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Studies on the genotoxicity of endosulfan in bacterial systems
Mutation Research 439 Ž1999. 63–67
Studies on the genotoxicity of endosulfan in bacterial systems K. Chaudhuri b, S. Selvaraj c , A.K. Pal a
a,)
Biophysics DiÕision, Saha institute of Nuclear Physics, 37, Belgachia Road, Calcutta-700 037, India b State Forensic Science Laboratory, 37 r 1 r 2 Belgachia Road, Calcutta-700 037, India c Inter UniÕersity Consortium for DAE Facilities, LB-8, Sector III, Saltlake, Calcutta-700 091, India Received 28 July 1998; revised 2 November 1998; accepted 11 November 1998
Abstract Endosulfan, an organochlorine pesticide, was subjected to the differential sensitivity assay in repair-deficient and repair-proficient strains of Escherichia coli K12, prophage l induction assay in WP2s Ž l . and mutation induction in E. coli K12. The induction of umu gene expression with endosulfan was studied also in Salmonella typhimurium TA1535rpSK1002 cells. The differential sensitivity assay revealed that the recA 13 strain was the most sensitive. Endosulfan induced prophage l in E. coli and umu gene expression in S. typhimurium cells; however, the extent of the effects were low. Endosulfan also induced a dose-dependent increase in forward mutations in E. coli K12 cells from ampicillin sensitivity to ampicillin resistance. Our studies indicate the genotoxic potential of endosulfan and the role of the recA gene in the repair of endosulfan-induced DNA damage. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Pesticide; Endosulfan; Genotoxicity
1. Introduction Pesticides, in addition to their intended effects, viz., the control of insects, weeds, or other pests are sometimes found to affect non-target organisms including man itself w1,2x. Because of the potential environmental impact connected with the introduction and heavy use of pesticides, it is urgent that the genotoxic potential of these agents be studied. Endosulfan, a member of the organochlorine class of pesticides, is used widely in the Indian subcontinent. It is acutely toxic to mammals, birds, fish and
)
Corresponding author
bees w3x. Endosulfan has also been found to induce neurotoxicity, renal toxicity, hepatotoxicity, haematologic toxicity, respiratory toxicity, and reproductive toxicity in mammals w1,4,5x. Many other organochloro compounds have been found to be mutagens w6x. Published genotoxicity studies with endosulfan are, however, limited in number. The purpose of the present study was to explore the genotoxicity of endosulfan using short-term bacterial assay systems. This study includes the differential sensitivity assay in Escherichia coli K12, prophage induction study in E. coli WP2s Ž l . strains, umu gene expression in Salmonella typhimurium strain TA 1535rpSK 1002 and mutation induction in E. coli K12 cells from ampicillin sensitivity to ampicillin resistance.
1383-5718r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 Ž 9 8 . 0 0 1 7 4 - 0
64
K. Chaudhuri et al.r Mutation Research 439 (1999) 63–67
2. Materials and methods
pyranoside ŽONPG. were obtained from Sigma, USA. All other chemicals used were of analytical grade.
