The cytotoxicity of malathion and essential oil of Nepeta crispa (lamiales: lamiaceae) against vertebrate and invertebrate cell lines

Introduction Pesticides are used as essential tools to control vector-borne diseases and agricultural pests and maintain quality and quantity crop production. Scientists attempt to use derived plant natural products due to environmental safety and low mammalian toxicity. Therefore, the cytotoxicity of malathion and Nepeta crispa essential oil against vertebrate L929 and invertebrate Sf9 cell lines were investigated. Methods About 2×103 cells were placed into the wells of a 96-well plate experiments. Then appropriate concentrations of malathion and N. crispa essential oil added to the wells. The cells were allowed to grow for 3-5 days and estimated the cell numbers. Control cell wells contained only cells with DMSO. All treatments and controls repeated at least four replicates. Results About 2×103 cells were placed into the wells of a 96-well plate experiments. Then appropriate concentrations of malathion and N. crispa essential oil added to the wells. The cells were allowed to grow for 3-5 days and estimated the cell numbers. Control cell wells contained only cells with DMSO. All treatments and controls repeated at least four replicates. Conclusion Plant essential oil not only had no negative effects but also had boosting effects on the L929 cell viability. Nepeta crispa essential oil had negative effects on the Sf9 cell viability with the differences that derived plant natural products containing environmentally friendly and readily biodegradable derivatives, hydrolyzing rapidly in nature and nearly having no destructive effects on mammals and environment.


Introduction
At present, pesticides with increasingly global marketing are used as an essential tool to control of vector-borne diseases and agricultural pests and maintain quality and quantity crop production. They also play a key role in the prevention and control of infectious diseases such as malaria, dengue, and filariasis [1]. Despite their importance for public health, there is concern about pesticides potential side effects. Exposure to insecticides has severe effects on reproductive performance in vertebrates. It may cause increasing rates of cryptorchidism and hypospadias male genital congenital anomalies in human populations. Various types of insecticide exposure may be a risk factor for cancers such as leukemia or lymphoma. The other undesirable effects of pesticides are may be direct toxicity to users, environmental pollution, ozone depletion, pesticide residues, and toxicity to non-target organisms. There is the link between humans who are occupationally in contact with insecticides and muscle fatigue, neurological diseases and psychotic disorders [2][3][4][5][6]. Insecticide resistance in arthropods of vectors of diseases and agricultural pests to synthetic insecticides has been considered as a substantial problem of the vector and pest management programs [7][8][9][10][11][12][13][14]. With great concern about environmental problems and human health of synthetic pesticides, scientists have attempted to use natural products derived from plants that are considered as an appropriate option for vector and pest management due to containing environmentally friendly and readily biodegradable derivatives [6,15,16]. Essential oils are fugitive oil compounds that are secondary metabolites of plants. Essential oils hydrolyze rapidly in nature and have less destructive effects on the environment because of their environmental safety and low mammalian toxicity [6,17]. Many studies have been done on insecticides effects of essential oils. Botanical insecticide compounds inhibit the activity of enzymes that are required to protect insects from oxidative stress, resistance to insecticides and other damage to insects [16,[18][19][20].

Many Iranian wild flowers have medicinal and insecticidal properties.
Several studies have been conducted on Lamiaceae family due to their toxic effects on various insect species. The family of Lamiaceae have high diversity and distribution in flora of Iran containing 46 genera and 420 species and sub-species [21]. Nepeta is a genus of Lamiaceae family that has been spread in many parts of the world, including Asia, Europe and North Africa. About of 280 annual or perennial worldwide species of Nepeta genus, there are 79 species present in Iran with of 38 species of native to the country [22,23]. In traditional medicine, Nepeta species are widely used as antispasmodic, anti-asthma, sedative, treatment of various digestive, neurological and respiratory diseases [24][25][26]. It's anti-viral, antiinflammatory and antioxidant properties also have been reported [27][28][29]   were drawn by clicking on graph line distribution and selecting "add trendline" option using Nasirian and Salehzadeh (2017a, b; 2019a, b) style [11,[37][38][39][40].

Results
Malathion cytotoxicity: Table 1 Figure 2). Table 2 also shows the results of descriptive analysis and Wilcoxon signedranks test between control and treatments and between treatments of cytotoxicity of malathion and essential oil of N. crispa against L929 and Sf9 cell lines. There was a significant difference between treatments of 10 -5 to 10 -3 malathion concentrations against L929 cell lines with control (P < 0.05) ( Figure 1 A and Table 2). There was also a significant difference between treatments of 10 -6 to 10 -3 malathion concentrations against Sf9 cell lines with control (P < 0.05) (Figure 1 B and Table 2). Although Wilcoxon signed-ranks test did not show a significant difference between treatments of 10 -8 and 10 -7 malathion concentrations against Sf9 cell lines with control (P > 0.05) ( Table 2).
While there was a significant difference at P < 0.001 level between treatments of 10 -8 and 10 -7 malathion concentrations against Sf9 cell lines with control (Figure 1 B).
Nepeta crispa essential oil cytotoxicity: the cytotoxicity of Nepeta crispa essential oil against L929 cell lines were gradually decreased with a moderately increasing slope in accordance with N. crispa essential oil concentrations from 10 -10 to 10 -3 µg-μL (Figure 1 C, Figure 2). While the cytotoxicity of N. crispa essential oil against Sf9 cell lines were strongly increased with an intensive increasing slope in accordance with N. crispa essential oil concentrations from 10 -10 to 10 -3 µg -μL (Figure 1 D, Figure 2).
Wilcoxon signed-ranks test revealed a significant difference between treatments of 10 -10 to 10 -4 N. crispa essential oil concentrations against L929 cell lines with control (P < 0.05) (Figure 1 C and Table 2).
Although Wilcoxon signed-ranks test did not show a significant difference between treatments of 10 -3 N. crispa essential oil concentration against L929 cell lines with control (P = 0.068) ( Table 2). While there was a significant difference at P < 0.001 level between treatments of 10 - cell lines (P < 0.05) ( Table 2). Even though there was a significant difference between treatments of 10 -3 malathion concentration with N. crispa essential oil at P < 0.001 level. Although Wilcoxon signedranks test did not show a significant difference between treatments of 10 -10 to 10 -3 malathion concentrations with N. crispa essential oil against Sf9 cell lines (P > 0.05). While there was a significant difference at P < 0.001 level between treatments of 10 -10 to 10 -7 malathion concentrations with N. crispa essential oil against Sf9 cell lines ( Table 2).

