Browsing by Subject "Scd1"

2,3,7,8-Tetrachlorodibentso-p-dioksin (TCDD) is the most potent congener of the group of dioxins. It forms as a byproduct or impurity in many chemical processes. TCDD is enriched in the food chain and people are exposed to it in small portions chronically mainly by nutrition. TCDD has many toxic impacts on humans and animals which are mediated by the cellular aryl hydrocarbon -receptor. In spite of decades of research the toxic mechanisms of TCDD are poorly known. By investigating the toxicity of TCDD with large doses in experimental animals we will gain more insight into its toxic mechanisms and into its risks to humans. An important form of acute toxicity of TCDD is the wasting syndrome in which rats eat less, lose weight and finally die or after a sublethal dose stay thinner than littermates. The mechanism by which TCDD causes the wasting syndrome is unclear. Two central enzymes that take part in the regulation of the body's energy balance are the adenosine monophosphate activated protein kinase (AMPK) and stearoyl-CoA-desaturase 1 (Scd1). Changes in the expression or the regulation of their action could well be related to the wasting syndrome. The goal of this experiment was to compare in liver the mRNA expression of AMPK, Scd1 and control genes which are widely used in quantitative PCR in dioxin-sensitive Long-Evans (L-E) and resistant Han/Wistar (H/W) rat strains after a large (lethal to the sensitive strain) TCDD dose at early phases of intoxication (days 1, 4 and 10). There was also a L-E rat group whose feed was restricted to mimic the wasting caused by TCDD. The goal of the feed restriction was to separate the possible primary effect of TCDD from a secondary effect, depletion of energy. RNA was isolated from liver samples and the cDNA was made from isolated RNA with M-MLV-derived reverse transcriptase enzyme using oligo dT and random hexamer primers. The mRNAs of Scd1-enzyme and the control genes in the liver samples were measured using real-time quantitative PCR and specific primers. At 10 days feed restriction lowered significantly the expression of AMPK mRNA in L-E-rats. At 4 and 10 days feed restriction lowered significantly also the expression of Scd1 mRNA; on day 10 the mRNA level of control rats was about 10,000-fold higher. At day 1 TCDD elevated the expression of Scd1-enzyme. TCDD did not cause a significant decrease in the expression of Scd1-enzyme on day 4 and on day 10 the decrease was significantly less, about 1/10 of the level in control rats. These findings suggest that either TCDD inhibits the strong decrease of AMPK and Scd1 caused by energy deficiency or it causes an induction of AMPK and Scd1 which in turn is countered by the weight loss caused by the wasting syndrome. Overall it seems that rats exposed to TCDD do not recognize the energy depletion and their hepatocytes do not turn on the energy sparing mode of metabolism. Feed restriction and TCDD affected clearly also the expression of the control genes GAPDH, Pgk1 and Bact which were believed to be stable. The use of control genes which are linked to the regulation of cellular energy balance is risky in long-term feed restriction experiments.