Dalively p65

---

NUKLEONIKA 2006;51(3):141

−

146

ORIGINAL PAPER

Introduction

DNA damage is a trigger of many pathological processes;

therefore, knowledge of the molecular mechanisms and

targets of antimutagens is important for understanding

their etiology and pathogenesis. Antimutagenic effects

may also be important from the point of view of normal

tissue protection during cancer radiotherapy. We have

earlier shown that the 1,4-dihydropyridine derivatives

are efficient antimutagens and DNA repair modulators

in vivo [3, 8, 9, 11, 12] and in vitro [15, 16]. In particular,

alkaline single cell gel electrophoresis (comet) assays

showed that DHP (sodium 3,5-bis-ethoxycarbonyl-2,6-

dimethyl-1,4-dihydropyridine-4-carboxylate, previously

designated as AV-153 [16]) reduced the number of

endogenously generated DNA strand breaks in untreated

human lymphocytes and human promyelocytic

leukemia HL-60 cells [16]. In HL-60 and Raji (human

B-lymphoblastic leukemia) cells exposed to 2 Gy of

γ

-radiation or 100 

µ

M hydrogen peroxide there was

a statistically significant increase in the single strand

break rejoining rate [16]. Another compound of

1,4-DHP series, glutapyrone (disodium salt of 2-(2,6-

dimethyl-3,5-diethoxycarbonyl-1,4-dihydropyridine-4-

carboxamido)-glutaric acid), protected against ethyl

methanesulfonate (EMS)-induced mutations in

Drosophila melanogaster and chromosome breakage

in mouse bone marrow [9, 11, 12].

Effects of an antimutagen

of 1,4-dihydropyridine series

on cell survival and DNA damage

in L5178Y murine sublines

Olga Dalivelya,

Natalya Savina,

Tatyana Kuzhir,

Iwona Buraczewska,

Maria Wojewódzka,

Irena Szumiel

O. Dalivelya, N. Savina, T. Kuzhir

Institute of Genetics and Cytology,

National Academy of Sciences of Belarus,

27 Akademicheskaya Str., Minsk 220072,

Republic of Belarus

I. Buraczewska, M. Wojewódzka, I. Szumiel

Department of Radiobiology and Health Protection,

Institute of Nuclear Chemistry and Technology,

16 Dorodna Str., 03-195 Warsaw, Poland,

Tel.: [phone], Fax: [phone],

E-mail: [e-mail]

Received: 20 April 2006

Accepted: 17 July 2006

Abstract  In a series of studies it was shown that 1,4-dihydropyridine derivatives (1,4-DHP) show antimutagenic and

anticlastogenic properties and accelerate repair of oxidant and ionising radiation generated DNA damage. Here, effects

of one of 1,4-DHP compounds (sodium 3,5-bis-ethoxycarbonyl-2,6-dimethyl-1,4-dihydropyridine-4-carboxylate denoted

as DHP) in X-irradiated L5178Y cells (murine lymphoma sublines, LY-R and LY-S) are reported. DHP treatment 1 h

before, during and after X-irradiation gave a radioprotective effect in double strand break (DSB) repair competent LY-R

cells: there was an increase in post-irradiation proliferation and cell viability as well as a slight acceleration of break

rejoining as measured by the neutral comet assay. In the radiosensitive LY-S cells with impaired non-homologous end-

joining system, the radioprotective effect was seen as enhanced growth and viability. There was, however, no effect on

the DSB repair rate. Notably, there was no dependence of the biological effects on DHP concentration in the range of

concentrations studied (1 nM 

−

 100 

µ

M), suggesting an all-or-none effect, as in cellular signaling induction observed in

radioadaptation or bystander effect. We assume that DHP acts by decreasing fixation of radiation inflicted DNA damage,

among others, by increasing the rate of DNA repair and enhancing the efficiency of checkpoint control. Direct

confirmation of this assumption is necessary.

