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

Pro-oxidant nature of CYP450-mediated pyrethroid metabolism needs further clarification

[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,

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 hepato-

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

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

release from CYP450 enzymatic complex by CYP450-inducers

because of superoxide and H2

298 Mitochondrial Diseases

carcinoma cell line HepG2 [49].

O2

CYP450 inducers should be evaluated with this type of side effect.

cytosolic cytochrome c, and cleaved procaspase-9 levels.

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

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 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 could be effective via their disruptive effect on Ca2+ balance. Ca2+ overload only can contribute to the formation of mtPTP; however, oxidative stress measured with excessive ROS formation and Ca2+ overload has a synergistic role in the formation of this pore to stimulate mitochondrial apoptosis [66].

Endoplasmic reticulum-mediated Ca2+ to mitochondria is necessary to adequate supply of reducing equivalents for oxidative phosphorylation because of enhanced phosphorylation of pyruvate dehydrogenase complex and activated AMPK (AMP-activated protein kinase) in the absence of this supply [79]. Giacomello et al. proposed a schema for anti- or pro-apoptotic proteins in ER-mediated Ca2+ supply to mitochondria [80]. Namely, Bax and other pro-apoptotic members of Bcl-2 family proteins enhance the ER Ca2+ load, and then mitochondria expose higher Ca2+ concentrations, mtPTP opens; while anti-apoptotic members of Bcl-2 cause the balanced Ca2+ concentration from ER stores; then apoptosis is inhibited, and the needed ATP levels are supplied enhancing the mitochondrial metabolism. According to Distelhorst and Bootman, under autophagy-promoting conditions, a mitochondrial Ca2+ transfer from ER protects the cells from death via adequate elimination of energy demands, while the excessive accumulation of Ca2+ via apoptosis-inducing chemicals and/or ROS triggers the irreversible apoptosis progression [81]. In fact, differential stimulation pathway of protein kinase C may result in the desensitization of inositol-1,4,5-triphosphate receptors via their phosphorylation by protein kinase C, which translocates to ER membranes in G-protein coupled protein subunit alpha s-cAMP pathway. In this way, desensitization of receptor to its ligand, inositol 1,4,5-triphosphate results in limited Ca2+ release from ER stores [82]. Enan and Matsumura have observed the translocation of protein kinase C from the cytosol to the membrane fraction in pyrethroid exposed rat brain synaptosomes [64]. Deltamethrin has caused the intracellular Ca2+ elevation, ROS formation, and mitochondrial apoptosis in HGB human glioblastoma cells; while these effects have been reversed by protein kinase C, ER Ca2+ pump, and inositol 1,4,5 formation inhibitors [83]. On the contrary, increased intracellular Ca2+ levels were not dependent on the phosphoinositide pathway in the effects of different pyrethroids in mouse primary neocortical neuron culture [55]. Therefore, tissue specificity and the dose-response curve of pyrethroid action on mitochondrial

Pyrethroid Insecticides as the Mitochondrial Dysfunction Inducers

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

301

Ca2+ supply from ER and apoptosis induction should be further investigated.

**affected by pyrethroid intoxication**

**4. Mitochondrial electron transport chain and energy production are** 

most pronounced effect has been observed in cypermethrin exposure, also [85].

Type I and type II pyrethroids could also be separated according to their toxic effects on different parts of the cell including mitochondria. Noncyano pyrethroid pyrethrin and permethrin increased the mitochondrial metabolic enzyme activities measured with the WST-1 method at low doses probably to support the bioenergetics needs of the cell in SH-SY5Y cells [84] while there is no or little effect on total ATP content. Mitochondrial enzyme activities and total ATP content have been decreased at higher doses. However, the most pronounced effect has been seen with an α-cyano compound cypermethrin starting with the low doses [84]. The same distinction could be done by their effect on human estrogen regulated breast cancer cell line (MCF-7). Coadministration of oestradiol has been potentiated the effects of these pyrethroids measured with total ATP and mitochondrial metabolic enzyme activities; but, the

According to the study of Gassner et al., permethrin and cyhalothrin caused the inhibition of complex I of electron transport chain in isolated rat liver mitochondria, and there are more than

Voltage-gated Ca2+-channel activation by allethrin has caused the mitochondrial cell death in rat Leydig cell tumor derived LC540 cells [67]. Allethrin exposure in these cell lines have resulted in the elevation of ROS, lipid peroxidation, intracellular Ca2+, cleaved PARP levels (executed by caspase-1), increased p53 gene expression, fluctuated SOD, CAT, GPx enzyme activities, and decreased mitochondrial membrane potential, Bcl-2, and pro-caspase-3 protein levels. It has been concluded that mitochondrial apoptosis by allethrin could be an important factor in decreased male fertility [67]. Similarly, allethrin exposure has caused the significant decrease in mitochondrial membrane potential and subsequent release of cytochrome c to the cytosol in the human corneal epithelial cell line [68]. Pro-apoptotic Bax expression has been increased, while anti-apoptotic Bcl-2 decreased, resulting in caspase-3 activation. Therefore, allethrin can trigger the mitochondrial apoptotic pathway in human corneal epithelial cells; although, they have not correlated their results with Ca2+ signaling.

An interesting support to these findings has been obtained with an estrogen receptor α and β binding studies of pyrethroids [69]. The studied chemicals have weak (fenvalerate) or no (permethrin, deltamethrin, and bifenthrin) binding capacity to estrogen receptor α, while permethrin has shown high affinity binding to estrogen receptor β. Lower but still strong binding to this protein has been observed with deltamethrin and fenvalerate, while bifenthrin has no binding capacity to this receptor. In another study, cypermethrin and permethrin exposure have increased the estrogen receptor α and β mRNA levels in TM4 mouse Sertoli cells to adapt decreased spermatogenic potential under pyrethroid toxicity [70]. Estrogen receptor β plays a role in preventing the mitochondrial apoptotic pathway and its suppression causes Bax activation, cytochrome c release, caspase 3 activation, and PARP cleavage [71].

Dissipation of mitochondrial membrane potential is an important event of apoptotic and necrotic cell deaths. It was observed in deltamethrin exposed rat primary hepatocytes with subsequent elevation of ROS, while programmed necrosis has been measured in these cells [72]. A common cell death sign or toxic insult starts a common cell death progression; but the ATP presence determines the type of cell death, apoptosis or necrosis [66]. Pro-apoptotic potential via the mitochondrial pathway of pyrethroids has been reported in many studies [47, 49, 73]; however, necrosis can also be occurred because of the ATP demand as was seen in the kidney of permethrin exposed rats [74] or in the heart of cypermethrin exposed frogs (*Rana cameroni*) [75].

Anti-apoptotic protein Bcl-xL interacts with VDACs to transfer Ca2+ into the mitochondria [76]. A continuous supply of Ca2+ into mitochondria via this way is necessary to maintain mitochondrial bioenergetics because of pyruvate, 2-oxoglutarate, and the NAD<sup>+</sup> -dependent isocitrate dehydrogenases, and three intramitochondrial tricarboxylic acid cycle (TCA) enzymes are stimulated by Ca2+ [77]. Anti-apoptotic members of Bcl-2 proteins (Bcl-2 itself, Bcl-xL, and Mcl-1) localized on the mitochondrial outer membrane and interact with the inositol-1,4,5-triphosphate receptors on the ER membrane to arrange the mitochondrial Ca2+ load during apoptotic signals and/or to enhance the mitochondrial metabolism for cellular resistance [76, 78].

Endoplasmic reticulum-mediated Ca2+ to mitochondria is necessary to adequate supply of reducing equivalents for oxidative phosphorylation because of enhanced phosphorylation of pyruvate dehydrogenase complex and activated AMPK (AMP-activated protein kinase) in the absence of this supply [79]. Giacomello et al. proposed a schema for anti- or pro-apoptotic proteins in ER-mediated Ca2+ supply to mitochondria [80]. Namely, Bax and other pro-apoptotic members of Bcl-2 family proteins enhance the ER Ca2+ load, and then mitochondria expose higher Ca2+ concentrations, mtPTP opens; while anti-apoptotic members of Bcl-2 cause the balanced Ca2+ concentration from ER stores; then apoptosis is inhibited, and the needed ATP levels are supplied enhancing the mitochondrial metabolism. According to Distelhorst and Bootman, under autophagy-promoting conditions, a mitochondrial Ca2+ transfer from ER protects the cells from death via adequate elimination of energy demands, while the excessive accumulation of Ca2+ via apoptosis-inducing chemicals and/or ROS triggers the irreversible apoptosis progression [81]. In fact, differential stimulation pathway of protein kinase C may result in the desensitization of inositol-1,4,5-triphosphate receptors via their phosphorylation by protein kinase C, which translocates to ER membranes in G-protein coupled protein subunit alpha s-cAMP pathway. In this way, desensitization of receptor to its ligand, inositol 1,4,5-triphosphate results in limited Ca2+ release from ER stores [82]. Enan and Matsumura have observed the translocation of protein kinase C from the cytosol to the membrane fraction in pyrethroid exposed rat brain synaptosomes [64]. Deltamethrin has caused the intracellular Ca2+ elevation, ROS formation, and mitochondrial apoptosis in HGB human glioblastoma cells; while these effects have been reversed by protein kinase C, ER Ca2+ pump, and inositol 1,4,5 formation inhibitors [83]. On the contrary, increased intracellular Ca2+ levels were not dependent on the phosphoinositide pathway in the effects of different pyrethroids in mouse primary neocortical neuron culture [55]. Therefore, tissue specificity and the dose-response curve of pyrethroid action on mitochondrial Ca2+ supply from ER and apoptosis induction should be further investigated.

could be effective via their disruptive effect on Ca2+ balance. Ca2+ overload only can contribute to the formation of mtPTP; however, oxidative stress measured with excessive ROS formation and Ca2+ overload has a synergistic role in the formation of this pore to stimulate mitochon-

Voltage-gated Ca2+-channel activation by allethrin has caused the mitochondrial cell death in rat Leydig cell tumor derived LC540 cells [67]. Allethrin exposure in these cell lines have resulted in the elevation of ROS, lipid peroxidation, intracellular Ca2+, cleaved PARP levels (executed by caspase-1), increased p53 gene expression, fluctuated SOD, CAT, GPx enzyme activities, and decreased mitochondrial membrane potential, Bcl-2, and pro-caspase-3 protein levels. It has been concluded that mitochondrial apoptosis by allethrin could be an important factor in decreased male fertility [67]. Similarly, allethrin exposure has caused the significant decrease in mitochondrial membrane potential and subsequent release of cytochrome c to the cytosol in the human corneal epithelial cell line [68]. Pro-apoptotic Bax expression has been increased, while anti-apoptotic Bcl-2 decreased, resulting in caspase-3 activation. Therefore, allethrin can trigger the mitochondrial apoptotic pathway in human corneal epithelial cells;

An interesting support to these findings has been obtained with an estrogen receptor α and β binding studies of pyrethroids [69]. The studied chemicals have weak (fenvalerate) or no (permethrin, deltamethrin, and bifenthrin) binding capacity to estrogen receptor α, while permethrin has shown high affinity binding to estrogen receptor β. Lower but still strong binding to this protein has been observed with deltamethrin and fenvalerate, while bifenthrin has no binding capacity to this receptor. In another study, cypermethrin and permethrin exposure have increased the estrogen receptor α and β mRNA levels in TM4 mouse Sertoli cells to adapt decreased spermatogenic potential under pyrethroid toxicity [70]. Estrogen receptor β plays a role in preventing the mitochondrial apoptotic pathway and its suppression causes

Bax activation, cytochrome c release, caspase 3 activation, and PARP cleavage [71].

drial bioenergetics because of pyruvate, 2-oxoglutarate, and the NAD<sup>+</sup>

Dissipation of mitochondrial membrane potential is an important event of apoptotic and necrotic cell deaths. It was observed in deltamethrin exposed rat primary hepatocytes with subsequent elevation of ROS, while programmed necrosis has been measured in these cells [72]. A common cell death sign or toxic insult starts a common cell death progression; but the ATP presence determines the type of cell death, apoptosis or necrosis [66]. Pro-apoptotic potential via the mitochondrial pathway of pyrethroids has been reported in many studies [47, 49, 73]; however, necrosis can also be occurred because of the ATP demand as was seen in the kidney of permethrin exposed rats [74] or in the heart of cypermethrin exposed frogs (*Rana cameroni*) [75]. Anti-apoptotic protein Bcl-xL interacts with VDACs to transfer Ca2+ into the mitochondria [76]. A continuous supply of Ca2+ into mitochondria via this way is necessary to maintain mitochon-

dehydrogenases, and three intramitochondrial tricarboxylic acid cycle (TCA) enzymes are stimulated by Ca2+ [77]. Anti-apoptotic members of Bcl-2 proteins (Bcl-2 itself, Bcl-xL, and Mcl-1) localized on the mitochondrial outer membrane and interact with the inositol-1,4,5-triphosphate receptors on the ER membrane to arrange the mitochondrial Ca2+ load during apoptotic

signals and/or to enhance the mitochondrial metabolism for cellular resistance [76, 78].


although, they have not correlated their results with Ca2+ signaling.

drial apoptosis [66].

300 Mitochondrial Diseases
