![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Departments of Veterinary Biochemistry (T.D., H.Y.) and Veterinary Physiology (H.I., S.K.), School of Veterinary Medicine, Rakuno Gakuen University, Hokkaido, Japan
(Received February 4, 2003; accepted May 9, 2003)
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
). The mean daily oral intake of nonylphenol by humans is
estimated to be 0.16 mg/day (Muller et
al., 1998). Nonylphenol has been shown to be a possible endocrine
disrupter due to its estrogenic effects in MCF7 cell proliferation assays
(Soto et al., 1991), binding
assays to the estrogen receptor (White et
al., 1994) and uterotropic assays in mice
(Shelby et al., 1996).
Exposure of male rainbow trout (Oncorhynchus mykiss) to four
different alkylphenolic chemicals, including nonylphenol, resulted in
synthesis of vitellogenin, a process normally dependent on endogenous
estrogens, and a concomitant inhibition of testicular growth
(Jobling et al., 1996). Male
and female ratios of Japanese medaka (Oryzias latipes) in a control
group (2:1) and a 100 µg/l nonylphenol treatment group (1:2) were reported
to be significantly different (Gray and
Metcalfe, 1997). Early neonatal exposure to nonylphenol has been
reported to cause dysfunction of postpubertal reproductive function in female
rats as well as disrupted development of gonads in male and female rats
(Nagao et al., 2000). It has
also been reported that nonylphenol administered orally at a dose of 50 mg/kg
of body weight induced a significant increase in uterine weight of prepubertal
rats and advanced the age of vaginal opening
(Laws et al., 2000).
Elucidation of the metabolism and fate of nonylphenol is important for
estimating the risks of the chemical for animals. Xenoestrogens such as
bisphenol A and diethylstilbestrol have been shown to be conjugated with
glucuronic acid by a UDP-glucuronosyltransferase isoform, UGT2B1, in the rat
liver (Yokota et al., 1999).
Nonylphenol has also been reported to be glucuronidated extensively in the
liver of animals such as rainbow trout
(Lewis and Lech, 1996;
Coldham et al., 1998; Thibaut
et al.,
1998a,b)
and rats (Moffat et al., 2001;
Yokota et al., 2002). Since
the liver is the main barrier against drugs in the body, identifying or
tracing the metabolites of nonylphenol is important for elucidation of the
disruptive effects of the compound on the reproductive system. In this study, we investigated the metabolism and kinetics of nonylphenol and those of other alkylphenols by means of liver perfusion, and we found that nonylphenol was delayed in excretion from the liver and that the length of the alkyl chain is a critical factor for the excretion rate and route out of the liver.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Animals. Male young adult Sprague-Dawley (SD) rats (8–13 weeks old) and MRP-2-deficient Eisai hyperbilirubinemic rats (EHBR) were used in all experiments. The rats were housed under standard conditions and given food and water ad libitum before use. The rats were handled according to the Laboratory Animal Control Guidelines of Rakuno Gakuen University based on the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health in the United States.
Expression of UDP-Glucuronosyltransferase 2B1. cDNA of rat
UDP-glucuronosyltransferase isoform UGT2B1 was obtained by reverse
transcriptase-polymerase chain reaction and expressed in yeast AH22 cells as
previously described (Yokota et al.,
1999). Nonylphenol glucuronidation was performed using yeast
microsome-expressed UGT2B1, and the resultant glucuronide conjugate was
analyzed by HPLC as described previously
(Yokota et al., 1999).
Surgical Procedure. The rats were anesthetized by intraperitoneal
injection of 60% urethane (0.3 ml/100 g of body weight). Whole liver perfusion
was performed according to the method reported by Sugano et al.
(1978) with slight
modifications (Inoue et al.,
2001). Briefly, after anesthesia, the abdomen was opened and the
liver, portal vein, bile duct, and inferior vena cava were exposed. The common
bile duct and the portal vein were cannulated with PE-10 and PE-50
polyethylene tubes (BD Biosciences, San Jose, CA), respectively, and
oxygenated Krebs-Ringer buffer, described below, was pumped through the liver
via the portal vein. The abdominal vena cava was incised immediately after
perfusion, and a dripping polyethylene tube (2 mm i.d., 3 mm o.d.) was
inserted. The thorax was then opened and the thoracic vena cava was ligated.
