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.

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.

TCDD exposure estimation for workers at a New Zealand 2,4,5-T manufacturing facility based on serum sampling data.

Employment in the manufacture of the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) is associated with potential exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and elevated serum lipid TCDD concentrations can be measured in workers for decades after terminated occupational exposure. As part of an epidemiological study of 1599 workers employed at a facility in New Plymouth, New Zealand that manufactured 2,4,5-T, serum TCDD concentrations measured in blood samples from 346 workers were used with work history records and a simple pharmacokinetic model in a linear regression to estimate dose rates associated with specific job exposure groups at the facility. The model was used to estimate serum TCDD concentration profiles over time for each individual in the full study group and accounted for 30% of the observed variance in TCDD concentrations in the serum donor subgroup. The model underestimated measured concentrations substantially for eleven individuals in the study group; examination of questionnaire data revealed a variety of activities apart from routine employment at the facility that may have contributed to the measured serum TCDD concentrations. Estimated serum TCDD concentrations were below 300 p.p.t. for all individuals in the cohort over the entire study time period, lower than estimates for other 2,4,5-T worker populations. This finding is consistent with occupational medicine records, which indicated that no cases of chloracne were ever diagnosed among workers employed on the site. The modeled exposures will be used in an evaluation of mortality patterns of workers at this facility.

Analysis of phenoxyacetic acid herbicides as biomarkers in human urine using liquid chromatography/triple quadrupole mass spectrometry.

Phenoxyacetic acids are widely used herbicides. The toxicity of phenoxyacetic acids is debated, but high-level exposure has been shown to be hepatotoxic as well as nephrotoxic in animal studies. An inter-species difference in toxic effects has been found, with dogs particularly susceptible. In this study a method using liquid chromatography/triple quadrupole mass spectrometry (LC/MS/MS) is described for the analysis of 4-chloro-2-methylphenoxyacetic acid (MCPA), and its metabolite 4-chloro-2-hydroxymethylphenoxyacetic acid (HMCPA), 2,4-dichlorophenoxyacetic acid (2,4-D), and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) in human urine. The urine samples were treated by acid hydrolysis to degrade possible conjugations. The sample preparation was performed using solid-phase extraction. Analysis was carried out using selected reaction monitoring (SRM) in the negative ion mode. Quantification of the phenoxyacetic acids was performed using [(2)H(3)]-labeled MCPA and 2,4-D as internal standards. The method was linear in the range 0.05-310 ng/mL urine and has a within-run precision of 2-5%. The between-run precision in lower concentration ranges was between 6-15% and between 2-8% in higher concentration ranges. The limit of detection was determined to 0.05 ng/mL. The metabolites in urine were found to be stable during storage at -20 degrees C. To validate the phenoxyacetic acids as biomarkers of exposure, the method was applied in a human experimental oral exposure to MCPA, 2,4-D and 2,4,5-T. Two healthy volunteers received 200 microg of each phenoxyacetic acid in a single oral dose followed by urine sampling for 72 h post-exposure. After exposure, between 90 and 101% of the dose was recovered in the urine. In the female subject, 23%, and in the male subject 17%, of MCPA was excreted as HMCPA.

Environmental fate and bioavailability of Agent Orange and its associated dioxin during the Vietnam War.

