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Apr 6, 2000 - Methyl parathion induced alterations in the level of mOlloamines, viz. norepinephrine, dopamine and serotonin were studied in discrete regions ...

Indian Jo urnal of Experimental Biology Vol. 39, March 200 I, pp. 276-279

Methyl parathion induced alterations in monoaminergic system of developing rat pups P Mahaboob Basha,' Shabana Begum 2* & Nayeemunnisa' 'Department of Zoology, Bangalore University, Bangalore 560 056, India 2Maharani's Science College for Women, Bangalore 560 001, India

Received 6 April 2000; revised 13 November 2000 Methyl parathion induced alterations in the level of mOll oa mines, viz. norepinephrine, dopamine and seroton in we re studied in di sc rete region s of developing central nervou s system of rat pups. A significant decrease in the level of mo noamines no ticed in methyl parathion toxicosis may be related to the altered neuronal activity and inefficiency, lead ing to depression and impairment in various be havioural activities. In contrast to AChE inhibition, monoamine oxidase (MAO) activi ty showed all increasing trend and it could cause deamination of catecholamines and accumulation of its metaboli tes. This suggests that an increased AChE inhibition may indirect ly stimulate MAO activity in developing rat pups ex posed to methy l parathion.

Insecticides mediate their actions via changes in the levels of second messengers. They have indirect effects on endogeneous neurohormone receptors, I membrane signal pathways 2 and release of neurotransmitters 3.4 . Statistical surveys reveal that neonates are more susceptib le to pesticide residues and are vIctIms of high percentage of fatal poi so nings 5.6 . Methyl parathion (o-o-d imethyl-onitrophenyl tri phosphate) an organophosphorous (OP) insecticide is reported to alter the turnover of catecholamines in different parts of brain7. Decreased levels of dopamine (DA), norepinephrine (NE) and serotonin (5-HT) have bee n reported in adult rats 7 13 upon chronic administration of OP compounds . . Biosy nthesis of neurotransmitter ACh decreased while its level in the discrete regions of developing centra l nervous system (CNS) during post natal life increased s. Since monoamines are involved in the transmitter activity of CNS alongwith ACh, it was considered desirable to study the effect of methyl monoamines, viz. dopamine, parathion on norepinephrine, and serotoni n in the developing rats during critical stages of CNS development. Developing (IS-and 21-day o ld) albino rat pups were used. They were injected intraperitonially with sublethal doses of methyl parathion (LDso.2mg and 3mg res pectively) and decapitated after 48 hr of insecticide administration. Discrete areas of brain and spinal cord were separated out for analysis at ODe. Levels of norepine phrine, dopamine and serotonin *Correspondent author


were measured by HPLC method . Standard norepinephrine bitartarate salt, dopa minehydroch loride, dopamine, serotonin, isoprotereno l hydrochloride and heptane sulfonic ac id were obtai ned from Sigma Chemical Co. Meth a nol (HPLC grade) was obtained from Spectroche mical s. Ana lytical grade EDT A, sodium acetate, orthophosphoric acid and perchloric acid (70 0/0) were obtained locally . The HPLC system consisted of delivery pump (model ERC-87 IO Erma Optical Works, Japan) a reverse phase analytica l co lumn Phenomenex, (3)1m ultra carb ODS), a degasser (ERC 3310, Erma Inc) , a recorder (Sekonic SS-IOO F) a nd a Fluorescence Spectrophotometer (Hitachi, Model 650-40, Japan) for detection. Samples were prepared and analysed by the procedure of Murai et al.l) with slight modification. The modified mobile phase, (pH 3.92) consisted of 0.02M sodi um acetate, 160/0 methano l (v/v), 0.1375 0/0 heptane su lfonic acid (w/v) and 0.1mM EDT A. Centrifuged supernatant samples (14,000 rpm) filtered through 0.45)1m membrane (Millipore) and IO0)11 of aliquot filtrate was injected onto the column. All separations were isocratic. Flow rate was set at 0.9mllmin. Isoproterenol was used as an internal standard. Levels of dopamine, norepinephrine and serotoni n after separation were detected at the excitation wave length o f 280nm and e mission wavelength of 315 nm keeping the slit width at IO for both excitation and emission. Tissue monoamine levels were calcu lated by comparing the


