**4. Mitochondrial electron transport chain and energy production are affected by pyrethroid intoxication**

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

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 40 regions of complex I as potential binding sites for pyrethroids because of their hydrophobic nature [86]. Inhibition of complex I may be related to ROS formation; but, it should be noted that complex I inhibitors can be divided into two groups as ROS producers and ROS production inhibitors [87]. Inhibition of complex I activity by permethrin has been caused a reduction in superoxide radical formation in striatum submitochondrial particles of rats [88]. Inhibition of succinate dehydrogenase activity, which has a role in TCA and in complex II, has been decreased after acute and subacute bifenthrin exposure in rat brain [89]. Deltamethrin has a major inhibition site between complexes II and III because of unaffected NADH dehydrogenase (complex I) and cytochrome c oxidase (complex IV) activities in the isolated rat liver mitochondrial preparation [90]. In this mitochondrial preparation, NADH oxidase, succinate oxidase, succinate dehydrogenase (complex II), NADH-cytochrome c reductase, and succinate cytochrome c reductase activities have been inhibited. Deltamethrin has also caused an inhibition of ADP-stimulated oxygen consumption and impaired the mitochondrial membrane potential [90].

matrix to maintain TCA or fatty acid production, and accumulation of oxaloacetate has caused the inhibition of malate dehydrogenase activity, a part of TCA of mitochondria in cypermethrin

Pyrethroid Insecticides as the Mitochondrial Dysfunction Inducers

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

303

Metabolomics approach is very effective to understand pyrethroid-induced metabolic changes. Reports show the metabolic shift to anaerobic fuel consumption and elevated fuel supply via gluconeogenesis to maintain energy levels in pyrethroid-induced stress conditions. For example, permethrin exposure increased urine lactate, acetate, 3-D-hydroxybutyrate, creatine, glycine, and formate while decreased citrate and 2-oxoglutarate levels in rats [99]. Elevated levels of urinary acetate and decreased TCA intermediates show the energy metabolism disorders. Similarly, Liang et al. reported that permethrin and deltamethrin exposure can cause the disturbance in energy metabolism via the enhanced rate of anaerobic glycolysis and fatty acid β-oxidation, and ketogenesis [100]. They found that these pyrethroids reduced the excretion of TCA intermediates and increased lactate, acetate, 3-D-hydroxybutyrate levels in treated rats. In another study, serum and urine metabolites levels have been changed by deltamethrin exposure, and then it was suggested that decreased utilization of pyruvate in TCA and consecutive anaerobiosis in exposed rats [101]. While a shift from aerobic respiration to anaerobiosis was also found in the brain of lambda-cyhalothrin exposed goldfish (*Carassius auratus*), a marked decrease has been observed in brain *N*-acetyl-aspartate levels, because of neuronal mitochondrial membrane damage via the ROS formation [102]. *N*-acetylaspartate is considered as a marker for mitochondrial dysfunction in neurons [103]. Higher levels of malate and alanine in cypermethrin exposed earthworms provide an argument for the increased gluconeogenesis and fueling the TCA for energy [104]. These effects have also been observed in the former studies [100–102]. However, as an opposite of these results, permethrin exposure has caused an increase in TCA intermediates and cellular fatty acids and a decrease in glutamate levels in rat neuroblastoma cell line B50 [105]. Increased fatty acid

β-oxidation should be a response to permethrin toxicity in these cells.

Voltage-dependent anion channels located in the mitochondrial outer membrane is the only way to supply TCA intermediates from cytosol to mitochondria, and its closure causes a metabolic shift [106]. However, urea generation is also operated in the mitochondrial matrix, and it requires a bulk of substrates such as ornithine, citrulline, adenine nucleotides, respiratory substrates, and other metabolites across the mitochondrial outer membrane in/out of mitochondria, possibly via VDACs [107]. The mitochondrial outer membrane is rich in VDACs that opens in normal operated mitochondria and mitochondrial hexokinase bounds to VDAC to orchestrate respiration, glycolytic pathway, and other metabolic pathways such as the pentose phosphate shunt [108]. We think that pyrethroids can be effective on these mitochondrial membrane proteins via their substrate and/or membrane docking interaction(s), finally causing a metabolic shift in exposed cells together with their electron transfer disorder effect on transport chain.

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

Reactive oxygen species reduces the oxygen consumption and decreases the fatty acid oxidation in adipocytes causing the lipid accumulation [109]. According to Chirumbolo and

exposed rats [89].

