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Experimental impact of aspirin
exposure on rat intestinal bacteria,
epithelial cells and cell line
Raj K Upreti, A Kannan and AB Pant
Abstract
Aspirin, a commonly used therapeutic non-steroidal anti-inflammatory drug (NSAID) is known to cause gastric
mucosal damage. Intestinal bacteria having a regulatory effect on intestinal homeostasis play significant role in
NSAID-induced intestinal injury. Bacteria and specific cell lines are considered to be suitable for toxicity
screening and testing of chemicals. Therefore, to evaluate and compare in vitro toxicity, cultures of rat intest-
inal epithelial cells (IEC), isolated bacteria and IEC-6 cell line were assessed for viability, morphometric analysis,
membrane transport enzymes and structural constituents for membrane damage, dehydrogenase activity test
for respiratory and energy producing processes and esterase activity test for intra- and extra-cellular degrada-
tion, following the post exposure to aspirin (0–50 mgmL
–1
). Similar pattern of dose-dependent changes in these
parameters were observed in three types of cells. Similar in situ effects on IEC validated the in vitro findings.
These findings indicate that higher aspirin concentrations may alter cellular functions of IEC and gut bacteria.
Furthermore, results suggest that gut bacteria and IEC-6 cell line can be used for the initial screening of
gastrointestinal cellular toxicity caused by NSAIDs.
Keywords
aspirin, acetyl salicylate, gut bacteria, intestinal epithelial cells, IEC-6 cell line
Introduction
Aspirin (acetylsalicylic acid) is one among the most
popular therapeutic non-steroidal anti-inflammatory
drug (NSAID) in the world. Now, it is being used
most frequently as a positive control to compare the
anti-inflammatory activity of new drug molecules.
The gastrointestinal (GI) effects are the most common
and predominant ones that are observed with the use
of aspirin and other NSAIDs. Numerous studies have
confirmed that aspirin has the ability to induce dam-
age in GI tract and promotes ulceration and bleeding
in GI tract.
1,2
Bacteria living within the intestinal
lumen are known to play an important role in host
homoeostasis. Aspirin and related compounds have
variety of effects on microorganisms and the enteric
bacteria have been shown to play a role in NSAID-
induced intestinal injury.
3-5
Furthermore, in vitro
studies using guinea pig and human gastric epithelial
cells in primary culture imply that NSAIDs cause gas-
tric mucosal damage through both necrosis and apop-
tosis of gastric mucosal cells.
6,7
However, available
literature is scanty regarding the interaction of aspirin
with intestinal bacteria and epithelial cell lines and
needs further exploration.
In vitro studies can be used to evaluate the effects
of a chemical/drug on specific bacterial population as
well as cell lines and that can provide data for further
detailed studies including hypothesis testing.
8
We
have earlier shown that facultative bacteria isolated
from rat gut and the intestinal epithelial cell line can
be used for preliminary screening of GI-tract cellular
toxicity caused by heavy metals.
9-11
The present study
was undertaken to compare the modulatory effects of
aspirin on cellular kinetics and morphological
Biomembrane Toxicology, Indian Institute of Toxicology
Research (National Laboratory of Council of Scientific &
Industrial Research, New Delhi-India), MG Marg, Lucknow –
226 001, India
Corresponding author:
Raj K Upreti, Biomembrane Toxicology, Indian Institute of
Toxicology Research, Post Box No. 80, MG Marg, Lucknow –
226 001, India
Email: upretirk@rediffmail.com
Human and Experimental Toxicology
29(10) 833–843
ªThe Author(s) 2010
Reprints and permission:
sagepub.co.uk/journalsPermissions.nav
DOI: 10.1177/0960327110363333
het.sagepub.com
changes in primary cultures of rat intestinal epithelial
cells, resident facultative intestinal bacteria and
immortal rat intestinal epithelial cell line (IEC-6).
