Mutagenic Activity of Rhodamine Dyes and Their Impurities As Detected by Mutation Induction in Salmonella and DMA Damage in Chinese Hamster Ovary Cells1

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

ABSTRACT The Sa/mone//a/mammalian-microsome test, as described Commercial rhodamine dyes 6G and B induce His* reversion by Ames ef al. (3), is used to detect bacterial mutagens. Many of these mutagenic chemicals also induce mutation and cancer mutations in Salmonella and single-strand breaks in Chinese in mammals. A high correlation between mutagenicity in this hamster ovary cells, as detected by alkaline sucrose sedimen test and carcinogenicity in mammals has been noted (15), tation. Aroclor 1254-induced rat liver homogenate (S9) is re although the percentage of correlation varies with the types of quired for production of genetic activity by these dyes. Rho chemicals chosen for testing (4, 5). The value of this test is damine 6G induces both frameshift and base substitution mu due in great part to the incorporation of a measure of mam tations, whereas rhodamine B induces only frameshift muta malian metabolism, in the form of a rat liver homogenate (S9) tions. Rhodamine 6G is genetically more active and more toxic in the assay system. Liver microsomes contain enzymes which than is rhodamine B in both the bacterial and mammalian can convert promutagens into genetically active mutagens, assays. Rhodamine 6G and B induce doublings of His* revert- detectable by His* reversion in different strains of Salmonella ants in Salmonella at the doses of 0.02 and 0.52 Â¿umol/plate (1). and shifts in the molecular weight of Chinese hamster ovary DMA at concentrations of 9 x 10~5 and 9 x 10"" M, respec Breakage of DNA in cultured cells of CHO, as detected by alkaline sucrose gradient sedimentation, is a procedure that tively. All genetic effects assayed demonstrate dose-related can be used with metabolic activation to detect chemicals with increases. Further testing of the pure dyes in Salmonella re potential genetic activity (12). Agents that cause such lesions vealed that rhodamine B loses most of its mutagenicity with in DNA are potential mutagens and/or carcinogens [see review purification, whereas rhodamine 6G does not. Impurities from by Roberts (19)]. The present method utilizes an assay for DNA commercial rhodamine B demonstrate the same extent of mu breakage in conjunction with an assay for cell survival. tagenicity as the commercial dye. Utilization of the tests in this study demonstrates the value of combining both microbial and mammalian experiments in mu INTRODUCTION tagenicity testing. The mammalian test for DNA damage can confirm the findings of the microbial test and simultaneously Rhodamine dyes have many uses as tracing agents in water can indicate the capability of a chemical to react with mam pollution and aerial pesticide spraying studies and as colors in malian DNA. Furthermore, the level of survival of mammalian drugs, cosmetics, textiles, and inks (9, 10). Rhodamine dyes cells at concentrations that induce DNA breaks allows specu 6G and B (Color Indexes 45160 and 45170) have been found lation as to the biological relevance of this damage. to be carcinogenic (22), but this study has been considered inadequate (9, 10). Rhodamine B has been shown to induce MATERIALS AND METHODS significantly higher than normal numbers of chromosomal bridges and fragments in anaphases and telophases of the Cell Culture. Salmonella typhimurium strains TA1535, broad bean, Vicia faba (13). In a preliminary screening test, TA1537, TA1538, TA98, and TA100, and media for bacterial rhodamine B also has been shown to induce mutations in testing were as described by Ames ef a/. (3). Salmonella (7). On the other hand, both rhodamine dyes 6G Monolayer cultures of wild-type CHO cells were maintained and B were found to cause no DMA damage in an in vitro in MEM supplemented with 10% FCS, nonessential amino bacterial test without metabolic activation (11 ). No other pub acids, and sodium pyruvate (Grand Island Biological Co., Bur lished evidence of mutagenicity, teratogenicity, or embryotox- lington, Ontario, Canada). Cells were grown at 37Â°, 5% CO2, icity caused by these dyes is available (9, 10). and high relative humidity. Cultures were routinely screened Due to their extensive use and potential mutagenicity and for the presence of Mycoplasma and found to be free of carcinogenicity, rhodamine dyes 6G and B were examined for contamination. All cell manipulations were carried out under induction of genetic effects using the Sa/mone//a/mammalian- microsome assay for mutation (3) and CHO3 cells in vitro for illumination from General Electric (Montreal, Quebec, Canada) gold fluorescent lamps (Model F40GO 6PK) which do not emit detection of DNA strand breakage.

