Clastogenicity, photo-clastogenicity or pseudo-photo-clastogenicity Genotoxic effects of zinc oxide

Mutation Research607(2006)215–224

Clastogenicity,photo-clastogenicity or pseudo-photo-clastogenicity: Genotoxic effects of zinc oxide in the dark,in pre-irradiated or simultaneously irradiated Chinese hamster ovary cells

Eric K.Dufour a,?,Tirukalikundram Kumaravel b,Gerhard J.Nohynek a,

David Kirkland b,Herv′e Toutain a

a L’Or′e al Research and Development,Worldwide Safety Department,92600Asni`e res,France

b COVANCE Laboratories Ltd,Oatley Road,Harrogate,North Yorkshire HG31PY,UK

Received26January2006;received in revised form5April2006;accepted12April2006

Available online21June2006

Abstract

Zinc oxide(ZnO),a widely used ingredient in dermatological preparations and sunscreens,is clastogenic in vitro,but not in vivo. Given that ZnO has an approximately four-fold greater clastogenic potency in the presence of UV light when compared with that in the dark,it has been suggested to be photo-clastogenic.In order to clarify whether this increased potency is a genuine photo-genotoxic effect,we investigated the clastogenicity of ZnO(mean particle size,100nm)in Chinese hamster ovary(CHO)cells in the dark (D),in pre-irradiated(PI,i.e.UV irradiation of cells followed by treatment with ZnO)and in simultaneously irradiated(SI,i.e.ZnO treatment concurrent with UV irradiation)CHO cells at UV doses of350and700mJ/cm2.The cytotoxicity of ZnO to CHO cells under the different irradiation conditions was as follows:SI>PI>D.In the dark,ZnO produced a concentration-related increase in chromosome aberrations(CA).In PI or SI CHO cells,ZnO was clastogenic at signi?cantly lower concentrations(approximately two-to four-fold)when compared with effective concentrations in the dark,indicating an increased susceptibility of CHO cells to ZnO-mediated clastogenic effects due to UV irradiation per se.The incidence of CA in SI or PI cells was generally higher than that in the dark.At similar ZnO concentrations,SI conditions generally produced higher CA incidence than PI conditions. However,when ZnO concentrations producing similar cytotoxicity were compared,CA incidences under PI or SI conditions were nearly identical.The modest increase in the clastogenic potency of ZnO following UV irradiation contrasts with the results observed with genuine photo-clastogenic agents,such as8-MOP,which may produce an increase in clastogenic potency of>15,000-fold under SI conditions.Our results provide evidence that,under conditions of in vitro photo-clastogenicity tests,UV irradiation of the cellular test system per se may produce a slight increase in the genotoxic potency of compounds that are clastogenic in the dark. In conclusion,our data suggest that minor increases in clastogenic potency under conditions of photo-genotoxicity testing do not necessarily represent a photo-genotoxic effect,but may occur due to an increased sensitivity of the test system subsequent to UV irradiation.

?2006Elsevier B.V.All rights reserved.

Keywords:Zinc oxide nanoparticles;CAS1314-13-2;Chinese hamster ovary cells;Clastogenicity;Photo-clastogenicity;Photo-genotoxicity Abbreviations:ZnO,zinc oxide nanoparticles;CA,chromosome aberration;CHO cells,Chinese hamster ovary cells;V-79cells,Chinese hamster V-79cells;GLP,good laboratory practice;PI,pre-irradiation or pre-irradiated;SI,simultaneous irradiation or simultaneously irradiated; 3T3NRU PT,neutral red uptake phototoxicity test on3T3mouse?broblasts

?Corresponding author.Tel.:+33147567905;fax:+33147564492.

E-mail address:edufour@1d26b30f76c66137ee0619d0(E.K.Dufour).

1383-5718/$–see front matter?2006Elsevier B.V.All rights reserved.

doi:10.1016/j.mrgentox.2006.04.015

216 E.K.Dufour et al./Mutation Research607(2006)215–224

1.Introduction

Zinc(Zn)is one of the most important trace ele-ments in the mammalian organism and is involved in homeostasis,immune response,oxidative stress,apop-tosis and ageing[1].Zn is also an essential component of mammalian androgen receptors,DNA repair enzymes and DNA or RNA synthesis,stability,proliferation and transcription[2,3].In animal models,Zn de?ciency has been associated with adverse reproductive effects includ-ing reduced fertility,foetal malformations and growth retardation in late pregnancy,whereas labour abnormal-ities,congenital malformations,and preterm labour have been reported in otherwise healthy women with low serum Zn concentrations[4].Zn and its salts were non-carcinogenic in rodents after inhalation,oral uptake or intra-peritoneal administrations,whereas Zn-de?ciency was shown to enhance the susceptibility of rodents to known carcinogens,such as4-nitroquinoline-N-oxide or cadmium.Finally,moderate dietary Zn-supplementation offered protection against carcinogenic substances[5].

Genotoxicity studies on Zn,its salts or its oxide(ZnO) had equivocal results.Although most in vitro and all in vivo tests were negative[5,6],Zn salts were reported to be slightly positive in in vitro genotoxicity tests,such as the Ames test or chromosome aberration(CA)test in cultured human lymphocytes[5].Similarly,results with Zn acetate in the Mouse Lymphoma Assay or in Chinese hamster ovary(CHO)cells were reported to be positive,albeit at high concentrations[7].Recently,the EU Scienti?c Committee on Toxicity,Ecotoxicity and the Environment concluded that Zn and its salts are not expected to be mutagens or carcinogens under the expected human exposure conditions[8].

