Control mice (= 3) received corn essential oil only. study we’ve utilized immunohistochemistry (IHC) staining with anti-BPDE-DNA antiserum (John et al., 2009; Pratt et al., 2007, 2011; truck Gijssel et al., 2002) to explore the localisation of BaP-derived DNA adducts inside the liver organ of HRN mice. Furthermore, we have researched BaP-DNA adduct development by 32P-postlabelling in a second mouse model, the P450 Reductase Conditional Null (RCN) mouse (Finn et al., 2007), to confirm results previously obtained in the HRN mouse model. 2.?Methods 2.1. Chemicals BaP (>96%) was purchased from SigmaCAldrich (St. Louis, MO). All other chemicals were of analytical purity or better. 2.2. Animal treatment All animal experiments were carried out under license in accordance with the law, and with local ethical approval. HRN (locus (= 3) for 1 day. Control mice (= 3) received corn oil only. Animals were killed 24 h after the single dose. Several organs (liver, lung, forestomach, glandular stomach, kidney, spleen and colon) were removed, snap frozen and stored at ?80 C until analysis. For IHC organ sections of the liver were fixed in PBS containing 4% paraformaldehyde, and subsequently subjected to paraffin embedding and sectioning. RCN (floxed mice (= 3) of BaP for 1 day. Control mice (= 3) received corn oil only. Animals were sacrificed 24h after the single CC0651 dose. Organs for 32P-postlabelling were collected as described above. 2.3. BaP-DNA adduct detection by 32P-postlabelling analysis Genomic DNA from whole tissue was isolated by a standard phenol-chloroform extraction method and DNA adducts were measured for each DNA sample using the nuclease P1 enrichment version of the 32P-postlabelling method as described CC0651 previously (Arlt et al., 2008; Phillips and Arlt, 2007). 2.4. BaP-DNA adduct detection by immunohistochemistry Rabbit polyclonal antibodies, elicited against BPDE-modified DNA (rabbit#30 bleed 6/30/78) (Poirier et al., 1980; Pratt et al., 2011; Weston et al., 1989), were employed for detection of dG-= 3) as described previously (Arlt et al., 2008). Control mice (= 3) received corn oil only. Hepatic microsomes from HRN and WT mice were isolated as EPLG6 reported. Pooled microsomal fractions were used for further analysis. Western blot CC0651 analysis of cytochrome lung, glandular stomach, spleen and colon) compared with WT mice (Fig. 1A). At the lower dose BaP-DNA adduct levels were substantially lower in all tissues, but as with the higher dose they were higher (~8-fold) in the livers of HRN mice than in WT mice (Fig. 1C). However, differences between HRN and WT mice in DNA adduct formation were not observed in extra-hepatic tissues with the lower dose of BaP. After oral administration of 12.5 or 125 mg/kg bw BaP, DNA adduct formation by BaP was overall lower compared to i.p. administration (compare Fig. 1B and ?andD).D). Again, DNA binding by BaP in the livers of HRN mice was higher relative to WT mice, but this effect was less pronounced (only ~2-fold) than after i.p. administration. Overall, no difference in DNA binding by BaP was observed in extra-hepatic tissues, independent of the dose, except for the colon in the higher dose group which showed 3-fold elevated DNA adducts in HRN mice relative to WT mice (Fig. 1B). Open in a separate window Fig. 1. Quantitative TLC 32P-postlabelling analysis of dG-= 3); each DNA sample was determined by two postlabelled analyses. Comparison was performed by <0.01 different from WT. RAL, relative adduct labelling. After treatment of RCN mice with a single i.p. dose of 125 mg/kg body weight BaP, the DNA adduct pattern on TLC again consisted of a single spot (dG-3-MC). Values are given as means SD (= 3);.