Chemical Pathology Research Division, Departments of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115
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ABSTRACT |
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We tested the hypothesis that
recognized gastroprotective agents exert direct protection against
ethanol-induced injury in isolated rat gastric mucosal cells in
vitro. If protection exists, we also wanted to identify
subcellular targets in the reversible and/or irreversible stages of
cell injury. Ethanol-induced cell injury was quantified by measuring
plasma membrane leakage (trypan blue exclusion and lactate
dehydrogenase release), mitochondrial integrity (succinic
dehydrogenase), and nuclear damage (ethidium bromide-DNA fluorescence).
Initial cell viability and responsiveness were estimated by the effects
of carbachol, carbachol + atropine, or
16,16-dimethyl-PGE2 on chief cell pepsinogen secretion.
Enriched parietal cells were stimulated by histamine, carbachol, or
histamine + IBMX. Preincubation of cells with PG, sucrose
octasulfate, or the sulfhydryl compounds N-acetylcysteine,
taurine, or cysteamine increased cell resistance 21% against
ethanol. Similar protection was found with low histamine
concentrations, but a higher concentration aggravated ethanol toxicity.
Other naturally occurring or synthetic gastroprotective agents offered
partial protection or aggravated ethanol-induced cell injury. Only a
few in vivo gastroprotective agents demonstrated in vitro direct
cytoprotection, which involved mainly the reversible stage of cell
injury (e.g., plasma membrane changes) and, less often, irreversible
(e.g., mitochondrial and nuclear) damage. Our findings also indicate
that a major part of the beneficial effect of gastroprotective agents
is expressed at the tissue level.
ethanol-induced cell injury; plasma membrane; mitochondria; nuclear damage; direct cytoprotection
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INTRODUCTION |
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THE TERM "CYTOPROTECTION" was introduced into gastrointestinal pathophysiology and pharmacology by A. Robert to describe the prevention of acute hemorrhagic gastric erosions produced by concentrated ethanol, hydrochloric acid, sodium hydroxide, sodium chloride, or boiling water by non-antisecretory doses of PGs in rats (28, 29). Although the basic biochemical mechanisms of gastric mucosal injury and protection remain unclear and controversial, the list of natural and synthetic "cytoprotective" compounds has been increasing. In vivo, chemically induced hemorrhagic mucosal lesions (HML) were also decreased by sulfhydryl (SH) compounds (10, 30, 39, 42), protease inhibitors (23, 39, 40), somatostatin (43), growth factors (14, 47), histamine (26), dopamine and related drugs (17, 27), gangliosides (35, 37), carotenoids (22), sucralfate and its derivatives (12, 36), antacids (24, 44), and spasmolytics (11, 32).
The original concept of cytoprotection has been criticized because of the incomplete protection by exogenous PG against chemically induced gastric HML. That is, histological and electron microscopic investigations revealed that only the deep hemorrhagic erosions could be reduced by PG or other cytoprotective compounds, whereas the surface cell damage was not decreased (10, 12, 16, 30, 44). Thus the terms "histoprotection" and "organoprotection" were suggested to reflect protection at the tissue and organ levels (41, 43). Tarnawski and colleagues (45) found a limited but statistically significant direct protection by PG of human isolated gastric glands against indomethacin- or ethanol-induced damage. Ivey and co-workers (31, 46) demonstrated that the chemically induced damage was diminished by PG and SH derivatives in cultured and transformed epithelial surface cells. Because the gastric mucosa contains numerous cell types, data are lacking concerning the possible direct protection of parietal, chief, and neuroendocrine cells by cytoprotective agents. Thus we tested the hypothesis that old and new cytoprotective compounds might exert direct protection on a mixed population of gastric mucosal cells (GMC). We also wanted to identify subcellular targets in the interaction of gastroprotective agents and ethanol. For this purpose, a method was adapted in our laboratory for the isolation of a mixed population of rat GMC with long viability and preserved membrane receptor sensitivity using minimal amounts of pronase and calcium-binding EGTA (13, 25). The new method not only includes the measurement of plasma membrane damage by the usual dye exclusion tests, e.g., by trypan blue (TB) exclusion, leakage of lactate dehydrogenase (LDH), or total protein from cytosol, but we can also assess mitochondrial integrity by measuring the activity of mitochondrial succinic dehydrogenase (SDH) and nuclear damage by fluorescence induced by ethidium bromide (EB)-DNA binding (25).
