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We have previously shown that trivalent chromium, and hexavalent chromium in the presence of one of its primary in vivo reductants, ascorbate, can bind to DNA and form interstrand crosslinks capable of obstructing replication. This effect was demonstrated in vitro by using Sequenase Version 2.0 T7 DNA polymerase; its parent enzyme, the unmodified T7 DNA polymerase; and Escherichia coli polymerase I large (Klenow) fragment; and it was demonstrated ex vivo by using Taq polymerase and DNA from chromium-treated human lung cells as template. This study was performed to determine whether DNA-bound chromium affects mammalian DNA polymerases in the same manner. Two mammalian enzymes, DNA polymerase alpha and DNA polymerase beta, were used. DNA polymerase alpha is a processive enzyme believed to be the primary lagging-stand synthetase, whereas DNA polymerase beta is a non-processive enzyme believed to function in DNA repair by filling single stranded gaps one base at a time. DNA polymerase arrest assays were performed with each of these enzymes to replicate DNA with toxicologically relevant levels of chromium adducts produced by either trivalent chromium or hexavalent chromium and ascorbate. Both enzymes responded to chromium-DNA damage by arresting replication, and the arrests increased in a dose-dependent manner. Furthermore, the guanine-specific pattern of arrests produced when an exonuclease-free preparation of DNA polymerase beta was used corresponded exactly to the arrest patterns produced in vitro by the exonuclease-free enzyme Sequenase and ex vivo by Taq polymerase. These results suggest that replication arrest may be a common response of polymerases to DNA-chromium lesions and provide a plausible mechanism for the inhibition of DNA synthesis and S-phase cell-cycle delay that occurs in mammalian cells treated with genotoxic chromium compounds.

Certain hexavalent chromium compounds are documented human carcinogens. Exposure of cells to particulate forms of chromium results in cell-enhanced dissolution of particles in the extracellular microenvironment and chronic production of chromium oxyanions, which are taken up by the cell through an anion transport system and are genotoxic and clastogenic. It was previously shown that apoptosis is the mode of cell death of nearly all of the Chinese hamster ovary cells (CHO-AA8 cell line), which die after high-dose, short-term treatments with soluble sodium chromate. In this report the mode of cell killing by particulate lead chromate and of low-dose continuous treaments of soluble sodium chromate designed to mimic conditions of ionic chromate uptake after lead chromate exposure was examined. CHO-AA8 cells were treated for 24 hr with doses of sodium chromate or lead chromate which cause a 50% decrease in survival in colony-forming effeciency assays. Longer treatments (up to 72 hr) at the same doses did not decrease survival further than the 24-hr exposure. Morphological changes indicative of apoptosis, as well as internucleosomal DNA fragmentation, were detectable by 24 hr after treatment with lead chromate or soluble sodium chromate. All of the cells killed by treatments with lead chromate particles underwent apoptosis as the mode of cell death and this was accurately modeled in cell culture by continuous treatments with low-dose soluble sodium chromate. Exposure of cells to hexavalent chromium compounds causes a spectrum of DNA damage which can be selectively altered by pretreatment of cells with antioxidant vitamins prior to chromium exposure. Here we show that ascorbate and alpha-tocopherol markedly inhibited the chromosomal aberrations induced by both particulate and soluble chromate compounds, even though chromium adduct levels were not decreased by either vitamin pretreatment. Cell survival assays showed that ascorbate, but not alpha-tocopherol, protected cells from apoptosis induced by sodium chromate. The results differentiate chromium-induced apoptosis from both chromosomal damage and adduct levels and suggest that other lesions sensitive to ascorbate but not tocopherol are the proximal inducing signal for chromium-induced apoptosis., (Copyright 1997 Academic Press.)

In 1992, a simple and sensitive assay for detecting DNA-protein crosslinks was developed [1]. In an effort to facilitate the greater use of the assay, a number of studies were conducted to evaluate its reliability and reproducibility. During this work, the assay was used to assess whether various metals and other compounds could induce crosslinks in cultured human lymphocytes (Epstein-Barr virus-transformed Burkitt's Lymphoma cell line). Potassium permanganate, mercury chloride, lead nitrate, magnesium perchlorate, aluminum chloride, and cadmium chloride did not induce DNA-protein crosslinks at either cytotoxic or non-cytotoxic levels. Copper sulfate, arsenic trioxide, and potassium chromate induced DNA-protein crosslinks only at cytotoxic concentrations. Acute lethality of the cells was assessed immediately after exposure to metals by trypan blue exclusion while long-term lethality was assessed by cell proliferation and trypan blue exclusion following an incubation period of 5 days after exposure to the metal compound. All metals exhibited more toxicity in the long-term lethality assay compared to the short-term assay. The cultured human lymphocytes treated with various doses of lead acetate, cadmium chloride, arsenic trioxide and copper sulfate, as well as cis-platinum and chromate, were sent to four different laboratories to compare the reliability and reproducibility of the DNA-protein crosslink assay. Depending on the chemical studied, there were quantitative differences in the results observed among the various laboratories using the assay. However, all laboratories generally showed that cis-platinum, chromate, arsenic trioxide and copper sulfate induced DNA-protein crosslinks at levels that produced acute cytotoxicity, whereas cadmium chloride and lead acetate did not.

