Association of UGT1A1 gene variants, expression levels, and enzyme concentrations with 2,3,7,8-TCDD exposure in individuals exposed to Agent Orange/Dioxin.

Among the congener of dioxin, 2,3,7,8-TCDD is the most toxic, having a serious long-term impact on the environment and human health. UDP-glucuronosyltransferase 1A1 (UGT1A1) plays a crucial role in the detoxification and excretion of endogenous and exogenous lipophilic compounds, primarily in the liver and gastrointestinal tract. This study aimed to investigate the association of UGT1A1 gene polymorphisms, expression levels, and enzyme concentration with Agent Orange/Dioxin exposure. The study included 100 individuals exposed to Agent Orange/Dioxin nearby Da Nang and Bien Hoa airports in Vietnam and 100 healthy controls. UGT1A1 SNP rs10929303, rs1042640 and rs8330 were determined by Sanger sequencing, mRNA expression was quantified by RT-qPCR and plasma UGT1A1 concentrations were measured by ELISA. The results showed that UGT1A1 polymorphisms at SNPs rs10929303, rs1042640 and rs8330 were associated with Agent Orange/Dioxin exposure (OR = 0.55, P = 0.018; OR = 0.55, P = 0.018 and OR = 0.57, P = 0.026, respectively). UGT1A1 mRNA expression levels and enzyme concentration were significantly elevated in individuals exposed to Agent Orange/Dioxin compared to controls (P < 0.0001). Benchmark dose (BMD) analyses showed that chronic exposure to 2,3,7,8-TCDD contamination affects the UGT1A1 mRNA and protein levels. Furthermore, UGT1A1 polymorphisms affected gene expression and enzyme concentrations in individuals exposed to Agent Orange/Dioxin. In conclusion, UGT1A1 gene polymorphisms, UGT1A gene expression levels and UGT1A1 enzyme concentrations were associated with Agent Orange/Dioxin exposure. The metabolism of 2,3,7,8-TCDD may influence UGT1A gene expression and enzyme concentrations.

Exposure to Agent Orange is associated with increased recurrence after surgical treatment of stage I non-small cell lung cancer.

OBJECTIVE: Approximately 3 million Americans served in the armed forces during the Vietnam War. Veterans have a higher incidence rate of lung cancer compared with the general population, which may be related to exposures sustained during service. Agent Orange, one of the tactical herbicides used by the armed forces as a means of destroying crops and clearing vegetation, has been linked to the development of several cancers including non-small cell lung cancer. However, traditional risk models of lung cancer survival and recurrence often do not include such exposures. We aimed to examine the relationship between Agent Orange exposure and overall survival and disease recurrence for surgically treated stage I non-small cell lung cancer. METHODS: We performed a retrospective cohort study using a uniquely compiled dataset of US Veterans with pathologic I non-small cell lung cancer. We included adult patients who served in the Vietnam War and underwent surgical resection between 2010 and 2016. Our 2 comparison groups included those with identified Agent Orange exposure and those who were unexposed. We used multivariable Cox proportional hazards and Fine and Gray competing risk analyses to examine overall survival and disease recurrence for patients with pathologic stage I disease, respectively. RESULTS: A total of 3958 Vietnam Veterans with pathologic stage I disease were identified (994 who had Agent Orange exposure and 2964 who were unexposed). Those who had Agent Orange exposure were more likely to be male, to be White, and to live a further distance from their treatment facility (P < .05). Tumor size distribution, grade, and histology were similar between cohorts. Multivariable Cox proportional hazards modeling identified similar overall survival between cohorts (Agent Orange exposure hazard ratio, 0.97; 95% CI, 0.86-1.09). Patients who had Agent Orange exposure had a 19% increased risk of disease recurrence (hazard ratio, 1.19; 95% CI, 1.02-1.40). CONCLUSIONS: Veterans with known Agent Orange exposure who undergo surgical treatment for stage I non-small cell lung cancer have an approximately 20% increased risk of disease recurrence compared with their nonexposed counterparts. Agent Orange exposure should be taken into consideration when determining treatment and surveillance regimens for Veteran patients.

