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. 2016 May 9;5(4):1160–1171. doi: 10.1039/c6tx00024j

Effects of multi-component mixtures of polyaromatic hydrocarbons and heavy metal/loid(s) on Nrf2-antioxidant response element (ARE) pathway in ARE reporter-HepG2 cells†

Sasikumar Muthusamy a,b, Cheng Peng a,b, Jack C Ng a,b,✉
PMCID: PMC6072108  PMID: 30090422

graphic file with name c6tx00024j-ga.jpgThe effect of mixtures of PAHs and heavy metal/loid(s) on the Nrf2 antioxidant pathway in HepG2-ARE cells was determined as an indicator of the oxidative stress response.

Abstract

Exposure to polyaromatic hydrocarbons (PAHs) and heavy metal/loid(s) has been demonstrated to induce an oxidative stress response in mammalian cells. The combined effect of PAHs and heavy metal/loid(s) on the oxidative stress response has not been reported extensively. The Nrf2 antioxidant response pathway plays an important role in cellular antioxidant defense against oxidative stress-induced cell damage. In this study, we have determined the combined effect of four PAHs (benzo[a]pyrene (B[a]P), naphthalene (Nap), phenanthrene (Phe) and pyrene (Pyr)) and three heavy metal/loid(s) (arsenic (As), cadmium (Cd) and lead (Pb)) on the Nrf2 antioxidant pathway using the ARE reporter-HepG2 cell line. The mixture study was carried out for binary, ternary, quaternary and seven-component combinations of PAHs and heavy metal/loid(s). Initially, individual dose responses for the PAHs (B[a]P, Nap, Phe and Pyr) and heavy metal/loid(s) (As, Cd and Pb), as well as their respective concentrations that induced an induction ratio of 1.5 (ECIR1.5), were determined. The luciferase assay system was used to quantify the induction of the Nrf2 antioxidant pathway. The individual dose response study showed that both PAHs and heavy metal/loid(s) activated the Nrf2 antioxidant pathway in ARE reporter-HepG2 cells. Among these chemicals, Cd was the most potent inducer, followed by B[a]P and As. Based on the individual dose response findings, PAHs and heavy metal/loid(s) were mixed at equipotent ratios using a fixed concentration ratio, and the effects of the mixtures of PAHs and heavy metal/loid(s) (binary to seven-component) on the Nrf2 antioxidant pathway were determined. The mixture effects were predicted by using the concentration addition (CA) model. Overall, the results showed that the multi-component mixtures of PAHs and heavy metal/loid(s) induced an oxidative stress response in ARE reporter-HepG2 cells, and that the CA model is an appropriate model to predict the interaction effect of these selected mixtures. A human cell line-based reporter gene assay system was successfully used to determine the mixture effects of two groups of common contaminants on oxidative stress response pathway.

Introduction

Cellular exposure to chemicals, either alone or in mixtures, causes an imbalance in reactive oxygen species (ROS) production, which may diminish the ability of cells to detoxify these ROS.1–3 Oxidative stress induced by environmental stressors is associated with epidemiological diseases such as cancers, lung diseases, neurodegenerative disorders, atherosclerosis, rheumatoid arthritis, diabetes, cardiovascular diseases, stroke and aging.4–6 Among the various classes of stressors, polyaromatic hydrocarbons (PAHs) and heavy metal/loid(s) are ubiquitous environmental pollutants of global concern. Both PAHs and heavy metal/loid(s) are known to cause a broad spectrum of toxic effects in humans.7,8

Chronic exposure to elevated levels of heavy metal/loid(s) like arsenic (As), cadmium (Cd) and lead (Pb), of which As and Cd are classified as Group I carcinogens by the IARC, can also cause adverse effects in the neurological, cardiovascular, hematological, gastrointestinal, musculoskeletal and immunological systems.9–11 Both the individual toxicity and human health risk assessment of these heavy metal/loid(s) have been extensively reviewed and reported by various international regulatory agencies, such as the WHO and US EPA. Oxidative stress is attributed as the unifying factor for metal/loid(s) toxicity.12–14 Heavy metal/loid(s) induced oxidative stress may result in lipid peroxidation and damage to cellular proteins and nucleic acids, leading to a variety of cellular dysfunctions including cell death.15,16

Similarly, some PAHs are known human carcinogens and cause developmental and immuno-toxicity. Most of the PAHs are indirect carcinogens and require metabolic activation to exert their toxicity. For example, benzo[a]pyrene (B[a]P), a potent Group I carcinogen, is metabolically activated by CYP1A1 and CYP1B1 enzymes.17 During the metabolism of B[a]P, free radicals are formed and these radicals can cause oxidative damage to DNA.18–20 Naphthalene (Nap) is classified as a 2B carcinogen and is associated with hemolytic anemia, cataracts and respiratory disorders.21 Oxidative stress plays an important role in naphthalene toxicity.22 Phenanthrene (Phe) and pyrene (Pyr) are classified as Group 3 carcinogens.23 There are no data available for Phe and Pyr toxicity to humans. These four PAHs are selected for this study because of their frequent occurrence at hazardous waste sites and potential human exposure. They are listed as priority pollutants by the US EPA.24 Naphthalene, Phe and Pyr are included in this study due to their common occurrence as mixtures with B[a]P and also to determine their potential interaction effect with B[a]P.

