Global alteration of gene expression in human keratinocytes by inorganic arsenic

Miguel A. Rea1,3, Jeff P. Gregg1, Qin Qin2, Marjorie A. Phillips2 and Robert H. Rice2,4

1 Department of Medical Pathology, University of California Davis Medical Center, 4645 Second Avenue, Research III Building, Rm 3300, Sacramento, CA 95817
2 Department of Environmental Toxicology, One Shields Avenue, University of California, Davis, Ca 95616-8588, USA

4 To whom correspondence should be addressed Email rhrice{at}ucdavis.edu


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Alteration of gene expression by inorganic arsenic has been studied in cultured human keratinocytes derived from normal epidermis, a premalignant lesion and a malignant tumor. The purpose was to find whether these cells displayed common alterations in gene expression that might elucidate the mechanism of arsenic action. Global analysis of ~12 000 genes by microarray showed that ~30% were expressed. Of these, transcription of a substantial fraction (up to 12%) was altered, nearly twice as many being suppressed as stimulated by 2-fold or more at 2 µM sodium arsenite or 6 µM arsenate, which did not affect cell growth. At 0.67 µM arsenite (50 p.p.b.), effects on transcription were less pronounced but clearly evident. Genes whose transcription was altered in common among all the treated keratinocytes included those induced by reactive oxygen, of which heme oxygenase-1 displayed the highest fold induction. Genes indicative of reactive oxygen generation were detected at the earliest time examined, raising the possibility this feature drives subsequent cellular responses. Unlike some agents that produced transient induction of heme oxygenase-1, arsenicals produced sustained induction. Comparison with other agents producing reactive oxygen in the cells, as reflected in heme oxygenase-1 induction, suggested cellular differentiation was suppressed by sustained but not transient generation of reactive oxygen. Sustained global changes in gene expression were seen in target cells treated chronically with inorganic arsenic at concentrations consumed by millions of humans in contaminated drinking water.

Abbreviations: hEP, normal human epidermal; HO1, heme oxygenase-1.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Inorganic arsenic is a known human carcinogen that targets the skin, bladder, lung and probably other sites (1). Most chronic exposure comes from drinking water, in some instances at high levels affecting large populations in underdeveloped regions of the world (2,3). Other sources of chronic exposure, including food dried using coal with high arsenic content (4), have also led to serious health problems. That the drinking water standard of 50 µg/l, in use until recently in the USA, was <10-fold below concentrations producing cancer and other major adverse health effects indicated a need for reducing the standard to provide greater protection of public health (5). A recent comprehensive analysis indicates the risk of cancer from exposure at low levels is even more serious than estimates leading the USEPA to propose a reduction in the standard to 10 µg/l (6).

Continuing uncertainty regarding the risk of exposure to inorganic arsenic at low levels derives from the inherent difficulty of epidemiological studies where the incidence of health effects due to exposure is low compared with the population background. Biochemical and cell biological approaches could help elucidate the mechanism by which arsenic produces cancer, and thereby help rationalize the shape of the dose–response curve at low concentration. Although experiments in this direction have produced a wealth of information about arsenic biotransformation and effects on a variety of cell types, a clear single mechanism of action has not emerged. Arsenic perturbs cells in numerous ways, including inducing chromosomal abnormalities, altering DNA repair or methylation patterns, increasing growth factor secretion and proliferation, inducing gene amplification, and producing oxidative stress (7). Suitable animal models for studying carcinogenesis of arsenic alone have proven elusive, indicating that it is not a strong tumor initiator, consistent with its ineffectiveness in giving point mutations (8). That it probably acts as a co-carcinogen, tumor promoter and/or tumor progressor is consistent with reports of mouse models for skin co-carcinogenicity with UV irradiation (9) and co-promotion with tetradecanoylphorbol acetate (10).

