Mapping the Mitochondrial Regulation of Epigenetic Modifications in Association With Carcinogenic and Noncarcinogenic Polycyclic Aromatic Hydrocarbon Exposure

Abstract

Polycyclic aromatic hydrocarbons (PAHs) refer to a ubiquitous group of anthropogenic air pollutants that are generated through incomplete carbon combustion. Although the immunotoxic nature of PAHs has been previously reported, the underlying molecular mechanisms of this effect are not fully understood. In the present study, we investigated the mitochondrial-mediated epigenetic regulation of 2 PAHs, carcinogenic (benzo[a]pyrene; BaP) and noncarcinogenic (anthracene [ANT]), in peripheral lymphocytes. While ANT exposure triggered mitochondrial oxidative damage, no appreciable epigenetic modifications were observed. On the other hand, exposure to BaP perturbed the mitochondrial redox machinery and initiated cascade of epigenetic modifications. Cells exposed to BaP showed prominent changes in the expression of mitochondrial microRNAs (miR-24, miR-34a, miR-150, and miR-155) and their respective gene targets (NF-κβ, MYC, and p53). The exposure of BaP also caused significant alterations in the expression of epigenetic modifiers (DNMT1, HDAC1, HDAC7, KDM3a, EZH2, and P300) and hypomethylation within nuclear and mitochondrial DNA. This further induced methylation of histone tails, which play a crucial role in the regulation of chromatin structure. Overall, our study provides novel mechanistic insights into the mitochondrial regulation of epigenetic modifications in association with PAH-induced immunotoxicity.

Keywords

air pollution mitochondrial redox signaling DNA methylation microRNAs histone modifications environmental health

Introduction

Air pollution has been reported as a primary contributor to the increasing global burden of diseases and a leading cause of death. According to the state of global air report (2019), air pollution resulted in 147 million years of healthy life lost and almost 4.9 million deaths in the year 2017.¹ Studies have also documented that such exposures may cause alterations within the genome and are associated with the increased risk of noncommunicable diseases including cancer.^2
-4 Polycyclic aromatic hydrocarbons (PAHs), the major component of vehicle emissions, industrial activities, and combustion of coal, are characterized as ubiquitous, toxic, and typical anthropogenic air pollutants which consist of 2 or more aromatic rings. These pollutants often exist as mixtures of compounds rather than occurring individually in the ambient air. Regardless of the chemical similarities among the class, the toxicological outcome of individual PAHs varies considerably. Benzo[a]pyrene (BaP) is classified as a group 1 carcinogen by the International Agency for Research on Cancer, whereas anthracene (ANT) in its native form is noncarcinogenic.⁵ Importantly, these compounds are known to affect vital immune functions through alterations in the mitogenic signaling pathways. Activation of mitogen-activated protein kinases cascade may not only lead to death of lymphoid cells but also suppresses essential immune responses.⁶ Although the specific molecular mechanisms underlying such immune modulation are not known, studies suggest that the cytochrome-P450-derived metabolites of PAH activate different oxidative and electrophilic signaling pathways in lymphoid and nonlymphoid cells. Mitochondria being the primary site of energy production are a major target of environmental pollutants.^2,7,8 The mitochondrial reactive oxygen species (ROS) and electrophilic metabolites are 2 critical regulators of mitochondrial-nuclear cross-talk, which directly influence cellular functionality and regulate epigenetic mechanisms that predominately involve DNA methylation, microRNAs (miRNAs) expressions, and post-translational histone modifications.^9
-11 These mechanisms act as sensitive readouts of the impacts induced by acute and chronic exposure to such chemicals. Increasing scientific investigations have also shown that epigenetic modifications play a vital role in the initiation and progression of different adverse health outcomes of environmental exposures.^12,13 Interestingly, cell-free epigenetic signatures including methylated nuclear DNA (nDNA) and mitochondrial DNA (mtDNA), post-translational modified histones and miRNA released into the circulation are considered as fingerprints of such ongoing cellular changes. However, concentration and molecular characteristics of these circulating biomolecules significantly differ with reference to physicochemical nature of the pollutant, quantum, and period of exposure. Therefore, deeper mechanistic understanding of epigenetic modifications may prove vital to establish minimal-invasive biomarkers for human health risk assessment of PAHs, a major contributor of air pollution.^14,15 We investigated the mitochondrial-mediated epigenetic regulation of 2 PAHs, carcinogenic (BaP) and noncarcinogenic (ANT), in peripheral lymphocytes. Mature lymphocytes that circulate in the blood were selected for this study as they can be obtained in a noninvasive fashion and have shown to reflect some of the epigenetic modifications induced by PAHs.^16,17

Materials and Methods

Reagents

Cells were treated with ANT (99%, CAS: 120-12-7) and Bap (97%, CAS: 50-32-8) procured from Alfa Aesar. For the preparation of the stock solution, chemicals were dissolved in the dimethyl sulfoxide (DMSO) from Sigma-Aldrich. The lymphocytes were isolated using HiSep solution and cultured with HiKaryoXL RPMI media from HiMedia Laboratories. For assessment of oxidative stress, CellROX Deep Red Flow Cytometry assay kit from Thermo Fischer Scientific and nuclear factor erythroid 2 related factor 2 (Nrf2) sandwich enzyme-linked immunosorbent assay (ELISA) kit from BIOCODON Technologies were used. Mitochondrial membrane potential was examined by using MitoProbe DilC₁ (5) assay kit from Thermo Fischer Scientific. Multicolor flow cytometric assay kits from BD Biosciences were used for evaluating apoptosis, DNA damage, cell proliferation, and activation of Caspase 3. The profiling of mitochondrial miRNAs (mitomiRs) was performed using miRNeasy Mini kit from QIAGEN and poly-A tailing kit obtained from Thermo Fischer Scientific. For relative gene expression analysis by quantitative real-time polymerase chain reaction (PCR), complementary DNA (cDNA), and GoTaq Q PCR Master Mix synthesis kits from Promega; KAPA SYBR FAST One-Step qRT-PCR kit from Merck KGaA; and Luna Universal Q Master Mix from New England Biolabs were used. The levels of DNA methylation were evaluated by using MethylFlash Global DNA Methylation ELISA Easy kit from Epigentek Group Inc and EpiJET Bisulfite Conversion kit from Thermo Fischer Scientific. The bovine serum albumin (BSA) used for preparation of the blocking buffer was purchased from HiMedia Laboratories. For assessment of histone modifications, Histone H3/H4 Modification Multiplex assay kits from Epigentek Group Inc and antibodies from Cell Signaling Technology were procured.