Association of PKLR gene copy number, expression levels and enzyme activity with 2,3,7,8-TCDD exposure in individuals exposed to Agent Orange/Dioxin in Vietnam.

2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) is the most toxic congener of dioxin and has serious long-term effects on the environment and human health. Pyruvate Kinase L/R (PKLR) gene expression levels and gene variants are associated with pyruvate kinase enzyme deficiency, which has been identified as the cause of several diseases linked to dioxin exposure. In this study, we estimated PKLR gene copy number and gene expression levels using real-time quantitative PCR (RT-qPCR) assays, genotyped PKLR SNP rs3020781 by Sanger sequencing, and quantified plasma pyruvate kinase enzyme activity in 100 individuals exposed to Agent Orange/Dioxin near Bien Hoa and Da Nang airfields in Vietnam and 100 healthy controls. The means of PKLR copy numbers and PKLR gene expression levels were significantly higher, while pyruvate kinase enzyme activity was significantly decreased in Agent Orange/Dioxin-exposed individuals compared to healthy controls (P < 0.0001). Positive correlations of PKLR gene copy number and gene expression with 2,3,7,8-TCDD concentrations were observed (r = 0.2, P = 0.045 and r = 0.54, P < 0.0001, respectively). In contrast, pyruvate kinase enzyme activity was inversely correlated with 2,3,7,8-TCDD concentrations (r = -0.52, P < 0.0001). PKLR gene copy number and gene expression levels were also inversely correlated with pyruvate kinase enzyme activity. Additionally, PKLR SNP rs3020781 was found to be associated with 2,3,7,8-TCDD concentrations and PKLR gene expression. In conclusion, PKLR copy number, gene expression levels, and pyruvate kinase enzyme activity are associated with 2,3,7,8-TCDD exposure in individuals living in Agent Orange/Dioxin-contaminated areas.

Atmospheric PCDD/F concentration and source apportionment in typical rural, Agent Orange hotspots, and industrial areas in Vietnam.

Vietnam has a double burden of dioxin from both industrial sources and historical sources. To evaluate the concentration of PCDD/Fs in ambient air in different areas of Vietnam and their possible sources, atmospheric samples were collected from three areas namely Son La (rural area) and Da Nang (harbor - Agent Orange hotspot area), and Ho Chi Minh City (metropolitan - industrial city). Vapor and solid phases of PCDD/Fs were collected and analyzed following the TO-9A sampling method. Principal Component Analysis and Positive Matrix Factorization model were applied to characterize the possible source. The average concentrations of PCDD/Fs were found to be 21.3 ± 13 fg I-TEQ/m3 in Son La (n = 32), 65.2 ± 34 fg I-TEQ/m3 in Da Nang (n = 16) and 139 ± 84 fg I-TEQ/m3 in Ho Chi Minh City (n = 8). The findings of this study targeted open burning (42%) and biomass burning (51%) as the major emission sources of PCDD/Fs in ambient air of Son La, Vietnam. Major possible sources of PCDD/Fs in Da Nang could be transportation activities (64%), however, the other factor (36%) was suspected to be contaminated with 2,3,7,8-TeCDD from Agent Orange. Most of PCDD/Fs emitted in Ho Chi Minh City related to industrial activities (93%).

Influence of dioxin exposure upon levels of prostate-specific antigen and steroid hormones in Vietnamese men.

Most studies on the relationship between Agent Orange and prostate cancer have focused on US veterans of the Vietnam War. There have been few studies focusing on the relationship between levels of prostate-specific antigen (PSA) and dioxins or steroid hormones in Vietnamese men. In 2009-2011, we collected blood samples from 97 men who had resided in a "dioxin hotspot" and 85 men from a non-sprayed region in Vietnam. Then levels of PSA, dioxins, and steroid hormones were analyzed. Levels of most dioxins, furans, and non-ortho polychlorinated biphenyls were higher in the hotspot than those in the non-sprayed region. Levels of testosterone, dehydroepiandrosterone, and estradiol differed significantly between the hotspot and the non-sprayed region, but there were no correlations between levels of PSA and steroid hormones and dioxins in either of the two regions. Our findings suggest that PSA levels in Vietnamese men are not associated with levels of dioxin or steroid hormones in these two regions.

