Vincristine exposure impairs skin keratinocytes, ionocytes, and lateral-line hair cells in developing zebrafish embryos
Introduction
Pharmaceutical contamination is a global challenge. Emissions from hospitals and drug manufacturers, and municipal wastewater are important sources of pharmaceutical contaminants (aus der Beek et al., 2016; Jureczko and Kalka, 2020; Rehman et al., 2015). Many published studies revealed evidence of environmental pollution by pharmaceuticals, including antineoplastic agents (Azuma et al., 2015; Besse et al., 2012; Ferrando-Climent et al., 2014; Heath et al., 2016; Jureczko and Kalka, 2020; Lin et al., 2014; Santos et al., 2017). This group of drugs is classified by the Anatomical Therapeutic Chemical (ATC) classification as Class L- Antineoplastic and Immunomodulating Agents (https://www.whocc.no/atc_ddd_index/?code = L&showdescription = yes) based on their therapeutic, pharmacological, and chemical properties. These drugs are resistant to biodegradation and cannot be efficiently removed during wastewater treatment, and concerns about their presence in the environment are rapidly increasing, primarily because of their known genotoxic, mutagenic, and carcinogenic effects (Jureczko and Kalka, 2020; Jureczko and Przystas, 2019).
Vincristine (VCR) is an anticancer chemotherapeutic drug that is listed on the World Health Organization’s List of Essential Medicines and is included in L01C Plant Alkaloids and Other Natural Products by the ATC classification. It inhibits mitosis at the metaphase through interacting with tubulin (Bates and Eastman, 2017) and has broad activities against acute lymphocytic leukemia, Hodgkin lymphomas, non-Hodgkin lymphomas, and childhood solid tumors including neuroblastomas, rhabdomyosarcomas, Wilms’ tumors, and Ewing sarcomas (Kothari et al., 2016). VCR is mainly metabolized via biliary elimination through feces (Jackson et al., 1978). Following an intravenous injection, 70 % of the parent compounds and metabolites were found in feces within 72 h, whereas only 10 % was excreted in urine within 24 h and very little thereafter (https://pubchem.ncbi.nlm.nih.gov/compound/5978) (Owellen et al., 1977). Because VCR is typically administered in an outpatient setting by an intravenous bolus injection, its metabolites can contaminate our living environment through the feces and urine of patients after they are discharged home. Several studies showed that VCR can be detected in hospital effluents, wastewater influents and effluents, and surface waters, with concentrations ranging 22.9∼49.1 ng/L. Kosjek et al. (2018) identified 11 biotransformation products of VCR and found that most of them retained typical vinca alkaloid structures, a finding that suggests that they may have potential pharmacological activity and toxicity. A previous study on the acute toxicity of VCR showed that the concentration for a 50 % maximal effect (EC50) for common duckweed was <10 mg/L, and it was classified as a toxic water contaminant (Jureczko and Przystas, 2019). However, additional information on the natural fate and ecotoxicological risks of VCR to aquatic biota is still very limited.
In humans, the toxicity associated with VCR is mostly neurological, with the commonly reported peripheral neuropathy. Although rare, cranial nerve paralysis was also reported (Broyl et al., 2010; Mahajan et al., 1981; Travis et al., 2014). Moreover, a few case reports revealed other VCR-associated toxicities including auditory (Kalcioglu et al., 2003; Lugassy and Shapira, 1990; Mahajan et al., 1981; Riga et al., 2007; Schweitzer, 1993), and renal tubulointerstitial injury (Hammond et al., 2002; Perazella, 2012; Shirali and Perazella, 2014). Peripheral neuropathy and other chemotherapy-related toxicities may cause patients to prematurely withdraw from cancer chemotherapy, and lead to long-term and late effects with cognitive, physical, and psychological consequences (Grewal et al., 2010; Postma et al., 1993). However, the mechanisms of VCR-related toxicities are still not well understood from either animal or human studies, and there is no proven way to prevent or treat these toxicities.
Zebrafish are vertebrates that have become important preclinical subjects over the past decade. In 2003, the National Institute of Health of the United States ranked zebrafish as the third most important experimental organism after mice and rats (https://www.fda.gov/consumers/consumer-updates/zebrafish-make-splash-fda-research). Zebrafish are particularly useful in high-throughput drug screening and so-called “predictive toxicology” due to their many advantages, including rapid reproduction, rapid embryonic development, optically transparent embryos, low maintenance costs, and the sharing of many common biological pathways with humans (Cassar et al., 2020; McGrath and Li, 2008).
