Molecular approach to evaluate the genotoxicity of glyphosate (roundup) using mosquito genome
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Abstract
Glyphosate, an active ingredient in Roundup is a broad spectrum, systemic and non -selective herbicide which is commonly used for eliminating weeds in agriculture and forest landscapes. The present studies deal with the evaluation of the genotoxic potential of Glyphosate with two different dose concentration of LD20 and LD40 on a mosquito Culex quinquefasciatus taken as an experimental model. For this, polymerase chain reaction technique (PCR) was used for detecting DNA damage by amplifying ribosomal DNA internal transcribed spacer 2 (ITS 2) region. The amplified products were sequenced and the results of treated and non-treated controls were compared by using Clustal W software programme. The results were studied in the form of transitions, transversions, deletions and additions of bases. The DNA band amplified from control stocks consisted of 440 bases while those
from LD20 and LD40 treated individuals were comprised of 423 and 468 bases respectively. The total number of mutations caused in LD20 treated stock was 205 out of which 68 were transitions, 90 transversions, 32 deletions and 15 additions. In case of LD40 treated individuals, as many as 221 bases had suffered mutations, out of which 66 were transitions, 90 transversions , 12 deletions and 41 additions. In both the cases the rate of transversions was higher than transitions. From these results it was evident that glyphosate has a potential to promote gene mutations in the individuals exposed to its semilethal doses.
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Keywords
Glyphosate, PCR, ITS 2, Culex quinquefasciatus
References
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Benachour, N., Sipahutar, H., Moslemi, S., Gasnier, C., Travert, C. and Seralini, G.E. (2007). Time and dose-dependent effects of Roundup on human embryonic and p l a c e n t a l cells and aromatase inhibition. Arch. Environ. Contam. Toxicol. 53: 126- 133.
Bolognesi, C., Bonatti, S., Degan, P., Callerani, E., Peluso, M., Rabboni, R., Roggeri, P. and Abbondandolo, A. (1997) Genotoxic activity of glyphosate and its technical formulation Roundup. J. Agric. Food Chem., 45:1957–1962.
Chaudhry, A. and Anand, P. K. (2004). Assessment of dominant- lethal effects of chlorpyrifos (CPF) using mosquito genetics. Pollution Research 23 (4): 767-771.
Chaudhry, A. and Anand, P. K. (2005). Evaluation of mutagenic potential of chlorpyrifos (CPF) using polytene chromosomes of Anopheles mosquitos. J. Env. Biology, 26 (1): 145-150.
Chaudhry, S., Neetu Dhanda, R. S. and Saluja, D. (2004). Random amplified polymorphic DNA polymerase chain reaction based differentiation of some species of the genus Anopheles (Culicidae: Diptera) J. Cytol. Genet. 5 (NS): 173- 183.
Chaudhry, S. and Sharma, M. (2006). RAPD- PCR based genomic characterization of three populations of Anopheles (Cellia) stephensi (Culicidae: Diptera) J. Cytol. Genet.7: 121-123.
Clements, A. N. (1994) The physiology of mosquito. New York, McMillan and Co.
Cox C. (1998). Glyphosate (Roundup). J. Pest Reform 18: 3-17.
Daruich, J., Zirulnik, F. and Gimenz, M. S. (2001). Effect of the herbicide glyphosate on enzymatic activity in pregnant rats and their fetuses. Environ. Res. 85: 226-231.
El Demerdash., F. M., Yousef, M. I. and Elagamy, E. I. (2001). Influence of paraquat glyphosate and cadmium on the activity of some serum enzymes and protein electrophoretic bahaviour ( in vitro ). J. Environ. Sci. Health B 36:29-42.
Evans, H. S. (1977). Molecular mechanism in the induction of chromosome aberrations In: D Scott BA Bridges and FH Sobels (Eds), Progress in Genetic Toxicology. Elseviser/ North Holland Biomedical Press, Amsterdam.
Finney, D. J. (1971). Probit Analysis. Cambridge Univ Press, Cambridge.
Gaulden, M. E. and Liang, J. C. (1982). Insect cells for testing clastogenic agents In: T C Hsu (Ed), Cytogenetic Assays of Environmental Mutagens. Allanheld Osmum, 107-135.
Gillet, J. W. (1970). The biological impact of pesticides in environment. Environment Health Sciences series. No. 1 Oregon State University, Corvallis.