In view of the uncertainty about its mechanism of action, present work concentrates on finding global effects of inorganic arsenic in human keratinocytes, a target cell type. Human epidermal cells resemble their natural state quite well in culture (11), although under optimal growth conditions they display some hyperproliferative character (12). Rather than the high concentrations often used in cell cultures in earlier studies, lower levels relevant to understanding pathological effects in humans are of particular interest. Well below the 10 µM limit estimated to be relevant for human epidemiological studies (13), the highest concentrations employed in present work produced no inhibition of growth over extended exposure times while markedly suppressing the differentiation program (14). Moreover, as the degree of neoplasia of the actual target cells is not known, cell lines derived from a malignant tumor and a premalignant lesion were compared with normal epidermal cells. All showed evidence of the generation of reactive oxygen species, a phenomenon evident at the earliest time point examined and possibly mediating changes in gene expression evident with chronic treatment.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell culture
Normal human epidermal (hEp), premalignant SCC12F2 and malignant SCC9 keratinocytes (15) were cultured using a feeder layer of lethally irradiated mouse 3T3 cells in a 3:1 mixture of Dulbecco-Vogt Eagle's modified and Ham's F12 media containing 5% fetal bovine serum, 5 µg/ml transferrin, 5 µg/ml insulin, 0.18 mM adenine, 20 pM triiodothyronine, 0.4 µg/ml hydrocortisone and antibiotics (16). The hEp cultures were also supplemented with 10 ng/ml cholera toxin upon inoculation and with 10 ng/ml epidermal growth factor starting at the first medium change. Cells were treated with either sodium arsenite (As +3) or sodium arsenate (As +5) at the indicated concentrations. For array experiments, arsenic treatment was initiated 3 days before confluence unless otherwise indicated and continued until 7–10 days after confluence. In other work, short-term treatments were employed with a variety of agents as indicated. The medium was changed at 3-day intervals until harvesting, at which time cultures were washed with isotonic phosphate-buffered saline, and the RNA was isolated by the phenol–guanidine thiocyanate–chloroform method (17) using Trizol (Invitrogen, Carlsbed, CA).

Expression profiling
Double-stranded cDNA was first synthesized starting with 10 µg of total RNA using the SuperScript Choice system (Invitrogen, Carlsbed, CA) and employing an HPLC purified oligo (dT)24 primer containing a T7 promoter (Proligo, La Jolla, CA). After clean up by phenol–chloroform extraction and ethanol precipitation, double-stranded cDNA was transcribed in vitro in the presence of biotin labeled nucleotides using T7 RNA polymerase (ENZO Diagnostics, Farmingdale, NY). cRNA was purified using RNeasy® mini spin silica columns (Qiagen, Valencia, CA) and fragmented at 94°C for 30 min in buffer (pH 8.1) containing 0.2 M Tris–acetate, 0.5 M potassium acetate and 0.15 M magnesium acetate. Fragmented cRNA (15 µg) was hybridized overnight at 45°C to U95Av2 arrays (Affymetrix, Santa Clara, CA). Hybridization was detected with streptavidin-labeled phycoerythrin using a confocal laser scanner (Affymetrix). The total intensity of each array was scaled to normalize for inter-array variations.

Expression analysis
Expression data were generated using Microarray Suite 5.0 (MAS 5.0) Affymetrix Gene Chip Software with a single chip for each exposure and cell line analyzed. MAS 5.0-generated expression data were filtered using the following conditions to identify differential expression. A particular transcript was considered to be induced only if its expression was >=80 intensity units (approximately twice the average background), its detection P-value was <=0.06, the difference of relative abundances between control and treatment for this transcript was >=80 intensity units, and the P-value indicative of changed expression was <=0.003. Similarly, a transcript was considered to be suppressed only when the signal of the corresponding untreated control transcript was >=80 intensity units and significantly detected (P <= 0.06), and the difference of relative abundances between control and treatment for the transcript was >=80 intensity units, and the P-value indicative of change was >=1–0.003. In general, only transcripts induced or suppressed by >=2 fold were considered as differentially expressed. These parameters permit identification of differentially expressed genes while limiting the number of false positives. For biological replicate experiments in vitro, the microarray platform employed here provides reproducible data with observed signal correlations of 0.98 to 0.99 and differential expression call (increase, decrease, no change) correlations of 0.89 to 0.91 (data not shown). Clustering (yielding self-organizing maps) was performed using the GeneCluster 1.0 software (18) with the usual normalization of expression levels across concentrations (mean = 0, SD = 1). Genes in clusters identified with arsenite treatment were clustered for arsenate treatment for comparison. The cluster expression data are available by email as an Excel file upon request (rhrice{at}ucdavis.edu); the genes in the clusters in Figure 3 are listed in a PDF file available at the website http://www.envtox.ucdavis.edu/Faculty_Personal_Web_Pages/rhrice.htm.



