Phenotyping for DNA repair capacity

Mutation Research 705 (2010) 107–129

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Mutation Research/Reviews in Mutation Research journal homepage: www.elsevier.com/locate/reviewsmr Community address: www.elsevier.com/locate/mutres

Review

Phenotyping for DNA repair capacity Ilse Decordier *, Kim Vande Loock, Micheline Kirsch-Volders Laboratorium voor Cellulaire Genetica, Vrije Universiteit Brussel, Belgium

A R T I C L E I N F O

A B S T R A C T

Article history: Received 27 December 2009 Received in revised form 10 May 2010 Accepted 10 May 2010 Available online 15 May 2010

The ability to repair DNA damage is strongly associated with the risk of cancer and other human diseases as it is essential for maintenance of genome stability. Moreover, DNA repair capacity is an important factor contributing to the inter-individual variability in mutagen exposure, cancer development and treatment through an individualized adjusted therapy. In addition to genotypes, functional phenotypic assays which integrate the different pathways provide useful tools to explore the role of DNA repair in cancer susceptibility. This review compares the presently available cellular DNA repair phenotype assays based on their characteristics, and discusses their advantages and limitations. Assays for assessment of DNA repair phenotype should be well characterized in terms of reliability, validity, sensitivity, inter- and intraindividual variability, and cancer predictivity. Our comparison reveals that the G1 and G2 challenge assays, although labour-intensive, can be considered as very useful assays to investigate DNA repair phenotype. They have been successfully applied to investigate repair capacity of both cancer patients and environmentally exposed populations, and can detect deficiencies in different repair pathways. Moreover, these assays allow to predict the cancer therapy responses and to investigate the cancer prognosis. Nevertheless, the choice of the assay depends on the scientific question addressed and on the objective of its application and more prospective studies are needed since the phenotype could reflect the pathophysiological alterations in the patient secondary to the disease. ß 2010 Elsevier B.V. All rights reserved.

Keywords: DNA repair phenotype Cancer risk Individual susceptibility

Contents 1. 2.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA repair assays based on the removal of DNA strand breaks or adducts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Assays based on the Comet assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1. Assessment of cellular strand break DNA repair capacity by following removal of induced DNA breaks on in vitro challenged cells of the donors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2. Assessment of cellular strand break DNA repair capacity by measuring incision capacity of intact donor cells, challenged in vitro in the presence or absence of repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.3. Assessment of cellular strand break DNA repair capacity by measuring incision capacity of extracts from donor cells incubated with mutagen-exposed reference cells using the comet assay – in vitro repair assays . . . . . . . . . 2.1.4. Advantages and disadvantages of assays based on the comet for the assessment of DNA repair phenotype. . . . . 2.2. The host cell reactivation assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. Advantages and disadvantages of the host cell reactivation assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Immunochemical measurement of DNA-adduct removal in donor cells incubated with adducts-inducing mutagens . . . . . DNA repair assays based on repair replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Unscheduled DNA synthesis assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.1. Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.2. Advantages and disadvantages of the UDS assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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* Corresponding author at: Laboratorium voor Cellulaire Genetica, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium. Tel.: +32 2 629 34 28; fax: +32 2 629 27 59. E-mail address: [email protected] (I. Decordier). 1383-5742/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.mrrev.2010.05.002

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5. 6. 7. 8. 9.

DNA repair assays based on induction of chromose breakage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. In vitro cytogenetic challenge assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.1. The G2-challenge CA assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.2. The G1-challenge CA assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.3. The G0- and G1-challenge MN assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . g-H2AX assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. In vitro repair assays assessing the functional activity of DNA repair enzymes in extracts from donor cells . Gene expression profiling of DNA repair genes by microarray analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA repair phenotype and predictivity for cancer risk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA repair phenotype and sensitivity to chemotherapy and radiotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction The ability to repair DNA damage is strongly associated with the risk of cancer and other human diseases such as neurodegenerative and inflammatory disorders and aging (for review [1]) as it is an ubiquitous defence mechanism that plays an essential role in cell survival and the maintenance of genome stability. DNA repair capacity is an important factor contributing to the inter-individual variability in response to mutagen exposure and cancer susceptibility [2]. Assessing this inter-individual variability in repair capacity is crucial in providing biomarkers not only for primary cancer prevention and early diagnosis but also for cancer treatment through individual adjusted radio- or chemotherapy. The discovery of millions of genetic polymorphisms in the human genome has sparked the hope that these variations in DNA sequences are the basis for inter-individual variations and will be useful for risk assessment and treatment of diseases, including cancer. One might consider that all genotypes of an individual can technically be described and, starting from this knowledge, extrapolate the potential functional activity also known as the phenotype. However, many factors can modulate this genotype– phenotype extrapolation including alternative splicing, gene silencing, post-transcriptional regulation, protein–protein interactions, and the fact that transcription of the genes is tissue and age dependent. Furthermore, environmental factors can modify both the DNA sequence (mutation) and/or regulation of transcription (epigenetic changes). If we consider a simple case, it is technically feasible to assess the functional activity of the protein corresponding to a given DNA sequence, as far as its function and target are known, this is a phenotype at molecular level. When the studied endpoint (e.g. repair of DNA strand breaks) is controlled by different gene products regulating a given pathway or different pathways, the complexity of the interactions is difficult to extrapolate from the knowledge of the individual molecular phenotypes, and a cellular phenotype integrating the interplay of the different enzymes and pathways might be a very useful information. Moreover it would allow to compare, for a same genotypic configuration, the efficiency of the functional activity of the gene product between different populations, e.g. between adults and newborns [3]. Ultimately, combination of the cellular phenotypes of different tissues would provide a realistic approach of the individual physiology and response to environmental changes. The cell has developed a network of DNA repair mechanisms, to ensure that the large variety of DNA lesions induced by exogenous and endogenous sources are effectively dealt with. In the human genome more than 130 genes have been found to be involved in these DNA repair systems. At least four, partly overlapping, main repair pathways operate in the removal of DNA lesions: base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR) and double-strand break repair (DSBR) [1,4] (Fig. 1).

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BER is the primary DNA repair pathway responsible for the correction of base lesions resulting from oxidative damage, alkylation, deamination and depurination/depyrimidination, as well as DNA single-strand breaks, two principal mechanisms are used: non-homologous end-joining (NHEJ) and homologous recombination (HR) (for review [5,6]). NER corrects a wide variety of DNA lesions, including chemically induced bulky adducts, intraand interstrand cross-links, UV-induced pyrimidine dimers and photoproducts. In addition, NER can also recognize oxidative damage that poses a special challenge for BER: cyclopurines (for review [6,7]). This repair pathway consists of two branches: global genome repair (GGR), which probes the genome for strand distortions, and transcription-coupled repair (TCR), which removes distorting lesions that block elongating RNA polymerases. DSBR performs the repair of DNA double-strand breaks induced by endogenous and exogenous attacks on the DNA backbone, as well as by the conversion of single-strand breaks into double-strand breaks during DNA replication (for review [6,8]). DNA damage that blocks the regular replication machinery involving DNA polymerase d/e (e.g., breaks and cross-links) can be repaired, bypassed by homologous recombination, which involves template switching and strand displacement, or bypassed by translesional synthesis (TLS), a specialized, relatively error-free (but still somewhat mutagenic) means of bypassing a specific subgroup of lesions (for review [6]). The MMR system is responsible for the removal of base

[(Fig._1)TD$IG]

Fig. 1. Overview of the different DNA lesions and their DNA repair systems (Adapted from Hoeijmakers et al. [6]), abbreviations: see text.

High No Ok (only assessed in pilot studies) Rol9-8022/ white light, Repair incisions

BER

Cell No extracts

Yes

Yes

Ok (only assessed in pilot studies) Environmentally/ Pilot studies occupationally exposed populations versus controls

No

No High No Contradictory Yes Yes No Intact cells NER UVC, PAHs Repair incisions

-Measuring incision capacity of (1) intact donor cells, challenged in the presence or absence of repair inhibitors Or (2) extracts from donor cells incubated with mutagenexposed reference cells

Cancer-cases versus controls Children versus adults

Environmentally/ Pilot studies, Contradictory occupationally larger studies exposed (n > 100) populations versus controls

No

No High No Contradictory

Repeat Reprod.

Environmentally/ Pilot studies, Contradictory larger studies occupationally exposed versus (n > 100) controls Yes Yes No Intact cells BER, DSBR X-rays, g-rays H2O2, bleomycin, styrene assay Oxidative base damage, SSB, DSB Assays based on the Comet -Following removal of induced DNA strand breaks on in vitro challenged cells of the donors

Reliability Size of study population

Criteria for testing

Study Synchr. Cryopr. HighIntact DNA Possible throughput population cells/cell cells repair possible pathway extracts required DNAdamaging

Method Table 1

2.1. Assays based on the Comet assay

Detected lesions

Specificity of the method

2. DNA repair assays based on the removal of DNA strand breaks or adducts

A sound approach to assess repair capacity is to evaluate the ability of a cell to remove damage which is induced experimentally

No

Validity Intra- and Heritability Predictive in twins for cancer interindividual

mismatches caused by spontaneous and induced base deamination, oxidation, methylation and replication errors (for review [9]). Although BER is the only DNA repair pathway to which no heritable diseases are associated, it is as important as the other pathways since it is involved in the repair of oxidative DNA damage that is a very common type of cellular stress. Moreover, polymorphisms in various genes involved in this DNA repair pathway have been associated with cancer [10–12]. Therefore, in addition to genetic polymorphisms in DNA repair genes, phenotypic markers of DNA repair have become very useful to study the role of particular genes in human cancers and to identify susceptible individuals. The development of functional assays which can assess individual’s DNA repair capacity is crucial for identifying high-risk subgroups in the general population. Application of phenotypic biomarkers have been shown to give more consistent results than those obtained using genotypic assays [13], results of which often indicate that variation in DNA repair capacity may have a substantial impact on cancer risk and treatment [14]. To date, an arsenal of assays has been developed to evaluate DNA repair capacity. These methods vary from assays based on the removal of DNA breaks or adducts, such as the comet assay measuring the removal of induced DNA breaks on in vitro challenged cells of the donors, to assays based on the induction of chromosome breakage such as the G2-challenge assay and in vitro assays measuring the DNA repair capacity in cell extracts. Moreover, researchers have recently also found their way to other molecular methodologies such microarray technology to assess gene expression profiles of various DNA repair genes [15]. Many of these assays have been used in epidemiologic studies for comparison of DNA repair capacity between cancer case subjects and healthy control subjects to assess the role of DNA repair in the development of human cancer or to investigate whether environmental exposed populations may have an increased cancer risk. Within the ECNIS (Environmental Cancer Risk, Nutrition and Individual Susceptibility) Network of Excellence we have attempted to review the state of the art methodologies presently available to assess cellular DNA repair phenotype with their design, results, advantages and disadvantages based on what is published in the openly available scientific literature to date. Therefore, the different assays were carefully analyzed and compared, based on the following criteria: methodology of the assay, which lesion(s) are detected, which DNA repair pathway is studied, which DNA-damaging agent is used, use of whole cells or cell extracts, possibility to use cryopreserved cells, need of synchronized cells, intra-individual variability (repeated experiments on the same donor on different times) and inter-individual variability of repair capacity investigated, heritability investigated in twins, application (cancer case versus control studies, environmental exposed versus control studies or others), potential for high-throughput analysis. A summary of this comparison is presented in Table 1. Some of the assays require in vitro culturing of PBMC or whole blood while others can be performed without in vitro culturing, an overview of the protocol for most of the assays is presented in Fig. 2(A and B). In addition, the predictivity of DNA repair phenotype assays for cancer risk and its application in the assessment of an individual’s sensitivity to cancer treatment are discussed.

