Cytogenomics of hexavalent chromium (Cr⁶⁺) exposed cells: A comprehensive review

In vitro studies

A549 cells, 300 μM potassium dichromate, 2 h exposure: In the first study on Cr⁶⁺ induced cellular gene expression modulation, Ye and Shi82 examined genomics in human lung type II epithelial A549 cell using microarray of 2400 genes and potassium dichromate as a source of Cr⁶⁺. They investigated the molecular basis of Cr⁶⁺ provoked ROS generation and the resultant oxidative stress, and found a significant dysregulation of 220 genes that were part of the pathways of oxidative stress, Ca²⁺ mobilization, energy metabolism, protein synthesis, cell cycle regulation, apoptosis, and carcinogenesis.

In stress response pathway (Table I), an upregulated transcription was seen in Cu / Zn superoxide dismutase (SOD), glutathione peroxidase, MT-IIA, MTF-1 (metal-regulatory transcription factor), p53, heat shock proteins (HSP60, HSP70, HSP75), and activating transcription factor-3 (ATF-3). The oxidative stress responsive proteins protected the correct conformation of newly formed proteins; their main function was to protect cells from ROS / oxidative stress and preserve the vitality of cells.

Table I Dysregulated genes of stress response pathway in Cr⁶⁺ exposed cells

In apoptosis pathway Ye and Shi82 reported upregulation of only the hSIAH1 (Siah E3 ubiquitin protein ligase 1) gene (Table II). Functionally, this apoptotic gene facilitated to label the protein for proteasomal degradation and the programmed cell death through induction of p53 signalling during Cr⁶⁺ induced stress.

Table II Dysregulated genes of apoptosis pathway in Cr⁶⁺ exposed cells

In cell cycle regulatory pathway (Table III), the upregulated genes transcribed protein products important for cell survival, cell polarization, cell growth and differentiation, G1 cell cycle arrest, and tumour suppressor function. By underexpressing the respective proteins, the downregulated genes dysregulated cell cycle control via slowdown of cyclin dependent kinase activation causing cell cycle arrest, and challenging epithelial cell integrity for apoptosis.

Table III Dysregulated genes of cell cycle pathway in Cr⁶⁺ exposed cells

In DNA repair and metabolism pathway (Table IV), only three genes were found to be dysregulated. The upregulated gene encoded photolyase that was involved in DNA repair. The downregulated CK2 (casein kinase 2) and cell division cycle (CDC)47 decreased the function of serine / threonine kinase and also required for DNA replication. The binding of CDK4 (cell cycle dependent kinase), CDK5 with CK2/CDC45 encoded a protein to regulate the signalling mechanisms in cell proliferation and growth.

Table IV Dysregulated genes of DNA repair, metabolism pathway in Cr⁶⁺ exposed cells

Among oncogenes (Table V), the upregulation of intracellular kinase, G-protein, Src, and MAPK showed their involvement in cell proliferation and differentiation. MAPK signalling regulated the proto-oncogene; activation of oncogenes by Cr⁶⁺ would support carcinogenesis.

Table V Dysregulated genes of oncogene pathway in Cr⁶⁺ exposed cells

In pathways of cellular energy metabolism and biosynthesis, a great majority of genes were found to be dysregulated. In energy metabolism pathway (Table VI), the upregulated genes would facilitate proton pumps for ATP synthesis, osmoregulation and active transport of molecules across cell membrane, nerve/muscle electrical excitability, and bioenergetics regulation. In biosynthesis pathway (Table VII), the upregulated genes would aid proteasomal degradation, biosynthesis dependent programmed cell function, and cell matrix homoeostasis.

Table VI Dysregulation genes of energy metabolism pathway in Cr⁶⁺ exposed cells

Table VII Dysregulated genes of biosynthesis pathway in Cr⁶⁺ exposed cells

In summary, these studies revealed that 300μM Cr⁶⁺ influenced the global cytogenomics and in particular regulated the stress, DNA repair, signalling system (Ca⁺⁺, G-protein), Src kinase, MAPK and CDK, oncogenes, bioenergetics, and cell cycle. Specificity of oncogene expression to Cr⁶⁺ exposure may be predisposed by the adenocarcinomas cell line based test system.

