CELL DAMAGE

Content
Components and functions of normal cell.
Cellular injury. Characteristics of the concept of injury.
Mechanisms and manifestations of damage of subcellular
structures: plasmatic membrane, mitochondria,
endoplasmatic reticulum, lysosomes, microtubules and
microphilaments, nucleus and cytoplasm.
Principles of classification of cell injuries.
Molecular mechanisms of cell injury.
Antioxidant mechanisms of cells.
Cell death.
Mechanisms of apoptosis.
Ageing.
 Cellular Pathology
All organ injuries start with structural
or molecular alterations in cells
concept began by Virchow in 1800's.
NORMAL CELL
 NORMAL CELL
Present day study of disease
attempts to understand how
cells react to injury.
at the subcellular or molecular level.
all cells share the basic organelles for the synthesis of:

proteins lipids carbohydrates energy

production
transport of ions and other substances.
to understand pathology, review normal structure and
function of cells.
you cannot appreciate the abnormal
before you understand the normal
 Plasma membrane
phospholipid bilayer with embedded
proteins / glycoproteins / glycolipids.
semipermeable membrane with pumps
for ionic / osmotic homeostasis
 Membrane damage
(main causes)
1. Lipid peroxidation (LPO), increased generation of free
radicals
2. Activation of phospholipases and lysosomal hydrolytic
enzymes
3. Membrane damage via amphiphilic, detergent substances
4. Cell swelling membrane tension, rupture
5. Inhibition of repair of the damaged membrane compounds
(blockage of de novo synthesis)
6. Immune complex influence the membrane macromolecules
7.Conformation disorders of macromolecules
Damage to lipid bilayer damage to phospholypase,
lipase activities of the membrane
 nuclear envelope /
nuclear pores Nucleus
chromatin
(euchromatin vs
heterochromatin)
nucleolus
(synthesis of
ribosomal RNA)
 Mitochondria
inner & outer membranes, cristae
intermembranous and inner matrix
compartments
oxidative phosphorylation (main source
of ATP)
 Endoplasmic reticulum (ER),
Ribosomes & Golgi Apparatus
Rough (RER) vs smooth (SER)
endoplasmic reticulum
Ribosomes (free in cytosol or
attached to RER)
Polysomes (threaded by
mRNA).
Condensing vacuoles/
secretory vesicles
 Enzymatic (acid Lysosome
hydrolases) digestion
of materials in the
cell
Endocytosis
Phagocytosis /
phagosome;
Pinocytosis /
pinocytotic vesicle;
Receptor-mediated
endocytosis
 Enzymes Peroxisome
(ex.
catalase,
oxidases) !
metabolism
of hydrogen
peroxide &
fatty acid
 Cellular functions
1. Movement muscle cells can generate forces that produce motion.
2. Conductivity is the main function of nervous cells. Conduction as a
response to a stimulus is manifested by a wave of excitation, an electrical
potential.
3. Metabolic absorption all cells take in and use nutrients and other
substances from their environment.
4. Secretion certain cells are able to synthesize new substances and secrete
them.
5. Excretion all cells are able to rid themselves of waste products resulting
from the metabolic breakdown of nutrients.
6. Respiration (oxidation) cells absorb oxygen which is used to transform
nutrients into energy in the form of ATP (in mitochondria)
7. Reproduction tissue growth occurs as cells enlarge and reproduce
themselves.
8. Communication is critical for all the other functions listed above enabling
the survival of the society of cells. Constant communication allows the
maintenance of a dynamic steady state.
 Cell Injury
Cell injury is defined as such a change in cell structure, metabolism,
physico-chemical properties and function which leads to impairment of its
vital activity

1. Cause intrinsic, extrinsic

infectious, non infectious
2. The type of influence.
direct mediated
acute chronic by the course
reversible irreversible
3. Manifestations of injury.
a. specific b. stereotypical (non specific)
4. In dependence on the pathogenically mechanisms of cells
damage divide on: a) violent (forced); b) cytopath(ogen)ic injury.
REVERSIBLE CELL INJURY
It occurs when
environmental
changes exceed the
capacity of the cell to
maintain normal
homeostasis.
If the stress is
removed in tissue or
if the cell withstand
the assault the injury
is reversible
 IRREVERSIBLE CELL INJURY
If the stress remains the severe, the cell
injury becomes irreversible and lead to cell
death
 Comparative analysis of reversible and irreversible cell injury
Reversible injury Inreversible injury
1. Mitochondrial oxygenation
1. Membrane damage
2. ATP
a. Loss of phospholipids
3. Na+K+ pump glycolysis
b. Cytoskeleton injury
lactate , pH
c. Free radical
4. Intracellular Na +, Ca2+ ,
extracellular K+
d. Lysis of lipids
a. nuclear chromatin
5. H20 shrinking 2. Ca2+ influx
b. lysosomal swelling
6. Acute cell swelling Increased Ca2+ load of mitochondria
Enlargement of
endoplasmic reticulum Uncoupling of oxidative phosphorilation

