Pollution-Induced Germ-line Mutations in Gull and Mouse Tandem - PowerPoint PPT Presentation

Pollution-Induced Germ-line Mutations in Gull and Mouse Tandem Repeat DNA Dr. James S. Quinn Biology Department McMaster University

DNA Damaging Agents In the Environment: - heritable mutations?

Hamilton Harbour Remedial Action Plan: Hamilton Harbour Remedial Action Plan: Hamilton Harbour Remedial Action Plan: Designated area of concern under IJC in 1978 Remediation goals established for chemical and biological state of the area Our lab contributed with an attempt to establish a Method to monitor germ-line mutations Heritable Mutations?

Contaminants Can Damage DNA body cells Sex cells Mutation Mutation ?? Cancer Unexposed offspring Exposed

Outline • Germline mutation monitoring • Pollution and minisatellite mutations • Experimental studies of ESTR mutations in mice • Implications and reactions • What can be done and is often not done (Hamilton’s legacy). • Future mutation work

Contaminant-Induced Heritable Mutations: Difficult to study Natural Populations Laboratory Studies • Comparisons • Enormous sample between exposed sizes and high and unexposed treatment doses populations lack statistical power • Do not reflect • Causal links exposure under between exposure ambient conditions and mutation difficult Few attempts have been made to study mutations to establish induced under ambient environmental conditions

Part 1: Germline mutation screening in herring gulls ( Larus argentatus ) on the Great Lakes

Dr. Carole Yauk

Mutation Detection: • Pedigree analysis • Multilocus DNA fingerprinting • Minisatellite loci • Non-parental bands = mutations Minisatellite DNA: • Non-coding, repetitive DNA • High spontaneous mutation rate

Hamilton Harbour Gulls Industrial core

Germline Minisatellite Mutation Rates in Herring Gulls: 0.025 Per-band mutation rate 0.02 0.015 Rural 0.01 0.005 0 Hamilton Kent Island Presqu'ile Chantry Island Yauk and Quinn (1996) PNAS 93: 12137- 12141.

Study Sites:

Yauk, Fox, McCarry and Quinn, 2000. Mutation Research 452:211-218

Mutation rate is negatively correlated with proximity to steel production: 2.5 0 0 1 x 2 e t a r n 1.5 o i t a t u 1 m d n a 0.5 b - r e P 0 Rural Urban 4 - 9km 2km 0km Distance from steel mills Yauk, Fox, McCarry and Quinn, 2000. Mutation Research 452:211-218

Germline Mutation Screening: Herring Gulls � Germline minisatellite mutation rates elevated at sites with integrated steel mills � Mutation rates negatively correlated with proximity to steel mills (Yauk and Quinn 1996 PNAS, Yauk et al. 2000 Mutat Res. )

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Are Other Organisms at Risk? What is the Route of Mutagen Exposure?

Germ-line mutation induction in other species • Elevated minisatellite mutation rates - humans living near Chernobyl and in Kazakhstan • Elevated microsatellite mutation rates - barn swallows living near Chernobyl

Potential Difficulties in Interpreting Comparisons Among Natural Populations: Underlying Isolating Route of Lifestyle / Behaviour Genetics Exposure Differences

Development of mouse-specific repetitive DNA markers (ESTRs) : • Acute gamma irradiation (Dubrova et al. 1998, 2000) • PCBs and diesel exhaust (Hedenskog et al. 1997) • ESTR markers more sensitive and efficient than previous rodent tests (e.g., Specific locus test)

Challenge: An experimental approach to detect heritable mutations caused by pollution. Sentinel Animals, Sensitive ESTR markers ambient conditions in lab mouse studies

Part 2: Germline mutation screening in sentinel rodents

Dr. Chris Somers

How Industrial By-Products Enter the Ecosystem in Hamilton Harbour: Airborne Emissions Fall Out Contact Cooling

Hamilton Harbour Gulls Mice Industrial core

Pier 25 Exposure Site: • Adjacent to steel industry mouse shed and busy highway M.O.E. • Close to herring gull nesting area • Long term air quality data Dofasco / Stelco

Freelton Exposure Site: mouse shed • ~30km north of Pier 25 •Removed from known point sources of pollution •Shed identical to one at Pier 25

Parents 1) Adults bred 6 weeks post-exposure 2) Complete families sampled for comparative Offspring DNA Fingerprinting

Mutation Detection: Pedigree DNA Profiling P 1 2 3 4 5 6 P • ESTR locus Ms6-hm 14.3kb (GGGCA) n 10.7kb • Chromosome 4 • Highly unstable in germline 6.2kb Mutations = changes in band size 4.7kb

Overall inherited mutation rates: 0.35 1.5X 0.30 Ms6-hm ) 0.25 E S 0.20 Hm-2 ± 0.15 ( e 0.10 t a r 0.05 n o 0.00 i t Rural Steel a t u m 0.08 d 0.07 n 2.0X a 0.06 b - 0.05 r MMS10 e 0.04 P 0.03 0.02 0.01 0.00 Rural Steel Somers, Yauk, White, Parfett and Quinn. 2002. PNAS 99: 15904-15907

