Department of Gynecology, Womens Hospital, Zhejiang University School of Medicine, Hangzhou 310006, Zhejiang, China
1 To whom correspondence should be addressed. e-mail: xinmei6{at}yahoo.com
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Abstract |
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Key words: bleomycin/chromatid/cytogenetics/endometriosis susceptibility/mutagen sensitivity
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Introduction |
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Studies from family investigations have demonstrated a higher incidence of endometriosis among familial cases compared with women without family history (Malinak et al., 1980; Simpson and Bischoff, 2002
). Familial tendency for endometriosis suggests an individual genetic susceptibility. Recent studies from molecular cytogenetic analysis have detected abnormal chromosomes including chromosomal gains or losses, loss of heterozygosity (LOH), clonal genetic changes and allelic imbalances in endometriotic cells (tissue) and endometriosis-derived cell lines (Jiang et al., 1998
; Gogusev et al., 2000a
,b; Sato et al., 2000
). These findings indicate that endometriosis can be considered as a polygenic/multifactorial disease with a possible genetic predisposition and with the involvement of environmental toxins, especially dioxins (TCDD) and polychlorodiphenyl compounds (PCBs), in its pathogenesis (Baranova et al., 1997
). In turn, research into the association of genetic susceptibility with environmental toxins may be useful to elucidate the undefined aetiology and pathogenesis of endometriosis.
Although chromosome breakage syndromes reflect extreme examples of the susceptibility syndrome, there may be, within the general population, individuals with latent genetic instability that can be unmasked by mutagen challenge in vitro. On the basis of this hypothesis, the mutagen sensitivity challenge assay has been used as a measure of constitutional genetic instability (net results of DNA repair capability and initial genetic instability) (Wu et al., 1998b). Numerous studies have shown that mutagen sensitivity is a good independent risk predictor for developing cancers (Spitz et al., 1993
; Wu et al., 1995a
). Yet, no correlation between mutagen sensitivity and risk of endometriosis, which exhibits some characteristics reminiscent of malignancy, has been reported.
Endometriosis is considered a benign disorder, but shares characteristics with malignancy such as abnormal morphology, deregulated growth of cells, cellular invasion and neoangiogenesis. The glandular epithelium occasionally displays cytological atypia and/or hyperplasia (Seidman, 1996), as well as DNA aneuploidy (Ballouk et al., 1994
). In vitro evidence exists for a monoclonal origin in endometriosis (Jiang et al., 1996
; Jimbo et al., 1997
). The progression of endometriotic lesions to frank malignancy can occur, although with a very rare incidence (Martini et al., 2002
). In this context, it is suggested that the development of endometriosis, like malignant diseases, might involve the acquisition of somatic genetic alterations in genes that regulate cell growth and differentiation.
In the present study, we investigated whether mutagen sensitivity, an index of constitutional genetic instability, was related to the risk of development of endometriosis. The mutagen sensitivity of peripheral lymphocytes from study subjects was determined by measuring the chromatid breaks induced by bleomycin in short-term culture. Bleomycin is a radiomimetic agent that causes single- and double-stranded breaks requiring base excision repair. The bleomycin sensitivity assay has been well established and widely used as an independent risk predictor for developing cancers of lung (Spitz et al., 1993; Wu et al., 1996
), liver (Wu et al., 1998a
) head and neck (Yu et al., 1999
; Zych et al., 2000
). In this study, we found that the mean number of chromatid breaks per cell induced by bleomycin in women with endometriosis was significantly higher than in women without endometriosis (P < 0.001).
