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.

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.

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.

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.

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.

[Toxicity of vehicle exhaust on BEAS-2B cells in vitro by air-liquid interface].

OBJECTIVE: To evaluate the toxic effect of vehicle exhaust( VE) on lung epithelial cells by air-liquid interface( ALI) method in vitro, and analyze the different toxicity of VE after being treated with 0. 2 µm filter. METHODS: VE were collected using20 liter Tedlar bags and their particulate matter( PM) number, surface and mass concentration were measured by particle size spectrometer for the interference of 0. 2 µm filter or non-filter. Four groups were included, which divided into blank control group, clean air group, filtered VE exposure group, non-filtered VE exposure group. The blank control group did not do any treatment; the clean air group was an artificial gas containing21% O_2 and 79% N_2; the filtered VE group( marked as f VE) was filtered using a 0. 2µm particle filter for VE. The VE group was used VE directly collected by air bag and marked as non-f VE. Except the blank control group, BEAS-2B cells were treated with clean air or VE by ALI method at a flow rate of 25 mL/min, 37 ℃ for 60 min in vitro. Cell relative viability was evaluated by CCK-8 assay. The reactive oxygen species( ROS)generation was determined via flow cytometry with 2', 7'-dichloro-dihydro-fluorescein diacetate( DCFH-DA) probe. Apoptosis and necrosis rate were measured using the commercial kit of Annexin V-FITC/PI by flow cytometry. RESULTS: In the non-f VE group, the PM of number, surface and mass concentration for 0. 5-10 µm diameters were 0. 24×10~3N/cm~3, 0. 29 ×10~3µm~2/cm~3 and 0. 19 µg/m~3, respectively, and for the PM of 10-500 nm diameters, they were 56 ×10~3N/cm~3、34. 53 ×10~8nm~2/cm~3 and 95ng/m~3, respectively. The PM of 0. 5-10 µm diameters in f VE group, their number, surface and mass concentration were less than 1 N/cm~3, 1 µm~2/cm~3 0. 001 µg/m~3, respectively. After filtration, the number, surface and mass concentration of PM in 10-500 nm diameters reduced by 89. 79%, 93. 57% and 90. 55%, respectively, as compared with non-f VE. In the clean air group, the cell relative viability, ROS generation, early apoptosis rate and late apoptosis and necrosis rate were( 90. 15 ± 4. 25) %, ( 1. 92 ± 0. 34)×10~5, ( 1. 09 ± 0. 48) % and( 8. 93 ± 3. 31) %, respectively. Compared with the clean air group, the cell relative viability, the ROS generation and the late apoptosis and necrosis rate of the two VE exposure groups were significantly different( all P < 0. 05). The cell relative viability of f VE exposure group were significantly higher than that in the non-f VE exposure group( t = 6. 331, P < 0. 001), and had no significant difference about the ROS generation[f VE ∶ non-VE =( 2. 94 ± 0. 21) ×10~5∶( 3. 32 ± 0. 49) ×10~5, t =-1. 252, P = 0. 279], early apoptosis rate [f VE∶ non-VE =( 1. 09 ± 0. 30) % ∶( 0. 99 ±0. 10) %, t = 0. 708, P = 0. 497] and late apoptosis and necrosis rate [f VE ∶ non-VE =( 21. 75 ± 10. 37) % ∶( 15. 32 ± 2. 74) %, t = 1. 347, P = 0. 242] between f VE and nonf VE exposure group( all P > 0. 05). CONCLUSION: Increased toxicity of human lung cells( BEAS-2B) in vitro were observed by ALI method at a flow rate of 25 mL/min, 37 ℃ for60 min. After using a 0. 2 µm filter, the toxicity was obviously decreased.

[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.

Occupational exposure to diesel engine exhaust and alterations in lymphocyte subsets.

