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Introduction

Nonmelanoma skin cancer (NMSC), including basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), is the most common skin cancer in non-Hispanic whites. Although NMSC-related deaths are rare, more than 1 million new NMSC cases are diagnosed annually in the United States, a number approximately equivalent to the total new cases of all other cancers combined (Jemal et al., 2006). NMSC is found primarily on sun-exposed sites, and a fair complexion and frequent exposure to sunlight are the primary risk factors (Armstrong et al., 1997; English et al., 1997; Strom and Yamamura, 1997; Armstrong and Kricker, 2001; Diepgen and Mahler, 2002). Another possible risk factor is suboptimal cellular DNA repair capacity, because patients with xeroderma pigmentosum have inherited defects in DNA repair and have a more-than-1,000-fold increased risk of sunlight-induced skin cancer (Kraemer et al., 1984; Kraemer et al., 1994; Lambert et al., 1995; Sarasin and Stary, 1997; Norgauer et al., 2003). This suggests that suboptimal DNA repair capacity could be a risk factor for skin cancer in the general population (Wei et al., 1993, 1994a, 1994b; Matta et al., 2003).

One key to the prevention of skin cancer is early identification of at-risk individuals and early detection of the disease. In that effort, biomarkers for genetic susceptibility to skin cancer are vital, but currently few biomarkers are available to serve this purpose. The mutagen sensitivity assay, which was developed by Dr T.C. Hsu (Cherry and Hsu, 1983), measures the number of mutagen-induced chromatid breaks per cell (b/c) in cultured primary peripheral blood lymphocytes. With bleomycin, a radiomimetic chemical, as a test mutagen, Dr Hsu and co-workers successfully used the assay to investigate genetic susceptibility to tobacco-related cancers (Cherry and Hsu, 1983; Spitz et al., 1989, 1995; Fireman et al., 1994; Cloos et al., 1996; Ankathil et al., 1999). Similarly, other test mutagens, such as 4-nitroquinoline-1-oxide (4-NQO), an UV-mimetic chemical (Nagao and Sugimura, 1976) that can cause bulky DNA adducts and chromosomal aberrations in exposed cells (Darroudi et al., 1989), are also used to determine genetic susceptibility to various types of cancer (Hsu et al., 1993a, 1993b; Gu et al., 1999).

In an early pilot study, Hsu et al. (1993a, 1993b) found that lymphocytes from melanoma patients were more susceptible to 4-NQO than those from head and neck cancer patients or healthy subjects. Therefore, we thought that in vitro 4-NQO-induced chromatid breaks in blood lymphocytes could also be used as a biomarker for studying genetic susceptibility to cutaneous malignancies with much simplified experimental procedures compared with those of the assay that uses UV as a mutagen. Since 4-NQO is a UV-mimetic and adduct-forming carcinogen that causes DNA damage uniquely repaired by global genome repair (Snyderwine and Bohr, 1992), it has been widely used in experimental research for cellular responses to DNA damage (Kyng et al., 2005), DNA repair (Ninomiya et al., 2004), and induced mutation spectrum in genomic (Muftuoglu et al., 2002) and mitochondrial DNA (Mambo et al., 2003).

However, no published studies, to our knowledge, have established an association between 4-NQO-induced mutagen sensitivity and risk of skin cancer in the general population. To further explore the feasibility of using 4-NQO as a mutagen substitute for UV and its relevance to host DNA repair capacity in lymphocytes, and to extend our earlier findings in the pilot study (Wei et al., 1996a), we tested our hypothesis in a larger, independent hospital-based case–control study of sporadic NMSC that 4-NQO-induced mutagen sensitivity is associated with the development of NMSC in the general population.

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Results

Characteristics of study subjects

We first examined the differences in the distributions of age, sex, and selected risk factors between the cases and the controls. The analysis included 191 cases (110 BCC and 81 SCC) and 176 cancer-free controls. All subjects were non-Hispanic whites. The distributions of age, sex, and selected risk factors for the cases and controls are presented in Table 1. The age range (meanSD) was 23–85 (59.014.1) years for BCC cases, 34–93 (65.512.2) years for SCC cases, and 23–83 (55.513.5) years for the controls. However, patients with BCC and SCC tended to be older than the controls. The proportion of men was higher in patients with BCC (62.7%) and SCC (88.9%) than in the controls (46.0%; Table 1). Since these differences could be due to possible biases in selecting the controls in a hospital setting, they were adjusted for in the following multivariate logistic regression analyses.