The main purpose of this study was to evaluate the possible direct cellular effects of established and newly synthesized gastroprotective compounds in isolated mixed rat GMC at the levels of the plasma membrane, mitochondria, or nuclei alone or against a moderate uniform cell injury after a short incubation with ethanol.
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MATERIALS AND METHODS |
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Reagents and Chemicals
The following reagents were purchased from Sigma Chemical (St. Louis, MO): N-acetyl-L-cysteine (NAC), L-alanine, D-arginine, L-arginine, atropine sulfate, brilliant cresyl blue (BCB), BSA, bovine hemoglobin, carbamycholine chloride (carbachol), DMSO, EB, EGTA, Folin and Ciocalteau's phenol reagent, fumaric acid, D-glucose, L-glutamine, glycine sodium salt, histamine dihydrochloride, HEPES, 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-diphenyltetrazolium chloride (INT), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), IBMX, MTT formazan, NAD+, Percoll, L(+)lactic acid, phenazine metasulfate (PMS), protease (type XXV, pronase E) from Streptomyces griseus, pyruvic acid sodium salt, Trizma base, TB, and urea. Cysteamine (2-aminoethanethiol) hydrochloride was purchased from Aldrich Chemical (Milwaukee, WI). 16,16-Dimethyl-PGE2 (dmPGE2) was a gift from Upjohn (Kalamazoo, MI), ganglioside GM1 from Angio-Medical (New York, NY), potassium sucrose octasulfate (SOS) from Marion Laboratories (Kansas City, MO), and KT1-32 from Kotobuki Seiyaku (Nagano, Japan), whereas pinaverium and KC-3-10667 were obtained from Kali-Chemie Pharma (Hannover, Germany). Taurine was purchased from National Biochemicals (Cleveland, OH). Protein measurement utilized the Bradford reagent (Pierce, Rockford, IL). Salts and other reagent grade chemicals were purchased from Fisher Scientific (Pittsburgh, PA).Preparation of Mixed GMC
GMC from one or two nonfasted Sprague-Dawley rats (180-210 g; Taconic Farms, Germantown, NY) were obtained by sequential incubation with a low concentration of pronase E in calcium-free medium (EGTA) as we previously described (13, 25). Cells were counted in an improved Neubauer counting chamber (Hemocytometer; Fisher Scientific), dispersed, and kept in warm (37°C) HEPES-buffered salt solution (0.159 M, pH 7.4) produced fresh in our laboratory with the following ingredients (in mM): 98.0 NaCl, 5.8 KCl, 2.54 NaH2PO4, 5.1 Na pyruvate, 6.9 Na fumarate, 2.0 L-glutamine, 24.5 HEPES-Na, 1.0 Tris base, 11.1 D-glucose, and 1.0 CaCl2 with 2.0 mg/ml (wt/vol) BSA. The volume of GMC suspension was adjusted to a density of 1.5 × 107 cells/ml for incubations, TB, and biochemical assays. The numbers of viable and nonviable cells indicating initial cell viability (CV) were also counted by TB exclusion test (% of unstained cells) (25).Secretory Studies
The responsiveness of rat mixed GMC and the membrane receptor sensitivity of both chief and parietal cells were also measured in separate experiments using secretory agents.Pepsinogen secretion.
Freshly isolated and dispersed (1 ml) rat GMC (5 × 106 cells/ml, CV = 94 ± 8%; chief cells = 42 ± 6%) were incubated with carbachol (101-10
4 M) or dmPGE2
(10
4-10
8M) alone as well as in
combination with carbachol (10
1M) + atropine
(10
4M) for 15 min at 37°C. Pepsinogen was determined by
a modified Berstad method at pH 2.0 (HCl-KCl buffer, 0.1 M) using 3.0 M
urea-denaturated bovine hemoglobin (1.0%) as substrate and incubated
for 10 min at 37°C (5). The enzymic products were
quantified with freshly diluted Folin and Ciocalteu's phenol reagent
spectrophotometrically (A = 578 nm) with a Gilford 2400-2
spectrophotometer, calculated from a pepsin (Merck, Rahway, NJ)
dose-response curve and expressed as micrograms of pepsin per minute
per 5 × 106 GMC.