Agent Orange Exposure and 2,3,7,8-Tetrachlorodibenzo-p-Dioxin (TCDD) in Human Milk.

Agent Orange was sprayed in parts of southern Vietnam during the U.S.-Vietnam war and was a mixture of two chlorophenoxy herbicides. The mixture was contaminated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD and other dioxins and furans are measurable in the milk of Vietnamese women. We explored whether the TCDD in milk from these women was from Agent Orange and whether lactational exposure can be a mode of transgenerational effects of TCDD from Agent Orange. A review of the world's literature on milk concentrations of polychlorinated compounds showed the presence of TCDD and other dioxins and furans in all countries that have been assessed. The congener profile of these chemicals, that is, the proportion of different congeners in the sample, can be used to assess the source of milk contamination. Measurements in most countries, including contemporary measurements in Vietnam, are consistent with non-Agent Orange exposure sources, including industrial activities and incineration of waste. Models and supporting human data suggest that TCDD from breastfeeding does not persist in a child past adolescence and that the adult body burden of TCDD is independent of whether the individual was breast- or bottle-fed as a child. These findings suggest that exposure to Agent Orange in Vietnam did not result in persistent transgenerational exposure through human milk.

Environmental fate and dietary exposures of humans to TCDD as a result of the spraying of Agent Orange in upland forests of Vietnam.

The fate and transport of 2,3,7,8-tetrachloro-p-dibenzodioxin (TCDD) released into the environment of South Vietnam (SVN) as a consequence of the aerial application of the herbicidal defoliant Agent Orange (AO) were simulated for a generic upland forest scenario and followed over a 50-year period (1965, 1968 and 1970 onwards). Modeled concentrations of TCDD in the environment were then used as inputs to a human exposure model, which focused on long-term exposures via the food chain. Intake rates and body burdens of TCDD were estimated for adult males over the course of the simulation period and compared to available biomonitoring data. One of the most important factors determining the magnitude of the simulated human exposure to TCDD was the fraction of the chemical deposited directly to soil (where it was assumed to have a degradation half-life of 10 or 15years) relative to the fraction assumed to remain on/in the forest canopy following the spray application (where it was assumed to have a degradation half-life of ≤48h). The simulated body burdens under the various scenarios considered were broadly consistent with the biomonitoring data from SVN collected in the mid-1980s to late 1990s. Taken together, the modeling results and empirical data suggest that highly elevated exposures to TCDD (i.e., body burdens in the several 100s of pg/g lipid range and greater) were not common among people inhabiting upland forest locations in SVN sprayed with AO and that peak and average body burdens were broadly similar to those of the general population of the U.S. in the 1970s and early 1980s. The model-based assessment is consistent with the 'hot spot' hypothesis i.e., potential exposures to TCDD linked to activities conducted on or near former bases where AO was stored are greater than potential exposures in areas subjected to aerial spraying.

Transport and bioaccumulation of polychlorinated dibenzo-p-dioxins and dibenzofurans at the Bien Hoa Agent Orange hotspot in Vietnam.

The Bien Hoa airbase (south of Vietnam) is known as one of the Agent Orange hotspots which have been seriously contaminated by Agent Orange/dioxin during the Vietnam War. Hundreds of samples including soil, sediment and fish were collected at the Bien Hoa Agent Orange hotspot for assessment of the environmental contamination caused by dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/Fs). The toxicity equivalency quotient (TEQ) concentration of PCDD/Fs in soil and sediment varied from 7.6 to 962,000 and 17 to 4860 pg/g dry wt, respectively, implying very high contamination of PCDD/Fs in several areas. PCDD/F levels in fish ranged between 1.8 and 288 pg/g TEQ wet wt and was generally higher than advisory guidelines for food consumption. 2,3,7,8-Tetrachlorinated dibenzo-p-dioxins (2,3,7,8-TCDD) contributed 66-99 % of TEQ for most of the samples, suggesting 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) from Agent Orange as the major source of the contamination. The vertical transport of PCDD/Fs was observed in soil column with high TEQ levels above 1000 pg/g dry wt (Vietnamese limit for necessary remediation activities- TCVN 8183:2009 (2009)) even at a depth of 1.8 m. The vertical transport of PCDD/Fs has probably mainly taken place during the "Ranch Hand" defoliant spray activities due to the leaks and spills of phenoxy herbicides and solvents. The congener patterns suggest that transports of PCDD/Fs by weathering processes have led to their redistribution in the low-land areas. Also, an estimate for the total volume of contaminated soil requiring remediation to meet Vietnamese regulatory limits is provided.

