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Assorted Toxins


Effect of enterotoxin on glutathione status in the intestinal mucosa.
Benard O; Balasubramanian KA
Wellcome Research Laboratory, Department of Gastrointestinal Sciences,
Christian Medical College Hospital, Vellore, India.
Indian J Biochem Biophys, 33(5):409-13 1996 Oct
The effect of luminal exposure of enterotoxins on the intestinal
mucosal glutathione (GSH) was studied in rat. Cholera toxin induced
fluid secretion and decreased mucosal GSH by 35% without altering
oxidized glutathione (GSSG) level. Toxin induced fluid secretion was
tested after mucosal GSH depletion by compounds such as diethyl maleate
(DEM) and buthionine sulfoximine (BSO) and thiol supplementation with
N-Acetyl cysteine (NAC). Fluid secretion was not altered by prior thiol
depletion or supplementation. Exposure of intestinal lumen to bacterial
endotoxin resulted in 25% decrease in mucosal GSH with two fold
increase in GSSG. Luminal exposure of Shiga toxin did not alter the
mucosal thiol. The level of other low molecular weight thiols, cysteine
and cystine was not altered by luminal exposure of any of these toxins.
These results show that although cholera toxin decreased the mucosal
GSH level, prior modulation of thiol status of the mucosa may not have
any effect on toxin-induced fluid secretion.

N-acetyl-L-cysteine is a pluripotent protector against cell death and
enhancer of trophic factor-mediated cell survival in vitro.
Mayer M; Noble M
Ludwig Institute for Cancer Research, London, United Kingdom.
Proc Natl Acad Sci U S A, 91(16):7496-500 1994 Aug 2
We have discovered that N-acetyl-L-cysteine (NAC) protects cells
against death induced by exposure to noxious stimuli and against
programmed cell death (apoptosis) associated with exposure to
inadequate amounts of trophic factors. NAC prevented glutamate-induced
death of oligodendrocytes and tumor necrosis factor alpha
(TNF-alpha)-induced death of oligodendrocytes and L929 fibroblasts.
Moreover, suboptimal doses of NAC plus ciliary neurotrophic factor
(which also protects oligodendrocytes against TNF-alpha-mediated
killing) acted synergistically to protect oligodendrocytes against
TNF-alpha-induced death. Protection against death by growth factor
deprivation was provided by the combination of (i) NAC, vitamin C, or
Trolox (a water-soluble analogue of vitamin E) with suboptimal
concentrations of protein trophic factors, (ii) NAC, vitamin C, or
Trolox with progesterone, and (iii) NAC with either vitamin C or
Trolox; these latter experiments suggest that the addition of tyrosine
kinase stimulators is not required to promote cell survival. In all
paradigms, NAC was either equally or more effective than the other
compounds examined. In light of the long history of therapeutic
application of NAC, we suggest that use of this compound may be of
interest in conditions where certain toxin-mediated forms of cell death
and/or apoptosis contribute significantly to disease.

Role of macrophage oxidative burst in the action of anthrax lethal
Hanna PC; Kruskal BA; Ezekowitz RA; Bloom BR; Collier RJ
Department of Microbiology and Molecular Genetics, Harvard Medical
School, Boston, Massachusetts 02115, USA.
Mol Med, 1(1):7-18 1994 Nov
BACKGROUND: Major symptoms and death from systemic Bacillus anthracis
infections are mediated by the action of the pathogen's lethal toxin on
host macrophages. High levels of the toxin are cytolytic to
macrophages, whereas low levels stimulate these cells to produce
cytokines (interleukin-1 beta and tumor necrosis factor-alpha), which
induce systemic shock and death. MATERIALS AND METHODS: Experiments
were performed to assess the possibility that the oxidative burst may
be involved in one or both of lethal toxin's effects on macrophages.
Toximediated cell lysis, superoxide anion and cytokine production were
measured. Effects of antioxidants and macrophage mutations were
examined. RESULTS: RAW264.7 murine macrophages treated with high levels
of toxin released large amounts of superoxide anion, beginning at about
1 hr, which correlates with the onset of cytolysis. Cytolysis could be
blocked with various exogenous antioxidants or with N-acetyl-L-cysteine
and methionine, which promote production of the endogenous antioxidant,
glutathione. Mutant murine macrophage lines deficient in production of
reactive oxygen intermediates (ROIs) were relatively insensitive to the
lytic effects of the toxin, whereas a line with increased oxidative
burst potential showed elevated sensitivity. Also, cultured blood
monocyte-derived macrophages from a patient with Chronic Granulomatous
Disease, a disorder in which the phagocyte's oxidative burst is
disabled, were totally resistant to toxin, in contrast to control
monocytes. CONCLUSIONS: These results imply that the cytolytic effect
of the toxin is mediated by ROIs. Additionally, cytokine production and
consequent pathologies showed partial dependence on macrophage ROIs.
Antioxidants moderately inhibited toxin-induced cytokine production in
vitro, and BALB/c mice pretreated with N-acetyl-L-cysteine or mepacrine
showed partial protection against lethal toxin. Thus ROIs are involved
in both the cytolytic action of anthrax lethal toxin and the overall
pathologic process in vivo.

