Research Article

A study of the toxicity of dimethoate on human lymphocytes and the ameliorative effect of root extract of Oroxylum indicum (L.) Vent

Richa Soni1, Asha Khanna2, Mamta Gokhale3*, Dayashankar Gautam4

1Department of Biotechnology, Pondicherry University, Pondicherry, India

2Retd.Head of Department Of Biotechnology, St.Aloysius College (Autonomous), Jabalpur, Madhya Pradesh, India

3Department of Biotechnology, St. Aloysius College (Autonomous), Jabalpur, Madhya Pradesh, India

4Department of Zoology, St. Aloysius College (Autonomous), Jabalpur, Madhya Pradesh, India

*For correspondence

Dr. Mamta Gokhale,

Department of Biotechnology, St. Aloysius College (Autonomous), Jabalpur, Madhya Pradesh, India.

Email: mamtashrirang@

gmail.com

 

 

 

 

 

 

 

Received: 28 June 2016

Revised: 20 July 2016

Accepted: 22 July 2016

ABSTRACT

Objective: To study the toxicity of dimethoate on human lymphocytes and the ameliorative effect of root extract of Oroxylum indicum (L.) Vent.

Methods: Human peripheral blood lymphocytes were separated from human blood by HiseptmLSM1077. They were exposed to 20,40,60 and 100 mM concentrations of dimethoate for 1 hour and the DNA damage was assessed by fluorescent comet assay. The toxicity of the insecticide was also tested by MTT assay.

Results: It was found that the number of cells with no damage decreased with the rise in concentration of dimethoate and there was a dose dependent increase in the number of damaged cells (9-98%). In MTT assay the viability of control cells was taken as 100% and after 2 hours of exposure to various concentrations of dimethoate, there was dose dependent decrease in viability. The viability of treated cells increased after incubation with 50% of the root extract of Oroxylum indicum (L.) Vent. However, no ameliorative effect was observed when the treated lymphocytes were incubated with crude root extract.

Conclusions: Thus the experiment supports the fact that 50% of root extract of Oroxylum has an ameliorative effect on the lymphocytes which were subjected to the dimethoate toxicity.

Keywords: Ameliorative effect, Human lymphocytes, Oroxylum indicum, Root extract, Dimethoat.

Introduction

The need to feed our growing population makes its necessary for the nation to increase its agricultural products. To achieve this, the use of more and more insecticides is evident in the agricultural practice. However, it is necessary to assess the effect of these insecticides on human beings. It is well known that an indiscriminate use of insecticides is a serious environmental threat. Almost every system of human body is affected by these chemicals, whether respiratory, reproductive, nervous or circulatory. There are four main groups into which these chemicals have been classified, viz. Organophosphates, Organochlorines, Carbamates and Pyrethroids. As majority of these chemicals are non- biodegradable, they tend to accumulate in the environment and also enter into the food chain. The most potent and important chemical class is of organophosphates, which act by inhibiting acetylcholinesterase hydrolysis of acetylcholine, resulting in acetylcholine accumulation in neuromuscular synapses.

Insecticides dimethoate was selected to observe its effect on human lymphocytes since it is widely used currently. Dimethoate is used against a broad range of insects such as thrips, aphids, mites, and whiteflies, and on a number of crops including citrus, cotton, fruit, olives, potatoes, tea, tobacco and vegetables. It is also permitted for the control of the flies in livestock accommodation, home gardens and food storage.Its trade names include Afidox (produced by Lucava), Cekutoate (Cequisa), Perfekthion (BASF), Rogor (Isagro), Dimezyl (Agriphar), Hilthioate (Hindustan), andTeeka (Nagarjuna (3.1%).[1]

The structural formula for dimethoate is given below.

Figure 1: O,O-dimethyl S-[2-(methylamino)-2-oxoethyl] dithiophosphate.

The LD50 of dimethoateis 60-385 mg/kg in rats, according to LD50 data quoted by WHO 2004, it falls in the category of mildly harzardous insecticide.

Plants have been a source of medicinal products since a long time. To date, approximately 100 species of plants have been examined and some active constituents isolated and identified, for instance several of the current chemotherapeutic drugs like vinblastine, methotrexate, taxol, and so forth, were first identified in plants.2 Oroxylum indicum (L.) Vent. tree has been used for the preparation of ayurvedic medicines since a long time. Every part of this tree contains metabolites that possess medicinal value with anticancer potential.3 Ameliorative effect of root extract of O. indicum was reported earlier against damage induced through tobacco extract in human lymphocytes4. In this experiment, damage was induced through Dimethoate (20 mM, 40 mM, 60 mM, 100 mM) in human lymphocytes and ameliorative effect of 70% ethanolic root extract (crude and 50%) was studied by MTT assay. The effect of the same concentrations of dimethoate was studied on human lymphocytes5 in which the parameters of damage were certain enzyme assays.