Zinc oxide(ZnO)is widely used in dermatological preparations and sunscreens[9].In the European Union, sunscreens are regulated under the EU Cosmetics Direc-tive and require an extensive safety dossier as described in the Notes of Guidance of the EU Scienti?c Committee on Cosmetics and Non-Food Products(SCCNFP).These Guidelines stipulate that UV?lters should be examined for genotoxicity,photo-toxicity,photo-sensitisation,as well as photo-genotoxicity in appropriate tests such as the photo-Ames and photo-CA tests[10].Accordingly, ZnO was tested in a series of assays,the results of which were recently reviewed[11].ZnO was photo-stable, non-photo-reactive,non-photo-(cyto)toxic(negative in the in vitro3T3NRU PT test),non-photo-irritant or-photo-allergenic in humans,and negative in the Ames or photo-Ames tests.The results of these different assays for photo-reactivity are summarised in Table1.Although ZnO was negative in vivo in the mouse bone marrow micronucleus test,it produced an increased incidence of CA in CHO and Chinese hamster V-79(V-79)cells at concentrations of814or10?g/mL and above,respec-tively.It was therefore rated to be in vitro clastogenic and, possibly,aneugenic.In the presence of UV irradiation, ZnO was clastogenic at approximately four-fold lower concentrations when compared with effective concen-trations in the dark,i.e.at195or3?g/mL and above in

Table1

Results of tests for in vitro or in vivo photo-reactivity of ZnO(unpublished data included in the industry safety dossier,reviewed in the SCCNFP opinion on ZnO[11])

Test/endpoint Test system/protocol Test material Result Rating

Photo-stability Absorption curves before

and after simulated solar

irradiation(30J/cm2)Sunscreen containing

15%uncoated micro?ne

ZnO

Similar absorption curves

before and after

irradiation

Photo-stable

Photo-reactivity Photo-catalytic oxidation

of liquid propane-2-ol to

propanone Micro?ne uncoated,

dimethicone-and

silica-coated ZnO

No signi?cant

photo-catalytic activity

Non-photo-reactive

In vitro photo-(cyto)toxicity Neutral Red uptake

phototoxicity test on

Balb/c3T3mouse

?broblasts Micronised uncoated ZnO Phototoxicity induction

factor equals1

Non-photo-(cyto)toxic

Photo-irritancy Single application to

human volunteers,

Hayes-test chambers

(occlusive patch effect)25%micronised uncoated

or dimethicone-coated

ZnO in cosmetic w/o

emulsion

No photo-irritant skin

reactions

Non-photo-irritant

Photo-allergy Six applications over3

weeks to human

volunteers,Hayes-test

chambers(occlusive

patch effect)25%micronised uncoated

or dimethicone-coated

ZnO in cosmetic w/o

emulsion

No photo-allergic skin

reactions

Non-photo-allergenic

E.K.Dufour et al./Mutation Research607(2006)215–224217 Table2

Results of in vitro genotoxicity/photo-genotoxicity tests on ZnO(unpublished data included in the industry safety dossier,reviewed in the SCCNFP opinion on ZnO[11])

Test Test system Test material Result Rating

Ames test S.typhimurium TA98,

100,1537and E.coli

WP2Micronised uncoated

ZnO formulated as a

10%emulsion

Negative Non-mutagenic

Photo-Ames test S.typhimurium TA98,

100,1537and E.coli

WP2Micronised uncoated

ZnO formulated as a

10%emulsion

Negative Non-photo-mutagenic

In vitro clastogenesis CHO cells Micronised uncoated

ZnO formulated as a

10%emulsion Positive at

≥814?g/mL

Clastogenic in vitro

In vitro photo-clastogenesis (750mJ/cm2)CHO cells Micronised uncoated

ZnO formulated as a

10%emulsion

Positive at

≥195?g/mL

Photo-clastogenic in vitro

In vitro clastogenesis V-79cells Aqueous suspension

of micronised

uncoated ZnO Positive at

≥10?g/mL

Clastogenic in vitro

In vitro photo-clastogenesis (375mJ/cm2)V-79cells Aqueous suspension

of micronised

uncoated ZnO

Positive at≥3?g/mL Photo-clastogenic in vitro

In vitro Comet test/photo-Comet test(230/450and 980mJ/cm2,HaCaT and V79cells,respectively)V-79cells and human

keratinocytes(HaCaT

cells)

Aqueous suspension

of micronised coated

ZnO

Weakly positive in

V-79cells,clearly

negative in HaCaT

cells

Equivocal

photo-genotoxicity in vitro

CHO or V-79cells,respectively.In contrast,the results of in vitro Comet and photo-Comet tests in human ker-atinocytes(HaCaT cells)and V-79cells were equivocal. The results of previous in vitro genotoxicity and photo-genotoxicity tests on ZnO are summarised in Table2.

