4 th Global Summit on Toxicology August 24

4 th 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

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

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

N-nitrosocompounds can be exogenous or endogenous N-nitroso compounds exogenous (found in the tobacco, food, etc. ) 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

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).

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

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)

Natural compounds having anticancer and / or antimutagenic activity chemopreventive substances

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

This lecture is taken from parts of the Ph. D 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)”

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

Work conditions p. H 2; T: room temperature (25 ºC) Molar ratio [Na. NO 2]/[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 [Na. NO 2] Green tea infusion: from 0 to 100 L Addition of antimutagen: before or after Na. NO 2 Biological studies Ames test Allium cepa test Physico-chemical studies: UV-Vis spectra

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

S. Typhimurium strains used in this work Strain Genotype TA 98 his. D 3052 Detecting type of mutagens Those that produce reading frameshifts Examples of mutagens * 2 -nitrofluorene * nitrosoderivatives of carcinogenic aromatic amines TA 100 his. G 46 Those that produce basepair substitution Na. N 3, aflatoxin B 1 Kristien Mortelmans, Errol Zeiger Mutation Research 455 (2000) 29– 60

Expression of results * Dose response curve: Nº revertants/plate vs. dose revertant colonies per plate Revertantes por * Reversion Coefficient (RC): Nº revertants of tested plate / Nº revertants of the control plate µg mutagen per plate 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

AMES TEST Substance to be tested S. typhimurium TA 98 or TA 100 strains 48 h; 37 ºC Top agar Minimal agarglucose medium revertant colonies Positive controls: 4 -Nitro-o-phenylenediamine, 2. 5 g/plate (TA 98) and sodium azide, 5 s g/plate (TA 100)

(a) (b) Photo of: a) spontaneous revertants (TA 100 strain) and b) revertants after treatment with the mixture Na. Stz-Na. NO 2 in acidic medium The same previous photo showing the manual counting of revertant colonies (a) (b)

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

Ascorbic acid did not show mutagenicity in the tested conditions in the Ames test with S. typhimurium TA 98 strain

Ascorbic acid did not show mutagenicity in the tested conditions in the Ames test with S. typhimurium TA 100 strain

Green tea extract (µL infusion/plate) did not show mutagenicity in the tested conditions in the Ames test with S. typhimurium TA 98 strain

Green tea extract (µL infusion/plate) did not show mutagenicity in the tested conditions in the Ames test with S. typhimurium TA 100 strain

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)

Na. Stz-Na. NO 2 HStz (Na. Stz as sodium salt)

AA ascorbic acid (HNO 2 + 1 e- + 1 H+ dehydroascorbic acid NO + H 2 O ) 2

Reaction mixture sulfathiazole-nitrite showed mutagenicity in the Ames test with S. typhimurium TA 98 strain

Reaction mixture sulfathiazole-nitrite showed mutagenicity in the Ames test with S. typhimurium TA 100 strain

Ascorbic acid added before nitrite to the reaction mixture inhibited the mutagenic activity of the mixture. strain TA 98; [AA max]/[Na. NO 2]: 1 AA was added before nitrite, i. e. : Na. Stz + HCl + AA + Na. NO 2 AA added before nitrite inhibited mutagenicity Na. Stz: 539 nmol/plate Na. NO 2 : 1740 nmol/plate Control with 1740 nmol AA/plate: 21 ± 3 revertants/plate

Ascorbic acid added to the reaction mixture sulfathiazole-nitrite previously formed inhibited the mutagenic activity of the mixture. strain TA 98; [AA max]/[Na. NO 2]: 1 AA was added after nitrite, i. e. : Na. Stz + HCl + Na. NO 2 + AA AA added after nitrite inhibited mutagenicity Na. Stz: 179 nmol/plate Na. NO 2 : 579 nmol/plate Control with 1740 nmol AA/plate: 21 ± 3 revertants/plate

Ascorbic acid added before nitrite to the reaction mixture inhibited the mutagenic activity of the mixture. Strain TA 100; [AA max]/[Na. NO 2]: 1. 5 AA was added before nitrite, i. e. : Na. Stz + HCl + AA + Na. NO 2 AA added before nitrite inhibited mutagenicity Na. Stz: 539 nmol/plate Na. NO 2 : 1740 nmol/plate Control with 1740 nmol AA/plate: 163 ± 9 revertants/plate

Ascorbic acid added to the reaction mixture sulfathiazole-nitrite previously formed inhibited the mutagenic activity of the mixture. strain TA 100; [AA max]/[Na. NO 2]: 1 AA was added after nitrite, i. e. : Na. Stz + HCl + Na. NO 2 + AA AA added after nitrite inhibited mutagenicity Na. Stz: 179 nmol/plate Na. NO 2 : 579 nmol/plate Control with 1740 nmol AA/plate: 106 ± 10 revertants/plate

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

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

Effect of green tea on the NAStz-Na. NO 2 reaction mixture

Green tea aqueous extract, without dilution, added before nitrite to the reaction mixture inhibited the mutagenic activity of the mixture from 10 L/plate (the first added); strain TA 98 GT was added before nitrite, i. e. : Na. Stz + HCl + GT + Na. NO 2 GT added before nitrite inhibited mutagenicity Na. Stz: 539 nmol/plate Na. NO 2 : 1740 nmol/plate Spontaneous revertants: 32. 00 4. 58

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 TA 98 GT was added after nitrite, i. e. : Na. Stz + HCl + Na. NO 2 + GT GT added after nitrite inhibited mutagenicity Na. Stz: 539 nmol/plate Na. NO 2 : 1740 nmol/plate

