By Author
  By Title
  By Keywords

November 1990, Volume 40, Issue 11

Original Article

BIOCHEMICAL ALTERATIONS IN THE LEVELS OF DNA, RNA AND PROTEIN IN DISCRETE AREAS OF RAT BRAIN FOLLOWING NUVACRON TOXICITY

S. Asif Zaidi  ( Department of Biochemistry, L.L. R.M. Medical College, Meerut- 250 004, India. )
Sangita Singh  ( Department of Biochemistry, L.L. R.M. Medical College, Meerut- 250 004, India. )
Lokesh Palni  ( Department of Biochemistry, L.L. R.M. Medical College, Meerut- 250 004, India. )
V.S. Singh  ( Department of Biochemistry, L.L. R.M. Medical College, Meerut- 250 004, India. )

ABSTRACT

The levels of DNA, RNA and Protein were estimated in cerebral hemisphere, cerebellum and brain stem of male albino rats with Nuvacron (0, 0, dimethyl -0 - 1 methyl 3 - methylamino - 3 - oxe - 1 - propenyl phosphate) 4 mg/kg body weight intraperitoneally (i.p.) daily for 10 days. The daily i.p. dose of Nuvacron depleted the level of DNA and protein in all brain regions. Increased level of RNA was observed in cerebral hemisphere, cerebellum and brain stem (JPMA40 :261, 1990).

INTRODUCTION

Organophosphorus insecticides are known to be cholinesterase inhibitors1. Tayyaba et al2 showed significant changes in the concentration of nucleic acids and protein in cerebral hemisphere, cerebellum and brain stem following organophosphate insecticide Metasystox administration. Hassan et at3 reported reduction in the DNA concentration after administration of organophosphate pesticide. Or­ganophosphate pesticide Dimecron a!ters the levels of DNA, RNA and protein in different regions of rat brain4. Neurotoxicity studies were therefore carried out to deter­mine whether Nuvacron alters the content of DNA, RNA and protein.

MATERIAL AND METHODS

Twelve male albino rats weighing 150 ± 20 g. were used for this study. The animals were allowed free access to pellet diet (Lipton India Ltd) and water. Animals were divided into two groups. Nuvacron (Hindustan Ciba-Geigy Ltd) 4mg/kg body weight was injected intraperitoneally for 10 days to 6 rats of the experimental group whereas the 6 animals of the control group received an equal amount of physiological saline concurrently. On the 11th day all rats were decapitated to dissect out cerebral hemisphere, cerebellum and brain stem.
Isolation and estimation of Nucleic Acids
The brain tissues were homogenised in chilled 10% trichloroacetic acid (TCA) solution. For the extraction of DNA and RNA, homogenates from various brain regions were taken in cooled centrifuge tubes and washed twice with chloroform-methanol (2:1) to remove lipids. Further, extraction of the residue was made with hot alcohol ether mixture (3: 1). The lipid free tissue was incubated with 1 N KOH at 37°C for 20 hrs to hydrolyse the RNA. The residue left after extraction of RNA was heated with 5% Te­chloroacetic acid (TCA) at 90°C for 15 minutes to extract DNA. The method of Dische5 was used for the estimation of DNA. RNA was estimated according to the method given by Volkin6. Protein estimation was made by the method of Lowry et al7.

RESULTS

The administration of Nuvacron to rats resulted in such signs as hyperexcitability to tactile stimuli, occasional fasciculations, convulsions, ataxia, loss of hair and loss of weight. After 7-8 days all the treated rats usually became lethargic. These signs intensified with the passage of time. The present investigation shows the effect of daily administration of 4 mg/kg body weight of Nuvacron (Monocrotophos) to male rats.

Table shows the changes in the level of DNA,RNA and protein in cerebral hemisphere, cerebellum and brain stem of rat. The daily i.p. dose of Nuvacron depleted the level of DNA and protein in all brain regions, whereas the level of RNA increased in cerebral hemisphere, cerebellum and brain stem.

