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.     

Teratogenic and toxicological examination of 2,4,5-trichlorphenoxyacetic acid in developing chick embryos

Practical grade 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), dissolved in dimethylsufoxide (DMSO), was injected into the air space of fertilized chicken eggs prior to incubation. Doses of 2,4,5-T administered were 12.5, 25, 50, 75, 100, and 125 mg/kg to determine herbicide toxicity on the first day of incubation. A similar group was studied on day 5 of incubation with doses of 2,4,5-T at 50, 75, 100 and 250 mg/kg. LD₅₀ was estimated to be 62 mg/kg on day zero and 68 mg/kg on day 5. Additional, embryos were exposed to 2,4,5-T at 50 mg/kg on day zero of gestation and sacrificed after 48 h of incubation. Serial sections were examined for teratological and developmental anomalies. None were found.     

Sequential histopathologic, hematologic, and blood chemistry changes induced in mice by a technical and a purified preparation of 2,4,5-trichlorophenoxyacetic acid.

Maternal mice were given 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) on days 6 through 14 of pregnancy in a tetratologic study at the National Center for Toxicological Research. Sick or moribund mice sacrificed after 4-8 doses of 120 mg/kg 2,4,5-T often showed severe myocardial lesions, hypocellularity of the bone marrow, and depletion of lymphocytes in the thymus, spleen, or lymph nodes. Healthy mice sacrificed on day 17, 11 days after treatment began, showed few or no severe lesions. To determine if lesions earlier in gestation contributed significantly to an increase in fetal abnormalities in the healthy 17-day survivors, dihybrid croos F2 pregnant and nonpregnant mice received by gavage 0, 60, or 120 mg/kg 2,4,5-T on days 6 through 14 of pregnancy. One group received a technical preparation containing 97.9 +/- 0.4% 2,4,5-T; another received a purified preparation containing 99 +/- 0.3% 2,4,5-T. Mice were sacrificed when they became moribund and at 6, 24, and 30 hr, as well as at 4, 6, 8, and 11 days after beginning treatment. Almost all mice given 60 mg/kg and many given 120 mg/kg 2,4,5-T appeared normal at sacrifice either early or late in pregnancy and showed little or no pathologic changes. Mice that became ill or moribund often showed severe lesions; few survived 11 days. Severe myocardial lesions were seen in 26 of 70 moribund mice fiven the technical 2,4,5-T and 24 of 33 given the purified preparation of 2,4,5-T. The moribund mice, particularly those given the purified compound, also showed a high incidence of lesions in other organs and marked hematological and blood chemistry changes. These findings indicate that the lesions are primarily due to 2,4,5-T rather than to impurities in the technical preparation; also impaired maternal health is not the primary cause of the increase in fetal abnormalities.     

Use of the periodic acid schiff stain to grade retarded fetal renal development in mice

In an earlier study, maternal mice were given by gavage 60–120 mg/kg 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) on days 6—14 of pregnancy and sacrificed on day 17. The Gomori stain revealed diminished alkaline phosphatase in the renal proximal tubules of fetuses exposed to 2,4,5-T during gestation indicating retarded renal functional and, probably, morphological development. Spare sections of the fetal kidneys from the earlier study were stained by the periodic acid-Schiff (PAS) procedure. This revealed, in exposed fetuses, a reduction of renal tubules with PAS-positive material in the brush borders comparable in incidence and distribution to those with alkaline phosphatase in the earlier study. These findings indicate that PAS can replace the Gomori stain as a screening procedure when retarded fetal renal development by a toxic agent is suspected. Unlike alkaline phosphatase, the PAS procedure is effective even after prolonged fixation in Bouin's solution which is commonly used in fetal studies.     

Teratogenic and Embryotoxic Effects of the Herbicides Di- and Trichlorophenoxyacetic Acids (2, 4D and 2, 4, 5-T)

Abstract: Commercial solutions of phenoxyacetic acids were tested for teratogenic effects in NMRI-mice. The Swedish product Hormoslyr 500-T contained only 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) while Hormoslyr 64 was a mixture of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-T (2:1). Subcutaneous injections were given from day 6 through day 14 of pregnancy and the animals were sacrificed on day 18. The number of resorbed embryos, living embryos with gross malformations, as well as internal and skeletal malformations were recorded. It was found that both preparations at the high dosage (110 mg/kg/day) were teratogenic and embryotoxic. At the low dose level (50 mg/kg/day) the 2,4,5-T solution was more harmful than the mixture of 2,4-D and 2,4,5-T. The risks of teratogenicity in human civilian use and the role of dioxins are discussed.     

Specificity of 2,4,5-trichlorophenoxyacetate on tetraethylammonium transport.

