Protection Against Chemically-Induced Oxidative Gastrointestinal Tissue Injury in Rats by Bismuth Salts

Protection Against Chemically-Induced

Oxidative Gastrointestinal Tissue Injury in

Rats by Bismuth SaltsDEBASIS BAGCHI, PhD, OWEN R. CARRYL, PhD, MINH X. TRAN, MANASHI BAGCHI, PhD,

PHILLIP J. VUCHETICH, ROGER L. KROHN, SIDDHARTHA D. RAY, PhD,

SEKHAR MITRA, PhD, and SIDNEY J. STOHS, PhD

Oxygen free radicals (OFR) are implicate d in the pathoge nesis of stress, chemically induce dgastric lesions, and gastrointe stinal injury. The concentration-de pendent scavenging abilitie sof bismuth subsalicylate (SBS), colloidal bismuth subcitrate (CBS), and selected OFRscavengers, including superoxide dismutase (SOD), catalase , mannitol, and allopurinol wereexamine d against biochemically or chemically generated superoxide anion, hydroxyl radical,and hypochlorite radical plus hypochlorous acid based on a chemiluminescence assay.Furthe rmore, both gastric (GM) and inte stinal mucosa (IM) were individually exposed invitro to these free radical generating systems, and the concentration-de pendent protectiveabilitie s of SBS and CBS against lipid peroxidation (LP) were compared with selected OFRscavengers. In addition, 24-hr fasted rats were orally treated with the necrotizing agents 0.6M HCl, 0.2 M NaOH, 80% ethanol, and aspirin (200 mg/kg) . The extent of tissue injury in theGM and IM was determined by assessing LP, DNA fragmentation, and membrane micro-viscosity. Dose- and time-dependent in vivo protective abilitie s of CBS (100 mg/kg) and SBS(15 mg/kg) were also assessed. Following incubations with superoxide anion and hydroxylradical generating systems in the presence of 125 mg SBS/lite r, approxim ately 47% and 61%inhibitions were observed in the chemilumine scence response , respective ly, while 48% and46% inhibitions were obse rved with 125 mg CBS/lite r. SBS and CBS exerted similar abilitie stowards hypochlorite radical plus hypochlorous acid. Approx. 3.1- and 3.7-fold increases inLP were obse rved in the GM and IM of rats following oral administration of 0.6 M HCl.Pretreatment of the rats with SBS and CBS decreased 0.6 M HCl-induced LP in the GM byapprox. 39% and 27% , respective ly, with similar decreases in LP in the IM. SBS exhibite dbetter protective abilitie s towards 0.6 M HCl and 0.2 m NaOH-induce d GM and IM injury ascompared to CBS. SBS and CBS provide d similar protection towards 80% ethanol-induce dgastric injury, while CBS exerted a superior protective ability towards aspirin-induce d gastricinjury. The results demonstrate that both SBS and CBS can scavenge reactive oxyge n speciesand prevent tissue damage produced by OFR.

KEY WORDS: bismuth subsalicylate ; bismuth subcitrate; catalase , allopurinol; mannitol; gastric mucosa; intestinal

mucosa; superoxide anion; hydroxyl radical; hypochlorite radical.

A growing body of evidence sugge sts that oxygen free

radicals are implicated in the pathogenesis of stress

and chemically induce d gastric lesions and gastroin-

testinal injury (1). Agents that ef® ciently scavenge

Manuscript received February 7, 1997; acce pted June 3, 1997.From the Department of Pharmaceuti cal and Administrative

Sciences, Creighton University School of Pharmacy and AlliedHealth Professions, Omaha, Nebraska.

Address for reprint requests: Dr. Sidney J. Stohs, School ofPharmacy and Allied Health Professions, Creighton University,2500 California Plaza, Omaha, Nebraska 68178.

Digestive Diseases and Sciences, Vol. 42, No. 9 (September 1997), pp. 1890 ± 1900

1890 Digestive Diseases and Sciences, Vol. 42, No. 9 (Septem ber 1997)

0163-2116/97/0900-1890$12.50/0 Ñ 1997 Plenum Publishing Corporation

oxygen free radicals have been shown to protect the

gastrointe stinal mucosa against noxious stimuli (2).

Bismuth compounds have been used as medicinal

agents for more than two centuries, with the ® rst

recorded medical use in 1785. Early applications in-

cluded their use as local demulscents for protective

activitie s on the skin and in the gastrointe stinal tract

to aid the healing of ulcers (3) as well as to treat and

prevent acute diarrhe a (4). Bismuth compounds have

been used to treat gastric duode nal ulcers, dyspepsia,

infectious diarrhe a, and parasitic infections (5). Bis-

muth compounds when administered for a short du-

ration to subje cts with normal renal and hepatic func-

tion are rare ly toxic (5) . Various bismuth salts

including subnitrate , subcarbonate , subgallate , subci-

trate , tartarate , and subsalicylate have been wide ly

used. However, the two bismuth compounds most

commonly used worldwide are colloidal bismuth sub-

citrate (CBS) and bismuth subsalicylate (SBS).

Colloidal bismuth subcitrate (CBS) has been used

successfully in the treatment of both gastric and du-

odenal ulcer diseases, eradication of Helicobacter py-

lori (a bacte ria responsible for various gastrointe stinal

problems), and various gastrointe stinal disorde rs (3,

6, 7). Bismuth subsalicylate (SBS) has been reported

to be effective in the prevention of trave ler’s diarrhe a,

infectious diarrhe a, dyspepsia, and several gastric dis-

orders. However, the mechanism of gastroprote ction

by these bismuth salts is not clearly unde rstood (8 ±

10) . We have hypothe sized that CBS and SBS may

provide gastroprote ction at least in part by attenuat-

ing gastrointe stinal oxidative stress.

The objective of the present studie s was to assess

the relationship between gastrointe stinal oxidative

stress and the comparative gastroprote ctive abilitie s

of CBS and SBS in both in vitro and in vivo models.

Reactive oxygen species including superoxide anion,

hydroxyl radical, and hypochlorite radical plus hypo-

chlorous acid were generated in vitro, and compara-

tive free radical scavenging abilitie s of CBS and SBS

were compared with selected oxygen free radical scav-

engers including superoxide dismutase , catalase,

mannitol, and allopurinol. Furthe rmore, both gastric

mucosa and intestinal mucosa were individually ex-

posed in vitro to these free radical generating systems,

and the concentration-de pendent protective abilitie s

of CBS and SBS were assessed by measuring produc-

tion of lipid peroxidation and compared with the

selected oxyge n free radical scavengers.

