spectral dependencies of killing, mutation, and ...biological, kansas city, mo.) plus 10% fetal calf...

[CANCER RESEARCH 41, 4916-4924, December 1981]

Spectral Dependencies of Killing, Mutation, and Transformation in

Mammalian Cells and Their Relevance to Hazards Caused by SolarUltraviolet Radiation1

F. Suzuki,2 A. Han, G. R. Lankas,3 H. Utsumi,4 and M. M. Elkind5

Mammalian Cell Biology Group, Division of Biological and Medical Research, Argonne National Laboratory, Argonne. Illinois 60439

ABSTRACT

Using germicidal lamps and Westinghouse sunlamps withand without filtration, the effectiveness of ultraviolet and near-

ultraviolet light in inducing molecular and cellular changes wasmeasured. Cell survival and the induction of resistance to 6-

thioguanine or to ouabain were measured with V79 Chinesehamster cells, cell survival and neoplastic transformation weremeasured with C3H mouse 10T1/2 cells, and the induction of

pyrimidine dimers containing thymine was measured in bothcell lines. The short-wavelength cutoff of the sunlamp emissionwas shifted from ~290 nm (unfiltered) to ~300 and -310 nm

by appropriate filters. Although it was found that the efficiencywith which all end points were induced progressively decreased as the short-wavelength cutoff was shifted to longer

wavelengths, the rates of decrease differed appreciably. Forexample, doses of near-ultraviolet light longer than ~300 nm

that were effective in mutating or in transforming cells wereineffective in killing them. In respect to pyrimidine dimer induction, several but not all cellular end points were induced bydose ratios of sunlamp light (short-wavelength cutoff, -290

nm) to germicidal lamp light (254 nm) in fairly close accord withthe doses required to produce equivalent proportions of dimers. However, for near-ultraviolet light having cutoffs at longer

wavelengths, the biological action observed was appreciablygreater than what would be predicted from the proportion ofdimers induced. From the latter observation, it is inferred thatincreasing intensities of short-wavelength ultraviolet light, as

would be expected from reductions in stratospheric ozonearound the earth, would result in smaller increases in biologicalaction, e.g., skin cancer, compared to current levels of actionthan would be predicted from an action spectrum completelycorresponding to that of a pyrimidine dimer induction spectrumin DMA.

INTRODUCTION

Non-ionizing radiation is biologically active. In addition to itsrole in photosynthesis and phototropism, in mammalian cells,

' Supported by the United States Department of Energy, Contract W-31-109-

ENG-38. and the United States National Cancer Institute. Grant CA 26984.Presented in part at the 71st Annual Meeting of the American Association forCancer Research, Inc., May 30, 1980, at San Diego, Calif. (11), and at the EighthInternational Congress of Photobiology, July 20 to 25, 1980, at Strasbourg,France (18).

2 On leave from Kanazawa University, Division of Radiation Biology, Kana-

zawa, Japan.3 Present address: Biodynamics, Mettlers Road, East Millstone, N. J. 08873.4 Present address: Radiation Biology Center, Kyoto University, Yoshida-Kon-

oecho, Sakyo-ku, Kyoto 606, Japan.5 To whom requests for reprints should be addressed.

Received June 15, 1981 ; accepted August 13, 1981.

light (and particularly UV light) is known to be cytotoxic (10,42), mutagenic (20), and oncogenic (2). The association between skin cancer and exposure to sunlight (33, 36), particularly in people suffering from xeroderma pigmentosum (31),and the sensitivity of cells derived from the latter individuals tocell killing and mutation by short-wavelength UV light (25) imply

that DMA targets and DNA repair processes are important inlight-induced effects (34, 35). In addition to activity resulting

from naturally occurring chromophores like DNA, polycyclicaromatic hydrocarbons (42) and porphyrins (8) are examplesof chemicals usually foreign to cells that are capable of photosensitizing mammalian cells.

The closeness of the 254 nm line from a germicidal lamp tothe DNA absorption peak at 265 nm and the relative inexpen-siveness of such lamps have led to their extensive use for thestudy of biological action thought to be associated with light-induced changes in the genome. In addition, the relative activityof luminous energy of wavelengths longer than 254 nm isreported to follow a DNA absorption spectrum for a number ofend points in bacteria and the induction of photoproducts inDNA (34).

Using monochromatic light in the UV-C6 and UV-B regions,

Rothman and Setlow (32) reported that the action spectrum ofcell killing of V79 Chinese hamster cells follows an inductionspectrum of pyrimidine dimers in DNA when the analysis isbased upon the relative number of quanta to reduce survival to10%; a similar set of survival data was reported with mouseL5178Y cells (21). In M3-1 Chinese hamster cells, an involvement of protein as well as nucleic acid was implicated for thewavelength dependence of cell killing (41 ), and a result similarto that reported with M3-1 cells was reported with mouse L-

cells (30). Chromatid aberrations in Chinese hamster cellsappear to represent action in protein as well as in DNA (6).More recently, it has been reported that the killing of nondivid-

ing normal human cells and xeroderma pigmentosum cells inculture also follows a DNA action spectrum in the UV-C andUV-B regions; the comparisons in this latter study were based

upon the relative number of quanta to reduce survival to 37%(23). However, for light in the UV-C and UV-B regions, cell

killing of Chinese hamster ovary cells does not appear tocorrelate with the induction of UV endonuclease-sensitive sites,

the latter being a measure of dimers in DNA (45).Although extensive measurements have been made with

