We have modelled the time-dependent dynamics of exosolar planets within the framework of a two-dimensional Cartesian model and the extended-Boussinesq approximation. The mass of the super-Earth models considered is 8 times the Earth's mass and the thickness of the mantle is 4700 km, based on a constant density approximation and a similar core mass fraction as in the Earth. The effects of depth-dependent properties have been considered for the thermal expansion coefficient, the viscosity and thermal conductivity. The viscosity and thermal conductivity are also temperature-dependent. The thermal conductivity has contributions from phonons, photons and electrons. The last dependence comes from the band-gap nature of the material under high pressure and increases exponentially with temperature and kicks in at temperatures above 5000 K. The thermal expansivity decreases by a factor of 20 across the mantle because of the high pressures, greater than 1 TPa in the deep mantle. We have varied the temperatures at the coremantle boundary between 6000 and 10,000 K. Accordingly the Rayleigh number based on the surface values varies between 3.5 × 10 7 and 7 × 10 7 in the different models investigated. Three phase transitions have been considered: the spinel to perovskite, the post-perovskite transition and the post-perovskite decomposition in the deep lower mantle. We have considered an Arrhenius type of temperature dependence in the viscosity and have extended the viscosity contrast due to temperature to over one million. The parameter values put us well over into the stagnant lid regime.

Our numerical results show that because of the multiple phase transitions and strongly depth-dependent properties, particularly the thermal expansitivity, initially most of the planetary interior is strongly super-adiabatic in spite of a high surface Rayleigh number, because of the presence of partially layered and penetrative convective flows throughout the mantle, very much unlike convection in the Earth's mantle. But with the passage of time, after several billion years, the temperature profiles become adiabatic. The notable influence of electronic thermal conductivity is to heat up the bottom boundary layer quasi-periodically, giving rise to strong coherent upwellingss, which can punch their way to the upper mantle and break up the layered convective pattern.

A c c e p t e d M a n u s c r i p t 1 Introduction

Up to now, well over 350 exosolar planets have been discovered and their habitability has been emphasized ( Haghighipohr and Raymond, 2007). According to the Extrasolar Planets Encyclopaedia (http//www.exoplanet.eu) and this list keeps on growing relentlessly. Most of these planetary bodies have been unveiled by Doppler analysis from the scattered light of the central star which reveals the wobbling of the star due to the gravitational pull of planets in close orbits [START_REF] Perryman | Extra-solar planets[END_REF][START_REF] Marcy | Observed Properties of Exoplanets: Masses, Orbits, and Metallicities[END_REF], Montgomery et al, 2008[START_REF] Bean | Detection of a third planet in the HD 74156 system using Hobby-Eberly telescope[END_REF]. Most of these planets found up to now are much larger than the Earth, typically, the size of Jupiter. But recently several 'superearth' planets estimated at 5 to 10 Earth masses, dubbed Superearths, have been uncovered, near the star Gliese 876, which have been estimated about 7 Earth masses. A nearby star Gliese 581, hosts several planets. One of these labeled planet-c has a mass estimated to be around five Earth masses (http://www.solstation.com/stars/gl581.htm). Recently both size and mass were obtained of a super-Earth planet of five Earth masses, in the CoRot-7 star system, resulting in the first density estimated of a terrestrial exoplanet, similar to Earth's mean density [START_REF] Queloz | The CoRoT-7 planetary system: two orbiting Super-Earths[END_REF].

These astronomical discoveries have elicited tremendous interest from the geophysical and also astrophysical communities (e.g. [START_REF] Kokubo | Formation of terrestrial planets from protoplanets[END_REF][START_REF] Bean | Detection of a third planet in the HD 74156 system using Hobby-Eberly telescope[END_REF] because planets of about 5 Earth masses are not much different from Earth in size, less than a factor two, whereas they have much higher internal pressure and interior temperature. Material behavior of solid state mantle silicates under extreme P, T condition differs from the behavior experienced under Earth like deep A c c e p t e d M a n u s c r i p t mantle conditions P < 136 GPa, T < 4000 K. In particular the perovskite phase MgSiO 3 may dissociate in the deep mantle of superearth planets (Umemoto et al., 2005). The endothermic phase transition corresponding to this mineral dissociation appears to have a strongly negative Clapeyron slope which could induce convective layering (e.g. [START_REF] Honda | Three-dimensional instabilities of mantle convection with multiple phase transitions[END_REF] leading to reduced cooling rate for the deep mantle and core. A somewhat similar configuration with an endothermic phase transition from spinel to perovskite could occur close to the core-mantle boundary of Mars at pressure values of about 24 GPa [START_REF] Breuer | Phase transitions in the martian mantle: implications for partially layered convection Earth Planet[END_REF].

Due to the increased surface gravity ( g ∼ 30ms -2 ) for a representative model planet of eigth times Earth's mass and outer radius (R ∼ 1.6R ⊕ ), and corresponding pressure gradient, the effective lithopheric strength of these super-earth planets is probably significantly higher than for Earth-like conditions. O'Neill and [START_REF] O'neill | Geological consequences of super-sized Earths[END_REF] have investigated this poignant effect on mantle dynamics where they found a clear trend towards stagnant lid convection for increasing planetary radius. This impacts the mantle convective regime and thermal history of the planet resulting in producing a higher internal temperatures and slower cooling of the interior.

Our simple density model is constrained by the given mass of the planet and we consider compressed mantle density up to 8267 kg/m 3 . Valencia et al. (2007a) investigated the internal structure of Super Earth planets using values for the internal temperature derived from parameterized convection models, leading to mantle temperatures that are not much higher than for the Earth's mantle.

Here we take a different tack and use the unknown temperature contrast across the mantle ∆T as a control parameter (e.g. van den Berg and Yuen, 1998) and we investigate a range of ∆T values from 6000 K to 10000 K. A tendency towards higher mantle temperature can be expected when the planet is in the stagnant lid regime as predicted by O'Neill and Lenardic (2007). At the same time the volumetric density of radiogenic heating scales with the compressed mantle density resulting in amplification of the internal heating for a given mass concentration of radioactive isotopes. In this way our model includes the (P, T ) range where [START_REF] Umemoto | Dissociation of MgSiO3 in the cores of gas giants and terrestrial exoplanets[END_REF] predict a new phase transition where the MgSiO 3 postperovskite phase dissociates into the oxides. This new phase transition is predicted to be endothermic with a strongly negative Clapeyron slope of -18 MPa/K and a strong impact on mantle circulation can be expected from this phase transition.

Very high pressure, as exists in a deep super-earth mantle, also strongly reduces thermal expansivity in the planetary mantle. This increases the phase buoyancy parameter of deep mantle phase boundaries and re-enforces the degree of convective layering induced by an endothermic phase boundary [START_REF] Christensen | Layered convection induced by phase transitions[END_REF]. Estimates based on a phonon thermodynamic model for the MgO-SiO 2 system, including the MgSiO 3 postperovskite phase, (Jacobs, M. and B. de Jong, 2007) predict a decrease in α across the mantle with of factor of twenty (M. Jacobs, 2007, personal communication). The role of pressure dependent α has been investigated for the Earth's mantle (Hansen et al. 1994, Matyska and[START_REF] Matyska | Lower mantle properties and convection models of multiscale plunes[END_REF] and precise values of the decrease of α across the mantle is still being investigated [START_REF] Katsura | P-V-T relations of MgSiO3 perovskite determined by in situ X-ray diffraction using a large-volume high-pressure apparatus[END_REF]. The stronger α contrast across a superearth mantle, considered here results in a larger tendency towards layering of deep mantle convection especially in the presence of the dissociation of post-perovskite.

Since the seminal experimental work on thermal conductivity by [START_REF] Hofmeister | Mantle values of thermal conductivity and the geotherm from phonon lifetimes[END_REF] the role played by thermal conductivity in mantle convection has received a rejuvenating interest [START_REF] Dubuffet | Effects of a realistic mantle thermal conductivity on the patterns of 3-D convection[END_REF][START_REF] Van Den Berg | The effects of variable thermal conductivity on mantle heat-transfer[END_REF], Dubuffett et al., 2002 ). But the thermal conductivity considered in those works has been concerned with modest temperature conditions. Thermal conductivity at high temperature conditions behave differently [START_REF] Umemoto | Dissociation of MgSiO3 in the cores of gas giants and terrestrial exoplanets[END_REF]. Above about 5000 K it is strongly impacted by an electronic contribution, which behaves exponentially in temperature and Therefore the deep mantle dynamics of planetary evolution under high temperature and very large pressure conditions, such as may be valid for super earth planets involves complex material behavior and may be rather different from an Earth-like case, where shallow tectonics may play a more prominent role. This makes the investigation of deep mantle dynamics in exosolar planets a very interesting subject for modelling studies within a comparative planetological framework.

Model description 2.1 Density model

As a first approximation, we will not consider density stratification due to self-compression, though we certainly recognize their importance in modifying flow structures in the deep mantle (e.g. [START_REF] Jarvis | Convection in a compressible fluid with infinite Prandtl number[END_REF]McKenzie, 1980, Zhang andYuen, 1996). Instead we apply a simple density model of a twolayer planet with uniform density of the mantle and core ρ m and ρ c respectively. A uniform mantle density is consistent with the use of an extended Boussinesq approximation (e.g. [START_REF] Christensen | Layered convection induced by phase transitions[END_REF] in our convection equations as explained in section 2.2. In Appendix A expressions are given for the mantle and core radius in terms the planetary mass, known from astronomic data, and the unknown core-mantle mass ratio X c . These expressions have been used to tabulate the planetary outer radius R(ρ c , ρ m ) for a relevant range of ρ c , ρ m values. This was carried out for models constrained by the planetary mass M = 8M ⊕ as in the recently discovered super-earth exosolar planet orbiting the star Gliese 876.

To constrain the model further, we make some necessary assumptions concerning the overall composition and state of core mantle differentiation. We assume the overall composition to be similar as for the Earth. We further assume that early core/mantle differentiation resulted in the same core-mass fraction as for Earth X c = 0.315. A contour diagram of planet outer radius R(ρ c , ρ m ) is shown in Fig. 1, for an Earth-like value of X c = 0.315.

Several special model cases are shown in Fig. 1, labeled m1, . . . , m5, indicated by separate symbols. Dimensional parameter values for these models are listed in Table 1.

Model m1 represents a uniform density case, ρ c = ρ m = ρ ⊕ , without core mantle differentiation, where the uniform density is equal to Earth's mean density, with an outer radius of twice the Earth radius.