Key words  1,4-dihydropyridine 

•

 DNA repair 

•

 neutral comet assay 

•

 L5178Y cells 

•

 cytotoxicity 

•

 radioprotective

effect

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142

O. Dalivelya et al.

The purpose of the reported investigation has been

to study the influence of an antimutagen of 1,4-dihydro-

pyridine series on cell survival and X-ray-induced DNA

damage in L5178Y murine sublines differing in radio-

resistance, repair of double strand breaks and metabolism

of poly(ADP-ribose) (reviewed in [17, 18]). The present

study is part of screening of 1,4-DHP derivatives

synthesized in the Latvian Institute of Organic Synthesis.

Materials and methods

Cell culture

Two sublines of murine lymphoma L5178Y, LY-R and

LY-S, were used, earlier characterized in detail [17, 19].

Cells of both sublines were maintained in logarithmic

growth phase in RPMI-1640 medium supplemented

with 10% heat inactivated fetal calf serum at 37°C and

5% CO

2

.

 

For estimation of cell growth and viability,

suspensions of cell density 25 

×

 10

3

/ml (for treatment

procedures without X-irradiation) and 100 

×

 10

3

/ml (for

irradiated cells) were used. The comet assay (single cell

gel-electrophoresis) was carried out at cell density no

less than 400 

×

 10

3

/ml.

Treatment procedures

Exponentially growing cells were irradiated on 60 mm

plastic Petri dishes with an X-ray machine at room tem-

perature, with the use of an X-ray machine (ANDREX,

Holger Andreasen, Denmark, 200 kVp, 5 mA, dose rate

1.2 Gy/min). Since the cell lines differ in radiosensitivity,

approximately equitoxic doses of X-rays were used for

irradiation: 1 Gy for LY-S and 2 Gy for LY-R cells. In

cell growth and viability tests, X-irradiated cells were

incubated at 37°C for 48 h. In the comet assay, cells of

both sublines were irradiated with 10 Gy of X-rays at

0°C for estimation of initial DNA damage and at 37°C

for repair analysis 15, 60 and 120 min after irradiation.

The 1,4-dihyropyridine derivative (DHP) at concen-

trations of 1 nM, 1 

µ

M and 100 

µ

M was added to the

culture medium 1 h before irradiation and present there

for the whole 48 h incubation in the growth and viability

tests and from 0 to 120 min after irradiation in the comet

assay. In micronuclei frequency determination, 1 h pre-

treatment before irradiation at concentrations of 1 nM

or 100 

µ

M DHP was carried out and DHP left to the

end of post-irradiation incubation. (1 mM DHP was used

to treat non-irradiated cells for comparison to control.)

Growth and viability tests

In growth tests, cell cultures, control and treated with

DHP at concentrations indicated, were grown from the

same cell density for 48 h. Relative cell numbers were

determined (ratio of cell number in the treated culture

to that in the control; direct count of morphologically

normal cells), as well as viability by the dye exclusion

test with trypan blue, as described below. Experiments

were repeated at least 2 times.

Viability of cells subjected to X-irradiation and/or

DHP treatment was estimated with the standard trypan

blue test. For this purpose, irradiated or non-irradiated

cells of both sublines were incubated for 48 h in the

presence or absence of DHP, whereupon the dye was

added at a final concentration of 0.2 mg/ml. The cells

were scored using a Buerker hemocytometer.

Neutral comet assay

The modified method of neutral comet assay (single

cell gel electrophoresis)  [23] was used for estimation

of DNA damage (predominantly DSB) and kinetics of

their rejoining. The cell suspensions (4 

×

 10

5

 cells/ml)

were mixed with an equal volume of low melting-point

agarose Type VII at a final concentration of 0.75%. Cell

suspensions were cast on microscope slides precoated

with 0.5% normal (regular) agarose Type IA. Samples

were covered with cover slips and left on ice for

solidification. Then, they were placed in a lysing buffer

(2.5 M NaCl, 100 mM EDTA, 10 mM Tris-HCl, 1%

N-lauroylsarcosine, pH 9.5) supplemented with 0.5%

Triton X-100 and 10% DMSO. Lysis was carried out

for 1

−

2 h at 4°C in the dark. After lysis, the slides were

washed three times with an electrophoresis buffer

(300 mM sodium acetate, 100 mM Tris-HCl, pH 8.3)