Risk factors for NMSC

Using a multivariate logistic regression analysis with adjustment for age and sex, we assessed the odds ratios (ORs) and 95% confidence intervals (CIs) to estimate the risk of NMSC associated with known risk factors, such as hair color, eye color, skin color, tanning ability, number of sunburns, freckling, dysplastic nevi, and family history of skin cancer. It was apparent that these risk factors were associated with a significantly increased risk for BCC (except for eye color and dysplastic nevi) and SCC (except for hair color, eye color, and dysplastic nevi) in this study population (Table 1). For example, the most pronounced risk factors were freckling (OR=3.71, 95% CI (2.13–6.44)) and family history of skin cancer (OR=2.94, 95% CI (1.63--5.28)) for BCC and one or more sunburns (OR=3.36, 95% CI (1.68–6.72)) and family history of skin cancer (OR=2.98, 95% CI (1.40–6.36)) for SCC. These risk factors were also correlated with each other, as expected. For example, hair color was statistically significantly correlated with eye color (r=0.218, P=0.004), skin color (r=0.223, P=0.003), tanning ability (r=0.210, P=0.006), number of sunburns (r=0.191, P=0.012), and freckling in the sun (r=0.216, P=0.005) in the controls (data not shown). Overall, the data suggested that these risk factors played a role in sporadic NMSCs in this study population.

4-NQO-induced chromatid breaks

We then examined the difference in the frequencies of 4-NQO-induced chromatid b/c between the cases and the controls. The number of simple chromatid breaks was scored from 50 well-spread metaphases for each subject and expressed as the number of chromatid b/c, as Lee et al. (1996) showed that the statistical efficiency of reading 50, compared with 100 metaphases was similar. Because the mean spontaneous b/c value derived from 50 metaphases of untreated cells was 0.02 (Wei et al., 1996b; Wang et al., 2000), which was more-than-10-fold less than that of 4-NQO treated cells (0.25 b/c in the controls), we used 4-NQO-induced b/c values only for statistical comparisons, as recommended by Hsu et al. (1989). Since the results of both log-transformed and untransformed data were very similar (data not shown), we presented the results of the untransformed data only for simplicity. The percentage distributions of 4-NQO-induced b/c values in BCC cases, SCC cases, and the controls are shown in Figure 1. It was apparent that the distributions of 4-NQO-induced b/c values in both BCC and SCC cases tended to be higher values than those of the controls. We did not perform a separate analysis of patients with a history of NMSC, because there was no statistical difference in b/c values between cases with and without previous NMSC (52 and 58 BCC cases, respectively, with meanSD, 0.390.34 vs 0.470.42 (P=0.322); 33 and 48 SCC cases, respectively, with 0.540.55 vs 0.400.32 (P=0.204)). The 4-NQO-induced b/c values were statistically higher in both BCC and SCC cases than in the controls (P<0.001 for both comparisons; Table 2).

After stratification by age, sex, and selected risk factors, the mean 4-NQO-induced b/c values remained statistically significantly higher in both BCC and SCC cases than in the controls for each subgroup (P<0.05), except for dysplastic nevi (P=0.457) for BCC and age younger than 55 years (P=0.123), blond or red hair (P=0.482), fair skin (P=0.073), and family history of skin cancer (P=0.082) for SCC. We suspected that these anomalies might be resulted from the smaller numbers of observations in the strata (Table 2). We also compared the differences in the strata in each of the subgroups, and found that only SCC patients with freckling in the sun as a child were borderline significantly sensitive to 4-NQO, as indicated by higher frequencies of chromatid breaks than SCC patients without freckling (P=0.053). Finally, we evaluated whether the sensitivity to 4-NQO was associated with tumor behavior, but we did not find any difference in 4-NQO-induced b/c values between 93 nonaggressive and 17 aggressive cases of BCC (meanSD, 0.430.39 vs 0.420.36, P=0.922) or between 48 nonaggressive and 33 aggressive cases of SCC (0.460.39 vs 0.460.49, P>0.99). Therefore, we did not consider nonaggressive and aggressive tumors separately in the following analyses.

