Cytochrome P450 2E1-enhanced Cisplatin Hepatotoxicity And Its Mechanisms

Cisplatin is one of the most potent anticancer drugs used in chemotherapy. In spite of its significant anticancer activity, the clinical use of cisplatin is often limited by its undesirable side effects such as nephrotoxicity. Hepatotoxicity can also occur when cisplatin is administered at high doses. Oxidative stress plays an important role in cisplatin-induced hepatotoxicity. Many cellular pathways have been suggested to contribute to induction of a state of oxidative stress. Cytochrome P450 2E1 (CYP2E1) is mainly expressed in liver and in small amounts in brain, kidney, lung, gastrointestinal tract, and lymphocytes. Due to being poorly coupled with NADPH-cytochrome P450 reductase, CYP2E1 exhibits enhanced NADPH oxidase activity and elevated rates of production of superoxide anion radical (O2.-) and hydrogen peroxide (H2O2), and in the presence of iron catalysts, produces powerful oxidants such as the hydroxyl radical (OH.-). It has been shown previously that CYP2E1 induction is involved in hepatotoxicity of iron and polyunstaturated fatty acids subsequent to ROS production, and elevated expression of CYP2E1 enhances liver injury induced by Fas agonist Jo2 and endotoxin. It is possible that cisplatin-induced oxidative stress and CYP2E1-mediated oxidative stress synergizes to produce stronger hepatotoxicity. In this study, the potentiation of ciplatin-induced hepatotoxicity by CYP2E1 was investigated.In order to study the enhancement of cisplatin toxicity by elevated expression of CYP2E1, mice model with elevated CYP2E1 induced by acetone was used. After drinking 2% acetone for 7 days, body weight had no change compared with control mice, CYP2E1 activity increased more than 2 times. Western blot analysis showed that CYP2E1 protein content also increased 2.5-fold. Cisplatin at 45mg/kg body weight can induce mouse hepatotoxicity 24 h after administration of cisplatin, serum ALT levels increased about 3-fold, and AST levels were elevated about 5-fold over control mice. The combination of acetone and cisplatin treatment further increased ALT levels about 2-fold and AST levels about 40%, over the cisplatin alone treatment. In liver sections from mice treated with cisplatin, degeneration and vacuolization but no necrosis was observed. However, in liver sections from mice treated with the combination of acetone and cisplatin, wildspread apoptotic cells, small necrotic foci, and occasional large necrotic areas were found. Apoptosis was detected by measuring activity of Caspase-3 and TUNEL staining. Cisplatin caused a 2-fold increase in activity of caspase-3, the combination of acetone and cisplatin caused a 50% further increase in caspase-3 activity; in liver sections from mice with cisplatin alone treatment, about 5% hepatocytes were positive TUNEL staining, and in liver sections from mice with cisplatin plus acetone treatment, positive TUNEL staining increased up to about 13% (P<0.05). Thus, the combination of acetone plus cisplatin produced greater liver injury than either cisplatin or acetone alone did. These results were confirmed by lower levels of transaminases and TUNEL staining after cisplatin treatment in CYP2E1 knockout mice, compared with wild type mice.We hypothesized that the enhancement of cisplatin-induced hepatotoxicity by acetone might be due to elevated oxidative stress produced by the combination of acetone-induced CYP2E1 plus cisplatin. Lipid peroxidation end products MDA and HAE increased in cisplatin- treated mice (P<0.05) and were slightly but significantly further increased by co-treatment with acetone and cisplatin (P<0.05 over values for cisplatin alone). Protein carbonyl formation, an early marker for protein oxidation, is weak in liver from control and acetone-treated mice, but stronger signals could be detected in liver from cisplatin-treated mice, and the strongest signals were detected in the group with the combination of cisplatin and acetone. Peroxynitrite (ONOO-), formed by the rapid reaction between NO. and O2.