[CANCER RESEARCH 41, 3179-3185, August 1981]0008-5472/81/0041-OOOOS02.00

Effect of Low-Density Lipoprotein on the Incorporation ofBenzo(a)pyrene by Cultured Cells1

Joyce F. Remsen and Rachel B. Shireman

Department of Biochemistry, J. Hillis Miller Health Center [J. F. P.], and Department of Food Science and Human Nutrition. Institute of Food and AgriculturalSciences [R. B. S.¡,University of Florida, Gainesville, Florida 32611

ABSTRACT

Benzo(a)pyrene added to human plasma in vitro associatedwith the plasma lipoproteins, especially the low-density fraction. The influence of plasma low-density lipoprotein on cellularuptake of benzo(a)pyrene was studied using WI-38, a human

embryonic lung fibroblast line, and GM 1915, a skin fibroblastline derived from a patient with homozygous familial hypercho-lesterolemia. The WI-38 cells were low-density-lipoprotein re

ceptor positive, and the familial hypercholesterolemia cellswere receptor negative by standard binding studies with 125I-

labeled low-density lipoprotein. Following 2 hr of incubation at37 or 4°,cell association of benzo(a)pyrene was determined

with benzo(a)pyrene bound to lipoprotein or added at the sameconcentration to serum-free medium or medium containingdelipidated serum. Uptake from delipidated or serum-free me

dium by both cell lines was linear with concentration, whileincorporation of benzo(a)pyrene bound to low-density lipopro

tein was much less and nonlinear at higher concentrations oflipoprotein. While low-density lipoprotein apparently influenced

the availability of benzo(a)pyrene to the cell, no differenceswere noted in the incorporation of benzo(a)pyrene by WI-38

and familial hypercholesterolemia cells. Thus, benzo(a)pyreneentered the cells from low-density lipoproteins despite the

absence of specific receptors, apparently by a rapid redistribution between the lipoprotein and cell membrane.

INTRODUCTION

B(a)P2 is a polycyclic aromatic hydrocarbon which is metab

olized by cells to derivatives which are mutagenic and carcinogenic. Chen ef al. (8) and Shu and Nichols (18) have demonstrated that B(a)P, a very hydrophobic compound, preferentially associates in human plasma with VLDL and LDL. Itassociates to a lesser extent with HDL and very little with serumalbumin in the presence of the lipoproteins. B(a)P exchangesamong the lipoprotein fractions with the largest percentageassociating with the VLDL and LDL.

B(a)P which enters the bloodstream is transported by chy-lomicrons and lipoproteins in vivo (19). Cultured cells includingskin fibroblasts, aortic smooth muscle, and lymphocytes havespecific plasma membrane receptors for the binding of LDL(12); it seemed plausible, therefore, that plasma lipoproteinsmight be important in the cellular incorporation of B(a)P byperipheral tissues.

1This work was supported by American Cancer Society Institutional Grant

ACS-79-069. Florida Agricultural Experiment Stations Journal Series Number2474.

2 The abbreviations used are: B(a)P. benzo(a)pyrene; VLDL, very-low-density

lipoproteins; LDL, low-density lipoproteins; HDL, high-density lipoproteins; FH.familial hypercholesterolemia; B(a)P-LDL, benzo(a)pyrene associated with LDL.

Received October 6, 1980; accepted May 12, 1981.

Two cell lines were selected to test the possible role of LDLin cellular uptake of B(a)P. One line, WI-38 lung fibroblasts, is

known to be capable of metabolizing B(a)P (1). The other line,GM 1915, is a fibroblast line from a patient with homozygousFH. These cells lack the LDL receptor. Thus, a comparativestudy of B(a)P incorporation of LDL receptor-positive and receptor-negative cells was possible.