Association between 4-NQO-induced b/c values and risk of NMSC

We next performed multivariate logistic regression analysis to calculate ORs and 95% CIs with adjustment for only age and sex to avoid attrition due to missing data for selected risk factors (Table 3). The 4-NQO-induced b/c values were fitted in the logistic regression model either as continuous or categorical variables. We found that the risk associated with an increment of 0.1 in the b/c value was 1.22 (95% CI (1.12–1.34)) for BCC and 1.26 (95% CI (1.12–1.41)) for SCC after adjustment for age and sex (Table 3). Using the median b/c value of the controls as the cutoff point, high b/c values were associated with a more-than-twofold increased risk for both BCC (OR=2.69, 95% CI (1.59–4.54)) and SCC (OR=2.46, 95% CI (1.28–4.71)) after adjustment for age and sex (Table 3). We also used 4-NQO-induced b/c tertile values of the controls as cutoff points to further evaluate the trend of the effects. We found that as the b/c values increased, the ORs increased for both BCC and SCC. Specifically, compared with the lower tertile, higher b/c values were associated with increased risk in a dose-dependent manner for both BCC (OR=1.50, 95% CI (0.72–3.09) for the middle tertile; OR=4.04, 95% CI (2.08–7.85) for the upper tertile; and SCC (OR=1.66, 95% CI (0.67–4.13) for the middle tertile; OR=4.72, 95% CI (2.07–10.75) for the upper tertile; P_trend <0.001 for both BCC and SCC; Table 3).

Multivariate analysis

Afterwards, we included all the selected risk factors in the multivariate logistic regression model for those who had provided complete information in a smaller data set (89 BCC cases, 67 SCC cases, and 126 controls). Using simultaneous adjustment for all variables listed in Table 4, we found that statistically significantly increased risk was associated with both age and sex in both BCC and SCC, but these could be adjusted as confounders due to possible selection bias in the hospital-based study. Most of the known sun-exposure-related risk factors were not significant in this model. It is likely that they were overadjusted by inclusion in the same model due to the correlation among them or limited sample size that may have caused reduction in study power. For example, lifetime sunburns was associated with a borderline significantly 2.5-fold increased risk for SCC but not for BCC (OR=1.35), whereas freckling was associated with 2.3-fold increased risk for BCC (OR=2.27, 95% CI (1.12–4.60); Table 4) but not for SCC (OR=1.45). However, family history of skin cancer remained a significant risk factor for both BCC (OR=2.75, 95% CI (1.29–5.87)) and SCC (OR=3.07, 95% CI (1.11–8.50)). More importantly, independent of these variables, risks associated with 4-NQO-induced b/c values greater than the control median remained higher for both BCC (OR=3.69, 95% CI (1.89–7.19)) and SCC (OR=4.02, 95% CI (1.68–9.60); Table 4). Therefore, these data suggested that in vitro sensitivity to 4-NQO was an independent risk factor for both BCC and SCC in this study population.

Interactions between 4-NQO-induced b/c and selected risk factors

We then assessed possible interactions between 4-NQO-induced chromatid breaks and each of the selected risk factors in multivariate logistic regression models. The hypotheses of additive and multiplicative interactions were tested when we included the interaction (or cross-product) terms (i.e., dichotomized mutagen sensitivity X each dichotomized risk factor) in a multivariate logistic regression model that included age, sex, main effect of mutagen sensitivity, and corresponding other risk factors. A departure from the multiplicative model was indicated if the odds ratio for the interactive term was greater than 1; however, there was no evidence of multiplicative interactions between mutagen sensitivity and any of selected risk factors. All hypotheses that failed to reject a multiplicative model were further tested in additive models, as assessed by the 95% confidence bounds. The following pairs of variables that were beyond the bounds suggested a possibility of a more-than-additive effect: freckling in the sun as a child and mutagen sensitivity (the P-value for the additive interactions was 0.002), tanning ability after prolonged sun exposure and mutagen sensitivity (p_interaction=0.026), and family history of skin cancer and mutagen sensitivity (p_interaction=0.049) for BCC, but not for SCC (data not shown).

