induction, inhibition, and biological properties of aryl ... · in the present paper, we report on...

[CANCER RESEARCH 39, 4612-4619, November 1979]0008-5472/79/0039-OOOOS02.00

Induction, Inhibition, and Biological Properties of Aryl HydrocarbonHydroxylase in a Stable Human B-Lymphocyte Cell Line, RPMI-17881

Harvey J. Freedman,2 Natalie B. Parker, Anthony J. Mannello, Mira L. Gurtoo,3 and Jun Minowada

Departments of Experimental Therapeutics [H. J. F., N. B. P., A. J. M.. H. L. GJ and Immunology [J. M.Â¡,Roswell Park Memorial Institute New York StateDepartment of Health, Buffalo. New York 14263

ABSTRACT

Aryl hydrocarbon hydroxylase was induced in the absenceof mitogens by several compounds in a stable, human B-lymphocyte cell line (RPMI-1788). Over the dose ranges testedand on molar basis the inducers, in decreasing order of potency, were 2,3,7,8-tetrachlorodibenzo-p-dioxin, dibenz(a,/7)-anthracene, 3-methylcholanthrene, benzo(a)pyrene, and 1,2-

benzanthracene. Potential inducers which, paradoxically, diminished basal aryl hydrocarbon hydroxylase, included 7,12-dimethylbenzanthracene, 2,5-diphenyloxazole, and chrysene.

Induction under optimal culture conditions ensured maximalactivities 3- to 4 fold above basal aryl hydrocarbon hydroxyl

ase. The characteristics of the induced [dibenz(a,A7)anthracene]and basal enzymes were found virtually identical. Both hadsimilar pH curves (optima at 8.25) and inhibitor specificity (a-and /5-naphthoflavones, metyrapone, and 2-diethylaminoethyl-2,2-diphenylvalerate in decreasing potency). Induced andbasal enzymes exhibited similar half-lives (41, 46 hr), apparent

activation energies (16.7, 16.6 kcal/mol), temperature optima(37-38, 38-39Â°), temperature-dependence of denaturation(range, 42-50Â°), and apparent Km's with benzo(a)pyrene (1.8,

0.8 /tM). The small difference in the apparent Kmwas related toenzyme concentration in the incubation rather than to thequality of the enzyme.

INTRODUCTION

AHH" is part of the microsomal mixed-function oxygenase

complex in endoplasmic reticulum of mammalian cells (10, 15).This substrate-inducible enzyme complex (15, 35) uses molec

ular O2, NADPH, and a specific cytochrome (28, 35) to metabolize many drugs, steroids, insecticides, and carcinogens (10,15). Polycyclic aromatic hydrocarbons, prevalent in the environment (9, 13, 54), are metabolically activated to reactiveepoxides which bind covalently to DMA (24, 43, 44, 56),possibly causing mutagenesis and carcinogenesis (24, 52, 53).A role for AHH in cancer etiology is suggested by a correlationbetween smoking and lung cancer (55) and between smokingand AHH induction (8). AHH occurs in many mammalian tissues

' Supported by Grants CA-13038, CA-17538, CA-05928. and CA-14413 from

the National Cancer Institute and Grant CTR-1253 from the Council for TobaccoResearch.

2 Supported by NIH Fellowship F-32-CA05928.3To whom requests for reprints should be addressed, at Grace Cancer Drug

Center, Roswell Park Memorial Institute, New York State Department of Health,666 Elm Street. Buffalo, N. Y. 14263.

' The abbreviations used are: AHH, aryl hydrocarbon hydroxylase; DBA,

dibenz(a,/i)anthracene; DMSO. dimethyl sulfoxide; PCS, fetal calf serum; RPMI,Roswell Park Memorial Institute; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin;DDT, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane; SKF-525A, 2-diethylami-noethyl-2,2-diphenylvalerate.

Received February 15, 1979; accepted August 13, 1979.

(6, 47, 50), including human monocytes (5) and mitogen-acti-

vated lymphocytes (7, 51).Lymphocyte AHH inducibility, increasingly studied since

1972, has been correlated with lung (26) and laryngeal (46)cancer susceptibility. Kellermann ef al. (25), Paigen ef al. (39,40), and Atlas ef al. (4) have reported on the inheritance oflymphocyte AHH inducibility; however, Paigen ef al. (38) recently observed no phenotypic difference between lung cancerpatient's progeny and a control population.

