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*3
* Department of Laboratory Medicine and Pathology,
Department of Surgery, and
** Department of Cell Biology and Neuroanatomy, University of Minnesota, Minneapolis, MN 55455-0385
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • genistein • enterocytes • Caco-2 • HT-29 • bacteria
|
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,
Lee et al. 1991
, Severson et al. 1989
).
The rationale for this is caused by the lower risk of breast, colon,
and prostate cancers observed in epidemiological studies of people from
Asia, where per capita soy consumption is 20–50 times greater than
that of people in the United States (Barnes 1995
,
Lee et al. 1991
, Severson et al. 1989
).
Diets enriched in soy protein have had beneficial effects in animal
models of carcinoma, including mammary, liver, colon, prostate, skin,
stomach, and bladder carcinomas; there is substantial evidence that
genistein is the primary anticancer agent in soy protein, and in recent
years, many investigators have switched to using purified genistein in
experimental studies (Barnes 1995
). Genistein also
inhibits the proliferation of a variety of human tumor cell lines in
culture (Barnes 1995
), and the role of genistein in
cancer treatment and prevention is an active area of research.
In an attempt to identify the mechanism by which genistein exerts its
inhibitory effect on tumor cells, Peterson (1995)
summarized a variety of cell functions that might be attributable to
genistein. Genistein inhibits angiogenesis, acts as a weak estrogen,
and functions as an antioxidant. Genistein inhibits the activity of
topoisomerase II, thus stabilizing the DNA-topoisomerase II complex and
facilitating double- and single-stand breaks in DNA, precipitating
growth inhibition or cell death. Genistein is often used in research
related to signal transduction because genistein markedly inhibits
protein tyrosine kinase activity, especially through the epidermal
growth factor receptor. It thus interferes with a variety of functions
that are necessary for cell growth and differentiation, such as protein
phosphorylation, enzyme activation, and second messenger generation and
transcription of immediate early genes in the nucleus. Although all of
these mechanisms can be deleterious to tumor cells, Peterson carefully
noted that, in tumor cells, the majority of these mechanisms are not
sensitive to physiological serum concentrations of genistein, estimated
to be <18.5 µmol/L (<5 µg/mL).
In addition to its potential role as an anti-tumor agent, there is
evidence that genistein may play a role in the prevention and/or
treatment of heart disease. There are numerous reports that dietary soy
protein is associated with lowered serum cholesterol in both humans and
experimental animals (Nagata et al. 1998
, Potter 1995
). In addition, genistein has been reported to inhibit
thrombin formation and platelet activation in vitro, suggesting that
genistein may modify the coagulation process and thus affect the
progression of cardiovascular disease (Wilcox and Blumenthal 1995
). However, data from a relatively small but well-designed
study involving 20 normocholesterolemic men indicated that prolonged
(28 d) administration of 60 soy protein/d elevated plasma genistein
with no noticeable alteration in the concentration of plasma
cholesterol or in platelet aggregation (Gooderham et al. 1996
). Thus, the potential usefulness of genistein as a
therapeutic or prophylactic agent in heart disease is unresolved, but
remains another active area of research.
Kops et al. (1997)
recently provided evidence for yet
another potential benefit of dietary genistein. Hydrolyzed soybean
isolates, developed for use in enteral nutrition products, were noted
to affect development of intercellular tight junctions in a colonic
adenocarcinoma cell line (namely C2BBe cells, a subclone of Caco-2
enterocytes), enhance the production of enterocyte brush border
enzymes, and inhibit the paracellular (between enterocytes) passage of
Salmonella typhi (but not S. typhimurium). It was
suggested that genistein might be used to enhance intestinal epithelial
barrier function and inhibit extraintestinal invasion of pathogenic
enteric bacteria.
Our laboratory is investigating the interactions of enteric bacteria
with cultured intestinal epithelial cells, namely Caco-2 and HT-29
enterocytes. Caco-2 and HT-29 cells have many features of
differentiated enterocytes, including apical microvilli, tight
junctions, structural polarity, ion conductance, and enzyme expression
(Huet et al. 1987
, Neutra and Louvard 1989
, Pinto et al. 1983
, Rousset 1986
). Caco-2 cells are spontaneously differentiated.