Ribosome order 3. Release of cytoplasmic enzymes (LDG)

Protein synthesis 4. Release of lysosomal enzymes

Impaired lipid deposition 5. Autophagy

The main causes of cell injury
Internal stresses

metabolic imbalances, nutritional deficiencies or

excesses
genetic abnormalities
acquired derangements hypoxia impairment in aerobic
tissue respiration, ischemia decrease in blood supply

External

physical agents (mechanical injury, high and low

temperature, radiation, electrical shock, sudden fluctuations of
the barometric pressure, acceleration, etc)
natural toxins, venoms
drugs, "chemicals" (Paracelsus) abundant oxygen,
increase in glucose, high doses of dietary salt, poisons,
insecticides, carbon monoxide, asbestos, drugs, social
stimulators, e.g. alcohol, narcotics.
 Cell injury signs
Morphological Functional

a. Swelling a. Decrease in function

b. Dystrophy b. Cellular
c. Thesaurismosis 1. Increase in permeability
2. Cytoplasmic enzymes
d. Dysplasia
leakage to the blood
e. Necrosis c. Metabolic derangements
f. Autolysis d. Injury mediators
e. Synthesis impairments
f. Electrolyte balance disorders
Molecular mechanisms of cell injury
1. Lipid:
1) free oxidation of lipids (FOL), Increased free radical
generation and lipid peroxydation oxidative stress
2) activating of phospholipase and
3) detergent action of free fat acids.
2. Impairment in calcium homeostasis (calcium stress)
3. Electrolyte-osmotic balance disorders
4. Acidosis (intracellular, extracellular)
5. Protein disorders enzymatic derangements
6. Nucleic acid disorders (transcription, translation, DNA
repair disorders) nucleic acid stress
7. Violation of power providing of cell.
 A common "final FREE RADICALS
pathway" in a variety of
forms of cell injury,
including injury brought
about by inflammatory
cells, is generation of
free radicals, i.e.,
molecular species with a
single unpaired electron
available in an outer
orbital.
Single free radicals
initiate chain reactions
which destroy large
numbers of organic
molecules
 Formation, Function, Types of Free Radicals
High Concentration
Ionizing Radiation of O2 Oxidase Reactions
Prod. Superoxide &
1. NADPH oxidase in the
Produces Hydroxyl FRs Hydroxyl FRs
PMN & monocyte cell
Prod. Hydrogen membrane
Damaged Peroxide (H2O2)
Mitochondria Myeloperoxidase
Prod. Superoxide FRs Hypochlorouse acid
Metals
2. Xanthine oxidase is a
(e.g. iron, copper)
ROS generate FRs
Drugs Prod. Hydroxyl FRs Prod. Superoxide FRs
(Fenton reaction)
(e.g. acetaminophen)
Conv. to acetaminophen Nitric Oxide
FRs attack a
FRs in the liver FRs prod. by macrophages molecule & steal
Carbon & endothelial cells its electron
tetrachloride Intima of elastic &
muscular arteries FRs : damage
Conv. to CCl3 FRs
membranes & DNA
in the liver LDL are oxidized by FRs,
lead to atherosclerotic pl.
 1. Oxidation of FREE-RADICAL
unsaturated fatty acids
in membranes ("lipid GENERATION
peroxidation", etc.)
* Basic biologists:
These are the same
reactions that make
unsaturated fats turn
rancid.
2. Cross-linking of
sulfhydryl groups of
proteins.
3. Genetic mutations Ionizing radiation: homolytic break of covalent
bonds in water, DNA and other biomolecules
ionizing
radiation
H2O OH + H
h
 Free radicals may be
generated in the
following ways:
1. By absorbing radiant energy (UV,
x-rays; striking water, these generate a
hydrogen atom and a hydroxyl radical;
when hydrogen peroxide contacts ferrous iron,
it is cleaved into two hydroxyl radicals (* the Fenton reaction).
2. As part of normal metabolism (for example, xanthine
oxidase and the P450 systems generate superoxide; our
white cells use free radicals to attack and kill invaders)
3. As part of the metabolism of drugs and poisons (the
most famous being CCl3.-, from carbon tetrachloride; even
O2 in high concentrations generates enough free radicals to
gravely injure the lungs).
 Reactive oxygen and nitrogen species
ROS/RNS
Free radical each molecule or its fragment, which can
exists independently and contains one or two unpaired
electrons
Reactive oxygen species - species, which contain one or
more oxygen atoms and are much more reactive than
molecular oxygen
superoxide radical

