Skip to main content
SpringerLink
Log in

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

  • Original Paper
  • Published:
Molecular & Cellular Toxicology Aims and scope Submit manuscript

Abstract

Cyanide is primarily a neurotoxin but its hepatotoxic and nephrotoxic potentials are also known. The present study reports the effect of alpha-ketoglutarate A-KG (2.5–20 mM; 0 min), a potential cyanide antidote on potassium cyanide (KCN; 1.25–20 mM) induced cytotoxicity in primary culture of rat hepatocytes. Cytotoxicity measured at various time points (0.5–24 h), was characterized by increased leakage of intracellular lactate dehydrogenase, alanine aminotransferase and aspartate aminotransferase, accompanied by diminished mitochondrial function (MTT assay), mitochondrial membrane potential (Rhodamine 123 assay), and ATP levels. However, lipid peroxidation (malondialdehyde assay) and DNA damage were not observed. In a separate study, levels of cyanide, AKG and thiocyanate were measured in the culture medium of hepatocytes, treated with KCN (5 mM) and/or A-KG (5 or 10 mM; 0 min), and in the serum of 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-KG interaction was best exhibited when both were added in equimolar dose in vitro. In rats, cyanide levels were significantly reduced by 1 and 2 g/kg A-KG. It can be concluded from the results that, (i) a very high dose of cyanide is required to produce cytotoxicity and other cellular perturbations in rat hepatocytes, (ii) cytotoxicity is independent of lipid peroxidation and DNA damage, (iii) A-KG provides significant protection against cyanide, particularly at equimolar dose in vitro, and (iv) a very high dose of A-KG is required for cyanide detoxification in vivo, suggesting that the dose of A-KG could be reduced by improving its bioavailability.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Pettersen, J. C. & Cohen, S. D. The effects of cyanide on brain mitochondrial cytochrome oxidase and respiratory activiies. J Appl Toxicol 13:9–14 (1993).

    Article  PubMed  CAS  Google Scholar 

  2. Borowitz, J. L., Kanthasamy, A. G. & Isom, G. E. Toxicodynamics of cyanide. In: Somani, S. M. (Eds.), Chemical Warfare Agents. Academic Press, CA, U.S.A. 20936 (1991).

    Google Scholar 

  3. Borowitz, J. L., Isom, G. E. & Baskin, S. I. Acute and chronic cyanide toxicity. In: Chemical warfare agents: Toxicity at low levels, Somani, S. M., Romano, J. A., (Jr.), (Eds.), CRC Press LLC, U.S.A., pp. 301–319 (2001).

    Google Scholar 

  4. Shou, Y., Gunasekar, P. G., Borowitz, J. L. & Isom, G. E. Cyanide-induced apoptosis involves oxidativestress-activated NF-κB in cortical neurons. Toxicol Appl Pharmacol 164:196–205 (2000).

    Article  PubMed  CAS  Google Scholar 

  5. Nicotera, P., Thor, H. & Orrenius, S. Cytosolic free Ca2+ and cell killing in hepatoma 1c1c7 cells exposed to chemical hypoxia. FASEB J 3:59–64 (1989).

    PubMed  CAS  Google Scholar 

  6. Ardelt, B. K., Borowitz, J. L. & Isom, G. E. Cyanideinduced lipid peroxidaion in different organs: subcellular distribution and hyderoperoxide generation in neuronal cells. Toxicology 89:127–137 (1994).

    Article  PubMed  CAS  Google Scholar 

  7. Johnson, J. D., Conroy, W. G., Burris, K. D. & Isom, G. E. Peroxidation of brain lipids following cyanide intoxication in mice. Toxicology 46:21–28 (1987).

    Article  PubMed  CAS  Google Scholar 

  8. Okolie, N. P. & Osagie, A. U. Differential effects of chronic cyanide intoxication on heart, lung and pancreatic tissues. Food Chem Toxicol 38:543–548 (2000).

    Article  PubMed  CAS  Google Scholar 

  9. Philbrick, D. J., Hopkins, J. B., Hill, D. C., Alexander, J. C. & Thomson, R. G. Effects of prolonged cyanide and thiocyanate feeding in rats. J Toxicol Environ Health 5:579–592 (1979).

    Article  PubMed  CAS  Google Scholar 

  10. Leuschner, J., Winkler, A. & Leuschner, F. Toxicokinetics aspects of chronic cyanide exposure in the rat. Toxicol Letts 57:195–201 (1991).

    Article  CAS  Google Scholar 

  11. Okolie, N. P. & Osagie, A. U. Liver and Kidney lesions and associated enzyme changes induced in rabbits by chronic cyanide exposure. Food and Chem Toxicol 37:745–750 (1999).