Study Design

The study was performed by using 2 PAHs, that is, BaP and ANT. For experiments, both chemicals were weighed and dissolved in the appropriate amount of DMSO to prepare 100 μM concentration stock solutions of both BaP and ANT. Further, this stock solution was diluted to identify an optimum dose for treatment so that the final concentration of DMSO in media is ≤0.1%. Based on earlier reports and our dose-dependent response analysis, we selected 10 µM as an optimum dose to carry out downstream experiments.¹⁸ Isolation of lymphocytes from whole blood was done by using HiSep, and the viability of the isolated cells was measured by using trypan blue exclusion dye. Then, 1 × 10⁶ viable cells were cultured with HiKaryoXL RPMI media in 5% CO₂ atmosphere at 37 °C for 24 hours.¹⁹ The cells were exposed to PAHs for 0 hours, 30 minutes, 1, 3, 6, 12, 24, 48, and 72 hours to assess different parameters, while DMSO (0.1%) treated cells were used as controls.

Measurement of Oxidative Stress

The generation of ROS was evaluated by using CellROX Deep Red Flow Cytometry assay kit. The CellROX Deep Red is a cell-permeable reagent that remains nonfluorescent in the reduced state, but upon oxidation shows a strong fluorogenic signal indicating the levels of ROS in cells. The assay was performed by following all the necessary instructions from the supplier. In brief, cells were harvested and incubated with the CellROX Deep Red reagent at a final concentration of 500 nM for 1 hour at 37 °C in dark. The cells were then analyzed through flow cytometry.⁸ For the examination of Nrf2 levels, a sandwich ELISA kit was used and the absorbance were recorded at 450 nm.²⁰ Briefly, the samples were premixed with the streptavidin-horseradish peroxidase (HRP)-labeled anti-Nrf2 antibody (provided in the kit) and added to the wells. After 1 hour incubation in dark, the wells were carefully washed to remove the unbounded samples and antibodies. Next, the substrate was added and the absorbance was measured at 450 nm using Spark multimode microplate reader (TECAN).

Evaluation of MtDNA and Membrane Integrity

The assessment of mitochondrial integrity was performed by evaluating the mtDNA copy number and membrane depolarization. Relative quantification of mtDNA copy number was performed through quantitative real-time PCR (Insta Q96) using 2 primer sets for amplification of mitochondrial NADH dehydrogenase ortholog 1 and nuclear gene human β-actin. The values were measured as the mtDNA to nDNA ratio for a given sample.²¹ Mitochondrial membrane potential was examined through MitoProbe DilC₁ (5) assay kit using flow cytometry (Attune NxT).² 1, 1′, 3, 3, 3′, 3′-Hexamethylindodicarbocyanine iodide, DilC₁ (5), is a cyanin dye which primarily accumulates within intact mitochondria. The intensity of the dye reduces following disruption of the membrane potential. For labeling, cells were suspended in prewarm phosphate buffered saline (PBS) and stained with 5 µL of DilC₁ (final concentration 50 nM) for 30 minutes. The cells were then washed, resuspended in 500 µL PBS, and analyzed through flow cytometry (Attune NxT, Thermo Fischer Scientific).

DNA Damage and Apoptosis

DNA damage response was examined by monitoring the levels of γH2AX using a multicolor flow cytometric tri test kit from BD Biosciences. In brief, cells were harvested and fixed for 30 minutes with 100 µL BD Cytofix/Cytoperm solution supplied within the kit. After which the cells were washed and incubated with the 100 µL BD Cytofix/Cytoperm Plus permeabilization buffer (provided in the kit) for 10 minutes on ice. Subsequently, the cells were washed and incubated with 5 µL of γH2AX specific antibody (provided in the kit) for 30 minutes. The mean fluorescence intensity of the cells was then analyzed through flow cytometry (Attune NxT).²² For assessment of mitochondria-mediated apoptosis, caspase-3 activity was measured in cells using the caspase-3 apoptosis kit. Briefly, the cells were washed and fixed for 20 minutes on ice. After fixation, the cells were washed with PBS and stained with phycoerythrin conjugated caspase-3 antibodies (supplied in the kit) for 30 minutes. The analysis of caspase-3 positive cells was performed by flow cytometry (Attune NxT).²¹

Profiling of mitomiRs Expression

To isolate mitomiRs, miRNeasy Mini Kit was used. Briefly, the cells were lysed and mixed with chloroform to perform phase separation. After which 1.5 volume of ethanol (absolute) was added to the separated aqueous phase and the blend was allowed to bind onto the RNeasy mini-column supplied within the kit. The columns were then subsequently washed and the elution of the bound miRNAs was done in fresh tubes by adding elution buffer. For downstream processing of the isolated mitomiRs, poly-A tailing was performed by using a reaction mixture comprising 5× EPAP buffer, MnCl₂, ATP solution, nuclease-free water, EPAP enzyme (provided in the kit), and mitomiRs. The reaction mixture was then incubated for 1 hour at 37 °C followed by the cDNA synthesis of the poly-A tailed miRNA through reverse transcription. The operating program was set at temperature cycle of 37 °C for 60 minutes, 42 °C for 60 minutes, 70 °C for 15 minutes, and 4 °C for 10 minutes. The prepared cDNA was then amplified using GoTaq Q PCR Master Mix and specific primers and the observed Ct values were analyzed for the determination of fold change.^23,24

