ß-naphthoflavone induction of CYP1A in brain of juvenile lake trout (Salvelinus namaycush Walbaum)
Department of Fisheries and Wildlife, Michigan State University, East Lansing, MI 48824, USA
* Author for correspondence (e-mail: liweim{at}msu.edu)
Accepted 3 February 2004
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Summary |
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Key words: lake trout, Salvelinus namaycush, CYP1A induction, brain, cytochrome P450, ß-naphthoflavone
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Introduction |
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The most intensively studied P450 genes in fish are CYP1As
(Nelson et al., 1996), which
are inducible by a wide variety of persistent contaminants found in the Great
Lakes, particularly polychlorinated biphenyls (PCBs) and dioxins. CYP1A
nucleotide sequences have been determined in rainbow trout
(Berndtson and Chen, 1994
;
Heilmann et al., 1988
), plaice
(Leaver et al., 1993
),
Atlantic tomcod (Roy et al.,
1995
), toadfish and scup
(Morrison et al., 1995
),
killifish (Morrison et al.,
1998
), red sea bream (Mizukami
et al., 1994
), sea bass (Stien
et al., 1998
) and, more recently, Atlantic salmon
(Rees et al., 2003
). Current
evidence shows that several CYP1A isoforms exist in fish
(Teramitsu et al., 2000
) and
they are distributed in tissues such as the liver, gut, kidney, gill and heart
(Goksøyr and Husoy,
1998
; Sarasquete and Segner,
2000
; Stegeman and Hahn,
1994
). Few immunocytochemistry studies show that constitutive
CYP1A proteins are present in the neurons and the endothelia of the fish brain
(Sarasquete and Segner, 2000
).
Inducible CYP1A proteins are found mainly in the pituitary cells and brain
endothelia in fish (Sarasquete and Segner,
2000
). The CYP1A-mediated in situ metabolism and cellular
toxicity of xenobiotics in the brain may have far-reaching consequences by
causing disruption of neuronal and neuroendocrine function
(Andersson et al., 1993
;
Huang et al., 2000
;
Morse et al., 1998
).
Many chemically different compounds induce de novo synthesis of
CYP1A protein (Nebert and Gonzalez,
1987; Nebert et al.,
1989
; Parkinson,
1995
; Stegeman and Hahn,
1994
). The inductive response in this subfamily is known to occur
via the high-affinity binding of aromatic hydrocarbons to an
intracellular receptor complex (the Ah receptor), involving the
90-kDa heat shock protein (Hsp90) and a nuclear translocation factor.
Translocation of the inducerreceptor complex to the nucleus results in
the transcriptional activation of the genes in the Ah battery
(Hoffman et al., 1991
;
Nebert and Jones, 1989
;
Nebert et al., 1989
);
therefore, levels of CYP1A mRNA and newly synthesized CYP1A proteins are
increased and the CYP1A proteins subsequently undergo processing, heme
insertion and folding to yield the catalytically active enzymes. Each of these
steps, i.e. mRNA, protein and catalytic activity, can be analyzed with a
suitable probe to detect induction
(Goksøyr and Förlin,
1992
). However, few studies have actually used all the above
assays to examine the transcription and translation of CYP1A in fish
simultaneously.
In the present study, we assessed the effects of sublethal
ß-naphthoflavone (BNF) exposure on both the distribution and dynamics of
CYP1A mRNA and protein in lake trout brain, using quantitative reverse
transcription polymerase chain reaction (Q-RT-PCR), in situ
hybridization and immunocytochemistry. BNF was chosen as the contaminant since
it is a well-known Ah receptor agonist and CYP1A inducer
(Smeets et al., 1999).
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Materials and methods |
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Experiment 1. Time-course Q-RT-PCR for BNF-induced CYP1A mRNA
Chemical exposure
Trout were not fed for 2 days prior to injections. Individuals were
randomly sampled and anesthetized by immersion in buffered 100 ng
l1 tricaine methanesulfonate (MS-222; pH 7.0; Sigma Chemical
Co., St Louis, MO, USA). Anesthetized fish were given an intraperitoneal
injection of either ß-naphthoflavone (BNF; Sigma; 50 mg
kg1 body mass) dissolved in corn oil (10 mg
ml1) or corn oil alone (corn oil was autoclaved and
sonicated prior to administration). Lake trout were divided into 40 liter
experimental aquaria where flow rate (0.5 l min1) and
temperature (12°C) were kept constant. Each aquarium received a group
(N=8) of either BNF-induced individuals or control individuals. One
control and one experimental group of lake trout were sampled prior to
injection (time zero) and after each exposure period of 2 h, 4 h, 8 h, 24 h, 2
days, 4 days, 8 days, 16 days and 32 days.
