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Alteration of glutathione S-transferase levels in Barrett’smetaplasia compared to normal oesophageal epitheliumSarah C. Cobbe, Gillian C. Scobie, Elizabeth Pohler, John D. Hayes,Neil M. Kernohan and John F. Dillon
Objective Oesophageal cancer associated with the
premalignant condition Barrett’s oesophagus has
increased in incidence over the last few years. Phase II
detoxifying enzymes, including glutathione S-transferases
(GSTs) protect the mucosa from carcinogens, which can
cause oxidative damage to cells. Therefore, a reduction in
these anti-oxidant enzymes can increase the risk of
carcinogenesis. The aim of this study was to compare the
extent of GST expression in normal oesophageal tissue,
Barrett’s oesophagus and oesophageal adenocarcinoma.
Design Antibodies raised against GST alpha, GST mu,
GST pi and microsomal GST were used to identify
expression of these proteins in tissue sections.
Method Paraffin-embedded sections were stained using
standard immunohistochemical techniques to
demonstrate the pattern of expression of GST proteins in
biopsy specimens. Twelve sections of Barrett’s metaplasia
and an equal number of specimens from normal
oesophageal tissue were examined, together with sections
from adenocarcinoma and normal gastric mucosa.
Results Expression of the GST enzymes appeared to be
reduced in Barrett’s tissue compared to normal
oesophageal tissue. Nuclear staining featured in some of
the normal tissue sections, but not in Barrett’s tissue.
Conclusion The reduction in GST expression suggested in
Barrett’s tissue is an interesting finding, as it is possible
that reduced expression of these detoxifying enzymes may
contribute to the risk of development of adenocarcinoma
in Barrett’s mucosa. Eur J Gastroenterol Hepatol 15:41–47
& 2003 Lippincott Williams & Wilkins
European Journal of Gastroenterology & Hepatology 2003, 15:41–47
Keywords: oesophagus, glutathione S-transferase, cancer, Barrett’soesophagus
Department of Molecular and Cellular Pathology, Biomedical Research Centre,University of Dundee, Dundee, UK.
Correspondence and reprint requests to Dr John Dillon, Ward 5 & 6, NinewellsHospital and Medical School, Dundee DD1 9SY, UK.Tel: +44 (0)1382 660 111; fax: +44 (0)1382 425 504;e-mail: [email protected]
Received 6 February 2002 Revised 2 August 2002Accepted 19 August 2002
IntroductionThroughout the world, the incidence of oesophageal
carcinoma has dramatically escalated over the past few
decades. In Scotland, the incidence of carcinoma has
increased by 2% per year in women and by 3% per year
in men over a 30-year period [1,2]. Epidemiological
data from the USA, Scandinavia, France, Switzerland,
Australia and New Zealand demonstrate that this is an
international trend [3–11].
The increase in incidence of oesophageal cancer has
coincided with an alteration in the most common
histological type of tumour found and the usual location
of these tumours within the oesophagus [12,13]. The
prevalence of oesophageal adenocarcinoma has now
surpassed that of squamous carcinoma in Western
Europe and the USA. In particular, cases of adenocarci-
noma associated with the premalignant condition
Barrett’s oesophagus have increased. The factors re-
sponsible for this alteration in geographical distribution
and increase in incidence are as yet unidentified.
Genetic susceptibility plays a role; however, this factor
alone cannot sufficiently explain the extent of the
increase in oesophageal tumours [14]. The relatively
short time period during which this increase has oc-
curred suggests the aetiology is likely to be environ-
mental.
Cells are protected against exposure to harmful environ-
mental xenobiotics by phase II drug detoxification
reactions. Such enzyme reactions increase the polarity
of xenobiotics, reducing their toxicity and promoting
subsequent excretion in the urine or bile. The suscept-
ibility of cells to oxidative damage by toxic chemicals is
increased if these phase II enzymes are not expressed
[15], and damage to key genetic material is a vital step
in the carcinogenic process.
The glutathione S-transferase (GST) isoenzymes are
phase II detoxifying enzymes, which were initially
described in 1961. They are dimeric proteins that
catalyse the conjugation of electrophilic chemicals with
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Original article 41
0954-691X & 2003 Lippincott Williams & Wilkins
reduced glutathione [16,17]. Since their discovery, sub-
stantial interest in the role of these enzymes in cancer
prevention has arisen. Two GST superfamilies exist
that comprise either cytosolic or membrane-bound
proteins. Evidence suggests that cytosolic GST in-
creases bodily resistance to carcinogens [18], products
of oxidative stress, environmental pollutants and certain
anti-cancer drugs [19]. In humans, these cytosolic
isoenzymes have been allocated into eight classes: GST
alpha (GSTA), mu (GSTM), pi (GSTP), sigma, theta
(GSTT), kappa, zet and omega [20–23].