Expression of MitomiR Target Genes

The assessment of target genes p53, MYC, and nuclear factor κβ (NF-κβ) was done in RNA isolated using the miRNeasy Mini kit as discussed elsewhere. Initially, the isolated RNA was subjected to cDNA synthesis using 1 µL random primers, 1 µL DNT mixture, RNA, and nuclease-free water to make up the volume up to 10 µL. The mixture was then incubated at 65 °C for 5 minutes. Later, 4 µL of 5X primscript buffer, 0.5 µL RNA inhibitor, 1 µL reverse transcriptase, and 4.5 µL nuclease-free water provided in the kit were added and subjected to a cycle of 37 °C for 60 minutes, 42 °C for 60 minutes, and 70 °C for 15 minutes in a Thermal cycler (GeneAmp PCR System 9700, Applied Biosystems) to synthesize cDNA. The 4 µL of synthesized cDNA was then mixed with 10 µL of Luna universal Q master mix, forward and reverse primers (1 µL each) and 4 µL of nuclease-free water. This was then subjected to 95 °C for 45 seconds followed by 35 cycles of 95 °C for 30 seconds, 53 °C for 30 seconds, and 68 °C for 15 seconds, and 1 cycle of 68 °C for 5 minutes.²⁵

Assessment of Epigenetic Modifiers

To examine the expression of different epigenetic modifiers, that is, histone demethylase (KDM3a), histone deactylases 1 (HDAC1), histone deactylases 7 (HDAC7), histone acetyltransferase (P300), and histone methyltransferase (EZH2), RNA was isolated using miRNeasy Mini kit as described elsewhere. Complementary DNA synthesis was performed as per the protocol discussed earlier. The steps of PCR cycle included 95 °C for 45 seconds; 35 cycles of 95 °C for 30 seconds; 53 °C for 30 seconds; 68 °C for 15 seconds; and 68 °C for 5 minutes. The quantification of DNA methyltransferase (DNMT1) was done using KAPA SYBR FAST One-Step qRT-PCR kit by real-time PCR (Insta Q96, HiMedia Laboratories).¹¹

DNA Methylation Levels

The quantification of 5-methylcytosine (5-mC) was performed using MethylFlash Global DNA Methylation (5-mC) ELISA Easy kit. In brief, 5-mC coating buffer comprising 100 ng DNA sample was used for the coating of wells. After the appropriate coating and blocking, specific antibodies were added to the wells followed by the addition of HRP (provided in the kit). The readings were then recorded at 450 nm by using Spark multimode microplate reader (TECAN).²¹ To examine global DNA methylation levels, LINE-1 methylation-specific PCR was performed as described previously. Briefly, 500 ng DNA sample was subjected to bisulfite conversion of cytosine to uracil using the EpiJET Bisulfite Conversion kit.² After thoroughly mixing the samples with the modification reagent, amplification was carried out at 98 °C for 10 minutes, followed by incubation at 60 °C for 150 minutes. Later, the amplified product was washed, desulphonated, and eluted in a step-wise manner. Subsequent amplification procedure was done using specific primer sets for methylated and unmethylated LINE-1 promoter region. The methylation levels of D1, D2, 12S, CYTB, and 16S region of mtDNA were also evaluated using a similar protocol.²

Assessment of Histone Modifications

The histone H3/H4 modification multiplex assay kits were used for quantification of the levels of histone modifications. Simultaneous measurement of 21 different histone H3 modifications and 10 histone H4 modifications were carried out through ELISA. The assay was done in triplicates for each sample, and absorbance was recorded at 450 and 630 nm on a Spark multimode microplate reader (TECAN)² for histone H3 and H4, respectively. Western blot analysis of H3 modifications was performed as per standard protocol as discussed elsewhere.²⁶ Initially, proteins were separated through sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred on a 0.2 µm pore-size nitrocellulose membrane (Bio-Rad). The membrane was then incubated with blocking buffer comprising 5% BSA in 1× PBS for 1 hour, washed, and incubated overnight with primary antibody at 4 °C. The HRP-labeled secondary antibody treatment was given for 2 hours. Subsequently, 3,3′,5,5′-tetramethylbenzidine substrate was used for blot development.

Results

Mitochondrial Stress and Membrane Integrity

Our results suggested that PAH exposure to cells induces significant generation of ROS. In comparison to the controls, the observed increase in mean fluorescence intensity (suggesting ROS generation) of the exposed cells was almost 3-fold higher. The maximum levels of ROS were observed in the cells exposed to BaP at 6 hours, while in ANT exposed cells, the generated ROS levels were highest at 3 hours (43.12% ± 4.98%), suggesting the early onset of ROS in ANT exposed cells. The values after 6 hours of BaP and ANT exposure were 41.25% ± 3.91% and 32.12% ± 4.15%, whereas the control values were 10.29% ± 1.86% (Figure 1A and Supplemental Figure 1A). Further, our results also indicated that PAH exposure causes significant disturbances in the mitochondrial integrity. The results of DilC₁ MitoProbe labeling showed that exposure to PAH causes mitochondrial membrane depolarization with maximum levels at 6 hours of the exposure. As compared with controls (4.13% ± 1.02%), the percentage of mean fluorescence intensity observed among ANT and BaP at 6 hours was 39.89% ± 3.91% and 23.32% ± 2.69%, respectively (Figure 1B and Supplemental Figure 1B). However, results of mtDNA copy number analysis did not show any significant alterations in cells exposed to PAHs. In comparison to controls (0.60 ± 0.023), the levels of mtDNA copy number were 0.54 ± 0.038 in ANT and 0.62 ± 0.046 in BaP following 6 hour treatment (Supplemental Figure 2).

Figure 1.

