[CANCER RESEARCH 41, 1789-1 793, May 1981]

Effect of Visible Light on Benzo(a)pyrene Binding to DMA of Cultured

Human Skin Epithelial Cells

Richard F. Camalier, Raymond Gantt, Floyd M. Price, Edward V. Stephens, Anne E. Baeck, William G. Taylor,and Katherine K. Sanford

Laboratory of Cellular and Molecular Biology, National Cancer Institute. NIH. Bethesda, Maryland 20205

ABSTRACT

The ubiquity of the photosensitive carcinogen benzo(a)py-

rene (BP) and visible light in the environment suggests thattheir interaction might lead to photoproducts harmful to humans. To test the combined impact of these two agents onhuman epithelial cells, binding of BP to cellular DMA wasassessed following treatment of cultures with BP and low-

intensity (4.6 watts/sq m) intermittent (12 hr daily, 3 to 5 days)cool white fluorescent light. Light exposure reduced the formation of covalent BP adducts 20-fold (from 150 to 7 pmol BPper mg DNA) in cells treated with 1 ¡igBP per ml and completelyinhibited cytotoxicity; even with 10/ig BPperml, light exposuremarkedly inhibited cytotoxicity. However, at low BP dosage(0.1 fig/ml), covalent adducts (2 pmol/mg DNA) to cellularDNA are produced and their formation is not influenced bylight. These adducts persisted for at least 7 days followingtreatment; this observation suggests that chronic low-level

exposure of human epithelium to BP may lead to an accumulation of DNA damage.

INTRODUCTION

It is generally known that the carcinogen, BP,' is an ubiqui

tous contaminant in large urban areas (22) and that lightdestroys BP. It is also known that visible fluorescent lightdamages chromosomal DNA (19, 21), induces mutations (3,14), and enhances neoplastic transformation in cultured cells(24). Because of the ubiquity of both visible light and BP in theenvironment and the possibility that light-sensitive BP may be

degraded to one or more reactive photoproducts, we haveexamined the combined impact of these 2 environmentalagents on cultured human epithelial cells. To test whether thiscombination of agents is detrimental, we have measured theeffect of visible light on the binding of BP to cellular DNA andobserved whether cytotoxicity is affected.

MATERIALS AND METHODS

Cell Culture. The procedure for initiating and maintainingprimary cultures of normal foreskin epithelial cells has beendescribed (23). Briefly, cultures were initiated by placing about8 1-mm square expiants (trimmed of dermis), into plastic T-25

flasks (Falcon Plastics, Oxnard, Calif.) coated with 0.5 ml horseserum (KC Biologicals, Lenexa, Kans.). The flasks were placedon end for 2 to 3 days in a 37° incubator to allow expiant

attachment. Subsequently, 2 ml of growth medium NCTC 168

' The abbreviations used are: BP. benzo(a)pyrene; 3-OH-BP. 3-hydroxy-

benzo(a)pyrene.Received May 6, 1980; accepted January 30, 1981.

with 10% horse serum and antibiotics were added; the flaskswere gassed with a humidified mixture of 10% CO2:90% airand incubated in the normal horizontal position. After outgrowthof epithelial cells (usually 3 to 6 days), culture medium wasrenewed every 2 to 3 days with increasing volumes to 5 ml asculture growth dictated. A sterile Pasteur pipet was used toremove the expiants after -10 days. The cultures were used

when 3 to 4 weeks old and approximately 75% confluent.Carcinogen and Light Treatment. Tritiated BP (15 to 40 Ci/

mmol; Amersham/Searle Radiochemical Centre, Amersham,England) and unlabeled BP (Gold Label; Aldrich Chemical Co.,Milwaukee, Wis.) were passed through a silica gel column (Bio-Sil A, 25 to 35 /¿m;Bio-Rad Laboratories, Rockville Center, N.Y.) with a 9- to 10-ml bed volume equilibrated with benzene

before use. The sample was dissolved in benzene, and 0.75 to1.0 ml was applied to the column and eluted with benzene.One-mi fractions were collected, and the 3 peak tubes (usually