Materials and Methods

Dimethoate 30% EC was purchased from the local insecticide dealer used for the study.

Isolation of lymphocytes from whole blood

Blood (2.5 ml) from healthy young male volunteer donor (approved by the institutional ethical committee) was collected in sterile EDTA vial. This was diluted with an equal volume of PBS (1X). HiSepTM LSM 1077 (Hi Media) (2.5 ml) was transferred aseptically into a centrifuge tube. This was then carefully overlaid with 5 ml of diluted blood. It was centrifuged at 400xg at room temperature (RT) for 30 min. Erythrocytes were sedimented and the lymphocytes formed a layer above the Hi Sep layer. Most of the supernatant was aspirated out and then the lymphocyte layer along with half of the Hi Sep layer was carefully aspirated into a separate centrifuge tube. It was then given two washes with isotonic PBS. Ten µl of the cell suspension were diluted with an equal amount of trypan blue solution, the viable cells were counted in a haemocytometer and appropriately diluted in TC 199 medium (Hi Media) supplemented with foetal bovine serum to give a final concentration of 3.33 x 104 cells ml-1.

Preparation of root extract from O. indicum

Root of O. indicum (L.) Vent. was collected from reserve forest area of Jabalpur (M.P), India. The dried roots were ground into a fine powder form. The root powder (1 g) was dissolved in 100 ml ethanol and was incubated in an orbital shaker at 120 rpm for 24 hours at 25±2ºC. After incubation period ethanolic crude root extract was filtered and taken as crude root extract (rec). Crude root extract was diluted to 50% with 70% ethanol and was taken as 50% rec.

MTT assay

This is a colorimetric assay that measures the reduction of 3-(4, 5-dimethylthiasol-2-yl)-2, 4,diphenyltetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase. The MTT enters the cells and passes into the mitochondria where it is reduced to an insoluble, coloured, formazan product. The cells are then solubilised with an organic solvent dimethyl sulfoxide (DMSO) and the released, solubilized formazan reagent is measured by spectrophotometer.

This study was performed according to Mosmann's basic technique with some modifications.6 Aliquots (120 µl) of the prepared lymphocyte suspension (3.33 x104 cells/ml) were seeded into a 96 well polystyrene tissue culture plate in multiples of 3 replicates. Readings were taken of 20 µl the three concentrations of dimethoate and of the root extract. One row containing only medium and cells served as a control. Aliquots of 20 µl of the dimethoate concentrations (20 mM, 40 mM, 100 mM) were added and incubated for 2 hours at 370C in humid incubator and the readings were taken.This served to provide the data for the damage done to the cells by the insecticide alone. Each concentration was tested in multiples of 3 replicates. Cells incubated in culture medium alone served as a control for cell viability which was taken as 100%. To some of the wells containing cells and medium and various concentrations of dimethoate, 20 µl of root extract was added. To other wells, 50% of the root extract was added and the plate was incubated over night at 370C.

After incubation, 20 µl aliquots of MTT solution (5 mg/ml in PBS) were added to each well and re-incubated for 2 hours at 370C. 60 µl of DMSO was then added to each well to dissolve the formazan crystals followed by incubation at 370C for 30 min. The culture plates were then placed in an ELISA microplate reader and absorbance was read at 600 nm. The amount of color produced was directly proportional to the number of viable cells. Before the procedure, the OD of the empty microplate wells was also noted. OD of various concentrations of dimethoate and root extracts was previously noted and the final OD was calculated after making the due adjustments for these two factors. All assays were performed in multiples of 3 replicates per dose.

Cell viability rate was calculated as the % of MTT absorption as follows:-

% survival = (Mean experimental absorbance/Mean control absorbance) × 100

Results of MTT were subjected to student's t test for statistical analysis.

COMET assay

The alkaline comet assay is a biomarker that is widely used to detect DNA damage induced by environmental chemical agents such as pesticides. When compared with other cytogenetic tests, such as sister chromatid exchange, micronuclei formation, and chromosomal aberration assays, the comet assay is the most rapid and sensitive method to evaluate the genotoxic agent in vitro as well as in vivo.

In this study, damage was produced in lymphocytes by varying concentrations (20, 40, 60 and 100 mM ethanolic solution) of dimethoate.