109

No

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Table 1 (Continued ) Specificity of the method Detected lesions

DNAdamaging

Method

Criteria for testing

Study Synchr. Cryopr. HighIntact DNA Possible throughput population cells/cell cells repair possible pathway extracts required

Size of study population

Reprod.

Host cell reactivation assay

Pyrimidine dimers, photoproducts, bulky adducts, intra-strand cross-links

Repeat

PAHs

NER

UV, BPDE

NER

Intact cells

No

Yes

Yes

Cancer-cases versus controls

Pilot studies, Needs further larger studies standardisation (n > 100)

Needs further No standardisation

NI

No

No

BPDE

NER

Intact cells

No

Yes

Yes

Cancer-cases versus controls

Pilot studies

Needs further standardisation

Needs further No standardisation

NI

No

No

NER UV, UVmimetic drugs Cisplatin, NAAF, Cyclosphosma mide

Intact cells

No

No

Yes

Environmentally/ Pilot studies occupationally exposed populations versus controls

Needs further standardisation

Needs further No standardisation

NI

No

No

Ok for bleomycin others need further standardisation

Yes Ok for bleomycin, others need further standardisation

High

Yes

Yes

Yes

High

No

Yes

Cancer-cases versus controls G1/G2-challenge CA assay

G0/G1-challenge MN assay

g-H2AX assay

Oxidative base g-rays damage

BER DSBR

SSB

Bleomycin UV NER

DSB

BPDE 4-NQO

DSB

Bleomycin

BER

g-rays NNK H2O2

DSBR

g-rays

BER

DSB

Intact cells

Yes

No

Yes

Environmentally/ Pilot studies, l arger studies occupationally (n > 100) exposed populations versus controls Cancer-cases versus controls

Intact cells

Yes

No

Yes

Cancer-cases versus controls

Pilot studies, High interlarger studies scorer and inter(n > 100) laboratory variability

High interscorer and interlaboratory variability

Intact cells

No

Yes

Yes

Cancer-cases versus controls

Pilot studies

Needs further standardisation

Needs further No standardisation

NI

No

No

Intact cells

No

Yes

Yes

Cancer-cases versus controls

Pilot studies

Needs further standardisation

Needs further No standardisation

NI

No

No

DSBR Microarray analysis

BER

NER MMS DSBR

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Immunochemical measurement of DNA-adduct removal Unscheduled DNA synthesis

Photoproducts bulky adducts DNA-adducts

Validity Intra- and Heritability Predictive in twins for cancer interindividual

Reliability

I. Decordier et al. / Mutation Research 705 (2010) 107–129

and a simple way to measure this is by the single cell gel electrophoresis or comet assay [16], which is a relatively simple, rapid and sensitive method for detecting DNA damage at the level of individual cells [17]. This is a fluorescence microscopic method to examine DNA damage and repair at individual cell level. In the

[(Fig._2)TD$IG]

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standard version of the comet assay, cells, with or without treatment with a test substance, are embedded in agarose, then lysed leaving supercoiled matrix-attached DNA in a nucleoid and subjected to an electric field. Small DNA fragments and free DNA loops can migrate away from the residual nucleus forming a

Fig. 2. Overview of the protocol with treatment schedule for the most common assays discussed in the present review. A. Assays require in vitro culturing of PBMC or whole blood; B. Assays which can be performed without in vitro culturing. (T; treatment; C: colcemide; H: harvest; Cyto-B: cytochalasin B).

[()TD$FIG]

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Fig. 2. (Continued ).

‘‘comet’’ tail when viewed by fluorescence microscopy, hence the name ‘‘Comet’’ Assay. The percentage of total fluorescence in the tail is related to the degree of DNA damage. This method was first introduced by Ostling and Johanson [18], who developed a neutral assay, in which cells embedded in agarose are placed on a microscope slide and lysed by detergents and high salt treatment. The liberated DNA is electrophoresed under neutral

conditions and stained with ethidium bromide. This technique only detects double-stranded DNA breaks. Since the introduction of the alkaline Comet assay in 1988 [17], a number of advancements have greatly increased the flexibility and utility of this technique for detecting various forms of DNA damage and DNA repair. In alkaline conditions, induced single- and double-strand breaks, alkali-labil sites and incomplete repair sites

[()TD$FIG]

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Fig. 2. (Continued ).

can be quantified. Several methods based on the comet assay have been developed to measure DNA repair capacity and are discussed in the following paragraphs. 2.1.1. Assessment of cellular strand break DNA repair capacity by following removal of induced DNA breaks on in vitro challenged cells of the donors 2.1.1.1. Studies. The comet can be used to follow the removal of DNA damage with time following in vitro challenging of lymphocytes with a DNA-damaging agent (Fig. 2A). In this assay ionising radiation or bleomycin are mostly used as challenging agent, inducing oxidative base damage, single-strand and doublestrand DNA breaks, and allows to investigate the BER and DSB repair capacity in the studied donors. This approach has been applied in several small pilot studies (varying from 6 to 45 cases and controls) to compare the DNA repair capacity between cancer patients and controls by challenging their PBMC with either ionising radiation [14,19,20–25]; bleomycin [26] or H2O2 [27]. Rajaee-Behbahani et al. [28] and Schmezer et al. [29] applied the bleomycin challenge comet assay and Saran et al. [30] the N-methyl, N-nitro, N-nitrosoguanidine

(MNNG) challenge comet assay in larger case-controls populations (more than 100 cases and controls) to investigate the altered DNA repair capacity in cancer patients. Using the comet assay, these studies found an impaired DNA strand break processing in cancer patients as compared to their healthy counterparts. This DNA repair assay using in vitro challenging with irradiation was also applied to study the radiosensitivity in cancer patients and to adjust radiotherapy [14,19,20,31,32]. These studies indicate that cancer patients may show an individual risk of radiation therapy toxicity and a risk of radiation-associated tumorigenesis. Although often used in small pilot studies, the challenging comet assay appears to be a useful tool to document the DNA repair phenotype in cancer patients, to identify susceptible individuals, e.g. children receiving radiotherapeutic treatment, and to allow a preventive surveillance for radiation-associated tumour development. This in vitro challenge assay has also been applied to assess the repair capacity of occupationally exposed populations. Our laboratory used the assay with ionising radiation [33,34] to assess the repair capacity of a small population worker of a nuclear power plant. It was demonstrated that workers repaired their DNA damage more efficiently as compared to the controls. The authors

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attributed this phenomenon to adaptive response by sub-chronic exposure to low dose of IR. This activation of the DNA repair machinery was also observed in other small studies [35–38], where a moderate increase of irradiation specific DNA repair rates was found among workers highly exposed to xenobiotics such as styrene, 1,3-butadiene (BD), lead or pesticides. We also evaluated the DNA repair capacity of individuals occupationally exposed to styrene [39] and observed a better in vitro repair capacity in workers after 1 h repair following challenge with styrene, while at higher level of residual DNA damage was found after 24 h of repair. On the other hand, in studies conducted to assess the repair capacity of workers exposed to mercury or city policemen and busdrivers exposed to polycyclic aromatic hydrocarbons (PAHs) [40–43] a significant deficiency in DNA repair in the exposed populations was observed. Challenging with H2O2 has been used by our team [3] in a pilot study to assess the genetic susceptibility of newborns for oxidative stress, for which we did not find significant differences as compared to their mothers. Mendez-Gomez et al. [44] evaluated the repair capacity of children exposed to arsenic and lead using the same challenge approach and found a decreased ability to repair H2O2-induced DNA damage. Simoniello et al. [45] oberved a similar DNA repair capacity in workers exposed to pesticide and in controls, using the challenge comet assay with H2O2. 2.1.1.2. Modifications of the method. The approach of assessing DNA repair capacity by measuring DNA damage removal with comet assay has also been modified by inclusion of purified enzymes to assess repair of oxidized bases [16]. After lysis the nucleoid DNA is incubated with a lesion-specific endonuclease which increases the number of breaks. Endonuclease III, which detects various oxidized pyrimidines or formamidopyrimidine DNA glycosylase (FPG, for 8oxoguanine and ring-opened purines Fapy-guanine) can be incorporated to measure specifically oxidative DNA damage [46]. In a recent study, Smith et al. [47] compared the ability of FPG, ENDOIII and hOGG1 to increase DNA strand breaks in the comet assay after treatment of mouse lymphoma cells with agent inducing oxidative or alkylating damage. They demonstrated that hOGG1 recognizes oxidative damage in the comet assay with greater specificity as compared to FPG or ENDOIII. On the other hand, Moller et al. (unpublished data) found a higher sensitivity for FPG as compared to hOGG1. Furthermore, in a recent ECVAG trial by Johansson et al. [48] using the method with FPG, the participating laboratories were succesfull in finding a dose-respons of oxidatively damaged DNA in coded samples. Repair of UV-induced cyclobutane pyrimidine dimers by nucleotide excision repair can be detected with the enzyme endonuclease V [49]. This method was applied on small populations of both cancer patients [50] and occupationally exposed individuals [51]. Rusin et al. [50] observed a less effective DNA repair in cancer cells from patients with metastasis than in cells from healthy controls. Fracasso et al. [51] performed DNA repair kinetics studies using this modified comet assay to monitor workers exposed to styrene and documented a drastic decrease in DNA repair activity as compared to controls. 2.1.2. Assessment of cellular strand break DNA repair capacity by measuring incision capacity of intact donor cells, challenged in vitro in the presence or absence of repair Alapetite et al. [52] applied the comet assay using in vitro challenging with UVC-light for prenatal diagnosis of Xeroderma Pigmentosum and Trichothiodystrophy in three families, and Møller et al. [53] to study the repair capacity of a small population of psoriasis patients with basal cell carcinoma. In this approach, the generation of DNA strand breaks following UV-C irradiation corresponds to DNA repair-mediated incisions, as UV-C itself does

