09-disruption of the circadian system by environmental factors

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Disruption of the circadian system by environmental factors: Effects of hypoxia,magnetic fields and general anesthetics agents

Yvan Touitou a,, Olivier Coste b, Garance Dispersyn c, Laure Pain c

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

a b s t r a c ta r t i c l e i n f o

Article history:Received 26 February 2010

Accepted 22 June 2010

Available online 6 July 2010

Keywords:

Rhythm desynchronization

Hypobaric hypoxia

Electromagnetic fields

General anesthetics

Propofol

Temperature circadian rhythm

Melatonin circadian rhythm

Cortisol circadian rhythm

Transmeridian flight

Jet lag

Fatigue

Circadian disruption

The biological clock of mammals is under the control of external factors, social life and the environment, andof 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 flights) 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 fields

and anesthetic agents whose importance has to be considered.

2010 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 929

1.1. Circadian synchronization and desynchronization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 929

1.2. Marker rhythms of the circadian time structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 930

2. Magnetic fields . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 931

2.1. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 931

2.2. Melatonin and the pineal gland in animal models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 931

2.3. Melatonin in humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 931

2.4. Endocrine and neuroendocrine systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 932

2.5. Biochemical and immuno-hematological variables in humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 932

2.6. Omics technologies application to EMF research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9323. Altitude and hypobaric hypoxia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 933

3.1. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 933

3.2. Influence of hypoxia in animal models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 933

3.2.1. Tolerance and intolerance to hypoxia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 933

3.2 .2. Hypoxia e ffect s on body temperature and locomotor activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 933

3.2.3. Hypoxia effects on the SCN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 933

3.3. Influence of hypoxia in humans. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 934

3.3.1. Hypoxia effects on body temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 934

Advanced Drug Delivery Reviews 62 (2010) 928945

Thisreview is part oftheAdvanced DrugDelivery Reviewstheme issue onChrono-Drug-DeliveryFocused on Biological Clock:Intra-andInter-Individual Variabilityo f Molecular Clock.

Corresponding author. Tel.: +33 1 47 02 32 05.

E-mail address: [email protected] (Y. Touitou).

0169-409X/$ see front matter 2010 Elsevier B.V. All rights reserved.

doi:10.1016/j.addr.2010.06.005

Contents lists available at ScienceDirect

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 e v i e r . c o m / l o c a t e / a d d r
http://dx.doi.org/10.1016/j.addr.2010.06.005http://dx.doi.org/10.1016/j.addr.2010.06.005http://dx.doi.org/10.1016/j.addr.2010.06.005mailto:[email protected]://dx.doi.org/10.1016/j.addr.2010.06.005http://www.sciencedirect.com/science/journal/0169409Xhttp://www.sciencedirect.com/science/journal/0169409Xhttp://unlabelled%20image/http://dx.doi.org/10.1016/j.addr.2010.06.005http://unlabelled%20image/mailto:[email protected]://dx.doi.org/10.1016/j.addr.2010.06.005


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3.3.2. Hypoxia effects on body temperature rhythm under constant routine conditions . . . . . . . . . . . . . . . . . . . . . . 934

3.3.3. Hypoxia effects on steroid hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 934

3.3.4. Hypoxia effects on rhythm markers: core body temperature and melatonin . . . . . . . . . . . . . . . . . . . . . . . . 934

3.3.5. Hypoxia effects on marker rhythms in long transmeridian flights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 935

3.4. Hypoxia effects on clock genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 936

4. General anesthetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 937

4.1. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 937

4.2. C ircadian timing and duration of action of general ane sthet ics agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 938

4.3. Anesthetic effects on restactivity and core body temperature cycles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 938

4.4. Anesthetic effects on hormonal secretions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9394.4.1. Steroid hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 939

4.4.2. Melatonin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 939

4.5. Anesthetics effects on clock genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 940

5. Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 941

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 942

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 N28 h, circadian if = 2028 h , and

ultradian if b20 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 modelsresults inthe 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 localizedto several regions of thebrain 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, bothof which drive our biologicalclock

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 timeof food intakein certain conditionsetc. that entrain

rhythms to 24 h [1]. In this respect, it is important to stress the

significance 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.shiftsof the peaktimeto an

earlier time) or phase delays (i.e shifts of the peak time to a later time).

This is named thePhaseResponse 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 examplesof rhythms with

a strong endogenous component which make them major markers of

the circadian synchronization of an organism [17]. As a final 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

thetemporal dissociation of biologicalclockfunctioningfrom thatof the

astronomical clock. It is either external when it is related to

environmental factors such as jet lag [18] and shift work [19], orinternal 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 flights (jet lag), night work, and shift work where

reversingor makingmajor changes to thetimingof our sociallife 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 modifications 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, hormone-

dependent 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 symp-

toms, 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 fixed 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 first hours of darkness using a dim light of 50 lx, the onset

of evening rise to be defined 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 profiles 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].

