Publications by authors named "David H Phillips"

191 Publications

Mutagenicity of N-hydroxy-4-aminobiphenyl in human TP53 knock-in (Hupki) mouse embryo fibroblasts.

Environ Mol Mutagen 2021 Apr 15;62(4):252-264. Epub 2021 Mar 15.

Department of Analytical, Environmental and Forensic Sciences, MRC-PHE Centre for Environment and Health, King's College London, London, UK.

TP53 harbors somatic mutations in more than half of human tumors with some showing characteristic mutation spectra that have been linked to environmental exposures. In bladder cancer, a unique distribution of mutations amongst several codons of TP53 has been hypothesized to be caused by environmental carcinogens including 4-aminobiphenyl (4-ABP). 4-ABP undergoes metabolic activation to N-hydroxy-4-aminobiphenyl (N-OH-4-ABP) and forms pre-mutagenic adducts in DNA, of which N-(deoxyguanosin-8-yl)-4-ABP (dG-C8-4-ABP) is the major one. Human TP53 knock-in mouse embryo fibroblasts (HUFs) are a useful model to study the influence of environmental carcinogens on TP53-mutagenesis. By performing the HUF immortalization assay (HIMA) TP53-mutant HUFs are generated and mutations can be identified by sequencing. Here we studied the induction of mutations in human TP53 after treatment of primary HUFs with N-OH-4-ABP. In addition, mutagenicity in the bacterial lacZ reporter gene and the formation of dG-C8-4-ABP, measured by P-postlabelling analysis, were determined in N-OH-4-ABP-treated primary HUFs. A total of 6% TP53-mutants were identified after treatment with 40 μM N-OH-4-ABP for 24 hr (n = 150) with G>C/C>G transversion being the main mutation type. The mutation spectrum found in the TP53 gene of immortalized N-OH-4-ABP-treated HUFs was unlike the one found in human bladder cancer. DNA adduct formation (~40 adducts/10 nucleotides) was detected after 24 hr treatment with 40 μM N-OH-4-ABP, but lacZ mutagenicity was not observed. Adduct levels decreased substantially (sixfold) after a 24 hr recovery period indicating that primary HUFs can efficiently repair the dG-C8-4-ABP adduct possibly before mutations are fixed. In conclusion, the observed difference in the N-OH-4-ABP-induced TP53 mutation spectrum to that observed in human bladder tumors do not support a role of 4-ABP in human bladder cancer development.
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http://dx.doi.org/10.1002/em.22429DOI Listing
April 2021

In vitro mutagenicity of selected environmental carcinogens and their metabolites in MutaMouse FE1 lung epithelial cells.

Mutagenesis 2020 12;35(6):453-463

Department of Analytical, Environmental and Forensic Sciences, MRC-PHE Centre for Environment and Health, King's College London, London, UK.

Chemicals in commerce or under development must be assessed for genotoxicity; assessment is generally conducted using validated assays (e.g. Tk mouse lymphoma assay) as part of a regulatory process. Currently, the MutaMouse FE1 cell mutagenicity assay is undergoing validation for eventual use as a standard in vitro mammalian mutagenicity assay. FE1 cells have been shown to be metabolically competent with respect to some cytochrome P450 (CYP) isozymes; for instance, they can convert the human carcinogen benzo[a]pyrene into its proximate mutagenic metabolite. However, some contradictory results have been noted for other genotoxic carcinogens that require two-step metabolic activation (e.g. 2-acetylaminofluorene and 2-amino-3-methylimidazo[4,5-f]quinoxaline). Here, we examined three known or suspected human carcinogens, namely acrylamide, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 4-aminobiphenyl (4-ABP), together with their proximate metabolites (i.e. glycidamide, N-OH-PhIP and N-OH-4-ABP), to aid in the validation of the FE1 cell mutagenicity assay. Assessments of the parent compounds were conducted both in the presence and absence of an exogenous metabolic activation mixture S9; assessments of the metabolites were in the absence of S9. The most potent compound was N-OH-PhIP -S9, which elicited a mutant frequency (MF) level 5.3-fold over background at 5 µM. There was a 4.3-fold increase for PhIP +S9 at 5 µM, a 1.7-fold increase for glycidamide -S9 at 3.5 mM and a 1.5-fold increase for acrylamide +S9 at 4 mM. Acrylamide -S9 elicited a marginal 1.4-fold MF increase at 8 mM. Treatment with PhIP -S9, 4-ABP ±S9 and N-OH-4-ABP -S9 failed to elicit significant increases in lacZ MF with any of the treatment conditions tested. Gene expression of key CYP isozymes was quantified by RT-qPCR. Cyp1a1, 1a2 and 1b1 are required to metabolise PhIP and 4-ABP. Results showed that treatment with both compounds induced expression of Cyp1a1 and Cyp1b1 but not Cyp1a2. Cyp2e1, which catalyses the bioactivation of acrylamide to glycidamide, was not induced after acrylamide treatment. Overall, our results confirm that the FE1 cell mutagenicity assay has the potential for use alongside other, more traditional in vitro mutagenicity assays.
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http://dx.doi.org/10.1093/mutage/geaa032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7846080PMC
December 2020

Mutagenicity of 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP) in human TP53 knock-in (Hupki) mouse embryo fibroblasts.

Food Chem Toxicol 2021 Jan 12;147:111855. Epub 2020 Nov 12.

Department of Analytical, Environmental and Forensic Sciences, MRC-PHE Centre for Environment and Health, King's College London, London, SE1 9NH, UK.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a possible human carcinogen formed in cooked fish and meat. PhIP is bioactivated by cytochrome P450 enzymes to form 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP), a genotoxic metabolite that reacts with DNA leading to the mutation-prone DNA adduct N-(deoxyguanosin-8-yl)-PhIP (dG-C8-PhIP). Here, we studied N-OH-PhIP-induced whole genome mutagenesis in human TP53 knock-in (Hupki) mouse embryo fibroblasts (HUFs) immortalised and subjected to whole genome sequencing (WGS). In addition, mutagenicity of N-OH-PhIP in TP53 and the lacZ reporter gene were assessed. TP53 mutant frequency in HUF cultures treated with N-OH-PhIP (2.5 μM for 24 h, n = 90) was 10% while no TP53 mutations were found in untreated controls (DMSO for 24 h, n = 6). All N-OH-PhIP-induced TP53 mutations occurred at G:C base pairs with G > T/C > A transversions accounting for 58% of them. TP53 mutations characteristic of those induced by N-OH-PhIP have been found in human tumours including breast and colorectal, which are cancer types that have been associated with PhIP exposure. LacZ mutant frequency increased 25-fold at 5 μM N-OH-PHIP and up to ~350 dG-C8-PhIP adducts/10 nucleosides were detected by ultra-performance liquid chromatography-electrospray ionisation multistage scan mass spectrometry (UPLC-ESI-MS) at this concentration. In addition, a WGS mutational signature defined by G > T/C > A transversions was present in N-OH-PhIP-treated immortalised clones, which showed similarity to COSMIC SBS4, 18 and 29 signatures found in human tumours.
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http://dx.doi.org/10.1016/j.fct.2020.111855DOI Listing
January 2021

Mutagenicity of acrylamide and glycidamide in human TP53 knock-in (Hupki) mouse embryo fibroblasts.