Fractions 7 to 9) contained 80 to 95% of the sample. Thesefractions were evaporated to dryness in a stream of nitrogenand dissolved in dimethyl sulfoxide, and 10 to 20 /il wereimmediately introduced into cultures containing 5 ml medium.All handling of BP was carried out in yellow filtered light andsubdued white light. The experimental cultures were illuminated at 37° with a desk lamp containing two 15-watt coolwhite fluorescent bulbs (Westinghouse, F15T8-CW) 40.6 cm

from the growth surface (4.6 watts/sq m) immediately afterintroduction of BP. The light irradJance was measured with anIL-700 Research Radiometer using a calibrated SEE 010 detector (International Light, Inc., Newburyport, Mass.). For 3 to5 days, cultures were exposed at 37° to alternating 12-hr

periods of light and dark (intermittent light regimen) withoutmedium renewal. In one experiment, an UF-III filter (Read

Plastics, Inc., Rockville, Md.), 1.5 mm thick, was used toexclude <400-nm wavelengths.

Measurement of BP Binding to DNA. Three to 5 days afterthe cultures were exposed to BP, the medium was aspiratedand the cell sheets were washed 3 times with phosphate-buffered saline (pH 7.4, 6.7 mM sodium-potassium phosphate,:0.85% NaCI solution). Two ml of 0.1 M Tris-HCI buffer, pH 8.2,

containing 0.02 M EDTA and 1% sodium dodecyl sulfate wereadded to the cell sheets for 15 to 20 min. The soluble andinsoluble cellular material was quantitatively transferred with a2-ml buffer rinse by pipet to a Corex centrifuge tube. Frequently, 3 to 5 cultures were combined in each Corex tube.Two volumes of cold ethanol were added to precipitate theDNA and other material. After at least 20 min at 0°,the tubes

were centrifuged at 8000 x g for 20 min, and the supernatantwas tested for radioactivity. If there were more than 300 dpm/ml, the precipitate was resuspended in the Tris-HCI buffer and

precipitated again. The precipitate was then suspended in 4 mlof 0.1 M Tris-HCI, pH 8.2, and CsCI (approximately 5.2 g) was

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R. F. Camalier et al.

added to give a density of 1.700 g/cu cm. Five ml of the well-

dispersed mixture were centrifuged in a Beckman Model SW65Ti rotor at 40,000 rpm for 48 hr, the fractions were collectedand diluted with 0.5 ml water, and the DMA region was locatedby absorbance at 260 nm and banded twice more in CsCIgradients. Every fraction of the third gradient was also countedin 10 ml Aguasol (New England Nuclear, Boston, Mass.) in aBeckman Model LS-250 scintillation spectrophotometer.

Fluorescent Assay of BP Metabolites. From each T-25

flask, with or without cells, 2.5 ml culture medium were transferred to a 15-ml Corex tube and determined essentially asdescribed previously (6, 30). Either BP or 3-OH-BP was added

to appropriate tubes (final concentration, 10 and 0.4 jug/ml,respectively) followed by 2 ml of acetone, and the contents ofeach tube were mixed well. Unbound BP, 3-OH-BP and otherBP metabolites were extracted with 4 ml n-hexane, and the

contents of each tube were again mixed well and centrifugedat room temperature to settle the emulsion formed. Three ml ofthe n-hexane layer were then transferred to a 13- x 100-mm

borosilicate disposable glass tube and mixed with 2 ml of 1 NNaOH. Subsequently, 1.0 ml of the NaOH layer was transferredto a 10- x 75-mm borosilicate disposable glass tube, and the

relative fluorescence was determined at 522 nm with an excitation wavelength of 396 nm.

RESULTS

Cytotoxicity of BP. Relatively low BP concentrations andlow-intensity light were used in these experiments to approach

exposure levels that might commonly be encountered in theenvironment. Fig. 1 shows clearly that cultures treated with 1tig BP per ml in the absence of light develop marked cytotox-

icity, whereas treated cells exposed to light appear as healthycell sheets with virtually no evidence of toxicity. Control cultures exposed to light in the absence of BP also show noevidence of toxicity. Even when a dose of 10 ftg BP per ml isused, only a slight cytotoxic effect is evident in the light-exposed cultures, whereas the shielded cells are severelydamaged.