not induce DNA breaks. It was found that psoriasis patients with basal cell carcinoma had more DNA repair incisions than noncancer patients [43]. Orlow et al. [54] used BPDE as challenging agent to evaluate the repair capacity of 108 lung cancer patients and found a reduced repair after BPDE-induced damage. The comet assay thus also allows the assessment of DNA strand break formation during excision repair, which leads to an increase in DNA migration (Fig. 2A). Based on this principle Gedik et al. [55] developed an adapted version of the earlier described DNA strand break repair assay, in which cells were challenged with UV-C and analyzed over a period of time for DNA repair incision with the comet assay in presence of the DNA polymerase inhibitor aphidicolin (APC). In the presence of the repair inhibitor APC, incomplete repair sites of UV-C lesions accumulate and lead to an additional increase of DNA migration, thus the DNA damage measured with the comet assay reflects the NER-dependent incision at sites of UV-C lesions [56,57]. The use of repair inhibitors has been proposed for human biomonitoring studies to increase the sensitivity of the comet assay [57,58]. Recently, our laboratory used this approach to develop a NER cellular phenotype assay to investigate the repair capacity (RC) by measuring the influence of the repair inhibitor APC on BPDEinduced DNA damage [59]. Since in vitro exposure to BPDE may lead to DNA strand breaks resulting from both direct interaction with DNA and incisions introduced by the repair enzymes, we aimed at discriminating between both types of breaks using the comet assay and quantified the DNA strand breaks after in vitro challenge of PBMC with BPDE in the presence or absence of the DNA polymerase inhibitor aphidicolin (APC) (Fig. 3). A low intraindividual and a high inter-individual variation in DNA repair capacity was observed, indicating the adequacy of the method. Crebelli et al. [58] used cytosine arabinoside (Ara-C) as repair inhibitor in a small study to assess the genetic effects of occupational exposure to PAHs and other agents of primary aluminium industry workers, the study did not highlight any statistically significant difference between the exposed and control workers. Another repair inhibitor used to assess NER capacity is novobiocin [60,61].

[(Fig._3)TD$IG]

Fig. 3. Diagram depicting the contribution of the different treatments to the measured DNA strand breaks resulting in the NER capacity in the total population (adapted from Vande Loock et al. [59]): (1) APC treatment alone results in a minor level of DNA damage; (2) DNA damage measured after treatment with BPDE consists of the direct DNA breaks and the NER incisions, a fraction of the induced DNA damage that is already repaired cannot be detected; (3) DNA damage measured after treatment with BPDE and APC consists of the APC effect, direct DNA breaks, the NER incisions and the remaining gaps resulting from the APC-blocked NER repair. The nucleotide excision repair capacity (RC) is defined as the amount of DNA strand breaks damage induced by BPDE in presence of APC, diminished with the damage induced by BPDE and APC alone.

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2.1.3. Assessment of cellular strand break DNA repair capacity by measuring incision capacity of extracts from donor cells incubated with mutagen-exposed reference cells using the comet assay – in vitro repair assays An alternative approach to assess cellular repair using the comet assay is an in vitro assay which is the converse of the normal enzymelinked comet assay. The principle of the assay is that an extract prepared from the cells of interest (e.g. human lymphocytes) is incubated with a substrate of DNA containing specific damage. The ability of the extract to introduce breaks in the DNA at damage sites (the initial step of repair) is detected with the comet assay [62]. In this method nucleoids derived from cells with a certain amount of specific DNA damage, namely oxidized bases introduced by visible light in the presence of the photosensitizer Ro 19-8022, act as a substrate and are incubated with lymphocyte extracts of unknown activity. The disappearance of DNA incisions with time, measured by the comet assay on the substrate nucleoids, is an index of the PBMC BER repair capacity. This methodology does not need stimulated, synchronized PBMC (Fig. 2B). This in vitro approach was applied to measure DNA repair of occupationally exposed workers [63–65]. Recently, Langie et al. [66] modified this repair assay in order to measure the capacity of human lymphocyte extracts to undergo nucleotide excision repair (NER). In this assay, gel-embedded nuclei from A549 cells pre-exposed to BPDE provide substrate DNA to assess the capacity of cell extracts from PBMC to cause incisions in BPDE-containing DNA-adducts. Gaiva˜o et al. [49] adapted this in vitro comet repair assay for the assessment of NER by using UVC-damaged substrate DNA. 2.1.4. Advantages and disadvantages of assays based on the comet for the assessment of DNA repair phenotype As described above, the comet assay allows DNA strand break detection at single cell level and is also being increasingly used in human biomonitoring. Moreover, in addition to the measurement of initially induced DNA damage, the comet assay can also be used to assess DNA strand break formation during excision repair, which also leads to an increase in DNA migration and makes the comet assay in all its modified forms a straightforward approach to study repair capacity. The comet assay has many advantages: it is a sensitive, reliable and rapid method which can be performed on both freshly isolated or cryopreserved cells; depending on the DNA-damaging agent used, different DNA repair pathways can be studied: BER, DSBR and NER; it does not require large numbers of cells. Assessment of cellular repair by inclusion of repair enzymes has the advantage of sensitivity, so that the cellular response to low levels of damage can be measured, as compared to the classic damage removal method, however, longer incubations are needed. Although visual scoring is still often used, results of the comet assay can be obtained by computer-based image analysis providing a faster analysis and high-throughput versions of the comet assay are under development and will allow determination of repair capacity in many samples simultaneously [67]. A limitation of the repair studies using the comet assay is the fact that it only provides an indirect indication for the speed of DNA strand break rejoining but not for repair fidelity [56]. Another shortcoming of the assay is its subjection to environmental and experimental variation. The high sensitivity of the comet methodology leads to high intra- and inter-individual variation, which can also vary in its turn between the different comet assaybased methods. A reliable methodology should be able to detect differences between individuals, resulting in significant inter-individual variation, but should show a minimal intra-individual variation. Using repair inhibitors, Cipollini et al. [61] found high intra- and inter-individual variation.

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This intra-individual variation in DNA damage measured with the comet assay after in vitro challenging of PBMC has also been reported by other studies [68]. As far as the in vitro DNA repair assays using cell extracts is concerned a good reproducibility [49,66] was observed. Nevertheless, these assays were only performed in rather small pilot studies. In general, the high sensitivity of the comet methodology leading to high intra- and inter-individual variation only allows predictions at population level. The comet assay is potentially very useful for identifying individuals with reduced repair capacity; therefore further efforts have to be addressed to improve its reproducibility. Another factor contributing to the improvement of the reproducibility is the inclusion of an internal standard. Although the protocols of the described versions of the comet assay are comparable between laboratories, still many variations exist in the different steps of the protocol. The use of an internal standard reduces inter-electrophoresis differences and allows comparisons between studies. Our laboratory developed and validated an internal standard for the comet assay using untreated and EMStreated K562 erythroleukemia cell line [69]. The use of untreated human PBMC or cell lines as negative controls and cells treated with genotoxic compounds as positive control has also been introduced by other laboratories [70–75]. Although these reference standards reduce variability, inclusion of a ‘‘real’’ internal standard by the use of specific cells in the same gel, clearly distinguished from the studied cells, would allow minimal variability and accurate inter-laboratory comparisons. Recently, Azqueta et al. [76] draw attention to a potential problem when using the in vitro repair assay using cell-free extracts: an increase in breaks caused by the extract from cells treated with a test compound may also be due to a direct effect of the test compound or to an increase in the activity of non-specific nucleases, therefore appropriate control experiments should always be performed. Recently the European Comet Assay Validation Group (ECVAG) has been established for the purpose of validation of the method and will focus on developping a set of ‘‘reference conditions’’, where a few of the most important steps of the methodology are kept constant, instead of introducing a standard operating procedure [77]. In addition, although many studies the usefulness of the comet challenge for the assessment of DNA repair capacity, most of these studies were only performed in pilot studies and small cohorts. In conclusion, the comet assay remains a highly adaptable assay that has been widely used and is capable of providing information on different types of DNA damage and the cell’s ability to repair the damage. Its application will increase with the development of high-throughput methods and fully automated comet analysis. 2.2. The host cell reactivation assay 2.2.1. Studies During the process of host cell reactivation intact cells repair damage localized to exogenous viral DNA. Athas et al. [78] developed the host cell reactivation (HCR) assay to measure nucleotide excision repair (NER) of UV-induced DNA damage in human isolated and PHA-stimulated PBMC using a transient expression vector harbouring the chloramphenicol acetyltransferase (CAT) reporter gene. In this in vitro assay, pCMVcat plasmid DNA damaged by the test agent is transfected into unexposed host cells and reactivation of the damaged plasmid is used as measure for the overall nucleotide excision repair capacity of the host cells. The assay can be performed on cryopreserved cells. The standard protocol involves isolation and freezing of lymphocytes. After thawing the PBMC they are cultivated for 72 h in presence of phytohaemagglutinin (PHA) followed by transfection. CAT-activity is measured after 40 h incubation, leaving a fixed time to allow

