Does doses and time of 2, 4-D application interfere in the physiology and wheat grains yield components?

The identification of the application stage and correct dose of 2,4-dichlorophenoxyacetic acid (2,4-D) herbicide is important so that wheat is not harmed. In view of this, the objective of this study was to evaluate the effect of 2,4-D doses applied at different development stages of wheat crop. The experiment was conducted in a randomized block design, arranged in a 4 × 5 factorial scheme, with four replications. In factor A, the application stages (before tillering, tillering, first node and booting) were allocated and the doses of 2,4-D (0, 349, 698, 1047 and 1396 g.ha­1) were allocated in factor B. The variables evaluated were phytotoxicity at 7, 14, 21, 28 and 35 days after application of the treatments (DAT), photosynthetic activity, CO2 internal concentration, stomatal conductance, efficient water use and carboxylation efficiency. The number of spikes·m­2, spike length and number of full and sterile grains were determined in the preharvest. Thousand grain mass, grain yield and hectoliter weight were determined after harvest. The results demonstrate that the herbicide caused phytotoxicity to wheat, being greater in increasing doses and mainly before tillering, causing grain sterility and decreased productivity. The other yield components did not present difference when increasing the dose and application in different stages as well as the physiological variables. The increase of the 2,4-D doses applied before tillering and in the booting stage caused linear decrease in wheat grain yield.

Genotoxicity evaluation of 2,4-D, dicamba and glyphosate alone or in combination with cell reporter assays for DNA damage, oxidative stress and unfolded protein response.

The current generation of carcinogenicity tests is often insufficient to predict cancer outcomes from pesticide exposures. In order to facilitate health risk assessment, The International Agency for Research on Cancer identified 10 key characteristics which are commonly exhibited by human carcinogens. The ToxTracker panel of six validated GFP-based mouse embryonic stem reporter cell lines is designed to measure a number of these carcinogenic properties namely DNA damage, oxidative stress and the unfolded protein response. Here we present an evaluation of the carcinogenic potential of the herbicides glyphosate, 2,4-D and dicamba either alone or in combination, using the ToxTracker assay system. The pesticide 2,4-D was found to be a strong inducer of oxidative stress and an unfolded protein response. Dicamba induced a mild oxidative stress response, whilst glyphosate did not elicit a positive outcome in any of the assays. The results from a mixture of the three herbicides was primarily an oxidative stress response, which was most likely due to 2,4-D with dicamba or glyphosate only playing a minor role. These findings provide initial information regarding the risk assessment of carcinogenic effects arising from exposure to a mixture of these herbicides.

Extracts from black carrot tissue culture as potent anticancer agents.

Black carrots contain anthocyanins possessing enhanced physiological activities. Explants of young black carrot shoots were cultured in Murashige and Skoog (MS) medium for callus initiation and were transferred to new MS medium supplemented with four different combinations of 2,4-dichlorophenoxyacetic acid and kinetin. Subsequently, the lyophilized calli and black carrot harvested from fields were subjected to ultrasound extraction with ethanol at a ratio of 1:15 (w:v). Obtained extracts were applied to various human cancer cell lines including MCF-7 SK-BR-3 and MDA-MB-231 (human breast adenocarcinomas), HT-29 (human colon adenocarcinoma), PC-3 (human prostate adenocarcinoma), Neuro 2A (Musmusculus neuroblastoma) cancer cell lines and VERO (African green monkey kidney) normal cell line by MTT assay. The highest cytotoxic activity was achieved against Neuro-2A cell lines exhibiting viability of 38-46% at 6.25 µg/ml concentration for all calli and natural extracts. However, a significantly high IC50 value of 170.13 µg/ml was attained in normal cell line VERO indicating that its natural counterpart is an ideal candidate for treatment of brain cancer without causing negative effects to normal healthy cells.

Transient effects of 2,4-dichlorophenoxyacetic acid (2,4-D) exposure on some metabolic and free radical processes in goldfish white muscle.

