Arsenic and Benzo[a]pyrene Differentially Alter the Capacity for Differentiation and Growth Properties of Primary Human Epidermal Keratinocytes

D. S. Perez*,{dagger}, L. Armstrong-Lea{dagger}, M. H. Fox{dagger}, R. S. H. Yang*,{dagger} and J. A. Campain*,{dagger},1

* Quantitative and Computational Toxicology Group, Center for Environmental Toxicology and Technology and {dagger} Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, Colorado 80523

Received June 10, 2003; accepted August 22, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Normal human epidermal keratinocytes (NHEK) have been chosen as an in vitro model to test the hypothesis that chemicals which alter or interfere in cellular differentiation will concomitantly induce growth perturbations and are, thus, potential carcinogens. In these studies, we have focused on two known skin carcinogens, arsenic and benzo(a)pyrene (BaP). Our results demonstrated that BaP inhibits terminal differentiation in NHEK, as measured by cross-linked envelope (CLE) formation, up to 5.8-fold in control and 1.7-fold in calcium (Ca2+)-treated cells. In comparison, arsenic decreased CLE formation 20-fold in control cells and 5.5-fold in Ca2+-treated NHEK. To characterize the effects of these agents on the growth rate and cell cycle distributions of NHEK, flow cytometric analysis was used. BaP at 2 µM increased proliferation rates by 29%. Altered cell-cycle distribution in BaP-treated cells indicated a more rapid progression through the cell cycle, possibly by a shortened G2 phase. In contrast, arsenic at 5 µM inhibited proliferation by 25%; growth arrest (9%) was also observed in NHEK treated with 2 mM Ca2+. Our findings suggest that, although both BaP and arsenic inhibit CLE production in NHEK, different mechanisms may be involved. Studies in progress will attempt to identify molecular markers involved in the observed chemical effects. These markers will facilitate a mechanistic understanding of how an altered balance between growth and differentiation may play a role in the transformation process in NHEK.

Key Words: keratinocytes; differentiation; proliferation; benzo[a]pyrene; arsenic; calcium.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The epidermis is characterized by a highly regulated balance between epithelial cell growth and differentiation, with a single basal layer of proliferating keratinocytes and stratified, overlying keratinized layers. Differentiation ultimately leads to a highly specialized, "dead-end" cell that cannot divide. However, within the basal compartment, cells can be changed by carcinogenic agents so that they are blocked from achieving the normal state of differentiation and are, instead, capable of indefinite proliferation. This imbalance between two such important and opposing processes has been linked to the onset of carcinogenesis.

The skin is a useful model system for understanding the signals that drive proliferation or differentiation, particularly with respect to alterations that may lead to cancer. Epidermal keratinocytes differentiate readily in culture and produce cross-linked envelopes (CLE), a complex protein structure that forms the protective barrier of the skin. This switch between proliferation and differentiation is regulated by phospholipase C and protein kinase C (PKC)-dependent pathways, as well as induction of tyrosine phosphorylation, among others (Denning et al., 1995Go; Filvaroff et al., 1990Go). Defects in terminal differentiation have been tied to transformation in keratinocytes, implying that targets for chemically-mediated carcinogenesis may well be pivotal signaling pathways involved in this process (Salnikow and Cohen, 2002Go). One way to approach this issue is through comparison of the alterations induced during differentiation by multiple skin carcinogens.

Arsenic is a potent human skin carcinogen. In fact, due to arsenic-comtaminated drinking water in many countries, development of neoplastic skin lesions has become a health problem of global proportions (Tseng, 1977Go). The exact mechanism involved in the transformation of keratinocytes by arsenic is unclear. Due to its clastogenic activity and inhibition of DNA repair, arsenic is presumed to act as a progressing agent during carcinogenesis (Leonard and Lauwerys, 1980Go; Vogt and Rossman, 2001Go). Arsenic has other effects on keratinocytes including: (1) altering expression of growth regulatory factors, (2) enhancing cell proliferation at low concentrations, and (3) inhibition of keratinization (Germolec et al., 1996Go; Kachinskas et al., 1994Go; Vega et al., 2001Go). It is likely that these effects are linked to one another and to the induction of skin cancer. Many investigators are currently attempting to address this issue through identification of important molecular switches that are negatively impacted by arsenic and which may render the skin a sensitive target to malignant transformation.

Benzo[a]pyrene (BaP) is among the Superfund Top 10 Priority Hazardous Substances and is a common constituent in petroleum products. BaP also represents a major class of heavy fraction hydrocarbons in mixtures such as coal tar, smoke, and automobile exhaust. There is, thus, potential for either environmental or occupational dermal exposure to BaP. The most likely source of dermal contact with toxic BaP levels would be in workers in the petroleum industry. Bioactivated BaP is a known skin carcinogen, acting as a mutagen and an initiating agent during transformation (Mager et al., 1977Go). Many of the earliest studies of BaP were carried out using the murine skin system (Lee et al., 1971Go). When applied dermally, BaP induces cytokinetic abnormalities and inflammation, followed by skin tumors (Albert et al., 1996Go). The chemical alters differentiation in multiple cell types (Edmondson and Mossman, 1991Go; Reiners et al., 1991Go), although keratinocytes themselves have not been tested. This activity is likely due to mutagenesis of proteins in crucial signal transduction paths. Clearly, much work remains to be done to clarify the relationship between BaP exposure, abnormalities in proliferation and differentiation, and development of neoplastic skin lesions.

The hypothesis around which our studies are centered is that chemicals which alter or interfere in differentiation in keratinocytes will also induce growth perturbations and are, thus, potential carcinogens. Although, all evidence points toward different primary mechanisms of action for BaP and arsenic, both induce skin cancer. It may well be that there are common pathways affected by the two chemicals, as well as chemical-specific effects. Work described herein compares the effects of arsenic and BaP on differentiation, growth rate, and cell cycle distribution in human keratinocytes. These studies should begin to address the issues of whether diverse chemicals with a common endpoint of skin carcinogenesis affect differentiation in a similar manner, and if so, whether cell growth characteristics are also impacted. In time, our findings should enlighten us as to exactly what is happening in cells treated with carcinogenic agents and which of these events may be involved in malignant transformation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
Sodium meta-arsenite (NaAsO2), calcium chloride (CaCl2), benzo[a]pyrene (C20H12), dimethyl sulphoxide (DMSO), pepsin, and bromodeoxyuridine (BrdU) were purchased from Sigma Chemical Co. (St. Louis, MO).

