Macedonian Journal of Medical Sciences

1 Biochemistry Department, Faculty of Basic Medical Sciences, Ladoke Akintola University of Technology, Ogbomoso, Nigeria; 2 Biochemistry Department, College of Medicine, University of Ibadan, Ibadan, Nigeria; 3 Anatomy Department, Faculty of Basic Medical Sciences, Ladoke Akintola University of Technology Ogbomoso, Ogbomoso, Nigeria; 4 Veterinary Microbiology and Parasitology Department, Faculty of Veterinary Medicine, University of Ibadan, Ibadan, Nigeria; 5 Molecular Biology and Biotechnology Division, Nigerian Institute of Medical Research Lagos, Lagos, Nigeria


Introduction
Lamivudine is a cytidine -analogue antiretroviral pro-drug. It is metabolically activated into 51-triphosphorylated derivative by cytosolic kinases in a step-wise manner in the liver. The actively metabolised drug inhibits polymerase gamma, the enzyme that catalyses the synthesis of mitochondrial DNA which may lead to mitochondrial dysfunction in the susceptible tissue [1] with the accompanied clinical effects. Oxidative stress, an imbalance between prooxidant and antioxidant levels favouring the former may arise profoundly as a result of mitochondrial dysfunction, underlies mechanism of a number of drug toxicities [2,3].
Lamivudine is largely excreted in the urine unchanged or to a minor extent as a trans-sulphoxide [4]. Although the drug has been reported to have limited toxicity relative to its pharmacologic counterparts [5], pancreatitis [6], polyneuropathy [7] and Parkinsonism [8] have been documented in patients on prolonged therapy.
Generally, much of the toxicologic information about this drug were obtained from clinical experience and the drug effects on liver especially following prolonged therapy at high doses, had not been precisely determined.
Therefore, the objective of this work was to establish toxic effects of the drug on rat liver at supratherapeutic doses and specifically the mechanism of the toxicity.

Materials and Methods
Standard solutions of the drug (Lamivir brand containing 150 mg lamivudine) were prepared in normal saline or in distilled water.

Embryonic in vitro study
This was carried out according to the method of Gabliks et al., [9]. An aliquot of 0.5 ml of the drug in normal saline equivalent to 4, 20, 100, 500 or 2,500 mg/kg was injected into the chorioallantoic sac of 10day old fertile embryonated egg (average weight 55 g) of Gallus domesticus, using 30 eggs per dose level. An aliquot of 0.5 ml normal saline only, injected into each embryonated egg of equal number served as the control. The response dose (RD50) was calculated from the data obtained using the normograph of Tint and Gillen [10].

Animal experiments
Four dose levels, 4, 20, 100, and 500 mg/kg [11,12], in maximum volume of 2ml. normal saline were adopted. The start dose was the recommended therapeutic dose for humans. The drug was administered orally in single doses to the adult female rats of Wistar strain. There were six rats per dose level. The control rats of equal number received 2ml. of normal saline only. Another group of rats on similar dose regimens were then placed on repeated (daily) oral administration. Both animal groups were observed for 14 and 45 days respectively in individual plastic animal cages. All the animals had access to normal chow and potable water ad libitum.

Sample preparations
Serum samples were prepared from the rat blood and stored in vacutainers. Livers were carefully removed rapidly, washed off blood as well as connective tissues with 1.15% ice-cold KCl solution. Homogenization of the sample took place in ice-cold 0.25 M sucrose solution buffered at pH 7.4 with 40 mM Tris.HCl with a Potter Elvherjem homogenizer. Each sample homogenate was centrifuged at 8000g for 10 minutes in 20% (w/v) buffered medium. The pellet so obtained was taken up in 10ml. of the medium and re-centrifuged. The supernatants were centrifuged at 12000g for 10 minutes to remove light mitochondria [13]. The final supernatants were combined and used as post-mitochondrial fraction for the biochemical analyses.

Analytical methods
In the sera, aspartate and alanine aminotransferases activities were determined using RANDOX (UK) Laboratories analytical kits while total protein concentrations were determined according to Lowry et al., [14], and malondialdehyde (MDA) concentration according to Yuda et al. [15]. Activity of glutathione S-transferase was measured using the method of Habig et al. [16], that of γglutamyltransferase (GGT) was by [17] while that of superoxide dismutase (SOD) was by [18], respectively in the hepatic post-mitochondrial fractions.

Histopathology
The harvested liver tissues of both the chicks and rats were fixed in formol saline for 24 hours and dehydrated thereafter using ethanol in ascending concentrations starting from 70%. The tissues were cleared using xylene and infiltrated with melted wax followed by embedding, sectioning, rehydration in descending grades of ethanol in that order and finally staining with hematoxylene and eosin. Histopathological studies were carried out on the prepared slides and examined under light microscope.

