ATG5 as biomarker for early detection of malignant mesothelioma.

OBJECTIVES: Malignant pleural mesothelioma (MPM) is an aggressive disease with grim prognosis due to lack of effective treatment options. Disease prediction in association with early diagnosis may both contribute to improved MPM survival. Inflammation and autophagy are two processes associated with asbestos-induced transformation. We evaluated the level of two autophagic factors ATG5 and HMGB1, microRNAs (miRNAs) such as miR-126 and miR-222, and the specific biomarker of MPM, soluble mesothelin related proteins (Mesothelin) in asbestos-exposed individuals, MPM patients, and healthy subjects. The performance of these markers in detecting MPM was investigated in pre-diagnostic samples of asbestos-subjects who developed MPM during the follow-up and compared for the three groups. RESULTS: The ATG5 best distinguished the asbestos-exposed subjects with and without MPM, while miR-126 and Mesothelin were found as a significant prognostic biomarker for MPM. ATG5 has been identified as an asbestos-related biomarker that can help to detect MPM with high sensitivity and specificity in pre-diagnostic samples for up to two years before diagnosis. To utilize this approach practically, higher number of cases has to be tested in order to give the combination of the two markers sufficient statistical power. Performance of the biomarkers should be confirmed by testing their combination in an independent cohort with pre-diagnostic samples.

Effects of extremely low-frequency magnetic fields on human MDA-MB-231 breast cancer cells: proteomic characterization.

Extremely low-frequency electromagnetic fields (ELF-MF) can modify the cell viability and regulatory processes of some cell types, including breast cancer cells. Breast cancer is a multifactorial disease where a role for ELF-MF cannot be excluded. ELF-MF may influence the biological properties of breast cells through molecular mechanisms and signaling pathways that are still unclear. This study analyzed the changes in the cell viability, cellular morphology, oxidative stress response and alteration of proteomic profile in breast cancer cells (MDA-MB-231) exposed to ELF-MF (50 Hz, 1 mT for 4 h). Non-tumorigenic human breast cells (MCF-10A) were used as control cells. Exposed MDA-MB-231 breast cancer cells increased their viability and live cell number and showed a higher density and length of filopodia compared with the unexposed cells. In addition, ELF-MF induced an increase of the mitochondrial ROS levels and an alteration of mitochondrial morphology. Proteomic data analysis showed that ELF-MF altered the expression of 328 proteins in MDA-MB-231 cells and of 242 proteins in MCF-10A cells. Gene Ontology term enrichment analysis demonstrated that in both cell lines ELF-MF exposure up-regulated the genes enriched in "focal adhesion" and "mitochondrion". The ELF-MF exposure decreased the adhesive properties of MDA-MB-231 cells and increased the migration and invasion cell abilities. At the same time, proteomic analysis, confirmed by Real Time PCR, revealed that transcription factors associated with cellular reprogramming were upregulated in MDA-MB-231 cells and downregulated in MCF-10A cells after ELF-MF exposure. MDA-MB-231 breast cancer cells exposed to 1 mT 50 Hz ELF-MF showed modifications in proteomic profile together with changes in cell viability, cellular morphology, oxidative stress response, adhesion, migration and invasion cell abilities. The main signaling pathways involved were relative to focal adhesion, mitochondrion and cellular reprogramming.

A 50 Hz magnetic field influences the viability of breast cancer cells 96 h after exposure.

BACKGROUND: The exposure of breast cancer to extremely low frequency magnetic fields (ELF-MFs) results in various biological responses. Some studies have suggested a possible cancer-enhancing effect, while others showed a possible therapeutic role. This study investigated the effects of in vitro exposure to 50 Hz ELF-MF for up to 24 h on the viability and cellular response of MDA-MB-231 and MCF-7 breast cancer cell lines and MCF-10A breast cell line. METHODS AND RESULTS: The breast cell lines were exposed to 50 Hz ELF-MF at flux densities of 0.1 mT and 1.0 mT and were examined 96 h after the beginning of ELF-MF exposure. The duration of 50 Hz ELF-MF exposure influenced the cell viability and proliferation of both the tumor and nontumorigenic breast cell lines. In particular, short-term exposure (4-8 h, 0.1 mT and 1.0 mT) led to an increase in viability in breast cancer cells, while long and high exposure (24 h, 1.0 mT) led to a decrease in viability and proliferation in all cell lines. Cancer and normal breast cells exhibited different responses to ELF-MF. Mitochondrial membrane potential and reactive oxygen species (ROS) production were altered after ELF-MF exposure, suggesting that the mitochondria are a probable target of ELF-MF in breast cells. CONCLUSIONS: The viability of breast cells in vitro is influenced by ELF-MF exposure at magnetic flux densities compatible with the limits for the general population and for workplace exposures. The effects are apparent after 96 h and are related to the ELF-MF exposure time.