MATERIALS AND METHODS

Growth of Cells. WI-38 cells (Flow Laboratories, Rockville,Md.) were grown in 5% CO2 in basal medium with Earle's salts

(Grand Island Biological Co., Grand Island, N. Y.), and FHfibroblasts, GM 1915 (Human Mutant Cell Repository, Camden,N. J.) in F-10 nutrient mixture (Grand Island Biological Co.),

both antibiotic free and supplemented with 10% fetal calf serum(Grand Island Biological Co.). For experiments, cells were usedin passages 10 to 19 for FH and 24 to 33 for WI-38. Thedifference in passage number (approximately equal to generations at 1:2 splits) was necessitated by the age at which thecultures were initially available. There was no obvious alterationin growth characteristics or morphology in either cell line withinthese passages. FH cell growth declined at passages greaterthan 22 to 23 and prevented their use at higher passagenumbers. Approximately 1 week prior to an experiment, cellstocks were trypsinized and seeded in 60-mm plastic Petridishes. This allowed sufficient time for cell monolayers to reachconfluence before an experiment. Approximately 24 hr beforeuse, the growth medium was replaced with 4 ml of Eagle'sbasal medium with Earle's salts or F-10 supplemented with

10% delipidated fetal calf serum. The medium containing delipidated serum served to maximize the number of LDL receptors(11). Both cell lines retained their usual morphology in thepresence of delipidated serum.

Preparation of Delipidated Fetal Calf Serum. Thawed fetalcalf serum was adjusted to a density of 1.2 g/ml with KBr, andthe lipoproteins floated by centrifugation at 40,000 rpm for 24hr in a T865 rotor in a Sorvall OTD-2 centrifuge. Under these

conditions, all of the lipoproteins were removed, and no cholesterol was detected by the cholesterol enzymatic assay (Kit350HDLB; Sigma Chemical Co.). The delipidated fetal calfserum was dialyzed extensively at 4°against buffer, 17.3 ITIM

Tris:130 ITIMNaCI:3.6 MIMKCI:1.2 mM CaCI2:1.2 ITIMMgSCX,pH 7.6 (3). Following dialysis, the serum was sterilized byfiltration through 0.20-fim pore size filters and stored at —20°

until use. The serum had approximately 3 mg protein per ml.Preparation of LDL. LDL was isolated from human plasma in

the presence of 0.02% sodium azide by differential densitypreparative ultracentrifugation between p1.006 and p1.06 (10).Isolated LDL was extensively dialyzed against 0.15 M NaCI:0.01 M sodium phosphate buffer at pH 7.4 and sterilized prior

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J. F. Remsen and R. B. Shireman

to use. Homogeneity was monitored by sodium dodecyl sulfate:polyacrylamide gel electrophoresis (14), and LDL concentration was determined by the method of Lowry ef al. (17).

Addition of B(a)P to LDL. [C-3H]B(a)P (Amersham/Searle

Corp., Arlington Heights, III.), specific activity, 37 Ci/mmol,was diluted with unlabeled B(a)P (Eastman Kodak Co., Rochester, N. Y.) to decrease the specific activity to 88 mCi/mmol.Radiopurity was greater than 97%. The B(a)P was adsorbedonto 3-mm glass beads from acetone by evaporating the acetone under nitrogen and then incorporated into purified LDL byincubation with LDL in phosphate-buffered saline (0.15 M NaCI:0.01 M sodium phosphate at pH 7.4) at 37°for 5 to 6 hr in the

dark (18). Uptake of B(a)P into LDL was quantitated by recovery of radioactivity and protein (17) following filtration to sterilize the LDL. The percentage of uptake of B(a)P by LDL variedfrom preparation to preparation, ranging from 3 to 13 pmolB(a)P per 100 ^g LDL. The [3H]B(a)P-LDL was stored at 4°and

used within a few days.The association of B(a)P with LDL was verified in several

ways, (a) The specific activity of [3H]B(a)P-LDL before and after

passage through a Sephadex G-75 column, 2.5 x 30 cm, inphosphate-buffered saline was determined. The column was

first calibrated with unlabeled LDL which eluted in the voidvolume; [3H]B(a)P-LDL also eluted in the void volume. Thespecific activities of [3H]B(a)P-LDL before and after chromatog-

raphy on the Sephadex G-75 column were the same. Free[3H]B(a)P did not elute in the void volume of the column; it

adsorbed to the Sephadex as less than 7% of the counts elutedin the included volume, (b) [3H]B(a)P-LDL, when chromato-

graphed on a 2- x 45-cm Sepharose 6B column in phosphate-

buffered saline, eluted in the included volume. The profile oftritium radioactivity coincided with that of the A28o, and thespecific activities of the peak fractions were the same (4 x 106