Low levels of GST correlate with increased sensitivity
of cells to damage by noxious substances, including
carcinogens [24]. This is demonstrated by the fact that
people lacking the GSTM1 gene (50% of the popu-
lation), and those lacking GSTT1 (16% of the popu-
lation), have an increased susceptibility to tumours of
the bladder, colon, skin and lung, especially if they are
heavy smokers [25,26]. Certain tumour types character-
istically over- or under-express GST [27–31]. GSTP1 is
notably increased in tumours of the colon, lung and
stomach and this rise often occurs during the pre-
neoplastic stage [30]. Therefore, GST levels may be
considered as surrogate markers for certain pre-neo-
plastic lesions; however, due to the ubiquitous distribu-
tion and variation in levels of GST, it is only useful in
clear cut cases such as the one described here.
In this study, we investigated the pattern of GST
expression in normal oesophageal tissue, Barrett’s tissue
and adenocarcinoma of the oesophagus to detect
whether these enzymes may be involved in the patho-
genic process. We selected a panel of four GST anti-
bodies: three for the cytosolic classes alpha, mu and pi,
and the fourth for a microsomal transferase. These
particular GSTs were selected because their expression
in other tumour types has already been investigated, as
mentioned above. Therefore, we felt it would be of
value to establish their behaviour within the oesopha-
gus. Tissue sections were stained using standard im-
munohistochemical techniques.
Materials and methodsMaterials
Chemicals, unless otherwise stated, were obtained from
AnalaR BDH chemicals (Poole, UK) or National Diag-
nostics (Atlanta, Georgia, USA). The primary antibodies
used were raised against GSTA1/2, GSTP1, GSTM1
and microsomal GST (MGST-1) [32]. Paraffin blocks
containing tissue samples were selected from the
pathology archive, Ninewells Hospital and Medical
School, Dundee. Section types used were from normal
oesophagus (n ¼ 8), Barrett’s oesophagus (n ¼ 11), oeso-
phageal adenocarcinoma (n ¼ 8), normal gastric tissue
(n ¼ 8), gastric carcinomas (n ¼ 2) and normal duode-
num (n ¼ 1).
Immunohistochemistry
Chosen tissue sections embedded in paraffin wax were
sliced at a thickness of 4 �m using a microtome (Leica
RM 2135, Leica, Nussloch, Germany). The sections
were resolubilized to allow the antibody access to the
antigens, and endogenous peroxidases were blocked for
10 min. Heat and citrate buffer were used to reduce
molecular bonding within the tissue.
The primary antibodies used were rabbit polyclonals
raised against the different GST subclasses and diluted
in phosphate-buffered saline (PBS) (Table 1). Normal
goat serum was applied prior to the antibody to reduce
non-specific binding.
The secondary antibody was anti-rabbit immuno-
globulin G (IgG) (Biogenex, San Ramon, California,
USA), which recognizes the rabbit primary polyclonal.
The secondary antibody system involved the formation
of a complex between a biotin molecule conjugated to
the secondary antibody and streptavidin, both diluted
1 : 25 in PBS.
To stain the sections, 3,39diaminobenzidine (DAB)
tetra hydrochloride (Sigma, Poole, Dorset, UK) (0.5 �g/
ml in PBS) was applied with 30% v/v hydrogen
peroxide (H2O2), followed by copper sulphate and
haematoxylin. The slides were then dehydrated,
mounted with distrene 80 dibutyl phthalate xylene and
examined microscopically.
Western blot analysis was used to demonstrate that the
GSTs were expressed in gastrointestinal tissue, and to
confirm the specificity of the immune cross-reactivity.
Rat liver and human stomach were probed by each
antibody, and bands correlating with the molecular
weight of GST were identified (Fig. 1). A second
isoform was identified when probing for GSTA1/2, and
degradation products were found with GSTM1 and
MGST-1. However, this should not have affected the
immunohistochemical techniques used in this study
because they were used only to define the cellular
localization of proteins. The disruptive nature of the
western blotting technique, during which cell lysis
occurs, can often result in degradation products.
