TDK conference 2019

Concerning the function of cytochrome P450 (CYP) enzymes in ruminants, limited data were available until now, especially regarding the extrahepatic tissues. Previously, the presence of these enzymes has been demonstrated in several animal species, not only in the liver, but also in the small intestines. So far, just a few CYP subfamilies were detected from rumen wall at mRNA level. Various metabolites can be produced by the ruminal microbial fermentation from orally ingested xenobiotics, and CYP enzymes, as a primary metabolic barrier, may have an important role in their metabolism. Hence, we investigated the activity of CYP1A2 and CYP2C9 isoenzymes in bovine hepatic and extrahepatic tissues.

Samples were gained from ruminal papillae, duodenal mucosa and processus caudatus of the liver from Aberdeen Angus beef cattle (n = 22) at the slaughterhouse of Mikofami Kft. in Zalaszentiván. The post-mitochondrial supernatant containing CYP enzymes was isolated after homogenization of tissue samples in phosphate buffer by a multi-step differential centrifugation. Specific activities of CYP1A2 and CYP2C9 enzymes were assessed by luminometric P450-Glo assays. BCA Assay Kit was applied to measure the total protein concentration of the samples in order to standardize the results. The amount of hemoglobin in the liver samples was determined with ammonia reagent. The enzyme activities measured in the liver were standardized to the total protein concentration excluding haemoglobin, whereas the CYP activity of the mucosal membrane of the rumen and intestines was standardized to the total protein concentration.

According to our results, activities of CYP1A2 and CYP2C9 were well detectable by the luminometric method. CYP1A2 activity was significantly higher than the activity of CYP2C9 in all three examined tissues. The difference between the activity of the investigated isoenzymes was the most remarkable in the case of liver samples, with nearly 100 times higher activity of CYP1A2 compared to that of CYP2C9. However, the activities of both examined isoenzymes were considerably lower in extrahepatic tissues compared to liver samples, but they were above the detection level, thus important data were gained by investigating these tissues as well. Our results suggest that the CYP1A2 isoenzyme may play an important role in the process of detoxification in cattle. meanwhile the CYP2C9 isoenzyme is suggested to play no central role in these processes. Valuable results have been obtained about the detection of detoxifying processes in cattle, hence we can state that our research group has successfully adapted the measurement procedure to detect CYP enzyme activity in ruminant samples.