Enrichment of parietal cells and measurement of
[14C]aminopyrine accumulation.
Freshly isolated and dispersed rat GMC (1-2 × 108) were separated in a Beckman 5.0 elutriation system
(600 rpm, 10°C) in Hanks' balanced salt solution supplemented with
0.1% (wt/vol) BSA. Parietal cells (60-65% of total GMC) were
collected by continuous Percoll density gradient centrifugation at a
density of 1.06 mg/ml (15). The implication of HCl
production in enriched rat parietal cells is based on accumulation of
14C-labeled aminopyrine ([14C]AP) in
acidic tubulovesicles of parietal cells on stimulation (3, 33,
34). Parietal cells (106/ml) were suspended in
Hanks' balanced salt solution (containing 106 cells/ml
0.1% albumin) and incubated at 37°C in an orbital shaker (100 oscillations/min). Incubations were carried out in a total volume of
1.0 ml with 0.05 Ci of [14C]AP in the absence or presence
of receptor stimuli such as carbachol (104 M), histamine
(10
4 M), IBMX (10
4 M), or their
combination. The washed cells were dissolved in 1.0 M NaOH, and
neutralized aliquots were used for liquid scintillation counting.
Treatment of Rat Isolated GMC
All compounds for incubation were freshly dissolved and diluted in HEPES-buffered salt solution containing albumin (0.2% wt/vol). The pH was adjusted to 7.4, and the agents were used immediately. Initially, 30% ethanol was freshly prepared and subsequently diluted in the reaction tube containing GMC and test substances. The final concentration was 15% ethanol (25).GMC (1.5 × 107 cells/tube) were tested for direct
cytoprotection by 60-min incubation (1.0 ml of cell suspension plus 1.0 ml of drug solution) before addition of 2.0 ml of 30% ethanol for 5 min. The tested compounds were also used in the parallel tubes without
ethanol treatment. The following gastroprotective compounds were tested
for direct cytoprotection: glycine
(103-10
6 M), D-arginine
(10
3-10
6 M), L-arginine
(10
3-10
6 M), L-alanine
(10
3-10
6 M), dmPGE2
(10
4-10
8 M), cysteamine
(10
2-10
4 M), NAC
(10
3-10
5 M), taurine
(10
3-10
5 M), histamine (2 × 10
3-2 × 10
7 M), GM1
(10
3-10
6 M), sucrose octasulfate
(10
2-10
4 M), nitecapone
(10
5-10
7 M), pinaverium
(10
3-10
6 M), and KC-10667
(10
3-10
6 M).
After incubations the cells and the supernatant containing ethanol were separated by careful centrifugation (500 g, 8 min). The cells were resuspended in another 2.0 ml of buffer and incubated for 10 min in a shaking water bath. The ethanol-free supernatant was used for measurement of LDH leakage. The cells were redispersed in 1.5 ml of buffer (107 cells/ml) and immediately distributed for simultaneous biochemical assays.
