|Year : 2016 | Volume
| Issue : 4 | Page : 929-935
Light and electron microscopic study on the effect of ketoconazole on the liver of adult male albino rats
Bothina L Mahmoud, Maisa A Kefafy, Rania I Yassien, Eman S Ahmad El-roghy MSc
Department of Histology, Faculty of Medicine, Menofyia University, Menoufiya, Egypt
|Date of Submission||03-Apr-2014|
|Date of Acceptance||30-Apr-2014|
|Date of Web Publication||21-Mar-2017|
Eman S Ahmad El-roghy
Sheibin El Kom, Menoufiya, 32511
Source of Support: None, Conflict of Interest: None
The aim of this study was to evaluate the structural, ultrastructural, and biochemical changes in albino rats' liver treated with different doses and durations of ketoconazole and the possible recovery after cessation of treatment.
Ketoconazole is an imidazole drug commonly used in treatment of fungal infection. It induces variable degree of hepatotoxicity.
Materials and methods
Fifty adult male albino rats were used in this study. The rats were divided into five groups. Group 1 was the control group. Group II (low dose treated) included 10 rats treated with ketoconazole at a dose of 40 mg/kg body weight orally for 7 days. Group III (high dose treated) included 10 rats treated with ketoconazole at a dose of 100 mg/kg body weight orally for 14 days. Group IV (arrested low dose) included 10 rats treated with low dose ketoconazole for 7 days followed by cessation of treatment for 7 days. Group V (arrested high dose) included 10 rats treated with high dose ketoconazole for 14 days followed by cessation of treatment for 14 days. At the end of each experimental period, animals were killed, blood samples were collected for biochemical study, and liver tissue samples were obtained for histological study.
Ketoconazole-treated rats showed destructive changes of liver tissue in the form of marked congested dilated blood vessels, hepatocyte necrosis and complete tissue loss, periportal fibrosis, and elevated serum transaminase. However, nearly complete recovery of liver tissue and improvement in serum enzymes occurred among those treated with low dose, whereas those treated with high dose still showed persistence of some changes.
Ketoconazole induced marked histological changes in the liver tissue especially with high dose.
Keywords: hepatocyte necrosis, hepatotoxicity, ketoconazole, liver
|How to cite this article:|
Mahmoud BL, Kefafy MA, Yassien RI, Ahmad El-roghy ES. Light and electron microscopic study on the effect of ketoconazole on the liver of adult male albino rats. Menoufia Med J 2016;29:929-35
|How to cite this URL:|
Mahmoud BL, Kefafy MA, Yassien RI, Ahmad El-roghy ES. Light and electron microscopic study on the effect of ketoconazole on the liver of adult male albino rats. Menoufia Med J [serial online] 2016 [cited 2020 Feb 16];29:929-35. Available from: http://www.mmj.eg.net/text.asp?2016/29/4/929/202534
| Introduction|| |
Drug-induced liver injury is a serious health problem that impacts on patients, physicians, pharmaceutical industry, and government regulators. In addition, it is the most common cause of acute liver failure in the USA, accounting for more than 50% of cases, and it is now the most common single adverse drug reaction leading to market removal of a drug . Human fungal infections have increased markedly in incidence and severity in recent years due to advances in surgery, cancer treatment, HIV epidemic, and critical care; all are accompanied by increase in the use of broad-spectrum antibiotics . Pharmacotherapy of fungal diseases has been revolutionized by introduction of the relatively nontoxic oral azole drug such as ketoconazole, which is used successfully in the treatment of fungal infection, but it may cause some hepatic damage . Ketoconazole is an azole-based broad-spectrum antifungal drug, which operates through inhibiting the biosynthesis of ergosterol in fungal cell wall, leading to increased cell permeability, and thus damaged mycetes. It is mainly used for treatment of systemic fungal infections such as candidiasis, blastomycetic dermatitis, coccidioidomycosis, histoplasmosis, and chromoblastomycosis . Ketoconazole has been used for treatment of prostate cancer with promising results . In addition, physicians use high-dose ketoconazole in women with advanced breast cancer resistant to conventional chemotherapy, ovarian hyperandrogenism syndrome, polycystic ovarian syndrome, hyperthecosis, acne, hirsutism, and amenorrhea . The incidence of hepatotoxicity induced by ketoconazole is the highest among the imidazole antifungals . However, there are few studies exploring the relationship between hepatotoxicity and dose and plasma concentrations of ketoconazole. Little is known about how the pharmacokinetic parameters change when liver injury occurs .
| Materials and Methods|| |
Fifty adult male albino rats weighing 130–150 g were used in this study. They were kept in the same environmental condition with free access to food and water ad libitum.
