Assessment of the hepatorenal and hematological parameters of rats exposed to graded doses of lysine acetylsalicylate

Lysine acetylsalicylate (LAS) is an analgesic used for the prevention of cardiovascular disease with few adverse though its effect on the hepatorenal and hematological parameters remains unclear. 45 mature male Wistar rats were equally divided into three groups. Group I, II, and III received daily intramuscular doses of 0.25 mL saline, 40, and 80 mg/kg b. wt. of LAS respectively for 2-weeks. After 2, 4, and 8 weeks from beginning of drug administration, 5 rats/group were assessed for hepatorenal function, complete blood-count, and histopathological findings of liver and kidney. LAS induced a significant increase (p ≤ 0.05) in serum levels of aspartate aminotransferase, alanine aminotransferase, urea, and creatinine in a dose-dependent manner, but a significant decrease (p ≤ 0.05) observed in the hematological parameters. Histopathological alterations were a dose-dependent manner, and persisted for 8-weeks at the higher dose. In conclusion, LAS induced reversible deleterious effects on hepatorenal functions and various hematological parameters in a dose-dependent manner.


Introduction
In human and veterinary medicine, the non-steroidal anti-inflammatory drugs (NSAIDs) are advised for alleviating inflammation, pain, and fever through inhibition of cyclooxygenases and prostaglandin synthesis [1,2]. In spite of great effort for improving the efficacy, safety, and potency of NSAIDs, adverse effects such as hepatorenal toxicity [3,4], gastrointestinal irritations [5], interference with hemostasis [3,4], cardiovascular [6,7] and reproductive problems are still common [4,[8][9][10]. Acetylsalicylic acid (ASA) or aspirin, obtained from the bark of the white willow tree, remains the most prescribed analgesics and antipyretics [2,11,12]. It is used for the prevention of heart strokes and attacks [13], myocardial infarctions [14,15] and colorectal cancer [16,17]. Prevalence studies in the United States showed a marked shift in use for the prevention of cardiovascular disease (CVD), which can be attributed to ASA use [18]. Among those 40 years and older, 23.4% reported taking daily doses of ASA for primary prevention of CVD, and out of these, 22.8% did so without a physician's order [18]. ASA at low doses irreversibly inhibits cyclooxygenase enzyme at platelets, preventing thromboxane's synthesis, thereby avoiding cardiovascular problems [19,20]. However, new studies questioned its overall benefit for primary prevention, as it is shown to increase the risk of major bleeding with no significant reduction in CVD risk and all-cause mortality [21,22].
Lysine acetylsalicylate (LAS) is water-soluble, making it useful for parenteral (i.m. and i.v.) administrations [23]. Only for a short period of time, ASA and LAS can be identified in the plasma as a result of its hydrolysis to salicylic acid (SAL) by carboxylesterases in plasma, liver, and red blood cells (RBCs) [24][25][26]. Because of this fast deacetylation, it was expected that the antiinflammatory effects of acetylsalicylate by-products are primarily attributed to SAL [27]. LAS applies a distinct analgesic effect compared to other agents and its bioavailability after i.m. administration was similar to i.v. and significantly exceeds orally administered agents [28].
The pharmacokinetic parameters of LAS were recorded and LAS was found to be widely distributed in all tissues; all of its pharmacokinetic characteristics were influenced by body weight, height, and body surface area. Repeated administration of high doses of LAS in patients with low body weights may give rise to toxic effects [29,30]. Toxicity studies showed no neurotoxicity after a chronic intrathecal administration of LAS in rats [31]. Furthermore, the role of LAS in preventing the toxicity of several materials such as paraquate [32] and aluminum was also observed [33]. LAS is used in various levels of prevention of heart diseases and stroke [34], presenting potent antiplatelet complex with fewer gastrointestinal adverse effects than aspirin [35]. Some revisions have compared aspirin with LAS in patients with persistent coronary artery disease and showed higher efficacy on platelet inhibition with LAS [1,[36][37][38].
There is not enough available data concerning the influence of LAS on hepatorenal and blood images. The present study was designed to investigate the effect of LAS on the complete blood count, serum levels of AST, ALT, urea and creatinine, as well as its effects on the histopathological findings in the liver and kidney. The hypothesis of this study was that subacute administration of LAS is either safe or adverse to mammals, and these effects are either reversible or irreversible using male rats as a model.