ResultsNormal oesophagus
Positive superficial staining and weak basal staining was
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Table 1 Dilution of glutathione S-transferase antibodies
Antibody Dilution
GSTA1/2 1:500GSTM1 1:100GSTP1 1:50MGST-1 1:100
42 European Journal of Gastroenterology & Hepatology 2003, Vol 15 No 1
observed with all the GST antibodies in the normal
oesophagus (Figs 2,3). The pattern of staining was
analogous for GSTP1 and GSTA1, suggesting that they
operate in similar areas of the oesophageal mucosa.
Nuclear staining was detected in a few of the normal
oesophageal sections for these two enzymes. However,
no particular pattern was identified in the distribution
and number of cells, with nuclear staining appearing to
be random (Fig. 2).
The GSTM1 gene is only present in 50% of the
general population [25]. Among the normal oesophageal
sections stained (n ¼ 7), one section was negative for
GSTM1 staining and one was equivocal. The other
normal oesophageal sections stained with GSTM1
showed weakly positive cytosolic staining which was
more superficially pronounced. Some nuclear staining
was witnessed, though not as consistently as with
GSTA1 and GSTP1.
Intense staining for MGST-1 was observed in the
superficial layers of the mucosa, as with the other
GSTs, in normal oesophageal tissue. No nuclear stain-
ing was identified.
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Fig. 1
Western blot demonstrating the presence of the glutathione S-transferases (GSTs) GSTP1, GSTA1, GSTM1 and MGST-1 in human gastric bodyand rat liver control. The normal molecular weight of GST is 29.8 kDa.
Fig. 2
Normal oesophagus stained with GSTA1 (310 objective). Cytoplasmicstaining is observed near the surface of the epithelium. Scatteredpositive nuclei are also present.
Fig. 3
Normal oesophagus stained with GSTM1 (310 objective).Cytoplasmic staining is evident throughout the epithelium, althoughnuclear immunoreactivity is not represented in this field.
GSTs in Barrett’s oesophagus Cobbe et al. 43
Barrett’s oesophagus
In general, staining with all the GSTs in Barrett’s
oesophagus was very weak or negative (Fig. 4). Faint
staining was, however, present in the very superficial
layers of some of the slides. The superficial differen-
tiated cells stained weakly in the cytoplasm, but to a
lesser degree than in normal oesophagus. Staining was
weaker proportional to the depth of the gland layer.
Intestinal metaplasia showed almost negative staining
in the crypts (Fig. 5). There was no nuclear staining
present in any of the Barrett’s tissue sections as
opposed to normal oesophagus.
Staining was decreased or mostly negative for both
GSTA1 and GSTP1, particularly in the Barrett’s intest-
inal metaplasia. This was also observed, though not so
convincingly, in the gastric metaplasia.
Of the 11 Barrett’s sections examined, six appeared to
express GSTM1, four were negative and one section
was equivocal. Staining, as with GSTA1 and GSTP1,
was more pronounced in the basal layers. Interestingly,
in one section, GSTM1 was expressed in the normal
squamous tissue on the slide; however, this was lost on
the Barrett’s tissue (Fig. 6).
The pattern of staining in Barrett’s oesophagus with
MGST-1 did not noticeably differ from that in normal
oesophageal tissue.
Oesophageal adenocarcinoma
With all the GST antibodies, staining of the tumour
cells was found to be heterogeneous, and we could
ascertain no consistent pattern (Fig. 7).
Normal gastric cells
In general, the gastric foveolar cells stained weakly,
especially with GSTP1. The specialized cells were
positive, as was noted with all the antibodies in the
selected panel.
Gastric carcinoma
With the intestinal-type carcinoma, all the GSTs stained
positively (Fig. 8). However, the antral diffuse-type
carcinoma stained noticeably less than the intestinal-
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Fig. 5
Barrett’s oesophagus stained with GSTP1 (320 objective). Intestinal-type epithelium with Barrett’s mucosa shows very low levels ofimmunoreactivity in the crypt epithelium, although weak cytoplasmicstaining persists in the gastric foveolar-type epithelium.
Fig. 4
Barrett’s oesophagus stained with GSTP1 (310 objective). Themetaplastic epithelium shows a markedly reduced level of staining withGSTP1 in comparison to the squamous epithelium. No nuclear stainingis present.
