Introduction

Air pollution, a pervasive mixture of chemicals and particulate matter (PM), is one of the greatest environmental risks to health. In 2019, the World Health Organization (WHO) estimated 11% of outdoor air pollution-related premature deaths were due to cancer within the respiratory tract [1].

Polycyclic aromatic hydrocarbons (PAHs) are among the chemicals found in the complex mixture of chemicals and PM in air pollution [2, 3]. Common sources of PAHs include household combustion devices, motor vehicles, industrial activities, and forest fires [2]. Exposure to airborne PAHs in both occupational and non-occupational settings were associated with the risk of developing breast and lung cancers [2,3,4,5,6,7,8]. Notably, a French prospective cohort study, of a large sample size with long-term exposure data of benzo(a)pyrene (BaP), showed significant association between airborne BaP exposure and overall breast cancer risk. The association was greater among women in menopausal transition and tobacco smokers [3]. Inevitably, the International Agency for Research on Cancer (IARC) classified BaP as a Group 1 carcinogen in humans, based on sufficient experimental evidence of carcinogenicity in animals and corroborated by consistent mechanistic evidence [9].

The IARC has also declared tobacco smoking to have sufficient and limited evidence in humans to cause lung and breast cancer, respectively [10]. Arguably, tobacco smoking is a good example of adverse health effects of exposure to chemicals mixture. This is because tobacco smoke contains more than 5,000 different chemicals, including PAHs, tobacco specific nitrosamines, aromatic amines, aldehydes, phenols, nitro compounds, volatile hydrocarbons, and other organic and inorganic chemicals [11]. Tobacco smokers who work at industrial facilities are at high risk of exposure to hydrocarbons mixture and the risks of breast and lung cancers have been shown to be greater among workers who smoke tobacco [3, 12]. Studies on the mechanism by which exposure to a mixture of hydrocarbons contributes to the development of breast and lung cancers are scarce and, indeed, a complex field to venture into. However, advances in toxicogenomics provide comprehensive databases on chemicals, genes, proteins, and diseases that one can utilize to gain insights into molecular pathways that chemical mixtures potentially influence in the development of a specific disease.

This article elucidates interactions of genes influenced by a mixture of carcinogenic hydrocarbons with those related to the development of breast and lung cancer. Importantly, the article demonstrates the capability of in silico data-mining for gauging probable molecular mechanisms of mixture-induced toxic effects. This may then assist in strategizing experimental studies to better understand the impact of airborne hydrocarbons in the development of breast and lung cancers. The findings of such studies would then contribute to the risk assessment of chemical mixtures to safeguard the health of people.

Methods

Selection of hazardous air pollutants

In 2019, Ismail et al. [13] undertook to prioritize the hazard classification of 188 chemicals in the Office of Environment Health Hazard Assessment (OEHHA) list of chemicals emitted from California refineries [14]. The prioritization was in accordance with the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS). The classifications considered were carcinogenicity (C), mutagenicity (M) and reproductive toxicity (R) from databases of nine countries. Out of the 188 chemicals, 67 were identified as carcinogens 1 A (known human carcinogen), 1B (presumed human carcinogen) or 2 (suspected human carcinogen) [13].

We confirmed the classification of these chemicals by referencing databases of six countries—Australia, European Union (EU), Japan, South Korea, Malaysia, and New Zealand—to reflect the latest classification. The reference databases (Table 1) were chosen as they were accessible in English on the open World Wide Web domain.

Table 1 Source of the UN GHS classification database
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From the revised list, chemicals with the most stringent carcinogenicity classification (1/1A/1B) (Suppl Table 1) were then screened for hydrocarbons, as they are common air pollutants and contained in tobacco smoke. These hydrocarbons were further analyzed for gene interactions in the development of breast and lung cancers. The molecular pathways potentially influenced by these genes were elucidated to gain insights on potential molecular pathways affected by hydrocarbons mixture.

Comparative Toxicogenomic database (CTD) analysis

The hydrocarbons were grouped into alkanes/alkenes, halogenated hydrocarbons, and PAHs. The linkages between these groups of hydrocarbons and cancers of the breast and lung, were explored by analyzing the chemical-gene/protein interactions obtained from the Comparative Toxicogenomic Database (CTD; https://ctdbase.org/). The analysis was based on data downloaded in July 2023. The CTD is a public domain database that allows the integration of data to provide a better understanding of the interactions between environmental chemicals, genes, and diseases [15]. Chemicals, chemical-phenotypes, gene ontology and chemicals-disease associations are the examples of information provided by the CTD. The search for genes associated with breast and or lung cancers was based on the CAS number of each individual carcinogenic hydrocarbon and inference network. The data-mining process flow is depicted in Fig. 1. The respective inference score and the reference links are in Supplementary Table 2.

