Insecticides

Insecticides

C H A P T E R 23 Insecticides Ramesh C. Gupta and Dejan Milatovic INTRODUCTION nontarget mammalian (including humans), avian, and wildlife sp...

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23 Insecticides Ramesh C. Gupta and Dejan Milatovic

INTRODUCTION

nontarget mammalian (including humans), avian, and wildlife species. Recently, Barr and Buckley (2011) stated that biomarkers of exposure from samples of human tissues, fluids, and excreta offer qualitative and quantitative evidence of pesticide exposure. These measurements are particularly useful in exposure research because they can highlight population-based exposure trends and improve estimates of pesticide exposure and dose. Biomarkers of effects include measurements of biochemical, physiological, or behavioral alterations that are a consequence of pesticide exposure. Lastly, biomarkers of susceptibility are measurements of an individual’s inherent ability to respond to insecticide exposures. These measurements include observations of molecular properties and functions, such as genetic polymorphisims and enzyme activities, which can affect the pharmacokinetics of insecticides, along with an individual’s biochemical disposition towards disease progression and repair. Insecticide toxicity can be acute, subacute, or chronic, depending on the duration of exposure and the dose involved. Thus, selection of a sensitive, accurate, and validated biomarker of exposure and effects appears to be a challenging task. This chapter describes biomarkers of exposure and effects of common insecticides in humans and animals.

Insecticides are of chemical or biological origin and are meant to control and kill insects. They have been used around the world for centuries. People are exposed to insecticides in their homes, offices, gardens, and workplaces, or through trace contaminants in food. Insecticides are used most extensively in agriculture, horticulture, and forestry. As a result, farmers, farm hands, and their families are maximally exposed to these chemicals. Insecticides are also used to control vectors, such as mosquitoes and ticks that are involved in spreading public health diseases, such as malaria, West Nile disease, Lyme disease, and others. In developing countries, insecticides are often involved in suicide attempts in humans, and malicious or accidental poisonings in pets, birds, and wildlife. Insecticides constitute a large number of chemicals of different classes, and they not only exert toxicity in insects, but in vertebrate mammals as well, through different mechanisms of action. Because of distinct differences in chemical structures, insecticides interact with different target and nontarget sites, including receptors, enzymes, and many other known and unknown molecules. Most insecticides are neurotoxicants as they target the nervous system, but they can also target other organs and systems in the body. The binding sites and adducts can be used as biomarkers of exposure and/or effects of insecticides. The insecticides are metabolized through different metabolic pathways and either parent compounds or their metabolites are often used as biomarkers of exposure. Insecticides can cause harmful health effects ranging from minor pain to death in

R. Gupta (Ed): Biomarkers in Toxicology. ISBN: 978-0-12-404630-6

ORGANOPHOSPHATES AND CARBAMATES Organophosphate (OP) and carbamate (CM) insecticides are often discussed together as anticholinesterase

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© 2014 Elsevier Inc. All rights reserved. DOI: http://dx.doi.org/10.1016/B978-0-12-404630-6.00023-3

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agents, because the insecticides of both classes inactivate the acetylcholinesterase (AChE) enzyme. AChE inhibiting OPs are esters of phosphoric or phosphonic acid, while AChE inhibiting CMs are esters of carbamic acid. Structurally, OPs are much more complex than CMs, and are categorized into 13 different types, such as phosphates, phosphonates, phosphorothioates, and phosphoramidates. During the last half-century, antiChE insecticides have gained wide popularity around the world due to two major factors: (1) lack of residue persistence in the environment and in mammalian systems, and (2) development of lesser resistance in insects compared to the organochlorine class of insecticides. Currently, hundreds of OPs and dozens of CMs are available on the market for their use as insecticides. Presently, OPs alone represent 50% of worldwide insecticide use. Being extremely toxic and due to a lack of species selectivity, anti-ChE insecticides pose serious threats to the health of nontarget mammalian (including humans), wildlife, avian, and aquatic species. Depending on the dose and duration of exposure, these insecticides may adversely affect various body organs and systems (nervous system, skeletal muscles, digestive, cardiovascular, respiratory, ophthalmic, reproductive, endocrine, dermal, immune, and others) at cellular and molecular levels.

Mechanism of toxicity Mechanism of action differs substantially in acute, intermediate, and chronic toxicity of anti-ChE insecticides. Acute clinical signs of OPs and CMs are primarily attributed to AChE inactivation at synapses in the brain and at neuromuscular junctions in skeletal muscles (Pope, 1999; Gupta, 2006; Satoh and Gupta, 2010). Physiologically, AChE is responsible for the hydrolysis of the neurotransmitter acetylcholine (ACh) and the termination of its biological activity within a microsecond. Both OPs and CMs react covalently with a serine residue in AChE in a similar manner to ACh. OPs and CMs inactivate AChE activity by phosphorylation and carbamylation, respectively, and differ quantitatively in rates of dephosphorylation and decarbamylation of inhibited AChE. It needs to be mentioned that the AChE enzyme can “age” with OPs and not with CMs. Following AChE inhibition, free acetylcholine (ACh) accumulates at the nerve endings of all cholinergic nerves and causes overstimulation of electrical activity. Inactivation of AChE activity . 70% leads to a toxic level of ACh accumulation at central and peripheral sites. The molecular interactions between OPs and AChE have been studied in much more detail than those between CMs and AChE, and have been described in recent publications (Timchalk, 2006; Gupta and Milatovic, 2012). Pavlovsky

