PHYTOTHERAPY RESEARCH, VOL. 5 , 130-133 (1991)

Inhibition of Clastogenic Effects of Nicotine by Chlorophyllin in Mice Bone Marrow Cells In Vivo

Soumitra Sen* and Archana Sharma Centre for Advanced Study in Cell and Chromosome Research, Department of Botany. University of Calcutta, 35 Ballygunge Circular Road, Calcutta 700 019. India

Geeta Talukder Vivckananda Institute of Mcdical Sciences. Calcutta 700026. India

The effect of chlorophyllin in modifying the clastogenic action of nicotine was tested in uiuo on mice bone marrow cells. Nicotine, when administered by gavage, induced chromosomal aberrations in frequencies directly proportional to the dose. Maximum effects were recorded at 6 h after exposure. Chlorophyllin, when given alone, was not clastogenic even at the highest concentration (1.50 mg/kg body wt). Simultaneous administration of nicotine and chlorophyllin with even lower doses (1.25 and 0.77 mg/kg body wt) reduced the frequency of chromosomal aberrations to the normal level. Chlorophyllin alone, given 2 h before nicotine, however, did not counteract the effects of nicotine. The use of green plant parts in modifying the genotoxicity of different agents may be related to the protective action of chlorophyllin.

Keywords: chlorophyllin; nicotine; anticlastogens; mice bone marrow chromosomes.

INTRODUCTION

The use of plants and plant products in modifying the action of genotoxic agents has received additional impetus following the reports of an inverse relationship between the consumption of vegetables and the inci- dence of cancer (Hirayama, 1981; Marshall et af., 1982; Pet0 et al., 1981; Winn et af., 1984). Chlorophyll and chlorophyllin are universally present in green plant parts and are known to be non-toxic (Hayatsu et al., 1988). Antimutagenic activity of these two chemicals has been demonstrated against a number of mutagenic agents by the Ames test (Ong et al., 1986).

The present investigation was undertaken to screen the action of chlorophyllin against the clastogenicity of nicotine-a potent clastogen-in bone marrow cells of mice in uiuo.

MATERIALS AND METHODS

Test chemicals and selection of dose

Nicotine (BDH, Poole, UK) was administered in two doses namely 0.77 and 1.10 mg/kg body wt according to the guidelines for in uiuo Assays in mice (Preston et af., 1987). The concentrations corresponded to 1/30 and 1/20 fractions of LD,,, dose (Merck Index, 1983). Chlorophyllin-sodium-copper salt, obtained from Sigma (St Louis, MO, USA), was dissolved in glass- distilled water. Three concentrations were used (0.77, 1.10 and 1.50mg/kg body wt). For the experiments,

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Table 1. Protocols followed for treatment Chemicals Composition

administered Set No. Imglkg body wt)

I. Nicotine alone IA 0.77 IB 1.10

II. Chlorophyllin IIA 0.77 alone llB 1.10

111. Chlorophyllin followed after IllA 0.77 2 h by nicotine 0.77

IV. Chlorphyllin IVA 0.77 T 0.77 and nicotine IVB 1 .I0 k 0.77 together

V. Negative control V Isotonic saline IV. Positive control VI Cyclophosphamide

25

laboratory bred male Swiss albino mice littermates, 8-10 weeks of age, with an average weight of 25 g were maintained on standard balanced diet (Hindustan Lever Limited, India) under standard conditions (tem- perature 28 k 2 "C; relative humidity 60 k 5 % , 12th photoperiod).

Experimental protocols

Five mice were used for each dose and for each sam- pling time. The mice were administered the chemicals orally by gavage according to the protocols described in Table 1. Isotonic saline was used as a negative control and cyclophosphamide (Sigma, St Louis, MO, USA) 25mg/kg body wt was administered i.p. as a positive control. The animals were killed 6,12, 18 and 24 h after treatment and bone marrow chromosome preparations were made by standard colchicine (2 mg/kg, i.p.)-

Received 21 August 1990 Accepted 3 October 1990

INHIBITION OF CLASTOGENIC EFFECTS OF NICOTINE BY CHLOROPHYLLIN 131

hypotonic (0.075 M) KCI-fixative (1 : 3 glacial acetic acid : ethanol)-flame drying schedule (Sharma and Sharma, 1980). et al. (1987).