Fig. 1
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Process flow for in-silico data-mining

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Identifying common genes for hydrocarbons mixture and breast and lung cancer development

The lists of genes extracted from the CTD were uploaded to an Excel spreadsheet. Further analysis was done with Cytoscape version 2.5.10—a free software package—to visualize, model and analyze molecular and genetic interaction networks [16].

Gene-gene interaction network construction

The complex gene-gene interactions network of the common genes between the hydrocarbons and the selected cancers was constructed with GeneMANIA, a free in silico tool (http://www.genemania.org) that provides a flexible interface to query genomic, proteomic, and gene function data [17, 18]. The tools’ dataset are from various publicly available databases, such as Gene Expression Omnibus (GEO) for co-expression data [19]; BioGRID for physical and genetic interaction data [20]; I2D for predicted protein interaction data [21]; and Pathway Commons for pathway and molecular interaction data [22,23,24,25]. The database has almost 2300 networks from eight different organisms that collectively contain nearly 600 million interactions covering almost 164,000 genes [18]. GeneMANIA generates networks from the data either directly or using an in-house analysis pipeline to convert profiles to functional association networks [26]. Co-expression networks were filtered (by default) to remove weak correlations [18]. In this study, Homo sapiens was selected as a target organism in GeneMANIA analysis.

Molecular pathways enrichment analysis

Pathway analysis was performed by Cytoscape ClueGO together with CluePedia plug-in version 2.5.10. The common genes found between hydrocarbons that are associated with the selected cancer development were inserted into the Load Marker List section. The Kyoto Encyclopedia of Genes and Genomes (KEGG), Reactome, and WikiPathways [27,28,29] databases were selected in the ClueGO settings to extract the list of pathways. Enrichment right-sided hypergeometric test was used for the enrichment with a Bonferroni step-down correction and a κ score of 0.3 to link the terms [30]. ClueGO plug-in integrates GO terms and KEGG/BioCarta pathways. The plug-in was used to visualize molecular pathways and gene ontology that are linked to the examined common genes connected to the selected cancers. The organism analyzed was set to Homo sapiens. The output results (gene-pathway interactions) are shown in Supplementary Figs. 1 & 2 that were used to construct Figs. 2 and 3.

Fig. 2
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Gene and molecular pathway interactions of hydrocarbons associated with breast cancer

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Fig. 3
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Gene and molecular pathway interactions of hydrocarbons associated with lung cancer

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Results

Classification revision

Among the 67 chemicals screened, the classifications of seven chemicals were revised to a more stringent category: from category 2 (suspected human carcinogen) to category 1/1A/1B (known human carcinogen/presumed human carcinogen). This revision was based on Japan’s most stringent classification. The seven chemicals are 1,1,2,2-tetrachloroethane (CAS No. 79-34-5), analine (CAS No. 62-53-3), anthracene (CAS No. 120-12-7), biphenyl (CAS No. 92-52-4), dichloromethane (CAS No. 75-09-2), methyl isobutyl ketone (CAS No. 108-10-1), and styrene (CAS No. 100-42-5) (Suppl Table 1). With the revision, 41 of the 67 chemicals are category 1/1A/1B carcinogens, of which 27 are hydrocarbons (Suppl Table 1). Among the 27 hydrocarbons, 25 were identified as alkanes, alkenes, halogenated hydrocarbons, and PAHs.

Comparative Toxicogenomic database analysis

Among the 25 hydrocarbons, nine were excluded from further toxicogenomics analysis for absence of curated data in the CTD. The nine chemicals are isobutane (CAS No. 75-28-5), n-butane (CAS No. 106-97-8), 1,1,2,2-tetrachloroethane (CAS No. 79-34-5), 1,2-dichloroethane (CAS No. 107-06-2), vinyl bromide (CAS No. 593-60-2), polychlorinated biphenyl (CAS No. 1336-36-3), benz(a)anthracene (CAS No. 56-55-3), biphenyl (CAS No. 92-52-4), and benzo(e)pyrene (CAS No. 192-97-2) (Suppl Table 2).

In the alkanes/alkenes group, only two of the four chemicals contained gene interactions data. Isoprene (2-methyl-1,3-butadiene (CAS No. 78-79-5)) affects 12 and 8 genes associated with breast and lung cancer, respectively, whilst 1,3-butadiene (CAS No. 106-99-0) affects 63 genes linked to breast cancer development and 48 genes linked to lung cancer (Suppl Table 2).

