BTEX in vitro exposure tool using human lung cells: trips and gains.

Cytotoxicity of benzene, toluene, ethylbenzene and xylenes (BTEX) to human lung cells was explored using three different exposure methods: Method 1 - in normal 96-well plates using DMSO as a carrier vehicle, we exposed (a) human lung carcinoma A549 cells, (b) A549 cells over-expressed with cytochrome P450 2E1 cells, and (c) normal lung fibroblast LL-24 cells to benzene, toluene, ethylbenzene and xylene individually and in a mixture which models car exhaust gases for between 1-88 h. We found that the order of the BTEX potency is benzeneCYP2E1 over-expressed A549 cells. A significant difference was found between inter-assay responses for all 24h exposures (P<0.005) suggesting a poor assay repeatability. No sign of potency increase was found from 6 to 72 h exposures. Method 2 - Using sealed vials to expose A549 cells to benzene, toluene and ethylbenzene, we observed a twenty-fold increase in their cytotoxicity, but also with no time-course effect. Method 3 - Using air exposed hanging-drop cell culture, we were able to see both an increase of demonstration of toxicity and a time-course effect from 1 to 12h exposure. We conclude that exposing cells in sealed and unsealed media using DMSO as a carrier vehicle was not suitable for BTEX exposure studies. Hanging-drop air exposure has more potential. It should be noted that if there are any changes in their exposure matrixes, its exposure mass distribution in cells could differ.

Mixture effects of benzene, toluene, ethylbenzene, and xylenes (BTEX) on lung carcinoma cells via a hanging drop air exposure system.

A recently developed hanging drop air exposure system for toxicity studies of volatile chemicals was applied to evaluate the cell viability of lung carcinoma A549 cells after 1 and 24 h of exposure to benzene, toluene, ethylbenzene, and xylenes (BTEX) as individual compounds and as mixtures of four or six components. The cellular chemical concentrations causing 50% reduction of cell viability (EC50) were calculated using a mass balance model and came to 17, 12, 11, 9, 4, and 4 mmol/kg cell dry weight for benzene, toluene, ethylbenzene, m-xylene, o-xylene, and p-xylene, respectively, after 1 h of exposure. The EC50 decreased by a factor of 4 after 24 h of exposure. All mixture effects were best described by the mixture toxicity model of concentration addition, which is valid for chemicals with the same mode of action. Good agreement with the model predictions was found for benzene, toluene, ethylbenzene, and m-xylene at four different representative fixed concentration ratios after 1 h of exposure, but lower agreement with mixture prediction was obtained after 24 h of exposure. A recreated car exhaust mixture, which involved the contribution of the more toxic p-xylene and o-xylene, yielded an acceptable, but lower quality, prediction as well.

Genotoxicity of hydroquinone in A549 cells.

Hydroquinone (HQ) is found in natural and anthropogenic sources including food, cosmetics, cigarette smoke, and industrial products. In addition to ingestion and dermal absorption, human exposure to HQ may also occur by inhaling cigarette smoke or polluted air. The adverse effects of HQ on respiratory systems have been studied, but genotoxicity HQ on human lung cells is unclear. The aim of this study was to investigate the cytotoxicity and genotoxicity of HQ in human lung alveolar epithelial cells (A549). We found that HQ induced a dose response in cell growth inhibition and DNA damage which was associated with an increase in oxidative stress. Cytotoxicity results demonstrated that HQ was most toxic after 24 h (LC50 = 33 µM) and less toxic after 1 h exposure (LC50 = 59 µM). Genotoxicity of HQ was measured using the Comet assay, H2AX phosphorylation, and chromosome aberration formation. Results from the comet assay revealed that DNA damage was highest during the earlier hours of exposure (1 and 6 h) and thereafter was reduced. A similar pattern was observed for H2AX phosphorylation suggesting that damage DNA may be repaired in later exposure hours. An increase in chromosomal aberration corresponded with maximal DNA damage which further confirmed the genotoxic effects of HQ. To investigate whether oxidative stress was involved in the cytotoxic and genotoxic effects of HQ, cellular glutathione and 8-Oxo-deoguanisone (8-Oxo-dG) formation were measured. A decrease in the reduced glutathione (GSH) and an increase oxidized glutathione (GSSG) was observed during the early hours of exposure which corresponded with elevated 8-Oxo-dG adducts. Together these results demonstrate that HQ exerts its cytotoxic and genotoxic effects in A549 lung cells, probably through DNA damage via oxidative stress.