hydroperoxyl radical

Free radicals
hydroxyl radical
ROS/RNS

nitric oxide

hydrogen peroxide
 Some characteristics of ROS
Half-
ROS Symbol Properties
life
poor oxidant, quite toxic & is deployed by the
Superoxide
O2- 10-6 s immune system to kill invading
radical
microorganisms.
Hydroperoxyl
HO2 stronger oxidant than O2-
radical
Hydrogen
H2O2 minits oxidant, diffuses across membranes
peroxide
Hydroxyl extremely reactive, diffuses only to very low
OH 10-9 s
radical distance

Alkoxyl radical LO 10-6 s less reactive than OH, but more than ROO

Peroxyl radical LOO 10-2 s weak oxidant, high diffusability

Singlet oxygen 1O 10-6 s powerful oxidizing agent

2
 Cellular sources
of ROS
myeloperoxidase transient metals
(oxidative burst: phagocytes) xanthine oxidase
hemoglobin
riboflavin
catecholamines

Cytochrome P450

electron
transport chain
oxidases
flavoproteins

lipid peroxidation
NADPH oxidase (oxidative burst: phagocytes)
Oxidative Stress and Oxygen Free Radicals
Superoxide anion (O2-)
may be formed via the
cytochrome P450 system,
found in hepatocytes,
which metabolizes many
drugs and toxins it can
be removed by superoxide
dismutase
Hydrogen peroxide
(H2O2) removed by
catalase or glutathione
peroxidase
Hydroxyl radical (.OH)
initiates lipid per-oxidation
and DNA damage
Pathologic Effects of Free Radicals
Hydroxyl radicals initiate lipid
peroxidation, leading to severe damage to
membranes, especially in mitochondria
Hydroxyl radicals cause oxidative
damage to proteins, which may damage
enzymes and structural proteins
Hydroxyl radicals can induce single- and
double-strand breaks in DNA, cross-linking,
and formation of adducts. This could lead to
defective transcription, accelerated cell aging,
or malignant transformation of the cell to a
cancerous phenotype
Formation of ROS and peroxynitrous acid in
phagocytic vacuole of phagocytes

SOD superoxid dismutase

MPO - myeloperoxidase
 Cellular sources of ROS - examples
e- e- + 2H+ e- + H+ e- + H+
Electron transport system:
O2 O2- H2O2 H2O + OH H2O

Autooxidation of hemoglobin: Hb(Fe2+)-O2 metHb(Fe3+) + O2-

Fenton reaction: Fe2+ + H2O2 Fe3+ + HO + OH-

Oxidative damage to lipids
Lipid peroxidation (LPO)
Formation of lipid (alkyl) radical initiated
by ROS:

ROS
LH + HO L + H2O

Alkyl radical react with O2 to produce

peroxyl radical:

L + O2 LOO

Peroxyl radical attacks another poly-

unsturated FA to produce new alkyl
radical and lipid peroxide:

LH + LOO L + LOOH
 Mechanisms of cell injury mediated by ROS and RNS
ROS a RNS

Lipids Proteins DNA

Lipid peroxidation Modification of aa, DNA damage

fragmentation and aa amino
aggregation of proteins
acids
Membrane damage

Loss of membrane Damage to Ca2+ and other Altered gene

integrity ion transport systems expression

Inability to maintain Depletion of ATP

normal ion gradients

Activation/deactivation of
various enzymes

Cell injury/
Cell death
 Antioxidants and
secondary defense
systems
Prevent ROS formation
Eliminate radicals by formation of
nonradicals or less reactive radicals
Repair dameged molecules and cell
structures
Expression of genes coding for
antioxidant enzymes

Antioxidants and secondary

defense systems

Enzyme Water-soluble
antioxidants Lipid-soluble

Nonenzymatic Endogenous
antioxidants Present in diet

Chelating agents
Enzymes of
repair and de
novo synthesis
of damaged
molecules
 Antioxidant system

I. Enzymatic II. Non enzymatic

a. Superoxide dismutase a. -tocopherol
(SOD)
b. Ascorbic acid
b. Catalase
c. Glutathione peroxidase ( c. Cysteine, mannitol,
glutathione, GSH) serotonin, selenium,
d. ubiquinone riboflavin, retinol,
carotinoids,
Glutathione system d. Reduced glutathione
Vitamins
Microelements (Selenium)
Amino acids with SH group

