Re: Autoimmunity, Brain & Beyon

[Guest guest]

" Previous in vivo studies conducted with T-2 toxin have demonstrated the

ability of trichothecenes to induce neurotoxic events when toxin was

injected into brain tissue directly [13, 14]. These studies suggested the

ability of trichothecenes to produce cytotoxic events, inflammation, and

apoptosis, which illustrate the potential immunopathological events that

have been witnessed in neurophysiological studies conducted with

trichothecenes in murine and rat models. T-2 toxin has been closely

evaluated for its effects in livestock and poultry [15, 16, 17, 18]. T-2

toxin is a common contaminant of feed for animals, and is associated with

lethargy, ataxia, emesis, and feed refusal in animals and humans [15, 16,

17, 18]. There is evidence of T-2 toxin transforming neurotransmitter

balance [17]. Increases in neurotransmitter concentrations, such as

catecholamines and serotonin are associated with loss of appetite [17].

These neurotransmitters are synthesized from specific amino acids, and the

transfer of these amino acids across the blood-brain barrier is highly

regulated by transport proteins [17]. When the BBB is compromised, studies

have demonstrated changes in the passage of these amino acids into the CNS,

leading to changes in neurotransmitter concentrations [17]. These studies

have demonstrated that the ingestion of T-2 toxin leads to changes in amino

acid permeability across the BBB, which could lead to the neurological

effects observed in animals exposed to trichothecene mycotoxins [17]. Other

animal studies conducted with trichothecenes have demonstrated the ability

of these agents to cause neurological events regardless of the

administration route [13].

Experimental evidence indicated that T-2 toxin administered intracerebrally

(i.c.) or subcutaneously (s.c.) resulted in similar events [13]. Rats

exposed via either route demonstrated depression of respiration and muscle

paralysis, followed by convulsions which led to death [13]. These results

demonstrated that regardless of the route of exposure, systemic distribution

of trichothecenes can reach the brain, resulting in neurological events

[13]. Further evidence showed that low levels of T-2 toxin were responsible

for the changes in the metabolism of brain biogenic monoamines, compared to

lethal doses [13]. Previous analyses have demonstrated that the disruption

of monoamine metabolism could alter food intake by altering hormone

secretion, peristaltic contractions, or thermal regulation.

The above studies provided evidence which suggested low levels of

trichothecenes were able to injure CNS activity and disrupt the integrity of

the BBB. There is also evidence that suggested the stimulation of

endothelial cells by trichothecenes led to pro-inflammatory activity. This

continuous stimulation of pro-inflammatory events appeared to further

aggravate the CNS in addition to the BBB.

In the present experiments, human vascular endothelial cells were exposed to

satratoxin H, LPS, and oxidative stress conditions to evaluate the cellular

pathways that were activated. Cells were also evaluated for additive

effects, due to exposure from satratoxin H and LPS, satratoxin H and H202.

The purpose of these experiments was to determine what effects low doses of

a

trichothecene mycotoxin from S.chartarum would induce in cells that compose

the BBB. The objective was to utilize HBCEC as an in vitro model to

determine the mechanism of toxicity produced by exposure to satratoxin H.

MATERIALS AND METHODS

A human brain capillary endothelial cell line (HBCEC) (Cambrex-Biowhittaker,

sville, MD), cell culture medium, and growth factors were purchased

from Cambrex-Biowhittaker (sville, land), Clonetics Division for

these experiments. Five x105 cells were cultured in 25 cm2 tissue culture

flasks (Corning Glass Works, Corning, NY). Cells were maintained in 5 ml of

endothelial cell growth medium (EGM-2MV) with the following growth factors:

2% (vol/vol) fetal bovine serum (FBS), human recombinant growth factor

(10ng/ml), EC growth supplement (12 µg/ml), hydrocortisone (1.0µg/ml), and

gentamicin (50 µg/ml) incubated with 5% CO2 at 37°C. The confluent cells

were sub-cultured on unsiliconized glass cover slips (25mm x 25mm) [19].

