Mutagenic Activity of Rhodamine Dyes and Their …...B or 75.2% for rhodamine 6G), and mutagen (14.28% for rhodamine B or 1.0% for rhodamine 6G). The optimal propor tion of S9 in the

[CANCER RESEARCH 39, 4412-441 7, November 1979]0008-5472/79/0039-OOOOS02.00

Mutagenic Activity of Rhodamine Dyes and Their Impurities as Detected

by Mutation Induction in Salmonella and DMA Damage in ChineseHamster Ovary Cells1

Earle R. Nestmann,2 George R. Douglas, Tibor I. Matula, Caroline E. Grant, and David J. Kowbel

Mutagenesis Section, Environmental and Occupational Toxicology Division, ¡E.R. N., G. R. D., C. E. G., D. J. K.J. and Drug Interactions Section, Drug ToxicologyDivision [T. I. M.], Health Protection Branch, Department of National Health and Welfare, Ottawa, Ontario, K1A OL2, Canada

ABSTRACT

Commercial rhodamine dyes 6G and B induce His* reversion

mutations in Salmonella and single-strand breaks in Chinesehamster ovary cells, as detected by alkaline sucrose sedimentation. Aroclor 1254-induced rat liver homogenate (S9) is re

quired for production of genetic activity by these dyes. Rhodamine 6G induces both frameshift and base substitution mutations, whereas rhodamine B induces only frameshift mutations. Rhodamine 6G is genetically more active and more toxicthan is rhodamine B in both the bacterial and mammalianassays. Rhodamine 6G and B induce doublings of His* revert-

ants in Salmonella at the doses of 0.02 and 0.52 ¿umol/plateand shifts in the molecular weight of Chinese hamster ovaryDMA at concentrations of 9 x 10~5 and 9 x 10"" M, respec

tively. All genetic effects assayed demonstrate dose-related

increases. Further testing of the pure dyes in Salmonella revealed that rhodamine B loses most of its mutagenicity withpurification, whereas rhodamine 6G does not. Impurities fromcommercial rhodamine B demonstrate the same extent of mutagenicity as the commercial dye.

INTRODUCTION

Rhodamine dyes have many uses as tracing agents in waterpollution and aerial pesticide spraying studies and as colors indrugs, cosmetics, textiles, and inks (9, 10). Rhodamine dyes6G and B (Color Indexes 45160 and 45170) have been foundto be carcinogenic (22), but this study has been consideredinadequate (9, 10). Rhodamine B has been shown to inducesignificantly higher than normal numbers of chromosomalbridges and fragments in anaphases and telophases of thebroad bean, Vicia faba (13). In a preliminary screening test,rhodamine B also has been shown to induce mutations inSalmonella (7). On the other hand, both rhodamine dyes 6Gand B were found to cause no DMA damage in an in vitrobacterial test without metabolic activation (11 ). No other published evidence of mutagenicity, teratogenicity, or embryotox-

icity caused by these dyes is available (9, 10).Due to their extensive use and potential mutagenicity and

carcinogenicity, rhodamine dyes 6G and B were examined forinduction of genetic effects using the Sa/mone//a/mammalian-microsome assay for mutation (3) and CHO3 cells in vitro for

detection of DNA strand breakage.

The Sa/mone//a/mammalian-microsome test, as described

by Ames ef al. (3), is used to detect bacterial mutagens. Manyof these mutagenic chemicals also induce mutation and cancerin mammals. A high correlation between mutagenicity in thistest and carcinogenicity in mammals has been noted (15),although the percentage of correlation varies with the types ofchemicals chosen for testing (4, 5). The value of this test isdue in great part to the incorporation of a measure of mammalian metabolism, in the form of a rat liver homogenate (S9)in the assay system. Liver microsomes contain enzymes whichcan convert promutagens into genetically active mutagens,detectable by His* reversion in different strains of Salmonella

(1).Breakage of DNA in cultured cells of CHO, as detected by

alkaline sucrose gradient sedimentation, is a procedure thatcan be used with metabolic activation to detect chemicals withpotential genetic activity (12). Agents that cause such lesionsin DNA are potential mutagens and/or carcinogens [see reviewby Roberts (19)]. The present method utilizes an assay for DNAbreakage in conjunction with an assay for cell survival.

