Mol Cell Toxicol (2012) 8:83-93DOI 10.1007/s13273-012-0011-y

Abstract Cyanide is primarily a neurotoxin but itshepatotoxic and nephrotoxic potentials are also known.The present study reports the effect of alpha-ketogluta-rate A-KG (2.5-20 mM; 0 min), a potential cyanideantidote on potassium cyanide (KCN; 1.25-20 mM)induced cytotoxicity in primary culture of rat hepato-cytes. Cytotoxicity measured at various time points(0.5-24 h), was characterized by increased leakage ofintracellular lactate dehydrogenase, alanine aminotrans-ferase and aspartate aminotransferase, accompaniedby diminished mitochondrial function (MTT assay),mitochondrial membrane potential (Rhodamine 123assay), and ATP levels. However, lipid peroxidation(malondialdehyde assay) and DNA damage were notobserved. In a separate study, levels of cyanide, A-KG and thiocyanate were measured in the culturemedium of hepatocytes, treated with KCN (5 mM)and/ or A-KG (5 or 10 mM; 0 min), and in the serumof rats given oral treatment of KCN (10 mg/kg) and/or A-KG (0.5, 1 or 2 g/kg; 0 min). Cyanide and A-KGinteraction was best exhibited when both were addedin equimolar dose in vitro. In rats, cyanide levelswere significantly reduced by 1 and 2 g/kg A-KG. Itcan be concluded from the results that, (i) a very highdose of cyanide is required to produce cytotoxicityand other cellular perturbations in rat hepatocytes, (ii)cytotoxicity is independent of lipid peroxidation andDNA damage, (iii) A-KG provides significant protec-

tion against cyanide, particularly at equimolar dose invitro, and (iv) a very high dose of A-KG is requiredfor cyanide detoxification in vivo, suggesting that thedose of A-KG could be reduced by improving its bio-availability.

Keywords Cyanide cytotoxicity, Hepatocytes, Alpha-ketoglutarate, Protection, Interaction

Cyanide causes non-competitive inhibition of the mito-chondrial cytochrome c oxidase, an end chain enzymeof cellular respiration1-3. This leads to diminishedcellular utilization of oxygen causing ATP depletion,lactic acidosis, increased intracellular calcium levels,and plasma membrane disruption2-4. Free radicaldamage is a major consequence of cyanide toxicitythat leads to lipid peroxidation, activation of protea-ses, lipases and xanthine oxidases, culminating in celldeath4-7.

Cyanide is primarily a neurotoxin but its differen-tial effects on heart, lung and pancreatic tissues havealso been reported8. Also, there are reports on hepatoto-xicity and nephrotoxicity of cyanide in different ani-mals following prolonged exposure9-12. We have earli-er shown cytototoxicity of cyanide in isolated ratthymocytes13, neuron-like PC12 (rat pheochromocy-toma)14,15, and LLC-MK2 (Rhesus monkey kidney epi-thelial) cells16. In these studies, the progression ofcyanide toxicity and mode of cell death greatly variedfor different cells. Further, we observed that isolatedrat hepatocytes, yet another cell type, were relativelyresistant to cyanide (our unpublished work). Since,hepatocytes play a crucial role in cyanide detoxifica-tion, we were interested to examine its cytotoxicity inisolated primary cultures of rat hepatocytes, which

ORIGINAL PAPER

Cytotoxicity of cyanide in primary culture of rat hepatocytesand its interaction with alpha-ketoglutarate

Rahul Bhattacharya1, Janardhanan Hariharakrishnan1, Ravindra M. Satpute1 & Rajkumar Tulsawani2

Received: 10 October 2010 / Accepted: 30 August 2011�The Korean Society of Toxicogenomics and Toxicoproteomics and Springer 2012

1Division of Experimental Therapeutics, Defence Research andDevelopment Establishment, Jhansi Road, Gwalior 474002, India2Division of Phytochemistry, Analytical Chemistry and Toxicology,Defence Institute of Physiology and Allied Sciences, Delhi 110 054,IndiaCorrespondence and requests for materials should be addressed to R. Bhattacharya ( rbhattacharya41@yahoo.com)

are very commonly used in vitro model for cytoto-xicity assays, particularly to delineate the hepatototo-xic potential of a chemical17,18.

