**5. Lipid metabolism is a target for pyrethroid-induced mitochondrial dysfunction**

Reactive oxygen species reduces the oxygen consumption and decreases the fatty acid oxidation in adipocytes causing the lipid accumulation [109]. According to Chirumbolo and Bjorklund, mitochondrial ROS formation and dysfunction could play a central role in the machinery of lipid accumulation via the interaction with AMPK and peroxisome proliferatoractivated receptor (PPAR) pathways [110]. Stressed cells accumulate lipids and enhance the hypoxic stimulus, and this occurs via AMPK-signaling pathways.

resulting in the inhibited translocation of PTEN-induced putative kinase 1 (PINK1) to defend cells against ROS formation by dysfunctional mitochondria. In mitochondrial damage conditions, PINK1 accumulation in outer membrane results with a selective autophagy [119]. Therefore, PINK1-dependent mitophagy is responsible for maintaining a healthy mitochondrial population for undesired excessive ROS formation [120, 121]. Exposure to deltamethrin has caused the apoptotic and autophagic death in rat pheochromocytoma cell line PC12 [122]. Although the autophagy inhibitor, 3-methyladenine exacerbated the deltamethrin toxicity, pre-treatment with autophagy inducer rapamycin and antioxidant *N*-acetylcysteine have

Pyrethroid Insecticides as the Mitochondrial Dysfunction Inducers

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

305

However, autophagy itself can be responsible for the ROS formation [123, 124]; therefore complex I-inhibition related cell death could be derived from PINK1-mediated mitophagy because of the inhibition of ROS formation and apoptosis via an antioxidant or PPAR-γ agonist treatment during mitochondrial autophagosome formation [124, 125]. Mitochondrial fusion can constitute a link between ROS formation and lipid accumulation. Downregulation of Mfn2 gene in human embryonic kidney cells 293 with siRNA caused triglyceride and ROS accumulation and decreased oxygen consumption [126]. Interestingly, impaired mitochondrial dynamics and dysfunctional autophagy can also be a cause *in vivo* triglyceride accumulation

Shen et al. [127] reported that mouse pre-adipocyte cells showed increased fat accumulation via AMPK/PPAR-γ intersection by deltamethrin exposure. Phosphorylated AMPK/AMPK (pAMPK/AMPK) ratio has been decreased, while PPAR-γ protein levels increased in these deltamethrin exposed cells. Permethrin has also caused similar changes in these cells with the elevation of triglyceride levels and decrement of *carnitine palmitoyltransferase 1-α* mRNA levels [128]. In this study, permethrin exposure decreased protein kinase B (Akt) and increased its activated phosphorylated forms (at Ser473 and Thr308) in C2C12 myotubes in the presence of insulin. Therefore, permethrin alters lipid metabolism in adipocytes and impaired glucose metabolism in myotubes and then increases the obesity and type-2 diabetes progression risks in exposed individuals. Authors discussed that these changes are related to mitochondrial Ca2+ and ROS formation. In another study, pAMPK levels have been increased with increased autophagosome formation and abnormal autophagy in cypermethrin treated rats and SH-SY5Y neuroblastoma cells [129]. Authors indicated that increased phosphorylation of AMPK shows the decreased AMP/ATP ratio via mitochondrial dysfunction. Although the above authors revealed the mitochondrial dysfunction-related adipogenesis, Xiao et al. [130] have defined an intracellular Ca2+- and ER stress-related adipogenesis in permethrin exposed

*Solute carrier family 25 member 25 (Slc25a25)* and *solute carrier family 2 member 1 (Slc2a1)* gene expressions have been affected by deltamethrin and cyfluthrin exposure in the cortical samples of rat brain *in vivo* with many other membrane proteins [131]. Slc25a25 serves as a solute carrier for adenine nucleotides in and from the mitochondrial inner membrane, while Slc2a1 is a major glucose transporter in the blood-brain barrier. These pyrethroids have also affected *pyruvate dehydrogenase kinase 4 (pdk4)* gene expression, which plays a role in glucose metabolism via inhibition of pyruvate dehydrogenase complex by phosphorylation.

increased the cell viability via the prevention of apoptosis progression.

in aged rat tissues [126].

mouse pre-adipocyte cells.

