Advanced Drug Delivery Reviews 62 (2010) 928945

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Advanced Drug Delivery Reviews

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a d d r

Disruption of the circadian system by environmental factors: Effects of hypoxia, magnetic elds and general anesthetics agents
Yvan Touitou a,, Olivier Coste b, Garance Dispersyn c, Laure Pain c
a b c

Unit de Chronobiologie, Fondation Ophtalmologique A. de Rothschild, 29 rue Manin, 75019-Paris, France Institut de recherches biomdicale des armes, Institut de mdecine navale (IRBA-IMNSSA), Toulon, France GRERCA, Inserm U666, Facult de Mdecine, 11 rue Humann, 67- Strasbourg, France

a r t i c l e

i n f o

a b s t r a c t
The biological clock of mammals is under the control of external factors, social life and the environment, and of internal genetic factors. When the biological clock of an individual is no longer in phase with its environment, either because there is no longer any harmony (desynchronization) between the two systems (shift work, night work, and transmeridian ights) or because the perception of signals in the environment is defective (blindness) or because of a pathology, disorders of the biological clock occur resulting in persistent fatigue, sleep disorders leading to chronic insomnia and mood disturbances that can cause depression. We review here new groups of factors that have been recently studied and that can be considered as potential disruptors of the circadian time structure. These factors are hypoxia, magnetic elds and anesthetic agents whose importance has to be considered. 2010 Elsevier B.V. All rights reserved.

Article history: Received 26 February 2010 Accepted 22 June 2010 Available online 6 July 2010 Keywords: Rhythm desynchronization Hypobaric hypoxia Electromagnetic elds General anesthetics Propofol Temperature circadian rhythm Melatonin circadian rhythm Cortisol circadian rhythm Transmeridian ight Jet lag Fatigue Circadian disruption

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Circadian synchronization and desynchronization . . . . . . . . . . 1.2. Marker rhythms of the circadian time structure . . . . . . . . . . . Magnetic elds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Melatonin and the pineal gland in animal models . . . . . . . . . . 2.3. Melatonin in humans . . . . . . . . . . . . . . . . . . . . . . . 2.4. Endocrine and neuroendocrine systems . . . . . . . . . . . . . . . 2.5. Biochemical and immuno-hematological variables in humans. . . . . 2.6. Omics technologies application to EMF research . . . . . . . . . . . Altitude and hypobaric hypoxia . . . . . . . . . . . . . . . . . . . . . . 3.1. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Inuence of hypoxia in animal models . . . . . . . . . . . . . . . 3.2.1. Tolerance and intolerance to hypoxia . . . . . . . . . . . . 3.2.2. Hypoxia effects on body temperature and locomotor activity. 3.2.3. Hypoxia effects on the SCN . . . . . . . . . . . . . . . . 3.3. Inuence of hypoxia in humans. . . . . . . . . . . . . . . . . . . 3.3.1. Hypoxia effects on body temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 929 929 930 931 931 931 931 932 932 932 933 933 933 933 933 933 934 934

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This review is part of the Advanced Drug Delivery Reviews theme issue on Chrono-Drug-Delivery Focused on Biological Clock: Intra-and Inter-Individual Variability of Molecular Clock. Corresponding author. Tel.: +33 1 47 02 32 05. E-mail address: yvan.touitou@chronobiology.fr (Y. Touitou). 0169-409X/$ see front matter 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.addr.2010.06.005

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3.3.2. Hypoxia effects on body temperature rhythm under constant routine conditions 3.3.3. Hypoxia effects on steroid hormones. . . . . . . . . . . . . . . . . . . . . 3.3.4. Hypoxia effects on rhythm markers: core body temperature and melatonin . . 3.3.5. Hypoxia effects on marker rhythms in long transmeridian ights . . . . . . . 3.4. Hypoxia effects on clock genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. General anesthetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Circadian timing and duration of action of general anesthetics agents . . . . . . . . . 4.3. Anesthetic effects on restactivity and core body temperature cycles . . . . . . . . . . 4.4. Anesthetic effects on hormonal secretions . . . . . . . . . . . . . . . . . . . . . . 4.4.1. Steroid hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.2. Melatonin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Anesthetics effects on clock genes . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Mammals are characterized by a temporal structure of complex biological rhythms covering a broad frequency spectrum (they are called infradian if the period N 28 h, circadian if = 2028 h , and ultradian if b 20 h), which are the object of multiple and highly complex interactions and which are present at all levels of organization: populations, individuals, organs and tissue cells, and sub-cellular organelles [1]. Circadian rhythms (period close to 24 h) are regulated in mammals by a main circadian clock in the suprachiasmatic nuclei (SCN) of the anterior hypothalamus. The SCN consists of a bilateral pair of small gray structures composed of about 10,000 neurons, located in the hypothalamus above the optic chiasma at the base of the third ventricle. Input pathways (e.g. light and social synchronizers) connect the circadian clock to the external environment while output pathways transfer circadian rhythmicity to physiologic, behavioral and biochemical parameters of the organism [25]. It receives the photoperiod signal directly from the retina by way of the retino-hypothalamic tract. The SCN is rhythmic in nature and the period of the rhythms generated in the SCN differs slightly from 24 h [6]. Its destruction in experimental animal models results in the disappearance of some circadian rhythms (melatonin, body temperature, sleepwake cycle), though not all of them, suggesting the existence of other biological clocks. A number of other clocks have been localized to several regions of the brain and to peripheral tissues, such as the liver, heart, muscle and kidneys [711]. These peripheral clocks, or at least some of them, appear to be coordinated by the SCN which is the only clock which directly receives the light-dark signal, the main synchronizer of the clock, but the hierarchization of clock functions and their interdependence are still poorly understood [2]. Coupling between the SCN and peripheral tissues might be achieved by nervous connections via the autonomic nervous system [4] or by humoral signals [3].

1.1. Circadian synchronization and desynchronization In humans, the two main synchronizers are the lightdark (LD) cycle and the sleepwake cycle, both of which drive our biological clock but do not create rhythms. Synchronization of biological rhythms is carried out through complex endogenous factors of genetic origin and by environmental factors or exogenous factors called synchronizers, of socioecological nature, such as the daynight alternation, the sleep wake cycle, the time of food intake in certain conditions etc. that entrain rhythms to 24 h [1]. In this respect, it is important to stress the signicance of sleep in the structuring of circadian rhythms [12], a function well demonstrated in humans through sleep deprivation experiments [13], as well as the importance of light in managing the human circadian system [14].

