IFN-γ and other serum cytokines in head and neck squamous cell carcinomas.

SUMMARY: Altered immune responses have been reported in head and neck cancer, and some of these responses have been associated with poor clinical outcomes. A multiple-array technology platform was used to simultaneously evaluate the levels of 25 cytokines. Pre-treatment serum levels were evaluated in 31 HNSCC patients and 6 healthy controls. The levels of 8 cytokines, specifically IL-1ra, IL-2, IL-5, IL-6, IL-8, IL-17, IFN-γ and IP-10, were significantly higher in patients than in controls. Among cancer patients we observed lower levels of IFN-γ and IL-7 in cases with nodal metastases compared to those with cN0 disease. We observed increases in the levels of some serum cytokines in HNSCC patients, as well as reductions in selected cytokines associated with regional progression. These findings provide an intriguing perspective on the development and validation of novel markers for follow-up evaluations and predictions of regional spreading in HNSCC patients.

In vitro evaluation of the potential toxic effects of palladium nanoparticles on fibroblasts and lung epithelial cells.

Palladium nanoparticles have been increasingly used in catalytic processes, wastewater treatment, electronics, and biomedicine. However, recent evidence proved that these nanoparticles are able to induce adverse effects both in in vitro and in vivo models. Nevertheless, molecular mechanisms underlying the toxic effects are still poorly understood. Therefore, this study aimed to investigate the potential toxicological mechanisms of palladium nanoparticles assessing their effects on normal diploid rat fibroblast and lung carcinoma human epithelial cell lines. Several endpoints such as cell growth, cell cycle progression, DNA damage, induction of apoptosis, reactive oxygen species production and expression of cell cycle regulatory proteins were evaluated. Results showed that palladium nanoparticles inhibited cell growth in a dose- and time-dependent manner in both cell lines, although with a more evident action on fibroblasts. Interestingly, inhibition of cell growth was not associated with the induction of apoptosis. Cell cycle progression was arrested in the G0/G1 phase and DNA damage was evident in both cell lines even if only a slight increase in the intracellular reactive oxygen species levels was detected. These findings provide valuable insight into understanding the molecular mechanisms responsible of palladium nanoparticles toxicity whose identification is essential to define an adequate risk assessment process.

Immunopathological characterization of cryptoglandular anal fistula: a pilot study investigating its pathogenesis.

AIM: The pathogenesis of cryptoglandular anal fistula (AF) is still under debate. Tissue inflammation could play a primary role. The pathological process of epithelial mesenchymal transition (EMT) might be involved but has never been investigated. METHOD: In a prospective pilot study, 12 patients with an AF had a fistulectomy. The excised track was divided into proximal (intrasphincteric) and distal (extrasphincteric) parts which were subjected to standard histopathological examination. The cytokines IL-8 and IL-1beta were analysed as markers of inflammation, while EMT was evaluated by expression of TGF-beta, Vimentin, Zeb-1, Snail and E-cadherin. The mRNA and protein expression of these molecules was investigated by real-time PCR (RT-PCR), Western blot analysis and immunohistochemistry and was compared with that of the normal adjacent tissue. RESULTS: Chronic inflammation and granulation tissue and a stratified epithelium were evident on standard histopathological examination. The cytokine IL-8 was more expressed in the proximal than the distal part of the track (fold increase 4.34 vs 3.60), while the reverse was found for IL-1beta (fold increase 1.33 vs 2.01); both were more intensely expressed compared with the normal anal mucosa. EMT was demonstrated, in both proximal and distal parts of the track, with an increase of TGF-beta, Vimentin, Zeb-1 and Snail and a mean decrease of E-cadherin. Western blot analysis and immunohistochemistry confirmed the protein expression. CONCLUSION: The study suggests that chronic inflammation is present in cryptoglandular fistulas. The inflammatory pattern might be different in the proximal than in the distal part of the fistula track. The cytokines IL-1beta and IL-8 could play a possible role in fistula formation. The study demonstrates for the first time the potential importance of EMT in the pathogenesis of cryptoglandular AF.

Expression of alpha-dystroglycan correlates with tumour grade and predicts survival in oral squamous cell carcinoma.

