Seminar Topic:
Mechanisms of Human Toxicity
Developed by Dr. Debra L. Forman
Senior Regional Toxicologist
Hazardous Waste Management Division
U.S. EPA, Region III
U.S. ENVIRONMENTAL PROTECTION AGENCY
REGION M
222
-------
Seminar Topic:
Mechanisms of Human Toxicity
Developed by Dr. Debra L. Forman
Senior Regional Toxicologist
Hazardous Waste Management Division
U.S. EPA, Region III
-------
Seminar: Mechanisms of Human Toxicity*
Syllabus
Time
Topic
Instructor
8:30 a.m.
Principles of Toxicology
Dr. Forman
9:45 a.m.
Toxicokinetics
Dr. Forman
10:30 a.m.
Break
10:45 a.m.
Mechanisms of Carcinogenicity
Dr. Forman
12:00 p.m.
Lunch
1:00 p.m.
The Hepatic System
Toxicology of the Liver
Dr. Forman
2:30 p.m.
The Hematopoeitic System
Toxicology of the Blood
Dr. Forman
3:45 p.m.
Break
4:00 p.m.
The Cardiovascular System
Toxicology of the Heart and Circulation
Dr. Forman
5:00 p.m.
Discussion and Review
Dr. Forman
'Developed and presented by Dr. Debra L Forman, Senior Regional Toxicologist, Hazardous Waste
Management Division, U.S. EPA, Region III, 841 Chestnut Street, Philadelphia, PA. 19107
-------
Mechanisms of Human Toxicity
Basic Principles of Toxicology
Instructor Dr. Forman
I. Introduction
A. What is Toxicology?
B. Mechanisms of Action
1. Direct
2. Indirect
C. Classification of Toxic Compounds
D. Poisons
II. Exposure
A. Routes of Entry
B. Duration and Frequency
1. Acute
2. Subchronic
3. Chronic
C. Concept of Threshold
D. Bioconcentration and Bioaccumulation
III. Dose Response
A. Cause and Effect is equivalent to Dose and Response
B. Relative potency, mechanisms of action
C. Distribution among responders
1. Hypersensitive
2. Resistant
3. Mean Dose
D. Measurement of the Response
1. Biochemical
2. Physiological
IV. Risk Assessment
A. Development of the RfD from dose response data
1. NOAEL No Observable Adverse Effect Level
2. LOAEL Lowest Observable Adverse Effect Level
3. Safety/Uncertainty factors
4. Modifying factors, based on professional
judgement.
B. The Hazard Index
-------
Toxicology is the study of the adverse effects
of substances on biological systems.
-------
Mechanisms of Action
Indirect:
CC1
Lipid peroxidation of cell membranes
and proteins leads to necrosis.
CC14 CClj +ci-
CC1. +Q2 cytochrome phase ( QC^QQ.
or: CCI4 CI 3C:
(carbene)
-------
Direct:
Cadmium
(Cd)
P04 Ca^
-~Lumen ~
1 f
glucose
>r
AA's
Depressed reabsorption
of amino acids, glucose
Ca+\ PO,.
Proximal Tubule
-------
Classification of Toxicants
Target Organ
Use
Source
Effects
Physical State
Chemistry
Poisoning Potential
occupational vs. environmental
\ /
biology and chemistry
-------
Toxicity Rating Chart (Poisoning Potential)
Probable Lethal Oral Dose for Humans
Rating
Dosage
(mg/kg)
For Average Adult
1. Practically nontoxic
2. Slightly toxic
3. Moderately toxic
4. Very toxic
5. Extremely toxic
6. Supertoxic
>15,000
5,000-15,000
500-5,000
50-500
5-50
<5
More than 1 quart
Between pint and quart
Between ounce and pint
Between tsp. and ounce
Between 7 drops and tsp.
A taste (< 7 drops)
-------
Approximate Acute LD50's of
Some Representative Chemical Agents
Agent mg/kg
Ethyl Alcohol 10,000
Elemental Selenium 6,700
Sodium Chloride (table salt) 4,000
Ferrous Sulfate 1,500
Pentobarbital Sodium 150
Sodium Selenite 7
Nicotine 1
d-Turbocurarine 0.5
Dioxin 0.001
Botulinum toxin 0.00001
-------
"All substances are poisons;
there is none which is not a
poison.
The right dose differentiates
a poison and a remedy."
Paracelsus (1493-1541)
-------
Airborne
Environmental Exposure
Pathways
I
Mobile Sources
(cars, etc.)
Other
Household
Sources
Household
Dust & Soil
yr
Industrial
Sources
*
Water
Soil
0*
Fishii
Household
Dust & Soil
Fishing
Drinking
Water
Swimming
I® li
Diet
Inhalation
Ingestion
Dermal Contact
Systemic Circulati
Peripheral tissues
I
X
Capillary Transfer
Utilization by Cells
Stomach
Small Intestine
Large Intestine
-------
Duration of Exposure
Acute
Subchronic
Chronic
-------
Toxicity depends on:
Dose Exposure
Threshold: Concentration
which elicits a systemic
toxic effect.
