Mutation Research, 244 (1990) 135-139 135 Elsevier

MUTLET 0350

Ca antagonist verapamil and tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induce chromosomal

aberrations in human lymphocytes

Joseph F r i e d m a n 1, F iore l la Shabta i 1, Ur i Sandowsk i 2, E h u d B a h a r a v 3 a n d Isaac H a l b r e c h t I

lB. Gattegno Research Institute, 2Emergency Department and 3Department of Medicine "B', Hasharon Hospital, Petah-Tiqva and Faculty of Medicine, Tel Aviv University (Israel)

(Accepted 28 November 1989)

Keywords: Ca antagonist; Chromosomal aberrations; 12-O-tetradecanoylphorbol-13-acetate; Ca ionophore; Supraventricular tachycar- dia

Summary

Lymphocytes from 8 healthy donors were cultured for 3 days in the presence of phytohemagglutinin. Ad- dition of the Ca antagonist verapamil or the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) increased chromosomal aberrations in comparison with control cultures. The effects of TPA and verapamil were additive. Although the Ca ionophore A23187 had no effect per se, it did abolish the effect of verapamil. Five patients with supraventricular tachycardia were treated clinically with verapamil for 1 week. Their lym- phocytes were cultured for 3 days in vitro in the presence of phytohemagglutinin. These lymphocytes showed higher chromosomal aberrations in comparison with lymphocytes isolated from the patients before treat- ment. The clinical significance is still unclear. We suggest that calcium ions may be necessary for the intact- ness of chromosomes of human lymphocytes and presumably of other cells.

Calcium is one of the body's second messengers: it relays electrical and chemical messages that ar- rive at a cell surface membrane to the biochemical machinery within the cell (Berridge and Irvine, 1984; Carafoli and Penniston, 1985; Evered and

Correspondence: Dr. Joseph Friedman, Department of Cell Biology, The Weizmann Institute of Science, 76100 Rehovot (Israel).

Whelan, 1986; Humes, 1984; Izant, 1983; Rasmussen, 1986). The calcium-binding proteins that reside in the plasma membrane of the cell and in the membranes of the endoplasmic reticulum and the mitochondria are the main regulators of the free calcium level in the cytosol and the organelles. They keep the normal concentration of Ca 2+ in the cell very low, around 10 - 7 M , while the concentration in the blood plasma is around 10-3 M. The normal very low Ca 2 + level means

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136

that relatively few ions need to be moved with a relatively small expenditure of energy, to regulate some biochemical reaction in the cell.

The currently available calcium channel blockers such as verapamil are potent and reasonably safe therapeutic agents in cardiovascular as well as other medical conditions (Braunwald, 1982; Fleckenstein, 1983; Murphy et al., 1983; Singh et al., 1978; Opie, 1984). Since they act not only on the cardiovascular system but also on a variety of other tissues, it is possible that they could cause damage to some biological functions by inhibiting the Ca 2 ÷ influx. Thus the present study was in- itiated primarily to follow the effect of verapamil on human lymphocytes and indeed we observed in- creased chromosomal aberrations.

12-O-Tetradecanoylphorbol-13-acetate (TPA), a tumor promoter, is known to activate protein kinase C (PKC) (Castagna et al., 1982; Nishizuka, 1984a,b). TPA increases the affinity of PKC for Ca 2 *, thereby permitting activation of the enzyme at resting intracellular Ca 2 ÷ levels. Cytosolic Ca 2 ÷ concentrations can be decreased in a few minutes by adding TPA, suggesting a stimulation of the Ca 2 +-transporting ATPase (Albert and Tashjian, 1985; Drummond, 1985; Lagast et al., 1984; Moolenaar et al., 1984; Sagi-Eisenberg et al., 1985; Tsien et al., 1982). Thus it was of interest to look at the effect of TPA and it was also found to in- crease chromosomal aberrations.

