Toxicity of particles derived from combustion of Ethiopian traditional biomass fuels in human bronchial and macrophage-like cells.

The combustion of traditional fuels in low-income countries, including those in sub-Saharan Africa, leads to extensive indoor particle exposure. Yet, the related health consequences in this context are understudied. This study aimed to evaluate the in vitro toxicity of combustion-derived particles relevant for Sub-Saharan household environments. Particles (< 2.5 µm) were collected using a high-volume sampler during combustion of traditional Ethiopian biomass fuels: cow dung, eucalyptus wood and eucalyptus charcoal. Diesel exhaust particles (DEP, NIST 2975) served as reference particles. The highest levels of particle-bound polycyclic aromatic hydrocarbons (PAHs) were found in wood (3219 ng/mg), followed by dung (618 ng/mg), charcoal (136 ng/mg) and DEP (118 ng/mg) (GC-MS). BEAS-2B bronchial epithelial cells and THP-1 derived macrophages were exposed to particle suspensions (1-150 µg/mL) for 24 h. All particles induced concentration-dependent genotoxicity (comet assay) but no pro-inflammatory cytokine release in epithelial cells, whereas dung and wood particles also induced concentration-dependent cytotoxicity (Alamar Blue). Only wood particles induced concentration-dependent cytotoxicity and genotoxicity in macrophage-like cells, while dung particles were unique at increasing secretion of pro-inflammatory cytokines (IL-6, IL-8, TNF-α). In summary, particles derived from combustion of less energy dense fuels like dung and wood had a higher PAH content and were more cytotoxic in epithelial cells. In addition, the least energy dense and cheapest fuel, dung, also induced pro-inflammatory effects in macrophage-like cells. These findings highlight the influence of fuel type on the toxic profile of the emitted particles and warrant further research to understand and mitigate health effects of indoor air pollution.

Genotoxicity by rapeseed methyl ester and hydrogenated vegetable oil combustion exhaust products in lung epithelial (A549) cells.

Biofuel is an attractive substitute for petrodiesel because of its lower environmental footprint. For instance, the polycyclic aromatic hydrocarbons (PAH) emission per fuel energy content is lower for rapeseed methyl ester (RME) than for petrodiesel. This study assesses genotoxicity by extractable organic matter (EOM) of exhaust particles from the combustion of petrodiesel, RME, and hydrogenated vegetable oil (HVO) in lung epithelial (A549) cells. Genotoxicity was assessed as DNA strand breaks by the alkaline comet assay. EOM from the combustion of petrodiesel and RME generated the same level of DNA strand breaks based on the equal concentration of total PAH (i.e. net increases of 0.13 [95% confidence interval (CI): 0.002, 0.25, and 0.12 [95% CI: 0.01, 0.24] lesions per million base pairs, respectively). In comparison, the positive control (etoposide) generated a much higher level of DNA strand breaks (i.e. 0.84, 95% CI: 0.72, 0.97) lesions per million base pairs. Relatively low concentrations of EOM from RME and HVO combustion particles (<116 ng/ml total PAH) did not cause DNA strand breaks in A549 cells, whereas benzo[a]pyrene and PAH-rich EOM from petrodiesel combusted using low oxygen inlet concentration were genotoxic. The genotoxicity was attributed to high molecular weight PAH isomers with 5-6 rings. In summary, the results show that EOM from the combustion of petrodiesel and RME generate the same level of DNA strand breaks on an equal total PAH basis. However, the genotoxic hazard of engine exhaust from on-road vehicles is lower for RME than petrodiesel because of lower PAH emission per fuel energy content.

Polycyclic aromatic hydrocarbon (PAH) exposure during pregnancy and child anthropometry from birth to 10 years of age: Sex-specific evidence from a cohort study in rural Bangladesh.

