The photogenotoxicity of titanium dioxide particles

Mutation Research 394 Ž1997. 125–132

The photogenotoxicity of titanium dioxide particles Yuzuki Nakagawa ) , Shinobu Wakuri, Kyoko Sakamoto, Noriho Tanaka Laboratory of Cellular Toxicology, Hatano Research Institute, Food and Drug Safety Center. 729-5 Ochiai, Hadano, Kanagawa 257, Japan Received 2 April 1997; revised 16 June 1997; accepted 20 June 1997

Abstract We employed a series of in vitro genotoxicity assays —a single cell gel ŽSCG. assay with mouse lymphoma L5178Y cells, a microbial mutation assay with Salmonella typhimurium, a mammalian cell mutation assay with L5178Y cells, and a chromosomal aberration assay with Chinese hamster CHLrIU cells— to evaluate the photogenotoxicity of titanium dioxide ŽTiO 2 . particles. Without UVrvisible light irradiation, TiO 2 particles exhibited no or weak genotoxicity. With irradiation, however, TiO 2 particles exhibited significant genotoxicity in the SCG and chromosomal aberration assays. Therefore, we concluded that TiO 2 particles are photogenotoxic. q 1997 Elsevier Science B.V. Keywords: Titanium dioxide; Photogenotoxicity; Chromosome aberration; Single cell gel assay

1. Introduction Titanium dioxide ŽTiO 2 . particles are widely used as a white paint pigment and as a cosmetics ingredient. TiO 2 particles show weak or no toxicity in vitro and in vivo w1–8x, although it is carcinogenic in rat lung w9x. Ultra fine TiO 2 particles catalyze the killing of bacteria w10,11x and cancer cells w12,13x by UVvisible ŽUVrvis. light, probably due to the generation of free radicals by photoexcited TiO 2 particles. In this study we investigated the genotoxicity of photoexcited TiO 2 particles Ž4 types. in four in vitro genotoxicity assays, a single cell gel electrophoresis ŽSCG. assay with mouse lymphoma L5178Y cells, a microbial mutation assay with Salmonella typhimurium, a mammalian mutation assay with

L5178Y cells, and a chromosome aberration assay with Chinese hamster CHLrIU cells.

2. Materials and methods 2.1. Titanium dioxide We used four types of titanium dioxide particles ŽCAS 13463-67-7.: p-25 Žanatase form, average size 0.021 mm, Nippon-Aerosil, Tokyo, Japan., WA Žanatase form, average size 0.255 mm, Wako, Tokyo, Japan., WR Žrutile form, average size 0.255 mm, Wako., and TP-3 Žrutile form, average size 0.42 mm, Fuji titan, Kanagawa, Japan.. 2.2. Cell culture

) Corresponding author. Tel.: q81 463 824751 ext. 431-433; Fax: q81 463 829627.

For the chromosome aberration assay, the Chinese hamster cell line CHLrIU was obtained from the

1383-5718r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 1 3 8 3 - 5 7 1 8 Ž 9 7 . 0 0 1 2 6 - 5

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Japanese Collection of Research Bioresources ŽJCRB. Bank. They were maintained in minimal essential medium ŽMEM; Nissui, Tokyo, Japan. supplemented with 10% fetal bovine serum ŽFBS; Filtron, Brooklyn, Australia.. For the SCG and mammalian cell mutation assays, the mouse lymphoma cell line L5178Yrtkqry clone 3.7.2C ŽL5178Y. was obtained from Dr. D. Clive ŽBurroughs Welcome Co., Research Triangle Park, NC, USA. through Dr. T. Sofuni ŽDivision of Genetics and Mutagenesis, National Institute of Health Sciences, Tokyo.. The L5178Y cells were maintained in RPMI 1640 medium ŽRPMI; Gibco BRL, Grand Island, NY, USA. supplemented with 1% Žwrv. sodium pyruvate, 0.05% Žwrv. pluronic F-68 ŽSigma, St. Louis, MO, USA., and 10% donor horse serum ŽHS; Intergen, New York, USA.. Both cell lines were cultured in the dark in a humidified incubator with an atmosphere of 5% CO 2 in air at 378C. 2.3. UV r Õis light irradiation We followed the standard irradiation procedures of the European Cosmetic Industry Association joint validation project which validated the method of neutral red assay with BALBrc 3T3 cells for the detection of photocytotoxicity w14x. The light source was a sunlight simulator ŽSOL 500, Dr Honle, Mar¨ tinsried, Germany. equipped with a filter ŽH1, Dr. Honle ¨ . that allowed 50% transmission at wavelength 335 nm. The spectra of generated UVrvis light was almost identical to natural sunlight, that is, the ratio of UVA to UVB light was about 25:1. The emitted energy was measured with a simple UVA meter Žtype no. 37, Dr. Honle ¨ . immediately before each irradiation. 2.4. SCG assay L5178Y cells were maintained at the cell density of 1 = 10 4 cells per ml to 5 = 10 5 cellsrml. For irradiation, they were suspended in Earle’s balanced salt solution ŽEBSS; Gibco BRL. at 2 = 10 6 cellsrml, and 0.5 mlrwell was placed into 24-well cluster dishes Ž143982, Nunc, Roskilde, Denmark..