The comet assay was performed in the following steps.

  1. 500 µl of the lymphocyte suspension was separated into 5 parts of 100 µl each in separate eppendorfs.
  2. 100 µl of cell culture medium 199+ FBS was added to each eppendorf and then 20, 40, 60 and 100 mM of dimethoate were added to 4 tubes and 5th was kept as a control.
  3. The basic technique of Dhawan et al.7 and Nandhakumar et al.8 was followed.
  4. The lymphocytes were incubated with the insecticide for 1 hour.
  5. The previously prepared slides coated with 1% NMPA were taken and a mixture of the treated lymphocytes with LMPA in (ratio of 8:10) was spread on the slides.
  6. Coverslips were placed on the slides and they were left on ice packs for 10 mins, then the coverslips were removed carefully.

After cooling at 40 C, the slides were immersed in precooled lysis solution for 40 min. They were then immersed in freshly prepared alkaline unwinding solution (pH>13). The slides were removed and immersed in electrophoresis buffer (pH>13). The electrophoresis run was for 30 min at 25 Volts. The slides were then stained by Ethidium bromide (20 µg/ml). Scoring was done on the fluroscence microscope and the damaged cells were categorized into eight types.9 Data from 100-200 cells of each concentration was collected and was compared to that of controls. The scoring was done visually as belonging to one of the eight predefined classes having a DNA damage of various classes ranging from 9-98%. Frequency of cells in each category was noted from each concentration.

Results and Discussion

MTT assay

In Table 1, the MTT results showed that there was a dose dependent drop in viability when the cells were treated with different concentrations of dimethoate after 2 hours of exposure. If the viability of untreated cells was taken as 100%, the viability at 20 mM was 96.08%, which fell to 85.08% when the concentration increased to 100 mM (significant at p<0.05). However, the ameliorative effect of 50% REC on cells damaged by 100 mM dimethoate was significant (p<0.01) as compared to the same treatment with 100% Rec. There was a further fall in viability when the cells were incubated with crude root extract in all the three concentrations of dimethoate.

There was however, an increase in viability when the cells treated with various concentrations of dimethoate were incubated with 50% of REC as compared to the viability with crude REC. This was due to the fact that crude REC was also toxic to the cells. Thus it was evident that at 20 mM toxicity of dimethoate, the 50% of REC had a great ameliorative effect hence the viability reached 98.75 which almost touched that of controls (100%).

Table 1: Effect of dimethotae and root extract (100% and 50%) of O. indicum (L.) Vent. on cell viability.

S.No Concentration of dimethoate (mM) Root extract (%) OD % of viability
1

Control (DM 0)

-

2.40

100

2

DM20

-

2.31

96.08

3

DM40

-

2.18

90.96

4

DM100

-

2.04

85.08

5

DM20

100

2.29

95.42

6

DM40

100

2.19

91.25

7

DM100

100

1.86

77.50*

8

DM20

50

2.37

98.75

9

DM40

50

2.21

92.08

10

DM100

50

2.09

87.08**

*significant at p<0.05;**significant at p<0.0

Comet assay

It is evident from the Table 2 that the controls showed a maximum no. of undamaged cells(77%) and there were no cells showing more damage than 9-45%. In lymphocytes treated with 20 mM of dimethoate, the maximum no. of cells were found to be in 9% damage category and only a few cells had more than 45% damage. In the 40 mM treatment category, the maximum number of cells showed 75% damage. All categories of damaged cells were seen although only a few had more than 91% damage. In the 60 mM exposed lymphocytes, 24.8% cells showed 75% damage and very few cells were in the 91-98% category. Lymphocytes exposed to 100 mM of the insecticide showed the least no. of normal cells (9.7%) and 27.1% cells were in the highly damaged category (Figure 3). The categories of the damage has been shown in Figure 2.

Table 2: Categories of damage in lymphocytes.

Conc. of dimethaote

No. of cells counted

0% No. of cells  (%)

9% No. of cells (%)

9-45% No. of cells (%)

45% No. of cells (%)

45-75% No. of cells (%)

75% No.ofcells (%)

91% No.ofcells (%)

91-98% No.ofcells (%)

Control (0 mM)

219

170(77%)

38(17.3%)

11(5.02%)

-

-

-

-

-

20 mM

106

24(22.6%)

43(40.5%)

29(27.3%)

6(5.6%)

2(1.8%)

2(1.8%)

-

-

40 mM

131

20(15.2%)

25(19%)

19(14.5%)

9(6.8%)

28(21.3%)

18(13.7%)

8(6.1%)

2(1.5%)

60 mM

205

40(19.5%)

71(34.6%)

29(14%)

12(5.8%)

-

51(24.8%)

-

2 (1.5%)

100 mM

195

19(9.7%)

30(15.3%)

-

20(10.2%)

37(18.9%)

36(18.4%)

26(13.3%)

27(13.8%)

Thus there was a progressive decrease in the no. of cells with no damage and an increase in cells with higher degree of damage as the concentration of the insecticide increased.

a) Control            b) 9%

c) 45%             d) 45-75%

e) 75%            f) 91%

g) 91-98%

Figure 2: Images of lymphocytes showing various categories (a - g) of DNA damage.