Mitochondrial oxidative stress and membrane depolarization. A, Graph showing the generation of reactive oxygen species in controls and cells exposed to ANT and BaP for 30 minutes, 1, 3, and 6 hours. Data are expressed as mean ± SE (n = 3), and P ≤ 0.05 was considered significant. B, Graph showing the loss of mitochondrial membrane potential upon exposure to ANT and BaP for 30 minutes, 1, 3, and 6 hours. Data are represented as mean ± SE values, and P ≤ 0.05 was considered statistically significant (n = 3). ANT indicates anthracene; BaP, benzo[a]pyrene; SE, standard error.

Levels of Nrf2

Quantification of the levels of Nrf2, an important regulator of oxidative stress which gets activated in response to pro-oxidants, was evaluated through ELISA. The levels of Nrf2 were significantly higher in both BaP- and ANT-exposed cells as compared to their respective controls. The recorded Nrf2 levels for cells exposed to BaP at 24 hours was 18.49 ± 2.14 pg/mL; while for ANT and controls, the levels were 28.39 ± 1.28 pg/mL and 2.9 ± 0.04 pg/mL, respectively (Figure 2).

Figure 2.

Levels of Nrf2. Graph demonstrating the levels of Nrf2 in controls and the cells exposed to ANT and BaP at different time points. Bars represent mean ± SE values, and P ≤ 0.05 was considered statistically significant (n = 3). ANT indicates anthracene; BaP, benzo[a]pyrene; Nrf2, nuclear factor erythroid 2-related factor 2; SE, standard error.

DNA Damage and Apoptosis

Increased accumulation of γH2AX and higher activation of caspase-3 in cells treated with PAHs suggested the ability of these pollutants to affect cellular integrity and proliferation. The mean fluorescence intensity for γH2AX reported at 6 hours among the ANT- and BaP exposed cells was 29.56% ± 3.45% and 21.04% ± 4.01%, respectively; whereas the control levels were 3.98% ± 0.04% (Figure 3A). Similarly, in comparison to controls, higher caspase-3 activity was also observed in the PAH exposed cells. The percentage caspase-3 activity observed at 1, 3, 6, 12, and 24 for ANT exposed cells was 2.35% ± 0.12%, 6.78% ± 0.89%, 18.92% ± 2.39%, 42.65% ± 3.97%, and 71.03 ± 6.91%, respectively; while for BaP exposed cells and controls, the observed caspase-3 activity at the similar time points was 1.89% ± 0.29%, 3.27% ± 0.82%, 6.52% ± 1.08%, 14.59% ± 2.36%, and 21.85% ± 3.92%, respectively; and 0.68% ± 0.08%, 1.03% ± 0.12%, 1.98% ± 0.19%, 3.83% ± 0.81%, and 5.97% ± 1.02%, respectively (Figure 3B).

Figure 3.

DNA damage and apoptosis. A, Graph illustrating the comparative assessment of H2AX phosphorylation in the controls and cells exposed to ANT and BaP at different time points. Data are expressed as mean ± SE, and P ≤ 0.05 was considered statistically significant (n = 3). B, Representative overlay histograms showing flow cytometric analysis of caspase-3 activation in the cells exposed to ANT and BaP for 6, 12, and 24 hours. The observed shift in the peak from control (blue) region suggests the increase in the caspase-3 activity among the exposed cells. ANT indicates anthracene; BaP, benzo[a]pyrene; SE, standard error.

Analysis of MitomiR and Targets

The results of expression analysis showed alterations in the mitomiR profiles of the BaP exposed cells. As compared to controls, downregulation of miR-24 and miR-155 and upregulation of miR-34a and miR-150 were observed in the cells exposed to BaP. The maximum downregulation and upregulation were observed in miR-24 and miR-155, respectively (Figure 4A). We further analyzed the effect of miRNA expression on their targets genes, that is, NF-κβ, MYC, and p53. Nuclear factor κβ, which gets activated upon oxidative stress and leads to the generation of an inflammatory response, is a homo- or heterodimeric complex. It acts as an inflammatory switch for the insinuation of epigenetic alterations While p53 and MYC are 2 regulatory genes that potentially regulate the process of cell cycle and apoptosis. Although expression of MYC, p53, and NF-κβ genes were upregulated in BaP treated cells, no significant changes were observed in cells exposed to ANT (Figure 4B).

Figure 4.

MitomiR and target analysis. The graphical representation of the expression profile of mitomiRs (A) and their targets, that is, MYC, p53, and NF-κβ (B), in the cells exposed to ANT and BaP for 72 hours. The fold change was calculated as 2^−ΔΔCT by identifying dC_T as the difference of internal control from their respective control and test values. ANT indicates anthracene; BaP, benzo[a]pyrene; NF-κβ, nuclear factor κβ.

Status of Epigenetic Modifiers

The analysis of epigenetic modifiers, that is, DNMT1, HDAC1, HDAC7, KDM3a, EZH2, and P300, showed marked alterations in BaP exposed cells. With respect to controls, the expression of DNMT1 was significantly lowered in the BaP exposed cells after 72 hours of exposure (Figure 5). Similarly, the levels of KDM3a, HDAC1, and HDAC7, P300, and EZH2 were also altered in cells exposed to BaP for 72 hours (Supplemental Figure 3).

Figure 5.

Epigenetic modifiers. Graph showing the real-time percentage expression of DNMT1 in controls and cells exposed to ANT and BaP for different time points. Data are expressed as mean ± SE, and P ≤ 0.05 was considered statistically significant (n = 3). ANT indicates anthracene; BaP, benzo[a]pyrene; SE, standard error.

DNA Methylation Profile

In cells treated with BaP for 72 hours, the methylation levels of D1, D2, 12S, CYTB, and 16S region of mtDNA were lower as compared to controls. However, no such change was observed in the cells exposed to ANT (Figure 6A). The mean value of 5-mC in BaP exposed cells at 72 hours was 0.54 ± 0.049, whereas the levels were 1.90 ± 0.098 and 2.18 ± 0.12 in ANT and control cells, respectively (Figure 6B). Similarly, the results of methylation-specific PCR showed differential expression of LINE-1 in cells exposed to BaP at 72 hours (Supplemental Figure 4). Our observations strongly indicated that BaP causes significant DNA hypomethylation, while no such changes were observed in ANT exposed cells.

