Review
Impact of the circadian clock on in vitro genotoxic risk assessment assays

https://doi.org/10.1016/j.mrgentox.2009.09.001Get rights and content

Abstract

Our society expects safety assessment for drugs, chemicals, cosmetics, and foods, which to date cannot be achieved without the use of laboratory animals. At the same time, society aims at refining, reducing, and (ultimately) replacing animal testing. As a consequence, much effort is taken to establish alternatives, such as toxicogenomics-based risk assessment assays on cultured cells and tissues. Evidently, the properties of cells in vitro will considerably differ from the in vivo situation. This review will discuss the impact of the circadian clock, an internal time keeping system that drives 24-h rhythms in metabolism, physiology and behavior, on in vitro genotoxic risk assessment. Our recent observation that DNA damaging agents can synchronize the circadian clock of individual cells in culture (and as a consequence the cyclic expression of clock-controlled genes, comprising up to 10% of the transcriptome) implies that the circadian clock should not be neglected when developing cell or tissue-based alternatives for chronic rodent toxicity assays.

Introduction

Carcinogenesis is a multi-step process in which genetic and epigenetic alterations enable cells to escape from regulated cell proliferation programs. A selective growth advantage of such cells (allowing clonal expansion), along with a loss of genomic stability (resulting in neoplastic transformation) ultimately results in the formation of primary and secondary (metastatic) tumors [1]. It is generally believed that the driving force behind tumor induction and progression is the induction of deletions and mutations, originating from DNA damage instigated by the exposure of the genome to dispense endogenous (i.e. reactive metabolic byproducts) and environmental (i.e. ionizing radiation, ultraviolet light, chemical compounds) agents [2]. To counteract the deleterious effects of DNA damage and faithfully duplicate their genome, cells have evolved an intricate network of genome care taking mechanisms, which include DNA damage signaling, cell cycle arrest, DNA repair, replicative senescence, and apoptosis, and which are collectively referred to as the DNA damage response (DDR) [3], [4], [5]. Malfunction of DDR pathways causes a mutator phenotype and predisposes to cancer, as well illustrated by genetic disorders like ataxia telangiectasia (AT; defective DNA damage signaling), xeroderma pigmentosum (XP; defective nucleotide excision repair), Fanconi anemia (defective cross-link repair), Li-Fraumeni, and familial retinoblastoma (defective cell cycle control) [6].

As cancer is the second leading cause of death in the Western society, and since pharmaceutical, chemical, cosmetic, and food industries are continuously developing new chemical compounds (adding up to the vast amount of natural environmental toxicants and carcinogens), it does not come as a surprise that our society demands such synthetic compounds to be tested for (geno)toxic and carcinogenic potential. To date, the only direct test for carcinogenic risk assessment of chemicals involves chronic exposure of rodents (or other animals, e.g. dogs) to various doses of the test compound (18–24 months; at least 50 animals per sex per dose) and analysis of tumor incidence and tumor type [7], [8]. As this chronic in vivo bioassay (apart from being time-consuming, expensive, and having limited sensitivity) uses large numbers of animals and has a severe impact on animal welfare, it also does not come as a surprise that the same society urges the scientific community to invest in the development of assays that reduce and refine, and preferably replace animal use (3-R principle).

An important step towards refinement has been made with the establishment of transgenic animal models such as the Xpa−/−/p53+/− mouse (deficient in nucleotide excision DNA repair and lacking one allele of the p53 tumor suppressor gene), which robustly responds in a very sensitive and discriminative manner to human genotoxic and non-genotoxic carcinogens within 9 months following initiation of treatment [9], [10]. More recently, the Xpa−/−/p53+/− mouse model has also been used in a short-term (14 days) exposure study, combined with a genome wide expression profiling using micro-array technology. This toxicogenomics approach allowed the identification of multigene gene-expression signatures as a highly predictive and discriminative biomarker for genotoxic and non-genotoxic carcinogens [11].

