Lisa Stevens, D.O.
Introduction
 Pathology
 Study (logos) of disease (pathos)

Structural, biochemical, and functional changes
 Cells, tissues, and organs that underlie disease
Introduction
 Four aspects of a disease process
 Cause (etiology)
 Mechanisms of its development (pathogenesis)
 Biochemical and structural alterations (molecular and
morphologic changes)
 Functional consequences of these changes (clinical
manifestations)
Etiology
 Two major classes
 Genetic
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Inherited mutations
Disease-associated gene variants
 Acquired
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Infectious
Nutritional
Chemical
Physical
Pathogenesis
 Sequence of events in the response of cells or tissues to
the etiologic agent
 From the initial stimulus to the ultimate expression of
the disease
 One of the main domains of pathology
Molecular and Morphologic Changes
 Structural alterations in cells or tissues
 Characteristic of a disease
 Diagnostic of an etiologic process
Functional Derangements and
Clinical Manifestations
 Functional abnormalities
 End results of genetic, biochemical, and structural
changes in cells and tissues
 Lead to the clinical manifestations (symptoms and signs)
Lead to the progression of disease (clinical course and
outcome)
Functional Derangements and
Clinical Manifestations
 Disease
 Starts with molecular or structural alterations in cells

Concept first put forth by Rudolf Virchow (19th century)
 Father of modern pathology
 Injury to cells and to extracellular matrix

Tissue and organ injury
 Determine the morphologic and clinical patterns of disease
Cellular Responses to Stress and
Noxious Stimuli
 Normal cell
 Confined to a narrow range of function and structure
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State of metabolism, differentiation, and specialization
Constraints of neighboring cells
Availability of metabolic substrates
 Maintains homeostasis (steady state)
Cellular Responses to Stress and
Noxious Stimuli
 Adaptations
 Reversible functional and structural responses

Usually due to physiologic stresses and pathologic stimuli
 Hypertrophy (increase in the size of cells)
 Hyperplasia (increase in the number of cells)
 Atrophy (decrease in the size and metabolic activity of
cells)
 Metaplasia (change in the phenotype of cells)
Cellular Responses to Stress and
Noxious Stimuli
 Cell injury
 Exposure to injurious agents or stress
 Deprivation of essential nutrients
 Compromised by mutations that affect essential cellular
constituents
 Reversible

Up to a certain point
 Irreversible injury and cell death

Stimulus persists
Cellular Responses to Stress and
Noxious Stimuli
 Cell death
 End result of progressive cell injury
 One of the most crucial events in the evolution of
disease
 Results from diverse causes
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Ischemia (reduced blood flow)
Infection
Toxins
Cellular Responses to Stress and
Noxious Stimuli
 Cell death
 Normal and essential process
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Embryogenesis
Maintenance of homeostasis
 Two principal pathways of cell death

Necrosis and apoptosis
Adaptations of Cellular Growth and
Differentiation
 Hypertrophy
 Increase in the size of cells

Results in an increase in the size of the organ
 No new cells, just larger cells
 Due to synthesis of structural components of the cells

Cellular proteins
Adaptations of Cellular Growth and
Differentiation
 Hypertrophy
 Physiologic or pathologic
 Cause
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Increased functional demand
Stimulation by hormones and growth factors
Adaptations of Cellular Growth and
Differentiation
 Hypertrophy
 Example: Striated muscle cells (heart and skeletal muscle)
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Limited capacity for division
Respond to increased metabolic demands
 Hypertrophy
Most common stimulus
 Increased workload
 Example: Bodybuilders "pumping iron"
 Increase in size of the individual muscle fibers
Hypertrophy
 Mechanisms
 Induced by linked actions
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Mechanical sensors
Growth factors
Vasoactive agents
 Two main biochemical pathways
 Phosphoinositide 3-kinase/Akt pathway
 Signaling downstream of G protein-coupled receptors
Hypertrophy
 Usually refers to increase in size of cells or tissues
 HOWEVER, a subcellular organelle may undergo
selective hypertrophy
 Example: Individuals treated with drugs (barbiturates)

Hypertrophy of the smooth endoplamic reticulum (SER) in
hepatocytes
 Adaptive response
 Increases the amount of enzymes (cytochrome P-450 mixed
function oxidases) available to detoxify the drugs
 Eventually, patients respond less to the drug
 May result in an increased capacity to metabolize other drugs
Hyperplasia
 Increase in the number of cells in an organ or tissue
 Results in increased mass of the organ or tissue
 May occur in the setting of hypertrophy
 Physiologic and pathologic
Hyperplasia
 Increase in the number of cells in an organ or tissue
 Physiologic hyperplasia
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Hormonal hyperplasia
 Increases the functional capacity of a tissue when needed
Compensatory hyperplasia
 Increases tissue mass after damage or partial resection
Hyperplasia
 Physiologic hyperplasia
 Hormonal hyperplasia
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Proliferation of the glandular epithelium of the female breast
 Puberty
 Pregnancy
 Compensatory hyperplasia
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Myth of Prometheus
Ancient Greeks recognized the capacity of the liver to
regenerate
Liver transplantation (donor)
Hyperplasia
 Pathologic Hyperplasia
 Caused by excesses of hormones or growth factors
acting on target cells
 Endometrial hyperplasia
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Abnormal hormone-induced hyperplasia
Common cause of abnormal menstrual bleeding
Hyperplasia
 Pathologic Hyperplasia
 Benign prostatic hyperplasia

Induced by responses to androgens
 Constitutes a fertile soil in which cancerous proliferation
may eventually arise
Atrophy
 Reduced size of an organ or tissue
 Results from a decrease in cell size and number
 Physiologic atrophy
 Common during normal development

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Embryonic structures
 Notochord
 Thyroglossal duct
Uterus
 Decreased size shortly after parturition
Atrophy
 Pathologic atrophy
 Depends on the underlying cause
 Local or generalized
 Common causes of atrophy
 Decreased workload (atrophy of disuse)

Muscle atrophy secondary to immobilization/bedrest
 Loss of innervation (denervation atrophy)
 Diminished blood supply
Atrophy
 Common causes of atrophy
 Inadequate nutrition

Profound protein-calorie malnutrition (marasmus)
 Use of skeletal muscle as a source of energy after other reserves
(adipose stores) have been depleted
 Loss of endocrine stimulation