In a separate set of experiments, 24-hr fasted fe-

male Sprague -Dawley rats were orally treated with

various necrotizing agents including 0.6 M hydrochlo-

ric acid (HCl), 0.2 M sodium hydroxide (NaOH),

80% ethanol, and aspirin (200 mg/kg) , and the extent

of tissue injury in the gastric mucosa as well as in the

inte stinal mucosa was determined by assessing lipid

peroxidation, DNA fragmentation, and membrane

microviscosity. Time-dependent in vivo protective

abilitie s of CBS (100 mg/kg) and SBS (15 mg/kg) were

also determined.

MATERIALS AND METHODS

In Vitro Generation of Oxygen Free Radicals. Oxygenfree radicals were generated as previously described (11) .To generate superoxide anion radical, xanthine (100 m M) in5 mM Tris HCl buffer was incubated with 8 mU/ml ofxanthine oxidase. The incubation mixture to generate hy-droxyl radical contained in a total volume of 2 ml, 5 mMTris HCl, 100 m M FeCl3, 100 m M EDTA, and 100 m Mxanthine. Xanthine oxidase (8 mU/ml) was added to initiatethe reaction and to produce hydroxyl radicals. Hypochloriteradicals plus hypochlorous acid were generated in 5 mMTris HCl buffer using 1 mM sodium hypochlorite .

Animals and Treatment. Female Sprague-Dawley rats(160 ± 180 g) were obtained from Sasco (Omaha, Nebraska).The animals were housed in a controlled environment of25°C with a 12-hr light and 12-hr dark cycle and wereacclimated for at least three to ® ve days before use. Allanimals were allowed free access to food (Purina RodentLab Chow No. 5001) and tap water. The animals werefasted for 24 hr prior to the administration of the necrotiz-ing agents, including 0.6 M HCl (12, 13) , 0.2 M NaOH (13),80% ethanol (14), and aspirin (200 mg/kg) (15) , which wereadministered orally using a feeding needle in a volume of 1ml. CBS (100 mg/kg) (14) or BSS (15 mg/kg) (16) wereadministered at two different time points. Groups of ani-mals were orally administered either CBS or SBS 30 minprior to or 30 min after the administration of the necrotiz-ing agents. Control animals received the vehicle. Animalswere killed 24 hr following oral administration of 0.6 M HCl(12, 13), while animals treated with either 0.2 M NaOH (12)or 80% ethanol (14) were killed at 1 hr after administrationof necrotizing agent. Animals treated with aspirin (200mg/kg) were killed 16 hr after aspirin treatment (15). Ani-mals were killed by decapitation. The gastric and intestinalmucosa were isolated from these animals as described byHeylings (17). An approval (ARC # 0225) from the Creigh-ton University Animal Research Committee was obtainedfor this project.

Chemicals. Colloidal bismuth subsalicylate (CBS) (BatchNo. 42270; Mfg date July 15, 1992) was obtained as agenerous gift from Yamanouchi Europe B.V. (The Neth-erlands). Bismuth subsalicylate (SBS) (Batch No. 0614; dateNovember 10, 1993) was generously provided by the Procter& Gamble Company (Cincinnati, Ohio). All other chemi-cals used in this study were obtained from Sigma ChemicalCo. (St. Louis, Missouri) and were of analytical grade or thehighest grade available.

Lipid Peroxidation . The formation of thiobarbituric acidreactive substances (TBARS) associated with gastric andintestinal mucosa from control and treated animals was

PROTECTION BY BISMUTH SALTS

1891Digestive Diseases and Sciences, Vol. 42, No. 9 (September 1997)

determined as an index of lipid peroxidation according tothe method of Buege and Aust (18) and Bagchi et al (19).Malondialdehyde was used as the standard. Absorbancevalues were measured at 535 nm and an extinction coef® -cient of 1.56 3 10

5/M/cm was used.

Chemiluminescence. Chemiluminescence as an index ofreactive oxygen species production was measured in a Chro-nolog Lumivette luminometer (Chronolog Corp., Philadel-phia, Pennsylvania). The assay was conducted in 3-ml glassminivials. The vials were incubated at 37°C prior to mea-surement, and the background chemiluminescence of eachvial was checked before use. Samples were preincubated at37°C for 15 min, and 4 m M luminol was added to enhancechemiluminescence. All additions to the vials as well aschemiluminescence counting procedures were performedunder dim lighting conditions. Results were examined ascounts per unit time minus background. Chemilumines-cence was monitored for 6 min at continuous 30-se c inter-vals (20).

DNA Fragm entation . Gastric and intestinal mucosa sam-ples were homogenized in lysis buffer (5 mM Tris HCl, 20mM EDTA, 0.5% Triton X-100, pH 8.0) . Homogenateswere centrifuged at 27,000 g for 20 min to separate intactchromatin in the pellets from fragmented DNA in thesupernatant fractions. Pellets were resuspended in 0.5 Nperchloric acid and 5.5 N perchloric acid was added tosupernatant samples to reach a concentration of 0.5 N.Samples were heated at 90°C for 15 min and centrifuged at1500 g for 10 min to remove protein. Resulting supernatantfractions were reacted with diphenylamine for 16 ± 20 hr atroom temperature, and absorbances were measured at 600nm. DNA fragmentation in control samples is expressed aspercent of total DNA appearing in the supernatant fraction.Treatment effects are reported as percent of control frag-mentation (21).

Membrane Microviscosity. Microviscosity of the gastricmucosa and intestinal mucosa from control and treatedanimals was determined as described previously (20). Thesamples were treated with 0.5 mM diphenyl hexatriene(DPH) in te trahydrofuran as the ¯ uorescent probe andincubated for 2 hr at 37°C (22) . Fluorescence polarizationas a measure of membrane microviscosity was determinedat 25°C within a thermostat cuvette holder in a Perkin-Elmer spectro¯ uorometer equipped with perpendicularand parallel polarizers, using an excitation wavelength of365 nm and an emission wavelength of 430 nm. Fluores-cence polarization and the apparent microviscosity werecalculated as described by Shintzky and Barrenholz (23).