Gurtoo ef al. (19, 21) elucidated properties and optimalassay conditions for lymphocyte AHH. However, even theoptimized assay had limitations, e.g., seasonal variations (40,42) and need for prior mitogen stimulation (7, 51), so that astable, immunologically defined (IgM-yielding) B-lymphocyteline (RPMI-1788) was selected as a tentative model for studyingAHH. Established in 1968 from a healthy 33-year-old male (31,32), this line was characterized by us with respect to AHH andgrowth properties (14). Favorable findings, e.g., high AHHactivity in the absence of mitogens, prompted our use of thisline as a biochemical model for studies of AHH. Furthermore,it was hoped that qualitative differences between the basal(constitutive) and the induced enzyme in kinetic and otherbiological properties would be of considerable epidemiologicalvalue in identifying the "high risk" inducible humans. With

these objectives in mind, various kinetic and biological properties of the basal and induced AHH were enumerated in theRPMI-1788 cell line which provided sufficient quantities of cells

for these studies instead of fresh lymphocytes which could notbe obtained in such quantities.

In the present paper, we report on the induction and inhibitionof AHH in the RPMI-1788 cells by various chemicals, as well

as on the kinetic, and biological properties of the enzyme inthese cells.

MATERIALS AND METHODS

Materials. Reagents and chemicals used included: 3-hy-droxybenzo(a)pyrene (gift of Dr. H. Gelboin, National CancerInstitute, Bethesda, Md.); benzo(a)pyrene, DBA, and spectro-photometric grades of acetone, n-hexane, and DMSO (Aldrich

Chemical Co., Milwaukee, Wis.); FCS and RPMI 1640 culturemedium powder (Grand Island Biological Co., Grand Island, N.Y.); Tris and quinine sulfate (Eastman Organic Chemicals,Rochester, N. Y.); penicillin G and streptomycin sulfate (Nutritional Biochemicals Corp., Cleveland, Ohio); NADH and NADPH(Sigma Chemical Co., St. Louis, Mo.); and reagent gradeMgCI?, NaCI solution and sucrose (Fisher Scientific Co., Pittsburgh, Pa.). The numerous chemicals tested as inducers andinhibitors of AHH are listed with their commercial sourcesunder "Induction and Inhibition Profiles."

Culture supplies and instruments used included plastic T-30

4612 CANCER RESEARCH VOL. 39

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AHH in a Human B-Lymphocyte Cell Line

(25 sq cm) Falcon culture flasks (Becton, Dickinson and Co.,Los Angeles, Calif.); 0.5-gallon glass roller bottles (No. 7730)

for spinner cultures (BÃ©licoGlass, Inc., Vineland, N. J.); and16- x 150-mm glass, round-bottomed screw-cap culture tubes(Kimble Division, Owens-Illinois Glass Co., Toledo, Ohio).

Cell Culture. Stock cell cultures were maintained at 37Â°in

0.5 gallon sealed glass roller (spinner) bottles containing RPMIMedium 1640 fortified with 5% FCS. Cells were kept in suspension by magnetically rotated stirring bars and in exponentialgrowth by daily replenishment of 50% of the medium.

Subcultures in Falcon flasks served to evaluate optimal in-ducer concentrations, cell densities, and enzyme half-lives.Optimal conditions derived previously (14) for culturing RPMI-1788 cells were adhered to; moreover, mitogen stimulation,proven unnecessary for AHH induction in these cells (14), wasomitted. The basic culturing protocol in brief (14) was: anexponentially growing stock culture was centrifuged for 5 minat 200 x g (1000 rpm with a 15-cm-radius head) and the cell

pellet was reconstituted to a density between 0.60 to 0.85 x106 cells/ml with fresh RPMI Medium 1640 containing 10%

FCS, penicillin (100 units/ml), and streptomycin (50 /ig/ml).Eight-mi aliquots of the cell suspension were incubated in T-30Falcon flasks with loose caps for 48 hr at 37Â°in 5% CO2 and

100% relative humidity. However, after the first 24 hr, selectedflasks received various inducers dissolved in 5 /il acetone orDMSO for final concentrations ranging from 0.1 to 30 /IM (DBAstandard was 0.3 /IM). Control cultures received 5 /il solvent.After 48 hr total incubation, cells from all flasks were stainedwith trypan blue, and the cells excluding the dye were considered to be alive. Flask contents were harvested by decantinginto 16- x 15-mm glass tubes and centrifuging (200 x g, 4Â°,

20 min). Supernatants were discarded, and cell pellets wereassayed for AHH activity immediately or after storage at -70Â°

for up to 2 weeks.Separate batch cultures of DBA-induced and matched con

trol cells were prepared in 0.5 gallon spinner bottles for comparative studies of induced and basal AHH. Culturing conditions optimal for AHH induction, given above, were used, andantibiotics, 10% FCS, and inducer (0.3 /IM DBA where needed)were added directly to spinner bottles.