and left in a fresh portion of the buffer for 1 h, then

placed in a horizontal gel electrophoresis unit filled with

a fresh electrophoresis buffer. The samples were

electrophoresed for 1 h at 14 V (0.5 V/cm, 7

−

8 mA) at

8°C in the dark. After electrophoresis, the slides were

rinsed with 0.4 M Tris (pH 7.5) and then stained with

1 

µ

M 4,6-diamidine-2-phenylindole dihydrochloride

(DAPI, 50 

µ

l per slide). For image analysis, pictures of

50 randomly selected comets per slide from two slides

in three separate experiments were captured at 200

×

magnification using an epifluorescence microscope

(Labophob-2, Nikon) equipped with a UV-1A filter

block. Image analysis was carried out using the Comet

v.3.1 (Kinetic Imaging Ltd., Liverpool, UK). The

measure of damage was the tail moment (fraction of

DNA in the tail times tail length). Data analysis was

based on the mean population response or on the

distributions of damage among cells. Statistical

evaluation and plots were prepared with Microsoft

Excel 2000 software.

Micronuclei frequency determination

Microscopic preparations for the cytokinesis block

micronucleus technique were made according to

Fenech and Morley [4]. Briefly, cell cultures were

incubated for 1 h with 1 nM or 100 

µ

M DHP,

X-irradiated  (1 Gy, LY-S; 2 Gy, LY-R cells) and

incubated for 16 h with 6 

µ

M/ml cytochalasin B (Sigma)

added immediately after irradiation. For micronuclei

scoring, cells were subjected to a hypotonic treatment

with 144 mM KCl solution (5 min, room temperature)

and fixed in a cold methanol-glacial acetic acid solution

(3:1). Microscopic preparations were made by dropping

cells on slides and staining with Giemsa. Non-irradiated

---

143

Effects of an antimutagen of 1,4-dihydropyridine series on cell survival and DNA damage...

cells were also treated in the same way with 1 

µ

M DHP

to compare with the non-treated control. Also in that

case, DHP was left for the whole 16 h incubation time.

The procedure was according to Fenech [4]. Two

microscopic slides were prepared for each experimental

point and 1000 or 2000 cells scored. The experiment

was repeated 3 times.

Results

Effects of DHP on cell viability and proliferation

Cytotoxicity of DHP at the concentrations of 1 nM,

1 

µ

M and 100 

µ

M was estimated in the trypan blue test

after 48 h cell incubation in the presence or in the

absence of DHP. Effect on cell proliferation was

estimated in a 48 h growth test on the basis of relative

cell numbers, as defined in the “Materials and methods”

section.

The results are presented in Table 1 (numerical data

and statistical significance from Student’s t test) and in

Fig. 1. In non-irradiated cells, DHP reduced the relative

cell number by 30% in LY-R cells and by 50% in LY-S

cells. The effect was independent of DHP concentra-

tion. In both LY sublines there was a slight decrease in

percentage of dead cells. Thus, altered viability level

could not affect proliferation; therefore, the decrease

in relative cell number apparently resulted from slowed

down cell cycle progression.

In contrast, there was an increase in relative cell

numbers in cell populations after exposure to X-rays

(LY-R cells, 2 Gy; LY-S cells, 1 Gy) by 34

−

53% in LY-R

cells and by about 14% in LY-S cells. In both LY

sublines, this protective effect was reflected in viability

increase, albeit to a different extent. The level of dead

Table 1. Effects of DHP (1,4-dihydropyridine derivative) on cell growth and viability of LY sublines after X-irradiation with

2 Gy (LY-R) or 1 Gy (LY-S)

Treatment

 Subline LY-R

Subline LY-S

Relative

 Dead cells

       t; P

*

Relative

  Dead cells

       t; P

*

cell number         [%]

cell number

         [%]

