Efficient hepatic uptake and concentrative biliary excretion of a mercapturic acid

Cheri A. Hinchman, James F. Rebbeor, and Nazzareno Ballatori

Department of Environmental Medicine, University of Rochester School of Medicine, Rochester, New York 14642

    ABSTRACT
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Abstract
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Procedures
Results
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References

The role of the liver in the disposition of circulating mercapturic acids was examined in anesthetized rats and in the isolated perfused rat liver using S-2,4-dinitrophenyl-N-acetylcysteine (DNP-NAC) as the model compound. When DNP-NAC was infused into the jugular vein (150 or 600 nmol over 60 min) it was rapidly and nearly quantitatively excreted as DNP-NAC into bile (42-36% of the dose) and urine (48-62% of dose). Some minor metabolites were detected in bile (<4%), with the major metabolite coeluting on HPLC with the DNP conjugate of glutathione (DNP-SG). Isolated rat livers perfused single pass with 3 µM DNP-NAC removed 72 ± 9% of this mercapturic acid from perfusate. This rapid DNP-NAC uptake was unaffected by sodium omission, or by L-cysteine, L-glutamate, L-cystine, or N-acetylated amino acids, but was decreased by inhibitors of hepatic sinusoidal organic anion transporters (oatp), indicating that DNP-NAC is a substrate for these transporters. The DNP-NAC removed from perfusate was promptly excreted into bile, eliciting a dose-dependent choleresis. DNP-NAC itself constituted ~75% of the total dose recovered in bile, reaching a concentration of 9 mM when livers were perfused in a recirculating mode with an initial DNP-NAC concentration of 250 µM. Other biliary metabolites included DNP-SG, DNP-cysteinylglycine, and DNP-cysteine. DNP-SG was likely formed by a spontaneous retro-Michael reaction between glutathione and DNP-NAC. Subsequent degradation of DNP-SG by biliary gamma -glutamyltranspeptidase and dipeptidase activities accounts for the cysteinylglycine and cysteine conjugates, respectively. These findings indicate the presence of efficient hepatic mechanisms for sinusoidal uptake and biliary excretion of circulating mercapturic acids in rat liver and demonstrate that the liver plays a role in their whole body elimination.

glutathione; organic anion transporters; mercapturic acid transport; liver detoxification

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

A VARIETY OF REACTIVE and potentially toxic chemicals are metabolized by binding to the tripeptide glutathione (GSH) and by conversion of these conjugates to the corresponding mercapturic acids, N-acetylcysteine S-conjugates (6, 8-11, 20). Mercapturic acid biosynthesis is mediated by a series of at least four enzymatic steps and three cell membrane transport events and is believed to require the interorgan shuttling of the metabolic intermediates (10, 20). The initial GSH conjugation is catalyzed by intracellular GSH S-transferases, whereas degradation of the resulting GSH S-conjugates to the cysteine S-conjugates is catalyzed by the sequential action of the ectoproteins gamma -glutamyltranspeptidase and dipeptidase. Cysteine S-conjugates are transported back into cells for acetylation by N-acetyltransferases to form the mercapturic acids, which are then exported from the cell. Mercapturic acids synthesized in the kidney may go directly into urine, a major route for their elimination (20), whereas those synthesized in the liver may be transported directly into bile (10, 11). Mercapturic acids synthesized in other tissues enter the bloodstream and may be cleared by kidney or liver, although the relative roles of these two organs in the disposition of circulating mercapturic acids have not been evaluated.

The route and mechanism of mercapturic acid disposition are determined to a large extent by the physicochemical properties of both the parent compound and the corresponding mercapturic acid. Compounds that are of high molecular weight and/or relatively hydrophobic are expected to be excellent substrates for hepatic organic solute transporters, but comparatively poor substrates for renal transporters. Conversely, smaller and more hydrophilic molecules should be more readily cleared by the kidneys and excreted into urine. Indeed, Petras and co-workers (19) recently reported that the relatively hydrophobic mercapturic acid of 4-hydroxynonenal, a cytotoxic lipid peroxidation product, is secreted by the isolated rat kidney largely into the blood circulation, with only a small amount going into urine. Another endogenous and relatively hydrophobic mercapturic acid is that derived from leukotriene C4 (N-acetyl-LTE4). Studies by Keppler and co-workers (13) indicate that LTE4 and N-acetyl-LTE4 are the most abundant cysteinyl leukotrienes in rat plasma and that these are cleared from the circulation by the liver.

