Elsevier

Environmental Pollution

Volume 231, Part 1, December 2017, Pages 1093-1103
Environmental Pollution

The cardiovascular toxicity of triadimefon in early life stage of zebrafish and potential implications to human health

https://doi.org/10.1016/j.envpol.2017.05.072Get rights and content

Highlights

  • Firstly we performed a systematic study about cardiovascular toxicity risk induced by triadimefon exposure using zebrafish.

  • Triadimefon-induced cardiovascular toxicity was characterized by abnormity and functional failure of cardiovascular system.

  • Down-regulation-related genes of ATPase, calcium channel, and cardiac troponin C may be the mechanism of the toxicity.

  • These findings can provide valuable information for health risk resulted from triadimefon.

Abstract

The health risk of triadimefon (TF) to cardiovascular system of human is still unclear, especially to pesticide suicides population, occupational population (farmers, retailers and pharmaceutical workers), and special population (young children and infants, pregnant women, older people, and those with compromised immune systems) who are at a greater risk. Therefore, firstly we explored the toxic effects and possible mechanism of cardiovascular toxicity induced by TF using zebrafish model. Zebrafish at stage of 48 h post fertilization (hpf) exposed to TF for 24 h exhibited morphological malformations which were further confirmed by histopathologic examination, including pericardial edema, circulation abnormalities, serious venous thrombosis and increased distance between the sinus venosus (SV) and bulbus arteriosus (BA) regions of the heart. In addition to morphological changes, TF induced functional deficits in the heart of zebrafish, including bradycardia and a significant reduced cardiac output that became more serious at higher concentrations. To better understand the possible molecular mechanisms underlying cardiovascular toxicity in zebrafish, we investigated the transcriptional level of genes related to calcium signaling pathway and cardiac muscle contraction. Q-PCR (quantitative real-time polymerase chain reaction) results demonstrated that the expression level of genes related to ATPase (atp2a1l, atp1b2b, atp1a3b), calcium channel (cacna1ab, cacna1da) and cardiac troponin C (tnnc1a) were significantly decreased after TF exposure. For the first time, the present study revealed that TF exposure had observable morphological and functional negative impacts on cardiovascular system of zebrafish. Mechanistically, this toxicity might result from the pressure of down-regulation of genes associated with calcium signaling pathway and cardiac muscle contraction following TF exposure. These findings generated here can provide information for better pesticide poisoning treatments, occupational disease prevention, and providing theoretical foundation for risk management measures.

Introduction

Triadimefon[TF:1-(4-chlorophenoxy)-3,3-dimethyl-1-(1H-1,2,4-triazol-1-yl)-2-butanone] is a registered broad-spectrum systemic sterol biosynthesis-inhibiting fungicide, which is extensively used to control rusts, mildews, fungal pests on fruits, vegetables, and ornamentals for more than two decades in China, and has a high solubility, strong stability and long half-life in water (Li et al., 2016, Liu et al., 2011). When fungicides are appropriately and scientifically utilized, they do not produce any serious health and environmental problems. However safety recommendations are not always followed, and undesirable high levels of residues are still detected in water, soils and food (Kong et al., 2016). It had been proved that high levels of TF residues were detected in soils, water and target agricultural products. For example, Papis and colleagues have reported that TF could be moderately absorbed in soils and hence maybe very mobile in the aquatic environment (Papis et al., 2006) and have been detected in water nearly 0.922 mg/L (Watschke et al., 2000). Furthermore, recent report showed that different pesticides were found in jujube ranged from 59.4 mg/kg to 2945.0 mg/kg, of which TF was found at a concentration of 1.0 mg/kg in fruits and 94.5 mg/kg in jujube soil (Liu et al., 2016a, Liu et al., 2016b), indicating that increased accumulation of pesticides in soil, water, and agricultural products pose serious threats to human health because of its wide application in agriculture and dietary intake through food consumption.

