Short-Term Biomarkers of Cigarette Smoke Condensate Tumor Promoting Po
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《毒物学科学杂志》
Research and Development, R. J. Reynolds Tobacco Company, Winston-Salem, North Carolina 27102
The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78229
ABSTRACT
Previous studies demonstrated that cigarette smoke condensates (CSCs) possessing significantly different tumorigenic potentials according to a standardized 30-week mouse skin tumor-promotion protocol could likewise be discriminated utilizing short-term indices of sustained hyperplasia and/or inflammation (G. M. Curtin et al., 2004, Toxicol. Sci. 81, 14–25). The current study employed a truncated initiation-promotion protocol to further evaluate CSC-induced hyperplasia, examining issues related to time course of induction, existence of a threshold and suitable dynamic range for detectable responses, and reversibility. Condensate application (9–36 mg "tar"/200-μl application, thrice-weekly for 3–15 weeks) induced treatment-related increases for epidermal thickness, proliferative index as assessed by 5-bromo-2'-deoxyuridine (BrdU) labeling, and ornithine decarboxylase (ODC) expression. Interestingly, observed increases for interfollicular BrdU labeling and ODC expression were partially reversed but still elevated upon cessation of promotion, while increases within the perifollicular epidermis remained elevated at a level similar to that observed during CSC application. In particular, assessments based on perifollicular ODC expression would appear to provide a greater opportunity for test article discrimination based on a rapid time to induction, a low threshold and expanded dynamic range of responses, and the potential to account for irreversible changes. These findings are particularly intriguing based on reports suggesting that ODC expression may be necessary for tumor promotion and that mouse skin tumors originate primarily within the perifollicular epidermis.
Key Words: tumor promotion; hyperplasia; ornithine decarboxylase; perifollicular epidermis.
INTRODUCTION
While mouse skin studies have contributed significantly to the delineation of multistage carcinogenesis into seemingly distinct but complementary phases (DiGiovanni, 1992; Slaga et al., 1987; Yuspa and Poirier, 1988), the complex interactions likely underlying initiation, promotion, and progression potentially complicate interpretation of experimental results. For instance, promotion would appear to entail the selective propagation of genetically altered (initiated) cells through the induction of a strong and persistent hyperplastic response. However, this view may be somewhat deficient in explaining the functional role of promoters, with this phase likely encompassing two discriminate processes (Rubin, 2001).
The sequential application of croton oil (the active ingredient being 12-O-tetradecanoylphorbol-13-acetate, TPA) and turpentine to initiated mouse skin has been reported to induce a synergistic response, suggesting a qualitative difference between these two promoters (Boutwell, 1964). Similar results were observed when substituting wound healing for turpentine, leading investigators to suggest that the carcinogenic process entailed: (1) initiation to a pre-cancer state, carried out by a mutagen and involving irreversible genetic change (i.e., ras mutation); (2) conversion of an initiated cell into a dormant tumor cell (induced by croton oil, first-stage promotion); and, (3) propagation of the dormant tumor cell population, dependent upon cellular proliferation (induced by turpentine or wound healing, second-stage promotion).
The existence of two discriminate processes during promotion is likewise supported by studies (Furstenberger et al., 1983) employing limited TPA application, followed by long-term promotion with the TPA analogue, 12-O-retinoylphorbol-13-acetate (RPA). Interestingly, RPA was observed to retain some promotional activity in initiated mouse skin even when its repeated application was delayed after TPA exposure. Moreover, short-term TPA exposure of non-initiated mouse skin, followed by 7,12-dimethylbenz[a]anthracene (DMBA) initiation (delayed to avoid complications associated with TPA-induced hyperplasia and inflammation) and subsequent long-term application of second-stage promoter resulted in a tumor response comparable to conventional TPA application (Furstenberger et al., 1985).
Although tumor promoters may not interact directly with DNA or function as mutagens, experimental evidence suggests that chromosomal effects may be induced as a consequence of promoter treatment. Increases in gaps and chromatid breaks, accompanied by intra- and inter-chromosomal exchanges were observed for cultured mouse keratinocytes following TPA treatment (Dzarlieva-Petrusevska and Fusenig, 1985). During subsequent studies, double minute chromosomes, the cytogenetic equivalent of gene amplification, were likewise observed (Petrusevska et al., 1988). Interestingly, treatments that inhibited tumor induction by TPA in initiated mouse skin similarly inhibited chromosomal aberrations, suggesting a causal relationship between the aberrations and the process of conversion. In terms of mouse skin, dermal application of the alkylating and clastogenic agent, methyl methanesulfonate (MMS) was reported to induce chromosome breaks and gaps in epidermal cells, with no initiating activity observed (Furstenberger et al., 1989). MMS would appear to be an effective first-stage promoter, but as a weak inducer of DNA synthesis it had to be accompanied by a second-stage promoter to exert a promoting effect comparable to TPA.
The existence of non-Ha-ras, DMBA-responsive target genes capable of increasing tumor development within the context of a classical initiation-promotion protocol is supported by studies using the Tg.AC mouse model (Owens et al., 1995). Based on the transgenic expression of oncogenic v-Ha-ras within the skin, this mouse model no longer requires DMBA initiation for tumor formation; moreover, the available initiated cell population (due to transgene expression) would be expected to far exceed that induced with a chemical initiator. Limited DMBA initiation and subsequent TPA promotion of Tg.AC mice was reported to induce a 10-fold increase in tumor-related endpoints (compared to acetone/TPA controls), the implication being that there exist additional target genes that cooperate with mutated Ha-ras to increase tumorigenicity.
Substantial experimental evidence suggests that cigarette smoke condensate (CSC) is most appropriately evaluated in terms of tumor promoting potential (Hoffman and Wynder, 1971; reviewed in Ilgren, 1992; Nesnow et al., 1985; Slaga et al., 1996; Curtin et al., 2004). The objective of the current study was to extend earlier findings regarding condensate-induced hyperplasia during mouse skin carcinogenesis, and to identify short-term markers that may be more reflective of the complete tumor promoting potential of CSC, i.e., in terms of both conversion and propagation.
MATERIALS AND METHODS
Participating laboratories.
R. J. Reynolds Tobacco Co. (RJRT; Winston-Salem, NC) was responsible for the generation, collection, and chemical characterization of cigarette smoke condensate, as well as the dermal tumor-promotion portion of the study. Veritas Laboratories, Inc. (Burlington, NC) collected and prepared tissues utilized during subsequent in vivo analyses. Analyses of paraffin-embedded skin sections were conducted in laboratories of the AMC Cancer Research Center (Denver, CO), which have recently relocated to The University of Texas Health Science Center at San Antonio (San Antonio, TX).
Test and control articles.
Experimental cigarettes (supplied by RJRT) were smoked, and the corresponding condensate collected and characterized as previously described (Meckley et al., 2004a,b). CSC dosing solutions were prepared by serial dilution of pooled condensate to predetermined "tar" concentrations using 8% water in high purity acetone. "Tar" is defined as the mass of wet total particulate matter minus the mass of nicotine and water.
DMBA, used as the chemical initiator for appropriate control and experimental groups, was obtained from Sigma Corporation (St. Louis, MO); dosing solution was prepared using high-purity acetone, and subject to concentration analysis prior to use. OPTIMA grade acetone was purchased from Fisher Scientific (Fair Lawn, NJ).
Experimental animals and tumor promotion protocol.
An Institutional Animal Care and Use Committee evaluated all animal procedures associated with the present study and assured that all proposed methods were appropriate.
Female SENCAR mice (ages 6–7 weeks) were received from the National Cancer Institute, Frederick Cancer Research and Development Center (Frederick, MD), and placed into quarantine for two weeks prior to initiation of animal dosing. Based on published reports specific to SENCAR mice (Miller et al., 1993; Wilson et al., 1994), topical application of promoter was performed during the telogen (resting) phase of the second hair cycle; hence, the potential for increased sensitivity associated with the anagen (sustained growth) cycle was minimized.
Procedures for animal quarantine (including health screens), identification, and assignment to control or experimental groups have been described previously (Meckley et al., 2004a,b). Mice were housed and cared for in accordance with the Institute of Laboratory Animal Research (ILAR), Commission on Life Sciences, National Research Council document, Guide for the Care and Use of Laboratory Animals (NRC, 1996), as detailed by Meckley et al. (2004a,b).
Animals were provided ad libitum access to certified PMI Rodent Diet 5002 feed (Purina Mills, Inc.; St. Louis, MO), presented as pellets; each feed lot was analyzed (by the supplier) for trace components and contaminants. Water originated from the municipal supply and was subsequently filtered through activated carbon and 5-μm particulate filters prior to delivery to the animals. Water was provided ad libitum via an automatic system, with analysis for trace components and contaminants performed semi-annually.
Experimental groups (N = 35) were initiated with a single, 200-μl application of 75 μg DMBA in acetone. Beginning one week after the application of DMBA, mice were promoted thrice-weekly (for 3, 6, 9, 12, or 15 weeks) with 0, 9, 18, 27, or 36 mg "tar" per 200-μl application (Fig. 1). Termination of promotion at 9 weeks for reversibility assessments coincided with the initial appearance of skin masses, which generally begin to develop following 10–12 weeks of condensate application. Condensate exposure levels were based on results from previous tumor promotion studies, with dosing performed as described by Meckley et al. (2004a,b); this included a two-week acclimation period for the highest dose of CSC employed. Among the control groups were animals that were initiated with either DMBA or acetone (vehicle control), followed by promotion with acetone (vehicle control for CSC).