Grisolia, C. K. (2002). A comparison between mouse and fish micronucleus test using cyclophosphamide, mitomycin C and various pesticides. Mutat. Res. 518: 145-150.
Jones, C. and Kortenkamp, A. (2000). RAPD library fingerprinting of bacterial and human DNA: application in mutation detection, teratogen, carcinogen. Mutagen. 20: 49-63.
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Lioi, M. B., Scarfi, M. R., Santoro, A., Barbieri, R., Zeni., O. and Salvemini, F. (1998). Cytogenetic damage and induction of pro- oxidant state in human lymphocytes exposed in vitro to glyphosate, vinclozolin, atrazine and DPX- E9636 Environ. Mol. Mutagen 32: 39-46.
Malik, J., Barry, G. and Kishore, G. (1989). The herbicide glyphosate. Biofactors, 2: 17-25.
McKelvey, U. J., Green, M. H., Schmezer, P., Pool- Zobet, B. L., De Meo, M. P. and Collins, A. (1993). The single cell gel electrophoresis assay (comet assay): A European review Mutat. Res., 288: 47- 63.
Menzer, R. E. (1987). Selection of animal models for data interpretation. In: Robert G. T.and Rodricks J. V. (eds). Toxic Substances and Human Risk Principles of Data Interpretation. National Institute of Health. Bethesda M. D. 1979 5-81.
OCDE (1998). Mammalian erythrocyte micronucleus test. No. 474. In: Addenum to OCDE Guidelines for Testing Chemicals. OCDE, France.
Pandrangi, R., Petras, M., Ralph, S. and Vrzoc. M. (1996). Alkaline single cell gel (comet assay) and genotoxicity monitoring using bullheads and carp. Environ. Mol. Mutagen 26: 345- 356.
Peluso, M., Munnia, A., Bolognesi, C. and Parodi, S. (1998). 32 P- Post labeling detection of DNA adducts in mice treated with the herbicide roundup Environ. Mol. Mutagen 31: 55-59.
Phillips, D. H. (1997). Detection of DNA modifications by the super 32 P- post labeling assay. Mutat. Res. 378: 550-554.
Rank, J., Jensen, A. G., Skov, B., Pedersen, L. H. and Jensen, K. (1993). Genotoxicity testing of the herbicide Roundup and its active ingredient glyphosate isopropylamine using the mouse bone marrow micronucles test Salmonella mutagenicity test and Allium anaphase- telophase test. Mutat. Res. 300: 29-36.
Richard, S., Moslemi, S., Sipahutar, H., Benachour, N. and Seralini, G.E. (2005). Differential effects of glyphosate and Roundup on human placental cells and aromatase. Environ. Health Perspect. 113: 716- 720.
Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular cloning: A laboratory manual (2nd ed). Cold Spring Harbor, New York.
Singh, K. R. P., Patterson, R. S., La Breque, G. C. and Razdan, R. K. (1975). Mass rearing of Cules pipiens fatigans Weid. J. Com. Dis. 7: 31-53.
Sobels F. H. (1974). The advantages of Drosophila for mutation studies. Mutat. Res. 26: 277-284.
Steinrucken, H. C. and Amrhein, N. (1980). The herbicide glyphosate is a potent inhibitor of 5- enolpyuvyl-shikimic acid-3- phosphate synthetase. Biochem. Biophys. Res. Commun. 94: 1207-1212.
Takahashi, M., Horie, M. and Aoba, N. (2001). Analysis of glyphosate and its metabolite minomethylphosphonic acid in agriculture products by HPLC. Shokuhin iseigaku Zasshi 42: 304-308.
Walsh, L. P., Mc Cormick, C., Martin, C. and Stocco, D . M. (2000). Roundup inhibits steroidogenesis by disrupting steroidogenic acute regulatory (StAR) protein expression Environ. Health Perspect 108: 769-776.
Williams, G. M., Kroes, R. and Munro, I. C. (2000). Safety evaluation and risk assessment of the herbicide Roundup and its active ingredient glyphosate for humans Regul Toxicol Pharmacol 31: 117-165.
Williams, J.G.K., Kubeklik, A.R., Livak, K.J., Rafalski, J.A. and Tingey, S.V. (1990). DNA polymorphisms amplified by arbitrary primers are useful genetic markers; Nucleic acids Research, 18: 6531-6535.