The ultimate aim is to entirely replace animal-based carcinogenic risk assessment assays by in vitro assays that make use of cultured tissue explants and/or cell lines, the latter requiring no animals at all. To this end, many efforts are undertaken worldwide to establish and validate transcriptomics-based high-throughput cellular assays for carcinogenic risk assessment. Evidently, in vitro culture systems have their own limitations and in vivoin vitro differences should be taken into account. For example, whereas in the intact organism, mammalian cells usually are exposed to oxygen levels ranging from 2 to 8% [12], cell culturing is often routinely performed at atmospheric oxygen levels (20%). Under such conditions, primary embryonic mouse fibroblasts will undergo a limited number of population doublings and enter a state of replicative senescence, after which some cells may escape and grow into a spontaneously immortalized cell line. This process is accompanied by an increase in spontaneous mutation frequency and a robust DNA damage response, which can be prevented by culturing the cells at 3% oxygen [13], [14], [15].

Another potential source of in vivoin vitro differences is the presence of an internal time keeping system, known as the circadian clock. Mammals are equipped with an internal light-entrained circadian clock that drives 24-h rhythms in metabolism, physiology, and behavior, and allows them to optimally anticipate the momentum of the day. Evidence is increasing that the intensity of adverse effects of exposure to environmental (e.g. pollutants) or therapeutic (e.g. anti-cancer drugs) genotoxic agents can depend on the time of exposure (e.g. morning vs. evening). The phenomenon of time-dependent changes in an organism's sensitivity to toxicants is referred to as chronotoxicity [16], [17]. Conversely, we have recently shown that genotoxic agents impinge on the circadian clock [18]. Below, following a general introduction on the circadian system, we will discuss the implications of the reciprocal link between genotoxic stress induced by DNA damage and the circadian clock, as well as its potential impact on in vitro genotoxic risk assessment assays.

Section snippets

The circadian system

The rotation of the earth around its axis imposes daily recurring changes to our environment, notably cyclic light-dark and temperature alternations. To anticipate to these solar day-night cycles, most organisms have developed an internal clock with a near 24-h periodicity, allowing them to optimally tune metabolic, physiological, and behavioral functions (e.g. the sleep-wake cycle, body temperature, blood pressure, hormone levels) to the special physiological needs the organism will have at

The circadian clock, cell cycle and DNA damage sensitivity

Interestingly, recent studies have shown that the cell cycle, as well as the DNA damage response that controls cell cycle progression under conditions of genotoxic stress, are connected to the circadian clock [5], [69], [70]. A circadian clock mediates an additional layer of control over cell cycle progression and it may well originate from the need of organisms to restrict replication of their genome (i.e. S-phase) to the moment of the day where the risk of exposure to environmental and

DNA damage and the circadian clock

Interestingly, we and others [18], [82] have shown that the connection between the mammalian clock and the DDR is reciprocal. When studying the response of cultured fibroblasts to DNA damage, we noticed to our great surprise that ionizing radiation is able to synchronize the circadian clock with an opposite phase angle, as compared to the rhythms elicited upon treatment of cells with known synchronizers such as serum shock, forskolin and dexamethasone (Fig. 2). This finding prompted us to

Consequences for in vitro genotoxic risk assessment assays

Evidently, our recent observation that DNA damaging agents can synchronize the circadian clock of individual cells in culture (and as a consequence the cyclic expression of clock-controlled genes, comprising up to 10% of the transcriptome) has implications for the development of transcription profiling based in vitro risk assessment assays.

Let us consider the set-up of a typical experiment for determination of the transcriptional response of cultured cells (e.g. fibroblasts, primary

Conflicts of interest

None.

Acknowledgements

This work was supported by grants from the Netherlands Organization of Scientific Research (ZonMW Vici 918.36.619), SenterNovem/Netherlands Genomics Initiative (“Netherlands Toxicogenomics Center”) and the European Community (“EUCLOCK” LSHG-CT-2006-018741) to GTJvdH.

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