The effect of polycyclic aromatic hydrocarbons on choline kinase activity in mouse hepatoma cells

Biochimica et Biophysica Acta, 1004 (1989) 274-277 Elsevier

274

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The effect of polycyclic aromatic hydrocarbons on choline kinase activity in mouse hepatoma cells Brian K. Paulson, T h o m a s J. Porter and C l a u d i a K e n t Department of Biochemistry, Purdue UniL~,ersity,West Lafayette, IN (U.S.A.) (Received 6 MArch 1989)

Key words: Choline kinas¢; Phosphatidylcholine synthesis; Aromatic hydrocarbon receptor

Choline klnase catalyzes the first rate-limiting step in the pathway of biosynthesis of phosphatldylcholine. This enzyme was shown previously to be induced in liver by treatment of rats with polyeyclic aromatic hydrocarbons (lshidate et al. (1980) Biochem. Blophys. Res. Commun. 96, 946-952). The present study was u~dertaken to determine whether choline kinase In the murine hepatonm cell line, Hepa lelcT, is inducible by aromatic hydrocarbons and, if so, whether this Induction is mediated by the aromatic hydrocarbon receptor. Treatment of Hepa 1c1¢7 cells with 10 ~M ,8-naphthoflatone resulted In a 1.6-fold increase of choline kinase activity, but no response was seen when the cells were exposed to either 5.0 pM benzolalpyrene or 1.0 nM 2~3,7,8-tetraddorodibenzo-p-doxin, both potent inducers of aryl hydrocarbon hydroxyhue. Cell line variants with either deficient or elevated aromatic hydrocarbon receptors showed no increase in choline kinase activity following treatment with any of the pflycyclic aromatic hydrocarbons. These resuL*s are not consistent with a role for the aromatic hydrocarbon receptor in increased choline kinase activity in Hepa lclc7 ceils.

The principal pathway for phosphatidylcholine biosynthesis in mammals involves incorporation of choline via phosphorylation by choline kinase (ATP: choline phosphotransferase, EC 2..7.1.32), conversion to CDPcholine by CTP:phosphochofine cytidylyitransferase, then transfer of phosphocholine to diacylslycerol by choline phosphotransferase [1]. Although considerable attention has been focused on the cytidylyltransferase as regulating this pathway, there is ample evidence that choline kinase is also a rate-determining enzyme for phosphatidylcholine biosynthesis and that its levels are modulated in vivo and in cell culture [2-6]. One treatment that has been shown to cause increased choline kinase activity is treatment of rats with certain polycyclic aromatic hydrocarbons ['7]. These compounds have been shown to cause induction of aromatic hydrocarbon hydroxylase, a cytochrome/'-450, via the aromatic hydrocarbon (Ah) receptor [81. This receptor is a member of the family of intracellular

Abbreviations: Ah, aromatic hydrocarbon; TCDD 2,3,7,8-teu'achlorodibenzy-p-dioxin: DMSO, die,ethyl sulfoxide. Correspondence: C. Kent, Department of Biochemistry, Purdue University, West Lafayette, IN 47907, U.S.A.

receptors in which binding of the ligand increases the affinity of the receptor for nucleus [9]. lshidate et al. [7] showed that hepatic choline kinase activity is increased up to 2-fold in rats by treatment with 3-methylcholanthrene, 3,4-benzola]pyrene, or /3naphthoflavone. The increase in choline kinase activity is the result of selective induction of a particular isozyme of choline kinase [I0], and the induction is semirive to prior treatment with actinomycin D or cycloheximide [7]. Ishidate et al. also demonstrated ,~hat the time course for cytochrome P-450 induction is the same as that for choline kinase induction [7]. These results suggest that the induction of choline kinase activity in rat liver may be mediated through the aromatic hydrocarbon receptor. Several murine hepatic cell lines with altered Ah receptors have been developed by Whitlock and coworkers [II-13]. Three types of variant cells have been isolated. In class I variants, the number of receptors or their binding properties are altered, but the receptors do show a ligand-induced increase in affinity for the nucleus. In class II variants, the receptors have normal binding properties, but are deficient in nuclear binding. A third type of variant has increased levels of Ah receptors. It occurred to us that, if choline kinase activity were inducible in Hepa lclc7 cells in response to