Humans have developed elaborate antioxidant defense mechanisms to protect cells against oxidative stress-induced damage.25 A major cellular defense mechanism against oxidative stress is the activation of antioxidant genes that are involved in the detoxification and elimination of reactive oxidants by enhancing cellular antioxidant capacity.26,27 Nrf2 (nuclear factor erythroid 2 (NFE2)-related factor 2) plays a pivotal role in protecting cells against oxidative stress through ARE-mediated expression and coordinated induction of antioxidant enzymes.28,29 Cellular exposure to electrophilic chemicals activates the Nrf2 antioxidant pathway and measurement of Nrf2 pathway induction is considered as a reliable indicator of oxidative perturbation. Heavy metals like As,30–32 Cd33–35 and Pb36,37 have been reported to activate the Nrf2 antioxidant pathway following their exposure and the Nrf2 antioxidant defense mechanism also plays a major role against B[a]P-induced carcinogenesis.38

PAHs and heavy metal/loid(s) often co-occur in the environment.39–41 Amongst these environmental pollutants, As, Cd, Pb and B[a]P are top priority pollutants.42 At elevated levels, these contaminants can cause serious health effects in humans and other organisms and oxidative stress is one of the common modes of action for these mixed contaminants. To the best of our knowledge, there are no studies reporting the effect of mixtures of PAHs and heavy metal/loid(s) on the oxidative stress response.

In this study, the ARE reporter-HepG2 cell line is used to determine the interaction effect on the Nrf2 antioxidant pathway, an indicator for the oxidative stress response. The liver is the major organ for environmental chemical metabolism and heavy metal/loid(s) are known to cause toxicity to liver cells.43 HepG2 cells have been extensively used for toxicological research and their inherent metabolic capacity is useful to determine the toxicity of chemicals like PAHs.44 These cells are highly differentiated and display many of the genotypic features of normal liver cells. In addition, the steady state maintenance of antioxidant defense is higher than that in primary hepatocytes.45,46 Hence, the HepG2 cell line is used as a model for studying the mechanisms of oxidative stress.

The objective of this study is to determine, for the first time, the effects of up to seven-component mixtures of PAHs and heavy metal/loid(s) on the Nrf2 antioxidant pathway using the ARE reporter-HepG2 cell line. The mixture effect is determined for binary, ternary, quaternary and seven-component mixtures of PAHs and heavy metal/loid(s).

Materials and methods

Chemicals

Cell culture medium MEM (minimum essential medium), trypsin-EDTA (0.25%), penicillin–streptomycin solution, Geneticin® selective antibiotic (G418 sulfate), non-essential amino acids, sodium pyruvate (100 mM) and fetal bovine serum (FBS) were purchased from Gibco® (Life Technologies, VIC, Australia). CellTiter96® Aqueous One solution cell proliferation assay system (G3581), luciferase assay system (E1501) and luciferase cell culture lysis 5× reagent (E1531) were purchased from Promega Corporation, Madison, WI, USA. Benzo[a]pyrene (B[a]P) (CAS number: 50-32-8), naphthalene (CAS number: 91-20-3), phenanthrene (CAS number: 85-01-8), pyrene (CAS number: 129-00-0), cadmium chloride (CAS number: 10108-64-2), lead acetate (CAS number: 6080-56-4), sodium arsenite (CAS number: 7784-46-5) and tert-butylhydroquinone (t-BHQ) (1948-33-0) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Cell line

The ARE reporter-HepG2 cell line (catalog # 60513) was purchased from BPS Bioscience Inc., CA, USA. This ARE reporter-HepG2 cell line contains a firefly luciferase gene under the control of ARE stably integrated into HepG2 cells. The reporter cells were maintained in a 75 cm2 culture flask containing MEM medium supplemented with 10% FBS, 1% non-essential amino acid, 1 mM sodium pyruvate, 1% penicillin/streptomycin and 600 μg per ml of Geneticin®. Cells were seeded into 96-well plates (Corning® 96-well flat clear bottom, sterile white polystyrene TC-treated microplates, Corning Life Sciences, NY, USA) at a density of 12 000 cells per well. Cells were incubated at laboratory room temperature (24 ± 1 °C) for 15 min following seeding for cell settling and incubated at 37 °C under 5% CO2 in a humidified incubator for 24 h.

