Evaluation of Genotoxicity in Complex PAH Mixtures in SoilEnvironmental Matrices Alexandra S. Long1, Paul A. White1,2
1 Department of Biology, University of Ottawa, Ottawa, ON 2Environmental Health Science and Research Bureau, Health Canada, Ottawa, ON
Presentation Outline
• Sample Extraction • Sample Fractionation
• Example #1 – Mutagenicity Assessment of Extracts from Contaminated Soil
• Example #2 - Mutagenicity Assessment of Extracts from Diesel and Biodiesel Exhaust
• Example #3 - Mutagenicity Assessment of Concentrated Urine Following Exposure to Wood Smoke
• Issues, problems, future prospects.
SAMPLE COLLECTION FOR ENVIRONMENTAL
EXTRACTION OF SOLUBLE ORGANIC
MATTER (SOF) FROM COMPLEX
Sample Extraction • Soxhlet Extraction
• Solid sample placed in extraction thimble • Solvent vapours condense over thimble, and dissolve
soluble portion of sample • Dissolved portion then re-enters distillation flask • Cycle can be repeated many times to concentrate extract
• Pressurized Fluid Extraction • Uses high temperature and pressure to force solvent
through an extraction thimble containing solid sample • High pressure keeps fluid below boiling point to allow high
penetration • Can combine with silica for in-cell fractionation
• Solid-Phase Extraction • Process to concentrate analytes from aqueous solutions • Sample applied to column containing solid (stationary
phase/bonded phase) • Normal phase – polar compounds retained • Reverse phase – non-polar compounds retained
FRACTIONATION OF COMPLEX ORGANIC
• 10% deactivated silica gel
• Analytes are sequentially eluted in appropriate solvent, according to polarity
Validation of Fractionation Procedure Modification of Lundstedt et al. 2003 ET&C 22(7) 1413-1420.
• Fraction 1 n-Hexane: Unpolar compounds such as alkanes • Fraction 2 n-Hexane/DCM: PAHs, alkyl-PAHs, O- and S-heterocyclics • Fraction 3 DCM: Nitro-PAHs, oxy-PAHs, N-heterocyclics • Fraction 4 – Methanol: Polar compounds
PAHs and alkyl-PAHs
Benzo[a]carbazole
Dibenzo[c,g,]carbazole
Dibenz[a,j]acridine
Solvent exchange into DMSO • TurboVap to evaporate solvent using Nitrogen gas
• N-Evap to complete solvent exchange to appropriate solvent for mutagenicity assessment (DMSO, etc.)
• Measure extractable organic matter (EOM) per unit sample (e.g., g soil, mg diesel PM)
GENOTOXICITY ASSESSMENT
• S9 microsomal mixture is the 9000×g supernatant fraction of liver homogenate from rats treated with Aroclor 1254.
• S9 contains P450 monooxygenases capable of simulating in vivo metabolism and activation of mutagenic PAHs.
Gene Mutation in Salmonella typhimurium “The Ames Test”
Reversion of histidine auxotrophs to wild type
The Ames Assay - Protocol Plate incorporation method • 0.01, 0.05 or 0.1 ml of the test solutions, 0.1 ml fresh bacterial culture and 0.5
ml sterile buffer (or S9) mixed with 2 ml overlay agar. • Mix and pour over surface of minimal agar. • Overlay is allowed to solidify.
Preincubation method • Test substance is preincubated with test strain and sterile buffer (or S9) for 20
min or more at 30-37oC prior to mixing and pouring. • Microsuspension version (Kado) – up to 10X more sensitive for certain sample
types (urine extracts, air PM extracts, etc.)
• Usually require 5 doses (half log intervals).
• Top dose should be somewhat toxic.
• Assess toxicity by observing background lawn.
• Plate incorporation test sometimes followed by preincubation test if negative.
Miscellaneous Details
YG1041† hisD3052 GCGC TA98(pYG233) pKM101- MucA/B, Amp
pYG233- Cnr, OAT, Kan
YG1042† hisG46 GGG TA100(pYG233) pKM101 – mucA/B, Amp
pYG233 – Cnr, OAT, Kan
Data and Results Reporting
• Equivocal results should be clarified by further testing using modified protocol(s).
• Concentration spacing is important.
• Solvent may be inappropriate.
• Negative results need to be confirmed on a case by case basis.
Criteria for a Positive Result: • Dose related or reproducible increase in the number of revertants in
at least one strain with or without S9. • Statistical analysis may be used as an aid, but should not be the only
criteria for a positive response. • 2-fold rule plus dose-related increase.
Epithelial cells derived from Muta™Mouse Lung
A convenient in vitro tool for assessment of mutagenicity.
5µm
Source: White et al., 2003. Environ Molec Mutagen 42:166- 184.
Muta™Mouse FE1 Cells
Epithelial lung cells isolated from adult male Muta™Mouse
Spontaneously immortalized cell line with characteristics of type I and type II alveolar cells.
