Jose Alfredo Guerrero

Pedro Castillo

Yacine Challal

Quadrotors Formation Control: A Wireless Medium Access Aware Approach

In this paper, the impact of the medium access protocols on the average consensus problem over wireless networks for a group of quadrotors is established. The stabilization of each helicopter is guaranteed using a simple and bounded nonlinear control strategy. We study the case of a group of quadrotors communicating over a wireless network considering both directed and undirected graphs of information flow. It turns out that the media access control (MAC) protocols have a direct impact in both convergence time and average consensus solution, i.e. the solution of the average consensus is no longer the average of the initial conditions. It will be shown that the solution for the average consensus problem over a wireless network depends directly on the MAC algorithm. Simulations are provided to demonstrate the theoretical results. In addition, to validate the control strategy some experimental tests have been carried out to control the yaw angle of two quadrotors.

I. INTRODUCTION

Small Unmanned Aerial Vehicle (UAV) teams are excellent candidates for improving efficiency and reducing risk in search and rescue missions in unknown or dangerous environments. The deployment of small UAV teams offers certain advantages over both individual vehicles and vehicles of larger size. Small vehicles can be more easily and cheaply deployed than larger ones, and can maneuver effectively in confined spaces. Size restrictions necessarily affect payload and range capabilities, but for applications in a finite domain that is not easily accessible, the small multi-vehicle platform can be quite advantageous for rescuers. In particular, the maneuverability and speed of small rotorcraft enable their use in wilderness areas over uneven and forested areas, as well as in and around buildings for fire or natural disaster rescue in urban environments.

In the particular case of multi-agent (multiple aerial vehicle) consensus problem, most of the literature is focused on modeling the information flow network using either fixed or switching topologies [START_REF] Olfati-Saber | Flocking for Multi-Agent Dynamic Systems: Algorithms and Theory[END_REF], [START_REF] Tanner | Stable Flocking of Mobile Agents, Part I: Fixed Topology[END_REF], [START_REF] Tanner | Stable Flocking of Mobile Agents, Part II: Dynamic Topology[END_REF], [START_REF] Lee | Stable Flocking of Multiple Inertial Agents on Balanced Graphs[END_REF], [START_REF] Gazi | Stability of an Asynchronous Swarm With Time-Dependent Communication Links[END_REF], among others. This means that the communication network does not suffer of time delay and packet drop problems. The main difference between fixed and switching topologies is that the switching topology case takes into account that every aerial vehicle has a limited range of interaction with its neighbors, i.e., the mobility of the aerial vehicles affects the information exchange topology. laboratory UMR 6599 CNRS-UTC, BP. 20529, 60200 Compiègne, France (jguerrer, castillo, ychallal)@hds.utc.fr. This work was supported by the SIRENE-CARNOT project.

The distributed nature of multiple robot control system over wireless networks represent an interesting research problem. Data loss, data corruption and time delay over lossy network are key factors that may lead to performance degradation and even cause instability. Recent work on networked control over noisy communication channels includes [START_REF] Sahai | The necessity and sufficiency of anytime capacity for control over noisy cummincation link[END_REF], [START_REF] Takikonda | Control over Noisy Channels[END_REF]. In most of the cases the packet drop phenomena is modeled as a random process without any specification of its probabilistic distribution [START_REF] Lin | Robust Consensus of Mu lti-Agent Systems with Noise[END_REF], [START_REF] Zhang | Robust Consensus for Uncertain Multi-agent Systems on Directed Communication Topologies[END_REF]. In [START_REF] Hu | Stability Robustness of Networked Control Systems with Respect to Packet Loss[END_REF], the authors consider the packet drop process as a Bernoulli process and develop stability conditions under these conditions. Another way to model the packet loss phenomena has been described in [START_REF] Ling | Soft Real-Time Scheduling of Networked Control Systems with Dropouts Governed by a Markov Chain[END_REF], where a Markov chain has been used to model the packet dropout process. This paper addresses the issue of packet loss as well as packet delay in a multi-agent aerial system considering a wireless network. For this end, we use the widely adopted network simulator (NS2) which provides a rich simulation environment modeling the different network communication layers: the physical layer (modulation, frequencies, signal and radio propagation models, wired and wireless channels, ...), link layer (different medium access control algorithms: TDMA, CSMA/CD, CSMA/CA, ALOHA, ...), routing layer (routing protocols over wired and wireless networks as well as mobile ad hoc networks), transport layer (TCP, UDP, RTP, etc.), and the application layer with a rich sample of applications for typical traffic generation scenarios. It will be assumed, for simplicity, that every agent in the multivehicle system broadcasts its information to its neighbors considering a fixed topology of information exchange, i.e. the mobility of the vehicles does not affect the information flow network. It is assumed that the neighbors of the i th -quadrotor are always in broadcasting range. Different network media access control protocols have been designed for wireless networks. Likewise, in this paper, we analyze the impact of the following algorithms on the multiple quadrotor average consensus problem: Carrier sense multiple access with collision avoidance (CSMA/CA) and time division multiple access (TDMA). This outline of the paper is as follows: A brief description of the multi-quadrotor dynamics and control over perfect communication links is introduced in section II. In Section III, the multi-quadrotor average consensus over wireless network analysis is presented. A stability analysis to improve the convergence of the consensus is proposed in section IV. Simulation results are illustrated in section V. And finally, conclusion and future work are discussed in section VI.

A. Quadrotor dynamic model and control scheme

Let us consider a group of N -quadrotor helicopters with the following dynamical model [START_REF] Lozano | Unmanned Aerial Vehicles Embedded Control[END_REF], [START_REF] Guerrero | Flight Formation of Multiple Rotorcrafts[END_REF]:

zi = F i cos(θ i ) cos(φ i ) -1; ψi = u ψi (1a) ÿi = F i cos(θ i ); sin(φ i ) φi = u φi (1b) ẍi = -F i sin(θ i ); θi = u θi (1c)
where (x i , y i , z i ) and (φ i , θ i , ψ i ) represent the position and orientation of the i th -quadrotor, respectively. F i denotes the thrust force vector and u ψi , u φi , u θi the generalized torques.

To stabilize the previous system, we use the following control laws,

F i = -σ b1 i (k 1i ż) -σ b2 i (k 2i (z i -z d i )) + 1 cos(φ i ) cos(θ i ) (2a)
u ψi = -σ b3 i (k 3i ψi ) -σ b4 i (k 4i (ψ i -ψ d i )) (2b)
u θi = -σ b5 i (k 5i θi ) -σ b6 i (k 6i θ i ) + σ b7 i (k 7i ẋi ) + σ b8 i (k 8i x i ) (2c)
u φi = -σ b9 i (k 9i φi ) -σ b10 i (k 10i φ i ) -σ b11 i (k 11i ẏi ) -σ b12 i (k 12i y i ) (2d)
where

|σ bm i (s)| < b mi is a saturation function k mi , b mi > 0; m = 1, ..., 12
; are constant, z d i and ψ d i are the desired altitude and heading for the i th -quadrotor, respectively. It was proved in [START_REF] Sanahuja | Stabilization of n integrators in cascade with bounded input with experimental application to a VTOL laboratory system[END_REF] that the nonlinear control laws in (2) guarantee the stabilization of the i th -quadrotor in closed loop system such that

lim t→∞ z i = z d i ; lim t→∞ x i = 0 lim t→∞ ψ i = ψ d i ; lim t→∞ y i = 0

B. From individual to collective behavior

In order to model the interactions among helicopters, a graph-based theoretical approach has been considered.

Notice that the closed-loop system, ( 1)-( 2), for the i thquadrotor can be expressed with the following kinematic model ẋi = u i ∀i = 1, .., N ;

where x i is the state vector and u i represents the control input vector.

Leader-based multi-quadrotor consensus is achieved using the following algorithm

u i = - j∈Ni (x i -x j ) (3)
where N i is the set of vehicles transmitting their information to the i th -quadrotor. Noticed that (3) ensures the consensus agreement in the sense of lim t→∞ |x i -

x j | = 0.
Hence, the position consensus among quadrotors yields

ẋ = -Lx ( 4
)
where L is the Laplacian matrix of the information exchange graph, more details see [START_REF] Guerrero | Flight Formation of Multiple Rotorcrafts[END_REF], [START_REF] Guerrero | Robust Consensus Tracking of Leader-Based Multi-Agent Systems[END_REF]. Therefore, the controllers in ( 2) can be improved to the case of multi-quadrotor consensus with the form

Fi = -σ b 1 i (k1 i ż) -σ b 2 i k2 i j∈N i (zi -zj) -z d i + 1 cos(φi) cos(θi) (5a)
τ ψ i = -σ b 3 i (k3 i ψi) -σ b 4 i   k4 i   j∈N i (ψi -ψj) -ψ d i     (5b)
τ θ i = -σ b 5 i (k5 i θi) -σ b 6 i (k6 i θi) + σ b 7 i (k7 i ẋi) + σ b 8 i   k8 i j∈N i (xi -xj)   (5c)
τ φ i = -σ b 9 i (k9 i φi) -σ b 10 i (k10 i φi) -σ b 11 i (k11 i ẏi) -σ b 12 i k12 i ˙ j∈N i (yi -yj) (5d)
which implies that

lim t→∞ |(z j -z i )| = z d i ; lim t→∞ |(x j -x i )| = 0 lim t→∞ |(ψ j -ψ i )| = ψ d i lim t→∞ |(y j -y i )| = 0
In order to illustrate the performance of the previous control strategy, simulations were carried, over the x-axis, out considering a 10-quadrotor platoon over a perfect communication network (no delay, no packet loss) with cyclic topology. The initial conditions are x i (0) = i, ∀i = 0, 1, ..., 9. Since the xand yaxis have a similar behavior, only the x-axis consensus performance graphs will be presented.

