Hexavalent Chromium-Induced Chromosome Instability Drives Permanent and Heritable Numerical and Structural Changes and a DNA Repair-Deficient Phenotype

Abstract

A key hypothesis for how hexavalent chromium [Cr(VI)] causes cancer is that it drives chromosome instability (CIN), which leads to neoplastic transformation. Studies show chronic Cr(VI) can affect DNA repair and induce centrosome amplification, which can lead to structural and numerical CIN. However, no studies have considered whether these outcomes are transient or permanent. In this study, we exposed human lung cells to particulate Cr(VI) for three sequential 24-hour periods, each separated by about a month. After each treatment, cells were seeded at colony-forming density, cloned, expanded, and retreated, creating three generations of clonal cell lines. Each generation of clones was tested for chromium sensitivity, chromosome complement, DNA repair capacity, centrosome amplification, and the ability to grow in soft agar. After the first treatment, Cr(VI)-treated clones exhibited a normal chromosome complement, but some clones showed a repair-deficient phenotype and amplified centrosomes. After the second exposure, more than half of the treated clones acquired an abnormal karyotype including numerical and structural alterations, with many exhibiting deficient DNA double-strand break repair and amplified centrosomes. The third treatment produced new abnormal clones, with previously abnormal clones acquiring additional abnormalities and most clones exhibiting repair deficiency. CIN, repair deficiency, and amplified centrosomes were all permanent and heritable phenotypes of repeated Cr(VI) exposure. These outcomes support the hypothesis that CIN is a key mechanism of Cr(VI)-induced carcinogenesis.Significance: Chromium, a major public health concern and human lung carcinogen, causes fundamental changes in chromosomes and DNA repair in human lung cells. Cancer Res; 78(15); 4203-14. ©2018 AACR.

Figure 1. Experimental Design for Clonal Expansion Study

This figure shows the experimental scheme for clonal expansion of Cr(VI)-treated cells. Cells were exposed to lead chromate for 24 h in three separate treatments. After each treatment, cells were seeded at colony forming density, cloned, expanded into cell lines and then retreated. Cell lines at each stage were evaluated for chromosomal changes, centrosome number, ability to repair DNA double strand breaks, and for growth in soft agar.

Figure 2. Repeated Exposure to Particulate Cr(VI) Induces Chromosome Instability

This figure shows the types of CIN observed in all metaphases examined as well as across generations. A) All CIN and all CIN across 3 generations. B) Structural CIN shown as the percent of structurally abnormal metaphases and total abnormal chromosomes in 100 cells accounting for metaphases with more than one damaged chromosome for all cells and across three generations. C) Numerical CIN in all cells and across 3 generations. D) Tetraploid and near tetraploid cells in all cells and across 3 generation. (1859 metaphases were analyzed for control clones and 1343 metaphases were analyzed for treated clones; 144, 431, and 1284 metaphases were analyzed in the G1, G2, and G3 generations in control cells, respectively. 160, 388, and 795 metaphases were analyzed in the G1, G2, and G3 generations in treated cells, respectively). *indicates statistically different from comparable control cells (p<0.05). E) Aneuploidy found in each treated clone. Analysis is based on 100 metaphases for each clone. WTHBF-6 shows the aneuploidy found in the parental cell line. C1-1 shows the highest level of aneuploidy found in the comparable control clones. Overall aneuploidy in treated clones was statistically higher than the control clones (p<0.05).

Figure 3. Chromosome Specific Effects Observed After Repeated Particulate Chromate Treatment

This figure shows the chromosome specific effects in all metaphases examined. A) Percent of metaphases with structural alterations to chromosomes. B) Percent of metaphases with missing chromosomes. C) Percent of metaphases with additional chromosomes.

Figure 4. Karyotype Pedigrees of Untreated and Treated Clones

This figure shows the karyotype designation for each of the control (A) and treated (B) clone families. All karyotypes were derived from at least 20 analyzed cells. Clone C1-1 and C51-1 had mixed populations of half normal and half abnormal karyotypes, the remaining 89 control clones were normal. Some treated clones had mixed cell populations, the number of cells in the brackets indicates the number of cells for each type of abnormality; a cp in front of the number means the karyotype is a composite of several karyotypic lines with related karyotypes but not all identical. Abnormal chromosomes are designated with an abbreviation (for example, der or t followed by the affected chromosome numbers (iso = isochromosome, t = balanced translocation, der = unbalanced translocation, add = additional material of unknown origin). Missing or additional chromosomes are indicated with a (-) or (+) sign. C) Representative karyotype of treated clones: T4-1 has derivative chromosome made up of chromosomes 1 and 9 and the complementary chromosome 9 is missing. T41-1; the derivative chromosome persists into the third generation; in addition, the third generation clone acquired an isochromosome 7. T1-1 has 75 chromosomes, 22 of which are structurally abnormal. All structurally abnormal chromosomes are indicated with arrows. Abnormal chromosomes include translocations, dicentric chromosomes, deletions and additions. There are also numerical defects; for example, there are 6 copies of chromosome 1 (4 normal and 2 abnormal), and there is only one copy of chromosomes 14 and 22. The analysis of clone T1-1 revealed every cell had a different chromosome complement and different structural abnormalities.

Figure 5. Cells Treated with Prolonged Particulate Cr(VI) Grow in Soft Agar

This figure shows the results of the soft agar assay. A) Images of the clones in agar. The first row shows a representative clone (T73-3) with the agar colonies stained in the dish. The second row of images show the cells under 10×, clone T41-1 did not form colonies in agar and only single cells are seen, clone T73-1 grew small colonies in agar, clone T23-2 grew large colonies in agar. B) Percent of all clones able to grow in agar. C) Percent of clones able to grow in agar by generation. *indicates statistically different from control clones (p<0.05).

Figure 6. Repeated Exposure to Particulate Cr(VI) Induces Centrosome Amplification, Spindle Assembly Checkpoint Bypass and DNA Repair Deficiency

These data show some of the potential underlying causes for CIN. Overall centrosome amplification in treated clones was statistically higher than the control clones (p<0.05). A) Percent of all clones with centrosome amplification. B) Percent of clones with centrosome amplification by generation. Analysis is based on 100 interphase cells for each clone. Experiments were done in triplicate. Overall SAC bypass in treated clones was statistically higher than the control clones (p<0.05). C) Percent of all clones with SAC bypass. D) Percent of clones with SAC bypass by generation. Analysis is based on 100 metaphases for each clone. E) Representative example of a control clone and a treated clone (background levels have been subtracted). F) Percent of clones with DNA repair deficiency. G) Percent of clones with DNA repair deficiency by generation. *indicates statistically different from control clones (p<0.05).