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repair completion in the host cells (Fig. 2A). The nucleotide excision repair (NER) pathway is responsible for removing UVinduced photoproducts, bulky adducts, cross-links and oxidative damage. The DNA repair capacity of an individual is defined as the ratio between the activity of the damaged reporter gene and that from the undamaged reporter gene. Several studies by Wei and colleagues have used the host cell reactivation assay in human PBMC in relatively large study populations (varying from 88 to 333 indivuduals) to investigate the role of DNA repair in susceptibility to sunlight-induced basal carcinoma [79–84]. Their findings provided evidence that a reduced repair capacity is one of the underlying mechanisms for sunlight-induced skin cancer in the general population. In addition, a study of a similar size, conducted by Landi et al. [85], suggests that DNA repair capacity, as measured with the host cell reactivation assay, is an important modifier of melanoma risk in the presence of other strong risk factors, such as a low propensity to tan and the presence of dysplastic nevi. The CAT assay has also been adapted by using cat reporter gene damaged by BPDE, which is a well known tobacco-activated carcinogen, to study how a reduced DNA repair capacity modulates susceptibility for lung cancer [86–91], breast cancer [92] and head and neck cancer [93,94]. These investigations involved both pilot studies [86,87,92,93] as well as larger cohorts [88,89–91,94]. Cheng et al. [93] hypothesized the existence of variation in DNA repair capacity among the general population and the risk of head and neck cancers. This was based on the observations that patients exhibited a suboptimal DNA repair capacity for removing BPDE-induced DNA-adducts, and the xeroderma pigmentosum paradigm of inherited defect in NER coupled with increased risk of cancer [95,96]. The authors confirmed and validated this association between DNA repair capacity and the risk of head and neck cancer. More recently, other adaptations have been applied to improve the applicability of the HCR. Qiao et al. [97] replaced the cat gene with the luciferase (luc) gene. It was demonstrated that the results of the LUC assay were comparable to those of the CAT assay in measuring DNA repair capacity for nucleotide excision repair. Moreover, the LUC assay shows less inter-assay variation, it requires fewer viable cells, is faster, not radioactive and less labour-intensive as compared to the CAT assay. This adapted version of the HCR assay using a luciferase reporter gene was applied by Matta et al. [98] in a relatively large population to demonstrate that a reduced DNA repair capacity is a susceptibility factor for nonmelanoma skin cancer. Ramos et al. [99] used the same method in a small population of breast cancer, patients. Other modifications of the host cell reactivation assay have been applied in pilot studies. Lin et al. [100] used a modified LUC assay to demonstrate that deficient DNA repair capacity of 4-ABP-induced DNA damage increases the risk for bladder cancer. Recently, Wang et al. [101] presented a modified HCR assay to determine the DNA repair capacity of human lymphocytes for alkylating DNA damage, in which they used dimethyl sulphate to induce alkylating DNA damage in the pCMVluc plasmid. 2.2.2. Advantages and disadvantages of the host cell reactivation assay The assay has the advantage of using a plasmid that is damaged and then transfected into the host cell rather than on direct damage to the host cell. This approach minimizes the cytotoxic effects of damaging agents to the host cells and the DNA repair capacity derived from the assay objectively reflects intrinsic cellular DNA repair capacity. Many studies applying the HCR assay used cryopreserved cells. This allows storage and accumulation of samples; many assays can be performed at one time, reducing the inter-experimental variation; culturing the samples at the same time avoids influence of unknown serum factors on DRC and stored

cells can be used later for assays of new biomarkers. Disadvantages of this approach are the following: cells are lost during cryopreservation and the assay needs multiple transfections of each sample, therefore the assay usually requires a high volume of blood, which makes it difficult to conduct large-scale molecular epidemiological studies. In addition, the host cell reactivation assay consists of many steps (preparing PBMC, treatment of the plasmid, transfection and measuring CAT enzyme activity) which can lead to high assay variation. Moreover, repair of DNA damage (e.g. adducts) in a plasmid transfected into cells has been shown to be substantially different from the process of repair of genomic damage [6]. In conclusion, the host cell reactivation assay is a relatively fast and simple assay, that allows indirect measurement of the extent and efficiency of the repair capacity of DNA damage such as photoproducts or adducts, but is not applicable to damaged plasmids containing single- or double-strand breaks which reduce the frequency of plasmid transfer [102] and not allowing distinction between repair-deficient and -proficient cells. In addition, the assay measures transcription-coupled repair at active genes, which is only a subset of total NER. Although the assay allows high-throughput analysis, the issues of inter- and intra-individual variation still need to be addressed adequately. It is important to standardize the assay and to apply it in larger well-designed case-control studies, with a close monitoring of the transfection efficiency. 2.3. Immunochemical measurement of DNA-adduct removal in donor cells incubated with adducts-inducing mutagens Motykiewicz et al. [103] developed an assay to evaluate nucleotide excision repair capacity which is based on the measurements of BPDE-DNA-adducts and which clearly estimates the individual’s sensitivity to a mutagen and its DNA repair capacity. With this method the level of BPDE-DNA-adducts are measured by means of immunohistochemistry with an anti-BPDEDNA antibody. The levels of adducts are measured immediately after exposure to BPDE to measure the initial DNA damage and after further culture of the treated cells in mutagen-free medium, to determine the DNA repair capacity (Fig. 2A). The rate of damage removal was considered as the measure of individual DNA repair efficiency. The authors established the test conditions in the XP lymphoblastoid cell line proficient (GM01989) and deficient (GM02485) in DNA repair and applied this new method in a pilot study analyzing 50 lymphoblastoid cell lines from sister discordant for breast cancer. The results of this study showed no significant difference in initial DNA damage between cases and controls but a significantly higher level of DNA-adducts in cases as compared to controls after culture in BPDE-free medium. The same group [104] expanded this pilot study to 137 new families where the same findings were observed. The advantage of using BPDE which is a direct-acting carcinogen is avoiding potential bias due to individual differences in efficiency of metabolism of the parent mutagen, B[a]P into the reactive form BPDE. Analysis of BPDE-adduct removal by immunofluorescence analysis has the potential for high-throughput analysis. Its feasibility on cryopreserved cells has not yet been investigated. To be applicable in large-scale studies, this method has to be further validated, especially evaluating the inter- and intraindividual variation and reliability. 3. DNA repair assays based on repair replication 3.1. Unscheduled DNA synthesis assay 3.1.1. Studies The unscheduled DNA synthesis (UDS) assay is used to assess a cell’s ability to perform global nucleotide excision repair. The UDS

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methodology was first described by Rassmussen and Painter [105] and its name originates from the fact that the method measures the DNA repair of cells in all stages of replication except S phase, when ‘‘scheduled’’ DNA synthesis of the entire genome is taking place. The assay allows the indirect evaluation of DNA repair through an elevated incorporation of [3H]-thymidine into the DNA of cultured mammalian cells during the repair of damage [106], and the length of time allowed for this incorporation is specific for repair of particular lesions. The uptake of [3H]-thymidine may be determined by autoradiography or by liquid scintillation counting. Nucleotide excision repair is the main DNA repair pathway removing helix-distorting DNA lesions, including UV-induced pyrimidine dimers and photoproducts, as well as chemically induced bulky adducts and intra-strand cross-links [107]. In addition this pathway can also be recruited for other types of DNA damage lesions that have not been repaired by BER or other DNA repair mechanisms. The UDS assay requires the analysis of living cells and has been applied on skin fibroblasts and human PBMC. Pero’s group [108,109] used the UDS assay combined with NAAAF challenging for biomonitoring of occupational exposure through which it was demonstrated that exposure to styrene [108], and to propylene oxide and ethylene oxide [109] are associated with a reduced DNA repair synthesis. Suppression of DNA repair capacity in workers exposed to ethylene oxide was also demonstrated by Mayer et al. [110]. The same methodology was applied to assess the relationship between reduced repair capacity and cancer risk. It was demonstrated that patients with or genetically predisposed to colorectal cancer showed a reduced capacity of DNA repair synthesis [111] and that, patients with colorectal polyps were deficient in carrying out DNA repair capacity [112]. Additionally, another study by Pero et al. [113], also using NA-AAF, concluded that a reduced UDS in mononuclear leukocytes is associated with a family history of cancer. Later on the UDS assay was used to assess the DNA repair capacity by using UV as challenging agent in patients with non-melanoma skin cancer and psoriasis [114], in breast cancer patients [115] and in healthy women having first-degree relatives with breast cancer [116]. In addition, this method was applied to study the DNA repair capacity of lymphocytes from smokers [117]. These studies demonstrated a lower rate of DNA repair in the studied populations, as compared to their control counterparts. All these studies described here were only performed in small populations. 3.1.2. Advantages and disadvantages of the UDS assay The UDS assay has the advantage to evaluate a cell’s capacity to perform NER on the entire genome, instead of only a subset, which is the case for the host cell reactivation assay that measures only transcription-coupled repair at active genes. In the UDS assay the degree of DNA repair is quantified by net grain count in nuclei over counts in cytoplasm on one by one bias. Although software programmes have been developed to reduce human subjectivity of the data analysis [118–120], it still suffers from subjective bias and is rather labour-intensive. Recently, Li et al. [121] developed an improved UDS methodology utilizing a dual-labelling where proliferating and DNA repairing cells were stained with [3H] thymidine whereas nuclei of living cells were stained with DAPI. This approach minimized the subjective bias issue and by the use of and automated image analysis system the procedure is simplified and the assay capacity increased. Other drawbacks of this method are its exposure of damaged cells to radioactivity and the fact the assay cannot be performed on cryopreserved cells. In summary, the UDS assay is a cell autonomous assay allowing one to look at the complex process of NER as a whole, but is rather technically complex, and needs more sensitivity and reliability. In addition, inter- and intra-individual variability has not been studied and this methodology has recently not been used anymore

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in epidemiological studies studying repair capacity in human tumours. 4. DNA repair assays based on induction of chromose breakage 4.1. In vitro cytogenetic challenge assays Several DNA repair assays consist of cytogenetic assays, in which chromosomal aberrations are analyzed after challenging cells in vitro with DNA-damaging agents. The rationale of these assays is that, in response to mutagen exposure, higher levels of genetic damage accumulate in people with less efficient DNA repair capacity as compared to normal individuals. This is supported by the observation of high mutagen-induced chromatid breaks in DNA repair deficiency syndromes such as Fanconi’s anemia, Ataxia Telangiectasia and Xeroderma Pigmentosum, Ataxia Telangiectasia (A-T), Nijmegen breakage syndrome (NBS) and A-T like disorder (ATLD) [122,123]. 4.1.1. The G2-challenge CA assay 4.1.1.1. Studies. In the G2-challenge chromosomal aberrations (CA) assay, often called the G2-mutagen sensitivity assay, the mean number of chromatid breaks per cell (b/c) at metaphase induced by mutagens in vitro in the late S-G2 phase of the cell cycle is measured and represents an in vitro indirect measure of DNA repair capacity for the estimation of individual susceptibility to cancer [124]. The G2-challenge CA assay requires synchronized cells and is performed on whole blood peripheral blood mononucleated cells (PBMC), stimulated with phytohemagglutinin (PHA) (Fig. 2A). The original assay was developed using the radiomimetic compound bleomycin [125], which is a clastogen mimicking the effects of radiation by generating free oxygen radicals capable of producing DNA single- and double-strand breaks after formation of a complex with DNA, ferrous ions (Fe2+), and oxygen, resulting in the release of oxygen radicals. Most of these lesions are repaired by BER and DSBR. This method has been applied to assess genetic susceptibility to cancer in both pilot studies [125–131], and in studies using larger populations (above 100 individuals) [132–135]. Later on the assay has been expanded to other etiologically important chemical mutagens, such as benzo[a]pyrene diol epoxide (BPDE) [116,134– 139], UV and its mimetic agent 4-nitroquinoline-1-oxide (4-NQO) [140,141], for which most of the described studies were performed in relatively large study populations of cancer-cases and controls. BPDE covalently binds to DNA and forms bulky adducts that necessitate nucleotide excision repair (NER). UV and 4-NQO induce photo-products that are mainly repaired by NER. The G2-challenge CA assay allows discrimination between individuals with cancer and healthy controls [124] and between individuals with cancer who develop secondary malignancies and those who did not [142–144]. It has also been demonstrated that the assay allows discrimination between individuals with a family history of cancer and those without [130,145,146]. In addition, joint effects between mutagen sensitivity and other risk factors such as tobacco smoking and alcohol consumption increase the predictive power of the assay [143,147]. In these studies, an increased number of patients with higher frequencies of chromosome aberrations compared to the controls is considered to be indicative of expression of mutagen sensitivity. Physical mutagen challenging, with UV-light and g-irradiation, can also been used in the G2-challenge CA assay [148–150]. The G2challenge CA assay using g-irradiation as challenging mutagen has also been used to specifically assess radiosensitivity, where higher yields of chromosomal damage correlate with radiosensitivity. The assay is often referred to as the G2-radiation assay and has proven to be a sensitive biomarker of chromosomal radiosensitivity in

