Hepatic Histological and Histochemical Alterations Induced by Rosuvastatin Therapeutic Doses.
Abstract The aim of the present study is to determine the potential toxicity of the therapeutic doses of rosuvastatin in Wistar albino rats. A total of 80 adult Wistar male albino rats were divided randomly into 4 groups. The control group (group 1) received oral gavage administration of normal saline (1 ml /kg/day for 90 consecutive days) while group II and group III received rosuvastatin (1.25 and 2.5 mg/kg/day respectively for 90 consecutive days). Group IV received only the vehicle (carboxymethyl cellulose, 0.25% mg/kg/day) for 90 consecutive days. Liver biopsy was taken form each rat under study for histological and histochemical examination. In comparison with respective control rats, ROSU-treated animals exhibited nuclear abnormalities, dilatation of blood sinusoids and central veins, inflammatory cell infiltration, necrosis, apoptosis, congestion and hydropic degeneration.
Also, ROSU exposure increased the activity of glucose-6-phosphate dehydrogenase with no change in the activity of alkaline phosphatase together with deletion in protein and glycogen hepatocytes content. The histological and histochemical alterations produced by ROSU might indicate hepatocytes insulation due to metabolic and structural disturbances caused by this drug. More histomorphological and ultrastructural investigations are needed for clear demonstration of rosuvastatin potential risk.
Key words: Rosuvastatin, statins, hepatic tissue, apoptosis, TUNEL assay, hydropic degeneration
Statins are a class of drugs used widely for the treatment of hyperlipidemia as well as for prevention of atherosclerosis and cardiovascular events with therapy duration of 30-120 days (Wainwright, 2005; Bjornsson et al., 2012). Statins reduce the level of circulating atherogenic lipoproteins by inhibition of hepatic 3-hydroxy-3- methylglutaryl coenzyme A (HMG-CoA) reductase (McTaggart, 2003; Guthrie and Martin, 2007). Pre- marketing biochemical clinical trials and initial toxicological studies in animals suggested that statins may cause hepatotoxicity, primarily elevations in serum aminotransferases levels induced with a need of liver enzymes monitoring (Veillard and Mach, 2002; Famularo et al., 2007). Although the therapeutic doses of lovastatin did not cause significant liver injury, they caused hepatocellular necrosis in rabbits when given in very high doses (MacDonald et al., 1988).
Similarly, high doses of simvastatin caused hepatocellular necrosis in guinea pigs (Horsmans et al., 1990). Statin-related drug-induced liver injury induced by atorvastatin, simvastatin, fluvastatin and others was reported in 1.2/100,000 users (Bjornsson et al., 2012; Russo et al., 2009).
Statins cause transaminitis with possible induction of acute liver failure, hepatitis and cholestasis (Vasudevan et al., 2005). One study found from the reports of the World Health Organization for deaths resulting from serious liver injury that most patients experienced liver injury after 3-4 months of statins therapy (Perger et al.,2003). However, the use of statins has been shown to improve liver abnormalities in patients with non- alcoholic fatty liver disease (Bjornsson et al., 2012; Hyogo et al., 2008). Some studies indicated that rosuvastatin ameliorated hepatic injury, inflammation, lipid perixodation, increased antioxidant enzymes activity and modulated immune response independent of lipid lowering effect (Awad and Kamel, 2010; Awad and El- Sharif, 2010).
Rosuvastatin is a relatively new statin as cholesterol-lowering drug, with liver is the main target organ (Nezasa et al., 2002; Famularo et al.,2007). Rosuvastatin is taken up by hepatocytes more selectively and more efficiently than other statins which has generated considerable controversy regarding its safety specially its probable potential hepatotoxicity (Famularo et al., 2007; Guthrie and Martin, 2007; Khan and Ibrahim, 2009).
Although statins hepatotoxicity is well recognized (Kaplowitz, 2004), rosuvastatin induced hepatotoxicity has been put into question (Bader,2010). Pre-marketing studies have suggested that rosuvastatin may have lesser potential to cause liver toxicity as compared with other statins (Davidson, 2007). On the otherhand, some studies and case reports showed that treatment with rosuvastatin might cause hepatotoxicity even with low doses or short time of treatment. With this limited data exist on rosuvastatin -induced liver injury, the present study was conducted to investigate the hepatic histological and histochemical alterations induced by therapeutic doses of this drug.