These glass cover slips were treated with a series of ethanol and water

washes, to remove the silicon coating, according to procedures published by

Simoni et. al., [19- 22]. Two coverslips were seeded per well in a 6-well

plate (Corning Inc.). In addition to coverslips, the cells were subcultured

in a series of 24-well plates until confluent (Corning Inc.). Experiments

were conducted on cells from either a fourth or fifth passage. Cell passages

were performed using the trypsin method recommended by Cambrex-Biowhittaker,

sville, MD. The cell lines were

tested by the supplier for human immunodeficiency virus (HIV), hepatitis,

Mycoplasma, bacteria, yeast, fungi, and smooth muscle α-actin expression.

The results of these tests were presented in Clonetics Certificate of

Analysis.

Prior to experimentation, satratoxin H samples were tested for endotoxin

contamination utilizing the QCL-1000 assay (Cambrex-Biowhittaker, Inc.,

sville, MD). If endotoxin removal was required, an affinity

chromatography method was utilized according to the manufacturer protocols

for Detoxi-Gel Endotoxin Removal Gel AffinityPak Prepacked Columns (Pierce,

Rockford, IL), followed by further evaluation using the QCL-1000 assay.

Previous in vitro studies have demonstrated that concentrations of 10ng/ml

of macrocyclic trichothecenes were able to induce significant increases in

cytokine synthesis and other cellular processes [24]. In the experiments

here, 1, 10, 100, 1000, and 5,000 ng/ml concentrations of satratoxin H were

evaluated. Prior to the addition of the compounds, the media in the 6 and

12-well plates were replaced with fresh medium. The samples were then

suspended in pyrogen-free, sterile water (Cambrex-Biowhittaker, Inc.,

sville, MD) and incubated with satratoxin H for 18 hours. Control

cells received an equal volume of water. A second subset of cells were

exposed to the concentrations of satratoxin H previously mentioned with the

addition of 250µM H2O2 or 50 EU/ml LPS to induce an inflammatory response.

A general cytotoxicity assay was conducted to determine which sample groups

were able to produce cytotoxic effects in cells. Lactate dehydrogenase

(LDH) concentrations were used to determine the cytotoxic effects of

satratoxin H on HBCEC. A commercial assay, LDH (DG-1340-UV) concentration

assay (Sigma Diagnostics), was utilized to determine the concentration of

LDH released by cells into the culture medium after an 18h incubation

period. The procedures described in the assay were followed to determine the

concentration of LDH released by cells due to cellular damage. Samples were

read spectrophotometrically at 340nm. This assay reflects cellular damage

induced by satratoxin H.

A series of immunofluorescent studies were conducted to evaluate

inflammatory events in EC due to satratoxin H exposure. Cells grown on

coverslips as described previously were washed with cold PBS twice, followed

by exposure to 3% formaldehyde in PBS for 20 minutes at RT [20]. The cells

were exposed to cold methanol, followed by a series of 4 washings with PBS

for a 30 minute period to permeabilize the cell membrane [20]. These cells

were then blocked against nonspecific antibody binding with 1% bovine serum

albumin (BSA) (Sigma Chemical Co.). Cells were exposed to monoclonal

antibody against human ICAM-1, VCAM-1, and P/E selectin for 1 hour at 37°C

(R & D Systems). The wells were washed 5 times with PBS, followed by a 1 hour

incubation with secondary antibody anti-mouse, fluorescein isothiocyanate

(FITC) conjugated F (ab') 2 (Boehringer Mannheim, Co., Biochemical Products,

Indianapolis, IN). Following a 30 minute incubation period, coverslips were

mounted onto slides, and later evaluated microscopically at 60x under oil-

emersion. This procedure demonstrated if satratoxin H was able to induce the

expression of adhesion molecule receptors. To quantitate the degree of

fluorescence, photographs of cells were scanned and evaluated with Scan

Analysis Software (Biosoft, Cambridge, UK). This procedure measured the

density of fluorescence in each picture and reported the results in units of

luminescence. The total area under a peak was used to determine the degree

of fluorescence per picture evaluated. Photographs from duplicate coverslips

under the same experimental conditions conducted in triplicate for each of

the adhesion molecule receptors were evaluated.

