Radical-induced oxidation of metformin H. Khouri 1 , F. Collin 1 , D. Bonnefont-Rousselot 2,3 , A. Legrand 2 , D. Jore 1 and M. Garde ` s-Albert 1 1 Laboratoire de Chimie Physique UMR 8601-CNRS, Universite ´ Paris 5, France; 2 Laboratoire de Biochimie Me ´ tabolique et Clinique, Faculte ´ de Pharmacie, Paris 5, France; 3 Laboratoire de Biochimie B, Ho ˆ pital de la Salpe ˆ trie ` re (AP-HP), Paris, France Metformin (1,1-dimethylbiguanide) is an antihyperglycae- mic d rug u sed t o norm alize g lucose concentrations in type 2 diabetes. Furthermore, antioxidant benefits have been reported in diabetic p atients treated with metformin. This work was aimed at studying the scavenging capacity of this drug against reactive oxygen species (ROS) like Æ OH and O ÆÀ 2 -free radicals. ROS were produced by gamma radio- lysis of water. The irradiated solutions of metformin were analyzed by UV/visible absorption spectrophotometry. It has been shown that hydroxyl free radicals react with met- formin in a concentration-dependent way. The maximum scavenging activity was obtained for concentrations of metformin ‡ 200 lmolÆL )1 , under our experimental condi- tions. An estimated value of 10 7 LÆmol )1 Æs )1 has been determined for the second order r ate co nstant k( Æ OH + metformin). Superoxide free radicals and hydro- gen peroxide do not initiate any oxidation on metformin in our in vitro experiments. Keywords: metformin; hydroxyl radical; antioxidant; radio- lysis. Metformin (MTF) (1 ,1-dimethylbiguanide, see structure in Fig. 1) is one of the most used oral antihyperglycaemic agents. It normalizes plasma glucose concentration without any stimulation of insulin production. It has been demon- strated that elevated glucose levels induce oxidative stress in diabetes, i.e. an imbalance between the production of oxidant species, particularly radical species, and the anti- oxidant defences [1]. This might partly explain the elevated risk factors for diabetic patients to develop cardiovascular complications [2,3]. This imbalance c an be detected by oxidative stress markers such as those of lipid peroxidation and protein oxidation. Previous in vivo and in vitro studies have demonstrated several antioxidant proper ties of m etfo rmin such as the inhibition of the formation of advanced glycation end products (AGEs) [4,5] that a re thought to be responsible for further diabetic complications, and the decrease in the formation of methylglyoxal, one of the precursors of AGEs [6]. Metformin improves liver antioxidant potential in rats fed a high-fructose d iet [ 7]. It has been observed that the administration of metformin in diabetic p atients ameliorates the a ntioxidant status. T his was shown by a decrease in lipid peroxidation [monitored by determining the production of thiobarbituric acid reactive substances (TBARS)] [8,9], a decrease in lipid peroxidation markers in both LDL and HDL [10], an increase in reduced glutathione (GSH) blood concentration ( usually low in d iabetic patients) [11] and in antioxidant enzyme activities (such as catalase and CuZn superoxide dismutase) [11]. Furthermore, clinical benefits against vascular complica- tions have been obtained, and protective e ffects against diabetic complications have been observed with metformin monotherapy [12]. Patients with type 2 diabetes receiving either metformin alone or accompanied by another treat- ment reduced by 40% the risk of developing f urther vascular complications compared to those receiving other treatments [12–14]. In order to improve the knowledge of M TF antioxidant mode of action, this work focused o n th e direct antioxidant properties of metformin in vitro against different oxygen- derived free radical species generated in aqueous solution by gamma radiolysis. Gamma radiolysis of water i s a well known m ethod that has many advantages, such as the homogeneous production of known quantities o f free radicals (as superoxide an ion O ÆÀ 2 or hydroxyl