Zaman, K., Macgill, R. S., Ahmad, S. and Pardini, R. S. (1994). An insect model for assessing mercury toxicity: Effects of mercury on antioxidant enzyme activities of the house fly Musca domestica and cabbage looper moth Trichoplusiani. Arch. Environ. Contam. Toxicol . 26(1): 114- 118.
Ausubel, F.M., Breut, R., Kingston, R.E., Moore, D.D., Sideman, J.G., Smith, J.A. and Struhl, K. (1999). Short protocols in molecular biology, John-Wiley and Sons Inc. N.Y., 4: 15.1-15.37.
Benachour, N., Sipahutar, H., Moslemi, S., Gasnier, C., Travert, C. and Seralini, G.E. (2007). Time and dose-dependent effects of Roundup on human embryonic and p l a c e n t a l cells and aromatase inhibition. Arch. Environ. Contam. Toxicol. 53: 126- 133.
Bolognesi, C., Bonatti, S., Degan, P., Callerani, E., Peluso, M., Rabboni, R., Roggeri, P. and Abbondandolo, A. (1997) Genotoxic activity of glyphosate and its technical formulation Roundup. J. Agric. Food Chem., 45:1957–1962.
Chaudhry, A. and Anand, P. K. (2004). Assessment of dominant- lethal effects of chlorpyrifos (CPF) using mosquito genetics. Pollution Research 23 (4): 767-771.
Chaudhry, A. and Anand, P. K. (2005). Evaluation of mutagenic potential of chlorpyrifos (CPF) using polytene chromosomes of Anopheles mosquitos. J. Env. Biology, 26 (1): 145-150.
Chaudhry, S., Neetu Dhanda, R. S. and Saluja, D. (2004). Random amplified polymorphic DNA polymerase chain reaction based differentiation of some species of the genus Anopheles (Culicidae: Diptera) J. Cytol. Genet. 5 (NS): 173- 183.
Chaudhry, S. and Sharma, M. (2006). RAPD- PCR based genomic characterization of three populations of Anopheles (Cellia) stephensi (Culicidae: Diptera) J. Cytol. Genet.7: 121-123.
Clements, A. N. (1994) The physiology of mosquito. New York, McMillan and Co.
Cox C. (1998). Glyphosate (Roundup). J. Pest Reform 18: 3-17.
Daruich, J., Zirulnik, F. and Gimenz, M. S. (2001). Effect of the herbicide glyphosate on enzymatic activity in pregnant rats and their fetuses. Environ. Res. 85: 226-231.
El Demerdash., F. M., Yousef, M. I. and Elagamy, E. I. (2001). Influence of paraquat glyphosate and cadmium on the activity of some serum enzymes and protein electrophoretic bahaviour ( in vitro ). J. Environ. Sci. Health B 36:29-42.
Evans, H. S. (1977). Molecular mechanism in the induction of chromosome aberrations In: D Scott BA Bridges and FH Sobels (Eds), Progress in Genetic Toxicology. Elseviser/ North Holland Biomedical Press, Amsterdam.
Finney, D. J. (1971). Probit Analysis. Cambridge Univ Press, Cambridge.
Gaulden, M. E. and Liang, J. C. (1982). Insect cells for testing clastogenic agents In: T C Hsu (Ed), Cytogenetic Assays of Environmental Mutagens. Allanheld Osmum, 107-135.
Gillet, J. W. (1970). The biological impact of pesticides in environment. Environment Health Sciences series. No. 1 Oregon State University, Corvallis.
Grisolia, C. K. (2002). A comparison between mouse and fish micronucleus test using cyclophosphamide, mitomycin C and various pesticides. Mutat. Res. 518: 145-150.
Jones, C. and Kortenkamp, A. (2000). RAPD library fingerprinting of bacterial and human DNA: application in mutation detection, teratogen, carcinogen. Mutagen. 20: 49-63.
Krishnan, K. S. (1964). A note on colonization of Culex. Bull. Wld. Hlth. Org. 31: 455- 456.
Lioi, M. B., Scarfi, M. R., Santoro, A., Barbieri, R., Zeni., O. and Salvemini, F. (1998). Cytogenetic damage and induction of pro- oxidant state in human lymphocytes exposed in vitro to glyphosate, vinclozolin, atrazine and DPX- E9636 Environ. Mol. Mutagen 32: 39-46.
