Drs Michael Gray and Kaye Kilburn Molds Mycotoxins and Public Health

Molds, Mycotoxins and
Public Health
Michael R. Gray, MD, MPH, CIME
Internal and Occupational Medicine
Emergency Medicine & Toxicology
In collaboration with
Kaye Kilburn, MD
Internal Medicine and Neurotoxicology
Robert Crago, Ph.D.
Psychology, Neurophysiology and
Neurotherapy
ImmunTox, LLC
PO Box 2050 Benson, Arizona 85602
11/11/02 APHA Philadelphia Property of ImmunTox, LLC 1

Case Study: Tucson Bank, 1994
• Bank Manager • 9–10 Other Employees
– Similar symptoms
– 49 y.o. Married Female
• Shortness of breath
– Symptoms
• Cognitive problems
• 18 month hx arthritis
(wheelchair bound) • Periorbital edema
• General malaise • Skin rashes limited to
exposed skin.
• Excessive fatigue
• Workplace related
• Cognitive dysfunction
w/ memory loss • Worsened on Mondays
• Difficulty concentrating • Improvement on
• Multiple other weekends & vacations
symptoms

Case Study: Tucson Bank, 1994
• Clinical Findings • Environmental findings
– Urticarial & granulomatous – Presence of multiple
skin changes filamentous molds
– Small airway obstruction & – Pipes leaking in common
reactivity wall to air handler &
– Immunologic hyper bathroom
activation & simultaneous – Elevated Stachybotrys
suppression spore counts detected in:
– Multiple inflammatory • Air and surface
symptoms • Highest on the manager’s
– Elevated EBV IgG desktop
antibodies • All employees passed this
desk to go to break room
– Cognitive deficits

Hidden Fungi

Historical Perspective
• Biblical Warning • Occupational Disease
– If your house be – Brown Lung Disease
contaminated with
plagues, molds, and – Farmer’s Lung
Leprosy, put the – Pigeon Breeder’s
contents in the middle Disease
and set it aflame – Yellow Rice Disease
--Leviticus
• Robigalia- April 25th
– 7th to 8th Century BC
– To protect trees & grains
from rusts or mildew

Toxic Effects of Mycotoxins in
Humans
• Trichothecenes
– “This family of mycotoxins causes multiorgan
effects including emesis and diarrhea, weight
loss, nervous disorders, cardiovascular
alterations, immunodepression, hemostatic
derangements, skin toxicity, decreased
reproductive capacity, and bone marrow
damage”
• Medical Aspects of Chemical and Biological
Warfare, Chapter 34: Trichothecene Mycotoxins,
1997

Fungi Producing
Trichothecene Toxins
• Fusarium
• Trichothecium
• Myrothecium
• Cephalosporium
• Stachybotrys
• Verticimonosporium
• Cylindrocarpon

Trichothecenes
• Major category of mycotoxins
• 1st case of mycotoxicosis
– 1932
– USSR
– Alimentary toxic aleukia
• 60% mortality
• Major chemical warfare agent
– Yellow Rain

Fungi Common in Sick Building
Syndrome
Alternaria Acremonium Epicoccum
Trichoderma Stachybotrys Penicillium

Top Twenty Symptoms (N=195)
Headache 38 45
Intense Fatigue 21 83
Spaciness 24 56
Memory Problem s 26 57
Nasal Sym ptom s 32 56
Throat Discomfort 31 34
Coughing 21 47
Lightheaded 29 33
Watery eyes 29 42 One a Month
Several Times a Month
Sinus Discomfort 26 51
Once a Week
Bloating 29 31 Several Times a Week
Daily to Almost Daily
Dizziness 36 20
Insom ina 27 30
Joint Discomfort 11 45
Weak Voice 21 28
Rash 17 34
Coordination Probs 19 28
Muscle Spas m 20 31
Slurred/Word Find 23 42
Cold Intolerance 12 36
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50 60 70of 80
ImmunTox,
90 100 110LLC
120 130 140 150 10
0
1
2
3
4
5
6
Cold Into
leran ce
Mus cle S
pa sm
Ras h
Coordin
ation pro
b lems
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We ak V o
ic e
Diz zines
s
Slurred /W
ord Find
ing
Bloati ng
Ins omnia
J oint Dis
c omfort
Lighthea
de d
Means
Throat d
is co mfor
t
Wa tery E
es y
Property of ImmunTox, LLC