2.1. Bacterial strains Bacterial strains used in this study were: Ži. E. coli K12 strains, AB1157: Žrepair proficient., AB 1886: Ž uÕrA 6., AB 2494: Ž lexA 1. and AB2463: Ž recA 13. obtained through the kind courtesy of Dr. Barbara J. Bachman, E. coli genetic stock centre, USA. The strains mentioned above are isogenic strains and their complete genotypes are provided by Dewitt and Adelberg w7x, Howard-Flanders et al. w8x, Howard-Flanders and Theriot w9x, and HowardFlanders w10x. The lambda lysogen used were a derivative of E. coli Brr, WP2s Ž l .: trp E65, uÕrA 155, and the indicator strain TH 008: Ž str r . were obtained through the kind courtesy of Dr. Virginia Houk, US Environmental Protection Agency, USA. The Salmonella typhimurium strain TA 1535rpSK 1002 carrying a umu CX- X lacZ fusion gene on multicopy plasmid was obtained through the kind courtesy of Dr. S. Nakamura, Japan. The detailed description of the strain is provided by Oda et al. w11x. 2.2. Bacterial media The culture media used were: Ž1. LB medium containing 0.5% yeast extract ŽDifco., 1% Tryptone ŽDifco., 1% NaCl in distilled water, pH adjusted to 7.2. Ž2. Nutrient Agar ŽNA. plates containing 1% Bacto Peptone ŽDifco., 0.8% Beef Extract Paste ŽDifco., 0.5% NaCl in distilled water and 1.4% Bacto Agar ŽDifco.. Ž3. Tryptone plates containing 1% Bacto Tryptone ŽDifco., 0.5% NaCl and 1.4% Bacto Agar ŽDifco. in glass distilled water supplemented with 100 mgrml Streptomycin. Ž4. Soft agar containing 0.7% Bacto agar ŽDifco. in distilled water. 2.3. Chemicals Endosulfan ŽCAS No: 115-29-7. commercial grade, tradename Hildan, was obtained from Hindustan Insecticides, India. The purity of the endosulfan as indicated by the manufacturer is 35% wrw and ‘inert’ ingredients are 65%. D-ampicillin, streptomycin sulphate, and O-nitrophenyl b-D-galacto-
2.4. Endosulfan treatment and assay of bacterial Õiability One milliliter aliquots of log phase cells were inoculated into 20 ml of culture media containing various concentrations of endosulfan, incubated at 378C with shaking in the dark for a fixed period of 2 h, and then immediately assayed for colony forming units Žc.f.u.. on pesticide free NA plates after appropriate dilution in saline Ž0.85% NaCl.. Colonies were scored after overnight incubation at 378C. 2.5. Prophage induction in E.coli WP2s (l ) strain by endosulfan One milliliter aliquots of log phase cells were inoculated into 4 ml of culture media containing different concentrations of endosulfan, incubated in the shaker bath for a fixed period of 2 h, and assayed for plaque forming units along with 0.3 ml of log phase cells in tryptone plates by the soft agar overlay method w12x. Plaques were scored after overnight incubation at 378C. 2.6. Assay for umu gene expression in Salmonella typhimurium cells The umu test was performed in general by the method of Oda et al. w11x. An overnight culture of S. typhimurium strain TA 1535rpSK 1002 was diluted 50-fold in LB medium Žsupplemented with 20 mgrml of D-ampicillin. and incubated with shaking at 378C until the optical density of the culture at 600 nm was 0.27. A 2.5-ml aliquot of culture was mixed with 0.1 ml of the test compound and the mixture was incubated in a shaker bath at 378C for a period of 2 h. b-galactosidase activity was determined by the method of Miller w13x. At least a 2-fold increase in b-galactosidase activity above the control level was required to produce a positive response w11x. 2.7. Assay for forward mutation One milliliter aliquots of AB1157 log phase cells were inoculated into 20 ml of culture media containing various concentration of endosulfan, and incu-
K. Chaudhuri et al.r Mutation Research 439 (1999) 63–67
65
bated with shaking at 378C for 2 h. The treated or untreated cells were properly diluted in saline and plated on NA plates with or without ampicillin. The ampicillin concentration used in this study was 3 mgrml. The spontaneous mutation frequency ŽMF. s and induced mutation frequency ŽMF. I was calculated as follows.
Ž MF. S s Ž No. of cfu at 0 mgrml endosulfan in ampicillin plates . r Ž No. of cfu at 0 mgrml of endosulfan in NA plates .
Ž MF. I s Ž No. of cfu at a fixed endosulfan conc. in ampicillin plates . r Ž No. of cfu at a fixed
Fig. 2. The prophage l induction profile of E. coli WP2s Ž l . cells after endosulfan treatment.
endosulfan conc. in NA plates . The mutation index ŽM.I.. at a fixed endosulfan concentration was calculated as
for each set of data, n1 and n 2 are the number of samples for ŽMF. I and ŽMF. S , respectively. Sp, the pooled standard deviation is calculated as follows:
M.I.s Ž Ž MF . I . r Ž Ž MF . S .
Sp s
The comparison of the induced mutation frequency ŽMF. I at different endosulfan concentration to the spontaneous mutation frequency ŽMF. S has been done by subjecting each set of data to Student’s t-test w14x employing the following formula. t s Ž Ž MF . I y Ž MF . S . r Ž Sp Ž 1rn1 q 1rn 2 .
1r2
.