Malik, J., Barry, G. and Kishore, G. (1989). The herbicide glyphosate. Biofactors, 2: 17-25.
McKelvey, U. J., Green, M. H., Schmezer, P., Pool- Zobet, B. L., De Meo, M. P. and Collins, A. (1993). The single cell gel electrophoresis assay (comet assay): A European review Mutat. Res., 288: 47- 63.
Menzer, R. E. (1987). Selection of animal models for data interpretation. In: Robert G. T.and Rodricks J. V. (eds). Toxic Substances and Human Risk Principles of Data Interpretation. National Institute of Health. Bethesda M. D. 1979 5-81.
OCDE (1998). Mammalian erythrocyte micronucleus test. No. 474. In: Addenum to OCDE Guidelines for Testing Chemicals. OCDE, France.
Pandrangi, R., Petras, M., Ralph, S. and Vrzoc. M. (1996). Alkaline single cell gel (comet assay) and genotoxicity monitoring using bullheads and carp. Environ. Mol. Mutagen 26: 345- 356.
Peluso, M., Munnia, A., Bolognesi, C. and Parodi, S. (1998). 32 P- Post labeling detection of DNA adducts in mice treated with the herbicide roundup Environ. Mol. Mutagen 31: 55-59.
Phillips, D. H. (1997). Detection of DNA modifications by the super 32 P- post labeling assay. Mutat. Res. 378: 550-554.
Rank, J., Jensen, A. G., Skov, B., Pedersen, L. H. and Jensen, K. (1993). Genotoxicity testing of the herbicide Roundup and its active ingredient glyphosate isopropylamine using the mouse bone marrow micronucles test Salmonella mutagenicity test and Allium anaphase- telophase test. Mutat. Res. 300: 29-36.
Richard, S., Moslemi, S., Sipahutar, H., Benachour, N. and Seralini, G.E. (2005). Differential effects of glyphosate and Roundup on human placental cells and aromatase. Environ. Health Perspect. 113: 716- 720.
Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular cloning: A laboratory manual (2nd ed). Cold Spring Harbor, New York.
Singh, K. R. P., Patterson, R. S., La Breque, G. C. and Razdan, R. K. (1975). Mass rearing of Cules pipiens fatigans Weid. J. Com. Dis. 7: 31-53.
Sobels F. H. (1974). The advantages of Drosophila for mutation studies. Mutat. Res. 26: 277-284.
Steinrucken, H. C. and Amrhein, N. (1980). The herbicide glyphosate is a potent inhibitor of 5- enolpyuvyl-shikimic acid-3- phosphate synthetase. Biochem. Biophys. Res. Commun. 94: 1207-1212.
Takahashi, M., Horie, M. and Aoba, N. (2001). Analysis of glyphosate and its metabolite minomethylphosphonic acid in agriculture products by HPLC. Shokuhin iseigaku Zasshi 42: 304-308.
Walsh, L. P., Mc Cormick, C., Martin, C. and Stocco, D . M. (2000). Roundup inhibits steroidogenesis by disrupting steroidogenic acute regulatory (StAR) protein expression Environ. Health Perspect 108: 769-776.
Williams, G. M., Kroes, R. and Munro, I. C. (2000). Safety evaluation and risk assessment of the herbicide Roundup and its active ingredient glyphosate for humans Regul Toxicol Pharmacol 31: 117-165.
Williams, J.G.K., Kubeklik, A.R., Livak, K.J., Rafalski, J.A. and Tingey, S.V. (1990). DNA polymorphisms amplified by arbitrary primers are useful genetic markers; Nucleic acids Research, 18: 6531-6535.
Zaman, K., Macgill, R. S., Ahmad, S. and Pardini, R. S. (1994). An insect model for assessing mercury toxicity: Effects of mercury on antioxidant enzyme activities of the house fly Musca domestica and cabbage looper moth Trichoplusiani. Arch. Environ. Contam. Toxicol . 26(1): 114- 118.
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Molecular approach to evaluate the genotoxicity of glyphosate (roundup) using mosquito genome. (2010). Journal of Applied and Natural Science, 2(1), 96-101. https://doi.org/10.31018/jans.v2i1.105