Arch Toxicol 2020 12 4;94(12):4173-4196. Epub 2020 Sep 4.

Department of Analytical, Environmental and Forensic Sciences, MRC-PHE Centre for Environment and Health, King's College London, London, SE1 9NH, UK.

Acrylamide is a suspected human carcinogen formed during high-temperature cooking of starch-rich foods. It is metabolised by cytochrome P450 2E1 to its reactive metabolite glycidamide, which forms pre-mutagenic DNA adducts. Using the human TP53 knock-in (Hupki) mouse embryo fibroblasts (HUFs) immortalisation assay (HIMA), acrylamide- and glycidamide-induced mutagenesis was studied in the tumour suppressor gene TP53. Selected immortalised HUF clones were also subjected to next-generation sequencing to determine mutations across the whole genome. The TP53-mutant frequency after glycidamide exposure (1.1 mM for 24 h, n = 198) was 9% compared with 0% in cultures treated with acrylamide [1.5 (n = 24) or 3 mM (n = 6) for 48 h] and untreated vehicle (water) controls (n = 36). Most glycidamide-induced mutations occurred at adenines with A > T/T > A and A > G/T > C mutations being the most common types. Mutations induced by glycidamide occurred at specific TP53 codons that have also been found to be mutated in human tumours (i.e., breast, ovary, colorectal, and lung) previously associated with acrylamide exposure. The spectrum of TP53 mutations was further reflected by the mutations detected by whole-genome sequencing (WGS) and a distinct WGS mutational signature was found in HUF clones treated with glycidamide that was again characterised by A > G/T > C and A > T/T > A mutations. The WGS mutational signature showed similarities with COSMIC mutational signatures SBS3 and 25 previously found in human tumours (e.g., breast and ovary), while the adenine component was similar to COSMIC SBS4 found mostly in smokers' lung cancer. In contrast, in acrylamide-treated HUF clones, only culture-related background WGS mutational signatures were observed. In summary, the results of the present study suggest that glycidamide may be involved in the development of breast, ovarian, and lung cancer.
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http://dx.doi.org/10.1007/s00204-020-02878-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7655573PMC
December 2020

Visualisation tools for dependent peptide searches to support the exploration of in vitro protein modifications.

PLoS One 2020 8;15(7):e0235263. Epub 2020 Jul 8.

Department of Chemistry, Oregon State University, Corvallis, OR, United States of America.

Dependent peptide searching is a method for discovering covalently-modified peptides-and therefore proteins-in mass-spectrometry-based proteomics experiments. Being more permissive than standard search methods, it has the potential to discover novel modifications (e.g., post-translational modifications occurring in vivo, or modifications introduced in vitro). However, few studies have explored dependent peptide search results in an untargeted way. In the present study, we sought to evaluate dependent peptide searching as a means of characterising proteins that have been modified in vitro. We generated a model data set by analysing N-ethylmaleimide-treated bovine serum albumin, and performed dependent peptide searches using the popular MaxQuant software. To facilitate interpretation of the search results (hundreds of dependent peptides), we developed a series of visualisation tools (R scripts). We used the tools to assess the diversity of putative modifications in the albumin, and to pinpoint hypothesised modifications. We went on to explore the tools' generality via analyses of public data from studies of rat and human proteomes. Of 19 expected sites of modification (one in rat cofilin-1 and 18 across six different human plasma proteins), eight were found and correctly localised. Apparently, some sites went undetected because chemical enrichment had depleted necessary analytes (potential 'base' peptides). Our results demonstrate (i) the ability of the tools to provide accurate and informative visualisations, and (ii) the usefulness of dependent peptide searching for characterising in vitro protein modifications. Our model data are available via PRIDE/ProteomeXchange (accession number PXD013040).
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0235263PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7343161PMC
September 2020

Enhanced DNA adduct formation by benzo[a]pyrene in human liver cells lacking cytochrome P450 oxidoreductase.

Mutat Res 2020 04 22;852:503162. Epub 2020 Feb 22.

Department of Analytical, Environmental and Forensic Sciences, MRC-PHE Centre for Environment and Health, King's College London, London, SE1 9NH, United Kingdom; NIHR Health Protection Research Unit in Health Impact of Environmental Hazards, King's College London in Partnership With Public Health England and Imperial College London, London, SE1 9NH, United Kingdom. Electronic address:

Diet is a major source of human exposure to polycyclic aromatic hydrocarbons (PAHs), of which benzo[a]pyrene (BaP) is the most commonly studied and measured. BaP has been considered to exert its genotoxic effects after metabolic activation by cytochrome P450 (CYP) enzymes whose activity can be modulated by cytochrome P450 oxidoreductase (POR), the electron donor to CYP enzymes. Previous studies showed that BaP-DNA adduct formation was greater in the livers of Hepatic Reductase Null (HRN) mice, in which POR is deleted specifically in hepatocytes, than in wild-type (WT) mice. In the present study we used human hepatoma HepG2 cells carrying a knockout (KO) in the POR gene as a human in vitro model that can mimic the HRN mouse model. Treatment to BaP for up to 48 h caused similar cytotoxicity in POR KO and WT HepG2 cells. However, levels of BaP activation (i.e. BaP-7,8-dihydrodiol formation) were higher in POR KO HepG2 cells than in WT HepG2 cells after 48 h. This also resulted in substantially higher BaP-DNA adduct formation in POR KO HepG2 cells indicating that BaP metabolism is delayed in POR KO HepG2 cells thereby prolonging the effective exposure of cells to unmetabolized BaP. As was seen in the HRN mouse model, these results suggest that cytochrome b, another component of the mixed-function oxidase system, which can also serve as electron donor to CYP enzymes along with NADH:cytochrome b redutase, contributes to the bioactivation of BaP in POR KO HepG2 cells. Collectively, these findings indicate that CYPs play a more important role in BaP detoxication as opposed to activation.
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http://dx.doi.org/10.1016/j.mrgentox.2020.503162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7184669PMC
April 2020

50 years existence and active participation of EEMS (now EEMGS) in the scientific community: A driver of European and international scientific collaborations for the protection of the environment and human health from genome stressors.

Mutat Res 2020 Feb - Mar;850-851:503132. Epub 2020 Jan 28.

Laboratory for Cell Genetics, Department Biology, Faculty of Sciences and Bio-engineering Sciences, Vrije Universiteit Brussel, Belgium.