Correlation between 4-NQO- and UV-induced b/c

For the subjects tested for 4-NQO sensitivity, their sensitivity to UV-B was also tested, and the data were published in a separate study (Wang et al., 2005). As 4-NQO is considered a UV-mimetic agent, we assessed the correlation between these two sets of mutagen sensitivity data as shown in Figure 2. There were 157 cancer-free control subjects whose data on both UV-induced b/c and 4-NQO-induced b/c values were available. However, we did not find evidence for a correlation between these two data sets (Figure 2).

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Discussion

In this case–control study, we found that sunlight-related risk factors played a role in both BCC and SCC, and that 4-NQO-induced mutagen sensitivity was an independent risk factor for both BCC and SCC in a dose–response manner in this study population. NMSC patients had significantly higher frequencies of 4-NQO-induced chromatid breaks that were associated with more-than-twofold increased risk for NMSC compared with the cancer-free controls. Therefore, a mutagen sensitivity assay using 4-NQO as the test mutagen may be used to assess genetic susceptibility to NMSC in the general population.

Although mutagen sensitivity was measured in cultured peripheral lymphocytes, high susceptibility to carcinogen-induced chromatid breaks has a clear genetic basis (Cloos et al., 1999; Tedeschi et al., 2004), and either spontaneous or in vitro-induced chromosomal aberrations by mutagens or carcinogens are relevant to cutaneous carcinogenesis in the target organ (Wei et al., 1996a; Lanza et al., 1997). The feasibility of procuring peripheral blood samples, the ease of using this in vitro 4-NQO mutagen sensitivity assay, and the small amount (2 ml) of blood needed for this assay make it a promising biomarker for assessing individual susceptibility to cancer. However, the usefulness of this assay in assessing susceptibility to cancers other than NMSC in the general population needs further investigation.

We previously published the data on UV-B-induced mutagen sensitivity and risk of NMSC in the same study population, in which we found that UV-B-induced chromatid breaks were associated with risk of developing BCC and SCC (Wang et al., 2005). However, we did not find the correlation between UV-B-induced b/c and 4-NQO-induced b/c in 157 control subjects who had been assessed by both of the assays. It is suggested that even though 4-NQO is similar to UV light in the molecular mechanisms of inducing mutagenesis such as base change, deletion, or phage induction ability, and repair in xeroderma pigmentosum fibroblastic cells such as DNA synthesis or colony-forming ability (Nagao and Sugimura, 1976), the formation of chromatid breaks induced by these two agents may have different mechanisms, especially in repair-proficient subjects. It is known that the UV-induced DNA lesions are mainly repaired by the transcription-coupled repair pathway, whereas the lesions induced by 4-NQO are repaired by the global genome repair pathway (Snyderwine and Bohr, 1992; Muftuoglu et al., 2002). However, no previously published report has investigated the correlation between damage to DNA and chromosomes induced by these two carcinogens in the general population. Therefore, our results remain to be validated by other investigators.

Although the exact mechanisms by which UV and 4-NQO induce chromosomal breaks are unknown, it is well known that during the repair of the lesions, repair enzymes can make more cuts on the damaged DNA strands (Marcon and Moens, 2005), which can cause double-strand breaks. A slow kinetics and suboptimal synapsis mechanism of back-up non-homologous end-joining of the double-strand breaks has been proposed (Iliakis et al., 2004), in which more time needed for exchanges through the joining of incorrect ends may cause the formation of chromosome aberrations. However, the mechanisms by which these lesions are converted into chromosome breaks are still poorly understood (Slijepcevic and Bryant, 1998). One of the mechanisms may be that the conversion is mediated by the enzyme telomerase, a possible mechanism called telomere capture that is intended to stabilize broken chromosomes in mammalian cells (Meltzer et al., 1993; Bosco and Haber, 1998).