Polycyclic aromatic hydrocarbons (PAHs) have endocrine disrupting properties and they cross the placental barrier, but studies on gestational exposure and child anthropometry are inconclusive. We aimed to elucidate the impact of early gestational PAH exposure on anthropometry from birth to 10 years of age in 1295 mother-child pairs from a nested sub-cohort of the MINIMat trial in Bangladesh. Several PAH metabolites [1-hydroxyphenanthrene (1-OH-Phe), Σ2-,3-hydroxyphenanthrene (Σ2-,3-OH-Phe), 4-hydroxyphenanthrene (4-OH-Phe), 1-hydroxypyrene (1-OH-Pyr), Σ2-,3-hydroxyfluorene (Σ2-,3-OH-Flu)] were quantified in spot urine collected around gestational week 8 using LC-MS/MS. Child weight and height were measured at 19 occasions from birth to 10 years. Multivariable-adjusted regression models were used to assess associations of maternal PAH metabolites (log2-transformed) with child anthropometry. The median concentration of 1-OH-Phe, Σ2-,3-OH-Phe, 4-OH-Phe, 1-OH-Pyr and Σ2-,3-OH-Flu was 1.5, 1.9, 0.14, 2.5, and 2.0 ng/mL, respectively. All maternal urinary PAH metabolites were positively associated with newborn weight and length and all associations were more pronounced in boys than in girls (p interaction for all <0.14). In boys, the strongest associations were observed with Σ2-,3-OH-Phe and Σ2-,3-OH-Flu for which each doubling increased mean birth weight by 41 g (95% CI: 13; 69 and 12; 70) and length by 0.23 cm (0.075; 0.39) and 0.21 cm (0.045; 0.37), respectively. Maternal urinary PAH metabolites were not associated with child anthropometry at 10 years. In longitudinal analysis, however, maternal urinary PAH metabolites were positively associated with boys' weight-for-age (WAZ) and height-for-age Z-scores (HAZ) from birth to 10 years, but only the association of 4-OH-Phe with HAZ was significant (B: 0.080 Z-scores; 95% CI 0.013, 0.15). No associations were observed with girls' WAZ or HAZ. In conclusion, gestational PAH exposure was positively associated with fetal and early childhood growth, especially in boys. Further studies are needed to confirm causality and to explore long-term health effects.

Phthalate levels in prenatal and postnatal bedroom dust in the SELMA study.

Phthalates are common in polyvinyl chloride (PVC) plastics and numerous consumer goods in our homes from which they can migrate and adhere to indoor dust particles. It is known that indoor dust exposure contribute to human phthalate intake; however, there is a lack of large studies with a repeated-measure design investigating how phthalate levels in indoor dust may vary over time in people's homes. This study investigated levels of seven phthalates and one alternative plasticiser di-iso-nonyl-cyclohexane-di-carboxylate (DiNCH) in bedroom dust collected prenatally around week 25 during pregnancy and postnatally at six months after birth, from 496 Swedish homes. Prenatal and postnatal phthalate levels were compared using correlation and season-adjusted general linear regression models. Over the nine-month period, levels of six out of seven phthalates were associated as indicated by a positive Pearson correlation (0.18 < r < 0.50, P < .001) and Lin's concordance correlation between matched prenatal and postnatal dust samples. Compared to prenatal levels, the season-adjusted postnatal levels decreased for five phthalates, whilst di-ethyl-hexyl phthalate (DEHP), di-2-propylheptyl phthalate (DPHP) and DiNCH increased. The results suggest that families with higher phthalate levels in bedroom dust during pregnancy are likely to remain among those with higher levels in the infancy period. However, all average phthalate levels changed over this specific nine-month period suggesting that available phthalate sources or their use were altered between the dust collections. Changes in home characteristics, family lifestyle, and phthalate replacement trends may contribute to explain the differences.

Lung function and self-rated symptoms in healthy volunteers after exposure to hydrotreated vegetable oil (HVO) exhaust with and without particles.

BACKGROUND: Diesel engine exhaust causes adverse health effects. Meanwhile, the impact of renewable diesel exhaust, such as hydrotreated vegetable oil (HVO), on human health is less known. Nineteen healthy volunteers were exposed to HVO exhaust for 3 h in a chamber with a double-blind, randomized setup. Exposure scenarios comprised of HVO exhaust from two modern non-road vehicles with 1) no aftertreatment system ('HVOPM+NOx' PM1: 93 µg m-3, EC: 54 µg m-3, NO: 3.4 ppm, NO2: 0.6 ppm), 2) an aftertreatment system containing a diesel oxidation catalyst and a diesel particulate filter ('HVONOx' PM1: ~ 1 µg m-3, NO: 2.0 ppm, NO2: 0.7 ppm) and 3) filtered air (FA) as control. The exposure concentrations were in line with current EU occupational exposure limits (OELs) of NO, NO2, formaldehyde, polycyclic aromatic hydrocarbons (PAHs), and the future OEL (2023) of elemental carbon (EC). The effect on nasal patency, pulmonary function, and self-rated symptoms were assessed. Calculated predicted lung deposition of HVO exhaust particles was compared to data from an earlier diesel exhaust study. RESULTS: The average total respiratory tract deposition of PM1 during HVOPM+NOx was 27 µg h-1. The estimated deposition fraction of HVO PM1 was 40-50% higher compared to diesel exhaust PM1 from an older vehicle (earlier study), due to smaller particle sizes of the HVOPM+NOx exhaust. Compared to FA, exposure to HVOPM+NOx and HVONOx caused higher incidence of self-reported symptoms (78%, 63%, respectively, vs. 28% for FA, p < 0.03). Especially, exposure to HVOPM+NOx showed 40-50% higher eye and throat irritation symptoms. Compared to FA, a decrement in nasal patency was found for the HVONOx exposures (- 18.1, 95% CI: - 27.3 to - 8.8 L min-1, p < 0.001), and for the HVOPM+NOx (- 7.4 (- 15.6 to 0.8) L min-1, p = 0.08). Overall, no clinically significant change was indicated in the pulmonary function tests (spirometry, peak expiratory flow, forced oscillation technique). CONCLUSION: Short-term exposure to HVO exhaust concentrations corresponding to EU OELs for one workday did not cause adverse pulmonary function changes in healthy subjects. However, an increase in self-rated mild irritation symptoms, and mild decrease in nasal patency after both HVO exposures, may indicate irritative effects from exposure to HVO exhaust from modern non-road vehicles, with and without aftertreatment systems.

