Analysis of Polycyclic Aromatic Hydrocarbons in Human Liver Cells

1. Determination of polycyclic aromatic hydrocarbons and its monohydroxilated metabolites in human liver cells using gas chromatography and high performance liquid chromatography with mass spectrometryVincent 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) NEPM - Health Investigation Level (HIL A) ΣPAHs (residental) = 300 mg/kg, B[a]P = 3 mg/kg

9. Overall project Exposure Effects UBM In vitro Cell culture Genotoxicity Cytoxicity ROS AhR Uptake Metabolism Bioavailability, Metabolism Bioaccessibility stomach & small intestine Extracellular Intracellular Simulated in vivo conditions Inform risk assessor, regulator, land owner

10. Which PAHs?

11. Methodology

12. Cell PAH uptake – solution

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

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. j.ng@uq.edu.au Associate supervisor: Dr. Cheng Peng The University of Queensland, National Research Centre for Environmental Toxicology (EnTox), 39 Kessels Road, Coopers Plains, Brisbane, Australia. c.peng@uq.edu.au Associate supervisor: Dr. Mary Fletcher The University of Queensland, Queensland Alliance for Agriculture and Food Innovation (QAAFI), 39 Kessels Road, Coopers Plains, Brisbane, Australia. m.fletcher@uq.edu.au Associate supervisor: Dr. Stephen Were Department of Agriculture and Fisheries (DAF), Biosecurity Queensland, 39 Kessels Road, Coopers Plains, Brisbane, Australia. Stephen.Were@daf.qld.gov.au

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: 3.1.1.11-12 Publications – 2015 Environ. Techol Innovation 2015 Mutagenesis 2016 Chemosphere UQ – UQI Scholarship and CRC CARE Scholarship UQ GSITA Scholarship Australian Govt. Greg Urwin Award

29. Thank you…