In the halogenated group, six of the twelve chemicals affect genes linked to breast or lung cancer genes. Tetrachlorodibenzodioxin (TCDD) (CAS No. 1746-01-6) interacts with the greatest number of genes related to breast cancer development (489 genes), followed by trichloroethylene (CAS No. 79-01-6; 230 genes), tetrachloroethylene (CAS No. 127-18-4; 40 genes), and dichloromethane (CAS No. 75-09-2; 35 genes). Both 1,2 dichloropropane (CAS No. 78-87-5) and 1,2-dibromoethane (CAS No. 106-93-4) affect only five genes (Suppl Table 2). Regarding genes linked to lung cancer development, TCDD, carbon tetrachloride (CAS No. 56-23-5), dichloromethane, and vinyl chloride (CAS No. 75-01-4) affect 250, 184, 25, and 16 genes, respectively, whilst 1,2-dichloropropane and 1,2-dibromoethane affect less than ten genes (Suppl Table 2).

Among the nine chemicals in the PAHs group, BaP (CAS No. 50-32-8) affects the greatest number of genes related to breast cancer development (492 genes), followed by anthracene (CAS No. 120-12-7; 12 genes) (Suppl Table 2). In regard to interactions with lung cancer-associated genes, BaP affects the greatest number of genes (259 genes), followed by benzo(b)fluoranthene (CAS No. 205-99-2; 52 genes), chrysene (CAS No. 218-01-9; 35 genes), dibenz(a, h)anthracene (CAS No. 53-70-3; 34 genes), and benzo(k)fluoranthene (CAS No. 207-08-9; 33 genes) (Suppl Table 2).

Genes interacted with hydrocarbons mixture that are connected to breast and lung cancers

The data-mining revealed 87 and 44 genes linked to breast and lung cancer, respectively, interacted with most of the investigated hydrocarbons (Suppl Table 3).

The mutual molecular pathways in the development of breast and lung cancer that are linked to these genes are aryl hydrocarbon receptor (AHR) pathway, apoptosis, chemical carcinogenesis, ferroptosis, fluid shear stress and atherosclerosis, lipid and atherosclerosis, miRNA in DNA damage response, Nrf2 pathway, nuclear receptors meta-pathway, and oxidative stress response (Suppl Figs. 1 & 2; Suppl Tables 4 & 5).

Molecular pathways involved in breast cancer but not in lung cancer development are androgen receptor signaling, DNA methylation, estrogen metabolism and signaling, and interleukin-10 (IL-10) anti-inflammatory signaling (Suppl Fig. 1 & Suppl Table 4).

Interleukin-3, 4, 13 and 17 (IL-3, IL-4, IL-13, IL-17) signaling pathways, p53 signaling, oxidative damage response, and SUMOylation are involved in the development of lung cancer (Suppl Fig. 2 & Suppl Table 5) but not breast cancer.

Gene-gene interaction network affected by the common genes

GeneMANIA Cytoscape predictive plug-in provides information on interaction types between the common genes. The interaction types include: (a) Co-expression—two gene products are linked if their expression levels are similar across conditions in a gene expression study; (b) Genetic interaction—two genes are functionally associated if one gene is affected by alterations that occur to the second gene; (c) Physical Interaction—two genes product are linked if they interact at protein level; (d) Co-localization—genes expressed in the same tissue or proteins found in the same location; (e) Interaction in shared protein domains; and (f) Interaction predicted by the server [18].

Complex networks encompassing the whole set of interactions between the common genes linked to breast and lung cancers are presented in Supplementary Fig. 3. Co-expression (47.33% of interactions) and physical interaction (40.18%) are the dominant interactions among the common genes in breast cancer development, followed by genetic interaction (2.90%), co-localization (2.74%), and interaction in shared protein domains (0.54%) (Table 2). In the case of lung cancer development, co-expression is the dominant interaction between the common genes (46.88%), followed by physical interaction (24.17%), shared protein domain (7.61%), co-localization (7.43%), and genetic interaction (3.04%) (Table 2).

Table 2 Type of gene interactions among the common genes linked to breast and lung cancer
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In gaining insights on the potential biological pathways that would be affected by exposure to a mixture of hydrocarbons, we focused on 16 genes common in the development of both cancers (Suppl Table 3). Among these genes, 12 are protein-coding genes and 4 are proto-oncogenes.