BEAS-2B cells, 10 μM sodium dichromate, 4 h exposure: Andrew et al83 investigated the cellular gene expression profile of human bronchial epithelial BEAS-2B cells using a limited microarray of 1200 genes. They examined changes in gene expression after acute exposure to 10 μM dose of the toxicant. Cr⁶⁺ was found to modulate a cluster of 44 genes (Tables I-V).

In stress pathway (Table I), none of the genes showed upregulation; only heat shock protein genes (hsp40, hsp60, hsp90) recorded downregulation. The molecular basis seemed to be the protein transport and chaperon like functions. Their downregulation indicated irregular trafficking of deformed proteins and the risk of apoptosis.

Downregulation of anti-apoptotic proteins (Table II), DIF-2, was also in line with above observation. Similarly, downregulation of anti-death protein ephrin type-A receptor 2 ECK (tyrosine kinase receptor) demonstrated predisposition to apoptosis.

The trend of apoptosis was also noticed by the downregulation of genes in cell cycle pathway (Table III). ERF1 (EGF response factor-1) encoded the transcriptional activator C2H2-type zinc-finger nuclear protein that was functionally tumour suppressor and important for cell growth and differentiation. Cyclin K protein regulated cyclin dependent kinase and RNA polymerase. LUCA 2 (lysosomal hyaluromidase 2) transcribed the hyaluronidase, a cell surface protein, associated with tumour suppression function and involved in cell growth, differentiation and migration. For cell cycle regulation in Cr⁶⁺ exposed cells, TNK1 (tysosine kinase 1) encoded the non-receptor type of tyrosine kinase that phosphorylated proteins downstream of Src kinase in intracellular signalling (Table III). YWHA1 (tyrozine 3-monoxygenase / tryptophan 5-monooxygenase activation protein) encoded the signal transducers; FGFR1 (fibroblast growth factor receptor 1) encoded the fibroblast growth factor receptor type of protein to modulate growth and differentiation. Their underexpression seemed to be in consonance with a shift of cell cycle into apoptosis mode or the dedifferentiation mode.

Cr⁶⁺ induced predisposition of cells to apoptosis or transformation in tumour phenotype was perceptible by the sluggish DNA repair and metabolism (Table IV) through downregulation of genes involved in DNA template elongation, DNA binding and replication, G₀ to G₁/S phase of cell cycle, the cell growth, senescence, and differentiation. In DNA repair group of genes, only HATB2 (histone acetyl-transferase B subunit) (aka RBBP7/ RBBP46) was upregulated; it was functionally involved, as histone acetyltransferase, to import the acetylated histone into nucleus and deposit on up-coming DNA chains for chromatin assembly. Its overexpression would support DNA synthesis and possibly also the cell growth and differentiation.

Among oncogenes (Table V), c-myc (myelocytomatosis), FRA1 (Fos like antigen 1), PKB/akt (murine thymone viral ancogene), PP-1a (protein phosphatise 1) and TNK1 (tyrosine kinase 1) were found to be downregulated. However, MAPKAP kinase was found to be upregulated. This protein helped in protein transport; its upregulation favoured transition of cells into S phase. The underexpression of c-myc (the transcription factor) seemed to down play the cell cycle progression and thus favoured apoptosis. Its erratic transcription is described in haematopoietic tumourigenesis9091. Gene FRA1 encoded the fos-like antigen. This protein zipped up with JUN proteins to form AP-1 transcription factor for regulation of cell proliferation, differentiation, and transformation. Downregulated PKB (AKT1) was a Ser/Thr protein kinase. Usually inactive in G₀ phase of cell cycle; it is activated by (platelet derived growth factor) PDGF through phosphatidylinositol 3-kinase and in this form it negatively regulated apoptosis by phosphorylation of the participating proteins. The downregulated TNK1 catalyzed phosphorylation of proteins downstream the membrane bound kinase. It is involved in negative regulation of cell growth and has tumour suppressor function.