Differentiation of HT-29 cells can be modulated by culture conditions.
When grown in the presence of glucose, HT-29 cells are relatively
undifferentiated, but when grown without glucose, HT-29 cells
differentiate into polarized absorptive enterocytes and mucus-secreting
goblet cells. Both Caco-2 and HT-29 cells were used to study enterocyte
interactions with a wide variety of bacterial species, and in vitro
interactions were shown to have in vivo relevance (Finlay and Falkow 1997
, Pucciarelli and Finlay 1994
). In
studies designed to test the effects of various signal transduction
agents on bacteria-enterocyte interactions, we noted that genistein
appeared to enhance intestinal epithelial barrier integrity of Caco-2
enterocytes, as well as inhibit bacterial internalization by Caco-2 and
HT-29 enterocytes. Herein we report that genistein appeared to
strengthen the integrity of Caco-2 enterocyte tight junctions, as
measured by transepithelial electrical resistance
(TEER),4
and that genistein exhibited a dose-dependent inhibitory effect on the
ability of both Caco-2 and HT-29 enterocytes to internalize several
species of enteric bacteria.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Salmonella typhimurium ATCC 14028, Listeria
monocytogenes ATCC 43249 serotype 1/2a, were obtained from the
American Type Culture Collection (Rockville, MD). Proteus
mirabilis M13, Escherichia coli M21, and E.
coli M14 are rodent isolates. These species of enteric bacteria
represent a spectrum of virulence, and these strains were used in
several in vivo studies of oral infectivity in rodents (Wells et al. 1987 and 1993
) as
well as in vitro studies of bacterial internalization by cultured
enterocytes (Wells et al. 1994, 1995, 1996a, 1996b, 1998
).
Cultured enterocytes.
Caco-2 and HT-29 cells were obtained from the American Type Culture
Collection and were cultivated in the absence of antibiotics according
to previously described procedures (Wells et al. 1994, 1995, 1996a, 1996b, 1998
). Briefly,
Caco-2 cells were cultivated in Dulbecco's Modified Eagle's Medium
supplemented with 15% fetal bovine serum and 4 mmol L-glutamine/L.
HT-29 cells were cultivated according to Huet et al. (1987)
in glucose-free Dulbecco's modified Eagle's medium
supplemented with 15% dialyzed fetal bovine serum, 4 mmol
L-glutamine/L, and 5 mmol galactose/L. All tissue culture reagents were
obtained from Sigma Chemical (St. Louis, MO). Caco-2 and HT-29 cells
were grown in 24-well plastic dishes. Caco-2 and HT-29 enterocytes were
seeded at 2 x 104 cells per well (2 cm2)
and incubated at 37°C in 9.5% CO2. These Caco-2 and
HT-29 cultures become confluent after 5–6 d. Caco-2 and HT-29
enterocytes were used after 15–18 d and after 21–24 d, respectively,
when these enterocytes were considered polarized and differentiated
(Huet et al. 1987
, Neutra and Louvard 1989
, Pinto et al. 1983
, Rousset 1986
). Mature Caco-2 and HT-29 cultures, containing
~106 enterocytes per well, were 
95% viable as
determined by the vital dyes trypan blue (0.36%) and propidium iodide
(20 mg/L). Caco-2 cells were used between passages nine and 64. HT-29
cells were used between passages 31 and 38, following adaptation to the
galactose-supplemented medium. Using transmission electron microscopy,
we have previously shown that these mature Caco-2 and HT-29 enterocytes
have characteristics of polarized cells, i.e., well-developed apical
microvilli, distinct apical and basolateral domains, and tight
junctions joining adjacent enterocytes (Wells et al. 1993, 1994, 1996a
).
Bacterial internalization by cultured enterocytes.
Enterocyte internalization of viable bacteria was assayed as described
(Wells et al. 1994, 1995, 1996a, 1996b, 1998
) with minor
modifications. Individual bacterial strains cultured overnight in
tryptic soy broth (Difco Laboratories, Detroit, MI) were washed twice
and diluted in the appropriate enterocyte tissue culture medium.