Materials and methods
Animals and chemicals
All the chemicals and reagents were purchased from
Sigma-Aldrich, E. Merck, Gibco, BRL and Hi-
Media, India. These were of analytical grade with
highest purity available. Acetyl salicylic acid
(aspirin) was used in the experiments by dissolving
it in minimum quantity of 1N NaOH and pH 7.4 was
adjusted by HCl. Dulbecco’s Modified Eagle’s
Medium (DMEM; pH 7.4) was used to make work-
ing concentrations from the stock solution. Healthy
adult male albino Wistar rats procured from the
Animal Breeding Facility of Indian Institute of
Toxicology Research, Lucknow, were used for the
isolation of intestinal bacteria, epithelial cells and
for in situ studies. The animals were housed indivi-
dually under standard animal house conditions with
natural light/dark cycle and a temperature of 25C
+2C. The standard animal food pellets and water
were given ad libitum. Clearance from the Animal
Ethical Committee of the Institute was obtained for
the use of animals.
Intestinal epithelial cell line (IEC-6)
IEC-6, normal rat small intestine cell line (ATCC
CRL 1592), was initially procured from National
Centre for Cell Sciences, Pune, India, and since then
has been maintained at our Institute. Monolayer’s of
cells were grown in DMEM supplemented with 5%
fetal bovine serum (FBS), 10 mgmL
-1
insulin, 100
mg L
-1
penicillin, 100 mg L
-1
streptomycin and 2.5
mg L
-1
fungizone (Invitrogen, Groningen, The Neth-
erlands) at 37C in a humidified atmosphere of 5%
CO
2
–95%air. Prior to the start of experiments, cells
were assessed for viability using trypan blue dye
exclusion test, and batches showing more then 95%
cell viability were used in the study. Investigations
were carried out using the cells of passages between
10 and 25.
Isolation and culture of rat intestinal bacteria and
epithelial cells (IEC)
The cecum was located by opening the abdomen with
full aseptic precautions and injected with 5.0 mL of
sterilized phosphate buffered saline (PBS) from one
end. After 2 min, fluid from the cecum was collected
and one loop-full was streaked out on nutrient agar
plate. After incubation for 24 hours, the bacterial
colonies formed were studied and the smears were
stained with Gram’s stain. The biochemical reactions
were put up and the bacteria were identified on the
basis of the ‘Bergey’s Manual of Determinative Bac-
teriology.’ The bacteria selected for present study
were, Escherichia coli, Pseudomonas sp. Lactobacil-
lus sp. and Staphylococcus sp. Furthermore, to isolate
the entire mixed population of resident intestinal bac-
teria, the cecum contents were filtered twice through
sterile glass wool to remove the intestinal debris and
the fecal matter. The mixed bacterial cell population
was harvested by centrifugation at 10,000gfor 20 min
at 4C, washed twice with desired media and
resuspended in the same media/buffer. The log phase
cultures of bacteria were inoculated in nutrient broth
containing (g L
1
) peptone, 2.0; beef extract, 3.0;
NaCl, 5.0 and were incubated at 37C on a rotatory
shaker. Intestinal epithelial cells (IEC) were isolated
and cultivated from overnight fasted rats, and cell via-
bility of IEC and IEC-6 following 24 hours in vitro
exposure to aspirin was carried out as described by
Upreti et al.
11
In vitro effect of aspirin on growth of bacteria
The test bacteria taken from 24-hour culture in
nutrient broth were inoculated in 10.0 mL of the same
medium until reaching an OD of 0.05 +0.01 at a
wavelength of 610 nm. Freshly prepared aspirin solu-
tions (pH 7.4) were added to the final concentrations
of 10–50 mgmL
1
in medium. These concentrations
were selected by taking into consideration the mini-
mum inhibitory concentration (MIC) value of the
aspirin. MIC is referred as the lowest concentration
of an antimicrobial, which inhibits the visible growth
of a microorganism after overnight incubation.