radical Æ OH), as well as the possibility to selectively produce one specific radical to be studied at a time [15–18]. Free radicals thus generated have been used to initiate one electron oxidation reaction(s) on metformin dissolved in water. In a previous work, we have identified the oxida- tion end-products of Æ OH-induced oxidation of m etformin [19]. Four products have been characterized (Fig. 1): methylbiguanide (MBG), a dimer o f M TF (diMTF), a hydroperoxide of MTF (MTFOOH) and 4-amino-2- imino-1-methyl-1,2-dihydro-1,3,5-triazine (4,2,1-AIMT). The generation of these oxidation end-products was shown to be dependent on the e xperimental conditions: MTFOOH is only produced under aerated conditions, while diMTF occurs only in nonaerated solutions, saturated with nitrogen protoxide. The two other products, MBG and 4,2,1-AIMT, have been found in both aerated Correspondence t o H. Khouri or F. Collin, L aboratoire de Chimie Physique, CNRS U M R 8601 universite ´ s Pa ris 5, 45 rue de s Sain ts- Pe ` res, 75270 Paris Cedex 06, Fr ance. F a x: + 33 1 42862213, Tel.: + 33 1 42862173, E- mail: h ania.khouri@univ-paris5.fr or fabrice.collin@univ-paris5.fr Abbreviations: 4,2,1-AIMT, 4-amino-2-imino-1-methyl-1,2-dihydro- 1,3,5-triazine; AGE, a dvanced glycation end p roducts; GSH , glut a - thione; MBG, methylbiguanide; MTF, metformin; ROS, reactive oxygen species; TBARS, thiobarbituric acid reactive substance. (Received 1 7 Au gust 2004, accepted 1 4 O ctober 2004) Eur. J. Biochem. 271, 4745–4752 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04438.x and nonaerated irradiated solutions of metformin. How- ever, o nly t wo radiation doses (50 and 300 Gy) and only one concentration of metformin (200 lmolÆL )1 ) h ave been studied [19]. In order to specify the Æ OH-induced oxidation mechanism of m etformin, we p resent in this paper t he effect of several radiation doses (from 52 to 627 Gy) on different metformin concentrations (from 4 to 500 lmolÆ L )1 ). UV/visible differential absorption spectra (where the reference is non-irradiated solutions) have been recorded as a function of the radiation dose. In addition, we have determined the initial slope of the curves [Dabsorbance k ¼ f(radiation dose)] which is propor- tional to the radiolytic yield (initial slope ¼ GÆDe k Æl, were G is the radiolytic yield, De k the differential molar extinction coefficient and l the optical path-length. Kinetic data have been obtained from the dilution curves {i.e. GÆDe k Æl ¼ f([metformin])}, allowing us to discuss the possible compe- tition of hydroxyl radicals between metformin and radio- lytically generated hydrogen peroxide. Materials and methods Chemicals All chemicals were purchased from Sigma (St Louis, MO, USA) except when mentioned. Metformin solutions (4–500 lmolÆL )1 )werepreparedin10mmolÆL )1 phosphate buffer NaH 2 PO 4 Æ2H 2 O (purchased from Prolabo, Manche- ster, UK) at pH 7. Ultra pure water (Maxima Ultra-pure Water, ELGA, resistivity 18.2 MW) was used to pre pare the solutions. Irradiations were carried out in test tubes that have been previously cleaned with hot TFD4 d etergent (Franklab S.A., France), rinsed thoroughly with ultra pure water,andthenheatedat400°C for 4 h to avoid any pollution by remaining organic compounds. Gamma radiolysis Radiolysis corresponds to the c hemical transformations of a solvent due to the absorption of ionizing radiations, which allows, w ithin a few nanoseconds, the production of a homogeneous solution of free radicals. In addition, this method allows selective generation of particular radicals from the solvent, and thus it is possible to s tudy their action towards the dissolved entities. Radiolytically generated free radicals are independent of the nature and of the concen- tration of the dissolved compound as long as its concentra- tion remains lower than or equal to 10 mmolÆL )1 [20]. Gamma r adiolysis was carried out by using an IBL 637 irradiator (CIS