Introduction. The cytochrome P450 (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 cytochrome P4503A subfamily have been reported in Limousine and Piedmontese cattle breeds (3). As an in-depth study, the expression of main P450-dependent drug metabolising enzyme (DME) activities (both at the catalytic and protein expression levels) were measured in the liver of three different meat cattle breed. Materials and Methods. Microsomal subcellular fractions were prepared, from the liver of male Charollais (C: n=10), Blonde d’Aquitaine (B: n=7) and Piedmontese (P: n=8) beef cattle, about 13-18 months old. Total P450 content and main P450-dependent DME activities were measured, by using known model substrates, according to previously published protocols (4, 5). Furthermore, the cytochrome P4501A (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) as reported elsewhere (3). The densitometric analysis of visualised bands was performed by ImageJ 1.34s, NIH, Bethesda, MD, USA). Data statistical analyses was performed by using the analysis of variance (ANOVA), followed by a post-test (Tukey-Kramer multiple comparisons test: GraphPad Instat® 3.06 for Windows, GraphPad Software Inc., San Diego, CA, USA). Results. Significant differences in the total liver P450 content were never found among breeds. Lower CYP1A, CYP2B and CYP2E1 DME activities were 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, lower significant CYP2B amounts were found in P and C vs B (P<0.01). No statistically significant differences were ever found in CYP1A, 2C and 3A contents. Discussion and Conclusion. It 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). It has been otherwise underlined how studies related to the expression of DME in farm animals might be very helpful in food safety assessment and drug registration requirements as well (4, 6). This 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 (6-10). Data obtained in the present study demonstrate that, mostly at the catalytical activity level, some differences exist between different cattle breeds, confirming what previously observed in liver CYP3A expression in P vs Limousine cattle (3). Actually, further immunological investigations to complete data for each P450 protein levels have been undertaken in our laboratory; this would permit the evaluation of linear correlation analysis between the individual CYP1A, 2B, 2C and 3A protein amounts and the respective model substrate catalytic activity, within and between the three different breeds. Furthermore, oncoming studies, whose major goal might be the confirmation of these preliminary results also at the gene expression levels, are foreseen. Reference. Honkakoski P. and Negishi M. Biochem. J. 2000; 347: 321-337. 2. Nebbia C. Vet. J. 2001; 161: 238-252. 3. Dacasto et al. Vet. Res. 2005; 36: 179-190. 4. Nebbia et al. Vet. J. 2003; 165: 53-64. 5. Purdon & Lehmann-McKeman J. Pharmacol. Toxicol. Methods 1997; 37: 67-73. 6. Sivapathasundaram S. et al. Biochem. Pharmacol. 2001; 62: 635-645. 7. Machala M. et al. Arch. Toxicol. 2003; 77: 555-560. 8. Sivapathasundaram S. et al. Toxicology 2003; 187: 49-65. 9. Sivapathasundaram S. et al. Comp. Biochem. Physiol. 2003; 134C: 169-173. 10. Szotáková B. et al. Res. Vet. Sci. 2004; 76: 43-51. Acknowledgements. This study was supported by a grant from Università degli Studi di Padova (60A08-8213/05: studi sull’espressione degli enzimi farmaco-metabolizzanti nel fegato di bovini appartenenti a razze diverse) to M.D and a two-years post-doctoral fellowship, from Università degli Studi di Torino, to M.C.

Expression of liver cytochrome P450 drug metabolising enzymes in different meat cattle breeds

GIANTIN, MERY;CAPOLONGO, FRANCESCA;DACASTO, MAURO
2006

Abstract

Introduction. The cytochrome P450 (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 cytochrome P4503A subfamily have been reported in Limousine and Piedmontese cattle breeds (3). As an in-depth study, the expression of main P450-dependent drug metabolising enzyme (DME) activities (both at the catalytic and protein expression levels) were measured in the liver of three different meat cattle breed. Materials and Methods. Microsomal subcellular fractions were prepared, from the liver of male Charollais (C: n=10), Blonde d’Aquitaine (B: n=7) and Piedmontese (P: n=8) beef cattle, about 13-18 months old. Total P450 content and main P450-dependent DME activities were measured, by using known model substrates, according to previously published protocols (4, 5). Furthermore, the cytochrome P4501A (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) as reported elsewhere (3). The densitometric analysis of visualised bands was performed by ImageJ 1.34s, NIH, Bethesda, MD, USA). Data statistical analyses was performed by using the analysis of variance (ANOVA), followed by a post-test (Tukey-Kramer multiple comparisons test: GraphPad Instat® 3.06 for Windows, GraphPad Software Inc., San Diego, CA, USA). Results. Significant differences in the total liver P450 content were never found among breeds. Lower CYP1A, CYP2B and CYP2E1 DME activities were 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, lower significant CYP2B amounts were found in P and C vs B (P<0.01). No statistically significant differences were ever found in CYP1A, 2C and 3A contents. Discussion and Conclusion. It 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). It has been otherwise underlined how studies related to the expression of DME in farm animals might be very helpful in food safety assessment and drug registration requirements as well (4, 6). This 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 (6-10). Data obtained in the present study demonstrate that, mostly at the catalytical activity level, some differences exist between different cattle breeds, confirming what previously observed in liver CYP3A expression in P vs Limousine cattle (3). Actually, further immunological investigations to complete data for each P450 protein levels have been undertaken in our laboratory; this would permit the evaluation of linear correlation analysis between the individual CYP1A, 2B, 2C and 3A protein amounts and the respective model substrate catalytic activity, within and between the three different breeds. Furthermore, oncoming studies, whose major goal might be the confirmation of these preliminary results also at the gene expression levels, are foreseen. Reference. Honkakoski P. and Negishi M. Biochem. J. 2000; 347: 321-337. 2. Nebbia C. Vet. J. 2001; 161: 238-252. 3. Dacasto et al. Vet. Res. 2005; 36: 179-190. 4. Nebbia et al. Vet. J. 2003; 165: 53-64. 5. Purdon & Lehmann-McKeman J. Pharmacol. Toxicol. Methods 1997; 37: 67-73. 6. Sivapathasundaram S. et al. Biochem. Pharmacol. 2001; 62: 635-645. 7. Machala M. et al. Arch. Toxicol. 2003; 77: 555-560. 8. Sivapathasundaram S. et al. Toxicology 2003; 187: 49-65. 9. Sivapathasundaram S. et al. Comp. Biochem. Physiol. 2003; 134C: 169-173. 10. Szotáková B. et al. Res. Vet. Sci. 2004; 76: 43-51. Acknowledgements. This study was supported by a grant from Università degli Studi di Padova (60A08-8213/05: studi sull’espressione degli enzimi farmaco-metabolizzanti nel fegato di bovini appartenenti a razze diverse) to M.D and a two-years post-doctoral fellowship, from Università degli Studi di Torino, to M.C.
File in questo prodotto:
Non ci sono file associati a questo prodotto.
Pubblicazioni consigliate

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11577/2469349
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus ND
  • ???jsp.display-item.citation.isi??? ND
social impact