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Fig. 3. Cluster analysis of expression patterns as a function of arsenic concentration in SCC12F2. Illustrated on the left are seven different patterns of expression (C1–C7) for arsenite, where the number of genes in each pattern is indicated. On the right are shown clusters of expression patterns for the same genes from cultures treated with arsenate.

 
Northern blotting
RNA was isolated as described above, fractionated on a 1% denaturing agarose gel, and transferred to nylon membranes. Blots were hybridized to 32P labeled cDNA probes for human involucrin as done previously (14) and human heme oxygenase-1 (HO1), prepared by RT–PCR from hEp and verified by sequencing, at 65°C in hybridization solution containing 7% SDS, 0.25 M sodium phosphate (pH 7.2), and 1 mM EDTA. Quantification was performed on a Molecular Dynamics SI PhosphorImager after which the values reported were corrected for unequal loading by normalization to the values for 18S RNA in each sample.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Gene expression profiling of arsenic-treated SCC9 cells
The malignant SCC9 line was treated with sodium arsenite (2 µM) or arsenate (6 µM). These concentrations, having no effect on cell growth or total protein per culture, were found empirically to produce nearly equivalent suppressive effects on differentiation under the conditions employed (14). Expression analysis showed that transcripts of ~30% of the genes surveyed (3576 of ~12 000) were judged to be present with no treatment. Of those present, 7% (254 transcripts) were judged to be either induced (87 transcripts) or suppressed (167 transcripts) at least 2-fold by treatment with arsenite. Results were similar with arsenate (5% induced or suppressed). In addition, 31 transcripts were absent in the untreated cultures but were induced by arsenite treatment. As illustrated in Figure 1, many more genes were suppressed than induced. The global nature of the changes is consistent with the treatment producing substantial perturbation of normal regulatory processes in the cells. A number of induced genes reflected an adaptive response to reactive oxygen including HO1 (11–32-fold), NAD(P)H quinone oxireductase (2.3–2.6-fold) and thioredoxin reductase (3-fold). With only a few possible exceptions, the degree of effect, positive or negative, was approximately the same for the two arsenic oxidation states, consistent with the major effect of arsenate arising from its reduction to arsenite in cells (19). The SCC9 genes with altered transcription, particularly those suppressed, included kinases, phosphatases, transcription factors and other participants in signal transduction pathways, many of which could plausibly contribute to altered properties such as loss of keratinocyte differentiation. Rather than study the action of each gene product individually, the focus was to identify those genes altered consistently among the different human keratinocytes employed.



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Fig. 1. Alteration of gene expression in SCC9 cultures treated with equally effective concentrations of sodium arsenite (2 µM) and arsenate (6 µM). The cultures were treated continuously for 2 weeks and harvested 10 days after reaching confluence. Only transcripts with the largest changes in expression are illustrated.

