Water Hardness Improves the Antioxidant Response of Zinc-Exposed Goldfish (Carassius auratus).

Zinc (Zn), a heavy metal, is an essential element in fish; however, exposure to high concentrations causes oxidative stress. Water hardness reduces oxidative stress reactions caused by heavy metals. To confirm the effect of water hardness on oxidative stress caused by Zn, goldfish were exposed to various Zn concentrations (1.0, 2.0, and 5.0 mg/L) and water hardness (soft (S), hard (H), and very hard (V)). The activity of superoxide dismutase (SOD) and catalase (CAT) in plasma increased with 1.0, 2.0, and 5.0 mg/L of Zn, and decreased with H and V water hardness. The levels of H2O2 and lipid peroxide (LPO) increased with Zn above 1.0 mg/L and decreased with H and V of water hardness. Caspase-9 mRNA expression in the liver increased after 7 and 14 days of Zn exposure and decreased with H and V water hardness. It was confirmed that DNA damage was less dependent on H and V water hardness. Based on the results of this study, at least 1.0 mg/L Zn causes oxidative stress in goldfish, and a high level of apoptosis occurs when exposed for more than 7 days. It appears that the oxidative stress generated by Zn can be alleviated by water hardness of at least 270 mg/L CaCO3. This study provides information on the relationship between the antioxidant response caused by heavy metals and water hardness in fish.

Synthetic microfiber exposure negatively affects reproductive parameters in male medaka (Oryzias latipes).

Microplastics not only accumulate in the bodies of fishes and cause damage to the organs, but also cause many other problems, such as reduced reproductive capacity, by acting directly or indirectly on the hypothalamus-pituitary-gonad axis (HPG axis). In this study, we investigated the changes in HPG axis-related genes in male medaka (Oryzias latipes) exposed to fiber-type microplastics. We confirmed the progression of vitellogenesis, a sign of endocrine disruption, in male fish. In the microfiber-exposed group, microfiber accumulation was confirmed in the gills and intestines. One week after exposure to two different concentrations of microfibers (500 and 1,000 fibers/L), the fish showed increased expression of gonadotropin-releasing hormone (GnRH) and luteinizing hormone receptor (LH-R) mRNA. From day 10 of exposure to the microfibers, there was an increase in the expression of the gonadotropin-inhibitory hormone (GnIH) mRNA and a decrease in the expression of GnRH and LH-R mRNA. There was an increase in the cytochrome P450 aromatase (CYP19a) mRNA expression and plasma estradiol (E2) concentration in the 1,000 fibers/L exposure group. High vitellogenin (VTG) mRNA expression was confirmed seven days after exposure in the 1,000 fibers/L group, which was consistent with the VTG mRNA expression signals detected in the liver using in situ hybridization. These results suggest that microfiber ingestion may cause short-term endocrinal disruption of the HPG axis in male medaka, which in turn may interfere with their normal maturation process.

Effects of microfiber exposure on medaka (Oryzias latipes): Oxidative stress, cell damage, and mortality.

Fiber-type microplastics are major anthropogenic contaminants of marine environments. They are released mainly during cloth washing and are discharged from wastewater treatment plants into aquatic environments. This study aimed to evaluate whether microfiber exposure causes oxidative stress and cell damage in medaka (Oryzias latipes Temminck and Schlegel 1846). Fish were exposed to one of two different concentrations (500 and 1000 fibers/L) of a polyester-based microfiber (MF) for 21 days, and the degree of cell damage and changes in expression of antioxidant enzymes were investigated. Fish survival decreased with increasing concentrations of MF. The expression levels of superoxide dismutase (SOD) and catalase (CAT) increased in MF-exposed groups compared to those in the control. SOD activity increased compared to the control group, and MF exposure induced a significant increase in both SOD activity and mRNA expression over time. CAT mRNA expression increased from day 10 onwards following exposure. Plasma malondialdehyde content increased significantly on day 7 of exposure in the 1000 fiber/L group and on day 10 in the 500 fiber/L group. Caspase-3 mRNA expression significantly increased until day 10 of exposure. A terminal transferase dUTP nick end labeling assay confirmed increased apoptosis, and a comet assay demonstrated that higher DNA damage occurred in response to increased MF concentration and exposure time. In conclusion, we confirmed that MF exposure affects antioxidant reactions in fish, thus inducing oxidative stress, apoptosis, and DNA damage. In addition, a comprehensive understanding of MF pollution in aquatic systems is urgently required.

