Description THE USE OF THE PROCESS OF INDUCTION OF DE NOVO

SYNTHESIS OF P-GLYCOPROTEIN (P-gp) IN THE TREATMENT OF XENOBIOTIC-INDUCED INTOXICATIONS

IN MAMMALS

Technical domain of the invention

The present invention concerns to the process of induction of de novo synthesis of P-glycoprotein (P-gp) to be used as a treatment of xenobiotic-induced intoxications in mammals and, in particular, of paraquat (PQ)-induced intoxications . It is shown, for the first time that the induction of de novo synthesis of P-gp, 2 hours after PQ exposure, led to a remarkable decrease of PQ accumulation in the lung to 40% of the group exposed only to PQ, and to an increase of its faecal excretion. It was possible to reverse the toxicity, as shown by several biochemical and histopathological biomarkers of toxicity in just 24 hours, constituting this invention an important step in the fight against PQ intoxications.

Antecedents of the invention

Paraquat dichloride (methyl viologen; PQ) is an effective and widely used herbicide, known to cause thousands of deaths in mammals from both accidental and voluntary ingestion, as well as from dermal exposure. The lethality is mainly the consequence of a severe pulmonary toxicity with the consequent respiratory failure, by proliferation of fibroblasts and excessive collagen deposition. The bad prognosis that usually results from this intoxication for which there is no available antidote, impelled the studies that conduct to this invention.

Since its introduction in agriculture in 1962 [1], the paraquat [(PQ: l,r-dimethyl-4,4'-bipyridylium ion: methyl viologen) figure 1], used as desiccant and defoliant in a variety of crops has caused thousands of deaths from both accidental and voluntary ingestion, as well as from dermal exposure [2-5]. Although it may be considered as one of the most toxic poisons frequently used for suicide attempts, it is readily available without restriction in several countries where it is registered. Depending on the ingested dose, different clinical patterns and outcomes have been observed in experimental animals and humans. PQ accumulates in the lung through a system for which the polyamines are the natural substrates [I]. In comparison to other organs, the lungs, and more specifically the alveolar epithelial and Clara cells, are endowed with a particularly active polyamine uptake system [6-10]. This is the main reason for the lung to constitute the target organ for PQ toxicity [I].

Briefly, the direct cellular toxicity of PQ is essentially due to its redox cycling (Fig.

2): PQ is reduced enzymatically, mainly by NADPH-cytochrome P-450 reductase [11] and NADH:ubiquinone oxidoreductase (complex I) [12, 13], to form PQ monocation free radical ( PQ ⁺ ). The PQ ⁺ is then rapidly re-oxidized in the presence of oxygen (which exhibits high concentrations in the lung), thus resulting in the generation of the superoxide radical (O ^" ) [14, 15]. This then sets in the well-known cascade leading to generation of reactive oxygen species (ROS), namely the hydrogen peroxide and the hydroxyl radical (HO) with the consequent deleterious effects characteristics of oxidative stress.Indeed, hydroxyl radicals [16, 17] have been implicated in the initiation membrane-damage by lipid peroxidation during the exposure to paraquat in vitro [16] as well as in vivo [15, 18] by attack on polyunsaturated lipids, depoly- merization of hyaluronic acid, inactivation of proteins and damage of DNA.

Nowadays, no antidote or effective treatment for PQ poisoning has been identified, the survival being mainly dependent on the amount ingested and the time elapsed until the patient is submitted to intensive medical measures to inactivate or to eliminate PQ, before its absorption and/or cellular uptake. These approaches, aimed to prevent PQ accumulation in the tissues, specially the lungs, include procedures such as induction of emesis and/or intestinal transit, gastric lavage, administration of oral absorbents, hemodialysis and hemoperfusion [19-23].

Besides these treatments, additional protective measures have also been adopted: (i) to prevent the generation of ROS, namely the effective iron chelation by desfer- rioxamine [24, 25]; (ii) to scavenge ROS including the maintenance of effective levels of antioxidants, such as vitamin-E [26]; (iii) those aimed to repair the ROS-induced lesions, particularly the maintenance of effective levels of reduced glutathione (GSH) by administrating λf-acetylcysteine [27, 28], and (iv) those aimed to reduce inflammation (normally administered few days after intoxication) by administration of dexamethasone (DX) [29, 30], methylprednisolone (MP) [31], cyclophosphamide (CP) [31] and NAC [27, 28].

