Analysis in the Rat of 4-Hydroxynonenal Metabolites Excreted in Bile

and HNA-lactone mercapturic acid conjugates. The fifth metabolite was isolated but remained unidentified. As previously observed for urinary eliminati...
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Chem. Res. Toxicol. 1999, 12, 887-894

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Analysis in the Rat of 4-Hydroxynonenal Metabolites Excreted in Bile: Evidence of Enterohepatic Circulation of These Byproducts of Lipid Peroxidation Alexia Laurent, Jacques Alary, Laurent Debrauwer, and Jean-Pierre Cravedi* Laboratoire des Xe´ nobiotiques, INRA, BP 3, 31931 Toulouse Cedex 9, France Received March 10, 1999

4-Hydroxynonenal (HNE) is a cytotoxic product resulting from the lipid peroxidation of membrane polyunsaturated fatty acids. In vitro, metabolism mainly leads to the corresponding alcohol (DHN), carboxylic acid (HNA), and the glutathione conjugate, whereas in vivo, mercapturic acid conjugates of HNE, DHN, HNA, and HNA-lactone and, more recently, dicarboxylic acids and related mercapturate conjugates were identified in urine of rats. In the study presented here, the identity of the HNE biotransformation products in the bile of rats following a single iv administration of [4-3H]HNE and the potential for enterohepatic recycling of HNE metabolites were investigated. The identity of metabolites was assessed by comparison of their HPLC retention times with those of the corresponding synthesized standards and by mass spectrometry analysis. Five metabolites were present in the bile; two of them corresponded to HNE- and DHN-glutathione conjugates. Two others metabolites were identified as DHNand HNA-lactone mercapturic acid conjugates. The fifth metabolite was isolated but remained unidentified. As previously observed for urinary elimination, the kinetic excretion of biliary metabolites exhibited a rapid metabolism of HNE in rats. Within 4 h of injection, the bile accounted for 19.5% ((2.8%) of the injected radioactivity, whereas only 3% was found in the feces within 48 h [Alary, J., et al. (1995) Chem. Res. Toxicol. 8, 34-39]. The extent of HNE enterohepatic recycling was estimated utilizing a modified version of the linked rat model in three animals. All rat recipients were found to have measurable levels of HNE metabolites in bile, confirming that HNE is likely to undergo enterohepatic recirculation in the rat. The extent of recycling was approximatly 7.7% of the total dose in this model. Two unknown metabolites were present in the bile of recipient rats and not found in the bile of donors rats, suggesting that intestinal microflora and/or intestinal mucosa could biotransform HNE-related compounds before or during the reabsorption process.

Introduction 4-Hydroxy-2,3-trans-nonenal is a major R,βunsaturated aldehyde produced in vivo during the peroxidation of n-6 polyunsaturated fatty acids such as arachidonic and linoleic acids (1). This compound, regarded as a “second toxic messenger” (1), is thought to be mainly responsible for the damage observed in free radical pathology, including cytotoxicity, genotoxicity, enzyme inactivation, and effects on cell proliferation and gene expression (2-7). The toxicity and atherogenicity of HNE have been attributed to covalent protein adducts mainly formed by 1,4-addition of nucleophilic sulfhydryl groups from proteins or peptides to the electrophilic double bond of HNE (Michael addition) even without benefit of enzymatic catalysis (8). In addition to protein adducts, clear evidence exists that HNE can form DNA adducts in vitro (9, 10). The possibility therefore exists (HNE)1

* To whom correspondence should be addressed. Telephone: +33 561 28 50 04. Fax: +33 561 28 52 44. E-mail: Jean-Pierre.Cravedi@ toulouse.inra.fr. 1 Abbreviations: HNE, 4-hydroxy-2,3-trans-nonenal; HNE-GSH, 4-hydroxynonenal-glutathione conjugate; DHN, 1,4-dihydroxynonene; DHN-GSH, 1,4-dihydroxynonene-glutathione conjugate; DHN-MA, 1,4-dihydroxynonene-mercapturic acid conjugate; HNA, 4-hydroxynonenoic acid; HNA-MA, 4-hydroxynonenoic-mercapturic acid conjugate; HNA-lactone-MA, hydroxynonenoic-lactone mercapturic acid conjugate.

