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-Dependent Induction of Liver Microsomal Esterification of Estradiol and Testosterone by a Prototypical Peroxisome Proliferator
Laboratory for Cancer Research, Department of Chemical Biology, College of Pharmacy, Rutgers, The State University of New Jersey (S.X., B.T.Z., V.T., A.H.C.), Piscataway, New Jersey 08854; Department of Pharmacology, University of North Carolina (I.R., R.T.), Chapel Hill, North Carolina 27599; Department of Veterinary Science, Center for Molecular Toxicology, Pennsylvania State University (J.M.P.), University Park, Pennsylvania 16802; and Laboratory of Metabolism, National Cancer Institute, National Institutes of Health (F.J.G.), Bethesda, Maryland 20892
Address all correspondence and requests for reprints to: Dr. Allan H. Conney, Laboratory for Cancer Research, Department of Chemical Biology, College of Pharmacy, Rutgers, The State University of New Jersey, 164 Frelinghuysen Road, Piscataway, New Jersey 08854-8020. E-mail: aconney{at}rci.rutgers.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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in the
induction of fatty acyl-coenzyme A:estradiol acyltransferase and fatty
acyl-coenzyme A:testosterone acyltransferase activities by
peroxisome proliferators, we fed 0.1% Wy-14,643 to female wild-type
and PPAR null mice for 11 d. The liver microsomal acyl-coenzyme
A:estradiol acyltransferase and acyl-coenzyme A:testosterone
acyltransferase activities were increased 4- to 5-fold in wild-type
mice fed Wy-14,643, but no increase was observed in null mice. These
results demonstrate that induction of acyl-coenzyme A:estradiol
acyltransferase and acyl-coenzyme A:testosterone acyltransferase
activities by a prototypical peroxisome proliferator is dependent on
PPAR. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peroxisome proliferators are a diverse class of compounds that
have been widely used, and they are of pharmaceutical, industrial, and
environmental importance. Examples of peroxisome proliferators include
hypolipidemic drugs, herbicides, plasticizers, and solvents (3, 4). Administration of peroxisome proliferators to rats and mice
results in an increase in the number and size of hepatic peroxisomes
and an increase in fatty acid-metabolizing enzymes, such as peroxisomal
fatty acyl-CoA oxidase and microsomal fatty acid 
-hydroxylase
(CYP450 4A) (3, 5). The induction of these enzymes by
peroxisome proliferators is known to result from an increased rate of
gene transcription mediated by PPAR, a member of the steroid hormone
receptor superfamily (6, 7). PPAR mediates the
activation of genes through dimerization with retinoid X receptor
and binding to cis-acting regulatory elements (peroxisome
proliferator response elements) upstream of promoter regions in target
genes (8, 9).
PPAR null mice (-/-) lack the expression of PPAR
protein (10). Administration of prototypical peroxisome
proliferators, such as clofibrate or Wy-14,643, to PPAR null mice
does not result in detectable hepatomegaly, peroxisome proliferation,
induction of the mRNA encoding the peroxisomal and microsomal
lipid-metabolizing enzymes, or hepatocarcinogenesis (responses that
were observed in PPAR wild-type mice) (6, 10). These
results indicate that PPAR is required for mediating the pleiotropic
response of rodents to peroxisome proliferators.
In the present study we evaluated the effects of peroxisome
proliferators on liver microsomal acyl-CoA:estradiol acyltransferase
and acyl-CoA:testosterone acyltransferase in PPAR wild-type (+/+)
and null (-/-) mice to determine whether the induction of these
enzyme activities is dependent on PPAR.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals
Female PPAR wild-type mice (+/+) and null mice (-/-) on an
Sv/129 genetic background were used. In the first experiment PPAR
wild-type (+/+) and null (-/-) mice were fed an AIN-76A diet or 0.5%
clofibrate (wt/wt) in an AIN-76A diet for 3 wk. Livers were removed for
the preparation of microsomes as described previously
(11). In a second experiment female C57 BL/6J mice and
female Sprague Dawley rats, purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN), were fed 0.5% clofibrate diet for 2
wk, and liver microsomes were prepared. In a third experiment PPAR
wild-type (+/+) and null (-/-) mice were fed 0.1% Wy-14,643 diet for
11 d. Livers were removed for the preparation of microsomes.
Protein concentrations were determined with the Bio-Rad Laboratories, Inc., assay method (Richmond, CA) according to the
supplier’s instructions, using BSA as a standard.
