Mancozeb exposure at sublethal concentration alters the transcription of the genes related to apoptosis in the adult zebrafish (Danio rerio) brain

 

Jaganathan Murugasan Kuppuswamy, Barathi Seetharaman*

Department of Biotechnology, School of Bioengineering, Faculty of Engineering and Technology,

SRM Institute of Science and Technology, Kattankulathur, Tamil Nadu – 603203, India.

 

ABSTRACT:

Mancozeb one of the most widely used dithiocarbamates fungicide in countries like India, Pakistan, and Brazil to protect the various crops. This present study aimed to evaluate the effects of mancozeb on the expression of mRNA of the related to the apoptosis pathway in the brain of the adult zebrafish (Danio rerio). The acute toxicity test was performed, and lethal concentration 50 (LC₅₀) values were estimated. The effect of sublethal concentrations of pesticides was evaluated by exposing to 1/4^th, 1/2^th, and 3/4^th of the LC₅₀ for 8 days. Significant changes in the level of mRNA expression of genes (Bcl-2, Bax, Casp-3, and casp-9) related to the apoptosis pathway were observed. Therefore, the finding of the present study shows that mancozeb has the potential to cause apoptosis in the brain of adult zebrafish.

 

 

 

INTRODUCTION:

Mancozeb (MZ) is dithiocarbamates fungicide, which is one of the top-most fungicides used to protect the crops against the fungal diseases¹. Even though MZ is low soil persistence, they rapidly degraded in the water to ethylene thiourea (ETU), which is highly toxic to living organisms². MZ is mildly toxic for vertebrates, but studies reported they are capable of causing neurotoxicity and genotoxicity³. MZ has a similar mechanism of action like organophosphate pesticide; they kill the pest by inhibits acetylcholinesterase (AChE) activity, which plays an important role in the normal transmission of nerve impulses^4–8. MZ is one of the most widely used fungicides in India, and there is only limited information is available on the toxic effects of its metabolite ethylene thiourea.

 

Agricultural workers were exposed to high concentrations of pesticides due to unsafe work practices, like mixing of pesticides with bare hands, spraying of pesticides without a mask, leakages from the container, and tanks of pesticide during the spraying operation⁹.

 

Moreover, follow-up studies of individuals who are exposed to high levels of these pesticides compounds have shown specific neurobehavioral changes such as anxiety, mood swings, emotional lability, depression, fatigue, irritability, drowsiness, and confu­sion. These neurobehavioral changes have been termed together as Chronic organophosphate induced neuropsychiatric disorder (COPIND) and also leads to neurodegenerative diseases^10,11.

 

Oxidative stress and free radical generation are the main culprits in neurodegeneration diseases and neuropsychiatric diseases such as Alzheimer's disease, Parkinson's disease, schizophrenia, and major depressive disorder^12–14. Pesticides can induce oxidative stress, by increasing the production and accumulation of free radicals in the cell and thereby alters the antioxidant defense mechanisms, including detoxification and scavenging enzymes^15–17. Free radicals produced by these pesticides attack glial cells and neurons and damage the cellular lipids, proteins, and nucleic acids by attacking chemical bonds in those molecules which trigger the apoptosis in neurons¹⁸. It is also reported that there is a complex interplay between the mechanism of energy metabolism, excitotoxicity, and oxidative damage that play an important role in neurodegenerative disease¹⁹. It is also reported that MZ exposure can induce genotoxicity and apoptosis in mouse peripheral blood mononuclear cells (PBMC) through reactive oxygen species (ROS) generation²⁰. The aim of the present study is to explore the apoptosis-inducing potential of MZ along with underneath molecular events using adult zebrafish. Its ability to alter the expression of some apoptotic pathway-related genes in the brain was assessed using adult zebrafish (Danio rerio). The genome of the zebrafish was well characterized, and this allows us to apply sophisticated molecular approaches to investigate mechanisms of toxicity^21–24. This information provides new insight into the toxicological mechanism of MZ.