Utilization of the tests in this study demonstrates the value ofcombining both microbial and mammalian experiments in mutagenicity testing. The mammalian test for DNA damage canconfirm the findings of the microbial test and simultaneouslycan indicate the capability of a chemical to react with mammalian DNA. Furthermore, the level of survival of mammaliancells at concentrations that induce DNA breaks allows speculation as to the biological relevance of this damage.

MATERIALS AND METHODS

Cell Culture. Salmonella typhimurium strains TA1535,TA1537, TA1538, TA98, and TA100, and media for bacterialtesting were as described by Ames ef a/. (3).

Monolayer cultures of wild-type CHO cells were maintained

in MEM supplemented with 10% FCS, nonessential aminoacids, and sodium pyruvate (Grand Island Biological Co., Burlington, Ontario, Canada). Cells were grown at 37°, 5% CO2,

and high relative humidity. Cultures were routinely screenedfor the presence of Mycoplasma and found to be free ofcontamination. All cell manipulations were carried out underillumination from General Electric (Montreal, Quebec, Canada)gold fluorescent lamps (Model F40GO 6PK) which do not emit

1 Preliminary reports of this work were presented at the 10th Annual Meeting

of the Environmental Mutagen Society. New Orleans, La., March 8 to 12, 1979.2 To whom requests for reprints should be addressed.

Received April 10, 1979; accepted July 25, 1979.

3 The abbreviations used are: CHO, Chinese hamster ovary; S9, supernatant

from centrifugea (9000 x g) liver homogenate used for metabolic activation;MEM, Eagle's minimal essential medium; FCS, fetal calf serum; DMSO, dimethyl

sulfoxide; BP, benzo(a)pyrene; 2-AAF, 2-acetylaminofluorene; dThd, thymidine,TLC, thin-layer chromatography.

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Mutagenicity of Rhodamine Dyes

light at wavelengths below 500 nm.Chemicals. Rhodamine B (Sigma Chemical Co., St. Louis,

Mo.; 90% purity) was dissolved in DMSO (Aldrich ChemicalCo., Milwaukee, Wis.) in one preliminary bacterial test. In allother bacterial experiments, stock solutions of rhodamine dyesB and 6G (ICI, Alderly Park, Cheshire, United Kingdom) wereprepared with sterile distilled H2O. For tests with CHO cells,rhodamine B was dissolved in MEM containing 2.5% PCS sincethe addition of water makes the treatment mixture hypotonie.Rhodamine 6G was difficult to dissolve in MEM so DMSO wasused with no apparent change in experimental results. BP(Aldrich Chemical Co.), 2-AAF (ICN K & K Laboratories, Inc.,

Plainview N.Y.), and aflatoxin B, (Makor Chem. Ltd., Jerusalem, Israel), dissolved in DMSO served as positive controls.

Bacterial Testing Procedures. The procedures were thosedescribed by Ames era/. (3). Aroclor 1254 (Analabs, Inc., N.Haven, Conn.) -induced and phénobarbital (BDH, Toronto,Ontario, Canada) -induced liver homogenates (S9) were pre

pared following the methods of Ames et al. (1, 3). Unlessotherwise specified, the abbreviation S9 by itself means thatthe rat liver was induced by Aroclor 1254. The graphs of Mohnand Ellenberger (17) are used to estimate the probability thatchemically induced mutation frequencies are different fromcontrol values.

Mammalian Testing Procedures. CHO cells were labeledovernight in 60-mm tissue culture dishes containing 8 x 10s

cells in MEM supplemented with 10% newborn calf serum,nonessential amino acids, sodium pyruvate, and 1.0 /iCi [3H]-dThd per ml (46 Ci/mmol) or 1.0 /¿Ci[14C]dThd per ml (50

mCi/mmol). The cells were chased with nonradioactive mediumfor 2 hr prior to treatment.