Hormone-responsive tissues (breast and reproductive organs)
 Pressure

Tissue compression for any length of time
Atrophy
 Mechanisms
 Results from decreased protein synthesis

Reduced metabolic activity
 Results from increased protein degradation in cells

Ubiquitin-proteasome pathway
 Responsible for the accelerated proteolysis
 Catabolic conditions (cancer cachexia)
Atrophy
 Accompanied by increased autophagy
 Increases in the number of autophagic vacuoles
 Autophagy ("self eating")
 Process in which the starved cell eats its own components
to survive
Atrophy
 Autophagic vacuoles
 Membrane-bound vacuoles that contain fragments of cell
components
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Vacuoles ultimately fuse with lysosomes
Contents are digested by lysosomal enzymes
Some cell debris (in the autophagic vacuoles) resist digestion
 Persist as membrane-bound residual bodies
 Lipofuscin granules (brown atrophy)
Metaplasia
 Reversible change
 One differentiated cell type replaced by another cell
type
 Adaptive substitution of cells (sensitive to stress)
 Cell types better able to withstand adverse environments
Metaplasia
 Most common epithelial metaplasia
 Columnar to squamous
 Occurs in the respiratory tract

Chronic irritation
 Cigarette smoker
 Normal PCCE replaced by stratified squamous epithelial
cells
 Lack of mucociliary elevator
 If persistent, may initiate malignant transformation in
metaplastic epithelium
Metaplasia
 Metaplasia from squamous to columnar type
 Barrett esophagus

Esophageal squamous epithelium is replaced by intestinal-like
columnar cells
 Influence of refluxed gastric acid
 Connective tissue metaplasia
 Formation of cartilage, bone, or adipose tissue
(mesenchymal tissues) in tissues that do not contain
these elements

Bone formation in muscle (myositis ossificans)
 Can occur following an intramuscular hemorrhage
Metaplasia
 Mechanisms
 Does not result from a change in the phenotype of an
already differentiated cell type
 Result of reprogramming


Stem cells (known to exist in normal tissues)
Undifferentiated mesenchymal cells present in connective
tissue
 Precursor cells differentiate along a new pathway
Practice Question
 A 43-year-old man has complained of mild burning substernal pain
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following meals for the past 3 years. Upper GI endoscopy is performed
and biopsies are taken of an erythematous area of the lower esophageal
mucosa 3 cm above the gastroesophageal junction. There is no mass
lesion, no ulceration, and no hemorrhage noted. The biopsies show the
presence of columnar epithelium with goblet cells. Which of the
following mucosal alterations is most likely represented by these
findings?
A. Dysplasia
B. Metaplasia
C. Hypertrophy
D. Hyperplasia
E. Ischemia
Practice Question
 A 19-year-old woman gives birth to her first child. She
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begins breast feeding the infant. She continues breast
feeding for almost a year with no difficulties and no
complications. Which of the following cellular processes
that began in the breast during pregnancy allowed her to
nurse the infant for this period of time?
A. Lobular hyperplasia
B. Stromal hypertrophy
C. Epithelial dysplasia
D. Steatocyte atrophy
E. Ductal epithelial metaplasia
Practice Question
 A study is performed involving the microscopic analysis of
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tissues obtained from surgical procedures. Some of these tissues
have the microscopic appearance of an increased cell size of
multiple cells within the tissue, due to an increase in the amount
of cytoplasm, with nuclei remaining uniform in size. Which of
the following conditions is most likely to have resulted in this
finding?
A. Uterine myometrium in pregnancy
B. Female breast at puberty
C. Liver following partial resection
D. Ovary following menopause
E. Cervix with chronic inflammation
Practice Question
 A 38-year-old man incurs a traumatic blow to his upper left
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arm. He continues to have pain and tenderness even after 3
months have passed. A plain film radiograph reveals a 4 cm
circumscribed mass in the soft tissue adjacent to the
humerus. The mass contains areas of brightness on the xray. Over the next year this process gradually resolves.
Which of the following terms best describes this process?
A. Dysplasia
B. Hyperplasia
C. Hypertrophy
D. Metaplasia
E. Neoplasia
Practice Question
 A 21-year-old woman has a routine Pap smear performed for a
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health screening examination. The pathology report indicates
that some cells are found cytologically to have larger, more
irregular nuclei. A follow-up cervical biopsy microscopically
demonstrates disordered maturation of the squamous
epithelium, with hyperchromatic and pleomorphic nuclei
extending nearly the full thickness of the epithelial surface. No
inflammatory cells are present. Which of the following
descriptive terms is best applied to these Pap smear and biopsy
findings?
A. Dysplasia
B. Metaplasia
C. Anaplasia
D. Hyperplasia
E. Aplasia
Practice Question
 A 3-year-old child has been diagnosed with ornithine
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transcarbamylase deficiency and has developed hepatic
failure. The left lobe of an adult donor liver is used as an
orthotopic transplant. A year later, the size of each liver in
donor and recipient is greater than at the time of
transplantation. Which of the following cellular alterations
is most likely to explain this phenomenon?
A. Metaplasia
B. Dysplasia
C. Hyperplasia
D. Anaplasia
E. Neoplasia
Lisa Stevens, D.O.
Causes of Cell Injury
 Oxygen deprivation
 Physical agents
 Chemical agents and drugs
 Infectious agents
 Immunologic reactions
 Genetic derangements
 Nutritional imbalances
Injurious
Stimuli
 Oxygen Deprivation
 Hypoxia

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
Deficiency of oxygen
Reduces aerobic oxidative respiration
Causes
 Reduced blood flow (ischemia)
 Inadequate oxygenation of the blood
 Cardiorespiratory failure
Injurious
Stimuli
 Oxygen Deprivation
 Hypoxia


