ี และสงิ่ แวดล้อมแห่งชาติ ครงที
ในการประชุมวิชาการโรคจากการประกอบอาชพ
ั้ ่ 8
th
(The 8 National Conference on Occupational and Environmental Diseases)
&
ี และสงิ่ แวดล้อมแห่งชาติ ครงที
การประชุมวิชาการนานาชาติดา้ นโรคจากการประกอบอาชพ
ั้ ่ 1
st
(The 1 International Conference on Occupational and Environmental Diseases)
ี และสงิ่ แวดล้อม
“สานพล ังข ับเคลือ
่ นนว ัตกรรมด้านโรคจากการประกอบอาชพ
ั
่ งคมที
เพือ
่ ก้าวทีม
่ น
่ ั คง สูส
ย
่ งยื
่ ั น”
Occupational and Environmental Health Innovation for
Health Sustainability
Session V
ี และสงิ่ แวดล้อม
เรือ
่ ง โรคมะเร็งจากการประกอบอาชพ
(Occupational and Environmental Cancer)
ดร.ประไพภ ัทร คล ังทร ัพย์
ี่ วชาญวิจ ัย
ผูเ้ ชย
สถาบ ันวิจ ัยวิทยาศาสตร์และเทคโนโลยีแห่งประเทศไทย (วว.)
prapaipat@tistr.or.th
1
้ หา:
เนือ
1) กลไกการเกิดมะเร็ง
ึ ษากลไกการเกิดมะเร็ง
2) ความก้าวหน้าของการศก
3) การทดสอบทางห้องปฏิบ ัติการทีเ่ กีย
่ วข้องก ับการ
ตรวจความผิดปกติของ Gene และ DNA
2
ห ัวข้อ 1.
“กลไกการเกิดมะเร็ง”
What causes
CANCER?
Copyright © 2017 TISTR
3
“สารพ ันธุพษ
ิ ”
Substances that cause damage to DNA
and GENE called
“Genotoxin”
4
Genotoxins cause DNA
damage…and lead to
Mutation and CANCER
5
Mutation




Permanent change in the
DNA sequence of a gene
Inherited or acquired
during lifetime
Single mutations are
often harmless but
multiple mutations can
results in cancer
What causes mutations in
DNA?
6
How do mutations cause cancer?




DNA RNA protein
Mutated DNA mutated RNA mutated
protein
Many mutations accumulated over time can
result in harmful changes in the cells
instructions
These mutations in genes result in mutations
in proteins that control the cell cycle
7
Cell cycle (ว ัฏจ ักรเซลล์)
Uncontrolled cell cycle = uncontrolled cell growth = tumor
Mutation can lead to…Cancer
Immortal cells
9
A physical or chemical agent
that causes mutation
is called “Mutagen”
Most cancer is caused by genetic mutations
often by a series of mutations.
Carcinogens = Mutagens
10
11
การกระจายต ัวของเซลล์มะเร็ง
Metastatic cancer
WHO Statistics
Year 2020 :
15 million people will die from cancer
Causes of Cancer
The etiology of cancer is multifactorial







Heredity
Immunity
Chemical
Physical
Viral
Bacterial
Lifestyle
Heredity
Genes isolated for several
classic familial cancer
syndromes:
 RB1 (retinoblastoma)
 APC (familial polyposis)
 Human Non Polyposis
Colon Cancer (HNPCC)
 BRCA 1&2 (breast cancer)
 p53 (many cancers)
Copyright © 2017 TISTR
15
Immunity
 HIV / AIDS
 Immunosuppression
Copyright © 2017 TISTR
16
Viruses
Microorganism
Cancer
 Human papilloma virus
 Cervical cancer
 Hepatitis B and hepatitis C viruses
 Liver cancer
 Human T-cell leukemia/lymphoma
virus
 Lymphoma and leukemia
 Human immunodeficiency virus
 Lymphoma and a rare cancer
called Kaposi's sarcoma
 Epstein-Barr virus
 Lymphoma
 Human herpes virus 8
 Kaposi's sarcoma
17
Bacterials
 Helicobactor pylori
 Other Parasites:
 Schistosoma spp
 Clonorchis sinensis
18
Chemicals