Procedures for daily observations (morbidity/mortality checks), as well as the collection of clinical signs, body weights, and mass tracking data were performed as previously described (Meckley et al., 2004a,b).
Collection of tissue from the chemical application site.
Skin samples were collected from the dosed area of each animal at the time of necropsy, i.e., 30 h following the last scheduled application of test article; approximately seven animals per group were sacrificed per treatment interval. Sections specified for histological evaluation were fixed in 10% neutral-buffered formalin.
Quantitative evaluation of epidermal hyperplasia.
Tissues utilized for histological evaluation were prepared using conventional paraffin sections and hematoxylin-eosin staining. Epidermal thickness was evaluated using an Olympus BX45 microscope (Melville, NY), with a minimum of 20 randomly selected sites (per animal) averaged; data is presented as the mean ± SD of seven animals (per treatment group).
Measurement of proliferative index.
For proliferative index analyses, mice were given an ip injection of BrdU (100 μl, 15 mg/ml 0.9% NaCl) 60 min prior to sacrifice. Tissue sections were immunostained with anti-BrdU antibody (1.0 μg IgG/ml) using the InnoGenex Mouse-on-Mouse Iso-IHC Kit (San Ramon, CA). Following deparaffinization and rehydration, slides were incubated in 1 mg/ml pepsin/0.1N HCl for 10 min at 37°C to enhance staining intensity; slides were then counterstained with hematoxylin and mounted in glycerol.
The number of BrdU-positive cells within the interfollicular epidermis (i.e., putative basal cells) was determined by counting an average of 500 cells for each tissue section; data is presented as the mean ± SD of three animals (per treatment group). For the perifollicular epidermis, cells adjacent to 4–6 hair follicles (predominantly 35–45 cells in length) were evaluated per animal, with data presented as the mean ± SD of three animals (per treatment group).
Immunohistochemical detection of ornithine decarboxylase expression.
For ODC expression analyses, tissue sections were immunostained using the mouse monoclonal antibody ODC Ab1 (Neomarkers, Lab Vision Corp.; Freemont, CA); this antibody recognizes the native ODC protein, and is reactive with human, rat, and mouse tissue. The number or percentage of ODC-positive cells in either the interfollicular or perifollicular epidermis was determined as previously detailed for proliferative index measurements.
Statistical analysis.
Data were analyzed using statistical tests available through the PATH/TOX computer software (version 4.2.2, Xybion Medical Systems; Cedar Knolls, NJ) and GraphPAD InStat software (San Diego, CA). Statistical procedures included means and SDs, Bartlett's test of homogeneity of variance, Dunnett's t-test, and Tukey-Kramer Multiple Comparisons Test; p values less than 0.05 were deemed statistically significant.
RESULTS
Clinical Observations and Body Weights for Control and Experimental Animals
Major abnormalities of the dosed skin, i.e., abnormalities observed within an experimental group at a frequency exceeding 10%, were limited to peeling skin. This clinical finding was noted for all animal groups promoted with CSC at concentrations 18 mg "tar"/application.
There were no significant differences (Dunnett's, p < 0.05) observed when comparing the group mean body weights of the CSC-promoted groups to the acetone-promoted (vehicle) control; hence, the experimental groups were not compared to each other.
Epidermal Thickness for Assessing Relative Hyperplastic Potential
Compared to acetone-promoted controls, CSC-dependent increases in epidermal thickness were observed at each time point associated with thrice-weekly application of promoter to DMBA-initiated mouse skin (Table 1). While increases observed at the lower CSC concentrations were enhanced with increased duration of promoter treatment, i.e., culminating in statistically significant (p < 0.05) increases for all applied doses following nine weeks of promotion, an effective dynamic range was not evident; specifically, statistically significant differences were not observed with increasing concentrations of CSC. Comparing the level of epidermal thickness for DMBA-initiated, acetone-promoted mouse skin versus that of the acetone-initiated control revealed that chemical initiation did not induce significant hyperplasia. Moreover, CSC promotion of acetone-initiated skin resulted in a substantial thickening of the epidermis, though this treatment regimen (acetone/CSC) fails to induce tumor formation in a manner different from the corresponding (acetone/acetone) controls (Meckley et al., 2004a).
While the statistical increases observed following nine weeks of CSC promotion (i.e., compared to acetone-promoted control) were generally retained during the six-week recovery period, treatment-related increases in epidermal thickness exhibited a tendency of continual decline in the absence of promoter application. The biological significance of these reductions (i.e., 27 to 38% for all dosing groups) remains unknown, but would appear to suggest reversibility of the CSC-induced changes in the absence of continued application. This conclusion is supported, in part, by the finding that DMBA-initiated mouse skin continually promoted with CSC (27 mg "tar"/application) exhibited statistically significant (p < 0.05) increases for epidermal thickness when compared to similarly promoted skin allowed six weeks to recover. Given the continual downward decline of epidermal thickening induced by CSC promotion of acetone-initiated control skin, there exists the possibility that this tissue eventually acclimates to the hyperplastic response induced by this promoter.
Proliferative Index for Assessing Relative Hyperplastic Potential
Proliferative index, assessed as a measure of BrdU labeling, was likewise used to evaluate the relative hyperplastic potential of CSC, with analyses conducted for both the interfollicular and perifollicular epidermis. Treatment-related increases for proliferative index were observed within the interfollicular epidermis at each time point associated with CSC promotion (Table 2, top panel). These increases were demonstrated to be statistically significant (p < 0.05) compared to acetone-promoted controls, and provided evidence of a reasonable dynamic range in terms of the elicited response.
Increased levels of BrdU labeling were retained during the six-week recovery period, with cessation of promoter treatment resulting in continued proliferative decline similar to that observed during assessments of epidermal thickness. Based on an experimental design that limited evaluations to six weeks recovery, the biological significance of these reductions (i.e., 20 to 38% for all dosing groups) remains unknown. Comparable levels of BrdU labeling were observed for mouse skin continually promoted with CSC (27 mg "tar"/application) and that similarly promoted but allowed six weeks to recover, with the trend of decreased labeling suggestive of tissue acclimation to promoter-induced hyperplasia. This conclusion is supported by results from the acetone/CSC group, as well as previous studies (Curtin et al., 2004) employing a similar experimental design.
Somewhat comparable results were obtained during an analysis of perifollicular epidermis (i.e., that region adjacent to and including the hair follicle) for CSC-induced increases in BrdU labeling (Table 2, bottom panel). Similar to that observed for the interfollicular epidermis, proliferative index was increased in a treatment-related and statistically significant (p < 0.05) manner during both promoter application and the corresponding six-week recovery period. While there was less evidence of an effective dynamic range in terms of CSC-induced changes, there was also less indication of tissue acclimation in response to the higher CSC application doses. Interestingly, CSC promotion of acetone-initiated skin resulted in substantial elevations in BrdU labeling for both the interfollicular and perifollicular epidermis. While the observed increases subsided even with continued promotion, the data effectively demonstrate that CSC possesses the potential to induce, at least transiently, the proliferation of non-initiated cells.
Ornithine Decarboxylase Expression for Assessing Epidermal Hyperplasia
ODC expression represented the last of the short-term indices used to assess the relative epidermal hyperplastic potential of CSC, with analyses likewise conducted for both the interfollicular and perifollicular epidermis. Dose-dependent increases for ODC expression were observed within the interfollicular epidermis at each time point associated with CSC promotion (Table 3, top panel). These increases were demonstrated to be statistically significant (p < 0.05) compared to acetone-promoted controls, and provided evidence of a reasonable dynamic range in terms of the elicited response.
Increased levels of ODC expression were retained during the six-week recovery period, with cessation of promoter application resulting in continued proliferative decline similar to that observed during assessments of epidermal thickness and interfollicular BrdU labeling. The notion of acclimation to CSC-induced hyperplasia is likewise supported by comparable levels of enzyme expression for mouse skin continually promoted with CSC (27 mg "tar"/application) and that similarly promoted but allowed six weeks to recover. Interestingly, ODC expression was elevated as a consequence of DMBA initiation, as evidenced by statistically significant (p < 0.05) increases for mouse skin initiated with DMBA and promoted with acetone compared to acetone-initiated control.
Somewhat comparable results were obtained during an analysis of perifollicular epidermis for CSC-induced increases in ODC expression (Table 3, bottom panel). Similar to that observed for the interfollicular epidermis, ODC expression was increased in a dose-dependent and statistically significant (p < 0.05) manner during both CSC promotion and the corresponding six-week recovery period. In fact, it would appear that ODC expression remained elevated following cessation of CSC promotion at each of the application doses tested, a finding that was unique to this series of experiments. Similarly unique was the finding that changes in perifollicular ODC expression induced during CSC promotion of acetone-initiated skin were significantly (p < 0.05) reduced compared to CSC promotion of DMBA-initiated skin.