et al. (2003) demonstrated that pyridostigmine, by increasing free ACh, enhances glutamatergic transmission in hippocampal CA1 neurons. This mechanism offers an explanation as to how pyridostigmine and other AChE inhibitors, including OP nerve agents and pesticides, cause epileptic discharge and excitotoxic damage. Evidence suggests that some of the ChE inhibitors directly interact with muscarinic ACh receptors (mAChRs) and nicotinic ACh receptors (nAChRs). The agonistic, antagonistic, potentiating, and inhibitory effects of ChE inhibitors on nAChRs were described by Smulders et al. (2003). These authors established that CM insecticides, which have a more potent interaction with nAChRs, are the less potent inhibitors of AChE; nAChRs are considered additional target sites, as they are involved in the toxicity of these insecticides. In addition to AChE and ACh receptors, OPs and CMs bind to other serine-containing esterases (such as butyrylcholinesterase and carboxylesterases), and proteases in serum and tissues. In a series of experiments, Gupta et al. (1991a,b, 1993, 1994a,b) demonstrated significant changes in creatine kinase (CK) and lactic dehydrogenase (LDH) as well as changes in the isozymes in rats following an acute exposure to carbofuran and methyl parathion. Leakage of these enzymes into the serum from target tissues was due to carbofuran- or methyl parathion-induced depletion of ATP in tissues (Gupta et al., 2000, 2001a,b). OP and CM insecticides also exert a myriad of toxic effects through multiple noncholinergic mechanisms soon after AChE inactivation (Gupta, 2004; Gupta et al., 2007; Zaja-Milatovic et al., 2009; Gupta and Milatovic, 2010, 2012; Terry, 2012). Among many noncholinergic mechanisms, activation of glutamatergic (NMDA receptors), adinosinergic, GABAergic, and monoaminergic systems appeared to be involved in the seizures and lethality associated with OP- or CM-induced poisonings (Solberg and Belkin, 1997; Choudhary et al., 2002; Dekundy and Kaminski, 2010; Slotkin and Seidler, 2012). OP-/CM-induced excitotoxicity for more than an hour can cause neurodegeneration and neuroinflammation in the cortex, amygdala, hippocampus, and some other brain regions involved in initiation and propagation of convulsions and seizures. The early morphological lesions include dendritic swelling of pyramidal neurons in the CA1 sector of the hippocampus. The AChE inhibitor-induced neuronal cell death is a consequence of a series of extra- and intracellular events leading to the intracellular accumulation of Ca12 ions and the generation of free radicals (Gupta et al., 2001a,b; Milatovic et al., 2010; Kazi and Oommen, 2012). OP- or CM-induced excessive production of free radicals causes oxidative and nitrosative stress to which the

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brain is especially vulnerable (Gupta et al., 2007, ZajaMilatovic et al., 2009; Gupta and Milatovic, 2010, 2012). Lipids are readily attacked by free radicals, resulting in the formation of a number of peroxidation products, such as F2-isoprostanes and F4-neuroprostanes, which are formed nonenzymatically. F4-neuroprostanes are specific biomarkers of oxidative damage to the neurons. These events, in addition to many more described elsewhere, cause irreversible destruction of cellular components, including proteins, DNA, and, particularly, mitochondria (Milatovic et al., 2006). Oxidative/nitrosative stress has been demonstrated to cause depletion of high-energy phosphates (ATP and phosphocreatine) by multiple mechanisms in the brain of rats acutely intoxicated with OP and CM insecticides (Gupta et al., 2001a,b; Gupta et al., 2007; Zaja-Milatovic et al., 2009). As mentioned earlier, in addition to the nervous system and skeletal muscles, OPs and CMs adversely affect many other body organs and systems and therefore additional mechanisms of action are involved in toxicity (Gupta, 2006; Satoh and Gupta, 2010; Gupta et al., 2011).

TABLE 23.1 List of common OP insecticides and their metabolites

Biomarkers

phosphates are derived from pesticides such as dichlorvos, monocrotophos, and dicrotophos, and diethyl phosphates are derived from pesticides such as chlorfenvinphos and paraoxon (Huen et al., 2012). These are the most commonly assayed metabolites for the exposure, biomonitoring, and risk assessment of OP pesticides (Duggan et al. (2003). Dulaurent et al. (2006) reported a LC-MS method for simultaneous determination of six DAPs in urine. DAP concentrations provide nonspecific information about exposure to a class of OPs, rather than a specific OP compound. Currently, among OPs, the most common metabolite measured is 3,5,6trichloropyridinol, a major metabolite of chlorpyrifos (Smith et al., 2012). Specific metabolites of malathion, such as malathion dicarboxylic acid and α and β isomers of malathion monocarboxylic acid, are also measured. Martinez and Ballesteros (2012) determined chlorfenvinphos and its metabolites (2-chloro-1-(2,4-dichloro-phenyl) vinyl ethyl hydrogen phosphate; 1-(2,4-dichlorophenyl) ethyl-β-D-glucuronic acid; 2,4-dichloromandelic acid; and 2,4-dichlorophenylethanediol glucuronide) in various tissues and fluids using GC-FID and GC-MS. Other commonly determined metabolites of OPs include 2-isopropyl-4-methyl-6-hydroxy-pyrimidine, a metabolite of diazinon; and 4-nitrophenol, a metabolite of parathion, methyl parathion, or EPN (Barr and Buckley, 2011); and 3-methyl-4-nitrophenol, a metabolite of fenitrothion (Okamura et al., 2012). Residues of several CMs are measured in serum, plasma, or whole blood as biomarkers of exposure to CMs. In general, CMs are unstable in blood, so their