The other models m2, . . . , m5 are located on the dashed line of constant ρ c /ρ m = 2.35. This ratio corresponds to the value for a two-layer model with homogeneous mantle and core, constrained by the Earth's total mass and moment of inertia factor. Model m2 is defined by setting the constraint R = 2 and ρ c /ρ m = 2.35. Substitution of the known parameters in the expression (15) for R c gives R c = 1.094. Substitution of the value for R c in the expression for ρ c [START_REF] Matyska | Lower mantle properties and convection models of multiscale plunes[END_REF] gives ρ c = 1.9246.

Finally substituting in the expression for the mantle density (17) gives ρ m = 0.81905. Models m3, . . . , m5 are obtained by applying an increasing uniform compression factor f > 1 to the core and mantle densities, keeping a constant ratio ρ c /ρ m = 2.35. f is defined to be the ratio of the core or mantle density of the different model cases listed in Table 1, m j , j = 1, . . . , 5, with respect to the corresponding core or mantle density of model m3.

A c c e p t e d M a n u s c r i p t

Figure 1: Planetary radius as a function of non-dimensional core and mantle density (scale value ρ ⊕ = 5511kgm -3 ) for models constrained by M = 8M ⊕ and X c = X c⊕ = 0.315. The dashed line represents the subset of models with increasing uniform compression factor f (see column 2 in Table 1) with a fixed density ratio ρ c /ρ m = 2.35. The labeled symbols m1, . . . , m5 refer to the model cases listed in Table 1. c ) and the values of the static pressure at the CMB and in the centre of the planet P (R c ) and P (0).

f R R c ρ m ρ c g(R) g(R c ) P (R c ) P (
The scaling of the surface gravity with the planet radius R for the models located on the dashed line in Fig. 1 is shown in Fig. 2 in the mantle and top of the core, for model case m4. The peak to peak variation of g is less than 10% in the mantle and we have used a uniform value of g equal to the surface value in our convection calculations. A relatively uniform mantle gravity value reflects the connection between our model and the Earth's mantle resulting from the given model assumptions, in particular for an Earth-like core mass fraction. Planets with a much smaller core mass fraction show a significantly

Convection model

The governing equations of the numerical model express conservation of mass momentum and energy in the extended Boussinesq formulation. We have also used a 2-D Cartesian model for a planetary body, which is another gross simplification. Symbols used are explained in Table 2.

∂ j u j = 0 (1)
-∂ i ∆P + ∂ j τ ij = Ra   αT - j Rb j Ra Γ j   δ i3 (2)
τ ij = η(T, P ) (∂ j u i + ∂ i u j ) (3)
DT Dt = ∂ j (κ(T, P )∂ j T ) + αDiw(T + T 0 ) + j γ j Rb j Ra Di DΓ j Dt (T + T 0 ) + Di Ra Φ + R H H(t) (4)
The continuity equation ( 1 , where E* is the activation free energy, R is the gas constant and T is the absolute temperature. Details of the parameterizations used are given in in Table 2. Energy transport is governed by [START_REF] Bunge | Low plume excess temperature and high core heat flux inferred from non-adiabatic geotherms in internally heated mantle circulation models[END_REF], where the righthand side terms are for thermal diffusion, adiabatic heating, latent heat of phase transitions, viscous dissipation and radiogenic internal heating respectively.

Multiple phase transitions are implemented using an extended Boussinesq formulation through phase parameter functions Γ j (P, T ), which parameterize the Clapeyron curves in the phase diagram. This is expressed in the equation of state [START_REF] Christensen | Layered convection induced by phase transitions[END_REF],

ρ = ρ 0   1 -α(T -T surf ) + j Γ j δρ j ρ 0   (5)
We apply a composite temperature and pressure dependent conductivity model which contains contributions from phonon (k lat ), photon (k rad ), [START_REF] Hofmeister | Mantle values of thermal conductivity and the geotherm from phonon lifetimes[END_REF] and electron k el transport [START_REF] Umemoto | Dissociation of MgSiO3 in the cores of gas giants and terrestrial exoplanets[END_REF].

k(T, P ) = k lat (T, P ) + k rad (T ) + k el (T ) (6)
The contributions of k lat and k rad are modified versions of an earlier work (van den Berg et al., 2005), which was based on the Hofmeister (1999) model.

k * lat = k 0 T surf T a × exp -4γ + 1 3 α(P )(T -T surf ) × 1 + K 0 P K 0 (7)
Symbols used in the parameterization of the conductivity are explained in Table 3. The parameterization of the pressure dependence of the phonon conductivity in [START_REF] Dubuffet | Controlling thermal chaos in the mantle by positive feedback from radiative thermal conductivity[END_REF] would make it the dominant contribution for Earth-like deep mantle conditions. In the present model applied to super-earth conditions we assume saturation of the phonon contribution above a temperature of about 4000 K where we apply a smooth truncation to an upper limit value of k trunc = 15 Wm -1 K -1 . To this end the original lattice component k * lat has been modified by introduction of a smooth truncation at a preset upper limit k trunc ,

k lat = (k * lat ) -2 + (k trunc ) -2 -1/2 (8)
The original polynomial representation of k rad of Hofmeister (1999) shows serious anomalies for temperatures above about 5000 K. Therefore we replaced this polynomial parameterization by a simple parameterization, where k rad (T ) is set to a uniform value of k rad = 2 Wm -1 K -1 in an operational temperature window and tapered off to zero on both sides of this window using harmonic (sine function) tapers with corner temperatures specified in Table 3.

The conductivity component from electron transport is parameterized as,

k el = k ep exp - E el k B T (9)
where k B is Boltzmanns constant and the activation energy is set to E el = 3 eV. The prefactor value for k ep , defined in Table 3, is obtained from an estimated reference conductivity at 10000 K of 40 The coupled equations ( 1), ( 2), ( 4), are solved on a 2-D rectangular domain of aspect ratio 2.5. Pa s

Wm -1 K -1 (Umemoto et al.,2006).
E Activation energy 3 • 10 5 J/mol V Activation volume 0.5 • 10 -6 m 3 /mol R Gass constant 8.3144 JK -1 mol -1 τ ij = ηe ij Viscous stress tensor - - Φ = ηe 2 Viscous dissipation function - α(z) = ∆α [c(1-z)+1] 3 Depth dependent thermal expansivity - ∆α = α(1)
Expansivity contrast across the layer 0.1-0.05

- c = ∆α 1/3 -1 - - α 0 Thermal expansivity scale value 4 • 10 -5 K -1 ρ Density - - ρ 0 Density scale value 8.267 kgm -3 c p Specific heat 1250 JK -1 kg -1 k Thermal conductivity - - k 0 Conductivity scale value 4.7 Wm -1 K -1 κ = k ρcp Thermal diffusivity - - g Gravitational acceleration 30 ms -2 Ra = ρ0α0g∆T h 3 κ0η0
Thermal Rayleigh number --

Rb j = δρj gh 3 κ0η0
Rayleigh number j th phase transition -δρ j Density increment j th phase transition -kgm -3

Γ j = 1 2 1 + sin π(z-z0j δz
Phase parameter j th phase transition --

z 0j = P (j) ref + γ j T -T (j) ref /(ρ 0 g) Depth j th phase

Results from numerical experiments

Six models with contrasting parameters will be investigated which show characteristic features for the extremely high temperature and pressure regime, which prevails in the deep mantle of a putative super-earth model with a mass eight times the Earth's mass. Three phase transitions involving the silicates are included in these models the parameters of which are listed in Table 4. These are an endothermic phase transition near 24 GPa corresponding to the transition from the γ spinel polymorph of olivine to perovskite and an exothermic transition near 125 GPa from perovskite to post-perovskite [START_REF] Tsuchiya | Phase transition in MgSiO3 perovskite in the earths lower mantle[END_REF]. The third transition from post-perovskite to a new extremely high pressure phase 3PO, which consists of periclase and silicon oxide, is strongly endothermic γ = -18 MPa/K and occurs at a reference pressure and temperature of 1000 GPa and 7000 K, i.e. near the core-mantle boundary (CMB) of the super-earth models, situated at 1.17 TPa, [START_REF] Umemoto | Dissociation of MgSiO3 in the cores of gas giants and terrestrial exoplanets[END_REF]. In the presentation of the modelling experiments, we have devoted our attention on the role of the 3PO phase transition in the dynamics of mantle convection. We investigate in particular the interplay of several thermal conductivity and thermal expansivity models with the 3PO phase transition controling the convective layering.

P ref T ref γ δρ/ρ 0 GPa K MPa/K % P
Our emphasis is to study the influence of the variations of physical properties on the style of exo-solar planetary convection under extreme conditions.

The characteristic parameters of the models being investigated, which are labeled A,B,C,D,E and F, are given in Table 5.

A The temperature at the CMB is kept at a constant value in these models. The unknown temperature contrast across the mantle ∆T is treated as a control parameter and we have investigated model behavior for the cases specified in Table 5 at three contrasting ∆T values, 6000, 8000, 10, 000 K respectively. Higher temperature contrasts than the estimated 4000 K for the Earth's mantle are in line with a higher volume/surface ratio of the larger planet investigated. Besides bottom heating, we have included internal heating from radioactive decay at a constant value of 2.5 × 10 -12 W/kg, resulting values of the internal heating number R H , defined in Table 2, vary between 10 and 17. We regard this range to be a speculative conjecture.

In Fig. 4 This dynamic episode can also be identified in a corresponding peak value of the VRMS curve shown in Fig. 16. Fig. 6c shows the temperature distribution of upward flowing tracers crossing the depth of 90% of the domain depth, about halfway the 3PO layer. There is a clear correlation with the (red) upward 3PO bondary crossings in frame (a). The temperature of the emerging hot upwellings at x = 1.2 and x = 1.8 increases towards the time of plume breakthrough t ∼ 1.1 Gyr. This is followed by a drop of the observed temperature and subsequent gradual increase in agreement with an episodic convective style.

Several physical effects interact in creating the dynamics of the 3PO layer. In particular the conductivity model and strongly endothermic phase transition, reinforced by a low value of the thermal expansivity, ∼ 5% of the surface value, play a role in controling the episodic behavior.

