Analysis of CYP1A1 induction in single cells of urothelial cell populations by flow cytometry

摘要: As carcinogenesis is a process starting at the single-cell level it desirable to study carcinogen-mediated effects in individual cells. A primary step chemically induced formation of reactive DNA-binding metabolites by cytochromes P450 (CYP). We applied indirect immunofluorescence stain CYP1A1 urothelial cells for quantification flow cytometry. Our studies were carried out with metabolically competent porcine urinary bladder epithelial (PUBECs) and human cell line 5637 which we have previously demonstrated mRNA induction polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (B[a]P) applying real-time RT-PCR. Flow cytometric analysis revealed that PUBEC fraction CYP1A1-induced increased B[a]P concentration. Furthermore, this effect was time-dependent, being more pronounced after 48 h than 24 h. However, could not be detected all analyzed treatment up 50 μM B[a]P. The reason remains unknown moment. Overall, B[a]P-treated divided into fractions clearly uninduced Another “unclear” one unclassifiable remained, as cytometry hampered B[a]P-related fluorescence. This ascribed phenolic formed are known fluoresce wavelengths above 500 nm, whereas does not. method permits detection protein large numbers cells, thereby providing an adequate basis statistical analyses. allows further insight metabolic competence single therefore valuable tool toxicological studies.