Certain circadian rhythms are more sensitive to exogenous factors than others. Depending upon the time a stimulus (e.g light) is applied, this stimulus can cause phase advances ( i.e. shifts of the peak time to an earlier time) or phase delays (i.e shifts of the peak time to a later time). This is named the Phase Response Curve (PRC) which corresponds to the phase shifts in the rhythm according to the time of stimulus presentation. The exogenous component of circadian rhythms thus plays an important part in the circadian rhythms of core body temperature, arterial blood pressure, pulse rate and bronchial diameter because the levels of these variables or functions are increased with physical and mental activities and decreased during sleep [1]. Similarly, we are aware of the role light plays in suppressing melatonin secretion [15], the effect of the timing of meals on particular circadian patterns [16] and the relationship between growth hormone (GH) and prolactin levels and sleep [in 1]. By contrast to the preceding examples, the circadian rhythms of plasma melatonin and cortisol are not very dependent upon exogenous factors; they are examples of rhythms with a strong endogenous component which make them major markers of the circadian synchronization of an organism [17]. As a nal result biological rhythms are of endogenous nature, genetically entrained by the synchronizers of the environment. When the body is no longer in phase with environmental signals, such as during isolation in caves or special laboratory facilities, all circadian rhythms persist but free-run with respect to environmental factors, meaning the period slightly differs from 24 h (slightly longer than 24 h, from 24.1 h to 25 h or sometimes shorter than 24 h) because the synchronizers of the environment no longer drive the period, and this is the reason that they are called circadian which means about a day [1]. This clock disorder leads to a desynchronization of the body because the resulting action of synchronizers on circadian rhythms to 24 h is no longer working. Desynchronization is therefore the expression of changes to the subject's normal synchronization, that is the temporal dissociation of biological clock functioning from that of the astronomical clock. It is either external when it is related to environmental factors such as jet lag [18] and shift work [19], or internal such as aging [20] and certain diseases [21]. Rhythm desynchronization occurs when there is a poor detection of the synchronizers by the biological clock which is observed in several situations: when synchronizers are completely suppressed, such as in experiments involving complete isolation from time references; when synchronizers are altered under conditions in which the biological clock and the environment run counter to each other, such as during transmeridian ights (jet lag), night work, and shift work where reversing or making major changes to the timing of our social life results in a dysfunctionning of the biological clock causing bodily disorders known as shift work intolerance [22]; when synchronizers are no longer detected, such as in blindness; in circadian disorders of the sleepwake cycle; and in a number of diseases involving modications of the rhythms or the appearance of a rhythm with an abnormal period or

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phase not found in healthy subjects such as depressive states, hormonedependent cancers i.e. breast, ovarian and prostate cancers [21], in alcoholic patients [23,24] amongst others. Regardless of the origin, desynchronization becomes manifest through atypical clinical symptoms, such as persistent fatigue, sleep disorders leading to chronic insomnia, and mood disorders that can cause depression and poor appetite [22]. 1.2. Marker rhythms of the circadian time structure A marker rhythm is a rhythmic variable characterizing the timing of the endogenous rhythmic time structure. It allows monitoring of the biologic timing of an organism and/or the timing of a related rhythm showing a xed time relation to the rhythm used as marker rhythm and provides information on the synchronization of indivi-

duals which is important for decisions about the timing of both treatment and diagnosis e.g. diseases and preventive healthcare [25]. Indeed marker rhythms can be altered in desychronized subject types as described above. A marker rhythm is a physiological variable with a prominent circadian rhythm such as body temperature, plasma cortisol whose circadian pattern is highly reproducible and reliable both on an individual basis and as a group phenomenon (Fig. 1) [17], plasma and salivary melatonin which are unaffected by masking factors (i.e. exogenous environmental factors masking the actual endogenous rhythms) other than bright light and which allow, when monitored during the rst hours of darkness using a dim light of 50 lx, the onset of evening rise to be dened by a marker known as Dim Light Melatonin onset (DLMO), white blood cells often used to follow the therapeutic effects of anticancer drugs, body temperature with its

Fig. 1. Circadian proles of plasma cortisol and melatonin in young healthy men. The circadian rhythm of the two hormones are highly reproducible from a day to another. Both are useful circadian markers of the time structure. From Selmaoui and Touitou [17].

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peak in the afternoon and a trough about 3 h before sleep onset, and ambulatory activity monitored using an actometer worn on the wrist. A marker rhythm is useful in the assessment of the rhythm synchronization of an organism and can be used as a tool in various circumstances for decision making e.g. time of sampling, timing of therapy, assessing therapeutic responses. The choice of a marker rhythm depends upon the aims of the research e.g white blood cells in cancer research, core body temperature in sports research, melatonin and cortisol in research dealing with shift work, though most often more than one marker rhythms is used to assess the rhythm synchronization of subjects under study. 2. Magnetic elds 2.1. Background The debate over the connection between electromagnetic elds (EMF) and cancer has become a hot issue in industrialized countries over the past years. The major sources of Extremely Low Frequency Fields (ELF) generation are electrical appliances, high power transmission and distribution lines. Due to the ubiquity of electrical appliances and apparatus in modern society, animals and humans live in an extremely complex electric and magnetic eld environment. Increasing concern in recent years about the effects of human exposure to electromagnetic elds (EMF) has been stimulated by a number of epidemiological studies reporting a possible link between magnetic elds and human diseases including leukemia [2629] and depression [30,31] although this soon gave rise to controversy [32,33]. Moreover, some in vivo studies suggest that magnetic elds may induce biological effects in organisms that could have deleterious consequences. In contrast to magnetic elds, there is little experimental or epidemiological basis for considering electric elds to be harmful for human health [34]. In this context the International Agency for Research on Cancer (IARC) classied ELF electric elds in category 3 which in the classication corresponds to inadequate evidence of deleterious effects and placed ELF magnetic elds in category 2B, corresponding to the category of agents that are possibly carcinogenic. It has to be noted that these extremely low frequency electric and magnetic elds are separate entities. 2.2. Melatonin and the pineal gland in animal models As early as 1963, the hypothesis was put forward that a decrease in the secretion of melatonin by the pineal gland might promote the development of breast cancer in humans [35]. The secretion of melatonin is known to be inhibited by light [15] which is the visible part of the EMF. The spectral sensitivity of the circadian system peaks between 450 and 480 nm. Moreover, the oncostatic properties of melatonin have been described [3537] as has its association with some depressive disorders [38,39] and with disorders of the circadian rhythm shown to generate neurobehavioral disturbances [40]. This resulted in the melatonin hypothesis as a tentative explanation for the occurrence of clinical disorders possibly related to exposure to EMF (50 Hz in Europe, 60 Hz in America) [41]. Exposure to EMF has also been shown to result in a reduction in pineal and plasma melatonin in various species and under photoperiodic conditions [4246]. The characteristics of the magnetic eld (linear or circular polarization), the animal species and, within a species, the strain have a role in determining the biologic response obtained. In order to compare short-term and long-term exposure effects, Selmaoui and Touitou [47] submitted rats to a 50 Hz sinusoidal magnetic eld during the night at different intensities for 12 h (from 14:00 h to 02:00 h) or repeatedly 18 h (from 14:00 h to 08:00 h) per day for 30 days. They found that a single 12 h exposure resulted in a signicant 30% decrease in melatonin levels and a 23% decline in pineal N-acetyltransferase activity (NAT), the key enzyme for

melatonin synthesis, only with the highest intensity used (100 T). The 30 days repeated exposure showed that while the 1 T intensity had no effects on pineal function, both 10 T and 100 T intensities resulted in a signicant 42% decrease in plasma melatonin levels; NAT activity was also decreased (Fig. 2) [47]. This study showed that the sensitivity threshold varies with the duration of exposure, thus suggesting that magnetic elds may have a cumulative effect upon pineal function [47]. No clear explanation exists for these various and contradictory results. A possible change in the spatial structure of the photoreceptor pigment rhodopsin due to the electric eld induced by the magnetic eld has been proposed. Magnetic elds might also change either the electrical activity of the pinealocytes or their ability to produce melatonin or both. 2.3. Melatonin in humans Research on the possible effects of magnetic elds in humans is important from a public health perspective since it is known that alterations in melatonin secretion (e.g. reduced amplitude or phase shift) are associated with clinical disorders such as fatigue, sleep and mood disorders, and altered vigilance, all of which are clinical signs of a desynchronization of circadian rhythms. We did not nd any effect on melatonin levels with short exposure of young healthy volunteers during night (9 h exposure) to a 50-Hz (10 T) magnetic eld [48]. This lack of an effect of acute exposure on

Fig. 2. Effects of chronic exposure of male rats to a sinusoidal 50-Hz magnetic eld ( from 1 to 100 T) on nocturnal pineal activity. The rats were exposed every day from 14:00 to 08:00 h for 30 days at three different intensities. Only 10 and 100 T were able to depress serum melatonin and pineal activity. No effect was observed on HIOMT activity. The asterisks indicate a signicant difference (p b 0.05) with control group (Ctrl). From Selmaoui and Touitou [47].

