Correction: Chromatin structure, transcriptional activity and DNA repair efficiency affect the outcome of chemotherapy in multiple myeloma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The exposome in practice: Design of the EXPOsOMICS project.

EXPOsOMICS is a European Union funded project that aims to develop a novel approach to the assessment of exposure to high priority environmental pollutants, by characterizing the external and the internal components of the exposome. It focuses on air and water contaminants during critical periods of life. To this end, the project centres on 1) exposure assessment at the personal and population levels within existing European short and long-term population studies, exploiting available tools and methods which have been developed for personal exposure monitoring (PEM); and 2) multiple "omic" technologies for the analysis of biological samples (internal markers of external exposures). The search for the relationships between external exposures and global profiles of molecular features in the same individuals constitutes a novel advancement towards the development of "next generation exposure assessment" for environmental chemicals and their mixtures. The linkage with disease risks opens the way to what are defined here as 'exposome-wide association studies' (EWAS).

Leptin, acylcarnitine metabolites and development of adiposity in the Rhea mother-child cohort in Crete, Greece.

OBJECTIVE: This study aims to investigate relations of serum leptin at age 4 with development of adiposity and linear growth during 3 years of follow-up among 75 Greek children and to identify serum metabolites associated with leptin at age 4 and to characterize their associations with adiposity gain and linear growth. METHODS: Linear regression models that accounted for maternal age, education and gestational weight gain and child's age and sex were used to examine associations of leptin and leptin-associated metabolites measured at age 4 with indicators of adiposity and linear growth at age 7. RESULTS: Each 1-unit increment in natural log-(ln)-transformed leptin corresponded with 0.33 (95% CI: 0.10, 0.55) units greater body mass index-for-age z-score gain during follow-up. Likewise, higher levels of the leptin-associated metabolites methylmalonyl-carnitine and glutaconyl-carnitine corresponded with 0.14 (95% CI: 0.01, 0.27) and 0.07 (95% CI: -0.01, 0.16) units higher body mass index-for-age z-score gain, respectively. These relationships did not differ by sex or baseline weight status and were independent of linear growth. CONCLUSIONS: These findings suggest that leptin, methylmalonyl-carnitine and possibly glutaconyl-carnitine are associated with weight gain during early childhood. Future studies are warranted to confirm these findings in other populations.

Chromatin structure, transcriptional activity and DNA repair efficiency affect the outcome of chemotherapy in multiple myeloma.

BACKGROUND: Melphalan is one of the most active chemotherapeutic agents in the treatment of multiple myeloma (MM). However, the mechanism underlying differential patient responses to melphalan therapy is unknown. METHODS: Chromatin structure, transcriptional activity and DNA damage response signals were examined following ex vivo treatment with melphalan of both malignant bone marrow plasma cells (BMPCs) and peripheral blood mononuclear cells (PBMCs) of MM patients, responders (n=57) or non-responders (n=28) to melphalan therapy. PBMCs from healthy controls (n=25) were also included in the study. RESULTS: In both BMPCs and PBMCs, the local chromatin looseness, transcriptional activity and repair efficiency of the transcribed strand (TS) were significantly higher in non-responders than in responders and lowest in healthy controls (all P<0.05). Moreover, we found that melphalan-induced apoptosis inversely correlated with the repair efficiency of the TS, with the duration of the inhibition of mRNA synthesis, phosphorylation of p53 at serine 15 and apoptosis rates being higher in responders than in non-responders (all P<0.001). CONCLUSIONS: Our findings provide a mechanistic basis for the link between DNA repair efficiency and response to melphalan therapy. Interestingly, the observation of these phenomena in PBMCs provides a novel approach for the prediction of response to anti-myeloma therapy.

Prediagnostic transcriptomic markers of Chronic lymphocytic leukemia reveal perturbations 10 years before diagnosis.

