Article
The Genetic Toxicity of Complex Mixtures of Polycyclic Aromatic Hydrocarbons: Evaluating Dose-Additivity in a Transgenic Mouse Model Alexandra S. Long, Christine L. Lemieux, Remi Gagne, Iain B Lambert, and Paul A White Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 06 Jun 2017 Downloaded from http://pubs.acs.org on June 7, 2017
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Environmental Science & Technology
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The Genetic Toxicity of Complex Mixtures of Polycyclic Aromatic Hydrocarbons:
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Evaluating Dose-Additivity in a Transgenic Mouse Model
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Alexandra S. Long1,2, Christine L. Lemieux3, Rémi Gagné2, Iain B. Lambert4, Paul A. White1,2
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Department of Biology, University of Ottawa, Ottawa, ON, K1N 6N5. 2Mechanistic Studies
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Division, Environmental Health Science and Research Bureau, Health Canada, Ottawa, ON,
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K1A 0K9. 3New Substances Assessment and Control Bureau, Health Canada, Ottawa, ON, K1A
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0K9. 4Department of Biology, Carleton University, Ottawa, ON, K1S 5B6.
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* Corresponding Author: Alexandra S. Long
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50 Colombine Driveway
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Tunney's Pasture A/L 0803A
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Ottawa, Ontario K1A 0K9 Canada
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Tel: 613-960-1606; Fax: 613-941-8530
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[email protected] 17 18
SHORT RUNNING TITLE: The Genotoxicity of Complex PAH Mixtures
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KEY WORDS: polycyclic aromatic hydrocarbon, benzo(a)pyrene, genotoxicity, mutagenicity,
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PAH mixtures, additivity, risk assessment
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ABSTRACT This study evaluates the risk assessment approach currently employed for polycyclic
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aromatic hydrocarbon (PAH)-contaminated media, wherein carcinogenic hazards are evaluated
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using a dose-addition model that employs potency equivalency factors (PEFs) for targeted
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carcinogenic PAHs. Here, Muta™Mouse mice were sub-chronically exposed to PAH mixtures
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(p.o.), and mutagenic potency (MP) values were determined for five tissues. Predicted dose-
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additive mixture MPs were generated by summing the products of the concentrations and MPs of
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the individual targeted PAHs; values were compared to the experimental MPs of the mixtures to
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evaluate dose-additivity. Additionally, the PEF-determined BaP-equivalent concentrations were
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compared to those determined using a bioassay-derived method (BDM) (i.e., an additivity-
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independent approach). In bone marrow, mixture mutagenicity was less than dose-additive and
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the PEF-method provided higher estimates of BaP-equivalents than the BDM. Conversely,
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mixture mutagenicity in site-of-contact tissues (e.g., small intestine) was generally more than
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dose-additive and the PEF-method provided lower estimates of BaP-equivalents than the BDM.
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Overall, this study demonstrates that dose-additive predictions of mixture mutagenic potency
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based on the concentrations and potencies of a small number of targeted PAHs results in values
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that are surprisingly close to those determined experimentally, providing support for the dose-
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additive assumption employed for human health risk assessment of PAH mixtures.
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Environmental Science & Technology
INTRODUCTION Polycyclic aromatic hydrocarbons (PAHs) are organic compounds produced by the
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incomplete combustion of organic material. PAH exposures characteristically involve complex
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mixtures in complex environmental matrices such as contaminated soil, urban air, vehicle
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exhaust, tobacco smoke, or contaminated water. Several PAHs have been shown to be genotoxic
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and/or carcinogenic in experimental animals, as well as carcinogenic to humans1, 2.
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Benzo(a)pyrene (BaP), the prototypical PAH, has been classified by the International Agency for
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Research on Cancer (IARC) as a Group 1 human carcinogen, with several additional PAHs listed
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as probable or possible human carcinogens (i.e., Group 2a or 2b)1. Additionally, several PAH-
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containing complex mixtures are Group 1 IARC carcinogens, including diesel exhaust3, air
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pollution4, tobacco smoke5, 6, and coal tar (CT)7.
