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Track 3: Genetic Toxicology

4th Global Summit on Toxicology August 24‐26, 2015 Philadelphia, USA. Track 3: Genetic Toxicology Chemopreventive action of L-ascorbic acid and green tea infusions on the acute toxicity and mutagenicity of reaction mixtures nitrite-sulfonamide Marcela Rizzotto* and Ana Pontoriero

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Track 3: Genetic Toxicology

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  1. 4th Global Summit on Toxicology August 24‐26, 2015 Philadelphia, USA Track 3: Genetic Toxicology Chemopreventive action of L-ascorbic acid and green tea infusions on the acute toxicity and mutagenicity of reaction mixtures nitrite-sulfonamide Marcela Rizzotto* and Ana Pontoriero Rosario National University, Faculty of Biochemistry and Pharmacy, Suipacha 531, 2000 Rosario, Argentina

  2. normal component of the organism Nitrite Cured meats (preservative), vegetables, fertilizers, contaminated drinking water

  3. Nitrite can react with nitrogen compounds in acidic media to give N-nitroso compounds, potentially mutagenic and carcinogenic nitrogen compounds (amines or amides of medicinal drugs or natural compounds) Nitrite acidic media N-nitroso compounds (N-NOC) Nitrosamines Nitrosamides increased risk of tumors of the central nervous system, gastrointestinal tract, bone increased risk of gastric, esophageal, nasopharyngeal, and bladder cancer

  4. N-nitrosocompounds can be exogenous or endogenous exogenous (found in the tobacco, food, etc.) N-nitroso compounds endogenous synthesized in the stomach Since the pioneering studies of Lijinsky et al., numerous papers dealing with the chemical and biological aspects of drug–nitrite interactions have been published. However, the problem of endogenous drug nitrosation is largely unrecognized. Giovanni Brambilla *, Antonietta Martelli, Mutation Research 635 (2007) 17–52

  5. Sulfonamides, or their derivatives are widely used for their various properties (antibacterial and antifungal both in human medicine and in veterinary, antidiabetic agents, diuretics, herbicides, analytical reagents, etc.), so it is common to find them as contaminants, either in the environment or in foods of animal and / or plants. In addition, these are among the antibacterian agents most widely used in the world, mainly because of its low cost, low toxicity and good antimicrobial activity).

  6. Some sulfa drugs in clinical use today sulfanilamide phthalylsulfathiazole sulfathiazole veterinary use sulfamethoxazole trimethoprim glibenclamide (glyburide) silver sulfadiazine

  7. Because sulfonamides possess amine and amide functions in their molecule, they can react with nitrite giving N-nitroso compounds, potentially mutagenic and / or carcinogenic. Primary amine Sulfathiazole Secondary Amide Glibenclamide (Glyburide)

  8. Natural compounds having anticancer and / or antimutagenic activity chemopreventive substances

  9. Inhibit nitrosation reactions by reaction with nitrite L-ascorbic acid Green tea (-) - Epigallocatechin-3-gallate (EGCG), the main polyphenol in green tea

  10. This lecture is taken from parts of the PhD work of Dr. Ana Pontoriero, , entitled: “Chemopreventive action of L-ascorbic acid   and green tea extracts on mutagenicity and acute toxicity of reaction mixtures sulfonamide-nitrite for the following sulfonamides or their derivatives: * sulfathiazole * complex sulfathiazole-cobalt(III) * glibenclamide (glyburide)”

  11. This talk will describe the work with sulfathiazole-nitrite mixture by the Ames test and glibenclamide-nitrite mixture by the Ames and Allium tests. For both mixtures we will analyze Uv-Vis spectra

  12. Work conditions pH  2; T: room temperature (25 ºC) Molar ratio [NaNO2]/[sulfonamide] 1: 3 [sulfonamide]: 0.5  10-4 M to 2.5  10-4 M Concentration of antimutagen L-ascorbic acid (AA): from 0 to  [NaNO2] Green tea infusion: from 0 to  100 L Addition of antimutagen: before or after NaNO2 Biological studies Ames test Allium cepa test Physico-chemical studies: UV-Vis spectra

  13. Ames Test Assay employing Salmonella typhimurium strains genetically modified for detection of mutagens MUTAGEN histidine independent (the strain grows without his: revertant colonies) His+ histidine dependents (without his the strain does not grow) His-