Statistical Analysis. Data for each group were subjectedto analysis of variance (ANOVA). ScheffeÂ ’s S method wasused as the post hoc test. The data are expressed as themean 6 SD of four to six individual incubations or animals.The level of statistical signi ® cance employed in all cases wasP , 0.05.

RESULTS

The in vitro ability of colloidal bismuth subcitrate

(CBS) and bismuth subsalicylate (SBS) to scavenge

bioche mically or chemically generated oxyge n free

radicals in comparison with selected free radical scav-

engers is presented in Table 1. Supe roxide anion,

hydroxyl radicals, and hypochlorite radical plus hypo-

chlorous acid were generated, and the production of

these free radicals was assessed by measuring en-

hanced chemilumine scence , which is a general assay

for the production of reactive oxyge n species (24) .

The response for superoxide anion was inhibite d 86%

by the addition of SOD plus catalase , while hydroxyl

radicals and the hypochlorite radical plus hypochlor-

ous acid generating system were inhibite d 84% and

77% following incubation with mannitol and allopuri-

nol, respectively (Table 1).

Three concentrations (5, 25, and 125 mg/liter) of

both CBS and SBS were assessed with respect to the

scavenging of these oxyge n free radicals. These con-

centrations were selected based on the previous work

of Salme la et al (25) . Approximate ly 3% , 19% , and

48% inhibitions were obse rved in the chemilumine s-

cence response following incubation of the superox-

ide anion generating system with 5, 25, and 125 mg/

lite r concentrations of CBS, respective ly, as compared

to control values (Table 1). Similar concentration

dependent inhibitions were observed with SBS fol-

lowing incubation with the superoxide generating sys-

tem (Table 1).

Following incubation of the hydroxyl radical gen-

erating system with 5, 25, and 125 mg/liter concentra-

tions of CBS, approximate ly 14% , 23% , and 46%

inhibitions in the chemiluminescence response were

obse rved, respectively, while at these same concen-

trations approximate ly 25% , 37% , and 61% inhibi-

tions in chemilumine scence response were obse rved

with SBS, respectively (Table 1).

In the hypochlorite radical plus hypochlorous acid

generating system, approximate ly 9% , 23% , and 51%

inhibitions were observed in the chemiluminescence

response following incubation with 5, 25, and 125

mg/liter concentrations of CBS, respectively, as com-

pared to control value s (Table 1). Similar inhibitions

were observed following incubation of the hypochlo-

rite radical plus hypochlorous acid generating system

with these three concentrations of SBS as compared

to control values (Table 1).

Gastric mucosa (1 mg protein/ml) and intestinal

mucosa (1 mg protein/ml) were individually exposed

to the various oxyge n free radical generating systems,

and the extent of mucosal injury was determined by

measuring lipid peroxidation. The production of thio-

barbituric acid reactive substances (TBARS) as an

index of lipid peroxidation is presented in Table 2.

Malondialde hyde (MDA) was used as the standard.

Following incubation of the superoxide anion gener-

BAGCHI ET AL


ating system with gastric mucosa and inte stinal mu-

cosa, approximate ly 2.3-fold increases in lipid peroxi-

dation were obse rved with both tissues as compared

to control value s. Supe roxide anion-induce d gastric

mucosal lipid peroxidation was inhibite d by 87% with

a combination of SOD (200 m g/ml) and catalase (200

m g/ml) , while with this combination of SOD plus

catalase approxim ate ly 100% protection was ob-

served against superoxide anion-induce d intestinal

mucosal lipid peroxidation (Table 2).

Hydroxyl radical increased lipid peroxidation in the

gastric mucosa and in the intestinal mucosa by ap-

proximate ly 2.8-fold as compared to control values.

Hydroxyl radical-induce d gastric mucosal and intesti-

nal mucosal lipid peroxidation were inhibite d by 80%

and 86% , respective ly, following incubation with

mannitol (1.25 m M), as compared to control value s.

Hypochlorite radical plus hypochlorous acid in-

creased lipid peroxidation by approximate ly 2.0- and

2.1-fold in gastric mucosa and intestinal mucosa, re-

spective ly. Following incubation with allopurinol

(1.47 mM), approximate ly 94% and 90% inhibitions

were obse rved in the gastric mucosal and intestinal

mucosa lipid peroxidation, respectively.

The concentration-de pendent comparative protec-

tive abilitie s of CBS and SBS against oxyge n free

radical-induce d gastric mucosal and intestinal muco-

sal lipid peroxidation are also presented in Table 2.

Concentration dependent protection against lipid

peroxidation was provide d by both CBS and SBS. At

TABLE 1. In Vitro GENERATION OF SUPEROXIDE ANION, HYDROXYL RADICAL, AND HYPOCHLORITE

RADICAL PLUS HYPOCHLOROUS ACID, AND INHIBITION BY COLLOIDAL BISMUTH SUBCITRATE (CBS) AND

BISMUTH SUBSALICYLATE (SBS) AND SELECTED FREE RADICAL SCAVENGERS*

Sam pleChem iluminescence response

(cpm)

Control 522 6 78a

CBS control

CBS (5 mg/lite r) 611 6 111a

CBS (25 mg/lite r) 747 6 181a

CGS (125 mg/lite r) 672 6 179a

SBS control

SBS (5 mg/lite r) 582 6 123a

SBS (25 mg/liter) 807 6 153b

SBS (125 mg/lite r) 702 6 108a,b

Superoxide anion

Xanthine (XN) 1 xanthine oxidase (XO) 8034 6 1112a

1 SOD (200 m g/ml) 1 catalase 1 (200 m g/ml) 1537 6 470b

1 CBS (5 mg/liter) 7652 6 954a

1 CBS (25 mg/lite r) 6628 6 1135a

1 CBS (125 mg/liter) 4450 6 529c

1 SBS (5 mg mg/liter) 7514 6 823a

1 SBS (25 mg/liter) 6706 6 624a

1 SBS (125 mg/liter) 4473 6 414c

Hydroxy radicalXN 1 FeCl3 1 EDTA 1 XO 9145 6 715a

1 Mannitol (1.25 m M) 1858 6 388b

1 CBS (5 mg/liter) 7969 6 1263a,d

1 CBS (25 mg/lite r) 7154 6 1151a,d

1 CBS (125 mg/liter) 5187 6 843c

1 SBS (5 mg/lite r) 7024 6 1037d

1 SBS (25 mg/liter) 5947 6 855c

1 SBS (125 mg/liter) 3851 6 402e

Hypochlorite radical plus hypochlorous acid

Sodium hydrochlorite 7845 6 1323a

1 Allopurinol (1.47 mM) 2218 6 439b

1 CBS (5 mg/liter) 7218 6 795a

1 CBS (25 mg/lite r) 6616 6 542a

1 CBS (125 mg/liter) 4124 6 815c

1 SBS (5 mg/lite r) 7431 6 670a

1 SBS (25 mg/liter) 6085 6 913a

1 SBS (125 mg/liter) 4434 6 513c

* The reactive oxygen species were generate d as described in the Materials and Methods section.