Figure 2 shows the consensus response. Observe in this figure that the convergence time is small and the solution of the average consensus over the x-axis is the average of the initial conditions, i.e., the platoon achieves consensus to 4.5. Thus, the previous nonlinear control laws guarantees the position synchronization of the quadrotor platoon. In literature, several simulations using the previous (similar or different) controllers have been carried out in order to prove the stability of the consensus or of the flight formation trajectory, see [START_REF] Fax | Information Flow and Cooperative Control on Vehicle Formations[END_REF], [START_REF] Lafferriere | Decentralized control of vehicle formations[END_REF], [START_REF] Dunbar | Distributed receding horizon control for multi-vehicle formation[END_REF], [START_REF] Chen | ISS-Based Robust Leader/Follower Trailing Control[END_REF]. Most of the assumptions in these works are considering wireless and perfect communication between the vehicles. In addition, the main results in flight formation are in general illustrated only in simulations.

As it is known, in a real multi-vehicle flight formation system, each aircraft collects information from its sensors and then exchange its information, employing wireless communication, with other autonomous vehicles in the network. Packet delay and packet loss in wireless becomes a major issue of study that must be taken into account when stabilizing multi-aerial vehicles. The goal of this work is to analyze, the impact of the wireless network communication in the real-time multi-vehicle consensus problem.

III. MULTI-AGENT CONSENSUS OVER WIRELESS NETWORKS

Let us consider the case of a N-quadrotor formation flying over a wireless communication channel. From the automatic control point of view, the key factors when using wireless channels are: end-2-end time delay, packet dropout rate, network connectivity and noise. Let us assume that the mobility of the agents does not affect the network connectivity and neglect the noise from sensors. Then, we focus our attention on the packet dropout rate and the end-2-end time delay problems. It has been shown in the communications literature [START_REF] Lian | Performance Evaluation of Control Networks: Ethernet, ControlNet and DeviceNet[END_REF], [START_REF] Liu | Wireless medium access control in networked control systems[END_REF] that both packet dropout rate and the end-2-end time delay are determined by the Medium Access Control protocols. Our study considers two common technologies for wireless communications: CSMA/CA used in IEEE802.11 and TDMA used in GSM.

A. CSMA/CA

This is a distributed random access algorithm used in many standards such as Wifi IEEE802.11. This scheme uses a carrier sense multiple access with collision avoidance (CSMA/CA) mechanism for resolving the problem of access to the communication medium. This implies that when a node detects a collision, it stops transmitting and waits for a random time before retransmitting. More precisely, the protocol CSMA/CA works as follows [22]:

1) a carrier sensing scheme is used 2) a data station that intends to transmit sends a jam signal, 3) after waiting a sufficient time for all stations to receive the jam signal, the data station transmits a frame 4) while transmitting, if the data station detects a jam signal from another station, it stops transmitting for a random time and then tries again. We distinguish two variants: with and without channel reservation. In CSMA/CA with channel reservation, the transmitter first transmits a RTS (Request to Send) for channel reservation, and waits for receiving a CTS (Clear To Send) from the destination before starting to send the data frame.

CSMA/CA is not suitable for real time communications, since it does not guarantee a upper bound for the delay before sending a data frame.

B. TDMA

This is a time slotted scheme used in many standards such as GSM. Accordingly with [22], TDMA (Time Division Multiple Access) is a collision-free multiple access technique whereby users share a transmission medium by being assigned and using (one at a time) time slots assigned previously.

TDMA is more suitable for real time communications, since it guarantees a upper bound delay before transmitting a data frame.

C. Network Analysis

To evaluate the performance of CSMA/CA and TDMA protocols on the quadrotor consensus problem, extensive simulations have been run using the network simulator NS2. As it is shown in Figures 3 and4, it is clear that the packet drops for TDMA protocol are almost null. We attribute the packet drops shown in Figure 3 to the synchronization phase during the simulation initialization. Unlike TDMA, CSMA/CA shows a higher rate of packet drops due to its random broadcasting nature, see Figure 4. CONFIDENTIAL. Limited circulation. For review only.

Preprint submitted to 2012 International Conference on Unmanned Aircraft Systems. Received February 8, 2012. Now, let us analyze the average end-2-end delay which gives an insight of the expected time delay on multiquadrotor systems. It is known that in real-time applications, after a large time delay, data may become useless. Therefore it is important to analyze the performance of MAC protocols such that the multi-quadrotor system will undergo the minimal time delay. On one hand, since TDMA is a time division multiple access technique to access the transmission medium, it is almost intuitively that the time delay, should show an almost constant rate. This can be confirmed by observing Figure 5. On the other hand, taking into account the packet drop rate at each quadrotor, it is expected that CSMA/CA would show a variable time delay and the maximum time delay is much higher than TDMA. The evolution of the average end-2-end time delay over time for the CSMA/CA protocol is shown in Figure 5. It is worth to mention that the end-2-end time delay has been obtained at Agent trace level, i.e. from application to application layer. IV. QUADROTOR CONSENSUS OVER WIRELESS NETWORKS In this section, we can introduce a Network-aware average consensus control for a multi-quadrotor system over a wireless network. By taking into account the phenomena discussed above, the packet dropout process will be considered as a source of time delays in the wireless network. As shown in [START_REF] Lian | Performance Evaluation of Control Networks: Ethernet, ControlNet and DeviceNet[END_REF], the end-2-end time delay τ is given by the difference T dst -T src (time at destination and time at source, respectively) which in turns depends on the preprocessing time T pre , wait time T wait , transmission time T tx and the post processing time T post . Since the nearest neighbor approach assumes that each vehicle communicates only with immediate neighbors that are in its radio range, and assuming that a packet loss implies the re-transmission of the data that has been lost, we consider the packet dropout process, as part of the transmission time delay which has been defined as the frame time T f rame and the propagation time T prop , more details see [START_REF] Lian | Performance Evaluation of Control Networks: Ethernet, ControlNet and DeviceNet[END_REF].

Then, we propose to use the following multi-quadrotor consensus control as in [START_REF] Chopra | Output Synchronization of Mechanical Systems[END_REF]

u i (t) = - j∈Ni (x i -x j (t -τ ji )) (6)
Thus, (3) becomes

F i = -σ b 1 i (k 1 i ż) -σ b 2 i k 2 i j∈N i (z i -z j (t -τ ji )) -z d i + 1 cos(φ i ) cos(θ i ) (7a)
τ ψ i = -σ b 3 i (k 3 i ψi ) -σ b 4 i   k 4 i   j∈N i (ψ i -ψ j (t -τ ji )) -ψ d i     (7b)
τ θ i = -σ b 5 i (k 5 i θi ) -σ b 6 i (k 6 i θ i ) + σ b 7 i (k 7 i ẋi ) + σ b 8 i   k 8 i j∈N i (x i -x j (t -τ ji ))   (7c)
τ φ i = -σ b 9 i (k 9 i φi ) -σ b 10 i (k 10 i φ i ) -σ b 11 i (k 11 i ẏi ) -σ b 12 i k 12 i ˙ j∈N i (y i -y j (t -τ ji )) (7d)
It was proved that all the states of each agent, using (2) in closed-loop system, goes to the desired values or to zero. Then for each agent ∃ a storage function

V i (x i ) > 0 such that Vi (x i ) = -S i (x i ) with S i (x i ) ≥ 0. Therefore, considering the following Lyapunov function V = N i=1 j∈N t t-τji x T j (χ)x j (χ)dχ + 2 N i=1 V i (8)
this implies that,

V = -2 N i=1 Si(xi)- N i=1 j∈N i (xj(t-τji)-xi) T (xj(t-τji)-xi) (9)
From the above it follows that

lim t→∞ |(x j (t -τ ji ) -x i )| = 0 (10)
From [START_REF] Hu | Stability Robustness of Networked Control Systems with Respect to Packet Loss[END_REF], it is clear that the solution of the average consensus to the average of the initial conditions depends on the network medium access algorithm, which in turns determines the values of the τ ji for each quadrotor. Figure 6 shows the performance of the average consensus considering τ ji = τ kj . It can be observed that the average consensus is the same than the one for the perfect communication case. The difference is that the convergence time for the equal time delay case is larger than for the perfect communication case.