Materials and methods

Growth and cytogenetic procedures Lymphocyte cultures were grown from the buffy

coats of freshly drawn heparinized venous blood samples from healthy donors. The cells were cultured in M-199 Hanks' salts base supplemented with 15070 heat-inactivated fetal calf serum, an- tibiotics and glutamine (Biological Industries, Beth Haemek, Israel). The division of lymphocytes was stimulated by 5 #g/ml phytohemagglutinin (PHA, reagent grade HA 15, Wellcome, England) for 68-72 h. Other agents were added concomitantly with PHA. TPA and the Ca ionophore A23187

(both from Sigma) were each prepared in a stock solution of 1 mg/ml ethanol and diluted with phosphate-buffered saline (PBS) prior to use. Verapamil HC1 (Ikacor) in a stock solution of 5 mM (Bayer, F.R.G.) was added directly to the cultures in the final concentration.

1 h prior to harvesting, 0.3/~g/ml colchicine was added. The cells were hypotonically treated with 2007o phosphate buffer solution, pH 7.0, for 12 min and fixed by acetic acid and methanol (1:3, v/v). 80-150 mitoses were examined for each experimen- tal point for each donor on coded slides, in a blind study. We followed aberrations such as achromatic lesions (gaps), isochromatid gaps and breaks, inter- changes, deletions and acentric fragments.

"In vivo studies" Patients with supraventricular tachycardia were

initially treated with 5-10 mg verapamil i.v. and then given 80 mg verapamil orally 3 times a day for 1 week. Venous blood samples were collected from these patients both before and after 1 week of treat- ment with verapamil. Lymphocytes were cultured in vitro for 3 days in the presence of P H A and studied cytogenetically.

Results

Fig. 1 shows the average results of 3 experiments in which different concentrations of verapamil were checked for their effect on chromosomal aberrations. Verapamil at a concentration of 3 × 10- 5 M was found to be optimal for inducing chromosomal aberrations. Table 1 shows the effect of this optimal concentration of verapamil on lym- phocytes from 8 healthy donors. In all the cases verapamil had a significant effect above the control values (ranging between a 1.5-fold and a 4.3-fold increase).

Table 2 shows that TPA caused a significant in- crease in chromosomal aberrations which was dose-dependent. Verapamil and TPA had an ad- ditive effect. In the control, TPA- and verapamil- treated cultures, the majority of aberrations (75-7907o) were achromatic lesions (gaps), 20-23 070

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Fig. 1. Induction of chromosomal aberrations in human lym- phocytes by different concentrations of verapamil. Mean values and standard deviations of 3 experiments. The aberrations in- clude gaps.

were breaks and only rarely were other kinds of

aberrations found. In verapamil-treated cultures acentric fragments which resulted from cen-

tromeric breaks were seen at low frequency. In order to check whether the effect of verapamil

on chromosomal aberrations is a consequence of reduced intracellular Ca 2+ , we added the Ca

ionophore A23187 which increases the Ca 2+ permeability of biological membranes. As shown

in Table 3, the Ca ionophore had no effect per se

on chromosomal aberrations but it reduced the

aberrations caused by verapamil to control values.

Table 4 shows that lymphocytes from 5

supraventricular tachycardia patients treated only with verapamil for 1 week have higher

chromosomal aberrations compared to lym- phocytes isolated before treatment. A few patients

137

(data not shown) who were treated with verapamil

for long periods (more than 3 years) showed a mild

and insignificant increase in chromosomal aberra- tions compared to the normal range observed in our laboratory. Since we do not have any data on

their chromosomal aberrations before the start of

treatment, these results have to be considered with

caution.

Discussion

The fact that Ca antagonists such as verapamil

are very useful drugs in the treatment of angina, ar-

rhythmia and hypertension does not rule out the

possibility that they may cause damage to some biological structures/functions.

In this study it was found that verapamil causes

chromosomal aberrations in PHA-stimulated human lymphocytes in vitro (Fig. 1, Table 1) and to

some extent in vivo (Table 4). The effect in vitro

was more pronounced than in vivo, and more ex-

periments are needed to evaluate the potential

damage of verapamil. Other Ca antagonists also

should be tested since many of them probably do not work in the same way as verapamil. The Ca

ionophore A23187 abolished the effect of

verapamil (Table 3), which supports our assump- tion that intracellular calcium is needed for

chromosomal intactness. Harker et al. (1986)

showed that verapamil potentiates selectively the cytotoxicity of antitumor agents which produce

DNA-strand scission. They suggested that verapamil may be acting to promote the formation

or inhibit the repair of such DNA breaks.

