ISPUB.com / IJTWM/4/2/9710
  • Author/Editor Login
  • Registration
  • Facebook
  • Google Plus

ISPUB.com

Internet
Scientific
Publications

  • Home
  • Journals
  • Latest Articles
  • Disclaimers
  • Article Submissions
  • Contact
  • Help
  • The Internet Journal of Third World Medicine
  • Volume 4
  • Number 2

Original Article

Biochemical Evaluation Of Hepatic Dysfunction As A Result Of Halofantrine Toxicity In Wistar Rats

H Nwanjo, G Oze, M Okafor

Keywords

halofantrine, hepatic dysfunction, toxicity

Citation

H Nwanjo, G Oze, M Okafor. Biochemical Evaluation Of Hepatic Dysfunction As A Result Of Halofantrine Toxicity In Wistar Rats. The Internet Journal of Third World Medicine. 2006 Volume 4 Number 2.

Abstract

Halofantrine is a phenanthrene methanol, belonging to the aryl-amino alcohol family, which is widely prescribed for the treatment of infections with chloroquine-resistant strains of Plasmodium falciparium. This study examined biochemical evaluation of hepatic dysfunction as a result of halofantrine toxicity in Wistar rats. Various concentrations of halofantrine (30mg/kg, 60mg/kg and 90mg/kg were administered to the three groups of Wistar rats. The fourth group of animals received distilled water (control). The body weight changes and the relative weight of the liver were measured. The serum hepatospecific markers such as aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP) activities and serum total bilirubin level were also estimated. The halofantrine treated groups had a significant increase in the relative weight of their livers (0<0.05) when compared with control. There was also significant increase in all the enzymes and total bilirubin (p<0.05) when compared with control. The results indicated that halofantrine might have hepatotoxic effect in Wistar rats.

 

Introduction

Malaria is an infectious diseased that continues to be associated with considerable morbidity and mortality and significant social and economic impact in developing countries. According to the World Health Organisation (WHO) Malaria is endemic in 91 countries, predominantly in Africa, Asia and Latin America with about 40% of the world's population at risk (WHO, 1996). It is caused by parasites that belong to the genius plasmodium with four different species namely Plasmodium falciparium, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae.

The resistance of these parasites especially Plasmodium falciparium to most antimalarial drugs is rampant and on the increase too. This is due to remarkable adaptability of the parasites to drugs and man's abuse of malaria drugs for prophylaxis and treatment of undiagnosed fever in endemic areas.

Halofantrine, a lipophilic phenanthrenemethanol belonging to the aryl amino alcohol is used for the treatment of acute uncomplicated multi-drug resistant malaria (Philips-Howard and Wood, 1996). It is schizonticidal with high degrees of activity against the asexual erythrocytic stage of malarial infections caused by single or mixed infections of Plasmodium falciparium or Plasmodium vivax. It has limited effect against the exoerythrocytic or gametocyte stages of malaria parasites, (SmithKline Beecham, 1998).

Clinical treatment with halofantrine is often accompanied by serious side effects such as abdominal pain, diarrhoea, prolongation of QTc interval and arrythmias that could be fatal. However, an increasing number of reports describing serious complications in the last few years have raised some doubt about the safety of halofantrine (Touze and Fourcarde, 1997) Halofantrine has been reported to be cardiotoxic (Nosten et al, 1993, Touze and Fourcarde, 1997). This may be similar to that of chloroquine, quinine and mefloquine by forming toxic complexes with ferritoporphyrin IX that damage the membrane of the parasite or organs. Also several studies have shown that other antimalarials such as chloroquine, and quinine are hepatoxic (Okonkwo et al, 1997, Debra and Megan, 1999).

Self-medication is especially common in developing countries like Nigeria and sometimes at dosages above the therapeutic dose. It has been shown that drugs that are effective in malaria treatment may cause damage to certain organs of the body, it is therefore important for drugs to be avoided and only to be safely administered when necessary. This study was therefore, carried out on the biochemical parameters as indices for assessing halofantrine-induced hepatotoxicity.

