Haemoglobin adducts of acrylonitrile and ethylene oxide in acrylonitrile workers, dependent on polymorphisms of the glutathione transferases GSTT1 and GSTM1

Fifty-nine persons with industrial handling of low levels of acrylonitrile (AN) were studied. As part of a medical surveillance programme an extended haemoglobin adduct monitoring [N-(cyanoethyl)valine, CEV; N- (methyl)valine, MV; N-(hydroxyethyl)valine, HEV] was performed. Moreover, the genetic states of the polymorphic glutathione transferases GSTM1 and GSTT1 were assayed by polymerase chain reaction (PCR). Repetitive analyses of CEV and MV in subsequent years resulted in comparable values (means, 59.8 and 70.3 μg CEV/1 blood; 6.7 and 6.7 μg MV/1 blood). Hence, the industrial AN exposures were well below current official standards. Monitoring the haemoglobin adduct CEV appears as a suitable means of biomonitoring and medical surveillance under such exposure conditions. There was also no apparent correlation between the CEV and HEV or CEV and MV adduct levels. The MV and HEV values observed represented background levels, which apparently are not related to any occupational chemical exposure. There was no consistent effect of the genetic GSTM1 or GSTT1 state on CEV adduct levels induced by acrylonitrile exposure. Therefore, neither GSTM1 nor GSTT1 appears as a major AN metabolizing isoenzyme in humans. The low and physiological background levels of MV were also not influenced by the genetic GSTM1 state, but the MV adduct levels tended to be higher in GSTT1- individuals compared to GSTT1 + persons. With respect to the background levels of HEV adducts observed, there was no major influence of the GSTM1 state, but GST- individuals displayed adduct levels that were about 1/3 higher than those of GSTT1+ individuals. The coincidence with known differences in rates of background sister chromatid exchange between GSTT1- and GSTT1 + persons suggests that the lower ethylene oxide (EO) detoxification rate in GSTT1- persons, indicated by elevated blood protein hydroxyethyl adduct levels, leads to an increased genotoxic effect of the physiological EO background.

Protection by methylproamine of irradiated human keratinocytes correlates with reduction of DNA damage

Purpose: The therapeutic ratio for ionising radiation treatment of tumour is a trade-off between normal tissue side-effects and tumour control. Application of a radioprotector to normal tissue can reduce side-effects. Here we study the effects of a new radioprotector on the cellular response to radiation. Methylproamine is a DNA-binding radioprotector which, on the basis of published pulse radiolysis studies, acts by repair of transient radiation-induced oxidative species on DNA. To substantiate this hypothesis, we studied protection by methylproamine at both clonogenic survival and radiation-induced DNA damage, assessed by γH2AX (histone 2AX phosphorylation at serine 139) focus formation endpoints. Materials and methods: The human keratinocyte cell line FEP1811 was used to study clonogenic survival and yield of γH2AX foci following irradiation (137Cs γ-rays) of cells exposed to various concentrations of methylproamine. Uptake of methylproamine into cell nuclei was measured in parallel. Results: The extent of radioprotection at the clonogenic survival endpoint increased with methylproamine concentration up to a maximum dose modification factor (DMF) of 2.0 at 10 μM. At least 0.1 fmole/nucleus of methylproamine is required to achieve a substantial level of radioprotection (DMF of 1.3) with maximum protection (DMF of 2.0) achieved at 0.23 fmole/nucleus. The γH2AX focus yield per cell nucleus 45 min after irradiation decreased with drug concentration with a DMF of 2.5 at 10 μM. Conclusions: These results are consistent with the hypothesis that radioprotection by methylproamine is mediated by attenuation of the extent of initial DNA damage.

Quantification of gammaH2AX foci in response to ionising radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming gammaH2AX(1). Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)(2). Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB(2,3). This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning approximately 2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete gammaH2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy(2). The loss of gammaH2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary(4-8). The disappearence of gammaH2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C(5,6). Further, removal of gammaH2AX by redistribution involving histone exchange with H2A.Z has been implicated(7,8). Importantly, the quantitative analysis of gammaH2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of gammaH2AX foci in gamma-irradiated adherent human keratinocytes(9)

Evaluation of the spatial distribution of γH2AX following ionizing radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as γH2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of γH2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of γH2AX as molecular marker of DSBs, a disparity in γ-irradiation-induced γH2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of γH2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between γH2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of γH2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3Dmodeling.