In the absence of guidelines on the interpretation of photo-genotoxicity test results,the clastogenic activity of ZnO at lower concentrations in the presence of UV light when compared with effective concentrations in the dark may be interpreted as a,albeit slight,photo-clastogenic activity.However,these results also raise intriguing questions.Since ZnO was non-photo-reactive, non-photo-toxic and non-photo-allergenic in vitro or in vivo(Table1),a genuine photo-genotoxic activity appears to have little biological plausibility[12,13].An alternative explanation for the observed effects could be that UV irradiation produced a higher susceptibility of the CHO or V-79cells to the intrinsic in vitro clastogenic activity of ZnO.Taking into account that in vitro tests for clastogenicity,such as CA or micronucleus tests in mammalian cells,are known to have poor speci?city,i.e. yield a high percentage of false-positive results[14,15], it is important to investigate whether an increased clasto-genic potency of a test compound in the presence of UV irradiation represents a genuine photo-genotoxic poten-tial or is an artefact,due to an increased sensitivity of the test system.

To this end,we tested ZnO for clastogenic activity under conditions of good laboratory practice(GLP)in CHO cells(a)in the dark,i.e.ZnO treatment in the absence of UV irradiation;(b)under the recommended conditions for photo-clastogenicity testing,i.e.simulta-neous UV irradiation(SI)and treatment with ZnO;and (c)under pre-irradiation conditions(PI),i.e.ZnO treat-ment after pre-irradiation of the cells with UV light.In order to establish a dose-response for potential effects, we performed the tests with the appropriate UV dose for photo-genotoxicity tests,i.e.a dose that yields a slight but reproducible photo-genotoxic effect[12,13],as well as a lower dose(700and350mJ/cm2,respectively).The aim of our study was to re-examine the photo-clastogenicity of a substance that is in vitro clastogenic in the dark. 2.Materials and methods

The protocol used in the present study complied with cur-rent recommendations for photo-genotoxicity testing[12,13].

2.1.Chemicals

Micro?ne uncoated ZnO(CAS RN1314-13-2),batch EHDE0402,was purchased from BASF AG,Ludwigshafen, Germany(Z-Cote?),and was>99%pure,with a particle size of less than200nm.8-Methoxypsoralen(8-MOP)was purchased from Sigma Aldrich Co.,Poole,UK,and4-nitroquinoline-

218 E.K.Dufour et al./Mutation Research607(2006)215–224

1-oxide(NQO)was purchased from Aldrich Chemical Co., Gillingham,UK.

2.2.Test protocols

2.2.1.UV irradiation

An Atlas Suntest?CPS solar simulator light source was used(Heraeus Equipment Limited,Brentwood,UK).The lamp irradiated six25-cm2culture?asks within an enclosed and temperature-controlled area.The temperature of the irradiation area was35?C and was maintained by a SunCool?Cooling unit attached to the Atlas system.The intensity of UV A and UVB was measured using a Dr.Gr¨o bel RM21UV meter.

A UV glass?lter was added to the lamp to remove the UVC component of the light(wavelengths less than290nm). The?ltering effect of the tissue culture?ask plastic and the growth medium covering the cells was measured and taken into account for the calculation of the delivered doses.The ratio of UVB:UV A irradiation delivered to the cells was1:30.

2.2.2.Rationale

To determine the effects of ZnO plus light on CA incidence, cells were treated with the selected range of ZnO concentra-tions.These were exposed to one or other of two UV doses (UV radiant exposures)either prior to(pre-irradiated cells) or simultaneously with ZnO treatment(simultaneously irradi-ated cells).The UV doses used were350and700mJ/cm2.The high UV dose was shown to cause a small increase in the inci-dence of cells containing structural aberrations excluding gaps according to internal data of the testing laboratory.Following calibration of the lamp,irradiation durations were calculated to obtain the required UV doses(3min41s and7min22s for the350and700mJ/cm2UV doses,respectively).The various concentrations of ZnO used were also tested in the absence of UV light.

2.2.

3.Controls

Medium at the appropriate volume was added to cultures designated as negative control.Controls were conducted with-out and with exposures to UV light.Additional cultures were treated with the positive control chemicals NQO(0.3?g/mL, in the absence of UV irradiation)or8-MOP(1.0?g/mL,in the absence and presence of UV irradiation).The positive control chemicals were dissolved in DMSO immediately prior to use.

2.2.4.Cell cultures

CHO cells,supplied by Dr.S.Galloway,West Point,PA, USA,are maintained at the testing laboratory in tissue culture ?asks containing McCoy’s5A medium including10%(v/v) foetal calf serum(FCS),and100?g/mL gentamycin.They are sub-cultured regularly to maintain low aberration incidences. Stocks of cells preserved in liquid nitrogen are reconstituted for each experiment so as to maintain karyotypic stability.The cells are screened for mycoplasma contamination.The modal chromosome number for the clone of CHO cells used at the testing laboratory is21.

Cell sheets were removed from stock cultures using trypsin/EDTA solution,and sub-cultured at a low density into25-cm2tissue culture?asks.The volume of?nal culture medium in each?ask was4.95mL.After1–3days of incuba-tion in an atmosphere of5%(v/v)CO2in air,and at37±1?C, cultures at a suitably low level of con?uence were selected for treatment.

2.2.5.Methods

The treatment scheme was as follows:

1.Non-irradiated group:cultures received no irradiation.

2.Pre-irradiated(PI)group:cultures received irradiation

2–3h(350mJ/cm2)or1–2h(700mJ/cm2)prior to ZnO treatment.

