artesunate, bone development, malaria, wistar rats
S Adebisi. The Toxicity Of Artesunate On Bone Develpments: The Wistar Rat Animal Model Of Malaria Treatment. The Internet Journal of Parasitic Diseases. 2008 Volume 4 Number 1.
Artesunate is presently well acclaimed a potent anti-malaria, and hitherto, un-resisted by the malaria parasite
Falciparum malaria is a mass killer that went out of control. The drug treatments for this potentially lethal infection that have been most widely recommended and provided over the past 50 years (i.e., chloroquine and sulfadoxine–pyrimethamine) no longer work in most tropical countries. Resistance to these drugs emerged in Asia and South America and spread to Africa. Replacing the failing chloroquine and sulfadoxine–pyrimethamine with effective drugs necessitate the artesunate invention 1 , 2
Pregnant women are a high-risk group in malaria attack, being more susceptible and likely to develop anemia, and if non-immune, are more likely to develop complications. Birth-weight is consistently reduced by malaria. Therefore, pregnant women desperately need effective and safe antimalarial treatments. The main concern surrounding the general deployment of the Artemisinin-based Combination Therapy (ACTs) is their safety in the first trimester of pregnancy 3 , 4 , 5 , 6 . Work by Chinese scientists in rodents and rabbits conducted in the 1970s indicated that early pregnancy exposure could induce fetal resorption. Recent reproductive toxicity studies have confirmed that this is a class effect of the compounds and is seen in all experimental animal species studied. It results from a specific inhibition of fetal erythropoeisis. Fetal resorption would result in early pregnancy loss. Much more worrying is the potential to cause developmental abnormality. In rodents and rabbits at doses close to human therapeutic doses, artemisinins given in a critical period in the early stage of gestation, may also cause limb deformation. In primates, doses of 12–30 mg/kg daily given continuously between days 20 – 50 postcoitum which is equivalent to 20–56 days in human pregnancy caused fetal resorption and mild long bone shortening, but no abnormalities were seen 7 .
In earlier studies, artesunate, an artemisinin antimalarial, had been reportedly embryolethal and teratogenic in rats, with the most sensitive days being 10 and 11 postcoitum 5 . Pregnant rats were administered a single oral dose of 17 mg/kg artesunate on days 10-11 and conceptuses were evaluated through day 14. Among other defects observed, delay in limb and tail development occurred by day 13. While embryos were viable through day 13, about 77% of embryos had died by day 14, presumably due to hypoxia and/or cardiac abnormalities. The same adverse effects were seen in the brain 8 , 9 and in a similar work to compare the developmental toxicity of structurally related artemisinins, dihdyroartemisinin, artemether, and arteether to that of artesunate after oral administration to rats 6
On the other hand, bones are rigid organs that form part of the endoskeleton of vertebrates. Bones function to move, support and protect the body; produce red and white blood cells and store minerals. The process of bone formation occurs in three stages, orchestrated by specialized bone cells that secrete and absorb materials as needed. First, a soft cartilage-based foundation is laid, upon which mature bone will solidify. Then, minerals containing calcium and phosphate are deposited throughout the foundation, creating a framework for the bone. Finally, this raw material is sculpted and hardened into bone. Errors in this process can result in developmental defects and bone diseases 10 , 11 . The present work examines the embryotoxic effects of artesunate on the developing bones.
Materials And Methods
Procurement of animals
20 healthy adult Wistar rats comprising of 20 females and 5 males were obtained from the animal holdings of Faculty of Pharmacy, Ahmadu Bello University, Zaria. The females rats were confirmed virgin, and that none of the rats had been previously used for any experimental studies, from the attendants. The animals were brought into, and kept in the animal holdings of the Department of Human Anatomy, and allowed to adapt to the new environment and further breeding. They were fed on rat pellets (Sander Agro Feeds Ltd., Ibadan), with clean and pure drinking water provided liberally. The room was kept clean and well ventilated. 60 female and 15 male rats from the bred were used for the study. Daily assessment of the weights was done until the animals weighed between 200g and 250g; they were subsequently divided into 3 groups: Control - A; and Experimental - B and - C; with 20 rats in each group. The rats were caged in fours with one male, for mating. Confirmation of gestation was done according to the method of Adebisi 12 .
Administration of drug
One sachet of the drug artesunate (Guilin Pharmaceutical Works, China), which contains 12 tablets was purchased from Famex Pharmacy in Samaru, Zaria, Nigeria. One tablet, which contains 50mg of the active ingredient was dissolved in 50mls of distilled water. According to Eweka and Adjene 9 , oral doses of 2mg/kg and 4mg/kg body weight were administered to pregnant rats in group B and C respectively from the 9th to the 11th day of gestation – being the active osteogenic period in this animal 13 . The group A rats were used as the control which received 2.0ml/kg body weight of distilled water on the same days.
