Fetotoxicity of 2,4-dichlorophenoxyacetic acid in rats and the protective role of vitamin E.

The present study was conducted to investigate the potential role of 2,4-dichlorophenoxyacetic acid (2,4-D) in inducing developmental toxicity and oxidative damage in pregnant rats and their fetuses as well as to assess the efficacy of vitamin E to prevent or alleviate such defects. Pregnant rats received 2,4-D (100 mg/kg bw) alone or in combination with vitamin E (100 mg/kg bw) daily from gestation day 1 to 19. The number of implantations, viable and resorbed fetuses and sex ratio were not statistically different among groups. However, fetuses maternally treated with 2,4-D were characterized by lower body weight and higher morphologic and skeletal defect rate. 2,4-D induced oxidative stress in the liver of mothers and fetuses which was indicated by a significant elevation of malondialdehyde level with reduction in catalase activity and total antioxidant capacity. Coadministration of vitamin E can counteract the deleterious effects of 2,4-D by successive reduction in the oxidative stress.

Bronchoconstriction induced by increasing airway temperature in ovalbumin-sensitized rats: role of tachykinins.

This study was carried out to determine the effect of allergic inflammation on the airway response to increasing airway temperature. Our results showed the following: 1) In Brown-Norway rats actively sensitized by ovalbumin (Ova), isocapnic hyperventilation with humidified warm air (HWA) for 2 min raised tracheal temperature (Ttr) from 33.4 ± 0.6°C to 40.6 ± 0.1°C, which induced an immediate and sustained (>10 min) increase in total pulmonary resistance (Rl) from 0.128 ± 0.004 to 0.212 ± 0.013 cmH2O·ml(-1)·s (n = 6, P < 0.01). In sharp contrast, the HWA challenge caused the same increase in Ttr but did not generate any increase in Rl in control rats. 2) The increase in Rl in sensitized rats was reproducible when the same HWA challenge was repeated 60-90 min later. 3) This bronchoconstrictive effect was temperature dependent: a slightly smaller increase in peak Ttr (39.6 ± 0.2°C) generated a significant but smaller increase in Rl in sensitized rats. 4) The HWA-induced bronchoconstriction was not generated by the humidity delivered by the HWA challenge alone, because the same water content delivered by saline aerosol at room temperature had no effect. 5) The HWA-evoked increase in Rl in sensitized rats was not blocked by atropine but was completely prevented by pretreatment either with a combination of neurokinin (NK)-1 and NK-2 antagonists or with formoterol, a ß2 agonist, before the HWA challenge. This study showed that increasing airway temperature evoked a pronounced and reversible increase in airway resistance in sensitized rats and that tachykinins released from the vagal bronchopulmonary C-fiber endings were primarily responsible.

Uncaria tomentosa extracts protect human erythrocyte catalase against damage induced by 2,4-D-Na and its metabolites.

The effect of ethanolic and aqueous extracts from leaves and bark of Uncaria tomentosa was studied, with particular attention to catalase activity (CAT - EC. 1.11.1.6). We observed that all tested extracts, at a concentration of 250 µg/mL were not toxic to erythrocyte catalase because they did not decreased its activity. Additionally, we investigated the protective effect of extracts on changes in CAT activity in the erythrocytes incubated with sodium salt of 2,4-dichlorophenoxyacetic acid (2,4-D-Na) and its metabolites i.e., 2,4-dichlorophenol (2,4-DCP) and catechol. Previous investigations showed that these chemicals decreased activity of erythrocyte catalase (Bukowska et al., 2000; Bukowska and Kowalska, 2004). The erythrocytes were divided into two portions. The first portion was incubated for 1 and 5h at 37°C with 2,4-D-Na, 2,4-DCP and catechol, and second portion was preincubated with extracts for 10 min and then incubated with xenobiotics for 1 and 5h. CAT activity was measured in the first and second portion of the erythrocytes. We found a protective effect of the extracts from U. tomentosa on the activity of catalase incubated with xenobiotics studied. Probably, phenolic compounds contained in U. tomentosa scavenged free radicals, and therefore protected active center (containing -SH groups) of catalase.

Adverse effects of 2,4-dichlorophenoxyacetic acid on rat cerebellar granule cell cultures were attenuated by amphetamine.

