In Vitro Enzyme Induction Experiment
In Vitro CYP1A2 Induction
Induction was detected. Cutoff used — not specified
- CYP1A2 4312402
Cell system HepG2 cell line
CYP1A2 activity was induced by BER at 0.5–4.5 μg/ml from 1.7 to 5.0 fold (SD estimated from Fig. 1)
Induction of CYP1A2 mRNA expression and protein level in HepG2 cells
CYP1A2 mRNA expression in HepG2 cells treated with BER exhibited an induction effect, which significantly increased by 1.47-, 5.90-, 11.71-, and 16.11-fold exposed to BER at 0.5, 1.5, 3.0, and 4.5 μg/ml (Fig. 2). Besides, CYP1A2 protein extracted from HepG2 cells which treated with BER exhibit a concentration dependent manner compared with the untreated by western blotting analysis (Fig. 2).
|Compound measured||Measurement||Value||Study sequence||Additional information||N replicates|
CYP1A2 mRNA level from HepG2 cells
Total RNA from cells was extracted and reverse transcripted, and the later relative quantification of CYP1A2 mRNA level was performed according to the manufacturer's protocols of the RNAisoTM Plus, PrimeScriptTM RT reagent Kit and SYBR Premix Ex TaqTM reagent (Takara, Kyoto, Japan). Gene-specific primers were designed as de- scribed previously (Table 1). BER was tested in four levels (4.5, 3, 1.5, 0.5 μg/ml), and 0.3% DMSO and BNF (10 μg/ml) was used as the ve- hicle and positive control. The polymerase chain reaction (PCR) was conducted using Thermal Cycler DiceTM TP800 real time system (Takara, Kyoto, Japan).
CYP1A2 protein expression from HepG2 cells
HepG2 cells were treated with different concentrations of BER (4.5, 3, 1.5, 0.5 μg/ml) for 48 h, and then were washed three times with ice- cold phosphate-buffered saline, and lysed in phosphate-buffered saline (1 ml) for 30 min, which contained 1% Triton X-100, 0.1% SDS, di- thiothreitol (1 mM), and phenylmethylsulfonyl fluoride (1 mM). After being scraped off the plate, the mixture was subjected to centrifugation at 21,000 × g for 5 min. Protein concentrations were determined by the BCA protein assay. In brief, total protein extracts (30 μg) were firstly separated on 10% SDS-polyacrylamide gel, secondly transferred to ni- trocellulose membranes (Bio-Rad Laboratories, Hercules, CA). Monoclonal CYP1A2 antibody and its secondary antibody were diluted in 5% non-fat dry milk in 1:200 and 1:5000 ratios, respectively. After incubation with nitrocellulose membranes, proteins interacting with antibodies were detected by an ODYSSEY infrared imaging system (LI- COR Biosciences, USA).
Total RNA was extracted and reverse transcripts
BCA protein assay