Chemical treatment

Stock dilutions of PAHs (B[a]P, Nap, Phe and Pyr) in DMSO and metal/loid(s) (As, Cd and Pb) in MilliQ water (18 MΩ cm) (Merck Millipore, VIC, Australia) were prepared. Working solutions were prepared in MEM medium and added to the plates containing the cultured cells with final concentration of vehicle (DMSO or MilliQ water) at 0.5% v/v.

Cytotoxicity assay

Initially, the cytotoxicity of the PAHs and heavy metal/loid(s) to ARE reporter-HepG2 cells were determined by measurement of cell viability using the MTS assay (CellTiter 96® aqueous one solution, Promega, Madison, WI, USA). The selected concentrations were 0, 1.56, 3.12, 6.25, 12.5, 25, 50 and 100 μM of B[a]P, Nap, Phe and Pyr; 0, 0.156, 0.312, 0.625, 1.25, 2.5 5, 10 and 20 μM of Cd; 0, 3.12, 6.25, 12.5, 25, 50, 100 and 200 μM of As and 0, 2.34, 4.68, 9.37, 18.75, 37.5, 75 and 150 μM of Pb. The working solutions containing the treatment chemicals and vehicle control were exposed in triplicate to the ARE reporter-HepG2 cells for 24 h.

Cytotoxicity was determined using the CellTiter 96® aqueous one solution. After a chemical treatment period of 24 h, the treatment medium was carefully aspirated using a multi-channel micropipette, then 20 μL of MTS reagent and 80 μL of DMEM were added to each well and incubated for another 2–3 h. The absorbance was measured at 490 nm in a microplate reader (FLUOstar Omega, BMG Labtech, VIC, Australia).

Cell viability was calculated as shown in eqn (1) after blanking.

graphic file with name c6tx00024j-t1.jpg 1

Determination of Nrf2 antioxidant pathway activation

Preliminary experiments showed that the Nrf2 antioxidant pathway activation in the ARE reporter-HepG2 cells was not proportional to dose levels at higher concentrations, and a substantial decrease in the Nrf2 pathway activation was observed at concentrations near the cytotoxic level (data not shown). Based on these observations, the selected concentrations for the individual dose response study on the Nrf2 antioxidant pathway were 0–5 μM of B[a]P; 0–15 μM of Nap, Phe and Pyr; 0–5 μM of As; 0–0.5 μM of Cd; and 0–10 μM of Pb. tert-Butylhydroquinone (t-BHQ, 0–20 μM) was used as a positive control. The treatment chemicals and controls were exposed to the ARE reporter-HepG2 cells for 24 h.

The Nrf2 pathway activation was quantified by using a luciferase assay system (catalogue # E1501) purchased from Promega Corporation, Madison, USA. The luciferase activity was determined as per the manufacturer's instructions. In brief, the growth medium was removed from the plates using a multi-channel micropipette after a treatment period of 24 h and the cells rinsed twice with phosphate buffered saline (PBS). Cell lysis buffer (1× lysis buffer was prepared from 5× lysis buffer), 20 μL per well, was added to each well and incubated for 5 min at room temperature. Then, luciferase assay reagent (luciferase assay buffer + lyophilized assay substrate), 100 μL per well, was added to the lysed cells and luminescence was quantified by using a microtiter plate reader.

The oxidative stress response was measured as the difference between ARE luciferase reporter expression in the chemical-treated groups compared to that of the vehicle control and was calculated using eqn (2).

graphic file with name c6tx00024j-t2.jpg 2

The results showed that none of the chemicals achieved a maximum induction of the Nrf2 antioxidant pathway. Based on these observations, the linear part of the concentration effect relationship was selected and the concentration that induced an IR of 1.5 (ECIR1.5) was determined, as described by Escher et al. (2013).47 In brief, the dose responses of the individual chemicals and the concentration that induces an induction ratio (IR) of 1.5 (ECIR1.5) was determined using a linear regression method. The individual concentration effect relationship was determined using eqn (3).

IR = 1 + slope × concentration 3

The ECIR1.5 was calculated using the linear regression method and using eqn (4).

graphic file with name c6tx00024j-t3.jpg 4

Mixture experiments

Mixture experiments were conducted for binary, ternary, quaternary and seven-component mixtures of PAHs and heavy metal/loid(s). All the mixture experiments were conducted at fixed ratio concentrations and chemicals were mixed at 1 : 1, an equipotent ratio based on their individual ECIR1.5 values. The mixtures were diluted in 1 : 3 serial dilutions six or seven times and a full concentration response study was carried out. The chemical treatment was carried out in triplicate for each concentration and two or three independent experiments were conducted for the mixture interaction studies. The details of the chemical mixtures and concentrations are provided in Table S1 of ESI.†

Prediction of mixture effects using the concentration addition (CA) model

Concentration addition (CA) and independent action (IA) models are commonly used for the prediction of chemical mixture toxicity48 and the CA model is used for chemicals with the same mode of action. The single dose response studies showed that both PAHs and heavy metal/loid(s) induced the Nrf2 antioxidant pathway (same mode of action) in ARE reporter-HepG2 cells. Hence, the CA model was used to predict the mixture effects as described by Escher et al. (2013),47 using eqn (5).

graphic file with name c6tx00024j-t4.jpg 5

ECIR1.5,CA is the concentration of the mixture, ECIR1.5,i is the concentration of component i, and pi is the molar concentration ratio of the ith component in the mixture.

The difference and quantitative relationship between the predicted and observed effect is determined using an index on prediction quality (IPQ).49

If the predicted value is greater than the observed value, the prediction quality is determined by using eqn (6).

graphic file with name c6tx00024j-t5.jpg 6

If the predicted value is less than the observed value, the prediction quality is determined by using eqn (7).

graphic file with name c6tx00024j-t6.jpg 7

An IPQ value of zero indicates an exact prediction by the reference models, while an IPQ value <0 indicates overestimation and a value >0 indicates an underestimation of mixture effects.

Statistical analysis

Data were presented as mean ± SD for the experimental and predicted values. Statistical analysis was carried out using Graphpad Prism version 6.00 for Windows (GraphPad software, Inc., CA, USA). Data was analyzed using “Student's t-test” and the significant difference between the experimental and CA-predicted values was evaluated at p < 0.05.

Results

Cytotoxicity of PAHs and heavy metal/loid(s)

The heavy metal/loid(s) were found to be more toxic to ARE reporter-HepG2 cells than the PAHs. Among the heavy metal/loid(s), Cd was more toxic, with an IC50 value of 3.36 μM, followed by As (IC50 = 72 μM) and Pb (IC50 = 108 μM). Benzo[a]pyrene was found to be toxic to HepG2 cells and a reduction in cell viability was observed at concentrations above 12.5 μM (max. of 30% at 25 μM). At 100 μM, B[a]P was found to precipitate in the medium during the 24 h exposure period. The other PAHs were found to be nontoxic to ARE reporter-HepG2 cells up to the maximum feasible (soluble) concentrations (Table S2 of ESI†).

Activation of Nrf2 antioxidant pathway by PAHs and heavy metal/loid(s)

The individual dose response studies showed that all the chemicals, including the four PAHs and three heavy metal/loid(s), activated the Nrf2 antioxidant pathway. Among these seven chemicals, Cd was the most potent inducer, with an ECIR1.5 of 0.58 μM. Both B[a]P and As have similar potency, with ECIR1.5 values of 0.93 and 1.1 μM, respectively. The ECIR1.5 values for the individual chemicals and the dose response curves are presented in the ESI (Table S2, Fig. S1 and S2† respectively).

Effect of mixtures of PAHs and heavy metal/loid(s) on the Nrf2 antioxidant pathway

The effects of binary to seven-component mixtures of PAHs and heavy metal/loid(s) on the activation of the Nrf2 antioxidant pathway in ARE reporter-HepG2 cells are given in Table 1. The experimental and predicted ECIR1.5 values, IPQ values and 95% confidence intervals (CI) for the experimental and CA-predicted values are presented in Table 1 and Fig. 1–6, which show the dose response effects of the multi-component mixtures of PAHs and heavy metal/loid(s) on the Nrf2 antioxidant pathway in ARE reporter-HepG2 cells.

Table 1. The multi-component mixtures of PAHs (benzo[a]pyrene (B[a]P), naphthalene (Nap), phenanthrene (Phe), and pyrene (Pyr)) and heavy metal/loid(s) (arsenic (As), cadmium (Cd), lead (Pb)) on activation of Nrf2 antioxidant pathway in ARE reporter-HepG2 cells.