Cell culture and culture reagents.
Cyropreserved normal human epidermal keratinocytes (NHEK) were purchased from the Clonetics Corp. (San Diego, CA). NHEK were grown in 5% CO2 at 37°C in defined Keratinocyte Growth Medium (KGM) (Clonetics Corp) containing bovine pituitary extract (BPE), human epidermal growth factor (hEGF), insulin, hydrocortisone, transferrin, epinephrine, and antibiotic, GA-1000, at proprietary concentrations as determined by the manufacturer.

Cytotoxicity assays.
Prior to beginning our studies on the effects of the chemical agents, BaP and arsenic, on keratinocyte differentiation and cell cycle distribution, cytotoxicity studies were carried out using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay as a measure of cell viability (Mosmann, 1983Go). For these assays, NHEK were exposed to increasing concentrations of either BaP or arsenic for 24 h. Viability, as measured by absorbance of cell lysates at 550 nm, of NHEK cells 96 h after exposure to various concentrations of BaP or arsenic, as compared to the appropriate controls (data not shown) was utilized to derive values for the lethal concentration (LC) resulting in 5, 20, and 50% death; these values were subsequently used in differentiation experiments and flow cytometric analysis. Our assumption in these studies was that the metabolic activation of BaP is necessary for its carcinogenic effects in skin and, thus, is also required for inducing alterations in growth and/or differentiation. As both preliminary studies in our lab and previous work (Kuroki et al., 1980Go) indicated that primary keratinocytes have intact metabolic capabilities for BaP, these assays were carried out without the addition of hepatic microsomal S9 fraction. The NHEK used in these studies were from pooled lots of cells from multiple donors and were obtained commercially in this form from Clonetics. One assumption made was that by pooling cells, metabolic capabilities for a large population would, on average, be more consistent from lot to lot than cells from single individuals. Cytotoxicity studies using NHEK from this manufacturer have supported this supposition in that results on many diverse chemicals (including BaP) compared closely between lots.

Differentiation assay.
Cross-linked envelope (CLE) formation as a measure of keratinocyte differentiation was quantified as described previously (Rice and Green, 1979Go). Briefly, NHEK were plated in 60-mm petri dishes at a density of 5000 cells/plate. When cells reached 60–70% confluency after approximately 5 days, they were treated for 24 h with the following chemicals and concentrations: BaP [0.2–2 µM], arsenic [0.5–5 µM], Ca2+ [0.5–3 mM], arsenic [0.5–5 µM] + Ca2+ [2 mM], and BaP [0.2–2 µM] + Ca2+ [2 mM]. Ca2+ was used as a positive control for induction of differentiation, thus acting to increase CLE production in NHEK. Experiments were also conducted where cells were treated at 20% confluency (2 days after plating) with the same chemicals and concentrations mentioned above. The results showed similar trends when compared to NHEK treated at 60% confluency. However, treatment at this earlier point produced a great amount of variability in cell growth in different treatment groups, which made it difficult to synchronize CLE counting for each group. The concentrations of BaP and arsenic utilized in these experiments corresponded to the range between the LC5 and the LC50 for these chemicals as determined in MTT assays for NHEK. The concentration of Ca2+ ranged from basal conditions (0.25 mM) that allowed proliferation of primary NHEK up to concentrations that were demonstrated, in our experiments, to induce maximum differentiation of exposed keratinocytes (2–3 mM). After the treatment period, cultures were re-fed with chemical-free medium and incubated an additional 2–3 days until they reached 90–100% confluency. At this point, NHEK cells were trypsinized, counted, and lysed with Lysis Buffer to analyze CLE formation. Lysis Buffer consisted of 1% sodium dodecyl sulfate (SDS), 1% ß-mercaptoethanol, 10 mM Tris hydrochloride, pH 8.0, and was added (1 ml) to each plate. CLE were visually quantified under a light microscope using a hemacytometer. Differentiation capacity of treated and control cultures was expressed as the ratio of CLE to cell number. Experiments to evaluate the nature of the dose-response for effects on CLE formation by Ca2+, arsenic, and BaP were also carried out. In these studies, Ca2+ concentrations ranged from 0.25 to 3 mM. Arsenic concentrations ranged from 0.125 to 5 µM. BaP concentrations ranged from 0.0016 to 5 µM. DMSO was utilized as a solvent control for BaP. The time course study was carried out by comparing CLE formation in cultures treated for 24 h with 2 mM Ca2+ as compared to control populations (0.25 mM Ca2+) at 7, 10, 13, and 15 days after plating.

Cell division rate measurement assay.
Determination of NHEK proliferation rates was carried out via BrdU labeling and propidium iodide staining followed by flow cytometric analysis (Steel, 1977Go; Wilson, 1994Go). Briefly, cells were plated in 100-mm petri dishes at a density of 10,000 cells/plate. When cells reached 10–15% confluency, they were treated with BaP [0.2, 1, 2 µM] or arsenic [0.5, 2, 5 µM] for 24 h and Ca2+ [2 mM] continuously. After the treatment period, cultures were re-fed with chemical-free medium and incubated until they reached 50–60% confluency, approximately 4 days. At this point, cells were pulse-labeled with 10 µM BrdU for 30 min. After washing to remove the BrdU, cells were cultured and fixed at 0, 2, and 4 h time points. The fixation and staining protocol was modified from that described by Larsen (1994)Go and Liao et al.(2001)Go Cells were filtered through 53 µm nylon mesh (Small Parts Inc., Miami Lakes, FL) prior to flow cytometric analysis.

Flow cytometric analysis.
Samples were analyzed with an Epics V cell sorter (Coulter, Miami, FL) interfaced to a Cicero data acquisition and display system (Cytomation Inc., Fort Collins, CO). Cells were illuminated by an argon ion laser at 488 nm (500 mW). FITC was measured at wavelengths between 515 to 530 nm, and PI was measured at wavelengths longer than 610 nm. The PI signal was gated on peak versus integral fluorescence to eliminate clumped cells. Thirty thousand cells were analyzed for each bivariate histogram.