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several pilot studies [151–153]. Many studies have demonstrated an increase in chromosome radiosensitivity in cancer patients as compared to controls [148,154–164]. Docherty et al. [165] could not confirm previous findings of increased chromosome radiosensitivity in breast cancer-cases as compared to controls, despite using a much larger and better-controlled study population as compared to the previous mentioned studies. Heritability for G2-mutagen sensitivity has been investigated in twin studies, indicating a clear genetic basis. Cloos et al. [144] estimated a 75% heritability in a twin study involving 25 pairs of monozygote twins and 14 pairs of dizygotes (twin pairs and siblings) after challenging with bleomycin and Tedeschi et al. [166] revealed a heritability of 58% studying nine monozygote and 10 dizygote twins for bleomycin sensitivity. In a more recent study by Wu et al. [167] the genetic heritability was evaluated in a larger population of 148 pairs of monozygotic twins, 57 pairs of dizygotic twins and 50 siblings. The authors found a heritability of 40.7%, 48.0%, 58.8% and 62.5% for bleomycin, benzo[a]pyrene diol, 4nitroquinoline-1-oxide and g-radiation, respectively. Evidence for heritability of cellular radiosensitivity (83.7%) using the G2mutagen sensitivity with g-radiation was also shown by Borgmann et al. [168]) in 15 monozygote twins. 4.1.1.2. Advantages and disadvantages of the G2-challenge CA assay. The bleomycin G2-challenge CA assay has been shown to be reliable and comparable across different laboratories. In a study by Cloos et al. [143] no differences between institutions in the distribution of bleomycin sensitivity in patients and controls were found. Recently, Erdei et al. [169] conducted an inter-laboratory comparison between two laboratories to address the concerns of the possible wide inter-individual, inter-observer variation and inter-laboratory variation and found a high correlation for all tests and no significant variation. However, it should be stressed that an adequate method to measure DNA repair capacity should be able to detect differences between individuals, resulting in significant inter-individual variation, but should show a minimal intraindividual variation. The radiation G2-challenge CA assay on the contrary, shows higher variations in yield and reproducibility, which makes interlaboratory comparisons difficult. Vral et al. [170,171] studied the intra- and inter-individual variability to assess the use of the assay for determining individual radiosensitivity and observed a significant intra-individual variability. Smart et al. [172] found a highly statistically significant inter-individual variability, by applying the assay on healthy controls, indicating that the assay can be used to examine the population profile of radiosensitivity. A shortcoming of the G2-challenge CA assay is the fact that chromatid breaks can only be detected in metaphase cells. Other DNA modifications such as point mutations and cross-links cannot be detected. In addition, since DNA damage is induced in late S-G2 phase, only double-strand breaks are measured and the significance of the role of DNA repair in G2 phase of the cell cycle in this assay is not well-understood. In addition, since the assay requires freshly cultured PBMC, the use of cryopreserved cells is not possible. This implies that samples cannot be accumulated, which would allow to perform many assays at once thereby reducing the inter-experimental variation. In conclusion, the G2-challenge CA assay is a relatively simple test which is widely used as phenotypic assay in cancer epidemiology and which has the flexibility to assess cancer risk related to exposures to different mutagens (reviewed in [173]). Its heritability has been estimated by classic twin studies, which makes it a well-established phenotypic susceptibility marker for cancer risk. However, it requires cytogenetic expertise to score and is time consuming and technically demanding for large-scale studies. Furthermore, the assay is carried out by challenging cells

with a test mutagen followed by measuring the DNA damage level immediately after treatment, this implies that a combination of mutagen sensitivity and DNA repair capacity is measured and moreover a rather general, non-specific impairment of the DNA repair machinery. In addition, the significance of the role of DNA repair in G2 phase of the cell cycle in this assay is not wellunderstood. Moreover, the definition of a mutagen sensitive phenotype is rather heterogenous in the literature: results of some studies are based on cut-off points of a certain percentile value (e.g. 25 or 75 percentile), while others are based on specific dichotomous chromatid break levels (e.g., b/c > 1.0) to compare sensitive versus non-sensitive subjects [123]. Therefore, additional studies are required to set up a standardized protocol and to validate the reliability of the assay, which would also allow automated scoring of chromatid breaks and enhance its applicability in highthroughput analysis. 4.1.2. The G1-challenge CA assay 4.1.2.1. Studies. Au et al. [174,175] developed the G1-challenge chromosome aberration (CA) assay to investigate mutagen sensitivity in populations exposed to environmental mutagen. It was hypothesized that chronic exposure to mutagens will not only lead to genotoxic effects, but also cause cells to have deficient DNA repair capacity due to modifications of DNA/proteins or mutations in DNA repair genes. For this method non-frozen PHA-stimulated synchronized PBMC cultures are challenged in vitro during the G1 phase of the cell cycle with physical agents such as X-rays, g-rays or UV light and the frequencies of chromosome aberrations are determined in the metaphase of the same cell cycle. The observed chromosome aberrations result from the induced DNA damage, oxidative base damage and single-strand and double-strand DNA breaks in case of X-rays and g-rays, and bulky adducts in case of UV light, that has been subjected to modification by the cellular DNA repair activities throughout the cell cycle (Fig. 2A). An increase in the levels of chromosome aberrations in cases as compared to controls reflects exposure-induced DNA repair deficiency. For example, chromosome deletions indicate the lack of or incomplete repair and translocations indicate errors in repair activities. Oxidative base damage, single-strand and double-strand DNA breaks induced by X-rays and g-rays require base excision repair (BER) and double-strand break repair (DSBR). UV light induces bulky adducts that are mainly repaired by NER. The G1-challenge CA assay with g-rays was performed in many studies, mostly performed on small populations to assess the induction of acquired susceptibility in exposed populations. It was demonstrated in cigarette smokers that smoking habit caused an abnormal DNA repair response to the challenge-induced damage [176], indicating an acquired susceptibility to the induction of DNA damage by environmental mutagens. Similar studies conducted for in residents of living nearby uranium mining and milling activities observed higher frequencies of cells with chromosome aberrations and deletion frequency than the control group [175,177], suggesting an abnormal DNA repair response in the target population. The G1-challenge assay was also used in several biomonitoring studies addressing the risk of urban air pollution, e.g. particle-associated PAHs. Schoolchildren in a high-density traffic area in Bangkok showed a significant reduced DNA repair capacity, measured as an increase in radiation-induced dicentric chromosomes and chromosome deletions per metaphase, as compared to children attending school in a provincial area [178]). In another study personal monitoring of traffic policemen demonstrated a significantly affected DNA repair capacity as compared to their office counterparts [179]. The assay was also applied to study the acquired susceptibility in occupational

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settings. Workers exposed to high concentrations of butadiene [174,180], benzene [181,182], styrene [183] and to mixed pesticides [184] showed higher frequencies of CA, indicating that the workers had an acquired susceptibility to environmental mutagens and thus were predicted to have increased risk of cancer. Navasumrit et al. [185] employed the assay to demonstrate the reduced DNA repair capacity of temple workers in Thailand that where occupationally exposed to incense smoke, containing high concentrations of PHA, benzene and 1,3-butadiene. On the other hand, in mothers that have given birth to children with neural tube defects [186], in workers exposed to very low concentration of benzene or in populations expsoed to average levels of air pollution [187] no defective DNA repair capacity was observed compared to the matched controls. These observations suggest that prolonged or excessive exposure to environmental pollutants, at high enough concentrations, is causally related to the induction of DNA damage and to the cellular repair mechanisms [179]. El-Zein et al. [188] used UV-G1-challenging to demonstrate that epidermodysplasia verruciformis (EV) patients which are know to have an increased risk for sunlight-induced skin cancer were deficient in the repair of chromosome aberrations induced by UVlight. Au and Salama [189] demonstrated that the assay is sensitive enough to identify small differences in DNA repair activities in a population by investigating the relationship between variant genotypes for genes involved in BER and NER and the expression of X-rays and UV-light induced chromosome aberrations in a healthy non-smoking population. 4.1.2.2. Advantages and disadvantages of the G1-challenge CA assay. The G1-challenge CA assay has several advantages as compared to the G2-mutagen sensitivity assay. The use of only physical DNA-damaging agents avoids the influence of individual variations such as differential cellular uptake and metabolism as compared to chemical agents [189]. In addition, in the G2challenge CA assay the time between exposure and observation is rather short (1–5 h). This implies that certain cell cycle phasedependent repair processes may not be activated resulting in an inadequate assessment of DNA repair capacity. Since exposure in the G1-challenge CA assay occurs in G1 and the frequencies of chromosome aberrations are determined in the metaphase of the same cell cycle, DNA repair activities that are occurring throughout the complete cell cycle can be assessed. Moreover, the G1challenge CA assay allows the detection of both chromosome-type (deletions, translocations) and chromatid-type (breaks and exchanges) of aberrations. Therefore, the assay has the unique feature to allow the identification of abnormal repair activities leading to the foramtion of chromosome translocations and deletions, rather than simply repair activities [190]. In addition, the assay has been shown to be a measure of repair deficiency and predictive of cancer [173]. However, similar to the G2-challenge CA assay the G1-challenge CA assay is also not able to detect DNA modifications such as point mutations and cross-links and cryopreserved PBMC cannot be used, since freshly cultured PBMC in whole blood cultures are required for the assay. In summary, G1-challenge CA assay is a phenotypic assay which covers more DNA repair mechanisms in a more adequate manner as compared to the G2-challenge CA assay and therefore may be of more general applicability (in particular for mixed exposures). However, it also requires cytogenetic expertise to score and is time consuming. It has only been used in a limited number of studies using small study populations. Additional studies are needed to study the inter- and intra-individual variability and to validate the reliability of the assay, which would also provide a standardized protocol for automated scoring and high-throughput analysis.

