Endophyte infected tall fescue (E+) is the base diet for nearly all beef cattle in the southern USA. It has been linked to a variety of toxicological conditions due to the presence of large numbers of ergot alkaloids....Endophyte infected tall fescue (E+) is the base diet for nearly all beef cattle in the southern USA. It has been linked to a variety of toxicological conditions due to the presence of large numbers of ergot alkaloids. This study was designed to investigate the effects of E+ seed extract and selected ergot alkaloids on the detoxification pathway by cytochrome P450 (CYP3A4) enzyme system. Tests were performed using the P450- Glo CYP3A4 enzyme activity kit (Promega, WI), according to the manufacturer’s manual. Luminescence was measured using a single tube TD20/20 luminometer. Endophyte infected tall fescue seed was extracted with 50/50 methanol/25 mM ammonium carbonate, cleaned and concentrated on Strata-X reversed phase column (Phenomenex). The extracts were evaluated on an HPLC, and then tested using a serial dilution method. Commercially available ergonovine (EN), ergocorine (ER), bromocryptine (BC) and ergocryptine (EC) were tested individually using 0 to 44 nM concentrations. Seed extract of E+ produced a significant (P P < 0.05) dose dependent manner with EC being most potent, followed by ER, BC, and then EN (70%, 40%, 30% and 10% at 44 nM concentration). The similarity of the inhibition curves of seed extract to that of the commercially available ergot alkaloids suggests a related mode of action and that the use of such ergot alkaloids and CYP3A4 assay is a good model to study the toxicity of tall fescue. Furthermore, it provides the foundation to identify the individual toxic components of purified endophyte infected tall fescue extract.展开更多
文摘Endophyte infected tall fescue (E+) is the base diet for nearly all beef cattle in the southern USA. It has been linked to a variety of toxicological conditions due to the presence of large numbers of ergot alkaloids. This study was designed to investigate the effects of E+ seed extract and selected ergot alkaloids on the detoxification pathway by cytochrome P450 (CYP3A4) enzyme system. Tests were performed using the P450- Glo CYP3A4 enzyme activity kit (Promega, WI), according to the manufacturer’s manual. Luminescence was measured using a single tube TD20/20 luminometer. Endophyte infected tall fescue seed was extracted with 50/50 methanol/25 mM ammonium carbonate, cleaned and concentrated on Strata-X reversed phase column (Phenomenex). The extracts were evaluated on an HPLC, and then tested using a serial dilution method. Commercially available ergonovine (EN), ergocorine (ER), bromocryptine (BC) and ergocryptine (EC) were tested individually using 0 to 44 nM concentrations. Seed extract of E+ produced a significant (P P < 0.05) dose dependent manner with EC being most potent, followed by ER, BC, and then EN (70%, 40%, 30% and 10% at 44 nM concentration). The similarity of the inhibition curves of seed extract to that of the commercially available ergot alkaloids suggests a related mode of action and that the use of such ergot alkaloids and CYP3A4 assay is a good model to study the toxicity of tall fescue. Furthermore, it provides the foundation to identify the individual toxic components of purified endophyte infected tall fescue extract.
