Evaluation of DNA damage in the esophageal mucosa and peripheral blood of patients with gastroesophageal reflux disease

Abstracts

RACIONAL: A doença do refluxo gastroesofágico é a afecção digestiva de maior prevalência. Os portadores podem apresentar na evolução algumas complicações, sendo o esôfago de Barrett a de maior importância, tendo em vista seu potencial de malignidade. Todavia os processos inflamatórios do trato gastrointestinal podem apresentar degeneração maligna. OBJETIVOS: Avaliar os possíveis danos do DNA em portadores de esofagite de refluxo gastroesofágico de vários graus e verificar a aplicação do ensaio Cometa na detecção dos mesmos. MÉTODOS: Foram estudados 25 pacientes distribuídos em quatro grupos: controle (n=5), esofagite leve (n=8), esofagite severa (n=5) e câncer (n=7). O ensaio Cometa foi realizado no sangue periférico (linfócitos) e biópsia do terço distal do esôfago. RESULTADOS: O ensaio Cometa detectou danos no DNA nos pacientes com esofagite leve e severa (sangue periférico e biópsia), sendo que na esofagite severa a intensidade dos danos foi maior (p<0,05). Os danos do DNA dos pacientes com esofagite severa e câncer não mostraram diferença significativa e a intensidade dos mesmos corresponde ao ensaio Cometa classe 4 (maior que 95% de danos). CONCLUSÕES: 1) As frequências de quebras do DNA da mucosa esofágica e linfócitos estão diretamente relacionadas ao grau de inflamação; 2) a esofagite severa apresenta praticamente a mesma frequência de danos no DNA do câncer esofágico; 3) o ensaio Cometa mostrou-se muito sensível para a detecção dos danos do DNA.

Esofagite péptica; Dano ao DNA; Ensaio em Cometa

ORIGINAL ARTICLE

^IDepartment of Genetics, Institute of Biosciences of Botucatu

^IIDepartment of Surgery and Orthopedics, School of Medicine, UNESP, Botucatu, SP, Brazil

Correspondence

ABSTRACT

BACKGROUND: The gastroesophageal reflux disease is the most prevalent digestive disorder. Patients with it may present some complications during its development, and Barrett's esophagus is the most important in view of its potential malignancy. However, the inflammatory processes of the gastrointestinal tract may show malignant degeneration.

AIM: To assess possible DNA damage in patients with gastroesophageal reflux esophagitis of various degrees and to evaluate the application of the Comet assay in its detection.

METHODS: Twenty-five patients were studied. They were divided into four groups: control (n=5), mild esophagitis (n=8), severe esophagitis (n=5) and cancer (n=7). The Comet assay was performed on peripheral blood cells (lymphocytes) and biopsy of the distal esophagus.

RESULTS: The Comet assay detected DNA damage in patients with mild and severe esophagitis (peripheral blood and biopsy), and damage intensity was greater in severe esophagitis (p<0,05). DNA damage in patients with severe esophagitis and cancer did not show significant difference, and its intensity corresponds to class-4 Comet assay (greater than 95% of damage).

CONCLUSIONS: 1) The frequencies of DNA breakage in the esophageal mucosa and lymphocytes are directly related to inflammation level; 2) severe esophagitis shows virtually the same DNA damage frequency as that of esophageal cancer; 3) the Comet assay showed to be very sensitive for DNA damage detection.

Headings: Esophagitis, peptic. DNA Damage. Comet assay.

INTRODUCTION

The gastroesophageal reflux disease (GERD) is an important disease of the gastrointestinal tract, in view of the high and increasing incidence, the intensity of symptoms and severity of complications, among which Barrett's esophagus (BE) stands out as the most important³. BE represents the last stage of esophagitis triggered by GERD, in which the distal squamous epithelium chronically exposed to gastroduodenal contents is replaced by metaplastic columnar epithelium¹⁵. The risk of developing cancer in BE patients is not well established and the rates range from 0.4 to 1.5%¹⁷ by year of follow-up.

However, any inflammation of the gastrointestinal tract predisposes to malignant degeneration⁵, a fact which may occur not only with the BE but in less advanced stages of esophagitis¹.

In addition, bacterial products released at the site of inflammation can cause DNA damage and these may represent the early stage of carcinogenesis¹².

Our objectives were to study the possible DNA damage in esophageal mucosa and peripheral blood of patients with reflux esophagitis in different degrees and verify the application of the Comet assay in the diagnosis of DNA damage and the risk of esophageal cancer.

METHOD

The design of this study was submitted to the Ethics Committee of the Faculdade de Medicina de Botucatu and approved (letter 187/2001). All patients signed a consent form.