BACKGROUND: B-cell lymphomas are a diverse group of hematological neoplasms with differential etiology and clinical trajectories. Increased insights in the etiology and the discovery of prediagnostic markers have the potential to improve the clinical course of these neoplasms. METHODS: We investigated in a prospective study global gene expression in peripheral blood mononuclear cells of 263 incident B-cell lymphoma cases, diagnosed between 1 and 17 years after blood sample collection, and 439 controls, nested within two European cohorts. RESULTS: Our analyses identified only transcriptomic markers for specific lymphoma subtypes; few markers of multiple myeloma (N = 3), and 745 differentially expressed genes in relation to future risk of chronic lymphocytic leukemia (CLL). The strongest of these associations were consistently found in both cohorts and were related to (B-) cell signaling networks and immune system regulation pathways. CLL markers exhibited very high predictive abilities of disease onset even in cases diagnosed more than 10 years after blood collection. CONCLUSIONS: This is the first investigation on blood cell global gene expression and future risk of B-cell lymphomas. We mainly identified genes in relation to future risk of CLL that are involved in biological pathways, which appear to be mechanistically involved in CLL pathogenesis. Many but not all of the top hits we identified have been reported previously in studies based on tumor tissues, therefore suggesting that a mixture of preclinical and early disease markers can be detected several years before CLL clinical diagnosis.

Progressive changes in chromatin structure and DNA damage response signals in bone marrow and peripheral blood during myelomagenesis.

The molecular pathways implicated in multiple myeloma (MM) development are rather unknown. We studied epigenetic and DNA damage response (DDR) signals at selected model loci (N-ras, p53, d-globin) in bone marrow plasma cells and peripheral blood mononuclear cells (PBMCs) from patients with monoclonal gammopathy of undetermined significance (MGUS; n=20), smoldering/asymptomatic MM (SMM; n=29) and MM (n=18), as well as in healthy control-derived PBMCs (n=20). In both tissues analyzed, a progressive, significant increase in the looseness of local chromatin structure, gene expression levels and DNA repair efficiency from MGUS to SMM and finally to MM was observed (all P<0.002). Following ex vivo treatment with melphalan, a gradual suppression of the apoptotic pathway occurred in samples collected at different stages of myelomagenesis, with the severity and duration of the inhibition of RNA synthesis, p53 phosphorylation at serine15 and induction of apoptosis being higher in MGUS than SMM and lowest in MM patients (all P<0.0103). Interestingly, for all endpoints analyzed, a strong correlation between plasma cells and corresponding PBMCs was observed (all P<0.0003). We conclude that progressive changes in chromatin structure, transcriptional activity and DDR pathways during myelomagenesis occur in malignant plasma cells and that these changes are also reflected in PBMCs.

Investigation of cell death induced by N-methyl-N-nitrosourea in cell lines of human origin and implication of RNA binding protein alterations.

Methylating agents, a widely used class of anticancer drugs, induce DNA methylation adducts, the most biologically significant being O(6)-methylguanine. The efficacy of these drugs depends on the interplay of three DNA repair systems: base excision repair (BER), methyl-directed mismatch repair (MMR) and direct damage reversal by O(6)-methylguanine-DNA methyltransferase (MGMT). An MGMT-inducible, MMR- and BER-proficient HeLa cell line was treated with different concentrations of N-methyl-N-nitrosourea (MNU), a model S(N)1 methylating agent, analogous to widely used methylating cancer chemotherapeutic drugs, under different expression levels of the repair enzyme (MGMT). MNU induced MGMT-dependent apoptotic cell death. In this particular cellular context, the induction of apoptosis was accompanied by modifications of the RNA binding protein poly(A)polymerase and significant down-regulation of the heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2. These results implicate alterations of the above mentioned RNA binding proteins in S(N)1 methylating agent-induced cell death and apoptosis, providing a possible perspective regarding their use as biomarkers of tumor resistance/sensitivity to chemotherapy.

Dietary acrylamide intake and risk of breast cancer in the UK women's cohort.