cpm/mg LDL) as that of the applied sample, (c) The associationof B(a)P with LDL was also verified by the profile of radioactivityobtained upon recentrifugation of the B(a)P-LDL in phosphate-

buffered KBr at p1.006 and p1.06. (cO B(a)P was minimallysoluble in the buffer alone. This was confirmed by adsorbing[3H]B(a)P onto glass beads as above, incubating at 37°with

phosphate-buffered saline, and then filtering as described forB(a)P-LDL. Less than 0.25% of the input radioactivity was

recovered in the filtrate.Preparation of [125I, 3H]B(a)P-LDL. 125l-labeled LDL was

prepared, and a portion of it was incubated with [3H]B(a)P as

above. The double-labeled LDL was filtered to sterilize; recovery of protein was measured, and B(a)P incorporation wasestimated.

Specific Binding of 125l-labeled LDL. To determine the specific binding of LDL to WI-38 and FH cells, 125l-labeled LDL was

prepared by the iodine monochloride method (4) and used ina modification of the cell binding assay as described by Goldstein and Brown (11). In brief, confluent cell monolayers wereincubated for 2 hr at 37 and 4°with 1"(-labeled LDL in the

presence or absence of competing excess unlabeled LDL (250iug/ml) in basal medium containing 10% delipidated fetal calfserum for both cell lines. Medium containing 125l-labeled LDL

was removed, and the monolayers were washed with phosphate-buffered saline. Bovine serum albumin was not used inthe phosphate-buffered saline wash as it did not affect removal

' R. B. Shireman. unpublished results.

of LDL but could interfere with the protein quantitation.3 Follow

ing the addition of 2 ml of 0.1 M NaOH, the radioactivity of thecell hydrolysate was quantitated by a Nuclear Chicago Model1185 gamma counter. Cell protein recovery was also measured(17), and the total uptake of LDL in ng/mg cell protein wascalculated. In some experiments, the nonspecific binding ofLDL (that measured in the presence of excess unlabeled LDL)was substracted from the total bound to obtain the specificbinding and uptake of LDL.

Measurement of B(a)P Associated with Cells. The incorporation of B(a)P by WI-38 and FH cells was quantitated by

incubating confluent cell monolayers in delipidated mediumcontaining [3H]B(a)P-LDL, in delipidated medium to which acomparable amount of [3H]B(a)P was added in a small volumeof acetone (maximum concentration, 0.2%), or [3H]B(a)P-LDLor [3H]B(a)P in serum-free medium. Hereafter, B(a)P addition

in acetone to medium will be referred to as free B(a)P. It isappreciated that the delipidated serum contains a variety ofproteins (about 3 mg/ml) and some residual lipid with whichthe B(a)P may associate. The specific activity of the B(a)P inthe LDL and added in acetone was identical in each experiment.After incubation, monolayers were processed as before. Todetermine cell protein, 0.2-ml aliquots were removed, and

Aquasol (New England Nuclear, Boston, Mass.) was added tothe remainder for measurement of radioactivity by a scintillationspectrometer (Beckman Instruments, Inc., Palo Alto, Calif.).Tritium radioactivity was corrected for quench to give comparable efficiencies for all samples.

Determination of 3H in the Presence of 125I.Comparison ofthe cellular incorporation of [3H]B(a)P with that of 125l-labeled

LDL was accomplished by using double-labeled B(a)P-LDL.Control samples contained only 125I;all samples were measuredfor 125Iactivity in a gamma counter. The samples were trans

ferred to plastic vials, an aliquot was removed for proteindetermination, and Aquasol was added to the remainder. Theamount of radioactivity appearing as tritium in the controlsamples containing only 125Iwas calculated and found to be

55% of the cpm as y-rays by the gamma counter. The samples

containing both isotopes were corrected by subtracting 55%of the cpm as y-rays from the cpm by liquid scintillation spec-

trometry. In all cases, the level of tritium from B(a)P was muchgreater than the 125I,so that any error in correction was minimal.