Enzyme assays
Enzyme assays for the esterification of estradiol or
testosterone by rat liver microsomes were carried out as described
previously (1). Incubation mixtures consisted of 10
µM 3H-labeled estradiol or
testosterone (
1–2 µCi), 100 µM oleoyl-CoA, together
with 5 mM magnesium chloride in 0.1 M sodium
acetate buffer (pH 5.0) in a final volume of 0.2–0.5 ml. For
preparation of the incubation mixture, radioactive estradiol or
testosterone in ethanol was added first and dried under nitrogen, and
then the remaining components of the incubation mixture (including
nonradioactive estradiol or testosterone in 2–5 µl ethanol) were
added. This procedure resulted in uniform distribution of radioactive
and nonradioactive steroid throughout the incubation mixture. The
reaction was initiated by the addition of hepatic microsomes (0.25–0.5
mg protein/ml). After incubation at 37 C for 30 min, the reaction was
arrested by placing the tubes on ice, followed by addition of ice-cold
sodium acetate buffer and ethyl acetate. The samples were vortexed
immediately and then centrifuged for 10 min at 3000 x
g. The organic upper phase was removed, and the extraction
was repeated a second time. The organic solvent extracts were combined
and evaporated to dryness under a stream of nitrogen. The resulting
residues were dissolved in 100 µl methanol and were analyzed by
HPLC.
HPLC methods
Measurement of esterified metabolites of estradiol and
testosterone was performed on a Spherisorb ODS column (5-µm particle
size; id, 250 x 4.6 mm). The HPLC system consisted of a Waters
600E solvent gradient programmer (Waters Corp., Milford,
MA), a Waters Lambda-Max model 481 UV detector (set at 280 nm), and a
radioactive flow detector (ß-ram from IN/US, Fairfield, NJ) with a
liquid cell (for 3H detection). The solvent
gradient used for elution of the oleoyl ester of estradiol was
described previously (1). The solvent system consisted of
acetonitrile/H2O with 0.1% acetic acid/methanol:
12 min isocratic at 30/6/64, 6 min with a no. 10 convex gradient to
60/0/40, 15 min isocratic at 60/0/40, 2 min with a no. 2 convex
gradient to 20/0/80, and 5 min isocratic at 20/0/80, and the column was
then returned to initial conditions over 15 min. The mobile phase for
elution of the oleoyl ester of testosterone was isocratic 100%
methanol for 20 min. The flow rate was 1.2 ml/min. The retention times
of the radioactive metabolites agreed exactly with the corresponding
UV-absorbing peaks. Incubation of 3H-labeled
estradiol or testosterone with oleoyl-CoA each resulted in a single
radioactive metabolite peak with a retention time of 27.6 or 11.6 min,
respectively, with the appropriate HPLC solvent systems described
above. Metabolite quantification was based on the amount of
radioactivity in the metabolite peak compared with the total
radioactivity collected from the HPLC column from each sample.
Statistical analysis
Data are presented as the mean ± SD.
Differences between means were assessed using a two-way ANOVA test,
Scheffé’s test or t test. The two-way ANOVA test and
Scheffé’s test were carried out using StatView software (Abacus
Concepts, Inc., Berkeley, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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wild-type (+/+) and null (-/-) mice were fed
0.5% clofibrate in an AIN-76A diet for 3 wk. Although the liver/body
weight ratio in wild-type mice was increased 45% after treatment with
clofibrate, the liver/body weight ratio in PPAR null mice was not
altered by clofibrate treatment (data not presented). Clofibrate
administration did not stimulate liver microsomal acyl-CoA:estradiol
acyltransferase activity in wild-type (+/+) or PPAR null (-/-)
mice (Table 1
). These results indicate
that clofibrate-induced increases in liver size in wild-type mice can
be dissociated from clofibrate-induced increases in hepatic
acyl-CoA:estradiol acyltransferase activity.
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). In Sprague Dawley rats, the liver/body weight
ratio was increased 18% (data not presented), but estradiol
esterification in liver microsomes was stimulated 900% by the
clofibrate treatment (Table 1). The results indicate a marked species
difference in sensitivity to clofibrate induction of acyl-CoA:estradiol
acyltransferase between rats and mice and also a strain difference in
inducibility between Sv/129 and C57BL/6J mice.
Effect of Wy-14,643 administration on the esterification of
estradiol and testosterone by liver microsomes from rats and wild-type
(+/+) and PPAR null (-/-) mice
The observation that administration of 0.5% clofibrate diet
stimulated liver microsomal acyl-CoA:estradiol acyltransferase in rats,
but not in wild-type (+/+) or PPAR null (-/-) mice (Table 1),
suggested that these mice are less sensitive to the induction of liver
microsomal acyl-CoA:estradiol acyltransferase by clofibrate than
rats (Table 1). Because of these observations we studied the effect of
a more potent peroxisome proliferator, Wy-14,643, on estradiol
esterification in rats and in wild-type and PPAR null mice. Feeding
0.1% Wy-14,643 to female rats for 7 d increased the liver
microsomal esterification of estradiol by about 5-fold (39.4 ±
4.9 vs. 189.2 ± 33.9 pmol estradiol-oleoyl ester
formed/mg protein/min; mean ± SD for three
control rats and four treated rats), and the liver microsomal
esterification of testosterone was increased about 6-fold (26.5 ±
2.9 vs. 168.6 ± 28.2 pmol testosterone-oleoyl ester
formed/mg protein/min; mean ± SD for two
control rats and four treated rats).
