Abstract
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.
Highlights
Statistical Analysis Results of the CYP450 enzyme assay were reported in luminescence
20 μM, resulted in a steep inhibition of CYP450 by ET and DHET suggesting that the sensitivity of assay was exceeded
Steer body weights were correlated with insulin-like growth factor 1 (IGF-1), ADG was not correlated to IGF-1
Summary
J. Darbyshire] infected with the endophytic fungus (Neotyphodium coenophialum) is responsible for the production of ergot alkaloids which have a significant economic impact on beef cattle. Darbyshire] infected with the endophytic fungus (Neotyphodium coenophialum) is responsible for the production of ergot alkaloids which have a significant economic impact on beef cattle Consumption of those mycotoxins is known to lower average daily gain, negatively impact reproductive traits, decrease parasite resistance, and reduce heat tolerance, all of which drive up the cost of production (for review see Strickland et al, 2011). Ergot alkaloids have an interesting relationship with animal cytochrome P450 (CYP) enzymes; CYP have been shown. CYP is a key enzyme in the biosynthetic pathway for ergot alkaloids (Haarmann et al, 2006)
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