Enzyme Assay. Harvested lymphocytes were assayed forAHH activity by the fluorometric assay (19) modified from aprevious method (35, 47). Briefly, pellets having 5 to 20 x 106

cells were suspended in 1 ml buffer mixture, pH 8.5, containing0.5 M Tris, 3 mM MgCI2, 0.2 M sucrose, 1.8 HIM NADPH, and1.4 mM NADH. Upon addition of benzo(a)pyrene (0.1 mw finalconcentration) and vigorous vortexing (5 sec), tubes wereincubated for 30 min at 37Â°,during which the reaction rate is

constant. The reaction was arrested with 4 ml acetone:hexane(1:3) followed by vigorous mixing (1 min), centrifugation (5 min,200 x g), separation, and extraction of the top organic phase(3 ml) with 0.5 ml of 1 N NaOH. The fluorescence of the alkalinephase, containing phenolic metabolites, was read on anAminco-Bowman spectrofluorometer (excitation 396 nm, emis

sion 522 nm) precalibrated with a quinine sulfate standard.Using 3-hydroxybenzo(a)pyrene for quantitation, AHH activityhas been expressed in units defined as pmol equivalents of 3-hydroxybenzo(a)pyrene formed per min per 106 viable cells.

Activities thus defined may be assumed identical to initialvelocities as long as 5 to 20 x 106 cells/ml were incubated for30 min (see "Results"). All experimental deviations are indi

cated in the chart legends. Inducibility ratios are calculated forexperimental purposes as the ratios of AHH activities in optimally DBA-induced cells to those in control cells.

Characterization of Basal and DBA-induced Hydroxylases.Basal and DBA-induced AHH in RPMI-1788 cells were separately characterized. The enzyme half-lives were determined

by studying the time course of the loss of the enzyme activityin culture. After an initial incubation in culture for 24 hr, batchcultures (100 ml volume, 0.65 x 106 cells/ml) were maximally

induced by exposure to 0.3 /IM DBA for 24 hr; then the cellswere harvested, washed, resuspended in RPMI Medium 1640enriched with 10% FCS, and subcultured in 8-ml portions in

Falcon flasks. Cultures for basal AHH were prepared by anidentical procedure except that no DBA was added. Duplicate8-ml cultures in Falcon flasks for AHH assay of both induced

and control cells were harvested immediately and also daily forthe next 5 days.

Other properties were determined using only matchedbatches of control cells with "inducibility ratios" of >3.0 to

ensure that at least 70% or more of the enzyme in the cellswas a consequence of induction. To elaborate pH profiles,benzo(a)pyrene was incubated with 10 x 106 cells in Tris

buffer mixture adjusted to pH values from 6.5 to 9.5; the AHHactivity was then determined. To establish rates of heat dena-turation, cells in 1-ml buffer mixture without nucleotides (see"Enzyme Assay") were subjected to various temperatures (0-70Â°) for different periods (7 to 25 min); after cooling on ice

and addition of the pyridine nucleotides and benzo(a)pyrene(0.1 mM), the heat-exposed cells were incubated at 37Â° and

assayed for residual AHH activity as above. To ascertain activation energies as well as optimal temperatures of catalysis,the cells (10 x 106 cells/ml in buffer mixture) were incubated

with saturating concentrations of benzo(a)pyrene at varioustemperatures (10-45Â°), and the activities were assayed andinitial velocities calculated. K,,,'s were evaluated by incubating

cells with various concentrations of benzo(a)pyrene (0.5 to 100fiM) and assaying AHH activity as above.

Induction and Inhibition Profiles. Over 20 compounds weretested as inducers of AHH in RPMI-1788 cells, while 6 were

tested as inhibitors of the induced and basal enzymes.In order to screen inducers, stock solutions (16 or 50 mM

where possible) and 5 serial dilutions were made for eachcompound, using acetone or DMSO as necessary. Each compound was then added, in at least 5 final concentrations (0.1to 10 jiiM) in duplicate, to 8-ml cultures in Falcon flasks inoculated 24 hr previously as described above (see "Cell Culture").

