ARYL HYDROCARBON HYDROXYLASE INDUCIBILITY A N D LY MPHOBLAST FORMATION IN LUNG CANCER PATIENTS

R. PRASAD l, N. PRASAD ' s 3 , J. E. HARRELL 1 , J. THORNBY 3, J . H. LIEM 3, P . T. HuDGlNS L, and J . TWAW Departments of ' ObstetrirJ & Gynecology ctnrl Radiology, Baylor College of Medicine, Hoiistoti. Texas 77030: and I/. A. Medicul Center, Houston, Texas 77211. USA

Aryl hydrocarbon hydroxylase ( A H H) inducibi- lity and lymphoblast formation were studied in lymphocytes from healthy control subjects and from lung cancer patients undergoing radiotherapy. The relationship between A H H inducibility and percentage lymphoblasts was statistically significant only for the pre-treatment patients (r = 0.598; p<0.05). In the control group and in patients undergoing radiotherapy the correlation between A H H inducibility and lyrnphoblast formation was positive but statistically it was not significant. Our data do not suggest a linear relationship bet- ween A H H inducibility and lymphoblart formation.

Aryl hydrocarbon hydroxylase (AHH) is a inicrosomal enzyme which metabolizes the polycyclic hydrocarbons. AHH activity can be induced in animals by several aromatic hydrocarbons (Nebert and Gelboin, 1969; Kouri et al., 1973; Benedict et al., 1973), as well as in human peripheral blood lymphocytes grown in culture in the presence of mitogens (Whitlock et al., 1972; Busbee et ul., 1972). In recent years speculation that the level of AHH inducibility in lymphoblasts is linked to susceptibility to lung cancer has stimulated a considerable amount of research on this enzyme (Bast et a/ . , 1976; Gurtoo etal. , 1975; Atlas et al., 1976; Paigen, et al., 1977a,b; McLemore et al., 1978). It has been rzported that AHH inducibility is directly proportional to the number of lymphoblasts in the culture (Kellermann rt at., 1Y73). This observation in turn led some investigators to question the relevance of the AHH inducibility test in lymphoblasts (Rao, 1974).

In order to further define the relationship between the AHH test and cancer we studied A H H in- ducibility in lymphocytes of lung cancer patients prior to any therapy, during and following radiation therapy. The AHH induction ratio and the percent- age of lymphoblast formation in cancer patients and in normal healthy controls were compared. We found a relationship between the AHH induction ratio and the number of lymphoblasts, but the relationship was not a linear one.

MATERIAL A N D METHODS

Thirteen patients with carcinoma of the lung, histologically confirmed, were included in this study. These patients were Caucasian males, ranging in age from 49 to 60 years, with no history of radio- therapy or chemotherapy prior to testing. All the lung cancer patients were routinely treated to anterior and posterior lung fields using E°Co unit. The typical radiotherapy treatment for lung cancer patients

consistcd of a total tumor dose of 4,050 rad given in a split course of 18 treatments as follows: ( I ) nine treatments, four treatments per week with an average tumor dose of 225 rad per treatment; (2 ) 2 weeks of rest period without treatment, and (3) nine treat- ments, four treatments per week with an average tumor dose of 225 rad per treatment. Lung cancer patients' blood was collected a t three separate times: prior to treatment; after the 9th treatment (2,025 rad cumulative); and after the 18th treatment (4,050 rad cumulative).

A control group of eight healthy volunteers was also included in the study. The healthy controls were of the same sex and race, were not on any medication, and were mainly hospital and college personnel. Blood was drawn only once from the control volun- teers.

After overnight fasting, peripheral blood was obtained from both the lung cancer patients and healthy volunteers. Heparin (30 unit/ml) was used as a n anticoagulant and blood samples were processed within 1 h of venesection.

