basic aspects and most commonly worldwide employed and validated in vitro assays leon f. stankowski,...
TRANSCRIPT
Basic Aspects and Most Commonly Worldwide Employed and Validated In Vitro Assays
Leon F. Stankowski, Jr., PhDConsultant, Genetic Toxicology
Genotoxicity
• OECD Guidelines• In Vitro Assays
• Mutation• Chromosome Aberration• Micronucleus
Agenda
Mutagen• agent that induces a change in the DNA
Clastogen• agent that induces chromosome breaks (structural
aberration)
Aneugen• agent that induces a change in chromosome number
(numerical aberration)
Some Terminology
Organisation for Economic Cooperation and Development
• 471 Bacterial Reverse Mutation Test
• 473 In Vitro Mammalian Chromosome Aberration Test
• 474 Mammalian Erythrocyte Micronucleus Test
• 475 Mammalian Bone Marrow Chromosomal Aberration Test
• 476 In Vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes
• 487 In Vitro Mammalian Cell Micronucleus Test
• 488 Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays
• 489 In Vivo Mammalian Alkaline Comet Assay
• 490 In Vitro Mammalian Cell Gene Mutation Test Using the Thymidine Kinase Gene
www.oecd.org
OECD Guidelines
Organisation for Economic Cooperation and Development
• 471 Bacterial Reverse Mutation Test
• 473 In Vitro Mammalian Chromosome Aberration Test
• 474 Mammalian Erythrocyte Micronucleus Test
• 475 Mammalian Bone Marrow Chromosomal Aberration Test
• 476 In Vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes
• 487 In Vitro Mammalian Cell Micronucleus Test
• 488 Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays
• 489 In Vivo Mammalian Alkaline Comet Assay
• 490 In Vitro Mammalian Cell Gene Mutation Test Using the Thymidine Kinase Gene
www.oecd.org
OECD Guidelines
• Apply to all testing situations• Some special modifications for human
pharmaceuticals• ICH - International Conference on Harmonisation of
Technical Requirements for Registration of Pharmaceuticals for Human Use
• ICH S2(R1)
www.ich.org
OECD Guidelines
• 471 Bacterial Reverse Mutation Test• S. typhimurium and E. coli tester strains• Reverse mutation to his or trp independence
• Basepair (bp) substitution – change in one base for another
• Frameshift (fs) mutation – insertion or deletion of one or a few a bases
Mutation Assays
• 471 Bacterial Reverse Mutation Test• Requires very specific change at G:C or A:T sites to
revert to his or trp independence• Five tester strains required
• TA1535 and TA100 (bp) and• TA98 (fs) and • TA1537 or TA97 or TA97a (fs) and• WP2 uvrA or WP2 uvrA (pKM101) or TA102 (bp)
• Engineered• deficient in DNA excision repair• error-prone DNA repair function• leaky cell walls
Mutation Assays
• 471 Bacterial Reverse Mutation Test• Treat 108 bacteria/plate (with limited his or trp)• 5+ dose levels
• 5000 µg/plate or solubility/toxicity limit (thinning of lawn)
• Positive controls (diagnostic)• Vehicle control• ±S9• All in triplicate• Incubate 48 hours• Count
• 2- or 3-fold dose-dependent increase?
Mutation Assays
Mutation Assays
CHO/HPRTHemizygous
+
GeneMutation
ChromosomeDamage
- -
MLA (TK+/-)Heterozygous
+
-
GeneMutation
ChromosomeDamage
-
-
-
-
From DeMarini et al. (1989)
• 490 Mouse lymphoma (MLA) and TK6• Autosomal• TK+/- TK+/- (TFTr)
• 476 CHO/HPRT assay• X-linked• Hprt+ Hprt- (TGr)
Mutation Assays
• HPRT and TK• Treat 2 - 3 x 106 cells• 4+ dose levels
• 2000 µg/mL or 10 mM or solubility/toxicity limit(10 – 20% RS or RTG)
• Positive and vehicle controls• ±S9• All in duplicate
Mutation Assays
• Subculture 2 – 7 days• Select mutants with TFT or TG• Concurrent cloning efficiency• Incubate 7 – 14 days• Count
• Significant, dose-dependent increase >95% control limit or GEF?
Mutation Assays
• 473 In Vitro Mammalian Chromosomal Aberration Test• HPBL, RPBL, CHO, CHL cells• Structural aberrations
• breaks/rearrangements (clastogens)
• Numerical aberrations• polyploidy/endoreduplication (aneugens)
Cytogenetics Assays
• 473 In Vitro Mammalian Chromosomal Aberration Test• Treat exponentially growing cells
• stimulate primary cultures with PHA
• 3+ dose levels • 2000 µg/mL or 10 mM or solubility/toxicity limit
(40 – 50% RPD, RICC, MI)
• Positive and vehicle controls• Short treatment ±S9• Extended treatment –S9 only• Single or duplicate
Cytogenetics Assays
• 473 In Vitro Mammalian Chromosomal Aberration Test• Harvest all at ~1.5 cell cycles after start of treatment
• Arrest cultures in metaphase (Colcemid or colchicine)
• Score 300 cells per dose level• Significant, dose-dependent increase >95% control limit?
Cytogenetics Assays
Break
Exchange
CHO CHO
Polyploidy
• 473 In Vitro Mammalian Chromosomal Aberration Test
Cytogenetics Assays
dicentric
HPBLbreak
HPBL
• 487 In Vitro Mammalian Cell Micronucleus Test• HPBL, RPBL, CHO, CHL, TK6 cells• Structural aberrations
• breaks/rearrangements (clastogens)
• Numerical aberrations• polyploidy/endoreduplication (aneugens)
Cytogenetics Assays
• 487 In Vitro Mammalian Cell Micronucleus Test
Cytogenetics Assays
Double Strand Breaks Spindle Fiber Dysfunction
MitoticCells
DaughterCells
i.e. Clastogens i.e. Aneugens
–CytoB
• 487 In Vitro Mammalian Cell Micronucleus Test
Cytogenetics Assays
Double Strand Breaks Spindle Fiber Dysfunction
Mitotic Cells
DaughterCells
i.e. Clastogens i.e. Aneugens
+CytoB
• 487 In Vitro Mammalian Cell Micronucleus Test• Treat exponentially growing cells
• stimulate primary cultures with PHA
• 3+ dose levels • 2000 µg/mL or 10 mM or solubility/toxicity limit
(50 – 60% RPD or RICC for cell lines, or CPBI or RI in primary cells and when using CytoB)
• Positive and vehicle controls• Short treatment ±S9• Extended treatment –S9 only• Single or duplicate
Cytogenetics Assays
• 487 In Vitro Mammalian Cell Micronucleus Test• Harvest all at ~1.5 – 2 cell cycles after start of treatment
• Arrest cultures in metaphase (Colcemid or colchicine)
• Score 2000 cells per dose level• Significant, dose-dependent increase >95% control limit?
Cytogenetics Assays
• 487 In Vitro Mammalian Cell Micronucleus Test• Mechanism of Action
• Fluorescent In Situ Hybridization (FISH)
• Antikinetochore staining (CREST)
Cytogenetics Assays
MN MN
Negative Positive
Cytogenetics Assays
• 487 In Vitro Mammalian Cell Micronucleus Test• Mechanism of Action
• 487 In Vitro Mammalian Cell Micronucleus Test
Cytogenetics Assays
Metaphase Cell C+ MN C– MN
Mice and human
Any species
Thank You!
Questions?
