Aryl Hydrocarbon Receptor Knockout Mice (AHR-/-) Exhibit Liver Retinoid Accumulation and Reduced Retinoic Acid Metabolism Fausto Andreola, Pedro M. Fernandez-Salguero, Maria V. Chiantore, et al. Cancer Res 1997;57:2835-2838. Published online July 1, 1997.
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ICANCERRESEARCH57, 2835—2838. July 15. 1997]
Advances in Brief Aryl Hydrocarbon
Receptor
Knockout
Mice (AHR')
Exhibit
Liver
Retinoid
Accumulation and Reduced Retinoic Acid Metabolism Fausto Andreola,'Pedro M. Fernandez-Salguero,'Maria V. Chiantore,MartinP. Petkovich,FrankJ. Gonzalez,and Luigi M. De Luca2 Division of Basic Sciences, National Cancer institute, NiH, Bethesda, Maryland 20892-4255 (F. A., P. M. F-S.. M. V. C.. F. J. G., L M. D. LI, and Cancer Research Laboratories. Queen ‘s University. Kingston, Ontario, Canada K7L 3N6 (M. P. P.]
Abstract
condition
Livers from aryl hydrocarbon receptor-null mice showed a 3-fold increase in retinoids and a 65% decrease in retinoic acid metabolism. Levels of expression of the retinoic acid 4-hydroxylase, P45ORAI, did not change, whereas cytochrome
P4501A2 levels were lower in the null mouse,
as shown earlier; however, this enzyme was found not to be active toward retlnolc acid. These data suggest that aryl hydrocarbon receptor controls retlnolc acid catabolism, through modulation of an unidentified target gene. Aldehyde dehydrogenases 1 and 2 were down-regulated markedly in the aryl hydrocarbon receptor-deficient mouse liver. 2,3,7,8-Tetrachlo rodlbenzo-p-dloxin
Induced
cytochrome
P4501A2
but not the aldehyde
dehydrogenases in wild-type mice, suggesting that aryl hydrocarbon re ceptor Is not Involved directly In the down-regulation of this gene. Trans glutalninase II, a retinoic acid-responsive gene product, was Increased 2-fold, consistent with the liver fibrosis phenotype observed In the null mice. These findings suggest a molecular connection between xenoblotlc activated receptor signaling and retinoid homeostasis.
of nutritional
vitamin
A deficiency
(7) and reversible
by
retinoids (8). Hepatic retinyl esters were found to be decreased by 3-methylcholanthrene, 7,l2-dimethylbenzanthracene, and benzo (a)pyrene (9). Symptoms of polychlorinated biphenyl exposure re semble those of vitamin A deficiency (10). Therefore, we tested the hypothesis that AHR deficiency might have some important conse quences on liver retinoid homeostasis and RA metabolism.
Materials and Methods Materials. All-trans-RA, retinol, and retinyl palmitate were purchased from Sigma Chemical Co. (St. Louis, MO). [1 l,12-3HIRA (specific activity,
5 1.2 Cilmmol) and 12,3-3H(N)lputrescinedihydrochlonde (specifIc activity, 44.4 Ci/mmol) were purchasedfrom NEN Life ScienceProducts(Boston,
MA). HPLC-gradeacetonitrile.methylenechloride.and methanolwere pur chased from Burdick and Jackson (Muskegon.
Ml), and 1-octanol (certified)
and hexane were purchased from Fisher Scientific (Fair Lawn. NJ). All of the solvents
used for HPLC were filtered through a 0.2-@xm nylon filter, and all of
the procedures were carried out under yellow light. HPLC. Retinylpalmitatewas detectedwith a Beckmanmodel I lOApump
Introduction
(Beckman Instruments, Palo Alto. CA) connected to a Knauer variable-wave length detector (Sonntek. Woodcliff Lake, NJ). A C- 130 guard column (Up
AHR3 is a member of the basic helix-loop-helix family of tran scription factors and is thought to mediate the toxic and carcinogenic effects of polycyclic aromatic hydrocarbons and dioxins such as TCDD. AHR activates a battery of target genes, including those
church
encoding P450s CYPIA1 and CYP1A2, by binding the AhRE up
acetonitrile.
