nervous system and cell damage by heavy metals and some other chemical compounds and its symptoms.

1. PRESENTATION
ON
NEUROTOXICITY & CYTOTOXICITY
PRESENTED BY
ANGOM BALESHWOR SINGH
AEMMA8-02

2. Father of Neurotoxicology
Toshio Narahashi

3. NEUROTOXICITY
• Any adverse change in the chemistry, structure
or function of the nervous system during
development or at maturity, following exposure
to a chemical , physical, biological influences.
• The toxic substance which causes neurotoxicity
is called neurotoxin.

4. NERVOUS SYSTEM
• Extremely complex
network, provides the
structural basis for
electrochemical
processes that are
responsible for multiple
functions.
• These functions are
sensation, memory,
speech, locomotion, &
unconscious hormonal
& autonomic
processes, transmit
information.

6. Peripheral Nervous System
• Consists of both sensory (transmit
information from sensory organs like eyes,
skin, ears to the CNS) & Motor neurons (
transmit information from CNS to muscles
and glands).
• The autonomic nervous system consists of
sensory & motor neurons that runs between
the CNS. It is responsible for monitoring &
regulating the internal environment.

7. Neuron

8. NEUROTRANSMISSION

9. NEUROTRANSMISSION

10. ACTION POTENTIAL

11. BLOOD BRAIN BARIER
• Protective network that filters blood flow to
the brain.
• Composed of dense and tightly packed layer
of endothelial cells
• Regulates the access of physiological
substances xenobiotics to CNS.
• Effectively impaired the transfer of charge,
high molecular weight compounds but not
lipid – soluble compounds.

12. CONT…
• Several transport mechanisms, like
monocarboxylic acid transporters or organic
anion transporters present in endothelial cells
mediate active transport through BBB.
• Whereas ABC transporters mediate the excretion
of compounds out of epithelial cells.
• Leaking of BBB makes brain vulnerable to toxins.
• Different potential points for entry of toxins are
such areas which are not completely protected by
BBB like hypothalamus, pineal body, motor &
sensory nerve terminals etc.

14. BBB can be broken down by
• Hypertension: High blood pressure opens the BBB
• Hyperosmolarity: High concentration of solutes can
open the BBB.
• Infection: Exposure to infectious agents can open the
BBB.
• Trauma, Ischemia, Inflammation, Pressure: Injury to
the brain can open the BBB.
• Development: The BBB is not fully formed at birth.
• Compounds like nitropropionate, dinitrobenzene, or
metronidazole harm endothelial cells , lead to porous
and permitting entry of toxin.

15. THE NERVOUS SYSTEM – SITE OF ATTACK
FOR TOXINS
• All part of CNS & PNS are potential for toxins.
• Most vulnerable due to complex and prolong
period of development.
• Exposure to neurotoxins mostly occurs through
breathing, ingestion along with food or water, or
contact through skin.
• Ionic & hydrophilic toxins mainly impair the PNS,
since they do not cross the BBB. Lipophilic agents
carried by special transport mechanism will enter
the Brain & effect the CNS.

16. Neurotoxic effect are usually classified
based on the structure of the nerve cell
that is impaired
• Hence , it can be differentiated as:
NEURONOPATHY
AXONOPATHY
DEMYELINATION

17. CELLULAR DAMAGE RESULTING IN
NEUROTOXICITY
• NEUROPATHY:
• Results when nerve cells or neurons are damaged or
destroyed.
• Subdivided into cytoplasmic, nuclear, & post synaptic.
• Loss of neuron to adult brain is irreversible.
• Damage to cell soma is followed by loss of axons & dendrites.
• Initial injury followed by apoptosis or necrosis.
• PNS neuron is possible for regeneration but not in CNS.
• Common toxin : exposure to lead, mercury, arsenic & thalium,
carbon tetrachloride, sniffing glue, CO etc

18. SPECIFIC NEUROTOXINS FOR
NEUROPATHY
Methylmercury :
• Best known neurotoxin associated with neuropathy.
• Cerebellar granular cells are most sensitive.
• Smaller neuron of visual & cerebellar cortex die after
intoxication, leading to central neuropathy.
• Symptoms:- sensory deficits followed by ataxia &
impaired motor coordination.

19. MPTP( 1-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine)
• Damages neurons in brain.
• Formed as a contaminant during the synthesis
MPPP(1-methyl-4-phenyl-4-propionoxypiperidine)
• MPTP results in an irreversible neurodegenerative
syndrome similar to parkinsonism.
• Active metabolite of MPTP,
methylphenylpyridinium(MPP+) selectively
induces neuropathy in dopaminergic neurons of
the substantia nigra, an area of brain important for
motor coordination.

