Mutagenic potential of toluidine blue evaluated in the Ames test

bfatufiorl Rewurch, 279 (I 902) 3SS-3SY (0 1991 Elsevier Science Publishers B.V. All rights reserved 0165-12lX/Y2/$OS.t11)

MUTGEN

01774

Mutagenic potential of toluidine blue evaluated in the Ames test Ann J. Dunipace ” Oral Health Rtwurch

‘, Rebecca

Beaven ‘, Timothy Noblitt and George Stookey a

‘, Yiming Li ‘, Susan Zunt b

Institute, 415 Lansing Street. Indianapolis. IN 56202 and ” lndiarta University School

uf Dentists.

1121 West Michigarl Street, Indianapolis. IN 46202 (U.S.A.1 (Received 23 August 1991) (Revision received 2.5 November (Accepted 5 December

Ke_words: Toluidine

blue. mutagenic potential:

Ames test: Thiazine

1991)

1991)

dye

Summary

Toluidine blue is a vital, metachromatic thiazine dye which is used as an adjunct in clinical examination for the early detection of asymptomatic recurrent or secondary primary carcinoma in individuals who are at high risk for developing oral cancer. Because available data on the mutagenicity of toluidine blue was limited and contradictory, this study was conducted to evaluate the mutagenic potential of toluidine blue in the in vitro Ames Salmonella test. Tester strains TA97a, TA98, TAlOO and TA102 were used. Toluidine blue was tested at concentrations of 0.1, 1.0, 10, 50, 100, 250 and 500 pg/plate, with and without S9 microsomal activation, and positive and negative controls were included. Results from tests without S9 showed a significant increase (p < 0.05) in number of revertants in TA102 and in TA97a with 50 and 100 p ; tolrlidine blue/plate, respectively. In tests with S9 activation. doses of toluidine blue ranging from I(! to 250 pg/plate induced dose-related increases in the number of revertants in all 4 strains. The results of this study indicate that toluidine blue has a mutagenic effect in the Ames test.

Vital staining of oral mucoxa with toluidine blue (tolonium chloride) is currently being used as an adjunct to routine clinical examination for the early detection of asymptomatic carcinoma in persons who are at high risk for developing oral cancer (Mashberg, 1981; Silverman et a1.,1984). Toluidine blue (Fig. 1) is a vital, metachromatic thiazine dye, with a molecular weight of 305.84, which is soluble in water and alcohol. Toluidine

blue binds with nuclear DNA and cytoplasmic RNA in addition to other tissue components including sulphates, phosphates and carboxylates,

Fig.

I.

Tolonium

chloride.

methylphenothiazin-S-ium Correspondence:

Dr. Ann J. Dunipace.

Oral Health

Institute, 415 Lansing Street, Indianapolis.

Research

IN 46202 (U.S.A.).

no-2-methylphenazathionium

3-Amino-7-(dimethylaminok2-

chloride:

3-amino-7-dimethyl-ami-

chloride: butene chloride: tolui-

dine blue 0; dimethyltoluthionine

chloride.

,rlt of which are increased in concentration in ::active or neopiastic tissue proliferation. It is believed that toiuidine blue-staining of the oral mucosa in individuals over 40 years of age, in those who consistently use alcohol and/or tobacco, or in patients with persistent ieukopiakia, previous premaiignant or cancerous oral lesions or a history of head and neck radiation, may be a valuable aid in clinical examination and early diagnosis of oral cancer (Mashberg, 1981; Siiverman et al.. 1984; Moyer et al., 1986; Miller et al., 19881. Toluidine blue-staining of the oral mucosa can be accomplished by a rinse technique, direct application of the dye or a two-step procedure which combines the rinse and direct application techniques. In both the rinse and the direct appiication procedures for screening oral soft tissue, toiuidine blue is used at a concentration of 1%. While daily human ingestion of 100-400 mg of toiuidine blue, for its antiheparin effect in controlling uterine bleeding, has been reported to cause minimai side effects of gastric distress, bladder tenesmus and occasional nausea and vomiting, and studies in dogs and humans have confirmed myocardiai toxicity at doses of 10 mg/kg, there have been no reports of toxicity or adverse reactions to oral rinsing or direct topical applications of a 1% toluidine blue solution (Mashberg, 1983). However, an earlier attempt by our laboratory to obtain toiuidine blue for a laboratory study led to the discovery that there was some question about whether or not toluidine blue had ever been officially approved by the FDA for human use and that, while use of this dye is commonly considered to cause minimal toxicity, the genotoxic potential of toiuidine blue has not been thoroughly tested. Limited information is available on the potential genotoxic activity of toluidine blue. From a 1978 study, MacPhee and Imray (1978) reported positive results when toiuidine blue was investigated in the Ames test in conjunction with other compounds, but details of this study were not provided so assessment of those results is not possible. In 1981, Mennigmann and Miiiler found that toiuidine blue failed to induce revertants in the bacterial tester strain TA1537 in the Ames test, and more recently, Ferguson and Baguiey