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PAHs induce somatic mutations in mammalian cells via DNA-binding of activated
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metabolites, subsequent incorrect or inadequate removal/repair of lesions, and fixation of
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mutations during replication3. Somatic mutations have been mechanistically and empirically
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linked to cancer, and genetic damage is considered an enabler of carcinogenesis8, 9. Reduction of
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exposures to known or suspected carcinogens, including PAHs and PAH mixtures, has been a
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focus of public health organisations for several decades, and a recent high-impact publication has
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re-emphasized the importance of environmental carcinogen exposures in determining the overall
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cancer risk10. Thus, efficient and effective assessment of the risks posed by PAH mixtures is
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critical for identifying exposure scenarios that can increase risk to human health; and moreover,
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interventions that can reduce risk.
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Hazard and risk assessments of complex environmental mixtures, including PAH
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mixtures in complex environmental matrices, are challenging since most available toxicological 3 ACS Paragon Plus Environment
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data pertain to individual compounds. Additionally, no two environmental mixtures are exactly
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the same, and the identity and toxicological properties of hazardous compounds in the mixtures
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remain largely unknown. Finally, there remains a paucity of data regarding possible interactions
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between known mixture components. Each of these issues hampers reliable hazard/risk
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assessment.
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To address these challenges, governmental organizations such as the US Environmental
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Protection Agency (USEPA)11 and Health Canada12 have published guidance documents for
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conducting human health risk assessments of chemical mixtures. Although examination of the
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actual mixture in question is ideal and preferred, it is unrealistic to toxicologically characterize
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all environmental mixtures. When whole mixture data are not available, component methods are
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recommended. Many component methods are based on dose-addition, which calculates the total
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hazard or risk of a mixture as the incremental sum of the contributions from a small number of
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targeted components.
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For estimating carcinogenic effects, the dose-addition model for PAHs employs several
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empirical assumptions. First, it is assumed that the genotoxic mixture components, when
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combined, act in a dose-additive manner. Unfortunately, there is a paucity of data to support the
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validity of this statement. Second, the model implies that the total hazard/risk posed by the
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mixture is effectively represented by the contributions from a handful of targeted chemicals that
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are included on governmental lists of prioritised pollutants, in this case the 7-8 PAHs that are
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probable, possible, or known human carcinogens1, 13, 14. This second assumption infers that
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application of a dose-addition model when only a few components are known implies that the
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total mixture hazard/risk can be adequately represented by the contributions of those known
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chemicals, which may be a significant uncertainty. To critically evaluate these assumptions for 4 ACS Paragon Plus Environment
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genotoxic PAHs, several studies have scrutinized the genotoxicity of complex PAH mixtures and
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simplified synthetic PAH mixtures. Unfortunately, the vast majority of these studies were
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conducted in vitro, and only examined highly simplified (e.g., binary) mixtures. A more
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thorough overview of PAH mixtures research is presented in our earlier works15-17.
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To estimate the incremental lifetime cancer risk posed by a PAH mixture, the dose-
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addition method assesses the concentrations of each targeted PAH, and then, using the
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appropriate compound-specific potency equivalency factors (PEFs), converts the concentration
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of each targeted PAH into equivalents of BaP, the reference PAH or index chemical. The total
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BaP-equivalent concentration in the mixture is then calculated as the sum of the converted
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incremental doses from each carcinogenic PAH. This PEF-driven assessment method (PEF-M) is
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appealing and convenient since it evaluates a small number of targeted PAHs, and does not
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require toxicological data for the specific mixture. However, regulatory agencies have
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recognized and highlighted limitations of human health risk assessment approaches based on a
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few PAHs and those that employ the assumption that doses scaled to BaP are additive11, 18, 19. A
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more thorough overview of PAH mixtures risk assessment approaches is presented in our earlier
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works15-17.
