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In vivo and in vitro reporters for androgen and Ah receptor responses to food components
Project Code: T09001
To develop GAL4 chimeric fusion receptors with the human Ah and AR receptors to act as reporters for human receptor activity both in cell lines and transgenic rats.
This should act as proof of principle of such “humanized” reporter assays in the detection of xeno-endocrine activity in foodstuffs.
Generation of reporter constructs. Chimeric receptors for Ah receptor, Androgen receptor and PPARδ have been generated by recombinant DNA technology. These have been validated in several in vitro cell models. chimeras extensive domain mapping had to be employed to generate functional chimeras of the androgen receptor. This domain mapping has revealed novel aspects of the molecular basis of androgen signaling. Stable cell lines expressing these chimeric receptors have been tested and shown to be sensitive reporters.
Generation of transgenic animals. We have generated a wide range of transgenic founder rats.
Ah receptor humanized rats: AhR/LacZ reporter dual transgene: three founders.
AhR single transgene: one founder, Lac Z single transgene: one founder. None of the founders obtained with the AhR and LacZ receptor dual transgenes have bred successfully, however we have successfully crossed the AhR and LacZ single transgene founders to generate fertile AhR/LacZ dual transgene animals. These were then tested in several experiments using 3-MC as a prototypic inducer, however these experiments were negative. Endogenous CYP1A1 induction was detected in AhR/LacZ rats treated with 3-MC confirming the efficacy of our treatment procedures. CYP1A1/LacZ reporter mice generated in our laboratory were used as positive controls for the induction and staining process. These CYP1A1/LacZ are highly sensitive in-vivo reporters for AhR activation activities in foodstuffs, with the important caveat that this system does not completely reflect the activity of the human receptor.
Androgen receptor humanized rats: AR/LacZ reporter dual transgene: five founders.
AR single transgene- two founders. Four AR/lacZ dual transgene lines were eventually bred successfully. These were tested for inducibility by testosterone. Initially we found that one line of the androgen receptor reporter rats displayed transgene-specific β-gal staining, which was weakly inducible with testosterone. The apparent induction by testosterone was not reproducible.
PPARδ humanized rats: PPARδ/LacZ reporter dual transgene: four founders.
Recent work with one of our PPARδ reporter lines has demonstrated successful inducibility by the fibrate drug, bezafibrate, in the intestine, kidney and spleen. This was achieved adding the compound to the rats’ food. This exciting finding provides proof of principle that the humanisation strategy is likely to work for the other receptors. The utility of the PPARδ humanised rat will allow us to make important observations regarding the role of this receptor in mediating responses to different dietary factors, and should provide new information on the role of dietary fats and colon cancer.
Generation of stable reporter cell lines. Stable cell lines for the AhR/GAL4 chimera were generated from CV-1 cells. These cell lines give a 5 fold inducibility wide a range of known AhR ligands, including 3-metylcholanthrene and β-napthoflavone.
Stable cell lines for the AR/GAL4 chimera were generated from PC-3 cells. These cell lines displayed a 4-5 fold response to androgen, however high concentrations of androgen were required for the response in these cell lines. Reporter cell lines were generated using the full length AR driving a MMTV enhancer-based reporter construct. These cell lines are highly sensitive to picomolar concentrations of androgens.
In conclusion: The concept of the utility of the GAL4/LBD chimera system for the generation of humanised reporter rats has been validated using the PPARδ reporter system. However, we have thus far been unable to successfully extend this to the androgen and Ah receptor system. The CYP1A1/LacZ reporter mouse generated in our laboratory is the only in vivo reporter for AhR ligands currently in existence. We have successfully generated reporter cell lines for the human AhR and human AR that are appropriate tools for the high throughput screening of foodstuffs for endocrine modulating activities and contamination by dioxins.
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