2,4-Dichlorophenoxyacetic acid (2,4-D), a worldwide-used herbicide, has been shown to produce a wide range of adverse effects in the health--from embryotoxicity and teratogenicity to neurotoxicity--of animals and humans. In this study, neuronal morphology and biochemical events in rat cerebellar granule cell (CGC) cultures have been analyzed to define some of the possible mechanisms involved in 2,4-D-induced cell death. For that purpose, amphetamine (AMPH) that has been shown to accelerate the recovery of several functions in animals with brain injury has been used as a pharmacologycal tool and was also investigated as a possible protecting agent. Addition of 2,4-D to CGC cultures produced a drastic decrease in cell viability, in association with an increased incidence of necrosis and apoptosis, and an increased level of reactive oxygen species, a decrease in glutathione content, and an abnormal activity of some enzymes with respect to the control group. The adverse effects of 2,4-D were partly attenuated in presence of AMPH. Some deleterious effects on several ultrastructural features of the cells, as well as the enhanced incidence of apoptosis, were partially preserved in AMPH-protected cultures as compared with those which were exposed to 2,4-D alone. The collected evidences (1) confirms the previously observed, deleterious effects of 2.4D on the same or a similar model; (2) suggests that the 2,4-D-induced apoptosis could have been mediated by or associated to an oxidative imbalance in the affected cells, and (3) shows some evidence of a protective effect of AMPH on 2,4-D-induced cell death, which could have been exerted through a reduction in the oxidative stress.

[Participation of dexamethasone and E and C vitamins in the modulation of the hepatotoxic effect induced by fomesafen and 2,4-D amino herbicides, in rats ]. / Participacion de la dexametasona y las vitaminas E y C en la modulacion del efecto hepatotoxico inducido por los herbicidas fomesafen y 2,4-D amino, en ratas.

The fomesafen and 2,4-D amine herbicide induce cytotoxic effects at hepatic level in rats, such as: hepatomegaly, hyperplasia and increase in the enzymes activity which participate in the processes of peroxisomal beta-oxidation of fatty acids. In this work, the effect of vitamin E and C was evaluated, as well as, the dexamethasone in the modulation of these hepatotoxic effects. Sprague-Dawley rats were treated with the herbicides and with the agents to be evaluated. The different treatments were given during 15 days orally route. The herbicides combined with the dexamethasone and antioxidant agents were administrated only and simultaneously with the herbicides. Once concluded the different treatment, the rats were weighed and sacrificed. It was evaluated the liver size and liver fragments were obtained to determine the enzymatic activity of Fatty Acyl CoA-oxidase (FACO) and cellular number. The results showed that the hepatomegaly induced by fomesafen was inhibited by the vitamins and by the dexamethasone, while any effect was not observed in the group of rats treated with 2,4-D amine. None of the agents modulated the FACO activity induced by herbicides in treated rats. However, the dexamethasone showed a protective effect in the hyperplasia induced by two herbicides. The hepatotoxic effects induced by the herbicides responded to a different mechanism due to the differences of the effects observed at the antioxidant agents. On the other hand, the inhibition of the cellular proliferation by the dexamethasone does not keep relation with the responsible mechanisms of inducing the oxidant stress into FACO activity. Under experimental conditions of this study, the use of these agents does not guarantee protection against the hepatotoxic effects induced by the herbicides.

Induction of apoptosis in human lymphocytes by the herbicide 2,4-dichlorophenoxyacetic acid.

Dimethylammonium salt of 2,4-dichlorophenoxyacetic acid (DMA-2,4-D) is a widely used herbicide that is considered moderately toxic. In the present study we found that DMA-2,4-D is able to cause apoptosis in peripheral blood lymphocytes of healthy individuals and Jurkat T cells. Apoptosis induced by DMA-2,4-D was dose and time dependent, independent of Fas, TNF receptor 1 or the aromatic hydrocarbon receptor, and involved disruption of the mitochondrial transmembrane potential and activation of caspase-9. ZVAD-FMK, a broad-spectrum inhibitor of caspases, blocked DMA-2,4-D-induced apoptosis completely. While an inhibitor of caspase-9, as well as caspase-9 and caspase-3 inhibitors in combination, strongly blocked DMA-2,4-D-induced apoptosis, an inhibitor of caspase-3 had a moderate inhibitory effect. Unlike Fas-mediated apoptosis, the initiator caspase, caspase-8, was not involved in DMA-2,4-D-induced apoptosis. Transfection of Jurkat cells with Bcl-2 prevented DMA-2,4-D-induced disruption of the mitochondrial transmembrane potential and led to a complete blockage of apoptosis. Our data indicate that DMA-2,4-D kills human lymphocytes by initiating apoptosis via a direct effect on mitochondria. The activation of caspases occurs downstream of mitochondrial damage, and the dysfunction of mitochondria appears to be sufficient for triggering all downstream events leading to apoptosis.

