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This is to certify that the

dissertation entitled

INDUCTION 0F LEUKOCYTE APOPTOSIS
BY TRICHOTHECENE MYCOTOXINS

presented by

Rebecca Lynne Uzarski

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Food Science/
Environmental Toxicology

 

 

Major professor

 

5'
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Date /’ 0

 

MS U is an Affirmative Action/Equal Opportunity Institution 0-12771

 

 

 

 

 

LIBRARY
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PLACE IN RETURN BOX to remove this checkout from your record.
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6/01 cJClFiC/DateDuepss-p. 15

INDUCTION or LEUKOCYTE APOPTOSIS BY TRICHOTHECENE MYCOTOXINS
By

Rebecca Lynne Uzarski

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Food Science and Human Nutrition

2002

ABSTRACT
INDUCTION or LEUKOCYTE APOPTOSIS BY TRICHOTHECENE MYCOTOXINS
By

Rebecca Lynne Uzarski

Trichothecenes are secondary fungal metabolites produced by food-bome and
environmental molds including F usarium, Stachybotrys, and Mvrothecium. Human and
animal exposure may occur via consumption of mold-contaminated grains, inhalation of
fungal spores present on water-damaged building materials, or airborne exposure during
suspected chemical warfare. Trichothecenes are potent translational inhibitors which
bind to the ribosomes of actively dividing leukocytes of the immune system. Exposure to
trichothecenes results in decreased leukocyte numbers and impaired resistance to
pathogens. The overall hypothesis of this dissertation is that trichothecene-induced
immune suppression is mediated in part by the induction of leukocyte death by apoptosis.
The specific objectives of these studies were to compare apoptosis induction by
trichothecenes and other naturally occurring translational inhibitors, determine the
physico-chemical components of the trichothecenes critical for the induction of
apoptosis, investigate the augmentation of trichothecene-induced cell death by
inflammatory agents, and determine the signal transduction mechanisms critical for
trichothecene-induced apoptosis. Comparisons of translational inhibitors suggested that
trichothecenes were among the most potent inducers of leukocyte apoptosis when
compared to other natural toxins. Multiple physico-chemical factors critical to transport

and ribosomal binding contribute to the apoptotic actions of the trichothecenes including

lipophilicity, electrostatic interactions, and molecular shape. Trichothecene-induced
leukocyte apoptosis may be augmented by signal components of stress and inflammation
such as TNF-a and F as ligand. Activation of mitogen activated protein kinases
(MAPKs) appears to be an early, critical step in the development of leukocyte apoptosis
induced by trichothecenes or potentiated by inflammatory agents. Taken together, these
studies contribute to our knowledge of the mechanism of leukocyte apoptosis by the
trichothecene mycotoxins and may facilitate risk assessment studies for this group of

natural toxins.

ACKNOWLEDGMENTS

I would like to thank Drs. James Pestka, John Linz, Jay Schroeder, and Norb
Kaminski for their guidance, support, and patience. I would like to acknowledge my
many friends and colleagues in the Pestka and Linz lab groups and members of the
Department of Food Science and Human Nutrition who have provided support and
assistance in many different forms. The Department of Food Science and Human
Nutrition, College of Agriculture and Natural Resources, Institute for Environmental
Toxicology, and the Society of Toxicology all provided funding for this research. A final

thank you goes to “The Dons” for both statistical and moral support.

iv

TABLE OF CONTENTS

LIST OF TABLES ..................................................... viii
LIST OF FIGURES ..................................................... ix
CHAPTER 1
Literature Review ....................................................... 1
General aspects of trichothecene mycotoxins ............................ 1
Environmental exposure to trichothecenes and adverse health effects ......... 1
Trichothecenes are immunotoxins ..................................... 6
Trichothecenes induce lymphocyte apoptosis ............................ 7
Trichothecene toxicity is potentiated by bacterial lipopolysaccharide ......... 8
Rationale ........................................................ 9
CHAPTER 2
Differential Induction of B Cell Cytotoxicity and Apoptosis By Trichothecene
Mycotoxins and Other Translational Inhibitors .......................... l 1
INTRODUCTION ................................................ l 1
MATERIALS AND METHODS ..................................... 13
Chemicals and reagents ...................................... 13
Cell lines and culture ........................................ l3
MTT conversion assay for cytotoxicity .......................... l4
Apoptosis assay ............................................ 14
Statistical analysis .......................................... 15
RESULTS AND DISCUSSION ..................................... 15
Induction of cytotoxicity and apoptosis by cycloheximide (CHX) ..... 15

Induction of cytotoxicity and apoptosis by trichothecene mycotoxins . . 17
Differential Induction of cytotoxicity and apoptosis by shiga like toxin 1

(SLTl) ............................................. 20

Differential Induction of cytotoxicity and apoptosis by ricin ......... 22

SUMMARY ..................................................... 23

CHAPTER 3

Induction of B Cell Cytotoxicity and Apoptosis by Environmental and F ood-bome

Trichothecenes: Quantitative Structure Activity Relationships ............. 27

INTRODUCTION ................................................ 27

MODEL DESIGN AND RATIONALE ............................... 3]

BIOLOGICAL TESTING .......................................... 34

Cell lines and culture ........................................ 34

Trichothecenes ............................................. 35

MTT conversion assay for cytotoxicity .......................... 35

Apoptosis assay ............................................ 36

Statistical analysis .......................................... 36

RESULTS ...................................................... 37

Trichothecene physical and chemical descriptors .................. 37

V

Trichothecene-induced B cell apoptosis and cytotoxicity ............ 37
Principal component analysis (PCA) of trichothecene-induced

cell death ........................................... 41
DISCUSSION ................................................... 45
CHAPTER 4
Potentiation of Trichothecene-Induced Cytotoxicity and Apoptosis
in Murine Leukocytes by TNF-a and F as Ligand ....................... 50
INTRODUCTION ................................................ 50
MATERIALS AND METHODS ..................................... 52
Chemicals and reagents ...................................... 52
Animals .................................................. 53
Primary cell culture ......................................... 53
MTT conversion assay for cytotoxicity .......................... 54
Apoptosis assay ............................................ 54
Statistical analysis .......................................... 55
RESULTS AND DISCUSSION ..................................... 55
Differential sensitivity of primary murine leukocytes to VT- , LPS-,
and VT plus LPS- induced apoptosis ...................... 55

Differential sensitivity of primary murine leukocytes to PGE2 -induced
apoptosis and cytotoxicity in the absence and presence of VT . . 59
Differential sensitivity of primary murine leukocytes to anti-IgM -induced
apoptosis and cytotoxicity in the absence and presence of VT . . 59
Differential sensitivity of primary murine leukocytes to DEX- induced
apoptosis and cytotoxicity in the absence and presence of VT . . 61
Differential sensitivity of primary murine leukocytes to anti-Fas-induced
apoptosis and cytotoxicity in the presence and absence of VT . . 66
Differential sensitivity of primary murine leukocytes to TNF-a-induced
apoptosis and cytotoxicity in the presence and absence of VT . . 68
Mechanism of TNF-a potentiation of VT-induced TH apoptosis involves
reactive oxygen species, intracellular Ca2+, mitogen activated

protein kinases, and caspase-3 ........................... 73
CONCLUSION .................................................. 76
CHAPTER 5
Mechanisms of Trichothecene-Induced Iurkat T Cell Apoptosis:
Dose Response and Potentiation by TNF-a ............................ 78
INTRODUCTION ................................................ 78
MATERIALS AND METHODS ..................................... 80
Chemicals and Reagents ..................................... 80
Cell Culture ............................................... 81
Apoptosis Assay ........................................... 81
MTT conversion assay for cytotoxicity .......................... 81
Spectrofluorometric detection of intracellular Ca2+ ................. 82
Spectrofluorometric detection of reactive oxygen species (ROS) ...... 82
Western blot analysis for mitogen activated protein kinase (MAPK)
and Bcl2 expression ................................... 83

vi

Fluorescence assay for caspase-3 activity ........................ 84

Statistical analysis .......................................... 84

RESULTS ...................................................... 85

Time course of VT-induced apoptosis ........................... 85

Intracellular calcium modulation by VT ......................... 87

Alterations in reactive oxygen species (ROS) by VT ............... 91

VT-induced activation of ERK 1/2 ............................. 93

VT-induced activation of p38 and INK 1/2 ....................... 97

Modulation of Bcl2 expression by VT .......................... 100

VT-induced activation of caspase—3 ........................... 104

DISCUSSION .................................................. 107
CHAPTER 6

Summary ............................................................ 113

LITERATURE CITED ................................................. 115

vii

LIST OF TABLES

Table 1.1 Type A and Type B trichothecene mycotoxin functional groups ........... 3
Table 2.1 The effective dose (ED50, ng/ml) resulting in 50% apoptosis and

cytotoxicity in WEHI-23l immature B cells exposed to several Type A,

Type B, and Type D trichothecenes ................................... 19

Table 2.2 Translational inhibitor dose (molarity) required to induce 50% apoptosis in
murine B cells ................................................... 26

Table 3.1 Physical and chemical properties generated for the trichothecene
mycotoxins ...................................................... 32

Table 3.2 Physical and chemical descriptor values selected for principal component
analysis ......................................................... 33

Table 3.3 Effective dose (EDso, ng/ml) resulting in 50% apoptosis or cytotoxicity
in three murine B cell lines following 18 h trichothecene mycotoxin exposure . 38

Table 3.4 Significant correlations observed between log transformed toxicity values
and physico-chemical descriptors of the trichothecene myctoxins ........... 40

Table 5.1 Dose response relationships of pharmacological inhibitors (1 h pre-
incubation) on VT-induced cytotoxicity measured by MTT conversion assay . . 89

Table 5.2 Dose response relationships of several pharmacological inhibitors (18 h co-
incubation) on VT-induced cytotoxicity measured by MTT conversion assay . 94

Table 5.3 Effect of pharmacological inhibitors on VT-induced caspase-3 activity . . . 106

Table 5.4 Effect of MAPK inhibitors on VT-induced caspase-3 activity .......... 106

viii

LIST OF FIGURES
Figure 1.1 Common tetracyclic scirpenol ring system of the trichothecene
mycotoxins ....................................................... 2
Figure 1.2 Type D (macrocyclic) trichothecene functional groups ................. 4

Figure 2.1 Cycloheximide-(CHX)-induced apoptosis and cytotoxicity in immature
and mature murine B cells .......................................... 16

Figure 2.2 Trichothecene-induced apoptosis and cytotoxicity in immature and mature
murine B cells ................................................... 18

Figure 2.3 Shiga Like Toxin 1 (SLT1)-induced apoptosis and cytotoxicity in mature
murine B cells ................................................... 21

Figure 2.4 Ricin-induced apoptosis and cytotoxicity in immature and mature murine
B cells ......................................................... 24

Figure 3.1 Principal component analysis (PCA) of 702/3 cytotoxicity (A) and
apoptosis (B) .................................................... 42

.. Figure 3.2 Principal component analysis (PCA) of CH3] cytotoxicity (A) and
apoptosis (B) .................................................... 43

Figure 3.3 Principal component analysis (PCA) of 70Z/3 log transformed
cytotoxicity (A) and apoptosis (B) .................................... 44

Figure 4.1 VT induced apoptosis (A) and cytotoxicity (B) in the primary murine
leukocyte cultures ................................................ 57

Figure 4.2 LPS-induced increases in splenocyte MTT conversion were inhibited by
VT ............................................................ 58

Figure 4.3 PGE2 induced apoptosis (A) and cytotoxicity (B) in primary murine
thymocyte cultures ................................................ 60

Figure 4.4 Anti-IgM induced apoptosis (A) and cytotoxicity (B) in primary murine
splenocyte cultures ................................................ 62

Figure 4.5 DEX induced apoptosis in primary murine thymocyte, splenocyte, and
Peyer’s patch cultures ............................................. 64

Figure 4.6 DEX-induced increases in splenocyte apoptosis were inhibited by VT . . . . 65
Figure 4.7 Anti-Fas induced apoptosis (A) and cytotoxicity (B) in primary murine

ix

thymocyte cultures ................................................ 67

Figure 4.8 Anti-Fas potentiated VT-induced apoptosis (A) and cytotoxicity (B) in
primary murine bone marrow cultures ................................. 69

Figure 4.9 TNF-a potentiated VT-induced apoptosis (A) and cytotoxicity (B) in
primary murine thymocyte cultures ................................... 71

Figure 4.10 VT plus TNF-a induced apoptosis signal mechanisms involve reactive
oxygen species, intracellular CA 2*, mitogen activated protein kinases, and

caspase-3 ....................................................... 74
Figure 5.1 Time course of VT-induced apoptosis in Jurkat T cells: dose dependence

(A) and potentiation by TNF-a (B) ................................... 86
Figure 5.2 Inhibition of VT-induced apoptosis by pharmacological agents .......... 88
Figure 5.3 Intracellular calcium modulation by VT ............................ 90
Figure 5.4 Elevation of reactive oxygen species (ROS) by VT ................... 92
Figure 5.5 VT-induced activation of ERK1/2 ................................ 95
Figure 5.6 VT-induced activation of p38 MAPK .............................. 98
Figure 5.7 VT-induced activation of JNKl/Z ................................ 101
Figure 5.8 Modulation of Bcl2 expression by VT ............................. 103
Figure 5.9 VT-induced activation of caspase-3 .............................. 105

CHAPTER 1

Literature Review

General aspects of trichothecene mycotoxins

F usarium, Stachybotrys, and Myrothecium produce biologically active secondary
fungal metabolites called trichothecenes (Bondy and Pestka, 2000). This group of
mycotoxins is comprised of 182 compounds (Grove 1993; 1996) characterized by a
tetracyclic scirpenol ring system (Figure 1.1) and further categorized into four subtypes
(A — D) based on the presence or absence of specific functional groups (Ueno, 1983).
Three of these subtypes, Type A, B, and D, are common in the air and soil and therefore,
pose the greatest health risk to humans and animals. Type A trichothecenes are
characterized by a hydroxy or acyl moiety at the R” R2, R3, or R5 position(s) and include
T-2 toxin and diacetoxyscirpenol (Table 1.1). Type B trichothecenes, such as
deoxynivalenol (DON) and nivalenol (NIV) possess a carbonyl at R5 in addition to the
Type A moieties previously described. The satratoxins are examples of Type D
(macrocyclic) trichothecenes which possess a diester or triester ring system at the R2 and

R3 positions (Figure 1.2).

Environmental exposure to trichothecenes and adverse health effects

Exposure to trichothecenes occurs via consumption of mold-contaminated grains,
inhalation of fungal spores present in heating ducts and building materials, and airborne
exposure during suspected chemical warfare (Peraica et al., 1999). Alimentary toxic
aleukia, the first recorded human mycotoxicosis attributed to the trichothecenes, occurred
in the Soviet Union between 1932 and 1947. F. sporotrichoides was isolated from 40%

I

 

 

 

 

Figure 1.1. Common tetracyclic scirpenol ring system of the trichothecene mycotoxins.
Functional group location is depicted by R,, R2, R3, R 4, and R5. Functional groups for

individual trichothecenes are listed in Table 1.1.

Table 1.1. Type A and Type B trichothecene mycotoxin functional groups.

 

 

Trichothecene Abbreviation R, R2 R3 R4 R5
Type A
T-2 Toxin A1 OH OAca OAc H ISVb
HT-2 Toxin A2 OH OH OAc H ISV
Diacetoxyscirpenol A3 OH OAc OAc H H
Acetyl T-2 Toxin A4 OAc 0A0 0A0 H ISV
T-2 Tetraol A5 OH OH OH H OH
T-2 Triol A6 OH OH OH H ISV
3'OH T-2 Toxin A7 OH OAc OAc H OCOCH2C(OH)(CH3)2
3'OH HT-2 Toxin A8 OH OH OAc H OCOCH2C(OH)(CH3)2
Neosolaniol A9 OH OAc OAc H OH
Isotrichodermin A10 H OAc H H H
Isotrichodermol A1 1 H OH H H H
3,15 Didecalonectrin A12 OH H OH H H
Verrucarol A13 H OH OH H H
Type B
Deoxynivalenol B 1 OH H OH OH =
Fusarenon-X B2 OH OAc OH OH =
Nivalenol B3 OH OH OH OH =
1 5-Acetyldeoxynivalenol B4 OH H OAc OH —O
3-Acetyldeoxynivalenol B5 OAc H OH OH =
4, 15 Diacetylnivalenol B6 OH OAc OAc OH =

 

aOAc: -OC=OCH3
bISV: -OC=OCH2CH(CH3)2

  

 

’ OH \ OH
\O \ CH3
CH3 OH
Verrucarin A (01) Satratoxin G (DZ)
\
OH \
OH
CH3
Satratoxin F (D4) Roridin A (D5)

Figure 1.2. Type D (macrocyclic) trichothecene functional groups. Type D abbreviations

are noted in parenthesis.

of grain samples. With a mortality rate of 60%, continuous consumption of the toxic
grain was a determinant in severity of the disease. Between 1960 and 1991, 53 outbreaks
of human food poisoning associated with moldy cereal occurred throughout China
affecting approximately 130,000 people (Li, 1999). Stachybotryotoxicosis in horses,
sheep, and cattle has been associated with consumption of Stachybotrys-infested hay and
straw (Bamburg et al., 1968). Moldy corn toxicosis has been recorded in pigs and cattle
in the Midwestern United States and linked to F usarium growth.

Recent attention has focused on the contribution of molds to indoor air illness and
specifically, the role of trichothecene-producing molds in indoor environments.
Trichothecenes are estimated to be between 10- and 40-times (FAQ/WHO, 2002; Peraica
et al., 1999) more toxic when inhaled. Both F usarium and Stachybotrys isolates have
been collected from ventilation systems of homes of allergen sensitized children
(Wickrnan et al., 1992). Stachybotrys has been found in water damaged homes, schools,
and public buildings where the occupants have reported building-related respiratory
disease (Mahmoudi and Gershwin, 2000). Thirty-seven cases of infant pulmonary
hemorrhage were reported and linked to water damaged homes containing Stachybotrjvs
in Cleveland, OH (Dearbom et al., 1999). Stachybotrys spores were isolated from lung
tissue of one child.

Trichothecenes have the potential to be used as chemical warfare agents. The
toxins are believed to exist in the arsenals of some countries (McGovern et al., 1999;
Heyndrickx et al., 1984; Zilinskas, 1997) and were reportedly used as chemical warfare
agents in South-East Asia between 1975 and 1981(Peraica et al., 1999).

Mycotoxin levels have not always been determined in food-borne and inhalation

exposures due to unsuitable diagnostic methods or lack of samples (Bamburg, 1972).

5

However, trichothecene levels were recently determined in samples from a food
poisoning outbreak in Anhui, China in 1991 (Li et al., 1999). Cereal samples contained
up to 51.4, 6.9, 2.5, and 2.5 mg/kg deoxynivalenol (DON, vomitoxin), nivalenol (NIV),
3-acetyl-DON, and 15-acety1-DON, respectively. T-2 toxin was detected at
concentrations up to 420 pg/kg in moldy rice implicated in a food-borne outbreak in
Zhejiang, China (Wang et al., 1993a). In addition, T-2 toxin, DON, and NIV are
routinely detected in raw agricultural products (Scott, 1997; WHO, 1990) and
commercial grain-based foods (Abouzied et al., 1991) intended for human consumption.

Of the trichothecenes, DON is among the most commonly encountered relative to
frequency and concentration in wheat, corn, and barley worldwide (Rotter et al., 1996).
DON levels can vary widely on a year-to—year and region-to-region basis due to weather
and storage conditions. DON at low levels (< 1 ppm) is frequently encountered but may
occasionally reach 5 to 20 ppm in processed foods (Abouzied et al., 1991).

The U. S. Food and Drug Administration has established a tolerance limit of 1
ppm DON for bran, flour, and germ targeted for human consumption. Health Canada
designated guidelines of 2 ppm DON in uncleaned soft wheat used for nonstaple goods
and 1 ppm for infant foods. Maximum tolerated levels of 0.5 ppm, 1 ppm, and 1 ppm
have been set by Austria, Hungary, and the Soviet Union, respectively (Pestka,
submitted). The Food and Agriculture Organization of the United Nations and World
Health Organization have recently recommended the development of DON equivalency

factors to assess human health risks from trichothecene exposure (F AO/W H0, 2002).