Bacterial growth was measured at different time inter-
vals up to 30 hours using turbidimetry at 610 nm.
Growth measurements were carried out in the absence
and presence of aspirin and the specific growth rate,
doubling time and number of generations of bacteria
was evaluated as described by Espigares and Maris-
can.
12
Viability was tested by colony forming units
(CFU) on agar plates. CFU count is an aggregation
of viable cells derived from single mother cell, which
represents to the measurement of one cell.
834 Human and Experimental Toxicology 29(10)
In vitro cell membrane studies
An approximately equal number of bacterial, IEC and
IEC-6 cells (2 10
6
cells mL
1
) were exposed to dif-
ferent concentrations of acetyl salicylate in the media
at 37C for 24 hours with constant shaking. Parallel-
untreated sets were also run under identical conditions
and served as controls. Following incubation, cells
were harvested by centrifugation at 10,000gfor
15 min. Intestinal epithelial cells were washed with
30 mM Tris buffer containing 2.5 mM EDTA, pH
8.1 and resuspended in the same buffer under cold
condition. Cells were disrupted by four 15-sec bursts
with an ultrasonic processor and centrifuged at 450g
for 10 min. To obtain the membrane fraction, the
sub-fractionation was carried out according to For-
stner et al.
13
Bacterial cell membrane was prepared
as described by Kumar and Upreti.
14
In situ studies
In order to validate the in vitro findings of intestinal
epithelial cells, rat intestinal loops were filled with
different concentrations of aspirin solutions and incu-
bated for 30 min in situ. The in situ studies were car-
ried out as described by Upreti et al.
11
Enzyme assays and biochemical estimations
Dehydrogenase (DHA) and esterase activity (EA)
tests were carried out as described by Liu
15
and Obst
and Holzapfel-Pschorn,
16
respectively. Alkaline
phosphatase (EC 3.1.3.1) was determined according
to Weiser
17
and Ca
2þ
-Mg
2þ
-ATPase (EC 3.6.1.3) as
described by Hidalgo et al.
18
Enzyme units were
defined as micromoles of product formed or liberated
per min under the assay conditions. Specific activity
was expressed as units per milligram of protein. Pro-
tein was determined according to Lowry et al.,
19
using
bovine serum albumin as standard. Carbohydrate
(total hexose) and sialic acid were, respectively, esti-
mated according to Roe
20
and Warren.
21
Total lipid
was extracted according to Folch et al.
22
Phospholi-
pids were quantified following digestion with 70%
perchloric acid and estimated according to the method
of Wagner et al.
23
Cholesterol was estimated accord-
ing to Zlatkis et al.
24
Morphometric analysis
Following the exposure of aspirin, live IEC and IEC-6
cells were examined under the phase-contrast
inverted microscope (Leica, Germany) to observe the
exposure-induced morphological changes as well as
the alteration in cell growth pattern. The changes were
quantified using automatic image analysis software
Lieca Q Win 500, hooked up with the microscope and
expressed in percentage area covered under the
microscopic fields.
Statistical analysis
The results are expressed as mean +SD and compar-
isons were made with appropriate controls employing
Student’s t-test. Probability values of less than
.05 were considered to be significant. The comparison
of the dose-dependent effects on different endpoints
was also made using the one-way ANOVA test.
Results
In vitro effect of aspirin on growth phase of rat
gut bacteria
The in vitro effect of aspirin on the growth profile of
rat gut bacteria is shown in Figure 1. Aspirin concen-
trations up to 20 mgmL
1
showed a more or less sim-
ilar growth profile throughout the growth phase as
that of respective controls in all bacteria, whereas,
30 and 50 mgmL
1
concentrations revealed slower
growth rate pattern. The presence of 50 mgmL
1
aspirin in medium retarded the growth rate of all bac-
teria significantly (p< .05) throughout the growth
phase. Further analysis of the data also revealed an
enhancement in doubling time as well as a decrease
in number of generations during the growth period
in all bacteria at 50 mgmL
1
concentration (data not
shown). Lactobacillus sp. and Staphylococcus sp.
belonging to gram-positive group showed higher
retardation (over 40%;p< .05) as compared to the
tested gram-negative and the mixed population of gut
bacteria.
