Reproductive and Developmental Effects of Sex-Specific Chronic Exposure to Dietary Arsenic in Zebrafish (Danio rerio).

The present study investigated the reproductive and developmental effects of sex-specific chronic exposure to dietary arsenic in zebrafish. Adult zebrafish (Danio rerio) were exposed to environmentally realistic doses of arsenic via diet [0 (control; no added arsenic), 30 (low), 60 (medium), and 100 (high) µg/g dry weight, as arsenite] for 90 days. Following exposure, arsenic-exposed females from each dietary treatment were mated with control males, and similarly, arsenic-exposed males from each dietary treatment were mated with control females. In females, arsenic exposure resulted in a dose-dependent decrease in reproductive performance (fecundity, fertilization success, and hatching success). Moreover, a dose-dependent increase in developmental toxicity (larval deformities and larval mortality) was observed with maternal exposure to arsenic. In contrast, in males, arsenic exposure also induced similar reproductive and developmental toxicity; however, the adverse effects were mainly evident only in the medium and high dietary arsenic treatment groups. We also examined the sex-specific effects of dietary arsenic exposure on the expression of genes that regulate the hypothalamus-pituitary-gonadal-liver (HPG-L) axis in fish. The gene expression results indicated the downregulation of HPG-L axis genes in females irrespective of the arsenic treatment dose; however, the reduced expression of HPG-L axis genes in males was recorded only in the medium and high arsenic treatment groups. These observations suggest that chronic arsenic exposure in either females or males causes reproductive and developmental toxicity in zebrafish. However, these toxic effects are markedly higher in females than in males. Our results also suggest that arsenic can act as an endocrine disruptor and mediate reproductive and developmental toxicity by disrupting the HPG-L axis in zebrafish.

Examining the subchronic (28-day) effects of aqueous Cd-BaP co-exposure on detoxification capacity and cardiac function in adult zebrafish (Danio rerio).

The present study aimed to examine the effects of environmentally relevant concentrations of cadmium (Cd) and Benzo[a]Pyrene (BaP) in the adult zebrafish (Danio rerio). To this end, fish were exposed to either 1 or 10 µg/L Cd or 0.1 or 1 µg/L BaP in isolation, or a co-exposure containing a mixture of the two toxicants. Our results showed extensive modulation of the expression of key antioxidant genes (GPx, SOD1, catalase), detoxifying genes (MT1, MT2, CYP1A1) and a stress biomarker (HSP70) differing between control, single toxicant groups and co-exposure groups. We additionally carried out histopathological analysis of the gills, liver, and hearts of exposed animals, noting no differences in tissue necrosis or apoptosis. Finally, we carried out ultrasonographic analysis of cardiac function, noting a significant decrease of E-wave peak velocity and end diastolic volume in exposed fish. This in turn was accompanied by a decrease in stroke volume and ejection fraction, but not cardiac output in co-exposed fish. The present study is the first to demonstrate that a subchronic aqueous exposure to a Cd-BaP mixture can extensively modulate detoxification capacity and cardiac function in adult zebrafish in a tissue-specific manner.

Modulation of Cd and BaP uptake rate during acute aqueous co-exposure in adult zebrafish (Danio rerio).

Cadmium and Benzo[a]pyrene are two toxicants of great environmental importance given their frequency and ability to cause extensive toxicity in aquatic organisms including fish. There is evidence that fish can modulate their respective uptake rate during simultaneous exposures, albeit the mechanism behind this is poorly understood. The present study aimed to examine this interaction by exposing adult zebrafish to either 89.3 nM Cd, 4.25 nM BaP or a combination of the two for 72 hrs prior to examining the uptake rate of either toxicant via short-term exposures (3-6 hrs) to radiotracers (109Cd and 14C-BaP). Our results showed that Cd uptake rate increased significantly in the gills when animals were pre-exposed to both toxicants simultaneously, resulting in an increased maximum uptake rate (Jmax). The increased Cd uptake rate did not correspond to increased expression of gill Cd transporters such as the epithelium calcium channel (ECaC) or the divalent metal transporter 1 (DMT1). Furthermore, BaP uptake rate increased significantly at the whole-body level when animals were exposed to both 5.03 nM 14C-BaP and 89.3 nM Cd concurrently. Additionally, we ran a time-course and observed BaP uptake rate is highest in the 6-12 hrs following the beginning of the exposure. Our results provide evidence that the increased bioaccumulation of Cd and BaP observed during co-exposures is at least in part due to an increase in uptake rate and is driven by separate mechanisms.

