Comparing cytotoxicity of propoxur and Nepeta crispa (Lamiales: Lamiaceae) essential oil against invertebrate (Sf9) and vertebrate (L929) cell lines

Background and Aim: Attempts to use the plant products are to be an appropriate option due to substantial concerns about human health and environmental problems of using synthetic pesticides. Therefore, the cytotoxicity of Nepeta crispa essential oil was compared with propoxur against invertebrate (Sf9) and vertebrate (L929) cell lines. Materials and Methods: The cell lines of Sf9 and L929 which were derived from the ovary glands of fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae) and mouse fibroblast cells, respectively, were obtained from the National Cell Bank of Pasteur Institute of Iran. About a number of 2 × 103 cells were placed into the wells of 96-well plate experiments. Then, appropriate concentrations of essential oil of N. crispa plant and propoxur added to the wells. The cells were allowed to grow for 3-5 days and estimated the numbers of cells. The cells of control experiment wells contained only cells with dimethyl sulfoxide. All control and treatment experiments repeated at least four replicates. Results: Propoxur had negative effects on the viability of both invertebrate (Sf9) and vertebrate (L929) cell lines. The cytotoxicity of propoxur against invertebrate (Sf9) and vertebrate (L929) cell lines was gradually increased in accordance with propoxur concentrations. The cytotoxicity of N. crispa essential oil against vertebrate (L929) cell line was gradually decreased in accordance with plant concentrations, while the cytotoxicity of N. crispa essential oil against invertebrate (Sf9) cell line was strongly increased in accordance with plant concentrations. Conclusion: Plant essential oil not only had no negative effects but also had boosting effects on vertebrate cell viability. Essential oil of N. crispa plant had negative effects on invertebrate cell viability with the differences that the products derived from plants possessing of biodegradable and environmentally friendly derivatives, hydrolyzing rapidly in nature, and nearly having no destructive effects on environment, humans, or the mammals.


Introduction
Attempts to use the plant products as natural pesticides usually based on plant essential oils are considered as a good candidate for the management of arthropod vectors of diseases and agricultural pests due to possessing of readily biodegradable and environmentally friendly derivatives [1][2][3][4][5]. There is a growing interest in the potential use of essential oils as a replacement for traditional herbicides and pesticides. Essential oils are complex natural products of plant origin exhibiting to have insecticidal, attractant, or repellent properties [6][7][8].
There are substantial concerns about human health and environmental problems of synthetic pesticides. Pesticides have potential side effects despite their importance to public health. Exposures to various types of insecticides may be a risk factor for cancers. Several undesirable effects of pesticides are maybe toxicity to non-target organisms, residues of pesticides, depletion of ozone layer, environmental pollution, and direct toxicity to users [2,[9][10][11][12]. Insecticide resistance in agricultural pests and vectors of diseases is also maybe as a substantial problem in management programs of the pests and vectors [7,[13][14][15][16][17][18][19].
One of the most natural aromatic plants in Iran is Nepeta crispa Willd. (Lamiales: Lamiaceae) that is popular in Iranian traditional medicine, especially with Hamadan Province people. N. crispa have insecticidal activity with antimicrobial and antifungal properties. The N. crispa plants are autochthonous of Iran climate, especially Hamadan Province [20][21][22].
Many studies have been conducted on the insecticidal properties of N. crispa plant, but a simultaneous comparative study about the cytotoxicity of essential oil of N. crispa with synthetic pesticides such as propoxur against the cell lines of invertebrates and vertebrates would be a particular of importance. Therefore, in this study, we compared the cytotoxicity of N. crispa essential oil with propoxur against invertebrates and vertebrates cell lines.

Ethical approval
The study was approved by the Ethics Committee of Hamadan  A total of 1000 g powder of shade-dried aerial parts of N. crispa were subjected to hydrodistillation using a Clevenger-type apparatus for 4 h. The essential oil was dehydrated over anhydrous sodium sulfate and transferred into amber-colored vials to store in a refrigerator at 4°C for further work.
To consider the cytotoxicity of essential oil of N. crispa plant and propoxur against invertebrate (Sf9) and vertebrate (L929) cell lines, about 2 × 10 3 cells/100 µl of culture medium were placed into the wells of a 96-well plate of treatment experiments and then appropriate concentrations of N. crispa essential oil and propoxur added to the wells. We allowed the cells to grow for 3-5 days and estimated the number of cells as described. Control cell wells contained only cells with 1µl/mL of DMSO. All treatment and control experiments repeated at least four replicates.

