* Intramural Microarray Center,
Laboratory of Molecular Toxicology, and
Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina 27709
Received April 11, 2002; accepted June 3, 2002
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ABSTRACT |
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Key Words: arsenic; keratinocytes; microarray; toxicity.
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INTRODUCTION |
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Although a number of arsenic-regulated genes and processes have been described (Rossman et al., 2001), many of the molecular events that occur following exposure to inorganic AsIII remain unknown. In addition, many studies examining the effects of arsenic are limited or may lack physiological relevance because they have been performed using high concentrations of AsIII. Many studies also have used established epithelial cell lines, nontarget cell types, or nonuniversally accepted animal models. Animals appear to be more resistant to the effects of arsenicals than humans, as evidenced by numerous studies exploring the effects of arsenicals in animals, including rodent (Kroes et al., 1974
), monkey (Thorgeirsson et al., 1994
), and dog (Byron et al., 1967
). The lack of a universally accepted animal model for inorganic arsenic carcinogenesis necessitates the use of representative normal human tissue such as normal human epidermal keratinocytes (NHEK) to investigate mechanism(s) of arsenic action. Normal human keratinocytes represent a primary in vivo target of arsenic, and thus provide a relevant and reasonable in vitro model in which to study the effects of arsenic on skin.
To better understand the gene expression response in NHEK, following AsIII exposure, we generated the transcriptional profile of NHEK following short-term, nontoxic AsIII exposure at multiple dose and time points. Two-fluor cDNA microarray analysis provided new insight into the genes and biological processes involved in the arsenic response in skin, and demonstrated the complexity of the AsIII transcriptional profile in NHEK. The importance of this study is two-fold. First, it demonstrates effects on global gene expression induced by AsIII that may contribute to carcinogenicity and/or toxicity. Secondly, the study was conducted using nontransformed, target-relevant, primary normal human keratinocytes. It is anticipated that this data will contribute to a more complete understanding of the toxic and carcinogenic mechanism(s) of action of AsIII in skin.
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MATERIALS AND METHODS |
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Cytotoxicity determination.
Neutral red dye, which is retained by viable cells, was used to investigate the viability status of NHEK as a result of AsIII treatment. NHEK were seeded into 96-well plates (2500 cells/well) and grown to approximately 60% confluence, at which time the media was replaced with (KBM) supplemented with AsIII (Sigma). Viability was assessed 24 h later by incubation in the presence of neutral red dye (50 µg/ml) for 3 h at 37°C. Media containing dye was removed and the cells were fixed in formaldehyde/CaCl2. Dye taken up by viable cells was extracted with ethanol/acetic acid prior to absorbency determination at 570 nm, using a microplate reader (Dynex Technologies, Inc., Chantilly, VA). Determination of cytotoxicity at the 24-h time point was done to define the toxicity status of the cells at the maximum treatment time. The 24-h point was selected because, at the density seeded, cells reached confluence by 48 or 72 h. To avoid confounding effects on gene expression that are associated with contact inhibition, NHEK were treated with AsIII at 5070% confluency. Measurements were made in triplicate wells and averaged for each concentration of AsIII.
Chip manufacturing and microarray analysis.
Sequence-verified human cDNA clones (Research Genetics, Huntsville, AL), termed the NIEHS human ToxChip, containing 1906 genes selected to represent major biochemical and toxicological processes, were used for this study. For a comprehensive list of all the clones on this chip, see dir.niehs.nih.gov/microarray/chips.htm and Nuwaysir et al., 1999. Methods for chip manufacturing were adapted from Duggan et al. (1999) and are also available at dir.niehs.nih.gov/microarray/methods.htm. Efforts to re-sequence clones as part of the validation process are an ongoing activity in the NIEHS microarray center. Updated gene annotations are made to the website listed above. Total RNA corresponding to a pool of 6 biological replicate experiments was isolated using QIAGEN RNeasy kits (Qiagen, Valencia, CA). cDNA targets were prepared from 37.575 mg of total RNA by oligo dT-primed polymerization using SuperScript II reverse transcriptase (Life Technologies, Gaithersburg, MD). Reverse transcription and labeling with the fluorescent dyes Cy3 or Cy5 (Amersham Pharmacia, Piscataway, NJ) was performed according to protocols available at dir.niehs. nih.gov/microarray/methods.htm.
