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Digestive Diseases and Sciences, Vol. 43, No. 3 (March 1998), pp.

646 ± 651

Impairment of Intestinal Mucosal


Antioxidant Defense System During
Salmonella typhimurium Infection
ALKA MEHTA, PhD, SURJIT SINGH, MD, and NIRMAL KUMAR GANGULY, MD

The mucosal pathology of Salm on ella typh im uriu m infe ction may in part be due to the
e xce ssive production of reactive oxyge n species (ROS). The in¯ ue nce of S. typhim urium
infection on the inte stinal mucosal antioxidant de fe nse system was inve stigate d. We inje cted
ligate d rat ile al loops with Salm on ella live culture or toxin. Afte r 18 hr of infe ction, the
animals were kille d and enterocytes isolate d from the ileal loops. The e nte rocyte -re duced
glutathione (GSH) content and activitie s of the enzyme s supe roxide dismutase (SO D),
glutathione pe roxidase (GSH-Px), catalase , glutathione -S-transfe rase (GST), glutathione
reductase (GR), and glucose -6-phosphate dehydroge nase (G6PDH) were spe ctrophotome tri-
cally estimated. The vitamin E and A contents were de te rmine d by high-pe rformance liquid
chromatograph y (HPLC). In both the Salm on ella live culture and toxin-tre ate d groups, the
e nterocyte GSH and vitamin E conte nts and activitie s of the enzymes SOD, GSH-Px,
catalase , GR, and G6PDH were signi® cantly de crease d as compare d to the control group.
Howeve r there was a signi® cant incre ase in the e nte rocyte activity of GST. The re was no
change in the vitamin A conte nt of the e nte rocytes. These ® ndings might indicate a de creased
e ndoge nous intestinal prote ction against ROS in S. typh im uriu m -mediate d infe ction, which
could contribute to the pathoge nesis of the disease.

KEY WORDS: antioxidants; intestine; Salm onella typhim urium ; glutathione.

Nontyphoidal Salm onella se rotype s such as S. typh i- A promine nt histological fe ature of S. typhim urium
m uriu m most often cause gastroe nteritis with wate ry inte stinal infe ction is a de nse in® ltration of polymor-
diarrhe a. There is gross structural damage to the phonucle ar cells in the epithe lium and the unde rlying
mucosa, which causes ge neralize d impairme nt of ab- lamina propria. A numbe r of studie s have shown that
sorption. Seve ral pote ntial virule nce factors of S. ty- white blood cells play an important role in the se cre-
phim urium may contribute to inte stinal mucosal dam- tory phe nomenon of S. typh im uriu m infection (5, 6).
age . These factors include e pithe lial invasion (1), In a recent study, Salm onella -e licite d e nte ritis has
synthe sis of an e nte rotoxin (2) and cytotoxin (3), and
bee n found to be cause d by only those strains that
induction of an in¯ ammatory response (4). Howe ver,
e xhibit transe pithe lial signaling to neutrophils (7).
the exact mechanisms by which S. typhim uriu m cause s
The dense accumulation of the in¯ ammatory le u-
mucosal damage are not well e stablishe d.
kocyte s in the inte stinal mucosa during S. typhi-
Manuscript re ce ive d May 27, 1997; acce pted Nove mber 1, 1997. m uriu m -mediate d infe ction sugge sts that these cells
From the De partme nts of Pediatrics and Expe rimental Me dicine
and Biotechnology, Postgraduate Institute of Medical Education &
participate in the pathoge ne sis and re solution of tis-
Re se arch, Chandigarh-160 012, India. sue damage by ge ne rating ROS and/or by re leasing
Address for re print reque sts: Dr. N.K. Ganguly, De partme nt of
E xperime ntal Medicine and Biotechnology, Postgraduate Institute
e nzymes. The ROS are highly toxic spe cie s and can
of Me dical Education & Research, Chandigarh-160 012, India. cause dam age to all the cellular compone nts, includ-