Express CYP1A1, as well as several GST isozymes
Can detect point & frameshift mutations, as well as small deletions
Well suited for comparison with in vivo Muta™Mouse results
FE1 cellsMutagen
Lambert et al., 2005
(ΔlacZ, galE-)
6 hour exposure period with serum-free media
72 hour fixation time prior to cell lysis and DNA extraction
PGal positive selection of lacZ mutants in GalE- E. coli
Results presented as Mutant Frequency at a given concentration
•Mutant Frequency - ratio of mutants to total plaques
Test (Ames, etc.)
mg soil eq./plate
Soil Total EOM
Test (Ames, etc.)
Potency = Rev/mg soil eq.
Concentration (mg/plate)
t s p
e r p
la t e
Salmonella Mutagenicity Testing
• Dead space filled with sand
• Extraction:
• Temp = 150C
Sample Fractionation & Solvent Exchange
• Fraction 1 (hexane) – aliphatic hydrocarbons
• Fraction 2 (3:1 hexane:DCM) – non-polar neutral fraction (i.e., PAHs, alkyl-PAHs, O- and S-heterocyclics)
• Fraction 3 (DCM) – semi-polar aromatics (aromatic amines, nitroarenes, N-heterocyclics and oxy-PAHs)
• TurboVap to evaporate solvent using Nitrogen gas
• N-Evap to complete solvent exchange to DMSO for mutagenicity assessment
• Measure the final volume of the extract
Levels of PAHs in Contaminated Soils from Wood Preservation, Coal Gasification, and Coke Production Sites in Sweden
Source: Lemieux et al. (2008) Environ Toxicol Chem 27:978-990.
Mutagenicity Assessment Ames/Salmonella Mutation Assay
Plate incorporation (standard) version
5 concentrations/sample, in triplicate
No S9
Score mutant colonies
72 hrs 37ºC
R e
ve rt
an ts
/p la
Potency = 27.4 rev/mg soil
R e
v e
rt a
n ts
/p la
Potency = 1.4 rev/mg soil
dose response curve
Observed Salmonella Mutagenicity
0
20
40
60
80
100
re v
e rt
a n
0
50
100
150
200
R e
v e
rt a
n ts
Potency = 9.3 rev/mg
Potency = 0.9 rev/mg
5 concentrations/sample, in duplicate
Tested without S9 only
72 hour recovery prior to cell lysis and DNA extraction
PGal positive selection of lacZ mutants in GalE- E. coli
FE1 cellsMutagen
(ΔlacZ, galE-)
Potency = 102.1
Potency = 5.9
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50
N e
t M
u ta
n t
Fr e
q u
e n
cy (x
1 0-5
Summary & Conclusions – Example #1 This Study Demonstrates that the Ames/Salmonella Assay is a useful tool to gain
mechanistic information about the nature of the mutagens in a particular sample/fraction
1. Both Fr 2 and Fr3 had substantial mutagenicity, although Fr2 (PAH-containing fraction) was generally more potent/mg soil
2. YG1041 gave a larger response, which is more sensitive to nitro-compounds & aromatic amines
3. Metabolic activation increased the potency, which indicates that the samples contain pro-mutagens
Additionally, the soil fractions were positive in a mammalian test system
1. Again, the PAH-containing fraction (Fr2) was more potent 2. The promutagens were able to be bioactivated by the mammalian metabolism,
and induce mutagenicity (i.e., no requirement for exogenous S9
Mutagenicity Assessment
Biodiesel Exhaust
Example #2
All Internal Combustion Engines Produce Undesirable Emissions
Regulated Emissions 1. Carbon Monoxide (CO) 2. Nitrogen Oxides (NOx, NO and NO2) 3. Hydrocarbons (THC) 4. Diesel Particulate Matter (PM)
Rapeseed methyl ester, rapeseed ethyl ester, used fry oil methyl ester, animal fat methyl ester, castor oil ethyl ester, hydrotreated vegetable oil, pure plant oils, soy methyl ester, olive oil methyl ester
CANOLA Canadian Oil Low Acid
Comparisons To Be Evaluated
FTP Heavy-duty Transient Cycle
0
500
1000
1500
2000
2500
1 101 201 301 401 501 601 701 801 901 1001 1101
Time seconds
Engine Speed
Dyno Torque
0
500
1000
1500
2000
2500
1 101 201 301 401 501 601 701 801 901 1001 1101
Time seconds
Engine Speed
Dyno Torque
Extract organics
Sample Processing for Examination of SOF (soluble organic fraction) of Diesel Particulates
Collect engine particulates
1. Non-polar neutral fraction
Evaluate mutagenicity of fractions
Sample S003 YG1041 +S9
Concentration (mg/plate)
t s p
e r p
la t e
duty Diesel Engine
Microsuspension (Kado) version
No S9
Score mutant colonies
72 hrs 37ºC
0 50 100 150 200 250 300 350 400 450
R e
ve rt
an ts
/p la
MutPot = 1.3 rev/µg
MutPot = 0.22 rev/µg
MutPot = 7.0 rev/µg
MutPot = 2.2 rev/µg
0
50
100
150
200
250
300
350
400
450
500
0 50 100 150 200 250 300 350 400 450
R e
ve rt
an ts
/p la
Summary & Conclusions – Example #2
This Study Demonstrates that the Ames/Salmonella Assay is useful for gaining mechanistic information about the test samples
1. Fraction 3 was substantially more mutagenic than Fr2, which indicates that the mutagenicity of these samples not primarily due to PAHs