Cr⁶⁺ exposure influenced the genes84 controlling cytoskeleton and cell junction boxes through adhesion/extracellular matrix. All the examined genes were downregulated. COLα2 (collagen α2) encoded pro-collagen to maintain the matrix integrity. CTNNA1 (catenin alpha 1) encoded the cadherins that in association with actin participated in cell differentiation. ITGB4 (integrin beta 4) encoded integrins to form cell-matrix or cell-cell adhesions. These proteins regulated cell integrity and shape. In aberrant expression state, integrins are key players in invasive carcinomas. Gene ZYX (zinc binding protein) encoded zyxin (the zinc binding protein) that accumulated in phosphorylated form at the cell surface making focal adhesion sites along the actin cytoskeleton and thus participating in signal transduction mechanisms. UPAR is a urokinase type plasminogen activator which regulates migration and invasion of cells and CI-B18 gene participates in cell adhesion process. Low expression of this gene could dysregulate the adhesion-molecule dependent changes favouring cell transformation.

The examination of genes regulating energy metabolism (Table VI) revealed the increased expression of glucose transport gene GLUT1 in Cr⁶⁺ exposed cells; it appeared to support major transport of aldoses including pentoses. A1ATR encoded the serine protease inhibitor that controlled inflammation; its downregulation would support inflammation essential for Cr⁶⁺ toxicity. Commensurate with apoptosis, downregulation of C1-B18 indicated the loss of cell differentiation potential and bioenergetics to regulate S phase of cell cycle.

Cr⁶⁺ exposure was shown to result in downregulation of several transcription regulators and suppressors (Table VII). A tone-down in level of these proteins could affect the S phase of the cell cycle requiring biosynthesis of proteins and thus influence cell proliferation and differentiation. This study83 suggested again the role of test dose-linked threshold switch which is crucial for Cr⁶⁺ toxicity in cells. However, a paucity of information on the observed cytogenomics vis-a-vis toxicity was notable. Cr⁶⁺ induced changes in transcription regulators and suppressors seen in the test system that lacks functional p53 and Rb gene showed the responsiveness of collateral genes and not the target gene to the toxicant.

BEAS-2B cells, 0.25 & 0. 5 μM potassium dichromate, 4 week exposure: In BEAS-2B cells, Sun et al84 for the first time reported the altered gene expression profile with respect to Cr⁶⁺ toxicity. They established Cr⁶⁺ transformed cell lines using 0.25 or 0.5 μM dose and 4-week long exposure condition and investigated the gene expression profile; the experimental conditions simulated the occupational exposure and the associated lung cancer. Microarray analysis using 28,869 gene array revealed the differential expression of genes recording >1.5 fold change in >1200 genes. A major group of genes was found to be commonly dysregulated in 0.25 or 0. 5μM Cr⁶⁺ transformed cell; and functionally associated to biosynthesis, apoptosis, cell junction desmocollin 2 (DSC2), desmocollin 3 (DSC3), prolyl endopeptidase (Prep), extracellular matrix ADAM (ADAM metallopeptidase domain 12), TIMP metallopeptiase inhibitor (TIMP3), matrix metalloproteinase-2 (MMP2), cysteine-rich secretory protein (CRISPLD2), cell adhesion (CDH6), cllaudin 1 (CLDN1), L1 cell adhesion molecule (LICAM), latrophilin 2 (LPHN2), absent in melanoma (AIM1), integrin alpha (ITGA), collagen type 4 alpha 1 (COL4A1), (COL5A1,2) and biglycan (BGN), laminin beta 1, gamma 2 (LAMB1, LAMC2), fibrullin (FBLN1, FBLN2). Mostly upregulated genes associated with cell junction, cell adhesion and extracellular matrix in Cr⁶⁺ transformed cells. Four genes namely latrophilin 2 (LPHN2), absent in melanoma 1 (AIM1), magtrix metallo peptidase 2 (MMP2), and cysteine-rich secretory protein LCCL domain containing 2 (CRISPLD2) were downregulated. Downregulated genes signified acquisition of carcinoma phenotype in Cr⁶⁺ exposed cells. Upregulation of cyclins in Cr⁶⁺ transformed cells in contrast to downregulation of TGFβ signalling system seemed to support cell transformation. Downregulated genes were related to integrins, collagens, laminin, and fibrullin components. HHIP (hedgehog interacting porotein) gene which antagonized hedgehog signalling pathways was underexpressed in these transformed cells. Study revealed the dysregulation of genes associated with cell adhesion, integrin receptor, cell matrix component, metalloproteinase indicating the loss of cell contact inhibition process of normal cells which is a cardinal change in cell transformation.