-, has been shown to nitrate free and protein-associated tyrosine residues and produce 3-nitrotyrosine(3-NT). No 3-NT signals were detected in liver sections from control and acetone-treated mice, weak signals could be detected in liver sections from cisplatin-treated mice, and the strongest signals were detected in the group with the combination of cisplatin and acetone. Similarly, the strongest signals for iron were detected in the group with the combination of cisplatin and acetone, weaker signals in cisplatin group, and no signals in control and acetone-treated mice.In order to examine the mechanisms by which CYP2E1enhances cisplatin toxicity, HepG2 cells expressing CYP2E1 (E47 cells) and control HepG2 cells not expressing CYP2E1 (C34 cells) were used. Incubation of E47 cells with cisplatin caused losses of cell viability in a time- and concentration-dependent manner. Exposure of C34 cells to cisplatin in the same concentrations and exposure times caused a much lower loss of cell viability. Concentrations of cisplatin e.g. 0.25 mM or duration of incubation e.g. 12 h which produced toxicity with the E47 cells had no effect with the C34 cells, thus indicating that E47 cells are more sensitive to cisplatin than C34 cells. Exposure of both cell lines to cisplatin caused a striking decrease in intracellular GSH with a somewhat more pronounced decline in E47 cells. Exposure of both cell lines to cisplatin caused a dramatic increase in ROS production with more elevation in E47 cells. These results suggest that exposure of E47 cells to cisplatin caused a more pronounced oxidative stress than that found with the C34 cells. To validate the role of enhanced oxidative stress in the CYP2E1 plus cisplatin-induced cytotoxicity, several intervention experiments were performed. BSO, which depletes GSH, enhanced the cisplatin-induced cytotoxicity both in E47 and C34 cells with the overall toxicity being much greater in the E47 cells. Glutathione ethyl ester (GSHE) can be taken up by cells and then metabolized into GSH within cells; DFO is a chelator of iron, which prevents iron-catalyzed formation of potent oxidants like hydroxyl radical; both of the antioxidants decreased loss of E47 cell viability. DAS, an inhibitor of CYP2E1, also protected against the loss of E47 cell viability induced by cisplatin, confirming the pivotal role of CYP2E1 in the enhanced sensitivity of E47 cells to cisplatin.In order to examine the role of MAPKs in cisplatin cytotoxicity, E47 or C34 cells were pretreated for 1 h with specific inhibitors of P38 MAPK, JNK, and ERK (SB203580, SP600125, and U0126, respectively), and then varying concentrations of cisplatin were added. After incubation for 24 h, MTT assays were performed to examine cytotoxicity. Among the three specific inhibitors of MAPK, only the ERK inhibitor U0126 decreased the loss of cell viability induced by cisplatin in both E47 and C34 cells, suggesting that ERK, but not P38 MAPK and JNK, played an important role in the cisplatin cytotoxicity. Indeed, cisplatin treatment caused a concentration-dependent ERK activation and U0126 inhibited this ERK activation in both C34 and E47 cells. However, E47 cells were more sensitive to cisplatin than C34 cells, but ERK activation had no significant difference. We examined the time course of ERK activation. Because 0.25 mM cisplatin led to cell death in E47 cells but not in C34 cells, 0.25 mM of cisplatin was selected for all following experiments. Cisplatin induced a sustained ERK activation in both C34 and E47 cells, but in E47 cells ERK activation occurred earlier. In order to test if earlier activation of ERK contributes to sensitiveness of E47 cells to cisplatin, U0126 at different times after cisplatin was added, and then cell viability was measured. Addition of U0126 at 16 h after cisplatin prevented cisplatin cytotoxicity in C34 cells but not in E47 cells. These results are consistent with the notion that sustained ERK activation contributed to cisplatin cytotoxicity both in E47 and C34 cells, and earlier ERK activation caused the E47 cells to be more sensitive to cisplatin.The relationship between glutathione and ERK activation in the actions of cisplatin in E47 cells was examined. U0126 partially protected against cell death induced by cisplatin alone or BSO plus cisplatin. BSO enhanced ERK