2. The most potent responses were observed for YG1041 without S9, which is indicative of contributions from nitro-compounds
Mutagenicity Assessment of
Concentrated Urine Following
Example #3
Exposure to combustion emissions associated with variety of adverse health effects
90% recently surveyed use temazcales • 1-2 times/week
Thompson et al. (2011) Int J Occup Environ Health 17:103- 112
Biomarkers & Biomonitoring
Pathophysiologic changes • Associated with specific exposures
Urinary Mutagenicity • Previously been shown to be useful biomarker
– Smokers
Study Population & Exposure Assessment
Unexposed (control)
Exposed 32 17 11 9 12
Total 41 20 15
• Creatinine • breakdown product of creatine phosphate in
muscle
• Creatinine adjustment of urine samples • Corrects for difference in glomerular filtration
(different urine flow rates) and hydration status
• Conducted via a colorimetric assay kit (Oxford Biomedical)
Smith-Steventsen, T et al.(2004) Norsk Epidemiology 14(2): 137-43;
Hydrolysis & Extraction
• 16 hours, 37°C
De-conjugated urinary metabolites extracted by solid phase extraction • C18 silica columns
• Eluted with methanol
Metabolism of benzo(a)pyrene
YG1041 Salmonella
S9 + cofactors
• Mutagenic potency calculated as slope of the concentration- response plot
Results
• 1.7 fold over pre- exposure
• 1.7-fold increase over control individuals
Note that dashed lines denote means and solid lines denote medians. * p=0.01; ** p<0.01
F MF C C
0
0.4
0.8
1.2
1.6
2
M u
ta ge
n ic
P o
te n
cy (r
ev er
ta n
ts /µ
m o
Urinary MP vs Time in Temazcal - All
Urinary MP is significantly correlated with time spent in temazcal
Urinary MP vs Time in Temazcal - Adults
Urinary MP is significantly correlated with time spent in temazcal
F MF
0
0.4
0.8
1.2
1.6
M u
ta ge
n ic
P o
te n
cy (r
e ve
rt an
ts /µ
m o
Summary & Conclusions – Example #3
This Study Demonstrates that Salmonella Urinary Mutagenicity Monitoring Can be Used to detect differences in Mutagen Exposure
1. Temazcal use contributed to an increase in urinary mutagenic activity. Post-exposure samples showed a significant 1.7-fold increase relative to pre-exposure samples, and unexposed controls
2. Urinary mutagenicity is empirically related to other measures of exposure such as duration of temazcal use
Issues to Consider
• Understand & appreciate the physical/chemical properties of the environmental matrix (i.e., soil, engine exhaust, urine).
• Understand & appreciate the source(s) of contamination, the physical/chemical nature of the contamination, as well as the kinetics and metabolism of the contaminants.
• Select effect endpoints, assay systems and exposure routes that are relevant and informative.
• Interpret the results in the proper context, and within the confines of the study design and the assay tools employed.
Interesting Research Questions…….
1. (A) In a study on environmental mutagens in complex environmental samples, name 3 important issues that must be carefully considered prior study initiation?
(B) What are the critical sample processing and preparation steps between collection and mutagenicity testing that ensure the generation of useful and reliable results??
2. Name two types of in vitro mutagenicity assays? What features of these assays need to be considered in relation to assessments of complex environmental samples and/or extracts of complex environmental samples?
3. Why would you employ enzymatic deconjugation during the preparation of human urine samples for mutagenicity testing?
4. Why is it important to relate the mutagenicity results back to the amount of bulk material (e.g., g dry weight soil, mL urine, etc.) necessary to induce the observed level of mutagenic activity?
ACKNOWLEDGEMENTS
Funding provided by
• Canada’s Federal Contaminated Sites Action Plan • Canadian Regulatory Strategy for Biotechnology • Program For Energy Research and Development (NRCan)
Temazcal Study was approved by:
• Health Canada’s Research Ethics Board • Office for the Protection of Human Subjects (UC Berkeley)
• Christine Lemieux • Iain Lambert • Staffan Lunstedt • Deborah Rosenblatt • Tak Chan • Nina Holland
• Paul Yousefi • Kirk Smith • Nick Lam • David DeMarini • Subjects in the temazcal study
We are very grateful to our Collaborators:
Holmsund, Umeå Wood preservation 1943 – 1983 Creosote, CCA1, zinc 3
Forsmo, Sollefteå Wood preservation 1933 – 1950 Creosote, CCA 2
Hässleholm Wood preservation 1946 – 1965 Creosote, CCA, zinc 2
Luleå Coke oven plant In operation Creosote, CCA, zinc 1
Husarviken, Stockholm
Gas works 1893 – 1972 Coal tar, heavy metals, cyanide 2
North Sweden Soil