Int. J . Cancer: 19, 378-382 (1977)

METABOLISM OF THE CARCINOGENIC HYDROCARBON BENZO(A)PYRENE IN HUMAN FIBROBLAST AND EPITHELIAL

PRODUCTS AND ARYL HYDROCARBON HYDROXYLASE ACTIVITY CELLS. 11. DIFFERENCES IN METABOLISM TO WATER-SOLUBLE

Hiroshi YAMASAKI, Eliezer HUBERMAN and Leo SACHS Department of Genetics, Weizmann Institute of Science, Rehovoth, Israel

Ary l hydrocarbon (benzo(a)pyrene) hydroxylase ( A H H) activity and metabolism o f benzo(a)pyrene t o water-soluble products were measured in cultures of body fibroblasts and kidney epithelial cells f rom different human embryos. A H H activity a t 24h after treatment wi th o r without benz(a)anthracene was determined in cultures from 23 embryos, and 3 days' accumulated metabolism of benzo(a)pyrene t o water soluble products was measured in cultures f rom 18 embryos. The body fibroblasts f rom the different embryos could be divided into three groups according t o the amount of water-soluble products, but not according t o the A H H activity. These three groups were not found by either assay in the cultures of kidney epithelial cells. In both fibroblast and epithelial cells, high metabolism t o water-soluble products was not necessarily associated with high A H H activity. The results extend our previous finding (Huberman and Sachs, 1973) of three presum- ably genetic groups for BP metabolism t o water- soluble products i n human fibroblast but not in epithelial cells and indicate that th is grouping was not found in these cells by measuring A H H activity.

Carcinogenic polycyclic hydrocarbons such as benzo(a)pyrene (BP) have to be converted by mixed function oxidases into metabolites that are biologi- cally active (Gelboin et al., 1969; Heidelberger, 1975; Huberman and Fogel, 1975; Huberman and Sachs, 1974, 1976).

The degree of metabolism may, therefore, be expected to play an important role in determining individual susceptibility to carcinogenesis by these compounds. We have previously shown that there is genetic heterogeneity of BP metabolism to water- soluble products in human fibroblast and epithelial cells and that fibroblasts from different embryos could be divided into three groups with different degrees of metabolism (Huberman and Sachs, 1973). It has also been reported that aryl hydrocarbon (BP) hydroxylase (AHH) activity in mitogen-activated human peripheral blood lymphocytes from different individuals can be divided into three groups (Kellerman et a!., 1973a, b).

Our present experiments were undertaken to extend our studies o n the grouping of human embryo fibroblasts with different degrees of BP metabolism to water-soluble products and to determine whether this grouping can also be detected by measuring AHH

activity. We have included in these experiments studies with body fibroblasts and kidney epithelial cells.

MATERIAL AND METHODS

Chemicals Randomly labelled tritiated benzo(a)pyrene (BP)

with a specific activity of 24 Ci/mmole was obtained from the Radiochemical Centre, Amersham, Bucks., England. SH-BP was dissolved in a final concentra- tion of 0.5 % dimethylsulfoxide (DMSO) and diluted with cold BP to a specific activity of 625 mCilmmole. Unlabelled BP, benz(a)anthracene (BA), and NADPH were obtained from Sigma (St. Louis, Mo., USA) and DMSO and n-hexane were from Fluka, A. G., Buchs, Switzerland, 3-Hydroxybenzo(a)- pyrene was kindly supplied by Dr. H. V. Gelboin (National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA). The BA was dissolved in acetone and 5Opl of this solution was added to the cell cultures containing 10 ml medium. This concentration of acetone showed no effect on aryl hydrocarbon (BP) hydroxylase (AHH) activity.