In vitro effect of aspirin on cell viability
Cell viability of bacteria, IEC and IEC-6 following 24
hours in vitro exposures to aspirin revealed a
concentration-dependent reduction. Aspirin concentra-
tion at 50 mgmL
1
showed 25%–35%non-viable cells
in three types of cell cultures; whereas, 200 mgmL
1
concentration caused more than 85%cell death (data
not shown).
Upreti R K et al.835
Effects of aspirin on DHA and EA tests
In vitro exposure of aspirin at 30 and 50 mgmL
1
was
found to be significantly effective in inhibiting DHA
activity in all the cell types used in the study. Among
the bacteria used, Pseudomonas sp. has shown signifi-
cant inhibition of DHA even at 20 mgmL
1
of aspirin
Figure 1. Growth phases of isolated rat gut bacteria in presence of aspirin. Data represents mean value from 3 to
5 experiments. SD has not been shown to avoid overcrowding. Variance was within a limit of 10%–15%.
836 Human and Experimental Toxicology 29(10)
exposure. Aspirin at lower doses (10 and 20 mgmL
1
)
did not cause significant changes under our experimen-
tal conditions. More or less similar trend was observed
for aspirin-induced EA inhibition at 30 and 50 mgmL
1
concentrations. However, aspirin at 20 mg/mL
1
was
found to be statistically significant inhibitory for EA
in E. coli (29.4 +3.2) and Pseudomonas sp.(22.5+
2.5). In general, all the cells – enteric bacteria, IEC and
IEC-6 – were responding to the aspirin exposure in a
dose-dependent manner for the inhibition of DHA and
EA (Table 1).
In vitro effect of aspirin on membrane enzymes
and constituents
The results of the influence of aspirin on membrane
enzymes and constituents of bacteria, IEC and IEC-
6 are summarized in Tables 2 and 3. Aspirin induced
alterations in the activity of alkaline phosphatase and
Ca
2þ
-Mg
2þ
-ATPase was observed in a synchronized
manner corresponding to the concentration. In
general, effect could reach to statistically significant
levels following the exposure of aspirin at 30 and
50 mgmL
1
. However, in case of Lactobacillus sp.
and IEC-6, changes in the activity of Ca
2þ
-Mg
2þ
-
ATPase were significant (p< .05) even at 20 mgmL
1
aspirin exposure. The basal activity and magnitude of
aspirin induced alterations for both alkaline phospha-
tase and Ca
2þ
-Mg
2þ
-ATPase was more or less similar
among the bacterial strains used except in case of gut
bacteria mixed population. The inhibition of Ca
2þ
-
Mg
2þ
-ATPase activity at 30 and 50 mgmL
1
were
32%and 48%for IEC and 42%and 56%for IEC-6,
respectively. Although the trend of the dose-response
was similar between the IEC and IEC-6 for these
enzymes, the comparative declines were statistically
higher in case of IEC-6. In general, IEC has shown
higher magnitude of activity of both the enzymes
i.e., alkaline phosphatase and Ca
2þ
-Mg
2þ
-ATPase
(Table 2).
Aspirin-induced significant decline of bacterial
membrane hexose content was observed ranging from
25%–50%at 30 mgmL
1
in different bacterial strains
used in the study. It was further increased up to 61%at
50 mgmL
1
concentration of aspirin. The inhibitory
effect was less pronounced at lower concentrations
of aspirin, especially in gram-negative bacteria as
compared to the gram-positive and the mixed bacter-
ial population. The decline in sialic acid content at
30 mgmL
1
was around 30%in most of the bacterial
populations and it was in the range of 41%–59%
following the exposure of 50 mgmL
1
aspirin.
