XRCC1 Protects against Particulate Chromate–Induced Chromosome Damage and Cytotoxicity in Chinese Hamster Ovary Cells

Abstract

Water-insoluble hexavalent chromium compounds are well-established human lung carcinogens. Lead chromate, a model insoluble Cr(VI) compound, induces DNA damage, chromosome aberrations, and dose-dependent cell death in human and Chinese hamster ovary (CHO) cells. The relationship between lead chromate-induced DNA damage and chromosome aberrations is unknown. Our study focus was on examining the role of XRCC1 in lead chromate-induced cytotoxicity and structural chromosomal aberrations in CHO cells. Three different cell lines were used: AA8 (parental), EM9 (XRCC1 mutant), and H9T3 (EM9 complemented with human XRCC1 gene). Cytotoxicity was significantly higher in EM9 cells when compared to AA8 and H9T3 cells, indicating that XRCC1 is important for protecting cells from lead chromate particles-induced cell death. The frequency of damaged metaphase cells was not affected by XRCC1 deficiency. However, the total amount of Cr(VI)-induced chromosome damage was exacerbated by XRCC1 deficiency, and the spectrum of damage changed dramatically. Chromatid and isochromatid lesions were the most prominent aberrations induced in all cell lines. XRCC1 was essential to reduce the formation of chromatid lesions but not for isochromatid lesions. In addition, XRCC1 deficiency resulted in a dramatic increase in the number of chromatid exchanges, indicating that XRCC1 is involved in protection from lead chromate-induced chromosome instability.

Homologous recombination repair protects against particulate chromate-induced chromosome instability in Chinese hamster cells

“…Generation of chromatid exchanges may lead to an increase in chromosome translocations, a hallmark of lung cancer [42][43][44]. Other published data suggest that Cr(VI)-induced DNA double strand breaks probably result from replication fork collapse during the repair of single strand breaks, crosslinks or adducts [26], consistent with our previous report showing that a deficiency in single strand breaks also leads to more complex chromosomal aberrations [33]. Any unrepaired double strand breaks then manifest as chromatid aberrations and exchanges in mitosis.…”

“…The relationship between dose and intracellular concentration was quantified using a separate regression analysis for each cell type. The resulting values and variance-covariance matrix were then used to estimate the differences among cell types at concentrations of 100, 500, 1000, and (for cytotoxicity only) 5000 [33]. All analyses were conducted using the SAS software package.…”

“…Determination of chromosomal damage was measured as previously described [32][33]. Briefly, logarithmically growing cells were treated with lead chromate for 24 h. Cells were then harvested, resuspended in hypotonic solution and fixed with Carnoy's fixative, dropped onto slides, stained with Giemsa, and cover slips were added.…”

“…Briefly, logarithmically growing cells were treated with lead chromate for 24 h. Cells were then harvested, resuspended in hypotonic solution and fixed with Carnoy's fixative, dropped onto slides, stained with Giemsa, and cover slips were added. Background damage levels were subtracted from each data point as previously described [32][33]. Chromosomes were analyzed using our published methods [8].…”

“…Analysis of the intracellular chromium ion levels was completed using an inductively coupled plasma-optical emission spectrometer (ICP-OES) as previously described [33]. Briefly, logarithmically growing cells were treated with lead chromate for 24 h. Cells were rinsed with PBS, followed by the addition of hypotonic and then 2% SDS.…”