3.Simultaneously irradiated(SI)group,350and700mJ/cm2:

cultures received irradiation simultaneously with ZnO treat-ment.

Prior to the start of treatment,the cell sheets from four?asks were removed using trypsin/EDTA and indi-vidual cell counts were obtained and a mean cell count calculated.This provided the starting(baseline)count for the calculation of cytotoxicity(expressed as decrease in population doublings relative to controls)at the time of cell harvest.

CHO cultures were established in25-cm2?asks and incu-bated at37±1?C in an atmosphere of5%(v/v)CO2in air, until approximately30–50%con?uent.

Following pre-irradiation,the medium was changed and ?asks incubated at37±1?C in an atmosphere of5%(v/v) CO2in air.The time between irradiation and ZnO treatment was between1and3h.PI?asks were treated with ZnO at the same time as the non-irradiated and SI?asks.

One set of quadruplicate cultures for each of the treatment regimes was treated with the medium and one set of duplicate cultures with ZnO(0.05mL per culture).After incubation in the dark for at least15min,?asks(SI group)were then exposed to the required UV dose.Flasks were kept in the incubator at37?C before and after irradiation.Three hours following addition of ZnO or control chemicals,cell monolayers from all cultures were washed with sterile saline,and re-fed with fresh McCoy’s 5A medium containing foetal calf serum and gentamycin. Cultures were then incubated at37?C for a further17h before harvesting.Approximately1.5h prior to harvest,colchicine was added to give a?nal concentration of approximately 1?g/mL to arrest dividing cells in metaphase.At the de?ned sampling times,monolayers of these cultures were removed using trypsin/EDTA and a measurement of cell counts/mL was performed on an aliquot of cell suspension using a Coulter counter.The remaining cell suspensions from each?ask were harvested and slides prepared for CA analysis using standard operating procedures.For the SI group,treatment and harvest times were staggered to ensure all?asks could be irradiated within3h of the addition of medium,ZnO or positive control.

E.K.Dufour et al./Mutation Research607(2006)215–224219

2.2.6.Selection of ZnO concentrations for light treatment

Concentrations used in the main experiment were selected on the basis of cytotoxicity(expressed as decrease in popula-tion doublings relative to controls),but slides were examined prior to analysis of CA to ensure suf?cient metaphases were available for analysis in the selected concentration range.Pop-ulation doublings(PD)were calculated for each concentration as follows:

PD=[log(N/Xo)]/log2

where N=mean?nal cell count/culture at each concentration; Xo=starting(baseline)count.

The numbers of cells/mL were measured in trypsinised samples of cell suspension using a Coulter counter.For each individual irradiation dose and condition,concentrations for analysis were selected to cover a cytotoxicity range from max-imum to little or no effect.

2.2.7.Scoring of aberrations

Where possible(and in the absence of a marked production of CA),100metaphases from each treatment were analysed blind for structural chromosome aberrations.When increased CA incidences were observed,slide analysis was halted when a minimum of10aberrant cells(cells with aberrations excluding gaps)per slide was observed.Only cells with19–23chro-mosomes were considered acceptable for analysis.Cells with more than23chromosomes were noted and recorded sepa-rately.Classi?cation of structural aberrations was based on the scheme described by ISCN[16].

Slide analysis was performed by competent analysts trained in the testing laboratory standard operating procedures.All analysts participating in this study were subject to the testing laboratory management and GLP control systems.

2.2.8.Analysis of results—treatment of data

The aberrant cells in each culture were categorised as fol-lows:

1.Cells with structural aberrations including gaps.

2.Cells with structural aberrations excluding gaps.

3.Polyploid,endoreduplicated or hyperdiploid cells.

The totals for category2in negative control cultures were compared with the contemporaneous testing laboratory neg-ative control(normal)ranges to determine whether the assay was acceptable or not.The proportions of aberrant cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test.

For each individual irradiation dose and condition,the pro-portion of cells in category2for each test treatment condition was compared with the proportion in concurrent negative con-trols using Fisher’s exact test.Probability values of p≤0.05 were accepted as signi?cant.The proportions of cells in cate-gories1,2and3were examined in relation to historical negative control(normal)ranges in order to assess biological signi?-cance.

Direct statistical comparison of the incidence of CA at var-ious ZnO concentrations under different irradiation conditions was not performed in this study.As aberrations are only usu-

Table3

Cytotoxicity results for ZnO in the dark,in pre-irradiated and simultaneously irradiated CHO cells,expressed as decrease in population doublings relative to controls

ZnO concentration (?g/mL)Cytotoxicity(%,decrease in population doubling)

No UV irradiation350mJ/cm2700mJ/cm2

Pre-irradiation Simultaneous

irradiation

Pre-irradiation Simultaneous

irradiation

0?(1.183)?(1.257)?(1.183)?(0.963)?(0.963)

27?4123?14?9

34?15?13?812?13

43?01319+0?4

5461413?1019

67?221?02931

84218?51438 1051336233158 1312528374162 1641051523890 2052156484334 2564453635878 3206374666152 4004488606688 5001007210010077

Figures represent the mean of two replicate cultures(four replicates for control cultures).Negative?gures represent increases in population doubling over respective control.Figures indicated in brackets for treatment without ZnO(0?g/mL)represent absolute values of population doubling.

