Determination of acrylamide-induced chick embryo brain glutathione S-transferases expression through enzyme activity and western blot

Sreenivasulu Dasari; Sailaja Gonuguntla; Balaji Meriga; Thyagaraju Kedam

doi:10.7324/JABB.2018.60108

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Abstract

Glutathione S-transferases (GSTs) are major detoxification enzymes which belong to Phase II defense enzymes; they can able to metabolize a variety of toxic chemical agents such as carcinogens, genotoxins, neurotoxins, and pesticides. Usually, GST will express when the living beings are encountered toxic chemical compounds. Acrylamide (ACR) is synthesized industrially and widely used in various industries. Usually, ACR formation occurs when food products prepared at high temperature. So that ACR is an environmental and food contaminant and it is well-proven neurotoxin. Due to highly mobile nature, birds that include poultry birds are main victims to xenobiotics (e.g., ACR) through food, water, and agricultural chemical formulas. In this study, ACR administered chick embryo brain GST activity level was assayed using 1-chloro-2,4-dinitrobenzene, and expression was assessed by western blot studies. The results show that the GST expression levels were increased in response to ACR by 24 and 48 h intervals. However, in 48 h interval, GST expression levels decreased slightly. Western blot studies also show similar pattern of GST expression. Immune blot studies showed similar GST band pattern as purification studies showed (our published work). In this study, enzyme activity and western blot analysis proved that the chick embryo brain GST was expressed more to detoxify ACR.

Keyword: Chick embryo brain glutathione S-transferases Acrylamide Glutathione S-transferases expression Western blot.

Citation:

Dasari S, Gonuguntla s, Meriga B, Kedam T. Determination of acrylamide-induced chick embryo brain glutathione S-transferases expression through enzyme activity and western blot. J App Biol Biotech. 2018;6(1):43-47. DOI: 10.7324/JABB.2018.60108

Copyright: Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license.