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Materials and methods |
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Cytogenetic analysis
In order to avoid subjective bias, a special technician at the genetics laboratory blind to the status of subjects was required to collect blood from all study subjects and conducted the standard procedure of cytogenetic analysis. First, blood was collected into heparinized tubes, transferred to the genetics laboratory and processed within 24 h. Afterwards, standard lymphocyte cultures were established as described previously (Zych et al., 2000). Briefly, 0.5 ml of whole blood was added to the growth medium consisting of 4.5 ml of RPMI 1640 medium (Gibco) supplemented with 15% fetal calf serum (Gibco), 1.5% phytohaemagglutinin (Gibco) and 100 U/ml each of penicillin and streptomycin (Shanghai Pharmaceutical Co., China). After the cells had been cultured for 3 days at 37°C with 5% CO2, they were incubated for 5 h with 30 mU/ml bleomycin (Chemical and Pharmaceutical Co. Ltd, Japan). To arrest the cells at metaphase, 100 µl of 50 µg/ml colcemid (Sigma, St Louis, MO) was added to the cultures 1 h before harvesting. This yielded cells in metaphase that were damaged by the bleomycin in the late SG2 phase of the cell cycle. The red blood cells were removed, and the lymphocytes were swollen in hypotonic solution (0.06 M KCl) and fixed in Carnoys fixative [3:1 (v/v) methanol:acetic acid]. After dropping the cells onto wet slides, the metaphase spreads were air dried and stained with Giemsa (Merck). All slides were number-matched to all study subjects and read by another experienced technician blind to the status of the subjects. For each subject, chromatid breaks were counted in 50 metaphases and expressed as chromatid breaks per cell. In the process of counting, only frank chromatid breaks or exchanges were recorded; chromatid gaps or attenuated regions were disregarded (Figure 1).
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Results |
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Discussion |
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There is increasing evidence that endometriosis, like neoplasias, may require the acquisition of somatic genetic alterations for its development. Molecular cytogenetic studies on endometriotic tissues and established endometriosis-derived cell lines have provided new evidence that acquired chromosome-specific alterations may be involved in endometriosis, possibly reflecting clonal expansion of chromosomally abnormal cells (Wu et al., 1995b). Molecular DNA studies examining the role of LOH in endometriotic lesions have identified that candidate tumor suppressor gene loci, including 5q, 6q, 9p, 11q and 22q, may play a role in the malignant transformation of endometriotic implants to endometrioid ovarian cancers (Bischoff and Simpson, 2000
). Recent evidence of mutations in the tumour suppressor gene in the endometrioid subtype of epithelial ovarian cancer has shown that somatic genetic alterations represent early events in the transformation of benign endometriotic cells (Thomas and Campbell, 2000
). In present study, we found that chromatid breaks induced by bleomycin in peripheral blood lymphocytes were seen mostly on chromosome 4 and 5, although some were seen on chromosome 1, 2, 6, 16 and 17. This result was in agreement with the reported bleomycin sensitivity test (Wu et al., 1995a
). It is implied that chromosome breakage depends not only on induced exogenous mutagen but also on individual inheritable susceptibility to environmental toxins.
Environmental contaminants including TCDD and PCBs have been suggested to play a role in the pathobiology of endometriosis (Baranova et al., 1997; Bruner-Tran et al., 1999
). In experimental animal models, TCDD and TCDD-like exposure promotes establishment of experimental endometriosis by interfering with the ability of progesterone to suppress endometrial matrix metalloproteinase (MMP) expression that is necessary for the establishment of ectopic lesions (Bruner-Tran et al., 1999
). TCDD induces many cytochrome P450 (CYP) isozymes, including CYP1A1, CYP1A2, CYP1B1, that hydroxylate 17
-estradiol to various catechols (Ricci et al., 1999
). Conversely, these induced CYP isozymes are largely responsible for TCDD metabolism (Inouye et al., 2002
; Shinkyo et al., 2003