-------
Concept of Threshold
threshold for
gross response
Chronic dose
accumulation of toxicant
\x Residual injury
threshold for
rFsidual injury
Dose
-------
Bioconcentration of Aroclor 1254
by Catfish
60,000
¦0 50,000
CB
LL
g 40,000
03
§ 30,000
O
c
o
§ 20,000
m
10,000
7 14 28 56 77
Water Exposure Time (days)
-------
Bioaccumulation in the Food Chain
Marsh hawk
Short-aarad owl
Tarraatrial
invert abratw
-------
The Dose-Response Relationship
( Log Dose
-------
Characteristics of
the Log Dose-Response Relationship
C\
-------
80
<3)
£ 60
a
CO
CD 40
CC
20
Log Dose
2k
O
c
0
3
CT
0)
Log Dose
[% Frequency = 100
-------
Derivation of RfD
from Dose Response
rt
I I I Mill I I I II
20 50 100 200 400 800 2000
Log Dose
NOAEL LOAEL
NOAEL
UFxMF
= RfD
UF intraspecies = 10
interspecies = 10
acute to chronic = 10
MF = professional judgement
-------
Approaches to the Measurement
of Toxic Effects
Biochemical Physiological
Liver Kidney Lung Heart
/ \ / \ / v. /\
SGPT, SGOT Maltase, trehalase irritation bronchospasm HR ECG
-------
Hazard Quotient Derivation
Actual Dose*
Theoretical Dose**
* calculated intake
** allowable RfD
-------
0.9
Hazard Index Calculation
Total Exposure
0.8
0.7
0.6
CD 0.5
"O
_c
-o 0-4 —
CO
N
CO 0.3
0.2
0.1
0
~ Cyanide (CN)
H Mercury (Hg)
¦ Hydrogen Sulfide (H^S)
Drinking Water Soil Ingestion
Inhalation of
Particulates
Dermal Contact
Exposure Pathway
-------
Mechanisms of Human Toxicity
Toxicokinetics
Instructor: Dr. Forman
I. Introduction
A. Exposure phase
B. Toxicokinetic phase
C. Toxicodynamic phase
II. Basic Concepts of Biological Transport
A. Cell Theory
B. Passive transport
1. simple diffusion
2. facilitated diffusion
3. filtration
C. Active transport
III. Basic Concepts of Toxicokinetics
A. Absorbtion
1. Gl tract
2. Skin
3. Lung
B. Distribution
1. Body Water compartments
2. Volume distribution
3. Storage
4. Blood-brain barrier
C. Excretion
1. Urinary
2. Biliary
3. Lung
D. Biotransformation
1. Sites of action
a. Liver
(1) microsomal enzymes
(2) biosynthetic enzymes
b. Other organs: kidney, lung
2. Rates of action
3. Bioactivation
-------
The Toxic Process
-------
Cell Membrane Structure
Phosphatidylcholine
x —i
I
o
0=P—or
I
o
. . ,1 "
CH,—CH —CH2
0
1
0
1
c=
1
0C=0
1
1
ch2
1
1
ch2
1
1
CH,
|
1
CHj
|
1
ch2
1
1
CH2
1
1
ch2
1
1
CH2
1
1
CH2
I
1
CH2
1
1
CHj
I
1
CH2
1
1
CHj
|
1
CH2
1
CHj
I
I
CH
II
1
CHj
1
II
CH
|
1
ch2
|
1
CH2
|
1
ch2
|
1
CH,
I
1
ch2
1
1
CHj
1
1
ch2
1
1
CHj
1
1
ch2
1
1
CHj
|
1
ch2
1
1
CHj
I
ch2
1
1
CHj
|
CHj
1
CHj
Polar head
Nonpolar tails
-------
Simple Diffusion
a. Lipid Soluble
b. Aqueous Soluble
Thickness
i
[Toxicant]
Flow
(D
Q
[Toxicant]
-------
Ionization of a Compound
COOH
(hydrophobic)
Benzoic Acid
coo©
(hydrophilic)
% Unionized 99.9
99
90
50
10
_L
0.1
pH
(hydrophilic)
% Unionized
0.1
I L
Aniline
10
I
so
(hydrophobic)
90
_L
99
J pH
-------
Other Modes of Transport
passive
active
Facilitated Diffusion
Filtration
Active Transport
Pinocytosis/Phagocytosis
-------
Carrier-Mediated Transport
• Facilitated diffusion
• Active transport
Transport Region
2
V/ \V
o g
EH6
0^,0
5
(6)
-------
Pinocytosis/Phagocytosis
w •• 1
• • • •
©
-------
Absorption in the GI Tract
• Ionization/passive diffusion
organic acids and bases
• Active transport
metals (ex: thallium, lead)
-------
Ionization in the GI Tract
PLASMA
pH 3 7.4
GASTRIC
JUICE
pH« 1.4
[l] HA
[tooo]
«\\\\\\\\\\\\\\\\\\\\\\\\v
LIPID MUCOSAL
xxx BARRIER
[l] HA < , A' [o.OOl]
TOTAL
(HA+A")
1001
1.001
HA . A" + H*
Nonionized Ionized
WEAK ACID, p/r*'4.4
-------
Respiratory System
-------
Compartmentalization of Total Body Water
Plasma (7.9%)
Interstitial (28.9%)
Extracellular
Intracellular (63.2%)
-------
Distribution of Toxicants
Dose
R,
Elimination
A. Plasma Protiens
B. Fat
C. Bone
-------
Plasma Proteins
/»> ++ —j + +
Ca , Cu , Zn