Quantitation of gammaH2AX foci in tissue samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming γH2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear γH2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of γH2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of γH2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, γH2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of γH2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of γH2AX foci in mouse tissues.

H2AX phosphorylation screen of cells from radiosensitive cancer patients reveals a novel DNA double-strand break repair cellular phenotype

BACKGROUND: About 1-5% of cancer patients suffer from significant normal tissue reactions as a result of radiotherapy (RT). It is not possible at this time to predict how most patients' normal tissues will respond to RT. DNA repair dysfunction is implicated in sensitivity to RT particularly in genes that mediate the repair of DNA double-strand breaks (DSBs). Phosphorylation of histone H2AX (phosphorylated molecules are known as gammaH2AX) occurs rapidly in response to DNA DSBs, and, among its other roles, contributes to repair protein recruitment to these damaged sites. Mammalian cell lines have also been crucial in facilitating the successful cloning of many DNA DSB repair genes; yet, very few mutant cell lines exist for non-syndromic clinical radiosensitivity (RS). METHODS: Here, we survey DNA DSB induction and repair in whole cells from RS patients, as revealed by gammaH2AX foci assays, as potential predictive markers of clinical radiation response. RESULTS: With one exception, both DNA focus induction and repair in cell lines from RS patients were comparable with controls. Using gammaH2AX foci assays, we identified a RS cancer patient cell line with a novel ionising radiation-induced DNA DSB repair defect; these data were confirmed by an independent DNA DSB repair assay. CONCLUSION: gammaH2AX focus measurement has limited scope as a pre-RT predictive assay in lymphoblast cell lines from RT patients; however, the assay can successfully identify novel DNA DSB repair-defective patient cell lines, thus potentially facilitating the discovery of novel constitutional contributions to clinical RS.

Reason of state and a new notion of fortune : Historicising the risk society

Since the 1980s the concept of risk has produced a large and diverse volume of sociological research. Ulrich Beck’s groundbreaking risk society thesis provides a particularly engaging contribution, since it seems that nearly every sociological account of risk engages with this work. For Beck, we are living in second modernity – a new epoch that breaks with pre-modernity and industrial society due to the centrality, incalculability and reflexivity of globalised risk. While Beck’s theory is compelling, a reading of other theorists such as Foucault (2007[1978]) and Hacking (1975,1990) suggests that a difficulty with Beck’s work is that in attempting to explain what is novel about risk in contemporary times, he too quickly passes over the complexities and ruptures of historical change that impact on the history and contingency of risk. This paper begins by presenting a brief analysis of the present state of risk by introducing Beck’s historical narrative of risk from pre-modernity to the risk society; it then outlines the challenges with the “risk as epoch” argument by considering a range of literature, which suggests risk has a more complex history than proposed by Beck; and finally it highlights the value in examining strategies of statecraft in early modern Europe, specifically Machiavelli’s The Prince (2008[1513]) and Giovanni Botero’s political treatise, Della Ragion di Stato (1956[1589]) – as a means of more thoroughly understanding how our current concept of risk emerges. In doing so, this paper seeks to open up new trajectories in the historicisation of risk for other interested scholars.

The Business of Unfinished Business : Reflections on Co-construction of Meanings in Research Encounters

My concern in this commentary is the discrepancy between cultural psychologists' theoretical claims that meanings are co-constructed by, with and for individuals in ongoing social interaction, and their research practices where researcher's and research participant's meaning-making processes are separated in time into sequential turns. I argue for the need to live up to these theoretical assumptions, by making both the initial research encounter and the researcher's later interpretation process more co-constructive. I suggest making the initial research encounter more co-constructive by paying attention to these moments when the negotiated flow of interaction between researcher and research participant breaks down, for it allows the research participant's meaning-making to be traced and makes the researcher's efforts towards meaning more explicit. I propose to make the later interpretation process more co-constructive by adopting a more open-ended and dialogical way of writing that is specifically addressed to research participants and invites them to actively engage with researcher's meaning-making.

