Impaired Liver Size and Compromised Neurobehavioral Activity are Elicited by Chitosan Nanoparticles in the Zebrafish Embryo Model

Abstract

The use of chitosan nanoparticles (ChNPs) in various biological and environmental applications is attracting great interest. However, potential side effects related to ChNP toxicity remain the major limitation hampering their wide application. For the first time, we investigate the potential organ-specific (cardiac, hepatic, and neuromuscular) toxicity of ChNPs (size 100–150 nm) using the zebrafish embryo model. Our data highlight the absence of both acute and teratogenic toxic effects of ChNPs (~100% survival rate) even at the higher concentration employed (200 mg/L). Although no single sign of cardiotoxicity was observed upon exposure to 200 mg/L of ChNPs, as judged by heartbeat rate, the corrected QT interval (QTc, which measures the time between the start of the Q wave and the end of the T wave in the heart's electrical cycle), maximum cardiac arrest, and ejection fraction assays, the same dosage elicited the impairment of both liver size (decreased liver size, but without steatosis and lipid yolk retention) and neurobehavioral activity (increased movement under different light conditions). Although the observed toxic effect failed to affect embryo survival, whether a prolonged ChNP treatment may induce other potentially harmful effects remains to be elucidated. By reporting new insights on their organ-specific toxicity, our results add novel and useful information into the available data concerning the in vivo effect of ChNPs.

AEO-7 Surfactant is “super Toxic” and Induces Severe Cardiac, Liver and Locomotion Damage in Zebrafish Embryos

“…Two compounds were employed as positive controls for general toxicity: diethylaminobenzaldehyde (DEAB, Sigma-Aldrich, Steinheim, Germany), an inhibitor of aldehyde dehydrogenases that causes signi cant pathologies and mortality in zebra sh embryos [25][26][27] and nanoparticles of zinc oxide (ZnO, diameter < 100 nm, catalog #721077-100G, Sigma-Aldrich, Steinheim, Germany) known to cause mortality and morphological deformities in zebra sh embryos and used previously as a positive control in toxicological studies [28][29][30]. In addition, in connection with the assays of neurotoxicity, 1-methyl-4phenyl-1,2,3,6-tetrahydropyridinehydrochloride (MPTP, Sigma-Aldrich, Steinheim, Germany), which causes permanent symptoms of Parkinson's disease in these same embryos, was used as the positive control [31].…”

“…Since different organs of the zebra sh become fully functional at different stages [26], acute toxicity, cardiotoxicity, and hematopoiesis were assessed once every 24 h for three days after initiating exposure at 24 hpf, and central nervous system (CNS) toxicity and hepatotoxicity following treatment from 96-120 hpf, as illustrated in Fig 1 [24,25,27,[35][36][37][38][39]. During early embryogenesis the protective chorion envelope around the embryos might interfere with uptake of the compounds being tested.…”

“…At 96 hpf, 15 embryos were transferred to each well of a 12-well plate and incubated for 24 h at 28 ºC with E3 medium (negative control),100 μM MPTP (positive control), or (iii) 0.8 or 3.2 μg/L AEO-7. Thereafter, each embryo was placed separately in a well on a 96-well plate for evaluation of locomotion utilizing the DanioVision device (Noldus Information Technologies, Wageningen, Netherlands) as described previously [27,46].…”

“…Hepatotoxicity was assessed in Tg[fabp10: RFP] transgenic AB zebra sh, which express red orescent protein (RFP) in their hepatocytes, allowing good-quality staining of the liver. At 96 hpf, the embryos were incubated for 24 h at 28 ºC with E3 medium (negative control), 1% ethanol (positive control) or 0.8 or 3.2 μg/L AEO-7, following which liver size (as an indication of necrosis and hepatomegaly) and yolk retention (as a re ection of hepatic lipid metabolism) were evaluated as described previously [26,27]. At 120 hpf, the liver of zebra sh embryos is fully developed [47].…”

“…Zebra sh have proven to be an excellent model for studying the cardiotoxic effects of xenobiotics [27,38], with an overall success rate for predicting cardiotoxic and non-cardiotoxic drugs of 100% according to some investigators [60,61] and a rating of excellent (>85%) for identifying cardiovascular toxins based on the criteria proposed by the European Center for the Validation of Alternative Methods [61]. The dorsal aorta (DA), the major axial artery in the trunk, is one of the vessels that forms rst during the early development of all vertebrates.…”