MATERIALS AND METHODS
A total of 80 health adult male Wistar albino rats (Rattus norvegicus) of the same age (8-10 weeks old) weighing 220-250 g of King Saud University colony were used. All animals were divided randomly into 4 groups (20 animals each), kept in the laboratory conditions for a period of 7 days for acclimatization and were fed with commercial rat pellets and drinking water ad libitum. The daily equivalent dose (mg/kg) of rosuvastatin was calculated on the basis of the surface area ratio according to standard methods (Reagan-Shaw et al., 2008). Accordingly, 1.25 and 2.5 mg/kg rosuvastatin (Crestor(r), AstraZeneca Pharmaceuticals LP, Wilmington) dissolved in normal saline containing sodium carboxymethyl cellulose (0.25%) were used as a daily equivalent therapeutic doses and administered orally for 90 consecutive days as follows: Group I (Control group): The members of this group were not subjected to rosuvastatin but to one ml normal saline/kg/day.
Group II: The members of this group received rosuvastatin (1.25 mg/kg/day). Group III: The members of this group received rosuvastatin (2.5 mg/kg/day). Group IV: The members of this group were exposed to the vehicle (0.25% carboxymethyl cellulose dissolved in one ml normal saline)/ kg/day.
Fresh portions of the liver from each rat were cut rapidly, fixed in neutral buffered formalin (10%), then dehydrated, with grades of ethanol (70%, 80%, 90%, 95% and 100%). Dehydration was then followed by clearing the samples in two changes of xylene. Samples were then impregnated with two changes of molten paraffin wax, then embedded and blocked out. Sections (4-5 um) were stained according to Bancroft and Stevens (1999), with the following conventional histological and histochemical stains: hematoxylin and eosin (H and E), periodic acid-Schiff (PAS) reagent, Best's carmine stain, Mallory trichrome and mercuric bromophenol blue. Histochemical reactions for alkaline phosphatase and glugose-6-phosphate dehydrogenase (G6PDH) were performed on fresh unfixed frozen sections. The demonstration of alkaline phosphatase was based on the Naphthol AS-BI method (c.f. Van Noorden and Fredriks,1992).
The specificity of the reaction for this enzyme was controlled by incubating a parallel set of sections in the incubating medium without substrate. Glugose-6-phosphate dehydrogenase detection was based on the lead method of Wachstein and Miesel (Bancroft and Stevens, 1999), and by using control consisted of parallel sections incubation in media lacking the substrate of the specified enzyme. The incubating medium consisted of NBT tetrazolium, disodium glucose-6-phosphate, nicotininamide adenine dinucleotide (NADP), sodium azide and phenazine methosulphate.
Apoptotic cells were detected by using terminal deoxynucleotidyl transferase (TdT)- mediated dUTP nick end labeling (TUNEL) detection kit (GenScript, USA), which is a sort of an immunohistochemical technique. The incubating medium consisted of proteinase K solution in PBS buffer, labeled Biotin/Streptavidin-HRP/DAB and terminal deoxynucleotidyl transferase. For negative controls, parallel sections were incubated in medium lacking terminal deoxynucleotidyl transferase.
All experiments were conducted in accordance with the guidelines approved by King Saud University Local Animal Care and Use Committee.
In comparison with the control group (Fig.1a), the liver of ROSU-treated rats had lost some hepatic architecture characteristics and demonstrated several histological and histochemical alterations.
Histological changes Occasional portal inflammatory cells infiltration mainly lymphocytes was seen in the periportal spaces of ROSU-treated rats (Fig. 1b). This infiltration was more prominent in rats received 2.5 mg than those received 1.25 mg of the drug.
Marked parenchymal necrosis was noticed in hepatocytes of ROSU-treated rats with eosinophilic cytoplasm (Fig. 1c). Apoptotic bodies in the form of intercellular rounded condensed eosinophilic bodies surrounded by clear holes were also seen (Fig. 1d).