TiO 2 particles were suspended in 0.5 ml EBSS and added to the wells Žtotal volume in each well: 1 ml.. After 1 h incubation in the dark, the cells were exposed to UVrvis light for 50 min at 0.2 mWrcm2 Ž0.61 Jrcm2 ., 0.4 mWrcm2 Ž1.25 Jrcm2 ., 0.8 mWrcm2 Ž2.50 Jrcm2 ., and 1.6 mWrcm2 Ž5 Jrcm2 .. Simultaneously, duplicate dishes were kept in the dark at room temperature as the control. A single culture was used for each treatment. After irradiation, the cells were suspended in 2 ml culture medium pre-cooled on ice, and 1 ml cell suspension was used for the SCG assay. The remaining 1 ml was mixed with 4 ml culture medium and incubated. After 20 h incubation, the viable cells were counted with a hemocytometer. The SCG assay was performed by the method of Singh et al. w15x as modified by Hartmann and Speit w16x. Briefly, test cells embedded in 0.5% low melting agarose ŽSeaPlaque GTG, FMC, Rockland, USA. were placed in ice-cold lysing solution Ž2.5 M NaCl, 100 mM Na 2-EDTA, 10% DMSO, 1% Triton X-100. for 2 h and then in ice-cold alkaline solution Ž1 mM Na 2-EDTA, 300 mM NaOH, pH 13.0. for 20 min to permit the DNA to unwind. Electrophoresis was conducted at 25 V constant voltage for 25 min at 88C. The slides were neutralized in ice-cold Tris buffer Ž400 mM, pH 7.2. for 30 min, stained with 50 ml ethidium bromide solution Ž2.0 mgrml., and analyzed by fluorescence microscopy. The tail length of 50 nuclei per treatment Ž25 nucleirslide. were measured Žfrom edge of nucleus to the tail end. with an ocular micrometer. 2.5. Chromosomal aberration assay We tested only p-25 TiO 2 particles. CHLrIU cells were seeded onto 35-mm plastic cell culture dishes Ž153066, Nunc. at 2 = 10 4 cellsrdish and cultured for 48 h. Just before irradiation, the culture medium was replaced by 1 ml EBSS. The TiO 2 particles were suspended in 1 ml EBSS and added to the dishes Žtotal volume in each dish: 2 ml.. After 1 h incubation in the dark, the cells were exposed to UVrvis light for 50 min at 0.4 mWrcm2 Ž1.25 Jrcm2 ., 0.8 mWrcm2 Ž2.50 Jrcm2 ., and 1.6 mWrcm2 Ž5 Jrcm2 .. Duplicate cultures were incubated at room temperature in the dark as controls. A

Y. Nakagawa et al.r Mutation Research 394 (1997) 125–132

single culture was used for each treatment. After irradiation, the cells were rinsed with EBSS and incubated in 2 ml MEM containing 10% FBS. After 20 h incubation and 2 h demecolcin Ž0.1 mgrml. treatment, chromosome slides were prepared by the conventional air-dry method. One hundred metaphase spreads with 22–26 chromosomes were examined for structural aberrations, and the number of polyploid cells was recorded. 2.6. Microbial mutation assay We carried out the Salmonellarmicrosome assays with p-25 TiO 2 particles by the standard plate incorporation method w17x using strains TA100, TA98, and TA102. Before irradiation, the TiO 2 particles were suspended in distilled water and added to 1.35 ml of a bacterial suspension. The mixtures were placed in 35-mm plastic cell culture dishes Ž153066, Nunc.; two dishes were used for each treatment. The UVrvis light irradiation was performed at 1.6 mWrcm2 for 10 min Ž1 Jrcm2 . or 50 minutes Ž5 Jrcm2 .. After irradiation, the suspension of bacteria and TiO 2 particles was mixed with top agar. The