 
Arsenic concentration dependence in SCC12F2
Changes in gene expression at 2 µM arsenite or 6.7 µM arsenate were similar in magnitude in SCC12F2 to those observed in SCC9 above. At 0.67 µM arsenite (equivalent to the former 50 p.p.b. drinking water standard in force for many years), the transcription of six genes was altered 3–16-fold. Under the less stringent criterion of a 2-fold change, the transcription of 22 genes was seen to be altered, the majority being suppressed. As seen in Figure 2, the effects produced by 0.67 µM arsenite were generally smaller but consistent with those produced by 2 µM arsenite. At concentrations of 0.2 µM or lower arsenite or 0.67 µM or lower arsenate no changes in gene transcription as high as 2-fold were detected except for a 2-fold suppression of the melanoma growth stimulatory activity gene observed as low as 0.2 µM arsenite. Cluster analysis of signal intensities illustrates the parallel action of arsenite and arsenate in seven patterns of response and the lack of substantial response below the two highest concentrations of each species of arsenic (Figure 3). At the highest concentrations, 6.7 µM arsenate altered the transcription of the same genes to virtually the same extent as 2 µM arsenite. The most sensitive marker for arsenic exposure was the increase in HO1, which was induced 16-fold at 0.67 µM and 169-fold at 2 µM arsenite.



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Fig. 2. Concentration dependence of gene expression alterations in premalignant keratinocytes. SCC12F2 cultures were treated continuously with 0.67 or 2 µM arsenite as indicated and harvested 10 days after reaching confluence. Illustrated are the 22 genes showing the highest responsiveness to 0.67 µM arsenite.

 
As shown in Figure 4 with SCC12F2, northern blotting corroborated the induction of HO1 and suppression of involucrin observed in microarray experiments. In each case arsenite had ~3-fold greater potency than arsenate. At the highest concentration tested (2 and 6.7 µM, respectively), each induced HO1 ~50-fold, whereas induction of ~6-fold was noted at the next highest concentration (0.67 and 2 µM, respectively). The concentration dependence of induction in SCC9, spontaneouly immortalized (SIK) and normal human epidermal cells was essentially the same (not shown). The differences in fold changes obtained in the northern versus microarray experiments probably reflect the relative difficulty of measuring the very low uninduced levels by either method. The highest concentrations of arsenic species tested suppressed involucrin mRNA levels to 20–40% of those in untreated controls. A lesser degree of suppression, to 70% of the untreated control value, was noted at the next lower concentrations, similar to previously reported findings with SCC9, SIK and hEp cells (14).



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Fig. 4. Northern blotting of HO1 and involucrin mRNA in premalignant keratinocytes. SCC12F2 cultures were treated with various concentrations of arsenite and arsenate under the same conditions as in Figures 2 and 3. The result presented, with duplicate samples at each concentration, is representative of five experiments with each gene.

 
Arsenate time course in hEp cells
Effects on keratinocyte differentiation were examined after treatment with arsenic for extended periods, typically 2 weeks, to permit maximal expression of differentiation markers in untreated control cultures. To examine more immediate effects, possibly enlightening as to perturbation of signalling pathways, transcriptional alterations in hEp treated with sodium arsenate (6 µM) were measured after treatment for 2, 5, 8 or 14 days. At the latest time point of treatment, corresponding to that chosen for the malignant and premalignant lines above, many more genes were altered at least 3-fold in expression in hEp (420 transcripts) than in SCC9 (85 transcripts) or in SCC12F2 (39 transcripts). As before, however, nearly twice as many of these were suppressed as induced.

The earliest time points examined were after 2 days of treatment starting either 2 days before confluence or at confluence. The only transcriptional effects (>=2 fold) detected before confluence were induction of 13 mRNAs (see ‘2 Pre’ in Table I), most indicative of the generation of reactive oxygen. Each transcript stayed elevated during the entire period of treatment. Study of stress response elements in the HO1 promoter has revealed most of these are in the battery of genes induced by reactive oxygen and regulated by activation of the Nrf2 transcription factor (20,21).