BACKGROUND: In 1996, the Committee on the Assessment of Wartime Exposure to Herbicides in Vietnam of the National Academy of Sciences' Institute of Medicine (IOM) issued a report on an exposure model for use in epidemiological studies of Vietnam veterans. This exposure model would consider troop locations based on military records; aerial spray mission data; estimated ground spraying activity; estimated exposure opportunity factors; military indications for herbicide use; and considerations of the composition and environmental fate of herbicides, including changes in the TCDD content of the herbicides over time, the persistence of TCDD and herbicides in the environment, and the degree of likely penetration of the herbicides into the ground. When the final report of the IOM Committee was released in October 2003, several components of the exposure model envisioned by the Committee were not addressed. These components included the environmental fate of the herbicides, including changes in the TCDD content over time, the persistence of TCDD and herbicides in the environment, and the degree of likely penetration of herbicides into the ground. This paper is intended to help investigators understand better the fate and transport of herbicides and TCDD from spray missions, particularly in performing epidemiological studies. METHODS: This paper reviews the published scientific literature related to the environmental fate of Agent Orange and the contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and discusses how this affected the potential exposure to TCDD of ground troops in Vietnam. Specifically, the mechanisms of dissipation and degradation as they relate to environmental distribution and bioavailability are addressed. RESULTS: The evaluation of the spray systems used to disseminate herbicides in Vietnam showed that they were capable of highly precise applications both in terms of concentrations sprayed and area treated. Research on tropical forest canopies with leaf area indices (a measure of foliage density) from 2 to 5 indicated that the amount of herbicide and associated TCDD reaching the forest floor would have been between 1 and 6% of the total aerial spray. Studies of the properties of plant surface waxes of the cuticle layer suggested that Agent Orange, including the TCDD, would have dried (i.e., be absorbed into the wax layer of the plant cuticle) upon spraying within minutes and could not be physically dislodged. Studies of Agent Orange and the associated TCDD on both leaf and soil surface have demonstrated that photolysis by sunlight would have rapidly decreased the concentration of TCDD, and this process continued in shade. Studies of 'dislodgeable foliar residues' (DFR, the fraction of a substance that is available for cutaneous uptake from the plant leaves) showed that only 8% of the DFR was present 1 hr after application. This dropped to 1% of the total 24 hrs after application. Studies with human volunteers confirmed that after 2 hrs of saturated contact with bare skin, only 0.15-0.46% of 2,4,5-T, one of the phenoxy acetic acid compounds that was an active ingredient of Agent Orange, entered the body and was eliminated in the urine. CONCLUSIONS: The prospect of exposure to TCDD from Agent Orange in ground troops in Vietnam seems unlikely in light of the environmental dissipation of TCDD, little bioavailability, and the properties of the herbicides and circumstances of application that occurred. Photochemical degradation of TCDD and limited bioavailability of any residual TCDD present in soil or on vegetation suggest that dioxin concentrations in ground troops who served in Vietnam would have been small and indistinguishable from background levels even if they had been in recently treated areas. Laboratory and field data reported in the literature provide compelling evidence on the fate and dislodgeability of herbicide and TCDD in the environment. This evidence of the environmental fate and poor bioavailability of TCDD from Agent Orange is consistent with the observation of little or no exposure in the veterans who served in Vietnam. Appreciable accumulation of TCDD in veterans would have required repeated long-term direct skin contact of the type experienced by United States (US) Air Force RANCH HAND and US Army Chemical Corps personnel who handled or otherwise had direct contact with liquid herbicide, not from incidental exposure under field conditions where Agent Orange had been sprayed.

Serum dioxin, chloracne, and acne in veterans of Operation Ranch Hand.

The possible relationship between exposure to Agent Orange and its contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin), during the Vietnam War and chloracne was investigated. The index subjects were veterans of Operation Ranch Hand, the unit responsible for aerial herbicide spraying in Vietnam from 1962 to 1971. Other Air Force veterans who served in Southeast Asia during the same period, but who were not involved with spraying herbicides, served as comparisons. None of the Ranch Hand veterans were diagnosed with chloracne; therefore, we restricted our analyses to acne. We found no meaningful or consistent association between dioxin exposure and prevalence of acne without or with regard to anatomical location. These results suggested that exposure of Ranch Hand veterans to dioxin was insufficient for the production of chloracne or perhaps the exposure may have caused chloracne that resolved and was currently undetectable.

Transport of 2,4,5-trichlorophenoxyacetic acid across the blood-cerebrospinal fluid barrier of the rabbit.