The concept of potential doubling time (Tpot) was proposed by Steel (1977)Go and can be used to estimate the proliferation alterations that occur following chemical treatment. The Tpot of individual cultures was calculated from cell labeling and flow cytometric data as described previously (Begg et al., 1985Go). Tpot is a cell division time that takes growth fraction, but not cell loss, into account (Steel, 1977Go; Wilson, 1994Go). Tpot was calculated with the equation: Tpot = {lambda}(TS/LI), where TS is the period of DNA synthesis, LI (labeling index) is the fraction of cells synthesizing DNA, and {lambda} is a correction factor for the nonlinear distribution of cells through the cell cycle (Steel, 1977Go; Wilson, 1994Go). Estimation of Tpot values relied upon distinction of cells within the four different stages of the cell cycle.

Statistical analysis.
Dunnett’s one-way analysis of variance (ANOVA) was used to compare the difference of treated to control groups (Tamhane et al., 2000Go). Treatment is significantly different if the p-value is 0.05 or less when compared to the appropriate control. Statistical regression models were produced for BaP, arsenic, and Ca2+ dose-dependency in NHEK by choosing a regression line, based on the highest correlation coefficient (r), that best fit the experimental data. These models were used to describe the relationship between increasing concentrations of BaP, arsenic, or calcium and effects on CLE formation as an endpoint of keratinocyte differentiation. From these regression models, we used the equation generated to compare the rate of change (i.e., the slope) to determine which chemicals had a more profound effect on CLE formation.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Arsenic and Benzo[a]pyrene Inhibit NHEK Differentiation in Vitro
Our primary objective in these studies was to characterize the effects of two diverse skin carcinogens, arsenic and BaP, on NHEK proliferation and terminal differentiation. From cytotoxicity analyses using the MTT assay, we chose three equitoxic concentrations of the two chemicals, which corresponded to their LC5, LC20, and LC50, to use in differentiation studies. Initial experiments were carried out in the absence of Ca2+ to measure the impact of chemical exposure on basal (or confluency-induced) differentiation in NHEK. In our hands, both arsenic and BaP were effective inhibitors of the differentiation process in this cell type (Fig. 1Go); at all three concentrations tested, arsenic or BaP-treated cells exhibited statistically significant reductions in CLE production as compared to the untreated NHEK culture. Our findings that arsenic maximally inhibited CLE formation by approximately 20-fold at the LC50, or 5µM, are in agreement with previous studies on the effects of the metal in differentiating keratinocytes (Jessen et al., 2001Go; Kachinskas et al., 1994Go). BaP, at equitoxic doses, was not as strong an inhibitor as arsenic, but decreased CLE formation at the LC5, LC20, and LC50 by 1.85-, 2.25-, and 5.80-fold, respectively. In our treatment protocol, cells were exposed to the chemicals acutely (for only 24 h) when they had reached approximately 60–70% confluence. It is highly likely that longer treatment, or treatment administered when the cultures were at low density and few cells were yet committed to differentiating, would have been even more effective at inhibiting the final formation of CLE in confluent cultures.



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FIG. 1. Effects of benzo[a]pyrene and arsenic on basal differentiation in NHEK. Cells were treated with increasing concentrations of BaP and arsenic for 24 h, and CLE were quantified as described in Materials and Methods. Ca2+, at 2 mM, was utilized as a positive control for induction of differentiation. Numbers of CLE for each treatment condition were standardized to cell number and are expressed as the mean of triplicates from 3 independent experiments ± SD. *Significantly different from the control using one-way ANOVA followed by Dunnett’s test, p < 0.05. **Signifies Ca2+ exposed cells being significantly different from control cells. (A) is graphical representations of the data in (B).

 
More detailed studies were then carried out to further explore the nature of the dose-response curves for arsenic- and BaP-mediated inhibition of CLE formation in NHEK. As expected, arsenic significantly (p < 0.05) perturbed CLE formation at all concentrations greater than 0.25 µM or the LC2 (Fig. 2Go); decreases in CLE production in treated versus control NHEK averaged from 1.6-fold to the maximum of 20-fold at 5 µM arsenic. BaP significantly perturbed NHEK differentiation at all concentrations >= 0.04 µM; this corresponds to a nontoxic concentration in this cell type. As seen in Figure 3Go, the inhibition of CLE formation ranged from 1.1- to 5.5-fold maximum at 5 µM BaP. The higher concentration of BaP required to achieve 5- to 6-fold inhibition of differentiation in this series of experiments versus those described above (5 µM versus 2 µM) is likely due to the lot of keratinocytes used. As NHEK cells are pooled populations of primary cells isolated from different individuals, they are characterized by intrinsic variations in sensitivity to toxic insult (Bae et al., 2001Go). For BaP, this may well be related to differences in metabolic capability in the different lots of cells. However, in all studies, regardless of the keratinocyte population used, we observed a highly consistent and dose-dependent inhibition of CLE formation by BaP. After regression analysis, a semilog model (y = b0 - b1 x log [concentration]) best predicted the experimental data for both BaP and arsenic.



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FIG. 2. Dose-dependent inhibition of CLE formation in NHEK by arsenic. NHEK were treated with increasing concentrations of arsenic for 24 h as indicated in the figure, and CLE were quantified as described in Materials and Methods. Control was untreated NHEK cultured in an identical manner. Numbers of CLE were standardized to cell number and are presented as the mean of triplicates from 8 independent experiments ± SD. *Significantly different from the control using one-way ANOVA followed by Dunnett’s test, p < 0.05. Statistical regression analysis was carried out to predict the best mathematical model for describing CLE formation as a function of chemical concentration. (A) is a graphical representations of the data in (B).

 


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FIG. 3. Dose-dependent inhibition of CLE formation in NHEK by benzo[a]pyrene. NHEK were treated with increasing concentrations of BaP for 24 h as indicated in the figure, and CLE were quantified as described in Materials and Methods. Controls were untreated NHEK and those treated with the DMSO solvent. Numbers of CLE were standardized to cell number and are expressed as the mean of triplicates ± SD from 16 independent experiments. *Significantly different from the control using one-way ANOVA followed by Dunnett’s test, p < 0.05. Statistical regression analysis was carried out to predict the best mathematical model for describing CLE formation as a function of chemical concentration. (A) is graphical representations of the data in (B).