Was studied 25 subjects (21 men and four women), aged between 15 and 85 years (mean: 57.32 ± 18.00 years) (Table 1).

After clinical assessment, patients underwent endoscopy and divided into four groups, depending on the endoscopic finding¹⁸ control (normal mucosa), mild esophagitis (reflux esophagitis grade I and II), severe esophagitis (esophagitis reflux grades III and IV) and cancer (squamous cell carcinoma).

During endoscopy four biopsies from the distal esophagus and 5 ml of peripheral blood were taken. Two samples from the biopsies were placed in Hanks balanced salt solution and the other in freezing midst.

All samples were sent for analysis of DNA damage using the Comet assay²⁰.

Esophageal mucosal cells and lymphocytes were isolated by digestion of proteins and collagenase, using the technique described by Pool-Zobel, et al.¹⁶. Then, the cells were subjected to the test of viability with ethidium bromide solution.

Determination of DNA damage

The technique used for determination of DNA damage was described by Singh, et al.²⁰, modified by Klaude, et al.¹¹, according to protocol Speit and Hartmann²².

The study of the slides allowed the blind analysis of 5000 cells, 50 per sheet totaling 200 cells per individual.

The cell analysis was made by the imaging system in epiflurescency microscope (Axiophot II Zeiss) and 400X magnification. Images were viewed with circular shapes (undamaged DNA - Class 0) and structures in the form of "Comets" (with DNA damage - Classes 1 to 4). The Comet assay grade 4 represents DNA damage in a percentage higher than 95%.

The length of each image means the distance of migration of damaged DNA strand. In each slide, the number of comets was multiplied by the value of its class, giving rise to the scores of each blade, which can range from 0 (no damage) to 200 (maximum damage). The scores were added to each slide, resulting in final scores.

The quantification of DNA damage of cells was performed by the Image Analysis System Internative Comet Assay II (Percentive Instruments). The parameters measured were the tail moment (tail length x tail intensity or frequency of DNA migrated) and total scores in groups.

For statistical analysis was used the Tukey test, which allowed the comparison of tail moment (CM) and total scores in the four groups.

RESULTS

Table 2 presents the averages evaluated parameters in the blood, according to groups and statistical tests.

Table 3 presents the means of the parameters evaluated in the second biopsy groups and statistical tests.

Considering the parameter of tail moment (Figure 1) was observed in biopsies significant difference between the control group, mild esophagitis, severe esophagitis and cancer, but the severe esophagitis and cancer groups showed no significant difference (p>0.05). Table 2 shows that in blood, control and mild esophagitis groups showed no significant difference when considering the tail moment parameter. However the values observed in these groups are smaller than those of severe esophagitis and cancer groups (p <0.05).

For scores (Figure 2) showed that when considering the biopsies, the four groups differ (Table 3). With respect to the blood, control groups, mild esophagitis and severe esophagitis are different (p <0.05), but the severe esophagitis and cancer groups showed no significant difference (Table 2).

DISCUSSION

In this research there was studied eight individuals with mild esophagitis and five with severe esophagitis. The average age of patients with severe esophagitis was 61.8 ± 18.07 years, higher than that observed in those with mild esophagitis (50.12 ± 18.58 years), suggesting that the esophageal mucosa suffered the harmful action of the stomach juice for a longer time, resulting in severe inflammation. A similar result was observed by Tseng, et al.²⁶, in epidemiological study conducted in China.

In addition to patients with esophagitis, were also studied seven patients with squamous cell carcinoma of the esophagus. In this group all patients were male, confirming literature data, according to which the disease has a predilection for male subjects^6,8,27. The age of patients with esophageal cancer (65.42 ± 14.08 years) does not differ from that by most authors^6,27. The patients in this group received no neodjuvant therapy (chemotherapy or radiotherapy) before collection of blood or biopsy of the distal esophagus, conduct that would cause DNA damage²³.

This research was conducted to evaluate DNA damage of the esophageal mucosa and peripheral blood of patients with gastroesophageal reflux esophagitis. DNA is not a stable molecule and is frequently exposed to various agents, natural or artificial, which may cause damage3. Under normal conditions, about 99% of the damaged DNA can be repaired, but about 1% can remain in the genome of the cell⁷. The unrepaired DNA damage can result in loss of genetic information, or interference with transcription and replication, and is therefore deleterious to the host⁴.

Another important aspect is that DNA damage can induce mutations^10,24. Such genetic instability may be linked to several diseases, including cancer^2,9.

Thus, detection of DNA damage is important to conduct research related to genetic toxicology and molecular epidemiology^19,25.

In this research was used the Comet assay or Single Cell Gel Eletrophoresis test indicator of genotoxicity, because quantifies DNA damage. The Comet assay was used by several researchers^12,14.