Cyanide is a reactive nucleophile known to interactwith the carbonyl moiety of various keto-acids likealpha-ketoglutarate (A-KG) to form a cyanohydrincomplex19. In light of this, various glycolytic sub-strates and keto-acid metabolites including A-KG wereevaluated for their cytoprotective effects against cya-nide in isolated rat hepatocytes20. Although, alpha-keto and aldehydic metabolites of carbohydrates andamino acids were found to confer protection, authorsproposed dihydroxyacetone and glyceraldehydes forfurther in vivo studies. Presently, A-KG is envisaged

as a potential antidote for cyanide poisoning21-23. Thepresent study addresses (i) cyanide-induced cytoto-xicity and other cellular perturbations in isolated rathepatocytes, (ii) role of lipid peroxidation and DNAdamage, (iii) cytoprotective effects of A-KG, and (iv)optimization of A-KG dose for cyanide detoxificationby assessing the interaction of A-KG and cyanide indifferent dose-combinations.

Effect of A-KG on cyanide-induced leakage ofintracellular enzymes from hepatocytes

Figures 1-3 show the effect of A-KG on potassiumcyanide (KCN) - induced leakage of intracellular lac-

84 Mol Cell Toxicol (2012) 8:83-93

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Figure 1. Leakage of lactate dehydrogenase from primaryculture of rat hepatocytes after exposure to: (A) differentconcentrations of KCN for 6 h and (B) 5.0 mM KCN for 6 h,in the presence or absence of 5.0 mM A-KG. Values are mean±SE of three different experiments. *Significantly differentfrom control; #significantly different from KCN at P⁄0.05(ANOVA-Student-Newman-Keuls).

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Figure 2. Leakage of alanine aminotransferase (ALAT) fromprimary culture of rat hepatocytes after exposure to: (A) differ-ent concentrations of KCN for 6 h and (B) 5 mM KCN for 6h, in the presence or absence of 5 mM A-KG. Values aremean±SE of three different experiments. *Significantly differ-ent from control; #significantly different from KCN at P⁄0.05 (ANOVA-Student-Newman-Keuls).

tate dehydrogenase (LDH), alanine aminotransferase(ALAT) and aspartate aminotransferase (ASAT). Fig-ure 1A shows that significant leakage of LDH occurredonly in 5 and 10 mM KCN after 6 h exposure. There-after, cells were exposed to 5 mM KCN in the presenceof equimolar dose of A-KG for 6 h. The leakage ofLDH caused by KCN was significantly prevented byA-KG (Figure 1B). Similar to LDH, leakage of ALATand ASAT was also prompted by 5 and 10 mM KCNalone. Further, leakage of ALAT and ASAT causedby 5 mM KCN was significantly resolved by simul-taneous treatment of 5 mM A-KG (Figures 2A-3B).Results indicate that addition of A-KG in equimolardose of KCN (1 : 1) afforded protection but when 5

mM KCN was challenged by 10 mM A-KG, no addi-tional protection was observed (data not shown).

Effect of A-KG on cyanide-induced mitochondrialdysfunction in hepatocytes

The hepatocytes were treated with KCN (1.25-20 mM)in the presence or absence of A-KG (2.5-20 mM) for0.5-24 h and mitochondrial integrity was measured byMTT assay. Significant decrease in MTT activity wasobserved after 2 h in 10 and 20 mM KCN while 5 mMwas toxic only after 6 h. A-KG per se was non-toxicto the cells but its 20 mM dose significantly reducedMTT activity after 24 h (data not shown). Thereafter,

Mol Cell Toxicol (2012) 8:83-93 85

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Figure 3. Leakage of aspartate aminotransferase (ASAT)from primary culture of rat hepatocytes after exposure to: (A)different concentrations of KCN for 6 h and (B) 5 mM KCNfor 6 h, in the presence or absence of 5 mM A-KG. Valuesare mean±SE of three different experiments. *Significantlydifferent from control; #significantly different from KCN atP⁄0.05 (ANOVA-Student-Newman-Keuls).