peak heights with that of peak height standards and with correc tion factor for the recovery of internal standard. The activity levels of monoami ne oxidase lo (MAO) were estimated by the method of Nachmias using dopamine as standard. The results were subjected to statistical analysis and the significance of difference between control and experimental mean was calculated by using Student's t test. Changes observed in the level of monoamines in discrete areas of CNS are presented in Table I. A significan t decrease in the level of dopamine, nor epinephrine and serotonin was observed in discrete regions of CNS during methyl parathion toxicosi s, in both the age groups studied. Contrarily an increase in the activity levels of MAO was also noticed. (Table 2). The mode of action of OP compounds is through inhibiting the enzyme cho linesterase (AChE) which causes accumu lation of ACh, thereby blocking transm ission of nerve impu lse. Increased levels of monoami nes in the brain tissue on exposure to OP


insecticides have been reported 11.12'. Contrarily diminution of monoamines following the dichlorvos treatment to rats is also reported 13 . However studies Table 2 - Changes in th e activity levels of monoamine ox idase (MAO) in the discrete areas ofCNS of control and methyl parathi on treated (MPT) rat pups [Values expressed as I.l moles of P-hydroxy phenyl acetaldehyde formed /g/hr are mean ±SD of 6 observations. Values in parenthesis indicate per cent increase over correspo ndin g contro ls] Reg ion Cerebral cortex Brain stem Cerebell um Spinal cord


15-da;ts-old 4A±0.18 19.8±0.32 (23A)b 5.2±0.16 6.0±0.23 (15A)" 5.8±0.34 19.9±OA6 (28 .I) b 6. 1 ±0.32 7.8 ±OA8 (27.86) b


8.2 ±OA6


7.8± OA6 ( II A)C


9. 8± 0.87 (36. I) C 10.6±0.99 (31.86) b

(36.7 ) c

8. 1 ±0.69

P values: "> 0.0 I; b> 0.005; C>0.00 I

Table I - Changes in the levels of norepinephrine (NE). dopam ine (DA) and serotonin (5-HT) in the discrete areas of CNS following methy l parathion treatment (MPT) in rat pups [Values expressed as ng/g wet wt of ti ss ue are mean ± SD of 6 observations. Values in the parenthesis indica te per cent decrease over corresponding controls.] Regio n







Cerebral Cortex



Brain Stem

463.0 ±32.6


171.0± 10.6

266.8± 18.9 (- 16. 10) C 469.5 ±38.9 (-2 1.05 ) C 135.3 ± 18.2 (-30.05) C

Spinal Cord


263.0±23.8 (-12 .04) C 350.2±23.8 (-24.3)C 123.6±9.6 (-27.7) C 49.0 ± 3.6 (- 19.67)"


598.0±48.6 193.0± 13.6 84.6±6.8


(-25.05) C


Cerebral cortex

181.2± 17.8

Brain stem

2 10.0± 18.9


76.8± 1.86

Spinal cord


150A± 14.9 (-16.68) C 170.6± 14.8 (-18.7)b 58.8 ± 1.32 (-23A3)C 40A±3.8 (-20A7)C

210.0± 17.6 260.4± 18A 96A±6.3

68.0 ± 3.9

166.2± 12.6 (-20.85) C 220.6± 19.6 (-15.28) c 68 .2 ±5.2 (-29.25)C 52.6±2.8 (-22.64) c



Cerebral Cortex


Brain Stem



123.0± 10.9

Spinal Cord


P values: ">0.0 1; "> 0.05; c>O.OO I; "NS

18 1.1 ± 13A (-27.56) C 318.6±23.6 (- 18.52)b 101.0 ± 7.9 (-17.87)b 76.8± 5.6 (- 13.3 1) "

312.0±20.6 460. 1± 22.8 160.6± 10.8 98 .0± 6.9

2 10.0±11.l (-32 .64) c 350.6± 18.3 (-23.79) C 122.6± 10.9 (-23.66) " 78.02 ± 5A (-20AO) "



Table 3 - C hanges in the levels of acetylc holine (ACh) and acetyl cholinesterase (AC hE) and cho line acetyl trans ferasl;! (C hAT) in disc rete areas of C NS of ra t pups after 48 hI's of methy l pa rathi o n trea tment

Region Cerebral Cortex Brain Stem Cerebe ll um Spinal corel

Ace tyl ch lo line (ACh) 15-day-old 21-day-old + 29.49 + 30.98 +28.08 +32. 19

+ 44.92 +39. 11 +23.63 +20.9 1

Acetyl cho lines terase (AChE) 15-day-old 2 1-day-old -38.23 - 31.78 -21.35 -30.9 1