The protein encoded by the 12 genes are baculoviral inhibitor of apoptosis (IAP) repeat-containing 5 (BIRC5), cyclin D1 (CCNDI), cytochrome P450 1B1 (CYP1B1), DNA methyltransferase 3 alpha (DNMT3A), estrogen receptor 1 (ESR1), glutathione S-transferase pi 1 (GSTP1), heme oxygenase 1 (HMOX1), interleukin 1 β (IL1B), interleukin 6 (IL6), transferrin receptor (TFRC), tumor necrosis factor (TNF), and tumor protein p53 (TP53). The 4 proto-oncogenes are c-Fos (FOS), Jun (JUN), HRas (HRAS) and Kras (KRAS) (Suppl Table 3).

Among the investigated hydrocarbons, 3 are associated with breast cancer development only: the halogenated hydrocarbon, trichloroethylene (TCE) and tetrachloroethylene, and anthracene, a polyaromatic hydrocarbon (Table 3). The chemical-gene interactions involved changes in mRNA and protein expression. All 3 chemicals do not interact with the HRAS and KRAS genes (Table 3).

Table 3 Chemical-gene interaction associated with breast neoplasms
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TCE interacted with the other 14 genes: it increased the expression of mRNA and/or protein of BIRC5 [31, 32], CCND1 [33], CYP1B1 [34], ESR1 [35], FOS [35], GSTP1 [35], HMOX1 [31, 35, 36], IL1B [37], IL6 [35], JUN [31, 35, 37], TNF [38], and TP53 [39]. It also decreased mRNA expression of CCND1 [40], CYP1B1 [36], DNMT3A [31, 41], IL6 [42], JUN [43], TFRC [35], TNF [44], TP53 [34], and decreased HMOX1, IL6 and TNF protein expression [38, 45] (Table 3). The potential biological pathways affected by TCE are AHR pathway, apoptosis, chemical carcinogenesis, DNA methylation, estrogen metabolism and signaling, ferroptosis, fluid shear stress & atherosclerosis, IL-10 anti-inflammatory signaling, lipid and atherosclerosis, miRNA in DNA damage, Nrf2 pathway, and oxidative stress response (Fig. 2).

Tetrachloroethylene interacted with only 5 genes (BIRC5, FOS, JUN, TNF, and TP53). It increased the expression of BIRC5 and TP53 mRNAs and TNF protein, as well as decreased the expression of FOS and JUN proteins (Table 3). The potential biological pathways affected by tetrachloroethylene are AHR pathway, apoptosis, estrogen signaling, ferroptosis, fluid shear stress & atherosclerosis, lipid and atherosclerosis, miRNA in DNA damage, nuclear receptors meta-pathway, and oxidative stress response (Fig. 2). Tetrachloroethylene ability to increase the expression of BIRC5 mRNA (Table 3) suggests deregulation of apoptosis as a potential mechanism affected by tetrachloroethylene in the development of breast cancer. This is because BIRC5—a member of the inhibitor of apoptosis (IAP) family—inhibits caspase activation, which leads to deregulation of apoptosis and increase cellular proliferation [46].

Anthracene interacted with only 3 genes (CCND1, CYP1B1, and ESR1). It increased the expression of CYP1B1 mRNA, as well as CCND1 and ESR1 protein (Table 3). The potential biological pathways affected by anthracene are AHR pathway, chemical carcinogenesis, estrogen metabolism and signaling, estrogen-dependent nuclear events, miRNA in DNA damage, and nuclear receptors meta-pathway (Fig. 2).

In the case of lung cancer, the interactions of carbon tetrachloride, vinyl chloride,dibenz(a, h)anthracene, benzo(b)fluoranthene, benzo(k)fluoranthene, and chrysene with the 16 genes, involved up- and down-regulation at mRNA and protein levels, gene polymorphism, and gene mutagenesis (Table 4).

Table 4 Chemical-gene interaction associated with lung neoplasms
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Carbon tetrachloride affected the up- and or down-regulation of all 16 genes at mRNA and or protein levels (Table 4). It increased the activity of DNMT3A, FOS, HMOX1, IL1B, IL6, JUN, and TNF. This implicates potential involvement of biological pathways associated with AHR pathway, apoptosis, chemical carcinogenesis, ferroptosis, fluid shear stress & atherosclerosis, oxidative stress response, and SUMOylation (Fig. 3).