Mechanism of miR-222 and miR-126 regulation and its role in asbestos-induced malignancy.

MiR-222 and miR-126 are associated with asbestos exposure and the ensuing malignancy, but the mechanism(s) of their regulation remain unclear. We evaluated the mechanism by which asbestos regulates miR-222 and miR-126 expression in the context of cancer etiology. An 'in vitro' model of carcinogen-induced cell transformation was used based on exposing bronchial epithelium BEAS-2B cells to three different carcinogens including asbestos. Involvement of the EGFR pathway and the role of epigenetics have been investigated in carcinogen-transformed cells and in malignant mesothelioma, a neoplastic disease associated with asbestos exposure. Increased expression of miR-222 and miR-126 were found in asbestos-transformed cells, but not in cells exposed to arsenic and chrome. Asbestos-mediated activation of the EGFR pathway and macrophages-induced inflammation resulted in miR-222 upregulation, which was reversed by EGFR inhibition. Conversely, asbestos-induced miR-126 expression was affected neither by EGFR modulation nor inflammation. Rather than methylation of the miR-126 host gene EGFL7, epigenetic mechanism involving DNMT1- and PARP1-mediated chromatin remodeling was found to upregulate of miR-126 in asbestos-exposed cells, while miR-126 was downregulated in malignant cells. Analysis of MM tissue supported the role of PARP1 in miR-126 regulation. Therefore, activation of the EGFR pathway and the PARP1-mediated epigenetic regulation both play a role in asbestos-induced miRNA expression, associated with in asbestos-induced carcinogenesis and tumor progression.

Exosomal transfer of miR-126 promotes the anti-tumour response in malignant mesothelioma: Role of miR-126 in cancer-stroma communication.

MiR-126 has been shown to suppress malignant mesothelioma (MM) by targeting cancer-related genes without inducing toxicity or histopathological changes. Exosomes provide the opportunity to deliver therapeutic cargo to cancer stroma. Here, a tumour stromal model composed of endothelial cells (HUVECs), fibroblasts (IMR-90 cells), non-malignant mesothelial cells (Met-5A cells) and MM cells (H28 and MM-B1 cells) was used. The cells were treated with exosomes from HUVECs carrying endogenous (exo-HUVEC) and enriched miR-126 (exo-HUVECmiR-126), and the uptake/turnover of exosomes; miR-126 distribution within the stroma; and effect of miR-126 on cell signalling, angiogenesis and cell proliferation were evaluated. Based on the sensitivity of MM cells to exo-HUVEC miR-126 treatment, miR-126 was distributed differently across stromal cells. The reduced miR-126 content in fibroblasts in favour of endothelial cells reduced angiogenesis and suppressed cell growth in an miR-126-sensitive environment. Conversely, the accumulation of miR-126 in fibroblasts and the reduced level of miR-126 in endothelial cells induced tube formation in an miR-126-resistant environment via VEGF/EGFL7 upregulation and IRS1-mediated cell proliferation. These findings suggest that transfer of miR-126 via HUVEC-derived exosomes represents a novel strategy to inhibit angiogenesis and cell growth in MM.

NOCTURNIN Gene Diurnal Variation in Healthy Volunteers and Expression Levels in Shift Workers.