The toxicity of TF have been reported by many studies. For example, several eco-toxicology studies have indicated that triazole fungicides were highly toxic to a number of nontarget organisms (Groppelli et al., 2005, Menegola et al., 2005). TF was suspected of having teratogenic potential on the basis of FETAX (Frog Embryo Teratogenesis Assay-Xenopus) analysis. For example, Groppelli and colleagues reported teratogenic effects in Xenopus after exposure of triazoles at the neurula stage, at the level of cartilages and muscles of the 1st and 2nd branchial arches (Groppelli et al., 2005). In addition, two previous studies reported that TF could alter thyroid hormone levels and related gene expression in zebrafish (Liu et al., 2011) and in Xenopus laevis tadpoles (Li et al., 2016) in the early life stage, respectively. Recent study also found that TF may have endocrine-disrupting effects on lizards (E.argus) by acting sensitively on sex steroid hormones and steroidogenic-related genes. In animal test, triazoles can specifically affect branchial development in rats when exposure was performed during neurulation phases (Menegola et al., 2000, Menegola et al., 2001) as well as thyroid tumors in rats and liver tumors in rats and mice (Wolf et al., 2006). Moreover, an increasing number of investigations also proved that TF was a type of cytochrome p450 enzyme inhibitor (Li et al., 2017). TF may have reproductive toxicity to animals and even human beings (Vinggaard et al., 1999, Vinggaard et al., 2000, Chu et al., 2016), which was related to its influence by blocking the synthesis of 17 β-estradiol (E2) from testosterone (Brande-Lavridsen et al., 2008).

However, all of the above toxicity focused on neurotoxicity, hepatotoxicity, reproduction toxicity, developmental toxicity, and endocrine-disrupting effects. There is no cardiovascular toxicity information available. According to news report (The news website, 2016), cardiovascular disease, which was produced by genetic factors and environmental factors as well as a combination of both of these factors, was a global public health problem with a high mortality and case fatality, and multiplies the risk for a number of complications and huge medical costs. Given the increased accumulation of TF in soil, water, and food, potential cardiovascular disease risk require desperately to be evaluated, particularly to pesticide suicides population, occupational population (farmers, retailers and pharmaceutical workers), and special population (young children and infants, pregnant women, older people, and those with compromised immune systems) who are at a greater risk. Sekhotha et al. (2016) reported that there was a close relationship between pesticides and cardiovascular diseases. But, adverse effects of TF on the cardiovascular system is largely unknown. Therefore, in the present study, we aimed to evaluate systematically and comprehensively if TF exposure produce the risk of cardiovascular disease.

However, how to find a good and reliable animal model system to study pesticides exposure-cardiovascular effects, clarify the toxic mechanism, and explore strategies to mitigate those adverse effects is an urgent problem to resolve. Thanks to an almost entirely sequenced genome and high conservation of gene function and many molecular pathways compared with humans (Zon and Peterson, 2005, Bakkers, 2011), the zebrafish (Danio rerio) has emerged as an invaluable and highly informative vertebrate model system for studying substance toxicity on development, particularly on the development of cardiovascular system. This transparent vertebrate offers numerous compelling experimental advantages for evaluating toxic effects not found in other model systems (Sarmah and Marrs, 2016). A number of laboratories have employed the zebrafish to investigate the toxic effects of model chemicals released into the environment on the development of cardiovascular system, including nanoparticles, pesticides and various organic pollutants (Asharani et al., 2011, Belair et al., 2001, Chakraborty et al., 2016, Gerlach et al., 2014, Henry et al., 1997, King-Heiden et al., 2012, Teraoka et al., 2002, McGee et al., 2013, Zhang et al., 2013). These studies highlighted the suitability of the zebrafish as an excellent model to study human cardiovascular diseases (Bakkers, 2011).

