|Year : 2011 | Volume
| Issue : 1 | Page : 74-79
Evaluation of carbamate insecticides as chemotherapeutic agents for cancer
Mohd. Amanullah1, Babu Y Hari2
1 Department of Biochemistry, College of Medicine, King Khalid University, Abha, Saudi Arabia
2 Department of Microbiology, Veterinary College, Nandinagar, Bidar, Karnataka, India
|Date of Web Publication||10-Feb-2011|
Department of Biochemistry, College of Medicine, King Khalid University, Abha
Background: Cancer chemotherapy has already been in practice by the use of toxins and some of the specific poisonous compounds of cyanide derivatives. Carbamate insecticides inhibit cellular metabolism including energy, protein, and nucleic acid metabolism, thereby, causing cell regression and death. Aim: Preliminary evaluation of three carbamate insecticides, namely, baygon, carbaryl, and carbofuran as chemotherapeutic agents for cancer is undertaken in the present study. Materials and Methods: The toxicity of carbamates on squamous cell carcinoma was assessed in-vitro using dye binding tests. Cells were grown in microtitration ELISA plates, as adherent cultures, for six hours, and then exposed to the drugs for 2, 4, 8, and 12 hours, and finally stained with neutral red, to assess the viable cell number, and with methylene blue for the determination of protein in the monolayer. Optical density was read in an ELISA reader. Statistical Analysis: The data obtained during the experiment was subjected to statistical analysis by using the student 't' test. Results: The results indicated that the percentage of the viable cell number reduced with an increase in the time of exposure of the drugs. Exposure of the tumor cells to the drugs for 12 hours detached them completely from the wells, and hence, all the cells were washed out. Exposure of the drugs prior to the establishment of the culture in-vitro resulted in the non-formation of the monolayer in the wells. Conclusions: Among the three drugs studied, the survival percent was least with carbaryl treatment followed by baygon, and with carbofuran treatment it was almost near to control group.
Keywords: Cancer, chemotherapy, carbamate, insecticides, baygon, carbaryl, carbofuran
|How to cite this article:|
M, Hari BY. Evaluation of carbamate insecticides as chemotherapeutic agents for cancer. Indian J Cancer 2011;48:74-9
| » Introduction|| |
Preliminary evaluation of the three carbamate insecticides, namely, baygon, carbaryl, and carbofuran, as chemotherapeutic agents for cancer, was undertaken in the present study. Carbamate insecticides are a group of compounds closely related to Organophosphate insecticides in chemical structure, mode of action, and many other properties. These compounds inhibit the enzyme acetylcholine esterase (AChE) in the neuromuscular junction and cause the death of the organism by neuromuscular paralysis.
The inhibition by carbamates can be selectively reversed by an appropriate dose of atropine. The hydroxyl (OH-) groups of the amino acid serine in the enzyme acetylcholine esterase are selectively inhibited by the formation of a covalent bond. The toxic effects observed in other tissues where the enzyme acetylcholine esterase is not prominent, is due to the inhibition of other enzymes containing serine moieties at the vital sites of the enzymes. This enzyme inhibition results in the attenuation of enzyme activity or total inactivation of the enzyme. Most of the enzymes of the energy metabolism (glycolysis), protein synthesis, and nucleic acid metabolism contain serine moieties at their active site or other sites, which are responsible for the enzyme action. Inhibition of these enzymes by carbamates results in energy deprivation to the cell, alteration in membrane permeability, depleted protein, and nucleic acid concentration, thereby causing cell stasis or death. Similarly, these carbamates inhibit the enzymes of cancer cells causing suspension in the growth of the tumor or tumor regression due to cell death.
Considering the action of carbamate insecticides on cell metabolism, this preliminary study was undertaken to explore the possibility of using three of the representative cabamates, namely, baygon, carbaryl, and carbofuran, in the treatment of cancer. The effects of these insecticides on normal cells namely hepatocytes, spleenocytes, and lymphocytes have been investigated and published elsewhere by the authors. , Cancer chemotherapy has already been in practice by use of toxins (Coley's toxins, dimethyl sulfoxide, hydrazine sulfate, podophyllotoxin) and some of the specific poisonous compounds of cyanide derivatives such as phenethyl isothiocyanate (PEITC) in the prevention of lung cancer.  Inhibitors of acetylcholine esterase (carbamates) are also in use for the treatment of other diseases, ,, and their safety index is indicated. ,,
| » Materials and Methods|| |
Evaluation of carbamate insecticides as cancer chemotherapeutic agents was conducted by the collection of tumor mass (squamous cell carcinoma) from an infected animal, histopathological examination of the tissue for confirmation of its necrotic nature, trypsinization and isolation of the cells from the tumor tissue, culturing of the cells as a monolayer in microtitration ELISA plates with 96 wells, exposure of the cell monolayer to the drugs, and finally subjecting the monolayer cells to dye binding tests for measurement of viable cell number and proteins in the monolayer, using an ELISA reader. Similar studies on other cell lines, namely, Myeloma cell lines and horn cancer cells have already been done and published by the first author. 