The present study examines the mechanism of disposition of circulating mercapturic acids by evaluating the ability of rat liver to take up, metabolize, and excrete S-2,4-dinitrophenyl-N-acetylcysteine (DNP-NAC), the mercapturic acid derived from 1-chloro-2,4-dinitrobenzene, a model xenobiotic.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials. HPLC grade acetonitrile was obtained from J. T. Baker Research Products (Phillipsburg, NJ). The 2,4-dinitrophenyl S-conjugates of GSH (DNP-SG), cysteine (DNP-Cys), cysteinylglycine (DNP-CG), and N-acetylcysteine (DNP-NAC) were synthesized as described previously (11). All other reagents were from Sigma Chemical (St. Louis, MO). Male Sprague-Dawley rats were obtained from Charles River Laboratories (Kingston, NY) and were fed ad libitum until time of experimentation.

HPLC analysis of bile, urine, and perfusate samples. DNP-SG and metabolites were detected and quantified as previously described (11). The HPLC system consisted of a Varian model 9012 liquid chromatograph system with a varichrom adjustable wavelength spectrophotometric detector (Varian, Sunnyvale, CA) and an HP 3394A integrator (Hewlett-Packard, Palo Alto, CA). Isocratic elutions were performed with a Bakerbond NP octadecyl (C18) column (4.5 × 250 mm, 5 µm; J. T. Baker Research Products) with a mobile phase of acetonitrile-water (25:75 with 0.1% H3PO4, vol/vol) and a flow rate of 1.0 ml/min. Compounds were detected at 365 nm and quantitated by the external standard method using the area under the peaks.

Urinary and biliary excretion in anesthetized rats. Male Sprague-Dawley rats (220-275 g) were anesthetized with 55 mg/kg pentobarbital sodium. A midline incision was made along the abdomen, and the bile duct and urinary bladder were isolated and cannulated with PE-10 and PE-50 tubing, respectively. The right jugular vein was cannulated with PE-50 tubing attached to a syringe containing 140 mM D-glucose. Body temperature was monitored and maintained at 37°C with a heat lamp controlled by a Tele-Thermometer and a rectal probe (Yellow Springs Instrument, Yellow Springs, OH). Additional anesthetic was administered intraperitoneally as required throughout the experiment. A mild water diuresis was induced in all animals by continuous intravenous infusion of 140 mM D-glucose at a rate of 3 ml/h. This technique has been found to induce a mild diuresis with glucose-free urine of low osmolality (22). Urine and bile samples were collected in 30-min intervals for 3.5 h, in ice-chilled tared tubes containing 0.4 ml of 10% perchloric acid. DNP-NAC was infused into the jugular vein over a 1-h interval (0.5-1.5 h) at doses of either 150 or 600 nmol dissolved in 140 mM D-glucose (3 ml/h). Urine and bile samples were filtered with Gelman 0.45-µm filters before HPLC analysis.

Isolated rat liver perfusions. Livers isolated from male Sprague-Dawley rats (251 ± 15 g body wt) were perfused as previously described (5, 11). Animals were anesthetized with 55 mg/kg pentobarbital sodium. A midline incision was made along the abdomen, and the bile duct was isolated and then cannulated with PE-10 tubing. A 14-gauge stainless steel cannula was inserted into the portal vein, and the liver was perfused with an oxygenated Krebs-Henseleit solution (37°C) containing heparin. After the thoracic vena cava was cannulated with PE-205 tubing, the liver was excised from the body cavity and placed inside the perfusion chamber. The perfusate solution was then changed to a Krebs-Henseleit buffer without heparin.

