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3 Biotech

, 8:438 | Cite as

Moderate levels of glyphosate and its formulations vary in their cytotoxicity and genotoxicity in a whole blood model and in human cell lines with different estrogen receptor status

  • L. K. S. De Almeida
  • B. I. Pletschke
  • C. L. Frost
Original Article

Abstract

In vitro studies were conducted to determine the short-term cytotoxic and genotoxic effects of pure glyphosate and two glyphosate formulations (Roundup® and Wipeout®) at concentrations relevant to human exposure using whole blood (cytotoxicity) and various cancer cell lines (cytotoxicity and genotoxicity). Pure glyphosate (pure glyph) and Roundup® (Ro) showed similar non-monotonic toxicological profiles at low dose exposure (from 10 µg/ml), whereas Wipeout® (Wo) demonstrated a monotonic reduction in cell viability from a threshold concentration of 50 µg/ml, when tested in whole blood. We evaluated whether using various cancer cells (the estrogen-E2-responsive HEC1A, MCF7 and the estrogen-insensitive MDA-MB-231) exposed to moderate doses (75–500 µg/ml) would indicate varied toxicity and results indicated significant effects in the HEC1A cancer cells. A non-monotonic reduction in cell viability was observed in HEC1A exposed to pure glyph (75–500 µg/ml) and proliferative effects were observed after exposure to Wo (75, 125 and 250 µg/ml). Genotoxicity assessment (test concentration 500 µg/ml) demonstrated DNA damage in the HEC1A and MDA-MB-231 cells. Adjuvants and/or glyphosate impurities were potential contributing factors of toxicity based on the differential toxicities displayed by Ro and Wo in human whole blood and the HEC1A cells. This study contributes to the existing knowledge about in vitro exposure to moderate concentrations of glyphosate or glyphosate formulations at cytotoxic and genotoxic levels. In addition, a suggestion on the relevance of the estrogen receptor status of the cell lines used is provided, leading to the need to further investigate a potential endocrine disruptive role.

Keywords

Glyphosate Roundup® Wipeout® Cytotoxicity Genotoxicity 

Abbreviations

EPSP

5-Enolpyruvylshikimate-3-phosphate synthase

EPA

Environmental Protection Agency

ER

Estrogen receptor

E-responsive

Estrogen responsive

Pure glyph

Pure glyphosate (99.5% purity)

Ro

Roundup®

Wo

Wipeout®

DI H2O

Deionised water

FITC

Fluorescein isothiocyanate

SEM

Standard error of the mean

TM

Olive tail moment

TL

Tail length (µm)

TDNA

Tail DNA (%)

WHO

World Health Organization

Notes

Acknowledgements

The authors would like to thank the Water Research Commission (WRC) of South Africa for funding. Conclusions drawn and opinions expressed are those of the authors and should not be attributed to the funding body. The authors wish to thank the reviewers for their valuable insight as it has improved the work.

Conflict of interest

All authors declare that they have no conflict of interest in the publication.