6 The abbreviations used are: UV-C, UV light of wavelengths shorter than

~290 nm (in this work, the primary emission from a 254 nm germicidal lamp);UV-B, light from ~290 to 330 nm (in this work, the emission Jrom unfilteredWestinghouse sunlamps); 6-TG, 6-thioguanine; OUA, ouabain; TT, thymine-thy-mine dimers; UT, uracil-thymine dimers; CT, cytosine-thymine dimers; 6-TG',resistance to 6-thioguanine; OUA1, resistance to ouabain; CC, cytosine-cytosine

dimers; PBS, isotonic phosphate-buffered saline, pH 7.4 (2).

4916 CANCER RESEARCH VOL. 41

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Solar UV and Action Spectra

bacterial DNA (28) and bacteria (e.g., see Ref. 44), fewer datahave been obtained with mammalian cells. Using light of discrete wavelengths, the induction of resistance to 5-bromode-

oxyuridine has been reported to follow a DMA pyrimidine dimerinduction spectrum (21). However, mammalian cells also canbe mutated by polychromatic UV-B light (20) or with fluorescent

light in the visible region (3, 20). In addition to tumor inductionin vivo (2), studies with cells have shown that transformationcan be induced in vitro by 254-nm light (5, 22, 27) or by a

polychromatic emission of longer wavelength (24).In this study, we present data for killing, mutation, and

neoplastic transformation in mammalian cells in culture inducedwith 254-nm UV-C light and polychromatic light sources in theUV-B region. We have used V79 Chinese hamster cells for cellkilling and mutation induction, and we have used 10T1/2 cells

(derived from a C3H mouse embryo) for cell killing and transformation induction. With both cell lines, we have measured theinduction of dimers containing thymine in DMA. All 3 cellularend points in mammalian cells are frequently associated withdamage induced in DMA, particularly when ionizing or non-

ionizing radiation is the inducing agent. The dose dependenciesof these effects have enabled us to inquire: (a) are the foregoing end points in mammalian cells related (e.g., are theyproduced by the same molecular lesion); and (b) does theirinduction follow a DMA action spectrum, i.e., a pyrimidine dimerinduction spectrum? In addition, by the use of filters thatprogressively reduce the intensity of the short-wavelengthemission from the polychromatic source of UV-B light that we

used, our results indicate the relative biological action to beexpected from the UV-B region of sunlight that is transmittedby the earth's ozone layer (12). This feature leads to inferences

about the biological importance of the increased intensities ofsolar UV that are to be expected from reductions in the ozonelayer around the earth (1 ).

MATERIALS AND METHODS

Cell Lines, Cell Cultivation, and Survival Assays. During the courseof this work and also the studies with non-ionizing radiations that

preceded and contributed to it (10, 14, 16, 42), several clones of V79Chinese hamster cells were used. The methods used for the cultivationof these clones and for the assessment of survival by colony formationhave been described and have remained essentially unchanged (10,14, 16, 42) except as noted below. Briefly stated, V79 cells werepassaged in a modified Eagle's minimal essential medium (K. C.

Biological, Kansas City, Mo.) plus 10% fetal calf serum (Biologos,Naperville, III.) and were incubated at 37Â° in a humidified atmosphere

containing 2% CO2. For survival and mutation assays, a suspension ofsingle cells was prepared, and, following incubation overnight to ensurethe resumption of asynchronous exponential growth, cells were irradiated. Cells were then incubated for colony formation or were resus-

pended and plated to reduce their concentration as the first step in the"phenotype expression period" prior to drug challenge (see below).

For all survival assays, with or without further manipulation of the cellsduring the expression period following irradiation, the medium was asnoted above except that the serum supplementation consisted of 2.5%fetal calf and 7.5% newborn calf sera (Biologos). This change had noeffect on survival.

Survival and transformation measurements were made with mouseC3H1OT1/2 cells, clone 8 (courtesy of Dr. C. Heidelberger, Los Angeles,

Calif.), as described previously (15, 17). Cells between the eighth and15th passages were used. Cells were passaged and assayed forsurvival in the same medium that was used for farming V79 cells, and

they were assayed for transformation in Eagle's basal medium (Grand

Island Biological Co., Grand Island, N. Y.) plus 10% fetal calf serum(Reheis Chemical Co., Kankakee, III.). Survival and transformationfrequencies after long exposure to filtered UV-B light were determined

from the same suspension of cells which was prepared immediatelyafter irradiation. Surviving fractions and their S.E.s were computed asfor V79 cells.