On the track of cell survival pharmaceuticals in the oligodendrocyte
type-2 astrocyte lineage.
Noble M; Mayer-Próschel M
Ludwig Institute for Cancer Research, London, United Kingdom.
Perspect Dev Neurobiol, 3(2):121-31 1996
The identification of compounds that can protect cells against death
induced by exposure to noxious stimuli and against programmed cell
death (apoptosis) associated with exposure to inadequate amounts of
trophic factors is of great interest in contemporary biology. We have
found that N-acetyl-L-cysteine (NAC) is able to promote cell survival
in these two distinct experimental paradigms of, respectively, "death
by murder" and "death by neglect." In the former case, NAC prevented
the death of oligodendrocytes induced by glutamate or tumor necrosis
factor-alpha (TNF-alpha), and also prevented TNF-alpha-induced death of
L929 cells. NAC also acted in synergy with ciliary neurotrophic factor
(CNTF) to prevent killing of oligodendrocytes by TNF-alpha. In analysis
of "death by neglect," NAC markedly enhanced the extent of spinal
ganglion neuron survival obtained with suboptimal concentrations of
nerve growth factor and of oligodendrocyte survival obtained with
suboptimal concentrations of CNTF or insulin-like growth factor-1.
Surprisingly, significant rescue of oligodendrocytes from apoptosis was
also observed with combinations of NAC with progesterone, vitamin C, or
Trolox, a water-soluble vitamin E analogue, although not with any of
these compounds applied individually. These results demonstrate that
cocktails of small molecules such as those we have studied may have
beneficial effects not predictable from the action of any individual
member of the cocktail. In light of the long clinical history of
therapeutic use of NAC and the other compounds identified in our
studies, we suggest that it may be of interest to examine use of NAC
alone, or combinations of NAC with the other small molecules we have
studied, in conditions in which certain toxin-mediated forms of cell
death or apoptosis contribute significantly to disease.

Protective role of sulfhydryl reagents in oxidant lung injury.
Patterson CE; Rhoades RA
Department of Physiology and Biophysics, Indiana University School of
Medicine, Indianapolis 46223.
Exp Lung Res, 14 Suppl():1005-19 1988
Recently there has been a great deal of interest in exploring possible
ways to protect the lung from oxidant damage. Since sulfhydryl
compounds are among the most important endogenous antioxidants, their
therapeutic use has been proposed. glutathione (GSH), the main
intracellular nonprotein sulfhydryl, plays an important role in the
maintenance of cellular proteins and lipids in their functional state.
With oxidant stress, GSH acts to protect cell constituents as evidenced
by increased turnover to GSSG, formation of mixed disulfides with
proteins, utilization of NADPH, and utilization of glucose in the
pentose pathway. When GSH is experimentally lowered (e.g., by protein
deficiency or with diethylmaleate) the toxic effects of oxidant stress
are exacerbated as evidenced by increased membrane and cell damage,
pulmonary edema, and mortality. Several recent investigations have
shown that sulfhydryl reagents (particularly N-acetyl cysteine, a
cell-permeable GSH precursor) can provide significant protection
against certain pulmonary toxins. N-acetyl cysteine reduced the lethal
effects of 100% O2 in rats by 65%. Therefore, the therapeutic potential
of sulfhydryl reagents in the treatment and prevention of oxidant
injury and the mechanisms involved are an important direction for lung