The treatment mixture contained S9 (7.1%), 20 mw 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.2

(4.77%), 0.5 M MgCI2 (0.238%), 3.3 M KCI (0.238%), 50 mwglucose-6-phosphate (2.38%), 40 rriM NADP+ (2.38%), dis

tilled H20 (6.67%), MEM with 2.5% PCS (61.9% for rhodamineB or 75.2% for rhodamine 6G), and mutagen (14.28% forrhodamine B or 1.0% for rhodamine 6G). The optimal proportion of S9 in the activation mixture was determined in preliminary experiments and does not affect the sedimentation properties of CHO DMA in alkaline sucrose gradients. Cells weresuspended in 1.4 ml of the treatment mix and treated for 1 hrin a 60-mm dish. After removal of the mixture, the cells werewashed 2 times in ice-cold, calcium- and magnesium-free,phosphate-buffered saline (KCI, 0.20 g/l; KH2PO4, 0.20 g/l;NaCI, 8.00 g/l; Na2HPO4-7H2O, 2.16 g/l) (Grand Island Biological Co.). Cells were removed from the culture dishes bygentle scraping and resuspended in 1 ml ice-cold phosphate-

buffered saline.Alkaline sucrose sedimentation was carried out according to

the method of Palcic and Skarsgard (18). Approximately 8 x103 [14C]dThd-labeled, control cells and 8 x 103 [3H]dThd-

labeled, treated cells were lysed on the gradients for 11 hr at20°and spun at 15,000 rpm for 6 hr in a Beckman SW50-1

rotor. Gradients were fractionated from the top into 0.2-ml

fractions, collected in miniscintillation vials, and counted in 5ml aquasol (New England Nuclear, Montreal, Quebec, Canada)after neutralization with 0.5 N HCI.

Calculation of molecular weights was carried out using themethod described by Palcic and Skarsgard (18) with a sedi

mentation constant derived from the sedimentation characteristics of bacteriophage T4 DNA. The cosedimentation of controland treated DNA greatly increases the precision of the breakage estimate.

Cell survival was estimated by determining colony-formingability at different dye concentrations. Approximately 180 cellswere plated in 60-mm tissue culture dishes in MEM:10% PCS

as above and allowed to attach for 4 to 5 hr. The cells werethen treated as above for 1 hr, washed gently with Hanks'

balanced salt solution 3 times and incubated for 6 to 7 days.Colonies were fixed in 3:1 ethanohglacial acetic acid, stainedwith hematoxylin, and counted.

Procedures for Purification of Rhodamine Dyes. Separationof subsidiary colors and impurities from rhodamine B wasperformed as described by Bell (6). For the purification ofrhodamine 6G, the developing solvent system was benzene:methanohammonia (80:20:0.2). The dyes were dissolved inmethanol and spotted in a band on each plate with a micropipet.Each TLC plate (20 x 20 cm; New England Nuclear) wasdivided into 3 portions. For rhodamine B they were: (a) themain color band (top color band), designated pure rhodamineB; (b) a small color band, just below the main color band; (c)remainder of plate containing all other subsidiary color bandsand UV visible bands, above and below the 2 major bands. Forrhodamine 6G, the 3 fractions were: (a) the main color band,close to the origin, designated pure rhodamine 6G; (b) a smallcolor band, just above the main color band; (c) remainder ofplate containing all other UV visible bands between the mainband and the solvent front.

Silica Gel H (250 /im), representing the 3 fractions, wasscraped and extracted repeatedly with methanol until little colorremained. The concentrations of the dyes recovered from theTLC plates were determined spectrophotometrically at 530 or547 nm for rhodamine B and 6G, respectively. Each portionwas dried under nitrogen and dissolved in equal volumes ofsterile distilled H2O for testing.

RESULTS

Induction of Mutation in Salmonella by Rhodamine B. Inpreliminary screening experiments with doses ranging from0.25 to 2.0 mg/plate, rhodamine B dissolved in DMSO inducedHis* reversions in strains TA1538 and TA98, but not in strains

TA1535, TA100, or TA1537. At 1.0 mg/plate, the rhodamine-

induced increases in yields of revertants compared to background were 4-fold with 50 /il S9 per plate and 11 -fold with 20

/il S9 per plate (data not shown).To confirm these findings, rhodamine B, dissolved in sterile

distilled H2O, was retested using differing amounts of Aroclor1254-induced S9 (Table 1, A) and also phenobarbital-inducedS9 (Table 1, P). The data are presented in Table 1 which showsthat rhodamine B is not mutagenic in strains TA1535 andTA100, which revert by base substitution mutation, nor in strainTA1537, revertible by frameshift mutation (1). Rhodamine B ismutagenic in strains TA1538 and TA98, which revert by aframeshift mutation (1). Rhodamine B-induced mutagenesis

occurs only with metabolic activation by either S9 preparation,A or P. Comparison of data for strain TA1538 in Columns Aand A(30), with 50 and 30 /il S9 per plate, respectively, showsthat reduction of S9 increases rhodamine B-induced mutagen-

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E. R. Nestmann et al.