Maintaining a multiplicity of infection of 100 (bacteria to enterocyte
ratio), 1 mL containing 108 viable bacteria was added to
each tissue culture well containing 106 confluent
enterocytes. Bacterial concentrations were determined by densitometry,
and confirmed by serial dilution followed by viable plate counts on
appropriate agar media. Bacteria were incubated with enterocytes for
1 h at 37°C. Enterocytes were then washed five times with Hanks
Balanced Salt Solution (HBSS), and tissue culture medium containing 50
mg gentamicin sulfate/L was added to kill residual viable extracellular
bacteria. After 2.5 h, epithelial cells were washed five times
with HBSS and lysed for 5 min with 1% Triton-X-100. Viable
intracellular bacteria were quantified following serial dilution and
incubation on appropriate agar media. Agar media consistently included
both colistin-nalidixic acid agar (Difco) supplemented with 5% sheep
blood and MacConkey agar supplemented with 10% lactose (Difco) to
verify the purity and concentration of the bacterial inocula, to verify
the absence of extracellular bacteria after incubation with gentamicin,
and to quantify the numbers of intracellular bacteria while verifying
the absence of bacterial contamination after epithelial cell lysis.
Bacterial adherence to cultured enterocytes.
Polyclonal antiserum to individual bacterial species was raised in
individual New Zealand White rabbits (Birchwood Valley Farms, Redwing,
MN) using standard methodology as previously described (Wells and Erlandsen 1991
). Preimmune rabbit antisera contained no
demonstrable antibodies to these bacteria as measured by indirect
immunofluorescence with affinity-purified, fluorescein-conjugated goat
anti-rabbit immunoglobulin G (Organoteknika-Cappel, West Chester, PA)
as the detector; optimal dilutions of primary rabbit antisera were
determined using similar methodology. To assay bacterial adherence to
cultured enterocytes, bacterial inocula were prepared as described
above, and 108 bacteria (pure culture) were incubated with
106 confluent enterocytes. After 1 h at 37°C,
enterocytes were washed five times with HBSS and bacterial adherence
was assayed by a modification of ELISA as described by Ofek et al. (1986)
. Washed enterocytes were lightly fixed for 5 min
with 0.05% paraformaldehyde, washed three times using HBSS with 5%
normal goat serum, then incubated with the appropriate primary rabbit
antiserum diluted 1:1000 or 1:2000 in HBSS with 5% goat serum. After
1 h at 37°C, enterocytes were again washed three times using
HBSS with 5% goat serum, and then incubated for 30 min at 37°C with
affinity purified horse radish peroxidase-conjugated goat anti-rabbit
IgG (whole molecule; Sigma). Enterocytes were washed three times with
HBSS, the blue color developed with 200 µL
3,3',5,5'-tetramethybenzidine-hydrogen peroxide color development
reagent (Sigma) for 30 min at room temperature, the reaction stopped
with 100 µL of 0.32 mol sulfuric acid/L, and the optical density read
at 450 nm in an ELISA plate reader (Molecular Devices, Sunnyvale, CA).
Control wells contained enterocytes that were not incubated with
bacteria, but were treated with all other reagents.
Effect of genistein on bacteria-enterocyte interactions.
To study the effects of 4',5,7-trihydroxyisoflavone (genistein; Sigma)
on bacterial internalization and bacterial adherence, enterocytes were
pretreated with various concentrations of genistein for 1 h prior
to addition of bacteria. Genistein was present throughout the bacterial
internalization and bacterial adherence assays. Stock solutions of 300
mmol genistein/L were maintained in dimethyl sulfoxide at -20°C and
diluted in appropriate tissue culture medium for use in experiments.
Preliminary experiments showed that pertinent concentrations of
dimethyl sulfoxide had no noticeable effect on bacterial
internalization and that pertinent concentrations of genistein had no
effect on bacterial viability. Genistein was used in concentrations
from 30 to 300 µmol/L because these concentrations are similar to
those used by others to study the effect of genistein on bacterial
internalization by cultured epithelial cells (Akeda et al. 1997
, Benjamin et al. 1995
, Bermudez and Young 1994
, Evans et al. 1998
, Rosenshine et al. 1992 and 1994
, Sandros et al. 1996
, Tang et al. 1998
).