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melatonin secretion has also been reported by several authors [49 51]. These data, however, do not rule out a possible effect with chronic exposure. We therefore conducted a study on workers exposed on a daily basis to magnetic elds both at work and at home (EDF electrical workers) for 1 to 20 years and we showed that this exposure did not lead to alterations in their melatonin secretion (Fig. 3) [52]. All of the clinical signs reported in some studies of people living near electric lines or substations thus do not appear to be associated with an alteration in their melatonin levels. The difference in the effects observed in animals and humans might be related to differences in the anatomical conguration of the pineal gland and the nocturnal rhythm of rodent activity [52]. Different sensitivity to magnetic elds between species can also play a role in these differences as it is known that some species perceive magnetic elds differently [53] and it is possible that some subjects are more sensitive to magnetic elds than others.

2.4. Endocrine and neuroendocrine systems A relationship between the pineal gland and the adrenal gland has been documented in vitro [54] as has the relationship between the pineal and other glands of the endocrine axis, e.g. thyroid, pituitary and gonads [5557]. In fact, depending on the time of day a single dose of melatonin administered to pinealectomized rats markedly affects their nocturnal thyroid activity [58]. Similarly, melatonin administered late in the afternoon for 10 days inhibits thyroid growth in mice and rats [59]. These and other similar results [6062] suggest that the pineal gland may be related to thyroid activities. Considering this hypothesis in light of ndings that exposure to electromagnetic elds appears to attenuate the nocturnal melatonin increase in experimental animals [47,6365], we wondered whether and in what ways electromagnetic eld exposure might affect the endocrine and neuroendocrine systems. We therefore examined the effects of electromagnetic elds on the adrenocortical system and the hypothalamo-pituitary-thyroid (HPT) axis. We found that the circadian rhythm of the HPT and hypothalamo-pituitary-adrenal axis (HPA) functions was not affected by acute exposure (9 h) to either a continuous or an intermittent 50-Hz magnetic eld [66]. Likewise, the circadian proles of Thyroxine Binding Protein (TBG) and TBK which represents TBG capacity in exposed subjects did not differ from those of the sham-exposed: both peaked during the day and reached their lowest levels during the dark phase. These results are consistent with those of animal experiments that have not found any clear effect of electric elds on the hypothalamicpituitarythyroid and adrenal axes [6769]. 2.5. Biochemical and immuno-hematological variables in humans We evaluated in young healthy healthy subjects the effects of nocturnal acute exposure to both continuous and intermittent magnetic elds (50-Hz, 10 T) on the circadian rhythm of biochemical variables in an exhaustive study concerning proteins, lipids, enzymes, electrolytes, nitrogen substances. We found no effect of magnetic elds whatever the condition of exposure on any of the studied variables, neither on their circadian pattern nor on their plasma levels [70].We also documented under the same conditions and on the same subjects hematologic and immunologic functions (hemoglobin, hematocrit, platelets, red blood cells, leukocytes, monocytes, and lymphocytes subpopulations) (CD3, CD4, CD8, NK cells and B cells). The results we obtained do not indicate that the magnetic elds had any effect on these variables [71]. The consequences on long-term exposure are not well known in humans. In rats, exposure up to 120 days did not affect the hematological variables [72,73]. The measurement of biogenic amines and their metabolites in blood and urine has become rmly established as a diagnostic indicator for some types of cancer, including phaechromocytoma, neuroblastoma and related neurogenic tumors; they are also considered to be markers of stress. We evaluated the nocturnal levels of urinary biogenic amines (adrenaline, noradrenaline, dopamine, dihydroxyphenylalanine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid). None of these amines was affected by exposure to magnetic elds [74]. 2.6. Omics technologies application to EMF research The application of high-throughput omics technologies to investigate the inuences of EMF on biological systems has been recently reviewed in a paper underlining the heterogeneity among the biological materials investigated e.g. blood cells/vessels, tissue cells, nerves and bacteria which makes it difcult to compare data from different research papers on this topic and to arrive at denite conclusions on the potential effects of EMF on biological systems [75]. We will give here some recently published examples of difculties in comparing EMF exposure to such heterogeneous biological materials.

Fig. 3. Comparative nocturnal plasma melatonin proles (A) and 6-sulfatoxymelatonin concentration ( 6SM; B) in the rst-void morning urine (20:00 to 08:00). This study was carried out in 15 healthy chronically (in the workplace and at home) exposed men (daily and for 1 to 20 years) to a 50-Hz magnetic eld in search of any cumulative effect from those chronic conditions of exposure. 15 healthy unexposed men served as controls. As shown here the exposed subjects experienced no change in the hormone levels or circadian prole of melatonin. From Touitou et al. [52].

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The expression of 3, 5 and 7 nicotinic receptor sub-unit genes in the SH-SY5Y neuroblastoma cell, a major component of the nicotinic cholinergic system implicated in various neurological disorders, has been found to be unaffected by exposure to 50-Hz EMF [76]. The expression of PHOX2A, PHOX2B and of their target gene dopamine-hydroxylase was not modied by EMF in a human neuroblastoma cell line SH-SY5Y [77]. The transcriptional response of human umbilical vein endothelial cells to various patterns and intensities of 50-Hz EMF failed to produce regulated candidate genes [78]. Proteomic methods used to investigate the effect of EMF exposure on SF767 human glioma cells revealed signicant alterations in the sot density of a subset of treated cells [79]. Most breast tumors become resistant to tamoxifen and it has been shown that EMF reduce the efcacy of tamoxifen in a manner similar to tamoxifen resistance; by exposing cells of the breast cancer line MCF-7 to EMF it has been found that EMF alter the expression of estrogen receptor cofactors, which for the authors may contribute to the induction of tamoxifen resistance in vivo [80]. Thus it is clear that direct comparisons of data from studies using different products are difcult. In addition, replication and control experiments with alternative technologies are needed, using the same endpoints and in different laboratories, which is not yet the case [75]. 3. Altitude and hypobaric hypoxia 3.1. Background A peripheral decit in oxygen (O2) delivery is called hypoxia. This decit may be related to different causes. Altitude is one of the main environmental causes of hypoxia in healthy subjects. Altitude leads to a decrease in barometric pressure (PB) with a negative exponential relation, whereas the inspired O2 fraction remains unchanged. The decrease in PB in turn leads to a decrease in O2 partial pressure in alveolar, arterial and tissue compartments and nally determines a hypobaric hypoxia. This problem is crucial in mountaineering activities and also in aeronautics to protect subjects against the deleterious effects of acute hypoxia. Although the physiological consequences of hypobaric hypoxia have been extensively studied in the last past century, studies dealing with the inuence of hypoxia on the circadian time structure including clock genes are rather few in both animals and humans. 3.2. Inuence of hypoxia in animal models 3.2.1. Tolerance and intolerance to hypoxia The existence of a circadian and circannual rhythm of tolerance to hypoxia in mice submitted to brief (20 min) and repeated episodes of severe hypoxia (FIO2 = 5%) in mice was suggested as early as 1978 [81]. Subsequently, differences in hypoxic tolerance according to the time of the day in rats synchronized by Light/Dark (L/D) cycle = 12:12 and submitted to a 10,500 m simulated altitude were reported [82]. Survival time was twice longer in rats exposed to severe hypoxia at 4 HALO (i.e. 4 h after light onset, corresponding to a rest phase) than at 16 HALO (i.e. 4 h after the beginning of the dark phase corresponding to an active phase). These differences were interpreted as a result of circadian variations in the capacity to mobilize and use carbohydrates reserves. These data were corroborated in mice [83]. The study showed there was circadian rhythm in the cerebral resistance to hypoxia, which can be shifted by the time of food presentation. The survival time was indeed longer during the light period than during the dark period under constant hypoxia, when the mice were fed ad libitum. This rhythm was completely reversed by restriction of food presentation from 09:00 h to 15:00 h, i.e. during the light phase. A negative correlation found between the survival times and the levels of core body temperature and of glycemia suggested once again that