EEMS and its successor Society EEMGS have provided a dynamic and successful platform to stimulate research and exchanges among the different actors involved in the protection of the environment and of human health from exposure to genome stressors. It includes basic, translational and applied research projects. This was possible due to the enthusiasm, creativity and support of scientists convinced of the importance of these issues. In the future young scientists will take over with new questions, new challenges, new technologies, new discoveries and new applications. A major challenge is the ethical questions emerging from the impressive potential of present genetic technologies capable of impacting the evolution of nature and humankind. The EEMGS, where academics, regulators and industries meet, should play a central role in these aspects, in particular in support of primary prevention and the establishment of internationally recognized guidelines. Collaboration with colleagues and other teams are of great importance to establish a stimulating open dialogue on scientific questions. However the key issues remain to do careful and rigorous research; to use logic and background knowledge; to define adequate experimental designs; to provide transparency in the protocols; to check repeatability of the results and to combine several statistical approaches in the quest to get to the truth. Among the many challenges ahead, re-evaluation of some key fundamental questions is necessary, such as the interplay between genetics and epigenetics, the existence of specific germ cell mutagens or the identification of the mechanisms leading to mutagen induced diseases. Translational and applied research will further include the development of systemic biomonitoring protocols, if possible in a single biological sample, the redaction of internationally harmonized guidelines but also the organization of platforms between geneticists and physicians open to all actors in the field. The creation of an independent European center to assess risk from exposure to mutagens, in particular in the light of the problematic of global warming might be very helpful.
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http://dx.doi.org/10.1016/j.mrgentox.2020.503132DOI Listing
April 2020

P-Postlabeling Analysis of DNA Adducts.

Methods Mol Biol 2020 ;2102:291-302

Department of Analytical, Environmental & Forensic Sciences, MRC-PHE Centre for Environmental & Health, King's College London, London, UK.

P-Postlabeling analysis is an ultra-sensitive method for the detection of DNA adducts, such as those formed directly by the covalent binding of carcinogens and mutagens to bases in DNA, and other DNA lesions resulting from modification of bases by endogenous or exogenous agents (e.g., oxidative damage). The procedure involves four main steps: enzymatic digestion of DNA sample; enrichment of the adducts; radiolabeling of the adducts by T4 kinase-catalyzed transference of P-orthophosphate from [γ-P]ATP; chromatographic separation of labeled adducts, and detection and quantification by means of their radioactive decay. Using 10 μg of DNA or less, it is capable of detecting adduct levels as low as 1 adduct in 10-10 normal nucleotides. It is applicable to a wide range of investigations, including monitoring human exposure to environmental or occupational carcinogens, determining whether a chemical has genotoxic properties, analysis of the genotoxicity of complex mixtures, elucidation of the pathways of activation of carcinogens, and monitoring DNA repair.
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http://dx.doi.org/10.1007/978-1-0716-0223-2_16DOI Listing
October 2020

The Impact of p53 on Aristolochic Acid I-Induced Gene Expression In Vivo.

Int J Mol Sci 2019 Dec 6;20(24). Epub 2019 Dec 6.

Department of Analytical, Environmental and Forensic Sciences, MRC-PHE Centre for Environment and Health, King's College London, London SE1 9NH, UK.

Exposure to aristolochic acid (AA) is linked to kidney disease and urothelial cancer in humans. The major carcinogenic component of the AA plant extract is aristolochic acid I (AAI). The tumour suppressor p53 is frequently mutated in AA-induced tumours. We previously showed that p53 protects from AAI-induced renal proximal tubular injury, but the underlying mechanism(s) involved remain to be further explored. In the present study, we investigated the impact of p53 on AAI-induced gene expression by treating , and mice with 3.5 mg/kg body weight (bw) AAI daily for six days. The Clariom™ S Assay microarray was used to elucidate gene expression profiles in mouse kidneys after AAI treatment. Analyses in Qlucore Omics Explorer showed that gene expression in AAI-exposed kidneys is treatment-dependent. However, gene expression profiles did not segregate in a clear-cut manner according to genotype, hence further investigations were performed by pathway analysis with MetaCore™. Several pathways were significantly altered to varying degrees for AAI-exposed kidneys. Apoptotic pathways were modulated in kidneys; whereas oncogenic and pro-survival pathways were significantly altered for and kidneys, respectively. Alterations of biological processes by AAI in mouse kidneys could explain the mechanisms by which p53 protects from or p53 loss drives AAI-induced renal injury in vivo.
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http://dx.doi.org/10.3390/ijms20246155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6940885PMC
December 2019

Characterising Mutational Spectra of Carcinogens in the Tumour Suppressor Gene Using Human Knock-in (Hupki) Mouse Embryo Fibroblasts.

Methods Protoc 2019 Nov 13;2(4). Epub 2019 Nov 13.

Department of Analytical, Environmental and Forensic Sciences, King's College London, London SE1 9NH, UK.

DNA in dividing cells is prone to mutagenesis, with mutations making key contributions to human disease including cancer. The tumour suppressor gene is the most frequently mutated gene in human tumours. Here, we present a robust protocol for studying mutagenesis utilising human knock-in (Hupki) mouse embryonic fibroblasts (HUFs). In the HUF immortalisation assay (HIMA), primary HUFs are treated with known or suspected carcinogens at 3% oxygen and then transferred to 20% atmospheric oxygen to induce senescence. Cells containing mutations (e.g., in ) that allow bypassing of senescence eventually emerge as immortalised clonal cell lines after 2-3 months of serial passaging. As not all immortalised HUF cells contain mutations, we developed a Nutlin-3a counter-screen to select for -mutated clones prior to sequencing. mutation spectra generated can be compared with those of human tumours recorded in the International Agency for Research on Cancer TP53 mutation database. Environmental mutagens that have demonstrated and validated the utility of the HIMA include ultraviolet radiation, aristolochic acid, and benzo[]pyrene. The mutation patterns induced by these mutagens in the HIMA corresponded to those found in human tumours from patients exposed to these mutagens. The approach presented helps to deepen our understanding of human cancer aetiology.
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http://dx.doi.org/10.3390/mps2040085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6961128PMC
November 2019

Deletion of cytochrome P450 oxidoreductase enhances metabolism and DNA adduct formation of benzo[a]pyrene in Hepa1c1c7 cells.

Mutagenesis 2019 12;34(5-6):413-420

Department of Analytical, Environmental and Forensic Sciences, MRC-PHE Centre for Environment and Health, King's College London, London, UK.

The environmental carcinogen benzo[a]pyrene (BaP) is presumed to exert its genotoxic effects after metabolic activation by cytochrome P450 (CYP) enzymes. However, studies using the Hepatic Reductase Null (HRN) mouse model, in which cytochrome P450 oxidoreductase (POR), the electron donor to CYP enzymes, is deleted specifically in hepatocytes, have shown that loss of hepatic POR-mediated CYP function leads to greater BaP-DNA adduct formation in livers of these mice than in wild-type (WT) mice. Here, we used CRISPR/Cas9 technology to knockout (KO) POR expression in mouse hepatoma Hepa1c1c7 cells to create an in vitro model that can mimic the HRN mouse model. Western blotting confirmed the deletion of POR in POR KO Hepa1c1c7 cells whereas expression of other components of the mixed-function oxidase system including cytochrome b5 (Cyb5) and NADH:cytochrome b5 reductase (which can also serve as electron donors to CYP enzymes), and CYP1A1 was similar in BaP-exposed WT and POR KO Hepa1c1c7 cells. BaP exposure caused cytotoxicity in WT Hepa1c1c7 cells but not in POR KO Hepa1c1c7 cells. In contrast, CYP-catalysed BaP-DNA adduct levels were ~10-fold higher in POR KO Hepa1c1c7 cells than in WT Hepa1c1c7 cells, in concordance with the presence of higher levels of BaP metabolite (e.g. BaP-7,8-dihydrodiol) in the medium of cultured BaP-exposed POR KO Hepa1c1c7 cells. As was seen in the HRN mouse model, these results suggest that Cyb5 contributes to the bioactivation of BaP in POR KO Hepa1c1c7 cells. These results indicate that CYP enzymes may play a more important role in the detoxication of BaP, as opposed to its bioactivation.
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http://dx.doi.org/10.1093/mutage/gez033DOI Listing
December 2019

The impact of p53 on aristolochic acid I-induced nephrotoxicity and DNA damage in vivo and in vitro.