Greater magnitude of decline in sialic acid shows high
vulnerability of gram-negative bacteria against
aspirin. Phospholipid contents did not reveal any
significant change in all the tested bacteria. Concomi-
tantly, a concentration-dependent decline in hexose
and sialic acid content was evident in IEC and
IEC-6. Highest tested dose of 50 mgmL
1
revealed
significant decline of 45%–50%in hexose and sialic
acid contents of both types of cells. Like bacteria, the
changes were also found to be non-significant for
membrane phospholipid content in both IEC and
IEC-6. In case of cholesterol content, significant
decline of 33%and 44%for IEC and 25%and 41%
for IEC-6 were evident at 30 and 50mgmL
1
concen-
trations of aspirin, respectively. In totality, similar
trend of responsiveness against aspirin exposure was
Table 1. Percentage inhibition of dehydrogenase (DHA) and esterase (EA) activity following in vitro exposure of aspirin
a
Concentration
(mgmL
1
)E. coli
Pseudo-
monas sp.
Lactobacillus
sp.
Staphylococcus
sp.
Gut bacteria mixed
population IEC IEC-6
DHA
10 5.5 +0.9 14.2 +2.1 6.6 +1.1 5.0 +0.8 6.3 +1.3 8.4 +1.4 7.9 +1.4
20 8.2 +1.4 34.1 +4.0
b
9.3 +1.3 5.5 +0.9 7.8 +1.0 15.0 +2.0 14.2 +1.9
30 41.3 +4.6
b
53.2 +5.9
b
25.9 +2.8
b
39.2 +4.4
b
32.5 +3.8
b
36.2 +4.2
b
40.5 +4.5
b
50 50.1 +5.4
b
59.0 +6.6
b
57.2 +6.2
b
48.4 +5.3
b
50.4 +5.6
b
47.6 +5.4
b
57.0 +6.1
b
EA
10 13.1 +1.7 13.6 +1.8 5.2 +0.8 7.0 +1.1 7.4 +1.0 6.9 +0.8 10.2 +2.0
20 29.4 +3.2
b
22.5 +2.5
b
6.9 +1.0 10.0 +1.4 13.2 +1.8 13.1 +1.8 16.1 +2.2
30 53.0 +5.8
b
29.7 +3.4
b
33.1 +3.7
b
34.2 +3.9
b
30.7 +3.5
b
29.6 +3.5
b
33.2 +3.8
b
50 58.1 +6.5
b
39.6 +4.7
b
44.4 +5.0
b
43.4 +4.7
b
40.6 +4.6
b
48.2 +5.5
b
41.8 +4.9
b
a
Values are percent inhibition mean +SD from three experiments.
b
p< .05.
Upreti R K et al.837
Table 2. In vitro effect of aspirin on membrane enzymes of rat intestinal bacteria, IEC and IEC-6
a
Concentration (mgmL
-1
)
Specific activity (Units/mg protein)
E. coli Pseudomonas sp. Lactobacillus sp. Staphylococcus sp. Gut bacteria mixed population IEC IEC-6
Alkaline phosphatase
0.0 2.49 +0.28 2.47 +0.25 .56 +0.27 2.45 +0.26 2.84 +0.30 4.90 +0.50 2.82 +0.29
20 2.34 +0.27 2.40 +0.28 2.14 +0.26 2.27 +0.27 2.46 +0.30 4.41 +0.49 2.64 +0.29
30 1.79 +0.19
b
1.69 +0.19
b
1.23 +0.15
b
1.52 +0.18
b
1.87 +0.21
b
3.53 +0.41
b
1.91 +0.23
b
50 1.41 +0.21
b
1.23 +0.19
b
1.03 +0.16
b
1.27 +0.15
b
1.45 +0.19
b
2.65 +0.32
b
1.38 +0.19
b
Ca
2þ
-Mg
2þ
-ATPase
0.0 0.321 +0.034 0.307 +0.033 0.301 +0.032 0.269 +0.028 0.326 +0.034 0.435 +0.045 0.323 +0.033
20 0.341 +0.039 0.301 +0.038 0.248 +0.027
b
0.237 +0.029 0.311 +0.035 0.383 +0.043 0.268 +0.027
b
30 0.198 +0.022
b
0.191 +0.024
b
0.160 +0.020
b
0.162 +0.020
b
0.225 +0.027
b
0.296 +0.036
b
0.187 +0.020
b
50 0.123 +0.015
b
0.114 +0.016
b
0.100 +0.016
b
0.124 +0.015
b
0.150 +0.021
b
0.226 +0.026
b
0.142 +0.017
b
a
Values are mean +SD from three experiments.
