Or protein analysis).To investigate whether BaP is capable of overcoming the repression of CYP1B1 induced by P90, PBMC were either pre-incubated with ultrafine P90 particles (32 g/ml) or remained untreated. After 30 min of incubation, BaP-conPBMC0 -20 -*BaP 0.1M ufP90 none*ufP90+BaP 0.1M BaP 1M BaP 10M*ufP90+BaP 1M*ufP90+BaP 10Mrel. CYP1B1 mRNA expression-60 -80 -100 -120 -140 -1000 -1500 -2000 -Compared to untreated A549 cells (-158 ?73) CYP1B1 mRNA levels were decreased after 22 h incubation with P90 by factor 4 (-707 ?347, p < 0.05) (Fig. 4C). LPS (-206 ?81) treatment shows no effect, whereas a slight effect was observed with fine TiO2 (-287 ?134) treatment. In cells incubated with both ultrafine P90 and fine TiO2, CYP1B1 transcript levels were decreased to -948 ?507. In the bronchial epithelial cell line Calu-3 the expression of CYP1B1 in untreated cells was 301 ?140 (Fig 4C). Ultrafine P90- (-2,820 ?1,546) and ultrafine P90/fine TiO2-treated cells (-3,106 ?1,988) showed a strong 10-Figure CYP1B1 induction in PBMC (BaP) in 5 between ultrafine P90 and benzo[a]pyrene Interference Interference between ultrafine P90 and benzo[a]pyrene (BaP) in CYP1B1 induction in PBMC. Cells remained untreated or pre-stimulated with ultrafine P90 (32 g/ml) for 30 min. Subsequently cells were stimulated with liposomes containing three different concentrations of BaP (0.1 M, 1 M, and 10 M) and incubated for 3 h. (n = 7 incubations from different donors, mean ?S.D.; * p < 0.05 compared to untreated control).Page 6 of(page number not for citation purposes)Particle and Fibre Toxicology 2009, 6:http://www.particleandfibretoxicology.com/content/6/1/taining liposomes were added in different concentrations (0.1 to 10 M) (Fig. 5). CYP1B1 mRNA in untreated cells (-29 ?18) was decreased by ultrafine P90 treatment (-1,178 ?1,011), on the other hand BaP induced this gene (0.1 M: -11 ?4.2; 1 M: -8.5 ?3.7; 10 M: -8.1 ?3.8). While at high BaP concentrations (1 and 10 M), ultrafine P90 could not reduce CYP1B1 mRNA, it was able to suppress the transcripts by factor 7 at 0.1 M BaP (p < 0.05).Effect of P90 on CYP1B1 protein levels in Calu-3 To investigate CYP1B1 protein levels we performed Western blot analysis with isolated microsomal protein of Calu-3 cells. In the blot shown in Fig. 6, the densitometric reading for CYP1B1 protein in Calu-3 decreased from 3,036,276 AU (untreated cells, 32 h) to 373,799 AU after ultrafine P90-treatment for 32 h. In average of 3 experiments CYP1B1 protein level in untreated cells was 2,721,541 ?379,059 AU and P90 treatment reduced this to 342,109 ?46,303 AU (Fig. 6). CYP1B1 mRNA levels analyzed at the same time point (32 h) and in the same cells used for Western blotting showed a decrease of transcript for ultrafine P90-treated cells (-314 ?144) compared to untreated cells (-3,551 ?1,889; p < 0.05) (Fig. 6, right panel).chrome P450 monooxygenases are involved in detoxification and toxification of xenobiotic substances. Toxification is caused by metabolic transformation of non or less toxic precursors into reactive intermediates. Cells may thus be influenced by two mechanisms/modes of action, Vorinostat they may either be protected or they may become more susceptible to other PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/17139194 inhaled substances by exposure to ultrafine P90. The strongest down-regulation of CYP1B1 expression after stimulation with particle mix of ultrafine P90 and fine TiO2 was observed in monocytes (60-fold, Fig. 1A). It is unclear at this point why CYP1B1 is so much more sensitive to.