**3. Prostate, ovary and breast**

The endocrine system of many vertebrate embryos seems to be particularly susceptible to a variety of substances or either natural or anthropogenic origin, including pesticides (Crews et al., 2000). However, there are few studies on developmental toxicology that focus on the 2,4-D's effects on hormone-sensitive organs such as the prostate, ovary and breast.

Free radicals are associated with oxidative stress and are also thought to play some significant roles in reproduction. Induction of oxidative stress by many environmental contaminants—such as pesticides—has also been pointed out during the last decade as a possible mechanism of some toxic effects on the reproductive system (Bagchi et al., 1992; Abdollahi et al., 2004). It is already known that reproductive cells and tissues will remain stable only when antioxidant and oxidant status are in balance (Lee et al., 2010). ROS levels are a double-edged sword, as long as they not only serve as key signal molecules in physiological processes, but also have a role in pathological processes involving the female reproductive tract (Agarwal et al., 2005).

On the other hand, there are diverse environmental chemical contaminants which can be potentially harmful to the mammary gland in association with estrogens. Oxidative catabolism of both estrogen and those compounds, a mechanism mediated by the same enzymes, generates reactive free radicals that can cause oxidative damage. Xenobiotic chemicals may exert their pathological effects through generation of reactive free radicals (Mukherjee et al., 2006).

There is growing evidence that free radicals can exert a wide spectrum of deleterious effects on the reproductive system and asocciated glands (Saradha et al., 2008). Thus, Pochettino et al. (2010) investigated the effect of 2,4-D on oxidative stress and antioxidative system and on some hormone-sensitive organs such as ventral prostate, ovaries and breasts, exposed to the herbicide during the pre- and the postnatal period, as described next (Pochettino et al., 2010).

#### **3.1 Prostate**

In rat ventral prostate, 2,4-D caused oxidative stress during the whole development, through a significant increase in lipid peroxides, hydroxyl radical levels and protein oxidation. Morevover, the antioxidant enzyme activity was increased at any age, as shown for Glutathione S-transferase (GST), catalase (CAT) and selenium-glutathione peroxidase (Se-GPx), with the exception of Se-GPx administered at the 90th postnatal day (PND 90). Nevertheless, at PND 90 a reduced activity of Glutathione Reductase (GR) was detected (Table 6).

GST is relevant to detoxification of endogenous compounds and xenobiotic substances such as environmental pollutants, drugs, and natural toxins (Pietsch et al., 2001; Padros et al., 2003; Cazenave et al., 2006). Several studies have demonstrated that enhanced GST activity by ROS in the testis could represent an adaptative response to oxidative stress, probably targeted to achieve a detoxification of peroxide-containing metabolites (Kaur et al., 2006).

As far as the testis is intimately related to the prostate, this interpretation looks coherent with the observed ROS-induced increase in GST activity in the prostate.


Hydroxy radical are expresed as 2,3 dihydroxybenzoic acid/salicilc acid rario; carbonyl groups and total thiols are expresed as micromol per miligram of protein; MDA is expresed as nanomol per microgram of protein. GST, CAT and GR activities are expresed as Units per miligram of protein; and Se-GPx is expresed as miliUnits per miligram of protein. Each value is the mean ± SEM. Values between brackets are % of increase () or decrease (); \*p < 0.05, n= 6/group. 70 mg 2,4-D/kg cw of mother. Abbreviations as in the text.

Table 6. Oxidative parameters in ventral prostate.

Therefore, the 2,4-D-induced increase in all ROS level, lipid peroxidation and protein oxidation may have caused some critical oxidative stress in ventral prostate. Nevertheless, the increased activity of some antioxidant enzymes in the prostate could have not been strong enough as to counteract the oxidative stress produced by the herbicide at different stages of rat development. Moreover, it is not a general rule that increase in oxidative species stimulates antioxidant activity (Celik & Tuluce, 2007).