Trichothecenes are immunotoxins

The common mechanism of action of all trichothecenes is inhibition of protein

6

synthesis following binding to the 60S ribosomal subunit. Ribosomal binding occurs
rapidly and drastically impacts cells that are actively dividing. Cells of the intestinal
mucosa and immune system are particularly sensitive to trichothecenes (Bondy and
Pestka, 2000). Ingestion of trichothecenes leads to gastrointestinal disorders including
nausea, vomiting, and diarrhea. Airborne exposure to this group of toxins results in
respiratory tract irritation, fatigue, and dermatitis. In addition, alterations of immune cell
function, dysregulation of the humoral immune response, and impairment of host
resistance to pathogens have been described for both exposure scenarios (Bondy and
Pestka, 2000; Nikulin etal., 1997; Nikulin et al., 1996). Symptoms described in
food-borne outbreaks and reported for suspected inhalation mimic symptoms of
experimental animals exposed to trichothecenes and provide a useful model for toxicity
studies.

DON, the most extensively studied trichothecene, may stimulate or suppress the
immune system dependent on dose and duration of exposure. Mice exposed to low
concentrations of DON exhibit increased cytokine production, increased serum IgA, and
IgA immune complex deposition in the kidney (Bondy and Pestka, 2000). The
development of IgA nephropathy in the mouse mimics human IgA nephropathy. Mice
exposed to high concentrations of DON have impaired host-resistance to challenge with

Listeria and Salmonella and decreased lymphocyte numbers.

Trichothecenes induce lymphocyte apoptosis
Cell death by apoptosis (Yang etal., 2000; Okumura et al., 2000; Shifrin and
Anderson, 1999) has been widely reported for the trichothecenes and is likely to

contribute to the observed immunotoxic effects. Apoptosis is a regulated form of cell

7

death that removes damaged or unnecessary cells. Scheduled cell death regulates
embryogenesis, maintains tissue homeostasis, and prevents autoimmunity (Dong et al.,
2002; Cohen et al., 1992; Koury et al., 1992). However, apoptosis may also result from
cellular stress (i.e. UV light, cytokines, and heat shock), chemical insult (Saini and
Walker, 1998), or ribotoxic stress resulting from ribosomal cleavage (Iordanov et al.,
1997) or ribosomal binding (Shifrin and Anderson, 1999) by translational inhibitors.
Down regulation of apoptosis in lymphocytes of the immune system may lead to
autoimmunity and inflammation whereas up-regulation of apoptosis may lead to immune

suppression and increased susceptibility to pathogens.

Trichothecene toxicity is potentiated by bacterial lipopolysaccharide

Signaling components of both stress (Albers et al., 1996; Pruett et al., 1993) and
inflammation (Luster et al., 2000;.Emmendoerffer et al., 2000) may play a critical role in
chemically-induced toxic responses. Recent studies indicate that exposure to low levels
of lipopolysaccharide (LPS), the biologically active component of gram negative
bacteria, can influence the magnitude of a toxic response to xenobiotic agents (Roth et
al., 1997). Increased hepatotoxic responses have been observed for carbon tetrachloride,
ethanol, and ally alcohol. Previous in vivo studies demonstrate increased trichothecene
toxicity in the presence of LPS. LD50 doses decreased and mortality increased in mice
exposed to Salmonella and T-2 toxin simultaneously (Tai and Pestka, 1988). Increased
lymphocyte apoptosis and mortality was observed in LPS and DON-treated mice (Zhou
et al., 2000; Zhou et al., 1999). The mechanisms involved in this synergistic toxicity
have not been elucidated. However, LPS induces its biological effects by stimulating

host cells to produce a variety of mediators including bioactive lipids (i.e prostaglandins),

8

glucocorticoids (i.e. corticosterone), and proinflammatory cytokines (i.e. TNF-a). These

mediators may interact with trichothecenes to alter immune cell responses.

Rationale
Despite the high potential for human exposure, little information exists on the role

of apoptosis in VT-induced immunosuppression and the signaling mechanisms involved

in apoptosis induction. The objectives of this research were as follows:

1. To compare the induction of apoptosis and cytotoxicity by several trichothecenes
and other translational inhibitors in three murine lymphocyte cell lines.

2. To determine the structural aspects of the trichothecenes that impart biological
activity in the induction of cell death in murine lymphocytes.

3. To investigate the potentiation of trichothecene-induced cell death by
inflammatory agents in primary murine lymphocytes.

4. To characterize the dose dependent induction of apoptosis and alterations in
apoptotic signal transduction mechanisms in human lymphocytes exposed to
trichothecenes.

5. To assess the potentiation of trichothecene-induced apoptosis and apoptotic signal
transduction mechanisms by TNF-a in a human lymphocyte model.

The aforementioned issues were addressed in the following five chapters.
Chapter 2 compares the induction of apoptosis and cytotoxicity by trichothecenes and
several
naturally occurring translational inhibitors in murine B lymphocytes. The B lymphocyte
lines studied represent phenotypically immature B cells (CH3 1, WEHI-231), and mature

B cells (CH12.LX). These cells are differentially sensitive to apoptotic agents. For

9

example, CH3] and WEHI-23l unlike CH12.LX, express surface IgM and are sensitive
to apoptosis induced by antigenic mimics such as anti-IgM (Warner et al., 1992). Both
immature and mature B cells express prostaglandin receptors, however, only immature
CH3] and WEHI-231 cells undergo apoptosis in response to PGE2 (Brown and Phipps,
1995). Finally, CH3], WEHI-231, and CH12.LX express FasR, however, mature B cells
are most susceptible to apoptosis induced by Fas ligand (Onel et al., 1995). Therefore,
differential sensitivity to translational inhibitors could be addressed using this
lymphocyte model system. Chapter 3 describes physical and chemical characteristics of
the trichothecenes that influence murine B lymphocyte apoptosis and cytotoxicity
induction and may be used to predict the toxicity of uncharacterized trichothecenes.
Chapter 4 investigates the potentiation of trichothecene-induced toxicity by inflammatory
agents in several primary murine lymphocytes. Chapter 5 compares dose response
induction of apoptosis and alterations in cell signal transduction mechanisms of
trichothecenes in human lymphocytes. In addition, the induction of apoptosis and
alterations in cell signaling in the potentiation of trichothecene-induced apoptosis by

TNF-oc are addressed. Chapter 6 summarizes these inter-related studies.

10

CHAPTER 2

Differential Induction of B Cell Cytotoxicity and Apoptosis By Trichothecene

Mycotoxins and Other Translational Inhibitors

INTRODUCTION

A number of natural toxins including trichothecene mycotoxins, shiga like toxins
(SLT), and ricin are potent translational inhibitors. Ribosomal binding by the
trichothecenes, secondary fungal metabolites produced by F usarium species, inhibits the
initiation/elongation step of protein synthesis. Both SLT, bacterial toxins produced by
Escherichia coli and Shigella dysenteriae, and ricin, a lectin produced by Ricinus
communis, cleave the ribosome to cause inhibition of protein synthesis. While these
three translational inhibitors are diverse in their mechanism of action, all induce cell
death by apoptosis (Chen etal., 1998; Griffiths et al., 1987; Kiyokawa etal., 2001; Yang
etaL,2000)

Apoptosis is a regulated form of cell death that removes damaged or unnecessary
cells. Scheduled cell death regulates embryogenesis, maintains tissue homeostasis and
prevents autoimmunity in the immune system (Dong etal., 2002; Cohen et al., 1992;
Koury et al., 1992). However, apoptosis may also result from cellular stress (i.e. UV
light, cytokines, and heat shock), chemical insult (Saini and Walker, 1998), or ribotoxic
stress resulting from ribosomal cleavage (Iordanov et al., 1997) or ribosomal binding
(Shifrin and Anderson, 1999) by translational inhibitors. Down-regulation of apoptosis

in lymphocytes of the immune system may lead to autoimmunity and inflammation

11

whereas upregulation of apoptosis may lead to increased susceptibility to pathogens.

Trichothecenes, SLT, and ricin pose a health risk to humans and animals exposed
to these toxins in food or the environment. Trichothecene mycotoxicosis has been linked
to consumption of F usarium-contaminated grain-based food and feeds (Bondy and
Pestka, 2000). Recent attention has focused on the contribution of molds to indoor air
illness through inhalation of trichothecene-producing spores (Smoragiewicz et al., 1993;
Johanning et al., 1996; Tuomi et al., 1998). The most common symptoms described for
trichothecene mycotoxicosis include vomiting, diarrhea, respiratory tract irritation,
leukopenia, and impaired immune function (Bondy and Pestka, 2000). Exposure to SLT
occurs through consumption of meat, apple juice, and vegetables contaminated with
toxin-producing E. coli (Sandvi g and vanDeurs, 2000). This group of toxins plays a
critical role in the pathogenesis of the epidemic form of hemolytic uremic syndrome
(HUS) which may ultimately lead to kidney failure. Following consumption of castor
beans or castor seed oil containing ricin, symptoms of gastroenteritis, hepatoxicity, and
teratogenicity were documented (Palatnick and Tenenbein, 2000; E1 Mauhoub etal.,
1983).

Clinical signs described following exposure to translational inhibitors may be
partially due to apoptosis induction. Gerrninal centers containing actively dividing B cell
lineages are particularly sensitive to protein synthesis inhibition. Of these lineages, the
immature B cell is the most susceptible to apoptosis (Carsetti et al., 1995; Garvy etal.,
1993). The objective of this study was to compare the sensitivity of three murine B cell
lines to apoptosis and cytotoxicity induction by several trichothecene mycotoxins, SLT-1,
and ricin to cycloheximide (CHX), a prototypical protein sythesis inhibitor commonly

used to study cell signal transduction mechanisms. The differential sensitivity observed

12

among immature (CH3], WEHI-23l) and mature (CH12.LX) B cell lineages to these
toxins may contribute to our understanding of mechanisms of toxicity of the

aforementioned translational inhibitors.

MATERIALS AND METHODS

Chemicals and reagents

Chemicals and reagents were purchased from Sigma Chemical Company (St.
Louis, MO) unless otherwise noted. Satratoxin G, satratoxin F, and satratoxin H were
obtained from Dr. Bruce Jarvis (University of Maryland, College Park, MD). 3'OH
metabolites were produced by the procedure of Yoshizawa et a]. (198 l; 1984).
Trichothecenes, cycloheximide (CHX), and ricin were dissolved in 100% ethanol and
diluted in RPMI-1640 supplemented with 100 U/ml penicillin, 100 pg/ml streptomycin,
50 11M 2-mercaptoethanol, 2 mM glutamine, 1 mM sodium pyruvate (Gibco Life
Technologies, Rockville, MD), and 1 mM non-essential amino acids (Gibco). F inal
ethanol concentration in the culture medium was less than 0.01% (v/v) which did not
induce apoptosis or cytotoxicity. SLT] (107 units/mg, Toxin Technology Inc., Sarasota,

FL) was dissolved in 0.01M PBS and diluted in supplemented RPMI-1640 medium.

Cell lines and culture

WEHI-23l and CH3] cells, phenotypically immature B cells, were obtained from
Dr. David Scott (American Red Cross Holland Laboratory, Rockville, MD). CH12.LX
cells, phenotypically mature B cells, were obtained from Dr. Geoffrey Haughton
(University of North Carolina, Chapel Hill, NC). Cell number and viability were

13

assessed microscopically by trypan blue dye exclusion using a hemocytometer (Stiles,
1986). Cells were cultured in supplemented RPMI-1640 medium with 5% (v/v)
heat-inactivated fetal bovine serum at a concentration of 5 x 105/ml in 200 til/well in 96
well flat bottom plates at 37°C in a 5% CO2 humidified incubator. Triplicate cultures
were treated with three concentrations of each translational inhibitor diluted in

supplemented RPMI-l640 for 18 h. All experiments were repeated (n=6).

MTT conversion assay for cytotoxicity

Following treatment, 20 pl of a 5 mg/ml solution of 3- (4,5 dimethylthiazol-
2-ul)-2,5.—diphenyl tetrazolium bromide (MTT) in 0.01 M PBS was added to each well for
the final 2 h of the 18 h incubation period (Marin et al., 1996). Microtiter plates were
centrifuged at 450 x g for 20 min and media were aspirated to minimize formazan crystal
loss. The resultant crystals were dissolved in 150 p1 dimethylsulfoxide (DMSO) for 15
min. Optical density was measured using a Vmax Microplate Reader (Molecular
Devices, Menlo Park, CA) at dual wavelengths, 570 nm and 690 nm. Percent MTT
inhibition was calculated as follows: ((1 - absorbance of treatment)/ absorbance of

control) x 100. Mean values were used to graphically determine ED50 values.

Apoptosis assay

Apoptotsis, as determined by cell morphology, was quantitated by fluorescence
microscopy as described (Duke and Cohen, 1995). Briefly, following trichothecene
treatment, microtiter plates were centrifuged at 450 x g for 10 min and 150 1.11 of
supernatant was removed. Five 11] of DNA staining reagent (acridine orange (100 pg/ml)

and ethidium bromide (100 pg/ml) in 0.01 M phosphate buffered saline (PBS)) was

14

added to each well. Cells were classified as apOptotic or normal based on chromatin
organization. A normal nucleus appeared large with an organized structure while an
apoptotic nucleus appeared small and highly condensed or fragmented. Percent apoptosis
was calculated as follows: number of cells with apoptotic nuclei/total cell number x 100.
Mean values were used to graphically determine the effective dose causing 50%

apoptosis (EDSO).

Statistical analysis

The data were analyzed using Systat version 5.0 (Evanston, IL). A Student's t test
was used to compare two groups while multiple groups were compared using a one way
analysis of variance (ANOVA) and a Tukey test for mean separation. A p value <0.05

Was considered statistically significant.

RESULTS AND DISCUSSION

Induction of cytotoxicity and apoptosis by cycloheximide (CHX)

Cycloheximide (CHX), an antibiotic produced by Streptomyces griseus, interferes
with the translocase enzyme to block the translocation step of protein synthesis. CHX is
widely used as a pharmacological tool to study cell signal trasduction mechanisms and
has been used to study superinduction of proteins (Zinck et al., 1995), to block apoptosis
(Takahashi et al., 2002; Liao et al., 2001), and to potentiate apoptosis (Li etal., 2001).
Induction of B cell cytotoxicity by CHX was evaluated at 18 h by the MTT conversion
assay. CHX significantly increased immature and mature B cell cytotoxicity (Figure
2.1). EDso values for cytotoxicity were 0.25, 0.33, and 0.15 pg/ml for CH3 1, CH12.LX,

15

Cycloheximide (uglml)

 

o 20 40 so so o 20 40 so so 100
Percent Apoptosis MTT Response

Figure 2.1. Cycloheximide-(CHX)-induced apoptosis and cytotoxicity in immature and
mature murine B cells. Murine CH3] (A, B), CH12.LX (C, D), and WEH1-231 (E, F) B
cells (5 X 105/ml) were exposed to CHX for 18 h prior to determination of apoptosis (A,
C, and E) and cytotoxicity (B, D, and F) as described. Asterisk indicates values

significantly different from vehicle control (p < 0.05).

16

and WEHI-231 cells, respectively. Induction of apoptosis at 18 h was evaluated by
fluorescence microscopy. CHX significantly increased apoptotic morphology in all cell
lines (Figure 2.1). ED50 values for apoptosis were 0.4, 0.15, and 2.0 pg/ml for CH3 1,
CH12.LX and WEHI-231 cells, respectively. These results suggest both immature and
mature B cells are similarly sensitive to cell death induced by CHX. The similarity in
apoptosis and cytotoxicity ED50 values suggests cell death was primarily due to
apoptosis. In previous studies, CHX-induced cell death was observed at higher doses (10
- 50 pg/ml) and later time points (24 - 72 h) in MDA-23 1, MCF-7, or Burkitt lymphoma
cells than reported here (Geier et al., 1996; Ishii et al., 1995; Geier et al., 1992)
suggesting murine B cells may be a more sensitive to CHX-induced cell death than these

aforementioned cell lines. However, ED50 values were not determined in these studies.

Induction of cytotoxicity and apoptosis by trichothecene mycotoxins

Trichothecene mycotoxins consist of 182 family members (Grove 1993; 1996)
divided into three subtypes based on the presence or absence of specific functional
groups (Figure 1.1, 1.2 and Table 1.1; Snyder, 1986). Ribosomal binding by the
trichothecenes inhibits protein synthesis in actively dividing cells (Bondy and Pestka,
2000). Induction of cytotoxicity and apoptosis by trichothecenes was evaluated at 18 h.
Deoxynivalenol (DON), a representative trichothecene, induced cytotoxicity and
apoptosis afier exposure to 100 - 1000 ng/ml and 1000 ng/ml respectively (Figure 2.2).
ED50 values for WEHI-231 cells were derived for 24 trichothecenes and are summarized
in Table 2.]. Similar values were calculated for CH12.LX and CH3] cells suggesting
immature and mature B cells are equally sensitive to trichothecene induced cell death

(summarized in Table 3.4).

17

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Table 2.1. The effective dose (EDso, ng/ml) resulting in 50% apoptosis and cytotoxicity
in WEHI-23l immature B cells exposed to several Type A, Type B, and Type D
trichothecenes.

 

 

Trichothecene Apoptosis (EDm) Cytotoxicity (EDm)
Type A
T-2 Toxin 2.0 0.8
HT-2 Toxin 6.5 2.75
Diacetoxyscirpenol 4.0 4.0
Acetyl T-2 Toxin 70 55
T-2 Tetraol >1000 4O
T-2 Trio] >1000 45
3’OH T-2 Toxin 250 10
3’OH HT-2 Toxin 1000 50
Neosolaniol 100 70
Isotrichodermol 800 650
Isotrichodermin 4000 700
3’15 Didecalonectrin 4500 1500
Verrucarol >10000 >10000
Type B
Deoxynivalenol 500 300
Fusarenon X 400 200
Nivalenol 475 400
15 AcDeoxynivalenol 500 300
3 AcDeoxynivalenol >10000 5000
4, 15 DiAcNivalenol 550 100
Type D
Verrucarin A 0.4 0.35
Satratoxin G 2.0 0.5
Satratoxin H 9.0 3.75
Isosatratoxin F 3.0 2
Roridin A 0.4 0.35

 

19

Trichothecenes demonstrated a characteristic rank toxicity in which Type D >
Type A > Type B (Table 2.1). Exceptions occurred when parent compounds, such as T-2
toxin, were compared to a less toxic form, such as T-2 tetraol and T-2 trio]. Cytotoxicity
values reported are similar to values previously reported for this group of translational
inhibitors (Ueno, 1983). ED50 values derived for cytotoxicity were generally lower than
values calculated for apoptosis. This suggests inhibition of MTT conversion may be
indicative of an earlier stage of apoptosis or cell growth inhibition without development
of a distinct apoptotic morphology by 18 h. No significant increase in necrotic cells was
noted by fluorescence microscopy. Differences in apoptotic and cytotoxic values were
not observed in CH3] or CH12.LX cell lines (Table 3.4) suggesting this may be a cell

line specific effect.

Differential Induction of cytotoxicity and apoptosis by shiga like toxin 1 (SLTI)

SLT] is a bacterial toxin. composed of two subunits. The B subunit binds to
surface glycolipids and the A subunit cleaves ribosomes and inhibits protein synthesis.
The induction of apoptosis by SLT] has been reported in several cell types and may
contribute to the development of HUS (Pijpers et_al., 2001; Nakao and Takeda, 2000;
Kodama et al., 1999). Induction of cytotoxicity and apoptosis by SLT] was evaluated in
murine B cells at 18 h (Figure 2.3). The ED50 value for CH12.LX cytotoxicity,
determined by MTT conversion assay, was 7 ng/ml. The dose resulting in 50%
apoptosis, measured using fluorescence microscopy, was 3 ng/ml. CH3] and WEHI-231
immature B cell viability were not significantly altered when treated with SLT]

concentrations up to 100 ng’ml.

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Differential sensitivity in B cell subpopulations to SLTl-induced cell death has
been observed in human B cells. Cohen et a]. (1990) observed most IgG- and
IgA-committed subsets were sensitive to SLTl-induced cell death while IgM- and
IgD-committed subsets were resistant. In contrast, we demonstrated sensitivity of IgM
expressing CH12.LX cells to SLTl-induced apoptosis. This differential sensitivity may
relate to glycolipid expression differences on cell surfaces rather than Ig expression.
Relatedly, Daudi cells are resistant to SLT] toxic effects due to deficient levels of SLT
glycolipid receptor Gb3/CD77 (Cohen et al., 1990) while renal tissue expresses the
highest levels of Gb3/CD77 thereby accounting for the high sensitivity of these cells to
SLT (Lingwood, 1996). It is possible that CH12.LX cells may express higher levels of
Gb3/CD77 than other IgM+ B cell subsets and explain the sensitivity of this cell line.
However, to our knowledge, cell surface expression of Gb3/CD77 on CH12.LX cells has
not been reported. SLT] uptake by a cell appears to be an early critical step in apoptosis
induction.