The liver was not excised. All experiments were performed in situ. While the
animals were still under anesthesia, euthanasia was performed by
exsanguination.
Liver Perfusion. Krebs-Ringer buffer (115 mM NaCl, 5.9 mM KCl, 1.2
mM MgCl2, 1.2 mM NaH2PO4, 1.2 mM
Na2SO4, 2.5 mM CaCl2, 25 mM
NaHCO3, 10 mM glucose) was used in all experiments. The buffer
solution was aerated by 95% O2 + 5% CO2, and the pH was
adjusted to 7.4. Ethanol containing alkylphenol was dispersed into the buffer
solution, and final concentration of ethanol was 1%. The substrate buffer
solution contained alkylphenol in a final concentration of 0.025 or 0.05 mM.
These buffer solutions were maintained in a water bath at 37°C. The
perfusion system consisted of a peristaltic pump (MP-32N; EYELA, Tokyo
Rikakikai Co. Ltd., Tokyo, Japan) and silicone tubes, as described in our
previous paper (Inoue et al.,
2001). The buffer solution was pumped at a constant rate of 30
ml/min, and the liver perfusion was carried out in a flow-through mode.
Preliminary perfusion was done for 15 min, and then the substrate buffer
solution was perfused for 5 min, followed by reperfusion of the Krebs-Ringer
solution. After perfusion of the substrate buffer, the excreted bile and
perfusate in the vein were collected at 5-min or 10-min intervals over a 1-h
period.
HPLC Analysis of Reaction Products. The bile was dissolved in
40-fold 50% acetonitrile, and perfusate was diluted with an equal volume of
acetonitrile; then, the perfusate mixtures were centrifuged for 10 min at
10,000g. The supernatant fractions were analyzed by HPLC (Shimadzu,
Tokyo, Japan) according to the method described previously
(Yokota et al., 1999;
Inoue et al., 2001).
Alkylphenols and its metabolites were eluted from HPLC by 75% acetonitorile
solution (acetonitrile/water/acetic acid, 75:25:0.1) for nonylphenol, 60%
solution for hexylphenol, 50% solution for butylphenol, and 35% solution for
ethylphenol. Recordings were made using a C-R8A integrator (Shimadzu, Tokyo,
Japan). Alkylphenols and its metabolites retained in the liver after the
perfusion were extracted with acetonitrile, the extractions were centrifuged
for 30 min at 25,000g, and the supernatants were analyzed by HPLC
with the same procedure described above. Alkylphenol-glucuronides were
quantitated by the amount of the deconjugated free alkylphenols after
ß-glucuronidase treatment of the reaction products as previously
described (Shibata et al., 2002).
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
; Coldham et al.,
1998; Thibaut et al.,
1998a,b)
(data not shown). A glucuronide conjugate of nonylphenol was produced by
UGT2B1 (Fig. 1D), indicating
that the main metabolite in the liver perfusion is NP-G. The peak of NP
glucuronide was confirmed to be not produced by AH22 yeast microsomes,
expressing no UGT2B1, and in the enzyme reactions where the substrate was
omitted. The Km value of the microsomal
UDP-glucuronosyltransferase was estimated to be 0.25 mM nonylphenol.
UDP-glucuronosyltransferase activities (Vmax) toward
nonylphenol and other alkylphenols in rat liver microsomes were assayed, and
the estimated enzymatic activities are shown in
Fig. 2.
UDP-glucuronosyltransferase activity toward nonylphenol showed about the same
value as that of ethylphenol. These alkylphenols were injected into the portal
veins of rats, and then the bile and perfusate in the veins were sampled and
analyzed by HPLC as previously described
(Inoue et al., 2001). The
resultant metabolites of alkylphenols were mainly glucuronide conjugates (data
not shown), as was the case for nonylphenol. The concentrations of the
glucuronide conjugates of alkylphenols in the bile and vein during liver
perfusion are shown in Fig. 3.