Light Sources and Irradiation. Germicidal lamps (General ElectricModel G15T8, 15 watts) were used for UV-C irradiation (principally

254 nm); the dose rate was 60 J/sq m/min as estimated from measurements with a Schwartz Model 800 thermopile plus a Keithley Model150B electrometer after subtracting the contribution from IR radiation(i.e., the signal from the thermopile obtained immediately after turningoff the lamps). For UV-B light, Westinghouse sunlamps (Model FS20,

20 watts) were used; the dose rate was 200 J/sq m/min measured asabove. Sunlamps filtered by 90-mm polystyrene Petri dish covers

(Falcon Plastics, Oxnard, Calif.), which were 0.94 mm thick, reducedthe dose rate to 130 J/sq m/min, whereas the use of 0.025-mm Mylar

films resulted in a dose rate of 129 J/sq m/min. Irradiation throughlarge dish covers or through Mylar films reduced the UV-B band widthmainly by progressively cutting off the short-wavelength end; i.e.,through dish covers, the emission band was shifted from ~290 to 330nm to ~300 to 330 nm, and, through Mylar films, it was shifted furtherto ~310 to 330 nm (see Ref. 42, Fig. 2, for the emission spectra).

V79 and 10T'/2 cells were irradiated while attached in 90-mm Petri

dishes and through ~10 ml of PBS at room temperature. For UV-C andunfiltered UV-B exposures, the dish covers were removed; no absorp

tion resulted from the intervening PBS solution. After irradiation, thebuffer was replaced by growth medium for colony formation, or thecells were suspended for appropriate dilution and further manipulationas required.

Mutation and Transformation Assays. V79 Chinese hamster cellswere challenged for resistance to 6-TG (5 ^g/ml) or to 2 mM OUA (bothfrom Sigma Chemical Co., St. Louis, Mo.) following a 6-day expression

period (43) which was found to be optimal. After irradiation, cells werepromptly suspended, diluted, and plated; this procedure was repeatedafter 3 days of growth. After another 3-day period, cells were sus

pended and plated: (a) in medium for colony formation; (i>) in mediumcontaining 2.5% fetal and 7.5% newborn calf sera plus 6-TG for the

determination of the proportion of cells that were presumptive mutantsat the hypoxanthine-guanine phosphoribosyltransferase locus; or (c)in medium lacking K+ and containing 10% fetal calf serum plus 2 mw

OUA for the determination of cells that were presumptive mutants atthe Na+-K*-ATPase locus.

Transformation frequencies of 1OT'/2 cells were determined as de

scribed previously (15, 17). In brief, after irradiation, cells were suspended and inoculated into 90-mm polystyrene dishes at concentra

tions close to 150 to 200 viable cells per dish. Cells were incubated for5 to 6 weeks in Eagle's basal medium plus 10% fetal calf serum with

medium changes as noted (15). Following staining for colony counting,the frequency of type 2 and 3 morphologically transformed coloniesper surviving cell was computed from the proportion of dishes containing no type 2 and 3 colonies. As discussed previously (15, 17), thisprocedure avoided the possibility of an overestimation of transformationfrequencies due to satellite formation as a result of the refeedingsrequired in the assay.

Dimer Induction. The induction of dimers containing thymine, i.e.,~f\ and (JY from CT, was measured in V79 and 10TVi cells and for all

the radiations used except for sunlamps filtered by Mylar. Inductionrates in V79 cells for germicidal lamps and for unfiltered sunlamps (seeTable 1) are taken from an earlier study (10).

The methods used for the new data reported here were essentiallythe same as those used earlier (10). That is, after the template labelingof the DNA in cells by growing them for 1 to 2 days in high-specific-activity [3H]thymidine, 6 to 24 Ci/mmol (Radiochemical Centre, Amer-

sham, England), cells were exposed to graded doses of UV, suspendedin PBS and frozen, and extracted with 10% trichloroacetic acid at 4Â°

DECEMBER 1981 4917



F. Suzuki et al.

Table 1

DimeryieldsCellsV7910TVÃŒRadiationuv-c'

UV-B"UV-B(filtered)'uv-c'

UV-B"UV-B(filtered)'Yield3

J-'m22.62

x 10'58.75 x 1CT72.80 X1CT82.14

X 10 56.16 X10~72.19

X 10~s(Corr.

coef.")(0.997)9

(0.995)9(0.915)(0.986)

(0.995)(0.997)%of

TTC75

675778

6758%

ofÃšV25

334322

3342%offV60

504064

5041%ofÃ©V40

5060365059Yielddose

ratio81.0

288201.0

32840

Total fractional yield of dimers containing thymine." Correlation coefficient of aJeast-squares fit to a linear regression.c Measured percentages of TT and o^ UT based upon 3H activity in dimers. UT_Â£omesfrom CT." Corrected percentage of TT and CT dimers, assuming that ÃšTrepresents CT, after accounting for the fact that

only the thymine of a CT dimer could be labeled. ^ ^e Ratio of doses to produce the same proportion of dimers based upon corrected percentages of TT and CT but not

including CC.' General Electric germicidal lamp, ~85% 254 nm.9 From Ref. 10." Westinghouse sunlamps (see Ref. 42 for spectral emission).' Westinghouse sunlamps filtered through a 100-mm-diameter polystyrene dish cover, 0.94 mm thick (see Ret. 42

for spectral emission).