Where, Ž MF . I and Ž MF . S are the mean values of the induced and spontaneous mutation frequencies
ž
Ž n1 y 1 . S12 q Ž n 2 y 1 . S 22
r Ž Ž n1 q n 2 y 2 .
1r2
1r2
/
.
The probability value Ž P value. corresponding to these t values thus calculated for Ž n1 q n 2 y 2. degrees of freedom is found out from the statistical table w15x.
3. Results The repair-deficient and repair-proficient strains of E. coli K12 were sensitive to endosulfan treat-
Fig. 1. Survival of different repair-deficient and repair proficient strains of E. coli K12 after endosulfan treatment for 2 h at 378C. `: AB 1157 Žrepair-proficient.; v: AB 1886 Ž uÕrA 6.; ^: AB 2494 Ž lexA 1.; ': AB 2463 Ž recA 13..
Fig. 3. Induction of umu gene expression by endosulfan in S. typhimurium strain TA 1535rpSK 1002, The ordinate represents the ratio of b-galactosidase units induced by endosulfan vs. that produced in the absence of endosulfan.
66
K. Chaudhuri et al.r Mutation Research 439 (1999) 63–67
ment to different degrees ŽFig. 1.. The AB 2463 Ž recA 13. strain was found to be the most sensitive. The D 37 values of different E. coli K12 strains were in the following order: AB1157: repair-proficient Ž28537 mgrml. ) AB 2494: lexA 1 Ž9360 mgrml. ) AB1886: uÕrA 6 Ž8125 mgrml. ) AB 2463: recA 13 Ž1708 mgrml.. Endosulfan was found to induce prophage l in lysogeneic E. coli WP2s cells. The prophage l induction was measured by the ratio IdrIo , Id being the plaque formers per ml produced by the pesticide treatment and Io being the corresponding value for spontaneous induction. Fig. 2 shows that the prophage l induction increased initially with the increased amount of endosulfan and then saturated at a endosulfan concentration of 100 mgrml. At higher concentrations, the induction level dropped. The maximum induction obtained was about 3.5-fold higher than the spontaneous induction level. Endosulfan induced umu gene expression in S. typhimurium strain TA1535rpSK 1002. The umu gene expression increased with increasing concentrations of endosulfan, and the maximum induction achieved was 4.2-fold higher than the spontaneous level ŽFig. 3.. Endosulfan also induced mutations in E. coli K12 AB 1157 cells from ampicillin sensitivity to ampicillin resistance ŽFig. 4.. The mutation index increased with increasing concentrations of the insecticide to a maximum increase of 14.4. In each case,
Fig. 4. Effect of endosulfan on the mutation index of E. coli AB 1157 cells from ampicillin sensitivity to ampicillin resistance after 2 h of incubation of the log phase cells at 378C with different concentrations of endosulfan.
the difference between the endosulfan induced mutation frequency and the spontaneous mutation frequency was significant at a level better than 0.05% Ž P - 0.0005..
4. Discussion Very few studies on the genotoxicity of endosulfan are available in the literature. This study established the genotoxic potential of endosulfan using a number of bacterial short-term assay systems. From the differential sensitivity assay, it is clear that excision, recombination, and SOS repair pathways are involved in the repair of endosulfan induced DNA damage. In the case of prophage l induction, endosulfan produced a 3.5 fold increase in plaque formers above the spontaneous level, indicating that endosulfan is a weak prophage inducer. DeMarini and Brooks w16x studied the induction of prophage l by a number of chlorinated organics. They indicated the response to be positive which produces more than two fold increase of plaque formers above the background level. Also the maximum induction obtained was at a much lower endosulfan concentration compared to the concentration needed for the survival assays. This is possibly due to the better permeability of the WP2s Ž l . strains. The induction of umu gene expression in S. typhimurium cells also showed a 4.2-fold increase of b-galactosidase activity at endosulfan concentrations comparable to those used for prophage l induction in E.coli WP2s Ž l . cells. The mutation induction by endosulfan treatment was very much significant ŽP - 0.0005. as is evident from the statistical analysis. From the survival assay, it is evident that the E. coli K12 uÕrA 6 and lexA 1 strains are much less sensitive compared to the recA 13 strain, which indicates that the recA gene plays a major role in the repair of endosulfan induced DNA damage. This pathway involved postreplication repair, which is error-prone in nature w17,18x. This is corroborated by the high mutation index obtained after different amounts of endosulfan treatment. These studies indicate the genotoxic potential of endosulfan in bacterial systems.