During the embryonic stage (beginning from the insemination of eggs to completion of absorption of the vitellus) in fish, the skin contains mostly keratinocytes, along with a few dispersed ionocytes, mucous-producing cells, and sensory cells (Eisenhoffer et al., 2017), which provide several essential functions, including physical protection, osmotic and ionic balance, gas exchange, and acid/ammonia excretion. Keratinocytes provide physical protection and a large surface area for O2/CO2 exchange. Mitochondrion-rich ionocytes are responsible for absorbing ions from ambient water (Horng et al., 2009, 2017; Hwang et al., 2011; Shen et al., 2011) and secreting acid and ammonia into the water (Horng et al., 2015; Liu et al., 2013; Shih et al., 2008; Wu et al., 2010).
Moreover, the lateral line is a peripheral sensory system in fish skin, which consists of several discrete sense organs (neuromasts) and the neurons that innervate them (Ghysen and Dambly-Chaudière, 2004). In each neuromast, there are about 10 hair cells whose function and morphology are similar to those of the human inner ear; they are responsible for detecting hydrodynamic signals and transducing these signals into electrical impulses (Chou et al., 2017; Germana et al., 2004; Yang et al., 2017), and they play important roles in a variety of behaviors such as schooling, predation, rheotaxis (Oteiza et al., 2017), and orientation. Zebrafish skin cells are ideal targets for determining the early sublethal effects of pollutants on aquatic animals, as these cells are directly exposed to the ambient environment. However, no studies have used zebrafish skin cells to test for VCR-related toxicities so far.
In this study, we examined the acute toxicities of waterborne VCR on zebrafish embryos at 0∼96 h post-fertilization (hpf) with endpoints including (i) changes in morphology, mortality, and hatching of embryos, and (ii) changes in the structure, number, and function of epithelial cells including ionocytes, keratinocytes, and lateral-line hair cells. We adopted a non-invasive, scanning ion-selective electrode technique (SIET) to measure Ca2+ influx of hair cells and acid secretion of ionocytes. The SIET was proven to be a powerful tool in our previous work measuring functional changes of these cells in vivo (Lin et al., 2006, 2015, 2019, 2020).
Section snippets
Zebrafish
Mature zebrafish (Danio rerio, AB strain, 10 months old) were provided by the Core Laboratory of Zebrafish at Taipei Medical University (Taipei, Taiwan) and were maintained in 120 × 45 × 45-cm tanks with a filtration system and thermal controller (with the water temperature maintained at 28 °C). Fertilized eggs were collected in the morning by mating three to five pairs of mature zebrafish, and were immediately transferred to artificial water (AW; 0.5 mM NaCl, 0.2 mM CaSO4, 0.2 mM MgSO4, 0.16
Effects of VCR on the percentage of mortality and hatching, heart rate, and morphology of zebrafish embryos
The cumulative mortality (%) of zebrafish embryos in different VCR concentrations (of 0, 1, 10, 15, 25, and 40 mg/L) at 0∼96 hpf are shown in Fig. 1A. No significant changes in mortality (%) were observed at VCR concentrations of 1 and 10 mg/L. In the 15- and 25-mg/L groups, mortality after hatching (%) had significantly increased at 72 hpf and had respectively reached 20 % (p < 0.01) and 60 % (p < 0.001) at 96 hpf. In the 40-mg/L group, the mortality (%) had dramatically increased to 70 % at
Discussion
A previous study showed that VCR is acutely toxic to common duckweed with an EC50 value of <10 mg/L (Jureczko and Przystas, 2019). In this study, a 96-h acute toxicity test on zebrafish embryos yielded an LC50 value of 20.6 mg/L, demonstrating the acute toxicity of VCR to fish. Another study examined the lethal and teratogenic effects of ten proteratogens on 0∼72-hpf zebrafish embryos by calculating the teratogenicity index (TI, defined as the quotient of the LC50 and EC50; a substance is
CRediT authorship contribution statement
Giun-Yi Hung: Conceptualization, Methodology, Writing - original draft, Funding acquisition. Po-Yen Chen: Investigation, Data curation. Jiun-Lin Horng: Conceptualization, Methodology, Formal analysis, Writing - review & editing. Li-Yih Lin: Conceptualization, Methodology, Formal analysis, Writing - review & editing, Funding acquisition.
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgments
This work was supported by grants from the Ministry of Science and Technology, Taiwan to Dr. Li-Yih Lin (MOST 107-2311-B-003-004-MY3) and to Dr. Giun-Yi Hung (MOST 108-2635-B-075-001). The funding source had no role in the study design, collection, analysis and interpretation of data, or writing of the manuscript.
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L.-Y. Lin and J.-L. Horng contributed equally to this study.