An Overview of Single-Cell Gel Electrophoresis ... - ACS Publications

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on January 14, 2016 | http://pubs.acs.org Publication Date: September 19, 2008 | doi: 10.1021/bk-2008-0993.ch003

Chapter 3

An Overview of Single-Cell Gel ElectrophoresisBased Dietary Human Intervention Trials for the Detection of DNA Protective Food Components 1

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Veronika A. Ehrlich , Christine Hoelzl , Franziska Ferk , Julia Bichler , Armen Nersesyan , Michael Kundi , and Siegfried Knasmüller 1

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Medical University of Vienna, Department of Medicine I, Division Institute of Cancer Research, Borschkegasse 8a, 1090 Vienna, Austria Medical University of Vienna, Center for Public Health, Kinderspitalgasse 15,1090 Vienna, Austria

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Single cell gel electrophoresis (SCGE- or comet-) assay is based on the determination of DNA-migration in an electric field and can be used in human intervention studies to investigate the impact of dietary factors on DNA stability. The use of this technique has yielded new and unexpected results concerning the antioxidant and chemoprotective properties of food components.

Integritiy and intact structure is a prerequisite for the optimal function of the DNA. Impaired DNA stability due to damage caused by reactive oxygen species (ROS), chemicals and lack of specific micronutrients plays a key role in a number of human diseases, such as cancer and neurodegenerative diseases as well as ageing (1-3). Therefore efforts have been made to identify dietary 18

© 2008 American Chemical Society In Functional Food and Health; Shibamoto, Takayuki, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.


19 components, which protect against DNA damage and their consequences. However, one of the shortcomings of currently used in vitro approaches is that they are mainly conducted under unphysiological conditions and do not provide information if chemoprotective chemicals are absorbed and metabolized in the body (4,5). Therefore, the results obtained in such trials cannot be extrapolated to the human situation. In order to draw conclusions if dietary components are effective in man, a number of techniques have been developed. Conventional methods such as chromosomal aberration analyses of metaphase cells and monitoring of micronuclei in peripheral blood cells are laborious and time consuming. Therefore, the single cell gel electrophoresis (SCGE- or comet-) assay, which is based on the measurement of DNA migration in an electric field as indicator for DNA damage is increasingly used in human studies. One of the advantages of this technique, in comparison to other genotoxicity assays is, that no cell divisions are required and that the experiments can be conducted with few cells. The development of automated computer aided analysis programs has facilitated the evaluation of comet formation (6,7) (commonly used endpoints are tail lengths, tail moments and percentage DNA in the tail). An overview on the methodological principle and guidelines for SCGE tests can be found in the articles of Tice (8) and Speit and Hartmann (9). The present paper will give a short overview on different comet assay protocols currently used in human studies and provide information about the current state of knowledge on the effects of dietary factors on DNA stability that were detected with these approaches.

Endpoints and Experimental Design of Human Studies The "classical" endpoint used in human SCGE studies is based on the protocol developed by Tice et al. (8). This procedure enables to measure endogenous formation of single and double strand breaks (SSBs and DSBs) and apurinic sites. Subsequently, Collins et al. (10) developed a modified protocol of the alkaline comet assay which enables the detection of oxidised DNA-bases by use of bacterial repair endonucleases. These enzymes, namely formamidopyrimidine glycosylase (FPG) and endonuclease III (ENDO III), recognize and remove oxidized purines and pyrimides by introducing additional breaks in the DNA and thereby increasing the tail intensity of the comets. The authors showed that the FPG-induced increase of tail intensity corresponds well with the formation of 8-hydroxydeoxyguanosine (8-OHdG) which is frequently used in human studies to monitor prevention of oxidative DNA damage (10). In order to draw conclusions on alterations of DNA sensitivity to reactive oxygen species (ROS) it is possible to treat isolated peripheral lymphocytes with ROS inducers (e.g. hydrogen peroxide or a-radiation) before and after the

In Functional Food and Health; Shibamoto, Takayuki, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.