References

  1. Abdullah MP, Dau J, Hong KS, Yew CH (1995) Improved method for the determination of glyphosate in water. J Chromatogr 697:363–369CrossRefGoogle Scholar
  2. Acuri A (2017) Glyphosate, forthcoming, In: Jessie H, Daniel J (ed) Objects of International Law', OUP, 2017. https://ssrn.com/abstract=3047054. Accessed 15 Sep 2018
  3. Adams BD, Furneaux H, White BA (2007) The micro-ribonucleic acid (miRNA) miR-206 targets the human estrogen receptor-α (ER-α) and represses ERα messenger RNA and protein expression in breast cancer cell lines. Mol Endocrinol 21:1132–1147CrossRefGoogle Scholar
  4. Amoros I, Alonso JL, Romaguera S, Carrasco JM (2007) Assessment of toxicity of a glyphosate-based formulation using bacterial systems in lake water. Chemosphere 67:2221–2228CrossRefGoogle Scholar
  5. Anadon A, Martinez-Larranaga MR, Martinez MA, Castellano VJ, Martinez M, Martin MT, Nozal MJ, Bernal JL (2009) Toxicokinetics of glyphosate and its metabolite aminomethyl phosphonic acid in rats. Toxicol Lett 190:91–95CrossRefGoogle Scholar
  6. Anderson D, Yu TW, McGregor DB (1998) Review: comet assay responses as indicators of carcinogen exposure. Mutagenesis 13:539–555CrossRefGoogle Scholar
  7. Antachopoulos C, Meletiadis J, Sein T, Roilides E, Walsh TJ (2007) Concentration dependent effects of caspofungin on the metabolic activity of Aspergillus species. Antimicrob Agents Chemother 51:881–887CrossRefGoogle Scholar
  8. Associated Press (2018) Jury awards $289M to man who blames roundup for cancer. Associated Press, New YorkGoogle Scholar
  9. Baylis DA (2000) Why glyphosate is a global herbicide: strengths, weaknesses and prospects. Pest Manag Sci 56:299–308CrossRefGoogle Scholar
  10. Benachour N, Sipahutar H, Moslemi S, Gasnier C, Travert C, Seralini GE (2007) Time and dose dependent effects of roundup on human embryonic and placental cells. Arch Environ Contam Toxicol 53:126–133CrossRefGoogle Scholar
  11. Benamu MA, Schneider MI, Sanchez NE (2010) Effects of the herbicide glyphosate on biological attributes of Alpaida veniliae (Araneae, Araneidae), in laboratory. Chemosphere 78:871–876CrossRefGoogle Scholar
  12. Benedetti AL, De Lourdes Vituri C, Trentin AG, Domingues MAC, Alvarez-Silva M (2004) The effects of sub-chronic exposure of Wistar rats to the herbicide Glyphosate-Biocarb®. Toxicol Lett 153:227–232CrossRefGoogle Scholar
  13. Beuret CJ, Zirulnik F, Gimenez MS (2005) Effect of the herbicide glyphosate on liver peroxidation in pregnant rats and their fetuses. Reprod Toxicol 19:501–504CrossRefGoogle Scholar
  14. Blasiak J, Trzeciak A (1998) Single cell gel electrophoresis (comet assay) as a tool for environmental biomonitoring. An example of pesticides. Pol J Environ Stud 7:189–194Google Scholar
  15. Bolognesi C, Bonatti S, Degan P, Gallerani E, Peluso M, Rabboni R, Roggieri P, Abbondandolo A (1997) Genotoxic activity of glyphosate and its technical formulation roundup. J Agric Food Chem 45:1957–1982CrossRefGoogle Scholar
  16. Buteau-Lozano H, Velasco G, Cristofari M, Balaguer P, Perrot-Applanat M (2008) Xenoestrogens modulate vascular endothelial growth factor secretion in breast cancer cells through an estrogen receptor-dependent mechanism. Endocrinol J 196:399–412CrossRefGoogle Scholar
  17. Calabrese EJ, Baldwin LA (2001) Hormesis: U-shaped dose responses and their centrality in toxicology. Trends Pharmacol Sci 22:285–291CrossRefGoogle Scholar
  18. Cartigny B, Azaroual N, Imbenotte M, Mathieu D, Vermeersch G, Goulle JP, Lhermitte M (2004) Determination of gyphosate in biological fluids by 1H and 31P NMR spectroscopy. Forensic Sci Int 143:141–145CrossRefGoogle Scholar
  19. Chuang CH, Hu ML (2004) Use of whole blood directly for single-cell gel electrophoresis (comet) assay In vivo and white blood cells for In vitro assay. Mutat Res 564:75–82CrossRefGoogle Scholar