Table 1His * reversion by rhodamine B in strains of Salmonella

Different amounts of rhodamine B were tested for mutagenic activity with Salmonella strains TA1535, TA100, TA1537, TA1538, and TA98,using methods described previously (3). Preparations of S9 were made using rat liver induced by Aroclor 1254 or by phenobarbitol.

No. of His*revertantsTA1535Compound

andmg (^moO/plate* APWater

8 6 685RhodamineB0.100(0.21)

8 9 6980.125(0.26)0.250(0.52)0.500

(1 .04) 8 9 6680.750(1.56)1

.000 (2.09) 7 6 4641.500(3.13)2.000(4.17)

24353.000(6.26)4.000

(8.35)BP0.010(0.04)

29e2-AAF0.050

(0.22) 6TA100

TA1537A

P -A98

99 5886

92 8867

94 6654

50 556

924486°

43e1330eA(30)

P12

8121199

61813

6914

4131028eTA

1538A

A(30)6

20308

234360e8

43e65e91e8

48e 166e190e5

158e223e272e282e302e

194e1176eTA98P

A(30)10

185017

11525026e

1476d146e46e

13 152e151e1

56e 5 184e215e105e378e1

284e 2254e

a The volume added per plate is 0.1 ml. Rhodamine B is dissolved and diluted in sterile, distilled water. BP and 2-AAF are dissolved in

DMSO.b -, numbers of revertants are means of quadruplicate plates without activation; A, A(30), numbers of revertants are means of duplicate

plates with 50 or 30 p\ of Aroclor 1254-induced S9 per plate; P, numbers of revertants are means of duplicate plates with 50 ¿ilof phenobarbitol-induced S9 per plate.

c Significantly increased over control, p < 0.01d Significantly increased over control, p < 0.05.

icity by more than 3-fold at the dose of 1 mg/plate with lesschange at other doses. The optimum amount of S9 per plateseems to be 30 ¡i\for rhodamine B-induced mutation, but BP

requires more S9 per plate (50 jul) for optimal mutagenesis (1 ).Although the mutagens used as positive controls (BP and 2-

AAF) induce more reversions in strain TA98 than in TA1538,rhodamine B is more mutagenic and less toxic in TA1538 thanin TA98.

Since the commercial rhodamine B used in this study isapproximately 90% pure, TLC was used to obtain a purifieddye. Table 2 shows the mutagenic responses of identicalvolumes of the 3 fractions obtained. Compared to 8-fold in

creases induced by commercial rhodamine B, the main bandand the major subsidiary band, Fractions 1 and 2, respectively,exhibit only slightly more than doublings of the backgroundfrequencies of His* reversions. However, Fraction 3 (the other

impurities) shows nearly the same mutagenicity as does commercial rhodamine B. Since the amount of impurities tested isadjusted to be the same as in an equal volume of the commercial dye, it is concluded that most of the mutagenic activity ofcommercial rhodamine B is due to contaminating impurities.

Cell Killing and DNA Damage in CHO Cells by RhodamineB. Commercial rhodamine B activated by S9 causes damagein single strands of DNA in CHO cells, as indicated by reductionin sedimentation velocity in alkaline sucrose gradients. Theseresults are plotted in Chart 1 which shows that lethality is highat concentrations producing little DNA damage. For example,the DOdose (9.6 x 10~" M) results in minimal DNA damage,less than 0.2 breaks/10s daltons.

Induction of Mutation in Salmonella by Rhodamine 6G.Table 3 shows results from 1 of 2 experiments assaying thepotential mutagenicity of rhodamine 6G in the Salmonella/microsome system. As in the case of rhodamine B, rhodamine

Table 2His * reversion by purified rhodamine B and impurities in Salmonella strain

TA 1538 with metabolic activation

The experimental conditions were similar to those described in Table 1.