DOSE (BOB), J-rrT2

200 400 600 800 1000 2000

i l i

UV-B (filtered, polystyrene)

DOSE(a DB), J-rrT2

O 200 400 600 800 IOOO I200 14004.0

0.01200

DOSE (â€¢), J-m"

UV-B (filtered. Mylar)

Chart 1. Survival curves of V79 Chinese hamster cells (A) and mouse C3H10T'/i cells (ÃŸ)exposed to different sources of UV light as follows: UV-C is the emissionfrom germicidal lamps (85% 254 nm); UV-B is the emission from Westinghouse sunlamps (~290 to 330 nm); UV-B (filtered, polystyrene) is UV-B light filtered bypolystyrene dish covers, 0.94 mm thick (-300 to 330 nm); and UV-B (filtered, Mylar) is UV-B light filtered by Mylar, 0.025 mm thick (-310 to 330 nm). P.E.. plating

efficiency; Ã‘ and N, multiplicity at the time of exposure; bars. S.E. in survival.

for 30 min the following day. The precipitate was then spun down,washed twice with 95% ethanol, dried at room temperature, and takenup in 97 to 100% formic acid (Eastman Kodak Co., Rochester, N. Y.).Hydrolysis of the DMA was performed using the method of Carrier andSetlow (4), and chromatography was as performed by Smith (37) andSutherland and Oliver (39). The fraction of dimers as TT or UT wasestimated from the relative positions of these dimer peaks to that of thethymine peak and the proportions of radioactivity contained in them.

RESULTS

Survival Curves, V79 and 10T1/2 Cells. Survival data for

both V79 Chinese hamster and mouse 10TV? cells, for several

of the light sources used in this study, are shown in Chart 1.These results were obtained with the particular clones of cellsthat were used in this study and are similar to data that havebeen already published (10, 42). With respect to V79 cells, wenote that, in other experiments performed in our laboratory,exposures of UV-B light up to at least 3000 J/sq m filtered bypolystyrene dish covers did not produce any killing.7 Presum

ably, even larger doses of UV-B light filtered by Mylar wouldnot have affected survival. Hence, although 10T1/z cells appearto be somewhat more sensitive to filtered UV-B light than do

7 F. M. Salih and L. D. Theriot, unpublished data.




UV-B (filtered, polystyrene)

2500

50 100 150DOSE(â€¢),J-rrT2

200 250

So/ar UV and Action Spectra

2500

50 100 150DOSE (â€¢),J-m'2

200 250

Chart 2. The induction in V79 Chinese hamster cells of resistance to 6-TG (5 ng/ml) in medium supplemented by 25% fetal and 7.5% newborn calf sera (A) orresistance to 2 HIM ODA in medium lacking K* but supplemented by 10% fetal calf serum (B). The same UV sources were used as in Chart 1. The pooled results of

several experiments are shown. Bars, S.E. in induction frequencies (per surviving cell).

V79 cells, a major loss in response in both cell lines resultsfrom a shift in the short-wavelength cutoff from ~290 to ~300

nm, i.e., from exposure to unfiltered sunlamps to exposure tosunlamps filtered by dish covers, respectively.

Mutation, 6-TG' and QUA'. Chart 2 shows the induction of

V79 cells resistant to 6-TG (5 fig/ml) or to 2 HIM ODA as a

function of dose of UV light. For both markers, the initialportions of the induction curves for UV-C and for UV-B lightappear linear, whereas the corresponding curves for UV-B light

filtered by polystyrene dish covers are initially concave upward.Although the shapes of the curves in Chart 2 for exposure toUV-B light filtered by Mylar may or may not be similar, it is clear

that, for both markers, a major loss in response results from ashift from dish cover to Mylar filtration. Further qualitativedifferences among the curves in Chart 2 will be evident fromthe discussion of Chart 4. However, it should be noted that allof the light sources that were used are ~10-fold more effectivein inducing resistance to 6-TG than to OUA.

Neoplastia Transformation. The dependence of transformation induction on dose for the different light sources isshown in Chart 3. The ordinate has a semilogarithmic scalebecause the induction data cover some 2 orders of magnitude.The shift in the scale of the ordinate of Chart 3 plus thesomewhat larger uncertainties make it difficult to compare theshapes of the transformation curves with the shapes of themutation curves. Nevertheless, the data in Chart 3 make clearthat, although UV-B light filtered by polystyrene dish covers is

quite effective in transforming cells (as with mutation induction),a large sector of effectiveness is lost by substituting Mylar filmsas filters in place of dish covers.

Dimer Induction. Table 1 shows in Column 3 the slopes of

5XICT3200

DOSE(B D I400 600

I), J-rrT2

800 IOOO I200 I400

I40

4XIO-5

60DOSE(Â»),

Chart 3. The induction of neoplastic transformation (frequency per survivingcell) in mouse C3H1OT'/zcells by the same light sources as in Chart 1. Since notransformants were observed for the 500-J/sq m dose of UV-B (filtered. Mylar),only an upper limit estimate can be made of the induction frequency for this dose.At the time that these data were obtained, the cumulative frequency of spontaneous transformants was 1.1 X 10~5.Bars, S.E. (uncertainties).

the linear regression lines fitted to measurements of the fractional yields of dimers containing thymine as a function of dosefor V79 and 10TV2 cells and for 3 of the 4 sources of UV light

DECEMBER 1981 4919



F. Suzuki et al.

60U)

w X

uj ~ 40

if< xg _- 30

Y ?io ^- 20

>-

^ IO

40

eninLuce â€”

v

30 -

i 20

IIOJ

ID

Sce

10

1.5 1.0 0.5 0.2 0.1 0.05

SURVIVING FRACTION

002 0.01 1.5 1.0 0.5 0.2 0.1 0.05

SURVIVING FRACTION0.02 0.01

Chart 4. A plot of the variation of resistance to 6-TG (5 /ig/ml) versus survival (A) or of resistance to 2 rnw OUA versus survival (B) for V79 Chinese hamster cellsexposed to the radiations used to obtain the data in Charts 2 and 1. respectively. As in Chart 2. the induced frequencies are per surviving cell. Bars, S.E.