Biointernational, Gif-sur-Yvette, France) of 137 Cs source, whose activity was % 22 2 TBq (6000 Ci). In our experiments the dose rate was 10.45 GyÆmin )1 .The dosimetry was determined by Fricke’s method [21], namely radio-oxidation of 1 mmolÆL )1 of iron(II) sulfate solution i n 0.4 molÆL )1 sulfuric acid (under an aerated atmosphere) taking k max (Fe 3+ ) ¼ 304 nm, e (304 nm) ¼ 2204 LÆmol )1 Æ cm )1 )at25°C, and a radiolytic yield of G(Fe 3+ ) ¼ 1.62 lmolÆJ )1 . Different radiation doses, ranging from 52 to 627 Gy, were delivered to 5 mL of the solution depend- ing on the time of the exposure to the c-ray source: the longer the time of the exposure, the higher the radiation dose. For each experimental set, 5 mL of non-irradiated solution was taken as a control. Water radiolysis by c-rays generates the free radical species e – aq , Æ OH, Æ H, and the molecular species H 2 and H 2 O 2 . U nder aerated conditions (oxygen c oncentration is about 0.2 mmolÆL )1 ), hydroxyl and superoxide radicals (resulting from the scavenging of e – aq and Æ H species) were simultaneously produced with radiolytic yields (G-values expressed in molÆJ )1 ) of 0.28 and 0.34 lmolÆJ )1 , respectively. In order to select only hydroxyl radicals, radiolysis was Fig. 1. Structures of the protonated form of metformin (1,1-dimethylbiguanide) and of the oxidation products generated from Æ OH attacks on metformin, according to [19]. 4746 H. Khouri et al. (Eur. J. Biochem. 271) Ó FEBS 2004 carried out in a nonaerated medium saturated with nitrogen protoxide (N 2 O). N 2 O scavenges hydrated electrons and converts them into hydroxyl radicals: as a result, Æ OH is produced with a final G-value of 0.56 lmolÆJ )1 ,thatistwice as high as the G-value in an aerated medium [20]. To selectively obtain superoxide anions, s odium formate (Pro- labo) was added to the solution at a concentration of 0.1 molÆL )1 in order to c onvert all radicals ( Æ OH, Æ Hand e – aq )intoO 2 Æ– radicals with a final G-value of 0.62 lmolÆJ )1 [20]. Analysis Detection of the oxidation products was achieved by spectrophotometric measurements with an UV/visible spec- trophotometer (Beckman DU 70). Samples were scanned from 200 nm to 300 nm. At p H 7 , metformin, like all biguanides, is present in its mono-protonated form (Fig. 1). Therefore, the p ossibility of r esonance gives to biguanides a characteristic absorption band at about 230 nm [22]. Beer– Lambert law was applicable on metformin within the studied range of concentrations (4–500 lmolÆL )1 ), and the molar extinction coefficient at 232 nm was found to be 12 300 ± 490 LÆmol )1 Æcm )1 ). Results Action of Æ OH/O ÆÀ 2 -free radicals A 450 lmolÆL )1 solution of metformin (in 10 mmolÆL )1 sodium phosphate buffer, pH 7) was irradiated at doses ranging from 52 to 627 Gy, with a dose rate of 10.45 GyÆmin )1 . The absolute absorption spectra (refer- ence ¼ phosphate buffer, 10 mmolÆL )1 , pH 7) a re presen- ted in Fig. 2A, as a function of the radiation dose. The non-irradiated solution shows a main absorption band at 232 nm c orresponding to the absorption of metformin [22]. As the radiation dose increased, the absorption at this wavelength decreased (illustrating the consumption of metformin) and two new bands were detected at 208 nm (intensified) and 258 nm, probably due to the generation of oxidation products. Differential absorption spectra (refer- ence ¼ non-irradiated metformin solution) allows us to better show the same phenomenon (Fig. 2B). The arrows in Fig. 2. UV/visible absorption spectra of metformin (450 lmo lÆL )1 ) as a function of the radiation dose (52–627 Gy) in aerated medium. (A) Absolute absorption spectra (refer- ence, phosphate buffer, 10 mmolÆL )1 ,pH7). (B) Differential absorption spectra (refer- ence, non-irradiated metformin solution). Optical path-length: l ¼ 0.2 cm, dose rate: I ¼ 10.45 GyÆmin )1 . The arrows in dicate th e decrease (disappearance) and the increase (appearance) in t he a bsorbance values as a function of the enhancing radiation dose. Ó FEBS 2004 Radical-induced oxidation of metformin (Eur. J. Biochem. 