Biomarkers for the evaluation of population health status 16 years after the intervention of arsenic-contaminated groundwater in Xinjiang, China.

The arsenicosis endemic area in the region of Kuitun and Chepaizi, Dzungaria district, Xinjiang, People Republic of China was the first identified arsenic endemic area in China where arsenic concentration of up to 850 µg/L in the groundwater was reported. An intervention was put in place in 1985 by government to provide an alternative water source at a centralized community level. Sixteen years on since the intervention, we evaluated the health status of 178 villagers from endemic and 179 villagers from control sites. Biomarkers in their urine, included arsenic, porphyrins and malondialdehyde (MDA) were measured and the prevalence of skin lesions was also assessed. The average urinary arsenic (117 ± 8.3 µg/g of creatinine) from the endemic-villages was significantly higher (p<0.001) than that of the controls (73.6 ± 3.2 µg/g of creatinine) while no significant difference was found in urinary porphyrins and malondialdehyde concentrations in the overall studies subjects from these two areas. However when the urinary arsenic was higher than 150 µg/g of creatinine, MDA and porphyrins were higher in the endemic-villagers compared to the controls. Fifty-one out of 178 people from the arsenic endemic area showed skin lesions related to arsenicosis but these were absent among villagers from the control site. Of particular concern, skin lesions related to arsenicosis were observed in 4 out of 9 subjects 16 years of age or younger who were from different villages and born after the completion of water intervention. Although sporadic exposure and/or voluntary drinking contaminated water were thought to be a contributor of arsenicosis after the water intervention, the contribution from other dietary arsenic intakes remain unclear.

Hanging drop: an in vitro air toxic exposure model using human lung cells in 2D and 3D structures.

Using benzene as a candidate air toxicant and A549 cells as an in vitro cell model, we have developed and validated a hanging drop (HD) air exposure system that mimics an air liquid interface exposure to the lung for periods of 1h to over 20 days. Dose response curves were highly reproducible for 2D cultures but more variable for 3D cultures. By comparing the HD exposure method with other classically used air exposure systems, we found that the HD exposure method is more sensitive, more reliable and cheaper to run than medium diffusion methods and the CULTEX(®) system. The concentration causing 50% of reduction of cell viability (EC50) for benzene, toluene, p-xylene, m-xylene and o-xylene to A549 cells for 1h exposure in the HD system were similar to previous in vitro static air exposure. Not only cell viability could be assessed but also sub lethal biological endpoints such as DNA damage and interleukin expressions. An advantage of the HD exposure system is that bioavailability and cell concentrations can be derived from published physicochemical properties using a four compartment mass balance model. The modelled cellular effect concentrations EC50cell for 1h exposure were very similar for benzene, toluene and three xylenes and ranged from 5 to 15 mmol/kgdry weight, which corresponds to the intracellular concentration of narcotic chemicals in many aquatic species, confirming the high sensitivity of this exposure method.

Race, ethnicity, and NIH research awards.

We investigated the association between a U.S. National Institutes of Health (NIH) R01 applicant's self-identified race or ethnicity and the probability of receiving an award by using data from the NIH IMPAC II grant database, the Thomson Reuters Web of Science, and other sources. Although proposals with strong priority scores were equally likely to be funded regardless of race, we find that Asians are 4 percentage points and black or African-American applicants are 13 percentage points less likely to receive NIH investigator-initiated research funding compared with whites. After controlling for the applicant's educational background, country of origin, training, previous research awards, publication record, and employer characteristics, we find that black applicants remain 10 percentage points less likely than whites to be awarded NIH research funding. Our results suggest some leverage points for policy intervention.

Antimony speciation and contamination of waters in the Xikuangshan antimony mining and smelting area, China.