However, such treatments are mainly palliatives and the fatality rate remains above 70%.

Summary of the invention

It was precisely this lacuna (the inexistence of antidote) in the treatment of PQ intoxications that impelled our study. It is objective of this invention to provide an effective solution to reduce the levels of PQ in the lung, and by this way its toxicity, increasing the percentage of success of the treatments and consequently the survival of the PQ-intoxicated patients. The present invention concerns to the process of induction of de novo synthesis of P-glycoprotein (P-gp) to be used as a treatment of mammals intoxicated with PQ (Fig. 3).

Although initially discovered by the capacity to confer human tumour cells

resistance to anticancer therapy, P-gp was found to be expressed in a polarized manner in a variety of normal organs and tissues such as lung, kidney (epithelium of the renal proximal tubule), small and large intestine (apical surface of mucosal cells), liver (biliary canalicular membrane of hepatocytes), lung, adrenal cortex, pancreatic ductulus, endometrium of pregnant uterus, capillaries of testes, and CD34 ⁺ bone marrow stem cells, and at high levels in the endothelial cells of brain capillaries [32-35

]•

Such a spatial distribution of this efflux transporter, has defined it as a functional importantelement in reducing the systemic exposure and specific tissue access of potentially harmful xenobiotics. What we describe, and for the first time, is precisely the process of de novo synthesis of P-gp in order to modify PQ toxicokinetics (Fig. 3).

In fact, the subject of our claims is precisely the opposite of the anticancer therapy, in which the main objective is to limit the drug efflux of the cells. What we developed was the induction of this transporter, as a way to eliminate PQ from its target organ (lung).

With the industrial application the invention here described, it is possible to decrease the lung PQ levels of intoxicated mammals to 40% of the levels in the PQ only exposed group in just 24 hours, leading to a reversion of the lung toxicity, constituting this invention an important step in the fight against PQ intoxications. Importantly, the described invention can be applied 2 hours after intoxication, conferring more realism in the application in human intoxications, since it reflects, in the majority of the cases, the necessary time to hospital arrival after PQ ingestion. The novelty concerning to this process is the induction of the P-gp de novo synthesis to eliminate rapidly toxic compounds out of the target cells, and thus decreasing their toxicity. That always occurs when drugs exhibiting the capacity to induced novo synthesis of P-gp are administered.

Brief description of the figures

Fig. 1.A Chemical structure of PQ and its salts available in the market.

Fig. 1.B Chemical structure of dexamethasone.

Fig. 2. Schematic representation of the mechanism of PQ toxicity. A. Cellular di- aphorases, SOD. Superoxide dismutase or spontaneously, CAT. Catalase, Gpx. Glutathione Peroxidase, Gred. Glutathione Reductase, PQ . Paraquat, PQ ⁺ . Paraquat cation free radical, FR. Fenton reaction, HWR. Haber- Weiss Reaction.

Fig. 3. Proposed scheme for the PQ efflux mediated by P-gp. Abbreviations: GR - glucocorticoid receptor, DNA - deoxyribonucleicacid, ATP - adenosine triphosphate, ADP - adenosine diphosphate, MDR - Multidrug resistance gene.

Fig. 4.1. Electron microscopy photographs of the lung tissue from 2 animals of control group, showing a normal pulmonary structure without evidences of alveolar

collapse, vascular congestion or cellular infiltrations. It is visible some pneumocytes type II (in A and B) and one alveolar macrophage (in B) (original magnification: A- 4.00Ox; B - 6.300Ox).

Fig. 4 JI. Electron microscopy photographs of the lung tissue from 2 animals of PQ only exposed group (25 mg/Kg, Lp.), both evidencing an altered alveolar structure, with alveolar collapse and numerous hypodense areas in the interstitial space, suggestive of a marked interstitial edema. In A, it is depicted 2 polymorphonuclear, one within a capillary and other in the interstitium; in B it is also notorious the mitochondrial swelling of type II pneumocytes (original magnification: A - 8.00Ox; B - 10.00Ox).