271) 4747 Fig. 2 indicate the variations in the absorption intensity of every characterized band as a function of the increasing radiation dose. Differential spectra highlight the previous observation: at 232 nm, the differential absorbance is decreasing (consumption of metformin), while it increases at 208 and 258 nm (formation of oxidation products). The differential absorbances at 232 nm and 258 nm have been reported as a fun ction of t he radiation dose (Fig. 3). At 232 nm, the differential absorbances decrease confirming the consumption of metformin as a function of the radiation dose (Fig. 3A). At 208 nm (not shown) and 258 nm (Fig. 3B), the differential absorbance increases with the radiation dose, indicating the simultaneous formation of one or more oxidation products. However, the 258-nm absorption band has been selected for this study, as non- irradiated metformin solution does not absorb at all at this wavelength; 258 nm is a characteristic wavelength of aromatic structure. In fact, Collin et al. [19] have identified 4,2,1-AIMT as one oxidation product of metformin (Fig. 1), which m ight be considered as the compound that absorbs at this particular wavelength. The other oxidation products identified (methylbiguanide and metformin peroxide) seem to share the same spectral characteristics as metformin since their chemical structures are very close. Similar analyses have been replicated for several metfor- min initial concentrations (4–500 lmolÆL )1 ). Initial slopes of the curves [DAbs k ¼ f(dose)], corresponding to GÆDe k Æl (where G is the radiolytic yield, De k the molar extinction coefficient and l the optical path-length) at 232 and 258 nm, respectively, have b een reported as a function of the initial concentration of metformin (Fig. 4A,B). These dilution curves give the evolution of GÆDe k (corrected for optical path-length l ¼ 1 cm) with the initial concentration of metformin. Both dilution curves at 232 nm and 258 nm exhibit the same profile, namely increasing values of GÆDe k at low metformin concentration (from 4 to 200 lmolÆL )1 ) followed by plateau values of GÆDe k at high metformin concentration (200–500 lmolÆL )1 ). Hence , these dilution curves exhibit two key areas. At the plateau, the value G.De k at 232 nm or 258 nm reaches a steady state, meaning that all free radicals ( Æ OH/O ÆÀ 2 ) produced by water radiolysis have reacted with metformin independently of its initial concentration (200–500 lmolÆL )1 ). The second key area is characterized by GÆDe k values that decrease as concentra- tions o f metformin decrease (200–4 lmolÆL )1 ). This latter phenomenon might be due to a competition between Fig. 3. Differential absorbances as a function of the radiation dose. [Metformin] ¼ 450 lmolÆL )1 , [phosphate b uffer] ¼ 10 mmolÆL )1 , pH 7, aerated medium. Reference ¼ non-irradiated metformin solu- tions. (A) 232 nm, (B) 258 nm. Op tical p ath-length: l ¼ 0.2 cm, dose rate: I ¼ 10.45 GyÆmin )1 . Uncertainties (RSD) h ave been calculated as being equal to 4%, at the 95% confidence level (2 r, n ¼ 3). Fig. 4. Dilution curves of metformin (GÆDe k as a function of the initial concentration of metformin), [phosphate buffer] = 10 mmolÆL )1 ,pH7, aerated medium. (A) 232 nm, (B) 258 nm – values are corrected for an optical path-length of 1 cm. Uncertainties (RSD) have been calculated as being equal to 4%, at the 95% confidence level (2 r, n ¼ 3). 4748 H. Khouri et al. (Eur. J. Biochem. 