Assessing the Toxicity of 17α-Ethinylestradiol in Rainbow Trout Using a 4-Day Transcriptomics Benchmark Dose (BMD) Embryo Assay.

There is an urgent demand for more efficient and ethical approaches in ecological risk assessment. Using 17α-ethinylestradiol (EE2) as a model compound, this study established an embryo benchmark dose (BMD) assay for rainbow trout (RBT; Oncorhynchus mykiss) to derive transcriptomic points-of-departure (tPODs) as an alternative to live-animal tests. Embryos were exposed to graded concentrations of EE2 (measured: 0, 1.13, 1.57, 6.22, 16.3, 55.1, and 169 ng/L) from hatch to 4 and up to 60 days post-hatch (dph) to assess molecular and apical responses, respectively. Whole proteome analyses of alevins did not show clear estrogenic effects. In contrast, transcriptomics revealed responses that were in agreement with apical effects, including excessive accumulation of intravascular and hepatic proteinaceous fluid and significant increases in mortality at 55.1 and 169 ng/L EE2 at later time points. Transcriptomic BMD analysis estimated the median of the 20th lowest geneBMD to be 0.18 ng/L, the most sensitive tPOD. Other estimates (0.78, 3.64, and 1.63 ng/L for the 10th percentile geneBMD, first peak geneBMD distribution, and median geneBMD of the most sensitive over-represented pathway, respectively) were within the same order of magnitude as empirically derived apical PODs for EE2 in the literature. This 4-day alternative RBT embryonic assay was effective in deriving tPODs that are protective of chronic effects of EE2.

Interactive effects of cadmium and Benzo[a]pyrene in adult zebrafish (Danio rerio) during short-term aqueous co-exposure.

Environmental water quality guidelines often work under the assumption that the toxicity of environmental pollutants is identical when present in isolation or in a complex chemical mixture. Thus, there is a crucial gap in our knowledge regarding how these toxicants interact and alter the toxicological effects in aquatic organisms. The present study examined the effects of acute (72-hr) aqueous exposures of Cadmium (Cd), a highly toxic non-essential trace metal, and Benzo[a]Pyrene (B[a]P), a prototypical polycyclic aromatic hydrocarbon (PAH) in adult zebrafish. Following a range-finding series of individual single-toxicant exposures, a second series was carried out using select concentrations in binary mixture exposures (using 5.8 or 22 µg/L for Cd; 0.44 or 1.07 µg/L for B[a]P). Our results demonstrated that tissue accumulation of both toxicants increased significantly in the presence of the second toxicant relative to single-toxicant exposures. Cd-only and B[a]P-only single toxicant exposures caused a significant downregulation of cytochrome p4501a (CYP1A1) and metallothionein-2 (MT2) mRNA in the gills, respectively, however binary co-exposures using both toxicants resulted in strong up-regulation of CYP1A1 and MT2. Additionally, co-exposures caused a strong induction of SOD1 and CAT mRNA transcript levels in the gill. The observed increase in body burden and transcript modulation did not translate into additive or more-than-additive toxic effects (oxidative stress) in zebrafish.

Differences in the subcellular distribution of cadmium and copper in the gills and liver of white sturgeon (Acipenser transmontanus) and rainbow trout (Oncorhynchus mykiss).

Recent studies have shown that white sturgeon (Acipenser transmontanus) are more resistant to cadmium (Cd) compared to rainbow trout (Oncorhynchus mykiss), whereas they are more sensitive than rainbow trout when exposed to copper (Cu). Differences in the subcellular distribution of metals among species could be one of the factors responsible for the differences in the sensitivity to metals. Although, subcellular distribution has been studied extensively in many species with many metals, its direct role in species-specific differences in the sensitivity has not been well studied. The objective of this study was to evaluate the role of subcellular distribution of metals in species-specific differences in the sensitivity to metals between sturgeon and trout. We compared the subcellular distribution of metals Cd and Cu in the cellular debris, heat-stable proteins, heat-denatured fraction, metal-rich granules, and organelles fractions from the gills and liver after exposure of juveniles of both species to 1.25 and 20 µg/L Cd and Cu for 8 days, respectively. Sturgeon diverted a higher amount of Cd towards biologically inactive metal pool (BIM) and a lower amount towards the biologically active metal pool (BAM) compared to trout in both tissues. This explained why sturgeon are able to tolerate a relatively higher exposure level to Cd compared to trout. For Cu, there was no statistically significant species-specific differences in the amounts diverted towards either BAM or BIM; hence, white sturgeon's greater sensitivity to Cu was not explained by its subcellular distribution strategies.