After the liver perfusion, alkylphenol-glucuronides were excreted only into
the bile (Fig. 3, A–D),
and only ethylphenol-glucuronide was excreted into both the bile and vein
within 60 min (Fig. 3E). When
0.05 mM butylphenol and hexylphenol were perfused, these alkylphenols were
also glucuronidated in the liver, but these glucuronides were not excreted
completely from the liver within 60 min of perfusion
(Fig. 3B). Trace amounts of
nonylphenol-glucuronide were detected only in the bile
(Fig. 3, C and D). The
alkylphenols remaining in the livers after 1 h of perfusion were extracted as
described under Materials and Methods. The amounts of alkylphenols in
the liver and that excreted into bile and vein during 1 h of perfusion are
shown in Fig. 4. Recovery of
alkylphenols after the perfusions were about 50 to 60%
(Fig. 4). One of the reasons
for this is that it was supposed that all other alkylphenol metabolites such
as hydroxylated alkylphenols and conjugated alkylphenols with sulfate or
glutathione remaining in the liver tissue were not extracted with acetonitrile
completely, and/or these metabolites were not detected in this HPLC system.
Unconjugated alkylphenols remained in the liver tissue, and small amounts of
free alkylphenols were detected in the bile
(Fig. 4). About 800 to 1000
nmol of injected nonylphenol could be conjugated as a glucuronide by the rat
liver within 1 h of perfusion, but most of the glucuronide and free
nonylphenol remained in the liver (Fig.
4). Other alkylphenols having shorter alkyl chains were excreted
smoothly into the bile as glucuronides
(Fig. 4). These results
indicated that alkylphenols with shorter (including C6) alkyl chains were
easily excreted into the bile as glucuronides and that only the glucuronide
conjugate of nonylphenol, which has a longer C9 alkyl chain, was not easily
excreted from the liver. The metabolites of butylphenol and hexylphenol in
liver perfusion of EHBR, which are deficient in xenobiotic conjugates
transporter MRP-2, are shown in Fig.
5. These alkylphenols were excreted into the vein as glucuronides,
and only slight amounts of the metabolites were observed in the bile
(Fig. 5A). Rapid excretion and
clearance of butylphenol-glucuronide from the liver were observed
(Fig. 5, A and B). The
cumulative biliary excretion of the alkylphenol-glucuronides, which were
produced in the liver, was severely impaired in EHBR compared with SD rats,
indicating that alkylphenol-glucuronides were transported to the bile by
MRP-2.
|
|
|
|
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
,b),
and other metabolites, including glucuronide conjugates of ring- or side
chain-hydroxylated nonylphenol (Coldham et
al., 1998), were detected. It has been reported that nonylphenol
was extensively metabolized to the glucuronide conjugate in rats
(Moffat et al., 2001;
Doerge et al., 2002). Doerge et
al. (2002) have reported that
two glucuronide metabolites (NP-glucuronide and p-nonyl-catechol
glucuronide) were detected in the rat liver and serum after oral
administration. We detected NP-glucuronide as a major peak with slight
shoulder peaks after the liver perfusion. Other metabolites may be contained
in the shoulder peaks. In this study, rat hepatocytes showed a significant
capability for glucuronidation of nonylphenol of about 800 to 1000 nmol/h by
liver perfusion, but the transport of the nonylphenol-glucuronide across the
canalicular membrane into the bile was delayed. These findings suggest that
nonylphenol glucuronidation is mediated by UDP-glucuronosyltransferase
isoform(s), such as UGT2B1, with high activity but that the resultant
glucuronide having a long (C9) alkyl chain could not be transported by
MRP-2.