Figure 7 shows the performance of the average consensus considering τ ji = τ kj . It can be observed that the solution of average consensus is different from the previous two cases: perfect communication case and equal time delays case. Based on the law of large numbers, the average of the results from a large number of simulations should be closed to the expected value. Figure 7 shows the consensus over time of the average of 1000 simulations. Now, lets take a look at the average consensus performance using a wireless network. The network scenario consisted of 10-quadrotors sharing information over a cyclic topology. A quadrotor application was developed, a UDP transport protocol was modified to exchange information with the quadrotor application and the MAC layer as shown in Figure 8. The following MAC algorithms: CSMA/CA and TDMA were analyzed using the network simulator NS2. Figures 9 and10 show the performance of the MAC protocols CSMA/CA and TDMA respectively. From Figures 9 and10, it is possible to observe that the solution for the average consensus as well as its convergence time are affected by the network media access control algorithm. It can be concluded that the average consensus using a CSMA/CA protocol presents a larger convergence time due to both packet drops and time delay in the end-2end transmission among quadrotors. Also, due to the fact that the access to the transmission medium is assigned randomly, it is evident that some quadrotors will transmit their positions to their neighbors before their counterparts. This implies that the difference between any two τ ji can be large enough such that some quadrotors will evolve faster than the others. Then, the quadrotor with the smallest τ ji will update its position at a highest rate than the others. Figure 11 shows the performance of the average consensus for a small platoon of 4 quadrotors using different medium access control algorithms such as: CSMA/CA, TDMA, constant time delay (τ ji > 0), and perfect communication.

The simulation results are summarized in Table I In order to validate the control strategy, some experimental tests have been carried out using CSMA/CA. To simplify the experiments we focus mainly in the yaw dynamic of the helicopter. The experiment is developed as follows; first the helicopters are stabilized at hover with ψ di = 0. Then, we changed manually this desired value with the control radio only for one helicopter. We begin changing ψ d for helicopter 1, later for helicopter 2, and so on. The main result is illustrated in Figure 12. Notice from this figure, the delayed response of the second/first helicopter when it follows the first/second quadrotor. More details of the platesformes see [START_REF] Lozano | Unmanned Aerial Vehicles Embedded Control[END_REF]. Fig. 12. Average consensus for the yaw subsystem. An external reference was given to the system in order to show the delay over the wireless network

VI. CONCLUSIONS AND FUTURE WORK

A nonlinear control based on saturation functions and a single integrator consensus control considering time delay for flight formation of mini rotorcraft was developed. We remark that TDMA is more suitable for real time communications than CSMA/CA, since it guarantees a upper bound delay before transmitting a data frame. CSMA/CA is less suitable suitable for real time communications, since it does not guarantee an upper bound for the delay before sending a data frame. Extensive simulations were run in order to show the performance of the developed control scheme.

Future work in this area includes experimental tests on mini rotorcraft using real-time embedded control systems and to develop an optimized medium access control algorithm for mobile robot systems.

Fig. 1 .

 1 Fig. 1. Quadrotor platoon II. MULTI-QUADROTOR CONSENSUS

Fig. 2 .

 2 Fig. 2. Multiple quadrotor consensus using perfect communication

Fig. 3 .

 3 Fig. 3. Packet drop using TDMA. Simulation time = 60 sec.

Fig. 4 .

 4 Fig. 4. Packet drop using CSMA/CA. Simulation time = 60 sec.

Fig. 5 .

 5 Fig. 5. End-2-End delay comparison between CSMA/CA and TDMA. Simulation time = 60 sec.

Fig. 6 .

 6 Fig. 6. Multiple quadrotor consensus with τ ji = τ kj

Fig. 7 .

 7 Fig. 7. Multiple quadrotor with τ ji = τ kj

Fig. 8 .Fig. 9 .Fig. 10 .

 8910 Fig. 8. Multiple quadrotor wireless network stack

Fig. 11 .

 11 Fig. 11. Comparison of convergence time for different MAC algorithms

  Quadrotor 1 Quadrotor 2

Preprint submitted to 2012 International Conference on Unmanned Aircraft Systems. Received February 8, 2012.


Minh Hien Pham

Hervé Rhi!!

!icolas Auzeil

Anne Regazzetti

Djamel Eddine Harami

Daniel Scherma!

Dr Guy G Chabot
email: guy.chabot@parisdescartes.fr

Identification and induction of cytochrome P450s involved in the metabolism of flavone 8 acetic acid in mice

The metabolism of flavone-8-acetic acid (FAA) has been hypothesized to be partly responsible for its potent anticancer activity in mice. The purpose of this study was to identify the mouse enzymes involved in FAA Phase I metabolism and evaluate their possible induction in vivo by FAA. Mouse microsomes metabolized FAA into 6 metabolites: 3',4'dihydrodiol-FAA, 5,6-epoxy-FAA, 4'-OH-FAA, 3'-OH-FAA, 3',4'-epoxy-FAA and 6-OH-FAA. Using Cyp-specific inhibitors (furafylline, Cyp1a2; α-naphthoflavone, Cyp1b1; tranylcypromine, Cyp2b9; quercetin, Cyp2c29; quinidine, 2d9; diethyldithiocarbamate, Cyp2e1; ketoconazole, Cyp3a11), the formation of 5,6-epoxy-FAA was mainly attributed to Cyps 1a2, 1b1, 2b9, 2c29 and 2e1, whereas the 3',4'-epoxy-FAA was formed by Cyps 2b9 and 3a11. The 4'-OH-FAA was generated by Cyps 1a2, 1b1, 2b9 and 2e1, and the 6-OH-FAA was formed by Cyps 1b1 and 2c9. Using the epoxide scavenger N-acetyl cysteine, 4'-OH-FAA, 3'-OH-FAA and 6-OH-FAA were shown to derive partly from non enzymatic isomerisation of their corresponding epoxides. The specific epoxide hydrolase inhibitor elaidamide allowed the confirmation that 3',4'-dihydrodiol-FAA was formed via the epoxide hydrolase. FAA treatment in vivo in mice led to a significant increase in the hepatic expression of Cyp1a2 (1.9-fold), 2e1 (2.1-fold), 2b10 (3.2-fold), 2d9 (2.3-fold) and 3a11 (2.2fold), as evaluated by qRT-PCR. In conclusion, several Cyps were shown to be involved in FAA metabolism, particularly Cyps 3a11 and 2b9 which were responsible for the formation of the principal metabolites (5,6-epoxy-FAA, 3',4'-epoxy-FAA), and that FAA could induce the expression of several Cyps after in vivo administration. The possible implication of these enzymes in the in vivo anticancer activity of FAA in mice is discussed.

I!TRODUCTIO!

The flavonoids are natural compounds possessing a large range of biochemical and pharmacological properties [START_REF] Middleton | The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer[END_REF] including antitumoral activities [START_REF] Lopez-Lazaro | Flavonoids as anticancer agents: structure-activity relationship study[END_REF]. Among the flavonoids identified as potential anticancer agents, flavone-8-acetic acid (FAA; 2-phenyl-8-(carboxymethyl)benzopyran-4-one; NSC-347512; LM975; Figure 1-A) emerged as an interesting lead because of its potent anticancer activity in mouse solid tumors [START_REF] Corbett | Activity of flavone acetic acid (NSC-347512) against solid tumors of mice[END_REF][START_REF] Plowman | Flavone acetic acid: a novel agent with preclinical antitumor activity against colon adenocarcinoma 38 in mice[END_REF][START_REF] Bibby | Unique chemosensitivity of MAC 16 tumours to flavone acetic acid (LM975, NSC 347512)[END_REF][START_REF] Pratesi | Response of chemically induced primary colon tumours of the mouse to flavone acetic acid (NSC 347 512)[END_REF] and also in human tumors transplanted in nude mice [7;8]. However, despite its impressive antitumor activity reported in murine models, FAA has not shown anticancer activity in humans [9;10]. This marked interspecies difference in FAA anticancer activity has been hypothesized to be due to a possible metabolic activation of this flavonoid in vivo in mice [11;12].

The mechanism of anticancer activity of FAA in mice is not fully elucidated. FAA is weakly cytotoxic in vitro but is active in vivo when the same cell lines are transplanted in mice [START_REF] Capolongo | Antiproliferative properties of flavone acetic acid (NSC 347512) (LM 975), a new anticancer agent[END_REF][START_REF] Drewinko | The activity of flavone acetic acid (NSC 347512) on human colon cancer cells in vitro[END_REF][START_REF] Schroyens | In vitro chemosensitivity testing of flavone acetic acid (LM975; NSC 347512) and its diethylaminoethyl ester derivative (LM985; NSC 293015)[END_REF]. In view of this discrepancy between in vivo and in vitro effectiveness, several indirect mechanisms of anticancer action have therefore been suggested: augmentation of natural killer activity and induction of interferon [START_REF] Hornung | Augmentation of natural killer activity, induction of IFN and development tumor immunity during the successful treatment of established murine renal cancer using flavone acetic acid and IL-2[END_REF]; antiangiogenic properties [START_REF] Lindsay | Effect of flavone acetic acid on endothelial cell proliferation: evidence for antiangiogenic properties[END_REF]; tumor blood flow shut down and induction of extensive hemorrhagic necrosis [3;17-20]. At the molecular level, FAA has been reported to induce DNA damage in Glasgow osteogenic sarcoma after in vivo administration, which could hint to an in vivo formation of reactive metabolites in mice [START_REF] Bissery | Flavone acetic acid (NSC 347512)-induced DNA damage in Glasgow osteogenic sarcoma in vivo[END_REF].