Materials And Methods

Animals

Twenty four Wistar rats bred in the Central Animal House of College of Medicine and Health Sciences, Imo State University, Owerri, Nigeria were used in the present study. They were maintained at a temperature range of 25oC to 30oC and a 12h light 12h dark cycle. They were fed with commercial growers mash, product of Tops Feeds Ltd, Sapele, Nigeria. Water and feed were provided ad libitum and this continued until the rats weighed between 200-300g.

Drugs

Halofantrine (HAL FAN) (20 mg/ml suspension) used in this study was the product of Smith Kline and French Laboratories, Nanterre, Cedar, France and was purchased from a standard pharmacy shop in Owerri, Nigeria. The drugs suspension was administered to the animals on the basis of their body weight. The suspension of halofantrine was administered orally using cannula.

Experimental designs

Animals were randomly assigned to 4 experimental groups (n = 6x4 groups) each having similar body weights.

Group I: (Control): Animals received distilled water Group II: Animals in this group were given 30 mg/kg halofantrine suspension in three divided doses. Group III: Animals in this group were given 60 mg/kg halofantrine suspension in three divided doses. Group IV: Animals in this group were given 90 mg/kg halofantrine suspension in three divided doses.

The drugs were administered orally for a period of 14 days. All the animals were allowed free access to food and water till the end of the experiment.

Blood Sample Collection

Twenty four hours after the last doses were administered, the animals were weighed and then anaesthetized with chloroform vapour, quickly brought out of the jar and sacrificed. Whole blood was collected by cardiac puncture from each animal into clean dry centrifuge tubes. The blood were allowed to stand for about 30 minutes to clot, and further centrifuged at 10,000 rpm for 5 minutes using Wisperfuge model 1384 centrifuge (Samson, Holland). Serum was separated from clot with Pasteur pipette into sterile serum sample tubes for the measurement of biochemical parameters. The liver from both control and test animals were removed and immediately washed with physiological saline and weighed.

Biochemical Analysis

Serum total bilirubin level was estimated based on Van den Berg reaction (Malloy and Everlyn, 1937). Diazotised sulphonilic acid (0.5ml) reacts with bilirubin in diluted serum (0.2ml serum + 1.8ml distilled water) and forms purple coloured azobilirubin, which was measured at 540 nm. Activities of serum aspartate transaminase (AST) and Alanine transaminase (ALT) were assayed by the method of Reitman and Frankel (1957). 0.2 ml of serum with 1ml of substrate (asparatate and ?-ketoglutarate) for AST, alanine and ?-ketoglutarate for ALT, in phosphate buffer pH 7.4) was incubated for an hour in case of AST and 30 minutes for ALT. 1ml of DNPH solution was added to arrest the reaction and kept for 20 minutes in room temperature. After incubation 1ml of 0.4N NaOH was added and absorbance was read at 540 nm. Activities expressed as IU/L.

Based on the method of King and Armstrong (1934) alkaline phosphates activities was assayed using disodium phenylphosphate as substrate. The colour developed was read at 680nm after 10 minutes and activities of ALP expressed as IU/L.

Statistical Analysis: statistical evaluation of data was performed by using one-way analysis of variance (ANOVA) followed by Duncan's multiple range test (DMRT) (Duncan, 1957).

Result

Table 1 shows the effect of halofantrine on body weight changes of Wistar rats and mean relative liver weight. The control and 30mg/kg halofantrine groups showed slight gain in body weight whereas the 60mg/kg and 90mg/kg halofantrine groups had slight loss in body weight. There was significant increase in the relative weight of the liver in 60mg/kg and 90mg/kg halofantrine groups (p< 0.05) when compared with the control.

Figure 1
Table 1: Mean body weight changes and relative liver weight before and after treatment with halofantrine in Wistar rats.

The changes in the mean value of serum bilirubin and serum hepatospecific markers in all the groups are shown in table 2. There was a significant increase in the levels of total and conjugated bilirubin concentrations and AST, ALT and alkaline phosphatase activities in both 60mg/kg and 90mg/kg halofantrine groups (p < 0.05) when compared with control while 30mg/kg halofantrine groups showed significant increase only in serum ALT and AST activities (p <0.05) when compared with the control. Two animals died in the 90mg/kg dose group while one animal died in 60mg/kg, no death was seen in 30mg/kg halofantrine group.