Exposure to TCDD from base perimeter application of Agent Orange in Vietnam.

Using recognized methods routinely employed by pesticide regulatory agencies, the exposures of military personnel that were mixer/loader/applicators (M/L/A) of Agent Orange (AO) for perimeter foliage at bases during the Vietnam War were estimated. From the fraction of TCDD in AO, absorbed dosage of the manufacturing contaminant was estimated. Dermal exposure estimated from spray drift to residents of the bases was calculated using internationally recognized software that accounted for proximity, foliar density of application site, droplet size and wind speed among other factors, and produced estimates of deposition. Those that directly handled AO generally had much higher exposures than those further from the areas of use. The differences in exposure potential varied by M/L/A activity, but were typically orders of magnitude greater than bystanders. However, even the most-exposed M/L/A involved in perimeter application had lifetime exposures comparable to persons living in the U.S. at the time, i.e., ~1.3 to 5 pg TCDD/kg bodyweight.

Agent orange herbicides, organophosphate and triazinic pesticides analysis in olive oil and industrial oil mill waste effluents using new organic phase immunosensors.

New immunosensors working in organic solvent mixtures (OPIEs) for the analysis of traces of different pesticides (triazinic, organophosphates and chlorurates) present in hydrophobic matrices such as olive oil were developed and tested. A Clark electrode was used as transducer and peroxidase enzyme as marker. The competitive process took place in a chloroform-hexane 50% (V/V) mixture, while the subsequent enzymatic final measurement was performed in decane and using tert-butylhydroperoxide as substrate of the enzymatic reaction. A linear response of between about 10nM and 5.0µM was usually obtained in the presence of olive oil. Recovery tests were carried out in commercial or artisanal extra virgin olive oil. Traces of pesticides were also checked in the oily matrix, in pomace and mill wastewaters from an industrial oil mill. Immunosensors show good selectivity and satisfactory precision and recovery tests performed in olive oil gave excellent results.

Levels of polychlorinated dibenzo-p-dioxins, dibenzofurans (PCDD/Fs) and dioxin-like PCBs in free range eggs from Vietnam, including potential health risks.

Chicken and duck eggs collected from three different areas in Vietnam were examined for polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs). These regions included a background area, an area sprayed with Agent Orange and the Bien Hoa airbase area where Agent Orange was handled by the US Army. The latter area now is inhabited and people keep their own laying hens. Egg samples were first screened with an in vitro reporter gene bioassay and a selection was analyzed by GC/HRMS. Samples from Bien Hoa airbase showed very high PCDD/F levels, up to 249 pg dioxin-equivalents (TEQ)/g fat, mainly due to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In the sprayed areas, levels (3.2-8.2 pg TEQ g(-1)) were comparable to those observed in background areas (3.2-8.2 pg TEQ g(-1) fat). The estimated average consumption of 22 g d(-1) of the highly contaminated eggs will result in a 2-fold exceedance of the current exposure limits for adults and 5-fold for children, even without considering other contaminated food sources. This indicates a potential health risk from consumption of these highly contaminated eggs, which were not yet considered as a source for exposure to PCDD/Fs of people living in the highly contaminated areas.

Agent Orange footprint still visible in rural areas of central Vietnam.