Chemiluminescence was measured in a Chronolog Lumivette Luminometer using 4 m M luminol. Eachvalue is the mean 6 SD of 4 ± 6 incubations. Values with different superscripts are signi ® cantly different

(P , 0.05) .



a concentration of 125 mg/liter, SBS inhibite d hy-

droxyl radical-mediated gastric and inte stinal mucosal

lipid peroxidation by 73% and 72% , respectively, as

compared to 30% and 28% , respective ly, for CBS. At

the same concentration, SBS inhibite d gastric and

inte stinal superoxide anion mediated injury by 94%

and 87% , respective ly, as compared to 90% and 70% ,

respectively, for CBS. Both CBS and SBS demon-

strated similar protective abilitie s (87± 92% inhibi-

tion) towards hypochlorite radical plus hypochlorous

acid-induce d mucosal lipid peroxidation.

Since lipid peroxidation occurs in response to free

radicals and reactive oxygen species, the in vivo effects

of 0.6 M HCl, 0.2 M NaOH, 80% ethanol, and aspirin

(200 mg/kg) were assessed on increased lipid peroxi-

dation in both gastric and inte stinal mucosa. The

production of TBARS as an index of lipid peroxida-

tion is presented in Table 3. The results indicate that

administration of 0.6 M HCl, 0.2 M NaOH, 80%

ethanol, and aspirin (200 mg/kg) increased gastric

mucosal lipid peroxidation by approximate ly 3.1-,

2.0-, 2.2-, and 3.0-fold, respective ly, while with these

same necrotizing agents approximate ly 3.7-, 2.1-, 2.5-,

and 3.5-fold increases, respective ly, were obse rved in

lipid peroxidation in the inte stinal mucosa (Table 3).

CBS and SBS were administe red intragastrically 30

min prior to or 30 min after the administration of the

necrotizing agents.

Pretreatment of the rats with CBS (15 mg/kg) and

SBS (100 mg/kg) decreased 0.6 M HCl-induce d gas-

TABLE 2. EFFECTS OF REACTIVE OXYGEN SPECIES ON GASTRIC AND INTESTINAL MUCOSAL LIPID PEROXIDATION , AND

CONCENTRATION -DEPENDENT PROTECTIVE ABILITIES OF COLLOIDAL BISMUTH SUBCITRATE (CBS) AND

BISMUTH SUBSALICYLATE (SBS)*

G roups

Lipid peroxidation [TBARS Conten t (nmol/m g protein)]

G astric mucosa % Control Intestinal mucosa % Control

Control 1.42 6 0.31a 1.51 6 0.32a

1 CBS (5 mg/lite r) 1.39 6 0.35a

1.67 6 0.29a

1 CBS (25 mg/liter) 1.71 6 0.23a 1.81 6 0.31a

1 CBS (125 mg/liter) 1.68 6 0.60a

1.76 6 0.18a

1 SBS (5 mg/liter) 1.65 6 0.35a 1.75 6 0.44a

1 SBS (25 mg/liter) 1.87 6 0.49a

1.86 6 0.30a

1 SBS (125 mg/lite r) 1.81 6 0.52a 1.87 6 0.17a

Superoxide anion gene rating systemSuperoxide anion 3.23 6 0.47a 228 3.42 6 0.52a 227

1 SOD 1 catalase 1.61 6 0.52b

113 1.42 6 0.34b

94

1 CBS (5 mg/liter) 2.83 6 0.40a 204 3.11 6 0.37a 186

1 CBS (25 mg/lite r) 2.34 6 0.40c

137 2.63 6 0.47a,c

145

1 CBS (125 mg/liter) 1.85 6 0.25c 110 2.28 6 0.45c 130

1 SBS (5 mg/lite r) 2.69 6 0.69a

163 2.97 6 0.22a

170

1 SBS (25 mg/liter) 2.36 6 0.42c 126 2.75 6 0.35a,c 148

1 SBS (125 mg/liter) 1.92 6 0.30c

106 2.12 6 0.37c

113Hydroxyl radical generating system

Hydroxyl radical 4.03 6 0.50a

284 4.29 6 0.39a

284

1 Mannitol (1.25 m M) 1.70 6 0.25b 120 1.72 6 0.24b 114

1 CBS (5 mg/liter) 3.43 6 0.41a,c

247 3.83 6 0.21a

229

1 CBS (25 mg/lite r) 3.11 6 0.55a 182 3.48 6 0.56a,c 192

1 CBS (125 mg/liter) 2.86 6 0.44c,d

170 3.03 6 0.32c

172

1 SBS (5 mg/lite r) 3.24 6 0.37a,c 196 3.51 6 0.23a,c 201

1 SBS (25 mg/liter) 2.86 6 0.51c,d

153 2.80 6 0.29d

151

1 SBS (125 mg/liter) 2.30 6 0.58d 127 2.40 6 0.47d 128

Hypochlorite radical plus hypochlorous acid generating systemOCl 1 HOCl 2.90 6 0.23a 204 3.11 6 0.40a 206

1 Allopurinol (1.47 mM) 1.50 6 0.23b

106 1.66 6 0.11b

110

1 CBS (5 mg/liter) 2.35 6 0.38a,d 169 2.68 6 0.48a,c 161

1 CBS (25 mg/lite r) 2.00 6 0.36c

117 2.24 6 0.19c

124

1 CBS (125 mg/liter) 1.81 6 0.42b,c 108 1.90 6 0.36b,c 108

1 SBS (5 mg/lite r) 2.57 6 0.54a,d

156 2.59 6 0.38a

148

1 SBS (25 mg/liter) 2.28 6 0.43d 122 2.38 6 0.44c 128

1 SBS (125 mg/liter) 2.01 6 0.40c,d

111 2.11 6 0.41c

113

* The reactive oxygen species were generated as described in the Mate rials and Methods section. Gastric or intestinal mucosa

(1 mg protein/ml) was added in each experime nt to asse ss the degree of lipid peroxidation underthe in¯ uence of these oxyge nfree radicals and the comparative protective abilities of CBS and SBS. The production of thiobarbituric acid reactive

sutstances (TBARS) was measure d as an index of lipid peroxidation. Each value presents the mean 6 SD of 4 ± 6 incubations.Values with different superscripts are signi® cantly different (P , 0.05) .

