Wall, Richard John
Potency and species specificity of aryl hydrocarbon receptor ligands.
PhD thesis, University of Nottingham.
The aryl hydrocarbon receptor (AhR) binds a wide range of structurally diverse compounds such as halogenated dibenzo-p-dioxins, dibenzofurans and biphenyls which are abundant in the environment. Activation of AhR leads to the regulation of a battery of xenobiotic enzymes including cytochrome P4501A1 (CYP1A1). The purely chlorinated compounds feature in the World Health Organisation’s (WHO) evaluation of dioxin-like compounds derived from a meta-analysis of previous potency data (toxic equivalency factors; TEFs), which is used to calculate the total toxic equivalence (TEQ).
The first aim of this work was to fully characterise the three most environmentally abundant mono-ortho-substituted polychlorinated biphenyls (PCBs; PCB 105, 118 and 156) including a re-evaluation of their putative antagonistic effects on AhR. Secondly, the effects of mixed halogenated compounds, currently not included in the TEQ estimation, were investigated as AhR agonists based on their environmental exposure and potency. Quantitative real-time PCR (qRT-PCR) was used to measure the AhR mediated induction of CYP1A1 mRNA in rat H4IIE and human MCF-7 cells. The three mono-ortho-substituted PCBs were shown to be antagonists of rat and human AhRs, an effect which is not currently included in the TEQ calculation. 2-bromo-3,7,8-trichlorodibenzo-p-dioxin (2-B-3,7,8-TriCDD) was found to be an AhR agonist that was 2-fold more potent than 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; considered one of the most potent in the environment). The majority of the other tested compounds were found to be within 10-fold less potent than TCDD and could therefore have a significant impact on the TEQ. A family of putative AhR agonists from AstraZencea were investigated and one of the compounds was shown to be a highly potent AhR agonist, 5-fold more potent than TCDD at inducing CYP1A1.
The results indicate approximately a 15-fold higher sensitivity of the rat cell line to the AhR agonists compared with the human cell line. It is not currently understood what confers these differences whether it is a difference in the mechanism of activation or purely as a result of differences in the AhR sequence. The mechanism of action is thought to be the same in both species and the associated proteins are both comparable. The amino acid sequences of the AhR, in both human and rat are quite similar but may play a significant role in the differences observed between species. Therefore in order to directly compare the rat and human AhRs, two novel cell line models were created using an inducible expression system to infect an AhR-deficient mouse cell line with a replication-defective virus containing either the rat or human AhR. The AhRs were activated with various compounds to induce mouse CYP1A1. The CYP1A1 mRNA was measured using qRT-PCR but showed that the two AhR genes were not expressed enough to produce a response detectable above the background CYP1A1 induction by the low levels of mouse AhR.
This research has shown that these dioxin-like compounds can have very different potencies at AhRs in different species so it is not always possible to predict the potency in humans from in vitro or rat in vivo toxicity data. Furthermore, it has identified compounds, such as 5F-203, which are significantly more potent in human compared to rat. This thesis provides information on the AhR species differences between human and rat that can be applied to risk assessment.
Thesis (University of Nottingham only)
||AhR, Aryl Hydrocarbon receptor, TCDD, mouse, human, rat, species differences, concentration response curve, cell culture, RNA, qRT-PCR, real time PCR, ligand binding
||R Medicine > RA Public aspects of medicine > RA1001 Forensic medicine. Medical jurisprudence. Legal medicine
Q Science > QP Physiology > QP501 Animal biochemistry
||UK Campuses > Faculty of Science > School of Biosciences
||04 Jun 2013 08:31
||13 Sep 2016 19:42
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