To separate these different effects we have investigated two contrasting models were the parameters were chosen to exclude a single effect. These are the constant conductivity model B and a variable conductivity model D with a zero Clapeyron slope of the 3PO transition. Fig. 7 shows results for both these models B (left) and D (right). Mass transport through the 3PO boundary is significantly smaller for the constant conductivity model B (Fig. 7 left hand) than for the variable conducivity model C (Fig. 6). This is quantified by the total number of phase boundary crossing events for the B model which is less then 50% of the C model value. This is in agreement with the snapshot of the temperature and streamfunction field shown in Fig. 4B, suggesting a layered convective flow regime for the bottom 3PO region with weak leakage through the boundary near x-coordinate 0.7 at the snapshot time 1.2Gyr.

The right hand frames of Fig. 7 show the result of removing the dynamical effect of the endothermic phase boundary by setting the Clapeyron slope to zero, while maintaining the variable thermal conductivity. The number of phase boundary crossings has strongly increased with respect to model C and the dynamics are controlled by stable cold downwellings instead of episodic hot plumes.

Comparison of the results of models B, C and D shows that the strongly endothermic phase transition is the essential ingredient for the layering of the flow in the 3PO region and that the effect of the highly variable conductivity is to produce a weakening of the convective layering and introduction of episodic behavior of the layering.

The latter effect can be understood by comparing the contrasting wavelength structure of the lateral variations of temperature in the 3PO layer between models B and C as shown in Fig. 4B,C.

The temperature field of the variable conductivity results is clearly dominated by longer wavelengths ( Matyska et al., 1994) than the corresponding constant conductivity snapshot. An increase in the dominant wavelength of lateral variations of temperature decreases the stability of phase induced convective layering [START_REF] Tackley | On the penetration of an endothermic phase-transition by upwellings and downwellings[END_REF].

The mechanism described above results from a more or less constant (in time) high effective conductivity value in the bottom layer including the phase boundary, comparable to a downward shift of the local Rayleigh number with well known impact on the phase induced convective layering (Christensen and Yuen, 1984). To test this explanation we did a model run, labeled E in Table 5, Overall the flow velocities are significantly smaller than in the previous cases for higher values of ∆T indicated by the streamfunction plots. The episodic hot plumes which occur for the C model at higher temperature contrasts are absent at the reduced ∆T = 6000 K. This is a result of the much smaller contribution of the electron conductivity for this case as illustrated in Fig. 18.

Because of their larger sizes,the exo-solar planet convection models are characterized by strongly depth-dependent properties and show clear signs of layered penetrative convection. We have therefore investigated the level of adiabaticity in the temperature distribution for two end-member values of the mantle temperature contrast ∆T of 10, 000K and 5000K respectively, for the model case C of Table 5. We have compared the instantaneous temperature profiles for the two models and compared them to a reference adiabat chosen by visually matching with the time dependent geotherm.

We also computed the deviation between the local temperature gradient and the adiabatic gradient

β = d < T > /dz -α(z)Di(< T > +T 0 ) ( 10
)
We display the results in Figure 12. Three frames are shown for each value of ∆T , 1) for the horizontally averaged temperature profile, 2) the temperature difference with the reference adiabat and 3) the deviation from the adiabatic gradient β. The reference adiabat which was chosen by trial and error visual matching with the geotherms is added as the red-dashed line in the left-hand temperature frame. The potential temperature for the cold case is 1500K and 2750K for the hot case. The results show that the difference between the geotherm and an 'equilibrium adiabat' decays with time for both the end-member cases, thus suggesting that with sufficient mixing of the early state an adiabatic profile will be obtained but only after a time interval of two or more billion years, which is longer for lower temperature contrast across the mantle.

It looks like in the initial spin-up of the model cold material is dumped and hot material rises.

Hot material is mixed rapidly in the upper half of the model as a result of the penetrative layering illustrated in the temperature and streamfunction snapshots of the previous Figures. This is illustrated by the rapid convergence of the geotherms to the reference adiabat. At the same time the bottom half is convecting much slower at least partly due to the high bottom conductivity, such that the bottom half is heating up slowly towards the adiabat. This trend is supported by the T -T a frames, showing convergence to the adiabat above a relatively substantial thermal boundary layer at the bottom with a thickness of 700 km. We give the corresponding results to Fig. 16 for the constant conductivity model B in Fig. 17.

These models are characterized by a penetrative convective regime with feeble convective vigor in the bottom half of the model and a low uniform conductivity, in agreement with low values of the surface heat flux, the rate of internal temperature and the rms velocity amplitudes.

Discussions and Concluding Remarks

Super-earth extrasolar planets are fundamentally different from the Earth in the pressure and temperature range of the different mantles and this particular aspect brings in more phase transitions and different heat transport mechanisms, unknown in the Earth's mantle and causes noticeable deviation from a thermal evolution predicted by parameterized convection [START_REF] Sharpe | Parameterized mantle convection and the Earth's thermal history[END_REF].

Our calculations address a number of important dynamical problems in thermal convection of super-Earth planets. However, we note that our model does have some shortcomings such as lack of density stratification, curvature from a spherical-shell model and also the assumption of a large core.

However, all of these factors will conspire to induce greater degrees of layering in mantle convection.

An interesting outcome from our study is the development of a super-adiabatic layer of around 700 km, which coincides with the location of the 3PO phase transition. This relatively thick superadiabatic layer will influence the amount of heat delivered from the core and the thermal evolution of the planet.

The major result from our work comes from the interplay between the strongly endothermic 3PO phase transition and the electronic thermal conductivity, which allows for the periodic build-up of thermal energy to be discharged across the bottom boundary layer in the form of coherent upwellings.

This represents a new type of episodic dynamics in which focussed plumes are periodically generated, by the steep rise in the exponential temperature dependence of the thermal conductivity.

We have shown that the high value of the thermal conductivity in the bottom thermal boundary layer is the critical ingredient creating this new dynamics. This was done by reproducing similar episodic dynamics with a purely depth dependent conductivity model defined from a time averaged conductivity profile of a model case with a fully temperature and pressure dependent conductivity.

This high degree of similarity shows that positive feedback from the temperature dependence of conductivity into the dynamics of plume formation is not a first order effect. This latter finding is somewhat similar to our earlier demonstration that significant delay in planetary secular cooling in models with temperature depedent phonon conductivity is mainly due to the average conductivity We further assume the mass fraction of an assumed metal core X c to be given. In the modelling experiments we set X c = 0.315, equal to the Earth value.

The model can then be formulated in terms of the volume average densities of the core ρ c and mantle ρ m . This way the outer planet radius R and the core radius R c can be expressed in terms of M , X c and ρ c , ρ m .

As a first approximation of cases with a self-compressing planet we consider several models which are related by a simple uniform compression factor, taken to be identical for the mantle and core. In the 2-D ρ c , ρ m parameter space these models are located on a line with fixed ρ c /ρ m . This parameterization can be used to study in a simple way the effect of compression on the internal gravity and pressure distribution.

In the following expressions are derived for the outer radius of the planet and its core in terms M, X c , ρ c , ρ m . For the total planetary mass we have,

M = M c + M m = X c M + (1 -X c )M (11)
The core radius is derived from the core mass fraction as,

R c = R 1 + ρ c ρ m 1 X c -1 -1/3 (12)
An expression for the outer radius is derived from the total mass as,

R 3 = 3M 4π X c 1 ρ c + (1 -X c ) 1 ρ m (13)
With the non-dimensionalization scheme,

M = M M 0 , R = R R 0 , ρ c = ρ c ρ 0 , M 0 = ρ 0 V 0 = ρ 0 4π 3 R 3 0 , we get, R 3 = M X c 1 ρ c + (1 -X c ) 1 ρ m (14)
For the special case of a uniform sphere with M = 1 this expression produces a unit non-dimensional

outer radius R = 1 with ρ c = ρ m = ρ 0 and ρ c = ρ m = 1.
For the core radius we get with the same non-dimensionalization scheme,

R c = R 1 + ρ c ρ m 1 X c -1 1/3 (15)
Explicit expressions for the core and mantle densities can be derived from the above for R and R c . show similar data for a modelrun with the same parameters as the variable conductivity model C except that the Clapeyron slope of the 3PO phase transition has been set to zero (right hand results).

ρ c = X c M R 3 c ( 16
)
ρ m = M (1 -X c ) R 3 -R 3 c ( 17
)
The number of up-and downward phaseboundary crossing events is 2506 and 2514 respectively.

A

  c c e p t e d M a n u s c r i p t becomes dominant in a composite conductivity model for these high temperatures (Umemoto et al., 2006).

  (a). The surface gravity increases non-linearly with the planetary radius as shown the left hand frame. Model case m4 with surface gravity 29.38 m/s 2 and a CMB pressure of 1.105 TPa has been used in the mantle convection experiments. At this CMB pressure the dissociation of post-perovskite can be expected to have taken place for sufficiently high temperature[START_REF] Umemoto | Dissociation of MgSiO3 in the cores of gas giants and terrestrial exoplanets[END_REF]. Fig.2(b)shows the radial distribution of the gravity accelleration g(r)

A c c e p t e d M a n u s c r i p t

  greater variation of g(r), with zero valued central gravity for a planet without a core. In general, low gravity values will impact the style of convection and may lead to increased layering in a similar way as a strong decrease of thermal expansivity with pressure from considerations of experimental equation of state[START_REF] Katsura | P-V-T relations of MgSiO3 perovskite determined by in situ X-ray diffraction using a large-volume high-pressure apparatus[END_REF].

Figure 2 :

 2 Figure 2: (a)(Left) surface gravity as a function of planet size (outer radius R) for the models constrained by M = 8M ⊕ , X c = X c⊕ = 0.315 and varying uniform compression with a fixed density ratio ρ c /ρ m = 2.35. Discrete symbols correspond to the models m1, . . . , m5 shown in Fig.1 and Table 1. (b)(Right) radial distribution of gravity accelleration for model m4.

  ) describes mass conservation of the incompressible fluid model. Conservation of momentum is expressed in the Stokes equation (2) based on the infinite Prandtl number assumption. The rheological constitutiive equation (3) defines the temperature and pressure dependent linear viscous rheology. Two types of viscosity models are applied in different model cases, an A c c e p t e d M a n u s c r i p t exponential parameterization in terms of layer viscosity contrasts and the temperature, commonly called the Frank-Kamentzky approximation and an Arrhenius type parameterization in terms of activation parameters of the form exp( E*/RT)

A c c e p t e d M a n u s c r i p t

  The different components of the composite conductivity model are shown for relevant temperature and pressure values in Fig. 3. Frames (a) and (b) show the distributions of the electron conductivity and radiative conductivity respectively which are independent of pressure. The electron conductivity becomes operational above a temperature of 5000 K. In the parameterization of k rad the upper cut-off temperature has been set at 5000 K. Frame (c) shows isobaric temperature distributions at four different pressure values, of the lattice conductivity component. The lattice conductivity has been truncated at high pressure at a value of 15 Wm -1 K -1 . The corresponding total effective conductivity values are shown in frame (d).