TB Dye Exclusion Test
The TB dye exclusion test was carried out as we described previously (25). Briefly, 106 cells (0.1 ml of cell suspension) were mixed with 0.1 ml of 0.4% TB, and 5 min later the number of stained (dead) and unstained (viable) cells were counted in a hemocytometer and the yield of viable cells and CV (% of unstained cells) were calculated.LDH Assay
LDH assay was carried out in samples of both supernatants alone and with 106 sonicated cells/0.1 ml (Sonifer cell disruptor 200; Branson, Danbury, CT). The colorimetric assay is based on reduction of 1.5 mM NAD+ to NADH catalyzed by LDH in the presence of lactate (50 mM) as substrate in a 1.0-ml final volume at 37°C in a 10-min incubation, as described previously (4). A color product was rapidly formed by reductions of PMS (1.6 mM) and INT (0.8 mM) in the same tube and measured spectrophotometrically at 500 nm. The specific LDH activity was measured and calculated as milliunits per minute per microgram of protein.Succinic Dehydrogenase Assay
Mitochondrial integrity was tested in 2 × 106 previously treated and redispersed GMC at 37°C after incubation (90 min) with MTT (2.4 mM) in buffer (pH 7.4) in 1.0 ml of total reaction volume. The color formazan product was quantified spectrophotometrically at 500 nm after its dissolution in 2.0 ml of DMSO (21). Specific activity of SDH was calculated as nanomoles of formazan per minute per microgram of protein.EB-DNA Fluorescence Assay
Nuclear damage of GMC was assessed by nuclear fluorescence of cells due to the EB-DNA binding as described previously (9). GMC suspension (1 ml containing 107 cells) was mixed with 2.0 ml of 25 µM EB solution, and fluorescence intensity was measured in a Perkin-Elmer fluorescence spectrophotometer (Hitachi Instruments) at 325-585 nm (excitation-emission). The results were expressed as arbitrary fluorescence units/107 cells.Protein Concentration
Protein concentration in the supernatants and sonicated cells was determined by Bradford's method (Bio-Rad Laboratories, Richmond, CA; Ref. 6). Absorption of samples at 595 nm was measured against blanks and known standards of BSA.Data Presentation and Statistical Evaluation
Data from three to eight experiments were pooled and expressed as means ± SE. Comparisons were performed by Student's t-tests (paired or unpaired) and nonparametric Mann-Whitney U-tests. Values were considered statistically significant at P < 0.05. ![]() |
RESULTS |
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Yield, Composition, and Viability Of Isolated Rat GMC
A large number of mixed GMC (1.2-1.6 × 108) can be isolated from a single rat glandular stomach. The ratio of parietal, chief, and mucous epithelial cells using supravital staining with 0.05% BCB was 23.0 ± 2.1, 42.1 ± 4.9, and 34.1 ± 3.7%, respectively. The average initial CV of 28 cell isolations was 90.5 ± 3.1% measured by TB dye exclusion. High CV (85-95%) was maintained for 7 h after cell isolation using our cell harvesting method and physiological solution at 37°C.Secretory Studies
Pharmacological investigations with chief cells in mixed rat GMC revealed that the muscarinic receptor agonist carbachol significantly stimulated pepsinogen secretion from 3.15 ± 0.56 to 7.50 ± 0.75 µg · min
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Uptake of [14C]AP in enriched (65%) rat parietal cells
was stimulated by either histamine or carbachol (P < 0.05). The histamine-induced increase in HCl production in parietal
cells was considerably potentiated by the phosphodiesterase inhibitor
IBMX (Fig. 2).
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Ethanol-Induced Cellular Injury: An Assay for Cytoprotection
The average values obtained by all methods used to measure indicators of damage before and after 5-min incubation with ethanol at 37°C are shown in Table 2. Ethanol (15% vol/vol) significantly decreased CV by TB dye exclusion from 90.3 ± 3.1 to 20.9 ± 1.4 (P < 0.001) and increased LDH leakage from cytosol into media via damaged cell membrane (P < 0.001). It induced severe mitochondrial and nuclear lesions as detected by marked decrease in SDH activity from 5.7 ± 0.3 to 1.5 ± 0.1 nmol formazan · min
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Effects of Thiol Compounds on Ethanol-Induced Cell Injury
NAC, taurine, or cysteamine alone did not induce any injury in mixed GMC (data not presented). The effects of 60-min incubation of isolated rat GMC with NAC against ethanol-induced cellular injury are shown in Fig. 3. Cell membrane damage quantified by TB dye exclusion and LDH leakage, mitochondrial SDH activity, and EB-DNA fluorescence revealed a mild (12-21%) protection against ethanol-induced damage (P < 0.05).
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As shown in Fig. 4, a similar
(10-20%) increase in cell resistance was detected after
60-min preincubation with taurine (2-aminoethanesulfonic acid) against
ethanol injury, and the difference was statistically significant with
LDH leakage and EB-DNA fluorescence (P < 0.05). The
protection of mitochondria and nuclei reached the level of significance
(P < 0.05).
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In contrast, cysteamine moderately aggravated (P < 0.05) the ethanol-induced cell membrane and mitochondrial damage
measured by TB exclusion and SDH, respectively. However, some
concentration-dependent nuclear protection (P < 0.01)
was measured by EB-DNA fluorescence. No major changes were found in LDH
leakage from cysteamine-treated GMC after incubation with 15% ethanol
(Fig. 5).
