Cytoskeletal perturbation induced by herbicides, 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)

To understand the mechanisms of toxicity of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), we have studied their effects on the cytoskeletal organization, particularly microtubules (MT) and microfilaments (MF), DNA synthesis, and the synthesis and composition of cytoskeletal proteins in mouse 3T3 cells. Exposure of cells to 2,4-D or 2,4,5-T resulted in a dose-dependent inhibition of DNA synthesis; 50% inhibition occurred at 2.21 mM and 0.90 mM for 2,4-D and 2,4,5-T, respectively. Furthermore, a strong synergistic inhibition of DNA synthesis was produced by mixtures (each having a total concentration of 1.25 mM) of 2,4-D with 2,4,5-T. Similarly, 2,4,5-T is more potent than 2,4-D in causing cytoskeletal perturbation as revealed by fluorescence microscopy. Treatment of cells with 2,4-D (2.5 mM) or 2,4,5-T (1.25 mM) for 20 h resulted in severe MT aggregation and the appearance of large bundles, which were organized in a rope-like structure in the former and a dramatic octopus-like pattern in the latter. Further, MT bundling is particularly severe in the cell center. Under these conditions, marked changes in MF organization also occurred as evidenced by clustering and crisscrossing of MF in the perinuclear region. A 1:1 mixture (final = 1.25 mM) of 2,4-D and 2,4,5-T, a formulation equivalent to Agent Orange composition, also induced a dramatic perturbation to the organization of MT and MF, resulting in the formation of ring-like structures. MT bundling is still apparent, especially around the outer edge of the "rings." MF are localized predominantly along the cell periphery, where they appear to be aggregated tightly forming patches. Surprisingly, the synthesis and composition of cytoskeletal proteins, which are resistant to detergent extraction but released by CaCl2, are essentially unaffected by 2,4-D or 2,4,5-T. These results suggest that the dramatic perturbation of the cytoskeletal morphology caused by these herbicides probably only results from a structural reorganization and redistribution of MT and MF.     

Effects of 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid on peroxisomal enzymes in rat liver

The effects of feeding 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) on the level of peroxisomal enzymes in rat liver were studied. The concentration of triglyceride in serum was decreased and the activity of cyanide-insensitive palmitoyl-CoA oxidation, catalase and carnitine acetyltransferase increased. However, the extent of the increase in the activity of these enzymes by treatment with 2,4-D was less pronounced than that by 2,4,5-T treatment. The administration of 2,4-D or 2,4,5-T increased the concentration of polypeptide with a mol. wt of 80,000 in the light mitochondrial fractions of the liver from the rats.     

In vitro uptake of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) by renal cortical tissue of rabbits and rats

The active transport of 2,4-D and 2,4,5-T by renal cortical slices of rats and rabbits has been characterized with in vitro techniques. Renal cortical slices, prepared from both species, accumulate 2,4-D and 2,4,5-T, although greater uptake, was noted with rabbit tissue. Nitrogen and various metabolic inhibitors reduced the uptakes. This indicates that these processes are energy dependent. The enhancement of accumulation by the addition of certain metabolic substrates, e.g. acetate, lactate, provides evidence that both organic acid herbicides are transported by the renal organic anion mechanism. Potassium-ion stimulation also supports this concept. It is concluded that the renal tubular transport by the organic anion mechanism may account for the relatively rapid disappearance of these compounds, which in turn may contribute to their low toxicity.     

Distribution of 2-methyl-4-chlorophenoxyacetic acid and 2,4-dichlorophenoxyacetic acid in male rats: evidence for the involvement of the central nervous system in their toxicity

The effects of different 2-methyl-4-chlorophenoxyacetic acid (MCPA) or 2,4-dichlorophenoxyacetic acid (2,4-D) pretreatments on the distribution of labeled [¹⁴C]MCPA or [¹⁴C]2,4-D given iv were studied in male rats. Single subcutaneous MCPA or 2,4-D pretreatment increased ¹⁴C activity in the brain, cerebrospinal fluid (CSF), liver, muscle, heart, or testis and decreased that in the plasma or kidney, while in the lung ¹⁴C activity remained approximately unchanged. Changes in the distribution of ¹⁴C activity as well as toxic signs such as myotonia and lethargy appeared within 0.5–1.5 hr after subcutaneous MCPA administration and disappeared in a few days. ¹⁴C activities in the brain and CSF of both saline-treated adult and 10- or 21-day-old animals were very low as compared to the other tissues, but in the treated animals these and also absolute MCPA concentration increased to about the level in the muscle or testis. Chronic MCPA exposure had only a slight effect on the distribution of ¹⁴C activity. The decreased binding of [¹⁴C]MCPA to plasma proteins caused by MCPA pretreatments may explain the increase of ¹⁴C activity in many tissues but not the high increase in the brain and CSF during intoxication. The results indicate that at large doses either the influx of MCPA and 2,4-D into the brain is highly increased or their efflux out of the brain is decreased. A potent increase in cerebral ¹⁴C activity coincided with the appearance of MCPA intoxication, which suggests that the central nervous system (CNS) is involved in the toxicity of chlorophenoxyacetic acids.     