Technical grade carbamate insecticides were obtained from Sigma-Aldrich Chemical Company, USA. Baygon (Propoxur) - Fluka and Riedel-de Haėn Catalog No. 45644; Carbaryl - Fluka and Riedel-de Haėn Catalog No. 45367 and Carbofuran - Fluka and Riedel-de Haėn Catalog No 45370. All these three were in crystalline form, in a 250 mg pack. All these were preliminary soluble in organic solvents. However, in order to avoid the effect of organic solvents on the cells in the culture, the solubility of these chemicals in water as reported by Kuhr and Dorough,  was used as an index for the preparation of different concentration gradients of the insecticides. Moreover, carbamates solubilized in organic solvents would never mix up with the hydrophilic culture media to reach the cell monolayer.
Baygon (97% purity) solubilizes in water at 20 o C making a 0.2% w/v solution, that is, 2000 ppm. Carbaryl (95% purity) solubilizes at 30 o C to 120 mg/liter, which forms a solution of 1200 ppm. Carbofuran (99%) solubilizes in water at 25 o C making a 700 ppm solution. Therefore, a full 250 mg of each of the three carbamates was poured into three different flasks containing one liter of deionized glass distilled water and kept at the specified temperature of each carbamate for one hour with constant stirring. Then, the excess and undissolved carbamate was allowed to settle down overnight and the upper 750 ml of liquid was collected into an amber colored bottle. Thus, a solution of 2000 ppm of baygon, 1200 ppm of carbaryl, and 700 ppm of carbofuran in water was prepared and then diluted appropriately in the culture media to make the final concentration of each of the carbamates to 400 ppm, for use in the present study.
The Minimal Essential Medium (MEM) supplemented with 10% fetal calf serum, was used as the culture media. The media was sterilized by filtering through a Seitz filter (stainless steel filter presses and non-asbestos filter sheets with nominal retention rates of 0.2 - 0.45 microns). In order to prevent further microbial growth a mixture of penicillin - G and streptomycin sulfate were added to the media, to make the final concentration of these antibiotics to 100 units/ml and 100 μg/ml, respectively. Geneticin at 50 μg/ml was also added to the media, to prevent the formation of excessive fibroblasts in the cell monolayer.
Squamous cell carcinoma [Figure 1] from the neck region of a non-descript bull, aged about one year, was collected during surgical excision at the Veterinary clinical complex.
In order to ascertain the nature of the tissue collected (neoplasm or otherwise), hisptopathology was carried out as per the method described by Peguet-Navarro.  In brief, the tissue was cut to the size of a 1 cm cube and fixed in 10% formalin for 24 hours. The tissue was then dehydrated by placing it in ascending concentrations of alcohol (50, 70, 80, 90, and 98% and absolute alcohol) for two hours in each dilution. Later the tissue was cleaned in xylene by placing it in 1 : 1 solution of xylene : alcohol for 30 minutes, and then three changes of pure xylene were given for an hour each. Paraffin infiltration of the tissue was done by incubating the xylene cleaned tissue in 1 : 1 solution of xylene : liquid paraffin (medium paraffin - melting point 60 o C) at 60 o C for two hours. The tissue was then given three changes of pure paraffin at 60 o C for two hours each. Finally the tissue was embedded, using metallic molds, in fresh paraffin, preheated to 60 o C. Thin sections of the tissue in the paraffin blocks were made using a microtome. The section was mounted on to a slide using egg white-glycerine solution and finally stained with Hematoxylin and Eosin [Figure 2] and [Figure 3].