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The current study aims to critically evaluate the assumption of dose additivity, as well as
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to determine whether contributions from other mixture components affect the observed mixture
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effects. Rodent cancer bioassays are extremely costly and, for a single chemical, take over two
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years to complete; therefore, since PAHs are carcinogens with a mutagenic mode-of-action1, we
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chose to employ in vivo mutagenicity as a surrogate endpoint for cancer. Previously, we
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published two studies that examined the mutagenic potency (MP) (i.e., the linear slope of the
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dose-response function) of 8 targeted PAHs deemed carcinogenic by the Canadian Council of 5 ACS Paragon Plus Environment
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Ministers of the Environment (i.e., CCME carcinogenic PAHs)20, fractions of organic extracts of
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PAH-contaminated soil, and matching synthetic mixtures that only contain the CCME
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carcinogenic PAHs in proportions identical to their presence in the soil fractions. Moreover, we
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compared the complex and synthetic mixture responses with those predicted based on the
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concentrations and potencies of the CCME carcinogenic PAHs examined. The first study
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employed a bacterial gene mutation assay15 and the later work used an in vitro mammalian cell
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gene mutation assay based on an immortalised cell line derived from the transgenic
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Muta™Mouse16, 17. In the bacterial study, we found less than dose-additive mixture results15, and
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in the in vitro study, we found both less than dose-additive and more than dose-additive mixture
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results, all of which were within 2-fold of the dose-additive prediction with a single exception16.
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The difference in the deviations from additivity between the bacterial and mammalian cell
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studies is presumably related to metabolic limitations, and this is discussed in more detail in
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Lemieux et al. (2015)16. In the current study, our aim was to expand on our earlier works by
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examining PAH mixture dose-additivity across several tissues using an in vivo model. Recently,
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we determined the in vivo MP of the eight CCME carcinogenic PAHs20, as well as the MP of a
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purified CT-based driveway sealcoat (CT-seal)21 following sub-chronic Muta™Mouse oral
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exposures. The data presented in these studies, plus new in vivo assessments of two CT
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preparations (i.e., CT-1 and CT-2), form the basis of the current follow-up study that employs in
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vivo genetic toxicity data to complete our evaluations of the dose-additivity assumption for
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PAHs. More specifically, we critically evaluate the dose-additivity paradigm by comparing
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observed MP values across five tissues for both the complex and synthetic PAH mixtures to
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those predicted using the incremental contributions of each PAH20. In order to evaluate possible
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contributions of other compounds present in the complex mixtures studied, we compared the 6 ACS Paragon Plus Environment
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complex mixture results to results obtained with synthetic mixtures only containing the eight
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CCME carcinogenic PAHs.
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Additionally, in order to examine the PEF-M for PAH mixtures, we previously developed
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a bioassay-derived method (BDM) that assesses the actual mutagenic activity of the complex
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mixture, and expresses that activity relative to the activity of BaP15, 17. This method calculates
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BaP-equivalent concentrations without employing a chemical-specific assumption of dose-
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additivity, and can be used as a tool to compare with the BaP-equivalent concentrations
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determined using the PEF-method. The use of the term “bioassay-derived method” refers to the
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fact that the BDM uses bioassay data for the actual mixture being evaluated. In contrast, the
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PEF-M does not. We employed this approach herein to scrutinise the assumption of dose-
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additivity underlying the human health risk assessment of PAH-containing mixtures.
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MATERIALS & METHODS
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Coal tar extraction
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CT-1, provided by the Electric Power Research Institute, is a dense non-aqueous phase
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liquid from a former manufactured gas plant (MGP) in the United States. CT-2, provided by the
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Canadian National Research Council, is from a Canadian coke production facility. Each CT
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sample was thoroughly mixed until homogenous, and an aliquot was removed and extracted in
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an identical manner as the CT-seal21, according to a method adapted from Wise et al.22. An
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aliquot of the extract was set aside for chemical analyses, and the remainder was used to prepare
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dosing solutions by first sonicating the eluate, and then diluting it in highly-refined olive oil
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(Sigma-Aldrich Canada, Oakville, ON). Dosing solutions, in mg equivalent (eq) weight of crude
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CT per ml, were prepared weekly and stored in amber glass vials.
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PAH content of coal tars by GC-MS 7 ACS Paragon Plus Environment
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The PAH content of the CTs was determined via GC-MS according to USEPA Method
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8270D. Analyses were conducted by a commercial laboratory as described previously21. The
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concentrations of a standard panel of 19 PAHs (including the 8 CCME carcinogenic PAHs18)
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were expressed as mg PAH/kg CT.