  20. Clair E, Mesange R, Travert C, Seralini GE (2012) A glyphosate-based herbicide induces necrosis and apoptosis in mature rat testicular cell in vitro and testosterone decrease at lower levels. Toxicol In Vitro 26:269–279CrossRefGoogle Scholar
  21. Clements C, Ralph S, Petras M (1997) Genotoxicity of select herbicides in Rana catesbeiana tadpoles using the alkaline single-cell gel DNA electrophoresis (comet) assay. Environ Mol Mutagen 29:277–288CrossRefGoogle Scholar
  22. Conolly RB, Lutz WK (2004) Nonmonotonic dose-relationships: mechanistic basis, kinetic modeling, and implications for risk assessment. Toxicol Sci 77:151–157CrossRefGoogle Scholar
  23. Coutinho CFB, Coutinho LFM, Mazo LH, Nixdorf SL, Camara CAP (2008) Short communication: Rapid and direct determination of glyphosate and aminomethylphosphonic acid in water using anion-exchange chromatography with coulometric detection. J Chromatogr A 208:246–249CrossRefGoogle Scholar
  24. De Liz Cavalli VL, Cattani D, Rieg CEH, Pierozan P, Zanatta L, Parisotto EB, Filho DW, Silva FRMB, Pessoa-Pureur R, Zamoner A (2013) Roundup disrupts male reproductive functions by triggering calcium-mediated cell death in rat testis and sertoli cells. Free Radic Biol Med 65:335–346CrossRefGoogle Scholar
  25. Denizot F, Lang R (1986) Rapid colorimetric assay for cell growth and survival: modification to the terazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods 89:271–277CrossRefGoogle Scholar
  26. Duramad P, Tager IB, Leikauf J, Eskenazi B, Holland NT (2006) Expression of Th1/Th2 cytokines in human blood after in vitro treatment with chlorpyrifos, and its metabolites in combination with endotoxin LPS and allergen. Der p1. J Appl Toxicol 26:458–465CrossRefGoogle Scholar
  27. Enciso M, Sarasa J, Agarwal A, Fernandez JL, Gosalvez J (2009) A two-tailed comet assay for assessing DNA damage in spermatozoa. Reprod BioMed Online 18:609–616CrossRefGoogle Scholar
  28. Ertel W, Kramer JP, Kenney J, Steckholzer U, Jarrar D, Trentz O, Schildberg W (1995) Downregulation of proinflammatory cytokines release in whole blood from septic patients. Blood 85:1341–1347PubMedGoogle Scholar
  29. Fairbairn DW, Olive PL, O´Neill KL (1995) The comet assay: a comprehensive review. Mutat Res 339:37–59CrossRefGoogle Scholar
  30. Frei E, Kuchenmeister F, Gliniorz R, Breuer A, Schmezer P (2001) N-nitrososdimethylamine is activated in microsomes from hepatocytes to reactive metabolites which damage DNA of non-parenchymal cell in rat liver. Toxicol Lett 123:227–234CrossRefGoogle Scholar
  31. Gasnier C, Dumont C, Benachour N, Clair E, Chagnon MC, Seralini GC (2009) Glyphosate-based herbicides are toxic and endocrine disruptors in human cell lines. Toxicology 262:184–191CrossRefGoogle Scholar
  32. Gasnier C, Benachour N, Clair E, Travert C, Langlois F, Laurant C, Decroix-Laporte C, Seralini GE (2010) Dig 1 protects against cell death provoked by glyphosate-based herbicides in human liver cell lines. J Occup Med Toxicol 5:1–13CrossRefGoogle Scholar
  33. Gehin A, Guillaume YC, Millet J, Guyon C, Nicon L (2005) Vitamins C and E reverse effect of herbicide-induced toxicity on human epidermal cells HaCaT: a biochemometric approach. Int J Pharm 288:219–226CrossRefGoogle Scholar
  34. George J, Shukla Y (2011) Review: pesticides and cancer: insights into toxicoproteomic-based findings. J Proteom 74:2713–2722CrossRefGoogle Scholar
  35. Glover JF, Irwin JT, Darbre PD (1988) Interaction of phenol red with estrogenic and antiestrogenic action on growth of human breast cancer cells ZR-75-1 and T-47-D. Cancer Res 48:3693–3697PubMedGoogle Scholar