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275 polycyclic aromatic hydrocarbons, this would be an ideal system for studying regulation of choline kJnase induction. We have, therefore, investigated the ability of polycyclic aromatic hydrocarbons to cause increased choline kinase in these cell lines. Benzo[a]pyrene, fl-naphthoflavone, dimethyl sulfoxide (DMSO) and minimum essential media (or modification) were obtained from Sigma Chemical Co., St. Louis, MO. Fetal bovine serum was from Whitakker M.A. Bioproducts, Inc., Walkerville, MD. Trypsin and gentamycin were from Gibco, Grand Island, NY. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was purchased from Chem Service, Inc., West Chester, PA. The cell stocks, Hepa lclc7, Bprcl, TAOclBprcl, BPrc4 and MUL 12, were generous gifts from Dr. James P. Whitlock, Department of Pharmacology, Stanford University School of Medicine, Stanford, CA. Cell stocks were maintained in 80-cm2 flasks (Nunc) in MEMa medium supplemented with 10% fetal bovine serum, at 37 *C in 5% CO2 and 100% relative humidity. Every 4 days, the cultures were treated with 0.05% trypsin and split 1 : 6. For experiments, cells were plated onto 100 mm dishes in medium supplemented with 10 /xg/ml gentamycin. Hepa lclc7 and MUL 12 cells were plated at 5.0-10 s cells/dish, the other cell types were seeded at a density of 1.0.106 cells/dish. 24 h after plating, a solution of polycyclic aromatic hydrocarbon in DMSO was added to the medium in the dish. Control cells received equivalent levels of DMSO (final concentration not greater than 1.0%). 16-20 h later, the medium was removed, the cells were washed three times with cold, buffered NaCI solution (137 mM NaCI, 1.5 mM KH2PO4, 6.6 mM Na2HPO4, 2.7 mM KCI, 0.91 mM CaCI2, 0.50 mM MgC12 (pH 7.4) and w e r e harvested on ice in 1.0 ml 10 mM Tris, 154 mM KCI (pH 7.5). The cells were lysed by sonicating for 10 s at 20% maximum output with a Model W-225 sonicator equipped with a microtip probe (Heat Systems Ultrasonics, Inc., Farmingdale, NY). Choline kinase activity was measured in cell homogenates, unless otherwise indicated, by the method of McCaman et al. [14] except that the assay volumes were adjusted upward to accomodate 45 /~1 of cell homogenate, the buffer was Tris-HCl, 100 mM, and 10 pl glacial acetic acid was used to halt the reaction. Aryl hydrocarbon hydroxylase activity was assayed by the method of Nebert and Gelboin [8]. Protein content of the cell homogenates was determined according to Lowry et al. [15]. Initial expela.ments to study the effects of polycyclic aromatic hydrocarbons on choline kinase activity involved treatment of parental Hepa lclc7 cells with varying concentrations of /3-naphthoflavone for 20 h. Choline kinase activity was increased up to 1.6-fold over the activity in control cells. The increase in activity was concentration-dependent, with near saturation at 10