 

MATERIALS AND METHODS:

Experimental fish:

Adult zebrafish (Danio rerio) wild-type of both sexes was purchased from a local pet shop. Before the exposure, the fishes have been acclimated in aerated tanks for two weeks. Zebrafish were fed twice a day with commercial fish food and kept at ambient temperature (28±1°C) with a photoperiod consisting of 14 h: 10 h light-dark cycle, pH 7.0±0.25, Dissolved Oxygen 8.5± 1.0 and conductivity 700–900μS.

 

Fish exposure and sample collection:

In this work, the experiment was carried out by using the commercial-grade formulations Mancozeb 75 WP (Skynzeb) manufactured by Skyn Crop Care Private Ltd., having 75% w/w of MZ as an active ingredient. The commercial formulation of mancozeb was used because only commercial preparations are used in agriculture. Pilot studies were performed to find the upper and lower limits of acute toxicity for MZ in adult zebrafish. Then acute toxicity test was conducted to determine the LC₅₀ MZ in adult zebrafish as per the OECD test guidelines²⁵.

 

Based on the acute toxicity test, fishes were exposed to three sub-lethal concentrations of MZ (0.69, 1.38, and 2.07mg/L) in dechlorinated water under a static renewal system for 8 days. Concentration corresponds to 1/4^th, 1/2nd, and 3/4^th of LC_50, and for control water without the toxicant was used, and each group consisted of 10 fishes and were maintained in triplicates. These concentrations were chosen because they are lower than lethal concentrations for adult zebrafish fish and also in humans, risk of acute intoxication by high doses of mancozeb is a major concern for agricultural and industrial workers, but the population at large can be exposed to mancozeb and other dithiocarbamates through residues in food²⁶. After the exposure, the fish were anesthetized on ice, and the brain was excised from the fish collected from each treatment group, resulting in five pooled samples for RNA extraction and mRNA transcription analysis.

Gene expression analysis:

Total RNA was extracted from zebrafish brain using TriSoln reagent (Merk-Genei, India) according to the manufacturer’s protocol. The M-MLV reverse transcriptase kit (NEB, BioLab, England) was used to synthesize cDNA. A 1µL of the RT product was used directly for real-time polymerase chain reaction (PCR). Quantitative real-time PCR amplifications were performed on a CFX96 Touch Real-Time PCR Detection System (Biorad, CA, USA) using the KAPA SYBR FAST qPCR Kits (Kapa, USA). The sample was analyzed according to the previous reports^27,28. The mRNA levels of the genes involved inthe apoptotic pathway including B-cell lymphoma/leukemia-2 gene (Bcl-2 ), Bcl-2 -associated X protein (Bax), caspase-3 (Casp3) and caspase-9 (Casp9) were analyzed to evaluate the effect of MZ on the expression of the apoptosis-related gene.Oligonucleotide primers of the genes mentioned were indicated in previous reports²⁹. The housekeeping gene beta-actin was used as an internal control. Quantification of the transcripts was performed using the 2 ^-ΔΔCt method.

 

Statistical analysis:

The data were checked for normality using the Kolmogorov-Smirnov test. Data were analyzed using one way ANOVA followed by Dunnet’s multiple comparisons test to compare means from experimental groups against a control group mean. The results are expressed as a mean ± standard deviation of the mean (SD). All statistical analyses were conducted using SPSS 13.0, and the level of significance was set at p < 0.05.

 

RESULT:

Pilot studies were performed to determine the 96 hrs LC50 of the fungicide MZ on adult zebrafish and found to be 2.76 mg/L. In this study, we investigated the effect of MZ on the mRNA expression of the genes involved in the apoptotic pathway. The adult zebrafish were exposed to different concentrations of MZ for 8 days. Total RNA was isolated and converted into cDNA. The cDNA subjected to qPCR using the primers for Bcl-2, Bax, Casp3, and Casp9.The result showed the expression of the gene encoding the anti-apoptotic protein Bcl-2 was significantly decreased as the concentration of the MZ increases (Fig. 1a). We also determined the mRNA expression of Bcl-2 associated X protein Bax, there was no significant change in the gene expressions at the concentration of 0.68 mg/L and 1.38 mg/L, but at the higher concentration 2.07 mg/L there was a significant increase in the mRNA expression of Bax gene when compared with the control in adult zebrafish brain (Fig. 1b).