BACKGROUND: The International Agency for Research on Cancer recently classified diesel engine exhaust (DEE) as a Group I carcinogen based largely on its association with lung cancer. However, the exposure-response relationship is still a subject of debate and the underlying mechanism by which DEE causes lung cancer in humans is not well understood. METHODS: We conducted a cross-sectional molecular epidemiology study in a diesel engine truck testing facility of 54 workers exposed to a wide range of DEE (ie, elemental carbon air levels, median range: 49.7, 6.1-107.7 µg/m(3)) and 55 unexposed comparable controls. RESULTS: The total lymphocyte count (p=0.00044) and three of the four major lymphocyte subsets (ie, CD4+ T cells (p=0.00019), CD8+ T cells (p=0.0058) and B cells (p=0.017)) were higher in exposed versus control workers and findings were highly consistent when stratified by smoking status. In addition, there was evidence of an exposure-response relationship between elemental carbon and these end points (ptrends<0.05), and CD4+ T cell levels were significantly higher in the lowest tertile of DEE exposed workers compared to controls (p=0.012). CONCLUSIONS: Our results suggest that DEE exposure is associated with higher levels of cells that play a key role in the inflammatory process, which is increasingly being recognised as contributing to the aetiology of lung cancer. IMPACT: This study provides new insights into the underlying mechanism of DEE carcinogenicity.

Reduced pulmonary function and increased pro-inflammatory cytokines in nanoscale carbon black-exposed workers.

BACKGROUND: Although major concerns exist regarding the potential consequences of human exposures to nanoscale carbon black (CB) particles, limited human toxicological data is currently available. The purpose of this study was to evaluate if nanoscale CB particles could be responsible, at least partially, for the altered lung function and inflammation observed in CB workers exposed to nanoscale CB particles. METHODS: Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), and Brunauer-Emmett-Teller were used to characterize CB. Eighty-one CB-exposed male workers and 104 non-exposed male workers were recruited. The pulmonary function test was performed and pro-inflammatory cytokines were evaluated. To further assess the deposition and pulmonary damage induced by CB nanoparticles, male BALB/c mice were exposed to CB for 6 hours per day for 7 or 14 days. The deposition of CB and the pathological changes of the lung tissue in mice were evaluated by paraffin sections and TEM. The cytokines levels in serum and lung tissue of mice were evaluated by ELISA and immunohistochemical staining (IHC). RESULTS: SEM and TEM images showed that the CB particles were 30 to 50 nm in size. In the CB workplace, the concentration of CB was 14.90 mg/m³. Among these CB particles, 50.77% were less than 0.523 micrometer, and 99.55% were less than 2.5 micrometer in aerodynamic diameter. The reduction of lung function parameters including FEV1%, FEV/FVC, MMF%, and PEF% in CB workers was observed, and the IL-1ß, IL-6, IL-8, MIP-1beta, and TNF- alpha had 2.86-, 6.85-, 1.49-, 3.35-, and 4.87-folds increase in serum of CB workers, respectively. In mice exposed to the aerosol CB, particles were deposited in the lung. The alveolar wall thickened and a large amount of inflammatory cells were observed in lung tissues after CB exposure. IL-6 and IL-8 levels were increased in both serum and lung homogenate. CONCLUSIONS: The data strongly suggests that nanoscale CB particles could be responsible for the lung function reduction and pro-inflammatory cytokines secretion in CB workers. These results, therefore, provide the first evidence of a link between human exposure to CB and long-term pulmonary effects.

Global and MGMT promoter hypomethylation independently associated with genomic instability of lymphocytes in subjects exposed to high-dose polycyclic aromatic hydrocarbon.