pM fl-naphthoflavone, indicating that this particular polycyclic aromatic hydrocarbon can increase choline kinase activity in these cells. When the same treatment was performed on BprcI cells, a variant with deficient nuclear binding of the Ah receptor, there was no increase in choline kinase activity upon treatment with fl-naphthoflavone (Table I). This result suggested that the Ah receptor may be involved in the induction of choline kinase in these cells. Subsequent results reported below, however, indicated otherwise. Benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are polycyclic aromatic hydrocarbons that bind tightly to the Ah receptor in Hepa lclc7 cells [16]. If the Ah receptor is mediating the increased choline kinase activity in these cells, then treatment with benzo[a]pyrene or TCDD should result in increased choline kinase activity. Table II shows that this was not the case. In fact, benzo[a]pyrene and TCDD were completely ineffective at inducing choline kinase activity under these conditions. Various concentrations of 3methylcholanthrene also failed to stimulate choline kinase activity in Hepa lclc7 cells (data not shown). Since benzo[a]pyrene, TCDD, and 3-methylcholanthrene are all strong ligands of the Ah receptor, these results suggested that the increased choline kinase activity was not mediated by this receptor. In order to determine whether the Ah receptors in these cells were still functioning as expected, aryl hydrocarbon hydroxylase assays were performed on samples of the same cell homogenates used for the choline kinase assays. All cell types responded in the manner expected. For example, aryl hydrocarbon hydroxylase activity in parental Hepa lclc7 cells increased an average of 25- and 46-fold in response to treatment with /3-naphthoflavone and TCDD, respectively. The variant BPrcl showed little or no aryl hydrocarbon hydroxylase activity with any treatment. These results indicated that the All receptors in the parent cells were still able to mediate induction of aryl hydrocarbon hydroxylase, as expected. TABLE I

Effect of [3-naphthoflavone on choline kinase activity in parental cells and a class H variant Parental mouse hepatoma cells (Hepa lclc7) and a class ll variant (Bprci) were treated with 10/~M/3-naphthoflavone (/JNF) in DMSO o r with DMSO alone for 20 h and the choline kinase activity wa:; assayed. Results are given as means :t: S.E. of duplicate assays on two plates for each treatment. Specific activity Cell type

DMSO

/]NF

Activation

Hepa lclc7 BP rcl

2.56+0.21 3.03 4- 0.09

4.17:t:0.18 a 2.82 + 0.49 b

1.~3x ..

a p < 0.001 vs. DMSO controls by one-way analy',is of variance. b No significant difference.

276 TABLE !1 Effect of bcnw[a]pyrene and 2,3,7,8.tetrachlorodibenzo.p.dioxin on choline kinase activity Hepa lclc7 cells were treated with 5.0/tM benzo[a]pyrene (B[a]P) or !.0 nM TCDD in DMSO or DMSO alone for 20 h and choline kinase activity was assayed. Results are given as means+S.E, of duplicate assays on the number of plates shown in parentheses. Differences in activity were not significant. Specific activity (nmol/min per mg protein) DMSO

B[a]P

2.56 :t:0,21

1.92 :!:0.48

2.67 :t: 1.00

(4)

(10)

(28)

i

-

TCDD

-

To further examine the effects of polycyclic aromatic hydrocarbons on choline kinase activity, the treatment with polycyclic aromatic hydrocarbons was repeated with two class I variants, BPrc4 and TAOclBprcl, and with a variant that has a high level of the Ah receptor, MUL 12 (Table HI). None of the variants showed increased choline kinase activity with any treatment. In conclusion, two lines of evidence indicate that the Ah receptor is not involved in induction of choline kinase activity in Hepa lclc7 cells. First, the only polycyclic aromatic hydrocarbon to cause increased choline kinase activity was/I-naphthoflavone. TCDD, the most potent ligand of the Ah receptor [17], was completely ineffective, as were two other well-established lipnds, b e ~ a ] p y r e n e and 3-methylcholanthrene. Second, even ~-naphthoflavone was unable to cause increased choline kinase activity in MUL-12, a variant with high Ah receptor activity.