different concentrations and sampling times on the clastogenicity of nicotine as recommended by Preston

RESULTS AND DISCUSSION Staining, scoring and statistical analysis

The slides were subsequently coded and stained in dilute Giemsa stain. 50 metaphase plates from each of the animals were scored for chromosome aberrations and 100 cells for mitotic index. Types of aberrations were recorded separately strictly in accordance with the schedule of Tice et af. (1987). All aberrations (chroma- tid gaps, chromosome gaps, chromatid breaks, chromo- some breaks. rearrangements) were considered equal-regardless of the number of breakages involved. Percentages of aberrant metaphase cells (excluding gaps) and number of chromosomal aber- rations (CA) per cell (excluding gaps) were computed. Level of significance was established at a=0.05 for statistical analysis. A 2-way ANOVA test followed by Duncan’s multiple range test (Sokal and Rohlf, 1981) was carried out to detect significant differences among

The different parameters screened were the frequen- cies of chromosomal aberrations per cell, (excluding gaps), damaged cells (excluding gaps) and mitotic index. The chromosomal aberrations induced per cell by nicotine alone excluding gaps, were mainly of the chromatid type, indicating effects at the G2 phase (Table 2). Two-way ANOVA test (Table 3), followed by’ Duncan’s multiple range test (Table 4) compared the effects of the two concentrations of nicotine at the three durations of exposure. The percentage of chro- mosomal aberrations was highest at 6 h after exposure to both doses of nicotine. It was significantly reduced at 12 h though it remained significantly higher than the control and the subsequent durations. At 18 and 24 h after exposure, the frequencies were significantly

Table 2. Chromosomal aberrations (CA) recorded in the bone marrow cells (250 cells per set) Period of exposure treatment set as Total CAI250 cells CAIcell % DC MI per Table 1 G’ G” B’ E CA DC lx+ SEMI (X+SEM) Ix+SD)

24 h V 3 0 2 0 0 2 0.008f0.011 0.802 1.095 2.09f0.16 VI 39 2 282 2 15 119 1.188t0.343 43.60k9.53 1.76f0.44

6 h IA 7 0 13 0 1 13 0.056k0.036 5.00f3.033 1.89k0.17 iB 24 0 60 0 0 53 0.240+0.100 21.20f8.075 1.78f0.35 IIC 4 0 2 0 0 2 0.010+0.011 1.0021.155 1.86f0.24 IllA 20 6 12 0 0 12 0.048f0.018 4.80f1.789 1.91 f0.31 IVA 1 1 0 9 0 0 8 0.036+0.017 3.20f 1.095 1.97k0.28 IVB 14 2 3 0 1 4 0.016f0.017 1.60k1.673 2.10f0.41

12 h IA 1 1 0 0 0 0 8 0.036f0.017 3.20f 1.095 1.92k0.23 IB 18 2 32 0 1 28 0.165f0.010 14.0021.633 1.86k0.37 IIC 3 0 1 0 0 1 0.005k0.010 0.50f1.000 2.16f0.26 IllA 10 3 6 0 0 6 0.030f0.011 3.00f1.155 2.00f0.15 IVA 5 1 3 0 2 4 0.02020.016 2.00f 1.633 1.88f0.61 IVB 6 1 3 0 0 3 0.015f0.010 1.50t1.000 1.94f0.15

18 h IA 17 0 5 0 0 5 0.020t0.014 2.00k1.414 1.78k0.34 IB 1 1 0 18 0 0 14 0.090t0.076 7.0024.761 2.10f0.17 IIC 3 1 3 0 0 3 0.015k0.019 1.50f1.915 1.89f0.29 IllA 10 0 4 0 0 4 0.020f0.016 2.002 1.633 1.78f0.28 IVA 9 0 3 0 0 3 0.01520.019 1.5021.914 2.05k0.31 IVB 1 1 0 3 0 0 3 0.015f0.019 1.50f1.914 1.87f0.27