The substrate specificity of MRP-2 has been studied by comparing the
transportation activity across the bile canalicular membrane in normal rats
with that in transport-deficient rats
(Paulusma et al., 1996) or in
EHBR, which have a hereditary defect in MRP-2
(Fernandez-Checa et al., 1992;
Takenaka et al., 1995;
Yamazaki et al., 1996). It has
been demonstrated that glutathione conjugates, glucuronides of xenobiotics,
sulfates of several bile acids, and some organic anions are substrates for
MRP-2, but the mechanism of the transport and the substrate specificity of the
transporter have not been clarified yet. In this study, it was found that
transportation activity of MRP-2 decreased with increase in the number of
alkyl chains of alkylphenols, suggesting that after MRP-2 recognizes or binds
to the glucuronic acid anion, the long alkyl chain of alkylphenol-glucuronide
disturbs the transportation across the membrane. The transporter, which
mediates the transportation of glucuronides into the vein from liver cells,
can transport only an ethylphenol-glucuronide having the shortest alkyl chain,
indicating that metabolic distribution and excretion of alkylphenol is caused
by the length of the alkyl chain. It has been shown that MRP-2 has
transportation activity of glucuronide conjugates of various xenobiotics
having large molecular mass (Yamazaki et
al., 1996), suggesting that inability of MRP-2 to transport
nonylphenol-glucuronide is due to the shape or hydrophobicity of the long (C9)
alkyl chain.
Finally, nonylphenol has been shown to reduce the level of CYP1A1
expression in murine Hepa-1c1c7 cells
(Jeong et al., 2001) and to
inhibit in vitro CYP1A1 activity in rat liver microsomes
(Lee et al., 1996) and
activities of human cytochrome P450s, including steroidogeneic CYP17
activities (Niwa et al.,
2002). Oral administration of nonylphenol decreased hepatic
testosterone hydroxylation and CYP2C expression level
(Laurenzana et al., 2002).
Estradiol binding to the estradiol receptor (ER) was significantly inhibited
by nonylphenol (Danzo, 1997).
These results suggest the possible inhibition of cytochrome P450s and ER
functions by nonylphenol, which delays excretion from the liver.
One of the toxicological implications for the delay of excretion of
nonylphenol and nonylphenol-glucuronide is estimated to the binding to and
inhibition of ER (Tabira et al.,
1999); another is the inhibition of MRP-2 which excretes various
glucuronides such as estradiol glucuronide
(Ito et al., 2001) and
bilirubin glucuronide (Kusuhara et al.,
1998). The delay of nonylphenol excretion is possible because of
the adverse effects of nonylphenol on rat reproductive systems
(Laws et al., 2000).
|
Footnotes |
|---|
Address correspondence to: Hiroshi Yokota, Department of Veterinary Biochemistry, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido, 069-8501 Japan. E-mail: h-yokota{at}rakuno.ac.jp
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  T. Daidoji, M. Ozawa, H. Sakamoto, T. Sako, H. Inoue, R. Kurihara, S. Hashimoto, and H. Yokota SLOW ELIMINATION OF NONYLPHENOL FROM RAT INTESTINE Drug Metab. Dispos., January 1, 2006; 34(1): 184 - 190. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Narukawa, H. Inoue, S. Kato, and H. Yokota GLUCURONIDATION OF 1-NAPHTHOL AND EXCRETION INTO THE VEIN IN PERFUSED RAT KIDNEY Drug Metab. Dispos., July 1, 2004; 32(7): 758 - 761. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
), butylphenol
(BP-G; 
), hexylphenol (HP-G; 
) and nonylphenol (NP-G; 
). The
profiles of nonylphenol-glucuronide excretion are shown at high scales in C
(0.025 mM) and D (0.050 mM). Only ethylphenol metabolites appeared in the
perfusate vein, and the profiles are shown in E [0.025 mM (
) and 0.050
mM (
) ethylphenol]. Parameters are shown as means ± S.E.
(n = 4 animals or n = 3 animals in ethylphenol
analysis).
). Data are represented in the upper box (A) when 0.025 mM alkylphneol
was injected, in the lower box (B) when 0.050 mM nonylphenol was injected.
About 30% of injected alkylphenols were absorbed to the inside of the silicon
tubes during the perfusions. Recovery was calculated with the tube absorption
of each alkylphenol. Parameters are shown as means ± S.E. (n =
4 animals or n = 3 animals in ethylphenol analysis).