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Effects of dmPGE2 and Ethanol
Concentration-dependent protection (15-21%) against ethanol-induced injury was found after 60-min incubation with dmPGE2 as measured by TB exclusion (P < 0.05), LDH release (P < 0.05), and changes in activity of SDH from 1.95 ± 0.11 to 3.31 ± 0.21 (P < 0.05). Only a slight increase (P > 0.05) in nuclear resistance was observed after incubation with dmPGE2 in ethanol-treated GMC (Fig. 6).
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Effects of SOS and Ethanol
Incubation of GMC for 60 min with SOS dose-dependently decreased the ethanol-induced LDH release and increased CV measured by TB dye exclusion; the differences reached statistical significance (P < 0.05) at 10
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Effects of Various Gastroprotective Compounds on Ethanol Toxicity
The effects of different agents that in vivo exert gastroprotection in isolated GMC with or without incubation with 15% ethanol are summarized in Table 3. Only the statistically significant changes in TB dye exclusion and biochemical assays are listed, indicating cell injury alone or aggravation of ethanol-induced cellular damage. Most of these compounds (i.e., GM1, nitecapone, pinaverium, KC-10667, and glycine) aggravated the ethanol-induced injury in GMC in a concentration-dependent fashion and, administered alone (i.e., without ethanol exposure), also exerted some direct cell toxicity in GMC. Ethanol-induced cell injury was aggravated by L-arginine, D-arginine, and L-alanine, but these compounds alone did not induce damage. Histamine had a biphasic effect in the rat GMC: low concentration protected but high concentration aggravated the ethanol-induced cell injury.
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DISCUSSION |
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Our study demonstrates that preincubation of isolated rat gastric mucosal cells with dmPGE2, SOS, or the SH compounds NAC, taurine, or cysteamine alone did not cause any measurable cell injury, and these agents slightly increased resistance (up to 21%) against ethanol-induced cellular damage. Similar protection was found with a low concentration of histamine, but at a higher concentration it aggravated ethanol toxicity. Partial or general aggravation of ethanol injury was detected on GMC membranes after preincubation with wide concentration ranges of amino acids such as L-arginine, D-arginine, glycine, L-alanine, and D-alanine or in vivo gastroprotective compounds such as nitecapone, ganglioside GM1, the spasmolytic pinaverium, and KC-10667. Some of these agents alone (i.e., without ethanol) exerted some direct cellular toxicity. Our results also revealed that the isolated chief cells (pepsinogen secretion) or enriched parietal cells (HCl production) have an excellent membrane receptor-mediated responsiveness to pharmacological stimuli such as cholinergic and histaminergic stimulators or PG. These results are in accordance with other studies in cultured and isolated parietal cells (3, 33, 34) or peptic cells (2, 8, 18, 19).
Acute gastric mucosal injury is a complex process because of the heterogenous structure and multiple functions of the stomach wall. In the pathways of direct and indirect chemical injury, vascular damage, inflammatory processes, free radicals, and proteases are involved (41, 45). The list of gastroprotective agents has been growing since the introduction of the concept of gastric cytoprotection, without a proportionate increase in our understanding of the mechanisms of gastric mucosal injury and protection. We nevertheless know that most of the mucosal protection is relative and indirect. Namely, despite the initial destruction of superficial epithelial cells in rats given ethanol intragastrically after pretreatment with gastroprotective agents, the histological integrity of the gastric mucosa is restored by rapid epithelial restitution and the organ structure and function are maintained.