This study aims to assess effects of 96 h goldfish exposure to 1, 10 and 100 mg/L of the herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D), on metabolic indices and free radical process markers in white muscle of a commercial fish, the goldfish Carassius auratus L. Most oxidative stress markers and antioxidant enzymes were not affected at 2,4-D fish treatment. 2,4-D fish exposure induced the elevated levels of total (by 46% and 40%) and reduced (by 77% and 73%) glutathione in muscles of goldfish of 10 mg/L 2,4-D and recovery (after 100 mg/L of 2,4-D exposure) groups, respectively. However, in muscles of 100 mg/L 2,4-D exposed goldfish these parameters were depleted (by 47% and 64%). None of investigated parameters of protein and carbohydrate metabolisms changed in white muscles of 2,4-D exposed fish, with exception of lactate dehydrogenase activity, which was slightly (by 11-15%) elevated in muscles of goldfish exposed to 10-100 mg/L of 2,4-D, but also recovered. Thus, the short term exposure of goldfish to the selected concentrations of 2,4-D does not substantially affect their white muscle, suggesting the absence of any effect under the environmentally relevant concentrations.

Assessing eco-toxicological effects of industrial 2,4-D acid iso-octylester herbicide on rat pancreas and liver.

We studied the eco-toxic and carcinogenic effects of a commonly used 2,4-D acid iso-octylester herbicide on rat liver and pancreas. The rats in Group 1 were fed a standard feed and the rats in Group 2 were fed with standard feed to which was added 200 mg/kg/day 2,4-D acid iso-octylester for 16 weeks. Azaserine, 30 mg/kg/body weight, was injected into rats of Groups 3 and 4 to investigate the effects of 2,4-D acid iso-octylester on the development of neoplasms. After feeding the rats with neoplasms in Group 4 with food including 200 mg/kg/day 2,4-D acid iso-octylester for 16 weeks, an autopsy was carried out on all animals. We found that 2,4-D acid iso-octylester caused the formation of atypical cell foci (ACF) in the pancreata and livers of rats. ACF that were formed experimentally by exposure to azaserine had increased diameter, volume and number of atypical cell foci/mm(2) and mm(3) after exposure to 2,4-D acid iso-octylester. Our observations indicated that this herbicide potentially is a cancer initiator.

Allergic reaction induced by dermal and/or respiratory exposure to low-dose phenoxyacetic acid, organophosphorus, and carbamate pesticides.

Several types of pesticides, such as organophosphates, phenoxyacetic acid, and carbamate have a high risk of affecting human health, causing allergic rhinitis and bronchial asthma-like diseases. We used our long-term sensitization method and a local lymph node assay to examine the allergic reactions caused by several types of pesticides. BALB/c mice were topically sensitized (9 times in 3 weeks), then challenged dermally or intratracheally with 2,4-D, BRP, or furathiocarb. One day post-challenge, the mice were processed to obtain biologic materials for use in assays of total IgE levels in serum and bronchoalveolar lavage fluid (BALF); differential cell counts and chemokine levels in BALF; lymphocyte counts and surface antigen expression on B-cells within regional lymph nodes (LNs); and, ex situ cytokine production by cells from these LNs. 2,4-D-induced immune responses characteristic of immediate-type respiratory reactions, as evidenced by increased total IgE levels in both serum and BALF; an influx of eosinophils, neutrophils, and chemokines (MCP-1, eotaxin, and MIP-1beta) in BALF; increased surface antigen expression on B-cells IgE and MHC class II production) in both auricular and the lung-associated LNs; and increased Th2 cytokine production (IL-4, IL-5, IL-10, and IL-13) in both auricular and the lung-associated LN cells. In contrast, BRP and furathiocarb treatment yielded, at most, non-significant increases in all respiratory allergic parameters. BRP and furathiocarb induced marked proliferation of MHC Class II-positive B-cells and Th1 cytokines (IL-2, TNF-alpha, and IFN-gamma) in only auricular LN cells. These results suggest that 2,4-D is a respiratory allergen and BRP and furathiocarb are contact allergens. As our protocol detected classified allergic responses to low-molecular-weight chemicals, it thus may be useful for detecting environmental chemical-related allergy.