The tritium counts were then corrected for quench.Reductive Methylation of 12Sl-labeled LDL. ' " l-labeled LDL

was modified by reductive methylation (20). In brief, 6 to 8 mg125l-labeled LDL per ml were dialyzed against 0.3 M sodium

borate buffer, pH 9.0. Addition of 1 mg sodium borohydride at0° was followed by 6 additions of 1 jtl of 37% aqueous

formaldehyde at zero time and subsequent 6-min intervals. The

reaction sequence was then repeated with readdition of sodiumborohydride and formaldehyde, followed by extensive dialysisat 4°against 0.15 M NaCI:0.01 % EDTA, pH 7.O. The extent of

modification of lysine residues was not determined. A portionof the 125l-labeled LDL was associated with B(a)P as describedabove. Reduced 125l-labeled LDL and [1251,3H]B(a)P-LDL were

compared to unmodified controls in cell binding assays.

RESULTS

Measurement of Specific Binding of 125l-labeled LDL. The

purpose of this experiment was to determine whether the lung

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B(a)P Uptake in Cultured Cells

fibroblast line (WI-38) possessed specific receptors for LDL.The specific binding of 125l-labeled LDL by WI-38 is shown in

Chart 1. Binding and internalization by WI-38 cells were assayed according to the classical protocol at both 37 and 4°

(11). WI-38 cells showed specific binding and uptake of LDL at37°, while at 4°, little bound LDL was internalized. These

results are similar to those reported for normal skin fibroblasts(6).

Incorporation of B(a)P by WI-38 Cells. The incorporation of[3H]B(a)P by WI-38 fibroblasts was compared at 37°and at 4°

after incubation for 2 hr with B(a)P-LDL or with free B(a)P in

delipidated medium. In Chart 2, the incorporation of B(a)P wascompared as a function of B(a)P-LDL concentration, expressedas ¡igLDL per ml of medium. The concentration of free [3H]-

B(a)P in the medium was adjusted to be comparable to thatassociated with the LDL in each experiment. The form in whichfree B(a)P exists when added in acetone to delipidated serumhas not been determined. It may associate with components ofthe delipidated serum such as albumin as suggested by theresults of other investigators (8, 18).

Incorporation was linear when cells were incubated at 37°

with free B(a)P as indicated in Chart 2. Less free B(a)P wasassociated with the cells at 4°, compared with 37°, but its

incorporation was also linear with concentration. This difference might be due to a difference in the rate of diffusion at the2 temperatures. Cellular incorporation of [3H]B(a)P from B(a)P-

LDL, however, reached an apparent saturation at higher concentrations. At 37 and 4°,the incorporation of B(a)P from LDL

was approximately the same, despite the fact that bound LDLwas not internalized at 4°(Chart 1).

A large percentage of the available B(a)P was incorporatedby the cells, ranging from 25 to 50% for free B(a)P and from 5to 25% for B(a)P-LDL. A higher percentage of available B(a)P

was incorporated at low concentrations, although the actualamounts in /jmol were, of course, less. Nonspecific binding of[3H]B(a)P-LDL to empty plastic Petri dishes was 0.5% or less,

while that of B(a)P in delipidated medium was 3 to 4%. For thisreason, only confluent monolayers were used. No metabolism

Io.— 600-3)

400-

I- 37°I- 4°

-t- -+- -f-

pg

20 30''I-LDL / ml medium

50

Chart 1. Specific binding of LDL to WI-38 cells. The specific binding of '"I-labeled LDL during 2 hr of incubation was determined at 37 and 4°for WI-38

fibroblasts by standard procedures of competition with excess (250 /jg/ml)unlabeled LDL. Nonspecific binding (and internalization at 37°) has been sub

tracted. The ng bound refers to that amount bound and internalized. Each pointis the average of duplicate samples in this and all other charts.

Chart 2. Incorporation of B(a)P by WI-38 cells at 37 and 4°. Confluentmonolayers of WI-38 fibroblasts were incubated for 2 hr at 37 or 4° withincreasing concentrations of [3H]B(a)P-LDL or added in a small amount of acetone

to medium containing delipidated serum. Comparable concentrations of B(a)Pwere used. The incorporation of radioactive B(a)P into cells was determined andexpressed as cpm//tg cell protein. The highest concentration of free B(a)P was13 UM, and there were 13 pmol B(a)P per 100 jig LDL per ml. This is arepresentative experiment from 4 experiments.

to water-soluble derivatives could be detected within the 2-hr

incubation period using cyclohexane extraction of the incubation medium.