Transport of the anionic herbicide, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) across the blood-cerebrospinal fluid barrier was investigated using the isolated choroid plexus of the adult rabbit in vitro and ventriculocisternal perfusion in vivo. In vitro, 2,4,5-T transport was effective, with tissue concentrations 20 times those in the medium after only 5 min of incubation with 1 microM 2,4,5-T. The tissue to medium ratios reached steady state by 20 min at approximately 45-fold. Uptake was energy dependent and inhibited by ouabain, phloridzin and several organic anions (probenecid, 2,4-dichlorophenoxyacetic acid and octanoate). Neither tyrosine (transported by a separate system) nor the neurotoxin, quinolinic acid, inhibited 2,4,5-T transport. Kinetic analysis yielded an apparent Km of 58 microM and Vmax of 111 nmol g-1 min-1 in the lateral ventricular choroid plexus with similar values (57 microM and 87 nmol g-1 min-1) in the fourth ventricular plexus. In vivo, the steady-state clearance of 2,4,5-T from the cerebrospinal fluid exceeded that of inulin and was reduced in a dose-dependent fashion by 2,4-dichlorophenoxyacetic acid and probenecid. Together, these data indicate that 2,4,5-T is cleared from the brain and cerebrospinal fluid by the organic anion transport system and that alterations in such transport may have a significant impact on the toxicity of this agent in the central nervous system.

Role of electron-donating cosubstrates in the anaerobic biotransformation of chlorophenoxyacetates to chlorophenols by a bacterial consortium enriched on phenoxyacetate.

A bacterial consortium that anaerobically mineralized phenoxyacetate, with transient production of phenol as an intermediate, was obtained from a methanogenic aquifer site near the Norman, OK municipal landfill. This consortium was able to convert the eight halogenated chlorophenoxyacetates tested to the corresponding chlorophenols. The chlorophenols were not subsequently metabolized. The addition of reduced substrates increased the rate of degradation of all chlorophenoxyacetates, with 78% of mono- and di-chlorinated substrates being transformed to chlorophenols in butyrate-amended cultures, compared to less than 37% transformed in unsupplemented cultures. Butyrate increased the transformation of 2,4,5-trichlorophenoxyacetate from 10% to 20%. An experiment evaluating the effects of several compounds on the side-chain cleavage reaction of 3-chlorophenoxyacetate showed that addition of compounds which readily act as hydrogen donors (butyrate, crotonate, ethanol, propionate, and hydrogen) resulted in 2 to 5 times the amount of 3-chlorophenoxyacetate transformed compared to controls with no amendment, formate had a slight stimulatory effect, and acetate and methanol had no effect. Butyrate addition also increased the rate of phenoxyacetate degradation, resulting in transient phenol accumulation not observed in butyrate-unamended controls. These results support the hypothesis that the side-chain cleavage of phenoxyacetate is a reductive process that is stimulated by the oxidation of reduced cosubstrates.

Dermal absorption of the phenoxy herbicides 2,4-D, 2,4-D amine, 2,4-D isooctyl, and 2,4,5-T in rabbits, rats, rhesus monkeys, and humans: a cross-species comparison.

Dermal absorption of the 14C-ring-labeled phenoxy herbicides 2,4-D [(2,4-dichlorophenoxy)acetic acid], 2,4-D amine (2,4-dichlorophenoxyacetic acid dimethylamine), 2,4-D isooctyl (2,4-dichlorophenoxyacetic acid isooctyl ester), and 2,4,5-T amine (2,4,5-trichlorophenoxyacetic acid trimethylamine) was examined following topical applications of the herbicides to the back of rabbits, the back and tail of rats, the forearm and forehead of rhesus monkeys, and the forehead of human volunteers. The effect of three pesticide vehicles (water, acetone, and Esteron LV96) was also investigated. The total percent dermal absorption was calculated from the mean percent urinary recoveries from the animal tests and corrected for nonurinary excretion of the radiolabel using data from intramuscular (im) injections. The human data are reported without im correction. The reliability of animal data for modelling human dermal absorption of pesticides is highlighted.

Distribution of three common chlorophenoxyacetic acid herbicides into the rat brain.

