Table 1.
Relative gene expressions in the intestine of TLR2-deficient that are changed at least 4-fold compared to wild-type mice.
Figure 1.
Expression of mRNA and protein production of hepatic and intestinal CYP1A1, CYP1A2, and CYP1B1.
(A) Gene expression levels for cytochrome P450 isoforms 1A1, 1A2 and 1B1 in the intestine and liver of mice that were either deficient for TLR2 (TLR2−/−) or not and that were either fed with chow soaked with corn-oil with benzo[a]pyrene (BaP) or without (n = 7–8 animals/group). Analyses were performed by qRT-PCR and statistical analysis was performed by two-way ANOVA followed by Tukey's post-hoc test. Values are given as means ± SEM; *(**/***): P<0.05 (0.01/0.001, resp.) vs. WT group without BaP feeding; †(††/†††): P<0.05 (0.01/0.001, resp.) vs. WT group with BaP feeding; ‡ (‡‡/‡‡‡): P<0.05 (0.01/0.001, resp.) vs. TLR2−/− group without BaP feeding; (B) Immunoblot analyses of protein expressions of CYP1A1 and CYP1A2 in the small intestine and the liver of mice that were either deficient for TLR2 (TLR2−/−) or not and were either fed with BaP for 3 weeks (100 mg/kg/day) or not. For all proteins investigated, lanes were loaded with 50 µg of microsomal protein per lane. Prostaglandin-endoperoxide synthase 2 (PTGS2) was used as a loading standard. (C) Densitometric evaluation of chemoluminescence of CYP1A bands: BaP feeding induced massively CYP1A1 protein production in the intestine of wild-type animals, but not in that of TLR2−/− mice. In contrast, CYP1A1 was induced moderately in the liver of TLR2−/− animals, but not in that of wild-type mice.
Table 2.
Hepatic and intestinal mRNA expression of AHR and ARNT in TLR2−/− and wild-type mice with and without BaP feeding.
Figure 2.
(A) Representative Western blots and (B) standardized protein expression of the aryl hydrocarbon receptor (AHR) and the aryl hydrocarbon receptor nuclear translocator (ARNT).
The protein expression in the intestinal mucosa of TLR2−/− mice and in that of WT mice (n = 5 animals/group) was normalized to α-actin. Statistical analysis was performed with Student's t-test. The expression of both proteins did not differ significantly between the genotypes (P = 0.394 for AHR and 0.737 for ARNT); CL: chemoluminescence.
Figure 3.
Representative HPLC chromatograms of the blood plasma extract of WT mice (A) and TLR2−/− mice (B).
In contrast to WT animals, mice that were deficient for TLR2 were incapable to eliminate BaP from their circulation after an over-night fast. The major peak in both chromatograms was caused by the internal standard chrysene (retention time: 7.5 min). Chromatograms of the blood plasma from TLR2−/− animals (n = 7 per group) showed a clear BaP peak (right chromatogram, retention time 11.5 min) after over-night (16 h) deprivation from BaP-charged chow, while the blood plasma of the WT animals was virtually free of BaP (left chromatogram, concentration below 0.05 ng/mL).
Figure 4.
Morphology of colon mucosa from TLR2−/− and wild-type mice with and without BaP feeding.
Histological view on methylene-blue stained colon mucosa of mice that were either deficient for functional TLR2 (or not and that were either fed with BaP for 21 days or not. (A) wild-type mice fed corn-oil soaked chow (control diet) (B) wild-type mice fed with BaP (C) TLR2−/− mice on control diet (D) TLR2−/− mice on BaP diet. In the two groups of mice that were fed with BaP, only the colon mucosa of TLR2−/− showed a dense surface coverage with polyps (D), indicating hyperproliferation of the colon mucosa tissue. Bar = 1 mm.