|Figure 2 :Histopathology of squamous cell carcinoma - showing the features of squamous cell carcinoma with malignant epithelial cells showing open type of nucleus with basophilic stained marked chromatin and epithelial outgrowth with epithelial pearls - H and E × 25|
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|Figure 3 :Histopathology of squamous cell carcinoma - the features of squamous cell carcinoma with malignant epithelial cells showing open type of nucleus with basophilic stained marked chromatin and epithelial outgrowth with epithelial pearls - H and E × 100|
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Isolation and culturing of the tumor cells was done as per the procedure described by Schmied,  with some modifications. The tumor mass was perfused thoroughly with Hank's buffer twice or thrice, to wash the contaminated blood. Two to five milliliters of trypsin (0.25%) were injected into the tumor mass and kept aside for 30 - 40 minutes at room temperature. Digestion was stopped by washing the material twice in ice-cold Hanks buffer. It was then cut into small pieces and minced in a Teflon tissue homogenizer to make a granular mixture. This mixture was then suspended in Hanks medium and filtered through a 40 μm mesh. Cell viability was determined by trypan blue dye exclusion. The cell concentration was adjusted to 4 x 10 5 cells/ml in the culture media, drugs were exposed for varying time intervals, and the effect of the drug was determined as follows:
(I) Viable cell number of the squamous cell carcinoma cells were made as per the protocol described by Fiennes. 
- Microtitration ELISA plates (five numbers) with 96 wells were taken and 200 μl of cells in the growth medium (containing 4 x 10 5 cells/ml) were added to each of the wells.
- Four out of five plates were covered with the plastic lid and incubated at 37 o C for six hours.
- The medium was removed and replaced with 200 μl of fresh medium containing carbamates at a final concentration of 400 ppm.
- Each of the plates was incubated at 37 o C for 2, 4, 8, and 12 hours, respectively, after inoculation of the carbamates.
- The fifth plate was used to inoculate the carbamates at the initiation of the experiment (0 hour observation), that is, without allowing the cells to accustom to the in-vitro (artificial) atmosphere.
- The medium was then removed and washed with 200 μl of PBS, thrice, to remove the drugs.
- Neutral red of 200 μl was added to each well of the plates and incubated for 90 min at 37 o C.
- The dye was removed and the cells were washed with two changes of warm PBS.
- The dye was extracted by adding 100 μl of absolute ethanol: 0.1 M citrate buffer, pH 4.2 (21.01 gm citric acid + 200 ml of 1 M NaoH per liter (A); 60 ml A + 40 ml 0.1 M HCl), mixed 1:1 v/v.
- This was agitated for 20 minutes at room temperature and (optical density) OD read at 540 nm in a standard ELISA plate reader.
OD of test / OD of standard x No. of cells taken in control
(II) Estimation of protein content of the squamous cell carcinoma cell monolayer was performed as per the procedure described by Pelletier. 
- Microtitration ELISA plates (five in number) with 96 wells were taken and 200 μl of cells in growth medium (containing 4 x 10 5 cells/ml) were added to each of the wells.
- Four out of five plates were covered with the plastic lid and incubated at 37°C for six hours.
- The medium was removed and replaced with 200 μl of fresh medium containing carbamates at the final concentration of 400 ppm.
- Each of the plates was incubated at 37°C for 2, 4, 8, and 12 hours, respectively, after inoculation of the carbamates.
- The fifth plate was used to inoculate the carbamates at the initiation of the experiment (0 hour observation), that is, without allowing the cells to get accustomed to the in-vitro (artificial) atmosphere.
- The medium was then removed and washed with 200 μl of PBS, thrice, to remove the drugs.
- Formalin in PBS, 200 μl of 10% v/v, was added, and the cells were fixed at room temperature for 10 minutes.
- Formalin was then removed and the cells were washed twice with 200 μl of borate buffer (0.01 M, pH 8.4, 3.8 g/liter in distilled water).
- One hundred microliters of methylene blue per well (1% w/v in borate buffer) were added and stained for 10 minutes, at room temperature.
- Mehtylene blue was removed and washed extensively with borate buffer, blotting the plate surface between washes. A minimum of five washes were made to remove the excess dye.
- The washed plates were left to dry at room temperature for 2 - 3 hours.
- Finally 200 μl of 0.1 M HCl was added to each well and the plate was kept on an automatic plate shaker for 10 minutes at room temperature, in order to solubilize the stain.
- OD was read in an ELISA reader at 660 nm.
Calculation:- Percent variation in protein content as compared to control = OD of test / OD of standard × 100
Results are expressed as means ± standard deviation (S.D.) or standard error mean (S.E.M). The data obtained during the experiment was subjected to statistical analysis by using the student 't' test. 
| » Results|| |
The detailed results are presented in [Table 1] and [Figure 4] and [Figure 5]. The percentage of the viable cell number reduced with an increase in the time of exposure of the drugs. Exposure of the tumor cells to the drugs for 12 hours detached them completely from the wells, and hence, all the cells were washed out. Among the three drugs studied, the survival percent was the least with carbaryl treatment followed by baygon and the minimum were affected by carbofuran treatment.
