Amie L. Holmes

Hexavalent Chromium-Induced DNA Damage and Repair Mechanisms

Hexavalent chromium is a commonly used industrial metal that has been shown to induce lung cancer in workers having long term exposure. In the particulate form, Cr(VI) dissolves slowly in vivo, leading to an extended exposure of lung cells. Hexavalent chromium is taken into the cell and rapidly reduced to Cr(V), Cr(IV), Cr(III), and reactive oxygen species. Cells treated with Cr(VI) are subject to several types of DNA damage resulting from this reduction, including base modification, single-strand breaks, double-strand breaks, Cr-DNA adducts, DNA-Cr-DNA adducts, and protein-Cr-DNA adducts. These types of damage, if left unrepaired or are misrepaired, can lead to growth arrest, cytotoxicity, and apoptosis, as well as mutations leading to neoplastic transformation and ultimately tumorigenesis. Here we review the current literature on Cr-induced DNA damage and its repair.

Chronic Exposure to Lead Chromate Causes Centrosome Abnormalities and Aneuploidy in Human Lung Cells

Hexavalent chromium [Cr(VI)] compounds are established human lung carcinogens. The carcinogenicity of Cr(VI) is related to its solubility, with the most potent carcinogens being the insoluble particulate Cr(VI) compounds. However, it remains unknown why particulate Cr(VI) is more carcinogenic than soluble Cr(VI). One possible explanation is that particulates may provide more chronic exposures to chromate over time. We found that aneuploid cells increased in a concentration- and time-dependent manner after chronic exposure to lead chromate. Specifically, a 24-hour lead chromate exposure induced no aneugenic effect, whereas a 120-hour exposure to 0.5 and 1 microg/cm2 lead chromate induced 55% and 60% aneuploid metaphases, respectively. We also found that many of these aneuploid cells were able to continue to grow and form colonies. Centrosome defects are known to induce aneuploidy; therefore, we investigated the effects of chronic lead chromate exposure on centrosomes. We found that centrosome amplification in interphase and mitotic cells increased in a concentration- and time-dependent manner with 0.5 and 1 microg/cm2 lead chromate for 120 hours, inducing aberrant centrosomes in 18% and 21% of interphase cells and 32% and 69% of mitotic cells, respectively; however, lead oxide did not induce centrosome amplification in interphase or mitotic cells. There was also an increase in aberrant mitosis after chronic exposure to lead chromate with the emergence of disorganized anaphase and mitotic catastrophe. These data suggest that one possible mechanism for lead chromate-induced carcinogenesis is through centrosome dysfunction, leading to the induction of aneuploidy.

Comparative genotoxicity and cytotoxicity of four hexavalent chromium compounds in human bronchial cells.

Hexavalent chromium (Cr(VI)) compounds are well-established human lung carcinogens. Solubility plays an important role in their carcinogenicity with the particulate Cr(VI) compounds being the most carcinogenic. Epidemiology and animal studies suggest that zinc chromate is the most potent particulate Cr(VI) compound; however, there are few comparative data to support these observations. The purpose of this study was to compare the genotoxicity of zinc chromate with two other particulate Cr(VI) compounds, barium chromate and lead chromate, and one soluble Cr(VI) compound, sodium chromate. The clastogenic effects of barium chromate and zinc chromate were similar, but lead chromate induced significantly less damage. The levels of DNA damage measured by gamma-H2A.X foci formation were similar for the three particulate chromium compounds. Corrected for chromium uptake differences, we found that zinc chromate and barium chromate were the most cytotoxic, and lead chromate and sodium chromate were less cytotoxic. Zinc chromate was more clastogenic than all other chromium compounds, and lead chromate was the least clastogenic. There was no significant difference between any of the compounds for the induction of DNA double strand breaks. All together, these data suggest that the difference in the carcinogenic potency of zinc chromate over the other chromium compounds is not due solely to a difference in chromium ion uptake and that the zinc cation may in fact have an important role in its carcinogenicity.

