[CANCER RESEARCH 35. 1235 1243. May 1975]

Properties, Inducibility, and an Improved Method of Analysis of ArylHydrocarbon Hydroxylase in Cultured Human Lymphocytes1

Hira L. Gurtoo, Natalie Bejba, and Jun Minowada

Department of Experimental Therapeutics. Grace Cancer Drug Center [H. L. G.. N. B.\. and Departments of Molecular Biologv |/V. B.\ and Immunology \J. M.\, Rosweli Park Memorial Institute. New York State Department o/ Health. Buffalo. ,\'ew York 14203

SUMMARY

The properties and inducibility of aryl hydrocarbonhydroxylase (AHH) in cultured human lymphocytes werestudied, and a sensitive method of analysis has beendeveloped. In agreement with other reports, peripheralblood lymphocytes per se had no activity and requiredpretreatment in culture with a mitogen for conversion tolymphoblasts to possess AHH activity. This activity had anabsolute requirement for reduced nicotinamide adeninedinucleotide phosphate. Under our conditions of incubation,reduced nicotinamide adenine dinucleotide (1.3 mM) orethylenediaminetetraacetic acid disodium salt (1 mvi),when added to reduced nicotinamide adenine dinucleotidephosphate-fortified incubation mixture, caused about 30%increase in activity and MnCl2 caused an appreciableinhibition. The reaction was linear with the number of cells,with up to 16.8 x IO6 cells contained in the incubationmixture, and had a pH optimum of 8.5. The enzyme wasfairly stable at -70°and retained about 90% of the original

activity for 15 days.The enzyme activity in the mitogen-stimulated lympho

cyte cultures appeared slowly and reached a maximum at 48to 72 hr of incubation at 37°,after which it considerably

decayed with time. Viability of the cells in culture alsodecayed considerably after 72 hr and reached about 50%level at 120 hr. The enzyme was inducible with 3-methyl-cholanthrene and dibenz(a,h)anthracene. Dibenz(a,h)an-thracene was a much more potent inducer than 3-methyl-cholanthrene and evoked a response in cells that would beconsidered noninducible with 3-methylcholanthrene.

By using purified lymphocytes and Rosweli Park Memorial Institute Medium 1640, providing larger surface areaduring culture, harvesting the cells at the time of minimumcell death and maximum AHH activity, and assaying AHHat its pH optimum of 8.5 instead of 7.6, it was possible toenhance the sensitivity of the assay an average of 17-fold.

INTRODUCTION

MMFO3 is an enzyme complex that is located in the

1This investigation was supported in part by Core Program GrantCA-13038, Erie County United Fund of New York, and an Instituteallocation from Dr. G. M. Murphy. Director. Rosweli Park MemorialInstitute. Buffalo, New York 14203.

2To whom reprint requests should be addressed.

Received October 29, 1974; accepted January 27, 1975.3The abbreviations used are: MMFO. microsomal mixed-function

endoplasmic reticulum of the cell and is involved in themetabolism of drugs, carcinogens, steroids, insecticides, andvarious other compounds (5). MMFO occurs principally inthe liver but is also found in other mammalian tissues. It canbe induced in vivo and in cell culture by treatment withvarious drugs, carcinogens, and insecticides (5, 9, 19). Morethan 200 compounds can influence the activities of thisenzyme complex and these have been divided into 2 generalclasses (5): phénobarbitaltypes, which enhance the formation of cytochrome P-450 and the activities of MMFOtowards several substrates; and PAH types, exemplified byMC and BP, which enhance the activities of MMFOtowards a limited number of substrates and stimulate theformation of a different type of cytochrome, cytochromeP-448.

The metabolism of PAH's is mediated by a component

enzyme system of the MMFO complex, commonly referredto as AHH. This enzyme system can be induced by severalPAH's and occurs in many mammalian tissues including

liver, lung, intestinal mucosa, thyroid, testis, adrenal cortex(1, 22), placenta (23), and skin (17). Several human tissuesare known to contain this inducible enzyme (3, 4, 11, 15).Although lymphocytes contain very little of this enzyme,activity appears after transformation to lymphoblasts inculture in the presence of a mitogen (3, 24). Kellermann etal. ( 13) studied the genetics of AHH in a human populationand reported that the inducibility of this enzyme wascontrolled by a single gene locus with 2 codominant alíeles.On the basis of their data, individuals were classified ashaving low, intermediate, or high inducibility of AHH. In asubsequent report (14), these investigators reported that asignificant positive correlation existed between the extent ofinducibility and susceptibility to lung cancer. In thesestudies, inducibility was measured as the ratio of theinduced AHH level in lymphoblasts after 24-hr exposure toMC to the level found in control cultures that were exposedto the solvent only.