Drugs and chemicals
LAS was obtained from ASPEGIC @ (900 mg/vial equal to 500 mg acetylsalicylate; Produced by AMRIYA Pharmaceutical Industries, Alexandria, Egypt under license of laboratories SYNTHELABO FRANCE, LE PLESSIS ROBIN-SON -FRANCE). All other chemicals used in this study were of analytical grade and were provided by the local suppliers.

Animals
A total of 45 adult male Wistar albino rats weighing approximately 170 ± 10 g each (8-week age) were used. The animals were housed in a room with a constant temperature (25 ± 2.0°C) and humidity (55 ± 5%) in polypropylene cages with alternating 12-h light/dark cycles and received a normal pellet diet specially prepared for laboratory rodents and water ad libitum. Before conducting the experiment, the animals were acclimatized for two weeks.

Experimental design and sample collection
Three groups were used in this study, and each group contained an equal number of rats. Group I, the control group, received only the vehicle (0.9% saline, 0.25 mL/rat), while Group II received 40 mg/kg b. wt. of LAS once daily via i.m. administration (calculated according to Paget and Barnes) [39], and Group III received 80 mg/kg b. wt. of LAS once daily. The duration of the experimental treatment was two weeks. For the sample collection directly after drug administration, 5 rats from each group were fasted for 12 h, anesthetized with light ether and sacrificed by decapitation for blood collection to assess the biochemical parameters. This was repeated after 4 and 8 weeks from the beginning of the experiment (sampling was as given in Table 1).
This design shows the sub-acute duration of LAS administration and the study of its actions in the subchronic period. Previous studies showed a variable duration of administration. In the present study the sampling was extended until the 8th week to investigate the possible recovery effects of LAS, which may extend to 3 months [33,40].

Blood analysis
Hemoglobin concentration was determined as cyanomethemoglobin using Sahl's method according to the method described by Benjamin [41]. Platelets, erythrocytes, and total leukocyte counts were performed using a double improved Neubauer hemocytometer (Dacie and Lewis) [42] As described briefly, total white blood cell (WBC) and red blood cell counts were determined in a Neubauer counting chamber after dilution of blood samples with specific diluents using the hemocytometer technique. Platelet count was estimated from Giemsa-stained blood smears and microscopically examined under high magnification.

Biochemical analysis
Total protein and albumin were measured as described previously using the biuret and bromocresol green methods by Lubran [43] and Doumas and Biggs [44], respectively. Serum protein reacts with cupric ions in an alkaline medium to produce a violet colour. The intensity of the colour, which has a maximum absorption at 540 nm, is proportional to the protein concentration. The globulin value was obtained by subtracting albumin from total protein values using the method described by Coles [45]. Serum AST, ALT, urea, and creatinine levels were measured using commercial kits from Diamond Diagnostic (Egypt).

Histopathological studies
Liver and kidney sections were collected immediately after decapitation. The sections were fixed in neutral formalin 10%, dehydrated in graded ethyl alcohol (50-100%), and embedded in paraffin wax. Then, the samples were cut into 4-5 μm thick and stained with H&E for microscopic investigation [46].

Statistical analysis
The obtained data were presented in the current study as means ± standard error (SE) and analyzed using ANOVA. Statistical significance was set to P ≤ 0.05. Significant differences between means were compared using Duncan multiple range tests, and calculations were carried out using the SAS system [47].Values within each raw not sharing a common superscript letter (a, b, and c) are significantly different (p < 0.05).

Ethical statement
The experimental protocol used in this study was carried out in accordance to the Guide for Care and Use of Animals, which was established by the National Research Council (NRC, 1995). This study was approved by the local university ethics committee for animal research.

Effect of Lysine acetylsalicylate on haematological pictures of male rats
Daily intramuscular injection of lysine acetylsalicylate at a dose of 80 mg/kg b. wt. for 14 days in adult male rats induced a significant decrease in the counts of red blood cells, white blood cells, platelets, and hemoglobin percent throughout the experimental period as compared with the other groups. The administration of 40 mg/kg bw. significantly decreased red blood cells and white blood cells after 2 and 4 weeks, but hemoglobin percent and platelet count significantly decreased only after two weeks from the beginning of its administration ( Table 2).

