Microorganisms including fungi can cause great harm and damage. They infect people, animals and plants, producing diseases that range in seriousness from mild infection to death.

Fluconazole, an orally active synthetic bis-triazole, is established worldwide as a leading antifungal agent [1]. In addition, it has an important role in the treatment and prophylaxis of fungal infections in immunocompromised adults [2,3].

Mechanistically, fluconazole inhibits the synthesis of ergosterol, which is an essential component of fungal cell membranes causing abnormalities in the membrane permeability, causing death to the cell [4]. Fluconazole has a favorable pharmacokinetic profile that includes a long serum half-time, which makes once-daily administration possible, more consistent absorption from the gastrointestinal tract than that of ketocnazole, excellent penetration into the cerebrospinal fluid, and elimination predominantly by renal mechanism [5]. Moreover, it is the standard agent for prophylaxis against invasive fungal infections of pancreas, liver and hematopoietic stem cell transplant recipients [6]. Fluconazole is also used in the treatment of oropharyngeal, oesophageal, vaginal or systemic candidiasis and for fungal skin infection [7]. In addition, Havlir et al. [8] pointed out that, administration of 200 mg daily dosage of fluconazole is effective in reducing deep fungal infection in patients with AIDS.

The successful use of fluconazole has been hampered by a variety of side effects including headache, hair loss, nausea, anorexia, eosinophilia and aspartate aminotransferase increases [9].

Guillaume et al. [10] described severe subacute liver damage occurring in a patient with AIDS who was receiving fluconazole maintenance therapy for a cryptococcosis. Pulmonary and hepatic toxicity due to nitrofurantoin and fluconazole treatment was also recorded by Linnebur and Parnes [11]. Severe hepatotoxicity has also been described by Rodriguez and Acosta [12] in patients undergoing itraconozole or fluconazole therapy. Essawy et al. [13] reported that force feeding toads with fluconazole at therapeutic dose level (0.26 mg/toad) resulted in pronounced alterations of blood cells and these alterations are more or less similar to those reported in human with leukaemia.

Some authors recommended the use of amphibians as quick biological test animals for screening the carcinogenicity and hazardous effects of chemicals and drugs [13-15]. This promotes us to study the possible carcinogenic activity of fluconazole in Egyptian toads Bufo regularis.

Sexually mature male and female toads, Bufo regularis (30-40g each), were used. The animals were collected by a regular supplier from El-Nozha district in Alexandria, Egypt. They were maintained in the laboratory at 22°C and fed earth worms once every 3 days. They were kept in large glass aquaria with small amounts of water that was changed twice daily.

The toads were divided into two groups of 100 toads each (50 males and 50 females). Each toad of the first group was force-fed daily for 20 weeks with 0.26 mg fluconazole dissolved in 0.5 ml amphibian saline. This dose represents what is equal to the human therapeutic dose. Fluconazole was obtained from Pfizer, Egypt under authority of Pfizer Inc., U.S.A. The toads of the second group served as controls and were force fed with 0.5 ml of amphibian saline.

Experimental toads and their controls were autopsied after different time interivals (each 2 weeks). Any affected organs or tumors present were quickly removed and fixed in Bouin’s fluid. The materials were then washed, dehydrated and embedded in paraffin wax and sectioned. The sections were stained with Ehrlich’s hematoxylin and counterstained with eosin for histopathological examinations.

Statistical analysis

In the present study, one -way analysis of variance (ANOVA) was used to assess the significant change in the percentage of mortality. Z-test was used for comparison of the percentage of tumor incidence between control and treated groups. The least significant difference (L.S.D) among groups was employed to test the significance of difference between any two groups.

I- Effect of fluconazoleon the mortality of toads

During the period of experiment, the number of deaths occuring among the experimental animals was recorded and the percent of mortality was calculated and tabulated after 20 weeks. (Table 1). It was found that only 6 out of 100 control toads (received amphibian saline) died during the experiment. Administration of fluconazole at a dose level of 0.26 mg/toad increased the mortality rate significantly by 16 %.

Table 1: Effect of administration of Fluconazole on the percentage of mortality of the Egyptian toad (Bufo regularis).

II- Histological observations

The architecture of the normal liver of toads (Figure 1A) is characterized by the presence of large number of hepatic acini that are arranged in clusters, closely packed and connected together by connective tissue. In addition, blood vessels, bile ductules and pigment granules are present.

