Introduction

Lead is known to be toxic, mutagenic and carcinogenic in human beings [₁]. The modern system of medicine uses chelation therapy to cure Pb toxicity [₂]. However, Ayurveda (Indian herbal medicine system) in Pb toxicity suggest the use of myrobalan (fruit of Terminalia chebula) [₃]. Our earlier study, showed the protective role of myrobalan towards Pb toxicity in mice [₄]. The present study, was undertaken to find out whether myrobalan can lower the cytogenetic effects of inorganic Pb in Allium test. The short term Pb exposure for 1 hr was used as Pb is known to disturb mitosis in Allium root tip cells [₅,₆,₇,₈].

Materials and Methods

Allum Cepa

Drug

Cultivation of Onions

Results

No morphological i.e., shape and color changes were noticed in the tips of roots of any group of bulbs. The root tips under NR for 72 hr did not grow following 1000, 3000 and 10,000 ppm Pb exposure for 1 hr (Table 1). DR for 72 hr also remained ineffective following 1 hr Pb exposure at 10,000 ppm, however, roots grew following Pb exposure at 1000 and 3000 ppm. The roots grew in both, NR and DR, more in DR following 1 hr Pb exposure at 100 ppm. The control roots grew during the corresponding recovery period.

Among the control root tips, MI did not change throughout the experiment (Table 2). Pb exposure for 1 hr at 1000, 3000 and 10,000 ppm, lowered MI. NR for 72 hr remained ineffective after 1000, 3000 and 10,000 ppm Pb exposure as cells of root tips appeared in interphase with hypertrophied nucleoli and did not resume mitotic course. DR could not revert mitostatic effect of lead at 10,000 ppm Pb exposure, however it was able to do so in roots expose to 1000 and 3000 ppm Pb during the 72 hr period of recovery. Following, 100 ppm Pb exposure, both NR and DR could overcome the mitostatic effect, more earlier in DR. Control root tips revealed no any mitostatic effect.

The observation of metaphases and anaphases after 1 hr exposure to growing roots indicates probable interference during transition of cells from prophase into metaphase (Table 3). The effect appeared dose dependent as following 100, 1000, 3000 and 10,000 ppm Pb exposure, 51%, 55%, 67% and 88% of the cells failed to show typical metaphase arrangement. No aberrations were noticed. Control root tips did not revealed any disturbed pro-metaphase transition.

Figure 1

Table 1: Mean root length (MRL) of onion bulbs grown following lead exposure at various concentrations

Root tips were initially grown in deionized water for 3 days and were then exposed at 0, 100, 1000, 3000 and 10,000 ppm lead nitrate for 1 hr. After lead exposure the root tips were washed with deionized water and were allowed to recover naturally (NR) or in presence of drug (DR) for 24, 48 and 72 hr. MRL is given in mm.

Values are shown as mean ± SEM of 20 roots in each group.

p > 0.05 ^{a, b, c.} MRL at 24, 48 and 72 hr vs MRL at 3 days of initial growth. ^{d, e.} MRL at 48 and 72 hr vs 24 hr. (—) is nil.

Figure 2

Table 2: Mitotic Index (MI) of onion roots following lead exposure at various concentrations

After 3 days of initial growth, MI was 48.97 ± 1.15.

p > 0.05. ^a, MI after 1 hr of Pb exposure vs MI after 3 days of initial growth. ^b,c,d. MI after 24, 48 and 72 hr of recovery vs MI after 1 hr Pb exposure.

^e. DR vs NR at 24 hr, 48 and 72 hr

^f. MI at 72 hr vs MI after 3 days of initial growth.

Figure 3

Table 3: Metaphases, anaphases and chromosomal aberrations following 1 hr lead exposures

After 3 days of initial growth, MI was 48.07 ± 1.51.

^a. MI after 1 hr of Pb exposure vs MI after 3 days of Initial growth.

^{b, c, d.} MI after 24, 48 and 72 hr of recovery vs MI after 1 hr of Pb exposure.

(—) Nil.

N and Ab is normal and abnormal.

Discussion

In Allium test, the important effect exhibited by Pb at a lower concentration was mitodepression and with higher concentrations, Pb exerted inhibition of prophase and finally resulted in the disappearance of mitosis. The mitostatic effect did not revert to normal condition i.e., cells did not reentered into mitosis from G1 during NR except among 100 ppm Pb exposed roots, however, recovery with DR could remove the mitostatic effect following 1000 and 3000 ppm Pb exposed roots. The effect of 10,000 ppm Pb exposure was not reverted during drug or in natural recovery process.

The earlier workers observed inhibition of root growth accompanied with spindle disturbing effect along with chromosomal aberrations following Pb exposures [₅,₆,₇,₈,₉,₁₀,₁₁,₁₂,₁₃]. In the present study, no chromosomal effects could be noticed. This may be due to the short Pb exposure time of 1 hr in the present study, while it was 24 hr or more in other studies. However, 1 hr exposure time was enough to find out early effects of Pb as within 1 hr Pb gets accumulated in the onion root cells [₅]. A temporary effect of Pb i.e., incompletely organised metaphase were seen after 1 hr Pb exposure, however, no abnormal anaphase was observed.

Pb has been shown to have varied effects. Pb may inhibit DNA repair processes [₁₄,₁₅], denature proteins [₁₆] and disturb the transcription process [₁₇]. Pb has been shown to induce lipid peroxidation [₁₈], alter membrane permeability [₁₉] and bind to SH-groups of tubulins in cell, disturbing typical arrangement of chromosomes at metaphase [₂₀]. Pb inhibits cell elongation, irrespective of mitotic activity due to changes in cell wall characteristics [₂₁]. Pb has been shown to compete with calcium ions, altering certain signal transduction processes [₂₂]. During the late G1 phase, restriction point gate opens in the presence of complex molecules at promoters for essential cell cycle genes [₂₃]. It appears that Pb may interfere with some of these processes, resulting in the mitostatic effect in onion root top cells and myrobalan may be able to counteract some of these activities.

Pb is removed from the cytoplasm of the cells in the process of detoxification [₅]. Root cell cortex wall is main storage site of lead [₂₄]. The ability of plants to bind heavy metals in cell wall and external polysaccharides has been shown [₂₅]. The detoxication takes place in the cytoplasm and cell wall within 12-24 hr, which may be responsible for the mitotic activity in root tips after low Pb intoxication.

Myrobalan may bind with the root tips as color changes were noticed from very pale to dark brown at higher concentration. It is suggested that myrobalan bind to Pb, making it unavailable, thereby reducing Pb toxicity. It is likely that Pb induced peroxidative damage may result in the mitostatic effect. Myrobalan has been shown to exert antioxidant and free radical scavenging activities [₂₆,₂₇], which may counteract the mitostatic effect of Pb.

The unrepaired DNA does not allow cells to go beyond G1 stage [₂₃]. The Pb induced DNA damage may be corrected by myrobalan as it has been shown to exert antimutagenic activity in Salmonella typhimurium against direct acting mutagens such as sodium azide and 4 nitro-o-diphenylenediamine [₂₈]. The antimutagenic activity of myrobalan was suggested to be due to hydrolysable tannins [₂₉]. The various plant components present in the extracts such as sulphahydryl and flavonoid compounds, gallic acid, ellagic acid, mucic acid, citric acid, reducing sugars and tannins etc. can modulate effects of environmental genotoxicants [₃₀]. As myrobalan possess many of these compounds, it may be able to reduce the cytotoxic effect of Pb.

Acknowledgement

Correspondence to

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