646 Digestive Diseases and Sciences, Vol. 43, No. 3 (March 1998)
0163-2116/98/0300-0646$15.00/0 Ñ 1998 Plenum Publishing Corporation
MUCO SAL DEFENSE IN INFECTION

ing lipids, structural and re gulatory prote ins, carbo- g/liter, KCl 0.2 g/liter, Na2 HPO 4 , 1.43 g/lite r, KH 2 PO 4 , 0.2
hydrate s, and DNA (8). Excessive ROS production g/liter, pH 7.3)] (16). The extract was centrifuged at 8000g
se e ms to play a part in a numbe r of dise ase s, including for 30 min, and the supernatant was concentrate d by ultra-
® ltration (PM-10 Amicon ® lters). It was then ® lter sterilized
disorde rs of the gastrointe stinal tract, as is shown in (0.2 m M ® lters) and stored at 4 °C. The toxin e xtract gave
animal models and some human studie s (9, 10) . positive results in rabbit ileal loop te st (17), rabbit skin
Mammalian cells are e quippe d with antioxidant permeability te st (18) , and e nzyme -linked immunosorbent
syste ms to combat fre e -radical-me diate d damage , and assay (19).
organs such as gastrointe stinal tract appe ar to be The presence of S-LT in our preparation was con® rmed
by addition of monoclonal antibodies (MAb) against chol-
particularly rich in the se substance s (11, 12) . In ge n-
e ra toxin (CT). Incubation with 1:1500 dilution of MAb (for
e ral, the antioxidants consist of none nzymatic sub- 30 min at 37 °C) complete ly neutralized the secretory activ-
stances such as vitamins A, C, and E; small mole cular ity of S-LT in the ligate d rabbit ileal loop.
weight compounds such as GSH; and thre e basic In the rat ileal loop assay, maximum ¯ uid accumulation
e nzymesÐ SOD, catalase , and GSH-Px (13, 14) . The was obtained when the animals we re injected intraluminally
oxidative damage , caused to the inte stinal epithe lium, with 1 ml of concentrated S-LT containing 3.6 mg protein
(volume/length 5 0.34 6 0.02) . This dose of toxin was used
will de pe nd on the balance betwee n the prooxidants in the subsequent study. Animals give n more concentrated
and the antioxidants. In some in¯ ammatory disorde rs toxin did not survive.
there is a comparative scarcity of antioxidant enzyme s Rat Ileal Loop Assay. Rats we re divided into three
such as SO D and catalase , which ampli® es the vul- groups of six rats e ach. A single ligate d ileal loop of 15 cm
ne rability of the colon towards the delete rious activity was constructed in e ach animal. The control group received
an intraluminal injection of ste rile PBS (1 ml, pH 7.2); the
of ROS (15) . Salmonella live-culture-tre ate d group receive d an intralu-
The hypothe sis of this study was that S. typh i- minal injection of 1 ml of PBS (pH 7.2) containing 10 CFU
7

