APOPTOSIS IS THE MODE OF CELL-DEATH CAUSED BY CARCINOGENIC CHROMIUM

The role of apoptosis in the mechanism of toxicity of hexavalent chrom ium, a human carcinogen, was investigated. Chinese hamster ovary (CHO) cells were treated with 150 or 300 mu M sodium chromate for 2 hr, dos es which decreased colony-forming efficiency to 53 and 5% of control, respectively. Cell growth was inhibited at least up to Day 8 after tre atment. DNA synthesis was inhibited to 30 and 19% of control at 1 hr a fter treatment, and did not begin to recover until Day 4 after treatme nt. Protein synthesis was inhibited by 52 and 60% in 150 and 300 mu M treated cells, respectively, 1 hr after treatment, and recovered to 14 2 and 93%, respectively, at 24 hr. Incubation of cells with nontoxic d oses of cycloheximide for 24 hr after treatment produced synergistic t oxicity with chromate in colony-forming efficiency assays. Ion gradien ts persisted to Day 2 as revealed by exclusion of trypan blue dye in 9 7% of treated cells. Fluorescence microscopy of acridine orange-staine d cells revealed morphological features of apoptosis including nuclear fragmentation in more than 90% of detached nonadherent cells and up t o 22% of adherent cells by Day 2 after treatment. Untreated cells rema ined morphologically normal. Transmission electron microscopy of chrom ate treated cells showed characteristic features of apoptosis includin g chromatin margination and fragmentation, and cytoplasmic condensatio n with intact membrane and organelle structure. Internucleosomal DNA f ragmentation (IDF) was delayed for at least 24 hr, whereafter it was d etected in both adherent and nonadherent cells through Day 5 after tre atment. These results indicate apoptosis as the mode of cell death cau sed by chromium and imply that apoptosis must be considered as a compo nent of chromium-induced multistage carcinogenesis. (C) 1994 Academic Press, Inc.