Figure 6.

DNA methylation profile. A, Graphical representation of the mitochondrial DNA methylation in the controls and cells exposed to ANT and BaP for 72 hours. Data are expressed as mean ± SE, and P ≤ 0.05 was considered significant as compared with controls. D1; D loop, D2; D loop 2. B, Graph showing the percentage 5-mC levels indicative of global DNA methylation in controls and cells exposed to ANT and BaP for 24, 48, and 72 hours. Data are expressed as mean ± SE (P ≤ 0.05). ANT indicates anthracene; BaP, benzo[a]pyrene; SE, standard error.

Histone Alterations

The evaluation of 21 specific H3 and 10 different H4 modifications showed significant alterations in the BaP exposed cells. In contrast, no changes were observed in cells treated with ANT (Figure 7A and B). Western blot analysis of H3K4me3, H3K4me9, H3K4me27, H3K4me36, and H3K4me79 showed marked hypermethylation in BaP treated cells (Supplemental Figure 5).

Figure 7.

Quantification of histone (H3 and H4) alterations. A, The levels of histone H3 modifications in controls and the cells exposed to ANT and BaP for 72 hours. Data are expressed as mean ± SE and P ≤ 0.05 was considered significant as compared with controls. B, The levels of histone H4 modifications in controls and the cells exposed to BaP for 72 hours. Data are expressed as mean ± SE and P ≤ 0.05 was considered significant. ANT indicates anthracene; BaP, benzo[a]pyrene; SE, standard error.

Discussion

Acute or chronic exposure to environmental pollutants significantly impairs immune cell functions which may contribute toward development of noncommunicable diseases. Such impairments are often mediated by metabolic changes that results from improper mitochondrial functioning as these semiautonomous organelles play a key role in regulating activation, differentiation, and survival of innate and adaptive cells. Previously, we have reported that exposure to different environmental toxins including air pollutants considerably affects mitochondrial redox machinery.^22,23 In the present study, we investigated the mitochondrial-mediated epigenetic regulation in peripheral lymphocytes following exposure to a carcinogenic (BaP) and noncarcinogenic (ANT) PAHs, an important class of air pollutant. Overall, our findings suggest that exposure to ANT induces mitochondrial toxicity with no significant epigenetic alteration, while BaP disturbs the redox machinery and initiates a cascade of aberrant epigenetic modifications that include changes in the expression of mitochondrial miRNAs, DNA hypomethylation, and modification in histone tails.

Moreover, mitochondria are the essential organelles that play a crucial role in the regulation of different cellular processes including ATP generation and apoptosis. Due to the extensive regulatory functioning, these organelles not only works as a cellular source of free radicals but also are more prone to electrophillic attack, radical injury, and dysfunction.^27
-29 Importantly, these oxidative bursts may cause deleterious effects on the mitochondrial genome as it lacks the protective structure of histones and an efficient repair system. In some circumstances, mitochondria compensate for the damage by inducing mtDNA replication. However, as defective mitochondria accumulate, it further leads to abnormal bioenergetic and replicative function.³⁰ The results observed herein showed significantly higher levels of ROS among the peripheral lymphocytes exposed to PAH (both BaP and ANT), which confirmed the potential of these compounds to stimulate mitochondrial stress. We also recorded considerable loss of mitochondrial membrane potential in the cells exposed to both BaP and ANT. Together, our findings demonstrated that noncarcinogenic PAHs not only disrupt mitochondrial integrity but cause loss of compensatory ability of the cell leading to apoptosis.

In order to understand the impact of excessive stress generated in the mitochondrial milieu on retrograde signaling, we examined the levels of Nrf2, an important mediator of mitochondrial-nuclear cross talk.³¹ Our results showed that in comparison to controls the levels of Nrf2 were significantly higher in the cells exposed to PAHs. These increased levels were maintained until 12 hours but declined subsequently suggesting the inability of cells to resist the stress. This was further confirmed by the higher accumulation of ƴH2AX in both ANT and BaP treated cells. An increase in the caspase-3 activity was also recorded in exposed cells; however, the activities were more pronounced in the cells exposed to ANT. Interestingly, the limited activation of caspases also indicated the probability that these mitochondria-induced genomic alterations in BaP exposed cells may further influence the epigenetic machinery.

Recent reports provide ample evidence that the mitochondrial niche serves as both source and regulatory target of the miRNAs that harbor within mitochondria.^32,33 In lymphocytes, these miRNAs form the basis of different signaling mechanisms engaged in the transcriptional regulation of different associated proteins. The miRNA panel used for our study comprised of 4 known mitomiRs, that is miR-24, miR-34, miR-150, and miR-155. These mitomiRs play an important role in the regulation of mitochondrial-derived inflammatory pathways considered vital for controlling protective immune responses; miR-24 and miR-150 have profound function in lymphocyte differentiation, whereas miR-155 controls the differentiation of CD4+ T cells into T helper type 1 (Th1), Th2, and Th17 subsets of helper T cells.^34
-36 A recent study suggested that miR-155 mediated gene regulation in immune cells greatly influences the cellular context of T and B lymphocytes.³⁷ The upregulation of miR-34a and miR-150 and downregulation of miR-24 and miR-155 in BaP treated cells were found to be associated with the expression profile of their respective target genes. We observed that exposure to BaP increases the expression of MYC, p53, and NF-κβ target genes, which are involved in a number of key cellular processes of stress-induced epigenetic alterations.