The assessment of DNA damage in peripheral blood (Table 2 - tail moment), showed that in control group and mild esophagitis values did not differ. However in severe esophagitis and cancer groups values are higher than those of the previous two groups (significant difference). In severe esophagitis and cancer groups observed values show no significant difference. When considering the parameter scores (Table 2) was observed than in control, mild esophagitis and severe esophagitis values differ, with most significant damage in the severe esophagitis group (143.6 ± 14.82) than in group mild esophagitis (42.5 ± 28.64). Severe esophagitis and cancer groups observed values show no significant difference (143.6 ± 14.82 X167.28 ± 22.60). The comparison of these results with the literature was hampered because no similar paper was found.

Table 3 shows that when analyzing DNA damage in esophageal mucosa, the values observed in control groups, mild esophagitis and severe esophagitis differed, being higher in the severe esophagitis group (4.96 ± 1.11) than in mild esophagitis (0.305 ± 0.37). The severe esophagitis and cancer groups were not significantly different (4.96 ± 1.11 X 5.24 ± 3.29). These results were observed when considering the tail moment parameter, in agreement to those published by Ladeira, et al.¹². These authors studied gastric mucosal biopsies of patients with mild and severe gastritis and found that the greater the severity of inflammation greater is the intensity of DNA damage.

In groups severe esophagitis and cancer DNA damage corresponds to the Comet assay class 4, with percentages above 95%.

When considering the parameter scores, the values observed in biopsies of patients control, mild esophagitis, severe esophagitis and cancer differ, being higher in severe esophagitis (220 ± 12.64) than in mild esophagitis (77.88 ± 28 ).

Inflammation, a variety of phagocytes (neutrophils, monocytes and macrophages) can generate free radicals in response to proinflammatory mediators²¹. Free radicals induce harmful effects to the cell's DNA and can induce DNA breaks and other genetic changes that are potentially carcinogenic³.

In this study, by analyzing the data obtained and taking into account the degree of mucosal inflammation, it can be seen that increasing the frequency of DNA breaks is related to increased intensity of mucosal inflammation, with significant differences between DNA damage in normal mucosa, mild esophagitis and severe cancer.

It should be noted that the Comet assay is a very sensitive technique that allows assessment of DNA damage in different cell populations, in addition to detecting differences in performance of the engine repair.

Based on these data, the authors suggest the possibility that inflammation itself exerts a strong genotoxic effect and consequent genomic instability in cells, increasing the likelihood of malignant degeneration.

CONCLUSIONS

The frequencies of DNA strand breaks in the esophageal mucosa and peripheral blood lymphocytes are directly related to the degree of inflammation; severe esophagitis presents almost the same frequency of DNA damage as in esophageal câncer; the Comet assay was highly sensitive for detecting DNA damage and appears to be promising as a prognostic test of susceptibility to malignancy.