20. PARKINSON’S DISEASE

21. Trimethyltin:-
• A potent biocide that selectively causes neuropathy in
the limbic system of the brain
• This poisoning is characterized by a marked
augmentation of excitability of hippocampal Neurons that
results in cell death.
METHANOL:
• Metabolic production of formic acid.
• It causes destruction of visual nerves, blindness.

22. LEAD
• Ubiquitous toxic metal
• Primary route of exposure is by ingestion
• Source is from lead-based paint, contaminated
drinking water, lead-glazed pottery
• Encephalopathy occurs at blood lead levels of
80-100 μg/dL.

23. CONT…
• Symptoms of encephalopathy include
lethargy, vomiting, irritability, loss of
appetite, and dizziness
– Progression of symptoms lead to ataxia,
reduced level of consciousness, which may
progress to coma and death
– Recovery is often associated with life-long
epilepsy, mental retardation, optic neuropathy,
blindness

24. CONT…
• Chronic toxicity affects PNS; Schwann cell
degeneration
• Mechanisms of toxicity include:
– Impairment of cell-cell connections
– Alterations in neurotransmitter levels
– Disrupts calcium metabolism

25. AXONOPATHIES
• Primary site of toxicity is axon
• Nerve damage classified as a central or peripheral distal
axonopathy.
• Distal axonopathy also known as dying back neuropathy.
• Distal swelling, Degeneration of axon, surrounding
myelin, but cell body remains intact
• Axonopathy is typically gradual in onset, affecting first
the distal regions of long axons & advancing slowly
towards the nerve’s cell body.
• Irreversible in CNS, but reversible in PNS
• Caused by CS2, acrylamide, gold, organophosphorous
esters.

26. DEMYELINATION:-
• Oligodendrocytes of the CNS & Schwann cells of the
PNS produce myelin, which forms layers that electrically
isolates the axons.
• Myelin damage may involve swelling of myelin, primary
demyelination, & secondary demyelination.
• Primary demyelination occurs as a consequence of a
direct attack of chemicals on myelin or myelin producing
cells. This damage is partly reversible.

27. • Secondary demyelination is an active process that
ultimately leads to the destruction of axons. It occurs as
a consequence of a traumatic destruction or toxic
axonopathy, where myelin is digested by macrophages.
• Diphtheria toxin causes segmental demyelination by
damaging the Schwann cell.
• Inorganic lead is a compound that causes peripheral
demyelination, & motor nerves are specially affected
• Triethyltin leads to myelin swelling.

28. EFFECTS OF NEUROTOXIN ON
NEUROTRANSMISSION
• Neurotransmission is especially susceptible to attack by
chemical at several sites
• Synapses are particularly sensitive to chemicals.
• Synaptic function requires the synthesis, storage, & release
of transmitters, the reaction of transmitters with receptors &
ultimately transmitter inactivation.
• Reduced inactivation results in an excess of transmitters,
which cause continuous depolarization of the nerve cell.
• Chemicals that activate receptors by mimicking transmitters
may have agonistic effects, antagonists occupy receptors and
inhibit the neurotransmitters to bind with the receptors

29. CHEMICALS WHICH EFFECT
NEUROTRANSMISSION
• Organophosphate derivatives:- many of them
are potent neurotoxins, functioning by irreversibly
inhibiting acetylcholinesterase. Inhibition of the
inactivation of acetylcholine leads to continuous
depolarization of post synaptic cholinergic receptors &
blocks neurotransmission.
• Domoic acid:- causes short term memory loss, brain
damage & death. It damages the hippocampus and
amygdaloid nucleus.
• Muscimol:- a specific agonist of GABA receptors.
Found in mushrooms.

30.  Botulinum toxin:- attacks one of the
fusion proteins at the neuromuscular
junction, preventing neurotransmitter
vesicles from anchoring to the synaptic
membrane to release acetylcholine. Lethal
dose 200-300pg/kg.
 Tetradotoxin:- Inactivates sodium
channels in nerve cell membranes, inhibits
the transmission of action potentials and
interrupts electrical signaling within
nerves.

31. CLINICAL SIGNS & SYMPTOMS INDUCED
BY NEUROTOXINS
• Hypoxia:- carbon monoxide and cyanide are responsible
for histotoxic hypoxia, the inability of the tissues to use
oxygen. Symptoms are inattentiveness, poor judgement,
memory loss etc.
• Ischemia:- shortage of blood supply to a particular
organ
• Encephalopathies:- Depending on the type and
severity of encephalopathy, common neurological
symptoms are loss of cognitive function, subtle
personality changes, inability to concentrate, lethargy,
and depressed consciousness.