Lymphocytes were isolated by Ficoll-Hypaque (specific gravity of 1.077) gradient method. Hepari- nized blood was diluted I :2 with phosphate-buffer- ed saline (PBS). t,'orty i i i l of diluted blood were layered over 10-ml aliquots of the Ficoll-Hypaque solution in 50-ml glass centrifuge tubes. The tubes were centrifuged a t 400 g for 40 min a t room teni- perature. The white buffy ring of lymphocyte-rich interphase fraction formed on the interface between the plasma and Ficoll-Hypaque column was collected in chilled 50-ml plastic centrifuge tubes containing 7.5 ml of PBS (Caz+ and Mg2+ free). The tubes were centrifuged (500 g ) for 10 min. The sedimented lymphocytes were washed twice with PBS and finally the cell pellet was re-suspended in 10 ml of F-13 medium. An aliquot of cell suspension was diluted to 0.07% with trypan blue solution and viable cells were counted in a hemocytometer. More than 95 % of the cells were lymphocytes and excluded trypan blue.

The lymphocyte suspension was then reconstituted with F-13 medium to a concentration of 0.5 Y: 10" cells/ml. Twenty ml of this suspension were then transferred to each of the four Falcon T-30 flasks.

Reprint requests should be addrcsscd to: Ilr. N. Prasad, Department of Radiology, Baylor College of Medicine, Houston, Texas 77030, LISA.

Received: September 1 1 , 1978 and in revised form December 22, 1978.

TA

BL

E I

AR

YL

HY

DR

OC

AR

BO

N H

YD

RO

XY

LA

SE (

AH

H)

IND

UC

IBIL

ITY

AN

D L

YM

PHO

BL

AST

FO

RM

AT

ION

IN

HE

AL

TH

Y C

ON

TR

OL

AN

D I

N L

UN

G

CA

NC

ER

PA

TIE

NT

S U

ND

ER

GO

ING

RA

DIO

TH

ER

APY

~~

~ ~

~~

Ary

l hy

droc

arbo

n hy

drox

ylas

e L

ymph

obla

st (%

)

0 2,

025

Stud

y G

roup

0

rad

2.02

5 ra

d 4,

050

rad

Indu

. (r

ad)

(Cad

) Ij

B '

Indu

. I/

B

Indu

. I/

B

4,05

0 (r

ad)

Patie

nt

1 2 3 4 5 6 7 8 9

0.46

9/0.

241

0.10

7/0.

067

1.20

0/0.

114

0.05

4/0.

007

0.06

4/0.

047

0.09

0/0.

044

0.95

4/0.

142

0.01

7/0.

006

0.70

0/0.

340

Con

trol

15

0.62

9/0.

292

16

0.52

6/0.

3 1 8

17

0.1

57/0

.118

18

0.16

6/0.

080

19

0.13

1/0.

102

20

0.52

2/0.

5 1 3

21

0.03

6/0.

018

22

0.1 51

/O. 1

3 1

1.9

1.6

10.6

8.0

1.4

2.1

6.7

2.9

2.1

2.2

1.8

1.3

2.1

1.3

1 .o

2.0

1.2

0.12

4/0.

097

0.18

4/0.

103

0.39

O/0

.150

0.25

1/0.

090

0.03

5/0.

023

0.51

0/0.

3 10

0.31

5/0.

131

0.02

2/0.

018

0.02

8/0.

023

I .3

1.8

2.6

2.8 1.5

1.7

2.4

1.2

1.2

0.05

7/0.

030

0.17

7/0.

091

0.08

0/0.

080

0.12

5/0.

045

0.33

3/0.

295

0.06

7/0.

064

0.08

1 /0.

041

0.12

1 /0.

094

0.1

17/0

. I07

1.9

1.9

1 .o

2.8

1.2

1.1

1.9

1.2

1.1

25

23

43

51

28

26

55

36

48

56

39

33

41

47

26

62

52

31

32

24

36

16

34

24

28

19

14

29

30

44

22

31

31

30

26

Patie

nts (

n =

13)

-

3.77

13.2

-

1.83

i~0

.6 '

-

1.57

10.6

37

.211

0.8

26.3

d~6.

8 28

.6*8

.0

Con

trol

(n =

8)

-

1.63

~0.

45 I

-

-

-

44.5

1 12

.1

-

-

p. z ci f n

I/B

, Ind

uced

/Bas

al; I

ndu,

Ind

ucib

ility

. - *

Val

ues w

ere o

btai

ned

for

13 pa

tient

s pr

ior

to r

adia

tion

the

rapy

and

for 9

of

I3 p

atie

nts

afte

r 2,

025

and

4,05

0 ra

d of

rad

iati

on (

cum

ulat

ive)

. - a

Mar

gina

l di

ffer

ence

3

be

twee

n pa

tient

and

con

trol

s (p

<O.lO

). - 'S

igni

fica

nt c

hang

e fr

om p

re-t

reat

men

t (p

<O.O

5). - j

Mar

gina

l cha

nge

from

pre

trea

tmen

t (p

t0.1

0).