Increased production of ROS + decreased activity of antioxidant

system = oxidative stress
 Antioxidant enzymes

ENZYME TISSUE SITE ______

Superoxide dismutase
Cu/Zn SOD primarily cytosol, nucleus
Mn SOD mitochondria
EC SOD extracellular fluid
Catalase peroxisomes
Glutathione peroxidase GPx cytosol, mitochondria
Glutathione reductase GRed cytosol, mitochondria
_________________________________________________
 Antioxidant enzymes
SOD scavenges superoxide radical:

SOD
O2- + O2- +
+ 2H H2O2 + O2
Catalase decomposes hydrogen peroxid in peroxisomes :

2H2O2 2H2O + O2
Glutathione peroxidase (GPx) decomposes H2O2 and lipid peroxides in
cytosol and mitochondria by help of GSH, NADPH and
glutathionereductase (GRed):

LOOH 2GSH NADP+

GPx GRed

H2O + LOH GSSG NADPH

 Nonenzymatic antioxidants
Endogenous antioxidants - Synthesized in the body
bilirubn
glutathione and other thiocompounds (thioredoxin)
uric acid
coenzyme Q (Ubichinon-10/Ubichinol-10)
lipooic acid
melatonin
sex hormones
2-oxoacids (pyruvate, 2-oxoglutarate)
dipeptides containig His (carnosine, anserine)
albumin (-SH groups)

Dietary antioxidants
ascorbic acid
vitamine E
carotenoids
flavonoids plant phenols (catechin, quercetin etc)