Inhibitors of the carrier-mediated influx of auxin in suspension-cultured tobacco cells.

Active auxin transport in plant cells is catalyzed by two carriers working in opposite directions at the plasma membrane, the influx and efflux carriers. A role for the efflux carrier in polar auxin transport (PAT) in plants has been shown from studies using phytotropins. Phytotropins have been invaluable in demonstrating that PAT is essential to ensure polarized and coordinated growth and to provide plants with the capacity to respond to environmental stimuli. However, the function of the influx carrier at the whole-plant level is unknown. Our work aims to identify new auxin-transport inhibitors which could be employed to investigate its function. Thirty-five aryl and aryloxyalkylcarboxylic acids were assayed for their ability to perturb the accumulation of 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene-1-acetic acid (1-NAA) in suspension-cultured tobacco (Nicotiana tabacum L.) cells. As 2,4-D and 1-NAA are preferentially transported by the influx and efflux carriers, respectively, accumulation experiments utilizing synthetic auxins provide independant information on the activities of both carriers. The majority (60%) of compounds half-inhibited the carrier-mediated influx of [14C]2,4-D at concentrations of less than 10 microM. Most failed to interfere with [3H]NAA efflux, at least in the short term. Even though they increasingly perturbed auxin efflux when given a prolonged treatment, several compounds were much better at discriminating between influx and efflux carrier activities than naphthalene-2-acetic acid which is commonly employed to investigate influx-carrier properties. Structure-activity relationships and factors influencing ligand specificity with regard to auxin carriers are discussed.

Renal organic anion secretion: evidence for dopaminergic and adrenergic regulation.

To examine possible regulatory control of renal proximal tubule organic anion secretion, winter flounder (Pleuronectes americanus) proximal tubule primary cultures were mounted in Ussing chambers. Unidirectional fluxes of [2,4-(14)C]dichlorophenoxyacetic acid were determined under short-circuited conditions. Phorbol 12-myristate 13-acetate (1 microM) caused a significant (P < 0.01) inhibition of net 2,4-dichlorophenoxyacetic acid secretion. Preincubation with staurosporine (1 microM) blocked the phorbol 12-myristate 13-acetate-induced decrease in secretion. Neither forskolin (10 microM) nor W-7 (20 microM) had any effect on net transport. Elevation of intracellular calcium activity with either A-23187 or thapsigargin produced a slight, transient decrease in transport. Addition of dopamine (1 microM) to the peritubular side, but not the luminal side, caused a significant (P < 0.01) decrease in net secretion. Both the alpha-adrenergic agonist oxymetazoline (10 microM) and depletion of intracellular Na+ transiently, but significantly (P < 0.05), increased net transport. The data indicate that renal organic anion excretion may be regulated through dopaminergic inhibition and alpha-adrenergic stimulation of net transepithelial secretion.

Chlorsulfuron blocks 2,4-D-induced cell enlargement and NADH oxidase in excised sections of soybean hypocotyls.

Elongation of soybean hypocotyl sections induced by the auxin herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D), was blocked by micromolar concentrations of the sulfonylurea herbicide chlorsulfuron (2-chloro-N-[[(4-methoxy-6-methyl-1,3,5-trizin-2- yl)amino]carbonyl]benzenesulfonamide). The inhibition at equimolar concentrations of 2,4-D and chlorsulfuron was overcome by a 10- or 100-fold excess of 2,4-D. Stimulation by 2,4-D of an 2,4-D-responsive NADH-oxidase activity of the soybean plasma membrane also was blocked by the presence of concentrations of chlorsulfuron equimolar to the 2,4-D. Chlorsulfuron alone was largely without effect on either cell elongation or the auxin-stimulated NADH-oxidase activity over a range of chlorsulfuron concentrations. The results show a clear correlation between inhibition of auxin-stimulated cell elongation and the inhibition of an auxin-stimulated component of a plasma membrane NADH-oxidase activity.