4

318 PRASAD ET AL.

The F-13 medium contained 20 % heat-inactivated fetal calf serum, antibiotics, 60 units/ml of heparin, and 3 % (v/v) phytohemagglutinin-M (all from Grand Island Biological Co., Grand Island, N. Y.). The flasks were incubated in a horizontal position at 37" C in a water-saturated atmosphere of 95% air and 5 % CO,. For all of the analyses we used the same lot of PHA, but two different lots of serum.

3-Methylcholanthrene (MC) was used to induce AHH activity. MC was bought from Eastmann Kodak Co., Rochester, N. Y., and recrystallized from ethanol. At 72 h of culture, 1 pl of 1.5 mM of MC in acetone per ml of medium was added to one (or more) of the culture flasks (induced culture), and lpl of acetone per ml of medium was added to another flask (control culture). The cells were incubated for an additional 24-h period. At harvest, a portion of the cell culture was used for viable cell count, which was performed by trypan blue dye exclusion tech- nique on a hemacytometer. The lymphoblast and total cell count were obtained using the Coulter Counter (Model ZBI). The rest of the cell suspension was centrifuged at 500 g for 10 min. The sedimented cells were then washed twice in PBS and resuspended in TM buffer, PH 8.0, to obtain a concentration of 1.8 x lo6 cells per 0.9 ml which was used for AHH assay.

AHH activity was measured by the fluorometric procedure described by Nebert and Gelboin (1968) and modified by Kellermann et al. (1973). In brief, the method was as follows: an aliquot of 0.9 ml of cell suspension (1.8 x lo6 cells per 0.9 ml TM buffer) was taken into each of the duplicate or triplicate tubes. To these cell suspensions, a 100-pl solution con- taining 0.7 mg each of NADH and NADPH (Sigma Chemical Co., St. Louis, Mo.)was added. Thereaction was started by the addition of 0.1 ,UM benzo(a)pyrene (BP) in 50 pl of acetone to each tube. The cells in the reaction mixture were incubated for 30 min at 37" C in a shaker bath. The reaction was terminated by the addition of 3 ml of an acetone:hexane mixture (1:3 ratio). The upper organic layer was then extracted with 0.5 ml of 1 N NaOH. The alkaline extract was transferred to a quartz micro- cuvet (0.9 ml capacity) and fluorescence was measured in an Aminco-Bowman spectrophoto- fluorometer at the excitation wavelength of 396 nm and emission 522 nm. The fluorescence readings were quantitated by comparing with the standard curve of 3-OH-BP. One unit of AHH activity is expressed as pmole equivalents of 3-OH-BP formed per min per 1.8 x lo6 viable cells. AHH inducibility is the ratio of induced and basal AHH activity. Calibration of fluorometer was performed before each run with a quinine sulfate standard and " minus-cell " blanks were run as references. The blanks were treated identically under the same experimental conditions as the other assay tubes. The data reported were obtained after subtracting the values for these minus-cell blanks.

RESULTS

The AHH inducibility and lymphoblast formation in healthy control and lung cancer patients under- going radiotherapy are shown in Table I. Percentage

lymphoblast formation and AHH inducibility were obtained for eight control subjects and 13 cancer patients. Values were obtained for all patients prior to radiation therapy, and for nine of the 13 patients after the 9th and 18th radiation treatments. Com- parison of AHH inducibility prior to radiation therapy indicated higher AHH inducibility in lung

. @ PRE4ADIATIDN (0 RADSl .

