Disruption of central and peripheral circadian clocks and circadian controlled estrogen receptor rhythms in night shift nurses in working environments.

Chronic disruption of circadian rhythms by night shift work is associated with an increased breast cancer risk. However, little is known about the impact of night shift on peripheral circadian genes (CGs) and circadian-controlled genes (CCGs) associated with breast cancer. Hence, we assessed central clock markers (melatonin and cortisol) in plasma, and peripheral CGs (PER1, PER2, PER3, and BMAL1) and CCGs (ESR1 and ESR2) in peripheral blood mononuclear cells (PBMCs). In day shift nurses (n = 12), 24-h rhythms of cortisol and melatonin were aligned with day shift-oriented light/dark schedules. The mRNA expression of PER2, PER3, BMAL1, and ESR2 showed 24-h rhythms with peak values in the morning. In contrast, night shift nurses (n = 10) lost 24-h rhythmicity of cortisol with a suppressed morning surge but retained normal rhythmic patterns of melatonin, leading to misalignment between cortisol and melatonin. Moreover, night shift nurses showed disruption of rhythmic expressions of PER2, PER3, BMAL1, and ESR2 genes, resulting in an impaired inverse correlation between PER2 and BMAL1 compared to day shift nurses. The observed trends of disrupted circadian markers were recapitulated in additional day (n = 20) and night (n = 19) shift nurses by measurement at early night and midnight time points. Taken together, this study demonstrated the misalignment of cortisol and melatonin, associated disruption of PER2 and ESR2 circadian expressions, and internal misalignment in peripheral circadian network in night shift nurses. Morning plasma cortisol and PER2, BMAL1, and ESR2 expressions in PBMCs may therefore be useful biomarkers of circadian disruption in shift workers.

Combined use of multiparametric high-content-screening and in vitro circadian reporter assays in neurotoxicity evaluation.

Accumulating evidence indicates that chronic circadian rhythm disruption is associated with the development of neurodegenerative diseases induced by exposure to neurotoxic chemicals. Herein, we examined the relationship between cellular circadian rhythm disruption and cytotoxicity in neural cells. Moreover, we evaluated the potential application of an in vitro cellular circadian rhythm assay in determining circadian rhythm disruption as a sensitive and early marker of neurotoxicant-induced adverse effects. To explore these objectives, we established an in vitro cellular circadian rhythm assay using human glioblastoma (U87 MG) cells stably transfected with a circadian reporter vector (PER2-dLuc) and determined the lowest-observed-adverse-effect levels (LOAELs) of several common neurotoxicants. Additionally, we determined the LOAEL of each compound on multiple cytotoxicity endpoints (nuclear size [NC], mitochondrial membrane potential [MMP], calcium ions, or lipid peroxidation) using a multiparametric high-content screening (HCS) assay using transfected U87 MG cells treated with the same neurotoxicants for 24 and 72 h. Based on our findings, the LOAEL for cellular circadian rhythm disruption for most chemicals was slightly higher than that for most cytotoxicity indicators detected using HCS, and the LOAEL for MMP in the first 24 h was the closest to that for cellular circadian rhythm disruption. Dietary antioxidants (methylselenocysteine and N-acetyl-l-cysteine) prevented or restored neurotoxicant-induced cellular circadian rhythm disruption. Our results suggest that cellular circadian rhythm disruption is as sensitive as cytotoxicity indicators and occurs early as much as cytotoxic events during disease development. Moreover, the in vitro cellular circadian rhythm assay warrants further evaluation as an early screening tool for neurotoxicants.

Airborne Microplastic Concentrations in Five Megacities of Northern and Southeast China.

Airborne microplastics (MPs) are receiving increasing attention due to their ubiquitous nature and the potential human health consequences resulting from inhalation. The limited data for airborne MP concentrations vary widely among studies (∼4 orders of magnitude), but comparisons are tenuous due to the inconsistent collection and detection/enumeration methodologies among studies. Herein, we used uniform methodologies to obtain comparable airborne MP concentration data to assess MP exposure intensity in five Chinese megacities. Airborne MP concentrations in northern cities (358 ± 132 items/m3) were higher than those in southeast cities (230 ± 94 items/m3) but of a similar order of magnitude, unlike previous studies. The majority (94.7%) of MPs found in air samples were smaller than 100 µm, and the main shape of airborne MPs was fragments (88.2%). Polyethylene, polyester, and polystyrene were the dominant polymers comprising airborne MPs. No consistent relationships were detected between airborne MP concentration and typical socioeconomic indices, and the spatial and diurnal patterns for airborne MPs were different from various components of air quality indices (PM2.5, PM10, etc.). These findings reflect the contrasting source/generation dynamics between airborne MPs and other airborne pollutants. Maximum annual exposure of humans to airborne MPs was estimated in the range of 1-2 million/year in these megacities, highlighting the need for additional research examining the human health risks from the inhalation of airborne MPs.

DNA methylome and transcriptome alterations and cancer prevention by curcumin in colitis-accelerated colon cancer in mice.

Inflammation is highly associated with colon carcinogenesis. Epigenetic mechanisms could play an important role in the initiation and progression of colon cancer. Curcumin, a dietary phytochemical, shows promising effects in suppressing colitis-associated colon cancer in azoxymethane-dextran sulfate sodium (AOM-DSS) mice. However, the potential epigenetic mechanisms of curcumin in colon cancer remain unknown. In this study, the anticancer effect of curcumin in suppressing colon cancer in an 18-week AOM-DSS colon cancer mouse model was confirmed. We identified lists of differentially expressed and differentially methylated genes in pairwise comparisons and several pathways involved in the potential anticancer effect of curcumin. These pathways include LPS/IL-1-mediated inhibition of RXR function, Nrf2-mediated oxidative stress response, production of NO and ROS in macrophages and IL-6 signaling. Among these genes, Tnf stood out with decreased DNA CpG methylation of Tnf in the AOM-DSS group and reversal of the AOM-DSS induced Tnf demethylation by curcumin. These observations in Tnf methylation correlated with increased and decreased Tnf expression in RNA-seq. The functional role of DNA methylation of Tnf was further confirmed by in vitro luciferase transcriptional activity assay. In addition, the DNA methylation level in a group of inflammatory genes was decreased in the AOM+DSS group but restored by curcumin and was validated by pyrosequencing. This study shows for the first time epigenomic changes in DNA CpG methylation in the inflammatory response from colitis-associated colon cancer and the reversal of their CpG methylation changes by curcumin. Future clinical epigenetic studies with curcumin in inflammation-associated colon cancer would be warranted.

World Trade Center (WTC) dust exposure in mice is associated with inflammation, oxidative stress and epigenetic changes in the lung.

Exposure to World Trade Center (WTC) dust has been linked to respiratory disease in humans. In the present studies we developed a rodent model of WTC dust exposure to analyze lung oxidative stress and inflammation, with the goal of elucidating potential epigenetic mechanisms underlying these responses. Exposure of mice to WTC dust (20µg, i.t.) was associated with upregulation of heme oxygenase-1 and cyclooxygenase-2 within 3days, a response which persisted for at least 21days. Whereas matrix metalloproteinase was upregulated 7days post-WTC dust exposure, IL-6RA1 was increased at 21days; conversely, expression of mannose receptor, a scavenger receptor important in particle clearance, decreased. After WTC dust exposure, increases in methylation of histone H3 lysine K4 at 3days, lysine K27 at 7days and lysine K36, were observed in the lung, along with hypermethylation of Line-1 element at 21days. Alterations in pulmonary mechanics were also observed following WTC dust exposure. Thus, 3days post-exposure, lung resistance and tissue damping were decreased. In contrast at 21days, lung resistance, central airway resistance, tissue damping and tissue elastance were increased. These data demonstrate that WTC dust-induced inflammation and oxidative stress are associated with epigenetic modifications in the lung and altered pulmonary mechanics. These changes may contribute to the development of WTC dust pathologies.

[The relationship between fat intake behavior and psychological impact factor in adolescent].

OBJECTIVE: To learn the dietary fat intake behavior status and influencing psychological factors in teenagers. METHODS: According to the multi-stage stratified cluster sampling method, students were recruited from middle and high schools in Hangzhou, Wuhan and Xi'an from March to May, 2012. Chinese version of adolescent dietary fat intake behavior of psychological measurement scales was utilized in field investigations with 3 448 effective questionnaires. Under the transtheoretical model, the status quo of teenagers fat intake behavior was analyzed and it shows the relationship between stages and psychological variables, including strategy of change, decision balance and self-efficacy. RESULTS: In the 3 448 participants, the proportion of girls were 52.4% (1 806/3 448) and boys were 47.6% (1 642/3 448), while the mean age was (14.85 ± 1.46). There are significant differences among distributed stages of behavior change, grouped by gender, grade and region (χ(2) values were 33.59, 20.53, 27.92, P < 0.001). In different gender groups, the number of boys in precontemplation came to the first and accounted for 24.2% (438/1 806), and the number of girls were more in contemplation and preparation stage, which accounted for 49.5% (813/1 642), and it was the least both in the action stage with the ratio 12.1% (218/1 806) and 14.7% (241/1 642) respectively; in different grade groups, the numbers of student in middle school and high school were the largest in contemplation and precontemplation stage, accounted for 24.9% (494/1 986) and 23.4% (343/1 462); and among different regions, the proportion in precontemplation, contemplation and preparation stage was not a few, and this ratio in Hangzhou, Wuhan, Xi'an area was 70.6% (801/1 135), 61.7% (649/1 052) and 68.8% (867/1 261), respectively. The number in action stage was the least of all, which were 144, 147, 168, respectively. Ordinal logistic mode showed that the four variables, including the scores of process of change, decision balance (pros), decision balance (cons) and self-efficacy, were factors which significantly affected the stages of behavior change, which showed a significant difference (F values were 155.12, 19.86, 44.59, 94.27, P < 0.001) in different stages. From precontemplation stage to action stage, the score of the process of change, decision balance (pros) and self-efficacy showed an upward trend, and achieved the highest score in the action stage (from 32.6 ± 12.8, 8.4 ± 2.9, 19.7 ± 6.9 to 48.4 ± 12.6, 10.1 ± 2.5, 25.9 ± 6.9, respectively); contrarily, the decision balance (cons) decreased with the development of behavioral stage (from 14.7 ± 4.4 to 12.2 ± 3.9). Post hoc comparison also found that the score of process of change, decision balance (pros) and self-efficacy after action stages were higher than those before action, while the score of decisional balance (cons) in the precontemplation stage was higher than that in the other 4 stages. CONCLUSION: Fat intake behavior in teenagers showed different stages, whose psychological factors had significant influences, with decision balance (pros) hindered the progress of stages of behavior change, while other three factors played a positive role.

[Effects of family and peer support upon the stages of health-related behavior in adolescent].

OBJECTIVE: To investigate the stages of health-related behaviors, family and peer support situation about adolescent in Wuhan, and to provide evidence for health intervention and health promotion. METHODS: Based on the principle of multi-stage stratified cluster sampling, extracted two junior middle school and two high school in two cities in Wuhan from March 2012 to May. A total of 1 200 students, which were part of the first and second grade class in middle school and high school, toke as the investigation object. The study surveyed following content, including the demographic situation, adolescent health-related behavior (physical activity, sedentary behavior, high-fat diet behavior, fruit and vegetable intaking behavior), stages of adolescent health related behavior (precontemplation, contemplation, preparation, action, maintenance) and adolescent support got from family and peer. The questionnaire was distributed in 1 200 copies, the valid questionnaires were 1 052 and the effective rate was 87.67%. Using χ² test to compare the different stages of change in health related behavior, t test was used to compare different groups of social support and using analysis of variance and post test to analyze social support of the four types behavior. RESULTS: In 1 052 students, there was 555 (52.8%) middle school students, 497 (47.2%) high school students, and 553 (52.6%) boys, 499 (47.4%) girls. In physical exercise and sedentary behavior, numbers of precontemplation was respectively accounting for 42.2% (444/1 200) and 28.4% (299/1 200). In high-fat diet behavior, the stages of preparation and maintain was occupied 46.7% (492/1 200). In fruit and vegetable intake behavior, there was 32.9% (346/1200) students in contemplation. Numbers in action stage was both less in the above four behavior, accounting for 6.2% (65/1 200), 8.9% (94/1 200), 14.0% (147/1 200) and 6.3% (66/1 200) separately. Adolescent peer support score respectively (2.9 ± 0.7), (2.8 ± 0.8) and (2.9 ± 0.9), which was higher than the family support scores ((2.7 ± 1.1), (2.5 ± 1.2) and (2.9 ± 1.2)) (t values were -8.72, -11.22 and -2.59, respectively. All P values were < 0.001) in physical exercise, sedentary behavior and high fat diet, but in the intake of fruits and vegetables behavior, family support score was higher than peer support (score was respectively (3.3 ± 0.9) and (3.5 ± 1.2); t < 9.97, P < 0.001). ANOVA and post hoc comparison showed that the score of family and peer support in the after-action stage was significantly higher than that the before-action stage in physical exercise, high-fat diet and fruit and vegetable intake behavior, while the differences in sedentary behavior was not significant. CONCLUSION: Youth health-related behaviors remained in its infancy, there was a certain impact on its stages which showed that the higher social support the more advanced stages of behavior.

[Evaluation on the Chinese version of adolescent fat intake behavior of psychological measurement scale and its reliability and validity].

OBJECTIVE: To develop a suitable fat intake behavior of psychological measurement scales for the Chinese adolescents and evaluate its validity and reliability. METHODS: According to the multi-stage stratified cluster sampling principle, a total of 3 600 junior students were recruited from the classes in 12 selected high schools in Hangzhou, Wuhan and Xi'an from March to May, 2012. Based on introducing and translating the original scale abroad, Chinese version of adolescent fat intake behavior of psychological measurement scales was utilized in field investigations. The reliability was assessed, using Cronbach's α and split-half reliability; while exploratory factor analysis used to test its validity, with entries-dimension correlation coefficient (IIC), correlation coefficient between the scores and the dimension, and the dimension of correlation coefficient test content validity. RESULTS: The valid subject of the study was 3 448(of whom males were 52.4% (1 806/3 448) and female were 47.6% (1 642/3 448)), while the mean age was (14.85 ± 1.46) years old. The internal consistency reliability (Cronbach's α) for total scale score and four domains were 0.922,0.933, 0.660, 0.773 and 0.869 respectively, whose split-efficacy reliability were separately 0.927, 0.933, 0.790, 0.624 and 0.889. Data from the exploratory factor analysis revealed the following dimensions:the entries were all inclusive, with the cumulative contribution rate at 59.453%, 56.062% and 52.668%, respectively. The results of IIC showed that in the four dimensions, the contained entries between Spearman correlation coefficient have statistically significant, with the r value range of 0.584-0.793, 0.665-0.818, 0.654-0.765 and 0.622-0.747 severely, while other dimensions from weak to moderate relationships, the r value ranged from -0.028 to 0.614. CONCLUSION: The reliability and validity of the adolescent fat intake behavior of psychological measurement scales (Chinese version) were good, and could be used to measure the fat intake behavior of psychological conditions in Chinese adolescents.

Advances in understanding the pathogenesis of post-traumatic epilepsy: a literature review.

Severe head trauma can lead to seizures. Persistent epileptic seizures and their progression are associated with the severity of trauma. Although case reports have revealed that early use of anti-seizure drugs after trauma can prevent epilepsy, clinical case-control studies have failed to confirm this phenomenon. To date, many brain trauma models have been used to study the correlation between post-traumatic seizures and related changes in neural circuit function. According to these studies, neuronal and glial responses are activated immediately after brain trauma, usually leading to significant cell loss in injured brain regions. Over time, long-term changes in neural circuit tissues, especially in the neocortex and hippocampus, lead to an imbalance between excitatory and inhibitory neurotransmission and an increased risk of spontaneous seizures. These changes include alterations in inhibitory interneurons and the formation of new, over-recurrent excitatory synaptic connections. In this study, we review the progress of research related to post-traumatic epilepsy to better understand the mechanisms underlying the initiation and development of post-traumatic seizures and to provide theoretical references for the clinical treatment of post-traumatic seizures.

Self-engineered binary nanoassembly enabling closed-loop glutathione depletion-amplified tumor ferroptosis.

Ferroptosis has emerged as a promising target for anticancer treatment, comprising iron-dependent lipid peroxidation and excessive accumulation of reactive oxygen species. Given that glutathione (GSH) overproduced in tumor cells antagonizes the cellular oxidation system, the reduction of GSH production has been extensively explored to induce ferroptosis. However, reducing GSH production alone is insufficient to trigger an intense lipid peroxidation storm. It is highly desirable to achieve systemic GSH depletion through simultaneous production and consumption intervention. Herein, we propose a bidirectional blockage strategy for closed-loop GSH depletion-amplified tumor ferroptosis. Sorafenib (Sor) and gambogic acid (GA) were elaborately fabricated as a self-engineered carrier-free nanoassembly without any nanocarrier materials. The PEGylated dual-drug nanoassembly enables favorable co-delivery and tumor-specific release of Sor and GA. Notably, a closed-loop GSH depletion is observed as a result of a Sor-induced decrease in GSH production and GA-accelerated GSH consumption in vitro and in vivo. As expected, this uniquely engineered dual-drug nanoassembly demonstrates vigorous antitumor activity in 4T1 breast tumor-bearing mice. This study presents a novel nanotherapeutic modality for ferroptosis-driven cancer treatment.

Assessing stroke rehabilitation degree based on quantitative EEG index and nonlinear parameters.

The assessment of motor function is critical to the rehabilitation of stroke patients. However, commonly used evaluation methods are based on behavior scoring, which lacks neurological indicators that directly reflect the motor function of the brain. The objective of this study was to investigate whether resting-state EEG indicators could improve stroke rehabilitation evaluation. We recruited 68 participants and recorded their resting-state EEG data. According to Brunnstrom stage, the participants were divided into three groups: severe, moderate, and mild. Ten quantitative electroencephalographic (QEEG) and five non-linear parameters of resting-state EEG were calculated for further analysis. Statistical tests were performed, and the genetic algorithm-support vector machine was used to select the best feature combination for classification. We found the QEEG parameters show significant differences in Delta, Alpha1, Alpha2, DAR, and DTABR (P < 0.05) among the three groups. Regarding nonlinear parameters, ApEn, SampEn, Lz, and C0 showed significant differences (P < 0.05). The optimal feature classification combination accuracy rate reached 85.3%. Our research shows that resting-state EEG indicators could be used for stroke rehabilitation evaluation.

Effects of Cyanobacterial Harmful Algal Bloom Toxin Microcystin-LR on Gonadotropin-Dependent Ovarian Follicle Maturation and Ovulation in Mice.

BACKGROUND: Cyanobacterial harmful algal blooms (CyanoHABs) originate from the excessive growth or bloom of cyanobacteria often referred to as blue-green algae. They have been on the rise globally in both marine and freshwaters in recently years with increasing frequency and severity owing to the rising temperature associated with climate change and increasing anthropogenic eutrophication from agricultural runoff and urbanization. Humans are at a great risk of exposure to toxins released from CyanoHABs through drinking water, food, and recreational activities, making CyanoHAB toxins a new class of contaminants of emerging concern. OBJECTIVES: We investigated the toxic effects and mechanisms of microcystin-LR (MC-LR), the most prevalent CyanoHAB toxin, on the ovary and associated reproductive functions. METHODS: Mouse models with either chronic daily oral or acute intraperitoneal exposure, an engineered three-dimensional ovarian follicle culture system, and human primary ovarian granulosa cells were tested with MC-LR of various dose levels. Single-follicle RNA sequencing, reverse transcription-quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, western blotting, immunohistochemistry (IHC), and benchmark dose modeling were used to examine the effects of MC-LR on follicle maturation, hormone secretion, ovulation, and luteinization. RESULTS: Mice exposed long term to low-dose MC-LR did not exhibit any differences in the kinetics of folliculogenesis, but they had significantly fewer corpora lutea compared with control mice. Superovulation models further showed that mice exposed to MC-LR during the follicle maturation window had significantly fewer ovulated oocytes. IHC results revealed ovarian distribution of MC-LR, and mice exposed to MC-LR had significantly lower expression of key follicle maturation mediators. Mechanistically, in both murine and human granulosa cells exposed to MC-LR, there was reduced protein phosphatase 1 (PP1) activity, disrupted PP1-mediated PI3K/AKT/FOXO1 signaling, and less expression of follicle maturation-related genes. DISCUSSION: Using both in vivo and in vitro murine and human model systems, we provide data suggesting that environmentally relevant exposure to the CyanoHAB toxin MC-LR interfered with gonadotropin-dependent follicle maturation and ovulation. We conclude that MC-LR may pose a nonnegligible risk to women's reproductive health by heightening the probability of irregular menstrual cycles and infertility related to ovulatory disorders. https://doi.org/10.1289/EHP12034.

Microplastic ingestion from atmospheric deposition during dining/drinking activities.

Human-health risks from microplastics have attracted considerable attention, but little is known about human-exposure pathways and intensities. Recent studies posited that inhalation of atmospheric microplastics was the dominant human-exposure pathway. Herein, our study identified that atmospheric microplastics ingested from deposition during routine dining/drinking activities represent another important exposure pathway. We measured abundances of atmospheric-deposited microplastics of up to 105 items m-2 d-1 in dining/drinking venues, with 90% smaller than 100 µm and a dominance of amorphous fragments rather than fibers. Typical work-life scenarios projected an annual ingestion of 1.9 × 105 to 1.3 × 106 microplastics through atmospheric deposition on diet, with higher exposure rates for indoor versus outdoor dining/drinking settings. Ingestion of atmospheric-deposited microplastics through diet was similar in magnitude to presumed inhalation exposure, but 2-3 orders of magnitude greater than direct ingestion from food sources. Simple mitigation strategies (e.g., covering and rinsing dishware) can substantially reduce the exposure of atmospheric deposition microplastics through diet.

Airborne microplastics in indoor and outdoor environments of a coastal city in Eastern China.

Microplastics (MPs) in marine and terrestrial environments have been intensively studied, but the dynamics of airborne MPs remains limited. Existing studies on atmospheric MPs are mostly derived from collection of atmospheric deposition, whereas direct measurements of airborne MPs are scarce. However, the abundance of airborne MPs is more relevant for evaluating human inhalation exposure risk. Herein, airborne MPs in indoor and outdoor environments from urban and rural areas of a coastal city in eastern China were investigated. MP concentrations (mean±SD) in indoor air (1583 ± 1180 n/m3) were an order of magnitude higher than outdoor air (189 ± 85 n/m3), and airborne MP concentrations in urban areas (224 ± 70 n/m3) were higher than rural areas (101 ± 47 n/m3). MPs smaller than 100 µm dominated airborne MPs, and the predominant shape of airborne MPs was fragments, as opposed to fibers. The larger MP size fractions contained a higher proportion of fibers, whereas the smaller size fractions were nearly exclusively composed of fragments. The health risk caused by ubiquitous airborne MPs should not be discounted as the maximum annual outdoor exposure of airborne MPs can reach 1 million/year, while indoor exposure may be even higher due to higher indoor airborne MP concentrations.

Analysis of the Transcriptome: Regulation of Cancer Stemness in Breast Ductal Carcinoma In Situ by Vitamin D Compounds.

Ductal carcinoma in situ (DCIS), which accounts for one out of every five new breast cancer diagnoses, will progress to potentially lethal invasive ductal carcinoma (IDC) in about 50% of cases. Vitamin D compounds have been shown to inhibit progression to IDC in the MCF10DCIS model. This inhibition appears to involve a reduction in the cancer stem cell-like population in MCF10DCIS tumors. To identify genes that are involved in the vitamin D effects, a global transcriptomic analysis was undertaken of MCF10DCIS cells grown in mammosphere cultures, in which cancer stem-like cells grow preferentially and produce colonies by self-renewal and maturation, in the presence and absence of 1α25(OH)2D3 and a vitamin D analog, BXL0124. Using next-generation RNA-sequencing, we found that vitamin D compounds downregulated genes involved in maintenance of breast cancer stem-like cells (e.g., GDF15), epithelial-mesenchymal transition, invasion, and metastasis (e.g., LCN2 and S100A4), and chemoresistance (e.g., NGFR, PPP1R1B, and AGR2), while upregulating genes associated with a basal-like phenotype (e.g., KRT6A and KRT5) and negative regulators of breast tumorigenesis (e.g., EMP1). Gene methylation status was analyzed to determine whether the changes in expression induced by vitamin D compounds occurred via this mechanism. Ingenuity pathway analysis was performed to identify upstream regulators and downstream signaling pathway genes differentially regulated by vitamin D, including TP63 and vitamin D receptor -mediated canonical pathways in particular. This study provides a global profiling of changes in the gene signature of DCIS regulated by vitamin D compounds and possible targets for chemoprevention of DCIS progression to IDC in patients.

Comparative genomics of susceptibility to mammary carcinogenesis among inbred rat strains: role of reduced prolactin signaling in resistance of the Copenhagen strain.

To elucidate the molecular basis for differential susceptibilities to mammary carcinogenesis, we compared the transcriptomes of normal mammary glands from pubescent female rats of the resistant Copenhagen (Cop) strain with those of the susceptible Fischer 344 (F344), August x Copenhagen Irish (ACI), Buffalo/N (Buf/N), Wistar-Furth (WF) strains and F1 (Cop x F344) progeny (F1). Gene expression profiles in mammary tissue within each rat strain were remarkably similar, indicating that gene expression was determined by genetic background. We next identified the subset of genes that were differentially expressed in all susceptible strains relative to the resistant Cop strain. Among these, the messenger RNAs encoding prolactin (Prl) and its cell surface receptor were significantly elevated in all susceptible strains. The expression levels of several Prl-regulated genes were also significantly elevated, indicating the presence of increased Prl signaling in mammary glands of all susceptible strains. Pathway analysis of gene expression profiles further identified the Prl-activated Jak/STAT-signaling pathway among the pathways that most distinguished sensitive rat strains from the resistant Cop rat. To test the hypothesis that reduced levels of the Prl signaling in mammary tissue partially contributed to the genetic resistance to mammary carcinogenesis, we used the neuroleptic drug, perphenazine, to transiently elevate serum Prl levels in the Cop strain. Whereas Cop rats are resistant to N-nitroso-N-methylurea (NMU)-induced mammary carcinogenesis, approximately 5% of pubescent Cop females treated with perphenazine and NMU exposure developed mammary adenocarcinomas with latencies comparable with those of sensitive strains. Together, these finding indicated that in the rat, the molecular mechanisms underlying genetic susceptibility to mammary carcinogenesis include de-regulation of Prl signaling.

Fucoxanthin Elicits Epigenetic Modifications, Nrf2 Activation and Blocking Transformation in Mouse Skin JB6 P+ Cells.

Nuclear factor erythroid-2-related factor-2 (Nrf2 or NFE2L2) is a master regulator of the anti-oxidative stress response, which is involved in the defense against many oxidative stress/inflammation-mediated diseases, including anticancer effects elicited by an increasing number of natural products. Our previous studies showed that the epigenetic modification of the Nrf2 gene plays a key role in restoring the expression of Nrf2. In this study, we aimed to investigate the epigenetic regulation of Nrf2 by astaxanthin (AST) and fucoxanthin (FX), carotenoids which are abundant in microalgae and seaweeds, in mouse skin epidermal JB6 P+ cells. FX induced the anti-oxidant response element (ARE)-luciferase and upregulated the mRNA and protein levels of Nrf2 and Nrf2 downstream genes in HepG2-C8 cells overexpressing the ARE-luciferase reporter. Both FX and AST decreased colony formation in 12-Otetradecanoylphorbol-13-acetate (TPA)-induced transformation of JB6 P+ cells. FX decreased the methylation of the Nrf2 promoter region in the JB6 P+ cells by the bisulfite conversion and pyrosequencing. Both FX and AST significantly reduced DNA methyltransferase (DNMT) activity but did not affect histone deacetylase (HDAC) activity in JB6 P+ cells. In summary, our results show that FX activates the Nrf2 signaling pathway, induces the epigenetic demethylation of CpG sites in Nrf2 and blocks the TPA-induced transformation of JB6 P+ cells, indicating the potential health-promoting effects of FX in skin cancer prevention.

Uncoupling genotoxic stress responses from circadian control increases susceptibility to mammary carcinogenesis.

We previously demonstrated that chemopreventive methylselenocysteine (MSC) prevents N-Nitroso-N-methylurea (NMU)-induced mammary carcinogenesis in the susceptible Fischer 344 (F344) rats by enhancing NAD+-dependent SIRT1 activity, restoring circadian expression of Period 2 (Per2) and circadian controlled genes. Here, we show that compared to the genetically resistant Copenhagen (COP) rat strain, mammary glands of the F344 rats have a 4-hour phase delay in circadian expression of Per2. Consequently, F344 rats failed to increase SIRT1 activity and circadian expression of Per2 and DDRR genes after exposure to NMU. Exposure of COP rats to NMU had the opposite effect, enhancing SIRT1 activity, increasing circadian expression of Per2 and DDRR genes. Significantly, SIRT1 activity and circadian expression of Per2 and DDRR genes in NMU-treated F344 rats on a chemopreventive regimen of MSC approximated those in NMU-treated COP rats. These results indicated that COP rats have an increased capacity to maintain NAD+-dependent SIRT1 activity under genotoxic stress. This contention was supported by increased stability of the period and phase of circadian locomotor activity in COP vs F344 rats exposed to changing light conditions. The increased sensitivity and rapid response of COP to changing light were correlated with the enhanced circadian response of this strain to carcinogen. Disturbance of circadian rhythm by jet lag also disrupted circadian expression of Per2 and DDRR genes, and accelerated mammary tumorigenesis in rodent models. These results suggested that uncoupling of DDRR responses from circadian control by environmental stresses and endogenous factors increases susceptibility to mammary carcinogenesis, possibly by inducing a promutagenic state.

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells.

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to sleep behavior. In vertebrates, circadian rhythm is controlled by a molecular oscillator that functions in both the suprachiasmatic nucleus (SCN; central pacemaker) and individual cells comprising most peripheral tissues. More importantly, disruption of circadian rhythm by exposure to light-at-night, environmental stressors and/or toxicants is associated with increased risk of chronic diseases and aging. The ability to identify agents that can disrupt central and/or peripheral biological clocks, and agents that can prevent or mitigate the effects of circadian disruption, has significant implications for prevention of chronic diseases. Although rodent models can be used to identify exposures and agents that induce or prevent/mitigate circadian disruption, these experiments require large numbers of animals. In vivo studies also require significant resources and infrastructure, and require researchers to work all night. Thus, there is an urgent need for a cell-type appropriate in vitro system to screen for environmental circadian disruptors and enhancers in cell types from different organs and disease states. We constructed a vector that drives transcription of the destabilized luciferase in eukaryotic cells under the control of the human PERIOD 2 gene promoter. This circadian reporter construct was stably transfected into human mammary epithelial cells, and circadian responsive reporter cells were selected to develop the in vitro bioluminescence assay. Here, we present a detailed protocol to establish and validate the assay. We further provide details for proof of concept experiments demonstrating the ability of our in vitro assay to recapitulate the in vivo effects of various chemicals on the cellular biological clock. The results indicate that the assay can be adapted to a variety of cell types to screen for both environmental disruptors and chemopreventive enhancers of circadian clocks.

Overexpression of 5-lipoxygenase and cyclooxygenase 2 in hamster and human oral cancer and chemopreventive effects of zileuton and celecoxib.

PURPOSE: Previous studies have suggested an important role of aberrant arachidonic acid metabolism, especially the cyclooxygenase (Cox) pathway, in oral carcinogenesis. However, it is unknown whether the 5-lipoxygenase (5-Lox) pathway contributes to oral carcinogenesis, and whether combination of inhibitors of both pathways may have synergistic or additive effects of chemoprevention. EXPERIMENTAL DESIGN: 5-Lox expression was examined in 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster and human oral cancer tissues by immunohistochemistry, and Cox2 expression was investigated in hamster oral tissues using in situ hybridization. Zileuton (a specific 5-Lox inhibitor) and celecoxib (a specific Cox2 inhibitor), either alone or in combination, were investigated for their chemopreventive effects on the DMBA-induced hamster model at the post-initiation stage through topical application. RESULTS: 5-Lox was overexpressed during oral carcinogenesis in hamsters and humans, as well as Cox2 in the hamster tissues. In a chemoprevention study using the post-initiation DMBA model, incidence of hamster oral squamous cell carcinoma was reduced from 76.9% (20 of 26) to 45.8% (11 of 24, P < 0.05) and 32.1% (9 of 28, P < 0.01) by 3% and 6% topical zileuton, respectively; and to 57.6% (15 of 26, P > 0.05) and 50% (12 of 24, P < 0.05) by 3% and 6% topical celecoxib, respectively. When used in combination, celecoxib and zileuton (3% of each) had an additive inhibitory effect on the incidence of squamous cell carcinoma (36%, 9 of 25, P < 0.01). Other pathologic variables and the levels of leukotriene B4 and prostaglandin E2 of the hamster tissues were reduced as well. CONCLUSIONS: The results clearly showed that both 5-Lox and Cox2 played important roles in oral carcinogenesis. Zileuton and celecoxib prevented oral carcinogenesis at the post-initiation stage through their inhibitory effects on arachidonic acid metabolism.