Chemical mixtures ECIR1.5 exp (μM) 95% CI ECIR1.5 CA (μM) 95% CI IPQ Observed maximum induction ratio
Binary mixtures
As + Cd 0.70 ± 0.24 0.09–1.3 0.90 ± 0.21 0.39–1.4 0.29 2.4
As + Pb* 1.63 ± 0.31 0.87–2.4 2.33 ± 0.21 1.8–2.9 0.43 2.4
Cd + Pb 2.2 ± 0.44 1.17–3.2 2.0 ± 0.21 1.52–2.6 –0.09 1.7
B[a]P + As 0.67 ± 0.25 0.05–1.3 1.04 ± 0.31 0.27–1.8 0.78 3.3
B[a]P + Cd 0.45 ± 0.16 0.04–0.9 0.80 ± 0.34 0.04–1.7 0.81 3.0
B[a]P + Pb 1.11 ± 0.34 0.26–2.0 2.13 ± 0.25 1.5–2.8 0.92 3.3
B[a]P + Nap 1.86 ± 0.45 0.70–3.0 1.76 ± 0.45 0.64–2.9 –0.06 2.5
B[a]P + Phe 2.1 ± 0.74 0.25–3.9 1.73 ± 0.37 0.81–2.7 –0.21 2.4
B[a]P + Pyr 1.56 ± 0.80 0.17–3.5 1.88 ± 0.54 0.54–3.2 0.21 3.3
Ternary mixtures
As + Cd + Pb 1.87 ± 0.55 0.50–3.2 1.70 ± 0.36 0.80–2.6 –0.09 2.3
B[a]P + As + Cd* 0.45 ± 0.16 0.05–0.9 0.79 ± 0.19 0.29–1.3 0.74 5.0
B[a]P + As + Pb* 1.22 ± 0.33 0.41–2.0 1.77 ± 0.32 0.97–2.6 0.45 3.4
B[a]P + Cd + Pb 1.00 ± 0.09 0.77–1.2 1.60 ± 0.27 0.93–2.3 0.60 3.3
B[a]P + Nap + Phe 1.5 ± 0.16 0.02–3.0 2.1 ± 0.83 0.6–3.5 0.39 4.4
B[a]P + Nap + Pyr 1.37 ± 0.23 0.13–3.4 1.94 ± 0.14 0.66–3.2 0.42 4.7
B[a]P + Phe + Pyr 1.34 ± 0.22 0.02–3.3 1.87 ± 0.05 1.4–2.33 0.40 4.8
Quaternary mixtures
B[a]P + As + Cd + Pb* 0.77 ± 0.12 0.46–1.1 1.47 ± 0.25 0.84–2.1 0.91 5.1
B[a]P + Nap + Phe + Pyr 1.96 ± 0.07 1.31–2.6 2.06 ± 0.29 0.14–4.6 0.05 4.6
All seven chemicals 2.06 ± 0.40 0.18–3.7 1.87 ± 0.06 1.3–2.4 –0.09 6.0
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Fig. 1. Dose response of binary mixtures of heavy metal/loid(s) for activation of Nrf2 antioxidant pathway in ARE reporter-HepG2 cells after 24 h exposure. IR – induction ratio; CA denotes dose response predicted by concentration addition model; EXP denotes experimental data. The experimental data were from three independent experiments in triplicate for each exposure concentration. Values are expressed as mean and dashed line indicates 95% confidence interval. As – arsenic, Cd – cadmium and Pb – lead.

Fig. 1

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Fig. 2. Dose response of binary mixtures of B[a]P and As, Cd or Pb for activation of Nrf2 antioxidant pathway in ARE reporter-HepG2 cells after 24 h exposure. IR – induction ratio; CA denotes dose response predicted by concentration addition model; EXP denotes experimental data. The experimental data were from three independent experiments in triplicate for each exposure concentration. Values are expressed as mean and dashed line indicates 95% confidence interval. As – arsenic, B[a]P – benzo[a]pyrene, Cd – cadmium and Pb – lead.

Fig. 2

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Fig. 3. Dose response of binary mixtures of B[a]P and Nap, Phe and Pyr for activation of Nrf2 antioxidant pathway in ARE reporter-HepG2 cells after 24 h exposure. IR – induction ratio; CA denotes dose response predicted by concentration addition model; EXP denotes experimental data. The experimental data were from three independent experiments in triplicate for each exposure concentration. Values are expressed as mean and dashed line indicates 95% confidence interval. B[a]P – benzo[a]pyrene, Nap – naphthalene, Phe – phenanthrene and Pyr – pyrene.

Fig. 3

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Fig. 4. Dose response of ternary mixtures of B[a]P, As, Cd and/or Pb for activation of Nrf2 antioxidant pathway in ARE reporter-HepG2 cells after 24 h exposure. IR – induction ratio; CA denotes dose response predicted by concentration addition model; EXP denotes experimental data. The experimental data were from three independent experiments in triplicate for each exposure concentration. Values are expressed as mean and dashed line indicates 95% confidence interval. As – arsenic, B[a]P – benzo[a]pyrene, Cd – cadmium and Pb – lead.

Fig. 4

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Fig. 5. Dose response of ternary mixtures of B[a]P, Nap, Phe and Pyr for activation of Nrf2 antioxidant pathway in ARE reporter-HepG2 cells after 24 h exposure. IR – induction ratio; CA denotes dose response predicted by concentration addition model; EXP denotes experimental data. The experimental data were from two independent experiments in triplicate for each exposure concentration. Values are expressed as mean and dashed line indicates 95% confidence interval. B[a]P – benzo[a]pyrene, Nap – naphthalene, Phe – phenanthrene and Pyr – pyrene.

