Protective Effect of Vitamin C against Behavioral, Histological Changes and Mortality Rate Induced by Paraquat in Drosophila melanogaster

Research Article

Austin Food Sci. 2020; 5(1): 1036.

Protective Effect of Vitamin C against Behavioral, Histological Changes and Mortality Rate Induced by Paraquat in Drosophila melanogaster

Viana JWM*, De Lima RCG, Silva JRL, Nunes RGS, Neto JE and Barros LM

Department of Biological Sciences, Regional University of Cariri (URCA), Crato, CE, Brazil

*Corresponding author: José WM Viana, Bachelor of Biological Sciences, Department of Biology, Regional University of Cariri, Crato, CE, Brazil

Received: April 19, 2020; Accepted: May 12, 2020; Published: May 19, 2020

Abstract

Paraquat (PQ) is known to cause fatal intoxications to humans and animals mainly by the generation of reactive species. However, none is known on the transgenerational toxicity of PQ and the effect of the supplementation of the standard antioxidant vitamin C (VIT C) to counteract PQ-induced toxicity in Drosophila melanogaster. Here, we investigated the possible protective effect of VIT C on behavioral, mortality rate, and histological changes in different generations of D. melanogaster following PQ exposure. Flies of both genders (P generation), of 1-3 days of age, were exposed separately at different concentrations of PQ (0.5, 2.5, 5 mg/g), and VIT C (25, 100, 200 mM) for 7 days (first exposure). The second generation of flies (F1) was exposed to the most toxic PQ concentration (2.5 mg/g) in the presence of vitamin C (25, 100, 200 mM) again for a week (second exposure), and the locomotor performance of flies resulting from this exposure was also evaluated (F2 generation). The number of death flies was recorded daily and their locomotor performance was evaluated by negative geotaxis assay. The histological analysis of the brain of flies was also performed. The results demonstrated that PQ significantly decreased the lifespan and altered the locomotor activity of flies. PQ (2.5 mg/g) promoted neurodegeneration evidenced by the appearance of vacuolated areas in brain of flies. However, treatment with VIT C decreased alterations in mortality rate and locomotor activity, as well as preventing neurotoxicity in the brain of PQ-exposed flies. Taken together, our result highlight VIT C as a potential therapeutic agent against PQ toxicity.

Keywords: Antioxidants; Paraquat (PQ); Vitamin C (VIT C); Flies; Toxicity.

Introduction

Herbicides have been used worldwide over the years to delay growth of weeds and eliminate phytopathologicalpragues that cause damage to agricultural plantations [1,2]. However, their use is known to have environmental impacts and cause health related problems [3]. Of particular pathological importance, herbicides are recognized as possible teratogenic, mutagenic and carcinogenic agents, as well as endocrine disruptors; and their long-term exposure can promote acute to chronic intoxication [4,5], leading to neurodegenerative diseases [6], and reproductive disturbances [7].

Paraquat (PQ) (1,1’dimethyl-4-4’-bipyridynium dichloride) is among the most widely used herbicides worldwide, and its action is increased when combined with the herbicide Diquat [8,9]. In spite of its wide use in agriculture, numerous studies have demonstrated that PQ cancause fatal intoxications for both humans and animals [10-19].

Substantial evidence from the literature indicate that PQ toxicity is mainly attributed to the increased production of superoxide anion, through oxidative stress induction mechanisms, generating more toxic reactive species, such as hydroxyl radicals (OH-) and hydrogen peroxide (H2O2), which have been associated with the depletion of endogenous antioxidant defense systems [2,19-22]. The accumulation of reactive species and the subsequent depletion of reducing agents create an environment of intense oxidative stress that can, consequently cause damage to the lipids, proteins, mitochondria and DNA, thus leading to the alteration of cellular functions [23-26].

Natural antioxidants have been reported to attenuate oxidative stress by eliminating free radicals and increasing endogenous antioxidant defences [27-31]. One of such compound is vitamin C (ascorbic acid), an important non-enzimatic antioxidant that acts as a potent reducing agent [32-36].

Over the past decades, the fruit fly, Drosophila melanogaster has considerable attracted the attention of scientists as an alternative model due to its 75% genetic similarity with mammals. In addition, it has been used to increase our understanding on the neuropathological and toxicological processes involved in the etiology of several diseases, such as Parkinson, Alzheimer, Cancer and chronic diseases [37-41]. Furthermore, Drosophila has a relatively fast reproductive cycle, small body size and easy maintenance in laboratory conditions [42].

Although vitamin C (VIT C) is well-known for its antioxidant activity, little is known about the effect of VIT C supplementation on the transgenerational transmission of Drosophila melanogaster after exposure to PQ. Therefore, this study aimed to investigate the possible protective effect of this vitamin C on behavioural, mortality rate, and histological changes in different generations of D. melanogaster following PQ exposure.

Material and Methods

Animals

Flies of both gender, aged 1 to 3 days old, were used in this study. They were obtained from the Laboratory of Biology and Toxicology (BIOTOX) of the Regional University of Cariri-URCA. The flies were kept in glass flasks containing culture medium constituted of 1 kg of corn meal, 50 g of ground soybeans, 50g of powdered milk, 50g of sugar and 10 g of salt. In addition, they were mainainedin a light/ dark cycle of 12h: 12h, under constant temperature and humidity (25 ± 15°C; 60% respectively), as described by Abolaji et al. [42] and adapted by Nunes and collaborators [40].