930 Y. Touitou et al. / Advanced Drug Delivery Reviews 62 (2010) 928945


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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 oftenmore than one marker rhythms is used to assess the rhythm

synchronization of subjects under study.

2. Magnetic fields

2.1. Background

The debate over the connection between electromagnetic fields

(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 trans-

mission 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 field environment.

Increasing concern in recent years about the effects of human

exposure to electromagnetic fields (EMF) has been stimulated by a

number of epidemiological studies reporting a possible link between

magnetic fields 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 fields may

induce biological effects in organisms that could have deleterious

consequences. In contrast to magnetic fields, there is little experi-

mental or epidemiological basis for considering electric fields to be

harmful for human health [34]. In this context the International

Agency for Research on Cancer (IARC) classified ELF electric fields in

category 3 which in the classification corresponds to inadequate

evidence of deleterious effects and placed ELF magnetic fields in

category 2B,corresponding to thecategoryof agentsthat are possibly

carcinogenic. It has to be noted that these extremely low frequencyelectric and magnetic fields are separate entities.

2.2. Melatonin and the pineal gland in animal models

As early as 1963, thehypothesis wasput 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 forthe 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 photope-

riodic conditions [4246]. The characteristics of the magnetic field

(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] submittedrats to a 50 Hz sinusoidal

magnetic field 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

significant 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 significant 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 fields 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 thephotoreceptor pigment rhodopsin due to the electric field induced by

the magnetic field has been proposed. Magnetic fields 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 fields 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.

Wedid not find anyeffect on melatonin levelswith short exposure

of young healthy volunteers during night (9 h exposure) to a 50-Hz

(10 T) magnetic field [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 field

( 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 significant difference (pb0.05) with

control group (Ctrl). From Selmaoui and Touitou [47].

http://image%20of%20fig.xn--1rc/


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melatonin secretion has also been reported by several authors [49

51]. These data, however, do notrule out a possible effectwith chronic

exposure. We therefore conducted a study on workers exposed on a

daily basis to magneticfields both at work and at home(EDFelectrical

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 effectsobserved in animals andhumans might be related to differences in the

anatomical configuration of the pineal gland and the nocturnal

rhythm of rodent activity [52]. Different sensitivity to magnetic fields

between species can also play a role in these differences as it is known

that some species perceive magnetic fields differently [53] and it is

possible that some subjects are more sensitive to magnetic fields 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 growthin 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 findings that exposure to

electromagnetic fields appears to attenuate the nocturnal melatonin

increasein experimentalanimals [47,6365], wewonderedwhetherand

in what ways electromagnetic field exposure might affect the endocrine

and neuroendocrine systems. We therefore examined the effects of

electromagnetic fields on the adrenocortical system and the hypotha-

lamo-pituitary-thyroid (HPT) axis. We found that the circadian rhythm

of the HPT and hypothalamo-pituitary-adrenal axis(HPA)functionswas

not affected by acute exposure (9 h) to either a continuous or an

intermittent 50-Hzmagneticfield [66]. Likewise,the circadianprofiles of

ThyroxineBindingProtein(TBG) andTBK which represents TBGcapacity

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 fields 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

magneticfields (50-Hz, 10 T) on thecircadian rhythmof biochemical

variables in an exhaustive study concerning proteins, lipids, enzymes,

electrolytes, nitrogen substances. We found no effect of magnetic

fields 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 sameconditions and on the samesubjects 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 fields 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 firmly 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, homova-nillic acid, and 5-hydroxyindoleacetic acid). None of these amines was

affected by exposure to magnetic fields [74].

2.6. Omics technologies application to EMF research

The application of high-throughput omics technologies to inves-

tigate the influences 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 difficult to compare data from

different research papers on this topic and to arrive at definite

conclusions on the potential effects of EMF on biological systems [75].

We will give here some recently published examples of difficulties in

comparing EMF exposure to such heterogeneous biological materials.

Fig. 3. Comparative nocturnal plasma melatonin profiles (A) and 6-sulfatoxymelatonin

concentration ( 6SM; B) in the first-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 magneticfield 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 profile of melatonin. From Touitou et al. [52].



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The expression of3, 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 modified 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 methodsused to investigate the effect of EMF exposure on SF767 human

glioma cells revealed significant 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 efficacy of

tamoxifen in a manner similar to tamoxifen resistance; by exposing

cells of the breastcancerline MCF-7 to EMF it has beenfound 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 difficult. 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 deficit in oxygen (O2) delivery is called hypoxia. This

deficit 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 finally 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 studiedin the last past century, studies dealing with the influence of hypoxia

on the circadiantime structureincluding clock genes are rather few in

both animals and humans.