20 intervention with putative protective food components (77-/5). Furthermore, modifications of the SCGE - assay were developed, which provide information on the impact of dietary and other factors on DNA-repair processes (11,16). In the last few years, we developed additional protocols for SCGE experiments with humans, which provide information if specific dietary factors affect DNA damage caused in lymphocytes by representatives of specific classes of genotoxic dietary carcinogens (e.g. acrylamide, furan, ochratoxin A, heterocyclic aromatic amines and nitrosamines). Due to lack of metabolizing (phase I) enzymes it is not possible to carry out studies with polycyclic aromatic hydrocarbons and aflatoxin B i , but it is feasible to conduct the experiments with DNA-reactive metabolites of these compounds (e.g. aflatoxin B epoxide, benzo(a)pyrene diolepoxide - BPDE). Figure 1 gives an overview on endpoints which can be monitored at present in human SCGE-studies. Most human SCGE trials were carried out as intervention trials (cross over or parallel design) which have the advantage that inter-individual variations can be minimized. Therefore, the number of participants required to obtain meaningful results is much lower as in comparative studies. Moller and Loft (77) defined standards for SCGE-trials, and postulated that the use of placebos and wash-out periods would increase the quality of such studies. In addition, we r

Figure 7. Endpoints used in SCGE-assays with humans.



21 include in our experiments run-in phases, in which the participants consume controlled diets (i.e. low amounts of foods that may interfere with the outcome of the study) and avoid excessive physical exercise. Other possible confounding factors which should be taken into consideration are age, sex, body mass index and smoking habits (18). In order to confirm the results of SCGE-measurements and to provide information about the mechanisms, responsible for the observed effects, additional parameters can be monitored: e.g. glutathione plasma levels, ROS-protective enzymes (SOD, GPx), total antioxidant capacity (TAC) and urinary isoprostane excretion in antioxidant studies. In experiments with adduct forming carcinogens, the activity of phase I and phase II enzymes can be determined by enzyme measurements or by use of microarrays (79).

The Current Database In 1997, Pool-Zobel and coworkers published the first results of a dietary intervention study in which the SCGE technique was used (20). In the following years, a number of studies were published, the results of these trials (until 2004) are summarized in the reviews of Moller and Loft (13, 17). Also in the last two years further studies were conducted: for example with specific vegetables, red berry juice, spices and carotenoids (see Table I). In all these investigations, DNA migration was analyzed in peripheral lymphocytes or leukocytes under standard conditions. Additionally enzyme (FPG, ENDO III) and ROS treatment were used to investigate endogenous and exogenous DNA-damage by ROS. In some of the trials, the study population included patients with increased endogenous DNA damage (e.g. with Diabetes type II). Typical examples for recent studies (i.e. after 2004) for dietary factors which caused prevention of DNA damage are listed in Table 1 (upper part); studies, in which no effects were detected are listed in the lower. Some of the trials yielded unexpected results: for example no protective effects were obtained in a large study conducted in Denmark (33), in which the participants consumed a mixed fruit and vegetable diet (600g/person/day). In contrast, significant protective effects were observed in a small trial with coffee (600ml/person/day), indicating that consumption of this beverage may contribute to a higher extent to prevention of oxidative DNA damage (30, 34). A very pronounced reduction of oxidised bases was also observed in a recent study with sumach (a traditional spice). Further experiments confirmed gallic acid as the active principle (Ferk et al., unpublished data). Strong protective effects were observed also with Brussels sprouts, and no correlation with their vitamin C contents was found (31), while in dietary interventions with blackcurrant juice or an anthocyanin-rich drink no reduction of oxidative damage was found (21); the lack of the effect may be due to the fact that anthocyans are not well absorbed in the GI tract.


22 Table I. Examples for Dietarylntervention Studies (2004 and newer) Based on the Comet Assay 1


Dietary factor Vitamin C (plain or slow releasing) 2x250mg/p/d

quercetin and quercetin-rich fruit juice lL/p/d (equals 18mg quercetin/p/d) carotenoids: lutein lycopene B-carotene 12mg/p/d each or 4mg each in a mix/p/d tomato-rich diet (8mg lycopene, 0.5mg 13-caro-tene, llmg Vitamin C/p/d) coffee 600ml/p/d (150ml metal filtered + 440ml paper filtered) coffee lL/p/d metal filtered Brussels sprouts 300g/p/d

high phenol extra virgin oil 50g/p/d (equals 30mg phenols/p/d) cruciferous and legume sprouts 113g/p/d

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Design of the study blinded, placebo controlled parallel intervention study n = 48

An Overview of Single-Cell Gel Electrophoresis ... - ACS Publications

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