3 The abbreviations used are: CHO, Chinese hamster ovary; S9, supernatant 1 Preliminary reports of this work were presented at the 10th Annual Meeting from centrifugea (9000 x g) liver homogenate used for metabolic activation; MEM, Eagle's minimal essential medium; FCS, fetal calf serum; DMSO, dimethyl of the Environmental Mutagen Society. New Orleans, La., March 8 to 12, 1979. 2 To whom requests for reprints should be addressed. sulfoxide; BP, benzo(a)pyrene; 2-AAF, 2-acetylaminofluorene; dThd, thymidine, Received April 10, 1979; accepted July 25, 1979. TLC, thin-layer chromatography.

4412 CANCER RESEARCH VOL. 39

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1979 American Association for Cancer Research. Mutagenicity of Rhodamine Dyes light at wavelengths below 500 nm. mentation constant derived from the sedimentation character Chemicals. Rhodamine B (Sigma Chemical Co., St. Louis, istics of bacteriophage T4 DNA. The cosedimentation of control Mo.; 90% purity) was dissolved in DMSO (Aldrich Chemical and treated DNA greatly increases the precision of the break Co., Milwaukee, Wis.) in one preliminary bacterial test. In all age estimate. other bacterial experiments, stock solutions of rhodamine dyes Cell survival was estimated by determining colony-forming B and 6G (ICI, Alderly Park, Cheshire, United Kingdom) were ability at different dye concentrations. Approximately 180 cells prepared with sterile distilled H2O. For tests with CHO cells, were plated in 60-mm tissue culture dishes in MEM:10% PCS rhodamine B was dissolved in MEM containing 2.5% PCS since as above and allowed to attach for 4 to 5 hr. The cells were then treated as above for 1 hr, washed gently with Hanks' the addition of water makes the treatment mixture hypotonie. Rhodamine 6G was difficult to dissolve in MEM so DMSO was balanced salt solution 3 times and incubated for 6 to 7 days. used with no apparent change in experimental results. BP Colonies were fixed in 3:1 ethanohglacial acetic acid, stained (Aldrich Chemical Co.), 2-AAF (ICN K & K Laboratories, Inc., with hematoxylin, and counted. Plainview N.Y.), and aflatoxin B, (Makor Chem. Ltd., Jerusa Procedures for Purification of Rhodamine Dyes. Separation lem, Israel), dissolved in DMSO served as positive controls. of subsidiary colors and impurities from rhodamine B was Bacterial Testing Procedures. The procedures were those performed as described by Bell (6). For the purification of described by Ames era/. (3). Aroclor 1254 (Analabs, Inc., N. rhodamine 6G, the developing solvent system was benzene: Haven, Conn.) -induced and phÃ©nobarbital (BDH, Toronto, methanohammonia (80:20:0.2). The dyes were dissolved in Ontario, Canada) -induced liver homogenates (S9) were pre methanol and spotted in a band on each plate with a micropipet. pared following the methods of Ames et al. (1, 3). Unless Each TLC plate (20 x 20 cm; New England Nuclear) was otherwise specified, the abbreviation S9 by itself means that divided into 3 portions. For rhodamine B they were: (a) the the rat liver was induced by Aroclor 1254. The graphs of Mohn main color band (top color band), designated pure rhodamine and Ellenberger (17) are used to estimate the probability that B; (b) a small color band, just below the main color band; (c) chemically induced mutation frequencies are different from remainder of plate containing all other subsidiary color bands control values. and UV visible bands, above and below the 2 major bands. For Mammalian Testing Procedures. CHO cells were labeled rhodamine 6G, the 3 fractions were: (a) the main color band, overnight in 60-mm tissue culture dishes containing 8 x 10s close to the origin, designated pure rhodamine 6G; (b) a small cells in MEM supplemented with 10% newborn calf serum, color band, just above the main color band; (c) remainder of nonessential amino acids, sodium pyruvate, and 1.0 /iCi [3H]- plate containing all other UV visible bands between