#### **3.2 Ovary**

320 Herbicides – Properties, Synthesis and Control of Weeds

The endocrine system of many vertebrate embryos seems to be particularly susceptible to a variety of substances or either natural or anthropogenic origin, including pesticides (Crews et al., 2000). However, there are few studies on developmental toxicology that focus on the

Free radicals are associated with oxidative stress and are also thought to play some significant roles in reproduction. Induction of oxidative stress by many environmental contaminants—such as pesticides—has also been pointed out during the last decade as a possible mechanism of some toxic effects on the reproductive system (Bagchi et al., 1992; Abdollahi et al., 2004). It is already known that reproductive cells and tissues will remain stable only when antioxidant and oxidant status are in balance (Lee et al., 2010). ROS levels are a double-edged sword, as long as they not only serve as key signal molecules in physiological processes, but also have a role in pathological processes involving the female

On the other hand, there are diverse environmental chemical contaminants which can be potentially harmful to the mammary gland in association with estrogens. Oxidative catabolism of both estrogen and those compounds, a mechanism mediated by the same enzymes, generates reactive free radicals that can cause oxidative damage. Xenobiotic chemicals may exert their pathological effects through generation of reactive free radicals

There is growing evidence that free radicals can exert a wide spectrum of deleterious effects on the reproductive system and asocciated glands (Saradha et al., 2008). Thus, Pochettino et al. (2010) investigated the effect of 2,4-D on oxidative stress and antioxidative system and on some hormone-sensitive organs such as ventral prostate, ovaries and breasts, exposed to the herbicide during the pre- and the postnatal period, as described next (Pochettino et al.,

In rat ventral prostate, 2,4-D caused oxidative stress during the whole development, through a significant increase in lipid peroxides, hydroxyl radical levels and protein oxidation. Morevover, the antioxidant enzyme activity was increased at any age, as shown for Glutathione S-transferase (GST), catalase (CAT) and selenium-glutathione peroxidase (Se-GPx), with the exception of Se-GPx administered at the 90th postnatal day (PND 90). Nevertheless, at PND 90 a reduced activity of Glutathione Reductase (GR) was detected

GST is relevant to detoxification of endogenous compounds and xenobiotic substances such as environmental pollutants, drugs, and natural toxins (Pietsch et al., 2001; Padros et al., 2003; Cazenave et al., 2006). Several studies have demonstrated that enhanced GST activity by ROS in the testis could represent an adaptative response to oxidative stress, probably targeted to achieve a detoxification of peroxide-containing metabolites (Kaur et al., 2006).

As far as the testis is intimately related to the prostate, this interpretation looks coherent

with the observed ROS-induced increase in GST activity in the prostate.

2,4-D's effects on hormone-sensitive organs such as the prostate, ovary and breast.

**3. Prostate, ovary and breast** 

reproductive tract (Agarwal et al., 2005).

(Mukherjee et al., 2006).

2010).

**3.1 Prostate** 

(Table 6).

The complex ovarian structure varies widely during differentiation. Free radicals play important regulating roles during the ovarian follicular cycle, possibly through inhibition of steroid production (Behrman et al., 2001). There is also a delicate balance between ROS and antioxidant enzymes in the ovarian tissues (Agarwal et al., 2005). Non-physiological effects of free radicals include premature ovarian follicular atresia via cell apoptosis. Many pesticides— e.g. the xenoestrogen pesticide methoxychlor — can induce oxidative stress and apoptosis in the ovary (Gupta et al., 2006). Moreover, clinical studies have reported increased levels of reactive oxygen species associated to a decreased female fertility (Agarwal et al., 2006).

Oxidative Stress as a Possible Mechanism

Table 8. Oxidative parameters in breast

against oxidants (Amstad et al., 1991).

glutathione disulfide (GSSG) to GSH (Kim et al., 2010).

gland is more sensitive to xenobiotics at these stages of development.

2,4-D induces cytotoxic effects and apoptosis in HepG2 cells.

**Hydroxyl radical** 

**Carbonyl groups** 

**Total Thiols** 

in the text.

