Genetic Toxicology

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Genetic Toxicology ???? ??????
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• Genetic toxicology
• Subspeciality of toxicology
• Involves the identification and analysis of
  agents with toxicity directed to the hereditary
  components of living organisms.
• Background
• Agents that induces alterations in nucleic acids
  and associated components at subtoxic exposure
  levels, resulting in modified hereditary
  characteristics or DNA inactivation, are
  classified as genotoxic.
• ?DNA repair system

Excision repair
1)
Base excision repair
?
Nucleotide excision repair
?
Recombinational repair
2)
SOS repair
3)
? Bacteria?? ?? ?? ???? ?? system
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Excision repair
1)
Nucleotide excision repair The mechanism of
excision repair of pyrimidine photodimers
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Excision repair
1)
Base excision repair DNA glycosylase
hydrolyze the glycosidic bonds of their
corresponding altered base
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2) Recombinational repair In recombination
repair, the gap in a newly synthesized DNA
strand opposite a damage site is filled by the
corresponding segment from its sister duplex
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3) SOS repair
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????? ?? ?? mutagen? UV light??. UV? ?? ??? DNA?
excision repair? recombinational repair? ?? ??
???? ????? ??? ?? ? repair system? DNA template?
???? ?? repair?? ???? DNA template? ???? repair?
? ?? ??? DNA ??? ??? ??? ??? ?? mutagenesis? ????
repair system? SOS repair system? ????? ??. ? SOS
system?? DNA recovery? DNA template ?? ????? ??
??? ???? ?? ??? ????? ? ?? ????? ???? ??. ? SOS
system? ???? ??? uvrA, uvrB, uvrD, recA, lexA,
ssb, ruv, recN, umuC, umuD
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Model for the SOS regulatory system
? SOS ??? ??. DNA ??? ???? RecA ???? ??? ?????
????? LexA ???? ????. LexA ???? ????? recA ????
DNA ?? ???? uvrA? uvrB? ??? ????. LexA? ?????? ??
???? ?????.
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2. Genotoxic substances usually have common
chemical or physical properties faciliating their
interaction with nucleic acids. 3. Mutagens
compose a subset of geneotoxics, demonstrated
experimentally to induce specific classes of
stable changes in, 1) the nucleotide sequence
of genes 2) chromosome structure 3)
chromosome number
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(No Transcript)
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• Materials Method
• Substrate method
• beta-galactosidase alkaline phosphatase (AP)
• ONPG (o-Nitrophenyl-b-D galactoside)
• beta-galactopyranoside
• ? beta-gal substrate
• 4-Nitroquinaline N-oxide Mutagen
• PQ37 strain

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PQ37 strain

• Deletion of the normal lac region
• The strain is made genetically more susceptible
  to
• genotoxic agents uvrA mutation
• Constitutive synthesis of alkaline phosphatase
• independent of SOS control

UmuC, UmuD misincorporated base? chain
elongation
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Method standard procedure for the SOS chromotest
1.  Culture PQ37 strain(3ml) at 37? for 18 h  
2.  1/10 dilution culture at 37? for 2 h
     (100 ? culture medium? 900 ? fresh medium??
?? ? 2108 bacteria/ml) 3.  Add 0, 0.5, 1.0,
2.0 ? mutagen(4-Nitroquinoline N-oxide)/???(ml),
respectively 4.  Shaking Incubation at 37? for 1
h 5.  1/10 dilution culture at 37? for 1 or 2 h 
  bacteria growth      (1 ml? culture medium? 9
ml fresh medium?? ??) 6.  ??? 0.2 ml?
ß-galactosidase???? ?? ? tube? ??    0   
1     2     3  
     1) Add  Strain(0.2 ml) (1)      2) Add  B
buffer(1.8 ml) (9)      3) 0.1 SDS (50?)
     4) voltex short time      5) 37??? 1520??
??      6) Add ONPG sol.(0.4 ml) (2)      7)
Incubation at R.T for 3090min      8) Reaction
time(t       )      9) Add 1M Na2CO3 (1 ml)
10) A420nm ??
             
            ? ONPG    0.4 ml  
            ? B buffer 1.8 ml  
            ? strain     0.2 ml  
 
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LB medium (Luria-Bertani Medium) per liter,
Distilled water Bacto-tryptone
10g Bacto-yeast extract
5g NaCl
10g B buffer Na2HPO4
16.1g
NaH2PO4 7H2O 5.5g
KCl
0.75g Sodium dodecyl sulfate (SDS)
1g b-mercaptoethanol
2.7ml OPNG solution (4mg/ml)
o-nitrophenyl-b-D-galactopyranoside 400mg
0.1M Na2HPO4 7H2O
61ml 0.1M NaH2PO4H2O
39ml
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Conclusion SOS ?????, DNA ??? ??? ????? ??? DNA
??? ??? ?? ?? ??? ??? ????? ?????. SOS system?
????? LexA ???? ?? ????, DNA ??? ??? ???? ???
????? RecA ? LexA? ???? ???. SOS system? DNA
????? ??? ??? ???? ?? ???? ????? ???? ??? ??? ??
????? ????. ??? ??? SOS regulatory system? ??????
??? ?? ?? ??? ?? DNA ?? ? ???? ?? ??? ???? ??.