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number of Hisq revertants on each plate was scored after 48 h incubation at 378C. 2.7. Mammalian cell mutation assay We conducted a mammalian cell mutation assay of p-25 TiO 2 particles using mouse lymphoma L5178Y cells. Prior to irradiation, the cells were suspended in EBSS and placed into 24-well cluster dishes at 5 = 10 6 cellsrwell. The TiO 2 particles were suspended in EBSS and added to the cell suspensions. After 1 h incubation in the dark, the cells were exposed to UVrvis light at 1.6 mWrcm2 for 50 min Ž5 Jrcm2 . and then suspended in culture medium. Cells Ž1.6 cellsrwell. were seeded onto 96-well cluster dishes to evaluate cytotoxicity. After 11–13 days, the number of wells without colony was recorded. In the mutation test, aliquots of irradiated cells were suspended in selection medium Žcontaining 3.0 mgrml of trifluorothymidine. and seeded to 96-well cluster dishes at 2000 cellsrwell Ž192 wells for one dose.. After 11–13 days, the number of empty wells and size of trifluorothymidine resistant colonies were recorded.

Fig. 1. Photogenotoxicity of four types of TiO 2 particles with or without UVrvis light irradiation Ž5 Jrcm2 . examined by SCG assay in L5178Y cells. a: length of DNA migration; b: surviving cell number relative to solvent control 24 h after treatment.

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3. Results

3.1. SCG assay Results of the SCG assays are shown in Fig. 1 and Fig. 2. DNA from cells exposed to p-25 and TP-3 without UVrvis light showed no increase in tail length whereas nuclei with irradiation showed a dose-dependent increase in mean tail length ŽFig. 1.. As shown in Fig. 2, mean tail length linearly increased with intensity of UVrvis light, when the concentration of p-25 particles was constant. Cell survival was reduced at doses that elicited photogenotoxicity ŽFig. 1 and Fig. 2.. DNA from cells exposed to WA showed a dose-dependent increase in mean tail length with and without UVrvis light exposure, but tail length was greater in the irradiated group. In contrast, exposure to WR particles caused no cell killing or enhanced DNA migration at any dose. Thus, two anatase and one rutile type of TiO 2 particles showed photo-enhanced DNA damage associated with decreased cell survival and the degree of photo-enhanced DNA migration was different in each case. Because the ultra-fine particles Žp-25. were the most potent, displaying activity at 12.5 mgrml with UVrvis light irradiation, we used them exclusively in subsequent experiments.

Fig. 3. Microbial mutagenicity of p-25 TiO 2 particles with or without irradiation in three S. typhimurium strains. No increase in revertant colony number was observed.

3.2. Microbial mutation assay Exposure of Salmonella typhimurium strains TA100, TA98, and TA102 to p-25 TiO 2 particles with or without UVrvis light irradiation induced no significant increase in the frequency of revertant colonies ŽFig. 3.. The negative response was confirmed in repeat experiment Ždata not shown.. 3.3. Mammalian cell mutation assay

Fig. 2. DNA damage Žas measured by SCG assay. in L5178Y cells exposed to 50 mgrml p-25 TiO 2 particles and to various UVA intensities. a: length of DNA migration; b: percent cell survival relative to solvent control 24 h after treatment.

L5178Y cells exposed to p-25 TiO 2 particles with 5 Jrcm2 UVrvis light irradiation showed no significant increase in the frequency of mutant colonies ŽTable 1.. The negative response was confirmed in an independent experiment Ždata not shown..

Y. Nakagawa et al.r Mutation Research 394 (1997) 125–132

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Table 1 L5178Y mouse lymphoma cell mutation assay with p-25 particles Concentration of TiO 2 Žmgrml. UVrvisq Ž5 Jrcm 0 250 500 750 1000 1250 1500 2000 UVrvisy : MMS 30 a

Relative survival at day 0 Ž%.