Comparison of B(a)P Incorporation by WI-38 and FH Fibroblasts. The uptake of B(a)P by LDL receptor-positive andreceptor-negative cells was compared.The incorporation of

free B(a)P was similar in both cell lines and was linear withconcentration (Chart 3), but the incorporation of B(a)P fromB(a)P-LDL was not concentration dependent at 50 fig/ml and

above for either cell line. The similarity in incorporation fromB(a)P-LDL by both cell lines was an unexpected result but was

confirmed in repeated experiments. Binding and internalizationof the LDL would be expected to carry associated B(a)P intothe WI-38 cells via the LDL receptor. Because the efficiency ofassociation of [3H]B(a)P with LDL varied from preparation to

preparation, the cpm (or mol) differs from experiment to experiment.

Uptake of free B(a)P by WI-38 cells from serum-free medium

was also linear with concentration and similar to B(a)P inmedium containing 10% delipidated serum (data not shown).Uptake from B(a)P-LDL in serum-free medium by both WI-38

and FH cells was increased slightly in the absence of serum(Table 1). Most experiments, however, were done with 10%delipidated serum, because after 2 hr in serum-free medium,

the cells were noticeably stressed. Unless otherwise noted,experiments were done with delipidated serum.

Kinetics of Incorporation of B(a)P by WI-38 and FH Cells.

Incorporation was followed as a function of time from 3 min to2 hr as shown in Chart 4. Because of processing time, no truezero-time value could be obtained. Curves for the incorporationof B(a)P added as B(a)P-LDL at 20 /jg/ml as a function of time

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J. F. Remsen and P. B. Shireman

12- D - LDL,*I-38O —LDL.FH 1915•- BlolP, Wl-38•- BlalP, FH1915

LDL ((jg/ml)Chart 3. Comparison of the incorporation of B(a)P-LDL and B(a)P from deli-

pidated medium at 37°by Wl-38 and FH cells. The experiment was conductedas described in Chart 2. The highest concentration of B(a)Pwas 3 ¡ÕM,comparableto B(a)P in LDL at 100 fig/ml. The specific acitivty was identical.

Table 1Cell uptake of B(a)P from B(a)P-LDL in serum-free medium or medium with

delipidated serumConfluent monolayers were incubated with 2 ml of medium as indicated for 2

hr at 37°.Monolayers were washed and processed as described in "Materialsand Methods."

CelllineWl-38FHB(a)P-LDL(fig/ml)51020501005102050100cpm/^g

cell1

0% Delipidatedserum7513425540054090146252391490protein8Noserum110205283563620105190306441565

a Values are the averages of duplicates.

by both cell lines were essentially superimposable. Incorporation from delipidated medium was also very similar. The slightlyhigher amount of B(a)P with FH cells may reflect the facts thatthese cells do not associate as closely as Wl-38 when confluent

and that there may be a small contribution from nonspecificbinding to plastic between cells.

Effect of B(a)P on I25l-labeled LDL Incorporation. To deter

mine the effect of the addition of B(a)P to LDL on the specificcellular binding, unlabeled B(a)P was associated with 125l-la-beled LDL, and its binding was compared with that of 125I-

labeled LDL alone in a standard assay. Because no excessLDL was added for competition, the curves shown in Chart 5represent total binding and internalization. The addition ofunlabeled B(a)P to LDL did not alter the concentration-dependent uptake of the 126l-labeled LDL by Wl-38 cells. The receptor-

negative FH cells showed a much decreased capacity forbinding of the LDL, and the presence of B(a)P did not influencethis binding. The relative behavior of the B(a)P could not befollowed in this experiment, but it indicates that the structure ofthe lipoprotein was not sufficiently perturbed to cause analteration in cellular binding and uptake of 126l-labeled LDL. On

the average, 2 molecules of B(a)P were present per LDLparticle.