Interestingly, surface binding of SLT] may initiate apoptosis independent of
ribosomal cleavage. The sequence of CD19 is similar to shiga B chain and binds to the
Gb3/CD77 receptor. The CD19-Gb3/CD77 complex is endocytosed and leads to the
induction of apoptosis in the absence of ribosomal cleavage (Sandvig and vanDeurs,
2000). Mori et a1. (2000) demonstrated that surface receptor binding in this pathway
leads to Gb3/CD77-IgM clustering, cross-linking, and activation of the antigen receptor

ligation mediated apoptosis pathway.

Differential Induction of cytotoxicity and apoptosis by ricin

Similar to SLT], ricin, a plant toxin, is composed of two subunits. The B subunit

22

binds to glycolipids or glycoproteins present in the cell membrane while the A subunit
cleaves ribosomal RNA to inhibit protein synthesis. One molecule of ricin has been
estimated to inactivate approximately 2000 ribosomes per minute (Sandvig and
vanDeurs, 2000). Induction of cytotoxicity and apoptosis by ricin was evaluated at 18 h
(Figure 2.4). ED50 values derived using the MTT conversion assay were 3.0, 0.05, and
4.0 jig/ml for CH3], CH12.LX, and WEHI-23l cells, respectively. Apoptotic
morphology was assessed by fluorescence microscopy. EDSo values for apoptosis
induction in CH3], CH12.LX, and WEHI-231 cells were 2.25, 0.03, and 4.0 pig/m1,
respectively. These results suggest mature B cells were more sensitive to ricin-induced
cell death than immature B cells. As with SLT], the differential sensitivity to ricin may
result from differences in glycolipid or glycoprotein expression that influence cellular
uptake of ricin. Similar ED50 values for apoptosis and cytotoxicity were calculated for
each individual cell line suggesting cell death was primarily due to apoptosis. A previous
study evaluating ricin-induced cell death reported approximately 60% death in MBA-231
cells following exposure to 250 pg/ml (Geier et al., 1996). This difference in dose may
reflect differences in cellular uptake of ricin which would be critical to the induction of
cell death.

SUMMARY

Trichothecenes, SLT, and ricin, natural toxins encountered in food and the
environment, pose a health risk to humans and animals. Upon exposure, these toxins
induce a ribotoxic stress response which may ultimately lead to apoptosis induction. This
study compared induction of cytotoxicity and apoptosis by the aforementioned natural

toxins to CHX, a translational inhibitor commonly used to study cell signaling events.

23

 

 

Ricin ( uglml)

 

o 20 40 so 80 o 20 40 so 80100
Percent Apoptosis MTT Response

Figure 2.4. Ricin-induced apoptosis and cytotoxicity in immature and mature murine B
cells. Murine CH3] (A, B), CH12.LX (C, D) and WEHl-231 (E, F) B cells (5 X 105/ml)
were exposed to ricin for 18 h prior to determination of apoptosis (A, C, and E) and
cytotoxicity (B, D, and F) as described. Asterisk indicates values significantly different

from vehicle controls (p < 0.05).

24

These results suggest lymphocytes of the immune system were particularly sensitive to
the toxic effects of translational inhibitors. Comparisons among the translational
inhibitors tested suggest Type D and Type A trichothecenes, ricin, and SLT] were more
potent inducers of apoptosis than CHX (Table 2.2). Type B, and Type A trichothecene
metabolites were less potent inducers of apoptosis when compared to CHX in this
immunotoxicity model. CHX and the trichothecenes tested demonstrated
non-preferential induction of cytotoxicity and apoptosis in immature or mature B cells
suggesting toxic effects of these natural toxins may be non-specific upon exposure.
However, mature B cells were more sensitive to ricin and SLTI-induced cytotoxicity and
apoptosis than immature B cells. This differential sensitivity may be due to glycolipid
and glycoprotein cell membrane expression which would result in variable cellular
uptake. Taken together, these data may be critical to identification and treatment
following accidental or deliberate exposure to the trichothecenes, SLT] or ricin. Up-
regulation of apoptosis in B lymphocytes may contribute to the impairment of the
immune response and other symptoms described following exposure to these naturally

occurring translational inhibitors.

25

Table 2.2. Translational inhibitor dose (molarity) required to induce 50% apoptosis in
murine B cell lines.

 

 

Translational Inhibitor Apoptosis (ED50)

Type D Trichothecenes 1 - 16 nM
Type A Trichothecenes 5 — 10 nM
Shiga Like .Toxin 1 10 nM
Ricin . 1 — 100 nM
Cycloheximide 0.5 uM
Type A Trichothecene Metabolites 0.5 — 50 uM
Type B Trichothecenes 1 — 2 11M

 

26

CHAPTER 3

Induction of B Cell Cytotoxicity and Apoptosis by Environmental and F ood-bome

Trichothecenes: Quantitative Structure Activity Relationships

INTRODUCTION

Trichothecene mycotoxins are naturally occurring environmental contaminants
produced by molds common in the air and soil. These fungal metabolites are
characterized by a tetracyclic scirpenol ring system (Figure 1.1) and categorized into four
subtypes based on the presence or absence of specific firnctional groups (Ueno, 1983).
Three of these subtypes, Type A, B, and D, pose the greatest health risk to humans and
animals and therefore, are the focus of this study. Type A trichothecenes are
characterized by a hydroxy or acyl moiety at R,, R2, R3, or R5 position(s) (Table 1.2).
Type B trichothecenes possess a carbonyl at R5 in addition to the Type A moieties. Type
D (macrocyclic) trichothecenes contain a diester or triester ring system at the R2 and R3
positions (Figure 1.2) (Snyder, 1986).

Historically, outbreaks of human and animal disease have been linked to
trichothecene- producing molds such as F usarium, associated with production of Type A
and B toxins, and Stachybotrys, associated with production of Type D toxins (reviewed
by Bamburg, 1968). Alimentary toxic aleukia in the Soviet Union and red mold disease
in Japan were linked to human consumption of F usarium contaminated grains.

Outbreaks of stachybotryotoxicosis in horses, sheep, and cattle have been associated with

consumption of Stachybotrys-infested hay and straw. Moldy com toxicosis has been

27

recorded in pigs and cattle in the Midwestern United States and linked to F usarium
growth.

Between 1960 - 199], 53 outbreaks of human food poisoning associated with
moldy cereal occurred throughout China (Li et al., 1999). Trichothecene levels were
recently determined in samples from a food poisoning outbreak in Anhui, China in 1991
(Li et al., 1999). Moldy cereal samples contained up to 51.4, 6.9, 2.5, and 2.5 mg/kg
deoxynivalenol (DON), nivalenol (NIV), 3-acetyl-DON, and 15-acetyl-DON,
respectively. Approximately 130,000 people were affected by gastrointestinal disorders
including nausea and vomiting. In addition, T-2 toxin was detected at concentrations up
to 420 ug/kg in moldy rice implicated in a food-borne outbreak in Zhejiang, China
(Wang et al., 1993a). Trichothecene levels have not always been determined in the
above mentioned outbreaks due to unsuitable diagnostic methods or lack of samples
(Bamburg, 1972). However, Type A (T-2 toxin) and Type B toxins (DON and NIV) are
routinely detected in raw agricultural products as well as commercial grain based foods
intended for human consumption (Scott, 1997; WHO, 1990; Abouzied et al., 1991).

Recent attention has focused on the contribution of molds to indoor air illness and
specifically, the role of trichothecene-producing molds in indoor environments. Both
F usarium and Stachybotrys isolates have been collected from ventilation systems of
homes of allergen sensitized children (Wickman et al., 1992). Stachybotrys has been
found in water damaged homes, schools, and public buildings where the occupants have
reported building-related respiratory disease (reviewed by Mahmoudi and Gershwin,
2000). Thirty-four cases of infant pulmonary hemorrhage were reported and linked to
water damaged homes containing Stachybotrys in Cleveland, OH (Dearbom et al., 1999).

Type A (T-2 toxin, HT-2 toxin, and diacetoxyscirpenol) and Type D (satratoxins and

28

verrucarins) trichothecenes have been identified in indoor air samples in which
respiratory disease has been reported (Smoragiewicz et al., 1993; Johanning et al., 1996;
Tuomi et al., 1998; Malrnoudi and Gershwin, 2000). Stachybon'ys was isolated from the
bronchoalveolar lavage fluid of one infant with pulmonary hemosideriosis but the
involvement of Type D trichothecenes was not determined (Elidemir et al., 1999).
Trichothecenes also have the potential to be used as chemical warfare agents and
are believed to exist in the arsenals of some countries (McGovern et al., 1999;
Heyndrickx et al., 1984; Zilinskas, 1997). The ability to predict toxicity of
trichothecenes would be of paramount importance to military medical personnel and
public health officials encountering these agents during military or terrorist attack.

Symptoms described in food-borne outbreaks (nausea, vomiting, diarrhea, and
leukopenia) and reported for suspected inhalation (respiratory tract irritation, fatigue,
dermatitis, impaired immune function, and nasal hemorrhage) mimic symptoms of
experimental animals exposed to trichothecene mycotoxins (Bondy and Pestka, 2000;
Nikulin etal., 1997; Nikulin et al., 1996). The primary target, both in vivo and in vitro,
of all trichothecenes is the 608 ribosomal subunit.

Ribosomal binding by trichothecenes occurs rapidly and blocks protein synthesis
in actively dividing immune cells in the bone marrow, spleen, thymus, and lymph nodes
(Ueno, 1983). Exposure to trichothecenes leads to alteration of immune cell function,
dysregulation of the humoral immune response, and impairment of host resistance to
pathogens (reviewed by Bondy and Pestka, 2000). Direct cytotoxicity (Hanelt et al.,
1994; Anderson et al., 1989; Yike et al., 1999) and induction of apoptosis (Yang et al.,
2000; Okumura et al., 2000; Shifrin and Anderson, 1999) are widely reported for this

group of compounds and likely to contribute to these immunotoxic effects. A general

29

rank order of toxicity is observed in which Type D > Type A > Type B (Ueno, 1983)
suggesting that the functional groups on the scirpenol ring system contribute greatly to
the biological action of this group of mycotoxins. Although the trichothecene family
contains 182 structurally related metabolites (Grove, 1993; 1996), the toxicity has been
well-characterized for only a few compounds. Recently, a joint committee on food
additives organized by the Food and Agriculture Organization of the United Nations and
World Health Organization recommended the development of DON equivalency factors
to assess human risk from trichothecene exposure (FAQ/WHO, 2001).

Quantitative structure activity relationship (QSAR) analysis may contribute to our
ability to understand trichothecene mechanism of action. QSAR links biological
properties of a chemical to its molecular structure (Barratt, 1998a). Consequently, a
hypothesis can be proposed to identify the physical or chemical parameters which are
crucial to the structure activity relationship of a group of chemicals. QSAR analysis is
commonly used to predict the toxic action of chemicals (Benigni and Andreoli, 1993;
Purdy, 1996; Kulkami et al., 2002), to predict receptor binding (Tong et al., 1997) or to
address mechanisms involved in a toxic endpoint (Lopez de Compadre, 1990).
Trichothecenes possess the structural similarity and the common mechanism of action (in
vivo and in vitro) necessary to develop a robust model (Barratt, 1998b). Such a model
can ultimately be used to predict the toxicity of untested trichothecenes. The goal of this
project was to apply QSAR using principal component analysis to identify the
physico-chemical characteristics which clearly separate the trichothecene mycotoxins
into subtypes (Type A, B, and D) and gain insight into trichothecene mechanisms of

action.

30

MODEL DESIGN AND RATIONALE

Three dimensional trichothecene structures were drawn and analyzed using Chem
3D version 4.0 (CambridgeSoft, Cambridge, MA) and Cache (Oxford Molecular Group
Inc., Beaverton, OR). Briefly, the dihedral driver was used to identify and access
conformational space. Allinger's molecular mechanics (MM2) force field parameters
were used to describe the energy of each molecule. The resultant stable conformation
with the lowest energy was selected following each pass. The geometry was
re-optimized and semi-empirical Austin Model 1 (AMI) calculations were implemented
using the closed shell wave function for the final optimization of each trichothecene
structure. Structures were systematically aligned and 34 physical and chemical
properties were calculated using Molecular Modeling Pro version 2.4 (WindowChem
Software Inc., Fairfield, CA; Table 3.1).

Variables were selected for further analysis based on (1) statistical correlation
between variables and (2) the relation of each variable to uptake, transport, and reactivity
properties relevant to biological activity. Several variables were highly correlated
(Pearson correlation, p < 0.05 (data not shown)) likely due to the similarities in variable
calculation. Eight variables were selected for further analysis (Table 3.2). Hansen
solubility parameter (SOL) and hydrophilic-lipophilic balance (HLB) are indicators of
membrane penetration or uptake of the molecule. Hansch Log P (Log P), Crippen Log P
(CRIP), and CNDO Log P (CNDO) are measurements of lipophilicity which influences
transportation of a molecule to the site of action. Kier connectivity index (KIER) is an
indicator of molecular size and shape and can reflect binding at the site of action.

Highest Occupied Molecular Orbital (HOMO) and Lowest Unoccupied Molecular

31

CNDO Log P‘I

Density

Hansch Log P

Hansen Hydrogen Bonding
Hansen Solubility Parameter

Hydrogen Bond Acceptor

' Hydrophilic Lipophilic Balance
Kier Connectivity 0

Kier Connectivity 2

Kier Connectivity Index 4

Table 3.1 Physical and chemical properties generated for the trichothecene mycotoxins.

Crippen’s Log P

Dipole Moment

Hansen Dispersion

Hansen Polarity

Highest Occupied Molecular
Orbital

Hydrogen Bond Donor

Hydrophilic Surface

Kier Connectivity ]

Kier Connectivity 3

Kier Shape Index Kappa 1

Lowest Unoccupied Molecular Molecular Depth
Orbital

Molecular Length Molecular Volume

Molecular Weight Molecular Width

Surface Area Valence 0

Valence 1 Valence 2

Valence 3 Valence Index 4

Water of Hydration Water Solubility

 

32

alProperties in bold type were selected for further analysis.

Table 3.2. Physical and chemical descriptor values selected for principal component

 

 

analysis.

Trichothecene“ SOLb HLBc LogP‘ CRIP‘ CNDOf Klee HOMOh LUMO‘
A1 18.065 13.641 3.103 0.846 6.724 11.601 -0.396 0.145
A2 19.381 13.815 0.457 0.716 5.965 10.879 -0.404 0.124
A3 17.035 15.076 0.943 0.275 5.037 9.773 -0.402 0.136
A4 24.222 14.599 2.118 0.975 7.699 12.203 -0.416 0.137
A5 26.172 12.680 -1.709 -O.897 3.012 8.980 -0.405 0.137
A6 20.264 13.187 -0449 0.587 4.779 10.567 -O.396 0.143
A7 22.012 14.433 1.152 1.248 5.918 12.020 -0.436 0.131
A8 18.753 14.039 0.993 1.118 4.931 11.237 -0437 0.129
A9 17.474 13.655 2.827 0.275 5.107 9.773 -0392 0.136
A10 21.427 8.955 1.385 1.444 3.871 7.752 -0421 0.135
All 18.306 11.226 2.511 1.573 4.740 8.389 -0435 0.144
A12 25.345 13.727 -0.427 -0.363 2.615 8.167 -0412 0.119
A13 22.054 12.400 -O.602 0.441 3.561 8.329 -0407 0.132
B] 24.600 16.077 -3.488 -0.050 3.181 9.323 -0.422 0.070
32 23.169 16.663 -3.845 -0545 3.839 10.428 -0.414 0.081
B3 26.716 16.407 -4.448 -O.674 2.974 9.700 -0425 0.069
B4 . 21.789 16.386 -2.582 0.079 4.077 9.557 -0.418 0.079
B5 22.992 16.386 -2335 0.079 3.988 9.923 -0411 0.075
B6 21.953 16.864 -3.469 -0.416 4.727 10.669 -0421 0.078
D1 20.779 13.699 3.161 1.966 7.453 12.259 -O.408 0.027
D2 22.249 14.223 1.757 0.382 7.260 15.890 -0415 0.047
D3 21.688 13.593 1.349 1.050 7.440 14.272 -O.416 0.056
D4 19.634 14.464 1.431 0.750 7.706 15.890 -0.420 0.048
D5 20.406 13.001 2.067 2.144 7.772 12.753 -0.404 0.061

 

‘Abbreviations as described in Table 1.1 and Fig 1.2.
bHansen Solubility Parameter

cHydrophilic Lipophilic Balance

dHansch Log P

”Crippen Log P

fCNDO Log P

gKier Connectivity Index

hHighest Occupied Molecular Orbital

iLowest Unoccupied Molecular Orbital

33

Orbital (LUMO) are indicators of electrophilic interactions and the ability of a molecule
to donate or accept electrons, respectively. HOMO and LUMO may be indicators of
specific binding at the site of action (Mekenyan and Veith, 1993). These variables were
again tested for collinearity using Gittins condition number (data not shown) (Gittins,

1985).

BIOLOGICAL TESTING

Relative toxicity of 24 trichothecene mycotoxins was determined using induction

of cell death by apoptosis or cytotoxicity in several cloned murine B cell lines as follows:

Cell lines and culture

70Z/3 cells, phenotypically pre-B lymphoblasts, were obtained from Dr. Richard
Schwartz (Michigan State University, E. Lansing, MI). CH3] cells, phenotypically
immature B cells, were obtained from Dr. David Scott (American Red Cross Holland
Laboratory, Rockville, MD). CH12.LX cells, phenotypically mature B cells, were
obtained from Dr. Geoffrey Haughton (University of North Carolina, Chapel Hill, NC).
Cell number and viability were assessed microscopically by trypan blue dye exclusion
using a hemocytometer (Stiles, 1986). Cell culture media and chemicals were purchased
from Sigma Chemical Company (St. Louis, MO) unless otherwise noted. Cells were
cultured at a concentration of 5 x 105/ml in 200 til/well in 96 well flat bottom plates at
37°C in a 5% CO2 humidified incubator. Cells were grown in RPMI-1640 medium
supplemented with 100 U/ml penicillin, 100 pg/ml streptomycin, 5011M

2-mercaptoethanol, 2 mM glutamine, 1 mM sodium pyruvate (Gibco Life Technologies,

34

Rockville, MD), and 1 mM non-essential amino acids (Gibco). CH3] and CH12.LX
media was supplemented with 5% (v/v) heat-inactivated fetal bovine serum (FBS,

Gibco). 70Z/3 media was supplemented with 10% (v/v) FBS.

Trichothecenes

Satratoxin G, satratoxin F, and satratoxin H were obtained from Dr. Bruce Jarvis
(University of Maryland, College Park, MD). 3'OH metabolites were produced by the
procedure of Yoshizawa et a1. (1981;1984). All other trichothecenes were purchased
from Sigma. Toxins were dissolved in 100% ethanol and diluted in RPMI-1640. F inal
ethanol concentration in the culture medium was less than 0.01% (v/v) which did not
induce apoptosis or cytotoxicity. Triplicate cultures were treated with toxin in lO-fold

dilutions or RPMI-1640 (control) for 18 11.

MTT conversion assay for cytotoxicity

Following trichothecene treatment, 20 1.11 of a 5 mg/ml solution of 3- (4,5
dimethylthiazol-2-ul)-2,S-diphenyl tetrazolium bromide (MTT, Sigma) in 0.01 M PBS
was added to each well for the final 2 h of the 18 h incubation period (Marin, et al.,
1996). Microtiter plates were centrifuged at 450 x g for 20 min and media were
aspirated to minimize formazan crystal loss. The resultant crystals were dissolved in 150
111 dimethylsulfoxide for 15 min. Optical density was measured using a Vmax
Microplate Reader (Molecular Devices, Menlo Park, CA) at dual wavelengths, 570 nm
and 690 nm. Percent MTT inhibition was calculated as follows: ((1 - absorbance of
treatment)/ absorbance of control) x 100. Mean values were used to graphically

determine ED50 values.

