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

Gianpiero Romano3, Alessandro Sgambato, Rita Mancini2, Giovanni Capelli1, Maria Rosaria Giovagnoli2, Giovanna Flamini, Alma Boninsegna, Aldo Vecchione2 and Achille Cittadini

Giovanni XXIII Cancer Research Center, Institute of General Pathology and
1 Institute of Hygiene, Catholic University School of Medicine, L.go F.Vito 1, 00168 Rome and
2 Cytopathology I Department of Experimental Medicine, University La Sapienza, Viale Regina Elena 324, 00161 Rome, Italy


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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.

Abbreviations: 8-OHdG, 8-hydroxy-2'-deoxyguanosine; AOD, average optical density; HGSIL, high-grade squamous intraepithelial lesion; HPV, human papillomavirus; LGSIL, low-grade squamous intraepithelial lesion; ROS, reactive oxygen species; SIL, squamous intraepithelial lesion.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The reactive oxygen species (ROS) formed during exposure to environmental oxidants and during endogenous metabolic processes, play an important role in the pathogenesis of cancer and other degenerative diseases (1). The ROS, such as superoxide anion radicals (O2•), hydrogen peroxide (H2O2), hydroxyl radicals (OH•) and singlet oxygen (1O2), can induce genotoxic damage, including single- and double-strand breaks, DNA–protein cross links, abasic sites and modified bases (2). Oxidation of the C8 of guanine is one of the most abundant type of oxidative DNA damage (3). The modified base 8-hydroxy-2'-deoxyguanosine (8-OHdG) is a mutagenic lesion in vivo and in vitro, leading to G-T and A-C substitutions (4). It is therefore suggested as a sensitive biomarker for molecular epidemiological assessment of cancer risk due to oxidative stress (57).

Several methods for measurement of 8-OHdG have been developed, including gas chromatography/mass spectroscopy (GC/MS), high-performance liquid chromatography with electrochemical detection (HPLC–EC), 32P-post-labelling (PPL) and immunoassays (812). Due to the detection method used, discrepancies in the measurement of 8-OHdG may arise. In fact, the hydrolysis and derivatization procedures needed for GC/MS can introduce artefacts leading to an overestimation of oxidative damage; whereas HPLC might underestimate 8-OHdG due to incomplete enzymatic digestion of DNA, and the production of hydroxyl radicals during PPL assay can cause artefactual formation of 8-OHdG from dG (13). For all these reasons, an immunoperoxidase method, using the monoclonal antibody 1F7, has recently been developed for 8-OHdG detection in single cells (14). This assay has shown sufficient sensitivity for oxidative damage detection in liver tissues from aflatoxin B1 (AFB1)-treated rats and oral mucosa cells from smokers and non-smokers (15).

In this study, after optimizing the method for the evaluation of oxidative DNA damage in human cells, we have used this technique to estimate the oxidative damage and its possible involvement in the development of cervical dysplasia.


    Materials and methods
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 Materials and methods
 Results
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Human subjects
Thirty-six women undergoing a routine Papanicolaou smear were enrolled in the study. After informed consent was obtained, cervical cells collected by scraping were spread on slides precoated with 0.2% poly-D-lysine. For each patient, two exocervical smears were prepared: the first one was used for PAH–DNA adduct immunohistochemical evaluation, and the second smear, stained by the Papanicolaou method, was utilized for cytological diagnosis. Samples were classified in one of three groups [negative, or low-grade or high-grade squamous intraepithelial lesion (LGSIL and HGSIL, respectively)] according to the Bethesda System (16). Women with cytological findings consistent with SIL were referred for colposcopy and biopsy. Cytopathological changes indicating human papillomavirus (HPV) infection were quantified on Papanicolaou-stained smears. For the subsequent analyses, patients were considered either HPV positive (serotype-independent) or negative. The numbers of koilocytes and dyskeratocytes were analysed in at least five different randomly chosen fields (200x magnification) and no less than 400 cells were counted.