Fig. 5

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Fig. 6. Dose response of quaternary and seven-component mixtures of PAHs and heavy metal/loid(s) for activation of Nrf2 antioxidant pathway in ARE reporter-HepG2 cells after 24 h exposure. IR – induction ratio; CA denotes dose response predicted by concentration addition model; EXP denotes experimental data. The experimental data were from two independent experiments in triplicate for B[a]P + Nap + Phe + Pyr and the seven-component mixture, and three independent experiments for B[a]P + As + Cd + Pb. Values are expressed as mean and dashed line indicates 95% confidence interval. As – arsenic, B[a]P – benzo[a]pyrene, Cd – cadmium, Nap – naphthalene, Phe – phenanthrene, Pb – lead and Pyr – pyrene.

Fig. 6

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Binary mixtures of PAHs and heavy metal/loid(s)

The binary mixtures of heavy metal/loid(s) (As + Cd, As + Pb and Cd + Pb), B[a]P + heavy metal/loid(s) (B[a]P + As, B[a]P + Cd and B[a]P + Pb) and B[a]P + PAHs (B[a]P + Nap, B[a]P + Phe and B[a]P + Pyr) showed varying potencies of activating the Nrf2 antioxidant pathway in ARE reporter-HepG2 cells (Table 1 and Fig. 1–3). The mixture of As + Cd showed a higher induction effect on the Nrf2 pathway compared to other heavy metal/loid(s) mixtures, with ECIR1.5 0.70 μM, followed by As + Pb (ECIR1.5 – 1.63 μM) and Cd + Pb (ECIR1.5 – 2.2 μM). A maximum induction ratio of 2.4 was observed with mixtures of As + Cd and As + Pb. In the case of B[a]P and heavy metal/loid(s) mixtures, B[a]P + Cd showed a higher induction effect (ECIR1.5 of 0.45 μM) compared to that of other mixtures and the maximum induction ratios observed for all three mixtures are almost equal (max. of 3.3). Among the binary mixtures of B[a]P + other PAHs, the mixture of B[a]P + Pyr had a higher induction effect (ECIR1.5 1.56 μM), and also showed a maximum induction ratio of 3.3, compared to those of the other B[a]P + PAHs mixtures. There was no significant difference between the experimental and predicted values for all the mixtures, except for the As + Pb mixture, and the IPQ value is less than one for all mixtures. There were overlaps of the 95% confidence intervals between the predicted and observed value for all the mixtures (Table 1).

Ternary, quaternary and seven-component mixtures of PAHs and heavy metal/loid(s)

The ternary mixture of B[a]P + Cd + As has a higher induction effect (ECIR1.5 0.45 μM), with a maximum induction ratio of 5, compared to the other ternary mixtures. In general, the induction effect is higher for the ternary mixtures of B[a]P + heavy metal/loid(s) compared to those of ternary mixtures of As + Cd + Pb and B[a]P + other PAHs. The ternary mixture of As + Cd +Pb showed a lower induction effect, with an ECIR1.5 of 1.87 μM. There was no significant difference between the observed and predicted effect for the ternary mixtures, except for B[a]P + As + Cd (p = 0.0088) and B[a]P + As + Pb (p = 0.003). The experimental values for these mixtures were less than the predicted values and therefore the mixture effect is underestimated.

The quaternary and seven-component mixtures also induced the Nrf2 antioxidant pathway with a maximum induction ratio of 6 observed with the seven-component mixture. There was no significant difference between the observed and predicted effect for these mixtures, except for B[a]P + As + Cd + Pb (p = 0.0131). The IPQ value is less than one for all mixtures (ternary to seven-component mixtures), with overlapping of the 95% confidence intervals observed between the predicted and observed values for all mixtures (Table 1).