BACKGROUND: No studies to date have demonstrated a clear association with breast cancer risk and dietary exposure to acrylamide. METHODS: A 217-item food frequency questionnaire was used to estimate dietary acrylamide intake in 33,731 women aged 35-69 years from the UK Women's Cohort Study followed up for a median of 11 years. RESULTS: In all, 1084 incident breast cancers occurred during follow-up. There was no evidence of an overall association between acrylamide intake and breast cancer (hazard ratio=1.08 per 10 µg day(-1), 95% CI: 0.98-1.18, P(trend)=0.1). There was a suggestion of a possible weak positive association between dietary acrylamide intake and premenopausal breast cancer after adjustment for potential confounders (hazard ratio=1.2, 95% CI: 1.0-1.3, P(trend)=0.008). There was no suggestion of any association for postmenopausal breast cancer (hazard ratio=1.0, 95% CI: 0.9-1.1, P(trend)=0.99). CONCLUSIONS: There is no evidence of an association between dietary acrylamide intake and breast cancer. A weak association may exist with premenopausal breast cancer, but requires further investigation.

Mutagenesis by man-made mineral fibres in the lung of rats.

The potential of two asbestos substitute mineral fibres--rock (stone) wool RW1 and glass wool MMVF10--to induce gene mutations, DNA strand breaks, inflammation and oxidative stress has been studied in rats. Male homozygous lamda-lacI transgenic F344 rats were intratracheally instilled with single doses of 1 and 2 mg/animal of fibres or with multiple doses of 2 mg/animal administered weekly on four consecutive weeks (8 mg in total). Exposure to RW1 fibres for 16 weeks significantly increased mutant frequency (MF) in the lung in a dose-dependent manner, while MMVF10 fibres did not exhibit any increase of MF at any dose. RW1 fibres gave a significant increase of MF at a dose of 1 mg. Four weeks after instillation, neither the single nor the multiple doses significantly increased MF for both fibre types. To investigate mechanisms for induction of mutations, other genotoxicity markers and parameters of inflammatory and oxidative damage were determined in relation to MF. A weak correlation of mutagenicity data with other genotoxicity parameters studied was observed. DNA strand breaks as measured by comet assay were increased in alveolar macrophages and lung epithelial cells of RW1 and MMVF10 treated rats. RWl fibres caused more extensive lung inflammation as measured by release of neutrophils into broncho-alveolar lavage fluid than MMVF10 fibres. The effects were observed 16 weeks post-exposure, indicating a persistence of the pathogenic process during the exposure period. Only minor differences in the extent of inflammatory processes were observed between the doses of 2 mg and 4 x 2 mg, suggesting that any threshold for inflammation lies below the dose of 2 mg. With the exception of the highest dose of MMVF10 fibres after 16 weeks of exposure, no significant increase of oxidative damage as measured by levels of malondialdehyde in lung tissue was observed. MMVF10 fibres caused weaker inflammation in the lung of rats and did not exhibit any mutagenic effect. We conclude that a weak but chronic inflammation (more likely than acute inflammation or direct oxidative damage) in the lung tissue of fibre treated rats characterized by moderate influx of inflammatory cells into BAL is probably responsible for the observed mutagenic effect of RW1 fibres.

Benzo[a]pyrene-enhanced mutagenesis by man-made mineral fibres in the lung of lamda-lacI transgenic rats.

In an attempt to examine the interaction of man-made mineral fibres with benzo[a]pyrene (B[a]P), homozygous X-lacI transgenic F344 rats were intratracheally treated with rock (stone) wool RWI and glass wool MMVF 10 fibres together with B[a]P. To analyze the induction of gene mutations by fibres and B[a]P in lung, single doses of 1 and 2 mg fibres/animal or multiple doses of 2 mg fibres/animal were administered weekly on 4 consecutive weeks (total dose 8 mg/animal). B[a]P (10 mg/animal) was administered either simultaneously with fibres (for single dose treatment with fibres) or together with the last fiber treatment (for multiple dose treatment with fibres). Animals were scarified 4 weeks after the last treatment. Benzo[a]pyrene administered simultaneously with RW1 fibres exhibited a strong synergistic effect on mutagenicity, the observed mutant frequency (MF) being more than three-fold higher than the net sum of the MF induced after separate administration of both agents. Our data suggest that DNA adducts induced by simultaneous B[a]P and fiber treatment lead to a strong increase in mutatant frequencies.