Immunoperoxidase detection of 8-OHdG
Slides were washed twice with PBS, treated with RNase (100 µg/ml; Sigma) for 1 h at 37°C and washed again. They were subsequently treated with proteinase K (10 µg/ml; Sigma) at room temperature. In order to denature DNA, the samples, after washing in PBS, were incubated with 4 N HCl for 10 min and with 50 mM Tris base for 5 min at room temperature. This method is essential for adduct evaluation, as demonstrated in previous studies (17). After washing with PBS, the samples were incubated with 0.3% H2O2 in methyl alcohol at room temperature for 30 min to quench endogenous peroxidase activity. Non-specific binding was blocked with 1.5% normal horse serum and incubation with the monoclonal antibody 1F7 (14) diluted 1:50 in horse serum was performed overnight at 4°C. The slides were then incubated with a biotinylated horse anti-mouse secondary antiserum, and the reaction was visualized with the ABC complex followed by diaminobenzidine (Vector Laboratories, Burlingame, CA). The samples were dehydrated in serial ethyl alcohol solutions and xylene and mounted with Permount (Fisher Scientific, Pittsburgh, PA). To minimize variations in the immunohistochemical assay, untreated control samples and 50 µg/ml AFB1-treated MCF-7 cells were stained together with each batch of samples.

To demonstrate staining specificity, a subset of slides from smokers were stained under various control conditions. For example, cells were pre-treated with DNase (Sigma; 100 µg/ml for 1 h at 37°C) before staining, stained omitting primary antiserum, or treated with a non-specific antibody, recognizing DNA damage produced by the photoactivated drug 8-methoxypsoralen (8-MOP, 8G1; 1:10 dilution of hybridoma supernatant) (18), or with the specific polyclonal antiserum preabsorbed with 8-OHdG (1 µg/µl for 20 min at room temperature).

A cell analysis system 200 microscope (Becton Dickinson, San Jose, CA) was used to measure the relative intensity of nuclear staining in 100 randomly selected cells. The area was explored in terms of 20–25 fields, within each of which four to five cells were selected, excluding those carrying morphologically degraded and pyknotic nuclei and the inflammatory cells. The image was obtained in black and white and the average optical density (AOD) was recorded. Results were expressed for each sample as the average of relative optical density multiplied by 1000 ± SD. This value (the estimate of 8-OHdG) was taken into account for the statistical analyses performed.

Definition of the standard dose–response curve
In order to develop a standard dose–response curve, MCF-7 breast adenocarcinoma cells, which are able to metabolize AFB1, were treated with increasing concentrations of AFB1 (0–50 µg/ml) dissolved in DMSO in complete RPMI medium. After 12 h the medium was removed and cells were recovered by treatment with trypsin for 5 min at 37°C. Cells were collected and centrifuged at 1300 r.p.m. for 10 min. The cell pellet was resuspended in sucrose buffer (0.25 M sucrose, 1.8 mM CaCl2, 25 mM KCl and 50 mM Trizma base pH 7.5) to a final ratio of 1x106 cells/ml. About 25 µl of cell suspension were added to 150 µl of carbowax-ethanol and cytospun at 300 r.p.m. for 5 min on slides precoated with 0.2% poly-D-lysine. Slides were air dried, fixed with 95% ethanol at –20°C and stored at –20°C until staining. The oxidative DNA damage was evaluated using the 1F7 antibody, which is specific for 8-OHdG (14).

Statistical analysis
To evaluate differences in 8-OHdG content among dysplasia stages, the one-way analysis of variance (ANOVA) was used. To evaluate differences in 8-OHdG among HPV+ and HPV women the unpaired t-test with equal variances was performed. Normality of AOD data was assessed by the Shapiro–Wilk W-test.

Moreover, in order to evaluate the increase in risk of progression through SIL grades as a function of 8-OHdG levels, and to evaluate the role of HPV and age as confounders, the polytomous logistic and the proportional odds models, extensions of the logistic regression analysis for unordered and ordered categorical responses were used. The proportional odds model was used because SIL grades can be considered ordinal categories, possible expression of a latent continuous process. In such cases, the literature suggests the appropriateness of the proportional odds model. Anyway, being aware of the strong proportionality assumptions of this model, we also tested the polytomous logistic model approach, which does not imply the ordinal nature of the outcome variable.

All the statistical analyses were performed with the Stata 6.0 statistical package (Stata Corp., College Station, TX).


    Results
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 Materials and methods
 Results
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 References
 
Definition of the standard dose–response curve
The means ± SD of the nuclear staining levels in MCF-7 cells for exposure to increasing concentrations of AFB1 are shown in Figure 1Go. Whereas the overall behaviour of the dose–response relationship appears to be curvilinear, this relationship shows a linear trend (Figure 1Go, inset) within the range of 8-OHdG values (30–125, see below) found in the human subject cervical cell samples (Pearson R = 0.975).