Fig. 4.m. Electron microscopy photographs of the lung tissue from 2 animals of the group intoxicated with PQ (25 mg/Kg, Lp.) and treated with dexamethasone (100 mg/Kg, Lp., 2 hours after intoxication) where it is possible to observe a tissue preserved structure. The signals of interstitial edema, although perceptible, are light, either in A as well as in B; in spite the alveolar macrophage present in B, it was not observed any leukocyte infiltration (original magnification: A - 5.000x; B - 6.30Ox).

Fig. 5.A % of wet lung weight/body weight.

Fig. 5.B Effect of the induction of de novo synthesis of P-gp in the lung PQ concentration. Results are expressed as μg of PQ/mg of protein.

Fig. 5.C Amount of PQ excreted by urine (mg of PQ/kg of body weight/26h).

Fig. 5 .D Amount of PQ excreted by faeces (mg of PQ/kg of body weight/26h)

Fig. 5 .E Concentration of MDA in lung in the different groups. Results are expressed in nmol of equivalents of MDA/mg of protein.

Fig. 5 .F Concentration of carbonyl groups in lung in the different groups. Results are expressed in nmol of equivalents of carbonyl groups/mg of protein.

Fig. 5 .G Pulmonary MPO activity in the different groups (U/g of protein).

Animals were treated as indicated and described in the Materials and Methods section. They were sacrificed 26 hours after PQ exposure (25 mg/kg). Each data in the graph is the mean + S.E.M. (standard error mean) of 8 animals. Key: (*) statistically difference with a significant interval of P <0.05; (**) statistically difference with a significant interval of P <0.01; (***) statistically difference with a significant interval of P <0.001; (ns) difference statistically non significant. P values were obtained by the non-parametric method of Kruskal-Wallis followed by the Dunn's test. Abbreviations: PQ - paraquat, Dex - dexamethasone, Ver - verapamil, MDA - malondialdehyde, MPO - myeloperoxidase, BW - body weight, WLW- wet lung weight.

Detailed description of the invention

The present invention concerns to the process of induction of de novo synthesis of P-glycoprotein (P-gp) to be used in the treatment of PQ mammal intoxications (Fig. 3).

P-gp (plasma membrane phosphoglycoprotein), a member of ATP-binding cassette (ABC) superfamily, was initially identified in tumour cells as an ATP-dependent transporter, which can export a wide variety of unmodified substrates out of the cell including Vinca alkaloids, colchicine, antibiotics and anthracyclines [29, 32, 36, 37] but also other anticancer drugs of natural or semi-synthetic origin, organic cations and diagnostic agents too [38]. This drug transport can occur against a concentration gradient, and it is independent of an electrochemical transmembrane potential or proton gradient [39] .It was possible, for the first time, to eliminate PQ from the lungs though the induction of this transporter, which pumped PQ out of the epithelial lung cells and bronchiolar Clara cells. The functionality of these cells is critic for life and it is strongly affected by PQ toxicity. The great advantage of this invention was to obtain a decrease of PQ lung levels and as consequence a decrease of PQ toxicity by continuous ROS generation.

In this invention we used a glucocorticoid drug - dexamethasone - in order to induce de novo synthesis of P-gp, and by this way to increase PQ efflux from its target organ. A number of reports exist noting dexamethasone induction of P-gp levels in liver, brain, and intestinal tissue and also in lung tissue [40], an effect that seems to be glucocorticoid concentration-dependent. This increase is rapid, since it was observed after only one day post-treatment [40].

Administering dexamethasone (100 mg/Kg, i.p.), two hours after rats PQ intoxication (25 mg/Kg, i.p.) it was possible to decrease the PQ lung levels to 40% of only PQ exposed animals in just 24 hours. The treatment with dexamethasone permitted also to stimulate PQ elimination by intestinal route (through induction of intestinal P-gp), thus contributing to lower the bloodstream levels by faecal excretion.

Subsequently, with the aim to validate our invention, it was performed a study with verapamil, a competitive inhibitor of P-gp, which blocked dexamethasone protective effects, and lead to an increase of PQ lung concentration (up to twice of the only PQ- exposed group in just 24 hours) and toxicity, indicating the important role of this transporter in PQ excretion.