il’X+!, also rsing the Ames test, reported that toiuidine bme was not genotoxic in bacterial strains TA1537, TA98 or TAlOO. However, both of these studies used fewer than the recommended tester strains, and there was no mention of the use of S9 activation in either study or of the doses at which toiuidine blue was tested. Because the available data on the mutagenicity of toiuidine blue were contradictory, this study was conducted to evaluate the genotoxic potential of toiuidine blue in the in vitro Arnes/microsome mutagenicity test. Materials and methods Chemicals

Toluidine Blue-O, (CAS Registry No. 92-31-91, sodium azide (NaN,, CAS Registry No. 26628-228) 1,8_dihydroxyanthraquinone (Danthron, CAS Registry No. 117-10-21, and revt.-butyi hydroperoxide (TBH, CAS Registry No. 75-91-12) were obtained from Sigma Chemical Company (St. Louis, MO). 4-Nitro-1,Zphenyienediamine (NPD, CAS Registry No. 99-56-91, 2-aminofluorene (2AF, CAS Registry No. 153-78-6) and dimethyi suifoxide (DMSO, CAS Registry No. 67-68-5) were purchased from Aldrich Company (Miiwaukee, WI). p-Biotin (CAS Registry No. 58-85-5) and t_-histidine monohydrate (CAS Registry No. 71-00-01) were from ICN National Biochemicais (Cleveland, OH). Agar (CAS Registry No. 900218-0) was from Difco Laboratories (Detroit, MI). Nutrient broth was obtained from Oxoid Ltd. (Basingstoke, England). Rat-liver S9, induced with Arocior 1254, was purchased from Litton Bionetits (Charleston, SC). Tester strains

Test strains of Salmonella, identified as TA97a, TA98, TAiOO and TA102 were kindly provided by Dr. Bruce N. Ames, Biochemistry Department, University of California at Berkeley. Immediately upon receiving, the tester strains were processed for confirmation of their genotype characteristics. Results of the confirmation tests verified the strains for their characters of histidine requirement, rfa mutation, uvrb mutation, R-factor and pAQ1 plasmid, and for ail assays,

257

fresh cultures were prepared from frozen permanents. Experimental design and test procedures

The procedures for confirmation of genotype characteristics of the strains and for the mutagenicity assays essentially followed those described previously (Ames, 1971; Ames et al., 1975; Maron and Ames, 1983). The test agent, toluidine blue (dissolved in water), in doses ranging from 0.1 to 500 pg/plate, was incorporated in 2 ml of top agar. Distilled, deionized water (DDW) served as the negative (solvent) control. The known mutagenic agents, NaN, and TBH (dissolved in water) and 2AF, NPD and danthron (dissolved in DMSO), were included as positive controls with the specific mutagen depending on the particular strain being tested. Each experiment was performed in triplicate, both with and without 10% S9 activation (50 ,4/plate), for each tester strain and chemical concentration using the standard plate incorporation method. S9 is a microsomal extract prepared from rat liver which has been induced with Aroclor 1254, and its inco~oration adds mammalian enzymes to this in vitro bacterial assay, increasing the sensitivity of the test (Ames et al., 1975). After incubation, all plates