|Table 1 :Effect of carbamate insecticides (400 ppm) on viable cell number and protein content (%) of squamous cell carcinoma cells cultured as adherent cultures in micro ELISA plates |
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|Figure 4 :Percent viable cell number at different time intervals due to the effect of different carbamates|
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|Figure 5 :Protein concentration (%) at different time intervals due to the effect of different carbamates|
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The protein content of the cell monolayer also followed the same trend, wherein, the least protein content was observed in the carbaryl treatment followed by baygon and carbofuran treatment, which had the least effect on the production of proteins in the cell monolayer. The results were statistically significant at P ≤ 0.01. When carbamates were inoculated in the media at the beginning (0 hour) of the culture, there was no formation of a monolayer even after 12 hours.
| » Discussion|| |
The purpose of selecting carbamate insecticides over organophosphates in the present study as agents for the treatment of cancer was that the inhibition of cholinesterase by carbamates is reversible. This inhibition can be selectively reversed by an appropriate dose of atropine. Hence, these can be used as therapeutic agents by administering atropine before treatment with carbamates. Therefore, the chemicals when used in chemotherapy of cancer shall be safe and less toxic as compared to organophosphates, because organophosphates have been seen to be mutagenic, teratogenic, and carcinogenic with varying effects on immunity and reproduction, and cause permanent lameness. However, carbamates are toxic to nerve and muscle tissues, but have a shorter duration of action and are reversible.  These compounds are rapidly detoxified and excreted, so their risk to warm-blooded animals is less than the other agents. 
Squamous cell carcinoma was trypsinized and grown as an adherent culture on micro ELISA plates. Both viable cell count and proteins in the monolayer were estimated after exposure to the drugs. Measurement of proteins in the monolayer was included as an indirect measure of cell count because during the procedure for viable cell count the cells were likely to be washed out along with the dye washings. Hence, the cells were fixed and then the proteins in the cells stained, minimizing cell loss during repeated washings.
The inhibition in the growth and multiplication of tumor cells noted in the present study clearly indicate that carbamate insecticides are potent inhibitors of cellular metabolism (both energy production and protein anabolism). However, the cell population did not cease completely, owing to their high survivability rates under stress. A preliminary evaluation of the drugs has been presented in this article. Other detailed results for evaluation of carbamate insecticides, as cancer chemotherapeutic agents, such as, viable cell counts, using trypan blue dye exclusion test, measurement of lactate dehydrogenase (LDH) leakage as an index of cell damage, effect of lactic acid, and protein production by the cells upon exposure to the drugs, as a manifestation of the cell metabolism, are published elsewhere by the authors. ,, Accordingly, it was found that carbamates bring about a considerable degree of inhibition in tumor cell growth and cell death / destruction / deformities. Hence, it can be concluded that carbamates can be good chemotherapeutic agents for cancer.
The efficacy and safety of carbamates ,, in the treatment of cancer in humans has to be worked out in detail. However, in animals, these drugs can be directly applied for the treatment, especially for peripheral tumors like horn cancer, squamous cell carcinoma, canine venereal transmissible tumors (CTVT), and mammary gland tumors. Carbamates can be conveniently used in large animals for cancer chemotherapy, because, about 250 mg/kg body weight of carbamates injected intravenously to cattle is non toxic.  This means that 250 g of carbamate can be safely injected at the site of the peripheral tumor in cattle and if atropine sulfate is pre-injected the dose can be further enhanced, thereby giving a good drug for treatment of peripheral cancers in cattle; also effective drugs for peripheral tumors are not available presently, for veterinary use. Similarly, it has been reported that in dogs, large doses of carbamates are non toxic, hence, they can be tried for treatment of canine venereal transmissible tumors (CTVT). If these drugs are found to be fruitful in the treatment of peripheral tumors then they can be tried on interior tumors by the use of 'smart-bombs' developed by the team of scientists  at the School of Chemistry, Tel Aviv University (TAU), Israel, for the delivery of drugs specifically to the tumor tissue.
Carbamate insecticides, not only inhibit the growth of tumor cells, but they also cause tumor cell destruction and death. Therefore, this study opens a new option for testing the feasibility of using carbamates, and carbaryl in particular, for the treatment of cancer. It seems, from the review of literature that no such attempt has been made to use carbamate insecticides in the treatment of cancer. While treating cancer with carbamates, the toxic effects on other cell types can be overcome by pre-injecting the antidote, atropine sulfate, as shown by Kimmarle  in his experiments, wherein he used 50 mg/kg atropine sulfate prior to the intraperitoneal dose of 100 mg/kg propoxur, without any neurotoxic signs. However, these chemicals are still not usable in treating human cancer. Furthermore, research and clinical trials on animals and then human beings is to be conducted before recommending them for treating human cancer.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]