that were used. In each case, a linear dependency fitted thedata quite well as suggested by the correlation coefficients(Table 1, Column 4). Measurements of dimer yields for sun-

lamps filtered by Mylar were not undertaken because it wasanticipated that excessively long exposures would be required.As will be noted later, the conclusions to which the data inTable 1 contribute do not require qualification as a consequence of our not having measured the dimer yields for UV-B

light filtered through Mylar films.Table 1, Columns 5 and 6, shows the percentages of ft and

ÃœT,based on 3H activity, of the total of the thymine-containing

dimers (4). In the next 2 columns, the origin of ÃœTdimers isaccounted formas well as the probability that only the thymineresidue of a CT dimer can be labeled. Finally, the yield doseratio in the last column gives the doses, relative to that requiredby UV-C, to produce the same proportion of TT + CT dimers.

Not accounted for in Table 1 is the dependence of the yieldof CC dimers on the light source that was used (as well as othertypes of light-induced base changes). Few data are available

on the wavelength dependence of the induction of Â¿Cdimersrelative to TT. Although with increasing wavelength we wouldexpect the proportion of CC dimers to increase, as does theproportion of CT dimers (see also Ref. 26), we would notexpect the wavelength dependence of the former to affect theyield ratios significantly in Table 1. From measurements madeby others,8 we would estimate that, for our UV-C radiation,

about 15% of the total yield of dimers would be due to CC andthat this percentage would rise to about 25% for UV-B filteredthrough a 0.94-mm polystyrene dish cover or a 0.25-mm Mylar

film. Hence, we would expect an accounting of CC dimerinduction to result in only a minor change in the yield doseratios in Table 1. The latter qualification should not significantlyaffect the arguments based upon dimer induction to be presented later.

Growing attached in plastic dishes, interphase 10T1/2 cells

and their nuclei are appreciably flatter than V79 cells and theirnuclei. This difference could contribute to the lower efficienciesof dimer induction in 10TVi cells versus V79 cells as the data

8 W. L. Carrier and J. D. Regan, personal communication.

in Table 1, Column 3, suggest. The latter differences are notlarge, nor are the nominal differences in the proportions of CTversus TT dimers in the 2 cell lines. For the purposes of thisstudy, therefore, we consider the yield dose ratios and theproportions of dimers induced by the different light sources notto be significantly different in these 2 cell lines.

ANALYSIS

Mutation and Transformation versus Survival. Various linesof argument, and some evidence, have supported an oftenmade association between the mechanisms of cell killing, mutation, and transformation. Since these end points were measured in effect with 4 different light sources, for purposes ofanalysis, it is useful to inquire if mutation and/or transformationis related to cell survival.

Chart 4A shows the frequencies (per surviving cell) of theinduction of cells resistant to 6-TG plotted as a function of the

surviving fraction of V79 cells. The data for this chart comefrom Chart 1 (corrected to single cells) and Chart 2. For theinduction of 6-TGr cells, in Chart 4A, it appears that a good

correlation exists for the biological effects produced by 2 quitedifferent radiations, UV-C and UV-B light. The nominal separation of the 2 curves between surviving fractions of from ~0.4to -0.07 is quite modest in view of the large differences in

dose required to produce equivalent biological effects. Thus,it appears that the mechanisms of induction of resistance to 6-TG and cell killing are similar when cells are exposed to UV-Cor unfiltered UV-B light. However, the curve for UV-B light

filtered by polystyrene dish covers clearly departs from theother 2 curves; this departure is to be expected since cells aremutated by this light source, although they are not killed.

Relative to the induction of OUAr cells, in Chart 4B, it is

apparent that essentially no correlations exist for the 3 lightsources considered. The implications of this last statement arediscussed later. (Note that the results in Charts 1 and 2obtained with UV-B light filtered by Mylar are not shown in

Chart 4, since this light source has little effect on mutation aswell as on cell survival.)

The variation of the transformation of 10TVfecells with survivalis plotted in Chart 5 (data from Charts 1 and 3). The results




with unfiltered UV-B light and UV-C light appear to lie close

enough to each other, in view of the uncertainties, and to beconsistent with similar mechanisms for the induction of bothend points, although the nominal separation of the curvesbecomes progressively greater (see also the discussion ofChart SB). However, the results with filtered UV-B light showthat, when intensities for wavelengths less than ~300 nm are

appreciably reduced by filtration, the 2 end points no longertrack together. In this latter respect, transformation and mutation induction show similar dependencies since, while cytotoxicaction is largely eliminated when the cutoff is below -300 nm,

transformation and mutation action are not.Biological Action versus Dimer Induction. Charts 4 and 5

permit inferences to be made about similarities, or the lack

5x10

4XIO-*0.5 0.2

SURVIVING FRACTION

Chart 5. A plot of the variation of the induction of transformation versussurvival of mouse C3H10T'/2 cells based upon the data in Charts 3 and 1,

respectively. Uncertainties in survival are omitted for clarity. Bars. S.E.


thereof, of mechanisms of action. With the next 2 charts, weinquire specifically to what extent the cellular end points measured reflect pyrimidine dimer induction in DMA.