Ketoconazole: it is available under the trade name Nizoral as a tablet of 200 mg, which is manufactured by Janssen-Cilag Pharmaceutica NV (Beerse, Belgium). It was dissolved in physiological saline and was given orally.
The rats were divided into five groups as follows:
Group I (control): it was composed of 10 adult rats. Four rats were kept without any treatment and other six rats were given physiological saline orally. Rats were killed as in the experimental group.
Group II (low dose treated): it was composed of 10 adult rats treated with ketoconazole at a dose of 40 mg/kg body weight orally daily for 7 days.
Group III (high dose treated): it was composed of 10 adult rats treated with ketoconazole at a dose of 100 mg/kg body weight orally daily for 14 days.
Group IV (arrested low dose): it was composed of 10 adult rats treated with ketoconazole at a dose of 40 mg/kg body weight orally daily for 7 days followed by cessation of treatment for 7 days.
Group V (arrested high dose): it was composed of 10 adult rats treated with ketoconazole at a dose of 100 mg/kg body weight orally daily for 14 days followed by cessation of treatment for 14 days.
At the end of each determined period, animals were killed, blood samples were collected, and liver tissues of each animal were excised. The tissues were prepared for light microscopic examination by H&E. , Masson's trichrome , and periodic acid Schiff's reaction (PAS)  and for ultrastructure examination using electron microscopy .
The animals' weight and the levels of alanine transaminase (ALT) and aspartate serum transaminase (AST) were measured in all groups. Thereafter, data were statistically analyzed using the Kruskal–Wallis test. Data were expressed as mean ± SE and analyzed using the Thaman test for comparison between all groups. Differences were regarded as nonsignificant when P value was greater than 0.05, significant when P value was less than 0.05, and highly significant when P value was less than 0.01.
| Results|| |
Light microscopic result
Group I (control)
Sections of this group showed the normal architecture of liver tissue with classic hepatic lobule. The central vein was located in the middle where hepatocytes plates were radiating, and they were separated by blood sinusoids. The hepatocytes were polygonal cells with vesicular nuclei and acidophilic cytoplasm. Some hepatocytes were binucleated. Portal tracts were found at an angle between three hepatic lobules and consisted of branch from hepatic artery, portal vein, and bile duct lined by cubical epithelium. Minimal collagenous fibers in the portal tract were seen in the sections stained by Masson trichome. PAS-stained section showed moderate reaction in the cytoplasm of the hepatocytes, where the reaction was more intense in the central hepatocytes than in peripheral ones [Figure 1].
|Figure 1: a-e) A photomicrograph of the control group (I) showing (a) classic hepatic lobule, central vein (CV) in middle, and hepatocytes plates separated by blood sinusoids (S) (H&E, ×400). (b) Hepatocytes with vesicular nuclei (N). Some are binucleated (→). Von Kupffer cells (KF) appear in blood sinusoids (H&E, ×1000). (c) Portal tract formed of bile ducts (D), hepatic artery (A), and portal vein (V) (H&E, ×400). (d) Minimal collagenous fibers (MT, ×400). (e) Moderate reaction in the endothelium of central vein (arrowhead) and in the hepatocyte cytoplasm, with unstained nuclei (→) (PAS, ×400). MT, Massonfs trichrome; PAS, periodic acid Schifffs reaction.|
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Group II (low dose treated)
Sections of this group showed disturbed irregular pattern of hepatic cords and irregular congested sinusoids. Central vein showed dilatation and congestion. The hepatocytes showed enlarged prominent nuclei and dispersed chromatin with faded nucleoli. Some hepatocytes showed necrosis and others showed cytoplasmic vacuolation. However, Von Kupffer cells showed hypertrophy. The changes in portal triad were dilated portal vein containing RBCs and high infiltration of mononuclear cells. Increased amount of collagenous fibers in the portal tract were seen in the sections stained by Masson trichrome. PAS-stained section showed strong reaction in the cytoplasm of most hepatocytes and in the endothelium of blood vessels [Figure 2].