stream regulatory elements. AHR knockout mice were found to be unresponsive to TCDD, given that the Cyplal and Cypla2 genes
tration) plus 0. 1% butylated hydroxytoluene,
were not inducible in the null mouse (1, 2). These mice are also
The analyses of retinol and RA were performed on a Partisil 10 octyl decyl sylane (ODS)-2 column (4.6 mm inside diameter X 25 cm; Whatman, Inc.,
resistant to the toxic effects of TCDD (3). Abnormal phenotypes have
been observed, including one-half to one-third liver size and liver fibrosis compared to wild-type mice and AHR-heterozygous mice. These observations suggest that the AHR plays a fundamental role in
cell and organ physiology and homeostasis and lend further support for the existence of an endogenous ligand. Retinoids are potent chemopreventive agents in several experimental systems of epithelial carcinogenesis induced by PAHs, including skin, lung, breast, pros
Scientific,
Oak Harbor,
WA) was used to protect
dichloromethane,
methanol,
analyses
of RA and retinol
in liver samples.
analyzer;
studies
with
Packard Instrument
acetonitrile
and
been reported to cause increased formation of benzo(a)pyrene DNA adducts in hamster tracheal epithelial cells (5) and to increase 3-meth ylcholanthrene-induced lung tumor formation in rats (6). Exposure of tracheal epithelial cells to PAHs and other carcinogens causes the formation of squamous metaplasia (6), a lesion also caused by the
(12), at a flow rate of 2.2 ml/min.
I These
two
authors
contributed
requests for reprints
equally
to
this
37. Room 3A-l7,
LCCTP, National Cancer Institute, NIH, 37 Convent Drive, Bethesda, MD 20892-4255.
Phone: (301) 496-2698; Fax: (301) 496-8709. 3 The abbreviations
used are: AHR,
aryl hydrocarbon
receptor;
TCDD,
model
pump
was
Co., Meriden.
1% ammonium
CT) for RA metabo
acetate
in water
(65:35
as the mobile phase, according to the procedure of Frolik et a!.
Extraction of Retlnolds from Liver. Liver (0.25 g) was homogenizedin 0.5 ml of salinewith a Polytronhomogenizer(Brinkman Instruments.West bury, NY). After the addition
of 5 volumes
(2.5 ml) of chloroform-methanol
(2: 1 concentration), the samples were mixed by Vortex for 2 mm and centri fuged at 3000 rpm for 10 mm. The organic phase was removed and evaporated to dryness under a slow stream of nitrogen. The residue was dissolved in 1 ml of ethanol for retinyl palmitate analysis and 250 pi of methanol for RA. To saponify
the lipids, an equal volume
of NaOH (0.1 M) was added to the latter
samples. The resulting mixture was incubated at 60°Cfor 30 mm and neutral ized by adding 1/10 of the volume of ice-cold HCI (0.5 M). An equal volume of chloroform-methanol (2: 1 concentration) was added and mixed, the lower organic phase was dried under nitrogen, the residue was dissolved in 500 @xl of
work.
should be addressed, at Building
The same
connected to a Gilson 116 UV detector (Gilson Medical Electronics, Middle ton, WI) and in series to a Radiomatic radioactivity flow detector ( 15OTR Flow
concentration)
2 To whom
(90: 15: 10:0. 1 concen
according to the procedure of
man model 1IOA pump was connected to a Waters 996 photodiode array detector (Waters Chromatography Division, Millipore, Milford, MA) for the
tate, bladder, and other systems (4). Dietary retinoid depletion has
18 U.S.C. Section 1734 solely to indicate this fact.
and 1-octanol
Clifton, NJ) fitted with a precolumn of Pellicular ODS (Whatman). A Beck
lism
charges. This article must therefore be hereby marked advertisement in accordance with
C- 18 (5-@xm)
Barua et a!. (11) at a flow rate of 1.2 mI/mm.
scintillation
Received 5/7/97; accepted 5/29/97. The costs of publication of this article were defrayed in part by the payment of page
a Waters
“Resolve― column (3.9 mm inside diameter X 30 cm). The detector was set at 325 nm for the detectionof retinyl palmitate. The mobile phaseconsistedof
acetonitrile, and lipids were extracted by adding an equal volume of hexane.
2,3,7,8-tetra
chlorodibenzo-p-dioxin; PAH, polycyclic aromatic hydrocarbon; RA, retinoic acid; HPLC, high-performance liquid chromatography; AHD, aldehyde dehydrogenase.