Global hypomethylation, gene-specific methylation, and genome instability are common events in tumorigenesis. To date, few studies have examined the aberrant DNA methylation patterns in coke oven workers, who are highly at risk of lung cancer by occupational exposure to polycyclic aromatic hydrocarbons (PAHs). We recruited 82 PAH-exposed workers and 62 unexposed controls, assessed exposure levels by urinary 1-hydroxypyrene, and measured genetic damages by comet assay, bleomycin sensitivity, and micronucleus assay. The PAHs in coke oven emissions (COE) were estimated based on toxic equivalency factors. We used bisulfite-PCR pyrosequencing to quantitate DNA methylation in long interspersed nuclear element-1 (LINE-1) and O(6)-methylguanine-DNA methyltransferase (MGMT). Further, the methylation alteration was also investigated in COE-treated human bronchial epithelial (16HBE) cells. We found there are higher levels of PAHs in COE. Among PAH-exposed workers, LINE-1 and MGMT methylation levels (with CpG site specificity) were significantly lowered. LINE-1, MGMT, and its hot CpG site-specific methylation were negatively correlated with urinary 1-hydroxypyrene levels (r = -0.329, p < 0.001; r = -0.164, p = 0.049 and r = -0.176, p = 0.034, respectively). In addition, LINE-1 methylation was inversely associated with comet tail moment and micronucleus frequency, and a significant increase of micronucleus in low MGMT methylation group. In vitro study revealed that treatment of COE in 16HBE cells resulted in higher production of BPDE-DNA adducts, LINE-1 hypomethylation, hypomethylation, and suppression of MGMT expression. These findings suggest hypomethylation of LINE-1 and MGMT promoter could be used as markers for PAHs exposure and merit further investigation.

Occupational exposure to diesel engine exhaust and serum levels of microRNAs in a cross-sectional molecular epidemiology study in China.

Diesel engine exhaust (DEE) is an established lung carcinogen, but the biological mechanisms of diesel-induced lung carcinogenesis are not well understood. MicroRNAs (miRNAs) are small noncoding RNAs that play a potentially important role in regulating gene expression related to lung cancer. We conducted a cross-sectional molecular epidemiology study to evaluate whether serum levels of miRNAs are altered in healthy workers occupationally exposed to DEE compared to unexposed controls. We conducted a two-stage study, first measuring 405 miRNAs in a pilot study of six DEE-exposed workers exposed and six controls. In the second stage, 44 selected miRNAs were measured using the Fireplex circulating miRNA assay that profiles miRNAs directly from biofluids of 45 workers exposed to a range of DEE (Elemental Carbon (EC), median, range: 47.7, 6.1-79.7 µg/m3 ) and 46 controls. The relationship between exposure to DEE and EC with miRNA levels was analyzed using linear regression adjusted for potential confounders. Serum levels of four miRNAs were significantly lower (miR-191-5p, miR-93-5p, miR-423-3p, miR-122-5p) and one miRNA was significantly higher (miR-92a-3p) in DEE exposed workers compared to controls. Of these miRNAs, miR-191-5p (ptrend  = .001, FDR = 0.04) and miR-93-5p (ptrend  = .009, FDR = 0.18) showed evidence of an inverse exposure-response with increasing EC levels. Our findings suggest that occupational exposure to DEE may affect circulating miRNAs implicated in biological processes related to carcinogenesis, including immune function.

Urinary Amino-PAHs in relation to diesel engine emissions and urinary mutagenicity.