Tissue collection and storage
After the appropriate exposure, lake trout were given an overdose of
MS-222. Whole brain tissue was excised with a pair of small forceps, submerged
in 0.5 ml RNALater© (Ambion, Austin, TX, USA) and immediately placed on
ice. Samples were then frozen at 80°C for long-term storage.
RNA isolation, quantification and storage
For isolation of total RNA, brain tissue was removed from RNALater©,
placed in 1 ml Trizol Reagent (Invitrogen Life Technologies, Carlsbad, CA,
USA) and homogenized. RNA pellets were reconstituted in varying amounts of
diethylpyrocarbonate-treated water (DEPC-H2O) dependent upon pellet
size. Genomic DNA was digested by incubation at 37°C with 1 µl
RNase-free DNase I (Roche Molecular Biochemicals, Mannheim, Germany) and 0.3
µl rRNasin (Promega Corp., Madison, WI, USA) per 100 µl total RNA. DNase
was inactivated by heating samples to 70°C for 10 min. Total RNA was
quantified (Sambrook et al.,
1989) using a GeneQuant pro RNA/DNA calculator (Amersham
Biosciences, Piscataway, NJ, USA). To verify that RNA concentrations and
dilutions were accurate, A260/A280 ratios were produced in triplicate, and a
quality assurance protocol was followed during spectrophotometer usage to
reduce the chances of pipetting error. Following RNA quantification, 3 µl
of each RNA sample was electrophoresed on a 1% ethidium bromide stained
agarose gel to check the integrity and density of 18S and 28S ribosomal RNA
bands (data not shown), increasing our confidence that each reverse
transcription reaction would receive the same amount of total RNA. For
long-term storage, RNA samples were supplemented with three volumes of 95%
ethanol, a 10% volume of 3 mol l1 sodium acetate and placed
at 80°C (Sambrook et al.,
1989
).
RT-PCR
Reverse transcription (all reagents were from Invitrogen Life Technologies)
was performed on all samples in a final volume of 20 µl containing a
1x concentration of First Strand Buffer, 0.01 mol l1
dithiothreitol, 1 mmol l1 of each deoxynucleotide
triphosphate, 2.5 µmol l1 oligo(dT), 5 units of MMLV
reverse transcriptase, 1 unit of rRNasin (Promega Corp.) and 100 ng of total
RNA. The reaction mixture was incubated at 37°C for 50 min and inactivated
at 70°C for 15 min. Then, 1 µl of the cDNA sample was spiked into a PCR
master mix. Each PCR reaction consisted of 12.5 µl of 2x TaqMan®
Universal PCR master mix (Applied Biosystems, Branchburg, NJ, USA), 300 nmol
l1 of each primer (forward WML158 5' CCA ACT TAC CTC
TGC TGG AAG C 3' and reverse WML159 5' GGT GAA CGG CAG GAA GGA
3'), 100 nmol l1 of the TaqMan® probe (WML160
5' TTC ATC CTG GAG ATC TTC CGG CAC TC 3') that contained a
3' TAMRA quencher and a 6-FAM fluorescent label at the 5' end, 1
µl of cDNA template, and deionized water to a final volume of 25 µl.
Reactions were then analyzed on an ABI 7700 real-time PCR thermalcycler
(Applied Biosystems) under the following conditions: 50°C for 2 min,
95°C for 10 min, and 40 cycles of 95°C for 15 s followed by 60°C
for 1 min. Amplification plots were generated, and CYP1A mRNA levels were
estimated against a standard curve.
Recombinant RNA standard and generation of standard curves
A CYP1A recombinant RNA standard was used to generate standard curves in
each set of reactions. The entire 160 samples were analyzed in two plates.