The vehicle was always 5 /il acetone or DMSO, except forTCDD which was added in 5 /il of p-dioxane at nM levels. One

pair of cultures received 5 /il solvent (controls) while anotherpair received 0.3 /IM DBA (internal standard) in every experiment. After 24 hr of exposure to the compounds, the cells werecounted, harvested, and assayed for AHH activity. The compounds screened as inducers were 1,2,3,4-DBA, 1,2,5,6-DBA,benzo(a)pyrene, benzo(e)pyrene, metyrapone, and phenan-threne (Aldrich Chemical Co.); benz(a)anthracene, 7,12-di-methylbenz(a)anthracene, 3-methylcholanthrene, chrysene,

DDT, and PPO (Eastman Organic Chemicals); testosterone,17/}-estradiol, and diethylstilbestrol (Calbiochem, La Jolla,Calif.); 1,2,3,4,5,6-hexachlorocyclohexane (lindane), chole-calciferol, n-octylamine, DL-isoproterenol, benzidine, sodium

cyclamate, and sodium saccharin (Sigma); and TCDD (gift of

NOVEMBER 1979 4613



H. J. Freedman et al.

Dr. A. Poland, University of Wisconsin, Madison, Wis.).In order to screen inhibitors, stock solutions and serial dilu

tions were made. Each compound was then added in 25-/Jaliquots (final concentrations, 1CT6 to 1CT3 M) followed by

benzo(a)pyrene (0.1 HIM) to 1-ml cell suspensions in buffermixture as described above (see "Enzyme Assay"). Duplicate

control tubes received the solvent. Activity of induced andbasal AHH was calculated in terms of specific AHH activity per106 viable cells, and the enzyme inhibition is expressed as

percentage of reduction from control activity. The compoundsstudied as inhibitors included: SKF-525A, a kind gift fromSmith, Kline and French Laboratories, Philadelphia, Pa.; a- andjÃŸ-naphthoflavones, metyrapone, 1,1,1-trichloropropene-2,3-

oxide, and cyclohexene oxide (Aldrich Chemical Co.).

RESULTS

Effect of Cell Density on AHH Activity and Inducibility.Chart 1A shows the dependence of AHH activity on cell density.When various numbers of cells were incubated, the productformation increased linearly up to 20 x 106 cells/ml, and

maximal product formation at all cell concentrations was muchless than 1% of the initial substrate concentration. The conditions of incubation, which ensured linearity (11) of reactionwith time (30 min) and cell concentration (up to 20 x 106 cells/

ml) were adhered to in all experiments.Chart 1B demonstrates the most suitable initial cell densities

for subculturing RPMI-1788 cells in order to maximize theinducibility ratio. When cells at increasing densities were incubated for 48 hr under conditions stated above (see "CellCulture"), induced AHH (0.3 /IM DBA) increased more rapidly

than basal AHH at first, but beyond 0.6 x 106 cells/ml the

increase was about equal. Consequently, the highest inducibility ratios (induced per basal AHH > 3) could be obtained byseeding subcultures with 0.60 to 0.85 x 106 cells/ml; this was

done henceforth to ensure relatively homogeneous basal andinduced enzymes.

Properties of Basal and DBA-induced AHH. The biologicalhalf-lives for the hydroxylases in RPMI-1788 cells were esti

mated from semilog plots of enzyme activity against time (Chart2). From the slopes (k1) of the least-squares lines fitted to

points beyond 24 hr following the 0.9% NaCI solution washes,namely,

y = 7.53 - 0.0074Ã•y = 1.33 - 0.0066f

(A)(B)

the ii/2 of decay, i.e., (2.303 log 2) /k or log 2/k' were

computed as 41 and 46 hr for induced and basal AHH, respectively. AHH activity was stimulated in both induced andbasal subcultures by virtue of fresh media necessitated by thewashes, and that exponential decay began only 24 hr later(33).

The pH profiles for both induced and basal AHH showedrelatively narrow ranges (2.5 pH units) and a common pHoptimum at 8.25 (Chart 3). Charts 4A and 4ÃŸdemonstratetemperature dependency and time courses, respectively, ofheat denaturation. When exposed to various temperatures for7 min, both enzymes showed increasingly rapid inactivation ina common range, 40-50Â°, with 50% inactivation at about 46Â°