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Figure 4. Mitochondrial integrity of primary culture of rathepatocytes as measured by MTT assay after exposure to:(A) different concentrations of KCN for 6 h and (B) 2.5 mMKCN for 6 h, in the presence or absence of 2.5 mM A-KG.Values are mean±SE of three different experiments. *Signi-ficantly different from control; #significantly different fromKCN at P⁄0.05 (ANOVA-Student-Newman-Keuls).

when cells were exposed to different doses of KCNfor 6 h, significant decrease in mitochondrial activitywas observed as compared to control cells, except 1.25mM KCN. No dose-dependent effect was observed(Figure 4A). Significant decrease in mitochondrialactivity caused by 6 h exposure of 2.5 mM KCN wasattenuated by equimolar dose of A-KG (Figure 4B).Further, a 20% increase in protection was observedwhen the dose of A-KG was enhanced from 2.5 to 5mM (data not shown).

Effect of A-KG on cyanide-induced changes in mitochondrial membrane potential (MMP) in hepatocytes

Figure 5 depicts the MMP of hepatocytes measuredby Rhodamine 123 fluorescence 6 h after treatmentwith 5 mM KCN. Cyanide-induced significant de-crease in MMP was blunted by simultaneous treat-ment of equimolar dose of A-KG. The MMP was notaltered by 1.25-2.5 mM KCN (data not shown).

Effect of A-KG on cyanide-induced changes in energy status of hepatocytes

Figure 6A shows that KCN (1.25-10 mM) causeddose-dependent decrease in cellular ATP content after6 h exposure. Further, simultaneous treatment of 2.5mM A-KG significantly prevented the diminution ofATP levels in cells caused by 6 h exposure of 2.5 mMKCN (Figure 6B). Although, the ATP levels in A-KGprotected cells were lower as compared to control

cells, the difference was not statistically significant.

Effect of cyanide on lipid peroxidation and DNAfragmentation in hepatocytes

Figure 7 reveals that hepatocytes treated with KCN(2.5-20 mM) for 6 h did not show any significant lipidperoxidation as compared to control cells when mea-sured by malondialdehyde (MDA) levels. Also, agarosegel electrophoresis of DNA extracted from the cellsexposed to KCN (1.25-20 mM) for 24 h did not showany DNA fragmentation as compared to control cells(Figure 8).

86 Mol Cell Toxicol (2012) 8:83-93R

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Figure 5. Mitochondrial membrane potential of primary cul-ture of rat hepatocytes as measured with Rhodamine 123 afterexposure to 5 mM KCN for 6 h, in the presence or absence of5 mM A-KG. Values are mean±SE of three different experi-ments. *Significantly different from control at P⁄0.05(ANOVA-Student-Newman-Keuls).

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Figure 6. ATP levels in primary culture of rat hepatocytesafter exposure to (A) different concentrations of KCN for 6 hand (B) 2.5 mM KCN for 6 h, in the presence or absence of2.5 mM A-KG. Values are mean±SE of three differentexperiments. *Significantly different from control; #signi-ficantly different from KCN at P⁄0.05 (ANOVA-Student-Newman-Keuls).