 
Effects of Calcium on NHEK Differentiation in Vitro
In addition to alterations in cell–cell and cell–matrix contact, increases in extracellular Ca2+ may serve as the most important signal for differentiation in keratinocytes. In order to more fully understand chemically-mediated perturbations in this process, we initially examined the normal dose-response and time course of Ca2+-induced formation of CLE in NHEK. For the former series of experiments, NHEK were exposed for 24 h to increasing concentrations of Ca2+, and CLE were quantified when cells reached confluency. Our studies demonstrated that Ca2+ concentrations ranging from 1 to 3 mM acted in a dose-dependent manner to induce statistically significant increases in differentiation in NHEK (Fig. 4Go). Although there was a detectable increase in CLE formation seen in cultures exposed to 0.5 mM Ca2+, as compared to cells grown under basal conditions of 0.25 mM Ca2+, this difference was not significant. The maximum increase in CLE formation (1.7-fold) was seen in cultures exposed to 2 mM Ca2+. After regression analysis, the power model (y = axb) yielded the best-fit description of the experimental Ca2+ dose-response data.



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FIG. 4. Dose-dependent induction of differentiation in NHEK by calcium. NHEK cells were treated with Ca2+, and CLE were quantified as described in Materials and Methods. Numbers of CLE as a function of Ca2+ concentration were standardized to cell number and expressed as a mean of triplicates from 6 independent experiments ± SD. Note the first data point shows basal levels of Ca2+ at normal culture conditions, which is a concentration of 0.25 mM. *Significantly different from the control using one-way ANOVA followed by Dunnett’s test, p < 0.05. Statistical regression analysis was carried out to predict the best mathematical model for describing CLE formation as a function of chemical concentration. (A) is a graphical representation of the data in (B).

 
Time-course analysis of CLE formation in response to Ca2+ exposure was also carried out in NHEK (Fig. 5Go). The purpose of this study was to determine the time of maximum CLE formation in NHEK after exposure to 2 mM Ca2+. This series of experiments demonstrated the following: (1) CLE production was time-dependent in both control and treated cultures; (2) in control populations, CLE formation increased at each time point, reaching a maximum at 15 days of 0.53 ± 0.065 CLE per cell; (3) Ca2+ substantially increased the rate at which the NHEK populations differentiated, with the first increase over control detectable by 7 days of culture and maximum CLE formation occurring at 10 days (0.63 ± 0.041 CLE per cell); and (4) the ratios of CLE in Ca2+-treated versus control cells at days 7, 10, 13, and 15 were 1.66, 1.84, 1.34, and 1.21, respectively. By 15 days in culture, there was no longer any statistically significant difference in differentiation between control and treated cells. This is likely due to the time-dependent increase in confluency in the control populations, which is a very effective inducer of terminal differentiation.



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FIG. 5. Time dependent effects of calcium on differentiation in NHEK. Cells were treated with 2 mM Ca2+ for increasing times, and CLE production was determined as described in Materials and Methods. Numbers of CLE were standardized to cell number and are shown as the mean of triplicates ± SD from 3 experiments. *Significantly different from the control using one-way ANOVA followed by Dunnett’s test, p < 0.05. (A) is a graphical representation of the data in (B).

 
Effects of Arsenic and Benzo[a]pyrene on Calcium-Induced Differentiation in NHEK
These studies were designed to explore whether BaP and/or arsenic were also inhibitory to differentiation induced by simultaneous treatment of NHEK cultures with 2 mM Ca2+. Treatment of cells was carried out for 24 h with Ca2+ and increasing concentrations of arsenic or BaP. The extent of CLE formation in the individual cultures was determined after cells reached 90–100% confluency or approximately 8 days. In general, the inhibitory effects of both BaP and arsenic on CLE formation were less striking in cells that were also exposed to high Ca2+, although both chemicals still demonstrated significant inhibition of the differentiation process. Under these conditions, only the two highest concentrations of BaP (LC20 and LC50) had a statistically significant inhibitory effect on differentiation when compared to Ca2+-induced cells. The maximum inhibition achieved by BaP was 1.7-fold at a concentration of 2 µM (Fig. 6Go). One possibility for these findings is that, by induction of proliferative arrest in treated cells, Ca2+ decreases the impact of arsenic and BaP on cell viability and, in turn, on other closely linked processes. This issue could be explored through more detailed analysis of the time-dependent cytotoxicity of these two chemicals in the presence and absence of Ca2+. Again, arsenic acted as a more effective inhibitor of differentiation in these cells, with a maximum decrease observed at 5 µM of 5.5-fold (Fig. 6Go) and significant inhibition of CLE formation when compared to control cells at all concentrations analyzed.



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FIG. 6. Effects of benzo[a]pyrene and arsenic on calcium-induced differentiation in NHEK. Keratinocytes were induced to differentiate by treatment with 2 mM Ca2+ in the presence of increasing concentrations of BaP or arsenic as indicated in the figure. Controls were untreated cells (control) and cells exposed only to Ca2+. CLE were quantified as described in Materials and Methods. Numbers of CLE were standardized to cell number and are expressed as the mean of triplicates from 3 experiments ± SD. *Significantly different from the Ca2+-treated control using one-way ANOVA followed by Dunnett’s test, p < 0.05. **Signifies Ca2+ exposed cells being significantly different from control cells. (A) is graphical representations of the data in (B).

 
Benzo[a]pyrene, Arsenic, and Calcium Alter Cell Cycle Distribution in NHEK
As proliferation and differentiation are opposing processes in keratinocytes, we were also interested in characterizing the cell cycle effects of BaP and arsenic, as compared to Ca2+ in this cell type. BrdU and propidium iodide staining and flow cytometric analysis were used to approach this issue. The resulting standard and bivariate DNA histograms allow visual observation of the dynamic changes in cell cycle distribution due to chemical exposure. Figure 7Go shows DNA histograms of NHEK cultures treated with 2 mM Ca2+, 5 µM arsenic, 2 µM BaP, and, for comparison, untreated control cells. It is apparent from this analysis that exposure of NHEK to arsenic under these conditions substantially altered the normal cell-cycle distribution, leading to both a G2 block and decreasing number of cells entering S-phase as compared to untreated controls; a graphical representation of the fraction of cells in treated populations versus the control in each phase of the cell cycle is shown in Figure 8Go. The effects of Ca2+ and BaP on cell cycle distribution were more subtle. A slight increase in the number of cells in G2/M, as well as a decrease in the number of early S-phase cells was seen in Ca2+-exposed populations when compared to controls. Cells exposed to BaP increased in G1/G0, with a decrease in G2/M cells and a slight decrease in the number of cells in S-phase. These changes were statistically significant at p values of 0.01 (for the impact of BaP on G1/G0) and < 0.05 (the rest), respectively.



