Combined toxic effects of cadmium and environmental microplastics in Aphanius fasciatus (Pisces, Cyprinodontidae).

Microplastics (MPs), plastic particles smaller than 5 mm in diameter, have received extensive attention as new environmental pollutants with still unexplored potential ecological risks. The main objective of the present study is to see if the concomitant exposure to MPs and Cd is more toxic than that to MPs or Cd separately in Aphanius fasciatus. Immature female were exposed to Cd and/or MPs for 21 days, and the subsequent effects were monitored by a combination of biochemical, histological and molecular toxicity markers. Exposure to Cd, but not to MPs, increased metallothioneins content and mRNA levels of the metallothioneins gene MTA both in liver and gills. In addition, we observed a significant oxidative stress response at histological, enzymatic (Catalase and Superoxide dismutase), non-enzymatic (proteins sulfhydryl and malondialdehyde) and gene expression levels to both toxicants in both tissues, particularly in gills, but no clear evidence for interaction between the two factors. Our results indicate a major effect of MPs on gills at different organizational levels. Finally, exposure to both MPs and Cd induced spinal deformities, although bone composition was only altered by the latter, whereas MTA mRNA bone levels were only increased realtive to controls in doubly-exposed samples. Interestingly, the simultaneous use of both pollutants produced the same effects as Cd and MPs alone, probably due to reduced bioavailability of this heavy metal.

Interference with zinc homeostasis and oxidative stress induction as probable mechanisms for cadmium-induced embryo-toxicity in zebrafish.

The present study was conducted to provide new insights into the mechanisms that may be responsible for cadmium (Cd)-induced toxicity in zebrafish larvae as well as the role of the trace element zinc (Zn) in reversing Cd harmful effects. For this purpose, zebrafish eggs were exposed to Cd or/and Zn for 96 h. The effects on morphological aspect; mortality rate; Cd, Zn, and metallothionein (MT) levels; oxidative stress biomarkers; as well as molecular expression of some genes involved in Zn metabolism (Zn-MT, ZIP10, and ZnT1) and in antioxidant defense system (Cu/Zn-SOD, CAT and GPx) were examined. Our results showed that Cd toxicity was exerted, initially, by an interference with Zn metabolism. Thus, Cd was able to modify the expression of the corresponding genes so as to ensure its intracellular accumulation at the expense of Zn, causing its depletion. An oxidative stress was then generated, representing the second mode of Cd action which resulted in developmental anomalies and subsequently mortality. Interestingly, significant corrections have been noted following Zn supplementation based, essentially, on its ability to interact with the toxic metal. The increases of Zn bioavailability, the improvement of the oxidative status, as well as changes in Zn transporter expression profile are part of the protection mechanisms. The decrease of Cd-induced MTs after Zn supplement, both at the protein and the mRNA level, suggests that the protection provided by Zn is ensured through mechanisms not involving MT expression but which rather depend on the oxidative status.

Exposure to microplastics leads to a defective ovarian function and change in cytoskeleton protein expression in rat.

Microplastics (MPs) are ubiquitous environmental contaminants; through their physicochemical properties, they can have potentially negative effects on the environment as well as on animal and human health. Studies addressing the toxicity of MPs on mammalian female reproduction are almost absent. Thus, the main objective of the present study was to assess the impact of oral exposure, during four estrous cycles, of 5 µm polystyrene-type microplastics (PS-MPs) on ovarian function in rats. Particles of PS-MPs were detected in the duodenum and, for the first time, in the different compartments of the ovarian tissue. The toxicity of accumulated PS-MPs was manifested by the reduced relative ovarian weights, by the alteration in the folliculogenesis and in the estrous cycle duration, and by the reduced serum concentration of estradiol. The defective ovarian function following PS-MP treatment might be due to the induction of oxidative stress, which has been proved by an increased malondialdehyde (MDA) concentration and an increased superoxide dismutase (SOD) and catalase (CAT) activities as well as a decreased protein sulfhydryl (PSH) level in the rat ovary. Importantly, by immunofluorescence and RT-PCR, we demonstrated a significant decrease in the expression of cytoskeletal proteins: α-tubulin and disheveled-associated activator of morphogenesis (DAAM-1) in the ovary of rats exposed to PS-MPs at proteomic and transcriptomic levels. Our results uncovered, for the first time, the distribution and accumulation of PS-MPs across rat ovary, revealed a significant alteration in some biomarkers of the ovarian function, and highlighted the possible involvement of MP-induced disturbance of cytoskeleton in these adverse effects.