Control (non-irradiated)   1

2.13 ± 0.20

1

  1.13 ± 0.13

  4.17; <0.001

DHP 1 nM

  0.72

2.43 ± 0.23

1.00; >0.05

0.51

  0.39 ± 0.19

  3.22; <0.001

DHP 1 

µ

M

  0.71

1.82 ± 0.23

1.03; >0.05

0.50

  0.36 ± 0.19

  3.35; <0.001

DHP 100 

µ

M

  0.65

1.66 ± 0.24

1.52; >0.05

0.48

  0.61 ± 0.20

  2.17; <0.05

X-irradiated

  1

5.30 ± 0.19

1

10.75 ± 0.13

23.70; <0.0001

DHP 1 nM

  1.34

4.31 ± 0.17

3.96; <0.001

1.21

  5.42 ± 0.12

29.61; <0.0001

DHP 1 

µ

M

  1.45

4.10 ± 0.16

4.14; <0.001

1.09

  6.69 ± 0.13

22.56; <0.0001

DHP 100 

µ

M

  1.53

4.72 ± 0.16

2.00; <0.05

1.13

  6.58 ± 0.13

23.17; <0.0001

   * Student’s test evaluation of the difference from control (non-irradiated) or radiation alone data; t value and probability (P). Mean

results from 3 experiments.

Fig. 1. Influence of DHP on dead cell percentage in LY-R and LY-S cells relative to the control level taken as 100% (non-

irradiated or X-irradiated cell populations).

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144

O. Dalivelya et al.

LY-R cells decreased by 10–20% only, that in LY-S

subline was reduced by up to 60%. As in the case of

unirradiated cells, there was no clear dose-dependent

relationship in the latter case: the lowest dose (1 nM)

was equally or even more effective than the higher doses

of DHP.

Effects of DHP on repair of X-ray-induced DNA

damage

Effect of DHP treatment in X-irradiated LY cells was

also studied with the use of the neutral comet assay. To

estimate the DHP influence on the endogenously

generated DNA damage, the dose of 1 

µ

M was chosen,

whereas in experiments with irradiated cells, the effect

of treatment with 1 nM and 100 

µ

M DHP was analysed.

The average tail moment value in LY-S cells (11.11 ±

0.37) exceeded that in LY-R cells (8.73 ± 0.1). DHP

treatment did not affect much these values (LY-S,

11.99 ± 0.38; LY-R, 9.96 ± 0.32). It must be taken into

consideration that tail moment value is a result not only

of the endogenously generated (“spontaneous”) DNA

breakage level, but also of chromatin conformation.

Kinetics of DNA damage induced by X-rays in LY-R

and LY-S cells treated or untreated with the 1,4-DHP

derivative is presented in Fig. 2. In cells of both sublines,

the main portion of induced DSBs was rejoined for the

first hour of cell incubation, with about 60% of DNA

damage being repaired during the first 15 min. In LY-R

cells (Fig. 2A), DHP decreased the level of X-ray-induced

DNA damage both immediately after irradiation and

during a later incubation. It was interesting to note that

the lowest and the highest doses of DHP were equally

efficient against X-rays in this subline. When the tail

moment value after X-irradiation alone was taken as

100%, then approximately 20% and more than 50% of

X-ray-induced DNA damage was reduced at 0 min and

60 min, respectively, indicating an increased rate of

DNA repair in the presence of DHP. It should be noted,

however, that although a tendency to accelerated repair

was reflected in these relative damage levels, the

differences between absolute tail moment values were

not statistically significant. Also, the residual damage

level was identical in DHP-treated and untreated cells.

In LY-S cells both 1 nM and 100 

µ

M DHP did not

alter the rate of break rejoining both in absolute

(Fig. 2B) and relative (not shown) tail moment values.

The level of control and control + 1 

µ

M DHP is

indicated in the figure and it can be seen that there

is no effect of DHP on tail moment in non-irradiated

cells of both sublines.

Effects of DHP on micronuclei frequency

To check whether the effect of DHP treatment on DNA

damage estimated with the comet assay is reflected in

chromosomal damage, we determined the frequency

of micronuclei in cells treated in a similar way as in

the other tests, except that the endpoint was generation

of micronuclei at 16 h after irradiation.