Arch Toxicol 2019 11 10;93(11):3345-3366. Epub 2019 Oct 10.

Department of Analytical, Environmental and Forensic Sciences, MRC-PHE Centre for Environment and Health, King's College London, London, SE1 9NH, UK.

Exposure to aristolochic acid (AA) is associated with human nephropathy and urothelial cancer. The tumour suppressor TP53 is a critical gene in carcinogenesis and frequently mutated in AA-induced urothelial tumours. We investigated the impact of p53 on AAI-induced nephrotoxicity and DNA damage in vivo by treating Trp53(+/+), Trp53(+/-) and Trp53(-/-) mice with 3.5 mg/kg body weight (bw) AAI daily for 2 or 6 days. Renal histopathology showed a gradient of intensity in proximal tubular injury from Trp53(+/+) to Trp53(-/-) mice, especially after 6 days. The observed renal injury was supported by nuclear magnetic resonance (NMR)-based metabonomic measurements, where a consistent Trp53 genotype-dependent trend was observed for urinary metabolites that indicate aminoaciduria (i.e. alanine), lactic aciduria (i.e. lactate) and glycosuria (i.e. glucose). However, Trp53 genotype had no impact on AAI-DNA adduct levels, as measured by P-postlabelling, in either target (kidney and bladder) or non-target (liver) tissues, indicating that the underlying mechanisms of p53-related AAI-induced nephrotoxicity cannot be explained by differences in AAI genotoxicity. Performing gas chromatography-mass spectrometry (GC-MS) on kidney tissues showed metabolic pathways affected by AAI treatment, but again Trp53 status did not clearly impact on such metabolic profiles. We also cultured primary mouse embryonic fibroblasts (MEFs) derived from Trp53(+/+), Trp53(+/-) and Trp53(-/-) mice and exposed them to AAI in vitro (50 µM for up to 48 h). We found that Trp53 genotype impacted on the expression of NAD(P)H:quinone oxidoreductase (Nqo1), a key enzyme involved in AAI bioactivation. Nqo1 induction was highest in Trp53(+/+) MEFs and lowest in Trp53(-/-) MEFs; and it correlated with AAI-DNA adduct formation, with lowest adduct levels being observed in AAI-exposed Trp53(-/-) MEFs. Overall, our results clearly demonstrate that p53 status impacts on AAI-induced renal injury, but the underlying mechanism(s) involved remain to be further explored. Despite the impact of p53 on AAI bioactivation and DNA damage in vitro, such effects were not observed in vivo.
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http://dx.doi.org/10.1007/s00204-019-02578-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6823306PMC
November 2019

Relationships between airborne pollutants, serum albumin adducts and short-term health outcomes in an experimental crossover study.

Chemosphere 2020 Jan 24;239:124667. Epub 2019 Aug 24.

MRC-PHE Centre for Environment & Health, Department of Analytical, Environmental & Forensic Sciences, School of Population Health & Environmental Sciences, Faculty of Life Sciences & Medicine, King's College London, Franklin-Wilkins Building, 150 Stamford Street, London, SE1 9NH, UK. Electronic address:

Exposure to air pollution can have both short-term and long-term effects on health. However, the relationships between specific pollutants and their effects can be obscured by characteristics of both the pollution and the exposed population. One way of elucidating the relationships is to link exposures and internal changes at the level of the individual. To this end, we combined personal exposure monitoring (59 individuals, Oxford Street II crossover study) with mass-spectrometry-based analyses of putative serum albumin adducts (fixed-step selected reaction monitoring). We attempted to infer adducts' identities using data from another, higher-resolution mass spectrometry method, and were able to detect a semi-synthetic standard with both methods. A generalised least squares regression method was used to test for associations between amounts of adducts and pollution measures (ambient concentrations of nitrogen dioxide and particulate matter), and between amounts of adducts and short-term health outcomes (measures of lung health and arterial stiffness). Amounts of some putative adducts (e.g., one with a positive mass shift of ∼143 Da) were associated with exposure to pollution (11 associations), and amounts of other adducts were associated with health outcomes (eight associations). Adducts did not appear to provide a link between exposures and short-term health outcomes.
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http://dx.doi.org/10.1016/j.chemosphere.2019.124667DOI Listing
January 2020

Protein Adductomics: Analytical Developments and Applications in Human Biomonitoring.

Toxics 2019 May 25;7(2). Epub 2019 May 25.

Environmental Research Group, Department of Analytical, Environmental and Forensic Science, School of Population Health and Environmental Sciences, King's College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, UK.

Proteins contain many sites that are subject to modification by electrophiles. Detection and characterisation of these modifications can give insights into environmental agents and endogenous processes that may be contributing factors to chronic human diseases. An untargeted approach, utilising mass spectrometry to detect modified amino acids or peptides, has been applied to blood proteins haemoglobin and albumin, focusing in particular on the -terminal valine residue of haemoglobin and the cysteine-34 residue in albumin. Technical developments to firstly detect simultaneously multiple adducts at these sites and then subsequently to identify them are reviewed here. Recent studies in which the methods have been applied to biomonitoring human exposure to environmental toxicants are described. With advances in sensitivity, high-throughput handling of samples and robust quality control, these methods have considerable potential for identifying causes of human chronic disease and of identifying individuals at risk.
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http://dx.doi.org/10.3390/toxics7020029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6631498PMC
May 2019

Impact of p53 function on the sulfotransferase-mediated bioactivation of the alkylated polycyclic aromatic hydrocarbon 1-hydroxymethylpyrene in vitro.

Environ Mol Mutagen 2019 10 4;60(8):752-758. Epub 2019 Jun 4.

Department of Analytical, Environmental and Forensic Sciences, MRC-PHE Centre for Environment and Health, King's College London, London, United Kingdom.