Both standard curves exhibited a correlation coefficient
(r2) of at least 0.995. Due to the high degree of sequence
identity among salmonid CYP1A genes (>95% by comparing CYP1A GenBank
sequences from brook trout, lake trout, Atlantic salmon and rainbow trout),
the cRNA standard was synthesized as follows. A 491 bp conserved region of the
CYP1A gene was amplified from an Atlantic salmon CYP1A clone (GenBank
Accession Number AF361643) using the following primers and conditions: forward
primer WML 169 5' TAA TAC GAC TCA CTA TAG GCT GTC TTG GGC TGT TGT GTA
CCT TGT G 3', reverse primer WML 170 5' TTT TTT TTT TTT TTT TTT
GGA GCA GGA TGG CCA AGA AGA GGT AG 3', conditions of 1 cycle at 94°C
for 4 min, 40 cycles at 94°C for 5 s and 72°C for 2 min, and 1 cycle
at 72°C for 5 min as added extension. The generated PCR product contained
a 5' T7 promoter, 454 bp of CYP1A sequence, including the region of the
real-time amplicon, and a poly dT tail at the 3' end. This product was
then diluted 1/100 with deionized water, re-amplified and up-scaled with the
same reaction conditions. The concentrated PCR product was cleaned using the
QIAquick® PCR Purification Kit (Qiagen, Valencia, CA, USA) and transcribed
using the Riboprobe In Vitro Transcription System (Promega Corp.)
according to standard protocol. The cRNA was then treated with RNase-free
DNase to remove excess DNA template and was subsequently extracted with
water-saturated (pH 4.9) phenol:chloroform (24:1). The aqueous phase was
isolated and extracted with chloroform:isoamyl alcohol (24:1) followed by an
overnight ethanol precipitation at 20°C. To remove free
nucleotides, the precipitated sample was spun for 10 min at 12 000
g, resuspended in 20 µl DEPC-H2O and filtered
through a NucAwayTM Spin Column (Ambion). The size and quality of the
cRNA standard was verified by analysis on an agarose gel and quantified at 260
nm using a spectrophotometer. This RNA standard was then used to generate
standard curves useful for the real-time Q-RT-PCR analysis of CYP1A (C. B.
Rees, J. E. Hinck, D. E. Tillitt and W. Li, manuscript submitted).
In order to quantify CYP1A levels, each plate of samples was normalized against a set of standard curve reactions. To generate standard curves, RT-PCR was carried out on a dilution series (1010103 molecules) of the CYP1A cRNA molecule. Amplification plots were analyzed on the ABI 7700, and Ct values for each of the reactions in the dilution series were calculated. Ct values were plotted against starting quantity of RNA template to generate the standard curve. Additional control reactions were also run on each plate including a no template (water) negative control, a no amplification (RNA) control, a negative reverse-transcription (water added) control and a CYP1A positive control by adding 1 µl of a plasmid containing a full-length CYP1A cDNA sequenced from lake trout (GenBank Accession Number AF539415) to the PCR reaction mixture.
Statistical analysis
All data were log-transformed to fulfill normality requirements and
analyzed using a two-way analysis of variance (Statistical Analysis Systems,
Cary, NC, USA). Simple effects were determined for each factor using the SLICE
procedure (Statistical Analysis Systems v. 8). All pairwise comparisons were
tested for significance using a TukeyKramer adjustment (Statistical
Analysis Systems v. 8).
Experiment 2. In situ hybridization for BNF-induced CYP1A mRNA in the brain
After a 4-day BNF induction (as described in experiment 1), 12 (6 control
and 6 induced) juvenile lake trout were anesthetized with 0.05% MS-222 and
perfused with 20 mlsaline and decapitated to excise their brains. The tissues
were fixed in 4% paraformaldehyde [in 0.1 mol l1 phosphate
buffer saline (PBS)] for 3 h. Following cryoprotection in 0.1 mol
l1 PBS (with 25% sucrose and 4% paraformaldehyde) overnight
at room temperature, the tissues were embedded in Tissue Tek O.C.T. compound
(Sakura Finetek, Torrance, CA, USA) and stored in a 80°C freezer.
The brain was sectioned into 20 µm slices using a Leica CM1850 cryostat,
adhered to Superfrost Plus microslides (Fisher, Orangeburg, NY, USA) and
stored at 80°C.
Synthesis of digoxingenin-labeled cRNA probes
The digoxingenin-labeled antisense RNA probe (500 bp)was generated from
lake trout CYP1A full-length cDNA clone using the Riboprobe In vitro
Transcription Systems (Promega). In brief, 2 µg of linearized vectors were
transcribed in the presence of 700 nmol digoxigenin-11-UTP. The cRNA was
collected by ethanol precipitation and resuspended in DEPC-H2O. The
sense RNA was prepared with a similar procedure and used as the negative
control.