the main dThd per ml (46 Ci/mmol) or 1.0 /Â¿Ci[14C]dThd per ml (50 band and the solvent front. mCi/mmol). The cells were chased with nonradioactive medium Silica Gel H (250 /im), representing the 3 fractions, was for 2 hr prior to treatment. scraped and extracted repeatedly with methanol until little color The treatment mixture contained S9 (7.1%), 20 mw 4- remained. The concentrations of the dyes recovered from the (2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.2 TLC plates were determined spectrophotometrically at 530 or (4.77%), 0.5 M MgCI2 (0.238%), 3.3 M KCI (0.238%), 50 mw 547 nm for rhodamine B and 6G, respectively. Each portion glucose-6-phosphate (2.38%), 40 rriM NADP+ (2.38%), dis was dried under nitrogen and dissolved in equal volumes of tilled H20 (6.67%), MEM with 2.5% PCS (61.9% for rhodamine sterile distilled H2O for testing. B or 75.2% for rhodamine 6G), and mutagen (14.28% for rhodamine B or 1.0% for rhodamine 6G). The optimal propor RESULTS tion of S9 in the activation mixture was determined in prelimi nary experiments and does not affect the sedimentation prop Induction of Mutation in Salmonella by Rhodamine B. In erties of CHO DMA in alkaline sucrose gradients. Cells were preliminary screening experiments with doses ranging from suspended in 1.4 ml of the treatment mix and treated for 1 hr 0.25 to 2.0 mg/plate, rhodamine B dissolved in DMSO induced in a 60-mm dish. After removal of the mixture, the cells were His* reversions in strains TA1538 and TA98, but not in strains washed 2 times in ice-cold, calcium- and magnesium-free, TA1535, TA100, or TA1537. At 1.0 mg/plate, the rhodamine- phosphate-buffered saline (KCI, 0.20 g/l; KH2PO4, 0.20 g/l; induced increases in yields of revertants compared to back NaCI, 8.00 g/l; Na2HPO4-7H2O, 2.16 g/l) (Grand Island Bio ground were 4-fold with 50 /il S9 per plate and 11 -fold with 20 logical Co.). Cells were removed from the culture dishes by /il S9 per plate (data not shown). gentle scraping and resuspended in 1 ml ice-cold phosphate- To confirm these findings, rhodamine B, dissolved in sterile buffered saline. distilled H2O, was retested using differing amounts of Aroclor Alkaline sucrose sedimentation was carried out according to 1254-induced S9 (Table 1, A) and also phenobarbital-induced the method of Palcic and Skarsgard (18). Approximately 8 x S9 (Table 1, P). The data are presented in Table 1 which shows 103 [14C]dThd-labeled, control cells and 8 x 103 [3H]dThd- that rhodamine B is not mutagenic in strains TA1535 and labeled, treated cells were lysed on the gradients for 11 hr at TA100, which revert by base substitution mutation, nor in strain 20Â°and spun at 15,000 rpm for 6 hr in a Beckman SW50-1 TA1537, revertible by frameshift mutation (1). Rhodamine B is rotor. Gradients were fractionated from the top into 0.2-ml mutagenic in strains TA1538 and TA98, which revert by a fractions, collected in miniscintillation vials, and counted in 5 frameshift mutation (1). Rhodamine B-induced mutagenesis ml aquasol (New England Nuclear, Montreal, Quebec, Canada) occurs only with metabolic activation by either S9 preparation, after neutralization with 0.5 N HCI. A or P. Comparison of data for strain TA1538 in Columns A Calculation of molecular weights was carried out using the and A(30), with 50 and 30 /il S9 per plate, respectively, shows method described by Palcic and Skarsgard (18) with a sedi that reduction of S9 increases rhodamine B-induced mutagen-

NOVEMBER 1979 4413

Table 1 His * 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. revertantsTA1535Compound No. of His*

TA1537A 1538A and mgPWater (^moO/plate* A A98 P- P12 A(30)6 (30)10 A 85Rhodamine 8 6 6 886 99 5 8121199 20308 185017 B0.100(0.21) 980.125(0.26)0.250(0.52)0.5008 9 6 867 92 8 234360e8 11525026e

43e65e91e8 680.750(1.56)1(1 .04) 8 9 6 654 94 6 61813 1476d146e46e 66e190e548e 1 641.500(3.13)2.000(4.17).000 (2.09) 7 6 4 56 50 5 6914 52e151e113 1