BAGCHI ET AL


tric mucosal lipid peroxidation by approxim ately 27%

and 39% , respective ly, as compared to control rats,

while unde r these same conditions similar decreases

in inte stinal mucosal lipid peroxidation were obse rved

(Table 3). Following posttreatment of the rats with

CBS and SBS, approxim ate ly 19% and 30% de-

creased in gastric mucosal lipid peroxidation were

obse rved, respective ly, while unde r these same con-

ditions approximate ly 14% and 25% decreases in

lipid peroxidation were observed in the intestinal

mucosa, respective ly, as compared to control value s

(Table 3).

Prior administration of CBS and SBS decreased 0.2

M NaOH-induce d gastric mucosal lipid peroxidation

by approxim ately 44% and 36% , respectively, as com-

pared to control value s, while these bismuth salts

decreased intestinal mucosal lipid peroxidation by

approxim ate ly 15% and 26% , respective ly, unde r

these same conditions (Table 3). Posttreatment of the

rats with CBS and SBS decreased 0.2 M NaOH-

induced gastric mucosal lipid peroxidation by approx-

imate ly 22% and 43% , respective ly, as compared to

control value s, while posttreatment with CBS and

SBS decreased intestinal mucosal lipid peroxidation

by approximate ly 36% and 33% , respective ly, unde r

these same conditions (Table 3).

Pretreatment of the rats with CBS and SBS re-

duced 80% ethanol-induce d gastric mucosal lipid per-

oxidation by approximate ly 10% and 23% , respec-

tively, as compared to control value s, while unde r

these same conditions CBS and SBS decreased intes-

tinal mucosal lipid peroxidation by approxim ately

11% and 13% , respective ly (Table 3). Administration

of CBS and SBS 30 min after the administration of

80% ethanol decreased gastric mucosal lipid peroxi-

dation by approximate ly 8% and 11% , respectively,

while unde r these same conditions approximate ly

11% and 21% decreases in intestinal mucosal lipid

peroxidation were obse rved, respectively, as com-

pared to control value s (Table 3).

Administration of CBS and SBS to rats 30 min

prior to the administration of aspirin (200 mg/kg)

decreased gastric mucosal lipid peroxidation by ap-

proximate ly 38% and 21% , respective ly, while similar

TABLE 3. NECROTIZING AGENT-INDUCED MODULATION OF LIPID PEROXIDATION IN GASTRIC AND

INTESTINAL MUCOSA, AND COMPARATIV E PROTECTIVE ABILITIES OF COLLOIDAL BISMUTH SUBCITRATE

(CBS) AND BISMUTH SUBSALICYLATE (SBS)*

G roup

Lipid peroxidation [TBARS (nm ol/mg protein)]

G astric mucosa Intestinal mucosa

Control 1.53 6 0.20a 1.33 6 0.11a

CBS only (1 hr) 2.70 6 0.44b

2.09 6 0.25b,c

CBS only (24 hr) 3.18 6 0.81b 2.63 6 0.50c

SBS only (1 hr) 2.40 6 0.37b

1.72 6 0.20b

SBS only (24 hr) 2.88 6 0.36b 2.14 6 0.55b

NaOH 0.2 M (1 hr) 3.11 6 0.25b

2.81 6 0.24c

CBS per NaOH 1.75 6 0.22a,c 2.38 6 0.22b,c

CBS post-NaOH 2.43 6 0.30b,c

1.79 6 0.14b

SBS pre-NaOH 2.00 6 0.26c 2.09 6 0.17b

SBS post-NaOH 1.78 6 0.19a,c

1.88 6 0.20b

HCl 0.6 M (24 hr) 4.79 6 0.47d 4.92 6 0.93d

CBS pre-HCl 3.52 6 0.69c

3.81 6 0.35c

CBS post-HCl 3.88 6 0.46c 4.24 6 0.40d,e

SBS pre-HCl 2.92 6 0.54b

3.11 6 0.38c

SBS post-HCl 3.34 6 0.54c 3.69 6 0.43c,e

Aspirin (200 mg/kg; 16 hr) 4.59 6 0.43d

4.71 6 0.30d

CBS pre-aspirin 2.84 6 0.34b,c 3.16 6 0.38c

CBS post-aspirin 3.29 6 0.70e

3.73 6 0.60e

SBS pre-aspirin 3.63 6 0.41e 3.89 6 0.86e

SBS post-aspirin 4.03 6 0.37e

3.97 6 0.67EtOH (80% , 1 hr) 3.40 6 0.98e 3.28 6 0.65c

CBS pre-EtOH 3.08 6 0.63b

2.91 6 0.50c

CBS post-EtOH 3.14 6 0.61b 2.93 6 0.53c

SBS pre-EtOH 2.62 6 0.29b

2.85 6 0.30c

SBS post-EtOH 3.01 6 0.38b 2.61 6 0.49c

* Female Sprague-Dawley rats were treated orally with either 0.6 m HCl, 0.2 M NaOH, 80% ethanol, or200 mg/kg aspirin. CBS or SBS was administered e ither 30 min prior to the administration of these

mecrotizing agents or 30 min after the administration of the necrotizing gents. Each value is the mean 6SD of 4 ± 6 animals. Malondialdehyde was used as the standard. Values with different superscripts are

signi® cantly different (P , 0.05) .



results were observed with respect to intestinal mu-

cosal lipid peroxidation (Table 3). Administration of

CBS and SBS to rats 30 min after the administration

of aspirin (200 mg/kg) decreased gastric mucosal lipid

peroxidation by approximate ly 28% and 12% , respec-

tively, as compared to control value s, while CBS and

SBS decreased intestinal mucosal lipid peroxidation

by approximate ly 21% and 15% , respective ly, unde r

the same conditions (Table 3).