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Synthetic mixture preparation
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Using the results of the chemical analyses, synthetic mixtures were prepared to match the
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composition of the 8 CCME carcinogenic PAHs in both CT-extracts, as well as the CT-seal
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extract. Synthetic mixture-1 (Syn-1) was prepared to match CT-1, Syn-2 was prepared to match
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CT-2, and Syn-3 was prepared to match CT-seal. Three concentrations of each mixture were
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prepared weekly in amber glass vials and dissolved in an appropriate volume of highly refined
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olive oil.
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Animal treatment
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Adult male Muta™Mouse mice were used for all in vivo exposures. As dietary intake
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provides the main source of human exposure to PAHs (i.e., for non-smokers who are not
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occupationally exposed)1, 23, an oral route of exposure was selected for this study. The dosing
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solutions were administered by oral gavage daily for 28 days, with a 3-day post-exposure
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sampling time as per OECD test guideline #48824. The administered doses of the complex
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mixtures were: 250, 500, and 1000 mg CT-eq/kg body weight (BW)/day of CT-1, 700, 1400, and
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2100 mg CT-eq/kg BW/day of CT-2, 1974, 3949, and 7897 mg CT-seal-eq/kg BW/day of the
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CT-seal. The doses of the synthetic mixtures were: 1092, 2184, 4368 mg CT-eq/kg BW/day for
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Syn-1; 700, 1400, and 2100 mg CT-eq/kg BW/day for Syn-2, and 1974, 3949, and 7897 CT-seal-
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eq/kg BW/day for Syn-3. Mice were euthanized and necropsied as previously described20, and
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glandular stomach (GS), small intestine (SI), liver, (Lv), bone marrow (BM), and lung (Lg) were 8 ACS Paragon Plus Environment
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immediately harvested and stored at -80ºC 20, 21. GS was used instead of forestomach as we were
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unable to obtain sufficient high-quality DNA from the latter. Mice were bred, maintained, and
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treated according to the Canadian Council for Animal Care Guidelines (available online at:
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http://www.ccac.ca/en_/standards/guidelines), and the Health Canada Animal Care Committee
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approved the protocols.
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Genomic DNA isolation
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Tissue samples were digested as described previously20. Genomic DNA was isolated
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from lysed tissues using a phenol/chloroform extraction procedure described previously25, 26.
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Isolated DNA was dissolved in TE buffer (10 mM Tris pH 7.6, 1 mM EDTA) and stored at 4°C.
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Mutant frequency analysis
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The PGal (phenyl-β-D-galactoside) positive selection assay was used to determine the
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lacZ mutant frequency (ratio of mutant plaque forming units (pfu) to total pfu) in DNA samples
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from GS, SI, Lv, BM, and Lg as previous described26-28.
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Statistical evaluation of the overall treatment effect
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The lacZ dose-response data were analysed in SAS v.9.1 (SAS Institute, Cary, NC).
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Initial analyses employed Poisson regression to examine the significance of the treatment, and
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Type 3 chi-squared analysis was used to assess goodness of fit20. Following pilot dose range-
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finding assays, doses were selected to yield distinctly linear dose-response functions whereby
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truncation (i.e., removal of a single high dose value) was rarely required. Should visual
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examination of the dose-response suggest the need to truncate the data series (i.e., remove the top
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dose), the restricted data were reanalysed. If reanalysis resulted in an improved chi-squared
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value, the truncated results were retained and used for MP determination, as described below.
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Truncation of dose-response data was only required for 1 of the 30 datasets examined. 9 ACS Paragon Plus Environment
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Experimental and dose-additive mutagenic potency MPs for the lacZ endpoint, which are defined as the slope of the linear portion of the
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dose-response functions, were determined using ordinary least-squares linear regression for the
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complex and synthetic mixtures in all tissues (expressed as mutant frequency x 10-5/mg CT (or
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CT-seal)-eq/kg BW/day). The observed MP values for individual PAHs in each tissue, expressed
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as mutant frequency x 10-5/mg PAH/kg BW/day, were recently published by our group20 (Table
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S1). A linear no-threshold response over the tested dose range was assumed. The MP values for
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individual PAHs were employed in our additivity calculation along with the measured PAH
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concentrations in the complex mixtures (in mg PAH/mg CT or CT-seal-eq). The dose-additive
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MP estimate for each PAH mixture, expressed as mutant frequency x 10-5/mg CT (or CT-seal)-
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eq/kg BW/day, was calculated as the sum of the converted incremental doses from each targeted
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PAH according to Equation 1. As noted above, the MPs were determined as non-thresholded,
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linear dose-response functions with constant potency over the dose range of interest.