Effect of A-KG on cyanide and thiocyanate levels in vitro

Figure 9 shows the levels of cyanide, A-KG and thio-cyanate in the culture medium of hepatocytes at differ-ent time points after addition of 5 mM KCN in the pre-sence or absence of simultaneous treatment of 5 or 10mM A-KG. About 75% recovery of cyanide was ob-served 5 min post treatment which progressively de-clined with time (Figure 9A). The levels of cyanide inKCN++A-KG treatments (1 : 1 or 1 : 2) remained sig-nificantly depleted as compared to KCN alone. How-ever, after 30 min cyanide levels in KCN++10 mM A-KG again increased and were significantly differentfrom KCN++5 mM A-KG at corresponding time points.In equimolar dose of KCN and A-KG (1 : 1), the levelof cyanide tapered to less than one-fifth of the origi-nal concentration 60 min post treatment. Figure 9Bshows that more than 50% and 95% recovery of A-KG was observed in 1 : 1 and 1 : 2 ratio of KCN and A-KG, respectively, 5 min after treatment. Although, notime-dependent loss of A-KG was observed, the re-covery of A-KG in 1 : 2 was significantly more as com-pared to 1 : 1 ratio after 30 min and 60 min. Under-standably, no significant levels of A-KG was measur-ed in KCN alone. Figure 9C reveals that the thiocya-nate levels in all the treatments marginally increasedwith time but there was no significant differenceamongst various treatments.

Effect of A-KG on cyanide and thiocyanate levels in vivo

Figure 10 shows the levels of cyanide, A-KG and

thiocyanate in the serum of rats after oral administra-tion of 10 mg/kg KCN, with or without simultaneoustreatment of 0.5, 1 or 2 g/kg A-KG. Cyanide levels inall the animals protected with A-KG were significant-ly lower as compared to unprotected animals at allthe time points (Figure 10A). Also, animals receiving1 and 2 g/kg A-KG showed significantly lower levelof cyanide as compared to 0.5 g/kg A-KG after 5 minof treatment. In all the treatments disappearance ofserum cyanide was time-dependent. Figure 10B showsthat A-KG levels in all the treatments progressivelyincreased up to 30 min and thereafter, markedly declin-ed by 60 min. Serum A-KG levels in rats treated with2 g/kg A-KG was significantly more as compared to0.5 and 1 g/kg A-KG at all the time points. Also, A-KG levels in 1 g/kg treatment were significantly moreas compared to 0.5 g/kg at 30 min. Figure 10C depictsthe levels of serum thiocyanate after various treat-ments. Thiocyanate levels progressively increased withtime in all the treatment groups and were not signifi-cantly different from each other. However, at 15 minthe levels in 2 g/kg A-KG was significantly elevatedas compared to other treatments, and all the A-KGtreated groups were significantly different from KCNalone at 60 min. This indicates that during the initialphase, thiocyanate production was more conspicuousin rats receiving 2 g/kg A-KG. The thiocyanate levels

Mol Cell Toxicol (2012) 8:83-93 87M

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Figure 7. Lipid peroxidation measured by malondialdehyde(MDA) levels in primary culture of rat hepatocytes after ex-posure to different concentrations of KCN for 6 h. Values areMean±S.E of three different experiments.

Figure 8. Agarose gel electrophoresis of DNA extracted fromprimary culture of rat hepatocytes exposed to different concen-trations of KCN for 24 h. The lanes represent: (1) 1 kb lad-der; (2) control; (3) 1.25 mM KCN; (4) 2.5 mM KCN; (5) 5.0mM KCN; (6) 10 mM KCN and (7) 20 mM KCN.

1 2 3 4 5 6 7

did not show much difference except the highest doseof A-KG.

Discussion

Cyanide is a neurotoxin but its toxic implications inother tissues are also documented8-12. Liver is consi-dered as one of the most commonly affected organsin pre-clinical toxicity studies. Therefore, primary cul-tures of rat hepatocytes serve as a suitable model toassess the cytotoxic potentials of poisons and hepato-

totoxicity in particular17,18,24. The work of Niknahadet al. (1984) refers to screening of various nutrientslike glycolytic substrates and keto-acid metabolitesagainst cytotoxicity of cyanide in isolated rat hepato-cytes20. Cytotoxicity was measured by trypan blue dyeexclusion (TBDE), cellular ATP levels and oxygenconsumption. Authors concluded that keto-acids andaldehytic metabolites of carbohydrates and amino acidsprotected cells from cyanide by more than one mecha-nism. This study was limited to screening of differentcompounds employing isolated rat hepatocytes. Wehad earlier shown the cytotoxicity of cyanide in single