Steady-state ¯ uorescence spectroscopy was utilized

to evaluate change s in membrane microviscosity as a

result of administration of the necrotizing agents 0.6

M HCl, 0.2 M NaOH, 80% ethanol, and aspirin (200

mg/kg) to rats, and the results are presented in Table

4. Membrane microviscosity is inversely proportional

to the membrane ¯ uidity. Following intragastric ad-

ministration of 0.6 M HCl, 0.2 M NaOH, 80% etha-

nol, and aspirin (200 mg/kg) , approxim ately 8.6-, 4.9-,

5.8-, and 7.4-fold increases, respective ly, in mem-

brane microviscositie s were obse rved in the gastric

mucosa, while unde r the same conditions approxi-

mate ly 13.5-, 9.6-, 9.1-, and 13.0-fold increases in

inte stinal mucosal membrane microviscositie s were

obse rved, respective ly.

Following administration of CBS and SBS 30 min

prior to the administration of 0.6 M HCl, approxi-

mate ly 48% and 68% decreases in membrane micro-

viscositie s were obse rved in the gastric mucosa, re-

spective ly, while CBS and SBS similarly decreased

membrane microviscositie s in the inte stinal mucosa

as compared to the control value s (Table 4). Post-

treatment of the rats with CBS and SBS decreased

gastric mucosal membrane microviscositie s by ap-

proximate ly 29% and 55% , respectively, as compared

to control value s, and decreased inte stinal mucosal

membrane microviscosities by approximate ly 47%

and 66% , respective ly, under the same conditions

(Table 4).

Following administration of CBS and SBS 30 min

prior to the administration of 0.2 M NaOH, approx-

imate ly 44% and 52% decreases in gastric mucosa

membrane microviscositie s were obse rved, respec-

tively, as compared to control values, while unde r the

same conditions approxim ate ly 59% decreases in

TABLE 4. NECROTIZING AGENT-INDUCED CHANGES IN MEMBRANE MICROVISCOSITY IN GASTRIC AND

INTESTINAL MUCOSA, AND PROTECTIVE ABILITIES OF COLLOIDAL BISMUTH SUBCITRATE (CBS) AND

BISMUTH SUBSALICYLATE (SBS)*

G roup

Membrane microviscosity (in poise)

G astric mucosa Intestinal mucosa

Control 0.18 6 0.01a 0.11 6 0.2a

CBS only (1 hr) 0.34 6 0.09b

0.28 6 0.03b

CBS only (24 hr) 0.35 6 0.05b 0.30 6 0.06b

SBS only (1 hr) 0.25 6 0.04c

0.18 6 0.03c

SBS only (24 hr) 0.28 6 0.07c 0.25 6 0.08b

NaOH 0.2 M (1 hr) 0.86 6 0.11d

1.01 6 0.15d

CBS pre-NaOH 0.48 6 0.06e 0.41 6 0.03e

CBS post-NaOH 0.60 6 0.03b

0.50 6 0.03f

SBS pre-NaOH 0.41 6 0.08e 0.42 6 0.03e

SBS post-NaOH 0.51 6 0.06e

0.51 6 0.05f

HCl 0.6 M (24 hr) 1.51 6 0.25g 1.42 6 0.28g

CBS pre-HCl 0.78 6 0.19d

0.61 6 0.09f,h

CBS post-HCl 1.08 6 0.13b 0.76 6 0.08h

SBS pre-HCl 0.49 6 0.12e,f

0.43 6 0.07e,f

SBS post-HCl 0.68 6 0.08f 0.48 6 0.07f

Aspirin (200 mg/kg; 16 hr) 1.31 6 0.46g

1.36 6 0.34g

CBS pre-aspirin 0.44 6 0.14b,e 0.43 6 0.07f

CBS post-aspirin 0.49 6 0.15e,f

0.45 6 0.11e,f

SBS pre-aspirin 0.61 6 0.12e,f 0.70 6 0.26h

SBS post-aspirin 0.70 6 0.14f

0.63 6 0.11f,h

EtOH (80% , 1 hr) 1.02 6 0.21h 0.95 6 0.22d

CBS pre-EtOH 0.37 6 0.09b

0.51 6 0.06f

CBS post-EtOH 0.45 6 0.12b,e 0.64 6 0.07f,h

SBS pre-EtOH 0.38 6 0.06b

0.49 6 0.08f

SBS post-EtOH 0.48 6 0.05b,e 0.60 6 0.08f,h

* Female Sprague-Dawley rats were orally treated with e ither 0.6 M HCl, 0.2 M NaOH, 80% ethanol, or200 mg/kg aspirin. CBS or SBS was administered e ither 30 min prior to the administration of these

necrotizing age nts or 30 min after the administration of the necrotizing agents. Each value is the mean 6SD of 4 ± 6 animals. Values with different superscripts are signi® cantly different (P , 0.05) .

BAGCHI ET AL


membrane microviscositie s were obse rved in intesti-

nal mucosa (Table 4). Following treatment of the rats

with CBS and SBS 30 min after the administration of

0.2 m NaOH, approximate ly 30% and 41% decreases

in membrane microviscositie s were obse rved in the

gastric mucosa, respective ly, as compared to control

rats, with approximate ly 51% and 49% decreases,

respectively, in the membrane microviscosity of the

inte stinal mucosa (Table 4).

Pretreatment of rats with CBS and SBS 30 min

prior to the administration of 80% ethanol resulted in

approximate ly 64% decreases in membrane microvis-

cositie s in the gastric mucosa, while decreasing intes-

tinal mucosal membrane microviscosity by approxi-

mate ly 46% and 49% , respective ly, as compared to

control rats (Table 4). Administration of CBS and

SBS to rats 30 min after the administration of 80%

ethanol decreased the membrane microviscositie s in

the gastric mucosa by approximate ly 56% and 53% ,

respectively, as compared to control animals, with

approximate ly 33% and 37% decreases, respective ly,

in membrane microviscositie s in the intestinal mucosa

(Table 4).

