1 Department of Medical Oncology, City of Hope Medical Center, and 2 Department of Biology, City of Hope, Beckman Research Institute, Duarte, CA 91010
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ABSTRACT |
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Gpx1 knockout (KO) mice had a higher number of regenerating
crypts in the jejunum than did Gpx2-KO or wild-type mice
analyzed 4 days after 10 Gy
-irradiation. Without
-irradiation,
glutathione peroxidase (GPX) activity in the jejunal and ileal
epithelium of Gpx1-KO mice was <10 and ~35%,
respectively, of that of the wild-type mice. Four days after exposure
to 11 Gy, GPX activity in wild-type and Gpx1-KO ileum was
doubled and tripled, respectively. However, jejunal GPX activity was
not changed. Thus the lack of GPX activity in the jejunum is associated
with better regeneration of crypt epithelium after radiation.
Gpx2 gene expression was solely responsible for the increase
in GPX activity in the ileum, since radiation did not alter GPX
activity in Gpx2-KO mice. The intestinal Gpx2
mRNA levels of Gpx1-KO and wild-type mice increased up to
14- and 7-fold after radiation, respectively. Although the Gpx1-KO jejunum had higher levels of PGE2 than
the wild-type jejunum after exposure to 0 or 15 Gy, these differences
were not statistically significant. Thus whether GPX inhibits PG
biosynthesis in vivo remains to be established. We can conclude that
the Gpx2 gene compensates for the lack of Gpx1
gene expression in the ileal epithelium. This may have abolished the
protective effect in Gpx1-KO mice against the radiation
damage in the ileum.
Gpx2 gene induction; microcolony survival assay; Gpx2-knockout mice; antioxidant protein 2; gene compensation
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INTRODUCTION |
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THE PREVAILING THEORY on the mechanisms of radiation-induced cell death identifies DNA single- and double-strand breaks as the direct damaging effect. Such damage is produced from direct interaction of the DNA with ionizing radiation or with the hydroxyl free radicals generated from radiolysis of water and other molecules. The cells with unrepaired DNA damage will die of necrosis or apoptosis (14). Although generation of H2O2 is an alternative route for hydroxyl free radical generation (42), there is little evidence that elevated Se-dependent glutathione peroxidase (GPX) activity protects cells from radiation damage (9, 25, 32, 35, 36).
The intestinal crypt epithelial cells are very sensitive to abdominal and pelvic radiation therapy. The actively dividing transitory epithelial cells are the most sensitive to radiation-induced injury. The damage in the mucosal epithelium can result in a variety of symptoms, including diarrhea, electrolyte imbalance, and sepsis. The survival of slowly proliferating crypt stem cells appears to play a central role in mucosal regeneration following injury (8, 31). Several mitogens enhance the survival of intestinal epithelial cells after radiation injury. These include keratinocyte growth factor (KGF), stem cell factor (SCF), and prostaglandins (PGs) (8, 11, 22).
KGF is a newly recognized member of the heparin-binding fibroblast growth factor family. Short-term pretreatment with KGF, and to a lesser extent SCF, from 2 h to 3 days before irradiation provides significant protection of the crypts (11, 22). The exact mechanism for the KGF or SCF enhancement of crypt survival is not clear. KGF is a potent inducer for a human analog of the antioxidant protein 2 (Aop2), which has a weak Se-independent GPX activity (13, 18, 27, 28, 37). Aop2 has no homology with the Se-dependent GPXs. Instead, Aop2 has homology with antioxidant protein 1 (Aop1) family, which used to be known as thiol-specific antioxidant protein (18). The human Aop2 is also known as non-Se GPX, 1-Cys peroxiredoxin, and KGF-regulated gene 1 (12, 13, 20, 21, 27). Aop2 appears to be ubiquitous and is highly expressed in the nasal epithelium, skin, and eye tissues including the ciliary body, retina, and iris (37). Whether Aop2 can contribute significantly to total GPX activity in intestinal epithelium and thus affect crypt hydroperoxide metabolism is not known. Therefore, we have included Aop2 in this study.