220 E.K.Dufour et al./Mutation Research 607(2006)215–224

ally detected at cytotoxic concentrations,and as cytotoxicity varied with treatment condition,we determined that compar-isons at equitoxic concentrations would be more informative.Therefore,at the high UV dose,we compared CA incidences under PI and SI conditions at ZnO concentrations that produced similar cytotoxicity,using Fisher’s exact test,i.e.84?g/mL PI (14%cytotoxicity)versus 54?g/mL SI (19%cytotoxic-ity),164?g/mL PI (38%cytotoxicity)versus 84?g/mL SI (38%cytotoxicity),256?g/mL PI (58%cytotoxicity)versus 105?g/mL SI (58%cytotoxicity),and 320?g/mL PI (61%cytotoxicity)versus 131?g/mL SI (62%cytotoxicity).

3.Results

3.1.Cytotoxicity of ZnO in the dark,in

pre-irradiated (PI)and simultaneously irradiated (SI)CHO cells

Cytotoxicity results of ZnO in the dark,in SI and PI cells are shown in Table 3,Fig.1(UV dose of 350mJ/cm 2)and Fig.2(700mJ/cm 2).In the dark,the cytotoxicity of ZnO was concentration-dependent,with 40–60%cytotoxicity (expressed as decrease in popu-lation doublings relative to controls)observed in the concentration range 256–320?g/mL.

In SI cells,the cytotoxicity was more pronounced than in the dark and related to the UV dose:40–60%cytotoxicity was observed in the concentration range 131–256?g/mL (350mJ/cm 2)or 84–131?g/mL (700mJ/cm 2).Cytotoxicity in SI cells increased over-all with ZnO concentrations,although a large vari-ability in ZnO cytotoxicity was observed at the

high

Fig.1.ZnO-induced cytotoxicity (%,expressed as decrease in popula-tion doublings relative to controls)in non-irradiated CHO cells (dark),pre-irradiated (PI)or simultaneously irradiated (SI)CHO cells (UV dose of 350mJ/cm 2).Small increases in population doubling at some treatments are shown as 0%

cytotoxicity.

Fig.2.ZnO-induced cytotoxicity (%,expressed as decrease in popula-tion doublings relative to controls)in non-irradiated CHO cells (dark),pre-irradiated (PI)or simultaneously irradiated (SI)CHO cells (UV dose of 700mJ/cm 2).Small increases in population doubling at some treatments are shown as 0%cytotoxicity.

UV dose (700mJ/cm 2)and at high ZnO concentrations (≥205?g/mL).

In PI cells,cytotoxicity of ZnO was concentration-dependent and comparable at the low UV dose (350mJ/cm 2)to that observed in SI cells (40–60%cytotoxicity was observed in the concentration range 105–205?g/mL).At the high UV dose (700mJ/cm 2)and at low ZnO concentrations (<205?g/mL),cyto-toxic effects of ZnO in PI cells were between those observed in the dark and SI cells,with 40–60%cytotoxic-ity observed in the concentration range 131–256?g/mL.At higher concentrations (≥205?g/mL),ZnO cytotoxi-city was similar in PI and SI cells.3.2.Clastogenicity of ZnO

For each individual irradiation dose and condition,the concentrations analysed for CA covered a range of cyto-toxicity from no or little to maximum effect.The clasto-genicity results of ZnO in the dark,in PI and SI cells are shown in Table 4,Fig.3(UV dose of 350mJ/cm 2)and Figs.4and 5(700mJ/cm 2).The types of structural aber-rations produced by ZnO were similar in the dark and under both irradiation conditions,and mainly consisted of chromatid deletions and exchanges (data not shown).3.2.1.Clastogenicity of ZnO in the dark

Treatment with ZnO in the dark produced a concentration-related increase in the proportion of cells with CA.The increases over control data were biolog-ically and statistically signi?cant at concentrations of ≥105?g/mL;the maximal CA incidence was 16%.The

E.K.Dufour et al./Mutation Research 607(2006)215–224221

Table 4

Clastogenicity results (%of cells with chromosome aberrations,excluding gaps)for ZnO in the dark,in pre-irradiated and simultaneously irradiated CHO cells ZnO concentration (?g/mL)

Proportion of cells with chromosome aberrations (%)No UV Irradiation

350mJ/cm 2700mJ/cm 2Pre-irradiation

Simultaneous irradiation Pre-irradiation Simultaneous irradiation 0121 2.5654 3.5––6a 12a 84 3.5––12c 25.5c 1055a 5.5b 11c 12.5c 25.5c 131 5.5b 10c 17.5c 27c 35c 16412c 11.5c 25c 24.5c 40.5c 25614c 23c 34.5c 27c 45c 32015.5c 25c 22.5c 36.5c 33c NQO 42.5c ––––8-MOP

2

2

84c

4.5

90c

Figures reported are mean of two replicate cultures (four replicates for control cultures);NQO:4-nitroquinoline-1-oxide (0.3?g/mL);8-MOP:8-methoxypsoralen (1.0?g/mL);–:not analysed.a p ≤0.05compared to concurrent controls.b p ≤0.01compared to concurrent controls.c p ≤0.001compared to concurrent controls.

positive control substance in the dark (NQO)produced a statistically signi?cant increase in CAs over control data,whereas 8-MOP was not clastogenic in the dark at the concentration tested (1.0?g/mL).