b
p< .05.
Table 3. In vitro effect of aspirin on membrane constituents of rat intestinal bacteria, IEC and IEC-6
a
Concentration (mgmL
-1
)
Specific activity (Units/mg protein)
E. coli Pseudomonas sp. Lactobacillus sp. Staphylococcus sp.
Gut bacteria
mixed population IEC IEC-6
Hexose
0.0 169 +17 174 +18 179 +19 162 +17 172 +18 200 +23 173 +18
20 164 +18 170 +20 136 +14
b
141 +16 158 +19 166 +19 149 +17
30 131 +15
b
115 +13
b
85 +9
b
95 +10
b
96 +11
b
132 +15
b
129 +14
b
50 73 +9
b
87 +10
b
68 +8
b
69 +8
b
71 +9
b
108 +12
b
86 +9
b
Sialic acid
0.0 10.3 +11 9.6 +1.0 7.7 +0.8 9.2 +0.9 9.6 +1.0 14.1 +1.4 21.2 +2.3
20 8.3 +12 10.1 +1.7 7.3 +0.9 7.6 +0.9 8.3 +1.0 12.4 +1.4 19.3 +2.2
30 7.2 +8
b
7.1 +0.7
b
6.2 +0.7
b
6.6 +0.7
b
6.8 +0.7
b
11.2 +1.1
b
15.5 +1.7
b
50 4.2 +5
b
5.4 +0.6
b
4.5 +0.5
b
5.3 +0.6
b
5.7 +0.6
b
7.1 +0.8
b
12.4 +1.4
b
Cholesterol
0.0 n.d. n.d. n.d. n.d. n.d. 46.2 +4.7 42.3 +4.3
20 – – – – – 41.1 +4.5 39.7 +4.5
30 – – – – – 30.9 +3.3
b
31.7 +<3.3
b
50 – – – – – 25.9 +2.9
b
24.9 +2.8
b
n.d. – not determined in bacteria.
a
Values are mean +SD from three experiments.
b
p< .05.
838
observed in bacterial cells and intestinal epithelium
cells (IEC) and IEC-6, however, the magnitude of
basal expressions and activities were reported at
higher side in IEC and IEC-6 than bacterial cells.
In situ effect of aspirin on IEC
In situ effects of aspirin on IEC membrane enzymes and
constituents following rat intestinal loop incubation are
summarized in Figure 2. A concentration-dependent
decrease in membrane alkaline phosphatase and
Ca
2þ
-Mg
2þ
-ATPase activities was observed. The
decrease in alkaline phosphatase at 20 mgmL
1
dose
was not statistically significant. The respective signifi-
cant decrease of 25%and 39%were evident at 30 and
50 mgmL
1
doses. As compared to the control, the sig-
nificant decline in Ca
2þ
-Mg
2þ
-ATPase activity was
20%,36%and 45%at 20, 30 and 50 mgmL
1
, respec-
tively. In general, the in situ inhibition pattern of both
the enzymes was similar to that of in vitro exposures
of aspirin on bacteria, IEC and IEC-6 cells. However,
the magnitude of percentage changes, particularly at
higher doses, was comparatively less. Membrane hex-
ose and sialic acid contents showed a significant
dose-dependent decline in all the tested aspirin concen-
trations. At 50 mgmL
1
concentration, it was 53%and
Figure 2. In situ effect of aspirin on membrane enzymes and constituents of rat intestinal epithelial cells. (A) Left axis
denotes specific activity for alkaline phosphatase and right axis for Ca
2þ
-Mg
2þ
-ATPase. (B) Left axis denotes hexose
content and right axis for sialic acid content. (C) Cholesterol and phospholipid. Values are mean +SD from three
experiments. *p< .05.