**4.** *In vitro* **studies** 

of Toxicity of the Herbicide 2,4-Dichlorophenoxyacetic Acid (2,4-D) 323

**Control** 2.61±0.11 3.04±0.11 4.31±0.45 **2,4-D** 2.65±0.44 3.49±0.52 4.49 ± 0.11

**Control** 19.25±0.82 28.57±3.86 59.38±10.69 **2,4-D** 21.34±5.47 23.31±5.49 57.37±14.89

**Control** 942±5 1072±77 3551±757 **2,4-D** 951±25 667±46\* (62%) 1560±226\* (56%)

**2,4-D** 70.65±7.48\* (34%) 139.2±17.94\* (96%) 217.8±18.95\* (37%)

**2,4-D** 10.41±1.91\* (40%) 13.54±0.92\*(30%) 32.35±5.98\* (55%)

**2,4-D** 62.55±1.57 81.27 ± 2.55\* (41%) 122.11±17.42\* (66%)

**2,4-D** 198±19\* (63%) 695±15\* (51%) 2257±474\* (60%)

**2,4-D** 7.39±1.54\* (53.6%) 60.02±9.05\* (34%) 76.02±10.95\* (67%)

**MDA Control** 52.62±1.57 71.07±4.68 158.41±2.59

**GST Control** 17.18±0.59 19.41±1.51 72.81±7.41

**CAT Control** 59.38±3.03 137.62±10.73 358.21±36.31

**Se-GPx Control** 538±44 1430±31 5596±1015

**GR Control** 15.94±0.91 90.75±5.51 228.81±14.31

The parameters are expresed as in Table 7. Each value is the mean ± SEM. Values between brackets are % of increase () or decrease (); \*p < 0.05, n= 6/group. 70 mg 2,4-D/kg cw of mother. Abbreviations as

Therefore, the decreased activity of anti-oxidative enzymes may decrease the protection

In that regard, Dimitrova et al. (1994) suggested that the superoxide radicals, either by themselves or after transformation to H2O2, stimulate cysteine oxidation and inhibit the activity of the enzymes. Furthermore, Regoli & Principato (1995) demonstrated that the flux of superoxide radicals inhibits CAT activity. Consequently, the decreased CAT activity might have reflected a flux of superoxide radicals promoted by 2,4-D. Moreover, GR also plays an important role in cellular antioxidant protection, catalyzing the reduction of

Thus, the decrease in thiol groups could reflect GSH depletion in the breast. Therefore, 2,4-D produced oxidative imbalance, mainly during puberty and adulthood, probably because the

It has been observed that 2.4-D concentrations of 1 to 2 mM impaired neurite outgrowth, disrupted the cytoskeleton, and disorganized the Golgi apparatus in cultured cerebellar granule cells (CGC) (Rosso et al., 2000). Futhermore, Kaioumuva et al. (2001b) have demonstrated that the dimethylamonium salt of 2,4-D (DMA 2,4-D) at 0.1 to 5 mM induces apoptosis in a dose- and time-dependent pattern in peripheral blood lymphocytes of healthy individuals and in Jurkat cells. Whereas, Tuschl & Schwab (2003) showed that 4 to 16 mM

**PND 45 PND 60 PND 90** 


The parameters are expresed as in Table 7. Each value is the mean ± SEM. Values between brackets are % of increase () or decrease (); \*p < 0.05, n= 6/group. 70 mg 2,4-D/kg cw of mother. Abbreviations as in the text.

Table 7. Oxidative parameters in ovary.

On analyzing the 2,4-D toxic effects on the ovary, Pochettino et al. (2010) found an increase in lipid peroxide (LPO) evidenced by augmented levels of malondialdehyde (MDA) and decrease antioxidant enzyme activity. These effects differed with age, while an increase in Se-GPx activity was exceptionally observed at all ages (Table 7). These effects could reflect the natural diversity of rat ovarian cell types at different ages. Another explanation would be the well-known, protecting effect of estrogens against apoptosis and oxidative stress in a variety of tissues and cells (Spyridopoulos et al., 1997; Tomkinson et al., 1997; Garcia-Segura et al., 1998; Pelzer et al., 2000). Estrogens increase all ovarian weight, follicular growth, and the mitotic index of granulose cells, and also control granulosa cell apoptosis (Richards, et al., 1980; Bendell & Dorrington, 1991) and have exerted varied antioxidant effects (Chatterjee & Chatterjee 2009). Further studies are needed to analyze the time-course of the effects observed.

#### **3.3 Breast**

Pocchetino et al. (2010) observed that 2,4-D increased MDA levels at all ages (Table 8). It is known that MDA reflects the extent of oxidant status and is considered a good marker of oxidative stress (Wen et al., 2006). Both, singlet oxygen and hydroyl radicals have a high potential to initiate free-radical chain reactions in lipid peroxidation (Celik & Tuluce, 2007). As the hydroyl radical level was unchanged in that study, 2,4-D could have stimulated LPO by increasing singlet oxygen levels. In addition, 2,4-D inhibited the activity of anti-oxidative enzymes such as CAT, Se-GPx, GR and GST (Table 9).