The acetonitrile (lower) phase, which contained the retinoids, was evaporated to dryness under N,, and the residue resuspended in 90 @xl of methanol. All of
2835
Downloaded from cancerres.aacrjournals.org on July 13, 2011 Copyright © 1997 American Association for Cancer Research
INCREASED LIVER RETINOIDS IN AHR-DEFICIENT MICE 5.-
(Ahr@'),
A
>
heterozygous
(Ahr@―) and homozygous
null (Ahr―)
mice
re
ceived a dose of 80 @xg/kgof body weight, because this dose of TCDD was shown to significantly increase RA metabolism in rat liver (13). The mice were
killed 3 days after injection, the livers were removed quickly and weighed, and C) a
hepatic microsomes RA Metabolism
Ii) Co
were incubated at 37°Cin a shaking water bath for 20 mm in 0.5 ml of 50 mM
were prepared as described. in Liver Microsomes. Microsomes
(300
@xgof protein)
Tris-HC1, 150 mMKC1,5 mi@i MgCl2,and a NADPH-regenerating system (2.5 units of glucose-6-phosphate
E
dehydrogenase,
500 nmol NADP,
and 0.5 p@mol
glucose-6-phosphate), pH 7.4, with 90 nM [3H]RA, according to Fiorella et a!. (13). Reactions were stopped in a mixture of dry ice and ethanol and lyophi lized. Each residue was extracted with 200 p1 of methanol, and the methanol
CO
0@
>@
phase was centrifuged in a desktop centrifuge for 3 mm to remove particulates. All of the samples were stored at —70°C until analyzed. Transglutaminase Assay. Liver samples were suspended in 2 volumes of 20 mMsodium phosphate (pH 7.2), 10 mMDli', 0.5 mr@i EDTA, and 50 @xg/ml phenylmethylsulfonyl fluoride and homogenized with a Polytron homogenizer. The homogenates were sonicated and spun at 14,000 rpm for 30 mm, and the supernatant was assayed for tissue transglutaminase by [2,3-3H(N)Jputrescine
ci) Ahr +1+
Ahr+/
5-
ci@
incorporation
>
into dimethyl casein (pH 9.0) at 28°Cfor 30 mm, according to
Lichti and Yuspa
(15). Protein
concentration
was determined
by the Bradford
C)
method (14).
C)
Isolation of Liver RNA and Northern Blot Analysis. RNA was isolated from liver homogenates in a guanidinium-thiocyanate solution after centnfu
C
gation
in a cesium
trifluoroacetate
gradient.
RNA
was extracted
in a 50:50
80
>@ > 60
CO
C
Co
5-
ci) > C) C)
@40@
C
I
2000
C
T
0
ci)
C.)
@5 20
1500
0@
0
1000
C
0.
ci@
Ahr+/-
r +/+ 500
Ahr-/
Fig. 2. AHR-null mice show a reduced capability to oxidize I3H]RA. 13H]RA was incubated for 20 mm with the liver microsomes, and the retinoids were extracted and
analyzed by HPLC as described in “Materials and Methods.― Solid columns. RA; open Ahr ÷1+
Ahr +1-
Ahr -/-
columns, oxidation products of RA. Differences between all of the groups were significant (P 0.05). Bars, SE (n 5 mice).
Fig. I . Liver retinoid accumulation in AHR' mice. Livers were homogenized and extracted as described in @‘Matetials and Methods.―HPLC analysis data demonstrate an increase in the concentration of retinyl palmitate (A), in which differences between
AHR' (P
(n = 5 mice), AHR@' (n
5), and AHR@'@(n
6000
3) were all significant
0.05). For RA (B) and retinol (C), differences between the AHR'
(n = 3),
AHR@' (n = 3), and AHR@'@(n = 3) were statistically significant between the AHR@'@ and AHR@ and between the AHR@' and AHR' . Bars, SE.
C ti)
i
@
the samples were filtered through a Microfilterfuge
Microsomes. @
tube (Rainin Instrument
Co., Woburn, MA) with a 0.2 @tm nylon filter and kept at —20°C until aliquots were analyzed. Mice were given a single i.p. injection of TCDD (80 @xg/kg
of body weight) in corn oil or vehicle alone, and, after 3 days, were sacrificed. The livers were homogenized in 10 m@Tris-HC1,250 mi@i sucrose (pH 7.4; 3 ml/g of tissue)
in a Polytron
homogenizer.