Diesel exhaust has long been of health concern due to established toxicity including carcinogenicity in humans. However, the precise components of diesel engine emissions that drive carcinogenesis are still unclear. Limited work has suggested that nitrated polycyclic aromatic hydrocarbons (NPAHs) such as 1-nitropyrene and 2-nitrofluorene may be more abundant in diesel exhaust. The present study aimed to examine whether urinary amino metabolites of these NPAHs were associated with high levels of diesel engine emissions and urinary mutagenicity in a group of highly exposed workers including both smokers and nonsmokers. Spot urine samples were collected immediately following a standard work shift from each of the 54 diesel engine testers and 55 non-tester controls for the analysis of five amino metabolites of NPAHs, and cotinine (a biomarker of tobacco smoke exposure) using liquid chromatography-mass spectrometry. An overnight urine sample was collected in a subgroup of non-smoking participants for mutagenicity analysis using strain YG1041 in the Salmonella (Ames) mutagenicity assay. Personal exposure to fine particles (PM2.5) and more-diesel-specific constituents (elemental carbon and soot) was assessed for the engine testers by measuring breathing-zone concentrations repeatedly over several full work shifts. Results showed that it was 12.8 times more likely to detect 1-aminopyrene and 2.9 times more likely to detect 2-aminofluorene in the engine testers than in unexposed controls. Urinary concentrations of 1-aminopyrene were significantly higher in engine testers (p < 0.001), and strongly correlated with soot and elemental carbon exposure as well as mutagenicity tested in strain YG1041 with metabolic activation (p < 0.001). Smoking did not affect 1-aminopyrene concentrations and 1-aminopyrene relationships with diesel exposure. In contrast, both engine emissions and smoking affected 2-aminofluorene concentrations. The results confirm that urinary 1-aminopyrene may serve as an exposure biomarker for diesel engine emissions and associated mutagenicity.

[Association between cytokines and trichloroethylene-induced hypersensitivity dermatitis].

OBJECTIVE: To detect the cytokines levels in serums of patients with trichloroethylene-induced hypersensitivity dermatitis and explore the effect biomarkers associated with this disease. METHODS: Twenty-two patients with TCE-induced hypersensitivity dermatitis, twenty-two healthy TCE-exposed workers from the same workshops with patients and twenty-two comparable unexposed controls were recruited in this study. Eight cytokines in serums from all subjects were detected using Liquid Suspended Biochip; the correlation among the eight cytokines including interleukin (IL)-1ß (IL-1ß), IL-5, IL-8, IL-10, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), macrophage chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1ß (MIP-1ß) and the correlation between IL-5 and eosinophil count were analyzed. RESULTS: The medians of levels of IL-1ß, IFN-γ, IL-5, IL-10, MCP-1, MIP-1ß, IL-8 among patients were 0.15, 80.13, 2.95, 6.45, 83.83, 1057.90, 440.22 pg/ml, respectively, which were higher than those among the TCE-exposed workers (0.09, 16.93, 0.11, 0.07, 28.75, 241.07, 28.26 pg/ml, respectively, all P values < 0.01) and unexposed controls (0.09, 3.14, 0.11, 0.07, 25.27, 209.64, 207.34 pg/ml, respectively, all P values < 0.01). The median of level of TNF-α among the patients was 13.26 pg/ml, which was significantly higher than that among TCE-exposed workers (4.87 pg/ml, P < 0.01) but not among unexposed controls; the median of level of IL-5 among the TCE-exposed workers was 0.11 pg/ml, which was significantly higher than that among the unexposed controls (0.11 pg/ml, P < 0.01). The median of levels of IL-8 among the unexposed controls was 207.34 pg/ml, which was significantly higher than that among the TCE-exposed workers (28.26 pg/ml, P < 0.01). In case group, except for correlation of TNF-α and IFN-γ, TNF-α and IL-5, the significant positive correlations were found among any two cytokines (r(IL-1ß,IFN-γ) = 0.500, r(IL-1ß,TNF-α) = 0.348, r(IL-1ß,MCP-1) = 0.537, r(IL-1ß,MIP-1ß) = 0.477, r(IL-1ß,IL-8) = 0.466, r(IL-1ß,IL-5) = 0.610, r(IL-1ß,IL-10) = 0.626, r(IFN-γ,MCP-1) = 0.460, r(IFN-γ,MIP-1ß) = 0.491, r(IFN-γ,IL-8) = 0.322, r(IFN-γ,IL-5) = 0.532, r(IFN-γ,IL-10) = 0.511, r(TNF-α,MCP-1) = 0.325, r(TNF-α,MIP-1ß) = 0.283, r(TNF-α,IL-8) = 0.430, r(TNF-α,IL-10) = 0.271, r(MCP-1,MIP-1ß) = 0.659, r(MCP-1,IL-8) = 0.526, r(MCP-1,IL-5) = 0.504, r(MCP-1,IL-10) = 0.614, r(MIP-1ß,IL-8) = 0.601, r(MIP-1ß,IL-5) = 0.451, r(MIP-1ß,IL-10) = 0.579, r(IL-8,IL-5) = 0.255, r(IL-8,IL-10) = 0.403, r(IL-5,IL-10) = 0.798, all P values < 0.05). The median of level of IL-5 among the patients with high eosinophils counts was 8.92 pg/ml, which was significantly higher than that among the patients with low eosinophils counts (1.04 pg/ml, P < 0.05). CONCLUSION: The abnormal production of IL-1ß, IFN-γ, TNF-α, IL-8, MCP-1, MIP-1ß, IL-5 and IL-10 was related with the pathogenesis of hypersensitivity dermatitis induced by TCE. These cytokines could be used as referential indexes in the early health surveillance and clinic disease treatment.