No other previous treatment proved to lower PQ lung concentrations or to stimulate its intestinal excretion. As referred above, the toxicity of PQ depends on its lung accumulation where PQ leads to an oxidative stress condition by continuous ROS generation.

The induction of P-gp with dexamethasone administration (100 mg/Kg of body weight), two hours after intoxication of rats with PQ(25 mg/Kg of body weight) it was possible to decrease the PQ lung levels to 40% of only PQ exposed animals in just 24 hours. This period between PQ exposition and dexamethasone administration was established taking into account the time that normally occurs in the human reality

between ingestion and the arrival of the intoxicated patient to the Emmergency Department to initiate the therapeutic.

Consequently, it was possible to reverse the pulmonary PQ toxicity, constituting this invention an important step against PQ mammal intoxications.

It was also possible to observe that the concomitant induction of P-gp of the intestinal epithelium permitted an increase of PQ excretion by intestinal route and c on- sequently of the organism.

Below it is described one example of a way to concretize the invention using as a mammal mode, the rat.

Reagents e drugs utilized

Paraquat, (methylviologen; l,l'-dimethyl-4,4'-bipyridinium dichloride), dex- amethasone

[( 11 β, 16α)-9-Fluoro- 11 , 17,21 -trihydroxy- 16-methylpregna- 1 ,4-diene-3,20-dione], (±)-verapamil hydrochloride (5-[(3,4 dimethoxyphenethyl) methylamino] - 2-(3,4-dimethoxyphenyl)-2-isopropylvaleronitrile hydrochloride), corn oil, 3,3',5,5'-Tetramethylbenzidine (TMB), 5-sulfosalicylicacid were all obtained from Sigma (St. Louis, MO, U.S.A.). The saline solution (NaCl 0.9%), sodium heparine (5000 UI/ml), sodium thiopental ( 0.5 g ) were purchased from B. Braun ( Lisbon , Portugal ). The sodium hydroxide (NaOH), sodium dithionite (Na S O ), 2-thiobarbituricacid (C 4 H4 N2 O2 S) and the trichloroacetic acid (Cl 3 CCOOH) were obtained from Merck ( Darmstadt , Germany ). All the reagents used were of analytical grade or of the highest grade available.

Treatment of animals - In vivo studies

The study was performed using (n=40) adults male Wistar rats obtained from Charles River S. A. ( Barcelona , Spain ), with a mean weight of 250 + 1Og. Animals were randomly distributed in number of two per polypropylene cage with a stainless steel net at the top and wood chips at the screen bottom, in a air-conditioned room (alternate cycles of 12 hours light and darkness, room temperature 22 + 2 ⁰ C and with a relative moisture of 50-60%) at least one week (quarantine) before starting the experience in order to acclimate to their surrounding conditions. Animals were allowed access to tap water and rat chow ad libitum during the quarantine period. Housing and experimental treatment of animals were in accordance with National Institutes of Health guidelines (ILAR 1996). The experiments complied with the current laws of Portugal.

After the quarantine period, animals were randomly divided into four groups of ten animals each. Every one of the animals was individually housed in metabolic cages where they stayed for 26 hours. Animals were fasted during the entire experimental period accordingly to other studies. Water was given ad libitum. The administrations of

vehicle (0.9% NaCl), PQ, dexamethasone and verapamil were all made i ntraperitoneally (i.p.) in an injection volume of 0.5 mL. The four groups were treated as follows (given doses were kg per body weight):

(I) Group I, n=10 (control): injected intraperitoneally (i.p.) with 0.5 mL of 0.9% NaCl. Animals were treated with two more administrations of 0.9% NaCl, one and two hours later, respectively;

(II) Group π, n=10 (PQ): injected i.p. with a dose of 25 mg/kg de PQ dissolved in 0.9% NaCl. Animals were treated with two more administrations of 0.9% NaCl, one and two hours later, respectively;

(HI) Group in, n=10 (PQ + Dex): injected i.p. with a dose of 25 mg/kg de PQ dissolved in 0.9% NaCl. Animals were treated with 0.9% NaCl and DEX (100 mg/kg), one and two hours later, respectively;

(IV) Group IV, n=10 (PQ + Ver + Dex): injected i.p. with a dose of 25 mg/kg de PQ dissolved in 0.9% NaCl. Animals were treated with verapamil (10 mg/kg) and dexamethasone (100 mg/kg), one and two hours later, respectively.