 

 

Fig. 1. The effect of Mancozeb on the mRNA expression. (a) Bcl-2 and (b) Bax in the adult zebrafish brain exposed to 0.69, 1.38, and 2.07 mg/L for 8days. Total RNA was isolated, followed by cDNA synthesis, and qPCR analysis b-actin was used as an internal control for normalization. The fold-change Bcl-2 and Bax were calculated relative to the control. * The asterisk represents statistical significance when compared to the controls, *p < 0.05, **p < 0.01, and ***p<0.001.

 

In addition to Bcl-2, and Bax, the genes in the caspase pathway, the mRNA level of genes such as Casp3 and Casp9 were also determined by qPCR (Fig. 2). The results showed that there was a significant decrease in the level of casp3 expression (Fig. 2a), but the mRNA expression of Casp9 there is no significance in the lower concentration but as the concentration increases the expression level of Casp9 mRNA significantly increases as the concentration increases (Fig. 2b).

 

Fig. 2. The effect of Mancozeb on the mRNA expression. (a) Casp-3 and (b) Casp-9 in the adult zebrafish brain exposed to 0.69, 1.38, and 2.07 mg/L for 8days. Total RNA was isolated, followed by cDNA synthesis, and qPCR analysis b-actin was used as an internal controlfor normalization. The fold-change Casp-3 and Casp-9 were calculated relative to the control. * The asterisk represents statistical significance when compared to the controls, *p < 0.05, **p < 0.01, and ***p<0.001.

 

DISCUSSION:

Mancozeb is the most widely used fungicide in India due to its low environmental persistence, but its metabolite ethylene thiourea is highly toxic. It is already reported that MZ exposure causes oxidative stress in an aquatic organism, which leads to the apoptotic cell death through the mitochondrial apoptosis pathway³⁰. Apoptosis is the process that can be defined as "gene-directed cellular self-destruction" or programmed cell death, and play an important role in the neurodegenerative diseases³¹. Here we report the pro-apoptosis potential of the MZ on the adult zebrafish brain. The result of the present study shows that the expression of Bcl-2 significantly reduced after exposure to MZ for 8 days. Bcl-2, which plays an essential role in promoting cell survival and inhibiting the actions of pro-apoptotic proteins. It regulates the antioxidant pathway at selected sites of ROS generation to prevent apoptosis and cellular damage³². It has been reported that oxidative stress can inhibit Bcl-2 mRNA expression in zebrafish ³³. It is also found that the expression of Bax is significantly increased. The Bcl-2 and Bax work oppositely in term of cell death, homodimer of Bax accelerates apoptosis and the heterodimer of Bax, and Bcl-2 polypeptides inhibit or delays cell death³⁴. Therefore, an increase in the homodimer of Bax resulting from the up-regulation of Bax and down-regulation of Bcl-2 could induce the release of cytochrome C from the mitochondria, which triggers the activation of Casp9 and other downstream caspases including Casp3^35,36. It is also observed that alteration in the expression of Casp3 and Casp9 after the exposure of zebrafish with MZ. Previously reported that environmental pollutants and pesticides have also shown the activation of caspase and their role in inducing apoptosis, which correlates with our results^28,29,37,38.

 

CONCLUSION:

In conclusion, our study shows that exposure to sublethal concentration of MZ alters the transcription of genes related to apoptosis. Thus, future studies aiming to correlate oxidative stress with changes in neurotransmitter signaling pathways that will bring novel opportunities to understand the role of MZ in neuropsychiatric disorders in vertebrates.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 18.12.2019           Modified on 02.02.2020

Accepted on 17.03.2020         © RJPT All right reserved