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.

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.

Quantification of Mg2+, Ca2+ and H+ transport by the gastrointestinal tract of the goldfish, Carassius auratus, using the Scanning Ion-selective Electrode Technique (SIET).

An in vitro gut-sac technique and the scanning ion-selective electrode technique (SIET) were used to characterize Mg2+, Ca2+, and H+ transport at both the mucosal and serosal surfaces of non-everted and everted gastrointestinal tissues obtained from Carassius auratus. As part of the study, two magnesium ionophores were compared (II vs. VI). Unfed animals displayed uniform transport of all ions along the intestine. Feeding resulted in elevated Mg2+ and Ca2+ transport when the gut lumen contained chyme however, under symmetrical conditions this increased transport rate was absent. Furthermore, zonation of divalent cation transport was present for both Ca2+ and Mg2+ under non-symmetrical conditions while the zonation remained for Ca2+ alone under symmetrical conditions. High dietary Mg2+ decreased absorption and induced secretion of Mg2+ in the posterior intestine. Uptake kinetics in the esophagus suggest large diffusive and/or convective components based on a linear relationship between Mg2+ transport and concentration and lack of inhibition by ouabain, an inhibitor of Na+-K+-ATPase. In contrast, kinetics in the rectum were suggestive of a low affinity, saturable carrier-mediated pathway. A decrease in Mg2+ and Ca2+ transport was observed in the posterior intestine (both at the mucosal and serosal surfaces) in response to ouabain. This impact was greatest for Ca2+ transport and when applied to the mucosal fluid and measured in everted preparations. In contrast a putative Mg2+ transport inhibitor, cobalt(III)hexamine-chloride, did not affect Mg2+ transport. This is the first study to use SIET approaches to study ion transport in the gut of teleost fish. This is also the first study to provide characterization of Mg2+ transport in the gut of C. auratus. Due to the limited selectivity of Magnesium ionophore II, subsequent studies of tissues bathed in physiological saline should be made using Magnesium Ionophore VI.

Identification of the putative goldfish (Carassius auratus) magnesium transporter SLC41a1 and functional regulation in the gill, kidney, and intestine in response to dietary and environmental manipulations.

While magnesium requirements for teleost fish highlight the physiological importance of this cation for homeostasis, little is known regarding the molecular identity of transporters responsible for magnesium absorption or secretion. The recent characterization of the vertebrate magnesium transporter solute carrier 41a1 (SLC41a1) in the kidney of a euryhaline fish has provided a glimpse of possible moieties involved in piscine magnesium regulation. The present study obtained a novel SLC41a1 coding sequence for Carassius auratus and demonstrated ubiquitous expression in all tissues examined. Transcriptional regulation of SLC41a1 in response to dietary and environmental magnesium concentrations was observed across tissues. Specifically, decreased environmental magnesium correlated with decreased expression of SLC41a1 in the intestine, whereas the gill and kidney were unaffected. Dietary magnesium restriction correlated with decreased expression of SLC41a1 in the intestine and gill, while again no effects were detected in the kidney. Finally, elevated dietary magnesium correlated with increased expression of SLC41a1 in the kidney, while expression in the intestine and gill remained stable. Plasma magnesium was maintained in all treatments, and dietary assimilation efficiency increased with decreased dietary magnesium. Consumption of a single meal failed to impact SLC41a1 expression, and transcript abundance remained stable over the course of digestion in all treatments. Transcriptional regulation occurred between 7 and 14days following dietary and environmental manipulations and short-term regulation (e.g. <24h) was not observed. Overall the data supports transcriptional regulation of SLC41a1 reflecting a possible role in magnesium loss or secretion across tissues in fish.