Underground emissions and miners' personal exposure to diesel and renewable diesel exhaust in a Swedish iron ore mine.

PURPOSE: Underground diesel exhaust exposure is an occupational health risk. It is not known how recent intensified emission legislation and use of renewable fuels have reduced or altered occupational exposures. We characterized these effects on multipollutant personal exposure to diesel exhaust and underground ambient air concentrations in an underground iron ore mine. METHODS: Full-shift personal sampling (12 workers) of elemental carbon (EC), nitrogen dioxide (NO2), polycyclic aromatic hydrocarbons (PAHs), and equivalent black carbon (eBC) was performed. The study used and validated eBC as an online proxy for occupational exposure to EC. Ambient air sampling of these pollutants and particle number size distribution and concentration were performed in the vicinity of the workers. Urine samples (27 workers) were collected after 8 h exposure and analyzed for PAH metabolites and effect biomarkers (8-oxodG for DNA oxidative damage, 4-HNE-MA for lipid peroxidation, 3-HPMA for acrolein). RESULTS: The personal exposures (geometric mean; GM) of the participating miners were 7 µg EC m-3 and 153 µg NO2 m-3, which are below the EU occupational exposure limits. However, exposures up to 94 µg EC m-3 and 1200 µg NO2 m-3 were observed. There was a tendency that the operators of vehicles complying with sharpened emission legislation had lower exposure of EC. eBC and NO2 correlated with EC, R = 0.94 and R = 0.66, respectively. No correlation was found between EC and the sum of 16 priority PAHs (GM 1790 ng m-3). Ratios between personal exposures and ambient concentrations were similar and close to 1 for EC and NO2, but significantly higher for PAHs. Semi-volatile PAHs may not be effectively reduced by the aftertreatment systems, and ambient area sampling did not predict the personal airborne PAHs exposure well, neither did the slightly elevated concentration of urinary PAH metabolites correlate with airborne PAH exposure. CONCLUSION: Miners' exposures to EC and NO2 were lower than those in older studies indicating the effect of sharpened emission legislation and new technologies. Using modern vehicles with diesel particulate filter (DPF) may have contributed to the lower ambient underground PM concentration and exposures. The semi-volatile behavior of the PAHs might have led to inefficient removal in the engines aftertreatment systems and delayed removal by the workplace ventilation system due to partitioning to indoor surfaces. The results indicate that secondary emissions can be an important source of gaseous PAH exposure in the mine.

Arsenic exposure and biomarkers for oxidative stress and telomere length in indigenous populations in Bolivia.