Proteomic analysis of serum in workers exposed to diesel engine exhaust.

Diesel engine exhaust (DEE) is classified as a Group 1 human carcinogen. Using a targeted proteomics approach, we aimed to identify proteins associated with DEE and characterize these markers to understand the mechanisms of DEE-induced carcinogenicity. In this cross-sectional molecular epidemiology study, we measured elemental carbon (EC) using a personal air monitor and quantified 1317 targeted proteins in the serum using the SOMAScan assay (SOMALogic) among 19 diesel exposed factory workers and 19 unexposed controls. We used linear regressions to identify proteins associated with DEE and examined their exposure-response relationship across levels of EC using linear trend tests. We further examined pathway enrichment of DEE-related proteins using MetaCore. Occupational exposure to DEE was associated with altered levels of 22 serum proteins (permutation p < .01). Of these, 13 proteins (CXCL11, HAPLN1, FLT4, CD40LG, PES1, IGHE.IGK..IGL, TNFSF9, PGD, NAGK, CCL25, CCL4L1, PDXK, and PLA2G1B) showed an exposure-response relationship with EC (p trend < .01), with serum levels of all but PLA2G1B declining with increasing air levels of EC. For instance, C-X-C Motif Chemokine Ligand 11 (CXCL11) showed the most significant association with DEE (ß = -0.25; permutation p = .00004), where mean serum levels were 4121.1, 2356.7, and 2298.8 relative fluorescent units among the unexposed, lower exposed (median, range : 56.9, 40.2-62.1 µg/m3 EC), and higher exposed (median, range of EC: 72.9, 66.9-107.7 µg/m3 EC) groups, respectively (p trend = .0005). Pathway analysis suggested that these proteins are enriched in pathways related to inflammation and immune regulation. Our study suggests that DEE exposure is associated with altered serum proteins, which play a role in inflammation and immune regulation.

Exposure to diesel engine exhaust and alterations to the Cys34/Lys525 adductome of human serum albumin.

We investigated whether exposure to carcinogenic diesel engine exhaust (DEE) was associated with altered adduct levels in human serum albumin (HSA) residues. Nano-liquid chromatography-high resolution mass spectrometry (nLC-HRMS) was used to measure adducts of Cys34 and Lys525 residues in plasma samples from 54 diesel engine factory workers and 55 unexposed controls. An untargeted adductomics and bioinformatics pipeline was used to find signatures of Cys34/Lys525 adductome modifications. To identify adducts that were altered between DEE-exposed and unexposed participants, we used an ensemble feature selection approach that ranks and combines findings from linear regression and penalized logistic regression, then aggregates the important findings with those determined by random forest. We detected 40 Cys34 and 9 Lys525 adducts. Among these findings, we found evidence that 6 Cys34 adducts were altered between DEE-exposed and unexposed participants (i.e., 841.75, 851.76, 856.10, 860.77, 870.43, and 913.45). These adducts were biologically related to antioxidant activity.