OBJECTIVE: The NOCTURNIN gene links nutrient absorption and metabolism to the circadian clock. Shift workers are at a heightened risk of overweight and of developing obesity and metabolic syndrome. This study investigates the diurnal variation of NOCTURNIN in healthy volunteers and its expression levels in rotational shift and daytime workers. METHODS: NOCTURNIN expression levels were evaluated in peripheral blood lymphocytes from 15 healthy volunteers at 4-hour intervals for 24 h. Metabolic parameters and NOCTURNIN expression were measured in workers engaged in shift and daytime work. RESULTS: In the group of volunteers NOCTURNIN expression showed diurnal variation, with a peak at 8:00 AM. NOCTURNIN expression was higher in shift workers than in daytime workers. Multivariate analysis confirmed the role of shift work as an independent factor affecting NOCTURNIN expression. Notably, its level correlated directly with body mass index and inversely with total energy expenditure. CONCLUSIONS: Measuring NOCTURNIN expression levels in human peripheral blood lymphocytes can improve investigations on the relationship between changes in circadian rhythm and metabolic disorders. Shift workers show higher NOCTURNIN levels than daytime workers.

BRCA1 and BRCA2 Gene Expression: Diurnal Variability and Influence of Shift Work

BRCA1 and BRCA2 genes are involved in DNA double-strand break repair and related to breast cancer. Shift work is associated with biological clock alterations and with a higher risk of breast cancer. The aim of this study was to investigate the variability of expression of BRCA genes through the day in healthy subjects and to measure BRCA expression levels in shift workers. The study was approached in two ways. First, we examined diurnal variation of BRCA1 and BRCA2 genes in lymphocytes of 15 volunteers over a 24-hour period. Second, we measured the expression of these genes in lymphocytes from a group of shift and daytime workers. The change in 24-hour expression levels of BRCA1 and BRCA2 genes was statistically significant, decreasing from the peak at midday to the lowest level at midnight. Lower levels for both genes were found in shift workers compared to daytime workers. Diurnal variability of BRCA1 and BRCA2 expression suggests a relation of DNA double-strand break repair system with biological clock. Lower levels of BRCA1 and BRCA2 found in shift workers may be one of the potential factors related to the higher risk of breast cancer.

Four-miRNA Signature to Identify Asbestos-Related Lung Malignancies.

BACKGROUND: Altered miRNA expression is an early event upon exposure to occupational/environmental carcinogens; thus, identification of a novel asbestos-related profile of miRNAs able to distinguish asbestos-induced cancer from cancer with different etiology can be useful for diagnosis. We therefore performed a study to identify miRNAs associated with asbestos-induced malignancies. METHODS: Four groups of patients were included in the study, including patients with asbestos-related (NSCLCAsb) and asbestos-unrelated non-small cell lung cancer (NSCLC) or with malignant pleural mesothelioma (MPM), and disease-free subjects (CTRL). The selected miRNAs were evaluated in asbestos-exposed population. RESULTS: Four serum miRNAs, that is miR-126, miR-205, miR-222, and miR-520g, were found to be implicated in asbestos-related malignant diseases. Notably, increased expression of miR-126 and miR-222 were found in asbestos-exposed subjects, and both miRNAs are involved in major pathways linked to cancer development. Epigenetic changes and cancer-stroma cross-talk could induce repression of miR-126 to facilitate tumor formation, angiogenesis, and invasion. CONCLUSIONS: This study indicates that miRNAs are potentially involved in asbestos-related malignancies, and their expression outlines mechanism(s) whereby miRNAs may be involved in an asbestos-induced pathogenesis. IMPACT: The discovery of a miRNA panel for asbestos-related malignancies would impact on occupational compensation and may be utilized for screening asbestos-exposed populations.

MiR-126 in intestinal-type sinonasal adenocarcinomas: exosomal transfer of MiR-126 promotes anti-tumour responses.

BACKGROUND: Intestinal-type sinonasal adenocarcinomas (ITACs) are aggressive malignancies related to wood dust and leather exposure. ITACs are generally associated with advanced stage at presentation due to the insidious growth pattern and non-specific symptoms. Therefore, biomarkers that can detect the switch from the benign disease to malignancy are needed. Essential for tumour growth, angiogenesis is an important step in tumour development and progression. This process is strictly regulated, and MiR-126 considered its master modulator. METHODS: We have investigated MiR-126 levels in ITACs and compared them to benign sinonasal lesions, such as sinonasal-inverted papillomas (SIPs) and inflammatory polyps (NIPs). The tumour-suppressive functions of MiR-126 were also evaluated. RESULTS: We found that MiR-126 can significantly distinguish malignancy from benign nasal forms. The low levels of MiR-126 in ITACs point to its role in tumour progression. In this context, restoration of MiR-126 induced metabolic changes, and inhibited cell growth and the tumorigenic potential of MNSC cells. CONCLUSIONS: We report that MiR-126 delivered via exosomes from endothelial cells promotes anti-tumour responses. This paracrine transfer of MiRs may represent a new approach towards MiR-based therapy.

DNA damage response in workers exposed to low-dose ionising radiation.

OBJECTIVE: Medical personnel using radiation for diagnosis and therapeutic purposes are potentially at risk of cancer development. In this study, the effect of ionising radiation (IR) exposure was evaluated as DNA damage response (DDR) in the circulating cells of occupationally exposed subjects. METHODS: The study population consisted of IR-exposed workers included both in group B (effective dose ranging between 0.04 and 6 mSv/year) and group A (probable effective dose exceeding 6 mSv/year), and the control group consisted of healthy individuals who had never been occupationally exposed to IR or other known carcinogenic agents. DNA damage (single-strand breaks, oxidised purine and pyrimidine bases) and DNA repair (t1/2, half time to repair DNA damage, amount of repaired DNA and DNA repair activity) were measured in lymphocytes using the comet assay. To evaluate the influence of IR doses and genetic predisposition to cancer, the enrolled population was stratified according to IR exposure level and family history of cancer. RESULTS: Increased DNA repair activity was found in IR-exposed group, and only subjects highly exposed to IR doses accumulated DNA damage in their circulating cells, thus supporting the hypothesis of 'radiation hormesis'. A significant increase in DNA damage accumulation and a reduced 8-oxoguanine glycosylase 1-dependent DNA repair activity were found in IR-exposed subjects with cancer cases across their family. CONCLUSION: Our results indicate that chronic exposure to a low dose of IR in occupational settings induces DDR in exposed subjects and may be mutagenic in workers with family history of cancer, suggesting that periodic surveillance might be advisable, along with exposure monitoring.

CRISPR/Cas9 and active genetics-based trans-species replacement of the endogenous Drosophila kni-L2 CRM reveals unexpected complexity.

The knirps (kni) locus encodes transcription factors required for induction of the L2 wing vein in Drosophila. Here, we employ diverse CRISPR/Cas9 genome editing tools to generate a series of targeted lesions within the endogenous cis-regulatory module (CRM) required for kni expression in the L2 vein primordium. Phenotypic analysis of these 'in locus' mutations based on both expression of Kni protein and adult wing phenotypes, reveals novel unexpected features of L2-CRM function including evidence for a chromosome pairing-dependent process that promotes transcription. We also demonstrate that self-propagating active genetic elements (CopyCat elements) can efficiently delete and replace the L2-CRM with orthologous sequences from other divergent fly species. Wing vein phenotypes resulting from these trans-species enhancer replacements parallel features of the respective donor fly species. This highly sensitive phenotypic readout of enhancer function in a native genomic context reveals novel features of CRM function undetected by traditional reporter gene analysis.

Exosomal miR-126 as a circulating biomarker in non-small-cell lung cancer regulating cancer progression.

Lung cancer is one of the leading causes of cancer-related deaths. It is diagnosed mostly at the locally advanced or metastatic stage. Recently, micro RNAs (miRs) and their distribution in circulation have been implicated in physiological and pathological processes. In this study, miR-126 was evaluated in serum, exosome and exosome-free serum fractions in non-small cell lung cancer (NSCLC) patients at early and advanced stages, and compared with healthy controls. Down-regulation of miR-126 was found in serum of advanced stage NSCLC patients. In healthy controls, circulating miR-126 was equally distributed between exosomes and exosome-free serum fractions. Conversely, in both early and advanced stage NSCLC patients, miR-126 was mainly present in exosomes. Different fractions of miR-126 in circulation may reflect different conditions during tumour formation. Incubation of exosomes from early and advanced NSCLC patients induced blood vessel formation and malignant transformation in human bronchial epithelial cells. On the other hand, exosome-enriched miR-126 from normal endothelial cells inhibited cell growth and induces loss of malignancy of NSCLC cells. These findings suggest a role of exo-miRs in the modulation of the NSCLC microenvironmental niche. Exosome-delivered miRs thus hold a substantial promise as a diagnostics biomarker as well as a personalized therapeutic modality.

Peripheral Skin Temperature and Circadian Biological Clock in Shift Nurses after a Day off.

The circadian biological clock is essentially based on the light/dark cycle. Some people working with shift schedules cannot adjust their sleep/wake cycle to the light/dark cycle, and this may result in alterations of the circadian biological clock. This study explored the circadian biological clock of shift and daytime nurses using non-invasive methods. Peripheral skin temperature, cortisol and melatonin levels in saliva, and Per2 expression in pubic hair follicle cells were investigated for 24 h after a day off. Significant differences were observed in peripheral skin temperature and cortisol levels between shift and daytime nurses. No differences in melatonin levels were obtained. Per2 maximum values were significantly different between the two groups. Shift nurses exhibited lower circadian variations compared to daytime nurses, and this may indicate an adjustment of the circadian biological clock to continuous shift schedules. Non-invasive procedures, such as peripheral skin temperature measurement, determination of cortisol and melatonin in saliva, and analysis of clock genes in hair follicle cells, may be effective approaches to extensively study the circadian clock in shift workers.

MicroRNA-126 induces autophagy by altering cell metabolism in malignant mesothelioma.

Autophagy favors both cell survival and cancer suppression, and increasing evidence reveals that microRNAs (MIRs) regulate autophagy. Previously we reported that MIR126 is downregulated in malignant mesothelioma (MM). Therefore, we investigated the role of MIR126 in the regulation of cell metabolism and autophagy in MM models. We report that MIR126 induces autophagic flux in MM cells by downregulating insulin receptor substrate-1 (IRS1) and disrupting the IRS1 signaling pathway. This was specific to MM cells, and was not observed in non-malignant cells of mesothelial origin or in MM cells expressing MIR126-insensitive IRS1 transcript. The MIR126 effect on autophagy in MM cells was recapitulated by IRS1 silencing, and antagonized by IRS1 overexpression or antisense MIR126 treatment. The MIR126-induced loss of IRS1 suppressed glucose uptake, leading to energy deprivation and AMPK-dependent phosphorylation of ULK1. In addition, MIR126 stimulated lipid droplet accumulation in a hypoxia-inducible factor-1α (HIF1α)-dependent manner. MIR126 also reduced pyruvate dehydrogenase kinase (PDK) and acetyl-CoA-citrate lyase (ACL) expression, leading to the accumulation of cytosolic citrate and paradoxical inhibition of pyruvate dehydrogenase (PDH) activity. Simultaneous pharmacological and genetic intervention with PDK and ACL activity phenocopied the effects of MIR126. This suggests that in MM MIR126 initiates a metabolic program leading to high autophagic flux and HIF1α stabilization, incompatible with tumor progression of MM. Consistently, MIR126-expressing MM cells injected into immunocompromised mice failed to progress beyond the initial stage of tumor formation, showing that increased autophagy has a protective role in MM.

Combined circulating epigenetic markers to improve mesothelin performance in the diagnosis of malignant mesothelioma.

OBJECTIVES: Malignant mesothelioma (MM) is a highly aggressive tumor with poor prognosis. A major challenge is the development and application of early and highly reliable diagnostic marker(s). Serum biomarkers, such as 'soluble mesothelin-related proteins' (SMRPs), is the most studied and frequently used in MM. However, the low sensitivity of SMRPs for early MM limits its value; therefore, additional biomarkers are required. In this study, two epigenetically regulated markers in MM (microRNA-126, miR-126, and methylated thrombomodulin promoter, Met-TM) were combined with SMRPs and evaluated as a potential strategy to detect MM at an early stage. MATERIALS AND METHODS: A total of 188 subjects, including 45 MM patients, 99 asbestos-exposed subjects, and 44 healthy controls were prospectively enrolled, serum samples collected, and serum levels of SMRPs, miR-126 and Met-TM evaluated. Logistic regression analysis was performed to evaluate the diagnostic value of the three biomarkers. Using this approach, the performance of the '3-biomarker classifier' was tested by calculating the overall probability score of the MM and control samples, respectively, and the ROC curve was generated. RESULTS AND CONCLUSION: The combination of the three biomarkers was the best predictor to differentiate MM patients from asbestos-exposed subjects and healthy controls. The accuracy and cancer specificity was confirmed in a second validation cohort and lung cancer population. We propose that the combination of the two epigenetic biomarkers with SMRPs as a diagnosis for early MM overcomes the limitations of using SMRPs alone.

Circadian Modulation of 8-Oxoguanine DNA Damage Repair.

The DNA base excision repair pathway is the main system involved in the removal of oxidative damage to DNA such as 8-Oxoguanine (8-oxoG) primarily via the 8-Oxoguanine DNA glycosylase (OGG1). Our goal was to investigate whether the repair of 8-oxoG DNA damage follow a circadian rhythm. In a group of 15 healthy volunteers, we found a daily variation of Ogg1 expression and activity with higher levels in the morning compared to the evening hours. Consistent with this, we also found lower levels of 8-oxoG in morning hours compared to those in the evening hours. Lymphocytes exposed to oxidative damage to DNA at 8:00 AM display lower accumulation of 8-oxoG than lymphocytes exposed at 8:00 PM. Furthermore, altered levels of Ogg1 expression were also observed in a group of shift workers experiencing a deregulation of circadian clock genes compared to a control group. Moreover, BMAL1 knockdown fibroblasts with a deregulated molecular clock showed an abolishment of circadian variation of Ogg1 expression and an increase of OGG1 activity. Our results suggest that the circadian modulation of 8-oxoG DNA damage repair, according to a variation of Ogg1 expression, could render humans less susceptible to accumulate 8-oxoG DNA damage in the morning hours.

Circadian gene expression and extremely low-frequency magnetic fields: an in vitro study.

It is well known that circadian clocks are mainly regulated by light targeting signaling pathways in the hypothalamic suprachiasmatic nucleus. However, an entrainment mediated by non-photic sensory stimuli was also suggested for peripheral clocks. Exposure to extremely low frequency (ELF) electromagnetic fields might affect circadian rhythmicity. The goal of this research was to investigate effects of ELF magnetic fields (ELF-MF) on circadian clock genes in a human fibroblast cell line. We found that an ELF-MF (0.1 mT, 50 Hz) exposure was capable of entraining expression of clock genes BMAL1, PER2, PER3, CRY1, and CRY2. Moreover, ELF-MF treatment induced an alteration in circadian clock gene expression previously entrained by serum shock stimulation. These results support the hypothesis that ELF-MF may be able to drive circadian physiologic processes by modulating peripheral clock gene expression.

Wood dust exposure induces cell transformation through EGFR-mediated OGG1 inhibition.

A high risk of neoplastic transformation of nasal and paranasal sinuses mucosa is related to the occupational exposure to wood dust. However, the role of occupational exposures in the aetiology of the airway cancers remains largely unknown. Here, an in vitro model was performed to investigate the carcinogenic effect of wood dusts. Human bronchial epithelial cells were incubated with hard and soft wood dusts and the DNA damage and response to DNA damage evaluated. Wood dust exposure induced accumulation of oxidised DNA bases, which was associated with a delay in DNA repair activity. By exposing cells to wood dust at a prolonged time, wood dust-initiated cells were obtained. Initiated-cells were able to form colonies in soft agar, and to induce blood vessel formation. These cells showed extensive autophagy, reduced DNA repair, which was associated with reduced OGG1 expression and oxidised DNA base accumulation. These events were found related to the activation of EGFR/AKT/mTOR pathway, through phosphorylation and subsequent inactivation of tuberin. The persistence in the tissue of wood dusts, their repetitious binding with EGFR may continually trigger the activation switch, leading to chronic down-regulation of genes involved in DNA repair, leading to cell transformation and proliferation.

Rotating-shift nurses after a day off: peripheral clock gene expression, urinary melatonin, and serum 17-ß-estradiol levels.

OBJECTIVE: Impairment of clock gene expression and changes in melatonin and 17-ß-estradiol levels may constitute biological alterations underlying the increased risk of breast cancer among shift workers. The aim of this study was to compare levels of selected core clock gene expression, 6-sulfatoxymelatonin (aMT6s), and 17-ß-estradiol between rotational shift work (SW) and daytime (DT) workers after a day off. METHODS: The cross-sectional study comprised 60 nurses with ≥2 years of SW and 56 permanent DT nurses. Transcript levels of circadian genes BMAL1, CLOCK, NPAS2, CRY1, CRY2, PER1, PER2, PER3, and REVERBα were determined by quantitative real-time polymerase chain reaction (PCR) in lymphocytes. All participants were tested in the early follicular phase of the menstrual cycle. Samples were collected at the beginning of the morning-shift after a regular night's sleep on a day off. Chronotype and sociodemographic characteristics were also evaluated. RESULTS: We found a significantly higher expression of BMAL1, CLOCK, NPAS2, PER1, PER2, and REVERBα and a lower expression of PER3, CRY1 and CRY2 among SW compared to DT nurses. SW participants did not demonstrate a significant difference in aMT6s levels, but they did show significantly higher 17-ß-estradiol levels compared to DT nurses. Multiple linear regression analysis confirmed the role of SW on expression of BMAL1 (ß 0.21, P=0.040), CLOCK (ß 0.35, P=0.008), NPAS2 (ß 0.30, P=0.012), PER1 (ß 0.33, P=0.008), PER2 (ß 0.19, P=0.047), PER3 (ß -0.27, P=0.012), CRY1 (ß -0.33, P=0.002), CRY2 (ß -0.31, P=0.005), REVERBα (ß 0.19, P=0.045), and on 17-ß-estradiol levels (ß 0.32, P=0.003). The analysis also confirmed the role of chronotype as an independent factor for PER1 (ß 0.48, P=0.001) and PER2 (ß -0.22, P=0.022) expression, and 17-ß-estradiol levels (ß 0.26, P=0.011). CONCLUSIONS: Rotating SW nurses show alterations in peripheral clock gene expression and 17-ß-estradiol levels at the beginning of the morning shift after a day off.

Occupational styrene exposure induces stress-responsive genes involved in cytoprotective and cytotoxic activities.

OBJECTIVE: The aim of this study was to evaluate the expression of a panel of genes involved in toxicology in response to styrene exposure at levels below the occupational standard setting. METHODS: Workers in a fiber glass boat industry were evaluated for a panel of stress- and toxicity-related genes and associated with biochemical parameters related to hepatic injury. Urinary styrene metabolites (MA+PGA) of subjects and environmental sampling data collected for air at workplace were used to estimate styrene exposure. RESULTS: Expression array analysis revealed massive upregulation of genes encoding stress-responsive proteins (HSPA1L, EGR1, IL-6, IL-1ß, TNSF10 and TNFα) in the styrene-exposed group; the levels of cytokines released were further confirmed in serum. The exposed workers were then stratified by styrene exposure levels. EGR1 gene upregulation paralleled the expression and transcriptional protein levels of IL-6, TNSF10 and TNFα in styrene exposed workers, even at low level. The activation of the EGR1 pathway observed at low-styrene exposure was associated with a slight increase of hepatic markers found in highly exposed subjects, even though they were within normal range. The ALT and AST levels were not affected by alcohol consumption, and positively correlated with urinary styrene metabolites as evaluated by multiple regression analysis. CONCLUSION: The pro-inflammatory cytokines IL-6 and TNFα are the primary mediators of processes involved in the hepatic injury response and regeneration. Here, we show that styrene induced stress responsive genes involved in cytoprotection and cytotoxicity at low-exposure, that proceed to a mild subclinical hepatic toxicity at high-styrene exposure.