Life-time exposure to waterborne copper IV: Sperm quality parameters are negatively affected in the killifish Poecilia vivipara.

In previous studies, we have shown that copper (Cu) is significantly accumulated in various tissues of killifish Poecilia vivipara following chronic exposure. Also, we showed that chronic metal exposure disrupted energy production and growth in this species. In the present study, we aimed to evaluate if chronic exposure to this metal could also affect reproductive parameters of P. vivipara males (sperm quality). In order to test that, newborn (<24 h-old) fish were exposed to two concentrations of waterborne Cu (5 and 9 µg/L) for 345 days. After exposure, fish were euthanized and the testes were collected for sperm analysis. We could observe that exposed animals had reduced sperm motility and period of motility. Also, the sperm of exposed fish had reduced plasma membrane integrity, mitochondrial functionality and DNA integrity when compared to sperm of control animals. It is suggested that the well-known association of Cu with elevated oxidative damage, endocrine disruption and energetic disturbance are involved with the observed outcomes. The results obtained in the present study show that chronic exposure to environmentally relevant concentrations of waterborne Cu caused reductions in all parameters used to evaluate sperm quality. Therefore, it is concluded that life-time exposure to this metal may disrupt fish reproduction and negatively affect the maintenance of its populations.

Life-time exposure to waterborne copper III: Effects on the energy metabolism of the killifish Poecilia vivipara.

Copper ions (Cu) are essential to life maintenance, nonetheless, elevated concentrations can be hazardous. Acute and sub-chronic toxic effects of this metal are well known and are usually related to enzymatic inhibition, elevated ROS production and dysfunction of energy metabolism. Despite that, chronic studies are extremely rare. Therefore, the aim of this study was to assess the effects of chronic exposure to 5, 9 and 20 µg/L Cu (28 ad 345 days) on the energy metabolism and survival of the killifish Poecilia vivipara. To accomplish that, we evaluated the activity of enzymes related to aerobic (pyruvate kinase (PK); citrate synthase (CS)) and anaerobic metabolism (lactate dehydrogenase (LDH)) in whole-body (28 days) or in gills, liver and muscle (345 days) of exposed fish. Additionally, whole-body oxygen consumption was evaluated in fish exposed for 28 days and hepatic and muscular expression of genes involved in mitochondrial metabolism (cox I, II and III and atp5a1) was assessed in animals exposed for 345 days. Finally, final survival was evaluated. Following 28 days, Cu did not affect survival neither enzyme activities. However, increased whole-body oxygen consumption was observed in comparison to control condition. After 345 days, 76.8%, 63.9%, 60.9% and 0% survival were observed for control, 5, 9 and 20 µg/L groups, respectively. Animals exposed to 5 and 9 µg/L had a significant reduction in branchial and muscular LDH activity and in hepatic PK activity. Also, exposure to 9 µg/L significantly increased hepatic CS activity. For gene expression, Cu down-regulated muscular cox II (9 µg/L) and III (5 and 9 µg/L), and up-regulated hepatic atp5a1 (9 µg/L). Findings reported in the present study indicate that chronic exposure to Cu induces tissue-specific responses in key aspects of the energetic metabolism. In gills and muscle, Cu leads to reduced energy production through inhibition of anaerobic pathways and mitochondrial respiratory chain. This effect is paralleled by an increased ATP consumption in the liver, characterized by the augmented CS activity and atp5a1 expression. Finally, reduced PK activity indicate that oxidative stress may be involved with the observed outcomes.

Life-time exposure to waterborne copper II: Patterns of tissue accumulation and gene expression of the metal-transport proteins ctr1 and atp7b in the killifish Poecilia vivipara.

The involvement of transporting proteins on copper (Cu) bioaccumulation was evaluated in the killifish Poecilia vivipara chronically exposed to environmentally relevant concentrations of waterborne Cu. Fish (<24 h-old) were maintained under control condition or exposed to different waterborne Cu concentrations (5, 9 and 20 µg/L) for 28 and 345 days in saltwater. Following exposure periods, Cu accumulation and the expression of genes encoding for the high affinity Cu-transporter (ctr1) and the P-type Cu-ATPase (atp7b) were evaluated. Whole-body metal accumulation and gene expression were evaluated in fish exposed to 28 days. Similarly, in fish exposed to 345 days, liver, gills and gut were also evaluated. No fish survival was observed after exposure to 20 µg/L for 345 days. Whole-body Cu accumulation was significantly higher in fish exposed to 20 µg/L Cu for 28 days and in fish exposed to 9 µg/L for 345 days in comparison to control animals. Similarly, tissue Cu accumulation was significantly higher in fish exposed to 9 µg/L for 345 days in comparison to control animal. However, no significant accumulation was observed in fish muscle. Following exposure for 28 days, whole-body ctr1 expression was slightly induced in fish exposed to 9 µg/L. In turn, no significant change in ctr1 expression was observed following exposure to Cu for 345 days. Differently, whole-body atp7b expression was markedly up-regulated in the whole-body of fish exposed Cu for 28 days and in tissues of fish exposed to Cu for 345 days. These findings indicate the expression of atp7b is more responsive to Cu accumulation in P. vivipara than ctr1 expression and, therefore, more suitable to be used as a biomarker of exposure to this metal. Also, we argue that the expression of atp7b is sustained at elevated levels for as much time as fish are maintained in Cu contaminated water.

Effects of life-time exposure to waterborne copper on the somatotropic axis of the viviparous fish Poecilia vivipara.

Reduced fish growth following chronic exposure to dissolved copper (Cu) is well reported in the literature. Nevertheless, information on the mechanism(s) involved in this process is scarce. Therefore, we evaluated growth, gene expression and concentrations of proteins related to growth regulation in the viviparous guppy Poecilia vivipara chronically exposed to dissolved Cu. Newborn (<24 h after birth) fish were kept under control conditions or exposed to environmentally relevant concentrations of Cu (5 and 9 µg/L) in salt water (24 ppt) for 345 days. After exposure, fish growth was evaluated based on body weight and length. Also, growth hormone (gh) mRNA expression was evaluated in brain, while growth hormone receptor 1 (ghr1) and 2 (ghr2) mRNA expressions were analyzed in brain, skeletal muscle and liver. In turn, insulin-like growth factor 1 (igf1) and 2 (igf2) mRNA expressions were evaluated in skeletal muscle and liver. Additionally, Gh concentration was assessed in brain, while Ghr concentration was evaluated in skeletal muscle and liver. Exposure to 9 µg/L Cu reduced fish body weigh and length. Metal exposure affected mRNA expression only in skeletal muscle. Reduced ghr2 mRNA expression was observed in guppies exposed to 5 and 9 µg/L Cu. Additionally, reduced igf1 and igf2 mRNA expressions were observed in guppies exposed to 9 µg/L Cu. However, no significant change in Ghr concentration was observed. The reduced ghr2 mRNA expression suggests that chronic Cu exposure induced an insensitivity of the skeletal muscle to Gh, thus resulting in reduced igf1 and igf2 mRNA expression which lead to reduced fish growth. These findings indicate that chronic exposure to dissolved Cu disrupts the somatotropic axis regulation, thus helping to elucidate the mechanism underlying the Cu-dependent inhibition of growth observed in the viviparous fish P. vivipara.