Phenotype of the HRN mouse

HRN™ mice live and breed normally and live a normal lifespan. This important finding allows these animals to be used for lead selection and development work in both short and long-term investigative studies in vitro and in vivo.  Although the mice exhibit no overt external phenotype their livers are enlarged and hyperlipidaemic exhibiting elevated cholesterol and fatty acid levels. In addition bile acid production is significantly reduced because of the role of the cytochrome P450 system in bile acid biosynthesis.  The HRN™ may therefore also be useful to investigate lipid metabolism.

The absence of cytochrome P450 reductase was confirmed by immunohistochemistry using labelled antibodies raised against the CPR protein. Figure 1 below compares immunohistochemically stained sections of liver from both HRN™ and normal mice that shows a lack of staining, and hence CPR protein, in the HRN™ mouse liver consistent with deletion of the CPR gene.

Figure 1. Immunohistochemistry of control and HRN mouse liver stained for cytochrome P450 reductase. Livers from adult male control and HRN™ mice were fixed in formalin/PBS, sectioned and immunostained with polyclonal antisera to POR. The brown staining in the control liver is indicative of the presence of CPR which is absent from the HRN™ liver section. Photomicrographs representative of each section taken at x4 magnification, bright field.

Confirmation that the P450 activity is attenuated in the liver is shown in figure 2, which shows the hydroxylation of testosterone in microsomes prepared from livers of male HRN™ mice compared to normal male mice. As testosterone is hydroxylated at three distinct sites (16β, 7α, and 6β) by three different cytochrome P450 isozymes (CYP2B, CYP2A. CYP3A respectively) it provides a good,common, substrate with which to compare CYP activity. The results confirm that the activity of all three CYPs are severely attenuated in microsomes from the HRN™ liver, demonstrating that the absence of CPR inactivates all CYP enzymes in that tissue.

Figure 2. Metabolism of testosterone in microsomal fractions from HRN and control mice. NADPH-dependent oxidation of testosterone is dramatically reduced in microsomal fractions from HRN livers.  The hydroxylation reflecting specific cytochrome P450 isozyme activity (expressed as percentages of control values) are: 6β-hydroxylation (CYP3A) <10%, 7α-hydroxylation (CYP2A) < 1%, 16β¬hydroxylation (CYP2B) ~ 0%.