The effect of 2,5-hexanedione on myelin protein zero expression, and its mitigation using Ginkgo biloba extract.

OBJECTIVE: To investigate the role of myelin protein zero (P(0)) in 2,5-hexanedione (2,5-HD)-induced peripheral nerve injury, and the protective effect of Ginkgo biloba extract (Egb761) on 2,5-HD-induced toxic peripheral neuropathy. METHODS: After 4 weeks of treatment with 2,5-HD at different doses (50, 100, 200, 400 mg/kg) in rats, changes in the levels of P(0) in rat sciatic nerves was investigated, and the effect of Egb761 on 2,5-HD-induced toxic peripheral neuropathy was studied. RESULTS: The blood-nerve barrier (BNB) permeability of the sciatic nerve increased, and the expression of P(0) mRNA and P(0) protein decreased in a dose-dependent manner after treatment with 2,5-HD for 4 weeks. Pretreatment with Egb761 protected against BNB interruption, and inhibited P(0) mRNA and protein reduction during 2,5-HD treatment. Pretreatment with Egb761 significantly reduced loss of body weight (P<0.01) and mitigated gait abnormalities (2.85±0.22) induced by 400 mg/kg 2,5-HD (P<0.01). It also reduced the signs of neurotoxicity induced by 2,5-HD. CONCLUSION: 2,5-HD inhibited the expression of P(0) in a dose-dependent manner, and this may be an important mechanism by which toxic peripheral neuropathy is induced by 2,5-HD. Egb761 has a protective effect against 2,5-HD-induced peripheral neurotoxicity in rats.

[Hypermethylation of O(6)-methylguanine-DNA methyltransferase in human bronchial epithelial cell induced by organic extracts of coke oven emissions].

OBJECTIVE: To elucidate the mechanism of carcinogenesis induced by coke oven emissions by investigating the cell genetic damage index and the methylation of O6-methylguanine-DNA methyltransferase (MGMT). METHODS: The human bronchial epithelial cell 16HBE was treated by 1 µmol/L B(a)P for 48 h, and then was exposed continuously to either 1‰ dimethyl sulfoxide (DMSO) or organic extracts of coke oven emission (OE-COE) for five days at the concentrations of 0, 2.5, 5.0, 10.0 and 20.0 µg/ml. The methylation-specific PCR (MSP-PCR), RT-PCR and immunoblotting were applied to detect the methylation status, changes of mRNA and protein of MGMT, respectively. Single cell gel electrophoresis was used to detect DNA damage induced by OE-COE. RESULTS: Compared with the control group (DMSO), there was a significant hypermethylation in all study groups, along with the suppression of mRNA and protein in a dose-dependent manner, and the gradation ratio of them was 1.0, 0.96, 0.96, 0.85, 0.32 and 1.0, 1.0, 1.1, 0.41, 0.52, separately. There was a significant DNA damage with a dose-effect relationship in all study groups (F = 41.22, P < 0.05), and the comet Olive tail moment was (2.98 ± 1.43), (4.76 ± 1.79), (10.09 ± 1.75), (11.38 ± 1.77), (11.67 ± 1.88). The further study found that the index of DNA damage was negatively correlated to the expression of MGMT mRNA and its protein. CONCLUSION: The DNA damage induced by COE might be associated with the suppression of MGMT caused by its hypermethylation.

[Analysis of subgroups of lymphocyte in peripheral blood among dermatitis medicamentosa-like of trichloroethylene patients and healthy exposed workers].