Vinyl chloride interacted with only 6 genes. In addition to down regulating HMOX1, IL1B, IL6, and TP53 at mRNA level, as well as decreasing HMOX1 protein expression, it increased mutagenesis of both KRAS [47,48,49,50] and TP53 [47,48,49] genes (Table 4). When KRAS gene is mutated, it becomes an oncogene that can transform normal cells into cancer cells [51], whilst TP53 mutations resulted in uncontrolled cell growth leading to cancer development [52]. Thus, the potential mechanism by which vinyl chloride contributes to the development of lung cancer is associated with disruption of normal cellular processes and promotion of tumorigenesis. In the case of TNF, vinyl chloride increased the mRNA and protein activity (Table 4), indicating potential impact in tumor microenvironment.

Among the 4 PAHs associated with lung cancer, benzo(b)fluoranthene and benzo(k)fluoranthene interacted with 11 of the 16 genes, whilst chrysene and dibenz(a, h)anthracene interacted with 5 and 4 genes, respectively (Table 4). The upregulation of DNMT3A mRNA was increased by benzo(b)fluoranthene but not by the other 3 PAHs (Table 4). Similarly, the regulations of KRAS mRNA and protein were unaffected by all 4 PAHs, except for benzo(b)fluoranthene increased the mutagenesis of KRAS gene [53, 54]. It also increased TP53 protein expression and affected its activity [55] (Table 4). This suggests that benzo(b)fluoranthene and vinyl chloride affected similar biological pathways in the development of lung cancer. A comprehensive overview of the interaction between the 16 genes and PAHs that are linked to lung cancer is shown in Fig. 3.

In the development of both cancers, TCDD and BaP interacted with all 16 genes by affecting the respective mRNA and protein expression and or protein activity (Table 5). BaP can also affect the methylation of BIRC5 3’UTR, GSTP1 promoter, HRAS and IL1B 5’UTR, and phosphorylation of TP53 protein [56,57,58]. The alkenes, isoprene and 1,3-butadiene increased the mutagenesis of both HRAS and KRAS genes [59], whilst BaP increased the mutagenesis of KRAS gene [53, 54, 60, 61] (Table 5). Isoprene also increased the expression of CCND1 protein (Table 5). This indicates similar mechanism of actions by which isoprene, 1,3-butadiene and BaP contribute to the development of both breast and lung cancers.

Table 5 Chemical-gene interaction associated with breast and lung neoplasmsŧ
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Discussion

In utilizing the in silico toxicogenomic data-mining approach—to explore molecular mechanisms by which exposure to hydrocarbons mixture affects cancer development—we identified 16 genes common in the development of breast and lung cancers that interact with most of the investigated hydrocarbons. Proteins encoded by these genes: BIRC5, CCND1, TNF, and the proto-oncogenes FOS, JUN, HRAS, and KRAS, have all been implicated in cell cycle regulation directly or indirectly. The other 9 genes, CY1B1, DNMT3A, ESR1, GSTP1, HMOX1, IL1B, IL6, TFRC, and TP53 encode proteins involved in xenobiotic metabolism, gene regulation, oxidative damage response, inflammatory response, iron homeostasis, regulation of cell signaling pathways, and DNA damage response.

It is noted that these common genes may interact with carcinogens other than the investigated hydrocarbons. For example, acetaldehyde (CAS No. 75-07-0), asbestos (CAS No. 1332-21-4), bis(2-ethyl hexyl) phthalate (CAS No. 117-81-7), cadmium (CAS No. 7440-43-9), and chromium (CAS No. 18540-29-9) interacted with most of the 16 common genes, except BIRC5, DNMT3A, HRAS and KRAS (Suppl Table 6). The latter 4 genes interacted with carbon tetrachloride, 1,3-butadiene, TCDD, and BaP in the development of both breast and lung cancers.

The chemical-gene interactions profile suggests complex crosstalk involving DNA damage, transcriptional and post-transcriptional regulations, as well as translational and post-translational regulations, that affect various biological pathways common to cancer development. These biological pathways include gene mutation, cell cycle progression, oxidative stress and damage responses, inflammatory responses, and DNA damage responses.

In our mapping of biological pathways for breast cancer, DNMT3A, an enzyme responsible for de novo DNA methylation, is shown to be involved in miRNA expression (Suppl Fig. 1). The crosstalk between DNA methylation and miRNA expression can drive the pathogenesis of a disease. For instance, miRNAs can influence DNA methylation patterns by targeting transcripts of proteins responsible for DNA methylation, such as DNMT3A. Conversely, the methylation of miRNA promoter regions can inhibit their transcription, affecting their ability to regulate gene expression. Such crosstalk has been shown to drive the hormone-dependent phenotype of breast cancer [62].