m uriu m infection might effe ct the antioxidant de fense of Salm onella; and the S-LT-treate d group, intraluminal
syste m of the enterocytes. Conside ring the e xce ssive injection of 1 ml of S-LT (3.6 mg protein). Afte r 18 hr of
ROS production as a result of neutrophil activation, infection, the animals were killed and loop segme nts e x-
cised quickly to me asure the various parame ters.
the antioxidant le vels of the e nte rocyte s could modify Isolation of En terocytes. For the isolation of e nterocytes,
pote ntial mucosal injury by the bacteria. Thus the aim loop segme nts isolated from control and e xpe rimental
of this study was to inve stigate the functional status of groups we re ¯ ushed with O 2 -saturate d PBS (pH 7.2) to
the antioxidants in enterocyte s during S. typhim urium force out the intestinal contents. The 15-cm long loop
infe ction. segme nts we re cut in half and the e nterocytes we re isolated
by the chelation e lution me thod (20) . The cells obtained
were sedimented, washed and suspended in Tris HCl buffer
MATERIALS AND METHODS (2 mM, pH 7.2), and puri® e d (21) . The viability of the cells
was checked by the trypan blue e xclusion test (22).
An im als. Male albino Wistar rats (100 ± 125 g) and male Enterocytes obtained were sonicated and a portion of the
Ne w Z e aland white rabbits (1.5± 3.0 kg) we re used for the cell suspension was centrifuged at 105,000 g for 1 hr. The
study. The animals we re checked for any bacte rial infection pellet constituted the cell me mbrane and the supernatant
by e xamination of three consecutive stool specimens by was the cytosol. The cytosol was used to measure the
both direct smear examination and formal e the r concentra- activitie s of SO D, catalase , GSH-Px, GST, GR, and
tion me thods. Animals fre e of any infection were used for G6PDH.
the study. Ass ay Meth od s. SO D activity was e stimated by the
Bacterial Strain . S. typhim urium strain 1402/84, obtained me thod of Kono (23). The reduction rate of nitrotetrazo-
from Central Re search Institute, Kasauli, was used for the lium blue (NBT) by superoxide radicals (O 2 z 2 ) was mon-
study. The strain was identi® ed by the standard biochemical itored at 550 nm utilizing the autooxidation of hydroxy-
2
tests and by serotyping. The colony forming units (CFU) of lamine hydrochloride as the source for O 2 z . SO D will
2
the strain were dete rmined by the spread plate technique. compete for O 2 z and decrease the reduction rate of NBT.
In the rat ligate d ileal loop te st, the maximum ¯ uid accu- The amount of SOD required to inhibit the rate of reduc-
mulation was obtained when the animals were injected tion of NBT by 50% was de® ned as one unit of activity.
intraluminally with 10 7 CFU of Salm on ella [volume of ¯ uid SOD activity was expressed as units of SOD per milligram
(ml)/length of the loop (cm) 5 0.38 6 0.08] . Therefore, this of protein.
dose of bacteria was used in the subsequent study. Animals The catalase activity was measured spectrophotometri-
give n a dose . 10 7 CFU did not survive. cally according to the method of Bee rs and Sizer (24) by
Preparation of S. typhim urium Toxin (S-LT). For the following the breakdown of hydrogen peroxide (H 2 O 2 ) at
preparation of S-LT, the bacterial cells harve ste d by cen- 240 nm.
trifugation from a 6 hr culture in Casamino ye ast extract The GSH-Px activity was dete rmined by following the
(incubated at 37 °C with longitudinal shaking; 100 oscilla- procedure of Paglia and V alentine (25) with H 2 O 2 as the
tions/min), we re e xtracte d with polymyxin B (Sigma) solu- substrate. The reduction of H 2 O 2 was coupled to NADPH
tion [6 g/liter phosphate buffe red saline (PBS); NaCl 8 oxidation via GR, and e nzyme activity was calculated as

Digestive Diseases and Sciences, Vol. 43, No. 3 (March 1998) 647
ME HTA ET AL

T ABLE 1. A NTIOXIDANT E NZYME A CTIVITIES IN E NTEROCYTES D URING S. typhim urium I NFECTION

G roup*

Antioxidant enzyme Control Live-culture-treated Toxin-treated

SOD (units/mg protein) 2.46 6 0.54 0.91 6 0.060 a


0.72 6 0.03 b
4.61 6 2.79 6 2.71 6
a a
Catalase (units/mg protein) 0.58 0.37 0.43
GSH-Px (nmol NADPH oxidized/min/mg
60.95 6 11.71 6 30.73 6
c b
protein) 6.99 1.39 4.98
GST (nmol product forme d/min/mg
19.49 6 36.99 6 40.36 6
b b
protein) 3.57 3.23 4.18

* Value s are expressed as mean 6 SEM of six different experiments; a, P , 0.05; b, P , 0.01; and c, P ,
0.001 as compared to the control group.