PGs are important mediators of epithelial integrity and function in the
gastrointestinal tract. Many PGs protect gastric and intestinal mucosa
from damaging agents, including -irradiation (15).
Cyclooxygenases (COXs) catalyze two key steps in PGs biosynthesis: oxygenation and cyclization of arachidonic acid to form
PGG2 and the reduction of the hydroperoxide of
PGG2 to form PGH2. Indomethacin, an inhibitor
of both COX1 and COX2, reduced the number of surviving crypts in the
irradiated mice (8). Although peroxynitrite, the coupling
product of nitric oxide and superoxide anion, activates COX in
activated macrophages in the presence of GPX (24),
hydroperoxides activate COX in vitro, an activity inhibited by GPX
(2, 23, 26). Whether GPX
inhibits PG biosynthesis in vivo has not been established. In this
study, we have investigated the effect of GPX activity on intestinal
crypt survival and PGE2 concentrations after exposure to a
high dose of
-irradiation. An inverse correlation between GPX
activity and either crypt survival or PGE2 concentration would support the inhibitory effect of GPX on PG synthesis in the
intestinal epithelium.
There are two major cellular Se-dependent GPX isozymes expressed in the intestinal epithelium: the classic ubiquitous GPX-1 encoded by the Gpx1 gene, and the epithelium-specific GPX-GI encoded by the Gpx2 gene (3, 10). Unlike the rather uniform distribution of GPX-1 in rat small intestine along the cephalocaudal axis, a higher level of Gpx2 gene expression was detected in the distal section of the mouse small intestine or the ileum (6, 10). We have estimated that GPX-1 contributed >80% of total GPX activity in mouse proximal small intestine, and GPX-1 and GPX-GI contributed ~50 and 35% of total GPX activity in the distal small intestine. Since these two isozymes have very similar substrate specificity, this apparent redundant gene expression suggests that the two isozymes may be regulated differently to serve different physiological functions.
In this manuscript, we describe the making of Gpx2 knockout
mouse lines. The homozygous Gpx2 knockout mice are
apparently normal. These mice with disrupted Gpx1 or
Gpx2 gene expression were analyzed for GPX activity and
crypt regeneration after exposure to -irradiation. We found that the
crypt survival is highly correlated with the lack of GPX activity. Our
result provides the first link between GPX activity and radiation
sensitivity in the intestinal epithelium. This study may provide a
reason for the development of GPX-specific inhibitors to be used as
protective agents on the intestinal epithelium during radiotherapy or
accidental exposure to radiation.
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EXPERIMENTAL PROCEDURES |
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Animals. The generation of mice with disrupted Gpx1 gene expression (Gpx1-KO) has been described previously (17). These homozygous Gpx1-KO mice on a mixed C57BL/6J (B6) and 129/Sv (129) genetic background (B6 × 129) were maintained by inbreeding. The wild-type control mice originated from heterozygous Gpx1-KO parents and were maintained similarly as a mixed B6 × 129 line. Breeding B6 × 129 Gpx1-KO with B6 for six generations generated the second Gpx1-KO subline in a B6 background. Southern blots were used for all genotyping. GPX assays were performed to confirm the phenotypes on the hemolysate from 25 µl of blood after rinsing the red blood cells with PBS. The relative specific activity was estimated as a fixed arbitrary ratio of activity to the A540 of Drabkin's reagent-treated hemolysate (1). Wild-type B6 mice were purchased from Jackson Labs (Bar Harbor, MA) and used as the controls for B6 Gpx1-KO mice.
