* Quantitative and Computational Toxicology Group, Center for Environmental Toxicology and Technology and
Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, Colorado 80523
Received June 10, 2003; accepted August 22, 2003
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ABSTRACT |
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Key Words: keratinocytes; differentiation; proliferation; benzo[a]pyrene; arsenic; calcium.
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INTRODUCTION |
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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., 1995; Filvaroff et al., 1990
). 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, 2002
). 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, 1977). 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, 1980
; Vogt and Rossman, 2001
). 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., 1996
; Kachinskas et al., 1994
; Vega et al., 2001
). 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., 1977). Many of the earliest studies of BaP were carried out using the murine skin system (Lee et al., 1971
). When applied dermally, BaP induces cytokinetic abnormalities and inflammation, followed by skin tumors (Albert et al., 1996
). The chemical alters differentiation in multiple cell types (Edmondson and Mossman, 1991
; Reiners et al., 1991
), 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.
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MATERIALS AND METHODS |
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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, 1983). 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., 1980
) 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, 1979). Briefly, NHEK were plated in 60-mm petri dishes at a density of 5000 cells/plate. When cells reached 6070% confluency after approximately 5 days, they were treated for 24 h with the following chemicals and concentrations: BaP [0.22 µM], arsenic [0.55 µM], Ca2+ [0.53 mM], arsenic [0.55 µM] + Ca2+ [2 mM], and BaP [0.22 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 (23 mM). After the treatment period, cultures were re-fed with chemical-free medium and incubated an additional 23 days until they reached 90100% 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, 1977; Wilson, 1994
). Briefly, cells were plated in 100-mm petri dishes at a density of 10,000 cells/plate. When cells reached 1015% 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 5060% 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)
and Liao et al.(2001)
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) 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., 1985
). Tpot is a cell division time that takes growth fraction, but not cell loss, into account (Steel, 1977
; Wilson, 1994
). Tpot was calculated with the equation: Tpot =
(TS/LI), where TS is the period of DNA synthesis, LI (labeling index) is the fraction of cells synthesizing DNA, and
is a correction factor for the nonlinear distribution of cells through the cell cycle (Steel, 1977
; Wilson, 1994
). Estimation of Tpot values relied upon distinction of cells within the four different stages of the cell cycle.
Statistical analysis.
Dunnetts one-way analysis of variance (ANOVA) was used to compare the difference of treated to control groups (Tamhane et al., 2000). 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.
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RESULTS |
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DISCUSSION |
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Although arsenic has previously been studied in terms of its effects on expression of individual differentiation-associated markers in keratinocytes (Jessen et al., 2001; Kachinskas et al., 1994
), 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)
involves inhibition of specific protein tyrosine phosphatases. Other mechanisms include suppression of p21 induction (Vogt and Rossman, 2001
), altered receptor function and/or expression, and interference in PKC signaling (Chen et al., 2000
).
The effect of arsenic on cell proliferation varies depending on both the cell type and concentration of the metal (Basu et al., 2001). 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., 2001
; Germolec et al., 1996
). 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., 1998
; States et al., 2002
). 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., 2002
).
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., 1991). 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, 1991
), 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., 1991
; Yoshimoto et al., 1980
). 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, 1980
; Puri et al., 2002
). 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, 1994). 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., 1997
). 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., 1983
). 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., 1991). Albert et al.(1991)
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., 2000
; Mudzinski, 1993
). Among cell cycle regulatory proteins potentially involved are p53 and p21WAF1 (Binkova et al., 2000
). 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., 1999
).
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.
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NOTES |
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