Acute toxicity Biomarkers for the monitoring of insecticides can typically be divided into three categories: biomarkers of exposure, effect, and susceptibility. Biomarkers of exposure to OPs and CMs may include determination of residue of parent compounds and/or their metabolites, and modified cells or their molecules (e.g. DNA and protein adducts) in biological tissue/fluids. Highly sophisticated and sensitive chromatographic, spectrometric, and other assays allow the confirmation and quantitation of OP/CM insecticides and their metabolites in body fluids and tissues at the ppm or ppb level. Jain (2006) described detailed aspects of sample selection, extraction, concentration, and analytical methods for detection of various OPs and CMs and their metabolites. Since these insecticides are unstable and metabolize in the body system very rapidly, it is highly likely that most often metabolites can be detected and not the parent compounds. Some commonly used OPs and their major metabolites that are used as biomarkers of OP exposure are listed in Table 23.1. Currently, a total of six dialkyl phosphate (DAP) metabolites are commonly identified and quantified, using GC-MS, as biomarkers of organophosphate pesticides exposure. Among these, there are three dimethyl phosphate metabolites (dimethylphosphate, dimethylthiophosphate, and dimethyldithiophosphate) and three diethyl phosphate metabolites (diethylphosphate, diethylthiophosphate, and diethyldithiophosphate). Dimethyl

Parent Compound

Specific Metabolite(s)

Azinphos-methyl/-ethyl 1,2,3-Benzotriazin-4-one Chlorfenvinphos 2-chloro-1-(2,4-dichlorophenyl)vinylethyl hydrogen phosphate, 1-(2,4-dichlorophenyl)ethyl-β-Dglucuronic acid Chlorpyrifos-methyl/ 3,5,6-Trichloro-2-pyridinol -ethyl Coumaphos 3-Chloro-4-methyl-7-hydroxypyrimidine Diazinon 2-Isopropyl-6-methyl-pyrimidin-4-ol Fenitrothion 3-Methyl-4-nitrophenol Isazofos, methyl/ethyl 5-Chloro-1,2-dihydro-1-isopropyl-[3H]1,2,4-triazol-3-one Malathion 2-[(Dimethoxyphosphorothioyl)sulfanyl] succinic acid, Malathion monocarboxylic acid, and Malathion dicarboxylic acid Methamidophos/ O,S-dimethyl hydrogen acephate phosphorothioate Parathion-methyl/4-Nitrophenol, also known as ethyl p-nitrophenol Pirimiphos-methyl 2-Diethylamino-6-methylpyrimidin-4-ol

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metabolites are often assayed. Metabolite(s) of carbaryl (1-naphthol), propoxur (2-isopropoxyphenol), and carbofuran (3-ketocarbofuran and 3-hydroxycarbofuran) are determined in serum or plasma for biomonitoring of CM insecticides. Some CM insecticides commonly measured in urine include aldicarb, carbofuran, carbosulfan, and pirimicarb. Carbaryl exposure is usually estimated based upon urinary measurements of 1-naphthol. However, 1-naphthol and 2-naphthol are also metabolites of naphthalene. Since the measurement of 1-naphthol does not distinguish these two pesticides, measurement of 4-hydroxycarbaryl glucuronide helps to circumvent this problem. In recent studies, OP and CM insecticides and their metabolites have also been measured in saliva. The data can be used as biomarkers of exposure, and for pharmacokinetics and pharmacodynamics (Timchalk, 2006; 2010; Timchalk et al., 2007; Smith et al., 2012). In two acute carbofuran poisoning cases, residues of carbofuran and its major metabolite 3-hydroxycarbofuran were detected in human hair (Dulaurent et al., 2011). Novel biomarkers of exposure to OP insecticides and nerve agents, and CM insecticides form their adducts with serine of butyrylcholinesterase and tyrosine of albumin, transferrin, tubulin, keratin, and other proteins (Grigoryan et al., 2009; Li et al., 2009, 2010; Stefanidou et al., 2009; Read et al., 2010; Schopfer et al., 2010; Tacal and Lockridge, 2010). These measurements can be directly related to the dose of an insecticide and are a function of insecticide exposure. Blood AChE inhibition is still considered one of the most sensitive and reliable biomarkers of exposure to OP and CM insecticides. Scientific and regulatory communities have identified and recognized RBC-AChE inhibition as a sensitive biomarker of exposure to OPs and CMs because it serves as a sensitive surrogate endpoint for the inhibition of brain AChE. In California, regulators specify monitoring blood ChE when using pesticides with toxicities , 50 mg/kg (Class I pesticides), and $ 50 and # 500 mg/kg (Class II pesticides). Examples are aldicarb, azinphos methyl (Class I pesticides) and malathion (Class II pesticide). In a recent experimental study, Reiss et al. (2012) stated that brain AChE inhibition is the most appropriate matrix for risk assessment of OP pesticides, such as chlorpyrifos. Muttray et al. (2005) found that EEG is possibly more sensitive than ChE inhibition in farmers exposed to low doses of methyl parathion. In recent years, egasyn-β-glucuronidase has also gained attention for being a sensitive biomarker of an acute OP exposure (Ueyama et al., 2010). In another study, liver prenylated methylated protein methyl esterase has been found as a sensitive enzyme in liver and brain to the exposure and effect of OPs. Perturbations in