Cell line number estimation
The base method for cell estimation is the Mossman method, which uses 3-(4,5-dimethylthiazol-2-yl)-2,5-difenyltetrazolium bromide (MTT, tetrazolium, compound). MTT is a quantitative coloring for living cells and cell proliferation, and it is a known method for in vitro cytotoxicity which measures the active metabolism of the cells. In this coloring solution, dehydrogenase enzyme reduced the MTT and produced blue formazan.
The wells of 96-well plates containing invertebrate (Sf9) and vertebrate (L929) cell lines were incubated with 10 µ MTT for 3 h at 36°C and 27°C, respectively. After the blue formazan and cells settled out and the supernatant was removed, 100 µ of DMSO was added to any well of 96-well plate, shaked for 15 min and then the absorbance of the solution read at 492 nm using ELISA reader [24].

Statistical analysis
The cytotoxicity trend lines of N. crispa essential oil and propoxur against invertebrate (Sf9) and vertebrate (L929) cell lines were estimated by Microsoft Excel version 2013. The trend lines were drawn by clicking on graph line distribution and selecting "add trendline" option using procedure described in previous studies [17][18][19][20][21][22][23][24][25][26][27][28].The equations and R-squared values of the cytotoxicity trend lines were also calculated by Microsoft Excel. IBM SPSS statistics data editor version 24 was used for any statistical analyses. Wilcoxon signed-rank test was used for comparing cytotoxicity of essential oil of N. crispa plant and propoxur between control and treatments, and cross treatments of N. crispa plant and propoxur. p<0.05 was considered statistically significant.

Cytotoxicity of N. crispa essential oil and propoxur
Tables-1 and 2 show the data of N. crispa essential oil and propoxur cytotoxicity against invertebrate (Sf9) and vertebrate (L929) cell lines, respectively. Figures-1 and 2 show the cytotoxicity of N. crispa essential oil and propoxur against invertebrate (Sf9) and vertebrate (L929) cell lines, respectively. Figure-3 shows the cytotoxicity trend lines of N. crispa essential oil (ng/μL) and propoxur (μg/μL) against invertebrate (Sf9) and vertebrate (L929) cell lines. Table-3 shows the results of descriptive analysis and Wilcoxon signed-rank test between control and treatments, treatments and cross treatments of cytotoxicity of essential oil of N. crispa plant, and propoxur against invertebrate (Sf9) and vertebrate (L929) cell lines.
The cytotoxicity of essential oil of N. crispa plant against invertebrate (Sf9) cell line was strongly increased with an intensive increasing slope in accordance with essential oil concentrations of N. crispa plant from 10 −10 to 10 −3 µg/μL ( Figures-1a and 3), while the cytotoxicity of essential oil of N. crispa plant against vertebrate (L929) cell line was gradually decreased with a moderately increasing slope in accordance with essential oil concentrations of N. crispa plant from 10 −10 to 10 −3 µg/μL ( Figures-1b  and 3). Wilcoxon signed-rank test revealed significant differences between the treatments of 10 −10 and 10 −4 essential oil concentrations of N. crispa plant against vertebrate (L929) cell line with control (p<0.05) ( Table-3 and Figure-1b). Wilcoxon signedrank test did not show a significant difference between the treatment of 10 −3 essential oil concentration of   Figure-1b). Wilcoxon signed-rank test did not show significant differences between the treatments of 10 −10 and 10 −3 essential oil concentrations of N. crispa plant against invertebrate (Sf9) cell line with control (p>0.05) ( Table-2 and Figure-1a),even though there were significant differences at p<0.001 level between the treatments of 10 −7 and 10 −3 essential oil concentrations of N. crispa plant against cell line of Sf9 (Figure-1a).
The cytotoxicity of propoxur against invertebrate (Sf9) and vertebrate (L929) cell lines was gradually increased with a relatively low decreasing slope in accordance with propoxur concentrations from 10 −10 to 10 −3 µg/μL (Figures-2a, b and 3). Wilcoxon signed-rank test did not show significant differences between the treatments of 10 −10 and 10 −3 concentrations of propoxur against invertebrate (Sf9) cell line with control (p>0.05) ( Table-3

Comparing of N. crispa essential oil and propoxur cytotoxicity
The cytotoxicity of essential oil of N. crispa plant against invertebrate (Sf9) cell line was strongly increased with an intensive increasing slope in accordance with essential oil concentrations of N. crispa plant, while the cytotoxicity of essential oil of N. crispa plant against vertebrate (L929) cell line was gradually decreased with a moderately increasing Wilcoxon signed-rank test did not show significant differences between separately treatments and cross treatments of N. crispa plant and propoxur concentrations against invertebrate (Sf9) and vertebrate (L929) cell lines, and invertebrate (Sf9) cell line, respectively (p>0.05), even though there were significant differences between cross treatments (10 −10 -10 −4 ) of N. crispa plant and propoxur concentrations against vertebrate (L929) cell line (p<0.05) ( Table-3).