K. Chaudhuri et al.r Mutation Research 439 (1999) 63–67
Acknowledgements The authors are thankful to Dr. Bikash Sinha, Director, SINP and Dr. S.N. Chintalapudi, Director, IUC-DAEF, Calcutta for extending their cooperation and necessary facilities. Thanks are also due to Mr. Arijit Pal, Biophysics Division of SINP, for assistance rendered by him in this work.
w7x w8x
w9x
w10x
References w11x w1x S.M. Naqvi, C. Vaishnavi, Bioaccumulative potential and toxicity of endosulfan insecticide to non-target animals, Comp. Biochem. Physiol. C 105 Ž1993. 347–361. w2x P.S. Wilkes, Gas-liquid chromatographic-mass spectrometric confirmation of endosulfan and endosulfan sulfate in apples and carrots, J. Assoc. Off. Anal. Chem. 64 Ž1981. 2108–2110. w3x C. Tomlin ŽEd.., Endosulfan Insecticide Acaricide, The Pesticide Manual, Crop Protection Publications, UK, 262, 1995, 388–390. w4x A. Pomes, K. Rodriguez-Farre, C. Sunol, Disruption of GABA-dependent chloride flux by cyclodienes and hexachlorocyclohexanes in primary cultures of cortical neurons, J. Pharmacol. Exp. Ther. 271 Ž1994. 1616–1623. w5x M.G. Wade, D. Desaulniers, K. Leingartner, W.G. Foster, Interactions between endosulfan and dieldrin on estrogenmediated processes in vitro and in vivo, Reprod. Toxicol. 11 Ž1997. 791–798. w6x M. Tzoneva, A. Kappas, V. Georgieva, R. Vachkova, V.
w12x w13x w14x w15x w16x
w17x
w18x
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Tziolas, On the genotoxicity of the pesticides endodan and kilacar in 6 different test systems, Mutat. Res. 157 Ž1985. 13–22. Dewitt, A.E. Adelberg, Genetics 47 Ž1962. 577. P. Howard-Flanders, R.P. Boyce, L. Theriot, Three loci in Escherichia coli K-12 that controls the excision of pyrimidine dimers and certain other mutagen products from DNA, Genetics 53 Ž1966. 1119–1136. P. Howard-Flanders, L. Theriot, Mutants of Escherichia coli K-12 defective in DNA repair and in genetic recombination, Genetics 53 Ž1966. 1137–1150. P. Howard-Flanders, Genes that control DNA repair and genetic recombination in Escherichia coli, Adv. Biol. Med. Phys. 12 Ž1966. 229–317. Y. Oda, S. Nakamura, I. Oki, T. Kato, H. Shinagawa, Evaluation of the new system ŽUMU-test. for the detection of environmental mutagens and carcinogens, Mutat. Res. 147 Ž1985. 219–229. A.K. Pal, S.N. Chatterjee, Induction of l prophage by furazolidone, Mutat. Res. 156 Ž1985. 69–75. J.H. Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1972. W.G. Gosset, Student, Biometrika 6 Ž1908. 1. R.A. Fisher, F. Yates, Statistical Tables for Biological, Agricultural and Medical Research, Oliver and Boyd, Edinburgh. D.M. DeMarini, H.G. Brooks, Induction of prophage lambda by chlorinated organics: Detection of single speciesrsingle site carcinogens, Environ. Mol. Mutagen. 19 Ž1992. 98–111. P.C. Hanawalt, P.K. Cooper, A.K. Ganesan, C.A. Smith, DNA repair in bacteria and mammalian cells, Annu. Rev. Biochem. 48 Ž1979. 783–836. P.C. Hanawalt, Perspectives on DNA repair and inducible recovery phenomena, Biochemie 64 Ž1982. 847–851.