271) Ó FEBS 2004 metformin and either phosphate buffer or hydrogen peroxide radiolytically generated, towards the action of Æ OH/O ÆÀ 2 radicals. To verify this assumption, the effect of various phosphate buffer concentrations (0.05, 0.5 and 5 mmolÆL )1 )atpH7 was studied in the presence of 50 lmolÆL )1 of metformin. After irradiation, solutions were analyzed by absorption spectrophotometry at 232 nm. Any change was observed i n the consumption of metformin, indicating that phosphate buffer did not compete at all with metformin towards Æ OH/O ÆÀ 2 -free radicals oxidation (data not shown). When hydrogen peroxide was added to the metfo rmin solutions, it was been first verified that there was no detectable effect of H 2 O 2 as an initiator of metformin oxidation in the absence of irradiation. However, under irradiation, there was a noticeable effect of the concentra- tion of H 2 O 2 (0.05, 0.5 and 5 mmolÆL )1 ) added to metformin (50 lmo lÆL )1 ) as shown in Fig. 5. These metformin–H 2 O 2 solutions were irradiated from 52 to 520 Gy and analyzed at 232 nm by absorption spectro- photometry. The consumption of metformin was gradually decreased by increasing the concentration of H 2 O 2 from 0.05 to 5 mmolÆL )1 regardless of the radiation dose. At 5 mmolÆL )1 H 2 O 2 , i t can be seen in Fig. 5 that m etformin was n ot consumed as the radiation dose increased, i.e. metformin no longer reacted with the radiolytically gener- ated free radicals. Action of O ÆÀ 2 radicals In order to study the effect of superoxide radicals as initiators of metformin oxidation, metformin solutions at different concentrations ranging from 50 to 100 lmolÆL )1 were irradiated in the presence o f sodium formate (0.1 mol ÆL )1 ). Under these conditions (0.1 molÆL )1 of sodium formate), O ÆÀ 2 radicals are the only radical species produced by water radiolysis with a formation yield of 0.62 lmolÆJ )1 , as described in Materials and methods. Metformin consumption was measured by absorption spectrophotometry at 232 nm. Under our experimental conditions, no detected effect of O ÆÀ 2 radicals on the initiation of metformin oxidation has been observed. This phenomenon implies that superoxide radicals would mainly dismutate in such conditions (k ¼ 6 · 10 5 LÆmol )1 Æs )1 at pH 7 [23]). Action of Æ OH radicals In order to study the action of Æ OH radicals on the initiation of metformin oxidation in nonaerated medium, different solutions of metformin (4–500 lmolÆL )1 )weresaturated with nitrogen protoxide (N 2 O). Under these conditions, Æ OH radicals are the main radical s pecies produced from water r adiolysis with a radiolytic yield of 5.6 · 10 )7 molÆJ )1 (see Materials and methods). The apparition of m etformin oxidation product(s) was followed by absorption spectro- photometry at 258 nm. In Fig. 6, for a metformin concentration of 500 lmolÆ L )1 , differential absorbances at 232 n m (Fig. 6A) and 258 nm ( Fig. 6B) have b een reported as a function of the radiation dose (from 52 to 627 Gy). The formation of oxidized product(s) exhibit the same profile as under a erated conditions (Fig. 3 A,B), confirming that Æ OH radicals are responsible for the initiation of metformin oxidation. Several metformin concentrations were studied under the same experimental conditions (nonaerated and N 2 O-satur- ated medium). The initial slope of the curves [DAbs k ¼ f(radiation dose)] allowed us to determine the GÆDe k values (corrected for an optical path-length l ¼ 1 cm). Dilution curves {GÆDe k ¼ f([MTF])} were plotted on Fig. 7. It can be observed that GÆDe k values increase with metformin initial concentration u p to 200 lmolÆL )1 and plate au va lues are reached for metformin initial concentrations supe rior to 200 lmolÆL )1 . At 232 and 258 nm (Fig. 7A,B, respectively), it can be noted that GÆDe k plateau values (13 ± 2 · 10 )4 and 6.5 ± 0.5 · 10 )4 , respectively) are twice as high as those obtained under aerated medium (6.5 ± 0.3 · 10 )4 , Fig. 4A and 3.2 ± 0.2 · 10 )4 , Fig. 4B). These observa- tions can be explained by the fact that Æ OH radicals have a formation yield under N 2 O atmosphere (0.56 lmolÆJ )1 ) twice as high as those of Æ OH radicals formed under aerated medium (0.28 lmolÆJ )1 ). However, the exact G-values of metformin oxidation products formation are not actually known. Fig. 5. Differential absorbance at 232 nm as a function of the radiation dose for me tformin solutions (50 lmolÆL )1 ) with or w ithout H 2 O 2 (0.05, 0.5 and 5 mmolÆL )1 ). [phosphate buf- fer] ¼ 10 mmolÆL )1 ,pH7,aeratedmedium, optical path-length: l ¼ 1 cm, do se ra te : I ¼ 10.45 GyÆmin )1 . (Reference, non- irradiated metformin solution). Uncertainties (RSD) have been calculated as b eing equal to 4%, at the 95% confidence level (2 r, n ¼ 3). Ó FEBS 2004 Radical-induced oxidation of metformin (Eur. J. Biochem. 