Mutagenesis by asbestos in the lung of lambda-lacI transgenic rats.

In order to get more insight into the mechanism of asbestos-related lung cancer, the mutagenic potential of asbestos was examined in vivo in rat lung. Groups of five transgenic lambda-lacI (Big Blue) rats were intratracheally instilled with single doses of 1 or 2mg, or with four weekly doses of 2mg, per animal of the amosite asbestos. Sixteen weeks after instillation, the mutation frequency was found to be increased in lung DNA by 2-fold at doses of 2 mg (P = 0.035) and of 4 x 2 mg (P = 0.007) amosite. No significant changes were observed after 4 weeks of exposure. In separate experiments, wild-type F344 rats were treated by the same regimen as described above and markers of inflammation, genotoxicity, cell proliferation and lung tissue damage were analysed. Our results indicate a weak but persistent inflammation and cell proliferation which possibly plays a major role in the observed mutagenic effect.

Benzo[a]pyrene-enhanced mutagenesis by asbestos in the lung of lambda-lacI transgenic rats.

To study the suspected mechanism of the interaction between tobacco smoking and asbestos exposure in the modulation of cancer risk, the mutagenic potential of asbestos in combination with the tobacco smoke carcinogen benzo[a]pyrene (B[a]P) was examined in vivo in the rat lung. B[a]P was administered intratracheally in one set of experiments, or by two daily intraperitoneal injections in another set of experiments, to lambdalacI transgenic rats, together with 1, 2 or 4 x 2 mg amosite in one experiment. In the first experiment, the combined action of amosite and B[a]P caused a synergistic (superadditive) increase of mutation frequency in the lung, as compared to groups treated only with asbestos or B[a]P. In the second experiment, i.p. treatment with B[a]P did not significantly alter the mutation frequency induced by amosite, neither after 4 nor after 16 weeks of exposure. The B[a]P-DNA adduct levels were unaffected by amosite co-treatment in both experiments. We assume that the synergistic increase of mutation frequency after intratracheal treatment was due to the mitogenic activities of B[a]P and of amosite. In conclusion, our findings indicate that a weak and delayed mutagenic effect of amosite in rat lung observed in another study was strongly enhanced by the concomitant action of B[a]P. The striking enhancement effect of B[a]P may provide a basis for understanding the suspected synergism of smoking on asbestos carcinogenesis.

Some lung cellular parameters reflecting inflammation after combined inhalation of amosite dust with cigarette smoke by rats.

Cellular changes were followed in lung cell suspensions after 175 day inhalation by rats of concentrations 30 mg/m3 or 60 mg/m3 of amosite asbestos every second day combined with daily exposure to cigarette smoke at 30 mg of total particulate matter (TPM)/m3 air. Concomitantly, lung inflammation was assessed by changes in the bronchoalveolar lavage fluid (BALF). A dose-dependent rise in the BALF inflammatory parameters was found. The rise of the proportion of binucleate (BNC) and multinucleate cells (MNC) in lung cell suspensions was also dose-dependent. It is concluded that, in the experimental assessment of effects of fibrogenic dusts, the number of BNC and of MNC in lung cell suspensions may serve as a useful semiquantitative biomarker of the inflammation.

Evaluation of bronchoalveolar lavage fluid cytotoxic parameters after inhalation exposure to amosite and wollastonite fibrous dusts combined with cigarette smoke.