BJ-hERT cells, 0-6, 9μM sodium chromate tetrahydrate, 4-24 h exposure: In this study, researchers developed a sub-population of telomerase-transfected human fibroblast (BJ-hTERT) called as B-5Cr which survived the lethal dose of Cr⁶⁺. These transgenic and apoptosis resistant cells had an increased growth potential85. A genotoxic dose of 0-6, 9 μM Cr⁶⁺ induced apoptosis in BJ-hERT cells but B-5Cr cells, that were resistant to apoptotic dose of 0-6, 9 μM Cr⁶⁺, ignored the apoptotic signal of secondary Cr⁶⁺ insult. In order to investigate the molecular basis of such a selective clonogenic cell survival response to Cr⁶⁺ , the analysis of gene expression was performed after exposure to secondary doses (0-6 and 9μM) of Cr⁶⁺ in B-5Cr and BJ-hTERT cells using human genome arrays. In apoptosis resistant transgenic B-5Cr cells, results revealed dysregulation of genes involved in cell cycle regulation, and apoptosis besides the dysregulation of genes in DNA repair irrespective of toxicant exposure period (Table IV). Cell cycle regulatory gene p21 (WAF1, i.e. cyclin-dependent kinase inhibitor-1) was upregulated in both cell populations. Those genes that up-regulated in BJ-hTERT but not in B-5Cr were the genes of pathways regulating growth arrest, DNA-damage-inducible and apoptosis involving GADD45, Caspase 3, MKP5, Myc, c-rel oncogene, VDAC (voltage dependent ion channel) gene. Genes that upregulated only in B-5Cr cells and not in BJ-hTERT cells included DNA repair endonuclease gene XPF (xeroderma pigmentosum group F), Collagenase type 4, Bcl-xL, and ligand induced apoptosis signalling receptor DR6. UV-RAG gene was upregulated in B-5Cr cells85. Pritchard et al (2005)85 revealed that most of the genes altered by Cr⁶⁺ in transgenic cells were involved in apoptosis, cell cycle and DNA repair pathways. The upregulation of GADD45, caspase 3 in BJ-hTERT cells and bcl-XL in B-5cr cells demonstrated the molecular basis for acquisition of apoptosis or clonogenic transformation potential following Cr⁶⁺ exposure in respective type of cells85. Upregulation of DNA repair gene in transgenic B-5cr cells indicated the possibility of clonogenic transformation of cells after DNA damage. The study85 suggested that cells with competent or upregulated DNA repair mechanism can withstand apoptosis stimulus. Together with ‘adequate-to-survive DNA repair’ status, clonal expansion of cells can occur; these cells can escape the cell death signal in presence of the upregulated DNA repair mechanism and can thus be selected clonogenically for tumourigenesis.

