Chemical structures of the pyrethroids that are mostly discussed in the current chapter.
Pyrethroids are used to decrease vector-based health concerns and to increase field yield against agricultural pests. Their metabolism is a concern to disrupt a cell’s homeostatic machinery via reactive oxygen species (ROS) production. They interact with lipid membranes to damage the fine balance between membrane lipids and membrane proteins, especially mitochondrial substrate transporters and electron carriers. Pyrethroids cause a shift in the metabolic energy production strategy, resulting in ROS production and intracellular lipid deposition. The change of open/closed conformation of some mitochondrial membrane proteins increases the vulnerability of mitochondria to Ca2+ ions. Membrane lipid fluidity change is also a concern because of permeability to the substrates and ions to produce energy and other substrates necessary for the cell. Pyrethroids can change the Ca2+ signaling and its interaction with ROS signals via disruption of the fine balance between endoplasmic reticulum and mitochondria. They can disrupt the mitochondrial DNA (mtDNA) via their hydrophobic nature or their ROS production capacity. In conclusion, mitochondria are the center of pyrethroid toxicity, and dysfunction of this organelle via pyrethroid toxicity plays an important role in the fate of cell. Their lipophilic and pro-oxidative nature together with Ca2+ homeostasis plays a synergistic role in this mitochondrial effect.
- reactive oxygen species
A pesticide has been described as an agent applied to kill, repel, or mitigate industry-, public health- and/or agriculture-related pests. They can also be used as plant growth regulator or nitrogen stabilizer. We use them to reduce the risk of decreased agricultural and industrial yield and prevent public health concerns such as vector-borne diseases, asthma and allergies, and microbial contamination (for more information: https://www.epa.gov/pesticides). Pesticides have entered into our lives more than 3000 years ago  and dried, ground Dalmatian pyrethrum flowers (contain natural pyrethrins) have been used against insect pests since ancient China. It has also been used in Europe more than 200 years ago against cockroaches, bedbugs, flies, and mosquitoes . A pyrethrin-derived synthetic pyrethroid allethrin has been synthesized in 1949 and entered the market in 1952 to use against household pests . To date, there are over 3500 pyrethroid-containing products registered .
The primary toxic effect of pyrethroids is on the voltage-gated sodium channels (VGSCs) like organochlorines such as DDT. The opening of these channels is extended by pyrethroid action and this causes the altered nerve function. According to their effect and chemical structure, pyrethroids divided into two types. Type I chemicals (allethrin, bifenthrin, bioresmethrin, permethrin, phenothrin, resmethrin, tefluthrin, and tetramethrin) do not contain a cyano group and they cause slowing in the closure of VGSCs. Therefore, the observed symptoms are tremors and seizures. On the contrary, Type II chemicals (cyfluthrin, cyhalothrin, cypermethrin, cyphenothrin, deltamethrin, fenpropathrin, fenvalerate, flucythrinate, flumethrin, fluvalinate, and tralomethrin) are the ones that are predominantly alpha-cyano-3-phenoxybenzyl alcohol esters and they cause a longer duration in the sodium current. The observed symptom is choreoathetosis accompanied by profuse salivation [5, 6]. Permethrin, a Type I pyrethroid, has not a disordering effect on polar head groups of phospholipids while it localizes within the hydrocarbon core . Because of its cyano group, cypermethrin, a Type II, localizes preferentially in the hydrophilic/hydrophobic region of the lipid plasma membrane, shows greater permanence and more fluidic effect on the membrane compared to permethrin . Therefore, the permanence of cypermethrin can be connected to the prolonged opening of sodium channels. This interaction could also be related to the more reduction of lipid-lipid interactions compared to Type Is; therefore, it decreases plasma membrane fluidity that is linked to the affected Na+-K+ ATPase activity to become the plasma membrane more permeable to the Na+ cations . Type I pyrethroids have a higher binding affinity to the protein of VGSCs  while they penetrate more easily into the cell. Although this is another issue for a review, the mutations observed on the VGSCs’ protein produce more resistant individuals against pyrethroid intoxication (for more information, see Silva et al. ). There is also a discrimination between these types according to their effects on calcium and chloride channels . Table 1 shows the chemical structures of pyrethroids that are mostly discussed in the current chapter.