The treatments in all groups were always conducted between 8:00 and 10:00 a.m.

Collection of urine and faeces during 26 h

Whole urines and animals faeces where collected during 26 hours.

Preparation of the lung homogenates

Twenty-six hours after PQ administration, anaesthesia was induced with sodium thiopental (60 mg/Kg, i.p.) and sodium heparin (1500 UI) was injected through the i.p. route. Animals were placed in the decubito supino position and tracheotomy and tracheal cannulation was done, followed by the immediate connection of the cannula to a mechanical ventilation system that supplied a tidal volume of 2 mL at a respiratory frequency of 60 r.p.m. The thorax was opened by two lateral transversal and one central longitudinal incision to expose the pulmonary artery. In 8 rats of each group, lungs were perfused in situ through the pulmonary artery with cold 0.9% NaCl for 3 min at a rate of 10 ml/min to be completely cleaned of blood. At the same time that this perfusion was initiated, a cut at the left wall ventricle was done to avoid overpressure.

Lungs were removed, cleaned of all major cartilaginous tissues of the conducting airways, pat-dried with gauze and weighted (for the evaluation of lung edema).

The weight was expressed in percentage of the wet weight/body weight (Fig. 5.A) and the lungs were processed as following:

(I) Right lungs were homogenized (Ultra-Turrax® Homogenizer) in ice-cold 50 mM phosphate buffer (KII PO4 + Na ₂ HPO4.II O) / 0.1% (v/v) Triton X-100 (pH 7.4). The homogenate was kept on ice, then centrifuged at 13.000 g, 4°C,for 10 min. Aliquots of the resulting supernatants were stored (-80 ⁰ C) for posterior quantification of the

pulmonary remaining PQ, determination of myeloperoxidase activity (MPO), carbonyl groups, quantification of the P-gp induction and protein.

(II) Left lungs were homogenized (Ultra-Turrax® Homogenizer) in trichloroacetic acid (TCA) 10%. The homogenate was kept on ice and then centrifuged at 13.000 g , 4 ⁰ C, for 10 min. Aliquots of the resulting supernatants were immediately used for evaluating the degree of lipid peroxidation of the samples.

Tissue processing for histological analysis

Two animals of each group were assigned to histological analysis. Lung samples were subjected to routine procedures for light microscopy (LM) and transmission electron microscopy (TEM) analysis. With the animals under anaesthesia, lung fixation was initiated in situ by perfusion through pulmonary artery, with 2.5% glutaraldehyde in 0.2 M sodium cacodylate buffer (pH 7.2 - 7.4) during 3 min. Subsequently, lungs were excised and sectioned into about 1 mm cubic pieces. After two washing steps, of 30 minutes each with buffer solution, the specimens were dehydrated in graded alcohol for 2 hours, and then embedded in Epon. Propylene oxide was the compound used in the dehydratation-impregnation transition. The inclusion phase lasted 2 days. All the procedures were done at 4°C, with exception of the inclusion phase, which was performed at 60 ⁰ C. Subsequent to the resin polymerization, semi-thin sections (thickening 1 μm) and ultra-thin sections (500 A of thickness) were prepared (Ultracut, Leica), respectively for LM and TEM analysis. The grids, mounted with the ultra-thin specimens sections, were double-contrasted with 0.5% saturated uranyl acetate aque- oussolution during 30 min and then with 0.2% lead citrate solution for 15 min. The slides, mounted with semi-thin sections, were stained with toluidine blue. Five slides and three grids from each animal (standing ten slides and six grids per group), were examined in a Zeiss Phomi III photomicroscope and in a transmission electronic microscope {Zeiss EM 10A).

The evaluation of structural and ultrastructural analysis alterations was performed and registered those deviant relatively to control group (Fig. 4.I-4.III).

Confirmation ofde novo synthesis of P-gp induction

The expression of P-gp in the lungs was quantified according to the methodology described by Demeule et al. [40].

