Abstract
Cadmium (Cd) and chromium(VI) (Cr(VI)) compounds are ubiquitous environmental carcinogens that have been associated with lung tumors. These two metals can induce genotoxicity, morphological transformation, signal transduction, and apoptosis in cultured mammalian cells. Reactive oxygen species (ROS) has been implied to mediate in the biological effects induced by Cd. Similarly, Cr(VI) carcinogenicity has been associated with its metabolic activation by intracellular reductants that generates reactive species, including Cr(V), Cr(IV), Cr(III), ROS, and other free radicals. Three major mitogen-activation protein kinases (MAPKs), i.e., JNK, p38, and ERK have been shown to regulate cell growth and apoptosis. This thesis has explored the roles of each MAPK in the growth arrest and apoptosis of CdCl2- or K2Cr2O7-treated cells using a human lung adenocarcinoma line, CL3. Cd decreased the ERK activity with concurrently transient activated-JNK at low cytotoxic dosages, whereas the three MAPKs were persistently activated at high cytotoxic dosages. Cr(VI) activated the three MAPKs in dose- and time-dependent manners providing this metal is existed in culture media. At equal cytotoxic dosages, Cd induces more activities of JNK and p38 with longer duration as compared with those activated by Cr(VI). Cross-talk of the MAPK signal pathways was observed in cells exposed to Cr(VI) but not Cd. The JNK activity induced by both metals was mediated through MKK7 but not MKK4. However, Cd but not Cr(VI) activated-JNK could transmit apoptotic signal. PD98059, an inhibitor of the ERK upstream activators MKK1/2, greatly enhanced the cytotoxicity and apoptosis of cells treated with low Cd dosages. SB202190, an inhibitor of p38, decreased the cytotoxicity and apoptosis induced by high Cd dosages. By contrast, neither SB202190 or PD98059 altered Cr(VI)-induced cytotoxicity. The results have suggested that JNK and p38 signals cooperatively participate in apoptosis induced by Cd and that the decreased ERK signal by low Cd dosages contributes to growth inhibition or apoptosis. Oppositely, the JNK, p38, and ERK although transiently activated by Cr(VI), do not correlate to Cr(VI) cytotoxicity. Furthermore, the roles of redox reactive species in the activation of MAPKs and cytotoxicities induced by these two metals have been investigated using modulators include 3-amino-1,2,4-triazole (3AT; a catalase inhibitor), catalase, H2O2, mannitol (a hydroxyl radical scavenger and a Cr(V) chelator), N-acetylcysteine (NAC), and vitamin C (ascorbate). 3AT that increases oxidative stress could block the activation of JNK by Cd but do not alter that induced by Cr(VI), suggesting catalase is critical for maintenance the JNK activity induced by Cd but not Cr(VI). 3AT also protected Cd cytotoxicity but enhanced Cr(VI) cytotoxicity. NAC that decreases oxidative stress also suppressed the JNK activity and cytotoxicity induced by Cd. The results have suggested that both increases and decreases oxidative stress can inhibit the ability of Cd to activate JNK and protect from cytotoxicity. Catalase enhanced the levels and duration of the Cd-activated JNK, supporting the results of 3AT. H2O2 additive increased the activities of p38 but not ERK induced by both metals. However, at high cytotoxic dosages H2O2 suppressed the JNK activation by Cd but enhanced that activated by Cr(VI). Mannitol did not affect the activation of three MAPKs by Cd, conversely, it reduced the p38 activation and increased the JNK and ERK activation by Cr(VI). These results have indicated that the p38 activation by the two metals is positively correlated to oxidative stress, while the activation of JNK by these two metals may be either increased or decreased by different oxidative species. Additionally, ascorbate markedly inhibited the JNK but did not affect the p38 and ERK activated in Cr(VI)-treated cells, suggesting that the redox cycle quenched by ascorbate is critical for Cr(VI) to activate JNK. Results presented here indicate that distinct mechanisms are involved in the activation of MAPKs in response to Cd and Cr(VI) to regulate cell growth and apoptosis despite that they both act as oxidative stress inducers and DNA damage agents.