were randomly coded with secondary numbers prior to examination so that the number of revertant colol,aies was determined without knowledge of treatm~rtt. The presence of a background lawn was assessed for each plate. Tke ns~a:; and standard deviation were calculated for the 3 plates in each group, and data were exatzined using Box’s method for homogeneity of variances followed by a one-way analysis of variance (ANOVA). Where variances were not homogeneous, the Welch test replaced the ANOVA. Multiple comparisons were made using Newman-Keul’s method to determine significant differences among the means for different doses. A response was considered positive if the number of induced histidine-dependent revertants was significantly increased over that of the negative control at p = 0.05. Results Data from the tests without S9 activation are summarized in Table 1. Significant increases (p < 0.05) in the number of revertants occurred with strain TA102 at 50 pg of toluidine blue/plate and with strain TA97a at 100 @g/plate, As evidenced by a sparse background lawn and fewer,

TABLE 1 MUTAGENICITY Test agent (@g/plate)

OF TOLUIDINE

BLUE WITHOUT S9 A~IVATION

Revertants per plate (mean fS.D.) TA97a a

DWW (100 $1 Toluidine Blue 0.1 1.0 10.0 50.0 100.0 250.0 500.0 Positive control ’

TA98

4742 37 181k 22h Ob 1113f271d

20*

TAlOZ

TAlOO 4

20f 3 20f 5 34+ 8 27+ 8 25+ 2 32+ 14 29+ 3_ 16734301

155+ 8

324k 24

142+21 188f28 I%+ 6 193+13 176+ 1.5 I 73+10 h Oh 1685 f 49

3441t: 19 279+ 17 33.5rt 29 519+ 22 207+100h’

2238i213

a Values within brackets are not statistically different (P > 0.05) as determined by Newman-Keul’s

analysis.

h Background lawn sparse and colonies relatively small. c 4-Nitro-1,2-phenyldiamine at 20 p&/plate for TA97a and TA98; sodium azide at 5 pg/plate at 100 pg/plate for TA102. d Value not statistically different from negative control (P > 0.05).

for TAlM); [err.-butyl hydroperoxide

TABLE 2 h~lUTAGENICITY OF TOLUIDINE Test agent (&Lg/plate)

BLUE WITH S9 AC’TIVATlON

Revertants per plate (mean k S.D.) TA97a

TA98

TAIMI

TAlO2

DDW 1160~1) Tohtidinc Blue I).1 1.0 10.0

ZJ

z]

-__

511.11 ittO. 250.0 S!lO.O Positive control

ii;]

403+ 45h Oh :\$I>+376

123+ 18 75+ # 43+ 9 3898k 112

200+ 2tl 1241 loh Oh 1045k 186

;;,j 1267; 554 + 104+ Oh 1271+

’ Values within brackets are not statistically different (P > 0.05) as determined by Newman-Keul’s analysis. ’ Background lawn sparse and colonies relatively smaller. ’ 2-aminofluorene at I5 rg/plate for TA97a. TA98 and TAIOO: 1.8-Dihydroxyanthraquinone at 50 pg/plate

smaller colonies,

toluidine

blue became toxic to and to TA97a and TAlOO

TA102 at 100 pg/plate at 250 pg/plate. Table 2 presents the results from tests with S9 activation_ Significant increases (p < 0.05) in the number of revertants occurred with strain TA102 at 10 and 50 pg/plate, with strain TA97a at 10, 100 and 250 pg/plate, with strain TA98 at 100 and 250 pg/plate, and with TAlOO at 100 pg/plate. In these tests, the toxic effects of toluidine blue became evident at 100 pg/plate for TA102, at 250 pg/plate for TAlOO and at 500 pg/plate for TA97a. Discussion

In each test the negative and positive controls gave acceptable numbers of revertants (Maron and Ames, 1983), and values obtained for the controls were consistent with values from previous studies conducted in this laboratory. Positive control agents caused a significant increase in the number of revertants compared to that of the negative control, thereby validating the methodology. Because toiuidine blue was visible in the top agar at the 4 highest doses, blind scoring was made more difficult, though every measure was taken during scoring to ensure non-biased results. At the 2 highest concentrations, the dye re-

170 182 50 h 3s

for TAlO2.