AIMS: Dystroglycan (DG) is a non-integrin adhesion molecule connecting the extracellular matrix to the actin cytoskeleton. Decreased expression of DG has been reported in several human cancers and related to tumour aggressiveness. METHODS: Expression of the alpha-DG subunit was evaluated by immunostaining in a series of oral squamous cell carcinoma (OSCC) and its relation with traditional prognostic indicators and with the clinical outcome of the patients was evaluated. RESULTS: Alpha-DG expression was easily detected in normal epithelium with a mean percentage of positive cells >80% but was undetectable in a significant fraction (59%) of OSCC. Loss of alpha-DG staining correlated with higher tumour grade (p = 0.04) and stage (p = 0.01), with nodal involvement (p = 0.001) and with an increased risk of recurrence (p = 0.002) and death (p = 0.004) in a univariate analysis, but it was not confirmed as an independent predictor of clinical outcome in a multivariate analysis. CONCLUSIONS: Loss of alpha-DG expression, which corresponds to loss of a functional DG complex, is a frequent event in human OSCC. Further studies are warranted on the role of this molecule in the entire multistep process of oral squamous tumorigenesis.

Post-translational modulation of CD133 expression during sodium butyrate-induced differentiation of HT29 human colon cancer cells: implications for its detection.

The CD133 molecule has been proposed as a surface marker of cancer stem cells in several human malignancies, including colon cancers. The function and the mechanisms regulating CD133 expression remain unknown. The HT29 human colon cancer cells undergo differentiation following treatment with various agents and represent a useful in vitro model of colon differentiation. This study evaluated the behavior of CD133 during sodium butyrate-induced differentiation of HT29 cells. Treatment with sodium butyrate induced a progressive decrease of CD133 expression, as assessed by flow cytometry using the AC133 monoclonal antibody. Indeed, expression of CD133, which was about 47% in untreated control cells, gradually decreased down to about 3% after 72 h in a time- and dose-dependent manner. No relationship was observed between CD133 protein evaluated by flow cytometry and mRNA expression level, and no changes were detected in the methylation status of the CD133 gene promoter during HT29 differentiation. Moreover, the expression of the CD133 protein, evaluated by Western blot analysis using a specific anti-CD133 antibody directed against the C-terminal intracytoplasmic region of human CD133 protein, did not correlate with flow cytometry results. Different results were also obtained using the two antibodies to analyze the expression of the CD133 molecule in human colon cancers. These findings demonstrate that membrane expression of the CD133 stem cell marker might undergo a complex regulation during differentiation of colon cells and suggest that HT29 cells are a useful in vitro model to study the mechanisms involved in this regulation which likely occurs at a post-transcriptional level.

Isolation and characterization of CD133+ cell population within human primary and metastatic colon cancer.

BACKGROUND: "Cancer stem cells" (CSC) have been identified as a minority of cancer cells responsible for tumor initiation, maintenance and spreading. Although a universal marker for CSC has not yet been identified, CD133 has been proposed as the hallmark of CSC in colon cancer. The aim of our study was to assess the presence of a CD133+ cell fraction in samples of colon cancer and liver metastasis from colon cancer and evaluate their potential as tumor-initiating cells. METHODS: Tissue samples from 17 colon cancers and 8 liver metastasis were fragmented and digested using collagenase. Cell suspensions were characterized by flow cytometry using anti-CD133, CD45 and CD31 antibodies. CD133+ cells were also isolated by magnetic cell sorting and their tumor-initiating potential was assessed versus the remaining CD133- fraction by soft-agar assay. RESULTS: Our results confirmed the existence of a subset of CD133+ tumor cells within human colon cancers. Interestingly, CD133+ cells were detectable in liver metastasis at a higher percentage when compared to primary tumors. Soft-agar assay showed that CD133+ cell fraction was able to induce larger and more numerous colonies than CD133-cells. CONCLUSION: Our findings data that the CD133+ colon cancer cells might play an important role in both primary tumors as well as in metastatic lesions thus warranting further studies on the role(s) of this subset of cells in the metastatic process.

Differential toxic effects of methyl tertiary butyl ether and tert-butanol on rat fibroblasts in vitro.