activation by cisplatin, and U0126 prevented this BSO-enhanced ERK activation. Pretreatment with 5 mM of GSHE blunted the E47 cell death induced by cisplatin alone or the enhanced cell death of cisplatin in combination with BSO. ERK activation was also inhibited by GSHE. Thus glutathione loss potentiated ERK activation by cisplatin in E47 cells. We next examined the glutathione level in order to validate the actions of BSO. After treatment with 0.25 mM of cisplatin, intracellular glutathione content decreased. However, U0126 had no effect on the glutathione level even though it protected against cisplatin toxicity. Much more striking glutathione loss was caused by cisplatin plus BSO, but U0126 did not increase glutathione levels, even though the U0126 partially protected against the potentiated toxicity produced by cisplatin plus BSO. These results suggest that the protection by U0126 is not related to elevating glutathione.Besides glutathione, ROS play an important role in cisplatin cytotoxicity, therefore we examined the relationship between ROS accumulation and ERK activation. Like ERK activation, cisplatin elevation of ROS accumulation occurred earlier in E47 cells than C34 cells. U0126 blocked ROS accumulation caused by cisplatin, even though glutathione levels were not restored. BSO alone did not lead to ROS accumulation, but it enhanced cisplatin-induced ROS accumulation. U0126 blocked this enhanced ROS accumulation even though U0126 did not restore the low glutathione levels. These results suggest that ERK activation and ROS accumulation are associated with each other.Cisplatin induces both necrosis and apoptosis. Since the MTT assay cannot distinguish the mode of cell death, experiments with PI staining were carried out to evaluate the mode of cell death caused by cisplatin. Necrosis was determined on the basis of positive PI staining (red color) of the nucleus, indicative of the loss of membrane integrity. After exposure to cisplatin alone, about 20% necrotic cell death was detected in E47 cells, but no necrosis was detected in C34 cells. U0126 decreased the cell necrosis induced by cisplatin in E47 cells. Apoptotic cells were detected after the cells were fixed followed by PI staining. Condensed or fragmented nuclei were counted as apoptotic cells. After exposure to cisplatin, about 50% apoptotic cells were detected in E47 cells, but less than 5% apoptotic cells was detected in C34 cells. Pretreatment with U0126 had no effect on cisplatin-induced apoptosis. Cisplatin-induced apoptosis is caspase- dependent. The activities of caspase-3, -8, and -9 increased after cisplatin treatment in both E47 and C34 cells, as compared to Controls. U0126 either did not inhibit or actually slightly increased activities of the three caspases in the presence of cisplatin in both E47 and C34 cells.Cisplatin induced much more necrosis in the presence of BSO. We evaluated the effect of BSO on the cisplatin-induced apoptosis. After treatment of E47 cells with BSO plus cisplatin, almost no apoptosis was detected. Thus, the BSO treatment switches cisplatin toxicity from an apoptotic mode of cell death to a necrotic mode. This switching may be due to the decrease in caspase activities found in the cisplatin plus BSO treated cells, in contrast to the increase in caspase activities found in cisplatin alone treated cells. The activities of caspases which were inhibited by cisplatin plus BSO were recovered after GSHE treatment, especially caspase-3. Interestingly, U0126 partially switched necrosis induced by cisplatin plus BSO back to apoptosis. While BSO plus cisplatin inhibited the activities of the three caspases, U0126 partially restored these activities, especially caspase-3 activity. Z-VAD-FMK, a pan-caspase inhibitor, inhibited the apoptosis produced by cisplatin alone; however, Z-VAD-FMK had no effect on the PI staining produced by cisplatin plus BSO. Caspases are cysteine proteases with a cysteine residue at the active site, and modification of the cysteine residue abrogates their catalytic activities. GSH can regulate the redox state within cells by forming reversible mixed disulphides with protein thiols to prevent irreversible oxidation of