Cypermethrin induces the *pyruvate kinase*, *glucose transporter*, *stearoyl-CoA desaturase-1*, *acyl-CoA oxidase*, and *carnitine palmitoyltransferase 1-α* mRNA levels in the liver of mice [111]. *PPAR-α* have also increased with increased pyruvate levels. In this study, hepatic free fatty acid transport genes have also been upregulated; then, cypermethrin is able to defect lipid metabolism and can cause the lipid accumulation (evidenced by increased lipid droplets in histologic sections) in this organ. An interesting situation is the overexpression of *stearoyl-CoA desaturase-1* gene because its activation is related to mitochondrial ROS generation, caspase-3 activation, and apoptotic cell death in the heart of rats fed with saturated fatty acid rich diet [112]. In this study, AMPK phosphorylation has been decreased with the overexpression of *stearoyl-CoA desaturase-1* gene. AMPK inactivation results in the activation of acetyl-CoA carboxylase. It increases malonyl CoA synthesis, and malonyl CoA reduces carnitine palmitoyltransferase activity to transport fatty acids into mitochondria for oxidation. Therefore, fatty acid oxidation is decelerated. It is known that mitochondrial fatty acid oxidation is an important ROS source [113]. However, mitochondria need fatty acids to maintain AMP/ATP ratio and to maintain its functions in physiological levels. Therefore, a subtle balance of fatty acid oxidation must be conducted. In this sense, PPAR (including all three forms) agonists upregulate the AMPK activity to mediate many physiological functions to protect cells from mitochondrial membrane potential change and ROS formation [114].

*Carnitine palmitoyltransferase-1 and PPAR-α* gene expressions have been upregulated by cypermethrin exposure in the liver of zebrafish (*Danio rerio*) with ROS activation [115]. The results reveal the importance of cypermethrin-induced oxidative stress on impaired fatty acid β-oxidation and mitochondrial dysfunction. PPAR-α is the most significant orchestrator of altered fatty acid metabolism in this process. Relation of pyrethroid-induced lipid accumulation and mitochondrial dysfunction has been conducted with some newer research. While Jin et al. [111] has not been found up or downregulated mRNA expression of *ppar-γ* with cypermethrin intoxication, Moustafa and Hussein [116] reported that lambda-cyhalothrin intoxication caused upregulation of *ppar-α* and *ppar-γ* transcripts in the liver of rats. Hepatic fat infiltration and periportal fatty changes have also been observed with an elevated ROS formation.

Cobalt chloride, a hypoxia mimetic agent, has caused downregulation of PPAR-γ, increased lipid accumulation, mitochondrial ROS production, and autophagy in mouse pre-adipocyte cells [117]. It is known that elevated levels of TNF-α can be found in dysfunctional neuronal cells. The high level exposure of TNF-α to mimic these cells has caused decreased PPAR-γ and AMPK proteins, ATP levels, and mitochondrial mass, while ROS levels and caspase-3 (an apoptotic executioner enzyme) increased in human neuronal stem cells [118]. Rosiglitazone, a PPAR-γ agonist, protected the cells from these adverse effects of TNF-α. Mitochondrial complex I activity has decreased in deltamethrin treated human dopaminergic neuroblastoma SH-SY5Y cells [73]. These cells had typical mitochondrial apoptotic signals. The authors revealed that the mitochondrial apoptosis was antagonized by PPARγ agonist rosiglitazone resulting in the inhibited translocation of PTEN-induced putative kinase 1 (PINK1) to defend cells against ROS formation by dysfunctional mitochondria. In mitochondrial damage conditions, PINK1 accumulation in outer membrane results with a selective autophagy [119]. Therefore, PINK1-dependent mitophagy is responsible for maintaining a healthy mitochondrial population for undesired excessive ROS formation [120, 121]. Exposure to deltamethrin has caused the apoptotic and autophagic death in rat pheochromocytoma cell line PC12 [122]. Although the autophagy inhibitor, 3-methyladenine exacerbated the deltamethrin toxicity, pre-treatment with autophagy inducer rapamycin and antioxidant *N*-acetylcysteine have increased the cell viability via the prevention of apoptosis progression.