Renal cortical slices from rats pretreated acutely with 2,4,5-trichlorophenoxyacetate (2,4,5-T) show a reduced ability to transport 2,4-dichlorophenoxyacetate (2,4-D) and tetraethylammonium (TEA). The depression in renal transport is dependent on the 2,4-D or TEA concentration in the incubation medium and is surmountable at high concentrations of 2,4-D or TEA. The steady-state transport of TEA by normal renal slices is inhibited by the organic anion 2,4,5-T, in a dose-related manner. Kinetic analysis of the effect of 2,4,5-T on TEA transport appears to demonstrate that 2,4,5-T is a competitive inhibitor of the steady-state accumulation of TEA. However, the initial influx of TEA was found to be unaffected by 2,4,5-T. The transport of another organic cation, N¹-methylnicotinamide, was depressed only modestly by 2,4,5-T.     

Specific teratogenic action of 2,4,5-trichlorophenoxyacetic acid on mouse and rat whole-embryo cultures

The effects of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) were studied at concentrations ranging from 0.17 to 3.52 m on post-implanted mouse and rat embryos cultured for 48 hr, either in the absence of any metabolic activation system or in the presence of mouse and rat S-9 mix. 2,4,5-T proved to be a potential teratogen, at 0.88 and 1.76 m , on mouse embryos in the absence of any metabolic activation system. In the presence of mouse S-9 mix, 2,4,5-T showed a high teratogenic potential at 0.17, 0.88 and 1.76 m . In contrast, in the presence of rat S-9 mix, 2,4,5-T induced structural defects only at 1.76 m . At 3.52 m , 2,4,5-T was 100% embryolethal with or without S-9 mix. On rat embryos, 2,4,5-T was potentially teratogenic only in the presence of mouse S-9 mix, causing a significant increase in dysmorphogenic effects at 0.17, 0.88 and 1.76 m . With or without rat S-9 mix, 2,4,5,-T was only embryolethal to rat embryos at 1.76 and 3.52 m . The abnormalities mainly involved the forebrain, the midbrain and the branchial arches. These results are consistent with the known in vivo embryotoxic action of this compound.     

A model for the dose-dependent pharmacokinetics of chiorophenoxy acid herbicides in the rat: The effect of enterohepatic recycling

A two-compartment pharmacokinetic model has been developed to describe the time course of chlorophenoxy acid herbicides, such as 2,4-dichlorophenoxy acetic acid (2,4-D), 2,4,5-trichlorophenoxy acetic acid (2,4,5-T), and 2- [2,4,5- trichlorophenoxy]propionic acid (2,4,5-TCPPA), in the plasma, urine, and bile of rats. The model assumes that saturable renal transport and extensive enterohepatic circulation are operative during the distribution and elimination of these compounds. The model was validated by comparing the predicted plasma concentrations and urinary and biliary excretion rates with those reported previously in the literature. The model is capable of predicting the changes in plasma and urinary elimination during bile duct cannulation and suggests that the nonlinear elimination of these compounds is dramatically influenced by enterohepatic circulation.     

Effects of the Herbicide 2,4,5-T on the Incorporation of 14C-Thymidine, 3H-Uridine and 3H-L-Leucine into L 929 Cells

Abstract: A rapid inhibition of the incorporation of ³H-L-leucine, ³H-uridine and ¹⁴C-thymidine into the macromolecular fraction of L 929 cells was found when 2.25 mM 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) was added to the incubation medium. The reduced incorporation of ³H-uridine and ¹⁴C-thymidine could be explained by an inhibitory effect of 2,4,5-T on the uptake of these compounds into the acid-soluble precursor pool, whereas a direct effect on protein synthesis was indicated. Kinetic analysis of the uptake of uridine into the acid-soluble pool indicated that 2,4,5-T inhibited the facilitated transport of this nucleoside into the cell. The overall results are interpreted as indicating that 2,4,5-T induced alterations in the permeability of the cellular plasma membrane.     

Effects of the Herbicide 2,4,5-Trichlorophenoxyacetic Acid on Growth and Morphology of L 929 Cells

Abstract: A dose-dependent inhibition of growth was found when monolayer cultures of L 929 cells were grown in the presence of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) in concentrations from 0.25 to 2.25 mM. At 1.75 and 2.25 mM an almost immediate cessation of growth was found. On removal of the herbicide (2.25 mM) after incubation for 3 or 6 days, cell multiplication was resumed after a lag period of about 24 hours. In the presence of 0.5 to 2.25 mM 2,4,5-T an accumulation of particles in the cytoplasm was observed, and on prolonged incubation in the presence of 2.25 mM 2,4,5-T the cells became rounded and detached from the substratum. By replacing the test medium by the control medium, however, the particles in the cytoplasm disappeared and the cells resumed their fibroblast-like structure.     