Incorporation of [1251,3H]B(a)P-LDL by Wl-38 and FH Cells.The relative incorporation of 125Iand [3H]B(a)P-LDL was stud

ied. Confluent monolayers of Wl-38 and FH cells were incubated for 2 hr at 37 and 4°with increasing concentrations of

K 6

5"

•- FH I9I5O - «1-38

8fo)P-LDL

BlalP

O 20 40 60

TimeIOO I2080

(mm)Chart 4. Kinetics of incorporation of B(a)P by Wl-38 and FH cells at 37°.The

kinetics of incorporation of free B(a)P or B(a)P-LDL at 20 jig LDL per ml and thecomparable B(a)P concentration (1.5 ¡IM)were followed up to 2 hr. The incorporation of 1 nmol of B(a)P was equivalent to 14,000 cpm.

.c•§I800-o._

I600-°

I400-

I200-"i1000-

om800-

3600-t

400-ö

20°--

•- LDL OO - LDL + B(a) P ^ ,'Wl-38 «Ils ^~' '0FH

cellsf'*K

f x"OQ'

/;P;

o'

— O-"? iiii

IO 20 30 40"I- LDL / mi medium

50

Chart 5. Effect of B(a)P on the cell incorpation of 125l-labeledLDL by Wl-38and FH at 37°.To determine whether B(a)P altered LDL and/or its binding anduptake, LDL was radioiodinated and then incubated with unlabled B(a)P underthe same conditions used in all previous preparations. The total cellular incorporation of the '"l-labeled LDL by both Wl-38 and FH was determined after a 2-

hr incubation.

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B(a)P Uptake in Cultured Cells

12Sl-labeled LDL or [1251,3H]B(a)P-LDL.

The curves in Chart 6A represent total binding and uptake ofdouble-labeled [1251,3H]B(a)P-LDL based on y counts. They areessentially identical to the curves for 125l-labeled LDL alone

(not shown). The FH cells showed a much depressed bindingof 125l-labeled LDL at both temperatures. LDL binding by Wl-

38 showed a significant temperature effect. As shown in Chart60, however, the incorporation of [3H]B(a)P from B(a)P-LDL

was essentially the same for both cell lines and both temperatures, confirming earlier results that incorporation of B(a)P isnot identical with binding of 125l-labeled LDL.

Reductive methylation of LDL significantly decreased thebinding of LDL to WI-38 cells at 37°, but the uptake of B(a)P

from reduced and control LDL was comparable (Table 2),indicating that specific binding of LDL to receptor-positive cellswas not essential for transfer of B(a)P from LDL to the cells.

Comparison of the Kinetics of Uptake of 12sl-labeled LDLand [3H]B(a)P. The time courses of cell uptake of LDL and

1600-

sl 1400-

1200

800

400

20 40 60 80pg '"I-LDL / ml medium

g 400-

1

S

1 200

100

LDL (pg/ml)Chart 6. Total binding of LDL by WI-38 and FH compared with incorporation

of B(a)P. LDL was iodinated with 125I.A portion of this LDL was then incubatedwith tritium-labeled B(a)P as before. Confluent monolayers of FH and WI-38 cellswere incubated with varying concentrations of 125l-labeled LDL with and without[3H]B(a)P for 2 hr. The incorporation of the 2 isotopes was compared at 37and 4°after correcting the 3H counts for rray effects. A, total binding of [125I,3H]B(a)P-LDL by the 2 cell lines. Curves for the binding of '"(-labeled LDL werethe same as those shown. B, incorporation of [3H]B(a)P from LDL by FH and WI-

38 cells. The data have been corrected for y emission.

Table 2Effect of reductive methylation of LDL on uptake of B(a)P by WI-38 cells

'•'l-labeledLDL was modified by reductive methylation of lysine residues asdescribed in "Materials and Methods" followed by incorporation of [3H]B(a)P.The LDL samples were incubated in duplicate for 1 hr at 37°with WI-38 cellsand processed as described in "Materials and Methods." The data belowcompare the uptake of I25I- and [3H]B(a)P from reduced and control LDL.