TreatmentNegative

control(H2O)CommercialrhodamineBFraction

1 (rhodamine B, purified mainband)Fraction

2 (small colorband)Fraction

3 (other impurities)6Amount/plate1.0

mg2.0mg1.0mg2.0mg0.1mí0.2mlc0.1mlc0.2

ml0His*

re

vertants/plate322157b173b5<57641"526112"171"

Numbers of revertants are means of duplicate plates. Amount of S9 addedis 30 fil/plate.

b Significantly increased over control, p < 0.01.e These volumes contain concentrations of these fractions that correspond to

1.0 and 2.0 mg of the purified main color band.d Significantly increased over control, p < 0.05.6 All other minor color and UV visible bands.

6G is mutagenic in strains TA1538 and TA98, but it is alsomutagenic in strains TA1537 and TA100. Thus, unlike rhodamine B, rhodamine 6G causes base substitution (in strainTA100) in addition to frameshift mutations. Rhodamine 6Grequires activation by S9 to induce mutation in Salmonella.Rhodamine 6G induces a doubling of revertants at only 3%(7.8 fig) of the dose required for a doubling by rhodamine B(250 jug) in strain TA1538. At its maximum, rhodamine 6Ginduces a 30-fold increase of revertants above background in

strain TA1538, whereas the maximal increase induced byrhodamine B is 9-fold. Rhodamine 6G shows toxicity at 500

/ig/plate in strain TA100 and at 1000 jug/plate in strainsTA1537, TA1538, and TA98, as indicated by the decreasing

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Mutagenicity of Rhodamine Dyes

numbers of revertants. Mutagens used as positive controls inTable 3, BP, 2-AAF, and aflatoxin B,, show the expected

mutagenic responses (16).To test the possibility that rhodamine 6G-induced mutagen-

icity is dependent upon photodynamic action, a solution ofrhodamine 6G was prepared in a darkened vial. Experimentswere performed in the absence of overhead illumination, withdaylight supplied by a north window 10 m behind. The plateswere in the dark for the duration of their incubation. The data,using strains TA100 and TA1538, are similar as in routineexperiments done with limited exposure of the dye or of theplates to light (Table 3).

tr=>

oOL

100 -

90 -

80 -

70

60

50

40

30

20

IO

IO"

2.0

1.8

1.6

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0

CET3m

orHo

IO"

RHODAMINE B CONCENTRATION (M)

Chart 1. Relationship between single-strand DNA damage (•)and colony-forming ability (O) in CHO cells treated with metabolically activated rhodamine Bfor 1 hr.

Rhodamine 6G was purified, and Table 4 shows the resultsof an experiment testing for mutagenicity of the 3 fractionsisolated in Salmonella strain TA1538 with S9. Compared withthe commercial dye, pure rhodamine 6G loses 22 to 23% of itsmutagenicity, but it still induces a 24-fold increase in revertants

over the control. Fraction 2 induces a tripling of revertants, andFraction 3 shows a 7-fold increase, about the same as for

Fraction 3 from the purification of rhodamine B. Due to themutagenic activity of Fraction 1, the purified dye, it is concludedthat pure rhodamine 6G is a mutagen.

Cell Killing and DNA Damage in CHO Cells by Rhodamine6G. Rhodamine 6G activated by S9 causes damage to singlestrands of DNA, as illustrated in Chart 2, which shows theeffect of rhodamine 6G in CHO cells and on CHO DNA. Although rhodamine 6G is approximately 10 times more potentin inducing DNA damage than is rhodamine B in these experiments, rhodamine B is much more toxic at concentrations

Table 4His" reversion by purified rhodamine 6G in Salmonella strain TA 1538 with

metabolic activationThe experimental conditions were similar to those described in Table 1.

TreatmentNegative

control (H2O)Commercial rhodamine6GFraction

1 (rhodamine 6G, purified main band)

Fraction 2 (small color band)

Fraction 3 (¡mpurities)cAmount/

plate0.1

ml125.0 /¿g250.0 /ig125.0 /ig250.0 /ig

0.1 ml"0.2 mlb0.1 ml"0.2 ml"His*

re

vertants/plate'119

306579235447

455961

142

Numbers of revertants are means of duplicate plates. Amount of S9 is 30iul/plate. All revertan! yields are significantly increased over control, p <0.01.

These volumes contain concentrations of these fractions that correspond to125 and 250 /ig of the purified main band.

c All other minor color and UV visible bands.