the rhythm of hypoxic tolerance was closely linked to the circadian rhythms of temperature and blood glucose in mice. All these results [82,83] suggest there is a phase opposition between hypoxic resistance rhythms on the one hand and core body temperature and blood glucose rhythms on the other hand. Resistance to chronic hypoxia is high during the light phase (i.e. the rest phase for the animals) with low levels of internal temperature and glycemia. On the contrary, resistance to chronic hypoxia is low during the dark phase (i.e. the active phase for the animals) with high levels of internal temperature and glycemia. A more recent study deals with the effect of acute hypoxia on ventilation and metabolism in newborn rats [84]. The authors showed that a brief exposure to normobaric hypoxia obtained by a decrease in the inspired oxygen fraction (FIO2 = 10 % for 15 min twice a day at 07:30 and 19:30 h) led to a disappearance of the nocturnal acrophase and a decrease in 24-h mean levels of ventilation (Ve) and aerobic metabolism (VO2). 3.2.2. Hypoxia effects on body temperature and locomotor activity In synchronized (L/D cycle = 12:12) adult rats submitted to a 5-day severe hypoxia (FIO2 = 10.5%), a disappearance of the circadian rhythm of temperature was observed [85]. Core body temperature rhythm reappeared by returning to normoxia without any phase shift. The amplitudes and 24-h mean levels of the circadian restactivity and energetic metabolism rhythms were dramatically decreased. In constant lightening conditions (L/L), which lead to free-running conditions for the circadian system, the circadian temperature rhythm also disappeared during similar hypoxic exposure and then reappeared without any apparent phase shift. The depression of circadian oscillations persisted with a bilateral sino-aortic denervation of the rats and was therefore considered independent of peripheral chemosensitivity [86]. All these results suggest that hypoxia had central effects more on hypothalamic thermic centres than on the circadian masterclock, classically located in the suprachiasmatic nuclei. Body temperature and locomotor activity were found to be differently affected, independently of the circadian clock, by a severe 63-h hypoxic exposure (F I O 2 = 10 %) in adult rats synchronized by L/D = 12:12 photoperiodic conditions [87]. Whereas body temperature presented several hypo and hyperthermic components, the activity level almost abolished at the beginning of the exposure progressively increased, but stayed at a lower level compared to the reference level [87]. A decrease in locomotor activity without alteration of the total duration of the active span in the golden hamster has been found when placed in constant darkness (D/D) and submitted to repeated severe hypoxic 3-h episodes (FIO2 = 8 %) for seven consecutive days at mid-subjective day for the animals (circadian time CT = 06:00-09:00) [88]. Placed in these conditions, the hamsters presented a cumulative phase delay of 46.4 min in their locomotor activity rhythm. The authors concluded that severe intermittent hypoxia may lead to a phase delay of circadian restactivity rhythm in free-running conditions, whereas no phase shift was reported in animal submitted to continuous severe hypoxia. Several factors may explain this difference: a continuous vs. intermittent hypoxic exposure, a masking effect which may act differently according to photoperiodic conditions of synchronization, a real phase delay which may be too small for being detected according to the sensitivity of the measures, and/or the statistical power of the experiment. 3.2.3. Hypoxia effects on the SCN Using a severe and longer duration (14 days) of continuous hypoxia (FIO2 = 10%) in rats alterations of the circadian rhythmicities of neurotransmitters in the SCN and the pineal gland [89]. Circadian variations of tissue concentrations of the vasoactive intestinal peptide (VIP), which plays a role in the output of the biological clock, were abolished in the SCN as were pineal 5-HIAA and serotonin which plays

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a key role in the synchronization of biological clock via nocturnal melatonin secretion. Moreover, a prolonged 8000-m exposure in a hypobaric chamber increased the expression of c-Fos (used as marker of neuronal activation) in the SCN of male adult Wistar rats [90]. Besides, the nitric oxide (NO) level generated by neuronal NO synthase (nNOS) increased in the SCN and may involve local hemodynamic changes (i.e. local vasodilatation leading to an increased blow ow) which may play a role in modulating the circadian rhythms as observed under hypobaric hypoxia at high altitude. These studies on animal model showed that continuous hypoxia alters the circadian proles of core body temperature and locomotor activity, with decreased 24-h mean levels and amplitudes [review in 91]. Phase shifts are not constantly observed though discrete phase delays have been reported for intermittent hypoxic exposure. This circadian effect of hypoxia may be unmasked in these experimental conditions by comparison with a continuous exposure. These alterations of the expression of circadian rhythms may be the impact of hypoxia on the SCN, considered as the main circadian clock. 3.3. Inuence of hypoxia in humans 3.3.1. Hypoxia effects on body temperature Few studies deal with the effects of hypoxia on human circadian time structure. Historically, Ashkenazi et al. in 1982 showed that a brief acute hypobaric hypoxia (2-3 min at the simulated altitude of 25,000 ft (~7600 m)) induced a phase delay of oral temperature, hand grip, and cognitive tasks rhythms in 3 pilots [92]. These delays persisted for 4 days after exposure (Fig. 4). Interindividual differences were found, during an expedition to Antarctica's mountains, in the adaptation ability of some variables (heart rate, body temperature, salivary sodium and potassium) of human circadian rhythms in response to changes in the photoperiod [93]. The circadian phase and amplitude of the studied rhythms were altered in subjects presenting a low resistance to hypoxia at the 30th day of the expedition with appearance of an ultradian component around 12 o'clock. By contrast, the circadian pattern was unaltered in subjects presenting a high resistance to hypoxia. However, some weeks later in the middle of the polar winter (i.e. during the polar night equivalent to constant darkness conditions), all subjects presented alterations in their rhythms, with the appearance of ultradian components [93]. 3.3.2. Hypoxia effects on body temperature rhythm under constant routine conditions Hypobaric hypoxic exposure might not be the unique factor involved in the circadian alterations observed in the two last studies performed in ecological conditions. Indeed, the effects of continuous hypoxia were examined during a controlled laboratory longitudinal study, using a constant routine protocol (total sleep deprivation in a standardized semi-recumbent posture, snacks every 2 h, under constant dim light conditions of 200 lx) in a climatic room (25 C) [94]. Normoxic exposure (FIO2 = 21%) as the standard was followed by hypoxic exposure (FIO2 = 13% obtained by adjunction of nitrogen in the climatic room, corresponding to an equivalent altitude of 3600 m), then by normoxic exposure for recovery evaluation (total of 3 experimental 28-h sessions). A marked decrease of the circadian amplitude of core body temperature rhythm and a profound alteration of the circadian rhythm of O2 consumption were observed whereas heart rate and arterial blood pressure rhythms remained unchanged. These results are concordant with studies on rodents reported above [86]. Finally, the authors conclude that the circadian alterations observed in humans may contribute to sleep disturbances observed by sleeping in altitude [94].