Livers were perfused initially with 80 ml of an oxygenated Krebs-Henseleit buffer containing 5 mM glucose, at a flow rate of 3.68 ± 0.35 ml · min-1 · g liver-1. Liver temperature was maintained at 37-37.5°C. After a 10-min control period, DNP-NAC (1, 5, or 25 µmol in 20 ml of Krebs-Henseleit buffer) was added to the recirculating perfusate for initial concentrations of 10, 50, or 250 µM. Perfusate samples (0.5 ml) were collected from the perfusate reservoir at regular intervals and placed into Microfuge tubes containing 50 µl of 69% perchloric acid. Additional, nonacidified 0.5-ml perfusate samples were removed from the reservoir every 10 min for the determination of lactate dehydrogenase (LDH) activity. Bile was collected into tared, ice-chilled Microfuge tubes containing 100 µl of 10% perchloric acid. Bile samples were diluted with 5% perchloric acid and filtered with Gelman 0.45-µm filters before analysis. Bile flow rates were determined gravimetrically, assuming a density of 1. Liver viability was monitored by perfusion pressure, bile flow rates, gross appearance, and the release of LDH into perfusate.

To examine the effects of sodium replacement and putative transport inhibitors on DNP-NAC uptake, livers were perfused single-pass with Krebs-Henseleit buffer. After a 10-min equilibration period, the perfusate was switched to Krebs-Henseleit buffer containing 3 µM DNP-NAC for a total of 120 s. After the first 60 s, putative inhibitors or Krebs-Henseleit buffer for control were infused just proximal to the portal vein cannula via a syringe infusion pump at a rate of 1.36 ml/min for 60 s. Concentrated stocks of inhibitors (25×) were prepared in Krebs-Henseleit buffer, and the pH was adjusted to 7.2-7.4 with NaOH. Effluent perfusate was collected in 30-s intervals (17-18 ml) in beakers containing 0.2 ml of 69% perchloric acid. The concentration of DNP-NAC in the influent and effluent perfusates was measured by HPLC. The amount of DNP-NAC removed from perfusate in the 30- to 60- and 90- to 120-s intervals was used to calculate DNP-NAC uptake. To examine the effects of sodium replacement, choline+ buffer containing 3 µM DNP-NAC was infused for 60 s after an initial 60-s infusion of regular Krebs-Henseleit buffer containing 3 µM DNP-NAC.

In vitro characterization of metabolites. To confirm that DNP-SG was being produced in livers treated with DNP-NAC, bile samples were treated with gamma -glutamyltranspeptidase (Sigma) and analyzed by HPLC. Bile samples previously collected in perchloric acid were neutralized by the addition of 2 M KHCO3. The resulting precipitate was allowed to settle to the bottom of the tube, and the supernatant was removed and placed into another tube. Purified bovine gamma -glutamyltranspeptidase was added to a concentration of 4.6 U/ml, in the presence of 100 mM Tris-glycylglycine buffer, pH 8.5, and this was incubated at 37°C for about 40 min. Controls were treated similarly but did not receive gamma -glutamyltranspeptidase. After incubation, samples were acidified with perchloric acid (5%), filtered, and reanalyzed by HPLC.

The spontaneous chemical conversion of DNP-NAC to DNP-SG was examined by incubating 50 µM DNP-NAC in Krebs-Henseleit buffer containing 5 mM GSH, 20 mM HEPES-Tris buffer, pH 7.5, and 0.5 mM acivicin, in the presence and absence of 1% rat liver homogenate at 37°C. Aliquots were removed at various time intervals, acidified with 5% perchloric acid, and injected into the HPLC.

