(CANCER RESEARCH 49, 6600-6604, December I, I989| Antimutagenic Effects of Taurine in a Bacterial Assay System1 Stewart A. Laidlaw,2 Marian F. Dietrich, Maria P. Lamtenzan, HernánI. Vargas, Jerome B. Block, and Joel D. Kopple Departments of Medicine fS. A. L., M. F. D., M. P. L., H. I. V., J. B. B., J. I). K.] and Public Health [S. A. L., J. D. K.J. Schools of Medicine and Public Health, llarhitr- VCLA Medical Center and the University of California Los Angeles, Terranee, California 90509 and I.os Angeles, California 90024 ABSTRACT ven, CT), danthrone and MNNG3 (Sigma). GES was kindly supplied by Dr. Ryan Huxtable. Taurine (2-aminoethanesulfonic acid) was evaluated as an antimu- Histidine-requiring mutant strains of Salmonella typhimurium were tagen in the Ames Salmonella tester strain assay. Taurine inhibited obtained from Dr. Bruce Ames. Several strains with sensitivity to mutagenesis by doxorubicin (-74%), bleomycin (-55%), mitomycin specific types of mutational events were used. Strain TA98 primarily C (—56%), and 2-aminofluorene (—52%), but not danthrone or detects frameshift mutations (10); strain TA 100 detects base pair sub benzo(a)pyrene, in strain I \ 102. In strain TA98, doxorubicin mutagen- stitution mutations (10), and strain TA 102 is particularly sensitive to icity, but not that of 2-aminofluorene or ben/o(a)pyrene, was inhibited mutations caused by oxidative damage (11, 12). The S9 microsomal by taurine. A'-Methyl-/V'-nitro-A'-nitrosoguanidine (-73%), but not fraction of homogenized rat liver was isolated by standard techniques dexon, mutagenicity was inhibited by taurine in strain TA100. Taurine (13, 14) and was included in assays of mutagens which require biological inhibited those mutagens against which it was effective in a dose-related activation to produce their mutagenic effects. fashion. Taurine was more effective in inhibiting doxorubicin mutagen The methods used in these studies were those described by Ames and icity in strain TA 102 than its analogues hypotaurine, /3-alanine, and coworkers. Briefly, the tester strains to be used in the assay were grown guanidinoethanesulfonic acid or alanine or glycine. The observed inhibi overnight in Oxoid nutrient broth (Difco Laboratories, Detroit, Ml) in tion may indicate a role for taurine in modulating the activity of oxidant an Orbit Environ-shaker (Lab-line, Melrose Park, IL). Compounds to species. be assayed for mutagenesis were mixed with 0.1 ml of the nutrient broth culture of bacteria, 0.5 ml of rat liver S9 mixture (when appro priate), and 2 ml of molten (45°C)top agar and immediately overlaid INTRODUCTION on Vogel Banner E Medium minimal glucose (2% w/v) plates contain Taurine (2-aminoethanesulfonic acid) is found in all tissues ing minimal liistilline (15), as described by Levin et al. (11). The plates were allowed to harden in the dark and were incubated inverted for 48 and biological fluids in animals and often is present in high h at 37°Cin a humidified incubator (Wedco, Inc., Silver Spring, MD). (HIM) concentrations ¡ntracellularly. Apart from its role as a Under the conditions described, there is a certain background level of conjugator of bile acids (1), its biological functions remain spontaneous revenants to the histidine-independent wild type. The poorly understood. Taurine has been implicated as a stabilizer degree of mutagenicity in response to a specific mutagenic agent is of excitable membranes (1, 2), an osmoregulatory agent (3), calculated as the number of revenants above the background level. and a modulator of neurotransmission (4). Recently, Wright et Revertan! colonies were counted using an Artek Model 880 plate al. (5) suggested that taurine may act as a general detoxifier for counter (Artek Systems, Farmingdale, NY). The range of concentra oxidant compounds, reacting with and removing chemically tions used for a given mutagen were chosen, on the basis of preliminary active free radical species. These species are generated at high testing, to produce the optimal mutagenic effect with minimal toxicity. rates in certain organs and cells, such as the heart, retina, and The toxicity of the compounds tested was assessed by whether the neutrophils, and can cause extensive chemical damage to lipids, number of spontaneous revenant colonies, in the presence of the proteins, carbohydrates, and nucleic acids. Taurine is present compound, fell in comparison to the number of colonies observed in in high concentrations in these cells and tissues (5, 6-8). the absence of the compound. Taurine or other amino acids, when Several known or suspected carcinogenic agents may cause present, were added to the tester strains at the same time as the mutagenic changes in DNA by promoting formation of reactive mutagen. free radical species (9). We therefore began a series of studies The effect of taurine on the mutagenicity of the various mutagens to examine whether taurine may protect against