Kupffer cell stimulation with Corynebacterium parvum reduces some
cytochrome P450-dependent activities and diminishes acetaminophen and
carbon tetrachloride-induced liver injury in the rat.
Raiford DS; Thigpen MC
Division of Gastroenterology, Vanderbilt University School of Medicine,
Nashville, Tennessee 37232.
Toxicol Appl Pharmacol, 129(1):36-45 1994 Nov
Chemical activation of Kupffer cells in vivo by vitamin A or latex
beads is associated with a worsening of hepatic injury induced by the
P450-dependent hepatotoxins acetaminophen (ACET) and carbon
tetrachloride (CCl4) and by the P450-independent toxin galactosamine
(GLN). Immunostimulants such as Corynebacterium parvum (CP) also
activate Kupffer cells, but do so while prompting release of soluble
mediators which depress microsomal oxidative activities in cultured
hepatocytes. Therefore, we sought to characterize the effects of CP on
hepatic injury in vivo due to ACET and CCl4 while employing GLN as a
control. Hepatic microsomal oxidative activity and glutathione (GSH)
disposition were examined since each influences susceptibility to
injury from ACET or CCl4. Rats were given CP 28 mg/kg i.v. 5 days
before challenge with hepatotoxicant. Hepatic injury was assessed 24 hr
after hepatotoxicant administration by measurement of serum alanine
aminotransferase (ALT) activity and review of histological sections.
Livers from parallel groups of rats were used to prepare microsomal and
cytosolic fractions, to measure tissue GSH, or for perfusion to assess
GSH efflux. Significant reductions in injury due to ACET or CCl4 were
observed while injury due to GLN was potentiated. Serum ALT levels
after ACET were 3000 +/- 620 in controls vs 170 +/- 45 IU/liter in the
CP-treated group and ALT levels after CCl4 were 3100 +/- 500 in
controls vs 1700 + 450 IU/liter in the CP-treated group. In contrast,
serum ALT levels after GLN were 920 +/- 230 in controls vs 1700 +/- 370
in the CP-treated group. Patterns of hepatic injury observed on
histological sections were those characteristic for each toxin and the
severity of injury correlated well with alterations in serum ALT levels
for each agent. Hepatic microsomal fractions from rats pretreated with
CP showed significantly diminished total cytochrome P450 content as
well as reduced activity for two P450IIE1 substrates, p-nitrophenol and
7-ethoxycoumarin. While sinusoidal efflux of GSH increased by 40% in
rats pretreated with CP and cytosolic glutathione-S-transferase
activity fell slightly, tissue GSH levels were unaffected. These data
demonstrate that CP decreases microsomal cytochrome P450 content,
reduces biotransformation of two P450IIE1 substrates, and diminishes
ACET- and CCl4-induced hepatic injury. In contrast, hepatic injury due
to the P450-independent toxin GLN was enhanced. Thus, chemical and
immune stimulation of Kupffer cells may result in divergent effects on
susceptibility to injury from individual hepatotoxins.