**Control** 3.65±0.26 1.89 ± 0.22 1.09 ± 0.13 **2,4-D** 8.75±0.89 1.98 ± 0.13 4.35 ± 0.53\* (93%)

**Control** 14.77±2.75 5.74 ± 0.13 6.22 ± 0.94 **2,4-D** 23.71±0.47\* (60%) 8.80±0.72\* (55%) 5.64±0.73

**Control** 1462±162 672±24 519±38 **2,4-D** 1360±176 676±39 537±22

**Control** 87.71±14.02 34.12±2.24 34.68±1.31 **2,4-D** 192.5±17.8\* (119%) 42.49±1.35\* (24%) 39.39±0.89\* (14%)

**Control** 38.51±0.41 10.59±0.81 10.99±0.18 **2,4-D** 25.15±1.37 (34.6%) 7.97±0.54\* (24.7) 9.91±0.57

**Control** 42.89±3.14 27.86±1.08 16.08± 0.42 **2,4-D** 43.41±0.67 15.34±0.43\* (44.9%) 16.38±0.71

**Control** 691±97 411±48 514±29 **2,4-D** 1622±117\* (135) 549±24\* (33%) 593±23\* (15%)

**Control** 14.48±3.44 17.15±1.67 28.64±2.31 **2,4-D** 12.67±2.61 16.88±1.45 19.62±1.75\* (31%)

The parameters are expresed as in Table 7. Each value is the mean ± SEM. Values between brackets are % of increase () or decrease (); \*p < 0.05, n= 6/group. 70 mg 2,4-D/kg cw of mother. Abbreviations as

On analyzing the 2,4-D toxic effects on the ovary, Pochettino et al. (2010) found an increase in lipid peroxide (LPO) evidenced by augmented levels of malondialdehyde (MDA) and decrease antioxidant enzyme activity. These effects differed with age, while an increase in Se-GPx activity was exceptionally observed at all ages (Table 7). These effects could reflect the natural diversity of rat ovarian cell types at different ages. Another explanation would be the well-known, protecting effect of estrogens against apoptosis and oxidative stress in a variety of tissues and cells (Spyridopoulos et al., 1997; Tomkinson et al., 1997; Garcia-Segura et al., 1998; Pelzer et al., 2000). Estrogens increase all ovarian weight, follicular growth, and the mitotic index of granulose cells, and also control granulosa cell apoptosis (Richards, et al., 1980; Bendell & Dorrington, 1991) and have exerted varied antioxidant effects (Chatterjee & Chatterjee 2009). Further studies are needed to analyze the time-course of the effects observed.

Pocchetino et al. (2010) observed that 2,4-D increased MDA levels at all ages (Table 8). It is known that MDA reflects the extent of oxidant status and is considered a good marker of oxidative stress (Wen et al., 2006). Both, singlet oxygen and hydroyl radicals have a high potential to initiate free-radical chain reactions in lipid peroxidation (Celik & Tuluce, 2007). As the hydroyl radical level was unchanged in that study, 2,4-D could have stimulated LPO by increasing singlet oxygen levels. In addition, 2,4-D inhibited the activity of anti-oxidative

**Hydroxyl radical** 

**Carbonyl groups** 

**Total Thiols** 

**MDA** 

**GST** 

**CAT** 

**Se-GPx** 

in the text.

**3.3 Breast** 

Table 7. Oxidative parameters in ovary.

enzymes such as CAT, Se-GPx, GR and GST (Table 9).

**GR** 

**PND 45 PND 60 PND 90** 


The parameters are expresed as in Table 7. Each value is the mean ± SEM. Values between brackets are % of increase () or decrease (); \*p < 0.05, n= 6/group. 70 mg 2,4-D/kg cw of mother. Abbreviations as in the text.

Table 8. Oxidative parameters in breast

Therefore, the decreased activity of anti-oxidative enzymes may decrease the protection against oxidants (Amstad et al., 1991).

In that regard, Dimitrova et al. (1994) suggested that the superoxide radicals, either by themselves or after transformation to H2O2, stimulate cysteine oxidation and inhibit the activity of the enzymes. Furthermore, Regoli & Principato (1995) demonstrated that the flux of superoxide radicals inhibits CAT activity. Consequently, the decreased CAT activity might have reflected a flux of superoxide radicals promoted by 2,4-D. Moreover, GR also plays an important role in cellular antioxidant protection, catalyzing the reduction of glutathione disulfide (GSSG) to GSH (Kim et al., 2010).

Thus, the decrease in thiol groups could reflect GSH depletion in the breast. Therefore, 2,4-D produced oxidative imbalance, mainly during puberty and adulthood, probably because the gland is more sensitive to xenobiotics at these stages of development.

