Asmita Samaer, Swatilekha Das, Bakul Pal, Sweta Das, Aninita Manal,Priyanka Biswas, Sujoy Ghosh, Shamim Hossain Manal, Priyanka Sow,Ruchira Das, Souvik Biswas, Ashis Kumar Panigrahi

aCytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani, Nadia, 741235, India

bCell and Developmental Biology Special, Department of Zoology, University of Kalyani, Kalyani, Nadia, 741235, India

cDepartment of Zoology, Dum Dum Motijheel College, Kolkata, 700074, India

dEco-Toxicology, Fisheries and Aquaculture Extn. Laboratory, Department of Zoology, University of Kalyani, Kalyani, Nadia, 741235, India

Keywords:

ABSTRACT Objectives: Pesticide toxicity has become one of the major environmental menaces affecting all types of life forms of the ecosystem. Pesticides get washed off from agricultural fields into nearby water bodies and enter the aquatic organisms. Their bio-accumulated form finally reaches the human race, through consumption of pesticide infested aquatic animals, causing several physiological dysfunctions. Hence it becomes necessary to find a therapeutic cure/a preventive measure to stop the health hazard issues of pesticide. With this projection a search for a phyto-based-product was made whose primary objective would be to lower the pesticidal toxicity in fish and simultaneously in the human race.Methods: In this study we tried to check whether the phyto-chemical, Chlorophyllin (CHL), known for its antigenotoxic, anti-oxidant activities, could render any kind of protection against Cypermethrin (CM) inducedtoxicity in fish model and mammalian cell line L6. Both the model L6 and fish were pre-treated with CHL prior to exposure of CM. Different scientific parameters like % cellular cytotoxicity, reactive oxygen species(ROS) generation, nuclear condensation, etc were checked to validate the possibility of CHL in protecting CMinduced toxicity.Results: The overall results revealed that pre-treatment with CHL could restrict the ROS generation leading to modulation in associated cytokine proteins expression NFkβ and IFNγ. Further, CHL lowered nuclear condensation and elevated expression of DNA repair proteins p53 and PARP, showing a kind of pre-activation of signalling cascades for overall protection against the severity of pesticidal toxicity.Conclusion: Thus, this phyto-based preventive approach would possibly solve many areas of human health issues related to pesticide toxicity in future.

1.Introduction

Plants and their bio-active compounds have been used over the years to cure a plethora of diseases. The subtle use of phyto-products in their appropriate amount in day to day lifestyle ranges from food, skin care,cosmetics, medicine, etc. The global scenario of choosing natural products over synthetic commodities have had a great shift off recently because of their affordability and limited side effects.

However, despite of rigorous monitoring, there is an alarming rate of increasing environmental pollution worldwide which may occur naturally (like arsenic, cadmium, etc contamination) or man-made (like pesticide) toxicity in different level of the ecosystem. Pesticides like cypermethrin, have been reported to induce DNA damage through cyclical amplification of ROS and activate inflammatory response factors like NFkβ, IFNγ, TNFα, etc. which brings about an impairment in signalling proteins p53 and PARP which are associated with the DNA repair system (Samadder et al., 2019; Singh et al., 2012, Huang et al.,2016).The effects of these perturbations have certainly crossed the threshold level in manifesting several health issues in human, therefore needing immediate therapeutic management to ensure that their existence is free from health hazard in the ecosystem.

The efficacy of phyto-products against myriads of diseases has been practiced from time long past. However, their resurgence use over synthetic products, having categorical and productive side effects,increased the popularity and choice of phyto-compounds in the present day. People often prefer several plant extracts, fruit juice and fruit pulp for healing purposes and often consume them as a regular precautionary substances for immunity and protection. Thus, this protective efficacy of phyto-product against the initiation and progression of certain diseases pave the way to evaluate the possibility of a phyto-derived compound Chlorophyllin to protect against the daily hazards of pesticide (toxicity)due to consumption of vegetables, fruits, fishes, etc.