    Article  CAS  Google Scholar 

  12. Sousa, A. B., Soto-Blanco, B., Guerra, J. L., Kimura, E. T. & Gorniak, S. L. Does prolonged exposure to cyanide promote hepatotoxicity and nephrotoxicity? Toxicology 174:87–95 (2002).

    Article  PubMed  CAS  Google Scholar 

  13. Bhattacharya, R. & Lakshmana Rao, P. V. Pharmacological interventions of cyanide-induced toxicity and DNA damage in isolated rat thymocytes and their protective efficacy in vivo. Toxicol Lett 119:59–70 (2001).

    Article  PubMed  CAS  Google Scholar 

  14. Satpute, R. M., Hariharakrishnan, J. & Bhattacharya, R. Alpha-ketoglutarate and N-acetyl cysteine protect PC12 cells from cyanide-induced cytotoxicity and altered energy metabolism. Neurotoxicol 29:170–178 (2008).

    Article  CAS  Google Scholar 

  15. Satpute, R. M., Hariharakrishnan, J. & Bhattacharya, R. Effect of alpha-ketoglutarate and N-acetyl on cyanide-induced oxidative stress mediated cell death in PC12 cells. Toxicol Ind Hlth 26:297–308 (2010).

    Article  CAS  Google Scholar 

  16. Hariharakrishnan, J., Satpute, R. M., Prasad, G. B. K. S. & Bhattacharya, R. Oxidative stress mediated cytotoxicity of cyanide in LLC-MK2 cells and its attenuation by Alpha-ketoglutarate and N-acetyl cysteine. Toxicol Letts 185:132–141 (2009).

    Article  CAS  Google Scholar 

  17. Ulrich, R. G. et al. Cultured hepatocytes as investigational models for hepatic toxicity: practical applications in drug discovery and development. Toxicol Letts 82/83:107–115 (1995).

    Article  CAS  Google Scholar 

  18. George, E., Hamilton, G. & Westmoreland, C. The use of in vitro models in hepatotoxicity testing. TEN 3:142–152 (1996).

    CAS  Google Scholar 

  19. Norris, J. C., Utley, W. A. & Hume, A. S. Mechanism of antagonising cyanide induced lethality by α-ketoglutaric acid. Toxicology 64:275–283 (1990).

    Article  Google Scholar 

  20. Niknahad, H., Khan, S., Sood, C. & O’Brien, P. J. Prevention of cyanideinduced cytotoxicity by nutrients in isolated rat hepatocytes. Toxicol Appl Pharmacol 128:271–279 (1994).

    Article  PubMed  CAS  Google Scholar 

  21. Bhattacharya, R. & Vijayaraghavan, R. Promising role of α-Ketoglutarate in protecting against the lethal effects of cyanide. Human Exp Toxicol 21:297–303 (2002).

    Article  CAS  Google Scholar 

  22. Bhattacharya, R., Lakshmana Rao, P. V. & Vijayaraghavan, R. In vitro and In vivo attenuation of experimental cyanide poisoning by alpha-kotoglutarate. Toxicol Letts 128:185–195 (2002).

    Article  CAS  Google Scholar 

  23. Bhattacharya, R. Alpha-ketoglutarate: A promising antidote to cyanide poisoning. In: Flora, S. J. S., Romano, J. S., Baskin, S. I., Sekhar, K. (Eds.), Pharmacological Perspectives of Toxic chemicals and Their Antidotes Narosa Publishing House (2004). New Delhi, India. pp. 411–430.

  24. Shoji, R., Sakoda, A., Sakai, Y., Utsuni, H. & Suzuki, M. A new assay for evaluating hepatotoxicity and cytotoxicity using LDL-uptake activity of Liver cells. J Health Sci 46:493–502 (2000).

    Article  CAS  Google Scholar 

  25. Ekwall, B., Clemedson, C., Ekwall, B., Ring, P. & Romert, L. EDIT: A new international multicentre programme to develop and evaluate batteries of in vitro tests for acute and chronic systemic toxicity. ATLA 27:339–349 (1999).

    Google Scholar 

  26. Bhattacharya, R., Lakshmana Rao, P. V., Bhaskar, A. S. B., Pant, S. C. & Dube, S. N. Liver slice culture for assessing hepatotoxicity of fresh water cyanobacteria. Hum Expt Toxicol 15:105–110 (1996).

    Article  CAS  Google Scholar 

  27. Niknahad, H., Khan, S. & O’ Brien, P. J. Hepatocyte injury resulting from the inhibition of mitochondrial respiration at low oxygen concentrations involves reductive stress and oxygen activation. Chemico-Biol Inter 98:27–44 (1995).