|Figure 2: (a-e) A photomicrograph of the low-dose group (II) showing (a) dilated congested portal vein (PV), distorted bile duct (D), congested sinusoid (S), and few focal of necrosis (NE) (H&E, ×400). (b) Dilated congested central vein (CV) and sinusoid (S), enlarged hepatocytes nuclei (N) with faded nucleoli (NOL), and hypertrophied Kupffer cells (KF) (H&E, ×1000). (c) Marked cellular infiltration (I) around the portal tract and hepatocytes cytoplasmic vacuolation (V) (H&E, ×400). (d) Increased amount of collagenous fibers (MT, ×400). (e) Strong reaction in most hepatocytes and in the endothelium of blood vessel (arrowhead), with unstained nuclei (→) (PAS, ×400). MT, Massonfs trichrome; PAS, periodic acid Schifffs reaction.|
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Group III (high dose treated)
Sections of this group showed massive destructive changes including irregular pattern of hepatocytes and highly dilated distorted central vein. The hepatocytes showed focal areas of necrosis and complete tissue loss. Focal area of enlarged abnormal hepatocytes with deeply acidophilic cytoplasm and indistinct nuclei that appeared as ghost-like cells was seen. Some hepatocytes of this group showed deeply stained pyknotic nuclei and marked cytoplasmic vacuolation. Von kupffer cells showed hypertrophy. The changes in portal triad were marked dilatation, distortion, and congestion of portal vein. There were bile duct dilatation and proliferation with cellular infiltration around the portal tracts. Excessive collagenous fibers in the portal tract were seen in the sections stained by Masson trichrome. PAS-stained section showed very strong reaction in most hepatocytes [Figure 3].
|Figure 3: (a-f) A photomicrograph of the high-dose group (III) showing (a) loss of hepatic architecture and distorted congested central vein (CV) with cellular infiltration (I) and focal area of tissue loss (→) (H&E, ×400). (b) Highly disturbed irregular pattern of hepatic cords with focal areas of ghost-like hypertrophied hepatocytes (F). Other hepatocytes show vacuolation (V) (H&E, ×400). (c) Marked cytoplasmic vacuolation (V) and enlarged Kupffer cells (KF) (H&E, ×1000). (d) Infiltration of mononuclear cells (I). Bile ducts (D) showing distortion, dilatation, and proliferation (H&E, ×400). (e) Excessive collagenous fibers deposition (MT, ×400). (f) Very strong reaction in the cytoplasm of most hepatocytes with negative staining of the hepatocytes nuclei (→) (PAS, ×400). MT, Massonfs trichrome; PAS, periodic acid Schifffs reaction.|
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Group IV (arrested low dose)
Sections of this group showed marked improvement of the histological picture. The liver architecture appeared nearly normal; however, some hepatocytes showed cytoplasmic vacuolation. Nearly minimal collagenous fibers in the portal tract were seen in the sections stained by Masson trichrome. PAS-stained section showed moderate reaction in most hepatocytes [Figure 4].
|Figure 4: (a-d) A photomicrograph of the arrested low-dose group (IV) showing (a) central vein (CV) located in the middle, and hepatocytes plates are separated by sinusoids. Some hepatocytes show cytoplasmic vacuolation (V) (H&E, ×400). (b) Portal tract with normal appearance (→) (H&E, ×400). (c) Minimal collagenous fiber (MT, ×400). (d) Moderate reaction in most hepatocytes with unstained nuclei (→) (PAS, ×400). MT, Massonfs trichrome; PAS, periodic acid Schifffs reaction.|
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Group V (arrested high dose)
Sections of this group showed moderate improvement of the histological picture, where the central vein showed moderate distortion and congestion. Some hepatocytes showed deeply stained pyknotic nuclei and others showed cytoplasmic vacuolation with focal area of complete tissue loss. Moderate congestion and dilatation of portal vein with some cellular infiltration around portal triad were noticed. Moderate amount of collagenous fibers in the portal tract were seen in the sections stained by Masson trichrome. PAS-stained section showed moderate reaction in the endothelium of central vein and in most hepatocytes; however, some hepatocytes showed strong reaction [Figure 5].