Chlorophyllin (CHL), a chlorophyll derivative salt was chosen for the purpose of our study. The anti-genotoxic, anti-carcinogenic (Das,Samadder, Mondal, et al., 2016; Das, Samadder, Das, et al., 2016; Gradecka-Meesters et al., 2011; Nagini et al., 2015; Vesenick et al., 2012),an wound healing accelerant (Tumolo & Lanfer-Marquez, 2018), and anti-diabetogenic (Patar et al., 2018) property of CHL was the platform of choosing this phyto-product which was found to be completely non-toxic at any dose in mammalian model.

Pesticides are often washed off from agricultural land and pollute the water bodies thereby affecting the aquatic ecosystem like the phytoplankton, zooplankton, fish, mollusca, etc. These pesticides are then accumulated in the body of human beings by consumption of fish,mollusc, zooplankton, etc through their daily diet, thereby leading to diverse health hazard which range from mild to severe depending on the susceptibility of the individual towards the chemical composition of the pesticide in use.

Therefore, the search for a phyto-based molecule to combat pesticide toxicity both in fish bio-system and in human race led us to undertake this present piece of work with the following objectives: (i) to assess the protective efficacy of CHL in CM-induced toxicity, (ii) to evaluate modulation, if any, in % cell viability, oxidative stress, nuclear condensation mitigated DNA damage and anti-oxidant enzyme profile in mammalian and/or fish muscle cells; (iii) to determine the possible signalling proteins involved in protecting CM-induced DNA damage and generation of ROS.

2.Materials and methods

2.1.Chemicals and reagents

The chemicals and reagents that were used for the study were of analytical grade, with ≥97% purity. Almost all of them were purchased from Sigma Aldrich. The exceptions have been mentioned where they appear in the text.

2.2.Cell culture of mammalian muscle cell line L6

Mammalian skeletal muscle cells, L6, were obtained from National Centre for Cell Science (NCCS, Pune, India). They were grown in a 5%carbon dioxide atmosphere at 37°C in Dulbecco’s modified Eagle’s Medium (DMEM). The medium was supplemented with 10% fetal bovine serum and 1% antibiotic. For all experimental studies, cells were plated separately; each plate was grown up to 80–90% confluency which served as the desired density for further investigation.

2.3.Selection of cypermethrin concentration in L6 cells

The sub-lethal concentration of 30 μM of cypermethrin (purchased from Pharma Agro Chemical, India 25 E.C.; permissible amount of acetone was used to dissolve cypermethrin) and a time period of 12 h incubation of its post administration was considered for this study. The selection of time and dosage of the pesticide was based on the report of Samadder et al. (2019).

2.4.Effective dose of chlorophyllin in L6 cells

CHL was dissolved in DMEM medium at different concentrations starting from 10 through 50 μg/ml. The L6 cells were pre-treated with 10, 20, 30, 40 and 50 μg/ml of CHL separately for 2 h (Rigonato et al.,2004) followed by exposure to standardized dose of CM (30 μM). Hence,two doses of chlorophyllin (CHL), 15 μg/ml and 30 μg/ml, were chosen from the range finding trial based on the difference in % of viable cells found in each case.

2.5.Acclimatization and maintenance of tilapia fish

Tilapia fishes were purchased from the local market and maintained at standard temperature and atmospheric conditions along with proper feeding in a small tank (Samadder et al., 2019). After around 15 days of acclimatization the experiments were set up by randomly selecting fish,irrespective of their sex, into different smaller tank as per standard practice.

2.6.Fish exposure to cypermethrin

An average weight of ～20-25 g b.w. of fishes were selected for this study. The sublethal dose of CM was found to be 0.4 μg/μl from our earlier range finding study by assessing their % cell viability in vital cells isolated form liver, muscle, kidney, testes and ovary. Therefore, fishes were exposed to 1/10th of this dose i.e., 0.04 μg/μl of CM via subcutaneous injection of 0.05 μl/g b.w. using 0.9% w/v of normal saline as the medium for injection of CM. Further, a post injection gap of 7 days was maintained for the development of damage in fish tissues induced by pesticide toxicity before sacrificing them for experimental studies(Samadder et al., 2019).