Ultimately, the CSC-induced ODC expression levels within the perifollicular epidermis represented some of the largest changes for indices employed to assess hyperplastic potential, approaching 10-fold increases compared to acetone-promoted controls. Moreover, the dynamic range of ODC expression evidenced within the perifollicular epidermis would likely provide the additional advantage of exhibiting statistically significant differences with increasing concentrations of CSC application. In contrast to findings for the interfollicular epidermis, there was no detectable increase for ODC expression as a consequence of DMBA initiation.
DISCUSSION
Histological observations of mouse skin treated topically with promoting agents have revealed that sustained epidermal hyperplasia plays a critical role in tumor promotion (Klein-Szanto and Slaga, 1981, 1982; Naito et al., 1987; Slaga et al., 1976; Spalding et al., 1993). Indeed, a qualitative correlation between sustained, potentiated epidermal hyperplasia and tumor promotion has been established for a number of structurally diverse chemicals (Argyris, 1985, 1989; DiGiovanni, 1992; Kruszewski et al., 1989; Naito et al., 1987; Walborg et al., 1998).
Postulating that the inherent tumorigenic activity associated with CSC is largely due to the selection and propagation of initiated cells, previous studies evaluated endpoints associated with sustained hyperplasia and/or inflammation in response to condensates possessing significantly different tumor-promoting potentials (Curtin et al., 2004). Attempts to correlate short-term endpoint data with both total tumors and percent tumor-bearing animals provided by the corresponding 30-week tumor promotion assay (Meckley et al., 2004b) yielded favorable results. Specifically, proliferative index and myeloperoxidase activity were highly correlated with total tumors and tumor-bearing animals (r > 0.95) following 4–12 weeks of promotion. Nonetheless, caution may be warranted when correlating these putative biomarkers with tumor formation based on the consideration that the condensate-induced changes ameliorated with continued test article application. Moreover, while difficult to assess using conventional in vivo testing models, condensate likely possesses genotoxic activity that is not directly related to Ha-ras mutation.
The current studies utilized condensate collected from a tobacco-burning cigarette, applied according to either a truncated or continued promotion schedule, in an attempt to identify biomarkers more reflective of the complex interactions underlying tumor promotion, i.e., accounting for both first- and second-stage promotion. Based on earlier results demonstrating that truncated condensate exposure induces a moderate tumor response even if terminated prior to tumor formation (Curtin et al., unpublished data), it was hypothesized that CSC promotion was capable of inducing irreversible change necessary for tumor development. Moreover, it was assumed that this change would likely cooperate with altered ras signaling, leading to dysregulated proliferative control and/or increased responsiveness to proliferative signaling.
Results from the current studies confirm that CSC possesses the potential for inducing statistically significant increases in epidermal thickness following promotion through nine weeks. However, epidermal thickness following cessation of CSC promotion was statistically reduced compared to that observed with continual promotion, with little evidence of the dynamic range of responses required for effective test article differentiation. Ultimately, data from successive studies suggest that CSC-induced increases in epidermal thickness are treatment-related, but reversible in the absence of continued promoter treatment or due to tissue acclimation (Curtin et al., 2004).
Within the interfollicular epidermis, CSC was observed to induce treatment-related and statistically significant increases in proliferative activity, assessed in terms of BrdU labeling. While a lower threshold and greater dynamic range of responses were evident earlier during the promotion regimen, BrdU labeling levels were found to exhibit downward trends following cessation of CSC exposure in a manner similar to that observed for epidermal thickness. Mouse skin continually promoted with CSC likewise exhibited an eventual downward trend similar to that observed in response to truncated promotion, indicating some level of tissue acclimation for the interfollicular epidermis. Assessments of BrdU labeling within the perifollicular epidermis provided less indication of tissue acclimation in response to the higher application doses of CSC, but also less evidence of an effective dynamic range in terms of induced changes. The observation that BrdU labeling tended to remain unchanged following cessation of promoter application, at least at the higher CSC concentrations tested, suggests that some portion of the perifollicular cell population was irreversibly committed to continued proliferation.
Ornithine decarboxylase expression, suggested by other laboratories to provide a promotion stimulus that is necessary for tumor formation (Clifford et al., 1995; Feith et al., 2001; Gilmour et al., 1992; O'Brien et al., 1997; Smith et al., 1998; Soler et al., 1998), was likewise examined within the interfollicular and perifollicular epidermis. For interfollicular epidermis, CSC was observed to induce dose-dependent and statistically significant increases in ODC expression, with cessation of promoter exposure leading to a downward trend similar to that observed for epidermal thickness and interfollicular BrdU labeling. The inability of continued promotion to induce ODC expression to a level different from that observed with truncated exposure was viewed as reaffirming the notion that some level of tissue acclimation occurs for the interfollicular epidermis during CSC promotion.
In contrast, CSC-induced ODC expression within the perifollicular epidermis tended to remain unchanged following cessation of promoter treatment, suggesting that some portion of the cell population was irreversibly committed to constitutive ODC expression in response to truncated promotion. While consistent with BrdU labeling data from the perifollicular epidermis, ODC expression provided the additional benefits of irreversibility at each of the CSC application doses examined and an expanded dynamic range of induced responses, i.e., 10-fold increases relative to vehicle controls, with statistically significant differences at each increased concentration of CSC. Recalling that an objective of these studies was to identify a putative biomarker that would account for genotoxic changes (i.e., first-stage promotion) likely contributing to tumor promoting potential and capable of complementing the effects of initiation, it may be noteworthy that ODC expression within the perifollicular epidermis was significantly reduced for skin treated with acetone and promoted with CSC compared to DMBA-initiated, CSC-promoted skin.
As to the relevance of these findings, CSC was demonstrated to induce proliferation and ODC expression in a dose-dependent and potentially irreversible manner within a region of the epidermis postulated to be the primary origin of mouse skin tumorigenesis. Support for the notion that most initiated cells reside within the perifollicular region was initially provided by studies in which cutaneous papillomas were effectively promoted in initiated mouse skin despite removal of the interfollicular epidermis (Argyris, 1980; Argyris and Slaga, 1981). These findings were confirmed using 5-fluorouracil, a treatment that causes extensive sloughing of interfollicular epidermis (Morris et al., 1997). More recently, it was reported that the interfollicular epidermis of abraded mice was observed to quickly regenerate from cells within the hair follicles (Morris et al., 2000). Subsequent promotion induced approximately half the number of papillomas of unabraded mice, suggesting that the targets for tumor development are stem cells found primarily within perifollicular epidermis.
Studies examining the contribution of ODC during tumor promotion (Gilmour et al., 1992), as well as the utility of K6/ODC Tg mice for studying tumorigenesis (O'Brien et al., 1997; Smith et al., 1998) similarly provide evidence that the perifollicular epidermis is central to tumorigenesis. Transgenic mice with ODC targeted to perifollicular keratinocytes were observed to no longer require promoter treatment for the development of tumors, while the targeting of ODC over-expression to both interfollicular and perifollicular keratinocytes failed to enhance DMBA-induced tumor formation (O'Brien et al., 1997). Moreover, the early development of spontaneous tumors in double transgenic mice, possessing elevated ODC directed to the outer root sheath cells of hair follicles (K6/ODC Tg) and carrying a v-Ha-ras transgene (TG.AC), suggests that ODC expression may be necessary for tumor promotion (Smith et al., 1998). Recent findings from our laboratory (Curtin et al., unpublished data) suggest a significant role for ODC expression during condensate-induced tumor formation. Specifically, SENCAR mice initiated with DMBA and provided 0.5% difluoromethylornithine (a specific inhibitor of ODC expression, presented in drinking water) during promotion with CSC (27 mg "tar"/application) developed 0.2 tumors/animal through 15 weeks of dosing, whereas similarly promoted mice not receiving the inhibitor developed 3 tumors/animal.
The current studies extend previous findings regarding the discrimination of tumor promoters using short-term indices of sustained hyperplasia (Curtin et al., 2004). While proliferative indices associated with the interfollicular epidermis effectively demonstrate treatment-related increases in response to CSC promotion, the biological relevance of these changes may be tempered by considerations related to reversibility of the elicited response (and/or tissue acclimation), as well as experimental evidence suggesting that the targets for skin tumor development are found primarily within perifollicular epidermis. In contrast, proliferative indices associated with the perifollicular epidermis, in particular ODC expression, provide the advantage of a low threshold and expanded dynamic range of responses with short-term CSC promotion. Based on experimental evidence demonstrating that this test article is likely incapable of effectively initiating mouse skin, statistical differences resulting from CSC promotion of initiated and non-initiated tissue may provide additional information regarding the induction of first- and second-stage promotion.
ACKNOWLEDGMENTS
Several of the authors, including the corresponding author, are employed by RJ Reynolds Tobacco Company (RJRT). In addition, this research was funded by RJRT; the funding organization does not have control over the resulting publication.
REFERENCES
Argyris, T. S. (1980). Tumor production by abrasion induced epidermal hyperplasia in the skin of mice. J. Invest. Dermatol. 75, 360–362.
Argyris, T. S. (1985). Regeneration and the mechanism of epidermal tumor promotion. CRC Crypt. Rev. Toxicol. 14, 211–258.
Argyris, T. S. (1989). Epidermal tumor promotion by damage in the skin of mice. In Progress in Experimental Tumor Research, Skin Carcinogenesis: Mechanisms and Human Relevance (T. J. Slaga, A. J. P. Klein-Santo, R. K. Boutwell, D. E. Stevenson, H. L. Spitzer, and D. D'Amato, Eds.), pp. 63–80. R. Less, New York.