The aim of this work was to compare the influence of amosite-asbestos and wollastonite fibrous dusts combined with cigarette smoke on chosen cytotoxic parameters of bronchoalveolar lavage fluid (BALF) in rats. Fisher 344 rats inhaled wollastonite or amosite fibrous dusts (60 or 30 mg x m(-3) air) one hour every two days combined with daily breathing of diluted mainstream tobacco smoke (30 mg of TPM x m(-3) air). The experiment lasted 6 months. After sacrifying the animals bronchoalveolar lavage (BAL) was performed and the viability and phagocytic activity of alveolar macrophages (AM), lactate dehydrogenase (LDH) and alkaline phosphatase activity (in the cell-free BALF), acid phosphatase (ACP) and cathepsin D activity (in cell-free BALF and BAL cell suspension) were examined. Exposure to amosite without tobacco smoke significantly decreased the viability of AM and increased the cathepsin D activity in BAL cells. Exposure to wollastonite significantly increased only the cathepsin D activity in BAL cells. Smoking significantly depressed the phagocytic activity of AM and amplified the amosite-induced increase of lysosomal enzyme activities--especially the activity of cathepsin D in BAL cells.

Impact of phase I or phase II enzyme polymorphisms on lymphocyte DNA adducts in subjects exposed to urban air pollution and environmental tobacco smoke.

Little is known about the impact of genetic variation on the genetic damage induced by urban air pollution or environmental tobacco smoke (ETS) in exposed populations. The levels of bulky DNA adducts ( 32P-postlabelling, nuclease P1 enrichment) and chromosomal aberrations were measured in lymphocytes of 194 non-smoking students living in the city of Athens, and the rural region of Halkida, Greece. In these individuals personal exposure to PAH was also measured. Furthermore, genetic polymorphisms were examined in cytochromes P450 1A1, 1B1, in the GSTM1, GSTP1 and GSTT1 as well as in microsomal epoxy hydrolase (EPHX) genes. Subjects with the CYP1*2A mutant genotype also suffering significant ETS exposure tended to exhibit higher adduct levels and % aberrant cells. In contrast, CYP1B1 polymorphisms seemed to have an impact on the DNA adduct levels only among individual with negligible ETS exposure. Subjects carrying both the CYP1*2A mutant genotype and the GSTM1 null genotype tended to have higher DNA adduct levels. A similar effect was also observed with the combined CYP1A1*2A/GSTP1 (Ile/Val) and the CYP1A1*2A/mEH "slow" polymorphisms. In both cases, the effect was more pronounced among subjects with higher levels of ETS exposure. Stepwise restriction of the observations to subjects characterised by (a) GSTP1 mutant, (b) GSTM1 null, (c) mEH "slow" (His139His) genotypes and (d) ETS exposure resulted in a significant trend of increasing DNA adduct levels only among individuals with at least one CYP1A1*2A mutated allele, illustrating the importance and complexity of gene-exposure and gene-gene interactions in determining the level of genotoxic damage on an individual levels.

Monitoring for DNA damage of humans occupationally exposed to methyl bromide.

BACKGROUND: Methyl bromide (MeBr) is a methylating agent, weak mutagen and possible animal carcinogen. A molecular epidemiological study to examine human exposure to, and consequent DNA damage by MeBr was conducted in an area where this agent is used extensively for soil sterilisation in greenhouses. MATERIALS AND METHODS: During the first part of the study, blood samples were collected from 21 persons within 24 hours after use of MeBr for greenhouse sterilisation, as well as from 19 non-exposed subjects. Personal air sampling was also carried out, indicating mean air concentrations for different subjects in the range 11-78 mg/m3. In the second part of the study, an attempt was made to examine professional applicators of MeBr who suffered particularly high exposures (mean exposures, based on personal monitoring 23-165 mg/m3). The levels of N7-methylguanine and O6-methylguanine, two DNA adducts known to be induced by MeBr, were assessed in blood leukocyte DNA. RESULTS: Concerning the first part, two subjects (one exposed and one control) were found to be positive for N7-methylguanine, while none of the blood samples analysed had detectable levels of O6-methylguanine. Among 6 such persons examined during the second part, 2 were found positive for N7-methylguanine while none was positive for O6-methylguanine. CONCLUSION: Within the detection power of this limited study, no significant evidence of induction of DNA damage in blood leukocyte DNA by MeBr was found.