(Chart 4/\). Similar time courses of inactivation prevailed forthe 2 enzymes, e.g., at 50Â°,>80% inactivation occurred in 15

min (Chart 46).Arrhenius plots for the 2 enzymes yielded practically parallel

lines for initial velocities below 37Â° (Chart 5). Therefore, the

apparent activation energies (Â£), being calculated from theslopes,

m = E/2.303R

where R is the gas constant (1.987 cal/Â°K/mol), were virtually

identical, namely, 16.7 and 16.6 kcal/mol for induced andbasal enzymes, respectively. Initial velocities declined at temperatures above 37Â°(Chart 5), in accord with our plots dem

onstrating heat denaturation (Charts 4/4 and 46). Therefore,such points are not relevant to the Arrhenius plots, but areincluded to demonstrate the similarity of temperature optima,namely 37-38Â° and 38-39Â°, for induced and basal AHH,

respectively.The effects of varying the substrate concentration upon the

initial velocities of the 2 hydroxylases is illustrated by theLineweaver-Burk plot in Chart 6/4. Although the maximal velocities differed considerably ( y-intercept), the apparent Km's were

similar, i.e., 1.8 and 0.8 fiM for the induced and the basalenzyme, respectively, as derived from the Lineweaver-Burkplot by the least-squares method (12). The 2-fold difference in

apparent Km between basal and induced AHH may not beintrinsic but related to differences in cellular enzyme concentrations, as the apparent Km's did not vary randomly in 4

experiments (Chart 66). In 2 experiments, using equal numbersof uninduced cells, the higher Km corresponded to the higherAHH activity, and a similar correlation prevailed in 2 otherexperiments with induced cells. Moreover, although apparent

Chart 1. A. dependence of AHH activity oncell density. Various numbers of uninducedcells were incubated with benzo(a)pyrene (BP)at 37Â° for 30 min in 1 ml Tris buffer mixture,pH 8.5. as described under "Enzyme Assay.

The rate of phenolic product formation wasmonitored fluorometrically. Points are meansof duplicate determinations. B. effect of celldensity on basal and DBA-induced AHH activities and inducibility ratios. Cell cultures,seeded at various densities, were incubatedfor 48 hr in FCS-enriched RPMI Medium 1640under conditions stated in text (see Cell Culture"). One set of cultures received 0.3 JIM

DBA, 24 hr before harvest, while an equivalentset served as controls (basal). After cells wereharvested. AHH activities were assayed andinducibility ratios calculated as described intext. Points are means of duplicate determinations. BP, benzo(a)pyrene.

5 IO I5 20CELL NUMBER(millions/ml)

0.2 0.4 0.6 0.8 LOINITIAL CELL DENSITY (mlllloni/ml)

0





5 r

â€¢Z4 0

WASH

24 48 72 96 120

HOURS AFTER WASH

Chart 2. Rates of decay of basal and DBA-induced AHH activities. After 24 hrinduction with 0.3 /ÃŒMDBA, the media of both induced and control (basal) cultureswere replaced with fresh 10% FCS-enriched medium following a 0.9% NaCIsolution wash. Thereafter, cells were harvested daily for the assay of residualAHH. Points are means of duplicate determinations. BP. benzo(a)pyrene.

Km's for uninduced cells were lower than those for induced

cells, the line joining points for the former seemed colinear withpoints for the latter and therefore was extrapolated with somejustification (35) as recommended for mutual depletion systems(48) (see "Discussion"). The extrapolated Kmat infinite enzyme

dilution would thus be virtually identical for basal and inducedAHH in RPMI-1788 cells.

Induction and Inhibition Profiles. The effects of potentialinducers on AHH activity in RPMI-1788 cells are summarized

in Table 1. When comparisons were made on the basis ofmolarity, the 6 potent inducers in increasing order of potencywere mainly polycyclic compounds, namely benz(a)anthra-cene, benzo(a)pyrene, 3-methylcholanthrene, 1,2,5,6-diben-zanthracene, 1,2,3,4-dibenzanthracene, and TCDD. Actually,

DDT, benzo(e)pyrene and sodium cyclamate showed borderline inducing capacity (<33% above controls) at 1 Â¡IMor higherconcentrations. Certain isomers [e.g., benzo(a)pyrene versusbenz(e)pyrene] but not others (1,2,3,4- versus 1,2,5,6-DBA)

showed significant differences in inducing ability. Degrees of

induction ranged from 133% (borderline) to over 500% ofcontrols, but even extraordinarily high concentrations did notsuffice to reveal true optima for benzo(a)pyrene.

On the other hand, some compounds, when administered tocultures in particular concentration ranges, significantly suppressed basal AHH. These miscellaneous compounds includedaromatic hydrocarbons (chrysene), steroids (estradiol), andcongeners (diethylstilbestrol). The effects of 6 potential inhibitors of AHH activity in RPMI-1788 cells are summarized in

Table 2. They are listed in decreasing rank order of inhibitoryeffects on induced AHH at 1 mM. Basal and DBA-induced AHH

exhibited virtually identical inhibition profiles except for theinhibition with SKF-525A. Although SKF-525A demonstrated

some inhibition of induced AHH (82% of control) at 1 rriM, lowerconcentrations inhibited neither basal nor DBA-induced en

zymes but, on the contrary, seemed to slightly potentiate both.