Importantly, the mitochondrial stress signals not only impact DNMT1 functioning but also are shown to be linked to mtDNA methylation. These signals facilitate translocation of DNMT1 to the mitochondria thereby influencing mtDNA gene transcription.^38
-40 Although association of BaP exposure with DNA methylation is known, the underlying mechanisms to establish the link between mitochondrial deregulation with changes in epigenetic machinery are missing.⁴¹ Our results suggested that BaP exposure causes significant alterations in the expression of epigenetic modifiers (DNMT1, HDAC1, HDAC7, KDM3a, EZH2, and P300), while no such changes were noticed in ANT treated cells. It is noteworthy that such aberrant expression of epigenetic modifiers is likely to trigger methylation and acetylation of nDNA and histones. Our results provided clear evidence of hypomethylation in D1, D2, 12S, CYTB, and 16S region of mtDNA. In addition, the global DNA methylation analysis using LINE 1 and 5-mC showed a significant hypomethylated profile in the BaP exposed cells. These findings not only highlight the functional link between mitochondrial and DNA methylation but also indicated the ability of BaP to induce long-term cellular changes. However, we did not observe any significant changes in methylation profiles of ANT treated cells. We consider that these observations are vital as the deregulated DNA methylation profile is known to be associated with immune disorders.⁴²

The intermediate mitochondrial metabolites are required for covalent modifications of N-terminal tails of histones.⁴³ Therefore, we assumed that mitochondria dysfunction after PAH exposure may induce alterations in the histone profile. In the present study, exposure to BaP caused significant histone H3 and H4 modifications, however no notable changes were observed following treatment with ANT. Together, these histone modifications in BaP exposed cells might influence gene expression by causing irreversible errors in the vital transcriptional machinery.

Conclusion

Overall, the study demonstrates that peripheral lymphocytes treated with PAHs disturb the functional integrity of mitochondrial machinery, and the effects were more pronounced in ANT treated cells as compared to BaP. Contrarily, the subtle changes in BaP exposure triggered a cascade of epigenetic changes mediated through mitochondrial retrograde signaling (Figure 8 and Table 1). Collectively, our results contribute to the current knowledge by demonstrating the differential immunotoxic potential of ANT and BaP. The findings further suggest that PAHs, at least in part, may be responsible for the adverse effects linked to immune function related to air pollution. As epigenetic modifications have regulatory impact on gene expression patterns, functional characterization of cell-free circulating signatures can be useful to establish minimal invasive biomarkers for air-pollution linked immune disorders. However, the use of such signatures in human health risk assessment studies will require careful validation in control and experimental settings.

Figure 8.

PAH-induced immunotoxicity. A novel insight into the possible role of mitochondria oxidative stress-induced epigenetic modifications in lymphocyte homeostasis following treatment with ANT and BaP. ANT indicates anthracene; BaP, benzo[a]pyrene; Cas 3, caspase-3; Cyt C, cytochrome c; DNMT, DNA methyltransferases; HDAC, histone deacetylase; miR, microRNA; PAH, polycyclic aromatic hydrocarbon; PARP, poly ADP ribose polymerase.

Table 1.

Differential Immunotoxic Potential of Anthracene and Benz[o]pyrene Following Exposure to Peripheral Lymphocytes.

Parameter	Targets	Observation
Parameter	Targets	ANT	BaP
Mitochondrial stress and membrane integrity	ROS	Time-dependent increase with maximum levels observed at 3 hours	Time-dependent increase with maximum peak observed at 6 hours
	Nrf2	Maximum increase observed at 6 hours	Maximum increase observed at 12 hours
	mtDNA	Nonsignificant decrease	Nonsignificant increase
	Membrane integrity	Time-dependent increase	Higher mitochondrial membrane depolarization
DNA damage and apoptosis	γH2AX activation	Increased levels of γH2AX with time	Levels of γH2AX comparatively lower than ANT
DNA damage and apoptosis	Caspase-3 activity	Levels of caspase-3 comparatively higher than BaP	Time-dependent increase with maximum levels at 24 hours
MitomiRs and target genes	miR-24, miR-155, miR-34a, miR-150	No significant alterations were observed	miR-24 and miR-155 were downregulated; and miR-34a and miR-150 upregulated
MitomiRs and target genes	MYC, p53, NF-κβ	No significant changes were noted	The expression of MYC, p53, and NF-κβ was upregulated
Epigenetic modifiers	DNMT1, HDAC1, HDAC7, KDM3a, EZH2, P300	No marked modifications were observed	Change in expression patterns were observed at 72 hours
DNA methylation	mtDNA methylation	No significant alterations were recorded	Hypomethylated profile of D1, D2, 12S, CYTB, and 16S regions
	5-methyl cytosine	No significant alterations were observed	Decreased levels of 5-methyl cytosine
	LINE-1	No significant changes were noted	Hypomethylation of LINE-1 observed
Histone modifications	H3 and H4	No significant alterations were observed	Significant modifications were noted

Abbreviations: ANT, anthracene; BaP, benzo[a]pyrene; DNMT, DNA methyltransferases; EZH2, histone methyltransferase; HDAC, histone deactylases; KDM3a, histone demethylase; mitomiRs, mitochondrial miRNAs; mtDNA, mitochondrial DNA; Nrf2, nuclear factor erythroid 2 related factor 2; P300, histone acetyltransferase; ROS, reactive oxygen species.

