Mapping the influence of hydrocarbons mixture on molecular mechanisms, involved in breast and lung neoplasms: in silico toxicogenomic data-mining.

BACKGROUND: Exposure to chemical mixtures inherent in air pollution, has been shown to be associated with the risk of breast and lung cancers. However, studies on the molecular mechanisms of exposure to a mixture of these pollutants, such as hydrocarbons, in the development of breast and lung cancers are scarce. We utilized in silico toxicogenomic analysis to elucidate the molecular pathways linked to both cancers that are influenced by exposure to a mixture of selected hydrocarbons. The Comparative Toxicogenomics Database and Cytoscape software were used for data mining and visualization. RESULTS: Twenty-five hydrocarbons, common in air pollution with carcinogenicity classification of 1 A/B or 2 (known/presumed or suspected human carcinogen), were divided into three groups: alkanes and alkenes, halogenated hydrocarbons, and polyaromatic hydrocarbons. The in silico data-mining revealed 87 and 44 genes commonly interacted with most of the investigated hydrocarbons are linked to breast and lung cancer, respectively. The dominant interactions among the common genes are co-expression, physical interaction, genetic interaction, co-localization, and interaction in shared protein domains. Among these genes, only 16 are common in the development of both cancers. Benzo(a)pyrene and tetrachlorodibenzodioxin interacted with all 16 genes. The molecular pathways potentially affected by the investigated hydrocarbons include aryl hydrocarbon receptor, chemical carcinogenesis, ferroptosis, fluid shear stress and atherosclerosis, interleukin 17 signaling pathway, lipid and atherosclerosis, NRF2 pathway, and oxidative stress response. CONCLUSIONS: Within the inherent limitations of in silico toxicogenomics tools, we elucidated the molecular pathways associated with breast and lung cancer development potentially affected by hydrocarbons mixture. Our findings indicate adaptive responses to oxidative stress and inflammatory damages are instrumental in the development of both cancers. Additionally, ferroptosis-a non-apoptotic programmed cell death driven by lipid peroxidation and iron homeostasis-was identified as a new player in these responses. Finally, AHR potential involvement in modulating IL-8, a critical gene that mediates breast cancer invasion and metastasis to the lungs, was also highlighted. A deeper understanding of the interplay between genes associated with these pathways, and other survival signaling pathways identified in this study, will provide invaluable knowledge in assessing the risk of inhalation exposure to hydrocarbons mixture. The findings offer insights into future in vivo and in vitro laboratory investigations that focus on inhalation exposure to the hydrocarbons mixture.

Cytochromes P450: Role in Carcinogenesis and Relevance to Cancers.

Cancer is a leading cause of mortality globally. Cytochrome P450 (CYP) enzymes play a pivotal role in the biotransformation of both endogenous and exogenous compounds. Various lines of evidence from epidemiological, animal, and clinical studies point to the instrumental role of CYPs in cancer initiation, metastasis, and prevention. Substantial research has found that CYPs are involved in activating different carcinogenic chemicals in the environment, such as polycyclic aromatic hydrocarbons and tobacco-related nitrosamines. Electrophilic intermediates produced from these chemicals can covalently bind to DNA, inducing mutation and cellular transformation that collectively result in cancer development. While bioactivation of procarcinogens and promutagens by CYPs has long been established, the role of CYP-derived endobiotics in carcinogenesis has only emerged in recent years. Eicosanoids derived from arachidonic acid via CYP oxidative pathways have been implicated in tumorigenesis, cancer progression and metastasis. The purpose of this review is to update the current state of knowledge about the molecular cancer mechanism involving CYPs with a focus on the biochemical and biotransformation mechanisms in the various CYP-mediated carcinogenesis and the role of CYP-derived reactive metabolites, from both external and endogenous sources, in cancer growth and tumor formation.

Cyp2a5 Promoter-based Gene Reporter Assay: A Novel Design of Cell-based Bioassay for Toxicity Prediction.