Bilirubin
Uric acid
Vitamin C
Adenosine
Tetracyclines
Chloramphenicol
Digitonin
Fatty acids
Suramin
Quinocrine
Penicillin
Salicylate
Para-aminosalicylate
Sulfonamides
Streptomycin
Acid dyes
Phenol red
Histamine
Triiodothyronine
Thyroxine
Barbiturates
Fe++ Steroid hormones
(Transferrin) (Transcortin)
Vitamin
Sialic acid
Thyroxine
Zn++, lipids
Cholesterol
Vitamins A, K, D, E
I
i
Cu++ (Ceruloplasmin)
Lithium carmine
Hemoglobin
(Haptoglobin)
-------
Toxicant Distribution
-------
Toxicant Elimination
10mg/kg
/s\
if 6
toxic levels
J 4
v subtoxic levels
f z\\,
i i i
"i\ "i —
Hours
-------
Biotransformation:
Phase I Reactions
Enzyme System
^ Predominantly
cytochrome p450
~
Lipophobic
Functional
Product
Group
Epoxide
A
Hydroxide
"OH
Sulfhydryl
SH
Hydroxylamine
H
-N-OH
Lipophilic Toxicant
passes through
cell membranes
-------
Biotransformation:
Phase II Reactions
Phase I
Reaction Product
+
Natural (endogenous)
Conjugating Agent
T
Conjugation Product
• Higher Polarity
• Greater Water Solubility
• Easily eliminated
-------
Absorption, Distribution and Excretion of Toxicants
Ingestion
Intraperitoneal
Subcutaneous
Intramuscular
¥———...J
l Blood ar
Dermal
and lymph
Feces
1
Kidney
Bladder
Urine
Lung
Alveoli
Expired air
Extracellular
fluid
Secretory
structures
Fat
Organs
Soft
tissue
Bone
Secretions
-------
Mechanisms of Human Toxicity
Mechanisms of Carcinogenicity
Instructor Dr. Forman
I. Introduction
A. Definition of Problem
1. Background
2. Incidence
3. Trends
B. Definition of Terms
1. End points
a. Tumor, tumorigenesis
b. Neoplasm, neoplastic, preneoplastic,
metastasis
2. Processes
a. Cancer, carcinogenesis, carcinogen
b Mutation, mutagenesis, mutagen
c. Genetic, epigenetic, genotoxic
3. Functional Units
a. Structural Genes: expressors, Oncogenes,
proto-oncogenes
b. Nonstructural Genes: suppressors
II. DNA as the Critical Target
A. Cell Structure (Review)
B. DNA Structure
1. Nucleotides
2. Genes
3. Chromosomes
C. Protein Synthesis
1. Translation
2. Transcription
3. Gene expression
D. Types of Mutations
1. Base changes (point)
2. Deletion/insertion (frameshift)
3. Chromosomal aberrations (rearrangements)
a. Duplication (recessive/dominant)
b. Nondisjunction
c. Translocation
-------
Mechanisms of Human Toxicity
Mechanisms of Carcinogenicity (Continued)
Instructor Dr. Forman
III. Theory of Carcinogenesis
A. 2-Stage Model
B. Multistage Model
IV. Mechanisms of Carcinogenesis
A. Chemical
B. Radiation
C. Virus
-------
Cancer is the result of one or more
persistent changes in the control of
genetic expression.
-------
Tabt* 1.1 Mortality far Laading Causa* of Daatfi, United Stataa—1983
Rank
Cause of Deatn
Numoer ol Deaths
Death Rate oer
'00.000 Population
Percent Qf
"otai Deairs
AJi Causes
2.019.201
743.4
100.0
i
Heart diseases
770.345
276.2
38 2
2
Cancer
442.986
169.0
21 9
3.
Cerebrovascular diseases
155.598
54.1
7 7
-J.
Accidents
92.488
36.5
-i 5
5.
Pneumonia ana influenza
55.854
19.2
2 8
6.
Chronic obstructive
'ung diseases
45.814
168
2.3
7
Diabetes meMtus
36.246
13 4
1 3
8.
Suicide
28.295
11 0
1 4
9.
Cirrhosis of liver
27 266
11 1
i 4
10.
Arteriosclerosis
26.371
8.6
' 3
11.
Homicide
20.191
78
1 0
12.
Diseases of nfancy'
19.310
9.0
1 0
Aaaotefl counesy of tne Amercan Cancer Society from Silveroerg. 1986 Used by oermisstoa
-------
Figure 18.—Relative Risks of Esophageal Cancer
in Relation to the Daily Consumption of
Alcohol and Tobacco
0-40g 41-00 g 81+ g-
Alcohol
Not*: Tha risk it 44.4 timaa graatar for individuals consuming 20 g or mora of
tobacco and 80 g or mora ot aiconot oar day (upoar ngnt Mock) tnan for in-
dhnduaia consuming littla or nona ot attnar dfug (lowar laft block). Ona ounca of
•tftyt alcorto* is approumataiy 23.4 grams, mus 40 grams is 1.7 oz. or aeproi-
imaiaty aqunatani 10 3 drinks.