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Table I. Gene transcription induced in hEp by treatment with 6 µM arsenatea

 
Several other patterns of expression were noted among genes that were induced. In one, induction was noted in all cultures treated starting at, but not before, confluence. Examples are metallothionein-1A, G and H (2.3–3.5-, 16–21- and 2.5-fold, respectively), serum amyloid (2–5.7-fold) and sequestosome 1 (p62), a protein 91% identical to the oxidative stress protein A170 in mouse (2.3–7.5-fold). In other patterns, induction occurred later, such as after 5 or 8 days of treatment (e.g. nicotinamide N-methyl transferase or epoxide hydrolase 1, respectively). These patterns could result from downstream effects of more immediate actions of arsenate.

Although gene induction was clearly evident, very few genes showed substantial suppression of transcription after 2 days of treatment of preconfluent cultures. When the treatment started at confluence, however, over 31 genes were seen to be suppressed by at least 3-fold after 2 days. By 5 days, the number reached 170 genes, and by 8 days it was 233 genes. By 14 days, the number was 89, suggesting longer-term adaptation. Among transcripts changing were structural proteins, enzymes, ion channels and transcription factors. Prominent among the numerous genes whose transcription was suppressed were those encoding differentiation markers as described previously (14,22). These were not expressed in untreated cultures by the first time point; therefore maximal suppression generally was not evident until at least 5 days of treatment. Consistent with earlier work, in the treatment period of 5–14 days profilaggrin was suppressed 27–97-fold, keratin 1 2.4–16-fold, keratin 4 9–30-fold, keratin 10 6–11-fold, keratinocyte transglutaminase 3–10-fold and involucrin 2.4–6.5-fold.

Arsenic-induced alterations in gene expression patterns were compared to find those that were common to the three cell samples employed. Among the genes induced, HO1 and NAD(P)H quinone oxireductase were strongly affected, indicating that reactive oxygen generation was a consistent feature of arsenic treatment (Table II). Small proline-rich protein 2 is known to be induced by UV light, nitroquinoline-1-oxide and inflammation (23,24) suggesting it was induced as well by reactive oxygen generated in the keratinocytes by arsenic. Transcriptional regulation of uridine phosphorylase has not been intensively studied, but present results raise the possibility it is also induced by oxidative stress.


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Table II. Alteration of genes in common among normal (hEp), premalignant (SCC12F2) and malignant keratinocytes (SCC9)a

 
Induction of HO1 versus suppression of differentiation
Present results showed induction of HO1 to be a highly sensitive indicator of arsenic exposure of the cells. To test how specific was the correlation between HO1 induction and suppression of differentiation, SCC9 cultures were treated with various other agents reported to produce reactive oxygen in cultured cells (Table III). Exposures were at maximally tolerated concentrations that did not reduce cell growth over a 2-week period. Among the agents tested, arsenate (6 µM), cadmium chloride (10 µM) or hemin (10 µM), produced a sustained induction of HO1 and also suppressed differentiation, judging by the involucrin content of the cultures, ordinarily reaching maximal levels by 10 days in untreated cultures (14). Although not as highly elevated after 10 days, the HO1 induction by hemin after 2 days (78 ± 8 fold) was undiminished. On the other hand, zinc chloride (100 µM) or glucose oxidase (30 ng/ml) produced only transient induction of HO1 and did not suppress involucrin content. (Exposure of the cells to glucose oxidase at 100 ng/ml induced HO1 ~20 fold after 8 h, but the cells did not survive long enough to measure involucrin mRNA.) Similarly, treatment of the cells with 1-chloro-2,4-dinitrobenzene (10 µM) gave detectable induction of HO1 by 4 h (~10 fold) that was not reproducibly above background by 8 h and had little effect on either HO1 or involucrin mRNA levels by 10 days. Treatment of the cells with t-butyl peroxide up to 10 mM did not detectably induce HO1 after 8 h or 10 days or affect involucrin expression (not shown). These observations suggest that with chronic treatment keratinocytes can accommodate to the reactive oxygen produced by the latter agents. Finally, the transition metal oxyanions vanadate (10 µM) and chromate (5 µM), as found previously (22), suppressed differentiation, but they produced no induction of HO1 evident at 8 h or 10 days. For each of the latter two agents, a 4 h time point showed essentially no induction in either SCC9 or hEp, whereas arsenite at 1 µM in hEp gave a 3-fold induction (data not shown). After 2 days of treatment, HO1 levels in vanadate and chromate-treated hEp cultures showed <2-fold induction, while induction in 1 µM arsenite- or 10 µM cadmium-treated cultures was 8- or 16-fold, respectively. At high concentrations (e.g. 200 µM), vanadate generates detectable reactive oxygen species in mouse epidermal cells (25), but at lower concentrations, similar to this study, has been reported not to induce intracellular oxidation (26). Chromate is well known to generate reactive oxygen in cells (27), but evidently not well enough at the low concentration employed in this work to be detectable by HO1 induction.