Effect of peroxisome proliferators on glutathione-dependent sulphobromophthalein excretion.

1. The effect of pretreatment of rats with the peroxisome proliferator 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) on sulphobromophthalein excretion from the isolated perfused rat liver has been investigated and compared with the effect of clofibrate which is also a peroxisome proliferator. 2. Rats fed 2,4,5-T at a dose of 0.25% in the diet showed a decrease in food intake, compared with controls and clofibrate-fed rats. 3. Treatment with either 2,4,5-T or clofibrate was associated with significant inhibition of the biliary excretion of unchanged, conjugated, and total sulphobromophthalein from perfused rat liver, compared with diet-matched controls. 4. There was a decrease in bile flow in the clofibrate-treated group, but not in the 2,4,5-T-treated group. 5. The results of the present study confirm previous studies that have shown an association between peroxisome proliferation treatment and inhibition of glutathione S-transferase-mediated sulphobromophthalein excretion.     

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.     

2,4,5-Trichlorophenoxyacetic Acid Influence on 2,6-Dinitrotoluene-Induced Urine Genotoxicity in Fischer 344 Rats: Effect on Gastrointestinal Microflora and Enzyme Activity

2,4,5-Trichlorophenoxyacetic acid (2,4,5-T) and 2,6-dinitrotoluene(2,6-DNT) are hazardous chemicals that have potential harmfuleffects. 2,6-DNT is recognized as a hepatotoxicant while 2,4,5-T,a component of Agent Orange, is also suspect. 2,6-DNT requiresboth oxidative and reductive metabolism to elicit genotoxiceffects. To determine what effect 2,4,5-T had on 2,6-DNT metabolism,intestinal enzymes, microbial populations, and urine mutagenicitywere examined during 2,4,5-T treatment. Weanling Fischer 344male rats were treated daily with 54.4 mg/kg 2,4,5-T by gavagefor 4 weeks. One, two, and four weeks after the initial 2,4,5-Tdose, rats were administered (po) 2,6-DNT (75 mg/kg) and urinewas collected for 24 hr in metabolism cages. Azo reductase,nitroreductase, ß-glucuronidase, dechlorinase, anddehydrochlorinase activities were examined concurrently. Treatmentof rats for 1 week reduced the transformation of 2,6-DNT tomutagenic urinary metabolites. This was accompanied by a decreasein the fecal anaerobic microorganisms. The elimination ofLactobacillusfermentum from the small intestine and cecum of treated animalsaccompanied a significant increase in oxygen-tolerant lactobacilliand other unidentified aerobic microorganisms. However, therewere no significant alterations in the intestinal enzyme activitiesexamined. By 2 weeks of 2,4,5-T treatment, microbiota and urinegenotoxicity returned to the levels observed in control animals.This trend continued for the duration of the experiment After2 weeks, while cecal nitroreductase and azo reductase activitiesincreased, small intestinal ß-glucuronidase activitydecreased. By 4 weeks, treated and untreated animal intestinalenzyme activities were indistinguishable. The transient increasein azo reductase and nitroreductase after treatment with 2,4,5-Tfor 2 weeks may have been counteracted by the reduced ß-glucuronidaseactivity, thus resulting in no change in 2,6-DNT-derived urinemutagenicity. However, other environmental chemicals, unaffectedby ß-glucuronidase, potentially could be activatedby 2,4,5-T exposure.     

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.     

Effects of 2,4,5-trichlorophenoxyacetic acid and quinolinic acid on 5-hydroxy-3-indoleacetic acid transport by the rabbit choroid plexus: Pharmacology and electron microscopic cytochemistry

2,4,5-Trichlorophenoxyacetic acid (2,4,5-T) reduced the uptake of 5-hydroxy-3-indoleacetic acid (5-HIAA) by the choroid plexus in a dose-related manner, while treatment with quinolinic acid at comparable concentrations did not inhibit 5-HIAA uptake. The role of carrier-mediated transport in the clearance of 5-HIAA from cerebrospinal fluid (CSF) was also evaluated in vivo by ventriculocisternal perfusion. Steady-state clearance of 5-HIAA from CSF exceeded that of inulin and was reduced competitively in the presence of 2,4,5-T. However, the clearance was not affected by quinolinic acid. The effect of 2,4,5-T on transport enzyme systems was also studied by electron microscopic cytochemistry. Na+-K+-ATPase and cytochrome oxidase activities in the choroid plexus were reduced by 2,4,5-T. Since this transport system in the choroid plexus is normally responsible for the excretion of the serotonin metabolite from the brain to the plasma, accumulation of endogenously produced organic acids in the CSF and the brain, secondary to reduced clearance by the choroid plexus, could be a contributing factor in the development of neurotoxicity.     