Effect of lysine acetylsalicylate on serum total protein, albumin, and globulin levels in male rats
Daily intramuscular injection of lysine acetylsalicylate at a dose of 40 mg/kg b. wt. for 14 days in adult male rats induced a significant decrease in total protein, albumin, and globulin levels after two weeks, while there was a non-significant effect on these parameters at 4 and 8 weeks after the injection (Table 3). Daily intramuscular injection of LAS at a dose of 80 mg/kg bw. for 14 days in adult male rats induced a significant decrease in total protein, albumin, and globulin levels after 2 and 4 weeks, while there was a non-significant effect on these parameters 8 weeks after the injection ( Table 3).

Effect of lysine acetylsalicylate on serum ALT, AST, urea, and creatinine levels in male rats
From Table 3, it can be observed that daily intramuscular injection of lysine acetylsalicylate at 40 or 80 mg/kg bw. for 14 days in adult male rats induced significant elevations in the serum levels of ALT, AST, urea, and creatinine after both 2 and 4 weeks from the beginning of treatment as compared with the control group. There were no significant effects on these parameters eight weeks after the start of treatment (Table 3).

Histopathological findings in the liver
Microscopically examined livers of rats administered with the vehicle showed a normal histological structure of hepatocytes that were radially arranged around the central veins ( Figure 1A). Two weeks post-LAS (40 mg/kg b. wt.), livers revealed multifocal to coalescent areas of vacuolar and hydropic degeneration of the hepatocyte cells, particularly in the periportal areas ( Figure 1B). Four weeks post-LAS (40 mg/kg b. wt.), livers revealed mild hydropic and fatty degeneration of hepatocytes ( Figure 1C). The degenerated hepatocytes exhibited a marked enlargement of the cells due to empty vacuoles that distended the cell cytoplasm. Eight weeks post-LAS (40 mg/kg b. wt.), the livers showed dilatation and congestion of the portal blood vessels with normal histological appearance of the hepatocytes ( Figure 1D). Two weeks post-LAS (80 mg/kg b. wt.), livers revealed focal areas of coagulative necrosis characterized by the retention of hepatic cord architecture and shrunken hepatocytes with hypereosinophilic cytoplasm and pyknotic nuclei. Multifocal areas of lytic necrosis, with loss of cord architecture, were replaced with erythrocytes and eosinophilic material (edema) ( Figure 1E). Four weeks after LAS treatment (80 mg/kg b. wt.), the livers of the examined treated rats revealed congestion of the central and portal veins with moderate mononuclear inflammatory cellular infiltration in the portal areas. Multifocal vacuolar and hydropic degeneration of the hepatocyte cells in the periportal zone of the hepatic lobules were characteristically observed. Moreover, small areas of lytic necrosis, with loss of cord architecture, were replaced by erythrocytes ( Figure 1F). Eight weeks post-LAS (80 mg/kg b. wt.), the livers showed the activation and enlargement of Kupffer cells with a normal architecture ( Figure 1G).