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

Green tea aqueous extract, diluted 1/10, added before nitrite to the reaction mixture inhibited the mutagenic activity of the mixture. Strain TA 98 GT was added before nitrite, i. e. : Na. Stz + HCl + GT + Na. NO 2 GT 1/10 added before nitrite inhibited mutagenicity Na. Stz: 539 nmol/plate Na. NO 2 : 1740 nmol/plate Spontaneous revertants: 32, 00 ± 8, 00

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 TA 98 GT was added after nitrite, i. e. : Na. Stz + HCl + Na. NO 2 + GT GT 1/10 added after nitrite inhibited mutagenicity Na. Stz: 539 nmol/plate Na. NO 2 : 1740 nmol/plate

glibenclamide (glyburide)

Increasing concentrations of glibenclamide did not influence the coefficient of reversion of S. typhimurium strains TA 98 and TA 100: glibenclamide did not show mutagenicity in the Ames test in the tested conditions

glibenclamide-Na. NO 2 mixture

Glibenclamide-nitrite reaction mixture did not show mutagenicity in the Ames test with S. typhimurium TA 98 strain in the tested conditions

Glibenclamide-nitrite reaction mixture did not show mutagenicity in the Ames test with S. typhimurium TA 100 strain in the tested conditions

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 TA 98 and TA 100 strain (RC 2) Na. NO 2/glibenclamide: 4/1; [glibenclamide]0: 10 m. M; [Na. NO 2]0: 40 m. M; p. H: 4

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

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. )

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

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. Na. NO 2 mixture. Conditions: room temperature, protected from light

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

The length of the roots as an index of toxicity and modifications in root consistency and shape (formation of tumors, hook roots, twisted roots) were observed as macroscopic parameters.

zone for measuring the mitotic index

The mitotic index was determined by scoring more than 5000 cells (1000 cells per slide). The mitotic index was calculated as the number of dividing cells (taking into account the following mitotic phases: meta-, and telophase) per 1000 observed cells. Mitotic index (increased or decreased) is an indicator of cytotoxicity

genotoxicity chromosomal aberrations Changes in the number and chromosome structure

Chromosomal aberration observed in Allium cepa meristematic cells exposed to chemical agents: a) normal anaphase b) anaphase with chromosome bridge (sulfathiazole-nitrite mixture) Staining reagent Schiff + acetic orcein; magnification: 400 X + zoom digital camera and / or computer

glibenclamide-Na. NO 2

Glibenclamide did not show influence on the root lenght of Allium cepa in the tested conditions (p value 0. 05)

Glibenclamide increased mitotic index (MI) of Allium cepa in the tested conditions (p value 0. 05)

Glibenclamide-nitrite mixture did not show influence on the root lenght of Allium cepa in the tested conditions (p value 0. 05)

Glibenclamide-nitrite mixture did not show influence on the mitotic index (MI) of Allium cepa in the tested conditions (p value 0. 05) comparing, mitotic index showed decrease in the mixture

Increasing amounts of ascorbic acid (AA) with a constant dose of the glibenclamide-nitrite mixture did not show influence on the mitotic index (MI) of Allium cepa (p value 0. 05), then, we may suggest that there was no production of cytotoxic substances with the addition of AA Mineral water (negative control): 31, 60 ± 4, 45. (a) sólo mezcla de reacción: [glibenclamida]0: 8, 10. 10 -5 M; [Na. NO 2]0: 2, 45. 10 -4 M; [HCl]0: 5, 98. 10 -5 M. I. M. : índice mitótico. AA: ácido L-ascórbico

Spectroscopic analysis of sulfa-nitrite mixtures and the antimutagens ascorbic acid (AA) and green tea

Ascorbic Acid AA solution was equimolar with nitrite

Sulfathiazole-nitrite mixture Na. Stz-Na. NO 2

Spectral pattern of sulfathiazole-nitrite mixture plus AA was the same both AA being added before or after the nitrite [AA] Abs 357 nm ( L)

Green tea (GT)

Na. Stz-Na. NO 2

Spectra obtained measuring total polyphenols in a green tea infusion, in the presence and absence of reaction mixture NAStz-nitrite

Spectral pattern of sulfathiazole-nitrite mixture plus GT was the same both GT being added before or after the nitrite green tea Abs 417 nm

Differential spectra of the reaction mixture with increasing amounts of green tea minus the sum of the spectra of the mixture and green tea alone showed increased of absorbance at 417 nm and decreased at 357 nm green tea Abs 417 nm Abs 357 nm

Electronic spectra of glyburide in the presence and absence of Na. NO 2 did not show evidence of reaction Transition due to nitrite

Summarizing …

Spectra analysis allows us to suggest the following: a) the reaction between sulfathiazole and nitrite occurs instantaneously in acidic media. b) the antimutagen (AA or aqueous green tea extract) reacts with the formed mutagens instantaneously and its action is independent of that it is added before or after the nitrite c) glibenclamide (commonly used antidiabetic) does not react with nitrite in the tested conditions, confirming the not observed mutagenicity in such mixtures by the Ames test.

Conclusions For everything mentioned above, both the AA as green tea appear to be effective antimutagens in mitigating mutagenicity of sulfa-nitrite mixtures in acid medium. While this study is only an initial stage, it is an auspicious to reach suggest that oral administration of a sulfonamide is conducted jointly with AA (vitamin C, which may or may not be added to the excipients) or with a cup of green tea to reduce the risk of sulfa reaction with nitrite in the acidic medium of the stomach.

South America I am from Rosario. Where is Rosario? ? Rosario Argentina

The “sulfa´s group”

thank you very much!!!