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Reference

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https://doi.org/10.1093/toxsci/49.2.156
2. Frova C. Glutathione transferases in the genomics era: New insights and perspectives. Biomol Eng 2006;23:149-69.
https://doi.org/10.1016/j.bioeng.2006.05.020
3. Konishi T, Kato K, Araki T, Shiraki K, Takagi M, Tamaru Y. Molecular cloning and characterization of alpha-class glutathione S-transferase genes from the hepatopancreas of red sea bream, Pagrus major. Comp Biochem Physiol C Toxicol Pharmacol 2005;140:309-20.
https://doi.org/10.1016/j.cca.2005.02.012
4. Dasari S, Ganjayi MS, Oruganti L, Balaji H, Meriga B. Glutathione S-transferases detoxify endogenous and exogenous toxic agents-minireview. J Dairy Vet Anim Res 2017;5:1-3.
https://doi.org/10.15406/jdvar.2017.05.00154
5. Hayes JD, Pulford DJ. The glutathione S-transferase supergene family: Regulation of GST and the contribution of the lsoenzymes to cancer chemoprotection and drug resistance Part II. Crit Rev Biochem Mol Biol 1995;30:521-600.
https://doi.org/10.3109/10409239509083492
6. Hayes JD, Flanagan JU, Jowsey IR. Glutathione transferases. Annu Rev Pharmacol Toxicol 2005;45:51-88.
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7. Liebler DC. Protein damage by reactive electrophiles: Targets and consequences. Chem Res Toxicol 2007;21:117-28.
https://doi.org/10.1021/tx700235t
8. Lopachin RM, Decaprio AP. Protein adduct formation as a molecular mechanism in neurotoxicity. Toxicol Sci 2005;86:214-25.
https://doi.org/10.1093/toxsci/kfi197
9. Norte AC, Ramos JA, Sousa JP, Sheldon BC. Variation of adult great tit Parus major body condition and blood parameters in relation to sex, age, year and season. J Ornithol 2009;150:651-60.
https://doi.org/10.1007/s10336-009-0387-1
10. Hegseth MN, Camus L, Helgason LB, Bocchetti R, Gabrielsen GW, Regoli F. Hepatic antioxidant responses related to levels of PCBs and metals in chicks of three Arctic seabird species. Comp Biochem Physiol C Toxicol Pharmacol 2011;154:28-35.
https://doi.org/10.1016/j.cbpc.2011.02.008
11. Pamplona R, Costantini D. Molecular and structural antioxidant defenses against oxidative stress in animals. Am J Physiol Regul Integr Comp Physiol 2011;301:R843-63.
https://doi.org/10.1152/ajpregu.00034.2011
12. Lopachin RM, Gavin T, Decaprio A, Barber DS. Application of the hard and soft, acids and bases (HSAB) theory to toxicant - Target interactions. Chem Res Toxicol 2012;25:239-51.
https://doi.org/10.1021/tx2003257
13. Schwöbel JA, Koleva YK, Enoch SJ, Bajot F, Hewitt M, Madden JC, et al. Measurement and estimation of electrophilic reactivity for predictive toxicology. Chem Rev 2011;111:2562-96.
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14. Rayburn JR, Friedman M. L-cysteine, N-acetyl-L-cysteine, and glutathione protect Xenopus laevis embryos against acrylamide-induced malformations and mortality in the frog embryo teratogenesis assay. J Agric Food Chem 2010;58:11172-8.
https://doi.org/10.1021/jf1023998
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https://doi.org/10.1016/S0161-813X(02)00025-6
17. Sumizawa T, Igisu H. Suppression of acrylamide toxicity by carboxyfullerene in human neuroblastoma cells in vitro. Arch Toxicol 2009;83:817-24.
https://doi.org/10.1007/s00204-009-0438-7
18. Alturfan AA, Tozan-Beceren A, Sehirli AO, Demiralp E, Sener G, Omurtag GZ. Resveratrol ameliorates oxidative DNA damage and protects against acrylamide-induced oxidative stress in rats. Mol Biol Rep 2012;39:4589-96.
https://doi.org/10.1007/s11033-011-1249-5
19. Lakshmi D, Gopinath K, Jayanthy G, Anjum S, Prakash D, Sudhandiran G. Ameliorating effect of fish oil on acrylamide induced oxidative stress and neuronal apoptosis in cerebral cortex. Neurochem Res 2012;37:1859-67.
https://doi.org/10.1007/s11064-012-0794-1
20. Zhu YJ, Zeng T, Zhu YB, Yu SF, Wang QS, Zhang LP, et al. Effects of acrylamide on the nervous tissue antioxidant system and sciatic nerve electrophysiology in the rat. Neurochem Res 2008;33:2310-7.
https://doi.org/10.1007/s11064-008-9730-9
21. Pourentezari M, Talebi A, Abbasi A, Khalili MA, Mangoli E, Anvari M. Effects of acrylamide on sperm parameters, chromatin quality, and the level of blood testosterone in mice. Iran J Reprod Med 2014;12:335-42.
22. Sen E, Tunali Y, Erkan M. Testicular development of male mice offspring's exposed to acrylamide and alcohol during the gestation and lactation period. Hum Exp Toxicol 2015;34:401-14.
https://doi.org/10.1177/0960327114542883
23. Sumner SC, Selvaraj L, Nauhaus SK, Fennell TR. Urinary metabolites from F344 rats and B6C3F1 mice coad ministered acrylamide and acrylonitrile for 1 or 5 days. Chem Res Toxicol 1997;10:1152-60.
https://doi.org/10.1021/tx9602123
24. Yu S, Son F, Yu J, Zhao X, Yu L, Li G, et al. Acrylamide alters cytoskeletal protein level in rat sciatic nerves. Neurochem Res 2006;31:1197-204.
https://doi.org/10.1007/s11064-006-9176-x
25. Habig WH, Pabst MJ, Jakoby WB. Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J Biol Chem 1974;249:7130-9.
26. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265-75.
27. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680-5.
https://doi.org/10.1038/227680a0
28. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications 1979. Biotechnology 1992;24:145-9.
29. Helm PA, Milne J, Hiriart-Baer V, Crozier P, Kolic T, Lega R, et al. Lake-wide distribution and depositional history of current-and past-use persistent organic pollutants in Lake Simcoe, Ontario, Canada. J Great Lakes Res 2011;37:132-41.
https://doi.org/10.1016/j.jglr.2011.03.016
30. Valko M, Morris H, Cronin MT. Metals, toxicity and oxidative stress. Curr Med Chem 2005;12:1161-208.
https://doi.org/10.2174/0929867053764635
31. Hu B, Deng L, Wen C, Yang X, Pei P, Xie Y, et al. Cloning, identification and functional characterization of a pi-class glutathione-S-transferase from the freshwater mussel Cristaria plicata. Fish Shellfish Immunol 2012;32:51-60.
https://doi.org/10.1016/j.fsi.2011.10.018
32. Limón-Pacheco JH, Gonsebatt ME. The glutathione system and its regulation by neurohormone melatonin in the central nervous system. Cent Nerv Syst Agents Med Chem 2010;10:287-97.
https://doi.org/10.2174/187152410793429683
33. Ballatori N, Krance SM, Notenboom S, Shi S, Tieu K, Hammond CL. Glutathione dysregulation and the etiology and progression of human diseases. Biol Chem 2009;390:191-214.
https://doi.org/10.1515/BC.2009.033
34. Fossi MC, Massi A, Lari L, Marsili L, Focardi S, Leonzio C, et al. Interspecies differences in mixed function oxidase activity in birds: Relationship between feeding habits, detoxication activities and organ chlorine accumulation. Environ Pollut 1995;90:15-24.
https://doi.org/10.1016/0269-7491(94)00098-X
35. Ronis MJ, Walker CH. The microsomal monooxygenase of birds. Revi Biochem Toxicol (USA)1989;10:301-84.
36. Testa B, Krämer SD. The Biochemistry of Drug Metabolism: Volume 2: Conjugations, Consequences of Metabolism, Influencing Factors. 1st ed., Vol. 2. (Zurich and Weinheim) New Jersey (USA): Wiley-VCH; 2010.
37. Ow YY, Stupans I. Gallic acid and gallic acid derivatives: Effects on drug metabolizing enzymes. Curr Drug Metab 2003;4:241-8.
https://doi.org/10.2174/1389200033489479
38. Rizvi SI, Maurya PK. Alterations in antioxidant enzymes during aging in humans. Mol Biotechnol 2007;37:58-61.
https://doi.org/10.1007/s12033-007-0048-7
39. Vyskocilová E, Szotáková B, Skálová L, Bártíková H, Hlavácová J, Boušová I. Age-related changes in hepatic activity and expression of detoxification enzymes in male rats. Biomed Res Int 2013;2013:408573.
https://doi.org/10.1155/2013/408573
40. LoPachin RM, Balaban CD, Ross JF. Acrylamide axonopathy revisited. Toxicol Appl Pharmacol 2003;188:135-53.
https://doi.org/10.1016/S0041-008X(02)00072-8
41. Takahashi M, Shibutani M, Nakahigashi J, Sakaguchi N, Inoue K, Morikawa T, et al. Limited lactational transfer of acrylamide to rat offspring on maternal oral administration during the gestation and lactation periods. Arch Toxicol 2009;83:785-93.
https://doi.org/10.1007/s00204-009-0418-y
42. Garey J, Paule MG. Effects of chronic oral acrylamide exposure on incremental repeated acquisition (learning) task performance in Fischer 344 rats. Neurotoxicol Teratol 2010;32:220-5.
https://doi.org/10.1016/j.ntt.2009.10.001
43. Dasari S, Gonuguntla S, Ganjayi M, Meriga B, Kedam T. Biochemical characterization of glutathione S-transferases purified from chick embryo brain. J Anim Poult Sci 2016;5:42-51.
44. Dasari S, Ganjayi MS, Meriga B, Kedam T. Developmental neurotoxicity of acrylamide: Defensive role of chick embryo glutathione s-trans-ferases. Adv Anim Vet Sci 2017;5:299-306.