  14. S. Typhimuriumstrains used in this work Kristien Mortelmans, Errol Zeiger Mutation Research 455 (2000) 29–60

  15. Expression of results * Dose response curve: Nº revertants/plate vs. dose * Reversion Coefficient (RC): Nº revertants of tested plate / Nº revertants of the control plate revertant colonies per plate Revertantes por RC  2, in the linear portion of the dose response curve, it means mutagenic materials Maron, D.M. and Ames, B. (1983) Mutation Research 113, 173-215 µg mutagen per plate

  16. AMES TEST Substance to be tested S.typhimurium TA98 or TA100 strains 48 h; 37 ºC Top agar revertant colonies Minimal agar-glucose medium Positive controls: 4-Nitro-o-phenylenediamine, 2.5 g/plate (TA98) and sodium azide, 5 g/plate (TA100)

  17. Photo of: a) spontaneous revertants (TA100 strain) and b) revertants after treatment with the mixture NaStz-NaNO2in acidic medium (a) (b) The same previous photo showing the manual counting of revertant colonies (b) (a)

  18. Effect of the antimutagens L-ascorbic acid (AA) and green tea extracts on the Ames test

  19. Ascorbic acid did not show mutagenicity in the tested conditions in the Ames test with S. typhimurium TA98 strain

  20. Ascorbic acid did not show mutagenicity in the in the tested conditions in the Ames test with S. typhimurium TA100 strain

  21. Green tea extract (µL infusion/plate) did not show mutagenicity in the tested conditions in the Ames test with S. typhimurium TA98 strain

  22. Green tea extract (µL infusion/plate) did not show mutagenicity in the tested conditions in the Ames test with S. typhimurium TA100 strain

  23. Formula used to calculate the % Inhibition of mutagenicity % of inh. = [(RCwithout AM - RCwith AM) / (RCwithout AM - 1)]  100 RC: reversion coefficient AM: antimutagen (AA or green tea)

  24. NaStz-NaNO2 HStz(NaStz as sodium salt)

  25. AA ascorbic acid dehydroascorbic acid (HNO2 + 1 e- + 1 H+ NO + H2O )  2

  26. Reaction mixture sulfathiazole-nitrite showed mutagenicity in the Ames test with S. typhimurium TA98 strain

  27. Reaction mixture sulfathiazole-nitrite showed mutagenicity in the Ames test with S. typhimurium TA100 strain

  28. Ascorbic acid added before nitriteto the reaction mixture inhibited the mutagenic activity of the mixture. strain TA98; [AA max]/[NaNO2]: 1 AA was added before nitrite, i.e.: NaStz + HCl + AA + NaNO2 AA added before nitrite inhibited mutagenicity NaStz: 539 nmol/plate NaNO2 : 1740 nmol/plate Control with 1740 nmol AA/plate: 21 ± 3 revertants/plate

  29. Ascorbic acid added to the reaction mixture sulfathiazole-nitrite previously formed inhibited the mutagenic activity of the mixture. strain TA98; [AA max]/[NaNO2]: 1 AA was added after nitrite, i.e.: NaStz + HCl + NaNO2 + AA AA added after nitrite inhibited mutagenicity NaStz: 179 nmol/plate NaNO2 : 579 nmol/plate Control with 1740 nmol AA/plate: 21 ± 3 revertants/plate

  30. Ascorbic acid added before nitriteto the reaction mixture inhibited the mutagenic activity of the mixture. Strain TA100; [AA max]/[NaNO2]: 1.5 AA was added before nitrite, i.e.: NaStz + HCl + AA + NaNO2 AA added before nitrite inhibited mutagenicity NaStz: 539 nmol/plate NaNO2 : 1740 nmol/plate Control with 1740 nmol AA/plate: 163 ± 9 revertants/plate

  31. Ascorbic acid added to the reaction mixture sulfathiazole-nitrite previously formed inhibited the mutagenic activity of the mixture. strain TA100; [AA max]/[NaNO2]: 1 AA was added after nitrite, i.e.: NaStz + HCl + NaNO2 + AA AA added after nitrite inhibited mutagenicity NaStz: 179 nmol/plate NaNO2 : 579 nmol/plate Control with 1740 nmol AA/plate: 106 ± 10 revertants/plate

  32. green tea Inhibitor of nitrosation reactions by reaction with nitrite. The aqueous green tea extract treated with nitrite in acid medium showed no mutagenicity in the Ames test