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FIG. 7. DNA histograms of NHEK exposed to benzo[a]pyrene, arsenic, and calcium. NHEK were treated with chemical effectors for 24 h, stained with propidium iodide, and analyzed by flow cytometry as described in Materials and Methods. DNA histograms demonstrate cell number versus DNA content of viable cells in treated and control populations as indicated in the figure and are representative of multiple (n = 8 ) experiments. Actual percentages in each phase were determined by computer analysis using Multicycle®. The concentrations of chemical effectors utilized were (B) 2 mM Ca2+, (C) 5 µM arsenic, and (D) 2 µM BaP.

 


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FIG. 8. Alterations in cell cycle distribution in NHEK populations treated with benzo[a]pyrene, arsenic, and calcium. NHEK were treated with chemical effectors for 24 h, stained with propidium iodide, and analyzed by flow cytometry as described in Materials and Methods. Cell percentages are expressed as the mean of triplicates ± SD from 7 experiments. Significant differences in the proportion of each population in the three (G1/G0, S, or G2/M) phases of the cell cycle were determined by one-way ANOVA followed by Dunnett’s test, **p < 0.01; *p < 0.05. (A) is graphical representations of the data in Table (B).

 
Bivariate histograms where obtained to explore chemical effects on the movement of cells 4 h after being labeled in S-phase with BrdU. When BrdU incorporation and DNA content in the chemically treated keratinocyte cultures were viewed simultaneously, we observed the following (Fig. 9Go): (1) in cultures treated with 2 µM BaP, BrdU-labeled cells progressed through G2 and mitosis and entered G1 during the 4 h labeling period (Fig. 9BGo); (2) this effect of BaP was dose dependent and was not detectable in cultures treated with lower concentrations or in the DMSO-solvent controls; and (3) in these latter populations, the majority of labeled cells were found in S-phase and G2/M. These observations indicate that BaP increased the rate at which keratinocytes proceeded through the cell cycle, a hypothesis in agreement with the Tpot data discussed below. An increased cell cycle rate was not seen in NHEK treated with either Ca2+ or arsenic (Figs 9C Go and 9DGo). In fact, although difficult to discern from bivariate histograms, in these latter populations the cell cycle rate was significantly decreased.



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FIG. 9. Bivariate histograms of control and chemically-treated NHEK. Cells were left untreated (A) or were exposed to 2 µM BaP (B), 2 mM Ca2+ (C), and 5 µM arsenic (D) as described in Materials and Methods. Staining with BrdU and propidium iodide, and analysis by flow cytometry were carried out as described in Materials and Methods. Representative histograms of chemically-treated and control populations demonstrate BrdU incorporation versus DNA content of viable cells from one of several experiments.

 
Chemical Effects Seen on NHEK Proliferation Rate
The results from flow cytometric analysis of cell cycle distributions were used to calculate the potential doubling times (Tpot) of cells treated with BaP, arsenic, and Ca2+ versus control populations (Fig. 10Go). As suggested by the histograms in Figures 7Go and 9Go, BaP, arsenic, and Ca2+ were all calculated to alter proliferative potential in treated NHEK. As expected from previous observations in the literature (Jensen et al., 1990Go), we observed a 3-h (9.2%) increase in Tpot in NHEK cells treated with 2 mM Ca2+ when compared to control cells. Arsenic at 2 and 5 µM had a similar effect, increasing Tpot in treated cells by 4.3 (12.5%) and 10.3 h (25.8%), respectively, as compared to controls. Interestingly, and in contrast to the decreased proliferative rates measured in Ca2+- and arsenic-treated cells, BaP at 0.2, 1, and 2 µM decreased Tpot by 5.4, 13.5, and 29.8%, respectively. This was equivalent to decreasing the Tpot by an average of 2.1, 3.6, and 15.1 h under the three treatment conditions.



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FIG. 10. Effects of benzo[a]pyrene, arsenic, and calcium on Tpot in NHEK. NHEK cells were treated with increasing concentrations of BaP or arsenic or with 2 mM Ca2+ for 24 h, stained with BrdU and propidium iodide, and analyzed by flow cytometry as described in Materials and Methods. Tpot for each culture was calculated as described by Steel (1977)Go and Wilson (1994)Go and is shown relative to that in untreated control cultures in terms of both change in h and percent change. Both arsenic (2 µM and 5 µM) and Ca2+ (2 mM) decreased proliferation rates, while BaP (1 µM and 2 µM) stimulated cell proliferation as compared to control NHEK. Twelve independent experiments were run. *Significant differences in Tpot were determined by one-way ANOVA followed by Dunnett’s test, p < 0.05. (A) is graphical representations of the data in (B).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Exposure to chemical carcinogens is thought to alter cellular signals that control the switch between keratinocyte division, differentiation, or apoptosis. However, the exact molecular and biochemical mechanisms involved in this process have not been clearly elucidated to date. In order to understand the impact of chemical exposure on keratinocyte function, it was first necessary to characterize normal differentiation in this cell type in vitro. It has previously been demonstrated that Ca2+ at concentrations in excess of 1 mM inhibits proliferation, induces stratification, and increases the formation of CLE in keratinocyte cultures (Rice and Green, 1979Go). Approximately this same concentration of Ca2+ leads to induction of involucrin and transglutamase, both of which play an intricate role in the terminal differentiation of this cell type. Our studies in vitro in NHEK support these findings and characterize the dose- and time-dependence of the induction of differentiation by Ca2+. The Tpot measurements also show a decreased rate of proliferation after Ca2+ treatment. Potential mechanisms for involvement of Ca2+ in differentiation include changes in cell adhesion and cell–cell contacts (Braga et al., 1995Go) and activation of a transmembrane Ca2+-dependent receptor (Denning et al., 2000Go).

Although arsenic has previously been studied in terms of its effects on expression of individual differentiation-associated markers in keratinocytes (Jessen et al., 2001Go; Kachinskas et al., 1994Go), our work is the first to demonstrate that the metal is a highly potent inhibitor of CLE formation as induced in vitro by confluence or the presence of exogenous Ca2+. One potential mechanism for the effects of arsenic on differentiation proposed by Kachinskas et al.(1994)Go involves inhibition of specific protein tyrosine phosphatases. Other mechanisms include suppression of p21 induction (Vogt and Rossman, 2001Go), altered receptor function and/or expression, and interference in PKC signaling (Chen et al., 2000Go).