Histopathological findings in the kidney
Microscopic examination of the kidneys of rats administered with the vehicle revealed normal histological appearance of the glomeruli and convoluted renal tubules (Figure 2A). Two weeks after LAS treatment (40 mg/kg b. wt.), the examined kidneys showed mild congestion of the renal blood vessels. In addition, the perivascular and periglomerular interstitium of the renal cortex was expanded by mononuclear inflammatory cells in moderate numbers, mostly lymphocytes and macrophages ( Figure 2B). Four weeks post-LAS (40 mg/kg b. wt.), the examined kidneys revealed degenerative changes characterized by vacuolar and hydropic degeneration of the epithelium lining. Degenerated tubular epithelial cells were swollen with vacuolated cytoplasm and the perivascular interstitium of the renal cortex was expanded by edema admixed with mononuclear inflammatory cells in moderate numbers, mostly lymphocytes and plasma cells ( Figure 2C). Eight weeks post-LAS (40 mg/kg b. wt.), the examined kidneys revealed degenerative changes in the lining epithelium of the proximal and distal convoluted tubules characterized by vacuolar and hydropic degeneration were commonly noticed. Two weeks post-LAS (80 mg/kg b. wt.), the kidneys showed marked pathological changes in the renal cortex. Glomerular tufts exhibited mesangial/endothelial cell necrosis with hypereosinophilic cytoplasm and pyknotic nuclei ( Figure 2E). Four weeks post-LAS (80 mg/kg b. wt.), the examined kidneys revealed shrinkage of the glomerular tuft and ectatic uriniferous spaces containing pale, eosinophilic, homogenous material within glomeruli. The lining epithelium of the proximal and distal convoluted tubules was swollen and exhibited vacuolar and hydropic degeneration ( Figure 2F). Eight weeks post-LAS (80 mg/kg b. wt.), the examined kidneys revealed large vesicular nuclei and pale basophilic cytoplasm in the regenerating tubules. Multifocally and predominantly around the glomeruli, the interstitium was expanded by aggregates of many lymphocytes and variable amounts of fibrosis. Occasionally, within glomeruli, uriniferous spaces were ecstatic and contained pale, eosinophilic, homogenous material (protein), as well as shrinkage of the glomerular tuft ( Figure 2G).