  33. Rich in catechins Catechins may react with nitrite in acidic media Green tea (-) epigallocatechin-3-gallate (EGCG), catechin present in greater proportion in green tea

  34. Effect of green tea on the NAStz-NaNO2 reaction mixture

  35. Green tea aqueous extract, without dilution, added before nitriteto the reaction mixture inhibited the mutagenic activity of the mixture from 10 L/plate (the first added); strain TA98 GT was added before nitrite, i.e.: NaStz + HCl + GT + NaNO2 GT added before nitrite inhibited mutagenicity NaStz: 539 nmol/plate NaNO2 : 1740 nmol/plate Spontaneous revertants: 32.00  4.58

  36. Green tea aqueous extract, without dilution, added to the reaction mixture sulfathiazole-nitrite previously formed inhibited the mutagenic activity of the mixture from 10 L/plate (the first added);strain TA98 GT was added after nitrite, i.e.: NaStz + HCl + NaNO2 + GT GT added after nitrite inhibited mutagenicity NaStz: 539 nmol/plate NaNO2 : 1740 nmol/plate

  37. As green tea aqueous extract without dilution, added to the reaction mixture before or after nitrite inhibited the mutagenic activity of the mixture from the first added, we decided to try it diluted 1/10

  38. Green tea aqueous extract, diluted 1/10, added before nitriteto the reaction mixture inhibited the mutagenic activity of the mixture. Strain TA98 GT was added before nitrite, i.e.: NaStz + HCl + GT + NaNO2 GT 1/10 added before nitrite inhibited mutagenicity NaStz: 539 nmol/plate NaNO2 : 1740 nmol/plate Spontaneous revertants: 32,00 ± 8,00

  39. Green tea aqueous extract, diluted 1/10, added to the reaction mixture sulfathiazole-nitrite previously formed inhibited the mutagenic activity of the mixture following a dose-response curve; strain TA98 GT was added after nitrite, i.e.: NaStz + HCl + NaNO2 + GT GT 1/10 added after nitrite inhibited mutagenicity NaStz: 539 nmol/plate NaNO2 : 1740 nmol/plate

  40. glibenclamide (glyburide)

  41. Increasing concentrations of glibenclamide did not influence the coefficient of reversion of S. typhimuriumstrains TA98 and TA100:glibenclamide did not show mutagenicity in the Ames test in the tested conditions

  42. glibenclamide-NaNO2 mixture

  43. Glibenclamide-nitrite reaction mixture did not show mutagenicity in the Ames test with S. typhimurium TA98 strain in the tested conditions

  44. Glibenclamide-nitrite reaction mixture did not show mutagenicity in the Ames test with S. typhimurium TA100 strain in the tested conditions

  45. Glibenclamide-nitrite reaction mixture prepared according to the conditions suggested by WHO for the study of a drug nitrosabilidad did not show mutagenicity in the Ames test with S. typhimurium TA98 and TA100 strain (RC  2) NaNO2/glibenclamide: 4/1; [glibenclamide]0: 10mM; [NaNO2]0: 40mM; pH: 4

  46. As glyburide-nitrite mixture in acidic media did not show mutagenic behavior in the Ames test, we decided to try it in the Allium cepa test

  47. Allium cepa test Biological assay using bulbs of Allium cepa L to determine phyto, cyto and / or genotoxicity of substances in liquid media (waters, effluents, several chemicals, medical drugs, etc.)

  48. Plant genotoxicity test (Allium cepa test) Since 1938, Allium cepa L (common onion) biological material has been widely used in laboratory tests, because of the rapid growth of its roots, the response of its genetic material to the presence of potentially cytotoxic and genotoxic substances in liquid media and a high correlation with mammal test systems. Another advantage of this test system is the presence of an oxidase enzyme system that is essential for promutagen evaluations, besides its low cost, simplicity and no sterile conditions . D. M. Leme, M. A. Marin-Morales, Mutat. Res. 2009, 682, 71– 81

  49. Equal-sized young bulbs of common Allium cepa were used. Onion bulbs (seven per dose) were kept in mineral water for 48 h, then exposed to glibenclamide and glibenclamide-NaNO2 mixture. Conditions: room temperature, protected from light

  50. Parameters evaluated in the Allium test Macroscopic parameters * Root elongation * Presence of anomalies as hooks, necrosis, tumors Microscopic parameters * Mitotic index * Chromosomal aberrations * Nuclear abnormalities * Micronucleus

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