HEPATOTOXIC EFFECT OF ITRACONAZOLE IN EXPERIMENTAL RATS

Itraconazole is a widely used antifungal drug. In situations such as managing patients with gastrointestinal basidiobolomycosis, third of which cases world-wide occur in the southwestern region of Saudi Arabia, prolonged treatment with this drug is required. Thus, this is study was designed to investigate the hepatotoxicity of long term administration of itraconazolein in Wistar rats. Two groups of rats were treated with itraconazole at doses of 5 and 10 mg kg-1, for 30 and 60 days, respectively. At the end of each period sera of rats were tested for liver enzymes (including ALT, ALP and γGTT, albumin and protein). Animals were sacrificed and livers were processed for histological examination. Compared to controls, all rats treated for 30 and 60 days showed significant elevation in the levels of liver enzymes. Histologically there was severe liver injury. Although itraconazole is a safe antifungal drug, prolonged treatment with this drug may lead to severe hepatitis and liver cell injury. Patients who require treatment with this drug for long periods (12 to 18 months) should have their liver functions periodically monitored.


INTRODUCTION
Itraconazole is a synthetic triazole fungicidal agent, was approved for use in 1992 in the USA and continues to be used widely as antifungal. Itraconazolehas a wide spectrum of activity against dermatophytes (Trichophyton, Epiderophyton and Microsporum spp.), moulds (Aspergillus spp.) and yeast (Candidia spp. and Cryptococcus neoformans) (Terrell, 1999).
Itraconazole acts primarily by inhibiting biosynthesis of ergosterol,which is an essential component of fungal cell membranes (Bailey et al., 1990;Zuckerman and Tunkel, 1994). The drugs bind to fungal cytochrome P450, preventing the conversion of lanosteroltoergosterol, which leads to abnormalities in cellmembrane activity and membrane-bound enzymeactivities causing fungal cell death (Bailey et al., 1990).
Adverse reactions to itraconazole includes skin eruptions, gastrointestinal upset, thrombocytopenia, headache, dizziness, edema of extremities, gynecomastia, hypokalemia and hypertriglyceridemia (Amichai and Grunwald, 1998). The level of serum transaminases increases in 1 to 5% of patients who receive continuous therapy (Persat et al., 2000;Gupta et al., 2002). Itraconazole has been used for 15 years in more than 50 million patients; serum enzymes elevations occurred in 1 to 5% of patients on continuous itraconazole therapy and 1.7 to 2% on pulse therapy; systematic hepatotoxicity is rare (Gupta et al., 2001). Symptomatic hepatitis rarely occurs and when it does, recovery generally ensues with the cession of medication (Hann et al., 1993). Talwalkar et al. (1999) reported a case of prolong cholestasis temporally associated with the use of itraconazole for oychomycosis. Peak bilirubin level of 32 mg dL −1 was documented approximately after two months after discontinuation of itraconazole therapy.
Reports on the pathological features of itraconazole induced hepatitis are still limited. Both cytolytic and cholestatic have been reported hepatitis to be caused by itraconazole (Legras et al., 2002). A report of itraconazole induced liver injury in three patients who all developed cholestatic pattern of injury with damage to the interlobular bile duct. In additiontwo of those patients developed ductopenia (Adriaenssens et al., 2001). Histological evaluation by Tuccori et al. (2008) and Srebrniki et al. (2005) showed massive panlobular Science Publications AJAVS necrosis. Both of their patients showed a pattern of cytolytic liver injury and both requires liver transplantation. In vivo study rats were treated intraperitoneally with itraconazoleexibited hepatocellular necrosis, degeneration of hepatocytes, bile duct hyperplasia, biliary cirrhosis and cell granuloma. The hepatotoxicity in rats was accompanied by a significant increase in ALP and ALT activities (Somchit et al., 2004).
However, Basidiobolomycosis is a rare fungal infection caused by Basidiobolusranarum a member of the order Entomophthorales found worldwide. Basidiobolomycosis usually causes subcutaneous infection, that mostly affects young males and transmitted through traumatic inoculation. Most cases has been reported from tropical and subtropical regions (Fahimazad et al., 2006).
Gastrointestinal Basidiobolomycosis (GIB) is rare more difficult to diagnose. However, (GIB) has been increasingly recognized; a review reported 44 cases worldwide including 19 from USA and 11 from Kingdom of Saudi Arabia (Vikram et al., 2012). A recent review reported 49 cases worldwide among these patients, 19 cases were pediatric mostly from Kingdom of Saudi Arabia (13 cases) most of them from, Aseer-Tohama and Jazan province, at the Southwest of the Kingdom of Saudi Arabia (Saleh et al., 2013). Lyon et al. (2001) reported seven cases of GIB. Al Jarie et al. (2003) reported six cases of pediatric GIB. Al Jarie et al. (2011) reviewed the records of five patients with GIB in the same region. Recently Saleh et al. (2013); reported three more pediatric patients all from the same geographic region.
Treatment of BIB included both surgical resection of fungal masses and prolong therapy of itraconzole (1 year to 18 months). Saleh et al. (2013) concluded that GIB is an emerging disease in Saudi Arabia. Most of pediatric GIB reported cases were from Southwest of Saudi Arabia which mandate further researching order to explore disease risk factors and better understanding of GIB. In this study, we aimed to investigate the hepatotoxic effect of long term-orally administered itraconzole in wistar rats.