2.7.Detection of cypermethrin in different tissues of fish

Presence of cypermethrin in different fish tissue homogenates like muscle, liver, kidney, testes and ovary were ascertained by following the spectrophotometric procedure of Janghel et al. (2007). The technique of alkaline hydrolysis of cypermethrin to cyanide ion was used in this case.Briefly, the cyanide ions formed at the end of hydrolysis further reacted with potassium iodide and leuco-crystal violet (4,4′,4′′-Methylidyne tris(N,N-dimethylaniline) was added to each sample. After completion of the reaction a crystal violet dye was formed in each sample which was measured at 595 nm, the λmaxof the dye. The concentration of cypermethrin was determined from the standard curve prepared by the same procedure prior to the experimental sample measurement.

2.8.Dose of chlorophyllin (CHL) in fish

The pre-treatment dose of CHL was selected as 0.5 mg/g b.w. (CHL I)and 1 mg/g b.w. (CHL II) after a range finding trial by assessing % viable cells in control and experimental sets and comparing the same. Each fish from their respective group was fed with either of the two doses of CHL through oral gavage once daily for consecutive 7 days prior to commencement of pesticide i.e., CM exposure.

2.9.Experimental design

Around twenty-four healthy tilapia fish irrespective of sexes were selected randomly from the rearing tank for the purpose of conducting the experiment. The fish were then subdivided into four groups, each group comprising six fishes.

Cells were isolated from liver, kidney, muscle tissues in a sterile ambience using DMEM medium (the same used forin vitroculture). The isolated cells were then centrifuged at 2000 g for 5 min after which the supernatant was discarded and the pellet was washed with sterilized phosphate buffered saline (PBS) and re-suspended in the same before plating them for different experiments. All the experiments were repeated thrice, independent of each other, before analyzing the data statistically.

Further, for cell culture, i.e.,in vitroL6 cell line model, similar control and experimental culture dishes were prepared and all the experiments were repeated thrice each of which was independent of each other.

The experiments were set in the following order forin vivoandin vitrostudies:

Group 1: Control (untreated)

Group 2: CM

Group 3: CHL(I)+CM

Group 4: CHL(II)+CM

2.10.% Cell viability assessment

3- (4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide(MTT) dye was used to determine the % of viable cells after seeding L6 cells (group 1 to 4) in 96 well plates in different group as stated in the experimental design. The data were collected in an ELISA plate reader as per standard practice (Samadder et al., 2012). Similarly, forin vivostudies liver, kidney and muscle cells were isolated from their respective tissues in control and experimental fish group 1 to 4. The cells were then plated in 96 well plate and % of viable cells were assessed by MTT assay.

2.11.Determination of reactive oxygen species generation and occurrence of nuclear condensation

Determination of generation of reactive oxygen species (ROS) and the occurrence of nuclear condensation were assessed as per standard practice (Samadder et al., 2011, 2016). The cells were grown to 70%confluency and washed with PBS (phosphate buffered saline). 1 μl of 100 mg/ml of 2′7′-dichloro dihydrofluorescein diacetate acetyl ester(H2DCF-DA) dye was added in each set and incubated in dark for 30min.After washing excess dye with PBS the cells were further counterstained with 0.5 μl of 100 mg/ml Hoechst dye for 30 min for assessment of nuclear condensation and observed under fluorescence microscope. For estimation of ROS in fish muscle tissues, the muscle cells were isolated in sterilized PBS and centrifuged at 2000 g. Near about 1000 cells were counted in each of control and experimental set and thereafter equal number of cells were stained with 1 μl of 100 mg/ml of H2DCF-DA. The number of fluorescing cells were counted microscopically.

2.12.Assessment of anti-oxidant enzyme profile in fish muscle: Catalase(CAT), lipid peroxidase (LPO) and superoxide dismutase (SOD)

The modulation in anti-oxidative enzymes like CAT, LPO and SOD were assessed using the muscle tissue extracted from the control and experimental fish group. Briefly, after sacrificing the fishes, their muscles were collected; equally weighed tissues were homogenized in phosphate buffered saline (PBS). The tissue extracts were then centrifuged at 10000 g for 30 min at 4°C after which the supernatant was collected and the standard protocols were followed: Ohakawa et al.(1979) for determining (LPO), Kakkar et al. (1984) for checking SOD activity and Maehly and Chance (1954) protocol for detecting any possible differences in CAT activity.