To isolate the mouse Gpx2 gene without interference from its pseudogene (5), intron primers were used for screening by PCR. Three positively identified P1 clones (nos. 8470, 8471, and 8472) were isolated from a genomic library of 129/Sv embryonic stem (ES) cells (Genome Systems, St. Louis, MO). A Hind III fragment (6.5 kb) isolated from the 8471 P1 clone was subcloned into pBluescript (Stratagene, La Jolla, CA) and sequenced (GenBank accession no. AJ249277). It contained 2 kb of 5' untranscribed region, 0.3 kb of exon 1, 2.2 kb of intron, 0.8 kb of exon 2, and 1.5 kb of 3' untranscribed region. The Gpx2-KO construct was made by substituting the exon 1 with the neo gene using the pPNT vector (40), which also contains a herpes simplex viral thymidine kinase gene cassette as shown in Fig. 1. The linearized pPNT-Gpx2-KO construct was transfected into W9.5 ES cells of the 129S3/SvImJ (129S3) mouse strain according to procedures previously described (39, 41). ES clones resistant to 0.3 mg/ml G418 and 2 µM ganciclovir were analyzed for the evidence of homologous recombination. Two correctly targeted clones, nos. 8 and 21, were injected into (CBA/CaJ-a/a × B6)F1 × B6 blastocysts, and resulting chimeric males were mated to B6 females to obtain germ line transmission of the mutation. The heterozygous Gpx2-KO mice were then intercrossed to generate mice homozygous for the mutation, i.e., Gpx2-KO mice. As controls, wild-type Gpx2+/+ mice from the same intercross were used. Thus all mice studied were of a mixed 129S3 × B6 genetic background. Occasionally, the B6 × 129 wild-type mice generated for the Gpx1-KO controls were used as the controls for the Gpx2-KO mice.
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Whole body irradiation.
All mice were maintained on a 12:12-h light/dark schedule and given
free access to standard lab mouse chow and water. Mice of both genders
at 8-16 wk of age were placed into a ventilated Plexiglas pie
container during exposure to -irradiation. All exposures were done
between 7:15 and 8:15 AM to minimize the potential variations caused by
circadian rhythm (19). The mice were exposed to a
60Co irradiator (Theratron-80 S/N 140; Atomic Energy of
Canada) at 51-56 cGy/min with a source-to-skin distance of ~80
cm. The exposure procedure and all other mouse work were done with the approval of the City of Hope Research Animal Care Committee. At certain
time points from 6 h to 7 days after exposure, mice were euthanatized by halothane (Halocarbon Labs, North Augusta, SC).
Crypt survival assay. Crypt microcolony survival was measured in animals terminated 4 days after irradiation following the described procedure (30, 43). Two 3-cm sections of the jejunum and the ileum starting from 1 cm distal to the pylorus end of stomach and 1 cm proximal to the cecum were excised. They were rinsed and fixed in Bouin's fixative for 2-3 h and, after rinsing 3 times with 50% ethanol, were equilibrated in 70% ethanol overnight. They were cut to 1-cm lengths and then embedded longitudinally in paraffin. They were then sectioned onto slides and stained with hematoxylin and eosin. The number of surviving crypts was scored from cross-sections. Two to four complete cross-sections were scored from each region of mice. Two to four mice exposed to each dose of radiation were analyzed in each experiment.
Assay of GPX activity in intestinal epithelium. The GPX activity was determined on mouse intestinal epithelium. Jejunal and ileal epithelial cells were isolated from two 12-cm sections (total small intestine is ~36 cm long) of the proximal and the distal small intestine (4, 10). Briefly, after the intestinal lumen was rinsed with buffer A containing PBS and 1 mM dithiothreitol, the epithelial cells were eluted three times with buffer B containing 1.5 mM EGTA in buffer A after incubation at 37°C for 10, 20, and 30 min. These isolated cells were pooled and washed 3 times in PBS. Four volumes of homogenate buffer containing 0.15 M Tris, pH 7.2, 0.1 M NaCl, 5 mM EGTA, and a cocktail of protease inhibitors were added to the packed cells. The cells were sonicated by five 1-s bursts with the use of a cell sonicator equipped with a microprobe (Branson Cell Disruptor 200; Branson Sonic Power, Danbury, CT). After centrifugation at 22,000 g for 30 min, GSH was added to an aliquot of the supernatant to 5 mM final concentration for the preservation of GPX-GI activity during freezing and thawing. The GPX activity was measured with 60 µM H2O2 and 3 mM GSH at pH 7.3. The protein concentration was determined with a BCA assay (Pierce Chemical, Rockford, IL) with bovine serum albumin as the standard.
Analysis of Southern and Northern blots.