In view of the poor cytotoxic activity of FAA in vitro and its potent anticancer activity in vivo in mice, the requirement for FAA metabolic activation has therefore been hypothesized [11;12]. In a search for potentially active FAA metabolites, we have previously identified six FAA phase I metabolites formed in vitro using mouse microsomes [START_REF] Pham | Identification of new flavone-8-acetic acid metabolites using mouse microsomes and comparison with human microsomes[END_REF]. The identified metabolites include three monohydroxylated derivatives (3'-OH-FAA, 4'-OH-FAA and 6-OH-FAA), two epoxides (3',4'-epoxy-FAA and 5,6-epoxy-FAA), and one dihydrodiol (3',4'-dihydodiol-FAA) (Figure 1-A) [START_REF] Pham | Identification of new flavone-8-acetic acid metabolites using mouse microsomes and comparison with human microsomes[END_REF]. We have also shown that human microsomes were unable to metabolize this compound to a significant extent, as compared with mouse microsomes [START_REF] Pham | Identification of new flavone-8-acetic acid metabolites using mouse microsomes and comparison with human microsomes[END_REF]. Although the mouse genome contains 72 putatively functional CYP genes compared to only 27 in humans [START_REF] Nelson | Comparison of cytochrome P450 (CYP) genes from the mouse and human genomes, including nomenclature recommendations for genes, pseudogenes and alternative-splice variants[END_REF], the cytochrome P450s (CYP) sub-families mostly involved in drug metabolism, i.e., the CYP1A, CYP2B, CYP2E, CYP3A and CYP4A appear to be roughly similar between mouse and man, although differences are observed in activity and also in inhibition studies [24;25].

Although mouse microsomes were shown to metabolize FAA, the enzymes responsible for their production are not presently identified. The purpose of the present study was therefore to identify the mouse enzymes involved in the formation of the principal FAA metabolites. The identification of the various mouse cytochrome P450s (Cyps, in lower case for mice) was accomplished using Cyp-specific inhibitors, and the implication of epoxide hydrolase was tested using elaidamide, a specific epoxide hydrolase inhibitor. In addition, because flavonoids have been shown to influence the expression of several Cyps after in vivo administration [START_REF] Hodek | Flavonoids-potent and versatile biologically active compounds interacting with cytochromes P450[END_REF], the influence of FAA treatment in mice on the expression of the principal hepatic Cyps was also investigated using quantitative RT-PCR. We have found that different sets of Cyps are involved in the formation of specific FAA metabolites and that FAA was shown to induce several hepatic Cyps after in vivo administration in mice.

MATERIALS A!D METHODS

Chemicals. Flavone-8-acetic acid (FAA, LM975, NSC347512, Fig. 1-A) and FAA mono-hydroxylated products at position 3' or 4' were kindly provided by Dr Jean-Jacques Berthelon (Merck-Lipha Santé, Lyon, France). The 6-OH-FAA was synthesized as previously described [START_REF] Pham | Identification of new flavone-8-acetic acid metabolites using mouse microsomes and comparison with human microsomes[END_REF] using appropriate starting material [START_REF] Dauzonne | A convenient synthesis of 3-chloro-3,4-dihydro-4hydroxy-3-nitro-2-phenyl-2H-1-benzopyrans[END_REF][START_REF] Dauzonne | Synthesis and in vitro cytotoxicity of a series of 3-aminoflavones[END_REF][START_REF] Bauvois | Synthesis and biological evaluation of novel flavone-8-acetic acid derivatives as reversible inhibitors of aminopeptidase N/CD13[END_REF]. The 3',4'-dihydrodiol-FAA, the 3',4'-epoxy-FAA and the 5,6-epoxy-FAA were generated in vitro using aroclor 1254 induced mouse microsomes according to the protocol described below. The following Cyp inhibitors were purchased from Sigma-Aldrich: furafylline (Cyp1a2), α-naphthoflavone (Cyp1b1), tranylcypromine (Cyp2c29, Cyp2c39), quercetin (Cyp2c29), quinidine (Cyp2d9), diethyldithiocarbamate (Cyp2e1) and ketoconazole (Cyp3a11). The specific epoxide hydrolase inhibitor elaidamide was synthesized and kindly provided by Dr Christophe Morisseau [START_REF] Morisseau | Inhibition of microsomal epoxide hydrolases by ureas, amides, and amines[END_REF]. Aroclor 1254 was obtained from Sigma-Aldrich (Saint Quentin Fallavier, France). All other chemicals were obtained from commercial suppliers and were of the highest purity available.

Preparation of aroclor induced mouse microsomes.

Aroclor-induced mouse microsomes were prepared according to the protocol described by Breinholt et al. [START_REF] Breinholt | In vitro investigation of cytochrome P450-mediated metabolism of dietary flavonoids[END_REF].

Female C57Bl/6 mice, 8 weeks of age were purchased from Janvier (Le Genest-St-Isle, France) and acclimated for a week in our animal facility and kept on a 12 h light/dark cycle with free access to food. Mice were injected intraperitoneally with aroclor-1254 (500 mg/kg body weight) dissolved in corn oil on day 0, and 5 days later the mice were sacrificed by cervical dislocation after a 24 h fasting period. All animal experiments complied with the French regulations concerning the protection of animals used for experimental and other scientific purposes (D2001-486), and with the European Commission regulations (OJ of ECL358 12/18/1986). The hepatic microsomes were prepared as described by Guengerich [START_REF] Guengerich | Microsomal enzymes involved in toxicology -Analysis and separation[END_REF]. Briefly, the livers were immediately excised and rinsed in ice-cold KCl 1.15 % solution, pooled and cut into small pieces with scissors. Four volumes of 0.1 M phosphate buffer (pH 7.4) were added and the livers homogenized using a Teflon potter at a speed of 50 rpm with 10 strokes. The liver homogenate was centrifuged at 15,000 × g for 20 min. After lipid elimination, the supernatant was ultra-centrifuged at 100,000 × g during 60 min, at 4°C. The pellet containing the microsomes were collected, covered by a phosphate buffer containing 20% glycerol, snap frozen on dry ice, and stored at -80°C until use. Total microsomal protein was determined using the Pierce BCA Protein Assay Reagent Kit (Rockford, IL,USA).

In vitro metabolism of FAA. Metabolism of FAA was carried out according to our previously described protocol [START_REF] Pham | Identification of new flavone-8-acetic acid metabolites using mouse microsomes and comparison with human microsomes[END_REF]. In brief, FAA (8 µg/ml final concentration) was incubated in 300 µl TRIS-HCL 0.1 M buffer, pH 7.4, containing 0.5 mg/ml microsomal protein, 1.6 mg/ml BSA, 5 mM MgCl 2 and a NADPH generating system corresponding to 1.3 mM. The incubation was carried out in opened 2 ml polypropylene tubes in a shaking water bath (150 oscillations/min) at 37°C. The reaction mixture was pre-warmed at 37°C for 3 min before the NADPH generating system was added and the incubation time was extended for an additional 60 min. The generation of metabolites was linear with time up to 2 h and protein concentration up to 2 mg/ml of microsomal protein, therefore the conditions used in the inhibition experiments (1 h and 0.5 mg/ml protein) were within the linear range. The incubation mixture was extracted twice with 800 µl ethyl acetate containing 2% acetic acid (v/v), the organic phase was evaporated under nitrogen and kept in dry form at -80°C, and then reconstituted with 300 µl of the HPLC mobile phase immediately prior HPLC analysis.

One hundred µl of the reconstituted residue were injected onto the Shimadzu HPLC system.

In these incubation conditions, mouse aroclor-induced microsomes could metabolize 4.3 ± 0.3 % of the FAA.

Determination of mouse Cyp enzymes involved in the FAA metabolism in vitro.

To determine mouse Cyps involved in the FAA metabolism, FAA metabolism by aroclorinduced mouse microsomes in vitro was carried out in absence or presence of various concentrations of specific Cyp inhibitors. The mouse Cyp orthologs corresponding to their human CYP counterparts are as follows [START_REF] Nelson | Comparison of cytochrome P450 (CYP) genes from the mouse and human genomes, including nomenclature recommendations for genes, pseudogenes and alternative-splice variants[END_REF]: Cyp1a2, CYP1A2; Cyp1b1, CYP1B1; Cyp2b9, CYP2B6; Cyp2c39, CYP2C19; Cyp2d9, CYP2D6; Cyp2e1, CYP2E1; Cyp3a11, CYP3A4.

The specific Cyp inhibitors used were: furafylline (Cyp1a2), α-naphthoflavone (Cyp1b1) [START_REF] Shimada | Selectivity of polycyclic inhibitors for human cytochrome P450s 1A1, 1A2, and 1B1[END_REF], tranylcypromine (Cyp2c29), quinidine (Cyp2d9) [START_REF] Harris | Metabolism of taxol by human hepatic microsomes and liver slices: participation of cytochrome P450 3A4 and an unknown P450 enzyme[END_REF], diethyldithiocarbamate (Cyp2e) [START_REF] Guengerich | Role of human cytochrome P-450 IIE1 in the oxidation of many low molecular weight cancer suspects[END_REF] and ketoconazole (Cyp3a11) [START_REF] Maurice | Effects of imidazole derivatives on cytochromes P450 from human hepatocytes in primary culture[END_REF]. Furafylline, α-naphthoflavone and ketoconazole were dissolved in DMSO, quercetin was dissolved in methanol, and tranylcypromine, quinidine, and diethyldithiocarbamate were dissolved in Tris-HCl buffer. Appropriate controls were run for the solvents which did not exceed 2% in volume in the control tube and in the test tube.

The choice of inhibitors range concentrations were based on Bogaards et al. [START_REF] Bogaards | Determining the best animal model for human cytochrome P450 activities: a comparison of mouse, rat, rabbit, dog, micropig, monkey and man[END_REF] (and references therein). The inhibitors were added to the incubation mixture containing microsomes 10 min before the addition of the NADPH generating system. The incubation and extraction conditions were carried out as described above for aroclor-induced mouse microsomes.