The homogenate
was centrifuged
at
10,000 x g for 10 mm. The pellet was discarded, and the supernatant was centrifuged at 110,000 x g for 1 h. The microsomal pellet was homogenized in 1 ml of the same buffer and stored at —70°C, according
Fiorella et a!. (1 3). The protein concentration binding
method
with BSA as standard
to the procedure
was determined
of
by the dye
(14).
TCDD Treatment. TCDD was administeredto 20-week-old male mice as a single i.p. injection in a final volume of 150 pi of corn oil. Wild-type
I
2 5000 4000
3000.
T
>
:@ 2000. Co
0) 1000.
C) I-
0. .
—I--
Ahr +1+
—I—
Ahr +1-
Ahr -I-
Fig. 3. Induction of tissue transglutaminase in livers of AHR knockout mice. TGase activity was assayed as described in “Materials and Methods.―Results were statistically different for AHR' and AHR@'@ genotypes and between AHR@' and AHR@ genotypes (n = 8 mice for each group), but not between AHR@' and AHR―@ genotypes. Bars. SE.
2836
Downloaded from cancerres.aacrjournals.org on July 13, 2011 Copyright © 1997 American Association for Cancer Research
INCREASED LIVER RETINOID5 IN AHR-DEFICIENT MICE
A
B AHR
+1+ +1-. -I-. AHD1 C
0 .@
10
(I) @
@--
A
AHD2
5—
>< LU
Actin
+1+
÷1-
-1-
Fig. 4. A, Northern blot analysis of mRNA encoding liver enzymes involved in RA synthesis. Liver total RNA (20 ag), isolated from AHR@'@,AHR@', and AHR' mice, was subjected to Northern blot analysis. The membrane was hybridized sequentially with the mouse AHD1 and AHD2 cDNAs. Mouse actin mRNA, which is not controlled by the AHR. was used to verify RNA integrity. B, following hybridization to each of the two probes, the membrane was exposed to Phosphor screens, and the signal was quantified. Data are presented as the mean level of expression; bars. deviation of each mouse with respect to the mean.
solution of phenol-chloroform
and precipitated
observation was the finding that vaccinia virus-expressed
with ethanol. Polyadenylated
RNA was isolated from total RNA by fractionation on an oligodeoxythymi dylate cellulose column (Pharmacia). Total RNA (20 @g) or 3 @.tg of polya denylated RNA were subjected to electrophoresis on a 1% agarose gel con taming 2.2 M formaldehyde. The RNA was blotted to Gene Screen Plus (DuPont) nylon membranes, fixed for 2 h at 80°Cunder vacuum, and prehy bridized at 65°Cfor 4 h in 0.5 Msodium phosphate buffer (pH 7.0), 1% BSA, 7% SDS, and 1 mM EDTA.
The membranes
in the same solution containing
were hybridized
overnight
1.5 X 106 cpm/ml of the corresponding
at 65°C cDNA
and CYP1A2
failed to metabolize
CYP1A1
RA (not shown).