Human red blood cell uptake and sequestration of arsenite and selenite: Evidence of seleno-bis(S-glutathionyl) arsinium ion formation in human cells.

Over 200 million people worldwide are exposed to the human carcinogen, arsenic, in contaminated drinking water. In laboratory animals, arsenic and the essential trace element, selenium, can undergo mutual detoxification through the formation of the seleno-bis(S-glutathionyl) arsinium ion [(GS)2AsSe]-, which undergoes biliary and fecal elimination. [(GS)2AsSe]-, formed in animal red blood cells (RBCs), sequesters arsenic and selenium, and slows the distribution of both compounds to peripheral tissues susceptible to toxic effects. In human RBCs, the influence of arsenic on selenium accumulation, and vice versa, is largely unknown. The study aims were to characterize arsenite (AsIII) and selenite (SeIV) uptake by human RBCs, to determine if SeIV and AsIII increase the respective accumulation of the other in human RBCs, and ultimately to determine if this occurs through the formation and sequestration of [(GS)2AsSe]-. 75SeIV accumulation was temperature and Cl--dependent, inhibited by 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid (H2DIDS) (IC50 1 ± 0.2 µM), and approached saturation at 30 µM, suggesting uptake is mediated by the erythrocyte anion-exchanger 1 (AE1 or Band 3, gene SLC4A1). HEK293 cells overexpressing AE1 showed concentration-dependent 75SeIV uptake. 73AsIII uptake by human RBCs was temperature-dependent, partly reduced by aquaglyceroporin 3 inhibitors, and not saturated. AsIII increased 75SeIV accumulation (in the presence of albumin) and SeIV increased 73AsIII accumulation in human RBCs. Near-edge X-ray absorption spectroscopy revealed the formation of [(GS)2AsSe]- in human RBCs exposed to both AsIII and SeIV. The sequestration of [(GS)2AsSe]- in human RBCs potentially slows arsenic distribution to susceptible tissues and could reduce arsenic-induced disease.

Comparative analyses of oxidative stress response and metallothionein induction in white sturgeon and rainbow trout during acute waterborne copper exposure.

Early life-stages of the endangered white sturgeon (Acipenser transmontanus) have been shown to be among the most sensitive fishes to aqueous copper (Cu) exposure. In a recent analogous study, we examined the role of whole-body Cu accumulation and Na homeostasis in species-specific differences between the sensitivity of white sturgeon and a common laboratory fish model, rainbow trout, to Cu. However, the potential roles of important mechanisms such as Cu-induced oxidative stress and/or metallothionein (MT) induction as potential drivers of sensitivity of white sturgeon to Cu have not been investigated to date. Here, rainbow trout and white sturgeon from three different early life-stages were exposed to waterborne Cu for 96 h, following which major antioxidant parameters, lipid peroxidation and MT gene expression were evaluated. Results indicated that during larval and swim-up life-stages, Cu induced oxidative damage in white sturgeon was greater than in rainbow trout. Moreover, baseline glutathione (GSH) was significantly greater in rainbow trout than white sturgeon. Observations also suggested that trout exceedingly relied on GSH to combat Cu-induced oxidative stress as they grew older. In contrast, sturgeon recruited an increasing level of MT to neutralize Cu-induced oxidative stress and/or Cu loading. In our recent study, we demonstrated that Na homeostasis is more susceptible to Cu in white sturgeon than in rainbow trout. Collectively, these findings indicate that the greater degree of oxidative damage in early life-stages, in addition to the higher magnitude of the disruption of Na homeostasis, contributes to the higher sensitivity of white sturgeon to Cu exposure.

Sensitivity of white sturgeon and rainbow trout to waterborne copper exposure: A comparative study of copper-induced disruption of sodium homeostasis.

Recent studies have demonstrated that white sturgeon are more sensitive to acute exposure to Cu than rainbow trout (Oncorhynchus mykiss), especially during early life-stages. However, the physiological mechanisms underlying this difference in sensitivity to Cu is not known. In the present study, we first confirmed the higher sensitivity (lower 96 h LC50 values) of white sturgeon to Cu at three different life stages (larva, swim-up, and juvenile) relative to their counterparts in rainbow trout. We also demonstrated that acute exposure to Cu (50 µg/L for 4.5 h) caused a significantly greater reduction in the rate of waterborne Na uptake in white sturgeon relative to that in rainbow trout across all three life-stages. In agreement with this observation, we also found that acute exposure to Cu (20 µg/L for 48 h) elicits a significantly greater decrease in whole body Na level in all life stages of white sturgeon compared to rainbow trout. In contrast, white sturgeon demonstrated a higher or similar level of Cu body burden relative to rainbow trout during acute Cu exposure (20 µg/L for 24 h), thereby indicating that Cu bioaccumulation is not a good indicator of its toxicity in these species. Overall, our study demonstrated that the differences in sensitivity to acute Cu exposure between white sturgeon and rainbow trout can be explained on the basis of differential effects of Cu on Na homeostasis.