4.0

@ AFTER 9th TREATMENT [CUMULATIVE 2025 RADS]

a 4.0

I 1 ' 0. *.- I

0.0 ' I 1 I I

i 8.0

@ AFTER 18th TREATMENT (CUMULATIVE 4050 RADS]

4 . 0 1 * I .* ; , , *.,

0.0 0 20 40 60 80

LYMPHOBLASTS I%] FIGURE 1 - Relationship between aryl hydrocarbon

hydroxylase (AHH) inducibility and lymphoblast for- mation in healthy control (x), and in lung cancer patients (0) : (a) prior to treatment; (b) after 9th treatment; and (c) after 18th treatment.

cancer patients than in the controls (pt0.10). All of the basal and induced AHH values for controls and most of the values for patients prior to radiation therapy represent averages of duplicate or triplicate determinations. Coefficients of variation were 0.25 for patients and 0.17 for controls. All of the lympho- blast data were obtained as the average of triplicate determinations. The coefficients of variation were 0.07 for patients and 0.03 for controls.

The patients evaluated after their 9th and 18th treatments had statistically significant decreases in AHH inducibility relative to their pre-radiation values. Similarly, patients had decreased values of percentage lymphoblast formation after the 9th

AHH AND LYMPHOBLAST IN LUNG CANCER 319

treatlnent (p <0.10) and after the 18th treatment (p <0.05). The relationship between AHH in- ducibility and percentage lymphoblasts is illustrated in Figure 1. The only statistically significant corre- lation coefficient for these two parameters was that for pre-treatmmt patients (r = 0.598; p<O.O5). In the control group and among patients undergoing radiation therapy, a positive correlation existed between AHH inducibility and lymphoblast forma- tion but this was not statistically significant (controls r = 0.399, p>0.05; patients after 9th: r = 0.399, p>O.O5; patients after 18th: r = 0.422, p>O.O5). This evidence does not indicate the existence of a linear relationship between percentage lymphoblast formation and AHH inducibility.

DISCUSSION

Reports o n lymphocyte response to PHA in lung cancer patients are conflicting. Recently Jett et al. (1978) have reported significantly lower lymphocyte transformation in lung cancer patients than in healthy controls. Similar findings have been reported by others (Ducos et al., 1970; Braeman and Deeley, 1975; Kerman and Stefani, 1977; Concannon et al., 1977; Rafla et al., 1978). We found no significant difference in the formation of lymphoblasts between hzalthy control subjects and cancer patients prior to radiation therapy. There were fewer subjects in our study than in the study reported by Jett et al. (1978). Also in our study lymphocyte transformation was evaluated by the morphology of the cells rather than by measuring the incorporation of [3H]thymidine. However, there are reports by others who did not find depressed lymphocyte transformation in lung cancer patients (Silk, 1967; Sutherland et al., 1971; Robinson et al., 1977; Barnes et al., 1975).

Our data indicate that lymphocyte transformation is significantly lowered in patients during radiation therapy. Such radiation-induced depression in lymphocyte transformation has been reported by others (Rafla et al., 1978; Braeman et al., 1974; Raben et al., 1976; Wara, 1977). It is speculated that radiation affects lymphocyte transformation

either by releasing some inhibitory factor into the patient’s serum, damaging the genetic mechanism of lymphocyte, or by destroying the greater pro- portion of the thymus-dependent (“ T ”-cell) lym- phocytes (Jenkins et al., 1973).

Our data do not show a linear relationship between lymphoblast formation and the AHH inducibility as found by Kellermann et al. (1973). The four patients (Fig. I) with the highest AHH inducibility ratios were among those with the highest percentage lymphoblasts. Except for these four patients (prior to treatment only), the evidence does not suggest a definitive relationship between the percentage lymphoblast formation and AHH inducibility. Similarly, although both percentage lymphoblast for- mation and AHH inducibility were reduced during radiation therapy, the changes were not significantly correlated. For example, in patient No. 1 (Table I) the formation of lymphoblasts dropped from 25 to 14% following the 18th radiation treatment, but his AHH inducibility did not change. On the other hand, in the case of patient No. 4, percentage lymphoblast formation decreased from 51 to 44% following therapy and AHH induction ratio decreased from 8.0 to 2.8. Furthermore, healthy controls with 47% to 56 % lymphoblast formation had AHH induction ratios of 1.3 to 2.2 whereas cancer patients with the same range of lyrnphoblast formation had induction ratios of 6.7 to 10.6. In another study we have compared the AHH inducibility and the PHA response in healthy control subjects over time and found no correlation between the two parameters (Prasad et al., 1978). These results suggest that AHH inducibility, although tested in lymphoblasts, is independent of the number of lymphoblasts formed. Probably it is the capability of lymphoblasts to produce AHH rather than their number that is relevant to AHH inducibility.