88 Mol Cell Toxicol (2012) 8:83-93C

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Figure 9. Levels of cyanide (A), A-KG (B) and thiocyanate(C) in the culture medium of primary culture of rat hepato-cytes after exposure to 5 mM KCN in the presence or absenceof simultaneous treatment of 5 mM KCN or 10 mM A-KG.The control baseline values were normalized in all the treat-ment groups. Values are mean±SE of three different experi-ments. *Significantly different from KCN; #significantly dif-ferent from KCN++5 mM A-KG at corresponding time pointsat P⁄0.05 (ANOVA-Student-Newman-Keuls).

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Figure 10. Levels of cyanide (A), A-KG (B) and thiocyanate(C) in the serum of rats after oral treatment with 10 mg/kgKCN (�0.75 LD50) in the presence or absence of simultane-ous treatment of 0.5, 1 or 2 g/kg A-KG. The control baselinevalues were normalized in all the treatment groups. Valuesare mean±SE of three different experiments. *Significantlydifferent from KCN; #significantly different from KCN++5mM A-KG at corresponding time points at P⁄0.05 (ANOVA-Student-Newman-Keuls).

cell suspension of rat thymocytes13 and its restorationby A-KG22. Thereafter, cyanide-induced cytotoxicityand altered energy metabolism, and its attenuation byA-KG and N-acetylcysteine (NAC) were shown inPC12 cells14. Further, the nature of cell death was de-lineated in PC12 cells and its response to treatmentswith A-KG and NAC was reported15. We further show-ed the oxidative stress mediated cytotoxicity of cyani-de in LLC-MK2 cells, in the presence or absence ofA-KG and NAC16. All these studies revealed that theprogression of cyanide toxicity and mode of cell deathwere dissimilar in different cells. In continuation ofthis, the present study was undertaken to elucidate thecytotoxicity of cyanide in primary cultures of rat hepa-tocytes and its response to simultaneous treatment ofA-KG. Antidotal efficacy of A-KG against experimen-tal cyanide poisoning has been amply documented21-23.

In the present study, cytotoxicity was characterizedby leakage of intracellular enzymes viz. LDH, ALATand ASAT. Mitochondrial integrity, energy status ofthe cells, MMP and lipid peroxidation were measuredby MTT assay, cellular ATP levels, incorporation ofcationic fluorescent dye (Rho 123) and MDA levels,respectively. DNA fragmentation was visualized byagarose gel electrophoresis of extracted DNA. Addi-tionally, the interaction of A-KG with cyanide wasassessed by the levels of cyanide, A-KG and thiocya-nate in vitro and in vivo. Leakage of LDH serves as anindex of compromised plasma membrane integrity,while leakage of ALAT and ASAT are also critical tocell viability and indicate hepatotoxic potential of achemical24-26. During our preliminary unpublishedwork, different doses of KCN (1.25-20 mM) wereadded to primary culture of rat hepatocytes for 0.5-24h, and cytotoxicity was measured by TBDE and leak-age LDH, ALAT, and ASAT. On the basis of TBDE,35-40% cell death occurred after 6 h in 5 mM KCN,which was accompanied by 40-60% leakage of theenzymes. The IC50 of KCN for TBDE and leakage ofLDH, ALAT and ASAT varied between 4.0 to 6.0 mMfor 6 h exposure period. However, the cytotoxicity of10-20 mM KCN was evident at 2 h itself, which pro-gressively increased with time, whereas 1.25-2.5 mMKCN did not produce appreciable cytotoxicity up to 6h. Data also revealed that when cells were exposed to2.5-10 mM A-KG alone, no cytotoxicity was observ-ed but 20 mM A-KG produced cytotoxicity after 24 hexposure. On the basis of fore going observations,1.25-10 mM KCN for 6 h exposure period was select-ed for our present cytotoxicity studies, and in a separ-ate study, 5 mM KCN was challenged by equimolardose of A-KG. However, effects of KCN on other para-meters occurred at different doses and this kind ofphenomenon has been observed by us during previ-