Cell cultures Cells were grown in Eagle's medium with a four-

fold concentration of amino acids and vitamins (H-21, Grand Island Biological Co. New York, N. Y., USA) with 10% fetal calf serum. Five ml medium were used in 50-mm plastic Petri dishes for the study of BP metabolism, or 10 ml medium in 100-mm Petri dishes for AHH activity. The cultures were incubated at 37" Cf0.5" C in a humidified incubator supplied with a constant flow of 10% CO, in air. Human embryos from induced abortions, aged 3-5 months, were kindly supplied by Dr. E. Caspi, Assaf Harofe Hospital, Israel. The embryos, after washing in phosphate-buffered saline (PBS), were dissected, the inner organs removed and the kidneys and body minced separately and washed twice with PBS. The minced tissue fragments were then stirred at 37" C in 0.25% trypsin solution (Difco, 1 :300), the cells collected every 10-15 min

Received: November 2, 1976.

BENZO(A)PYRENE METABOLISM IN HUMAN CELLS 379

and the fragments digested down to the last piece. Cells were seeded at 2 x lo6 to 5 x lo6 cells per 50-mm Petri dish, or 5 x lo6 to 15 x lo6 cells per 100-mm Petri dish. Medium was changed 3-5 days after sezding and the cells were subcultured at confluency. The kidney cultures contained more than 95 % epithelial cells while the body cultures contained more than 95 % fibroblasts (Huberman and Sachs, 1973). BP metabolism and the enzyme assay were determined in secondary cultures, 1-2 weeks after their initiation.

Measurement of AHH activity The AHH activity was assayed when the cultures

were confluent. AHH was determined 24 h after treatment with or without 4.3 benz(a)anthracene. AHH activity in control and treated cells was assayed as previously described (Gelboin et al., 1969; Yamasaki et al., 1975). The reaction mixture for the standard assay of AHH activity contained 50 pmoles of Tris HCI buffer, p a 7.5, 0.6 pmoles of NADPH, 3 pmoles of MgCI,, 0.1 ml of cell hom- ogznates (0.5 to 2.0 mg of protein) and 62.5 nmoles of benzo(a)pyrene (added in 50 pl ethanol, just before starting the reaction). The total volume was 1.0 ml. After 30 min incubation with shaking in air at 37" C,

the reaction was stopped with 1 ml of acetone, and the hydrocarbons- were extracted with 5 ml of n- hexane. After the mixture had been stored overnight at 4" C, 4 ml of the hexane layer were transferred to another tube and the hydroxy derivatives of benzo- (a)pyrene were extracted with 2 ml of 1 N NaOH. To the control tube, 1 ml of acetone was added before the addition of benzo(a)pyrene. The hydroxylase activity was linear up to 30 min with 0.5 to 2.0 mg of protein. Fluorescence of the base-extractable metabolites was measured with a fluorometer, with excitation at 396 nm and emission at 522 nm. A unit of AHH activity was defined as that amount catalyzing the formation of hydroxylated products causing fluorescence equivalent to that of 1 pmole of 3-hydroxybenzo(a)pyrene. The protein was measured according to Lowry with bovine serum albumin as a standard (Lowry et al., 1951). Three to five 100-mm Petri dishes of secondary cultures were used for triplicate determinations of enzyme activity and the amount of protein.

Measurement of BP metabolism to water-soluble products

The cells of confluent cultures were treated for 3 days with 3H-BP at 1 pg/ml in a final concentration

I o4

E

t a w ti 3

I T

FIGURE 1 Metabolism of benzo(a)pyrene

(BP) to water-soluble products by cultured cells from different em- bryos: (a) 0 , body fibroblasts; (b) 0, kidney epithelial cells. Water- soluble products were determined after 3 days' treatment of confluent cultures with %H-BP.

EMBRYO NUMBER

380 YAMASAKI ET AL.

of 0.5% DMSO, and parallel Petri dishes were treated with the same amount of unlabelled BP for cell counts. Two to four 50-mm Petri dishes were separately tested after the 3-day treatment. Extraction of water-soluble metabolites from 3H-BP and measurement of radioactivity were carried out as previously described (Huberman et al., 1971 ; Huberman and Sachs, 1973). The water-soluble products from BP are given in pmoles per lo6 cells per 3 days.