|Figure 5: (a-d) A photomicrograph of the arrested high-dose group (V) showing (a) vacuolated hepatocytes cytoplasm (V). Some hepatocytes show pyknotic nucleus (PK) and area of tissue loss (→) (H&E, ×400). (b) Moderate congestion and dilatation of portal vein (PV) and some cellular infiltration (I). Some hepatocytes show indistinct nuclei (→) (H&E, ×400). (c) Moderate amount of collagenous fibers (MT, ×400). (d) Moderate PAS reaction in the endothelium of CV (arrowhead) and in most hepatocytes; however, some hepatocytes show strong reaction with unstained nuclei (→) (PAS, ×400). MT, Massonfs trichrome; PAS, periodic acid Schifffs reaction.|
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Electron microscopic result
Electron microscopic examination of ultrathin sections of the control group showed hepatocyte with euchromatic double nuclei containing distinct nucleolus and peripheral heterochromatin and euchromatin. The cytoplasm contained numerous rounded mitochondria, secondary lysosome, regular rough and smooth endoplasmic reticulum, and electron-dense glycogen granule. Long microvilli were projecting toward blood sinusoid, which contained red blood cell [Figure 6]a and [Figure 6]b.
|Figure 6: (a–f) An electron micrograph from a section in the liver of (a) the control group showing hepatocyte with euchromatic double nuclei (N), numerous mitochondria (M), secondary lysosomes (LY), regular rough and smooth endoplasmic reticulum (rER), and glycogen granules (G) (×1500). (b) The control group showing part of the nucleus (N), numerous mitochondria (M), regular rER, and glycogen granules (G). Long microvilli (arrows) projecting toward sinusoid (S) containing red blood cell (RBC) (×3000). (c) The low-dose group showing irregular nuclear membrane (N). Cytoplasm contains numerous rough endoplasmic reticulums (rER), lipid droplets (L), secondary lysosome (LY), glycogen granules (G), and lamellar bodies (arrowhead) (×3000). (d) The high-dose group showing shrunken nucleus (N). Cytoplasm contains multiple lipid droplets (L), degenerated mitochondria (M), lamellar bodies (arrowhead), glycogen granules (G), and short irregular microvilli (arrows) project in sinusoid containing Kupffer cell (KF) (×2000). (e) The arrested low-dose group showing normal appearance of hepatocyte nucleus (N), rounded mitochondria (M), rough endoplasmic reticulum (rER), secondary lysosomes (LY), few lipid droplets (L), and long microvilli (arrow) project into sinusoid (S) (×2500). (f) Liver of the arrested high-dose group showing two hepatocytes; both contain pyknotic nuclei (N), many lipid droplets (L), deformed mitochondria (M), and sinusoid containing Kupffer cell (KF) and RBC (×1000).|
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Liver section of the low-dose group (II) showed hepatocyte containing part of the nucleus with irregular nuclear membrane and numerous rough endoplasmic reticulum with increased granularity. In addition, there were some lipid droplets, secondary lysosome, and electron-dense glycogen granules. Lamellar bodies with variable sizes were seen [Figure 6]c.
Liver section of the high-dose group (III) showed hepatocyte with shrunken indented nucleus. The cytoplasm was packed with multiple lipid droplets of variable sizes and contained degenerated, dense, deformed mitochondria with loss of their cristae. There were lamellar bodies and variable-sized dense glycogen granules. Short irregular microvilli were seen projecting toward blood sinusoid, which showed the presence of Von Kupffer cell [Figure 6]d.
Liver section of the arrested low-dose group (IV) showed hepatocyte containing part of nucleus. The cytoplasm showed numerous rounded mitochondria, rough endoplasmic reticulum, secondary lysosomes, and few lipid droplets. Long microvilli were seen projecting into blood sinusoid [Figure 6]e.
Liver section of the arrested high-dose group (V) showed two hepatocytes; both contained pyknotic nuclei, many lipid droplets of variable sizes, and numerous deformed mitochondria. Blood sinusoid showed Von Kupffer cell and red blood cell [Figure 6]f.