Because of the potential significance of this work ( 14), weundertook detailed studies of the characteristics and theinducibility of AHH in cultured lymphocytes. This reportdescribes the results of these studies as well as the development of a more sensitive procedure for assaying AHHactivity in cultured lymphocytes.

oxygenase: PAH, polycyclic aromatic hydrocarbon; MC. 3-methylcholanthrene: BP. benzo(o)pyrene; AHH, aryl hydrocarbon hydroxylase; DBA.diben/(a.h)anthracene: FCS, fetal calf serum; RPMI, Rosweli ParkMemorial Institute: 3-OH-BP. 3-hydroxyben/o(a)pyrene.

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H. L. üurtoo et al.

MATERIALS AND METHODS

Chemicals. BP and DBA (97%) were obtained fromAldrich Chemical Co., Milwaukee, Wis. BP was purified byrecryslalli/.ution from benzene and then by thin-layer chro-matography on silica gel plates (0.25 mm; E. Merck,Darmstadt, Germany) developed with benzene:hexane(1:15, v/v) solvent system. MC was obtained from EastmanOrganic Chemicals, Rochester, N. Y. Phytohemagglutininwas obtained from Difco Laboratories, Inc., Detroit, Mich.,and heat-inactivated PCS and pokeweed mitogen were theproducts of Grand Island Biological Company, GrandIsland, N. Y. RPMI Medium 1640 was prepared in thislaboratory. Penicillin G potassium and streptomycin sulfatewere obtained from Nutritional Biochemicals Corporation,Cleveland, Ohio. Hypaque sodium and Ficoll were suppliedby Winthrop Laboratories, New York, N. Y., and byPharmacia Fine Chemicals, Upssala, Sweden, respectively.Plastic culture flasks were obtained from Becton, Dickinsonand Company, Los Angeles, Calif. Fifteen-mi glass culture tubes were obtained from Kimble Products Division,Owens-Illinois Glass Company, Toledo, Ohio. Spectralgrade acetone and hexane were supplied by Aldrich Chemical Co., Milwaukee, Wis., and J. T. Baker Chemical Co.,Phillipsburg, N. J., respectively. NADPH and NADHwere the products of Sigma Chemical Co., St. Louis, Mo.,and Calbiochem, San Diego, Calif., respectively. Fluorescence measurements were made on the Aminco-Bowmanspectrofluorometer. 3-OH-BP was used as the referencestandard for the fluorescence determinations and was kindlyprovided as a gift by Dr. Harry Gelboin of the NationalCancer Institute.

Cell Culture. All cultures were prepared under sterileconditions and in all studies blood from apparently healthyvolunteers was used. No volunteer was currently on anymedication. In a typical assay, 15 to 20 ml of blood werewithdrawn aseptically in heparinized tubes and kept at roomtemperature, and within 3 hours the tlood was diluted with1 or 2 volumes of RPMI Medium 1640. Eight to 10 ml ofthe diluted blood were layered over a 3-ml column ofHypaque-Ficoll (specific gravity, 1.080) gradient (2) contained in each of the three 15-ml conical graduated centrifuge tubes. The tubes were centrifuged at 1400 rpm at roomtemperature for 30 min and the lymphocyte-rich interphasefraction, forming a white fluffy ring on the top of theHypaque-Ficoll column, was collected from several tubesand placed in a 35-ml conical centrifuge tube. Thirty ml ofRPMI Medium 1640 were then added, and the tube wascentrifuged at 1000 rpm for 5 min to sediment the lymphocytes free of platelets that remained in the supernatant. Thelymphocyte pellet was resuspended in 15 ml of RPMIMedium 1640 containing 10% heat-inactivated FCS andcentrifuged again at 1000 rpm for 5 min. The cell pelletcontained more than 95% lymphocytes, and the yield rangedfrom 30 to 40% of the total lymphocytes in blood. This yieldcould be substantially increased [57 ±15%;n = 6] (numberof individuals in the study), when the sedimented blood inthe Hypaque-Ficoll-containing tubes was pooled, diluted 1:1with RPMI Medium 1640, and resedimented on another set

of Hypaque-Ficoll columns. The purified, washed lymphocytes, obtained after single or double sedimentation of theblood, were then resuspended in 33 ml of RPMI Medium1640 containing 10% heat-inactivated FCS; penicillin, 100/tg/ml; streptomycin, 50 jug/ml: and phytohemagglutininand pokeweed mitogen; both of which were diluted 1:100with the medium. Eight ml of this suspension were thentransferred to each of the 4 Falcon T-30 (30 ml) flasks,unless otherwise stated. The flasks were incubated inhorizontal position (to provide a larger surface area) at 37°

for 48 hr in an atmosphere of 5% CO2 and 100% relativehumidity. At 48 hr, 5 n\ of MC or DBA in acetone, to a finalconcentration of 1 to 2.5 /uM,were added to each of the 2flasks (induced cultures), and to each of the other 2 flasks 5¿¿Iof acetone (control culture) were added. Incubation wascontinued for an additional 24-hr period, then the cells wereharvested by centrifugation, and, unless stated otherwise, analiquot was counted for viable lymphoblasts by the standardtrypan blue due exclusion procedure.