Determination of toxicity of subacute treatment of some plant growth regulators on rats.

The effects of some plant growth regulators (PGRs), 2,3,5-triiodobenzoic acid (TIBA), Naphthaleneacetic acid (NAA) and 2,4-Dichlorofenoxyacetic acid (2,4-D), at sublethal concentrations on antioxidant defense system [glutathione peroxidases (GPx), reduced glutathione (GSH), glutathione reductase (GR), glutathione-S-transferase (GST) and catalase (CAT)], immune potential enzymes [adenosine deaminase (ADA) and myeloperoxidase (MPO)], and lipid peroxidation content [Malondialdehyde, (MDA)] were investigated in lung and speen tissues of rats. Sprague-Dawley albino rats were exposed to 0, 50, or 100 ppm (parts per million) TIBA, NAA, or 2,4-D in drinking water ad libitum for 25 days continuously. According to the results, MDA concentration significantly increased in the tissues treated with 100 ppm dosage of NAA or 2,4-D without any change in the tissues of rats treated with both dosage of TIBA. The GSH depletion in the spleen tissue of rats treated with both the dosage of NAA and 2,4-D were found to be significant. Also, GSH level in the spleen was significantly reduced with 100 ppm of 2,4-D and NAA. The activity of antioxidant enzymes were also seriously affected by PGRs; GPx significantly decreased in the lung of rats treated with both dosages of the PGRs, whereas GPx activity in the spleen were significantly increased with 100 ppm dosage of 2,4-D and NAA. On the other hand, CAT activity significantly decreased in the lung of rats treated with both dosages of NAA, 100 ppm of 2,4-D and 50 ppm of TIBA, and also in the spleen treated with 50 ppm NAA and 2,4-D. The ancillary enzyme GR activity significantly decreased in the spleen with both doses of the PGRs, also in the lung treated with both dosages of 2,4-D, 50 ppm of NAA and 100 ppm of TIBA. The drug metabolizing enzyme GST activity significantly reduced in the lung of rats treated with both dosages of the PGRs and also in the spleen treated with 100 ppm dosage of 2,4-D and TIBA and 50 ppm of NAA. Meanwhile, immune potential enzyme MPO activity significantly increased in the spleen of rats treated with both doses of NAA and TIBA whereas ADA activity significantly decreased in the spleen of rats treated with 100 ppm dose of NAA and TIBA. The observations presented led us to conclude that the administrations of subacute NAA, 2,4-D, and TIBA promote MDA content, inhibit the antioxidative defense system and activate or inhibit immune potential enzymes in the rat's spleen and lung tissues. These data suggest that PGRs produced substantial organ toxicity in the lung and spleen during the period of a 25-day subacute exposure.

Effects of long-term exposure of the red swamp crawfish Procambarus clarkii to a mixture of two herbicides, 2,4-dichlorophenoxyacetic acid and monosodium methanearsonate, and associated human health risks.

2,4-dichlorophenoxyacetic acid and monosodium methanearsonate are often sold in commercial mixtures. Bioconcentration studies have been performed for each of these herbicides individually, but little information exists concerning long-term exposure to a mixture of these herbicides. The following study examined the uptake of arsenic in crawfish after long-term exposure to this mixture, and the health risks associated with consumption of these crawfish. Bioconcentration and depuration experiments using a 50:50 by concentration mixture of the two herbicides, with and without surfactant, were performed to quantify how much arsenic is concentrated in the edible tissue of the crawfish. Of the three tissues (muscle, gill, and hepatopancreas) sampled hepatopancreas bioconcentrated the highest amount of arsenic. Surfactant significantly reduced this uptake but did not affect bioconcentration of arsenic into other tissues. Surfactant had no effect on depuration of arsenic from any of the tissues. Cooking lowered hepatopancreatic arsenic content, possibly as a result of structural changes in the hepatopancreas. Assessment of the human health risk associated with consuming these crawfish showed an exposure dose at the high end of consumption that was approximately twice the reference dose for arsenic. Cancer risks were averaged at approximately 7 extra tumors in a population of 10,000 and 6 extra tumors in a population of 10,000 resulting from a lifetime consumption of crawfish exposed to the herbicide mixture without and with surfactant, respectively.