The body weights of the animals were significantly decreased after low-dose treatment and highly significantly decreased after high-dose treatment. Arrested low-dose treatment showed nonsignificant decrease, but still significant decrease was observed after arrested high-dose treatment compared with control ([Table 1]).
|Table 1 Descriptive statistics for mean and SD of body weight in different groups|
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Serum transaminase results showed significant increase after low-dose treatment but highly significant increase with high-dose treatment. Arrest of treatment showed nonsignificant increase after low-dose treatment, but still significant increase was observed after high-dose treatment compared with control ([Table 2] and [Table 3]).
|Table 2 Descriptive statistics for mean and SD of the AST level in different rats groups|
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|Table 3 Descriptive statistics for mean and SD of the ALT level in different rats groups|
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| Discussion|| |
Systemic fungal infections are becoming a serious problem in clinical practice. These infections are usually life-threatening, especially for the immune-compromised patients . Commonly prescribed antifungal agents can lead to high risks for nephrotoxicity and hepatotoxicity in clinic patients. Ketoconazole is an imidazole derivative commonly prescribed for both superficial and systemic treatment of mycotic infections. However, a high dosage of ketoconazole can cause fatal hepatic necrosis . Hepatotoxicity induced by ketoconazole is correlated to dose, maximum plasma concentration, and clearance . However, ketoconazole-induced hepatotoxicity has been documented in rats .
This study was carried out to throw more light on the structural, ultrastructural, and biochemical changes in ketoconazole-treated liver in adult male albino rats. Animals were administered ketoconazole orally at two different doses – 40 mg/kg/day according to Shaikh et al.  and 100 mg/kg/day according to Padmapriya et al.  – for 1 and 2 weeks, respectively. Thereafter, animals were left without treatment for similar period to notice the possible recovery.
Animals of the low-dose group were drowsy, weak, and had loss of appetite and weight, with nil mortality rates. However, animals of the high-dose group showed worse general condition with mortality rates of 30%. These symptoms were previously reported by Tahir and Nour  who stated that hypomagnesemia produced by azole might be attributed to the loss of appetite. Animal death was recorded by Allen et al.  after ketoconazole treatment, and this death could be attributed to hepatotoxicity. Casely et al.  stated that the mechanism by which ketoconazole induces hepatotoxicity is through its primary metabolite N-deacetyl ketoconazole, which is largely responsible for downregulation of two hepatic genes – serum amyloid A and hepcidin. Amin  reported that N-deacetyl ketoconazole is the major metabolite that undergoes further metabolism by the flavin-containing monooxygenase to form potentially toxic reactive metabolites. He also reported that it induces massive DNA fragmentation, increase in lipid peroxidation, and depletion of glutathione in rat liver. Shaikh et al.  stated that the mechanism of injury was suspected as a reaction of ketoconazole or its metabolite N-deacetyl ketoconazole, which is more cytotoxic. Ketoconazole has also beenreported to mediate hepatotoxicity through chelating glutathione with active toxic metabolites.
At autopsy, the liver of the high-dose group was pale yellow with a friable consistency; the same gross picture was documented previously by Binev et al. . However, low-dose-treated liver appeared congested and enlarged, similar to the finding of Hassan et al. .
Administration of low and high doses of ketoconazole caused considerable structural and ultrastructural changes that were more exaggerated with high dose. The main effect was primarily on the vascular bed, where there was marked congestion and dilatation of the central and portal veins and hypertrophy of the hepatocytes with cytoplasmic vacuolation. Mononuclear infiltrating cells were also noticed; these changes were documented previously by Kemeir .
Hassan et al.  explained that the developed vascular dilatation could be related to hepatic congestion that may have developed during course of treatment or as a result of direct action of ketoconazole on the vessels wall, or it may be due to back pressure in the portal tract as a result of fatty infiltration. However, the cellular infiltrations detected in the present study could be similar to those explained by Sakr  who stated that the leukocytic infiltrations were considered as a prominent response of the body tissue facing any injurious impacts.
Increased cytoplasmic granularity of the hepatocytes was also observed, and this finding was reported by Shaikh et al.  who attributed this change to disorganization of the arrangement of ribosome on the rough endoplasmic reticulum.
