Lentiviral-mediated RNAi knockdown yields a novel mouse model for studying Cyp2b function

Abstract

There are few in vivo knockout models available to study the function of Cyp2 members involved in the metabolism of endogenous and exogenous chemicals. These models may help provide insight into the cytochrome P450s (CYPs) responsible for the detoxification and activation of drugs, environmental toxicants, and endobiotics. The aim of this work is to produce a potent Cyp2b-knockdown (KD) mouse for subsequent study of Cyp2b function. We made a quintuple Cyp2b-KD mouse using lentiviral-promoted short hairpin RNA (shRNA) homologous to all five murine Cyp2b subfamily members (Cyp2b9, 2b10, 2b13, 2b19, and 2b23). The Cyp2b-KD mice are viable, fertile, and without obvious gross abnormalities except for an increase in liver weight. Expression of the three hepatic Cyp2b members, 2b9, 2b10, and 2b13, is significantly repressed as demonstrated by quantitative real-time PCR and Western blotting. The constitutive androstane receptor activator, 1,4-Bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), was used to determine if shRNA-mediated Cyp2b10 repression could be outcompeted by Cyp2b10 induction. TCPOBOP-treated Cyp2b-KD mice show 80-90% less Cyp2b protein expression than TCPOBOP-treated wild-type (WT) mice, demonstrating that Cyp induction does not outcompete the repressive function of the shRNA. Untreated and TCPOBOP-treated Cyp2b-KD mice are poor metabolizers of parathion compared with WT mice. Furthermore, Cyp2b-KD mice are sensitive to parathion, an organophosphate insecticide primarily metabolized by Cyp2b enzymes, when compared with WT mice. In summary, we designed an shRNA construct that repressed the expression and activity of multiple Cyp2b enzymes. We foresee that this novel Cyp2b-KD mouse model will significantly improve our understanding of the role of Cyp2b enzymes in chemical sensitivity and drug metabolism.

FIG. 1.

siRNA target areas for mouse Cyp2b genes. There are five areas of the mouse Cyp2b subfamily that are sufficiently conserved so that all the Cyp2bs could potentially be knocked down by the same siRNA. siRNA scales estimate a greater than 70% knockdown (shown in parenthesis^a) of Cyp2b expression using two of these siRNAs (shown in gray). shRNA constructs were made to KD2 and KD3 and inserted into the pRNAT-U6.2/lenti vector. NCBI accession numbers are provided next to the name of each Cyp2b gene. Numbers on the right indicate sequence length near the area of homology.

FIG. 2.

Hepatic Cyp2b expression in WT and Cyp2b-KD mice as demonstrated by QRT-PCR (A) and Western blots (B). Data were expressed as mean ± SEM (n = 5–8). Statistical significance was determined by Student’s t-test using the GraphPad Prism 4.0 software package. Significant differences at *p < 0.05, **p < 0.01, and ***p < 0.001. WT = wild-type, KD = Cyp2b-KD mice, M = male, and F = female. Expression was not quantified in the Western blots because of the low expression of Cyp2bs in the male mice and Cyp2b-KD female mice.

FIG. 3.

Hepatic Cyp2b expression in WT and Cyp2b-KD mice treated with the Cyp2b10 inducer, TCPOBOP. (A) RNA expression of Cyp2b9, Cyp2b10, and Cyp2b13 as measured by QRT-PCR (n = 5–8). (B) Protein expression of hepatic Cyp2b subfamily members (n = 3–4). Data were expressed as mean ± SEM. Statistical significance was determined by Student’s t-test using the GraphPad Prism 4.0 software package. *p < 0.05 and **p < 0.01. WT = wild-type, KD = Cyp2b-KD mice, M = male, and F = female.

FIG. 4.

Hepatic expression of CAR in WT and Cyp2b-KD mice treated with TCPOBOP or corn oil (carrier) as measured by QRT-PCR (A) or Western blots (B, C). (A) WT mice are shown in white bars, and Cyp2b-KD mice are shown in black bars. Statistical significance was determined by ANOVA followed by Dunnett’s post hoc test with the GraphPad Prism 4.0 software package. Western blots in corn oil (B) or TCPOBOP-treated mice (C). Statistical significance of the Western blots was determined by Student’s t-test. An asterisk indicates significant difference with a p < 0.05.

FIG. 5.

Histopathology of WT and Cyp2b-KD (KD) mice treated with corn oil or TCPOBOP (TC). (A) Histopathology was measured as described in the “Materials and Methods” section using a combined histopathology score from several different measures. Asterisks indicate statistical differences (*p < 0.05; **p < 0.01; and ***p < 0.001) as determined by a two-way ANOVA followed by a Bonferroni posttest (n = 3). (B) Female WT mouse showing hyperplasia after TC treatment (200×). (C) Female Cyp2b-KD mouse showing no discernable hyperplasia after TC treatment (200×).

FIG. 6.

Microsomal metabolism of parathion. Microsomes from control and TCPOBOP-pretreated WT and Cyp2b-KD mice were prepared and incubated with 20μM parathion for 60 min. The formation of parathion’s relatively nontoxic metabolite, PNP, and its toxic metabolite paraoxon (POXON) was quantified by HPLC as described in the “Materials and Methods” section. (A) PNP and (B) POXON formation in microsomes from untreated WT and CYP2b-KD mice. (C) PNP and (D) POXON formation in microsomes from TC-pretreated WT and Cyp2b-KD mice. A white bar indicates WT mice, and a black bar indicates Cyp2b-KD mice. Significant differences in the formation of the metabolites between WT and Cyp2b-KD mice were assessed by ANOVA followed by Tukey’s multiple comparison test using the GraphPad Prism 4.0 software package. The letter “a” indicates a significant difference between WT and Cyp2b-KD males (p < 0.01), and the letter “b” indicates a significant difference between WT and Cyp2b-KD females (p < 0.01).

FIG. 7.

Increased toxicity of parathion in Cyp2b-KD mice compared with WT mice. (A) WT male mice treated with parathion. (B) Cyp2b-KD male mice treated with parathion. (C) WT female mice treated with parathion. (D) Cyp2b-KD female mice treated with parathion. (E) Overall, differential toxicity to parathion in Cyp2b-KD mice compared with WT mice. A significant increase in toxicity to parathion was observed in Cyp2b-KD mice as determined by the Kruskal-Wallis nonparametric test for independent variables followed by Dunn's post hoc test using the GraphPad Prism 4.0 software package. Severity of toxicity: 0 = not toxic; 1 = eye leakage; 2 = lethargy/tremors; 3 = morbidity; and 4 = death (p < 0.05). WT = wild-type, KD = Cyp2b-KD mice, M = male, and F = female.