Aphanius fasciatus: a molecular model of scoliosis?

STUDY DESIGN: Observational study of Killifish with spinal deformities OBJECTIVE: To evaluate the morphology and molecular biology of Aphanius fasciatus with severe spine deformities. Idiopathic Scoliosis affects 3% of the population and is an abnormal three-dimensional curvature of the spine with unknown cause. The lack of a model system with naturally occurring spinal curvatures has hindered research on the etiology of IS. METHODS: The Mediterranean killifish Aphanius fasciatus, collected from the coast of Sfax (Tunisia), which has an inborn skeletal deformity was chosen. We used morphologic features to evaluate the severity of scoliosis according to the different types and performed a biochemical analysis using factors previously studied in humans (estradiol, melatonin and Insulin Growth Factor 1 "IGF-1"). RESULTS: We have detected relevant molecular deviations that occur in Killifish deformities and the fish with severe scoliosis are smaller and less old than the ones with milder scolioses. Furthermore, a significant change in levels of ovarian estradiol, liver IGF-1 and brain melatonin was noted between deformed and normal fish. CONCLUSIONS: Aphanius fasciatus could be used as a molecular model system to study the etiology of IS in humans as the characterization of the Aphanius fasciatus scoliosis syndrome has revealed morphological and biochemical parallels to IS. However, it is important to note the limitations of the proposed model, including the short lifespan of the fish. LEVEL OF EVIDENCE: III.

Effect of melatonin and folic acid supplementation on the growth performance, antioxidant status, and liver histology of the farmed gilthead sea bream (Sparus aurata L.) under standard rearing conditions.

The present study aimed to investigate the effect of dietary of melatonin (MLT) and folic acid (FA) administrations on growth performance, antioxidant status, and liver histological structure of juvenile gilthead sea bream, Sparus aurata L. under standard rearing conditions. Four diets were considered: a basal diet considered a control and three diets supplemented with 40 mg/kg of melatonin (MLT), 2 mg/kg of folic acid (FA), and with the mixture of melatonin and folic acid (MLT + FA). Each diet was randomly allocated to triplicate groups of fish (mean initial weight was 2.99 ± 0.55 g) for 41 days. The obtained results clearly indicated that the melatonin-supplemented diet decreased significantly the growth performance parameters (final body weight, weight gain rate, and specific growth rate) and IGF-1 level of the gilthead sea bream, while the folic acid-supplemented diet has no significant effect on these parameters. The mixture supplementation of melatonin and folic acid has no significant effect on the growth parameters due to the possible interaction between melatonin and folic acid effects. Furthermore, fish fed with all experimental diets showed significantly higher superoxide dismutase activity (SOD) and protein sulfhydryl level (PSH) and lower lipid peroxidation level (TBARS) and catalase activity (CAT) which confirm their powerful antioxidant role. The acetylcholinesterase activity (ACHE) decreased in fish fed with all experimental diets. The underlying mechanisms of driving melatonin and folic acid to reduce acetylcholinesterase activity require further studies. The histological structure of liver of control S. aurata fish shows severe hepatic lipid accumulation in large vacuoles that diminished after dietary individual or mixture folic acid and melatonin supplementations over 41 days. This work proved that 2 mg/kg of dietary folic acid has a positive effect on the growth performance, oxidative stress defense, and hepatic lipid accumulation reduction in the gilthead sea bream fish. Under our experimental conditions, melatonin failed to improve the growth indexes WGR, SGR, and IGF-I. This study recommends the diet supplementation with a dose lower than 2 mg/kg of food due to the observed effects on tissue ACHE activity.

First evidence on protective effect of exogenous melatonin supplementation against disruption of the estrogenic pathway in bone metabolism of killifish (Aphanius fasciatus).

This study was carried out to investigate the effects of exposure to estrogen antagonist nafoxidine hydrochloride (NH) and/or melatonin (Mlt) on certain bone metabolism parameters in killifish Aphanius fasciatus, a species suggested to be a suitable model for studying spinal deformities such as scoliosis. Immature females of A. fasciatus receiving 10 µg/L NH and/or 100 µg/L of Mlt were used and were sacrificed 30 days after the treatment. The spinal column, brain, and liver were collected and analyzed by various histological, biochemical, chemical, and molecular investigations. NH exposure increased frequency of histological alterations and caused signs of spinal column demineralization such as significant decrease in the percentage of nonorganic components content and calcium concentration. These changes were accompanied by decreased alkaline phosphatase activity (AP), hepatic insulin growth factor-1 (IGF-1) content, and, interestingly, cerebral Mlt concentration. Concomitant treatment with Mlt and NH enhanced expression of the gene encoding the Mlt receptor "mtnr1aa"and significantly restored the normal skeletal histology and the normal metabolism bone parameters. Our data suggest that disturbance of estrogen pathway in A. fasciatus induces cerebral Mlt depletion and, then, causes skeletal tissue alterations and bone demineralization and that exogenous Mlt supplementation has a protective effect. Thus, estrogen receptor antagonists and Mlt become important compounds to consider for the accurate prediction and assessment of bone physiology and spinal deformities in fish.

