comet assay - sperm - medik.cz · 2010-05-05 · comet assay. environmental health perspectives...
TRANSCRIPT
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outline
● introduction● protocol + new applications● studies● conclusion
● maybe some extras?
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outline
● introduction● protocol + new applications● studies● conclusion
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basic info
● Single cell gel electrophoresis● DNA damage measured on single cells● simple, inexpensive, sensitive and flexible● any type of cell● few cells needed● uniform protocol● labour-intensive● quantitative
for original contact the author:[email protected]
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DNA damage
● environmental and drug genotoxicity, genetic diseases and lifestyle
● sperm (Sakkas 2010)– apoptosis during
spermiogenesis (50%)
– sperm chromatin remodelation (nicks)
– ROS– endogenous caspases and endonucleases
Berra 2010
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history
● 1984 – Ostling & Johanson– neutral version
● 1988 – Singh– alkaline version
● 1989 – Singh– sperm (prot K)
● 1999 – Lewis (Steele, Hughes, Donnelly)– sperm (DTT+LIS)
Matějovičová
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outline
● introduction● protocol + new applications● studies● conclusion
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overview
● fixation of cells in agarose gel● lysis of cell membrane and cytoplasm● decondensation● unwinding● elfo● staining● acquisition● scoring of comets● data analysis
cometassayindia.org
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slide preparation & lysis
● suspension of cells in LMP agarose● on pretreated slides● lysis in salt & detergent
comics.vitamib.com
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decondensation
● proteinase K (Singh 1989) – long incubation
● DTT + LIS (Lewis 1999)
● RNase + prot K (Duty 2003)
– very long incubation
● DTT (Schmid 2007)● future NaOH
wikipedia
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11/31
unwinding & electrophoresis
● 300mM NaOH → pH = 13,6– double strand, single strand,
alkali-labile sites (abasic)
wikipediascifair.org
for original contact the author:[email protected]
12/31
acquisition
● non- X integrated camera X automatic system● sensitive to dynamic range
– saturation, background, detection limit
for original contact the author:[email protected]
for original contact the author:[email protected]
13/31
scoring
● commercial X opensource X visual scoring● end-points
– tail DNA %– tail length– tail moment– other central
moments
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data analysis
● statistically very weak– skewed (lognormal)– median + IQR– 95%, GM
● rule of small numbers– hierarchical replicates
(biological, technical)– 25x2x2(x3)
● experimental unit– biological replicate
PLK1APLK
2
PLE1APLE2
CLK1ACLK2
CLE1ACLE2
PSK1APSK2
PSE1APSE2
CSK1ACSK2
CSE1ACSE2
0
20
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80
100
TD%
0123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960616263646566676869707172737475767778798081828384858687888990919293949596979899100
0
5
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25
histogram
for original contact the author:[email protected]
for original contact the author:[email protected]
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new applications
● oxidative damage– 8-oxoguanine– FPG (purines)– endoIII (pyrimidines)
● Comet-FISH– leukocytes– wcpFISH (X)– locus specific
(p53, AZF)● crosslinks, repair
wikipedia
Rapp 2000
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outline
● introduction● protocol + new applications● studies● conclusion
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male fertility studies
● dsb X total damage (Alvarez 2005 + Cui 2000)– oxidative damage precedes DNA breaks
● early X late paternal effect (Tesarik 2004, 2005)– TUNEL (s+dsb) correlates with 8c embryo death
● ROS (rev Agarwal 2008)– smoking, inflammation– PUFA– 8-oxoguanine correlates
with male infertility(Shen 1999)and pregnancy loss (Loft 2003) wikipedia
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other studies
● diseases – DM Type I has higher 8-oxoguanine
concentration (Agbaje 2008)● drugs treatment
– antioxidant treatment increases decondensation (Ménézo 2007)
● occupational exposure– acrilonitrile (Xu 2003), phthalates (Duty 2003)
● environment and lifestyle– age and caffeine intake (Schmid 2007)
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other studies
● Hughes 1996– similar baseline damage in asthenozoospermic,
normospermic infertile and fertile– change after pretreatment with X-ray or H2O2– fertile group contains a resistant subpopulation
of sperm● Steele 2000
– different cryopreservation have similar damage levels in testicular sperm
– ejaculated sperm has more damaged DNA than testicular sperm
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outline
● introduction● protocol + new applications● studies● conclusion
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summary
● one of many tests for DNA damage● irreplaceable
– ROS, Comet-FISH– at different levels of sensitivity– total damage (dsb, ssb, als)
● delicate– quantitative analysis– statistically weak– almost impossible to validate
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literature
● Sakkas, Denny, and Juan G Alvarez. Sperm DNA fragmentation: mechanisms of origin, impact on reproductive outcome, and analysis. Fertility and Sterility 93, March 2010, 1027-1036.
● Berra, Carolina Maria et al. Plasmid DNA damage induced by singlet molecular oxygen released from the naphthalene endoperoxide DHPNO2 and photoactivated methylene blue. Química Nova 33, 2010.
● Ostling, O, and K J Johanson. Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochemical and Biophysical Research Communications 123, August 1984, 291-298.