Genomic DNA was isolated from ES cells and 1 cm of mouse tails
following the described procedure (34). Genotyping of the Gpx1 or Gpx2 genes was done by Southern blot
analysis on the BamH I or Apa I digested genomic
DNA. After resolving 10 µg of DNA per sample on 0.7% agarose gels,
the DNA was denatured with 0.25 M HCl for 10 min and then transferred
onto Zeta Probe blotting membranes (Bio-Rad, Richmond, CA) in 0.4 N
NaOH overnight. Total RNA was isolated from 60 mg (~0.6 cm) of small
intestine. After homogenization by polytron in the lysis buffer, the
total RNA was isolated with the RNeasy kit (Qiagen, Valencia, CA). Ten
micrograms of RNA per sample were resolved in a formaldehyde-denaturing
agarose gel containing 1.3% agarose and then transferred to a Zeta
Probe blotting membrane with 10× standard saline citrate
(34). The hybridization and washing was performed at
62°C in buffers that had high concentrations of SDS, as recommended
by the manufacturer. All presented mRNA levels were normalized against
-actin mRNA levels. Similar results were obtained with normalization
against 28S rRNA, which was also probed initially and later
periodically. Quantification of the Gpx1 and Gpx2
mRNA levels was done with phosphor imaging after 6- to 24-h exposure
(Molecular Dynamics, Sunnyvale, CA).
Measurement of PGE2 and protein levels. The mice backcrossed to B6 for 7-8 generations were used for this study. About 0.12 g of the jejunum (a 4-cm segment from 1 cm distal to the stomach) and 0.1 g of the ileum (a 4-cm segment from 1 cm proximal to cecum) were processed to measure PGE2 concentrations. These two sections of small intestine were rinsed with PBS and then snap frozen in liquid nitrogen. Without thawing, they were homogenized in 1 ml of 100% methanol containing 1 mM indomethacin to inhibit COX activity in a prechilled 10 ml cylinder with a polytron. The homogenization buffer was also prechilled on dry ice. After centrifugation at 10,000 g for 15 min, 0.5 ml of supernatant was added to 10 ml of H2O, pH 3. A trace amount of 3H-PGE2 (~10,000 dpm) with a high specific activity was added to monitor for the yield through organic extraction. Ten milliliters of diluted supernatant was passed through an activated C-18 Sep-Pak cartridge (Waters, Milford, MA) for solid-phase extraction. The column was washed with 5 ml H2O followed by 5 ml hexane. Prostaglandins were eluted in 5 ml of ethyl acetate containing 1% methanol. Half a milliliter was counted for radioactivity, and another 0.1 ml was lyophilized and resuspended in 0.1-0.5 ml of an enzyme immunometric assay buffer supplied by the manufacturer (Cayman Chemical, Ann Arbor, MI). Duplicate PGE2 assays were performed on an aliquot of each sample with enzyme immunoassay.
Protein concentrations in the clarified methanol extract were determined with BCA assay (Pierce). Bovine serum albumin was used as a standard. The interference of indomethacin was accounted for by assaying the homogenization buffer. The value obtained from the homogenization buffer was subtracted from all sample values. We estimated that 0.5 µg of protein had been extracted from 1 mg of wet tissue.Statistical analysis. All statistical analysis (ANOVA) was done with a two-tailed Student's t-test using Microsoft Excel 97. A P value of <0.05 was considered significant.
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RESULTS |
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Generation of Gpx2-KO mice. The target for Gpx2 gene disruption was the 300 bp of exon 1, which contained the UGA codon for the selenocysteine amino acid residue (Fig. 1A). A 2-kb DNA sequence 5' to the translation start site and 2.2 kb of mouse Gpx2 intron were generated by PCR and then inserted 5' and 3' to a 1.8-kb neo gene cassette and flanking sequence in pPNT vector. The 5' end of the neo cassette had an Apa I recognition site. Thus, after homologous recombination, Apa I digestion resulted in a 4.9-kb fragment from the disrupted Gpx2 gene and a 7-kb fragment from the wild-type Gpx2 gene when probed with the exon 2 probe (Fig. 1B). The ~14 kb of Apa I fragment appeared to contain the Gpx2 pseudogene (5). Lanes 1, 2, and 3 of the Southern blot in Fig. 1B contain mouse DNA isolated from a wild-type, a heterozygous Gpx2-KO, and a homozygous Gpx2-KO mouse, respectively. Expression of the Gpx2 mRNA in each type of mouse is shown in Fig. 1C. No Gpx2 mRNA was detectable in homozygous Gpx2-KO mice when the Gpx2 exon 1 was used as the probe. A Gpx2 mRNA of higher molecular weight was detected in the Gpx2-KO mice when the Gpx2 exon 2 was used as the probe.