Drugs and Chemicals
The drug (Itraconazole) was purchased from Saudi Arabian Japanese Pharmaceuticals Company, Jeddah, Saudi Arabia, ansqaspepared by dissolving in drinking water. All the biochemical kits were purchased from Human Diagnostics (Wiesbaden, Germany).

Animals
White male albino rats 8 weeks old and weighing between 230-250 g were used in the current study. Rats were supplied by the animal house at the Medical School of King Khalid University. The rats were housed in standard plastic cages (6 rats/cage) in an environmentally controlled room with a constant temperature of 25-27°C and 12 h light/dark cycle. The rats were fed a "standard lab diet" and given ad libitum access to food and water. All rat experimental procedures were performed in compliance with the regulations of Ethical Committee in the College of Medicine at King Khalid University (Abha, Saudi Arabia) and were performed in agreement with the Principles of Laboratory Animal Care, advocated by the National Society of Medical Research and the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH Publication No. 85, Rev, 1985).

Experimental Design
Rats were divided in 3 major groups and were treated as follows orally: • Control group (n = 12): Received a daily dose of drinking water (5 mL kg −1 ) • Treaded group one (TG1, n = 12): Were 12 rats given (Itraconazole) in a final dose of 5 mg kg −1 and were further subdivided into 2 subgroups each of 6 rats: A-TG1a: Given the drug for 30 consecutive days • TG1b: Given the drug for 60 consecutive days • Treated Group two (TG2, n = 12): Were 12 rats given (Itraconazole) in a final dose of 10 mg kg −1 and were further subdivided into 2 subgroups each of 6 rats: A-TG2a: Given the drug for 30 consecutive days • TG2b: Given the drug for 60 consecutive days

Blood and Tissue Sampling
On the last day of each period (1 or 2 months), 6 rats of each groups were subjected to overnight fasting and then were anaesthetized using light diethyl ether. Blood samples were immediately taken from the eye orbital vein and placed in plain tubes to clot at room temperature. Following centrifugation at 4000 rpm for 10 min, serum was collected and stored at -20°C until analyses. Immediately after blood collection, animals were killed by decapitation. Livers were quickly collected for routine histological examination.

Serum Biochemical Analysis
Serum samples were analyzed to measure the levels of total protein, total albumin, alanine Aminotransferase Science Publications AJAVS (ALT), aspartate Aminotransferase (AST), Gamma Glutamyltransferase (GGT). All analyses were performed with commercially kits (Human Diagnostics Wiesbaden, Germany) according to manufacturer's instructions.

Histological Studies
Histological examination of liver samples was carried out by routine histological procedures. Tissue fixation was carried out, immediately after the experimental procedure, with 10% neutral buffered formaldehyde solution (pH 7.0). Processing was carried out as per the schedule for dehydration, clearing and paraffin infiltration and then the collected tissue was embedded and blocded out. Paraffin sections 5 µm were mounted on clean glass slides coated with Mayer's egg albumin and were stained with hematoxyline and eosin 3. RESULTS

Biochemical Findings
The treatment of the rats with (Itraconazole) daily at doses of both (5 and 10 mg kg −1 ) for 1 month and 2 months days resulted in significant (p<0.001, for all) increases in serum activities of aspartate Aminotransaminase (AST) and alanine Aminotransferase (ALT) and gamma-Glutamyltransferase (γ-GTT) in rats ( Fig. 1) with a concomitant significant reduction in total protein and albumin when compared to control rats received the vehicle (Fig. 2).
The ANOVA analysis showed that serum increases in the levels of these enzymes and the reduction of total protein and albumin were significantly more profound in rats administered (Itraconazole) at the dose of 10 mg kg −1 at both 1 month and 2 months treatment when compare to the group of rats administered the drug at a dose of 5 mg kg −1 with the same periods. After one month treatment, the percentage of increase in the levels of ALT, AST and GTT were 51.5±2.34% versus 204.67±10.56, 46.21±4.56% versus 100.56±5.67 and 85.76±6.54% versus 196.21±8.76%, respectively and the decreases in total protein and total albumin were -31.67±3.45% versus -43.75±2.34% and 28.56±1.45 versus 42.2±3.67% when doses of 5 and 10 mg kg −1 were compared to each, respectively. However, after 2 months treatments, all the percents were deteriorated more with both treatments. The increase in the levels of these hepatic enzymes were 112±5.67% versus 324±8.98, 60±6.32% versus 145±7.32% and 159±10.11% versus 233±11.45%, respectively and the decrease in the levels of total proteins and albumin were 37±2.32% versus 56.25±3.12% and 41.66±4.12% versus 51.65±3.44% when doses of 5 and 10 mg kg −1 were compared.