To determine whether the inhibition of cytotoxicity is due tovisible light or near UV, cells treated with 1 jug BP per ml wereexposed through an UF-III filter, which excludes wavelengths

<400 nm. The equivalent inhibition by filtered and unfilteredlight shows that the effective wavelength(s) is in the visiblerange.

Effect of Visible Light on BP Binding to Cellular DNA. Thecytotoxic effect of BP is generally attributed to the formation ofcovalent bonds with cellular macromolecules, including DNA,after metabolic activation by the mixed-function oxidases (4,7-9), although 3-OH-BP has been reported to be directly

cytotoxic (10). In view of our findings that fluorescent lightinhibits BP cytotoxicity, we determined the influence of light oncovalent binding of BP to cellular DNA.

In the first experiment, cultures were treated with 1 /¿gofradioactive BP per ml, with and without intermittent light exposure. After treatment, cells were harvested in sodium dode-

cyl sulfate; ethanol was added, and the precipitate was bandedin CsCI by isopyknic centrifugation. Table 1 shows that intermittent light reduces the covalent binding of BP to cellular DNAmore than 20 times. This result is consistent with the knownphotosensitivity of BP. However, at a dose of 0.2 fig BP per ml,light reduced covalent binding only 2-fold, from 6.6 to 3.2 pmol

bound per mg DNA. Further, in cultures treated with 0.1 fig BPper ml, covalent binding of BP to cellular DNA is essentially

Fig. 1. Protective influence of intermittent, low-intensity fluorescent light exposure after treatment with BP. Primary human foreskin epithelial cells were primed for24 hr with a noncytotoxic dose of BP (0.1 fig/ml) and then treated for 3 days with a cytotoxic dose level (1.0 fig/ml). One-half of the cultures were also subjected toalternating 12-hr periods of light and dark during the treatment period, while the other cultures were kept shielded. In light-exposed cultures, virtually no changes inmorphology are seen (A) in contrast to shielded cultures in which a frank cytotoxic response is apparent (ß).Unstained culture, x 200.

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Light and BP Toxicity in Human Epithelial Cells

Table 1

Effect of visible light on BP binding to cellular DNA

pmol BP/mg DNA

Experiment Shielded Intermittent light

129162

5.877.82

0.16 -

0.00

FRACTION NUMBER

Chart 1. Effect of light on BP binding to cellular DNA. Fractions collected fromthe final CsCI gradient demonstrate equivalent adduci formation in light-exposedand shielded cells treated with low concentrations of BP. Cells treated withradioactive BP (0.1 ¡ig/m\; 12 Ci/mmol) were exposed to light, harvested, andbanded 3 times in CsCI, as described in "Materials and Methods." and compared

with shielded controls. Radioactivity, when added to cells immediately beforeprocessing, did not appear in the final gradient. Peak areas were calculated aftersubtracting background determined from the dashed lines drawn beneath thepeaks. Arrows, fractions included in the calculation. O, radioactivity; •,absorb-ance at 260 nm. Top, shielded cells; bottom, light-exposed cells.

identical in shielded and light-exposed cultures, i.e., 1.1 and

1.3 pmol/mg DNA, respectively (Chart 1). Values ranging from1.0 to 4.4 pmol were obtained in different experiments; thisvariation probably resulted from individual differences amongdonors (13, 26), since within an experiment with tissue fromthe same donor the range of variation in BP binding to DNAfound is <30%. Thus, at cytotoxic level of BP (1 jug/ml), lightdramatically reduces binding to DNA while at low noncytotoxicconcentrations (0.1 jug/ml), light has no apparent effect although measurable binding occurs.