Figure 2
Table 2: Mean values of activities of serum AST, ALT, ALP and levels of bilirubin in normal and experimental rats.

Discussion

The observed increase in the relative weight of the liver in this study indicates that the drug might have toxic effect on this organ. It has been reported that increase or decrease in either absolute or relative weight of an organ after administering a chemical or drugs is an indication of the toxic effect of that chemical (Simons et al, 1995).

The results of this study revealed that halofantrine might have deleterious effects on the liver of Wistar rats. Serum AST, ALT, ALP and bilirubin are the most sensitive markers employed in the diagnosis of hepatic damage because they are cytoplasmic enzymes released into circulation after cellular damage (Sallie et al, 1991). The increased activities of AST, ALT, ALP and the level of bilirubin in serum indicate halofantrine - induced hepatocellular damage. Liver enzymes are usually raised in acute hepatoxicity but tend to decrease with prolonged intoxication due to damage to the liver cells (Cornellius, 1979). This was confirmed by an earlier study (Obi et al, 2004), in which the marked elevations of hepatic marker molucules were reported. Other antimalarials such as chloroquine (Pari and Murugavel, 2004), amodiaquine (Farombi et al, 2000) and quinine (Debra and Megan, 1999) are also reported to induce hepatic damage.

It has been suggested that halofantrine by virtue of its lyophilic character (Hurberstone et al, 1996), can be expected to permeate biomembraneous barriers (Bloom and Fawceth, 1973). Pretreatment with halofantrine has evidently been shown to inhibit Na+-K+-ATPase and Ca2+- Mg2+-ATPase pumps. Also many of the cell proteases and phospholipase are activated in the presence of calcium ions. The activation of phospholipases has a damaging effect on the membrane, causing release of free fatty acids and phospolipids. These are toxic and can also initiate arachidonic acid production resulting in production of free radicals (Murphy et al, 1983). It has been reported that halofantrine pre-treatment enhances the levels of nicotinamide adenine diphosphate (NADPH), cytochrome C reductase and cytochrome Gs, both enzymes being involved in metabolism of endogenous and exogenous compounds (Voznessensky and schenkman, 1994). The enhancement of these two enzyme systems will obviously lead to accumulation of free radical oxygen species, since the enzymes have been shown to generate superoxide radical (Gibson and Sket, 1992). These may in part explain the level of bilirubin in serum manifested in the halofantrine induced hepatocellular damage in Wistar rats.

This study has established the hepatoxic potential of high doses of halofantrine in Wistar rats. The dose used in this work is high in comparison with the therapeutic dose levels in humans because small laboratory animals eliminate drugs faster than humans (Laumann et al, 1995). From this study, however, it is found that halofantrine induced hepatocellular damage at high concentrations causes increase in serum hepatospecific markers in Wistar rats.

Correspondence to

Harrison Ugo Nwanjo,Ph.D, Department of Medical Lab. Science, Imo State University, Owerri, Nigeria. E-mail – harrisonnwanjo@yahoo.co.uk GSM-08033525389