Periodical examination of the liver of toads force-feed with fluconazole at a dose level of 0.26 mg/toad daily for 20 weeks revealed several pathological changes including development of tumors. The liver tumors began to appear 10 weeks after the initiation of feeding and the incidence of induced tumors was 8 %. The tumors are well differentiated hepatocellular carcinomas in which the nodules appear as localized mass (Figure 1B - Figure 1D) occupied by crowded, irregular and poorly differentiated cells. Cells exhibit loss of polarity and display varying degrees of pleomorphism. Nuclei are enlarged, spherical or oval in outline and some of them are hyperchromatic. Nucleoli are prominent and mitotic figures are also encountered (Figure 1D). The central part of the tumor mass exhibits necrosis (Figure 1B).

Figure 1a-d: Light micrographs of sections of toad’s liver’ (Haematoxylin and Eosin stain): a- showing the architecture of normal liver. Hepatocytes are arranged in closely packed hepatic acini.Arrows point at pigment granules. Bv: blood vessel. (X 100). b- showing tumor nodule (T) in the liver of fluconazoletreated toad. Nc: Necrotic cells. (X 100). c- showing a high power view of focal liver lesion. Note large vesicular nuclei (arrow’s heads) and prominent nucleoli of tumor cells. (X 400). d- showing tumor cells with pleomorphic, irregular vesicular nuclei with prominent nucleoli. Arrows point at multiple round and abnormal mitotic figures. (X 1000).

The hepatic parenchmal cells surrounding the tumor nodules are disorganized and lose their uniform arrangement into hepatic acini. Hepatocytes adjacent to the nodules appear compressed.

In addition to hepatic neoplastic lesions, liver cells of some fluconazole treated toads showed feathery degenerated and vacuolated cytoplasm, proliferation of bile ducts, fatty changes, dilated blood vessels and congested sinusoids (Figure 2a – Figure 2d).

Figure 2a-d: light micrographs of sections of liver of fluconazole-treated toads (Haematoxylin and Eosin stain): a-showing multiple intra and extracellular bile thrombi (B). The liver cells show feathery cytoplasm. (Degenerative changes), (X 200). b- Showing marked fatty changes (degenerative changes) with prominent central vein and preserved architecture. (X 200). c- Showing markedly dilated congested blood vessels (Bv) congested sinusoids (S) and multiple bile thrombi (B). (X 200). d- Showing proliferation of bile duct (Bd). (X 100).

Moreover, stomach and Ileum of 4 toads showed focal mucosal ulceration and in the fat body of 4 experimental toads, increase cellularity with replacement of fat cells by another cellular infiltrate was recorded (Table 2). The cellular infiltrate has the same structure as the liver focal lesion (metastatic deposits from liver mass). No pathological changes were detected in the liver or other organs of any toad of the control group during 20 weeks.

In the present study, oral administration of fluconazole produced several histopathological changes in the liver of toads. The prominent and good finding was a focal hepatic lesion, which was frequently found (8%). This mass of lesion is composed of neoplastic cells with the histological signs of malignancy.

The carcinogenic effect of the antifungal drugs has been investigated in different species of animals. EL-Mofty et al. [14,16] showed that dietary exposure to griseofulvine produced hepatocellular carcinomas in toads and breast neoplasia and lung tumors in mice. Also, induction of leukaemia by the antifungal drugs, griseofulvine , nizoral and fluconazole has been documented [13,17,18].

In a two years carcinogenicity study in Spragne-Dawley rats, fluconazole decreased mammary fibroadenomas in females and increased hepatic adenomas in males [19]. Rosita et al., [20] showed that low concentrations of fluconazole induced hepatotoxicity in primary culture system of rat hepatocytes. Pulmonary and hepatic toxicity due to nitrofurantoin and fluconazole treatment was also recorded by Linnebur and Parnes [11]. Severe hepatotoxicity has also been described by Rodriguez and Acosta [12] in patients undergoing itraconozole or fluconazole therapy Olin [21] observed an increased incidence of hepatocellular adenomas in male rats treated with fluconazole at dose levels of 5 and 10 mg/kg/day. Treatment-related side effects affecting the liver and biliary system were also reported in children received multiple doses of fluconazole [22].

The principal mechanism of action of the triazoles is to inhibit cytochrome P450 enzymes in fungal organisms. Because these enzymes are also integral components of the smooth endoplasmic reticulum and of the inner mitochondrial membrane, Guillaume, et al. [10] hypothesized that the mitochondrial hepatic abnormalities could be related to some fluconazole–cytochrome P450 enzyme interaction and the structural alterations in liver cells are associated with mitochondria impairment. On the other hand, Somicht et al. [23] showed that inhibition of cytochrome P450 may increase the hepatotoxicity induced by azole antifungal drugs which have been demonstrated to inhibit cytochrome P450.

The present results are very important because many patients in our country (Egypt) use extensively fluconazole as a highly effective antibiotic for the treatment of various types of fungal infections. Finally, we recommended that, antifungal drugs in general, are not to be used haphazardly in a massive scale and whenever used, patients must always be carefully scrutinized.