ous studies as well14-16.The present study revealed that cyanide remarkably

increased leakage of LDH, ALAT and ASAT in thesame order. It suggests that the hepatocytes were acute-ly injured, the cell membrane integrity was compro-mised and the cytosolic enzymes leaked out from thecells. Cyanide also diminished the energy levels ofthe cells, accompanied by mitochondrial dysfunction.Cyanide-induced ATP depletion affects mitochondrialfunction, and disrupts cellular calcium homeostasisleading to activation of Ca2++-dependent plasma mem-brane phospholipases and proteases. This impairs theplasma membrane integrity and makes it leaky27. Mito-chondrial dysfunction leading to hepatocyte injurycould be attributed to cellular perturbations arising outof both energy deficit and oxidative stress caused bygeneration of reactive oxygen species (ROS)27. In thepresent study, ROS levels were not measured but therewas no evidence of lipid peroxidation. Ardelt et al.(1994) also observed that cyanide causes lipid peroxi-dation in mouse brain but not in liver6. Perhaps forthis reason liver damage has not been associated withacute cyanide intoxication28. Moreover, cyanide me-tabolism is largely facilitated by rhodanese, a mito-chondrial enzyme abundantly present in hepatocytes29.Possibly, for this reason the hepatocytes are moreresistant to cyanide as compared to other cells. Thisis evident by the fact that the dose of cyanide requiredto cause cytotoxicity in hepatocytes was very high ascompared to other cells14-16. We observed similar phe-nomenon in our previous studies with isolated rat thy-mocytes. The difference in sensitivity of cyanide canbe attributed to the fact that primary cultures are richin enzymatic and non-enzymatic antioxidants as com-pared to cell lines which usually undergo several pass-age13,21. Also, nutritional status of hepatocytes is close-ly related to its tolerance to hypoxia and hepatotoxicagents as it has been observed that hepatocytes iso-lated from fasted rats were much more susceptible tocyanide, and it largely depended on the glycogen re-serve of the cells20. Cyanide-induced activation of en-donucleases is known to play crucial role in oligonu-cleosomal cleavage of DNA. Single strand breaks ofDNA can also be generated through Ca2++-dependentmechanism in cells exposed to oxidative stress30. Wehave earlier observed oligonucleosomal cleavage ofDNA, characterizing apoptotic type of cell death fol-lowing cyanide exposure in thymocytes and LLC-MK2cells, while PC12 cells were found to undergo necrosisor apoptosis depending on the dose and duration ofcyanide exposure14-16. However, in the present study,we did not observe any kind of DNA damage, possib-ly due to resistance of the cells to oxidative stress.