Finite element methods are

  used (van den Berg et al., 1993) based on the SEPRAN finite element package (Segal and Praagman, 1994) to solve the equations. The boundary conditions used are: free slip, impermeable conditions on all boundaries. The temperature at the horizontal boundaries are constant with a fixed temperature contrast ∆T across the layer driving thermal convection. Zero heat-flux conditions apply to the vertical boundaries. By prescribing a constant temperature at the CMB in our model we have not included thermal coupling between the mantle and the core heat reservoir. The initial condition for the temperature for all model runs was obtained from a non-dimensional temperature snapshot of a time dependent convection run with representative parameters. Profiles of the initial temperature are shown in the results section in Fig. 12. The finite element mesh consists of 45,451 nodal points, 151 points vertical and 301 points in the horizontal direction. The nodal points span 22,500 quadratic triangular elements for the Stokes equation. Each Stokes element is subdivided in four linear triangles for the energy equation. The time-dependent energy equation drives the time integration which is based on a predictor corrector method employing an implicit Euler predictor and a Crank-Nicolson corrector step in a second order correct integration scheme. An adaptive time step has been used equal to half the Courant time step.

  = ∂ j u i + ∂ i u j strain rate tensor -e = [1/2e ij e ij ] 1/2 Second invariant of strain rate -w Vertical velocity aligned with gravity -η exp (T, z) = η 0 exp(cz -bT ) Exponential viscosity model -Pa s b = ln(∆η T ), c = ln(∆η P ) where ∆η T = 300, ∆η P = 100 are viscosity contrasts due to T and P η 0 Viscosity scale value 2.5 • 10 23 Pa s η Arr (P, T ) = B exp E+P V RT Arrhenius viscosity model -Pa s B Viscosity prefactor 4.98 • 10 17

  representative snapshots of the temperature field and corresponding streamfunction field are shown for model cases A,B,C defined in Table5, with the same temperature contrast ∆T = 10, 000 K. Instantaneous positions of the three phase boundaries are indicated by the white lines. The depths of the first two phase transitions are much smaller than in the Earth's mantle due to the much greater pressure gradient resulting from the high density (ρ = 8.29 kg/m 3 ) and gravity acceleration g = 29.4 ms -2 derived in section 2.1. Phase boundary topography is also directly affected by the large pressure gradient of the exosolar planet. This is the reason that the peak to peak amplitude of the topography on the 3PO phase boundary is limited to a value of 170 km in spite of the fact that the relevant Clapeyron slope has a very high magnitude of 18 MPaK -1 .The different degree in the style of layered convection near the bottom 3PO phase boundary can be clearly discerned in these frames, in particular in the distribution and spacing of the contour lines in the streamfunction. Model A with a decrease factor of thermal expansivity across the mantle ∆α = 0.1 shows limited interaction between the endothermic 3PO phase boundary, with hot plumes crossing the phase boundary in several locations. A whole mantle flow pattern is indicated by the complete circuits of the streamfunction with highest velocities related to three cold descending flows emerging from the top boundary.The middle row of snapshots for model B with, ∆α = 0.05, illustrates a penetrative convective flow regime in the streamfunction with flow concentrated in the top half of the mantle and the bottom circulation is being driven by the convection cells in the shallow mantle. As in the A model the flow is driven by the cold downwellings. The temperature snapshots reveal an increased layering at the 3PO boundary at the bottom, as indicated by the mushroom shaped plumes, which show only limited penetration of the deepest phase boundary. The increase in layering between models A and B is related to the smaller value of the thermal expansivity near the 3PO phase transition resulting in a higher effective phase buoyancy parameter that scales inversely with respect to α[START_REF] Christensen | Layered convection induced by phase transitions[END_REF]. Similar layered penetrative convection was observed in[START_REF] Breuer | Phase transitions in the martian mantle: implications for partially layered convection Earth Planet[END_REF] in a model for the Martian mantle.

A c c e p t e d M a n u s c r i p t

  Fig.16 later in this section is reflected in the clusters of hot upward boundary crossing events that emerge at several x-locations. The two event clusters at t ∼ 1.1 Gyr and dimensionless x-coord ∼ 1.2 and 1.8 are associated with two plumes breaking through the 3PO boundary shown in Fig.4.

A c c e p t e d M a n u s c r i p t

  figure shows the number of upward crossing events accumulated in 100 Myr time bins normalized by the total number of particles as an estimate of the fractional volume flux involved. The maximum flux values coiincide with the episodic clusters of hotplumes breaking through the boundary. Up to 5% of the mantle volume crosses the 3PO boundary per 100 Myr for this particular case.

4 .

 4 Fig. 10 shows the representative snapshots of temperature and streamfunction for different model runs with a temperature contrast ∆T = 8000 K. The contrast between the B and C models has become smaller compared to Fig. 4 due to the smaller value of the conductivity near the bottom. This is a result of the reduced temperature and the strong temperature dependence of the electronic conductivity component which is dominant in the results shown in Fig. 4. Model results for a smaller temperature difference of ∆T = 6000 K are shown in Fig. 11. The same distinction holds between the A,B and C model cases as in the previous two figures with a whole mantle convection regime for the A case and penetrative convection for the models B and C.

Fig. 13 1

 131 Fig.13shows horizontally averaged profiles of temperature, viscosity and the thermal conductivity for models A,B and C. The value of the temperature contrast is ∆T = 10, 000K in all cases

Fig. 18 shows

 18 Fig. 18 shows the overall breakdown of the composite conductivity for the different ∆T values used in the convection results. The corresponding temperature profiles are shown in Fig. 18a. The controlling effect of temperature and pressure on the different components of k is clearly illustrated. The electron thermal conductivity k el becomes operational at T > 5000K (see Fig. 3). Since the radiative component k rad tapers off for T > 5000K, there is a thresholding effect and represents an effective switch from k rad to k el at this temperature. k el is the dominant mechanism near the bottom boundary layer for the model with ∆T = 10, 000K. The results presented sofar show clearly that an episodic type of deep mantle dynamics is produced by the interplay of thermal expansivity, thermal conductivity and the Clapeyron slope of the 3PO phase transition. O'Neill and Lenardic (2007) have argued that super-Earth type terrestrial planets have a larger propensity for stagnant lid dynamics of the upper mantle as a result of the high pressure gradient in the shallow leading to surpressing brittle deformation mechanisms. This may have an impact also on episodic deep mantle dynamics. In the following we therefore investigate the impact of a stagnant lid on the model results. Stagnant lid convection occurs for sufficiently high viscosity contrasts around 10 5 to 10 6 (e.g. Moresi and Solomatov, 1995) To investigate the effects of stagnant lid convection in our models we have used an Arrhenius parameterization of the viscosity, defined in Table 2, in a model, labeled F in Table 5, with identical thermophysical parameters as in model C and a temperature contrast across the mantle ∆T = 10000 K. Snapshots of depth profiles of relevant quantities for model F are given in Fig 19. The stagnant lid character of model F is illustrated in the profiles of the viscosity and the amplitude of the velocity field. The viscosity profiles show more than six orders of viscosity variation and a stagnant lid of about 300 km thickness. This is in agreement with the velocity profiles showing an effectively rigid outer surface and rapid increase of the flow velocity in the mantle below the lid. Temperature and conductivity profiles aer similar to the corresponding profiles of model C shown in Fig. 13, except for a systematic increase of mantle temperature over time due to the decrease in in mantle cooling capacity in the presence of the stagnant lid. Time series of global quantities for model F are shown in Fig. 20, corresponding to similar data for model C shown in Fig. 16. The episodic nature of the mantle dynamics for model F is most clearly illustrated by the Vrms curve in Fig. 20c which is much like the corresponding curve for the C model and ∆T = 10000 K. Differences between results of the two models are; shorter time scale for model F, related to a combination of higher temperature and conductivity and lower viscosity in the deep mantle, compared to model C. Further, the mantle temeperature increases more, in the stagnant lid case F, as a consequence of the thermal resistance of the stagnant lid.A snapshot of the temperature field from the results of the F model case is shown in Fig.21for a model time of 1.24 Gyr, close to the first of a double peak in Vrms, corresponding to a plume breaking through the 3PO phase boundary, visible in the snapshot. Compared to the corresponding mobile lid results in Fig.4c(model C) and Fig.9(model E), cold downwellings are less significant in Fig.21as a result of the different stagnant lid conditions. This further illustrates the fact that the

  profile characterized by a sub-lithospheric conducitvity minimum in the shallow mantle creating a low conductivity zone (LCZ) (van den[START_REF] Van Den Berg | The combined influences of variable thermal conductivity, temperature-and pressure-dependent viscosity and core-mantle coupling on thermal evolution[END_REF].Furthermore we have shown that the episodic plume dynamics occurs both in a mobile lid mantle convection regime obtained with an exponential parameterization of the viscosity, with modest tempareture dependence, and a stagnant lid regime, obtained with an Arrhenius type viscosity with more than six orders of viscosity variation due to temperature. The latter case is considered more representative for exosolar planets of the super-Earth type (O'Neill and Lenardic,2007). A stagnant lid regime of planetary convection with variable viscosity may be linked to plate tectonics through a plastic bifurcation(Trompert and Hansen, 1997) and may play a role also in exosolar planets(Valencia et al., 2007b). The robustness of the episodic mantle dynamics is an important result since it underlines the fact that parameterized convection models which do not capture episodicity are inadequate to model thermal evolution of large super-Earth type exosolar planets. Future work would need to incorporate both realistic equation of states[START_REF] Umemoto | Dissociation of MgSiO3 in the cores of gas giants and terrestrial exoplanets[END_REF], Valencia et al., 2009 ) and sphericity. But the major findings of electronic thermal conductivity should still be valid under these circumstances.

  We consider a simple two layer model of a planet consisting of a homogeneous core and mantle with contrasting uniform densities ρ c and ρ m respectively. Assuming a uniform density mantle is consistent with the use of an extended Boussinesq formulation of the mantle convection equations applied in our modelling experiments. The two layer model is set up for a planet of given mass M .