35

Apoptosis assay

Apoptotic cells were quantitated by fluorescence microscopy as described by
Duke and Cohen (1995). Briefly, following trichothecene treatment, microtiter plates
were centrifuged at 450 x g for 10 min and 150 pl of supernatant was removed. Five 11]
of DNA staining reagent (acridine orange (100 pg/ml) and ethidium bromide (100 ug/ml)
in 0.0] M phosphate buffered saline (PBS)) was added to each well. Cells were
classified as apoptotic or normal based on chromatin organization. Briefly, a normal
nucleus appears large with an organized structure while an apoptotic nucleus appears
small and highly condensed or fragmented. Percent apoptosis was calculated as follows:
number of cells with apoptotic nuclei/total cell number x 100. Mean values were used to

graphically determine the effective dose causing 50% apoptosis (EDSO).

Statistical analysis

Modeled physico-chemical parameters were compared between trichothecene
subgroups using a one-way analysis of variance (ANOVA) and a Tukey test for mean
separation. Pearson correlation was used to identify correlations between each
physico-chemical parameter and apoptosis or cytotoxicity using Systat, version 5.0
(Evanston, IL). The ability of these parameters and toxicity to differentiate between
trichothecene subgroups was assessed using principal components analysis (PCA). All
data sets were tested for multivariate normality and log transformations were used as
needed. Characteristics of the trichothecenes which acted as surrogates for toxicity were
then identified. PCA was performed using Statistical Analysis Software (SAS) version

6.12.

36

RESULTS

Trichothecene physical and chemical descriptors

Physical and chemical variables calculated using Molecular Modeling Pro version
2.4 were compared between trichothecene subgroups (Table 3.3). Type B trichothecene
HLB measurements (16.46 :1: 0.11) were significantly higher (p<0.01) than both Type A
(13.19 i 0.45) and Type D (13.80 i 0.26) subgroups while Log P calculations (-3.36 :1:
0.32) were significantly lower (p<0.01). Mean Log P values for Type A and Type D
were 0.946 :1: 0.41 and 1.953 d: 0.33, respectively. CRIP measurements of Type B toxins
(-0.255 i 0.14) were also significantly lower (p<0.01) than both Type A (0.634 :t 0.20)
and Type D (1.258 :t 0.34). CNDO and KIER measurements of Type D trichothecenes
(7.526 d: 0.09 and 14.213 i 0.76, respectively) were significantly higher (p<0.01) than
both Type A (4.920 :t 0.40 and 9.975 i 0.43, respectively) and Type B (3.80 :l: 0.26 and-
9.933 :t 0.21) values. LUMO values for Type A (0.134 i 0.002), Type B (0.076 :t
0.002), and Type D (0.048 i 0.006) trichothecenes were all significantly different from
each other (p<0.01). No significant differences were noted between subgroups for SOL
and HOMO calculations. These data suggest: (1) one subgroup can be separated from
the remaining groups on the basis of lipophilicity (Log P, CRIP, CNDO) (2) molecular
branching is greater in Type D than Type A and B toxins (KIER) and (3) trichothecene

subgroups exhibit a broad range of ability to accept electrons (LUMO).

Trichothecene-induced B cell apoptosis and cytotoxicity
Previous studies suggest that germinal centers of the spleen and Peyer's patch are

sensitive to DON-induced apoptosis, thus B cells are highly susceptible (Zhou et al.,

37

Table 3.3. Effective dose (ED50, ng/ml) resulting in 50% apoptosis or cytotoxicity in
three murine B cell lines following 18 h trichothecene mycotoxin exposure.
CH12.LX

Apoptosis Cytotoxicity Apoptosis Cytotoxicity Apoptosis Cytotoxicity

Trichothecenea
A1
A2
A3
A4
A5
A6
A7
A8
A9
A1 0
A1 1
A12
A1 3
B 1
B2
B3
B4
B5
B6
D1
D2
D3
D4
D5

aTrichothecene abbreviations as described in Table 1.1 and Fig. 1.2. Results are

702/3

4.5
8.0
40
400
400
25
150
3.5
45
500
400
600
>10000
400
450
375
550
>10000
60
1.5
3.0
0.5
3.0
4.0

0.6
3.0
40
250
150
30
45
2.5
25
200
100
300
>10000
90
200
275
125
2000
30
0.85
4.25
0.7
5.75
3.0

CH3]

2.0
3.5
20
400
250
20
15
3.25
30
400
250
500
>10000
400
450
450
450
3000
40
0.4
2.75
0.35
2.5
0.4

representative of duplicate experiments (n=6).

38

1.5
3.0
25
500
200
25
30
3.0
50
350
250
550
>10000
225
250
350
225
5000
40
0.35
3.25
0.3
2.75
0.35

3.0
3.5
30
350
350
10
70
3.5
35
350
50
350

>1 0000
400
400
400

400

4000

40
2.0
2.0
1.0
3.0
3.25

2.5
3.5
20
250
150
10
20
3.5
25
350
30
300
>10000
250
325
325
375
3500
40
3.25
4.0
1.25
2.75
3.0

2000). Induction of apoptosis or general cytotoxicity was assessed for 24 trichothecenes
in three B lymphocyte cell lines representing three distinct lineages. The cell lines tested
represent pre-B (70Z/3), immature (CH31), and mature (CH12.LX). Of these lineages,
the immature B cell is the most susceptible to apoptosis (Carsetti, 1995; Garvy, 1993).
The effective doses resulting in 50% apoptosis or cytotoxicity (EDSO) were graphically
determined for these cell lines and these data are summarized in Table 3.4.

Apoptosis and cytotoxicity exhibited a general rank toxicity in which Type D >
Type A > Type B. Exceptions to this rank toxicity occurred when considering the
metabolized forms of the parent compounds. For example, subsequent reductions in ED50
values from 10 to 100-fold were observed when T-2 toxin was compared to several of its
metabolites (Table 3.3). However, cytotoxicity values reported here are similar to values
previously reported for this group of toxins (Ueno, 1983). ED50 values for apoptosis and
cytotoxicity were similar suggesting that cell death was primarily due to apoptosis. ED50
values for apoptosis and cytotoxicity were similar in all three cell lines suggesting that
the level of differentiation did not influence B cell susceptibility to trichothecene-induced
cell death.

Pearson Correlation indicated that no significant (p<0.05) relationship existed
between trichothecene-induced cell death and any single physical or chemical descriptor
(data not shown). Interestingly, when toxicity values were log transformed to reduce the
effect of outliers and to ensure the data approximated a normal distribution, significant
correlations were observed with Log P, SOL, KIER, CNDO, and CRIP (Table 3.4).
CNDO and KIER showed the strongest correlations with log transformed toxicity. The

correlation with log transformed apoptosis was slightly higher than with log transformed

39

Table 3.4. Significant correlations observed between log transformed toxicity values and
physico-chemical descriptors of the trichothecene mycotoxins.

70Z/3
Apoptosis Cytotoxicity Apoptosis Cytotoxicity Apoptosis Cytotoxicity

CH3]

CH12.LX

Log P3

r -0.502 —0.496 -0.548 -0.510 -0.560 -0.571
p 0.012 0.014 0.006 0.016 0.014 0.011
SOL

r 0.488 0.555 0.527 0.507 0.527 0.555
p 0.016 ' 0.005 ' 0.008 0.011 0.008 0.005
KIER

r -0.750 -0.690 —0.762 -0.751 -0.716 -0.681
p 0.001 0.001 0.001 0.001 0.001 0.001
CNDO

r -0.834 -0.786 -0.859 -0.844 -0.824 -0.805

p 0.001 0.001 0.001 0.001 0.001 0.001
CRIP

r 0.440 0.416 0.503 0.488 0.493 0.509
p 0.032 0.043 0.012 0.016 0.014 0.011

 

aPhysico-chemical abbreviations as described in Model Design and Rationale.

40

cytotoxicity for these descriptors. Multivariate techniques were used to simultaneously

examine physico-chemical descriptor values and cell death by apoptosis or cytotoxicity.

Principal component analysis (PCA) of trichothecene-induced cell death

The effects of physicochemical parameters and the induction of apoptosis and
cytotoxicity on separating trichothecene subtypes were assessed using PCA. Vector
loadings of the first PC of each analysis indicated that Log P, CRIP, and CNDO
weighted heavily in structuring both the 70Z/3 pre-B cell apoptosis (0.500, 0.462, and
0.463, respectively) and cytotoxicity (0.504, 0.469, and 0.439, respectively) data (Figures
3.1 A and 3.1 B). LUMO and KIER weighted heavily (-0.524 and 0.514, respectively)
for the apoptosis and (-0.515 and 0.504, respectively) cytotoxicity data sets in the second
PC in each analysis. Similarly, vector loadings of the first PC indicated that Log P,
CRIP, and CNDO also weighted heavily in CH3] immature B cell apoptosis (0.500,
0.466, and 0.442, respectively) and cytotoxicity (0.499, 0.465, and 0.441, respectively)
(Figures 3.2A and 3.28). LUMO and KIER weighted heavily (-0.514 and 0.499,
respectively) for the apoptosis and (-0.517 and 0.502, respectively) cytotoxicity data sets
in the second PC in each analysis for CH3] cells. Finally, Log P, CRIP, and CNDO
weighted heavily in the first PC of each analyses using CH12.LX mature B cell apoptosis
(0.500, 0.465, and 0.440, respectively) and cytotoxicity (0.501, 0.466, and 0.440,
respectively) (data not shown). Again, LUMO and KIER weighted heavily in the
second PC, (-0.518 and 0.508, respectively) for the apoptosis and (-0.517 and 0.505,
respectively) cytotoxicity data sets in each analysis. The combination of PC] and PC2
explained more than 65% of the total variation of these data. When apoptosis and

cytotoxicity were log transformed, results were similar. In each analysis of log

41

 

 

 

 

 

 

A HLB KIER
3‘ : CNDO
2 ‘ ‘ 1 D2
D4
. 36 D5
1 B3 91 . A, D3
3 ; A3
0 -..$Q.L. ........................ .. ................... ‘ ...................................................
1 9A2 A1 CRIP
A 12 A5 A8 LOG P
N -2 I
*5 A10
2 _3 - A13 A 1
E 4 TOX LUMO HOMO
O :
.3 3 ~
g 1 D2
a 2 ‘ D4
_ Be 05
1 33 g -1 B4 . .. A3 03
o . .SQL ................ Bs .......................... = ‘ A7 ............................................ L"
A2 CRIP
1 - 9
- A5 A8 LOG P
-2 -1
A13 A10
-3 qTOX . A
'4 l l 7 I LUMIO V HOMO!
-4 -3 -2 -1 0 1 2 3 4

Principal Component 1

Figure 3.]. Principal component analysis (PCA) of 70Z/3 cytotoxicity (A) and apoptosis
(B). PCA was performed on 8 physicochemical characteristics of the trichothecenes
generated using molecular modeling and toxicity data. PC] explained 37.7% and 38.3%
while PC2 explained 27.6% and 27.0% of the variation in the cytotoxicity and apoptosis
data sets respectively. Lipophilic values were weighted heavily in PC1, while LUMO
weighted heavily in PC2 in each analysis. KIER and CNDO acted in conjunction with

toxicity and could possibly serve as a measure of toxicity.

42

 

 

 

 

 

4 HLB KIER
3 q A CNDO
2‘ 1 D2
D4
86 D5
1 - 83
851 34 . A3 03
0 .SQL ..................... 85 ..................... ‘ A7 ....................................................
A2 CRIP
-1 ~ ‘ 9 A1
Aé‘lz A5 A8 LOG P
N '2 I
e A10
2 _3 1 A13
a A
g 41 TOX % LUMO HOMO
E; HLB ; KIE'
.9 B 5 CNDO
E 3 “
8- 1 02
2 ..,
D4
1- Ba 1 36 05
BE B4 - A3 D3
0 -SQL ......................... ....................... y 7 ..................................................
A2 CRIP
-1 -1 A 9
A 12 A5 A8 LOG P
-2 ~
-3 ~ A13 1 AA10
4 TOX LUMO HOMO
-4 -3 -2 -1 0 1 2 3 4

Principal Component 1

Figure 3.2. Principal component analysis (PCA) ofCH31 cytotoxicity (A) and apoptosis
(B). PCA was performed on 8 physicochemical characteristics of the trichothecenes
generated using molecular modeling and toxicity data. PC] explained 38.1% and 38.0%
while PC2 explained 27.4% and 27.6% of the variation in the cytotoxicity and apoptosis
data sets respectively. Lipophilic values were weighted heavily in PC], while LUMO
weighted heavily in PC2 in each analysis. KIER and CNDO acted in conjunction with

toxicity and could possibly serve as a measure of toxicity.

43

 

 

 

 

 

 

4 E
A LUMO A1 HOMO
3 ‘ 1 1o LOG P
2 ~ CRIP
A13 :
rox 55 A8
1 ‘ 0412 .. A1
: / [A2
0 -1 ............................................................. :‘ ........ ‘ ...................................................
SOL = i 1- . D3
2;: ,2 - B3 86 D4 CNDO
g 5 02
s ’3'
g 4 HLB KIER
m E
E» B LUMO A11 HOMO
E 3- 1 o LOG P
2 ~ CRIP
TOX A13 5 A8
1 i *1 5
2 3 1 3
: , z”
o .. .............................................................. ........................................................
: 5 D3
_, SOL B4 _ 5 .
3%1 D4
,2 B3 36 CNDO
'102
-3 ~ ;
4 . . HLB . . KIER,
-4 -3 -2 -1 0 1 2 3 4

Principal Component 1

Figure 3.3. Principal component analysis (PCA) of 702/3 log transformed cytotoxicity
(A) and apoptosis (B). PCA was performed on 8 physicochemical characteristics of the
trichothecenes generated using molecular modeling and toxicity data. PC] explained
43.3% and 43.6% while PC2 explained 28.2% and 28.6% of the variation in the
cytotoxicity and apoptosis data sets respectively. Lipophilic values were weighted
heavily in PC], while LUMO weighted heavily in PC2 in each analysis. KIER and
CNDO acted in conjunction with toxicity and could possibly serve as a measure of

toxicity.

44

transformed apoptosis and cytotoxicity, CNDO and/or Log P weighted heavily in PC]
and LUMO weighted heavily in PC2 (Figures 3.3 A, 3.3 B, and data not shown). PC 1
and PC2 combined always explained more than 72% of the variation in data sets using
log transformed toxicity. Collectively, these results suggest that the trichothecenes
exhibit a broad range of lipophilicity and LUMO binding values that, when combined,
account for the major portion of the variation in the data set. Log P and LUMO can
divide trichothecenes into clear subgroups without overlap, suggesting these two
descriptors combined are good toxicity predictors for unknown trichothecenes.
Graphical display of PCA permitted a two-dimensional visualization of
physicochemical descriptors in relation to toxicity. Thus, descriptors with the greatest
influence on toxicity were identified based on the relationship of eigenvectors. Log P
and LUMO played a major role in structuring these data sets and separating trichothecene
subgroups, but had less influence on the toxic action of these compounds to 70Z/3 cells.
However, KIER and CNDO acted in conjunction with apoptosis (Figures 3.1A, 3.3A)
and cytotoxicity (Figures 3.1B, 3.38) therefore potentially acting as surrogates in
separating the data. Therefore, KIER and CNDO could possibly serve as predictors of
toxicity. Similar relationships were noted for CH3] (Figures 3.2A and 3.28) and

CH12.LX cells (data not shown).

DISCUSSION

The worldwide occurrence and potential health hazard presented by the
trichothecene mycotoxins requires an improved understanding of the mechanisms of

toxic action of these compounds to facilitate risk assessment. The ability of

45

trichothecenes to inhibit protein synthesis and induce cell death of lymphocytes is central
to the observed immunotoxicity (Ueno, 1983). A previous study indicated B
lymphocytes in germinal centers of the spleen and Peyer's patch are particularly sensitive
to trichothecene-induced apoptosis (Zhou, et. al 2000). Therefore, our model was
designed using apoptosis and cytotoxicity of several B lymphocyte lineages as a measure
of toxicity. To our knowledge, this is the first report of trichothecene-induced cell death
in B lymphocytes and physico-chemical characteristics of individual trichothecene
mycotoxins.

This study suggests that several factors may contribute to the apoptotic and
cytotoxic actions of the trichothecenes. These compounds exhibit a broad range of
lipophilicity, which correlated with log transformed toxicity. Therefore, when the data
approached a normal distribution, toxicity increased with increasing lipophilicity.
However, lipophilicity did not correlate with non-transformed data, indicating
lipophilicity alone was not a good indicator of toxicity when outliers, or less toxic
trichothecenes such as verrucarol and 3-Ac-DON, were included in the analysis.
Lipophilicity was always weighted heavily in PCl of each analysis, reinforcing that the
trichothecenes exhibit a broad range of lipophilic values.

In a previous study addressing trichothecene structure-activity relationships Ramu
et al. (1989) concluded that no clear correlation existed between toxicity and lipophilicity
in the cytotoxic actions of several trichothecenes. In contrast, Grove and Mortimer
(1969) demonstrated diminished cytotoxicity with decreased lipophilicity in a HEp2 cell
model system. Both studies focused on Type A compounds and included less toxic
outliers. Thompson and Wannemacher (1986) compared toxicity of 19 trichothecenes

both in vitro and in vivo. In this study, the most potent trichothecenes possessed acetate

46

groups or hydrocarbon chains, which strongly influence lipid solubility. They suggest
the variable potency of the trichothecenes is due to the rate of transport into the cell with
other properties contributing to toxic action such as factors influencing ribosomal
binding and protein synthesis inhibition.

LUMO was significantly different for all trichothecene subgroups but did not
significantly correlate with toxicity data. In using PCA, LUMO always weighted heavily
in PC2. The importance of LUMO in shaping the data set suggests that electrostatic
interactions, specifically, the ability to accept electrons, may be important for the toxic
action of these compounds. These data support previous studies which demonstrated that
the C9-10 double bond and C 12-13 epoxide, common to all trichothecenes, are necessary
for biological activity (Wei and McLaughlin, 1974; Ehlrich and Daigle, 1987). The
C9-10 double bond and C 12-13 epoxide may be important for determining trichothecene
conformation or may participate directly in binding to the ribosomes.

As LUMO values decrease, the binding affinity of a compound increases.
Interestingly, trichothecene LUMO values suggest that Type D compounds have greater
binding affinities than Type B compounds, which in turn, have greater binding affinities
than Type A. Mekenyan and Veith (1993) observed an interdependence between LUMO
and Log P in which compounds became more dependent on Log P with increasing
LUMO values (decreased binding affinity). A similar relationship may apply to the
trichothecenes. Type D compounds are very reactive and very lipophilic suggesting that
they are efficiently transported to the ribosome and bind with a high affinity. Type A
compounds are less reactive, but still relatively lipophilic indicating they are reaching the
target site in sufficient quantities to bind to the ribosomes. As the Type A parent

compounds are further metabolized, they become more soluble and may not reach the site

47

of action. Type B compounds are reactive, but very soluble suggesting less of the toxin
reaches the target site but binds effectively when it does.

KIER values for the Type D trichothecenes were significantly higher than Type A
and B values and demonstrated a significant (p<0.05) correlation with log transformed
toxicity. KIER did not correlate with the non-transformed toxicity data suggesting KIER
alone is not a good predictor of toxicity when outliers, or less toxic trichothecenes such
as verrucarol and 3- Ac-DON, were included in the analysis. Trichothecenes can be
divided into two groups based on the step of protein synthesis blocked. Wei and
McLaughlin (1974) suggested functional group size may contribute to the ability of the
toxin to block the initiation or elongation-termination step of protein synthesis. Bamburg
(1968) noted that the elongation/termination inhibitors were unsubstituted or possessed
only small substituents. Interestingly, KIER values for the initiation inhibitors previously
characterized (Ueno, 1983) and modeled here ranged from 9.7 - 12.0, while
elongation/termination inhibitors ranged from (7.8 - 8.4). These data suggest branching
of the molecule is important for binding and may predict the step of protein synthesis a
trichothecene blocks.

In summary, no single physico-chemical characteristic of the trichothecene
mycotoxins correlated with apoptosis or cytotoxicity. Lipophilicity (Log P, CRIP,
CNDO) and molecular branching (KIER) correlated with log-transformed toxicity values.
Therefore, log transformation of the toxicity data would be necessary when using
lipophilicity and molecular branching to predict toxicity of less toxic outliers in the data
set, such as 3-Ac-DON and verrucarol. Using PCA analysis, the relationship of the
physicochemical vectors to toxicity was demonstrated. These results indicated CNDO

and KIER had the greatest influence in separating the data set on the basis of toxicity.