Causes, continued
 Decreased oxygen-carrying capacity of the blood
 Anemia
 Carbon monoxide poisoning
 Severe blood loss
Depending on the severity
 Cells may adapt, undergo injury, or die
Injurious
Stimuli
 Physical Agents
 Mechanical trauma
 Extremes of temperature (burns and deep cold)
 Sudden changes in atmospheric pressure
 Radiation
 Electric shock
Injurious
Stimuli
 Chemical Agents and Drugs
 Chemicals (too many to list)
 Glucose or salt in hypertonic concentrations
 Oxygen at high concentrations
 Trace amounts of poisons
 Environmental and air pollutants
 Insecticides, herbicides
 Industrial and occupational hazards
 Recreational drugs (alcohol)
 Therapeutic drugs
Injurious
Stimuli
 Infectious Agents
 Submicroscopic viruses to the large tapeworms
 Rickettsiae
 Bacteria
 Fungi
 Higher forms of parasites
Injurious
Stimuli
 Immunologic Reactions
 Injurious reactions to endogenous self-antigens

Several autoimmune diseases
 Immune reactions to external agents


Microbes
Environmental substances
Injurious
Stimuli
 Genetic Derangements
 Severe defects

Congenital malformations associated with Down syndrome
 Chromosomal anomaly
 Subtle defects

Decreased life span of red blood cells
 Single amino acid substitution in hemoglobin in sickle cell
anemia
 Variations in the genetic makeup

Influence the susceptibility of cells by chemicals and other
environmental insults
Injurious
Stimuli
 Nutritional Imbalances
 Protein-calorie deficiencies

Underprivileged populations
 Deficiencies of specific vitamins
 Self-imposed problems

Anorexia nervosa
 Nutritional excesses


Excess of cholesterol
Obesity
Morphologic Alterations
 Sequential morphologic changes in cell injury
 Reversible injury
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Generalized swelling of the cell and its organelles
Blebbing of the plasma membrane
Detachment of ribosomes from the ER
Clumping of nuclear chromatin
 Associations
 Decreased generation of ATP
 Loss of cell membrane integrity
 Defects in protein synthesis
 Cytoskeletal damage
 DNA damage
Reversible Injury
 Two features of reversible cell injury
 Cellular swelling
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
Cells are incapable of maintaining ionic and fluid homeostasis
Result of failure of energy-dependent ion pumps in the plasma
membrane
 Fatty change
 Hypoxic injury
 Various forms of toxic or metabolic injury
 Manifested by the appearance of lipid vacuoles in the cytoplasm
 Hepatocytes and myocardial cells
Reversible
Injury
 Morphology
 Cellular swelling
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First manifestation of almost all forms of injury to cells
Difficult morphologic change to appreciate with the light
microscope
 More apparent at the level of the whole organ
Pallor, increased turgor, and increase in weight of the organ
Microscopic examination
 Small clear cytoplasmic vacuoles (distended and pinched-off
ER)
 Hydropic change or vacuolar degeneration
Reversible Injury
 Ultrastructural changes
 Plasma membrane alterations

Blebbing, blunting, and loss of microvilli
 Mitochondrial changes
 Swelling
 Appearance of small amorphous densities
 Dilation of the ER
 Detachment of polysomes
 Intracytoplasmic myelin figures may be present
 Nuclear alterations
 Disaggregation of granular and fibrillar elements
Irreversible Cell Injury and Cell
Death
 Continuous damage
 Cell injury becomes irreversible

Cell cannot recover and it dies
 Two principal types of cell death
 Necrosis

Severe membrane damage


Lysosomal enzymes enter the cytoplasm and digest the cell, and cellular
contents leak out
Always a pathologic process
 Apoptosis

Cell's DNA or proteins are damaged beyond repair
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Cell kills itself by nuclear dissolution, fragmentation of the cell without
complete loss of membrane integrity, and rapid removal of the cellular
debris
Serves many normal functions
Not necessarily associated with cell injury
Necrosis
 Morphologic appearance
 Result of denaturation of intracellular proteins and
enzymatic digestion of the lethally injured cell
 Necrotic cells

Unable to maintain membrane integrity
 Contents leak
 Elicits inflammation in the surrounding tissue
Necrosis
 Necrotic cells
 Increased eosinophilia in hematoxylin and eosin (H & E)
stains

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Loss of cytoplasmic RNA (which binds the blue dye, hematoxylin)
Denatured cytoplasmic proteins (which bind the red dye, eosin)
 Glassy homogeneous appearance
 Loss of glycogen particles
 Digestion of cytoplasmic organelles---vacuolated cytoplasm
(moth-eaten)
 Dead cells

Replaced by large, whorled phospholipid masses (myelin figures)
 Derived from damaged cell membranes
 Phospholipid precipitates
 Phagocytosed by other cells
 Further degraded into fatty acids
Necrosis
 Necrotic cells
 Nuclear changes
 Due to nonspecific breakdown of DNA
 Karyolysis
 Fading of the basophilia of the chromatin
 A change that presumably reflects loss of DNA because of
enzymatic degradation by endonucleases
 Pyknosis
 Nuclear shrinkage
 Increased basophilia
 Chromatin condenses into a solid, shrunken basophilic mass
 Karyorrhexis
 Pyknotic nucleus undergoes fragmentation
 Nucleus in the necrotic cell totally disappears (1 or 2 days)
Patterns
of Tissue Necrosis
 Coagulative necrosis
 Architecture of dead tissues is preserved for a span of a few
days
 Tissue displays a firm texture
 Eosinophilic, anucleate cells persist for days or weeks
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Removed by phagocytosis of the cellular debris by infiltrating
leukocytes
Digestion of the dead cells by the action of lysosomal enzymes of
the leukocytes
 Example:
 Ischemia caused by obstruction in a vessel may lead to coagulative
necrosis of the supplied tissue
 Localized area
 Infarct
Patterns of Tissue Necrosis
 Liquefactive necrosis
 Characterized by digestion of the dead cells

Resulting in transformation of the tissue into a liquid viscous
mass
 Seen in focal bacterial infections
 Occasionally seen in fungal infections
 Creamy yellow
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Dead leukocytes
Purulent matter (aka pus)
 Hypoxic death of cells in the CNS
Patterns
of
Tissue
Necrosis
 Gangrenous necrosis
 Not a specific pattern of cell death
 Commonly used in clinical practice
 Applied to a limb (usually lower leg)

Lost its blood supply and has undergone necrosis (typically
coagulative necrosis)
 Involving multiple tissue planes
 Add in a bacterial infection

More liquefactive necrosis
 Because of the actions of degradative enzymes in the bacteria
and the attracted leukocytes
 Wet gangrene
Patterns of Tissue Necrosis
 Caseous necrosis
 Encountered most often in foci of tuberculous infection
 “Caseous" (cheeselike)