prenylated protein metabolism are involved in OPinduced noncholinergic toxicity, since prenylated proteins play a role in cell signaling, proliferation, differentiation, and apoptosis (Lamango, 2005). Alterations in these enzymes can be used as biomarkers of OP exposure or effects, but none of these can reveal the identity of a particular OP insecticide. Additionally, multiple noncholinergic mechanisms are involved in OP and CM induced developmental neurotoxicity that occurs at levels of OPs far below those that cause AChE inhibition (Slotkin, 2006; Bouchard et al., 2011; Slotkin and Seidler, 2012). Using an NMR-based metabonomic approach, Liang et al. (2012) reported alterations of urinary profiles of endogenous metabolites in rats subacutely exposed to propoxur. Interestingly, in many of the investigations cited here, noncholinergic alterations occurred at doses below those that induce AChE inhibition. These noncholinergic endpoints can be used as biomarkers of OP/CM-induced effects.

Intermediate syndrome Humans exposed to large doses of an OP (methamidophos, fenthion, dimethoate, monocrotophos, etc.) or a carbamate (carbofuran) insecticide can suffer from intermediate syndrome (IMS), after the acute cholinergic crisis. IMS usually occurs 24 96 h after exposure to the insecticide due to insufficient or lack of oxime therapy and severe and prolonged AChE inhibition. IMS is clearly a separate entity from acute toxicity and delayed neuropathy, and it is characterized by acute respiratory paresis and muscular weakness, primarily in the facial, neck, and proximal limb muscles. IMS is also accompanied by generalized weakness, cranial nerve palsies, depressed deep tendon reflexes, ptosis, and diplopia. These symptoms may last for several days or weeks, depending on the insecticide involved. It needs to be pointed out that despite severe AChE inhibition, muscle fasciculations and mAChRs-associated hyper-secretory activities are absent. Multiple mechanisms appear to be involved in IMS, such as AChE inhibition, oxidative stress, nAChR mRNA expression, and changes in repetitive nerve stimulation (RNS). In addition, electrophysiological abnormalities occur at the neuromuscular junctions in patients with IMS. All these parameters can be used as biomarkers of IMS induced by OPs or CMs (De Bleecker, 2006).

Delayed toxicity Exposure to some OPs can cause another type of toxicity known as OP-induced delayed polyneuropathy (OPIDP). Symptoms of OPIDP appear after symptoms

ORGANOPHOSPHATES AND CARBAMATES

of acute cholinergic crisis and IMS subside, that is, 2 3 weeks after a single-dose exposure. Symptoms of this syndrome include tingling of the hands and feet, followed by sensory loss, progressive muscle weakness, and flaccidity of the distal skeletal muscle of the lower and upper extremities, and ataxia. Pathogenesis of OPIDP involves phosphorylation (inhibition) of neuropathy target esterase (NTE) enzyme and its “aging” in peripheral nerves (Gupta and Milatovic, 2012). NTE is a large polypeptide of 1327 amino acids, a membrane bound esterase with a molecular weight of 155 kDa. Its physiological role has not yet been established. Large epidemics of OPIDP occurred in the United States, Morocco, Fiji, and India from ingestion of triortho-cresyl phosphate (TOCP) affecting tens of thousands of people. OPIDP has also been caused by certain OP pesticides, such as leptophos, dichlorvos, fenthion, trichloronat, trichlorfon, methamidophos, and chlorpyrifos (Lotti and Moretto, 2005; Jokanovic et al., 2011). Although the severity of OPIDP does not appear to correlate with the degree of aged NTE, NTE inhibition and its aging are still considered as the best understood mechanisms and biomarkers (Mangas et al., 2012).