mained undissolved for a period of time, probably allowing some of the bacteria to be exposed to very high concentrations while others were exposed to very little of the chemical initially. Localized areas of cell death may have resulted where undissolved toluidine blue was concentrated. It is possible that these conditions affected the growth of the bacteria leading to some high standard deviations. Where positive results of mutagenicity were obtained, the greatest number of revertants were induced at either 50 or 100 rug/plate. The use of S9 activation resulted in a greater number of revertants than when S9 was not used. The sensitivity of the test in detecting the mutagenic effect of toluidine blue is apparently greater with metabolic activation which serves to create test conditions more similar to those of mammalian metabolism. Studies previously reported in the literature make no mention of testing toluidine blue in the Ames test with the newer tester strains TA97a and TA102 or with S9 activation. This is of concern because the most striking results of this study were found with these strains and when S9 was used. Tester strains T,497a and TA102 were not yet available at the time Mennigmann and M,i.iller (1981) reported that toluidine blue was non-mutagenic in the Ames test when tested with strain TA1537. However, at the time Ferguson

and Baguley (1988) conducted their investigation of toluidin~ blue and reported negative results in strains TA1537, TAN and TAiOO, ail of which were tested without S9 activation, strains TA97a and TA102 as well as the S9 microsome preparation were available and shoufd have been incorporated into their study. The results of this study and of other investigations illustrate the importance of testing a substance in all four standard strains, both with and without S9 activation. As approximately 80% of carcinogens prove to be mutagenic in the Ames test, this in vitro assay is useful for prescreening potential carcinogens (Maron and Ames, 1983). However, mutagen~ci~ assays that are currently available each have strengths and weaknesses, and the mutagenic potential of an agent cannot be reliably determined by any single genotoxic test procedure (de Serres and Matsushima, 198’7).The data obtained from this study indicate that toluidine blue is mutagenic under these test conditions; however, it is important that these positive results be followed by a series of comprehensive, in vivo investigations to further assess the mutagenic potential of toluidine blue.

Ames, 5. (1971) The detection of chemicaf mutagens with enteric bacteria, in: A. Holiaender (Ed.), Chemical Muta-

gens, Principles and Methods of Their Detection. pp. 267-2x7. Ames. B.N.. J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmon&a/ mammalian-microsome mutagenicity test, Mutation Res.. 34.3 I 7-364. de Serres. F.J. and T. Matsushima 11987) Meeting report: deployment of short-term assays for environmental mutagens and carcinogens. Mutation Res., 182. 173-184. Ferguson, L.R.. and B.C. Baguley (1988) Verapamit as a co-mutagen in the Salmonel~a/mamm~~ian microsome mutagenicity test, Mutation Res., 209, 57-62. MacPhee. D.G., and F.P. Imray (1978) Frameshift, Mutagenesis by methyfene blue plus visible light: implications for the Ames test, Mntation Res., .53,359-360. Maron. D.M., and B.N. Ames f 19831 Revised methods for the Salmonella mutagenicity test, Mutation Res.. 113.173-215. Mashherg. A. (1981) Reevaluation of toluidine blue application as a diagnostic adjunct in the detection of asymptomatic oral squamous carcinoma, Cancer, 46. 758-763. Mashberg, A. (1983) Final evaluation of tolonium chloride rinse for screening of high-risk patients with asymptom~tic squamous carcinoma, J. Am. Dent. Assoc.. 10% 319-323. Menni~mann, H.D., and W, Miilter f1981f Dependence of the mutagenic power of heteroatomic dyes on their DNAbase-pair specific@, Mutation Res., 91, 183-191. Miller, R-L., B.W. Simms and A.R. Goutd fl988) Toiuidine blue staining for detection of oral premal~~nant lesions and carcinomas, J. Oral. Pathol., 17, 73-78. Moyer, G.N., G.M. Taybos and G.B. Pelleu G.B.. (1986) Toluidine blue rinse: potential for benign lesions in early detection of oral neopptasms. J. Orat. Med.. 411% 1I I-1 13. Silverman, S., C. Migliorati and J. Barbosa fi984f Toluidine blue staining in the detection of precancerous and malignant lesions. Oral Surg. Oral Med. Oral Pathot.. 57. 3793x2.