Mutagenicity of Steviol and Its Oxidative Derivatives in Salmonella typhimurium TM677.

Abstract

Stevioside1) is natural non-caloric sweetener from Stevia rebaudiana BERTONI, which has been used for a long time in Paraguay and Brazil. Purified stevioside is an odorless powder approximately 250 to 300-fold sweeter than sucrose, and is composed of steviol, a diterpenic hydroxy carboxylic acid and three glucose molecules. At present, stevioside is widely used as a non-caloric sugar substitute, and in various kinds of drinks and food products in many countries. In Japan alone, approximately 200 t of S. rebaudiana extract are consumed each year. Stevioside has been subjected to various assessments for safety, and to date, no serious toxic effects have been reported. 2,3) Stevioside and the crude extract of S. rebaudiana have been determined as non-mutagenic in many bacterial test systems, such as some test strains of Salmonella typhimurium, Escherichia coli, and Bacillus subtillis, either in the presence or absence of metabolic activation, which is mostly derived from the rat liver S9 fraction. 4) However, it is known that stevioside is converted to its aglycone, steviol, by intestinal bacteria when orally administered to rats. 5) Pezzuto et al. 6) demonstrated that steviol causes a dose-dependent positive response in the forward mutation assay using S. typthimurium TM677 in the presence of a metabolic activation system (Aroclor-induced rat liver S9 fraction).Recently Suttajit et al. 7) showed that stevioside, at a high dosage (50 mg/plate), possesses a weak mutagenic activity S9 fraction. Since this observed mutagenic effect could be caused by the impurities present in stevioside. Matsui et al. 8) examined seven mutagenicity tests using bacteria (reverse mutation assay, forward mutation assay, umn test and rec assay), cultured mammalian cells (chromosomal aberration test and gene mutation assay), and mice (micronucleus test). Consequently, the aglycone, steviol, was shown to produce dose-related positive responses with the same mutagenicity, i.e. forward mutation assay using S. typhimurium TM677, and chromosomal aberration test using Chinese hamster lung fibroblast cell (CHL) line, and gene mutation assay using CHL. Herein, we wish to identify the genuine mutagenic active substance that is mutagenic toward S. typhimurium in the absence of S9 in steviol derivatives. The present paper describes the mutagenicity (forward mutation assay) studies, which were carried out with steviol and several of its oxidative derivatives.We report that steviol (2), steviol-16a,17-epoxide (3), 15-hydroxy-steviol (4), 15-oxo-steviol (5), steviol methyl ester (6), 16-oxo-steviol methyl ester (7), 3,16-seco-13-oxo-steviol methyl ester (8), 3,16-seco-13-hydroxy-steviol methyl ester (9), and steviol methyl ester 8,13-lactone (10) are mutagenic toward S. typhimurium strain TM677, in the presence of a 9000ϫg supernatant fraction (S9) obtained from the liver of Aroclor 1254-pretreated rats. While only steviol methyl ester 8,13-lactone (10) showed mutagenicity toward the same strain in the presence and in the absence of S9, it is int...

Steviol Glucuronide as Excretion Product of Stevioside in Human Volunteers: Lack of Carcinogenic Properties of Steviol Glycosides and Steviol

“…However, stevioside and even steviol were inactive in various other TA strains of Salmonella typhimurium with or without S9 mix at doses up to 5 mg/plate (TA 97, 98, 100, 102 and 104), in Escherichia coli WP2 wv/vl/pKMlOl and in the rec-assay using Bacillus subtilis even when activation S9 mix was present (99% purity, 13; 96% purity, 14). The direct mutagenic activity of 15-oxo-steviol was refuted by (19), but confirmed by (17). The activity of steviol in Salmonella typhimurium TM677 was very low and was only about 1/3000 that of 3,4-benzopyrene and that of steviol methyl ester 8,13 lactone was 1/24500 that of furylfuramide (17).…”

“…The direct mutagenic activity of 15-oxo-steviol was refuted by (19), but confirmed by (17). The activity of steviol in Salmonella typhimurium TM677 was very low and was only about 1/3000 that of 3,4-benzopyrene and that of steviol methyl ester 8,13 lactone was 1/24500 that of furylfuramide (17). Although a weak activity of steviol and some of its derivatives was found in the very sensitive S. typhimurium TM677 strain, the authors concluded that the daily use of stevioside as a sweetener is safe.…”

“…Mutagenic effects of steviol, the aglycone of stevioside, and/or its metabolites were reported in the forward mutation assay using Salmonella typhimurium TM677 (12; concentrations tested: 0,0.1,0.5, 1,2.5 and 10 mg/ml; [14][15][16][17]. After metabolic activation, it was shown that so far unknown steviol metabolites caused mutations in Salmonella typhimurium TM677, i.e.…”

Stevia in de strijd tegen cariës

“…Bij gebruik als zoetstof zijn geen nadelige effecten op de voortplanting beschreven, noch teratogene, carcinogene of allergische effecten (Geuns, 2003;EFSA 2010). In in vitro-onderzoek is een genotoxisch effect beschreven van steviol en enkele van diens metabolieten in een zogenoemde voorwaartse mutatie-assay in Salmonella typhimurium TM677 (Pezzuto et al, 1985;Terai et al, 2002). Dit effect lijkt zich echter niet zonder meer naar de in vivo-situatie te vertalen: slechts 1 onderzoek heeft een in vivo genotoxisch effect beschreven bij dieren met behulp van een zogenoemde 'komeettest' (Nunes et al, 2007).…”

Stevia’s (Stevia rebaudiana B.) degree of acceptability on a chamomile (Matricaria chamomilla L.) drink infusion

“…Teniendo en cuenta que preocupaciones de genotoxicidad relacionados con la ingesta de steviol glucósidos, tienden a perpetuarse en la literatura, realizaron: (1) revisión de los estudios de genotoxicidad que constituyen fuentes de incertidumbre, (2) revisión adicional de estudios publicados desde 2008, que pueden dilucidar un potencial de genotoxicidad en seres humanos, y (3) evaluación de la necesidad de realizar estudios adicionales de genotoxicidad in vivo. Terai et al (2002) sostienen TM677 posee alta especificidad, y en este sentido no es apropiada para la evaluación de la seguridad de salud humana. Asimismo Brusick (2008) ha sostenido que la dosis de steviol, produce citotoxicidad excesiva (aproximadamente 100% en el gen de ensayo de mutación y >50% de muerte celular en el ensayo de aberración cromosómica).…”