2.13.Estimation of expression of NFkβ, IFNγ, p53 and PARP by ELISA assay

Fig. 1.Detection of cypermethrin content in different fish tissues at 595 nm wavelength using UV-Vis spectrophotometer.

Fig. 2.Assessment of % cell viability in vitro and in vivomodel: 2a: Graphical representation % cellular viability shown in L6 cells in vitro. ＊＊p <0.01 vs CM,＊p <0.05 vs CM were considered significant for Student’s t-test. 2b: % Cellular viability assessment shown by graphical representation in different cells isolated from respective tissues of fish. ＊＊p <0.01 vs CM, ＊p <0.05 vs CM were considered significant for Student’s t-test.

Expressions of NFkβ, IFNγ, p53 and PARP proteins were analyzed using anti-NFkβ, anti-IFNγ, anti-p53 and anti-PARP primary antibodies(Santa Cruz Biotechnology, Inc., USA) and enzyme conjugated secondary antibodies (Sigma Aldrich, USA) following the standard protocol of Paul et al. (2011). The absorbance was quantified at 404 nm wavelength in an ELISA reader. Briefly, after quantifying the total protein content in each group of fish and L6 cell line the proteins were equated and the number of cells corresponding to the equal content of protein in each set were seeded in 96 well ELISA plate for bothin vitroandin vivo(isolated fish muscle cells) studies for determining the differences in protein expression, if any, in control and experimental set. In case ofin vitrostudy NFkβ, IFNγ, p53 and PARP proteins were checked and in case ofin vivoassessment, p53 and PARP protein expression were evaluated in isolated fish muscle cells as per standard practice of indirect ELISA (Das, Samadder, Mondal, et al., 2016; Das, Samadder, Das, et al.,2016).Student’s t-test. Additionally, one way ANOVA was performed along with Student’s t-test for ELISA.

Fig. 3.Determination of ROS and DNA damage: Fluorescence microscopic evaluation for ROS (reactive oxygen species) generation and nuclear condensation along with measurement of fluorescence intensity in different L6 cell set.

2.14.Statistical analysis

All the data presented in tables and figures were mean values of three independent experiments which have been statistically analyzed by

Table 1 Determination of the modulatory effect of chlorophyllin (CHL) against cypermethrin (CM)-induced toxicity in fish: Data representing % fluorescing cells treated with DCFDA for detecting ROS, assessment of total protein content (μg/ml), absorbance (a.u) for p53 and PARP proteins by ELISA. ＊＊＊p <0.001 vs CM,＊＊p <0.01 vs CM, ＊p <0.05 vs CM were considered significant for Student’s ttest.

3.Results

3.1.Spectrophotometric detection of cypermethrin from different tissues in fish

The amount of cypermethrin which were detected from different fish tissues were presented as % showing highest in liver, then in muscle and kidney followed by lowest in testes and ovary tissues (Fig. 1).

3.2.Assessment of % cell viability

% of viable cells were found to be greater in CHL pre-treatment +CM administered set of cells when compared to CM treated group of cells in both mammalian muscle cellsin vitro(Fig. 2a) and fish liver, kidney and muscle cellsin vivo(Fig. 2b).

3.3.Evaluation of ROS generation and nuclear condensation

CM treatment showed an increase in both ROS generation and nuclear condensation when compared to that of control. However, cells pre-treated with CHL and then exposed to CM showed an inhibition in ROS generation and reduction in the formation of condensed nucleus(Fig. 3). Similar results were also obtained in fish muscle cells where CHL pre-treatment could prevent the extent of ROS generation by CM exposure as compared to experimental set where fish were exposed to CM pesticide without any pre-treatment with CHL (Table 1).