Fluorescent intensities of the printed DNA targets were measured using the Axon 4000 scanner (Axon, Foster City, CA) and Array Suite (Scanalytics, Fairfax, VA) software was used to perform data acquisition and image analysis. Images corresponding to Cy3 and Cy5 fluorescent dyes were analyzed as previously described in Chen et al. (1997), using the ArraySuite v2.0 extensions of the IPLab image-processing software package (Scanalytics). This program allows location of targets on the array, measures local background for each target, and subtracts it from the target intensity value, and it identifies differentially expressed genes using a probability-based method.
Intensity values corresponding to each gene on the cDNA microarray chips from the Cy3 and Cy5 channels were represented as a ratio of AsIII-exposed to time matched-control cells. For each individual chip, genes altered in a statistically significant manner at the 95% confidence level were determined (Chen et al., 1997). Replicate intensity values for each gene were documented from four independent hybridizations for each time and dose. A dye reversal procedure was employed, where two intensity values for a given gene on the chip were generated from instances where a sample was tagged with Cy3, and two more values from when the same sample was tagged with Cy5 to ensure minimization of error due to fluor-associated bias. Genes that indicated fluor bias or high variation were not considered for further analysis. Genes, significantly detected in three or four out of four arrays, were identified based upon a binomial distribution using MicroArray Project System (MAPS) (p
0.00048) (Bushel et al., 2001
). GeneSpring software (Silicon Genetics, Redwood City, CA) was used to perform the self-organizing maps (SOM) procedure on a set of genes constituting the union of all genes altered in a statistically significant manner from every dose and time point.
Thymidine incorporation assay.
Keratinocytes were seeded into 24-well tissue culture plates (4000 cells/well) in KBM+ and grown to 5060% confluence. Media were switched to fresh (KBM) for AsIII treatment (triplicate wells for each concentration of AsIII). Forty-six-h after addition of AsIII, cells were labeled for 2 h with 2 µCi/ml [3H]thymidine (Amersham Pharmacia) and washed several times with ice-cold Hanks balanced salt solution. Several washes were performed with 10% trichloroacetic acid (Mallinckrodt, Phillipsburg, NJ) and radioactivity was eluted using 0.3 N NaOH. Thymidine incorporation into DNA was determined by scintillation counting. All measurements were performed in triplicate and repeated at least twice.
Quantification of mitotic index.
Keratinocytes were treated as previously described. Following a 6-, 8-, or 24-h incubation with different concentrations of AsIII, keratinocytes were fixed on 12-well plates by the gentle addition of cold methanol. After 10 min, methanol was removed and the plates were air-dried and stored at 4°C until staining with 0.1 µg/ml 4,6-diamidino-2-phenylindole (DAPI). DAPI-stained cells were examined by fluorescence microscopy. The percentage of mitotic cells (the mitotic index) was determined from counts of a minimum of 2000 cells relative to controls. All measurements were performed in replicates of 6 and repeated at least two times.
Northern blots.
Total RNA was isolated from NHEKs treated with 0.005, 0.5, 5 µM AsIII for 24 h as previously described, using the RNeasy midi-prep system. An additional sample of RNA was derived from NHEK pretreated for 2 h with 5 mM N-acetylcysteine (NAC) (Sigma, St. Louis, MO) and exposed to 5 µM AsIII. Twenty-µg aliquots of total RNA from these samples were fractionated by formaldehyde/agarose gel electrophoresis. Following transfer to nylon membranes, membrane-bound RNA was hybridized to 32P-labeled probes for human Protein kinase C delta, Fibroblast growth-factor receptor 4, MSH-5, cdc25b, p53, serine/threonine kinase 25,or NAD(P)H quinone oxidoreductase, all prepared using random-primers methodology (Invitrogen, Carlsbad, CA). 32P-labeled probes were derived from the same cDNA template as that spotted on the glass cDNA microarray. Individual membranes were exposed to phosphorimaging screens, and band intensity was quantified using the NIH Image software package for the purpose of graphing and comparing to microarray data.
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RESULTS |
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To further confirm the effect of AsIII on proliferation, mitotic indices were determined in NHEK following AsIII exposure. The number of cells undergoing mitosis, measured by DAPI staining, was increased compared to control (0 µM), following exposure to 0.005 and 0.5 µM AsIII. A decrease in the mitotic index at 5 µM AsIII at 6 and 8 h of exposure (Fig. 1C) (also at 2 and 4 h, data not shown) suggested a G2/M arrest. However, 24 h after exposure to 5 µM AsIII, cells undergoing mitosis were present in appreciably higher numbers relative to time-matched controls.