Dilatation and congestion of hepatic sinusoids became evident in the in the liver of ROSU-treated rats (Fig. 1e). This alteration was more prominent in rats received 2.5 mg of the drug than those received 1.25 mg and was associated with hepatocytes necrosis.
The hepatic tissue of the ROSU-treated rats showed dilated central vein in all drug treated members (Fig. 1f).
Occasional perivascular edema was seen in the hepatic triads of rats exposed to ROSU administration (Fig. 2a). This change appeared in all treated groups. This alteration appeared in some hepatocytes of rats received 2.5 mg ROSU but rarely seen in those received 1.25 mg ROSU (Fig.2b). The insulted hepatocytes exhibited poorly delineated nuclei.
Variable nuclear abnormality was mainly seen in the hepatocytes of rats received 2.5 mg ROSU and to a lesser extent in the those received 1.25 mg ROSU. These abnormalities included marked binucleation, anisokaryosis, karyolysis, karyorrhexis and karyopyknosis. Some pyknotic hepatocytes of rats exhibited clumping and condensation chromatin condensation in the periphery of the nuclei together with irregularity nuclear membranes. Hepatocytes showed nuclei disappearance while others exhibited fragmentation or dissolution of their nuclei (Figs. 2c-d).
Occasional autolytic changes and cytoplasm eosinophilia were observed in some hepatocytes especially in those with indistinct cell membranes (Fig. 2e). Hepatocytes cytoplasm swelling and vacoulation were seen specially at high dose ROSU treated rats (Fig. 2f).
Histochemical changes Compared with liver of control group, ROSU administration has produced significant reduction in liver glycogen specially in rats received 2.5 mg ROSU (Figs. 3a-c). Hepatocytes in the perivenous zones were more affected the those surround the periportal spaces.
Compared with liver of control group, ROSU administration has produced considerable reduction in hepatocytes protein in both group of rats received 1.25 mg and 2.5 mg ROSU respectively (Fig. 3d-f).
A marked increase in the activity of G6PDH was seen in the liver of ROSU treated rats. The activity of this enzyme was more pronounced in rats received 2.5 mg ROSU than those received 1.25 mg of the drug (Figs. 3g-i). In the control livers, this enzyme was mainly localized in the bile canalicular membranes of hepatocytes the liver. The activity of the enzyme was demonstrated clearly in the adventitia coat of blood vessels and was not affected by ROSU treatment.
None of the above histological and histochemical alterations were detected in the liver of the control rats or those received carboxymethyl cellulose.
Apoptosis detection Compared with liver of control group, apoptotic cells were observed in some hepatocytes of the liver of ROSU-treated groups as seen by Tunel assay (Figs. 4a,b). No portal fibrosis or cirrhosis were detected due to chronic therapeutic doses of rosuvastatin in the liver of any member of the treated groups over the entire period of the study.
Statins are metabolized mainly by the liver and increase aminotransferases levels with hepatic potential toxicity that might be attributed to alteration of the hepatocyte cellular membrane rather than direct liver injury (Veillard and Mach, 2002; Clarke and Mills, 2006).
The results of the present work showed inflammatory cells infiltration in the hepatic tissue due to ROSU chronic exposure. This may suggest that ROSU could interact with proteins and enzymes of the hepatic interstitial tissue by interfering with the antioxidant defense mechanism and leading to reactive oxygen species (ROS) generation which in turn may imitate an inflammatory response (Johar etal., 2004).
Cell necrosis induced by ROSU chronic exposure as shown by the present work was described previously by other studies which investigated other members of statins (MacDonald et al., 1988; Corsini et al., 1996). Necrosis is produced as a result of cell degeneration accompanied by organelles swelling and amorphous eosinophilic cytoplasm followed by shrinking and dissolution of nuclei (Campos-Pereira et al., 2012). The seen necrosis induced by ROSU may be due to statins effect on permeability of hepatocytes cell membrane that lead to depletion of cholesterol. Also, this might indicate swelling of some organelles such as mitochondria or by oxidative stress on these cells by glutathione depletion.