Derived from the friable white appearance of the area of
necrosis
 Microscopic examination
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Collection of fragmented or lysed cells
Amorphous granular debris enclosed within a distinctive
inflammatory border
 Characteristic of a focus of inflammation known as a
granuloma
Patterns of Tissue Necrosis
 Fat necrosis
 Term that is well fixed in medical parlance

Does not denote a specific pattern of necrosis
 Focal areas of fat destruction
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Release of activated pancreatic lipases into the substance of
the pancreas and the peritoneal cavity
 Microscopic examination
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Foci of shadowy outlines of necrotic fat cells
Basophilic calcium deposits
Inflammatory reaction
Patterns of Tissue Necrosis
 Fibrinoid necrosis
 Special form of necrosis
 Seen in immune reactions involving blood vessels
 Complexes of antigens and antibodies

Deposited in the walls of arteries
 Microscopic examination

Deposits of these "immune complexes" and fibrin
 Bright pink and amorphous appearance (“fibrinoid”)
Mechanisms
of
Cell
Injury
 Principles that are relevant to most forms of cell
injury
 Cellular response to injurious stimuli
 Depends on the nature of the injury, its duration, and its
severity
 Small doses of a chemical toxin or brief periods of ischemia
may induce reversible injury
 Large doses of the same toxin or more prolonged ischemia
 Instantaneous cell death
 Slow, irreversible injury leading in time to cell death
 Consequences of cell injury depend on the type, state,
and adaptability of the injured cell
Mechanisms of Cell Injury
 Principles that are relevant to most forms of cell injury
 Cell injury results from different biochemical mechanisms
acting on several essential cellular components
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Mitochondria
Cell membranes
DNA in nuclei
 Any injurious stimulus may simultaneously trigger multiple
interconnected mechanisms that damage cells

Difficult to ascribe cell injury in a particular situation to a single or
even dominant biochemical derangement
ATP
 ATP is produced in two ways
 Major pathway (mammalian cells)

Oxidative phosphorylation of adenosine diphosphate
 Reaction that results in reduction of oxygen by the electron
transfer system of mitochondria
 Second pathway

Glycolytic pathway
 Generates ATP in the absence of oxygen
 Uses glucose derived either from body fluids or from the
hydrolysis of glycogen
Depletion of ATP
 ATP depletion and decreased ATP synthesis
 Associated with both hypoxic and chemical (toxic)
injury
 Major causes
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Reduced supply of oxygen and nutrients
Mitochondrial damage
Actions of toxins (e.g., cyanide)
ATP
 High-energy phosphate in the form of ATP
 Required for virtually all synthetic and degradative
processes within the cell
 Depletion of ATP to 5% to 10% of normal levels

Widespread effects on many critical cellular systems
Depletion
of
ATP
 Effects on critical cellular systems
 Activity of the plasma membrane energy-dependent sodium
pump is reduced


Failure of this active transport system causes sodium to enter and
accumulate inside cells and potassium to diffuse out
The net gain of solute is accompanied by isosmotic gain of water,
causing cell swelling, and dilation of the ER
 Cellular energy metabolism is altered
 Reduced supply of oxygen to cells (i.e. ischemia)
 Oxidative phosphorylation ceases
 Decrease in cellular ATP
 Increase in adenosine monophosphate
 Glycogen stores are rapidly depleted
Depletion
of
ATP
 Effects on critical cellular systems
 Failure of the Ca2+ pump leads to influx of Ca2+

Damages intracellular organelles
 Prolonged or worsening depletion of ATP

Structural disruption of the protein synthetic apparatus
occurs
 Manifested as detachment of ribosomes from the rough ER
 Dissociation of polysomes
 Consequent reduction in protein synthesis
Depletion
of
ATP
 Effects on critical cellular systems
 Oxygen or glucose deprivation

Proteins may become misfolded
 Trigger a cellular reaction (unfolded protein response)
 Cell injury and even death
 Irreversible damage to mitochondrial and lysosomal
membranes

Cell necrosis
Mitochondrial Damage
 Mitochondria
 Cell's suppliers of life-sustaining energy in the form of
ATP
 Critical players in cell injury and death
 Damaged by:
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Increases of cytosolic Ca2+
Reactive oxygen species
Oxygen deprivation
 Mutations in mitochondrial genes are the cause of some
inherited diseases
Mitochondrial
Damage
 Formation of a high-conductance channel in the
mitochondrial membrane
 Mitochondrial permeability transition pore

Opening of this conductance channel leads to the loss of
mitochondrial membrane potential
 Resulting in failure of oxidative phosphorylation and
progressive depletion of ATP
 Necrosis of the cell
Mitochondrial
Damage
 Mitochondria
 Sequester proteins between their outer and inner
membranes

Capable of activating apoptotic pathways
 Cytochrome c and caspases (indirectly activate apoptosisinducing enzymes)
 Increased permeability of the outer mitochondrial membrane
 Leakage of these proteins into the cytosol
 Death by apoptosis
Calcium Homeostasis
 Calcium ions are important mediators of cell injury
 Cytosolic free calcium
 Normally maintained at very low concentrations (∼0.1
μmol)
 Intracellular calcium is sequestered in mitochondria and
the ER
 Increased cytosolic Ca2+ activates a number of
enzymes
 Deleterious cellular effects
 Phospholipases (membrane damage)
Calcium Homeostasis
 Increased cytosolic Ca2+ activates a number of enzymes
 Proteases (break down both membrane and cytoskeletal
proteins)
 Endonucleases (responsible for DNA and chromatin
fragmentation)
 ATPases (hastening ATP depletion)
 Increased intracellular Ca2+ levels
 Induction of apoptosis

Direct activation of caspases and by increasing mitochondrial
permeability
Free
Radicals
 Free radicals
 Chemical species that have a single unpaired electron in an
outer orbit
 Energy created by this unstable configuration is released
through reactions with adjacent molecules