Sinus D
isc omfort
Coughin
g
Spa cine
ss
Mem ory
Problem
s
Nas al S
ympto ms
Headac h
e
156 Exposed Patient’s Compared to 28 Referent Patient’s
Fatigu e
Exposed
Compared Symptom Profiles
Non Exposed
11
Historical Perspective
• Spring 1998
– CDC MMWR 9 cases
hemorrhagic pneumonitis in Infants
• Stachybotrys chartarum found in lungs
– Stachybotrys chartarum (atra) present
in homes
– In National Survey Ruth Etzel, MD, Ph.D.
finds 115 cases of hemosiderosis associated
with mold exposure (mortality approx. 15%)

Stachybotrys chartarum
• Isolated from lung of a child with
pulmonary hemosiderosis
– Strain JS5106
– Stachyrase A
• New Chymotrypsin-like Serine proteinase
• Cleaves major protease inhibitors,
• Several active peptides, and
• Collagen
• All above found in the lung
– Infection and Immunity, Jan 2002, p 419-423

Immune Effects
• Increased B-cells
• Excessive T-cell activation
– CD3+CD26+(TA1) excess
– CD3+HLA-DR+ excess
• Suppressor cell activation
– CD8+CD38+ excess
– CD8+HLA-DR+ excess
– Increased Interleuken 2 Receptors on T-cells

Immune Activation Effects
Exposed
Exposed Patients
Patients to
to Normal
Normal Ranges
Ranges for
for Activation
Activation Markers
Markers
50.000
50.000
45.000
45.000
40.000
40.000
35.000
35.000
30.000 Exposed
Exposed Mean
Mean
30.000
Min Normal
Min Normal
25.000
25.000
Mean
Mean Normal
Normal
20.000
20.000 Max
Max Normal
Normal
15.000
15.000
10.000
10.000
5.000
5.000
0.000
0.000
RR+
RR+
22))
++
lss)
++
+
+
)
388
266
el ll
I LL
D3
D2
Ce
--DD
--DD
++((I
CCD
CCD
BB C
LAA
LAA
255
HHL
HHL
8++
D2
++((
33++
D8
CCD
CD
3++
8++
2200
CCDD
D3
D8
%%C
CD
CD
CCDD
5++
%%C
%%C
5
%%
CDD
%%
C
%%

Immune Depression Effects
%CD3-CD16+CD56+ Natural %CD8+CD11b+ Human
Killer Cells as % Tot Lymph Leuko Compliment Receptors
20.0 45.0
20 45
18 40
16 35
14 12.5
30 25.0
12
25
10 7.7 20
8
5.0 15
6
10 5.0 5.3
4
5
2
0
0
Low Exposed Mean High Low Exposed Mean High
Normal Mean Normal Normal Normal Mean Normal Normal

Immune Suppression
Lymph Stimulation by Concanavillin Stimulation
Phytohemagglutinin (PHA)
400
200 300
150
200
100
100
50
0
Low Normal Exposed Mean Normal High Normal
0
Low Nor ma l Expose d Me a n Me a n Nor ma l High Nor ma l
Mean

Immune Effects
• Increased markers of autoimmunity
– Anti CNS myelin antibodies
– Anti PNS myelin antibodies
– Anti smooth muscle antibodies
– Anti nuclear antibodies