ACKNOWLEDGEMENT

This investigation was supported in part by research funds from the Veterans Administration Medical Center, Houston, Texas.

INDUCT’IBILITE DE L’ARYL HYDROCARBURE HYDROXYLASE ET FORMATION DE LYMPHOBLASTES CHEZ DES SUJETS ATTEINTS DE CANCER D U POUMON

L’inductibilite de l’aryl hydrocarbure hydroxylase (AHH) et la formation de lymphoblastes ont Bt6 6tudi6es dans les lymphocytes de t6moins normaux et de sujets atteints de cancer du poumon soumis B une radiothtrapie. Le rapport entre I’inductibilite de I’AHH et le pourcentage de lymphoblastes n’Ctait statistiquement significatif que pour les sujets non encore trait& (r = 0.598; p <0.05). Chez les temoins et chez les malades en radiotherapie, la correlation entre I’inductibilite de I’AHH et la formation de lymphoblastes etait positive, mais n’avait pas de signification statistique. D’aprbs ces rksultats, i l ne semble pas y avoir de rapport linbaire entre l’inductibilitk de 1’AHH et la formation de lymphoblastes.

REFERENCES

ATLAS, S . A., VESELL, E. S., and NEBERT, D. W., Genetic control of interindividual variations in the inducibllity of aryl hydrocarbon hydroxylase in cultured human lympho- cytes. Cancer Res. , 36, 4619-4630 (1976).

BARNES, E. W., FARMER, A, , PENHALE, W. J., [RVINE, W. J., ROSCOE, P., and HORNE, N . W., Phytohemagglutillin-induced lymphocyte transformation in newly presenting patients with primary carcinoma of the lung. Cancer, 36, 187-193 (1975).

BAST, R. C., OKUDA, T. , PLOTKIN, E., TARONE, R. , RAPP, H. J., and GELBOIN, H. V., Development of an assay for aryl hydrocarbon (benzo(a)pyrene) hydroxylase in human peripheral blood monocytes. Cancer Res., 36, 1967-1974 (1976). BENEDICT, W. F., CONSIDINE, N. , and NEBERT, D. W., Geneticdifferences in aryl hydrocarbon hydroxylaseinduction and benzo(a)pyrene tumorigenesis in the mouse. Mol. Phar- macof., 9, 266-277 (1973).

320 PRASAD ET AL.

BRAEMAN, I., BIRCH, A., and DEELEY, T. J., Depression of in-vitro lymphocyte reactivity after radical radiotherapy. Ann. clin. Res., 6 , 338-340 (1974). BRAEMAN, J., and DEELEY, T. J., The lyniphocyte response and prognosis i n cancer of the lung. Brit. J. Radio/., 48, 668-669 (1975). BUSBEE, D. L., SHAW, C. R., and CANTRELL, E. T.. Aryl hydrocarbon hydroxylase induction in human leukocytes. Science, 178, 315-316 (1972). CONCANNON, J. P., DALBOW, M. H., ENG, C. P., and CONWAY, J ., Immunoprofile studies for patients with bi onchogenic carcinoma. I. Correlation of pre-therapy studies with stage of disease. Int. J. radiat. Oncol. Biol. Phys. , 2,447-454 (1977). Ducos, I., MIGUERES, J., COLOMBIER, P., KESSOUS, A., and POUJOULET, N., Lymphocyte response to PHA in patients with lung cancer. Lancet, I, 1111-1112 (1970). GURTOO, H. L., BEJBA, N., and MINOWADA, J., Properties, inducibility, and an improved method of analysis of aryl hydrocarbon hydroxylase in cultured human lymphocytes. Cancer Res., 35, 1235-1243 (1975). JENKINS, V. K., OLSON, M. H., and ELLIS, H. N., In-vitro methods of assessing lymphocyte transformation in patients undergoing radiotherapy for bronchogenic cancel. Texas Rep. Biol. Med., 31, 19-28 (1973). JETT, J. R., MOSES, H. L., BRANUM, E. L., TAYLOR, W. F., and FONTANA, R.S., Bmzo(a)pyrene metabolism and blast transformation in peripheral blood mononuclear cells from smoking and nonsmoking populations and lung cancer patients. Cancer, 41, 192-200 (1978). KELLERMANN, G., CANTRELL, E., and SHAW, C. R., Variations in extent of aryl hydrocarbon hydroxylase induction in cultured human lymphocytes. Cancer Res., 33, 1654-1656 (1973). KERMAN, R. H., and STEFANI, s. s., Phytohoniagglutinin stimulation of lymphocytes in lung cancer patients. Oncology,