In the event that inflammatory pathways are activated, the permeability of

the BBB can increase to traffic cells across the BBB. In addition,

substances that are toxic to HBCEC may increase the permeability across the

BBB due to programmed cell death. During these events cell shrinkage can

occur which can lead to increased permeability across the BBB. To evaluate

whether satratoxin H is able to increase permeability across the BBB, the

passage of radio-labeled 125I- albumin (Diagnostic Products Corp., Los

Angeles, CA) was evaluated. Cells were cultured in 75 cm2 tissue culture

flasks with EGM-2MV (Cambrex-Biowhittaker, sville, MD), in humidified

conditions with 5% CO2, until they reached confluence, and were subcultured

in 24 well plates tissue culture (TC) treated, 0.4µm polycarbonate membrane

cell culture devices (Whatman Inc., Clifton, NJ). A monolayer of cells grown

on 0.4µm polycarbonate membranes in 24 well plates were exposed to 125I-

albumin and monitored every 15min for the

diffusion rate of albumin across the monolayer. Radioactivity was measured

using a scintillation machine as counts per unit (CPU).

Endothelial cells on unsiliconized glass coverslips were evaluated after

exposure to satratoxin H and control conditions for apoptosis utilizing

Apoptosis Detection Kit Annexin V-FITC (APO-AP) (Sigma, St. Louis, MO).

Early apoptotic events were determined by monoclonal antibody (anti-PS

fluorescein conjugate) binding to phosphatidylserine (PS) translocated from

the inner cell membrane to the outer membrane. Late apoptotic events were

observed by the translocation of propidium-iodide (PI) through compromised

cellular and nuclear membranes. PI is able to bind to DNA fragments upon

translocation to the nucleus. The detection of immunofluorescent staining

was evaluated microscopically (Olympus-IX71 Confocal Microscope, Leeds Inst.

Inc., Irving, TX). The software program used to view fluorescence was

Metamorph® (Universal Imaging Corp., Dowington, PA). These assays

demonstrated the concentration of satratoxin H able to induce apoptosis in

HBCEC.

To quantitate the degree of apoptosis in cells, an ELISA method was used to

determine the levels of cytochrome C from cellular extracts. Cytochrome C is

maintained in the mitochondria of healthy cells. In the event of programmed

cell death, cytochrome C is released from the mitochondria into the cytosol

where it activates pathways associated with apoptosis. The ELISA for the

evaluation of cytochrome C was conducted according to protocols described in

the manual provided by R & D Systems.

During an inflammatory response, and early apoptotic events cells are under

oxidative stress, which leads to the production of lipid radicals and lipid

peroxidation. Previous studies have demonstrated that lipid radicals are

able to inhibit anti-apoptotic genes which allow a cell to enter into

apoptosis. In these experiments, the presence of oxidative stress was

evaluated by measuring lipid hyperperoxides (LOOH) and reduced glutathione

(GSH) levels according to established methods [21, 22].

Established methods were utilized to measure oxidative stress conditions to

determine the concentration of GSH [21]. In a normal cell, GSH is unable to

cross the nuclear membrane, but during oxidative stress the oxidized form of

GSH, oxidized glutathione (GSSG), is able to cross this structure. GSSG is

able to decrease the binding of p60/p65 complex of NF- κB to DNA, which

reduces the pro-inflammatory cascade that is activated by NF- κB. The

intracellular GSH levels were measured from cell homogenates [21]. The

reaction of GSH with 5, 5' dithiobis and sulfhydryl compounds leads to a

color change, producing a yellow pigment. The samples were read

spectrophotometrically at 412nm (Sigma Chemical Co., St Louis, MO). Results

were compared to a standard curve using GSH in nanomoles per milligram of

protein. Protein concentrations for the cell homogenate fractions were

determined using a BCA Protein Assay Kit (Pierce, Rockford, IL).

