A'edah Abu-Bakar

Regulation of CYP2A5 gene by the transcription factor nuclear factor (erythroid-derived 2)-like 2

We have previously shown that cadmium, a metal that alters cellular redox status, induces CYP2A5 expression in nuclear factor (erythroid-derived 2)-like 2 wild-type (Nrf2+/+) mice but not in the knockout (Nrf2–/–) mice. In the present studies, the potential role of Nrf2 in cadmium-mediated regulation of Cyp2a5 gene was investigated in mouse primary hepatocytes. Cadmium chloride (CdCl2) caused a time-dependent induction of the CYP2A5 at mRNA, protein, and activity levels, with a substantial increase observed within 3 h of exposure. Immunoblotting showed cadmium-dependent nuclear accumulation of Nrf2 within 1 h of exposure. Cotransfection of mouse primary hepatocytes with Cyp2a5 promoter-luciferase reporter plasmids and Nrf2 expression plasmid resulted in a 3-fold activation of Cyp2a5 promoter-mediated transcription relative to the control. Deletion analysis of the promoter localized the Nrf2 responsive region to an area from –2656 to –2339 base pair. Computer-based sequence analysis identified two putative stress response elements (StRE) within the region at positions –2514 to –2505 and –2386 to –2377. Chromatin immunoprecipitation and electrophoretic mobility shift assays showed that interaction of the more proximal StRE with Nrf2 was stimulated by CdCl2. Finally, site-directed mutagenesis of the proximal StRE in Cyp2a5 promoter-luciferase reporter plasmids abolished Nrf2-mediated induction. Collectively, the results indicate that Nrf2 activates Cyp2a5 transcription by directly binding to the StRE in the 5′-flanking region of the gene. This acknowledges Cyp2a5 as the first phase I xenobiotic-metabolizing gene identified under the control of the StRE-Nrf2 pathway with a potential role in adaptive response to cellular stress.

Mitochondrial targeting of bilirubin regulatory enzymes: an adaptive response to oxidative stress

The intracellular level of bilirubin (BR), an endogenous antioxidant that is cytotoxic at high concentrations, is tightly controlled within the optimal therapeutic range. We have recently described a concerted intracellular BR regulation by two microsomal enzymes: heme oxygenase 1 (HMOX1), essential for BR production and cytochrome P450 2A5 (CYP2A5), a BR oxidase. Herein, we describe targeting of these enzymes to hepatic mitochondria during oxidative stress. The kinetics of microsomal and mitochondrial BR oxidation were compared. Treatment of DBA/2J mice with 200mgpyrazole/kg/day for 3days increased hepatic intracellular protein carbonyl content and induced nucleo-translocation of Nrf2. HMOX1 and CYP2A5 proteins and activities were elevated in microsomes and mitoplasts but not the UGT1A1, a catalyst of BR glucuronidation. A CYP2A5 antibody inhibited 75% of microsomal BR oxidation. The inhibition was absent in control mitoplasts but elevated to 50% after treatment. An adrenodoxin reductase antibody did not inhibit microsomal BR oxidation but inhibited 50% of mitochondrial BR oxidation. Ascorbic acid inhibited 5% and 22% of the reaction in control and treated microsomes, respectively. In control mitoplasts the inhibition was 100%, which was reduced to 50% after treatment. Bilirubin affinity to mitochondrial and microsomal CYP2A5 enzyme is equally high. Lastly, the treatment neither released cytochrome c into cytoplasm nor dissipated membrane potential, indicating the absence of mitochondrial membrane damage. Collectively, the observations suggest that BR regulatory enzymes are recruited to mitochondria during oxidative stress and BR oxidation by mitochondrial CYP2A5 is supported by mitochondrial mono-oxygenase system. The induced recruitment potentially confers membrane protection.

Balancing benefits of human genetic research against civic concerns: Essentially Yours and beyond – the case of Australia

Large human genetic databases, especially those that are biomedical and forensic, have emerged since the completion of the Human Genome Project. However, this development has occurred in a time of intense public ambivalence to life science and genomics innovations. Controversies revolve around genetic modification, stem cell technologies and human genetic databases. Debate about databases focuses on how to balance the benefits from genetic research against civic concerns, typically, privacy and unfair discrimination and, more recently, public trust. In 1989, Australian jurisdictions began developing regulatory standards for human genetic databases but from the start these lacked uniformity and adequate scope. Enduring concerns led to a widescale public inquiry (2001-2003), which produced the Essentially Yours report. However, while the Australian government supports many of the reports recommendations, civic concerns remain as policy responses are checkered. In this special report, we reflect on the debate, the rise of the inquiry, its recommendations and policy responses, and competency and trust in regulation.

Cyp2a5 Promoter-based Gene Reporter Assay: A Novel Design of Cell-based Bioassay for Toxicity Prediction

The murine cytochrome P450 2a5 (Cyp2a5) gene is regulated by complex interactions of various stress‐activated transcription factors (TFs). Elevated Cyp2a5 transcription under chemical‐induced stress conditions is achieved by interplay between the various TFs – including as aryl hydrocarbon receptor (AhR) and nuclear factor (erythroid‐derived 2)‐like 2 wild‐type (Nrf2) – at the ‘stress‐responding’ cluster of response elements on the Cyp2a5 promoter, as well as through mRNA stabilization mediated by interaction of the stress‐activated heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) with the 3′‐UTR of the CYP2A5 mRNA. We designed a unique toxicity pathway‐based reporter assay to include regulatory regions from both the 5′ and the 3′ untranslated regions of Cyp2a5 in a luciferase reporter plasmid to reflect in vivo responses to chemical insult. Human breast cancer MCF‐7 cells were stably transfected with pGL4.38‐Cyp2a5_Wt3k (wild‐type) or mutant – pGL4.38‐Cyp2a5_StREMut and pGL4.38‐Cyp2a5_XREMut – reporter gene to monitor chemical‐induced cellular response mediated by AhR and Nrf2 signalling. The recombinant cells were treated with representative of AhR agonist, polycyclic aromatic hydrocarbons, brominated flame retardant, fluorosurfactant, aromatic organic compound and metal, to determine the sensitivity of the Cyp2a5 promoter‐based gene reporter assays to chemical insults by measuring the LC50 and EC50 of the respective chemicals. The three assays are sensitive to sublethal cellular responses of chemicals, which is an ideal feature for toxicity pathway‐based bioassay for toxicity prediction. The wild‐type reporter responded well to chemicals that activate crosstalk between the AhR and Nrf2, whilst the mutant reporters effectively gauge cellular response driven by either Nrf2/StRE or AhR/XRE signalling. Thus, the three gene reporter assays could be used tandemly to determine the predominant toxicity pathway of a given compound.

Toxicity of Cadmium and Cellular Stress: Bilirubin a Potential Endogenous Substrate of Cytochrome P450 2A5

We have estimated of the maximum radiation dose received from consuming an oyster at Hiroshima following the A-bomb detonation in 1945