Loss of pre-B and IgM(+) B cells in the bone marrow after exposure to a mixture of herbicides.

This study determined alterations to bone marrow B-cell populations after in vivo exposure to a mixture containing the herbicides 3,4-dichloropropionanilide (propanil) and 2,4-dichlorophenoxyacetic acid (2,4-D) and compared them to the effects of exposure to the individual herbicides. Propanil and 2,4-D are postemergent herbicides that are sold commercially as a mixture. The individual herbicides or the mixture containing propanil and 2,4-D were administered intraperitoneally to C57Bl/6 female mice at doses from 50 to 200 mg herbicide/kg body weight. The mixtures were given in a 1:1 ratio. Flow cytometric analysis was performed to quantitate bone marrow B-cell populations at 1, 2, 7, and 14d posttreatment. Mixture treatment decreased pre-B and immunoglobulin (Ig) M(+) B-cell populations at all doses by 2 d postexposure. The cell populations were still decreased at 7d posttreatment. In contrast, exposure to the individual herbicides only caused decreases in the pre-B and IgM(+) B-cell populations 7d after exposure to the high doses. Previous studies have demonstrated that corticosterone levels are increased by exposure to propanil. Therefore, the glucocorticoid hormone, corticosterone, was investigated as a possible mediator of cell loss in the bone marrow. Treatment with the glucocorticoid receptor antagonist, RU 486, however, did not prevent cell loss in the bone marrow of mice exposed to the mixture of propanil and 2,4-D. This study demonstrates that pre-B and IgM(+) B-cell populations are decreased after exposure to propanil, 2,4-D, or the mixture containing propanil and 2,4-D. Exposure to the mixture had greater toxic effects than the individual herbicides on bone marrow pre-B and IgM(+) B-cell populations, emphasizing the need to study mixture interactions.

Isolation and expression of a novel starch-storing cell-specific gene containing the KH RNA binding domain from tobacco-cultured cells BY-2.

In cultured Bright Yellow-2 tobacco (Nicotiana tabacum) cells, the depletion of 2,4-dichlorophenoxyacetic acid (2,4-D) in the culture medium induces amyloplast development. This differentiation also includes a decrease in cell multiplication, and an increase in cell size. These changes were primarily triggered by the depletion of 2,4-D, and accelerated by the addition of benzyladenine (BA). Three cDNAs were identified whose transcript levels are specifically increased during differentiation of starch-storing cells using the differential display method, and designated as starch-storing cell induced genes (SCI genes). One of these cDNAs, SCI2 encodes a 285 amino acids long protein with a KH RNA-binding domain. A database search revealed that this protein showed similarity to respective domains of mammalian quaking proteins. 2,4-D addition, which can convert starch-storing cells into dividing cells, to starch-storing BY-2 cells, immediately decreases the SCI2 transcript level, suggesting that SCI2 may have some role in starch-storing cell differentiation in BY-2 cells.

Review of 2,4-dichlorophenoxyacetic acid (2,4-D) epidemiology and toxicology.

