Evaluation of non-invasive biomonitoring of 2,4-Dichlorophenoxyacetic acid (2,4-D) in saliva.

The objective of this study was to evaluate the potential for non-invasive biomonitoring of 2,4-Dichlorophenoxyacetic acid (2,4-D) in saliva. Using an in vitro rat salivary gland epithelial cell (SGEC) system, a collection of experiments investigating chemical protein binding, temporal and directional transport, as well as competitive transport with para-aminohippuric acid (PAH), a substrate for renal organic anion transporters, was conducted to identify cellular transport parameters required to computationally model salivary transport of 2,4-D. Additionally, a physiological protein gradient was implemented to mimic physiologically relevant concentrations of protein in rat plasma and saliva, and under these conditions the transfer of 2,4-D was markedly slower, driven by increased protein binding (i.e. reduced free 2,4-D species available to cross salivary barrier). The rate of transfer was directly proportional to the amount of unbound 2,4-D and demonstrated no indication of active transport. An in vivo assessment of 2,4-D exposure in rats revealed non-linear protein binding in plasma, indicating saturated protein binding and increased levels of unbound 2,4-D species at higher doses. A strong correlation between 2,4-D concentrations in saliva and unbound 2,4-D in plasma was observed (Pearson correlation coefficient = 0.95). Saliva:plasma 2,4-D ratios measured in vivo (0.0079) were consistent within the linear protein binding range and expected 2,4-D levels from occupational exposures but were significantly different than ratios measured in vitro (physiological conditions) (0.034), possibly due to 2,4-D concentrations in saliva not being at equilibrium with 2,4-D concentrations in blood, as well as physiological features absent in in vitro settings (e.g. blood flow). We demonstrated that 2,4-D is consistently transported into saliva using both in vitro and in vivo models, making 2,4-D a potential candidate for human non-invasive salivary biomonitoring. Further work is needed to understand whether current sensor limits of detection are sufficient to measure occupationally relevant exposures.

Preparation, Characterization and Intracellular Imaging of 2,4-Dichlorophenoxyacetic Acid Conjugated Gold Nanorods.

Visualizing the biodistribution of pesticides inside living cells is great importance for enhancing targeting of pesticides. Here we reported for the first time that gold nanorods (Au NRs) with size of 39.4 nm x 11.3 nm could be used as a fluorescent tracer to examine the distribution of a typical herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D), in tobacco bright yellow 2 (BY-2) cells. The nanostructures of hybrid materials were analyzed by using Raman spectra and X-ray photoelectron spectroscopy (XPS), including spectra assignments and electronic property. These data revealed 2,4-D has successfully conjugated MP-Au NRs according to Raman and XPS. The biodistribution of the conjugates inside BY-2 cells was directly examined at 12 and 24 h by the two-photon microscopy. The intensity of two-photon luminescence (TPL) inside cells demonstrated that the conjugates could be localized and excluded by BY-2 cells. Thus, this labeling approach opens up new avenues to the facile and efficient labeling of pesticides.

Estimating absorbed dose of pesticides in a field setting using biomonitoring data and pharmacokinetic models.

Linking biomarker data to pharmacokinetic (PK) models permits comparison of absorbed dose with a toxicological benchmark, which is an important step to understanding the health implications of pesticide exposure. The purpose of this analysis was to evaluate the feasibility of reconstructing the absorbed dose of two pesticides using PK models developed from biomarker data in a study of occupational application of these compounds. Twenty-four-hour urine samples were collected from farmers 24 h before through 96 h after a typical application of chlorpyrifos or 2,4-D. PK models were used to link the amounts found in urine samples to absorbed dose. Modeled total body dose estimates (in micrograms) were compared to measured dose from time 0-96 h. Despite the complexities surrounding the interpretation of biomonitoring data from a field setting, the models developed as part of this analysis accurately estimated the absorbed dose of 2,4-D and chlorpyrifos when collection of urine samples was largely complete. Over half of the farmers were excluded from modeling due to suspected noncompliance with urine collection or confounding exposure events, which highlights the importance of these issues for designing and interpreting biomonitoring data in future studies. Further evaluation of PK models in scenarios using single void samples is warranted for improving field-based dose assessments.