Fig. 4. Acute hypoxia exposure as phase shift inducer (mean times of acrophase occurrence SD). Daily shifts were observed in the hour of highest level of some physiological parameters on the left part and of the time needed to accomplish some cognitive tasks on the right part. Black arrow indicate day of exposure. From Ashkenazi et al. [92].

3.3.3. Hypoxia effects on steroid hormones The plasma cortisol circadian prole was not modied in the course of 79-h exposure at an altitude of 4350 m in a group of climbers, except for an increase in the 24-h mean level [95]. Nevertheless, the cycle of cortisol was established with only four measures per day which does not allow precise detection of phase changes. In our laboratory, we recently showed that plasma cortisol 24-h cycles were altered by diurnal 8-h exposure to mild hypobaric exposure, simulating the conditions during a prolonged ight in a pressurized cabin [96]. An initial drop in plasma cortisol was observed under hypobaric hypoxia with a rebound of secretion just after the hypoxic exposure, whereas the phase and the 24-h mean levels remained unchanged (Fig. 5). By contrast, testosterone and gonadotrophin patterns were not signicantly altered in the same acute experimental conditions [97]. 3.3.4. Hypoxia effects on rhythm markers: core body temperature and melatonin We documented the effects of diurnal 8-h hypobaric exposure on circadian markers (body temperature, plasma melatonin, and urine 6sulfatoxymelatonin) and on sleep through studies at two simulated altitudes i.e. 8000 ft (~2400 m) and 12,000 ft (~3600 m), corresponding to maximal cabin altitudes for pressurized large cabins xed by aeronautical regulations in civil and military aviation respectively.

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Fig. 5. Alterations of cortisol secretion circadian patterns during altitude exposure. An initial fall of plasma cortisol was observed under hypobaric hypoxia with a rebound of secretion just after the hypoxic proof, whereas the phase and the 24-h mean level remained unchanged. From Coste et al. [92].

Cabin altitudes of civil large aircrafts usually reach 5000 to 6,000 ft in the ancient planes, and nearly 8000 ft in recent ones. By contrast, military large aircrafts are still less pressurized. A delay in the evening decline of core body temperature was initially observed mainly during the 12,000 ft exposure (Fig. 6) [98]. In a second experiment, this delay persisted also during recovery, suggesting a real phase delay and not only a transient physiological effect of hypoxia on body temperature (Fig. 7) [98,100]. In parallel, a decrease in the nocturnal peak of plasma melatonin was initially described (Fig. 8) [99] followed by a signicant decrease in nocturnal urine 6-sulfatoxymelatonin values during recovery (Fig. 9). The alterations were particularly marked in the youngest subjects and were once again compatible with a phase delay of melatonin in response to hypoxic exposure [99,100]. Moreover, no signicant alterations of immune circadian proles (immunoglobulins A, G and M and CD4 CD8 lymphocytes counts) were detected in the same experimental conditions [101]. All these results strongly suggest

that such exposure leads to a real phase delay of about 1 h, with a moderate but signicant impact on recovery sleep. We therefore concluded that prolonged mild hypoxia, acting on the circadian time structure, may contribute to post-ight fatigue, independently of the jet lag phenomenon related to quick crossing of several time zones during a transmeridian ight [102]. Some data in rodents indicates that the functioning of the SCN and the pineal gland, main actors of the circadian system, are impacted by hypoxic exposure [88,89] and very recent genetic studies on clock genes contribute to providing novel clues concerning the role of hypoxia in the circadian system.

3.3.5. Hypoxia effects on marker rhythms in long transmeridian ights As described above, the effects of mild hypobaric hypoxia on circadian markers (core body temperature, plasma cortisol, and plasma melatonin) were studied in aeronautical settings [98102].

Fig. 6. Effects of an 8-h diurnal exposure to mild hypoxia on core body temperature circadian patterns. A delay in the evening decline of body temperature was initially observed mainly during the 12,000 ft exposure, suggesting a physiological effect of hypoxia on body temperature or a possible phase shift of temperature rhythm. From Coste et al. [98].

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Fig. 7. Effects of an 8-h diurnal exposure to mild hypoxia on core body temperature circadian patterns during recovery. Delayed evening decline of body temperature and delayed shift of thermal trough observed during both altitude exposure and during recovery suggested a real phase delay of body temperature rhythm. From Coste et al. [98].

Long transmeridian ights provoke clinical complaints in passengers and crewmembers, mostly commonly fatigue, sleep and mood disorders, and occasionally digestive signs. These symptoms are related to the desynchronization of the body's circadian rhythms and make up a symptom complex referred to as jet lag [review in 103]. Most studies dealing with the biological attributes of jet lag and circadian time structure in humans do not take into account any other ight factors [104,105]. Moreover, post-ight fatigue in pilots, associated with a decrease in cognitive performance, has been described after long ights, regardless of whether time zones are crossed, and in this regard no differences have been found between transmeridian and northsouth ights [106110]. Environmental cabin factors may therefore be involved and contribute to post-ight fatigue by altering circadian patterns independently of the number of time zones crossed. Of these factors, hypobaric hypoxia may be especially important. Moreover, the effects of mild hypobaric hypoxia may have been different with other clock time exposures, like with a night exposure for example. 3.4. Hypoxia effects on clock genes In the nineties, a so-called Clock gene was described in the mouse suprachiasmatic nuclei [111,112]. This gene encoded a

protein containing basic helixloophelix (BHLH) and PAS (PER ARNTSIM) domains [113]. The PAS domain is an important structural feature for the genes involved in circadian rhythmicity, whereas basic helixloophelix transcription factors BHLHB2 (also referred as DEC1/Eip1/SHARP-2/Stra13/Clast5) and BHLHB3 (also referred as DEC2/SHARP-1/SHARP1) are regulated by many extracellular stimuli, and particularly by hypoxia [114]. BHLHB2 and BHLH3 are known to play pivotal roles in multiple signalling pathways, including cell differentiation, growth cell, apoptosis, oncogenesis, immune system, circadian rhythms and sleep homeostasis. Indeed, the Fu group recently published results dealing with a specic mutation of DEC2 linked with the very short-sleeper phenotype [115]. Multiple members of the basic helix-loop-helix/PAS including clock genes and hypoxic-inducible factor-1 alpha (hif-1) family were found to be expressed in the suprachiasmatic nuclei, suggesting a rich array of potential interactions relevant to the regulation of the suprachiasmatic circadian clock [112]. Moreover, it has been shown that exposure to hypoxia leads to increased PER1 and CLOCK protein levels in the mouse brain [116]. Based on co-immunoprecipitation experiments showing a protein-protein interaction between PER1 and sub-unit of HIF-1, it has been suggested that hypoxic effects observed on clock genes may be

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Fig. 8. Effects of an 8-h diurnal exposure to mild hypoxia on plasma melatonin circadian patterns. Decrease of plasma melatonin nocturnal peak at both studied altitudes was observed, especially in the younger subjects. From Coste et al. [99].