Argyris, T. S., and Slaga, T. J. (1981). Promotion of carcinomas by repeated abrasion in initiated skin of mice. Cancer Res. 41, 5193–5195.
Boutwell, R. K. (1964). Some biological aspects of skin carcinogenesis. Prog. Exp. Tumor Res. 4, 207–250.
Clifford, A., Morgan, D., Yuspa, S. H., Soler, A. P., and Gilmour, S. (1995). Role of ornithine decarboxylase in epidermal tumorigenesis. Cancer Res. 55, 1680–1686.
Curtin, G. M., Hanausek, M., Walaszek, Z., Mosberg, A. T., and Slaga, T. J. (2004). Short-term in vitro and in vivo analyses for assessing the tumor promoting potentials of cigarette smoke condensates. Toxicol. Sci. 81, 14–25.
DiGiovanni, J. (1992). Multistage carcinogenesis in mouse skin. Pharmacol. Ther. 47, 63–128.
Dzarlieva-Petrusevska, R. T., and Fusenig, N. E. (1985). Tumor promoter 12-O-tetradecanolyphorbol-13-acetate (TPA)-induced chromosome aberrations in mouse keratinocyte cell lines: A possible genetic mechanism of tumor promotion. Carcinogenesis 6, 1447–1456.
Feith, D. J., Shantz, L. M., and Pegg, A. E. (2001). Targeted antizyme expression in the skin of transgenic mice reduces tumor promoter induction of ornithine decarboxylase and decreases sensitivity to chemical carcinogenesis. Cancer Res. 61, 6073–6081.
Furstenberger, G., Kinzel, V., Schwarz, M., and Marks, F. (1985). Partial inversion of multistage tumorigenesis in the skin of NMRI mice. Science 230, 76–78.
Furstenberger, G., Sorg, B., and Marks, F. (1983). Tumor promotion by phorbol esters in skin: Evidence for a memory effect. Science 220, 89–91.
Furstenberger, G., Schurich, B., Kaina, B., Petrusevska, R. T., Fusenig, N. E., and Marks, F. (1989). Tumor induction in initiated mouse skin by phorbol esters and methyl methanesulfonate: Correlation between chromosome damage and conversion (stage 1 of tumor promotion) in vivo. Carcinogenesis 10, 749–752.
Gilmour, S. K., Robertson, F. M., Megosh, L., O'Connell, S. M., Mitchell, J., and O'Brien, T. G. (1992). Induction of ornithine decarboxylase in specific subpopulations of murine epidermal cells following multiple exposures to 12-O-tetradecanoylphorbol-13-acetate, mezerein and ethyl phenylpropriolate. Carcinogenesis 13, 51–56.
Hoffman, D., and Wynder, E. (1971). A study of tobacco carcinogenesis. XI. Tumor initiators, tumor accelerators, and tumor promoting activity of condensate fractions. Cancer 27, 848–864.
Ilgren, E. B. (1992). Initiation and Promotion in Skin or Liver Neoplasia: A 65-year Annotated Bibliography of International Literature. CRC Press, Boca Raton, FL.
Klein-Szanto, A. J. P., and Slaga, T. J. (1981). Numerical variation of dark cells in normal and chemically induced hyperplastic epidermis with age and efficiency of tumor promoter. Cancer Res. 41, 4437–4440.
Klein-Szanto, A. J. P., and Slaga, T. J. (1982). Effects of peroxides on rodent skin: Epidermal hyperplasia and tumor promotion. J. Invest. Dermatol. 79, 30–34.
Kruszewski, F. H., Naito, M., Naito, Y., and DiGiovanni, J. (1989). Histologic alterations produced by chrysarobin (1,8-dihydroxy-3-methyl-9-anthrone) in SENCAR mouse skin. Relationship to skin tumor promoting activity. J. Invest. Dermatol. 92, 64–71.
Meckley, D. R., Hayes, J. R., Van Kampen, K. R., Mosberg, A. T., and Swauger, J. E. (2004a). A responsive, sensitive, and reproducible dermal tumor promotion assay for the comparative evaluation of cigarette smoke condensates. Regul. Toxicol. Pharmacol. 39, 135–149.
Meckley, D. R., Hayes, J. R., Van Kampen, K. R., Ayres, P. H., Mosberg, A. T., and Swauger, J. E. (2004b). Comparative study of smoke condensates from 1R4F cigarettes that burn tobacco versus ECLIPSE cigarettes that primarily heat tobacco in the SENCAR mouse dermal tumor promotion assay. Food Chem. Toxicol. 42, 851–863.
Miller, S. J., Wei, Z.-G., Wilson, C., Dzubow, L., Sun, T.-T., and Lavker, R. M. (1993). Mouse skin is particularly susceptible to tumor initiation during early anagen of the hair cycle: Possible involvement of hair follicle stem cells. J. Invest. Dermatol. 101, 591–594.
Morris, R. J., Coulter, K., Tryson, K., and Steinberg, S. R. (1997). Evidence that cutaneous carcinogen-initiated epithelial cells from mice are quiescent rather than actively cycling. Cancer Res. 57, 3436–3443.
Morris, R. J., Tryson, K. A., and Wu, K. Q. (2000). Evidence that the epidermal targets of carcinogen action are found in the interfollicular epidermis or infundibulum as well as in the hair follicles. Cancer Res. 60, 226–229.
Naito, M., Naito, Y., and DiGiovanni, J. (1987). Comparison of the histological changes in the skin of DBA/2 and C57BL/6 mice following exposure to various promoting agents. Carcinogenesis 8, 1807–1815.
National Research Council (1996). Guide for the Care and Use of Laboratory Animals. Institute of Laboratory Animal Research, Commission on Life Sciences, Washington, DC.
Nesnow, S., Triplett, L., and Slaga, T. (1985). Studies on the tumor initiating, tumor promoting, and tumor co-initiating properties of respiratory carcinogens. In Carcinogenesis, Vol. 8 (M. Mass et al., Eds.), pp. 257–277. Raven Press, New York.
O'Brien, T. G., Megosh, L. C., Gilliard, G., and Soler, A. P. (1997). Ornithine decarboxylase over-expression is a sufficient condition for tumor promotion in mouse skin. Cancer Res. 57, 2630–2637.
Owens, D. M., Spalding, J. W., Tennant, R. W., and Smart, R. C. (1995). Genetic alterations cooperate with v-Ha-ras to accelerate multistage carcinogenesis in TG.AC transgenic mouse skin. Cancer Res. 55, 3171–3178.
Petrusevska, R. T., Furstenberger, G., Marks, F., and Fusenig, N. E. (1988). Cytogenetic effects caused by phorbol ester tumor promotion in primary mouse keratinocyte cultures: Correlation with the convertogenic activity of TPA in multistage skin carcinogenesis. Carcinogenesis 9, 1207–1215.
Rubin, H. (2001). Synergistic mechanisms in carcinogenesis by polycyclic aromatic hydrocarbons and by tobacco smoke: A bio-historical perspective with updates. Carcinogenesis 22, 1903–1930.
Slaga, T. J., Budunova, I. R., Gimenez-Conti, I. R., and Aldaz, C. M. (1996). The mouse skin carcinogenesis model. J. Investig. Dermatol. Symp. Proc. 1, 151–156.
Slaga, T. J., O'Connell, J., Rotstein, J., Patskan, G., Morris, R., Aldaz, M., and Conti, C. (1987). Critical genetic determinants and molecular events in multistage skin carcinogenesis. Symp. Fundam. Cancer Res. 39, 31–34.
Slaga, T. J., Scribner, J. D., Thompson, S., and Viaje, A. (1976). Epidermal cell proliferation and promoting ability of phorbol esters. J. Natl. Cancer Inst. 57, 1145–1149.
Smith, M. K., Trempus, C. S., and Gilmour, S. K. (1998). Co-operation between follicular ornithine decarboxylase and v-Ha-ras induces spontaneous papillomas and malignant conversion in transgenic skin. Carcinogenesis 19, 1409–1415.
Soler, A. P., Gilliard, G., Megosh, L., George, K., and O'Brien, T. G. (1998). Polyamines regulate expression of the neoplastic phenotype in mouse skin. Cancer Res. 58, 1654–1659.
Spalding, J., Nomma, J., Elwell, M. R., and Tennant, R. (1993). Chemically induced skin carcinogenesis in a transgenic mouse line (TG-AC) carrying a v-Ha-ras gene. Carcinogenesis 14, 1335–1341.
Walborg, E. F., DiGiovanni, J., Conti, C. J., Slaga, T. J., Freeman, J. J., Steup, D. R., and Skisak, C. M. (1998). Short-term biomarkers of tumor promotion in mouse skin treated with petroleum middle distillates. Toxicol. Sci. 45, 137–145.
Wilson, C., Cotsarelis, G., Wei, Z.-G., Fryer, E., Margolis-Fryer, J., Ostead, M., Tokarek, R., Sun, T.-T., and Lavker, R. M. (1994). Cells within the bulge region of mouse hair follicle transiently proliferate during early anagen: Heterogeneity and functional differences of various hair cycles. Differentiation 55, 127–136.