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Table III. Induction of heme oxygenase (HO1) and suppression of involucrin (Inv)a

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Present work is pursuant to earlier findings that inorganic arsenic suppresses the differentiation program of cell types such as keratinocytes (22) and adipocytes (28). Subsequent reports suggest this phenomenon reflects perturbation of key elements in the differentiation program of each cell type (14,29). In view of the large number of genes whose regulation now is seen to be substantially perturbed, the effect on differentiation may constitute only a fraction of a more global perturbation. Thus, differentiation markers may not be targeted preferentially; their responses may simply reflect a large-scale perturbation of cell regulation. Reports of arsenic action in cultured rat liver cells (30) or human fibroblasts (31) also showed significant effects on transcription of a large fraction of the 568–588 genes surveyed. While the degree of neoplasia is uncertain in the human epidermal cells that arsenic targets to develop into cancer, the global effects seen across the spectrum from normal to malignant keratinocytes suggest any stage is susceptible. Indeed, considerably more genes were affected in the normal than pre-malignant and malignant cells, possibly reflecting decreased regulatory flexibility in the latter from oncogene activation and loss of cell-cycle checkpoints and tumor suppressors. Although inorganic arsenic is a poor mutagen/carcinogen alone, it could increase the susceptibility of normal human keratinocytes as a co-mutagen/co-carcinogen in the presence of other agents as demonstrated in mouse models (9,10).

Among current theories of arsenic action in target cells, present results showing induction of a battery of oxidative stress-responsive genes support a role for generation of reactive oxygen species as a source of global effects. The time course study using hEp indicates this is an early result of arsenic treatment and, in the simplest interpretation, could drive subsequent adverse cellular responses. The earliest effects may be the most critical for elucidating the mechanism of arsenic action. Few other effects on gene transcription were detected at the earliest treatment time (2 days) but many were obvious after at least 5 days of treatment, notably transcriptional suppression. The hypothesis merits exploration that transcription is sensitive to reactive oxygen of those genes suppressed in common among the malignant, premalignant and normal keratinocytes studied after longer exposure periods.

Arsenic is well known to produce oxidative stress in model systems as judged by induction of stress proteins (32) and response of a fluorescent reporter (33), and in mice (34), although the concentrations generally employed in such studies were beyond human tolerance. However, recent reports show that humans chronically ingesting drinking water with high arsenic concentrations have increased reactive oxidants and lower antioxidant capacity in the blood (35,36). Hence, generation of reactive oxygen species plausibly contributes to or is a major cause of adverse human effects from arsenic exposure. Present work indicates this is important even at 0.67 µM (50 p.p.b.), emphasizing the importance of elucidating the molecular basis of arsenic action. Reduction of arsenate to arsenite has been demonstrated in cells and could contribute to redox cycling (32). This reduction occurs in keratinocytes (19), and no convincing evidence for distinct effects of the arsenate versus arsenite species was obtained in the present experiments, suggesting they act by the same mechanism to produce the downstream effects. Arsenite may stimulate endogenous formation of reactive oxygen in a variety of possible ways, and was seen to induce a small but consistent increase (1.5–2-fold) in xanthine oxidase in present work.