0.5

Chart 3. Effect of pH on basal and DBA-induced AHH activities. AHH activityof a constant number of DBA-induced and uninduced cells was assayed atdifferent pH's in Tris-HCI buffer. Points are means of duplicate determinations.

BP. benzo(a)pyrene.

1.2Chart 4. A, heat denaturation profiles of

basal and DBA-induced AHH activities. A constant number of cells was preincubated at thespecified temperature for 7 min in buffer mixture, then the reduced pyridine nucleotidesand the substrate benzo(a)pyrene (BPÃŒwereadded and cells assayed as described in thetext, at 37Â°. Points are means of duplicate

determinations. B, time courses of heat denaturation of DBA-induced and basal AHH activities. After preincubation of cells in the absence of cofactors or substrate for the timesand at the temperatures indicated, to eachbuffered cell suspension were addedbenzo(a)pyrene (BP) and reduced pyridine nucleotides; the mixture was assayed for residualactivity. Points are means of duplicate determinations. A. DBA-induced cells; B, uninduced

INDUCED (DBA)

20 40 60PRE-INCUBATION TEMPERATURE^ )

IOO 5 IO

PRE-INCUBATION TIME (minulÂ«Â«)

NOVEMBER 1979 4615




DISCUSSION

In view of the significance of AHH for polycyclic aromatichydrocarbon metabolism and the need for a readily availablesource of human enzyme, we have extensively characterizedboth basal and induced forms of the enzyme in a fast-growing,human B-lymphocyte line. By screening many microsomal enzyme inducers and inhibitors for effects on RPMI-1788 cell

AHH, TCDD was found to be the most potent inducer, just asfor fresh lymphocyte AHH (27), followed by 5 polycyclic aromatic hydrocarbons known to induce AHH in liver cells (9, 16,37) and fresh lymphocytes (7, 19, 51). The insecticide DDT,known to stimulate hepatic drug metabolism (23) and AHH (16)as well as cholecalciferol and sodium cyclamate, caused bor-

TEMPERATURE PC)

3.1 3.3 3.5IO"5/ TEMPERATURE PK"')

Chart 5. Arrhenius plots for basal and DBA-induced AHH activities in RPMI-1788 cells. Tris-butfered cell suspensions were incubated with benzo(a)pyrene

(BP) at various temperatures, and the initial velocities ot hydroxylation weredetermined as described in the text. The activation energies were calculatedfrom the slopes of the lines below 37Â°; points above 37Â° showing decliningvelocities serve to demonstrate the temperature optima (37-39Â°). Points are

means of duplicate determinations.

derline degrees of induction. Some compounds, e.g., metyra-pone, diethylstilbestrol, testosterone, and 17/S-estradiol, which

depressed AHH, are known inhibitors of AHH in vivo or incertain cell culture systems (18, 34). It might be argued thatsome of these or other hydrophobic agents depressed AHHbecause they were retained by the lipoidal and microsomemembranes (18, 49) and could not be removed by washing;this explanation does not appear likely because, unless thesechemicals or their metabolites caused irreversible inactivationof the enzyme, the dilution effect produced when the cells wereresuspended in the incubation was so great as to have completely eliminated inhibition due to reversible interaction between the enzyme and the compounds tested. This contentionis further supported by the observation that cholecalciferol andn-octylamine, which are documented AHH inhibitors (1, 18),failed to suppress the AHH in RPMI-1788 cells.

Although at present there is no evidence to suggest that thepolycyclic aromatic hydrocarbon-inducible cytochrome P-448

in rodent liver is similar to that induced in extrahepatic tissues,Goujon ef al. (18) reported that in rodent liver cholecalciferoland n-oclylamine preferentially inhibit cytochrome P-450-linked AHH rather than cytochrome P-448-linked AHH. Thisconsideration suggests that the so-called "basal" AHH is pri

marily cytochrome P-448 linked. The inhibition profiles of Table2 corroborate this and, moreover, suggest that DBA-inducedand basal AHH in RPMI-1788 B-cells may be identical andequivalent to cytochrome P-448-associated AHH. Thus, a- andyS-naphthoflavones, known to be specific inhibitors of cytochrome P-448-linked AHH (1, 18, 49), blocked basal and

induced activities strongly and about equally. On the otherhand, metyrapone and SKF-525A, specific inhibitors of cytochrome P-450-linked AHH (1,18), only minimally inhibited both

activities. The seemingly enhanced AHH activity at suboptimaldoses of SKF-525A is a common observation with some lipo-

philic inhibitors, which, according to Goujon era/. (18), mightbind to nonspecific membrane sites rendering them less available for the substrate benzo(a)pyrene; addition of benzo(a)-

pyrene then might allow more rapid hydroxylation.By and large, the properties of basal versus DBA-induced

