Gender-related differences in basal DNA damage in lymphocytes of a healthy Indian population using the alkaline Comet assay

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Abstract

The Comet assay, a sensitive, rapid and non-invasive technique, measures DNA damage in individual cells and has found wide acceptance in epidemiological and biomonitoring studies to determine the DNA damage resulting from lifestyle, occupational and environmental exposure. The present study was undertaken to measure the basal level of DNA damage in a normal, healthy Indian male and female population. Out of the 230 volunteers included in this study, 124 were male and 106 were female. All the individuals belonged to a comparable socio-economic background and aged between 20 and 30 years. They were also matched for their smoking and dietary habits. The period of sample collection was also matched. The results revealed a statistically significant higher level of DNA damage in males when compared to females as evident by an increase in the Olive tail moment [3.76±1.21 (arbitrary units) for males as compared to 3.37±1.47 for females (P<0.05)], tail DNA (%) [10.2±2.96 for males as compared to 9.40±2.83 for females (P<0.05)] and tail length (μm) [59.65±9.23 for males and 49.57±14.68 for females (P<0.001)]. To our knowledge, this report has, for the first time demonstrated significant differences in the basal level of DNA damage between males and females in a normal healthy Indian population.

Introduction

Biological markers of DNA damage (e.g. micronuclei formation, gene mutations and chromosomal aberrations) have been used extensively for human biomonitoring studies [1], [2], [3], [4], [5]. The last decade has seen an increase in the use of the single cell gel electrophoresis or the Comet assay, which measures DNA damage in individual cells [6], [7], [8], [9], [10], [11], [12], [13], [14], [15]. This method, within a short time, has found wide usage in epidemiological and biomonitoring studies in humans, to determine DNA damage, as a result of lifestyle or due to occupational and environmental exposures [6], [7], [8], [9], [10], [11], [12], [13], [14], [15]. The role of DNA integrity and damage is central to the development of cancers and most human cancers are associated with DNA instability [16]. The Comet assay is thus proving to be a sensitive method for monitoring variability in genomic instability, which may be associated with cancer risk.

DNA damage can be enhanced by exposure to various chemicals [17], [18], environmental pollutants [9], [19], steroid hormones [20] and radiation [21], [22], [23]. An increase in DNA damage has been reported to be associated with infections, diseases [12], dietary habits, lifestyle [7], [8], [10], [13] and advancing age [24], [25].

Although, most of the human cancers occur in both sexes, some of them are exclusive to males or females. A report of the “Surveillance, Epidemiology and End Results” programme of the National Cancer Institute, USA, revealed that in 50 countries, where data were available, males predominantly showed an increase in the overall cancer incidence compared to females [26].

Our earlier study has shown that amongst Indian men belonging to the upper middle socio-economic group of society, the extent of DNA damage is dependent on dietary and smoking habits and age [10]. Since the Indian population comprises several ethnic groups whose health, dietary habits as well as environmental exposures are different, an attempt was made to gather information regarding the status of DNA damage in normal, healthy male and female volunteers living in an urban environment. The present study includes the data of these 124 males and 106 females.

Section snippets

Chemicals

Agarose, low melting agarose (LMA) and RPMI-1640 were procured from BRL Life Technologies Inc. (Gaithersburg, MD, USA). Phosphate buffered saline (PBS, Ca2+ Mg2+ free) and Triton X-100 were obtained from Hi Media Pvt. Ltd., India.

Ethidium bromide (EtBr), ethylenediaminetetraacetic acid (EDTA) disodium salt, Tris buffer, Trypan blue and Histopaque 1077 were obtained from Sigma, St. Louis, USA. All other chemicals were obtained locally and were of AR grade.

Collection of samples

The volunteers were first year students

Results

The volunteers were categorised according to their age, smoking and eating habits as presented in Table 1. The cell viability for all the 230 samples at the time of experiment always exceeded 95%.

Discussion

Several studies representing different countries have shown that males have a higher incidence of overall, major cancers compared to females. Cancers of the oral cavity, stomach, colon and rectum, show nearly a two-fold higher incidence in males [26]. Prostate and breast cancers are most frequently diagnosed in males and females, respectively, followed by lung cancer, colon cancer, bladder cancer and malignant lymphomas. Lung cancers show a seven-fold higher mortality rate in males than in

Acknowledgements

A.D. wishes to thank CSIR for funding (HRD-YSA-99/project/99). M.B. is grateful to CSIR for awarding the Junior Research Fellowship. The authors wish to acknowledge the valuable contribution of Ms. Sonali Mehrotra, DAVV, Indore, as part of her MSc (biotechnology) summer project. We wish to thank all the volunteers who participated in this study. The technical support of Messrs. Kanhaiya Lal, B.S. Pandey and Rajesh Mishra is also acknowledged.

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