D05
Hepatic cytochrome P450‐mediated metabolism of new fluorescent substrates in cats

Abstract

INTRODUCTIONThe cytochrome P450 superfamily constitutes a multigene membrane-bound enzyme system which catalyses oxidations of both xenobiotics and relevant endogenous compounds [1]. Several constitutional factors (gender, age, species, strain, pathological and physiological conditions) might contribute, together with induction and inhibition phenomena (which are classified as exogenous or xenobiotic-dependent factors) to the modulation of the overall biotransformation capacity [2]. In a previous study, differences in the expression of the liver the cytochrome P450 3A subfamily was reported in Limousine and Piedmontese cattle breeds [3]. The expression of main P450dependent drug metabolising enzyme (DME) activities (both at the catalytic and protein expression levels) were measured in the liver of three different breeds of beef cattle. MATERIALS AND METHODSMicrosomal subcellular fractions were prepared, from the liver of male Charollais (C: n = 10), Blonde d'Aquitaine (B: n = 7) and Piedmontese (P: n = 8) cattle, about 13-18 months old. Total P450 content and main P450-dependent DME activities were measured using known model substrates, according to previously published protocols [4,5]. In addition, cytochrome P450 1A (CYP1A), 2B (CYP2B), 2C (CYP2C) and 3A (CYP3A) apoprotein levels were measured in pooled microsomes by immunoblotting (using polyclonal antibodies directed toward the respective rat and/or human P450 isoforms) [3]. The densitometric analysis of visualised bands was performed by ImageJ 1.34s (NIH). Statistical analysis of data was performed using ANOVA, followed by a post-test (Tukey-Kramer multiple comparisons test: GraphPad InstatÒ 3.06 for Windows). RESULTSSignificant differences in the total liver P450 content between breeds were not found. Lower CYP1A, CYP2B and CYP2E1 DME activity was noticed in P (P < 0.05, P < 0.01, P < 0.001, depending from the substrate used) vs. C or A (P < 0.05, P < 0.001). At the protein level, significantly lower amounts of CYP2B were found in P and C vs. B (P < 0.01). No statistically significant differences in CYP1A, 2C and 3A content were found. DISCUSSIONIt is known that several non-pharmacogenetic factors, such as age, gender, species, disease factors or exposure to environmental pollutants, might contribute to the expression and regulation of hepatic P450 in domestic animals [2]. Studies related to the expression of DME in farm animals might be very helpful in food safety assessment and also for drug registration [4,6]. This is of particular importance in cattle, which represent an important source of animal-derived food products. In this respect, studies on DME expression in ruminants have already been published [6][7][8][9][10]. Data obtained in the present study demonstrate that, mostly at the catalytical activity level, some differences exist between different breeds of cattle, confirming what has been observed previously in liver CYP3A expression in P vs. Limousine cattle [3]. Further immunological investigations for each level of P450 protein have already be...