References

r-0. Adjene, J.O., Agoreyo, F.O. (2003). Effect of halofantrine (Halfan) on the histology of the ovary of mature female Wistar rat. Afri. J. Reprodu. Health. 7(1): 113-120.
r-1. Bloom, W., Fawcett, D.W. (1975). Textbook of histology. 10th Edition. Company, Philadelphia. Pp 805-857
r-2. Cornelius, C.E. (1979). Biochemical evaluation of hepatic function in dogs. J. Am. Hosp. Ass. 15: 25-29.
r-3. Debra, K.F., Megan, N.L. (1999). Quinine-induced hepatotoxicity. Ann. Pharmacother.33:32-34.
r-4. Duncan, B.D. (1957). Multiple range tests for correlated and heteroscedastic means. Biometrics. 13: 359-364
r-5. Gibson, G. and Skett, P. (1992). Enzymes and free radical generation. In: Introduction to drug metabolism. Chapman and Hall, London. Pp 223 –228.
r-6. Humberstone, A.J., Porter, C.J., and Chairman, W.N. (1996). Antimalaral in dermatology. J. Pharm. Sci. 85: 525-529.
r-7. King, E. J and Armstrong, A.R. (1934). Determination of serum and bile phosphatase activity. J. Canad. Med. Assoc. 31: 376-378
r-8. Laumann, H., Lullmann-Rauch, R., Wassermann, O. (1995). Drug induced phospholipodosis Crit Rev. Toxicol. 4: 185-218.
r-9. Malloy, E. Evelyn, K. (1957). Colorimetric method for the determination of serum oxaloacetic and glutamic pyruvate transaminase. Am. J. Clin. Pathol. 28: 56-63.
r-10. Murphy, E., Aitcon, J. F., Horres, C.R., Lieberman, M. (1983). Halofantrine enhances the level of NADPH cytochrome reductase. Am. J. Physiol. 245: 316-321.
r-11. Nosten, F., Ter Kulie, F.O., Luxemburger, C. Woodrow, C., Kyle, D.E., chongsulphajaisiddhi, T., white, N. J. (1993). Cardiac effects of antimalarial treatment with halofantrine Lancet: 34 (8852): 1054-1056.
r-12. Obi, E., Orisakwe, O. E., Asomugha, L.A., Udemezue, O.O. (2004). The Hepatotoxic effect of halofantrine in guinea pigs. Indian J. pharmacy. 36(5): 303-305
r-13. Okonkwo, C.A., Agomo, P.U., Mafe, A.G. Akundele S.K. (1997). A study of the hepatoxicity of chloroquine (SN-7618) in mice.Nig. Qt. J. Hosp. Med: 7:183-187.
r-14. Orisakwe, O. E., Obi, E., Udemezue, O. O. (2003). Effect of halofantrine on testicular architecture and testosterone level in guinea pigs. Enr. Bull.Drug Res.11:105-109.
r-15. Pari, L. and Amali, R.D. (2005). Protective role of tetrahydrocurcumin (THC) an active principle of tumeric on chloroquine induced hepatoxicity in rats. J. Pharm. Pharmaceut. Sci. 8(1): 115-123.
r-16. Philips- Howard, P.A., and Wood, D. (1996). Halofantrine in the treatment of multi-drug resistant malaria. Drug safety. 14(3): 141-142.
r-17. Reitman, S., Frankel, S.A. (1957). Colorimetric method for the determination of serum oxaloacetic and glutamic pyruvate transaminase. Am. J. clin. Pathol. 28:56-63.
r-18. Sallie. R., Tredger, R.S., Williams, R. (1991). Drugs and the liver. Biopharmaceutical Drug Dispos, 12: 251-259.
r-19. Simons, J.E., Yany, R.S., Berman, F. (1995). Evaluation of the nephrotoxicity of complex mixture containing organics and metals. Advantages and disadvantages of the use of real-world complex mixture. Environ. Health Prospect. 103 (supple. 1): 67-71
r-20. SmithKline Beccham, (1998). Halofantrine in Gillis M.C editor. CPS Compendium of pharmaceuticals and specialities 33rd edition, Ottawa, Canadan Pharmacists Association. P. 689-700.
r-21. Tonze, J.E. and Fourcade, L. (1997). Is halofantrine still advisable in Malaria? Annals Trop. Med. Parasitol. 91: 867 –873.
r-22. Voznessensky, A.I. and Schenkman, J.B. (1994). Enhancement of Cytochrome Gs by Halofantrine. T. Bio. Chem. 269: 15724-15731.
r-23. World Health Report. (1996). The Director General, fighting disease, fostering development, World Health Report, General ISSN 1020-6311.

Author Information

Harrison Ugo Nwanjo, Ph.D.
Department of Medical Lab. Science, Imo State University

Gabriel Oze, M.Sc.
Department of Medical Biochemistry, Imo State University

Matthew Chidi Okafor, Ph.D.
Department of Medical Biochemistry, Imo State University

Download PDF

Your free access to ISPUB is funded by the following advertisements:

 

 

BACK TO TOP
  • Facebook
  • Google Plus

© 2013 Internet Scientific Publications, LLC. All rights reserved.    UBM Medica Network Privacy Policy

Close

Enter the site

Login

Password

Remember me

Forgot password?

Login

SIGN IN AS A USER

Use your account on the social network Facebook, to create a profile on BusinessPress