**Oxidative Stress as a Possible Mechanism of Toxicity of the Herbicide 2,4- Dichlorophenoxyacetic Acid (2,4-D)**

Bettina Bongiovanni1, Cintia Konjuh1,

Arístides Pochettino1 and Alejandro Ferri2

*1Laboratorio de Toxicología Experimental, Departamento de Ciencias de los Alimentos y Medio Ambiente; 2Departamento de Química Analítica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina* 

#### **1. Introduction**

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904.

Chlorophenoxy herbicides are widely used in agriculture and forestry, for the control of broad-leaved weeds in pastures, cereal crops, as well as along public rights of way. Structurally, these herbicides consist of a simple aliphatic carboxylic acid moiety attached to a chlorine-substituted aromatic ring via an ether linkage. One of the most commonly used herbicides of this type is 2,4-dichlorophenoxyacetic acid (2,4-D) (Fig. 1). In congruence with the similitude between its molecular structure and that of the plant hormone indole-acetic acid, 2,4-D acts as a plant growth regulator that can interfere with normal hormonal action and plant growth (Munro et al., 1992).

Fig. 1. Structure of the 2,4-Dichlorophenoxyacetic Acid.

2.4-D was synthesized for the first time in 1941 and commercially marketed in the United States (U.S.) in 1944 (IARC, 1986) and worldwide since 1950 (Munro et al. 1992). The widespread use of 2,4-D as a domestic herbicide and as a component of Orange Agent encouraged the study of its toxicity.

Human exposure to chlorophenoxy herbicides may occur through inhalation, skin contact or ingestion. The predominant route for occupational exposure to 2,4-D has been the absorption of spills or aerosol droplets through the skin.

Several studies have shown that doses of 50, 70 or 100 mg/kg body weight (bw)/day of 2,4- D produce a wide range of toxic effects on the embryo and on the reproductive and neural

Oxidative Stress as a Possible Mechanism

in some brain regions (Rosso et al., 2000).

which was not observed in well-nourished animals.

1998; Bukowska et al., 2003; Duchnowicz et al., 2002).

(GSH) content (Tables 3, 4 & 5, respectively) (Ferri et al., 2007).

**2.1 Metals and monoamines levels** 

2003).

for each brain region.

**2.2 Oxidative stress** 

CNS.

of Toxicity of the Herbicide 2,4-Dichlorophenoxyacetic Acid (2,4-D) 317

In the last two decades many different alterations have been reported in neonatal rats exposed to 2,4-D through breast milk, at a dose producing no overt signs of toxicity in dams. Alterations in astroglial cytoarchitecture and neuronal function (Brusco et al., 1997) as well as neuro-behavioral changes were observed in pups and adult rats after an early exposition to the herbicide (Bortolozzi et al., 1999, 2001). Other reported effects in neonate rats were a deficit in myelin lipid deposition (Konjuh et al., 2008) and changes in the ganglioside pattern

Studies in well-fed or undernourished rat offsprings showed that the mechanisms for the induction of the above effects would include some changes in brain monoaminergic system (Ferri et al., 2000) and in iron (Fe), copper (Cu) and zinc (Zn) brain levels (Ferri et al.,

Importantly, the combination of neonatal undernourishment plus mothers' exposure at 2,4- D low dose (70 mg/kg bw) induced a higher modification of the measured parameters than those induced by undernourishment or 2,4-D exposure alone. The data showed a different pup's brain areas susceptibility to the 2,4-D effects and an increased vulnerability to the herbicide, including an increased mortality at a higher dose (100 mg/kg bw), a feature

In addition, the results suggest that malnutrition or exposure to 2,4-D exert their effects independently (Tables 1 & 2) (Ferri et al., 2003) and the fact that the alterations observed are very different according to the area involved, reinforces the idea of a selective susceptibility

Different studies suggest some functional relationships between the oxidative status of the Central Nervous System (CNS) and the protecting level of catecholamines (Kumiko et al., 2001) and metals, like Fe and Cu, the major generators of reactive oxygen species –ROS- in Alzheimer's disease (Huang et al., 1999), related with a decreased glutathione (GSH) content (Dringer, 2000) and also involved in Fenton's and Haber Weiss' redox reactions . (Halliwell & Gutteridge, 1998; Milton, 2004). Other data have shown that 2,4-D affects the redox chain, thus altering cell energetic metabolism and redox balance (Palmeira et al., 1994; Sulik et al.,

In rat pups, exposure to 2,4-D through breast milk induced a number of changes in different brain areas, such as disparate changes in the activity of some protective enzymes, an increase in reactive oxygen species (ROS) levels, and a depletion of reduced glutathione

Therefore, as long as a high oxygen consumption by the CNS increases its sensitivity to oxidative stress (Emerit et al., 2004), the observed changes in the levels of metal ions and neurotransmitters, particularly catecholamines, as well as the oxidative status imbalance, would point out oxidative stress as one possible mechanism of adverse 2,4-D effects on the

systems in animal (mostly rat) and human models (Rosso et al., 2000; Barnekow et al., 2001; Charles et al., 2001). Doses of 50 mg/kg bw/day of 2,4-D have been reported to increase ventral prostate weight in rats. Treatment of human prostate cancer cell cultures with 10 nM 2,4-D enhanced the androgenic activity of dihydroxytestosterone (DHT) on cell proliferation and transactivation (Kim et al., 2005). In cultured chinese-hamster ovary cells, 2.0 to 10.0 µg/ml 2,4-D were reported to produce DNA damage and sister chromatid exchange (Gonzalez et al., 2005). Importantly, although the 2,4-D toxicity in low doses is controversial, the U.S. Environmental Protection Agency (U.S. EPA, 2006) established a LD50 of 639 mg/kg based on rat studies.

There could be particular situations in which the susceptibility of a population exposed to environmental pollutants can be dangerously enhanced. This may be the case for many rural populations subjected to some specific nutritional deficiencies, as often observed in developing countries. Such situation may be worthy of attention during the development stage, especially concerning the endocrine and nervous systems.

It has been recently found that 2,4-D administered to lactating rats can pass to suckling pups, an can also inhibit the suckling-induced hormone release in the mother. Thus, gestational and lactational periods –including the neonatal and prepubertal stages– seem to be particularly favorable for the induction of 2,4-D effects in rodents (Stürtz et al., 2000; 2006).