48

Taken together, this study suggests CNDO and KIER, or possibly a combination of these
variables, may provide the most accurate prediction of trichothecene apoptosis and
cytotoxicity. Thus, the transportation of trichothecenes to the ribosome and molecular

branching, which impacts binding, are critical for trichothecene-induced toxicity.

49

CHAPTER 4

Potentiation of Trichothecene-Induced Cytotoxicity and Apoptosis

in Murine Leukocytes by TNF-oc and Fas Ligand

INTRODUCTION

Vomitoxin (VT, deoxynivalenol), a trichothecene mycotoxin, is a naturally
occurring environmental contaminant produced by molds commonly infesting cereal
grains. .Trichothecenes are not significantly reduced or removed from grains during
milling and processing procedures (Wolf-Hall et al., 1999) and may enter finished food
products at ppm levels (Abouzied et al., 1991). Exposure to this group of toxins leads to
alteration of immune cell function, dysregulation of the humoral immune response, and
impairment of host resistance to pathogens (Bondy and Pestka, 2000). The toxicity of
trichothecenes, including vomitoxin, is attributed to ribosomal binding and subsequent
inhibition of protein synthesis in actively dividing immune cells of the bone marrow,
spleen, thymus, and lymph nodes (Ueno, 1983). Induction of apoptosis in these tissues
by the trichothecenes (Islam et al., 1998; Shinozuka et al., 1997) is likely to contribute to
the observed immunosuppressive effects.

Historically, outbreaks of human disease have been linked to
trichothecene-producing molds such as F usarium (Bamburg, 1968). Alimentary toxic
aleukia in the Soviet Union and red mold disease in Japan were linked to human
consumption of F usarium contaminated grains. Between 1960 - 1991, 53 outbreaks of

human food poisoning associated with moldy cereal occurred throughout China (Li et al.,

50

1999). Trichothecene levels were recently determined in samples from a food poisoning
outbreak in Anhui, China in 1991 (Li et al., 1999). Moldy cereal samples contained up to
1.5 mg/kg VT. Approximately 130,000 people were affected by gastrointestinal
disorders including nausea, vomiting, diarrhea, and leukopenia. Symptoms described in
food-home outbreaks mimic symptoms of experimental animals exposed to trichothecene
mycotoxins (Bondy and Pestka, 2000).

Components of both stress (Albers et al., 1996; Pruett et al., 1993) and
inflammatory (Luster et al., 2000; Emmendoerffer et al., 2000) signaling pathways play a
critical role in chemically-induced toxic responses. Recent studies indicate that
exposure to low levels of lipopolysaccharide (LPS), the biologically active component of
gram negative bacteria, can influence the magnitude of a toxic response to xenobiotic
agents. Increased hepatotoxic responses have been observed for carbon tetrachloride,
ethanol, and ally alcohol (Roth et al., 1997). Previous in vivo studies demonstrate
increased trichothecene toxicity in the presence of LPS which lowered LDso doses and
increased mortality in T-2 toxin treated mice (Tai and Pestka, 1988) and increased
lymphocyte apoptosis and mortality in VT-treated mice (Zhou et al., 2000; Zhou et al.,
1999). The mechanisms involved in this synergistic toxicity have not been elucidated.

LPS induces its biological effects by stimulating host cells to produce a variety of
mediators including bioactive lipids (i.e. prostaglandins), glucocorticoids (i.e.
corticosterone), and proinflammatory cytokines (i.e. TNF-a). These mediators may
interact with VT to alter immune cell responses. For example, two chemicals may
interact in an additive manner in which the combined effect is equal to the sum of the
two, while synergism is defined by an effect that is greater than the sum. Potentiation

occurs when one chemical does not elicit an effect, but when combined makes the second

51

chemical more toxic. Finally, an antagonistic interaction occurs when one chemical
reduces or inhibits the effect of another (Casarett et al., 1996).

The objective of this study was to determine if cells exposed to low levels of the
trichothecene VT could interact with LPS or other mediators of stress and inflammation
including prostaglandin E2 (PGEZ), anti-immunoglobulin, glucocorticoids, Fas ligand, or

TNF-a in the induction of apoptosis and cytotoxicity in primary leukocyte cultures.

MATERIALS AND METHODS

Chemicals and reagents

Chemicals and reagents were purchased from Sigma Chemical Company (St.
Louis, MO) unless otherwise noted. VT was dissolved in 100% ethanol and diluted in
RPMI-l640 medium supplemented with 100 U/ml penicillin, 100 pg/ml streptomycin,
SOpM 2-mercaptoethanol, 2 mM glutamine, 1 mM sodium pyruvate (Gibco Life
Technologies, Rockville, MD), 1 mM non-essential amino acids (Gibco), and 10 % (v/v)
fetal bovine serum (FBS, Gibco). Final ethanol concentration in the culture medium was
less than 0.01% (v/v) which did not induce apoptosis or cytotoxicity. LPS (Salmonella
typhimurium), PGEZ, dexamethasone (DEX, water-soluble) ethylenediaminetetraacetic
acid (EDTA), and N-acetyl-L-cysteine (NAC) were dissolved in supplemented
RPMI-l640. Anti-IgM (Pharrningen, San Diego, CA), anti-Fas (Pharrningen), and
TNF-ct (R&D Systems, Minneapolis, MN) were dissolved in 0.01 M phosphate buffered
saline (PBS, pH 7.2) and diluted in supplemented RPMI-l640. Final concentration of
TNF-a (20 ng/ml) in culture was equivalent to 1000 U/ml. BAPTA-AM, PD98059,
38203580, and Caspase 3 Inhibitor I were purchased from Calbiochem (San Diego, CA)
and dissolved in cell culture grade dimethylsulfoxide (DMSO). Final DMSO

52

concentration in cell culture media was less than 0.1% (v/v) which did not induce

apoptosis or cytotoxicity.

Animals

All animal handling was conducted humanely in accordance with the
recommendations established by the National Institutes of Health and approved by
Michigan State University Laboratory Animal Research Committee. Female B6C3F1
mice (8 - 10 weeks) were obtained from Charles River (Portage, MI). Animals were
housed three per cage and acclimated 1 week at the MSU Laboratory Animal Resources
Facility in a humidity and temperature controlled room with a 12 h light and dark cycle.

Mice were provided standard rodent chow and water ad lib.

Primary cell culture

Mice were euthanized by cervical dislocation and thymus (TH), spleen (SP),
femurs containing bone marrow (BM), and Peyer's patch (PP) were immediately
removed. Single cells were released from TH, SP, and PP by forcing the tisstre through a
40 pm nylon mesh Collector Tissue Sieve (Bellco Glass Inc., Vineland, NJ) and then
submerged into cold RPMI-1640 medium. BM was flushed from the femur using
RPMI-l640. SP and BM cell suspensions were treated with erythrocyte lysing buffer
containing 0.83% (w/v) ammonium chloride, 0.1% (w/v) potassium bicarbonate, and
0.0037% (w/v) EDTA for 2 min at room temperature and centrifuged twice at 450 x g
for 10 min. Cell number and viability were determined using a hemacytometer (Stiles,
1986). All cells (1 x 106/ml) were cultured in supplemented RPMI-l640 medium at

37°C in a 5% CO2 humidified incubator in 96 well plates in a final volume of 300

53

til/well. Cells were incubated with VT, each mediator, or VT plus each mediator for 18 h
prior to apoptotic and cytotoxic measurements. Inhibitors or chelators (1 pM) were
pre-incubated with cells for 2 h and removed prior to the addition of VT, TNF-a, or VT
plus TNF-a in fresh medium. All experiments were repeated and results represent mean

values from a minimum of four mice.

MTT conversion assay for cytotoxicity

Following treatment, 20 p1 of a 5 mg/ml solution of 3- (4,5 dimethylthiazol-
2-ul)-2,5- diphenyl tetrazolium bromide (MTT) in 0.01 M PBS was added to each well
for the final 4 h of the 18 h incubation period (Marin et al., 1996). Microtiter plates were
centrifuged at 450 x g for 20 min and media was aspirated to minimize formazan crystal
loss. The resultant crystals were dissolved in 150 pl DMSO for 15 min. Optical density
was measured using a Vmax Microplate Reader (Molecular Devices, Menlo Park, CA) at
dual wavelengths, 570 nm and 690 nm. Percent control response was calculated as
follows: ((1-absorbance of treatment/absorbance of control) x 100). Mean values were

used to graphically determine ED50 values when possible.

Apoptosis assay

Apoptotic cells were quantitated using fluorescent DNA binding dyes as
described by Duke and Cohen (1995). Briefly, following treatment, microtiter plates
were centrifuged at 450 x g for 10 min and 250 pl of supernatant was removed. Five p1
of DNA staining reagent (acridine orange (100 pg/ml) and ethidium bromide (100 pg/ml)
in 0.01 M PBS) was added to each well and gently mixed. Cells were incubated on ice

until sample analysis. A fluorescence micrOSCOpe was used to count approximately 200

54

cells under 400 x magnification, 400/490 nm excitation and 520 nm emission. Cells were
classified as apoptotic or normal based on chromatin organization. A normal nucleus
appeared large with an organized structure whereas apoptotic nuclei appeared small and
highly condensed or fragmented. Percent apoptosis was calculated as follows: ((number
of cells with apoptotic nuclei/total cell number) x 100). Mean values were used to

graphically determine the effective dose causing 50% apoptosis (ED50) when possible.

Statistical analysis

The data were analyzed using Systat version 5.0 (Evanston, IL). A Student's t test
was used to compare two groups while multiple groups were compared using a one way
analysis of variance (ANOVA) and a Tukey test for mean separation. Samples receiving
two treatments were analyzed for additivity, synergy/potentiation, or antagonsim by
randomly combining single treatment replicates to calculate an expected mean additive
response with variance. This calculated value was compared to actual co-treated samples
using a Mann Whitney Rank Sum test. A p value < 0.05 was considered statistically

significant.

RESULTS AND DISCUSSION
Differential sensitivity of primary murine leukocytes to VT- , LPS—, and VT plus LPS-
induced apoptosis
Induction of apoptosis (Yang etal., 2000; Okumura et al., 2000; Shifrin and
Anderson, 1999) and cytotoxicity (Hanelt et al., 1994; Anderson et al., 1989; Yike et al.,
1999) are widely reported for the trichothecene mycotoxins and likely contribute to their

toxic effects. Sensitivity of unstimulated primary leukocyte cultures prepared from TH,

55

SP, BM, and PP to VT was assessed. Induction of apoptosis at 18 h was evaluated by
fluorescence microscopy and based on cell morphology. Apoptosis was significantly
increased (p<0.05) by VT (250, 500 ng/ml) in SP cultures (Figure 4.1A). VT (500
ng/ml) also significantly increased TH and BM apoptotic morphology. Morphological
changes were not noted in VT-treated PP cells. Concurrent with apoptosis induction,
increased cytotoxicity was Observed by MTT assay in SP and BM leukocytes exposed for
18 h to 250 ng/ml VT and all leukocytes exposed to 500 11ng (Figure 4.1B). The
present study demonstrates that several primary murine leukocytes are sensitive to
VT-induced cytotoxicity and apoptosis.

Sepsis (Oberholzer et al., 2001; Ayala et al., 1996) or administration of LPS to
mice (Zhang et al., 1993; Zhang et al., 1994) results in apoptosis in lymphocytes of the
BM, SP, and TH. In thepresent study, LPS concentrations up to 20 pg/ml did not alter
apoptotic morphology in any of the primary leukocyte cultures (data not shown).
However, all three LPS doses tested significantly increased MTT conversion, indicative
of a proliferative response, in SP (Figure 4.2). Zhou et al. (2000) reported a synergistic
increase in lymphocyte apoptosis in mice co-exposed to LPS and VT. As previously
stated, LPS did not directly induce apoptosis in murine TH, SP, BM, or PP leukocyte
cultures. However, VT inhibited the LPS-induced proliferative responses in SP
leukocytes, but did not significantly alter apoptotic morphology in the co-treated cultures.
The ability to inhibit protein synthesis by VT (Ueno, 1983) may have contributed to this
reduced proliferative response. These results suggest the in vivo induction of apoptosis
in immune tissues exposed to LPS alone or LPS and VT is not due to LPS directly, but
may require a secondary mediator. Possible interactions between prostanoids,
immunoglobulins (as antigen mimics), glucocorticoids, Fas, and TNF-a signaling

components with VT were further investigated.

56

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Figure 4.2. LPS-induced increases in splenocyte MTT conversion were inhibited by VT.
Splenocytes were exposed to VT (0 - 500 ng/ml), LPS (0 - 20 pg/ml) or both for 18 h.
Cytotoxicity was measured by conversion of MTT during the final 4 h in culture. Data
(n=4) represent the mean percent control response i SEM. 'A Student’s t test was used
to determine values significantly different from the untreated control and bvalues

significantly different from the respective LPS control (p<0.05).

58

Differential sensitivity of primary murine leukocytes to PGE2 ~induced apoptosis and
cytotoxicity in the absence and presence of VT

Prostanoids, including PGEZ, increase in response to acute inflammation prior to
leukocyte recruitment (Tilley et al., 2001). PGE2 can activate or inhibit effector cell
functions depending on stimuli and target cell. Receptors for prostanoids are expressed
on TH, SP, BM, and macrophage cells. The ability of PGE2 to induce apoptosis in
immature B lymphocytes (Brown et al., 1992) and immature and mature T cells (Pica et
al., 1996; Mastino et al., 1992) suggests prostanoids may be capable of interacting with
chemical agents to alter immune cell responses by selectively affecting cell death.
Sensitivity of unstimulated primary leukocyte cultures prepared from TH, SP, BM, and
PP to PGE2 was assessed. Microscopic analysis of PGEz-induced apoptosis indicated a
significant increase (p<0.05) in TH cultures exposed to 20 pM for 18 h (Figure 4.3A).
Decreased MTT conversion (increased cytotoxicity) was observed in TH cultures
exposed to 0.2 pM (75.7 i 1.1%), 2 pM (60.0 i3.3%), and 20 pM (57.2 i 5.0%) PGE2
(Figure 4.3B). No significant changes in apoptosis or cytotoxicity were observed in SP,
BM, or PP leukocyte cultures. Apoptosis in TH exposed to a combination of VT at
concentrations up to 500 ng/ml and PGE2 was not statistically different than either TH
single treatment (data not shown). The addition of VT to these cultures did not
significantly alter TH apoptotic or cytotoxic responses. These results suggest VT and

PGE2 signaling pathways do not collaborate in primary leukocyte signaling.

Differential sensitivity of primary murine leukocytes to anti-IgM -induced apoptosis and
cytotoxicity in the absence and presence of VT

Antigen receptor ligation has been used extensively as a model of clonal B cell

59

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deletion (Hasbold and Klaus, 1990; Joseph et al., 1995; Gottschalk and Quintans, 1995).
Following anti-IgM crosslinking, B cells undergo cell cycle arrest and apoptosis. This
process appears to be regulated by the B cell microenvironment (Modigliani et al., 1997)
and a significant increase in ceramide levels is of critical importance (Gottschalk and
Quintans, 1995). Sensitivity of unstimulated primary leukocyte cultures prepared from
TH, SP, BM, and PP to anti-IgM (as an antigen mimic) was assessed. Anti-IgM (1
pg/ml) significantly increased SP leukocyte apoptosis (Figure 4.4A) and decreased MTT
conversion (78.9 :1: 5.3%, p<0.05; Figure 4.48). Apoptotic morphology and cytotoxicity
were not altered in TH, BM, or PP cultures. The combination of VT and anti-IgM was
not significantly different than either SP single treatment (data not shown). These results
suggest VT and antigen-dependent signaling pathways do not interact in primary

leukocytes.

Differential sensitivity of primary murine leukocytes to DEX- induced apoptosis and
cytotoxicity in the absence and presence of VT

Endogenous increases in glucocorticoids can result from both inflamr‘natory
conditions (Cabrera et al., 2000) and cell stress resulting from chemical exposure (Pruett
et al., 1993). Glucocorticoids can both inhibit (Bailly-Maitre et al., 2001) and induce
(Krishna et al., 1995; Andreau et al., 1998) apoptosis. The induction of lymphocyte
apoptosis by glucocorticoids has been well characterized (Gruber et al, 1994; Krishna et
al., 1995; Andreau et al, 1998) and provides a model in which two apoptotic inducers
may interact.

Sensitivity of unstimulated primary leukocyte cultures prepared from TH, SP,
BM, and PP to DEX was assessed. Apoptosis induction by DEX was observed for TH,

61

 

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SP, and PP cultures (Figure 4.5). The effective doses resulting in 50% (ED50) apoptosis
for TH and SP were 23 and 2 nM, respectively. Maximum apoptosis observed in PP
cultures was 43.77 i 2.0%. BM apoptosis was not observed at concentrations of DEX up
to 5 uM (data not shown). ED50 values for TH and SP cytotoxicity were 12 and 30 nM,
respectively (data not shown). In a previous study, Pestka et. al (1994) demonstrated in
vitro that DEX (100 nM)-induced SP apoptosis was significantly reduced by a high dose
of VT (50,000 ng/ml). In the present study, induction of SP apoptosis by 10 nM DEX
was significantly inhibited by 500 ng/ml VT (Figure 4.6). Interestingly, MTT-conversion
results for VT plus DEX treated SP leukocytes was not significantly affected even though
apoptosis was inhibited (data not shown). These data suggest metabolic activity
(mitochondrial conversion of MTT) was arrested for at least a portion of the incubation
period. It is not clear if the development of apoptosis was completely inhibited or just
delayed in this model system.

The opposing effects on apoptosis by DEX and VT may involve NFKB.
DEX-induced inactivation of NFKB has been described in both T cells (Ray and
Prefontane, 1994; Scheinman et al., 1995) and B cells (Donjerkovic et al., 2000) while
VT activates NFKB in T cells model (Ouyang et al., 1996). Wu et a1. (1996)
demonstrated the necessity of NF KB inhibition in the induction of B cell apoptosis. Here,
apoptosis was induced in WEHI-23l B cells and normal murine splenic B cells by
preventing the degradation of the inhibitor of NF KB, interfering with NFKB binding, and
microinjecting IKB-a-GST protein. VT may be similarly upregulating NFKB and

inhibiting or delaying DEX-induced SP apoptosis.

63

 

 

 

 

 

 

 

 

 

 

 

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Figure 4.5. DEX induced apoptosis in primary murine thymocyte, splenocyte, and Peyer’s
patch cultures. Primary murine leukocyte cultures were exposed to DEX (0 - 100 nM) for
18 h. Percent apoptosis was assessed by fluorescence microscopy. Data (n=4) represent
the mean i SEM. 'A Student’s t test was used to determine values significantly different
from the respective tissue control value (p < 0.05). TH - thymocytes; SP - splenocytes;

BM - bone marrow; PP - Peyer’s patch.

64

 

 

 

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Splenocytes were exposed to VT (0 - 500 ng/ml), DEX (0 - 100 nM) or both for 18 h.
Percent apoptosis was assessed by fluorescence microscopy. Data (n=4) represent the
mean percent control response i SEM . ‘A Student’s t test was used to determine values
significantly different from the untreated control, bvalues significantly difierent from the
respective DEX control, and cvalues significantly different from the respective VT control

(p<0.05).

65

Interestingly, the use of glucocorticoids in vivo has been investigated in the
pharmacological treatment of trichothecene toxicity. Matsuoka and Kubota (1987)
inhibited fusarenone-x induced gastro-intestinal permeability with hydrocortisone. In
addition, DEX administration significantly increased the LDSO value of T-2 toxin in mice
(Fricke and Jorge, 1991) and lethality in rats (Harvey et al., 1994). In the latter studies,
the high dose required to reduce trichothecene toxicity suggests glucocorticoids were
acting indirectly by reducing the inflammatory response rather than a direct

receptor-mediated mechanism.

