Toxic effects of triclocarban on larval zebrafish: A focus on visual dysfunction
Graphical abstract
Introduction
Triclocarban (TCC) is an antimicrobial agent widely used in personal care products. After application, TCC could enter the aquatic environment through direct or indirect wastewater discharge (Chen et al., 2014). The environmental residues of TCC have been well documented (Chen et al., 2018; Chen et al., 2014; Yun et al., 2020). In China, TCC concentrations in surface water from the Xiaoqing River were up to 382 ng/L (Wang et al., 2014). As a result, the biological effect of TCC has received increasing attention in recent years.
Many reports have demonstrated that TCC disrupts the endogenous hormonal balance, thereby contributing to reproductive system diseases (Chung et al., 2011; Dong et al., 2018; Yun et al., 2020). Cao et al. (2020) found the estrogenic disruption effect of TCC through the binding affinity of TCC to estrogen-related receptor γ. In addition, TCC showed in vitro antagonist activities on thyroid receptors (Kenda et al., 2020). This result attracted our attention because thyroid hormone signaling is responsible for the specification of retinal cone photoreceptors, which enable daytime, color, and high-acuity vision (Eldred et al., 2018; Volkov et al., 2020). Moreover, some thyroid hormone disrupting chemicals were reported to cause adverse effects on eye development of zebrafish (Baumann et al., 2019; Dong et al., 2014). It seems reasonable to hypothesize that TCC is likely to perturb visual development. Caioni et al. (2021) found the adverse impact of TCC on eye size and melanogenesis during zebrafish early embryonic development. However, there is a lack of information regarding the consequences of TCC on visual function.
Vision is the most essential sensory system, which is closely related to a series of behavioral activities, such as foraging and avoiding predators (Carvalho et al., 2002). It should be noted that fish live in the aquatic environment and do not have eyelids, suggesting that fish eyes are directly exposed to water-borne pollutants and thereby suffer a higher probability of accumulation and injury (Burreau et al., 2000; Chen, 2020). A previous report found that exposure to 2,2’,4,4’-tetrabromodiphenyl ether (BDE-47), which has the ability to disrupt the thyroid hormone system of zebrafish larvae at multiple exposure levels, will lead to abnormality in vision development and visually-mediated behaviors in larval zebrafish (Parsons et al., 2019, Xu et al., 2017). Visual impairment is fatal to wildlife, reflecting in the inability to escape predators and to hunt for food (Liu et al., 2018). Compared with those for reproductive toxicity (Rochester et al., 2017; Zou et al., 2021), however, the research on the ocular toxicity of pollutants is quite limited.
Zebrafish (Danio rerio) is a commonly used model organism (Howe et al., 2013). Two notable characteristics, including similar retinal structures to humans and the rapid development of visual system at early embryonic stages, make zebrafish most suitable for ocular toxicological studies (Chen, 2020). As a reflex that appears in zebrafish, optokinetic response (OKR) occurs in response to a visual stimulus on the retina, which is mediated visually. This behavioral assay is to record eye saccades of larval zebrafish, which are evoked by the presentation of moving vertical black and white stripes (Easter and Nicola, 1997). Since the head is immobilized and the eyes are moveable during the assay, OKR is considered to better represent visual functions than other visually-mediated behaviors that may be caused by a compromised locomotor activity (Chen, 2020), enabling direct testing of our hypothesis that TCC could impair visual system. In order to explore the mechanism of TCC-induced visual deficits, the changes in the thickness and cell density of retinal layers were observed by hematoxylin and eosin (H&E) stained sections. Also, molecular biology techniques, such as quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) and immunofluorescence, were used to detect the expression of critical opsin genes and photoreceptor protein in the retina. This study aimed to propose a novel target organ impaired by TCC and to reveal insights into the mechanism of TCC-induced ocular toxicity.
Section snippets
Chemicals and reagents
Triclocarban (TCC, 99.0% purity, CAS: 101-20-2) was purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). A stock solution of TCC at a concentration of 10 g/L was prepared in dimethyl sulfoxide (DMSO, 99% purity). Agarose (CAS: 9012-36-6) was obtained from Mich Scientific. Carboxymethyl cellulose (CMC, CAS: 9004-32-4) and anti-rhodopsin 1D4 antibody were purchased from Sigma-Aldrich (Shanghai, China) and Abcam (Shanghai, China), respectively. Fluorescence-conjugated secondary antibody and
Actual TCC contents in exposure solution
The concentrations of TCC in the exposure solution with embryos were measured at the initiation (day 0) and end (day 6) of exposure (Table S5). We found that the actual TCC concentrations were similar to and lower than the corresponding nominal values at day 0 and day 6, respectively. For the control experiment without embryos, TCC contents did not change significantly every day (Table S5). These findings suggest the strong bioaccumulation of TCC in biological tissues, which has been reported
Discussion
Changes in an organism's phenotype result from the influence of environmental factors. As shown in Fig. 1a, there is almost no difference in the larval zebrafish body among different groups, but the zebrafish larvae in the L- and H-groups appear to be microphthalmia. These findings agree with previous studies regarding the developmental toxicity of TCC on zebrafish (Tal et al., 2017; Xu et al., 2021). Some thyroid hormone disrupting chemicals, such as propylthiouracil and tetrabromobisphenol A,
Conclusions
In the present study, we found that exposure to environmentally relevant concentrations of TCC did cause phenotypic defects of the visual system, such as significantly decreased eye size, retinal ganglion cell density, OKR, and phototactic response. Also, TCC could reduce ONL thickness with the down-regulation of critical opsin genes and rhodopsin. Furthermore, the results of visually-mediated behavioral and opsin gene transcription analysis showed that TCC affected the sensitivity of zebrafish
Declaration of Competing Interest
There are no conflicts to declare.
Acknowledgments
The present study was funded by the National Natural Science Foundation of China (42177254), the Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program (2017BT01Z032), National Key Research and Development Project (2019YFC1804604), Guangdong Provincial Key Laboratory of Chemical Pollution and Environmental Safety (2019B030301008), Science and Technology Planning Project of Guangdong Province (2020B1212030008). We thank China Zebrafish Resource Center (CZRC) for
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