Chromium(III) complexes currently being sold as dietary supplements were tested for their ability to cause chromosomal aberrations in Chinese hamster ovary cells. Complexes were tested in soluble and particulate forms. Chromium picolinate was found to produce chromosome damage 3-fold to 18-fold above control levels for soluble doses of 0.050, 0.10, 0.50, and 1.0 mM after 24 h treatment. Particulate chromium picolinate doses of 8.0 micrograms/cm2 (corresponding to a 0.10 mM solublized dose) and 40 micrograms/cm2 (0.50 mM) produced aberrations 4-fold and 16-fold above control levels, respectively. Toxicity was measured as a decrease in plating efficiency relative to controls. The above treatments produced > or = 86% survival for all doses except 1.0 mM chromium picolinate, which produced 69 +/- 10% survival. Chromium nicotinate, nicotinic acid, and chromium(III) chloride hexahydrate did not produce chromosome damage at equivalent nontoxic doses. Damage was inferred to be caused by the picolinate ligand because picolinic acid in the absence of chromium was clastogenic. Data are evaluated in terms of their relevance to human exposure based on pharmacokinetic modeling of tissue accumulation and are discussed in terms of literature reporting toxic effects of picolinic acid.

Hexavalent chromium (Cr) compounds are respiratory carcinogens in humans and animals. Treatment of Chinese hamster ovary cells with 150 and 300 microM 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Particulate lead chromate is a highly water-insoluble cytotoxic and carcinogenic agent, but its mechanism of action remains obscure. We investigated its effects on DNA damage in CHO cells after a 24-h exposure using alkaline or neutral filter elution and cytogenetic studies. Concentrations (0.08, 0.4 and 0.8 micrograms/cm2), which reduced the colony-forming efficiency of CHO cells to 94, 50 and 10%, respectively, produced dose-dependent DNA single-strand breaks and DNA-protein crosslinks, but no DNA double-strand breaks or DNA-DNA crosslinks were observed. The single-strand breaks were absent from cells given a 24-h recovery period after removal of the treatment medium, even though most of the particles remained adhered to cells and to the culture dish. In contrast, both the DNA-protein crosslinks and chromosomal aberrations persisted even after the 24-h recovery period. These results suggest that the mechanism of the particle-induced early DNA single-strand breaks may be different from DNA-protein crosslinks and the lesions leading to chromosomal aberrations, or alternatively, that the repair of single-strand breaks is more efficient than the repair of DNA-protein crosslinks in the unavoidable continuing presence of carcinogen. These results also suggest that the chromosome damage may be related to the persistent DNA-protein crosslinks, and further confirm the genotoxic activity of carcinogenic lead chromate particles.

Hexavalent chromium compounds are carcinogenic to humans, are potent inducers of tumors in experimental animals, and can neoplastically transform cells in culture. In this study, the effects of sodium chromate on the expression of the 78-kDa glucose-regulated protein (GRP78) gene and on general transcription were investigated with respect to the DNA damage induced by this agent. DNA single-strand breaks, DNA-protein cross-links, and chromium-DNA adducts were present in CHO cells immediately after 2 h of treatment with sodium chromate. Subsequently, these types of damage were repaired at different rates. Single-strand breaks were essentially repaired after 8 h. By 24 h posttreatment, no cross-links remained in cells exposed to 30 or 150 microM chromate, although cells treated with the 300-microM concentration still contained cross-links at that time. DNA-chromium adducts remained unrepaired for at least 32 h. The moderate constitutive level of GRP78 mRNA was not affected by chromate. Chromate did, however, suppress induction of this gene by tunicamycin in a concentration-and time-dependent manner. Thirty micromolar sodium chromate (96% survival), which caused the least DNA damage, had no effect on GRP78 induction, general RNA synthesis, or mRNA synthesis. Induction of GRP78 was suppressed immediately and 12 h after treatment with 150 microM chromate (54% survival), although there was a partial recovery of induction at 24 h after treatment, which correlated with the repair of DNA-protein cross-links. In contrast, both total cytoplasmic RNA and mRNA synthesis were suppressed by approximately 60-75% for at least 32 h by 150 microM chromate. At the 300-microM concentration (8% survival), where DNA-protein cross-links persisted beyond 24 h, GRP78 induction was totally suppressed for at least 24 h, while total RNA and mRNA synthesis were suppressed by 80-90% for at least 32 h. Overall, 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-chromium adducts. These results indicate that chromate exerts differential effects on the induction of the GRP78 gene and on general transcription.