David H. Phillips

Polycyclic aromatic hydrocarbons in the diet

Polycyclic aromatic hydrocarbons (PAHs), of which benzo[a]pyrene is the most commonly studied and measured, are formed by the incomplete combustion of organic matter. They are widely distributed in the environment and human exposure to them is unavoidable. A number of them, such as benzo[a]pyrene, are carcinogenic and mutagenic, and they are widely believed to make a substantial contribution to the overall burden of cancer in humans. Their presence in the environment is reflected in their presence at detectable levels in many types of uncooked food. In addition, cooking processes can generate PAHs in food. PAHs can also be formed during the curing and processing of raw food prior to cooking. Several studies have been carried out to determine the levels of exposure to PAHs from representative human diets, and the proportion of the overall burden of environmental exposure to PAHs that is attributable to the diet. In most cases, it is concluded that diet is the major source of human exposure to PAHs. The major dietary sources of PAHs are cereals and vegetables, rather than meat, except where there is high consumption of meat cooked over an open flame. More recently, biomonitoring procedures have been developed to assess human exposure to PAHs and these have also indicated that diet is a major source of exposure. Exposure to nitro-PAHs through food consumption appears to be very low.

Fifty years of benzo(a)pyrene

It is fifty years since the publication of the report on the isolation, from coal tar, and identification of the potent chemical carcinogen benzo(a)pyrene, the culmination of over 10 years of research instigated and directed by E.L. Kennaway. The events leading to that discovery are of interest in themselves. Subsequent progress in unravelling the metabolic fate of polycyclic aromatic hydrocarbons has contributed to our understanding of the mechanism of chemical carcinogenesis.

Oxidative DNA damage mediated by copper(II), iron(II) and nickel(II) Fenton reactions: evidence for site-specific mechanisms in the formation of double-strand breaks, 8-hydroxydeoxyguanosine and putative intrastrand cross-links

The role of metal ion-DNA interactions in the Fenton reaction-mediated formation of putative intrastrand cross-links, 8-hydroxydeoxyguanosine (8-OHdG) and single- and double-strand breaks was investigated. Salmon sperm DNA and pBluescript K+ plasmid were incubated with hydrogen peroxide and either copper(II), iron(II), or nickel(II), which differ in both their affinity for DNA and in the spectrum of oxidative DNA damage they induce in Fenton reactions. EDTA was included in these incubations according to two different strategies; the first (strategy 1) in which DNA and metal ions were mixed prior to the addition of EDTA, the second (strategy 2) in which EDTA and metal ions were mixed prior to the addition of DNA. The formation of the putative intrastrand cross-links, monitored by 32P-postlabelling, was not affected by the addition of between 10 microM and 5 mM EDTA to the copper(II) Fenton reaction according to strategy 1. In contrast, the level of cross-links declined significantly upon inclusion of 20 microM EDTA and above when added according to strategy 2. Similarly, formation of these lesions declined in the iron(II) Fenton reaction more dramatically upon addition of 5 mM EDTA when added according to strategy 2 compared to strategy 1, while the yield of cross-links formed in the nickel(II) Fenton reaction declined equally with both strategies with up to 25 mM EDTA. The formation of single- and double-strand breaks was investigated in plasmid DNA by agarose gel electrophoresis and subsequent densitometry. The formation of linear DNA in the iron(II) Fenton reaction decreased dramatically upon inclusion of EDTA according to strategy 2, while no such decline was observed using strategy 1. In contrast, the formation of linear DNA in the copper(II) Fenton reaction decreased upon inclusion of EDTA according to both strategies. A decrease in the formation of open-circular DNA was also observed upon inclusion of EDTA according to both strategies; however this decrease occurred at a lower EDTA concentration in strategy 2 (100 microM) compared to strategy 1 (200 microM), and the level of open-circular DNA reached a lower level (8. 5% compared to 24.2%). The nickel(II) Fenton reaction generated only open-circular DNA, and this was completely inhibited upon addition of 25 microM EDTA according to both strategies. There was less formation of 8-OHdG in the copper(II) and iron(II) Fenton reactions when EDTA was added according to strategy 2 than according to strategy 1. These results suggest that a site-specific mechanism is involved in the formation of double-strand breaks and, to a lesser extent, 8-OHdG and the putative intrastrand cross-links, while the formation of single-strand breaks is more likely to involve generation of hydroxyl radicals in solution.