The sustained generation of reactive oxygen plausibly could result in the suppression of differentiation by arsenic. This scenario is supported by the action of cadmium or hemin, for which such an effect has not been reported previously. As cadmium has been seen to induce a global change in gene expression in yeast that is distinct from oxidative stress (37), its effects could reflect more than one mode of action. Moreover, the effectiveness of vanadate and chromate in the absence of HO1 induction indicate that suppression may occur by other means. This can be rationalized readily in the case of vanadate in view of its biological activity as a protein tyrosine phosphatase inhibitor (38). If the generation of reactive oxygen contributes to the carcinogenicity of arsenic, the means by which this downstream effect occurs remains to be elucidated. The suppression of certain genes could have an important role. For example, among the genes suppressed in common among the keratinocytes studied (Table II) were those encoding insulin growth factor-like binding protein 3, which functions as a pro-apoptotic intermediary (39), and TNF-related apoptosis-inducing ligand. In fact, regardless whether the changes were secondary, resulting from downstream effects of chronic treatment such as oxidative stress or altered DNA methylation, they could plausibly contribute to development of neoplastic behavior. Generation of reactive oxygen as a fundamental driver toward neoplasia is not incompatible with other proposed mechanisms of arsenic action that may occur as a consequence or independently, including inactivation of the p53 DNA repair pathway (40).

Stimulation of replication through increased production or secretion of growth factors such as GM-CSF, TGF-alpha and TNF-alpha as reported by others (10,41) has been proposed to contribute to tumor promotion. A very recent microarray study reported arsenite simultaneously increases redox-related gene expression and proliferation while decreasing transcription of certain DNA repair genes (42). Present experiments did reveal increases in transcription of heparin-binding EGF-like growth factor (3–16-fold), PDGF (13-fold) and a small increase in TGF-alpha (1.9-fold), as well as ornithine decarboxylase (3–4-fold). In addition to the large number of transcripts observed to change, a substantial number of other genes were present at low levels and therefore not observed to change, including proto-oncogenes and tumor suppressor genes that may contribute to biological effects. However, no stimulation of proliferation has been observed in the cultures, perhaps not discernible in present conditions of optimized growth.

In the absence of a suitable animal model and a mechanistic basis with which to justify extrapolation of health effects to low doses, considerable controversy has surrounded setting a drinking water standard for inorganic arsenic. A contribution to rationalizing regulatory decisions may come from examining the concentration dependence of transcriptional effects on a wide range of genes. Present experiments in a human target cell type show that arsenite at the former drinking water standard of 50 p.p.b. (0.67 µM) has clear effects on gene expression, as reported previously for suppression of differentiation (14), and provide evidence for generation of reactive oxygen. Although blood concentrations of inorganic arsenic do not correspond directly to the concentrations in drinking water (35), urine concentrations are close to those in the drinking water, while methylated arsenicals in the urine are higher still (36). Pentavalent methylated arsenicals can be reduced in vivo to trivalent forms that may contribute substantially to toxic effects (4345). Inasmuch as human epidermal cells appear to conduct little arsenic methylation in culture (45), the results reported here may underestimate the combined effect of the total metabolite pool to which cells are exposed. Nevertheless, finding a transcriptional no effect level in target cells provides a potentially valuable criterion for estimating maximal safe exposure levels. In this regard, HO1 could be useful, being responsive at all times of treatment tested and the most sensitive marker for arsenic exposure in the cultured keratinocytes.


    Notes
 
3Present address: Centro de Estudios Académicos sobre Contaminación Ambiental, Universidad Autónoma de Querétaro, Cerro de las Campanas s/n, Querétaro, Qro., C.P. 76080, México Back


    Acknowledgments
 
This work was supported by US Public Health Service grants 2R01 AR27130, 2P42 ES04699, 2P30 ES05707, CONACYT agreement 92991, and the Universidad Autónoma de Querétaro.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received July 9, 2002; revised January 2, 2003; accepted January 21, 2003.