The effects of low pH and high water temperature on oxidative stress and cell damage in juvenile olive flounder Paralichthys olivaceus: comparison of single and combined environmental conditions.

The use of fossil fuels by anthropogenic activities causes ocean acidification and warming, and these changes in the marine environment can negatively affect the metabolism, growth, and survival of fish. In the present study, we evaluated the ability of olive flounder Paralichthys olivaceus to cope with future marine environmental changes by investigating the oxidative stress (cortisol, HSP70), antioxidant enzyme (superoxide dismutase; SOD, catalase; CAT) activity, and apoptosis (caspase-3) after exposure to control conditions (20 °C and pH 8.1), warming (30 °C) and acidification (pH 7.5) conditions, and a combined environment (30 °C and pH 7.5) for 28 days. Under warming conditions, increased oxidative stress, activity of antioxidant enzymes, and apoptosis were observed. Acidifying conditions showed negative effects at the beginning of exposure, but these effects were offset over time. Even in a combined environment of acidification and warming, negative effects were seen only at the beginning of exposure and were not sustained. In conclusion, the effects of acidification on oxidative stress, antioxidant response, and apoptosis in P. olivaceus did not exceed the effects of warming. These results suggest that P. olivaceus can cope with the predicted future acidifying environment.

Water hardness alleviates the stress response caused by waterborne zinc in goldfish Carassius auratus.

In this study, the combined effect of waterborne Zn and water hardness on the stress response in the goldfish Carassius auratus was investigated. Goldfish were exposed to Zn concentrations of 0.5, 1.0, and 3.0 mg/L and water hardness of 90, 270, and 450 mg/L CaCO3 for 1, 3, 7, and 14 d. After exposure, it was determined that higher the Zn concentration, the more obvious the stress response. However, the stress response reduced with increasing water hardness. An increase in the Zn concentration caused stress responses in fish according to the increase in the mRNA expressions of corticotropin-releasing hormone and adrenocorticotropic hormone and cortisol level in the hypothalamus-pituitary-interrenal axis. The expression of these factors was the highest on day 7 and decreased on day 14. Furthermore, to evaluate the stress change in the liver tissue, we analyzed alanine aminotransferase, aspartate aminotransferase, and heat shock protein 70 concentrations to determine the damage caused by Zn and the change in water hardness. Immunohistochemistry staining for Na+/K+-ATPase in the gills showed that the gill activity was inhibited by Zn, and an increase in water hardness could improve Na+/K+-ATPase. In conclusion, we found that increasing water hardness is a successful method to reduce the stress response in goldfish caused by Zn.

Toxicity response to benzo[α]pyrene exposure: Modulation of immune parameters of the bay scallop, Argopectenirradians.

Bay scallops were exposed to four BaP concentrations (0.5, 1.0, 10 and 50 µg/L) for 72 h to elucidate their immune response. Immune parameters were evaluated by measuring nitric oxide (NO) levels in hemolymph. Additionally, we measured peptidoglycan recognition proteins (PGRP), fibrinogen-domain-containing protein (FReDC1), metallothionein (MT), and heat shock protein (HSP) 70 mRNA expression in digestive diverticula. NO as well as FReDC1 and MT expression in each BaP group increased significantly over time except for the BaP 0.5 group. The PGRP and HSP70 mRNA expression in the BaP 50 group increased in the range 6-24 h and then decreased. In situ hybridization also confirmed that there was higher MT mRNA expression in the BaP 50 group than in the control group at 72 h. Our results suggest that higher levels of BaP dampened scallop immune responses, while simultaneously reducing their ability to cope with oxidative stress and DNA damage. BaP exposure can be considered a potential immune inducer in bay scallop.

Water Hardness Can Reduce the Accumulation and Oxidative Stress of Zinc in Goldfish, Carassius auratus.