Yuspa, S. H., and Poirier, M. C. (1988). Skin carcinogenesis. Adv. Cancer Res. 50, 25–69.(Geoffrey M. Curtin, Margaret Hanausek, Z)
The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78229
ABSTRACT
Previous studies demonstrated that cigarette smoke condensates (CSCs) possessing significantly different tumorigenic potentials according to a standardized 30-week mouse skin tumor-promotion protocol could likewise be discriminated utilizing short-term indices of sustained hyperplasia and/or inflammation (G. M. Curtin et al., 2004, Toxicol. Sci. 81, 14–25). The current study employed a truncated initiation-promotion protocol to further evaluate CSC-induced hyperplasia, examining issues related to time course of induction, existence of a threshold and suitable dynamic range for detectable responses, and reversibility. Condensate application (9–36 mg "tar"/200-μl application, thrice-weekly for 3–15 weeks) induced treatment-related increases for epidermal thickness, proliferative index as assessed by 5-bromo-2'-deoxyuridine (BrdU) labeling, and ornithine decarboxylase (ODC) expression. Interestingly, observed increases for interfollicular BrdU labeling and ODC expression were partially reversed but still elevated upon cessation of promotion, while increases within the perifollicular epidermis remained elevated at a level similar to that observed during CSC application. In particular, assessments based on perifollicular ODC expression would appear to provide a greater opportunity for test article discrimination based on a rapid time to induction, a low threshold and expanded dynamic range of responses, and the potential to account for irreversible changes. These findings are particularly intriguing based on reports suggesting that ODC expression may be necessary for tumor promotion and that mouse skin tumors originate primarily within the perifollicular epidermis.
Key Words: tumor promotion; hyperplasia; ornithine decarboxylase; perifollicular epidermis.
INTRODUCTION
While mouse skin studies have contributed significantly to the delineation of multistage carcinogenesis into seemingly distinct but complementary phases (DiGiovanni, 1992; Slaga et al., 1987; Yuspa and Poirier, 1988), the complex interactions likely underlying initiation, promotion, and progression potentially complicate interpretation of experimental results. For instance, promotion would appear to entail the selective propagation of genetically altered (initiated) cells through the induction of a strong and persistent hyperplastic response. However, this view may be somewhat deficient in explaining the functional role of promoters, with this phase likely encompassing two discriminate processes (Rubin, 2001).
The sequential application of croton oil (the active ingredient being 12-O-tetradecanoylphorbol-13-acetate, TPA) and turpentine to initiated mouse skin has been reported to induce a synergistic response, suggesting a qualitative difference between these two promoters (Boutwell, 1964). Similar results were observed when substituting wound healing for turpentine, leading investigators to suggest that the carcinogenic process entailed: (1) initiation to a pre-cancer state, carried out by a mutagen and involving irreversible genetic change (i.e., ras mutation); (2) conversion of an initiated cell into a dormant tumor cell (induced by croton oil, first-stage promotion); and, (3) propagation of the dormant tumor cell population, dependent upon cellular proliferation (induced by turpentine or wound healing, second-stage promotion).
The existence of two discriminate processes during promotion is likewise supported by studies (Furstenberger et al., 1983) employing limited TPA application, followed by long-term promotion with the TPA analogue, 12-O-retinoylphorbol-13-acetate (RPA). Interestingly, RPA was observed to retain some promotional activity in initiated mouse skin even when its repeated application was delayed after TPA exposure. Moreover, short-term TPA exposure of non-initiated mouse skin, followed by 7,12-dimethylbenz[a]anthracene (DMBA) initiation (delayed to avoid complications associated with TPA-induced hyperplasia and inflammation) and subsequent long-term application of second-stage promoter resulted in a tumor response comparable to conventional TPA application (Furstenberger et al., 1985).
Although tumor promoters may not interact directly with DNA or function as mutagens, experimental evidence suggests that chromosomal effects may be induced as a consequence of promoter treatment. Increases in gaps and chromatid breaks, accompanied by intra- and inter-chromosomal exchanges were observed for cultured mouse keratinocytes following TPA treatment (Dzarlieva-Petrusevska and Fusenig, 1985). During subsequent studies, double minute chromosomes, the cytogenetic equivalent of gene amplification, were likewise observed (Petrusevska et al., 1988). Interestingly, treatments that inhibited tumor induction by TPA in initiated mouse skin similarly inhibited chromosomal aberrations, suggesting a causal relationship between the aberrations and the process of conversion. In terms of mouse skin, dermal application of the alkylating and clastogenic agent, methyl methanesulfonate (MMS) was reported to induce chromosome breaks and gaps in epidermal cells, with no initiating activity observed (Furstenberger et al., 1989). MMS would appear to be an effective first-stage promoter, but as a weak inducer of DNA synthesis it had to be accompanied by a second-stage promoter to exert a promoting effect comparable to TPA.
The existence of non-Ha-ras, DMBA-responsive target genes capable of increasing tumor development within the context of a classical initiation-promotion protocol is supported by studies using the Tg.AC mouse model (Owens et al., 1995). Based on the transgenic expression of oncogenic v-Ha-ras within the skin, this mouse model no longer requires DMBA initiation for tumor formation; moreover, the available initiated cell population (due to transgene expression) would be expected to far exceed that induced with a chemical initiator. Limited DMBA initiation and subsequent TPA promotion of Tg.AC mice was reported to induce a 10-fold increase in tumor-related endpoints (compared to acetone/TPA controls), the implication being that there exist additional target genes that cooperate with mutated Ha-ras to increase tumorigenicity.
Substantial experimental evidence suggests that cigarette smoke condensate (CSC) is most appropriately evaluated in terms of tumor promoting potential (Hoffman and Wynder, 1971; reviewed in Ilgren, 1992; Nesnow et al., 1985; Slaga et al., 1996; Curtin et al., 2004). The objective of the current study was to extend earlier findings regarding condensate-induced hyperplasia during mouse skin carcinogenesis, and to identify short-term markers that may be more reflective of the complete tumor promoting potential of CSC, i.e., in terms of both conversion and propagation.
MATERIALS AND METHODS
Participating laboratories.
R. J. Reynolds Tobacco Co. (RJRT; Winston-Salem, NC) was responsible for the generation, collection, and chemical characterization of cigarette smoke condensate, as well as the dermal tumor-promotion portion of the study. Veritas Laboratories, Inc. (Burlington, NC) collected and prepared tissues utilized during subsequent in vivo analyses. Analyses of paraffin-embedded skin sections were conducted in laboratories of the AMC Cancer Research Center (Denver, CO), which have recently relocated to The University of Texas Health Science Center at San Antonio (San Antonio, TX).
Test and control articles.
Experimental cigarettes (supplied by RJRT) were smoked, and the corresponding condensate collected and characterized as previously described (Meckley et al., 2004a,b). CSC dosing solutions were prepared by serial dilution of pooled condensate to predetermined "tar" concentrations using 8% water in high purity acetone. "Tar" is defined as the mass of wet total particulate matter minus the mass of nicotine and water.
DMBA, used as the chemical initiator for appropriate control and experimental groups, was obtained from Sigma Corporation (St. Louis, MO); dosing solution was prepared using high-purity acetone, and subject to concentration analysis prior to use. OPTIMA grade acetone was purchased from Fisher Scientific (Fair Lawn, NJ).
Experimental animals and tumor promotion protocol.
An Institutional Animal Care and Use Committee evaluated all animal procedures associated with the present study and assured that all proposed methods were appropriate.
Female SENCAR mice (ages 6–7 weeks) were received from the National Cancer Institute, Frederick Cancer Research and Development Center (Frederick, MD), and placed into quarantine for two weeks prior to initiation of animal dosing. Based on published reports specific to SENCAR mice (Miller et al., 1993; Wilson et al., 1994), topical application of promoter was performed during the telogen (resting) phase of the second hair cycle; hence, the potential for increased sensitivity associated with the anagen (sustained growth) cycle was minimized.
Procedures for animal quarantine (including health screens), identification, and assignment to control or experimental groups have been described previously (Meckley et al., 2004a,b). Mice were housed and cared for in accordance with the Institute of Laboratory Animal Research (ILAR), Commission on Life Sciences, National Research Council document, Guide for the Care and Use of Laboratory Animals (NRC, 1996), as detailed by Meckley et al. (2004a,b).
Animals were provided ad libitum access to certified PMI Rodent Diet 5002 feed (Purina Mills, Inc.; St. Louis, MO), presented as pellets; each feed lot was analyzed (by the supplier) for trace components and contaminants. Water originated from the municipal supply and was subsequently filtered through activated carbon and 5-μm particulate filters prior to delivery to the animals. Water was provided ad libitum via an automatic system, with analysis for trace components and contaminants performed semi-annually.
Experimental groups (N = 35) were initiated with a single, 200-μl application of 75 μg DMBA in acetone. Beginning one week after the application of DMBA, mice were promoted thrice-weekly (for 3, 6, 9, 12, or 15 weeks) with 0, 9, 18, 27, or 36 mg "tar" per 200-μl application (Fig. 1). Termination of promotion at 9 weeks for reversibility assessments coincided with the initial appearance of skin masses, which generally begin to develop following 10–12 weeks of condensate application. Condensate exposure levels were based on results from previous tumor promotion studies, with dosing performed as described by Meckley et al. (2004a,b); this included a two-week acclimation period for the highest dose of CSC employed. Among the control groups were animals that were initiated with either DMBA or acetone (vehicle control), followed by promotion with acetone (vehicle control for CSC).