that HNE reacts with DNA in vivo, causing mutational events similar to those reported in vitro (11). Moreover, the fact that alkenals inhibit certain DNA repair systems (12) would increase the magnitude of the mutagenic and genotoxic effect of HNE. In recent years, much effort has been spent on identifying the metabolic products of HNE and understanding its detoxification processes. The rapid biotransformation of this cytotoxic aldehyde in less reactive compounds could play a key role in the defense system of various tissues against toxic substances arising from free radicalinduced lipid peroxidation. In vitro, several studies carried out with rat liver subcellular fractions and hepatocytes have demonstrated that the main metabolites of HNE were the corresponding alcohol, carboxylic acid, and the glutathione conjugate (13-16). In vivo, identification of urinary metabolites in the rat after iv administration of HNE yielded at least four different mercapturic acids (17): 1,4-dihydroxynonane-mercapturic acid (DHN-MA) which appeared to be the major urinary metabolite, 4-hydroxynonenal-mercapturic acid (HNE-MA), the mercapturic acid of 4-hydroxynonenoic acid (HNA-MA), and the corresponding lactone (HNAlactone-MA). These metabolic pathways were confirmed by De Zwart et al. (18) in the rat after ip administration of HNE. In both cases, 48 h after the dose had been

10.1021/tx9900425 CCC: $18.00 © 1999 American Chemical Society Published on Web 09/03/1999

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administered, the total recovery of administered radioactivity was 60-80%, whereas only 3% was excreted in the feces with iv administration and 18% found in this excreta following ip injection. The possible role of biliary excretion in this discrepancy was hypothesized (18). Very recently, additional polar metabolites corresponding to dicarboxylic acids and related mercapturate conjugates were found in urine of rats and mice treated with HNE (19, 20), confirming the complexity of the biotransformation pathways of HNE in vivo. To extend our understanding of the metabolic fate of this aldehyde in mammals, we investigated the biliary metabolites of HNE in the rat. For this purpose, an HPLC system was developed to allow the separation and the isolation of the biotransformation products, and peak identification was performed by mass spectrometry analysis. Furthermore, in this study we treated rats with cannulated bile ducts with the bile collected from rats previously injected with HNE to determine the existence of enterohepatic circulation for HNE metabolites and to determine any contribution this enterohepatic circulation has to the overall metabolism of HNE.

Materials and Methods Chemicals. Standard HNE was kindly provided by H. Esterbauer (Department of Biochemistry, University of Graz, Graz, Austria). It was supplied as HNE diethyl acetal dissolved in chloroform and was stored at -20 °C until it was required. Just prior to use, HNE was prepared from its diethyl acetal derivative by 1 mM HCl hydrolysis over the course of 1 h at room temperature. HNE diethyl acetal was synthesized according to the method of Esterbauer and Weger (21), and HNE was liberated as for the standard. [4-3H]HNE diethyl acetal was synthesized at CEA, Service des molecules marque´es CEN (Saclay, France), according to the method developed in our laboratory (22). Its specific activity was 222 GBq/mmol, and its radiochemical purity, as determined by radio-HPLC, was 95%. Raney nickel and sodium borohydride were purchased from Aldrich (Saint Quentin Fallavier, France), and Helix pomatia β-glucuronidase was from Sigma (Saint Quentin Fallavier, France). Glutathione was from Boehringer Manheim (Meylan, France). All chemicals, solvents, and reagents for the preparation of buffers and HPLC eluents were the highest grade commercially available from Merck (Nogent-sur-Marne, France). Ultrapure water from the Milli-Q system (Millipore, Saint Quentin en Yvelines, France) was used for HPLC eluent preparation. Syntheses of Standards. HNE-GSH was synthesized according to the procedure of Uchida and Stadtman (6). HNE was reacted with a stoichiometric amount of GSH for 12 h at 37 °C in 1 mL of 50 mM sodium phosphate (pH 7.2). The sample was loaded on a Supelclean LC-18 cartridge (Supelco Park, Bellefonte, PA) and eluted with 5 mL of methanol. The methanolic fraction was evaporated to dryness and taken up in 500 µL of methanol. The same procedure was used to prepare [3H]HNE-GSH from [3H]HNE. DHN-GSH was synthesized by sodium borohydride reduction of HNE-GSH as previously described (6). After 1 h at 37 °C, the remaining borohydride was decomposed by adding 1 M HCl. The reaction mixture was purified according to the procedure used for HNE-GSH. 1,4-Dihydroxynonane was obtained by treating DHN-GSH with Raney nickel (6). DHN-MA was synthesized according to the procedure of Alary et al. (17). Briefly, HNE was reacted with a stoichiometric amount of N-acetylcysteine for 72 h at 50 °C. The resulting HNE-MA was reduced by sodium borohydride. The same procedure was used to prepare [3H]HNE-MA from [3H]HNE.