158e223e272e282e302e 24353.000 924486Â° 4131028eTA 84e215e105e378e156e 5 1 (6.26)4.000 (8.35)BP0.010(0.04)

29e2-AAF0.050 43e1330eA(30) 194e1176eTA98P

(0.22) 6TA100 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 Â¿ilofphenobarbitol- induced S9 per plate. c Significantly increased over control, p < 0.01 d Significantly increased over control, p < 0.05.

icity by more than 3-fold at the dose of 1 mg/plate with less Table 2 His * reversion by purified rhodamine B and impurities in Salmonella strain change at other doses. The optimum amount of S9 per plate seems to be 30 Â¡i\for rhodamine B-induced mutation, but BP TA 1538 with metabolic activation The experimental conditions were similar to those described in Table 1. requires more S9 per plate (50 jul) for optimal mutagenesis (1 ). re Although the mutagens used as positive controls (BP and 2- vertants/plate322157b173b5<57641"526112"171" AAF) induce more reversions in strain TA98 than in TA1538, TreatmentNegative rhodamine B is more mutagenic and less toxic in TA1538 than (H2O)Commercialcontrol in TA98. BFraction rhodamine mg2.0 mg1.0 Since the commercial rhodamine B used in this study is band)Fraction1 (rhodamine B, purified main mg2.0 approximately 90% pure, TLC was used to obtain a purified mg0.1 band)Fraction2 (small color mÃ0.2 dye. Table 2 shows the mutagenic responses of identical mlc0.1 volumes of the 3 fractions obtained. Compared to 8-fold in 3 (other impurities)6Amount/plate1.0 mlc0.2 creases induced by commercial rhodamine B, the main band ml0His* and the major subsidiary band, Fractions 1 and 2, respectively, Numbers of revertants are means of duplicate plates. Amount of S9 added is 30 fil/plate. exhibit only slightly more than doublings of the background b Significantly increased over control, p < 0.01. frequencies of His* reversions. However, Fraction 3 (the other e These volumes contain concentrations of these fractions that correspond to 1.0 and 2.0 mg of the purified main color band. impurities) shows nearly the same mutagenicity as does com d Significantly increased over control, p < 0.05. mercial rhodamine B. Since the amount of impurities tested is 6 All other minor color and UV visible bands. adjusted to be the same as in an equal volume of the commer cial dye, it is concluded that most of the mutagenic activity of commercial rhodamine B is due to contaminating impurities. 6G is mutagenic in strains TA1538 and TA98, but it is also Cell Killing and DNA Damage in CHO Cells by Rhodamine mutagenic in strains TA1537 and TA100. Thus, unlike rhoda B. Commercial rhodamine B activated by S9 causes damage mine B, rhodamine 6G causes base substitution (in strain in single strands of DNA in CHO cells, as indicated by reduction TA100) in addition to frameshift mutations. Rhodamine 6G in sedimentation velocity in alkaline sucrose gradients. These requires activation by S9 to induce mutation in Salmonella. results are plotted in Chart 1 which shows that lethality is high Rhodamine 6G induces a doubling of revertants at only 3% at concentrations producing little DNA damage. For example, (7.8 fig) of the dose required for a doubling by rhodamine B the DOdose (9.6 x 10~" M) results in minimal DNA damage, (250 jug) in strain TA1538. At its maximum, rhodamine 6G less than 0.2 breaks/10s daltons. induces a 30-fold increase of revertants above background in Induction of Mutation in Salmonella by Rhodamine 6G. strain TA1538, whereas the maximal increase induced by Table 3 shows results from 1 of 2 experiments assaying the rhodamine B is 9-fold. Rhodamine 6G shows toxicity at 500 potential mutagenicity of rhodamine 6G in the Salmonella/ /ig/plate in strain TA100 and at 1000 jug/plate in strains microsome system. As in the case of rhodamine B, rhodamine TA1537, TA1538, and TA98, as indicated by the decreasing

