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
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ABSTRACT |
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
-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
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INTRODUCTION |
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
-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.
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EXPERIMENTAL PROCEDURES |
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
-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
-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
-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.
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RESULTS |
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).
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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.
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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.
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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
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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).
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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.
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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.
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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.
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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
-glutamyltranspeptidase and then reanalyzed by HPLC. This enzyme
should remove the
-glutamyl moiety from the GSH
S-conjugate and lead to the formation
of the cysteinylglycine S-conjugate (DNP-CG). Incubation with
-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.
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DISCUSSION |
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
-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
-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
-glutamyltranspeptidase and dipeptidase activities. Previous studies
have demonstrated that DNP-Cys is a substrate for cysteine conjugate
-lyase enzymes, producing free sulfhydryl compound (DNP-SH) (21).
The latter is methylated by
S-methyltransferase to form
DNP-S-CH3 (21).
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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.
 |
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