Comparative cytotoxic responses of cultured avian and rodent aortic
smooth muscle cells to allylamine.
Ramos KS; Thurlow CH
Department of Physiology and Pharmacology, College of Veterinary
Medicine, Texas A&M University, College Station 77843-4466.
J Toxicol Environ Health, 40(1):61-76 1993 Sep
The present studies were designed to compare the acute cytotoxic
responses of cultured avian and rodent aortic smooth muscle cells
(SMCs) to allylamine (AAM), a selective vascular toxin. SMCs were
isolated from male Japanese quail or Sprague-Dawley rats and
established in culture by standard procedures. Cellular glutathione
(GSH) content and lactate dehydrogenase (LDH) leakage were used as
indices of cytotoxicity. Exposure of avian and rodent SMCs in primary
culture to AAM (0.2-200 microM) for 4 h was associated with a
significant reduction in cellular GSH and enzyme leakage in cultures of
both cell types. Increased exposure time to 24 h further depleted
cellular GSH levels and enhanced the leakage of LDH in primary cultures
of avian SMCs. In contrast, enhanced LDH leakage occurred without
further GSH depletion in primary cultures of rodent SMCs upon exposure
to AAM for 24 h. Removal of serum did not modulate the cytotoxic
response profile of primary cultures of avian SMCs treated with 200
microM AAM, but was associated with marked elevation in cellular GSH
levels and significant LDH leakage in rodent SMC cultures. The
cytotoxic responses to 0.2-200 microM AAM in secondary cultures of
avian SMCs were comparable to those observed in primary culture. In
contrast, AAM-induced enzyme leakage did not consistently correlate
with changes in GSH content in subcultured rodent SMCs. Challenge with
200 microM acrolein (ACR) for 4 h reduced the GSH content in avian, but
not rodent, subcultures of SMCs. However, significant LDH leakage
occurred in subcultures of both cell types upon exposure to ACR.
Although hydrogen peroxide (H2O2) did not modulate GSH levels in avian
or rodent cultures, leakage of LDH was observed in rat SMCs challenged
with 200 microM H2O2. Removal of serum did not alter the cytotoxic
responses of avian subcultures to 200 microM AAM for 24 h, but fully
prevented cytotoxicity in rodent subcultures. These data suggest that
potentially significant variations in the sequence of events leading to
injury may exist between quail and rat aortic SMCs. These differences
may contribute to the enhanced avian susceptibility to AAM-induced
aortic injury in vivo.

Effects of a cysteine precursor, L-2-oxothiazolidine-carboxylate,
nutritional status, and sex on tissue glutathione and hepatic
GSH-utilizing enzymes of CD-1 mice [published erratum appears in Res
Commun Chem Pathol Pharmacol 1988 Sep;61(3):432]
Moslen MT; Harper BL; Roy D
Department of Pathology, University of Texas Medical Branch, Galveston
Res Commun Chem Pathol Pharmacol, 61(1):49-63 1988 Jul
Objectives of this study were to compare the effects of sex,
nutritional status and L-2-oxothiazolidine carboxylate (OTC) treatment
on tissue constituents frequently involved in responses to chemical
toxins. Four groups of adult CD-1 mice were studied: fed females, fed
males, fasted males, and fasted males three hours after treatment with
OTC (10 mmoles/kg, sc). Female fed mice were found to differ from male
fed mice as follows: lower tissue GSH in liver and kidney but not lung;
lower hepatic microsomal cytochrome P-450 content and cytosolic GSH
transferase activities, particularly using CDNB as substrate; and
higher hepatic GSH peroxidase but similar GSSG reductase activities.
Overnight fasting was associated with a decrease in hepatic and renal
GSH and hepatic cytochrome P-450. OTC treatment was only found to
increase hepatic GSH and decrease renal GSH. Thus in fasted CD-1 male
mice, the intracellular cysteine precursor, OTC, has an apparently
selective effect on tissue GSH contents without confounding effects on
hepatic GSH utilizing or restoring activities.