BACKGROUND: Women living in the Bolivian Andes are environmentally exposed to arsenic, yet there is scarce information about arsenic-related effects in this region. Several biomarkers for telomere length and oxidative stress (mitochondrial DNA copy number, mtDNAcn; 8-Oxo-2'-deoxyguanosine, 8-oxo-dG; and 4-hydroxy nonenal mercapturic acid, 4-HNE-MA) have been previously linked to arsenic, and some of which are prospective biomarkers for cancer risk. OBJECTIVE AND HYPOTHESIS: To evaluate associations between arsenic exposure and telomere length, mtDNAcn, 8-oxo-dG, and 4-HNE-MA in Bolivians. Arsenic exposure was hypothesized to be positively associated with all four toxicity biomarkers, particularly in individuals with a less efficient arsenic metabolism. METHODS: The study encompassed 193 indigenous women. Arsenic exposure was assessed in urine as the sum of inorganic arsenic metabolite concentrations (U-As) measured by HPLC-HG-ICP-MS, and in whole blood as total arsenic (B-As) measured by ICP-MS. Efficiency of arsenic metabolism was evaluated by a polymorphism (rs3740393) in the main arsenic methylating gene AS3MT measured by TaqMan allelic discrimination, and by the relative fractions of urinary inorganic arsenic metabolites. Telomere length and mtDNAcn were determined in peripheral blood leukocytes by quantitative PCR, and urinary 8-oxo-dG and 4-HNE-MA by LC-MS/MS. RESULTS: U-As and B-As were associated with longer telomeres and higher mtDNAcn, particularly in women with a less efficient arsenic metabolism. Urinary 8-oxo-dG and 4-HNE-MA were positively associated with U-As, but only 4-HNE-MA was associated with B-As. Arsenic metabolism efficiency did not have a clear effect on the concentrations of either of these biomarkers. CONCLUSION: Bolivian women showed indications of arsenic toxicity, measured by four different biomarkers. Telomere length, mtDNAcn, and 4-HNE-MA were positively associated with both U-As and B-As. The association of arsenic exposure with telomere length and mtDNAcn was only present in Bolivian women with a less efficient metabolism. These findings call for additional efforts to evaluate and reduce arsenic exposure in Bolivia.

Low-level exposure to polycyclic aromatic hydrocarbons is associated with reduced lung function among Swedish young adults.

BACKGROUND: Exposure to polycyclic aromatic hydrocarbons (PAHs) has been linked to adverse pulmonary effects. However, the impact of low-level environmental PAH exposure on lung function in early adulthood remains uncertain. OBJECTIVES: To evaluate the associations between urinary PAH metabolites and lung function parameters in young adults. METHODS: Urinary metabolites of pyrene, phenanthrene, and fluorene were analysed in 1000 young adults from Sweden (age 22-25 years) using LC-MS/MS. Lung function and eosinophilic airway inflammation were measured by spirometry and exhaled nitric oxide fraction (FeNO), respectively. Linear regression analysis was used to evaluate associations between PAH metabolites and the outcomes. RESULTS: Median urinary concentrations of 1-OH-pyrene, ∑OH-phenanthrene, and ∑OH-fluorene were 0.066, 0.36, 0.22 µg/L, respectively. We found inverse associations of ∑OH-phenanthrene and ∑OH-fluorene with FEV1 and FVC, as well as between 1-OH-pyrene and FEV1/FVC ratio (adjusted P < 0.05; all participants). An increase of 1% in ∑OH-fluorene was associated with a decrease of 73 mL in FEV1 and 59 mL in FVC. In addition, ∑OH-phenanthrene concentrations were, in a dose-response manner, inversely associated with FEV1 (B from -109 to -48 compared with the lowest quartile of ∑OH-phenanthrene; p trend 0.004) and FVC (B from -159 to -102 compared with lowest quartile; p-trend <0.001). Similar dose-response associations were also observed between ∑OH-fluorene and FEV1 and FVC, as well as between 1-OH-pyrene and FEV1/FVC (p-trend <0.05). There was no association between PAH exposure and FeNO, nor was there an interaction with smoking, sex, or asthma. CONCLUSION: Low-level PAH exposure was, in a dose-response manner, associated with reduced lung function in young adults. Our findings have public health implications due to i) the widespread occurrence of PAHs in the environment and ii) the clinical relevance of lung function in predicting all-cause and cardiovascular disease mortality.

Indoor phthalate exposure and contributions to total intake among pregnant women in the SELMA study.

Phthalates are widely used in consumer products. Exposure to phthalates can lead to adverse health effects in humans, with early-life exposure being of particular concern. Phthalate exposure occurs mainly through ingestion, inhalation, and dermal absorption. However, our understanding of the relative importance of different exposure routes is incomplete. This study estimated the intake of five phthalates from the residential indoor environment for 455 Swedish pregnant women in the SELMA study using phthalate mass fraction in indoor dust and compares these to total daily phthalate intakes back-calculated from phthalate metabolite concentrations in the women's urine. Steady-state models were used to estimate indoor air phthalate concentrations from dust measurements. Intakes from residential dust and air made meaningful contributions to total daily intakes of more volatile di-ethyl phthalate (DEP), di-n-butyl phthalate (DnBP), and di-iso-butyl phthalate (DiBP) (11% of total DEP intake and 28% of total DnBP and DiBP intake combined). Dermal absorption from air was the dominant pathway contributing to the indoor environmental exposure. Residential exposure to less volatile phthalates made minor contributions to total intake. These results suggest that reducing the presence of low molecular weight phthalates in the residential indoor environment can meaningfully reduce phthalate intake among pregnant women.