Statistical analysis. Data are expressed as means ± SD. Student's t-test was used to determine significant differences between means at P < 0.05.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Biliary and urinary excretion of DNP-NAC after intravenous administration to rats. DNP-NAC administered into the jugular vein of anesthetized rats was rapidly and nearly quantitatively excreted unchanged into bile and urine at both dose levels tested, 150 and 600 nmol (Fig. 1). Biliary and urinary excretion reached a peak during the infusion interval (0.5-1.5 h), and most of the DNP-NAC was cleared within 1 h of stopping the infusion (Fig. 1). At the lower dose 42% of the total dose was recovered in bile and 48% in urine, whereas at the higher dose these values were 36 and 62%, respectively. Some minor metabolites were detected in bile (<4%), with the major metabolite coeluting with DNP-SG, whereas DNP-NAC accounted for essentially all of the dose recovered in urine (data not shown). These data indicate that both liver and kidney play an important role in the clearance of this circulating mercapturic acid. To examine the mechanism for the hepatobiliary clearance of DNP-NAC, additional studies examined uptake into the isolated perfused rat liver.


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Fig. 1.   Biliary and urinary excretion of S-2,4-dinitrophenyl-N-acetylcysteine (DNP-NAC) in rats given either 150 (A) or 600 (B) nmol of DNP-NAC intravenously over 1-h interval (0.5-1.5 h). Rats were anesthetized, catheters were placed in the jugular vein, urinary bladder, and bile duct, and mild diuresis was induced by continuous intravenous infusion of 140 mM D-glucose at a rate of 3 ml/h. Urine and bile samples were collected in 30-min intervals for 3.5 h, in ice-chilled tared tubes containing 0.4 ml of 10% perchloric acid. Values are means ± SD (n = 3 at each dose).

Rapid clearance of DNP-NAC from isolated rat liver perfusate. Rat livers perfused with 100 ml of recirculating Krebs-Henseleit buffer containing DNP-NAC at initial concentrations of either 10, 50, or 250 µM efficiently removed this mercapturic acid from perfusate, with half-time values of ~7, 12, and 18 min, respectively (Fig. 2). After 35 min of perfusion only a small fraction of the DNP-NAC dose remained in perfusate.


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Fig. 2.   Time course of DNP-NAC removal from recirculating rat liver perfusate. Livers were perfused with 100 ml of KrebsHenseleit buffer containing 5 mM D-glucose and initial DNP-NAC concentrations of 10 (n = 4), 50 (n = 3), or 250 µM (n = 5). Values are means ± SD.

A comparison of the 5-min uptakes of DNP-NAC at different initial concentrations revealed that it was a linear function of extracellular concentration (Fig. 3), indicating that uptake is not saturable at concentrations up to 250 µM. However, because of the rapid uptake of DNP-NAC (Fig. 2), 5 min does not represent a true initial rate of uptake. Nevertheless, these data demonstrate a high capacity for DNP-NAC transport. Uptake was considerably higher than that of DNP-SG and was comparable to that of bile acids or other cholephilic organic anions (Fig. 3) (12).


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Fig. 3.   Concentration dependence of initial rates of DNP-NAC and S-2,4-dinitrophenylglutathione (DNP-SG) uptake by rat liver. Amount removed from recirculating perfusate at 5 min after addition of these compounds was taken as amount taken up by liver. Values are means ± SD of 3-5 experiments at each concentration.

To characterize the transport system responsible for hepatic uptake of this organic anion, additional studies examined the effects of sodium replacement and of putative transport inhibitors on DNP-NAC uptake (Table 1). For these experiments livers were perfused single pass with 3 µM DNP-NAC in the presence and absence of various inhibitors, and the concentration of DNP-NAC in the influent and effluent perfusates was measured by HPLC. Livers were exposed to the indicated concentrations of inhibitors for only 60 s. There was no significant increase in LDH release from this brief exposure.

                              
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Table 1.   Effects of Na+-free (choline+) medium and putative transport inhibitors on DNP-NAC uptake by perfused rat liver

Control livers removed 72 ± 9% of the DNP-NAC from the perfusate during single-pass perfusion. When sodium was replaced with choline+ there was a small decrease in DNP-NAC uptake, but this was not statistically significant (Table 1). Sodium replacement also produced a decrease in bile flow and an increase in perfusion pressure (data not shown), indicating indirect effects on hepatobiliary function.