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4.1.3. The G0- and G1-challenge MN assay 4.1.3.1. Studies. The use of micronuclei (MN) as a measure of early genotoxic effects has become a standard assay in human biomonitoring studies, but can also be used to measure DNA repair. Micronuclei can be found in dividing cells as small, extranuclear bodies resulting from chromosome breaks (leading to acentric fragments) and/or whole chromosomes that did not reach the spindle poles during cell division. Micronuclei represent therefore a measure of both chromosome breakage and chromosome loss [191–193]. The cytokinesis-block micronucleus (CBMN) assay is the most extensively used method for measuring MN in cultured human lymphocytes because scoring is specifically restricted to cells that have completed one nuclear division and are recognized by their binucleated appearance [194–196]. MN, which occur in cells that have completed at least one mitosis, are scored in these binucleated cells [194]. In a classical in vitro CBMN test, human lymphocytes are cultured in the presence of PHA to stimulate mitosis. After 44 h, cytochalasin B is added to the culture and cells are harvested at 72 h (Fig. 2A). The CBMN assay has become an established biomarker for genomic instability. Combination of fluorescence in situ hybridization (FISH) using probes for pancentromeric, regions with the cytokinesis-block micronucleus assay allows discrimination between micronuclei containing a whole chromosome (centromere positive micronucleus) and an acentric chromosome fragment (centromere negative micronucleus) [197,198]. Besides its capacity to detect clastogenic and aneugenic events, the CBMN assay can provide additional measures of genotoxicity and cytotoxicity: nucleoplasmic bridges (NPB, a marker of chromosome rearrangement) and nuclear buds (NBUD), a marker of gene amplification) [199]. Although the MN assay is mainly used to assess DNA damage, micronuclei analysis can also be applied to assess DNA repair capacity, when taken into account only MN resulting from chromosome breaks, since MN containing whole chromosomes are not eliminated by repair processes. MN harbouring chromosomal fragments may result from direct double-strand DNA breakage, conversion of SSBs into DSBs after cell replication, or inhibition of DNA synthesis. Since structural chromosomal damage leading to the formation of MN involves acentric fragments, MN frequency should be linked with the level of unrepaired DNA double-strand breaks at the time of mitosis. The CBMN method has been mostly applied in small studies to measure susceptibility after in vitro challenging G0-lymphocytes with ionising radiation [161,200–202] and H2O2 [201] and is therefore referred to as the G0 micronucleus assay (G0MNT). It was demonstrated that subjects who developed breast cancer and their relatives were more sensitive than controls to the DNA-damaging effect of ionising radiation as shown by micronucleus frequency [200], the same chromosomal radiosensitivity was observed by Baeyens et al. [202] who compared breast cancer patients with a known or putative genetic predisposition to a group of healthy women. In addition, breast cancer patients also show elevated radiation-induced MN frequencies compared to oesophageal cancer patients, indicating a higher contribution of radiosensitivity-related genes to the development of breast cancer [203]. Rothfuss et al. [201] observed this radiosensitivity in 10 of 11 cases of BRCA1 mutation carriers, which was indicative of a defect in double-strand break repair. Using this G0MNT challenge assay with ionising radiation, Varga et al. [204] also demonstrated a significant difference between sporadic breast cancer patients and controls. Kotsopoulos et al. [205], on the contrary, found no significant difference in DNA repair capacity using the same methodology between healthy BRCA1 mutation carriers and healthy mutation-negative females as controls.

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Using the G1-challenge MN assay, El-Zein et al. [206] showed a differential sensitivity in a relatively large population of lung cancer patients and healthy controls to nicotine-derived nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). The repair kinetics for NNK-induced genetic damage has not been fully understood but may involve several DNA repair pathways, including base excision and nucleotide excision repair pathways [207]. Lymphocytes from lung cancer patients and controls were challenged in vitro in G1 with NNK and chromosomal damage endpoints (micronuclei, nucleoplasmic bridges and nuclear buds) were scored in the CBMN assay. Both spontaneous and NNKinduced micronuclei frequencies were significantly higher in lung cancer patients as compared with controls. Recently, it was demonstrated that inclusion of micronuclei in mononucleated cells to the assay, improves the positive and negative predictive value for disease status in the same case-control study for lung cancer [208]. Similar to the G2-challenge CA assay, the G1-challenge MN assay using bleomycin was shown to have a high genetic component towards its sensitivity, which was revealed in a twin study by Tedeschi et al. [166]. Moreover, this study demonstrated that micronuclei analysis in old male twins, selected on basis of age, life-style and habits provided a higher power as compared to the analysis of chromosome aberrations in the G2-challenge CA assay to unreveal the genetic basis of mutagen sensitivity [166]. In addition, a pilot study performed with first-degree relatives by Burrill et al. [209] suggested that radiosensitivity in this assay may be an inherited characteristic associated with predisposition to breast cancer. 4.1.3.2. Advantages and disadvantages of the G0- and G1-challenge MN assay. Compared to the previous described CA assays, the CBMN assay has several advantages: scoring of MN is easier and faster than the determination of chromosome aberrations and it does not require metaphase cells. Another advantage of the CBMN assay lies in its recent automation [210]. The implementation of automated methods for MN detection allows the analysis of a large number of cells and the exclusion of subjective judgment and individual scoring skills, since often high inter-laboratory and inter-scorer differences occur with manual scoring. However, if the assay is not combined with FISH, one portion of the observed MN is due to DNA repair defects, those containing chromosome breaks. Furthermore, exchanges arisen by mistakes in DNA repair such as balanced translocations may be missed by this assay. Compared to the comet assay that only indirectly indicates the speed of DNA strand break rejoining, the cytogenetic challenge assays described in this review provide a measure for impaired fidelity of strand break rejoining which can lead to chromosome aberrations and micronuclei and might therefore be better suited for the assessment of mutagen sensitivity [56]. On the other hand, although these assays provide information on the frequencies of chromatid breaks remained in the cells, they do not always offer a clear molecular mechanisms by which the breaks have been formed or repaired and are therefore an indirect measurement of DNA repair capacity. This is especially the case when the challenging mutagens used induce DNA lesions that are repaired by NER. During the repair of these lesions, repair enzymes can make more cuts on the damaged DNA strands [211] leading to double-strand breaks or chromosomal breaks. A slow kinetics and suboptimal synapsis mechanism of back-up non-homologous endjoining of the double-strand breaks has been suggested in which more time needed for exchanges through the joining of incorrect ends may cause the formation of chromosome aberrations [212]. However, the exact mechanisms by which these lesions are converted into chromosome breaks remain to be elucidated. A

possible mechanism could be telomere capture that is intended to stabilize broken chromosomes in mammalian cells and that is mediated by the enzyme telomerase [213,214]. 4.2. g-H2AX assay One of the earliest responses to DNA double-strand breaks is the carboxy-terminal phosphorylation of the histone g-H2AX by the DNA-activated kinases ATR, ATM and DNA-PK, yielding g-H2AX that forms foci flanking the regions of the double-stranded breaks (reviewed [215]). g-H2AX foci can be counted by immunofluorescence staining using g-H2AX specific antibodies (Fig. 2B). Enumerating g-H2AX foci can be used to measure the repair of radiation-induced double-strand breaks [216,217]. Up to date, only few studies used the g-H2AX assay to study the relation between cancer susceptibility and DNA repair, and most of them focussed on radiation-sensitivity of human ATM heterozygotes [218] and unaffected parents from retinoblastoma patients [219]. In the study by Kato et al. [218] two versions of the g-H2AX assay were applied, to examine the sensitivity to ionising radiation of human ATM heterozygotes and showed a DNA double-strand break rejoining defect in ATM heterozygotes. One version involved the scoring of g-H2AX foci in non-cycling contact inhibited fibroblasts after low dose-rate irradiation. In the second version of the assay, the G2/M g-H2AX assay, g-H2AX foci on chromosomes of mitotic cells are analyzed shortly after high dose-rate irradiation of the cells in G2. This G2/M g-H2AX assay can be considered as a reminiscent of the G2 radiation assay. Application of these two methodologies provides an elegant way to discriminate between NHEJ capacity and other G2-related damage processing systems. The low dose-rate g-H2AX assay requires the use of non-cycling G0 or G1 cells, that do not proliferate during prolonged exposures and reflects only the contribution to DSB rejoining from the NHEJ system which operates throughout the cell cycle, while in S- and G2 also other processes are know to mediate cellular radiation responses. Cells deficient for NHEJ would be hypersensitive in both the low dose-rate G0 assay and the G2/M assay, and deficiencies in G2-dependent repair systems would be as sensitive in the G2/M assay but not in the low dose-rate G0 assay. Recently, Kotsopoulos et al. [205] examined the potential of the g-H2AX assay as a biomarker of DNA repair capacity to discriminate between women with and without BRCA1 mutations. However, they could not find any significant differences in DNA repair capacity between BRCA1 mutation carriers and non-carrier controls, indicating that this assay is not likely to be useful in the identification of this high-risk group. Several reports described a flow cytometry-based method for gH2AX detection in fixed peripheral human blood cells [219–222] that was recently optimized by Ismail et al. [223] for non-fixed cells, which could be stored at 80 8C for extended periods without loss of the g-H2AX signal. With this method the authors found a significant inter-individual variation in the g-H2AX response. 5. In vitro repair assays assessing the functional activity of DNA repair enzymes in extracts from donor cells Another more biochemical approach to measure DNA repair capacity of oxidative DNA damage involves the incubation of cell extracts with an oligonucleotide containg one 8-oxoguanine residue and radiolabeled with 32P at the 50 end of the DNA strand containing the lesion. This enzymatic OGG activity assay was developed by Paz-Elizur et al. [224]. In this assay OGG activity removes the modified guanine, leading to the formation of an abasic site, which is then cleaved by AP lyase activity of the enzyme or by the action of AP endonucleases present in the protein extract. The alteration in size on cleavage of the phosphodiester bond at the