32. NEUROTOXICITY DIAGNOSTIC TEST
• Identification of single endpoint is problematic due to
complexity and integrative nature of NS.
• As a result effects are measured at multiple levels of NS
organisation, including behavioral, neurophysiological,
neurochemical, and neuroanatomical levels.
• Historically, morphological methods have been used to
detect , however, assessments of neurotoxic potential
can be enhanced by combination of morphological and
functional data. It consist of primary and secondary
prevention.
• It may in vivo or in vitro.
• Testing strategy for neurotoxicity was given in 1983 in
risk assessment paradigm.

33. TEST STRATEGY FOR
NEUROTOXICITY
• 1st tier testing: It is to determine whether a chemical has
the potential to produce any neurotoxic effect.
• 2nd tier testing: concerned with the characterization of
neurotoxicity, such as the type of structural or
functional damage produced and degree and location
of neuronal loss. During this test quantitative study of
relation between applied dose & delivery dose and
between dose &biologic response test are involved.
• 3rd tier testing: It is the study of mechanism of action of
chemical agents.

34. CURRENT IN VITRO PROCEDURES
Bioassay test:
• It is a test method employed in measuring response of
living animal or plant tissue to the toxicity of chemical
contaminants.
• Certain no. of individuals of sensitive species are
exposed to specific conc. of the contaminant for
specific period to examine the toxic effect.
• It may be of several types :
• Acute toxicity test(24-96hrs)
• Chronic toxicity test(7 days)
• Static renewal test(exposure to fresh solution
at every 24hr interval)
• Flow through test
• Wide range toxicity test

35. • Result are analysed in terms of LC 50 and
EC 50.
• LC50 is standard measure of toxicity of
surrounding medium that will kill half of the
sample population of a specific test animals
in a specific period through exposured via
inhalation.
• EC50 is statistically derived conc. of a
substance in an environment medium
expected to produce a certain effect in 50%
of test organisms in a given population under
a definite set of conditions.

36. Place the test organism in
test chamber
Test solution constantly
replenished
Test animal is exposed to
96hrs in flow through
system
Estimation of concentration
Response relation
Observation biological
physical & chemical

37. HISTOPATHOLOGY
• It is the most important in vitro test diagnostic of neurotoxicity in
tissue level.
• The histological specimen can be prepared as whole mount,
sections and smears.
• It occurs through several steps:
a) Taking samples
b) Fixation
c) Alcohol washing
d) Clearing
e) Embedding
f) Sectioning
g) Staining
h) observation

38. CURRENT IN-VIVO PROCEDURE
• Neurotoxic effects on complex integrative
functions – motor performance, sensory
acuity, memory, and cognitive process can be
detected only in vivo.
BEHAVIORAL ASSESSMENT:
• Chemical induced functional alternations of
nervous system( sensory, motor, autonomic,
and cognitive function) are measured by
behavioral techniques.

39. FUNCTIONAL OBSERVATIONAL
BATTERIES
• FOBs is a neurobehavioral assessment tool
describing various behaviourlogical and
activity.
• Functional observational batteries (FOBs) are
designed to detect and measure overt
neurotoxic effect.
• FOBs test are essentially clinical examination
that detect the presence or absence, and in
some cases the relative degree, of specific
neurologic signs.

40. Test procedure:
Animal selection:
 Species or strain: laboratory rat or mouse
 Age : young adult( at least 42 days old)
 Sex: equal no. of each sex are required for each
dose level.
 Female shall be nonpregnant
Number of animals:
 At least 8 of each sex
 Control group is required

41. • Minimal list of observation :
• Any unusual response w.r.t body position, activity
level, coordination of movement
• Any unusual behavior like walking backwards,
headflicking, circling ,etc
• The presence of :
• Convultion
• Tremor
• Increase level of salivation
• Unusual respiration
• Diarrhoea
• Excessive or diminished urination
• Forelimb or hind limb grip strength
• Data reporting and evaluation

42. CYTOTOXICITY
• Cytotoxicity is the degree to which an agent
or substance has specific destructive action
on certain cells. It is the quality to being toxic
to cells.
• Cells exposed to a cytotoxic compound can
respond in a number of ways.
• The cells may undergo necrosis, other way
they can activate a genetic program of
controlled cell death.

43. INDICATORS OF CYTOTOXICITY
o The main effect of toxicant exposure to cell is
cell death. It may be programmed cell
death(apoptosis) or necrosis.
Types of cell death occur will depend on:
Concentration of toxicant
Exposure time of toxicant
Dose of particular toxicant.