To determine whether the increase in hepatic RA is biologically meaningful,
expression
of type II transglutaminase
activity,
the gene
for which contains a RA response element (16), was examined. This activity was increased in homogenates from AHR-null livers as com pared to wild-type mouse liver (Fig. 3). Because AHD1 and AHD2 are involved in RA synthesis, we
probe labeled by random priming (Pharmacia) with a [32P]dCTP.The filters were washed at 65°Cfor 30 mm in 2X SSC (1X SSC: 0.15 Msodium chloride, 0.015 Msodium citrate), 0.5% SDS and two times at 65°Cfor 30 mm each in
AHR+/-i
AHR-/
a solution of 0.15 M NaCI, 0.015 M sodium citrate, and 0.5% SDS. The membranes
@
were exposed
for 2 h to Phosphor
screens
(Eastman
Kodak),
oil
and
the screens were developed using a Phosphorlmager (Molecular Dynamics). The signals obtained were quantified by volume integration using software provided by the manufacturer. Results
TCDD
oil
TCDD
tt@T t. @-..° 0
In an attempt to determine the biochemical basis of the liver fibrosis
II
phenotype found in AHR@ mice, levels of RA, retinol, and retinyl palmitate were measured in liver. Remarkably, the AHR@' mice
showed a 3-fold increase in the concentration of retinyl palmitate from 600 to 2000 ;Lg/g liver (Fig. 1A). The heterozygote showed an increase to intermediate value (1250 @g/g).A 3-fold accumulation in
CYP1A2
RA and retinol was found in livers of the AHR' genotype com pared to controls (Fig.!, B and C, respectively). No significant statis tical difference was observed in RA and retinol levels between AHR@@and AHR@ mice. Livers
from
AHR@
mice
showed
a marked
decrease
Actin
in their
ability to metabolize BA compared to AHR@@and AHR―@micro Fig. 5. Regulation of the AHD2 and CYP1A2 in wild-type (AHR@)
somes (Fig. 2). Interestingly, in vivo pretreatment with TCDD did not induce RA metabolism in null or wild-type mice, suggesting that a
(AHR@)
P450 in the CYP1A family is not involved. Consistent with this
were developed by use of a Phosphorlmager.
mouse
liver.
Total
RNA
(20
@sg) isolated
from
mice
administered
and AHR-null TCDD
was
subjected to Northern blot analysis using the AHD2 and CYP1A2 cDNAs. The signals
2837
Downloaded from cancerres.aacrjournals.org on July 13, 2011 Copyright © 1997 American Association for Cancer Research
INCREASED LIVER RETINOIDS IN AHR-DEFICIENT MICE
investigated the expression of their mRNA by Northern blot analysis.
A marked reduction (8—10-fold)in the expression of AHD1 and AHD2 mRNAs was found in AHR' mice versus AHR@' and AHR@'@mice (Fig. 4). The observed down-regulation of the AHD1 and AHD2 in AHR' mice could result from diminished transcrip tion rates
due to the lack of AHR,
if direct
transcriptional
total RNA from control
type AHR@'@ and AHR' using
AHD2
cDNA
(oil) and TCDD-treated
In conclusion, this work demonstrates a large increase in retinyl palmitate,
control
would occur, as shown previously for CYP1A2 (1). Alternatively, the high levels of RA found in AHR' mice could control the synthesis of RA through an inhibitory feedback mechanism acting on AHD1 and AHD2. Direct transcriptional control by AHR would imply that the expression of these genes would be TCDD inducible. To address this question,
The observed large increase in the concentration of retinyl palmitate and RA and the resulting increase in transglutaminase II, transforming growth factor /3, and collagen (not shown) might well be responsible for the observed liver fibrosis found in the AHR-null mouse.
The mouse
CYPIA2
S., Nebert,
(1), expression
of CYPIA2
in AHR'
mice
F. J. Aryl-hydrocarbon
was
Discussion
a preneoplastic
system
receptor-deficient
mice are resistant
to 2,3,7,8-tetrachlorod
Academic Press, 1983. 7. Sporn, M. B., and Newton, D. L. Chemoprevention of cancer with retinoids. Fed. Proc.. 38: 2528—2534, 1979. 8. Huang, F. L.. Lancilloui, F., and Dc Luca, L. M. Retinoids in epithelial differentiation
TCDD administration to rats greatly reduces retinyl palmitate liver levels. Moreover, in vivo and in vitro studies have shown that expo metaplasia,
F. J. Immune
6. Nettesheim. P., and Marchok. A. Neoplastic development in airway epithelium. in: G. Klein and S. Weinhouse (eds.), Advances in Cancer Research, pp. 1—70. New York:
The work of Brouwer et a!. (17) and Chen et a!. (18) has shown that
squamous
S., Ward, J. M., and Gonzalez,
ibenzo-p-dioxin-induced toxicity. Toxicol. AppI. Pharmacol., 140: 173—179,1996. 4. Hong, W. K., and Lotan, R. Retinoids in Oncology. New York: Marcel Dekker, 1993. 5. Genta, V. M., Kaufman, D. G.. Hams. C. C., Smith. J. M.. Spom. M. B., and Saffiotti, U. Vitamin A deficiency enhances binding of benzo(a)pyrene to tracheal epithelial DNA. Nature (Lond.), 247: 48—49, 1974.