Epoxide hydrolase inhibition. Because the 3',4'-dihydrodiol-FAA was likely generated from the hydolysis of the 3',4'-epoxy-FAA by the epoxide hydrolase, we determined the role of this enzyme using elaidamide, an epoxide hydrolase inhibitor [START_REF] Morisseau | Inhibition of microsomal epoxide hydrolases by ureas, amides, and amines[END_REF], at final concentrations of 2, 4 and 10 µM.

!on enzymatic transformations. Non-enzymatic transformations during FAA metabolism incubation were determined by analysis of transformation of FAA metabolites in PBS buffer. FAA metabolites extract was reconstituted in 300 µl PBS buffer incubated at 37°C in a shaking water bath and injected onto the Shimadzu HPLC system at time 0 and 60 min.

Evaluation of direct or indirect hydroxylation.

In order to determine if FAA monohydroxylates were formed directly from FAA or indirect via epoxidation [START_REF] Guengerich | Cytochrome P450 oxidations in the generation of reactive electrophiles: epoxidation and related reactions[END_REF], FAA metabolism by aroclor-1254 induced mouse microsomes was carried out in presence of the epoxide scanvenger N-acetyl cysteine (NAC) according to the protocol described by Bu et al. [START_REF] Bu | Human in vitro glutathionyl and protein adducts of carbamazepine-10,11-epoxide, a stable and pharmacologically active metabolite of carbamazepine[END_REF]. In brief, NAC was added to the the pre-warmed incubation mixture at a final concentration of 7.5 mM. FAA metabolites and NAC adducts were further analyzed on the LC/MS systems described below.

HPLC UV and HPLC UV MS conditions. Two HPLC systems were used during this project. The first, HPLC-UV system was a Shimadzu CLASS-VP ® (version 5.3), equipped with a C 18 column (Beckman Ultrasphere ODS, 5 µm; 4.6 × 25 mm) thermostated at 20°C, and a UV detector set at 300 nm. The mobile phase consisted of a solution of 26% methanol, 19% acetonitrile, 55% aqueous acetic acid (2%), at a flow rate of 1 ml/min. The second system of LC-MS/MS consisted of a Surveyor  HPLC equipped with a UV diode array detector and was coupled with a mass spectrometer (LCQ-Advantage  , ThermoElectron). The mass spectrometer consisted of an electrospray ionization system used in positive ion polarity mode and a quadrupole ion trap mass analyzer. The operating parameters where as follows: the spray needle voltage was set at 4.0 kV and the spray was desolvated with a nitrogen sheath gas. Electrospray capillary voltage was 4.0 V, nitrogen was used as auxilliary gas and the capillary temperature was 250°C. A C 18 reversed phase column (EC. 250/2 Nucleodur Sphinx RP, 5 µm, 250 mm X 2.1 mm, Macherey-Nagel) thermostated at 30°C was used. The metabolites were resolved using the following tertiary gradient: from 0 to 25 min: 7-24% methanol; 4-13 % acetonitrile; 89-63 % TFA 0.1 %. From 25 to 40 min: 24-26 % methanol; 13-14 % acetonitrile; 63-60 % TFA 0.1 %. From 40 to 42 min: 26-45 % methanol; 14-22 % acetonitrile; 60-33 % TFA 0.1 %. From 42 to 47 min: 45 % methanol; 22 % acetonitrile; 33 % TFA 0.1 %. From 47 to 49 min: 45-7 % methanol; 22-4 % acetonitrile; 33-89 % TFA 0.1 %. And, finally from 49 to 75 min: 7 % methanol; 4 % acetonitrile; 89 % TFA 0.1 %. Flow rate was 0.2 ml/min. Volume of injection was 50 µl. The Xcalibur ® software was used for data acquisition and analysis.

In vivo treatment with standard Cyp inducers and FAA. Female C57Bl/6 mice, 8 weeks of age were purchased from Janvier (Le Genest-St-Isle, France) and acclimated for a week in our animal facility and kept on a 12 h light/dark cycle with free access to food. The mice were injected i.p. (intraperitoneally) in a volume of 0.2 ml with the following inducers at the indicated doses and schedules (five mice per group): Control A mice received 0.9% saline i.p. for 3 consecutive days; Control B mice received corn oil i.p. on 3 consecutive days; 3-Methylcholanthrene, 80 mg/kg, i.p., suspended in corn oil was administered for 3 consecutive days (Monday, Tuesday, Wednesday) [START_REF] Pan | Use of a novel real-time quantitative reverse transcriptionpolymerase chain reaction method to study the effects of cytokines on cytochrome P450 mRNA expression in mouse liver[END_REF], β-naphthoflavone, 80 mg/kg/day, i.p., suspended in corn oil, for 3 consecutive days [START_REF] Martignoni | An in vivo and in vitro comparison of CYP gene induction in mice using liver slices and quantitative RT-PCR[END_REF]; Dexamethasone, 100 mg/kg/day, i.p., suspended in corn oil, for 3 consecutive days [START_REF] Martignoni | An in vivo and in vitro comparison of CYP gene induction in mice using liver slices and quantitative RT-PCR[END_REF]; for all the previous treatments, the mice were sacrificed on the fifth day after an overnight fasting. Aroclor 1254 (500 mg/kg, dissolved in corn oil) was administered on day 1, and the mice were sacrificed on the 5th day after an overnight fasting [START_REF] Breinholt | In vitro investigation of cytochrome P450-mediated metabolism of dietary flavonoids[END_REF]. Phenobarbital was administered in drinking water (0.1 %) for 10 days, and the mice were sacrificed on the 11th day after an overnight fasting. For FAA, two treatment schedules were tested: FAA treatment A, consisted of one i.p. one dose on day 1 (180 mg/kg), and the mice were sacrificed on day 5 after an overnight fasting [3;4] FAA treatment B included 3 i.p. administration (at 180 mg/kg each) on days 1, 3 and day 5, and the mice were sacrificed on day 9 after an overnight fasting [3;4]. Immediately upon sacrificing the mice (CO 2 ), the liver was quickly removed, rinsed with ice cold PBS, and aliquots of each liver lobe were immediately transferred to a tube containing a RNAlater solution (QIAGEN) and were kept at 4°C for further extraction of total RNA. Quantitative RT PCR. Extraction of total RNA was accomplished using RNable (Eurobio), and one microgram of total RNA, as measured with Ribogreen measurement, was reverse transcribed in a final volume of 20 µl containing 4 µL of 5 X RT buffer, 20 units of RNasin RNase inhibitor (Promega), 10 mM DDT, 100 units of Superscript II RNase Hreverse transcriptase (Invitrogen), and 3 mM random hexamers (Invitrogen). The samples were incubated at 20°C for 10 min, 42 °C for 30 min, and 99 °C for 5 min. Real-time PCR reactions were carried out using ABI PRISM 7900HT Sequence Detection System (Applied Biosystems) in a 384-well, clear optical reaction plate with optical adhesive covers (Applied Biosystems). The PCR primers for the genes of interest were designed with the Oligo6 software using the GenBank reference sequences (Table 1). They were chosen in such a manner to be in distinct exons, to ensure a specific cDNA versus genomic DNA amplification, and their specificity was assessed using the NCBI BLAST website. Reactions were run in a 5 µl volume in duplicate, with 2 µL of cDNA solution, 2.5 µL of Power SYBR Green Master Mix (Applied Biosystems) and 0.5µL of 10 µM primers solution. The PCR program was: 95°C for 10 min, followed by 45 cycles of (15 seconds at 95°C; 1 min at 60°C).

Cycle threshold values were obtained from the ABI PRISM 7900 SDS software. For each PCR well, raw fluorescence data were exported and a curve-fitting method was applied to evaluate the individual efficiencies [START_REF] Rutledge | Sigmoidal curve-fitting redefines quantitative real-time PCR with the prospective of developing automated high-throughput applications[END_REF]. For each target the efficiency was considered as shared for all wells. This efficiency was set as the mean of the fitted individual efficiencies.

Individual Ct values exported from the SDS software were used to calculate the relative expression level of target t in sample S:

s t s Ct t eff ) , ( s t, L - =
, Ns being the normalization factor for sample S, here corresponding to the total cDNA level as measured with Oligreen. Oligreen fluorescence measurements were carried out using the ABI PRISM 7900HT Sequence Detection System. Reactions were run in a 10 µl volume in triplicate, with 5µL of cDNA solution and 5 µL of Oligreen stock solution (Invitrogen) 200x diluted in TE buffer.

Fluorescence was continuously read at 80°C for 30 sec, a condition which ensures exclusive cDNA measurement [START_REF] Rhinn | One-step quantification of single-stranded DNA in the presence of RNA using Oligreen in a real-time polymerase chain reaction thermocycler[END_REF].