SOURCE. National Instituta on Aleonoi adum »nd Alcoholism <2i7).
-------
6000
o) 5000
0
c/>
5 4000
1
CO
2 3000
Z
2000
z 1000
0 10 20 30 40 50 60 70 80
Age at Diagnosis
Figure 1.4 Cancer is a disease of the old—as illustrated
by the absolute numbers of new cases in 1978. {Source:
National Cancer Institute. SEER program)
-------
trillion
billion
million ¦
According to some-views tms isa
' period of variaDie length during
wtven a running cattle with immune
. system is a "draw"Selection"
, may produce immune-resistant
variants curing this penod.
nost dies ot cancer or
complications <
demise ot neooiasm it new oiood
.^supply is not esiaonsned
*
¦Cancer Cell is Created
Time ¦
Up to 30 or 40 Years
The generalized natural history of a neoplasm
Stages of carcinoma
development. A single initially
transformed cell begins to
proliferate, giving rise to a
preneoplastic eeO population
idyspUsiaJ which progresses
first to carcinoma in situ and
than to invash* carcinoma.
-------
Carcinogenic Processes
Cancer carcinogenesis carcinogen
Mutation
Genetic
mutagenesis mutagen
genotoxic
-primary,
ultimate or
initiator
carcinogen
-pro- or
pre- carcinogen
epigenetic
-cocarcinogen
-promotor
-solid state
carcinogen
f
-------
Carcinogenic Endpoints
• Tumor tumorigenesis
• Neoplasm neoplastic preneoplastic
• Metastasis
"7
-------
Functional Units
• Structural genes/ Expressors
• Oncogene, proto-Oncogene:
Capable of expressing
malignancy.
• Non-structural genes/ Supressors
• Regulate the inherited
predisposition to cancer.
-------
DNA is the critical target.
-------
Evidence That Carcinogens Initiate
Cancer via Alterations in DNA
• 90% of all carcinogens are mutagens
• Defects in DNA repair result in increased
carcinogenesis
3
• Chromosomal deletions predispose cancer
• Ultimate carcinogens form DNA adducts
• Some chemical carcinogens have base
specificity
-------
The Cell
smooth
endoplasmic
reticulum
Goigi complex
riDosomes
cytooiasmic
vesicle
mitochondrion
pore complex
rough
endoplasmic
reticulum
^secretory vesica
cytoplasm
nuclear
envelope
nucleolus
plasma
mem crane
Figure 2.1 The parts of a "typical" animal call
-------
Structure of DNA
Phosphoric Nitrogenous Base
Sugar Acid
Deoxyribose
• 0 D
Adenine Thymine
8
Gnanin Cytosine
r
u
A
Nucleotide
II
K
n
n
A
Double Helix
-------
ONA
NUCLEUS
(rflNA)
H | rflNAA
¦ / aiMmotM
P j
CYTOPLASM
ONA
growing
oraram cna
tflNAmotaeuin
cany arnno i
ton
Figure 2.13 The synthesis of protein by the ceM. The genetic code carried in the ONA of the nucleus
is "transcnbed" in the nucleus into messenger RNA (mRNA). Then mRNA moves to a nbosome m the
cytoplasm where ammo acids earned on transfer RNA (tflNA) molecules are Bonded together to form
protetns. The seouence of ammo acids is determined by the sepuence of nucleotide bases m the
mRNA. each triplet" calling for a matching tflNA molecule, each of which carries a specific amino
acid.
-------
glycoprotein
protein
glycoprotein
/
protein
protein
- amino aeid
cham
OOOO " carfiofrydrate
unit
Figure Z2 The fluid mosaic mode) of the cell membrane. Integral proteins form part of the mem-
brane. being deeply imbedded in the lipid bilayer. Other proteins, called penoherat protwfs. are
more loosely bound to the outer or inner membrane surfaces.
-------
Mutations
1 2 3
point mutation frameshifts
-------
regulator gene
\
regulator i
operator oft"
ooeron
gene a gene o gene c
no transection
ooerator "on"
^ Regulator I ^ ^ gene a gene d gene c
1 1 1 1 i ' 1 i ¦ ' 1 1 '
mRNAs transcnoed
inducer
^ regulator il ^ ^ gene a ^ gene e ^ gene f
mfiNAa transcnbed
gene d gene e
no tranacnpoon
ngure 2.14 Regulation of tranacnption. In certain simple organisms, gene expression is regulated by
me ooeron mecftanwn illustrated here. One Kind of regulator produces an active repressor (e) mat
must be inactivated by an "inducer" substance before tne ooerator can be switched "on" (b). in
anotner kind of regulator system, me regulator gene produces an inactive repressor substance (e)
mat meat be converted to me active form by a repressing substance if me ooeron is to be turned off" (d).
(
-------
Steps in Carcinogenesis
A. Initiation
( 1st genetic event
hyperplasia?