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8-oxoguanine is detected by conventional gelelectrophoresis and visualisation of the 32P-labelled fragments. This methodology was applied to study whether reduced oxidative DNA damage repair is associated with lung cancer [224,225] and with squamous cell carcinoma of the head and neck [226]. Gackowski et al. [227] using a similar assay also found a decreased OGG activity in non-small lung cancer patients as compared to their healthy counterparts. An alternative in vitro fluorometric assay developed by Kreklau et al. [228] allows quantitative measurement of alkylation repair [O(6)-methylguanine DNA methyltransferase (MGMT) and methylpurine DNA glycosylase (MPG) activities], and oxidative DNA damage repair [hOGG1 and human abasic endonuclease (APE1) activities] from a single cell extract. This approach involves preparation of cell extracts in a common buffer, in which all of the DNA repair proteins are active and the use of fluorometrically labelled oligonucleotide substrates containing DNA lesions specific to each repair protein. However, a possible complication in applying such an approach to DNA repair assays in general and to hOGG1 assay in particular, is the recognition of the fluorescent tag as a ‘‘DNA lesion’’ by cellular proteins [224]. Nevertheless, the potential advantages of the fluorometric method in terms of simplification and potential for high-throughput analysis warrant the effort to apply it to large-scale epidemiology assays. Machella et al. [229] assessed the DNA end-joining (EJ) capacity in cell lines derived from sisters discordant for breast cancer to investigate whether individual differences in double-strand break repair are a significant risk factor. The EJ capacity was measured in an in vitro phenotypic assay on nuclear extracts from lymphoblasts by testing the ability of extracts to join together monomers of a linearized plasmid substrate either with sticky ends (EcoRI) or blunt ends (HincII). The results revealed that an inefficient DNA repair capacity is associated with breast cancer susceptibility. Maksimenko et al. [230] designed a method to follow repair incisions in molecular beacons by an extract of in vitro challenged donor cells. This assay measuring base excision repair activity is based on the fluorescence quenching mechanism of molecular beacon. A molecular beacon is a single-stranded oligonucleotide probe containing a sequence complementary to a target that is flanked by self-complementary termini, and carries a fluorophore and a quencher at the 50 - and 30 -ends [229]. Tyagi and Kramer [231] developed a single-stranded DNA oligonucleotide labelled with a 50 -fluorescein (F) and a 30 -Dabcyl (D), the fluorophore, F, is held in close proximity to the quencher, D, by the stem–loop structure design of the oligonucleotide. Following removal of the modified base or incision of the oligonucleotide, the fluorophore is separated from the quencher and fluorescence can be detected as a function of time. 6. Gene expression profiling of DNA repair genes by microarray analysis With the development of chip technology (microarrays and proteomics) it is possible to screen the effects of exposure at the molecular level by high-throughput testing methods. A microarray is a small device, like a microscope slide, with thousands of different known DNA sequences immobilized at different addresses on the surface. Sample preparation starts with the isolation of total RNA containing messenger RNA representing a quantitative copy of genes expressed at the time of sample collection. mRNA is extracted from a sample of interest and a control and are then separately converted into complementary DNA (cDNA) using a reverse-transcriptase enzyme. Next, each cDNA (sample and control) are labelled with a different fluorescent dyes (e.g. Cy3 and Cy5). During the hybridization the labelled cDNA (from the sample and the control), purified and then hybridized against the cDNA molecules spotted on the glass slide. Image

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acquisition and data analysis is the final step of microarray experiments (Fig. 2B). Microarray analysis allows the identification of a complete gene expression profile and thus also to investigate changes in the expression of DNA repair genes involved in different DNA repair pathways. Recently, several studies have applied this method to detect gene expression changes in DNA repair-related genes. A study by Cloos et al. [15] aimed at revealing the pathways involved in mutagen sensitivity by microarray analysis of bleomycinexposed lymphoblastoid cells from individuals with a high and low mutagen sensitivity. They found a considerable number of genes involved in biological processes, such as cell growth and maintenance, proliferation, cell cycle regulation and DNA repair. Comparison of insensitive and sensitive individuals showed other differentially expressed genes involved in signal transduction and cell growth/maintenance. Fachin et al. [232] monitored the gene expression profile using microarrays in lymphocytes from radiation workers and observed changes in the expression levels of several genes involved in diverse DNA repair pathways. In a study by Colombo et al. [233] the gene expression profiles of human tumour and non-tumour larynx tissues were compared. Among the genes showing statistically significantly differences between the tumour and non-tumour samples, also genes involved in DNA repair were identified. Federico et al. [234] applied microarray analysis to study the effects of 131I radiotherapy in thyroid carcinoma patients. The authors could detect significant changes in the expression profiles of DNA repair genes. Microarray analysis has the advantage to analyse a complete gene expression profile, it can provide information on all different DNA repair pathways. Nevertheless, this method still shows some important drawbacks: no general standardized approach exists for analysis of the data, which require robust bio-informatics systems, sample storage and preparation are critical for the reproducibility, and some host factors such post-transcriptional modifications are not taken into account. 7. DNA repair phenotype and predictivity for cancer risk Aberrations of many cellular functions play a role in the etiology of cancer, among those DNA repair processes are essential in maintaining genomic integrity and protection against cancer. Several studies have shown that individuals with DNA repair capacity below the population mean can be at risk to develop various kinds of cancer. During the last decades there has been a world-wide accelerating increase of cancer incidence. Therefore there is an urgent need for reliable risk prediction tools for estimating individual probability of cancer, allowing early diagnosis. The accumulation of genetic changes may lead to genetic instability, which may result into cancer. This genetic instability can be mediated through chromosomal changes and has therefore the potential to be cytogenetically detectable [208]. It has been demonstrated that chromosome aberration frequencies [235–238] and micronuclei frequencies [238,239] are the two cytogenetic endpoints providing strong evidence for the association between the extent of chromosomal damage and cancer risk. Up to now, only for these two cytogenetic endpoints used to assess DNA repair phenotype the predictivity for cancer risk has been extensively investigated: the G1- and G2-challenge CA and the G1-challenge MN assay. Measuring chromosomal aberrations after challenging with DNA-damaging agents has been extensively applied to assess DNA repair capacity (the G1/G2-challenge CA). This methodology has been successfully used in numerous epidemiologic studies as an indicator of individual predisposition for a variety of cancers, (as described in Section 2.1.1). Besides retrospective studies, also

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prospective studies have provided further support in the evidence that mutagen sensitivity is a risk factor for cancer development [143,144,147,240]. Micronuclei frequencies in peripheral blood lymphocytes have been extensively used as a biomarker of chromosomal damage, genomic stability and cancer risk, integrating acquired mutations and genetic susceptibility. The G1-challenge micronucleus assay was evaluated by El-Zein et al. [206,208] to predict lung cancer risk. Lymphocytes from lung cancer patients and controls were challenged in vitro in G1 with nicotine-derived nitrosamine 4(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and chromosomal damage endpoints (micronuclei, nucleoplasmic bridges and nuclear buds) were scored in the CBMN assay. In lymphocytes from lung cancer patients a significantly higher sensitivity to NNK was observed in terms of micronuclei, nucleoplasmic bridges and nuclear buds, as compared to the controls. Moreover, the use of the CBMN assay was evaluated to predict cancer risk based on the number of chromosomal endpoints defined by percentile cut points in controls. Probabilities of being a cancer patient were 96%, 98% and 100% when using the 95th percentiles of spontaneous and NNK-induced micronuclei, nucleoplasmic bridges, and nuclear buds, respectively. These studies are the first to validate the use of the CBMN assay, with NNK as challenge mutagen, in a case-control study by testing the sensitivity of this genomic instability biomarker as a predictor of lung cancer risk. Recently, Iarmarcovai et al. [241] performed a meta-analysis evaluating and summarizing the available evidence linking cancer status and MN in peripheral blood lymphocytes. This review revealed (i) a higher baseline MN frequency in untreated cancer patients; (ii) an increased MN frequency in cancer patients after radiation treatment; and (iii) an inverse association with their radiation dose in the challenge assay performed to test radiosensitivity in cancer patients. These results substantiate the evidence that MN play a role in different steps of carcinogenesis. Nevertheless, prospective studies using the G1-challenge MN assay would provide further support to its predictivity for cancer risk. Important is the fact that these challenge assays are the only DNA repair assays whose heritability has been studied in classical twin studies [142,166,167]. These twin studies demonstrated that mutagen sensitivity is an inherited characteristic, suggesting strong genetic determinants of the mutagen-induced chromosome damage. Since it has been stated that the expression of mutagen sensitivity, as measured by the challenge assay, is based on the interaction between mutagen exposure and individual susceptibility [242]; identification of genes involved in individual susceptibility and determination of the segregation pattern of these genes could contribute to a better insight into the assessment of cancer risk. Several studies have demonstrated that bleomycin sensitivity is partly affected by polymorphisms of DNA repair genes and of bleomycin hydroxylase (BLXH) [243–246]. These findings corroborate the genetic basis for bleomycin sensitivity as demonstrated in twin studies [142,167,166]. Further studies are needed to investigate the relationship between genotypes and phenotypes for DNA repair and to adequately validate the challenge assays for their cancer predictivity: studying the influence of multiple polymorphisms and on the sensitivity for bleomycin, for other DNA-damaging agents that induce similar types of DNA damage as bleomycin but are not affected by BLHX (such as ionising radiation) and for compounds inducing other types of DNA damage (such as BPDE) could also contribute to a valuable predictivity of the assay. In addition, an important advantage of these challenge assays as phenotypic marker in relation to cancer risk is that they assess an activity mechanistically important in tumorigenesis and integrates several host factors (i.e., different DNA repair mechanisms)