4414 CANCER RESEARCH VOL. 39

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1979 American Association for Cancer Research. Mutagenicity of Rhodamine Dyes numbers of revertants. Mutagens used as positive controls in Rhodamine 6G was purified, and Table 4 shows the results Table 3, BP, 2-AAF, and aflatoxin B,, show the expected of an experiment testing for mutagenicity of the 3 fractions mutagenic responses (16). isolated in Salmonella strain TA1538 with S9. Compared with To test the possibility that rhodamine 6G-induced mutagen- the commercial dye, pure rhodamine 6G loses 22 to 23% of its icity is dependent upon photodynamic action, a solution of mutagenicity, but it still induces a 24-fold increase in revertants rhodamine 6G was prepared in a darkened vial. Experiments over the control. Fraction 2 induces a tripling of revertants, and were performed in the absence of overhead illumination, with Fraction 3 shows a 7-fold increase, about the same as for daylight supplied by a north window 10 m behind. The plates Fraction 3 from the purification of rhodamine B. Due to the were in the dark for the duration of their incubation. The data, mutagenic activity of Fraction 1, the purified dye, it is concluded using strains TA100 and TA1538, are similar as in routine that pure rhodamine 6G is a mutagen. experiments done with limited exposure of the dye or of the Cell Killing and DNA Damage in CHO Cells by Rhodamine plates to light (Table 3). 6G. Rhodamine 6G activated by S9 causes damage to single strands of DNA, as illustrated in Chart 2, which shows the 100 - 2.0 effect of rhodamine 6G in CHO cells and on CHO DNA. Al though rhodamine 6G is approximately 10 times more potent 90 - 1.8 in inducing DNA damage than is rhodamine B in these experi

80 - 1.6 ments, rhodamine B is much more toxic at concentrations

CE T3 70 1.4 m Table 4 His" reversion by purified rhodamine 6G in Salmonella strain TA 1538 with 60 1.2 tr => metabolic activation 50 1.0 The experimental conditions were similar to those described in Table 1. o re 40 0.8 r vertants/ o H TreatmentNegative plate'119 OL o plate0.1 30 0.6 control (H2O) ml Commercial6GFraction rhodamine 125.0 /Â¿g 306 20 0.4 250.0 /ig 579 1 (rhodamine 6G, purified main band) 125.0 /ig 235 IO 0.2 250.0 /ig 447 0.1 ml" Fraction 2 (small color band) 45 0.2 mlb 0 59 Fraction 3 (Â¡mpurities)cAmount/ 0.1 ml" IO" IO" 61 0.2 ml"His* 142 RHODAMINE B CONCENTRATION (M) Numbers of revertants are means of duplicate plates. Amount of S9 is 30 iul/plate. All revertan! yields are significantly increased over control, p <0.01. Chart 1. Relationship between single-strand DNA damage (â€¢)and colony- These volumes contain concentrations of these fractions that correspond to forming ability (O) in CHO cells treated with metabolically activated rhodamine B 125 and 250 /ig of the purified main band. c All other minor color and UV visible bands. for 1 hr.

Table 3 His * reversion by rhodamine 6G in strains of Salmonella The experimental conditions were similar to those described in Table 1. revertants/plate6Compound No. of His*

1535TA1537A TA 100 TA98A4 1538 and (ig dumol)/plateaWaterRhodamine A6 A115 - A20 115261d1011 108 101620383d7 324867d96"152"5 50586998d156d188" 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

476d9 7 114 26tf71 5 277"3 206"288d 736d514 110 "1091"1d 2 31 477d6 350d566o 1021d24011 16 37\d2 731d1 575d954"311"378rf2254d495d 389d0000428"1300e*Dark 72"0 945d0 93d194d1506*49Dark 793rf38"TA

(0.22)Aflatoxin Bi0.1TA

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

NOVEMBER 1979 4415

1.0 R, N-R, 0.9 Rj-OOC 0.8 0.7

0.6 0.5 o 0.4 > o 0.3 z 0.2 RI234B(CH2CHj)2HHer6GCH2CHjCH3CH2CH3HCI 0.1 0 Â¿ â€¢'' -:â€¢ i3 IQ