Taken together, the NHEK viability and proliferation data suggest that there were no apparent confounding effects on cellular growth status (e.g., apoptosis, contact inhibition) that would complicate gene expression analysis and interpretation at the AsIII doses and time points tested.
Arsenic-Mediated Gene Expression Changes
NHEK exposed to AsIII (0.0055 µM) were collected for gene expression array measurements. Self-organizing maps (SOM), representing gene clusters that responded in time- and dose-dependent manner, were used to explore patterns of AsIII-modulated gene expression. A dose-time-response curve, generated from reproducibly changed gene expression indicated that there was an increase in detectable gene expression changes as the time and dose of AsIII exposure increased (Fig. 2). Specific gene expression changes (Table 1) indicated a majority of changes might be annotated to functional categories related to proliferation, DNA repair, and oxidative stress.
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Finally, in the present study, gene expression relevant to proliferative signal transduction was altered in response to AsIII treatment (Table 1, Fig. 3C). These changes include the downregulation of several protein tyrosine phosphatases and differentiation genes (Fig. 3C
), as well as an increase in protein kinase C delta expression and several growth factors (Fig. 3C
). These changes may contribute to the increased proliferation in NHEK, as measured by elevated [3H]thymidine incorporation after 48 h of AsIII exposure (Fig. 1B
).
Gene Expression Validation
We validated, via Northern blotting, the expression profile of 7 genes that are associated with oxidative stress, DNA repair, and proliferation from samples exposed to 0.0055 µM at 24 h. Comparison of data from cDNA microarray and Northern blotting demonstrated a high level of correlation between the two methods (Fig. 4), where the induction or repression of each gene was confirmed across multiple samples. Microarray measurements are typically only semi-quantitative, with compression of values occurring at high fold changes. The Northern-blot measurements are likely to provide better quantitation, but generally the quantitative measurements with the two approaches shows a high degree of correlation (Amundson et al., 1999
).
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DISCUSSION |
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The induction of oxidative stress-related genes by AsIII in this study suggests that oxidative stress generation occurs in AsIII-exposed NHEKs. Several in vivo and in vitro studies indicate that arsenic produces reactive oxygen species (ROS) such as the dimethylarsinic peroxy radical via dimethylarsine, a metabolite of dimethylarsinic acid (DMA), which in turn is the dimethylated metabolite of AsIII (Yamanaka and Okada, 1994). While high levels of ROS might be predictive of increased tumor formation in rodents, carcinogenesis has not been demonstrated in vivo with arsenic exposure solely (Kroes et al., 1974
). Therefore, generation of ROS, while having a cooperative role, is not likely the primary, or the sole, mechanism of arsenic carcinogenicity. Our gene expression data suggest that DNAprotein damage resulting from AsIII induced oxidative stress, direct physical interaction of AsIII with cellular enzymes, or mimicking of phosphate by AsV does not occur at lower doses. At higher doses, where increased DNA damage would be predicted to occur, SOD1 and p21 were upregulated.
The management of oxidative stress is the job of numerous cellular enzymes. One of these enzymes, NAD(P)H quinone oxidoreductase (NQO1), is a flavoprotein that catalyzes the reduction of quinones, quinone imines, and azo-dyes, thereby protecting cells against free radical and ROS-mediated mutagenicity and carcinogenicity (Jaiswal, 1994). Elevated NQO1 activity and gene expression also were observed in both preneoplastic tissues and established tumors (Cresteil and Jaiswal, 1991
). In our study, NQO1 expression was robustly elevated following exposure of NHEK to AsIII. The significance of this finding is increased by a study demonstrating that arsenic can enhance TCDD-inducible NQO1 gene expression, indicating that co-exposure to arsenic and AHR ligands (which are prevalent in the environment) may disrupt the regulation of phase I and phase II detoxification genes (Maier et al., 2000
). The latter may lead to imbalances in gene expression that have important consequences in skin toxicity and cancer.