Lipid hydroperoxides (LOOH) were evaluated in cell homogenate fractions

using previously established methods [21, 22]. This assay is able to

directly measure the LOOH concentration Samples were mixed with SDS-acetate

buffer, pH3.5, and aqueous solution of thiobarbituric acid. The reaction

mixture was heated at 95°C for 90 minutes. After cooling, the red-colored

complex was extracted with n-butanol-pyridine [21, 22]. The data were

measured spectrophotometrically at 532 nm, and compared to a standard curve

[21, 22]. Results were expressed in nanomoles of LOOH per microgram of cells

[21, 22].

Statistical analysis (α= 0.05) was performed using Sigma Stat, a statistical

software program designed by Jandel (SPSS), to analyze the data using

one-way analysis of variance (ANOVA) of each experimental group with the

controls. Normality of the data was determined using a Kolmogorov-Smirnov

normality test. If data did not meet normality, a non-parametric

Kruskal-Wallis ANOVA would have been applied. If data were normal, and ANOVA

demonstrated significance, a post hoc test, Tukey test (a modified t test),

was used to make multiple comparisons to determine which experimental groups

demonstrated significance compared to the controls and between experimental

groups. Results were graphed using Sigma Plot, a graphical program designed

by Jandel (SPSS).

RESULTS

In these experiments HBCEC were evaluated for the expression of inflammatory

and apoptotic events from the exposure of satratoxin H, LPS, and H202.

Negative controls were exposed to sterile, pyrogen-free water, and positive

controls were exposed to 50EU/ml LPS and 250µM H202 in a volume of 20µl.

These cells were incubated for a period of 18h at 37°C with 5% CO2 in 6 well

plates and unsiliconized coverslips. The evaluation of LDH levels

demonstrated significant (P<0.05) cytotoxic events in cells exposed to

1000ng/ml SH and additive conditions (10ng/ml + LPS and 10ng/ml + H2O2).

These results can be seen in Figure 2.

Cells were later evaluated for the expression of adhesion molecule receptors

(ICAM, VCAM, P/E selectin) expressed in the event of inflammation. Figures

3, 5, and 7 demonstrate immunofluorescent results from the expression of

ICAM, VCAM and P/E selectin. Cells that expressed the adhesion molecules

receptors on the surface expressed were bound by Ab conjugated to the green

fluorescent stain FITC. A live video-camera attached to the microscope was

used to photograph three sections of each slide. A total of 6 slides per an

experimental group were evaluated. Pictures of the cells were further

evaluated to quantitate the density of fluorescence using Scan Analysis

Software (BioSoft, Cambridge, UK). Figures 4, 6, and 8 demonstrate the

degree of fluorescence produced by cells. The total area beneath a peak was

used to determine the intensity of

receptor expression for each sample group. Figure 4 demonstrates a

significant increase (P< 0.05) in the expression of ICAM on cells exposed to

satratoxin H 100ng/ml, 1000ng/ml, and 10ng/ml + H202. VCAM expression was

significantly greater (P<0.05) than control cells when exposed to 100ng/ml

SH, LPS, H202, 10ng/ml + LPS, and 10ng/ml + H202. A significant expression

(P< 0.05) of P/E selectin was detected in HBCEC cells exposed to 100ng/ml,

1000ng/ml, LPS, H202, 10ng/ml + LPS, and 10ng/ml + H202.

Further evidence of damage to the BBB, is EC cell shrinkage which leads to

greater permeability across the BBB. To evaluate whether satratoxin H is

able to induce HBCEC shrinkage, a monolayer of cells grown on

0.4µmpolycarbonate membranes in 24 well plates were exposed to 125I-

albumin and

monitored every 15min for the diffusion rate of albumin across the

monolayer. There was a significantly greater rate (P< 0.05) of diffusion

across cells exposed to 100ng, and 1000ng/ml of SH. A significant (P< 0.05)

additive effect was observed in cells exposed to 10ng/ml + LPS and 10ng/ml +

H202. These results are seen in Figure 9.