Bjorklund, mitochondrial ROS formation and dysfunction could play a central role in the machinery of lipid accumulation via the interaction with AMPK and peroxisome proliferatoractivated receptor (PPAR) pathways [110]. Stressed cells accumulate lipids and enhance the

Cypermethrin induces the *pyruvate kinase*, *glucose transporter*, *stearoyl-CoA desaturase-1*, *acyl-CoA oxidase*, and *carnitine palmitoyltransferase 1-α* mRNA levels in the liver of mice [111]. *PPAR-α* have also increased with increased pyruvate levels. In this study, hepatic free fatty acid transport genes have also been upregulated; then, cypermethrin is able to defect lipid metabolism and can cause the lipid accumulation (evidenced by increased lipid droplets in histologic sections) in this organ. An interesting situation is the overexpression of *stearoyl-CoA desaturase-1* gene because its activation is related to mitochondrial ROS generation, caspase-3 activation, and apoptotic cell death in the heart of rats fed with saturated fatty acid rich diet [112]. In this study, AMPK phosphorylation has been decreased with the overexpression of *stearoyl-CoA desaturase-1* gene. AMPK inactivation results in the activation of acetyl-CoA carboxylase. It increases malonyl CoA synthesis, and malonyl CoA reduces carnitine palmitoyltransferase activity to transport fatty acids into mitochondria for oxidation. Therefore, fatty acid oxidation is decelerated. It is known that mitochondrial fatty acid oxidation is an important ROS source [113]. However, mitochondria need fatty acids to maintain AMP/ATP ratio and to maintain its functions in physiological levels. Therefore, a subtle balance of fatty acid oxidation must be conducted. In this sense, PPAR (including all three forms) agonists upregulate the AMPK activity to mediate many physiological functions to protect cells from

*Carnitine palmitoyltransferase-1 and PPAR-α* gene expressions have been upregulated by cypermethrin exposure in the liver of zebrafish (*Danio rerio*) with ROS activation [115]. The results reveal the importance of cypermethrin-induced oxidative stress on impaired fatty acid β-oxidation and mitochondrial dysfunction. PPAR-α is the most significant orchestrator of altered fatty acid metabolism in this process. Relation of pyrethroid-induced lipid accumulation and mitochondrial dysfunction has been conducted with some newer research. While Jin et al. [111] has not been found up or downregulated mRNA expression of *ppar-γ* with cypermethrin intoxication, Moustafa and Hussein [116] reported that lambda-cyhalothrin intoxication caused upregulation of *ppar-α* and *ppar-γ* transcripts in the liver of rats. Hepatic fat infiltration

and periportal fatty changes have also been observed with an elevated ROS formation.

Cobalt chloride, a hypoxia mimetic agent, has caused downregulation of PPAR-γ, increased lipid accumulation, mitochondrial ROS production, and autophagy in mouse pre-adipocyte cells [117]. It is known that elevated levels of TNF-α can be found in dysfunctional neuronal cells. The high level exposure of TNF-α to mimic these cells has caused decreased PPAR-γ and AMPK proteins, ATP levels, and mitochondrial mass, while ROS levels and caspase-3 (an apoptotic executioner enzyme) increased in human neuronal stem cells [118]. Rosiglitazone, a PPAR-γ agonist, protected the cells from these adverse effects of TNF-α. Mitochondrial complex I activity has decreased in deltamethrin treated human dopaminergic neuroblastoma SH-SY5Y cells [73]. These cells had typical mitochondrial apoptotic signals. The authors revealed that the mitochondrial apoptosis was antagonized by PPARγ agonist rosiglitazone

hypoxic stimulus, and this occurs via AMPK-signaling pathways.

304 Mitochondrial Diseases

mitochondrial membrane potential change and ROS formation [114].

However, autophagy itself can be responsible for the ROS formation [123, 124]; therefore complex I-inhibition related cell death could be derived from PINK1-mediated mitophagy because of the inhibition of ROS formation and apoptosis via an antioxidant or PPAR-γ agonist treatment during mitochondrial autophagosome formation [124, 125]. Mitochondrial fusion can constitute a link between ROS formation and lipid accumulation. Downregulation of Mfn2 gene in human embryonic kidney cells 293 with siRNA caused triglyceride and ROS accumulation and decreased oxygen consumption [126]. Interestingly, impaired mitochondrial dynamics and dysfunctional autophagy can also be a cause *in vivo* triglyceride accumulation in aged rat tissues [126].

Shen et al. [127] reported that mouse pre-adipocyte cells showed increased fat accumulation via AMPK/PPAR-γ intersection by deltamethrin exposure. Phosphorylated AMPK/AMPK (pAMPK/AMPK) ratio has been decreased, while PPAR-γ protein levels increased in these deltamethrin exposed cells. Permethrin has also caused similar changes in these cells with the elevation of triglyceride levels and decrement of *carnitine palmitoyltransferase 1-α* mRNA levels [128]. In this study, permethrin exposure decreased protein kinase B (Akt) and increased its activated phosphorylated forms (at Ser473 and Thr308) in C2C12 myotubes in the presence of insulin. Therefore, permethrin alters lipid metabolism in adipocytes and impaired glucose metabolism in myotubes and then increases the obesity and type-2 diabetes progression risks in exposed individuals. Authors discussed that these changes are related to mitochondrial Ca2+ and ROS formation. In another study, pAMPK levels have been increased with increased autophagosome formation and abnormal autophagy in cypermethrin treated rats and SH-SY5Y neuroblastoma cells [129]. Authors indicated that increased phosphorylation of AMPK shows the decreased AMP/ATP ratio via mitochondrial dysfunction. Although the above authors revealed the mitochondrial dysfunction-related adipogenesis, Xiao et al. [130] have defined an intracellular Ca2+- and ER stress-related adipogenesis in permethrin exposed mouse pre-adipocyte cells.

*Solute carrier family 25 member 25 (Slc25a25)* and *solute carrier family 2 member 1 (Slc2a1)* gene expressions have been affected by deltamethrin and cyfluthrin exposure in the cortical samples of rat brain *in vivo* with many other membrane proteins [131]. Slc25a25 serves as a solute carrier for adenine nucleotides in and from the mitochondrial inner membrane, while Slc2a1 is a major glucose transporter in the blood-brain barrier. These pyrethroids have also affected *pyruvate dehydrogenase kinase 4 (pdk4)* gene expression, which plays a role in glucose metabolism via inhibition of pyruvate dehydrogenase complex by phosphorylation. Therefore, pyrethroids can be effective on cells at different levels of metabolism. In a similar manner, permethrin caused a significant elevation of *pdk4* and *phosphoenolpyruvate carboxylase (pepck)* gene transcripts in the muscle and liver of mice, respectively [132]. Permethrin exposure displayed similar results [133] that were seen in the study of Kim et al. [128]. In addition, phosphorylated Akt at Thr308 and glucose transporter 4 (glut4) protein levels have been decreased in the muscle; therefore, authors concluded that permethrin can alter the glucose and lipid metabolism via an AMPK-dependent pathway and produce insulin resistance and obesity risk in exposed groups. In contrast, insulin-stimulated Akt phosphorylation has been decreased by permethrin in pAMPK-independent and the ERK-dependent manner in C2C12 myotubes, and this mechanism could be a reason for insulin resistance development [134]. Therefore, the exact mechanism of lipid accumulation in different cell types may use different pathways; however, we believed that ER-mitochondria axis and their relation in Ca2+ and ROS signaling are the main curators of these effects of pyrethroids.

pyrethroid action. For example, fenvalerate has not interacted with mitochondrial membrane proteins measured with intrinsic protein fluorescence, mainly by tryptophan fluorescence quenching in the isolated mitochondria from *Helicoverpa armigera* larvae (cotton bollworm) [141]. Because of its hydrophobic nature, deltamethrin has increased the mitochondrial membrane rigidity in the isolated rat liver mitochondrial preparation and this can cause the impaired transport of different ions between cytosol and mitochondrial matrix [90]. Permethrin has caused a decrease in mitochondrial membrane fluidity and this could be a reason for a bioenergetic crisis in the cell because of irregular energy transduction in striatum submitochondrial particles of rats [88]. Mitochondrial membrane fluidity at the hydrophilichydrophobic region of the bilayer has decreased, while fluidity in the hydrophobic core increased in the heart of 300-day old rats exposed the permethrin between 6 and 21 days of their life [142]. Moreover, decreased cholesterol levels in mitochondrial membranes have been observed while it increased in the plasma membrane of heart cells. Therefore, these observations and pro-oxidative properties of permethrin could cause the altered cardiac ultrastructure and function. This effect of permethrin has also been found in Leydig cells of mice testis as discussed above [137]. As an integral membrane protein, VDAC interacts with membrane cholesterol [143] and ATP synthesis, ATP/ADP exchange by adenine nucleotide translocator (ANT) at the inner membrane, ATP/ADP and metabolite exchange by VDAC can

Pyrethroid Insecticides as the Mitochondrial Dysfunction Inducers

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

307

Effects of pyrethroids are not limited to mitochondrial membranes because of fluidity decline in the hydrophobic core of cypermethrin exposed rat erythrocyte plasma membrane [144]. Similar fluidity decline has been observed in deltamethrin exposed common carp (*Cyprinus carpio*) erythrocyte plasma membranes [145]. Phosphatidylethanolamine, phosphoglyceride, phosphatidic acids, and cardiolipin levels were decreased, making the membrane more rigid and less permeable. Decreasing these components can cause oxidative stress and cell membrane ageing. Interestingly, cardiolipin is an exclusive component of the inner mitochondrial membrane, and it plays a significant role in governing the mitochondrial bioenergetics processes (interaction with respiratory chain proteins and substrate carriers) and dynamics [146]. Cardiolipin reduction has been observed via ROS-induced lipid peroxidation in nerve growth factor-deprived rat sympathetic neurons and this has caused the loss of mitochondrial density [147]. As a membranous structure, the same finding may be observed with mitochondrial preparations, but it is an issue for further studies. It has been concluded that high lipophilicity and pro-oxidative potential of pyrethroids can affect the biological membranes with their

While 18 kDa translocator protein (TSPO; formerly known as peripheral benzodiazepine receptor) ligands PK 11195 and Ro5-4864 are anti-apoptotic in the concentrations close to their TSPO affinity, they can also be pro-apoptotic agents at higher levels [148, 149]. It has been evidenced that pyrethroids can bind and interact with TSPO [150, 151], located on the mitochondrial outer membrane and participates to cholesterol transport as a cholesterol channel into mitochondria collectively with VDAC and ANT [152, 153]. Many type I and type II pyrethroids can bind this protein on rat brain membranes, while fluvalinate and fenvalerate have poor potency [154]. Furthermore, *cis*-permethrin has decreased the mRNA levels of *tspo* in mice testis [137]. In the study of Vadhana et al. [142], mitochondrial cholesterol levels have

be affected by associated membrane composition [93].

functional proteins to mediate the dysfunctional mitochondria.

Affected lipid metabolism by pyrethroids has also been observed in other studies including fish and mammals [135, 136].