Laurent et al. [3H]HNA was synthesized by sodium chlorite oxidation of HNE (23). A mixture of [3H]HNE and HNE (13 µmol) was dissolved in 500 µL of HCl (1 mM) and was added with 100 µL of an aqueous solution of sulfamic acid (15 µmol) and 100 µL of sodium chlorite (15 µmol) in water, for 3 h at room temperature. The solution was extracted three times with 2 mL of chloroform. The chloroform extract was treated with 5 mL of sodium bicarbonate solution (0.1 M). The organic layer was discarded, and then the aqueous phase was acidified to pH 2 and extracted with CHCl3. HNA-lactone-MA was synthesized by reaction of HNA with N-acetylcysteine, according to the method of Alary et al. (17). The identity of these standard compounds was confirmed by FAB-MS in the negative ion mode. Incubation of DHN with Glutathione S-Transferases. GST’s activity toward 1-chloro-2,4-dinitrobenzene was determined by the method of Habig et al. (24). A 2 mL aliquot of 0.1 M phosphate buffer (pH 6.5) containing 1 mM GSH, DHN (216 kBq), and 0.25 µg of rat liver cytosolic glutathione S-transferases (10.6 × 106 nmol of S-2,4-dinitrophenylglutathione min-1 mg of protein-1) purified by GSH affinity chromatography was incubated for 2 h at 25 °C. After protein precipitation with 8 mL of ice-cold methanol, the aqueous phase was adjusted to pH 2.5. The sample was loaded on a Supelclean LC-18 cartridge, and after the cartridge had been washed with dilute phosphoric acid (pH 2.5), the sample was eluted with 15 mL of methanol. An aliquot of the methanolic fraction was evaporated to dryness, taken up with water/acetonitrile (95:5, v/v), and analyzed by HPLC. Animal Treatments. Three separate experiments were conducted, using male Wistar rats weighing approximately 250 g. In each case, the animals were anesthetized with a urethan (Prolabo, Fontenay-sous-Bois, France) intraperitoneal injection (1.2 g/kg) and were kept anesthetized for the duration of the experiment. In experiment I, for 12 rats in which the bile duct was cannulated using PE 10 surgical tubing, a combination of 2 mg of HNE and [3H]HNE was subsequently injected into the penis vein to a final specific activity of 0.83 MBq/mg. Bile from nine rats was quantitatively collected on ice for 4 h after administration of the dose to facilitate the production of sufficient quantities of metabolites needed for identification purposes. Concurrently, bile from three rats was collected at 15 min intervals for 1.5 h postinjection to establish a time course excretion curve. In experiment II, for six rats, 1.18 MBq (0.83 µg) of [3H]HNE was injected into the penis vein. Three rats had bile duct cannulas as described for experiment I, and the bile was collected at 1 h intervals for 6 h postinjection. Bile volumes were measured, and an aliquot was sampled for radioactivity counting. Remaining bile samples were pooled, divided in three equivalent volumes, shock-frozen in liquid nitrogen, and stored at -80 °C until they were used in experiment III. Concurrently, the three other rats were Sham operated, but without choledoch cannulation. After 4 h, animals were sacrificed, blood samples were collected via the jugular vein, and residual urine was withdrawn from the bladder with a 5 mL syringe. Tissues (liver, kidney, and fat) were sampled in triplicate (ca. 200 mg each) post-mortem from the Sham-operated group. Remaining carcasses containing unsampled viscera and digestive contents were homogenized using a mincing machine. Five aliquots of this homogenate were sampled. Samples were analyzed for tritium residue content following oxidation in a Tri-Carb model 306 oxidizer (Packard Instrument Co., Downers Grove, IL). The total residue concentrations were calculated from the specific activity and radioactivity per mass unit. The extent of HNE enterohepatic recycling was estimated utilizing a modified version of the linked rat model. Generally, the linked rat model is prepared from a bile duct-cannulated rat by placing the distal free end of the bile cannula into the duodenum of a second rat, allowing bile to flow directly from the bile duct of the donor rat into the duodenum of the recipient rat (25). In our case (experiment III), donor rat bile was not

