Environmental Risk Assessment of Oxaliplatin Exposure on Early Life Stages of Zebrafish (Danio rerio)

Abstract

Pharmaceuticals are actually identified as a threat to the ecosystem. Nowadays, the growing consumption of antineoplastic agents has been related to their continuous input in natural environments. These substances can interfere with physiological and biochemical processes of aquatic species over their entire life cycle. Oxaliplatin (OXA) is a widely used chemotherapeutic agent to treat colon or rectal cancer. This study was aimed to evaluate the developmental toxicity of the OXA exposure. To this end, zebrafish embryos were incubated with 0.001, 0.1, 0.5 mg/L OXA. At different timepoints mortality rate, hatching rate, developmental abnormalities, histological analysis, oxidative stress and mRNA expression of gene related to oxidative stress were evaluated. Our results showed that OXA exposure can induce increased mortality and developmental abnormalities reducing the hatching rate. Histological analysis demonstrated that OXA induced liver, intestine, muscle and heart injury. Superoxide dismutase and catalase activities were significantly increased after OXA exposure demonstrating its oxidative effects. The mRNA expression levels of apoptosis-related genes (caspase-3, bax and bcl-2) were significantly upregulated by OXA exposure. In conclusion, we highlighted that OXA exposure led to a dose-related developmental toxicity, oxidative stress and apoptosis.

Keywords: ROS; cell death; developmental toxicity; oxaliplatin.

Figure 1

Embryo phenotypes, mortality and hatching rate after 24 to 96 h of exposure to OXA. (A) The embryo phenotypes in the unexposed and OXA-exposed groups. (B) The mortality rate in zebrafish embryos exposed to OXA. (C) The hatching rate in zebrafish embryos exposed to OXA. The asterisk denotes a statistically significant difference when compared with the CTRL: *** p < 0.001 versus control.

Figure 2

Effects of OXA exposure on activities of SOD (A) and CAT (B), in the larval zebrafish. Embryonic zebrafish was exposed to OXA for 96 hpf. Data are expressed as the mean ± SEM of three replicates (about 10 larvae per replicate). The asterisk denotes a statistically significant difference when compared with the CTRL: * p < 0.05, *** p < 0.001 versus control.

Figure 3

Histopathological changes in the hearts, livers, intestines, and muscles of zebrafish larvae exposed to OXA at 96 hpf. LV = Liver; HE = Heart. Data are presented as means ± SEM or medians with interquartile ranges for non-parametric data of 10 larvae for each group. Scale bars 40× magnification.

Figure 4

The OXA exposure effects on cell death zebrafish embryos. At 96 hpf, OXA 0.001, 0.1, 0.5 mg/L exposure, the levels of cell death were observed and photographed by a fluorescence microscope after staining with acridine orange (A). Percentage of cell death histogram (B). The results are expressed as mean of three independent experiment data. Related gene expression levels of apoptotic pathway in zebrafish embryos exposed to OXA at concentrations of 0.001, 0.1, 0.5 mg/L at 48 hpf (C). The fold change from the CTRL group is used to reflect the mRNA expression levels. * p < 0.05, ** p < 0.01, *** p < 0.001 versus control. Scale bars 4× magnification.