Developing rat brain monoamine levels following in utero exposure to a mixture of 2,4-dichlorophenoxyacetic and 2,4,5-trichlorophenoxyacetic acids

Pregnant rats were gavaged with a 1:1 mixture of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) at 0 (G0), 50 (G 50) and 100 (G 100) mg/kg per day on gestational days 6-15. Treatment significantly (P less than 0.05) delayed ontogeny of dopamine (DA), but not norepinephrine (NE) levels, in the thalamus-hypothalamus on postnatal day 7; in the pons-medulla on days 7,9 and 15; and in the olfactory lobes on day 9. On day 25, serotonin (5-HT) levels were significantly decreased in the pons-medulla of G 100 rats, whereas 5-hydroxyindoleacetic acid (5-HIAA) levels decreased in the thalamus-hypothalamus and pons-medulla of G 50 and G 100 rats.     

Induction of cytosolic and microsomal epoxide hydrolases and proliferation of peroxisomes and mitochondria in mouse liver after dietary exposure to p-chlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid

The effects of dietary exposure to 0.125% (w/w) p-chlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid or 2,4,5-trichlorophenoxyacetic acid on the content of peroxisomes and levels of certain xenobiotic-metabolizing enzymes in mouse liver have been investigated. In agreement with the literature on rat liver 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid were found to cause extensive proliferation of peroxisomes (as judged by the total levels of "mitochondrial" protein, carnitine acetyltransferase, cyanide-insensitive palmitoyl-CoA oxidation and catalase) in mouse liver. On the other hand, exposure to p-chlorophenoxyacetic acid did not significantly affect any of these parameters. As with certain other peroxisome proliferators, 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid increased total cytochrome oxidase activity as well. In addition, dietary exposure to 2,4-dichlorophenoxyacetic acid and to 2,4,5-trichlorophenoxyacetic acid resulted in increases in the activities of cytosolic and microsomal epoxide hydrolases in mouse liver and generally less pronounced increases in the total cytosolic glutathione transferase activity and microsomal content of cytochrome P-450. In the case of cytochrome P-450, this process can be said to be a true induction (i.e. the amount of enzyme protein is increased), because the assay procedure for cytochrome P-450 measures holoenzyme amount. Immunoquantitation demonstrated that this was also the case for the changes in cytosolic epoxide hydrolase. The dramatic differences in proliferation of peroxisomes and induction of xenobiotic-metabolizing enzymes seen here with compounds differing relatively little in structure may indicate that a receptor mechanism of some kind is involved.     

Ontogeny of swimming behavior and brain catecholamine turnover in rats prenatally exposed to a mixture of 2,4-dichlorophenoxyacetic and 2,4,5-trichlorophenoxyacetic acids

Rats exposed in utero on gestational days 6-15, to nonfetotoxic and grossly nonteratogenic mixtures (50 or 100 mg/kg) of 2,4-D/2,4,5-T as found in Agent Orange (but without significant contamination with 2,3,7,8 tetrachloro-p-dioxin) manifested subtle developmental neurotoxicity. Maturation of swimming behavior was significantly delayed on postnatal day 7 in both treatment groups. The concentration of norepinephrine (NE) in whole brain was significantly increased on postnatal day 15 in both treatment groups, whereas the concentration of dopamine (DA) was increased on postnatal day 15 at 100 mg/kg. The turnover and efflux rate constant of DA in whole brain were significantly reduced whereas the turnover time increased on postnatal day 3. The efflux rate constant for NE decreased and the turnover time increased significantly on postnatal day 15 at 100 mg/kg. These data indicate the value of ontogenic assessment following exposure to small doses, which result in functional alterations in the absence of overt toxic signs.     

