Table 1.
Why women experience more adverse drug reactions than men?
Table 2.
EC50 values for cell viability.
Table 3.
EC50 values for toxicity parameters.
Fig 1.
Cell viability curves for Diclofenac, Chlorpromazine, Acetaminophen, and Verapamil.
5000 human primary hepatocytes pooled from either 12 male donors (M), or 12 pre-menopausal female donors (3F), or 12 post-menopausal female donors (4F) were seeded in 96 well plates and exposed to 8 serial concentrations (using a dilution factor of 1:2) of Diclofenac (from 13.6 to 1750 uM), Chlorpromazine (from 1.9 to 250 uM), Acetaminophen (from 0.3 to 35 uM), or Verapamil (from 7.8 to 1000 uM). Decrease in cell number after 30 min, 2h, or 5h exposure to drugs was assessed by lysing the cells with CellTiter-Glo Reagent and measuring the ATP levels released. Assays were run in triplicates. Results are expressed as mean ±SEM. Dose response curves were plotted using GraphPad Prism.
Fig 2.
Cell viability curves for Omeprazole and Caffeine.
5000 human primary hepatocytes pooled from either 12 male donors (M), or 12 pre-menopausal female donors (3F), or 12 post-menopausal female donors (4F) were seeded in 96 well plates and exposed to 8 serial concentrations (using a dilution factor of 1:2) of Omeprazole (from 1.9 to 250 uM), or Caffeine (from 39 to 5000 uM). Cell viability after 30 min, 2h, or 5h exposure to drugs was assessed by lysing the cells with CellTiter-Glo Reagent and measuring the ATP levels released. Assays were run in triplicates. Results are expressed as mean ±SEM. Data were plotted using GraphPad Prism.
Fig 3.
Fluorescence images of human primary hepatocytes.
5000 untreated and treated primary hepatocytes derived from M, 3F, and 4F donors were stained for 30 min using Hoechst 33342 and either TMRE and TOTO3 (a-f), or DHE (g-l), or ER tracker red (m-r), or Fluo-4 (s-x) dyes and imaged using the Cellomics ArrayScan VTI. A 10x objective was used to collect 10 images per well with the filter set XF93. Primary hepatocytes treated for 3h with 125 uM Chlorpromazine or untreated and stained with Hoechst 33342 (blue), TMRE (red), and TOTO3 (green) are reported in a-f. Primary hepatocytes treated with 1750 uM Diclofenac for 30 min or untreated and stained with Hoechst 33342 (blue) and DHE (red) are reported in g-l. Primary hepatocytes treated for 5h with 0.78 uM Acetaminophen or untreated and stained with Hoechst 33342 (blue) and ER tracker (red) are reported in m-r. Primary hepatocytes treated for 5h with 250 uM Omeprazole or untreated and stained with Hoechst 33342 (blue) and Fluo-4 (green) are reported in s-x.
Fig 4.
Nuclear intensity dose response curves after 4h treatment with Diclofenac, Verapamil, Caffeine, Acetaminophen, Chlorpromazine, and Omeprazole.
5000 human primary hepatocytes pooled from either 12 male donors (M), or 12 pre-menopausal female donors (3F) or 12 post-menopausal female donors (4F) were seeded in 96 well plates and exposed to 8 serial concentrations (using a dilution factor of 1:2) of Diclofenac (from 13.6 to 1750 uM), Acetaminophen (from 0.3 to 35 uM), Verapamil (from 7.8 to 1000 uM), Chlorpromazine (from 1.9 to 250 uM), Caffeine (from 39 to 5000 uM), or Omeprazole (from 1.9 to 250 uM) for 30 min, 1h, 2h, 3h, 4h, and 5h. Hepatocytes nuclei were stained with Hoechst 33342 and increase in nuclear intensity was measured at different time points using the Cellomics ArrayScan VTI platform and the Target Activation Bioapplication v.4. GraphPad Prism-derived dose response curves obtained treating primary hepatocytes for 4h with Diclofenac, Verapamil, Acetaminophen, Chlorpromazine, and Omeprazole are reported. Statistically significant sex differences (P value<0.05) comparing the EC50 values from the three groups of hepatocytes (M vs 3F vs 4F) were observed only with primary hepatocytes treated for 4h with Diclofenac (P value = 0.0090) or with Verapamil (P value = 0.0144). Diclofenac and Verapamil resulted more toxic in 4F hepatocytes. No statistically significant nuclear intensity modifications were obtained comparing M, 3F, and 4F hepatocytes treated with Acetaminophen (P value = 0.7471), Chlorpromazine (P value = 0.6578), and Omeprazole (P value = 0.7574). Caffeine did not give any increase in nuclear intensity. Assays were run in triplicates. Results are expressed as mean ±SEM.
Fig 5.
Cytotoxicity dose response curves.
5000 human primary hepatocytes pooled from either 12 male donors (M), or 12 pre-menopausal female donors (3F), or 12 post-menopausal female donors (4F) were seeded in 96 well plates and exposed to 8 serial concentrations of Diclofenac (from 13.6 to 1750 uM), Chlorpromazine (from 1.9 to 250 uM), Acetaminophen (from 0.3 to 35 uM), and Verapamil (from 7.8 to 1000 uM). ROS formation (DHE dye), endoplasmic reticulum status (ER tracker red dye), mitochondrial damage (TMRE dye), and plasma membrane permeability (TOTO3 dye) were measured at different time points (30 min, 2h, 3h, 4h, 5h) using the Cellomics ArrayScan VTI platform and the Target Activation Bioapplication v.4. Representative dose-response curves are shown for drugs having a P value<0.05 (M vs 3F vs 4F) in Table 3 (Diclofenac, Chlorpromazine, Acetaminophen, and Verapamil). Three replicates were tested and represented using GraphPad Prism. Results are expressed as mean ±SEM.