The scientific evidence in humans and animals relevant to cancer risks, neurologic disease, reproductive risks, and immunotoxicity of 2,4-D was reviewed. Despite several thorough in vitro and in vivo animal studies, no experimental evidence exists supporting the theory that 2,4-D or any of its salts and esters damages DNA under physiologic conditions. Studies in rodents demonstrate a lack of oncogenic or carcinogenic effects following a lifetime dietary administration of 2,4-D. Epidemiologic studies provide scant evidence that exposure to 2,4-D is associated with soft tissue sarcoma, non-Hodgkin's lymphoma, Hodgkin's disease, or any other cancer. Overall, the available evidence from epidemiologic studies is not adequate to conclude that any form of cancer is causally associated with 2,4-D exposure. There is no human evidence of adverse reproductive outcomes related to 2,4-D. The available data from animal studies of acute, subchronic, and chronic exposure to 2,4-D, its salts, and esters show an unequivocal lack of systemic toxicity at doses that do not exceed renal clearance mechanisms. There is no evidence that 2,4-D in any of its forms activates or transforms the immune system in animals at any dose. At high doses, 2,4-D damages the liver and kidney and irritates mucous membranes. Although myotonia and alterations in gait and behavioral indices are observed after overwhelming doses of 2,4-D, alterations in the neurologic system of experimental animals are not observed with the administration of doses in the microgram/kg/day range. It is unlikely that 2,4-D has any neurotoxic potential at doses below those required to induce systemic toxicity.

Unique renal tubule changes induced in rats and mice by the peroxisome proliferators 2,4-dichlorophenoxyacetic acid (2,4-D) and WY-14643.

Peroxisome proliferators are non-mutagenic carcinogens in the liver of rodents, acting both as initiators and promoters. The National Toxicology Program (NTP) conducted a study of several peroxisome proliferators (PPs), including Wyeth (WY)-14643 as a prototypical PP and 2,4-dichlorophenoxyacetic acid (2,4-D) as a weak PP, in Sprague-Dawley rats. B6C3F1 mice, and Syrian hamsters. In the kidney, an unusual change was observed in the outer stripe of the outer medulla, especially in rats treated with 2,4-D or WY-14643. This change was characterized by foci of tubules that were partially or completely lined by basophilic epithelial cells with decreased cytoplasm and high nuclear density. Changes typical of chronic nephropathy such as interstitial fibrosis or basement membrane thickening were not associated with these foci. Results of immunohistochemical staining for catalase and cytochrome P-450 4A in the kidney indicated increased staining intensity in renal tubular epithelial cells primarily in the region where the affected tubules were observed: however, the altered cells were negative for both immunohistochemical markers. Ultrastructurally, affected cells had long brush borders typical of the P3 tubule segment. The most distinguishing ultrastructural change was a decreased amount of electronlucent cytoplasm that contained few differentiated organelles and, in particular, a prominent reduced volume and number of mitochondria; changes in peroxisomes were not apparent. In addition to the lesion in rats, mice treated with the highest dose of 2,4-D, but not WY-14643, manifested similar renal tubular changes as seen by light microscopy. Neither chemical induced renal tubular lesions in hamsters. Hepatocellular changes characteristic of PPs were present in all 3 species treated with WY-14643, but not 2,4-D. These results indicate that the rat is the species most sensitive to the nephrotoxic effects of PPs and there is a site specificity to this toxicity related to areas of PP-related enzyme induction. Although 2,4-D is considered a weak PP for the liver, it was the most effective at inducing renal lesions, indicating that the toxic potency of various PPs will depend on the target organ.

The effect of exposure to a commercial 2,4-D herbicide formulation during gestation on urethan-induced lung adenoma formation in CD-1 mice.

Female CD-1 mice were exposed to a commercial amine formulation of 2,4-dichlorophenoxyacetic acid (2,4-D) on days 6-16 of gestation in drinking water at concentrations ranging from 0 to 1.0% of the formulated product, equivalent to approximately 0-650 mg/kg/d expressed as the amine derivative. The effect of 2,4-D on urethan-induced pulmonary adenoma formation was evaluated in female offspring 19 w after birth. Urethan-induced sleeping times observed following ip injection of 1.5 mg urethan/g bw 7 w after birth were not altered by 2,4-D (p = 0.10), indicating that 2,4-D did not affect the rate of urethan elimination. 2,4-D exposure did not affect the number of tumors produced (p = 0.58), but did reduce the mean tumor diameter in the highest dose group (p < 0.01). This minor antineoplastic activity of 2,4-D may be related, in part, to inhibitory effects of 2,4-D on various enzymatic or metabolic pathways, essential for cellular growth and tissue development. Since exposure to 2,4-D during pregnancy had little impact of tumor production, it is unlikely that persistent alteration to developing immune cells involved in the cell-mediated immunosurveillance mechanisms occurred. The subtle alteration in tumor size and the mild impairment of growth in the offspring were observed almost exclusively in the highest treatment group. Since this level of exposure is well in excess of those associated with normal application of 2,4-D, the hazard to non-target mammalian populations appears minimal.