Heavy metal accumulation and histomorphological alterations in Aphanius fasciatus (Pisces, Cyprinodontidae) from the Gulf of Gabes (Tunisia).

The present study illustrates an analysis of histological changes; cadmium (Cd), copper (Cu) and zinc (Zn) accumulation; and metallothionein (MT) levels in normal and deformed Mediterranean killifish, Aphanius fasciatus (Pisces, Cyprinodontidae), collected from unpolluted (S1) and polluted areas (S2) in the Gulf of Gabes in Tunisia. Metal determination in water and sediment showed that the concentrations were significantly higher (p < 0.0001) in S2 compared to S1. Deformed fish showed a significantly higher accumulation of Cd, Cu, and Zn and high levels of MTs in their tissues compared to normal ones. Histopathological investigations revealed greater changes in gills, kidney, liver, and bone tissues of fish from the polluted area than those recorded in fish from the reference area. In comparison to normal fish of the polluted area (S2), tissue alterations were more developed in deformed specimens of this site. A possible relationship between metallic pollution, incidence of spinal deformities, and histological changes in A. fasciatus in the polluted site was discussed.

Comparative study of longevity, growth, and biomarkers of metal detoxication and oxidative stress between normal and deformed Aphanius fasciatus (Pisces, Cyprinodontidae).

This study compared fish longevity, growth, and oxidative stress between normal and deformed Aphanius fasciatus collected from the Gulf of Gabes. For this purpose, fish were collected from control (S1) and polluted (S2) sites in Tunisian coast and percentages of spinal deformities were determined. Oxidative stress biomarkers were also compared between normal and deformed fish, including levels of protein sulfhydryl (SH), malondialdehyde (MDA), and metallothioneins (MT), as well as activities of superoxide dismutase (SOD) and alkaline phosphatase (AP). Levels of heavy metals (Cd, Cu, and Zn) and polycyclic aromatic hydrocarbons (PAH) were also measured. Data showed that the highest occurrence of deformities was observed in younger fish and decreased significantly thereafter with age. Chemical analysis demonstrated high levels of heavy metals and PAH at polluted sites compared to a reference location. In deformed fish, a growth perturbation marked by a decrease of growth rate and condition index (CI), a high accumulation of Cd was noted. In addition, oxidative stress marked by changes in the studied biomarkers was observed. Taken together, oxidative stress, diminished longevity, and a disturbance of growth may be considered to be responsible factors contributing to spinal deformities.

Effects of cadmium and high temperature on some parameters of calcium metabolism in the killifish (Aphanius fasciatus).

This study aims to investigate the influence of high temperature on cadmium (Cd) toxicity in Aphanius fasciatus (Pisces: Cyprinodontidae). For this reason, Cd, mineral, and organic content in the vertebral column as well as the histological structure of gills and bone were compared in fishes exposed for 30 days to Cd (2 mg/L CdCl2) and/or high temperature (26 °C). Cd exposure caused a negative correlation between Cd and Ca concentrations (r = 0.98, p < 0.05), as well as a significant decrease in inorganic components (p < 0.05) and ash weight/dry weight ratio (p < 0.05) in the vertebral column. These changes were accompanied by an increased frequency of histological alterations in gills and bone. Concomitant treatment with Cd and high temperature increases Cd accumulation and Ca depletion in the skeletal tissue and increases the frequency and the severity of histological alterations. These results confirm that temperature increases Cd toxicity and needs to be taken into account for the accurate prediction and assessment of Cd-induced spinal deformities in fish.

Possible chemical causes of skeletal deformities in natural populations of Aphanius fasciatus collected from the Tunisian coast.