One of the new possibilities for assessing the phenomenon of direct gastric cytoprotection is to investigate in vitro the possible protection of isolated GMC by naturally occurring or exogenous compounds against chemically induced and measurable cell damage. We have recently developed and optimized morphological and biochemical methods for harvesting mixed populations of rat GMC to investigate reversible and irreversible cell damage and protection at the level of cell membranes in organelles (13, 25). Selective and parallel assessment of chemical injury and protection of plasma membrane, mitochondria, and nuclei can be investigated with sensitive measurements such as TB exclusion, LDH leakage, SDH activity, or EB-DNA binding. These targets were selected because of their critical roles in reversible and irreversible cell injury. That is, the extent of plasma membrane and mitochondrial damage is currently accepted as the rate-limiting step between reversible and irreversible cell damage, whereas nuclear damage is an indicator of cell death by either necrosis or apoptosis (7, 38). This short incubation (5 min) of rat GMC with a low concentration of ethanol induced reversible and/or irreversible cell membrane, mitochondrial, or nuclear injury that can be measured by morphological and biochemical assays (25). The short exposure is meant to imitate the in vivo conditions when fluids like ethanol are rapidly emptied from the stomach and mucosal lesions develop within minutes (10, 16, 20, 40). Our previous experiments revealed only minimal or no protection of isolated GMC against ethanol injury after preincubation with PG for 30 min, whereas the present results indicate a moderate increase in cellular resistance of GMC after 1-h incubation with dmPGE2.
The SH compounds constitute one of the groups of endogenous mediators of acute gastroprotection (10, 20, 30, 39, 42, 44). Only a moderate but statistically significant direct gastroprotection was observed in rat GMC against ethanol-induced cytotoxicity by preincubation with NAC (in all organelles examined) or taurine (in mitochondria and nuclei). Taurine is a metabolite of L-cysteine. In vivo, it protects several organs against chemical injury, and we recently found gastroprotection against ethanol in rats (unpublished data). Cysteamine, another SH-containing agent, has also been shown to exert in vivo gastroprotection (42). Romano et al. (31) demonstrated that cysteamine has a direct protective effect in vitro against damage induced by taurocholate or indomethacin in gastric epithelial monolayers derived from a human cell line. In our study with freshly isolated rat GMC, cysteamine did not induce significant enhancement of cell resistance.
SOS, a derivative of sucralfate, had a moderate protective effect against ethanol injury in isolated rat GMC. This direct cellular protection might be one of the mechanisms of acute gastroprotection by sucralfate (36).
GM1, a ganglioside and sialic acid-containing glycosphingolipid, exerts acute gastric mucosal protection against ethanol (37, 48). Gangliosides play a role in the regulation of transmembrane signaling, cellular differentiation and proliferation, membrane fluidity, and ion transport (35). In the present study, exogenous GM1 was not able to protect against ethanol-induced damage in isolated GMC.
Amino acids such as L- or D-arginine, glycine, and D- or L-alanine are either modulators of neural transmissions or sources of vasoactive nitric oxide. They are also gastroprotective against ethanol injury in rats (unpublished data), and they reduce tubular cell damage in the kidney (1). In our study they did not have any direct cytoprotective effect in rat GMC against ethanol.
Our findings show the following. 1) A large number of mixed GMC with high and long viability and preserved membrane receptor sensitivity can be isolated from rat stomach. 2) Selective examination of ethanol injury and protection of plasma, mitochondrial, and/or nuclear membranes may be demonstrated biochemically and morphologically. 3) Only a slight or moderate concentration-dependent protection was detected by thiol compounds, dmPGE2, or SOS. Of the few chemicals that exerted direct cytoprotection in vitro (e.g., NAC, taurine, dmPGE2, SOS), all decreased plasma membrane damage as revealed by TB exclusion and/or LDH release, whereas only taurine and NAC diminished nuclear damage, i.e., cell death. This implies that it is much easier to offer protection against the reversible than against the irreversible stage of cell injury. 4) In contrast, a large number of naturally occurring or synthetic in vivo gastroprotective compounds have no direct cytoprotective effects against ethanol cytotoxicity in a mixed population of GMC. 5) Consequently, minimal or no correlation could be demonstrated between in vitro cytoprotection and in vivo gastroprotection by most compounds investigated. These findings indicate that a major part of the beneficial effect of gastroprotective agents seems to be mediated via complex mechanisms at the tissue and organ levels.
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ACKNOWLEDGEMENTS |
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We thank Joanne M. Jenkins, Ken Green, and Gabor Nagy for technical assistance and acknowledge the contributions of Eileen Holman and Rozalia Nagy in the preparation of the manuscript.
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. Szabo, Pathology & Laboratory Medicine Service, 5901 East 7th St., VA Med. Ctr., Long Beach, CA 90822 (E-mail: sandor.szabo{at}med.va.gov).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 29 July 1999; accepted in final form 14 June 2000.
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