controls (up to 35-fold) following exposure to βNF. Based upon both AhRs having similar abundances of transcript in target organs of DLC toxicity, both AhRs being up-regulated following exposure to βNF, and both AhRs having greatest similarity to AhRs known to bind DLCs, it is hypothesized that both AhR1 and AhR2 of white sturgeon might mediate effects of DLCs in this species. Since current risk assessments are based on data derived largely from highly divergent fishes within the Salmonidae, presence of two functional AhRs in white sturgeon, one of which has greatest similarity to AhRs of birds, might have significant implications for the sensitivity of sturgeons to DLCs compared to other fishes.

Assessment of the sensitivity of three North American fish species to disruptors of steroidogenesis using in vitro tissue explants

There is concern regarding exposure of aquatic organisms to chemicals that interfere with the endocrine system. One critical mechanism of endocrine disruption is impairment of steroidogenesis that can lead to altered hormone levels, altered or delayed sexual development, and ultimately reproductive failure. With the current large gap in knowledge and a high degree of uncertainty regarding the sensitivity of fishes native to northern ecosystems to endocrine disrupting chemicals (EDCs), the aim of this study was to develop an in vitro gonadal explant assay enabling the assessment of EDCs on sex-steroid production in wild fish species native to North America. Northern pike (Esox lucius), walleye (Sander vitreus), and white sucker (Catostomus commeroni) were sampled from a reference location in Lake Diefenbaker, Saskatchewan, Canada, at spawn and multiple post-spawn time points. Gonads were excised and immediately exposed for 24h to a model inducer (forskolin) or inhibitor (prochloraz) of steroidogenesis in L-15 supplemented media. Furthermore, seasonal profiles of plasma 11-ketotestosterone (11-KT) and 17-β estradiol (E2) concentrations were characterized. Enzyme-linked immunosorbent assays were used to quantify hormone concentrations in plasma and media. The seasonal profile of plasma hormones was significantly correlated with basal in vitro hormone production. Gonad tissue exposed to forskolin showed a concentration-dependent increase in E2 and a general increase in 11-KT. Gonad tissue exposed to prochloraz resulted in a decrease of concentrations of 11-KT and E2. These results illustrated that gonadal tissue is undergoing steroidogenesis in an in vitro setting that is comparable to in vivo hormone profiles, and which is responsive to chemical exposure in a concentration-dependent manner. The seasonal time point during which gonad explants were excised and exposed had an impact on the potency and magnitude of responses, resulting in a seasonal effect on sensitivity. Male and female white sucker showed greatest sensitivity to forskolin, while male and female walleye showed greatest sensitivity to prochloraz. Also, gonad explants from these species were found to have greater sensitivity than responses previously reported for in vitro explants of other fish species such as the fathead minnow (Pimephales promelas), and stable cell lines currently used as screening applications to detect chemicals that might disrupt the endocrine system. Therefore, current approaches that use stable cell lines or tissue explants from standardized small bodied laboratory species might not be protective of some wild fish species. Future research is required that investigates whether this in vitro gonadal explant assay is predictive of in vivo effects in wild species of fishes.

Differences in Activation of Aryl Hydrocarbon Receptors of White Sturgeon Relative to Lake Sturgeon Are Predicted by Identities of Key Amino Acids in the Ligand Binding Domain

Dioxin-like compounds (DLCs) are pollutants of global environmental concern. DLCs elicit their adverse outcomes through activation of the aryl hydrocarbon receptor (AhR). However, there is limited understanding of the mechanisms that result in differences in sensitivity to DLCs among different species of fishes. Understanding these mechanisms is critical for protection of the diversity of fishes exposed to DLCs, including endangered species. This study investigated specific mechanisms that drive responses of two endangered fishes, white sturgeon (Acipenser transmontanus) and lake sturgeon (Acipenser fulvescens) to DLCs. It determined whether differences in sensitivity to activation of AhRs (AhR1 and AhR2) can be predicted based on identities of key amino acids in the ligand binding domain (LBD). White sturgeon were 3- to 30-fold more sensitive than lake sturgeon to exposure to 5 different DLCs based on activation of AhR2. There were no differences in sensitivity between white sturgeon and lake sturgeon based on activation of AhR1. Adverse outcomes as a result of exposure to DLCs have been shown to be mediated through activation of AhR2, but not AhR1, in all fishes studied to date. This indicates that white sturgeon are likely to have greater sensitivity in vivo relative to lake sturgeon. Homology modeling and in silico mutagenesis suggests that differences in sensitivity to activation of AhR2 result from differences in key amino acids at position 388 in the LBD of AhR2 of white sturgeon (Ala-388) and lake sturgeon (Thr-388). This indicates that identities of key amino acids in the LBD of AhR2 could be predictive of both in vitro activation by DLCs and in vivo sensitivity to DLCs in these, and potentially other, fishes.

