Iranian Journal of Pharmacology and Therapeutics Razi Institute for Drug Research (RIDR) of Iran University of Medical Sciences and Health Services (IUMS) ISSN: 1735-2657 Vol. 4, Num. 2, 2005, pp. 100-104

Iranian Journal of Pharmacology & Therapeutics, Vol. 4, No. 2, 2005, pp. 100-104

Prevention of Acetaminophen-Induced Mitodepression with Myrobalan (Fruit of Terminalia chebula) in Allium cepa Model

HEMANT SINGH RATHORE and POOJA CHOUBEY

Cell Biology Unit, School of Studies in Zoology, Vikram University, Ujjain, India.

Address correspondence to: Dr. H.S. Rathore, School of Studies in Zoology, Vikram University, Ujjain, India. E-mail: hemant2005hemant@sancharnet.in

Received April 24, 2005; Revised July 28, 2005; Accepted July 30, 2005

Code Number: pt05022

ABSTRACT

Allium cepa bulbs were grown in pure tap water (group I), in seven concentrations of acetaminophen (7.81, 15.62, 31.25, 62.50, 125, 250, 500, and 1000 ppm) in the presence (group II) and absence of my-robalan (fruit of Terminalia chebula) at a fix concentration of 0.10 mg/mL. Parameters of study were mean root length, mitotic index, abnormal mitosis and chromosomal aberrations and morphology of root. Aceta-minophen at all concentrations except 1000 ppm where roots did not grow at all, significantly inhibited root growth and declined mitotic index, effect appeared concentration dependent (group II). In the presence of myrobalan (group III) acetaminophen-induced mitodepression could be checked significantly. No morphological i.e. shape and color changes, abnormal mitosis and any type of chromosomal aberrations could be detected in any group. Probable protective role of myrobalan is discussed.

Keywords: Allium cepa, Mitodepression, Acetaminophen, Myrobalan, Terminalia chebula

Acetaminophen (paracetamol) has earned a promi-nent place as a common house hold analgesic which is available without prescription in several parts of the world [ 1 ]. Genotoxicity of acetaminophen is also on record and it is suggested that use of paracetamol may contribute to an increase in the total burden of genotoxic damage in man [ 2 ]. Ayurved (ancient Indian system of herbal medicine) recommends life time use of myro-balan alone or in Trifla (meaning three fruits: T. che-bula, T. belirica, Emblica officinalis) to maintain gen-eral good health of human beings because it is safe and possesses antioxidant and antimutagenic properties [ 3 - 5 ]. Myrobalan alone does not exert any ill effects in Allium test [ 6 ]; however, acetaminophen does so [ 7 - 8 ].

Aim of this study was to find out whether myrobalan could reduce, nullify or intensify cytotoxic effect of acetaminophen in Allium root tip cells when adminis-tered simultaneously.

MATERIALS AND METHODS

Allium cepa

Dry healthy onion bulbs 1.5 to 2.0 cm in diameter were obtained from local market.

Test Herbal Drug

Myrobalan, dried young nuts of Terminalia chebula were procured from local herbal medicine shop, and were gently backed for few minutes and cooled. Swollen nuts were ground to a fine powder. A recent study (Sharma, 2003) revealed lack of mitostatic effect of myrobalan on mitosis in Allium test at 0.10 mg/mL [ 6 ]; therefore this concentration was selected for the present study.

Test Chemical

Acetaminophen (paracetamol) under trade name 'medimol' dispersible 500 mg B.P. tablets made by synchem laboratories, Baroda (Gujrat) was used. Each tablets was dissolved in known amount of luke warm tap water to prepare suspensions of known concentra-tions.

Experimental Design

Experiments were planned as per standard protocol for Allium test [ 9 ]. Onions were descaled and placed on, twelve test tubes filled with pure tap water, (Group I, controls). Second series of 12 test tubes bearing onions were filled with suspension of each concentration of acetaminophen (Group 2, acetaminophen exposed). Third series of test tubes bearing onions were also filled with different concentrations of acetaminophen suspension but each contained myrobalan powder at 0.10 mg/mL level (Group III, acetaminophen plus myrobalan exposed). All solutions were changed every 24 hrs. Af-ter 48 hr two onions out of twelve in each series with most poorly growing roots were removed. Same day i.e. after 48 hours distal 2 mm of five roots were cut off from five individual bulbs from each series, and fixed in acetic acid - ethyl alcohol (1:3 v/v) for chromosomal study. Every time fixation was done at a fix time 11:00 a.m.

After 72 hrs total lengh of the 05 root bundles in each series of each onion was measured to record mean root length.

Squashing and Observations of Slides

Root tips were squashed using N-HCl and 2% aceto-carmine stain. Four fields from each slide were observed to cover 50 cells in each i.e. total 200 cells per slide, 3000-4000 cells were observed for each group of onion. Mitotic index was calculated as percentage of dividing cells. Slides were also observed to find out mitotic arrest, chromosomal aberrations, fragments, abnormal orientation, lagging chromosomes and poly-ploidy etc.

Statistics

Experiments were repeated five times. Students’t’ test was applied at 5% level of significance.