4-Hydroxynonenal Metabolites Excreted in Bile diverted directly into the duodenum of the recipient rat but rather collected into tubes and later delivered to recipient rats by intraduodenal infusion. Three bile duct-cannulated rats (recipient rats) were prepared as described for experiment I. A sample of bile (ca. 1 mL) from donor rats previously treated with [3H]HNE (see experiment II) was infused into the duodenum of each receiving animal via the end part of the choledoch canal. The duration of the infusion was approximately 10 min, and the radioactivity that was injected amounted to 166 kBq per rat. Bile was collected at 1 h intervals for 6 h postinjection. Unless otherwise indicated, bile samples were filtered through a 50 kDa omega ultrafiltration membrane (microconcentrators, Filtron Technology Corp., Northborough, MA) and stored at -20 °C until analysis. Radioactivity Determination. Aliquots of bile, urine, and tissues samples were counted in a Tricarb 4430 scintillation counter (Packard Instrument Co.) with Ultima Gold as the scintillation cocktail (Packard Instrument Co.). HPLC. The HPLC system consisted of two 420 Kontron pumps with a 491 gradient former and a Spectra Physics UV 150 detector set at 223 nm. Radioactivity detection was carried out with a radiomatic Flo-one A-200 instrument with Flo-scint 2 as the scintillation cocktail (Packard Instrument Co.). HPLC separations were performed on a Spherisorb ODS 2 Column (5 µm, 25 cm × 0.46 cm) from Shandon (Eragny, France), protected by a precolumn (5 µm, 1 cm × 0.46 cm, Spherisorb ODS 2). Collection of radioactive peaks for MS analysis was carried out with a Gilson (Villiers-Le-Bel, France) model 202 fraction collector. The gradient elution system for the separation of biliary metabolites of HNE was as follows. Eluent A contained 95% ammonium acetate (20 mM) adjusted to pH 3.5 with acetic acid and 5% acetonitrile. Eluent B consisted of 30% aqueous buffer as described above and 70% acetonitrile. Solvents were delivered at a flow rate of 1 mL/min as follows: from 0 to 3 min, 100% A; from 3 to 15 min, a linear gradient from 100 to 72% A; from 15 to 30 min, 72% A; from 30 to 35 min, a linear gradient from 72% A to 100% B; from 35 to 40 min, 100% B; and from 40 to 41 min, a linear gradient from 100% B to 100% A. Mass Spectrometry. Peak identification was carried out by FAB-MS. After filtration, pooled bile samples from nine rats were directly injected into the HPLC system and the metabolites were collected. The eluate was evaporated to dryness, and each fraction residue (20-300 µg) dissolved in 100 µL of methanol was stored at -20 °C until mass spectrometry analysis. Mass spectra were obtained on a Nermag R-10-10H (Delsi Nermag Instruments, Argenteuil, France) single-quadrupole mass spectrometer working in the negative ion mode. FAB experiments were carried out with an M-Scan FAB gun (M-Scan Ltd., Ascot, U.K.) using xenon gas for bombardment at an accelerating voltage of 8 kV, with a discharge current of 1-2 mA. Samples were prepared by mixing 1 µL of the sample solution (1 µg/µL in methanol) with 1 µL of the Magic Bullet matrix (5:1 dithiothreitol/dithioerythritol, v/v). For GC/MS analyses, the instrument was coupled to a Delsi DI 200 (Delsi Nermag Instruments) gas chromatograph fitted with a BPX5 (25 m × 0.22 mm i.d. × 0.25 µm) capillary column (SGE, Villeneuve St. Georges, France). The samples were injected in the splitless mode. The following temperature program was used: 50 °C for 50 s, then from 50 to 230 °C at a rate of 25 °C/min, and from 230 to 280 °C at a rate of 5 °C/min. Helium was used as the carrier gas at a flow rate of 1 mL/min with a backpressure of 0.8 bar. Mass spectra were generated by electron impact ionization at 70 eV with an emission current of 200 µA, at a source temperature of 220 °C. The injector and interface temperature was 270 °C. Calculations. Calculations of the biliary half-life of elimination were carried out via nonlinear regression using GraphPad Prism software. Correlation coefficients (R) were calculated as a measure of the strength of the fit between the data and the curve. A Student’s t test was used to test for overall differences