Upreti R K et al.839
51%, respectively. Like in vitro exposures,
phospholipid contents did not show any significant
alteration. Cholesterol content revealed a similar
dose-dependent decline pattern as observed in case of
IEC and IEC-6 in vitro exposures.
Morphometric analysis
Highlights of the morphological changes in IEC and
IEC-6 are summarized in Figures 3 and 4. The analy-
sis was made to study the total covered area by the cell
mass under the given field. Then these values were
calculated by comparing the values with the area cov-
ered by untreated cells. The adverse effects on cell
physiology were apparent at 30 mgmL
1
aspirin con-
centration in both IEC-6 and IEC cells, respectively
(76%and 82%area of control). This was further
intense with the increase in aspirin concentrations viz,
44%and 56%of control at 50 mgmL
1
in IEC-6 and
IEC cells, respectively. The total covered area analy-
sis indicated that the exposures at 30 mgmL
1
and
higher concentrations used in the study were able to
pose the statistically significant reduction in cell size
and mitotic index as evidenced by the formation of
protuberances and spindle-shaped structures. The
concentrations of aspirin up to 20 mgmL
1
did not
show significant cytotoxic effects. Dose-dependent
changes in the cell number and morphology were seen
and image analysis shows that number of cells got
decreased significantly following the exposure of
aspirin at 30 mgmL
1
concentration onwards. Mor-
phological distortion in the cells was also seen with
number of blabbing, spindle-shaped structures,
nuclear condensation and cell debris (Figure 4).
Discussion
The results of the present study demonstrate the simi-
larities in the pattern of majority of in vitro toxic
effects of aspirin on the resident intestinal bacteria,
IEC-6 cell line and the intestinal epithelial cells of
rats. Growth of bacteria is a common parameter to
study the response to toxic insults, as it also reflects
the viability of the bacterial population. In the present
study, the growth profile of gut bacteria in general
showed an aspirin concentration-dependent inhibi-
tion. A similar pattern of a concentration- and time-
dependent decrease in the number of generations in
all the isolated bacterial populations was also evident
following the exposure of aspirin. Concomitantly, the
viability pattern of the IEC and IEC-6 also revealed a
similar aspirin concentration-dependent cell death.
Morphometric analysis of both the cell type also
showed the accordance with the findings of other end-
points studied. Dose-dependent reduction in cell
growth by aspirin and other NSAIDs on intestinal and
non-intestinal tumor cell lines and decreased cell via-
bility in primary cultures of gastric mucosal cells have
been shown in vitro studies.
6,25,26
An inhibition of DHA and EA represents intracel-
lular toxicity, where DHA is linked to the respiratory
and energy-producing processes, and EA involved in
intra- and extracellular degradation of organic sub-
stances is considered as an indicator of general hetero-
trophic activity of the cell.
9,11,27
Role of mitochondria
in aspirin-induced gastric injury in gastric epithelial
cells is well documented.
7
Statistically similar trend
of a decrease in the levels of DHA and EA in bacterial
cells, IEC and IEC-6 cell line in the present study sug-
gest that the mode of intracellular interaction; meta-
bolism and toxic responses may have similarities in
the cellular pathways of aspirin in these three types
of cell systems.
Intestinal epithelial cells are one of the major sites
of aspirin interaction and it has been documented that
aspirin produces a dose-related GI toxicity.