Transdermal absorption of the herbicide 2,4-dichlorophenoxyacetic acid is enhanced by both ethanol consumption and sunscreen application.

Xenobiotics absorption is a health concern and skin is a major exposure site for many of these chemicals. Both alcohol consumption and topical sunscreen application act as transdermal penetration enhancers for model xenobiotics. The effect of combining these two treatments on transdermal absorption of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was therefore examined. Skin from rats ingesting low (1.5 g/kg) medium (4.3 g/kg) or high (6 g/kg) ethanol doses or saline control was treated with a commercially available sunscreen containing titanium dioxide and octyl methoxycinnimate and transdermal absorption of 2,4-D was monitored. Ethanol increased penetration by a factor of 1.9, 2.0 and 2.5 for animals treated with 1.5, 4.3 and 6 g/kg respectively, demonstrating an ethanol-induced dose response. Sunscreen application to skin from ethanol gavaged rats caused 2,4-D absorption above that induced by ethanol alone by an additional factor of 1.3, 2.1 and 2.9 for 1.5, 4.3 and 6 g/kg respectively. Comparing 2,4-D transdermal absorption after exposure to both ethanol and sunscreen with a theoretical value (sum of penetration after ethanol or sunscreen treatment) demonstrates that these two treatments enhance additively at the higher doses tested. Results of this study emphasize the importance of limiting excessive alcohol consumption in individuals with potential herbicide exposure rather than discouraging the use of sunscreens, since the consequences of UV-induced skin cancer are far more series than the risks that would be associated with observed increases in chemical exposure.

The renal-specific transporter mediates facilitative transport of organic anions at the brush border membrane of mouse renal tubules.

The renal secretion of organic anions across the proximal tubules is achieved by a coordination of uptake and efflux transporters. This study reports the expression, localization, and functional properties of mouse renal-specific transporter (RST). Mouse RST mRNA is predominantly expressed in the kidney and localized on the brush border membrane of mouse kidney proximal tubules. Mouse RST-expressing HEK293 cells exhibited saturable uptake of p-aminohippurate (Km approximately 234 microM), which was increased by an increase in K(+) concentration or in the presence of Ba(2+) and ouabain and decreased by diethylpyrocarbonate, a histidine modifier. An increase in K(+) concentration enhanced the uptake of benzylpenicillin, 2,4-dichlorophenoxyacetate, and dehydroepiandrosterone sulfate, suggesting polyspecific substrate specificity of mouse RST. Vectorial transport of 2,4-dichlorophenoxyacetate was observed in the basal-to-apical direction in rat organic anion transporter 3-expressing LLC-PK1 cells (rOat3-LLC); however, coexpression of mouse RST in rOat3-LLC caused a 1.3-fold increase in the basal-to-apical transport. In addition, the basal-to-apical transport of benzylpenicillin and urate was 3- and 2.5-fold greater than that in the opposite direction in the double-transfected cells, respectively, whereas their transepithelial transport in vector- or rOat3-LLC was symmetrical. Furthermore, the basal-to-apical transport of benzylpenicillin was saturable and reduced by increasing extracellular K(+) concentration and ouabain. These results suggest that mouse RST mediates the efflux of organic anions including urate and works as exit for organic anions in the proximal tubules. In addition to the kidney, mouse RST was detected in the brain capillaries and the choroid plexus, and it may also play a role in efflux transport of organic anions across the barriers of the central nervous system.

Active ingredients in sunscreens act as topical penetration enhancers for the herbicide 2,4-dichlorophenoxyacetic acid.