Procedures for daily observations (morbidity/mortality checks), as well as the collection of clinical signs, body weights, and mass tracking data were performed as previously described (Meckley et al., 2004a,b).
Collection of tissue from the chemical application site.
Skin samples were collected from the dosed area of each animal at the time of necropsy, i.e., 30 h following the last scheduled application of test article; approximately seven animals per group were sacrificed per treatment interval. Sections specified for histological evaluation were fixed in 10% neutral-buffered formalin.
Quantitative evaluation of epidermal hyperplasia.
Tissues utilized for histological evaluation were prepared using conventional paraffin sections and hematoxylin-eosin staining. Epidermal thickness was evaluated using an Olympus BX45 microscope (Melville, NY), with a minimum of 20 randomly selected sites (per animal) averaged; data is presented as the mean ± SD of seven animals (per treatment group).
Measurement of proliferative index.
For proliferative index analyses, mice were given an ip injection of BrdU (100 μl, 15 mg/ml 0.9% NaCl) 60 min prior to sacrifice. Tissue sections were immunostained with anti-BrdU antibody (1.0 μg IgG/ml) using the InnoGenex Mouse-on-Mouse Iso-IHC Kit (San Ramon, CA). Following deparaffinization and rehydration, slides were incubated in 1 mg/ml pepsin/0.1N HCl for 10 min at 37°C to enhance staining intensity; slides were then counterstained with hematoxylin and mounted in glycerol.
The number of BrdU-positive cells within the interfollicular epidermis (i.e., putative basal cells) was determined by counting an average of 500 cells for each tissue section; data is presented as the mean ± SD of three animals (per treatment group). For the perifollicular epidermis, cells adjacent to 4–6 hair follicles (predominantly 35–45 cells in length) were evaluated per animal, with data presented as the mean ± SD of three animals (per treatment group).
Immunohistochemical detection of ornithine decarboxylase expression.
For ODC expression analyses, tissue sections were immunostained using the mouse monoclonal antibody ODC Ab1 (Neomarkers, Lab Vision Corp.; Freemont, CA); this antibody recognizes the native ODC protein, and is reactive with human, rat, and mouse tissue. The number or percentage of ODC-positive cells in either the interfollicular or perifollicular epidermis was determined as previously detailed for proliferative index measurements.
Statistical analysis.
Data were analyzed using statistical tests available through the PATH/TOX computer software (version 4.2.2, Xybion Medical Systems; Cedar Knolls, NJ) and GraphPAD InStat software (San Diego, CA). Statistical procedures included means and SDs, Bartlett's test of homogeneity of variance, Dunnett's t-test, and Tukey-Kramer Multiple Comparisons Test; p values less than 0.05 were deemed statistically significant.
RESULTS
Clinical Observations and Body Weights for Control and Experimental Animals
Major abnormalities of the dosed skin, i.e., abnormalities observed within an experimental group at a frequency exceeding 10%, were limited to peeling skin. This clinical finding was noted for all animal groups promoted with CSC at concentrations 18 mg "tar"/application.
There were no significant differences (Dunnett's, p < 0.05) observed when comparing the group mean body weights of the CSC-promoted groups to the acetone-promoted (vehicle) control; hence, the experimental groups were not compared to each other.
Epidermal Thickness for Assessing Relative Hyperplastic Potential
Compared to acetone-promoted controls, CSC-dependent increases in epidermal thickness were observed at each time point associated with thrice-weekly application of promoter to DMBA-initiated mouse skin (Table 1). While increases observed at the lower CSC concentrations were enhanced with increased duration of promoter treatment, i.e., culminating in statistically significant (p < 0.05) increases for all applied doses following nine weeks of promotion, an effective dynamic range was not evident; specifically, statistically significant differences were not observed with increasing concentrations of CSC. Comparing the level of epidermal thickness for DMBA-initiated, acetone-promoted mouse skin versus that of the acetone-initiated control revealed that chemical initiation did not induce significant hyperplasia. Moreover, CSC promotion of acetone-initiated skin resulted in a substantial thickening of the epidermis, though this treatment regimen (acetone/CSC) fails to induce tumor formation in a manner different from the corresponding (acetone/acetone) controls (Meckley et al., 2004a).
While the statistical increases observed following nine weeks of CSC promotion (i.e., compared to acetone-promoted control) were generally retained during the six-week recovery period, treatment-related increases in epidermal thickness exhibited a tendency of continual decline in the absence of promoter application. The biological significance of these reductions (i.e., 27 to 38% for all dosing groups) remains unknown, but would appear to suggest reversibility of the CSC-induced changes in the absence of continued application. This conclusion is supported, in part, by the finding that DMBA-initiated mouse skin continually promoted with CSC (27 mg "tar"/application) exhibited statistically significant (p < 0.05) increases for epidermal thickness when compared to similarly promoted skin allowed six weeks to recover. Given the continual downward decline of epidermal thickening induced by CSC promotion of acetone-initiated control skin, there exists the possibility that this tissue eventually acclimates to the hyperplastic response induced by this promoter.
Proliferative Index for Assessing Relative Hyperplastic Potential
Proliferative index, assessed as a measure of BrdU labeling, was likewise used to evaluate the relative hyperplastic potential of CSC, with analyses conducted for both the interfollicular and perifollicular epidermis. Treatment-related increases for proliferative index were observed within the interfollicular epidermis at each time point associated with CSC promotion (Table 2, top panel). These increases were demonstrated to be statistically significant (p < 0.05) compared to acetone-promoted controls, and provided evidence of a reasonable dynamic range in terms of the elicited response.
Increased levels of BrdU labeling were retained during the six-week recovery period, with cessation of promoter treatment resulting in continued proliferative decline similar to that observed during assessments of epidermal thickness. Based on an experimental design that limited evaluations to six weeks recovery, the biological significance of these reductions (i.e., 20 to 38% for all dosing groups) remains unknown. Comparable levels of BrdU labeling were observed for mouse skin continually promoted with CSC (27 mg "tar"/application) and that similarly promoted but allowed six weeks to recover, with the trend of decreased labeling suggestive of tissue acclimation to promoter-induced hyperplasia. This conclusion is supported by results from the acetone/CSC group, as well as previous studies (Curtin et al., 2004) employing a similar experimental design.
Somewhat comparable results were obtained during an analysis of perifollicular epidermis (i.e., that region adjacent to and including the hair follicle) for CSC-induced increases in BrdU labeling (Table 2, bottom panel). Similar to that observed for the interfollicular epidermis, proliferative index was increased in a treatment-related and statistically significant (p < 0.05) manner during both promoter application and the corresponding six-week recovery period. While there was less evidence of an effective dynamic range in terms of CSC-induced changes, there was also less indication of tissue acclimation in response to the higher CSC application doses. Interestingly, CSC promotion of acetone-initiated skin resulted in substantial elevations in BrdU labeling for both the interfollicular and perifollicular epidermis. While the observed increases subsided even with continued promotion, the data effectively demonstrate that CSC possesses the potential to induce, at least transiently, the proliferation of non-initiated cells.
Ornithine Decarboxylase Expression for Assessing Epidermal Hyperplasia
ODC expression represented the last of the short-term indices used to assess the relative epidermal hyperplastic potential of CSC, with analyses likewise conducted for both the interfollicular and perifollicular epidermis. Dose-dependent increases for ODC expression were observed within the interfollicular epidermis at each time point associated with CSC promotion (Table 3, top panel). These increases were demonstrated to be statistically significant (p < 0.05) compared to acetone-promoted controls, and provided evidence of a reasonable dynamic range in terms of the elicited response.
Increased levels of ODC expression were retained during the six-week recovery period, with cessation of promoter application resulting in continued proliferative decline similar to that observed during assessments of epidermal thickness and interfollicular BrdU labeling. The notion of acclimation to CSC-induced hyperplasia is likewise supported by comparable levels of enzyme expression for mouse skin continually promoted with CSC (27 mg "tar"/application) and that similarly promoted but allowed six weeks to recover. Interestingly, ODC expression was elevated as a consequence of DMBA initiation, as evidenced by statistically significant (p < 0.05) increases for mouse skin initiated with DMBA and promoted with acetone compared to acetone-initiated control.
Somewhat comparable results were obtained during an analysis of perifollicular epidermis for CSC-induced increases in ODC expression (Table 3, bottom panel). Similar to that observed for the interfollicular epidermis, ODC expression was increased in a dose-dependent and statistically significant (p < 0.05) manner during both CSC promotion and the corresponding six-week recovery period. In fact, it would appear that ODC expression remained elevated following cessation of CSC promotion at each of the application doses tested, a finding that was unique to this series of experiments. Similarly unique was the finding that changes in perifollicular ODC expression induced during CSC promotion of acetone-initiated skin were significantly (p < 0.05) reduced compared to CSC promotion of DMBA-initiated skin.
Ultimately, the CSC-induced ODC expression levels within the perifollicular epidermis represented some of the largest changes for indices employed to assess hyperplastic potential, approaching 10-fold increases compared to acetone-promoted controls. Moreover, the dynamic range of ODC expression evidenced within the perifollicular epidermis would likely provide the additional advantage of exhibiting statistically significant differences with increasing concentrations of CSC application. In contrast to findings for the interfollicular epidermis, there was no detectable increase for ODC expression as a consequence of DMBA initiation.