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Figure 1. Biliary excretion of total radioactivity 4 h after iv administration of [3H]HNE (1.18 MBq, 0.83 µg) in donor rats (a) and after intraduodenal infusion in recipient rats of bile from donor rats (b) (see Materials and Methods for details). Values are means ( SD from three rats. (p < 0.05) in relative excretion levels between groups treated with low and high doses.

Results Excretion of the radioactivity following an iv injection of [3H]HNE occurred rapidly in the bile. Within 1 h of administration of 0.83 µg and 2 mg of this compound, 13.1 ( 3.5 (Figure 1a) and 13.9 ( 2.4% (experiment I, data not shown) of the dose was eliminated via this route, respectively; after 4 h, these values reached 19.6 ( 4.8 and 19.5 ( 2.8%, respectively (data not shown). No significant difference was observed between the two groups. The time courses of excretion of radioactivity in the bile of rats treated with a low dose of [3H]HNE are presented in Figure 1. Panel a of the figure corresponds to data from experiment II (see Materials and Methods) i.e., to bile-cannulated rats given an iv dose of labeled HNE. Under these experimental conditions, the radioactivity was excreted in bile with a t1/2 of 0.66 h and the elimination followed first-order kinetics. The data were fit (R ) 0.999) with the equation y ) 36.83e-1.047x + 0.087. The data presented in Figure 1b are those obtained from experiment III, i.e., from rats treated by duodenum infusion with the bile collected from donor rats previously treated iv with [3H]HNE (see Materials and Methods). In this case, the biliary excretion of total radioactivity appeared to follow first-order elimination kinetics. The half-life of elimination (t1/2) was 5.71 h, and the data were fit (R ) 0.997) with the equation y ) 2.029e-0.121x - 0.450. On the basis of these equations, the total amount of radioactivity excreted in the bile of donor rats and

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Table 1. HNE Residues in Various Tissues of Rats 4 h after iv Administration of [3H]HNE (1.18 MBq, 0.83 µg)a tissue

concentration

tissue

concentration

blood fat liver

0.53 ( 0.15 0.24 ( 0.08 2.83 ( 0.71

kidney whole bodyb

1.88 ( 0.62 0.47 ( 0.15

a Values are expressed as nanograms of HNE equivalents per gram of tissue and are means ( SD from three rats. b Whole body corresponded to homogenized rat carcasses containing digestive contents and viscera, including unsampled blood, fat, liver, and kidney.