In the case of lung cancer, the function of DNMT3A may be modified by SUMOylation a process of attaching and detaching small proteins called Small Ubiquitin-like Modifier (SUMO) to and from target proteins. This may lead to changes in the methylation of genes involved in cell growth and division, potentially contributing to uncontrolled cell proliferation. SUMOylation of other target proteins has been shown to enhance lung cancer metastasis [63].

The nature of chemical interaction in a mixture of hydrocarbons, such as additive, synergistic, potentiation, and antagonism cannot be discerned from this study due to inherent limitations of the study approach. However, the differences in chemical-gene interactions observed among the hydrocarbons provide insights into potential impact of exposure to hydrocarbons mixture.

For example, TCE—a halogenated hydrocarbon associated with increased risk of breast cancer in male and female workers [64, 65]—may potentiate the risk of lung cancer from exposure to dibenz(a, h)anthracene and the risk of both breast and lung cancer from exposure to BaP. The potential mechanism for such potentiation is increased DNA damage through DNA adduct formation and increased cellular proliferation through deregulation of apoptosis. TCE upregulates TP53 protein expression [39], as well as BIRC5 mRNA and protein expression [31, 32]. Elevated cellular TP53 protein has been shown to increase bioactivation of PAHs, such as dibenz(a, h)anthracene and BaP, by the enzyme cytochrome P450 1A1 (CYP1A1), which resulted in the elevation of DNA adduct levels [66]. BIRC5, on the other hand, inhibits caspase activation, which leads to deregulation of apoptosis and increase cellular proliferation [39].

In the case of vinyl chloride co-exposed with dibenz(a, h)anthracene and BaP, the DNA adduct formation via p53-dependent CYP1A1 bioactivation of the two PAHs, may be reduced as vinyl chloride is known to increase mutagenesis of the TP53 gene [48, 49].

Chemical carcinogenesis

Chemical carcinogenesis that pivots on the AHR pathway appears to be the bridge linking the development and progression of breast and lung cancers. AHR plays a "double-edged sword" that promotes or suppresses tumorigenesis, depending on cell and tissue context and mode of AHR activation. In breast cancer, AHR shapes the tumor microenvironment and modifies immune tolerance [67], whilst in lung cancer, AHR is involved in the regulation of cell proliferation, angiogenesis, inflammation, and apoptosis [68].

AHR is a multi-functional transcription factor activated by a variety of ligands, such as BaP, benz(a)anthracene, TCDD, and metabolites of tryptophan, heme and arachidonic acid, indigoids, and equilenin (reviewed in [69]). These ligands can be agonist, antagonist or selective AHR modulators [70]. Upon ligand binding, the cytosolic AHR-ligand complex is translocated into the nucleus where it heterodimerizes with the aryl hydrocarbon nuclear transporter (ARNT) before binding to the xenobiotic/dioxin response elements (XREs/DREs) in the promoter of target genes and triggers their expression [71]. These genes are involved in many physiological functions, such as xenobiotic metabolism [71], immune response [72], cell cycle and proliferation [73, 74], lipid metabolism [75, 76], tumor promotion [77, 78], and negative regulation of AHR pathway [76]. Perturbations of these physiological functions have been shown to be associated with cancer development and progression, which suggests a complex role of AHR in chemical carcinogenesis.

In xenobiotic metabolism, AHR activates transcriptional up-regulation of the cytochrome P450 1A1 (CYP1A1) and CYP1B1 genes. Most of the investigated hydrocarbons are linked to these two genes. Some are substrates for both enzymes. For example, the first step of BaP hydroxylation to BaP-7,8-epoxide, and the final epoxidation step to form BaP-7,8-dihydrodiol-9,10-epoxide (BPDE) are catalyzed by CYP1A1 and CYP1B1 enzymes in the lung and breast tissues [79,80,81], BPDE is a highly genotoxic metabolite that binds to deoxyguanosine at position N-2 to form DNA adducts [82]. Cigarette smoke was reported to induce CYP1A1 and CYP1B1 expressions in lung tissue of smokers and of lung cancer patients (both smokers and non-smokers) [83,84,85], which correlates with increased levels of BPDE and DNA adducts [86,87,88,89,90]. Bulky BaP-like DNA adducts were also detected in breast cancer patients [91, 92]. PAHs reactive metabolites are known to cause point mutations in RAS proto-oncogenes, such as codon 13 and codon 61 of the HRAS gene (reviewed in [93]). These observations suggest that the AHR/CYP450-dependent DNA adducts formation is a likely pathway to be affected by exposure to hydrocarbons mixture in the development of breast and lung cancers.

