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• W2205910700 abstract "Dioxins (polychlorinated dibenzo-p-dioxins, dibenzofurans and dioxin-like polychlorinated biphenyls) are pervasive and biomagnifying environmental contaminants, to which people are generally exposed through foods of animal origin; in Finland, a major source is some fatty fish species of the Baltic Sea. The dioxins, especially the most potent of them, the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), are often called “super poisons”, based on their extreme acute toxicity to laboratory animals and feared long-term effects in humans. Children, before and after birth, are especially susceptible to this toxicity, possibly induced by picogram daily exposures, while the risks to adults are less certain and appear, for example in Finland, to be outweighed by the beneficial effects of fish consumption. Furthermore, the most important endpoints of dioxin toxicity have not been fully untangled, and even the mechanisms of the most sensitive developmental effects have not been thoroughly elucidated. After gaining access to the body, the dioxins bind to a specialised intracellular aryl hydrocarbon receptor (AHR), directly influencing the expression of a substantial number of genes. The AHR, a transcription factor with no established endogenous ligand, first “converges” the dioxin effects, but then diverges them depending on the affected species and even the strain, as well as the gender, age and developmental stage of the individual. This divergence is exemplified in the dramatic variability of the acute toxicity of TCDD even within one species: Han/Wistar (H/W) rats tolerate over 1000-fold larger dioxin doses than the Long–Evans (L–E) strain, almost solely depending on a minor structural difference in the ARH. In addition, TCDD toxicity develops slowly and is manifested differently in various laboratory animals. A unifying feature of acute TCDD intoxication in many species, however, is a dramatic feed intake reduction and weight loss, termed the wasting syndrome, which seems to be related to direct or indirect derangement of the central regulation of feeding. Clarification of the mechanisms behind the wasting syndrome would thus yield information on an important facet of acute dioxin toxicity and open the way to using TCDD as a molecular tool to study the physiology of feed intake and body weight regulation. In this research, a straightforward approach was selected to tackle the pathophysiological challenge of wasting. The aforementioned strain difference was utilised to address the AHR function, and gene expression measurements were targeted at the hypothalamus, the essential food intake and body weight regulator in the brain. In the first part of the thesis research, alterations in the AHR signalling cascade and some related proteins as well as food intake-related neuropeptide neurotransmitters and their receptors were measured using quantitative reverse transcription PCR (RT-qPCR). In the latter, methodological part, some intricacies of the RT-qPCR technique were considered: The reproducibility of the poorly controllable reverse transcription (RT) of RNA to cDNA is a major determinant of the reliability of expression analysis, and toxicological studies involving TCDD have particular difficulty in discovering treatment-unaffected stable mRNA transcripts, reference genes, that can especially be used to reduce RT variation. In the two expression studies, the mRNA levels of AHR-related proteins and feeding regulatory factors were measured from hypothalamic blocks at 6, 24 or 96 hours after TCDD doses that generated only a minor reduction in feed intake in the resistant H/W rats, but induced a marked reduction in feed intake and body weight (and would have eventually led to lethality) in the sensitive L–E strain. In the two methodological experiments, a set of stable reference genes was first sought among the extensive and divergent expression changes induced by AHR activation, 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. There were small constitutive differences in the hypothalamic mRNA expression of some AHR signalling cascade molecules between L–E and H/W rats, but these were most likely not causally related to the development of the wasting syndrome. A functional AHR signalling cascade seems to be present in the hypothalamus, as shown by the pronounced induction of cytochrome oxidases and Ahrr, an AHR signalling repressor protein. However, the magnitude and importance of cytochrome induction in neurones should be determined, employing anatomically more detailed sampling. The lack of any drastic upor downward changes in hypothalamic neuropeptide or receptor mRNA following TCDD treatment and the stability of hypothalamic reference gene expression speak against a severe cytotoxic effect on, or permanent hyperexcitation of the cells taking part in eating regulation. A late elevation of some orexigenic (feeding inducing) factors brought about by TCDD seems to be a compensatory reaction to body weight loss. Notably, the employed hypothalamic block sampling may cause expression changes confined to a localised point and/or circadian time to elude detection, and the complex neurophysiology and anatomy of the hypothalamus may lead to reciprocal cancellation or dwindling of alterations. In addition, the somewhat divergent feeding and energy balance regulation between L–E and H/W rats also warrants further research, and as a more general question, the importance of the hypothalamic neuropeptides in relation to other systems in eating regulation has not been definitively resolved. The number of genes displaying an acceptable steadiness of expression in the face of lethal TCDD toxicity is small; four transcripts (Actb, Gapdh, Pgk1 and Sdha) were satisfactory for the hypothalamus, and besides the expression constancy, RT stability was found to have a strong influence on the usability of the potential reference genes. Hence, the use of only one steadily expressed but unstably reverse transcribing reference gene (Actb) has most likely not induced marked bias in the results attained in the hypothalamic expression studies, but it may have inadvertently increased variation. Furthermore, RT variance markedly exceeded qPCR variance, stressing the importance of replication at the RT level, while the practical consequences of differences in the reproducibility of the individual qPCR enzymes appear to be of little significance. On the contrary, salient differences were noted between replicate PCR runs, and these should be taken into account in the design and data analysis of RT-qPCR experiments. Finally, the use of linear hierarchical models and Bayesian inference was found to offer the possibility to build a coherent statistical model of the whole RT-qPCR experiment with normalisation over all expression measurements, thereby maximising the use of the data." @default.
• W2205910700 created "2016-06-24" @default.
• W2205910700 creator A5061353165 @default.
• W2205910700 date "2013-06-08" @default.
• W2205910700 modified "2023-09-28" @default.
• W2205910700 title "TCDD-induced changes in the expression of selected hypothalamic feeding-regulatory genes and mRNA quantification using reverse transcription qPCR" @default.
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