Induction of Internucleosomal DNA Fragmentation by Carcinogenic Chromate: Relationship to DNA Damage, Genotoxicity, and Inhibition of Macromolecular Synthesis

Hexavalent chromium (Cr) compounds are respiratory carcinogens in humans and animals. Treatment of Chinese hamster ovary cells with 150 and 300 μM sodium chromate ( Na2CrO4) for 2 hr decreased colony-forming efficiency by 46 and 92%, respectively. These treatments induced dose-dependent internucleosomal fragmentation of cellular DNA beyond 24 hr after chromate treatment. This fragmentation pattern is characteristic of apoptosis as a mechanism of cell death. These treatments also induced an immediate inhibition of macromolecular synthesis and delayed progression of cells through S-phase of the cell cycle. Cell growth (as evidenced by DNA synthesis) was inhibited for at least 4 days and transcription remained suppressed for at least 32 hr. Many of the cells that did progress to metaphase exhibited chromosome damage. Chromate caused the dose-dependent formation of DNA single-strand breaks and DNA-protein cross-links, but these were repaired 8 and 24 hr after removal of the treatment, respectively. In contrast, Cr-DNA adducts (up to 1/100 base-pairs) were extremely resistant to repair and were still detectable even 5 days after treatment. Compared with other regions of the genome, DNA-protein cross-links and Cr adducts were preferentially associated with the nuclear matrix DNA of treated cells, which was 4.5-fold enriched in actively transcribed genes. Chromium adducts, formed on DNA in vitro at a similar level to that detected in nuclear matrix DNA, arrested the progression of a DNA polymerase in a sequence-specific manner, possibly through the formation of DNA-DNA cross-links. Total RNA and mRNA synthesis and induction of expression of the inducible GRP78 gene were suppressed in a concentration- and time-dependent manner by chromate. The effects of chromate on GRP78 induction correlated most closely with the presence of DNA-protein cross-links but suppression of total RNA and mRNA synthesis correlated with the presence of DNA-Cr adducts in cells. These results suggest that the persistent Cr-DNA adducts may be responsible for the protracted cell cycle delay and transcriptional inhibition caused by chromate. Escape from apoptosis may be one of the steps involved in chromate-induced carcinogenesis.