in one measurement. Nevertheless, one should be cautious with the interpretation of the cancer association, since the cells obtained from individuals with newly diagnosed cancer may also reflect the consequences of the cancer instead of only the host’s susceptibility. Often only retrospective studies were performed, this implies that one cannot exclude a potential problem/bias in discriminating between cause and result. When groups of cancer patients and healthy individuals are compared, a lower DNA repair capacity in cancer patients can be interpreted as either by predisposition, or an effect of the cancer. Therefore, the cancer predictivity value of a DNA repair phenotype assay should be evaluated in prospective studies with a nested case-control design, for which DNA and PBMC samples from a suitable cohort would be collected and stored, rather then in retrospective studies. At some time in the future, patients with disease could be identified and matched with healthy controls from within the same cohort. As far as the other DNA repair phenotype assays described in this review, some epidemiological evidence for cancer predictivity has only been reported for the host cell reactivation assay. It was demonstrated that suboptimal DNA repair capacity using the host cell reactivation assay using BPDE as DNA-damaging agent was associated with lung cancer risk [86,90]. Subsequent studies by Gorlova et al. [247] and Spitz et al. [248] confirmed this finding using larger sample sizes. However, due to the fact that the host cell reactivation assay is a relatively costly and time-consuming method, DNA repair capacity assessed by this assay has not yet been prospectively validated as a cancer risk predictor. 8. DNA repair phenotype and sensitivity to chemotherapy and radiotherapy The different DNA repair assays discussed in the present manuscript are mainly used in epidemiologic studies to compare DNA repair capacity between cancer case subjects and controls subjects to evaluate whether a deficient DNA repair machinery contributes to the development of cancer or to investigate whether environmental exposed populations may have an increased cancer risk. Nevertheless, application of these DNA repair phenotype assays can also provide valuable information regarding the intrinsic DNA characteristics of cancer cells and their responses to chemotherapy or radiotherapy, thereby contributing to establish an individualized adjusted therapy, since the anti-cancer activity of many chemotherapeutic compounds or ionising radiation is directly linked to the ability to induce DNA damage in tumour cells and tissues. Moreover it is known that a percentage of cancer patients develop secondary tumours after being treated with cytostatic drugs, therefore the assays measuring DNA repair capacity could not only be used to measure the sensitivity of cells to anti-neoplastic drug as well as their ability to subsequently repair the induced damage but also to evaluate the persistence of the genetic damage induced by such treatments. Many factors can influence the tumour response to treatment, such as the type of tumour, the type of treatment used, the oxygen level and the specific genetic make-up of the individual patient [249]. With this wide variability in patient response, an assay that provides an accurate prediction of the tumour response on individual basis would be a very valuable asset in the cancer treatment. Most of the assays described here have not been widely employed yet as a tool to predict the individual’s tumour sensitivity to antineoplastic drugs or radiation. The suitability of such an assay for the prediction of tumour sensitivity depends on its sensitivity, simplicity, rapidity and the ability to perform it on different cell types and tissues.

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The most common approach involves the indirect measurement of the efficacy of the cancer treatment by assessing the DNA damage and repair in surrogate tissue, mostly PBMC from the cancer patient who is undergoing the treatment. Although the collection of PBMC is less invasive compared to target tissue cells and they are the surrogate cells of choice in studies one should take into account that PBMC may not necessarily reflect the response of the tumour cells. DNA repair assays such as the in vitro cytogenetic challenge assays and the comet assay could be suitable candidates to assess the cancer patient’s sensitivity to cancer treatment. It has been suggested that G2-challenge assay can be used as a biomarker for the prediction of responses and outcomes in chemotherapy and radiotherapy. Lo´pez de Mesa et al. [250] evaluated the chemotherapy induced genetic instability in pediatric cancer patients using bleomycin-induced mutagen sensitivity. The cytogenetic assays are relatively simple, inexpensive and robust assays, and have the potential to be adequate for prediction of tumour sensitivity. However, they need synchronized cells and cannot be performed on just a few cells from a biopsy. The comet using in vitro challenging with irradiation has been applied to study the radiosensitivity in PBMC of cancer patients and to adjust radiotherapy [14,19,20]. Recently, McKenna et al. [249] reviewed the potential use of the comet assay in the clinical management of cancer. It is a simple, quick and relatively inexpensive test carried out on single cell suspensions, and thus both on PBMC and cells extracted from a biopsy, making it a an appropriate method for clinical testing of cancer treatment sensitivity. The assay has been successfully applied to demonstrate that radiosensitivity can be measured in various tumour cell lines and to assess the sensitivity of human cancer cell lines to drugs routinely used in chemotherapy such as cisplatin, tamoxifen, mitomycin C (reviewed in [249]). However, more research on the reliability, reproducibility and validity is needed to make the comet assay a predictive technique to assess the individual response of chemo-/radiotherapy. Much research has also been focussed on gene expression analysis by microarray technology to asses the genetic signature of tumour cells thereby providing an indication for an individual’s response to cancer treatment [251]. Nevertheless, they are still some limitations concerning the interpretation of the gene expression data regarding its predictivity for cancer treatment, and other DNA repair phenotype assays are still necessary to validate the findings of this method. 9. Discussion The individual susceptibility to mutagen exposure and therefore to cancer risk is influenced by a complex interplay between genetic and environmental factors. This implies that information on the genotype (single nucleotide polymorphisms) may not be informative enough and functional phenotypic assays are needed to provide useful tools to explore the role of DNA repair in the etiology of cancer. Moreover, in addition to genotyping, phenotypic markers, would also help to estimate differences in genetic diversity among different ethnic groups. Over the past two decades, several methodologies for the assessment of DNA repair capacity have been developed. The importance of measuring DNA repair capacities is reflected by the exponentially increasing number of studies on DNA repair, both in cancer-case-control or environmental exposed-control studies. In this review we aimed at describing and evaluating the different methodologies presently developed to determine DNA repair capacity phenotypes with their design, results and, advantages and disadvantages.

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Assays for phenotypical assessment of DNA repair capacity in epidemiological studies should fullfil the following criteria: i) The reliability of a new assay should be established across laboratories, providing multi-laboratory validation of the method. Reliability consists of both reproducibility and repeatability and concerns the extent to which a measurement yields the same results on repeated experiments. As reviewed in the present manuscript, the issue of reproducibility and inter-versus intra-individual variation has only been addressed in detail for the different approaches of the comet assays and the cytogenetic challenge assays. ii) A new method should be validated, this should not be confused with reliability. Validity can be defined as the lack of systematic error measurement when a measurement is compaired with a standard, a reference method representing the ‘‘truth’’. iii) The sensitivity and specificity has to be established and verified in large studies. In addition, the minimal sample size and number of assays per donor should be well-established to provide enough statistical power before starting a larger study. Moreover, correctly matched controls in terms of number, age, gender and smoking should be included. iv) They should be well characterized in terms of inter-versus and intra-individual variability. v) They should the potential for cancer risk predictivity. Therefore, not only retrospective studies should be performed, since this implies that one cannot exclude a potential problem/bias in discriminating between cause and result. When groups of cancer patients and healthy individuals are compared, a lower DNA repair capacity in cancer patients can be interpreted as either by predisposition, or an effect of the cancer. Therefore, ideal would be to perform prospective studies with a nested case-control design, for which DNA and PBMC samples from a suitable cohort would be collected and stored. vi) DNA repair phenotype assays should have the potential for high-throughput analysis. Recent advances in image analysis allow automation of several methodologies. vii) The tissue specificity should be well-established before using surrogate tissues or cell lines. Most assays described are conducted on peripheral blood mononucleated cells (PBMC), instead of target cells such as lung or liver cells, although the latter is an important issue of feasibility in large population studies. PBMC can be seen as reflecting the overall body environment to which they are exposed since they are circulating throughout the body. However, the question remains whether the biological effects observed in these surrogate cells also occur in the target cells and whether the observed effects can predict cancer or not. Therefore, combination of the cellular phenotypes of different tissues would provide a realistic approach of the individual determination of DNA repair capacity. Moreover, regarding the suitability of DNA repair assays a diagnostic tools in clinical cancer management, the methods should be applicable to any cell type taken from a biopsy. viii) The optimal use of fresh or frozen cells/tissues should be addressed. Some studies have already investigated the feasibility of cryopreserved PBMC for the mutagen sensitivity assay [252] and the comet assay [32,253,254]. Although some of the assays are suitable to be performed on frozen material, this will represent an important change in the study protocols and will need validation with many samples stored for different periods of time. ix) The choice of the assay does also depend on whether researchers are studying the DNA repair phenotype at

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population level or at individual level. Studies at population level are mainly applied for biomonitoring and have other requirements as compared to studies at the individual level which are mainly focussing on cancer therapy. x) In addition, the mutagen studied and the type of DNA lesions it induces will also influence the choice of the assay, since not all methods allow investigating each DNA repair pathway. Moreover, different mutagens may affect cells through different molecular mechanisms and thereby activating different repair pathways. This also implies that a person who is sensitive to one mutagen may be resistant to another. Furthermore, a growing body of evidence suggest that the different repair process can act as a ‘‘back-up’’ system for each other and certain lesions can be repaired by different repair pathways. For example, besides BER, oxidized DNA base lesion can also be repaired by NER, where the lesion-containing oligonucleotide is removed [255]; or by MMR when the oxidized nucleotides escape to nucleotide pool sanitation by nucleotidyl hydrolases and are incorporated into DNA [256]. In addition, in vitro experiments suggest that BER may also be involved in the repair of bulky BPDE-adducts [257]. Therefore it may be necessary to use a battery of mutagens and a panel of complementary assays to refine our ability to assess DNA repair capacity and to identify subpopulations at high-risk for developing cancer. Although they require living and dividing cells, the cytogenetic challenge assays, and especially the G1- and G2-challenge assay can be considered as one of the most useful assays to investigate DNA repair phenotype. The major advantages of using CA or MN are that they are easy to perform and the results are very robust. They have been successfully applied to assess repair capacity in both cancer patients and in environmentally exposed populations. Depending on the challenging agent used, they can detect deficiencies in different repair pathways. Moreover, these assays allow to predict the cancer therapy responses and to investigate the cancer prognosis. In addition, their usefulness as a functional biomarker in biomonitoring of exposed populations has been validated (reviewed in [179]). Nevertheless the choice of the assay depends on the objective of its application: for primary prevention and biomonitoring at population level or cancer therapy at individual level, but also on the scientific question of the researchers and on which exposure they want to address. Furthermore, it is not possible to investigate all different DNA repair pathways with one single method. As far as cancer predictivity is concerned, only the cytogenetic challenge assays have been shown to be predictive [123,206], but are labourintensive. Moreover, it should be taken into account that the phenotype could reflect the pathophysiological alterations in the patient secondary to the disease. Therefore, prospective studies are needed, which could avoid these problems since subjects are studied prior to the onset of cancer. Conflict of interests A conflicting interest exists when professional judgement concerning a primary interest (such as patient’s welfare or the validity of research) may be influenced by a secondary interest (such as financial gain or personal rivalry). It may arise for the authors when they have financial interest that may influence their interpretation of their results or those of others. Examples of potential conflicts of interest include employment, consultancies, stock ownership, honoraria, paid expert testimony, patent applications/registrations, and grants or other funding.

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