Abstract
Abstract Cr(VI) compounds are ubiquitous environmental toxicants that have been evaluated as human carcinogens. Cr(VI) genotoxicity and carcinogenicity are highly associated with the generation of reactive species including Cr(V), Cr(IV), Cr(III) and reactive oxygen species during its intracellular reduction. De-regulation of cell cycle progression is an important issue leading to cancer formation. However, the mechanism that Cr(VI) interferes with cell cycle progression is not fully understood. In this thesis, we have investigated the effects of Cr(VI) on cell cycle checkpoint control and genotoxicities of Cr(VI)-treated cells synchronized before and after DNA replication using human non-small lung carcinoma CL3 cells. In asynchronous cells, Cr(VI) exposure caused the delay of cell cycle at S phase. The Cr(VI)-elicited S phase delay was also observed in G1 phase cells collected from counterflow centrifugal cell elutriation system. Cr(VI) did not cause significant G1 delay. Cells were synchronized at G1/S border or early G2 phase using aphidicolin, exposed to 20 mM of Cr(VI) for 2 h, kept culture for 2-10 h and subjected for flow cytometry analysis. A significant G2/M delay were observed in both Cr(VI)-treated G1/S or early G2 cells. Mitotic figure analysis using an inverted microscope confirmed that Cr(VI) delays M phase entry. Using western blotting and immunocomplex kinase activity analysis techniques, we have observed that the activation of cdc2 kinase is delayed for 2 h, and the degradation of cyclin B1 and deactivation of cdc2 kinase are also delayed for 4 h. The results suggest that Cr(VI) delays G2 enter into M phase and the exit of M phase. Cr(VI) induced similar levels of cytotoxicity and genotoxicity in G1/S and early G2 cells. Ascorbic acid pretreatment could enhance the effect of Cr(VI) on the delay of G2/M phase and degradation of cyclin B1 but not the activity of cdc2 kinase in Cr(VI)-treated G1/S cells, suggesting ascorbic acid enhances the delay of M phase exit induced by Cr(VI). Ascorbic acid alone slightly increased micronucleus formation in both untreated and Cr(VI)-treated cells. Together, Cr(VI) causes cell cycle delay at S, G2 and M phases, however, it does not induce G1 arrest, which can allow abnormal replication of damaged-DNA and subsequently leading to gene deletion and carcinogenesis.