DISCUSSION
Histological observations of mouse skin treated topically with promoting agents have revealed that sustained epidermal hyperplasia plays a critical role in tumor promotion (Klein-Szanto and Slaga, 1981, 1982; Naito et al., 1987; Slaga et al., 1976; Spalding et al., 1993). Indeed, a qualitative correlation between sustained, potentiated epidermal hyperplasia and tumor promotion has been established for a number of structurally diverse chemicals (Argyris, 1985, 1989; DiGiovanni, 1992; Kruszewski et al., 1989; Naito et al., 1987; Walborg et al., 1998).
Postulating that the inherent tumorigenic activity associated with CSC is largely due to the selection and propagation of initiated cells, previous studies evaluated endpoints associated with sustained hyperplasia and/or inflammation in response to condensates possessing significantly different tumor-promoting potentials (Curtin et al., 2004). Attempts to correlate short-term endpoint data with both total tumors and percent tumor-bearing animals provided by the corresponding 30-week tumor promotion assay (Meckley et al., 2004b) yielded favorable results. Specifically, proliferative index and myeloperoxidase activity were highly correlated with total tumors and tumor-bearing animals (r > 0.95) following 4–12 weeks of promotion. Nonetheless, caution may be warranted when correlating these putative biomarkers with tumor formation based on the consideration that the condensate-induced changes ameliorated with continued test article application. Moreover, while difficult to assess using conventional in vivo testing models, condensate likely possesses genotoxic activity that is not directly related to Ha-ras mutation.
The current studies utilized condensate collected from a tobacco-burning cigarette, applied according to either a truncated or continued promotion schedule, in an attempt to identify biomarkers more reflective of the complex interactions underlying tumor promotion, i.e., accounting for both first- and second-stage promotion. Based on earlier results demonstrating that truncated condensate exposure induces a moderate tumor response even if terminated prior to tumor formation (Curtin et al., unpublished data), it was hypothesized that CSC promotion was capable of inducing irreversible change necessary for tumor development. Moreover, it was assumed that this change would likely cooperate with altered ras signaling, leading to dysregulated proliferative control and/or increased responsiveness to proliferative signaling.
Results from the current studies confirm that CSC possesses the potential for inducing statistically significant increases in epidermal thickness following promotion through nine weeks. However, epidermal thickness following cessation of CSC promotion was statistically reduced compared to that observed with continual promotion, with little evidence of the dynamic range of responses required for effective test article differentiation. Ultimately, data from successive studies suggest that CSC-induced increases in epidermal thickness are treatment-related, but reversible in the absence of continued promoter treatment or due to tissue acclimation (Curtin et al., 2004).
Within the interfollicular epidermis, CSC was observed to induce treatment-related and statistically significant increases in proliferative activity, assessed in terms of BrdU labeling. While a lower threshold and greater dynamic range of responses were evident earlier during the promotion regimen, BrdU labeling levels were found to exhibit downward trends following cessation of CSC exposure in a manner similar to that observed for epidermal thickness. Mouse skin continually promoted with CSC likewise exhibited an eventual downward trend similar to that observed in response to truncated promotion, indicating some level of tissue acclimation for the interfollicular epidermis. Assessments of BrdU labeling within the perifollicular epidermis provided less indication of tissue acclimation in response to the higher application doses of CSC, but also less evidence of an effective dynamic range in terms of induced changes. The observation that BrdU labeling tended to remain unchanged following cessation of promoter application, at least at the higher CSC concentrations tested, suggests that some portion of the perifollicular cell population was irreversibly committed to continued proliferation.
Ornithine decarboxylase expression, suggested by other laboratories to provide a promotion stimulus that is necessary for tumor formation (Clifford et al., 1995; Feith et al., 2001; Gilmour et al., 1992; O'Brien et al., 1997; Smith et al., 1998; Soler et al., 1998), was likewise examined within the interfollicular and perifollicular epidermis. For interfollicular epidermis, CSC was observed to induce dose-dependent and statistically significant increases in ODC expression, with cessation of promoter exposure leading to a downward trend similar to that observed for epidermal thickness and interfollicular BrdU labeling. The inability of continued promotion to induce ODC expression to a level different from that observed with truncated exposure was viewed as reaffirming the notion that some level of tissue acclimation occurs for the interfollicular epidermis during CSC promotion.
In contrast, CSC-induced ODC expression within the perifollicular epidermis tended to remain unchanged following cessation of promoter treatment, suggesting that some portion of the cell population was irreversibly committed to constitutive ODC expression in response to truncated promotion. While consistent with BrdU labeling data from the perifollicular epidermis, ODC expression provided the additional benefits of irreversibility at each of the CSC application doses examined and an expanded dynamic range of induced responses, i.e., 10-fold increases relative to vehicle controls, with statistically significant differences at each increased concentration of CSC. Recalling that an objective of these studies was to identify a putative biomarker that would account for genotoxic changes (i.e., first-stage promotion) likely contributing to tumor promoting potential and capable of complementing the effects of initiation, it may be noteworthy that ODC expression within the perifollicular epidermis was significantly reduced for skin treated with acetone and promoted with CSC compared to DMBA-initiated, CSC-promoted skin.
As to the relevance of these findings, CSC was demonstrated to induce proliferation and ODC expression in a dose-dependent and potentially irreversible manner within a region of the epidermis postulated to be the primary origin of mouse skin tumorigenesis. Support for the notion that most initiated cells reside within the perifollicular region was initially provided by studies in which cutaneous papillomas were effectively promoted in initiated mouse skin despite removal of the interfollicular epidermis (Argyris, 1980; Argyris and Slaga, 1981). These findings were confirmed using 5-fluorouracil, a treatment that causes extensive sloughing of interfollicular epidermis (Morris et al., 1997). More recently, it was reported that the interfollicular epidermis of abraded mice was observed to quickly regenerate from cells within the hair follicles (Morris et al., 2000). Subsequent promotion induced approximately half the number of papillomas of unabraded mice, suggesting that the targets for tumor development are stem cells found primarily within perifollicular epidermis.
Studies examining the contribution of ODC during tumor promotion (Gilmour et al., 1992), as well as the utility of K6/ODC Tg mice for studying tumorigenesis (O'Brien et al., 1997; Smith et al., 1998) similarly provide evidence that the perifollicular epidermis is central to tumorigenesis. Transgenic mice with ODC targeted to perifollicular keratinocytes were observed to no longer require promoter treatment for the development of tumors, while the targeting of ODC over-expression to both interfollicular and perifollicular keratinocytes failed to enhance DMBA-induced tumor formation (O'Brien et al., 1997). Moreover, the early development of spontaneous tumors in double transgenic mice, possessing elevated ODC directed to the outer root sheath cells of hair follicles (K6/ODC Tg) and carrying a v-Ha-ras transgene (TG.AC), suggests that ODC expression may be necessary for tumor promotion (Smith et al., 1998). Recent findings from our laboratory (Curtin et al., unpublished data) suggest a significant role for ODC expression during condensate-induced tumor formation. Specifically, SENCAR mice initiated with DMBA and provided 0.5% difluoromethylornithine (a specific inhibitor of ODC expression, presented in drinking water) during promotion with CSC (27 mg "tar"/application) developed 0.2 tumors/animal through 15 weeks of dosing, whereas similarly promoted mice not receiving the inhibitor developed 3 tumors/animal.
The current studies extend previous findings regarding the discrimination of tumor promoters using short-term indices of sustained hyperplasia (Curtin et al., 2004). While proliferative indices associated with the interfollicular epidermis effectively demonstrate treatment-related increases in response to CSC promotion, the biological relevance of these changes may be tempered by considerations related to reversibility of the elicited response (and/or tissue acclimation), as well as experimental evidence suggesting that the targets for skin tumor development are found primarily within perifollicular epidermis. In contrast, proliferative indices associated with the perifollicular epidermis, in particular ODC expression, provide the advantage of a low threshold and expanded dynamic range of responses with short-term CSC promotion. Based on experimental evidence demonstrating that this test article is likely incapable of effectively initiating mouse skin, statistical differences resulting from CSC promotion of initiated and non-initiated tissue may provide additional information regarding the induction of first- and second-stage promotion.
ACKNOWLEDGMENTS
Several of the authors, including the corresponding author, are employed by RJ Reynolds Tobacco Company (RJRT). In addition, this research was funded by RJRT; the funding organization does not have control over the resulting publication.
REFERENCES
Argyris, T. S. (1980). Tumor production by abrasion induced epidermal hyperplasia in the skin of mice. J. Invest. Dermatol. 75, 360–362.
Argyris, T. S. (1985). Regeneration and the mechanism of epidermal tumor promotion. CRC Crypt. Rev. Toxicol. 14, 211–258.
Argyris, T. S. (1989). Epidermal tumor promotion by damage in the skin of mice. In Progress in Experimental Tumor Research, Skin Carcinogenesis: Mechanisms and Human Relevance (T. J. Slaga, A. J. P. Klein-Santo, R. K. Boutwell, D. E. Stevenson, H. L. Spitzer, and D. D'Amato, Eds.), pp. 63–80. R. Less, New York.