Methyl tertiary butyl ether (MTBE) is the most widely used motor vehicle fuel oxygenate since it reduces harmful emissions due to gasoline combustion. However, the significant increase in its use in recent years has raised new questions related to its potential toxicity. In fact, although available data are somehow conflicting, there is evidence that MTBE is a toxic substance that may have harmful effects on both animals and humans and an unresolved problem is the role played by MTBE metabolites, especially tertiary butyl alcohol (TBA), in determining toxic effects due to MTBE exposure. In this study, the toxic effects of MTBE have been analyzed on a normal diploid rat fibroblast cell line (Rat-1) and compared to the effects of TBA. The results obtained suggest that both MTBE and TBA inhibit cell growth in vitro but with different mechanisms in terms of effects on the cell cycle progression and on the modulation of cell cycle regulatory proteins. In fact, MTBE caused an accumulation of cells in the S-phase of the cell cycle, whereas TBA caused an accumulation in the G0/G1-phase with different effects on the expression of cyclin D1, p27Kip1, and p53. Moreover, both MTBE and TBA were also shown to induce DNA damage, as assessed in terms of oxidative DNA damage and nuclear DNA fragmentation, that appeared to be susceptible of repair by the cell DNA-repair machinery. In conclusion, these findings suggest that both MTBE and TBA can exert, by acting through different molecular mechanisms, important biological effects on fibroblasts in vitro. Further studies are warranted to shed light on the mechanisms responsible for the observed effects and on their potential significance for the in-vivo exposure.

Dystroglycan expression is reduced during prostate tumorigenesis and is regulated by androgens in prostate cancer cells.

Prostate cancer, the most frequently diagnosed cancer in Western men, can display a high variability in term of clinical aggressiveness and prognosis and none of the available markers is able to accurately predict its clinical course. Dystroglycan (DG), a non-integrin adhesion molecule, is a complex formed by two subunits, alpha- and beta-DG, which bind to extracellular matrix molecules and cytoskeleton, respectively. DG expression is frequently reduced in human cancers and has been related to tumor grade and aggressiveness. This study investigated the role of DG in human prostate tumorigenesis and its suitability as a prognostic marker. The expression level of extracellular alpha-DG subunit was frequently reduced in human prostate cancer cell lines and primary tumors and the percentage of positive tumor cells was significantly further decreased in vivo following androgen ablation therapy (median = 1%) compared to pre-treatment samples (median = 28%). A significant relationship was observed between alpha-DG staining on the post-treatment samples and tumor recurrence. A dose- and time-dependent decrease of DG expression also occurred in human prostate cancer cells following treatment with the anti-androgen flutamide. Stable expression of an exogenous DG cDNA in the LNCaP human prostate carcinoma cell line resulted in a marked inhibition of both anchorage-dependent and independent growth and of the in vivo tumorigenicity. These findings confirm and extend previous evidence that disturbances in the function of the DG complex might contribute to the definition of the malignant behavior of prostate cancer cells and suggest that androgens might regulate DG expression in these cells.

Analysis of dystroglycan regulation and functions in mouse mammary epithelial cells and implications for mammary tumorigenesis.

Abnormalities in the interactions of cells with the extracellular matrix (ECM) play an important role in the development and progression of many types of cancer and are a hallmark of malignant transformation. The dystroglycan (DG) complex is a transmembrane glycoprotein that forms a continuous link from the ECM to the actin cytoskeleton, providing structural integrity and perhaps transducing signal, in a manner similar to integrins. Deregulated expression of DG has been reported in a variety of human malignancies and related to tumor differentiation and aggressiveness. In breast cancer, reduced DG expression has been associated with patient survival and with loss of differentiation of tumor cells. Limited data are available on DG physiology in epithelial cells. In this study, we used the HC11 spontaneously immortalized murine mammary epithelial cells to study DG function(s) and regulation in normal cells. We found that expression of DG protein and mRNA is cell-cycle and cell-density regulated in these cells. Moreover, expression of both DG subunits increased upon lactogenic differentiation of the HC11 cells. The turnover of cell-surface-expressed DG was evaluated in the same cells and half-life of DG subunits was evaluated to be about 12 h. DG-specific small inhibitory RNAs were used to analyze the effects of a reduced expression of DG in these cells. Cells in which DG expression was suppressed were growth inhibited, accumulated in the S-phase of the cell cycle, failed to undergo lactogenic differentiation, and displayed an increase in the percentage of apoptotic cells. Moreover, changes were observed in the expression and/or activity of several molecules involved in cell growth control. These results demonstrate that DG expression is tightly regulated in normal mammary epithelial cells and support the hypothesis that DG is involved in several functions other than structural integrity in these cells. This finding provides new insight into the roles played by DG in epithelial cell physiology and will contribute to our understanding of its involvement in the process of epithelial cell transformation.

Lycopene induces apoptosis in immortalized fibroblasts exposed to tobacco smoke condensate through arresting cell cycle and down-regulating cyclin D1, pAKT and pBad.