Effect of chlorophenoxyacetic acid herbicides on glycine conjugation of benzoic acid.

1. 2,4-Dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) (0.1-0.5 mmol/kg i.p.) delayed the disappearance of injected benzoate from blood and diminished the urinary excretion of the formed benzoylglycine, but elevated the blood levels of benzoylglycine in rat, suggesting that these herbicides interfere with both the formation and the renal transport of benzoylglycine. 2. Inhibition of the renal excretion of benzoylglycine by 2,4-D or 2,4,5-T (0.5 mmol/kg i.p.) was directly demonstrated in rat injected with benzoylglycine. 3. Inhibition of benzoylglycine formation from benzoic acid by 2,4-D or 2,4,5-T (0.5 mmol/kg i.p.) was directly demonstrated in renal pedicles-ligated rats injected with benzoate. 4. Neither 2,4-D nor 2,4,5-T influenced the hepatic concentrations of ATP, coenzyme A (CoA) or glycine; therefore, it is unlikely that they inhibit glycine conjugation of benzoic acid by diminishing the availability of co-substrates. 5. Although the chlorophenoxyacetic acids did not appear to be a substrate for the mitochondrial acyl-CoA synthetases, both 2,4-D and 2,4,5-T diminished the activity of benzoyl-CoA synthetase (but not that of benzoyl-CoA:glycine N-acyltransferase) in solubilized hepatic mitochondria. These findings suggest that 2,4-D and 2,4,5-T impair benzoylglycine formation in rat by inhibiting benzoyl-CoA synthetase.     

Relationship between 2,4,5-trichlorophenoxyacetate and the renal organic base transport system.

At concentrations which did not alter tissue oxygen consumption, 2,4,5-trichlorophenoxyacetate (2,4,5-T) increased the rate of tetraethylammonium (TEA) efflux from renal slices by 35–130 per cent. The efflux of N¹-methylnicotinamide (NMN) was unaffected by 2,4,5-T. The steady-state transport of 2,4,5-T by renal slices was not altered by competitive or irreversible inhibitors of the base transport system. These data and others indicate that, despite 2,4,5-T-TEA interaction, 2,4,5-T is not transported totally or even to a great extent by the renal organic base secretory mechanism. A model is presented that is consistent with all the observations.     

In vitro uptake of organic ions by renal cortical tissue of rats treated acutely with 2,4,5-trichlorophenoxyacetic acid.

The effect of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) on renal cortical function has been studied in male adult rats. Significant decreases in organic acid and organic base transport were measured when rats were pretreated with 2,4,5-T 24 hr before in vitro analysis of renal function. Experiments in which injections of p-aminohippurate were given showed no effect on organic acid or organic base transport by renal cortical slices. The data are interpreted to mean that pretreatment with 2,4,5-T has a depressive influence on the transport of some organic ions. Adult animals which were treated daily with 2,4,5-T accumulated a large body store of this herbicide during the first 6 days of administration. However, by 9 days the animals excreted essentially the dose of 2,4,5-T administered, and by 16 days the herbicide accumulated during the first 6 days had been almost totally eliminated. This chronic excretion pattern may explain why only moderate toxicity has been reported for this herbicide.     

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.     

The in vitro evaluation of the toxicities of three related herbicide formulations containing ester derivatives of 2,4,5-T and 2,4-D using sub-mitochondrial particles

The aim of this investigation was to determine the contribution made by the different components of herbicide formulations to the overall toxicity of the formulations. Three related herbicide formulations were chosen. The first, Agent Orange, consisted only of the butyl esters of 2,4,5-T and 2,4-D. The second was Agent Orange diluted with diesel fuel and the third formulation tested was a tree and blackberry killer, which consisted of the butyl ester of 2,4,5-T, the ethyl ester of 2,4-D, diesel fuel and two surfactants. The potential toxic effects of these three formulations were evaluated by determining their inhibitory effects on the oxidative functions of submitochondrial particles prepared from beef heart mitochondria. The effective concentration that caused a 50% inhibition of the activities of the submitochondrial particles was determined for all three formulations. When the toxicity of the individual components of these formulations was evaluated, it was established that the so-called 'inert' components i.e. diesel fuel and surfactants contributed approximately 50% of the overall toxicity of the complete formulations. Hence the results confirm the importance of evaluating the toxicity of complete formulations, rather than only focussing on the active components. While cellular and sub-cellular assays cannot account for pharmacokinetic and pharmacodynamic changes that may affect the toxicity of xenobiotics, the sub-mitochondrial particle test is useful as an initial screening assay.