RESULTS

FAA metabolism using aroclor induced mouse microsomes. Figure 1-A presents the structures of FAA metabolites formed by aroclor-induced mouse microsomes, and Figure 1-B depicts a typical HPLC-UV chromatogram of FAA and its major phase I metabolites. The parent compound FAA eluted at 17.1 min, whereas the six previously identified metabolites [START_REF] Pham | Identification of new flavone-8-acetic acid metabolites using mouse microsomes and comparison with human microsomes[END_REF], that were all more hydrophilic than FAA, eluted at the following retention times in this reversed-phase HPLC system (Figure 1 Mouse Cyp enzymes involved in FAA metabolism. In order to identify the mouse Cyps involved in FAA metabolism, incubations were carried out in absence or presence of increasing concentrations of the Cyp-specific inhibitors presented in Table 2. The possible interference between FAA metabolites and inhibitors (or their possible metabolites) was also assessed by incubating the inhibitors alone with mouse microsomes.

Furafylline, a Cyp1a2 inhibitor, was tested in the concentration range of 5, 10, 20 and 50 µM. In absence of furafylline, all the above identified FAA metabolites were detected (Fig. 2-A). In presence of furafylline (Rt, 5.2 min), which did not interfere with the elution profile of FAA metabolites (Fig. 2-B), all the FAA metabolites were reduced up to maximal values of 36 to 57% at the maximal inhibitor concentration used (50 µM) (Table 2). However, because high inhibitor concentrations may lead to unspecific inhibition, the intermediate 10 µM concentration appears as a better indicator of specific inhibition (above 20%) for the formation of metabolites 5,6-epoxy-FAA and 4'-OH-FAA.

The Cyp1b1 inhibitor α-naphthoflavone was tested at final concentrations ranging from 5 to 20 µM (Table 2). Most FAA metabolites were highly inhibited in presence of αnaphthoflavone and the reduction of metabolite areas was also inhibitor concentrationdependent with values of 70 to 89% at the maximal concentration of inhibitor (20 µM). Of interest, concerning the 3',4'-epoxy-FAA, a biphasic effect was observed, as the low concentrations of the inhibitor (5 and 10 µM) led to an increase in the production of 3',4'epoxy-FAA, whereas higher concentrations produced a maximum inhibition of 40% (Table 2). As indicated above, because high concentrations of inhibitor may be involved in unspecific inhibition, it appears that at the intermediate concentration of 10 µM, the following metabolites were inhibited by more than 20%: 3',4'-dihydrodiol-FAA; 5,6-epoxy-FAA; 4'-OH-FAA; and 6-OH-FAA.

The involvement of Cyp2b9 and Cyp2c39 was determined using the inhibitor tranylcypromine. All the metabolites achieved more that 40% inhibition at the the intermediate 50 µM concentration of the inhibitor, with the exception of the 6-OH-FAA (Table 2).

Quercetin, a Cyp2c29 inhibitor, led to a 52% inhibition of the 3',4'-dihydrodiol-FAA, a 73% inhibition of the 5,6-epoxy-FAA and a 85% inhibition of the 6-OH-FAA at the highest concentration tested of 30 µM (Table 2). At the intermediate concentration of 20 µM, only 5,6-epoxy-FAA and 6-OH-FAA were mostly inhibited with 48% and 53% inhibition, respectively. The chromatographic interference of quercetin with the elution of 4'-OH-FAA and 3', 4'-epoxy-FAA in our HPLC conditions precluded the detection of these metabolites with this inhibitor. However, the indirect production of these metabolites via the measurable production of the 3',4'-dihydrodiol-FAA, probably generated via the 3',4'-epoxy-FAA, is an indirect evidence of an inhibition of these B ring metabolites.

The involvement of Cyp2d9 was determined using quinidine as the inhibitor. Our results demonstrated that Cyp2d9 was not significantly involved in the formation of any metabolite even at a relatively high inhibitor concentration of 10 µM which is twice the usual concentration employed for this inhibitor [START_REF] Bogaards | Determining the best animal model for human cytochrome P450 activities: a comparison of mouse, rat, rabbit, dog, micropig, monkey and man[END_REF] (Table 2).

In presence of diethyldithiocarbamate, a Cyp2e1 inhibitor, the production of 5,6epoxy-FAA and 4'-OH-FAA was significantly inhibited (>20%) at an intermediate concentration of this inhibitor (100 µM) (Table 2).

The contribution of Cyp3a11 to the production of FAA metabolites was determined with the specific inhibitor ketoconazole at final concentrations ranging from 2 to 40 µM. In presence of low (2 µM) to moderate (4 µM) concentrations of this inhibitor, only the 3',4',dihydiol-FAA was highly inhibited by 75% and 88%, respectively, indicating that Cyp3a11 was indeed involved in the formation of the 3',4,-epoxy-FAA intermediate metabolite (Table 2).

Determination of direct or indirect monohydroxylation of FAA.

Because the monohydroxylated FAA metabolites could be derived from 2 metabolic routes, i.e., a direct hydroxylation or an hydroxylation via an epoxidic intermediate [START_REF] Guengerich | Cytochrome P450 oxidations in the generation of reactive electrophiles: epoxidation and related reactions[END_REF], we next evaluated the contribution of each pathway to their formation. The metabolism of FAA was conducted in absence or presence of the epoxide scavenger N-acetylcysteine (NAC). Because NAC adducts are very hydrophilic, we had to use gradient elution conditions in order to separate the adducts from the other metabolites as described in Materials and Methods.

In absence of NAC, FAA was metabolized by aroclor-induced mouse microsomes into several metabolites as shown in Figure 3 In presence of the scavenger NAC, all metabolite peaks were reduced (Figure 3-B).

However, the 2 epoxides and the 3',4'-dihydrodiol-FAA peaks were more dramatically reduced compared to the other peaks (Figure 3 To ascertain that the NAC adducts were produced directly with the two FAA epoxides, and not from FAA or its monohydroxylated metabolites, we performed control incubations of FAA with microsomes, but without NAC (Figure 3-D) which did not show any peak at m/z 460 [M+H] + . In addition, control incubations with FAA and its monohydroxylated derivatives (i.e., 3'-, 4'-, and 6-OH-FAA) with NAC, but without microsomes, did not yield any molecular ion at m/z 460 (results not shown), indicating that epoxide formation is required for the appearance of m/z peaks at 460. Because the presence of NAC was also accompanied by a complete disappearance of 3',4'-dihydrodiol-FAA, it was therefore deduced that the latter was derived from its corresponding 3',4'-epoxy-FAA.

As mentioned above, it was observed that in presence of NAC, the 4'-OH-FAA, the 3'-OH-FAA and the 6-OH-FAA, were significantly reduced. The 4'-OH-FAA and the 3'-OH-FAA peaks were reduced by 86 and 67%, respectively, whereas the 6-OH-FAA metabolite was diminished by 40% (Figure 3-B). However, because these peaks were not completely suppressed in presence of the scavenger NAC, this is an indication that direct oxidation of FAA to the monohydroxylated metabolites is also taking place.

!on enzymatic transformation of the epoxides to their corresponding monohydroxylated FAA derivatives. Because the two FAA epoxides were found unstable and that they apparently were transformed into their corresponding mono-hydroxylated derivatives in the incubation conditions (pH 7.4, 37°C), we next explored the possible contribution of their non-enzymatic transformations to their corresponding phenolic compounds. To do so, FAA metabolite extracts were reconstituted in PBS buffer and incubated at 37°C for 1h. We observed that the peak areas of the 5,6-epoxy-FAA decreased by 47%, whereas the 3',4'-epoxy-FAA decreased by 15%. In parallel, their corresponding phenols, i.e., the 6-OH-FAA peak was doubled (100 % increase), the 4'-OH-FAA was increased by 70%, and the 3'-OH-FAA was increased by 171%. It is of interest that the formation of the 5-OH-FAA was not observed in this experiment. It was also noteworthy that the 3',4'-dihydrodiol-FAA peak area was not altered significantly, indicating that this dihydrodiol is not generated by simple chemical hydrolysis of the corresponding parent epoxide, but could be derived from an enzymatic hydrolysis.

Involvement of microsomal mouse epoxide hydrolase in the formation of 3',4'

dihydrodiol FAA. To study the possible involvement of mouse epoxide hydrolase in the formation of the 3',4'-dihydrodiol-FAA, we used elaidamide, a specific epoxide hydrolase inhibitor. In absence of elaidamide, all the FAA metabolites were formed, as presented in

Effect of FAA treatment on the expression of the principal Cyp enzymes in mice.

Because some flavonoids have been reported to modulate the expression of several enzymes, we next explored the effects of an in vivo treatment of FAA in mice on the expression of the principal hepatic Cyps using quantitative RT-PCR. For comparison purposes, the following standard inducers were also included in these experiments: phenobarbital, β-naphthoflavone, 3-methylcholanthrene, dexamethasone and aroclor 1254. The results are presented in Table 3 along with those of FAA after one or three in vivo treatments.

As expected, phenobarbital significantly induced the expression of Cyps 2b10, 2d9 and 3a11, by a factor of 3.2-, 2.5-and 1.6-fold, respectively (Table 3). Also as expected, βnaphthoflavone, 3-methylcholanthrene and aroclor were all potent inducers of several Cyps of the family 1 (Cyp1a1, 1a2, 1b1) (Table 3). Dexamethasone could also induce the following Cyps: Cyp1a1 (24-fold), 1a2 (4-fold), 1b1 (3.1-fold), 2b10 (1.7-fold), 2d9 (18-fold) and 3a11

(3.6-fold).

Concerning the FAA, one treatment (180 mg/kg i.p.) unexpectedly led to a marked reduction of Cyp 1a1, 1a2 and 1b1 transcripts (Table 3). However, after 3 FAA treatments, the following Cyps were significantly induced: Cyp1a2 (1.9-fold), 2e1 (2.1-fold), 2b10 (3.2fold), 2d9 (2.3-fold), 3a11 (2.2-fold) (Table 3).