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Fig. 1. Dose–response curve of 8-OHdG increase in MCF-7 cells treated with 0, 0.78, 1.56, 3.12, 6.25, 12.5, 25 and 50 µg/ml AFB1. The linear relationship in the range up to 200 relative staining units (AOD) is shown in the inset (which is an extract of the largest plot, as indicated by dashed box) (Pearson's R = 0.975).

 
Pre-absorption of primary antiserum with 8-OHdG (1 µg/µl) before use decreased relative staining intensity of cells treated with 50 µg/ml AFB1 from 355 ± 91 to 43 ± 13. Pre-treatment of slides with DNase decreased staining of the same cells to 40 ± 8. Substitution of primary antiserum with a non-specific antiserum recognizing DNA damage produced by 8-MOP decreased the staining level of 50 µg/ml AFB1-exposed cells to control cell levels (data not shown).

Cervical cell samples
Representative positive and negative nuclear staining for 8-OHdG in cervical cells is shown in Figure 2Go. The specificity of the reaction was assessed by comparing the AOD of a positive sample (125 ± 36) to that obtained for the same sample after pre-adsorption of the primary antiserum with 8-OHdG (1 µg/µl): nuclear staining of this sample decreased to 21 ± 7. Moreover, pre-treatment of the same sample with DNAse decreased staining to 18 ± 10. Finally, staining with an unrelated antiserum recognizing DNA damage produced by 8-MOP was 25 ± 7, whereas after omission of the primary antiserum it was 18 ± 9 (data not shown).



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Fig. 2. Immunohistochemical staining for 8-OHdG in cervical cells. Staining of cells from (A) subject 11 (AOD ± SD = 37 ± 5) and (B) subject 32 (AOD ± SD = 112 ± 20). Magnification x400.

 
Nuclear staining levels showed differences between cytologic groups (Table IGo). The distribution of AOD values was compatible with normality within each group, as assessed by the Shapiro–Wilk W-test.


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Table I. Summary statistics for the three cytologic groups
 
The analysis of variance (ANOVA) showed significant differences in the means of the 8-OHdG level among normal, LGSIL and HGSIL (P < 0.001). Comparison of the three groups showed statistical significant difference between normal and HGSIL (P < 0.001, Bonferroni corrected) and between LGSIL and HGSIL (P = 0.003, Bonferroni corrected), but not between normal and LGSIL (P = 0.17, Bonferroni corrected).

Grouping observations by HPV status, mean relative staining was 67.1 ± 23.3 in the 21 HPV and 68.5 ± 27.4 in the 15 HPV+ patients. As these 8-OHdG levels were compatible with the normal distribution, the unpaired t-test with equal variances was performed, which showed no significant difference in 8-OHdG levels between HPV+ and HPV subjects (P = 0.8).

The distribution of 8-OHdG levels in HPV+ and HPV subjects categorized according to histological diagnosis is shown in Figure 3Go.



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Fig. 3. Box-plot of relative staining intensity of 8-OHdG in cervical cells by dysplasia class and HPV status. The lower and upper edges of the boxes are the 25 and 75 percentiles, respectively. Median values are shown by the line within the box. The width of each box is proportional to sample number. Whiskers represent adjacent values. On the y-axis are shown the highest and lowest values of the whole sample and the median values of each group.

 
To evaluate the increase of risk of SIL grade progression for increasing 8-OHdG levels, both polytomous and proportional odds regression models were run. The results of both models are presented in Table IIGo, in which the odds ratios (ORs) for a 15 unit increase in 8-OHdG level are shown. Both models confirm an increase in the risk of progression through SIL grades for increasing levels of 8-OHdG.


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Table II. Results of the polytomous and proportional odds models
 
The ORs estimated for 8-OHdG AOD in the polytomous logistic model either considering the LGSIL versus normal (OR 2.34, P = 0.052) and the HGSIL versus LGSIL (OR 3.17, P = 0.020) were similar and comparable with the combined OR in the proportional odds model (OR 3.34, P < 0.001). These findings, together with the similar log likelihood values obtained for the two models (–27.93 versus –28.21) support the applicability of the proportional odds model assumptions in this case. The proportional odds models have higher statistical power than the corresponding polytomous ones, and therefore they also produce a more reliable result in the presence of a small sample size, as in this case. Therefore, we can assert that the OR = 3.34 (95% confidence interval 1.77–6.29) can be considered the best estimate of the increasing risk for 15 unit increase in 8-OHdG through all the process from normal to HGSIL.