The tumor suppressor p53, encoded by TP53, is known as the "guardian of the genome." Sulfotransferases (SULTs) are involved in the metabolism of alkylated polycyclic aromatic hydrocarbons such as 1-hydroxymethylpyrene (1-HMP), which is a known substrate for SULT1A1. To investigate the impact of TP53 on the metabolic activation of 1-HMP, a panel of isogenic human colorectal HCT116 cells having TP53(+/+), TP53(+/-), or TP53(-/-) were treated with 10 μM 1-HMP for 24 hr. 1-HMP-DNA adduct formation was determined by ultraperformance liquid chromatography-tandem mass spectrometry analysis, which quantified two nucleoside adducts N -(1-methylpyrenyl)-2'-deoxyguanosine and N -(1-methylpyrenyl)-2'-deoxyadenosine. 1-HMP treatment resulted in significantly (~40-fold) higher DNA adduct levels in TP53(+/+) cells than in the other cell lines. Higher levels of 1-HMP-induced DNA adducts in TP53(+/+) cells correlated with higher basal expression of SULT1A1/3 in this cell line, but 1-HMP treatment showed no effect on the expression of this protein. These results indicate that the cellular TP53 status is linked to the SULT1A1/3-mediated bioactivation of 1-HMP, thereby broadening the spectrum of p53's targets. Environ. Mol. Mutagen., 60:752-758, 2019. © 2019 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/em.22299DOI Listing
October 2019

A Compendium of Mutational Signatures of Environmental Agents.

Cell 2019 05 11;177(4):821-836.e16. Epub 2019 Apr 11.

Academic Department of Medical Genetics, School of Clinical Medicine, University of Cambridge, Cambridge CB2 9NB, UK; MRC Cancer Unit, University of Cambridge, Cambridge CB2 0XZ, UK; Wellcome Trust Sanger Institute, Hinxton CB10 1SA, UK. Electronic address:

Whole-genome-sequencing (WGS) of human tumors has revealed distinct mutation patterns that hint at the causative origins of cancer. We examined mutational signatures in 324 WGS human-induced pluripotent stem cells exposed to 79 known or suspected environmental carcinogens. Forty-one yielded characteristic substitution mutational signatures. Some were similar to signatures found in human tumors. Additionally, six agents produced double-substitution signatures and eight produced indel signatures. Investigating mutation asymmetries across genome topography revealed fully functional mismatch and transcription-coupled repair pathways. DNA damage induced by environmental mutagens can be resolved by disparate repair and/or replicative pathways, resulting in an assortment of signature outcomes even for a single agent. This compendium of experimentally induced mutational signatures permits further exploration of roles of environmental agents in cancer etiology and underscores how human stem cell DNA is directly vulnerable to environmental agents. VIDEO ABSTRACT.
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http://dx.doi.org/10.1016/j.cell.2019.03.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6506336PMC
May 2019

Application of hepatic cytochrome b/P450 reductase null (HBRN) mice to study the role of cytochrome b in the cytochrome P450-mediated bioactivation of the anticancer drug ellipticine.

Toxicol Appl Pharmacol 2019 03 25;366:64-74. Epub 2019 Jan 25.

Department of Analytical, Environmental and Forensic Sciences, MRC-PHE Centre for Environment and Health, King's College London, London, United Kingdom. Electronic address:

The anticancer drug ellipticine exerts its genotoxic effects after metabolic activation by cytochrome P450 (CYP) enzymes. The present study has examined the role of cytochrome P450 oxidoreductase (POR) and cytochrome b (Cyb5), electron donors to P450 enzymes, in the CYP-mediated metabolism and disposition of ellipticine in vivo. We used Hepatic Reductase Null (HRN) and Hepatic Cytochrome b/P450 Reductase Null (HBRN) mice. HRN mice have POR deleted specifically in hepatocytes; HBRN mice also have Cyb5 deleted in the liver. Mice were treated once with 10 mg/kg body weight ellipticine (n = 4/group) for 24 h. Ellipticine-DNA adduct levels measured by P-postlabelling were significantly lower in HRN and HBRN livers than in wild-type (WT) livers; however no significant difference was observed between HRN and HBRN livers. Ellipticine-DNA adduct formation in WT, HRN and HBRN livers correlated with Cyp1a and Cyp3a enzyme activities measured in hepatic microsomes in the presence of NADPH confirming the importance of P450 enzymes in the bioactivation of ellipticine in vivo. Hepatic microsomal fractions were also utilised in incubations with ellipticine and DNA in the presence of NADPH, cofactor for POR, and NADH, cofactor for Cyb5 reductase (Cyb5R), to examine ellipticine-DNA adduct formation. With NADPH adduct formation decreased as electron donors were lost which correlated with the formation of the reactive metabolites 12- and 13-hydroxy-ellipticine in hepatic microsomes. No difference in adduct formation was observed in the presence of NADH. Our study demonstrates that Cyb5 contributes to the P450-mediated bioactivation of ellipticine in vitro, but not in vivo.
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http://dx.doi.org/10.1016/j.taap.2019.01.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6382462PMC
March 2019

The potential of omics approaches to elucidate mechanisms of biodiesel-induced pulmonary toxicity.

Part Fibre Toxicol 2019 01 8;16(1). Epub 2019 Jan 8.

Department of Analytical, Environmental and Forensic Sciences, MRC-PHE Centre for Environment & Health, School of Population Health and Environmental Sciences, Franklin-Wilkins Building, King's College London, London, SE1 9NH, UK.

Background: Combustion of biodiesels in place of fossil diesel (FD) has been proposed as a method of reducing transport-related toxic emissions in Europe. While biodiesel exhaust (BDE) contains fewer hydrocarbons, total particulates and carbon monoxide than FD exhaust (FDE), its high nitrogen oxide and ultrafine particle content may still promote pulmonary pathophysiologies.

Main Body: Using a complement of in vitro and in vivo studies, this review documents progress in our understanding of pulmonary responses to BDE exposure. Focusing initially on hypothesis-driven, targeted analyses, the merits and limitations of comparing BDE-induced responses to those caused by FDE exposure are discussed within the contexts of policy making and exploration of toxicity mechanisms. The introduction and progression of omics-led workflows are also discussed, summarising the novel insights into mechanisms of BDE-induced toxicity that they have uncovered. Finally, options for the expansion of BDE-related omics screens are explored, focusing on the mechanistic relevance of metabolomic profiling and offering rationale for expansion beyond classical models of pulmonary exposure.

Conclusion: Together, these discussions suggest that molecular profiling methods have identified mechanistically informative, novel and fuel-specific signatures of pulmonary responses to biodiesel exhaust exposure that would have been difficult to detect using traditional, hypothesis driven approaches alone.
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http://dx.doi.org/10.1186/s12989-018-0284-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6504167PMC
January 2019

Bulky DNA adducts, microRNA profiles, and lipid biomarkers in Norwegian tunnel finishing workers occupationally exposed to diesel exhaust.

Occup Environ Med 2019 01 13;76(1):10-16. Epub 2018 Nov 13.

Section for Toxicology and Biological Work Environment, Department of Chemical and Biological Work Environment, National Institute of Occupational Health, Oslo, Norway.

Objectives: This study aimed to assess the biological impact of occupational exposure to diesel exhaust (DE) including DE particles (DEP) from heavy-duty diesel-powered equipment in Norwegian tunnel finishing workers (TFW).