modulated by HIF-1 [116]. A real cross-talk seems to exist between hypoxic and circadian pathways with a cooperative role for HIF-1 and CLOCK proteins in transcriptional activation of target genes like the vasopressin gene [117]. Hypoxic broblasts showed an alteration of Stra13 and Dec2 circadian gene expression, two circadian transcriptional regulators that are overexpressed and no longer rhythmic in hypoxic broblasts [118]. This effect is associated with a loss of circadian expression of the clock genes Rev-erb and Bmal1 and the clock-controlled Dbp because STRA 13 and DEC2 proteins both antagonize CLOCK: BMAL1 dependent transactivation of the Rev-erb and Dbp promoters. Moreover, an intermittent hypoxia, like that occurring in sleep apnea syndromes may alter clock gene expression via an inammatory mechanism, i.e. an increase in interleukin-6 (IL-6) [119]. All these studies suggest that hypoxia may act on specic transcriptors, which impact the expression of circadian clock genes. Thus, hypoxia has not only physiologic effects but also a circadian effect. This latter effect may also depend on the clock time of exposure, as many other synchronizers also called Zeitgebers. Finally, the fact that DEC2 is also involved in sleep

length control in mammals may explain that hypoxia may have some consequences on recovery sleep after hypoxic exposure via a circadian effect, as recently described [98100]. 4. General anesthetics 4.1. Background Clinical research on circadian rhythms in patients after their surgery suggests that both general anesthetics and surgery can alter the circadian time structure since patients complain of fatigue and sleep disorders suggestive of a disturbance of the circadian restactivity cycle [120]. General anesthesia can be described as a pharmacologic state involving amnesia, immobility, unconsciousness, and analgesia with the aim of creating a state of sensory deprivation to induce a lack of motor reaction to stimuli and to obtain explicit amnesia [121]. Two kinds of general anesthetics are commonly used in human surgery practice: intravenous agents (propofol, ketamine, etomidate, and thiopental) and inhaled gases (sevourane, isourane, halothane, and desurane). In the days following general anesthesia, patients

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Fig. 9. Urinary patterns of 6-sulfatoxymelatonin (6-SM) of healthy subjects of two age groups during three consecutive 24 h cycles D0 (reference), D1 (diurnal hypoxic 8-h exposure from 08:00 to 16:00 h), and D2 (recovery) under two experimental conditions (8000 ft and 12,000 ft). The urinary excretion of 6-SM was expressed as a ratio of the urinary excretion of creatinine (g of 6-SM/mmol of creatinin). A signicant decrease of nocturnal excretion of 6-sulfatoxymelatonin occurred only in the 2228 year group whereas the excretion remained stable in the 2938 year group. From Coste et al. [100].

often complain of sleep and mood disorders, and loss of vigilance. Since anesthesia is not uncoupled from surgery, it is difcult to dissociate the proper effects on the circadian time structure of general anesthetics from those of surgery which include amongst others pain, tissue injury, surgical stress and immobilization in bed. The disruptive effects of general anesthesia on circadian time structure might have considerable consequences in humans and sustain post-operative wakesleep disorders.

important to take it in account in any the research dealing with the effects of general anesthetics on biological rhythms. 4.3. Anesthetic effects on restactivity and core body temperature cycles It was rst shown in rats studied in constant darkness( which is a condition reecting the endogenous component of the clock) and anesthetized near the restactivity transition point with propofol that the drug induced a 1-h phase advance of the rest activity rhythm in these specic laboratory conditions [125]. We thus found it worthwhile to examine whether general anesthesia with propofol can impact the rat circadian temporal structure by disturbing circadian restactivity and body temperature rhythms under normal lightdark conditions closer to light/dark conditions in humans (lightdark 12:12 h). Propofol was administered at three different Zeitgeber times: ZT6 (middle of the rest period), ZT10 (2 h before the beginning of activity period), ZT16 (4 h after the beginning of activity period). Zeitgeber is a chronobiologic term which can be dene as follows: under standard lightdark cycles (12 h of light/12 h of darkness), the time of lights on denes Zeitgeber time zero (ZT 0) and the time of lights off denes Zeitgeber time twelve (ZT 12). We found that on the day after anesthesia, propofol induced a signicant 60- to 80-min phase advance of both restactivity and body temperature rhythms and that this shift persisted the second day after anesthesia [130]. In addition, the amplitudes of both restactivity and body temperature rhythms were decreased on the rst and second days after anesthesia. These results were obtained

4.2. Circadian timing and duration of action of general anesthetics agents It is well-established in rodents and rabbits that the efcacy of anesthesia was better and/or the duration longer with anesthetic administration during the rest span compared to activity span for pentobarbital [123], ketamine [124] and more recently for propofol [122,125]. This daynight difference in the effect seems to be independent of liver cytochrome P-450 content and activity [126] which may suggest that these differences in drug-effects are related to circadian differences in the sensitivity of the central nervous system, though circadian rhythms in liver enzymes involved in drug metabolism may also play a role [127]. Variations in the duration of action of general anesthetics could also be related to circadian variations in the post-synaptic GABA receptors, peak activity and maximal receptor-binding afnity of which occurs during the rest span [128,129] . Whatever the endogenous origin of this daynight difference in the effects of anesthetics, it is

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whatever the time of administration of propofol. However, the effects are more important at ZT10 than at ZT6 and ZT16. Indeed, the phase shift induced by propofol at ZT6 and ZT16 is around 60 min whereas it is around 80 min when propofol is administered at ZT10. These data suggest that general anesthesia may act as an external factor that could disturb the circadian time structure. Ketamine anesthesia during the rest period (ZT3/ZT4) has also been shown to perturb the circadian rhythms of both body temperature and general locomotor activity on the day of anesthesia but with a return to basal values on the day after anesthesia [131]. In recent studies by our laboratory on the effects of propofol on the circadian time structure in humans, we aimed at looking for the effects of this anesthetic on patients, in their real-life conditions, without any surgery [132]. Our patients received short-duration general anesthesia with propofol for ambulatory colonoscopy, a medical act without surgical intervention, and went back home some hours after anesthesia. The propofol anesthesia for colonoscopy was performed between 8 a.m. and noon. We showed that diurnal rest was increased on the day of anesthesia and the following 24 h whereas nocturnal sleep was unchanged [130]. We also showed, using non-parametric analysis, a decrease in the strength of coupling of the rhythm to supposedly stable environmental factors and increased fragmentation of the rhythm after anesthesia. The restactivity rhythm was less strongly coupled to Zeitgebers in the days following anesthesia with a decrease in interdaily stability parameters (the strength of coupling the rhythm to supposed stable environmental Zeitgebers) and increased interdaily variability (the extent and transition between rest and activity) during the 72 h following general anesthesia [132] (Fig. 10). We demonstrated here that synchronization to local time is impaired for some days by light anesthesia with propofol. In our study and in the literature, anesthesia was performed in the morning; thereby we still do not know if the anesthesic effects on circadian rhythms are dependent of the treatmenttime because there is no data on anesthesia performed during the night in humans. Whether such effects are also present with other anesthetics is unknown though a recent study on sevourane (administered during the rest period) showed a decrease in the expression of the clock gene per2 which is involved in the regulation of the clock in rodents [135]. Among the few studies focusing on the effects of general anesthesia on restactivity and body temperature rhythms, it has been shown, in healthy human volunteers, that isourane (at subanesthetic dose given between 9:30 and 10:30 a.m.) decreases the temperature circadian amplitude on the day of anesthesia without any phase-shift [136]. These observed effects could be due in fact to a residual decrease activity induced by anesthesia in the following hours and a subsequent decrease in the heat production.

