In Vitro CYP1A2 Induction CSV JSON

In Vitro Enzyme Induction Experiment

In Vitro CYP1A2 Induction

Induction was detected.  Cutoff used — not specified

acetaminophen 1125315

  • CYP1A2 4312402

Cell system HepG2 cell line

Results

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

Experimental Conditions

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).

Enzyme activity

200,000 cells/mL

76 h

Total RNA was extracted and reverse transcripts

48 h

BCA protein assay

72 h

Phenacetin O-deethylation