Alcohol
Asbestos
Wood dust
Rubber, plastics, dyes
Tar / bitumen
Aflatoxin
Alkylating agents
Tobacco
19
Lifestyle & Psychological Factors
 Stress has been
implicated in increased
susceptibility to several
types of cancers
 Sleep disturbances,
diet, or a combination
of factors may weaken
the body’s immune
system
20
Occupational and Environmental Factors








Asbestos
Nickel
Chromate
Benzene
Arsenic
Radioactive substances
Cool tars
Herbicides/pesticides
21
Smoking
 Single biggest
cause of cancer
 25-40% smokers
die in middle age
 9 in 10 lung
cancers
 Know to cause
cancer in 1950
22
Industrial Pollution
23
Physical causes
 Ultraviolet radiation
 Sunlight
 Certain industrial sources
 Radiation
 Radon
 Cancer treatment
24
Factors Believed to Contribute to
Global Causes of Cancer
Copyright © 2017 TISTR
25
ห ัวข้อ 2.
ึ ษา
ความก้าวหน้าของการศก
กลไกการเกิดมะเร็ง
26
DNA Repair
Nucleotide excision repair (NER)
is a particularly important
DNA repair mechanism.
DNA damage occurs constantly
because of chemicals e.g.
 intercalating agents
 radiation
 mutagens
27
Ultraviolet (UV) radiation-induced DNA damage
DNA exposed to ultraviolet (UV) radiation results in covalent
dimerization of adjacent pyrimidines, typically thymine residues
called thymine dimers
Before
After
28
These pyrimidine dimers distort the sugar phosphate
backbone and prevent proper replication and transcription.
29
DNA Repair :
Nucleotide Excision Repair (NER)
30
Xeroderma pigmentosum (XP) disease
is a rare genetic disease.
It is characterized by
sensitivity to light
(UVA, UVB and UVC)
31
Xeroderma pigmentosum (XP)
Symptoms :
 pigment changes
(lots of spots on skin)
 premature skin aging
 malignant tumor development
 Eyes sensitive to sunlight
(eye cancer)
32
ห ัวข้อ 3.
การทดสอบทางห้องปฏิบ ัติการ
ทีเ่ กีย
่ วข้องก ับการตรวจความผิดปกติ
ของ Gene และ DNA
Copyright © 2017 TISTR
33
APPLICATION OF
GENOTOXICITY TESTING
2
Organisation for Economic Co-operation and Development
Genotoxicity Tests
A standard battery of tests
set by Regulatory Authorities,
are used to determine
the mutagenicity &
carcinogenicity
OECD : Section 4 Health Effect
35
5
In vitro Genotoxicity Tests
1.
2.
3.
4.
Ames test
Chromosome aberration (CA)
Micronucleus (MN)
Comet assay
9
Ames Test
10
The Bacterial Reverse Mutation (Ames) Assay
Detects relevant genetic changes & majority of
genotoxic rodent carcinogens.
Salmonella typhimurium
Detect one of the genes involved in
histidine biosynthesis
Escherichia coli
Detect one of the genes involved in
tryptophan biosynthesis
11
Methodology :
Test for mutagenicity
Control : no mutagen
12
Ames’ Evaluation / Analysis
A
B
Revertant bacteria (His+)
14
Chromosome Aberration
Test
15
Principle :
The purpose of in vitro chromosome aberration
test…is to identify agents (mutagen-carcinogen) that
cause structural chromosome aberrations in culture
mammalian cells.
Structure aberrations
may be of two type:
• Chromosome
• Chromatids
16
Chromosomal aberration using
“Metaphase analysis”
17
Microscopic examination
19
Type of chromosome aberrations
(50 metaphase/slide)
Dicentrics are usually clonogenically lethal because of segregation
problems that arise during anaphase of mitosis.
20
Chromosomal Lesions
Chromatid gap (CG)
Chromatid break (CB)
chromosomal rearrangements
Exchange
Deletion
Dicentric
chromosome
Ring
Dislocalisation
21
Micronucleus Test
22
Micronucleus (MN) test
Micronucleus (MN) …is formed during the
metaphase/anaphase transition stage
“ It may arise from a whole
lagging chromosome or an
acentric chromosome
fragment detaching from a
chromosome after breakage
which do not integrate in the
daughter nuclei”
23
Cell stage for CA and MN assays
Chromosome
aberration
Micronucleus
assay
24
Micronucleated (MN) cells of human lymphocytes
(TK6) treated with mitomycin C (MMC)
with one micronucleus
with two micronuclei
29
Automated micronucleus detection
Software that
automatically scans
slides prepared for the
micronucleus assay;
results are shown in
a cell gallery and are
summarized with a
data histogram
Used for genotoxicity
evaluation (potential
for an agent to induce
DNA damage)
30
COMET TEST
Single Cell Gel Electrophoresis (SCGE)
33
Comet assay
 Single cell gel electrophoresis (SCGE)
assay (developed in the mid 1980 ; Östling
and Johanson)
 Analysis of :