  688

Figure 3 :

 3 Figure 3: Components of the composite condivity model. Frames (a) and (b) show the distributions of the electron conductivity and radiative conductivity respectively which are independent of pressure. Frame (c) shows the lattice conductivity, at different values of pressure, using a truncation value at high pressure of 15W m -1 K -1 . Frame (d) shows the corresponding effective conductivity. Color coding of the pressure values P i , 0, 390, 780, 1170GP a is indicated in the plot legend.

Figure 4 :A c c e p t e d M a n u s c r i p t 690 Figure 5 :

 46905 Figure 4: Snapshots of non-dimensional temperature (left) and streamfunction (right) for different models A,B,C defined in Table 5, for the same temperature contrast across the layer ∆T = 10, 000K. White lines indicate the instantaneous position of the three phase boundaries included in the model.

Figure 6 :

 6 Figure 6: (a) phase boundary crossing events at the 3PO transition displayed by the horizontal and time coordinates of passive tracers monitoring the local mineral phase for model C. Red (1864) and blue (1877) symbols represent upward and downward crossings respectively. (b) upward mass flux through the 3PO boundary estimated by (100 Myr) time binned values of the number of upward phase boundary crossings normalized by the total number of tracers in the domain. Red dots indicate results based on the full data set of 2000 tracer particles. The black crosses showing test results computed from half the data set indicate that a sufficient number tracers were used. (c) Color coded temperature values of upward monitor crossings of the depthlevel 0.9 times the layer depth, inside the 3PO layer, for model C. The number of upward crossing events is 1387. A rainbow palette has been used indicating temperature variation between 7000 K and 10,000 K. The hotter cores of plumes can be distinguished from the cooler off-central parts of the upwellings. Episodic broadening of the upwelling flows is in agreement with the episodic dynamics of the 3PO layer which is reinforced by a positive feedback related to the temperature dependence of the conducitvity.

Figure 7 :

 7 Figure 7: Similar results as in Fig.6 the uniform conductivity model B (left). The number of upand downward phase boundary crossing events is 772 and 774 respectively. The two rightand frames

  693

Figure 8 :Figure 9 :

 89 Figure 8: (a) (3PO)phase boundary crossing events for model E with purely depth dependent conductivity showing similar results as in Fig.6a. The number of upward and downward events is 1819 and 1821 respectively. (b) Conductivity depth profile used for model E shown in red, computed by timeaveraging k(z, t) profiles from model C shown in black.

Figure 10 :

 10 Figure 10: Temperature and streamfunction snapshots for models similar to Fig. 4 but with a temperature contrast ∆T = 8000K across the mantle. The contrast between the B and C models has become significantly smaller due to the much smaller value of the conductivity near the bottom. This is a result of the reduced temperature and the strong temperature dependence of the electronic conductivity component which is dominent in the results shown in Fig. 4.

Figure 12 :

 12 Figure 12: Vertical profiles illustrating the time dependent degree of adiabaticity of the mantle for two endmember model cases, of type C defined in Table 5, with different temperature contrast across the mantle, ∆T = 10, 000K top row (a),(b),(c) and ∆T = 5000K bottom row (d),(e),(f ). The lefthand column (a),(d) shows snapshots of the horizontally averaged temperature at different time instances indicated in the legend. The red dashed line indicates an adiabatic temperature profile with potential (surface) temperature of 2750K (a) and 1500K (d) visually selected to overlap with the evolving geotherms. The middle column (b) and (e) shows snapshots of the difference between the mantle temperature profile and the reference adiabat. The righthand column shows corresponding profiles of the deviation of the local temperature temperature gradient from the adiabatic value defined in (10).

Figure 13 :

 13 Figure 13: Horizontal average depth profiles of temperature (left column) viscosity (middle) and thermal conductivity (right), for three models A (bottom row), B (middle row) and C (top row) shown in Fig.4. The temperature contrast across the mantle is fixed at ∆T = 10, 000K for all cases. The Clapeyron curve of the 3PO phase is indicated by the straight black line in frames (a),(d),(f ).

Figure 14 :Figure 15 :

 1415 Figure 14: Similar profiles as in Fig. 13 but now for a temperature contrast across the mantle ∆T = 8000K.

Figure 16 :

 16 Figure 16: Timeseries of (a) bottom heat flux, (b) volume average temperature and (c) root-meansquared velocity for the variable conductivity models C. Three values of the mantle temperature contrast ∆T are considered, 6000 (blue), 8000 (black) and 10,000 K (red).

M a n u s c r i p t November 9 , 2009 35 702Figure 17 :Figure 18 :

 9351718 Figure 17: Similar timeseries as in Fig. 16 for the constant conductivity models B with thermal expansivity contrast ∆α = 0.05. These uniform k models show no global warming and much lower bottom heat flux than shown in Fig. 16. The low Vrms values are in agreement with penetrative convection without large scale mass transport through the 3PO interface.

Figure 19 :

 19 Figure 19: Depth profiles of horizontally averaged temperature (a), conductivity (b), viscosity (c), and magnitude of velocity (d) for three snapshots labeled by the model time value in frame (a). The Clapeyron curve of the 3PO transition is drawn in the lefthand frame to indicate the approximate location of the phase boundary. The viscosity varies by six orders of magnitude due to temperature in the shallower part of the model. This results in stagnant lid convection as illustrated by the vertical distribution of the velocity amplitude indicating an approximately 300 km thick immobile lid.

  707

Figure 20 :

 20 Figure 20: Time series of global quantities for the stagnant lid model F: (a) CMB heatflow, (b) volume average temperature and (c) rms velocity amplitude. The dynamics of model F is similar as in model C, as characterized by the episodic behavior of hot plumes breaking through the 3PO phase boundary, producing sharp peaks in Vrms. Model F shows sharper peaks than model C which is related to the greater viscosity contrast due to temperature and lower viscosity in the bottom region near the 3PO transition.

Table 1 :

 1 Dimensional parameters of models m1 . . . m5, corresponding to the discrete symbols in Fig.1. The table includes the surface-and core-mantle boundary values of the gravity accelleration g(R) and g(R

	0)

Table 2 :

 2 Physical parameters

	transition	-	-

Table 3 :

 3 Parameters describing the conductivity model.

Table 4 :

 4 Parameters of the phase transitions.

	1 23.6 2000	-1.8	7.5
	P 2 125 2750	7.2	1.5
	P 3 1000 7000	-18	2

Table 5 :

 5 Characteristic parameters of the convection models investigated.

		Expansivity contrast conductivity model Clapeyron slope 3PO Viscosity type
	Model	∆α	k(T, P )	γ MPa/K
	A	0.1	k 0	-18	exponential
	B	0.05	k 0	-18	exponential
	C	0.05	k lat + k rad + k el	-18	exponential
	D E F	0.05 0.05 0.05	M k lat + k rad + k el k(z) a n u s c r i p t 0 exponential -18 exponential k lat + k rad + k el -18 Arrhenius
		c c e p t e d

November 9, 2009

Acknowledgements

We thank discussions with Phil Allen, Michel Jacobs, Radek Matyska, Joost van Summeren, Diane Valencia, Renata Wentcovitch and K. Umemoto. Constructive reviews of Craig O'Neil and an anonymous reviewer were very helpful for improving the manuscript. This research has been supported by the ITR grant from National Science Foundation given to the VLAB at the University of Minnesota. Computational resources were provided through the Netherlands Research Center for Integrated Solid Earth Science (ISES 3.2.5).


J Stefanska

M Sokolowska

A Sarniak

A Wlodarczyk

Z Doniec

D Nowak

MD Rafal Pawliczak
email: rafal.pawliczak@csk.umed.lodz.pl

APOCYNIN DECREASES HYDROGEN PEROXIDE AND NIRTATE CONCENTRATIONS IN EXHALED BREATH IN HEALTHY SUBJECTS

Keywords: apocynin, NADPH oxidase, ROS, NOS, EBC

The imbalance between reactive oxygen species (ROS) synthesis and antioxidants might be involved in the pathogenesis of many inflammatory diseases. NADPH oxidase, an enzyme responsible for ROS production, may represent an attractive therapeutic target to inhibit for the treatment of these diseases.

Apocynin is an inhibitor of activation of NADPH oxidase complex present in the inflammatory cells.

In double blind, placebo controlled, cross-over study, we investigated the effect of nebulized apocynin on ROS synthesis in 10 nonsmoking healthy volunteers. Apocynin (6 ml of 0.5 mg/ml) was administered by nebulization and its effects on H 2 O 2 , NO 2 -and NO 3 -generation were assessed after 30, 60 and 120 minutes by collecting exhaled breath condensate (EBC) samples using an EcoScreen analyzer. Additionally, respiratory parameters have been evaluated, utilizing spirometry and DLCO. We also analyzed peripheral blood differential counts and NO 2 -serum level, cough scale control and blood pressure as safety parameters.

Apocynin caused reduction of H 2 O 2 concentration in EBC as compared to placebo, after 60 min. of inhalation (0.18 μM vs. 0.31 μM, p<0.05) as well as after 120 min. (0.2 μM vs. 0.31 μM, p<0.05). Similarly, apocynin significantly decreased concentration of NO 3 ¯ as compared to placebo, after 60 and 120 min. (6.8 μM vs. 14.4 μM and 6.5 μM vs. 14.9 μM respectively,

Introduction

Asthma is a chronic airway disease, characterized by airway inflammation and hyperresponsiveness [START_REF] Kharitonov | Reproducibility of exhaled nitric oxide measurements in healthy and asthmatic adults and children[END_REF]. Human airways from healthy subjects contain cells that are able to release reactive oxygen species (ROS) such as hydrogen peroxide (H 2 O 2 ) and peroxynitrite [START_REF] Nowak | Exhalation of H2O2 and thiobarbituric acid reactive substances (TBARs) by healthy subjects[END_REF][START_REF] Ricciardolo | Reactive nitrogen species in the respiratory tract[END_REF], i.e. polymorphonuclear leukocytes (PMNs), eosinophils or macrophages.