There is a lot of interest in the health benefits of dietary carotenoids and on the relationship of these compounds with smoke. In particular, it is unknown if the enhanced cancer risk observed in smokers following beta-carotene supplementation can be also found using other carotenoids. Here, we studied the effects of the tomato carotenoid lycopene on molecular pathways involved in cell cycle progression, apoptosis and survival in immortalized RAT-1 fibroblasts exposed to cigarette smoke condensate (TAR). Lycopene (0.5-2.0 microM) inhibited cell growth in a dose-and time-dependent manner, by arresting cell cycle progression and by promoting apoptosis in cells exposed to TAR. The arrest of cell cycle was independent of p53 and of 8-OH-dG DNA damage and related to a decreased expression of cyclin D1. Moreover, the carotenoid up-regulated apoptosis and down-regulated the phosphorylation of AKT and Bad in cells exposed to TAR. Such an effect was associated to an inhibition of TAR-induced expression of Cox-2 and hsp90, which is known to maintain AKT activity. This study suggests that lycopene, differently from beta-carotene, can exert protective effects against cigarette smoke condensate.

Redox regulation of cell proliferation by pyrrolidine dithiocarbamate in murine thymoma cells transplanted in vivo.

Pyrrolidine dithiocarbamate (PDTC) is a synthetic compound largely used in cell biological studies and known to exert either antioxidant or pro-oxidant effects. Recently, its antitumoral activity has been proposed on the basis of its antioxidant and proapoptotic effects. In the present study, we evaluated the effect of increasing i.p. doses of PDTC on the growth of a strain of highly malignant thymoma cells inoculated in the peritoneum of inbred Balb/c mice. PDTC treatment increased the number of thymoma cells in a dose-dependent manner, enhancing the percentage of proliferating tumor cells. PDTC exerted regulatory effects on cell cycle distribution, decreasing the expression of cell cycle inhibitors. Alterations in the production of intracellular reactive oxygen species, levels of oxidized glutathione, and intracellular levels of the redox-active metals iron and copper were also observed. The above results represent the first evidence that PDTC may induce in vivo cell proliferation in a murine thymoma cell model. In addition, we suggest that the ability of PDTC to bind and transport metals inside the cell and its pro-oxidant property may be factors underlying its effects on thymoma cell proliferation and cell cycle distribution.

DNA oxidative damage during differentiation of HL-60 human promyelocytic leukemia cells.

DNA oxidative damage was measured in human promyelocytic leukemia HL-60 cells, in the same cells committed to granulocytic differentiation with dimethyl sulfoxide (DMSO) or all-trans-retinoic acid (RA) and in mature human peripheral granulocytes (HPG). DNA damage was evaluated as single strand breaks and 8-OHdG adducts, measured by single cell electrophoresis or by monoclonal antibodies, respectively. The basal levels of either marker of DNA damage were higher in undifferentiated HL-60 cells than in HPG and DMSO- or RA-differentiated cells. Treatment with H(2)O(2) increased 8-OHdG formation in all cells, but the levels of DNA damage remained higher in undifferentiated cells as compared to the differentiated ones. Three lines of evidence suggested that the higher levels of DNA damage observed in undifferentiated cells were at least in part attributable to a reduced detoxification of reactive oxygen species (ROS). First, undifferentiated cells were shown to accumulate higher levels of dichlorodihydrofluorescein-detectable ROS than HPG and DMSO- or RA-differentiated cells. Second, undifferentiated HL-60 cells were characterized by reduced levels of GSH and lower GSH/GSSG ratios as compared to the differentiated cells. Third, pretreatment of undifferentiated HL-60 cells with antioxidants such as alpha-tocopherol or beta-carotene suppressed the elevation of ROS and the formation of 8-OHdG induced by H(2)O(2). Further evidence for the importance of the oxidant/antioxidant balance was obtained by modulating the iron-catalyzed decomposition of H(2)O(2) to hydroxyl radicals in undifferentiated HL-60 cells. In fact, pretreatment with FeSO(4) increased the formation of 8-OHdG induced by H(2)O(2), whereas pretreatment with the iron chelator deferoxamine produced the opposite effect. These results illustrate correlations between the oxidant/antioxidant balance and DNA damage and suggest that the capability of a cell population to withstand oxidative stress and DNA damage may depend on its degree of differentiation.

Resveratrol, a natural phenolic compound, inhibits cell proliferation and prevents oxidative DNA damage.