Levels of polychlorinated dioxins/furans (PCDD/PCDF) in selected environmental samples (soils, sediments, fish, and farm animals) were analyzed from the area of Phong My commune (Thua Thien-Hue province, Vietnam). This area was affected by Agent Orange spraying during the Vietnam war (1968-1971). Whereas PCDD/PCDF content in soil and sediment samples is relatively low and ranges between 0.05 and 5.1 pg WHO-TEQ/g for soils and between 0.7 and 6.4 pg WHO-TEQ/g for sediments, the PCDD/PCDF content in poultry muscle and liver in most cases exceeded the maximum permissible limit of dioxin content per unit fat mass. In some cases of soil and sediments samples, 2,3,7,8-TCDD represented more than 90% of the total PCDD/PCDF, which indicates Agent Orange as the main source.

Post-Vietnam military herbicide exposures in UC-123 Agent Orange spray aircraft.

BACKGROUND: During the Vietnam War, approximately 20 million gallons of herbicides, including ~10.5 million gallons of dioxin-contaminated Agent Orange, were sprayed by about 34 UC-123 aircraft that were subsequently returned to the United States, without decontamination or testing, to three Air Force reserve units for transport operations (~1971-1982). In 1996, observed dioxin contamination led to withdrawal of these UC-123s from public auction and to their smelting in 2009. Current Air Force and Department of Veterans Affairs policies stipulate that "dried residues" of chemical herbicides and dioxin had not lead to meaningful exposures to flight crew and maintenance personnel, who are thus ineligible for Agent Orange-related benefits or medical examinations and treatment. Sparse monitoring data are available for analysis. METHODS: Three complementary approaches for modeling potential exposures to dioxin in the post-Vietnam war aircraft were employed: (1) using 1994 and 2009 Air Force surface wipe data to model personnel exposures and to estimate dioxin body burden for dermal-oral exposure for dried residues using modified generic US Environmental Protection Agency intake algorithms; (2) comparing 1979 Air Force 2,4- dichlorophenoxyacetic acid and 2,4-5-trichlorophenoxyacetic acid air samples to saturated vapor pressure concentrations to estimate potential dioxin exposure through inhalation, ingestion and skin contact with contaminated air and dust; and (3) applying emission models for semivolatile organic compounds from contaminated surfaces to estimate airborne contamination. RESULTS: Model (1): Body-burden estimates for dermal-oral exposure were 0.92 and 5.4pg/kg body-weight-day for flight crew and maintainers. The surface wipe concentrations were nearly two orders of magnitude greater than the US Army guidance level. Model (2): measured airborne concentrations were at least five times greater than saturated vapor pressure, yielding dioxin estimates that ranged from 13.2-27.0pg/m(3), thus supporting the likelihood of dioxin dust adsorption. Model (3): Theoretical models yielded consistent estimates to Model 2, 11-49pg/m(3), where the range reflects differences in experimental value of dioxin vapor pressure and surface area used. Model (3) results also support airborne contamination and dioxin dust adsorption. CONCLUSIONS: Inhalation, ingestion and skin absorption in aircrew and maintainers were likely to have occurred during post-Vietnam use of the aircraft based on the use of three complementary models. Measured and modeled values for dioxin exceeded several available guidelines. Deposition-aerosolization-redeposition homeostasis of semivolatile organic compound contaminants, particularly dioxin, is likely to have continually existed within the aircraft. Current Air Force and Department of Veterans Affairs policies are not consistent with the available industrial hygiene measurements or with the widely accepted models for semivolatile organic compounds.

Dioxin/POPs legacy of pesticide production in Hamburg: part 1--securing of the production area.