Apoptotic events were observed in addition to inflammation. An annexin V

apoptotic detection assay (Sigma, St. Louis, MO) was utilized to evaluate

apoptosis. In the event of early apoptosis, phosphatidylserine (PS)

expressed on the inner cell membrane is flipped to the outer surface of the

cell membrane as an indication of apoptosis. In the detection assay,

secondary Ab conjugated to FITC (green) binds to PE on the surface of cells

in the event of apoptosis. Late

stages of apoptosis consist of chromatin fragmentation and permeability of

the nuclear membrane. In the event of late stages of apoptosis, propidium

iodide (red) binds to damage chromatin material. These events are observed

in Figure 10. Compared to the negative control cells that received water,

cells exposed to 100ng/ml SH, and 1000ng/ml SH, and LPS demonstrated early

and late stages of apoptosis, whereas the control cells did not have a red

stain in the nucleus of the cell. To further evaluate apoptosis, cytochrome

C levels from cell extracts were evaluated using an ELISA method. These

results demonstrated that a significantly increased amount (P< 0.05) of

cytochrome C was released from cells exposed to 10ng/ml, 100ng/ml, LPS,

10ng/ml + LPS, and 10ng/ml + H202. These results can be seen in figure 11.

An additional indicator of apoptosis is oxidative stress. In the event of

oxidative stress, glutathione (GSH) acts as a reducing agent against

reactive oxygen species (ROS) such as lipid radicals and peroxides. However,

if GSH levels in a cell are insufficient to compensate for the degree of

oxidative stress, both apoptotic and inflammatory pathways are further

activated. To determine whether mycotoxins increased oxidative stress levels

in HBCEC, a quantitative method was used to determine the levels of GSH

present in cell extracts exposed to various experimental conditions. The

results demonstrated a significant decrease (P> 0.05) in the concentration

of GSH (µg/ml) in cells exposed to 100ng/ml SH, 1000ng/ml SH, LPS, H202,

10ng/ml + LPS, 10ng/ml + H202. These results are seen in Figure 12. The

production of lipid peroxidation,

further demonstrates the degree of oxidative stress induced on HBCECs.

Results from the thiobarbituric acid assay (T-BARS) demonstrated that there

was a significant increase (P>0.05) in lipid peroxidation when cells were

exposed LPS, H2O2, moderate concentrations of SH (100ng/ml and 1000ng/ml),

and additive conditions (10ng/ml + LPS and 10ng/ml + H2O2). These results

can be seen in Figure 13.

.....

CONCLUSIONS Results from the adhesion mole receptor expression on HBCEC

demon

lecu

strate that satratoxin H levels of 100ng/ml and 1000ng/ml are able to induce

inflammatory pathway activation alone. Additive effects are demonstrated

with very low concentrations of SH, such as 10ng/ml in the presence of

inflammatory agents such as LPS and H202. Similar concentrations of the

mycotoxin are able to induce apoptotic pathways leading to the activation of

early stages of apoptosis in the presence of 100ng/ml SH, however evidence

of late stages of apoptosis are observed with 1000ng/ml and 10ng/ml + LPS or

10ng/ml + H202. These results demonstrate the ability of satratoxins to

induce apoptotic pathways at the same concentrations that inflammatory

pathways are being activated. This suggests that low levels of inflammation

and apoptotic events can be induced in the presence of moderate levels of

SH, and low levels of SH are able to induce similar events in the presence

of other inflammatory agents and oxidative stress conditions, as

demonstrated by the levels of GSH and cytochrome C in cell extracts. In

addition, the ability of the mycotoxins to induce cell shrinkage at moderate

to low levels of SH demonstrate the potential ability of these agents to

compromise the integrity of the BBB which could lead to further neurological

damage from mycotoxins or other harmful agents. The presence of lipid

peroxidation in cells exposed to moderate concentrations of SH and additive

conditions, further demonstrates the ability of the mycotoxins to amplify

cellular

damage through the indirect production of lipid radicals and other ROS. The

results further suggest that low to moderate levels of SH are able to induce

inflammatory and apoptotic pathways that amplify the cellular damage by the

continuous activation of these biological pathways.