DISCUSSION

The results of this experiment establish a regional heterogeneity in the levels of nucleic acids and protein in the rat brain following experimental Nuvacron toxicity. Significant depletion of DNA was observed in the cerebral hemisphere, whereas the reduction in the level of DNA was considerably less in the cerebellum. Tayyaba et al2 sug­gested that the reduction in DNA level maybe attributed to the increased DNase activity in discrete areas of rat brain following organophosphate pesticide Metasystox ad­ministration. Since the average amount of DNA per diploid nucleus is constant for all normal tissues of the body, including brain tissue, measurement of DNA provides a convenient method for estimating the total cell population in various regions of the brain, or in the entire brain8. It shows that amounts of DNA in white matter are ap­proximately equal to those in the cortex or caudate nucleus9-11 and that regional differences in the amount of brain DNA are relatively small. Different regions of the brain have different DNA concentrations. The maximum concentration of DNA was observed in cerebellum. Mihailovic et al12. Palladin13 Grenell14 and May and Grenell15 also suggested that the cerebellum shows excep­tionally great amounts of DNA in cats, rabbits and rats respectively. Large total amounts of DNA in the cerebel­lum reflect the extreme cell density of the cerebellar granular layer. Hassan et al3 reported reduction in the DNA concentration after administration of organophos­phate pesticide. This reduction maybe a result of degenera­tive changes in neuron and nerve fibres following or­ganophosphate pesticide toxicosis. Changes in the amount of DNA can be used to detect whether toxic agents affect cellular proliferation and cell death. The earliest effects of toxic substances on DNA will probably involve: (a) the disturbance of DNA repair enzymes, (b) the intercalation of the substance into the DNA molecule, or (c) the direct binding of the substance to the nucleic acid moiety or its associated chromesomal proteins. Since this mode of action will ultimately interfere with the functional integrity of the central nervous system, it offers great promise for the identification of toxic compounds. Alteration in the level of DNA following organophosphate Dichlorvos intoxication was observed by Paolo & Fini16. According to Dean17 Metasystox, a sulphur containing organophosphate dtple­ted the level of DNA. Chromatolysing neurons in the chick, following organophosphate intoxication have been repor­ted by Janzik & Glees18. Nerve cells from different nuclei of the brain vary greatly in total RNA content. The amount of RNA in gray matter usually exceeds that in white matter, similarly, the ratio of RNA to DNA in pay matter is usually larger than that in white matter9,10,12,14. In this study the RNA level was increascd in all regions of rat brain. This may be due to the changes in the activity of enzymes which control the synthesis and turnover of nucleic acids. According to Heath19, demyelination is a feature of poisoning by many organophosphate pesticides and it results in the increased production of RNA. The rate of RNA and protein synthesis in the brain is very high and nerve cell functioning is dependent on protein metabolism. The total protein level was reduced in cerebral hemisphere, cerebellum and brain stem. It may be explained on the basis of higher breakdown of proteins due to increased proteolytic activity, which ultimately leads to the reduction in the protein content of the brain tissue. Further studies with the help of histochcmical proce­dure and estimation of lysosomal enzymes may lead to better understanding of the mechanism of organophos­phate intoxication.

REFERENCES

1. Coppage, D.L., Matthews, E., Cook, G.H. and Knight, J. Brain acetylcholine sterase inhibition in fish as a diagnosis of Environ­mental poisoning by Malathion, 0, 0-dimethyl S - (1,2 - dicar­bethoxyethyl) phosphorodithioate. Pesticide Biochern. PhysioL, 1975; 5 : 536.
2. Tayyaba, K., Hassan, M., Islam, F. and Khan, N.H. Organophos­phate pesticide metasystox- induced regional alterations in brain nucleic acid metabolism. Indian J. Exp. BioL,1981; 19: 688.
3. Hassan, M., Maitra, S.C. and Ali, S.F. Organophosphate pesticide DDVP induced alterations in the rat cerebellum and spinal cord an electron microscope study. Exp. Pathol., 1979; 17: 88.
4. Zaidi, S.A., Pande, S. and Singh, V.S. Effect of Organophosphate pesticide Dimecron (Phosphamidon) on the level of DNA, RNA and Protein of rat brain. Orient J. Chern., 1988; 4 : 400.
5. Dische, Z. Estimation of DNA. Mikrocbern., 1930; 8 : 4.
6. Volkin, E. and Cohn, W.E. Estimation of nucleic acids in methods of biochemical analysis. (ed. EdGlich, Inter Science publishers Inc. New York), 1964, pp. 287-305.
7. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, RJ. Protein measurement with the folinphenol reagent. J. Biol. Chem., 1951; 193: 265.
8. Heller, I.H. and Elliott, K.A. Desoxyribonucleic acid content and cell density in brain and human brain tumors. Canad. J. Biochem., 1954; 32 : 584.
9. Bodian, D. and Dziewiatkowski, D. The disposition of radioactive phosphorus in normal, as compared with regenerating and degenerating nervous tissue. J. CelluL Comp. Physiol., 1950; 35:155.
10. Logan, J.E., Mannell, W.A. and Rossiter, RJ. Estimation of nucleic acids in tissue from the nervous system. Biochern. J., 1952 ; 51: 470.
11. Elliott, K.A.C. and FIeller, I.H. Metabolism of neurons and glia, in metabolism of the nervous system. Edited by D. Richter, Oxford, Pergamon Press, 1957, p. 286.
12. Mihailovic, L., Jankovic, B.D., Petkovic, M. and Mancic, D. Dis­tribution of DNA and RNA in different regions of cat’s brain. Experientia, 1958; 14: 9.
13. Palladin, A.V. Metabolism of nucleic acids in the brain during its development, in biochemistry of the developing nervous system. edited by H. Waelsch, New York, Academic Press, 1955, p. 177.
14. Grenell, it 0. Nucleic acid content of cerebral neurons. Neurology (Minneap.), 1958; 8 (Suppl.): 56.
15. May, L. and Grenell, R.G. Nucleic acid content of various areas of the rat brain. Proc. Soc. Exp. BioL Med., 1959; 102: 235.
16. Paolo, P. and Fini, A. Damage by dichlorvos of human lymphocyte DNA. Tumori, 1980; 66 : 425.
17. Dean, B.J. The effect of dichlorvoson cultured human lymphocytes. Arch. Toxicol., 1972; 30 : 75.
18. Janzik, HI-I. and Glees, P. Chromatolysing spinal neurons in the chick following tri-cresylphosphate (TCP) intoxication. Ada NeuropathoL, 1966; 6: 303.
19. Heath, D.F. Organophosphorus poisons, anticholinesterase and related compounds. London, Pergamon, 1961, p. 238.

Journal of the Pakistan Medical Association has agreed to receive and publish manuscripts in accordance with the principles of the following committees: