**2. Reactive oxygen formation and its relation to the biotransformation of pyrethroids**

Pyrethroids are the esters of acids like chrysanthemic acid, halo-substituted chrysanthemic acid, and 2-(4-chlorophenyl)-3-methyl butyric acid and alcohols like allethrolone and 3-phenoxybenzyl alcohol and they mostly contain more than one asymmetric carbon atom [3]. The stereoisomeric nature plays a significant role in the biotransformation of some pyrethroids like fenvalerate [23]. This can also contribute to their toxic effect. For example, different stereoisomeric forms of permethrin have caused the increase in intracellular reactive oxygen species (ROS) and lipid peroxidation levels and decrease in superoxide dismutase (SOD) and catalase (CAT) activities in rat pheochromocytoma cells (PC12); but this effect is enantioselective, and the most effective stereoisomer is 1*R*-*trans*-permethrin [24].

Pyrethroid biotransformation in mammals including human consists oxidation, ester hydrolysis (both are called as Phase I reactions), and conjugation with endogenous molecules (Phase II reactions) [3, 25, 26]. Oxidation reactions are catalyzed by isoforms of cytochrome P450s (CYP450s), and ester bonds are hydrolyzed by carboxylesterase(s) [26].

The produced metabolites can be more potent endocrine disruptors than parent compound for humans [27]. Romero et al. found that CYP450-mediated oxidation products of deltamethrin (2'-OH and 4'-OH deltamethrin) are more toxic than the parent compound measured with cell viability, lipid peroxidation, and nitric oxide formation on human dopaminergic neuroblastoma SH-SY5Y cells [28]. Moreover, abnormal locomotor activity observed in prenatal deltamethrin exposure has been associated with increased expression of CYP450 enzymes in the offsprings of rats [29]. However, the pyrethroids are commonly used as a replacement of organophosphate and organochlorine insecticides because of their low mammalian toxicity at the first time of their popularity. The low toxicity has been attributed to their rapid metabolism in mammals [18]. For this reason, their metabolism considered as a detoxification because of rapid clearance from the body [25, 30]. Most of the metabolites are highly hydrophilic, and then rapidly excreted via urine and feces. Some of the metabolites from *R*-cyano-3-phenoxybenzyl alcohol derivative pyrethroids, however, shows incomplete excretion and have longer bioretention in skin and stomach [25, 26]. Moreover, some of the conjugation metabolites are lipophilic and participate in toxicity reactions [25]. The biotransformation to hydrophilic compounds may also be a source of their toxicity in mammals as described below.

A single dose of cypermethrin and/or fenvalerate has caused the increase in SOD and CAT activities and in lipid peroxidation levels in the erythrocytes of rats [31]. As specified, noncyano (Type I)—cyano (Type II) discrimination can also be observed in oxidative stress-inducing potential of these chemicals. For example, permethrin (a Type I) disturbed the antioxidant defense more than cypermethrin (a Type II) in the erythrocytes of treated rats [8]. Because of its cyano group, cypermethrin shows longer permanence in the membrane, while permethrin can pass easily from this lipid bilayer with its lipophilic nature to reach more readily to cellular subcompartments such as endoplasmic reticulum (ER) membranes that contain CYP450s. Although the presence of α-cyano group decreases the hydrolysis rate of ester bond [32], this group decomposes to cyanides and aldehydes to produce free radicals [33]. Endogenously formed superoxide anion radical is dismutated to hydrogen peroxide (H2 O2 ) spontaneously or a SOD-catalyzed reaction. The formed H2 O2 is degraded to water via CAT in peroxisomes and/or glutathione peroxidases (GPx) in the cytosol, mitochondria, nucleus, and also in peroxisomes [34, 35]. Although the H2 O2 is not assessed as a ROS, it can act as a substrate for hydroxyl radical formation via a metal (it is mostly iron) catalyzed reaction if it cannot convert to water efficiently. Hydroxyl radical is the strongest radical capable of oxidizing DNA, cellular membrane lipids, and proteins, and there is no effective agent to escape them in the cell [35]. The most important intracellular iron source is the active site of CYP450s because of their iron content in the catalytically active center [36–40].

children associated with detectable levels of pyrethroid metabolites in the urine; therefore, abnormalities in the dopamine system that is more threatening for boys may be a result of growing use of pesticides, especially pyrethroids [20]. Urinary pyrethroid residues have been correlated with increased chronic heart disease in nonoccupational exposed Chinese people [21]. Occupational exposure to pyrethroids, for example, in the textile industry, is also an

**Table 1.** Chemical structures of the pyrethroids that are mostly discussed in the current chapter.

IUPAC name: [cyano-(3-phenoxyphenyl) methyl]

IUPAC name: [(S)-cyano-(3-phenoxyphenyl) methyl]

(1R,3R)-3-(2,2-dibromoethenyl)-2,2-dimethylcyclopropane-1-carboxylate

CAS No: 52315-07-8

CAS No: 52918-63-5

National Center for Biotechnology Information. PubChem Compound Database; CID = 11,442, https://pubchem.ncbi.

National Center for Biotechnology Information. PubChem Compound Database; CID = 5,282,227, https://pubchem.ncbi.

National Center for Biotechnology Information. PubChem Compound Database; CID = 40,326, https://pubchem.ncbi.

National Center for Biotechnology Information. PubChem Compound Database; CID = 2912, https://pubchem.ncbi.nlm.

National Center for Biotechnology Information. PubChem Compound Database; CID = 40,585, https://pubchem.ncbi.

3-(2,2-dichloroethenyl)-2,2-dimethylcyclopropane-1-carboxylate

Pyrethroids are the esters of acids like chrysanthemic acid, halo-substituted chrysanthemic acid, and 2-(4-chlorophenyl)-3-methyl butyric acid and alcohols like allethrolone and 3-phenoxybenzyl alcohol and they mostly contain more than one asymmetric carbon atom [3]. The stereoisomeric nature plays a significant role in the biotransformation of some pyrethroids like fenvalerate [23]. This can also contribute to their toxic effect. For example, different stereoisomeric forms of permethrin have caused the increase in intracellular reactive

important issue throughout the world [22].

nlm.nih.gov/compound/11442 (accessed June 18, 2018).

nlm.nih.gov/compound/5282227 (accessed June 18, 2018).

nlm.nih.gov/compound/40326 (accessed June 18, 2018).

nlm.nih.gov/compound/40585 (accessed June 18, 2018).

nih.gov/compound/2912 (accessed June 18, 2018).

Cypermethrin4 (Type II)

296 Mitochondrial Diseases

Deltamethrin5 (Type II)

1

2

3

4

5

**biotransformation of pyrethroids**

**2. Reactive oxygen formation and its relation to the** 

Pro-oxidant nature of CYP450-mediated pyrethroid metabolism needs further clarification because of superoxide and H2 O2 release from CYP450 enzymatic complex by CYP450-inducers [35–40]. Pro-oxidative toxicity of pyrethroids has been reported in mammalian studies. Raina et al. suggest that the induction of oxidative stress in dermal cypermethrin exposed rats should be related to its biotransformation via CYP450-catalysis [41]. Metofluthrin, a known carcinogenic agent at high doses, induces mainly CYP2B isoforms and increases oxidative stress via the increase of reduced glutathione (GSH) levels (a well-known cellular antioxidant molecule) in rats [42]. Without an induction of apoptosis, the authors conclude that the metofluthrin has reversible effects, and it may be noncarcinogenic for a human. On the contrary, deltamethrin and permethrin exposure has caused the induction of caspase 3/7 activities; therefore, it has been concluded that oxidative potentials of pyrethroids can trigger the apoptosis in human HepG2 cells and primary hepatocytes [43]. Deltamethrin and permethrin have also caused the stimulation of mRNA transcripts of CYP1A1, CYP3A4, and CYP2B6 isoforms and CYP3A4 protein levels. NADPH-dependent microsomal ROS formation has been observed in the liver of etofenprox exposed rats, and it has been concluded that observed lipid peroxidation and DNA oxidation in the liver should be related with CYP2Binduction by etofenprox exposure [34]. CYP450-mediated cytosolic and/or mitochondrial ROS formation [44, 45] might cause cell death [46], and we conclude that CYP450 activation via pyrethroid exposure might cause mitochondrial damage and cell death. Therefore, CYP450 inducers should be evaluated with this type of side effect.

such as subtoxic levels of ROS. There is a fine balance between these two signaling systems and dysfunction in either of these systems can affect another one. Therefore, this situation is harmful or a signal for defense for a cell [52]. As stated in the review of Chirumbolo and Bjørklund [53], we believed that pyrethroids can exert their toxicity via the induction of ROS on ER membranes via CYP450 activity and uncontrolled Ca2+ release from ER stores (and/or intracellular flux), which are used to conduct a fine balance between the ER and mitochondria deciding the autophagy or apoptosis. In this sense, we try to explain the mitochondrial effects of pyrethroids

Pyrethroid Insecticides as the Mitochondrial Dysfunction Inducers

http://dx.doi.org/10.5772/intechopen.80283

299

considering their oxidative stress-inducing potential and Ca2+ homeostasis of the cell.

from ER stores or via Ca2+ influx triggers the ROS formation and cell death [58, 60].

1,4,5-triphosphate levels in rat brain slices in the presence of neomycin or LiCl [64].