AHH in the RPMI-1788 cells were found nearly identical. As tokinetics in cell culture, basal and induced hydroxylases dem-

l 2 4l/CBENZOfalPYRENEK/tir1)

0.1 0.2 0.3 0.4TOTAL ENZYME CONCENTRATION

Chart 6. A. Lineweaver-Burk plots of DBA-induced and uninduced AHH. Initial velocities of the uninduced and the DBA-induced AHH were obtained at variablebenzo(a)pyrene (BP) concentrations as indicated. The regression lines obtained by the method of least squares were fitted to the experimental points. Each point isa mean of duplicate determinations. 8, the dependence of Kâ€ž'son total enzyme concentration. The apparent Km and Vmaxvalues from 2 experiments with uninduced

cells (closed bars) and induced cells (open bars) were used to construct this chart. The apparent Vmaxwas normalized to take into account the number of cells (inmillions) used in each experiment. Thus, the total enzyme concentration (AHH activity) is the product of apparent Vmâ€ž,and the cell number (in millions).





Table 1Induction of AHH activity in RPMI-1788 lymphocytes by various compounds

Cell cultures were exposed for 24 hr to various concentrations of each compound, in duplicate, thenharvested and assayed for AHH activity as detailed in "Materials and Methods.

AHH activity (% ofcontrol)Inducer1

,2,3,4-DBA"1,2,5.6-DBA7,12-DMBAB(a)A3MCDDTSodium

cyclamateSodiumsaccharinLindaneCholecalciferoln-OctylaminePL-lsoproterenolMetyraponeChrysenePPOBenzidinePhenanthreneDESB1

70-EstradiolTestosteroneBenzo(a)pyreneBenzo(e)pyreneTCDD0.1

/IM3102501008515910093122128129100123104252978122115947530

nu1970.3

/IM30035082801671271279711111011495128020829096717560

nM2951

/IM2753005585160123110931111101141048101865836373820.3

/IM1401203

UM275250482401401301009397811149562202057525880633flM22011010/IM275303051271331278611010510972862029332768597030

/IM51080Optimal

inducerconcentration80.200.3010.000.6010.00Ietu'2.000.03

Interpolated as required and calculated only for time apparent optima yielding >33% increase overbasal activities.

6 1.2,3.4-DBA, dibenzanthracene; 1,2,5.6-DBA is dibenz(a./>)anthracene; DMBA, dimethylbenzanthra-

cene; B(a)A, benz(a)anthracene; 3MC, 3-methylcholanthrene; lindane, 1,2.3.4.5.6-hexachlorocyclohexane:DESB, diethylstilbestrol; TCDD. 2,3,7,8-tetrachlorodibenzo-p-dioxin.

'" Inducer concentration yielding double the basal activity (interpolated as required).

onstrated optimal synthesis in a common cell density range(0.6 to 1.0 x 106 cells/ml) as well as similar half-lives of

subsequent disappearance of the activity (46 and 41 hr, respectively). Moreover, basal and induced AHH exhibited similarpH optima (both 8.25), temperature optima (37-39Â°), apparent

activation energies (16.6, 16.7 kcal/mol), temperature dependence of denaturation (range, 42-50Â°), and apparent Km's

with benzo(a)pyrene (0.8, 1.8 jtM).Even the differences in Kmseemed not intrinsic but possibly

attributable to some unknown effects related to total enzymeconcentration in the incubation. It is common when studyingmulticomponent, membrane-bound enzymes that are not com

pletely purified (29) to find that the level of enzyme concentration somewhat affects the Km; this has, for example, beenattributed in the case of AHH to nonspecific membrane bindingof some substrate (35) and in the case of epoxide hydrase tocompetition with substrate by a lipid cofactor (29). Pending theresolution of technical difficulties in solubilization and purification of complex, membrane-bound enzymes like AHH, "apparent" enzymatic measurements are made from crude cell

homogenates or microsomal pellets (1, 16, 19, 35, 37).Other investigators have found differences between control

and polycyclic aromatic hydrocarbon-induced AHH, mainly in

liver and fetal cells. Thus, in comparison to the uninducedenzyme, the induced enzyme may (2, 3, 20) or may not (16)have a lower Km and exhibits different substrate (10) andinhibitor specificities (1, 18, 49) and a somewhat differentspectrum of metabolites from benzo(a)pyrene (41). InducedAHH also has different spectral (2, 28), immunological (36,

Table 2Inhibition of AHH activity in RPMI-1788 lymphocytes by various compounds

Suspensions of both control and DBA-induced cells were treated in duplicatewith each inhibitor (3 concentrations) and with 0.1 mM benzo(a)pyrene. Theywere then incubated and assayed for residual AHH activity under conditionsdetailed in "Materials and Methods." Each value is a mean of 2 or 3 determina

tions.

