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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.
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
G roup*
* 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.
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
*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
650 Digestive Diseases and Sciences, Vol. 43, No. 3 (March 1998)
MUCO SAL DEFENSE IN INFECTION
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Digestive Diseases and Sciences, Vol. 43, No. 3 (March 1998) 651