Histopathological Findings
Light microscopic examination of livers of control rats showed normal hepatic architecture, normal central vein (Fig. 3a) and normal portal tract (Fig. 3b)  Examination of livers rats (TG 1.a) that received itraconazole (5 mg kg −1 ) for 30 days showed a preserved hepatic architecture, normal central vein, few sinusoids congested with blood and prominent Kupffer cells (Fig. 4a).
The portal tract in this group was normal (Fig. 4b).
Rats treated with the same dose of the drug for 60 days (TG1b) showed severe congestion and dilatation of the central vein and the hepatic sinusoids, increase in the number of Kupffer cells and distorted hepatic architecture (Fig. 5a). Furthermore, the portal area showed congestion, dilatation, thickening of the wall of the portal vein and disruption and dilatation of the bile ductules and few inflammatory cells infiltration and prominent Kupffercells (Fig. 5b). Light microscopy of livers of rats that received itraconazole at a dose of 10 mg for 30 days (TG 2a) showed thickening of the central vein wall and dilated sinusoids were congested with blood. Increase in the number Kupffercells was also observed (Fig. 6a) and the portal vein was congested with blood and the bile ductules were disrupted and few inflammatory cellular infiltration (Fig. 6b). Livers of rats treated with the same dose (10 mg kg −1 ) for 60 days (TG 2b) showed dilated central vein, disrupted endothelial lining, distorted hepatic architecture and prominent Kupffercells (Fig. 7a). Severe congestion of the portal vein, marked thickening of its wall and dilated biliary ductules that were infiltrated with inflammatory cells (Fig. 7b).

DISCUSSION
The present study clearly documents the profound hepatotoxicity of itraconazole in experimental rats. In a similar study, Somchit et al. (2004) treated the rats with Itraconazole intraperitoneally for 14 days and found that rats exhibited severe hepatocellular necrosis, degeneration of hepatocytes and cell granuloma. The hepatotoxicity in rats was accompanied by a significant increase in ALP and ALT activities. Although elevated liver enzymes and low albumin and protein were documented in this study, no hepatocellular necrosis or degeneration of hepatocytes were observed.
We treated experimental rats for a longer period (30 and 60 days) in order to simulate the prolonged treatment of fungal diseases such as gastrointestinal blasidiobolomycosis, a disease about a third of which reported world-wide occurs in southwestern region of Saudi Arabia. The liver cell injury as revealed by histology was mainly vascular and resulted in extreme dilatation and congestion of blood in the central veins and portal veins accompanied by thickening of their walls, congestion and dilation of blood sinusoids. In addition, we also report the decrease in plasma proteins (both total protein and albumin). These findings were not reported in the earlier study (Somchit et al., 2004). Here too, we also document an increase to GGT. In this study, toxicity of itraconazole seems to involve obstructive elements as evident by disruption of bile ducts in the portal tracts.
Since Gastrointestinal Basidiobolomycosis (GIB) is endemic in southwestern Saudi Arabia and the disease requires prolonged treatment for a period of 12 to 18 months with itraconbazole (the drug of choice), treating physicians should be advised to regularly monitor the liver enzymes and may need to adjust the dosage if necessary. The severity of liver injury due to itraconazole has been reported to range from mild and transient enzyme elevations to severe acute hepatitis that may require transplantation or may even lead to death (Talwalkar et al., 1999;Srebrniki et al., 2005).

CONCLUSION
Our study showed that prolonged orally administered itraconazole is hepatotoxic and leads to severe hepatitis and liver cell injury. Patients who require treatment with this drug for long periods (12 to 18 months) should have their liver functions periodically monitored.

ACKNOWLEDGMENT
The researcher wishes to thanks Mr. Mahmoud A. Alkhateeb and Mr. Riyad M. Alessa for their contribution in the experimental work of the current study.