Diversity of TMPRSS2-ERG fusion transcripts in the human prostate

TMPRSS2-ERG gene fusions have recently been reported to be present in a high proportion of human prostate cancers. In the current study, we show that great diversity exists in the precise structure of TMPRSS2-ERG hybrid transcripts found in human prostates. Fourteen distinct hybrid transcripts are characterized, each containing different combinations of sequences from the TMPRSS2 and ERG genes. The transcripts include two that are predicted to encode a normal full-length ERG protein, six that encode N-terminal truncated ERG proteins and one that encodes a TMPRSS2-ERG fusion protein. Interestingly, distinct patterns of hybrid transcripts were found in samples taken from separate regions of individual cancer-containing prostates, suggesting that TMPRSS2-ERG gene fusions may be arising independently in different regions of a single prostate.

The 32P-postlabeling assay for DNA adducts.

32P-postlabeling analysis is an ultrasensitive method for the detection and quantitation of carcinogen–DNA adducts. It consists of four principal steps: (i) enzymatic digestion of DNA to nucleoside 3′-monophosphates; (ii) enrichment of the adduct fraction of the DNA digest; (iii) 5′-labeling of the adducts by transfer of 32P-orthophosphate from [γ-32P]ATP mediated by polynucleotide kinase (PNK); (iv) chromatographic or electrophoretic separation of the labeled adducts or modified nucleotides and quantitation by measurement of their radioactive decay. The assay requires only microgram quantities of DNA and is capable of detecting adducts at frequencies as low as 1 in 1010 nt, making it applicable to the detection of events resulting from environmental exposures, or experiments using physiological concentrations of agents. It has a wide range of applications in human, animal and in vitro studies, and can be used for a wide variety of classes of compound and for the detection of adducts formed by complex mixtures. This protocol can be completed in 3 d.

Platelet and osteoclast β3 integrins are critical for bone metastasis

Mice with a targeted deletion of β3 integrin were used to examine the process by which tumor cells metastasize and destroy bone. Injection of B16 melanoma cells into the left cardiac ventricle resulted in osteolytic bone metastasis in 74% of β3+/+ mice by 14 days. In contrast, only 4% of β3–/– mice developed bone lesions. Direct intratibial inoculation of tumor resulted in marrow replacement by tumor in β3–/– mice, but no associated trabecular bone resorption as seen inβ3+/+ mice. Bone marrow transplantation studies showed that susceptibility to bone metastasis was conferred by a bone marrow-derived cell. To dissect the roles of osteoclast and platelet β3 integrins in this model of bone metastasis, osteoclast-defective src–/– mice were used. Src-null mice were protected from tumor-associated bone destruction but were not protected from tumor cell metastasis to bone. In contrast, a highly specific platelet aggregation inhibitor of activated αIIbβ3 prevented B16 metastases. These data demonstrate a critical role for platelet αIIbβ3 in tumor entry into bone and suggest a mechanism by which antiplatelet therapy may be beneficial in preventing the metastasis of solid tumors.

Tumour necrosis factor-alpha mediates tumour promotion via a PKC alpha- and AP-1-dependent pathway.

Tumour necrosis factor-α (TNF-α) deficient mice (TNF-α−/− mice) are resistant to skin carcinogenesis. Cellular signalling via the transcription factor complex AP-1 is thought to play a key role in tumour promotion. The induction of a specific subset of AP-1 responsive genes thought to be important for tumour development, namely GM–CSF, MMP-9 and MMP-3, was suppressed in TNF-α−/− compared to wild-type mouse skin in response to the tumour promotor TPA. The differential induction of these genes correlated with a temporal shift in AP-1 activation and c-Jun expression in TNF-α−/− compared to wild-type epidermis. The major receptor for TPA-induced signalling in basal keratinocytes, PKCα, was also differentially regulated in wild-type compared with TNF-α−/− epidermis. A marked delay in TPA-induced intracellular translocation and downregulation of PKCα was observed in TNF-α−/− epidermis, which correlated with the deregulated TPA-induced AP-1 activation and c-Jun expression. The frequency of DNA adduct formation and c-Ha-ras mutations was the same in wild-type and TNF-α−/− epidermis after DMBA treatment, suggesting that TNF-α was not involved in tumour initiation. These data suggest that the pro-inflammatory cytokine TNF-α is a critical mediator of tumour promotion, acting via a PKCα- and AP-1-dependent pathway. This may be one mechanism by which chronic inflammation increases susceptibility to cancer.

