Animal models in the neurotoxicology of 2,4-D

Abstract

2,4-D is a selective pre- and postemergence herbicide used for several crops. It is hazardous for the environment and risk for humans; therefore, several studies attempt to evaluate its effects and consequences of its use. The nervous system is supposedly a target for this herbicide, and this comprehensive review gathers the information about animal models that have been used for the study of the neurotoxicity of 2,4-D. The studies used several methods to evaluate the neurotoxicity of this herbicide, most of which used rodents, mainly rats, two used fish, and one used chicken eggs. The main behavioral effect observed concerned alterations in locomotor patterns and reduced motor activity. Biochemical analysis showed decreased levels of serotonin (5-HT) and increased levels of its metabolites and increased or decreased levels of DA and its metabolites depending on the brain area analyzed. Hypomyelination is also a possible effect of 2,4-D when the exposure occurs during the proliferation and development of the oligodendrocytes. The worst neuropathologic effects were observed in fish. Since most studies focused on the neurotoxicity of 2,4-D in rodents, the effect it may have on other species and groups of animals, especially with different physiology, is unclear and it should be researched.

Introduction

2,4-D is the International Organization for Standardization name for 2,4-dichlorophenoxyacetic acid. 2,4-D is a selective herbicide, first synthesized in 1941, that acts as an auxin, a plant growth hormone. It is absorbed by roots and leaves, causing excessive growth which leads to death, being more effective in broad-leaved weeds. ^{1 –3} It has pre- and postemergence uses for crops such as rice, coffee, sugarcane, corn, and soybean. ^4,5

It is classified in Brazil as extremely toxic (class I) and highly soluble in water and soil, and this is a hazardous product for the environment (class III) and a risk for humans. ^3,5 The US Environmental Protection Agency considers the 2,4-D to have moderate toxicity to birds and mammals and to be slightly toxic to fish. It is a light-yellow powder used in various commercial formulations, of which the most used is the dimethylamine salt. ^{4 –6}

2,4-D toxicity has been tested in several animal models and many human intoxications have been reported. The nervous system is supposed to be one of the target organs of chlorophenoxy herbicides, such as 2,4-D, and the effects related to loss of motor functions, lethargy, and behavioral alterations have been described. ^{7 –9} The objective of this literature review is to analyze data in respect of the neurotoxicity of 2,4-D in animal models.

Literature review

The research was carried out using three scientific databases: Periódicos CAPES, PubMed, and SciELO. Several searches were made for titles and abstracts containing several permutations of keywords, which were 2,4-D; 2,4-Dichlorophenoxyacetic; neurotoxic; neurotoxicity; and nervous system. The inclusion factors were studies evaluating the toxicity of 2,4-D and the use of animal models. The exclusion factors were scientific papers that did not use live animals (e.g. using cell cultures) and studies that did not address the effect of 2,4-D on the nervous system.

We found 41 articles (excluding redundant titles) and 12 of them fit the criteria stated above. ^{1,2,7 –16} Of the 12 selected articles, 9 studied the effect of 2,4-D on rodents of different strains (CD-1 mice, ⁷ Wistar, ^{8,10 –13} Fischer 344, ^1,14 and Sprague-Dawley rats ¹⁵ ), one of them also used New Zealand white rabbits. ⁷ Two studies evaluated the effect of this herbicide in fish, zebra fish (Danio rerio) ¹⁶ and guppy (Poecilia reticulata), ⁹ and one study used chicken eggs. ²

Oral administration at doses ranging from 60 mg/kg to 300 mg/kg in Wistar rats resulted in reduced locomotion and rearing frequencies as well as reduced motor activity and increased immobility. The effects increased the higher the dose used. ¹⁰ When administered via gavage in corn oil to Fischer 344 rats, at doses of 15–250 mg/kg, the highest dose (250 mg/kg) resulted in incoordination and alterations in gait, mainly on the first day and limb stiffness as well as reduced motor activity, only in the first day. ⁷

For 1 year, 2,4-D was available ad libitum in the diets (weekly for 13 weeks and then every 4 weeks) of Fischer 344 rats at doses of 5–150 mg/kg/day. The effects were observed mostly in females of the highest dose group. The grip strength of the forelimbs increased and there were no differences in motor activity. The retinas of females from the larger dose group had bilateral retinal degeneration at the end of the study, characterized by the loss of photoreceptor cell layers. There were no pathological changes in the nervous system. ⁷

The maternal behavior of Wistar nulliparous rats under 2,4-D oral exposure during the gestational period was evaluated. The doses were approximately 15, 25, and 50 mg/kg/day. The appearance of the dams and the number of pups and their survival were not affected. However, some maternal behaviors were disrupted, such as decreased licking and nursing puppies, totally annulling the licking of the anogenital region of the pups at all doses; latency and length of retrieval of pups; dams leaving the nest; and increased time spent out of it. ¹¹