### **4.** *In vitro* **studies**

It has been observed that 2.4-D concentrations of 1 to 2 mM impaired neurite outgrowth, disrupted the cytoskeleton, and disorganized the Golgi apparatus in cultured cerebellar granule cells (CGC) (Rosso et al., 2000). Futhermore, Kaioumuva et al. (2001b) have demonstrated that the dimethylamonium salt of 2,4-D (DMA 2,4-D) at 0.1 to 5 mM induces apoptosis in a dose- and time-dependent pattern in peripheral blood lymphocytes of healthy individuals and in Jurkat cells. Whereas, Tuschl & Schwab (2003) showed that 4 to 16 mM 2,4-D induces cytotoxic effects and apoptosis in HepG2 cells.

Oxidative Stress as a Possible Mechanism

**CAT** 30.97 ± 1.26 15.80 ± 1.23\*

**Se-GPx** 9.71 ± 1.20 39.75 ± 2.90\*

culture for 24 or 48 h in presence or ausence of 1 mM 2,4-D.

or decrease (); \*p < 0.001, n= 10/group. Abbreviations are indicated in the text.

**Enzimes** 

al., 1999).

2003).

(Bongiovanni et al., 2011).

of Toxicity of the Herbicide 2,4-Dichlorophenoxyacetic Acid (2,4-D) 325

**(Zn,Cu) SOD** 10.43 ± 1.23 10.56 ± 1.45 8,49 ± 1,20 7.05 ± 1.65 **(Mn) SOD** 4.45 ± 0,88 5.97 ± 1.90 2,86 ± 1,90 1.98 ± 1.00

Parameters are expresed as Units per miligram of protein. Values between brackets are % of increase ()

On using a PC-12 cell model, other authors have been previously shown that a depletion of mitochondrial and cytoplasmatic GSH results in increased ROS levels, disruption of the mitochondial transmembrane potential, rapid loss of mitochondial function, decrease in the ATP concentration, and eventually a higher cell death rate (Nieminen et al., 1995; Wüllner et

Therefore, the alteration in oxidative parameters suggest that the possible mechanisms of chlorophenoxy herbicide toxicity could involve dose-dependent cell membrane damage, uncoupling of oxidative phosphorylation, acetylcoenzyme disruption (Bradberry et al., 2000), and an indirect disruption of mitochondrial transmembrane potential which may lead to caspase inactivation (Kaioumova et al., 2001a). Mitochondrial structural modifications and increased permeability of the pores were also reported in association with a ROS increase (Belizário et al., 2007). In contrast, other studies suggest that 2,4-D cytotoxic effects are exerted by apoptosis induction via a direct effect on mitochondria (Tuschl & Schwab,

In this regard, Bongiovanni et al. (2011), in agreement with De Moliner et al. (2002), demonstrated that 2,4-D induces apoptosis and necrosis in CGC. While De Moliner et al. (2002) showed that 2,4-D-induced apoptosis is associated with and increase in caspase-3 activity preceded by cytochrome-c release from mitochondria, the quantification of ultrastructural changes showed that 1 mM 2,4-D stimulated neuronal death. As much as 49% of necrotic cells and 20% of apoptotic cells were observed, while only 31% of CGC presented normal growth with respect control group (p<0.001; Fig. 3 compared with Fig. 4)

Table 10. CAT, SODs and GPx activities (means ± SEM) in rat cerebellar granule cells in

**24 h 48 h Control 1 mM 2,4-D Control 1 mM 2,4-D** 

( 49%) 15.82 ± 1.59 6.52 ± 0.83\*

( 309%) 12.73 ± 1.75 33.73 ± 4.31\*

( 59%)

(165%)

In rat CGC, either 1 or 2 mM 2,4-D induced similar increases of cellular death. The herbicide decreased significantly mean neuronal survival (46.4%) after 48 h, while no affect was observed after 24 h of treatment (Bongiovanni et al., 2007, 2011) (Fig. 2).

Fig. 2. Effect of 2,4-D on rat cerebellar granule cell viability. Cell cultures were incubated for 24 or 48 h in presence or ausence of 1 mM 2,4-D. Values are means ± SEM; \* indicates p< 0.001 vs. control group; n= 10/group.

Bongiovanni et al. (2007, 2010) studied oxidative stress as a possible mechanism of toxicity aiming to elucidate the mechanism of death induction by 2,4-D. Oxidative stress parameters were altered: ROS level and Se-GPx activity increased whereas CAT activity decreased at both treatment times (24 and 48 h). GSH content was reduced only after 48 h of 2,4-D treatment. However, neither Mn-SOD nor Cu,Zn-SOD activities nor reactive nitrogen species (RNS) levels were affected (Tables 9 & 10). Interestingly, although the oxidative parameters evaluated were modified at the two time-limits studied, the cell viability only decreased at 48 h of treatment. This finding could be explained by a time dependency of this latter alteration.