  36. Hartmann A, Speit G (1997) The contribution of cytotoxicity to DNA-effects in the single cell gel test (Comet assay). Toxicol Lett 90:183–188CrossRefGoogle Scholar
  37. Hartmann A, Agurell E, Beevers C, Brendler-Schwaab S, Burlinson B, Clay P, Collins A, Smith A, Speit G, Thybaud V, Tice RR (2003) Recommendations for conducting the In vivo alkaline comet assay. Mutagenesis 18:45–51CrossRefGoogle Scholar
  38. Hokanson R, Fudg R, Chowdhary R, Busbee D (2007) Alteration of estrogen-regulated gene expression in human cells induced by the agricultural and horticultural herbicide glyphosate. Hum Exp Toxicol 26:747–752CrossRefGoogle Scholar
  39. Hovhannisyan GG (2010) Review: fluorescence in situ hybridization in combination with the comet assay and micronucleus test in genetic toxicology. Mol Cytogenet 17:3–11Google Scholar
  40. Hsiang YH, Hertzberg R, Hecht S, Liu LF (1985) Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J Biol Chem 280:14873–14878Google Scholar
  41. Jagetia GC, Rao SK (2011) Assessment of radiation-induced DNA damage by comet assay cultured HeLa cells treated with guduchi (Tinospora cordifolia Miers) before exposure to different doses of γ-radiation. Res Pharmaceutical Biotechnol 3:93–103Google Scholar
  42. Kim YH, Hong JR, Gil HW, Song HY, Hong SY (2013) Mixtures of glyphosate and surfactant TN20 accelerate cell death via mitochondrial damage induced apoptosis and necrosis. Toxicol In Vitro 27:191–197CrossRefGoogle Scholar
  43. Klinger MHF, Jelkmann W (2002) Role of blood platelets in infection and inflammation. J Interferon Cytokine Res 22:913–922CrossRefGoogle Scholar
  44. Kojima H, Takeuchi S, Niiyama K, Kobayashi K (2004) Screening for estrogen and androgen receptor activities in 200 pesticides by In vitro reporter gene assays using Chinese hamster ovary cells. Environ Health Perspect 112:524–553CrossRefGoogle Scholar
  45. Koller VJ, Furhacker M, Nersesyan A, Misik M, Eisenbauer M, Knasmueller S (2012) Cytotoxic and DNA-damaging properties of glyphosate and Roundup in human-derived buccal epithelial cells. Arch Toxicol 86:805–813CrossRefGoogle Scholar
  46. Krakauer T (2002) Stimulant-dependent modulation of cytokines and chemokines by airway epithelial cells: cross talk between pulmonary epithelial and peripheral blood mononuclear cells. Clin Diagn Lab Immunol 9:126–131PubMedPubMedCentralGoogle Scholar
  47. Kreutz LC, Barcellos LJG, De Faria Valle S, De Oliveira Silva T, Anziliero D, Dos Santos ED, Pivato M, Zanatta R (2011) Altered hematological and immunological parameters in silver catfish (Rhamdia quelen) following short term exposure to sub-lethal concentration of glyphosate. Fish Shellfish Immunol 30:51–57CrossRefGoogle Scholar
  48. Kumaravel TS, Jha AN (2006) Reliable comet assay measurements for detecting DNA damage induced by ionizing radiation and chemicals. Mutat Res 605:7–16CrossRefGoogle Scholar
  49. Kwiatkowska M, Huras B, Bukowska B (2014) The effect of metabolites and impurities of glyphosate on human erythrocytes (in vitro). Pestic Biochem Physiol 109:34–43CrossRefGoogle Scholar
  50. Larsen K, Najle R, Lifschitz A, Virkel G (2012) Effects of sub-lethal exposure of rats to the herbicide glyphosate in drinking water: glutathione transferase enzyme activites, levels of reduced glutathione and lipid peroxidation in liver, kidney and small intestine. Environ Toxicol Pharmacol 34:811–818CrossRefGoogle Scholar
  51. Lin N, Garry VF (2000) In vitro studies of cellular and molecular developmental toxicity of adjuvants, herbicides, and fungicides commonly used in Red River Valley, Minnesota. J Toxicol Environ Health 60:423–439CrossRefGoogle Scholar
  52. Lummato MM, Di Fiori E, Sabatini SE, Cacciatore LC, Cochon AC, Del Carmen M, De Molina R, Juarez AB (2013) Evaluation of biochemical markers in the golden mussel Limnoperna fortunei exposed to glyphosate acid in outdoor microcosms. Ecotoxicol Environ Saf 95:123–129CrossRefGoogle Scholar