Lymphocyte yields were calculated from differential cellcounts and total white cell counts done on whole blood.Following a single sedimentation of blood on Hypaque-Ficoll gradients, lymphoblast yields for 6 individuals at theend of 72 hr culture ranged from 1.4 to 3.0 x IO6(mean ±S.D.; 2.01 ±0.66 x IO6) viable lymphoblasts/culturederived from 5 ml of blood. Following 2 sedimentationsdone sequentially, the lymphoblast yields for 6 otherindividuals ranged from 2.22 to 8 x IO6(mean ±S.D.; 4.48±2.36 x IO6)viable lymphoblasts/culture derived from 5ml blood. Usually a single sedimentation was used to purifythe lymphocytes from the blood.

In some experiments batch cultures were prepared asfollows. Lymphocytes were purified from a large batch ofblood (over 100 ml) by sedimentation on Hypaque-Ficoll,washed as before, and then cultured in various volumes ofmedium not exceeding 50 ml. The rest of the procedure wassimilar to that described above.

For comparison of the RPMI method with that reportedby Busbee et al. (3), 40 ml of blood were withdrawn from anindividual. From 20 ml, lymphocytes were isolated andcultured to give 4 cultures as described above; from theremaining 20 ml, the leukocytes were isolated and culturedby the method of Busbee et al. (3) to give 4 final cultures, 2induced and 2 control.

The DNA in some cultures was assayed by the colorimetrie method of Richards (20).

Enzyme Assay. As described above, 4 cultures, 2 inducedand 2 control, were derived from 15 to 20 ml of blood in theRPMI method. The cells from each culture flask wereharvested and assayed for AH H activity by the fluorometricprocedure described by Nebert and Gelboin (18) andmodified by Busbee et al. (3), which was further improved toobtain optimal activity. The 2 modifications included assayat pH 8.5, instead of at 7.6, and the use of higherconcentrations of NADPH and NADH. Briefly, the methodwas as follows. Cells were incubated in Tris (0.05 M):MgCl2(3 mM):sucrose (0.2 M) buffer mixture (total volume, 1 ml)containing NADPH (1.7 HIM)and NADH (1.3 HIM),unlessstated otherwise. The pH of the buffer was 8.5, unless stated

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AH H in Cultured Human Lymphocytes

otherwise. The reaction was started with the addition of BP(0.1 HIM)in 50 n\ acetone to each tube. The incubation wascarried out at 37°for 30 min, at the end of which thereaction, mixture in each tube was extracted with 4 ml ofhexane:acetone mixture (3:1). The organic phase was separated and extracted with 0.5 ml of l N NaOH which wasused for reading its fluorescence at an excitation wavelength of 396 nm and an emission wavelength of 522 nm.The fluorescence was quantitated by comparison with thestandard curve obtained with various dilutions of 3-OH-BP. Unless otherwise stated, activity is expressed as pmoleequivalents of 3-OH-BP formed per min per IO6 viablelymphoblasts or per culture. The fluorometer was calibrated with a quinine sulfate standard before each run and"minus-cell" blanks and "zero-time-plus-cell" blanks were

run as references. The results reported in this paper wereobtained after subtracting the values for minus-cell blanks,which were treated under conditions identical with the control incubations but lacked cells. Zero-time-plus-cellblanks showed no activity; the reaction in these incubationswas stopped immediately after the addition of BP.

RESULTS

Viability of Lymphocytes during Culture. In the studiesdescribed by Kellermann et al. (13, 14), leukocytes werecultured for 72 hr, exposed to the inducer or the solvent foran additional 24 hr, and assayed for AHH. For determination of the effect of time in culture on the viability oflymphoblasts, 9 samples of blood from different subjectswere studied. The results showed that at 72 hr in culture asignificant percentage of cells, 78 ±4% (mean ±S.D.),were still viable. At 96 and 120 hr only 61 ±7 and 50 ±11%, respectively, of the cells were viable. In 10 othersubjects tested, the number of viable lymphoblasts wasessentially identical in cultures with or without the addedinducer, MC. Viability determination by the dye exclusiontest did not provide a useful evaluation of the functionalstatus of these cells with respect to AHH activity. Althoughby 96 hr about 60% of the cells were still viable, aconsiderable decrease in the specific activity of AHH after72 hr in culture was observed, as is described below.