recipient rats can be estimated to be 19.96 and 7.71% of the administered dose, respectively. Accordingly, the extent of intestinal reabsorption of HNE-related compounds can be evaluated as (7.71/19.96) × 100 ()38.57%) of the first-pass biliary excretion, which corresponds to 7.7% of the total administered dose. The 3H concentrations in tissues were obtained following iv administration of [3H]HNE (experiment II) and were recorded as nanograms of HNE equivalents per gram of tissue, based on a specific activity of 1.42 MBq/ µg (Table 1). Four hours after administration, the higher residue concentrations were found in liver and kidney. However, the recovery per organ corresponded to 6.17 ( 1.53, 0.73 ( 0.24, and 23.00 ( 7.42% of the administered dose in the liver, the kidney, and the whole body, respectively (data not shown). Bile samples from the rats treated with [3H]HNE or with bile collected from donor animals were filtered on 50 kDa membranes prior to chromatography. Less than 5% of the total radioactivity was retained during this procedure. HPLC analyses of bile from [3H]HNE-treated rats allowed the separation of HNE metabolites and showed five peaks designated A-E (Figure 2a). No qualitative and quantitative difference was observed with the metabolic profile of rats treated with the high dose of [3H]HNE (2 mg) (data not shown). Total recovery of HPLC-injected radioactivity was controlled for each bile analysis and averaged 90%. Enzymatic treatment with H. pomatia juice left the profile unchanged, suggesting that neither glucuronide nor sulfate conjugates were present in the bile. Metabolite A accounted for 0.8 ( 0.4% of the administered dose. This compound coeluted with standard DHN-GSH (tR ) 17.2 min). Characterization of metabolite A was achieved by negative FAB-MS. The FAB spectrum (Figure 3a) showed quasi-molecular ions [M H]- at m/z 464 and [M - 2H + Na]- at m/z 486, together with fragment ions at m/z 446 and 468, corresponding to [M - H - H2O]- and [M - 2H + Na - H2O]-, respectively. Comparison of the mass spectrum of metabolite A with that of reference standard DHN-GSH confirmed that metabolite A was DHN-GSH. Metabolite B accounted for 0.6 ( 0.2% of the injected dose. This compound coeluted with the standard HNEGSH (tR ) 17.7 min). The negative FAB-MS spectrum of metabolite B (Figure 3b) exhibited quasi-molecular ions [M - H]- at m/z 462 and [M - 2H + Na]- at m/z 484, as well as fragment ions at m/z 444 and 466 corresponding to the same ion species as described for metabolite A. These data confirmed that metabolite B was HNE-GSH. Metabolite C accounted for 6.2 ( 1.9% of the administered dose. This metabolite had the same retention time as standard DHN-MA (tR ) 19.8 min). After treatment with Raney nickel, metabolite C gave a compound which

Figure 2. Typical HPLC profiles of [3H]HNE biliary metabolites collected 0-4 h after iv injection of [3H]HNE (1.18 MBq, 0.83 µg) (a) and of biliary metabolites obtained 0-6 h after intraduodenal infusion in recipient rats of bile from donor rats (b) (see Materials and Methods for details).

coeluted with standard 1,4-dihydroxynonane (tR ) 31.3 min). Characterization of metabolite C was performed by FAB-MS in the negative ion mode. The FAB spectrum exhibited an intense [M - H]- quasi-molecular ion at m/z 320, which is in agreement with that of DHN-MA (Figure 4a). In addition to this molecular ion, the spectrum exhibited characteristic fragments at m/z 162 and 191 which can be interpreted as resulting from the cleavage of both thioether bonds with charge retention on the sulfur atom. All these data demonstrated that metabolite C was DHN-MA. Metabolite D represented 2.3 ( 0.8% of the administered dose. It exhibited the same retention time as HNAlactone-MA (tR ) 27 min). The nature of metabolite D was confirmed by FAB-MS analyses in the negative ion mode that showed a [M - H]- ion at m/z 316 (Figure 4b). Moreover, the spectrum exhibited an intense ion at m/z 512, but a control bile sample collected and analyzed under similar conditions also exhibited this ion (data not shown). Thus, the ion at m/z 512 can be attributed to an endogenous substance. All these mass spectral data confirmed the identity of metabolite D as the HNAlactone-MA. Metabolite E accounted for 0.6 ( 0.2% of the administered dose. This metabolite had a retention time of 37 min, whereas the retention time of HNE is 35 min. Metabolite E is therefore less polar than HNE. Methylation of this metabolite did not affect its retention time (data not shown), suggesting the absence of a derivatizable acidic group in the parent compound. Under our experimental conditions, metabolite E coeluted with standard HNA methyl ester, but due to the low amount of available material and to the difficulty of purifying this metabolite, we were not able to confirm this structure by GC/MS or LC/MS at this time.