  53. Luo L, Wang F, Zhang Y, Zeng M, Zhong C, Xiao F (2017) In vitro cytotoxicity assessment of roundup (glyphosate) in L-02 hepatocytes. J Environ Sci Health Part B 52:410–417CrossRefGoogle Scholar
  54. Manas F, Peralta L, Raviolo J, Ovando HG, Weyers A, Ugnia L, Gonzalez Cid M, Larripa I, Gorla N (2009) Genotoxicity of AMPA, the environment metabolite of glyphosate assessed by the comet assay and cytogenetic tests. Ecotoxicol Environ Saf 72:834–837CrossRefGoogle Scholar
  55. Marchiosi R, De Lourdes Lucio Ferrarese M, Bonini EA, Fernandes NG, Ferro AP, Filho OF (2009) Glyphosate-induced metabolic changes in susceptible and glyphosate-resistant soybean (Glycine max L.) roots. Pestic Biochem Physiol 93:28–33CrossRefGoogle Scholar
  56. Markaverich B, Mani S, Alejandro MA, Mitchell A, Markaverich D, Brown T, Velez-Trippe C, Murchison CO, Malley B, Faith R (2002) A novel endocrine-disrupting agent in corn with mitogenic activity in human breast and prostatic cancer cells. Environ Health Perspect 110:169–177CrossRefGoogle Scholar
  57. Martinez A, Reyes I, Reyes N (2007) Cytotoxicity of the herbicide glyphosate in human peripheral blood mononuclear cells. Biomedica 27:594–604CrossRefGoogle Scholar
  58. Mesange R, Bernay B, Seralini GE (2012) Ethoxylated adjuvants of glyphosate-based herbicides are active principles of human cell toxicity. Toxicology 313:122–128CrossRefGoogle Scholar
  59. Mladinic M, Berend S, Vrdoljak AL, Kopjar N, Radic B, Zeljezic D (2009) Evaluation of genome damage and its relation to oxidative stress induced by glyphosate in human lymphocytes in vitro. Environ Mol Mutagen 50:800–807CrossRefGoogle Scholar
  60. Moller P, Knudsen LE, Loft S (2000) The comet assay as a rapid test in biomonitoring occupational exposure to DNA-damaging agents and confounding factors. Cancer Epidemiol Biomarkers Prevention 9:1005–1015Google Scholar
  61. Motulsky H, Christopoulos A (2003) Fitting models to biological data using linear and nonlinear regression: a practical guide to curve fitting. Graphpad Software Inc, San Diego. http://www.graphpad.com. Accessed 15 Mar 2014
  62. Nowak I, Shaw LM (1995) Mycophenolic acid binding to human serum: characterization and relation to pharmacodynamics. Clin Chem 41:1011–1017PubMedGoogle Scholar
  63. Olorunsogo OO, Bababunmi EA, Bassir O (1979) Effect of glyphosate on rat liver mitochondria in vivo. Bull Environ Contam Toxicol 22:357–364CrossRefGoogle Scholar
  64. Paul V, Pandey R (2017) Is the herbicide glyphosate really safe? Curr Sci 112:11–13CrossRefGoogle Scholar
  65. Peixoto F (2005) Comparative effects of the roundup and glyphosate on mitochondrial oxidative phosphorylation. Chemosphere 61:1115–1122CrossRefGoogle Scholar
  66. Pieniąžek D, Bukowska B, Duda W (2004) Comparison of the effect of Roundup ULTRA 360 SL pesticide and its active compound glyphosate on human erythrocytes. Pestic Biochem Physiol 79:58–63CrossRefGoogle Scholar
  67. Richard S, Moslemi S, Siphatur H, Benachour N, Seralini GE (2005) Differential effects of glyphosate and roundup on human placental cells and aromatase. Environ Health Perspect 113:716–720CrossRefGoogle Scholar
  68. Soso AB, Barcellos LJG, Ranzani-Paiva MJ, Kreutz LC, Quevedo RM, Anziliero D, Lima M, Da Silva LB, Ritter F, Bedin AC, Finco JA (2007) Chronic exposure to sub-lethal concentration of glyphosate-based herbicide alters hormone profiles and affects reproduction of femal Jundia (Rhamdia quelen). Environ Toxicol Pharmacol 23:308–313CrossRefGoogle Scholar
  69. Steinmann HH, Dickeduisberg M, Theuvsen L (2012) Uses and benefits of glyphosate in German arable farming. Crop Protect 42:164–169CrossRefGoogle Scholar