Apoptosis is a sort of programmed cell suicide that is highly regulated and executed via activation of specific signaling pathways. This abnormality is usually accompanied by particular morphological alterations such as DNA fragmentation, nuclear condensation, and formation of apoptotic bodies which are then engulfed by macrophages or neighboring cells without initiating an inflammatory response, death or disruption to the surrounding tissue (Pollack and Leeuwenburgh, 2001; Pollack et al., 2002; Kluck et al., 1997). The mitochondrion plays a central role in regulating apoptosis by cytochrome c release into the cytosol, which then forms an "apoptosome". Some reports indicated that statins induce apoptosis by an increase caspase-9 and caspase-3 activty together with pyknosis, chromatin marginalization, and formation of dense bodies (Campos-Pereira et al., 2012).
Cell apoptosis induced by ROSU as shown by the present work is in agreement with other findings (Guijarro et al., 1998; Rabkin and Kong, 2003; Erl, 2005; Kaufmann et al., 2006; Westwood et al., 2006). All tested lipophilic statins induced apoptosis of skeletal muscle cells while hydrophilic ones induced hepatic cells (Kaufmann et al., 2006).
The seen nuclear abnormalities induced by chronic exposure to ROSU such as marked binucleation, karyorrhexis, karyolysis and apoptosis might indicate hepatocytes cytotoxicity (Tolbert et al., 1992; Celik et al., 2003; Unal et al., 2005). Nuclear degeneration starts with pyknosis, followed by karyorrhexis and karyolysis lead to cell necrosis (Zamzami and Kroemer, 1999; Biradar et al., 2012). The results of the present work showed marked and more frequent hepatocytes binucleation in the animal exposed to ROSU than the control ones. This alteration may represents a consequence of cell injury usually seen in regenerating cells and might be due to increased cellular activity and nuclear interruption in the mechanism of ROSU detoxification (Gerlyng1 et al., 2008). These nuclear changes were dose dependent where nuclear damage was more prominent in the liver of rats received 2.5 mg ROSU than the ones received 1.25 mg.
Hepatocytes nuclear abnormalities were reported by other studies used high dose of lovastatin (MacDonald and Halleck, 2004). The distortion and swelling of hepatocytes together with the central vein dilatation might indicate that ROSU may affect the cell membrane permeability of hepatocytes and blood vessels endothelial lining. Hepatocytes swelling due to ROSU exposure as seen in the present study might lead to cellular transporters adaptation (Johnson, 1995). Ischaemic or pharmacologic disruption of cellular transporters can cause swelling of parenchyma of the liver cells leading to hepatotoxicity (Ajibade et al., 2012). ROSU may have acted as toxins to the hepatocytes, affecting their cellular integrity and causing defect in membrane permeability and cell volume homeostasis. These together with the hypertrophy of hepatocytes as observed in the treated rats may indicate support the cytotoxic effect of ROSU.
The present study showed that ROSU induced reduction in hepatocytes protein content. This might be due to effect of rosuvastatin on enzymes involved on protein synthesis and to the effect on the cytosol Ca2+ that might mediate variety of deleterious effects on the hepatocytes ribosomes and the rough endoplasmic reticulum (Kumar et al., 2005; El-Daly, 2011). Rosuvastatin has been noted to produce transient proteinuria while its high doses have been associated with cases of renal failure accompanied by severe proteinuria (Guthrie and Martin, 2007; Kostapanos et al., 2010).
Administration of ROSU caused depletion of glycogen stores in the hepatocytes of ROSU treated rats. This alteration might be due to the effect of ROSU on glucose absorption or on the enzymes involved in the process of glycogenesis or/and glycolysis (Kumar et al., 2005). Hepatocytes in the perivenous zones were more affected the those surround the periportal spaces. This might indicate that glycolysis was more affected than glycogenesis by rosuvastatin.
The results obtained in the present investigation reveal that the therapeutic doses of rosuvastatin induced considerable histological and histochemical alterations in the liver of rats. Further studies are recommended to be carried out to corroborate these findings.
The present project was supported by the Research Center, College of Science, King Saud University.
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Corresponding author: email@example.com 0030-9923/2013/0001-0149 dollar 8.00/0 Copyright 2013 Zoological Society of Pakistan
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|Author:||Al-Doaiss, Amin A.; Alarifi, Saud A.; Jarrar, Bashir M.|
|Publication:||Pakistan Journal of Zoology|
|Date:||Feb 28, 2013|
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