Effects of Columbia River water on early life-stages of white sturgeon (Acipenser transmontanus)

The white sturgeon (Acipenser transmontanus) population that resides in the Columbia River in British Columbia (BC), Canada, has suffered recruitment failures for more than three decades. During the summers of 2008 and 2009, studies were performed to determine whether exposure to water downstream of a metal smelter in Trail, BC affected survival or growth of early life-stages of white sturgeon through 60+ days post-fertilization (dpf). In both years, there were no significant differences in survival of fish that were exposed to water from downstream compared to water from upstream of the smelter. At 20-21dpf, average mortality was 2.4 percent and 12 percent in upstream water for 2008 and 2009, respectively, which was similar to the average mortality of 3.8 percent and 7.2 percent in downstream water for 2008 and 2009, respectively. Relatively great mortality after 20-21dpf complicated analysis of the subchronic exposure, but use of a survival analysis indicated that the average fish died at 25-29dpf, regardless of whether the water to which they were exposed came from upstream or downstream of the smelter. In addition, measured concentrations of metals in river water were less than the threshold for adverse effects on early life stages of white sturgeon. Based upon these analyses, it is not likely that current concentrations of metals in the Columbia River in southern BC are adversely affecting survival of early life stages of white sturgeon larvae.

Identification and response to metals of metallothionein in two ancient fishes: White sturgeon (Acipenser transmontanus) and lake sturgeon (Acipenser fulvescens)

White sturgeon (Acipenser transmontanus) are among the most sensitive species of fishes to Cu, Cd, and Zn, but there is no information about sensitivity of lake sturgeon (Acipenser fulvescens). To begin to elucidate molecular mechanism(s) of sensitivity of sturgeons to metals a cDNA encoding metallothionein (MT) was amplified from livers of white sturgeon (WS-MT) and lake sturgeon (LS-MT), and expression in response to Cu, Cd, or Zn was characterized in liver explants from each species. The primary structure of WS-MT and LS-MT contained 20 cysteine residues, which is the same as MTs of teleost fishes. However, the primary structure of WS-MT and LS-MT contained 63 amino acids, which is longer than any MT identified in teleost fishes. Abundance of transcripts of WS-MT in explants exposed to 0.3, 3, 30, or 100 μg/L of Cu was 1.7-, 1.7-, 2.1-, and 2.6-fold less than in controls, respectively. In contrast, abundances of transcripts of WS-MT were 3.3- and 2.4-fold greater in explants exposed to 30 μg/L of Cd and 1000 μg/L of Zn, respectively. Abundance of transcripts of LS-MT was not significantly different at any concentration of Cu, Cd, or Zn. MT is hypothesized to represent a critical mechanism for detoxification of metals. Therefore, results of this study suggest that sensitivity of sturgeons to exposure to Cu, Cd, or Zn might be a result of the relatively lesser maximal response of MT to metals. The study also suggestslake sturgeon might be more sensitive than white sturgeon to metals.

High Conservation in Transcriptomic and Proteomic Response of White Sturgeon to Equipotent Concentrations of 2,3,7,8-TCDD, PCB 77, and Benzo[a]pyrene

Adverse effects associated with exposure to dioxin-like compounds (DLCs) are mediated primarily through activation of the aryl hydrocarbon receptor (AHR). However, little is known about the cascades of events that link activation of the AHR to apical adverse effects. Therefore, this study used high-throughput, next-generation molecular tools to investigate similarities and differences in whole transcriptome and whole proteome responses to equipotent concentrations of three agonists of the AHR, 2,3,7,8-TCDD, PCB 77, and benzo[a]pyrene, in livers of a nonmodel fish, the white sturgeon (Acipenser transmontanus). A total of 926 and 658 unique transcripts were up- and down-regulated, respectively, by one or more of the three chemicals. Of the transcripts shared by responses to all three chemicals, 85% of up-regulated transcripts and 75% of down-regulated transcripts had the same magnitude of response. A total of 290 and 110 unique proteins were up- and down-regulated, respectively, by one or more of the three chemicals. Of the proteins shared by responses to all three chemicals, 70% of up-regulated proteins and 48% of down-regulated proteins had the same magnitude of response. Among treatments there was 68% similarity between the global transcriptome and global proteome. Pathway analysis revealed that perturbed physiological processes were indistinguishable between equipotent concentrations of the three chemicals. The results of this study contribute toward more completely describing adverse outcome pathways associated with activation of the AHR.

Linking Oxidative Stress and Magnitude of Compensatory Responses with Life-Stage Specific Differences in Sensitivity of White Sturgeon (Acipenser transmontanus) to Copper or Cadmium

Sensitivity of white sturgeon (Acipenser transmontanus) to copper (Cu) or cadmium (Cd) has been shown to significantly differ as a function of life-stage. This study investigated oxidative stress, metal homeostasis, and associated compensatory responses as potential mechanisms of this sensitivity pattern in three early life-stages. Sturgeon were most sensitive to Cu at 15 days post hatch (dph), which was accompanied by a significant increase in lipid peroxidation (LPO). Genes involved with amelioration of oxidative stress were significantly less inducible at this stage than in older, less sensitive fry. At 48 dph, acute lethality of sturgeon exposed to Cd was greatest and body LPO was significantly induced by 3.5-fold at 5 μg Cd/L. Moreover, there was a small but significant increase in antioxidative responses. At 139 dph, sturgeon were most tolerant to Cu and Cd and accumulation of these metals was least. Also, expression of metallothionein (MT) and apoptotic genes were greatest while expression of metal transporters was reduced and concentration of LPO was not different from controls. Our results suggest that life-stage specific sensitivity of white sturgeon to metals is complex, encompassing differences in the ability to mount compensatory responses important for metal homeostasis and combating oxidative stress and concomitant damages.