Chronic toxicity Following chronic exposure to low levels of OPs, pesticide application workers, greenhouse workers, agricultural workers, and farm residents reveal a relatively consistent pattern of neurobehavioral deficits (Rohlman et al., 2011; Lein et al., 2012). Patients can suffer from chronic OP-induced neuropsychiatric disorder (COPIND) with symptoms of anxiety, depression, difficulty in concentrating, memory impairment, and others (Jamal and Julu, 2002; Pancetti et al., 2007; Ross et al., 2010; Chen, 2012; Payan-Renteria et al., 2012). Pancetti et al. (2007) described the role of an enzyme, acylpeptide hydrolase, in cognitive processes, which are usually compromised following chronic OP exposure. Mixers, loaders, applicators, and flaggers, but not field workers themselves, are tested for blood ChE activity, if they work with pesticides for 7 days or more within a 30-day period (Wilson et al., 2005). Some studies demonstrated a link between neurobehavioral performance and current biomarkers of OP exposure including blood ChE activity and urinary metabolites of OPs. Arguably, other studies suggested that these biomarkers are neither predictive nor diagnostic of the neurobehavioral effects of chronic pesticide exposure, and biomarkers of neuroinflammation and oxidative stress need to be included, in addition to blood ChE and urinary metabolites (Banks and Lein, 2012). Pancetti et al. (2007) described acylpeptide hydrolase as a more sensitive enzyme than AChE,

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and therefore proposed it as a biomarker of OP exposure and associated cognitive deficits. In addition, dystonic reactions, schizophrenia, cogwheel rigidity, choreoathetosis and EEG changes have been reported with high-dose exposure. These extrapyramidal symptoms are thought to be due to the inhibition of the AChE in the human extrapyramidal area. Psychosis, delirium, aggression, hallucination and depression may also be seen during recovery from the cholinergic syndrome. Schizophrenic and depressive reactions with severe memory impairment and difficulties in concentration are observed in workers exposed to these pesticides, and RBC-AChE is used as a biomarker of these effects. Recently, Middlemore-Risher et al. (2010) in an experimental study observed that repeated exposures to low-level chlorpyrifos caused impairments in sustained attention and increased impulsivity in rats. Speed et al. (2012) examined hippocampal synaptic transmission and pyramidal neuron synaptic spine density in mice treated with a non-signs producing dose of chlorpyrifos (CPF, 5 mg/kg/day for 5 consecutive days) early (2 7 days) and late (3 months) after the last injection. Increased synaptic transmission was found in the CA3-CA1 region of the hippocampus of CPF-treated mice 2 7 days after the last injection. In contrast, 3 months after CPF treatment, a 50% reduction in synaptic transmission occurred due to a 50% decrease in CA1 pyramidal neuron synaptic spine density. Findings suggested that progressive synaptic abnormalities occurred leading to persistent brain damage, despite the absence of cholinergic toxicity. Rats acutely intoxicated with a signs-producing dose of carbofuran (1.5 mg/kg, sc) or DFP (1.5 mg/kg, sc) showed marked decreases in dendritic length and spine density in the CA1 region of hippocampal pyramidal neurons due to oxidative/nitrosative stress and depletion of high-energy phosphates (Gupta et al., 2007; ZajaMilatovic et al., 2009). Both AChE inhibitors also caused significant increases in PGE2, a marker of neuroinflammation. Of course, cytokines and C-reactive protein can also be used as biomarkers of inflammation (Lein et al., 2012). OPs and CMs produce a variety of reproductive and developmental toxicity in humans and animals. Following in utero exposure to OP/CM insecticides, brain damage can occur in children in an absence of cholinergic toxicity. Biomarkers for reproductive and developmental effects of these insecticides are described in detail by Gupta et al. (2011). In an electrophysiological study, Nio and Breton (1994) investigated the effects of paraoxon and physostigmine on rabbit pyramidal cells firing pattern and hippocampal theta rhythm. The results revealed that paraoxon and physostigmine have a rather similar

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influence on the septo-hippocampal pathway and also suggested that paraoxon could act within local hippocampal circuitry through other systems than the cholinergic system exclusively. In similar studies, Desi and Nagymajtenyi (1999), Papp et al. (2004), and Narahashi (2006) investigated electrophysiological effects of various OPs and CMs, suggesting the involvement of noncholinergic mechanisms. In a preliminary study, De Luca et al. (2006) showed the electromyographic signal as a presymptomatic indicator of OPs in the body, using DFP as a test substance. These parameters can be used as biomarkers of morphological and electrophysiological changes in CNS and PNS, and early indicators of behavioral alterations. A human genetic polymorphism of paraoxonase-1 (PON-1) in detoxification of several OP insecticides, including the active metabolite of chlorpyrifos, chlorpyrifos-oxon, is well established, resulting in the expression of PON-1 activity within a segment of the population. PON-1 and cytochrome P450 can be used as biomarkers of genetic polymorphism in regard to OP toxicity (Costa et al., 2006; Furlong et al., 2006; Huen et al., 2012; Lein et al., 2012). In a recent study, Huen et al. (2012) found that, compared to their mothers, newborns have much lower quantities of the detoxifying PON-1 enzyme, suggesting that infants may be especially vulnerable to organophosphate pesticide exposure.

CHLORINATED HYDROCARBONS Chlorinated hydrocarbon or organochlorine insecticides are classified into three groups: (1) dichlorodiphenylethanes (DDT, dicofol, methoxychlor, and perthane), (2) hexachlorocyclohexanes (benzene hexachloride, chlordane, lindane, mirex, and toxaphene); and (3) chlorinated cyclodienes (aldrin, dieldrin, endrin, endosulfan, and heptachlor). The first use of the chlorinated hydrocarbons or organochlorines was for dielectrics and as fire retardants. The first use of these compounds as insecticides occurred when benzene was added to liquid chlorine in the field and it was noted that the product killed insects. Cyclodienes, such as aldrin and dieldrin, used as insecticides, became available for use in the 1940s. Dichlorodiphenyltrichloroethane (DDT) became available during WWII and was used extensively as an insecticide worldwide. Because of their persistence in the environment and in biological systems, most insecticides of this group have been eliminated from use today. Endosulfan and lindane are the most biodegradable organochlorines and are still used today.

Mechanism of toxicity There are at least two different mechanisms of action for organochlorine insecticides (Narahashi, 1987; Ensley, 2012a). DDT type insecticides affect the peripheral nerves and brain by slowing Na1 influx and inhibiting K1 efflux, resulting in excess intracellular K1 in the neuron, which partially depolarizes the cell. The threshold for another action potential is decreased leading to premature depolarization of the neuron. The aryl hydrocarbons and cyclodienes, in addition to decreasing action potentials, may inhibit the post-synaptic binding of GABA (Bloomquist and Soderlund, 1985). The cyclodiene-induced hyperactivity of the CNS and convulsions can be explained based on their structural resemblance to the GABA receptor antagonist picrotoxin. The cyclodienes act by competitive inhibition of the binding of GABA, an inhibitory neurotransmitter at its receptor, causing stimulation of the neurons. Both GABAA and GABAB receptors play a vital role in mammalian toxicity. GABAA present in the mammalian synapse are ligand gated chloride ion channels. In the human brain, the GABAA receptor consists of four or five hydrophobic domains, namely M1, M2, M3, M4, and ˚ M5. The five M2 domains are arranged to form a 5.6 A diameter ion channel. GABAB receptors present in mammals are coupled to calcium and potassium channels and the action of this neurotransmitter is mediated by G-proteins. Following its release in the synapse, GABA diffuses to the presynaptic terminal of another nerve, where GABA binds to its GABAA receptors. The binding of GABA to its receptor causes chloride ions to enter the synapse, leading to hyperpolarization of the terminal, which inhibits the release of other neurotransmitters. Due to such inhibition, postsynaptic stimulation of other nerves by other neurotransmitters, such as acetylcholine, is reduced. As a consequence of inhibition of GABA, there is no synaptic downregulation and there can be excessive release of other neurotransmitters. CNS symptoms in animals poisoned by chlorinated cyclodienes include tremors, convulsions, ataxia, and changes in EEG patterns. The CNS symptoms could be due either to: (1) inhibition of the Na1/K1-ATPase or the Ca21/Mg21-ATPase activity, which can then interfere with nerve action or release of neurotransmitters, and/or (2) inhibition of the GABA receptor function. In a recent study, Mladenovic et al. (2010) reported that lipid peroxidation may contribute to the neurotoxic effects of lindane in early acute intoxication and that behavioral manifestations correlate with lipid peroxidation in the rat brain hippocampus, which is one of the sites for initiation and propagation of seizures. The nervous system of the developing organism appears to be more vulnerable to the toxicity of organochlorine insecticides, and multiple mechanisms are

PYRETHRINS AND PYRETHROIDS

involved. DDT and related compounds produce a direct effect on the motor fibers and on the motor area of the cerebral cortex or they act as endocrine disruptors in the hypothalamic-hypophysis-thyroid axis. Neonatal exposure to DDT causes a significant reduction in the density of muscarinic receptors in the cerebral cortex of mice. These cholinergic receptors have a direct involvement in the process of neuronal excitement and inhibition. Following chronic exposure, DDT causes disruption of the thyroid system. DDT or its metabolites alter the production of thyroidal hormone and its availability to target tissues, given that the insecticide blocks glucuronidation. Thyroid hormone is known to play a pivotal role in the development of the cerebral cortex. This hormone serves as a signal for neuronal differentiation and maturation as well as participates in neuronal migration and proliferation, synaptogenesis, and myelinization. Developmental exposure to dieldrin has been shown to alter the dopamine system and increase neurotoxicity in an animal model of Parkinson’s disease. The dopamine transporter (DAT) and the vesicular monoamine transporter 2 (VMAT2) play an integral role in maintaining dopamine homeostasis and alteration of their levels during development could result in increased vulnerability of dopamine neurons later in life. Prenatal exposure of mice during gestation and lactation to low levels of dieldrin (0.3, 1 or 3 mg/kg every 3 days), caused a dose-dependent increase in protein and mRNA levels of DAT and VMAT2 in their offspring at 12 weeks of age (Richardson et al., 2006). In a recent investigation, Kamel et al. (2012) suggested that amyotrophic lateral sclerosis risk may be associated with use of organochlorine pesticides.

Biomarkers Residues of organochlorine insecticides and their metabolites can be detected at ppb levels in human and animal tissues and fluids (blood, urine and milk). GC and GC-MS are most commonly used to confirm and quantitate the residues of organochlorines and their metabolites. Biomonitoring studies have revealed that these insecticides are widely distributed throughout the body and deposit in fat and fatty tissue. Residues have been found in various human reproductive tissues, including amniotic fluid, blood stream, maternal blood, umbilical cord blood, breast milk, colostrum, placenta, semen, and urine (Malik et al., 2011). Recent studies suggest that the organochlorine endosulfan causes acetylcholinesterase (AChE) inhibition, neurotoxicity, and brain function impairment in rat, rabbit, and zebrafish (Mor and Ozmen, 2010; Silva de Assis et al., 2011; Pereira et al., 2012).