We investigated the changes in toxicity stress in goldfish, Carassius auratus, under exposure to different concentrations of Zn and water hardness for 14 days. We analyzed the changes in water hardness and Zn accumulation after exposure. To investigate the stress levels, the expression of metallothionein, caspase-3 activity, NO activity, and total antioxidant capacity were detected. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were also performed to measure apoptosis in the liver. The results showed that compared to the control group, a more significant difference in the accumulation of Zn in body stress markers (metallothionein, caspase-3 activity, NO activity, and total antioxidant capacity) were observed with increasing Zn concentration and exposure time. Notably, at the same Zn concentration and exposure time, lower stress levels were discovered in the samples under harder water conditions. Finally, the TUNEL assay showed that Zn accumulation caused apoptosis and high water hardness could reduce the apoptosis. In conclusion, we found that high water hardness can influence the absorption of Zn, and alleviating the hardness levels can reduce the toxicity stress caused by Zn.

Temporal Changes in Physiological Responses of Bay Scallop: Performance of Antioxidant Mechanism in Argopecten irradians in Response to Sudden Changes in Habitat Salinity.

Changes to habitat salinity may induce oxidative stress in aquatic organisms. The effect of salinity on the antioxidant function of bay scallops was investigated at 55, 70, 85 and 120% of seawater salinity (SW), with 100% SW as the control. The scallops were sampled 0, 6, 12, 24, 48 and 72 h after the salinity change to measure superoxide dismutase (SOD), catalase (CAT), hydrogen peroxide (H2O2), and lipid peroxidation (LPO) levels, as well as apoptosis in the digestive diverticula and/or hemolymph. The SOD immunohistochemistry and apoptotic response were assessed at 55% and 120% SW at 12 h. Antioxidant expressions at 55% and 70% SW peaked at 24 h or 48 h, and then decreased. At 120% SW, they increased with exposure time. The H2O2 and LPO levels at each SW increased significantly with time. A comet assay also revealed that changes in salinity increased the rate of nuclear DNA damage in all the salinity groups. Thus, variations in salinity result in significant physiological responses in bay scallops. A change in habitat salinity of 15% or more produces oxidative stress that cannot be resolved by the body's antioxidant mechanism, suggesting that excessive generation of reactive oxygen species can lead to cell death.

Exogenous cortisol and red light irradiation affect reproductive parameters in the goldfish Carassius auratus.

Reproductive hormones play essential roles in the control of reproduction and gonadal maturation in fish. The purpose of this study was to determine the effects of cortisol administration (10 µg/g or 50 µg/g) or red light irradiation at two intensities (0.5 W/m2 or 1.0 W/m2) on the reproductive hormones in goldfish (Carassius auratus). The effects of different treatments were analyzed by determining the mRNA expression levels of gonadotropin-inhibitory hormone receptor (GnIH-R), chicken gonadotropin-releasing hormone (cGnRH-II), salmon GnRH (sGnRH), FSHß, LHß, and plasma testosterone and the level of 17ß-estradiol for 48 h. Additionally, by double immunofluorescence staining, we detected the expression of both GnIH and GnRH in the diencephalons of goldfish brains. The mRNA expression of GnIH-R was significantly higher in the cortisol group and red light-irradiated group from 3 to 48 h than in the control group. Additionally, the mRNA levels of cGnRH-II, sGnRH, FSHß, LHß, testosterone, and 17ß-estradiol were significantly lower in the cortisol group than in the other groups from 3 to 48 h. These results indicated that both cortisol and red light-emitting diode (LED) light increased GnIH expression and inhibited GnRH expression. In particular, red light irradiation suppressed reproductive responses as much as the cortisol treatment at 48 h. Thus, it could be an alternative method for suppressing reproductive responses in future aquacultures.

Exposure of bay scallop Argopecten irradians to micro-polystyrene: Bioaccumulation and toxicity.

Marine microplastic pollution poses a threat to aquatic organisms, including bivalves. In this study, we investigated the accumulation of microplastics and their elicited antioxidant stress response in the bay scallop Argopecten irradians. Scallops were exposed to 1 µm diameter micro-polystyrene (MP) beads at 10, 100, and 1000 beads/mL concentrations for a 7 day period. Bead presence in the digestive diverticula and defense responses in the digestive diverticula and hemolymph were measured at 1, 3, 5, and 7 days. The activity and expression of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) and H2O2 in the digestive diverticula and/or hemolymph of scallops increased with microplastic concentration and exposure duration. These results suggest that microplastics can accumulate in the digestive diverticula of A. irradians, and that exposure to microplastics induces oxidative stress in bivalves. It is likely that exposure to high concentrations of micro- or nano-sized plastic particles could potentially have adverse effects in bivalves.