Discussion

Oxidative stress has been implicated in the pathophysiology of various systemic diseases and mechanisms of action of chemical toxicity. Most importantly, oxidative stress plays a crucial role in carcinogenesis1,50 and environmental agents are one of the main exogenous sources for ROS production. Measurement of the oxidative stress response is a sensitive endpoint for chemical exposure and the various methods used for its determination include direct measurement of the ROS, oxidative damage to biomolecules and detection of antioxidant levels.51,52 Most of these methods are technically laborious and lack specificity and the National Research Council (NRC, 2007 53) recommends the development of rapid and economical cell-based and high throughput assays to determine the perturbation of cellular response using in vitro assays for better understanding of human diseases. Measurement of stress response pathways (Nrf2 antioxidant pathway) has been identified as a toxic pathway indicator53 and human cell-based reporter gene assays have been developed to measure the Nrf2 antioxidant pathway as a means of monitoring the oxidative stress response. These assays measure the ARE activation using the luciferase reporter gene, which is preferred as a screening tool due its rapidness and stable transfection, which helps to define modes of action.36,54 This bioassay has been used for screening pharmaceutical molecules for Nrf2 activation55 and profiling of environmental chemicals.56 The Nrf2 luciferase assay is designed to measure changes in the transcriptional activity of Nrf2, where the Nrf2 binds to ARE and regulates genes involved in cytoprotection. The Nrf2-responsive luciferase construct monitors the increase or decrease in the transcriptional activity of Nrf2 and activity of the ARE pathway. Therefore, the changes of luciferase expression in the chemical-treated cells provide a sensitive measure of changes in the Nrf2 activity. Methods such as real time PCR can provide information about gene expression and steady-state level of transcription, which is influenced by transcriptional activity and mRNA instability.36 Thus, cell-based assays are preferred to monitor the Nrf2 pathway activity and hence chemically induced changes in oxidative stress response.

The Nrf2-reporter gene assays have been developed using various immortalized cell lines, including HEK293T, MCF7, A172, A549, HepG236 and Huh cell lines.57 The results from these studies have shown that Nrf2 activity profiles vary between the cell lines, due to the origin of tissues, cellular subtype and culture conditions. Thus, the results obtained by using a particular cell line (HepG2 cells) can be correlated with the response of the liver in the body, although the results do not necessarily provide a complete picture of biological response and other factors like changes in temperature, pH, and luciferase buffer may also affect the luminescence signal.

In this study, we have determined the effect of up to seven-component mixtures on the Nrf2 antioxidant pathway using ARE reporter-HepG2 cells. The results showed that both heavy metal/loid(s) and PAHs activated the Nrf2 antioxidant pathway. The role of the Nrf2 antioxidant pathway in heavy metal/loid(s) and B[a]P toxicity has been reported.58,59 This present study shows that non-carcinogenic PAHs like Nap, Phe and Pyr can also induce a positive response in activation of the Nrf2 antioxidant pathway. There are no studies available in the literature reporting the effects of these PAHs on the Nrf2 antioxidant pathway.

Our results also show that multi-component mixtures of PAHs and heavy metal/loid(s) displayed various degrees of activity on the Nrf2 antioxidant pathway in ARE reporter-HepG2 cells. In the case of binary mixtures, the B[a]P + Cd mixture had a higher induction effect compared to those of the other combinations. Among the binary mixtures, B[a]P with heavy metal/loid(s) showed higher induction of the Nrf2 pathway compared to mixtures of metal/loid(s) and B[a]P + PAHs and a similar trend was observed with ternary mixtures, where B[a]P + Cd + As showed a higher induction effect than those of the other ternary mixtures. The observed ECIR1.5 value for the seven-component mixture was higher than those of lower order mixtures (binary and ternary). This could be due to the mixtures of both potent inducers (Cd, B[a]P, As and Pb) and less active chemicals (Nap, Phe and Pyr). Various reports indicated that combined exposure of As, Cd and/or Pb, or in combination with other metals, increased the oxidative stress response compared to their individual responses.60–62 Cd was found to enhance the Nrf2 antioxidant pathway and total glutathione level of B[a]P when compared to those of B[a]P alone.63 Arsenic and Pb also have a synergistic effect on the oxidative stress response in combination with B[a]P.64,65 These studies did not use any prediction model to determine the interaction and interpretation was based on statistical difference between individual and mixture groups. There are no detailed reports available for these mixtures at higher order (ternary mixture and above). For the first time, we report here oxidative stress response data for ternary, quaternary and seven-component mixtures containing four PAHs and three heavy metal/loid(s).

CA and IA models are commonly used to predict mixture toxicity, and these models are used for chemicals with similar and dissimilar modes of action respectively. We have used only the CA model in this study to predict the mixture effect on the Nrf2 antioxidant pathway as the individual PAHs and metal/loids showed the same mode of action (Nrf2 pathway activation). The CA model is the preferred reference model for risk assessment of mixtures consisting of both similar and dissimilar acting chemicals66 and this model is considered as a general solution for mixture risk assessment.67 This prediction model (CA) has been used to predict the mixture effect of different classes of chemicals, including pharmaceuticals and pesticides, on the oxidative stress response using the AREc32 cell line.47 The mixtures of pharmaceuticals and pesticides showed induction activity in the AREc assay and the mixture effect is well predicted by the CA model.