RHODAMINE 6G CONCENTRATION (M) Chart 2. Relationship between single-strand DNA damage (â€¢)and colony- forming ability (O) in CHO cells treated with metabolically activated rhodamine Chart 3. This diagram was drawn after those presented by the International Agency for Research on Cancer (9, 10) showing the similarity in structure (A) of 6G for 1 hr. rhodamine dyes 6G and B and a table (ÃŸ)of the differences between these compounds. producing equivalent DMA damage with rhodamine 6G. For example, the DMA damage seen at the D0 dose of rhodamine whereas purified rhodamine B shows litte mutagenicity. The B (9.6 x 10~4 M) corresponds to a concentration of rhodamine relevance of these findings to human safety remains to be 6G which reduces colony forming ability by only 20% (80% evaluated; however, with data from the present work as sup survival). The D0 dose for rhodamine 6G is 1.75 x 10~4 M. portive evidence,4 rhodamine 6G recently was designated for All procedures with CHO cells were carried out under gold carcinogenicity testing in the National Cancer Institute Bioas- say Program.5 fluorescent lighting, which contains few, if any, wavelengths of light below 500 nm, to reduce chances of photochemical The low toxicity of rhodamine 6G in CHO cells, relative to reactions within tissue culture cells or with the dyes. Thus, it is the amount of DNA damage, suggests that this compound may unlikely that the activity observed with CHO cells caused by constitute a genetic hazard to mammals, especially if similar the dyes was due to interaction with light. results are found in mammalian studies done in vivo. However, on the same basis, rhodamine B exhibits less potential as a DISCUSSION genetic hazard. Since the mutagenicity of rhodamine B in Salmonella is due mainly to impurities, it is possible that the Levels of public exposure to rhodamine dyes 6G and B high relative lethality of rhodamine B is caused by toxic effects appear to be limited to contamination of water through their of the pure fraction that may not be related to damage to DNA. use as tracing agents or to their use in products such as Determinations of estimates of genetic risk to humans of these cosmetics, drugs, inks, textiles, leather, etc. (9, 10). Occupa dyes must await the outcome of further work using in vivo tional exposures may be more significant since contact with mammalian studies. large quantities of these dyes is routine in water pollution and Since purified rhodamine 6G is highly mutagenic and purified aerial spraying studies, in plumbing, and in the manufacture of rhodamine B shows very little genetic activity, the chemical these dyes. Production figures for these dyes, quoted by the structures of these closely related dyes may offer a clue to International Agency for Research on Cancer (9, 10), are their dissimilar actions. Chart 3 shows the basic rhodamine minimal estimates based only on disclosed reports of produc (xanthene) structure and the different constituents for these 2 tion. World-wide production of rhodamine B for the period dyes. Rhodamine B has a quaternary nitrogen with a positive 1972 to 1975 exceeded 2.2 x 106 kg, with western European charge (Ri) and a carboxyl group at position R3, having no net manufacturers accounting for 90% of this amount. Total dis charge at neutral pH. On the other hand, at pH 7, rhodamine closed production of rhodamine 6G in the United States and 6G has a net positive charge and thus is more likely to cross Japan exceeded 2.8 x 105 kg for the 2-year period 1974- the cell membrane. However, both dyes are sufficiently polar 1975, with 90% of this figure made in the forms of phospho- molecules to dissolve in water, so that these differences may molybdic and phosphotungstic salts of rhodamine 6G. be inconsequential. Since metabolic activation of rhodamine The present results show that commercial rhodamine dyes 6G by rat liver enzymes is required for its mutagenicity, this 6G and B are genetically active in short-term tests using molecule must be a substrate for an activating enzyme. The cultured bacterial and mammalian cells. Compared to rhoda mine B, rhodamine 6G is more mutagenic, able to induce a doubling of revertants per plate in Salmonella strain TA1538 at 4 Presented to the Chemical Selection Subgroup of the Clearinghouse on Environmental Carcinogens, October 24, 1978. 3% of the dose and producing 10 times the DNA damage in 5 R. A. Griesemer. memo to Executive Secretary. Chemical Selection Working CHO cells. Purified rhodamine 6G is potently mutagenic, Group, Novembers, 1978.