Blood-brain barrier damage by 2-methyl-4-chlorophenoxyacetic acid herbicide in rats

The integrity of blood-brain barrier (BBB) to serum proteins was studied in rats acutely intoxicated by 2-methyl-4-chlorophenoxyacetic acid (MCPA). Intoxication was produced by giving sodium salt of MCPA sc in single doses of 100, 250, and 500 mg/kg at various intervals before killing the animals. Evans blue, immunohistochemistry of serum proteins, and electron microscopy were used to study the barrier. Extravasations of Evans blue-albumin complex indicating BBB damage were observed with the 250 and 500 mg/kg doses. Lateral cerebral cortex and medulla oblongata were the tissues most consistently affected. Most severe extravasations occurred 4.5 hr after administration of 250 mg/kg MCPA. At 24.5 hr, no extravasation of Evans blue could be demonstrated, which suggests reversibility of the damage. The immunohistochemical demonstration of endogenous albumin or immunoglobulin-G verified the same localizations of the BBB damage. Electron microscopy of the damaged brain areas revealed increased amounts of vesicles in the endothelial cells and local swellings of the basal laminae of the capillaries. As indicated by the extent of extravasation of serum components into the brain, BBB damages appeared within 1.5 hr and showed a marked recovery in 24.5 hr. The damages coincided with the toxic symptoms of the herbicide. Thus the central nervous system was affected by MCPA intoxication.     

Renal handling of 2-methyl-4-chlorophenoxyacetic acid (MCPA) in rats

Following i.p. administration of various doses of 2-methyl-4-chlorophenoxyacetic acid (MCPA), ca. 50% is excreted during a 5-h diuresis experiment. After i.p. administration of MCPA, virtually no distribution occurs (Vrel = 18% of the body weight). Renal excretion of MCPA can be accelerated by inhibition of its renal tubular reabsorption. The distinct inhibition of renal excretion of MCPA by simultaneous administration of probenecid or p-aminohippurate (PAH) indicates the active tubular transport of MCPA; this transport process can be stimulated by treatment of rats with triiodothyronine. Active tubular transport of MCPA was confirmed by measurement of MCPA accumulation in renal cortical slices, both under aerobic and anaerobic conditions. Accumulation of MCPA under anaerobic conditions indicates an additional passive uptake and binding of MCPA in kidney tissue in accordance with the high degree of binding to plasma albumin (85%).     

Distribution of 2,4,5-trichlorophenoxyacetic acid in the mouse fetus

The herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) readily crosses the placenta in the mouse on gestational day 13. Concentrations of 2,4,5-T in maternal tissues and fetuses were obvious at 30 min, highest at 8 h, diminished by 24 h and almost entirely eliminated by 48 h. Autoradiographs of the whole fetus showed that initially 2,4,5-T was mainly in the highly vascularized tissues such as liver followed by the skin, eyes and ventricles of the brain. At 48 h, there was a general distribution of 2,4,5-T in the skeletal muscles. None was detected in the palate. The excretion rate in the mouse was approx. 60 gm/h; about 52% of the dose was recovered unchanged in the urine in 24 h.     

Comparative studies on the embryotoxicity of 2-methyl-4-chlorophenoxyacetic acid, mecoprop and dichlorprop in NMRI mice

From the group of herbicidal phenoxy carbonic acids, 2-methyl-4-chlorophenoxyacetic acid (MCPA; 0-500 mg/kg), 2-(4-chloro-2-methyl-phenoxy) propionic acid (mecoprop/MCPP; 0-700 mg/kg) and 2-(2,4-dichlorophenoxy) propionic acid (dichlorprop/2,4-DP; 0-500 mg/kg) as well as the dextrorotatory compounds of MCPP (MCPPD; 0-500 mg/kg) and 2,4-DP (2,4-DPD; 0-500 mg/kg) were studied in NMRI mice after oral administration between days 6-15 of pregnancy. All five substances proved to be embryotoxic and teratogenic in varying intensity. MCPA proved to be most effective: it was embryotoxic from doses of 100 mg/kg and teratogenic from 200 mg/kg. The remaining compounds (MCPP, MCPPD, 2,4-DP, 2,4-DPD) were embryotoxic from doses of 300 mg/kg and caused malformations of the skeleton from 400 mg/kg. The embryocidal and teratogenic potencies of the dextrorotatory components of MCPA and 2,4-DP exceeded those of the corresponding racemates. Influences of MCPPD and 2,4-DPD upon postimplantative loses, frequency of cleft palates and wavy ribs appeared already at dosages being 100-200 mg/kg below those of the racemates given to the respective groups of experimental animals. Additional alterations of the skeleton were observed which did not occur following administration of the racemic mixtures: deformed centrums of thoracic vertebra and exencephaly.     

A fatal intoxication from ingestion of 2-methyl-4-chlorophenoxyacetic acid (MCPA)

We report a fatal intoxication case by the ingestion of an herbicide, 2-methyl-4-chlorophenoxyacetic acid (MCPA). A 23-year-old male was found dead in his car. The forensic autopsy revealed no remarkable morphological changes. However, in a toxicological screening test, MCPA was qualitatively detected from the extracts of stomach contents. Then MCPA in the extract of each body fluid and organ tissue was determined by gas chromatography-mass spectrometry with trimethylsilyl-derivatization as follows (microg/g): 888.3 in the heart blood, 578.1 in the peripheral blood, 52.2 in the urine, 770.9 in the brain, 1362 in the right lung, 1135 in the liver, 755.5 in the right kidney, and 10,200 in the stomach contents. These data strongly suggested that the male orally ingested MCPA. Moreover, p-chloro-o-cresol (4-chloro-2-methylphenol) was also determined in the body fluids and organ tissues, suggesting a metabolite of MCPA. From these toxicological data, together with autopsy findings, the cause of his death was diagnosed as acute MCPA poisoning.     