The murine cytochrome P450 2a5 (Cyp2a5) gene is regulated by complex interactions of various stress-activated transcription factors (TFs). Elevated Cyp2a5 transcription under chemical-induced stress conditions is achieved by interplay between the various TFs - including as aryl hydrocarbon receptor (AhR) and nuclear factor (erythroid-derived 2)-like 2 wild-type (Nrf2) - at the 'stress-responding' cluster of response elements on the Cyp2a5 promoter, as well as through mRNA stabilization mediated by interaction of the stress-activated heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) with the 3'-UTR of the CYP2A5 mRNA. We designed a unique toxicity pathway-based reporter assay to include regulatory regions from both the 5' and the 3' untranslated regions of Cyp2a5 in a luciferase reporter plasmid to reflect in vivo responses to chemical insult. Human breast cancer MCF-7 cells were stably transfected with pGL4.38-Cyp2a5_Wt3k (wild-type) or mutant - pGL4.38-Cyp2a5_StREMut and pGL4.38-Cyp2a5_XREMut - reporter gene to monitor chemical-induced cellular response mediated by AhR and Nrf2 signalling. The recombinant cells were treated with representative of AhR agonist, polycyclic aromatic hydrocarbons, brominated flame retardant, fluorosurfactant, aromatic organic compound and metal, to determine the sensitivity of the Cyp2a5 promoter-based gene reporter assays to chemical insults by measuring the LC50 and EC50 of the respective chemicals. The three assays are sensitive to sublethal cellular responses of chemicals, which is an ideal feature for toxicity pathway-based bioassay for toxicity prediction. The wild-type reporter responded well to chemicals that activate crosstalk between the AhR and Nrf2, whilst the mutant reporters effectively gauge cellular response driven by either Nrf2/StRE or AhR/XRE signalling. Thus, the three gene reporter assays could be used tandemly to determine the predominant toxicity pathway of a given compound.

Tumour suppressor protein p53 regulates the stress activated bilirubin oxidase cytochrome P450 2A6.

Human cytochrome P450 (CYP) 2A6 enzyme has been proposed to play a role in cellular defence against chemical-induced oxidative stress. The encoding gene is regulated by various stress activated transcription factors. This paper demonstrates that p53 is a novel transcriptional regulator of the gene. Sequence analysis of the CYP2A6 promoter revealed six putative p53 binding sites in a 3kb proximate promoter region. The site closest to transcription start site (TSS) is highly homologous with the p53 consensus sequence. Transfection with various stepwise deletions of CYP2A6-5'-Luc constructs--down to -160bp from the TSS--showed p53 responsiveness in p53 overexpressed C3A cells. However, a further deletion from -160 to -74bp, including the putative p53 binding site, totally abolished the p53 responsiveness. Electrophoretic mobility shift assay with a probe containing the putative binding site showed specific binding of p53. A point mutation at the binding site abolished both the binding and responsiveness of the recombinant gene to p53. Up-regulation of the endogenous p53 with benzo[α]pyrene--a well-known p53 activator--increased the expression of the p53 responsive positive control and the CYP2A6-5'-Luc construct containing the intact p53 binding site but not the mutated CYP2A6-5'-Luc construct. Finally, inducibility of the native CYP2A6 gene by benzo[α]pyrene was demonstrated by dose-dependent increases in CYP2A6 mRNA and protein levels along with increased p53 levels in the nucleus. Collectively, the results indicate that p53 protein is a regulator of the CYP2A6 gene in C3A cells and further support the putative cytoprotective role of CYP2A6.

Mitochondrial targeting of bilirubin regulatory enzymes: An adaptive response to oxidative stress.