Table 3His * reversion by rhodamine 6G in strains of Salmonella

The experimental conditions were similar to those described in Table 1.No. of His*revertants/plate6Compound

and (igdumol)/plateaWaterRhodamine

6G3.9(0.01)7.8(0.02)15.6(0.03)31.3(0.07)62.5(0.13)125.0(0.26)250.0(0.52)500.0(1.04)1000.0(2.09)BP10.0(0.04)2-AAF50.0

(0.22)AflatoxinBi0.1TA

1535 TA 100TA1537A

A6

11 108115261d10

7 114476d914 110736d511 161021d240

389d0000428"1300e*Dark

-A115

7101620383d

526tf711d 2 31"1091"

37\d272"0

793rf38"TA

1538TA98A4

324867d96"152"5

277"3477d6731d1945d0

93d194d1506*49Dark

A20

50586998d156d188"

206"288d350d566o

575d954"311"378rf2254d495d

The volume added/plate is 0.1 ml. Rhodamine 6G is dissolved in sterile, distilled water. BP, 2-AAF, and aflatoxinBi are dissolved in DMSO.

Numbers of revertants are means of duplicate plates. Amount of S9 for results in the Aroclor and "Dark" columns

is 30 /il/plate.c -, without activation; A. Aroclor; Dark, experiments were performed in subdued light." Significantly increased over control, p < 0.01.

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E. R. Nestmann et al.

1.0

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0

o>

oz

¿ •'' -:• i3 IQ

RHODAMINE 6G CONCENTRATION (M)

Chart 2. Relationship between single-strand DNA damage (•)and colony-forming ability (O) in CHO cells treated with metabolically activated rhodamine6G for 1 hr.

producing equivalent DMA damage with rhodamine 6G. Forexample, the DMA damage seen at the D0 dose of rhodamineB (9.6 x 10~4 M) corresponds to a concentration of rhodamine

6G which reduces colony forming ability by only 20% (80%survival). The D0 dose for rhodamine 6G is 1.75 x 10~4 M.

All procedures with CHO cells were carried out under goldfluorescent lighting, which contains few, if any, wavelengths oflight below 500 nm, to reduce chances of photochemicalreactions within tissue culture cells or with the dyes. Thus, it isunlikely that the activity observed with CHO cells caused bythe dyes was due to interaction with light.

DISCUSSION

Levels of public exposure to rhodamine dyes 6G and Bappear to be limited to contamination of water through theiruse as tracing agents or to their use in products such ascosmetics, drugs, inks, textiles, leather, etc. (9, 10). Occupational exposures may be more significant since contact withlarge quantities of these dyes is routine in water pollution andaerial spraying studies, in plumbing, and in the manufacture ofthese dyes. Production figures for these dyes, quoted by theInternational Agency for Research on Cancer (9, 10), areminimal estimates based only on disclosed reports of production. World-wide production of rhodamine B for the period1972 to 1975 exceeded 2.2 x 106 kg, with western European

manufacturers accounting for 90% of this amount. Total disclosed production of rhodamine 6G in the United States andJapan exceeded 2.8 x 105 kg for the 2-year period 1974-

1975, with 90% of this figure made in the forms of phospho-molybdic and phosphotungstic salts of rhodamine 6G.

The present results show that commercial rhodamine dyes6G and B are genetically active in short-term tests using

cultured bacterial and mammalian cells. Compared to rhodamine B, rhodamine 6G is more mutagenic, able to induce adoubling of revertants per plate in Salmonella strain TA1538 at3% of the dose and producing 10 times the DNA damage inCHO cells. Purified rhodamine 6G is potently mutagenic,

R, N-R,

Rj-OOC

RI234B(CH2CHj)2HHer6GCH2CHjCH3CH2CH3HCI

Chart 3. This diagram was drawn after those presented by the InternationalAgency for Research on Cancer (9, 10) showing the similarity in structure (A) ofrhodamine dyes 6G and B and a table (ß)of the differences between thesecompounds.

whereas purified rhodamine B shows litte mutagenicity. Therelevance of these findings to human safety remains to beevaluated; however, with data from the present work as supportive evidence,4 rhodamine 6G recently was designated for

carcinogenicity testing in the National Cancer Institute Bioas-say Program.5

The low toxicity of rhodamine 6G in CHO cells, relative tothe amount of DNA damage, suggests that this compound mayconstitute a genetic hazard to mammals, especially if similarresults are found in mammalian studies done in vivo. However,on the same basis, rhodamine B exhibits less potential as agenetic hazard. Since the mutagenicity of rhodamine B inSalmonella is due mainly to impurities, it is possible that thehigh relative lethality of rhodamine B is caused by toxic effectsof the pure fraction that may not be related to damage to DNA.Determinations of estimates of genetic risk to humans of thesedyes must await the outcome of further work using in vivomammalian studies.