Inhalation of hydrogenated vegetable oil combustion exhaust and genotoxicity responses in humans.

Biofuels from vegetable oils or animal fats are considered to be more sustainable than petroleum-derived diesel fuel. In this study, we have assessed the effect of hydrogenated vegetable oil (HVO) exhaust on levels of DNA damage in peripheral blood mononuclear cells (PBMCs) as primary outcome, and oxidative stress and inflammation as mediators of genotoxicity. In a randomized cross-over study, healthy humans were exposed to filtered air, inorganic salt particles, exhausts from combustion of HVO in engines with aftertreatment [i.e. emission with nitrogen oxides and low amounts of particulate matter less than 2.5 µm (approximately 1 µg/m3)], or without aftertreatment (i.e. emission with nitrogen oxides and 93 ± 13 µg/m3 of PM2.5). The subjects were exposed for 3 h and blood samples were collected before, within 1 h after the exposure and 24 h after. None of the exposures caused generation of DNA strand breaks and oxidatively damaged DNA, or affected gene expression of factors related to DNA repair (Ogg1), antioxidant defense (Hmox1) or pro-inflammatory cytokines (Ccl2, Il8 and Tnfa) in PBMCs. The results from this study indicate that short-term HVO exhaust exposure is not associated with genotoxic hazard in humans.

Inflammation, oxidative stress and genotoxicity responses to biodiesel emissions in cultured mammalian cells and animals.

Biodiesel fuels are alternatives to petrodiesel, especially in the transport sector where they have lower carbon footprint. Notwithstanding the environmental benefit, biodiesel fuels may have other toxicological properties than petrodiesel. Particulate matter (PM) from petrodiesel causes cancer in the lung as a consequence of delivery of genotoxic polycyclic aromatic hydrocarbons, oxidative stress and inflammation. We have reviewed articles from 2002 to 2019 (50% of the articles since 2015) that have described toxicological effects in terms of genotoxicity, oxidative stress and inflammation of biodiesel exhaust exposure in humans, animals and cell cultures. The studies have assessed first generation biodiesel from different feedstock (e.g. rapeseed and soy), certain second generation fuels (e.g. waste oil), and hydrogenated vegetable oil. It is not possible to rank the potency of toxicological effects of specific biodiesel fuels. However, exposure to biodiesel exhaust causes oxidative stress, inflammation and genotoxicity in cell cultures. Three studies in animals have not indicated genotoxicity in lung tissue. The database on oxidative stress and inflammation in animal studies is larger (13 studies); ten studies have reported increased levels of oxidative stress biomarkers or inflammation, although the effects have been modest in most studies. The cell culture and animal studies have not consistently shown a different potency in effect between biodiesel and petrodiesel exhausts. Both increased and decreased potency have been reported, which might be due to differences in feedstock or combustion conditions. In conclusion, combustion products from biodiesel and petrodiesel fuel may evoke similar toxicological effects on genotoxicity, oxidative stress and inflammation.

Influence of Airborne Particles' Chemical Composition on SVOC Uptake from PVC Flooring-Time-Resolved Analysis with Aerosol Mass Spectrometry.

We sampled ammonium sulfate particles and indoor particles of outdoor origin through a small chamber covered with polyvinyl chloride flooring. We measured the uptake of semivolatile organic compounds (SVOCs) by the airborne particles in real time. The particles acquired SVOC mass fractions up to 10%. The phthalate ester (di(2-ethylhexyl)phthalate) (DEHP), a known endocrine disruptor, contributed by approximately half of the sorbed SVOC mass. The indoor particles acquired a higher DEHP fraction than laboratory-generated ammonium sulfate aerosol. We attribute this increased uptake to absorption by organic matter present in the indoor particles. Using a thermodenuder to remove volatile components, predominantly organics, reduced the SVOC uptake. Positive matrix factorization applied to the organic mass spectra suggests that hydrocarbon-like organic aerosol (typically fresh traffic exhaust) sorbs DEHP more efficiently than aged organic aerosol. The SVOC uptake is one of the processes that modify outdoor pollution particles after they penetrate buildings, where the majority of exposure occurs. Particles from indoor sources, typically dominated by organic matter, will undergo such processes as well. Aerosol mass spectrometry improves the time resolution of experimental investigations into these processes and enables experiments with lower, relevant particle concentrations. Additionally, particle size-resolved results are readily obtained.

Fluorene exposure among PAH-exposed workers is associated with epigenetic markers related to lung cancer.