B. Promotion
2nd genetic event
tumor stem cell
differentiation
(g)
multiplication
carcinoma
in situ
Invasion
I'
Metastasis
-------
The 2-Stage Model of Carcinogenesis
AGENT CEli MORPHOLOGY CELL CHARACTERISIIC5
CARCINOGEN
INITIATOR
PROMOTER
NORMAL CELL
altered
DNA
INITIATED CELL
gene
activation
DIVIDING TUMOR
CELLS
I
Phenotype of normal cell.
Genotype of tumor ceil,
but repressed.
Restraining influence of
differentiated cell system
overcome: immunological,
hormonal, etc.
VISIBLE TUMOR
cakm-
SMUtS
innarm
iKONflHl
CAKM-
6HHIS
wowtjoi
COlVttSiOl
Nttiurm
NMMAl
CEli
»»
ill
3
r
IIITUTU
CUL
•
I
i
i
a
s
4
-v
1 -
J
2
•
i 1
W
J 1
Jl s!
ils|i
2^ li
OQMAIT
TUMI
cai
4
m
a
a
1 |
51 Js «**!
3 ^ 1-
6MWM
lUMOt
\l
-------
THE BASELINE RISK ASSESSMENT:
ASSESSING TOXICITY
UNIT CANCER RISK
R.-*1 — in
Si; JO7S/60
-------
THE BASELINE RISK ASSESSMENT
ASSESSING TOXICITY
EPA RISK ASSESSMENT GUIDELINES
WEIGHT OF EVIDENCE CLASSIFICATION
GROUP A HUMAN CARCINOGEN
GROUP B PROBABLE HUMAN CARCINOGEN
GROUP C POSSIBLE HUMAN CARCINOGEN
GROUP D 1NOTCLASSIFIABLE
PHiiS
lief
/X- -
V; fi: '* "
% f « >1 i »<:
x > - . •: ft- *• •
Aftv ¦' • & ,¦¦ ¦
"J-17
SF-J076/61
-------
Chemical Carcinogenesis
\
Activation
\
A Karat tons in-
ONA
ElaetrephUie
Raactam .
!»~»
ANA
Otna* Can
Components
Biochamicat CaN
Laaton(a) Preparation
». Initiation
CarcinoQan
Livar - Raaiatant
Hapatocyta
- Othar Typaa (?)
Skin • A Narad
ummwrnmaon
-------
Organ Specificities of Some Carcinogens
Ethylnitrosourea
Formaldehyde
Methoxy-m-Phenylene, Diamine
Vinyl Chloride
2-Acetylaminofluorene
Bis(chloromethyl) Ether
2-N aphthy lamine
Glioma, Astrocytoma
Nasopharyngeal Carcinoma
Lymphoma
Hepatic Angiosarcoma
Hepatocellular Carcinoma
Oat Cell Carcinoma of the Lung
Bladder Carcinoma
-------
RADON (Rn)
• 25 known isotopes of Radon (Rn)
• 3 occur naturally in the environment
219Rn, 220Rn and 222Rn
• Each arises as a natural radioactive
decay product of Uranium (U) or
Thorium (Th).
-------
RADIOACTIVE DECAY OF ^Rn
-------
DISTANCE RANGE OF Rn PROGENY
GEL
7-15/im
MUCUS
LAYER
25-50/im
CILIATED
SECRETORY
-------
Viral Carcinogenesis
Call transformation and
progeny virus release
% e®
-------
Mechanisms of Human Toxicity
The Hepatic System
Toxicology of the Liver
Instructor: Dr. Forman
I. Introduction
A. Anatomy
1. microanatomy
2. vascular anatomy
B. Function Anatomy: The Liver Lobule
1. classical liver lobule
2. portal lobule
3. hepatic acinus
II. General Classes of Liver Injury
A. Metabolic
1. direct hepatocyte damage
a. necrosis
b. cirrhosis
2. accumulation of lipids/phospholipids
a. steatosis
b. crystalloid inclusions
B. Secretion of Bile/Cholestatic injury
1. bilirubin transport
2. conjugation
3. bile stasis due to duct damage
III. Specific Types of Liver Injury
A. Necrosis
1. The Necrogenic Process
a. toxicokinetics
b. metabolic activation/deactivation
c. formation of necrotic form
d. interaction with cellular constituents
2. Mechanism of Necrogenic Action
a. P450 enzyme system
b. Phase I/Phase II reactions
c. inhibitors of necrosis/ability to repair
3. Necrogenic forms
a. Aflatoxin B,
b. Parathion
c. CCI4
-------
Mechanisms of Human Toxicity
The Hepatic System
Toxicology of the Liver (Continued)
Instructor: Dr. Forman
B. Steatosis
1. Lipid metabolism
a. triglycerides
b. fatty acids
c. lipoproteins
d. protein synthesis and membrane function
2. Mechanism of Steatosis
a. Lipid transport
b. fatty acid uptake
c. fatty acid synthesis
d. fatty acid oxidation
3. Ethanol Toxicity
-------
The Hepatic System
-------
Liver Anatomy
bile Ajct
lymph,
vessel
portal triad
^ixHonal tonrpk*
intracellular pace
-------
Volumetric Composition of the
Rat Liver Parenchyma41
Hepatocytes
77.8
*
1.15
Nuclei
7.6
0.50
Cytoplasm
70.2
1.13
Sinusoidal cells
6.3
Hh
0.49
Endothelial cells
2.8
0.19
Nuclei
0.44
0.08
Cytoplasm
2.36
*
0.08
Kupffer cells
2.1
0.31
Nuclei
0.39
0.07
Cytoplasm
1.71
0.07
Fat-storing cells
1.4
0.19
Nuclei
1.12
0.04
Cytoplasm
1.12
*
0.04
Spaces
15.9
0.75
Disse space
4.9
0.35
Sinusoidal lumen
10.6
£
0.45
Bile canalkuli
0.43
*
0.05
'Data from Blouin (1977). Values are percentages %
S.E.