44. MAIN IMPACTS OF CYTOTOXICITY
1. Membrane alterations
2. Mitochondrial changes
3. Protease cascade
4. DNA fragmentation

45. 1.Membrane alterations-
In the early stages of apoptosis, changes
occur at the cell surface and plasma
membrane.
2. Mitochondrial changes-
During apoptosis mitochondrial
permeability is altered and apoptosis
specific protease activators are released
from mitochondria

46. 3.Protease cascade-
Signals leading to the activation of caspases,
which play a pivotal role in the initiation and
execution of apoptosis induced by various
stimuli.
4. DNA fragmentation-
The biochemical hallmark of apoptosis is the
fragmentation of the genomic DNA, an
irreversible event that commits the cell to die.

48. CELLULAR RESPONSE TO
TOXICANT EXPOSURE
• Toxicants can disrupt the molecular control
elements that regulate constitutive cell death
process.
• Main response to cell to toxicant exposure are
apoptosis and necrosis.
• It is currently believed that toxicant-stimulated cell
death represents a dose dependent continuum.
• With lower dose exposure stimulating apoptosis
and higher doses leading to a threshold, after
which necrosis predominates.

49. Some major toxicants are-
1. Heavy metals
2. Organochlorines
3. Environmental estrogen (xenoestrogen &
phytoestrogen)
4. Pulp mill effluents
5. Irradiation
6. Thermal stress and
7. Carcinogens

50. HEAVY METALS
• Heavy metals have specific ability to
introduce cell death in fish tissues, like Hg,
Cu, Cd lead, zinc, Arsenic, Tributyltin etc.
• Examples –
1) Cu exposure cause epithelial cell death in
Cyprinus carpio.
2) Cd exposure introduce hepatocyte
apoptosis in Rainbow trout.
3) Trybutyltin (>1µM) lead to mitochondrial
swelling and alternation to red blood cells
and leulocytes in rainbow trout.

51. ORGANOCHLORINES
• It is a large group of pesticides and other
synthetic organic compounds with chlorinated
aromatic hydrocarbon.
• They cause variety of toxic effects including-
a) Cardiovascular dysfunction
b) Edema
c) Reduced reproductive capacity etc.

52. ENVIRONMENTAL ESTROGEN
• It has examined that exposure to
environmental estrogen may be mediated
by up- or down-regulation of cell death.
• Example - 17α – ethinylestradiol (EE).
• It’s exposure cause- Increase in testicular
cell death in adult males.

53. PULP MILL EFFLUENTS
• It is the effluents come or discharge from paper
industry to the aquatic environments.
• They are complex mixture containing natural wood
sterols, metals, aromatic hydrocarbons and if
chlorine bleaching of pulp occurs, chlorinated
hydrocarbons.
• Can cause necrosis on epithelial cells, reduced
reproductive capacity etc.

54. IRRADIATION
It can potentially induce apoptosis or
necrosis in cells.
THERMAL STRESS
Recent evidence demonstrating that heat
shock can induce cell death in a variety of
fish tissues.

55. CARCINOGENS
• Exposure to some toxicants has been shown
to have reverse effect.
• Example – exposure of gulf killifish to the
suspected carcinogen N-methyl-N-nitro-N –
nitrisoguanidine (MNNG) resulted in a
decreased incidence of hepatocyte
apoptosis.
• Resulting to be a potential contributing factor
to the suspected carcinogenicity of MNNG.

56. CELL DETECTION METHODS
1. Dye exclusion methods
2. MTT assay
3. TUNEL assay
4. Comet assay

57. 1.DYE EXCLUSION METHOD
Dye used are-
1. Trypan blue.
2. Propidium iodide (Fluorescent dye)
• Viable (intact plasma membrane) and
dead (damaged plasma membrane) cells
can be differentiate by this method.

58. 2. MTT ASSAY
• Colorimetric assay for
assessing cell metabolic
activity.
• Cellular oxidoreductase
enzymes are capable of
reducing the tetrazolium
dye MTT 3-(4,5-
dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium
bromide to its
insoluble formazan, which
has a purple colour.

59. 3. TUNEL ASSAY
• Here we used terminal dideoxynucleotidyl
transferase (TdT).
• Which incorporate with hapten-tagged
nucleotides into the 3’-strand breaks that
occur in DNA during apoptosis.
• If these nucleotides are coupled to a
fluorescent molecule,
• The apoptotic cells can be analyzed by flow
cytometry

62. REFERENCE
• Toxicology & Risk Assessment: A comprehensive Introduction.
Edited by:- Helmut Greim & Robert Snyder
John wiley & sons, Ltd
• www.nap.edu/read/1801
• www.khanacademy.org/science/biology/human-biology/neuron-
nervous-system/a/overview-of-neuron-structure-and-function
• https://en.wikipedia.org/wiki/Cytotoxicity
• Antony W. et al, (2005) Environmental Toxicology, Cell death,
investigation and appElsevier B.V. Page no. 316 to 322.
• Toxicological Sciences, Volume 119, Issue 1, 1 January 2011, Pages
3–19

63. Thank You