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In general, residue of organochlorine insecticides and/or their metabolites in blood/serum, fat, and urine are used as biomarkers of exposure. In the case of endosulfan, AChE inhibition can also be used as a biomarker of toxicity.

PYRETHRINS AND PYRETHROIDS Pyrethrins and pyrethroids (synthetic pyrethrins) have a wide variety of applications in agriculture, public and animal health, and residential settings throughout the world. Pyrethroid insecticides have also been used for disinsection of commercial aircraft (Wei et al., 2012). Pyrethrins were first developed as insecticides from extracts of the flower heads of Chrysanthemum cinerariaefolium, whose insecticidal potential was appreciated in ancient China and Persia. There are six naturally occurring pyrethrins (pyrethrins I and II, cinerins I and II, and jasmolins I and II). Because of their rapid decomposition by light and air, synthetic derivatives were developed, and are commonly referred to as pyrethroids (Anadon et al., 2009). The first generation pyrethroids were developed in the 1960s, including bioallethrin, tetramethrin, resmethrin, and bioresmethrin. By 1974, a second generation of more persistent compounds was developed, including permethrin, cypermethrin, and deltamethrin. These insecticides have significantly greater mammalian toxicity compared to first generation pyrethroids. Later, some other pyrethroids, such as fenvalerate, lambda-cyhalothrin, and beta-cyfluthrin, were discovered. Because of their high insecticidal potency, broad-spectrum activity, relatively low mammalian toxicity, lack of environmental persistence, and less insect resistance, pyrethroids have gained much success in the recent past, and now account for more than 25% of the global insecticide market. Using a recent nomenclature, pyrethroids are of two types and produce two syndromes through multiple mechanisms of action. Type I pyrethroids are those which lack α-cyano-3-phenoxybenzyl moiety and give rise to the tremor syndrome (T syndrome). A few examples of type I are pyrethrin I, allethrin, bioallethrin, tetramethrin, resmethrin, cismethrin, phenothrin, and permethrin. These insecticides cause severe fine tremors, marked reflex hyperexcitability, sympathetic activation, and paresthesia (with dermal exposure). Type II pyrethroids are those which contain α-cyano-3-phenoxybenzyl moiety and cause the choreoathetosis/salivation (CS) syndrome. A few examples of type II pyrethroids include cyphenothrin, cypermethrin, deltamethrin, fenvalerate, cyfluthrin, cyhalothrin, flucynthrate, and esfenvalerate. These insecticides produce

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profuse watery salivation, coarse tremors, increased extensor tone, moderate reflex hyperexcitability, sympathetic activation, choreoathetosis, seizures, and paresthesia (dermal exposure). The clinical signs of type I and type II pyrethroids appear to be largely independent of the route of administration. Trans- and cis- isomers of fluorocyphenothrin confusingly produce type I and type II effects, respectively. Some pyrethroids, such as fenpropathrin, have been classified as hybrids and appeared to show a mixture of types I and II effects (T and CS). All pyrethroids cause a reduction in motor activity. Reduced locomotor activity in the rat after oral gavage dosing has been used to quantify pyrethroid neurotoxicity. Unfortunately, the effect is nonspecific and does not readily allow conclusions to be made about mechanism of action (Gammon et al., 2012). Overall, type I pyrethroids with a trans substituted acid moiety have a much lower mammalian toxicity than the corresponding cis isomers. This is partly due to the more rapid ester hydrolysis of trans rather than for cis isomers. For type II pyrethroids, there is much less difference in toxicity between trans and cis isomers, suggesting a similar target site for type II pyrethroids. Both pyrethroid classes have a similar range of mammalian toxicity, but for commercial pesticides, type II pyrethroids such as deltamethrin and cypermethrin are generally more toxic than type I pyrethroids such as permethrin (Ray and Forshaw, 2000). A survey of the general US population has revealed that exposure to pyrethroid insecticides is widespread and that children may have higher exposures than adolescents and adults (Barr et al., 2010). In general, young animals and children are more susceptible than adults to pyrethroid toxicity, and the focus of ongoing research is to explore to which degree children are more susceptible than adults. Flight attendants of commercial aircrafts disinsected with pyrethroids have complains of irritation of the skin and mucosa, sore throat, vomiting, abdominal pain, headache, dizziness, and nausea (Murawski, 2005; Sutton et al., 2007; Wei et al., 2012). It needs to be pointed out that pyrethrins and pyrethroids also exert reproductive and developmental toxicity in animals and humans (Malik et al., 2011).

