Florfenicol (FF) is one of the widest used broad-spectrum antibiotics in aquaculture. While it is well known that cytochrome P450 superfamily CYP 3A metabolizes a myriad of drugs in the terrestrial animals, whether or not this is true for florfenicol metabolism in Nile Tilapia remains unknown. The study investigated multi-organ CYP 3A gene regulation and hepatic CYP 3A enzyme activity of Nile Tilapia at two dosages and two rearing temperatures. The results suggested that CYP 3A is involved in the FF metabolism in Nile Tilapia. FF at regulatory dosage could inhibit CYP 3A activity both at genetic and enzymatic levels
The tilapias were orally administered with either 5 or 15 mg/kg FF in 5 consecutive days when rearing at either 25 or 30°C. The CYP 3A gene expression levels (relative to the control group) in the liver, gill and kidney were measured by qPCR on day 1 and 5. The results indicated that overall, FF at recommended dosage (15 mg/kg) significantly inhibited CYP 3A gene expression in all 3 organs; except for the group with lower dosage (5 mg/kg) at lower temperature (25°C), which exhibited an upregulation of the gene level. When inhibition occurs, it started after the first dose (day 1). (Fig. 1)
To elucidate whether hepatic CYP 3A enzyme activity was also inhibited by FF, the enzyme activity assay was first established using CYP 3A-specific substrate ketoconazole (KTZ) to define CYP 3A enzymatic activity. The liver S9 was prepared and tested with the same concentrations (0.125, 0.25, 0.5 mM) of FF or KTZ at the presence of BFCOD. The result suggested that FF showed approximately 60% of the inhibitory binding ability defined by KTZ (Fig. 2), proving the association of FF to CYP 3A enzyme.
Finally, after FF administration, the hepatic CPY 3A enzyme activities were in general decreased by 50% at both dosages and temperatures, suggesting the inhibitory effect of FF on CYP 3A at protein level (Fig. 3).
In conclusion, the current study demonstrated the association of FF metabolism through CYP 3A enzyme and revealed the mainly inhibitory effects of FF on CYP 3A activity both at the genetic and the enzymatic levels. The inhibition could be both dose and temperature dependent.