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Cancer risk assessment of airborne PAHs based on in vitro mixture potency factors Kristian Dreij, Ase Mattsson, Ian Jarvis, Hwanmi Lim, Jennie Hurkmans, Johan Gustafsson, Christoffer Bergvall, Roger Westerholm, Christer Johansson, and Ulla Stenius Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b02963 • Publication Date (Web): 26 Jun 2017 Downloaded from http://pubs.acs.org on June 28, 2017
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Cancer risk assessment of airborne PAHs based on in vitro mixture potency
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factors
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Kristian Dreija*, Åse Mattssona, Ian WH Jarvisa1, Hwanmi Limb, Jennie Hurkmansc, Johan
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Gustafssonb, Christoffer Bergvallb, Roger Westerholmb, Christer Johanssonb,c, Ulla Steniusa
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a. Institute of Environmental Medicine, Karolinska Institutet, 171 77 Stockholm, Sweden
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b. Department of Environmental Science and Analytical Chemistry, Stockholm University, 106 91 Stockholm, Sweden
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c. Environment and Health Administration, 104 20 Stockholm, Sweden
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*corresponding author: Kristian Dreij, Institute of Environmental Medicine, Karolinska
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Institutet, Box 210, 171 77 Stockholm, Sweden, +46-8-52487566,
[email protected] 14 15
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London SE1 9NH, United Kingdom
present address: Division of Analytical and Environmental Sciences, King’s College London,
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Abstract
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Complex mixtures of polycyclic aromatic hydrocarbons (PAHs) are common environmental
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pollutants associated with adverse human health effects including cancer. However, the risk
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of exposure to mixtures is difficult to estimate and risk assessment by whole mixture potency
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evaluations has been suggested. To facilitate this, reliable in vitro based testing systems are
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necessary. Here, we investigated if activation of DNA damage signaling in vitro could be an
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endpoint for developing whole mixture potency factors (MPFs) for airborne PAHs. Activation
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of DNA damage signaling was assessed by phosphorylation of Chk1 and H2AX using
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western blotting. To validate the in vitro approach, potency factors were determined for seven
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individual PAHs which were in very good agreement with established potency factors based
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on cancer data in vivo. Applying the method using Stockholm air PAH samples indicated
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MPFs with orders of magnitude higher carcinogenic potency than predicted by established in
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vivo-based potency factors. Applying the MPFs in cancer risk assessment suggested that 45.4
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(6% of all) cancer cases per year in Stockholm are due to airborne PAHs. Applying
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established models resulted in < 1 cancer case per year, which is far from expected levels. We
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conclude that our in vitro based approach for establishing MPFs could be a novel method to
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assess whole mixture samples of airborne PAHs to improve health risk assessment.
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Introduction
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The World Health Organization (WHO) has estimated 3.7 million deaths world-wide from
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outdoor air pollution in 2012. If indoor air pollution (households cooking over coal, wood or
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biomass stoves) was included total air pollution is responsible for 7 million premature deaths
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annually, making air pollution the single largest environmental health risk.1 Outdoor air
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pollution is classified as carcinogenic to humans by the International Agency for Research on
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Cancer (IARC)2 and it is estimated that 6% of deaths due to air pollution is attributed to lung
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cancer while stroke and ischemic heart disease are the most common causes.1 Air pollution is
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comprised of a large array of different chemicals associated with particulate matter (PM)
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including polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and
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metals, which all may contribute to the lung cancer risk.3 A large number of PAHs that are
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associated with PM in ambient air have been shown to play an important role in causing lung
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cancer in humans.3-5
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Cancer risk assessment of environmental PAHs is generally performed by one of two different
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approaches: using a surrogate compound for the whole mixture or component based factors
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(relative potency factors, RPFs or toxic equivalency factors, TEFs).6 For air, benzo[a]pyrene
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(B[a]P) has been suggested as a surrogate for the entire PAH content7 and a European target
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value of 1 ng B[a]P/m3 has been set for the protection of human health.8 In the second
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approach the different components of the PAH mixture are assigned RPFs based on animal
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cancer data from which cancer risk estimates relative to B[a]P are derived. However, both
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these approaches are likely to misestimate the actual health risks of human exposure to
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complex mixtures.9-11 For example, they do not take possible non-additive mixture effects into
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account, several PAHs have not been assigned a RPF, and many PAHs with low
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environmental concentration but high carcinogenic potential, e.g. dibenzo[def,p]chrysene
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(DBC, also known as dibenzo[a,l]pyrene) and benz[j]aceanthrylene (B[j]A), are not routinely ACS Paragon Plus Environment
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measured.12-13 Excluding the often very potent high-molecular weight PAHs when assessing
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health risks associated with atmospheric PAH exposure probably leads to a significant
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underestimation of the risks.14-16 To overcome these limitations in risk assessment of PAH
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mixtures whole mixture potency evaluations have been suggested.17-18 This approach has the
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least uncertainty in relation to the impact of interaction effects and unknown mixture
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constituents. However, it requires site specific data on the PAH mixture which might not
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always be available and is potentially limiting from an economical and ethical perspective in
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relation to the required extensive in vivo testing. There is therefore a major need for a reliable
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in vitro based testing and assessment system for whole mixture approaches.