REFERENCES

• 1. Andreollo NA, Lopes LR, Coelho-Neto JS. Doença do refluxo gastroesofágico: qual a eficácia dos exames no diagnóstico? Arq Bras Cir Dig. 2010; 23(1):6-10.
• 2. Ames BN. Mutagenesis and carcinogenesis: endogenous and exogenous factors. Env Mol Mutagen 1989; 14: suppl 16. p. 66-77.
• 3. Baik SC, Youn HS, Chung ME, Lee WK, Cho MJ, Ko GH, Park CK, Kasai H, Rhee K. Increased oxidative DNA damage in infected human gastric mucosa. Câncer Res 1996; 56: 1279-82.
• 4. Bernstein C, Bernstein H. Aging, sex and DNA repair. New York. Academic Press 1991. p. 15-25.
• 5. Boland CR, Luciani MG, Gasche C, Goel A. Infection, inflammation and gastrointestinal cancer. Gut 2005; 54: 1321-31.
• 6. Cho SH, Shim HJ, Lee SR, Ahn JS, Yang DH, Kim YK, Nam TK, Lee JJ, Kim HJ, Chung IJ. Concurrent chemoradiotherapy with S - 1 and cisplatin in advanced esophageal cancer. Diseases of the esophagus 2008; 21: 697-703.
• 7. Frei B. Reactive oxygen species and antioxidants vitamins: mechanisms of action. Am J Med 1994; suppl. 3A: 5S - 13S.
• 8. Henry MACA, Lerco MM, Oliveira WK. Câncer do esôfago em pacientescom megaesôfago chagásico. Arq Gastroent 2007; 44: 151-5.
• 9. Higginson, J. Changing concepts in câncer prevention: limitations and implications for future research in environmental carcinogenesis. Cancer Res 1988; 48: 1381-89.
• 10. Kings JS, Philips JW, Morgan WF. The role DNA Double strand break rejoining in chromosome damage in repair. In: Natarajan AT. Chromosomal Alteration. Berlin: Spring - Verlag 1994. p. 65-5.
• 11. Klaude M, Eriksson S, Nygren J, Ahnstion GN. THe comet assay: mechanisms and technical considerations. Mut Res 1996; 363: 89-96.
• 12. Ladeira MSP, Rodrigues MAM, Salvadori DMF, Queiroz DMM, Freire, Maia DV. DNA damage in patients infectes by Helicobacter pylori. Cancer Epidemiol Biomarbers Prev 2004; 13: 631-7.
• 13. Moraes-Filho JP, Cecconello I, Gama-Rodrigues JJ, Paula Castro L, Henry MACA, Meneghelli V, Quigley E. Brazilian consensus on gastroesophageal reflux disease: propasals for assessment, classification and manegement. Amer J Gastroent 2002; 97: 241-8.
• 14. Olliver JR, Hardie LJ, Gong YY, Dexter S, Chalmers D, Harris KM, Wild CP. Risk factors, DNA damage, an disease progression in Barrett's esophagus. Cancer Epidemiol Biomarkers Prev 2005; 14: 620.
• 15. Pisegna, J, Holtmann G, Howdwn CW, Katelaris PH, Sharma P, Spechler S. Review article: oesophgeal coplications and consequences of persistent gastro-oesophageal reflux diecase. Aliment Pharmacol Ther 2004; 20(suppl 9): 47-56.
• 16. Pool-Zobel BL, Bub AH, Wollowski I, Rechmmer G. Consumption of vegetables reduces genetic damage in humans: first results of human intervention trial with carotenoid a rich foods. Carcinogesis 1997; 18: 1847-50.
• 17. Reis LFL, Gomes LI, Brentani RR. Biologia molecular no câncer. In: Gama-Rodrigues JJ, Mchado MCC, Rasslan S. Clínica Cirúrgica. Barueri, SP - Manole, 2008, p. 1636-43.
• 18. Savary G e Miller M. The esophagus. Handbook and atlas of endoscopy. Solothum: Verlog Gassmann, 1978; p. 135-42.
• 19. Schottenfeld D e Beebe-Dimmer J. Chronic inflammation a common and important factor in the pathogenesis of neoplasia. CA Cancer J Clin 2006; 56: 69-83.
• 20. Singh NP, MCCoy MT, Tice RR, S chneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 1988; 175: 184-91.
• 21. Sokol RJ, Hoffenberg EJ. Antioxidantes na doença gastrointestinal pediátrica. Clinicas Pediatr AM Norte, Rio de Janeiro, 1997, v. 8, p. 457-72.
• 22. Speit G, Hartmann A. The contribution of excision repair to the DNA effects in the alkaline single cell get test (comet assay). Mutagenesis 1995; 10: 555-9.
• 23. Tercioti Jr V, Lopes LR, Coelho Neto JS, Andreollo NA. Aspectos atuais da terapêutica neoadjuvante no carcinoma epidermóide de esôfago. Revisão da literatura. Arq Bras Cir Dig 2009; 22: 33-40.
• 24. Terzoudi GI, Pantelias GE . Conversion of DNA damage into chromosome damage in response to cell cycle regulation of chromatin condensation after irradiation. Mutagenesis 1997; 14: 271-6.
• 25. Tice RR. The single cell gel/comet assay: a microgel electrophoretic technique for detection of DNA damage and repair in individual cells. In: Phillis DH, Venis S. Env Mol Mutag, Oxford, Bios Scientific Publishers, 1995. p. 315-333.
• 26. Tseng PH, Lee YC, Chiu HM, Huang SP, Liao WC, Chen CC, Wang CC, Wang HP, Wu MS, Lin JT. Prevalence and clinical characteristics of Barret's esophagus in a Chinese general population. J Clin Gastroenterol 2008; 42: 1074-8.
• 27. Valladares G, Bredt CG, Dias Lan LC, Souza Filho ZA, Tomasich FDS, Malafaia O. Esofagogastrectomia com linfadenectomia em dois campos no câncer do esôfago torácico. Rev Col Brás Cir 2008; 35: 374-81.

Evaluation of DNA damage in the esophageal mucosa and peripheral blood of patients with gastroesophageal reflux disease

Edilene Lúcia Bertolozzo^I; Dertia Villalba Freire-Maia^I; Mauro Masson Lerco^II; Marcelo Sady Plácido^I; Maria Aparecida Coelho de Arruda Henry^I

Publication Dates

• Publication in this collection
  19 Jan 2011
• Date of issue
  Dec 2010

History

• Received
  08 June 2010
• Accepted
  04 Oct 2010