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Figure 3. Negative FAB mass spectra of [3H]HNE biliary metabolites corresponding to DHN-GSH (a) and HNE-GSH (b). Ions at m/z 273 and 307 are due to the FAB matrix.

Figure 4. Negative FAB mass spectra of [3H]HNE biliary metabolite corresponding to DHN-MA (a) and HNA-lactoneMA (b). Ions at m/z 153, 273, and 307 are due to the FAB matrix.

The metabolic profile obtained from the bile of recipient rats (experiment III) exhibited some major differences in comparison with the profile of the bile of donnor rats (Figure 2b). We did not find HNA-lactone-MA in the samples, and only traces, if any, of glutathione conjugates (HNE-GSH and DHN-GSH) were detected. Surprisingly, two additional peaks were present but remained unidentified (tR ) 23.1 and 25.5 min). The time course of excretion of [3H]HNE metabolites in bile is shown in Figure 5. We clearly observe two groups of metabolites, DHN-MA and HNA-lactone-MA, for which the maximal excretion occurred 60 min after the treatment, whereas the major fraction of the glutathione conjugates and metabolite E was excreted 30 min after treatment. Incubation of [3H]DHN with cytosolic glutathione Stransferases and glutathione did not result in the formation of DHN-GSH (data not shown). These results suggest that DHN, unlike HNE, is not a substrate for soluble GSTs.

Figure 5. Time course excretion of [3H]HNE metabolites in bile following iv administration of [3H]HNE (1.66 MBq, 2 mg). Values are mean ( SD from three rats. A is DHN-GSH, B HNE-GSH, C DHN-MA, D HNA-lactone-MA, and E unknown.

Discussion The fast metabolism of HNE was reported in several in vitro (13, 15, 16, 26, 27) and in vivo sudies (17, 18), and it is generally agreed that this feature plays an important part in the protection of biological systems against lipid peroxidation products. Our work demonstrates the efficiency of this defense mechanism since the same rate of biliary excretion was observed in the lowdose experiment, mimicking the free HNE physiological values found in rat and human plasma (28), and when a

2000-fold higher dose was administered. Our results are in accordance with the data for urine excretion reported by Alary et al. (17), who showed that more than 40% of the administered radioactivity was eliminated in the urine within the first 2 h when rats were injected iv with a low dose of HNE (700 ng), this percentage reaching 67% within 48 h. In contrast, these authors found only 3% of the radioactivity in the feces 48 h after treatment. This percentage seems to be in apparent contradiction with our experiment in which ca. 20% of the dose was excreted in bile 4 h after injection. This difference suggests that

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Scheme 1. Proposed Metabolic Pathways for the in Vivo Biotransformation of HNEa

a

Urinary metabolites are those reported by Alary et al. (17).

the major part of HNE biliary metabolites could be reabsorbed from the gastrointestinal tract either directly or after biotransformations by the intestinal flora. A modified linked rat model was employed to confirm enterohepatic circulation in three animals. All three recipient rats were found to have measurable levels of HNE metabolites in their bile, following intraduodenal delivery of bile collected from donor rats receiving a 0.83 µg iv dose of [3H]HNE. The presence of HNE metabolites in the bile of recipient rats confirms that HNE is likely to undergo enterohepatic recirculation in the rat. To estimate quantitatively the degree of enterohepatic recirculation taking place under our experimental conditions, the calculated rate of biliary elimination of radioactivity in the recipient rats was compared to the corresponding value in the donor rats. This suggests that the extent of recycling was approximately 7.7% of the total dose in this model. Furthermore, since two metabolites were present in the bile of recipient rats and were not found in the bile of donor rats, it would appear that intestinal microflora and/or intestinal mucosa could biotransform HNE-related compounds before or during the reabsorption process. Such a phenomenon was previously described for other types of glutathione conjugates. For example, the radioactivity measured in the feces of rats 48 h after administration of [14C]propachlor, a substrate of GSTs, amounted to 19% of the dose (29). In contrast, when the bile duct was cannulated, 66% of the radioactivity was excreted via this route. The discrepancy between the amount of radioactivity excreted in the bile and the percentage recovered from the feces was explained by an enterohepatic circulation of the mercapturic acid and cysteine conjugates. In that case, the mercapturic acid conjugate and its precursors were excreted in the bile and became substrates for the intestinal microflora. The products of bacterial metabolism were then partly reabsorbed into