Mutational signatures associated with tobacco smoking in human cancer

Assessing smoke damage in cancer genomes We have known for over 60 years that smoking tobacco is one of the most avoidable risk factors for cancer. Yet the detailed mechanisms by which tobacco smoke damages the genome and creates the mutations that ultimately cause cancer are still not fully understood. Alexandrov et al. examined mutational signatures and DNA methylation changes in over 5000 genome sequences from 17 different cancer types linked to smoking (see the Perspective by Pfeifer). They found a complex pattern of mutational signatures. Only cancers originating in tissues directly exposed to smoke showed a signature characteristic of the known tobacco carcinogen benzo[a]pyrene. One mysterious signature was shared by all smoking-associated cancers but is of unknown origin. Smoking had only a modest effect on DNA methylation. Science, this issue p. 618; see also p. 549 Tobacco smoke causes cancer through mutational processes that are more complex than previously thought. Tobacco smoking increases the risk of at least 17 classes of human cancer. We analyzed somatic mutations and DNA methylation in 5243 cancers of types for which tobacco smoking confers an elevated risk. Smoking is associated with increased mutation burdens of multiple distinct mutational signatures, which contribute to different extents in different cancers. One of these signatures, mainly found in cancers derived from 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 clocklike mutational process. Smoking is associated with limited differences in methylation. 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 smoking-related cancer types.

STrengthening the reporting of OBservational studies in Epidemiology—Molecular Epidemiology (STROBE-ME): an extension of the STROBE statement

Advances in laboratory techniques have led to a rapidly increasing use of biomarkers in epidemiological studies. Biomarkers of internal dose, early biological change, susceptibility and clinical outcomes are used as proxies for investigating interactions between external and / or endogenous agents and body components or processes. The need for improved reporting of scientific research led to influential statements of recommendations such as the STrengthening Reporting of OBservational studies in Epidemiology (STROBE) statement. The STROBE initiative established in 2004 aimed to provide guidance on how to report observational research. Its guidelines provide a user-friendly checklist of 22 items to be reported in epidemiological studies, with items specific to the three main study designs: cohort studies, case-control studies and cross-sectional studies. The present STrengthening the Reporting of OBservational studies in Epidemiology - Molecular Epidemiology (STROBE-ME) initiative builds on the STROBE statement implementing nine existing items of STROBE and providing 17 additional items to the 22 items of STROBE checklist. The additions relate to the use of biomarkers in epidemiological studies, concerning collection, handling and storage of biological samples; laboratory methods, validity and reliability of biomarkers; specificities of study design; and ethical considerations. The STROBE-ME recommendations are intended to complement the STROBE recommendations.

Mutagenicity testing for chemical risk assessment: update of the WHO/IPCS Harmonized Scheme

Since the publication of the International Programme on Chemical Safety (IPCS) Harmonized Scheme for Mutagenicity Testing, there have been a number of publications addressing test strategies for mutagenicity. Safety assessments of substances with regard to genotoxicity are generally based on a combination of tests to assess effects on three major end points of genetic damage associated with human disease: gene mutation, clastogenicity and aneuploidy. It is now clear from the results of international collaborative studies and the large databases that are currently available for the assays evaluated that no single assay can detect all genotoxic substances. The World Health Organization therefore decided to update the IPCS Harmonized Scheme for Mutagenicity Testing as part of the IPCS project on the Harmonization of Approaches to the Assessment of Risk from Exposure to Chemicals. The approach presented in this paper focuses on the identification of mutagens and genotoxic carcinogens. Selection of appropriate in vitro and in vivo tests as well as a strategy for germ cell testing are described.