Chronic Exposure to Zinc Chromate Induces Centrosome Amplification and Spindle Assembly Checkpoint Bypass in Human Lung Fibroblasts

Hexavalent chromium (Cr(VI)) compounds are known human lung carcinogens. Solubility plays an important role in its carcinogenicity with the particulate or insoluble form being the most potent. Of the particulate Cr(VI) compounds, zinc chromate appears to be the most potent carcinogen; however, very few studies have investigated its carcinogenic mechanism. In this study, we investigated the ability of chronic exposure to zinc chromate to induce numerical chromosome instability. We found no increase in aneuploidy after a 24 h exposure to zinc chromate, but with more chronic exposures, zinc chromate induced concentration- and time-dependent increases in aneuploidy in the form of hypodiploidy, hyperdiploidy, and tetraploidy. Zinc chromate also induced centrosome amplification in a concentration- and time-dependent manner in both interphase and mitotic cells after chronic exposure, producing cells with centriolar defects. Furthermore, chronic exposure to zinc chromate induced concentration- and time-dependent increases in spindle assembly checkpoint bypass with increases in centromere spreading, premature centromere division, and premature anaphase. Last, we found that chronic exposure to zinc chromate induced a G2 arrest. All together, these data indicate that zinc chromate can induce chromosome instability after prolonged exposures.

The cytotoxicity and genotoxicity of soluble and particulate cobalt in human lung fibroblast cells

Cobalt exposure is increasing as cobalt demand rises worldwide due to its use in enhancing rechargeable battery efficiency, super-alloys, and magnetic products. Cobalt is considered a possible human carcinogen with the lung being a primary target. However, few studies have considered cobalt-induced toxicity in human lung cells. Therefore, in this study, we sought to determine the cytotoxicity and genotoxicity of particulate and soluble cobalt in human lung cells. Cobalt oxide and cobalt chloride were used as representative particulate and soluble cobalt compounds, respectively. Exposure to both particulate and soluble cobalt induced a concentration-dependent increase in cytotoxicity, genotoxicity, and intracellular cobalt ion levels. Based on intracellular cobalt ion levels, we found that soluble cobalt was more cytotoxic than particulate cobalt while particulate and soluble cobalt induced similar levels of genotoxicity. However, soluble cobalt induced cell cycle arrest indicated by the lack of metaphases at much lower intracellular cobalt concentrations compared to cobalt oxide. Accordingly, we investigated the role of particle internalization in cobalt oxide-induced toxicity and found that particle-cell contact was necessary to induce cytotoxicity and genotoxicity after cobalt exposure. These data indicate that cobalt compounds are cytotoxic and genotoxic to human lung fibroblasts, and solubility plays a key role in cobalt-induced lung toxicity.

Comparative cytotoxicity and genotoxicity of particulate and soluble hexavalent chromium in human and sperm whale (Physeter macrocephalus) skin cells

Chromium (Cr) is a global marine pollutant, present in marine mammal tissues. Hexavalent chromium [Cr(VI)] is a known human carcinogen. In this study, we compare the cytotoxic and clastogenic effects of Cr(VI) in human (Homo sapiens) and sperm whale (Physeter macrocephalus) skin fibroblasts. Our data show that increasing concentrations of both particulate and soluble Cr(VI) induce increasing amounts of cytotoxicity and clastogenicity in human and sperm whale skin cells. Furthermore, the data show that sperm whale cells are resistant to these effects exhibiting less cytotoxicity and genotoxicity than the human cells. Differences in Cr uptake accounted for some but not all of the differences in particulate and soluble Cr(VI) genotoxicity, although it did explain the differences in particulate Cr(VI) cytotoxicity. Altogether, the data indicate that Cr(VI) is a genotoxic threat to whales, but also suggest that whales have evolved cellular mechanisms to protect them against the genotoxicity of environmental agents such as Cr(VI).

Cytotoxicity and Genotoxicity of Hexavalent Chromium in Human and North Atlantic Right Whale (Eubalaena glacialis) Lung Cells

Humans and cetaceans are exposed to a wide range of contaminants. In this study, we compared the cytotoxic and genotoxic effects of a metal pollutant, hexavalent chromium [Cr(VI)], which has been shown to cause damage in lung cells from both humans and North Atlantic right whales. Our results show that Cr induces increased cell death and chromosome damage in lung cells from both species with increasing intracellular Cr ion levels. Soluble Cr(VI) induced less of a cytotoxic and genotoxic effect based on administered dose in right whale (Eubalaena glacialis) cells than in human (Homo sapiens) cells. Whereas, particulate Cr(VI) induced a similar cytotoxic effect but less of a genotoxic effect based on administered dose in right whale cells than in human cells. Differences in chromium ion uptake explained soluble chromate-induced cell death but not all of the soluble chromate-induced chromosome damage. Uptake differences of lead ions could explain the differences in particulate chromate-induced toxicity. The data show that both forms of Cr(VI) are less genotoxic to right whale than human lung cells, and that soluble Cr(VI) induces a similar cytotoxic effect in both right whale and human cells, while particulate Cr(VI) is more cytotoxic to right whale lung cells.