Frequency of mutant coloniesr10 6 cells

Percentage of small colony

100 66.1 94.4 100.0 112.6 138.5 107.6 78.5

128.0 77.1 83.1 85.6 96.1 122.0 79.7 93.9

41.2 39.0 35.5 28.6 51.3 38.4 35.8 39.2

110.9

709.0

45.7

2.

a

Methyl methanesulfonate ŽCAS 66-27-3. was used as the positive control.

Table 2 Chromosome aberrrations in CHLrIU cells exposed to various concentrations of TiO 2 particles Žp-25. and 5Jrcm2 UVrvis light irradiation

UVrvisq Ž5 Jrcm2 .

Ofloxacin

f

UVrvisy

Ofloxacin a

Types and numbers of chromosome aberration

b

Concentration of TiO 2 Žmgrml.

Cell survival Ž%. a

Observed

ctg

csg

ctb

cte

csb

cse

mul

Structural aberration Ž%.

0 0.78 1.56 3.13 6.25 12.50 25.00 50.00 28.50

100 99.3 66.9 42.6 44.1 39.2 46.6 3.0 37.9

100 100 100 100 100 100 100 100 100

0 1 1 3 3 0 3 0 4

0 0 0 0 0 0 0 0 0

1 0 2 6 8 7 9 8 131

0 0 5 6 3 7 8 13 126

1 0 0 0 0 1 1 1 0

0 1 0 0 1 1 0 0 0

0 0 0 0 0 0 0 2 9

9) 7 10 ) 11) ) 15 ) ) 65 ) )

10 ) 10 ) ) 15 ) ) 64 ) )

0 25.00 50.00 100.00 200.00 400.00 800.00 28.50

100 102.1 73.3 61.0 44.5 20.1 5.0 92.7

100 100 100 100 100 100 100 100

0 2 0 0 1 0 0 0

0 0 0 0 0 0 1 0

0 1 0 0 0 2 1 0

0 0 0 1 0 0 0 0

0 0 0 0 0 0 0 0

0 0 0 0 1 1 0 0

0 0 0 0 0 0 0 0

0 3 0 1 2 3 2 0

0 1 0 1 1 3 1 0

TAG 2 2 7

c

% of Polyploid

d

TA 2 1 6 8 5

0.20 0 0 0.20 0.60 0.40 0.40 N.T. e 1.60 ) 0.20 0.20 0.20 0 0 0.60 0 0.20

Surviving cell number relative to the solvent control at 24 h after treatment. Types of chromosomal aberration: chromatid gap Žctg., chromosome gap Žcsg., chromatid break Žctb., chromatid exchange Žcte., chromosome exchange Žcse., metaphase with uncountable complex aberrations Žmul.. c % of metaphases with chromosomal aberrations with gap ŽTAG. and without gap ŽTA.. ) p - 0.05, ) ) p - 0.01 ŽFisher exact probability test.. d % of polyploidy based on 500 observed cells. ) p - 0.05 ŽFisher exact probability test.. e Poliploidy was not analysed at this concentration, because not enough cells Ž500. were obtained to observe. f Ofloxacin ŽCAS 82419-36-1. was used as the positive control. b

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Table 3 Chromosome aberrations in CHLrIU cells exposed to 50 mgrml TiO 2 Žp-25. and various UVArvis light intensities UVA energy ŽJrcm2 .

Cell survival Ž%. a

Types and numbers of chromosome aberration

b

Structural aberration Ž%.

c

Observed

ctg

csg

ctb

cte

csb

cse

mul

TAG

TA

% of Polyploid d

Without TiO 2 0 100 5.00 88.8

100 100

0 1

0 0

1 1

0 0

0 0

0 0

0 0

1 2

1 1

0 0

With TiO 2 Ž50 mgrml. 0 60.7 1.25 48.0 2.50 12.0 5.00 5.2

100 100 100 100

0 1 1 1

0 0 0 0

0 1 3 9

0 0 2 7

0 0 0 0

0 0 0 0

0 0 0 0

0 2 4 11) )

0 1 4 10 ) )