α-Hexachlorocyclohexane (HCH), ß-HCH, and γ-HCH (lindane) were recently included as new persistent organic pollutants (POPs) in the Stockholm Convention. Therefore, the chemicals need to be globally addressed, including the disposal of historic wastes. At most sites, the approximately 85% of HCH waste isomers were dumped. At a former lindane factory in Hamburg and some other factories the HCH, waste was recycled producing residues with high polychlorinated dibenzo-p-dioxin and dibenzofuran (PCDD/PCDF) levels. The soil and ground water under the former pesticide factory was/is highly contaminated with HCH (260 tons), chlorobenzenes (550 tons), and PCDD/PCDF (6 kg toxic equivalents (TEQ)). This contamination did not result from disposal operations but from spillages and leakages during the 30 years of the factory's production history. A containment wall has been constructed around the production area to prevent the dispersal of the pollutants. The ground water is managed by a pump and treat system. Over the last 15 years, approximately 10-30 tons of this pollution reservoir has been pumped and incinerated. For the contaminated production buildings, specific assessment and demolition technologies have been applied. In addition to their HCH waste isomer deposition, former lindane/HCH productions need to be assessed for possible recycling practice of HCH and related PCDD/PCDF contamination of the production area and buildings. Since such recycling activities have taken place at several factories in different countries, the experience of assessment and management of the described production area and contaminated buildings could be valuable. Such assessment could be addressed within the frame of the Stockholm Convention.

The dioxin/POPs legacy of pesticide production in Hamburg: part 2--waste deposits and remediation of Georgswerder landfill.

α-HCH, ß-HCH, and γ-HCH (lindane) were listed as persistent organic pollutants in the Stockholm Convention. Therefore, they need to be globally addressed including the wastes remaining from historic use and production. While at most lindane production sites the unintentionally produced 85% HCH waste isomers have been deposited, at a former pesticide factory in Hamburg-Moorfleet HCH waste isomers have been recycled from 1953 to 1984 by thermal decomposition to chlorobenzenes and resulted in high polychlorinated dibenzo-p-dioxin/polychlorinated dibenzofuran (PCDD/PCDF)-contaminated residues. The management of the PCDD/PCDF-contaminated waste from the former pesticide factory in Hamburg has been assessed and quantified. Based on past accredited PCDD/PCDF measurements, the registered 3,700 tonnes of disposed thermal HCH decomposition residue contained 333 to 854 kg of PCDD/PCDF toxicity equivalent (I-TEQ) in 53-102 tonnes total sum of PCDD/PCDF. The wastes have been deposited together with other wastes in landfills in Hamburg and other parts of Germany. For the Georgswerder landfill (Hamburg), where approximately 50% of the PCDD/PCDF is disposed, current and previous situation and remediation activities are described. While PCDD/PCDF leaching from the landfill is controlled and incinerated, more water soluble organochlorines (vinyl chloride, cis-1,2-dichlorethene, chlorobenzenes) and benzene remain as a challenge for groundwater management. A comprehensive aftercare program has been established and will need to be operated by future generations including renewal of containment systems. Former lindane/HCH productions need-in addition to HCH deposits-to be assessed for possible recycling practice of HCH and related PCDD/PCDF-containing deposits. This could systematically be addressed within the Stockholm Convention implementation.

Use of a bisphenol-A imprinted polymer as a selective sorbent for the determination of phenols and phenoxyacids in honey by liquid chromatography with diode array and tandem mass spectrometric detection.

An extraction-preconcentration procedure based on the use of a molecularly imprinted polymer (MIP) as selective sorbent has been developed for the determination of several phenolic compounds (bisphenol-A, bisphenol-F and 4-nitrophenol) and phenoxyacid herbicides (2,4-D, 2,4,5-T and 2,4,5-TP) in honey samples. Liquid chromatography with diode array detection (LC-DAD) and electrospray ionisation-ion trap mass spectrometry (LC-IT-MS) were used for the separation, identification and quantification of these analytes. The molecularly imprinted polymer was obtained by precipitation polymerisation with bisphenol-A (BPA) as template and 4-vinylpyridine as the functional monomer. The behaviour of this sorbent was compared with those of other materials frequently used in SPE. The selectivity of the BPA-MIP for the target analytes was tested in samples containing other pesticides in common use. The recoveries achieved for all six compounds were in the 81-96% range. By applying the proposed procedure prior to LC-IT-MS, the limits of detection achieved in commercial honey samples were in the 0.1-3.8 ng g(-1) range, with relative standard deviations of 12-24%.