Homologous Recombination Repair Signaling in Chemical Carcinogenesis: Prolonged Particulate Hexavalent Chromium Exposure Suppresses the Rad51 Response in Human Lung Cells

The aim of this study was to focus on hexavalent chromium, [Cr(VI)], a chemical carcinogen and major public health concern, and consider its ability to impact DNA double strand break repair. We further focused on particulate Cr(VI), because it is the more potent carcinogenic form of Cr(VI). DNA double strand break repair serves to protect cells against the detrimental effects of DNA double strand breaks. For particulate Cr(VI), data show DNA double strand break repair must be overcome for neoplastic transformation to occur. Acute Cr(VI) exposures reveal a robust DNA double strand break repair response, however, longer exposures have not been considered. Using the comet assay, we found longer exposures to particulate zinc chromate induced concentration-dependent increases in DNA double strand breaks indicating breaks were occurring throughout the exposure time. Acute (24 h) exposure induced DNA double strand break repair signaling by inducing Mre11 foci formation, ATM phosphorylation and phosphorylated ATM foci formation, Rad51 protein levels and Rad51 foci formation. However, longer exposures reduced the Rad51 response. These data indicate a major chemical carcinogen can simultaneously induce DNA double strand breaks and alter their repair and describe a new and important aspect of the carcinogenic mechanism for Cr(VI).

Human lung cell growth is not stimulated by lead ions after lead chromate-induced genotoxicity

Chromate compounds are known human lung carcinogens. Water solubility is an important factor in the carcinogenicity of these compounds with the most potent carcinogenic compounds being water-insoluble or ‘particulate’. Previously we have shown that particulate chromates dissolve extracellularly releasing chromium (Cr) and lead (Pb) ions and only the Cr ions induce genotoxicity. Pb ions have been considered to have epigenetic effects and it is thought that these may enhance the carcinogenic activity of lead chromate, perhaps by stimulating Cr-damaged cells to divide. However, this possibility has not been directly tested. Accordingly, we investigated the ability of Pb ions to stimulate human lung cells and possibly force lead chromate-damaged cells to grow. We found that at concentrations of lead chromate that induced damage, human lung cells exhibited cell cycle arrest and growth inhibition that were very similar to those observed for sodium chromate. Moreover, we found that soluble Pb ions were not growth stimulatory to human lung cells and in fact induced progressive mitotic arrest. These data indicate that lead chromate-generated Cr ions cause growth inhibition and cell cycle arrest and that Pb does not induce epigenetic effects that stimulate chromate-damaged cells to grow.

The impact of homologous recombination repair deficiency on depleted uranium clastogenicity in Chinese hamster ovary cells: XRCC3 protects cells from chromosome aberrations, but increases chromosome fragmentation.

Depleted uranium (DU) is extensively used in both industry and military applications. The potential for civilian and military personnel exposure to DU is rising, but there are limited data on the potential health hazards of DU exposure. Previous laboratory research indicates DU is a potential carcinogen, but epidemiological studies remain inconclusive. DU is genotoxic, inducing DNA double strand breaks, chromosome damage and mutations, but the mechanisms of genotoxicity or repair pathways involved in protecting cells against DU-induced damage remain unknown. The purpose of this study was to investigate the effects of homologous recombination repair deficiency on DU-induced genotoxicity using RAD51D and XRCC3-deficient Chinese hamster ovary (CHO) cell lines. Cells deficient in XRCC3 (irs1SF) exhibited similar cytotoxicity after DU exposure compared to wild-type (AA8) and XRCC3-complemented (1SFwt8) cells, but DU induced more break-type and fusion-type lesions in XRCC3-deficient cells compared to wild-type and XRCC3-complemented cells. Surprisingly, loss of RAD51D did not affect DU-induced cytotoxicity or genotoxicity. DU induced selective X-chromosome fragmentation irrespective of RAD51D status, but loss of XRCC3 nearly eliminated fragmentation observed after DU exposure in wild-type and XRCC3-complemented cells. Thus, XRCC3, but not RAD51D, protects cells from DU-induced breaks and fusions and also plays a role in DU-induced chromosome fragmentation.