In zebrafish, the heart is the first organ to develop and function and a beating heart forms by 22 hpf. By 48 hpf, the cardiovascular system is fully functional and exhibits a complex repertoire of ion channels and metabolic processes (Thisse and Zon, 2002).

Herein, in this study, 48 hpf zebrafish embryos were employed as the test organism, and the effects of TF on the cardiovascular system were studied systematically including malformation assessment (pericardial edema, circulation defects, hemorrhage, thrombogenesis, SV-BA distance and histopathology) and function assessment (heart rate, atria/ventricular ratio and cardiac output). Previous studies reported that Ca-dependent signaling are associated with heart failure, and they can alter the expression of some key Ca regulatory proteins involved in excitation-contraction coupling and their regulation by kinases and phosphatases (Bers and Guo, 2005, Kim et al., 2008). In addtion, Ca2+-ATPase is responsible for the regulation of Ca2+ uptake into the sarcoplasmic reticulum (Xu et al., 2009). Furthermore, troponin complex is a component of cardiac muscle thin filaments, and is often utilized in medicine as a target for some cardiotonic drugs used in the treatment of heart failure (Katrukha, 2013). To get an insight into the possible mechanisms of TF on cardiovascular system of zebrafish, so we performed the transcriptional profiles analysis of genes related to calcium signaling pathway and cardiac muscle contraction. Our results obtained here may provide information for TF-exposure cardiovascular disease risk, particularly for better pesticide poisoning treatments, occupational disease prevention, and providing theoretical foundation for risk management measures.

Section snippets

Chemicals

96% triadimefon was bought from Jiangsu Sword Agrochemicals Co., Ltd. Stock solutions were prepared in 100% dimethyl sulfoxide (DMSO; Amresco, Solon, OH,USA) and stored in the dark at 4 °C. 4% paraformaldehyde in phosphate-buffered saline was brought from Aladdin holdings (group) Co., Ltd (Shanghai, China). O-dianisidine was obtained from Sigma (St. Louis, MO, USA). Trizol reagent, reverse transcriptase kit and the SYBR Green system were purchased from Takara (Dalian, China).

Zebrafish husbandry

Wild-type AB strain

MNLC, LC10 and LC50

The mortality of embryos in each group at the end of exposure time (24 h) was recorded. Then TF-induced zebrafish mortality curve, which was presented in Fig. 1, was fitted in Origin 9.0. On the basis of mortality curves, MNLC, LC10 and LC50 of TF were estimated and calculated as 37.4, 42.5 and 47.2 μg/mL, respectively. Therefore 1/4 MNLC (9.4 μg/mL), 1/2 MNLC (18.7 μg/mL), MNLC (37.4 μg/mL), LC10 (42.5 μg/mL) and LC50 (47.2 μg/mL) were setup to study the cardiovascular toxicity of IF in

Discussion

Because of its widespread use, triadimefon (TF) is ubiquitous and accumulated in the environment, high levels of residues have been detected in soil, water bodie, and food (Liu et al., 2016a, Liu et al., 2016b, Papis et al., 2006, Watschke et al., 2000), which increases the risk to human health via food chain day-by-day, especially to pesticide suicides population, occupational population (farmers, retailers and pharmaceutical workers), and special population (young children and infants,

Conclusion

To the best of our knowledge, in this study, we was the first to explore systematically and comprehensively the detailed cardiovascular toxicity induced by TF using zebrafish model and found that TF induced serious and typical cardiovascular toxicity characterized by morphologic abnormity (pericardial edema, thrombosis and increased SV-BA distance) and functional failure (bradycardia and reduced cardiac output). Down-regulation of ATPase-related genes (atp2a1l, atp1b2b, atp1a3b), calcium

Conflict of interest statement

The authors declare that they have no competing interests.

Acknowledgments

This study was financially supported by Zhejiang Provincial Science & Technology Planning Project of China (no. 2015C02019) and Zhejiang Provincial Natural Science Foundation of China (no. Z12C140006).

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