Protein quantification

Protein quantification was performed accordingly to the method of Lo wry et al. [41 ] using bovine serum albumin as standard.

Quantification of PQ in the lung

Aliquots of right lung supernatants were treated with 5-sulfosalicylic acid (5% in final volume) to precipitate proteins and then centrifuged ( 13.000 g , 4° C for 10 min). The resulting supernatant fractions from lung, urine and faeces were alkalinized with

NaOH IO N (pH >9) and then gently mixed with few crystals of a reductant (sodium dithionite) to give the blue colour, characteristic of the PQ cation radical.

PQ quantification was carried out using a rapid, simple method based on second- derivative spectrophotometry [42-44] using a Shimadzu model UV7VIS 160 double- beam with a built-in microcomputer and a quartz cell with an optical path length of 1.0 cm . No interference was observed in the zero-order and second-derivative spectrum of the blank. The data of a zero-order spectrum obtained by scanning from 500 to 380 nm with a 0.5 nm bandwidth was stored in the machine and then differentiated with four nm of differential wavelength to give a second derivative spectrum. A qualitative and quantitative analysis of reduced paraquat was made at the amplitude peaks of 396-403 nm of the second-derivative spectrum. The calibration curve in the 0.2-8 μg/ml range obeys Lambert-Beer's law. The samples were diluted in order to fall into the reference range of the standard curve.

Using these experimental conditions, the intra- and inter-day coefficients of variation showed values lower than 5% and the detection limit of the method was 100 ng/mL.

The results were expressed in μg of PQ/mg of protein (Fig. 5.B).

Urine and faeces samples processing

Faeces were processed in order to quantify the intestinal elimination of PQ. The faeces homogenized were treated with 5-sulfosalicylic acid (5% in final volume) and then centrifuged ( 13.000 g , 4 ⁰ C, for 20 min). The supernatant fraction was again centrifuged five times more to obtain, as most as possible, a clean supernatant.

Concerning the urine samples, these were centrifuged ( 13.000 g , 4 ⁰ C, for 10 min).

The quantification of PQ in faeces and whole urine was performed accordingly to the described for the lungs.

Results were expressed in mg of PQ/kg of body weight/ 26 h (Fig. 5.C e 5.D).

Lipid peroxidation (LPO)

LPO is a process of unsaturated lipid oxidative damage caused by ROS. In an oxidative stress situation, a failure of the endogenous antioxidants defense mechanisms could occur with formation of ROS, capable of oxidative attack of membranes lipids [45]. It involves the formation and propagation of lipid radicals, the uptake of oxygen, a rearrangement of double bounds in unsaturated lipids, and the eventual destruction of membrane lipids, producing a variety of breakdown products, including alcohols, ketones, aldehydes, and ethers [46]. The LPO degree was here evaluated by the method of the reactive substances to thiobarbituric acid (TBARS) [47]. Briefly, this method has as principle, the reaction of one molecule of malondialdehyde (MDA) or MDA- like substances with two of 2-thiobarbituric acid (TBA) and formation of a pink

relatively stable complex having an absorption maximum at 535 nm. An aliquot of the left lung supernatant was added to TBA (1%) (1:1; v/v). The mixture was heated for 10 minutes in a boiling water bath. After cooling, the flocculent precipitate was removed by centrifugation at 1000 g during 10 minutes and the measuring of absorbancy was made at 535 nm.

Results were expressed nmol of MDA/mg proteins using an extinction coefficient of 1.56 ' 10 ⁵ M ¹ Cm ^"1 (Fig. 5.E).

Quantification of protein carbonyl groups

ROS are thought to be involved in many physiological and pathological processes and are known to oxidatively modify besides lipids, also DNA, carbohydrates and proteins. One such modification is the addition of carbonyl groups to amino acid residues in proteins. Free radical damage to proteins has been implicated in the oxidative inactivation of several key metabolic enzymes. Oxidatively modified proteins accumulate in different pathological conditions, including inflammatory diseases [48]. The oxidation of proteins is caused by interaction of proteins with ROS which can be generated by enzyme catalyzed redox reactions [49]. Fragmentation of polypeptide chains, increased sensitivity to denaturation, formation of protein-protein cross-linkages as well as modification of amino acids side chains to hydroxyl or carbonyl derivatives are possible outcomes of oxidation reactions [50].