Alteration and Activation of BP by Light. The finding thatlight did not reduce the amount of BP bound to DNA at 0.1 /ig/ml in the same way that it reduced the binding at 1.0 /¿g/mlissurprising if the mechanism of inhibition of BP adducts is simpledestruction of BP by light. To ascertain the rate of BP destruction by light in complex medium supplemented with serum, wemeasured the percentage of radioactivity remaining in theaqueous phase after ethyl acetate extraction at daily intervals.Chart 2 shows that after the first day, nearly 2 times thefractional amount of BP is destroyed at 1.0 /ig compared with0.1 /¿g/ml(49% or 0.49 jug/ml versus 27% or 0.025 /ng/ml,

respectively). After 4 days, the curves seem to be convergingnear 70% of the radioactivity (69 and 58%) remaining in theaqueous layer. These data represent a minimum rate of BPalteration, since BP could be converted to compounds whichstill partition largely in the ethyl acetate phase. We concludefrom this experiment that low-intensity light converts BP to awater-soluble form(s) or perhaps induces binding to water-

soluble macromolecules and that the rate of conversion issomewhat faster at 1.0 jug BP per ml than at 0.1 fig BP per ml.

Although high-intensity light has been shown to induce bind

ing of BP to DNA in a simple salt solution (28), it is relevant todetermine whether the BP altered by low-intensity light, asshown above, also binds to DNA in the complex serum-con

taining culture medium. Accordingly, 0.1 /ig radioactive BP perml was incubated in culture medium with calf thymus DNA at37° for 48 hr, with or without continuous illumination. In the

presence of light, there was a large increase, about 20-fold, in

the radioactivity associated with DNA (Chart 3). The bound BPproduct is covalently linked because the isolation procedureinvolving phenol extraction, ethanol precipitation, and multipleCsCI banding procedures would remove noncovalently boundmaterial. Thus, fluorescent light can induce covalent binding ofBP to DNA in complex culture medium and without metabolicconversion by cellular mixed-function oxidases.

Effect of Light on BP Monooxygenase Activity. Light exposure of culture medium generates toxic products (25), someof which can induce BP monooxygenase (18). These observations suggest that light would enhance cytotoxicity in culturestreated with BP. In contrast, the preceding studies with humanepithelial cells showed that light did not produce observablecytotoxicity in the absence of BP and inhibited cytotoxicity incultures treated with BP. This inhibition of cytotoxicity by lightcould result from direct or indirect inactivation of BP-metabo-

lizing enzymes, interference with their induction, or destructionof cytotoxic metabolites.

DO

Chart 2. Effect of visible light on the ethyl acetate extraction of radioactivityfrom BP in culture medium. Two sets of 5 T-25 pl.istic culture flasks eachcontaining 5 ml of culture medium (NCTC 168 plus 10% horse serum) andradioactive BP (0.1 or 1.0 /jg/ml) were exposed to continuous cool whitefluorescent light (4.6 watts/sq m). At 1-day intervals, an exposed and a shieldedcontrol flask were extracted 3 times with an equal volume of ethyl acetate. Thepercentage remaining in the aqueous phase was calculated by dividing theradioactivity left in the aqueous layer by the sum of that in the aqueous andorganic layers (combined) multiplied by 100. O, flasks containing 1.0 fig BP perml exposed to light; •,shielded control; D, flasks containing 0.1 fig BP per mlexposed to light; •shielded control.

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R. F. Camalier et al.

06

12 16 O 4

FRACTION NUMBER

Chart 3. Light-induced covalent binding of radioactivity from BP to purifiedcalf thymus DNA in culture medium. A plastic T-25 flask containing 5 ml culture

medium (NCTC 168 plus 10% horse serum), DMA (250 ^g/ml). and radioactiveBP (0.1 /ig/ml) was exposed to cool white fluorescent light (4.6 watts/sq m) for48 hr. Two volumes of ethanol were then added to the exposed flask and ashielded control. After 1 hr at 0°the precipitate was collected and dissolved inTris-HCI buffer containing 1% sodium dodecyl sulfate, phenol extracted, repre-cipitated with ethanol, dissolved in Tris buffer, and banded 3 times in CsCI, asdescribed in "Materials and Methods." •,absorbance at 260 nm; O, radioactiv

ity. Left, shielded control; right, medium exposed to light.