The conceptual basis for Chart 6 is the following. For a givendose of UV-C light, 2 or more induced changes (survival,

mutation, and/or transformation) have been measured in eachcell line. Further, for each of these induced changes, thecorresponding doses of near-UV light having the same effect

may be obtained from the curves in Charts 1 to 3. Thus, inChart 6, the ordinate is the ratio of the dose of near-UV light(i.e., filtered or unfiltered UV-B light) to the dose of UV-C lightrequired to produce the same effect plotted as a function ofthe UV-C dose. Also shown in each chart are the dimer yield

dose ratios taken from the last column in Table 1. Thus, byexamining the dose ratio as a function of UV-C dose, one maydetermine if the induction of a particular end point reflects therelative efficiencies of pyrimidine dimer induction over a broadrange of biological effect (10, 45) as opposed to particularlevels of effect (6, 15, 21, 23, 30, 41 ).

In Chart 6, the horizontal dashed lines are the yield doseratios from Table 1 for unfiltered sunlamps and for sunlampsfiltered by dish covers. The lower 3 curves in Chart 6/4 refer tothe former, while the upper 2 refer to the latter radiation.Throughout the range of UV-C dose considered, the circles

and closed squares lie close to each other and not far abovea dose ratio of 28. (If an increase in the relative yield of CCdimers with wavelength were accounted for, the separation ofthe latter 2 sets of data from the dimer yield dose ratio wouldbe further increased.) In contrast, consistently and appreciablysmaller doses of unfiltered UV-B light are required to induceOUAr cells.

Even less concordance between the dose ratio to induceequal proportions of TT plus CT dimers and equal biologicaleffect is evident for UV-B light filtered by polystyrene dish

covers in Chart 6A. Data for survival are not plotted because,for V79 cells, reductions in survival were not observed (Chart1). It is a possibility that doses of filtered UV-B light some 800

2000

IOOO

- 820

3 IOO

10

V79-B3IOH

UV-B

Surv.6-TG'

QUA'

Dimerratio

a28

UV-B

(polystyrene)

A

A

820

10 15DOSE, UV-C,

20 25

IOOOG

/Â¿.840XZ5

1Ã«vâ€¢z.0

100CSor32

inB

IOT'/2CELLSUV-B

UV-B UV- Br(polystyrene)

(Mylar) â€¢

k Surv. o D- ^^~ Trans. /surv. â€¢ â€¢ A -

Dinner ratio 32840:

"J-o- -^L____:â€”

â€¢---.

i.i.i.30 10 20 30 40

DOSE, UV-C, J-m~2

Chart 6. Comparisons of the doses of UV-B or doses of UV-B filtered by polystyrene (dish covers) or by Mylar films to produce the same effect in V79 cells (A) orin 10T'A cells (B) as UV-C light. The dashed lines give the dimer yield ratios from Table 1. The data for survival (Surv.) come from Chart 1; those for the induction of6-TG' and QUA' cells come from Chart 2; and those for the induction of transformed (Trans.) cells come from Chart 3.

DECEMBER 1981 4921



F. Suzuki et al.

times greater than the doses of UV-C light that were used might

kill equal proportions of cells. However, to induce equal proportions of 6-TG' cells or of OUAr cells, filtered UV-B doses

reduced some 2-fold or some 8-fold, respectively, are required

as compared to the doses to produce equal proportions ofpyrimidine dimers. Hence, both of these mutations are inducedwith appreciably greater frequency than would be predicted onthe assumption that pyrimidine dimers in DMA are the causativelesion in DMA. (Note that dose ratios are not plotted for resultswith V79 cells obtained with UV-B light filtered by Mylar. For

mutation as well as for cell survival, the biological changesinduced with the latter radiation are not sufficiently large topermit inferences to be drawn with confidence.)

Relative to transformation of 10TV2 cells, Chart 68 showsresults qualitatively similar to those just discussed for V79cells. Dose ratios for cell survivals (open circles) lie close to,but also above, the Ã•T plus CT dimer yield dose ratio forunfiltered UV-B light. However, except for low frequencies oftransformation (S4 x 10~4 per survivor) induced by unfiltered

UV-B light, all of the remaining ratios in Chart 66 lie appreciablybelow the respective dimer yield dose ratios. The disparity fortransformation induced by unfiltered UV-B becomes progres

sively greater with increasing effect, a trend similar to thatnoted in Chart 5. For UV-B light filtered by polystyrene dishcovers, the disparity is about 2-fold even at high levels ofsurvival (open squares), and the disparity is some 10-fold atlow as well as at high frequencies of transformation. For UV-B

light filtered by Mylar, it is clear that appreciably smaller ratiosof dose are required to transform cells (triangles) than whatone would infer should be needed to induce dimers. The basisfor this statement is that the dose ratios for equivalent levels oftransformation lie below the dimer yield dose ratio for UV-B

light filtered by polystyrene dish covers. Since Mylar shifts theshort-wavelength cutoff about 10 nm (i.e., from ~300 nm fordish cover filtration to ~310 nm for Mylar filtration; Ref. 42,

Fig. 2), one would expect the doses for equal dimer yields tobe appreciably greater for UV-B light filtered by Mylar versusUV-B light filtered by polystyrene, both relative to UV-C light.