Biomarkers of genotoxicity of urban air pollution. Overview and descriptive data from a molecular epidemiology study on populations exposed to moderate-to-low levels of polycyclic aromatic hydrocarbons: the AULIS project.

Epidemiologic studies indicate that prolonged exposure to high pollution levels is associated with increased risk of cancer, especially lung cancer. However, under conditions of moderate or low air pollution, epidemiologic evidence does not permit reliable conclusions. Biomarker-based population studies may serve as complementary tools providing a better understanding of the relative contribution of ambient atmospheric pollution to the overall genotoxic burden suffered by city dwellers. However, past efforts to apply biomarkers to studies of low levels exposure to urban air pollution have given inconclusive results, partly because of the absence of adequate data on personal exposure, covering a time-window which is appropriate for the biomarkers being examined, as well as a battery of biomarkers reflecting different stages of the carcinogenic process. In the present paper, the potential of biomarker-based population studies to aid the assessment of the genotoxic and carcinogenic effects of urban air pollution is reviewed by reference to the achievements and limitations of earlier reported studies. The design and methodology adopted in a recently completed large-scale population study, carried out in the context of the European Union Environment and Climate Programme, known by the short name of AULIS project, is discussed and descriptive statistics of the main findings of the project are presented. These findings indicate that for cohorts suffering moderate-to-low exposures to airborne particulate-bound polycyclic aromatic hydrocarbons (PAHs), no simple correlation with biomarkers of genotoxicity existed and suggest that additional factors made a significant contribution to the overall genotoxic burden.

Biomarkers of genotoxicity of air pollution (the AULIS project): bulky DNA adducts in subjects with moderate to low exposures to airborne polycyclic aromatic hydrocarbons and their relationship to environmental tobacco smoke and other parameters.

The levels of bulky DNA adducts were measured by (32)P-post-labelling in lymphocytes of 194 non-smoking students living in the city of Athens and the region of Halkida, Greece, once in the winter and again in the following summer. Personal exposures to particulate-bound polycyclic aromatic hydrocarbons (PAH) were significantly higher in Athens subjects during both seasons. There was hardly any diagonal radioactive zone in the pattern of DNA adducts observed. Highest adduct levels were observed in a sub-group of subjects living in or near the Halkida Institute campus, which was located in rural surroundings with a minimal burden of urban air pollution. The remaining Halkida subjects had intermediate levels, while Athens subjects showed the lowest levels. This trend, which was observed over both monitoring seasons, consistently paralleled the variation in three markers of exposure to environmental tobacco smoke (ETS), namely (i) declared times of exposure to ETS during the 24 h prior to blood donation, (ii) plasma cotinine levels and (iii) chrysene/benzo[g,h,i]perylene ratios in the profile of personal PAH exposure. Furthermore, among the Halkida campus area subjects (but not the remaining subjects) positive correlations were observed between DNA adducts and (i) measured personal exposures to chrysene or benzo[a]pyrene, (ii) time of declared ETS exposure and (iii) chrysene/benzo[g,h,i] perylene ratios. These correlations suggest that, for a group suffering minimal exposure to urban air pollution, exposure to ETS was a significant determinant of the observed DNA damage. Gender had a consistent and significant effect on adduct levels (males having higher levels), which remained significant even after multiple regression analysis. Habitual consumption of roasted meat was significantly associated with an enhancement of adduct levels and the effect was strengthened when only individuals unexposed to ETS were taken into consideration. No significant effects were observed for other dietary parameters or factors reflecting exposure to air pollution.