Reactive oxygen species have a strong impact on homeostasis and are thought to play an important role in inflammation in asthma and COPD [START_REF] Masood | Reversal of oxidative stress-induced anxiety by inhibition of phosphodiesterase-2 in mice[END_REF]. ROS play a key role in initiation as well as amplification of inflammation in asthmatic airways. When imbalance between reactive oxygen species concentration and the endogenous antioxidant system emerges, oxidative stress occurs. Oxidant-antioxidant imbalance leads to pathophysiological effects associated with asthma such as vascular permeability, mucus hypersecretion, smooth muscle contraction, and epithelial shedding [START_REF] Masood | Reversal of oxidative stress-induced anxiety by inhibition of phosphodiesterase-2 in mice[END_REF][START_REF] Adcock | New targets for drug development in asthma[END_REF]. The concentrations of nitric oxide and hydrogen peroxide in exhaled air are increased in asthmatics and it is considered to reflect the state of airway inflammation [START_REF] Ko | Are exhaled breath condensates useful in monitoring asthma?[END_REF]. ROS in the airways come from various enzymes expressed in airway cells.

The EBC collection used in this study is a safe method of gaining information regarding respiratory fluids. Since the pattern of breathing is normal, it is safer than FVC measurements, which may provoke bronchospasm in some asthmatic patients. It is noted that some people tend to hyperventilate especially at the beginning of EBC collection, but this has not led to any adverse event [START_REF] Horvath | Exhaled breath condensate: methodological recommendations and unresolved questions[END_REF]. Though the methodology of EBC has not yet been standardized, and thus may constitute a some methodological bias that may hamper a solid interpretation of the data, it is still very useful and accurate method to gather research material. In the respiratory system H 2 O may be released both from inflammatory cells and epithelial cells. Hydrogen peroxide is a putative marker of airway inflammation in exhaled air. Stimulated alveolar macrophages from asthmatics generate more reactive oxygen species than alveolar macrophages from healthy subjects [START_REF] Rysz | Increased hydrogen peroxide concentration in the exhaled breath condensate of stable COPD patients after nebulized N-acetylcysteine[END_REF].

M A N U S C R I P T A C C E P T E D ARTICLE IN PRESS

The main ROS, is superoxide, generated through one-electron reduction of oxygen molecule.

It reacts with enzymes, producing a series of other ROS, including H 2 O 2 . Reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is a principal enzyme responsible for the superoxide production [START_REF] Zalba | Oxidative stress in arterial hypertension: role of NAD(P)H oxidase[END_REF]. NADPH oxidase (EC 1.6.3.1) is a complex, containing p47phox and p67phox subunits, membrane-bound cytochrome b558 (composed of gp91phox or its homologues), p22phox, and a small G protein rac [START_REF] Cotter | Effect of the NAD(P)H oxidase inhibitor, apocynin, on peripheral nerve perfusion and function in diabetic rats[END_REF]. It generates superoxide anion after activation, i.e. aggregation of all subunits. The agent that is able to inhibit the release of superoxide anion by NADPH oxidase by inhibiting activation NADPH oxidase itself is apocynin (4-hydroxy-3methoxyacetophenone, acetovanillone) [11]. It blocks migration of p47phox to the mitochondrial membrane, pivotally involved in initiating assembly of the functional NADPH oxidase complex. Apocynin is a naturally occurring agent, isolated from roots of plants Picrorhiza kurroa and Apocynum cannabinum. It is an acetophenone with a molecular weight of 166.17 and forms needles upon crystallization from water. It possesses a faint vanilla odor and has a melting point of 115°C and has very low toxicity [START_REF] Luchtefeld | Dose formulation and analysis of diapocynin[END_REF]. Apocynin reveals multiple biological aptitudes, such as neuroprotective features [START_REF] Tang | Apocynin attenuates cerebral infarction after transient focal ischaemia in rats[END_REF][START_REF] Wang | Apocynin protects against global cerebral ischemia-reperfusion-induced oxidative stress and injury in the gerbil hippocampus[END_REF], beneficial effects on arteriosclerosis and hypertension [START_REF] Holland Jaaj | Prevention of atherosclerosis using NADPH oxidase inhibitors[END_REF][START_REF] Meyer | A central role for the endothelial NADPH oxidase in atherosclerosis[END_REF][START_REF] Hamilton | NAD(P)H oxidase inhibition improves endothelial function in rat and human blood vessels[END_REF], inhibition of cancer cells migration [START_REF] Klees | Apocynin derivatives interrupt intracellular signaling resulting in decreased migration in breast cancer cells[END_REF] and scavenger properties [START_REF] Heumuller | Apocynin is not an inhibitor of vascular NADPH oxidases but an antioxidant[END_REF]. Particularly, it displays powerful antiinflammatory and antioxidant effects in a variety of cell and animal models as well as in human studies [START_REF] Hu | Apocynin but not L-arginine prevents and reverses dexamethasone-induced hypertension in the rat[END_REF][START_REF] Peters | Effect of apocynin on ozone-induced airway hyperresponsiveness to methacholine in asthmatics[END_REF]. There is only one paper characterizing nebulized apocynin influence on humans [START_REF] Peters | Effect of apocynin on ozone-induced airway hyperresponsiveness to methacholine in asthmatics[END_REF].

Through inhibiting superoxide production, apocynin may affect decrease of H 2 O 2 , NO 2 ¯ and NO 3 ¯ generation, as O 2 ¯· is a pivotal element of ROS and NOS formation [START_REF] Kawasaki | O2 and reactive oxygen species (ROS) detoxification complex composed of O2-responsive NADH:rubredoxin oxidoreductase -flavoportein A2 -desulfoferrodoxin operon enzymes, rubperoxin, and rubredoxin in Clostridium acetobutylicum[END_REF][START_REF] Timoshenko | NO-dependent regulation of lectin-and menadione-induced H2O2 production by cells from pleural effusions of lung cancer patients and by immune cells[END_REF][START_REF] Neill | Nitric oxide evolution and perception[END_REF]. Nitric oxide (NO), is generated by nitric oxide synthase (NOS, EC 1.14.13.39), from L-arginine. In physiological concentrations, half-life time of endothelium-derived NO particle is about 1-30 s. In vivo, NO is easily oxidized to NO 2 -, which half-life in blood is 110s, and afterwards, transforms into stable nitrate NO 3 -(half-life time = 8 h) [START_REF] Goligorsky | Endothelial nitric oxide synthase: from structure to function in one aspartic substitution[END_REF][START_REF] Bryan | Methods to detect nitric oxide and its metabolites in biological samples[END_REF]. In some study, significant correlations between EBC nitrite and selected lung function parameters, symptoms and airway hyper-reactivity is presented [START_REF] Straub | Hall GI Correlation of nitrites in breath condensate and lung function in asthmatic children[END_REF] while in other, that EBC nitrite was a good indicator for asthma severity [START_REF] Robroeks Cm Vdkk | Exhaled nitric oxide and biomarkers in exhaled breath condensate indicate the presence, severity and control of childhood asthma[END_REF]. Exhaled nitric oxide has the potential to be a valuable marker of inflammation at different lung depths, making it attractive as a noninvasive monitoring tool.

The most probable mechanism of the inhibitory effect of apocynin is the reactivity of apocynin radical with thiol compounds. Additionally, apocynin needs to be activated (oxidized) in the presence of H 2 O 2 and myeloperoxidase (MPO), abundant in neutrophils, to

M A N U S C R I P T A C C E P T E D ARTICLE IN PRESS 4
inhibit the NADPH oxidase [START_REF] Vejrazka | Apocynin inhibits NADPH oxidase in phagocytes but stimulates ROS production in non-phagocytic cells[END_REF]. The oxidation effects in conversion to a dimer, which has been shown to be more efficient than apocynin itself monomer [START_REF] Klees | Apocynin derivatives interrupt intracellular signaling resulting in decreased migration in breast cancer cells[END_REF].

Through inhibiting source of ROS, apocynin has, among others, strong antiinflammatory properties. By preventing generation of the most serious radical -O 2 ¯·, it obviates formation of other ROS as well as RNS (reactive nitrogen species). Hence, it may play important role in treatment of airway inflammatory disease, like bronchial asthma [START_REF] Peters | Effect of apocynin on ozone-induced airway hyperresponsiveness to methacholine in asthmatics[END_REF].

Although therapeutic strategies focusing on the elimination of superoxide have been developed in animal models and in clinical cases, the effects of such reagents are still unclear [11,[START_REF] Masamune | NADPH oxidase plays a crucial role in the activation of pancreatic stellate cells[END_REF][START_REF] Muijsers | Apocynin and 1400 W prevents airway hyperresponsiveness during allergic reactions in mice[END_REF][START_REF] Lafeber | Apocynin, a plant-derived, cartilage-saving drug, might be useful in the treatment of rheumatoid arthritis[END_REF]. Therefore, in this study, we investigated whether inhalation of apocynin may influence hydrogen peroxide, nitrite and nitrate concentrations in airways in healthy subjects.

Simultaneously, we analyzed safety parameters and adverse events of nebulized apocynin in the applied dose.

Material and methods

Study Design

The study had a double-blind, placebo-controlled, cross-over design, consisted of 2 visits, separated 30 to 60 days. If during the first visit the drug was used, in the second visit a subject nebulized placebo or vice versa. The summary of the study design with the detailed characteristics of the performed analyses are shown in Fig. 1a. and 1b. Before and after procedure safety measures (arterial blood pressure, heart rate value, and cough scale) were performed.

The appropriate methods have been chosen for the determination of H 2 O 2 , NO 2 -and NO 3 -in EBC to detect a significant difference in a healthy non-smoking population. The methods are imperfect, but accurate enough to display the differences between the state of airways before and after apocynin nebulization.

The study protocol was approved by the local Ethics Committee (no. RNN/12/08/KE) and written consent was obtained from every subject prior to the study.

Subjects

Ten healthy, nonsmoking volunteers participated in the study (mean age 32 years, 6 men, 4

women, no atopy). This number of subjects is typical for phase I RCTs (randomized clinical trials) [START_REF] Peters | Effect of apocynin on ozone-induced airway hyperresponsiveness to methacholine in asthmatics[END_REF][START_REF] Zhao | The relationship between oxidative stress and acid stress in adult patients with mild asthma[END_REF][START_REF] Jorres | The effect of 1 ppm nitrogen dioxide on bronchoalveolar lavage cells and inflammatory mediators in normal and asthmatic subjects[END_REF]. The volunteers had not suffered from any infectious diseases including upper respiratory tract infections for at least 3 months prior to the study (exclusion criterion).

They were free of any medication and routine physical examination was normal (inclusion

M A N U S C R I P T A C C E P T E D ARTICLE IN PRESS 5
criterion). The study has begun at 8 a.m. The volunteers were asked not to do any exercise for one day. They were free of any medication and routine physical examination was normal.

