Annual Meeting of the Japanese Society of Toxicology

We addressed roles of the pregnane X receptor (PXR, also steroid X receptor SXR) and the aryl hydrocarbon receptor (AHR) in regulation of cytochrome P450 CYP2 and CYP3 genes in developing zebrafish exposed to potential agonists for the PXR and AHR. Zebrafish embryos were exposed to 5-pregnen-3β-ol-20-one (pregnenolone) or carrier (DMSO) for 24 h, starting at 48 hours post-fertilization (hpf), and then harvested at day 3. We also exposed one-day-old embryos to 3,3’,4,4’,5-pentachlorobiphenyl (PCB126) or DMSO for 24 h and then held in clean water until day 4. Pregnenolone caused a concentration-dependent increase in mRNA expression of PXR, some of the CYP2AAs, as well as CYP3A65 and CYP3C1, all of which peaked at 3 µM and then declined. PCB126 also upregulated the transcript levels of those genes in most cases in a concentration-dependent manner. We next examined the role of the PXR and AHR in the modified expression of those potential target genes by using morpholino antisense oligonucleotides (MO) to block initiation of the translation. Treatment of embryos with PXR-MO, but not control-MO, partially inhibited pregnenolone-induced expression of PXR, CYP2 and CYP3 genes. Similarly, AHR2-MO treatment blocked PCB126-induced transcript expression of PXR and some CYP2 and CYP3 genes. The present study shows that PXR not only is self-upregulated, but also upregulated via activation of AHR2 in zebrafish embryos. Selected zebrafish CYP2 and CYP3 genes appear to be under the regulation of PXR and AHR2. They include some CYP2AAs that were first identified in zebrafish.

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Here, we aimed to enhance rat hepatocyte functions and durations in culture through improving both oxygen supply and its actual concentrations at the cell level. Rat primary hepatocytes were cultured in sandwich culture method with Matrigel on PDMS (Polydimethylsiloxane) membrane under various O₂ concentrations (20%-O₂(+), 10%-O₂(+), 5%-O₂ (+)) to investigate their effects on the performances of culture. In parallel, TCPS (Tissue culture treated-polystyrene) cultures were carried out as control groups (20%-O₂(-), 10%-O₂(-), 5%-O₂ (-)).
Cellular level O₂ tensions (pO₂) on PDMS membranes were almost at the equilibrium concentrations in the incubator: 18-20%, 9-10% and 4-5% under the cultures of 20%-O₂(+), 10%-O₂(+) and 5%-O₂ (+), which were remarkably lower on TCPS surfaces. HIF staining confirmed that hepatocytes were normoxic under 20%-O₂(+) and 10%-O₂(+) but hypoxia under other cultures. Albumin secretion increased greatly under 10%-O₂(+) from Day 2 and maintained at the highest level throughout the culture. CYP1A1/2 activity also increased greatly from Day 8 under 10%-O₂(+) and 5%-O₂(+) and maintained at a higher level on Day 14 under 10%-O₂(+). PCR array analysis illustrated that expression levels of various drug-metabolism genes under 20%-O₂(+) and 10%-O₂(+) were closest to those seen in the isolated rat liver before inoculation. These results demonstrate that it is important to satisfy cellular oxygen demand at in vivo-like physiological oxygen concentrations in in vitro hepatocyte culture and oxygen permeable membrane provide a simple methodology to realize this condition for in vitro toxicological research.

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