nanomoles of NADPH oxidized per minute per milligram RESULTS


of protein.
The activity of GST was dete rmined spectrophotometri- Evalu ation of Isolation of En terocytes. Our cell
cally with 1-choloro-2,4-dinitrobenzene as the substrate ac- pre parations were found to be compose d of . 90 ±
cording to the method of Habig e t al (26). An extinction 95% epithe lial cells whe n e xamine d unde r the phase -
coef® cient of 9.6 mM2 1 cm 2 1 was used to calculate the
e nzyme activity, which was expressed as nanomoles of con-
contrast microscope as well as whe n staine d pre para-
jugate formed per minute per milligram of protein. tions were examine d microscopically. Examination of
The GSH content was measured in the trichloroace tic the ileal re mnants showed that the in¯ ammatory and
acid e xtracts of the cell sonicates by the me thod of Moron mese nchymal cells re maine d in place almost intact.
e t al (27) . The interaction of GSH with 5,5 9 -dithiobis-( 2- Cell viability was . 90% in enterocytes isolate d from
nitrobenzoic acid) was me asured spectrophotome trically at
the control rats.
412 nm. The results were e xpressed as micromoles of GSH
per milligram of protein. Estim ation of Antioxid an ts. Table 1 shows the ac-
For the estimation of vitamin E and A leve ls, the samples tivitie s of the antioxidant e nzyme s in e nte rocytes iso-
were prepared according to the me thod of Thurnham et al late d from the control, S. typh im uriu m live -culture -
(28) . In brief, the cell sonicate was mixe d with sodium tre ated, and S-LT-tre ate d groups. The enterocyte
dodecylsulfate (SDS), deproteinized with e thanol, and e x- SO D, catalase , and GSH-Px activitie s were de crease d
tracte d with heptane . The organic layer was evaporated to
dryness; reconstitute d with mobile phase (acetonitrile ± by 63% , 39.5% , and 80.8% , re spe ctive ly, in the live -
me thanol± chloroform, 47:47:6 by v/v/v), and analyzed by culture -tre ated group and by 70.7% , 41.2% , and
HPLC. Separation was achieved with C 18 m Bondapak col- 49.6% , re spective ly, in the toxin-tre ate d group as
umn (3.9 3 30 cm) using the solvent system ace tonitrile± compare d to the control group. Howeve r, the entero-
me thanol± chloroform (47:47:6), with a ¯ ow rate of 1.8 cyte GST activity was incre ase d by 89.8% and
ml/min. Vitamins E and A were dete cte d at 292 and 325
nm, respectively, by the dete ctor. 107.1% , re spective ly, in the Salm onella live -culture -
Enzym es of Glutation e Cycle. The GR enzyme activity and S-LT-tre ate d groups as compare d to the control
was me asured by the me thod of Carlberg and Manne rvik group.
(29) , where, in the presence of the e nzyme , the hydrogen of Table 2 shows the GSH conte nt and activitie s of
NADPH is transfe rred to the oxidized glutathione (GSSG). GR and G6PDH in e nte rocyte s isolate d from the
The resulting decrease in the absorbance at 340 nm due to
the oxidation of NADPH was me asured spectrophotometri- control, Salm onella live -culture -tre ated, and S-LT-
cally and the result was e xpressed as nanomoles of NADPH tre ated groups. The GSH conte nt was decrease d by
oxidized per minute per milligram of protein. 48.8% and 49.3% , respe ctive ly, in the Salm onella
G6PDH Assay. The G6PDH activity was measured by the live -culture - and toxin-tre ate d groups as compare d to
me thod of Bhatn agar e t al (30) by using glucose-6- the control group. The GR and G6PDH activitie s also
phosphate as the substrate. The reduction of NADP 1 to
NADPH was spectrophotometrically followed at 340 nm, showe d a 39.3% and 43.4% de crease re spective ly in
1 the e nte rocyte s isolate d from the Salm onella live -
and the result was expressed as nanomoles of NADP
reduced per minute per milligram of protein. culture -tre ated group and a 36.7% and 47.6% de -
Protein Estim ation . The protein e stimation was done crease, re spe ctive ly, in the toxin-tre ate d group as
according to the me thod of Lowry et al (31) by using bovine compare d to the control group.
serum albumin as the standard.
Statis tical An alysis. All the results we re expressed as Table 3 shows the vitamin E and A conte nts of the
me an 6 SEM . The data we re analyzed by Student’ s t test e nte rocyte s isolate d from the control, Salm on ella live -
(unpaired). P , 0.05 was take n as signi® cant. culture -tre ated, and S-LT-tre ate d groups. V itamin E

648 Digestive Diseases and Sciences, Vol. 43, No. 3 (March 1998)
MUCO SAL DEFENSE IN INFECTION

T ABLE 2. GSH CONTENT , GR AND G6PDH E NZYME A CTIVITIES IN E NTEROCYTES D URING S. typhim urium I NFECTION

Enzym e activity

G SH content G R (nm ol NADPH G 6PDH (nm ol NADP


G roup (nm ol/m g protein) oxidized/m in/m g protein) reduced/m in/m g protein)

Control 2.23 6 0.05 55.92 6 6.12 66.12 6 7.02


Live culture
1.14 6 33.96 6 37.41 6
c a b
treated 0.08 6.52 3.90
Toxin treated 1.13 6 0.19 c 35.37 6 5.52 a 34.62 6 5.19 b

*V alues are e xpressed as me an 6 SEM of six different observations; a, P , 0.05; b, P , 0.01; and c, P , 0.001 as compared to
the control group.