Hybridization
Tissue sections were brought to room temperature, treated with proteinase K
(20 µg ml1 in PBS) for 5 min and post fixed for 15 min in
4% paraformaldehyde (in 0.1 mol l1 PBS). Sections were
rinsed three times for 10 min each in PBS before a 2 h incubation in
prehybridization solution, containing 50% deionized formamide, 1x
Denhart's solution, 750 mmol l1 sodium chloride, 25 mmol
l1 ethylenediaminetetraacetic acid (EDTA), 25 mmol
l1 piperazine-N,N'-bis-2-ethanesulfonic acid
(PIPES), 0.25 mg ml1 calf thymus DNA, 0.25 mg
ml1 poly A acid and 0.2% sodium dodecyl sulfate (SDS).
Sections were then hybridized with antisense or sense RNA probes in
hybridization solution (prehybridization solution with 5% dextran sulfate) at
60°C for 1620 h. After hybridization, sections were washed three
times for 10 min each in 2x SSC [containing 0.3% polyoxyethylenesorbitan
monolaurate (Tween-20)] followed by three washes in 0.2x SSC (containing
0.3% Tween-20) at 65°C.
Immunovisualisation of digoxigenin
For detection of digoxigenin-labeled probes, the sections were blocked for
1 h in 4% dry milk, 2% bovine albumin and 0.3% triton. The sections were
incubated for 3 h with alkaline phosphatase-conjugated sheep-anti-digoxigenin
Fab fragments (1:1000 in blocking solution; Boehringer Mannheim, Indianapolis,
IN, USA) followed by nitroblue tetrazolium chloride and 5-bromo-4-chloro-3
indolyl phosphate substrate (NBT/BCIP; Boehringer Mannheim) for 2030
min, and counterstained with Nuclear Fast Red (Vector Laboratories,
Burlingame, CA, USA).
Experiment 3. Immunocytochemistry for BNF-induced CYP1A protein in the brain
Some sections from experiment 2 were selected for immunocytochemistry
study. Sections were washed in Tris buffer saline (TBS: 50 mmol
l1 Tris buffer, 150 mmol l1 NaCl, pH 7.2)
three times (5 min each) in between each step. All the procedures followed the
manufacturer's instruction. Unless otherwise specified, all the reactions were
performed at room temperature. Sections were reacted with 0.01%
H2O2 (DAB substrate kit; Vector) for 10 min to eliminate
the endogenous peroxidase activity, followed by avidin- and then
biotin-blocking solutions for 10 min each (AvidinBiotin Blocking Kit;
Vector) to eliminate endogenous biotin. Sections were incubated at 4°C
overnight in the primary antibody solution (1:200 rabbit-anti-rainbow trout
CYP1A, CP-226; Cayman Chemical, Ann Arbor, MI, USA) in TBS with 0.05% Triton
X-100 and normal goat serum (Vectastain ABC kit for rabbit IgG; Vector).
Sections were reacted with the biotinylated secondary antibody
(goat-anti-rabbit, Vectastain ABC kit; Vector) for 2 h, incubated in ABC
solution (Vectastain ABC kit; Vector) for 2 h, reacted with
3,3'-diaminobenzidine and NiCl2 (DAB substrate kit; Vector)
for 15 min, counterstained with hematoxylin (Sigma) for 5 min, dehydrated
through an ethanol series (70%, 95%, 100%; 2 min each), clarified twice by
xylene (5 min and 10 min) and covered with glass using DPX mounting media
(Sigma).
To examine the specificity of the antibody used for immunocytochemistry,
western blot was performed. The brain and liver tissues of non-treated and
BNF-treated lake trout were homogenized separately in 200 µl ice-cold 10
mmol l1 Tris buffer (pH 7.4 containing 25 µg
ml1 leupeptin, 5 µg ml1 aprotonin, 40
µg ml1 phenylmethylsulfonyl fluoride, 50 µg
ml1 benzamidine and 0.5 µg ml1
pepstatin) at 0°C. Protein concentration was determined using a DCA
protein analysis kit (Pierce, Rockford, IL, USA). 25 µg of protein were
then applied to 10% acrylamide/N,N'-methylene-bisacrylamide
(29:1 mix; Bio-Rad Laboratories, Hercules, CA, USA) SDS-PAGE at 150 V for45
min (Laemmli, 1970). Gels were
transferred to polyvinylidene difluoride (PVDF) membranes (Immobion-P;
Millipore, Billerica, MA, USA) by electroblotting. The PVDF membranes were
then blocked with 5% (w/v) nonfat dry milk in TBST (20 mmol
l1 Tris-HCl, 150 mmol l1 NaCl, 0.04% Tween
20) overnight. The PVDF membranes were incubated in the primary antibodies
(1:200, Cayman) in 5% nonfat dry milk for 1 h. After washing three times with
TBST, the PVDF membranes were then reacted with goat-anti-rabbit antibody
conjugated with horseradish peroxidase (Pierce) at a 1:10 000 dilution for 1
h. Protein signal was detected by chemiluminescence using the SuperSignal West
Pico Chemiluminescent kit (Pierce).