Two ES-transfectant cell lines were used to create two independent mouse knockout lines, designated as nos. 8 and 21. No difference can be detected between these two lines based on Southern blot, Northern blot, and activity assays (data not shown). Therefore, we have combined the results obtained from these two lines.Crypt survival.
Whether Gpx1 and Gpx2 gene expression could have
any effect on the survival of crypt epithelium after exposure to
-irradiation was analyzed in the jejunum and the ileum (Fig.
2). The number of cryptlike foci of the
surviving epithelial cells was scored on the cross-sections of
intestine 4 days after exposure. Each epithelial focus represents
survival of one or more clonogenic stem cells able to regenerate a
crypt. Figure 2 shows the jejunum of B6 × 129 Gpx1-KO
and wild-type mice after exposure to 0, 15, 17, and 20 Gy. Although
-irradiation destroyed most of the epithelial cells,
Gpx1-KO mice had more surviving stem cells proliferating to
form regenerative foci than Gpx2-KO mice and wild-type mice (Fig. 3).
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Elevation of GPX activity.
To determine whether the crypt survival correlated with GPX activity,
we analyzed the GSH-dependent H2O2-reducing
activity in the jejunum and ileum. As shown in Fig.
4, the change in GPX activity was
monitored during the first 7 days after exposure to 11 Gy. Kinetics of
activity change were studied at 11 Gy since this is at the threshold of
crypt sterilization. The jejunal epithelium of Gpx1-KO mice
had very low GPX activity, in contrast to the high level of GPX
activity in the wild-type mice throughout the 7 days. On the contrary,
the ileal epithelium of Gpx1-KO mice had ~35% of the GPX
activity in the wild-type mice before exposure to -irradiation. Four
days after irradiation, GPX activity in the ileal epithelium of
Gpx1-KO and wild-type mice had quadrupled and doubled,
respectively. The ileal GPX activity in the exposed Gpx1-KO
mice was higher than that in the unexposed wild-type mice. One each of
B6 and B6 × 129 Gpx1-KO mice and two B6 × 129 wild-type mice were assayed at each time point as shown in Fig. 4.
Since we did not find any difference in GPX activity between these two lines of Gpx1-KO mice, we have combined the data.
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Elevation of the Gpx2 mRNA levels after -irradiation.
To determine whether the increase in GPX activity correlated with the
increase of the Gpx2 mRNA level, but not the Gpx1
mRNA level, the intestinal RNA was analyzed on Northern blots.
Total RNA was isolated from the middle small intestine (distal jejunum) of wild-type mice between 0 and 6 days after exposure to 11 Gy. As
shown in Fig. 6, the Gpx2 mRNA
levels in wild-type mice were tripled 3-4 days after exposure,
whereas the Gpx1 mRNA levels did not change significantly.
The Aop2 mRNA level was doubled after irradiation, although
the relative abundance of the Aop2 mRNA was 10-fold lower
than the Gpx2 mRNA. The Gpx2 and Aop2
mRNA were maintained at the elevated levels afterwards. The kinetics of
Gpx2 mRNA level change in the Gpx1-KO small
intestine was also analyzed (Fig. 7).
Three days after exposure to 11 Gy, the Gpx2 mRNA levels
increased 15-fold and the Aop2 mRNA level increased 2-fold.
Unlike the wild-type mice, the increase of Gpx2 and
Aop2 mRNA levels in the Gpx1-KO mice was
transient. The mRNA levels returned to near basal levels 6 days after
exposure.
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Effect of Gpx1 gene expression on PGE2 levels in the
small intestine.