KOURI, R. E., RATRIE, H., and WHITMIRE, C. R., Evidence for a genetic relationship between methylcholanthrene- induced subcutaneous tumors and inducibility of aryl hydrocarbon hydroxylase. J. nat. Cancer Insf. , 51, 197-200 (1973). MCLEMORE,T. L., MARTIN, R. R.,PICKARD, L. R.,SPRINGER, R. P., WRAY, N. P., TOPPELL, K. L., MATTOX. K. L., GUINN, G. A., CANTRELL, E. T., and BUSBEE, D. L., Analysis of aryl hydrocarbon hydroxylase activity In human lung tissue, pulmonary macrophages and blood lymphocytes. Cancer, 41,

34, 10-12 (1977).

2292-2300 (1978).

NEBERT, D. W., and GELBOIN, H. V., Substrate-inducible microsomal aryl hydrocarbon hydroxylase i n mammalian cell culture. I . Assay and properties of induced enzyme. J . h id . Chem.. 243. 6242-6249 (1968). NEBERT, D. W., and GFLROIN, H. V., The in wivo and in vitro induction of aryl hydrocarbon hydroxylase in mani- malian cells of different species, tissues, stiains, and develop- ment and hormonal states. Arch. Biochem. Biophys., 134,

PAIGEN, R., GURTOO, H. L., MINOWADA, J., HOUTEN, L.,

HAYNER, N. T., Questionable relation of aryl hydrocarbon hydioxylase t o lung-cancer risk. N. Engl. J. Med., 297,

PAIGEN, B., MINOWADA, J., GURTOO, H. L., PAIGEN, K., PARKER, N. B., WARD, E., HAYNER, N. T., BROSS, 1. D. J . . BOCK, F., and VINCENT, R., Distribution of aryl hydrocarbon hydroxylase inducibility in cultured human lymphocytes. Cancer Res., 37, 1829-1837 (19176). PRASAD, N., PRASAD, R., HARRELL, J. E., THORNBY, J., and FAHR, L., Relationship between mitogen response and aryl

76-89 (1969).

VINCENT, R., PAIGEIY, K., PARKER, N. B., WARD, E., and

346-350 (1977~~) .

hydrocarbon hydroxylase in cultured- human lymphocytes. Life Sci., 23, 247-252 (1978). RABEN, M., WALACH, N., GALILI, U., and SCHLESINGER, M., The effect of radiation therapy on lymphocyte subpopulations in cancer patients. Cancer, 37, 1417-1421 (1976). RAPLA, S.,, YANG, S. J., and MELEKA, F., Changes in cell- mediated immunity in patients undergoing radiotherapy. Cancer, 41, 1076-1086 (1978). RAO, L. G. S., AHH inducibility and lung cancer. Lancet, 1, 1228 (1974). ROBINSON, E., BARTAL, A,, COHEN, Y., HAASZ, R., and MEKORI, T., Treatment of lung cancer by radiotherapy, chemotherapy, and methanol extraction residue of BCC (MER). Cancer, 40, 1052-1059 (1977). SILK, M., Effect of plasma from patients with carcinoma o n in-vitro lymphocyte transformation. Cancer, 20, 2088-2089 (1967). SUTHERLAND, R. M., INCH, W. R., and MCCREDIE, J. A., Phytohemagglutinin (PHA)-induced transformation of lym- phocytes from patients with cancer. Cancer, 27, 574-578 (1971). WARA, W. M., Immunosuppression associated with radiation therapy. Int. J. Radiat. Oncol. Biol. Phys. , 2, 593-596 (1977). WHITLOCK, J. P., JR., COOPER, H. L.. and GELBOIN, H. V., Aryl hydrocarbon (benzo(a)pyrene) hydroxylase is stimulated in human lymphocytes by mitogens and benz(a)anthiacene. Science, 177, 3 15-3 16 ( I 972).