OBJECTIVES: Exposure to high-molecular-weight polycyclic aromatic hydrocarbons (PAHs) may cause cancer in chimney sweeps and creosote-exposed workers, however, knowledge about exposure to low-molecular-weight PAHs in relation to cancer risk is limited. In this study, we aimed to investigate occupational exposure to the low-molecular-weight PAHs phenanthrene and fluorene in relation to different cancer biomarkers. METHODS: We recruited 151 chimney sweeps, 19 creosote-exposed workers and 152 unexposed workers (controls), all men. We measured monohydroxylated metabolites of phenanthrene and fluorene in urine using liquid chromatography coupled to tandem mass spectrometry. We measured, in peripheral blood, the cancer biomarkers telomere length and mitochondrial DNA copy number using quantitative PCR; and DNA methylation of F2RL3 and AHRR using pyrosequencing. RESULTS: Median PAH metabolite concentrations were higher among chimney sweeps (up to 3 times) and creosote-exposed workers (up to 353 times), compared with controls (p<0.001; adjusted for age and smoking). ∑OH-fluorene (sum of 2-hydroxyfluorene and 3-hydroxyfluorene) showed inverse associations with percentage DNA methylation of F2RL3 and AHRR in chimney sweeps (B (95% CI)=-2.7 (-3.9 to -1.5) for F2RL3_cg03636183, and -7.1 (-9.6 to -4.7) for AHRR_cg05575921: adjusted for age and smoking), but not in creosote-exposed workers. In addition, ∑OH-fluorene showed a 42% mediation effect on the inverse association between being a chimney sweep and DNA methylation of AHRR CpG2. CONCLUSIONS: Chimney sweeps and creosote-exposed workers were occupationally exposed to low-molecular-weight PAHs. Increasing fluorene exposure, among chimney sweeps, was associated with lower DNA methylation of F2RL3 and AHRR, markers for increased lung cancer risk. These findings warrant further investigation of fluorene exposure and toxicity.

Particle characterization and toxicity in C57BL/6 mice following instillation of five different diesel exhaust particles designed to differ in physicochemical properties.

BACKGROUND: Diesel exhaust is carcinogenic and exposure to diesel particles cause health effects. We investigated the toxicity of diesel exhaust particles designed to have varying physicochemical properties in order to attribute health effects to specific particle characteristics. Particles from three fuel types were compared at 13% engine intake O2 concentration: MK1 ultra low sulfur diesel (DEP13) and the two renewable diesel fuels hydrotreated vegetable oil (HVO13) and rapeseed methyl ester (RME13). Additionally, diesel particles from MK1 ultra low sulfur diesel were generated at 9.7% (DEP9.7) and 17% (DEP17) intake O2 concentration. We evaluated physicochemical properties and histopathological, inflammatory and genotoxic responses on day 1, 28, and 90 after single intratracheal instillation in mice compared to reference diesel particles and carbon black. RESULTS: Moderate variations were seen in physical properties for the five particles: primary particle diameter: 15-22 nm, specific surface area: 152-222 m2/g, and count median mobility diameter: 55-103 nm. Larger differences were found in chemical composition: organic carbon/total carbon ratio (0.12-0.60), polycyclic aromatic hydrocarbon content (1-27 µg/mg) and acid-extractable metal content (0.9-16 µg/mg). Intratracheal exposure to all five particles induced similar toxicological responses, with different potency. Lung particle retention was observed in DEP13 and HVO13 exposed mice on day 28 post-exposure, with less retention for the other fuel types. RME exposure induced limited response whereas the remaining particles induced dose-dependent inflammation and acute phase response on day 1. DEP13 induced acute phase response on day 28 and inflammation on day 90. DNA strand break levels were not increased as compared to vehicle, but were increased in lung and liver compared to blank filter extraction control. Neutrophil influx on day 1 correlated best with estimated deposited surface area, but also with elemental carbon, organic carbon and PAHs. DNA strand break levels in lung on day 28 and in liver on day 90 correlated with acellular particle-induced ROS. CONCLUSIONS: We studied diesel exhaust particles designed to differ in physicochemical properties. Our study highlights specific surface area, elemental carbon content, PAHs and ROS-generating potential as physicochemical predictors of diesel particle toxicity.

Determination of genomic N3-methylthymidine in human cancer cells treated with nitrosamines using capillary electrophoresis with laser-induced fluorescence.