This study attempt to quantify and identify skeletal deformities in natural populations of Aphanius fasciatus collected from the Tunisian coast and tends to found a possible relationship between these anomalies and several types of pollutants presents in the environment. Fish were collected from one reference area (S1: coast of Luza) and three polluted areas (S2: industrialized coast of Sfax, S3: coast of Khniss and S4: Hamdoun'Oued). Various patterns of skeletal deformities were diagnosed using double staining technics, and the levels of heavy metals (Cd, Cu and Zn), various polycyclic aromatic hydrocarbons (PAHs) and estrogenic compounds were determined in water and sediment from different sites. Spatio-temporal variation of the spinal deformities frequencies in A. fasciatus show that high incidence of spinal anomalies has been recorded in population collected from S2 in comparison to three other ones. Morphological results indicated that skeletal deformities of adult A. fasciatus were grouped into 13 categories that described abnormalities on spines, vertebrae, arcs and mandibles. A total of 1025 abnormalities were quantified. The results of chemical analysis showed that the levels of heavy metals and PAHs were significantly higher in S2 than in S1, S3 and S4. High level of estrogenic activity was observed only in S4. A possible correlation between environmental exposures to a mixture of pollutants in coastal waters in S2 and spinal deformities in A. fasciatus was suggested.

Structural and molecular analysis of pollution-linked deformities in a natural Aphanius fasciatus (Valenciennes, 1821) population from the Tunisian coast.

The Eastern coast of Tunisia hosts a mosaic of populations of the Mediterranean killifish A. fasciatus, an excellent monitoring species for the analysis of environmental impact in coastal Mediterranean areas. Among these populations, fish from the Sfax area, in the Gulf of Gabès, are known for presenting a high prevalence of different physiological alterations, including spinal deformation. Compared to a reference clean area, Luza, Sfax water and sediments are polluted with heavy metals (20 to 40 times more Cd, for example) and other compounds like PAHs (10 times higher levels). Histological analyses of deformed fish from this area showed an abnormal abundance of collagen fibers infiltrated in the spinal area, either as a physiological mechanism to alleviate the consequences of the deformation of the bones or as a secondary effect of the same pathological/toxicity mechanism inducing bone deformation. Deformed fish from Sfax showed elevated (4-5 fold) levels of transcripts from the collagen COL1A2 gene in bone tissue, compared to either non-deformed animals from the same location or normal animals from the reference Luza site. These results are consistent with an overexpression of collagen in the bone tissue, in agreement with the histological results. We propose that the deformation observed in the Sfax Aphanius population is a degenerative process occurring in adult animals and probably terminal, as it results in severe limitations on the fish capacity to swim, to catch food, and ultimately to survive.

Influence of cadmium exposure on growth and fecundity of freshwater mosquitofish Gambusia affinis: in situ and in vivo studies.

This study aims to investigate the effect of cadmium (Cd) exposure on growth and fecundity of mosquitofish Gambusia affinis. For this purpose, two natural populations of pregnant females of G. affinis captured from two sites were differently contaminated with Cd (S1 present Cd levels 5-fold higher than S2) and a sublethal exposure to 0.4 mg CdCl(2)/L (10% of LC(50)) during 56 days was conducted in vivo. The length-weight regression revealed a significant difference in the growth between these two populations. A significant difference in fecundity was also noted between the two populations. Indeed, the embryo numbers in pregnant females captured from S1 are significantly higher than those noted in pregnant females from S2 (21.17±5 and 7.97±2.12, respectively; p<0.05). Following Cd exposure, we noted a growth perturbation resulting in lower values of both indices BWG and SGR following 7 and 21 days (-5.21 and -1.18 for BWG, and -2.09 and -0.46 for SGR, respectively) and a recuperation of growing weight at 42 and 56 days (1.32 and 1.71 for BWG, and 0.45 and 0.54 for SGR, respectively). For CF index, we observed a significant difference (p<0.05) between control and Cd groups at 7 and 21 days of exposure, and at 21 and 56 days respectively for HSI and GSI indices. Furthermore, Cd contents in both tissues (liver and yolk sac) and fractions (cytosolic and membrane) are significantly different between groups during experimentation. In addition, the Cd contents noticed in the liver membrane fraction are significantly higher than those noted in the yolk sac tissue. The MTs levels revealed a significant difference between the control and Cd groups. In liver tissue, a significant difference was noted, in MTs levels, during the Cd exposure (7, 21, 42, and 56 days) while in the yolk sac tissue the difference was noted at 42 days of exposure. Taken together, these results imply the potential negative effect of Cd on physiological status of G. affinis as evidenced by decreasing growth and fecundity rate.

Evaluation of environmental impact on natural populations of the Mediterranean killifish Aphanius fasciatus by quantitative RNA biomarkers.