). It is suggested that individuals with reduced activities of metabolic detoxification enzymes caused by genetic polymorphisms would have an increased sensitivity to environmental toxins. Recent evidence has shown that chromosome breakage and aberrations are associated with genes encoding polymorphic metabolic detoxification enzymes including glutathione S-transferase M1 (GSTM1) (Poli et al., 1999
; Norppa, 2001
), N-acetyltransferase 2 (NAT2) (Norppa, 2001
) and CYP 1A1/1A2 (Poli et al., 1999
; Abdel-Rahman et al., 2000
). In cultured human lymphocytes, the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK) induced a significantly higher level of chromosome aberration in cells with the CYP2E1 WT/*5B genotype compared with cells with the CYP2E1 WT/WT (Abdel-Rahman et al, 2000
). The individuals who lack the GSTM1 gene have a higher level of DNA adducts and chromosome damage detected in lymphocytes, and the GSTT1-null individuals have an increased baseline level of sister chromatid exchanges (SCEs) in lymphocytes. In addition, subjects having the NAT2 slow acetylator genotype also have an increased baseline frequency of lymphocyte chromosome aberrations (Norppa, 2001
). As a result, genetic polymorphisms may be important in explaining individual variation in genotoxic response observed in genetic toxicology tests with human cells. Baranova et al. (1997
) reported that endometriosis patients had a higher percentage of null GSTM1 genotype and NAT2 slow acetylator genotypes compared with controls. A recent study of genetic polymorphism analysis has found that the CYP19 VNTR (TTTA)(10) allele as well as the combined genotype CYP1A1 m1 polymorphism and GSTM1 null deletion were associated with the risk of endometriosis development (Arvanitis et al., 2003
). In this context, it is speculated that gene polymorphic metabolic detoxification enzymes GSTM1, NAT2 and CYP1 may affect bleomycin sensitivity in short-term cultured lymphocytes from our study participants. Interestingly, Kocabas et al. (2000
) reported that null GSTM1 genotype, null GSTT1 and NAT2 slow acetylator genotype associated with the elevated risk of endometriosis development does not influence bleomycin sensitivity. In contrast, genetic polymorphisms of X-ray repair cross-complementing 1 (XRCC1), a DNA repair gene, affect cell sensitivity to bleomycin (Tuimala et al., 2002
). Conse quently, whether gene polymorphic metabolic detoxification enzymes GSTM1, NAT2 and CYP1 impact on bleomycin sensitivity in patients with endometriosis warrants further investigation. Also, it is indicated that sensitivity to bleomycin may have high heritability, reflecting individual capacity to repair DNA lesions, and endometriosis may arise as result of somatic DNA alterations occurring in a multistep process, analogous to the origin of neoplasias.
Recent studies have shown that individuals who smoke heavily and have a history of tobacco dust exposure have an increased percentage of chromosome aberrations in lymphocytes compared with healthy non-smokers (Kao-Shan et al., 1987; Umadevi et al., 2003
). In short-term cultured lymphocytes, the mean number of chromatid breaks per cell induced by bleomycin was significantly higher in smokers than in non-smokers (Strom et al., 1995
). However, in our current study, all participants were non-smokers, and we did not find any statistical correlations between passive smoking in the home and workplace and chromatid breaks per cell. The possibility that the small sample size and light passive smoking in this study might affect our results should be considered. Since ageing has an important influence on DNA damage and repair due to a gradual accumulation of aberrant cells in both the stem cells of bone marrow and peripheral lymphocytes resulting from a decreased efficiency in the recognition and repair of induced damage or an increase in accumulated exposure to environmental clastogenic agents, the cells from older individuals should exhibit an increase in the amount of chromosome aberrations (Carbonell et al., 1996
). However, our data showed no significant correlation between chromatid breaks per cell and age. This discrepancy may be relative to the age distribution selected in our participants. Since endometriosis may arise as a result of somatic DNA alterations occurring in a multi-step process analogous to the origin of neoplasia, it is suggested that patients with late-stage endometriosis might be more sensitive to bleomycin than those with early-stage endometriosis. However, our data did not support this hypothesis. Of course, one possibility is that bleomycin sensitivity may represent an effect rather than a cause of endometriosis. Therefore, further studies are necessary to determine these correlations.