Resveratrol (3,4',5-trihydroxystilbene) is a naturally occurring phenolic compound which is present at high levels in wine and has been recently proposed as a potential cancer chemopreventive and chemoterapeutic agent. In this study, we evaluated the antiproliferative activity of resveratrol on a panel of cell lines of various histogenetic origin, including normal rat fibroblasts and mouse mammary epithelial cells compared to human breast, colon and prostate cancer cells. The concentration of resveratrol inhibiting cell growth by 50% (IC(50)) ranged from about 20 to 100 microM. At such concentration, we were unable to detect a significant increase in the apoptotic index in most of the cell lines analyzed. We also studied the effects of resveratrol on cell cycle distribution. The most striking effect was a reduction in the percentage of cells in the G2/M phase which was most frequently associated with an increase of cells in the S phase of the cell cycle. We also found that resveratrol is able to prevent the increase in reactive oxygen species (ROS) following exposure to oxidative agents (i.e. tobacco-smoke condensate (TAR) and H(2)O(2)). Resveratrol also reduced nuclear DNA fragmentation, as assessed by single cell gel electrophoresis (comet test). Taken together our results suggest that resveratrol can act as an antimutagenic/anticarcinogenic agent by preventing oxidative DNA damage which plays a pivotal role in the carcinogenic activity of many genotoxic agents.

Isolation of normal epithelial cells adapted to grow at nonphysiological concentration of magnesium.

Extracellular magnesium (Mg) depletion inhibits the growth of the HC11 normal mammary epithelial cells. In this study we found that an acute increase in extracellular Mg generally exerts a positive effect on the growth of these cells. We also isolated and characterized two derivatives adapted to grow and proliferate at nonphysiological concentration of Mg. The growth properties of the HC-LMg cells at 25 microM Mg were comparable to those of the parental HC11 cells in the regular medium (0.5 mM Mg) despite an increased expression of the CDK inhibitor p27(Kip1). They also showed a reduced dependence from serum to grow. The HC-HMg cells have been adapted to grow and proliferate at an increased (45 mM) Mg concentration. Cell total Mg content was 19.6, 9.7, and 20.1 nmol/mg protein in the HC11, HC-LMg, and HC-HMg cells, respectively. Thus, we have isolated derivatives of normal epithelial cells which are able to maintain Mg content in a physiological range in the face of different extracellular concentration gradients and will be a valuable tool for further studies on the regulation of Mg homeostasis in eukaryotic cells.

8-hydroxy-2'-deoxyguanosine in cervical cells: correlation with grade of dysplasia and human papillomavirus infection.

In this study, the 8-hydroxy-2'-deoxyguanosine (8-OHdG) level was assessed in human cervical cells by an immunoperoxidase method and was related to the presence of human papillomavirus (HPV) infection and precancerous lesions. After optimizing the immunohistochemical method of detecting oxidative DNA damage in whole cells, we have used this technique to estimate the oxidative damage in cervical cells collected during a routine PAP test. The analysis of variance (ANOVA) of the data from human samples showed significant differences in the 8-OHdG content among normal, low-grade and high-grade squamous intraepithelial lesion (SIL, HGSIL and LGSIL, respectively; P < 0.001). In the comparison of the three groups, statistically significant differences were detected between normal SIL and HGSIL (P < 0.001) and between LGSIL and HGSIL (P = 0.003), whereas no statistically significant difference was found between normal SIL and LGSIL (P = 0.1). Grouping observations by HPV status, no significant difference was detected in 8-OHdG levels between HPV(+) and HPV(-) subjects (P = 0.8). The polytomous and proportional odds models, extensions of the logistic regression analysis, showed that the effect of 8-OHdG levels in rising the risk of dysplasia was roughly constant through SIL grades. In conclusion, the immunoperoxidase method, applied to single human cervical cells, provides clear evidence that significant differences exist in 8-OHdG content between normal and dysplastic cells and that oxidative DNA damage might play an important role in cervical carcinogenesis.

Evaluation of 8-hydroxydeoxyguanosine in human oral cells: the importance of tobacco smoke and urban environment.