The Mediterranean killifish, Aphanius fasciatus (Valenciennes, 1821) represents an excellent sentinel species for analysis of environmental impact in coastal areas. Quantitative changes on liver mRNA levels of five stress-related genes--metallothionein (metal exposure), vitellogenin, (estrogenic effects), cytochrome P4501A (CYP1A, dioxin-like compounds), superoxide dismutase (oxidative stress) and HSP70 (general stress)--were tested as markers of exposure to pollutants both in natural populations and in experimental treatments with model effectors. Analysis of fish from a metal-contaminated site in the Tunisian coast (Sfax) indicated that fish presenting spinal deformities (eight times more frequent at this site than in reference sites) showed increased levels of hepatic CYP1A mRNA levels compared to non-deformed fish from the same site or from the reference Louza site, suggesting a combined impact by metals and organic pollutants. Due to the strategy used in their design, these quantitative RNA markers will likely be useful to analyze environmental impacts on other related fish species.

Influence of high temperature on cadmium-induced skeletal deformities in juvenile mosquitofish (Gambusia affinis).

The aim of this study was to assess the effect of high temperature on cadmium (Cd)-induced skeletal deformities in juvenile Mosquitofish, Gambusia affinis. For this purpose, 188 juveniles (1 day old) were equally divided into the control group, which was maintained in Cd-free water at 24 degrees C, and three treated groups exposed either to Cd (0.4 mg/l as Cd Cl(2)) at 24 degrees C, to high temperature (32 degrees C), or to Cd at 32 degrees C for 30 days. The results showed that Cd exposure at 24 degrees C significantly increased the Cd accumulation (P < 0.0001) in the whole tissues of juveniles as well as the incidence of skeletal deformities (P < 0.01) compared with control animals. Exposure to high temperature also led to a significant increase in the incidence of skeletal deformities (P < 0.01) with respect to the control group. Interestingly, our results showed that the combined exposure to Cd and high temperature led to a more significant increase in Cd accumulation and in the frequency of spinal deformities than exposure to Cd or high temperature alone. These results confirm that temperature increases Cd toxicity and needs to be taken into account for the accurate prediction and assessment of Cd-induced spinal deformities in fish.

Involvement of cd bioaccumulation in spinal deformities occurrence in natural populations of Mediterranean killifish.

The aim of this study was to investigate the possible influence of environmental exposure to cadmium (Cd) on the spinal deformities occurrence in the Mediterranean killifish, Aphanius fasciatus (Pisces: Cyprinodontidae). For this purpose, some indicators of skeletal bone mineralization, Cd, and calcium (Ca) concentrations in spinal column as well as bioaccumulation of Cd from the water and the sediment have been compared in normal and deformed fish collected from polluted (S1) and nonpolluted (S2) areas in the Gulf of Gabès in Tunisia. When compared to the normal fish, the deformed fish showed signs of spinal column demineralization such as significant decrease in the ash weight/dry weight ratio, percentage of nonorganic components content, and Ca concentration. Cd concentrations in spinal column and liver were significantly higher in deformed fish than in normal fish. A highly significant negative correlation (r = -0.915, p < 0.01) between Cd and Ca concentrations was noted in spinal column of deformed fish. Bioaccumulation factors of Cd in the liver from the water and the sediment in deformed fish were also significantly higher (p < 0.0001) than in normal fish from S1 and S2. These findings suggest that the ability to accumulate large amount of Cd may represent a potential risk to induce spinal deformities in natural populations of Mediterranean killifish.

Association of spinal deformities with heavy metal bioaccumulation in natural populations of grass goby, Zosterisessor ophiocephalus Pallas, 1811 from the Gulf of Gabès (Tunisia).

The present study illustrates an analysis of spinal deformities associated with metal accumulation in natural populations of Zosterisessor ophiocephalus derived from polluted (S1) and unpolluted (S2) areas in the Gulf of Gabès in Tunisia. Three basic types of spinal deformities were detected: kyphosis, scoliosis and lordosis. These basic deformities frequently co-occur. Spinal deformities were observed in 10.72% of the total examined fish (n = 494). Deformed fish were 3.85 times more frequent in S1 than in S2. In both sexes, the highest occurrence of deformities was observed in the 111-120 mm class decreasing thereafter with fish length. Hepatic concentrations of cadmium (Cd), copper (Cu) and zinc (Zn) were significantly higher in Z. ophiocephalus from S1 than those from S2. Comparisons between fish in each site showed that liver concentrations of Cd and Zn were significantly higher in deformed fish than in normal fish. The relationship between metals accumulation and observed spinal deformities as well as the suitability of this kind of studies for environmental monitoring are discussed.