Discussion
Based on the results of the study, propoxur had negative effects on the viability of both invertebrate (Sf9) and vertebrate (L929) cell lines. These results confirm the reports of the previous studies that concluded insecticides decreased growth of the invertebrate and vertebrate cell lines [29,30]. Compared with the propoxur treatment experiments, the highest rate of cell viability was observed in the control group which did not receive any toxic agent.The viability of cell lines which were exposed to the different concentrations of propoxur was lower than control group and decreased with increasing concentrations of propoxur. The negative effects of propoxur on the viability of both invertebrate (Sf9) and vertebrate (L929) cell lines were also confirmed by Wilcoxon signed-rank test by observing significant differences between the treatments of 10 −5 -10 −3 and 10 −8 -10 −3 concentrations of propoxur with control group against the invertebrate (Sf9) and vertebrate (L929) cell lines, respectively, at p<0.05 or p<0.001 levels ( Table-3).
Unlike propoxur, essential oil of N. crispa plant did not have negative effects on the viability of vertebrate (L929) cell line. Compared with the control Available at www.veterinaryworld.org/Vol.12/November-2019/3.pdf   group, the highest rate of cell viability was observed in the experiment treatments which were treated with essential oil of N. crispa plant. The viability of vertebrate (L929) cell line which was exposed to different essential oil concentrations of N. crispa plant was higher than control group and increased with increasing essential oil concentrations of N. crispa plant. The boosting effects of experiments of N. crispa plant on the viability of vertebrate (L929) cell line were also confirmed by Wilcoxon signed-rank test by observing significant differences between the treatments of 10 −10 and 10 −3 essential oil concentrations of N. crispa plant with propoxur treatments of 10 −10 -10 −3 concentrations and control group against the vertebrate (L929) cell line at p<0.05 or p<0.001 levels. In addition to some previous benefits of the natural products deriving from natural plants [31,32], this boosting effects of essential oil of N. crispa plant on the viability of cell line of vertebrates may be considered as the new benefits of the products deriving from natural plants.
Like propoxur, essential oil of N. crispa plant had negative effects on the viability of invertebrate (Sf9) cell lines with the differences that the products deriving from natural plants possessing of readily biodegradable and environmentally friendly derivatives, hydrolyzing rapidly in nature, and nearly having no destructive effects on environment, humans, or the mammals [1,2]. In addition, the application of essential oil concentrations of N. crispa plant (ng/μL) was 1000 folds lower than concentrations of propoxur (μg/μL). The viability of invertebrate (Sf9) cell lines which were exposed to essential oil concentrations of 10 −9 and 10 −7 -10 −3 N. crispa plant was lower than control group and decreased with increasing essential oil concentrations of N. crispa plant.
The negative effects of essential oil of N. crispa plant on the viability of invertebrate (Sf9) cell lines were also confirmed by Wilcoxon signed-rank test by observing a significant difference between the treatments of 10 −7 and 10 −3 essential oil concentrations of N. crispa plant with control group against the invertebrate (Sf9) cell lines at p<0.001 level. In addition to that, the food chain and ecosystem contamination with pesticides, non-target organism extinction, insecticide resistance of insects, mutation, and pollution of water and soil have been critical problems using pesticides in the past decades. However, with the application of the products deriving from natural plants, there is no concern about their potential side effects such as toxicity to non-target organisms, residues of pesticides, depletion of ozone layer, environmental pollution, and direct toxicity to users. We will not encounter probably some psychotic disorders, neurological diseases and fatigue of muscles or cancers, and face up to severe insecticide effects on performance of vertebrate reproductive system [2,[9][10][11][12]. Some studies  did not encounter a substantial problem of insecticide resistance in arthropods of the vectors of diseases and agricultural pests [7,[13][14][15][16][17][18][19].

Conclusion
Based on the results of the study, propoxur had negative effects on the viability of both invertebrate and vertebrate cell lines. Unlike propoxur, essential oil of N. crispa plant did not have negative effects on the viability of vertebrate cell line. The viability of vertebrate cell line was increased with increasing essential oil concentrations of N. crispa plant. This boosting effect of N. crispa plant on the viability of vertebrate cell line may be considered as the new benefits of the products deriving from natural plants.