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[1]
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As with our previous in vitro work, statistical comparisons of the calculated dose-additive
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MPs with corresponding experimentally-observed mixture MPs were not conducted due to the
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large variance associated with the predicted dose-additive potency values that are attributable to
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the individual variances associated with the contributions of each individual PAH29. The tissue-
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matched MP of each synthetic PAH mixture was compared to that of the corresponding complex
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mixture using the two-tailed Student t-test (p ≤ 0.05). 10 ACS Paragon Plus Environment
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BaP-equivalent concentrations: Potency equivalency factor method and bioassay-derived
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method
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Routine assessment of incremental lifetime cancer risk for oral exposure to PAH-
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contaminated complex matrices (e.g., soil) requires quantitative information pertaining to (1) the
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level of PAH contamination (i.e., the concentrations of targeted PAHs), (2) PEFs to convert PAH
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concentrations to a BaP-equivalent concentration, (3) ingestion rate, (4) exposure duration, (5)
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lifespan, (6) body weight, and (7) the BaP oral cancer slope factor. For this study, we only
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calculated the BaP-equivalent concentrations, as the remaining factors would be constants for a
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given PAH mixture. The PEF-M (Equation 2), which assumes dose-additivity, calculates total
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BaP-equivalents for each of the examined mixtures as the sum of the products of PAH
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concentration and their compound-specific cancer PEFs. The maximum possible PEF ranges
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were calculated using the highest and lowest published PEF values (derived from in vivo data
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only) for each PAH, obtained from 13 sources14, 30-41 cited in18, and two additional sources42, 43
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(Table S2).
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[2]
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In contrast, the BDM approach (Equation 3 below), which provides tissue-specific BaP-
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equivalents for each of the examined mixtures, calculates total BaP-equivalents (in mg BaP/mg
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CT or (CT-seal)-eq) as the ratio of the experimental Muta™Mouse MP of a mixture (as mutant
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frequency x 10-5/mg CT (or CT-seal)-eq/kg BW/day) to the Muta™Mouse MP of BaP (as mutant
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frequency x 10-5/mg BaP/kg BW/day) in the same tissue (Equation 3). By definition, this
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approach does not assume additivity of prioritised mixture components. 11 ACS Paragon Plus Environment
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[3]
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The BDM was employed to calculate BaP-equivalents, in mg BaP/mg eq. CT or CT-seal,
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for the 3 complex and 3 synthetic mixtures, and 95% confidence limits were calculated as the
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product of the standard error and the t value for the appropriate number of degrees of freedom44.
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The BDM-derived BaP-equivalent concentration values were then compared with those
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calculated using the traditional PEF-M. More specifically, to determine whether there are
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meaningful differences between the BDM- and PEF-derived BaP-equivalent concentration
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values, the upper or lower 95% confidence limits of the BDM-derived BaP equivalent
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concentrations were visually compared with the maximum or minimum PEF-M-derived BaP-
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equivalent concentrations calculated from the range of PEF values examined.
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RESULTS
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PAH content of the coal tar and coal tar-based sealcoat
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PAH concentrations in the CTs and CT-seal are summarised in Table S3. The results of
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the chemical analysis of CT-1 revealed that it is composed of 28% analysed PAHs by weight,
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including 6.0% CCME carcinogenic PAHs, and nearly 1% BaP. Analysed PAHs made up 19%
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of CT-2, with 1.8% of the material being CCME carcinogenic PAHs, and 0.30% BaP. Analysed
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PAHs made up 4.4% of CT-seal, with 0.96% being CCME carcinogenic PAHs, and 0.19% BaP.
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The lacZ mutagenic potency of the complex and synthetic PAH mixtures
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The Poisson regression results indicate that all six mixtures induced a significant increase in lacZ mutant frequency in all tissues examined (Figure S1, Table S4, p