Since purified rhodamine 6G is highly mutagenic and purifiedrhodamine B shows very little genetic activity, the chemicalstructures of these closely related dyes may offer a clue totheir dissimilar actions. Chart 3 shows the basic rhodamine(xanthene) structure and the different constituents for these 2dyes. Rhodamine B has a quaternary nitrogen with a positivecharge (Ri) and a carboxyl group at position R3, having no netcharge at neutral pH. On the other hand, at pH 7, rhodamine6G has a net positive charge and thus is more likely to crossthe cell membrane. However, both dyes are sufficiently polarmolecules to dissolve in water, so that these differences maybe inconsequential. Since metabolic activation of rhodamine6G by rat liver enzymes is required for its mutagenicity, thismolecule must be a substrate for an activating enzyme. The

4 Presented to the Chemical Selection Subgroup of the Clearinghouse on

Environmental Carcinogens, October 24, 1978.5 R. A. Griesemer. memo to Executive Secretary. Chemical Selection Working

Group, Novembers, 1978.

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Mutagenicity of Rhodamine Dyes

most obvious difference between the dyes in terms of metabolicactivation is the presence of a secondary amino group inrhodamine 6G. N-Hydroxylation of similar compounds, such as2-AAF and other aromatic amines, is known to result in highly

mutagenic metabolites (2). A test of this hypothesis awaitsfurther experimentation with an enzyme-free hydroxylation sys

tem (20, 21), as has been shown to activate promutagens inyeast (14).

Other compounds of the same basic xanthene structure(Chart 3) can act as direct mutagens, e.g., phloxine in Esche-

richia coli (11 ) and 2 other xanthenes in S. typhimurium (8).Rhodamine 6G, requiring metabolic activation, is a potentmutagen, purified rhodamine B is weakly mutagenic with activation, and erythrosine (8) is a nonmutagen. Further studies ofrelated compounds are underway for a better understanding ofthe chemical and biochemical bases for the mutagenic activityof these dyes.

ACKNOWLEDGMENTS

We thank Drs. G. Becking, I. Chu, R. H. Haynes, and L. Ritter for usefuldiscussions. Dr. B. Ames for providing bacterial strains, and Dr. R. Worton forthe gift of CHO cells. This work was initiated under contract with Bio-ResearchLaboratories, Limited, Senneville, Quebec.

REFERENCES

1. Ames, B. N., Durston, W. E., Yamasaki, E., and Lee, F. D. Carcinogens aremutagens: a simple test system combining liver homogenates for activationand bacteria for detection. Proc. Nati. Acad Sei. U. S. A., 70: 2281-2285,1973.

2. Ames, B. N., Gurney, E. G.. Miller, J. A., and Bartsch, H. Carcinogens asframeshift mutagens: metabolites and derivatives of 2-acetylaminofluoreneand other aromatic amine carcinogens. Proc. Nati. Acad. Sci. U. S. A.. 69.3128-3132, 1972.

3. Ames, B. N., McCann, J., and Yamasaki, E. Methods for detecting carcinogens and mutagens with the Sa/mone//a/mammalian-microsome mutagen-icity test. Mutât.Res.. 31: 347-364, 1975.

4. Andrews, A. W., Thibault, L. H., and Lijinsky, W. The relationship betweencarcinogenicity and mutagenicity of some polynuclear hydrocarbons. Mutât.Res.. 5Õ. 311-318, 1978.

5. Andrews, A. W., Thibault. L. H., and Lijinsky. W. The relationship betweenmutagenicity and carcinogenicity of some nitrosamines. Mutât. Res., 51:319-326. 1978.