To determine whether Gpx1 gene expression affects PG
biosynthesis in the small intestine, we have measured PGE2
concentrations in jejunum and ileum after exposure to 15 Gy. As shown
in Fig. 10, B6 Gpx1-KO
jejunum had a higher level of PGE2 than the wild-type jejunum after exposure to 0 and 15 Gy. The basal PGE2
concentration in the Gpx1-KO jejunum was 21.0 ± 8.5 vs. 15.5 ± 10 pg PGE2/µg protein in the wild-type
jejunum. The induced PGE2 concentration in the
Gpx1-KO jejunum was 215 ± 76 pg PGE2/µg
protein after exposure to 15 Gy compared with 146 ± 41 pg
PGE2/µg protein in the wild-type jejunum. The
Gpx1-KO ileum also had a higher basal level of
PGE2 (38 ± 9 pg PGE2/µg protein) than
the wild-type ileum (20 ± 10.5 pg PGE2/µg protein).
However, the irradiated Gpx1-KO ileum had the same level of
PGE2 (101 ± 57 pg PGE2/µg protein) as
the exposed wild-type ileum (114 ± 64 pg PGE2/ µg
protein). Nevertheless, none of the differences are statistically
significant. Only the increase of PGE2 level by
-irradiation in both the jejunum and ileum of the Gpx1-KO
and the wild-type mice is statistically significant.
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DISCUSSION |
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We have previously shown that both GPX-1 and GPX-GI are the major
GPX activities in the intestinal epithelium of mouse and rat. The mouse
jejunal and ileal epithelia have different levels of GPX-1 and GPX-GI
activity (6, 10). To study their function in
the crypt epithelium after exposure to -irradiation, we have generated Gpx2-KO mice. Similar to the Gpx1-KO
mice, the Gpx2-KO mice have lower levels of GPX activity in
the gastrointestinal tract than the wild-type mice, and there is no
unusual phenotype in the Gpx2-KO mice under normal
conditions (17). GPX-1 contributes to 80% and 50% of
total GPX activity in the jejunum and the ileum, respectively. Four
days after exposure to high-dose
-irradiation, the jejunum of both
B6 and B6 × 129 Gpx1-KO mice has higher numbers of
surviving crypt foci than the wild-type controls. The ileum of the
B6 × 129 but not B6 Gpx1-KO mice is also more
resistant to radiation. Thus radiation resistance can be associated
with lack of GPX activity. The levels of resistance found in the
Gpx1-KO mice are comparable to the protection afforded by
KGF and PG mimetics (9-11).
It was previously reported that radiation injury decreases the number
of surviving crypts in the jejunum. Indomethacin, an inhibitor of COX,
further decreases the number of crypt foci. The indomethacin response
can be blocked by dimethyl-PGE2 (8). In vitro
studies have shown that GPX activity inhibits activation of COX, the
rate-limiting enzyme for PGE2 biosynthesis (2, 23). Radiation increases the PGE2
concentration ninefold in the jejunum of the Gpx1-KO and
wild-type mice and about threefold in the ileum of the
Gpx1-KO and wild-type mice. With the exception of the
irradiated ileum, the Gpx1-KO small intestine has higher levels of PGE2 than wild-type small intestine does. Perhaps
because of the large variation of PGE2 levels in samples,
the differences are not statistically significant. Thus we cannot make
any conclusions about the in vivo effect of GPX activity on the
PGE2 level. Our PGE2 measurements are
comparable to the reported data in FVB/N mice (8). This
suggests that the PGE2 level is similarly regulated by
-irradiation in these two strains of mice.
Although the Gpx2 mRNA was expressed in the jejunal epithelium of the wild-type and Gpx1-KO mice, it did not result in a significant increase of GPX activity, probably due to the lack of GPX-GI production. The juncture between the regions of the intestine showing activity increases was between 33 and 50% of the length of the intestine from the duodenum (data not shown). The lack of GPX-GI activity in the jejunum may be due to the fact that GPX-GI is ultrasensitive to cellular redox status. GPX-1 is already sensitive to oxidative damage (16, 29, 44), and we have found that GPX-GI is more labile than GPX-1 (4). Thus the lack of GPX activity in the jejunum of the Gpx1-KO mice may be due to the rapid inactivation of GPX-GI in this tissue in the absence of GPX-1. In the wild-type mouse jejunum, the high level of GPX-1 activity would have masked the increase of GPX-GI activity.