The intracellular level of bilirubin (BR), an endogenous antioxidant that is cytotoxic at high concentrations, is tightly controlled within the optimal therapeutic range. We have recently described a concerted intracellular BR regulation by two microsomal enzymes: heme oxygenase 1 (HMOX1), essential for BR production and cytochrome P450 2A5 (CYP2A5), a BR oxidase. Herein, we describe targeting of these enzymes to hepatic mitochondria during oxidative stress. The kinetics of microsomal and mitochondrial BR oxidation were compared. Treatment of DBA/2J mice with 200mgpyrazole/kg/day for 3days increased hepatic intracellular protein carbonyl content and induced nucleo-translocation of Nrf2. HMOX1 and CYP2A5 proteins and activities were elevated in microsomes and mitoplasts but not the UGT1A1, a catalyst of BR glucuronidation. A CYP2A5 antibody inhibited 75% of microsomal BR oxidation. The inhibition was absent in control mitoplasts but elevated to 50% after treatment. An adrenodoxin reductase antibody did not inhibit microsomal BR oxidation but inhibited 50% of mitochondrial BR oxidation. Ascorbic acid inhibited 5% and 22% of the reaction in control and treated microsomes, respectively. In control mitoplasts the inhibition was 100%, which was reduced to 50% after treatment. Bilirubin affinity to mitochondrial and microsomal CYP2A5 enzyme is equally high. Lastly, the treatment neither released cytochrome c into cytoplasm nor dissipated membrane potential, indicating the absence of mitochondrial membrane damage. Collectively, the observations suggest that BR regulatory enzymes are recruited to mitochondria during oxidative stress and BR oxidation by mitochondrial CYP2A5 is supported by mitochondrial mono-oxygenase system. The induced recruitment potentially confers membrane protection.

Function and regulation of the Cyp2a5/CYP2A6 genes in response to toxic insults in the liver.

The mouse hepatic cytochrome P450 (CYP) 2A5 and its human orthologue CYP2A6 catalyse the metabolism of a number of drugs and toxins, such as halothane and aflatoxin B1. The enzymes are named "Coumarin 7-hydroxylase" and "Nicotine Hydroxylase", respectively, by virtue of their high affinity and specific activity towards these compounds. Bilirubin, the breakdown product of haem, has been suggested to be the endogenous substrate for both enzymes. Uniquely, CYP2A5 and CYP2A6 are induced during pathological conditions associated with liver injury when the function of most other CYP enzymes is compromised, which suggests an exceptional mode of regulation of the corresponding genes. Regulation of these genes is indeed complex where the promoters interact with multiple stress-activated transcription factors. The Cyp2a5 promoter contains a "stress-responding" cluster of binding motifs, which interact with major mediators of toxic insults including nuclear factor-E2 p45-related factor 2 (Nrf2) and aryl hydrocarbon receptor (AhR). These interactions are crucial in the up-regulation of the genes under stress conditions. Additionally, elevated transcription is also achieved through mRNA stabilisation mediated by interaction of the stress activated heterogenous ribonucleoprotein A1 (hnRNP A1) with the 3'UTR of the CYP2A5/CYP2A6 mRNA. The up-regulation via enhanced transcription combined with mRNA stabilisation, as seen in some of the stress situations, leads to a particularly strong, fast and persistent response. This review brings together knowledge obtained from studies in our laboratories and others' on regulation of Cyp2a5/CYP2A6 genes in response to toxic insults and toxicological significance of their catalytic activities that may provide clues to a functional role of the enzymes in relation to liver toxicity.

Metabolism of bilirubin by human cytochrome P450 2A6.

The mouse cytochrome P450 (CYP) 2A5 has recently been shown to function as hepatic "Bilirubin Oxidase" (Abu-Bakar, A., et al., 2011. Toxicol. Appl. Pharmacol. 257, 14-22). To date, no information is available on human CYP isoforms involvement in bilirubin metabolism. In this paper we provide novel evidence for human CYP2A6 metabolising the tetrapyrrole bilirubin. Incubation of bilirubin with recombinant yeast microsomes expressing the CYP2A6 showed that bilirubin inhibited CYP2A6-dependent coumarin 7-hydroxylase activity to almost 100% with an estimated K(i) of 2.23 µM. Metabolite screening by a high-performance liquid chromatography/electrospray ionisation mass spectrometry indicated that CYP2A6 oxidised bilirubin to biliverdin and to three other smaller products with m/z values of 301, 315 and 333. Molecular docking analyses indicated that bilirubin and its positively charged intermediate interacted with key amino acid residues at the enzyme's active site. They were stabilised at the site in a conformation favouring biliverdin formation. By contrast, the end product, biliverdin was less fitting to the active site with the critical central methylene bridge distanced from the CYP2A6 haem iron facilitating its release. Furthermore, bilirubin treatment of HepG2 cells increased the CYP2A6 protein and activity levels with no effect on the corresponding mRNA. Co-treatment with cycloheximide (CHX), a protein synthesis inhibitor, resulted in increased half-life of the CYP2A6 compared to cells treated only with CHX. Collectively, the observations indicate that the CYP2A6 may function as human "Bilirubin Oxidase" where bilirubin is potentially a substrate and a regulator of the enzyme.

