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.     

Toxicological implications of pesticides: their toxic effects on seeds of food plants.

Pesticides are widely used for the protection of economic crops from a variety of noxious pests. The repeated and indiscriminate uses and the extreme stability of certain pesticides have led to their accumulation in plants, animals, soils and sediments, thus effecting widespread contamination of the environment. Soil contaminants are especially serious because they can inhibit or impair the seed germination of our food and feed crops. Seeds can come in close contact with pesticides through processes such as prematurity application, fumigation, seed dressings, and seed treatments. Several reports have indicated the toxic effects of pesticides on seed germination. Possible mechanisms of the toxic action on pesticides during the germination of seeds have been discussed with emphasis on biochemical, histological, and cytological alterations. Bioassay procedures employing seed germination as a smiple, feasible, economical, time-saving indicator of toxicity have been described briefly. Attention is then drawn to the possible potential health hazards arising from the presence of pesticidal chemicals in food plants since the toxicological implications of long term exposure to pesticides are often more far-reaching.     

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.     

Effects of anionic xenobiotics on rat kidney: I—tissue and mitochondrial respiration

The polar 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) metabolite, 2,2-bis p-chlorophenyl)acetic acid (DDA), and the phenoxyacetic acid herbicides, 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), were previously shown to be accumulated to high levels in liver and kidney via the organic acid transport system, raising the possibility of organ-specific toxicity secondary to transport. In these studies, accumulation of DDA was shown to depress oxygen consumption by renal cortical slices at high doses (0.1 and 1 mM). Isolated renal and hepatic mitochondria were uncoupled by low doses of DDA (5–10 nmoles/mg mitochondrial protein). Maximal uncoupling was seen at 50–70 nmoles/mg. 2,4-D and 2,4,5-T also produced uncoupling, but at doses of 70 nmoles/mg or higher. All agents were more effective with α-ketoglutarate or pyruvatemalate as substrate than with succinate. With succinate as substrate (but not α-ketoglutarate or pyruvate-malate), all three agents also depressed State 3 (ADP-stimulated) respiration. Again. DDA was more effective than 2,4-D or 2,4,5-T. These results suggest that accumulation of these or other anionic xenobiotics may lead to toxicity in those tissues possessing the organic anion transport system.     

The fate of 2,4-dichlorophenoxyacetic acid (2,4-D) following oral administration to man

The pharmacokinetic profile of 2,4-D is defined in man. Five male human volunteers ingested a single dose of 5 mg/kg 2,4-D without detectable clinical effects. Concentration of 2,4-D were determined in plasma in 3 of 5 subjects and in urine in all subjects at intervals after ingestion. The elimination of 2,4-D from plasma in all subjects occurred by an apparent first-order rate process with an average half-life ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) of 11.6 h. All subjects excreted 2,4-D in the urine with an average $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 17.7 h. Excretion occurred mainly as 2,4-D (82.3%) with smaller amounts excreted as a 2,4-D conjugate (12.8%). Essentially all of the 2,4-D was absorbed from the gastrointestinal tract in man. Clearance of 2,4-D from the plasma and excretion from the body are first-order rate processes. There was no evidence that 2,4-D would accumulate following repeated administration.     

Evaluation of renal function in saccharin treated rats

Renal function and transport have been examined in male and female rats at various times during a 6-month exposure to sodium saccharin. The animals were maintained on special diets containing 0%, 1%, 5% or 7.5% sodium saccharin. Development patterns for renal transport and renal function in the control animals were normal. The 5% and 7.5% concentrations of saccharin caused a reduction in the renal slice steady-state accumulation of p-aminohippurate (PAH) and tetraethylammonium (TEA) which became significant after the animals were 30 days of age or older. Enhanced sodium excretion was observed in every age group of animals tested when these animals consumed sodium saccharin concentrations ? 1% in the diet. At 60 days of age, increased urine volume, a decrease in urine osmolality, and increased potassium excretion were also found. All effects of saccharin on renal function and transport were promptly and completely reversible when the animals were removed from the saccharin diet for as little as 24 h. The effects of saccharin on PAH transport apparently were attributable to competitive inhibition. Although effects on TEA transport and other renal function parameters were not defined as to mechanism, the effects were as readily reversible as those on PAH when saccharin was removed from the diet.     