cysteine residues under oxidative stress conditions. Therefore, the possibility was considered that after GSH depletion by BSO, irreversible oxidation of cysteine residues under oxidative stress induced by cisplatin could inactivate caspases, and explain why apoptosis was not detected in the cisplatin plus BSO treated cells. In the contrary, GSHE pretreatment may prevent oxidation of or restore cysteine residues and thereby switch necrosis by cisplatin plus BSO to apoptosis. This recovery of caspase activities resulted in a switch of the cisplatin plus BSO toxicity from necrosis to apoptosis in the presence of GSHE.As mentioned above, cisplatin increased ROS production, and this increase could be further elevated by cisplatin plus BSO treatment; U0126 and GSHE both lowered the elevated ROS produced by cisplatin alone or cisplatin plus BSO, which may account for their protection against the cisplatin and cisplatin plus BSO toxicity. To more directly evaluate the role of ROS in cisplatin toxicity, especially the mode of cisplatin toxicity, the effect of several antioxidants was evaluated. The necrosis produced by the combination of cisplatin plus BSO was strongly prevented by treatment with a scavenger of superoxide (MnTMP), a synthetic vitamin E analogue which is a powerful inhibitor of lipid peroxidation (Trolox), and by an iron chelator (DFO). These antioxidants were as effective as GSHE in lowering the cisplatin plus BSO necrosis. Thus oxidative stress plays a key role in the cisplatin-induced necrosis. However, the antioxidants did not mimic GSHE in promoting a switch of cisplatin plus BSO toxicity from necrosis to apoptosis, suggesting that GSHE is not promoting this switch by acting as an antioxidant. The likely reason that GSHE could restore the apoptosis might be related to the increase in the intracellular GSH level was increased by GSHE but not by the other antioxidants. However, as mentioned above, U0126 had no effect on intracellular glutathione content, although it restored or promoted activities of caspases in the presence of cisplatin plus BSO. It is proposed that there must be another thiol resource which can replace glutathione to adjust the intracellular redox status. We found that expression of thioredoxin (TRX), a redox active protein which also regulates cellular redox status, decreased after treatment with cisplatin. BSO strikingly decreased TRX expression in the presence of cisplatin. U0126 restored this decreased TRX expression induced by cisplatin alone or in combination with BSO. GSHE could also promote the recover of the inhibited TRX expression produced by cisplatin alone or in combination with BSO. To prove the role of TRX, TRX siRNA was applied to decrease the expression of TRX. After CP-induced caspase-3 activity was inhibited by pretreatment with BSO, U0126 failed in restoring the inhibited caspase-3 activity when TRX expression was inhibited by TRX siRNA. It is conjectured that in this model, TRX might replace GSH to maintain caspases in their active state when GSH was depleted by BSO, and that the ability of U0126 to prevent necrosis but not apoptosis is due to the U0126-mediated increase in TRX levels.In conclusion, both in vitro and in vivo results indicate that elevated CYP2E1 enhances cisplatin-induced hepatotoxicity, and the mechanism may involve increased production of ROS and oxidative stress. In CYP2E1- overexpressing HepG2 cells, cisplatin induces earlier and sustained activation of ERK, ERK activation promotes production of ROS and in turn ROS activates ERK, which contributes to enhancement of cisplatin cytotoxicity. Cisplatin induces both necrosis and apoptosis in HepG2 cells; cisplatin-induced apoptosis is caspase-dependent; ERK activation promotes necrosis but not apoptosis in HepG2 cells. Glutathione depletion by BSO switches cisplatin-induced apoptosis to necrosis, glutathione supplement can switch necrosis induced by cisplatin plus BSO back to apoptosis, but non-thiol antioxidants cannot.Thioredoxin can replace glutathione to promote caspase activation and switch necrosis induced by cisplatin plus BSO back to apoptosis. ERK inhibitor U0126 can prevent this switch through elevating thioredoxin.