of Fisc us et al. 14 have sugges ted OP induced acc umul ati on of ACh to stimul ate cholinergic exci tati on whi ch in turn may dep lete th e levels of monoamines. The present stu dy corroborates the reported res ults revea ling a reduction in the level of mo noamines in various regions of CNS in methyl parathi on tox icity. Norepinephrine con taining neurons have bee n implicated in a wide variety of central functi ons. Lack of norepinephrine at its receptors res ults in depress ion where as oppos ite situati ons, resul ting in over stimu lati on of norepinephrine receptors, result in mani a l5 . 16 . In the light of the sign ificant dec rease fo und in norepinephrine levels in discrete regions of CNS (Table I) it is suggested th at methy l parathi on induced stress may lead to depression in the developing mammal. Since alterations occurring in monoamines are associated with depressive reacti ons, it is probable th at methyl parathi on acts on ~-ad re n erg i c receptors and brings about changes in the levels of monoami nes . Dopamine containing neurons are also involved in a wide variety of central functions viz. locomoti on, feed ing, drinkin g and other behav iours 16. 17, while the involvement of serotoni n has been suggested in the regul ati on of di verse physiological processes such as sleep, aggression, sexuality, temperature, res ponse to pain and regulati on of neuroendocrine function 18-20. In the present study significant decline observ ed in dopam ine levels more so in cerebellum in preference to other areas, and a significant reducti on in th e level of serotonin in cerebral cortex in preference to other areas (though a general decrement is noticed) may suggest the impairment of vari ous behavioural activi ties in whi ch these monoamines are in volved. Increased acti vity of MAO was found to cause dea mi nati on of dopamine and acc umulation of its metabo lite in the brain regions of fish ex posed to phosalone 12. These studi es substantiate the present fi ndings since methy l parathi on ad ministrati on en hanced the MAO act ivity and decreased th e monoamine levels in the disc rete reg ions of CNS of deve lopi ng rat pu ps (Table I & 2). A marked depleti on

-35. 11 -2 1. 72 - 28.07 - 25.79

C ho li ne acetyl tra;lsfcra se (ChAT) 21-el ay-olel 15-day-o ld -1 5.94 -1 3.89 - 23.43 - 20.9 1

-28 .9 1 -:14.89 -20.13 - 21.7 1

of dopamine and norepinephrine in cerebellu m is refl ected by a mark ed elevati on of MAO in th at reg ion. Concom itantl y, brain stem showed a small dopamine decrement and MAO increment. This diffe rence can be ex pl ained on the ground that gray matter ri ch cerebellum is likely to be affec ted by a tox icant more severely as compared to white matter. However, in the cerebral cortex whi ch is also a gray matter ri ch region, a comparati vely lower diminuti on of monoamines was observed. This contrad icti on may be ex plained by the hi gher lipid content of cerebrum as compared to cerebellum 2 1. 22. Stimulation in the ac ti vity level of MAO lead ing to suppression of monoaminergic system in the presen t study may poi nt to the altered neuronal ac ti vi ty and ineffici ency induced by meth yl parathi on exposure. In contrast to AChE inhibition fo llowing insecti cide ex pos ure (Table 3), an elevati on in the ac ti vity level of MAO may cause deaminati on of catecholamines and accumul ati on of its metabolites leading to neuro logical disorders assoc iated with imbalance in the levels of biogeni c amines. Further, it can be stated that in methyl parathi on tox icosis, the cholinergic system seem to be the first affected and th is int urn affects the monoaminergic system. References I Katz E J, Co rtes V I, Eldefrawi M E & Eld efraw i A T, C hl o rpyrifos, parath io n and th eir oxons bind 10 and dese nsiti ze a ni cotin ic ace ty l choline recepto r: Re levance to their tox icities, Toxicol Appl p/wrlllacol , 146 (1997) 227 . 2 So ng X, Seid ler J L, Saleh J L, Z hang J Podille S & Slotkin T A, Cellular mechani sms fo r develo pme ntal tox icology of chl o rpyrifos: Targettin g the ade ny l cyclase sig nalin g cascade, Toxicol Appl P/w rlllacol, 145 ( 1997) 158. 3 Liu J & Pope C N. Comparit ive presy napti c ne uroc hemical changes in rat striatum fo ll ow ing ex pos ure to chl o rpyrifos or parathi o n, J Toxicol Ell viroll Hlrh . 53 ( 1998) 53 1. 4 Uppa l R P, Garg B 0 & A hmed A, Effect of malath ion and DDT o n the ac ti o n of so me tranq uil izers o n learni ng and memo ry traces in rats, Illdiall J Exp Bioi, 21 ( 1983) 6 17. 5 Ro ny Math ew, Joe Verg hese, Venkataram:l n B V & Joseph T , Effec t o f inh a lat io n o f insec ti cide spray o n lea rn ing and memo ry in rats, Illdiall J Exp Bioi. 27,3 (1989) 258. 6 Mahaboob Basha P & Nayeemunnisa, Effec t of methyl





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