To test an effect on induction, cultures were preinduced for24 hr with low noncytotoxic levels of BP (0.1 /ig/ml) to inducethe enzymes and then treated with higher levels (1.0 and 10.0/¿g/ml)and intermittent light. If light exposure protects againstcytotoxicity of BP by inhibiting induction of the enzymes, thenBP should induce a cytotoxic effect in preinduced culturesexposed to light. No such cytotoxicity was observed. Therefore, the light must inactivate the enzymes after induction ordestroy enzyme metabolites.

To test whether light in the presence of BP inhibits activity ofthe mixed-function oxidases, fluorescence of the phenol metabolites of BP, principally 3-OH-BP, was measured. In Experiment 1 (Table 2), 21-day cultures were incubated for 3 to 4days in the absence or presence of BP (1 mg/ml) and intermittent light. Relative fluorescence in light-treated cultures was

<5% of that in shielded controls. Thus, light treatment eitherinactivates the monooxygenases or destroys the fluorescentmetabolites.

To test the latter alternative, 3-OH-BP (0.4 /¿g/ml)was in

cubated in culture medium for 3 to 4 days in the presence orabsence of intermittent light (Table 2, Experiment 2). Clearly,in the absence of cells, light exposure destroys 3-OH-BP and

probably other fluorescent BP metabolites as well.To test whether light also inhibits activity of the monooxy

genases, sets of three 21-day cultures were treated with BP(1 jig/ml) to induce the enzymes; one set was exposed tointermittent light and the other was shielded. A third control setwas not treated with light or BP. After 2 days of incubation,cells were harvested and assayed for monooxygenase activityby a procedure similar to that described (6, 30). Light exposuredecreased enzyme activity to <10% of that in the shielded BP-

treated controls and was equivalent to the untreated set. Theseresults indicate that light in the presence of BP inhibits monooxygenase activity as well as destroying the principal fluorescent metabolite 3-OH-BP.

Stability of BP-DNA Adducts. Although light exposure apparently does not influence the amount of covalent binding of

BP to cellular DNA at noncytotoxic BP levels, light couldconceivably so affect the sites of BP binding as to interfere withDNA repair. Accordingly, cells were exposed to 0.1 fig radioactive BP per ml for 5 days in the presence and absence ofintermittent light; BP was removed, and cultures were incubated for an additional 7 days in the dark to assess the extentof repair. Table 3 shows that, with or without intermittent light,virtually the same amount of BP remained covalently bound toDNA 7 days after terminating BP-light treatment. Apparently,these BP-DNA adducts are not removed during a 7-day repair

period.

DISCUSSION

This report describes the effects of environmental levels ofcool white fluorescent light on the binding of BP to DNA ofhuman epithelial cells in culture. At cytotoxic BP concentrations, low levels of visible light strongly inhibit both cytotoxicityand adduct formation. This inhibition is consistent with thegenerally known fact that light destroys BP. Paradoxically, wefound that light induced BP binding to purified calf thymus DNAunder similar conditions. This finding might be explained by theobservation of Guenthner ef al. (12) that most of the reactiveBP electrophiles formed outside the cell nucleus do not bind toDNA because the numerous nucleophiles encountered in thecytoplasm trap the BP electrophiles. Thus, the purified calfthymus DNA dispersed throughout the medium is more accessible to the reactive BP species produced by light than is thenuclear DNA surrounded by cytoplasm. A second anomaly isthat BP at low noncytotoxic concentration forms adducts to thesame extent in the presence or absence of light. Therefore,some adduct formation occurs by a light-insensitive process

that is detected only with a low noncytotoxic dose. This processmay be important because the low concentration of BP usedapproaches environmental levels; furthermore, because theadducts persist for at least 7 days, they may accumulate withprolonged BP exposure.