Fischer CDF 344 rats underwent dermal exposure for 2 h/day 5 days/week, of approximately 150 mg (corresponding to approximately 34.6 mg/kg, according to the pretreatment weight average: 230.4 ± 3.3 g) during 2 weeks and 111 mg (corresponding to approximately 19.7 mg/kg, according to the pretreatment weight average: 177.4 ± 2 g) for 3 weeks and there were no neuropathologic consequences and the grip strength was not affected. ¹⁴

Unilateral intracerebral administration was tested in three regions of the brain of Wistar rats. The results of 100 μg 2,4-D in the striatum were decreased exploratory and motor activities, postural deviation, and moderate ipsilateral circling response. The same amount (100 μg) was injected in the accumbens, which also resulted in decreased exploratory and motor activities, circling response was not observed, but the total turn numbers were reduced; in the medial forebrain bundle (MFB), 50 μg was injected and alterations in locomotion activity were not observed but ipsilateral circling response was observed. ⁸

Behavioral changes were observed in guppies after the exposure to 2,4-D. When administered a dose of 15 mg/L, less general activity and grouping at the aquarium corners were observed after 24 h. At a dose of 30 mg/L, after 12 h of treatment, there was shortness of breath, sudden rotations, and jumping. The effects were more severe at the highest dose of 45 mg/L, and almost 6 h after dosing, loss of equilibrium, sudden movements, grouping around the aeration area, and color loss were observed. ¹⁵

The accumulation and regional distribution of 2,4-D was quantified by labeled [¹⁴C] 2,4-D in New Zealand white rabbits, Sprague-Dawley rats, and CD-1 mice. It was more marked when there was a pre-exposure to the herbicide, with greater effect in increasing doses. A pre-exposure dose of 250 mg/kg injected into the saphenous vein of rats and application of 8 μCi/kg of 2,4-D increased [¹⁴C] presence in the brain and cerebrospinal fluid, as well as in the liver, testes, lungs, heart, and muscles, but activity decreased in the kidneys and plasma, and myotomy and lethargy were also observed in the animals. ¹⁵ For both mice and rabbits (intraperitoneally injected with pretreatment of 0, 40, 80, or 160 mg/kg, the last dose only in rabbits, and treatment of 50 μCi/kg of [¹⁴C] 2,4-D), the areas with greatest accumulation of 2,4-D were the brain stem, cerebellum, and frontal cortex, and the smaller were the hypothalamus and caudate nucleus. In pregnant female mice, the concentration found in the fetal brain was higher than in the maternal but smaller when compared with other tissues and blood plasma. ⁷

It has been found that 2,4-D can be detected in the brain and serum of exposed animals even at a small dose of 10 mg/kg. Through samples of the cerebrospinal fluid and choroid plexus of New Zealand white rabbits, the source of the accumulation of 2,4-D in the brain was checked. There were two hypotheses, increased entry in the brain or failure in its elimination. The blood–brain barrier permeability did not change in CD-1 mice, indicating another mechanism, which was better explained in the rabbits by the competitive inhibition of 2,4-D elimination via active transport in the anionic system of the choroid plexus. ^10,7 This mechanism is likely the same in fish and other species. However, in early development, the susceptibility of 2,4-D infiltration in the brain of embryos is possibly higher until the blood–brain barrier is fully developed. Eutherian animals are protected by the mother during this period, but oviparous animals may be more vulnerable.

In neurochemical studies, 2,4-D at an oral dose of 200 mg/kg in Wistar rats did not affect the levels of dopamine (DA) and homovanillic acid (HVA) in the striatum; however, serotonin (5-HT) levels decreased and 5-hydroxyindoleacetic acid (5-HIAA), a serotonin metabolite, increased after 4-h exposure. In the brain stem, only 5-HIAA levels increased. This indicates an increase in the functional activity of serotonin after the application of 2,4-D. ¹⁰ Maternal behavior alterations in Wistar rats orally exposed to 15, 25, and 50 mg/kg/day are associated with decreased 5-HT and increased DA levels in the arcuate nucleus as well as decreased serum prolactin levels. ¹¹

With the injection of 100 μg of 2,4-D in the striatum of Wistar rats, similar effects were observed concerning 5-HT, though there was a slight increase in HVA in the injected hemisphere. There were no changes in monoamine levels. In rats injected with 100 μg of 2,4-D in the accumbens, HVA and 5-HT increased in the injected hemisphere, whereas in the other hemisphere injected with the vehicle, the levels decreased. When injected into the MFB (50 μg), levels of DA and its metabolites, HVA and dihydroxyphenylacetic acid, in the striatum decreased, 5-HT and 5-HIAA also decreased, but only after 7 days. Injection in the MFB caused more effects on the striatum than intrastriatal application. ⁸