Immune Effects (n=195)
Autoimmune
Autoimmune Antibodies
Antibodies Patients
Patientsw/
w/Pos
PosAutoantibodies
Autoantibodiesof
of195
195Total
TotalPatients
Patients
120
120 40
40
100
100 Pts 35
35
Pts at
at 256
256
80 Pts at 128 30
30
80 Pts at 128 Pts
25 Pts atat40
40
60
60 Pts
Pts at
at 64
64 25
Pts
Pts atat80
80
40 Pts at 32 20
20
40 Pts at 32 Pts
Pts atat160
160
Pts
Pts at
at 16
16 15
15
20 Pts
20 Pts atat320
320
10
10
00
CNS CNS CNA Periph Periph Periph 55
CNS CNS CNA Periph Periph Periph
Myelin
Myelin Myelin
Myelin Myelin
Myelin Nerve
Nerve Nerve
Nerve Nerve
Nerve 00
IgG IgA IgM IgG IgA IgM Anti-nuclear
IgG IgA IgM IgG IgA IgM Anti-nuclearAb
Ab Anti-Smooth
Anti-SmoothAb
Ab

Factors affecting respirability
• Particulate size
– Greater than 5
microns
• Non-respirable
• Except if aspect ratio >
1:3
– 5 microns to 0.005
micron
• Considered respirable
• Reaches alveoli
• Passive filtration is
useless

Pulmonary Effects of Spores
Smokers and Nonsmokers
45
40 41
35
35
30 31
29 29
25
20
15
14
10
5
0
Critical V Severe Severe Moderate Mild Normal
<30 30 - 45 46 - 55 56 - 65 66 - 80 >80
Degree of Small Airway Obstruction Percentage of Predicted FEF 75%

Pre and Post Bronchodilator FEF 75%
Combined Smokers and Non-Smokers
80
70
60
50
40
30
20
10
0
Critical Very severe Severe Moderate Mild
Pred Pre FEF75% Pred post FEF75%

Pre Dilator Degree of Severity
Smokers vs. Nonsmokers
25
24 24 25
23
20 21
15
17
14
10
10
5 8
4 4 2
0
Critical V Severe Severe Moderate Mild Normal
<30 30 - 45 46 - 55 56 - 65 66 - 80 >80
Nonsmokers Smokers

Mean Pre and Post FEF 75%
Nonsmokers vs. Smokers
80
60
40 Nonsmokers
Smokers
20
0
Pre FEF75% Post FEF75%

Pre and Post FEF 75% by Smoke Pack
Years
70
60
50
40
30
20
10
0
> 20 Years 11 to 20 6 to 10 1 to 5
Pred Pre FEF75% Pred post FEF75%

Pulmonary Mycotoxicosis
• Pulmonary Nodules
– Associated with intense chest
pain
– Aspergillus common, but not
only cause
– Presence across fissure line
implies “kissing” lesions
reflecting exodigestion—
secretion of digestive
enzymes
– Antifungals effective, but often
induce toxicity (liver, bone
marrow, kidneys vulnerable)

Symptom Profiles – Dr. Crago
Low in energy
Headaches
Remembering things
Trouble conc
Worrying
Blank Mind
Double-check
Feeling weak
Feeling tense
Feeling blocked Pt Answering 2

Pt Ansering 3
Easily annoyed Pt Answering 4
Dif making decisions
Trouble falling asleep
Feeling blue
Slowly for correctness
Nervousness
Muscle soreness
Hot/cold spells
Tingling
Loss sexual interest
0 3 6 9 12 15 18 21 24

Kilburn Neurotox Data
• The following data is Dr. K. Kilburn’s
analysis of 43 mycotoxin exposed patients
and 202 non-exposed referent subjects as
precent of predicted (PFTs), means,
Standard Deviation (SD), and P values by
analysis of variance

Demographic Data
43 exposed Mean +/_ SD 202 referent Mean +/-SD P value
Age 48.2 +/_ 13.9 46.6 +/- 20.6 0.624

(years)
Education (years) 15 +/- 2.6 12.9 +/- 2.3 0.0001

Reaction Times (ms)
Simple 104.0 +/_ 6.6 99.9 +/- 3.7 0.0001
Choice 102.6 +/- 6.6 100.0 +/- 3.7 0.0001
Reaction Tim e
104
102
100
98
96
Simple Reaction Choice Reaction
Time (ms) Time (ms)
P=0.0001 P=0.0001