-------
The Functional Liver Lobule
-------
Liver Injury
tumors
metabolic capabilities
I
direct
hepatocyte
damage
\
—i
accumulation
of lipids or
phospholipids
necrosis
cirrhosis
/
steatosis
(fatty liver)
metabolic
and
cholestatic
injury
lamellated
crystalloid
inclusions
secretion of bile
I
cholestatic
injury
bilirubin conjugation bile
transport stasis
and duct
damage
-------
Liver Necrosis
Envtromtmal
Hopatonocragonic
ChamtcH
Absorption
<< Activation—
Inactivation
Excration
Uiiimaie
"iecrog»fi:c
form
t
Diract ana
Indirect
infractions
-------
Necrotic Liver Injury
metabolic capabilities
I
tumors direct
hepatocyte
damage
/ \
necrosis cirrhosis
i \
zonal diffuse massive
I
1
accumulation
of lipids or
phospholipids
/ \
secretion of bile
I
begins with
disruption of
membrane
I
lipid
peroxidation
iirciiiuiaiic
/ lipid \
J metabolic
and
cholestatic
injury
steatosis
(fatty liver)
lamellated
crystalloid
inclusions
bilirubin
transport
cholestatic
injury
conjugation
bile
stasis
and duct
damage
Ca++
homeostasis
binding of
proteins
to cellular
macromolecules
-------
Necrotic Liver Injury
CeJ Injury
Ultrastructural Characteristics
Transition Point:
Point of No Return:
Necrosis:
Dilated endoplasmic reticulum
Condensed mitochondria
Condensed nuclear chromatin
Increased cell cytoplasmic volume (swelling)
Blebs on plasma membrane
Formation of apical vacuoles
Decreased numbers of polysomes
Altered cytoskeletal organization
All of the above features plus swelling (low
amplitude) and amorphous (flocculent) densities
of mitochondria
Empirically defined in the various tissues.
However, no structural or biochemical definition is
yet available
High-amplitude swelling of mitochondria
Large flocculent densities of mitochondria
Massive swelling of cell, electron lucent cytosoi
Marked clumping of nuclear chromatin
Discontinuities and vesiculation of membranes
Formation of cell fragments
Phagocytosis of cells by resident macrophages and/
or inflammatory cells
-------
Ways to Prevent Chemically Induced Liver Necrosis
Inhibition of metabolic activation
Chemical trapping of reactive metabolites
* Increasing inactivating biotransformations
Inhibition of lipid peroxidation
Modulation of reversible late stages of chemically induced liver injury
-------
P450 enzyme system = MFO
0"/
~
e'
NADPH
NADPH-P450
Reductase
(FAD, FMN)
e-
Cytochromebg
• NADPH-P450 Reductase and P450
• membrane bound
• sensitive to phospholipid environment
-------
Hepatic Biochemistry
Phase I Reactions
• oxidation
• hydroxylation
• reduction
• hydrolysis
Increased water solubility
Phase II Conjugation
• glucoronate
• glutathione
• sulfate
• acetate
Decreases biological activity
Increases water solubility
Facilitates excretion
-------
Hepatonecrogenic Chemical Reactive Metabolite
Activation Enzyme
o o
o o
cnO
O^'OCH,
Aflatosin 01
°conO
0^0^-^ OCH,
Epoxide
P-450
CiHj0\n
CjHjP
Partthbn
S :
Atomic sulfur
P-4SQ
CHCLj
a
o=c'
Phosgon
Pi&O
-------
Extent of necrogenic effect depends on:
• intensity of metabolic activation
• reactivity of product
• natural defenses—or ability to repair
-------
P450 enzyme system = MFO
. *
ev
~
~
s
e" NADPH-P450
—~ Reductase
(FAD, FMN)
/
Cytochrome b i
limit NADPH
decreases P450
add substrates with high affinity for P450
-------
Defenses and Ability to Repair
Free Radical Attack
• Scavengers
• SOD
• glutathione
• catalase
• Nutritional antioxidants
• Vitamin E
• Vitamin A-P450 inhibitor
Binding to cellular macromolecules
• DNA/RNA repair ability
-------
Mechanism of Steatosis
• inhibition of lipid transport
• increased uptake of fatty acids
• increased synthesis of fatty acids
• decreased oxidation of fatty acids
-------
Lipid Metabolism and Distribution
BLOOD
Lipoprotein
^Non-ester ified
fatty acids
Chylomicrons
A.