markedly lower compared to wild-type mice.
sure to PAH (I 9) induces
D. W., Rudikoff,
impairment and hepatic fibrosis in mice lacking the dioxin-binding Ah receptor. Science (Washington DC). 268: 722—726,1995. 2. Schmidt. J. V., Su, G. H., Reddy, J. K., Simon, M. C., and Bradfield, C. A. Characterization of a murine Ahr null allele: involvement of the Ab receptor in hepatic growth and development. Proc. Natl. Acad. Sci. USA, 93: 6731—6736, 1996. 3. Femandez-Salguero, P. M., Hilbert, D. M., Rudikoff, S., Ward, J. M., and Gonzalez,
was used as a
CYP1A2 transcripts in the Ahr@ mice (Fig. 5). As expected from work
with
I. Femandez-Salguero. P., Pineau. T., Hilbert. D. M.. McPhail. T., Lee. S. S., Kimura,
positive control to show TCDD-mediated induction in AHR@'@ mice. TCDD failed to induce AHD2 transcripts, whereas it clearly induced previous
mice. Together
References
wild
mice were analyzed by Northern blot
as a probe.
retinol, and RA in the livers of AHR@
the observed retinoid reduction as a consequence of carcinogen PAH and TCDD exposure, our findings suggest a molecular connection between the xenobiotic-activated Al-IR signaling and retinoid homeostasis.
lesion
and tumorigenesis. in: C. 0. Enwonwu (ed). Diet. Nutrition and Cancer. Vol. IV, pp.
also caused by vitamin A deficiency (20), and that RA in the culture medium of hamster tracheas inhibits the carcinogen-induced meta
9. Chen, L. C.. Berberian, I., Glauert, H. P., Robertson, L. W., and Chow, C. K. Altered
plastic response (8). Very recent work has shown that TCDD strongly inhibits RA induction of retinoic acid receptor fJ and of type II cellular
10. Kimbrough, R. D. The toxicity of polychlorinated polycyclic compounds and related
RA binding protein transcripts during palate cell development (21),
11. Barua. A. B.. Furr, H. C., Janick-Buckner. D., and Olson, J. A. Simultaneous analysis
suggesting an interaction of these signal transduction Remarkably, a 3-fold increase in retinyl palmitate,
of individual carotenoids, retinol, retinyl esters and tocopherols in serum by isocratic non-aqueous reverse phase HPLC. Food Chem., 46: 419—424, 1993. 12. Frolik, C. A.. Tavela, 1. E., Peck, G. L., and Sporn, M. B. High-pressure liquid chromatographic determination of l3-cis-retinoic acid and all-trans-retinoic acid in human plasma. Anal. Biochem.. 86: 743—750,1993. 13. Fiorella, P. D., Olson, J. R., and Napoli, J. L. 2,3,7,8-Tetrachlorodibenzo-p-dioxin induces diverse retinoic acid metabolites in multiple tissues of the Sprague-Dawley
185—201. Nashville:
liver microsomes
were less efficient
pathways. RA, and retinol than the AHR@'@
and AHR@' microsomes in oxidizing RA, and expression of mRNAs encoding catabolizing P450s (CYP1A2) showed a profound reduction in knockout
mice
and lack of inducibility
by TCDD,
RA catabolism
through
another
gene.
as demonstrated
Interestingly,
the
AHR' genotype did not show any effect on the expression of the recently cloned P45ORAI (22). The marked reduction in RA oxidation in AHR' mice may account for the 3-fold increase in RA, which could control its own synthesis through an inhibitory feedback mech anism. Increased RA levels could cause a strong down-regulation of the RA-synthesizing enzymes, as suggested by our findings that the
AHR'
livers showed a marked reduction in the AHD1 and AHD2
mRNAs. In addition, the lack of inducibility of AHD2 transcripts by TCDD, which clearly induced CYP1A2 transcripts in the AHR@'@ but not in AHR' mice, excludes a direct involvement of AHR on
AHD expression. Taken together, these results are consistent with the hypothesis
of a RA-mediated
inhibitory
feedback
mechanism
acting
on AHD1 and AHD2, possibly resulting in the 3-fold increase of retinol levels observed in AHR knockout mice. RA has been also shown to have a regulatory effect on retinol metabolism. It can inhibit retinol oxidation in ferret livers (23) and increase
its esterification
in human
keratinocytes
and rat livers
inducing lecithin:retinol acyltransferase activity (24, we can assume that the high levels of RA in AHR' responsible not only for the accumulation of retinol, sequestration from the oxidative pathway through the esterification to retinyl palmitate.