TABLE, i l l

of [ J ~ t ~ , ben:o[a]pyr~e and TCDD on choline kinase at~lfy in cell lines wltk altt,nvl aron~le hydrocarbon receptors ! variant ~ (BPro4 and TAOclBWcl), a class !! variant (BW'cl). and a variant with a hish activity Ah re,,~,ptor (MUL 12) were exposed to 10 ttM B-naphthoflavone (,gNF), 5.0 /tM bem~alpyrane (l~alP) or 1.0 nM TCDD in DMSO or DMSO alone for 20 h and choline kinase activity was measuged, Values are 8iven as means:l: S,E.. of duplicate asuffs, the number of plates siren in parentheses. There was no significant increase in choline kinase activity with at~v treatment in any of the cell lines. Spcgifg activity (nmol/min per rrq; protein) Cell type

DMSO

BNF

BPr¢4

6.94+1.26

6.24:E0.11 6.20+0.$5

TAOcl

MUL 12 BPrcl

B[aIP

TCDD 6.03 :l: 0.01

(8)

(2)

(6)

(2)

6.60 +0.43

4.93+0.37

4.74:!:0.58

4.21+0.10

(6)

(4)

(6)

(2)

6.174-0.43 6.38+1.24

3.60+0.09

5.67+0.45

(6)

(2)

(4)

(2)

3.33 + 0.63 (14)

Z87 4-0.61 (10)

3.35 + 0.46 (4)

3.57 + 0.78 (8)

We cannot say whether the induction of choline kinase in rat liver is mediated by the Ah receptor Induction in vivo may not necessarily be map~fested in cultured cells. It is also possible that induction of choline kinase in rat liver is mediated by the hew, toxic effects of polycyclic aromatic hydrocarbons [17]. Carbon tetrachloride, a chemically dissimilar hepatotoxic agent, causes induction of rat hepatic choline kinase activity similar to that seen upon treatment with 3-methylcholanthrene [10]. The induction of choline kinase in rat liver, therefore, may be due to a hep~tc.~to~Jc e f f ~ ~ot mediated by the Ah receptor. The mechanism for increased choline kinase activity in parental Hepalclc7 cells in response to/~-naphthoflavone has not been identified. Possibly a different protein is mediating this induction. For example, a protein binding polycyclic aromatic hydrocarbon that is distinct from the Ah receptor has been identified and characterized [18-20]. This protein has a high affinity for benzo[a]pyrene and/Lnaphthoflavone" but not for TCDD. In the course of our experiments on choline kinase in Hepa lclc7 cells, we found that benzo[a]pyrene was a strong inducer of aryl hydrocarbon hydroxylase activity in the class I variant cells, while TCDD had only the sfight effect expected for this class of Ah receptor mutation. The existence of multiple proteins that bind polycyclic aromatic hydrocarbons suggests that the increase in choline kinase activity in Hepa 1clc7 cells treated with fl-naphthoflavone might be mediated by a protein distinct from the Ah receptor. Thus, while Hepa lclc7 cells are not a suitable system for studying induction of choline kinase via the Ah receptor, these cells may be aseful for studying the roles of other proteins responsive to polycyclic aromatic hydrocarbons. The authors with to thank Dr. James P. Whitlock for the cell lines and advice. We also thank Dr. Gary Perdue for helpful discussions and Ms. Joanna Giordano for technical assistance. This work was supported by American Cancer Society Grant BC 577. References 1 Kennedy, E.P. (1986) The biosynt~lesis of phospholipids. In: (Op denKamp, J.A.F, Roelofsen, B. and Wirtz, K.W.A., eds.), Lipids and Membranes: Past, Present, and Future. Elsevier, Amsterdam. 2 Warden, C.H. and Friedkin, (1985) M. J. Biol. Chem. 260, 6006-6011. 3 0 k a , T. and Perry, J.W. (1979) Develop. Biol. 68, 311-~18. 4 Weinhold, P.A., Skinner, R.S. and Sanders, R.D. (1973) Biochim. Biophys. Acta 326, 43-51. 5 Vigo, C. and Vance, D.E. (1981) Biochem. J. 200, 321-326. 6 Infante, J.P. and Kinsella, J.E. (1978) Biochem. J. 176, 631-633. 7 Ishidate, K.~ Tsuruoka, M. and Nakazawa, Y. (1980) Biochem. Biophys. Res. Commun. 96, 946-952. 8 Nebert, D.W. and Gelboin, H.V. (1968) J. Biol. Chem. 243, 6242-6249.

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