  70. Stoddard MB, Pinto V, Keiser PB, Zollinger W (2010) Evaluation of whole blood cytokine release assay for use in measuring endotoxin activity of group b Neisseria meningitides vaccines made from lipid A acylation mutants. Clin Vaccine Immunol 17:98–107CrossRefGoogle Scholar
  71. Thongprakaisang S, Thiantanawat A, Rangkadilok N, Suriyo T, Satayavivad J (2013) Glyphosate induces human breast cancer cells growth via estrogen receptors. Food Chem Toxicol 59:129–136CrossRefGoogle Scholar
  72. Tice RR, Agurell E, Anderson D, Burlison B, Hartmann A, Kobayashi H, Miyamae Y, Rojas E, Ryu E, Sasaki YF (2000) Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen 35:206–221CrossRefGoogle Scholar
  73. Ündeğer Ü, Başaran N (2005) Effects of pesticides on human peripheral lymphocytes in vitro: induction of DNA damage. Arch Toxicol 79:169–176CrossRefGoogle Scholar
  74. Vandenberg LN, Colborn T, Hayes TB, Heindel JJ, Jacobs DR, Lee DH, Shioda T, Soto AM, Vom Saal FS, Welshons WV, Zoeller RT, Myers JP (2012) Review: hormones and endocrine-disrupting chemicals: low dose effects and nonmonotonic dose responses. Endocr Rev 33:378–455CrossRefGoogle Scholar
  75. Ventura C, Nunez M, Miret N, Lamas DM, Randi A, Venturino A, Rivera E, Cocca C (2012) Differential mechanisms of action are involved in chlorpyrifos effects in estrogen-dependent or -independent breast cancer cells exposed to low or high concentrations of the pesticide. Toxicol Lett 213:184–193CrossRefGoogle Scholar
  76. Vera-Candioti J, Soloneski S, Larramendy ML (2013) Evaluation of the genotoxic and cytotoxic effects of glyphosate-based herbicides in the ten spotted live-bearer fish Cnesterodon decemmaculatus (Jenyns 1842). Ecotoxicol Environ Saf 89:166–173CrossRefGoogle Scholar
  77. Vock EH, Lutz WK, Hormes P, Hoffmann HD, Vamvakasa S (1998) Discrimination between genotoxicity and cytotoxicity in the induction of DNA-double strand breaks is cells treated with etoposide, melphalan, cisplatin, potassium cyanide, Triton X 100, and γ-radiation. Mutat Res 413:83–94CrossRefGoogle Scholar
  78. Vom Saal FS, Welshon S (2006) Large effects from small exposures II. The importance of positive controls in low-dose research on bisphenol A. Environ Res 100:50–76CrossRefGoogle Scholar
  79. Wang X, Kilgore MW (2002) Signal cross-talk between estrogen receptor alpha and beta and the peroxisome proliferator-activated receptor gamma 1 in MDA-MB-231and MCF-7 breast cancer cells. Mol Cell Endocrinol 194:123–133CrossRefGoogle Scholar
  80. Woodburn TA (2000) Glyphosate: production, pricing and use worldwide. Pest Manag Sci 56:309–312CrossRefGoogle Scholar
  81. World Health Organization (WHO) (2004) Summary statement: glyphosate and AMPA in drinking water. WHO guidelines for drinking water quality, 3rd edn. WHO, GenevaGoogle Scholar
  82. Wylie I (2015) Glyphosate is a ‘probably carcinogenic’ herbicide. Why do cities still use it? The Rockefeller Foundation, New York, pp 1–9Google Scholar
  83. Yared E, McMillan TJ, Martin FL (2002) Genotoxic effects of oestrogens in breast cells detected by the micronucleus assay and the comet assay. Mutagenesis 17:345–352CrossRefGoogle Scholar
  84. Yue Y, Zhang Y, Zhou L, Qin J, Chen X (2008) In vitro study on the binding of herbicide glyphosate to human serum albumin by optical spectroscopy and molecular modeling. J Photochem Photobiol B 90:26–32CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • L. K. S. De Almeida
    • 1
  • B. I. Pletschke
    • 1
  • C. L. Frost
    • 2
  1. 1.Department of Biochemistry and MicrobiologyRhodes UniversityGrahamstownSouth Africa
  2. 2.Department of Biochemistry and MicrobiologyNelson Mandela UniversityPort ElizabethSouth Africa

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