The effect of arsenic on cell proliferation varies depending on both the cell type and concentration of the metal (Basu et al., 2001Go). Studies in keratinocytes demonstrated that low levels of arsenic or an arsenic-containing metal mixture significantly stimulated growth factor production and proliferation, while levels of arsenic associated with toxicity decreased cell division rates (Bae et al., 2001Go; Germolec et al., 1996Go). In our studies, Tpot increased (or proliferative rate decreased) in cultures treated with arsenic at 2 µM and higher, at least partly due to a G2/M phase delay, as has been seen with other cell types (Ma et al., 1998Go; States et al., 2002Go). One interesting observation from our studies was that arsenic was substantially more effective at inhibiting differentiation than proliferation. One possibility suggested by these findings is that, at lower concentrations, arsenic acts to stimulate proliferation and inhibit differentiation in primary keratinocytes, both potentially carcinogenic insults. At higher concentrations, cytotoxicity may lead ultimately to cell cycle blocks and apoptosis. Recent clinical studies in therapy of acute promyleocytic leukemia and ovarian carcinoma, among others, have taken advantage of the anticancer activity of arsenic (Li et al., 2002Go).

We have also been able to demonstrate that BaP had substantial impacts on both cell proliferation and differentiation in human keratinocytes. As seen with arsenic, inhibition of differentiation was observed at much lower concentrations of BaP than were alterations in proliferation. The effect of BaP on differentiation was more substantial in the absence of exogenous Ca2+ than in its presence. This finding is interesting, given the suggestion that in the murine system, induction of differentiation by Ca2+ increases the metabolic capacity of keratinocytes and thus increases activation of PAHs (Reiners et al., 1991Go). To date, there have been no published studies examining the effect of BaP on primary keratinocyte differentiation in vitro. However, in related work (Edmondson and Mossman, 1991Go), treatment of hamster tracheal epithelial cells with BaP in vitro leads to alterations in expression of multiple differentiation-related cytokeratins. The fact that BaP is capable of altering the differentiation program of committed cells may explain, in part, why exposure of the tracheal epithelium to BaP leads to metaplasia and squamous cell carcinoma in the lung (Chopra et al., 1991Go; Yoshimoto et al., 1980Go). Mutagenic alteration of regulatory molecules involved in growth and differentiation is the most likely mode of action for BaP in exposed cells. In previous studies, other genotoxic chemicals have been demonstrated to alter differentiation in skin (Huberman, 1980Go; Puri et al., 2002Go). However, one must not discount the possibility that additional, epigenetic mechanisms may also exist. In this case, metabolic activation may not be crucial, or even necessary, for the observed effects of the chemical.

One potential mechanism for the effects of BaP on differentiation is the inhibitory effect it can exert on PKC activity in certain cell types (Ou and Ramos, 1994Go). From several lines of study, it appears that different PKC isoforms are involved in steps during both early and late differentiation and that it is the ratios and locations of these different isoforms within a keratinocyte that may be important (Lee et al., 1997Go). Initiated keratinocytes derived from exposure to chemicals such as MNNG and DMBA display a resistance to differentiating agents due to alterations in the PKC pathway (Kulesz-Martin et al., 1983Go). Identification of other molecular targets altered in these cells, as well as the signal transduction pathways they are involved in, will greatly aid our understanding of the processes of growth, differentiation, and/or carcinogenesis in this cell type.

One thing that is clear from our studies is that cultures treated with BaP also progressed through the cell cycle more quickly, with a shorter S phase or G2/M or both. Although in our current studies, stimulatory effects on proliferation and inhibition of differentiation by BaP were generally detected within a day of each other, we did not determine the exact time course of these two events. One possibility is that by stimulating proliferative pathways in NHEK, BaP effectively opposes terminal arrest and subsequent differentiation. Alternatively, by first rendering a substantial population of keratinocytes resistant to signals that normally induce differentiation, BaP may effectively increase the number of cycling cells and, thus, decrease the potential doubling time as compared to untreated cultures. It will be enlightening to carry out detailed kinetic analyses of the two events to see if such a relationship can be defined.

Although there have been no descriptions of the effects of BaP on cell cycle kinetics in primary keratinocytes in culture, previous studies examining this issue have been carried out in the murine skin system (Reiners et al., 1991Go). Albert et al.(1991)Go demonstrated that, while the carcinogen was highly toxic to keratinocytes in vivo, it also acted to increase the labeling index in surviving cells. In these, and other, studies, proliferation correlated closely with tumor formation under the different exposure scenarios. In contrast, in other cell types in vitro, BaP has been shown to induce DNA adducts and a delayed S-phase, with or without a G2/M arrest (Jeffy et al., 2000Go; Mudzinski, 1993Go). Among cell cycle regulatory proteins potentially involved are p53 and p21WAF1 (Binkova et al., 2000Go). Although we are currently uncertain as to why our findings in keratinocytes contrast with these previous studies, there are many possible explanations, including the use of different cell types and experimental protocols. Additionally, it is possible that the population of cells surviving BaP may be genetically altered in such a way that they proliferate more rapidly. One potential scenario is due to the mutational loss of a crucial cell cycle checkpoint function. Similar effects have been seen with caffeine or 2-aminopurine (Ford et al., 1999Go).

The long-term goal of our research program is the development of accurate and efficient risk assessment strategies for environmentally relevant chemicals and chemical mixtures. One approach is through use of in vitro cell culture systems and biologically based dose-response models depicting toxic endpoints. The human keratinocyte cell system is useful for the study of potential carcinogens. As the cells undergo normal differentiation in culture, it is possible to explore the link between disruptions in this cellular process and malignant transformation. In studies described herein, we have demonstrated that, while the known human carcinogens, arsenic and BaP, are both effective inhibitors of differentiation in primary keratinocytes in vitro, they exert interesting, but opposite, effects on cell growth. We are currently carrying out microarray and Real Time RT-PCR analysis on NHEK cultures treated with arsenic or BaP to compare the important molecular alterations that are occurring under these different exposure conditions. Interactive effects of arsenic and BaP in this system may, in the future, be a productive avenue of exploration, as these two chemicals do occur together in environmentally prevalent mixtures, such as those at hazardous waste sites. A better understanding of the balance between growth and differentiation and how it is altered by defined chemicals or chemical mixtures during carcinogenesis will aid immeasurably in the risk assessment process.