Treatment schedule

Exposure of parent generation (P) flies to PQ and VIT C: To test the effect of different concentrations of PQ and VIT C, the flies, regardless of gender, were exposed for 7 days (1 week) to the diet supplemented with PQ in increasing concentrations of 0.5, 2.5 and 5.0 mg/g diet, or VIT C in increasing concentrations (25, 100 and 200 mM), and the count of the number of dead flies (mortality rate) was recorded daily at the same time.

For this first experiment, the flies were divided into seven groups (30 flies for each group in triplicate), six of which received the following treatment: Group I - PQ 0.5 mg/g; Group II - PQ 2.5 mg/g; Group III - PQ 5.0 mg/g; Group IV - VIT C 25 mM; Group V - VIT C 100 mM; Group VI - VIT C 200 mM. The flies in the control group received neither VIT C nor PQ.

At the end of the 7-days period, the surviving flies from the P generation underwent locomotor activity test (negative geotaxis assay) and later discarded. However, the resulting pupae from the P generation were allowed to hatch to constitute the F1 generation (2nd generation). Of note, the locomotor activity of flies of the F1 generation was also performed for comparative purpose.

PQ exposure and treatment with VIT C in F1 generation flies: Based on the preliminar result obtained from PQ exposure to P generation about the mortality rate, the concentration of 2.5 mg/g of diet was selected to investigate the possible protective effect of vitamin C against the transgenerational contamination caused by PQ exposure. For this purpose, 30 flies (per group, in triplicate) of both gender of F1 generation were exposed to diet supplemented with 2.5 mg PQ/g of diet with or without (PQ alone) different concentrations of VIT C (25, 100 and 200 mM). The control diet did not received VIT C or PQ.

For the second experiment, the flies were divided into five groups, four of which received treatment: Group I - PQ (2.5 mg/g); Group II - PQ (2.5 mg/g) + VIT C (25 mM); Group III - PQ (2.5 mg/g) + VIT C (100 mM); and Group IV - PQ (2.5 mg/g) + VIT C (200 mM). The daily count of the number of dead flies from the F1 generation was performed during the second 7-day exposure periodand later submitted to negative geotaxis essay. Then, the flasks were emptied, leaving the pupae of this second exposure to hatchin order to form the F2 generation of flies (3rd generation). The F2 generation of flies were then submitted to negative geotaxis assay after completing one week for comparative purposes.

Nagative geotaxis assay (evaluation of locomotor activity): The locomotor performance of the P, F1 and F2 generations flies was investigated by the negative geotaxis assay, which is based on the evaluation of the locomotor activity of the flies. Briefly, the flies were anesthetized on ice, and 10 flies of each flask were placed into vertically oriented glass tubes and allowed to recover for 10 min. The flies were then shaken down to the bottom of the tubes and the number of flies that climbed the 5 cm distance in 6 seconds, as well as those that remained below the marked line in the tube (5 cm), were recorded for analysis. The assay was repeated three times with 3 min interval between the assays, according to experimental procedures previously described by Kiss et al. [43] and adapted by Nunes et al. [40].

Histological analysis of the brain of Drosophila melanogaster

The F1 generation flies were selected to perform the histological analysis in order to know if the increasing concentrations of VIT C (25, 100 and 200 mM) prevented the PQ neurotoxicity in the most toxic concentration (2.5 mg/g). For this, some samples were immediately immersed in 10% buffered formaldehyde and fixed for 24 h, then washed in a buffer solution, dehydrated in alcohol in an increasing series of concentrations (50-100%) and included in glycol methacrylate (historesin) (Historesin Leica®).

Further, 2 μm cuts were done in microtome (Leica®) model RM 2245 equipped with glass razor. The histological sectionswere distended in distilled water and placed on laminas, dried in a buffer at 60°C for 1 min and submitted to toluidine blue staining technique. The laminas in triplicate per animal were analyzed under a light microscope Leica®DM500 and photographed by Leica EC3 locked to the microscope and to Leica® Application Suite (LAS) EZ for histological analysis, according to procedures previously described by Kretzschmar et al. [44], Kosmidis et al. [45] and Dutta et al. [46].

Statistical analysis

Two-way ANOVA followed by Benferroni test was performed using statistical software GraphPad Prism version 6.0, to find out significant variations among different groups of different generations. The results were expressed as mean ± SEM and considered statistically significant when p < 0.05.

Results

As depicted in (Figure 1), the exposure of generation P flies to PQ at concentrations of 2.5 and 5 mg/g of diet caused a significant increase in the mortality rate when compared to the control (p < 0.05), with the higher mortality at concentration of 2.5 mg/g (Figure 1A). However, 0.5 mg/g of PQ (group I) did not cause any effect on the survival of flies when compared with the control (p > 0.05), and the other groups exposed to the PQ (groups II and III) (Figure 1A and 1B). A similar effect was observed with the highest concentration of VIT C (200 mM) used (group VI) when compared to the control (p> 0.05). Throughout the 7-day exposure period, it is possible to notice that this concentration of VIT C exerted a greater protective activity in the survival rate of generation P flies compared to the other groups exposed to the VIT C (groups IV and V) (Figure 1A). The cumulative number of generation P dead flies, obtained at the end of the period of exposure to PQ and VIT C separately, shows that there was a slight reduction in the mortality rate with the increase in the concentration of VIT C when compared to the control (p > 0.05; Figure 1B).