Water samples from Xikuangshan (China), the world largest antimony (Sb) mine with a Sb mining and smelting history of more than 200 years, were analyzed. These water samples ranged from stream water in the vicinity of the mining and smelting area that received seepage from ore residues to the underground mine-pit drainage. The concentrations of total Sb, Sb (III) and Sb (V) of the samples were determined by HPLC-ICP-MS. In addition, water pH and concentrations of major cations and anions were analyzed. All 18 samples demonstrated total Sb concentrations with ppm levels from 0.33 ppm to 11.4 ppm, which is two to three orders of magnitude higher compared to the typical concentration of dissolved Sb in unpolluted rivers (less than 1 ppb). This is probably the first time that such high Sb contents have been documented with complete environmental information. Distribution of total Sb and Sb species was investigated, taking into account the respective local environment (in the mining area or close to the smelter, etc.). Sb (V) was the predominant valence in all 18 samples. Only trace levels of Sb (III) were detected in 4 of the 18 samples. Geochemical speciation modeling showed the dominant species was Sb(OH)(6)(-). It is also probably the first time that such high Sb contents have been documented in the natural environment with Sb speciation distribution information. Several potential oxidation pathways are also discussed that might have facilitated the oxidation of Sb (III) in the natural environment. Signs of intoxication were observed among local mine workers with extensive exposure to different forms of Sb for a long period of time.

Arsenicosis status and urinary malondialdehyde (MDA) in people exposed to arsenic contaminated-coal in China.

The current arsenic exposure condition, arsenicosis prevalence, urinary arsenic and MDA (malondialdehyde) concentrations in people were studied. The study area, a village in Xing Ren County in Guizhou Province, PR China, is a coal-borne arsenicosis endemic area that was identified several decades ago. The residents in Xing Ren have been using coal containing high arsenic levels all their life. Urinary arsenic levels of villagers were 192.2+/-22 microg/g creatinine (n=113) in the coal-borne endemic area (Xing Ren county) and were significantly higher than 63.6+/-5.9 microg/g creatinine (n=30) in a neighbouring control site (a village in Xing Yi county). The urinary MDA concentrations of villagers from the endemic area were also significantly higher compared to those of the control area. There was a strong correlation between age and urinary arsenic and MDA concentrations in the endemic area of Xing Ren; urinary arsenic and MDA levels decreased with age. Fifty out of 113 (44.3%) villagers in the endemic area had arsenicosis symptoms and the prevalence in villagers older than 40 y was 100% in male (92.2% overall). Urinary MDA concentration was significantly higher in people with arsenicosis symptoms in the endemic areas. Oxidative stress (urinary MDA concentration) was strongly related to arsenic exposure but not to the age and smoking habit. Higher urinary arsenic and MDA levels in younger villagers from the endemic area suggest that they are having a higher exposure to coal-borne emitted arsenic because they spend more time indoor. There is an urgent need to develop proper intervention methods in the Guizhou endemic areas in order to reduce the risk to the local communities who are still using arsenic contaminated-coal.

Urinary porphyrins as biomarkers for arsenic exposure among susceptible populations in Guizhou province, China.

Coal is widely used in PR China. Unfortunately, coal from some areas in Guizhou Province contains elevated levels of arsenic. This has caused arsenicosis in individuals who use arsenic-contaminated coal for the purposes of heating, cooking and drying of food in poorly ventilated dwellings. The population at risk has been estimated to be approximately 200,000 people. Clinical symptoms of arsenicosis may include changes of skin pigmentation, hyperkeratosis of hand and feet, skin cancers, liver damage, persistent cough and chronic bronchitis. We analyzed the porphyrin excretion profile using a HPLC method in urine samples collected from 113 villagers who lived in Xing Ren district, a coal-borne arsenicosis endemic area and from 30 villagers from Xing Yi where arsenicosis is not prevalent. Urinary porphyrins were higher in the arsenic exposed group than those in the control group. The correlation between urinary arsenic and porphyrin concentrations demonstrated the effect of arsenic on heme biosynthesis resulting in increased porphyrin excretion. Both uroporphyrin and coproporphyrin III showed significant increases in the excretion profile of the younger age (<20 years) arsenic-exposed group, suggesting that porphyrins could be used as early warning biomarkers of chronic arsenic exposure in humans. Greater increases of urinary arsenic and porphyrins in women, children and older age groups who spend much of their time indoors suggest that they might be at a higher risk. Whether elevated porphyrins could predict adverse health effects associated with both cancer and non-cancer end-points in chronically arsenic-exposed populations need further investigation.