Cell counts, DNA contents, and AHH activities ofduplicate cultures, either induced or uninduced, were almostalways within a 10% variation. Cell counts at the end of a24-hr induction period were within 15% of the countsobtained on cultures at the time the inducer or acetone wasadded.

AHH Activity during Culture. Blood from 9 subjects,different from the ones used above, was used in thesestudies. Cultured lymphocytes were harvested at the timepoints indicated in Table 1 and then assayed for AHH in abuffer mixture of pH 7.6.

On Day 0, i.e., on the day of purification of lymphocytesfrom blood, no detectable AHH activity was found, especially during the 1st few hr in culture. The activity appearedslowly and reached a maximum at 48 to 72 hr in culture,after which it generally dropped precipitously (Table 1). Ingeneral, the kinetics of enzyme activity was similar wheninduced cultures that had received MC or DBA 24 hr prior

Table I

Kinetics of AHH activity in lymphocytes cultured in the presence orabsence of an inducer

Lymphocytes were prepared from 40 to 60 ml blood obtained from eachindividual. For 3 time points 12 cultures, in 15-mlglass culture tubes, wereprepared. Six cultures received 5 f¡\of acetone in each, and each of theother 6 received MC (2.5 n\i) or DBA (2.5 ¿iM)in 5 n\ acetone. Inducer oracetone was added 24 hr prior to harvest. Harvested cells were assayed forAHH activity in Tris:MgCI2: sucrose buffer mixture (pH 7.6) containingBP (0.1 mM)and NADPH (1.7 mM).The cell number in the cultures rangedfrom 1to 6 x 10scells. Each value is the mean of duplicate determinations.

AHH activity(pmoles 3-OH-BP formed/10' cells/min)

Subject123456789CultureControl

cellsDBA cellsRatioControl

cellsDBA cellsRatioControl

cellsDBA cells

RatioControl

cellsDBA cellsRatioControl

cellsMC cellsRatiosControl

cellsMC cellsRatioControl

cellsMC cellsRatioControl

cellsMC cellsRatioControl

cellsMC cellsRatio48°0.137

0.3622.6000.129

0.4003.1000.054

0.1923.5000.029

0.1505.2000.021

0.0291.4000.037

0.0671.8000.035

0.0401.1000.030

0.0652.2000.0

II0.008

0.70072°0.183

0.6333.4000.121

0.8296.8000.100

0.1701.7000.100

0.4004.0000.027

0.0331.2000.027

0.0542.0000.071

0.1922.7000.025

0.0873.5000.029

0.0632.20096°0.070

0.4967.0000.037

0.2637.0000.054

0.3175.9000.071

0.3214.5000.017

0.0211.2000.004

0.0215.0000.012

0.0453.7000.008

0.0253.1000.012

0.0625.200

" Number of hr in culture.

to harvest were tested. Maximum response to the inducergenerally occurred when cells in culture were exposed to theinducer at 48 hr and harvested at 72 hr, although someexceptions were noted. The results suggested that "resting"

lymphocytes have little AHH activity and, in order for theenzyme activity to appear, these cells must undergo blas-togenesis in the presence of a mitogen. These observationsagree with earlier reports (24).

In Table 1 it can also be seen that the ratio of the inducedto control value depended on the number of days cells werein culture. In general, DBA was found to be a better inducerthan MC.

Requirements and Kinetics of the AHH Reaction. Asshown in Table 2, the enzyme system had an absolute

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H. L. Gurtoo et al.

Table 2Requirements for AH H activity

Lymphocytes were purified from 60 ml of blood and then cultured eitherin batch cultures or in separate flasks, in the presence of the inducer MC.Details have been described in the text and in the legend of Chart 1. Thecultured cells in different flasks were harvested, pooled, and suspended in asmall volume of 0.25 M sucrose, 100-^1 aliquots of which were assayedfor AHH activity at pH 8.5. as shown. Details of the assay have beendescribed in the text. Bach value is the mean of duplicate determinations,and the results of a typical experiment are given below.

Conditions of incubation

3-OH-BP formed(pmoles/O.I ml cell suspension/min)

Complete"+ NADH (1.3 HIM)+ EDTA(I PIM)-NADPH

1.061.611.610.00

" Incubation mixture contained Tris:MgCl2:sucrose buffer. 0.1 ml ofpooled cell suspension. NADPH (1.7 mM), and BP (0.1 mM).

requirement for NADPH. Synergism of drug oxidation byNADH in the presence of NADPH is known (6). Indifferent experiments, the addition of NADH (1.3 mM) tothe incubation mixture enhanced AHH activity by about30%. Activity was decreased by 70 to 90% when NADH (2.6mM) was substituted for NADPH. EDTA (1 mM), whichhas been reported to inhibit AHH activity in hamster fetalcell cultures by about 30% (18), stimulated AHH activityin cultured human lymphocytes by about 30%.

