ARGT-related inhibition of GlcNAc-1-P transferase activity in different tissues of sheep and rats.

Abstract

AbstractThe inhibition of the enzyme N-acetylglucosamine-1-phosphate transferase (GPT) by corynetoxins (CTs) is a primary biochemical lesion associated with Annual Ryegrass Toxicity (ARGT) and its tunicamycins (TMs)-based experimental model. The determination of microsomal GPT activity in different tissues provides an indirect way to assess the toxicodynamics of TMs. However, this can be subject to variation depending on the conditions of the collection and storage prior to analysis. This study was conducted to determine the stability of enzyme in sheep and rat liver tissue stored at -80°C. The presence of CTs/TMs in tissues other than the liver which can adversely affect the endogenous microsomal GPT was also determined along with the native GPT activity for rough and total microsomal fractions from various tissues of sheep. Liver samples were collected from four male rats and three 18-month-old ewes. The GPT activity of the rough microsomal fraction was determined in fresh tissue samples and samples stored for 4 months at -80°C. To determine the effects of CTs/TMs on the GPT of different tissues, the specific GPT activities for the total, smooth and rough microsomal fractions isolated from liver, kidney, heart, lung, muscle and brain were assessed. Groups of 5 sheep were also exposed to 40.5 to 4.5 µg TMs/kg/bw/day in their diet over a 12-month period while groups of 20 mice were similarly exposed to 121.5 or 13.5 µg TMs/kg/bw/day in their diet over a 9- and 12-month period, respectively. The results showed that liver samples can be successfully stored at -80°C for at least 4 months without any adverse effect on the GPT activity. However, the GPT stability of freeze-stored liver microsomes is not so reliable but does increase if the protein concentration is greater than 3 mg/ml, resulting in a loss of less than 10% of initial activity over a 4-week period. The GPT activity in sheep liver microsomes was affected to a far greater extent in vivo by TMs than activity in other tissues and is therefore the tissue of choice as an indicator of tunicaminyluracil glycolipid exposure. A dose-dependent effect on liver rough microsomal GPT activity was observed in rats and sheep in both genders when exposed to low levels of dietary TMs. There was no evidence of any compensating upregulation of GPT activity back to levels consistent with untreated animals in either study.