Agricultural workers are encouraged to use sunscreen to decrease the risk of UV-related skin cancer. Our previous studies have shown certain commercial sunscreens to be penetration enhancers. The focus of this project is to determine whether active ingredients in sunscreen formulations (i.e., the UV absorbing components and insect repellants for the sunscreen/bug repellant combinations) also act as dermal penetration enhancers for herbicides in vitro. The total percentages of 2,4-dichlorophenoxyacetic acid (2,4-D) penetrating through hairless mouse skin in 24 h ranged from 54.9 +/- 4.7 for the no sunscreen control to 86.9 +/- 2.5 for padimate-o. Of the active ingredients tested (7.5% octyl methoxycinnamate, 7% octocrylene, 0.6% oxybenzone, 5% homosalate, 5% octyl salicylate, 8% padimate-o, 10% sulisobenzone, and 9.5% and 19% N,N-diethyl-m-toluamide [DEET]), all but octocrylene led to a significant increase in total 2,4-D penetration as compared to the control (P < 0.05), and only octocrylene and oxybenzone did not significantly decrease the corresponding lag time. Octyl salicylate (P < 0.01) and octyl methoxycinnimate (P < 0.05) significantly increased the 3H2O penetration across mouse skin, indicating physical damage to the stratum corneum. Additional studies demonstrated that the penetration enhancement seen across hairless mouse skin also occurred with human skin. Thus, the active ingredients of sunscreen formulations enhance dermal penetration of the moderately lipophilic herbicide 2,4-D.

Comparative metabolism of 2,4-dichlorophenoxyacetic acid (2,4-D) in rat and dog.

1. There is a significant species difference in the toxicity of 2,4-dichlorophenoxyacetic acid (2,4-D). The oral no overall adverse effect level (NOAEL) for chronic toxicity of 2,4-D in rat is 5 mg kg(-1) day(-1) and in dog is 1 mg kg(-1) day(-1). The maximum tolerated dose (MTD) in rat is 150 and 75 kg(-1) day(-1) for male and females, respectively. The MTD in dog is 7.5 mg kg(-1) day(-1) for males and females. 2. In an attempt to explain the increased sensitivity to 2,4-D in dog, male and female rats and dogs were orally dosed with either 5 or 50 mg kg(-1) 14C-2,4-D. The rates and routes of excretion were investigated along with plasma toxicokinetics and biotransformation of the compound. 3. Elimination of the radioactive dose of 2,4-D from rat plasma was significantly faster than in dog. The approximate t(1/2) were 1.3-3.4 h for rat and 99-134 h for dog following a 5 or 50 mg kg(-1) dose, respectively. This led to large differences in the calculated AUC(0-infinity) 21-57 microg eq. h g(-1) for rat and 4889-5298 microg eq. h g(-1) for dog at 5 mg kg(-1), and 122-2358 microg eq. h g(-1) for rat and 34,110-44,296 microg eq. h g(-1) for dog at 50 mg kg(-1)). 4. In rat, the major route of excretion was in the urine. Excretion was essentially complete after 24 h for the low dose and after 48 h for the high dose. For dog, elimination was incomplete over the sampling period with only about 50% of the dose recovered. Urine was the principal route of excretion at the low dose, but about equal amounts were excreted in urine and faeces at the high dose over 120 h. 5. In rat, 2,4-D was unmetabolized and excreted in urine as the parent compound. In dog, the dose was excreted mainly following metabolism. 2,4-D in dog was conjugated forming the taurine, serine, glycine, glutamic acid, cysteine, sulphate and glucuronide conjugates, plus an unidentified metabolite, which were excreted in urine. Plasma, however, only contained unmetabolized 2,4-D. 6. The results show that the body burden of 2,4-D in dog is significantly higher than in rat for an equivalent dose, which is consistent with the increased sensitivity of dog to 2,4-D toxicity.

Effects of active sunscreen ingredient combinations on the topical penetration of the herbicide 2,4-dichlorophenoxyacetic acid.