the blood, while the other part was eliminated in the feces. Bakke and co-workers (30) demonstrated that mercapturic acid and cysteine conjugates could be reabsorbed from the gastrointestinal tract without the involvement of the intestinal flora. In this study, although the enterohepatic cycle may have increased the length of duration of HNE metabolites in the body, the toxicological consequences of this phenomenon are probably limited. If we assume that HNE is the main reactive compound, only the cleavage of the GSH conjugate of HNE by retro-Michael reaction could result in HNE formation during biliary recycling. However, the low amount of HNE-GSH eliminated in bile should limit the toxicological significance of this pathway. Identification of biliary metabolites confirmed that hepatic GSH plays a major role in the metabolic fate of HNE (31). The study performed by Alary et al. (17) had shown the presence in urine of rats treated with HNE of four mercapturic acid conjugates, namely, HNE-MA, DHN-MA, HNA-MA, and HNA-lactone-MA. The presence in bile of DHN-GSH and HNE-GSH was thus expected and confirms the metabolic pathways proposed by these authors. However, no trace of HNA glutathione conjugate, the likely precursor of the HNA-lactone-MA, was found. In addition, we found in bile DHN-MA and HNA-lactone-MA the major biliary mercapturic acid metabolites of HNE. However, no formation of HNE and the HNA-mercapturic acid conjugate was observed in this study (Scheme 1). The presence in bile of the DHNand HNA-lactone mercapturic acid conjugates supports the fact that glutathione conjugates are cleaved by γ GT and glycine dipeptidase within bile ducts and then acetylated by acetyltransferases and acetyl-CoA in the hepatocytes (32-34). In accordance with Figure 5 and since no HNA-GSH was detected in bile, we could suppose that HNAlactone-MA originates from HNE-GSH. The same origin may be proposed for the formation of DHN-GSH. Moreover, incubation of DHN with cytosolic glutathione

4-Hydroxynonenal Metabolites Excreted in Bile

S-transferases and glutathione did not lead to the formation of DHN-GSH, suggesting that DHN was not a substrate for the cytosolic glutathione S-transferase (18). This observation supports the hypothesis that the DHNglutathione conjugate may be formed by the action of cytosolic glutathione S-transferase and GSH on HNE followed by the reduction of the aldehyde group of HNEGSH. Aldehyde reductases which are able to metabolize aldehydes to their corresponding alcohols (35) could be involved in the reduction of the aldehyde group of HNEGSH. A possible role of microsomal glutathione Stransferase in the conjugation of DHN cannot be excluded. These pathways were not hypothetized by Alary et al. (17) in their proposal for the in vivo formation of urinary mercapturic acid of HNE and related compounds. Additional studies are warranted to explore the possible routes for the formation of DHN-MA and HNA-lactoneMA. As expected, the conjugation of HNE to glutathione seems to be the major antioxidative defense system. However, it has been suggested that GSH conjugates of R,β-unsaturated aldehydes formed via Michael addition may undergo reversible conjugation with glutathione (36). In this situation, GSH could be considered a vehicle for the transport of reactive compounds from the tissue in which they are formed to some other site in which they are released (37). Among alkenals, this finding was first proposed for acrolein (38), but in that case, the retroMichael reaction occurred with the corresponding Nacetyl conjugate rather than from the GSH adduct. The reversibility in GSH conjugate formation of 4-hydroxypentenal was then hypothetized by Baillie and Kassahun (39). It will be important to establish whether reversible GSH conjugation plays a role in the transport of HNE in vivo. This study provides further evidence for the role of the glutathione and mercapturic acid pathway in the biotransformation of HNE and demonstrates that bile is a significant excretion route for the corresponding metabolites. In addition, our work clearly indicates that HNE metabolites can be absorbed from the gastrointestinal tract and partly resecreted in the bile after having undergone further biotransformation. Whether such metabolites would be biotransformed by the gut microflora and/or by the intestinal wall remains a topic for future research.

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