Fig 1.
APAP-induced DNA fragmentation.
J774.2 macrophages and HepG2 cells were cultured and treated with APAP (10 μmol/ml) for 18 hours after treatment with or without 200 μM DAS for 24 h as described in Materials and Methods. DNA fragmentation was visualized by 0.5μg/ml ethidium bromide staining of DNA fragments separated on 1.5% agarose gel. DNA breakdown was also visualized by using a single cell comet assay according to the vendor’s protocol. The slides were examined at x100 magnification using an Olympus fluorescence microscope. Images of 50 randomly selected nuclei were analyzed per slide. Typical results from 3 such experiments have been shown.
Fig 2.
Apoptosis in J774.2 macrophages and HepG2 cells were measured after APAP and DAS treatment using Flow cytometry as described in the vendor’s protocol using the Becton Dickinson FACSCantoII analyzer. Apoptotic cells were estimated by the percentage of cells that stained positive for Annexin V-FITC. Representative histograms of flow cytometric results are shown.
Fig 3.
Effect of APAP on ROS production.
Intracellular production of ROS was measured using lucigenin coupled method and chemiluminescence was measured using Turner’s luminometer as described in Materials and Methods [24–28]. The values expressed are mean ±SEM for at least three determinations. Asterisks (*) indicate significant difference (p ≤0.05) from control values, # indicate significant difference (P ≤0.05) from APAP-treated group.
Fig 4.
Effect of APAP on GSH metabolism.
J774.2 macrophages andHepG2 cells were treated with APAP and DAS alone or in combination and GSH levels in the mitochondria and post-mitochondrial supernatant (PMS) were measured by enzymatic method as described in the Materials and Methods (4A). Total GST-conjugating activity was measured using CDNB as a substrate (4B). Microsomal GST activity was measured using CDNB as a substrate in the presence of NEM as an activator of membrane-bound microsomal GST (4C). Ethacrynic acid was used as substrate to measure GSTpi isoenzyme (4D) and 4-HNE was used to measure GSTA4-4 isoenzyme (4E) as described before [24–28]. GSH-Px activity was measured using cumene hydroperoxide as a substrate (4F). Results are expressed as mean ± SEM of three determinations. Asterisks (*) indicate significant difference (p ≤0.05) from control values, # indicate significant difference (p ≤0.05) from APAP-treated group.
Fig 5.
Effect of APAP on CYP450 activities.
J774.2 macrophages and HepG2 cells were treated with APAP and DAS as described above and post mitochondrial supernatant was used to measure CYP2E1 (5A), CYP3A4 (5B), CYP1A1 (5C) and CYP1A2 activities as described in the Materials and Methods. The values expressed are mean ±SEM of three determinations. Asterisks (*) indicate significant difference (p≤0.05) from untreated control cells, # indicate significant difference (p≤0.05) from APAP-treated group.
Fig 6.
Effect of APAP on ATP production, membrane potential, mitochondrial matrix enzyme, aconitase and mitochondrial respiratory complexes.
APAP treated cells were lysed and ATP was measured by the luciferase-dependent chemiluminescence assay as described in the vendor’s protocol (6A). The mitochondrial membrane potential was measured by using a mitotracker fluorescent cationic dye according to the manufacturer’s protocol (6B). % reduction in mitochondrial membrane potential is shown in a typical histogram taking an average of at least three experiments. Aconitase activity was assayed in J774.2 macrophages and HepG2 cells after treatment with APAP and DAS alone or in combination (6C). Activities of mitochondrial respiratory enzyme complexes I and IV were measured in freshly isolated mitochondria from APAP-treated cells using ubiquinone, and cytochrome c respectively as substrates as described in the Materials and Methods (6D). The values are expressed as mean ± SEM of three determinations. Asterisks (*) indicate significant difference (p≤0.05) from untreated control cells. # indicate significant difference (p ≤0.05) from APAP-treated group.
Fig 7.
Expression of GST isoenzyme, CYP450 isoenzyme MDR1 proteins.
Proteins (50μg) from PMS extract from APAP and DAS treated J774.2 macrophages and HepG2 cells were separated on 12% SDS-PAGE and transferred on to nitrocellulose paper by Western blotting as described in the Materials and Methods. GST pi, GSTA4-4 and microsomal GST (MGST1-1) proteins were visualized using specific antibodies against these proteins (Fig 7A). The expression of CYP isoenzymes was visualized using isoenzyme specific antibodies against CYP2E1, CYP3A4, CYP1A1 and CYP 1A2 (Fig 7B). MDR1 protein expression was measured using specific antibody against the protein (Fig 7C). Beta-actin was used as loading control. Representative Western blots from three experiments are shown. R.I gives the relative intensity of the protein compared to the control untreated cells as 1.0. Molecular weight is expressed in kDa.