Norethindrone causes cellular and hepatic injury in zebrafish by compromising the metabolic processes associated with antioxidant defence: Insights from metabolomics
Graphical abstract
Introduction
Over the last few decades, the consumption of progestins (i.e. synthetic progesterone) has globally increased due to their high effectiveness for birth control and many other medical applications (Stefanick, 2005; Market Data Forecast, 2020). Therefore, progestins often enter and contaminate aquatic environments in the municipal and agricultural areas via the excreta of humans and livestock (Chang et al., 2009; Shen et al., 2018), especially when sewage treatment plants are not yet designed for efficient removal of progestins (Fent, 2015; Chiu et al., 2016). Since progestins can persist and accumulate in aquatic environments, there is growing concern about their potential adverse effects on aquatic organisms (Besse and Garric, 2009; Fent, 2015; Kumar et al., 2015), particularly considering that the market demand for progestins is predicted to keep escalating in the future (Market Data Forecast, 2020).
One of the most commonly used progestins is norethindrone (NET), which is typically applied for contraception, hormone therapy and treatment of menstrual disorders (Apgar and Greenberg, 2000). In view of the extensive use for medical applications, NET has been widely detected in various water bodies at noticeable concentrations (e.g. 53 ng/L in wastewater, Viglino et al., 2008; 143 ng/L in runoff, Liu et al., 2014; 872 ng/L in stream water, Kolpin et al., 2002). As a synthetic hormone, NET has been shown to trigger endocrine disrupting effects on aquatic organisms (e.g. change in sex ratio and reduction in reproductive output, Paulos et al., 2010; Hou et al., 2019), which probably diminish their populations. Apart from endocrine disruption, recent studies reveal that NET, particularly at high concentrations, can lead to inflammatory response and inflict cellular damage (Hou et al., 2020; Liang et al., 2020), suggesting that NET can pose more deleterious effects on aquatic organisms than previously thought. Yet, the mechanism underlying the cellular damage mediated by NET remains largely unexplored.
Cellular damage is usually associated with elevated oxidative stress due to the accumulation of reactive oxygen species (ROS), where their production through mitochondria can be induced by environmental stressors (e.g. pollutants and thermal stress, Leung et al., 2019; Rodriguez-Dominguez et al., 2019; Wang et al., 2021). This suggests that NET may undermine the metabolic processes responsible for antioxidant defence that scavenges excess ROS, eventually resulting in cellular damage. Indeed, endocrine disrupting chemicals can lead to metabolic disruption directly by modulating metabolic pathways or indirectly by interacting with specialized metabolic receptors (Casals-Casas and Desvergne, 2011; Gore et al., 2015). To decipher how NET modifies a variety of metabolic processes in organisms, endogenous metabolites (i.e. end products of metabolic processes) can be examined because their profile (i.e. metabolome) can act as metabolic fingerprints that allow identification of the specific metabolic processes (e.g. protein synthesis, fatty acid synthesis, lipid metabolism, etc.) altered by environmental stressors (Messerlian et al., 2017). For instance, Cao and Wang (2016) applied a metabolomic approach to study the impacts of metal pollution on oysters and found that osmotic regulation and energy metabolism of oysters were disturbed by heavy metals. Compared with traditional biomarkers, such as physiological and biochemical parameters, metabolomes offer a higher level of sensitivity to detect the effects of environmental stressors on organisms in addition to the more comprehensive information provided (Long et al., 2020). If NET can disrupt the metabolic processes associated with antioxidant defence, links would be found among certain metabolites, antioxidant defence and cellular damage.
Here, we assessed how NET affects the antioxidant defence of zebrafish and the histological features in the liver, followed by determining the change in the metabolome. Zebrafish was used as a model organism because its liver metabolism and immune response are similar to those of many animals, including humans. The liver was examined because it offers many vital physiological functions (e.g. antioxidant defence and detoxification), but is primarily attacked by ROS (Vascotto and Tiribelli, 2015), meaning that liver health is a good proxy for the fitness of organisms. We hypothesized that zebrafish could tolerate the potential adverse effects of NET at low concentrations (50 ng/L), but its antioxidant defence (SOD and GSH) would be impaired at high concentrations (500 ng/L), resulting in cellular damage (MDA) and hepatic injury (ALT and histological features). When the liver was injured, we expected that the expression of metabolites associated with antioxidant defence would be downregulated. By integrating metabolomics with conventionally measured biomarkers, the findings herein can provide a comprehensive understanding of how endocrine disrupting chemicals can threaten aquatic organisms through disruption of metabolic pathways, implying that these chemicals can pose deleterious effects other than endocrine disruption as previously shown.
Section snippets
Experimental design
Adult zebrafish Danio rerio (body length: 3.68 ± 0.18 cm; wet weight: 0.38 ± 0.09 g) were purchased from a fish market in Guangzhou, China. In the laboratory, their body shape and markings on body surface were examined to identify and collect the females (Kimmel et al., 1995). The female zebrafish (denoted as “fish” hereafter) were then maintained in 40 L aquaria containing 30 L dechlorinated tap water with continuous aeration under stable conditions (pH: 7.50 ± 0.05; temperature:
Results
After the 72-day exposure, the survival rate of fish was > 95% and was unaffected by NET, regardless of the concentration (Fig. 1). The number of normal cells in the liver was not significantly affected by NET at 50 ng/L (∼495 cells/mm2 vs. ∼500 cells/mm2 for the control), but reduced by ∼58% at 500 ng/L (∼212 cells/mm2) (Fig. 2a). The number of small vacuoles was slightly increased by NET at both 50 and 500 ng/L, while large vacuoles were only observed at 500 ng/L (Fig. 2b–d). The liver cells
Discussion
The widespread occurrence of NET has raised substantial concern about its impacts on the populations of aquatic organisms (Besse and Garric, 2009; Fent, 2015). In this study, we found that NET at environmentally relevant concentrations did not cause acute mortality of zebrafish, but led to liver damage and impaired antioxidant defence by disrupting metabolic pathways, which may have repercussions for the fitness and survival in the long term.
Conclusion
Progestins are well-known for their endocrine disrupting effects on aquatic organisms, such as causing masculinization and altering mating behaviour (Hou et al., 2020; Liang et al., 2020; Chen et al., 2021); however, they can also inflict cellular damage, indicating their toxic effects. Here, we revealed that NET, a commonly used progestin for medical applications, did not cause acute mortality at environmentally relevant concentrations, but it weakened antioxidant defence and hence resulted in
Author contributions
XW, LH and JYSL conceived and designed the experiments. XW, ZT, SC, LH and LG conducted the experiments and collected the data. ZT, XL and DK analysed the data. XW and ZQ drafted the paper. JYSL statistically analysed the data, provided data visualization, critically reviewed and revised the paper.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This research was funded by the Natural Science Foundation of Guangdong Province, China (Grant No. 2017A030313141 and 2018A030313478).
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