LDL(/ig/ml)10

2050125l-labeled

LDL

(ng/mg cellprotein)Control454

10311562Reduced81

143359[3H]B(a)P

(ng/mg cellprotein)Control154

269343Reduced143

210344

B(a)P by WI-38 and FH cells were compared at 37° using

double-labeled LDL. As seen in Table 3, the rate of cellular

incorporation of the 2 isotopes differed in both cell lines. Thedecrease in the 3H:126I ratio for WI-38 was attributed to the

continued binding and internalization of LDL, while the B(a)Predistributed between the cells and the medium LDL (see Table4 below). In FH cells, there was an overall increase in the ratiowith time because only nonspecific uptake of LDL occurred,and little 125Iradioactivity was present, while tritium radioactivty

increased. Thus, the time courses for incorporation of LDL andB(a)P differ with B(a)P showing a more rapid uptake at earlytimes than LDL. In experiments performed at 4°,the ratios of

the 2 cell lines were similar to each other and higher than at37°due to the lower amount of LDL bound to the cells (data

not shown).Release of B(a)P by WI-38 Cells. The B(a)P associated

previously with cells could be removed during posttreatmentincubation in various media (Table 4), indicating that some timeis necessary for the carcinogen to be internalized for metabolism. The kinetics of removal of B(a)P by media was essentiallythe same whether associated with cells from B(a)P-LDL or freeB(a)P in medium with delipidated serum.

DISCUSSION

The uptake of B(a)P by cells exposed to B(a)P-LuL has beencompared with the uptake of B(a)P in the absence of LDL. Ithas been reported previously that the tumorigenesis producedby polynuclear aromatic hydrocarbons is influenced by thecoinstillation of adsorptive particles along with the carcinogens(13). Furthermore, Lakowicz and coworkers (15, 16) foundthat adsorption of B(a)P to several types of particulates enhanced its rate of uptake into lipid vesicles. Since LDL is avehicle for transport of B(a)P in the plasma, uptake rates incultured cells were used to determine the ability of the lipopro-tein to deliver this carcinogen to cells. In addition, since LDL isdegraded in the lysosome, we thought that there might be adifference between the metabolism of the LDL-associated carcinogen and the free carcinogen. Other factors have beenshown to influence the relative amounts of the metabolitesformed (9) and therefore could alter the levels of damage toDNA and other cell components by affecting the formation ofB(a)P diol-epoxides.

Based on earlier experimental evidence, a model has beenproposed (5) for the binding of LDL, ¡nternalization of the entireparticle followed by hydrolysis of the protein, and release ofthe lipids. If the cellular incorporation of B(a)P were dependentonly upon the binding and uptake of the LDL, the temperature

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J. F. Remsen and P. B. Shireman

Table 3Comparative uptake of LDL and B(a)P at 37°

Monolayers of Wl -38 and FH cells were exposed to [1251,3H]B(a)P-LDL or 125I-

labeled LDL in duplicate for the times indicated. Cells were processed, andradioactivity was determined as described in "Materials and Methods.'

TimeCellline(min)WI-38

361020FH

361020125l-labeledLDL(cpm/mgprotein)5121,2151,4821,7813041,070822605[3H]B(a)P(cpm/mgprotein)44,95076,350100,400104,00042,19781,95281,17288,7003H:'"1

ratio886368581397799147

Table 4Removal of B(a)P from WI-38 cells

Monolayers of WI-38 cells were exposed to B(a)P-LDL, 20 /ig/ml, or acomparable concentration of B(a)P in delipidated medium for 30 min and thenwashed with buffer, and 2 ml of fresh medium were added for 2 hr of incubation.Medium composition was as indicated. Calculation of percentage released wasbased on total B(a)P in the cells plus the posttreatment incubation medium.

%releasedTreatmentB(a)P-LDLB(a)PMediumLDL,

50 fig/ml10%DelipidatedserumNo

additionLDL,

50fig/ml10%DelipidatedserumNo

addition60

min7558373553120min7962479623

and concentration-dependent cellular uptake of LDL and B(a)P

should be parallel under all incubation conditions. Furthermore,cells lacking specific receptors should have a decreased incorporation of both LDL and B(a)P and thus might be "protected" from B(a)P. When the 2 cell lines were exposed to

B(a)P in LDL, however, the incorporation of the carcinogenwas similar for both lines (Chart 3) despite the absence of LDLreceptors in the FH cells (Chart 6). Also, although the cellularuptake of 125l-labeled LDL is much less at 4°than at 37°(Chart6A), the uptake of [3H]B(a)P from LDL was approximately the

same at both temperatures for both cell lines (Chart 6ß).Thisis interpreted as indicating that, in 2 hr, there were an extensiveredistribution and equilibration of B(a)P from LDL into theplasma membrane of the cells.