Differential sensitivity of primary murine leukocytes to anti-Fas-induced apoptosis and
cytotoxicity in the presence and absence of VT

Fas ligand (F asL) and Fas receptor (FasR) are upregulated following sepsis
(Ayala et al., 1998) and administration of LPS to mice (Kitamura et al., 2001). Binding
of FasL to FasR results in the induction of apoptosis in thymocytes and B cells of BM,
SP, and PP (Nilsson et al., 2000; Ayala et. al., 1998; Nishimura et al., 1997; Kakinuma et
al., 1999). In the present study, sensitivity of unstimulated primary leukocyte cultures
prepared from TH, SP, BM, and PP to anti-F as was assessed. A significant increase in
apoptotic morphology was noted in TH cell cultures exposed to anti-F as (1 ug/ml) for 18
h. Whereas apoptosis in SP, BM, or PP were unaffected (Figure 4.7A). In addition,
MTT conversion was significantly decreased in TH treated with 0.1 ug/ml (56.6 i 4.8%)
and 1 pg/ml anti-F as (48.6 i 2.4%; Figure 4.7B) A previous in vitro study required
pre-treatment of primary SP cultures with LPS to sensitize the cells to F as mediated

apoptosis (Watanabe et al., 1995).

66

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Apoptosis and cytotoxicity for VT (up to 500 ng/ml) and anti-Fas combined were
not significantly different than the single-treatment TH cultures (data not shown). In
contrast, the combination of VT (500 ng/ml) and anti-Fas (0.1 or 1 ug/ml) induced a level
of apoptosis in BM leukocytes (Figure 4.8A) that was significantly greater than either
single treatments alone (p<0.05). Percentages of apoptosis for the untreated control,
anti-Fas ( l ug/ml), VT (500 ng/ml), and the combination were 17.6%, 15.1%, 23.7%, and
32.0% respectively. The expected mean additive increase over the untreated control after
exposure to the combined toxins was 6.1%. Statistical analysis of the apoptosis data
indicated that anti-F as potentiated VT-induced BM apoptosis. This interaction was also
observed for combinations of lower doses of VT (250 ng/ml) and anti-Fas (0.1 ug/ml).
M'IT conversion by BM cultures treated with VT (250 ng/ml) and anti-Fas (1 ug/ml)
were significantly lower than each individual treatment (p<0.05). However, the
combination treatments with 500 ng/ml VT were not statistically different from VT alone
(Figure 4.88). A similar potentiation of apoptosis was reported in murine granulosa
cells co-treated with the translational inhibitor cycloheximide (CHX) and anti-Fas
antibody (Quirk et al., 1998). Cells treated with anti-F as alone did not undergo
apoptosis. This potentiation of apoptosis may result from the upregulation of FasR by
CHX, demonstrated both in vivo (Kimura and Yamamoto, 1997) and in a T cell model
(Tang et al., 1999), which would sensitize the cells to Fas-mediated apoptosis. Similarly,

VT may upregulate FasR and sensitize BM cells to Pas-mediated apoptosis.

Differential sensitivity of primary murine leukocytes to TNF-a-induced apoptosis and
cytotoxicity in the presence and absence of VT

TNF-a is a potent proinflammatory cytokine produced by lymphocytes and other

68

 

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cells in response to inflammation or environmental stress (Baud and Karin, 2001).

TNF-a induces a variety of cell responses including proliferation, differentiation, and
apoptosis. The induction of apoptosis by TNF-a depends on recruitment of several
signaling proteins to TNFRI including TNFRl-associated death domain protein
(TRADD), receptor-interacting protein 1 (RIPl), Fas-associated death domain protein

(F ADD), TNF-receptor associated factor 2 (TRAF2), and TNF-related apoptosis inducing
ligand (TRAIL). Sensitivity of unstimulated primary leukocyte cultures prepared from
TH, SP, BM, and PP to TNF-a-induced apoptosis was assessed in the present study.
TNF-a (0.8 - 20 ng/ml) did not significantly increase apoptosis or cytotoxicity in any
primary leukocyte cultures after 18 h (data not shown). However, the combination of VT
(250 or 500 ng/ml) and TNF-a (4 or 20 ng/ml) significantly increased TH apoptosis
(Figure 4.9A). The observed apoptosis was significantly higher than apoptosis induced
by either mediator alone (p<0.05). Percentages of TH apoptosis for the untreated control,
VT (500 ng/ml), TNF-a (20 ng/ml), and the combination were 8.8%, 13.8%, 10.1%, and
40.5% respectively. The expected mean additive increase over the untreated control after
exposure to the combined toxins was 6.3%. Statistical analysis of the apoptosis data
indicated TNF-a potentiated VT-induced TH apoptosis (p<0.05). This interaction was
also observed for the combination of lower doses of VT (250 ng/ml) and TNF-a (4
ng/ml). MTT conversion by TH cultures treated with VT (250 or 500 ng/ml) and TNF-a
(20 ng/ml) was significantly lower than each individual treatment (Figure 4.9B). Percent
inhibition of MTT conversion for VT (500 ng/ml), TNF-a (20 ng/ml), and the
combination was 7.7%, 3.6%, and 42.6% respectively. The expected mean additive
inhibition over the untreated control after exposure to the combined toxins was 11.3%.
Statistical analysis of MTT inhibition indicated TNF-a potentiated VT-induced

70

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71

cytotoxicity (p<0.05). The combination of VT and TNF-a had no significant effect on
SP, BM, or PP apoptosis or cytotoxicity.

Apoptotic responses to TNF-a are thought to be dependent on the inhibition of
genes involved in proliferation and activation responses by RNA or protein synthesis
inhibitors (Baud and Karin, 2001). However, NFKB is required for activation-induced
cell death of CD4+CD8+ thymocytes (Hettmann et al., 1999; Bessho et al., 1994), and the
IkB kinase complex is essential for protection of T lymphocytes against anti-F as and
TNF-a-induced apoptosis (Senftleben et al., 2001). The upregulation of NFKB by VT
(Ouyang et al., 1996) may be contributing to the increased apoptosis observed in TH
leukocytes co-treated with TNF-a and VT. Recently, the upregulation of TRAIL by
NFKB has been demonstrated in T cells (Siegmund et al., 2001; Baetu et al, 2001).

Cycloheximide induces T cell death in a FADD/caspase-S dependent manner
without Fas or TNF receptor I interaction (Tang et al., 1999). Similarly, VT-induced
apoptosis may be FADD-dependent. FADD is a common component of both TNF-a and
Fas death receptor pathways and may explain the potentiation of apoptosis in both TNF-a
plus VT treated TH and anti-Fas plus VT treated BM leukocytes. In addition, LPS
induces endothelial cell apoptosis in a FADD-dependent manner independent of TNF-
a/F as death receptor involvement (Choi et al., 1998). The induction of a
FADD-dependent pathway may explain the in vivo induction of apoptosis by LPS plus
VT (Zhou et al., 2000). While the receptor-proximal events of death receptor pathways
are well defined, the role of other signal components, such as mitogen activated protein
kinase (MAPK) activation are reportedly cell-type specific (Baud and Karin, 2001). We
investigated signal events involved in the potentiation of VT induced apoptosis by

TNF-a in TH leukocyte cultures using several pharmacological inhibitors.

72

Mechanism of TNF-a potentiation of VT-induced TH apoptosis involves reactive oxygen
species, intracellular Ca”, mitogen activated protein kinases, and caspase-3

The potentiation of VT-induced apoptosis by TNF-a provides a model in which
an inflammatory component significantly increases the immunotoxic response of a
chemical. This model was further investigated to determine the upstream mechanisms
involved in TH ap0ptosis. For these studies, cells were pretreated with each signaling
inhibitor and the percent apoptosis was compared to the VT plus TNF-a treated control
cultures and the untreated control.

N-acetyl-cysteine (NAC) replenishes intracellular glutathione, protecting the cell
against the production of reactive oxygen species. NAC blocks apoptosis induced by
TNF-oz (Talley et al., 1995) and cycloheximide plus TNF-ct co-treated cells (Cossarizza
et al., 1995). A significant decrease in intracellular glutathione was reported in
cycloheximide-treated Jurkat cells (Chiba et al., 1996). In the present study, VT plus
TNF-a-induced TH apoptosis was significantly reduced by NAC (10.9 at 0.5%; Figure
4.10). This value was not significantly different than the untreated control value (8.8 :1:
0.3%). Previous studies indicate NAC blocks NFKB and INK activation by TNF-a
(Shrivastava and Aggrarwal, 1999) and NF KB resulting from Fas ligation (Giardina etal.,
1999)

Ca2+ mobilization may influence apoptosis in a cell-type specific manner. For
example, elevation of intracellular Ca2+ induces the appearance of phosphatidylserine on
the membrane of apoptotic Jurkat cells (Hampton et al., 1996) but is not necessary for
TNF-a-induced apoptosis in a rat/mouse T cell hybridoma (Denecker et al., 1997). In
the present study, intracellular Ca2+ chelator BAPTA-AM significantly reduced VT plus
TNF-a TH apoptosis (10.9 i 0.9%; Figure 4.10). This value was not significantly

73

1

Control

VT + TNF-a 9.
NAC
Bapta-AM
EDTA
PD98059
88203580

 

Caspase-3

0
0"
A
O
_x
U"

20 25 30

Percent Apoptosis

Figure 4.10. VT plus TNF-a induced apoptosis signal mechanisms involve reactive
oxygen species, intracellular Ca 2" , mitogen activated protein kinases, and caspase-3.
Thymocyte cultures were pre-treated 2 h with 1 uM of each inhibitor and exposed to VT
(500 ng/ml), TNF-a (20 ng/ml), or both for 18 h. Percent apoptosis was assessed by
fluorescence microscopy. Data (n=6) represent the mean i SEM. aA student’s t test was
used to determine values significantly different from the untreated control or bVT plus

TNF-a positive control (p<0.05).

different than the untreated control value (8.8 d: 0.3%). Extracellular Caz’ chelator
EDTA did not significantly alter TH apoptosis. In a previous study, FADD-DN T cells
demonstrated an impaired release of intra-cellular, but not extra-cellular Caz‘ (Hueber et
al., 2000). Interestingly, intracellular Ca2+ and ROS produced during TNF-a-induced
apoptosis are interdependent (K0 et al., 2000). Production of ROS in BAPTA/AM
treated fibroblasts was delayed while removal of ROS blocked the TNF-a mediated
intracellular Ca2+ increases and apoptosis. Taken together, these results suggest that both
ROS and intracellular Ca2+ may be necessary for VT plus TNF-a-induced apoptosis and
these signals may be intertwined.

MAPKs are a group of serine/threonine specific protein kinases that connect cell
surface signals through kinase cascades to transcription factors and ultimately modulate
gene expression. c-Jun-N-terminal kinase (JNK), p3 8/SAPK, and ERK 1/2 are critical
regulators of cell survival in Fas and TNF-a treated cells (Roulston et al., 1998; Tran et
al., 2001). In the present study, PD98059, an inhibitor of MEKl and subsequently ERK
1/2, inhibited VT plus TNF-a induced apoptosis (12.5 :1: 0.5%), however, this value was
significantly higher than control levels. Activation of ERK 1/2 by TNF-a is commonly
associated with proliferation or activation of cells (McLeish et al., 1998). Recently,
Bommhardt et al. (2000) demonstrated a role for ERK in thymocyte negative selection.
In contrast, Tran, et al (2001) demonstrated that ERK activation could override apoptotic
signals from death receptors. Interestingly, Davies et a1. (2000) demonstrated that
PD98059 (50 mM) can reduce both MEKl and p38 activity to approximately 85% of the
control value. In addition, Salh et al. (2000) described JNK inhibition by PD98059 in
HT29 cells. Therefore, the role of ERK in VT plus TNF-a-induced apoptosis of TH is

presently unclear.

75

The activation of p38 in response to TNF-ct is biphasic, and both independent and
dependent on caspase activation (Roulston et al., 1998; Herr et al., 1999). Apoptosis in
the presence of SB203580, a highly selective inhibitor of p38, was significantly
decreased to control levels (11.1 d: 1.2%) in VT plus TNF-a co-treated thymocytes.
Caspase activation is critical in the induction of apoptosis by TNF-cc (Belka etal., 2001;
Aggarwal et al., 2000; MacFarlane et al., 2000). Caspase-3 inhibitor 1, a specific but
reversible inhibitor, reduced apoptosis to 15.8 i 1.1%, which was significantly higher
than control values. These results suggest p38 and Caspase-3 may play critical roles in

VT plus TNF-oc-induced TH apoptosis.

CONCLUSION

Apoptosis is a mechanism of cell death which contributes to homeostasis in the
immune system by removing self-reactive lymphocytes (Cohen et al., 1992). Negative
selection of lymphocytes has been well characterized in the thymus (T cells) and
germinal centers of the SP and PP (B cells). Immune cell apoptosis may also result from
chemical insult. Signaling components of both cellular stress and inflammation may
interact with a chemical agent to alter immune cell responses. This study demonstrates
that VT, a potent protein synthesis inhibitor, could inhibit or enhance immune cell
apoptosis and cytotoxicity as exemplified by (l) VT inhibition of LPS-induced
proliferation and DEX-induced apoptosis in SP leukocytes and (2) Potentiation of
VT-induced apoptosis in BM leukocytes by anti-F as and TH leukocytes by TNF-a. The
potentiation of VT-induced apoptosis by TNF-a was fiirther characterized and

pharmacological inhibition studies suggest critical roles for ROS, intracellular Ca”,

76

p3 8/SAPK, and caspase activation. Future studies will begin to decipher the role of these

mediators in the potentiation of VT-induced apoptosis by TNF-a.

77

CHAPTER 5

Mechanisms of Trichothecene-Induced Jurkat T Cell Apoptosis:

Dose Response and Potentiation by TNF-oz

INTRODUCTION

F usarium, Stachybotrys, and Myrothecium produce biologically active fungal
metabolites called trichothecenes (Bondy and Pestka, 2000). Human and animal
exposure to trichothecenes may occur through consumption of contaminated grains or
inhalation of mold spores. The most common trichothecene contaminant of grain crops
in the United States and Canada is vomitoxin (VT, deoxynivalenol) (Rotter et al., 1996).
VT is resistant to milling, processing, and baking and may readily enter cereal-based
food products intended for human consumption (Abouzied et al., 1991).

Leukocytes of the immune system are particularly sensitive to VT and the
trichothecenes. Trichothecenes are immunosuppressive at high doses resulting from both
inhibition of protein synthesis and induction of leukocyte death by apoptosis in the bone
marrow, lymph nodes, spleen, and thymus. (Bondy and Pestka, 2000). Apoptosis is a
critical regulatory pathway of the immune system that restricts excessive inflammation
and prevents autoimmunity through activation of death receptors (Dong, et al., 2002). In
addition, apoptosis may result from cellular stress (i.e. UV light, viral infection,
cytokines, and heat shock) or chemical insult (Saini and Walker, 1998). Recently,

apoptosis induction by ribotoxic stress resulting from ribosomal cleavage (Iordanov et

78

 

al., 1997) or ribosomal binding (Shifrin and Anderson, 1999) by translational inhibitors
has been described.

Despite the high potential for human exposure, little information exists on the role
of apoptosis in VT-induced immunosuppression and the mechanisms involved in
apoptosis induction. Induction of apoptosis is characterized by exposure to extracellular
stimuli, such as VT, and the initiation of a signaling pathway. The pathway triggered is
largely dependent on the specific stimulus and the type of target cell (Saini and Walker,
1998).

Mitogen activated protein kinases (MAPKs) are centrally important in
determining cellular responses to extracellular stimuli. MAPKs are divided into three
distinct, but partially overlapping signaling pathways that include ERKl/Z (p44/p42),
JNKl/2 (p54/p46), and p3 8. Phosphorylation and subsequent activation of transcription
factors by MAPKs leads to. expression of genes critical for apoptosis induction including
signaling components of death receptor pathways (Hsu etal., 1999; Paris et al., 1998).
Activation of MAPKs has been previously reported and linked to apoptosis induction by
trichothecenes (Yang et al., 2000; Shifrin and Anderson, 1999).

As previously reported in Chapter 4, anti-Fas antibody in murine bone marrow
cells and TNF-a in murine thymocytes potentiated VT-induced apoptosis. Previous in
vivo studies in our lab demonstrated increased trichothecene-induced apoptosis and
mortality in the presence of lipopolysaccharide (LPS), the biologically active component
of gram negative bacteria (Zhou et al., 2000; Zhou et al., 1999). The mechanisms
involved in this synergistic toxicity have not been elucidated. The potentiation of
VT-induced apoptosis by TNF-a was significantly reduced by pharmacological inhibitors

of MAPKs. These results suggest death receptor pathways may converge or cooperate

79

 

with VT-induced cell signal mechanisms in some cell types and this process may involve
MAPKs.

The objective of this study was to characterize VT-induced apoptosis in a human
T cell model focusing on both dose-reponse effects and potentiation by the
proinflammatory cytokine TNF-a. In addition, the relationship between these effectors
and activation of second messengers (i.e. intracellular calcium and reactive oxygen
species), upregulation of three MAPK family members, Bel-2 expression, and caspase

substrate cleavage was investigated.

MATERIALS AND METHODS

Chemicals and Reagents

Chemicals and reagents were purchased from Sigma Chemical Company (St.
Louis, MO) unless otherwise noted. VT, anisomycin (AN), and monensin were dissolved
in 100% ethanol and diluted in supplemented RPMI-l640 medium. Final ethanol
concentration in the culture media was less than 0.01% (v/v) which did not induce
apoptosis or cytotoxicity. TNF-a (R & D Systems, Minneapolis, MN) was dissolved in
0.01 M phosphate buffered saline (PBS, pH 7.2) and diluted in supplemented RPMI-1640
medium for a final concentration in culture of 1000 U/ml or 20 ng/ml. Diphenylene-
iodonium chloride (DPI), BAPTA-AM, PD98059, 88203580, and Caspase 3 Inhibitor I
were purchased from Calbiochem (San Diego, CA) and dissolved in cell culture grade
dimethylsulfoxide (DMSO). Final DMSO concentrations in cell culture media was less

than 0.1% (v/v) which did not induce apoptosis or cytotoxicity.

80

 

——::-= 3.-...- a: - v “"’"‘""' '

Cell Culture

Jurkat T lymphocytes (American Type Cell Culture, Manassas, VA) were grown
in RPMI-l640 medium supplemented with 100 U/ml penicillin, 100 pg/ml streptomycin,
50 pM 2-mercaptoethanol, 2 mM glutamine, 10 mM HEPES (Sigma Chemical
Company), 1 mM sodium pyruvate, 1 mM non-essential amino acids, and 10% (v/v) fetal
bovine serum (FBS, Gibco Life Technologies, Rockville, MD). Cell number and
viability was assessed by trypan blue dye exclusion using a hemocytometer (Stiles,

1986).

Apoptosis Assay

Jurkat cells (5 x 105/ml) were cultured in 96 well plates (300 [Ll/well) as
described. Apoptotic cells were quantitated by fluorescence microscopy as described by
Duke and Cohen (1995). Microtiter plates were centrifuged at 450 x g for 10 min and
250 pl of supernatant was removed. Five pl of DNA staining reagent (acridine orange
(100 pg/ml) and ethidium bromide (100 ug/ml) in 0.01 M PBS) was added to each well.
Cells were classified as apoptotic or normal based on chromatin organization. A normal
nucleus appeared small and highly condensed or fragmented. Percent apoptosis was

calculated as follows: number of cells with apoptotic nuclei/total cell number x 100.

MTT conversion assay for cytotoxicity

Following treatment, 20 pl of a 5 mg/ml solution of 3- (4,5 dimethylthiazol-
2-ul)-2,5-diphenyl tetrazolium bromide (MTT) in 0.01 M PBS was added to each well for
the final 4 h of the 18 h incubation period (Marin, etal., 1996). Microtiter plates were

centrifuged at 450 x g for 20 min and media was aspirated to minimize formazan crystal

8]

 

loss. The resultant crystals were dissolved in 150 pl DMSO for 15 min. Optical density
was measured using a Vmax Microplate Reader (Molecular Devices, Menlo Park, CA) at
dual wavelengths, 570 nm and 690 nm. Percent control response was calculated as

follows: ((1-absorbance of treatment/absorbance of control) x 100).

