Figure 1.
Cell viability and membrane integrity in SH-SY5Y cells exposed to oxaliplatin.
Cells (2×105 cells/well) were exposed 24 h to oxaliplatin (OXA) (2–200 µM). Cell viability (redox potential) was evaluated with Alamar blue and membrane integrity was evaluated with neutral red tests (A). Cell viability which is indicative of mitochondrial metabolism was evaluated with MTT test (B). Values are the mean ± S.E.M. expressed as percentage of the control, five different assays per group. *: statistically different (p<0.05) from the mean values in control cells.
Figure 2.
Oxidative stress and mitochondrial activity in SH-SY5Y cells exposed to oxaliplatin.
Cells (2×105 cells/well) were exposed 24 h to oxaliplatin (OXA) (2–200 µM). Oxidative stress was evaluated by Reactive Oxygen Species (ROS) production using dihydroethidium and DCF-DA tests (A) and Nitric Oxide (NO) production evaluated as nitrite content (B) by the Griess reaction. Mitochondrial activity was evaluated by determining mitochondrial membrane potential (Δφm) (C) using JC-1 test and mitochondrial levels of negatively charged phospholipids, mainly cardiolipin, using nonyl acridine orange test (D). Values are the mean ± S.E.M. expressed as percentage of the control, five different assays per group. *: statistically different (p<0.05) from the mean values in control cells.
Figure 3.
P2X7 receptor activation, chromatin condensation and caspase-3 activity in SH-SY5Y cells exposed to oxaliplatin.
Cells were exposed 24 h to oxaliplatin (OXA) (2–200 µM). P2X7 receptor activation (A) was evaluated using YOPRO-1 test. The resulting fluorescence was viewed with a microscope (B). Chromatin condensation (C) was evaluated using Hoechst 33342 test. Caspase-3 proteolytic activity (D) in lysates of cells was evaluated using the apoTargetTM Caspase-3 Protease assay. In these tests, cells were exposed or not to a 30-min pre-treatment with the specific P2X7 receptor antagonist Brilliant Blue G (BBG; 10 µM) prior to OXA for an additional 24 h. Values are the mean ± S.E.M. expressed as percentage of the control, five different assays per group. *: statistically different (p<0.05) from the mean values in control cells.
Table 1.
Cytokines and PGE2 release in oxaliplatin-treated cells after acetaminophen, ibuprofen or N-acetyl cysteine treatment.
Table 2.
Viability and oxidative stress in RAW 264.7 cells exposed 24 h to oxaliplatin (25–200 µM).
Table 3.
P2X7R activation, chromatin condensation and caspase-3 activity in RAW 264.7 exposed 24 h to oxaliplatin (25-200µM).
Figure 4.
Cell viability and oxidative stress in oxaliplatin-treated SH-SY5Y cells pre-treated by protective drugs.
Cells (2×105 cells/well) were exposed for 24 h to oxaliplatin (OXA) (50, 100 or 200 µM) after a 30-min pre-treatment either with acetaminophen (AAP, 50 µM), ibuprofen (IBU, 1 µM) or N-acetyl cysteine (NAC, 1 mM). Cell viability (redox potential) was evaluated with Alamar blue (A). Oxidative stress was evaluated by ROS production using dihydroethidium (B) and DCF-DA (C) tests and nitric oxide (NO) production evaluated as nitrite content (D) by the Griess reaction. Lipid peroxidation (E) was determined using the thiobarbituric acid (TBA) method at 200 µM OXA. Mitochondrial activity was evaluated by determining mitochondrial membrane potential (Δφm) (F) using JC-1 test and mitochondrial levels of negatively charged phospholipids, mainly cardiolipin, using nonyl acridine orange test (G). Values are the mean ± S.E.M. expressed as percentage of the control, five different assays per group. Significance of differences: OXA alone versus control, *p<0.05; AAP, or IBU, or NAC versus OXA alone: $ p<0.05.
Figure 5.
Chromatin condensation, P2X7R activation and caspase-3 activity in oxaliplatin-treated SH-SY5Y cells pre-treated by protective drugs.
Cells (2×105 cells/well) were exposed for 24 h to oxaliplatin (OXA) (50, 100 or 200 µM) after a 30-min pre-treatment either with acetaminophen (AAP, 50 µM), ibuprofen (IBU, 1 µM) or N-acetyl cysteine (NAC, 1 mM). Chromatin condensation (A) was evaluated using Hoechst 33342 test and P2X7 receptor (P2X7R) activation (B) using YOPRO-1 test. The apoTargetTM Caspase-3 Protease assay was used for the in vitro determination of caspase-3 proteolytic activity (C) in lysates of SH-SY5Y cells as described by the manufacturer's instructions. Values are the mean ± S.E.M. expressed as percentage of the control, five different assays per group. Significance of differences: OXA alone versus control, *p<0.05; AAP, or IBU, or NAC versus OXA alone: $ p<0.05.
Figure 6.
Behavioral assessment of cold hyperalgesia in oxaliplatin-treated C57BL/6 mice using the cold plate test.
Mice were repeatedly injected i.p. with 7 mg/kg oxaliplatin (OXA) at days 1, 2, 5 and 6 (28 mg/kg cumulated dose). The latency of first jump was used to evaluate the painful response at 2°C (A), values represent mean ± SEM, * p<0.01. A cut off was set at 3 min in order to avoid tissue damages (B); results are presented as Kaplan-Meier survival curve, * p<0.001.
Figure 7.
Oxidative stress and mitochondrial activity in oxaliplatin-treated C57BL/6 mice.
Mice were repeatedly injected i.p. with 7 mg/kg oxaliplatin (OXA) at days 1, 2, 5 and 6 (28 mg/kg cumulated dose; n = 10). Oxidative stress was evaluated by ROS production using dihydroethidium (A) and DCF-DA (B) tests and NO content (C) by the Griess reaction. Values are the mean ± S.E.M. expressed as percentage of the control (n = 8). *: statistically different (p<0.05) from the mean values in control mice.
Figure 8.
Chromatin condensation, P2X7 receptor activation and caspase-3 activity in oxaliplatin-treated C57BL/6 mice.
Mice were repeatedly injected i.p. with 7 mg/kg oxaliplatin (OXA) at days 1, 2, 5 and 6 (28 mg/kg cumulated dose; n = 10). Mitochondrial activity was evaluated by determining mitochondrial membrane potential (A) using JC-1 test and mitochondrial levels of negatively charged phospholipids (B) using nonyl acridine orange test. Chromatin condensation (C) was evaluated using Hoechst 33342 test and P2X7 receptor activation (D) using YOPRO-1 test. The apoTargetTM Caspase-3 Protease assay was used for the in vitro determination of caspase-3 proteolytic activity (E) in lysates of brain mitochondrial homogenates as described by the manufacturer's instructions. Values are the mean ± S.E.M. expressed as percentage of the control (n = 8). *: statistically different (p<0.05) from the mean values in control mice.
Figure 9.
Hypothesis of pathophysiological mechanisms of OXA-induced pain neuropathy.
OXA-induced pain neuropathy seems mediated by cell death P2X7 receptor activation and mitotoxicity which induce apoptosis. Symbols: inhibiting pathway; LPO: lipoperoxydation; AAP: acetaminophen; IBU: ibuprofen; NAC: N-acetyl cysteine.