The present study was also undertaken to elucidate

Mol Cell Toxicol (2012) 8:83-93 89

the interaction of cyanide and A-KG added in differ-ent ratios in culture medium or administered in differ-ent doses in rats, so that bio-availability of A-KGcould be ascertained to optimize its dose for cyanideantagonism. Cyanide is rapidly transported in thebody by blood and about 60% is bound to plasma pro-teins, a small amount is present in the RBCs, and theremainder present as free cyanide31. Therefore, anytherapeutic agent would be of immense value if itcould rapidly sequester free cyanide ion in the body.Norris et al. (1990) injected various molar ratios ofA-KG and cyanide into high pressure liquid chroma-togram and demonstrated that addition of cyanidereduced the peak area of A-KG at a molar ratio greaterthan 1 : 5. Also, blood from naive mice was spikedwith A-KG and cyanide, and A-KG was found to re-duce the peak area of hydrogen cyanide released intothe head space at molar ratios of ¤1 : 2.5. Effect ofcyanide on the ultraviolet spectrum of A-KG was alsodetermined as an indication of binding at 316 nm19. Inthe present study, addition of cyanide and A-KG inmolar ratios of 1 : 1 and 1 : 2, depleted free cyanide inculture medium and the ratio of 1 : 1 was found to bebetter than 1 : 2. This indicates that excess of A-KGdid not enhance the binding and it remained in themedia. The elevated A-KG levels after 60 min can beascribed to delayed dissociation of A-KG from cyano-hydrin complex, while the remaining cyanide was per-haps internalized into the cells and was not availablein the medium. The cyanide profile in in vivo studieswas almost similar to in vitro studies, indicating theconsumption of cyanide by A-KG, while the A-KGlevels were dissimilar. An increased recovery of freeA-KG after 30 min perhaps coincides with its C maxin serum. The in vivo experiments also revealed thatthe bio-availability of A-KG in serum did not com-mensurate with the dose of A-KG administered orallyin rats. At 2 g/kg A-KG, the distribution half-life, eli-mination half-life and C max of A-KG were found tobe 0.35 h, 0.53 h and 36.9μg/mL, respectively32. Also,when A-KG was administered lumenally to pigs, onlya small percentage was found to appear in the portalcirculation, possibly due to mucosal metabolism andlimited absorption33. Intestinal absorption of A-KG isalso known to be limited in young pigs largely due tosubstantial first-pass gastrointestinal metabolism34.Approximately 80% of cyanide is usually eliminatedas thiocyanate, a reaction enzymatically mediatedthrough rhodanese28,29. The thiocyanate profile in invitro system was quite different from in vivo model.This can be attributed to the fact that in vitro systemis a static model, where the internalization of the poi-son is limited, considering that rhodanese is locatedin mitochondria. Also, there could be diminished

turnover of the enzyme in isolated cells and also limit-ed passive entry of cyanide into the cells. Therefore,thiocyanate formation is very limited. In contrast, invivo model is a dynamic system and enzyme-sub-strate interaction is more efficient as there are severalthiol groups present in the body which mediate cya-nide detoxification. Therefore, depleted cyanide levelscorrelated with elevated thiocyanate levels. Although,no dose response effect of A-KG was observed in vivo,certainly 2 g/kg dose is considered to be better than 1g/kg dose20,21. The protective effects of A-KG can beattributed to interaction of A-KG with cyanide to formcyanohydrin complex but other protective mechanismof A-KG cannot be excluded14-16,20,35. In the biologi-cal system, A-KG gets converted into glutamine whichis a precursor for glutathione synthesis and this mayexplain additional protection afforded by A-KG35.Further, antioxidant properties of A-KG have also beendiscussed in our previous studies14-16.

In conclusion, the present study reveals that (i) avery high dose of cyanide is required to produce cyto-toxicity in primary culture of rat hepatocytes, (ii) thecytotoxicity is independent of oxidative stress and DNAdamage, (iii) equimolar dose of A-KG is required toafford maximum protection in vitro, and (iv) exces-sively high dose of A-KG is required for cyanide de-toxification in vivo. Further studies should be takenup in reducing the dose of A-KG without sacrificingits antidotal efficacy.

Materials & Methods

Chemicals and reagents

KCN was purchased from E. Merck, Germany andA-KG (disodium salt), William’s E medium, fetal calfserum (FCS), HEPES sodium salt (4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid, sodium salt), MTT(3-4,5-dimethyl thiazol-Z-yl)-2,5-diphenyl tetrazoli-um), rhodamine 123 and other molecular biologygrade chemicals were from Sigma-Aldrich Co. (St.Louis, Missouri, USA). Hank’s balanced salt solu-tion (HBSS), phosphate buffered saline (PBS) and othercell culture ingredients were from HiMedia (Mumbai,India). All the solutions were prepared fresh in tripledistilled water (TDW).