3.2.2.Clastogenicity of ZnO in simultaneously irradiated (SI)CHO cells

In SI cells,treatment with ZnO produced a more pro-nounced increase in CA incidences when compared

with

Fig.3.ZnO-induced incidence (%)of chromosome aberrations (CA,excluding gaps)in non-irradiated CHO cells (dark),pre-irradiated (PI)or simultaneously irradiated (SI)CHO cells (UV dose of 350mJ/cm 2).those in the dark.CA incidences increased with ZnO con-centration (except at the highest concentration tested of 320?g/mL)and UV dose,and reached biological (i.e.outside historical control range)and statistical signi?-cance at concentrations of ≥105?g/mL (350mJ/cm 2)or ≥54?g/mL (700mJ/cm 2).The maximal CA inci-dences were 35%and 45%at the low and high UV dose,respectively.The positive control in the presence of UV light,8-MOP,produced a statistically

signi?cant

Fig.4.ZnO-induced incidence (%)of chromosome aberrations (CA,excluding gaps)in non-irradiated CHO cells (dark),pre-irradiated (PI)or simultaneously irradiated (SI)CHO cells (UV dose of 700mJ/cm 2).

222 E.K.Dufour et al./Mutation Research607(2006)

215–224

Fig.5.Incidence(%)of chromosome aberrations(CA,excluding gaps)versus cytotoxicity(%,expressed as decrease in population doublings relative to controls)in non-irradiated CHO cells(dark), pre-irradiated(PI)or simultaneously irradiated(SI)CHO cells(UV dose of700mJ/cm2).Small increases in population doubling at some treatments are shown as0%cytotoxicity.CA incidences under PI or SI conditions were compared at concentrations that produced similar cytotoxicity(14%vs.19%,38%vs.38%,58%vs.58%,and61%vs. 62%,respectively)using the Fisher’s exact test.No statistically signif-icant differences in the incidence of CA between PI and SI cells were found.

increase in CAs over control data,with similar CA inci-dences observed at both UV doses.

3.2.3.Clastogenicity of ZnO in pre-irradiated(PI) CHO cells

Similar to the results in SI cells,CA incidences in PI cells increased with ZnO concentration and UV dose.At similar ZnO concentrations,CA incidences in PI cells were generally between those observed in the dark and those in SI cells,except at the low UV dose in the low concentration range(≤164?g/mL)where they tended to be similar to those observed in the dark.Moreover, at the highest concentration(320?g/mL),the propor-tion of cells with CAs was similar in PI and SI cells. The increases in CA incidence in PI cells over con-trol data were biologically and statistically signi?cant at the same concentrations as for SI cells,i.e.at concen-trations of≥105?g/mL(350mJ/cm2)or≥54?g/mL (700mJ/cm2).The maximal CA incidences observed were25%and37%at the low and high UV dose,respec-tively,and were between those observed in the dark and those in SI cells.When CA incidences at the high UV dose were compared at ZnO concentrations inducing similar cytotoxicity,PI and SI conditions produced a nearly identical increase in CA incidence(Fig.5).In contrast to the results observed in SI cells,treatment with8-MOP did not produce any increased CA incidence under PI conditions.

4.Discussion

Our results suggest an interaction of competing parameters,all of which may have affected the photo-(cyto)toxicity and/or photo-clastogenicity of ZnO:(a) the intrinsic cytotoxicity and clastogenicity of ZnO in the dark,(b)the effects of pre-or simultaneous irradia-tion,(c)the protective effect of ZnO,which is an ef?cient UV?lter and(d)the repair of UV-induced damage prior to ZnO treatment in pre-irradiation conditions.

Current recommendations for photo-genotoxicity testing recommend a UV irradiation dose that produces a slight,but reproducible photo-genotoxic effect[12,13]. Therefore,under the conditions of our study,700mJ/cm2 was an appropriate irradiation dose,which also corre-sponded to the conditions of the previous test in CHO cells,which used750mJ/cm2(Table2).The cytotoxicity results of our study suggest that,under conditions of pre-irradiation(PI)and simultaneous irradiation(SI)of CHO cells,the toxicity of ZnO was enhanced when compared to that after incubation in the dark.SI generally produced more severe cytotoxic effects when compared with those observed under PI conditions,except at high ZnO con-centrations where a similar cytotoxicity was observed after PI and SI,probably due to protection by ZnO against UV-induced damage under SI conditions.

In the dark,the concentration-related increase in CA incidence con?rmed that ZnO is clastogenic under con-ditions of standard in vitro clastogenicity tests.Consis-tent with the results of previous experiments,our data show that under conditions of photo-genotoxicity test-ing(SI conditions),ZnO produces clastogenic effects of similar severity at concentrations approximately two-to four-fold lower than in the dark,which may sug-gest photo-clastogenicity.However,a similar shift in effective clastogenic concentrations(increased clasto-genic potency)was observed under PI conditions where CHO cells were irradiated in the absence of ZnO.Addi-tionally,ZnO treatment under SI conditions produced a higher maximal effect(CA incidence)than in the dark (increased clastogenic activity),and a similar increase was observed under PI conditions.Finally,there were no qualitative differences under dark,PI or SI condi-tions in the clastogenic effects observed,i.e.types of structural aberrations produced(chromatid deletions and exchanges).These?ndings indicate that the observed increased clastogenic potency and activity under UV

E.K.Dufour et al./Mutation Research607(2006)215–224223

light is unrelated to photo-activation of ZnO,but a con-sequence of an increased susceptibility of CHO cells to ZnO-mediated clastogenic effects due to UV irradiation of cells per se.