Inducible bilirubin oxidase: a novel function for the mouse cytochrome P450 2A5.

We have previously shown that bilirubin (BR), a breakdown product of haem, is a strong inhibitor and a high affinity substrate of the mouse cytochrome P450 2A5 (CYP2A5). The antioxidant BR, which is cytotoxic at high concentrations, is potentially useful in cellular protection against oxygen radicals if its intracellular levels can be strictly controlled. The mechanisms that regulate cellular BR levels are still obscure. In this paper we provide preliminary evidence for a novel function of CYP2A5 as hepatic "BR oxidase". A high-performance liquid chromatography/electrospray ionisation mass spectrometry screening showed that recombinant yeast microsomes expressing the CYP2A5 oxidise BR to biliverdin, as the main metabolite, and to three other smaller products with m/z values of 301, 315 and 333. The metabolic profile is significantly different from that of chemical oxidation of BR. In chemical oxidation the smaller products were the main metabolites. This suggests that the enzymatic reaction is selective, towards biliverdin production. Bilirubin treatment of primary hepatocytes increased the CYP2A5 protein and activity levels with no effect on the corresponding mRNA. Co-treatment with cycloheximide (CHX), a protein synthesis inhibitor, resulted in increased half-life of the CYP2A5 compared to cells treated only with CHX. Collectively, the observations suggest that the CYP2A5 is potentially an inducible "BR oxidase" where BR may accelerate its own metabolism through stabilization of the CYP2A5 protein. It is possible that this metabolic pathway is potentially part of the machinery controlling intracellular BR levels in transient oxidative stress situations, in which high amounts of BR are produced.

Regulation of CYP2A5 gene by the transcription factor nuclear factor (erythroid-derived 2)-like 2.

We have previously shown that cadmium, a metal that alters cellular redox status, induces CYP2A5 expression in nuclear factor (erythroid-derived 2)-like 2 wild-type (Nrf2+/+) mice but not in the knockout (Nrf2-/-) mice. In the present studies, the potential role of Nrf2 in cadmium-mediated regulation of Cyp2a5 gene was investigated in mouse primary hepatocytes. Cadmium chloride (CdCl2) caused a time-dependent induction of the CYP2A5 at mRNA, protein, and activity levels, with a substantial increase observed within 3 h of exposure. Immunoblotting showed cadmium-dependent nuclear accumulation of Nrf2 within 1 h of exposure. Cotransfection of mouse primary hepatocytes with Cyp2a5 promoter-luciferase reporter plasmids and Nrf2 expression plasmid resulted in a 3-fold activation of Cyp2a5 promoter-mediated transcription relative to the control. Deletion analysis of the promoter localized the Nrf2 responsive region to an area from -2656 to -2339 base pair. Computer-based sequence analysis identified two putative stress response elements (StRE) within the region at positions -2514 to -2505 and -2386 to -2377. Chromatin immunoprecipitation and electrophoretic mobility shift assays showed that interaction of the more proximal StRE with Nrf2 was stimulated by CdCl2. Finally, site-directed mutagenesis of the proximal StRE in Cyp2a5 promoter-luciferase reporter plasmids abolished Nrf2-mediated induction. Collectively, the results indicate that Nrf2 activates Cyp2a5 transcription by directly binding to the StRE in the 5'-flanking region of the gene. This acknowledges Cyp2a5 as the first phase I xenobiotic-metabolizing gene identified under the control of the StRE-Nrf2 pathway with a potential role in adaptive response to cellular stress.

Balancing benefits of human genetic research against civic concerns: Essentially Yours and beyond - the case of Australia.