Because DNP-NAC is an anionic amino acid derivative it may be a substrate for either the sinusoidal organic anion or amino acid transporters. As shown in Table 1, DNP-NAC uptake was unaffected by L-glutamate, L-cystine, or L-cysteine, indicating that amino acid transporters are not involved. GSH at a concentration of 1 mM produced a small decrease in DNP-NAC uptake, although this decrease was not statistically significant (Table 1). In contrast, compounds that are substrates or known inhibitors of the recently cloned sinusoidal organic anion transporters oatp1 and oatp2 (14-17) significantly inhibited DNP-NAC uptake. Bilirubin ditaurate, probenecid, DIDS, and dibromosulfophthalein markedly decreased uptake (Table 1). Taurocholate had no significant effect. Uptake was also unaffected by N-acetyl derivatives of L-cysteine, L-glutamate, and L-methionine but was inhibited by another mercapturic acid (N-acetyl-S-benzyl-L-cysteine) and by two DNP-derivatized amino acids (Table 1). The latter three compounds are anionic and somewhat hydrophobic, properties that are shared by substrates of the sinusoidal organic anion transporters (14-17).

Concentrative biliary excretion of DNP-NAC. Most of the DNP-NAC removed from the perfusate was excreted unchanged into bile, with biliary DNP-NAC concentrations as high as 10-12 mM in livers perfused with 250 µM DNP-NAC (Fig. 4A). This concentrative biliary excretion of the unchanged mercapturate was associated with a marked dose-dependent choleresis (Fig. 4B).


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Fig. 4.   Time course for biliary excretion of DNP-NAC (A) and corresponding changes in bile flow (B). Values are means ± SD (n = 4, 3, and 5 for 10, 50, and 250 µM DNP-NAC, respectively).

DNP-NAC itself accounted for most (~75%) of the total dose excreted into bile (Fig. 5); however, a number of metabolites were also found. A compound that coeluted with DNP-SG on HPLC analysis was the most abundant metabolite, accounting for up to 17% of the dose recovered in bile (Fig. 5). Compounds that coeluted with DNP-Cys and DNP-CG were next in abundance, and three unknown compounds were also found in bile in relatively small amounts (altogether accounting for <4% of the total dose recovered in bile; Fig. 5). The proportion of the dose excreted in bile as the mercapturate decreased and that of DNP-SG and DNP-Cys increased, with increasing dose (Fig. 5).


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Fig. 5.   Relative amounts of DNP-NAC and metabolites excreted into bile. Bile was collected for a total of 35 min after administration of either 10, 50, or 250 µM DNP-NAC. DNP-Cys and DNP-CG, S-2,4-dinitrophenyl derivatives of cysteine and cysteinylglycine. Values are means ± SD.

The time course for biliary excretion of DNP-NAC metabolites is shown in Fig. 6. DNP-SG was the most abundant biliary metabolite at all three doses, reaching concentrations of ~0.2, 0.5, and 2.5 mM in livers perfused with 10, 50, and 250 µM DNP-NAC, respectively. At the 10-µM dose, the unknown metabolites made up a significant fraction of the total biliary metabolites, particularly at the latter bile collection intervals (Fig. 6A).


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Fig. 6.   Time course of biliary excretion of DNP-NAC metabolites from rat livers perfused with either 10 (A), 50 (B), or 250 µM (C) DNP-NAC. Values are means ± SD.

Only a small fraction of total metabolites was found in the recirculating perfusate (Figs. 7 and 8). No metabolites were detected at the lowest dose of DNP-NAC. At the higher doses, DNP-Cys was the predominant metabolite, with concentrations reaching ~10 µM with 250 µM DNP-NAC (Fig. 7). Small amounts of DNP-SG and unknown compounds were also observed in perfusate (Fig. 7). Total recovery of dose (i.e., bile, perfusate, and liver) was ~80% at all three doses of DNP-NAC (Fig. 8). The fate of the other 20% is unknown but may be due in part to metabolism to compounds that cannot be detected in our assay system or to binding or compartmentation within the liver.


