Embryotoxicity of fluconazole on developing chick embryos

Background Fluconazole is a first-generation triazole used as an antifungal treatment for skin, hair, and nail infections. The study aimed to assess the embryotoxicity and teratological effects of fluconazole on chick embryos. Fertilized eggs were divided into four groups: two experimental groups treated with different concentrations of fluconazole (0.1 ml/egg, 0.2 ml/egg) containing 12 and 11 eggs, respectively, one group treated with distilled water (10 eggs), and a control group (10 eggs) left untreated. The liquid form of fluconazole was administered on the 4th day of incubation, and observations were made on the 9th day. Results There were different anomalies observed in them like hydrocephaly, microcephaly shorting of the beak, agenesis, Amelia, micromelia, anophthalmia, microphthalmia, and kyphosis. There were also observed the morphometric measurements with a difference of significant ( p < 0.001) and ( p < 0.01) in CR length, body weight, head circumference, eye circumference, forelimb, and hind limb. Different vital organ defects were also observed in histological studies. Fluconazole in various combinations has been found to cause embryotoxicity and teratological consequences in chick embryos. Conclusion The current study showed that fluconazole is teratogenic in creating chick undeveloped organisms. The embryotoxic impacts were recorded on structural morphometric and graphic levels.

mutations (Vale et al., 2015).An in vitro susceptibility study using the 1997 National Committee for Clinical Laboratory Standards showed that 92% of Candida albicans isolate 5-fluorocytosine and 90% were at risk of fluconazole and itraconazole.Local variability in the frequency of fluconazole and itraconazole was observed.Isolates from the northwest and southeast regions were more immune to fluconazole and itraconazole than those from the northeast and southwest regions (Pfaller et al., 1998;Bilal, 2021b).
Fluconazole is a specific inhibitor of ergosterol, a steroid found only in the fungal cell membrane.Fluconazole is rapidly dispersed in body fluids and is absorbed by the gastrointestinal tract.Fluconazole is marketed as Brazilian capsules (Corrêa & Salgado, 2011).Natural antimicrobial qualities, which differ greatly among medications and include antibacterial, antiviral, and antifungal properties, are a common feature of all immunosuppressants.It has been demonstrated that immunosuppression alters the composition of the gut microbiome and that variations in the microbiome's composition are related to inflammation such as uveitis (Bilal, 2021a;Jones, 2020).
Fungal infections, both in the upper and lower extremities, are a major problem for severely impaired patients who are transmitted to the bone marrow.From the beginning of the type of conditioning until the neutrophil count returned to 1000 per microliter, suspected toxins or planned fungal infections were suspected or proven, and fluconazole or a placebo was given for prevention.Fluconazole, given as a way to prevent recipients of bone marrow transplants, reduces the risk of both systemic and superficial fungal infections (Wadaan et al., 2023;Goodman et al., 1992).
Fluconazole is derived from imidazole which is used to treat fungal infections locally and systemically.Fluconazole is not recommended for oral use because of its many side effects.It is used in topical gels for the treatment of skin fungal infections (Helal et al., 2012).
Effect of fluconazole prophylaxis on cases of fungal infections and death in critically ill surgical patients.In the intensive care unit, fluconazole was compared with placebo for fungal infections (SICU).Although prophylactic fluconazole medications to prevent mycoses in SICU patients appear to reduce the incidence of these diseases, they do not appear to increase survival (Shorr et al., 2005).Azoles antifungal has anti-leishmanial action.Fluconazole is a drug used to treat leishmania for severe cutaneous leishmaniasis (Alrajhi et al., 2002;Bilal et al., 2021aBilal et al., , 2021b)).
Cryptococcosis is a fungal infection caused by Cryptococcus neoformans, a yeast-treated fungus found worldwide.In patients with AIDS, cryptococcosis is rarely treatable, and fluconazole is the preferred treatment for life-threatening depression (nutrition).Long-term stress treatment with fluconazole has been linked to improved azole resistance in cryptococcal infections in AIDS patients (Aller et al., 2000).
Infectious fungal infections are associated with a higher rate of morbidity and mortality in premature babies.Prophylactic fluconazole was tested for its effectiveness in preventing fungal rot and invasive infections in children with very low birth weights.In newborns with a birth weight of less than 1000 g, prophylactic dosing of fluconazole during the first six weeks of life helps prevent fungal rot and invasive fungal infections (Kaufman et al., 2001;Iftikhar et al., 2021).
