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

The role of apoptosis in the mechanism of toxicity of hexavalent chromium, a human carcinogen, was investigated. Chinese hamster ovary (CHO) cells were treated with 150 or 300 mu M sodium chromate for 2 hr, doses which decreased colony-forming efficiency to 53 and 5% of control, respectively. Cell growth was inhibited at least up to Day 8 after treatment. DNA synthesis was inhibited to 30 and 19% of control at 1 hr after treatment, and did not begin to recover until Day 4 after treatment. Protein synthesis was inhibited by 52 and 60% in 150 and 300 mu M treated cells, respectively, 1 hr after treatment, and recovered to 142 and 93%, respectively, at 24 hr. Incubation of cells with nontoxic doses of cycloheximide for 24 hr after treatment produced synergistic toxicity with chromate in colony-forming efficiency assays. Ion gradients persisted to Day 2 as revealed by exclusion of trypan blue dye in 97% of treated cells. Fluorescence microscopy of acridine orange-stained cells revealed morphological features of apoptosis including nuclear fragmentation in more than 90% of detached nonadherent cells and up to 22% of adherent cells by Day 2 after treatment. Untreated cells remained morphologically normal. Transmission electron microscopy of chromate treated cells showed characteristic features of apoptosis including chromatin margination and fragmentation, and cytoplasmic condensation with intact membrane and organelle structure. Internucleosomal DNA fragmentation (IDF) was delayed for at least 24 hr, whereafter it was detected in both adherent and nonadherent cells through Day 5 after treatment. These results indicate apoptosis as the mode of cell death caused by chromium and imply that apoptosis must be considered as a component of chromium-induced multistage carcinogenesis. (C) 1994 Academic Press, Inc.