Supplemental Material

Supplemental_Material - Mapping the Mitochondrial Regulation of Epigenetic Modifications in Association With Carcinogenic and Noncarcinogenic Polycyclic Aromatic Hydrocarbon Exposure

Supplemental_Material for Mapping the Mitochondrial Regulation of Epigenetic Modifications in Association With Carcinogenic and Noncarcinogenic Polycyclic Aromatic Hydrocarbon Exposure by Arpit Bhargava, Roshani Kumari, Surbhi Khare, Ruchita Shandilya, Pushpendra Kumar Gupta, Rajnarayan Tiwari, Akhlaqur Rahman, Koel Chaudhury, Irina Yu Goryacheva and Pradyumna Kumar Mishra in International Journal of Toxicology

Footnotes

Author Contributions

Bhargava, A. contributed to acquisition and drafted the manuscript; Kumari, R. contributed to acquisition and analysis and critically revised the manuscript; Khare, S. contributed to acquisition and critically revised manuscript; Shandilya, R. contributed to acquisition and analysis and critically revised the manuscript; Gupta, P.K. contributed to acquisition and critically revised manuscript; Tiwari R. contributed to interpretation and critically revised manuscript; Rahman, A. contributed to acquisition and critically revised manuscript; Chaudhury, K. contributed to interpretation and analysis and critically revised the manuscript for important intellectual contribution; Goryacheva, I.Y. contributed to interpretation and analysis and critically revised the manuscript for important intellectual contribution; Mishra, P.K. contributed to conception and design, contributed to acquisition, analysis, and interpretation, drafted the manuscript, and critically revised the manuscript for important intellectual contribution. All authors gave final approval and agree to be accountable for all aspects of work ensuring integrity and accuracy.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the financial assistance received from the Ministry of Human Resource and Development (MHRD) and Indian Council of Medical Research (ICMR), Government of India, New Delhi, to the laboratory of Professor Pradyumna Kumar Mishra.

ORCID iD

Pradyumna Kumar Mishra

References

Health Effects Institute. State of Global Air 2019. Special Report. Health Effects Institute; 2019. Accessed December 12, 2019. https://www.stateofglobalair.org/sites/default/files/soga_2019_report.pdf

Bhargava

Shukla

Bunkar

, et al. Exposure to ultrafine particulate matter induces NF-κβ mediated epigenetic modifications. Environ Pollut. 2019;252(2):39–50. doi:10.1016/j.envpol.2019.05.065

Gonzales

Shiao

.MTHFR Gene polymorphism-mutations and air pollution as risk factors for breast cancer: a metaprediction study. Nurs Res. 2017;66(4):152–163. doi:10.1097/NNR.0000000000000206

Bai

Shin

Burnett

, et al. Exposure to ambient air pollution and the incidence of congestive heart failure and acute myocardial infarction: A population-based study of 5.1 million Canadian adults living in Ontario. Environ Int. 2019;132(6):105004. doi:10.1016/j.envint.2019.105004

International Agency for Research in Cancer (IARC). 2019. Monographs on the Identification of Carcinogenic Hazards to Humans. Report of the Advisory Group to Recommend Priorities for the IARC Monographs during 2020–2024. IARC; 2019. Accessed August 19, 2019. https://monographs.iarc.fr/news-events/advisory-group-report-on-priorities-for-the-iarc-monographs-during-2020-2024/

Burchiel

Luster

. Signaling by environmental polycyclic aromatic hydrocarbons in human lymphocytes. Clin Immunol. 2001;98(1):2–10. doi:10.1006/clim.2000.4934

Mishra

Raghuram

Panwar

Jain

Pandey

Maudar

. Mitochondrial oxidative stress elicits chromosomal instability after exposure to isocyanates in human kidney epithelial cells. Free Radic Res. 2009;43(3):718–728. doi:10.1080/10715760903037699.

Mishra

Bunkar

Raghuram

Khare

Pathak

Bhargava

. Epigenetic dimension of oxygen radical injury in spermatogonial epithelial cells. Reprod Toxicol. 2015;52(9):40–56. doi:10.1016/j.reprotox.2015.02.006

Shukla

Bunkar

Kumar

, et al. Air pollution associated epigenetic modifications: Transgenerational inheritance and underlying molecular mechanisms. Sci Total Environ. 2019;656(2):760–777. doi:10.1016/j.scitotenv.2018.11.381

10.

Bunkar

Bhargava

Khare

Mishra

. Mitochondrial anomalies: driver to age associated degenerative human ailments. Front Biosci (Landmark Ed). 2016;21(6):769–793.

11.

Mishra

Raghuram

Jain

Khare

Pathak

. Mitochondrial oxidative stress-induced epigenetic modifications in pancreatic epithelial cells. Int J Toxicol. 2014;33(4):116–129. doi:10.1177/1091581814524064

12.

Desiderio

Longo

Parrillo

, et al. Epigenetic silencing of the ANKRD26 gene correlates to the pro-inflammatory profile and increased cardio-metabolic risk factors in human obesity. Clin Epigenetics. 2019;11(7):181. doi:10.1186/s13148-019-0768-0

13.

Gruzieva

Yousefi

, et al. Prenatal particulate air pollution and DNA methylation in newborns: an epigenome-wide meta-analysis. Environ Health Perspect. 2019;127(4):57012. doi:10.1289/EHP4522.

14.

Bhargava

Bunkar

Aglawe

, et al. Epigenetic biomarkers for risk assessment of particulate matter associated lung cancer. Curr Drug Targets. 2018;19(6):1127–1147. doi:10.2174/1389450118666170911114342.

15.

Bhargava

Khare

Bunkar

, et al. Cell-Free circulating epigenomic signatures: non-invasive biomarker for cardiovascular and other age-related chronic diseases. Cur Pharm Des. 2017;23(3):1175–1187. doi:10.2174/1381612822666161027145359

16.

Woodson

Mason

Choi

, et al. Hypomethylation of p53 in peripheral blood DNA is associated with the development of lung cancer. Cancer Epidemiol Biomarkers Prev. 2001;10(4):69–74.

17.

Pavanello

Pesatori

Dioni

, et al. Shorter telomere length in peripheral blood lymphocytes of workers exposed to polycyclic aromatic hydrocarbons. Carcinogenesis. 2010;31(2):216–221. doi:10.1093/carcin/bgp278

18.

Rezazadeh Azari

Mohammadian

Pourahmad

, et al. Individual and combined toxicity of carboxylic acid functionalized multi-walled carbon nanotubes and benzo a pyrene in lung adenocarcinoma cells. Environ Sci Pollut Res Int. 2019;26(5):12709–12719. doi:10.1007/s11356-019-04795-x

19.