The present study shows there was no significant difference between the observed and predicted ECIR1.5 values for 15 out of the 19 mixtures and an overlap of the 95% confidence interval between experimental and predicted values was observed for all mixtures. The observed ECIR1.5 for the mixture of As + Pb, ternary mixtures of B[a]P + Cd + As and B[a]P + As + Pb, and the quaternary mixture of B[a]P and heavy metal/loid(s) showed significant differences with the CA prediction and the ECIR1.5 values for these mixtures were less than the predicted values, suggesting that the CA model under-predicted the interaction effect for these mixtures. A closer examination of the predicted response showed that the CA model tends to underestimate the interaction effect for most of the mixtures at lower concentrations. The predicted response of the binary mixtures of B[a]P + heavy metal/loid(s), As + Cd, Cd + Pb and B[a]P + other PAHs was underestimated for lower order combinations, and the same trend of underestimation at lower concentrations was observed for ternary, quaternary and seven-component mixtures. For a few mixtures, like binary mixtures of B[a]P + heavy metal/loid(s), ternary mixtures of B[a]P + Cd+ As, B[a]P + Nap + Pyr, B[a]P + Phe + Pyr and B[a]P + As + Cd + Pb, the effect was underestimated at greater concentrations. An IPQ compares the difference between observed effects and those predicted by models (CA and IA) and indicates the accuracy of predictions of the models.49 An IPQ value of <0 or >0 indicates an over or underestimation, respectively, of mixture effects and values of –1 and +1 indicate over or under prediction by the prediction models. In our study, the IPQ value is close to zero for 5 out of the 19 mixtures, less than 0.5 for 8 out of the 19 mixtures and less than 1 for the remaining six mixtures, which indicates acceptable agreement between the predicted and observed effects. In the case of the mixtures which showed significant differences between the predicted responses by the CA model, the IPQ values are less than 1 for all four mixtures and overlaps of the 95% CI intervals between predicted ECIR1.5 values were observed. This indicates acceptable agreement between the observed and predicted effects. Based on these findings, we can conclude that the CA model can be used to predict the interaction effect between PAHs and heavy metal/loid(s) on the Nrf2 antioxidant pathway. In general and with the exceptions stated above, concentration addition may be appropriate for the risk assessment of B[a]P, Nap, Phe, Pyr, As, Cd and Pb mixtures.

Conclusions

A human cell line-based reporter gene assay system (ARE reporter-HepG2 cells) has been successfully used to determine the effect of chemical mixtures on the oxidative stress response. This is the first report on the effects of individual and up to seven-component mixtures of PAHs (B[a]P, Nap, Phe and Pyr) and heavy metal/loid(s) (As, Cd and Pb) on the Nrf2 antioxidant pathway in ARE reporter-HepG2 cells. Individual and multi-component mixtures of PAHs and heavy metal/loid(s) activated the Nrf2 antioxidant pathway in ARE reporter-HepG2 cells. The CA model appears to be an appropriate model to predict the effect of these selected mixtures on the oxidative stress response pathway. The effect of PAH and heavy metal/loid(s) mixtures on the oxidative response pathway can be utilized as an adjunct tool to inform health risk assessment. However, its adoption can be strengthened by the incorporation of a suite of other biological endpoints (AhR activation, cytotoxicity and genotoxicity), which forms part of our ongoing research.

Abbreviations

ARE

Antioxidant response element

As

Arsenic

ATSDR

Agency for Toxicological Substances and Disease Registry

B[a]P

Benzo[a]pyrene

CA

Concentration addition

Cd

Cadmium

DMSO

Dimethyl sulfoxide

ECIR1.5

Concentration that induces an induction ratio of 1.5

EDTA

Ethylene diamine tetra acetic acid

EPA

Environmental Protection Agency

EXP

Experimental values

h

Hour or hours

HEK

Human embryonic kidney cells

HepG2

Human hepatocellular carcinoma cell

IA

Independent action

IARC

International Agency for Research on Cancer

IPQ

Index on prediction quality

IR

Induction ratio

Nap

Naphthalene

Nrf2

Nuclear factor erythroid 2 (NFE2)-related factor 2

MCF-7

Michigan cancer foundation-7

min

Minute or minutes

MTS

Tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]

PAHs

Polyaromatic hydrocarbons

Pb

Lead

PBS

Phosphate buffered saline

Phe

Phenanthrene

Pyr

Pyrene

ROS

Reactive oxygen species

μM

Micromolar

mM

Millimolar

WHO

World health organization

Conflict of interest

There is no conflict of interest.

Supplementary Material

Click here for additional data file. (568.5KB, pdf)

Acknowledgments

This research is funded by the Cooperative Research Centre for Contamination Assessment and Remediation of the Environment (CRC-CARE) (Project No. 3.1.01.11-12). UQ UQI and CRC-CARE PhD top-up scholarship to SM are acknowledged. Entox is a partnership between Queensland Health and the University of Queensland.

Footnotes

†Electronic supplementary information (ESI) available. See DOI: 10.1039/c6tx00024j

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