Sacrifice of animals
Two rats were sacrificed each day from day 12 to day 21 by chloroform inhalation and the foetuses retrieved through maternal abdominal incision. The abdominal walls were opened and the full extent of both uterine horns exposed promptly. Before opening either horn, however, a careful count was made of both the foetal swellings and of the metrial glands. Metrial glands are highly vascularised, yellowish nodules, which are found along the meso-metrial margins of the uterine horns where they mark any original implantation site, whether the embryo or the foetus associated with that site survive or not. Records of litter size, number resorbed, dead, living or malformed foetuses were taken. The retrieved foetuses were subsequently weighed and fixed in 10% formol saline. Under stereoscopic microscope, detailed morphological examinations were done to observe any distortion of a gross nature. The foetuses were processed in alizarin red S stains according to the methods of Dix 14 with sodium alizarin sulphonate, (BDH chemicals Ltd., Poole, England), to monitor and score the daily sequence and extent of calcification in the bones. Photographs of the specimens were taken on Nikkon-M-35 597767 camera.
Procedures for bone mineral analysis
The total skeletal calcium and phosphorus contents were determined by the following wet digestion methods in the Soil Science Department, Ahmadu Bello University, Zaria, following these procedures:
The dried bone was ground in to powder, and weighed out 0.5 to 1.0g of the ground sample in to the digestion tube into which was added 25ml of the HNO3 and mix the contents. Place the tube into the digestion block inside a fume hood and set the temperature control of the digestor to 150 ° C. Digest for 1 ½ hours. Increase the temperature to 230 ° C and digest for another 30 minutes. Reduce the digestor temperature back to 150 ° C. Add 1ml of the HCl to the tube and heat the content at 150 ° C for about 30 minutes. Switch off the digestor. Remove the tubes and add about 30 ml. of distilled water to the tube within a few minutes. Add more water to the tube and mix to make up to mark on the tube and mix the content, filter content if necessary or make up to 100ml volumetric flask. Transfer 10ml aliquot from digest into a 50ml volumetric flask. Add 10ml of ammonium molybdate – ammoniumvandase reagent. After 30 minutes read intensity of colour at 470ml wavelength on spectrophotometer to determine calcium level in the solution; and also the phosphorus level, colorimetrically using atomic absorption spectrophotometer SP 192 (PYE UNICAM).
Assessment of bone calcification
Following alizarin red S staining, various skeletal dysgenesis were seen as indicated by impairment of, and retarded calcification, severest in the group C rats. These exhibited non-calcified palate, vertebrae, spine and arches; skull, sternabrae and ribs. Group B foetuses exhibited few cases of missing ribs, vertebrae, phalanges, non-ossified tarsals and metacarpals (Figs. 1 and 2).
Daily monitor of the sequence of calcification shows that there was delay of about 36 to 48 hours or more in the group B rat bones, and delay of about 12 to 24 hours in the group C foetal bones, as compared with the control rat timing; the longest delay being in the skull and limb bones (Tables 1.1 - 1.3).
Assessment of foetal rat morphology
Structural defects observed in the ethanol rat foetuses include microcephally, adactyly, stunted of growth and stature and wrinkled skin. Death in the extremely malformed foetuses in the experimental rats occurred (Table 2).
Assessment of bone mineral values
Calcium and Phosphorus are expressed as milligram per gram dry bone weights. In the group C, the calcium content was low through out the foetal age. Likewise the phosphorus, the levels apparently plateaued through the gestational days and were significantly lower than the control and group B (Table 3). Calcium level was relatively higher in the control and group B foetuses than in the group C through out the gestational days. Phosphorus contents were higher in the control than group B rats. In the group C, the mineral levels remained relatively lowest throughout the foetal days.
Artesunate is a potent blood schizonticide agent for plasmodium falciparum 15 . It is effective against Plasmodium falciparum resistant to all other antimalarial drugs. It does not have hypnozoiticidal activity. Artesunate has been reported to clear fever in patients with severe falciparum malaria 16 - 25 hours after parenteral administration. Artemisinin and its derivatives inhibit erythropoiesis in the early foetus, this could possibly explain the paleness, wrinkle skin observed and fetal resorption seen in the experimental animals. In its malarial combat quest, artesunate binds tightly to parasitized erythrocyte membranes 16 . The functional group responsible for antimalarial activity of artesunate is endoperoxide bond. Release of an active oxygen species from this bond kills the malaria parasite if accumulated in the erythrocytic cells. Artesunate has demonstrated the fastest clearance of all anti-malarials currently used and acts primarily on the trophozite phase, thus preventing progression of the malaria disease. It is converted to active metabolite dihydroartemesinin that then inhibits the sarcoplasmic/endoplasmic reticulum Calcium ATPase encoded by