24 h IA 10 1 3 0 0 3 0.012t0.011 1.205 1.095 1.80k0.30 IB 12 2 31 1 0 31 0.124+0.026 12.40f2.608 1.67f0.27 IIC 8 1 2 0 0 2 0.008f0.011 0.802 1.905 1.86f0.29 IllA 7 0 3 0 0 3 0.015f0.010 1.502 1.000 1.7750.36 IVA 8 0 3 0 0 3 0.01520.019 1.50k1.915 2.09f0.38 IVB 8 0 2 0 0 2 0.010+0.011 1.00f1.155 1.92f0.42

G’, G”=chromatid and isochromatid gap; 6’. B”=chrornatid and isochromatid break; CR = chromosomal rearrangement; DC = damaged cells; CA = chromosomal aberration; MI = mitotic index; X = mean value; SEM =standard error of the mean; SD=standard deviation. Number of animals used five in all sets.

132 SOUMITRA SEN, ARCHANA SHARMA AND GEETA TALUKDER

Table 3. Two-way ANOVA test showing significant differ- ences among the various treatment groups and sampling time

Degree of Sum of Mean sum Source of freedom squares of squares variation (dfl (ssl IMSSJ F

Treatment 6 0.0608 0.0101 20.02" Duration 3 0.0049 0.001 6 3.28' Error 18 0.0091 0.0005

' Significant at F=O.O5 level

higher than control but were lower than that at 12 h. The higher dose of nicotine gave similar results.

Chlorophyllin was administered in three doses and in no case were chromosomal aberrations recorded in frequencies higher than the control value, shown by the negative control. Therefore, this compound was not toxic to chromosome or cell division.

Chlorophyllin, when administered to the mice 2 h before nicotine, could not protect against the clasto- genic activity of nicotine in the bone marrow cells. However, when chlorophyllin was administered simul- taneously with nicotine to the animals by gavaging, it

Table 4. Duncan's multiple range test

IIC" IVB V IVA IllA IA IB 0.038 0.056 0.080 0.086 0.113 0.124 0.610

Duration

18h 2 4 h 12h 6 h 0.195 0.200 0.286 0.426

Straight lines indicate that there is no significant difference between the points at p = 0.05. a Chlorophyllin was administered in three doses (0.77, 1.10 and 1.50 mglkg body wt) and in no case where chromosomal aberration could be recorded were the freqeuncies higher than the control value. CAlcell values of the 1.50 mglkg body wt set is used for statistical analysis.

Treatment sets

was able to inhibit clastogenicity of nicotine to a signifi- cant level even with the lowest dose used of chlorophyl- lin (0.77mg). The middle dose of chlorophyllin (1.15 mg/kg) drastically reduced the frequency of chro- mosomal aberrations induced by nicotine and the value became the same as the negative control. Therefore, when administered simultaneously, chlorophyllin is able to protect completely against the clastogenicity of nicotine in mouse bone marrow cells. Mitotic index was not affected significantly by nicotine.

Lai el a f . (1980) had correlated the inhibitory action of crude plant extracts against the mutagenic activity of a variety of chemicals with their chlorophyll content. Both chlorophyll and chlorophyllin were found to be nontoxic and their antimutagenic property against known mutagens was attributed to a heat-stable princi- ple (Lai, 1979; Arimoto et a f . , 1980; Ong et af., 1986).

Previously, we have reported the modulatory action of fresh green leaves of Piper betfe L. against the cytotoxic effects of the nut of Areca catechu L. and leaf of Nicotiana tabacum L. used in betel quid chewed in India (Sen, 1987; Sen et a f . , 1987; 1989a-d; 1990; 1991). The present report indicates the possible role of chlorophyllin in antagonizing the clastogenic effects in these complex mixtures.

Chlorophyll has been found to be an antioxidant (Sato et a f . , 1984). Chlorophyllin, on the other hand, had been reported to be able to scavenge free radicals, to bind to the active groups of mutagens and adsorb or absorb toxic compounds (Dhir, 1989; Hayatsu et al . , 1988; Sharma, 1990). One or more of these activities may be involved in the inhibition of nicotine clastogeni- city by chlorophyllin.

Acknowledgements

The authors are grateful to the Council of Scientific and Industrial Research for financial assistance and to Professor A. K. Sharma, Programme Coordinator of the Centre for Advanced Study, for facilities and encouragement.

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