free radical- tested was examined both over a range of mutagen concentrations and at several different taurine concentrations. The effect of taurine on each induced cellular damage. In this study, we used the Ames mutagen was assayed in separate experiments. Each combination of Salmonella bacterial assay system to assess the ability of taurine taurine and mutagen was examined using duplicate plates, and the net to reduce the mutagenic capacity of various known carcinogens. number of revenants reported is the average of these duplicate plates minus the average number of spontaneous revenants. In each assay series, duplicate plate incubations were performed in parallel to the test MATERIALS AND METHODS plate incubations to confirm the mutagenic sensitivity of the tester Chemicals were obtained as follows: taurine (U.S. Biochemical Cor strain used (using a standard mutagen) and to check for toxicity of the poration, Cleveland, OH); glycine, alanine. fi-alanine, and hypotaurine vehicles (water or dimethyl sulfoxide) used to administer the mutagen (Sigma Chemical Co., St. Louis, MO); doxorubicin (Adriamycin; Adria and/or taurine or other amino acids. In addition, at frequent intervals, Laboratories, Columbus, OH); bleomycin (Mead Johnson, Evansville. incubations were performed to confirm the phenotypic characteristics IN); mitomycin C (Bristol-Meyers, Syracuse, NY); benzo(a)pyrene and of each tester strain. The characteristics evaluated were the histidine 2-AF (Aldrich Chemical, Milwaukee, WI); dexon (Analabs. New Ha- requirement, the incidence of spontaneous reversion, the continued exhibition of ampicillin resistance, confirmation of the rfa character, Rcccived 6/13/89; revised 8/16/89; accepted 9/5/89. indicating a partial loss of the bacterial lipopolysaccharide barrier, by The costs of publication of this article were defrayed in part by the payment crystal violet sensitivity (13), and of continued sensitivity to UV light, of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. indicating the absence of a functioning uvrßgene, a functional loss 1Supported by IR01 DK33112 (to J. D. K.). the UCLA Clinical Nutrition characteristic of the strains used (16). Research I'nit 5P01 CA427IO (to S. A. L., J. B. B.), and Eisenhower Medical Data were expressed as (a) the net number of revenants per plate Center. Rancho Mirage. CA (to H. I. V.). Some of the data in this paper were presented at the Annual Meeting of the Federation of American Soeicties for (i.e., the measured number of revenant colonies in the presence of the Experimental Biology. Las Vegas. NV, in May 1987. tested substance minus the number of spontaneous revenants) or (/>) 1To whom requests for reprints should be addressed, at Division of Nephrology and Hypertension. Harbor-UCLA Medical Center. 1000 W. Carson Street. ' The abbreviations used are: MNNG, A'-methyl-.V'-nitro-.V-nitrosoguanidine; Terranee. C'A 90509. 2-AF, 2-aminofluorene: GES. guanidinoethanesulfonic acid. 6600 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1989 American Association for Cancer Research. TAURINE AND MUTAGENESIS 600 the percentage inhibition of reversion, i.e., o— ono tau •—«tau,50mg/plate Rm ~ Rm x 100 500 * »tau,10mg/plate 3 * »tau,1mg/plate .0- 400-• where Rmis the number of revenants in the presence of a mutagen and R„,is the number of revenants in the presence of the mutagen and taurine or other tested compound. RESULTS •00 Taurine itself produced no toxic or mutagenic effects with Adriamycin (/¿g/plate) any bacterial tester strain examined; this was observed with Fig. 1. Effect of increasing concentrations of taurine (tau) on the dose response taurine concentrations from 50 to 50,000 /¿g/plate.As reported of revenants to increasing doses of doxorubicin in Strain TA 102. The scale of previously (17-20), mutagenicity was observed for doxorubicin the abscissa is logarithmic (range, 10-100 ^g doxorubicin/plate); the scale on the (Adriamycin), bleomycin, mitomycin C, 2-AF, dexon, dan- ordinate is arithmetic. Net revertants/plate refers to the number of revenant bacterial colonies observed in the presence of the mutagen minus the number of throne, and benzo(a)pyrene in appropriate strains. The degree revertan! colonies observed when no mutagen was added. Data points represent of mutagenicity observed was dose dependent for each mutagen. the mean of duplicate plate determinations. The figure is representative of the results obtained in several (at least two) replications of the experiment. Using bacterial strain TA 102, the mutagenicity of doxorubi cin, bleomycin, mitomycin C, and 2-aminofluorene was inhib 800 ited by taurine in a dose dependent manner (Table 1; Figs. 1- o—o no tau • «tau,10000ug/p 3). The inhibition tended to be greater at lower concentrations 700 »—»tau,5000ug/p 600 » »tau,1000ug/p of the mutagen and sometimes was not evident at higher mu a otau, 500ug/p tagen levels and/or lower taurine concentrations.