Species and life-stage specific differences in cadmium accumulation and cadmium induced oxidative stress, metallothionein and heat shock protein responses in white sturgeon and rainbow trout.

Understanding the mechanistic basis of differences in the sensitivity of fishes to metals is important for developing informed ecological risk assessment approaches for metals. Whole body metal accumulation, metallothionein induction, oxidative stress and associated antioxidant response, as well as heat shock proteins (mainly HSP70) are known to play important roles in determining the toxicity of metals in fish. Hence, in this study we have cross-evaluated these parameters as a function of Cd exposure across different life-stages of two evolutionarily distinct fish species, namely rainbow (Oncorhynchus mykiss) trout and white sturgeon (Acipenser transmontanus). These two species have been shown to differ significantly in their physiological and apical responses to Cd exposure. The findings of the present study suggest that species-specific differences in the sensitivity to Cd could partially be explained by HSP70 gene response and oxidative damage biomarkers. However, not all the parameters studied in this study could explain the life-stage specific differences universally and were limited to only some life-stages. Based on the observations in the present study and other recent studies, it is apparent that species- and life-stage specific differences in the sensitivity to Cd and possibly other metals is a complex phenomenon and could be driven by multiple toxicokinetic and toxicodynamic factors.

Investigating the Role of Ionoregulatory Processes in the Species- and Life-Stage-Specific Differences in Sensitivity of Rainbow Trout and White Sturgeon to Cadmium.

There are huge variations in life-stage- and species-specific sensitivities among the fishes to the exposure with metals; however, the physiological mechanisms underlying these differences are not well understood to date. This study revealed significant life-stage-specific (larval, swim-up, and juvenile) and species-specific differences between two evolutionary distant species of fishes, rainbow trout ( Oncorhynchus mykiss) and white sturgeon ( Acipenser transmontanus), following acute exposures to Cd. Although the 96 h LC50 of Cd was similar in both species at the larval stage, trout demonstrated an increased sensitivity to Cd at later life stages as compared to sturgeon. Moreover, exposure to Cd disrupted calcium (Ca) uptake and whole body Ca levels in trout by a greater degree relative to that in sturgeon regardless of life stage. Finally, white sturgeon demonstrated a lower affinity for Cd uptake relative to the more sensitive rainbow trout. This infers a differential nature of the interaction between Cd and Ca transport pathways in the two species and partially explains the differences in Cd sensitivity between rainbow trout and white sturgeon described previously. Overall, our results suggest that species- and life-stage-specific differences in sensitivity to waterborne Cd in fish are likely a function of the interplay between Cd uptake and Cd-induced disruption of Ca homeostasis.

Expression stability and selection of optimal reference genes for gene expression normalization in early life stage rainbow trout exposed to cadmium and copper.

Gene expression analysis represents a powerful approach to characterize the specific mechanisms by which contaminants interact with organisms. One of the key considerations when conducting gene expression analyses using quantitative real-time reverse transcription-polymerase chain reaction (qPCR) is the selection of appropriate reference genes, which is often overlooked. Specifically, to reach meaningful conclusions when using relative quantification approaches, expression levels of reference genes must be highly stable and cannot vary as a function of experimental conditions. However, to date, information on the stability of commonly used reference genes across developmental stages, tissues and after exposure to contaminants such as metals is lacking for many vertebrate species including teleost fish. Therefore, in this study, we assessed the stability of expression of 8 reference gene candidates in the gills and skin of three different early life-stages of rainbow trout after acute exposure (24h) to two metals, cadmium (Cd) and copper (Cu) using qPCR. Candidate housekeeping genes were: beta actin (b-actin), DNA directed RNA polymerase II subunit I (DRP2), elongation factor-1 alpha (EF1a), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), glucose-6-phosphate dehydrogenase (G6PD), hypoxanthine phosphoribosyltransferase (HPRT), ribosomal protein L8 (RPL8), and 18S ribosomal RNA (18S). Four algorithms, geNorm, NormFinder, BestKeeper, and the comparative ΔCt method were employed to systematically evaluate the expression stability of these candidate genes under control and exposed conditions as well as across three different life-stages. Finally, stability of genes was ranked by taking geometric means of the ranks established by the different methods. Stability of reference genes was ranked in the following order (from lower to higher stability): HPRT

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.