1. Determination of polycyclic aromatic hydrocarbons and its monohydroxilated metabolites in human liver cells using gas chromatography and high performance liquid chromatography with mass spectrometry Vincent Lal PhD candidate 14th April 2017

2. Overview Background Methodology Results and discussion Conclusions
Acknowledgements

3. Over 3 million contaminated sites
world wide
Potential risk (human health)
Remediation of sites
contaminant
time
cost

4. Australian HRA guidelines
Environmental Health Risk Assessment
(EHRA)
National Environment Protection Measure
(NEPM)
Soil based Health Investigation Level (HILs)

5. Real estate issue?
Former gasworks site in Sydney, Australia

6. Human health risk assessment
Soil (HIL based on land-use + contaminant) – NEPM
Site specific (soil properties)
Soil particle size ( < 250 µm)

7. Remediation gasworks site - Toowoomba. Cost: $20,000,000

8. PAHs in gasworks contaminated soil and sediment (Australia and beyond)
Country
Matrix
Value
(mg/kg)
Reference
Australia
Soil
335 – 8645
Thavamani et al. 2011
United States of America
Koganti et al. 1998
United Kingdom
Sediment
75500
Goodfellow et al. 2001
Khalil et al. 2006
Kreitinger et al. 2007
France
Haeseler et al. 1999
Stroo et al. 2000
2 - 68
Cave et al. 2010
NEPM - Health Investigation Level (HIL A) ΣPAHs (residental) = 300 mg/kg, B[a]P = 3 mg/kg

9. Simulated in vivo conditions
Overall project
Compounds
TEF*
IARC
Pyrene
0.001 3
Naphthalene 2B
Phenanthrene
Benzo[a]pyrene
1.0 1
Pb/Pb(C2H3O2)2·3H2O 2A
As/NaAsO2
Cd/CdCl2
Exposure
Effects
UBM
stomach & small intestine
Bioaccessibility
Extracellular
In vitro Cell culture
Intracellular
Genotoxicity
Cytoxicity
ROS
AhR
Uptake
Metabolism
Bioavailability, Metabolism .
Simulated in vivo conditions
Inform risk assessor, regulator, land owner

10. Which PAHs? Compound Rings IARC Benzo[a]pyrene 5 1 Pyrene 4 2B
Phenanthrene
Naphthalene 3 2

11. Methodology

12. Cell PAH uptake – solution

14. Methodology overview (PAHs + OH-PAHs)
PAH dose: 5µM
Solution: Pure compound
HepG2 (human liver cells)

19. Compound (N = 10)
Recovery (%)
1 min
RSD (%)
2 min
Naphthalene 5
Phenanthrene 10 7
Pyrene
Benzo[a]pyrene 16 8
1-OH Naphthalene
2-OH Naphthalene
1-OH Phenanthrene
2-OH Phenanthrene
3-OH Phenanthrene
4-OH Phenanthrene
1-OH Pyrene
3-OH Benzo[a]pyrene 20

20. Compound (N = 10)
LOD (pg mg-1)
Naphthalene
1-OH Naphthalene
2-OH Naphthalene 5
0.1
Phenanthrene
1-OH Phenanthrene
2-OH Phenanthrene
3-OH-Phenanthrene
4-OH Phenanthrene
Pyrene
1-OH Pyrene
Benzo[a]pyrene
3-OH Benzo[a]pyrene 10

21. Results and discussion

22. Uptake – conc dependent (p < 0.05 )
- instantaneous indicating
passive diffusion
- Maximum 3 – 6 h
plateau up to 24 h
- Phase 1 metabolite
3-OH B[a]P, indicating
metabolic activation
- Continued to increase over
exposure period

24. Key findings
This is the first application of using a QuEChERS approach
with an in vitro analysis using the HepG2 cells (biological matrix)
Modified QuEChERs procedure is adaptable, simple and is
efficient in the assessment of PAHs and OH-PAHs in
small amount of biological samples, useful in risk assessment
studies
Spike recovery experiment indicated method was sufficient to
test for major metabolites of PAHs

25. Uptake of PAHs into HepG2 cells was significantly influenced in a
concentration dependent manner
Almost instantaneous uptake of PAHs – passive diffusion
Metabolic capacity of HepG2 resulted in formation of major
metabolites that can be quantified and be useful biomarkers
in vitro studies

26. Acknowledgement Principal supervisor: Prof. Jack Ng.
The University of Queensland, National Research Centre for Environmental
Toxicology (EnTox), 39 Kessels Road, Coopers Plains, Brisbane, Australia.
Associate supervisor: Dr. Cheng Peng
Toxicology (EnTox), 39 Kessels Road, Coopers Plains, Brisbane, Australia.
Associate supervisor: Dr. Mary Fletcher
The University of Queensland, Queensland Alliance for Agriculture and Food
Innovation (QAAFI), 39 Kessels Road, Coopers Plains, Brisbane, Australia.
Associate supervisor: Dr. Stephen Were
Department of Agriculture and Fisheries (DAF), Biosecurity Queensland, 39
Kessels Road, Coopers Plains, Brisbane, Australia.

27. Other colleagues and advisors
Dr. Sasikumar Muthusamy
Dr. Qing (Summer) Xia
Patrick Segel, Dr. Ken Tong and DAF team
Eugene inorganics QHFSS
QAEHS staff and students
Prof. Jochen Muller (UQ) and Prof. Trevor Penning Groups (Upen)

28. Funding for this project is provided by CRC CARE
Project number:
Publications – Environ. Techol Innovation
2015 Mutagenesis
Chemosphere
UQ – UQI Scholarship and CRC CARE Scholarship
UQ GSITA Scholarship
Australian Govt. Greg Urwin Award

29. Thank you…