Recent evidence also suggests that chromatid breaks are not simply the expanded DNA double-strand breaks, and that there is a lack of correlation between reduced DNA double-strand break rejoining and disappearance of chromatid breaks. As a result, a modified Revell's model has been proposed that in less than 20% of the total breaks, both spontaneous and radiation-induced chromatid breaks result from DNA double-strand breaks signaling and rearrangement processes from within large looped chromatin domains (Bryant, 1998), and that the rest may result from intrachromatid rearrangements, including a very small proportion involving complete excision of a looped domain (Bryant et al., 2004). Nevertheless, whether different DNA repair mechanisms are involved in the formation of the UV- and 4-NQO-induced chromatid breaks remains to be determined.

There were some observable differences in the sensitivity to in vitro exposure to 4-NQO not only between BCC and SCC but also within each cancer site. For example, an apparently higher level of 4-NQO-induced chromatid breaks was observed in young BCC but in old SCC patients, in BCC patients with blond or red hair but in SCC patients with black or brown hair. This may reflect the fact that genetic susceptibility (i.e., early age onset or having some genetic traits) plays a major role in BCC, whereas overexposure to sunlight (i.e., accumulative dose characterized by older age and no genetic traits) is the major cause for SCC (Armstrong and Kricker 2001). However, there were also some inconsistencies in the findings. For example, an apparently higher level of 4-NQO-induced chromatid breaks was also observed in both BCC and SCC patients who had no history of sunburns, had freckling, or had not reported a family history of skin cancer. These results may also reflect instability of the estimates, because the sample size of a given subgroup or stratum was substantially reduced in the stratification, suggesting a larger study may be needed to verify these findings.

In summary, we found that the frequency of 4-NQO-induced chromatid breaks was significantly higher in NMSC cases than in the controls. This higher frequency was associated with a more-than-twofold increased risk for both BCC and SCC, and a dose–response relationship was found between the tertile of mutagen sensitivity and the risk for both BCC and SCC, independent of other known risk factors. These findings suggest that in vitro 4-NQO-induced mutagen sensitivity does not appear to be correlated to that induced by UV, which also reflects susceptibility to NMSC. However, we did not have accumulative sun exposure data for each subject, which may have an effect on DNA repair phenotype. However, the 4-NQO-induced frequency of chromatid breaks values was 10 times higher than that in the untreated cells, suggesting that sun-induced chromatid breaks in lymphocytes may be negligible. Furthermore, we used the in vitro treatment with the same conditions for cases and controls and adjusted possible sun-exposure confounders (sunburn, freckling, and tanning ability) in the multivariate logistic models. Therefore, the sunlight-induced chromatid breaks should not have, if any, a major effect on the b/c values we used for comparison. Finally, because of the inherent bias in selecting study subjects in a hospital-based case–control study, such as potential selection bias in sunlight exposure and self-reported skin lesions, and the relatively small sample size of this study, the findings need further validation by other independent, larger or prospective studies.

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Materials and Methods

Study population

The study included patients with BCC and SCC who registered at the Departments of Dermatology and Head and Neck Surgery at The University of Texas M.D. Anderson Cancer Center and DermSurgery Associates from July 1996 to August 2000. There were no restrictions on age, sex, or ethnicity. Controls were reportedly cancer-free healthy people selected from genetically unrelated visitors who were accompanying cancer patients to the clinics at M.D. Anderson Cancer Center or came to M.D. Anderson for prostate cancer screening. The exclusion criteria were previous chemotherapy or radiotherapy, any metastasis, and any cancer history other than NMSC for case subjects and any blood transfusion in the last 6 months for all participants. Informed consent was obtained from all participants, and a standardized, self-administered questionnaire was used to collect demographic data and data on risk factors, such as natural hair color, eye color, skin color, history of sunlight exposure (including freckling in the sun as a child, tanning ability, and number of sunburns), medical history, and family history of first-degree relatives with any cancer. Each participant donated 20 ml of blood, which was collected with heparinized tubes. This analysis included only non-Hispanic whites. The study was conducted according to the Declaration of Helsinki Principles, and the study protocol was approved by the institutional review board of M.D. Anderson.