Fig. 6
Normal oesophageal mucosa positively staining for GSTM1, withnegative glandular epithelium in the same field.
44 European Journal of Gastroenterology & Hepatology 2003, Vol 15 No 1
type carcinoma with all of the antibodies tested. Some
areas were found to be completely negative (Fig. 9).
Normal duodenum
A section of normal duodenum was stained to see
whether the pattern of staining in the intestinal meta-
plasia resembled that of the normal intestinal mucosa.
Strongly positive staining was found with all the GSTs,
in the specialized cells as expected but also in the
epithelium.
DiscussionThe purpose of this study was to investigate whether
the GST family are involved in the tumourigenic
process from normal oesophagus to Barrett’s oesopha-
gus, and finally to adenocarcinoma. The general trend
witnessed suggests that metaplastic tissue may have
lower levels of GST compared to normal oesophagus.
Since the GSTs are protective detoxifying enzymes,
this decrease in expression could facilitate the expres-
sion of adenocarcinoma on a background of Barrett’s
metaplasia.
The normal tissue sections studied were extracted from
regions surrounding oesophageal lesions, which were
likely to have been exposed to noxious compounds.
Therefore, the strongly positive superficial staining ob-
served may be a protective mechanism induced by the
chemical stress to which oesophageal cells are often
directly exposed.
The GSTP1 antibody demonstrated the most convin-
cing reduction in staining in Barrett’s tissue. GSTP1 is
a widely distributed member of the cytosolic GST
family, and increased levels of this enzyme have been
implicated as a tumour marker in other cancers, such as
colon, skin and lung [30]. However, in this study, it
appears that a lack of GSTP1 in premalignant tissue
may allow progression to malignancy.
A number of sections of normal and Barrett’s oesopha-
gus did not stain for GSTM1, which would be expected
since only 50% of the population have the gene to
encode this protein [25]. This finding supports our
confidence in the specificity of the antibodies. Interest-
ingly, in one slide containing normal and Barrett’s
tissue adjacent to one another, staining for the mu
antibody was present in the former but was lost in the
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Fig. 7
A poorly differentiated intestinal-type adenocarcinoma of theoesophagus (320 objective). Patchy staining is seen with GSTP1.
Fig. 8
Intestinal-type gastric carcinoma stained with GSTA1 (320 objective).Although stained with a different antibody to that used in Figure 6, thisimage is representative and demonstrates that intestinal-typecarcinomas express higher levels of GST enzymes than the diffusesubtypes of gastric adenocarcinoma.
Fig. 9
Diffuse-type gastric carcinoma stained with GSTM1 (320 objective).Signet ring cells show reduced levels of staining with the GST incomparison to intestinal-type adenocarcinoma.
GSTs in Barrett’s oesophagus Cobbe et al. 45
latter. The loss of expression of GSTM1 suggests that
this may be a factor in the metaplastic process, though
this is speculative as it was only observed on one slide.
Normal gastric and duodenal sections were studied to
detect any similarities in GST expression between
these tissues and Barrett’s gastric and intestinal meta-
plasia. Gastric tissue and gastric metaplasia showed
similar weak staining; however, normal duodenal sec-
tions were markedly positive, unlike the pale staining
of the intestinal metaplasia. This result is in accordance
with the fact that intestinal-type metaplasia is more
prone to undergoing malignant change.
The expression of GSTs in oesophageal adenocarcioma
is complex, with variable changes identified for each
subclass. Furthermore, within individual tumours, het-
erogeneity of staining with each individual antibody
was noted. A larger study is necessary to reconcile these
changes in protein expression with the development of
oesophageal adenocarcinoma. This was initially thought
to contradict the theory that decreased levels of these
detoxifying enzymes allowed the progression of the
malignant process. However, it is well known that
tumours often express proteins in a dysregulated and
heterogeneous manner, which may explain the increase
in GST in the neoplastic stage.
Previous literature generally validates the pattern of
staining we identified in normal and Barrett’s tissue.