Several mechanisms by which AHR modulates the cell cycle have been proposed to account for the pro-/anti-proliferative action of AHR agonists observed with tumor cells in vitro [67, 68, 78]. One of the proposed ligand-activated mechanisms involved transcriptional upregulation of the CDKN1B gene by agonist-activated AHR binding to the gene’s promoter region [94, 95]. However, this mechanism has not been demonstrated in the development and progression of either breast or lung cancer. Being an inhibitor of cyclin-dependent kinase activity, increased CDKN1B activity limits phosphorylation of retinoblastoma protein (Rb), resulting in restriction of E2F-dependent gene expression and progression through the cell cycle [70]. In the absence of ligand, AHR complexed with cyclin D and the cyclin-dependent kinases CDK4/6 to promote cell cycle progression in human breast cancer cells [96]. TCDD, the atypical AHR agonist, dissociates the AHR/cyclinD/CDK complex to induce cell cycle arrest [96]. This contradictory role of AHR may reflect the impact of exposure to hydrocarbon mixtures on cell proliferation, as most of the investigated hydrocarbons are AHR agonists with different affinity to the receptor [70]. Similar contradictory effects of AHR on cell cycle progression were also observed in human lung cancer cells [97]. The impact of exposure to mixtures of 2-methyl-1,3-butadiene, carbon tetrachloride, TCDD, and BaP on this pathway may contribute to the development of breast and or lung cancer as these hydrocarbons interacted with CDKN1B gene (Suppl Table 2).  

The mechanisms by which AHR shapes the tumor microenvironment are unclear, but it has been proposed that systemic and tumor-localized generation of endogenous AHR ligands heightened AHR expression/activity, which may establish a pro-inflammatory yet immune-suppressive tumor micro-environment. This favors tumor survival and escapes from immune surveillance, which results in tumor progression [70]. Indeed, AHR overexpression that is correlated with elevated expression of inflammatory markers, including interleukin-8 (IL-8), has been observed in human breast tumors [98]. IL-8 has been identified as a critical gene that mediates breast cancer invasion and metastasis to the lungs [99]. The involvement of IL1B—one of the 16 common genes identified in this study—in this mechanism has not been elucidated in both breast and lung cancer development and progression. Our mapping, however, showed IL1B is involved in modulating the IL-17 signaling pathway, lipid and atherosclerosis pathway, and fluid shear stress and atherosclerosis pathway in both breast and lung cancer development. It is plausible that the impact of PAHs mixture on these pathways may involve AHR activation, as fluid shear stress in endothelial cells has been shown to modulate CYP1A-dependent AHR activation [100, 101], but the mechanism of activation remains unclear [67, 68].

Adaptive responses to cellular damage

AHR activation has also been shown to be associated with the oxidative stress response pathway. For example, exposure of estrogen receptor (ER) positive breast cancer cells to low doses of PAHs mixture activated AHR and overexpressed CYP1 isoforms, which correlated with increased expression of antiapoptotic and antioxidant proteins [102].

Besides catalyzing the biotransformation of PAHs to DNA damaging reactive metabolites, CYP1A1 and CYP1B1 catalyze the oxidation of estradiol (E2) to 2-hydroxyestradiol and 4-hydroxyestradiol, which subsequently undergo one-electron oxidation to produce unstable semiquinones (SQs) intermediates [103], potential mutagens that can damage DNA [104, 105]. Additionally, redox cycling can occur, where the SQs can pass their unpaired electron to molecular oxygen, forming a superoxide anion and restoring the catechol. Superoxide anion can then be metabolized to other reactive oxygen species (ROS), including hydrogen peroxide (H2O2) [103, 105]. Another contributor of ROS in breast cancer cells is the expression of CYP2E1, which increased significantly in breast tumors and adjacent tissues [106]. CYP2E1 also regulates autophagy, stimulates stress in the endoplasmic reticulum, and suppresses the metastatic potential of breast cancer cells [107], indicative of the protective role of CYP2E1.

Excessive ROS can cause DNA damage, as well as lipid and protein oxidation, which triggers an oxidative stress response that involves activation of the NRF2-KEAP1 signaling pathway. This pathway modulates the expression of genes encoding antioxidant proteins, such as superoxide dismutase and HMOX1. The latter is involved in the maintenance of cellular homeostasis by catalyzing the oxidation of heme to carbon monoxide, biliverdin, and ferrous iron. These biologically active compounds participate in cellular protection by reducing oxidative injury, attenuating the inflammatory response, inhibiting cell apoptosis, and regulating cell proliferation [108]. In mouse, HMOX1 activity increased tumor growth and angiogenic potential, as well as decreased apoptosis in lung cancer progression [109], whilst in rat and human breast cancer cells, HMOX1 activity inhibits proliferation [110].