Spectrofluorometric detection of intracellular Ca”

Intracellular Ca2+ was measured as described by McConnack and Cobbold
(199]). Cells (1 x 107) were resuspended in 0.75 ml of incubation media (120 mM
NaCl, 20 mM HEPES, 4.7 mM KCI, 1.2 mM KHZPO4, 1.2 mM MgSO,, 1.25 mM CaCIz,
10 mM glucose, pH 7.4 containing 2% (w/v) bovine serum albumin (BSA)) and
combined with 0.25 ml of loading solution (containing a final concentration of 0.025%
pluronic F 127 and 20 pM Fluo-3-AM in incubation buffer) for 30 min at 35’C water
bath. Cells were then washed three times at 450 x g in incubation media without BSA
and diluted to a concentration of 2 x 106/m1. Cells (150 pl/well) were aliquoted into
Fluoronunc 96 well plates (Fisher Scientific, Pittsburgh, PA). Background fluorescence
was recorded (485 nm excitation, 534 nm emission) using a CytoFluor II microwell
fluorescence reader and the CytofluorII software Version 2.0 (Biosearch Incorporated,
Bedford, MA). When baseline fluorescence stabilized, ionomycin (positive control), VT,
TNF-a, or combination treatments were added and fluorescence intensity was recorded at

several time points.

Spectrofluorometric detection of reactive oxygen species (ROS)
Intracellular levels of reactive oxygen species (ROS) were monitored

spectrofluorometrically (Kim et al., 1996) as described above for intracellular Ca2+

82

detection. Briefly, cells were resuspended in 0.75 ml of incubation media and combined
with 0.25 ml of loading solution (containing dichlorofluorescin diacetate (DCF DA) in
incubation buffer) for 15 min at 35°C water bath. Cells were then washed three times as
described, diluted to a concentration of 2 x 106/m1, and aliquoted into fluoronunc 96 well
plates. When baseline fluorescence stabilized, H202 (positive control), VT, TNF-a, or
combination treatments were added and fluorescence intensity was recorded at several

time points.

Western blot analysis for mitogen activated protein kinase (MAPK) and Bcl2 expression
Phosphorylation of MAPKs or alterations in Bcl2 were assessed by Western
blotting using phospho-p44/p42 ERK, phospho-p38 MAPK, phospho-JNK specific rabbit
IgG antibody (Cell Signaling, Beverly, MA) or Bcl2 (Santa Cruz Biotech, Santa Cruz,
CA) as previously described (Yang et al., 2000). Briefly, cells were suspended in lysis
buffer (10 mM Tris pH 7.4, 1 mM sodium orthovanadate, 1% (w/v) SDS) on ice for 30
min and sonicated. Lysates were centrifuged at 4000 x g for 5 min at 4C and protein
concentration in the supernatant was determined using a Bio-Rad Protein Assay kit
(Bio-Rad Laboratories, Melville, NY). Total cell lysates (10 pg) were resolved using
10% (w/v) SDS-PAGE. Gels were electrophorhetically transferred to polyvinylidene
difluoride membrane (PVDF). Membranes were probed with the primary antibodies
(1:1000 dilution) described above and stripped per manufacturer's protocol and probed
with p44/p42 MAPK specific antibody (Santa Cruz Biotech), p38 MAPK specific
antibody (Cell Signaling) and INK specific antibody (Santa Cruz Biotech) diluted 1:1000
and recognizing both phosphorylated and unphosphorylated forms of each MAPK to

verify total MAPK protein levels. To verify loading for Bclz, membranes were stripped

83

as described and probed with actin specific antibodies diluted 1:2000 (Cell Signaling).
After incubation with horseradish peroxidase-conjugated anti-rabbit IgG antibodies
diluted 1:2000 (Cell Signaling), the membrane was developed with a chemiluminescence

detection kit (Amersham, Arlington Heights, IL).

Fluorescence assay for caspase-3 activity

DEVD-specific caspase activity was measured as previously described (Shifrin
and Anderson, 1999). Cells (5 x 105/ml) were resuspended in 0.1 ml of lysis buffer (20
mM HEPES, pH 7.1, 1% (v/v) Triton X 100, 10 mM KCI, 1.5 mM MgC12, 1 mM EDTA,
1 mM EGTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 5 pg/ml
pepstatin, 10 pg/ml leupeptin, 2 pg/ml aprotinin, 25 ug/ml N-acetyl-leu-Ieu-norleucinal
(Calbiochem)), incubated on ice for 10 min, vortexed for 5 s and lysates were clarified
for 10 min at 4000 x g at 4°C. DEVD-specific activity was determined by mixing 50 pg
of protein with 0.2 ml of reaction buffer (100 mM HEPES, pH 7.1, 10% sucrose, 0.1%
CHAPS, 10 mM dithiothreitol, 0.1 mg/ml bovine serum albumin and 2 pM
DEVD-AMC) and incubating at 30°C for 20 min. Caspase activity was calculated by
measuring fluorescence of released AMC with excitation at 360 nm and emission at 460

nm.

Statistical analysis

The data were analyzed using Systat version 5.0 (Evanston, IL). A Student's t test
was used to compare two groups while multiple groups were compared using a one way
analysis of variance (ANOVA) and a Tukey test for mean separation. A p value < 0.05

was considered statically significant. Samples receiving two treatments were analyzed

84

for synergy/potentiation by randomly combining single treatment replicates to calculate
an expected mean additive response with variance. This calculated value was compared
to actual co-treated samples using a Mann Whitney Rank Sum test. A p value < 0.05 was

considered statistically significant.

RESULTS

Time course of VT-induced apoptosis

VT induces apoptosis in both murine and human leukocytes in vitro (Yang et al.,
2000; Shifrin and Anderson, 1999) and in vivo (Zhou et al., 2000). The dose dependent
induction of apoptosis in Jurkat cells over a 24 h period was characterized. Apoptotic
cell morphology, assessed by fluorescence microscopy, was significantly increased over
the untreated controls by 6 h of exposure to VT (500 - 1000 ng/ml, Figure 5.1 A). This
elevation in apoptosis was observed throughout the remaining 24 h sample period. VT
(100 ng/ml) did not significantly alter Jurkat cell morphology up to 24 h of exposure.

Similarly, a significant increase in apoptosis was observed in cells exposed to VT
plus TNF-a between 6 - 24 h of exposure (Figure 5.1 B). The observed apoptosis was
significantly higher than apoptosis induced by either mediator alone between 15 to 24 h.
VT (250 ng/ml) alone significantly increased apoptosis between 15 - 24 h, while TNF-a
had no effect. Percentages of Jurkat apoptosis at 18 h for the untreated control, VT (250
ng/ml), TNF-a (20 ng/ml), and the combination were 2.1%, 18.2%, 0.8%, and 40.2%
respectively. The expected mean additive increase over the untreated control after
exposure to the combined toxins was 16.9%. Statistical analysis of the apoptosis data
indicated TNF-a potentiated VT-induced Jurkat apoptosis (p<0.05). The signal

85

050550: 0-0.2... 00.0 ...> 0:0 55%: on.

0-...Z... .2500: 03. ...> 0:09:00 00 000: 003 <>OZ<0 .2500 00525: 00: 50:0 50:0..00 35000550 000.0> 055:0:00 00 000:
003 :00: 0 0.50005 <.. .me 0 0005 000 50850: GHE 000m. .2 5-9 0.0205. 05.0 000.:0> :0 000008000 000000083. .00 00000000
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0:00 000:3 Am. 0-0.20 >0 50.5.5002. 000 $0.. 0050000000 0000 0:00 .. 50:3 5 0.0000000 000005-...> .0 00:08 05.... ...m 00%.“.

 

 

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86

transduction mechanisms involved in the induction of apoptosis by VT alone and in the

presence of TNF-oz were further investigated.

Intracellular calcium modulation by VT

Disruption of calcium homeostasis is frequently associated with cell injury and
apoptosis in a variety of tissues (Penninger and Kroemer, 1998). A rapid, transient
increase of intracellular Ca2+ was reported following exposure to the trichothecene T-2
toxin in HL—60 cells prior to the induction of apoptosis (Yoshino et al., 1996).

Apoptosis induction was further characterized using pharmacological inhibitors.
Jurkat cells were treated with each inhibitor for l h prior to the addition of VT (500
ng/ml) 0r co-incubated for 18 h. The intracellular calcium chelator, BAPTA-AM (10 — 25
M) did not significantly influence VT (500 ng/ml)-induced apoptosis or cytotoxicity
(Figure 5.2 A). In contrast, the potentiation of VT (250 ng/ml)-induced apoptosis by
TNF-a could be significantly reduced following a 1 h pretreatment with BAPTA-AM (10
— 25 mM; Figure 5.2 B). In addition, BAPTA-AM (25 mM) significantly decreased VT
plus TNF-a-induced cytotoxicity (Table 5.1).

The role of Ca” in VT and VT plus TNF-a-induced apoptosis was further
assessed. Jurkat cells were loaded with FLUO-3-AM, a fluorogenic calcium probe.
Following loading, cells were treated with VT, TNF-ct, VT plus TNF-at, or ionomycin as
a positive control. VT, TNF-a, or VT plus TNF-a did not significantly alter fluorescence
intensity following 10 min of exposure (data not shown, Figure 5.3). Ionomycin induced
a rapid increase in fluorescence intensity that peaked at 7 min of exposure and was

maintained through 10 min. The results suggest intracellular calcium increases did not

87

..0:500 0>.:.000 0250000: 00. 50.... 50:0»..0 >.500.-..:w.0 00:.0> 055:0:00 0. 000: 003 :00. 0 0.50005 .0... .EMm
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0- ”.2 ... 00.0 25%.: 03. ...> :0 A< ..0:500 020.000 0500 00m. ..> 50:58 0>.:0w05 0.005 0: 0000000 000 50m0 .00.w0.0005:000

0000 00.3 0 . 00.00080 0:03 0:00 50:3 .050w0 .00.w0.0005:000 >0 0.0000000 000:0=.-..> .00 00.00.00. .N.m 0:0wr.

0.00.0000. 500.00

om ov om ON 0.. o om ov on em 0.. o

. . . . . _ . . . . .

 

 

0-0000000
owmmommw
0000000

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.. ._ i 500 000

500 002
m. m .0

 

 

 

 

 

 

88

Table 5.1. Dose response relationships of pharmacological inhibitors (1 h pre-incubation)
on VT-induced cytotoxicity measured by MTT conversion assay.

 

 

 

 

 

 

Treatment VT (500 ng/ml) VT (250 ng/ml) + TNF-0t (20 ng/ml)
Percent Control Response Percent Control Response
DPIOuM 238:2.4 35.5: 1.7
DPI 10 uM 252:0.3 45.2i0.7°
DP125 uM 24.6i 1.8 41.0i2.4
DPI 50 pM 12.5 i 0.4 49.2 i 3.0‘
BAPTA-AM 0 uM 23.2 i 0.9 35.3 i 1.6
BAPTA-AM 10 uM 21.9 i 0.9 39.1 i 2.0
BAPTA-AM 25 uM 24.8 i 0.7 46.9 i 0.8‘
BAPTA-AM 50 pM 25.6 i 2.2 31.1 i 0.8
PD98059 0 uM 29.9 i 4.5 52.7 i 2.8
PD9805910 uM 38.1 i- 3.7 58.7 3:12
PD98059 25 pM 37.0 :34 57.4 i 2.8
PD98059 50 pM 32.1 :12 54.1 i 4.3
SB203580 0 pM 32.9 i 0.7 50.4 i 2.8
SB203580 10 11M 51.0 i 5.7a 65.7 i 4.8“
SB203580 25 pM 58.4 : 4.0a 86.1 i 7.2‘
SB203580 50 uM 48.9 i 4.4“ 84.5 i 1.7'
Monensin 0 uM 25.5 i 0.8 40.1 i 3.2
Monensin 10 uM 25.4 i1.0 35.6 i1.6
Monensin 25 uM 22.5 i 1 45.0 i 0.8
Monension 50 uM 21.1 i 0.2 29.6 i 0.9

 

Data (n=3) represent mean percent control response i SEM. 3A Student’s t test was used
to determine values significantly different from the respective untreated control.

89

 

ION

Relative Fluorescence
N
l

 

 

 

Time (min)

Fig 5.3. Intracellular calcium modulation by VT. Jurkat cells were loaded with F LUO-3-
AM as described in Materials and Methods. When background fluorescence was stable,
ionomycin(1 uM, positive control), VT (250 ng/ml), TNF-a (20 ng/ml), or VT (250
ng/ml) plus TNF-a (20 ng/ml) was added. Fluorescence intensity was monitored using a

Cytofluor II microwell plate reader. Data (n=3) represents mean i SEM.

9O

occur as an early signal in VT (250 ng/ml)-, TNF-a-, or VT plus TNF-a-induced

apoptosis.

Alterations in reactive oxygen species (ROS) by VT

Reactive oxygen species (ROS) are generated during mitochondrial respiration
and have been implicated in cellular signaling including the induction of apoptosis (Davis
et al., 2001). Suneja et al. (1989) reported free radical generation following oral
exposure to trichothecenes in a rat model. In the present study, apoptosis induction by
VT (500 ng/ml) was significantly reduced by pretreatment with DPI (10 pM), an
irreversible inhibitor of NADPH oxidoreductase (Figure 5.2 A). However, cytotoxicity
was significantly increased when cells were co-incubated with DPI (50 pM) and VT (500
ng/ml) (Table 5.1). The potentiation of VT (250 ng/ml)-induced apoptosis by TNF-a
could be significantly reduced following a 1 h pretreatment with DPI (10 pM; Figure 5.2
B). In addition, DPI (10, 50 pM) significantly decreased VT plus TNF-a-induced
cytotoxicity (Table 5.1).

To further assess the role of ROS, Jurkat cells were loaded with DCF DA, a
fluorogenic oxygen radical probe. Following loading, cells were treated with VT (0 -
1000 ng/ml), TNF-a, VT (250 ng/ml) plus TNF-a, or H202 as a positive control. VT,
TNF-a, or VT plus TNF-a did not significantly alter fluorescence intensity following 5
min of exposure (data not shown, Figure 5.4). H202 induced a rapid increase in
fluorescence intensity. The results suggest generation of ROS did not occur as an early

signal in VT- or VT plus TNF-a-induced apoptosis.

91

 

H202

an 2 T
o
C
0
o
(D
93
o
2
u.
a)
._>.
:5
a)
CI 1 #
VT+TNF

 

 

 

l l l l r I
-6 -4 -2 0 2 4 6 8

Time (min)

Figure 5.4. Elevation of reactive oxygen species (ROS) by VT. Jurkat cells were loaded
with DCFDA as described in Materials and Methods. When background fluorescence was
stable, H202 (1 uM, positive control), VT (0 - 10 pg/ml), TNF-a (20 ng/ml), or VT (250
ng/ml) plus TNF-a (20 ng/ml) was added. Fluorescence intensity was monitored using a

Cytofluor II microwell plate reader. Data (n=3) represents mean :h SEM.

92

VT-induced activation of ERK 1/2

The MAPKs, ERKl/Z, have been linked to the induction of negative selection in
thymocytes (Bommhardt et al., 2000), apoptosis induced by loss of anchorage (Zugasti et
al., 2001), and activation-induced cell death in Jurkat cells (van den Brink, et. al, 1999).
Several trichothecenes induce ERKl/2 (Yang et al., 2000). In the present study MEKI
inhibitor PD98059 (10 pM) did not significantly influence VT-induced apoptosis when
cells were pre-treated for l h. Similarly, pre-incubation with PD98059 (10 - 50 pM) did
not alter VT-induced cytotoxicity. However, co-incubation with 0.1 uM PD98059
protected Jurkat cells from VT-induced cytotoxic effects (Table 5.2).

Western blot analysis of VT treated cells demonstrated ERK 2 phosphorylation
between 0.25 - 4 h in cells treated with VT 250 and 500 ng/ml (Figure 5.5 A). ERK 2
was up-regulated between 0.25 - 1 h in cells treated with VT (1000 ng/ml). VT (100
ng/ml), a dose that does not alter apoptotic cell morphology, did not significantly change
ERKl/Z phosphorylation up to 4 h after treatment. Comparison of the treatments at 0.25,
1, and 2 h did not demonstrate dose-dependent phophorylation of ERK 2 (data not
shown, Figure 5.5 B) possibly due to maximal activation by 0.25 h.

The potentiation of VT (250 ng/ml)-induced apoptosis by TNF—a could be
significantly reduced following a 1 h pretreatment with PD98059 (10 pM; Figure 5.2 B).
Pre-incubation with PD98059 did not significantly alter VT plus TNF-a-induced
cytotoxicity. However, 18 h co-incubation with PD98059 (0.1 pM) resulted in a
significant decrease in MTT-based cytotoxicity for VT plus TNF-a treated cells (Table
5.2). Western blot analysis of VT plus TNF-a treated cells demonstrated ERK 2
phosphorylation was up-regulated between 0.25 - l h (Figure 5.5 A). TNF-a (20 ng/ml)
did not activate ERK1/2 at any time point tested (0 - 4 h, data not shown). Total ERKl/Z

93

Table 5.2. Dose response relationships of several pharmacological inhibitors (18 h co-
incubation) on VT-induced cytotoxicity measured by MTT conversion assay.

 

 

Treatment VT (500 ng/ml) VT (250 ng/ml) + TNF-a (20 ng/ml)
. Percent Control Response Percent Control Response
No Treatment 7 64.35 : 2.50 74.93 : 4.39
DPI 1 pM 64.92 : 6.01 57.41 : 8.97
DPIO.11uM 61.65:8.71 83.48: 12.15
PD980591uM 51.55 : 2.85 58.52 : 5.13
PD98059 0.1 uM 80.94 : 3.853' 104.41 : 5.36“
SB203580 1 uM 53.99 : 6.71 58.06 : 3.44
SB203580 0.1 uM 68.82 : 10.99 79.38 : 7.21
Monensin 1 uM 37.37 : 0.46 67.97 : 5.29
Monensin 0.1 uM 74.04 : 5.05 78.00 : 6.06

 

Data (n=3) represent mean percent control response :1: SEM. 3'A Student’s t test was used to
determine values significantly different from the respective untreated control.

94

Figure 5.5. VT-induced phosphorylation of ERK1/2 . Lysates (10 pg) from Jurkat cells
were incubated in the absence and presence of VT (100 - 1000 ng/ml), TNF-a (20 ng/ml),
or VT (250 ng/ml) plus TNF-a (20 ng/ml) for O - 4 h (A). Dose dependent increases in p-
ERK after 15 min of exposure (B) were investigated by Western blot analysis using
phospho-specific ERKl/Z antibodies as described. Anisomycin (AN) was used as a
positive control. The blots were stripped and total ERK levels were confirmed using
p44/p42 specific antibodies recognizing both phosphorylated and unphosphorylated
forms (B, second panel). Two bands were detected corresponding to ERKl/Z. Results

are representative of two experiments.

95

Time(h)
0 0.25 0.5

VTlOO M1:

p-ERK 1
p-ERK 2

i -_ ERK]
W250 ”Cu-a 3: 313mm

 

cuss-:2
VTlOOO ---==t
VT+TNF-a Mt}: :3

VT 500

,0
fig .5

96

 

ERK 1
KERK 2

ERK 1
KERK 2

ERK 1
KERK 2

ERK l
KERK 2

ERK 1
Ken 2

(phosphorylated and unphosphorylated) was confirmed to be unchanged by the various
treatments in Figure 5.5 B (second panel). These results suggest a role for ERK 2 in VT

and VT plus TNF-a-induced apoptosis.

VT-induced activation of p38 and JNK 1/2

Activation of p38 and JNK MAPK is associated with stress stimuli such as UV
radiation, heat shock, cytokines, and osmotic shock (Kortenjann and Shaw, 1995).
Trichothecenes induce a ribotoxic stress response that activates both p38 and JNK
(Shifrin and Anderson, 1999; Yang et al., 2000). Shifrin and Anderson (1999)
demonstrated phosphorylation of p38 and JNK substrate c-Jun after 2 h of exposure to 3
ug/ml VT. Yang, et a1 (2000) could not detect p38 or JNK phosphorylation at a low VT
(10 ng/ml) dose, but inhibited apoptosis induced by a high dose of VT (2.5 - 5 pg/ml)

. I with SBZO3580.

In the present study, SB203580 (10 - 50 pM), a selective inhibitor of p38 MAPK
kinase, significantly reduced VT-induced apoptosis and cytotoxicity when pre-incubated
with cells 1 h prior to the addition of toxin. Western blot analysis of p38 indicated a
prolonged phosphorylation of p38 between (0 - 4 h) in cells treated with 250 - 1000
ng/ml VT (Figure 5.6 A). VT (100 ng/ml) transiently increased p-p38 with maximal
phosphorylation at l h. A dose dependent increase in p-p38 was observed after 2 h
exposure (Figure 5.6 B). Total p38 (phosphorylated and unphosphorylated) was
confirmed to be unchanged in Figure 5.6 B (second panel).