Inorganic or organic chemicals-proteins, lipids, carbohydrates,
nucleic acids
 Reactive oxygen species (ROS)
 Type of oxygen-derived free radical
 Produced normally in cells during mitochondrial respiration and
energy generation
 Degraded and removed by cellular defense systems
 Produced in large amounts by leukocytes, particularly neutrophils
and macrophages
Generation
of
Free
Radicals
 The reduction-oxidation reactions that occur during
normal metabolic processes
 Absorption of radiant energy (ultraviolet light, x-rays)
 Ionizing radiation can hydrolyze water into •OH and
hydrogen (H) free radicals
 Rapid bursts of ROS
 Produced in activated leukocytes during inflammation
 Enzymatic metabolism of exogenous chemicals or
drugs
Generation
of
Free
Radicals
 Transition metals such as iron and copper donate or
accept free electrons during intracellular reactions
(Fenton reaction (H2O2 + Fe2+ → Fe3+ + OH + OH
 Nitric oxide
 Important chemical mediator that can act as a free
radical
 Generated by endothelial cells, macrophages, neurons,
and other cell types
Removal
of
Free
Radicals
 Free radicals are inherently unstable and generally decay
spontaneously
 Cells have developed multiple nonenzymatic and
enzymatic mechanisms to remove free radicals
 Minimize injury
 Iron and copper can catalyze the formation of ROS
 Levels of these reactive metals are minimized by binding of
the ions to storage and transport proteins (e.g., transferrin,
ferritin, lactoferrin, and ceruloplasmin), thereby minimizing
the formation of ROS
 Enzymes acts as free radical-scavenging systems
Pathologic Effects of Free
Radicals
 Three reactions
 Lipid peroxidation in membranes
 Presence of O2, free radicals may cause peroxidation of lipids within
plasma and organellar membranes
 Oxidative damage is initiated when the double bonds in
unsaturated fatty acids of membrane lipids are attacked by O2derived free radicals
 Oxidative modification of proteins
 Free radicals promote:
 Oxidation of amino acid side chains
 Formation of protein-protein cross-linkages (e.g., disulfide
bonds)
 Oxidation of the protein backbone
 Lesions in DNA
 Single- and double-strand breaks in DNA
 Cross-linking of DNA strands
 Formation of adducts
Membrane Damage
 Several biochemical mechanisms may contribute to
membrane damage
 Reactive oxygen species
 Decreased phospholipid synthesis
 Increased phospholipid breakdown
 Cytoskeletal abnormalities
Membrane
Damage
 Consequences
 The most important sites of membrane damage during cell injury

Mitochondrial membrane damage



Plasma membrane damage




Opening of the mitochondrial permeability transition pore leading to
decreased ATP
Release of proteins that trigger apoptotic death
Loss of osmotic balance and influx of fluids and ions
Loss of cellular contents
Cells may also leak metabolites
 Vital for the reconstitution of ATP, thus further depleting energy
stores
Injury to lysosomal membranes




Leakage of their enzymes into the cytoplasm
Activation of the acid hydrolases in the acidic intracellular pH of the
injured cell
Activation of these enzymes leads to enzymatic digestion
Cells die by necrosis
Lisa Stevens, D.O.
Ischemic and Hypoxic Injury
 Most common type of cell injury in clinical medicine
 Studied extensively
 Humans
 Experimental animals
 Culture systems
 Hypoxia
 Reduced oxygen availability
 Occurs in a variety of clinical settings
Ischemic and Hypoxic Injury
 Ischemia
 Supply of oxygen and nutrients is decreased
 Because of reduced blood flow

Consequence of a mechanical obstruction in the arterial
system/reduced venous drainage
 Compromises the delivery of substrates for glycolysis
Ischemic and Hypoxic Injury
 Ischemic tissues
 Aerobic metabolism compromised
 Anaerobic energy generation stopped


Glycolytic substrates are exhausted
Glycolysis is inhibited
 Accumulation of metabolites
 Ischemia tends to cause more rapid and severe cell and
tissue injury than does hypoxia in the absence of
ischemia
Mechanisms
of(post-hypoxia
Ischemic
Cell Injury
 Sequence of events
or ischemia)
 Oxygen tension within the cell decreases
 Loss of oxidative phosphorylation
 Decreased generation of ATP
 Failure of the sodium pump
 Loss of potassium
 Influx of sodium and water
 Cell swelling
 Influx of Ca2+
 Progressive loss of glycogen
 Decreased protein synthesis
 Functional consequences may be severe at this stage
Mechanisms
of Ischemic Cell Injury
 Example:
 Heart muscle ceases to contract within 60 seconds of
coronary occlusion
 Loss of contractility does not mean cell death
 Continued hypoxia

Worsening ATP depletion
 Further deterioration
 Cytoskeleton disperses
Loss of ultrastructural features (microvilli and the formation
of blebs)
 Myelin figures (degenerating cellular membranes)


Seen within the cytoplasm (in autophagic vacuoles) or
extracellularly
Mechanisms of Ischemic Cell
Injury
 Example:
 Continued hypoxia (continued from previous slide)
 Cytoskeleton disperses

Mitochondria—swollen

Due to loss of volume control in these organelles
ER remains dilated
 Entire cell is markedly swollen

Increased concentrations of water, sodium, and chloride
 Decreased concentration of potassium

 If oxygen is restored, all of these disturbances are
reversible!!!!
Mechanisms of Ischemic Cell
Injury
 If ischemia persists, irreversible injury and
necrosis ensue!!!!
 Irreversible injury
 Severe swelling of mitochondria
 Extensive damage to plasma membranes (giving rise
to myelin figures)
 Swelling of lysosomes
 Large, flocculent, amorphous densities develop in
the mitochondrial matrix
Mechanisms of Ischemic Cell
Injury
 Example:
 Myocardium

Irreversible injury can be seen as early as 30 to 40 minutes
after ischemia
 Massive influx of calcium into the cell (ischemic zone)
 Death is mainly by necrosis, but apoptosis also contributes
 Apoptotic pathway is activated by release of proapoptotic molecules from leaky mitochondria
 Cell's components are progressively degraded
 Widespread leakage of cellular enzymes into the
extracellular space
 Dead cells replaced by large masses (myelin figures)
 Either phagocytosed by leukocytes
 Degraded further into fatty acids

Calcification of fatty acid residues
Mechanisms of Ischemic Cell
Injury
 Despite many investigations
 No reliable therapeutic approaches for reducing the
injurious consequences of ischemia in clinical situations
 Most useful strategy in ischemic (and traumatic) brain
and spinal cord injury