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1. Eaton DL, Bammler TK. Concise review of the glutathione S-transferases and their significance to toxicology. Toxicol Sci 1999;49:156-64. https://doi.org/10.1093/toxsci/49.2.156

2. Frova C. Glutathione transferases in the genomics era: New insights and perspectives. Biomol Eng 2006;23:149-69. https://doi.org/10.1016/j.bioeng.2006.05.020

3. Konishi T, Kato K, Araki T, Shiraki K, Takagi M, Tamaru Y. Molecular cloning and characterization of alpha-class glutathione S-transferase genes from the hepatopancreas of red sea bream, Pagrus major. Comp Biochem Physiol C Toxicol Pharmacol 2005;140:309-20. https://doi.org/10.1016/j.cca.2005.02.012

4. Dasari S, Ganjayi MS, Oruganti L, Balaji H, Meriga B. Glutathione S-transferases detoxify endogenous and exogenous toxic agents-minireview. J Dairy Vet Anim Res 2017;5:1-3. https://doi.org/10.15406/jdvar.2017.05.00154

5. Hayes JD, Pulford DJ. The glutathione S-transferase supergene family: Regulation of GST and the contribution of the lsoenzymes to cancer chemoprotection and drug resistance Part II. Crit Rev Biochem Mol Biol 1995;30:521-600. https://doi.org/10.3109/10409239509083492

6. Hayes JD, Flanagan JU, Jowsey IR. Glutathione transferases. Annu Rev Pharmacol Toxicol 2005;45:51-88. https://doi.org/10.1146/annurev.pharmtox.45.120403.095857