Transepithelial electrical resistance and filamentous actin.
Transepithelial electrical resistance can be used to monitor changes in
epithelial cell culture integrity that was presumably caused by
loosening of the tight junctions (Hildago et al. 1989
).
TEER of Caco-2 and HT-29 enterocytes were studied with the Millicell
Electrical Resistance System (Millipore, Bedford, MA) using enterocytes
cultivated 15–18 d and 21–24 d, respectively, on Falcon 0.45 µm
cyclopore membranes with 0.3 cm2 surface area (Becton
Dickinson, Lincoln Park, NJ). All electrical resistance readings were
recorded after subtracting the average resistance of two membranes in
the absence of enterocytes, i.e., membranes equilibrated overnight in
tissue culture medium. Because TEER values often vary among individual
enterocyte cultures, the electrical resistance value was recorded for
each membrane before and after experimental treatment, and the
percentage decrease from baseline was calculated for each membrane.
Positive controls included incubation of enterocytes in the presence of
calcium-free medium and in the presence of 1 mg/L cytochalasin D
(Sigma), as previously described (Wells et al. 1995 and 1998
).
The distribution of filamentous actin in the enterocyte cytoskeleton
was observed using the methodology of Howard and Meyer (1984)
with minor modifications. Enterocyte cultures were
incubated at 37°C for 30 min with 0.8 µmol fluorescein-labeled
phalloidin/L (Sigma) suspended in 5% buffered formalin containing
0.1 g lysophosphatidyl choline/L (Sigma), then washed three times
with HBSS, mounted in phosphate buffered saline:glycerin (one part:nine
parts) containing 1 g/L p-phenylenediamine (Sigma) at pH 8, and viewed
by epifluorescent microscopy.
Statistical analysis.
To quantify bacterial internalization by enterocytes, each bacterial
strain was tested in at least three separate assays, performed on
different days, each assay representing the average of triplicate
tissue culture wells. Bacterial numbers were converted to
log10 prior to statistical analysis. The lower limit of
assay detection was 50 bacteria or 1.7 log10; values below
this limit were assigned a value of 1.7. Differences in numbers of
internalized bacteria and differences in TEER measurements (percentage
change from baseline) were analyzed by one-way ANOVA followed by
Fisher's test for significant differences. The correlation coefficient
(r) was used to assess linear relationships between the
concentration of genistein and the numbers of internalized bacteria as
well as the percentage change in TEER. For an individual bacterial
strain, differences in adherence between control and genistein-treated
enterocytes were analyzed by paired t-test. All statistical
analyses were performed with StatView 4.5 (Abacus Concepts, Berkeley,
CA), and P 
0.05 was considered significant.
|
RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The effect of various concentrations of genistein on Caco-2
internalization of L. monocytogenes, S. typhimurium, P.
mirabilis, E. coli M21, and E. coli M14 is presented in
Fig. 1.
Enterocytes pretreated with 300 µmol/L genistein internalized
significantly fewer L. monocytogenes, S. typhimurium, E.
coli M21, and E. coli M14 than enterocytes not
pretreated with genistein. The effect of genistein on bacterial
internalization appeared dose dependent, as noted by r
values of 0.65–0.79 (P < 0.01) for the effect of
increasing concentrations of genistein on Caco-2 internalization of
L. monocytogenes, S. typhimurium, E. coli M21, and E.
coli M14 (Fig. 1)
.
|
).Although HT-29 cells generally were less permissive for bacterial
uptake (Fig. 2)
compared to Caco-2 cells (Fig. 1)
, the pattern of
bacterial internalization was similar. Enterocytes pretreated with 300
µmol/L genistein internalized significantly fewer L.
monocytogenes, S. typhimurium, and P. mirabilis than
enterocytes not pretreated with genistein. In addition, the
relationship between the concentration of genistein and the numbers of
internalized L. monocytogenes, S. typhimurium, and P.
mirabilis appeared dose dependent with correlation coefficients
(r) of 0.6 (P 0.01) for each of these
three bacterial strains. It should be noted that the two E.
coli strains were relatively noninvasive in HT-29 cells, with
internalization approaching the lower limit of assay detection (1.7
log10), thus prohibiting interpretation of the effect of
genistein on bacterial internalization.
|
95% viable, similar to the viability of untreated
enterocytes. Effect of genistein on bacterial adherence to Caco-2 and HT-29 enterocytes.