3.4.Determination of modulation of anti-oxidative enzyme profile in fish muscles: lipid peroxidase (LPO), superoxide dismutase (SOD) and catalase(CAT) on pre-treatment with CHL

The overall result from anti-oxidative enzyme profile clearly revealed that CHL pre-treatment prior to CM exposure could alter the enzyme activities; CHL could restrict CM-induced elevation of LPO level and simultaneously inhibited pesticide-induced diminish in SOD and CAT activities (Fig. 4).

3.5.Quantification of modulation in protein expression

The expression of different proteins like NFkβ, IFNγ, p53 and PARP protein were modulated in the experimental groups (Gr 3 and Gr 4)where CHL was administered first followed by CM exposure. Pretreatment with CHL showed a restriction in NFkβ and IFNγ expression while a greater degree of elevation was noticed in p53 and PARP protein expression in CHL+CM treated set of mammalian cellsin vitro(Fig. 5).Similar results were also obtained from the results of protein expression of p53 and PARP observed in muscle cellsin vivo.Additionally, there was a distinct difference in the total protein content in each group of the experimental sets. CHL pre-treatment could restrict the loss of total protein content when the fish were exposed to CM, thereby showing a kind of protection towards pesticide induced protein depletion(Table 1).

Fig. 4.Anti-oxidant enzyme profile assessment: Graphical representation of Lipid peroxidase (A), Superoxide dismutase (B) and Catalase (C) in control and experimental set in fish muscle. ＊＊＊p <0.001 vs CM, ＊＊p <0.01 vs CM, ＊p <0.05 vs CM were considered significant based on Student’s t-test.

4.Discussion and conclusion

Fig. 5.Protein expression associated with ROS and DNA damage repair signaling cascade: Modulatory effects of CHL in altering CM induced changes in the expression of signal proteins NFkB, IFNγ, p53 and PARP by ELISA. ＊＊＊p <0.001 vs CM, ＊＊p <0.01 vs CM, ＊p <0.05 vs CM were considered significant only after completion of Student’s t-test.

The issue of pesticide toxicity affecting every life existing in the ecosystem has become an area of great concern specially when it interferes with human fitness leading to a variety of health hazards in today’s life style. The overall findings of our study emphasis on this particular issue where our prime concern lies within the scope of exploring plant products as an environment friendly chemical to combat the severity of pesticide toxicity (Molavia et al., 2014; Samadder et al.,2019). Phyto-products are the only most reliable non-toxic/limitedly toxic source of medicinal resource which has been used by mankind for a long time. In the present study the preventive potential of the phyto-based product, chlorophyllin (CHL), was assessed against pesticide toxicity through well established scientific protocols. The idea of our work encompass two major aspects, the very first being to deal with the issue that pesticide after being washed off from agricultural landforms contaminate the surrounding water bodies affecting wide range of aquatic life form (including fishes) on the first hand. Secondly, the affected but unidentified aquatic animals being consumed by the human race in their daily diet makes them more susceptible to the bioaccumulation and biomagnifications effects of these pesticides. Therefore, in this study the selection of the phyto-chemical chlorophyllin(CHL) was made keeping certain points in mind: i) CHL should be able to act on both types of organism, the fish, (being the food of millions worldwide) and human race, ii) CHL should have a wide range of the protective efficacy against variety of toxic substances induced dysfunction/diseases and iii) CHL should be able to interact and modulate several upstream and downstream bio-factors for withholding the magnitude of damages imparted by the toxic substances in experimental models. The overall results portray that CHL could qualify all the issues and gave us a purview towards understanding the molecular signalling mechanism which aided in its process of protection from cypermethrin(CM) toxicity in mammalian skeletal muscle cells(in vitro)and fish tissues (in vivo).

In our earlier studies we found that CM induces generation of reactive oxygen species (ROS) which in turn disturbed the normal functioning and balance of the overall anti-oxidative enzyme homeostasis(Samadder et al., 2019). This result corroborated with the observations reported by other group(s) of researcher who pointed out clearly that cypermethrin altered the oxidative stress-mediated JNK/ERK signalling cascades (Singh et al., 2012, Huang et al., 2016). In our present study pre-treatment of mammalian and fish muscle cells with chlorophyllin(CHL) followed by exposing them to CM resulted in inhibiting in ROS generation and its accumulation as compared to CM treated alone, i.e.,without CHL-pre-treatment. The restriction in ROS level in turn altered several inflammatory signalling molecules and cytokines like NFkβ and IFNγ thereby reducing the cyclical amplification of ROS. Furthermore,modulation in the anti-oxidant enzyme profile observed in CHL pre-treatment series possibly demarcates the numerous route of molecular and biochemical protection which has been “switched on” during the pre-treatment period with CHL prior to pesticide exposure.