Although X-irradiation was carried out with

approximately equitoxic doses, the response of LY

sublines considerably differed because of different

duration of the G2 arrest: 4 h per Gy in LY-R cells and

11 h per Gy in LY-S cells as well as the apoptosis

proneness [19]. This is reflected in the % of binucleated

cells in the DHP-treated and X-irradiated cell

populations (Fig. 3). A difference could be seen between

LY sublines, in agreement with the radiation sensitivity,

but DHP + X treatment did not alter the percentage

of binucleated cells as compared with X alone. Thus,

the progression through the cell cycle leading to

successful mitosis did not seem to be affected by the

treatment.

For micronuclei frequency, only preliminary data are

available which, however, are consistent with the comet

assay results. Also here, the effect did not depend on

DHP concentration, so, the results for 1 nM and

100 

µ

M were pooled. With micronuclei frequency after

X-irradiation alone taken as 100%, the relative fre-

quency (%) for DHP + X treated LY-R cells was 72.92

± 1.77 (mean ± standard deviation) and this decrease

was significant (chi square test, 

χ

2

 = 8.7); for LY-S cells

the decrease was not significant (68.23 ± 10.42).

Fig. 2. Time course of rejoining of X-ray-induced DNA breaks in LY-R (A) and LY-S (B) cells untreated or treated with

1 nM or 100 

µ

M DHP (present 1 h before irradiation and during the whole repair interval). The results for both concentrations

were pooled because of lack of dependence on DHP concentration. The level of control and control + 1 

µ

M DHP is indicated.

The differences between tail moment values obtained for DHP treated and untreated X-irradiated cells are not statistically

significant.

A

B

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145

Effects of an antimutagen of 1,4-dihydropyridine series on cell survival and DNA damage...

Discussion

The present study is part of screening of DHP deriva-

tives synthesized in the Latvian Institute of Organic

Synthesis. So far, a pronounced antimutagenic and anti-

clastogenic activity was noted in studies on Drosophila

melanogaster, murine tissues, human lymphocytes and

a variety of human cancer cell lines [3, 8, 9, 11, 15, 16].

There were, however, few attempts at defining the

mechanism of action of this group of compounds, except

that accelerated DNA repair was seen, as estimated with

the alkaline comet assay after treatment with hydrogen

peroxide or methyl methane-sulfonate (MMS) as well

as after 

γ

-irradiation [16].

In studies with Drosophila melanogaster, the anti-

mutagenic efficiency of 1,4-DHP derivatives was shown

to depend on their electron-donating activity [8]; hence,

they may act as antioxidants against spontaneous

mutations. However, the protective effect against EMS

supports the assumption that DHP derivatives also

modulate DNA repair [11, 12, 15, 16], and indeed,

accelerated rejoining of single strand breaks was

observed [16]. Moreover, DHP considerably stimulated

synthesis of poly(ADP-ribose) polymers and the

increase correlated with an increase in DNA repair rate

in hydrogen peroxide treated cells [15]. As discussed

below, other possible targets of 1,4-DHP derivatives

include progression through the cell cycle, DNA damage

repair and fixation processes (notably, both dependent

on poly(ADP-ribosylation)) and calcium ion homeostasis.

The observations on LY cells, reported above,

indicate that the effect on DSB repair may be important

in the radioprotective activity, since 

−

 as summarised in

Table 2 

−

 this effect was weaker in LY-S cells that are

DSB repair-defective (reviewed in [20]). Nevertheless,

some protection was observed even in these cells,

indicated by determinations of relative cell number and

viability tests. It seems of importance that DHP consi-

derably slowed down proliferation of non-irradiated

cells. Moreover, this effect was independent of DHP

concentration (see Fig. 1). In another study, DHP treat-

ment did not alter the distribution in cell cycle phases

[16], thus, apparently not causing any arrest at a specific

checkpoint; neither did it exert any lethal effect, as

judged from the viability tests. Hence, it was plausible

to assume that DHP facilitated or enhanced this aspect

of the cellular response to DNA damage that consisted

in arrest in cell cycle progression. The arrest depends

on a complicated signaling system (review in [13]) and

exerts a protective effect by favoring repair and preventing

damage fixation. Nevertheless, the percent-age of

binucleated cells (Fig. 3) did not reflect any change in

the cycling compartment of the cell populations under

examination. This aspect of DHP action awaits further

investigation with the use of other incubation intervals

for cytochalasin B treatment, as well as the use of flow

cytometry to assess cell cycle progression.