The DNA adduct 8-hydroxydeoxyguanosine (8-OHdG) has been widely used as a sensitive biomarker for oxidative damage. To investigate the role of environmental factors on oxidative DNA damage formation, the level of 8-OHdG was determined in oral cells from 109 healthy volunteers by an immunohistochemical method. A statistically significantly higher content of 8-OHdG was detected in oral cells from smokers (111 +/- 55, n = 38) compared with non smokers (78 +/- 48, n = 71), (p < 0.01). Moreover, subjects living in an urban area showed a higher level of oxidative damage with respect to those living in a countryside-suburban area (99 +/- 53, n = 58 vs. 78 +/- 51, n = 51), (p = 0.03). No significant association was detected between 8-OHdG in oral cells and other variables such as passive smoke, oral infections, alcohol or vitamin intake and grilled food consumption. This work suggests that tobacco smoke and environmental exposure to pollutants lead to a measurable increase of oxidative damage in oral cells and confirms that the immunoperoxidase method is an appropriate approach for epidemiological analyses.

Polycyclic aromatic hydrocarbon-DNA adducts in cervical smears of smokers and nonsmokers.

OBJECTIVES: The aim of this study was to detect polycyclic aromatic hydrocarbon-DNA (PAH-DNA) adducts in single cervical cells collected during a routine Papanicolaou smear and to relate this carcinogen exposure dose marker to smoking habit. METHODS: An immunohistochemical assay, using a polyclonal antiserum raised against benzo[a]pyrene diol epoxide-DNA adducts, was performed to evaluate PAH-DNA adducts in cervical cells collected from 16 volunteers who smoked at least 20 cigarettes/day and 16 nonsmokers. RESULTS: The mean adduct level, determined as relative staining intensity by an optical density image analyzer, was significantly higher in smokers compared to nonsmokers (AOD x 1000 +/- SD = 98 +/- 32 and 73 +/- 25, respectively) (P = 0.04). CONCLUSIONS: These results demonstrate that this immunohistochemical assay, much simpler than other methodologies used to evaluate PAH-DNA adducts in cervical tissue, is sufficiently sensitive for quantitative adduct evaluation in single epithelial cervical cells, as already verified for other exfoliated material. This work thus confirms that tobacco smoke is a risk factor for genotoxic damage generation in cervical cells and indicates a procedure likely adaptable to a large population screening.

Biological characterization of central and peripheral primary non small cell lung cancers (NSCLC).

BACKGROUND: Non Small Cell Lung Carcinomas (NSCLC) comprise 90% of all lung carcinomas. Studies have demonstrated a preferential central (bronchus-derived) localization for squamous cells, whereas adenocarcinomas are frequently peripheral (bronchiolo-alveolus derived). It has been suggested that exposure to carcinogenic insults including cigarette smoke, may induce different types of tumors in different locations. MATERIALS AND METHODS: Forty one NSCLC patients staged according to WHO and TNM were considered for localization and biological parameters (p53 expression, cell ploidy and S-phase). RESULTS: p53 overexpression was found more frequently in central than in peripheral tumors (69% vs 39%) (p = 0.074). Central tumors were more aneuploid (69%) than peripheral ones (46%) (p = 0.03) No difference in smoking habit was observed in the two groups. CONCLUSIONS: Our results suggest that there is no apparent biological difference between these two groups of NSCLCs, and that the smoking does not play a role in either histotype determination or biological behavior.

Evaluation of polycyclic aromatic hydrocarbon-DNA adducts in exfoliated oral cells by an immunohistochemical assay.

Polycyclic aromatic hydrocarbon-DNA adducts were evaluated in oral cells from 98 healthy volunteers by an immunohistochemical method using a specific antiserum against benzo(a)pyrene-DNA adducts revealed by the immunoperoxidase reaction. Mean adduct content, determined as relative staining intensity by absorbance image analyzer, was significantly higher in the cells from tobacco smokers compared with nonsmokers (330 +/- 98, n = 33 versus 286 +/- 83, n = 64, respectively) with a P = 0.013 obtained by two-sample t test with equal variances. We found that in the smoker group, the PAH-DNA adduct content increases with the number of cigarettes. Thus, the relative staining intensity was 305 +/- 105 in the group smoking 1-10 cigarettes/day (n = 16), 347 +/- 77 in the 11-20 group (n = 14), and 386 +/- 112 in the group smoking more than 20 cigarettes/day (n = 3; P = 0.03 by nonparametric test for trend). No significant association was detected between PAH-DNA adducts in oral cells and variables such as residential area, oral infections, alcohol or vitamin intake, grilled food consumption, and professional activity. This work confirms and extends previous data suggesting that this immunohistochemical method might be used as a valuable dosimeter of genotoxic damage in a carcinogen-exposed population, although further studies are needed to verify the applicability of the test in high-risk populations other than smokers.