Large human genetic databases, especially those that are biomedical and forensic, have emerged since the completion of the Human Genome Project. However, this development has occurred in a time of intense public ambivalence to life science and genomics innovations. Controversies revolve around genetic modification, stem cell technologies and human genetic databases. Debate about databases focuses on how to balance the benefits from genetic research against civic concerns, typically, privacy and unfair discrimination and, more recently, public trust. In 1989, Australian jurisdictions began developing regulatory standards for human genetic databases but from the start these lacked uniformity and adequate scope. Enduring concerns led to a widescale public inquiry (2001-2003), which produced the Essentially Yours report. However, while the Australian government supports many of the report's recommendations, civic concerns remain as policy responses are checkered. In this special report, we reflect on the debate, the rise of the inquiry, its recommendations and policy responses, and competency and trust in regulation.

Evidence for induced microsomal bilirubin degradation by cytochrome P450 2A5.

Oxidative metabolism of bilirubin (BR) -- a breakdown product of haem with cytoprotective and toxic properties -- is an important route of detoxification in addition to glucuronidation. The major enzyme(s) involved in this oxidative degradation are not known. In this paper, we present evidence for a major role of the hepatic cytochrome P450 2A5 (Cyp2a5) in BR degradation during cadmium intoxication, where the BR levels are elevated following induction of haem oxygenase-1 (HO-1). Treatment of DBA/2J mice with CdCl(2) induced both the Cyp2a5 and HO-1, and increased the microsomal BR degradation activity. By contrast, the total cytochrome P450 (CYP) content and the expression of Cyp1a2 were down-regulated by the treatment. The induction of the HO-1 and Cyp2a5 was substantial at the mRNA, protein and enzyme activity levels. In each case, the up-regulation of HO-1 preceded that of Cyp2a5 with a 5-10h interval. BR totally inhibited the microsomal Cyp2a5-dependent coumarin hydroxylase activity, with an IC(50) approximately equal to the substrate concentration. The 7-methoxyresorufin 7-O-demethylase (MROD) activity, catalyzed mainly by the Cyp1a2, was inhibited up to 36% by BR. The microsomal BR degradation was inhibited by coumarin and a monoclonal antibody against the Cyp2a5 by about 90%. Furthermore, 7-methoxyresorufin, a substrate for the Cyp1a2, inhibited BR degradation activity by approximately 20%. In sum, the results strongly suggest a major role for Cyp2a5 in the oxidative degradation of BR. Secondly, the coordinated up-regulation of the HO-1 and Cyp2a5 during Cd-mediated injury implicates a network of enzyme systems in the maintenance of balancing BR production and elimination.

Acute cadmium chloride administration induces hepatic and renal CYP2A5 mRNA, protein and activity in the mouse: involvement of transcription factor NRF2.

Modulation of the cytochrome P450 (CYP) monooxygenase system by cadmium was investigated in male, adult DBA/2J mice treated with a single dose (16 micromol/kg body weight, i.p.) of cadmium chloride (CdCl2). Total CYP content of liver and kidney microsomes decreased maximally (56% and 85%, respectively) 24 and 18 h, respectively, after CdCl2 treatment. Progressive increases of hepatic coumarin 7-hydroxylase (COH) activity; indicative of CYP2A5 activity, relative to the total CYP content were seen at 8 h (2-fold), 12 h (3-fold), 18 h (12-fold), and 24 h (15-fold). Similar changes were seen in the kidney. Liver and kidney CYP2A5 mRNA levels increased maximally 12 and 4 h after treatment and decreased to almost half 6 h later. In contrast, kidney and liver CYP2A5 protein levels increased maximally at 18 and 24 h. The CYP2A5 mRNA levels in the kidney and liver increased after Cd treatment in Nrf2 +/+ but not in Nrf2 -/- mouse. This study demonstrates that hepatic and kidney CYP2A5 is upregulated by cadmium with a somewhat faster response in the kidney than the liver. The strong upregulation of the CYP2A5 both at mRNA and enzyme activity levels, with a simultaneous decrease in the total CYP concentration suggest an unusual mode of regulation of CYP2A5 in response to cadmium exposure, amongst the CYP enzymes. The observed decrease in the mRNA but not in protein levels after maximal induction may suggest involvement of post-transcriptional mechanisms in the regulation. Upregulation of CYP2A5 by cadmium in the Nrf2 +/+ mice but not in the Nrf2-/- mice indicates a role for this transcription factor in the regulation.