271) 4749 Discussion According to our experimental results, it seems that neither superoxide radicals nor hydrogen peroxide react with metformin, but that Æ OH radicals are the only species initiating metformin oxidation. Knowing that Æ OH-free radicals can abstract one electron (charge transfer) or one H atom, or add to a double bond, we may assume that Æ OH- free radicals can abstract an H atom from the CH 3 groups and/or from the N–H between the N(CH 3 ) 2 and NH 2 . Hydroxyl radicals can also add to the C¼NH double bonds (giving nitrogen-centred free radicals). It can be noted that, because of the conjugation of the nitrogen electron pair [of NH 2 ,NHandN(CH 3 ) 2 ]withtheC¼NH double bonds, the charge transfe r process of Æ OH abstracting an electron from the nitrogen electron pair seems rather unfavourable. Scheme 1 summarizes the radical-induced oxidation of metformin. MTF Æ symbolizes the Æ OH-induced radical of metformin. Once metformin radicals are produced, they might undergo various reactions leading to different oxida- tion products [19]. In the presence of oxygen, metformin radical may react with oxygen molecules leading to peroxy radicals which could be reduced (maybe by superoxide radicals) to give metformin hydroperoxide (MTFOOH), whereas in the absence of oxygen, metformin radicals would tend to dimerize (diMTF). The occurrence of these latter compounds is oxygen dependent [19]. Another two oxida- tion end-products have been observed by Collin et al., i.e. MBG and 4,2,1-AIMT [19] whose mechanisms of forma- tion are unknown. In order to specify the different steps of the proposed mechanism, additional results would be necessary, mainly the quantification of the oxidation products. The observed progressive inhibition of metformin oxida- tion, in the presence of added hydrogen peroxide, would come from the reaction of Æ OH radicals with H 2 O 2 .An estimated value of the second order rate constant of k( Æ OH + MTF) could be determined, by comparing the initial rates of Æ OH radical with hydrogen peroxide [relation (1)] or with metformin [relation (2)]. vð Æ OH þ H 2 O 2 Þ¼kð Æ OH þ H 2 O 2 Þ½ Æ OH½H 2 O 2  0 ð1Þ vð Æ OH þ MTFÞ¼kð Æ OH þ MTFÞ½ Æ OH½MTF 0 ð2Þ It is well known that the rate constant of Æ OH radicals with H 2 O 2 is close to 10 7 LÆmol )1 Æs )1 [24]. For the highest Fig. 6. Differential absorbances as a function of the radiation dose. [Metformin] ¼ 500 lmolÆL )1 , [phosphate b uffer] ¼ 10 mmolÆL )1 , pH 7, N 2 O-saturate d solutions. Reference, non-irradiated metformin solution.(A)232nm,(B)258nm.Opticalpath-length:l¼ 0.2 cm, dose rate: I ¼ 10.45 GyÆmin )1 . Uncertainties (RSD) have been cal- culated as being equal to 17% (A) and 8% (B), at the 9 5% confidence level (2 r, n ¼ 3). Fig. 7. Dilution curves of metformin (GÆDe k as a function of the initial concentration of metformin), [phosphate buffer] = 10 mmolÆL )1 ,pH7, N 2 O-saturated solutions. (A) 232 nm, (B) 258 nm – values are correc- ted for an optical path-length of 1 cm. Uncertainties (RSD) have been calculated as being equal to 17% (A) and 8% (B), at the 95% con- fidence le vel ( 2 r, n ¼ 3). 4750 H. Khouri et al. (Eur. J. Biochem. 