Methods: TFW (n=69) and referents (n=69) were investigated for bulky DNA adducts (by P-postlabelling) and expression of microRNAs (miRNAs) (by small RNA sequencing) in peripheral blood mononuclear cells (PBMC), as well as circulating free arachidonic acid (AA) and eicosanoid profiles in plasma (by liquid chromatography-tandem mass spectrometry).

Results: PBMC from TFW showed significantly higher levels of DNA adducts compared with referents. Levels of DNA adducts were also related to smoking habits. Seventeen miRNAs were significantly deregulated in TFW. Several of these miRNAs are related to carcinogenesis, apoptosis and antioxidant effects. Analysis of putative miRNA-gene targets revealed deregulation of pathways associated with cancer, alterations in lipid molecules, steroid biosynthesis and cell cycle. Plasma profiles showed higher levels of free AA and 15-hydroxyeicosatetraenoic acid, and lower levels of prostaglandin D and 9-hydroxyoctadecadienoic acid in TFW compared with referents.

Conclusion: Occupational exposure to DE/DEP is associated with biological alterations in TFW potentially affecting lung homoeostasis, carcinogenesis, inflammation status and the cardiovascular system. Of particular importance is the finding that tunnel finishing work is associated with an increased level of DNA adducts formation in PBMC.
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http://dx.doi.org/10.1136/oemed-2018-105445DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6327869PMC
January 2019

Mutational spectra and mutational signatures: Insights into cancer aetiology and mechanisms of DNA damage and repair.

Authors:
David H Phillips

DNA Repair (Amst) 2018 11 24;71:6-11. Epub 2018 Aug 24.

MRC-PHE Centre for Environment and Health, King's College London, UK; NIHR Health Protection Research Unit in Health Impact of Environmental Hazards at King's College London in Partnership with Public Health England, Department of Analytical, Environmental and Forensic Sciences, School of Public Health and Environmental Sciences, Faculty of Life Sciences and Medicine, King's College London, UK. Electronic address:

Reporter gene assays, in which a single mutation from each experiment can contribute to the assembly of a mutation spectrum for an agent, have provided the basis for understanding the mutational processes induced by mutagenic agents and for providing clues to the origins of mutations in human tumours. More recently exome and whole genome sequencing of human tumours has revealed distinct patterns of mutation that could provide additional clues for the causative origins of cancer. This can be tested by examining the mutational signatures induced in experimental systems by putative cancer-causing agents. Such signatures are now being generated in vitro in a number of different mutagen-exposed cellular systems. Results reveal that mutagens induce characteristic mutation signatures that, in some cases, match signatures found in human tumours. Proof of principle has been established with mutational signatures generated by simulated sunlight and aristolochic acid, which match those signatures found in human melanomas and urothelial cancers, respectively. In an analysis of somatic mutations in cancers for which tobacco smoking confers an elevated risk, it was found that smoking is associated with increased mutation burdens of multiple different mutational signatures, which contribute to different extents in different tissues. One of these signatures, mainly found in tissues directly exposed to tobacco smoke, is attributable to misreplication of DNA damage caused by tobacco carcinogens. Others likely reflect indirect activation of DNA editing by APOBEC cytidine deaminases and of an endogenous clock-like mutational process. The results are consistent with the proposition that smoking increases cancer risk by increasing the somatic mutation load although direct evidence for this mechanism is lacking in some cancer types. Thus, next generation sequencing of exomes or whole genomes is providing new insights into processes underlying the causes of human cancer.
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http://dx.doi.org/10.1016/j.dnarep.2018.08.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6219445PMC
November 2018

The impact of p53 function on the metabolic activation of the carcinogenic air pollutant 3-nitrobenzanthrone and its metabolites 3-aminobenzanthrone and N-hydroxy-3-aminobenzanthrone in human cells.

Mutagenesis 2018 10;33(4):311-321

Department of Analytical, Environmental and Forensic Sciences, MRC-PHE Centre for Environment and Health, King's College London, London, UK.

The tumour suppressor p53, encoded by TP53, is a key player in a wide network of signalling pathways. We investigated its role in the bioactivation of the environmental carcinogen 3-nitrobenzanthrone (3-NBA)found in diesel exhaust and its metabolites 3-aminobenzanthrone (3-ABA) and N-hydroxy-3-aminobenzanthrone (N-OH-3-ABA) in a panel of isogenic human colorectal HCT116 cells differing only with respect to their TP53 status [i.e. TP53(+/+), TP53(+/-), TP53(-/-), TP53(R248W/+) or TP53(R248W/-)]. As a measure of metabolic competence, DNA adduct formation was determined using 32P-postlabelling. Wild-type (WT) p53 did not affect the bioactivation of 3-NBA; no difference in DNA adduct formation was observed in TP53(+/+), TP53(+/-) and TP53(-/-) cells. Bioactivation of both metabolites 3-ABA and N-OH-3-ABA on the other hand was WT-TP53 dependent. Lower 3-ABA- and N-OH-3-ABA-DNA adduct levels were found in TP53(+/-) and TP53(-/-) cells compared to TP53(+/+) cells, and p53's impact was attributed to differences in cytochrome P450 (CYP) 1A1 expression for 3-ABA whereas for N-OH-3-ABA, an impact of this tumour suppressor on sulphotransferase (SULT) 1A1/3 expression was detected. Mutant R248W-p53 protein function was similar to or exceeded the ability of WT-p53 in activating 3-NBA and its metabolites, measured as DNA adducts. However, identification of the xenobiotic-metabolising enzyme(s) (XMEs), through which mutant-p53 regulates these responses, proved difficult to decipher. For example, although both mutant cell lines exhibited higher CYP1A1 induction after 3-NBA treatment compared to TP53(+/+) cells, 3-NBA-derived DNA adduct levels were only higher in TP53(R248W/-) cells but not in TP53(R248W/+) cells. Our results show that p53's influence on carcinogen activation depends on the agent studied and thereby on the XMEs that mediate the bioactivation of that particular compound. The phenomenon of p53 regulating CYP1A1 expression in human cells is consistent with other recent findings; however, this is the first study highlighting the impact of p53 on sulphotransferase-mediated (i.e. SULT1A1) carcinogen metabolism in human cells.
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http://dx.doi.org/10.1093/mutage/gey025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6180618PMC
October 2018

Hepatic DNA damage in harbour porpoises (Phocoena phocoena) stranded along the English and Welsh coastlines.

Environ Mol Mutagen 2018 08 3;59(7):613-624. Epub 2018 Jul 3.

Department of Analytical, Environmental and Forensic Sciences, MRC-PHE Centre for Environment and Health, King's College London, London, United Kingdom.

