Mutation Research/Genetic Toxicology and Environmental Mutagenesis
Carcinogenic lead chromate induces DNA double-strand breaks in human lung cells
Introduction
Exposure to hexavalent chromium [Cr(VI)] has been known for more than a century to be associated with induction of cancer in humans, especially bronchial carcinoma, causing an 18–80-fold increased risk of lung cancer [1], [2], [3], [4]. However, all Cr(VI) compounds are not equally potent as carcinogens. In particular, the water-insoluble (particulate) Cr(VI) salts are more potent carcinogens than the water soluble ones [2], [3], [4], [5]. For example, only the particulate compounds are consistently tumorigenic in experimental animals [2], [3], [4] and only the particulates induce neoplastic transformation of C3H10T1/2 mouse embryo cells [5]. Further data from pathological studies of workers with Cr(VI)-induced lung cancers indicate that Cr(VI)-induced tumors originate in bronchial cells at the site of bifurcations where Cr(VI) particles are most likely to impact and persist. These observations strongly support the conclusion that particulate Cr(VI) compounds are the carcinogenic compounds [6], [7]. However, the carcinogenic mechanisms of particulate Cr(VI) are poorly understood and the full spectrum of particulate Cr(VI)-induced genotoxic damage has not been established.
Most studies using particulate Cr(VI) have focused on lead chromate (LC) as a model compound for the particulate salts [8], [9]. Studies in human lung cells showed that LC particles partially dissolve outside the cell releasing chromate and Pb ions, which enter the cell [10], [11]. The internalized Cr ions then cause DNA damage including adducts, single strand breaks and chromosomal aberrations, cytotoxicity and growth inhibitory effects [10], [11], [12], [13], [14], [15], [16], [17]. The internalized Pb ions have no apparent effect [10], [16], [17]. However, the full spectrum of damage induced by particulate Cr(VI) has not been determined as its ability to induce DNA double strand breaks (DSBs) has not been investigated. DSBs are of particular concern because they are most likely to induce genomic and chromosomal instability, which are hallmarks of Cr(VI)-induced tumors [18]. DSBs are particularly dangerous because, in addition to the potential for mutations to develop at their site of occurrence, incorrect DSB repair can lead to chromosomal translocations that generate chromosome instability [19].
Only two studies have considered soluble Cr(VI)-induced DSBs [20], [21]. Each reported positive results; however, both studies considered only very acute exposures (0.5–3 h) with high concentrations and neither determined if the DSBs occur in human lung cells. Specifically, Wakeman et al. [21] treated HeLa cells with potassium chromate for 30 min and Ha et al. [20] treated primary human skin fibroblasts (HSF) for 1–3 h. Particulate chromate requires some time to dissolve and cannot achieve these levels in such a short period of time [8], [10], [16]. It remains to be seen if chromate induces DSBs at the lower and more chronic exposures that occur with particulate compounds and if these effects occur in human lung cells. Accordingly, the purpose of this study was to improve our current understanding of particulate Cr(VI) and study the induction and response to particulate chromate-induced DSBs in human lung cells.
Section snippets
Chemicals and reagents
Lead chromate, sodium chromate, demecolchicine, d-ascorbic acid and potassium chloride (KCl) were purchased from Sigma (St. Louis, MO). Giemsa stain was purchased from Biomedical Specialties Inc. (Santa Monica, CA). Crystal violet, methanol and acetone were purchased from J.T. Baker (Phillipsburg, NJ). Dulbecco's minimal essential medium and Ham's F-12 medium (d-MEM/F-12) were purchased from Mediatech Inc. (Herndon, VA). Cosmic calf serum (CCS) was purchased from Hyclone, (Logan, UT). Gurr's
Cytotoxicity and clastogenicity of lead chromate particles
The aim was to determine if lead chromate induces DSBs and to determine the initial cellular response to that damage. Thus, the intent was to select concentrations of lead chromate that induced DNA damage, but that were not completely cytotoxic. We selected concentrations over a range of cytotoxicity from low to high toxicity in WTHBF-6 cells (Table 1). Concentrations of 0.1, 0.5, 1 and 5 μg/cm2 induced 85, 60, 36 and 3% relative survival, respectively. This cytotoxicity assay measures the
Discussion
Particulate chromate compounds target fibroblasts and epithelial cells at bronchial bifurcation sites in human lungs [7], [25]. These particulates are potent carcinogens because they persist in the lungs for extended periods of time and induce genotoxic damage [26], [27], [28], [29]. However, the full spectrum of damage induced by these compounds has not been determined. Specifically, the ability of particulate chromates to induce DNA DSBs had not been investigated. DSBs are one of the most
Acknowledgements
We thank Shawn Holt and Lynne Elmore for their expertise and assistance in creating the hTERT immortalized cell line; Dan Swett for technical assistance with the apoptosis assay; Jonathan Moreland for technical assistance with the cell cycle arrest assay; and Geron Corporation for the use of the hTERT materials. This work was supported by NIEHS grant ES10838 (J.P.W.), the Maine Center for Toxicology and Environmental Health at the University of Southern Maine, NIEHS grant ES013301 (B.X), NIOSH
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2022, Toxicology and Applied PharmacologyCitation Excerpt :Aromatic amines and hexavalent chromium are found in similar exposure settings, for example both chemicals are found in paints, pigments, dye, rubber industry, and tobacco smoke (ATSDR (Agency for Toxic Substances and Disease Registry), 2016; ATSDR (Agency for Toxic Substances and Disease Registry), 2012a; ATSDR (Agency for Toxic Substances and Disease Registry), 2012b; ATSDR (Agency for Toxic Substances and Disease Registry), 2005; Sinha et al., 2000; Zheng and Lee, 2009). Hexavalent Chromium (Cr[VI]) is a known human lung, nasal, and esophageal carcinogen found in both occupational settings and as an environmental contaminant (Wise Sr et al., 2002; Wise et al., 2017; Xie et al., 2007; Xie et al., 2005; Speer and Wise Sr., 2018; Wise and Wise Sr., 2018). Cr(VI) is also a well-established genotoxic agent and is used for studying lung cancer and genotoxicity in human lung cell lines (Speer and Wise Sr., 2018).