The 4th dimension of in vitro systems - Time to level up.

Various in vitro model systems have been established over the last decades to understand physiological processes, the causalities of diseases and the response of humans to environmental and industrial chemicals or therapeutic drugs. Common to all is a limited biological significance due to the impairment of functionality, for instance by the lack of physiological 3D tissue architecture or the loss of fundamental regulatory mechanisms including the circadian rhythm. The circadian rhythm is an adaption of living organisms to rhythmic environmental changes of the day-night cycle and coordinates behavior as well as various crucial physiological processes in a 24-hour pattern. Here, we discuss the impact of integrating circadian regulation in experimental approaches and toxicological assessments to improve the biological relevance of the obtained results. In particular, it is known for some time that an ongoing disruption of the circadian rhythmicity is associated with an increased risk for cardiovascular disease, metabolic dysfunction or cancer. In the context of health recovery, the importance of circadian control mechanism is recognized by chronopharmacological concepts to increase the efficiency of pharmacological treatment strategies. Despite the undeniable circadian dependency and the biological relevance of manifold cellular and molecular processes, the impact of circadian regulation is hardly considered in a wide range of biomedical and toxicological research areas. Reactivating the circadian regulation holds the promise to enhance the biological relevance and reliability of in vitro approaches. In the context of human health protection the implementation of a circadian regulation will subsequently generate advanced physiologically relevant in vitro approaches and allows an improved toxicological assessment of health risks. In addition, the establishment of circadian disruption as a novel toxicological endpoint will provide a better understanding of toxicological mode of actions of environmental and industrial chemicals or drugs and enlarge the knowledge of disease development.

Restoring circadian synchrony in vitro facilitates physiological responses to environmental chemicals.

BACKGROUND: The growing requirement of hazard and risk assessment of environmental chemicals and the efforts to minimize animal testing, increases the demand for innovative and predictive in vitro test systems in toxicology, reflecting the physiological conditions of human nature. Here, an elemental factor regulating a variety of physiological processes is the day-night rhythm. This circadian rhythm, describing a biological oscillation with a 24-h period is hardly acknowledged in toxicology and test method development. Whilst, in animals or humans the entire organism exhibits a rigorous cellular circadian synchrony, in conventional in vitro systems each cell follows its own rhythm, due to the absence of appropriate synchronizing signals. OBJECTIVE: Here we investigated whether circadian synchronization of human cells in an in vitro system improves the cellular response and, thus, increases the sensitivity of the test system. Since the circadian regulation of metabolism is particularly well understood, and dioxin and dioxin-like compounds are of major concern for environmental health we focused on the ubiquitous drug metabolizing detoxification system mediated by the aryl hydrocarbon receptor (AHR). METHODS: To this end, we applied various prototypical AHR activators onto different human cell lines under non-synchronized or circadian synchronized conditions and determined the dose response on representative endogenous target genes. RESULTS: Remarkably, the cellular response dynamic upon chemical treatment was substantially enhanced in circadian synchronized cells and followed a rhythmic expression pattern. This broader dynamic range was associated with a strikingly higher induction of AHR target genes and the corresponding enzymatic activity, thereby rather mimicking the in vivo situation. CONCLUSION: Our findings indicate that a synchronized circadian rhythm in a cell culture based test system can improve the physiological relevance of an appropriate in vitro method by reflecting the biological in vivo situation more closely. Accordingly, it is a promising tool to facilitate the wide acceptance of in vitro methods in the field of regulatory toxicology and to further optimize the toxicological assessment of environmental chemicals.