The distribution of three common 14C-labelled chlorophenoxyacetic acid herbicides (2,4-dichlorophenoxyacetic acid or 2,4-D, 2-methyl-4-chlorophenoxyacetic acid or MCPA, 2,4,5-trichlorophenoxyacetic acid or 2,4,5-T) into the different brain areas was studied in rats pretreated with toxic doses of the herbicides (238-475 mg/kg). Also, their binding to proteins in rat plasma was determined in vitro by increasing the concentrations of chlorophenoxyacetic acids in the incubate from 0 to 1 mg/ml. Both 2,4-D and MCPA pretreatments increased brain concentrations of 14C-labelled herbicides more markedly than 2,4,5-T pretreatments did. No essential differences were found in the distribution between the different brain areas. Protein-unbound fractions of 2,4-D and MCPA in the plasma were clearly higher than those of 2,4,5-T but the highest herbicide concentration increased the protein-unbound fraction of 2,4,5-T more (7-13-fold) than of 2,4-D and MCPA (5-fold). The results suggest that the greater increase in the penetration into the brain of 2,4-D and MCPA than of 2,4,5-T during their intoxication is due to some factors other than the changes in their binding to plasma proteins and mere enhanced diffusion through the blood-brain barrier.

Pharmacokinetics of 2,4,5-T in mice as a function of dose and gestational status.

A pharmacokinetic study was conducted in CD-1 mice with the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as a function of dose (15, 30, 60, and 90 mg/kg, iv) and gestational status (nonpregnant, day 6, 10, 13, and 17 of gestation, and postpartum). Analysis for 2,4,5-T and its metabolites was based on an electron-capture gas-liquid chromatographic method. Pharmacokinetic stimulation of the blood, urine, and feces data from each mouse was performed on an analog-digital hybrid computer system based on a two-compartment model with parallel, first-order elimination kinetics. Data analysis demonstrated dose-independent kinetics for most pharmacokinetic parameters but a gestational status-dependence. There was a tendency, as indicated by an increase in the biologic half-life and AUC and decrease in clearance and total percent recovery, for pregnant animals to eliminate 2,4,5-T more slowly as gestation progressed, resulting in potentially increasing fetal exposure during the later stages of pregnancy.

The pharmacokinetics of chlorinated phenoxy acid herbicides: a literature review.

The chlorinated phenoxy acid herbicides (CPAHs) appear to have similar pharmacokinetics. They are rapidly and almost completely absorbed from an oral dose. They distribute to other tissues and are highly protein-bound in the plasma. The CPAHs are rapidly eliminated unchanged in the urine by an active process in the kidneys. Increasing doses apparently influence absorption, metabolism, distribution and elimination of the CPAHs so that biological effects are increased. Combinations of CPAHs are likely to result in additive or potentiated biological effects. Data suggest that CPAH toxicosis may be alleviated by treatment with fluids and bicarbonate to increase urinary pH and volume, thereby increasing excretion.

Cloning, physical mapping and expression of chromosomal genes specifying degradation of the herbicide 2,4,5-T by Pseudomonas cepacia AC1100.

A genomic library of total DNA of Pseudomonas cepacia AC1100 was constructed on a broad-host-range cosmid vector pCP13 in Escherichia coli AC80. A 25-kb segment was isolated from the library which complemented a Tn5-generated, 2,4,5-trichlorophenoxyacetic acid-negative (2,4,5-T-) mutant, P. cepacia PT88. This mutation was partially characterized and appeared to be lacking functional enzyme required for metabolism of an intermediate of the 2,4,5-T pathway, recently identified as 5-chloro-1,2,4-trihydroxybenzene [Chapman et al., Abstr. Soc. Environ. Toxicol. Chem. USA 8 (1987) 127]. A simple colorimetric assay was developed to detect the presence of this active enzyme in intact cells and was used to determine the expression of complementing genes. Subcloning experiments showed that a 4-kb BamHI-PstI fragment and a 290-bp PstI-EcoRI fragment, separated by 1.3-kb, were required for complementation. Both fragments are identified to be chromosomal in origin. Hybridization studies using the subcloned fragments revealed that in addition to a Tn5 insertion, mutant PT88 contained an extensive chromosomal deletion accounting for its 2,4,5-T- phenotype. The cloned fragments did not show homology to plasmid DNAs carrying degradative genes for toluene, naphthalene and 3-chlorobenzoate.