In different experiments, carried out either at pH 7.6 or8.5, the reaction generally occurred with constant velocityonly for 30 min of incubation. Because of this and the factthat the total amount of the product formed never exceeded0.4% of the total substrate concentration, a condition underwhich reactions are considered linear (7), a 30-min incubation period was used in all assays. Other investigators (3, 13,14) have used a similar period of incubation for assayingAHH in cultured human lymphocytes.

Effect of Acetone and Freezing on AHH Activity. Acetonewas used as a solvent for BP and the required concentrationof BP was dissolved in 50 ^1 of acetone. Because of thepossibility that acetone, by altering the microsomal membrane lipids, might alter the AHH activity, it was considered to be necessary to test the effect of various acetoneconcentrations in the assay medium on AHH activity. In 3different experiments, it was found that AHH activity in thepresence of 10, 30, or 50 n\ acetone was essentially similar;however, it was reduced by about 10 to 20% when 70 ¿/Iofacetone were used as the solvent for BP.

Recent evidence has shown that MnCl2 prevents thedegradation of cytochrome P-450 by blocking NADPH-mediated lipid peroxidation of the microsomal membranes(16, 21). At low concentrations (2 mM and lower), MnCl2has also been shown to cause a stimulation of AHH inhamster fetal culture cells (18). We studied the effect ofMnCl2 on AHH activity in cultured human lymphocytesand the results are shown in Chart 1. MnCl2 did notstimulate the reaction but instead produced a substantialinhibition (Chart 1) at the various levels tested.

Chart 2 shows the dependence of AHH activity on thenumber of control and induced cells in the reaction mixture.

Linearity of this reaction was observed up to 16.8 x IO6cells, and this was the same for both the MC-induced andcontrol cells.

Freezing cell pellets at -20° resulted in a substantial loss

of activity. This was prevented to a large extent when cellpellets were frozen at -70°. At this temperature, the

enzyme remained fairly stable for 15 days and retainedabout 90% of the original activity.

Effect of pH on AHH Activity. The pH optimum forAHH activity in hamster fetal cell cultures has beenreported to be about 7.5 (18), and most assays of thisenzyme in animal liver preparations have been performed atapproximately this pH (10). Similar pH has also been used

Chart 1. Effect of MnCl2on AHH activity. Lymphocytes were purifiedfrom 100 ml of blood obtained in 20-ml portions from 5 normal subjects.Lymphocytes were purified from each portion of blood separately. Thepurified lymphocytes were cultured in several flasks in such a way that eachflask had lymphocytes from 5 ml blood and 4 flasks were derived from eachindividual. Each culture received 5 n\ MC (2.5 JIM)24 h prior to harvestThe harvested cells were pooled and suspended in a small volume of 0.25 Msucrose. 100-^1aliquots of which were assayed in duplicate for AHH in thepresence of different concentrations of MnCI2. Assays were carried out atpH 8.5 as described in the text: points, means of duplicate determinations.

5.0

? 4.0

Tg 2.0

AÕ3^ LO

42 8.4

CELL NUMBER

12.6.10«

168

Chart 2. Relationship between AHH activity and cell number. Cellswere prepared in batch cultures as described in the text. MC-inducedcultures received MC (1.5 MM)24 hr prior to harvest, whereas controlcultures received acetone alone. Harvested cells were suspended in a smallvolume of 0.25 Msucrose and different aliquots of these suspensions wereassayed for AHH activity at pH 8.5.

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A HH in Cultured Human Lymphocytes

to assay AHH activity in human lymphocyte cultures (3, 13,14). Chart 3 shows the pH profile of AHH activity incultured human lymphocytes. Both the uninduced andMC-induced cells have an optimal pH at 8.5. In differentexperiments, the increase in activity at pH 8.5 in comparison to pH 7.5 ranged from 200 to 400%.

Dose-Response Relationship between the Inducers andAHH Activity. The effects of different concentrations of theinducer, MC or DBA, on the inducibility of AHH incultured lymphocytes were studied in 2 subjects. The resultsof these studies are shown in Chart 4. In the subjects tested,concentration of either inducer for optimal induction rangedfrom I to 3 /IM; however, the concentration of MC abovethis level was slightly toxic as indicated by a slight drop inactivity in the subject designated MC #4. Although thesamples from 2 subjects tested differed about 6-fold in theirbasal AHH activity, the enzyme in samples obtained fromeither subject was inducible at all concentrations of DBA.On the other hand, enzyme in samples from the subjectdesignated MC #4 was inducible with MC, whereas in theother subject, MC #3, a small decrease in AHH activity wasobserved at most of the concentrations of MC. Also, it wasobserved that DBA was a much more potent inducer thanMC was and evoked a response in cells that would beconsidered noninducible if tested with MC alone.