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Results |
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Experiment 2. Distribution of BNF induced CYP1A mRNA in brain
Fig. 2 shows histological
sections of lake trout midbrain in control fish and in fish subjected to a
4-day BNF exposure. As shown in Fig.
2A, no CYP1A mRNA was detectable in control lake trout midbrain.
After a 4-day exposure to BNF, CYP1A mRNA was highly induced and universally
expressed in the midbrain, mainly in the endothelia and rarely in the glia.
Fig. 2B illustrates a higher
magnification of mRNA-positive glia that were in direct contact or close
vicinity to the blood vessels. No CYP1A mRNA-positive cells showed neuronal
morphology in the samples examined. In other brain regions, no detectable
CYP1A mRNA was found in the control fish whereas BNF-induced CYP1A mRNA
expression was evenly distributed throughout the whole brain (data not
shown).
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Experiment 3. BNF induced CYP1A immunoreactivity in specific brain regions
Fig. 3 illustrates
constitutive expression of CYP1A proteins in glial cells. CYP1A immunoreactive
glial cells course along and directly contact the blood vessels in the control
juvenile lake trout.
|
Figs 4, 5 demonstrate that BNF induces CYP1A immunoreactivity in selected regions of lake trout brain. As shown in Fig. 4, CYP1A immunoreactivity was clearly induced in the olfactory bulb and the valvula of the cerebellum, a folded structure tucked in the ventricle within the optic tectum. BNF appeared to increase CYP1A immunoreactivity in endothelia, glia, neurons and the nerve fibers in the olfactory bulb (Fig. 4). In some fish, BNF also increased CYP1A immunoreactivity in endothelia, glia and neurons in the valvula of the cerebellum (Fig. 4). However, increased CYP1A immunoreactivity in this brain region showed individual variation. On the contrary, control and BNF-treated juvenile lake trout showed similar constitutive CYP1A immunoreactivity in endothelia, glia and neurons in the tectum mesencephali (part of the midbrain), corpus cerebelli longitudinalis, and torus an accessory cerebelloid structure that lies at the medial edge of the optic tectum in the midbrain (Fig. 5).
|
|
Fig. 6 demonstrates that hemorrhage depresses CYP1A immunoreactivity in the brain. As shown in Fig. 6A, BNF-treated fish with hemorrhages contained depressed CYP1A immunoreactivity in the brain compared with the control and BNF-treated fish without hemorrhages. Fig. 6B indicates that CYP1A immunoreactivity-depressed brain regions contained sporadic hemorrhage sites.
|
Western blot analysis showed that CYP1A antisera specifically identified a protein at 65 kDa and revealed a robust increase of this protein in the liver tissues of BNF-exposed fish (data not shown).
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Discussion |
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CYP1A protein was constantly produced at a low level in the brain and was only induced in specific brain regions such as the olfactory bulb and the valvula of the cerebellum. It is advantageous to have highly inducible CYP1A proteins in the olfactory bulb and the valvula of the cerebellum since both regions are readily accessed by xenobiotic compounds, as described below.
The olfactory bulb serves the first protection line in the brain for
xenobiotics. The peripheral olfactory system is readily exposed to a wide
variety of xenobiotic compounds since the olfactory receptor neurons
(via their apical dendrites) are in direct and continuous contact
with the external environment. In addition, olfactory receptor neurons
innervating the olfactory bulbs provide direct access to the central nervous
system for certain toxicants (Gottofrey
and Tjalve, 1991; Hastings and
Evans, 1991
). Therefore, CYP protein in the olfactory bulbs should
be constitutively expressed and also highly inducible in response to any acute
xenobiotic exposure. Indeed, the presence of constitutive CYP1A1 in fish
olfactory systems has been confirmed
(Andersson and Goksøyr,
1994
; Monod et al.,
1994
,
1995
;
Ortiz-Delgado et al., 2002
),
and CYP1A1 immunoreactivities and enzyme activities are induced in the
olfactory organs in fish exposed to xenobiotics
(Goksøyr and Förlin,
1992
; Smolowitz et al.,
1992
).