We found no evidence for a disturbance in LDL particlesthemselves sufficient to alter the cellular binding response. Thebinding and incorporation of 125l-labeled LDL were the same

with and without B(a)P present. The binding assay, however,might not be sufficiently sensitive to detect minor differences.B(a)P has been found to affect both phospholipid synthesisand distribution by exchange between rat liver microsomes andmitochondria in vitro (2), presumably by disorganizing the lipidbilayer.

In contrast to lipid vesicle uptake rates with talc and otherparticipates (16), cells incorporated significantly less B(a)Pfrom LDL as compared with that of free B(a)P in delipidatedmedium (Chart 3) or serum-free medium. The incorporation ofB(a)P from delipidated medium was linear with concentrationup to 13 ¡IM,and it probably associated with the hydrophobicportion of the cell membrane by simple diffusion. Our data

indicate that cells which are directly exposed to B(a)P beforeit reaches the bloodstream may accumulate much higher concentrations than those exposed to B(a)P-LDL, since the uptakeof B(a)P from B(a)P-LDL appears to be limited (Charts 3 and4). Although the uptake of 125l-labeled LDL is temperature

dependent, the uptake of B(a)P from LDL was not as temperature dependent over the 2-hr incubation period, indicating afairly rapid equilibration, even at 4°.Other investigators have

shown that pyrene exchanges among HDL particles in msec(7). In these experiments, the transfer of B(a)P from LDL tocells was rapid, reaching a plateau in 10 to 20 min at 37°(Chart 4). This pattern is not similar to that of 125l-labeled LDL

uptake which requires approximately 2 hr of incubation at 20/ig/ml to reach maximal uptake (11 ). After 20 min, a continuousexchange of B(a)P between LDL and cells probably occurs(Table 4). The difference in behavior of B(a)P and 125l-labeled

LDL is further emphasized by comparison of the ratio of incorporation by the WI-38 and FH cells as a function of concentra

tion (see Chart 6) and of time (Table 3). Comparison of the 2cell lines and 2 temperatures indicates that the FH cells associated much more extensively with [3H]B(a)P compared with125l-labeled LDL. If B(a)P entered cells only as a function of thereceptor, both cell types should have the same 3H:125Iratio for

a given concentration of LDL. In addition, modification of LDLto reduce specific binding to receptor-positive cells did not

affect cell uptake of B(a)P (Table 2).Partitioning of B(a)P between the cell and LDL explains in

part why the B(a)P uptake from LDL appears to saturate athigher concentrations of LDL. In vivo, assuming transfer andredistribution, B(a)P could enter and leave cells constantly.Experiments are underway to detemine the time course withwhich B(a)P progresses from the plasma membrane to theendoplasmic reticulum for metabolism.

In summary, the plasma transport of B(a)P by LDL seems tobe important in that LDL influences the availability of B(a)P tocells. There appears to be a rapid and extensive redistributionof the carcinogen between LDL in medium and cultured cells,so that any contribution of B(a)P entry by receptor-mediated

uptake of LDL cannot be detected in these experiments. Thishas been demonstrated in several ways: (a) similarity in cellularuptake of B(a)P, but not LDL, at 4 and 37°; (b) similarity of

uptake of B(a)P, but not LDL, in receptor-positive and receptor-

negative cell lines; (c) the kinetics of uptake of LDL and B(a)Pdiffers; and (d) reductive modification of LDL significantly decreases LDL binding and uptake but not that of B(a)P. Inconclusion, specific binding of LDL to receptors does notappear to be essential for the incorporation of B(a)P into cells.

ACKNOWLEDGMENTS

The authors thank Walter Jones for preparation of the graphs and MelissaMichaels for typing the manuscript.

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AUGUST 1981 3185

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1981;41:3179-3185. Cancer Res   Joyce F. Remsen and Rachel B. Shireman 

)pyrene by Cultured CellsaBenzo(Effect of Low-Density Lipoprotein on the Incorporation of

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