Parameters are expresed as micrograms per miligram of protein. Values between brackets are % of increase () or decrease (); \*p < 0.001, n= 10/group. Abbreviations are indicated in the text.

Table 9. ROS, RNA and GSH levels (means ± SEM) in rat cerebellar granule cell in culture for 24 or 48 h in presence or ausence of 1 mM 2,4-D.

In rat CGC, either 1 or 2 mM 2,4-D induced similar increases of cellular death. The herbicide decreased significantly mean neuronal survival (46.4%) after 48 h, while no affect was

\*

**Control 1 mM 2,4-D** 

Fig. 2. Effect of 2,4-D on rat cerebellar granule cell viability. Cell cultures were incubated for 24 or 48 h in presence or ausence of 1 mM 2,4-D. Values are means ± SEM; \* indicates

Bongiovanni et al. (2007, 2010) studied oxidative stress as a possible mechanism of toxicity aiming to elucidate the mechanism of death induction by 2,4-D. Oxidative stress parameters were altered: ROS level and Se-GPx activity increased whereas CAT activity decreased at both treatment times (24 and 48 h). GSH content was reduced only after 48 h of 2,4-D treatment. However, neither Mn-SOD nor Cu,Zn-SOD activities nor reactive nitrogen species (RNS) levels were affected (Tables 9 & 10). Interestingly, although the oxidative parameters evaluated were modified at the two time-limits studied, the cell viability only decreased at 48 h of treatment. This finding could be explained by a time dependency of this

**RNS** 7.45 ± 1.13 8.23 ± 1.85 4.82 ± 0.27 6.05 ± 0.47

**GSH** 2.408 ± 0.09 2.225 ± 0.09 1.508 ± 0.061 1.125 ± 0.031\*

Parameters are expresed as micrograms per miligram of protein. Values between brackets are % of increase () or decrease (); \*p < 0.001, n= 10/group. Abbreviations are indicated in the text.

Table 9. ROS, RNA and GSH levels (means ± SEM) in rat cerebellar granule cell in culture

**24 h 48 h Control 1 mM 2,4-D Control 1 mM 2,4-D** 

( 123%) 2.28 ± 0.35 4.13 ± 0.32\*

( 81%)

( 25%)

observed after 24 h of treatment (Bongiovanni et al., 2007, 2011) (Fig. 2).

p< 0.001 vs. control group; n= 10/group.

**ROS** 1.03 ± 0.25 2.30 ± 0,22\*

for 24 or 48 h in presence or ausence of 1 mM 2,4-D.

latter alteration.

**Parameters** 


Parameters are expresed as Units per miligram of protein. Values between brackets are % of increase () or decrease (); \*p < 0.001, n= 10/group. Abbreviations are indicated in the text.

Table 10. CAT, SODs and GPx activities (means ± SEM) in rat cerebellar granule cells in culture for 24 or 48 h in presence or ausence of 1 mM 2,4-D.

On using a PC-12 cell model, other authors have been previously shown that a depletion of mitochondrial and cytoplasmatic GSH results in increased ROS levels, disruption of the mitochondial transmembrane potential, rapid loss of mitochondial function, decrease in the ATP concentration, and eventually a higher cell death rate (Nieminen et al., 1995; Wüllner et al., 1999).

Therefore, the alteration in oxidative parameters suggest that the possible mechanisms of chlorophenoxy herbicide toxicity could involve dose-dependent cell membrane damage, uncoupling of oxidative phosphorylation, acetylcoenzyme disruption (Bradberry et al., 2000), and an indirect disruption of mitochondrial transmembrane potential which may lead to caspase inactivation (Kaioumova et al., 2001a). Mitochondrial structural modifications and increased permeability of the pores were also reported in association with a ROS increase (Belizário et al., 2007). In contrast, other studies suggest that 2,4-D cytotoxic effects are exerted by apoptosis induction via a direct effect on mitochondria (Tuschl & Schwab, 2003).

In this regard, Bongiovanni et al. (2011), in agreement with De Moliner et al. (2002), demonstrated that 2,4-D induces apoptosis and necrosis in CGC. While De Moliner et al. (2002) showed that 2,4-D-induced apoptosis is associated with and increase in caspase-3 activity preceded by cytochrome-c release from mitochondria, the quantification of ultrastructural changes showed that 1 mM 2,4-D stimulated neuronal death. As much as 49% of necrotic cells and 20% of apoptotic cells were observed, while only 31% of CGC presented normal growth with respect control group (p<0.001; Fig. 3 compared with Fig. 4) (Bongiovanni et al., 2011).