Parental Origin of Mutations: Single locus ESTR markers 0.40 Paternal Maternal ) E 0.35 S ± ( 0.30 e t a 0.25 r n o 0.20 i t a t 0.15 u m 0.10 d n a 0.05 b - r 0.00 e P Rural Steel * Elevation in Hamilton mice is due mostly to mutations inherited through the paternal germline Somers, Yauk, White, Parfett and Quinn. 2002. PNAS 99: 15904-15907

Conclusions from Exp 1: • Ambient air at Steel site induced heritable germ cell changes in exposed adult mice • Male germline more susceptible than female

Next Steps? • Replication of the findings • Narrowing the field

Protecting sentinel mice from particulate matter: HEPA filtration Ambient Air Removes: -99.99% > 0.3µm -99.97% > 0.1µm

Two-way ANOVA (Env. Exposure per family, Paternal and maternal single locus ESTR rates) Paternal Maternal Source df F -value P -value F - P - value value Exposure 1, 69 7.22 0.0090 3.68 0.0590 site 1, 69 8.03 0.0060 0.07 0.7948 HEPA- filtration 1, 69 13.79 0.0004 1.60 0.2098 Interaction

Overall germline mutation rates: 0.30 0.25 Ms6-hm 0.20 Per-band mutation rate (±SE) 0.15 Hm-2 0.10 0.05 0.00 Rural Rural Rural Steel Steel Steel HEPA male HEPA male 0.05 0.04 0.04 0.03 0.03 0.02 MMS10 0.02 0.01 0.01 0.00 Rural Rural Rural Steel Steel Steel HEPA male HEPA male Somers, McCarry, Malek, and Quinn. 2004. Science 304: 1008-1010

Paternal and Maternal Mutation Rates Paternal Maternal 0.40 ) E 0.35 S ± 0.30 ( e t 0.25 a r n 0.20 o i 0.15 t a t u 0.10 m 0.05 d n a 0.00 b - r e Rural Rural Rural male Steel Steel Steel male P HEPA HEPA Paternal germline significantly affected Somers, McCarry, Malek, and Quinn. 2004. Science 304: 1008-1010

Pier 25 Freelton Pier 25 Freelton Pier 25 Freelton Mean Daily TSP (g) 0.12 0.1 0.08 0.06 0.04 0.02 0 Pier 25 Freelton High - volume air sampling: 24 hour period High- -volume air sampling: 24 hour period volume air sampling: 24 hour period High

What might be causing mutations? Exposure year Total PAH conc. (ng/m 3 ) 200 117.6 ng/m 3 150 100 50 0 1994 1995 1996 1997 1998 1999 PAHs from coal combustion are a good candidate group

Air chemistry: PAH testing *Pyrene Benzo[k]fluoranthene Benzo[a]fluorene Benzo[j]fluoranthene Benzo[b]fluorene *Benzo[e]pyrene Benzo[b]naphtho[2,1-d]thiophene *Benzo[a]pyrene Benzo[ghi]fluoranthene *Perylene Benzo[c]phenanthrene Indeno[1,2,3-cd]pyrene Benzo[b]naphtho[2,3-d]thiophene *Dibenz[a,c]anthracene Benz[a]anthracene Picene Cyclopenta[cd]pyrene Benzo[ghi]perylene *Chrysene *Coronene Benzanthrone Dibenzo[a,e]pyrene Benz[a]anthracene-7,12 dione Dibenzo[a,i]pyrene Benzo[b]fluoranthene Dibenzo[a,h]pyrene

Total PAHs: TSP 30 171x PAH concentration (ng/m 3 ) Rural Steel 25 75x 20 40x 15 10 10x 3x 5 4x 0 0 1 to 3 4 to 9 10 to 18 19 to 23 24 Daily hours downwind of industrial core Weighted average: 33-fold difference between sites; Rural = 0.4ng/m3, steel = 13.4ng/m3

Implications?

Possible Human Health Risks to the unborn • Genetic diseases influenced by tandem repeat DNA: • Cancers associated with Hras oncogene • Type I Diabetes • Type of Epilepsy • Huntington’s disease • Fragile X syndrome • Possible Changes in “coding” genes (requires demonstration that ESTR assay reflects similar changes in genes)

Around your home

Around Your City • Leaves filter particulate air pollution • Protect forests, plant trees • Limit road construction and urban sprawl • Walk, bike, bus, or car pool

Around your city/province/country

Paternal Maternal Source df F -value P -value F - P - value value 1, 69 7.22 0.0090 3.68 0.0590 Exposure site HEPA- 1, 69 8.03 0.0060 0.07 0.7948 filtration Interaction 1, 69 13.79 0.0004 1.60 0.2098 Timing? Parental investment in Gametes?

Future Research (collaboration with Carole Yauk and others) • In utero exposures – maternal mutations • Stability of mutations to F 2 • DNA expression in exposed individuals – protection of germ-line DNA • Possible genomic instability