Cypermethrin and fenvalerate have rescued the tsBN7 (a temperature sensitive cell type) cells from apoptotic death with elevated temperature compared to cyclosporine A, a mitochondrial membrane permeability transition pore (mtPTP) inhibitor [65]. According to the authors, elevation in cytosolic Ca2+ is at the core of the formation of mtPTP, and these pyrethroids

Deltamethrin can inactivate the VGSCs. Downregulation of gene transcripts of these proteins in deltamethrin exposed human SK-N-AS neuroblastoma cells has also been observed with an intracellular Ca2+ elevation and calpain activation-mediated pathway [61]. Therefore, this situation causes the ER stress-related nonmitochondrial apoptotic pathway in human SK-N-AS neuroblastoma cells by deltamethrin [62]. According to this model, deltamethrininduced VGSC opening has been caused Ca2+ overload and activation of ER stress pathway engaging calpain and caspase-12 without an increase in cytosolic cytochrome c levels (an indicator for mitochondrial apoptotic pathway). In this way, resultant sodium influx via opening the VGSCs can activate the phosphatidylinositol turnover; the intermediates formed via this turnover will activate protein kinase C and the Ca2+ release from internal stores [63]. Deltamethrin can activate directly the protein kinase C enzyme at its very low dose [64]. According to the authors, "*deltamethrin has a direct-action site likely to be on protein kinase C, an inositol polyphosphates-independent Ca*2+ *triggering site (e.g., ryanodine receptor and ER stores), and/or phosphoprotein phosphatase.*" Interestingly, deltamethrin was able to increase the inositol

Cellular Ca2+ stores can be a target for pyrethroid action and pyrethroid-mediated intracellular Ca2+ load could be related to mitochondrial changes. For example, early life exposure to permethrin increased the intracellular Ca2+ influx in the heart of permethrin exposed rats [54]. Pyrethroids can activate the dose-dependent Ca2+-influx in the tetrodotoxin-sensitive pathway (a specific inhibitor of VGSCs) with different potencies and efficacies in mouse primary cortical neurons [55]. However, the changes in Ca2+ dynamics could not always be dependent on VGSCs, at least for bifenthrin at nanomolar concentrations in mouse primary cortical neurons [56]. In fact, pyrethroids can modify voltage-gated Ca2+ channels at concentrations similar to VGSCs, and Type IIs are more potent to induce Ca2+ influx according to voltage- and patchclamp electrophysiological and *in situ* functional studies [57]. High intracellular Ca2+ levels can cause damage to mitochondria [58, 59], and changes in intracellular Ca2+ levels via release

**3. Cellular Ca2+ stores and pyrethroids**

Deltamethrin exposure has caused early ROS formation and subsequent decrease in GSH levels, Bcl-2 protein expression, and mitochondrial membrane potential and increase in Bax, p38 MAPK expressions, and caspase-3 activity in isolated splenocytes from mice [47]. Similar results have also been found in the brain of deltamethrin exposed rats [48]. The number of apoptotic cells has been decreased by *N*-acetylcysteine, a well-known antioxidant agent, while buthionine sulfoximine, a GSH depleting agent, worsened the effects [47]. Therefore, when redox balance favors the ROS formation, it could be the main curator of mitochondrial dysfunction and related cell death. Not only synthetic ones but also natural pyrethrins can cause ROS formation and related mitochondrial dysfunction and apoptosis in human hepatocarcinoma cell line HepG2 [49].

In fact, cells can die because of the ER stress-dependent pathways in pyrethroid intoxication. For example, Zhao et al. have suggested nonmitochondrial apoptotic pathway with an extracellular route [50]. According to their model, fenvalerate acts as an endocrine disruptor through the induction of apoptosis of mice germ cells. Fas/FasL-directed caspase-8 activation has caused the germ cell apoptosis without the change in Bcl-2, Bax, mitochondrial and cytosolic cytochrome c, and cleaved procaspase-9 levels.

Interestingly, ER and mitochondria have multiple contact sites called mitochondria-ER associated membranes with a characteristic set of proteins. From these domains, not only Ca2+ but also ROS-mediated signals may be transmitted to the mitochondria after ROS-based ER stress (for more details, see [51]). On these domains, inositol-1,4,5-triphosphate receptors interact with voltage-dependent anion channels (VDACs) on the outer membrane of mitochondria to transfer Ca2+. As an important second messenger, Ca2+ interacts with other signaling systems such as subtoxic levels of ROS. There is a fine balance between these two signaling systems and dysfunction in either of these systems can affect another one. Therefore, this situation is harmful or a signal for defense for a cell [52]. As stated in the review of Chirumbolo and Bjørklund [53], we believed that pyrethroids can exert their toxicity via the induction of ROS on ER membranes via CYP450 activity and uncontrolled Ca2+ release from ER stores (and/or intracellular flux), which are used to conduct a fine balance between the ER and mitochondria deciding the autophagy or apoptosis. In this sense, we try to explain the mitochondrial effects of pyrethroids considering their oxidative stress-inducing potential and Ca2+ homeostasis of the cell.