RESULTS

Benzo (a)pyrene metabolism to water soluble products Benzo(a)pyrene (BP) metabolism to water-soluble

products was determined in confluent cultures of body fibroblasts and/or kidney epithelial cells from 18 different embryos. Different amounts of water- soluble products from BP were produced by the body fibroblast cultures from different embryos (Fig.1). The highest metabolism of BP was observed in fibroblasts from embryo No. 13 which metabolized 2200 pmoles BP per lo6 cells per 3 days, while the lowest metabolism of BP was observed in fibroblasts from embryo No. 7 which metabolized 5.5 times less than those from embryo No. 13.

The fibroblasts from the 15 different embryos tested can be divided, as in previous experiments with 10 other embryos (Huberman and Sachs, 1973), into three groups according to their ability to metabolize BP into water-soluble products. One group, with the lowest efficiency, included two embryos with an average of 450 pmoles BP per loR cells per 3 days. The second group included 11 embryos with an average of BP metabolism which was twice as high as in group 1. The highest metabolism of BP, observed in fibroblasts from two embryos, was about five times higher than the metabolism of fibroblasts in group 1.

BP metabolism was also tested in confluent cultures from kidneys which contained more than 95% epithelial cells. These cultures also showed a heterogeneity in BP metabolism. However, the kidney cells from the different embryos did not show any clustering into groups. High metabolism of BP in kidney cultures from an embryo was not necessarily associated with high metabolism in the fibroblast cultures from the same embryo.

Aryl hydrocarbon [benzo (a)pyrenel hydroxylase Aryl hydrocarbon [benzo(a)pyrene] hydroxylase

(AHH) activity was measured in body fibroblast cultures and in kidney epithelial cultures from 23 different human embryos of which 15 were also tested for metabolism of BP into water-soluble products. This was undertaken in order to determine whether differences in BP metabolism to water- soluble products in the different embryos were

associated with differences in AHH activity. Enzyme activity was determined 24 h after treatment with or without benz(a)anthracene (BA). At this time AHH activity in the BA-treated cultures was at an optimal level (Fig. 2). The AHH activity in control cultures which received medium only ranged from 0.6 to 7.5 units per mg protein. AHH activity after treatment with BA was induced in all the cultures of body fibroblast and kidney epithelial cultures. AHH activity after BA treatment ranged in the different embryos from about 4 units up to about 20 units per mg protein in fibroblast cultures and up to about 40 units in kidney cultures. High AHH activity in the fibroblast cultures from an embryo was not neces- sarily associated with high activity in the kidney epithelial cultures from the same embryo. Although there was no clustering into groups in the fibroblast cultures of the 20 different embryos according to their AHH activity, there was a group of kidney epithelial cultures where 4 out of 16 embryos showed a high AHH activity. These results are summarized in Figure 3. No correlation could be observed be- tween high AAH activity and high metabolism of BP into water-soluble products.

DISCUSSION

The present experiments extend our previous finding of three groups, presumably genetic, for

I I I I I I

v_. , , , x

9 18 27 36 45 0

HOURS

FIGURE 2 Aryl hydrocarbon [benzo(a)pyrene J hydroxylase activity

in cultured cells at different times with or without treat- ment with 4.3 /AM benz(a)anthrancene (BA): (a) body fibroblasts from embryo No. 21 ; (b) kidney epithelial cells from embryo No. 23. 0 and 0 , BA treated; x, not treated with BA.

BENZO(A)PYRENE METABOLISM IN HUMAN CELLS

I

II 8 5 4 17 16 20 21 18 12 6 15 16 13 14 9 20 21 I I I I I I I

381

0.1‘

FIGURE 3 Aryl hydrocarbon [benzo(a)-

pyrene] hydroxylase (AHH) activity in cultured cells from different embryos: (a) body fibroblasts; (b) kidney epithelial cells. AHH activity was determined after 24 hours’ treatment with or without 4.3 ,UM benz(a)anthracene. (BA). and 0 , BA treated; x not treated with BA.