Discussion
In recent years, insect insecticide resistance, ecosystem and food chain pesticide contamination, extinction of non-target organisms, mutation, and soil and water pollution has been critical problems. the experiments of malathion treatments, the highest rate of cell viability was observed in the control group which did not receive any toxic agent. While, the viability of cell lines which were exposed to the different concentrations of malathion was lower than control group and decreased with increasing malathion concentrations (Figure 1 A,B, Figure 2). The negative effects of malathion on the viability of both L929 and Sf9 cell lines were also confirmed by Wilcoxon signed-ranks test by observing a significant difference between treatments of 10 -5 to 10 -3 and 10 -8 to 10 -3 malathion concentrations with control group against the L929 and Sf9 cell lines, respectively at P < 0.05 or P < 0.001 levels (Figure 1 A,B) and Table 2.
Unlike malathion, essential oil of Nepeta crispa did not have negative effects on the viability of L929 cell lines. Compared with the control group, the highest rate of cell viability was observed in the experiment treatments which were treated with N. crispa essential oil. The viability of L929 cell lines which were exposed to different concentrations of N. crispa essential oil was higher than control group and increased with increasing N. crispa essential oil concentrations (Figure 1 C, Figure 2). The boosting effects of N. crispa essential oil on the viability of L929 cell lines were also confirmed by Wilcoxon signed-ranks test by observing a significant difference between treatments of 10 -10 to 10 -3 N. crispa essential oil concentrations with treatments of malathion 10 -10 to 10 -3 concentrations and control group against the L929 cell lines at P < 0.05 or P < 0.001 levels (Figure 1 C) and  [6,15]. In addition, the application of N. crispa essential oil concentrations (ng -μL ) was 1,000 folds lower than malathion concentrations (μg -μL ). The viability of Sf9 cell lines which were exposed to concentrations of 10 -9 and 10 -7 to 10 -3 N. crispa essential oil was lower than control group and decreased with increasing N. crispa essential oil concentrations (Figure 1 D, Figure 2). The negative effects of N. crispa essential oil on the viability of Sf9 cell lines were also confirmed by Wilcoxon signed-ranks test by observing a significant difference between treatments of 10 -7 to 10 -3 N. crispa essential oil concentrations with control group against the Sf9 cell lines at P < 0.001 level (Figure 1 D). But with application of the natural products deriving from plants, there is no concern about their potential side effects like direct toxicity to users, environmental pollution, ozone depletion, pesticide residues and toxicity to non-target organisms. We will not face up to severe insecticide effects on vertebrate reproductive performance, and encounter probably some type of cancers or muscle fatigue, neurological diseases and psychotic disorders [2][3][4][5][6]. Maybe no longer encounter to a substantial problem of the pest and vector management programs due to insecticide resistance in arthropods of the agricultural pests and vectors of diseases [7][8][9][10][11][12][13][14].

Conclusion
Pesticides are used as an essential tool to vector-borne diseases and agricultural pests, and maintain quality and quantity crop production.
With great concern about environmental problems and human health On the other hand there is no concern about plant essential oil potential side effects like direct toxicity to users, environmental pollution, ozone depletion, pesticide residues, and toxicity to nontarget organisms with application of the natural products deriving from plants. We will not also encounter probably some type of cancers or muscle fatigue, neurological diseases, and psychotic disorders by applying the derived natural products of plants. As well as maybe no longer encounter to a substantial problem of the vector and pest management programs due to insecticide resistance in arthropods of the vectors of diseases and agricultural pests.

What is known about this topic
• Scientists attempt to use derived plant natural products due to environmental safety and low mammalian toxicity; • Nepeta crispa (lamiales: lamiaceae) is one of the most aromatic plants in Iran which is popular in Iranian traditional medicine.

What this study adds
• Plant essential oil not only had no negative effects but also had boosting effects on the L929 cell viability.
• Nepeta crispa essential oil had negative effects on the Sf9 cell viability with the differences that derived plant natural products containing environmentally friendly and readily biodegradable derivatives, hydrolyzing rapidly in nature and nearly having no destructive effects on mammals and environment.

Acknowledgments
We are thankful to Moshkfam Fars Company, Shiraz, Iran, for supplying malathion insecticide. We also acknowledge the help of