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We recently showed that the potency of DBC and B[j]A to activate DNA damage signaling in
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vitro through the proteins Checkpoint kinase 1 (Chk1) and H2A histone family, member X
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(H2AX) relative to that of B[a]P was in very good agreement with published in vivo-based
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RPFs.12,19 Chk1 and H2AX are both involved in the cellular DNA damage response
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controlling early cell cycle regulation and genomic stability maintenance.20-21 The
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applicability of using activation of H2AX as an in vitro marker for genotoxic potency of other
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groups of compounds is also supported by previous studies.22-24 Based on this we proposed
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that activation of DNA damage signaling could be a relevant endpoint in vitro for developing
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mixture potency factors for environmental PAH samples.9,19 In the present study we further
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ascertain the validity of this approach by comparing our in vitro based system with RPFs
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obtained from cancer data in vivo. Subsequently we apply this methodology to assign mixture
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potency factors (MPFs) for whole mixture air PAH samples and investigate the feasibility of
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applying these in cancer risk assessment of PAH air pollution in Stockholm, Sweden.
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Materials and Methods
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Chemicals and reagents ACS Paragon Plus Environment
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Caution: PAHs are carcinogenic and experimental handling must be carried out under special
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safety conditions such as those outlined in the National Cancer Institute guidelines.
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Phenanthrene
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benzo[a]anthracene (B[a]A) and benzo[b]fluoranthene (B[b]F) were obtained from Sigma-
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Aldrich (Stockholm, Sweden). Pyrene (Pyr) and dibenz[a,h]anthracene (DB[a,h]A) were
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supplied by Merck and Chem Service, respectively. Cell culture reagents were supplied by
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Gibco (Life Technologies, Stockholm, Sweden). Antibodies used for Western blotting were
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phospho-Chk1 (Ser317; pChk1) and phospho-H2AX (Ser139; γH2AX) from Cell Signaling
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Technology (Beverly, MA), and Cdk2 (M2) and secondary anti-rabbit antibodies from Santa
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Cruz (Santa Cruz, CA).
(Phen),
benzo[a]pyrene
(B[a]P),
dibenzo[def,p]chrysene
(DBC),
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Air sampling
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The air particulate matter (PM) samples were collected at two sites, one at the roof top (22
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meters above the ground) of the Arrhenius laboratory, Stockholm University (STHLM, N
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59.36561, E 18.05941) and the other from a car tunnel, Söderledstunneln (TUNNEL, N
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59.311138, E 18.073036), running under one of the main islands of Stockholm city center.
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The sampling from the roof top site has been previously described.25 In brief, an in-house
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built pump device (average flow rate: 974 L/min) was used to collect total PM on a
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fluorocarbon-coated glass fiber filter (Ø = 235 mm, Fiberfilm Filters, Pallflex, Pall
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Corporation, Putnam, CT, USA) with sampling on a weekly basis for 10 weeks from January
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31 to April 5 during 2013. Total air volume sampled was 97727 m3 and total PM was 655 mg.
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For the tunnel sampling, PM10 (PM diameter