content was decreased by 55.4% and 50.4% , respe c- the de ple tion of the thiol groups. The oxidative stre ss
tive ly, in e nte rocyte s isolate d from the Salm onella induce d by the RO S ge ne rate d from both the se
live -culture - and toxin-tre ate d groups as compare d to source s would impair cellular antioxidants, favoring
the control group. The re was no signi® cant change in cell damage or de ath.
the vitamin A conte nt of the e nte rocyte s. SO D functions in the dismutation of O 2 z 2 to
H 2O 2 . The rele vance of SO D-mediate d tissue prote c-
DISCUSSION tion against injury re late d to ROS has bee n shown in
The re sults of the pre sent study show that during S. an e xpe rime ntal colitis model, whe re in¯ ammation
typhim urium infe ction, the enterocyte vitamin E and was appre ciably re duced by tre atme nt with SO D. In
GSH conte nts and enzymatic activitie s of SOD, GSH- the pre se nt study, the signi® cant decrease in the SO D
Px, catalase , GR and G6PDH are impaire d; the GST activity might be cause d by more inactivation through
activity is enhance d, and the vitamin A content re - local conce ntration of the enzyme . A similar de crease
mains unalte re d. As antioxidants are esse ntial in pro- in the SO D activity of the inte stinal mucosa has bee n
te cting the cells against ROS (12, 32) , the e nte rocyte s re porte d during ische mia± repe rfusion injury (34) and
isolate d from rats tre ated with Salm onella live culture in¯ ammatory bowel disease (35) .
or S-LT may be more susce ptible to oxidative dam- GSH-Px and catalase function in concert to remove
age . This impairme nt may be explaine d on the basis the H 2 O 2 from the cells. Direct inhibition of GSH-Px
of the de le terious e ffect of ROS on the antioxidants. and catalase by the ROS has be en shown in a pre vious
The ROS in S. typh im uriu m live culture /S-LT-treate d study (12) . With an impairm ent in the activitie s of
2
rat ile um may originate from the enzyme NADPH SO D, catalase , and GSH-Px, O 2 z and H 2O 2 will not
oxidase in the in® ltrating neutrophils and/or the e n- be ef® cie ntly detoxi® ed and these can react in an iron-
zyme xanthine oxidase (XO) prese nt in the entero- or coppe r-catalyze d Habe r-We iss re action and form
cytes (33) . Activate d neutrophils se crete various e n- the very re active hydroxyl (.OH) radical. The .O H can
zyme s and ROS as by products of phagocytosis. These initiate the peroxidation of the membrane lipids to a
various e nzymes and metabolite s can induce se ve re faste r e xtent, than can be controlle d by the cells,
tissue damage . The e nzyme XO e xists in normal there by re sulting in comple te de struction of the mem-
he althy cells pre dominantly as a NAD 1 -re ducing xan- brane . V itamin E is the major chain-bre aking antiox-
thine de hydroge nase and can be conve rte d to an idant in the biome mbrane s that can inte rfe re with the
oxyge n-radical-produ cing form (XO) e ither by the propagation of lipid pe roxidation (36) . With a de -
prote ase s re le ase d from the in¯ ammatory cells or by crease in the vitamin E conte nt, the membrane de -
struction may be furthe r e nhance d. A signi® cant in-
T ABLE 3. V ITAMIN E AND A L EVELS IN E NTEROCYTES DURING S. crease in the GST activity in the tre ated groups may
typhim urium I NFECTION* aid in the de toxi® cation of the lipid peroxide s, prob-
Vitamin E Vitam in A
ably to compe nsate for the fre e -radical-me diate d
G roup (ng/m g protein) (ng/m g protein) damage .
GSH is the most important cellular low-mole cular-
Control 31.91 6 5.94 2.19 6 0.59
Live -culture-treate d 14.24 6 2.78
a
1.86 6 0.56 weight thiol that prote cts the cell from fre e-radical-
Toxin-treate d 15.81 6 1.17 a 2.79 6 0.32 mediate d damage . GSH may act e ithe r by prote cting
*V alue s are e xpressed as me an 6 SEM of six differe nt e xperime nts; cells from lipid pe roxidation or by prote cting prote in
a, P , 0.05 as compare d to the control group. sulfhydryl groups from be coming irre ve rsibly oxidize d

Digestive Diseases and Sciences, Vol. 43, No. 3 (March 1998) 649
ME HTA ET AL

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