Playing Pranks: Traditions, communities & co-creative cultural performance practice

Pranks, hoaxes and practical jokes are co-creative cultural performance practices that appear across times, contexts and cultures. These practices include everyday play amongst families, friends and coworkers, entertainment programs such as Prank Patrol, Punked or Scare Tactics, and aesthetic and activist pranks perpetrated by situationist artists, guerrilla artists, and, most recently, culture ‘jammers’ or ‘hackers’ intent on turning capitalist systems back on themselves. Although it can, in common usage, describe almost any show off behaviour, a prank in the strictest definition of the term is a performance that deploys a very specific set of strategies. It is an act of trickery, mischief, or deceit, that must be taken as real, and momentarily cause real fear, anger or worry for an unwitting spectator-become-performer, who is meant to play along until the trick is revealed and their response can be represented back to the prankster, other spectators, or society as a whole, either for the sake of entertainment or for the sake of commentary on a cultural phenomenon. A prank, in this sense, deliberately blurs the boundaries between daily and dramatic performance. It creates a moment of uncertainty, in which both the prankster’s ability to be creative, clever, or culturally astute, and the prankee’s ability to play along, discern the trick, discern the point of the trick, and, in the end, be duped, be a good sport, or even play/pay the prankster back, are both put to the test. In this paper, I consider a number of pranking traditions popular where I am in Australia, from the community-building pranks of footballers, bucks parties and ‘drop bear’ tales told to tourists, to the more controversial pranks of radio shock jocks, activists and artists. I use performance, spectatorship and ethical theory to examine the engagement between prankster, pranked spectator, and other spectators, in this most distinctive sort of community-driven performance practice, and the way it builds and breaks status, social and other sorts of relationships within and between specific communities.

BRCA1 deficiency exacerbates estrogen-induced DNA damage and genomic instability

Germline mutations in BRCA1 predispose carriers to a high incidence of breast and ovarian cancers. BRCA1 functions to maintain genomic stability through critical roles in DNA repair, cell cycle arrest and transcriptional control. A major question has been why BRCA1 loss or mutation leads to tumors mainly in estrogen-regulated tissues, given that BRCA1 has essential functions in all cell types. Here we report that estrogen and estrogen metabolites can cause DNA double strand breaks (DSB) in estrogen receptor-α negative breast cells and that BRCA1 is required to repair these DSBs to prevent metabolite-induced genomic instability. We found that BRCA1 also regulates estrogen metabolism and metabolite-mediated DNA damage by repressing the transcription of estrogen-metabolising enzymes, such as CYP1A1, in breast cells. Lastly, we used a knock-in human cell model with a heterozygous BRCA1 pathogenic mutation to show how BRCA1 haploinsufficiency affects these processes. Our findings provide pivotal new insights into why BRCA1 mutation drives the formation of tumours in estrogen-regulated tissues, despite the general role of BRCA1 in DNA repair in all cell types.

DNA damage in response to an Ironman triathlon

The major aims of this study were to investigate the effect of an Ironman triathlon on DNA migration in the single cell gel electrophoresis assay, apoptosis and necrosis in the cytokinesis-block micronucleus cytome assay with lymphocytes and on changes of total antioxidant capacity in plasma. Blood samples were taken 2 days (d) before, within 20 min, 1 d, 5 d and 19 d post-race. The level of strand breaks decreased (p<0.05) immediately after the race, then increased (p<0.01) 1 d post-race and declined (p<0.01) until 19 d post-race. Apoptotic and necrotic cells decreased (p<0.01) and the total antioxidant status increased (p<0.01) immediately after the race. The results indicate that ultra-endurance exercise does not cause prolonged DNA damage in well-trained male athletes.