College,
I991.
(PAHs).
chemicals. CRC Cdt. Rev. Toxicol., 2: 445—498, 1974.
rat. Toxicol. AppI. Pharmacol., 134: 222—228,1995.
previously (I). However, vaccinia-expressed CYPIAI and 1A2 failed to metabolize RA (not shown), leading to the conclusion that AHR influences
Medical
tissue levels of vitamin A by selected polycyclic aromatic hydrocarbons Polycyclic Aromatic Compounds, 4: 173—182,1994.
was found in the AHR-null mice compared to wild-type mice. More over, Ahr@
Meharry
by
25). Therefore, mice might be but also for its activation of its
14. Bradford. M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72: 248—254.1976. 15. Lichti. U., and Yuspa, S. H. Modulation of tissue and epidermal transglutaminases in mouse epidermal cells after treatment with I2-O-tetradecanoylphorbol-l3-acetate and/or retinoic acid in @‘ivo and in culture. Cancer Res., 48: 74—81, 1988. 16. Nagy, L., Saydak, M., Shipley, N., Lu, S., Basilion, J. P., Yan, Z. H., Syka, P., Chandraratna. R. A.. Stein, J. P., Heyman. R. A.. and Davies, P. J. Identification and characterization of a versatile retinoid response element (retinoic acid receptor re sponse element-retinoid X receptor response element) in the mouse tissue transglu taminase gene promoter. J. Biol. Chem., 271: 4355—4365, 1996. 17. Brouwer, A., van den Berg, K. J., and Kukler, A. Time and dose responses of the reduction in retinoid concentrations in C57BL/Rij and DBA/2 mice induced by 3.4,3',4'-tetrachlorobiphenyl. Toxicol. AppI. Pharmacol., 78: 180—189,1985. 18. Chen, L. C., Berberian. I., Koch, B.. Mercier, M., Azais-Braesco, V., Glauert, H. P.,
Chow, C. K., and Robertson, L. W. Polychlorinated and polybrominated biphenyl congeners and retinoid levels in rat tissues: structure-activity relationships. Toxicol. Appl. Pharmacol., 114: 47—55,1992. 19. Lasnitzki, I. Hypovitaminosis A in the mouse prostate gland cultured in chemically defined medium. Exp. Cell Res., 28: 40—51, 1962. 20. Lancillotti, F., Darwiche. N.. Cdli, G., and Dc Luca, L. M. Retinoid status and the
control of keratin expression and adhesion during the histogenesis of squamous metaplasia of tracheal epithelium. Cancer Res., 22: 6144—6152, 1992. 21. Weston, W. M.. Nugent. P., and Greene, R. M. Inhibition of retinoic-acid-induced
gene expression by 2.3,7,8-tetrachlorodibenzo-p-dioxin. Biochem. Biophys. Res. Commun., 207: 690—694.1995. 22. White, J. A., Guo, Y. D., Baetz. K.. Beckett-Jones, B., Bonasoro, J., Hsu, K. E., Dilworth, F. J.. Jones, 0.. and Petkovich, M. Identification of the retinoic acid inducible all-trans-retinoic acid 4-hydroxylase. J. Biol. Chem.. 271: 29922—29927, 1996. 23. Wang, X. D., Krinsky, N. I., and Russell, R. M. Retinoic acid regulates retinol metabolism via feedback inhibition of retinol oxidation and stimulation of retinol esterification in ferret liver. J. Nutr., 123: 1277—1285,1993. 24. Kurlandsky, S. B., Duell, E. A., Kang, S., Voorhees, J. J., and Fisher, G. J. Auto regulation of retinoic acid biosynthesis through regulation of retinol esterification in
human keratinocytes. J. Biol. Chem.. 271: 15346—15352.1996. 25. Matsuura, T., and Ross. A. C. Regulation of hepatic lecithin: retinol acyltransferase activity by retinoic acid. Arch. Biochem. Biophys., 301: 221—227.1993.
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