● Singh, N P, M T McCoy, R R Tice, and E L Schneider. A simple technique for quantitation of low levels of DNA damage in individual cells. Experimental Cell Research 175, March 1988, 184-191.
● Singh, N P et al. Abundant alkali-sensitive sites in DNA of human and mouse sperm. Experimental Cell Research 184, October 1989, 461-470.
● Steele, E K, N McClure, R J Maxwell, and S E Lewis. A comparison of DNA damage in testicular and proximal epididymal spermatozoa in obstructive azoospermia. Molecular Human Reproduction 5, September 1999, 831-835.
● Duty, Susan M et al. The relationship between environmental exposures to phthalates and DNA damage in human sperm using the neutral comet assay. Environmental Health Perspectives 111, July 2003, 1164-1169.
● Schmid, T E et al. The effects of male age on sperm DNA damage in healthy non-smokers. Human Reproduction (Oxford, England) 22, January 2007, 180-187.
● Rapp, A, C Bock, H Dittmar, and K O Greulich. UV-A breakage sensitivity of human chromosomes as measured by COMET-FISH depends on gene density and not on the chromosome size. Journal of Photochemistry and Photobiology. B, Biology 56, July 2000, 109-117.
● Alvarez, Juan G. The predictive value of sperm chromatin structure assay. Human Reproduction (Oxford, England) 20, August 2005, 2365-2367.
● Cui, J, E H Holmes, T G Greene, and P K Liu. Oxidative DNA damage precedes DNA fragmentation after experimental stroke in rat brain. The FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology 14, May 2000, 955-967.
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literature
● Tesarik, Jan, Ermanno Greco, and Carmen Mendoza. Late, but not early, paternal effect on human embryo development is related to sperm DNA fragmentation. Human Reproduction (Oxford, England) 19, March 2004, 611-615.
● Tesarik, Jan. Paternal effects on cell division in the human preimplantation embryo. Reproductive Biomedicine Online 10, March 2005, 370-375.
● Agarwal, Ashok, Kartikeya Makker, and Rakesh Sharma. Clinical relevance of oxidative stress in male factor infertility: an update. American Journal of Reproductive Immunology (New York, N.Y.: 1989) 59, January 2008, 2-11.
● Shen, H M, S E Chia, and C N Ong. Evaluation of oxidative DNA damage in human sperm and its association with male infertility. Journal of Andrology 20, December 1999, 718-723.
● Loft, Steffen et al. Oxidative DNA damage in human sperm influences time to pregnancy. Human Reproduction (Oxford, England) 18, June 2003, 1265-1272.
● Agbaje, I M et al. Increased concentrations of the oxidative DNA adduct 7,8-dihydro-8-oxo-2-deoxyguanosine in the germ-line of men with type 1 diabetes. Reproductive Biomedicine Online 16, March 2008, 401-409.
● Ménézo, Yves J R et al. Antioxidants to reduce sperm DNA fragmentation: an unexpected adverse effect. Reproductive Biomedicine Online 14, April 2007, 418-421.
● Xu, De-Xiang et al. Exposure to acrylonitrile induced DNA strand breakage and sex chromosome aneuploidy in human spermatozoa. Mutation Research 537, May 2003, 93-100.
● Hughes, C M, S E Lewis, V J McKelvey-Martin, and W Thompson. A comparison of baseline and induced DNA damage in human spermatozoa from fertile and infertile men, using a modified comet assay. Molecular Human Reproduction 2, August 1996, 613-619.
● Steele, E K, N McClure, and S E Lewis. Comparison of the effects of two methods of cryopreservation on testicular sperm DNA. Fertility and Sterility 74, September 2000, 450-453.
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outline
???● maybe some extras?
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extras
● staining + quantitative analysis
● other tests for DNA damage
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staining
● brightness index (extinction coef x quant. gain)– SYBR Green 35– EB (PI) 2.8 (0.6)
● future– head with DAPI/TATAA– tail (total human genomic probe + Texas red)
bdbiosciences.com
27/31
quantitative analysis
● dynamic range = (saturation limit – background) / detection limit
for original contact the author:[email protected]
for original contact the author:[email protected]
28/31
dynamic range
● saturation limit– bit depth– exposition time– loss of information
Dhawan 2009
for original contact the author:[email protected]
for original contact the author:[email protected]
29/31
dynamic range
● saturation limit
Dhawan 2009
for original contact the author:[email protected]
for original contact the author:[email protected]
for original contact the author:[email protected]
30/31
other tests
● DNA damage– flow cytometry – SCSA, TUNEL, ISNT– fluo microscopy – AOT, DBD-FISH– trans microscopy – SCDT, TB
● protamination– FCM (ChrA3), trans microscopy (AB), molbio
(protein separation)● ROS damage
– FCM (DCFH), HPLC+MS (8oxoG),
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REAL END●
THANK YOU foryour ATTENTION
● Dhawan, Alok, and Royal Society of Chemistry (Great Britain). The comet assay in toxicology. [Cambridge UK]: Royal Society of Chemistry, 2009.