Therefore, despite being too small to provide definite
conclusions on the subject, this study highlights possi-
ble mechanisms that could explain the pathological
process occurring in Barrett’s metaplasia. There has
been a limited number of studies of phase II enzyme
levels in Barrett’s oesophagus, thus the present study is
in a relatively new field of research. Investigations
performed by van Lieshout et al. in the Netherlands in
1999 demonstrated low levels of GST activity in human
Barrett’s epithelia as opposed to normal oesophagus
[33]. They studied Barrett’s tissue samples retrieved
from human oesophageal endoscopies and compared
them to normal tissue. Their study is comparable with
the present one as both have utilized human samples
from endoscopic biopsy and used sensitive immuno-
logical techniques to identify the induction of enzymes.
van Lieshout et al. identified a 35% reduction in GST
enzyme activity in Barrett’s tissue compared to the
normal oesophagus. They also studied the isoenzymes
present by densometrical analysis following western
blotting and reported GSTP and GSTM to be de-
creased by 30% and 24% respectively in Barrett’s
tissue. These results suggest a decrease similar to the
one that we witnessed using immunohistochemistry.
However, van Lieshout et al. claimed that GSTA was
considerably raised, by 625%, in Barrett’s tissue com-
pared to normal tissue. Conversely, we found that, like
GSTP1, GSTA1/2 was decreased in Barrett’s tissue.
This disparity could be explained by differences in the
antibodies used, rather than the actual quantity of
protein detected.
Compton et al. also reported a similar decrease in
GSTP expression in Barrett’s tissue using immuno-
histochemistry [34]. In addition, with northern blot
analysis they observed reduced GSTP1 mRNA levels
in Barrett’s tissue compared to either normal oesopha-
gus or adenocarcinoma samples. This is relevant to our
study as we also observed a decrease in Barrett’s tissue,
coupled with a further increase in adenocarcinoma
tissue.
However, in a further study, van Lieshout et al. used
immunohistochemistry to investigate the expression of
GSTA1 and GSTP1 in normal oesophagus, Barrett’s
oesophagus and adenocarcinoma [35]. In contrast to
their previous study, they described an increase in
GSTP1 expression between normal and Barrett’s oeso-
phagus as opposed to a decrease. They also claimed
that GSTA1 was not present in normal oesophagus, but
was markedly increased in Barrett’s, then reduced again
in adenocarcinoma. Differences in the technique and
antibodies used probably account for these discrepan-
cies, which further experimentation is required to
resolve.
Nuclear staining with GSTs has been previously
reported in the oesophagus and in other tissues such as
the uterus and skin [35–37]. The nuclear staining we
observed in normal oesophagus is possibly a defensive
reaction to the threat of carcinogens or reflux of acid or
bile in the oesophagus. Potential reasons for GSTs to
enter the nucleus are to protect the DNA or to act as
transcription factors by triggering gene expression.
Before this hypothesis can be proved, a more substan-
tial panel of normal tissue samples extracted from the
area around a lesion should be compared with normal
tissue from volunteers without oesophageal pathology.
The fact that nuclear staining was witnessed in a small
number of normal sections suggests that the transmis-
sion of the GST into the nucleus is part of a stress
response, and its presence depends on which challenges
the cells were exposed to prior to being collected. It is
possible that prolonged and multiple episodes of acid
stress may desensitize the phase II response, though
this theory requires investigation.
One of the drawbacks of using immunohistochemistry
is that it is a semi-quantitative technique, therefore
levels of staining give only an impression of the amount
of protein present as opposed to an exact figure. The
positive staining seen in the immunohistochemical
procedure was authenticated by negative control slides
without visible brown staining. This proved that the
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
46 European Journal of Gastroenterology & Hepatology 2003, Vol 15 No 1
level of background staining seen in the slides was
minimal; however, the problem with using polyclonal
antibodies is that they can undergo nonspecific binding.
The use of pre-immune sera from the rabbit in which
the antibodies were raised would have helped distin-
guish further nonspecific staining, but this was not
available in our study. Thus, the results witnessed may
not be entirely specific, although the technique is
highly sensitive. Visual observations are not quantita-
tive and can give no absolute evidence. However,
overall impressions can be drawn from the alterations in
patterns and obvious changes in staining intensity.
In the future, further research is required, using a
substantially larger number of sections for immuno-
histochemistry. However, immunohistochemistry has
definite limitations, and therefore more sophisticated
techniques using fresh tissue samples would yield
results of higher quality. This would also produce a
more accurate reflection of the tumourigenic process invivo.
AcknowledgementsS.C.C. is very grateful to all the staff of the Gastro-
enterology Laboratory in Ninewells Hospital for allow-
ing her to work there, and for all their useful advice on
experimental techniques.
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GSTs in Barrett’s oesophagus Cobbe et al. 47