ABSTRACT
OBJECTIVE To investigate the expression differences of seven cytochrome P450 (CYP) isoforms between L02, LX-2 and HepG2 cells and identify the most suitable cell type for different CYP researches. METHODS L02, LX-2 and HepG2 cells were treated with omeprazole (Ome), dexamethasone(Dex), phenobarbital sodium (Phe), isoniazid (Iso) and rifampicin (Rif) at 5, 10, 20 and 40 μmol • L-1. Cell viability was analyzed by CCK-8 assay. The gene expressions of CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 in LX-2 cells were assessed by real-time quantitative PCR (RT-qPCR). The protein expressions of these seven CYP isoforms in L02, LX-2 and HepG2 cells were assessed by Western blotting. Furthermore, CYP3A4 activity in the three types of cells treated with Rif 5, 10, 20 and 40 μmol • L-1 for 24 h was validated by Luciferin-PFBE. RESULTS Cell viabilities of all the three hepatocytes were over 80% when exposed to Ome, Dex, Phe, Iso and Rif (≤40 μmol • L-1) for 24 h. According to the RT-qPCR, Phe could significantly enhance the gene expressions of CYP2B6 (P<0.05) and CYP2D6 (P<0.01) in LX-2 cells. The results of Western blotting exhibited protein expression differences of seven CYP isoforms between L02, LX-2 and HepG2 cells treated with Ome, Dex, Phe, Iso and Rif (≤40 μmol-L-1) for 24 h. CYP2C9 [Integrated absorbance (IA)=1.58±0.07], CYP2C19 (IA= 0.95±0.03) and CYP3A4 (IA=1.29±0.05) had higher expression abundances in L02 cells, CYP2B6 (IA= 1.48±0.01) and CYP2D6 (IA=1.46±0.02) in LX-2 cells, and CYP1A2 (IA=1.62±0.19) and CYP2E1 (IA= 1.49±0.10) in HepG2 cells. Additionally, CYP3A4 activity in L02, LX-2 and HepG2 cells could not be up-regulated by Rif. CONCLUSION The most suitable cell type for different CYP researches is L02 for CYP2C9, CYP2C19 and CYP3A4, LX-2 for CYP2B6 and CYP2D6, HepG2 for CYP1A2 and CYP2E1, respectively.
ABSTRACT
Two novel approaches are described, in which metabolically competent human derived cells were used for the detection of genotoxic effects of environmental carcinogens. In the first, human hepatoma (Hep G2) cells were used for micronucleus and single cells were used for micronucleus and single cell gel electrophoresis (SCGE) assays. These cells have retained the activities of phase I and phase II enzymes which are usually lost during cultivation. We demonstrated that these cells are suitable for the detection of the genotoxic effects of representatives of various classes of DNA-reactive procarcinogens such as benzo(a) pyrene (B(a)P), 2-amino-3-methylimidazo-[4,5-f]-quinoline (IQ), cyclophosphamide (CP), and N-nitrosodimethylamine (NDMA), isatidine and aflatoxin B1 (AFB1). Furthermore, we found that these tests also detect the mutagenic effects of rodent carcinogens such as safrole and hexamethylphosphoramide (HEMPA), which give negative results in conventional in vitro procedures. Additional experimental series showed that genotoxicity assays with Hep G2 cells are also useful for the detection of co- and antimutagens, in particular for compounds which act via induction of activating and detoxifying enzymes. In the second approach, a protocol for stable co-cultivation sandwich cultures with primary human hepatocytes was used. The cultivation of the cells under organotypical conditions leads to an extension of their life span and results in an improved expression of drug metabolising enzymes. Two different experimental models were developed: In the first, the induction of HPRT mutations in V-79 cells was used as an endpoint, in the seconds, single strand breaks were measured in human K562 cells in SCGE assays. Experiments which were carried out with B(a)P and 7,12-diemethylbenz(a)anthracene as model compounds indicate that in both systems positive results are obtained. In conclusion, our data show that tests with human Hep G2 cells as well as sandwich cultures with primary human liver cells are useful for the detection of environmental carcinogens and probably reflect their effects in humans better than conventional in vitro assays with metabolically incompetent cells which are currently used in most mutagenicity studies.