One level at which persistent organic pollutants (POPs) and polycyclic aromatic hydrocarbons PAHs) can exert damage is by causing DNA strand-breaks or nucleotide base modifications, which, if unrepaired, can lead to embryonic mutations, abnormal development and cancer. In marine ecosystems, genotoxicity is expected to be particularly strong in long-lived apex predators due to pollutant bioaccumulation. We conducted P-postlabeling analyses optimized for the detection and quantification of aromatic/hydrophobic DNA adducts in the livers of 40 sexually-mature North Atlantic harbour porpoises (Phocoena phocoena) stranded along the English and Welsh coastlines. We examined hepatic tissue to search for inflammatory and preneoplastic lesions and examine their association with adduct levels. Adducts were found in all porpoises (mean: 17.56 ± 11.95 per 10 nucleotides), and were higher than levels reported for marine vertebrates from polluted sites. The pollutants causing the induced DNA adducts could not be further characterized. Hepatic DNA damage did not correlate with levels of blubber POP concentrations (including total polychlorinated biphenyl [PCBs], dichlorodiphenyltrichloroethane [DDT] and dieldrin); PAH concentrations were not available for the present study. However, DNA damage predicted occurrence of inflammatory and preneoplastic lesions. Further, our data showed a reduction in hepatic DNA adduct levels with age in the 40 animals examined while POP concentrations, particularly PCBs, increased with age. Using a different dataset of 145 mature male harbour porpoises confirmed that higher contaminant levels (total PCBs, DDT and dieldrin) are found in older animals. The reduction in hepatic DNA adduct levels in older animals was in accordance with other studies which show that suppression of hepatic CYP1A enzyme activity at high PCB concentrations might impact on CYP1A-mediated DNA adduct formation of PAHs which are ubiquitous environmental pollutants and readily metabolized by CYP1A to species binding to DNA. In summary, our study shows that pollutant-induced DNA damage is prevalent in harbour porpoises from UK waters and may lead to detectable sub-lethal hepatic damage. Environ. Mol. Mutagen. 59:613-624, 2018. © 2018 The Authors Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.
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http://dx.doi.org/10.1002/em.22205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6174976PMC
August 2018

The role of cytochrome P450 enzymes in carcinogen activation and detoxication: an in vivo-in vitro paradox.

Carcinogenesis 2018 07;39(7):851-859

Department of Analytical, Environmental and Forensic Sciences, MRC-PHE Centre for Environment and Health, King's College London, Franklin-Wilkins Building, London, UK.

Many chemical carcinogens require metabolic activation via xenobiotic-metabolizing enzymes in order to exert their genotoxic effects. Evidence from numerous in-vitro studies, utilizing reconstituted systems, microsomal fractions and cultured cells, implicates cytochrome P450 enzymes as being the predominant enzymes responsible for the metabolic activation of many procarcinogens. With the development of targeted gene disruption methodologies, knockout mouse models have been generated that allow investigation of the in-vivo roles of P450 enzymes in the metabolic activation of carcinogens. This review covers studies in which five procarcinogens representing different chemical classes, benzo[a]pyrene, 4-aminobiphenyl (4-ABP), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, 2-amino-9H-pyrido[2,3-b]indole and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, have been administered to different P450 knockout mouse models. Paradoxically, while in-vitro studies using subcellular fractions enriched with P450 enzymes and their cofactors have been widely used to determine the pathways of activation of carcinogens, there is evidence from the in-vivo studies of cases where these same enzyme systems appear to have a more predominant role in carcinogen detoxication rather than activation.
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http://dx.doi.org/10.1093/carcin/bgy058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6124610PMC
July 2018

A novel method for source-specific hemoglobin adducts of nitro-polycyclic aromatic hydrocarbons.

Environ Sci Process Impacts 2018 Mar 21. Epub 2018 Mar 21.

Columbia Center for Children's Environmental Health, Department of Environmental Health Sciences, Mailman School of Public Health, Columbia University, 722 W. 168th St., 12th Floor, New York, NY 10032, USA.

Polycyclic aromatic hydrocarbons (PAH) are ubiquitous air pollutants associated with negative impacts on growth, development and behavior in children. Source-specific biological markers of PAH exposure are needed for targeting interventions to protect children. Nitro-derivatives of PAH can act as markers of exposure to diesel exhaust, gasoline exhaust, or general combustion sources. Using a novel HPLC-APCI-MS/MS detection method, we examined four hemoglobin (Hb) adducts of nitro-PAH metabolites and the Hb adduct of a benzo[a]pyrene (BaP) metabolite in 22 umbilical cord blood samples. The samples were collected from a birth cohort with comprehensive data on prenatal PAH exposure, including prenatal personal air monitoring and DNA adducts in maternal and umbilical cord blood. Using non-parametric analyses, heat maps, and principal component analysis (PCA), we analyzed the relationship between the five Hb adducts and previous PAH measurements, with each measurement representing a different duration of exposure. We found that Hb adducts derived from several diesel-related nitro-PAHs (2-nitrofluorene and 1-nitropyrene) were significantly correlated (r = 0.77, p ≤ 0.0001) and grouped together in PCA. Nitro-PAH derived Hb adducts were largely unrelated to previously collected measures of exposure to a number of PAH parent compounds. These measures need to be validated in a larger sample.
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http://dx.doi.org/10.1039/C7EM00522ADOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6150855PMC
March 2018

Erratum to "Gene expression profiles modulated by the human carcinogen aristolochic acid I in human cancer cells and their dependence on TP53" [Toxicol. Appl. Pharmacol. 232(1) (2008) 86-98].

Toxicol Appl Pharmacol 2018 04 20;344:75. Epub 2018 Feb 20.

Section of Molecular Carcinogenesis, Institute of Cancer Research, Brookes Lawley Building, Cotswold Road, Sutton, Surrey SM2 5NG, UK. Electronic address:

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http://dx.doi.org/10.1016/j.taap.2018.02.010DOI Listing
April 2018

The impact of chemotherapeutic drugs on the CYP1A1-catalysed metabolism of the environmental carcinogen benzo[a]pyrene: Effects in human colorectal HCT116 TP53(+/+), TP53(+/-) and TP53(-/-) cells.

Toxicology 2018 04 19;398-399:1-12. Epub 2018 Feb 19.

Department of Analytical, Environmental and Forensic Sciences, MRC-PHE Centre for Environment and Health, King's College London, 150 Stamford Street, London SE1 9NH, United Kingdom; NIHR Health Protection Research Unit in Health Impact of Environmental Hazards at King's College London in partnership with Public Health England, London, 150 Stamford Street, London SE1 9NH, United Kingdom. Electronic address:

Polycyclic aromatic hydrocarbons such as benzo[a]pyrene (BaP) can induce cytochrome P450 1A1 (CYP1A1) via a p53-dependent mechanism. The effect of different p53-activating chemotherapeutic drugs on CYP1A1 expression, and the resultant effect on BaP metabolism, was investigated in a panel of isogenic human colorectal HCT116 cells with differing TP53 status. Cells that were TP53(+/+), TP53(+/-) or TP53(-/-) were treated for up to 48 h with 60 μM cisplatin, 50 μM etoposide or 5 μM ellipticine, each of which caused high p53 induction at moderate cytotoxicity (60-80% cell viability). We found that etoposide and ellipticine induced CYP1A1 in TP53(+/+) cells but not in TP53(-/-) cells, demonstrating that the mechanism of CYP1A1 induction is p53-dependent; cisplatin had no such effect. Co-incubation experiments with the drugs and 2.5 μM BaP showed that: (i) etoposide increased CYP1A1 expression in TP53(+/+) cells, and to a lesser extent in TP53(-/-) cells, compared to cells treated with BaP alone; (ii) ellipticine decreased CYP1A1 expression in TP53(+/+) cells in BaP co-incubations; and (iii) cisplatin did not affect BaP-mediated CYP1A1 expression. Further, whereas cisplatin and etoposide had virtually no influence on CYP1A1-catalysed BaP metabolism, ellipticine treatment strongly inhibited BaP bioactivation. Our results indicate that the underlying mechanisms whereby etoposide and ellipticine regulate CYP1A1 expression must be different and may not be linked to p53 activation alone. These results could be relevant for smokers, who are exposed to increased levels of BaP, when prescribing chemotherapeutic drugs. Beside gene-environment interactions, more considerations should be given to potential drug-environment interactions during chemotherapy.
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http://dx.doi.org/10.1016/j.tox.2018.02.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6593262PMC
April 2018

EXPOsOMICS: final policy workshop and stakeholder consultation.