Exhaled Breath Condensate (EBC) collection EBC was collected using a modification of the method described previously by Doniec et al. [START_REF] Doniec | Passive smoking does not increase hydrogen peroxide (H2O2) levels in exhaled breath condensate in 9-year-old healthy children[END_REF], and regarding ERS Task Force on EBC [START_REF] Horvath | Exhaled breath condensate: methodological recommendations and unresolved questions[END_REF]. The subjects breathed spontaneously through a mouthpiece for 20 min. Each subject wore a nose clip during this procedure. EBC was collected by using a condenser (EcoScreen; Erich Jaeger Viasys; Hoechberg, Germany) that yielded nongaseous components of expiratory air. Subjects breathed through a mouthpiece connected to the condenser, and were asked to breathe at a normal frequency and tidal volume, wearing a nose clip, for a period of 20 min. Approximately 2 ml of condensate was collected and immediately stored at -80°C in the laboratory for maximum 7 days, until the procedure of H 2 O 2, NO 2 -/NO 3 -measurement [START_REF] Szkudlarek | Exhaled hydrogen peroxide correlates with the release of reactive oxygen species by blood phagocytes in healthy subjects[END_REF].

Apocynin inhalation 6 ml of apocynin of total dose 3 mg (0.5 mg/ml dissolved in sterile 0.9% NaCl as the study drug) [START_REF] Peters | Effect of apocynin on ozone-induced airway hyperresponsiveness to methacholine in asthmatics[END_REF] has been nebulized for 15-20 min. through the mouthpiece with using of a nose clip. A nebulizer Pulmo Aide AP-50 (DeVilbiss; Richmond, VA) was used (mass median aerosol diameter 3.1 μm, output 0.3 ml/min.).

Determination of hydrogen peroxide (H 2 O 2 ) in EBC

The H 2 O 2 concentration in EBC was measured according to the method applied previously by Nowak et al. [START_REF] Nowak | Exhalation of H2O2 and thiobarbituric acid reactive substances (TBARs) by healthy subjects[END_REF] Briefly, 600 μl of EBC was mixed with 600 μl of HRP solution (1 U/ml) containing 100 mM homovanillic acid and was incubated for 60 min. at 37°C. Then, the sample was mixed with 150 μl 0. The absorbance was measured at 562 nm utilizing a microplate reader Sunrise (Tecan, Männedorf Germany). The concentration of NO 2 -in the samples was calculated utilizing the NO 2 -standard curves [START_REF] Dziedzic | Comparison of cadmium and enzyme-catalyzed nitrate reduction for determination of NO2-/NO3-in breath condensate[END_REF].

As NO 2 -and NO 3 -are present on every laboratory surface, including glassware and pipette tips, precautions were taken to avoid contamination of the sample. Any material that might come in contact with EBC, including devices used for collection, processing and assaying EBC was thoroughly rinse with highly pure (distilled/de-ionised) water [START_REF] Horvath | Exhaled breath condensate: methodological recommendations and unresolved questions[END_REF].

Determination of nitrate (NO 3 - ) in EBC
We modified the method described by Dziedzic et al. [START_REF] Dziedzic | Comparison of cadmium and enzyme-catalyzed nitrate reduction for determination of NO2-/NO3-in breath condensate[END_REF], for measurement of NO The frequency of coughing was assessed using a 10 cm coughing visual analogue scale with the extremes marked 1 -'I never cough' and 10 -'I am coughing all the time'. The visual analogue scales were administered before and after apocynin/placebo nebulization [START_REF] Leidy | The breathlessness, cough, and sputum scale: the development of empirically based guidelines for interpretation[END_REF].

Statistical analysis

The data from the study were analyzed utilizing Statistica software package (v.

Results

Safety measures

There were no significant changes in blood pressure, heart rate value and cough scale before and after apocynin or placebo application (p>0.05). No serious and non-serious adverse events were observed throughout the study.

Furthermore, we have analyzed an effect of apocynin inhalation on some parameters of blood differential counts (e.g.: white blood cells, red blood cells, hemoglobin, hematocrit, platelets, lymphocytes, monocytes and granulocytes) (Table 1). Neither apocynin nor placebo nebulization influenced blood differential counts.

Inhalation of apocynin or placebo aerosol had any effect neither on respiratory parameters (FEV 1 , FVC, PEF), nor on DLCO parameters (TLCOc/VA), (p>0.05), (Table 1). Apocynin caused significant changes in NO 3 -concentration in EBC as well. Moreover, we observed a significant decrease of NO 3 -concentration 60 and 120 min. after apocynin inhalation (Fig. 2c.), whereas placebo caused augmentation of NO 3 -concentration in these timepoints (6.8 μM after apocynin vs. 14.4 μM after placebo and 6.5 μM vs. 14.9 μM respectively, p<0.05). Before placebo inhalation, the concentration of nitrate was higher than before apocynin nebulization.

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Serum -NO 2 -concentration

The analysis of the whole group of volunteers showed that there is no nitrite concentration change in serum of healthy subjects after apocynin nebulization (p>0.05).

Discussion

In this study, we investigated the effect of nebulized apocynin administration at the dose of 3 mg on simultaneously hydrogen peroxide, nitrite and nitrate generation as well as safety parameters in healthy subjects. Using EBC technique we have confirmed that apocynin decreases H 2 O 2 exhalation. Apocynin caused a significant decline of H 2 O 2 concentration after 60 minutes of inhalant administration, not causing any side-effects. Furthermore, the inhibitory potency of this drug last at least 2h after apocynin nebulization.

The use of apocynin as an inhibitor of the activation of the NADPH oxidase complex is based on the inhibition of the assembly process, as the migration of the p47phox component to the membrane is impeded in its presence [START_REF] Barbieri | Apocynin prevents cyclooxygenase 2 expression in human monocytes through NADPH oxidase and glutathione redox-dependent mechanisms[END_REF]. It is also known that the oxidation of apocynin plays an important role in its inhibitory effect. apocynin revealed a potential value to limit peroxynitrite formation in inflammatory conditions in vivo [11]. Among others, in airways, there are some pivotal sources of superoxide radical -NADPH oxidase in inflammatory cells [START_REF] Matthiesen | Inhibition of NADPH oxidase by apocynin inhibits lipopolysaccharide (LPS) induced up-regulation of arginase in rat alveolar macrophages[END_REF], inhibited by apocynin, mitochondrial sources [START_REF] Zmijewski | Participation of mitochondrial respiratory complex III in neutrophil activation and lung injury[END_REF][START_REF] Poderoso | The formation of peroxynitrite in the applied physiology of mitochondrial nitric oxide[END_REF] and arachidonic acid (AA) metabolism [START_REF] Hii | Regulation of the NADPH oxidase activity and anti-microbial function of neutrophils by arachidonic acid[END_REF].

Peters et al investigated the effect of nebulized apocynin on ozone-induced bronchial hyperresponsiveness in vivo in humans. They demonstrated that apocynin reduced ozoneinduced airway hyperresponsiveness in mild asthmatics [START_REF] Peters | Effect of apocynin on ozone-induced airway hyperresponsiveness to methacholine in asthmatics[END_REF]. Furthermore, Hougee et al.

observed that oral administration of apocynin had inhibitory effects on cyclooxygenases similar to the non-steroidal anti-inflammatory drug (NSAID) ibuprofen [START_REF] Hougee | Oral administration of the NADPH-oxidase inhibitor apocynin partially restores diminished cartilage proteoglycan synthesis and reduces inflammation in mice[END_REF]. Nowak et al showed that only one third of never smoked subjects continuously exhale detectable amounts of H 2 O 2 [START_REF] Nowak | Exhalation of H2O2 and thiobarbituric acid reactive substances (TBARs) by healthy subjects[END_REF], and additionally, that exhalation of H 2 O 2 by healthy never smoked subjects correlates with the ability of blood phagocytes to produce ROS in general [START_REF] Szkudlarek | Exhaled hydrogen peroxide correlates with the release of reactive oxygen species by blood phagocytes in healthy subjects[END_REF]. Nevertheless, our results stay in agreement with Guatura's report [START_REF] Guatura | Increased exhalation of hydrogen peroxide in healthy subjects following cigarette consumption[END_REF], demonstrating that healthy nonsmoking subjects exhale detectable concentration of hydrogen peroxide. We showed here that it can be subsequently reduced by apocynin, not evoking any harmful effects. Such results may suggest that apocynin might be used to alleviate inflammatory process in some diseases.

Both H 2 O 2 and NO are associated with oxidative stress, which is defined as an imbalance between oxidants and antioxidants, and is thought to be involved in airway inflammation and respiratory diseases [START_REF] Psathakis | Exhaled markers of oxidative stress in idiopathic pulmonary fibrosis[END_REF] H 2 O 2 is generated from superoxide anion by the action of superoxide dismutase in neutrophils, eosinophils, macrophages and epithelial cells, and in particular, by eosinophils in asthma [START_REF] Ueno | Inflammatory markers in exhaled breath condensate from patients with asthma[END_REF][START_REF] Horvath | Combined use of exhaled hydrogen peroxide and nitric oxide in monitoring asthma[END_REF][START_REF] Conner | Lactoperoxidase and hydrogen peroxide metabolism in the airway[END_REF] Exhaled nitric oxide (eNO) is a marker of airway inflammation [START_REF] Kharitonov | Reproducibility of exhaled nitric oxide measurements in healthy and asthmatic adults and children[END_REF]. Although NO is produced in the airways by a variety of cells, large amounts may be generated from those involved in the acute and chronic inflammatory responses [START_REF] Puthucheary | Exhaled nitric oxide is decreased by exposure to the hyperbaric oxygen therapy environment[END_REF]. NO plays a role in pulmonary host defence mechanisms, and is thought to have both bactericidal and bacteriostatic effects [START_REF] Hucke | Nitric oxidemediated regulation of gamma interferon-induced bacteriostasis: inhibition and degradation of human indoleamine 2,3-dioxygenase[END_REF].

Nitric oxide is difficult to measure because it is a free radical which reacts rapidly with oxygen, superoxide, water, thiols, amines, and lipids to form products with biochemical activities ranging from bronchodilation to cytotoxicity [START_REF] Gaston | The biology of nitrogen oxides in the airways[END_REF][START_REF] Liu | Exhaled breath condensate as a method of sampling airway nitric oxide and other markers of inflammation[END_REF]. Nitrates and nitrites are products of nitric oxide metabolism, which can be detected in EBC.