Methylating substances alter DNA by forming N3-methylthymidine (N3mT), a mutagenic base modification. To develop a sensitive analytical method for the detection of N3mT in DNA based on capillary electrophoresis with laser-induced fluorescence detection (CE-LIF), we synthesized the N3mT-3'-phosphate as a chemical standard. The limit of detection was 1.9 amol of N3mT, which corresponds to one molecule of N3mT per 1000 normal nucleotides or 0.1%. With this method, we demonstrated that the carcinogenic nitrosamine N'-nitrosonornicotine (NNN) induced N3mT in the human lung cancer cell line A549. Treatment with NNN also caused an elevated degree of 5-hydroxymethylcytidine (5hmdC) in DNA, while the methylation degree (i.e. 5-methylcytidine; 5mdC) stayed constant. According to our data, NNN could, via yet unknown mechanisms, play a role in the formation of N3mT as well as 5hmdC. In this study we have developed a new sensitive analytical method using CE-LIF for the simultaneous detection of the three DNA modifications, 5mdC, 5hmdC and N3mT.

Sensitive detection of hydroxymethylcytosine levels in normal and neoplastic cells and tissues.

A new sensitive analytical method using capillary electrophoresis with laser induced fluorescence (CE-LIF) was applied for the simultaneous detection of DNA methylation and hydroxymethylation levels in human cancers of different origin. DNA hydroxymethylation, measured as 5-hydroxymethylcytosine (5hmC) levels, was decreased in gliomas with mutation in the isocitrate dehydrogenase 1 (IDH1) gene when compared to IDH1-wildtype gliomas. Independent from IDH1 mutation, 5hmC levels were decreased in lung carcinomas when compared to normal lung tissue. Reduced DNA hydroxymethylation was also observed upon dedifferentiation in cultured murine embryonic fibroblasts. Our data show that reduced DNA hydroxymethylation is related to cellular dedifferentiation and can be detected in various types of cancers, independent from the IDH1 mutation status. Quantitative determination of altered 5hmC levels may therefore have potential as a biomarker linked to cellular differentiation and tumorigenesis.

DNA methylation of the cancer-related genes F2RL3 and AHRR is associated with occupational exposure to polycyclic aromatic hydrocarbons.

Some polycyclic aromatic hydrocarbons (PAH) are known carcinogens and workplace PAH exposure may increase the risk of cancer. Monitoring early cancer-related changes can indicate whether the exposure is carcinogenic. Here, we enrolled 151 chimney sweeps, 152 controls and 19 creosote-exposed male workers from Sweden. We measured urinary PAH metabolites using LC/MS/MS, the cancer-related markers telomere length (TL) and mitochondrial DNA copy number (mtDNAcn) using qPCR, and DNA methylation of lung cancer-related genes F2RL3 and AHRR using pyrosequencing. The median 1-hydroxypyrene (PAH metabolite) concentrations were highest in creosote-exposed workers (8.0 µg/g creatinine) followed by chimney sweeps (0.34 µg/g creatinine) and controls (0.05 µg/g creatinine). TL and mtDNAcn did not differ between study groups. Chimney sweeps and creosote-exposed workers had significantly lower methylation of AHRR CpG site cg05575921 (88.1 and 84.9%, respectively) than controls (90%). Creosote-exposed workers (73.3%), but not chimney sweeps (76.6%) had lower methylation of F2RL3 cg03636183 than controls (76.7%). Linear regression analyses showed that chimney sweeps had lower AHRR cg05575921 methylation (B = -2.04; P < 0.057, adjusted for smoking and age) and lower average AHRR methylation (B = -2.05; P < 0.035), and non-smoking chimney sweeps had lower average F2RL3 methylation (B = -0.81; P < 0.042, adjusted for age) compared with controls. These cancer-related markers were not associated with urinary concentrations of PAH metabolites. In conclusion, although we found no associations with PAH metabolites in urine (short-term exposure), our results suggest dose-response relationship between PAH exposure and DNA hypomethylation of lung cancer-related loci. These findings indicate that further protective measures should be taken to reduce PAH exposure.

The impact of p53 function on the metabolic activation of the carcinogenic air pollutant 3-nitrobenzanthrone and its metabolites 3-aminobenzanthrone and N-hydroxy-3-aminobenzanthrone in human cells.