To determine if the inhibitory effect of genistein on bacterial
internalization was related to inhibition of bacterial adherence,
enterocytes were pretreated with 300 µmol/L genistein, and bacterial
adherence was assayed by ELISA. Using this method, absolute numbers of
adherent bacteria are not determined; adherence is measured as optical
density units, permitting assessment of relative adherence only. In
addition, because the optical density is dependent on the intensity of
the color reaction, which is dependent on antibody affinity,
comparisons between bacterial strains are also not appropriate. The
effect of enterocyte pretreatment with 300 µmol/L genistein on
adherence of L. monocytogenes, S. typhimurium, P. mirabilis,
and E. coli M21 to Caco-2 and HT-29 enterocyes is presented
in Fig. 3.
Genistein pretreatment was associated with greater adherence of
L. monocytogenes on HT-29 enterocytes and with greater
adherence of S. typhimurium on both HT-29 and Caco-2
enterocytes; for all other pair-wise combinations, genistein had no
significant effect (Fig. 3)
. Thus, although pretreatment of enterocytes
with 300 µmol/L genistein was generally associated with lower
bacterial internalization (Figs. 1 and 2)
, this decreased
internalization was not associated with decreased bacterial adherence
(Fig. 3)
.
|
Incubation of HT-29 enterocytes for 1 h with 300 µmol/L genistein had no noticeable effect on TEER (data not shown) or on the distribution of filamentous actin, as observed by reaction with fluorescein-labeled phalloidin (not shown). However, 300 µmol/L genistein did have a noticeable effect on these measurements using Caco-2 enterocytes.
The TEER of Caco-2 cultures incubated with 0, 30, 100, and 300 µmol/L
genistein, 1 mg/L cytochalasin D, and calcium-free medium is shown in
Fig. 4.
As expected (Wells et al. 1995 and 1998
), 1-h incubation in calcium-free medium or in
cytochalasin D-supplemented medium was marked by decreased Caco-2 TEER.
In contrast, incubation of Caco-2 enterocytes with 0, 30, 100, or 300
µmol/L genistein increased TEER with a strong, positive dose response
(r = 0.9). The mechanism responsible for the slight
decrease in TEER following addition of 0 µmol/L genistein is unclear,
but is likely because of a transient alteration in pH associated with
exposure of enterocytes to fresh tissue culture medium.
|
represents typical observations of control and treated Caco-2
enterocytes, based on observations of over 100 microscopic fields in
duplicate enterocyte cultures.
|
and shows the expected decrease in TEER. As shown in Fig. 6
B, 1-h incubation of either 108 S.
typhimurium or E. coli M21 with Caco-2 enterocytes also
resulted in the expected decrease in TEER, but 1-h preincubation with
300 µmol/L genistein abrogated this decrease in TEER, and there was a
noticeable increase in TEER following 1-h incubation of bacteria with
genistein-treated Caco-2 enterocytes with either S.
typhimurium or E. coli M21.
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|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Rosenshine et al. (1992, 1994)
noted that pretreatment
of either HeLa cervical epithelial cells, Henle-407 enterocytes, or
A431 cells (an epidermal cell line that overexpresses the epidermal
growth factor receptor) with up to 250 µmol/L genistein had no effect
on bacterial adherence or on epithelial invasion of S.
typhimurium, but genistein inhibited the internalization of a
genetically manipulated E. coli carrying a plasmid-encoded
inv gene (invasion gene) from Yersinia
enterocolitica. Sandros et al. (1996)
noted that
500 µmol/L genistein was associated with decreased internalization of
Porphyromonas gingivalis by KB oral epithelium. Tang et al. (1998)
observed decreased uptake of L.