Although some personal lifestyle factors including menstrual characteristics, dysmenorrhoea, previous abortion, age and smoking have been reported to be associated with endometriosis risk (Cramer and Missmer, 2002), only dysmenorrhoea in our study was found to have a higher incidence in cases than in controls when using logistic regression analysis. On further analysis, dysmenorrhoea was not correlated with chromatid breaks per cell when we categorized it as presence or absence. To date, endometriosis is only diagnosized pathologically, and thus it is difficult to recruit control participants. In this study, we used 14 patients with an ectopic pregnancy and five patients with an ovarian simple cyst (cysts with a thin wall and clear liquid) as the control group, and these might not be the ideal control subjects. However, if we use healthy participants as the control group, we cannot exclude endometriosis patients among them without undergoing surgery. Which healthy population participants are most appropriate as the non-endometriosis control group needs further investigation. In any case, our result with regard to mean chromatid breaks per cell in controls agreed with previous reported results (Carbonell et al., 1996
). In summary, this was the first study, to our knowledge, to evaluate the correlation between mutagen sensitivity and the risk of development of endometriosis. The preliminary data suggested that sensitivity to bleomycin-induced chromatid breaks in lymphocytes correlated with the risk of endometriosis development. Although the differences between cases and controls were statistically significant, the small sample size necessitates that this finding be validated in a larger study. More data are also needed to determine whether this sensitivity applies to the general endometriosis population.
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Acknowledgements |
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References |
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American Fertility Society (1995) Revised American Fertility Society classification of endometriosis. Fertil. Steril., 43, 351352.
Arvanitis, D.A., Koumantakis, G.E., Goumenou, A.G., Matalliotakis, I.M., Koumantakis, E.E. and Spandidos, D.A. (2003) CYP1A1, CYP19, and GSTM1 polymorphisms increase the risk of endometriosis. Fertil. Steril., 79, 702709.[CrossRef][ISI][Medline]
Ballouk, F., Ross, J.S. and Wolf, B.C. (1994) Ovarian endometriotic cysts. An analysis of cytologic atypia and DNA ploidy patterns. Am. J. Clin. Pathol., 102, 415419.[ISI][Medline]
Baranova, H., Bothorishvilli, R., Canis, M., Albuisson, E., Perriot, S., Glowaczower, E., Bruhat, M.A., Baranov, V. and Malet, P. (1997) Glutathione S-transferase M1 gene polymorphism and susceptibility to endometriosis in a French population. Mol. Hum. Reprod., 3, 775780.[Abstract]
Baranova, H., Canis, M., Ivaschenko, T., Albuisson, E., Bothorishvilli, R., Baranov, V., Malet, P. and Bruhat, M.A. (1999) Possible involvement of arylamine N-acetyltransferase 2, glutathione S-transferases M1 and T1 genes in the development of endometriosis. Mol. Hum. Reprod., 5, 636641.
Bischoff, F.Z. and Simpson, J.L. (2000) Heritability and molecular genetic studies of endometriosis. Hum. Reprod. Update, 6, 3744.
Bischoff, F.Z., Heard, M. and Simpson, J.L. (2002) Somatic DNA alterations in endometriosis: high frequency of chromosome 17 and p53 loss in late-stage endometriosis. J. Reprod. Immunol., 55, 4964.[CrossRef][ISI][Medline]
Bruner-Tran, K.L., Rier, S.E., Eisenberg, E. and Osteen, K.G. (1999) The potential role of environmental toxins in the pathophysiology of endometriosis. Gynecol. Obstet. Invest., 48, 4556.[CrossRef][ISI][Medline]
Carbonell, E., Peris, F., Xamena, N., Creus, A. and Marcos, R. (1996) Chromosome aberration analysis in 85 control individuals. Mutat. Res., 370, 2937.[ISI][Medline]
Cramer, D.W. and Missmer, S.A. (2002) The epidemiology of endometriosis. Ann. NY Acad. Sci., 955, 1122.
Gogusev, J., Bouquet de Joliniere, J., Telvi, L., Doussau, M., Stojkoski, A. and Levardon, M. (2000a) Cellular and genetic constitution of human endometriosis tissues. J. Soc. Gynecol. Invest., 7, 7987.[CrossRef][ISI][Medline]
Gogusev, J., Bouquet de Joliniere, J., Telvi, L., Doussau, M., du Manoir, S., Stojkoski, A. and Levardon, M. (2000b) Genetic abnormalities detected by comparative genomic hybridization in a human endometriosis-derived cell line. Mol. Hum. Reprod., 6, 821827.