Human PBMC cells, 0.2-10 μM sodium dichromate, 18 h exposure: In a study to identify biomarkers for Cr⁶⁺ exposure using human PBMC, change in gene expression was probed as the early markers using human gene chip of 18,000 transcripts86. Researchers selected PBMC as the test system and test dose of 0.2 μM Cr⁶⁺ to analyze gene expression alteration in immunoregulatory pathways. The test dose was biologically effective as it decreased chemokine secretion; the dose of 0.2 μM was preferred over 10 μM dose that elicited an increase in chemokine secretion and was comparatively higher in view of the investigators. A cluster of 1,659 genes was found to be significantly altered by 0.2 μM Cr6. Genes pertaining to pathways of apoptosis, cell cycle regulation, and immune systems were found to be dysregulated (Tables II, III, VIII). Low expression of many immunoregulatory genes like CD163, MRC, CD93, CD14, SLAMF8, and STAB1 was noticeable (Table VIII). Several pro-apoptotic genes cell death protein gene R1PK1, apoptosis inducing serine/threonine protein kinase STK17B, death inducer obliterator DIDO1 were upregulated and the anti-apoptotic gene BIRC1 was downregulated indicating predisposition to apoptosis after Cr⁶⁺ exposure (Table II). The upregulation of cyclins (also seen in humans45), CDC proteins, cyclin dependent kinases, G2/M transition regulatory protein, and downregulation of growth arrest proteins supported observation of the role of thresholds in Cr⁶⁺ toxicity (Table III). The downregulation of genes encoding protein involved in vital functions like anti-inflammation process revealed the onset of inflammation after Cr⁶⁺ exposure, which strengthened the process of Cr⁶⁺ programmed cell death. The study86 indicated the modulation of many vital pathways by Cr⁶⁺ including - the immune response, intracellular signalling, apoptosis, along with the cellular metabolism, RNA transport and binding, biogenesis and organelle organization, and transition metal binding. Cr⁶⁺ induced changes seen in immunoregulatory gene expression may be non specific to toxicant as the test system was immune cell.

Table VIII Dysregulated genes of immune system pathway in Cr⁶⁺ exposed cells

Human fibroblast cells, 5μM Potassium dichromate, 16h exposure: Investigations, using human dermal fibroblast cells and human Ref-8V2 Sentrix bead chip array87, revealed the global gene expression profile of Cr⁶⁺ exposed cells. Relation between dermatitis and Cr⁶⁺ is well known but the gene expression study in this aspect was done for the first time by Sellamuthu et al87. Dermal fibroblasts were cultured and exposed to 5μM (LC₅₀ value) Cr⁶⁺ concentration. Total RNA was isolated for microarray study. Several apoptosis linked genes involved in p53 signalling pathways were found to be overexpressed, suggesting that apoptosis was p53 dependent. The pro-apoptotic genes were noted to be upregulated and the anti-apoptotic genes downregulated. A cluster of 1153 genes was found to be significantly altered (>1.8 fold). More than 200 dysregulated genes showed relation to the programmed cell death. Besides apoptosis, differentially expressed genes also belonged to cell death, cell viability and survival. This study emphasized on genes involved in apoptosis. Interestingly 300 genes participating in cancer pathway were found to be differentially expressed. Apart from cancer, potential of Cr⁶⁺ to impact pathways of inflammation, immuno-regulatory system, endocrine system, metabolism, and genetic disorder of skin was also noticeable; genes were found to be involved in cell function of growth and differentiation, signalling mechanism, transport, cell cycle regulation, protein metabolism, and cell development. These results validated the development of threshold for apoptosis after Cr⁶⁺ exposure in human fibroblasts.

In vivo study

Sprague-Dawley rats, 0.25 mg/kg b. wt., sodium dichromate for 3 consecutive days: Izzotti et al88 conducted a study on Sprague-Dawley rats after administering sodium dichromate; test dose was administered intratracheally and repeatedly. Gene expression profile was investigated in rat liver and lung using 216 gene nylon arrays. A cluster of 56 genes was found to be upregulated 3-fold in lung compared to liver (Tables II-VI); none of the examined genes was downregulated. Biological annotation analysis of the upregulated genes revealed their roles in pathways like stress response, DNA repair and metabolism, energy metabolism, biosynthesis, apoptosis, oncogene and cell cycle. In stress response genes (Table I), the observed upregulation in Cu/Zn SOD, HSP-70, and damaged protein degradation enzyme was similar to earlier observation82.