AHH activity as % of the activity of the uninduced or in

duced cells

Inhibitora-NFÂ°ÃŸ-NFMetyraponeSKF-525ATCPOCHOCelltypeUninduced

cellsInducedcellsUninduced





cellsInducedcellsIHM72657368103100118111109901109510*

MM1513686210392123116107958095103(iM3614613989761118296919996

NF, naphthoflavone; TCPO. 1,1,1-trichloropropene 2,3-oxide; CHO, cyclo-hexene oxide.

45), and electron paramagnetic resonance properties (36) thanthe truly constitutive enzyme. In contrast, few (if any) differences have been found, in our hands, between basal and theinduced AHH either in fresh cultured lymphocytes (22) or in

NOVEMBER 1979 4617




the RPMI-1788 cells (this report). We feel that this paradox is

explained by the culturing medium used. Because increasingenrichment of RPMI Medium 1640 with PCS progressivelyelevated basal but not polycyclic aromatic hydrocarbon-in

duced activity (14) and because the inhibitor sensitivities ofboth basal and induced AHH in the RPMI-1788 cells were akinto that for polycyclic aromatic hydrocarbon-induced P-448-linked AHH (this report), it may be concluded that factors inPCS induce AHH in supposedly "control" lymphocytes. This is

consistent with reported effects of PCS on fresh human lymphocytes (1, 21) and the reported presence of undialyzable,unknown inducers of AHH in the sera (17). Therefore, theallegedly basal AHH in uninduced cultured lymphocytes may,in reality, consist largely of induced, P-448-associated AHH.

Nevertheless, of greater significance for projected applications is the similarity in induced AHH between fresh and RPMI-1788 lymphocytes. AHH's from these 2 cell types have shown

significant similarities in enzymological properties: the inducedenzyme from RPMI-1788 cells and fresh lymphocytes, respec

tively, exhibited pH optima of 8.25 and 8.5 (19), apparent Kmvalues of 2 and 4 Â¡IM,and similar inhibition and inductionspecificities (22). The mean inducibility ratio with DBA for theRPMI-1788 cell AHH was 3.2 (14), whereas the range for the

fresh lymphocytes was 1.5 to 5 (21, 25, 40). Finally, DBAinduced AHH about twice as much as did 3-methylcholanthrenein the RPMI-1788 cells (see "Results") and in fresh lympho

cytes (19).Only 2 qualitative differences have been apparent between

the 2 cell types. For one, fresh lymphocytes had no detectableAHH without prior stimulation by phytohemagglutinin and poke-weed mitogen (7, 19, 51), whereas RPMI-1788 B-cells had

measurable AHH activity without prior mitogen exposure (14).For another, unlike fresh cells, the RPMI-1788 cells proliferatedrapidly, doubling daily with appropriate changes of medium(14). Admittedly, these differences might reflect adaptivechanges to long-term culture, as incorporation of the Epstein-Barr viral genome (31) and insensitivity to some B-cell (e.g.,

pokeweed) mitogens (14).Notwithstanding adaptations to establishment of culture, the

RPMI-1788 cell line is nonmalignant (31). This, plus the com

bined advantages of high AHH activity and inducibility in theabsence of mitogens, of unlimited availability, and of relativelyinvariant samples in the absence of donors, clearly renders thiscell line a suitable biochemical model for studies on microsomalmixed-function oxygenases and metabolically related enzymes.

Finally, the present studies lead to the important conclusionthat close similarities, shown here, between the enzymologicaland various other biological properties of the basal and theinduced AHH limit any epidemiological application of thesecharacteristics to identify the so-called high risk, high-AHH-

inducibility phenotype.

ACKNOWLEDGMENTS

The authors wish to thank Sandi Randazzo and Christina Turley for theirassistance in the preparation of this manuscript.

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1979;39:4612-4619. Cancer Res Harvey J. Freedman, Natalle B. Parker, Anthony J. Marinello, et al. Cell Line, RPMI-1788Hydrocarbon Hydroxylase in a Stable Human B-Lymphocyte Induction, Inhibition, and Biological Properties of Aryl

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