TABLE 1

INDUCTION OF CHROMOSOMAL ABERRATIONS IN HUMAN LYMPHOCYTES IN THE PRESENCE OF VERAPAMIL 3×10 -5 M

Mitoses with aberrations (%)

Donor 1 2 3 4 5 6 7 8

Control 9.5 (2.2) 15.4 (3.3) 8.3 (2.4) 8.4 (1.7) 5.8 (1.2) 8.0 (2.0) 12.6 (2.8) 9.5 (2.1) Verapamil 18.9 (8.1) 23.1 (9.3) 36.0 (11.2) 25.2 (7.5) 16.1 (4.7) 19.3 (6.3) 18.9 (5.7) 15.7 (3.4)

The values out of parentheses include gaps, the values in parentheses exclude gaps.

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TABLE 2

INDUCTION OF CHROMOSOMAL ABERRATIONS IN

HUMAN LYMPHOCYTES BY TPA AND VERAPAMIL

TABLE 3

Mitoses with

aberrations (%)a Mitoses with

Control 9.5 _+ 3.2 - aberrations (%)a

TPA 100 ng/ml 18.6 _+ 3.0 Control 9.8 _+ 2.9

TPA 250 ng/ml 27.8 _+ 4.4 Verapamil 18.9 _+ 3.3

Verapamil 3 × 1 0 - 5 M 18.9 _+ 3.1 Verapamil + A23187 0.1 #g/ml 9.8 _+ 2.7

TPA 100 ng/ml + verapamil 3 x 10-SM 37.3 + 5.5 Verapamil + A23187 0.2 #g/ml 11.2 + 3.0

aMean values and standard deviations are based on 3 ex- A23187 0.2 ttg/ml 8.4 + 2.5

periments. The aberrations include gaps.

THE EFFECT OF THE Ca IONOPHORE A23187 ON

VERAPAMIL (3x10 -5 M)-INDUCED CHROMOSOMAL

ABERRATIONS IN HUMAN LYMPHOCYTES

aMean values and standard deviations of 3 experiments. The aberrations include gaps.

In accordance with some authors (Birnboim, 1982; Emerit and Cerutti, 1981; Emerit et al., 1983) and in contrast to others (Kinsella et al., 1983) we found that TPA increases chromosomal aberra- tions in PHA-stimulated human lymphocytes. Emerit et al. (1983) showed that antioxidants sup- press the clastogenic action of TPA, which in- dicates the intermediacy of active oxygen. Another mechanism which may contribute to the increased chromosomal aberrations induced by TPA could result from stimulation of the Ca pump. We have found recently that TPA increases the activity of Ca ATPase 5-fold in PHA-stimulated human lym- phocytes (Friedman et al., 1988). The increase in Ca ATPase activity, which is considered to func- tion as a Ca pump (Lichtman et al., 1981), is con- sistent with the results of others who found that TPA decreases the intracellular Ca 2÷ in a few minutes, suggesting stimulation of a calcium extru- sion mechanism (Albert and Tashjian, 1985;

Drummond, 1985; Lagast et al., 1984; Moolenaar et al., 1984; Sagi-Eisenberg et al., 1985; Tsien et al., 1982).

The intactness of chromosomes probably depends on the neutralization of the negative phosphate charges in the DNA. Considering that only one-half of the negative charges are neutraliz- ed by histones, there is much room for polyamines and metal ions to complete the neutralization. In- deed, inhibition of polyamine synthesis caused chromosomal aberrations in Chinese hamster ovary cells (Pohjanpelto and Knuutila, 1984). Others have found the importance of Ca 2 + for the stability and structure of chromosomes (Stef- fensen, 1961; Lewis and Laemmli, 1982; Moyne and Harper, 1982). More studies should be under- taken to understand the complicated relationship between chromosome structure/function and Ca 2 + regulation.

TABLE 4

COMPARISON OF CHROMOSOMAL ABERRATIONS IN HUMAN LYMPHOCYTES OF PATIENTS WITH SUPRA- VENTRICULAR TACHYCARDIA BEFORE AND AFTER TREATMENT WITH VERAPAMIL IN VIVO

Mitoses with aberrations (%)a

Patient 1 2 3 4 5

Before treatment 11.2 8.7 7.7 8.5 6.3

After treatment 17.5 18.2 I0.1 11.2 7.7

aThe aberrations include gaps.

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Communicated by R.J. Preston