Studies on the genotoxicity of endosulfan in bacterial systems K. Chaudhuri b, S. Selvaraj c , A.K. Pal a
a,)
Biophysics DiÕision, Saha institute of Nuclear Physics, 37, Belgachia Road, Calcutta-700 037, India b State Forensic Science Laboratory, 37 r 1 r 2 Belgachia Road, Calcutta-700 037, India c Inter UniÕersity Consortium for DAE Facilities, LB-8, Sector III, Saltlake, Calcutta-700 091, India Received 28 July 1998; revised 2 November 1998; accepted 11 November 1998
Abstract Endosulfan, an organochlorine pesticide, was subjected to the differential sensitivity assay in repair-deficient and repair-proficient strains of Escherichia coli K12, prophage l induction assay in WP2s Ž l . and mutation induction in E. coli K12. The induction of umu gene expression with endosulfan was studied also in Salmonella typhimurium TA1535rpSK1002 cells. The differential sensitivity assay revealed that the recA 13 strain was the most sensitive. Endosulfan induced prophage l in E. coli and umu gene expression in S. typhimurium cells; however, the extent of the effects were low. Endosulfan also induced a dose-dependent increase in forward mutations in E. coli K12 cells from ampicillin sensitivity to ampicillin resistance. Our studies indicate the genotoxic potential of endosulfan and the role of the recA gene in the repair of endosulfan-induced DNA damage. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Pesticide; Endosulfan; Genotoxicity
1. Introduction Pesticides, in addition to their intended effects, viz., the control of insects, weeds, or other pests are sometimes found to affect non-target organisms including man itself w1,2x. Because of the potential environmental impact connected with the introduction and heavy use of pesticides, it is urgent that the genotoxic potential of these agents be studied. Endosulfan, a member of the organochlorine class of pesticides, is used widely in the Indian subcontinent. It is acutely toxic to mammals, birds, fish and
)
Corresponding author
bees w3x. Endosulfan has also been found to induce neurotoxicity, renal toxicity, hepatotoxicity, haematologic toxicity, respiratory toxicity, and reproductive toxicity in mammals w1,4,5x. Many other organochloro compounds have been found to be mutagens w6x. Published genotoxicity studies with endosulfan are, however, limited in number. The purpose of the present study was to explore the genotoxicity of endosulfan using short-term bacterial assay systems. This study includes the differential sensitivity assay in Escherichia coli K12, prophage induction study in E. coli WP2s Ž l . strains, umu gene expression in Salmonella typhimurium strain TA 1535rpSK 1002 and mutation induction in E. coli K12 cells from ampicillin sensitivity to ampicillin resistance.
1383-5718r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 Ž 9 8 . 0 0 1 7 4 - 0
64
K. Chaudhuri et al.r Mutation Research 439 (1999) 63–67
2. Materials and methods
pyranoside ŽONPG. were obtained from Sigma, USA. All other chemicals used were of analytical grade.
2.1. Bacterial strains Bacterial strains used in this study were: Ži. E. coli K12 strains, AB1157: Žrepair proficient., AB 1886: Ž uÕrA 6., AB 2494: Ž lexA 1. and AB2463: Ž recA 13. obtained through the kind courtesy of Dr. Barbara J. Bachman, E. coli genetic stock centre, USA. The strains mentioned above are isogenic strains and their complete genotypes are provided by Dewitt and Adelberg w7x, Howard-Flanders et al. w8x, Howard-Flanders and Theriot w9x, and HowardFlanders w10x. The lambda lysogen used were a derivative of E. coli Brr, WP2s Ž l .: trp E65, uÕrA 155, and the indicator strain TH 008: Ž str r . were obtained through the kind courtesy of Dr. Virginia Houk, US Environmental Protection Agency, USA. The Salmonella typhimurium strain TA 1535rpSK 1002 carrying a umu CX- X lacZ fusion gene on multicopy plasmid was obtained through the kind courtesy of Dr. S. Nakamura, Japan. The detailed description of the strain is provided by Oda et al. w11x. 2.2. Bacterial media The culture media used were: Ž1. LB medium containing 0.5% yeast extract ŽDifco., 1% Tryptone ŽDifco., 1% NaCl in distilled water, pH adjusted to 7.2. Ž2. Nutrient Agar ŽNA. plates containing 1% Bacto Peptone ŽDifco., 0.8% Beef Extract Paste ŽDifco., 0.5% NaCl in distilled water and 1.4% Bacto Agar ŽDifco.. Ž3. Tryptone plates containing 1% Bacto Tryptone ŽDifco., 0.5% NaCl and 1.4% Bacto Agar ŽDifco. in glass distilled water supplemented with 100 mgrml Streptomycin. Ž4. Soft agar containing 0.7% Bacto agar ŽDifco. in distilled water. 2.3. Chemicals Endosulfan ŽCAS No: 115-29-7. commercial grade, tradename Hildan, was obtained from Hindustan Insecticides, India. The purity of the endosulfan as indicated by the manufacturer is 35% wrw and ‘inert’ ingredients are 65%. D-ampicillin, streptomycin sulphate, and O-nitrophenyl b-D-galacto-