Following the administration of CBS and SBS 30

min prior to the administration of aspirin (20 mg/kg) ,

approximate ly 66% and 53% decreases in membrane

microviscositie s were obse rved in the gastric mucosa,

respectively, with similar decreases in membrane mi-

croviscositie s in aspirin-induced intestinal mucosa

(Table 4). Oral administration of CBS and SBS 30

min after the administration of aspirin (200 mg/kg)

decreased membrane microviscositie s in the gastric

and intestinal mucosa by amounts similar to the pre-

treatment with CBS and SBS (Table 4).

Programme d cell death (apoptosis) is a selective

process of physiological cell de le tion that plays a

major role in developmental biology and in the main-

tenance of homeostasis in vertebrates. Fragmentation

of nuclear DNA is a biochemical hallmark of apopto-

sis. DNA fragmentation in the gastric and intestinal

mucosa induce d by the necrotizing agents 0.6 M HCl,

0.2 M NaOH, 80% ethanol, and aspirin (200 mg/kg)

are shown in Table 5, with increases of approximate ly

2.4-, 2.3-, 2.3-, and 2.5-fold occurring in gastric mu-

cosal DNA fragmentation values, respective ly, as

compared to control values. Under the same condi-

TABLE 5. NECROTIZING AGENT-CAUSED DNA FRAGMENTATION IN GASTRIC AND INTESTINAL MUCOSA, AND PROTECTIVE

ABILITIES OF COLLOIDAL BISMUTH SUBCITRATE (CBS) AND BISMUTH SUBSALICYLATE (SBS)*

G roup

DNA fragm entation (% con trol)

G astric mucosa

G astric mucosa

(% control) Intestinal Mucosa

Intestinal mucosa

(% control)

Control 4.11 6 0.57a 100 3.77 6 0.43a 100

CBS only (1 hr) 5.18 6 0.66c

126 5.38 6 0.49b

143CBS only (24 hr) 5.37 6 0.41b 131 5.21 6 0.52b 138

SBS only (1 hr) 4.92 6 0.43a

120 4.73 6 0.59b

125SBS only (24 hr) 5.48 6 0.59a 133 5.13 6 0.81b 136

NaOH 0.2 M (1 hr) 9.41 6 0.70c

229 9.56 6 0.48c

254CBS pre-NaOH 7.28 6 0.60d,e 177 7.49 6 0.76d,f 199

CBS post-NaOH 7.77 6 0.93d

189 7.50 6 0.66d,f

199SBS pre-NaOH 6.21 6 0.43e 151 6.47 6 0.69d 172

SBS post-NaOH 6.46 6 0.75c

157 6.81 6 0.53d

181HCl 0.6 M (24 hr) 9.95 6 0.98c 242 11.00 6 1.04e 292

CBS pre HCl 7.55 6 0.69d

184 7.90 6 0.52f

210CBS post HCl 7.64 6 1.23d 186 8.09 6 0.42f 215

SBS pre HCl 6.51 6 0.50e

158 6.63 6 0.79d

176SBS post HCl 6.60 6 0.84e 161 6.96 6 0.85d 185

Aspirin (200 mg/kg; 16 hr) 10.33 6 0.61c

251 11.44 6 1.06e

303CBS pre-aspirin 6.42 6 0.58e 156 7.64 6 1.35d 203

CBS post-aspirin 6.67 6 1.04e

162 7.69 6 0.93c

204SBS pre-aspirin 7.88 6 1.17d 192 8.51 6 0.63c 226

SBS post-aspirin 8.27 6 0.81d

201 8.82 6 0.44c

234EtOH (80% ; 1 hr) 9.25 6 0.45c,d 225 9.37 6 0.61c 249

CBS pre-EtOH 6.77 6 0.96e

165 6.48 6 1.36d

172CBS post-EtOH 7.11 6 0.90e 173 6.95 6 0.61d 184

SBS pre-EtOH 7.03 6 0.76e

171 6.80 6 0.60d

180SBS post-EtOH 7.06 6 0.66e 172 6.84 6 0.49d 181

* Female Sprague -Dawley rats were orally treated with either o.6 M HCl, 0.2 M NaOH, 80% ethanol, or 200 mg/kg aspirin. CBSor SBS was administered either 30 min prior to the administration of these necrotizing agents or 30 min after the

administration of the necrotizing age nts. Each value is the mean 6 SD of 4 ± 6 animals. Values with different superscripts aresigni® cantly different (P , 0.05) .



tions the necrotizing agents caused 2.9-, 2.5-, 2.5-, and

3.0-fold increases in DNA fragmentation in the intes-

tinal mucosa, respective ly, as compared to control

value s (Table 5).

The comparative protective abilitie s of CBS and

SBS towards necrotizing agent-induce d gastric and

inte stinal mucosal DNA damage are presented as

percent of controls in Table 5. The administration of

both CBS and SBS before and after ethanol treat-

ment in animals resulted in similar decreases in DNA

damage in both gastric and intestinal mucosa. In

animals given eithe r 0.2 M NaOH or 0.6 M HCl, SBS

pre- and posttreatment provide d 20 ± 30% greater

protection in both gastric and inte stinal mucosa

against DNA fragmentation than CBS. In these stud-

ies, SBS provide d 20 ± 25% greater protection against

DNA damage in gastric mucosa than in intestinal

mucosa. In animals given aspirin (200 mg/kg) , approx-

imate ly 30% greater protection against DNA damage

was provide d by CBS in gastric mucosa as compared

to the inte stinal mucosa. Greater protection was pro-

vided when CBS was given 30 min prior to the aspirin.

DISCUSSION

Bismuth preparations are commonly used to treat a

varie ty of gastrointe stinal disorde rs including peptic

ulce rs, dyspepsia, infectious diarrhea, and parasitic

infections. Agents that provide protection against gas-

tric mucosal injury by a mechanism other than inhi-

bition or neutralization of gastric acid have been

described as cytoprotectants (10) . Several mecha-

nisms of gastric cytoprotection have been propose d.

However, the mechanism associated with cytoprote c-

tin by bismuth salts is not clearly unde rstood.