Synthetic antioxidants
N-acetylcystein (scavenger of ROS), deferoxamine (chelator),
alopurinol (inhibitor of XO), acetyl salicylic acid (feritine synthesis)
 Vitamin E
(fat-soluble)
1) Prevents lipid
peroxidation in cell
membranes;
2) Neutralizes
oxidized LDL
Vitamin C (water-
soluble)
1) Neutralizes FRs
produced by
pollutants and
cigarette smoke
Smokers have
levels of Vit.C
because they are
used up in
neutralizing FRs
derived from cigarette
smoke.
2) Best neutralizer of
hydroxyl FRs.
 Oxidative Stress
Absorption of radiant energy with sufficient
energy to initiate the radiolysis of water, i.e.,
ionizing radiation, leads to the following
reaction:
. .
H O H + OH
2
 ACCUMULATION OF OXYGEN-
DERIVED FREE RADICALS
(OXIDATIVE STRESS)
Cell injury induced by free radicals, particularly reactive
oxygen species, is an important mechanism of cell damage in
many pathologic conditions, such as chemical and radiation
injury, ischemia-reperfusion injury (induced by restoration of
blood flow in ischemic tissue), cellular aging, and microbial
killing by phagocytes.
Free radicals are 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, such as inorganic or organic chemicals
proteins, lipids, carbohydrates, nucleic acidsmany of which
are key components of cell membranes and nuclei.
Moreover, free radicals initiate autocatalytic
reactions, whereby molecules with which they react
are themselves converted into free radicals, thus
propagating the chain of damage.
 ACCUMULATION OF OXYGEN-DERIVED
FREE RADICALS (OXIDATIVE STRESS)
Reactive oxygen species (ROS) are a type of oxygen-derived free
radical whose role in cell injury is well established.
ROS are produced normally in cells during mitochondrial respiration
and energy generation, but they are degraded and removed by
cellular defense systems.
Thus, cells are able to maintain a steady state in which free radicals may
be present transiently at low concentrations but do not cause damage.
When the production of ROS increases or the scavenging systems are
ineffective, the result is an excess of these free radicals, leading to a
condition called oxidative stress.
Oxidative stress has been implicated in a wide variety of pathologic
processes, including cell injury, cancer, aging, and some degenerative
diseases such as Alzheimer disease.
ROS are also produced in large amounts by leukocytes, particularly
neutrophils and macrophages, as mediators for destroying microbes, dead
tissue, and other unwanted substances.
Therefore, injury caused by these reactive compounds often accompanies
inflammatory reactions, during which leukocytes are recruited and
activated.
 Reperfusion Injury and
Activated Oxygen
Toxic oxygen species are
generated, not during the
ischemia itself, but during
reperfusion, hence the term
reperfusion injury
This has clinical relevance,
since reperfusion of heart
muscle is commonly achieved
with per-cutaneous angioplasty.
Patients more than 20
minutes post-infarction
are at risk for reperfusion
injury.
Reperfusion Injury and Activated Oxygen
Generation of oxygen free radicals occurs from
parenchymal and endothelial cells and from infiltrating
leukocytes
Reactive oxygen species can further damage
mitochondrial membranes, which precludes generation of
ATP and leads to cell death
Ischemic injury is associated with inflammation, as a
result of the production of cytokines and increased
expression of adhesion molecules by hypoxic parenchymal
and endothelial cells
Recent data suggest that activation of the
complement pathway may contribute to ischemia-
reperfusion injury.
The complement system is involved in host defense and is
an important mechanism of immune injury. Knockout mice
lacking several complement proteins are resistant to this
type of injury.
Mechanisms of membrane damage in
ischemia and reperfusion
Cellular response to ischemia. ATP production by mitochondria relies on an adequate supply of oxygen
and of energy substrates such as glucose. Mitochondrial function is therefore compromised soon after
failure of blood supply, resulting in failure of production of ATP. One consequence of lack of ATP is failure
of ATP-dependent membrane pumps, which normally pump sodium (and with it water) out of cells. Failure
of membrane ion pumps leads to accumulation of sodium and water in the cell cytoplasm, with disruption
of internal membrane systems. Failure of internal membrane pumps also allows free calcium to enter the
cytosol, where it activates many destructive enzyme systems. Structural damage to internal membranes
and the cytoskeleton, coupled with lack of ATP, leads to impairment of key synthetic pathways, including
those of protein synthesis. Rupture of lysosomes and intracellular liberation of powerful hydrolytic
enzymes, active at a low pH, brings about further cellular dissolution.
 Summary:
Reperfusion generates free radicals from
parenchymal, endothelial, and
inflammatory cells in the injured tissue,
often producing more cellular injury than
the initial ischemia, largely due to
membrane damage
Be able to identify grossly and
microscopically: Myocardial infarction,
renal infarction (pale infarct), gangrene
 Activating of membrane
phospholipase
The surplus activating of
phospholipase A2 has an important
value in pathogenesis of cell damage.
This enzyme carries out breaking up of
phospholipids of cell diaphragms to
a) unsaturated fatty acids and
b) lysophospholipids.
Unsaturated fatty acids, in particular
arachidonic acid, under act of certain
enzymes transform to biologically
active matters - eicosanoids.
Lysophospholipids have ability to
create a micelle and they are very
strong detergents. High concentration
of ions of Ca2+ in a cytoplasm is the
basic reason of activation of
phospholipase A2.
 Detergents action of surplus of free fatty acids
Free fatty acids in large concentrations similarly as lysophospholipids
damage bimolecular lipid layer of cellular membranes.
It is possible to select 4 basic mechanisms of increase of maintenance
of free fatty acids in a cell:
1) Entering of free fatty acids is increased cell in the presence of high
level of lipids in blood, that is observed during activating of processes of
lipolysis in fatty tissues (stress, diabetes).
2) Formation of free fatty acids is increased in lysosomes (at
atherosclerosis).
3) Liberation of free fatty acids is increased from phospholipids of
cellular membranes under act of phospholipases.
4) The use of free fatty acids is broken by a cell as energy sources
(diminishing of enzymes is a beta-oxidization and to the Krebs cycle
during hypoxia).
 Ca2+ - homeostasis disorders
1. Increased entrance
a. Hypercalcia
b. Impaired barrier function of the membranes
(increase in peroxidation processes)
2. Impaired efflux Ca-accumulation
a. Ca-pump disorder, Ca-channels impairments
disorders in synaptic plasticity
b. Ca2+- Na+ exchange mechanism disorder
Calcium functions as a messenger for Calcium
the release of many intracellular enzymes.
Normally, intracellular calcium levels are
mechanisms
kept extremely low compared with
extracellular levels. These low
intracellular levels are maintained by
energy-dependent, membrane-
associated calcium/magnesium
(Ca2+/Mg2+) ATPase exchange systems.
Ischemia and certain toxins lead to an
increase in cytosolic calcium because
of increased influx across the cell
membrane and the release of calcium
stored in the mitochondria and
endoplasmic reticulum.
The increased calcium level activates a
number of enzymes with potentially
damaging effects.
The enzymes include the phospholipases
responsible for damaging the cell
membrane, proteases that damage the
cytoskeleton and membrane proteins,
ATPases that break down ATP and hasten
its depletion, and endonucleases that
fragment chromatin.
 The increase of
concentration of
Ca2+ ions causes in
a cytoplasm:

a) contraction of
fibrillar structures
of cell
(myofibrillar);

b) activating of
phospholipase A2

c) violation of
connection between
the processes of
oxidization and
phosphorylation.
Cytosolic free calcium is a potent destructive agent.
 Pathogenetic effects of Ca stress