The attempt to adjust the models for HPV status in multivariate models produced unreliable estimates, due to low number of observations for some covariate patterns. Moreover, age did not act as a confounder, the age distribution of subjects being somewhat similar in the three groups, as can be seen in Table IGo.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cervical carcinoma is the third most frequent female genital cancer (21). Cervical carcinogenesis is a multistep process, developing through stages that can be histologically recognized. Invasive cervical cancer is preceded by dysplastic lesions that are associated with HPV infection (22). Although the viral infection plays an important role, the exact mechanism for the development of cervical cancer is still unknown.

In the present study the extent of the endogenous oxidative DNA damage in human cervical cells was assessed by an immunoperoxidase method and was related to the presence of HPV infection and SIL. The immunoperoxidase technique has the advantage that it allows oxidative DNA damage determination without the risk of artificial production of 8-OHdG during the DNA extraction and hydrolytic processes (23). Moreover, the morphological approach allows the detection of adducts in specific cell types, permitting exclusion of unrelated, for example inflammatory, cells. The specificity of the method was tested in vitro by treating MCF-7 cells with AFB1, which was reported to induce oxidative DNA damage in a time- and dose-dependent manner (24). A dose-related increase in the detectable oxidative damage was observed in MCF-7 cells, and the 50 µg/ml AFB1-treated cells showed an 8-OHdG content 6-fold more elevated with respect to untreated controls. The MCF-7 cells were used to measure 8-OHdG increase following exposure to AFB1 because these cells are known to be able to metabolize and activate AFB1 and they have been used previously for similar purposes (15). However, we also performed similar experiments with the Rat-1 cells (normal diploid rat fibroblasts) exposed to increasing doses of H2O2, and obtained a similar linear dose–response relationship with the same antibody within the range of values observed in cervical cells (data not shown). Moreover, four different approaches were performed to demonstrate the specificity of immunoperoxidase reaction: (i) substitution of primary antiserum with a non-specific antibody; (ii) pre-absorption of primary antiserum with antigen; (iii) pre-treatment with DNase; and (iv) omission of specific primary antiserum. In all these conditions the relative nuclear staining of cervical cells decreased to background level.

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. In particular, the mean relative staining was elevated ~2-fold in samples with HGSIL compared with normal. These data are in agreement with other studies reporting increased levels of 8-OHdG in neoplastic cells with respect to normal cells in breast, kidney, lung, brain, stomach and colon cancers (25,26).

It is well known that HPV infection plays a critical role in etiology of dysplasia of the cervix. Our data show similar levels of 8-OHdG in patients with and without HPV infection, suggesting that the link between HPV and cervical dysplasia may be independent of oxidative damage.

Polytomous and proportional odds models, extensions of the logistic regression, showed that the risk associated with increasing 8-OHdG levels appears to be roughly constant through all the process from normal to HGSIL.

Our data do not allow us to definitely rule out that the increase in 8-OHdG level is just a consequence, rather than a cause, of the dysplastic state. A mechanism has been proposed for cervical carcinogenesis involving the HPV proteins E6 and E7 that are able to interact with, and inactivate, the p53 and pRb tumor suppressor proteins (27). Much evidence suggests, however, that other mechanisms may synergize with HPV infection in cervical cell transformation (28) and it has been proposed that oxidative damage induced by diversely originating ROS might be one of them (28,29). It is also possible that the increase in 8-OHdG level is the consequence of an unknown non-HPV molecular event. However, we believe that the observed relationship between oxidative DNA damage and the development of cervical SIL retains its importance whatever the cause of the increase in 8-OHdG level might be.

Studies on rat tissues have reported that oxidative damage to DNA could be a major cause of the physiological changes associated with aging (30). Our data show no correlation between patient's age and oxidized base adducts in DNA, in agreement with other studies performed on human breast and brain tissues (3133).

In conclusion, our results show that a link exists between oxidative DNA damage and the progression of cervical dysplasia and suggest that oxidative stress might play an important role in this disease. However, they require confirmation on a larger sample in order to better understand their implications in terms of cervical cancer prevention.


    Notes
 
3 To whom correspondence should be addressed Email: crogxxiii{at}rm.unicatt.it Back


    Acknowledgments
 
The authors would like to thank Prof. David Wypij, Department of Biostatistics, Harvard School of Public Health, MA, for his valuable suggestions about the statistical methods and the presentation of the results in this paper. This work has been supported in part by a grant from MURST 1998 (EX 40%).


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received May 5, 1999; revised February 15, 2000; accepted February 25, 2000.