Animals and treatments

Thirty two male Wistar rats weighing 125-150 g forthe in vivo studies and 18 male rats weighing 300-350g for the preparation of primary culture of hepatocyteswere obtained from the animal facility of Defence Re-search and Development Establishment (DRDE), Gwa-

90 Mol Cell Toxicol (2012) 8:83-93

lior (India). Animals were housed in polypropylenecages and had access to water and standard chow diet(Ashirwad Brand, Chandigarh, India) ad libitum. Ani-mals were maintained in controlled environmental con-ditions of ambient temperature (22±2�C) and relativehumidity of 40-60%, in a 12 : 12 light/dark cycle. Thestudy was approved by the Institutional Ethical Com-mittee on Animal Experimentations. Thirty two ratswere divided into eight groups of four each as follows:(1) Control, (2) 10 mg/kg KCN (�0.75 LD50), (3) 0.5g/kg A-KG, (4) 1.0 g/kg A-KG, (5) 2.0 g/kg A-KG,(6) KCN++0.5 g/kg A-KG, (7) KCN++1.0 g/kg A-KG,and (8) KCN++2.0 g/kg A-KG. Both KCN and A-KGwere administered orally in a volume ⁄10 mL/kg bodyweight, using a 16 gauge oral feeding cannula (HSE-Harvard, Germany). Control animals received equi-valent amount of TDW. Animals were anesthetizedby ether and blood was collected from the retro orbi-tal plexus at given time points for various biochemi-cal estimations.

Isolation of primary culture of rat hepatocytes and treatments

Hepatocytes were isolated from rats following thetwo step perfusion method of Berry and Friend36. Brief-ly, the liver was perfused retrogradely with perfusionbuffer containing collagenase and CaCl2, and the isolat-ed hepatocytes were diluted with William’s E mediumsupplemented with FCS, penicillin-streptomycin, in-sulin, dexamethasone, HEPES and CaCl2. Cells wereincubated in culture plates at a density of 1×105 cells/mL in humidified atmosphere of 5% CO2++95% air at37�C. Viability of cells ranged between 95-98%, asdetermined by TBDE37. After 24 h of incubation aconfluent monolayer of flattened hepatocytes was ob-served. At this point of time the culture medium wasdecanted and replaced with HBSS (pH 7.4). There-after, cells received various treatments, and aliquotsof cell suspension were drawn at indicated time pointsfor various assays.

Biochemical assays

The leakage of intracellular LDH, ALAT and ASATinto extracellular medium was measured using Eco-line diagnostic kits (Merck India Ltd., Mumbai, India).To assess the mitochondrial integrity of the cells, MTTassay was performed as discussed by Mosman (1983)38.Cellular ATP content was measured by biolumine-scence method using Calbiochem kit (Darmstadt, Ger-many). The MMP was measured by incorporation ofa cationic fluorescent dye (Rhodamine123) in theintact cells following the method of Jiang and Acosta(1993)39. The fluorescent intensity of the dye decreases

quantitatively in response to dissipation of the MMP40.The MDA levels were measured as an index of lipidperoxidation, using Calbiochem kit. Cyanide andthiocyanate levels in cell culture medium and serumwere determined by Orion 9458 BN and Orion 9606BN ion selective electrodes (Thermo Electron Corp.,USA), respectively. Prior to cyanide estimation thesamples were subjected to microdiffusion in Conwaycells as per the method of Yagi et al. (1990)41. A-KGwas estimated by the method of Williamson and Cor-key (1969)42.

Agarose gel electrophoresis

DNA fragmentation study was performed by extract-ing intracellular DNA as discussed by Gong et al.(1994)43. Cells (2×107/mL) were grown in 25 cm2 cul-ture flasks and exposed to various treatments for 24h. The isolated DNA was electrophoresed on 1.2%agarose gel impregnated with ethidium bromide. 1 kbDNA served as standard.

Statistical analysis

Each in vitro experiment consisted of three separateplates from the same culture which were averagedtogether (n==1). Each experiment was conductedthrice and the results were expressed as mean±SE ofthree experiments. For the in vivo studies, the resultswere expressed as mean±SE (n==4). The statisticalanalysis was performed using one-way analysis ofvariance (ANOVA) followed by Student-Newman-Keuls multiple comparison test. Statistical signific-ance was drawn at P⁄0.05 using SigmaStat software(SPSS Inc., USA).

Acknowledgements Authors thank Dr. R. Vijayara-ghavan, Director, DRDE, Gwalior for his support andencouragement.

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