Pathological stimuli

Mitochondria Ca2+ Endoplasmic

Ca2+ reticulum Ca2+

Increase of cytosolic Ca2+

ATP-ase Phospholipase Proteinase Endonuclease

Decrease Reduction of Destruction of Segmentation of

ATP phospholipids membrane and nuclear chromatin
cytoskeleton
proteins
 Electrolyte-osmotic
mechanism of cell
damage.
It connect with Na+ and K+ ions.
Aligning the concentrations of ions of
Na+ and K+ on either side of cell
membrane (multiplying maintenance
of Na+ and degree of maintenance of
K+ is in a cytoplasm) in the basis
can have two mechanisms:
1) strengthening of ions diffusion
through a cell membrane from
extracelullar concentration and
electric gradient;
2) violation of mechanisms of active
transport of Na+ and K+ (Na-K-
pump).
The first mechanism will be realized in
the conditions of violations water-
electrolyte exchange (hypernatremia,
hypokaliemia) and violation of barrier
function of cell membrane (increase of
its ionic permeability).
Disorders of function of Na-K-
pump can be conditioned the
deficit of P in a cell,
multiplying maintenance of
cholesterol in lipids bilayer of
membrane (for example, at
atherosclerosis), by the action
of a number of specific
inhibitors Na-K-ATP-elements
(for example, strophanthine
(ouabaine)).
A change in maintenance of
ions of Na+ and K+ is caused:
a) loss of electric potential of
cellular diaphragm;
b) it was swollen cells
(oedema);
c) osmotic injury of cellular
membranes, which is
accompanied the increase of
their permeability.
 Water-ion balance impairment
Na+ - K+ pump disorder leads to:

1. Rest potential impairment changes

in threshold, action potential,
impulse transduction
2. Swelling of the cell
3. Osmotic tension of the membrane
4. Impairment of membrane barrier
function
5. Impaired electrogenesis (ECG, EEG)
 As the cell membrane is highly permeable to water and water
follows the osmotic gradient, the cell depends on osmotic
equilibrium to maintain its volume.
In order to counterbalance the high intracellular concentration of
proteins, amino acids, and other organic substrates, the cell
lowers the cytosolic ionic concentration. This is done by
Na+/K+-ATPase, which pumps Na+ out of the cell in exchange
for K+.
Normally the cell membrane is only slightly permeable for Na+,
but highly permeable for K+, so that K+ diffuses out again.
This K+-efflux creates an inside negative potential which drives
Cl out of the cell. In this ionic shift, which uses up adenosine
5"-triphosphate (ATP), reduction of the cytosolic concentration
of Na+ and Cl (adding up to ca. 230 mosm/L) is much greater
than the rise in cytosolic K+ concentration (ca. 140mosm/L).
Reduction in intracellular Na+ concentration by Na+/K+-
ATPase is necessary not only to avoid cell swelling, but also
because the steep electrochemical gradient for Na+ is utilized
for a series of transport processes.
The Na+/H+ exchanger eliminates one H+ for one Na+, while
the 3 Na+/Ca2+ exchanger eliminates one Ca2+ for 3 Na+.
Na+-bound transport processes also allow the (secondarily)
active uptake of amino acids, glucose, etc. into the cell.
Lastly, depolarization achieved by opening the Na+ channels
serves to regulate the function of excitable cells, e.g. the signal
processing and transmission in the nervous system and the
triggering of muscle contractions.
 As the activity of Na+-transporting carriers and channels
continuously brings Na+ into the cell, survival of the cell requires
the continuous activity of Na+/K+-ATPase.
This intracellular Na+ homeostasis may be disrupted if the
activity of Na+/K+-ATPase is impaired by ATP deficiency
(ischemia, hypoxia, hypoglycemia).
The intracellular K+ decreases as a result, extracellular K+
rises, and the cell membrane is depolarized.
As a consequence, Cl enters the cell and the cell swells up.
These events also occur when the energy supply is compromised,
or when Na+ entry exceeds the maximal transport capacity of the
Na+/K+-ATPase.
Numerous endogenous substances (e.g., the neurotransmitter
glutamate) and exogenous poisons (e.g., oxidants) increase the
entry of Na+ and/or Ca2+ via the activation of the respective
channels.
The increase in intracellular Na+ concentration not only leads to
cell swelling, but also, via impairment of the 3Na+/Ca2+ exchanger,
to an increase in cytosolic Ca2+ concentration.
Ca2+ produces a series of cellular effects; among others it
penetrates into the mitochondria and, via inhibition of mitochondrial
respiration, leads to ATP deficiency.
 Mechanism
of acidosis
Reasons of cell acidosis:
1) there is the surplus entering of
H+ ions in cell from a extracelullar
environment;
2) formation of sour products in a
cell during glycolysis activating
(lactic acid lactate), violations of
Krebs cycle (carbons acids),
hydrolitic breaking
up(disintegration) phospholipids
of cellular membranes (free fat
acids, phosphoric acid) and
others;
3) violation of fastening of free H+
ions is as a result of insufficiency
of the buffer systems of cell;
4) violation of move out H+ ions
from a cell. Reason of this is
disorders of Na-H-exchange
mechanism, and also in the
conditions of the broken local
circulation of blood in tissue.
 Mechanism
of acidosis.
Conclusions of acidosis of cell:
a) change conformation of
protein molecules with
violation of them function and
properties;
b) increase of permeability of
cellular membranes;
c) activating enzymes of
lysosomes.
If there is a lack of O2, energy
metabolism switches to
anaerobic glycolysis.
The formation of lactic acid,
which dissociates into lactate
and H+, causes cytosolic
acidosis that interferes with
the functions of the
intracellular enzymes, thus
resulting in the inhibition of
the glycolysis so that this last
source of ATP dries up.
 Proteins Mechanisms
The proteins mechanisms of cell damage
contain:
1) inhibition enzymes (reverse and irreversible);
2) denaturation, that violation of native structure of
albumins molecules as a result of conditioned the
break connections of changes of the second or
tertiary structures of proteins;
3) proteolisis, that is carried out under the action of
lysosomal enzymes.
Proteins used in diagnosis of tissue damage
by blood testing
 The nucleic mechanisms
Its conditioned with violations
of nucleic acids (DNA, RNA).
Its disturbance of replication,
transcription and translation
processes.
Thus, universal mechanisms
of increase of permeability of
cellular membranes are:
1) activating of FOL;
2) activating of phospholipases;
3) osmotic breaking up
membranes;
4) adsorption of albumens is on
a membrane.
The damage of
mitochondrias is
accompanied:
1) oppressing the
processes of the
cellular breathing,
2) violation of
connection between
the processes of
oxidization and
phosphorilation.
DAMAGE TO DNA AND PROTEINS
Cells have
mechanisms that
repair damage to
DNA, but if this
damage is too severe
to be corrected (e.g.,
after exposure to DNA
damaging drugs,
radiation, or oxidative
stress), the cell
initiates a suicide
program that results
in death by apoptosis.
A similar reaction is
triggered by
improperly folded
proteins, which may
be the result of
inherited mutations or
external triggers such
as free radicals.
Because these
mechanisms of cell
injury typically cause
apoptosis, they are
discussed later in the
chapter.
 Before concluding our discussion of the mechanisms of cell injury, it is useful to
consider the possible events that determine when reversible injury becomes
irreversible and progresses to cell death. The clinical relevance of this question is
obviousif we can answer it we may be able to devise strategies for preventing cell
injury from having permanent deleterious consequences.
The clinical relevance of this question is
obviousif we can answer it we may be
able to devise strategies for preventing cell
injury from having permanent deleterious
consequences. However, the molecular
mechanisms connecting most forms of cell
injury to ultimate cell death have proved
elusive, for several reasons. The point of
no return, at which the damage becomes
irreversible, is still largely undefined, and
there are no reliable morphologic or
biochemical correlates of irreversibility. Two
phenomena consistently characterize
irreversibilitythe inability to reverse
mitochondrial dysfunction (lack of
oxidative phosphorylation and ATP
generation) even after resolution of the
original injury, and profound
disturbances in membrane function. As
mentioned earlier, injury to lysosomal
membranes results in the enzymatic
dissolution of the injured cell that is
characteristic of necrosis.
 DAMAGE TO DNA AND
PROTEINS
Leakage of intracellular proteins through the
damaged cell membrane and ultimately into the
circulation provides a means of detecting tissue-
specific cellular injury and necrosis using blood
serum samples.
Cardiac muscle, for example, contains a
specific isoform of the enzyme creatine
kinase and of the contractile protein
troponin;
Liver (and specifically bile duct epithelium)
contains an isoform of the enzyme alkaline
phosphatase; and hepatocytes contain
transaminases.
Irreversible injury and cell death in these tissues
are reflected in increased levels of such proteins
in the blood, and measurement of these
biomarkers is used clinically to assess damage
to these tissues.
After damage to mitochondrial membranes there is failure of ATP production and loss
of the normal membrane potential of the mitochondrion.
The mitochondrial membrane pores (PTPC megachannels) open and release proteins
into the cytosol, which can cause apoptosis, as described below.
If many mitochondria in a cell fail, causing a catastrophic reduction in ATP production,
the cell will die by a non-apoptotic route.
 Mechanisms of cell death.
Terminology:

Necrosis: Morphologic changes seen in

dead cells within living tissue.
Autolysis: Dissolution of dead cells by the
cells own digestive enzymes. (not seen)
Apoptosis: Programmed cell death.
Physiological, for cell regulation.
 Mechanisms of cell death
Apoptosis is a pathway of cell death that is
induced by a tightly-regulated suicide program in
which cells destined to die activate enzymes that
degrade the cells own nuclear DNA and nuclear
and cytoplasmic proteins
Developmental morphogenesis
Radiation
Immune system regulation
Viral infections
Cancers
Toxins
The process was recognized in 1972 by the distinctive morphologic
appearance of membrane-bound fragments derived from cells, and
named after the Greek designation for falling off.
 General biochemical mechanisms
Defects in plasma membrane permeability.
Oxygen deprivation or generation of reactive
oxygen species (free radical).
Loss of calcium homeostasis.