Physiological and oxidative stress response of goldfish Carassius auratus induced by a light dimming system.

Light is an essential factor for organisms and affects the endocrine and stress regulation of fish in nature. However, sudden changes in light and dark conditions in artificial environments can negatively impact fish. In the present study, to evaluate the physiological and oxidative stress responses of goldfish (Carassius auratus) exposed to two different light conditions, sudden light changes and slowly dimming light changes for 24 h, we analyzed the mRNA expression and activity of stress indicators [corticotropin-releasing hormone (CRH) and pro-opiomelanocortin (POMC)], levels of plasma cortisol and glucose, mRNA expression of glucocorticoid receptor (GR), and activity of plasma oxidative stress indicators (superoxide dismutase and catalase). Consequently, the mRNA expressions and activities of CRH and POMC, plasma levels of cortisol and glucose, and mRNA expression of GR were found to be significantly increased during the light changes, particularly in the control group. Additionally, plasma levels of cortisol and glucose in the control group were significantly higher than those in the dimming group during the light changes. However, no significant differences in mRNA expression levels and activities of antioxidant enzymes both in the control and dimming groups were observed. These results indicate that dimming light induces less stress than sudden changes in light.

Effects of blue light spectra on retinal stress and damage in goldfish (Carassius auratus).

There have been a number of studies on the negative effects of blue light exposure in various species; however, little information is available on the impacts of blue light intensity and duration on fish. We investigated the effects of blue light spectra on stress in the retinas of goldfish, using a blue (460 nm) light-emitting diode (LED) at three intensities (0.5, 1.0, and 1.5 W/m2). The experiment was conducted for 4 weeks, and sampling was performed at intervals of 1 week. We measured changes in the expression of cortisol, and the concentrations of hydrogen peroxide (H2O2), melanin-concentrating hormone receptor (MCH-R), and caspase-3 in the retinas of goldfish. In addition, we measured histological changes in the retina. We used a transferase dUTP nick end labeling (TUNEL) assay to evaluate the apoptotic response to blue LED spectra. Levels of cortisol, H2O2, MCH-R, and caspase-3 increased with exposure time and light intensity. Histological analysis revealed that the thickness of melanin granules increased with exposure time and light intensity. The progressive TUNEL assay revealed many apoptotic cells after exposure to blue LED light, increasing with exposure time and light intensity. Irradiation with blue light for longer than 1 week induced increased retinal stress and may induce apoptosis in the retinas of goldfish, even at a low intensity.

Effects of light-emitting diode spectra on the vertebrate ancient long opsin and gonadotropin hormone in the goldfish Carassius auratus.

We determined the molecular mechanism underlying the environmental (photoperiodic) regulation of sexual maturation in fish, we examined the expression of sexual maturation-related hormones and vertebrate ancient long opsin (VAL-opsin) in goldfish (Carassius auratus) exposed to different light spectra (red and green light-emitting diodes). We further evaluated the effect of exogenous gonadotropin hormone (GTH) on the expression of VAL-opsin under different light conditions. Our results demonstrated that the expression of GTHs was higher in the fish exposed to green light, and VAL-opsin levels were increased in the fish receiving GTH injection. Therefore, we have uncovered a molecular mechanism underlying the environmental (light)-induced trigger for sexual maturation: VAL-opsin is activated by green light and GTH, which promotes the expression of sexual maturation genes.

Effect of green light spectra on the reduction of retinal damage and stress in goldfish, Carassius auratus.

We investigated the effect of light spectra on retinal damage and stress in goldfish using green (530 nm) and red (620 nm) light emitting diodes (LEDs) at three intensities each (0.5, 1.0, and 1.5 W/m(2)). We measured the change in the levels of plasma cortisol and H2O2 and expression and levels of caspase-3. The apoptotic response of green and red LED spectra was assessed using the terminal transferase dUTP nick end labeling (TUNEL) assay. Stress indicator (cortisol and H2O2) and apoptosis-related genes (caspase-3) decreased in green light, but increased in red light with higher light intensities over time. The TUNEL assay revealed that more apoptotic cells were detected in outer nuclear layers after exposure to red LED over time with the increase in light intensity, than the other spectra. These results indicate that green light efficiently reduces retinal damage and stress, whereas red light induces it. Therefore, red light-induced retina damage may induce apoptosis in goldfish retina.