Adverse events (AEs) and drug interactions are associated with fluconazole treatment in newborns and other groups of children.The most common side effects are hepatotoxicity and gastrointestinal toxicity.It is important to remember that drug interactions with fluconazole can cause serious side effects (Egunsola et al., 2013).
Ketoconazole and fluconazole are two of the antifungal drugs currently available.With the dispersion of the tablet, the absorption of ketoconazole depends on the pH of the stomach.The effects of height are induced by cimetidine on pH in fluconazole and bioavailability-related ketoconazole (Blum et al., 1991b).
In patients with advanced HIV infection, Candida esophagitis is still a major source of infection.Fluconazole is well known as the most effective treatment for this condition.In individuals with advanced HIV infection and confirmed Candida esophagitis, caspofungin has been shown to be equally effective and well tolerated as fluconazole (Villanueva et al., 2002).
In patients who were immune to biopsy-proven esophageal candidiasis, voriconazole was shown to be at least as effective as fluconazole (Ally et al., 2001).Amphotericin B (AMB) is a common treatment for fungal endophthalmitis, which can cause blindness.AMB has an anti-angiogenic effect on the chick chorioallantoic membrane (CAM) model in vivo.According to the findings, AMB has the potential to be used as an anti-angiogenic drug.Because this is the first CAM study, further research on animals and humans is needed (Gokce et al., 2016).
Three clinical locations for Candida Krusei underwent investigations into the mechanisms of fluconazole resistance.Ergosterol is the primary sterol of Candida krusei, and fluconazole inhibits 14-demethylase in the body, according to biological sterol analysis, which was higher in the presence of fluconazole than in the absence of the drug.When compared to Candida albicans strains that are vulnerable to fluconazole, the 14-demethylase activity in C. krusei cell extracts was 16 to 46 times more resistant to suppression (Orozco et al., 1998).
Fluconazole is a product of a research program aimed at developing the drug an extensive antifungal that can be used to treat infectious diseases and the system.It led to the development of a novel number from 1,3-bistriazole-2-arylpropan-2-ol which showed promise in various models of fungal infections (Bilal et al., 2022a(Bilal et al., , 2022b;;Iftikhar et al., 2021).Fluconazole, a 2,4-difluoro phenyl analog with a suitable combination of antifungal efficacy, pharmacokinetic properties, aqueous solubility, and safety profile, has been selected for development (Richardson et al., 1990).Fluconazole, an antifungal, has been found to be a potent inhibitor of cytochrome P450 (P450) 2C9, a key enzyme involved in converting the powerful anticoagulant warfarin into inert hydroxy warfarin metabolites in vivo.Fluconazole may interact with any drug whose approval is controlled by P450s 2C9, 3A4, and other unknown isoforms.Overall, the findings clearly support the notion that human in vitro microsomal data can be used to predict metabolically based in vivo drug interactions (Kunze et al., 1996).
The aims and objectives of the present study were to check embryotoxicity and teratological effect of fluconazole on chick embryos.

Methods
Embryotoxicity tests were performed on freshly fertilized eggs of the white leghorn.Eggs were purchased at the Lahore Veterinary Research Center.All eggs were divided into 4 groups, A B C, and D. They were all stored in an incubator filled with alcohol.On the fourth day, the two groups were treated with different fluconazole, concentrations were 0.1 ml/egg and 0.2 ml/egg.One group was controlled, and the other was treated with 0.1 ml of distilled water.
Without considering the size of the eggs, all the eggs were randomly selected then all the eggs were washed with alcohol on a piece of cotton and marked in groups.
Fluconazole was found to be stable.The concentration of the chemical used in the 15 mg stock solution was adjusted by dissolving 10 ml of concentrated water used in 0.1 ml and 0.2 ml in the stock solution (to make a dose, for a dose in liquid form).
With the aid of a scalpel, a small hole was completed in the egg case outside group D. To protect the shell membrane, special care was performed.0.1 ml of each fluconazole was injected into an egg sac with an insulin syringe.The whole process was done in a sterile state.The hole in the eggshell was covered with melted paraffin wax.