7. Liebler DC. Protein damage by reactive electrophiles: Targets and consequences. Chem Res Toxicol 2007;21:117-28. https://doi.org/10.1021/tx700235t

8. Lopachin RM, Decaprio AP. Protein adduct formation as a molecular mechanism in neurotoxicity. Toxicol Sci 2005;86:214-25. https://doi.org/10.1093/toxsci/kfi197

9. Norte AC, Ramos JA, Sousa JP, Sheldon BC. Variation of adult great tit Parus major body condition and blood parameters in relation to sex, age, year and season. J Ornithol 2009;150:651-60. https://doi.org/10.1007/s10336-009-0387-1

10. Hegseth MN, Camus L, Helgason LB, Bocchetti R, Gabrielsen GW, Regoli F. Hepatic antioxidant responses related to levels of PCBs and metals in chicks of three Arctic seabird species. Comp Biochem Physiol C Toxicol Pharmacol 2011;154:28-35. https://doi.org/10.1016/j.cbpc.2011.02.008

11. Pamplona R, Costantini D. Molecular and structural antioxidant defenses against oxidative stress in animals. Am J Physiol Regul Integr Comp Physiol 2011;301:R843-63. https://doi.org/10.1152/ajpregu.00034.2011

12. Lopachin RM, Gavin T, Decaprio A, Barber DS. Application of the hard and soft, acids and bases (HSAB) theory to toxicant - Target interactions. Chem Res Toxicol 2012;25:239-51. https://doi.org/10.1021/tx2003257

13. Schwöbel JA, Koleva YK, Enoch SJ, Bajot F, Hewitt M, Madden JC, et al. Measurement and estimation of electrophilic reactivity for predictive toxicology. Chem Rev 2011;111:2562-96. https://doi.org/10.1021/cr100098n

14. Rayburn JR, Friedman M. L-cysteine, N-acetyl-L-cysteine, and glutathione protect Xenopus laevis embryos against acrylamide-induced malformations and mortality in the frog embryo teratogenesis assay. J Agric Food Chem 2010;58:11172-8. https://doi.org/10.1021/jf1023998

15. LoPachin RM. The changing view of acrylamide neurotoxicity. Neurotoxicology 2004;25:617-30. https://doi.org/10.1016/j.neuro.2004.01.004

16. Sickles DW, Stone JD, Friedman MA. Fast axonal transport: A site of acrylamide neurotoxicity? Neurotoxicology 2002;23:223-51. https://doi.org/10.1016/S0161-813X(02)00025-6

17. Sumizawa T, Igisu H. Suppression of acrylamide toxicity by carboxyfullerene in human neuroblastoma cells in vitro. Arch Toxicol 2009;83:817-24. https://doi.org/10.1007/s00204-009-0438-7

18. Alturfan AA, Tozan-Beceren A, Sehirli AO, Demiralp E, Sener G, Omurtag GZ. Resveratrol ameliorates oxidative DNA damage and protects against acrylamide-induced oxidative stress in rats. Mol Biol Rep 2012;39:4589-96. https://doi.org/10.1007/s11033-011-1249-5

19. Lakshmi D, Gopinath K, Jayanthy G, Anjum S, Prakash D, Sudhandiran G. Ameliorating effect of fish oil on acrylamide induced oxidative stress and neuronal apoptosis in cerebral cortex. Neurochem Res 2012;37:1859-67. https://doi.org/10.1007/s11064-012-0794-1

20. Zhu YJ, Zeng T, Zhu YB, Yu SF, Wang QS, Zhang LP, et al. Effects of acrylamide on the nervous tissue antioxidant system and sciatic nerve electrophysiology in the rat. Neurochem Res 2008;33:2310-7. https://doi.org/10.1007/s11064-008-9730-9

21. Pourentezari M, Talebi A, Abbasi A, Khalili MA, Mangoli E, Anvari M. Effects of acrylamide on sperm parameters, chromatin quality, and the level of blood testosterone in mice. Iran J Reprod Med 2014;12:335-42.

22. Sen E, Tunali Y, Erkan M. Testicular development of male mice offspring's exposed to acrylamide and alcohol during the gestation and lactation period. Hum Exp Toxicol 2015;34:401-14. https://doi.org/10.1177/0960327114542883

23. Sumner SC, Selvaraj L, Nauhaus SK, Fennell TR. Urinary metabolites from F344 rats and B6C3F1 mice coad ministered acrylamide and acrylonitrile for 1 or 5 days. Chem Res Toxicol 1997;10:1152-60. https://doi.org/10.1021/tx9602123

24. Yu S, Son F, Yu J, Zhao X, Yu L, Li G, et al. Acrylamide alters cytoskeletal protein level in rat sciatic nerves. Neurochem Res 2006;31:1197-204. https://doi.org/10.1007/s11064-006-9176-x

25. Habig WH, Pabst MJ, Jakoby WB. Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J Biol Chem 1974;249:7130-9.

26. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265-75.

27. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680-5. https://doi.org/10.1038/227680a0

28. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications 1979. Biotechnology 1992;24:145-9.

29. Helm PA, Milne J, Hiriart-Baer V, Crozier P, Kolic T, Lega R, et al. Lake-wide distribution and depositional history of current-and past-use persistent organic pollutants in Lake Simcoe, Ontario, Canada. J Great Lakes Res 2011;37:132-41. https://doi.org/10.1016/j.jglr.2011.03.016

30. Valko M, Morris H, Cronin MT. Metals, toxicity and oxidative stress. Curr Med Chem 2005;12:1161-208. https://doi.org/10.2174/0929867053764635

31. Hu B, Deng L, Wen C, Yang X, Pei P, Xie Y, et al. Cloning, identification and functional characterization of a pi-class glutathione-S-transferase from the freshwater mussel Cristaria plicata. Fish Shellfish Immunol 2012;32:51-60. https://doi.org/10.1016/j.fsi.2011.10.018

32. Limón-Pacheco JH, Gonsebatt ME. The glutathione system and its regulation by neurohormone melatonin in the central nervous system. Cent Nerv Syst Agents Med Chem 2010;10:287-97. https://doi.org/10.2174/187152410793429683

33. Ballatori N, Krance SM, Notenboom S, Shi S, Tieu K, Hammond CL. Glutathione dysregulation and the etiology and progression of human diseases. Biol Chem 2009;390:191-214. https://doi.org/10.1515/BC.2009.033

34. Fossi MC, Massi A, Lari L, Marsili L, Focardi S, Leonzio C, et al. Interspecies differences in mixed function oxidase activity in birds: Relationship between feeding habits, detoxication activities and organ chlorine accumulation. Environ Pollut 1995;90:15-24. https://doi.org/10.1016/0269-7491(94)00098-X

35. Ronis MJ, Walker CH. The microsomal monooxygenase of birds. Revi Biochem Toxicol (USA)1989;10:301-84.

36. Testa B, Krämer SD. The Biochemistry of Drug Metabolism: Volume 2: Conjugations, Consequences of Metabolism, Influencing Factors. 1st ed., Vol. 2. (Zurich and Weinheim) New Jersey (USA): Wiley-VCH; 2010.

37. Ow YY, Stupans I. Gallic acid and gallic acid derivatives: Effects on drug metabolizing enzymes. Curr Drug Metab 2003;4:241-8. https://doi.org/10.2174/1389200033489479

38. Rizvi SI, Maurya PK. Alterations in antioxidant enzymes during aging in humans. Mol Biotechnol 2007;37:58-61. https://doi.org/10.1007/s12033-007-0048-7

39. Vyskocilová E, Szotáková B, Skálová L, Bártíková H, Hlavácová J, Boušová I. Age-related changes in hepatic activity and expression of detoxification enzymes in male rats. Biomed Res Int 2013;2013:408573. https://doi.org/10.1155/2013/408573

40. LoPachin RM, Balaban CD, Ross JF. Acrylamide axonopathy revisited. Toxicol Appl Pharmacol 2003;188:135-53. https://doi.org/10.1016/S0041-008X(02)00072-8

41. Takahashi M, Shibutani M, Nakahigashi J, Sakaguchi N, Inoue K, Morikawa T, et al. Limited lactational transfer of acrylamide to rat offspring on maternal oral administration during the gestation and lactation periods. Arch Toxicol 2009;83:785-93. https://doi.org/10.1007/s00204-009-0418-y

42. Garey J, Paule MG. Effects of chronic oral acrylamide exposure on incremental repeated acquisition (learning) task performance in Fischer 344 rats. Neurotoxicol Teratol 2010;32:220-5. https://doi.org/10.1016/j.ntt.2009.10.001

43. Dasari S, Gonuguntla S, Ganjayi M, Meriga B, Kedam T. Biochemical characterization of glutathione S-transferases purified from chick embryo brain. J Anim Poult Sci 2016;5:42-51.

44. Dasari S, Ganjayi MS, Meriga B, Kedam T. Developmental neurotoxicity of acrylamide: Defensive role of chick embryo glutathione s-trans-ferases. Adv Anim Vet Sci 2017;5:299-306.