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Fig. 7.   DNP-NAC metabolites found in recirculating perfusate after administration of 250 µM DNP-NAC. No metabolites were detected at lowest dose (10 µM), and only low levels of metabolites were detected at 50 µM dose. Values are means ± SD, n = 5.


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Fig. 8.   Relative recovery of dose in perfusate, liver, and bile at 35 min after DNP-NAC administration. Values are means ± SD.

In vitro characterization of metabolites. To confirm the identity of the DNP-SG peak in bile, bile samples collected from livers perfused with DNP-NAC were treated with gamma -glutamyltranspeptidase and then reanalyzed by HPLC. This enzyme should remove the gamma -glutamyl moiety from the GSH S-conjugate and lead to the formation of the cysteinylglycine S-conjugate (DNP-CG). Incubation with gamma -glutamyltranspeptidase resulted in the disappearance of the DNP-SG peak and an increase in the DNP-CG peak, confirming the presence of DNP-SG in bile (data not shown).

To examine the mechanism by which the DNP-SG was being formed, DNP-NAC (50 µM) was incubated in protein-free Krebs-Henseleit buffer in the presence and absence of 5 mM GSH (Fig. 9). In the presence of GSH some of the DNP-NAC was spontaneously converted to DNP-SG, indicating a retro-Michael type of chemical reaction (1, 7). Over a 2-h period ~30% of the DNP-NAC was converted to DNP-SG. Addition of freshly prepared rat liver homogenate (1% final concentration) did not further accelerate this conversion (Fig. 9). The high GSH concentration in both liver and bile (5-10 mM), the high DNP-NAC concentration in bile, and the relatively high biliary pH (7.6-8.0) all favor this exchange reaction.


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Fig. 9.   Spontaneous conversion of DNP-NAC to DNP-SG in presence of glutathione (GSH). Loss of DNP-NAC (A) and formation of DNP-SG (B) were followed by HPLC analysis under the following incubation conditions: 1) DNP-NAC in Krebs-Henseleit buffer containing 20 mM HEPES-Tris, pH 7.5, and 0.5 mM acivicin, 2) DNP-NAC in same buffer supplemented with 5 mM GSH, and 3) DNP-NAC in buffer supplemented with 5 mM GSH and 1% rat liver homogenate. Data are expressed as means ± SD, n = 3 for each.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

The present study demonstrates that rat liver plays a significant role in the clearance of a representative mercapturic acid, DNP-NAC, from the bloodstream. When DNP-NAC was administered intravenously, 36-42% was recovered in bile and the balance was recovered in urine, indicating that both liver and kidney participate in its clearance. Biliary and urinary excretion was rapid, with essentially all of the dose excreted within 1 h of DNP-NAC administration.

Rapid hepatic uptake of DNP-NAC was confirmed in studies with the isolated perfused rat liver, which indicated that livers were able to remove >70% of the DNP-NAC from the sinusoidal circulation during single-pass perfusion with 3 µM DNP-NAC. Uptake was independent of sodium and was inhibited by modulators of the oatp family of transporters (14-17), indicating that DNP-NAC is a substrate for an oatp-related sinusoidal organic solute transporter. Uptake was nearly completely blocked by 0.2 mM bilirubin-ditaurate, 0.5 mM probenecid, and 0.1 mM DIDS, known inhibitors of oatp1 (14, 15). Uptake was also inhibited by N-acetyl-S-benzyl-L-cysteine, a mercapturic acid, and by two DNP-derivatized amino acids (Table 1), compounds whose physicochemical properties make them likely substrates for sinusoidal multispecific organic anion transporters (15). Our recent studies in Xenopus oocytes expressing oatp1 substantiate the present findings by demonstrating that DNP-NAC is a low-affinity substrate for oatp1 (unpublished observations). However, additional studies are needed to examine whether DNP-NAC is also a substrate for other hepatic organic solute transporters and the role of these sinusoidal transporters in the clearance of structurally distinct mercapturic acids.