6. Bell, S. Thin layer Chromatographie determination of subsidiary dyes in D &C red no. 14 and D & C red no. 37. J. Assoc. Off. Anal. Chem., 57. 961-

965. 1974.7. Brown, J. P.. Dietrich, P. S., and Bakner, C. M. Mutagenicity testing of some

drug and cosmetic dye lakes with the Sa/mone//a/mammalian microsomeassay. Mutât.Res., 66. 181-185. 1979.

8. Brown, J. P.. Roehm. G. W., and Brown, R. J. Mutagenicity testing ofcertified food colors and related azo, xanthene and triphenylmethane dyeswith the Sa/mone//a/microsome system. Mutât. Res., 56: 249-271, 1978.

9. International Agency for Research on Cancer. Rhodamine B. IARC Monogr.,16: 221-231. 1978.

10. International Agency for Research on Cancer. Rhodamine 6G. IARC Monogr., »6:233-239, 1978.

11. Kada, T.. Tutikawa, K.. and Sadaie. Y. In vitro and host-mediated "rec-assay" procedures for screening chemical mutagens; and phloxine, a mu

tagenic red dye detected. Mutât.Res.. 16: 165-174. 1972.12. Laishes, B. A., and Stich, H. F. Repair synthesis and sedimentation analysis

of DMA of human cells exposed to dlmethylnitrosamine and activated di-methylnitrosamine. Biochem. Biophys. Res. Commun., 52. 827-833, 1973.

13. Landa. Z.. Klouda, P., and Pleskotova. D. The mutagenic effects of fluoro-chromes. In: J. Veleminsky and T. Gichner (eds.). Induction of Mutationsand the Mutation Process, pp. 115-122. Prague: Publishing House of theCzechoslovak Academy of Sciences. 1965.

14. Mayer, V. W. Mutagenic effects induced in Saccharomyces cerevisiae bybreakdown products of 1-naphthylamine and 2-naphthylamine formed in anenzyme-free hydroxylation system. Mutât.Res.. )5. 147-153, 1972.

15. McCann, J., Choi, E., Yamasaki, E., and Ames. B. N. Detection of carcinogens as mutagens in the Salmonella/microsome test: assay of 300 chemicals. Proc. Nati. Acad. Sei. U. S. A., 72: 5135-5139. 1975.

16. McCann. J., Spingarn, N. E.. Kobori, J., and Ames, B. N. Detection ofcarcinogens as mutagens: bacterial tester strains with R factor plasmids.Proc. Nati. Acad. Sei. U. S. A., 72 979-983. 1975.

17. Mohn, G. R., and Ellenberger, J. The use of Escherìchia coli K12/343/113(A) as a multi-purpose indicator strain in various mutagenicity testingprocedures. In: B. J. Kilbey, M. Legator, W. Nichols, and C. Ramel (eds.).Handbook of Mutagenicity Test Procedures, pp. 95-118. Amsterdam: El-sevier/North-Holland Biomedicai Press, 1977.

18. Palcic. B., and Skarsgard, D. The effect of oxygen on DNA single-strandbreaks produced by ionizing radiation in mammalian cells. Int. J. Radiât.Biol., 21: 417-433, 1972.

19. Roberts, J. J. The repair of DNA modified by cytotoxic mutagenic andcarcinogenic chemicals. In: J. T. Lett, and H. Adler (eds.). Advances inRadiation Biology, Vol. 8, pp. 211-436. New York: Academic Press, Inc.,1978.

20. Udenfriend. S.. Clark, C. T.. Axelrod, J.. and Brodie. B. B. Oxidativeproperties of ascorbic acid. Fed. Proc., 11: 300-301. 1952.

21. Udenfriend, S.. Clark, C. T.. Axelrod, J.. and Brodie, B. B. Ascorbic acid inaromatic hydroxylation. I. A model system for aromatic hydroxylation. J.Biol. Chem.. 208. 731-739. 1954.

22. Umeda. M. Experimental study of xanthene dyes as carcinogenic agents.Gann, 47: 51-78. 1956.

NOVEMBER 1979 4417

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1979;39:4412-4417. Cancer Res   Earle R. Nestmann, George R. Douglas, Tibor I. Matula, et al.   in Chinese Hamster Ovary Cells

and DNA DamageSalmonellaDetected by Mutation Induction in Mutagenic Activity of Rhodamine Dyes and Their Impurities as

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