DISCUSSIO!

Although the metabolism of FAA has been hypothesized to be involved in its mechanism of anticancer activity in mice [11;12], the enzymes involved in FAA metabolism are presently unknown. The purpose of the present work was therefore to identify the principal phase I enzymes involved in FAA metabolism in vitro. In addition, because several flavonoids have been reported to be involved in the modulation of the expression of CYP enzymes [START_REF] Hodek | Flavonoids-potent and versatile biologically active compounds interacting with cytochromes P450[END_REF], we were also interested to evaluate the possible effect of FAA treatment on the expression of the main Cyps expressed in mouse liver.

The principal FAA metabolites formed in vitro using aroclor-induced mouse microsomes consisted of one dihydrodiol (3',4'-dihydrodiol-FAA), three monohydroxylated metabolites (3'-OH-FAA, 4'-OH-FAA, 6-OH-FAA), and two epoxides (5,6-epoxy-FAA, 3',4'-epoxy-FAA). Of these metabolites, the two epoxides 5,6-epoxy-FAA and 3',4'-epoxy-FAA were the predominant ones representing 36% and 27% of the total metabolites area, respectively.

Using Cyp-specific inhibitors, we have shown that the production of several FAA metabolites could be significantly reduced, thus providing novel information concerning the Cyp families and sub-families that are mostly involved in the formation of these metabolites.

A summary of the principal mouse Cyps involved in FAA metabolism is presented in Figure Because monohydroxylation of phenols can also be generated through epoxide intermediates [42;43], we also evaluated the indirect production of the three monohydroxylated metabolites via an intermediate epoxide using N-acetyl cysteine as an epoxide scavenger. In presence of NAC, the 4'-OH-FAA, the 3'-OH-FAA and the 6-OH-FAA, were significantly reduced, indicating that a significant percentage of these hydroxylated metabolites were indeed derived from the non enzymatic isomerization of their corresponding epoxides. The involvement of epoxide hydrolase in the formation of the 3',4'dihydrodiol-FAA was also confirmed using elaidamide, a specific inhibitor of this enzyme [START_REF] Morisseau | Inhibition of microsomal epoxide hydrolases by ureas, amides, and amines[END_REF].

We were next interested to assess the influence of FAA on the expression of the principal hepatic Cyps after an in vivo administration in the mouse, because the expression of these enzymes can indeed be influenced by flavonoids [START_REF] Hodek | Flavonoids-potent and versatile biologically active compounds interacting with cytochromes P450[END_REF]. Using quantitative RT-PCR, FAA was surprisingly found to have a dual effect, i.e., the repression of some Cyps after only one treatment, and the induction of other Cyps after three treatments. Concerning the repression of the expression of Cyps of the 1a subfamily after one in vivo treatment, this rather unexpected result may be due to the fact that some flavonoids may act as ligand of the aryl hydrocarbon receptor (AhR) at low concentrations and repress the transcription of the Cyp1a gene [START_REF] Reiners | Suppression of cell cycle progression by flavonoids: dependence on the aryl hydrocarbon receptor[END_REF][START_REF] Gasiewicz | Analysis of structural requirements for Ah receptor antagonist activity: ellipticines, flavones, and related compounds[END_REF][START_REF] Lu | Substituted flavones as aryl hydrocarbon (Ah) receptor agonists and antagonists[END_REF].

For the Cyp1b subfamily no evident mechanism is presently available to explain its repression.

The induction of Cyps 1a2, 2e1, 2b10, 2d9 and 3a11 after three in vivo FAA treatments was also interesting. High concentrations of flavonoids have been previously

shown to function as AhR agonists and may therefore activate the transcription of the Cyp1a gene. Since FAA is an unsubstituted flavonoid, it is remarkable that similar unsubstituted flavonoids, e.g., flavone and alpha-naphthoflavone, were also found to induce CYPs 1A1, 1A2 and 2B1/2 [44;47]. For the other Cyp subfamilies (i.e., 2d, 2e and 3a), there is no published data that we are aware of, concerning the induction of these Cyps by flavonoids.

In conclusion, we have found that several mouse Cyps are involved in the formation of FAA phase I metabolites. The two main metabolites formed in vitro were the 5,6-epoxy-FAA and the 3',4'-epoxy-FAA which were mainly formed by Cyps 1a2, 1b1, 2b9, 2c39, 2c29, and 2e1, for the former, and Cyps 2b9, 2c39, 3a11 for the latter. The formation of 3'-OH-FAA and 4'-OH-FAA were mainly derived from the 3',4'-epoxy-FAA, whereas 6-OH-FAA was mainly formed directly (60%) by Cyps 1b1 and 2c29. After an in vivo administration of FAA in mice, the expression of Cyps 1a1, 1a2 and 1b1 were repressed after one treatment, whereas three treatments led to an induction of Cyps 1a2, 2e1, 2b10, 2d9 and 3a11.

The enzymes identified in FAA metabolism, as well as their in vivo induction could probably play a role in the remarkable anticancer activity of FAA observed in mice. Although only phase I enzymes were studied in the present work, the involvement of other enzymes, particularly phase II enzymes, could also play a role in FAA anticancer activity because some flavonoids have recently been reported to exert their antiproliferative activity through sulfate conjugation [START_REF] Meng | Activation of aminoflavone (NSC 686288) by a sulfotransferase is required for the antiproliferative effect of the drug and for induction of histone γ-H2AX[END_REF]. c Fold-induction relative to their respective controls (saline or corn oil). Mean ± SEM of 5 independent determinations. -.

Legends for Figures

-.

  -B): 3',4'-dihydrodiol-FAA (Rt 4.1 min); 5,6-epoxy-FAA (Rt 7.6 min); 4'-OH-FAA (Rt 9.0 min); 3'-OH-FAA (Rt 10.0 min); 3',4'-epoxy-FAA (Rt 11.2 min); and 6-OH-FAA (Rt 12.3 min). Among these metabolites, the two FAA epoxides (5,6-epoxy-FAA and 3',4'-epoxy-FAA) were the predominant ones, representing 36.3 ± 1.1 % and 26.7 ± 1.5 % of the total metabolite area, respectively (as evaluated at 300 nm). The 3',4'-dihydrodiol-FAA, the 4'-OH-FAA, the 3'-OH-FAA and the 6-OH-FAA, represented 14.0 ± 0.6 %, 6.8 ± 0.6 %, 1.6 ± 0.6 % and 14.5 ± 1.0 % (n = 3) of the total metabolite area, respectively.

  -A. In these highly resolutive gradient elution conditions, FAA metabolites were eluted at the following retention times (Rt): 3',4'-dihydrodiol-FAA = 28.5 min; 5,6-epoxy-FAA = 39.7 min; 4'-OH-FAA = 43.9 min; 3'-OH-FAA = 45.6 min; 3',4'-epoxy-FAA = 47.4 min; 6-OH-FAA = 49.1 min; and, FAA = 56.6 min (Figure 3-A).

  -B). In parallel to the decrease of two epoxides, we also observed the formation of 4 new peaks at Rt of 26.4, 29.7, 30.2 and 31.5 min (Figure 3-B). Mass spectrum analysis of these 4 new peaks presented the same molecular ion m/z 460 [M+H] + which corresponded to the molecular ion of conjugation of one FAA epoxide (MW 296) with a molecule of NAC (MW 163) (Figure 3-C).

Figure 4 -

 4 Figure 4-A. The first metabolite peak at Rt of 4.1 min was the 3',4'-dihydrodiol-FAA. In

5 .

 5 It can be noted that for the cycle A oxidation, 5,6-epoxidation was best afforded by Cyps 1a2, 1b1, 2b9, 2c39, 2c29, and 2e1, whereas for the 6-hydroxylation, Cyps 1b1 and 2c29 were the most effective. For cycle B epoxidation (3',4'-epoxy-FAA), Cyp 2b9/2c39 and 3a11 were the principal Cyps involved. For the formation of 4'-OH-FAA, Cyps 1a2, 1b1, 2b9 and 2e1 were the most effective.

Figure 1 .

 1 Figure 1. A) Structure of flavone-8-acetic acid (FAA) and its phase I metabolites formed in

Figure 2 .

 2 Figure 2. HPLC-UV chromatogram showing FAA metabolism in absence (A), or presence of

Figure 3 .

 3 Figure 3. HPLC-UV chromatogram analysis (gradient elution) for the determination of direct

Figure 4 .

 4 Figure 4. HPLC-UV chromatograms depicting the involvement of epoxide hydrolase in the

Figure 5 .

