The effect of a short-term oral administration of potassium cyanide (KCN) (200 ppm in diet) with or without sodium nitrite (NaNO2) pretreatment on rat brain microsomal Ca2* ATPase was investigated. The specific activity value of the enzyme significantly decreased (p\u3c0.05) by 50% compared with control and by 63% for KCN-treated rats compared with KCN-treated rats pretreated with NaNO;,. There was no significant difference at the h = 0.05 level between the values obtained for the control and KCN-treated rats pretreated with NaNO,. These results show both that feeding lowers brain microsomal Ca2f-ATPase activity and that NaNO, has a protective role (antidote function) in that respect

Adenuga, G.. A.

Odunuge, Odutayo O

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Stephen F. Austin State UniversitySFA ScholarWorksFaculty Publications Chemistry and Biochemistry1997Sodium Nitrite Alone Protects the Brain o _i_Microsomal Ca -ATPase Against PotassiumCyanide-induced Neurotoxicity In RatsOdutayo O. OdunugeStephen F Austin State University, odunugaoo@sfasu.eduG.. A. AdenugaFollow this and additional works at: http://scholarworks.sfasu.edu/chemistry_facultypubsPart of the Biochemistry Commons, and the Chemistry CommonsTell us how this article helped you.This Article is brought to you for free and open access by the Chemistry and Biochemistry at SFA ScholarWorks. It has been accepted for inclusion inFaculty Publications by an authorized administrator of SFA ScholarWorks. For more information, please contact cdsscholarworks@sfasu.edu.Recommended CitationOdunuge, Odutayo O. and Adenuga, G.. A., "Sodium Nitrite Alone Protects the Brain o _i_ Microsomal Ca -ATPase AgainstPotassium Cyanide-induced Neurotoxicity In Rats" (1997). Faculty Publications. Paper 7.http://scholarworks.sfasu.edu/chemistry_facultypubs/7Bioscience Reports, Vol. 17, No. 6, 1997Sodium Nitrite Alone Protects the Braino _i_Microsomal Ca -ATPase Against PotassiumCyanide-induced Neurotoxicity In RatsO. O. Odunuga1*3 and G. A. Adenuga2Received May 30, 1977The effect of a short-term oral administration of potassium cyanide (KCN) (200 ppm in diet) with orwithout sodium nitrite (NaNO2) pretreatment on rat brain microsomal Ca2* ATPase was investigated.The specific activity value of the enzyme significantly decreased (p<0.05) by 50% compared withcontrol and by 63% for KCN-treated rats compared with KCN-treated rats pretreated with NaNO;,.There was no significant difference at the h = 0.05 level between the values obtained for the controland KCN-treated rats pretreated with NaNO,. These results show both that feeding lowers brainmicrosomal Ca2f-ATPase activity and that NaNO, has a protective role (antidote function) in thatrespect.KEY WORDS: Brain; microsomes; Ca2*-ATPase; potassium cyanide; sodium nitrite.INTRODUCTIONMany peasant communities in the developing countries fed on agriculturalproduce (plant and animal) that contain toxic substances even after processing.Among the staples in Nigerian diet for instance are cassava (Manihata) andbitter-leaf (Amygdaline vernonia). Cassava contains a vyanogenic glycoside-Linamarin, while bitter-leaf contains another cyanogenic glycoside-amygdaline.1,2The consumption of cassava as the major source of carbohydrate has beenassociated with the cyanide-induced neurological disorder known as TropicalAtaxic Neuropathy.2Toxin-induced cell death resulting from pathological increase in intracellularfree calcium ion concentration is known to be caused by a number of mechanismsincluding direct interference with Ca2t-transporter.5 The calcium transporter thatplays a central role in the fine regulation of calcium ion concentration is the1 Laboratory for Biomembrane Research, Department of Biochemistry, College of Medicine.University of Ibadan, Ibadan, Nigeria.2 Department of Biochemistry, Ogun State University, P.M.B. 2002, Ago-Iwoye. Nigeria.x To whom correspondence should be addressed.5430144-8463/97/1200-0543$12.50/0 © 1997 Plenum Publishing Corporation544 Odunuga and Adenugaendoplasmic/sarcoplasmic reticulum Ca:' -ATPasc otherwise known as the mic-rosomal Ca2<-ATPase/ It has been suggested that the Ca21-ATPase could heused as a target for toxins and drugs.41 In an earlier work," we observed thatcyanide, specifically potassium cyanide (KCN), depresses the activity of the brainmicrosomal Ca2 + -ATPase in rats.Sodium nitrite (NaNO 2 ) in combination with sodium thiosulphate is a widelyused antidote for cyanide poisoning.7 Amazingly, even sodium nitrite itself is atoxitant.'s In this paper, we report that sodium nitr i te alone protects the brainmicrosomal Ca2' -ATPase against KCN-induced neurotoxicity.MATERIALS AND METHODSAll chemicals used for these investigations were the purest quality available.Fifteen (15) male Wistar rats obtained from the animal house of the Faculty ofBasic Medical Sciences, Ogun State University, Ago-Iwoye, Nigeria, were usedfor this experiment. All animals were fed on rat pellets (obtained from PfizerProducts Plc., Lagos) and maintained under laboratory conditions for a weekbefore the experiment commenced. They were allowed feed and water ad libitumbefore and during the experiments.The rats were divided into three groups based on the treatment given: groupA (control), group B (KCN-treated) and group C (KCN/NaNO2-treated). of five(5) animals each. Group A received no treatment and thus served as control.Animals in group B were given diet containing KCN (200 ppm). Group C animalswere pretreated with water containing NaNO2 (200 ppm) for seven (7) days andlater received diet containing KCN (200 ppm). The allowable level of n i t r i t e usedfor preservation of foods in the developing countries is around 200 ppm." Thethree groups were fasted overnight and allowed their various diets and water fortwo days.The rats were sacrificed at the end of the feeding period and their brainsquickly removed and kept at 4°C in ice-cold buffer. Homogenates of the rat brainswere prepared using potter-Elvehjem homogenizer. The post mitochondrialsupernatant (PMS) was obtained as earlier described by Famulski and Carafoli(1982)'' except that centrifugation at 100,000 X g for 1 hour was not necessary.The PMS was used for enzyme assay. Brain microsomal Ca:' -ATPase activitywas assayed as described by Adenuga et al. (1992).4RESULTSThe brain microsomal CaJ ' -ATPase activities of rats after the administrationof 200 ppm KCN in diet with and without nitrite pretreatment are shown inTables 1-3. No significant difference was observed in the mean body weights ofthe animals in each of the three groups before and after treatments. The brainmicrosomal Ca2 "-ATPase activities of KCN-treated rats decreased significantly(p<0.05) to about 50% that of the control group (Table 1). Also, significantlydecreased Ca24-ATPase activities were observed for KCN-treated rats (p<().()5)Protection of Ca2' -ATPase 545Table 1. Effect of KCN on rat brain microsomal Ca2' -ATPase activityGroupControlKCN-treatedBody Weight (g)160.3± 1.28165.4± 1.43Ca2 ' -ATPaseactivities(umoles Pi/mgProtein/hr.)2.07 ±0.211.03 ±0.18** Significantly different from control at p<0.05.Table 2. Combined effect of KCN and NaNO2 on rat brainmicrosomal Ca2' -ATPaseGroupControlKCN/NaNO2-treatedBody weight (g)160.3 ± 1.28163.2 ±1.41Ca2*- ATPaseactivities(jumoles Pi/mgProtein/hr.)2.07 ±0.211.63 ±0.24* Significantly different from control at P<0.05.Table 3. Effect of NaNO2 pretreatment on KCN-induced inhibi-tion of rat brain microsomal Ca2 '- ATPase activityGroupKCN-treatedKCN/NaNO2-treatedBody weight (g)165.4± 1.43163.2 ±1.41Ca21- ATPaseactivities(//.moles Pi/mgProtein/hr.)1.03 ±0.181.63 ±0.24** Significantly different at P<0.05.in contrast to KCN-treated rats pretreated with NaNO2 (Table 3). However, nosignificant difference was observed between the Ca2+-ATPase activities ofKCN-treated animals pretreated with NaNO2 and that of the control group(p > 0.05) (Table 2).DISCUSSIONCyanide is known to inhibit cytochrome oxidase at the active site and thisblocks the terminal event in electron transport. This single site of action isresponsible for the rapid and often fatal toxic effects of cyanide. As a result ofcyanide inhibition, oxidative phosphorylation is compromised and there is