Notably, the NRF2-KEAP1 signaling pathway is linked to ferroptosis, one of the common pathways in the development of breast and lung cancer mapped out in this study.

Ferroptosis

Ferroptosis is a non-apoptotic programmed cell death, which has gained traction as a new target for treating tumors [111]. It is regulated by a complex signaling pathway that is dependent on lipid peroxidation and iron accumulation [111, 112]. Evidence that supports the potential physiological roles of ferroptosis in tumorigenesis resides in the way it is induced in cancer cells. This includes activation of the RAS–RAF–MEK–ERK pathway and induction in cancer cells with mutant RAS, as well as dependency on iron, which is known to be important for cancer cell proliferation (reviewed in [112]). Induction of ferroptosis has been shown to suppress tumor growth, but ferroptotic damage favors tumor growth by triggering inflammation-associated immunosuppression in the tumor microenvironment [112]. Therefore, the three key features of ferroptosis: iron accumulation, increased lipid peroxidation and inability to efficiently reduce lipid peroxidases, must be well regulated to strike the delicate balance of survival and damage in tumorigenesis.

Little is known about ferroptosis role in breast and lung cancer progression, and the impact of exposure to hydrocarbons mixture on such association. However, several studies have found important correlations between mutations in tumor suppressor gene and proto-oncogene, TP53 and RAS, and in genes encoding proteins involved in stress response pathways. One of these pathways is the NRF2 signaling pathway [112], which is one of the common molecular pathways identified in this study.

Depending on the pathological condition, the transcription factor NRF2 serves as either an anti- or pro-ferroptotic activator. Under oxidative stress conditions, NRF2 complexed with its chaperon protein to bind to the ARE (Antioxidant Response Element) on the promoter region of its target genes with anti- or pro-ferroptotic functions. An example of iron-related NRF2 target gene that promotes ferroptotic cascade is HMOX1, which catalyzes the cleavage of heme to form biliverdin, carbon monoxide, and ferrous iron (Fe2+) [113]. Chemical-induced ferroptotic cell death driven by increased HMOX1 expression was observed in HT-1080, neuroblastoma and glioblastoma cell lines [114,115,116]. An example of NRF2 acting as anti-ferroptotic activator is in its regulating the expression of enzymes responsible for glutathione synthesis, as well as preventing lipid peroxidation and reducing oxidized CoQ10, a key membrane antioxidant (GPX4 and FSP1) [113]. Notably, GSTP1 has been shown to be involved in tumor development through the ferroptosis pathway [117] and was suggested to be a novel negative regulator of ferroptosis that may play an important role in lung cancer radiotherapy by inhibiting ferroptosis [118]. Crosstalk mechanisms between the RAS–RAF–MEK–ERK pathway and the NRF2 signaling pathway, with the involvement of GSTP1 in ferroptosis during tumorigenesis in breast and lung cells, and impact of exposure to halogenated and polyaromatic hydrocarbons on the crosstalk mechanisms have yet to be explored.

The role of AHR and NRF2 in regulating ferroptosis in breast and lung cancer cells is unclear, but AHR has been shown to promote the development of non-small cell lung cancer (NSCLC) by inducing the expression of SLC7A11, a key regulator of ferroptosis [119].

In sum, as most hydrocarbons are AHR ligands, the impact of inhalation exposure to hydrocarbons mixture on these physiological functions is complex, given the distinct classes of AHR ligands: agonist, antagonist and selective AHR modulators [70]. However, this study revealed an important role of AHR in being the bridge linking the development and progression of breast and lung cancers as it is involved (directly and or indirectly) in the regulation of biological pathways mapped out in this study. Notably, the mechanism by which IL1B regulates IL-8—a critical gene that mediates breast cancer invasion and metastasis to the lungs [99]—and the role of AHR in such mechanism, is worth pursuing.

Conclusion

Within the inherent limitations of in silico toxicogenomics associated tools, we were able to elucidate the molecular pathways of breast and lung cancer development potentially affected by exposure to hydrocarbons mixture. In silicon data-mining depends on the online sources and the quality of the interactions present in them. Complex molecular pathways were obtained by drawing statistical associations between chemical-gene-disease relationships. Therefore, dose-response relationship, interaction profile of hydrocarbons mixture, route and duration of exposure to the investigated hydrocarbons mixture, along with individual sensitivity of exposed subjects, cannot be drawn from this study. In conclusion,  our findings should be regarded as insights into future in vivo and in vitr laboratory investigations that focus on inhalation exposure to the hydrocarbons mixture.