Nuclear orientation is one of the key constituents of optimum functioning of cells and tissues. In our study, fish treated with CM, induced nuclear condensation in muscle cells, while the same condensation of nucleus was found to be arrested in the CHL pre-treated fish group. CHL is known to have a strong binding and base stacking property within the grooves of the DNA (Das, Samadder, Mondal, et al., 2016; Das,Samadder, Das, et al., 2016) which possibly allow only a weak interaction with other substances/toxicants (in this case) with the DNA.Thus, exposure to CM after being pre-treated with CHL resulted in less nuclear condensation and DNA damage and could aid in diminishing overall cell/tissue damage in differentin vitroandin vivomodel systems.

Further, to examine the possible involvement of any signalling proteins with the process of restoration of DNA damage and diminish of nuclear condensation we proceeded with the investigation of the modulatory role of CHL on DNA damage repair protein PARP and p53.Results of the protein expression study revealed that CHL pre-treatment could pre-activate and/or over-express the PARP and p53 proteins even greater than their normal expression. These two proteins are known to be closely associated with the DNA repairing process and thus their elevated expression i.e., “switched on” state/pre-activation condition in CHL pre-treated cells serve the purpose of possible protection/prevention against severe DNA damage. The underlying mechanism might be orchestrated with the fact that due to strong CHL-DNA interaction the damages made by weak bonds of toxicant-DNA interaction get readily repaired with the “switched on”/pre-activated repair machinery and thus prepares the cells to combat against any further toxicant effects.

Therefore, the overall results of the present study suggest that CHL could be an essential beneficiary phyto-based molecule for its possible role in preventing cellular damage against pesticide toxicity thereby aiding in maintaining optimum homeostatic condition of the cells for their proper functioning. Thus, the use of this phyto-based, non-toxic,easily affordable, biodegradable and most importantly environmental friendly molecule, CHL, would help to restore a harmony between different levels of ecosystem ranging from aquatic fishes to human.Thus, this particular study should encourage the extensive use of these phyto-based protective compound(s) which would be necessary for therapeutic management of pesticide toxicity globally.

Funding

SERB (DST) (ECR/2017/000355), UGC-BSR-starup grant, University of Kalyani (PRG and DST-PURSE).

Credit authorship statement

Asmita Samadder: Conceptualization, Methodology, Writing - review & editing, literature, Final Editing. Swatilekha Das: Data curation,Visualization, Writing - original draft, preparation. Bakul Pal: Data curation, Visualization, Writing - original draft, preparation. Sweta Das:Data curation, Visualization, Writing - original draft, preparation.Anindita Mandal: Data curation, Visualization, Writing - original draft,preparation. Priyanka Biswas: Data curation, Visualization, Writing -original draft, preparation. Sujoy Ghosh: Data curation, Visualization,Writing - original draft, preparation. Shamim Hossain Mandal: Data curation, Visualization, Writing - original draft, preparation. Priyanka Sow: Investigation, and, Validation, Critical statement of outcome confirmation, Writing - review & editing. Ruchira Das: Investigation,and, Validation, Critical statement of outcome confirmation, Writing -review & editing. Souvik Biswas: Software. Ashis Kumar Panigrahi:Supervision.

Declaration of competing interest

None to declare.

Acknowledgement

Grateful acknowledgements are extended to SERB (DST) (ECR/2017/000355), UGC-BSR Start-up grant for providing research funds which was used partly to accomplish this piece of work. Authors thank University of Kalyani (PRG and DST-PURSE) for their minor research funding which was used for the purpose of this work. PS is grateful to UGC for her research fellowship.