Transient induction of hypothermia (core body temperature
to 92°F)
 Reduces the metabolic demands of the stressed cells
 Decreases cell swelling
 Suppresses the formation of free radicals
 Inhibits the host inflammatory response
Ischemia-Reperfusion Injury
 Restoration of blood flow to ischemic tissues
 Promotes recovery of cells (reversibly injured)
 Certain circumstances
 Blood flow is restored to cells that have been ischemic but
have not died

Paradoxical injury is exacerbated
 Proceeds at an accelerated pace
 Reperfused tissues may sustain loss of cells in addition to the
cells that are irreversibly damaged at the end of ischemia


Ischemia-reperfusion injury
Clinically important
 Contributes to tissue damage during myocardial and cerebral
infarction and following therapies to restore blood
Ischemia-Reperfusion Injury
 Reperfusion injury occur
 New damaging processes are set in motion during
reperfusion


Causes the death of cells that might have recovered otherwise
Several proposed mechanisms
 Damage may be initiated during reoxygenation
 Increased generation of reactive oxygen and nitrogen species

Cellular antioxidant defense mechanisms may be
compromised by ischemia
 Accumulation of free radicals
Mediators of cell injury (calcium) may also enter reperfused
cells
 Damages various organelles


Produced in reperfused tissue as a result of mitochondrial
damage
Ischemia-Reperfusion Injury
 Ischemic injury
 Associated with inflammation


Result of the production of cytokines
Causes additional tissue injury
 Activation of the complement system
 May contribute to ischemia-reperfusion injury
 Involved in host defense
 Important mechanism of immune injury
Chemical (Toxic) Injury
 Chemical injury remains a frequent problem in clinical
medicine
 Major limitation to drug therapy
 Many drugs are metabolized in the liver
 Frequent target of drug toxicity
 Toxic liver injury

Most frequent reason for terminating the therapeutic use or
development of a drug
Chemical (Toxic) Injury
 Chemicals induce cell injury
 Direct injury

Combining with critical molecular components
 Example: Mercuric chloride poisoning
 Mercury binds to the sulfhydryl groups of cell membrane
proteins
Causes increased membrane permeability and inhibition of ion
transport
 Damage is usually to the cells that use, absorb, excrete, or
concentrate the chemicals


Cells of the gastrointestinal tract and kidney
Apoptosis
 Pathway of cell death
 Induced by a tightly regulated suicide program
 Cells destined to die activate enzymes that degrade the cells'
own nuclear DNA and nuclear and cytoplasmic proteins
 Cells break up into fragments (apoptotic bodies)
 Contain portions of the cytoplasm and nucleus
 Plasma membrane of the apoptotic cell and bodies remains
intact


Structure is altered
Tasty targets for phagocytes
 Dead cell and its fragments are rapidly devoured
 Before the contents have leaked out
 Cell death by this pathway does not elicit an inflammatory
reaction in the host
Apoptosis
 Death by apoptosis
 Normal phenomenon that serves to eliminate cells that
are no longer needed
 Maintains a steady number of various cell populations in
tissues
 Causes of Apoptosis
 Involution of hormone-dependent tissues upon
hormone withdrawal




Endometrial cell breakdown during the menstrual cycle
Ovarian follicular atresia in menopause
Regression of the lactating breast after weaning
Prostatic atrophy after castration
Apoptosis
 Causes of Apoptosis, continued
 Cell loss in proliferating cell populations to maintain a
constant number (homeostasis)




Immature lymphocytes in the bone marrow
Thymus that fails to express useful antigen receptors
B lymphocytes in germinal centers
Epithelial cells in intestinal crypts
Apoptosis
 Causes of Apoptosis, continued
 Elimination of potentially harmful self-reactive
lymphocytes

Before or after they have completed their maturation
 Prevent reactions against one's own tissues
 Death of host cells that have served their useful purpose


Neutrophils in an acute inflammatory response
Lymphocytes at the end of an immune response
Apoptosis in Pathologic
Conditions
 Apoptosis eliminates cells that are injured beyond
repair without eliciting a host reaction
 Thus limiting collateral tissue damage
 Death by apoptosis is responsible for loss of cells in a
variety of pathologic states
 DNA damage

Radiation, cytotoxic anticancer drugs, and hypoxia
 Production of free radicals
Apoptosis in Pathologic
Conditions
 Death by apoptosis is responsible for loss of cells in
a variety of pathologic states
 Accumulation of misfolded proteins

Improperly folded proteins
 Mutations in the genes encoding these proteins
 Damage caused by free radicals
 Accumulation of these proteins in the ER
 ER stress
Apoptosis in Pathologic
Conditions
 Death by apoptosis is responsible for loss of cells in
a variety of pathologic states
 Cell death in certain infections

Viral infections
 Apoptosis is induced by the virus (as in adenovirus and
HIV infections) or by the host immune response (as in
viral hepatitis)
 Pathologic atrophy in parenchymal organs after duct
obstruction

Pancreas, parotid gland, and kidney
Morphology of Apoptosis
 Cell shrinkage
 Smaller in size
 Cytoplasm is dense
 Organelles are more tightly packed

Recall that in other forms of cell injury, an early feature is cell
swelling, not shrinkage
 Chromatin condensation
 Most characteristic feature of apoptosis
 Chromatin aggregates peripherally, under the nuclear
membrane, into dense masses of various shapes and
sizes
 Nucleus itself may break up, producing two or more
Morphology of Apoptosis
 Formation of cytoplasmic blebs and apoptotic bodies
 Extensive surface blebbing
 Fragmentation into membrane-bound apoptotic bodies
 Phagocytosis of apoptotic cells or cell bodies
 Macrophages
Biochemical Features of
Apoptosis
 A specific feature of apoptosis is the activation of
several members of a family of cysteine proteases
 Caspases

Two properties of this family of enzymes
 The “c" refers to a cysteine protease
 The "aspase" refers to the unique ability of these enzymes
to cleave after aspartic acid residues
Biochemical Features of
Apoptosis
 The caspase family
 Divided functionally into two groups


Initiator
 Caspase-8 and caspase-9
Executioner
 Caspase-3 and caspase-6
 Exist as inactive pro-enzymes, or zymogens, and
must undergo an enzymatic cleavage to become
active
 The presence of cleaved, active caspases is a marker
for cells undergoing apoptosis
Mechanisms of Apoptosis
 Process of apoptosis
 Divided