2.4. Endosulfan treatment and assay of bacterial Õiability One milliliter aliquots of log phase cells were inoculated into 20 ml of culture media containing various concentrations of endosulfan, incubated at 378C with shaking in the dark for a fixed period of 2 h, and then immediately assayed for colony forming units Žc.f.u.. on pesticide free NA plates after appropriate dilution in saline Ž0.85% NaCl.. Colonies were scored after overnight incubation at 378C. 2.5. Prophage induction in E.coli WP2s (l ) strain by endosulfan One milliliter aliquots of log phase cells were inoculated into 4 ml of culture media containing different concentrations of endosulfan, incubated in the shaker bath for a fixed period of 2 h, and assayed for plaque forming units along with 0.3 ml of log phase cells in tryptone plates by the soft agar overlay method w12x. Plaques were scored after overnight incubation at 378C. 2.6. Assay for umu gene expression in Salmonella typhimurium cells The umu test was performed in general by the method of Oda et al. w11x. An overnight culture of S. typhimurium strain TA 1535rpSK 1002 was diluted 50-fold in LB medium Žsupplemented with 20 mgrml of D-ampicillin. and incubated with shaking at 378C until the optical density of the culture at 600 nm was 0.27. A 2.5-ml aliquot of culture was mixed with 0.1 ml of the test compound and the mixture was incubated in a shaker bath at 378C for a period of 2 h. b-galactosidase activity was determined by the method of Miller w13x. At least a 2-fold increase in b-galactosidase activity above the control level was required to produce a positive response w11x. 2.7. Assay for forward mutation One milliliter aliquots of AB1157 log phase cells were inoculated into 20 ml of culture media containing various concentration of endosulfan, and incu-
K. Chaudhuri et al.r Mutation Research 439 (1999) 63–67
65
bated with shaking at 378C for 2 h. The treated or untreated cells were properly diluted in saline and plated on NA plates with or without ampicillin. The ampicillin concentration used in this study was 3 mgrml. The spontaneous mutation frequency ŽMF. s and induced mutation frequency ŽMF. I was calculated as follows.
Ž MF. S s Ž No. of cfu at 0 mgrml endosulfan in ampicillin plates . r Ž No. of cfu at 0 mgrml of endosulfan in NA plates .
Ž MF. I s Ž No. of cfu at a fixed endosulfan conc. in ampicillin plates . r Ž No. of cfu at a fixed
Fig. 2. The prophage l induction profile of E. coli WP2s Ž l . cells after endosulfan treatment.
endosulfan conc. in NA plates . The mutation index ŽM.I.. at a fixed endosulfan concentration was calculated as
for each set of data, n1 and n 2 are the number of samples for ŽMF. I and ŽMF. S , respectively. Sp, the pooled standard deviation is calculated as follows:
M.I.s Ž Ž MF . I . r Ž Ž MF . S .
Sp s
The comparison of the induced mutation frequency ŽMF. I at different endosulfan concentration to the spontaneous mutation frequency ŽMF. S has been done by subjecting each set of data to Student’s t-test w14x employing the following formula. t s Ž Ž MF . I y Ž MF . S . r Ž Sp Ž 1rn1 q 1rn 2 .
1r2
.
Where, Ž MF . I and Ž MF . S are the mean values of the induced and spontaneous mutation frequencies
ž
Ž n1 y 1 . S12 q Ž n 2 y 1 . S 22
r Ž Ž n1 q n 2 y 2 .
1r2
1r2
/
.
The probability value Ž P value. corresponding to these t values thus calculated for Ž n1 q n 2 y 2. degrees of freedom is found out from the statistical table w15x.
3. Results The repair-deficient and repair-proficient strains of E. coli K12 were sensitive to endosulfan treat-
Fig. 1. Survival of different repair-deficient and repair proficient strains of E. coli K12 after endosulfan treatment for 2 h at 378C. `: AB 1157 Žrepair-proficient.; v: AB 1886 Ž uÕrA 6.; ^: AB 2494 Ž lexA 1.; ': AB 2463 Ž recA 13..