Figure 3: Percentage of lymphocytes in various damage categories.

Mechanism of action of organophosphorus and carbamate insecticides was studied previously.10 Pesticides are a very important group of environmental pollutants used in intensive agriculture for protection against diseases and pests. The estimated annual application is more than 4 million tons, but only 1% of this reaches the target pests. While their use improves the quantity of agricultural products it potentially affects their quality, as pesticides may enter human diet.

Since dimethoate is one of the most frequently used organophosphates these days, this work of toxicity assay of this insecticide on human lymphocytes was taken up. The methods of determination of damage to lymphocytes used were comet assay and MTT assay.

The effect of dimethoatein lymphocytes were studied by Gargouri et al.5 They divided the lymphocytes into two groups. The first group was incubated for 4 hrs at 37ᵒC with different concentrations of dimethoate (0, 40, 60 and 100 mM). The second group was pre incubated for 4 hrs at 37ᵒC with quercitin. They found that dimethoate caused a significant increase in malondialdehyde levels and increase in superoxide dismutase and catalase activities in lymphocytes at different concentrations. Quercetin preincubated lymphocytes showed a significant protection against the cytotoxic effect induced by dimethoate. Ameliorative effect of O. indicum metabolites on tobacco extract induced cell damage in human lymphocytes was previously studied by Gokhale et al.4 It was found that the survival rate of damaged lymphocytes was enhanced after root extract treatment. The aqueous treatment showed better ameliorative effect than n-butanol extract.

In the present study, lymphocytes were incubated with 20, 40, 60 and 100 mM of dimethoate for 1 hour. Taking into consideration the number of cells in different categories of damage (ranging from no damage to 91-98%), it was found that number of damaged cells in the various categories showed a dose dependent increase.

Similar work has been done by other workers on organophosphates and pyrethroids. Blasiak and Trzeciak1 studied the effects of Malathion and Isomalathion (25 and 200 mM ethanolic solution) on lymphocytes after 1 hour exposure by comet assay, they found that isomalathion caused a maximum increase in Comet length at 200 mM as compared to the control. The increase was dose dependent. The toxic effect of exposure to tobacco dust on lymphocytes of bidi rollers was studied by comet assay by Khanna et al.11 They found that an increase in the duration of exposure to tobacco dust leads to an increase in comet tail length as compared to non exposed controls.

The effect of cypermethrin on human lymphocytes was studied by Suman et al.12 The comet tail length analysis showed a progressive increase with an increase in concentration. The comet length in control cells was 3.29 µm and at 3.84 µM, it was 26.67 µm. The cytotoxic effects of cypermethrin were determined by MTT assay by these workers. They found a dose dependent cytotoxic effect on lymphocytes on exposure of 2 hours.

Conclusions

The lymphocytes were treated with 20, 40, 60 and 100 mM of dimethoate for 1 hour and the comet assay was performed. The cell damage was assessed by fluorescent microscopy and it was found that there was a progressive decrease in the cells with no damage and an increase in cells with greater damage as the concentration of the insecticide increased. For the MTT assay, the toxic effect of the same concentrations of dimethoate was studied after 2 hours exposure. The viability ranged from 96.08% at 20 mM to 85.08% at 100 mM. The viability fell further after an overnight incubation with crude root extract, but it showed a rise when incubated with 50% of root extract (rising to 98.5% which almost touches the value for control untreated cells). Thus the experiment shows that 50% of the root extract exhibits an ameliorative capacity to reverse the cellular damage caused by the insecticide.

Acknowledgements

The authors are grateful to Principal and Management of St. Aloysius College (Autonomous), Jabalpur (M.P) India for providing lab facility for the work. The authors are thankful to Department of Science and Technology for supporting this research through research project (vide reference no. SR/WOS-A/LS-514/2013).

Funding: Department of Science and Technology (vide reference no. SR/WOS-A/LS-514/2013)

Conflict of interest: None declared

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