One possible explanation for the light-insensitive process is

that some BP or BP derivatives concentrate preferentially inlipophilic areas of the cell where the microenvironment protects

Table 2Effect of intermittent light on BP and 3-OH-BP with and without cells

Experiment12BP 3-OH-BP Light CellsFlasks+

- + +5-t-+5-1-5-"

- - +3-b+2+

- -t-3+

3--1- + - 3Relative

fluorescence4.7±0.45a112.0+

18.21.9±0.223.0-«-

1.73470.0±7.073.3

±0.29445.0±111.21.2

± 2.15

Average ±S.D.' BP or 3-OH-BP added just before extraction. See "Materials and Methods.

Table 3

Persistence of BP Adducts to Cellular DNA

Light+Day2.3

1.91pmol

BP/mgDNA5

Day 122.0

5 1.98

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Light and BP Toxicity in Human Epithelial Cells

them from light-induced chemical alterations and/or enhances

their reactivity with DMA. One such microenvironment is present in mitochondria. In fact, Graffi (11) has shown that poly-

cyclic hydrocarbons accumulate in mitochondria. Further, Allenand Coombs (1) found that BP adducts to mitochondrial DNAwere 50 times more frequent than those to nuclear DNA intertiary mouse embryo cultures. In addition, Backer and Weinstein (2) showed that the BP metabolite, 7,8-dihydroxy-9,10-

epoxybenzo(a)pyrene formed 40 to 90 times more adducts tomitochondrial than did nuclear DNA in 3 rodent cell lines.These effects are attributed to the lipid microenvironment ofmitochondrial DNA which tends to concentrate the BP metabolite. Pertinent to our observation that the DNA adducts formedat the noncytotoxic level of BP are not repaired are 2 reportswhich indicate the absence of excision repair in mammalianmitochondria. Absence of excision repair was shown in bothhuman and rodent cells for UV-induced pyrimidine dimers (5)and for N-methyl-N'-nitro-N-nitrosoguanidine- and 4-nitroqui-

noline 1-oxide-induced DNA damage in rat ascites cells (16).A second possible explanation for the light-insensitive proc

ess is that a small amount of BP is tightly bound to a lipophilicregion of a serum protein(s) and protected from light-induced

change. If so, with a low concentration of BP, the fractionprotected from light would be larger than with a high concentration. Therefore, with a low concentration, the overall rate ofdestruction by light would be slower. The data of Chart 2 areconsistent with this explanation in that the low concentration ofBP is destroyed substantially more slowly than is the highconcentration.

In contrast to the present observations on the prevention ofBP cytotoxicity by light in mass culture, cytotoxic effects ofblack light on BP-treated Chinese hamster cells in a plating

assay have been reported (15, 17). Several factors couldexplain these divergent results. Undoubtedly, the use of a near-

UV light induces a different spectrum of photoproducts ascompared to visible light. Also, cells at low density, as in aplating assay, are more sensitive to toxic substances such ashydrogen peroxide which is generated in culture medium exposed to both near-UV and visible light (20); these effects areless apparent in mass culture (24, 25, 29). Further, cell typescan differ in sensitivity; for example, human fibroblasts aremore sensitive to visible light than are epithelial cells (27).

Fluorescent light is shown not only to destroy BP and 3-OH-

BP but also to inhibit intracellular activity of BP monooxygenaseand presumably other enzymes of the mixed-function oxidase

system. This inhibition is associated with enzyme inactivationrather than with simple inhibition of induction, since cells prein-

duced with BP lack activity after light exposure. From ourresults, we cannot distinguish between direct photoinactivationof the enzyme or indirect inactivation by production of photo-

products of BP, BP derivatives, or other components.

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1. Allen, J. A., and Coombs. M. M. Covalent binding of polycyclic aromaticcompounds to mitochondrial and nuclear DNA. Nature (Lond.). 287: 244-245. 1980.

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3. Bradley, M. O., and Sharkey, N. A. Mutagenicity and toxicity of visiblefluorescent light to cultured mammalian cells. Nature (Lond.). 266. 724-

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)pyrene Binding to DNA ofaEffect of Visible Light on Benzo(

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