It is tempting to assume that 1,4-DHP derivatives,

which belong to well known voltage-dependent Ca

2+

channel blockers act through modulation of calcium ion

transport. There are some older positive reports, e.g.

diltiazem, a benzothiazepine calcium channel blocker,

alone or in combination with 1,4-DHP blockers,

protected against bone marrow damage (cytogenetic

damage, cell death) and mortality in whole body

irradiated mice [5

−

7]. Nevertheless, survival studies with

human cell lines (HeLa and MDA-MB-231) involving

blocking of the radiation-induced increase in cyto-

plasmic calcium concentration with another member

of 1,4-DHP family, nifedipine, did not reveal a

Table 2. Summary of DHP effects in LY cells, X-irradiated with approximately equitoxic doses (LY-R, 2 Gy; LY-S, 1 Gy)

Feature examined after DHP pre-treatment and X-irradiation

LY-R

LY-S

DNA breakage rejoining

Slightly increased

Unchanged

Micronuclei frequency

Decreased by 27%

Unchanged

Early lethal effect (48 h growth)

Decreased by 43%

Decreased by 14%

Viability (48 h after irradiation)

Enhanced by 20%

Enhanced by 60%

Fig. 3. Percentage of binucleated cells after 16 h incubation with cytochalasin B. (A) LY-R cells irradiated with 2 Gy X-rays

without or with DHP treatment at the concentrations indicated (DHP present 1 h before irradiation and during the whole

incubation interval). (B) LY-S cells irradiated with 1 Gy X-rays and treated as above. The differences between values obtained

for DHP treated and untreated X-irradiated cells are not statistically significant.

A

B

---

146

O. Dalivelya et al.

connection between the radiation effects on cellular

Ca

2+

 homeostasis and cell survival [21]. Other more

recent data indicate that calcium is required for ionising

radiation-induced cell cycle regulation and protein

kinase C activation, but the increase in cytoplasmic

calcium concentration is not a universal cellular response

to irradiation [10]. Finally, glutapyrone, unlike classical

1,4-DHPs, lacks calcium antagonistic or agonistic

properties, whereas it has an antimutagenic and anti-

neoplastic activity [9, 11, 12, 22].

The lack of dependence of the biological effects on

DHP concentration in the range of concentrations

studied (1 nM 

−

 100 

µ

M), suggests an all-or-none effect,

as in cellular signaling induction observed in radio-

adaptation [2, 18] or bystander effect (reviews in [1, 14,

17]. It should be added that radioadaptation has recently

been interpreted as a result of decrease in damage

fixation rather than repair stimulation [18]. Although

at present only an indirect support for such assumption

is available, it may be speculated that in the case of DHP

the antimutagenic effect is based on the same principle.

Furthermore, damage fixation is poly(ADP-ribose)

polymerase-dependent and its activity is modulated by

DHP [15]. The reported results must be treated as

preliminary, but they warrant further investigations

concerning the mechanisms of action of DHP in cells

with damaged DNA.

Acknowledgment  The authors express gratitude to

colleagues from the Laboratory of Membrane Active

Compounds and 

β

-Diketones of the Latvian Institute of

Organic Synthesis who provided the 1,4-DHP derivative

for investigation, and to Prof. Gunars Duburs for helpful

consultations. The authors from the Institute of Genetics

and Cytology NAS of Belarus were supported by the grant

of Physicians for Social Responsibility/International

Physicians for the Prevention of Nuclear War (PSR/

IPPNW), Switzerland. The authors IB, MW and IS were

supported by the Ministry of Education and Science

statutory grant to the Institute of Nuclear Chemistry and

Technology.

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