OBJECTIVE: To study the effects of trichloroethylene (TCE) to lymphocyte subsets among exposed workers, and explore the early immunological effect biomarkers for prevention of hypersensitivity dermatitis induced by TCE. METHODS: Twenty-eight patients with TCE-induced hypersensitivity dermatitis, 56 healthy TCE-exposed workers from the same workshops with patients, and 28 comparable unexposed controls were recruited in this study. The total lymphocyte count and the major lymphocyte subsets including T cell, CD4(+) T cell, CD8(+) T cell, B cell, NK cell in peripheral blood were measured by Flow Cytometer analysis and Standard blood count analysis. RESULTS: The total lymphocyte count and T cell, CD4(+) T cell, CD8(+) T cell among patients (median at 2810.00, 1846.17, 831.87, 904.05 cell counts/µl blood) were significantly increased compared with TCE-exposed workers (median at 2101.00, 1218.59, 643.87, 482.81 cell counts/µl blood, Z = -3.19, -4.96, -3.22, -4.99, P < 0.001) and unexposed controls (median at 1900.00, 1223.60, 558.60, 325.80 cell counts/µl blood, Z = -3.30, -4.46, -3.45, -5.03, P < 0.001), the NK cell and CD3(+)CD4(+)/CD3(+)CD8(+) ratio among patients (median at 255.50 cell counts/µl blood and 1.11) were significantly decreased compared with the unexposed controls (median at 642.60 cell counts/µl blood and 1.96, Z = -3.56 and -3.11, P < 0.01). Meanwhile, for the exposed workers, the CD8(+) T cell (median at 482.81 cell counts/µl blood) was significantly increased and the NK cell and CD3(+)CD4(+)/CD3(+)CD8(+) ratio (median at 318.76 cell counts/µl blood and 1.27) were significantly decreased compared with unexposed controls (median at 325.80 and 642.60 cell counts/µl blood and 1.96, Z = -2.63, -3.52, -2.29, P < 0.05). CONCLUSION: Occupational exposure to TCE could affect the lymphocyte subsets, especially T cell and NK cell. The total lymphocyte count, T cell and CD4(+) T cell might be effect biomarkers for subjects with hypersensitivity dermatitis among TCE-exposed workers.

[Association between polymorphisms of metabolic genes and telomere length in workers exposed to polycyclic aromatic hydrocarbon].

OBJECTIVE: To investigate the association between the polymorphisms of metabolic genes and telomere length of genomic DNA in peripheral blood of workers exposed to polycyclic aromatic hydrocarbons (PAHs). METHODS: One hundred and forty five coke-oven workers exposed to PAHs and sixty eight non-exposed medical staffs were recruited in this study. Urinary 1-hydroxypyrene (1-OHP) served as the internal exposure dose of PAHs for all subjects. Relative telomere length (RTL) of genomic DNA in peripheral blood was used as telomere length and measured by real-time PCR. Polymorphisms of metabolic genes were detected by PCR-based methods. RESULTS: Compared with control group, the exposure group shown a decreased RTL (1.10 +/- 0.75 vs 1.43 +/- 1.06, P < 0.05). In the coke-oven workers, after adjusting the sex, age, cigarettes per day and urinary 1-OHP, RTL (1.25 +/- 0.93) of workers with CT genotype at the CYP1A1 3801 T > C was significantly longer than that (0.93 +/- 0.51) of workers with TT genotype (P < 0.05). RTL (0.90 +/- 0.58) of individuals with the Tyr/His genotype at mEH Tyr113His was significantly shorter than that (1.24 +/- 0.90) of individuals with the Tyr/Tyr genotype (P < 0.05). RTL (1.02 +/- 0.64) of individuals with the CT genotype at AHR rs10250822 was significantly shorter than that (1.36 +/- 1.14) of individuals with the CC genotype (P < 0.05). RTL (0.93 +/- 0.54) of individuals with the AT genotype at AHR rs10247158 was significantly shorter than that (1.19 +/- 0.84) of individuals with the AA genotype (P < 0.05). CONCLUSION: The results of present study suggested that PAHs exposure could induce the shorted RTL, CYP1A1, mEH, AHR polymorphisms might influence the change of telomere length of genomic DNA in peripheral blood of workers exposed to PAHs.