**dysfunction**

A discrepancy has been found compared to the results presented by Braguini et al. [90]. Cytochrome c oxidase activity has decreased within different time series in deltamethrinexposed rat brains *in vivo* [91]. In these *in vivo* mitochondrial preparations, deltamethrin has caused a decrease in mitochondrial cytochrome c levels, mitochondrial membrane permeability transition, and mitochondrial membrane potential. These changes can result in a mitochondrial apoptosis and may reveal the neurotoxic action of pyrethroids. However, succinate cytochrome c reductase activity has not changed, while cytochrome c oxidase activity increased in the liver of deltamethrin-intoxicated rats *in vivo* [92]. In these liver preparations, biotransformation enzymes of pyrethroids have also not changed. In addition to their ROS inducing by ER-bound CYP450 activities, pyrethroids can disturb the electron transfer on the transport chain and can cause the altered ATP levels and ROS formation to induce mitochondrial dysfunction and sequential death.

Metabolic shift determined by increased lactate levels are observed in tumor cells although they are grown in oxygenic cultures, and this can be a strategy to avoid oxidative stress and apoptosis induction [93]. Pyrethroid intoxication causes a metabolic shift through the oxidative phosphorylation to anaerobic glycolysis and altered lipid and protein metabolism *in vivo*. Several pyrethroids have decreased the hepatic protein levels, increased hepatic lactate dehydrogenase, blood and plasma urea levels in rats [94, 95]. Authors have concluded that pyrethroids are able to stimulate metabolic shift from oxidative phosphorylation to anaerobic glycolysis. A support for these observations has been obtained in the muscle and heart of cypermethrin exposed rats [96]. It has caused the decreased succinate dehydrogenase while increased glucose-6-phosphate dehydrogenase and lactate dehydrogenase activities reflecting the anaerobiosis. Decreased succinate dehydrogenase activity indicates the inadequate substrate supply for TCA [96]. A similar metabolic shift due to succinate dehydrogenase and malate dehydrogenase inhibition with increased lactate formation and lactate dehydrogenase activity has also been observed in cypermethrin-intoxicated fish *Labeo rohita* [97] or in fenvalerate-intoxicated fish *Oreochromis niloticus* [98].

Hepatic aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase activities, total lipids, phospholipids, free fatty acids, and cholesterol levels have increased, while glycogen and total protein levels decreased in beta-cyfluthrin exposed rats [95]. Aminotransferases produce oxaloacetate and pyruvate intermediates that are transported into the mitochondrial matrix to maintain TCA or fatty acid production, and accumulation of oxaloacetate has caused the inhibition of malate dehydrogenase activity, a part of TCA of mitochondria in cypermethrin exposed rats [89].

40 regions of complex I as potential binding sites for pyrethroids because of their hydrophobic nature [86]. Inhibition of complex I may be related to ROS formation; but, it should be noted that complex I inhibitors can be divided into two groups as ROS producers and ROS production inhibitors [87]. Inhibition of complex I activity by permethrin has been caused a reduction in superoxide radical formation in striatum submitochondrial particles of rats [88]. Inhibition of succinate dehydrogenase activity, which has a role in TCA and in complex II, has been decreased after acute and subacute bifenthrin exposure in rat brain [89]. Deltamethrin has a major inhibition site between complexes II and III because of unaffected NADH dehydrogenase (complex I) and cytochrome c oxidase (complex IV) activities in the isolated rat liver mitochondrial preparation [90]. In this mitochondrial preparation, NADH oxidase, succinate oxidase, succinate dehydrogenase (complex II), NADH-cytochrome c reductase, and succinate cytochrome c reductase activities have been inhibited. Deltamethrin has also caused an inhibition of ADP-stimulated

A discrepancy has been found compared to the results presented by Braguini et al. [90]. Cytochrome c oxidase activity has decreased within different time series in deltamethrinexposed rat brains *in vivo* [91]. In these *in vivo* mitochondrial preparations, deltamethrin has caused a decrease in mitochondrial cytochrome c levels, mitochondrial membrane permeability transition, and mitochondrial membrane potential. These changes can result in a mitochondrial apoptosis and may reveal the neurotoxic action of pyrethroids. However, succinate cytochrome c reductase activity has not changed, while cytochrome c oxidase activity increased in the liver of deltamethrin-intoxicated rats *in vivo* [92]. In these liver preparations, biotransformation enzymes of pyrethroids have also not changed. In addition to their ROS inducing by ER-bound CYP450 activities, pyrethroids can disturb the electron transfer on the transport chain and can cause the altered ATP levels and ROS formation to induce mitochon-