However,ZnO treatment under SI conditions pro-duced greater CA incidences than those observed under PI conditions,except at the highest ZnO concentra-tion where they were comparable under both PI and SI conditions.These?ndings are not surprising,taking into account the well-known production of chromosome aberrations secondary to cytotoxicity[17,18]and the more pronounced cytotoxic effects observed under SI conditions when compared with those under PI con-ditions.The comparison of CA incidences obtained at ZnO concentrations that produced similar cytotoxicity clearly shows that UV irradiation under both PI and SI conditions produced a similarly increased susceptibility of the cells to the intrinsic clastogenic activity of ZnO (Fig.5).Possibly,at the highest ZnO concentration,the ZnO-mediated UV protection became more prominent, resulting in similar clastogenic responses under both PI and SI conditions.At the low UV dose(350mJ/cm2) and in the low concentration range,effects of ZnO under PI conditions were not signi?cantly different from those observed in the dark.Possibly,the2–3-h gap between pre-irradiation at this lower UV dose and ZnO treatment allowed recovery of CHO cells,as rapid repair by mam-malian cells of UV-induced DNA and cellular damage has been well described[13].However,at the higher UV dose,the1–2-h gap between pre-irradiation and ZnO treatment may not have been suf?cient to allow recov-ery,as the clastogenic responses were very similar under PI and SI conditions at this higher UV dose.Additional studies would be required to assess the role of cell recov-ery after pre-irradiation.

A clastogenic compound has been de?ned as a sub-stance that produces structural changes of chromosomes [19].Tentative guidelines on photo-genotoxicity/photo-clastogenicity testing have de?ned photo-genotoxicity as the property of a compound to induce genotoxic effects when irradiated with UV and/or visible light [12,13].However,current guidelines offer no guidance for classi?cation of in vitro clastogenic substances that become more potent or active in the presence of UV light.

In vitro clastogenicity tests are notorious for high rates of false-positive results.Given that more than55% of all substances tested yielded positive results in these tests[15],the de?nition of in vitro photo-genotoxicity for substances that are clastogenic in the dark requires urgent attention,particularly when taking into account the absence of validated in vivo tests that could dis-tinguish genuine from pseudo-photo-clastogens.Possi-

bly,such substances should also be investigated in pre-

irradiated cells as described in the present study.

However,increased clastogenic potency in the pres-

ence of UV light may indeed represent genuine photo-

clastogenic effects.For example,8-MOP,a possible

human photo-co-carcinogen[20]and a standard pos-

itive control substance in photo-genotoxicity/photo-

clastogenicity tests,was reported to be clastogenic in

CHO cells in the dark at concentrations of≥251?g/mL.

In the presence of UV light,8-MOP produced an

increased CA incidence at concentrations as low as

0.016?g/mL,i.e.at a concentration approximately

16,000-fold lower than in the dark[21].These results

suggest that,in the presence of UV,the potency of gen-

uine photo-genotoxic agents may increase by more than

four orders of magnitude.Given that the increase in

clastogenic potency in the presence of UV light was ≥4000-fold lower for ZnO relative to that of8-MOP, this provides additional support to the view that the

phenomenon observed with ZnO has little in common

with genuine photo-genotoxicity.Moreover,in our study,

the results obtained with ZnO contrasted with those

observed with8-MOP,which produced increases in CA

incidences under SI conditions,but not under PI condi-

tions.

Finally,it has been recognised that photo-stability

and lack of photo-toxic or photo-allergenic activity of a

substance are strong indicators that it is unlikely to pos-

sess photo-genotoxic properties[12,13].Given that ZnO

is photo-stable,non-photo-reactive,non-photocytotoxic

(negative in the in vitro3T3NRU PT test),non-photo-

irritant and non-photo-allergenic,a genuine photo-

genotoxic activity of ZnO appears to be biologically

implausible.

Overall,prior and simultaneous treatments with UV

increased the susceptibility of CHO cells to the clasto-

genic activity of ZnO.Given that under conditions of pre-

irradiation,ZnO was not exposed to UV,our results do

not support the view that a slightly increased clastogenic

potency under conditions of standard photo-genotoxicity

tests represents a genuine photo-clastogenic effect,but

indicate that these effects are a consequence of an

increased sensitivity of the test system due to UV irra-

diation.Whereas direct genotoxic effects of UV light

are well known,this is the?rst report describing UV-

mediated increased sensitivity of a mammalian geno-

toxicity test system.Although our results describe such

an effect for a single substance only,they suggest

that the de?nition of photo-genotoxicity for substances

that are clastogenic in the dark may require further

validation.

224 E.K.Dufour et al./Mutation Research607(2006)215–224

Acknowledgements

The authors wish to thank Susan Riley(COV ANCE Laboratories Ltd,Harrogate,UK)for her help in design-ing this study.

References

[1]M.Stefanidou,C.Maravelias,A.Dona,C.Spiliopolou,Zinc:a

multipurpose trace element,Arch.Toxicol.80(2006)1–9. [2]D.M.Robins,Androgen receptors and molecular mechanism of

male-speci?c gene expression,Novartis Found.Symp.268(2005) 42–52.