Statistical analyses
One-way analysis of variance (ANOVA) with Dunnett multiple comparisons unpaired t-test was employed. Data were considered significant when p<0.05.

Results and Discussion
The response dose (RD 50 ), the drug dose that killed/caused unhatching of half the population of the chick embryos was calculated as 427 mg/kg (data not shown). No drug-induced mortality among the rats was recorded. Body-weight gain was comparatively reduced in rats on repeated administrations (Tables 1) OA Maced J Med Sci. 3 which might be drug -related (r = -0.822). There were no statistically significant changes in the activities of the serum enzymes as well as protein concentration in rats dosed with single oral administrations when compared with the control or with 4 mg/kg (data not shown). However, the increased serum enzymes activities accompanied with reduced serum protein concentration in rats repeatedly exposed to the drug were statistically significant at 500 mg/kg only, when compared with the control ( Table 2). The specific activities of hepatic GST and GGT in rats were found to increase with applied doses ( Table 3). The specific activities of both enzymes were significantly increased by as little a dose as 20 mg/kg when compared with the control. Also, the hepatic concentration of MDA and specific activity SOD increased in a similar fashion as GST and GGT (Table 4). In comparison with the control, the increased concentration of hepatic MDA was significant as from 100 mg/kg whereas the mean specific activity of SOD was significantly increased by the 20 mg/kg dose (Table 4). The mean ratio of the liver-weight to the total body-weight among the rats repeatedly exposed to the drug increased with the dose. The effect of 500 mg/kg was very significant (Table 5). The results of the histopathologic investigations of the liver tissues of both the chick embryos and the rats following the administration of lamivudine are presented in Figures 1 to 6 and 7 to 11 respectively. It was revealed that as little a dose as 4 mg/kg, (the human therapeutic equivalent of the drug), produced lymphoid aggregates in the chick embryo livers but 20 mg/kg dose produced similar effects in the rats exposed repeatedly to the drug.     The fertile avian embryo is highly sensitive and susceptible to the toxicity of xenobiotics. In addition, the presence of phases 1 and 2 drugmetabolising enzymes in the chicken embryo [19,20] informed our use of the egg titration/embryonation technique for our in vitro toxicity studies. The LD 50 427 mg/kg, the dose responsible for the death of half the population of the embryos, informed our choice of 500 mg/kg as the terminal dose in the animal experiments, and the dose differential in multiples of five as applied is in accord with the geometric progression requirement of moving average method of LD 50 determination [11].
The observed reduction in body-weight gain among the rats on repeated doses relative to the control or to those on single doses could be associated with the drug [21]. Body-weight is known to be determined by complex mechanisms regulating energy balance. A number of neurotransmitter systems acting in several hypothalamic nuclei are pivotal to the regulation of body fat stores. Reduced adipose tissue has been foremost cause of low bodyweight [22]. Abnormal fat metabolism or lipodystrophy has been reported as a consequence of mitochondrial dysfunction associated with many of the nucleoside/nucleotide reverse transcriptase inhibitors [23].
The increased activities of serum enzymes resulted from their release into blood circulation following structural damage to the liver cells by the drug 24]. The low levels of total protein in sera of these animals reflected negative protein turn-over which occurs in pathologic livers [25]. The results of the serum analyses in rats on single doses seemed to show animal recovery from the boost of the drug doses [26].
GGT and GST, both GSH-dependent enzymes, are found in large amounts in the liver. Their respective increases in mean specific activities sustained by GSH supply, was probably an adaptive response to the oxidative challenge occasioned by the drug.
MDA is a product of lipid peroxidation. Lipid peroxidation is a widely accepted mechanism of cellular injury and death. The elevated MDA levels in the rat livers suggested onset of oxidative stress presumably by GSH depletion [27]. The increased specific activity of SOD concerned with superoxide dismutation, might be an adaptive response to the sustained oxidative challenge by the drug.
The observed increase in liver-weight/bodyweight ratio could be connected with lipodystrophy, abnormal fat metabolism; a clinical presentation in NRTI-exposed patients, associated with mitochondrial dysfunction [27]. Further work is needed to confirm lipodystrophy in lamivudineexposed subject whereas data here presented could be a seminal contribution towards quantitative proof of lamivudine-induced lipodystrophy.
In conclusion, the result of the investigations suggested hepatotoxic potentials of lamivudine at high doses (≥ 100 mg/kg) on prolonged administration. Oxidative stress appeared to be the drug mechanism of the toxicity in rat liver. According to the reported British Toxicological Society rating [28], 500 mg 3TC/kg repeatedly taken orally for such periods of time, was ‛harmful' to the female Wistar rat.