anincreased demand on glycolysis.10 This results in ATP depletion and the effectsinclude: interference with membrane integrity, ion pumps, and protein synthesis,loss of cell function and perhaps cell death. Our earlier work6 showed that546 Odunuga and Adenugacyanide disrupts Ca2+ homeostasis by inhibiting the microsomal Ca2+-ATPaseactivity. However, it is yet to be investigated if this inhibition is by a directinteraction with the enzyme molecule or as a result of ATP depletion.Sodium nitrite in combination with sodium thiosulphate is generally used asan antidote against cyanide poisoning because of its ability to mediate theoxidation of haemoglobin in vivo to form methaemoglobin. This haemoglobinvariant is ineffective in carrying oxygen to tissues, but has a high affinity forcyanide and removes it from ferricytochrome oxidase enzyme that has beeninhibited by binding of cyanide. Additional treatment with thiosulphate results inelimination of the cyanide.7Interestingly, nitrite is known to inhibit platelet aggregation by virtue of itsconversion to nitric oxide.10 Furthermore, it is believed that nitrites interact withtransport proteins thereby interfering with normal receptor-ligand interactions.10It however appears that nitrite reverses the effect of cyanide on the microsomalCa2+-ATPase by its ability to free ferricytochrome oxidase enzyme from cyanideinhibition. Perhaps as a result, adequate energy is generated and the pumpresumes its normal function. It is possible though that the net positive effect ofnitrite in the system overwhelms its seemingly toxic tendencies. It is not yet clearwhether nitrite could ameliorate cyanide poisoning by influencing the enzyme-cyanide interaction, if at all there is any.REFERENCES1. Oyanuga. V. A. (1968) Roots, tubers and green leaves. In: Nigeri's foods and feeding stuffs: Theirchemistry and nutr i t ive value. V. A. Oyenuga (ed.). 3rd Edition. Ibadan University Press. Ibadan.Nigeria, 20-27.2. Osuntokun, B. O. (1968) An ataxic neuropathology in Nigeria: A clinical, biochemical andelectrophysiological study. Brain 91:215.3. Carafoil, E. (1967) Intracellular calcium homeostasis. Ann. Rev. Biochem.. 56:395.4. Adenuga. G. A., Bababunmi, E. A. and Hendrikse, R. G. (1992) Depression of the Ca2'-ATPaseactivity of the rat liver endoplasmic reticulum by the liver tumor promoters, l.l,l-trichloro-2,2-bis(p-chlorophenyl) ethane and phenobarbital. Toxicology 71:1-6.5. Olorunsogo, O. O. and Bababunmi, E. A. (1993) Plasma membrane ion-motive ATPaseapplicable to toxicological studies. In: (Uned.), Chemical safety: Metabolism and Toxicity(Collected Lectures). International programme on chemical safely by World Health Organisation,Vol. 41, 101-112.6. Odunuga. O. O., Odukoya, A. M. and Adenuga, G. A. (1996) Depression of the brainmicrosomal Ca2 + -ATPase activity by potassium cyanide. Afr. J. Med. Pharm. Sci. (In Press).7. Rumack, B. H. and Lovejoy, F. H. Jr. (1990) Clinical Toxicology. In: M. O. Amddur, J. Doull andC. K. Klaassen (eds.) Toxicology: The Basic Science of Poisons. 4th Edition, Pergamon. U.S.A.,933p.8. Schwerin, F. T., Rostenstein, R. and Smith, R. P. (1983) Cyanide prevents the inhib i t ion ofplatelet aggregation by nitroprusside, hydroxylamine and axide. Thromb., Haemostas. 50:780-783.9. Famulski, K. and Carafoli, E. (1982) Ca~ '- transporting act ivi ty of membrane fractions isolatedfrom the post mitochondrial supernatant of rat liver. Celt Calcium, 3:263.10. Klaassen, C. D. and Eaton. D. L. (1990) Principle of Toxicology. In: M. O. Ambdur. J. Doull andC. K. Klaassen (eds.) Toxicology: The Basic Science of Poisons. 4th Edition, Pergamon, U.S.A..276p.11. Gross, R. L. and Newberne, P. M. (1977) Natura l ly occuring toxic substances in food. Clin.Pharm. and Ther. 22:680-698.

Sodium Nitrite Alone Protects the Brain o _i_ Microsomal Ca -ATPase Against Potassium Cyanide-induced Neurotoxicity In Rats

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Sodium Nitrite Alone Protects the Brain o _i_ Microsomal Ca -ATPase Against Potassium Cyanide-induced Neurotoxicity In Rats

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