Because glutathione is central in the arsenic detoxification scheme, via its involvement in methylation, scavenging of free radicals, and direct binding to AsIII, we hypothesized that elevated levels of GSH would attenuate changes in gene expression mediated by AsIII in NHEK, an effect that has been largely unexplored in this model system. NAC, which was used to elevate intracellular GSH prior to AsIII exposure and subsequent gene expression analysis, attenuated many of the effects of AsIII on gene expression. Interestingly, the attenuation was not specific to oxidant stress-related genes but also genes associated with DNA repair and proliferation, suggesting a relationship between oxidant stress and DNA repair and proliferation in response to AsIII exposure.
In addition, there was evidence of suppression of DNA repair genes in response to AsIII treatment. The exact mechanism involved in the downregulation of DNA repair gene expression is currently unknown; however, p53 suppression might play a role in this process (Smith et al., 2000). p53 controls several aspects of the cell cycle that allow for DNA repair, and it also has been implicated directly in the regulation of DNA repair genes (Zhu et al., 2000
). A previous report showed a decrease in p53 protein levels in immortalized keratinocytes (HaCaT) in response to AsIII exposure (Hamadeh et al., 1999
) and hypermethylation of a p53 promoter region was observed in A549 cells exposed to AsIII (Mass and Wang, 1997
). Arsenic-related basal cell carcinomas were found to express lower p53 staining compared to control patients exhibiting sporadic cell carcinomas (Boonchai et al., 2000
). These data indicate that p53 downregulation occurs at both the transcription and protein level following arsenic exposure. p53 transcript suppression may also have consequences on cell cycle progression and G2/M checkpoint function that might lead to altered damage repair.
Repression of DNA repair enzyme gene expression is one of the novel findings in this report. Although DNA repair alterations have been cited in conjunction with arsenite exposure (Hartwig et al., 1997; Rossman et al., 2001
), the mechanism of repair inhibition has been attributed to the physical affinity/binding of AsIII to vicinal dithiol groups inherent to the repair enzymes. However, in this report we show that AsIII works at the transcriptional level to repress a suite of DNA repair enzyme genes (Table 1). This suggests that arsenic may work at more than one level to deregulate DNA repair that, in combination with other events, contributes to toxicity or cancer. The downregulation of DNA (cytosine-5-)-methyltransferase 1 occurred in a dose- and time-dependent fashion following AsIII treatment (Fig. 3B
), and was of particular interest because altered biological (DNA and protein) methylation is proposed to contribute to arsenic carcinogenicity. While a decrease in methyltransferase 1 message was evident at higher doses (2.5 and 5 µM), a slight, yet statistically significant increase was detected at lower doses (0.005 and 0.5 µM). The role DNA methyltransferase plays in arsenic-induced toxicity and cancer remains unclear; however, the linearity of the dose response at 8 and 24 h (0.96 and 0.98 respectively) (Fig. 3B
) suggests a strong relationship between AsIII treatment and repression of DNA methyltransferase. This data is consistent with the dose- and time-dependent downregulation of HMT1 (hnRNP methyltransferase, S. cerevisiae)-like 2, which is responsible for the methylation of arginine residues on proteins, and may help to explain the reduction in monomethylarginine levels that occur following arsenic treatment of 3T3 fibroblasts (Wang et al., 1992
). Data in this report reveal that AsIII (at low doses) increases cellular proliferation in NHEK as measured by an increase in cellular mitoses and [3H]thymidine incorporation. These observations confirm the effects of arsenic on cell proliferation observed in rodent cells and human fibroblasts and keratinocytes (Trouba et al., 1999
; Vega et al., 2001
). Our data suggest that the enhanced proliferation associated with AsIII exposure may be mediated in part by multiple mechanisms, including alterations in cell cycle regulators such as Cyclin G1, whose upregulation is associated with proliferating tissue or rapidly dividing cells (Zhu et al., 1997
), the downregulation of negative regulators of proliferation, such as protein tyrosine phosphatase signaling (Afshari and Barrett, 1994
), TGF-ß (Gniadecki, 1998
) or TNF-
signaling pathways (Detmar and Orfanos, 1990
).
