Cattle are a very important part of the human food chain. Administration of veterinary drugs and other xenobiotic compounds to cattle can often result in the accumulation of metabolic residues in edible tissues that can potentially affect humans through the food chain. An understanding of drug metabolism in this species is vital to ensure safe use of drugs in cattle and eventually the provision of safer animal derived food products to man. Sulfation catalysed by sulfotransferases (SULTs) is an important phase 2 drug metabolising reaction. It is not only involved in the detoxification of drugs and xenobiotics but also in the bioactivation of procarcinogens. It is also important in the metabolism of several drugs used routinely in cattle. However, very little work has been carried out on SULTs in cattle. A variety of in vitro tools are available to study drug metabolising enzymes (DMEs) like SULTs. These include tissue microsomes, cytosol, recombinant enzymes and isolated cells such as the hepatocytes.Recombinant SULTs are important tools that can be used for the study of isoform specific drug biotransformation, drug-drug interactions and the effect of genetic polymorphisms on the activity of specific isoforms. As the liver is the major drug metabolising organ in the body, it is essential to study expression and activity of cytosolic liver sulfotransferases. Hepatocytes contain DMEs and drug transporters along with all the necessary cofactors that represent in vivo conditions. This makes hepatocytes a better representative of in vivo conditions as compared to microsomes, cytosol or recombinant enzymes. In this study we have characterised sulfotransferases in cytosol, recombinant enzymes and hepatocytes.Antibodies previously raised against human sulfotransferase isoforms were used in the detection of cytosolic bovine sulfotransferases. Probe substrates established for activity with human SULTs were used for assessing the activity of recombinant and cytosolic bovine sulfotransferases. Cytosol was prepared from 8 male livers and 12 female livers (8 untreated and 4 treated with an exogenous progestin). SULT1B1, SULT1E1 and SULT2A1 were detected in bovine liver cytosol. Expression of SULT2A1 in the bovine liver was sex specific with males expressing almost twice as much SULT2A1 compared to the females. However, no activity was detected with dehydroepiandrosterone (DHEA) which is used as a probe substrate for SULT2A1 in humans. Pregnenolone is metabolised by SULT2A1 and SULT2B1 in humans. Activity towards this substrate was detected in the bovine liver, however no sex related differences in activity were observed. 4-nitrophenol liver, however no sex related differences in activity were observed. 4-nitrophenol is metabolised by several members of the SULT1 family in humans such as SULT1A1, SULT1B1 and SULT1C. 17ß-estradiol is a probe substrate for human SULT1E1. Activity was detected with 4-nitrophenol in male and female bovine livers. Male liver cytosol followed Michaelis-Menten kinetics whereas the female liver cytosol displayed partial substrate inhibition. This suggests that different enzymes have been involved in the biotransformation of 4-nitrophenol in the male and female liver. Activity towards 17ß-estradiol in the female liver was almost 4 times higher than in the male liver.Recombinant bovine sulfotransferases (SULT1A1, SULT1B1, SULT1E1 and SULT2A1) were expressed in E. coli. All bovine SULTs except SULT2A1 were expressed in the soluble fraction. Like human SULT1A1, bovine SULT1A1 also displayed partial substrate inhibition, however the extent of inhibition (as seen with the Ki values) was lower compared to human SULT1A1. Bovine SULT1B1 followed Michaelis-Menten kinetics with 4-nitrophenol. Substrate specificity profiling carried out with equal amounts of bovine SULT1A1 and SULT1B1 revealed that SULT1B1 was better at sulfating phenolic compounds as compared to bovine SULT1A1. SULT1A1 is highly expressed in the human liver and is the major enzyme involved in drug metabolism in the human liver. This might not be the case in cattle given that SULT1B1 was found to be better at sulfation than SULT1A1 and expression of SULT1A1 was not detected in the bovine liver using antibodies. Human SULT1E1 is known to metabolise 17ß-estradiol with a very high affinity and with a Km in the low nanomolar range. Comparatively, bovine SULT1E1 metabolised 17ß-estradiol with a lower affinity, in the micromolar range.Expression and activity of bovine sulfotransferases differed from human sulfotransferases and some of the differences could be attributed to key amino acid residue substitutions in the active site of the bovine SULTs. For example, substitution of Phe141 in human SULT1E1 to Leu141 in bovine SULT1E1 restricts the ability of bovine SULT1E1 to form strong van der Waals interactions with the substrate due to loss of an aromatic hydrocarbon ring. This could explain the reduced affinity of bovine SULT1E1 for 17ß-estradiol. Substitutions of small uncharged residues with large charged ones in the active site of bovine SULT2A1 could have unforeseeable effects that could result in the formation of an insoluble protein. Substitutions in the active site of bovine SULT1A1 that bind the second molecule of 4-nitrophenol could be responsible for the reduced partial substrate inhibition effects observed in comparison to human SULT1A1. In order to further validate some of these findings it would be necessary to perform additional experiments that involve mutating the substituted residue to the original one as found in the human/mouse counterpart and looking for restoration of original properties. The work was extended to investigate conjugative metabolism of the steroid hormone 17ß-estradiol and its stereoisomer 17a-estradiol in microsomes, cytosol and cryopreserved hepatocytes all prepared from bovine liver. It was found that glucuronidation was the main route for estradiol metabolism in cattle since large amount of glucuronide metabolites were detected in microsomes and cryopreserved hepatocytes. In comparison no sulfate metabolites were detected in cytosol and hepatocytes.We now have a better understanding of some of the important phase 2 drug metabolism pathways in cattle.
|Date of Award
|Michael Coughtrie (Supervisor)