Mitochondrial

damage.
Chemical injury

Genetic variation
 Apoptosis
Receptors Fas, (Apo1, Trigger action of cytokines and
CD95), TNF hormones
RIP
FAXD
TRADD Provider molecules of Causes: moderate injury during the
apoptotic signal to the cell
entrance of O2, different actions

Conduction of intracellular Repression of

apoptotic signal (Bad1) Expression of apo- apoptosis blockaders
ptotic genes (bax, BCL-2, X
p53, Ced-3, p21)

Activation of cellular 1) Activation of death receptors-DR,

cysteine photolytic 2) Mitochondria dependent pathway: Output
caspases + nucleases of Ca, proteins, cytochrome C

Lysis of cellular proteins

Fragmentation of nucleus and cytoplasm Activated
Apoptotic bodies caspases
Autophagocytosis
Without inflammation
 Morphologic features of Apoptosis

1. Cell shrinkage.
2. Nuclear chromatin condensation
and fragmentation
3. Apoptotic bodies formation
4. Phagocytosis of apoptotic bodies
by adjacent cells or macrophages.
5. Intacted membrane.
 Role of apoptosis in physiology and pathology
1. During embryogenesis ( implantation, organogenesis, growth,
metamorphosis)
2. In adults hormone dependent involution (during menstrual cycle,
menopause, atrophy of prostate and breasts)
3. Destruction of cells in the reproducing cellular populations (epithelial
cells of intestine )
4. Cell death in tumor (regression)
5. Death of neutrophils in the active inflammatory process
6. Death of immune cells (B,T- lymphocytes)
7. Death of cytotoxic T- lymphocytes
8. Pathological atrophy in the parenchimatous organs
9. During the some viral infections (hepatitis)
10. Temperate action of various noxious factors
Confocal 3d images of nuclei from nonapoptotic (A) and
apoptotic (B) cells stained with PI

A B
 Morphogenesis of cell injury

Atrophy - physiological, pathological

Types - local, general, dysfunctional, due to
compression, blood circulation, neurogenous

Necrosis, necrobiosis (protein dystrophy)

Autolysis, caryorhexis, caryolysis, plasmorrhexis,

plasmolysis, demarcation zone, inflammation
around the necrosis
 Morphology of necrotic cells
Cell and nuclear swelling
Vacuolization of cytoplasm
Patchy chromatin condensation
Mitochondrial swelling
Plasma membrane rupture
Dissolution of chromatin
Attraction of inflammatory cells
Relationships between sublethal and lethal cell damage. Following sublethal
damage a cell may recover or, with persistence of the damaging stimulus, cell death
may result. The sequential structural changes of cell death are termed 'necrosis'.
Comparison of cell death by apoptosis and necrosis
Relationships between sublethal and lethal cell damage. Normal cells that are subject to a damaging
stimulus may initiate apoptosis or may become sublethally damaged. If the stimulus abates, cells
may recover by resynthesis of proteins and elimination of damaged components. If a damaging
stimulus continues, either cells die through apoptosis or, when critical cell damage takes place,
mainly through critical lack of ATP, cells die and undergo necrosis. Massively damaging stimuli, e.g.
great heat or strong acids, cause immediate coagulation of proteins and death of cells.
 Adaptive responses resulting in increased tissue mass. Increased functional demand
or endocrine stimulation are what usually cause hypertrophy and hyperplasia. These
new patterns of growth are stable while the causative stimulus persists, but once it is
removed the tissue returns to a normal pattern of growth.
Pathology
of elderly
 Ageing:

Progressive time related loss of

structural and functional
capacity of cells leading to
death

Senescence, Senility, Senile

changes.
Ageing of a person is intimately
related to cellular ageing.
 Factors affecting ageing:
Stress Diminished stress
Infections response.
Diseases Diminished immune
response.
Malnutrition
Good health.
Accidents
 Cellular mechanisms of
ageing
Cross linking proteins &
DNA.
Accumulation of toxic by-
products.
Ageing genes.
Loss of repair
mechanism.
Free radicale injury
Telomerase shortening.
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Thank you for attention!