Bhargava

Khare

Bunkar

Lenka

Mishra

. Role of mitochondrial oxidative stress on lymphocyte homeostasis in patients diagnosed with extra-pulmonary tuberculosis. Cell Biol Int. 2016;40(3):166–176. doi:10.1002/cbin.10549

20.

Mishra

Khan

Bhargava

, et al. Regulation of isocyanate-induced apoptosis, oxidative stress, and inflammation in cultured human neutrophils: isocyanate-induced neutrophils apoptosis. Cell Biol Toxicol. 2010;26(3):279–291. doi:10.1007/s10565-009-9127-9

21.

Panwar

Raghuram

Jain

, et al. Cell cycle deregulation by methyl isocyanate: Implications in liver carcinogenesis. Environ Toxicol. 2014;29(2):284–297. doi:10.1002/tox.21757.

22.

Gupta

Bhargava

Kumari

, et al. Impairment of mitochondrial-nuclear cross talk in lymphocytes exposed to landfill leachate. Environ Health Insights. 2019;13(10):1178630219839013. doi:10.1177/1178630219839013

23.

Bhargava

Tamrakar

Aglawe

, et al. Ultrafine particulate matter impairs mitochondrial redox homeostasis and activates phosphatidylinositol 3-kinase mediated DNA damage responses in lymphocytes. Environ Pollut. 2018;234:406–419. doi:10.1016/j.envpol.2017.11.093

24.

Raghuram

Pathak

Jain

, et al. Molecular characterization of isocyanate-induced male germ-line genomic instability. J Environ Pathol Toxicol Oncol. 2010;29(6):213–234.

25.

Raghuram

Pathak

Jain

, et al. Molecular mechanisms of isocyanate induced oncogenic transformation in ovarian epithelial cells. Reprod Toxicol. 2010;30(8):377–786. doi:10.1016/j.reprotox.2010.05.087

26.

Panwar

Jain

Khan

, et al. Imbalance of mitochondrial-nuclear cross talk in isocyanate mediated pulmonary endothelial cell dysfunction. Redox Biol. 2013;1(3):163–171. doi:10.1016/j.redox.2013.01.009

27.

Byun

Baccarelli

. Environmental exposure and mitochondrial epigenetics: study design and analytical challenges. Hum Genet. 2014;133(4):247–257.

28.

Matilainen

Quirós

Auwerx

. Mitochondria and Epigenetics - Crosstalk in Homeostasis and Stress. Trends Cell Biol. 2017;27(3):453–463.

29.

Weinhouse

. Mitochondrial-epigenetic crosstalk in environmental toxicology. Toxicology. 2017;391(1):5–17.

30.

Hou

Zhu

Zhang

, et al. Airborne particulate matter and mitochondrial damage: a cross-sectional study. Environ Health. 2010;9(4):48. doi:10.1186/1476-069X-9-48.

31.

Pardo

Shemesh

, et al. Nrf2 protects against diverse PM2.5 components-induced mitochondrial oxidative damage in lung cells. Sci Total Environ. 2019;669(7):303–313. doi:10.1016/j.scitotenv.2019.01.436

32.

Bienertova-Vasku

Sana

Slaby

. The role of microRNAs in mitochondria in cancer. Cancer Lett. 2013;336(5):1–7. doi:10.1016/j.canlet.2013.05.001

33.

Borralho

Rodrigues

Steer

. MicroRNAs in mitochondria: an unexplored niche. Adv Exp Med Biol. 2015;887(3):31–51. doi:10.1007/978-3-319-22380-3_3

34.

Baltimore

Boldin

O’Connell

Rao

Taganov

. MicroRNAs: new regulators of immune cell development and function. Nat Immunol. 2008;9(4):839–845. doi:10.1038/ni.f.209

35.

Ghisi

Corradin

Basso

, et al. Modulation of microRNA expression in human T-cell development: targeting of NOTCH3 by miR-150. Blood 2011;117:7053–7062. doi:10.1182/blood-2010-12-326629

36.

Cho

Nguyen

, et al. A novel miR-24-TCF1 axis in modulating effector T cell responses. J Immunol. 2017;198(3):3919–3926. doi:10.4049/jimmunol.1601404

37.

Hsin

Loeb

Leslie

Rudensky

. The effect of cellular context on miR-155-mediated gene regulation in four major immune cell types. Nat Immunol. 2018;9(6):1137–1145. doi:10.1038/s41590-018-0208-x

38.

Saini

Mangalhara

Prakasam

Bamezai

RNK

. DNA methyltransferase 1 (DNMT1) isoform 3 methylates mitochondrial genome and modulates its biology. Sci Rep. 2017;7(3):525. doi:10.1038/s41598-017-01743-y

39.

Cuyàs

Fernández-Arroyo

Verdura

, et al. Metformin regulates global DNA methylation via mitochondrial one-carbon metabolism. Oncogene. 2018;37(5):963–970. doi:10.1038/onc.2017.367

40.

Sharma

Pasala

Prakash

. Mitochondrial DNA: epigenetics and environment. Environ Mol Mutagen. 2019;60(2):668–682. doi:10.1002/em.22319

41.

Duan

, et al. Global and MGMT promoter hypomethylation independently associated with genomic instability of lymphocytes in subjects exposed to high-dose polycyclic aromatic hydrocarbon. Arch Toxicol. 2013;87(1):2013–2222. doi:10.1007/s00204-013-1046-0

42.

Guo

, et al. Alterations of global DNA methylation and DNA methyltransferase expression in T and B lymphocytes from patients with newly diagnosed autoimmune thyroid diseases after treatment: a follow-up study. Thyroid. 2018;28(5):377–385. doi:10.1089/thy.2017.0301

43.

Salminen

Kauppinen

Hiltunen

Kaarniranta

. Krebs cycle intermediates regulate DNA and histone methylation: epigenetic impact on the aging process. Ageing Res Rev. 2014;16(4):45–65. doi:10.1016/j.arr.2014.05.004

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