Sunscreen use can reduce the incidence of certain skin cancers. However, a number of commercially available formulations have been shown to enhance the transdermal penetration of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). Most of the active ingredients used in these compounds can individually act as penetration enhancers. Commercial sunscreens frequently contain multiple active ingredients in order to provide broad sunscreen protection. The purpose of this study was therefore to examine the effect of these active ingredient combinations on the transdermal absorption of 2,4-D in vitro. All six of the combinations tested resulted in increased cumulative penetration (P <0.01) and faster lag times (P <0.05). The 2,4-D cumulative penetration in the presence of the OFF! Deepwoods combination was significantly greater than the absorption with either the individual ingredients or their average (P <0.05). A systematic study designed to isolate the chemicals responsible for this enhancement demonstrated that with UV absorbers DEET synergistically increased the 2,4-D penetration and that DEET's cumulative enhancement properties correlate with its concentration. By contrast, octocrylene significantly slowed the lag time when used in combinations and was the only active ingredient that showed any antagonistic effects on 2,4-D penetration. Because none of the active ingredient combinations were able to inhibit dermal uptake of 2,4-D, it seems that proper selection of inert ingredients may be the most feasible solution for reducing penetration enhancement.

Sunscreens containing physical UV blockers can increase transdermal absorption of pesticides.

People are encouraged to wear sunscreens because of their effectiveness at reducing the risk of skin cancer. The dermal penetration of the herbicide 2,4-D can be enhanced by commercial formulations containing chemical ultraviolet (UV) absorbers, the absorbers themselves and the insect repellent DEET. This work has been extended to determine whether commercially available sunscreens containing the physical UV absorbers titanium dioxide (TiO2) or zinc oxide (ZnO) enhance the transdermal absorption of pesticides. Hairless mouse skin was pretreated with either commercially available sunscreens or the UV absorbers themselves, dissolved in phenyl trimethicone. In vitro permeability studies were performed with the pesticides 2,4-D, paraquat, parathion or malathion. The data demonstrate that pretreatment with five of the nine sunscreens tested increased the transdermal absorption of 2,4-D (P<0.05). Transdermal studies using paraquat, parathion and malathion pretreated with a representative sunscreen all demonstrated significant penetration enhancement when compared to controls (P<0.05). Repeated 2,4-D and sunscreen applications resulted in either no change between pulses or an increase in absorption after the second pulse depending on the washing regimen. Examining penetration of individual UV absorbers formulated in phenyl trimethicone showed that that ZnO can impede 2,4-D penetration and TiO2 had no effect. Combining UV absorbers in the presence of trimethicone resulted in 'sunscreens' that could actually inhibit 2,4-D penetration. Inert ingredients therefore control the increased absorption seen in commercial sunscreen products and this enhancement can be eliminated by substituting phenyl trimethicone as the solvent. Sunscreen use must still be encouraged even with the undesirable side effect of increased penetration through the skin.

Sunscreens can increase dermal penetration of 2,4-dichlorophenoxyacetic acid.

Agricultural workers are encouraged to wear sunscreen to reduce their risk of skin cancer. These workers are also exposed to herbicides during the course of their day. The skin is the major source of chemical exposure in agriculture. The purpose of this work is to determine the effect of sunscreen use on the transdermal absorption of a model herbicide, 2,4-dichlorophenoxyacetic acid. Hairless mouse skin was pretreated with one of nine commercially available sunscreens purchased at a local drug store. The herbicide 2,4-dichlorophenoxyacetic acid was placed on top of the epidermis in an in vitro diffusion chamber for 24 hours. The total penetrating through the skin in 24 hours ranged from 39.1 +/- 1.7% for the no sunscreen control to 81.0 +/- 2.8% for Neutrogena Oil Free Sunscreen. Of the nine sunscreens tested, six led to a significant enhancement of total 2,4-dichlorophenoxyacetic acid penetration as compared to the control (p < 0.01). Careful selection of sunscreen during pesticide application could reduce potential exposure.

The development of a new method to estimate total daily dose of pesticides in professional turf applicators following multiple and varied exposures in occupational settings.