Argyris, T. S., and Slaga, T. J. (1981). Promotion of carcinomas by repeated abrasion in initiated skin of mice. Cancer Res. 41, 5193–5195.
Boutwell, R. K. (1964). Some biological aspects of skin carcinogenesis. Prog. Exp. Tumor Res. 4, 207–250.
Clifford, A., Morgan, D., Yuspa, S. H., Soler, A. P., and Gilmour, S. (1995). Role of ornithine decarboxylase in epidermal tumorigenesis. Cancer Res. 55, 1680–1686.
Curtin, G. M., Hanausek, M., Walaszek, Z., Mosberg, A. T., and Slaga, T. J. (2004). Short-term in vitro and in vivo analyses for assessing the tumor promoting potentials of cigarette smoke condensates. Toxicol. Sci. 81, 14–25.
DiGiovanni, J. (1992). Multistage carcinogenesis in mouse skin. Pharmacol. Ther. 47, 63–128.
Dzarlieva-Petrusevska, R. T., and Fusenig, N. E. (1985). Tumor promoter 12-O-tetradecanolyphorbol-13-acetate (TPA)-induced chromosome aberrations in mouse keratinocyte cell lines: A possible genetic mechanism of tumor promotion. Carcinogenesis 6, 1447–1456.
Feith, D. J., Shantz, L. M., and Pegg, A. E. (2001). Targeted antizyme expression in the skin of transgenic mice reduces tumor promoter induction of ornithine decarboxylase and decreases sensitivity to chemical carcinogenesis. Cancer Res. 61, 6073–6081.
Furstenberger, G., Kinzel, V., Schwarz, M., and Marks, F. (1985). Partial inversion of multistage tumorigenesis in the skin of NMRI mice. Science 230, 76–78.
Furstenberger, G., Sorg, B., and Marks, F. (1983). Tumor promotion by phorbol esters in skin: Evidence for a memory effect. Science 220, 89–91.
Furstenberger, G., Schurich, B., Kaina, B., Petrusevska, R. T., Fusenig, N. E., and Marks, F. (1989). Tumor induction in initiated mouse skin by phorbol esters and methyl methanesulfonate: Correlation between chromosome damage and conversion (stage 1 of tumor promotion) in vivo. Carcinogenesis 10, 749–752.
Gilmour, S. K., Robertson, F. M., Megosh, L., O'Connell, S. M., Mitchell, J., and O'Brien, T. G. (1992). Induction of ornithine decarboxylase in specific subpopulations of murine epidermal cells following multiple exposures to 12-O-tetradecanoylphorbol-13-acetate, mezerein and ethyl phenylpropriolate. Carcinogenesis 13, 51–56.
Hoffman, D., and Wynder, E. (1971). A study of tobacco carcinogenesis. XI. Tumor initiators, tumor accelerators, and tumor promoting activity of condensate fractions. Cancer 27, 848–864.
Ilgren, E. B. (1992). Initiation and Promotion in Skin or Liver Neoplasia: A 65-year Annotated Bibliography of International Literature. CRC Press, Boca Raton, FL.
Klein-Szanto, A. J. P., and Slaga, T. J. (1981). Numerical variation of dark cells in normal and chemically induced hyperplastic epidermis with age and efficiency of tumor promoter. Cancer Res. 41, 4437–4440.
Klein-Szanto, A. J. P., and Slaga, T. J. (1982). Effects of peroxides on rodent skin: Epidermal hyperplasia and tumor promotion. J. Invest. Dermatol. 79, 30–34.
Kruszewski, F. H., Naito, M., Naito, Y., and DiGiovanni, J. (1989). Histologic alterations produced by chrysarobin (1,8-dihydroxy-3-methyl-9-anthrone) in SENCAR mouse skin. Relationship to skin tumor promoting activity. J. Invest. Dermatol. 92, 64–71.
Meckley, D. R., Hayes, J. R., Van Kampen, K. R., Mosberg, A. T., and Swauger, J. E. (2004a). A responsive, sensitive, and reproducible dermal tumor promotion assay for the comparative evaluation of cigarette smoke condensates. Regul. Toxicol. Pharmacol. 39, 135–149.
Meckley, D. R., Hayes, J. R., Van Kampen, K. R., Ayres, P. H., Mosberg, A. T., and Swauger, J. E. (2004b). Comparative study of smoke condensates from 1R4F cigarettes that burn tobacco versus ECLIPSE cigarettes that primarily heat tobacco in the SENCAR mouse dermal tumor promotion assay. Food Chem. Toxicol. 42, 851–863.
Miller, S. J., Wei, Z.-G., Wilson, C., Dzubow, L., Sun, T.-T., and Lavker, R. M. (1993). Mouse skin is particularly susceptible to tumor initiation during early anagen of the hair cycle: Possible involvement of hair follicle stem cells. J. Invest. Dermatol. 101, 591–594.
Morris, R. J., Coulter, K., Tryson, K., and Steinberg, S. R. (1997). Evidence that cutaneous carcinogen-initiated epithelial cells from mice are quiescent rather than actively cycling. Cancer Res. 57, 3436–3443.
Morris, R. J., Tryson, K. A., and Wu, K. Q. (2000). Evidence that the epidermal targets of carcinogen action are found in the interfollicular epidermis or infundibulum as well as in the hair follicles. Cancer Res. 60, 226–229.
Naito, M., Naito, Y., and DiGiovanni, J. (1987). Comparison of the histological changes in the skin of DBA/2 and C57BL/6 mice following exposure to various promoting agents. Carcinogenesis 8, 1807–1815.
National Research Council (1996). Guide for the Care and Use of Laboratory Animals. Institute of Laboratory Animal Research, Commission on Life Sciences, Washington, DC.
Nesnow, S., Triplett, L., and Slaga, T. (1985). Studies on the tumor initiating, tumor promoting, and tumor co-initiating properties of respiratory carcinogens. In Carcinogenesis, Vol. 8 (M. Mass et al., Eds.), pp. 257–277. Raven Press, New York.
O'Brien, T. G., Megosh, L. C., Gilliard, G., and Soler, A. P. (1997). Ornithine decarboxylase over-expression is a sufficient condition for tumor promotion in mouse skin. Cancer Res. 57, 2630–2637.
Owens, D. M., Spalding, J. W., Tennant, R. W., and Smart, R. C. (1995). Genetic alterations cooperate with v-Ha-ras to accelerate multistage carcinogenesis in TG.AC transgenic mouse skin. Cancer Res. 55, 3171–3178.
Petrusevska, R. T., Furstenberger, G., Marks, F., and Fusenig, N. E. (1988). Cytogenetic effects caused by phorbol ester tumor promotion in primary mouse keratinocyte cultures: Correlation with the convertogenic activity of TPA in multistage skin carcinogenesis. Carcinogenesis 9, 1207–1215.
Rubin, H. (2001). Synergistic mechanisms in carcinogenesis by polycyclic aromatic hydrocarbons and by tobacco smoke: A bio-historical perspective with updates. Carcinogenesis 22, 1903–1930.
Slaga, T. J., Budunova, I. R., Gimenez-Conti, I. R., and Aldaz, C. M. (1996). The mouse skin carcinogenesis model. J. Investig. Dermatol. Symp. Proc. 1, 151–156.
Slaga, T. J., O'Connell, J., Rotstein, J., Patskan, G., Morris, R., Aldaz, M., and Conti, C. (1987). Critical genetic determinants and molecular events in multistage skin carcinogenesis. Symp. Fundam. Cancer Res. 39, 31–34.
Slaga, T. J., Scribner, J. D., Thompson, S., and Viaje, A. (1976). Epidermal cell proliferation and promoting ability of phorbol esters. J. Natl. Cancer Inst. 57, 1145–1149.
Smith, M. K., Trempus, C. S., and Gilmour, S. K. (1998). Co-operation between follicular ornithine decarboxylase and v-Ha-ras induces spontaneous papillomas and malignant conversion in transgenic skin. Carcinogenesis 19, 1409–1415.
Soler, A. P., Gilliard, G., Megosh, L., George, K., and O'Brien, T. G. (1998). Polyamines regulate expression of the neoplastic phenotype in mouse skin. Cancer Res. 58, 1654–1659.
Spalding, J., Nomma, J., Elwell, M. R., and Tennant, R. (1993). Chemically induced skin carcinogenesis in a transgenic mouse line (TG-AC) carrying a v-Ha-ras gene. Carcinogenesis 14, 1335–1341.
Walborg, E. F., DiGiovanni, J., Conti, C. J., Slaga, T. J., Freeman, J. J., Steup, D. R., and Skisak, C. M. (1998). Short-term biomarkers of tumor promotion in mouse skin treated with petroleum middle distillates. Toxicol. Sci. 45, 137–145.
Wilson, C., Cotsarelis, G., Wei, Z.-G., Fryer, E., Margolis-Fryer, J., Ostead, M., Tokarek, R., Sun, T.-T., and Lavker, R. M. (1994). Cells within the bulge region of mouse hair follicle transiently proliferate during early anagen: Heterogeneity and functional differences of various hair cycles. Differentiation 55, 127–136.
Yuspa, S. H., and Poirier, M. C. (1988). Skin carcinogenesis. Adv. Cancer Res. 50, 25–69.(Geoffrey M. Curtin, Margaret Hanausek, Z)