Uptake of phenoxyacetic acid derivatives into Caco-2 cells by the monocarboxylic acid transporters

The uptake mechanism of phenoxyacetic acid (PA) and its chlorine derivatives, 4-chlorophenoxyacetic acid (4-CPA), 2,4-dichlorophenoxyacetic acid (2,4-D), and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), was investigated using Caco-2 cells. The cells were incubated with PA, 4-CPA, 2,4-D or 2,4,5-T at pH 6.0 and 37 °C. The order of uptake and lipophilicity expressed by n-octanol partition coefficients were PA < 4-CPA < 2,4-D < 2,4,5-T. Incubation at 4 °C or at pH 7.4 significantly decreased these uptake. Furthermore, pretreatment with the protonophore, carbonylcyanide-p-(trifluoromethoxy) phenylhydrazone, or coincubation with benzoic acid, a typical substrate for the proton-linked monocarboxylic acid transporters (MCTs), significantly decreased the uptake of all compounds. The initial uptake rates of all compounds except PA were apparently saturable, suggesting the involvement of a carrier-mediated process. The order of uptake clearance of the compounds was the same as the order of their uptake and lipophilicity. Preloading of cells with benzoic acid significantly increased their uptake except for PA. These results suggest that the uptake of PA, 4-CPA, 2,4-D and 2,4,5-T from the apical membrane of Caco-2 cells is mediated via common MCTs shared, at least in part, with benzoic acid, and the increase in lipophilicity due to the chlor-substitution may increase uptake via the MCTs.     

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.     

Developmental toxicity studies in rats and rabbits on 2,4-dichlorophenoxyacetic acid and its forms.

The potential for 2,4-D and its salts and esters to induce developmental toxicity was investigated in rats (8 studies) and rabbits (7 studies). Maternal toxicity associated with exposure was dependent on the dose level expressed as 2,4-D acid equivalents. The severity of the maternal effect was correlated to the 2,4-D acid-equivalent dose, with increasing dose levels that exceeded renal clearance causing increasingly more severe maternal effects. In both species, maternal body weight effects began to be manifested at dose levels of 30 mg 2,4-D acid equivalent/kg/day. At higher dose levels (50-75 mg/kg/day in rats and 75-90 mg/kg/day in rabbits), body weights and feed consumption were more severely affected. At dose levels > or =90 mg/kg/day in rats, clinical signs of toxicity (ataxia, muscular stiffness, and decreased motor activity) and mortality were noted. The no-observed-adverse-effect level (NOAEL) for maternal toxicity in both species across the family of 2,4-D salts and esters was approximately 10 mg/kg/day. Significantly decreased fetal body weights and increased fetal variations were seen in rats only at maternally toxic dose levels in excess of 90 mg/kg/day acid equivalent. At maternally toxic doses in rabbits, embryonal and fetal development were essentially unaffected. There were no effect on maternal reproductive measures such as litter size, resorption rates, or fetal body weights, and there was no evidence of teratogenic activity. In summary, equivalent toxicity of the salts and esters is consistent with rapid and complete metabolic conversion to 2,4-D acid. No adverse fetal effects were noted at dose levels that did not also produce evidence of maternal toxicity or exceed renal clearance of 2,4-D indicating that the developing rat and rabbit fetus were not uniquely sensitive to 2,4-D and its forms.     

Teratogenic effects of methyl parathion in developing chick embryos.

The effect of methyl parathion (MP) on developing chick embryos was investigated. The embryos were exposed at different stages of development to various doses of MP via the yolk sac route. This resulted in retarded growth which included reduced body weight and reduced body length and length of leg bones. Other teratogenic signs observed were short neck, muscular hypoplasia of legs, abdominal hernias and haemorrhagic spots in brain and upper body. These findings suggest that MP was a teratogen when injected into the yolk sacs of developing embryos.     