Acetaminophen-induced toxicity to human epidermoid cell line A431 and
hepatoblastoma cell line Hep G2, in vitro, is diminished by silymarin.
Shear NH; Malkiewicz IM; Klein D; Koren G; Randor S; Neuman MG
Division of Dermatology, Sunnybrook Health Science Centre, Ont.,
Skin Pharmacol, 8(6):279-91 1995
The skin and liver may be targets for cytotoxicity induced by oxidative
drug metabolites. We used human epidermoid A431 cells and human
hepatoblastoma Hep G2 cells as the experimental model. The aim of the
study was to investigate and evaluate the effect of silymarin on
acetaminophen (APAP)-induced toxicity under controlled conditions.
Silymarin is known to be a potent antioxidant that diminishes toxicity
induced by a variety of other hepatotoxins (e.g. Amanita phaloides,
algae's toxins, carbon tetrachloride). glutathione (GSH) depletion was
enhanced by adding to the medium buthionine sulfoximine
[L-buthionine-(S,R)-sulfoximine, BSO]. Cells were incubated with
high-concentration 5-20 mM APAP or alpha-(minimum essential medium for
2-24 h to evaluate the drug's ability to reduce cytoviability.
Viability was then quantitated by metabolism of the tetrazolium dyes
(MTT) and neutral red (NR). Cytoviability was 100% for controls. For
Hep G2 treated for 24 h with 20 mM, APAP viability was 56.0% by MTT and
62.5% by NR. BSO-treated cells showed an enhanced cytotoxicity,
determined by both assays. Administration of 0.5 mM silymarin reduced
cytotoxicity significantly. In A431 cells, treatment with 20 mM APAP
reduced viability by 57% (MTT) and 69% (NR) versus control (100%). BSO
further decreased viability. Since incubation with silymarin showed
significant protection against APAP toxicity, it can be considered a
cytoprotective agent in this in vitro model of drug toxicity. GSH
concentrations in both cell lines decrease significantly after exposure
to 20 mM APAP, or 0.5 mM versus control (p < 0.05), and increased (p <
0.001) if incubated with APAP and silymarin. The protective effect
could be through mitochondrial membrane stabilization and/or an
increase in available GSH.

Biliary excretion of glutathione and glutathione disulfide in the rat.
Regulation and response to oxidative stress.
Lauterburg BH; Smith CV; Hughes H; Mitchell JR
J Clin Invest, 73(1):124-33 1984 Jan
Regulation of the biliary excretion of reduced glutathione (GSH) and
glutathione disulfide (GSSG) and responses to selected model toxins
were examined in male Sprague-Dawley rats. In control and
phenobarbital-pretreated rats in which the intrahepatic concentration
of GSH was modulated by the administration of diethyl maleate or
acetaminophen, the biliary concentration of GSH was consistently lower
than, but directly proportional to, the intrahepatic concentration of
GSH. Furthermore, increments in bile flow produced by the infusion of
sulfobromophthalein (BSP)-glutathione were associated with proportional
increases in the biliary excretion of GSH, suggesting that GSH passes
into bile passively along a concentration gradient. In contrast, GSSG
appears to be secreted into bile against a steep concentration
gradient. An increased hepatic production and biliary excretion of GSSG
resulted from the administration of t-butyl hydroperoxide. Measurement
of biliary GSSG and BSP during a constant infusion of the GSH adduct of
BSP indicated that GSSG shares a common excretory mechanism with GSH
adducts. Diquat, nitrofurantoin, and paraquat also markedly stimulated
the biliary excretion of GSSG. On a molar basis, these compounds
generated much more GSSG than a direct substrate for glutathione
peroxidase such as t-butyl hydroperoxide, indicating that the compounds
undergo redox-cycling with concomitant production of hydrogen peroxide.
Aminopyrine (0.8 mmol/kg) also significantly increased biliary GSSG.
This increase, however, was associated with a proportional increase in
bile flow and in the biliary excretion of GSH such that the GSSG/GSH
ratio in bile did not change. Acetaminophen and chloroform, two
compounds generating electrophilic metabolites that deplete
intrahepatic GSH, led to a progressive decrease in the biliary
excretion of GSH and GSSG. Furosemide and dimethylnitrosamine, the
electrophilic metabolites of which do not deplete hepatic GSH,
minimally altered biliary GSH and GSSG. Similarly, carbon tetrachloride
and iproniazid, which yield organic radical metabolites that can
peroxidize membrane lipids, did not increase the biliary excretion of
GSSG. This finding indicates that membrane-bound lipid hydroperoxides
may not be good substrates for glutathione peroxidases. The measurement
of the biliary excretion of GSSG and of the GSSG/GSH ratio in bile is a
sensitive index of oxidative stress in vivo and thus complements other
in vivo parameters for the study of reactive intermediates of
xenobiotics such as the determination of covalent binding, the
formation of lipid hydroxy acids, and the depletion of intracellular


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