Metabolic shift determined by increased lactate levels are observed in tumor cells although they are grown in oxygenic cultures, and this can be a strategy to avoid oxidative stress and apoptosis induction [93]. Pyrethroid intoxication causes a metabolic shift through the oxidative phosphorylation to anaerobic glycolysis and altered lipid and protein metabolism *in vivo*. Several pyrethroids have decreased the hepatic protein levels, increased hepatic lactate dehydrogenase, blood and plasma urea levels in rats [94, 95]. Authors have concluded that pyrethroids are able to stimulate metabolic shift from oxidative phosphorylation to anaerobic glycolysis. A support for these observations has been obtained in the muscle and heart of cypermethrin exposed rats [96]. It has caused the decreased succinate dehydrogenase while increased glucose-6-phosphate dehydrogenase and lactate dehydrogenase activities reflecting the anaerobiosis. Decreased succinate dehydrogenase activity indicates the inadequate substrate supply for TCA [96]. A similar metabolic shift due to succinate dehydrogenase and malate dehydrogenase inhibition with increased lactate formation and lactate dehydrogenase activity has also been observed in cypermethrin-intoxicated fish *Labeo rohita* [97] or in

Hepatic aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase activities, total lipids, phospholipids, free fatty acids, and cholesterol levels have increased, while glycogen and total protein levels decreased in beta-cyfluthrin exposed rats [95]. Aminotransferases produce oxaloacetate and pyruvate intermediates that are transported into the mitochondrial

oxygen consumption and impaired the mitochondrial membrane potential [90].

drial dysfunction and sequential death.

302 Mitochondrial Diseases

fenvalerate-intoxicated fish *Oreochromis niloticus* [98].

Metabolomics approach is very effective to understand pyrethroid-induced metabolic changes. Reports show the metabolic shift to anaerobic fuel consumption and elevated fuel supply via gluconeogenesis to maintain energy levels in pyrethroid-induced stress conditions. For example, permethrin exposure increased urine lactate, acetate, 3-D-hydroxybutyrate, creatine, glycine, and formate while decreased citrate and 2-oxoglutarate levels in rats [99]. Elevated levels of urinary acetate and decreased TCA intermediates show the energy metabolism disorders. Similarly, Liang et al. reported that permethrin and deltamethrin exposure can cause the disturbance in energy metabolism via the enhanced rate of anaerobic glycolysis and fatty acid β-oxidation, and ketogenesis [100]. They found that these pyrethroids reduced the excretion of TCA intermediates and increased lactate, acetate, 3-D-hydroxybutyrate levels in treated rats. In another study, serum and urine metabolites levels have been changed by deltamethrin exposure, and then it was suggested that decreased utilization of pyruvate in TCA and consecutive anaerobiosis in exposed rats [101]. While a shift from aerobic respiration to anaerobiosis was also found in the brain of lambda-cyhalothrin exposed goldfish (*Carassius auratus*), a marked decrease has been observed in brain *N*-acetyl-aspartate levels, because of neuronal mitochondrial membrane damage via the ROS formation [102]. *N*-acetylaspartate is considered as a marker for mitochondrial dysfunction in neurons [103]. Higher levels of malate and alanine in cypermethrin exposed earthworms provide an argument for the increased gluconeogenesis and fueling the TCA for energy [104]. These effects have also been observed in the former studies [100–102]. However, as an opposite of these results, permethrin exposure has caused an increase in TCA intermediates and cellular fatty acids and a decrease in glutamate levels in rat neuroblastoma cell line B50 [105]. Increased fatty acid β-oxidation should be a response to permethrin toxicity in these cells.

Voltage-dependent anion channels located in the mitochondrial outer membrane is the only way to supply TCA intermediates from cytosol to mitochondria, and its closure causes a metabolic shift [106]. However, urea generation is also operated in the mitochondrial matrix, and it requires a bulk of substrates such as ornithine, citrulline, adenine nucleotides, respiratory substrates, and other metabolites across the mitochondrial outer membrane in/out of mitochondria, possibly via VDACs [107]. The mitochondrial outer membrane is rich in VDACs that opens in normal operated mitochondria and mitochondrial hexokinase bounds to VDAC to orchestrate respiration, glycolytic pathway, and other metabolic pathways such as the pentose phosphate shunt [108]. We think that pyrethroids can be effective on these mitochondrial membrane proteins via their substrate and/or membrane docking interaction(s), finally causing a metabolic shift in exposed cells together with their electron transfer disorder effect on transport chain.