The tumour suppressor p53, encoded by TP53, is a key player in a wide network of signalling pathways. We investigated its role in the bioactivation of the environmental carcinogen 3-nitrobenzanthrone (3-NBA)found in diesel exhaust and its metabolites 3-aminobenzanthrone (3-ABA) and N-hydroxy-3-aminobenzanthrone (N-OH-3-ABA) in a panel of isogenic human colorectal HCT116 cells differing only with respect to their TP53 status [i.e. TP53(+/+), TP53(+/-), TP53(-/-), TP53(R248W/+) or TP53(R248W/-)]. As a measure of metabolic competence, DNA adduct formation was determined using 32P-postlabelling. Wild-type (WT) p53 did not affect the bioactivation of 3-NBA; no difference in DNA adduct formation was observed in TP53(+/+), TP53(+/-) and TP53(-/-) cells. Bioactivation of both metabolites 3-ABA and N-OH-3-ABA on the other hand was WT-TP53 dependent. Lower 3-ABA- and N-OH-3-ABA-DNA adduct levels were found in TP53(+/-) and TP53(-/-) cells compared to TP53(+/+) cells, and p53's impact was attributed to differences in cytochrome P450 (CYP) 1A1 expression for 3-ABA whereas for N-OH-3-ABA, an impact of this tumour suppressor on sulphotransferase (SULT) 1A1/3 expression was detected. Mutant R248W-p53 protein function was similar to or exceeded the ability of WT-p53 in activating 3-NBA and its metabolites, measured as DNA adducts. However, identification of the xenobiotic-metabolising enzyme(s) (XMEs), through which mutant-p53 regulates these responses, proved difficult to decipher. For example, although both mutant cell lines exhibited higher CYP1A1 induction after 3-NBA treatment compared to TP53(+/+) cells, 3-NBA-derived DNA adduct levels were only higher in TP53(R248W/-) cells but not in TP53(R248W/+) cells. Our results show that p53's influence on carcinogen activation depends on the agent studied and thereby on the XMEs that mediate the bioactivation of that particular compound. The phenomenon of p53 regulating CYP1A1 expression in human cells is consistent with other recent findings; however, this is the first study highlighting the impact of p53 on sulphotransferase-mediated (i.e. SULT1A1) carcinogen metabolism in human cells.

Inhalation and Dermal Uptake of Particle and Gas-Phase Phthalates-A Human Exposure Study.

Phthalates are ubiquitous in indoor environments, which raises concern about their endocrine-disrupting properties. However, studies of human uptake from airborne exposure are limited. We studied the inhalation uptake and dermal uptake by air-to-skin transfer with clean clothing as a barrier of two deuterium-labeled airborne phthalates: particle-phase D4-DEHP (di(2-ethylhexyl)phthalate) and gas-phase D4-DEP (diethyl phthalate). Sixteen participants, wearing trousers and long-sleeved shirts, were under controlled conditions exposed to airborne phthalates in four exposure scenarios: dermal uptake alone and combined inhalation + dermal uptake of both phthalates. The results showed an average uptake of D4-DEHP by inhalation of 0.0014 ± 0.00088 (µg kg-1 bw)/(µg m-3)/h. No dermal uptake of D4-DEHP was observed during the 3 h exposure with clean clothing. The deposited dose of D4-DEHP accounted for 26% of the total inhaled D4-DEHP mass. For D4-DEP, the average uptake by inhalation + dermal was 0.0067 ± 0.0045 and 0.00073 ± 0.00051 (µg kg-1 bw)/(µg m-3)/h for dermal uptake. Urinary excretion factors of metabolites after inhalation were estimated to 0.69 for D4-DEHP and 0.50 for D4-DEP. Under the described settings, the main uptake of both phthalates was through inhalation. The results demonstrate the differences in uptake of gas and particles and highlight the importance of considering the deposited dose in particle uptake studies.

Isolation and characterization of a respirable particle fraction from residential house-dust.

Indoor air pollution has caused increasing concern in recent years. As we spend most of our lives indoors, it is crucial to understand the health effects caused by indoor air pollution. Household dust serve as good proxy for accessing indoor air pollution, especially smaller dust particles that can pass into the lungs are of interest. In this study we present an efficient method for the isolation of dust particles in the respirable size range. The respirable fraction was recovered from vacuum cleaner bags, separated by stepwise sieving, followed by characterization for size, morphology, surface area, organic content and elemental composition. The respirable fraction was obtained in a yield of 0.6% with a specific surface area of 2.5m2/g and a Mass Median Aerodynamic Diameter of 3.73 ± 0.15µm. Aluminum and zink were the dominating metals measured in the dust, whereas the major mineral components were found to be silicon dioxide and calcium carbonate. The fraction of organic matter in the dust was measured to be 69 ± 1%. The organic matrix contained bacterial and fungi and a presence of skin fragments. We present here an efficient and fast method for the isolation of dust particles in the respirable size range. That is of considerable value due to the need for large quantities of respirable particle fractions to conduct toxicological studies and risk assessment work.