Naganuma et al (8) demonstrated that bismuth

subnitrate signi® cantly decreased the le thal toxicity,

cardiotoxicity, and bone marrow toxicity of adriamy-

cin. In a subsequent study, Nakagawa et al (26) dem-

onstrated that pretreatment with bismuth nitrate sig-

ni® cantly prevented the clastoge nicity of adriamycin,

cyclophosphamide , cisplatin, and L-phenylalanine

mustard. Since a signi® cant increase in tissue concen-

trations of metallothion ein is obse rved following

treatment with bismuth subnitrate , the authors con-

cluded that the ability of bismuth subnitrate to reduce

toxicity of adriamycin and clastogenicity of various

antineoplastic drugs may be ascribed to the induction

of metallothione in. These authors concluded that the

protective effect of metallothione in might be due to

its ability to scavenge free radicals or inhibit their

formation. However, recent studie s by Klaasen (per-

sonal communication) have demonstrated that the

toxicity of adriamycin is not alte red in metallothio-

ne in transgenic mice or metallothione in null (knock-

out) mice. Furthermore , the effect of bismuth on

adriamycin toxicity did not differ between these ge-

netically altered mice and control animals. Thus, it

appears that metallothione in may not be involve d in

the chemoprote ctive effects of bismuth salts.

Forsell (9) propose d that bismuth acts as a mild

irritant and induces liberation of endoge nous prosta-

glandins in mucosa, and the prostaglandins maintain

blood ¯ ow and prevent vascular injury caused by

irritants as ethanol. This hypothe sis has not been

con® rmed.

Although bismuth salts are widely used as gastro-

inte stinal chemoprote ctants, and reactive oxygen spe-

cies are be lieved to participate in many gastrointe sti-

nal disorders (1), no studies have been conducte d to

assess the abilitie s of bismuth salts to scavenge reac-

tive oxyge n species or the abilitie s of bismuth salts to

provide protection against reactive oxygen species-

induced tissue damage . The present studies have as-

sessed the abilitie s of colloidal bismuth subcitrate

(CBS) and bismuth subsalicylate (SBS) to scavenge

various forms of reactive oxygen species and prevent

tissue damage in both in vitro and in vivo systems.

Furthermore , the radical-scave nging abilitie s of these

two bismuth salts were compared with commonly

used scavengers of reactive oxyge n species.

Initial studie s (Table 1) indicated that both CBS

and SBS produce d concentration-de pendent inhibi-

tions in the chemically or bioche mically generated

production of superoxide anion, hydroxyl radical, and

hypochlorite radical plus hypochlorous acid in vitro.

In these studies, SOD plus catalase effective ly inhib-

ited superoxide anion, mannitol effective ly inhibite d

hydroxyl radical, and allopurinol effective ly scavenged

hypochlorite radical plus hypochlorous acid. Thus,

these results suggest that both bismuth salts exhibit

the ability to scavenge or quench various oxyge n free

radicals or prevent the ir formation.

In order to determine whether these bismuth salts

could prevent oxidative tissue damage to gastric and

inte stinal mucosa, mucosal tissues were incubate d

with reactive oxygen species generating systems in the

presence and absence of the bismuth salts (Table 2).

The results clearly demonstrate that both bismuth

salts produce concentration-de pendent inhibition of

lipid peroxidation in gastric and intestinal mucosa,

which was produce d by using a superoxide anion

generating system, a hydroxyl radical generating sys-

tem, and a hypochlorite radical plus hypochlorous

BAGCHI ET AL


acid generating system. CBS and SBS produce similar

effects with respect to the inhibition of lipid peroxi-

dation in the presence of the superoxide anion gen-

erating system and hypochlorite radical generating

system. In the presence of the hydroxyl radical gen-

erating system, SBS appeared to produce a small but

nonstatistically greater protective effect as compared

to CBS at the highe st concentration of each that was

used. The results of this study (Table 2) clearly dem-

onstrate that these oxygen free radical generating

systems can produce lipid peroxidation in vitro when

incubate d with gastric and inte stinal mucosa, and

coincubation with eithe r CBS or SBS provide s con-

centration-de pendent inhibition of lipid peroxidation.

Thus, the results indicate that these bismuth salts can

provide protection against oxygen free radical in-

duced tissue injury in gastrointe stinal mucosa.

In order to determine whether CBS and SBS could

provide chemoprotection in vivo, these bismuth salts

were administered eithe r 30 min before or 30 min

after administration of the necrotizing agents 0.2 M

NaOH, 0.6 M HCl, 80% ethanol, and aspirin (200

mg/kg). The effects of these necrotizing agents on

lipid peroxidation (Table 3), membrane microviscos-

ity (Table 4), and DNA fragmentation (Table 5) in

gastric and intestinal mucosa were assessed. The re-

sults demonstrated that these necrotizing agents pro-

duced 2.0 to 3.7-fold increases in lipid peroxidation in

gastric and intestinal mucosa under the conditions

that were employed (Table 3), while unde r the same

conditions, increases in membrane microviscositie s as

great as 13.5-fold were observed (Table 4). Further-

more, these necrotizing agents produced 2.3- to 3.0-

fold increases in DNA fragmentation in gastric and

inte stinal mucosa (Table 5). The results also clearly

demonstrated that administration of CBS and SBS

before and after necrotizing agents partially pre-

vented increases in lipid peroxidation, membrane mi-

croviscositie s, and DNA fragmentation.

The results of the in vitro and in vivo studie s that

have been conducte d support the hypothe sis that the

cytoprotective abilitie s of bismuth salts as SBS and

CBS at least in part involve the abilitie s of these

bismuth salts to scavenge or prevent the formation of

reactive oxyge n species and prevent oxidative tissue

damage by reactive forms of oxyge n. The current

studie s do not preclude the possibility that bismuth

salts may induce prostaglandin formation, which may

provide partial protection through various regulatory

mechanisms (9). Since the bismuth salts that were

studied provide protection both in vitro and in vivo, it

is doubtful that these bismuth salts are acting through

an indirect mechanism, as for example, the induction

of metallothione in.

In summary, the results of the present study have

provide d extensive information on the possible mech-

anism of bismuth salts as chemoprote ctants, as well as

the possible role of reactive oxyge n species in stress-

related oxidative tissue damage in gastric and intesti-

nal mucosa.

ACKNOWLEDGMENTS

These studies were supported in part by a grant from theProcter & Gamble Company. The authors thanks Ms. Lu-Ann Schwery for technical assistance.

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BAGCHI ET AL


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Protection Against Chemically-Induced Oxidative Gastrointestinal Tissue Injury in Rats by Bismuth Salts