monocytogenes using HeLa cells pretreated with 250 µmol/L
genistein, and Benjamin et al. (1995)
reported that 50
µmol/L genistein inhibited HeLa cell internalization of an
enteroaggregative strain of E. coli. Invasion of corneal
epithelial cells by Pseudomonas aeruginosa was inhibited in
the presence of 200 µmol/L genistein (Evans et al. 1998
). Using Caco-2 enterocytes pretreated with up to 200
µmol/L genistein, Akeda et al. (1997)
reported a
dose-dependent inhibition of internalization of several invasive
strains of Vibrio parahemolyticus. And, although bacterial
adherence appeared unaffected, internalization of Mycobacterium
avium was inhibited using HEp-2 laryngeal epithelial cells and
HT-29 enterocytes pretreated with 100–300 µmol/L genistein
(Bermudez and Young 1994
). Thus, using a variety of
bacterial species and a variety of epithelial cell lines, a repeated
observation has been that genistein inhibits bacterial internalization
by cultured epithelial cells, without inhibiting bacterial adherence.
Our data are consistent with the above-cited studies, with one
exception. We noted that genistein inhibited S. typhimurium
internalization by Caco-2 and HT-29 enterocytes, while
Rosenshine et al. (1992 and 1994)
reported
genistein had no effect on S. typhimurium internalization by
HeLa, Henle-407, and A431 cells. This difference is not surprising
because inhibitors of bacteria internalization may have markedly
different effects in different cell lines, and data obtained using
Caco-2 and HT-29 enterocytes may differ from data obtained using other
types of epithelia (Wells et al. 1998
). Nonetheless, the
effect of genistein on bacterial adherence and internalization reported
in the present study was generally consistent with data reported by
other investigators using other bacterial species and other cell lines.
An important caveat pertinent to essentially all studies involving the
biologic effects of genistein (including the present study) is that
nonphysiologic concentrations of genistein are needed to induce the
biologic effect. Based on phamacokinetic calculations involving daily
intake of isoflavones, gut absorption, distribution to peripheral
tissues, and excretion, Barnes et al. (1996)
concluded
that blood isoflavone concentrations rarely exceed 1–5 µmol/L. As
described above, the inhibitory effect of genistein on bacterial
internalization by epithelial cells occurs at much higher
concentrations, ranging from 50 to 500 µmol/L. Interestingly, in
summarizing the effects of genistein on in vitro growth of tumor cell
lines, Peterson (1995)
observed that few of the
postulated mechanisms for genistein-induced inhibition of tumor cell
growth are effective at the physiologic serum concentration of
genistein, and he commented that nonspecific mechanisms of action will
likely take place if genistein, or any drug, is used at abnormally high
concentrations. Curiously, in spite of the high doses of genistein
required to achieve an effect, and in spite of the multiple biological
effects of genistein, most investigators have concluded that
genistein-induced inhibition of bacterial internalization provides
evidence for the involvement of signal transduction in this process,
specifically protein tyrosine kinase activity.
In the present study, the concentrations of genistein associated with
decreased bacterial internalization were also associated with enhanced
junctional integrity of Caco-2 enterocytes, reflected by increased TEER
(Fig. 4)
. This positive effect of genistein on TEER appeared sufficient
to overcome decreased TEER induced by bacterial contamination of the
enterocyte culture (Fig. 6)
and appeared associated with rearrangement
of actin filaments concentrated in the perijunctional areas of the
enterocytes (Fig. 5)
, although a cause and effect relationship between
increased TEER and perijunctional actin rearrangement cannot be
established at this time. Consistent with these findings, Rao et al. (1997)
reported that pretreatment of Caco-2 enterocytes
with 300 µmol/L genistein for 30 min inhibited a
H2O2-induced decrease in TEER; this effect of
genistein was dose dependent, with 30 µmol/L causing minimal
inhibition and 100 µmol/L inhibiting 36–42% of the
H2O2-induced decrease in TEER. Although
Rao et al. (1997)
tested only Caco-2 enterocytes, we
tested the effect of genistein on HT-29 enterocyes as well and noted
that genistein had no noticeable effect on HT-29 TEER or on
distribution of HT-29 actin filaments. Nonetheless, our data and those
of Rao et al. (1997)
are compatible with the hypothesis
that genistein may have a barrier-sustaining effect on the integrity of
the human intestinal epithelium. The conflicting data obtained with
HT-29 enterocytes does not eliminate the possibility of in vivo
relevance of observations with Caco-2 enterocytes, but serves as a
reminder that data obtained from transformed cell lines should be
interpreted with caution and should be tested for in vivo relevance.