Initiation phase
 Caspases become catalytically active
Execution phase
 Caspases trigger the degradation of critical cellular
components
 Two pathways


Intrinsic (mitochondrial)
Extrinsic (death-receptor initiated)
Mechanisms
of Apoptosis
 The Intrinsic (Mitochondrial)
Pathway of
Apoptosis
 Major mechanism of apoptosis in all mammalian
cells
 Result of increased mitochondrial permeability
 Result of release of pro-apoptotic molecules (death
inducers) into the cytoplasm
 leads to activation of the initiator caspase-9
Mechanisms of Apoptosis
 The Extrinsic (Death Receptor-Initiated) Pathway of
Apoptosis
 Initiated by engagement of plasma membrane death
receptors on a variety of cells
 Death receptors are members of the TNF receptor family

Contain a cytoplasmic domain involved in protein-protein
interactions that is called the death domain
 Delivering apoptotic signals
 Leads to activation of the caspase-8 and -10
Mechanisms of Apoptosis
 The Execution Phase of Apoptosis
 Two initiating pathways converge to a cascade of caspase
activation

Mediates the final phase of apoptosis
 Enzymatic death program is set in motion by rapid and
sequential activation of the executioner caspases

Caspase-3 and -6
 Act on many cellular components
Mechanisms of Apoptosis
 Removal of Dead Cells
 Formation of apoptotic bodies breaks cells up into "bitesized"

Edible for phagocytes
 Healthy cells

Phosphatidylserine is present on the inner leaflet of the
plasma membrane
 Apoptotic cells

Phospholipid "flips" out and is expressed on the outer layer of
the membrane
 Recognized by several macrophage receptors
 Cells that are dying by apoptosis secrete soluble factors that
recruit phagocytes
Clinico-pathologic Correlations
 Examples of Apoptosis
 Growth Factor Deprivation



Hormone-sensitive cells deprived of the relevant hormone
Lymphocytes that are not stimulated by antigens and
cytokines
Neurons deprived of nerve growth factor die by apoptosis
 DNA Damage

Exposure of cells to radiation or chemotherapeutic agents
Autophagy
 Process in which a cell eats its own contents
 Survival mechanism in times of nutrient deprivation
 Starved cell lives by cannibalizing itself and recycling
the digested contents
 Intracellular organelles and portions of cytosol are first
sequestered from the cytoplasm in an autophagic
vacuole
 Subsequently fuses with lysosomes to form an
autophagolysosome

Cellular components are digested by lysosomal enzymes
Intracellular
 Manifestation of Accumulations
metabolic derangements in cells
 Intracellular accumulation of abnormal amounts of
various substances

Stockpiled substances fall into two categories
 Normal cellular constituent
 Water, lipids, proteins, and carbohydrates
 Accumulates in excess
 Abnormal substance
 Exogenous (mineral or products of infectious agents)
 Endogenous (product of abnormal synthesis or
metabolism)
 May be harmless to the cells
 Occasionally they are severely toxic
 Located in either the cytoplasm (frequently within
phagolysosomes) or the nucleus
Intracellular
Accumulations
 Attributable to four
types of abnormalities
 A normal endogenous substance is produced at a
normal or increased rate, but the rate of metabolism
is inadequate to remove it

Fatty change in the liver and reabsorption protein droplets
in the tubules of the kidneys
 An abnormal endogenous substance, accumulates
because of defects in protein folding and transport
and an inability to degrade the abnormal protein
efficiently


Accumulation of mutated α1-antitrypsin in liver cells
Various mutated proteins in degenerative disorders of the
central nervous system
Intracellular
Accumulations
 Attributable to four
types of abnormalities
 A normal endogenous substance accumulates
because of defects, usually inherited, in enzymes
that are required for the metabolism of the
substance

Storage diseases (genetic defects in enzymes involved in
the metabolism of lipid and carbohydrates, resulting in
intracellular deposition of these substances)
 An abnormal exogenous substance is deposited and
accumulates because the cell has neither the
enzymatic machinery to degrade the substance nor
the ability to transport it to other sites

Accumulations of carbon particles and nonmetabolizable
chemicals (silica)
Lipids
 All major classes of lipids can accumulate in cells
 Triglycerides
 Cholesterol/cholesterol esters
 Phospholipids

Components of the myelin figures found in necrotic cells
Lipids
 Steatosis (Fatty Change)
 Abnormal accumulations of triglycerides within
parenchymal cells
 Seen in the liver

The major organ involved in fat metabolism
 Occurs in heart, muscle, and kidney
Lipids
 Steatosis (Fatty Change)
 Causes


Toxins, protein malnutrition, diabetes mellitus, obesity, and
anoxia
Most common causes of significant fatty change in the liver
(developed countries)
 Alcohol abuse
 Nonalcoholic fatty liver disease
 Associated with diabetes and obesity
Lipids
 Mechanisms for triglyceride accumulation in the liver
 Free fatty acids from adipose tissue or ingested food are
normally transported into hepatocytes


Esterified to triglycerides, converted into cholesterol or
phospholipids, or oxidized to ketone bodies
Excess accumulation of triglycerides within the liver may
result from excessive entry or defective metabolism and export
of lipids
 Such defects are induced by alcohol
 Hepatotoxin that alters mitochondrial and microsomal
functions

Leading to increased synthesis and reduced breakdown of lipids
Lipids
 Morphology
 Fatty change is most often seen in the liver and heart
 Appears as clear vacuoles within parenchymal cells

Intracellular accumulations of water or polysaccharides (e.g.,
glycogen) may also produce clear vacuoles
 Identification of lipids requires the avoidance of fat
solvents commonly used in tissue preparation


Prepare frozen tissue sections of either fresh or aqueous
formalin-fixed tissues
Sections may then be stained with Sudan IV or Oil Red-O
 Orange-red color to the contained lipids
Lipids
 Gross examination--Liver
 Mild fatty change may not affect the gross appearance
 Progressive accumulation


Organ enlarges and becomes increasingly yellow
Extreme instances, the liver may weigh two to four times
normal
 Bright yellow, soft, greasy organ
 Gross examination—Heart