One of the most commonly used non-radiochemical methods for determining carbonyl content in proteins is a spectrophotometric method which uses 2,4-dinitrophenylhydrazine (DNPH). Carbonyl groups (ketones and aldehydes) react with DNPH to form 2,4-dinitrophenylhydrazone and the amount of hydrazone formed is quantitated spectrophotometrically [51].

Carbonyls were determined according to Levine et al. [51], with slight modifications. Sample proteins were precipitated with ice-cold TCA (final concentration 10 % v/v). After a 10-min incubation period at 4 ⁰ C, samples were centrifuged at 11,000 g for 3 min. The protein pellet was re-suspended in 0.5 ml of 10 mM DNPH/2 M HCl. Blanks were prepared using 500 μl of each sample and 0.5 ml of 2 M HCl without DNPH. Both the samples and blanks were run in duplicate. Samples and blanks were vortexed continuously at room temperature for 1 hour. The samples were re- precipitated with 0.5 ml of 20 % of TCA, centrifuged at 11,000 g for 3 min. After centrifugation, the supernatant was discharged and 1 ml of ethanol-ethylacetate (1/1; v/v) solution was added to each tube to remove free DNPH reagent, aided by mechanical disruption of the pellet by vortexing. Tubes were allowed to stand for 10 min and then spun again (15 min at 11,000 g ) repeating the washing procedure two times more for a total of three washes.

After the final wash, the resulting pellet was re-suspended in 1 ml of 6 M guanidine

hydrochloride with 20 mM potassium dihydrogen phosphate (pH 2.3). To speed up the solubilisation process, the samples were incubated in at 37°C water bath for 30-60 min. The final solution was centrifuged ( 3000 g , 5 min) to remove insoluble material. The carbonyl content was calculated from the absorbance measurement at 380 nm and an absorption coefficient ε=22,000 M ^~ cm ^" [52]. The absorbance difference of the DNPH-treated samples versus the HCl blanks was determined, and the protein carbonyl content was expressed as nmol of DNPH incorporated per mg of protein (Fig. 5.F ).

Mieloperoxida.se (MPO) activity

The extension of lung inflammation in the different treatments was evaluated by MPO activity determination accordingly to the method followed by Suzuki et al. and Andrews et al. [53, 54], with slight modifications. MPO is a heme enzyme which activity reflects indirectly the lung PMN (polymorphonuclear) leucocytes infiltration, since MPO is located within the primary azurophil granules of PMN leucocytes. The activity was determinate spectrophotometrically at 655 nm measuring the 3,3',5,5'-tetramethylbenzidine (TMB) oxidation by MPO/H O system. When oxidized, TMB yields a blue-green colour. Briefly, the supernatants were initially submitted to three cycles of snap freezing. The assay mixture consisted of 50 μl of supernatant fraction, 50 μl of TMB (final concentration 7.5 mM ) dissolved in DMSO. Reading was initiated by addition of 50 μl of H O (final concentration 1.5 mM ) dissolved in phosphate buffer (Na ₂ HPO ₄ ^H ₂ O 50 mM , pH 5.4). The rate of MPOM O system catalyzed oxidation of TMB was followed by recording the increase of at 655 nm at 37 ⁰ C absorbance during 3 minutes. Considering the linear phase of the reaction, it was determinate the absorbance changed per minute. One enzyme unit was defined as the amount of enzyme capable to reduce one μl de H O /min under assay conditions.

Results were expressed in enzyme units/g of protein, using the extinction coefficient of 3.9 ' 10 ⁴ M ¹ Cm ^"1 (Fig. 5.G).

Statistical analysis

Results are expressed as mean + S.E.M. (standard error of the mean). Statistical comparison between groups was estimated using the non-parametric method of Kruskal-Wallis followed by the Dunn's test. In all cases, P values lower than 0.05 were considered statistically significant.

The results obtained and presented in Fig. 1 to 5 demonstrate the importance of the invention process here described, in the pulmonary elimination of PQ and on its intestinal excretion, which results in a strong decrease of toxicity confirmed by the decreased of the several toxicological parameters evaluated.

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