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

Overview Background Methodology Results and discussion Conclusions Acknowledgements

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

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

Real estate issue? Former gasworks site in Sydney, Australia

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

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

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 0.57 - 3120 Koganti et al. 1998 United Kingdom Sediment 75500 Goodfellow et al. 2001 147 - 1234 Khalil et al. 2006 4 - 5700 Kreitinger et al. 2007 France 507 - 6277 Haeseler et al. 1999 7 - 1040 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

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  

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

Methodology

Cell PAH uptake – solution

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

Compound (N = 10) Recovery (%) 1 min RSD (%) 2 min Naphthalene 75 - 85 5 85 - 90 Phenanthrene 80 - 95 10 85 - 105 7 Pyrene Benzo[a]pyrene 75 - 80 16 85 - 102 8 1-OH Naphthalene 2-OH Naphthalene 70 - 85 80 - 98 1-OH Phenanthrene 2-OH Phenanthrene 3-OH Phenanthrene 4-OH Phenanthrene 70 - 90 85 - 100 1-OH Pyrene 80 - 90 80 - 100 3-OH Benzo[a]pyrene 70 - 75 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

Results and discussion

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

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

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

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 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

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)

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

Thank you…