3.2. Influence 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 synchronizedby 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 theotherhand. Resistance

to chronic hypoxia is high duringthe light phase (i.e. therest phase for

the animals) with lowlevels 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 ofinternal 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 con-

stant 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 oscilla-

tions 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 (FIO2 = 1 0 % ) i n a du lt r at s

synchronized by L/D= 12:12 photoperiodic conditions [87].Whereas body temperature presented several hypo and hyper-

thermic 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 %) forsevenconsecutive 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 submittedto 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 condi-

tions 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 SCNas were pineal5-HIAA andserotonin 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 vasodilatationleading to an increased blowflow)which may

play a role in modulating the circadian rhythms as observed underhypobaric hypoxia at high altitude.

These studies on animal model showed that continuous hypoxia

alters the circadian profiles 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. Influence 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 componentaround 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 astandardized 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].

3.3.3. Hypoxia effects on steroid hormones

Theplasmacortisolcircadianprofilewasnotmodifiedinthecourse

of79-hexposureatanaltitudeof4350 minagroupofclimbers,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 flight in a pressurized cabin [96]. Aninitial 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 signifi-

cantly altered in the sameacute experimentalconditions [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 6-

sulfatoxymelatonin) and on sleep through studies at two simulated

altitudes i.e. 8000 ft (~2400 m) and12,000 ft (~3600 m),corresponding

to maximal cabin altitudes for pressurized large cabins fixed by

aeronautical regulations in civil and military aviation respectively.

Fig. 4. Acute hypoxia exposure as phase shift inducer (mean times of acrophase

occurrenceSD). 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].



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Cabin altitudes of civil large aircraftsusually 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 significant

decrease in nocturnal urine 6-sulfatoxymelatonin values during

recovery (Fig. 9). The alterations were particularly marked in the

youngest subjects andwere onceagaincompatible witha phase delay ofmelatonin in response to hypoxic exposure [99,100]. Moreover, no

significant alterations of immune circadian profiles (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 significant impact on recovery sleep.

We therefore concluded that prolonged mild hypoxia, acting on the

circadian time structure, may contribute to post-flight fatigue, inde-

pendentlyof the jet lagphenomenon related to quick crossing of several

time zones during a transmeridian flight [102]. Some data in rodents

indicates that the functioning of the SCN and the pineal gland, main

actorsof thecircadian system, areimpactedby hypoxicexposure[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 flights

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

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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Long transmeridian flights provoke clinical complaints in passen-

gers 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

flight factors [104,105]. Moreover, post-flight fatigue in pilots,

associated with a decrease in cognitive performance, has beendescribed after long flights, regardless of whether time zones are

crossed, and in this regard no differences have been found between

transmeridian and northsouth flights [106110]. Environmental

cabin factors may therefore be involved and contribute to post-flight

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 specific 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

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].

http://image%20of%20fig.xn--60c/


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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 fibroblasts

showed an alteration of Stra13 and Dec2 circadian gene expres-

sion, two circadian transcriptional regulators that are over-

expressed and no longer rhythmic in hypoxic fibroblasts [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

inflammatory mechanism, i.e. an increase in interleukin-6 (IL-6)

[119].

All these studies suggest that hypoxia may act on specific

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 thiopen-

tal) and inhaled gases (sevoflurane, isoflurane, halothane, and

desflurane). In the days following general anesthesia, patients

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].



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often complain of sleep and mood disorders, and loss of vigilance.

Since anesthesia is not uncoupled from surgery, it is difficult 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.

4.2. Circadian timing and duration of action of general anesthetics

agents

It is well-established in rodents and rabbits that the efficacy ofanesthesia 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 recep-

tors, peak activity and maximal receptor-binding affinity of which

occurs during the rest span [128,129] . Whatever the endogenous

origin of this day

night difference in the effects of anesthetics, it is

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 first shown in rats studied in constant darkness( which

is a condition reflecting 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 specific 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 conditionsin 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 define as follows: under standard lightdark

cycles (12 h of light/12 h of darkness), the time of lights on

defines Zeitgeber time zero (ZT 0) and the time of lights off

defines Zeitgeber time twelve (ZT 12). We found that on the day

after anesthesia, propofol induced a significant 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 first and

second days after anesthesia. These results were obtained

Fig. 9. Urinary patterns of 6-sulfatoxymelatonin (6-SM) of healthy subjects of two age groups during three consecutive 24 h cycles D 0 (reference), D1 (diurnal hypoxic 8-h exposure

from 08:00to 16:00 h),and D2 (recovery) under twoexperimental conditions(8000 ftand 12,000 ft). Theurinary excretionof 6-SM wasexpressed as a ratio of theurinary excretion

of creatinine (g of 6-SM/mmol of creatinin). A significant 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].

http://image%20of%20fig.xn--u4c/


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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 ofanesthesia 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.