0 0 0 0

a

Surviving cell number relative to the UVrvisy control after 24-h incubation. Types of chromosomal aberration: chromatid gap Žctg., chromosome gap Žcsg., chromatid break Žctb., chromatid exchange Žcte., chromosome exchange Žcse., metaphase with uncountable complex aberrations Žmul.. c % of metaphases with chromosomal aberrations with gap ŽTAG. and without gap ŽTA.. ) ) p - 0.01 ŽFisher exact probability test.. d % of polyploidy based on 100 cells observed. b

3.4. Chromosomal aberration assay In the absence of UVrvis light irradiation, p-25 TiO 2 particles did not increase chromosome aberration frequency at any dose tested. However, cells treated with p-25 TiO 2 particles and 5 Jrcm2 UVrvis light showed a dose-dependent increase in chromosome aberrations ŽTable 2.. The principal structural aberrations induced were chromatid breaks and chromatid exchanges, while polyploidy was increased 2- to 5-fold for doses of 6.25–50 mgrml. At 50 mgrml p-25 TiO 2 particles, UVrvis light caused a dose-dependent increase in chromatid breaks and exchanges ŽTable 3.. The cytotoxic responses were different for cells exposed to UVrvis light ŽIC 50 : 2.7 mgrml. and not exposed ŽIC 50 : 146.7 mgrml.. Thus p-25 TiO 2 particles was more than 50-times cytotoxic in the presence of UVrvis light than in the absence of it.

4. Discussion In vivo toxicity studies have demonstrated that inhalation of TiO 2 particles induces pulmonary inflammation in rats w1,2x, and chronic exposure results in tumors such as bronchioalveolar adenomas or

keratinizing squamous cell carcinomas in rats w9x. On the other hand, oral w4x or parenteral w5x administration of TiO 2 particles indicated no toxicity symptoms or carcinogenicity in rats. An in vivo micronucleus study indicated a weak positive effect in mice w6x. There have been several reports on in vitro systems such as the chromosomal aberration assay w8x, SCE assay w8x, microbial mutation assay w8x, micronucleus assay w7x, and mouse lymphoma assay w3,8x, these data showed negative results but the studies did not include UVrvis light irradiation. It has been suggested that TiO 2 particles induce free radical formation in surrounding molecules under conditions of UVrvis light irradiation w18x, thereby inducing photodynamic cell death in bacteria w10,11x or cultured mammalian cancer cells w12,13x. In this study, we used the SCG assay and chromosomal aberration assay and showed that photoexcited TiO 2 particles induced primary DNA damage and structural chromosome aberrations in cultured mammalian cells. These genotoxic activities depended on TiO 2 dose and UVrvis light intensity. Without UVrvis light irradiation, only high concentrations of WA TiO 2 particles induced primary DNA damage, but no significant cytotoxic response was observed. Because primary DNA damage induced by TiO 2 particles and UVrvis light irradiation is accompa-

Y. Nakagawa et al.r Mutation Research 394 (1997) 125–132

nied by cytotoxicity, WA TiO 2 particles probably caused non photodynamic DNA damage through a different mechanism. We did not observe the induction of gene mutations by photoexcited TiO 2 particles in microbial or mammalian cell systems. Therefore, it was suggested that the DNA lesion catalyzed by photoexcited TiO 2 particles resulted in chromosomal aberration rather than gene mutations. Another possibility is that our experimental conditions detecting the gene mutations could have been not for optimal operation. Additional experiments are needed to resolve this apparent non-concordance between mutagenicity and clastogenicity. Some ongoing work with anti-oxidant and free radical scavengers has suggested that the photogenotoxicity of photoexcited TiO 2 particles may be attributable to their oxidizing activity Žin preparation.. In vivo photogenotoxicity assays are needed to evaluate human cancer risk posed by photoreactive environmental mutagens that are detected by in vitro assays. At present, however, the carcinogenic risk to humans of photodynamic substances is not well understood. Thus it is important to develop and validate in vivo test systems as well as in vitro assays for photogenotoxicity.

w3x

w4x

w5x

w6x

w7x

w8x

w9x

w10x

Acknowledgements This study is a communication by the Mammalian Mutagenicity Study Group ŽMMS. of the Mammalian Mutagenicity Society of Japan. We thank H. Ono for his advice on conducting this study. This work was supported by a Grant for Health Earth and Amenity Research Toward 21 ŽHEART 21. Promotion Fund from the Ministry of Health and Welfare ŽMHW. of Japan.

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