Oxidative Stress as a Possible Mechanism

abreviations in Fig. 3.

balance of CGC *in vitro* (Bongiovanni et al., 2007, 2011).

or 0.5 mM melatonin in CGC cultures (Bongiovanni et al., 2007).

were observed by electron microscopy (Fig. 4 compared with Fig. 5).

of Toxicity of the Herbicide 2,4-Dichlorophenoxyacetic Acid (2,4-D) 327

In these studies, melatonin and amphetamine were used as phamacological tools aiming to improve the analysis of oxidative stress as a mechanism of toxicity, by assessing whether these compounds could be effective in preventing the toxic effect of 2,4-D in the redox

A remarkable body of evidence indicates that melatonin exerts antioxidative protection in cell culture and *in vivo* systems (Pandi-Perumal et al., 2006). Regarding to 2,4-D toxicity, the oxidative stress induced by 1 mM 2,4-D was counteracted by the concomitant addition of 0.1

On the other hand, amphetamine has constistently been reported to accelerate the recovery of several functions in animals and humans with brain injury (Goldstein, 2000; Martinsson & Eksborg, 2004). Amphetamine was also shown to stimulate both the dendritic growth in the ventral tegmental area (Mueller et al., 2006) and the neurotrophic and neuroplastic responses after brain damage (Moroz et al., 2004; Adkins & Jones 2005). However, few data are available regarding any possible protective effect of amphetamine. In this regard, Bongiovanni et al., (2011) demonstrated that 1 or 10 µM amphetamine reverted the 2,4-Dinduced apoptosis and oxidative stress in CGC. Nevertheless, amphetamine alone induced no significant changes with respect to the control culture. Noteworthy, at 1 μM AMPH plus 2,4-D, 39% of the cells were normal; 53% were necrotic, and 8% showed apoptosis. At 10 μM AMPH plus 2,4-D, 57% of the cells were normal, 43% were necrotic, and no apoptotic cells

Fig.5. Electron photomicrographies showing the ultrastructural cytoplasmatic characteristics of cerebellar granular cells after 2,4-D and 10 µM AMPH addition to the medium for 48 h. a– b. Cells present more conserved morphology (nucleus and cytoplasm) than those treated with 2,4-D alone (Cf Figs. 4a, b). Bars correspond to 1 µm. c. The cell shows mitochondria and Golgi cisterns more preserved than those of the cells treated with 2,4-D alone (Cf Fig. 4c). Bars correspond to 600 nm. AC apoptotic cell, NC necrotic cell, V vacuole and other

The collected evidence would indicate a protective effect of melatonin and amphetamine against 2,4-D-induced cell death, possibly due to an inhibition of the oxidative mechanisms, as judged by the close relationship between ROS and apotosis induction (Carmody &

Fig. 3. Electron photomicrographies showing cerebellar granular neurons cultured in a control medium (NaCl 0.9%) for 48 h. a–b. Cell morphology is preserved (nucleus with laxe chromatin, dense chromatin patch close to the nucleus envelope, scarce cytoplasm, and the presence of neurites). Bars correspond to 1 µm in (a) and 160 nm in (b); c. Cells show preserved ultrastructural characteristics (Golgi apparatus, polyribosomes and mitochondrial characteristics of normal granular cerebellar cells). Bars correspond to 320 nm in (c). C cytoplasm, CC dense chromatin, G Golgi apparatus, LC laxe chromatin, M mitochondria, N nucleus, NM nuclear membrane, P polyribosome, PM plasmatic membrane.

Fig. 4. Electron photomicrographies showing the ultrastructural cytoplasmatic characteristics of cerebellar granular cells after 2,4-D addition to the medium for 48 h. a–b. An apoptotic cell (nuclear fragmentation and very dense chromatinic accumulus), a necrotic cell (cytoplasm very scarce, no nucleus), and cells with scarce cytoplasm and small nucleus are shown, allowing comparison with the control group (Cf Figs. 3a, b). Bars correspond to 1 µm. c. A cell with cytoplasmatic protutions, vacuoles, disorganization of the cytoplasmatic reticulum, distended cisterns of the Golgi apparatus, and mitochondrial swelling. Bars correspond to 400 nm. AC apoptotic cell, NC necrotic cell, V vacuole, and other abreviations in Fig. 3.