Inouye, K., Shinkyo, R., Takita, T., Ohta, M. and Sakaki, T. (2002) Metabolism of polychlorinated dibenzo-p-dioxins (PCDDs) by human cytochrome P450-dependent monooxygenase systems. J. Agric. Food Chem., 50, 54965502.[CrossRef][ISI][Medline]
Jiang, X., Hitchcock, A., Bryan, E.J., Watson, R.H., Englefield, P., Thomas, E.J. and Campbell, I.G. (1996) Microsatellite analysis of endometriosis reveals loss of heterozygosity at candidate ovarian tumor suppressor gene loci. Cancer Res., 56, 35343539.[Abstract]
Jiang, X., Morland, S.J., Hitchcock, A., Thomas, E.J. and Campbell, I.G. (1998) Allelotyping of endometriosis with adjacent ovarian carcinoma reveals evidence of a common lineage. Cancer Res., 58, 17071712.[Abstract]
Jimbo, H., Hitomi, Y., Yoshikawa, H., Yano, T., Momoeda, M., Sakamoto, A., Tsutsumi, O., Taketani, Y. and Esumi, H. (1997) Evidence for monoclonal expansion of epithelial cells in ovarian endometrial cysts. Am. J. Pathol., 150, 11731178.[Abstract]
Kao-Shan, C.S., Fine, R.L., Whang-Peng, J., Lee, E.C. and Chabner, B.A. (1987) Increased fragile sites and sister chromatid exchanges in bone marrow and peripheral blood of young cigarette smokers. Cancer Res., 47, 62786282.[Abstract]
Kocabas, N.A., Karahalil, B., Karakaya, A.E. and Sardas, S. (2000) Influence of GSTM1 genotype on comet assay and chromosome aberrations after induction by bleomycin in cultured human lymphocytes. Mutat. Res., 469, 199205.[ISI][Medline]
Malinak, L.R., Buttram, V.C. Jr., Elias, S. and Simpson, J.L. (1980) Heritage aspects of endometriosis. II. Clinical characteristics of familial endometriosis. Am. J. Obstet. Gynecol., 137, 332337.[ISI][Medline]
Martini, M., Ciccarone, M., Garganese, G., Maggiore, C., Evangelista, A., Rahimi, S., Zannoni, G., Vittori, G. and Larocca, L.M. (2002) Possible involvement of hMLH1, p16(INK4a) and PTEN in the malignant transformation of endometriosis. Int. J. Cancer, 102, 398406.[CrossRef][ISI][Medline]
Norppa, H. (2001) Genetic polymorphisms and chromosome damage. Int. J. Hyg. Environ. Health, 204, 3138.[ISI][Medline]
Poli, P., Buschini, A., Candi, A. and Rossi, C. (1999) Bleomycin genotoxicity alteration by glutathione and cytochrome P-450 cellular content in respiratory proficient and deficient strains of Saccharomyces cerevisiae. Mutagenesis, 14, 233238.
Ricci, M.S., Toscano, D.G., Mattingly, C.J. and Toscano, W.A., Jr (1999) Estrogen receptor reduces CYP1A1 induction in cultured human endometrial cells. J. Biol. Chem., 274, 34303438
Sato, N., Tsunoda, H., Nishida, M., Morishita, Y., Takimoto, Y., Kubo, T. and Noguchi, M. (2000) Loss of heterozygosity on 10q23.3 and mutation of the tumor suppressor gene PTEN in benign endometrial cyst of the ovary: possible sequence progression from benign endometrial cyst to endometrioid carcinoma and clear cell carcinoma of the ovary. Cancer Res., 60, 70527056.
Seidman, J.D. (1996) Prognostic importance of hyperplasia and atypia in endometriosis. Int. J. Gynecol. Pathol., 15, 19.[ISI][Medline]
Shinkyo, R., Sakaki, T., Ohta, M. and Inouye, K. (2003) Metabolic pathways of dioxin by CYP1A1: species difference between rat and human CYP1A subfamily in the metabolism of dioxins. Arch. Biochem. Biophys., 409, 180187.[CrossRef][ISI][Medline]
Simpson, J.L. and Bischoff, F.Z. (2002) Heritability and molecular genetic studies of endometriosis. Ann. NY Acad. Sci., 955, 239251.