BMC Public Health 2018 02 15;18(1):260. Epub 2018 Feb 15.

Barcelona Institute for Global Health (ISGlobal), Barcelona, Spain.

The final meeting of the EXPOsOMICS project "Final Policy Workshop and Stakeholder Consultation" took place 28-29 March 2017 to present the main results of the project and discuss their implications both for future research and for regulatory and policy activities. This paper summarizes presentations and discussions at the meeting related with the main results and advances in exposome research achieved through the EXPOsOMICS project; on other parallel research initiatives on the study of the exposome in Europe and in the United States and their complementarity to EXPOsOMICS; lessons learned from these early studies on the exposome and how they may shape the future of research on environmental exposure assessment; and finally the broader implications of exposome research for risk assessment and policy development on environmental exposures. The main results of EXPOsOMICS in relation to studies of the external exposome and internal exposome in relation to both air pollution and water contaminants were presented as well as new technologies for environmental health research (adductomics) and advances in statistical methods. Although exposome research strengthens the scientific basis for policy development, there is a need in terms of showing added value for public health to: improve communication of research results to non-scientific audiences; target research to the broader landscape of societal challenges; and draw applicable conclusions. Priorities for future work include the development and standardization of methodologies and technologies for assessing the external and internal exposome, improved data sharing and integration, and the demonstration of the added value of exposome science over conventional approaches in answering priority policy questions.
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http://dx.doi.org/10.1186/s12889-018-5160-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5815236PMC
February 2018

Editorial: 40th Anniversary of UKEMS.

Authors:
David H Phillips

Mutagenesis 2017 12;32(6):543-544

King's College London.

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http://dx.doi.org/10.1093/mutage/gex038DOI Listing
December 2017

Genotoxicity of fine and coarse fraction ambient particulate matter in immortalised normal (TT1) and cancer-derived (A549) alveolar epithelial cells.

Environ Mol Mutagen 2018 05 25;59(4):290-301. Epub 2018 Jan 25.

Department of Analytical, Environmental and Forensic Sciences, MRC-PHE Centre for Environment and Health, King's College London, London, United Kingdom.

Human exposure to airborne particulate matter (PM) is associated with adverse cardiopulmonary health effects, including lung cancer. Ambient PM represents a heterogeneous mixture of chemical classes including transition metals, polycyclic aromatic hydrocarbons (PAHs) and their derivatives such as nitro-PAHs, many of which are classified as putative carcinogens. As the primary site of human exposure to PM is the lungs, we investigated the response of two alveolar epithelial cell lines, the tumour-derived A549 and newly described TT1 cells, to fine and coarse PM collected from background and roadside locations. We show that coarse PM elicits a genotoxic response in the TT1 cells, with the strongest signal associated with the background sample. This response could be recapitulated using the organic extract derived from this sample. No responses were observed in PM-challenged A549 cells. Fine PM failed to elicit a genotoxic response in either cell line despite the higher PAH concentrations within this fraction. Consistent with the lack of a simplistic association between PM PAH content and the observed genotoxic response, TT1 cells treated with benzo[a]pyrene (BaP) demonstrated no increase in the selected markers. In contrast, a pattern of response was observed in TT1 cells challenged with 3-nitrobenzanthrone (3-NBA) similar to that with coarse PM. Together, these data illustrated the suitability of the TT1 cell line for assessing PM-induced genotoxicity and challenge the contention that fine roadside PM poses the higher cancer risk. Furthermore, the response to 3-NBA and not BaP suggests a major contribution of nitro-PAHs to the overall toxicity of PM. Environ. Mol. Mutagen. 59:290-301, 2018. © 2018 The Authors Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.
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http://dx.doi.org/10.1002/em.22166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5947684PMC
May 2018

Cytochrome b impacts on cytochrome P450-mediated metabolism of benzo[a]pyrene and its DNA adduct formation: studies in hepatic cytochrome b /P450 reductase null (HBRN) mice.

Arch Toxicol 2018 Apr 24;92(4):1625-1638. Epub 2018 Jan 24.

Department of Analytical, Environmental and Forensic Sciences, MRC-PHE Centre for Environment and Health, King's College London, Franklin-Wilkins Building, 150 Stamford Street, London, SE1 9NH, UK.

Benzo[a]pyrene (BaP) is an environmental pollutant that, based on evidence largely from in vitro studies, exerts its genotoxic effects after metabolic activation by cytochrome P450s. In the present study, Hepatic Reductase Null (HRN) and Hepatic Cytochrome b /P450 Reductase Null (HBRN) mice have been used to study the role of P450s in the metabolic activation of BaP in vivo. In HRN mice, cytochrome P450 oxidoreductase (POR), the electron donor to P450, is deleted specifically in hepatocytes. In HBRN mice the microsomal haemoprotein cytochrome b , which can also act as an electron donor from cytochrome b reductase to P450s, is also deleted in the liver. Wild-type (WT), HRN and HBRN mice were treated by i.p. injection with 125 mg/kg body weight BaP for 24 h. Hepatic microsomal fractions were isolated from BaP-treated and untreated mice. In vitro incubations carried out with BaP-pretreated microsomal fractions, BaP and DNA resulted in significantly higher BaP-DNA adduct formation with WT microsomal fractions compared to those from HRN or HBRN mice. Adduct formation (i.e. 10-(deoxyguanosin-N-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-BaP [dG-N-BPDE]) correlated with observed CYP1A activity and metabolite formation (i.e. BaP-7,8-dihydrodiol) when NADPH or NADH was used as enzymatic cofactors. BaP-DNA adduct levels (i.e. dG-N-BPDE) in vivo were significantly higher (~ sevenfold) in liver of HRN mice than WT mice while no significant difference in adduct formation was observed in liver between HBRN and WT mice. Our results demonstrate that POR and cytochrome b both modulate P450-mediated activation of BaP in vitro. However, hepatic P450 enzymes in vivo appear to be more important for BaP detoxification than its activation.
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http://dx.doi.org/10.1007/s00204-018-2162-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5882632PMC
April 2018