Incubation
The incubator used for this test has a capacity of about 100-150 eggs.Eggs were placed on incubator shelves.Every incubator shelf was covered with a layer of cotton and sterilized with alcohol.The temperature was set to 37-38, and by placing a water beaker in the incubator humidity was maintained.Eggs were changed twice a day.Eggs were screened if an odor was found and discarded.

Recovery
It is done on the ninth day of incubation.The eggs were removed from the incubator and opened on the wide side in a bowl filled with water.Albumin was released.With the help of a blunt force yolk, the ball was removed.The embryo was separated from the cocoon in an enamel dish and added water.All obtained eggs were fixed with a Bouin's solution (picric acid, acetic acid, and formalin) for 48 h.All embryos were washed with 70% and 90% alcohol for histology (Fig. 1).

Morphological observations
Morphometric observations include a measurement of CR length, recording of head weight and eye circumference, and organ size of the retracted chicks.

Data analysis
Complete data were subject to statistical data.Average values were obtained for all groups in all parameters with the help of Microsoft Excel.All values were analyzed.

Morphological studies
To assess the embryotoxic effects of fluconazole, all embryos from the experimental groups were compared to those from the control group in morphometric and functional assessments.

Control group
The fetus of this group was well formed into neck regions, heads, and stems.The head had well-formed brain segments and a long neck.The larvae had large, bulky eyes.The mouth was also quite distinct with a diverse higher jaw.The shaft region is in fine form.The spine and heart rates of all embryos were common.The protruding front legs and hind legs were extant in the shaft area, digital establishment had activated in some cases.It was recorded that the hind limbs have very different numbers and show a much more advanced stage of growth compared to the previous numbers.All embryos had neatly folded and distinct tails.Although several abnormalities have been observed in embryos of experimental groups treated with fluconazole (Figs. 2, 3, Table 1).

Treatment 1
A total of 9 embryos were recovered in a set.In total, 2 embryos were abnormal.Morphological and teratological effects in this set were less as compared to other experimental groups.There was microcephaly in 9% of embryos.Hydrocephaly was in 2% of embryos, 5% of embryos had shorting of beak while 7% had Agenesis.Most embryos had prominent eyes while 1 % had Anophthalmia and 5% had Microphthalmia.There was 14% Amelia and 20% micromyelia in the forelimb.In the hind limb, there was 12% Amelia and 18% micromelia.The neck was well developed while 6% kyphosis was observed.

Treatment 2
A total of 5 embryos were recovered in this group and 4 were malformed.20% microcephaly was observed in embryos.35% agenesis was also observed in them.Microphthalmia was observed in 39% of embryos.42% showed Amelia and 39% micromyelia observed in forelimb, while in hind limb there were 55% Amelia and 39% micromyelia observed.

Morphometric studies Control group
The crown rump length of the embryos averaged 13.70 ± 1.14 mm and the body weight of the embryos averaged 44.18 ± 17.30 mm.The head circumference of the embryos averages 5.88 ± 0.50 mm.The eye circumference of embryos averages 2.65 ± 0.25 mm.The forelimb and embryos average 3.15 ± 0.26 mm and the hind limb length averages 2.77 ± 0.40 mm (Figs. 5, 6, 7).

Experimental group Treatment 1(D.W)
The crown rump length of the embryos of the set was an average of 13.05 ± 1.91 mm.The body weight of embryos averages 27.36 ± 11.17 mm.Head circumference of embryos averages 5.98 ± 0.22 mm.The eye circumference of embryos averages 2.86 ± 0.68 mm.The forelimb length of embryos averages 2.86 ± 0.45 mm.The hind limb length of embryos averages 3.03 ± 0.35 mm.

Treatment 2 (0.1 ml/egg)
The crown rump length of this group averaged 10.86 ± 0.59 mm.The body weight of embryos averages