Spitz, M.R., Fueger, J.J., Halabi, S., Schantz, S.P., Sample, D. and Hsu, T.C. (1993) Mutagen sensitivity in upper aerodigestive tract cancer: a casecontrol analysis. Cancer Epidemiol. Biomarkers Prev., 2, 329333.[Abstract]
Strom, S.S., Wu, S., Sigurdson, A.J., Hsu, T.C., Fueger, J.J., Lopez, J., Tee, P.G. and Spitz, M.R. (1995) Lung cancer, smoking patterns, and mutagen sensitivity in Mexican-Americans. J. Natl Cancer Inst. Monogr., 18, 2933.[Medline]
Thomas, E.J. and Campbell, I.G. (2000) Molecular genetic defects in endometriosis. Gynecol. Obstet. Invest., 50, 4450.[CrossRef][ISI][Medline]
Tuimala, J., Szekely, G., Gundy, S., Hirvonen, A. and Norppa, H. (2002) Genetic polymorphisms of DNA repair and xenobiotic-metabolizing enzymes: role in mutagen sensitivity. Carcinogenesis, 23, 10031008.
Umadevi, B., Swarna, M., Padmavathi, P., Jyothi, A. and Reddy, P.P. (2003) Cytogenetic effects in workers occupationally exposed to tobacco dust. Mutat. Res., 535, 147154.[ISI][Medline]
Wu, X., Delclos, G.L., Annegers, J.F., Bondy, M.L., Honn, S.E., Henry, B., Hsu, T.C. and Spitz, M.R. (1995a) A casecontrol study of wood-dust exposure, mutagen sensitivity, and lung cancer risk. Cancer Epidemiol. Biomarkers Prev., 5, 583588.
Wu, X., Hsu, T.C., Annegers, J.F., Amos, C.I., Fueger, J.J. and Spitz, M.R. (1995b) A casecontrol study of nonrandom distribution of bleomycin-induced chromatid breaks in lymphocytes of lung cancer patients. Cancer Res., 55, 557561.[Abstract]
Wu, X.F., Spitz, M.R., Delclos, G.L., Connor, T.H., Zhao, Y., Siciliano, M.J. and Hsu, T.C. (1996) Survival of cells with bleomycin-induced chromosomal lesions in the cultured lymphocytes of lung cancer patients. Cancer Epidemiol. Biomarkers Prev., 5, 527532.[Abstract]
Wu, X., Gu, J., Patt, Y., Hassan, M., Spitz, M.R., Beasley, R.P. and Hwang, L.Y. (1998a) Mutagen sensitivity as a susceptibility marker for human hepatocellular carcinoma. Cancer Epidemiol. Biomarkers Prev., 7, 567570.[Abstract]
Wu, X., Gu, J., Amos, C.I., Jiang, H., Hong, W.K. and Spitz, M.R. (1998b) A parallel study of in vitro sensitivity to benzo[a]pyrene diol epoxide and bleomycin in lung cancinoma cases and controls. Cancer, 83, 11181127.[CrossRef][ISI][Medline]
Yu, G.P., Zhang, Z.F., Hsu, T.C., Spitz, M.R. and Schantz, S.P. (1999) Family history of cancer, mutagen sensitivity, and increased risk of head and neck cancer. Cancer Lett., 146, 93101.[CrossRef][ISI][Medline]
Zych, M., Schlade-Bartusiak, K., Chorostkowska, A., Stembalska, A., Krecicki, T. and Sasiadek, M. (2000) Bleomycin-induced chromosome aberrations in head and neck cancer patients analyzed by classical cytogenetics and FISH. Cancer Lett., 152, 123127.[CrossRef][ISI][Medline]
Submitted on February 21, 2003; resubmitted on May 6, 2003; accepted on June 12, 2003.