Discussion
LAS (a water-soluble form of aspirin administered via injection) and aspirin induce similar anti-inflammatory activity in vivo. The study hypostasis was that subacute administration of LAS is either safe or adverse to  mammals, and these effects are either reversible or irreversible using male rats as a model, and the outcome of the study seems to support and contribute to our hypothesis. To the best of our knowledge, this is the first study to show the influence of a sub-acute administration of LAS on complete blood count, liver, and kidney function. LAS (80 mg/kg, b. wt. i.m. for two weeks) induced a significant decrease in the counts of red blood cells, white blood cells, platelets, and hemoglobin percent throughout the experimental period. The addition of 40 mg/kg b. wt. significantly decreased red blood cells and white blood cells after 2 and 4 weeks, while the hemoglobin and platelet counts were significantly reduced after 2 weeks from the beginning of its administration. It is well known that blood is a composite of 2-phase fluid: formed components and plasma. The formed components contain red blood cells, white blood cells, and reticulocytes (platelets). The main task of red blood cells is to carry and supply oxygen to the outlying tissues within the body. However, the complex fluid dynamics occurring in microvessels are mainly manipulated by the mechanical and flow properties of red blood cells [48]. Hence, modifications within the biomechanical properties of red blood cells probably cause a decrease in oxygen and supplement supply to marginal tissues [49]. White blood cells, besides their essential role in the immune system and inflammation, play a role in modulating the blood stream [50].
Aspirin at low doses was reported to have no significant effects on the number of red blood cells [51]. Meanwhile, a decrease in the white blood cell number associated with a remarkable decrease in the count of monocytes possibly indicates that the phagocytic function of the body has been affected by aspirin [52]. Aspirin can cause anemia and reduction in the blood oxygen-carrying capacity, and hence the amount of oxygen delivered to marginal tissues [53] through a significant decrease in the values of PCV and Hb [54]. Additionally, our results are in accordance with Meischer; [55] Rainsford; [56] Navratil et al. [57], who stated that low doses of aspirin significantly decrease red blood cells and white blood cell counts, and attributed this reduction to the inhibitory action on bone-marrow hematopoiesis caused by aspirin. Many authors have reported the mechanisms of these effects, and Meischer [55] and Rainsford [58] stated that aspirin induced potential acetylation of bone marrow macromolecules, causing blood disorders. Sarkar et al., [59] Tomoda et al., [60] Modi, and Merchant [61] reported that aspirin modifies the membrane permeability of red cells by stopping ion-dependent ATPase and cholinesterase activity. Dacie and Lewis [62] reported that intravascular hemolysis could be a major contributing factor to the decline in red cell number.
LAS induced significant elevations in serum levels of ALT and AST after 2 and 4 weeks from the beginning of treatment. Eight weeks after the beginning of treatment, there were no significant effects on these parameters. These findings are correlated with those observed by Vyas et al. [54] where the activities of AST and ALT were significantly elevated by aspirin. Increased activity of AST and ALT is an indication of hepatic damage; [63] this reflects that LAS has hepatotoxic effects. These toxic effects could result from the inhibition of prostaglandin synthesis induced by NSAIDs, which results in vasoconstriction and diminished organ perfusion, which plays a role in tissue abnormalities [64]. In addition to compromising the cellular defense system against reactive molecule attacks, increased serum levels of ALT and AST seem to have profound impacts on the normal function of hepatocytes. The liver is well known as a primary target organ for the metabolism of drugs and hence more susceptible to toxicity. In these cases, liver toxicity might be a consequence of idiosyncratic metabolic reactions, as the toxic metabolites bind to cell proteins within the hepatocytes, leading to aberrations [65].
Our results were in accordance with Prescott [66], who stated that aspirin and salicylates have been recognized as potentially hepatotoxic. In addition, Ebong et al. [67] observed a significant increase in the levels of ALT and AST levels upon aspirin exposure. According to Ingram [68], the elevation of both transaminase enzymes is presumed to result from leakage, damage, or necrotic liver cells. These results are in agreement with Doxy [69], who reported that levels of ALT and AST were increased following liver damage. These results agree with those of Tiefenbich and Wichner [70], who reported an increase in liver transaminase levels; these enzymes show an early rise in almost all diseases of the liver and remain elevated for 2-6 weeks in the presence of the disease. Additionally, Bush [71] reported that liver damage is associated with increases in serum levels of both AST and ALT. In two-and four-weeks' post-administration, AST and ALT levels showed a pronounced increase at 80 mg/kg compared to 40 mg/kg, which indicates that the damage is dose-dependent.
The current study showed that injection of LAS induced significant elevations in serum levels of urea and creatinine after both 2 and 4 weeks from the beginning of treatment. This finding may be due the fact that the administration of NSAIDs to susceptible individuals might cause renal failure due to a reduction in glomerular filtration rate and renal plasma flow, as explained by Ejaz et al., [72]. The mechanism of action of NSAIDs involves the prevention of vasodilator prostaglandin synthesis from the arachidonic acid metabolism pathway. This in turn causes vasoconstriction and a drop in glomerular capillary pressure, leading to a rapid decrease in the glomerular filtration rate, which is characterized by increased serum levels of creatinine and urea. Gaspari et al. [73] reported that urea and creatinine are metabolic waste products, which are easily filtered via the glomeruli of the kidneys; therefore, their serum levels are commonly used as indicators of renal toxicity, as stated by Perrone et al. [74] The effects of these drugs may be due to functional impairment of renal function by acetylsalicylic acid mediated by inhibition of prostaglandin synthesis from arachidonic acid. The elevation in urea and creatinine levels reflects the state of glomerular filtration and kidney damage, supported by Coles [45]. Eight weeks after the beginning of treatment, there were insignificant effects on these parameters, indicating recovery from the adverse effects.
The results of the present study demonstrated that injection of LAS for two weeks induced a remarkable reduction in serum albumin level after 2 and 4 weeks from the beginning of the experimental period, which is consistent with the results of Karima et al. [75] and Mendez et al. [76], who reported that a significant decline in albumin production in the liver appears to be associated with a potential impairment of hepatocellular function stimulated by severe inflammation as well as subsequent sepsis. These consequences were confirmed by the histopathological findings in the examined liver and kidney, where congestion of both the central and portal veins and the detrimental alteration of the hepatocytes in the form of vacuolar and hydropic degeneration were observed. Eight weeks after LAS, the livers showed activation and enlargement of Von Kupffer cells with normal architecture. Moreover, the examined kidneys revealed large vesicular nuclei and pale basophilic cytoplasm in the regenerating tubules. These findings correlated and matched with the levels of AST, ALT, creatinine, urea, and other studied parameters, indicating the recovery from the adverse effects of LAS after 8 weeks from the beginning of its administration. It could be concluded that LAS is a beneficial drug for humans and must be used within therapeutic doses and for a specific period. It induced dose-dependent adverse effects on liver and kidney functions, complete blood count, and protein profiles. Recovery from these effects was dose-dependent.