Dibutyl phthalate disrupts energy metabolism and morphology in the gills and induces hepatotoxicity in zebrafish.

This study investigated the acute effects of dibutyl phthalate (DBP) exposure on energy metabolism and gill histology in zebrafish (Danio rerio). The in vitro incubation of gill tissue with 10 µM DBP for 60 min altered tissue energy supply, as shown by decreased lactate content and lactate dehydrogenase (LDH) activity. Higher concentrations of DBP (100 µM and 1 mM) increased lactate content and LDH activity; however, they blocked glucose uptake, depleted the glycogen content in cellular stores, and induced injury to the gills, as measured by LDH release to the extracellular medium. In addition, in vivo exposure of fish to 1 pM DBP for 12 h induced liver damage by increasing alanine aminotransferase (ALT) and gamma-glutamyl transferase (GGT) activities. Gill histology indicated hyperemia, lamellar fusion, lamellar telangiectasis, and necrosis. Data indicate that acute exposure of zebrafish gills to the higher DBP concentrations studied induces anaerobic cellular activity and high lactate production, causing gill damage, diminishing cell viability, and incurring liver dysfunction.

Influence of the aquatic environment and 1α,25(OH)2 vitamin D3 on calcium influx in the intestine of adult zebrafish.

We investigated the effects of environment calcium challenge and 1α,25(OH)2 vitamin D3 (1,25-D3) on 45Ca2+ influx in the intestine of zebrafish (ZF). In vitro45Ca2+ influx was analyzed using intestines from fed and fasted fish. ZF were held in water containing Ca2+ (0.02, 0.7, 2.0 mM) to analyze the ex vivo45Ca2+ influx in the intestine and for histology. Intestines from fish held in water with Ca2+ were incubated ex vivo to characterize ion channels, receptors, ATPases and ion exchangers that orchestrate 45Ca2+ influx. For in vitro studies, intestines were incubated with antagonists/agonist or inhibitors to study the mechanism of 1,25-D3 on 45Ca2+ influx. Fasted ZF reached a plateau for 45Ca2+ influx at 30 min. In vivo fish at high Ca2+ stimulated ex vivo45Ca2+ influx and increased the height of intestinal villi in low calcium. In the normal calcium, 45Ca2+ influx was maintained by the reverse-mode Na+/Ca2+ (NCX) activation, Na+/K+-ATPase pump and sarco/endoplasmic reticulum calcium ATPase (SERCA) pump. However, Ca2+ hyperosmolarity is supported by L-type voltage-dependent calcium channels (L-VDCC), transient receptor potential vanilloid subfamily 1 (TRPV1) and Na+/K+-ATPase activity. The calcium challenge causes morphological alteration and changes the ion type-channels involved in the intestine to maintain hyperosmolarity. 1,25-D3 stimulates Ca2+ influx in normal osmolarity coordinated by L-VDCC activation and SERCA inhibition to keeps high intracellular calcium in intestine. Our data showed that the adult ZF regulates the calcium challenge (per se osmolarity), independently of the hormonal regulation to maintain the calcium balance through the intestine to support ionic adaptation.

Calcium influx and spermatogenesis in the testis and liver enzyme activities in the zebrafish are rapidly modulated by the calcium content of the water.

This study investigated the effects of varying environmental Ca2+ concentrations on the influx of Ca2+ to the testis, testicular morphology, and liver enzymes in the zebrafish. Adult zebrafish (Danio rerio) were held in water containing low (0.02 mM), control (0. 7 mM) or high (2 mM) Ca2+ concentrations for 12 h. Testes were then incubated in vitro with 0.1 µCi/mL 45Ca2+ to measure Ca2+ influx at 30 and 60 min and qualitative and quantitative testicular histological analyses were conducted. In addition, activity of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyl transpeptidase (GGT), enzymes that indicate tissue damage, were evaluated in the liver. The testes from zebrafish exposed in vivo to low (0.02 mM) and high (2 mM) Ca2+ content water had a higher Ca2+ influx than the control group after 30 min of incubation, and at 60 min (high Ca2+ group only). There were morphological changes in the testes from the low and high Ca2+ groups including spermatozoa distributed in dense agglomerates and apoptotic cells. Furthermore, zebrafish exposed to high Ca2+ containing water had an increased density of haploid cells (spermatids and spermatozoa). In addition, both low and high Ca2+ water affected liver function by increasing ALT and GGT activities. Collectively, these studies show that alterations in calcium homeostasis in the testis, stimulation of the spermatogenic wave and hepatic injury were rapid responses to changes in the concentration of Ca2+ in the water.

In vivo and in vitro short-term bisphenol A exposures disrupt testicular energy metabolism and negatively impact spermatogenesis in zebrafish.

This study investigated the in vitro and short-term in vivo effects of Bisphenol A (BPA) on testicular energy metabolism and morphology in the zebrafish (Danio rerio). Testes were incubated in vitro for 1 h or fish were exposed in vivo to BPA in the tank water for 12 h. Testicular lactate, glycogen and cholesterol were measured and 14C-deoxy-d-glucose uptake and activity of lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined. In addition, testis samples from the in vivo exposures were subject to digital analysis of testicular cells using Ilastik software and the Pixel Classification module and estimation of apoptosis by Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) immunohistochemical analysis. Our results from in vitro studies showed that BPA at 10 pM and 10 µM decreased testicular lactate content, glycogen content and LDH activity, but increased testicular AST activity. In addition, only BPA at 10 pM significantly decreased testicular ALT activity and cholesterol content. However, 14C-deoxy-d-glucose uptake was not changed. Furthermore, our results from in vivo studies showed that 10 pM BPA but not 10 µM BPA reduced testicular content of lactate and glycogen. In addition, both BPA concentrations decreased AST activity, whereas only BPA at 10 µM reduced ALT activity. However, LDH activity was not changed. Additionally, both concentrations of BPA induced spermatocyte apoptosis and a decrease in the proportion of the surface area of spermatids and spermatozoa. Collectively these data suggest that short-term BPA exposure affects energy metabolism and spermatogenesis in male zebrafish.

Exposure to a Brazilian pulp mill effluent impacts the testis and liver in the zebrafish.

While many studies have shown that pulp mill effluents can affect ovarian physiology in fish, far fewer studies have considered the effects in males. We conducted a lab study to examine the effects of effluent from a Brazilian pulp and paper mill on hepatic and testicular morphology and various aspects of testicular physiology in the zebrafish Danio rerio. Males were exposed to lab water (control) or 4% effluent for 14 days. Effluent exposure did not affect testis size as measured by the gonadosomatic index, but contributed to morphological changes in the seminiferous tubules. The number of cysts with histopathological changes was elevated in effluent-exposed fish and the number of cysts containing spermatids was significantly reduced. The testis of effluent exposed fish had reduced levels of lactate, elevated lactate dehydrogenase activity, increased levels of reactive oxygen species and reduced levels of phosphorylated P38 mitogen-activated protein kinase (pP38 MAPK). Separate studies showed that the addition of lactate to testicular tissue incubated in vitro increased the activation of P38 MAPK. Effluent exposure also increased vacuolization, necrosis, apoptosis, hyperemia, and fat infiltration of the hepatocytes. Collectively, we provide evidence of short term effects of pulp mill effluent on testicular and hepatic physiology and biochemistry in the zebrafish.