single strand break (SSB)
double strand break (DSB)
alkali-labile site (ALS) of DNA
incomplete excision repair sites in
eukaryotic individual cell
 Quantification of the denatured DNA
fragments migrating out of the cell
nucleus during electrophoresis.
28
Basic Steps of Comet assay
1
2
 Cells: TK6 (human lymphoblasts)
 24 h-treated cells with the sample
 Alkaline lysis solution (pH  13) to
produce single-stranded DNA
 Wash and re-suspended in 50 µM
H2O2 for 5 min
Slide layer
3
6

DNA will be visualized by
fluorescence microscopy

Analyzed by Comet assay
III® software
 25V, 300mA under
alkaline condition to
produce comets
5
 Ethidium bromide (DNA-binding dye)
4
DNA damage : Comet scoring
 50 comet cells/ slide
 Tail length (TL) = the distance
of DNA migration measured
from the center of the nucleus
towards the end of the tail
 Tail moment (TM) = The product
of distance and normalized
intensity integrated over the tail
length, (Lx % DNAx).
A damage measure combining the
amount of DNA in the tail with the
distance of migration (severity of
DNA damage)
30
When cell is scored an
intensity profile is
displayed next to the
cell and the comet is
silhouetted by a
pseudo-colour overlay.
The data obtained from the analysis is
transferred to a spreadsheet in Microsoft Excel
50
10 Rules to Avoid Cancer
1.
2.
3.
4.
Don’t smoke
Don’t smoke
Don’t smoke
Avoid exposure to other known carcinogens,
including aflatoxin, asbestos and UV light.
5. Enjoy a healthy diet, moderate in calories, salt and
fat, and low in alcohol.
6. Eat fresh fruit and vegetables several times a day
7. Be physically active and avoid obesity.
8. Have vaccination against, or early
detection/treatment of, cancer causing chronic
infections.
9. Have the right genes
10. Have good luck !
57
58
“Telomere Instability”
assay
59
Prof.Elizabeth H. Blackburn
University of California San Francisco (UCSF)
The Nobel Prize in Physiology or Medicine 2009
60
Telomeres
Telomeres are distinctive structures
found at the ends of our chromosomes.
They consist of the same short DNA
sequence repeated over and over again.
This sequence is usually repeated
about 3,000 times and can reach up
to 15,000 base pairs in length.
In humans the telomere
sequence is TTAGGG
Telomere abnormalities can lead to CANCER !!!
61


Sodium nitrate when ingested forms a potential carcinogen, nitrosamine
Sodium nitrate is still used because it is effective in preventing botulism
62
ไนเตรตและไนไตรท์ก ับมะเร็ง
ื้ แบคทีเรียมบางชนิดในนา้ ลาย
เกิดจากไนไตรท์ปริมาณสูงทาปฏิกริ ย
ิ าก ับเชอ
ท าปฏิก ริ ย
ิ าก ับสารเมีน
(amine) ในอาหาร ท าให้เ กิด
“สารไนโตรซามีน
(nitrosamine)”
63
Methods :
Telo-centro FISH
 Telomere specific probe labeled with CY3
 Centromere labeled with FITC
 Chromosome stained by DAPI
Telomere
Centromere
Chromosome
Copyright © 2017 TISTR
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Copyright © 2017 TISTR
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Carcinogen
66
“ด้วยความขอบคุณ”
67