[The effect of the amine salt of 2,4-dichlorophenoxyacetic acid on the cluster- and colony-forming capacity of the bone marrow and on the mononuclear phagocyte system]. / Vliianie aminnoi soli 2,4-dikhlorfenoksiuksusnoi kisloty na klaster- i kolonieobrazuiushchuiu sposobnost' kostnogo mozga i sistemu mononuklearnykh fagotsitov.

In experiments on 124 noninbred white rats weighing 180-200 g the influence of the intragastric administration of amino of 2,4-dichlorophenoxyacetic acid in doses of 2.0 and 20 mg/kg daily on the characteristics of their mononuclear phagocyte system was studied. The study revealed a decrease in the number of monocytic precursors in the marrow, an increase in their capacity for colony formation, the inhibited cell function on the periphery of the system simultaneously with the enhancement of oxidizing processes. The metabolic activation of cells was accompanied by their functional insufficiency, which led to the activation of monocytopoiesis and the increased migration of monocytes to peripheral blood.

Comparative subchronic and chronic dietary toxicity studies on 2,4-dichlorophenoxyacetic acid, amine, and ester in the dog.

Forms of 2,4-dichlorophenoxyacetic acid (2,4-D) are herbicides used in the control of a wide variety of broadleaf and woody plants. Subchronic toxicity studies in dogs were conducted on three forms of 2,4-D: the parent form, 2,4-D acid (ACID); 2,4-D dimethylamine salt (DMA); and 2,4-D 2-ethylhexyl ester (2-EHE). The three studies were designed to allow for comparison of the toxicity of the three forms. Doses in the subchronic studies, on an acid equivalent basis, were 0, 0.5 (ACID only), 1.0, 3.75, and 7.5 mg/kg/day. Treatment related findings in the three studies included reductions in body weight gain, and food consumption, and minor increases in blood urea nitrogen, creatinine, and alanine aminotransferase. The data from the three subchronic studies demonstrated the comparable toxicity of ACID, DMA, and 2-EHE and support a subchronic no observed adverse effect level (NOAEL) of 1.0 mg/kg/day for all three forms. Due to the similarity in toxicity of the three forms of 2,4-D, a 1-year chronic toxicity study was performed on the parent ACID to fully characterize the potential toxicity of 2,4-D in the dog. ACID was well tolerated at doses of 0, 1.0, 5.0, and 7.5 mg/kg/day. The clinical pathology alterations were similar to those seen in the subchronic studies and were not progressive. The histopathology alterations observed were not severe in nature and the no observed effect level in the chronic study was determined to be 1.0 mg/kg/day. There was no indication of any immunotoxic or oncogenic response in the studies. In conclusion, the findings of these studies indicate comparable toxicity among representative forms of 2,4-D and their generally low toxicity following subchronic and chronic dietary exposure in the dog.

The effects of Tordon 202c exposure on urethan-induced lung adenoma formation in female CD-1 mice.

Female CD-1 mice were exposed to Tordon 202c, a herbicide containing 2,4-dichlorophenoxyacetic acid and picloram, in the drinking water for 15 w at concentrations ranging from 0 to 0.3% of the product formulation. After 3 w of the 15-w treatment period, the mice received 1.5 mg/g urethan ip. Pulmonary adenoma production was evaluated 12 w later. Tordon 202c exposure produced a dose-dependent increase in tumor number, but had no effect on tumor size. Urethan-induced sleeping times which reflected the rate of urethan metabolism or excretion were altered, but a specific dose-related effect which could be correlated with tumor production was not observed. This suggests that Tordon 202c exposure influences adenoma formation by immunological mechanisms rather than by causing indirect effects on urethan metabolism or excretion.

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.