[3]F.D.Urnov,1d26b30f76c66137ee0619d0ler,Y.L.Lee, C.M.Beausejour,J.M.

Rock,S.Augustus,A.C.Jamieson,M.H.Porteus,P.D.Gregory, M.C.Holmes,Highly ef?cient endogenous human gene correc-tion using designed zinc-?nger nucleases,Nature435(2005) 646–651.

[4]K.Mahomed,D.K.James,J.Golding,R.McCabe,Zinc sup-

plementation during pregnancy:a double blind randomised con-trolled trial,Br.Med.J.299(1989)826–830.

[5]US Environmental Protection Agency,Toxicological review of

zinc and compounds,EPA/635/R-05/002,2005.Available at: 1d26b30f76c66137ee0619d0/iris/toxreviews/0426-tr.pdf.

[6]Anonymous,Zinc Environmental Health Criteria,vol.221,IPCS,

World Health Organisation,Geneva,Switzerland,2001.

[7]E.D.Thompson,J.A.McDermott,T.B.Zerkle,J.A.Skare,B.L.B.

Evans,D.E.Cody,Genotoxicity of zinc in4short term mutagenic-ity assays,Mutat.Res.223(1989)267–272.

[8]EU Scienti?c Committee on Toxicity,Ecotoxicity and the envi-

ronment,opinion on the results of the risk assessment of zinc metal,zinc chloride,zinc sulphate,zinc distearate,zinc phos-phate and zinc oxide,adopted by the CSTEE during the39th Plenary Meeting of10September2003(C7/GF/csteeop/zincs/ 100903D(03)).Available at:ec.europa.eu/food/fs/sc/sct/ out197en.pdf.

[9]T.Maier,H.C.Korting,Sunscreens—which and what for?Skin

Pharmacol.Physiol.18(2005)253–262.

[10]The Scienti?c Committee on Cosmetic Products and Non-Food

Products intended for consumers,The SCCNFP’S notes of guid-ance for the testing of cosmetic ingredients and their safety evaluation(5th revision),adopted by the SCCNFP during the 25th Plenary Meeting of20October2003(SCCNFP/0690/03).

Available at:ec.europa.eu/health/ph risk/committees/sccp/ documents/out242en.pdf.[11]The Scienti?c Committee on Cosmetic Products and Non-Food

Products intended for consumers,Opinion concerning zinc oxide (Colipa no S76),adopted by the SCCNFP during the24th Plenary Meeting of24–25June2003(SCCNFP/0649/03).Available at: ec.europa.eu/health/ph risk/committees/sccp/documents/ out222en.pdf.

[12]E.Gocke,L.M¨u ller,P.J.Guzzie,S.Brendler-Schwaab,S.Bulera,

C.F.Chignell,L.M.Henderson,A.Jacobs,H.Murli,R.

D.Sny-

der,N.Tanaka,Considerations on photochemical genotoxicity: report of the International Workshop on Genotoxicity Test Proce-dures Working Group,Environ.Mol.Mutagen.35(2000)173–184.

[13]S.Brendler-Schwaab,A.Czich,B.Epe,E.Gocke,B.Kaina,L.

M¨u ller,D.Pollet,D.Utesch,Photochemical genotoxicity:prin-ciples and test methods.Report of a GUM task force,Mutat.Res.

566(2004)65–91.

[14]M.Ishidate,M.C.Harnois,T.Sofuni,A comparative analysis of

data on the clastogenicity of951chemical substances tested in mammalian cell cultures,Mutat.Res.195(1988)151–213. [15]D.Kirkland,M.Aardema,L.Henderson,L.Muller,Evaluation

of the ability of a battery of3in vitro genotoxicity tests to dis-criminate rodent carcinogens and non-carcinogens.I.Sensitivity, speci?city and relative predictivity,Mutat.Res.584(2005)1–256.

[16]ISCN:An International System for human Cytogenetic Nomen-

clature,Recommendations of the international standing commit-tee on human cytogenetic nomenclature,Editor Felix Mitelman, S.Karger,Switzerland,1995.

[17]D.Scott,S.M.Galloway,R.R.Marshall,M.Ishidate Jr.,D.Bru-

sick,J.Ashby,B.C.Myhr,Genotoxicity under extreme culture conditions.A Report from ICPEMC Task Group9,Mutat.Res.

257(2001)147–204.

[18]S.M.Galloway,Cytotoxicity and chromosome aberrations in

vitro:experience in industry and the case for an upper limit on toxicity in the aberration assay,Environ.Mol.Mutagen.35(2000) 191–201.

[19]ICH Topic S2A:genotoxicity:guidance on speci?c aspects

of regulatory genotoxicity tests for pharmaceuticals,approved September1996.Available at:1d26b30f76c66137ee0619d0/LOB/media/ MEDIA493.pdf.

[20]R.S.Stern,1d26b30f76c66137ee0619d0ird,The carcinogenic risk of treatments for severe

psoriasis,Cancer73(1994)2759–2764.

[21]H.Murli,M.Aardema,1d26b30f76c66137ee0619d0wlor, C.Spicer,Photoclasto-

genicity—an improved protocol,its validation,and investigation of the photogenotoxicity of DMBA,Environ.Mol.Mutagen.40 (2002)41–49.

本文来源：https://www.bwwdw.com/article/uvje.html