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.     

Induction of rat hepatic long-chain acyl-CoA hydrolases by various peroxisome proliferators

Induction of cytosolic long-chain acyl-CoA hydrolases was investigated in rat liver after administration of various peroxisome proliferators and related compounds. Treatment of rats with di-(2-ethylhexyl)-phthalate, di-(2-ethylhexyl)-adipate or tiadenol induced hydrolases I and II, while acetylsalicylic acid induced only hydrolase II. Among the various phenoxyacetic acid derivatives and related compounds, 2,4,5-trichlorophenoxyacetic acid, 2-(4-chlorophenoxy)-2-methylacetic acid, 2-(2-chlorophenoxy)-2-methylpropionic acid and clofibric acid induced both hydrolases I and II, whereas 2, 4-dichlorophenoxyacetic acid induced only hydrolase II. All nine of the above-mentioned inducers also markedly increased the activity of peroxisomal beta-oxidation. Other compounds tested (2-chlorophenoxyacetic acid, 4-chlorophenoxyacetic acid, 4-chlorophenol, phenoxyacetic acid and phenoxy-2-methylacetic acid) were ineffective as inducers. These results suggest that inducers of acyl-CoA hydrolase II also enhance peroxisomal beta-oxidation activity, but do not necessarily induce acyl-CoA hydrolase I. The structure-inducing activity relationships of these compounds are discussed.     

Teratogenic and antiteratogenic effects of nicotinamide derivatives in chick embryos

Teratogenic and antiteratogenic effects of nine nicotinamide analogs in chick embryos were investigated. Further, the teratisms of 6-aminonicotinamide (6-AN), nicotinamide analogs, and an organophosphate (diazinon) were compared. White leghorn chick embryos were used. Agents were injected into the yolk of eggs on d 3 of incubation. Morphological observations were made on d 17 of incubation. Chemical names for compounds I to IX are: I, 6-dimethylaminonicotinamide; II, 6-diethylaminonicotinamide; III, 6-methylamino-3-(N-methyl)-nicotinamide; IV, 6-dimethylamino-3-pyrimidine carboximide; V, 6-(dimethylamino)-nicotinic acid; VI, 6-chloro-3-[N-(5-diethylamino)-2-pentyl]-nicotinamide; VII, 6-mercaptonicotinamide; VIII, [N-acetyl-N'-(3-pyridyl)-carbonyl]-hydrazine; IX, nicotinamide 1-N-oxide. The LD50 values in mumol per egg were as follows: 6-AN, 0.073; compound II, 0.23; compound III, 1.11; compound I, 1.32; compound VI, about 3. Compounds IV, V, VII, VIII, and IX showed no toxicity or lethality at the highest doses tested (10 mumol/egg). Among the nine nicotinamide analogs, compounds I, II, and III, which have an amino group at the 6-position of the pyridine ring, were teratogenic. Their teratogenic signs were similar to those caused by 6-AN: they showed growth retardation, anteriorly directed short legs, and coarse, dense feathering. The teratogenic effects of compounds I, II, and III were prevented by pretreatment with nicotinamide, as were the effects of 6-AN and diazinon. Among the nine analogs, only compound VIII had an antiteratogenic effect against the diazinon-induced micromelia (in which the cardinal signs were tibiotarsal angulations and poor feathering). For teratisms produced by 6-aminonicotinamide analogs and organophosphates, nicotinamide was an effective antiteratogenic agent. However, some differences in the malformations induced by both types of agents were found. We suggest that the addition of a 3-acetylpyridine type to the nicotinamide-related teratisms (6-AN type, 3-acetylpyridine type, and organophosphate type) will provide a clearer distinction among the types.     