Kinetics of Inductionof AHH Activity. The data shown inChart 5 confirmed the previous observations (Chart 4) thatDBA was several times more potent than MC as an inducerof AHH activity in cultured human lymphocytes. While theoverall AHH activity in the uninduced (control) culture didnot appreciably change for 24 hr after the addition of 5 u\acetone (which were added at 48 hr in culture), there was a

Chart 3. Effect of pH on AHH activity. In each case, cells were derivedfrom 80 to 100 ml of blood obtained from several normal donors in 15- to20-ml portions. Lymphocytes were purified from each portion of bloodseparately and cultured as described in the legend of Chart 1.The cells wereharvested, pooled, and suspended in a small volume of 0.25 Msucrose, and100-/J aliquots of this suspension were assayed for AHH activity at eachpH in duplicate. Points, means of duplicate determinations. Controlcultures received acetone and MC-induced cultures, MC.I and MC.2(which were prepared from different batches of blood), received MC (2.5JIM)24 hr prior to harvest. The pH value refers to the pH of the buffer thatwas of adequate capacity to buffer any alterations in pH caused by NADHaddition.

10 30CONC OF MC ANO DBA lo.hl IN

Chart 4. Dose-response relationship between the concentration (cone.)of the inducer and AHH activity. For each subject, lymphocytes werepurified from blood and cultured in a batch culture as described in the text.At the end of 48 hr of culture. 5-ml portions from the flask were transferredto each of the several 15-ml culture tubes in such a way that for each point 4tubes were made, 2 receiving a variable concentration of MC and the other2 receiving a variable concentration of DBA. Two additional tubes, whichreceived acetone alone, served as controls. Twenty-four hr later, the cellswere harvested and assayed for AHH activity at pH 8.5. Point, mean ofduplicate determinations. In the case of the subjects designated MC #3 andMC #4, 2.9 x 10*and 3.0 x 10"cells, respectively, were used in each assay.

Note the scales on the 2 ordinales are different and represent the 2 subjectsemployed in this study.

DBA-INDUCED,

12 24 36HOURS IN CULTURE POST INDUCER

Chart 5. Kinetics of the induction of AHH during culture. Lymphocytes were purified from blood and cultured in batch cultures for 48 hr. Atotal of 6 batch cultures were made. At the end of 48 hr of incubation 2flasks received acetone alone, 2 received MC (1.5 ^M), and the other 2received DBA (1.5 /xM). Thereafter. 5-ml portions, each containingapproximately 3 x 10*cells, from each flask were removed at the indicated

time points and centrifuged to obtain the cells which were assayed forAHH at pH 8.5. Point, mean of duplicate determinations.

considerable loss of activity beyond 24 hr, and at 36 hrabout 65% of the basal AHH activity was lost. In thepresence of either inducer, AHH activity in these culturesincreased with time, and optimal activity was reached at 30hr after the addition of the inducer and thereafter activitydecreased. DBA-induced cultures showed higher en/ymaticactivity than MC-induced cultures did at all time periods

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H. L. Gurtoo et al.

tested. Since basal activity in control cultures remainedessentially unchanged only for 24 hr after the addition of thesolvent and since appreciable induction was obtained at 24hr after addition of the inducer, the induction period in allstudies was terminated at 24 hr.

Comparison between the KI'M I Method and the Method

of Busbee et al. (3) for the Measurement of AHH Activity.Since in studies on the genetics of AHH in normal peopleand lung cancer patients Kellermann et al. (13, 14) used themethod of Busbee et al. (3) for measuring AHH activity incultured leukocytes, we compared the method of Busbee et

al. (3) with the RPMI method in order to determine thesensitivity of the RPMI method relative to the method ofBusbee et al. (3). The results shown in Table 3 indicated thatAHH activity, both the total activity per 30 min per culturederived from 5 ml blood and the specific activity (activityper IO6cells per min), obtained by the RPMI method wasmany times higher than that obtained by the method ofBusbee et al. (3). This was true for both the control and theinduced activities. The control activity was 17 times higherby the RPMI method, although the increase for individualsamples ranged from 10- to 60-fold higher. The average

Table 3Comparison between the RPMI method and ¡hemethod of Busbee et al. (3) for the measurement of AHH activity in cultured

lymphocytesForty ml blood were withdrawn from each individual and split into two 20-ml portions. Lymphocytes purified from 1 portion were

cultured and assayed at pH 8.5, as described in the text. The other portion was used for the preparations of leukocytes which werecultured and assayed by the method of Busbee et al. (3). Four final cultures, each culture derived from 5 ml of blood, were used ineach method for each individual. Two of these received MC ( 1.5 ^M) in each and Ihe other 2 each received acetone 24 hr prior toharvest. Each value is the mean of duplicate determinations. Average cell count of 4 cultures derived from a 20-ml portion of bloodwas used to calculate the specific activity. We have found this method more accurate than using cell counts that have been done onceonly on a culture. Furthermore, preliminary studies showed that cell numbers and DNA contents vary within 10% in 4 culturesderived from 20 ml blood and cultured side by side.