The rapid clearance of DNP-NAC by the liver indicates that hepatocytes may play a more important role in the disposition of mercapturic acids than previously envisioned. Although there is considerable evidence for a major role of the kidney in mercapturic acid excretion (20), the role of the liver has not been documented. Mercapturic acids that are synthesized in extrarenal tissues are released into blood plasma and are thought to be delivered to the kidney for excretion in urine (20). In contrast, the present findings indicate that the liver may play a key role in the clearance of some mercapturic acids.

The DNP-NAC removed from the sinusoidal circulation of the perfused rat liver was promptly excreted into bile in high concentrations, indicating an active hepatobiliary transport process. The concentrative biliary excretion of DNP-NAC and its metabolites was associated with a dose-dependent increase in bile flow. Biliary DNP-NAC concentrations were between 6 and 12 mM at the 250 µM dose, whereas liver levels were comparatively low (<0.3 mM at 35 min of perfusion; Fig. 8). The transport system responsible for this concentrative biliary secretion of DNP-NAC is not known, although this anion may be a substrate for mrp2, the ATP-dependent canalicular organic solute transporter (16, 18). Additional studies are needed to examine this possibility.

Interestingly, the major biliary metabolite of DNP-NAC was DNP-SG, which appears to be formed from the spontaneous and reversible reaction of GSH with DNP-NAC, a retro-Michael conversion (1). A diagram illustrating the putative biochemical pathways of hepatic DNP-NAC metabolism is provided in Fig. 10. The retro-Michael reaction between GSH and DNP-NAC probably occurs both in liver and bile, owing to the high concentrations of these substances at these sites. The high biliary pH (7.6-8.0) also favors this exchange reaction. The DNP-SG formed within hepatocytes will be rapidly transported into bile by mrp2 (4), where it will be degraded by the ectoproteins gamma -glutamyltranspeptidase and dipeptidase, to form DNP-CG and DNP-Cys, respectively (2, 3, 11). As shown in Fig. 10, DNP-Cys can also be generated from DNP-NAC in a reaction catalyzed by intracellular acylases. Conversely, DNP-Cys can be acetylated by intracellular N-acetyltransferase to regenerate DNP-NAC. Previous studies have demonstrated that additional minor hepatic metabolites of DNP-Cys are the free thiol (DNP-SH) and the S-methyl-thiol (DNP-S-CH3) (21), which are produced by the enzymes cysteine conjugate beta -lyase and S-methyltransferase, respectively (Fig. 10).


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Fig. 10.   Pathways of hepatic DNP-NAC metabolism. GSH conjugate (DNP-SG) appears to be formed by retro-Michael reactions between GSH and DNP-NAC and possibly between GSH and DNP-Cys. Cysteine S-conjugate is formed by 2 pathways: 1) deacetylation of DNP-NAC by intracellular acylases or 2) sequential degradation of GSH S-conjugate (DNP-SG) by gamma -glutamyltranspeptidase and dipeptidase activities. Previous studies have demonstrated that DNP-Cys is a substrate for cysteine conjugate beta -lyase enzymes, producing free sulfhydryl compound (DNP-SH) (21). The latter is methylated by S-methyltransferase to form DNP-S-CH3 (21).

Collectively, these reactions lead to the formation of multiple metabolites, some of which may be more reactive (toxic) than the parent compounds (9, 20). Toxicity is also determined by the fate of the metabolic products within the intestine. Biliary metabolites may undergo further metabolic bioactivation in the intestinal lumen, they may be reabsorbed into the portal circulation, or they may be excreted in feces. The relative balance between these competing pathways will determine both the disposition of the chemicals and their propensity to produce cellular injury.

    ACKNOWLEDGEMENTS

This work was supported in part by the National Institutes of Health Grants ES-06484 and DK-48823, Center Grant ES-01247, and Toxicology Training Program Grant ES-07026.

    FOOTNOTES

Address for reprint requests: N. Ballatori, Dept. of Environmental Medicine, Box EHSC, Univ. of Rochester School of Medicine, Rochester, NY 14642.

Received 15 September 1997; accepted in final form 27 May 1998.

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

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Am J Physiol Gastroint Liver Physiol 275(4):G612-G619
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