Thus, for cell killing and the induction of 6-TG' cells, the

increases in the doses of unfiltered sunlamp light are largerthan, but still approximately correlated with, the reduced efficiency for the induction of dimers by this radiation comparedto 254 nm UV-C light. There may also be concordance between

pyrimidine dimer ratios and low frequencies of transformationfor unfiltered UV-B and UV-C light. However, all of the remaining comparisons in Chart 6 indicate that smaller doses of near-

UV light are required to induce changes than would be predicted based upon dimer yield dose ratios. Accounting for thespectral dependence of CC induction would not require asignificant qualification of the foregoing statement. Thus, weinfer that a lesion(s) in addition to the pyrimidine dimer playsan increasingly important role in the wavelength interval of-290 to ~310 nm if action in DMA is responsible for the

biological change.

DISCUSSION AND CONCLUSIONS

The work of a number of investigators has clearly establishedthat mammalian cells can be killed, mutated, and transformedby non-ionizing radiation. In our study, the use of several

different sources of light to induce these end points, plus

measurements of the induction of thymine-containing dimers inDNA, permits us to draw a number of new inferences andconclusions.

To facilitate the discussion, it is useful to begin with acharacterization of the UV sources that we used. Germicidallamps, our UV-C light, constitute essentially a line source at

254 nm. This is close to the maximum in the DNA absorptionspectrum, 265 nm (34), which is very likely the basis for therelative efficiency with which germicidal lamps induce a numberof biological effects. Unfiltered sunlamps produce a spectrumin which the intensity distribution drops at ~290 nm to 30% of

its maximum intensity, at 313 to 315 nm. Filters consisting of90-mm polystyrene dish covers (0.94 mm) shift the 30% intensity of sunlamps to -300 nm, and films of Mylar (0.025 mm)shift the 30% intensity to ~310 nm. Although sunlamps with or

without filters are polychromatic, as noted above, they may becharacterized by short-wavelength cutoffs at ~290, ~300, and~310 nm for sunlamps without filters, with dish covers, and

with Mylar films, respectively (see Ref. 42). Thus, the latter 3sources enable an assessment to be made of the loss of light-induced activity as the short-wavelength cutoff is progressivelyshifted to longer wavelengths. The near-UV region traversed isthe region where the earth's ozone layer is effective in absorb

ing the shorter-wavelength emissions from the sun (e.g., see

Ref. 29, Fig. E.1).Our results with V79 and 10TV? cells show that the relative

effectiveness of the near-UV sources drops off more rapidly for

cell killing than for mutation or transformation as the cutoff isshifted from ~290 to -300, to -310 nm. From this lack of

concordance, it follows that the mechanism of cell killing mustbe different from that of mutation and transformation, a conclusion similar to one reached by Zelle ef al. (45) but in apparentcontradiction to the views of Doniger ef al. (7) who used Syrianhamster embryo cells for killing and transformation induced bymonochromatic UV light. The biological action lost in shiftingthe cutoff from -290 to -300 nm effectively eliminates lethalityover dose ranges in which cells are mutated and transformedwith relatively high probability. If a very effective repair processis invoked to account for the inefficiency of cell killing due tolight of wavelength longer than -300 nm, then clearly, this

process must be error prone.Next, comparing the effects of germicidal lamps and unfil

tered sunlamps, we note that the mechanism of action is similarfor cell killing and the induction of 6-TG' cells (Chart 4/4) butdissimilar for cell killing and the induction of QUA' cells (Chart

46). Since 6-TG resistance probably results from lesions that

break chromosomes (40) as well as from those that inducepoint mutations, whereas OUAr probably reflects the latter

process alone (13), we may infer that cell lethality results froma molecular pathology that includes chromosome breakage(and rearrangement) for wavelengths up to -300 nm. The

relationship between transformation in 10TV2 cells and survivalcould be consistent with chromosomal breakage (and rearrangement) in view of the uncertainties, particularly in thetransformation measurements (Chart 5). However, when comparisons are made between mutation and transformation versus survival for filtered UV-B light, it becomes clear from Charts

4 and 5, as already noted, that these end points do notcorrelate. Hence, for wavelengths between -290 and 300 nm,whatever the nature of the lesions may be that result in 6-TGand OUA resistance or in transformation, they are handled by




fr"

a cell much differently than are the lesions affecting survival.The additional point evident in Charts 2 and 3 is that quanta ofwavelengths longer than -310 nm are very inefficient in inducing mutations, although some effectiveness is still observed fortransformation.