Balance Sway Speed (cm/sec)
Eyes Opened 140.5 +/_ 49.9 100.2 +/- 2.5 0.0001
Eyes Closed 168.7 +/- 102.7 103.1 +/- 26.7 0.0001
200
150
100
50
0
Eyes Opened Eyes Closed
P=0.0001 P=0.0001

Blinking Reflex Latency R-1 (ms)
Right 113.9 +/_ 10.8 99.4 +/- 14.6 0.0001
Left 115.1 +/- 11.4 96.4 +/- 13.2 0.0001
120
115
110
105
100 4 3 EXPOSED M EAN
95 2 0 2 REFERENT M EAN
90
85
Right Lef t
P=0.0001 P=0.0001

Color Perception Score
Right 69.6 +/_ 40.8 102.6 +/- 51.1 0.0001
Left 75.8 +/- 48.8 102.6 +/- 51.1 0.002
120
100
80
60 4 3 EXPOSED MEAN
40 2 0 2 R EFERENT MEAN
20
0
Right Left P=0.002
P=0.0001

Visual Performance
Right 87.5 +/_ 23.9 100.0 +/- 22.8 0.006
Left 90.0 +/- 20.9 101.1 +/- 21.7 0.008
105
100
95
43 EXPOSED MEAN
90
202 REFERENT MEAN
85
80
Right Left
P=0.006 P=0.008

Grip Strength (lbs)
43 exposed Mean +/-SD 202 referent Mean +/-SD P value
Right 91.6 +/_20.0 99.3 +/- 17.5 0.012
Left 86.8 +/_ 22.5 99.1 +/- 17.5 0.0001
100
98
96
94
92
90
43 EXP OS ED MEAN
88 202 REFERENT MEAN
86
84
82
80
Right P=0.012 Lef t
P=0.0001

Verbal Recall
Immediate 76.7 +/_ 23.4 99.8 +/- 31.1 0.0001

Delayed 68.7 +/- 38.2 99.9 +/- 41.3 0.0001
100
90
80
70
60
50 43 EXPOSED MEAN
40 202 REFERENT MEAN
30
20
10
0
Immediate Delayed
P=0.0001 P=0.0001

Information, Pictures, Similarities
Information 88.9 +/_ 34.7 101.5 +/_ 39.4 0.052
Picture Completion 78.8 +/_ 30.7 99.3 +/_ 32.2 0.0002
Similarities 95.5 +/_ 25.8 98.1 +/_ 41.2 0.691
120
100
80
60
40
20
0
Information Picture Simularities
P=0.052 Completion P=0.691
P=0.0002

Profile of Mood States
43 exposed 202 referent P value
Mean +/_ SD Mean +/-SD
Score 59.7 +/- 41.8 21.0+/- 31.6 0.0001
Tension 16.1 +/- 8.5 9.1+/- 5.8 0.0001
Depression 14.6 +/- 12.2 8.1+/- 9.3 0.0001
Anger 13.4 +/- 10.1 8.3+/- 7.9 0.0003
Fatigue 15.2 +/- 7.3 7.6+/- 6.1 0.0001
Vigor 10.3 18.3 0.0001

+/- 6.7 +/- 6.3
Confusion 12.9+/- 6.9 6.1+/- 4.5 0.0001

Mechanism for Neurotoxic
Effects of Micotoxins
• Fumonisin B1 (FB1) enzyme inhibitor
blocking biosynthesis of sphingolipids and
accumulations of sphiganine
• Resultant brain hemorrhage
(leukoencephalomalacia) (Wilson, 1992)
• Sterigmatocystin interferes with
sphyngosine metabolism: an essential
component of neuronal membranes
• Tremulo- and Vomitoxins are mycotoxins

Fungal Effects
• Neurological Component
– Cognitive dysfunction • Attention Deficit Disorders
• Confusion and – Suspect intrusion of
spaciness paroxysmal increased
• Memory loss brain wave frequency
and activity
• Spatial disorientation
• Dyslexia
• Seizures