In another experiment female wild-type (+/+) mice and PPAR null
(-/-) mice were fed 0.1% Wy-14,643 diet for 11 d. The
liver/body weight ratio in wild-type mice was increased about 3-fold by
Wy-14,643 administration; in contrast, the liver/body weight ratio in
PPAR null mice was not altered by Wy-14,643 administration (data not
presented). Liver microsomal formation of the oleoyl ester of estradiol
or testosterone was increased 4- to 5-fold in wild-type mice treated
with Wy-14,643. In contrast, the liver microsomal esterification of
estradiol and testosterone was not stimulated in PPAR null (-/-)
mice treated with Wy-14,643 (Table 2).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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wild-type mice on an Sv/129
background. Liver microsomal acyl-CoA:estradiol acyltransferase
activity was stimulated by only 90% in female C57BL/6J mice. In
contrast, similar clofibrate administration to female Sprague Dawley
rats resulted in increased liver microsomal acyl-CoA:estradiol
acyltransferase activity by 900% (Table 1). These results
indicate that sensitivity to induction of liver microsomal
acyl-CoA:estradiol acyltransferase by clofibrate is species dependent;
mice are less sensitive than rats. Several studies have reported
different sensitivities to peroxisome proliferators between mice and
rats (12, 13, 14). The different responses to clofibrate
administration of mice and rats observed in this study could be
partially explained by different pharmacokinetics or elimination rates
of clofibrate in mice and rats. The elimination half-life of clofibric
acid (the active form of clofibrate) was 2 h in mice and 5–8 h in
rats (15). Since mice and rats have comparable levels of
hepatic PPAR mRNA (16), the different responses to
clofibrate administration in the two species may not be explained by
the relative abundance of the PPAR. Other possible reasons for the
different sensitivities of rat and mouse liver microsomal
acyl-CoA:estradiol acyltransferase to induction by clofibrate may
include different regulatory mechanisms for the target genes and/or
different esterase activities for hydrolyzing the estradiol fatty acid
esters in rats and mice.
In another experiment, wild-type and PPAR null mice were treated
with Wy-14,643, a more potent peroxisome proliferator (PPAR agonist)
than clofibrate. Liver microsomal acyl-CoA:estradiol acyltransferase
activity and acyl-CoA:testosterone acyltransferase activity were 4- to
5-fold higher in wild-type (+/+) mice treated with dietary Wy-14,643,
but no increase was observed in PPAR null (-/-) mice (Table
2). These studies indicate that the induction of liver microsomal
esterification of estradiol and testosterone by peroxisome
proliferators is dependent on PPAR.
The induction of fatty acyl-CoA oxidase and CYP450 4A by peroxisome
proliferators is known to result from an increased rate of gene
transcription mediated by PPAR (10). Administration of
clofibrate or gemfibrozil to rats induces liver microsomal
acyl-CoA:estradiol acyltransferase activity without altering its pH
optimum or Km value (1). These
observations suggest that administration of clofibrate or gemfibrozil
increased the level of the same fatty acyl-CoA:estradiol
acyltransferase enzyme that is present in microsomes from untreated
rats. Further studies are needed to determine whether clofibrate or
gemfibrozil administration stimulates transcription of the fatty
acyl-CoA:estradiol acyltransferase gene, enhances the stability of the
corresponding mRNA, facilitates the translation of the corresponding
mRNA, and/or inhibits the breakdown of the fatty acyl-CoA
acyltransferase protein.
During the course of our studies, we found that treatment of mice with
Wy-14,643 increased the esterification of testosterone (Table 2).
Unpublished observations in our laboratory indicate that treatment of
rats with clofibrate also stimulates the liver microsomal
esterification of testosterone, corticosterone, pregnenolone,
and dehydroepiandrosterone. The physiological significance of the
effect of clofibrate administration to enhance the esterification of
several steroid hormones is unknown.
In summary, the results of our studies indicate that the induction of
liver microsomal acyl-CoA:estradiol acyltransferase and microsomal
acyl-CoA:testosterone acyltransferase activities by a prototypical
peroxisome proliferator is dependent on PPAR.
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Footnotes |
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2 William M. and Myrle W. Garbe Professor of Cancer and Leukemia
Research.
Abbreviations: CoA, Coenzyme A; CYP450, cytochrome P450; Wy-14,643, [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid.
Received December 19, 2000.
Accepted for publication April 17, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. J Natl Cancer Inst 90:1702–1709 isoform of the peroxisome proliferator-activated
receptor gene in mice results in abolishment of the pleiotropic effects
of peroxisome proliferators. Mol Cell Biol 15:3012–3022[Abstract]
(PPAR)
cDNA cloned from guinea-pig liver encodes a protein with similar
properties to the mouse PPAR: implications for species differences
in responses to peroxisome proliferators. Arch Toxicol 72:169–177[CrossRef][Medline]This article has been cited by other articles:
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  J. C. Corton and P. J. Lapinskas Peroxisome Proliferator-Activated Receptors: Mediators of Phthalate Ester-Induced Effects in the Male Reproductive Tract? Toxicol. Sci., January 1, 2005; 83(1): 4 - 17. [Abstract] [Full Text] [PDF]
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