4.4. Anesthetic effects on hormonal secretions 4.4.1. Steroid hormones In humans, it has rst been shown that the levels of plasma cortisol, a glucocorticoid hormone synthesized by the adrenal cortex exhibiting marked circadian variations [137,138], are decreased during the time of a surgery (in the morning) using propofol as an anesthetic [120]. Evidently, this does not allow the actual effects of general anesthesia to be separated from the surgical act itself. Contradictory data have been published on the effects of sevourane on cortisol secretion after operation which was found to be either increased for up to 12 h after surgery [139] or decreased 2 to 4 h after surgery [140]. These contradictory data may be explained by differences in patients' recruitment, timing of anesthesia and surgery, drug administration before surgery and the age of patients, amongst other factors. The type of surgery (cataract, cardiac surgery, haemorrhoidectomy..) may also be involved in these differences. With regard to laboratory animals, propofol (administration time not precised) was shown to increase the secretion of corticosterone (the main glucocorticoid in rodents), soon (5 min) after the injection with a parallel increase in B-endorphins which suggested that propofol stimulates the release of both CRF and ACTH resulting in an increased corticosterone secretion [141]. Different and contradictory data have been described with two other anesthetics, i.e. ketamine and thiopental, both of which did not affect corticosterone secretion in rabbits during anesthesia but in contrast increased hormone secretion during the rst 60120 min recovery period [142]. The inhibitory effects of propofol on adrenal steroidogenesis have been evidenced in vitro during the rst step of steroid synthesis [143] between cholesterol and pregnenolone. We recently showed in rats that general propofol anesthesia independent of the time-of-day of its administration (injections at ZT6, ZT10 and ZT16) induces a dramatic increase in corticosterone secretion during the early recovery period without effect on ACTH secretion which excludes a primary stress-like activation of the hypothalamo-pituitary-adrenal axis [133] (Fig. 11). Since propofol is one of the most commonly used anesthetics, its effects on the biological system is of particular relevance and it is of interest to note that a link between post-operative high levels of plasma cortisol and post-operative cognitive dysfunction has been suggested [144]. 4.4.2. Melatonin General anesthesia and surgery are associated with sleep disorders during the post-operative period. As shown above, general propofol anesthesia impacts the circadian time structure and particularly the restactivity cycle in rats [130]. Since interactions between melatonin and the restactivity cycle are well known [145], we examined very recently in our laboratory general propofol anesthesia effects on melatonin secretion in vivo on the circadian prole of plasma melatonin [134]. For the in vivo experiments, rats were exposed to L/D 12:12 conditions and anesthetized with propofol around their peak of melatonin secretion (Zeitgeber Time16) and trunk blood samples were collected according to 7 Zeitgeber Times to assess propofol effects on circadian melatonin secretion. We showed that in vivo propofol disrupts melatonin by signicantly decreasing its secretion (2228%) immediately after the wake up from anesthesia and then increasing signicantly melatonin secretion 20 h after anesthesia (38%) (Fig. 12) [134]. The results suggest that this increase may be due to a shift in the circadian rhythm of melatonin which is coherent with our data of a phase advance of the rest-activity and temperature rhythms in rats given propofol [130]. Propofol acts via a positive modulation of the inhibitory function of the neurotransmitter Gamma-aminobutyric acid (GABA) through type-A GABA receptors which are expressed in many brain areas, including the outputs from SCN [146]. GABA seems to be involved in transmitted signals from the SCN to the paraventricular nucleus stimulation of melatonin synthesis

Fig. 10. Changes in the interdaily stability (IS) and intradaily variability (IV) parameters (expressed as arbitrary units (mean value SD)) for the 72 h period following propofol. From Dispersyn et al. [132].

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Fig. 11. 24 h propofol anesthesia effects on corticosterone and ACTH secretions. (A) 24 h prolonged effects of a single propofol or control injection on rats (n = 72) corticosterone concentrations at one injection time (ZT16). (B) 24 h post propofol anesthesia or intralipids effects on ACTH concentrations at one injection point (ZT16). For (A) and (B), values are mean SEM. Asterisks denote signicant differences (p b 0.05) to mean values between propofol and Intralipids treatment. From Dispersyn et al. [133].

from the pineal gland. The effects of GABA infusion are dependent upon the circadian stage [147]. Recent data showed that GABAergic agonists administered during the mid-subjective day induce a phase advance of circadian rhythm and modify the expression of clock genes per1 and per2 within the SCN [148,149]. This result has been obtained only at one circadian time of GABAergic agonist administration, so we do not know if this result can be obtained at other circadian times. One hypothesis is that propofol, a GABA agonist, has an inhibitory effect on the outputs from SCN to the paraventricular nucleus. The alternative mechanism might be a direct effect on clock genes expression within the SCN, inducing a phase advance of the circadian rhythms. Cosinor analysis (a procedure for the analysis of biological rhythms based on the tting of a cosine wave to the raw data) suggested that propofol anesthesia induces a phase advance of the circadian secretion of melatonin. Our results demonstrate that melatonin secretion is disrupted during the 24 h following propofol anesthesia. In conclusion of all the animals and humans studies, it appears that general anesthesia induces a desynchronization of the circadian time structure during the post-anesthesia period. The importance of the desynchronization is dependent on the time of administration of the general anesthetic. Indeed, the desynchronization of the circadian rhythms is bigger when the anesthesia is performed during the rest

period than during the activity period. However, the precise cellular mechanisms involved in circadian clock desynchronization induced by general anesthesia remain unknown. 4.5. Anesthetics effects on clock genes The circadian clock (suprachiasmatic nucleus, SCN) is regulated at the gene level. The transcription of clock genes generates oscillations; these are generated by an auto-feedback loop system in which the transcription of clock genes is suppressed by clock gene products that play a central role in oscillation [150]. The main clock genes identied at this time are Period (Per), Cryptochrome(Cry), Clock and BMAL1. To date, few data on the effects of general anesthesia on clock genes as well in human than in animal model are available. However, some studies show that general anesthetics (inhaled gases as well as intravenous) impact the expression of several clock genes in rat brains during rest period [150152]. Firstly, concerning inhalation anesthetics, it was shown that sevourane impacts the expression of clock gene Per 2 by decreasing its expression level in the whole brain when rats are anesthetized during light period (6-h anesthesia), even if it has no impact on the other clock genes studied (Per1, Clock, Bmal1, Per3,

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Fig. 12. . Effects of propofol anesthesia or control treatment on melatonin secretion at one Zeitgeber time of injection (ZT16) on peripheral melatonin secretion. Values are given as mean SD. * Signicant differences (P b 0.05) to mean values between propofol and control. From Dispersyn et al. [134].

Rev-erb-alpha) [152]. These data were collected in rats still anesthetized under 6-hour administration of sevourane. Unfortunately this study provides no data on the long-lasting effect of sevourane on Per2 expression level after awakening from anesthesia. However, another study showed that the Per2 gene expression level was decreased 24 h after anesthesia with sevourane during the rest period in rat whole brain [151] and thus demonstrated a long-lasting effect of anesthesia at the molecular level. The issue with these two studies is that the results are obtained on whole brain and do not allow the localization of gene expression. Thus the differences in gene expression do not reect the expression levels of these genes in the suprachiasmatic nucleus (SCN), which is the primary circadian pacemaker in mammals. Secondly, concerning intravenous anesthetics, a similar experimental approach showed that a 6-hour administration of propofol during the rest period decreased Per2 gene expression levels in rat whole brain [150]. Dbp and tef (composites of the core oscillating loops in the SCN) brain expression levels were equally decreased by propofol anesthesia. The issue in this study is the same as that in the two previous ones, as data were obtained on the whole brain. Indeed, the authors acknowledge that the data obtained can result from the summation of both suppression and activation in different neurons and do not reect the expression of these genes in SCN [150]. Finally, opioids are often used in combination with anesthetics, to set a balanced anesthesia, and appear to modify the photic responsiveness of the circadian pacemaker as assessed by clock genes. Indeed, the administration of the opioid fentanyl decreased light-induced Per1 gene expression during night in Syrian hamsters in the SCN [153]. The effects of anesthetics associated with opioids remain unknown. In conclusion, inhaled as well as intravenous general anesthetics act on the main clock genes like Period gene even if studies are rare and performed on the whole brain of rats, and not yet on the SCN. 5. Conclusions and perspectives Most living beings change their behavior on a daily basis (24-h), with rhythmicity a fundamental property of living matter. Biological