The association between increased tight junctional integrity and
decreased bacterial internalization by Caco-2 enterocytes could be
important. We have previously noted the separation of individual
enterocytes using mature confluent cultures incubated 1 h in a
calcium-free medium (Wells et al. 1995
), in a medium
containing purified toxin secreted by enterotoxigenic strains of
Bacteroides fragilis (Wells et al. 1996b
), or
in a medium containing cytochalasin D (Wells et al. 1998
). These three treatments did not affect enterocyte
viability, but these three treatments were associated with increased
internalization of the same strains of S. typhimurium, P.
mirabilis, and E. coli used in the present study.
Electron microscopic visualizations of bacterial cells that were
preferentially adherent to the exposed enterocyte lateral surface
accompanied these increased numbers of intracellular bacteria. The fact
that calcium-free medium, B. fragilis enterotoxin, and
cytochalasin D are disparate treatments with similar outcomes (i.e.,
exposure of the enterocyte lateral membrane and increased bacterial
internalization) argues strongly that the lateral enterocyte surface
might be the preferred site of internalization for some strains of
enteric bacteria. Thus, if the integrity of the enterocyte tight
junctions is increased and contaminating bacteria are less able to open
the junctions and expose the lateral enterocyte surface, decreased
bacterial internalization might be the expected outcome. Therefore,
although the genistein-induced decrease in bacterial internalization by
enterocytes occurred at nonphysiologically high concentrations of
genistein, we noted that decreased internalization was associated with
increased TEER (of Caco-2 enterocytes), indirectly supporting the
hypothesis that the enterocyte lateral surface might be the preferred
site of internalization for some species of enteric bacteria.
Although maximum achievable plasma levels of genistein are likely well
below those associated with the effects of genistein on bacterial
internalization by enterocytes, this does not eliminate the possibility
that dietary genistein might play a role in modulating endocytosis of
enteric bacteria at the enterocyte surface. In addition, there is
anecdotal, in vivo evidence that dietary soy, and perhaps genistein,
might play a role in inhibiting extraintestinal invasion of enteric
bacteria. Using a mouse model of endotoxin-induced bacterial
translocation (Wells et al. 1992
), we previously
reported that a soy-containing liquid diet was associated with
decreased extraintestinal invasion of normal enteric flora (such as
E. coli and Enterococcus sp.) from the intestinal
lumen to the draining mesenteric lymph nodes. Using rats with burn
wounds, dietary soy was not only associated with decreased bacterial
translocation to the mesenteric lymph nodes, but was also associated
with maintenance of the integrity of the intestinal mucosa
(Nakamura et al. 1997
). Unfortunately, intestinal
concentrations of genistein were not measured in either of these in
vivo studies involving the effect of dietary soy on extraintestinal
invasion of enteric bacteria.
Assuming the results from the present study have relevance in humans,
i.e., assuming 300 µmol/L genistein inhibits bacterial endocytosis by
human enterocytes in vivo, it would be difficult to ingest enough
soybeans to obtain this concentration in the intestinal lumen.
Consumption of 35 g soybeans/d (typical intake for Tiawanese) is
comparable to a genistein intake of only ~185 µmol/d (Barnes 1995
). However, administration of purified genistein as a
dietary supplement might be effective in elevating the concentration of
intestinal genistein and might aid in reducing the incidence of
systemic infection caused by invading intestinal flora. Such dietary
supplementation might decrease the incidence of extraintestinal
invasion of normal enteric bacteria in high risk patients, such as
postsurgical patients, trauma patients, immunosuppressed cancer
patients, and organ transplant recipients (Berg 1995
).
Data from the present study indicate that this hypothesis should be
tested in a relevant in vivo model.
|
FOOTNOTES |
|---|
1 This work was supported in part by Public Health
Service grant AI 23484 from the National Institutes of Health.
2 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
4 Abbreviations used: HBSS, Hanks baseline salt
solution; TEER, transepithelial electrical resistance.
Manuscript received August 19, 1998. Initial review completed October 21, 1998. Revision accepted November 30, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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