    NOTES
 
1 To whom correspondence should be addressed at Center for Environmental Toxicology and Technology, Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO 80523. Fax: (970) 491-8304. E-mail: julie.campain{at}colostate.edu. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Albert, R. E., Miller, M. L., Cody, T. E., Barkley, W., and Shukla, R. (1991). Cell kinetics and benzo[a]pyrene-DNA adducts in mouse skin tumorigenesis. Prog. Clin. Biol. Res. 369, 115–122.[Medline]

Albert, R. E., Miller, M. L., Cody, T. E., Talaska, G., Underwood, P., and Andringa, A. (1996). Epidermal cytokinetics, DNA adducts, and dermal inflammation in the mouse skin in response to repeated benzo[a]pyrene exposures. Toxicol. Appl. Pharmacol. 136, 67–74.[CrossRef][ISI][Medline]

Bae, D. S., Gennings, C., Carter, W. H., Jr., Yang, R. S., and Campain, J. A. (2001). Toxicological interactions among arsenic, cadmium, chromium, and lead in human keratinocytes. Toxicol. Sci. 63, 132–142.[Abstract/Free Full Text]

Basu, A., Mahata, J., Gupta, S., and Giri, A. K. (2001). Genetic toxicology of a paradoxical human carcinogen, arsenic: a review. [Review] [183 refs]. Mutat. Res. 488, 171–194.[CrossRef][ISI][Medline]

Begg, A. C., McNally, N. J., Shrieve, D. C., and Karcher, H. (1985). A method to measure the duration of DNA synthesis and the potential doubling time from a single sample. Cytometry 6, 620–626.[ISI][Medline]

Binkova, B., Giguere, Y., Rossner, P., Jr., Dostal, M., and Sram, R. J. (2000). The effect of dibenzo[a,1]pyrene and benzo[a]pyrene on human diploid lung fibroblasts: The induction of DNA adducts, expression of p53 and p21(WAF1) proteins and cell cycle distribution. Mutat. Res. 471, 57–70.[ISI][Medline]

Braga, V. M., Hodivala, K. J., and Watt, F. M. (1995). Calcium-induced changes in distribution and solubility of cadherins, integrins and their associated cytoplasmic proteins in human keratinocytes. Cell Adhes. Commun. 3, 201–215.[ISI][Medline]

Chen, N. Y., Ma, W. Y., Huang, C., Ding, M., and Dong, Z. (2000). Activation of PKC is required for arsenite-induced signal transduction. J. Environ. Pathol. Toxicol. Oncol. 19, 297–305.[Medline]

Chopra, D. P., and Joiakim, A. P. (1991). Alterations in sugar residues in squamous metaplasia in hamster tracheal explants induced by benzo(a)pyrene and its reversal by retinoic acid. In vitro Cell Dev. Biol. 27A, 229–233.[ISI]

Denning, M. F., Dlugosz, A. A., Cheng, C., Dempsey, P. J., Coffey, R. J., Jr., Threadgill, D. W., Magnuson, T., and Yuspa, S. H. (2000). Cross-talk between epidermal growth factor receptor and protein kinase C during calcium-induced differentiation of keratinocytes. Exp. Dermatol. 9, 192–199.[CrossRef][ISI][Medline]

Denning, M. F., Dlugosz, A. A., Williams, E. K., Szallasi, Z., Blumberg, P. M., and Yuspa, S. H. (1995). Specific protein kinase C isozymes mediate the induction of keratinocyte differentiation markers by calcium. Cell Growth Differ. 6, 149–157.[Abstract]

Edmondson, S. W., and Mossman, B. T. (1991). Alterations in keratin expression in hamster tracheal epithelial cells exposed to benzo[a]pyrene. Carcinogenesis 12, 679–684.[Abstract]

Filvaroff, E., Stern, D. F., and Dotto, G. P. (1990). Tyrosine phosphorylation is an early and specific event involved in primary keratinocyte differentiation. Mol. Cell. Biol. 10, 1164–1173.[ISI][Medline]

Ford, H. L., and Pardee, A. B. (1999). Cancer and the cell cycle. [Review] [56 refs]. J. Cell. Biochem. Suppl. 32–33, 166–172.[CrossRef]

Germolec, D. R., Yoshida, T., Gaido, K., Wilmer, J. L., Simeonova, P. P., Kayama, F., Burleson, F., Dong, W., Lange, R. W., and Luster, M. I. (1996). Arsenic induces overexpression of growth factors in human keratinocytes. Toxicol. Appl. Pharmacol. 141, 308–318.[CrossRef][ISI][Medline]

Huberman, E. (1980). The induction of mutation and differentiation in mammalian cells by chemicals which initiate or promote tumor formation. Dev. Toxicol. Environ. Sci. 8, 121–132.[Medline]

Jeffy, B. D., Chen, E. J., Gudas, J. M., and Romagnolo, D. F. (2000). Disruption of cell cycle kinetics by benzo[a]pyrene: inverse expression patterns of BRCA-1 and p53 in MCF-7 cells arrested in S and G2. Neoplasia 2, 460–470.[CrossRef][ISI][Medline]

Jensen, P. K., Norgard, J. O., Knudsen, C., Nielsen, V., and Bolund, L. (1990). Effects of extra- and intracellular calcium concentration on DNA replication, lateral growth, and differentiation of human epidermal cells in culture. Virchows Arch. B Cell Pathol. Incl. Mol. Pathol. 59, 17–25.[ISI][Medline]

Jessen, B. A., Qin, Q., Phillips, M. A., Phillips, D. L., and Rice, R. H. (2001). Keratinocyte differentiation marker suppression by arsenic: mediation by AP1 response elements and antagonism by tetradecanoylphorbol acetate. Toxicol. Appl. Pharmacol. 174, 302–311.[CrossRef][ISI][Medline]

Kachinskas, D. J., Phillips, M. A., Qin, Q., Strokes, J. D., and Rice, R. H. (1994). Arsenate perturbation of human keratinocyte differentiation. Cell Growth Differ. 5, 12235–1241.