Adipose
tissue
Gut
Acetyl CoA
Fatty acids
*
Glycerol
Protein Synthesis
Lipid acceptor protein
Phospholipids
LIVER
-------
Ethanol Toxicity
BLOOD
Lipoprotein
p|_*Non-«sterifled
fatty acids
Chylomicrons
A.
Adipose
tissue
Gut
Acetyl CoA
Protein Synthesis
I ,
LIpM acceptor protein
fatty Acids
/
Glycerol' f \
(Triglycerides)
Phospholipids
LIVER
-------
15
COMTMt
ITMANOt
0
•
9
A
A
-------
Chronic ethanoi consumption
Increased acetaldehyde concentration
Mitochondrial
impairment
Decreased acetaldehyda
metabolism
-------
Mechanisms of Human Toxicity
The Hematopoeitic System
Toxicology of the Blood
Instructor Dr. Forman
I. Introduction
A. Plasma
B. Formed Elements
1. RBC's: Oj carrying capacity
2. WBC's: defenses and immune function
3. Platelets: clotting
C. Hemoglobin
1. Structure
2. Suitability to oxygen carrying
3. Carriage of C02
II. Toxic Responses of the Blood
A. Anemia
B. Leukemia
C. Hypoxemia/Hypoxia
III. Blood toxicants
A. Lead
1. Description and Sources
2. Toxicokinetics
3. Mechanism of Action
a. disruption of heme production
b. neurologic deficits
c. alterations in Vitamin D metabolism
d. immunosuppressive responses
B. Benzene
1. Description and Sources
2. Toxicokinetics
3. Mechanism of action
a pancytopenia
b. immunosuppression
-------
Composition of Circulating Blood
Plasma
Leukocytes
>-ci® n alf
G®<
o°e
)«_ *3®
Erythrocytes
-------
Composition of Circulating Blood
Plasma
• water
• inorganic salts
• amino acids
• vitamins
• hormones
• lipid
• clotting factors
• plasma proteins
Formed Elements
• white blood cells
(WBC's, leukocytes)
• platelets
(thrombocytes)
• red blood cells
(RBC's, erythrocytes)
-------
HB Structure and O2
Carrying Capacity of Blood
chain
[3 chain
chain
a chain
Heme group
imidazole
-021| 02
-o2/|[o2
0T0
| o21 02|
99
°2|°
y
"°21[,0:
03
o,
02
02
02
02
-------
Carriage of C02
,90
< S ' A '• W * M
f z ' •/ -
i * f >
, C» ~>, S
-------
Oxygen Transport
1.
Peripheral Tissues
Alveolus
0, + Hb—>Hb02
Interstitial Space
-------
Toxic Responses of the Blood
Lack of RBC
Function
/ \
inadequate shape
heme changes
production
Lack of RBC
Number
bone marrow genetic abnormalities
/ \ / \
maturation production fragility
shape
• Anemia
• Leukemia
• Hypoxemia/Hypoxia
-------
Sources of Lead
• Natural
• Mining/Industry
• Auto Emissions
-------
Utilization of Lead Products
in the U.S. (1984)
• Batteries
72%
• Gasoline additives
7%
• Pigments & ceramics
6%
• Ammunition
4%
• Solder
2%
• Other
9%
total tonnage used/year = 1,207,033
Source: Air Quality Criteria for Lead (1986)
-------
Pathways of Lead
from the Environment to Man
Absorption: Ingestion 10% adults
40% children
Inhalation 90%
-------
Distribution of Lead in
Bodv Tissues
Body Burden:
Blood«soft tissue«bone
Tt Tt Ti
16-28 days 30-130 days 10 years
-------
Mechanism of Toxicity of Lead
RtOUCTIOM OF
HEME I00T FOOL
T
ERYTHROPOIETIC
EFFECTS
Heme Molecule
imidazole
O,
CARDIOVASCULAR
AND
CEREBRAL DYSFUNCTION
-------
-------
Sources of Benzene
• Natural
• Industrial Sources
petroleum refineries
chemical manufacturers
• Consumer Sources
glues
household cleaners
paint strippers/art supplies
tobacco smoke
-------
Toxicokinetics of Benzene
Absorption:
inhalation 50%
ingestion **90%
dermal contact
Distribution:
blood
bone marrow *
fat
liver
Elimination:
exhalation 12-50%
(unchanged form)
Tr>15hrs.
urine *>33%
(metabolites)
Ty-24 hrs.