The evaluation of absorbed dose of pesticides in humans requires a knowledge of the kinetics and dynamics of the compound. In some circumstances, data that allow for the estimation of dose may be available from human volunteer studies, although often, it will be based on results from animal studies. If human metabolism data are available, estimates of dose may be more accurate, but it should be recognized that pesticide exposure in an occupational setting may differ from that in a controlled laboratory study. In this study, data from previously published studies are used to evaluate the urinary excretion of 2,4-dichlorophenoxyacetic acid (2,4-D), following single dermal applications to human volunteers. These studies are evaluated with the objective of determining the best method of predicting total absorbed dose following multiple and varied exposures in occupational settings. Further, an alternative to laboratory-controlled human volunteer studies is presented. Data from a third previously published biological monitoring study on six professional pesticide applicators over a 2-week period were used to generate estimates of the urinary excretion of the pesticide 2,4-D that would result from a single dose. The method used to estimate the urinary excretion parameters is a variation of an overlay technique used in pharmacology, and may provide information on the kinetics of other pesticides when it is not possible to conduct human studies. The generated estimates of 24-h urinary excretion of 2,4-D over a 6-day period were remarkably similar to those obtained in controlled studies. Finally, a method was developed to use the generated estimates to determine total absorbed dose of pesticides for an independent group of 95 professional pesticide applicators. This method requires information on the amount of pesticide used for 6 days prior to the collection of two, 24-h urine samples.

2,4-Dichlorophenoxyacetic acid influx is mediated by an active transport system in Chinese hamster ovary cells.

The influx of 2,4-dichlorophenoxyacetic acid (2,4-D) into Chinese hamster ovary (CHO) cells was studied. The cells mainly took up but did not metabolize the undissociated form of the herbicide. The uptake of 2,4-D was carried out against a concentration gradient and was inhibited by sodium azide and dinitrophenol. The results presented here show that the herbicide influx was an active, energy dependent process. (Na+ + K+)ATPase does not seem to be involved because ouabain, an inhibitor of the enzyme, did not affect the 2,4-D uptake.

Canine exposure to herbicide-treated lawns and urinary excretion of 2,4-dichlorophenoxyacetic acid.

A recent study by Hayes et al. (J. Natl. Cancer. Inst., 83: 1226-1231, 1991) found an increased risk of malignant lymphoma associated with exposure to 2,4-dichlorophenoxyacetic acid (2,4-D) in pet dogs. We conducted a study to determine the extent to which dogs absorb and excrete 2,4-D in urine after contact with treated lawns under natural conditions. Among 44 dogs potentially exposed to 2,4-D-treated lawns an average of 10.9 days after application, 2,4-D concentrations greater than or equal to 10.0 micrograms/l were found in 33 dogs (75%) and concentrations of > or = 50 micrograms/l were found in 17 (39%). Among 15 dogs with no known exposure to a 2,4-D-treated lawn in the previous 42 days, 4 (27%) had evidence of 2,4-D in urine, 1 at a concentration of > or = 50 micrograms/l. The odds ratio for the association between exposure to a 2,4-D-treated lawn and the detection of > or = 50 micrograms/l 2,4-D in urine was 8.8 (95% confidence interval, 1.4-56.2). Dogs exposed to lawns treated within 7 days before urine collection were more than 50 times as likely to have 2,4-D at concentrations > or = 50 micrograms/l than dogs with exposure to a lawn treated more than 1 week previously (odds ratio = 56.0; 95% confidence interval, 10.0-312.2). The highest mean concentration of 2,4-D in urine (21.3 mg/l) was found in dogs sampled within 2 days after application of the herbicide.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of the mosquito repellent DEET and long-wave ultraviolet radiation on permeation of the herbicide 2,4-D and the insecticide DDT in natural rubber gloves.

Studies were conducted to determine the effect of a commonly used insect repellent, DEET (N,N-diethyl-m-toluamide), on the permeability of rubber gloves used as chemical protective clothing (CPC) by pesticide applicators. Glove permeation analysis was conducted with an automated in vitro diffusion analysis (AIDA) method employing an in-house, flow-through permeation cell design. Permeation of 14C-ring-labeled 2,4-D (2,4-dichlorophenoxyacetic acid) in natural rubber glove material was 2.4 +/- 1.81% at 48 hr after treatment of the glove with 2,4-D applied with DEET; this was not significantly different (Student's t-test; p less than 0.05) from 3.2 +/- 3.46% permeation of 2,4-D observed without DEET. Similarly, there was no significant difference between the permeation of pp'-DDT (1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane) applied with DEET (11.7 +/- 5.02%) and without DEET (11.4 +/- 4.86%) to natural rubber glove material. Scanning electron microscopy of the natural rubber glove material, however, demonstrated disruption of the surface ultrastructure following a 24-hr treatment with DEET. The AIDA analysis also suggested that exposure of the glove material to long-wave ultraviolet (UVA) radiation enhanced the glove permeability to 2,4-D (6.2 +/- 0.73% [+UVA]; 0.3% +/- 0.14% [-UVA]) but had no effect on the permeation of DDT. Because the CPC of pesticide applicators is commonly exposed to solar UVA, this finding may raise concerns about the efficacy and safety of CPC in general.