3-OH-BP formed (pmoles)

Method of Busbee et al. RPMI method

Subject123456789101112"

Blood" BloodCultureControl

MCControl

MCControl

MCControl

MCControl

MCControl

MCControl

MCControl

MCControl

MCControl

MCControl

MCControl

MCsedimented

sedimentedTotal

activity/ Specific activity/culture/30 min IO6cells/min2.90

4.381.32

2.203.11

4.353.85

4.263.00

8.0012.522.64.52

5.3819.9

21.06.27

6.164.33

3.75once

on Hypaque-Ficolltwice on Hypaque-Ficoll0.018

0.0270.006

0.0100.093

0.1320.042

0.0470.060

0.1670.119

0.2150.049

0.0570.247

0.2600.192

0.1880.071

0.062Induced:

control1.511.671.401.102.671.811.191.051.000.87to

purify lymphocytes for thisto purify the lymphocytes forTotal

activity/culture/30min74.7

104.776.2

141.565.6

145.394.1

167.625.9

37.1112.1174.770.6

236.1183.3

395.861.1

65.679.2

137.2130.0247.8107.2

163.7method,

this method.Specific

activity/10«cells/min1.51

2.111.64

3.041.43

1.671.352.410.90

1.001.35

2.100.36

1.210.74

1.590.92

0.980.83

1.431.21

2.311.15

1.76Induced:

control1.41"1.86"2.21"1.78°1.43°1.56°3.34"2.16s1.07"1.73"1.90"1.52"

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AH H in Cultured Human Lymphocytes

control activity per culture per 30 min was 75 ±29 (mean ±S.D.) pmoles of 3-OH-BP formed in 6 individuals whoselymphocytes were purified by a single sedimentation ofblood on the Hypaque-Ficoll gradients. This is comparedwith a value of 105 ±46 in 6 other individuals whoselymphocytes were purified by sedimentation of the bloodtwice on the Hypaque-Ficoll gradients. On the other hand,an average value of 6 ±6 pmoles of 3-OH-BP formed percontrol culture per 30 min for 10 individuals was obtainedby the method of Busbee et al. (3). The specific activities indifferent samples (see Table 3) obtained by the method ofBusbee et al. compare well with those reported by Keller-mann et al. (12).

Based on the extent of AH H inducibility in culturedlymphocytes, i.e., the ratio between the induced and thecontrol level of AHH, Kellermann et al. (12, 13) haveclassified humans into 3 classes: (a) low with a ratio up to2.70; (b) intermediate with a ratio up to 3.6; and (c) highwith a ratio greater than 3.6. It can be seen from Table 3that 9 of 10 subjects could be classified as low by eithermethod. However, one individual (Subject 7) with a ratio of3.3 by the RPMI method would be classified as intermediate, but the same individual falls in the low inducibilitygroup when assayed by the other procedure. The reason forthis discrepancy is not apparent to us.

Studies on Stable Lymphocyte Cell Lines. Because of thedifficulties associated with obtaining large qunatities ofblood for various studies on AHH, it would be very useful tohave stable cell lines with significant AHH activity thatcould be used in studies on methodology, on the metabolismof PAH's, and also as standard references for comparison

and standardization. We have tested 10 such cell lines forAHH activity and the results are shown in Table 4. The dataindicated that at least 3 cell lines, SN1006, AN-8, andRPMI-1788, had significant activity that could make themuseful in future studies.

DISCUSSION

The characteristics of AHH in cultured human lymphocytes described here and those reported by Whitlock et al.(24) suggest that this activity belongs to the class of enzymescommonly referred to as MMFO's that utilize a hemo-protein, cytochrome (P-450 or P-448), for catalysis of theenzymatic activity. Various factors and conditions influencing the activity of this enzyme in cultured lymphocytes werestudied. These included cofactor and manganese requirements; culture conditions; pH optimum; and the effects ofsolvent, freezing, and inducers.

The results indicate that an appreciable basal AHHactivity can be measured in mitogen-stimulated culturedhuman lymphocytes derived from as little as 5 ml of blood,provided the cells are cultured under optimal conditionsand assayed at the stage of optimal AHH activity andminimum cell death using an optimal pH of 8.5 instead of7.6. In the method described by Busbee et al. (3) and usedby Kellermann et al. (13, 14) in studies on the geneticsof AHH in normal people and lung cancer patients, cul-

Table 4A HH activity in stählelymphocyte cell lines and its response to induction

Stable lymphocyte cell lines obtained from the cell bank of RPMI wereprepared in batch cultures as described in the text. Three cultures for eachcell line were prepared; 24 hr prior to harvest I culture received acetone, Ireceived MC (1.5 /IM),and I received DBA (1.5 ¿iM).Each culture was splitinto 2 equal portions, and cells were harvested from each portion andassayed at pH 8.5 as described in the text. The number of cells perincubation ranged from 4 to 25 x 10* cells. Each value is the mean ofduplicate determinations.