Fig. 3. Electron photomicrographies showing cerebellar granular neurons cultured in a control medium (NaCl 0.9%) for 48 h. a–b. Cell morphology is preserved (nucleus with laxe chromatin, dense chromatin patch close to the nucleus envelope, scarce cytoplasm, and the presence of neurites). Bars correspond to 1 µm in (a) and 160 nm in (b); c. Cells show

nucleus, NM nuclear membrane, P polyribosome, PM plasmatic membrane.

Fig. 4. Electron photomicrographies showing the ultrastructural cytoplasmatic

in Fig. 3.

characteristics of cerebellar granular cells after 2,4-D addition to the medium for 48 h. a–b. An apoptotic cell (nuclear fragmentation and very dense chromatinic accumulus), a necrotic cell (cytoplasm very scarce, no nucleus), and cells with scarce cytoplasm and small nucleus are shown, allowing comparison with the control group (Cf Figs. 3a, b). Bars correspond to 1 µm. c. A cell with cytoplasmatic protutions, vacuoles, disorganization of the cytoplasmatic reticulum, distended cisterns of the Golgi apparatus, and mitochondrial swelling. Bars correspond to 400 nm. AC apoptotic cell, NC necrotic cell, V vacuole, and other abreviations

preserved ultrastructural characteristics (Golgi apparatus, polyribosomes and mitochondrial characteristics of normal granular cerebellar cells). Bars correspond to 320 nm in (c). C cytoplasm, CC dense chromatin, G Golgi apparatus, LC laxe chromatin, M mitochondria, N

In these studies, melatonin and amphetamine were used as phamacological tools aiming to improve the analysis of oxidative stress as a mechanism of toxicity, by assessing whether these compounds could be effective in preventing the toxic effect of 2,4-D in the redox balance of CGC *in vitro* (Bongiovanni et al., 2007, 2011).

A remarkable body of evidence indicates that melatonin exerts antioxidative protection in cell culture and *in vivo* systems (Pandi-Perumal et al., 2006). Regarding to 2,4-D toxicity, the oxidative stress induced by 1 mM 2,4-D was counteracted by the concomitant addition of 0.1 or 0.5 mM melatonin in CGC cultures (Bongiovanni et al., 2007).

On the other hand, amphetamine has constistently been reported to accelerate the recovery of several functions in animals and humans with brain injury (Goldstein, 2000; Martinsson & Eksborg, 2004). Amphetamine was also shown to stimulate both the dendritic growth in the ventral tegmental area (Mueller et al., 2006) and the neurotrophic and neuroplastic responses after brain damage (Moroz et al., 2004; Adkins & Jones 2005). However, few data are available regarding any possible protective effect of amphetamine. In this regard, Bongiovanni et al., (2011) demonstrated that 1 or 10 µM amphetamine reverted the 2,4-Dinduced apoptosis and oxidative stress in CGC. Nevertheless, amphetamine alone induced no significant changes with respect to the control culture. Noteworthy, at 1 μM AMPH plus 2,4-D, 39% of the cells were normal; 53% were necrotic, and 8% showed apoptosis. At 10 μM AMPH plus 2,4-D, 57% of the cells were normal, 43% were necrotic, and no apoptotic cells were observed by electron microscopy (Fig. 4 compared with Fig. 5).

Fig.5. Electron photomicrographies showing the ultrastructural cytoplasmatic characteristics of cerebellar granular cells after 2,4-D and 10 µM AMPH addition to the medium for 48 h. a– b. Cells present more conserved morphology (nucleus and cytoplasm) than those treated with 2,4-D alone (Cf Figs. 4a, b). Bars correspond to 1 µm. c. The cell shows mitochondria and Golgi cisterns more preserved than those of the cells treated with 2,4-D alone (Cf Fig. 4c). Bars correspond to 600 nm. AC apoptotic cell, NC necrotic cell, V vacuole and other abreviations in Fig. 3.

The collected evidence would indicate a protective effect of melatonin and amphetamine against 2,4-D-induced cell death, possibly due to an inhibition of the oxidative mechanisms, as judged by the close relationship between ROS and apotosis induction (Carmody &

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Cooter, 2001). While apoptosis and necrosis present some early features that may be common to both, mithocondrial disorders could be irreversibly compromised in necrotic, but not in apoptotic neurons (Nicotera & Leist, 1997). This could explain why amphetamine decrease apoptosis but not necrosis in 2,4-D-treated cells.

In summary, 2,4-D would induce necrosis and apoptosis, the latter being possibly mediated by an oxidative imbalance.