Amongst apoptosis related genes (Table II), overexpression of proteins catalyzing the pro-apoptotic activity was novel and included Bcl-XL, Bcl-2 associated death promoter, cyclins, CDC-like kinase, CDC phosphatase, cyclin dependent kinase regulators, microtubule constituents, Ser/Thr protein kinase specific to G1 to S phase transition of cell cycle. Proteins interacting with Rb gene product to allow mitosis, and protein kinase complex for progression of G1 phase of cell cycle were also overexpressed.

This study88 revealed upregulation of several genes encoding DNA metabolizing enzymes (Table IV) like DNase, topoisomerases, multifunctional DNA repair enzyme, DNA polymerase alpha/beta/delta1, telomerase associated protein. The changes seemed to be crucial for DNA repair process through endonucleolytic activity, changing topology of DNA, forming constituent of the ribonucleoprotein complex responsible for telomerase activity, and identification and repair of apurinic/apyrimidinic sites.

Cr⁶⁺ upregulated two oncogenes - ras and c-jun terminal kinase gene (Table V), which encoded cell transforming proteins. Their increased levels could help in growth of transformed cells. The upregulation of genes involved in energy metabolism was also noted (Table VI). Their protein products (namely flavin containing monooxygenases, epoxide hydrolases, Cytochrome b5 reductase, acyl-coenzyme A dehydrogenase, aldehyde dehydrogenase, thiosulphate sulphurtransferase) played critical role in influencing the metabolism of xenobiotics, fatty acid (long chain and very long chain), cholesterol, various aldehydes, lipid peroxides, corticosteroids, neurotransmitters, and sulphur containing proteins. In summary, this study revealed that Cr⁶⁺ administration caused selective changes locally at the site of administration albeit similar as seen in cultured cells82. Altered gene expression was seen in Cr⁶⁺ metabolism, stress response, DNA repair, signalling pathways, apoptosis and cell cycle regulation. The study, although limited, was contributory in understanding the mechanisms of Cr⁶⁺ toxicity and suggested an involvement of thresholds in Cr⁶⁺ toxicity.

B6C3F1 mouse, (0, 0.3, 4, 14, 60, 170 or 520 mg/l) sodium dichromate dehydrate for 7 & 90 days: Recently, Kopec et al89 conducted the study on B6C3F1 female mice to investigate the key events of Cr⁶⁺ induced tumour formation in vivo. Dose dependent gene expression profile was examined after 7 and 90 days of regular exposure to Cr⁶⁺ in drinking water. Mouse intestinal epithelial gene expression was investigated using mouse 4X44K whole genome oligonucleotide microarray containing 21307 genes. After seven days, the differentially expressed genes exhibited the comparable expression profiles at ≤14 or ≥60 mg/l dose. A cluster of 6562 unique differentially expressed genes was identified having >1.5 fold change at one or more doses in duodenum at 8^th day. Using the same data filtering criteria, cluster of 4448 unique differentially expressed genes in intestinal samples was noticed at 8^th day, and clusters of 4630 and 4845 were detected in duodenum and jejunum, respectively at day 91. In long term exposure study, the differentially expressed genes exhibited the dissimilar expression profile. Genes in duodenum and jejunum, responding to range of Cr⁶⁺ concentrations (0.3-150 mg/l) participated in functions, e.g. immunoregulation, oxidative stress, cell cycle, growth, proliferation, DNA damage / repair: only the selected intestinal genes that were differentially expressed following exposure to 0.3-520 mg/l sodium dichromate dehydrate have been listed in Tables I, III, V, VIII. Activation of oxidative stress responsive genes MT2, MTF-1, Gpx (glutathione peroxidase) and Sod was seen; Ye and Shi82 also observed changes in similar genes. In DNA repair pathway, base and nucleotide excision repair gene Apex1 (base & nucleotide excision repair gene) Mlh1, Msh2 (Mut S protein homology 2), and Msh6 (Mut S protein homology 6). A comparison of changes in gene expression after 7 or 90 days of exposure to toxicant revealed overlaps of gene expressions. Taken together, the study showed that oxidative stress and the cytotoxicity were the early effects of Cr⁶⁺ exposure and that the differentially expressed genes were associated with oxidative stress, cell cycle and immuno-regulation pathways.