Mycotoxin Effects
• Neurological • Pulmonary
– Behavioral changes – Small airway
– Cognitive changes obstruction
– Ataxia – Hypersensitivity
– Convulsions pneumonitis
– Multiple sclerosis – Hemorrhagic
pneumonitis
– Optic neuritis
• Cardiovascular
• Immune suppression
– Vascular permeability
• Interferes with protein
synthesis

Mycotoxin Intoxication
• Acute and Chronic Mycotoxicosis can cause
serious and sometimes multisystem diseases
with severe and sometime fatal outcomes.
• The possibility of Mycotoxin intoxication should
be considered when an acute disease occurs in
several persons when there is no evidence of
infection with a known etiologic agent, and no
improvement in the clinical picture follows
treatment.

Assays for Trichothecenes
• Protein Translation assay in Airborne Particulates
– Rapid, inexpensive assay using firefly luciferase has
been developed to detect and quantify the fungus-
derived toxicity of airborne particulates
– Entire testing procedure can be accomplished in
2 hours
– Authors demonstrated T-2 toxin, Satratoxin, and DON
inhibit luciferase mRNA in a cell free rabbit
reticulocyte system
– Results are highly reproducible
– Applied and Environmental Microbiology, Jan, 1999, p. 88-94

Symptoms of Sick Building
Syndrome
• Head, Eyes, Nose, • Gastrointestinal
Throat – Nausea & Vomiting
– Headache
– Diarrhea
– Dizziness
– Constipation
– Dry eyes
– Watery eyes • Respiratory
– Itchy eyes – Cold & flu symptoms
– Epistaxis – Cough
– Stuffy nose
– Rhinorrhea
– Alopecia

Fungal Exposure & Health
Effects
• Pathogenic infections
• Allergic illnesses
from fungi
• Hypersensitivity
pneumonitis

Conclusion
• Acute and Chronic Mycotoxicosis can cause
serious and sometimes multisystem diseases
with severe and sometime fatal outcomes
• The possibility of Mycotoxin intoxication should
be considered when an acute disease occurs in
several persons when there is no evidence of
infection with a known etiologic agent, and no
improvement in the clinical picture follows
treatment

Afterword:
Be Not Afraid
To Look For
Zebras
--Anon

Molds, Mycotoxins and
Human Health
Michael R. Gray, MD, MPH, CIME
Internal and Occupational Medicine
Emergency Medicine & Toxicology
In collaboration with
Kaye Kilburn, MD
Internal Medicine and Neurotoxicology
Robert Crago, Ph.D.
Psychology, Neurophysiology and
Neurotherapy
ImmunTox, LLC
PO Box 2050 Benson, Arizona 85602

Drs Michael Gray and Kaye Kilburn Molds Mycotoxins and Public Health

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Molds, Mycotoxins and

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Alternaria Acremonium Epicoccum

Trichoderma Stachybotrys Penicillium

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Property of ImmunTox, LLC

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Degree of Small Airway Obstruction Percentage of Predicted FEF 75%

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Pred Pre FEF75% Pred post FEF75%

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Pred Pre FEF75% Pred post FEF75%

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Feeling blocked Pt Answering 2

Dif making decisions

Trouble falling asleep

Slowly for correctness

Loss sexual interest

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Age 48.2 +/_ 13.9 46.6 +/- 20.6 0.624

Education (years) 15 +/- 2.6 12.9 +/- 2.3 0.0001

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Eyes Closed 168.7 +/- 102.7 103.1 +/- 26.7 0.0001

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Immediate 76.7 +/_ 23.4 99.8 +/- 31.1 0.0001

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Information 88.9 +/_ 34.7 101.5 +/_ 39.4 0.052

Picture Completion 78.8 +/_ 30.7 99.3 +/_ 32.2 0.0002

Similarities 95.5 +/_ 25.8 98.1 +/_ 41.2 0.691

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Depression 14.6 +/- 12.2 8.1+/- 9.3 0.0001

Anger 13.4 +/- 10.1 8.3+/- 7.9 0.0003

Fatigue 15.2 +/- 7.3 7.6+/- 6.1 0.0001

Vigor 10.3 18.3 0.0001

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