Kulesz-Martin, M., Kilkenny, A. E., Holbrook, K. A., Digernes, V., and Yuspa, S. H. (1983). Properties of carcinogen altered mouse epidermal cells resistant to calcium-induced terminal differentiation. Carcinogenesis 4, 1367–1377.[ISI][Medline]

Kuroki, T., Nemoto, N., and Kitano, Y. (1980). Metabolism of benzo[a]pyrene in human epidermal keratinocytes in culture. Carcinogenesis 1, 559–565.[ISI][Medline]

Larsen, J. K. (1994). Measurement of cytoplasmic and nuclear antigens. In Flow Cytometry: A Practical Approach, pp. 93–117. Oxford University Press, Oxford.

Lee, P. N., and O’Neill, J. A. (1971). The effect both of time and dose applied on tumour incidence rate in benzopyrene skin painting experiments. Br. J. Cancer 25, 759–770.[ISI][Medline]

Lee, Y. S., Dlugosz, A. A., McKay, R., Dean, N. M., and Yuspa, S. H. (1997). Difinition by specific antisense oligonucleotides of a role for protein C{alpha} in expression of differentiation markers in normal and neoplastic mouse epidermal keratinocytes. Mol. Carcinog. 18, 44–53.[CrossRef][ISI][Medline]

Leonard, A., and Lauwerys, R. R. (1980). Carcinogenicity, teratogenicity and mutagenicity of arsenic. Mutat. Res. 75, 49–62.[ISI][Medline]

Li, D., Du, C., Lin, Y., and Wu, M. (2002). Inhibition of growth of human nasopharyngeal cancer xenografts in SCID mice by arsenic trioxide. Tumori 88, 522–526.[ISI][Medline]

Liao, K. H., Gustafson, D. L., Fox, M. H., Chubb, L. S., Reardon, K. F., and Yang, R. S. (2001). A biologically based model of growth and senescence of Syrian hamster embryo (SHE) cells after exposure to arsenic. Environ. Health Perspect. 109, 1207–1213.[ISI][Medline]

Ma, D. C., Sun, Y. H., Chang, K. Z., Ma, X. F., Huang, S. L., Bai, Y. H., Kang, J., Liu, Y. G., and Chu, J. J. (1998). Selective induction of apoptosis of NB4 cells from G2+M phase by sodium arsenite at lower doses. Eur. J. Haematol. 61, 27–35.[ISI][Medline]

Mager, R., Huberman, E., Yang, S. K., Gelboin, H. V., and Sachs, L. (1977). Transformation of normal hamster cells by benzo(a)pyrene diol-epoxide. Int. J. Cancer 19, 814–817.[ISI][Medline]

Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55–63.[CrossRef][ISI][Medline]

Mudzinski, S. P. (1993). Effects of benzo[a]pyrene on concanavalin A-stimulated human peripheral blood mononuclear cells in vitro: inhibition of proliferation but no effect on parameters related to the G1 phase of the cell cycle. Toxicol. Appl. Pharmacol. 119, 166–174.[CrossRef][ISI][Medline]

Ou, X., and Ramos, K. S. (1994). Benzo[a]pyrene inhibits protein kinase C activity in subcultured rat aortic smooth muscle cells. Chem. Biol. Interact. 93, 29–40.[CrossRef][ISI][Medline]

Puri, P. L., Bhakta, K., Wood, L. D., Costanzo, A., Zhu, J., and Wang, J. Y. (2002). A myogenic differentiation checkpoint activated by genotoxic stress. Nat. Genet. 32, 585–593.[CrossRef][ISI][Medline]

Reiners, J. J., Jr., Cantu, A. R., and Pavone, A. (1991). Distribution of constitutive and polycyclic aromatic hydrocarbon-induced cytochrome P-450 activities in murine epidermal cells that differ in their stages of differentiation. Prog. Clin. Biol. Res. 369, 123–135.[Medline]

Rice, R. H., and Green, H. (1979). Presence in human epidermal cells of a soluble protein precursor of the cross-linked envelope: Activation of the cross-linking by calcium ions. Cell 18, 681–694.[ISI][Medline]

Salnikow, K., and Cohen, M. D. (2002). Backing into cancer: effects of arsenic on cell differentiation. [letter; comment.]. [Review] [31 refs]. Toxicol. Sci. 65, 161–163.[Abstract/Free Full Text]

States, J. C., Reiners, J. J., Jr., Pounds, J. G., Kaplan, D. J., Beauerle, B. D., McNeely, S. C., Mathieu, P., and McCabe, M. J., Jr. (2002). Arsenite disrupts mitosis and induces apoptosis in SV40-transformed human skin fibroblasts. Toxicol. Appl. Pharmacol. 180, 83–91.[CrossRef][ISI][Medline]

Steel, G. G. (1977). Growth kinetics of tumors: cell population kinetics in relation to the growth and treatment of cancer. In Basic Theory of Growing Cell Populations, pp. 57–85. Clarendon Press, Oxford.

Tamhane, A. C., and Dunlop, D. D. (2000). Multiple Comparisons of Means, pp. 475–476. Prentice-Hall, Upper Saddle River, NJ.

Tseng, W. P. (1977). Effects and dose–response relationships of skin cancer and blackfoot disease with arsenic. Environ. Health Perspect. 19, 109–119.[ISI][Medline]

Vega, L., Styblo, M., Patterson, R., Cullen, W., Wang, C., and Germolec, D. (2001). Differential effects of trivalent and pentavalent arsenicals on cell proliferation and cytokine secretion in normal human epidermal keratinocytes. Toxicol. Appl. Pharmacol. 172, 225–232.[CrossRef][ISI][Medline]

Vogt, B. L., and Rossman, T. G. (2001). Effects of arsenite on p53, p21 and cyclin D expression in normal human fibroblasts—a possible mechanism for arsenite’s comutagenicity. Mutat. Res. 478, 159–168.[ISI][Medline]

Wilson, G. D. (1994). Flow Cytometry: A Practical Approach. In Analysis of DNA-Measurement of Cell Kinetics by the Bromodeoxyuridine/Anti-Bromodeoxyuridine Method, pp. 137–156. Oxford University Press, Oxford.

Yoshimoto, T., Inoue, T., Iizuka, H., Nishikawa, H., Sakatani, M., Ogura, T., Hirao, F., and Yamamura, Y. (1980). Differential induction of squamous cell carcinomas and adenocarcinomas in mouse lung by intratracheal instillation of benzo(a)pyrene and charcoal powder. Cancer Res. 40, 4301–4307.[Abstract]





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