Contrary to jejunal Gpx2 gene expression, the increase of
Gpx2 mRNA level in the ileal epithelium does result in an
increase of GPX activity. After exposure to 10 Gy, the GPX activity
level in Gpx1-KO ileum is higher than that found in the
unexposed wild-type mice. This provides the first evidence that the
Gpx2 gene can compensate for the lack of Gpx1
gene expression. Unlike the Gpx1 gene, of which mRNA
level is constitutively expressed in most tissues, the Gpx2
mRNA level is highly regulated. In addition to this effect of
radiation, all-trans retinoic acid can increase Gpx2 mRNA level 10-fold, as shown in MCF-7 breast cancer
cells (7). Since
-irradiation of MCF-7 cells did not
increase Gpx2 mRNA levels (data not shown), and maximal
levels of Gpx2 mRNA in small intestine occurred at 3-4
days postirradiation, this suggests that the increase in mRNA levels is
not a direct response to radiation. Rather, it is a response to some
component of secondary injury. The kinetics of Gpx2 mRNA
changes is similar to the changes observed with Cox1 in the
intestine after exposed to high-dose
-irradiation (8).
This suggests that the Gpx2 and the Cox1 genes
are coregulated in this exposure.
Although we have found that most GPX activity in the intestinal
epithelium is contributed by GPX-1 and GPX-GI under normal conditions,
the recent identification of Aop2 has raised the question of its
possible contribution to GPX activity after exposure to -irradiation. The Aop2 gene is normally expressed at a
low level in the intestinal epithelium but is highly expressed in the
eye (13, 27, 37). It has been
shown in keratinocytes that the Aop2 gene is highly
inducible by KGF, which can also increase crypt survival. We can rule
out any significant contribution of Aop2 to total GPX activity in the
intestinal epithelium on the basis of the following observations:
1) Aop2 mRNA level was increased only onefold
after exposure to high-dose
-irradiation; 2) Aop2 has low
GPX activity, and 3) no increase in GPX activity occurred in
Gpx2-KO mice after exposure to radiation.
Our study elucidates a function for Gpx1 gene expression in
the intestinal epithelial cells after exposure to -irradiation. The
fact that the Gpx1-KO and the wild-type ileum have similar sensitivity to
-irradiation is most likely a result of the
compensatory effect of Gpx2 gene expression. To study the
function of the Gpx2 gene in the ileal epithelium against
high-dose radiation, the crypt survival in Gpx1 and
Gpx2 double knockout mice should be analyzed in the future.
Considering the beneficial effect of voiding GPX activity in the crypt epithelium against radiation damage, it may be highly desirable to develop GPX-specific inhibitors as therapeutic reagents. Gold (I)-containing compounds, including aurothioglucose and the antiarthritic drug auranofin, are potent inhibitors for several selenocysteine-containing enzymes (33, 38). Unfortunately, aurothioglucose is a much more potent inhibitor for thioredoxin reductase and less effective on GPX-1 when administered intraperitoneally. To our knowledge, there are no GPX-specific inhibitors reported.
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ACKNOWLEDGEMENTS |
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We thank Heather Adams for maintaining mouse colonies, Helen Sun for processing mouse tissue samples for histological analysis, Sabine Werner at the Swiss Federal Institute of Technology for providing the mouse Aop2 cDNA clone, and Jason D. Morrow at Vanderbilt University for advice on the PGE2 assay.
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FOOTNOTES |
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This work was supported by American Heart Association Grant 9960042Y (F.-F. Chu). PhosphorImager was funded by National Science Foundation Grant BIR-9220534.
Address for reprint requests and other correspondence: F.-F. Chu, Dept. of Medical Oncology, City of Hope Medical Center, 1500 E. Duarte Road, Duarte, CA 91010-3000 (E-mail: fchu{at}coh.org).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 15 November 1999; accepted in final form 8 March 2000.
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