CelllineMOLT-3CCRF-HSB-2B4I1-4SNI006B-21AL-1OgunAN-8RPM1-1788B-23-2SampleControl

MCDBAControl

MCDBAControl

MCDBAControl

MCDBAControl

MCDBAControl

MCDBAControl

MCDBAControl

MCDBAControl

MCDBAControl

MCDBAActivity/

IO6cells/min"<

0.004< 0.004< 0.0040.056

0.0310.0600.014

0.0070.0210.067

0.0570.087<0.004

< 0.004< 0.0040.004

< 0.0040.0040.005

<0.0040.0050.088

0.0670.1470.079

0.0600.1250.031

0.0630.036

1Activity as equivalent of pmoles of 3-OH-BP formed per IO6cells per

tures were induced at 72 hr and harvested at 96 hr. Although no data are available on the kinetics of AHHactivity in the cells cultured under these conditions, itappears that the low specific activity of the cells studiedwas probably due to an excessive number of degenerating cells that may still be counted as viable on a dyeexclusion basis. When the RPMI method of AHH analysis of cultured lymphocytes was compared with the methodof Busbee et al. (3), there was a marked elevation (average, 17-fold) in the enzyme activity of lymphocytes derived from 5 ml of blood. Similar results were obtained whenthe 2 methods were compared on the basis of specific AHHactivity. The enhancement in assay sensitivity has signifi-

MAY 1975 1241

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H. L. Guríooet al.

canee with regard to the accurate analysis of basal AHHlevels in cultured human lymphocytes. According to Keller-mann et al. (13, 14) it is the fold inducibility, i.e., the ratio ofinduced to basal activity, that is important in the determi-tion of susceptibility to lung cancer or in the assignment ofindividuals to various AHH-genetic groups. Any error inthe measurement of the basal activity would be reflected inthe alteration of the inducibility ratio and thus would resultin an improper assignment of individuals to various AHHinducibility groups. Kellermann et al. (14) have reportedthat individuals in high- and intermediate-AHH-inducibilitygroups have a 16- and 10-fold higher risk of getting lungcancer than those in the low-inducibility group. By enhancing the assay sensitivity, as done in the RPMI method, theerror associated with the measurement of basal levels isconsiderably minimized.

AHH activity measured in hamster fetal cell cultures hasbeen reported to have a pH optimum of 7.5 (18), whereas inthe cultured human lymphocytes the optimal pH was 8.5which was the same for both the noninduced and theMC-induced cells.

The studies presented here are in accord with thosereported by Whitlock et al. (24) in showing that restinglymphocytes have practically no AHH activity and that thisactivity is acquired on transformation of these cells tolymphoblasts by pretreatment in culture with a mitogen.This means that the lymphocytes, which constitute arelatively readily available human tissue, have the geneticpotential for expressing AHH activity in vitro undersuitable culture conditions. This makes them well suited forstudies on human MMFO's since similar studies in vivo are

not possible and are influenced by age, sex, nutritional,hormonal, and diurnal variations (5) and by exposure tocarcinogens (8), pharmacologically active drugs, and insecticides (5). The capacity to induce the enzyme with variousinducers, the individual variability in the extent of thisinducibility, the absence of enzyme activity in restinglymphocytes and individual variability in the response tomitogen activation in culture, and the sensitivity of theAHH assay as described in this paper offer advantages forstudying the genetics and mechanism of induction ofmicrosomal enzymes and the capacity for the metabolism ofdrugs, steroids, and carcinogenic compounds in humans.

ACKNOWLEDGMENTS

We are very grateful to Dr. G. M. Murphy, Director of RPMI, for hisinterest in this work and for providing "seed money" to support it initially.

We also thank Dr. E. Mihich and Dr. K. Paigen for their support andinterest and for providing valuable criticism during the preparation of thismanuscript. Our thanks are also due to Dr. B. Paigen and Dr. C. Dave fortheir criticism of this manuscript. We are also very grateful to Dr. E.Cohen for providing us with blood samples. Finally, we wish to acknowledge the skilled assistance of Nancy Hayner in the cell culture work.

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AH H in Cultured Human Lymphocytes

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1975;35:1235-1243. Cancer Res   Hira L. Gurtoo, Natalie Bejba and Jun Minowada 

LymphocytesAryl Hydrocarbon Hydroxylase in Cultured Human Properties, Inducibility, and an Improved Method of Analysis of

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