The potentiation of VT (250 ng/ml)-induced apoptosis by TNF-a could be
significantly reduced following a 1 h pretreatment with SBZO3580 (10 pM, Figure 5.2
B). SB203580 (10 - 50 pM) significantly decreased VT plus TNF-a-induced

97

Figure 5.6. VT-induced phosphorylation of p38 MAPK. Lysates (10 pg) from Jurkat
cells were incubated in the absence and presence of VT (100 - 1000 ng/ml), TNF-a (20
ng/ml), or VT (250 ng/ml) plus TNF-a (20 ng/ml) for 0 - 4 h (A). Dose dependent
increases in p-p38 after 2 h of exposure (B) were investigated by Western blot analysis
using phospho-specific p38 MAPK antibodies as described. Anisomycin (AN) was used
as a positive control. The blots were stripped and total p38 levels were confirmed using
p38 specific antibodies recognizing both phosphorylated and unphosphorylated forms (B,
second panel). One band was detected corresponding to p38. Results are representative

of two experiments.

98

Time (h)
0 0.25 0.5 1 2 4
VT 100 ‘ - m ‘— p-p38
VT 250 i“- . «— p-p38
VT 500 M <— p-p38

VT 1000 w <— p.p33

VT + TNFa --- r— .- <—— p-p38

 

99

cytotoxicity (Table 5.1). A prolonged activation of p38 between (0 - 4 h) was observed
in cells treated with VT plus TNF-a (Figure 5.6 A). In addition, the combination of VT
plus TNF-a increased p-p38 expression greater than either individual treatment alone
(Figure 5.6 B).

Phosphorylation of JNKl/Z was observed between 0.25 - 2 h following VT (500 -
1000 ng/ml) exposure. VT (250 ng/ml) transiently increased p-INK (0.25 - 1 h) while
VT (100 ng/ml) induced activation at 1 h (Figure 5.7 A). A dose dependent activation of
JNK was demonstrated following 2 h VT exposure (Figure 5.7 B). Total JNK
(phosphorylated and unphosphorylated) was confirmed to be unchanged in Figure 5.7 B
(second panel).

VT plus TNF-0L transiently increased p-JNK (0.25 - 1 h). Induction of IN K at 2 h
by VT plus TNF-a resembled the individual VT (250 ng/ml) treatment. TNF-a did not
induce significant phosphorylation of JNK at any time point tested (0 — 4 h). These
results support a role for stress-activated p38 and JNK in VT and VT plus

TNF-a-induced T cell apoptosis.

Modulation of Bcl2 expression by VT

Bcl2 family of proteins encompasses both anti-apoptotic and pro-apoptotic
members that control the involvement of the mitochondria in the apoptotic process by
controlling the formation of pores in the membrane (Tsujimoto and Shimizu, 2000). Bcl2
expression in VT-treated Jurkat cells was further assessed. Bcl2 expression was not
significantly altered in cells exposed to VT (500 ng/ml) or VT plus TNF-a (data not
shown) Bcl2 levels in cells treated with 1000 ng/ml VT were unchanged between 0 - 15 h

(Figure 5.8). Levels dropped significantly at 18 h which corresponded to approximately

100

Figure 5.7. VT-induced phosphorylation of JNK1/2. Lysates (10 pg) from Jurkat cells
were incubated in the absence and presence of VT (100 - 1000 ng/ml), TNF-a (20 ng/ml),
or VT (250 ng/ml) plus TNF-a (20 ng/ml) for 0 - 4 h (A). Dose dependent increases in p-
JNK after 2 h of exposure (B) were investigated by Western blot analysis using phospho-
specific JNK antibodies as described. Anisomycin (AN) was used as a positive control.
The blots were stripped and total JNK levels were confirmed using JNK1/2 specific
antibodies recognizing both phosphorylated and unphosphorylated forms (B, second
panel). Two bands were detected corresponding to JNK 1/2. Results are representative

of two experiments.

10]

A Time (h)
0 0.25 0.5 l 2 4
-

m".

p—JNK 1

VT 100 . p-JNK 2
- - in u? p-JNKl

VT 250 __ - - p-JNK 2
b“ '5‘" p-JNK 1

p-JNK 1

HHMHH
:2:

VTIOOO 0.9.- p-JNK2
an - - p-JNKl
VTJ’TNF“ —.. p-JNK2
B
,0
Q
N O Q Q
§ 3 0 .2 § 9' ,5
0 .0 0 0 0 5 5 0
“fund-i“ -fl‘“ " -- 4-P-JNK1

’01-.-- ---- 4— p-JNKZ

.... ~ - 0‘ 4— JNK2

102

Time (h)

<—— Bcl2

 

Figure 5.8. Modulation of Bcl2 expression by VT. Lysates (10 pg)
from Jurkat cells were incubated in the absence or presence of VT
(1000 ng/ml) up to 18 h. Alterations in Bcl2 expression were analyzed
by Western blot analysis. One band was detected corresponding to

Bclz. Results are representative of two experiments.

103

70% apoptotic morphology in the cell population. The results suggest Bcl2 did not play a
role in VT-induced or VT plus TNF-a-induced apoptosis and support a previous study
(Okumura et al., 2000) in which Bcl2 levels were not altered in T-2 toxin induced

apoptosis.

VT-induced activation of caspase-3

Activation of caspases during apoptosis results in the cleavage of cellular
substrates critical for the development of an apoptotic morphology (Cohen, 1997).
Several trichothecene mycotoxins activate caspases (Shifrin and Anderson, 1999). In the
present study, Caspase-3 Inhibitor I (10 pM), a reversible Caspase-3 inhibitor, effectively
reduced VT-induced apoptosis and VT plus TNF-ct induced apoptosis (Figure 5.2).
Caspase activation was not elevated following 1 h exposure to VT (data not shown).
However, significant increases in caspase activation were noted at 3 and 5 h exposure to
VT (500 - 1000 ng/ml; Figure 5.9 A) and VT plus TNF-a (Figure 5.9 B). Caspase
activation by VT plus TNF-ct was significantly greater than either individual treatment
alone. Caspase substrate cleavage remained elevated at 7 h post-exposure (data not
shown). VT (100 ng/ml) and TNF-or alone did not induce measurable caspase substrate
cleavage.

The ability of pharmacological inhibitors to block caspase activation was
evaluated. Monensin, DPI, and BAPTA-AM did not significantly alter caspase substrate
cleavage at 3 h (Table 5.3). MAPK inhibitors SB203580 and PD98059 did not reduce
VT (500 ng/ml) caspase activation (Table 5.4). However, VT plus TNF-a-induced
caspase fluorescence was significantly reduced by SBZO3580. These results support

previous studies indicating a role for caspase activation in trichothecene-induced

104

 

VT100

VT 500
VT 1000

 

 

 

VT 100
VT 500

VT 1000

 

 

 

TNF-a
VT 250

VT + TNFOL

 

TNF-01
VT 250

VT + mm 8b

 

O 1 2 3
Relative Fluorescence

Figure 5.9. VT-induced activation of Caspase-3. Jurkat cells were treated with VT (0 -
1000 ng/ml), TNF-a (20 ng/ml), or VT (250 ng/ml) + TNF-a (20 ng/ml) for 3 or 5 h. Cell
lysates (50 pg) were incubated with fluorogenic caspase substrate DEVD-AMC (2 pM) as
described. Fluorescence intensity was measured using a Cytofluor II microwell plate
reader. Data (n=6) represents mean fluorescence intensity : SEM. ’ A Student’s t test
was used to determine values significantly different from the untreated control. bAN OVA

was used to compare VT (250 ng/ml), TNF-a (20 ng/ml), and VT plus 'I NF-a treatments.

105

Table 5.3. Effect of pharmacological inhibitors on VT-induced caspase-3 activity.

 

 

Treatment VT (500 ng/ml) VT (250 ng/ml) + TNF-or (20 ng/ml)
Relative Fluorescence Relative Fluorescence
No Treatment 1.6 :1: 0.09 1.7 x 0.09
Monensin 10 pM 1.77 : 0.02 1.67 : 0.01
DPI 40 pM 1.63 : 0.04 1.58 : 0.09
BAPTA-AM 5 pM 1.85 : 0.04 1.8 : 0.03

 

Table 5.4. Effect of MAPK inhibitors on VT-induced caspase-3 activity.

 

 

Treatment VT (500 ng/ml) VT (250 ng/ml) + TNF-or (20 ng/ml)
Relative Fluorescence Relative Fluorescence
No Treatment 1.46 : 0.04 1.3 : 0.04
SB230580 10 pM 1.3 i 0.02 0.95 : 0.01
PD98059 10 pM 1.56 :1: 0.01 1.61 : 0.04

 

106

apoptosis (Nagase et al., 2001; Shifrin and Anderson, 1999) that is partially mediated by

p38 MAPK.

DISCUSSION

Vomitoxin (VT) is the most frequently encountered food-borne trichothecene
worldwide (Rotter et al., 1996). Trichothecenes can impair immune function and
exposure may increase an individual's susceptibility to Candida, Salmonella, and Listeria
(Bondy and Pestka, 2000). TNF-a, a cytokine released under conditions of inflammation
or environmental stress, may play a role in pathogen susceptibility following VT
exposure. The potentiation of VT-induced apoptosis by TNF-oz, suggests the possibility
for ribotoxic stress response and cytokine signaling pathways to converge and contribute
to immunosuppression. In addition, this interaction provides a model in which toxic
effects of a chemical may be amplified by an immune or inflammatory response at the
cellular level. This study addressed the kinetics of the second messengers intracellular
Caz’and ROS, MAPK activation, Bcl2 expression, and caspase enzymatic activity in the
development of VT-induced T cell apoptosis to gain insight into mechanisms by which
VT alters immune function and contributes to the manifestation of immune suppression.

Induction of intracellular Caz’and ROS has been implicated as both an early signal
to activate MAPKs (Seo et al., 2001; Yu et al., 2000) or a late signal as a result of
mitochondrial permeability (Castedo et al., 1996)) in the induction of apoptosis. Neither
intracellular Ca2+ nor ROS increased during early exposure to VT. However,
pre-incubation with BAPTA-AM significantly reduced VT plus TNF-a-induced

apoptosis and cytotoxicity. Pre-incubation with DPI significantly reduced both VT (500

107

ng/ml) and VT plus TNF-a induced apoptosis. It is most likely that calcium and ROS
may be increasing at a later time point during the apoptotic process. Our results differ
from a previous report that the trichothecene T-2 toxin transiently increased intracellular
calcium between 3 - 5 min of exposure in HL-60 cells (Yoshino et al., 1996). The reason
for this difference may be stimulus dependent, cell-type specific effect, or due to assay
sensitivity.

Intracellular Caz’and ROS are frequently associated with the development of
irreversible damage to the mitochondria. Initially, increases in oxidant production by the
mitochondria can no longer be inactivated by endogenous antioxidants (Chakraborti, et
al, 1999). This stimulates a cascade of events involving calcium release from the
mitochondria that stimulates calcium dependent proteases, nucleases, and phospholipases
critical in the development of an apoptotic morphology. Pore formation in the
mitochondrial membrane is mediated by the Bcl2 protein family. In the present study,
Bcl2 levels were not altered by VT treatment suggesting another member of the Bcl2
family may be important in trichothecene-induced apoptosis. In support of these
findings, Okumura et al. (2000) could not demonstrate a role for Bcl2 in T-2 toxin
induced apoptosis.

Pore formation is followed by the release in cytochrome c and activation of the
caspase family. Fluorogenic caspase substrate cleavage was significantly increased by
VT following 3 h of toxin exposure and throughout the 7 h sample period. Caspase
activation by 3 h is in agreement with previous studies in which caspase activation was
increased by several trichothecenes by 3 h (Nagase et al., 2001; Shifrin and Anderson,

1999). Caspase activation induced by VT plus TNF-a at 3 and 5 h was significantly

108

increased over either individual treatment alone suggesting caspase activation may be a
signaling step where ribotoxic stressors and cytokine signaling pathways converge.

MAPK phosphorylation appears to be an early, critical signal for the development
of VT-induced apoptosis. Phosphorylation of several transcription factors by the MAPKs
may be critical to cellular responses to stress or apoptosis (Widmann et al., 1999). For
example, ERKl/2 activates Elk-l and nur 77(Pavlovic etal., 2000; Widmann et al.,
1999), p38 activates ATP-2 (Cho et al., 2001) and JNK activates c-Jun (Faris et al., 1998;
Behrens et al., 2001). Induction of human T cell apoptosis by VT or VT plus TNF-a
was associated with the up-regulation of ERK1/2, p38, and JNKl/Z MAPKs. Levels of
VT (100 ng/ml) that did not increase apoptosis did not increase ERK1/2 phosphorylation
and caused only a transient increase in p38 and INK l h after exposure. These data
suggest prolonged activation of MAPKs and the resultant transcriptional activation may
be critical to apoptosis induction.

A protective effect of ERK1/2 in a mouse macrophage model of VT-induced
apoptosis was previously described (Yang et al., 2000). In that study, when MEKl and
subsequently ERK1/2, was blocked by pretreatment with 40 pM PD98059, VT (2.5 and 5
mg/ml)-induced apoptosis was drastically increased. In the present study, VT (250
ng/ml) plus TNF-a induced apoptosis was significantly reduced by pretreatment with 10
pM PD98059. However, cytotoxicity measured by MTT conversion assay was not
recovered in cells pretreated with 10 — 50 pM. This suggests development of apoptotic
morphology may have been delayed, but not reversed. Co-incubation with PD98059 (0.1
pM) resulted in a significant reversal of cytotoxicity in both VT (500 ng/ml) and VT plus
TNF-a treated cells. Data presented here suggests ERK 2 may contribute to the

induction of apoptosis under conditions of low toxin concentrations. Exposure to 2.5 — 5

109

pg/ml VT (2.5 — 5 times greater than the highest concentration used in this study) may
have caused rapid, severe damage that triggered ERK activation as a cell survival
mechanism (Yang et al., 2000). Up-regulation of ERK 2 has been commonly associated
with T cell antigen receptor ligation and activation-induced T cell death (Izquierdo et al.,
1994; Izquierdo et al., 1993). However, previous reports of the inhibition of p38 (Davies.
et al., 2000) and JNK (Salh, et al., 2000) by PD98059 require further confirmation of the
role of ERK in this apoptotic model.

Prolonged phosphorylation of p38 and JNK are more commonly associated with
stress stimuli and apoptosis (Chen et al., 1996). Shifrin and Anderson (1999)
demonstrated up-regulation of p38 and JNK for several trichothecenes. Yang et al.
(2000) reported a correlation between stress-activated protein kinases (i.e. p38 and JNK)
and apoptosis induced by trichothecenes. Similarly, we observed prolonged activation of
p38 and JNK for levels of VT that induced apoptosis and this activation was dose
dependent at 2 h. In addition, p38 phosphorylation was significantly increased in VT
plus TNF-a over either individual treatment at 2 h, suggesting p38 may represent a point
where ribotoxic stressors and cytokine signaling pathways converge. Low levels of VT
that did not induce apoptosis triggered only a transient increase in p38 and INK
detectable at 1 h. Also in agreement with Yang et al. (2000), 88203580 could partially
block VT-induced apoptosis suggesting p38 could contribute to trichothecene-induced
apoptosis. The role of JNK in VT-induced T cell apoptosis has not been deciphered, but
will require use of a dominant negative mutant JNK kinase 1 (SEKl) or the recently
available pharmacological inhibitor of JNK.

While the role of ERK 2, p38, and JNK1/2 in T cell apoptosis is not completely
understood, all three phosphorylate Elk-1 (Zinck et al., 1995; Shao et al., 1998) and

110

NF KB (Lee, et al., 1997; Schwenger, et al., 1998; Zhao and Lee, 1999), common
transcription factors associated with apoptosis. Elk-1 regulates c-fos transcription and
prolonged activation by protein synthesis inhibitors anisomycin and cycloheximide is
mediated by ERK, p38, and JNK (Zinck, et al., 1995). NFKB is induced by VT (Ouyang
et al., 1996) and TNF-a (Kasof et al., 2001). The up-regulation of death receptor 6
(DR6) by TNF-a was recently demonstrated to occur via NF KB activation (Kasof et al.,
2001). The ability of all three MAPKs to activate Elk-l and NFKB suggests a point of
signaling convergence in which inhibition by pharmacological agents may be overcome
by the remaining active MAPKs. This may explain the partial inhibition observed when
cells were pre-incubated with PD98059 or SB203580.

MacFarlane et a1. (2000) presented a kinetic model of receptor (TRAIL and Fas)
and chemical (lactacystin)—induced apoptosis in Jurkat T cells. In this model,
receptor-induced apoptosis resulted in a rapid onset of apoptotic morphology. TRAIL
induced 70% apoptosis by 2 h while Fas induced 70% apoptosis by 8 h. Chemical-
induced apoptosis reached 70% by 18 h following lactacystin exposure. Caspase-3
activation in TRAIL- and Pas-mediated apoptosis was maximal by l and 2 h',
respectively. Chemical-induced caspase activity significantly increased by 12 h
post-exposure. MacFarlane et a1. (2000) reported maximal p38 and INK phosphorylation
l h after caspase induction by TRAIL and 2 h after caspase induction by Fas. In the
chemical model of apoptosis, p38 and JNK phosphorylation peaked at 8 h, prior to
caspase activation. Neither model of apoptosis could be inhibited by 88203580 leading
the authors to conclude p38 does not play a critical role in the induction of apoptosis in
Jurkat T cells. Similar to chemical-induced apoptosis, VT apoptotoic morphology

peaked at 18 h, and phosphorylation of MAPKs was demonstrated prior to caspase

lll

enzyme activity. In contrast to the chemical-induced apoptotsis, caspase-3 activity
increased much earlier (3 h) and S8203580 significantly inhibited the development of
apoptotic morphology. Taken together, these data suggest ribotoxic stressors may
present a third kinetic model of apoptotic development in Jurkat T cells and this pathway
may involve components of both death receptor and chemical induced pathways

presented by MacFarlane et al. (2000).

112

CHAPTER 6

Summary

Toxins encountered in food and the environment pose a health risk to humans and
animals. The unscheduled induction of apoptosis by these compounds may contribute to
immune suppression by decreasing the number of leukocytes in the immune system and
decreasing host-resistance to pathogens. Comparisons made here among naturally
occurring translational inhibitors suggest trichothecene mycotoxins are among the most
potent inducers of apoptosis and cytotoxicity in lymphocytes.

The worldwide occurrence of the trichothecenes and the potential for their use as
biological warfare agents requires an improved understanding of the mechanism of action
of this group of toxins- The studies presented here identified several structural
characteristics of the trichothecenes critical for biological activity. These factors
influence both cellular transport and binding of these compounds to the site of action and
may facilitate risk assessment studies to predict the toxicity of the untested compounds in
this group of mycotoxins.

Deoxynivalenol (DON, vomitoxin, VT) is the most common food-borne
trichothecene (Rotter et al., 1996). Despite the high potential for human exposure, little
information exists on the role of apoptosis and cytotoxicity in DON-induced immune
suppression. Recent studies demonstrated increased DON toxicity in the presence of
bacterial lipopolysaccharide (LPS; Zhou et al., 2000; Zhou et al., 1999). The
mechanisms involved in the potentiation of DON toxicity by LPS have not been
elucidated, but suggest trichothecene toxicity may be augmented by signal components

involved in cell stress and inflammatory responses. The studies presented here

113

demonstrated a potentiation of DON induced apoptosis and cytotoxicity by TNF-a and
Fas ligand. The present studies suggest cell signaling by translational inhibitors such as
DON may cooperate with death receptor or cytokine signal pathway components to
increase lymphocyte cell death.

Mitogen activated protein kinases (MAPKs) are serine/threonine specific protein
kinases that connect cell surface signals through kinase cascades to transcription factors.
These studies suggest that prolonged activation of MAPKs is crucial for the induction of
apoptosis by DON and the potentiation of DON-induced apoptosis by TNF-a. The
phosphorylation of transcription factors by MAPKs drives gene expression. The
identification of transcription factors activated by DON through MAPK cascades should
be the focus of future studies. Taken together, these studies have contributed to our
mechanistic understanding of trichothecene toxicity and may facilitate risk assessment

studies for this group of natural toxins.

ll4

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