I 1 1 1 1 1 I I I 1 l l I

t r

benzo(a)pyrene (BP) metabolism to water-soluble products in cultured fibroblasts in human embryos (Huberman and Sachs, 1973; Kellerman et al., 19736). This grouping was not observed in cultures of kidney epithelial cells. The results of 3 days’ accumulated metabolism to water-soluble products were compared with the measurement of aryl hydrocarbon (BP) hydroxylase (AHH) activity at 24 h after treatment with benz(a)anthracene. The present data indicate that this measurement of AHH activity differed from the measurement of water- soluble products, in that it did not show the three groups in the human fibroblast cultures. Embryos with BP metabolism to water-soluble products did not necessarily show high AHH activity. It has, however, been reported that measurement of AHH activity showed three genetic groups in mitogen- activated human peripheral blood lymphocytes (Kellerman et al., 1973~). Our results from three groups by measuring water-soluble products but not by determining AHH activity, which measured phenol production from BP, indicate that AHH

activity is presumably not the rate-limiting step in the metabolism to water-soluble products in cultured fibroblasts. We have indeed shown that in these cells the phenols represent only a small fraction compared to the water-soluble products (Huberman and Sachs, 1973). The water-soluble products are most likely the result of enzymatic conjugation with glutathione, sulfuric or glucuronic acid, of metabolites such as epoxides and dihydrodiols in addition to the phenols. Our present results reinforce previous conclusions (Huberman and Sachs, 1973; Kellerman et al., 1973~1, 6) that there are three groups of individuals with different levels of BP metabolism and indicate that measurement of AHH activity at one moment of time did not detect these groups in fibroblasts.

ACKNOWLEDGEMENTS

We are indebted to Miss Sara Etkin for skillful technical assistance. This work was supported by Contract no. N01-CP-02217 with the National Cancer Institute, National Institutes of Health.

METABOLEME DWN HYDROCARBURE CANCERIGCNE, LE BENZO(A)PYR~NE, DANS LES CELLULES EPlTHf!LlALES ET LES FIBROBLASTES H UMAINS.

II. DIFFERENCES DU Mf!TABOLlSME E N PRODUITS HYDROSOLUBLES ET ACTlVlTE DE L’ARYL-HYDROCARBURE HYDROXYLASE

L’activiti de I’aryl-hydrocarbure hydroxylase ( A H H) du benzo(a)pyr&ne et le m6tabolisme de cet hydrocarbure en produits hydrosolubles ont i t 6 mesuris dans des cultures de fibroblastes et de cellules 6pith6liales rinales provenant de divers embryons humains. L’activit6 A H H a 6 t i diterminie 24 h apr&s le traitement avec ou sans benz(a)anthrac&ne dam des cultures provenant de 23 embryons et le m6tabolisme

382 YAMASAKI ET AL.

du benzo(a)pyrbne en produits hydrosolubles qui s’est op6r6 en.trois jours a 6te mesur6 dans des cultures provenant de 18 embryons. Lesfibroblastes du corps des divers embryons ont pu Etre divis6s en trois groupes sur la base de la quantit6 de produits hydrosolubles, mais pas de I’activit6 A H H . Aucune des deux epreuves n’a permis de retrouver ces trois grouper dans les cultures de cellules Bpithhliales r6nales. Dans les deux types de cellules, un fort m6tabolisme en produits hydrosolubles n’6tait pas nbcessalrernent associ6 a une grande activite AH H. Ces resultats concordent avec nos constatations pr6c6dentes (Huberman e t Sachs, 1973), selon lesquelles iI existerait pour le rnitabolisme du BP en produits hydrosolubles, trois groupes probable- ment gbnetiques dans lesflbroblastes humains, mais pas dans les cellules 6pith6liales; i l s indiquent 6galement que la mesure de I’activit6 A H H ne permet pas de deceler les trois groupes dans ces cellules.

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LOWRY, 0. J., ROSEBROUGH, N. J. , FARR, A. L., and RANDALL, R. J.. Protein measurement with the folin phenol reagent. J. biol. Chem., 193,265-275 (1951).

YAMASAKI, H., HUBERMAN, E., and SACHS, L., Regulation of aryl hydrocarbon (benzo(a)pyrene) hydroxylase activity in mammalian cells. Induction of hydroxylase activity in No- 0 2 ’ -dibutyryl adenosine 3’5’-monophosphate and amino- phylline. J . biol. Chem., 250, 7766-7770 (1975).

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