    Article  CAS  Google Scholar 

  28. Way, J. L. Cyanide intoxication and its mechanism of antagonism. Ann Rev Pharmacol Toxicol 24:451–481 (1984).

    Article  CAS  Google Scholar 

  29. Baskin, S. I., Horowitz, A. M. & Neally, E. W. The antidotal action of sodium nitrite and sodium thiosulphate against cyanide poisoning. J Clin Pharmacol 32:368–375 (1992).

    PubMed  CAS  Google Scholar 

  30. Trump, B. F. & Berezesky, I. K. Calcium-mediated cell injury and cell death. FASEB J 9:219–228 (1995).

    PubMed  CAS  Google Scholar 

  31. Ryan, J. G. Cyanide. In: Emergency Toxicology, 2nd edn., Viccellio, P. (Ed.), Lippincott-Raven Pub. Philadelphia, pp. 969–978 (1998).

    Google Scholar 

  32. Patra, D. Serum kinetics study of α-ketoglutarate in Wistar rats following single oral administration. A project report submitted by Jai Research Foundation, Valsad, India to DRDE, Gwalior. Personal communication (Study No. 1573), pp. 1–27 (2005).

  33. Buddington, R. K., Pajor, A., Buddington, K. K. & Pierzynowski, S. Absorption of alpha-ketoglutarate by the gastrointestinal tract of pigs. Comp Biochem Physiol A Mol Integr Physiol 132:215–220 (2004).

    Article  Google Scholar 

  34. Lambert, B. D. et al. First-pass metabolism limits the intestinal absorption of enteral alpha-ketoglutarate in young pigs. J Nutr 136:2779–2784 (2006).

    PubMed  CAS  Google Scholar 

  35. Cynober, L. A. The use of α-ketoglutarate salts in clinical nutrition and metabolic care. Clin Nutr Metab Care 2:33–37 (1999).

    Article  CAS  Google Scholar 

  36. Berry, M. N. & Friend, D. S. High yield preparation of isolated rat parenchymal cells. J Cell Biol 43:506–520 (1969).

    Article  PubMed  CAS  Google Scholar 

  37. Bhattacharya, R., Lakshmana Rao, P. V., Parida, M. M. & Jana, A. M. Antidotal efficacy of antioxidants against cyanide poisoning in vitro. Def Sci J 49:55–63 (1999).

    CAS  Google Scholar 

  38. Mosman, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Meth 65:55–63 (1983).

    Article  Google Scholar 

  39. Jiang, T. & Acosta, D. An in vitro model of cyclosporine-induced nephrotoxicity. Toxicol Appl Pharmacol 20:486–495 (1993).

    CAS  Google Scholar 

  40. Johnson, L. V., Walsh, M. L. & Chen, L. B. Localization of mitochondria in living cells with rhodamine 123. Proc Natl Acad Sci 77:990–994 (1980).

    Article  PubMed  CAS  Google Scholar 

  41. Yagi, K., Ikeda, S., Schweiss, J. F. & Homan, S. M. Measurement of blood cyanide with microdiffusion method and ion-specific electrode. Anesthesiology 73:1028–1031 (1990).

    Article  PubMed  CAS  Google Scholar 

  42. Williamson, J. R. & Corkey, B. E. α-ketoglutaratedetermination with glutamate dehydrogenase. Methods Enzymol 13:455–458 (1969).

    Google Scholar 

  43. Gong, J., Trganos, F. & Darzynkiewicz, Z. A selective procedure for DNA extraction from apoptotic cells applicable for gel electrophoresis and flow cytometry. Anal Biochem 218:314–319 (1994).

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Division of Experimental Therapeutics, Defence Research and Development Establishment, Jhansi Road, Gwalior, 474002, India

    Rahul Bhattacharya, Janardhanan Hariharakrishnan & Ravindra M. Satpute

  2. Division of Phytochemistry, Analytical Chemistry and Toxicology, Defence Institute of Physiology and Allied Sciences, Delhi, 110 054, India

    Rajkumar Tulsawani

Authors
  1. Rahul Bhattacharya
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Janardhanan Hariharakrishnan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ravindra M. Satpute
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Rajkumar Tulsawani
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Rahul Bhattacharya.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bhattacharya, R., Hariharakrishnan, J., Satpute, R.M. et al. Cytotoxicity of cyanide in primary culture of rat hepatocytes and its interaction with alpha-ketoglutarate. Mol. Cell. Toxicol. 8, 83–93 (2012). https://doi.org/10.1007/s13273-012-0011-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13273-012-0011-y

Keywords

Search

Navigation