Effects of diazinon on nucleotide and amino acid contents of chick embryos. Teratogenic considerations

The effects of diazinon (DZN), an organophosphorus (OP) insecticide, and nicotinamide (Nam) on the pyridine nucleotide, purine and pyrimidine ribonucleotides, and free alpha amino acid contents of chick embryos were determined. The teratogen (DZN) and/or the anti-teratogen (Nam) were administered by the intravitelline route to chicken eggs at day 3 of incubation, and nucleotide and amino acid analyses were made on acid-soluble extracts of homogenates of the embryos at day 10. The results show that the amounts of both the oxidized and reduced forms of NAD and NADP were decreased by the insecticide and restored by Nam. The amounts of the purine and pyrimidine ribonucleotides in the embryos were also decreased by DZN but their changes were proportionately not as great as those of the pyridine nucleotides. The levels of the purine and pyrimidine ribonucleotides were also wholly or partially restored by Nam. Neither DZN nor Nam had any effect on the "energy charge" of the embryos. The levels of free tryptophan (TRP) and histidine (HIS) were decreased by DZN while the levels of threonine (THR) and aspartic acid (ASP) were increased. All other amino acid levels remained virtually unchanged in response to DZN or Nam. Based upon these findings, a possible involvement of TRP in OP insecticide-induced micromelia, parrot beak, and abnormal feathering in chick embryos is considered.     

Pesticides and other chemical residues in Dutch total diet samples (June 1976-July 1978)

Over a period of 2 yr 126 different food items of a market basket of 16-18-yr old males were purchased every 2 months. The foodstuffs were prepared as for eating, and were combined in 12 commodity groups. Twelves samples of each food group were homogenized and analysed for 78 different chemicals, including pesticides, PCBs, bromine, heavy metals, arsenic and selenium. Thirty-four of these chemicals were detected in the various samples and the means and ranges of residue concentrations found in each food group are reported. Most chemicals were found in concentrations below the Dutch residue tolerance limits, the two exceptions were omethoate and carbendazim. Using the concentrations found in the total diet samples the daily intakes of the various chemicals were calculated. The daily intake figures were evaluated with the aid of the Acceptable Daily Intakes (ADI) recommended by FAO/WHO. For practically all chemicals examined the mean and the maximum intakes were well below the ADI.     

Interaction of chlorophenoxyalkyl acid herbicides with rat-liver glutathione S-transferases

The in vitro interaction of four chlorophenoxyalkyl (CPA) acid herbicides with rat-liver glutathione S-transferase (GST) was studied using reduced glutathione and 1-chloro-2,4-dinitrobenzene as substrates. Inhibition of GST activity by the CPA acids in crude extracts was dose dependent. Ring substitution and side-chain length were shown to be of importance in determining the extent of GST inhibition. While GST AA, an isoenzyme of GST, was stimulated by two CPA acids, each of the other GST isoenzymes (A, B, C, E and M) was inhibited, to different degrees. Kinetic studies revealed a mixed type inhibition of the isoenzymes. Conjugates of CPA acids with glutathione were not formed. These results indicate that CPA acids interact with GST by binding directly to these proteins, possibly at a different locus from that of the substrate. The binding of CPA acids to GST may, therefore, have a protective function against these herbicides.     

Tissue-specific induction of oxidative stress in goldfish by 2,4-dichlorophenoxyacetic acid: Mild in brain and moderate in liver and kidney

This study investigated the effects of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) on free radical-related processes in tissues of goldfish given 96 h exposures to 1, 10 or 100 mg/L of 2,4-D as well as 96 h recovery from the 100 mg/L treatment. In liver, 2,4-D exposure increased levels of protein carbonyls and lipid peroxides by 36–53% and 24–43%, respectively, but both parameters reverted during recovery, whereas in brain glutathione status improved in response to 2,4-D. Lipid peroxide content in kidney was enhanced by 40–43% after exposure to 2,4-D with a decrease during recovery. Exposure to 2,4-D also reduced liver acetylcholinesterase activity by 31–41%. The treatment increased catalase activity in brain, but returned it to initial levels after recovery. In kidney, exposure to 100 mg/L of 2,4-D caused a 33% decrease of superoxide dismutase activity. Thus, goldfish exposure to 2,4-D induced moderate oxidative stress in liver and kidney and mild oxidative stress in brain.