Dioxins are pervasive environmental contaminants, to which people are generally exposed through foods of animal origin. Children, before and after birth, are especially susceptible to their toxicity, while the risks to adults seem quite small at current exposure levels. The dioxins bind to a specialised intracellular AH receptor, directly influencing the expression of a substantial number of genes and resulting to variable toxicity depending on the affected species and the strain, as well as the gender and age of the individual. This is exemplified in the dramatic variability of the acute toxicity of the most potent dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD]) within one species: H/W rats tolerate over 1000-fold larger doses than the L E strain. On the other hand, one of the few unifying features of acute TCDD intoxication in many laboratory species is a dramatic feed intake reduction and weight loss, termed the wasting syndrome.
In this research, the pathophysiological challenge of wasting was tackled in two studies by measuring the effect of one dose of TCDD on hypothalamic mRNA levels of AHR-related proteins and feeding regulatory factors in the resistant H/W and sensitive L E rats. In the latter two experiments the perviously-employed quantitative reverse transcription PCR (RT-qPCR) methodology was refined: A set of stable reference genes for RT-qPCR was first sought, and then the identified reference genes were employed in a study comparing the robustness of various RT and qPCR enzymes and mapping the RT-qPCR variation sources.
The small constitutive and TCDD-induced differences in the hypothalamic mRNA expression of some AHR signalling cascade molecules between L E and H/W rat are most likely not causally related to the wasting syndrome. However, a functional AHR signalling cascade appears to be present in the hypothalamus. The lack of any drastic changes in hypothalamic neuropeptide or receptor mRNA following TCDD treatment speak against a severe cytotoxic effect on, or permanent hyperexcitation of the cells. Notably, the employed hypothalamic block sampling might miss expression changes confined to a localised point and omits the various extra-hypothalamic systems in eating regulation. The future
studies should thus be targeted to individual nuclei, but to also have a wider scope of eating regulation both inside and outside CNS.
The number of genes displaying an acceptable steadiness of expression in the face of lethal TCDD toxicity is small and besides this the RT stability has a strong influence on the usability of the potential reference genes. Furthermore, RT variance markedly exceeds qPCR variance, stressing the importance of replication at the RT level. Finally, linear hierarchical models and Bayesian inference offer an efficient way to build a coherent statistical model of the whole RT-qPCR
experiment maximising the use of the data.
|Tila||Julkaistu - 2013|
|OKM-julkaisutyyppi||G5 Tohtorinväitöskirja (artikkeli)|
- 413 Eläinlääketiede