 5 Figure 5. Schematic summary of proposed phase I metabolic pathways using aroclor-

Figure 1

 1 Figure 1 Pham et al.

FAA 6

 6 Figure 1 Pham et al.

FAA 6

 6 Figure 2 Pham et al

  Figure 2 Pham et al

Table 1 . Primers used for the determination of mouse cytochrome P450s (Cyp) mR!A

 1

				PCR product	GenBank
	Cyp gene Forward primer	Reverse primer
				(bp)	number
	Cyp1a1	867-GGGAGGTTACTGGCTCTGGA	1077-TGGACATTGGCATTCTCGT	220	NM_009992
	Cyp1a2	961-CCCGAGGAGAAGATTGTCAAC 1102-CGATCCCTGCCAACCAC	158	NM_009993
	Cyp1b1	1315-TGCCTGCCACTATTACGGA	1440-GTCCCTCCCCACAACCTG	143	NM_009994
	Cyp2b10 937-GGCTTCCTGCTCATGCTC	1020-CGGTCATCAAGGGTTGGT	101	NM_009999
	Cyp2d9	1173-CGCTTTGGGGACATTGTT	1257-GTTGGGGAGGAGGATCGT	102	NM_010006
	Cyp2e1	786-CACTGGACATCAACTGCCC	947-ATGAGAATCAGGAGCCCATA 181	NM_021282
	Cyp3a11 328-GTCACAGACCCAGAGACGAT	553-TTTGCCTTCTGCCTCAAGTA	245	NM_007818

Table 2 . Determination of mouse cytochrome P450s (Cyps) involved in the metabolism of FAA using Cyp specific inhibitors a

 2 SEM of 3 independent determination. c ND, not determined, because of inhibitor peak interference with these metabolites. Bold characters indicate intermediate inhibitor concentrations.

	Inhibitor (Cyp inhibited)	Inhibitor Conc. (µM)	Percent inhibition 3',4'-di-hydrodiol-FAA 5,6-epoxy-FAA	4'-OH-FAA	3', 4'-epoxy -FAA	6-OH-FAA
		5	-3 ± 5 b	19 ± 3	7 ± 4	-2 ± 5	-3 ± 8
	Furafylline	10	6 ± 6	34 ± ± ± ± 13	29 ± ± ± ± 10	5 ± 8	19 ± 12
	(Cyp1a2)	20	14 ± 8	42 ± 6	35 ± 3	27 ± 5	28 ± 5
		50	36 ± 1	57 ± 2	52 ± 3	54 ± 3	52 ± 6
		5	10 ± 1	5 ± 5	5 ± 4	-29 ± 6	16 ± 1
	α α α α naphthoflavone	10	50 ± ± ± ± 1	21 ± ± ± ± 9	32 ± ± ± ± 20	-18 ± 11	53 ± ± ± ± 1
	(Cyp1b1)	15	76 ± 2	42 ± 4	62 ± 15	11 ± 2	73 ± 1
		20	89 ± 3	76 ± 14	70 ± 15	40 ± 16	84 ± 4
		25	4 ± 10	23 ± 3	13 ± 4	10 ± 1	-17 ± 31
	Tranylcypromine	50	45 ± ± ± ± 8	52 ± ± ± ± 2	44 ± ± ± ± 5	42 ± ± ± ± 4	10 ± 34
	(Cyp2b9, Cyp2c39)	75	68 ± 1	68 ± 2	57 ± 2	59 ± 2	29 ± 33
		100	75 ± 3	74 ± 3	64 ± 7	64 ± 3	37 ± 32
		10	-21 ± 7	26 ± 6	ND c	ND	25 ± 12
	Quercetin	20	13 ± 7	48 ± ± ± ± 11	ND	ND	53 ± ± ± ± 8
	(Cyp2c29)	25	35 ± 11	62 ± 11	ND	ND	76 ± 4
		30	52 ± 7	73 ± 6	ND	ND	85 ± 4
		10	1 ± 4	17 ± 13	-7 ± 13	-5 ± 9	-23 ± 11
	Quinidine	50	7 ± 4	20 ± 4	5 ± 4	-7 ± 1	-26 ± 3
	(Cyp2d9)	100	15 ± 8	27 ± 1	13 ± 7	-16 ± 5	-17 ± 3
		200	39 ± 2	35 ± 5	24 ± 5	-13 ± 0	5 ± 10
		50	-61 ± 12	1 ± 5	17 ± 7	-66 ± 26	-3 ± 4
	Diethyldithiocarbamate	100	-53 ± 8	24 ± ± ± ± 5	31 ± ± ± ± 7	-67 ± 16	4 ± 10
	(Cyp2e1)	200	-29 ± 20	49 ± 6	54 ± 2	-62 ± 46	34 ± 5
		300	28 ± 15	75 ± 4	71 ± 9	11 ± 17	62 ± 2
		2	75 ± 3	17 ± 8	-40 ± 6	-82 ± 7	-16 ± 2
		4	88 ± ± ± ± 1	16 ± 1	-20 ± 20	-101 ± 10	-9 ± 11
	Ketoconazole (Cyp3a11)	10	94 ± 1	41 ± 5	-32 ± 31	-62 ± 9	2 ± 13
		20	96 ± 1	61 ± 4	25 ± 6	0 ± 7	35 ± 7
		40	97 ± 1	85 ± 1	62 ± 11	59 ± 2	78 ± 1
	a Microsomal incubation were carried out as described in Materials and Methods section in absence or
	presence of the indicated inhibitor concentrations. The 3'-OH-FAA is not shown because it represented
	only about 2% of total metabolites. The mouse Cyp orthologs correspondance with the human CYPs
	are as follows (mouse Cyp, human CYP): Cyp1a2, CYP1A2; Cyp1b1, CYP1B1; Cyp2b9, CYP2B6;
	Cyp2c39, CYP2C19; Cyp2d9, CYP2D6; Cyp2e1, CYP2E1; Cyp3a11, CYP3A4.

b Mean ±

Table 3 . Quantitative RT PCR determination of the principal cytochrome P450s (Cyp) induced by standard inducers and flavone 8 acetic acid (FAA) a

 3

	Inducer	1a1 b	1a2	1b1	2e1	2b10	2d9	3a11
	Phenobarbital	0.07 c ± 0.04 0.75 ± 0.1 0.2 ± 0.09 1 ± 0.2	3.2 ± 0.7 2.5 ± 0.4 1.6 ± 0.1
	β-naphthoflavone	450 ± 330	30 ± 9	28 ± 8	1.1 ± 0.05 1.6 ± 0.2 2.4 ± 0.5 1.2 ± 0.3
	3-methylcholanthrene 11000 ±2900 180 ± 17	680 ± 120 1.5 ± 0.1	2.4 ± 0.7 2.9 ± 0.4 1.5 ± 0.4
	Dexamethasone	24 ± 16	4 ± 2	3.1 ± 1.7 0.78 ± 0.02 1.7 ± 0.2 18 ± 3.9	3.6 ± 1.0
	Aroclor 1254	7800 ± 2900 200 ± 39	370 ± 89 0.4 ± 0.1	8.4 ± 1.4 6.0 ± 1.4 5.5 ± 1.6
	FAA 1 treatment	0.003 ±0.001 0.08 ± 0.01 0.1 ± 0.01 0.6 ± 0.1	0.5 ± 0.08 0.5 ± 0.1 0.7 ± 0.1
	FAA 3 treatments	0.6 ± 0.3	1.9 ± ± ± ± 1.1	1.1 ± 0.3 2.1 ± ± ± ± 0.5	3.2 ± ± ± ± 1.5 2.3 ± ± ± ± 0.7 2.2 ± ± ± ± 0.4
	CYP): Cyp1a2, CYP1A2; Cyp1b1, CYP1B1; Cyp2b9, CYP2B6; Cyp2c39, CYP2C19; Cyp2d9,
	CYP2D6; Cyp2e1, CYP2E1; Cyp3a11, CYP3A4.

a Levels of Cyp transcripts were determined by quantitative RT-PCR as described in the Materials and Methods section.

b

The mouse Cyp orthologs correspondance with the human CYPs are as follows (mouse Cyp, human

' epoxy FAA 6 OH FAA Time (min) UV Detector Response at 300 nm (mV)

		3',4' dihydrodiol FAA 3',4' dihydrodiol FAA 3',4' dihydrodiol FAA	3',4' dihydrodiol FAA 3',4' dihydrodiol FAA 3',4' dihydrodiol FAA
		Furafylline Furafylline Furafylline
		5,6 epoxy FAA 5,6 epoxy FAA 5,6 epoxy FAA	5,6 epoxy FAA 5,6 epoxy FAA 5,6 epoxy FAA
	Pham et al	4' OH FAA 3' OH FAA 3',4' epoxy FAA 6 OH FAA 3',4Figure 2 4' OH FAA 3' OH FAA 4' OH FAA 3' OH FAA 3',4' epoxy FAA 6 OH FAA 4' OH FAA 3' OH FAA 3',4' epoxy FAA 6 OH FAA 4' OH FAA 3' OH FAA 6 OH FAA 3',4' epoxy FAA 4' OH FAA 3' OH FAA 3',4

' epoxy FAA 6 OH FAA Time (min) UV Detector Response at 300 nm (mV)

			3',4' dihydrodiol FAA	3',4' dihydrodiol FAA
			Furafylline
			5,6 epoxy FAA	5,6 epoxy FAA
	Pham et al	Figure 2	3',4' epoxy FAA 6 OH FAA 4' OH FAA 3' OH FAA	4' OH FAA 3' OH FAA	3',4

' epoxy FAA 6 OH FAA Time (min) UV Detector Response at 300 nm (mV)

Acknowledgements. We thank Dr Christophe Morisseau for synthesizing and providing us with elaidamide, a specific epoxide hydrolase inhibitor. Supported by the Institut National de la Santé et de la Recherche Médicale (Inserm), the Centre National de la Recherche Scientifique (CNRS), and the Institut National du Cancer (INCa, Boulogne Billancourt, France).