The valvula (small folding doors) of the cerebellum in the lake trout is a
folded structure tucked in the ventricle within the optic tectum
(Butler and Hodos, 1996). The
function of the valvula in the lake trout is not known, but in weak electric
fish such as mormyrids it plays a major role in electroreception
(Butler and Hodos, 1996
). Since
the whole valvula is immersed in the ventricle, it is more readily exposed to
xenobiotics that can penetrate the bloodbrain barrier and enter the
cerebrospinal fluid. Clearly, it is advantageous to have inducible CYP1A
protein in this brain area to detoxify xenobiotics.
Constitutive CYP1A immunoreactivity in endothelia, glia and neurons was
observed in juvenile lake trout brain, which was consistent with the
immunohistochemical studies by Smolowitz et al.
(1991) and Stegeman et al.
(1991
) in that CYP1A proteins
were localized at vascular endothelia of the fish brain. Other studies in fish
also provided the evidence that cerebral CYP1A immunoreactivity was not
restricted to the endothelia but was also localized in neuronal tissue
(Reinecke and Segner, 1998
;
Sarasquete et al., 1999
).
Since our results indicate that most of the glial cells that showed CYP1A
immunoreactivity were attached to the blood vessels, it is likely that these
glial cells can absorb xenobiotic chemicals from the blood vessels and they
constitutively produce CYP1A proteins to serve a protective function by
eliminating xenobiotics from the central nervous system.
It is surprising that some BNF-treated juvenile lake trout showed depressed
CYP1A immunoreactivity in the brain compared with the control fish. In these
fish, we found sporadic hemorrhage sites. It is known that BNF mimics the
effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in altering
local brain circulation (Dong et al.,
2002). TCDD has been shown to cause multifocal hemorrhages in
zebrafish and lake trout embryos (Andreasen
et al., 2002
; Spitsbergen et
al., 1991
; Toomey et al.,
2001
). It is evident that 4-day BNF treatment caused multifocal
hemorrhages in some juvenile lake trout; however, these pathological effects
varied among individuals.
Studies in other species also indicated that hemorrhage may depress
BNF-induced CYP1A immunoreactivity. Local hemorrhages in the brain trigger the
inflammatory response (Perry et al.,
1993; Rothwell et al.,
1996
) in which glial cells, specifically astrocytes and microglia,
become activated and stimulate the acute phase immuno-response, which
downregulates microsomal CYP protein level in cultured cells, humans and other
animals (Nicholson and Renton,
2001
; Paton and Renton,
1998
; Renton et al.,
1999
; Renton and Nicholson,
2000
; Shimamoto et al.,
1998
; Stanley et al.,
1991
). Most cytokines that are found to decrease basal CYP
production can counteract Ah receptor-mediated increase of CYP1A
protein and its associated EROD activity. Our discovery of depressed CYP1A
immunoreactivity in the BNF-treated fish with hemorrhages was consistent with
the CYP1A immunodepression in the inflammatory response.
BNF differentially induced CYP1A mRNA and protein in juvenile lake trout
brain. CYP1A mRNA was not constantly produced in the brain. Once CYP1A mRNAs
were induced, they distributed universally throughout the endothelia of the
whole brain. Occasionally, induced CYP1A mRNAs were found in the glial cells.
On the contrary, CYP1A protein was constantly produced at a very low level in
endothelia, glia and neurons in lake trout brain. Only specific brain regions
showed increased CYP1A immunoreactivity, and the increase was not as robust as
that of the CYP1A mRNA. This may be due to the immunological privilege of the
brain (Perry et al., 1993;
Rothwell et al., 1996
). The
mechanism to depress CYP1A protein apparently requires protein synthesis since
treatment with cycloheximide in combination with Ah-receptor agonist
led to superinduction of CYP1A mRNA
(Abdel-Razzak et al., 1994
). It
is likely that CYP1A protein level is more tightly regulated in the brain than
is CYP1A mRNA. Therefore, BNF can induce CYP1A mRNA universally in the brain
endothelia whereas CYP1A protein only increased in specific cells and in
specific brain regions. On the contrary, in other tissues such as livers, a
transient CYP1A mRNA induction could be followed by a prolonged induction of
CYP1A protein level (Kloepper and Stegeman,
1992
,
1994
).
To summarize, BNF differentially induces CYP1A mRNA and protein expression in juvenile lake trout brain. The induction of CYP1A mRNA is universally distributed throughout the endothelia of the whole brain while the increase of CYP1A protein is less robust and area specific. BNF may induce hemorrhage in some individuals and may cause the immunodepression of CYP1A protein in the brain.
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Acknowledgments |
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