Discussion
Fluconazole belongs to a class of drugs called azole antifungals.The most common cause of fungal infections is a yeast called candida.It's a drug that's used to treat serious systemic candidal infections (Saag and Dismukes 1988).It was reported that intravenous fluconazole 400 to 800 mg daily for 5 weeks followed by intraperitoneal fluconazole 150 mg daily for 2 days induced a prolonged QT interval with torsades de points in a 59-year-old female with Candida albicans peritonitis.Because of the time course of events and the fact that the patient was not receiving any additional QT-prolonging medicines, the authors believe that QT prolongation was a direct effect of fluconazole (Khazan & Mathis, 2002;Wassmann et al., 1999); Iftikhar et al., 2023).
Fluconazole prescription during the first trimester is not completely contraindicated, according to data published in the recent decade.Many of the instances studied have methodological flaws, such as a lack of cautionary reporting of patterns of usage throughout pregnancy, a lack of controlling for confounding factors, the potential impacts of recall bias, and a lack of data on suspected deformities in abortions (Bellantuono et al., 2013).
Anxiety and panic disorders are routinely treated with fluconazole in women of reproductive age.It is difficult to find information about its safety during pregnancy.Rayburn et al. (2002) for example, conducted a postmarketing evaluation of 542 cases of first-trimester fluconazole exposure and found no evidence of an increase in spontaneous abortion.
The present reading was conducted to gauge the effect of the embryotoxicity of fluconazole on hatchling embryos.Different concentration was given to the fertilized eggs on the 4th day of incubation and recovery was done on the 9th day of incubation.After the recovery of the chick embryos, they were subjected to morphometric, morphological, and histological observation.Morphometric analysis revealed substantial (p < 0.001 and p < 0.01) reductions in CR length, body weight, head, and ocular circumferences in the current study.These deformities were seen in all groups treated with varying concentrations of fluconazole, indicating that fluconazole, when taken in low doses, poses a risk of causing teratogenicity and embryotoxicity.The results of the current study were also observed by.Winker's (2007) found an elevated risk of preterm birth and low birth weight in infants whose mothers were given fluconazole during their pregnancy.Infants exposed early in pregnancy had a substantially higher prevalence of relatively major congenital abnormalities.Similar observations were made by Takzare et al. (2011) on rat fetuses revealing that there was a substantial difference.
In the current study, the treated embryos had a significant reduction in head circumference and body weight when compared to the control group.These findings were in line with (Blum et al., 1991a;Iqbal et al., 2002) those of a study in which 10 cases of newborns were    and Hill, 2003;Rayburn et al., 2002).
Staying away from fluconazole during pregnancy and lactation would be prudent.tostay away from the likely gamble of congenital surrenders, doctors recommend fluconazole as monotherapy at the least successful measurements for the briefest conceivable length fixation ought to be benzodiazepine treatment among pregnant or lactating ladies includes involving these medications at the most minimal dose for the briefest conceivable term, keeping away from use during the principal trimester, and keeping away from multidrug regimens (Friedberg and Gartner, 1990;Iqbal et al., 2002).

Conclusions
The current study showed that fluconazole is teratogenic in creating chick undeveloped organisms.The embryotoxic impacts were recorded on structural morphometric and graphic levels.The morphological and morphometric analysis showed various peculiarities.Likewise, histological outcomes uncovered the underdevelopment of numerous organs of chick undeveloped organisms.Consequently, keeping in view, the previously mentioned discoveries, it is laid out that fluconazole has embryotoxic and teratogenic potential, and to that end, this medication ought to be totally kept away from in pregnant ladies, and in its place, different strategies ought to be considered to adapt to melancholy and uneasiness, for example, mental help by performing quieting exercises like intercession.Anyway, it very well may be involved under outrageous conditions in the least conceivable fixation and not without the oversight of the doctor.

Fig. 1
Fig. 1 Scheme representing the application of fluconazole to fertilized eggs on the 9th day of incubation

Fig. 6 Fig. 8
Fig. 6 A composite macro-photograph representing the embryo of group A smoked with the lowest dose (0.1 ml/egg) of fluconazole, showing a number of defects in the development of body parts on the 9th day of gestation, microcephaly (Mc), agenesis of the beak (Ag), Amelia (Am) and micromelia (Mm)

Fig. 10
Fig. 10 Graphic representation shows the conclusion of fluconazole on the eye size of chick embryos recovered at 9 days of gestation

Fig. 12
Fig. 12 Graphic representation shows the results of fluconazole on the size of the hind limb of chick embryos recovered at 9 days of gestation

Fig. 13
Fig. 13 Graphic representation shows the outcome of fluconazole on the body weight of chick embryos recovered at 9 days of gestation

Table 1
Embryotoxic effects of different concentrations of fluconazole on 9th days of old chick embryos

Table 3
Morphological modifications induced in 9 days of developing chick embryos treated with a concentration of fluconazole