28
Aspirin
side effects in intestine may be attributed to the initial
biochemical modifications in the epithelial cell mem-
brane. These alterations can then lead to activation of
a variety of inhibition responses and hence cause
Figure 3. Morphometric analysis of cultured intestinal
epithelium cells (IEC) and intestinal epithelium cell line
(IEC-6) following the exposure of various concentrations
of aspirin. Values are presented as percentage control area
covered in the microscopic fields. Other details are as
given in the text. *p< .05.
840 Human and Experimental Toxicology 29(10)
intestinal toxicity. Dose-dependent inhibition of
membrane alkaline phosphatase and Ca
2þ
-Mg
2þ
-
ATPase following aspirin exposure as observed in the
present investigation in intestinal bacteria, IEC and
IEC-6 cells could lead to the impairment of uptake
and transport of vital ions. Aspirin and its biotrans-
formed derivative salicylate have been reported to
alter the ATPase activity in vitro on direct incubation
as it binds to cell membrane due to its lipophilic
nature and alters membrane viscosity.
29
Khokher and
Figure 4. Representative microphotographs of intestinal epithelium cells (IEC) and intestinal epithelium cell line (IEC-6)
demonstrating the morphological alterations and growth pattern following the exposure of various concentrations of
aspirin. Original magnification is 400.
Upreti R K et al.841
Dandona
30
have correlated the in vitro inhibitory
effects of aspirin on osteoblast function due to the
inhibition of alkaline phosphatase activity in human
osteoblast cultures. The similarity in the inhibition
pattern of membrane marker enzymes in three types
of cells in present study further suggests the involve-
ment of common steps in the pathways of the metabo-
lism/toxicity of aspirin in these cells.
Similar pattern of significant decline in membrane
hexose and sialic acid content of all bacteria, IEC and
IEC-6 with higher tested concentrations of aspirin in
the present study suggests an alteration in the integrity
of the membrane of these three types of cells.
31,32
The
concentration of cholesterol, another important mem-
brane constituent of eukaryotic cells, also showed sig-
nificantly similar dose-dependent decline in IEC and
IEC-6, suggesting an altered fluidity of membrane
with higher dose exposures in these two types of cells.
However, there was no significant change in phospho-
lipid content of IEC, IEC-6 and the bacterial cell
membrane.
Mammalian gut bacteria have important metabolic,
trophic and protective functions and the commensal
host-bacteria relationship study in the intestine has
shown that the differentiation of epithelial cells is
greatly affected by interaction with resident bac-
teria.
33
The resident gut bacteria and intestinal epithe-
lial cells are known to interact in harmony.
9,34
Present
findings of more or less similar aspirin concentration-
dependent effects in all the tested parameters of
intestinal bacteria and epithelial cells further
strengthen the dynamic relationship between eukar-
yotic and prokaryotic cells in GI tract. In addition, the
in vitro findings on the effect of aspirin on IEC mem-
brane enzymes and constituents were also validated
following in situ exposures using rat intestinal loop
model. On comparison, results further revealed more
or less similar effects at each endpoint. This indicates
that interaction and metabolism of aspirin under
physiological conditions is likely to be similar at cell
culture conditions.
35
In general, in vitro models employing specific cells
in culture including bacteria and cell lines are suitable
for the study of toxicity and metabolism of chemicals/
drugs. The suitability is due to their easy handling,
quicker response and the presence of most of bio-
chemical pathways similar to those present in the cells
of higher animals.
9-11,36-38
Similarities in most of the
tested responses and effects of aspirin between IEC,
IEC-6 cell line and bacteria as observed in the present
study further strengthen the notion that gut bacteria
and the IEC-6 cell line can be used as an alternate
to animals for screening of GI cellular toxicity caused
by new NSAIDs.
Acknowledgements
The authors are grateful to The Director, IITR, Lucknow,
for his keen interest in the study. This work was supported
by Net Work project (NWP 0034) of Council of Scientific
& Industrial Research (CSIR), New Delhi, India.
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