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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:
External
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
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
ROS
LH + HO L + H2O
L + O2 LOO
LH + LOO L + LOOH
Mechanisms of cell injury mediated by ROS and RNS
ROS a RNS
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
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
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):
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
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
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