RESULTS

Mean Root Length

All tested concentrations of acetaminophen (except at 1000 ppm where roots did not grow at all) caused significant inhibition in the growth of roots (Gr. II) in comparison to control (Gr. I). A comparison between Gr. II and Gr. III (acetaminophen + myrobalan) revealed that myrobalan could check acetaminophen induced root growth inhibition at all concentrations ( Table 1 ).

Morphology, Color and Shape of Root Tips

Morphology i.e. color and shape of Allium cepa root tips cultivated in all test concentrations of acetamino-phen alone (Gr. II) or acetaminophen plus myrobalan (Gr. III) did not reveal any change from controls (Gr. I) ( Table 2 ).

Mitotic Index

In comparison to controls (Gr. I), all test concentra-tions of acetaminophen (Gr. II) significantly lowered mitotic index i.e. percentage of dividing cells. This mi-todepression effect appears dose dependent. Even in the presence of myrobalan (Gr. III) acetaminophen exterted its inhibitory action on mitosis but effect was found significantly less pronounced indicating preventive ac-tion of myrobalan against acetaminophen ( Table 3 ).

Abnormal Mitosis

No chromosomal aberrations and abnormal mitosis could be seen in root tip cells of onions in any groups ( Table 4 ).

DISCUSSION

Earlier workers observed cytotoxicity, spindle dis-turbing effect and chromosomal effects of acetaminophen (paracetamol) in animal, human and plant cells [ 10 - 19 ]. Results of the present study reveal cytotoxicity i.e. mitodepression mitostatic (low mitotic index to no cell growth at all) but no chromosomal effect. This de-scripency might be due to use of comparatively lower concentrations in the present study of acetaminophen than those used by other workers.

A careful persual of results indicate two findings for discussion, firstly probable mechanism of action of acetaminophen in root tip cells for lowering mitosis and secondly probable protective action of myrobalan.

Acetaminophen can bind irreversibly to DNA, can cause DNA break, can inhibit both replicative DNA synthesis and repair DNA synthesis [ 1 ]. It can block DNA replication by inhibiting deoxyribonucleotide (n NTP) synthesis in several mammalian cell types [ 18 ]. Also, acetaminophan is topoisomerase II poison [ 20 ]. It can increase membrane permeability, can deplete cellular glutathione level and can cause lipid peroxidation and can bind with proteins [ 10 , 21 ]. During late G1 restriction point gate opens in the presence of complex molecules at promotors of essential cell cycle genes and unrepaired DNA does not allow cells to go beyond G1 stage [ 22 ]. It appears that acetaminophen might have interfered in some complex manner at DNA and/or gene level resulting in mitodepression- mitostatic condition in Allium root tip cells.

Cytotoxicity of N-acetyl-p-benzoquinone imine (NAPQI) a common intermediate of paracetamol in cultured rat hepatocytes could be prevented fully by the addition of N-acetylcysteine, GSH or ascorbate during the exposure period [ 10 ]. Cytotoxicity of acetaminophen was prevented by curcumin, a natural constituent of Curcuma longa in rat hepatocytes [ 23 ]. Curcumin protected against paracetamol induced lipid peroxida-tion, increased cellular GSH and lowered LDH leakage. Crude extract of seaweed, Sargussum polycysturn (brown alga) could afford protection against acetamino-phan induced lipid peroxidation through their free radi-cal scavenging property [ 24 ]. It is likely that aceta-monophen induced peroxidative damage could result in the decline in mitosis but if myrobalan possesses anti-oxidant properties it can reduce toxic effects of aceta-minophen. Infact myrobalan has been shown to exert antioxidant and free radical scavenging activities [ 4 , 25 - 26 ].

Unrepaired DNA does not allow cells to go beyond G1 stage [ 22 ] and acetaminophen may damage DNA [ 2 ]. It is likely that acetaminophen induced DNA damage could have been remedied by myrobalan because it is known to exert antimutagenic activity in bacteria against direct acting mutagens sodium azide and 4-nitro-o-phenylene diamine, [ 27 ] later this property was attributed to tannins [ 28 ].

The Allium root cells also posses certain enzymes, the mixed function oxidases like that of mammalian hepatocytes that can activate promutagens to mutagens [ 9 ]. In case of metal toxicity detoxification in root cells takes place in the cytoplasm and cell wall within 12-24 hr. which was held responsible for the mitotic activity at low concentration exposures [ 29 ]. Similar action to-wards acetaminophen at low concentrations in the pre-sent case can not be ruled out.

Individual plant components like sulfhydryl and fla-vonoid compounds, gallic acid, ellagic acid, mucic acid, citric acid, reducing sugars and tannins can modulate effects of many genotoxicants [ 30 ], myrobalan possesses many of such compounds [ 31 ], which can be held responsible for reducing cytotoxic effects of acetaminophen in Allium cepa root tip cells in the present case.

ACKNOWLEDGEMENTS

Head of department gave departmental facilities. We thank Prof. Dr. Bernad Mechler, Department of Devlopmental Genetics, Deutsches Krebsforschungzentrum, Heidelberg, Germany and Dr. G. Fiskesjo, Institute of Genetics, University of Lund, Sweden for providing literature.

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