Inferences about the nature of the action spectrum may bemade from the curves in Chart 6. In respect to transformation,it is noted first that the dashed parts of the curves for unfilteredUV-B light (as well as for UV-B light through dish covers)

correspond to the plateau regions of the respective inductioncurves in Chart 3. Dose ratios determined for induction frequencies in these regions are likely to be appreciably moreuncertain than ratios corresponding to lower frequencies. Withthe preceding qualification in mind, the end points cell killing inboth cell lines, 6-TG resistance Â¡nV79 cells, and transformationin 10T'/2 cells all appear to require increases in the dose ofunfiltered UV-B, compared to 254 nm light, approximately inagreement with the dime yield dose ratios. Particularly for cellkilling and the induction of 6-TG resistance, the foregoing

appears to be true over a large range of biological effect.However, except perhaps for transformation, all of the resultsjust noted lie above the dose ratio for TT plus CT induction. Anaccounting for the wavelength dependence of other photole-

sions and photoproducts in DNA that might be produced moreefficiently by UV-B compared to UV-C radiation, e.g.,alkaline-

labile lesions that give rise to strand breaks (9), photoproductsof the 5,6-dihydroxydihydrothymine type (19), and C*Cdimers,

would result in a further disparity between the induction of thecellular end points from changes induced in DNA. Strandbreaks are produced with much lower efficiency than dimers(9, 10), and such is probably also the case for the induction ofring-saturated products of pyrimidines for wavelengths shorterthan ~310 nm (19). An accounting for Â¿Cdimers, however,could reduce the dimer yield ratio by â€”8%.Thus, while the

agreements may not be perfect, they are close enough andover a broad enough range of effect to suggest that dimerformation plays a principal role in cell killing, the induction of6-TG resistance, and transformation for 254 nm and unfiltered

sunlamp light.The consistencies evident in the foregoing serve to highlight

the inconsistencies contained in the following. The dose ratiocurve for the induction of OUA resistance by unfiltered UV-B

light is clearly different from the dimer yield curve and thecurves for survival and induced resistance to 6-TG in Chart 6/4.

Essentially all of the comparisons between dimer yields andcellular end points in Chart 6, A and ÃŸ,for filtered UV-B light

indicate that lesions in addition to dimers in DNA must beinvolved. We may expect that the latter statement applies evento the induction of transformation by UV-B light filtered by

Mylar films, since the dose ratio to produce an equal yield ofpyrimidine dimers is bound to be even larger than it is forsunlamps filtered by dish covers. We are led to conclude,therefore, that, although a DNA pyrimidine dimer action spectrum could account in part for the cell changes induced byunfiltered sunlamps compared to germicidal lamps, for wavelengths greater than ~300 nm, such is not the case. In partic

ular, appreciably more mutation and transformation are observed for wavelengths greater than -300 nm, and this is also

the case in respect to transformation for wavelengths greaterthan ~310 nm, than can be accounted for by pyrimidine dimer

formation. Consequently, if pyrimidine dimers are the principal


photolesion induced in DNA by quanta in the 290 to 310 nmregion, it is likely that mutation and transformation inductioncannot be entirely attributed to absorption in DNA. Our resultsalso suggest that a chromophore other than, or in addition to,DNA is involved even for an end point-like mutation, which

probably results ultimately from action registered in DNA.The inferences and conclusions contained in the foregoing

discussion agree qualitatively with those of Zelle era/. (45) butare in essential disagreement with the conclusions of Donigeret al. (7) in respect, at least, to the correlations that theyreported between the wavelength dependencies of killing,transformation, and pyrimidine dimer induction. The discrepancies between the latter results and ours may reflect essentialdifferences in the cells and methods used. However, we alsonote the important difference that our light sources in the near-

UV region were polychromatic, whereas Doniger ef al. (7) usedmonochromatic light. Taken at face value, therefore, one mightinfer that the molecular and/or biological effects of polychromatic light cannot be a simple integration of the effects produced by monochromatic light weighted by appropriate relativeintensities. Further, it would appear that the interactive biological effects resulting from broad-spectrum near-UV light may

be synergistic.For wavelengths of near-UV light corresponding to those not

entirely absorbed by the earth's ozone layer, i.e., 290 to 310

nm (12), the evidence that mutation and transformation aremore readily induced compared to pyrimidine dimers indicatesthat a modification may be required in estimates that are madeof the relative increases in biological hazards that would resultfrom reductions in stratospheric ozone (1, 29, 38). It is veryprobable that increases in cell killing, mutation, and transformation would result from such reductions (e.g., see Ref. 29,Fig. D.1). However, the increases in action compared to whatcurrently exists would be less than predicted on the assumptionthat absorption in DNA (assumed in this work to be indicatedby pyrimidine dimer induction) is responsible for biologicalaction for wavelengths in the region of the cutoff due to thecurrent ozone layer. The reason for this is that biological action,at least in the form of mutation and transformation, probablyalready exceeds what would be expected from a DNA absorption spectrum. Hence, a shift in the current ozone cutoff towardshorter wavelengths (e.g., see Ref. 29, Fig. E.1) would resultin a smaller relative increase in biological action than expected.

Our experiments were significantly facilitated by the use ofpolychromatic sources of near-UV light rather than monochro

matic light or solar radiation. Also, the results obtained withsuch sources can be expected to simulate what would resultfrom the UV-B region of sunlight. To develop a more compre

hensive understanding of the wavelength dependence of theinduction of various cellular end points, monochromaticsources should also be used. However, because of the possibility that interactive action might be produced by differentwavelengths, as we have noted, studies that include polychromatic sources remain useful and important.

ACKNOWLEDGMENTS

We wish to acknowledge the competent technical assistance of E. Buess, J.Dainko. C-M. Chang-Liu, and M. Uchic.

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spectral dependencies of killing, mutation, and ...biological, kansas city, mo.) plus 10% fetal calf...

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