Well-trained, healthy triathletes experience no adverse health risks regarding oxidative stress and DNA damage by participating in an ultra-endurance event

Also physical exercise in general is accepted to be protective, acute and strenuous exercise has been shown to induce oxidative stress. Enhanced formation of free radicals leads to oxidation of macromolecules and to DNA damage. On the other hand ultra-endurance events which require strenuous exercise are very popular and the number of participants is continuously increasing worldwide. Since only few data exists on Ironman triathletes, who are prototypes of ultra-endurance athletes, this study was aimed at assessing the risk of oxidative stress and DNA damage after finishing a triathlon and to predict a possible health risk. Blood samples of 42 male athletes were taken 2 days before, within 20 min after the race, 1, 5 and 19 days post-race. Oxidative stress marker increased only moderately after the race and returned to baseline after 5 days. Marker of DNA damage measured by the SCGE assay with and without restriction enzymes as well as by the sister chromatid exchange assay did either show no change or deceased within the first day after the race. Due to intake during the race and the release by the cells plasma concentrations of vitamin C and α-tocopherol increased after the event and returned to baseline 1 day after. This study indicates that despite a temporary increase in some oxidative stress markers, there is no persistent oxidative stress and no DNA damage in response to an Ironman triathlon in trained athletes, mainly due to an appropriate antioxidant intake and general protective alterations in the antioxidant defence system.

Forecasting outcomes in tennis matches using within-match betting markets

Klaassen and Magnus (2003) provide a model of the probability of a given player winning a tennis match, with the prediction updated on a point-by-point basis. This paper provides a point-by-point comparison of that model with the probability of a given player winning the match, as implied by betting odds. The predictions implied by the betting odds match the model predictions closely, with an extremely high correlation being found between the model and the betting market. The results for both men’s and women’s matches also suggest that there is a high level of efficiency in the betting market, demonstrating that betting markets are a good predictor of the outcomes of tennis matches. The significance of service breaks and service being held is anticipated up to four points prior to the end of the game. However, the tendency of players to lose more points than would be expected after conceding a break of service is not captured instantaneously in betting odds. In contrast, there is no evidence of a biased reaction to a player winning a game on service.

Learning Chinese as a Heritage Language: An Australian Perspective

Summary: This book explores the fascinating topic of heritage language learning, looking in particular at Chinese Australians' learning of Chinese. The author studies the investment, challenges and benefits of heritage language learning across varied contexts including school, work, home and in the community. The book investigates how Chinese Australians navigate and negotiate their Chineseness and how resources are used to support their learning. The book is based on a mixed methods study which uses Bourdieu's sociological theory, and offers implications for sociologists of language and education, Chinese heritage language learners and teachers, as well as language and cultural policy makers. Review: This book is a compelling account of the habitus of Chineseness in a world of mobility. It offers up a plethora of insights into the implication of heritage language learning in the constitution of Chinese identity; it makes available a sophisticated mixed methods approach for using the thinking tools of Pierre Bourdieu; it adds to these tools a nuanced cultural dimension. Karen Dooley, Queensland University of Technology, Australia In our increasingly trans-migratory world, language can be central to cultural identity. Dr Mu’s research breaks new ground by adapting Bourdieu’s insights to examine how cultural identity (‘Chinese-ness’) is linguistically learned and practiced in Australia. His remarkable book will interest educators and researchers grappling with how language pertains to identity. Tom Strong, University of Calgary, Canada A thought-provoking, highly engaging work that has deftly shown how Bourdieusian framework can be applied in the research field of literacy education and Heritage Language learning. A must-read for those interested in overseas Chinese communities and Heritage Language learning across various immigrant communities in general! Liang Du, Beijing Normal University, China

Mobile gene silencing in Arabidopsis is regulated by hydrogen peroxide

In plants and nematodes, RNAi can spread from cells from which it is initiated to other cells in the organism. The underlying mechanism controlling the mobility of RNAi signals is not known, especially in the case of plants. A genetic screen designed to recover plants impaired in the movement but not the production or effectiveness of the RNAi signal identified RCI3, which encodes a hydrogen peroxide (H2O2)-producing type III peroxidase, as a key regulator of silencing mobility in Arabidopsis thaliana. Silencing initiated in the roots of rci3 plants failed to spread into leaf tissue or floral tissue. Application of exogenous H2O2 reinstated the spread in rci3 plants and accelerated it in wild-type plants. The addition of catalase or MnO2, which breaks down H2O2, slowed the spread of silencing in wild-type plants. We propose that endogenous H2O2, under the control of peroxidases, regulates the spread of gene silencing by altering plasmodesmata permeability through remodelling of local cell wall structure, and may play a role in regulating systemic viral defence.