Department of Animal Science, University of Arkansas Fayetteville, AR, USA.
Front Chem. 2015 Mar 11;3:16. doi: 10.3389/fchem.2015.00016. eCollection 2015.
Determining a grazing animal's susceptibility to ergot alkaloids has been a research topic for decades. Our objective was to determine if the Promega™ P450-Glo assay could be used to indirectly detect ergot alkaloids or their metabolites in urine of steers. The first experiment validated the effects of ergot alkaloids [0, 20, and 40 μM of ergotamine (ET), dihydroergotamine (DHET), and ergonovine (EN)] on human CYP3A4 using the P450-Glo assay (Promega™ V9800). With this assay, luminescence is directly proportional to CYP450 activity. Relative inhibition of in vitro cytochrome P450 activity was affected (P < 0.001) by an interaction between alkaloids and concentration. That interaction resulted in no concentration effect of EN, but within ET and DHET 20 and 40 μM concentrations inhibited CYP450 activity when compared with controls. In experiment 2, urine was collected from Angus-sired crossbred steers (n = 39; 216 ± 2.6 days of age; 203 ± 1.7 kg) after grazing tall fescue pastures for 105 days. Non-diluted urine was added to the Promega™ P450-Glo assay, and observed inhibition (3.7 % ± 2.7 of control). Urine content of total ergot alkaloids (331.1 ng/mg of creatinine ± 325.7) was determined using enzyme linked immunosorbent assay. Urine inhibition of CYP450 activity and total alkaloids were correlated (r = -0.31; P < 0.05). Steers were genotyped at CYP450 single nucleotide polymorphism, C994G. Steer genotype affected (P < 0.03) inhibition of CYP450 activity by urine; heterozygous steers had the least amount of CYP450 inhibition suggesting that genotyping cattle may be a method of identifying animals that are susceptible to ergot alkaloids. Although, additional research is needed, we demonstrate that the Promega™ P450-Glo assay is sensitive to ergot alkaloids and urine from steers grazing tall fescue. With some refinement the P450-Glo assay has potential as a tool for screening cattle for their exposure to fescue toxins.
几十年来，确定放牧动物对麦角生物碱的易感性一直是一个研究课题。我们的目标是确定 Promega™ P450-Glo 测定法是否可用于间接检测牛尿中的麦角生物碱或其代谢物。第一个实验使用 P450-Glo 测定法（Promega™ V9800）验证了麦角生物碱[0、20 和 40 μM 的麦角胺（ET）、二氢麦角胺（DHET）和麦角新碱（EN）]对人 CYP3A4 的影响。在此测定法中，发光与 CYP450 活性直接成正比。生物碱浓度之间的相互作用（P < 0.001）影响体外细胞色素 P450 活性的相对抑制作用。该相互作用导致 EN 没有浓度效应，但在 ET 和 DHET 中，与对照组相比，20 和 40 μM 浓度抑制了 CYP450 活性。在实验 2 中，从放牧梯牧草牧场 105 天后的安格斯杂交牛（n = 39；216 ± 2.6 天龄；203 ± 1.7 kg）收集未经稀释的尿液。将未经稀释的尿液加入 Promega™ P450-Glo 测定法中，观察到抑制作用（对照的 3.7%±2.7）。使用酶联免疫吸附测定法测定尿液中总麦角生物碱的含量（331.1ng/mg 肌酐±325.7）。CYP450 活性和总生物碱的尿液抑制作用呈相关性（r = -0.31；P < 0.05）。对 CYP450 单核苷酸多态性 C994G 进行了牛基因分型。牛基因型（P < 0.03）影响尿液中 CYP450 活性的抑制作用；杂合子牛的 CYP450 抑制作用最小，这表明对牛进行基因分型可能是识别易受麦角生物碱影响的动物的一种方法。尽管还需要进一步的研究，但我们证明 Promega™ P450-Glo 测定法对放牧梯牧草的牛尿中的麦角生物碱敏感。经过一些改进，P450-Glo 测定法有可能成为筛选牛接触梯牧草毒素的工具。
