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. 2021 Feb 17;10(2):439.
doi: 10.3390/foods10020439.

In Vivo Genotoxicity Evaluation of a Stilbene Extract Prior to Its Use as a Natural Additive: A Combination of the Micronucleus Test and the Comet Assay

Concepción Medrano-Padial  1 , María Puerto  1 , Ana Isabel Prieto  1 , Nahúm Ayala  2 , Pauline Beaumont  3 , Caroline Rouger  3 , Stéphanie Krisa  3 , Silvia Pichardo  1
Affiliations

In Vivo Genotoxicity Evaluation of a Stilbene Extract Prior to Its Use as a Natural Additive: A Combination of the Micronucleus Test and the Comet Assay

Concepción Medrano-Padial et al. Foods. .

Abstract

Genotoxic data of substances that could be used as food additives are required by the European Food Safety Authority. In this sense, the use of an extract from grapevine shoots containing a stilbene richness of 99% (ST-99), due to its antioxidant and antibacterial activities, has been proposed as an alternative to sulfur dioxide in wine. The aim of this work was to study, for the first time, the in vivo genotoxic effects produced in rats orally exposed to 90, 180, or 360 mg ST-99/kg body weight at 0, 24, and 45 h. The combination of micronucleus assay in bone marrow (OECD 474) and standard (OECD 489) and enzyme-modified comet assay was used to determine the genotoxicity on cells isolated from stomach, liver, and blood of exposed animals. The ST-99 revealed no in vivo genotoxicity. These results were corroborated by analytical studies that confirm the presence of stilbenes and their metabolites in plasma and tissues. Moreover, to complete these findings, a histopathological study was performed under light microscopy in liver and stomach showing only slight modifications in both organs at the highest concentration used. The present work confirms that this extract is not genotoxic presenting a good profile for its potential application as a preservative in the wine industry.

Keywords: comet assay; genotoxicity; in vivo; micronucleus; rat; stilbenes.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
The level of DNA damage measured on cells isolated from liver, stomach, and blood of male and female rats exposed to ST-99 as the formation of strand breaks (SBs) by the standard comet assay (a), and oxidative DNA damage as Endo III-sensitive sites (b) and FPG-sensitive sites (c) by the modified comet assay. The levels of DNA strand breaks and oxidized pyrimidines/purines are expressed as % DNA in tail. All values are expressed as mean ± SD. The significant levels observed are * p < 0.05, ** p < 0.01, or *** p< 0.001.
Figure 2
Figure 2
(a) Plasma and (b) liver concentrations of resveratrol (resv), total resveratrol-glucuronides (resv-G), ε-viniferin (ε-vin), and total ε-viniferin-glucuronide (ε-vin-G) 3 h after final dose of 360 mg ST-99/kg b.w Data are expressed as mean ± SD in ng/mL and ng/g for plasma and liver, respectively (n = 10).
Figure 3
Figure 3
Histopathological changes in the liver of rats exposed to ST-99. Normal hepatic parenchyma is observed in negative control group (A). Detail of hepatic cordons of rat exposed to the positive control (B), showing glycogenic degeneration (arrow) and abundant polyploid hepatocytes (circle). Rats exposed to 90 and 180mg/kg ST-99 showed an apparently normal liver parenchyma (C,D). Rats exposed to 360 mg/kg ST-99 exhibited slight presence hepatic apoptosis throughout the lobule (circle) and glycogenic degeneration (arrow) (E,F).
Figure 4
Figure 4
Histopathological changes in the stomach of rats exposed to ST-99. Apparently normal gastric mucosa is observed in negative control group (A). In positive control rats (B) gastric mucosa with processes of desquamative necrotic gastritis (circle) are observed. Rats exposed to 90 mg/kg ST-99 showed an apparently normal gastric mucosa (C). Unaltered gastric mucosa are observed in rats treated with 180 mg/kg ST-99 (D). Rats exposed to 360 mg/kg ST-99 exhibited desquamative catarrhal gastritis (circle) (E,F).
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