Thepropofol anesthesia forcolonoscopy 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 demonstratedhere 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

anesthesiceffects on circadian rhythms are dependent of the treatment-

time 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 sevoflurane (administeredduring

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 isoflurane (at subanesthetic dose given

between 9:30 and 10:30 a.m.) decreases the temperature circadian

amplitudeon the dayof anesthesia without any phase-shift [136]. These

observed effects could be due in fact to a residual decrease activity

induced by anesthesia in thefollowinghours anda subsequent decrease

in the heat production.

4.4. Anesthetic effects on hormonal secretions

4.4.1. Steroid hormones

In humans, it hasfirstbeen shown thatthe levelsof plasmacortisol,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 beenpublished on the effects of sevoflurane 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

beforesurgeryand theage of patients, amongst other factors. Thetypeof

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 contradic-

tory 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 thefirst 60120 min recovery period [142].

The inhibitory effects of propofol on adrenal steroidogenesis have

been evidenced in vitro during the first 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 ofplasma 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 profile 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 bloodsamples were collected according to 7 Zeitgeber Times to assess

propofol effects on circadian melatonin secretion. We showed that in

vivo propofol disrupts melatonin by significantly decreasing its

secretion (2228%) immediately after the wake up from anesthesia

and then increasing significantly 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

(expressedas arbitraryunits(mean valueSD)) forthe 72 h periodfollowing propofol.

From Dispersyn et al. [132].



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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 rhythmand modifythe 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 notknow if this resultcan be obtained at other circadiantimes. One

hypothesis is that propofol, a GABA agonist, has an inhibitory effect onthe 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 fitting 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 theanimals andhumans 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 identified

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 theexpression of several clock genes in ratbrains

during rest period [150152].

Firstly, concerning inhalation anesthetics, it was shown that

sevoflurane 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,

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 significant differences (pb0.05) to mean values between propofol and Intralipids treatment. From Dispersyn et al. [133].



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Rev-erb-alpha) [152]. These data were collected in rats still

anesthetized under 6-hour administration of sevoflurane. Unfortu-

nately this study provides no data on the long-lasting effect of

sevoflurane 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 sevo-

flurane 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 reflect

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 experi-

mental 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 reflect 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 photicresponsiveness 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 anes-

thetics 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 ofcircadian 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 modifications

in photoperiod and outside temperature.

To ascertain whether an organism is synchronized (or desyn-

chronized), 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 anendogenous 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 hypothalamo-

pituitary-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

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 asmean SD. * Significant differences (Pb0.05) to mean values between propofol and control. From Dispersyn et al. [134].



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had reliable cortisol rhythms. Although the circadian rhythms of

these hormones are endogenously generated by the SCN, numer-

ous 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 totallyblind 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, pharmaco-

logical 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 fields to which everybody is exposed

throughout the year, hypobaric hypoxia encountered in flight, and

general anesthetics agents currently used for surgery.

External desynchronization linked to jet lag has been the object of

numerous studies. Long duration transmeridian flights cause a

number of clinical problems in passengers and crew, who complain

mostly of fatigue, sleep and mood disorders and sometimes of gastro-

intestinal 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 flight factors into account,

though it has been shown that post-flight fatigue is associated with

decreased cognitive performance described after long haul flights,

independently of the time zones crossed [106110].

Environmental cabin factors may therefore be involved and

contribute to post-flight 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 at12,000 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 significantly decreased

the nocturnal peak of melatonin, the latter effect being age-

dependent.

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 ampli-

tudes [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 circadianrhythms 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 post-

flight 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 secretionhas thereforebeen putforward in some

epidemiologic studies as another possible mechanism. With regard tomagnetic fields, we showed huge differences in their effects in

humans when compared to rodents. Using a short 50 Hz exposure we

found a significant 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 significantly decreased plasma and pineal melatonin

and pineal NAT with an exposure for one month which strongly

suggests a cumulative effect of magnetic fields in rats. By contrast we

failed to find any effects on plasma melatonin, cortisol and immune

functionsin humans eitheron 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]. Themelatonin hypothesis put forward in

some epidemiologic studies as an explanation to the increase of the

relative risk of cancer in peopleexposed to EMF cantherefore 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 structureduring the 48 to 72 h post-anesthesia period. However,

the precise cellular mechanisms involved in circadian clock desyn-

chronization 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.

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