Toxicity and distribution of 2-methyl-4-chlorophenoxyacetic acid (MCPA) in developing chick embryos

The toxicity of the herbicide Erbitox E30, a commercial formulation of 2-methyl-4-chlorophenoxyacetic acid (MCPA) containing 28% MCPA as sodium-potassium salt and 72% of unknown ingredients, was tested on chick embryos. Sterile aqueous solutions of MCPA were injected into the air chamber at doses of 0, 1.5, 3.0, 6.0, 9.0, or 10.5 mg/egg on day 0 or on day 4 of incubation. The mortality rate for the embryos treated on day 0 of incubation was high in the first 5 days, low from 5-12 days and again increased by 15 days. The 15-day LD50 was 4.4 mg/egg (95% C.I. 3.7-5.3 mg/egg). HPLC analysis of albumen and yolk showed that concentrations of MCPA in the albumen were detectable at 5 min, highest at 7 days and markedly diminished by 14 days of incubation; a significantly lower concentration of MCPA was found in the yolk throughout the incubation period, except at 14 days when the yolk concentration was 4 times higher than the albumen concentration. At 15 days of incubation, MCPA was evenly distributed in the tissues of the embryo. MCPA was more toxic to 4-day embryos; concentrations above 6.0 mg/egg were lethal to all embryos within the first week of incubation. The 15-day LD50 for treatment on day 4 of incubation was 2.8 mg/egg (95% C.I. 2.5-3.2 mg/egg). The liver was affected by treatment with MCPA, being green in treated embryos. However, histological examination revealed few changes in the liver parenchyma.     

The metabolism and distribution of 2,4,5-trichlorophenoxyacetic acid in female rats

[1-¹⁴C]-2,4,5-Trichlorophenoxyacetic acid (2,4,5-T) was fed to pregnant and non-pregnant female rats at various dosages, and expired air, urine, feces, internal organs and tissues were analyzed for radioactivity. During the first 24 hr, 75 ± 7% of the radioactivity was excreted in the urine and 8.2 ± 4.6% in the feces. No ¹⁴C was found in the expired air. There was no significant difference in the rate of elimination between the pregnant and nonpregnant rats, or among the dosages used. Radioactivity was detected in all tissues, with the highest concentration being found in the kidney. The maximum concentration of radioactivity in all tissues was generally reached between 6 to 12 hr after po dosing and then started to decline rapidly. Radioactivity was also detected in the fetuses and in the milk. The average biological half-life of 2,4,5-T in the organs was 3.4 hr for the adult rats and 97 hr for the newborn.     

Ca2+ homeostasis alterations induced by 2,4-dichlorophenoxyacetic butyl ester and 2,4-dichlorophenoxyacetic acid on avian skeletal muscle

Fertilized hen eggs were treated externally with 2,4-dichlorophenoxyacetic butyl ester (2,4-D b.e.) (3.1 mg/egg) before the start of the incubation. Actomyosin and sarcoplasmic reticulum adenosine triphosphatase (ATPase) activities from leg and complexus muscles of chicks hatched from treated eggs were measured. No significant variations were detected in the ATPase activities of actomyosin, but the sarcoplasmic reticulum Mg2(+)-activated ATPase and Ca2+, Mg2(+)-activated ATPase were inhibited 50 and 38% respectively. 45Ca2+ uptake into soleus muscle was increased by the 2,4-D b.e. treatment. The compartmental analysis of 45Ca2+ uptake kinetics showed increases in Ca2+ fluxes in sarcolemma and mitochondria and in the mitochondrial calcium pool. Isolated soleus muscles were treated with 2,4-dichlorophenoxyacetic acid (2,4-D) or 2,4-D b.e. [14C]2,4-D reached it highest level in these muscles after 1 hr of treatment. The in vitro treatment with 2,4-D or 2,4-D b.e. increased 45Ca2+ uptake into the muscles. 2,4-D b.e. produced greater alterations than 2,4-D. The compartmental analysis of the 45Ca2+ uptake kinetics also showed increases of the mitochondrial Ca2+ pool and Ca2+ fluxes through sarcolemma and mitochondria. These results led to a hypothesis based on Ca2+ permeability alterations for explaining the myopathic actions of these phenoxyherbicides.