Hepatocytes necrosis and complete tissue damage were detected in this study, and this is in agreement with the study by other investigators such as Shaikh et al. , who stated that the azole group increases the mitotic cross-overs that affect chromosome segregation by an indirect mechanism, which is possibly related to induced structural chromosomal damage, and often lead to tissue damage especially to the liver. However, hepatocytes necrosis in the present work was explained by Shaikh et al. , who stated that the toxic metabolites damage the smooth endoplasmic reticulum, mitochondria, and plasma membrane.
Enlarged prominent nuclei and dispersed chromatin with faded nucleoli detected after low-dose treatment were similar to those found by Shaikh et al. , and these could be explained as by Liu et al.  who reported that the azole groups increase mitotic figures in the hepatocytes.
This study revealed an increase in the number and size of Kupffer cells, and this was in harmony with the study by Hashem . This increase could be attributed to defense mechanism of the liver macrophages against ketoconazole toxicity.
There was an increase in collagen fibers deposition around the portal tract, which was dose-dependent and more pronounced after high-dose ketoconazole treatment. Such finding confirms the previous observation by Shaikh et al.  who stated that the major source of increased collagen is Ito cells in space of Disse, which transform into myofibroblast-like cells because of toxic injury, which in turn increase fibrosis.
There was increased deposition of glycogen granules detected by PAS stain, more pronounced after high-dose treatment. This finding was confirmed by electron microscopic study and could be attributed to marked damage to the hepatocytes, failing to metabolize such granules.
Electron microscopic study confirmed the light microscopic finding, where there were degenerated mitochondria with loss of their cristae and the nuclei displaying signs of pyknosis and karyolysis; these changes were documented by Elshennawy and Aboelwafa  and could be a sign of cell degeneration. Mitochondrial changes were explained by Essawy et al.  who stated that azoles inhibit cytochrome P450 enzymes in fungal organisms. As these enzymes are also integral components of the inner mitochondrial membrane, the mitochondrial hepatic abnormalities could be related to some azole–cytochrome P450 enzyme interaction, and the structural alterations in the liver cells are associated with mitochondrial impairment. The changes that occurred in the nucleus (pyknosis and karyolysis) could be explained by the study by Escajeda et al.  who proved that ketoconazole is a cytochrome P450 inhibitor that causes apoptosis through p53-dependent and other pathways in specific cancerous cell lines.
Foamy cytoplasmic vacuoles detected by light microscope were actually lamellar bodies and lipid droplets. This finding indicates that ketoconazole induced phospholipidosis; according to Asaoka et al. , numerous microsomes and a major metabolite N-deacetyl ketoconazole are considered to be responsible for ketoconazole-induced phospholipidosis. However, Hashem  attributed this lipid infiltration as a defense mechanism by which hepatocyte attempts to collect all toxic compounds invading the cell in these vacuoles before excretion.
Cytoplasmic vacuolations, irregular rough endoplasmic reticulum, and irregular short microvilli toward blood sinusoid were noticed; these changes were detected by Hashem .
Increased cytoplasmic granularity detected in present study by light microscope was confirmed by electron microscopic study that revealed numerous rough endoplasmic reticulums with increased granularity; this finding is in harmony with the study by Shaikh et al.  who attributed this change to disorganization of the arrangement of ribosomes on the rough endoplasmic reticulum.
The serum transaminase activities of both AST and ALT were increased in dose-dependent manner of ketoconazole; these biochemical results were observed by many investigators such as Asaoka et al. . According to Mohan , the elevation of serum enzymes AST and ALT usually indicates hepatocytes necrosis with enzymes released from the cells to the serum. Thus, elevated enzymes confirm the hepatocytes necrosis in this study.
After cessation of treatment, there was nearly complete recovery of the liver tissue among those treated with low dose, whereas those treated with high dose still showed persistence of some changes. The degree of recovery of the damaged tissue increased progressively with low dose, and this was in harmony with the theory of regeneration, where Haussinger  stated that the liver is the most internal organ having large capacity of regeneration. However, persistence of some alterations with high dose may indicate the need for more time for recovery to occur.
| Acknowledgements|| |
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3]