Weight of the evidence on the human carcinogenicity of 2,4-D.

The phenoxy herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) is widely used to control the growth of weeds and broadleaf plants. We convened a panel of 13 scientists to weigh the evidence on the human carcinogenicity of 2,4-D. The panel based its findings on a review of the toxicological and epidemiological literature on 2,4-D and related phenoxy herbicides. The toxicological data do not provide a strong basis for predicting that 2,4-D is a human carcinogen. Although a cause-effect relationship is far from being established, the epidemiological evidence for an association between exposure to 2,4-D and non-Hodgkin's lymphoma is suggestive and requires further investigation. There is little evidence of an association between use of 2,4-D and soft-tissue sarcoma or Hodgkin's disease, and no evidence of an association between 2,4-D use and any other form of cancer. Scientists on the panel were asked to categorize 2,4-D as a "known," "probable," "possible," or "unlikely" carcinogen or as a noncarcinogen in humans. The predominant opinion among the panel members was that the weight of the evidence indicates that it is possible that exposure to 2,4-D can cause cancer in humans, although not all of the panelists believed the possibility was equally likely: one thought the possibility was strong, leaning toward probable, and five thought the possibility was remote, leaning toward unlikely. Two panelists believed it unlikely that 2,4-D can cause cancer in humans.

[Adsorption of pesticides on synthetic adsorption polymers]. / Adsorpcja pestycydów na syntetycznych polimerach adsorpcyjnych.

On the basis of adsorption of selected pesticides on synthetic adsorption polymers it has been shown that both the adsorption balances as well as kinetics and dynamics of the adsorption strongly depend on the concentration of hydrogen ions of the liquid phase. The dependence of adsorption isotherms on pH value was demonstrated with sufficient accuracy by resorting to the models having been suggested on the ground of IAS theory and the potential one. The kinetic investigations have revealed that the surface diffusion proceeds extremely rapidly in pH ranges that lodge between sorptive pK value and the isoelectric point of the adsorbent surface. The known solutions of transportation and balance equations, which in this case were transferred on the pH-dependent adsorption processes, were implemented for simulating the breakthrough curves. The investigation, covering the effect of the presence of heavy metals in a solution exerted on the pesticides adsorption properties, has shown that the formation of model-pesticide type connections improves the adsorption properties of pesticides to a lesser or greater extent.

Behavioral changes in rats fed a diet containing 2,4-dichlorophenoxyacetic butyl ester.

Oral administration of 2,4-dichlorophenoxyacetic butyl ester (2,4-Dbe) at a dose of 69 mg/kg/day to nulliparous females had no deleterious effects on either open field (OF) and rotarod performance. By contrast, dams treated with 2,4-Dbe during pregnancy exhibited impairments of OF activity, rotarod performance and improved active avoidance learning (AAL) retention. Administration of 2,4-Dbe to 90-day-old intact male rats depressed spontaneous OF activity, acquisition of conditioned avoidance responses (CARs) and rotarod endurance, but improved AAL performance. Castration itself impaired performance in the rotarod test, and improved AAL, but did not alter OF activity significantly. The effects of castration were reversed by exogenous testosterone. In gonadectomized rats, 2,4-Dbe prevented the reversal of the effect of testosterone on the influence of castration on behavior if given concomitantly with the testosterone. However, when the 2,4-Dbe treatment started seven days after testosterone, the 2,4-Dbe effects on OF, rotarod and AAL behaviors were reinstated. Thus, testosterone appears to be important for causing the toxic effects of 2,4-Dbe in rats.