Fig. 3. Induction of umu gene expression by endosulfan in S. typhimurium strain TA 1535rpSK 1002, The ordinate represents the ratio of b-galactosidase units induced by endosulfan vs. that produced in the absence of endosulfan.
66
K. Chaudhuri et al.r Mutation Research 439 (1999) 63–67
ment to different degrees ŽFig. 1.. The AB 2463 Ž recA 13. strain was found to be the most sensitive. The D 37 values of different E. coli K12 strains were in the following order: AB1157: repair-proficient Ž28537 mgrml. ) AB 2494: lexA 1 Ž9360 mgrml. ) AB1886: uÕrA 6 Ž8125 mgrml. ) AB 2463: recA 13 Ž1708 mgrml.. Endosulfan was found to induce prophage l in lysogeneic E. coli WP2s cells. The prophage l induction was measured by the ratio IdrIo , Id being the plaque formers per ml produced by the pesticide treatment and Io being the corresponding value for spontaneous induction. Fig. 2 shows that the prophage l induction increased initially with the increased amount of endosulfan and then saturated at a endosulfan concentration of 100 mgrml. At higher concentrations, the induction level dropped. The maximum induction obtained was about 3.5-fold higher than the spontaneous induction level. Endosulfan induced umu gene expression in S. typhimurium strain TA1535rpSK 1002. The umu gene expression increased with increasing concentrations of endosulfan, and the maximum induction achieved was 4.2-fold higher than the spontaneous level ŽFig. 3.. Endosulfan also induced mutations in E. coli K12 AB 1157 cells from ampicillin sensitivity to ampicillin resistance ŽFig. 4.. The mutation index increased with increasing concentrations of the insecticide to a maximum increase of 14.4. In each case,
Fig. 4. Effect of endosulfan on the mutation index of E. coli AB 1157 cells from ampicillin sensitivity to ampicillin resistance after 2 h of incubation of the log phase cells at 378C with different concentrations of endosulfan.
the difference between the endosulfan induced mutation frequency and the spontaneous mutation frequency was significant at a level better than 0.05% Ž P - 0.0005..
4. Discussion Very few studies on the genotoxicity of endosulfan are available in the literature. This study established the genotoxic potential of endosulfan using a number of bacterial short-term assay systems. From the differential sensitivity assay, it is clear that excision, recombination, and SOS repair pathways are involved in the repair of endosulfan induced DNA damage. In the case of prophage l induction, endosulfan produced a 3.5 fold increase in plaque formers above the spontaneous level, indicating that endosulfan is a weak prophage inducer. DeMarini and Brooks w16x studied the induction of prophage l by a number of chlorinated organics. They indicated the response to be positive which produces more than two fold increase of plaque formers above the background level. Also the maximum induction obtained was at a much lower endosulfan concentration compared to the concentration needed for the survival assays. This is possibly due to the better permeability of the WP2s Ž l . strains. The induction of umu gene expression in S. typhimurium cells also showed a 4.2-fold increase of b-galactosidase activity at endosulfan concentrations comparable to those used for prophage l induction in E.coli WP2s Ž l . cells. The mutation induction by endosulfan treatment was very much significant ŽP - 0.0005. as is evident from the statistical analysis. From the survival assay, it is evident that the E. coli K12 uÕrA 6 and lexA 1 strains are much less sensitive compared to the recA 13 strain, which indicates that the recA gene plays a major role in the repair of endosulfan induced DNA damage. This pathway involved postreplication repair, which is error-prone in nature w17,18x. This is corroborated by the high mutation index obtained after different amounts of endosulfan treatment. These studies indicate the genotoxic potential of endosulfan in bacterial systems.
K. Chaudhuri et al.r Mutation Research 439 (1999) 63–67
Acknowledgements The authors are thankful to Dr. Bikash Sinha, Director, SINP and Dr. S.N. Chintalapudi, Director, IUC-DAEF, Calcutta for extending their cooperation and necessary facilities. Thanks are also due to Mr. Arijit Pal, Biophysics Division of SINP, for assistance rendered by him in this work.
w7x w8x
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