feces
-------
Mechanism of Action: Benzene
Benzene
intermediate formation ~ -g metabolites
of free radicals phenol, hydroquinone
Benzene epoxide
)/ excreted in urine
attack on Bone Marrow stem cells
Pancytopenia:
— Depressed Maturation of
RBC's WBC's Thrombocytes
t ~r
Decreased circulating Impaired morphology
volumes and function
necrosis
of bone (especially lymphocytes)
marrow tissue
}
/\ clotting problems
" ^
Anemia Immune Leukopenia
fatty deficiencies
infiltration X
of bone
marrow tissue Leukemia
increased repository for benzene ~ Benzene
-------
Immune Function
tissue antigens
I
T, Ig molecules
B lymphocyte
virus
neutralization enhanced
phagocytosis
Antibody secreting
plasma cell
7 \
induce
cytotoxicity
of
foreign cell
(lysis)
complement
activation
-------
Mechanisms of Human Toxicity
The Cardiovascular System
Toxicology of the Heart and Circulation
Instructor Dr. Forman
I. Introduction
A. Structure and Function
1. Conducting cells
2. Contracting cells
B. Excitation/Contraction Coupling
1. t-tubule System
2. Role of Calcium
II. Electrical Properties of the Heart
A. Generation of an Electrocardiogram (ECG)
B. Concept of Refractoriness
1. Absolute
2. Effective
3. Relative
4. Supranormal
C. Einthoven's Triangle
D. Description of the ECG
1. p wave, QRS complex, t wave
2. Heart Rate
a tachycardia
b. bradycardia
ill. Cardiotoxic Responses
A. Arrhythmias (Chronotropic Responses)
1. direct: perturbation of ion channel
2. indirect: perturbation of membrane composition
B. Ventricular Contractility (Inotropic Responses)
1. direct: effects on muscle function
2. indirect: effects on ion channels
IV. Cardiac Toxicants
A. Cardiac Glycosides
B. Environmental Risk Factors in Cardiovascular Disease
1. Ethanol
2. Carbon Monoxide
-------
CONDUCTION ELEMENTS
Right Atrium
Left Atrium
Bundle of HIS
Right Ventricle —
Left Ventricle
-------
CONTRACTILE ELEMENTS
vSr-
DEEP BULBO SPIRAL
DEEP SINO SPIRAL
-------
EXCITATION/CONTRACTION COUPLING:
T-Tubul«
MORPHOLOGIC
SYSTEM
Transversa
Tubular .
System
Lateral Sac,
Sarcomere
MECHANISM
Transmits
Depolarizing
Impulse
Throughout
Muscle Fiber
Calcium Release,
Actin-Myosin
Interaction In
Presence Of
Calcium
PHVSlQLOqtC
EVENT
Excitation
Link
I
Contraction
Sarcoplasmic Calcium Removal
Reticulum From Relaxation
Tubules Actin-Myosin
Complex
-------
RECORDING AN ECG
Bipolar
Unipolar
Lead A
Unipolar
Lead B
-------
THE REFRACTORY PERIOD
mV 0-
—
\ »
& V HP
-------
EINTHOVEN'S TRIANGLE
RA
/
V
Vla
'LL
-------
CARDIAC VECTORS
-------
THE ELECTROCARDIOGRAM (ECG)
R
-------
Cardiotoxic Responses
Arrhythmias
• Direct —perturbation of ion channels
ex: cardiac glycosides
• Indirect —perturbation of membrane fluidity
ex: ethanol
Ventricular Contractility
• Direct —perturbation of muscle function
ex: myocardial ischemia, ethanol
• Indirect —perturbation of ion balance
ex: cardiac glycosides
-------
EXAMPLES
OF ARRHYTHMIAS
• + P T-P
Atrial Tachycardia Rate — 230 beats/min
Normal Rhythm
Compensatory
P A p A Pause p
Atrial Premature Excitation
P + T
P P
Atrial Flutter
Ventricular Tachycardia
Atrial Fibrillation
Ventricular Premature Excitation
Ventricualr Fibrillation
First Degree
Block
P
s^-
Second Degree Block
Complete Block
P I P + T
rs
-------
DIRECT EFFECTS OF
CARDIAC GLYCOSIDES:
ARRHYTHMOGENICITY
A. NORMAL
Extracellular
B. AFTER DIGITALIS
Extracellular
-------
INDIRECT EFFECTS OF
CARDIAC GLYCOSIDES:
MYOCARDIAL CONTRACTILITY
Cardiac glycosides or aglycones
i
Inhibition of Na*. K*-ATPase
1
Inhibition ot Na pump
1
Increase in intracellular Na
i
Reduced calcium extrusion via Na-Ca exchange mechanism
and possibly
calcium release from mitochondria
i
Calcium overload
-------
ETHANOL
TOXICITY
T Ca
phospholipid
protein
>
amphfphilt
*
-------
MYCARDIAL ISCHEMIA
HYPERTENSION
CORONARY
ATHERO-
SCLEROSIS
ISCHEMIA
(MYOCARDIAL INFARCTION)
REDUCED MYOCARDIAL PERFORMANCE
HEART FAILURE
DEATH
-------
ENVIRONMENTAL RISK FACTORS IN
CARDIOVASCULAR DISEASE
Coronary arteriosclerotic heart disease
Carbon disulphide*
Carbon monoxide*
Fibrogenic dusts
Heavy metals
Hypertensive agents
Lead
Cadmium
Physical agents
Noise
Radiation
Heat
Cold
Non-atheromatous ischaemic heart disease
Nitrates*
Cardiac arrhythmias
Hydrocarbons*
Fluorocarbons*
Direct myocardial tissue damage
Cobalt*
Antimony
Arsenic
Arsine
Yellow phosphorus
•Well documented.
-------
CARBON MONOXIDE
TOXICITY
CO or deprivation of 02 (hypoxia)
i r
Hypoxia of vascular wall
Conversion of Hb to carboxy Hb
Damage to vascular wall
Increased permeability to macromolecules
from blood
Development of Atherosclerosis
uptake of lipid
edema
------- |