rhythms are characterized by their ability to be entrained by external environmental cues, mostly consequences of the earth's revolution around its axis (lightdark cycle). For living organisms, the L/D cycle is transposed in biological representations, the circadian rhythms, genuine mirrors of an adaptation feature to constantly changing surroundings. Many biological functions show circadian rhythms, that is their temporal variation may be considered as a cyclic function with a periodicity ranging between 20 and 28-h. The persistence of a circadian rhythmicity in an environment without any known external time cues suggests that there is an internal time-keeping system called the biological clock which, in mammals, is the SCN that is responsible of many aspects of circadian rhythmicity [13]. The daily rhythmicity is thus the result of the combined action of the endogenous biological clock(s) and environmental time cues. Our organism is synchronized when it works in harmony (in synchrony) with environmental factors called synchronizers which include the lightdark cycle, the sleepwake cycle, meal schedules, and seasonal factors related to modications in photoperiod and outside temperature. To ascertain whether an organism is synchronized (or desynchronized), we need the use of circadian markers allowing the circadian time structure of this organism to be determined. Motor activity, body temperature, plasma cortisol and melatonin patterns are important marker rhythms. Locomotor activity and core body temperature are often used in experimental protocols [154156]. The circadian rhythm of body temperature is generated by an endogenous component but is also dependent on motor activity [157,158]. Cortisol and melatonin are also among the main biological marker rhythms. The circadian rhythm of cortisol is driven by the endogenous oscillator situated in the hypothalamic suprachiasmatic nuclei (SCN) [159]. Entrainment of the cortisol rhythm by the lightdark cycle is mediated by the eyes and transmitted to the SCN, which drives the basal hypothalamopituitary-adrenal (HPA) rhythmicity. The circadian rhythm of melatonin is also generated by the SCN [159] and entrained by the lightdark cycle via the retino-hypothalamic tract. This raises a question about the relation between the rhythms of melatonin and cortisol. This relation has been studied in blind subjects and seems to be highly correlated [160]. We found in healthy subjects that those subjects with a slightly variable melatonin circadian rhythm

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had reliable cortisol rhythms. Although the circadian rhythms of these hormones are endogenously generated by the SCN, numerous external factors can affect the stability of their rhythmicity [161]. The use of marker rhythms allows investigation of rhythm desynchronization which occurs when the biological clock is no longer in step with its surroundings, in situations such as jet lag, shift work, night work but also in some (but not all) elderly people, in some diseases including depression and cancer and in totally blind persons [review in 1]. 24-h measurements of such marker rhythms as motor activity, body temperature, plasma cortisol and melatonin provide information on the rhythm desynchronization of individuals and are thus important from a physiologial, pharmacological and therapeutics points of view. Indeed, marker rhythms can be altered in all those circumstances linked to environmental disruption but also in circumstances not directly related to the environment like those observed with endogenous stress e.g. due to depression, blindness, aging desynchronization or with the use of certain medications. In this review we have studied three different kinds of factors able to provoke a disruption of the circadian time structure, namely magnetic elds to which everybody is exposed throughout the year, hypobaric hypoxia encountered in ight, and general anesthetics agents currently used for surgery. External desynchronization linked to jet lag has been the object of numerous studies. Long duration transmeridian ights cause a number of clinical problems in passengers and crew, who complain mostly of fatigue, sleep and mood disorders and sometimes of gastrointestinal symptoms, which all are considered to be related to circadian desynchronization [review in 103]. Most studies which have examined the biological effects of jet lag on circadian rhythms in human beings have not taken any other ight factors into account, though it has been shown that post-ight fatigue is associated with decreased cognitive performance described after long haul ights, independently of the time zones crossed [106110]. Environmental cabin factors may therefore be involved and contribute to post-ight fatigue by changing the circadian time structure independently of the number of time zones crossed. Recently, we showed that an 8-h diurnal exposure to hypoxia at 12,000 ft may induce a transient phase delay of circadian core body temperature rhythm in young healthy men [98100]. We also found that exposure to mild hypoxia changes the expression of cortisol circadian pattern, with an initial fall in cortisol and a secondary rebound of secretion which followed the alterations in autonomic balance assessed by heart rate variability, and signicantly decreased the nocturnal peak of melatonin, the latter effect being agedependent. In animal models, we showed that continuous hypoxia alters the expression of circadian markers, core body temperature and locomotor activity, with decreased 24-h mean levels and amplitudes [review in 91]. Phase shifts are not constantly observed though discrete phase delays have been reported for intermittent hypoxic exposure. These alterations of the expression of circadian rhythms may be the impact of hypoxia on the SCN. Some data in rodents indicates that both the SCN and the pineal gland, main actors of the circadian system, are impacted by hypoxic exposure [88,89] and very recent data on clock genes contribute to strongly suggest a role of hypoxia in the circadian system. Independently of the number of time zones crossed i.e. independently of jet lag, environmental cabin factors may therefore be involved in postight fatigue by altering circadian time structure. Of these factors, hypoxia may be especially important [102]. The debate is, however, still open between a physiological effect of hypoxia leading to alterations of circadian rhythms and a real impact of hypoxia on circadian structure itself. During the past 20 years several laboratories have explored the possibility that EMF induce biological effects in search of an

explanation to some (though not all) epidemiologic studies reporting an association between exposure to EMF and the incidence of cancer [162]. Numerous studies have looked for evidence that EMF have genotoxic or epigenetic activity. These studies have found no replicated evidence that EMF have the potential to either cause or contribute to cancer [163]. Melatonin, a neurohormone produced by the pineal gland has been shown to have oncostatic properties. The decline in melatonin secretion has therefore been put forward in some epidemiologic studies as another possible mechanism. With regard to magnetic elds, we showed huge differences in their effects in humans when compared to rodents. Using a short 50 Hz exposure we found a signicant decline with the highest exposure (100 T) of rat plasma and pineal melatonin and a decreased activity of rat pineal NAT, the key enzyme for melatonin synthesis. This effect was found to be dose- and time-dependent since 10 T had no effect on short exposure but signicantly decreased plasma and pineal melatonin and pineal NAT with an exposure for one month which strongly suggests a cumulative effect of magnetic elds in rats. By contrast we failed to nd any effects on plasma melatonin, cortisol and immune functions in humans either on short exposure (9 h exposure) or under chronic exposure of up to 20 years in workers exposed both during their work and at home [52]. The melatonin hypothesis put forward in some epidemiologic studies as an explanation to the increase of the relative risk of cancer in people exposed to EMF can therefore be ruled out. EMF do not alter human circadian time structure even after chronic exposure for 20 years. Some medications may affect the circadian system. With regard to medications we explored the effects of a general anesthetic agent, propofol. In all animal and human studies, we found that general anesthesia for 20 min induces a desynchronization of the circadian time structure during the 48 to 72 h post-anesthesia period. However, the precise cellular mechanisms involved in circadian clock desynchronization induced by general anesthesia remains unknown, though inhaled as well as intravenous general anesthetics act on main clock genes like Period gene even if the studies are rare and performed on the whole brain of rat and not yet on the SCN. References
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