Other events that may contribute to the enhancement of cell proliferation following arsenic exposure include the activation/dysregulation of mitogen and stress-activated protein (MAP) kinase pathways (Trouba et al., 2000). Data demonstrate that PKC delta, a gene upregulated by AsIII in NHEK in our study, plays an important role in AsIII-induced AP-1 activation in JB6 cells through different MAP kinase pathways (e.g., ERKs, JNKs, and p38 kinases) (Huang et al., 2001
). The latter, along with data in the present report, suggest that long-term alterations in PKC delta/Ca2+-related signaling may contribute to abnormal cell growth and differentiation in the skin, the result of AsIII exposure. Indeed, this is consistent with our observations of decreased expression of differentiation-associated genes (Seewaldt et al., 1997
) such as retinoic acid-responsive protein, retinoic acid-induced RIG precursor, and retinoic acid receptor
1 in AsIII-treated cells. The data in the current report suggests another novel mechanism by which arsenic may influence MAP kinase- and stress-related signal transduction pathways. Ste20-homologous proteins (e.g., STK25) are implicated in mammalian MAP kinase pathways as important transducers of oxidant-mediated signals from the p21 family of GTPases (Brown and Kitchin, 1996
), and are activated by cellular stress (Pombo et al., 1997
). We found that NHEK STK25 expression is elevated following AsIII exposure. At present, the long-term consequences of AsIII-induced STK25 gene expression in NHEK is unknown. However, our data implicate STK25 in the transduction of AsIII-mediated mitogenic and stress signals; signals that may contribute to skin carcinogenesis and toxicity
To infer the mechanism of arsenic carcinogenicity from a relatively short duration of exposure has limitations. Long-term exposure of NHEK to AsIII is challenging due to the onset of differentiation, which makes it difficult to monitor long-term genotypic and phenotypic events that are consequences associated with alterations in gene expression. The gene expression alterations induced by AsIII in NHEKs can cooperatively lead to transforming events pertinent to carcinogenesis. This hypothesis is supported by the increase in several transformation or tumor associated biomarkers in AsIII-treated NHEKs. For example, gro-1 oncogene, v-yes oncogene, tumor-associated signal transducer 2, tumor associated antigen L6, capping protein, and MM-1 were observed to be induced in a dose- and time-dependent manner. The downregulation of cellular adhesion-related gene products, including laminin ß 2, metalloproteinase 1 and integrin ß 4, was also observed. Dysregulation of cellular adhesion has been documented in several cancers (Ota et al., 2001). Transcripts for Gro 1, tumor-associated antigen L6, and capping protein are abundantly expressed in both tumorigenic (Pam 212) and metastatic (Pam LY and LU) cell lines derived from BALB/c keratinocytes, as compared to primary keratinocytes (Dong et al., 2001
). A reduction in laminin ß 2, DNA polymerase delta, and matrix metalloproteinase 1 transcript levels also were found in Pam 212 and Pam LY/LU cell lines when compared to the primary keratinocytes, a phenomenon that we also observed as a result of AsIII treatment. The relevance of these alterations to the carcinogenic action of AsIII is supported by the fact that squamous cell carcinoma is the same endpoint observed in human populations exposed to relatively high levels of arsenic in drinking water (Boonchai et al., 2000
; Leonard and Lauwerys, 1980
). Evidence that AsIII modulates the expression of these genes in a similar fashion suggests that the consequences of these mechanistic alterations are associated with similar transformation events as reported by Dong and coworkers (2001).
The main observations of this study, the simultaneous increase in gene expression indicative of oxidative stress, decrease in transcript levels of DNA repair enzymes, and increase in cell proliferation gene expression, suggest an integrative mode of action of arsenic. The novelty of the results presented here is that AsIII concomitantly modulates gene expression associated with increased proliferation, decreased DNA repair, and increased oxidative stress in nontransformed NHEK.
The data presented in the current report gives rise to multiple testable hypotheses regarding gene expression that may contribute to or play a role in arsenic toxicity and carcinogenesis. The use of specific pharmacological inhibitors and/or gene knockout models will provide invaluable tools for assessing the relationship between arsenic-induced gene expression, toxicity, and cancer. Further studies will also aim at distinguishing arsenic-specific effects from responses to other metals. These studies will, ultimately, help to integrate specific gene expression alterations with phenotypic endpoints germane to the carcinogenicity potential of arsenic and may lead to better biomarkers of exposure/effects.
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ACKNOWLEDGMENTS |
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NOTES |
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2 Present address: Amgen, Inc., One Amgen Center Drive, Thousand Oaks, CA 91320-1799.
3 To whom correspondence should be addressed at Amgen, Inc., One Amgen Center Drive, Mailstop 5-1-A, Thousand Oaks, CA 91320-1799. E-mail: cafshari{at}amgen.com.
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