4416 CANCER RESEARCH VOL. 39

most obvious difference between the dyes in terms of metabolic 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- activation is the presence of a secondary amino group in 965. 1974. rhodamine 6G. N-Hydroxylation of similar compounds, such as 7. Brown, J. P.. Dietrich, P. S., and Bakner, C. M. Mutagenicity testing of some 2-AAF and other aromatic amines, is known to result in highly drug and cosmetic dye lakes with the Sa/mone//a/mammalian microsome assay. MutÃ¢t.Res., 66. 181-185. 1979. mutagenic metabolites (2). A test of this hypothesis awaits 8. Brown, J. P.. Roehm. G. W., and Brown, R. J. Mutagenicity testing of further experimentation with an enzyme-free hydroxylation sys certified food colors and related azo, xanthene and triphenylmethane dyes tem (20, 21), as has been shown to activate promutagens in with the Sa/mone//a/microsome system. MutÃ¢t. Res., 56: 249-271, 1978. 9. International Agency for Research on Cancer. Rhodamine B. IARC Monogr., yeast (14). 16: 221-231. 1978. Other compounds of the same basic xanthene structure 10. International Agency for Research on Cancer. Rhodamine 6G. IARC Mon ogr., Â»6:233-239, 1978. (Chart 3) can act as direct mutagens, e.g., phloxine in Esche- 11. Kada, T.. Tutikawa, K.. and Sadaie. Y. In vitro and host-mediated "rec- richia coli (11 ) and 2 other xanthenes in S. typhimurium (8). assay" procedures for screening chemical mutagens; and phloxine, a mu Rhodamine 6G, requiring metabolic activation, is a potent tagenic red dye detected. MutÃ¢t.Res.. 16: 165-174. 1972. 12. Laishes, B. A., and Stich, H. F. Repair synthesis and sedimentation analysis mutagen, purified rhodamine B is weakly mutagenic with acti of DMA of human cells exposed to dlmethylnitrosamine and activated di- vation, and erythrosine (8) is a nonmutagen. Further studies of methylnitrosamine. Biochem. Biophys. Res. Commun., 52. 827-833, 1973. 13. Landa. Z.. Klouda, P., and Pleskotova. D. The mutagenic effects of fluoro- related compounds are underway for a better understanding of chromes. In: J. Veleminsky and T. Gichner (eds.). Induction of Mutations the chemical and biochemical bases for the mutagenic activity and the Mutation Process, pp. 115-122. Prague: Publishing House of the of these dyes. Czechoslovak Academy of Sciences. 1965. 14. Mayer, V. W. Mutagenic effects induced in Saccharomyces cerevisiae by breakdown products of 1-naphthylamine and 2-naphthylamine formed in an ACKNOWLEDGMENTS enzyme-free hydroxylation system. MutÃ¢t.Res.. )5. 147-153, 1972. 15. McCann, J., Choi, E., Yamasaki, E., and Ames. B. N. Detection of carcino We thank Drs. G. Becking, I. Chu, R. H. Haynes, and L. Ritter for useful gens as mutagens in the Salmonella/microsome test: assay of 300 chemi discussions. Dr. B. Ames for providing bacterial strains, and Dr. R. Worton for cals. Proc. Nati. Acad. Sei. U. S. A., 72: 5135-5139. 1975. the gift of CHO cells. This work was initiated under contract with Bio-Research 16. McCann. J., Spingarn, N. E.. Kobori, J., and Ames, B. N. Detection of Laboratories, Limited, Senneville, Quebec. carcinogens as mutagens: bacterial tester strains with R factor plasmids. Proc. Nati. Acad. Sei. U. S. A., 72 979-983. 1975. REFERENCES 17. Mohn, G. R., and Ellenberger, J. The use of EscherÃ¬chia coli K12/343/ 113(A) as a multi-purpose indicator strain in various mutagenicity testing 1. Ames, B. N., Durston, W. E., Yamasaki, E., and Lee, F. D. Carcinogens are procedures. In: B. J. Kilbey, M. Legator, W. Nichols, and C. Ramel (eds.). mutagens: a simple test system combining liver homogenates for activation Handbook of Mutagenicity Test Procedures, pp. 95-118. Amsterdam: El- and bacteria for detection. Proc. Nati. Acad Sei. U. S. A., 70: 2281-2285, sevier/North-Holland Biomedicai Press, 1977. 1973. 18. Palcic. B., and Skarsgard, D. The effect of oxygen on DNA single-strand 2. Ames, B. N., Gurney, E. G.. Miller, J. 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NOVEMBER 1979 4417

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1979 American Association for Cancer Research. Mutagenic Activity of Rhodamine Dyes and Their Impurities as Detected by Mutation Induction in Salmonella and DNA Damage in Chinese Hamster Ovary Cells

Earle R. Nestmann, George R. Douglas, Tibor I. Matula, et al.

Cancer Res 1979;39:4412-4417.

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