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10 Since simultaneous NO 2 -and NO 3 -measurements provide better indication of NO production in respiratory system, in our study both of those parameters were investigated [START_REF] Dziedzic | Comparison of cadmium and enzyme-catalyzed nitrate reduction for determination of NO2-/NO3-in breath condensate[END_REF].

Unexpectedly, there were considerable differences in NO 2 -and NO 3 -concentrations after apocynin as well as after placebo nebulization. Though NO 2 -concentration slightly increased after 30 minutes of inhalation and after 60 min it decreased, the same effect has been observed when apocynin and placebo were nebulized. These results confirm previous data [START_REF] Vejrazka | Apocynin inhibits NADPH oxidase in phagocytes but stimulates ROS production in non-phagocytic cells[END_REF], which displayed that, apocynin at first stimulates ROS formation, however, after a certain period, the inhibition of ROS yield occurs. The same trend might appear in case of placebo because of the activation of alveolar phagocytes caused by mild exertion connected with EBC collection, or because of nebulization effect. NO 2 -may also originate from alveolar epithelium [START_REF] Lundeberg | Nitric oxide in exhaled air[END_REF]. Still, there is not much data showing EBC time point study. Nevertheless, Bodini et al., [START_REF] Bodini | Flunisolide decreases exhaled nitric oxide and nitrotyrosine levels in asthmatic children[END_REF] also observed that the levels of several analyzed parameters, i.e. nitrotyrosine, in EBC changed after placebo treatment at the two time points of the study. These results might suggest that there is a need to evaluate an influence of EBC collection on inflammatory parameter levels as an effort which might accompany EBC collection may also have an influence on EBC contents.

Furthermore, our results suggest, that apocynin causes significant decrease of NO 3 concentration. Apocynin entailed gentle growth of NO 3 -level, as reported before [START_REF] Vejrazka | Apocynin inhibits NADPH oxidase in phagocytes but stimulates ROS production in non-phagocytic cells[END_REF], but then it firmly reduced it. These data confirm antiinflammatory capabilities of apocynin, which have been claimed in literature. Additionally, no adverse effects occurred during and after apocynin nebulization.

According to Szkudlarek et al. [START_REF] Szkudlarek | Exhaled hydrogen peroxide correlates with the release of reactive oxygen species by blood phagocytes in healthy subjects[END_REF], ability of blood polymorphonuclear leukocytes (PMNs) to produce ROS, may mirror H 2 O 2 release from cells localized in the close neighborhood of lower airways epithelial lining fluid. Nevertheless, this rule is not reflected in case of nitrite.

Our research displayed, that there was no significant alteration of NO 2 -concentration in blood serum after apocynin inhalant application. Additionally, no changes in blood differential counts have been noticed. This may suggest that nebulized apocynin does not diffuse into the blood, and acts locally [START_REF] Lafeber | Apocynin, a plant-derived, cartilage-saving drug, might be useful in the treatment of rheumatoid arthritis[END_REF][START_REF] Stolk | Characteristics of the inhibition of NADPH oxidase activation in neutrophils by apocynin, a methoxy-substituted catechol[END_REF].

Apocynin did not affect respiratory parameters as well. No lung functional tests parameters have been changed after apocynin administration. Peters et al, [START_REF] Peters | Effect of apocynin on ozone-induced airway hyperresponsiveness to methacholine in asthmatics[END_REF] also showed that the change in FEV 1 immediately followed the ozone exposure in asthmatics, was not significantly different between apocynin and placebo group.

The fact that apocynin has no influence on DLCO might show that it not causes changes in airways gas diffusion. These findings may have important clinical implications for the safety We are aware of several limitations that apply to our study. The study contained relatively small group of subjects (n=10), however, there are many significant studies on such few objects. Additionally, we shortly intend to continue extend our research.

We are also aware, that except for nitrite concentration, nitrate and hydrogen peroxide should have been investigated in serum. Nevertheless as apocynin showed not to infiltrate into blood and influence on NO 2 -concentration, we concluded that it is case might be omitted.

One of the current limitations of EBC measurements is the low concentration of many biomarkers so that their measurement is limited by the sensitivity of assays.

One relative disadvantage of EBC measurements is that they require a subsequent analysis and it is likely that there will be important advances in on-line detection of particular biomarkers using sensitive biosensors. On the other hand, an EBC collection allows detecting several biomarkers at one time, without harmful actions to the patients. It also could become a useful tool for monitoring and screening of healthy individuals for possible early pulmonary tissue damage. EBC collection is a completely noninvasive way of sampling the respiratory tract that can be repeated frequently within short intervals without adverse events with good reproducibility in EBC volume and mediator concentration for several tested markers Moreover, the fact that there is not EBC collection standard might have triggered differences in research, dependently on technique and equipment applied [START_REF] Horvath | Exhaled breath condensate: methodological recommendations and unresolved questions[END_REF]. Therefore, we adapted a modification of the method described previously by Nowak et al. [START_REF] Nowak | Exhalation of H2O2 and thiobarbituric acid reactive substances (TBARs) by healthy subjects[END_REF], as in this study results have been repetitive.

The significant limitations in our study are the differences in baselines before nebulization.

Ideally, baseline values should not differ. The possible explanation of this fact might be variability among individuals. Nevertheless, the concentrations difference is not statistically significant.

In conclusion, we displayed that 3 mg of nebulized apocynin decreased H 2 O 2 and NO 3 concentrations in EBC of healthy subjects. Nebulized apocynin was well tolerated and no adverse events were observed throughout the study. Thus, it might be a valuable antiinflammatory agent, acting locally in airways with very low toxicity and neutral to other physiological parameters. Nevertheless, there is a need to evaluate the antiinflammatory and safety potential of apocynin, not only in healthy subjects but also in the state of disease.

  stay in agreement with literature, as apocynin has been shown to act as a strong antiinflammatory agent. Muijsers et al. showed that apocynin inhibited peroxynitrite formation in murine macrophages model. Though this experiment was performed in vitro,

  inflammatory diseases like asthma or COPD. Nevertheless, further investigations, involving patients with airway inflammatory diseases, are needed, especially as apocynin does not cause any severe and non-severe adverse effects.

  Figures legend:

  μmol/l) were prepared freshly in deionized water and kept on ice prior to use. 60 μl of NaNO 2 standard or EBC were dispensed in duplicate. Control wells contained 60 μl of deionized water. The volume was made up to 100 μl with phosphate-buffered saline (PBS) pH 7.2. For colour development, the samples were incubated for 10 min. at room temperature with 40 μl of Griess solution A (58.07 mmol/l sulphanilamide, Sigma, St. Louis, MO, USA in 2 M sulphuric acid) and then with 40 μl of Griess solution B (38.58 mmol/l naphthylethylenediamide dihydrochloride in deionized water; Sigma, St. Louis, MO, USA).

		ARTICLE IN PRESS
	12.5
		M A N U S C R I P T
	(Norwalk, CT, USA). Excitation was at 312 nm and emission was measured at 420 nm. The lower limit of H 2 O 2 detection was 0.08 μM. The intra-assay variability was 1-2.1% for A C C E P T E D
	standard solutions of 0.05-0.75 μM H 2 O 2 . Data were expressed in μM [2].
	Determination of nitrite (NO 2	-) in EBC
	Determination of NO 2
		6

1 M glycine-NaOH buffer (pH 12.0) with addition of 25 mM EDTA. The homovanillic acid oxidation product as a measure of the amount of H 2 O 2 was determined spectrofluorimetrically using a Perkin Elmer Luminescence Spectrometer LS-50B -with Griess solution was performed by micromethod, carried out in 98-well plates, according to Griess' method

[START_REF] Dziedzic | Comparison of cadmium and enzyme-catalyzed nitrate reduction for determination of NO2-/NO3-in breath condensate[END_REF]

. NO 2 -standards (concentration range: 0.5-

  Doganay et al. [38]. 100 μl of serum was added in duplicate to an Eppendorf tube. Control wells contained 100 μl of deionized water. The samples were mixed with 50 μl 10 min.) and with 125 μl of Griess B solution. Afterwards, 250 μl of 10 % TCA were added, shaked vigorously, and centrifuged for 15 min., 20,000 x g. The absorbance was measured by microplate reader Sunrise (Tecan, Männedorf Germany).

	ARTICLE IN PRESS
	(incubated for Safety parameters
	[37]. Determination of nitrite (NO 2 The concentration of NO 2 -in serum was measured according to the method of Griess, -) in serum A C C E P T E D described by M Peripheral blood differential counts were measured using an ABX Micros OT 45 system (Horiba ABX, Montpellier, France). Master-Laboratory Screen (Jaeger Master Screen Body System, Wuerzburg, Germany) was used for lung functional tests including forced vital capacity (FVC), forced expiratory volume in the first second (FEV1), single breath carbon monoxide diffusing capacity corrected for hemoglobin and alveolar volume (TLCOc), A N U S C R I P T according to ERS/ATS standards (2006).
	7

3

-using the NADPH-nitrate reductase (EC 1.6.6.2 from Aspergillus species, Sigma, St. Louis, MO, USA). Sixty microliters of EBC was added in duplicate to a flat bottom 96 well plate. Control wells contained 60 μl of deionized water. The samples were mixed with 30 μl of NADPHnitrate reductase (250 mU/ml dissolved in deionized water) and 10 μl of NADPH solution (0.625 mg/ml in deionized water, Sigma, St. Louis, MO, USA). The plates were incubated for 30 min. at room temperature. Then, the samples were mixed with Griess solutions and the absorbance was measured by microplate reader Sunrise (Tecan, Männedorf Germany). NO 3 concentration was calculated utilizing the method described previously by Dziedzic et al.

of PBS (PH 7.2), 50 μl of NADPH-nitrate reductase (250 mU/ml in deionized water) and 17 μl of NADPH solution (0.625 mg/ml in deionized water). The plates were incubated for 30 min. in room temperature. Then, the samples were mixed with 125 μl of Griess A solution

Table 1 .

 1 Apocynin effects on safety parameters in group of 10 healthy subjects. PFTs parameters data shown are the median±1 st QR; comparison of FEV 1 , PEF and FVC values are presented. No significant effect to PFTs parameters has been observed. Peripheral blood differential counts are shown as the median±1 st QR. No significant differences were found (p>0.05). No effects to blood pressure, heart rate value and cough have been observed.

	9.5±8.6	9.3±9.0	9.4±8.2	9.6±8.3

Conflict of Interest:

The authors declare that there are no conflicts of interest.