271) Ó FEBS 2004 hydrogen peroxide concentration (5 mmolÆL )1 ), a quasi- total inhibition of metformin (50 lmolÆL )1 ) oxidation (Fig. 5) has been observed, involving a reaction rate of it Æ OH radicals with H 2 O 2 at least 10 times higher than those of Æ OH radicals with metformin [relation 3]. vð Æ OH þ H 2 O 2 Þ > 10  vð Æ OH þ MTFÞð3Þ From relations 1–3, it can be deduced that the second order rate constant [k( Æ OH + metformin)] is lower than 10 8 LÆmol )1 Æs )1 ). Therefore, this rate constant is likely of the same order of magnitude (% 10 7 LÆmol )1 Æs )1 ) than that of hydrogen peroxide with Æ OH radicals. It is worth mention- ing that this value is rather weak for a reaction involving hydroxyl radicals whose k-values are usually diffusion controlled, and approximately e qual to 1 0 9 )10 10 LÆmol )1 Æs )1 [24]. Accordingly, metformin exhibits a relat- ively weak radical scavenging capacity against Æ OH radicals in vitro. In the radiolysis solutions, H 2 O 2 could come from different pathways: (i) from Æ OH radical recombination (in the spurs) giving H 2 O 2 with a G-value of 0.7 · 10 )7 molÆJ )1 (this production being independent of the presence of metformin); (ii) from O ÆÀ 2 (in equilibrium with HO Æ 2 ) radical dismutation (in homogeneous phase) leading to H 2 O 2 with a G-value of (3.4 · 10 )7 )/2 m olÆJ )1 , i.e. (Ge – aq +G H )/2, in the case where O 2 Æ– radicals do not react neither with metformin nor with the metformin radical, and (iii) from O ÆÀ 2 radical oxidation of metfo rmin radical giving H 2 O 2 with a G-value of 3.4 · 10 )7 molÆJ )1 . H 2 O 2 concentration in the radiolysis solution is propor- tional to G(H 2 O 2 ) and to the radiation dose ([H 2 O 2 ] ¼ G(H 2 O 2 ) · dose). For example, at 50 Gy (which is a dose where G-value can be determined), t he following H 2 O 2 concentration can be calculated: 3 .5 lmolÆL )1 [pathway (i)], 1 2 lmolÆL )1 [pathway (i) + (ii)] or 20.5 lmolÆL )1 [pathway (i) + (iii)]. Such H 2 O 2 concentrations are similar to the lowest concentrations of metformin (from 4 to 50 lmolÆL )1 ). Hence, the hypothesis of a competition of Æ OH radicals between H 2 O 2 and metformin is plausible providing that the rate constants [k( Æ OH + H 2 O 2 )and k( Æ OH + metformin)] be of the same order of magnitude [i.e. % 10 7 LÆmol )1 Æs )1 ]. In agreement with these consider- ations, it can be proposed that the decrease of GÆDe k values at low m etformin concentration ( 4–200 lmolÆL )1 )(Figs4 and 7) would come from the competition of Æ OH radicals between metformin and radiolytically generated hydrogen peroxide. Conclusion We have investigated the antioxidant properties of metfor- min against Æ OH and O ÆÀ 2 -free radicals produced by water gamma radiolysis. Metformin aqueous solutions (from 4 to 500 lmolÆL )1 ) were analyzed by UV/visible absorption spectroscopy. We have shown that metformin does not scavenge O ÆÀ 2 radicals, but is able to react with Æ OH radicals. However, under our experimental condition s, the Æ OH-induced oxidation of metformin depended on its initial concentration because of the possible competitive reaction of Æ OH radicals with radiolytically generated H 2 O 2 . Moreover, we have determined an estimated value of 10 7 LÆmol )1 Æs )1 ) for the second order r ate constant o f the reaction of Æ OH radicals with metformin. Our results obtained with an in vitro model allow assuming that metformin, at a molecular level, is not a very good scavenger of reactive oxygen species. 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(1988) Critical review of rate constants for reactions of hydrated electrons, hydrogen atoms and hydroxyl radicals in aqueous solution. J. Phys. Chem. Ref. Data 17, 513–887. 25. Ewiss, S.A. & Abdel-Rahman, M.S. (1995) Effect of metformin on glutathione and m agnesium in n ormal an d stre tozotocin- indu ced diabetic rats. J. Appl. T oxicol. 15, 387–390. 4752 H. Khouri et al. (Eur. J. Biochem. 271) Ó FEBS 2004 . [23]). Action of Æ OH radicals In order to study the action of Æ OH radicals on the initiation of metformin oxidation in nonaerated medium, different solutions of metformin. unfavourable. Scheme 1 summarizes the radical-induced oxidation of metformin. MTF Æ symbolizes the Æ OH-induced radical of metformin. Once metformin radicals are produced,