Mutagen sensitivity assay

In this assay, mutagen sensitivity was expressed as the number of 4-NQO-induced chromatid b/c after 24 hours of treatment (Hsu et al., 1993a). Briefly, short-term cultures of 1 ml of fresh whole blood were established in 9 ml of RPMI 1640 medium supplemented with 20% fetal bovine serum with a final concentration of 112.5 g/ml phytohemagglutinin (Remel, Lenexa, KS) to stimulate T-lymphocyte growth. After 48 hours of culture, the cells were treated with 4-NQO at a final concentration of 10 M and were allowed to grow for another 23 hours before being treated with colcemid (Gibco BRL, Carlsbad, CA) at 0.06 g/ml to induce mitotic arrest 1 hour before harvesting. We used conventional chromosome harvesting procedures: the cells were treated for 15 minutes with 60 mM hypotonic KCl solution and fixed three times for 5 minutes, each with freshly prepared methanol:acetic acid (3:1 v/v), after which air-dried slides were prepared as previously described (Cherry and Hsu, 1983). All the slide preparation procedures were performed by one of us (Ping Xiong). The slides were then stained with 4% Giemsa (Biomedical Specialties, Santa Monica, CA) for 7 minutes. All slides were evaluated for chromosomal aberrations by the late Dr Hsu, who was masked to the case–control status of the subjects. The number of simple chromatid breaks was scored from 50 well-spread metaphases per blood sample.

Statistical analysis

The number of chromatid b/c was analyzed as a continuous variable. Student's t-test was used to compare the mean number of chromatid b/c between groups. As the chromatid b/c were not normally distributed, we also performed Student's t-test for log-transformed data. We used the median and tertile of chromatid b/c in the control group as the cutoff values to calculate the OR. Correlation analysis was used to explore the relationships between sun exposure variables. Univariate and multivariate logistic regression analyses with adjustment for age and sex were performed to calculate the adjusted OR and 95% CI for each variable of interest. Some subjects did not provide information on some variables (such as hair color, eye color, skin color, tanning ability, number of sunburns, freckling, dysplastic nevi, and family history of skin cancer), and these variables were treated as missing data in the analysis. Afterward, we included all the selected variables in the multivariate logistic regression analyses for subjects who provided complete information. A more-than-multiplicative interaction was suggested when OR₁₁>OR₁₀ OR₀₁, in which OR₁₁=the OR when both factors were present, OR₀₁=the OR when only factor 1 was present and OR₁₀=the OR when only factor 2 was present (Kleinbaum et al., 1982). To assess evidence for departure from a multiplicative model, we modeled interaction terms between variables using standard unconditional logistic regression. We were specifically interested in searching for interactions indicating a more-than-multiplicative relationship (i.e., interaction terms from the logistic regression with positive coefficients), because these interactions identify subgroups of individuals who may be at particularly high risk for developing NMSC. We were also interested in identifying departures from additive models. Empirically, a more-than-additive interaction was indicated if OR₁₁>OR₁₀+OR₀₁-1. When the test for multiplicative interaction was not rejected, further tests for additive interaction were performed by a bootstrapping test of goodness-of-fit of the null hypothesis of an additive model with no interaction against an alternative hypothesis that allows an additive interaction. To perform the hypothesis test for additive models, we implemented bootstrapping using Stata 8.2 (StataCorp LP, College Station, TX). All statistical tests were two-sided, and P<0.05 was considered statistically significant. We analyzed all data, except for additive models, using SAS software (version 8e; SAS Institute, Cary, NC).

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Conflict of Interest

The authors state no conflict of interest.

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Acknowledgments

This work was supported in part by the National Institutes of Health National Cancer Institute Grants P01 CA 68233 (G.L.C.), R01 CA 100264 (Q.W.), and the National Institute of Environmental Health Sciences Grant R01 ES11740 (Q.W.). We thank Margaret Lung for assistance in recruiting study participants, Yijue Zhao for laboratory assistance, Monica Domingue, and Joanne Sider for assistance in preparing the manuscript, and David Galloway for scientific editing.