Investigation of Toxicity Induced by Chemicals and Their Mechanisms using Zebrafish Vertebrate Animal Model

Gaur, Himanshu and Bhargava, Anamika (2019) Investigation of Toxicity Induced by Chemicals and Their Mechanisms using Zebrafish Vertebrate Animal Model. PhD thesis, Indian institute of technology Hyderabad.

Full text not available from this repository. (Request a copy)

Abstract

Human population is constantly exposed to health risks by exposure to chemicals belonging to various classes such as pesticides, pollutants and chemicals used in agriculture, food industries etc. It is important to keep use of such chemicals under continuous observation and their effects must be re-evaluated in the light of changing conditions of use and new scientific information (Council directive 89/107/EEC). Major part of my thesis is focused on investigating toxic effects and mechanisms of action of one such environmental pollutant, glyphosate, a non-selective herbicide. In this thesis, mechanistic toxicology of sodium benzoate (SB), a common food preservative, has also been investigated. I have used zebrafish vertebrate animal model for toxicity studies included in my thesis and therefore I have also established a zebrafish research lab at IITH. Glyphosate is a non-selective herbicide. Results included in my thesis indicate significant acute toxicity in zebrafish embryos exposed to glyphosate concentrations 50 µg/mL and above. LD50 for glyphosate was calculated to be 66.04 ± 4.6 µg/mL after 48 hours of exposure. Developmental toxicity due to glyphosate was also observed as physical malformations in zebrafish embryos such as yolk sac edema, pericardial edema, body bending and eye defects. Glyphosate induced cardiotoxicity above 50 µg/mL concentrations as indicated by reduced heartbeat of zebrafish embryos in a dose and time dependent manner. To analyze heartbeat, we developed a simple yet powerful method of heartbeat analysis from the untethered, unanesthetized and unlabeled zebrafish larva using ImageJ (open-source) software. We named our semi-automatic method as ZebraPace for Zebrafish Precise Algorithm for Cardiac-rhythm Estimation. Our method is truly cost-effective, uses algorithm which involves fewer steps without the requirement of cumbersome image processing and programming steps. It is also highly reproducible across researchers. The decrease in the heartbeat of zebrafish embryos upon exposure to glyphosate was accompanied by a reduction in the gene expression of cacna1c (L-type calcium channel) and ryr2a (Ryanodine receptor) indicating alterations in the proteins involved in excitation-contraction coupling and calcium signaling. A change in calcium signaling upon glyphosate exposure was also evident by the upregulation of hspb11 gene (heat shock protein) in our experiments. Of note, hspb11 has been used as a marker to detect changes in cytosolic calcium. Therefore, taken together our results indicate that glyphosate may be inducing cardiotoxicity by changes in calcium signaling. In addition, in our experiments, glyphosate reduced the generation of nitric oxide (NO). NO signaling and calcium signaling are tightly linked and any alteration in one will lead to changes in the other. Glyphosate exposure to adult zebrafish resulted in a reduced reproduction potential indicated by less number of successful mating between treated zebrafish. We observed developmental defects and cardiotoxicity in the progeny of adult zebrafish exposed to glyphosate at a concentration of 10 µg/mL. It is important to note that, the obtained progeny was not exposed to glyphosate. Taken together, these results indicate that glyphosate induced toxicity can be carried to the next generation. Swimming behaviour was also affected in adult zebrafish exposed to glyphosate at a concentration of 10 µg/mL. The speed of swimming was increased which was interrupted with long durations of freezing in between as compared to control adult fishes. Treated adult zebrafish also showed anxiety-like behaviour as indicated by reduced exploration of the center of the open field test tank. I have also investigated mechanistic toxicology of SB. Results included in my thesis indicate that SB induced biochemical (oxidative stress) and behavioural (anxiety-like behaviour) abnormalities in developing zebrafish larva. LD50 of SB induced toxicity was approximately 400 ppm after 48 hours of SB exposure. Our data strongly supports harmful effects of SB at increasing doses and suggests caution in the excessive use of this preservative in processed and convenience foods. Overall, results in my thesis indicate that both glyphosate and SB can cause significant toxicity and we suggest caution in the excessive use of chemicals which have the potential to cause toxicity.

[error in script]

IITH Creators: