Granulysin-mediated reduction of PDZRN3 induces Cx43 gap junctions activity exacerbating skin damage in trichloroethylene hypersensitivity syndrome.

Trichloroethylene (TCE)-induced hypersensitivity syndrome (THS) has been a concern for many researchers in the field of environmental and occupational health. Currently, there is no specific treatment for THS, leaving patients to contend with severe infections arising from extensive skin lesions, consequently leading to serious adverse effects. However, the pathogenesis of severe skin damage in THS remains unclear. This study aims to investigate the specific danger signals and mechanisms underlying skin damage in THS through in vivo and in vitro experiments. We identified that cell supernatant containing 15 kDa granulysin (GNLY), released from activated CD3-CD56+NK cells or CD3+CD56+NKT cells in PBMC induced by TCE or its metabolite, promoted apoptosis in HaCaT cells. The apoptosis level decreased upon neutralization of GNLY in the supernatant by a GNLY-neutralizing antibody in HaCaT cells. Subcutaneous injection of recombinant 15 kDa GNLY exacerbated skin damage in the THS mouse model and better mimicked patients' disease states. Recombinant 15 kDa GNLY could directly induce cellular communication disorders, inflammation, and apoptosis in HaCaT cells. In addition to its cytotoxic effects, GNLY released from TCE-activated NK cells and NKT cells or synthesized GNLY alone could induce aberrant expression of the E3 ubiquitin ligase PDZRN3, causing dysregulation of the ubiquitination of the cell itself. Consequently, this resulted in the persistent opening of gap junctions composed of connexin43, thereby intensifying cellular inflammation and apoptosis through the "bystander effect". This study provides experimental evidence elucidating the mechanisms of THS skin damage and offers a novel theoretical foundation for the development of effective therapies targeting severe dermatitis induced by chemicals or drugs.

Alterations to biomarkers related to long-term exposure to diesel exhaust at concentrations below occupational exposure limits in the European Union and the USA.

BACKGROUND: We previously found that occupational exposure to diesel engine exhaust (DEE) was associated with alterations to 19 biomarkers that potentially reflect the mechanisms of carcinogenesis. Whether DEE is associated with biological alterations at concentrations under existing or recommended occupational exposure limits (OELs) is unclear. METHODS: In a cross-sectional study of 54 factory workers exposed long-term to DEE and 55 unexposed controls, we reanalysed the 19 previously identified biomarkers. Multivariable linear regression was used to compare biomarker levels between DEE-exposed versus unexposed subjects and to assess elemental carbon (EC) exposure-response relationships, adjusted for age and smoking status. We analysed each biomarker at EC concentrations below the US Mine Safety and Health Administration (MSHA) OEL (<106 µg/m3), below the European Union (EU) OEL (<50 µg/m3) and below the American Conference of Governmental Industrial Hygienists (ACGIH) recommendation (<20 µg/m3). RESULTS: Below the MSHA OEL, 17 biomarkers were altered between DEE-exposed workers and unexposed controls. Below the EU OEL, DEE-exposed workers had elevated lymphocytes (p=9E-03, false discovery rate (FDR)=0.04), CD4+ count (p=0.02, FDR=0.05), CD8+ count (p=5E-03, FDR=0.03) and miR-92a-3p (p=0.02, FDR=0.05), and nasal turbinate gene expression (first principal component: p=1E-06, FDR=2E-05), as well as decreased C-reactive protein (p=0.02, FDR=0.05), macrophage inflammatory protein-1ß (p=0.04, FDR=0.09), miR-423-3p (p=0.04, FDR=0.09) and miR-122-5p (p=2E-03, FDR=0.02). Even at EC concentrations under the ACGIH recommendation, we found some evidence of exposure-response relationships for miR-423-3p (ptrend=0.01, FDR=0.19) and gene expression (ptrend=0.02, FDR=0.19). CONCLUSIONS: DEE exposure under existing or recommended OELs may be associated with biomarkers reflective of cancer-related processes, including inflammatory/immune response.

Association between diesel exhaust exposure and mitochondrial DNA methylation.

OBJECTIVES: Diesel exhaust is an established human carcinogen, however the mechanisms by which it leads to cancer development are not fully understood. Mitochondrial dysfunction is an established contributor to carcinogenesis. Recent studies have improved our understanding of the role played by epigenetic modifications in the mitochondrial genome on tumorigenesis. In this study, we aim to evaluate the association between diesel engine exhaust (DEE) exposure with mitochondrial DNA (mtDNA) methylation levels in workers exposed to DEE. METHODS: The study population consisted of 53 male workers employed at a diesel engine manufacturing facility in Northern China who were routinely exposed to diesel exhaust in their occupational setting, as well as 55 unexposed male control workers from other unrelated factories in the same geographic area. Exposure to DEE, elemental carbon, organic carbon, and particulate matter (PM2.5) were assessed. mtDNA methylation for CpG sites (CpGs) from seven mitochondrial genes (D-Loop, MT-RNR1, MT-CO2, MT-CO3, MT-ATP6, MT-ATP8, MT-ND5) was measured in blood samples. Linear regression models were used to estimate the associations between DEE, elemental carbon, organic carbon and PM2.5 exposures with mtDNA methylation levels, adjusting for potential confounders. RESULTS: DEE exposure was associated with decreased MT-ATP6 (difference = -35.6%, P-value = 0.019) and MT-ATP8 methylation (difference = -30%, P-value = 0.029) compared to unexposed controls. Exposures to elemental carbon, organic carbon, and PM2.5 were also significantly and inversely associated with methylation in MT-ATP6 and MT-ATP8 genes (all P-values < 0.05). CONCLUSIONS: Our findings suggest that DEE exposure perturbs mtDNA methylation, which may be of importance for tumorigenesis.

Functional and structural characteristics of HLA-B*13:01-mediated specific T cells reaction in dapsone-induced drug hypersensitivity.

BACKGROUND: Severe cutaneous adverse drug reactions (SCARs) are a group of serious clinical conditions caused by immune reaction to certain drugs. The allelic variance of human leukocyte antigens of HLA-B*13:01 has been strongly associated with hypersensitivities induced by dapsone (DDS). T-cell receptor mediated activation of cytotoxic T lymphocytes (CTLs) has also been suggested to play an essential role in pathogenesis of SCARs. However, HLA-B*13:01-DDS-TCR immune synapse that plays role in drug-induced hypersensitivity syndrome (DIHS) associated T cells activation remains uncharacterized. METHODS: To investigate the molecular mechanisms for HLA-B*13:01 in the pathogenesis of Dapsone-induced drug hypersensitivity (DDS-DIHS), we performed crystallization and expanded drug-specific CTLs to analyze the pathological role of DDS-DIHS. RESULTS: Results showed the crystal structure of HLA-B*13:01-beta-2-microglobulin (ß2M) complex at 1.5 Å resolution and performed mutation assays demonstrating that I118 or I119, and R121 of HLA-B*13:01 were the key residues that mediate the binding of DDS. Subsequent single-cell TCR and RNA sequencing indicated that TCRs composed of paired TRAV12-3/TRBV28 clonotype with shared CDR3 region specifically recognize HLA-B*13:01-DDS complex to trigger inflammatory cytokines associated with DDS-DIHS. CONCLUSION: Our study identified the novel p-i-HLA/TCR as the model of interaction between HLA-B*13:01, DDS and the clonotype-specific TCR in DDS-DIHS.

Aberrant mitochondrial DNA methylation and declined pulmonary function in a population with polycyclic aromatic hydrocarbon composition in particulate matter.

Air pollution exposure has been found to be associated with epigenetic modification of the mitochondrial genome, which could subsequently induce adverse health outcomes. However, very limited studies exist regarding the association between fine particulate matter (PM2.5) exposure and pulmonary function at the molecular level of mitochondrial epigenetic changes. This study aimed to investigate the association of platelet mitochondrial DNA (mtDNA) methylation with occupational PM2.5 exposure and pulmonary function. First, 768 participants were occupationally exposed to polycyclic aromatic hydrocarbon (PAH)-enriched PM2.5 in a coke-oven plant in East China. The levels of PM2.5, PAH components bound to PM2.5, and urinary PAH metabolites in the workplace environment were measured as an internal dose, respectively. mtDNA methylation was measured by bisulfite pyrosequencing of two genes of ATP synthase (MT-ATP6 and MT-ATP8). Mediation analysis was conducted to evaluate the role of mtDNA methylation in pulmonary alteration induced by PAH. A decreasing trend of platelet mtDNA methylation was observed with increase in PM2.5 exposure across all participants. As an important PAH metabolite in urine, 1-hydroxypyrene (1-OHP) was significantly negatively associated with FEV1/FVC (Forced Expiratory Volume in 1s/Forced Vital Capacity) ratio. The participants with high serum folate levels (≥10 nmol/L) showed positive association between MT-ATP6 methylation and FEV1/FVC ratio. Mediation analysis suggested that MT-ATP6 methylation mediated the significant association of urinary 1-OHP with FEV1/FVC. Our findings suggested the methylation of platelet mitochondrial gene MT-ATP6 and FEV1/FVC to be negatively associated with PM exposure. Platelet mtDNA methylation acted as an intermediary between PAH exposure and lung function decline. The mitochondrial epigenetic regulation in platelets, in response to PM exposure, might be involved in subsequent progress of abnormal pulmonary function.

Total pancreatectomy and islet cell autotransplantation: a 10-year update on outcomes and assessment of long-term durability.

BACKGROUND: Total pancreatectomy and islet cell autotransplantation (TPIAT) offers an effective, lasting solution for the management of chronic pancreatitis up to 5-years post-operatively. Our aim was to assess durability of TPIAT at 10-years. METHODS: Patients undergoing TPIAT for chronic pancreatitis eligible for 10-year follow-up were included. Primary outcomes, including endocrine function and narcotic requirements, were reported at 5-, 7.5-, and 10-years post-operatively. RESULTS: Of the 231 patients who underwent TPIAT, 142 met inclusion criteria. All patients underwent successful TPIAT with an average of 5680.3 islet equivalents per body weight. While insulin independence tended to decrease over time (25.7% vs. 16.0% vs. 10.9%, p = 0.11) with an increase in HbA1C (7.6% vs. 8.2% vs. 8.4%, p = 0.09), partial islet function persisted (64.9% vs. 68.0% vs. 67.4%, p = 0.93). Opioid independence was achieved and remained durable in the majority (73.3% vs. 72.2% vs. 75.5%, p = 0.93). Quality of life improvements persisted, with 85% reporting improvement from baseline at 10-years. Estimated median overall survival was 202.7 months. CONCLUSION: This study represents one of the largest series reporting on long-term outcomes after TPIAT, demonstrating excellent long-term pain control and durable improvements in quality of life. Islet cell function declines over time however stable glycemic control is maintained.

Liver glycogen-induced enhancements in hypoglycemic counterregulation require neuroglucopenia.

Iatrogenic hypoglycemia is a prominent barrier to achieving optimal glycemic control in patients with diabetes, in part due to dampened counterregulatory hormone responses. It has been demonstrated that elevated liver glycogen content can enhance these hormonal responses through signaling to the brain via afferent nerves, but the role that hypoglycemia in the brain plays in this liver glycogen effect remains unclear. During the first 4 h of each study, the liver glycogen content of dogs was increased by using an intraportal infusion of fructose to stimulate hepatic glucose uptake (HG; n = 13), or glycogen was maintained near fasting levels with a saline infusion (NG; n = 6). After a 2-h control period, during which the fructose/saline infusion was discontinued, insulin was infused intravenously for an additional 2 h to bring about systemic hypoglycemia in all animals, whereas brain euglycemia was maintained in a subset of the HG group by infusing glucose bilaterally into the carotid and vertebral arteries (HG-HeadEu; n = 7). Liver glycogen content was markedly elevated in the two HG groups (43 ± 4, 73 ± 3, and 75 ± 7 mg/g in NG, HG, and HG-HeadEu, respectively). During the hypoglycemic period, arterial plasma glucose levels were indistinguishable between groups (53 ± 2, 52 ± 1, and 51 ± 1 mg/dL, respectively), but jugular vein glucose levels were kept euglycemic (88 ± 5 mg/dL) only in the HG-HeadEu group. Glucagon and epinephrine responses to hypoglycemia were higher in HG compared with NG, whereas despite the increase in liver glycogen, neither increased above basal in HG-HeadEu. These data demonstrate that the enhanced counterregulatory hormone secretion that accompanies increased liver glycogen content requires hypoglycemia in the brain.NEW & NOTEWORTHY It is well known that iatrogenic hypoglycemia is a barrier to optimal glycemic regulation in patients with diabetes. Our data confirm that increasing liver glycogen content 75% above fasting levels enhances hormonal responses to insulin-induced hypoglycemia and demonstrate that this enhanced hormonal response does not occur in the absence of hypoglycemia in the brain. These data demonstrate that information from the liver regarding glycogen availability is integrated in the brain to optimize the counterregulatory response.

Elevated Alu retroelement copy number among workers exposed to diesel engine exhaust.

BACKGROUND: Millions of workers worldwide are exposed to diesel engine exhaust (DEE), a known genotoxic carcinogen. Alu retroelements are repetitive DNA sequences that can multiply and compromise genomic stability. There is some evidence linking altered Alu repeats to cancer and elevated mortality risks. However, whether Alu repeats are influenced by environmental pollutants is unexplored. In an occupational setting with high DEE exposure levels, we investigated associations with Alu repeat copy number. METHODS: A cross-sectional study of 54 male DEE-exposed workers from an engine testing facility and a comparison group of 55 male unexposed controls was conducted in China. Personal air samples were assessed for elemental carbon, a DEE surrogate, using NIOSH Method 5040. Quantitative PCR (qPCR) was used to measure Alu repeat copy number relative to albumin (Alb) single-gene copy number in leucocyte DNA. The unitless Alu/Alb ratio reflects the average quantity of Alu repeats per cell. Linear regression models adjusted for age and smoking status were used to estimate relations between DEE-exposed workers versus unexposed controls, DEE tertiles (6.1-39.0, 39.1-54.5 and 54.6-107.7 µg/m3) and Alu/Alb ratio. RESULTS: DEE-exposed workers had a higher average Alu/Alb ratio than the unexposed controls (p=0.03). Further, we found a positive exposure-response relationship (p=0.02). The Alu/Alb ratio was highest among workers exposed to the top tertile of DEE versus the unexposed controls (1.12±0.08 SD vs 1.06±0.07 SD, p=0.01). CONCLUSION: Our findings suggest that DEE exposure may contribute to genomic instability. Further investigations of environmental pollutants, Alu copy number and carcinogenesis are warranted.

Polycyclic aromatic hydrocarbons in particulate matter and serum club cell secretory protein change among schoolchildren: A molecular epidemiology study.

Airborne particulate matter (PM) is a complex mixture containing various kinds of harmful components. Exposure to air PM is associated with childhood respiratory disease, but epidemiological data are limited concerning the circulating respiratory injury protein on the etiology of childhood respiratory disease. Specifically, the role of PM toxic components or its biological effective dose (adduct) in respiratory injury remains unclear. To demonstrate the dose-response relationship and the main mechanism on circulating club cell secretory protein (CC16) from PM compositions among children, we enrolled 273 boarding schoolchildren in China, including 110 and 163 children of whom were in the low- and high-PM exposed areas, respectively. In this study, we measured the internal exposure levels, including serum polycyclic aromatic hydrocarbons (PAH) adduct, urinary metals, and AhR expression, and detected the serum CC16 level as a lung injury marker. Environmental tobacco exposure in children was assessed by urinary cotinine. We found that significantly higher levels of serum CC16, benzo[a]pyridin-7,8-dihydroglycol-9,10-epoxide (BPDE)-albumin adduct, urinary molybdenum, selenium, arsenic, cadmium and barium, and lower level of AhR expression in high-PM exposed group. There was a good association between serum BPDE-albumin adduct and CC16 (ß = 0.222, P = 0.006). There was no association on urinary metals and serum CC16. BPDE-albumin adduct was directly associated with serum CC16 alternation [direct effect = 0.2044, 95% confidence interval (CI) = (0.0426, 0.36)]. PM could cause serum CC16 increased in children. PAH and its adduct might play a key role in lung injury during PM exposure.

Genetic structure of the endangered Irrawaddy dolphin (Orcaella brevirostris) in the Gulf of Thailand.

The Irrawaddy dolphin (Orcaella brevirostris) is an endangered, small cetacean species which is widely distributed in rivers, estuaries, and coastal waters throughout the tropical and subtropical Indo-Pacific. Despite the extensive distribution of this species, little is known of individual movements or genetic exchange among regions in Thailand. Here, we evaluate the genetic diversity and genetic structure of O. brevirostris in the eastern, northern and western Gulf of Thailand, and Andaman Sea. Although phylogenetic relationships and network analysis based on 15 haplotypes obtained from 32 individuals reveal no obvious divergence, significant genetic differentiation in mitochondrial DNA (overall FST = 0.226, P < 0.001; ΦST = 0.252, P < 0.001) is apparent among regions. Of 18 tested microsatellite loci, 10 are polymorphic and successfully characterized in 28 individuals, revealing significant genetic differentiation (overall FST = 0.077, P < 0.05) among the four sampling sites. Structure analysis reveals two inferred genetic clusters. Additionally, Mantel analysis demonstrates individual-by-individual genetic distances and geographic distances follow an isolation-by-distance model. We speculate that the significant genetic structure of O. brevirostris in Thailand is associated with a combination of geographical distribution patterns, environmental and anthropogenic factors, and local adaptations.

[Effect of ERK/JNK cell signaling pathway in carbon black induced cytokine IL-6 and IL-8 expression changes].

OBJECTIVE: To investigate the roles of extracellular signal-regulated kinase(ERK)/c-Jun amino-terminal kinase(JNK) signaling pathway on the expression of interleukin-6(IL-6) and interleukin-8(IL-8) in human embryonic lung fibroblasts(HELF) induced by carbon black. METHODS: HELFs were cultured in RPMI-1640 medium containing 0, 15, 30, 60, 120 or 240 µg/mL carbon black for 24 h, and the appropriate dose of carbon black was determined by MTT assay result HELFs were divided into three groups: HELFs, HELFs transfected with ERK dominant negative mutant plasmid(DN-ERK) and HELFs transfected with JNK dominant negative mutant plasmid(DN-JNK). 100 µg/mL carbon black was used to treat HELFs(CB), DN-ERK HELFs(CB-DN-ERK), DN-JNK HELFs(CB-DN-JNK), and HELFs without any black carbon treatment were considered as control group. At 16 h after carbon black treatment, scanning electron microscope(SEM) was used to observe HELFs morphology and whether there were carbon black particless. At 0, 1, 2, 4, 8, 12, 24 and 36 h, the enzyme linked immunosorbent assay(ELISA) was used to detect CB and control groups HELFs IL-6 and IL-8 expression levels, whereas CB-DN-ERK and CB-DN-JNK HELFs were detected only at 24 h. RESULTS: SEM result showed no carbon black particles were observed in CB group HELFs, whereas their surface projections were increased. The CB group HELFs IL-6 expression levels at 2 h(44. 86±3. 65 ng/L) and 4 h(76. 52±3. 15 ng/L) were significantly lower than those of the control group(96. 78±2. 82 and 147. 32±3. 26 ng/L)(P<0. 05), whereas the IL-6 expression levels were significantly higher than those of the control group(105. 54±6. 10, 101. 27±5. 84 and 97. 15±5. 12 ng/L) at 16 h(202. 64±7. 20 ng/L), 24 h(200. 38±6. 20 ng/L) and 36 h(183. 54±4. 54 ng/L)(P<0. 001). At 24 h(136. 75±3. 81 ng/L) and 36 h(149. 12±2. 74 ng/L), the CB group IL-8 expression levels were significantly higher than those of the control group(75. 16±2. 84 and 73. 44±2. 15 ng/L)(P<0. 001). Compared with CB group HELFs, CB-DN-ERK and CB-DN-JNK groups HELFs had significantly lower IL-6 and IL-8 expression levels(P<0. 05). CONCLUSION: While carbon black induced HELFs IL-6 and IL-8 expression levels changes, ERK and JNK may upregulate IL-6 and IL-8 expression levels.

[Roles of ERK/JNK in carbon black induced AP-1 cell signaling pathway changes].

OBJECTIVE: To investigate the role of ERK/JNK in the alteration of activator protein-1(AP-1) signaling pathway in human embryonic lung fibroblasts(HELFs) induced by carbon black. METHODS: HELFs were cultured in RPMI 1640 medium containing 0, 15, 30, 60, 120 or 240 µg/mL carbon black for 24 h, and the appropriate dose of carbon black was determined by MTT assay result. HELFs were divided into three groups: HELFs, HELFs transfected with ERK dominant negative mutant plasmid(DN-ERK) and HELFs transfected with JNK dominant negative mutant plasmid(DN-JNK). 100 µg/mL carbon black was used to treat HELFs(CB), DN-ERK HELFs(CB-DN-ERK), DN-JNK HELFs(CB-DN-JNK), and HELFs without any treatment were considered as control group. At 0, 1, 2, 4, 8, 12, 24 and 36 h of CB and control groups HELFs, the western blot was used to detect ERK, p-ERK, JNK, p-JNK, p38, p-p38, c-Jun, p-c-Jun, c-Fos, p-c-Fos protein expression levels, and AP-1 activity was detected by luciferase method. Whereas CB-DN-ERK and CB-DN-JNK HELFs were detected only at 24 h. RESULTS: Compared with the protein expression levels at 0 h, CB group HELFs ERK and p-ERK protein expression increased at each time point, whereas p38 protein expression decreased. AP-1 activity of CB group HELFs was declined to the lowest at 8 h(0.72±0.12), and upregulated to the peak at 36 h(1.38±0.11). CB group HELFs c-Fos, p-c-Fos and c-Jun protein expression levels at each time point from 1 h to 24 h were greater than those of 0 h, and p-c-Jun protein expression levels at 1 h, 2 h, 4 h, 8 h, 36 h were also greater than those of 0 h. CB group HELFs AP-1 activity, ERK, p-ERK, JNK, p-JNK, p38, p-p38, c-Jun, p-c-Jun, c-Fos, p-c-Fos protein expression levels changes followed biphasic patterns. There were no statistically significant differences in AP-1 activity between CB group HELFs(1.03±0.10) and CB-DN-ERK group(1.02±0.04) or CB-DN-JNK group(1.09±0.10) HELFs(t=0.16, P=0.88; t=0.73, P=0.50). However, compared with CB group HELFs, c-Fos(t=5.31, P=0.01), p-c-Fos(t=4.33, P=0.01), p-c-Jun(t=10.95, P& lt; 0.01)in CB-DN-JNK group, and c-Fos protein expression levels in CB-DN-ERK group(t=42.72, P& lt; 0.01)were significantly decreased. CONCLUSION: While carbon black induces HELFs increased protein expression levels of ERK, p-ERK, c-Jun, p-c-Jun, c-Fos and p-c-Fos, JNK may upregulate c-Fos, p-c-Fos, p-c-Jun protein expression levels, and ERK may upregulate c-Fos protein expression level.

Utility evaluation of HLA-B*13:01 screening in preventing trichloroethylene-induced hypersensitivity syndrome in a prospective cohort study.

OBJECTIVES: Trichloroethylene (TCE) -induced hypersensitivity syndrome (TIHS) is a potentially life-threatening disease. Several genetic susceptibility biomarkers have been found to be associated with TIHS, and this systematic prospective study has been conducted to evaluate the utility of these genetic susceptibility biomarkers in preventing the disease. METHODS: The newly hired TCE-exposed workers were recruited from March 2009 to October 2010. HLA-B*13:01 genotyping and 3-month follow-up procedure were conducted. All workers were monitored for adverse reaction by telephone interview every week. The workers with early symptoms of TIHS were asked to go to the hospital immediately for further examination, diagnosis and treatment. The medical expense record data of patients with TIHS were collected for cost-effectiveness analysis in 2018. RESULTS: Among 1651 workers, 158 (9.57%) were found to carry the HLA-B*13:01 allele and 16 (0.97%) were diagnosed with TIHS. HLA-B*13:01 allele was significantly associated with an increased TIHS risk (relative risk=28.4, 95% CI 9.2 to 86.8). As a risk predictor of TIHS, HLA-B*13:01 testing had a sensitivity of 75%, a specificity of 91.1% and an area under curve of 0.83 (95% CI 0.705 to 0.955), the positive and negative predictive values were 7.6% and 99.7%, respectively. The incidence of TIHS was significantly decreased in HLA-B*13:01 non-carriers (0.27%) compared with all workers (0.97%, p=0.014). Cost-effectiveness analysis showed that HLA-B*13:01 screening could produce an economic saving of $4604 per TIHS avoided. CONCLUSIONS: Prospective HLA-B*13:01 screening may significantly reduce the incidence of TIHS and could be a cost effective option for preventing the disease in TCE-exposed workers.

The effects of CS@Fe3O4 nanoparticles combined with microwave or far infrared thawing on microbial diversity of red seabream (Pagrus major) fillets based on high-throughput sequencing.

The present study investigated the effects of CS@Fe3O4 nanoparticles combined with microwave or far infrared thawing on microbial diversity of red seabream (Pagrus major) fillets in terms of thawing loss, pH, TVB-N, classical microbiological enumeration and high-throughput sequencing, and the same parameters were also studied for 24 h after thawing. Four thawing methods were used: microwave thawing (MT), far-infrared thawing (FT), CS@Fe3O4 nanoparticles combined with microwave thawing (CMT) and CS@Fe3O4 nanoparticles combined with far-infrared thawing (CFT). The results showed that CFT and CMT had lower values of pH and TVB-N compared to the FT and MT. Based on conventional microbial count analysis, CFT and CMT samples also maintained lower TVC, pseudomonas and LAB counts. Using high-throughput sequencing analysis, Compared with FT and MT, CFT and CMT samples showed a significant decrease in the proportion of the Pseudomonadaceae flora. However, the proportion of Pseudomonas, Bacillaceae and Thermaceae also increased significantly after 24 h of storage, which indicated that become the predominant microbiota in red seabream (Pagrus major) fillets.

Time-course effects of antioxidants and phase II enzymes on diesel exhaust particles-induced oxidative damage in the mouse lung.

Mechanisms responsible for diesel exhaust particle (DEP)-induced toxicity in respiratory disorders are poorly understood, recent experimental and controlled exposure studies suggested that oxidative stress might be involved. To investigate the time-course effects DEP on nuclear factor erythroid 2-related factor 2 (Nrf2), a key regulator in cellular adaptive antioxidant response, mice were intratracheal instilled with 100 µg DEP/mouse and sacrificed after 30 min, 6 h, 12 h, 24 h, 48 h, and 72 h. We measured reactive oxygen species (ROS) as well as Nrf2 and antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and phase II enzymes including heme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase-1 (NQO1), glutamate-cysteine ligase catalytic subunit (GCLC), glutamate-cysteine ligase modifier subunit (GCLM) in the lungs. Additionally, histopathological changes were examined. At 6 h, ROS peaked, most of the enzymes were activated, and the histology showed the lungs were damaged. At 12 h, ROS returned to normal level and CAT activity decreased, while protein expression of Nrf2, HO-1, NQO1, GCLC, and GCLM increased, and the lungs were recovering from damage. After 24 h, ROS started to decrease and Nrf2 showed a decreasing trend at both gene and protein levels, while the lung damage had been entirely restored. These results suggested that a single exposure to DEP induce transient oxidative stress in the lungs, with time-dependent effects on Nrf2 and antioxidant enzymes and phase II enzymes.

Polycyclic aromatic hydrocarbons exposure and hematotoxicity in occupational population: A two-year follow-up study.

BACKGROUND: Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental and occupational pollutants. To date, the effect and mechanism by which PAHs exposure impaired hematopoietic system remains unclear. METHODS: We examined the capability of PAHs to disrupt hematopoiesis in a study of 639 male participants in China by measuring complete blood counts (CBC) in 2013 and 2014. Gas chromatography-mass spectrometry (GC/MS) method was used to measure airborne levels of PAHs and benzene. We measured 1-hydroxypyrene (1-OHP), S-phenylmercapturic acid (SPMA) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) in urinary by ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method. RESULTS: We found decreased dose-response of white blood cells, eosinophils, monocytes and lymphocytes with increased PAHs exposure in two consecutive years. We did not find association between benzene with CBC in our study. After stratification analysis by smoking status, the findings were highly consistent. White blood cells, monocytes and red blood cell counts were decreased in high urinary 8-OHdG group. CONCLUSIONS: Our study showed that PAHs could impair the hematopoietic system independently, and oxidative stress might play an important role in potential hematotoxicity.

Declining Pulmonary Function in Populations with Long-term Exposure to Polycyclic Aromatic Hydrocarbons-Enriched PM2.5.

This study assesses the effects of long-term exposure to ambient air pollutants on inflammatory response and lung function. We selected 390 male coke oven workers with exposure to polycyclic aromatic hydrocarbons (PAHs) and fine particulate matter (PM2.5) and 115 control workers. The average duration in the exposed group was 9.10 years. The total amount of PAHs was more enriched in PM2.5 which collected from the coke oven workshops compared with the control areas. Correspondingly, the internal PAHs exposure indicated by urinary 1-hydroxypyrene (1-OHP) in the exposure group increased 25.7-fold compared to that of the control group. Moreover, the increasing level of urinary 1-OHP was associated with the decrease of forced expiratory volume in 1 s to forced vital capacity ratio (FEV1/FVC). In non-current smokers of exposure group, inverse correlation of 1-OHP with FEV1/FVC was also found. Particularly, an exposure duration-dependent decline in FEV1/FVC and mean forced expiratory flow between 25% and 75% of forced vital capacity (FEF25-75%) indicated that small airways were functionally obstructed. Furthermore, the increasing serum high-sensitivity C-reactive protein (hs-CRP) was correlated with the decline in pulmonary function in all subjects. These findings provide a clue that long-term exposure to PAHs-enriched PM2.5 impairs pulmonary function in occupational population.

Occupational exposure to diesel engine exhaust and alterations in immune/inflammatory markers: a cross-sectional molecular epidemiology study in China.

The relationship between diesel engine exhaust (DEE), a known lung carcinogen, and immune/inflammatory markers that have been prospectively associated with lung cancer risk is not well understood. To provide insight into these associations, we conducted a cross-sectional molecular epidemiology study of 54 males highly occupationally exposed to DEE and 55 unexposed male controls from representative workplaces in China. We measured plasma levels of 64 immune/inflammatory markers in all subjects using Luminex bead-based assays, and compared our findings to those from a nested case-control study of these markers and lung cancer risk, which had been conducted among never-smoking women in Shanghai using the same multiplex panels. Levels of nine markers that were associated with lung cancer risk in the Shanghai study were altered in DEE-exposed workers in the same direction as the lung cancer associations. Among these, associations with the levels of CRP (ß= -0.53; P = 0.01) and CCL15/MIP-1D (ß = 0.20; P = 0.02) were observed in workers exposed to DEE and with increasing elemental carbon exposure levels (Ptrends <0.05) in multivariable linear regression models. Levels of a third marker positively associated with an increased lung cancer risk, CCL2/MCP-1, were higher among DEE-exposed workers compared with controls in never and former smokers, but not in current smokers (Pinteraction = 0.01). The immunological differences in these markers in DEE-exposed workers are consistent with associations observed for lung cancer risk in a prospective study of Chinese women and may provide some insight into the mechanistic processes by which DEE causes lung cancer.

Long-term exposure to diesel engine exhaust induces primary DNA damage: a population-based study.

OBJECTIVES: Diesel engine exhaust (DEE) is a ubiquitous environmental pollutant and is carcinogenic to humans. To seek early and sensitive biomarkers for prediction of adverse health effects, we analysed the components of DEE particles, and examined the genetic and oxidative damages in DEE-exposed workers. METHODS: 101 male diesel engine testing workers who were constantly exposed to DEE and 106 matched controls were enrolled in the present study. The components of DEE were analysed, including fine particulate matter (PM2.5), element carbon (EC), nitrogen dioxide (NO2), sulfur dioxide (SO2) and polycyclic aromatic hydrocarbons (PAHs). Postshift urine samples were collected and analysed for 1-hydroxypyrene (1-OHP), an internal exposure marker for DEE. Levels of DNA strand breaks and oxidised purines, defined as formamidopyrimidine-DNA glycosylase (FPG) sites in leucocytes, were measured by medium throughput Comet assay. Urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) was also used to determine the level of oxidative stress. RESULTS: We found higher levels of PM2.5, EC, NO2, SO2 and PAHs in the diesel engine testing workshop and significantly higher urinary 1-OHP concentrations in exposed subjects (p<0.001). Compared with controls, the levels of parameters in normal Comet and FPG-Comet assay were all significantly higher in DEE-exposed workers (p<0.001), and in a dose-dependent and time-dependent manner. There were no significant differences between DEE-exposed workers and controls in regard to leucocyte FPG sensitive sites and urinary 8-OHdG levels. CONCLUSIONS: These findings suggest that DEE exposure mainly induces DNA damage, which might be used as an early biomarker for risk assessment of DEE exposure.

[Optimization of primary hepatocytes model and study on the cytotoxicity of styrene and styrene oxide].

OBJECTIVE: To establish a model in vitro for primary cultured mouse hepatocytes with high viability and function, and evaluate the acute toxicity of the primary hepatocytes exposed to the chemicals such as styrene and styrene oxide (SO). METHODS: Based on the classical method, the two-step collagenase digestion method was optimized by reverse and intermittent perfusion, restriction of digestion time as well as purification of percoll liquid. Hepatocytes were isolated from BALB/C mouse by an improved isolated method and then cultured in monolayer and sandwich configuration. The primary cultured hepatocytes model was assessed by various indexes including cell morphology, cell viability, intracellular glycogen granules, as well as albumin (ALB), lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and blood urine nitrogen (BUN) levels in the supernatant. In addition, the primary cultured hepatocytes were treated with various concentrations from 0.2 to 25 micromol/L of styrene and styrene oxide during different time from 3 to 48 hours. The cytotoxicity induced by the two toxicants was assessed by CCK-8 and LDH assays. RESULTS: On average, the isolation using this improved method resulted in the cell viability of (90.3 +/- 5.2) %, the cell purity of (95.3 +/- 4.2)% and the yield of (2.4 +/- 0.9) x 10(7) viable cells. More than 90% cells showed a typical morphological feature of hepatocytes in sandwich configuration within 7 days, and contained a large number of glycogen granules on the third day. The ALB secretion, ALT and LDH leakage and BUN synthesis as well as cell viability fluctuated during 8 days, and they stayed at stable levels between 3 to 7 days in sandwich configuration. But they fluctuated during 6 days in monolayer configuration. In comparison with the monolayer configuration, the levels of ALB and BUN were distinctly increased and the levels of LDH and ALT were significantly decreased in sandwich configuration. The levels of ALB [ (1.42 +/- 0.20) g/L ] and BUN [(1.97 +/- 0.22) mmol/L] as well as cell viability were the highest, while the levels of LDH [ (7.30 +/- 2.33) U/L] and ALT [ (6.51 +/- 1.86) U/L] were the lowest in sandwich configuration on the third day. The relative low cytotoxicity and high cell survival rate ( more than 90%) were shown in treated hepatocytes with styrene and styrene oxide within 6 hours by CCK-8 and LDH measurements, and there was no distinct difference in the determination of cytotoxicity between the two methods. With the prolonged exposure time, the cell survival rate was lower by CCK-8 assay (less than 85%) than the one by LDH assay. The relative obvious cytotoxicity and low cell survival rate (about 85%) by CCK-8 method were revealed in treated cells with 5 micromol/L of styrene and styrene oxide for 24 hours, but there was no significant difference between CCK-8 and LDH assays. With the increase of the concentrations, the cell survival rate was lower by CCK-8 assay (less than 80%) compared with LDH assay. CONCLUSION: The improved two-step collagenase digestion method combination with sandwich culture method might maintain the morphology and function of primary cultured mouse hepatocytes for seven days. The cytotoxic effects of styrene and styrene oxide might be accurately evaluated by means of primary cultured hepatocyte model from 3 to 7 days. The chemicals might have major adverse effects on the functions of the organelles in hepatocytes such as mitochondria, but little influence to the cell membrane damage.