Grossly apparent bands of yellowed myocardium
 Alternating with bands of darker, red-brown, uninvolved
myocardium (tigered effect)
Cholesterol and Cholesterol
Esters
 Most cells use cholesterol for the synthesis of cell
membranes
 Without intracellular accumulation of cholesterol or
cholesterol esters
 Accumulations manifested histologically by
intracellular vacuoles are seen in several pathologic
processes
 Atherosclerosis

Atherosclerotic plaques
 Smooth muscle cells and macrophages within the intimal layer
of the aorta and large arteries are filled with lipid vacuoles,
most of which are made up of cholesterol and cholesterol
Cholesterol and Cholesterol
Esters
 Xanthomas
 Intracellular accumulation of cholesterol within
macrophages (acquired and hereditary
hyperlipidemic states)
 Clusters of foamy cells are found in the subepithelial
connective tissue of the skin and in tendons
 Cholesterolosis
 Focal accumulations of cholesterol-laden
macrophages in the lamina propria of the
gallbladder
 Niemann-Pick disease, type C
 Lysosomal storage disease
 Caused by mutations affecting an enzyme involved
in cholesterol trafficking
Proteins
 Intracellular accumulations of proteins
 Appear as rounded, eosinophilic droplets, vacuoles, or
aggregates in the cytoplasm
 Reabsorption droplets in proximal renal tubules

Seen in renal diseases associated with protein loss in the urine
 May be normal secreted proteins that are produced in
excessive amounts

Plasma cells engaged in active synthesis of immunoglobulins
Proteins
 Defective intracellular transport and secretion of
critical proteins
 α1-antitrypsin deficiency
 Emphysema
 Accumulation of cytoskeletal proteins
 Microtubules, thin actin filaments ,thick myosin
filaments, and intermediate filaments


Alcoholic hyaline is an eosinophilic cytoplasmic inclusion in
liver cells and is composed predominantly of keratin
intermediate filaments
Neurofibrillary tangle found in the brain in Alzheimer disease
contains neurofilaments and other proteins
 Aggregation of abnormal proteins
 Deposits can be intracellular, extracellular, or both
Hyaline Change
 Alteration within cells or in the extracellular space
 Gives a homogeneous, glassy, pink appearance
 Widely used as a descriptive histologic term rather
than a specific marker for cell injury
 Produced by a variety of alterations
 Does not represent a specific pattern of accumulation
Glycogen
 Readily available energy source stored in the
cytoplasm of healthy cells
 Excessive intracellular deposits of glycogen
 Seen in patients with an abnormality in either glucose or
glycogen metabolism
 Appear as clear vacuoles within the cytoplasm
 Dissolves in aqueous fixatives
 Tissues are best fixed in absolute alcohol
 Staining with Best carmine or the PAS reaction

Rose-to-violet color to the glycogen
Pigments
 Pigments are colored substances, some of which are
normal constituents of cells (melanin)
 Others are abnormal and accumulate in cells only
under special circumstances
 Exogenous pigments (coming from outside the body)
 Carbon (coal dust)
 Ubiquitous air pollutant of urban life
 Accumulations of this pigment blacken the tissues of the
lungs (anthracosis) and the involved lymph nodes
 Tattooing
 Localized, pigmentation of the skin
 Pigments inoculated are phagocytosed by dermal
macrophages
Pigments
 Endogenous pigments (synthesized within the body
itself)
 Lipofuscin
 Insoluble pigment
 Also known as lipochrome or wear-and-tear pigment
 Composed of polymers of lipids and phospholipids in
complex with protein
 Not injurious to the cell or its functions
 Telltale sign of free radical injury and lipid peroxidation
 Yellow-brown, finely granular cytoplasmic, often perinuclear,
pigment in tissue sections
 Seen in cells undergoing slow, regressive changes
 Prominent in the liver and heart of aging patients or patients
with severe malnutrition and cancer cachexia
Pigments
 Melanin
 Endogenous, non-hemoglobin-derived, brown-black
pigment
 Formed when the enzyme tyrosinase catalyzes the
oxidation of tyrosine to dihydroxyphenylalanine in
melanocytes
 The only endogenous brown-black pigment
Pigments
 Hemosiderin
 Hemoglobin-derived, golden yellow-to-brown, granular
or crystalline pigment
 Serves as one of the major storage forms of iron
 Represents aggregates of ferritin micelles
 Seen normally in mononuclear phagocytes of the bone
marrow, spleen, and liver

Actively engaged in red cell breakdown
Pigments
 Iron pigment appears as a coarse, golden, granular
pigment
 Within the cell's cytoplasm
 Visualized in tissues by the Prussian blue
histochemical reaction
 Underlying cause is the localized breakdown of red
cells
 Hemosiderin is found initially in the phagocytes in the
area
 Systemic hemosiderosis
 Mononuclear phagocytes of the liver, bone marrow,
Pigments
 Bilirubin
 Normal major pigment found in bile
 Derived from hemoglobin
 Contains no iron
Pathologic Calcification
 Abnormal tissue deposition of calcium salts, together
with smaller amounts of iron, magnesium, and other
mineral salts
 Two forms of pathologic calcification
 Dystrophic calcification
 Local deposition in dying tissues
 It occurs despite normal serum levels of calcium and in the
absence of derangements in calcium metabolism
 Encountered in areas of necrosis
 Coagulative, caseous, or liquefactive type
 Metastatic calcification
 Deposition of calcium salts in otherwise normal tissues
 Hypercalcemia secondary to some disturbance in calcium
Pathologic Calcification
 Morphology
 Calcium salts




Basophilic, amorphous granular, clumped appearance
Intracellular or extracellular, or in both locations
Over time, heterotopic bone may be formed in the focus of
calcification
Lamellations (psammoma bodies)
 Present in benign and malignant conditions
Cellular Aging
 Result of a progressive decline in cellular function and
viability
 Caused by genetic abnormalities and the accumulation
of cellular and molecular damage due to the effects of
exposure to exogenous influences
 Aging is a regulated process that is influenced by a
limited number of genes
 Aging is associated with definable mechanistic
alterations
Cellular
Aging
 The known
changes that contribute to cellular
aging
 Decreased cellular replication
 Accumulation of metabolic and genetic damage
 Cellular life span
 Determined by a balance between damage resulting
from metabolic events occurring within the cell and
counteracting molecular responses that can repair
the damage