Whether neonatal exposure of 2,4-D throughout lactation alters the myelination of Wistar rats was evaluated. In rats, oligodendrocytes develop after birth and the deposition of myelin occurs rapidly over 2 weeks. 2,4-D was administered in a dose of 100 mg/kg/day in three different periods, 9–25, 9–15, and 15–25 postnatal days. A decrease in total lipids in the brain was observed in the groups that were exposed up to the 25th day, and their composition changed, the cholesterol ester levels highly increased by 263% and phospholipids (38.6% measuring phosphate) and free fatty acid (62.2%) levels decreased, which may alter the fluidity and stability of cell membranes. ¹²

There was hypomyelination in the above rats as a result of reduced galactolipids. ¹² Wistar rats treated with subcutaneous injections of 2,4-D (doses 70 and 100 mg/kg every 48 h in the dorsal region of the neck tested in several groups starting treatment at 7 or 12 postnatal days and ending on days 17 or 25) also showed a significant decrease in ganglioside and DNA levels in groups treated with higher doses for longer times. ¹³

The hypomyelination was further explored by De Moro et al. ⁹ using 2,4-D butyl ester in fertilized chicken eggs. The contamination was topical with 3.1 mg/egg. No changes in development and brain appearance were observed. The deposition of galactolipids has decreased 30–40% due to changes in the level of cerebrosides (42–55%) and sulfatides (32–37%). The cholesterol content, the total number of proteins, and activity of the enzyme CNPase, which is one of the important markers for the synthesis or demyelination of myelin, were significantly lesser than the control over development. The DNA content decreased at first but increased significantly from day 14, resulting in a decreased protein/DNA ratio.

When the treatment is done on chicken eggs from the 15th day, there is no change in the myelin content. ⁹ All this suggests that the vulnerable period is the proliferation and development of the oligodendrocytes, which is a possible target for the action of 2,4-D, either by reducing their capacity for lipid synthesis or by destroying formed myelin. ^9,12,13

Zebra fish is a widely used animal model, and Ton et al. ¹⁶ aimed to establish it as a model for neurotoxicity, testing some compounds in embryos of the species, including 2,4-D. Embryos exposed 6 h postfertilization (hpf), up to 48 or 96 hpf at doses of 50–75 μM (23.87–35.81 mg/L), presented decreased motility, slow heart rate (at 50 μM), and edema of the heart (at 75 μM). These were also present at a dose of 200 μM (95.51 mg/L), as well as short body and hemorrhage. The neurotoxic effects were increased apoptosis, disrupted motor neuron growth, and reduction of axon projections to the optic tectum. 2,4-D is slightly teratogenic in Zebra fish, indicating that its neurotoxicity is specific because it is not toxic for other animals. ¹⁶

There were behavioral and neurotoxic effects on the spinal cord of guppies at three doses (15, 30, and 45 mg/L), exposed for 72 and 96 h. Survival rates after 96 h were 30, 60, and 90%, respectively, and 100% for control. The effects observed were loss of neurons, edema, degeneration of Nissl corpuscles, pyknotic nuclei, gliosis, intercellular spaces, and vacuolization. At the lower doses (15 and 30 mg/L), these effects were mainly mild or absent at 72 h, whereas in 96 h, most became moderate. At the highest dose (45 mg/L), the effects were mild and moderate, and at 72 h, most became severe at 96 h, except vacuolation (moderate). ²

The route of exposure may affect the severity of the effects, given that dermal exposure did not result in any problems for the nervous system, even though there was a prolonged time exposure (weeks), compared with a single oral exposure, which resulted in alterations, mainly behavioral. The dose and animal used are also variables to be considered. The experimental doses varied from 5 mg/kg to 300 mg/kg for oral, dermal, and injected administration in rodents, while the exposure was topical in chicken eggs. For experiments in fish, the dilution of the herbicide was made in water (varying from 15–95 mg/L). Intracerebral administration used very low doses (50–100 μg), and considering the different exposures and doses, it is hard to make a correlation of its effects with other types of administration.

Conclusion

The effect of 2,4-D is dose-dependent, the effects increasing with dose. The main neurotoxic effects observed for 2,4-D were reduced motor activity; alterations in locomotor patterns and disrupted behaviors; hypomyelination; changes in lipids, protein, and DNA levels; changes in serotonin (5-HT), DA, and associated compounds; and histopathological changes.

Labeled 2,4-D was used to show its accumulation and distribution, being more present in the brain stem, cerebellum, and frontal cortex. Its entrance and accumulation in the brain was better explained by anionic system transport and no change in the blood–brain barrier was detected.

Biochemical studies indicated that the levels of 5-HT usually decreased while its metabolite levels increased and that the levels of DA and its metabolites increased or decreased in different brain regions.

Hypomyelination was present when the organisms were exposed during myelination. This was evidenced by a reduction in the levels of lipids due to 2,4-D, some of which are important in myelination. Protein and DNA levels may possibly be reduced as an effect of the herbicide.

Each species may respond differently to exposure to the herbicide, causing different effects. Most neuropathologic effects were observed in fish, which appears to be more sensible to 2,4-D. Among the studied animal models, the majority were rodents, being necessary studies in other species and groups of animals, since they have different metabolism and physiology.