Long-term health effects of pesticides such as their developmental and reproductory, endocrine disruption, neurobehavioral, carcinogenic, and immunological ones besides their acute impact have been considered by many scientists and regulatory services such as WHO, FAO, USEPA, and ECHA for many years. Currently, we experience the pesticides via drinking water, soil, food, and air. Directly ingesting of pesticide products can be assessed as a suicide action, but millions of acute poisoning cases occur in every year worldwide . Although the pyrethroid insecticides are less persistent in the environment compared to organochlorines, they are highly lipophilic with their high octanol/water partition coefficient (Kow) . Therefore, dietary exposure to these compounds trigger the safety concerns . Indoor application to control household pests is also another path for human exposure.
Significant pyrethroid residues have been found in drinking water, human breast milk, and cow milk in a sample location of South Africa where indoor residual spraying was applied for malaria control compared to a mountain population . Malaria control or agricultural applications have caused pyrethroid accumulation such as cypermethrin, lambda-cyhalothrin, esfenvalerate/fenvalerate, and permethrin in breast milk from Brazil, Colombia, and Spain mothers . However, the residues never exceeded the maximum daily intake levels. Babina et al. reported that more than one chemical and simultaneous exposure to organophosphate and pyrethroids was common in South Australian preschool children . Barr et al. surveyed the U.S. population with 5046 samples between the period of 1999 and 2002 to detect pyrethroid residues in urine samples, and they concluded that pyrethroid exposure is widespread in the U.S. population and children probably have higher exposure risk compared to adolescents and adults . Exposure to pyrethroids in the levels common in Canadian children’s urine has been associated with parent-reported behavioral anomalies . A sex-dependent attention-deficit/hyperactivity disorder has been found in U.S. 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 . Urinary pyrethroid residues have been correlated with increased chronic heart disease in nonoccupational exposed Chinese people . Occupational exposure to pyrethroids, for example, in the textile industry, is also an important issue throughout the world .
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 . The stereoisomeric nature plays a significant role in the biotransformation of some pyrethroids like fenvalerate . 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
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) .
The produced metabolites can be more potent endocrine disruptors than parent compound for humans . 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 . Moreover, abnormal locomotor activity observed in prenatal deltamethrin exposure has been associated with increased expression of CYP450 enzymes in the offsprings of rats . 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 . 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
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 . 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 . 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 , this group decomposes to cyanides and aldehydes to produce free radicals . Endogenously formed superoxide anion radical is dismutated to hydrogen peroxide (H2O2) spontaneously or a SOD-catalyzed reaction. The formed H2O2 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 H2O2 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 . The most important intracellular iron source is the active site of CYP450s because of their iron content in the catalytically active center [36, 37, 38, 39, 40].
Pro-oxidant nature of CYP450-mediated pyrethroid metabolism needs further clarification because of superoxide and H2O2 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 . 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 . 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 . 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 CYP2B-induction by etofenprox exposure . CYP450-mediated cytosolic and/or mitochondrial ROS formation [44, 45] might cause cell death , 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.
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 . Similar results have also been found in the brain of deltamethrin exposed rats . The number of apoptotic cells has been decreased by
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 . 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 ). 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 . As stated in the review of Chirumbolo and Bjørklund , 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.
3. Cellular Ca2+ stores and pyrethroids
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 . 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 . 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 . 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 patch-clamp electrophysiological and
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 . Therefore, this situation causes the ER stress-related nonmitochondrial apoptotic pathway in human SK-N-AS neuroblastoma cells by deltamethrin . According to this model, deltamethrin-induced 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 . Deltamethrin can activate directly the protein kinase C enzyme at its very low dose . According to the authors, “
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 . 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 .
Voltage-gated Ca2+-channel activation by allethrin has caused the mitochondrial cell death in rat Leydig cell tumor derived LC540 cells . 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 . 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 . 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 . 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 . 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 .
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 . 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 . 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  or in the heart of cypermethrin exposed frogs (
Anti-apoptotic protein Bcl-xL interacts with VDACs to transfer Ca2+ into the mitochondria . A continuous supply of Ca2+ into mitochondria via this way is necessary to maintain mitochondrial bioenergetics because of pyruvate, 2-oxoglutarate, and the NAD+-dependent isocitrate dehydrogenases, and three intramitochondrial tricarboxylic acid cycle (TCA) enzymes are stimulated by Ca2+ . 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 . Giacomello et al. proposed a schema for anti- or pro-apoptotic proteins in ER-mediated Ca2+ supply to mitochondria . 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 . 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 . Enan and Matsumura have observed the translocation of protein kinase C from the cytosol to the membrane fraction in pyrethroid exposed rat brain synaptosomes . 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 . 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 . Therefore, tissue specificity and the dose-response curve of pyrethroid action on mitochondrial Ca2+ supply from ER and apoptosis induction should be further investigated.
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  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 . 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 .
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 . 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 . Inhibition of complex I activity by permethrin has been caused a reduction in superoxide radical formation in striatum submitochondrial particles of rats . 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 . 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 . 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 .
A discrepancy has been found compared to the results presented by Braguini et al. . Cytochrome c oxidase activity has decreased within different time series in deltamethrin-exposed rat brains
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 . Pyrethroid intoxication causes a metabolic shift through the oxidative phosphorylation to anaerobic glycolysis and altered lipid and protein metabolism
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 . 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 .
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 . 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 . 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 . While a shift from aerobic respiration to anaerobiosis was also found in the brain of lambda-cyhalothrin exposed goldfish (
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 . 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 . 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 . 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 dysfunction
Reactive oxygen species reduces the oxygen consumption and decreases the fatty acid oxidation in adipocytes causing the lipid accumulation . 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 proliferator-activated receptor (PPAR) pathways . Stressed cells accumulate lipids and enhance the hypoxic stimulus, and this occurs via AMPK-signaling pathways.
Cypermethrin induces the
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 . 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 . 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 . 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 . 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 . Although the autophagy inhibitor, 3-methyladenine exacerbated the deltamethrin toxicity, pre-treatment with autophagy inducer rapamycin and antioxidant
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 . Interestingly, impaired mitochondrial dynamics and dysfunctional autophagy can also be a cause
Shen et al.  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
6. Mitochondrial membrane structures and dynamics in pyrethroid intoxication
Another effect of pyrethroids is on the structural integrity and dynamics of mitochondria observed in histopathological studies.
Pyrethroids can pass and interact with biological membranes because of their lipophilic nature . As mitochondrion stand its membranous structures, mitochondrial membranes and other proteins in addition to the electron transport proteins are candidate structures for 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
Effects of pyrethroids are not limited to mitochondrial membranes because of fluidity decline in the hydrophobic core of cypermethrin exposed rat erythrocyte plasma membrane . Similar fluidity decline has been observed in deltamethrin exposed common carp (
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 . Furthermore,
7. Mitochondrial DNA and pyrethroids
There are very few studies on the mitochondrial DNA (mtDNA) alterations induced by pyrethroids in vertebrates. According to the results of Wang and Zhao  study, mtDNA somatic mutation frequency has been increased in the lung tissue of pesticide exposed (including pyrethroids) fruit growers. They have concluded that the increased frequency of mtDNA mutations may result from ROS formation, and the frequency has somewhat like cancer patients’ tissues. Because of the adjacency of mtDNA to possible ROS formation centers in mitochondria , pyrethroid-induced mtDNA mutations could be linked to their ROS inducing potentials. In cypermethrin exposed zebrafish larvae, ROS induction has been augmented, while
Pyrethroids bifenthrin, cypermethrin, and deltamethrin have increased ρ-mutation frequency in
In conclusion, pyrethroids can perform their toxic action via their oxidative potentials including unbalanced Ca2+ flux in/out of the organelles and cells. Mitochondria might be the most vulnerable organelle for pyrethroid toxicity. Pyrethroids probably can change the interaction of mitochondrion and ER to create an imbalance between the fine equilibrium of ROS and Ca2+ signals. This affects the form of cellular metabolic energy production, accumulation of lipids and other metabolites, and cell death type. Pyrethroids can also change the mitochondrial membrane structures to affect their ability for metabolism and ROS production capacity. These effects may be related to the endocrine disruption, diabetic, dopaminergic, and obesity-induction potential of pyrethroids that are observed in exposed individuals as exemplified in the above sections such as altered lipid metabolism and cholesterol delivery into the mitochondria. However, there are many gaps that must be solved, such as, interaction with mitochondrial membrane proteins, specific mutagenesis caused by pyrethroid molecule and mtDNA interaction, etc.