Mechanism of toxicity The primary effects of pyrethroids in mammalian species include neurotoxicity and neurobehavioral toxicity that result primarily from hyperexcitation of the nervous system. Type I syndrome involves action in the CNS and PNS, while type II syndrome (CS) involves primarily the CNS. Hyperexcitation of the nervous system is caused by repetitive firing and depolarization of

the nervous system. The primary site of action of pyrethroids is the sodium channels of cells, but they also affect chloride and calcium channels. These insecticides slow the opening and closing of the sodium channels, ultimately leading to the excitation of the cell. An increase of sodium in the sodium channels results in a cell which is in a stable and hyper-excitable state. The duration of the sodium action potential is much shorter for type I pyrethroids than for type II. While type I pyrethroids result in primarily repetitive charges, cell membrane depolarization is the main mechanism of toxic action exerted by type II pyrethroids. The effect of type I pyrethroids on sodium channels in nerve membranes is similar to those produced by DDT-type insecticides. The direct action of pyrethroids on sensory nerve endings leads to paresthesia, as they cause repetitive firing (Ensley, 2012b). If not all, most of the neurotoxic effects of pyrethroids are due to their interaction at voltage-gated sodium channels. The degree of hyperexcitability is doserelated, but the nature of this excitability is pyrethroid structure-dependent. Insect sodium channels are 100 times more sensitive than rat brain sodium channels. The lower sensitivity of mammalian sodium channel isoforms compared with insect channels to pyrethroids offers a mechanistic explanation for its species selective toxicity. Of course, it has become clear that besides insensitive mammalian sodium channels (e.g. Nav 1.2 and Nav 1.7), more sensitive isoforms also exist (e.g. Nav 1.3, Nav 1.6 or Nav 1.8). Recently, McCavera and Soderlund (2012) provided direct evidence for the preferential binding of deltamethrin and tefluthrin (but not S-bioallethrin) to Nav 1.6Q3 channels in the open state and implied that the pyrethroid receptor of resting and open channels occupies different conformations that exhibit distinct structure activity relationships. Interestingly, the Nav 1.6 isoform is not only a target for developmental neurotoxic effects, but also is a likely target for the central neurotoxic effects of pyrethroids in adult animals. Voltage-gated chloride and calcium channels have been implicated as additional sites of action (Soderlund, 2012). Voltage-sensitive chloride channels are found in nerves, muscles, and salivary glands, and are modulated by protein kinase C. The function of chloride channels is to control cell excitability. The decrease in the chloride-open channel state produced by type II pyrethroids serves to increase excitability and therefore to synergize pyrethroid actions on the sodium channel. While calcium channels may contribute in some way to the action of at least some pyrethroids, there is no basis at present to identify effects on calcium channels as an essential mechanism of pyrethroid intoxication (Shafer and Meyer, 2004). Recently, Gammon et al. (2012) suggested that different clinical signs associated with type I

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and type II syndromes result from effects on different channel isoforms or a combination of them. Interestingly, Ali (2012) has demonstrated involvement of oxidative stress in pathogenesis of neurotoxicity by type II pyrethroid lambda-cyhalothrin in rats. For further details on molecular mechanisms in neurotoxicity and neurobehavioral toxicity of pyrethrins and pyrethroids, readers are referred to Wolansky and Harrill (2007), Soderlund (2012), and Van Thriel et al. (2012).

Biomarkers Pyrethrins and pyrethroids are structurally diverse, and understanding their chemistry and toxicology plays a vital role in the development of biomarkers. Significant advancements in analytical chemistry and toxicology have led to the development of biomarkers to assess biomonitoring in the environment and exposure in the general population (Sudakin, 2006; Barr et al., 2010; Gammon et al., 2012). Future challenges in the application of these biomarkers in epidemiological studies are being explored, as there is a need for improved understanding of the toxicokinetics and pharmacodynamics of pyrethroids in mammalian species, including humans. Pyrethroids are of low to moderate toxicity due to their moderate absorption (40 60%) and rapid metabolism following oral administration (Anand et al., 2006; Barr et al., 2010; Gammon et al., 2012). Oxidases and esterases, primarily in the liver, metabolize pyrethroids at varying rates. The most rapidly metabolized pyrethroids have the lowest toxicity. Based on experimental data, Gammon et al. (2012) have described that brain or blood plasma concentrations of parent pyrethroids correlate with acute toxicity and that metabolites (especially hydrolytic products) generally have little or no effect on neurotoxicity. In the context of biomarkers, most studies have investigated urine as the analytical matrix (Barr et al., 2010). Leng et al. (1996) and Leng and Gries (2005) detected metabolites of some pyrethrins and pyrethroids (permethrin, cypermethrin, cyfluthrin, and deltamethrin), using GC-MS, in the urine of pesticide applicators. Using the same methodology, Wei et al. (2012) assayed metabolites of these pyrethroids in the urine of commercial flight attendants. Elflein et al. (2003) also used GC-MS to detect metabolites of some other commonly used pyrethroids, including allethrin, resmethrin, phenothrin, and tetramethrin in human urine. HPLC and LC-MS-based methods have also been employed to determine the residues of pyrethroids and their metabolites in body tissues and fluids (Baker et al., 2004; Anand et al., 2006; Kim et al., 2006; Barr et al., 2010). Recently, Ishibashi et al. (2012) developed a highthroughput assay for cypermethrin and tralomethrin

using supercritical fluid chromatography-tandem mass spectrometry. Metabolites of some of the commonly used pyrethrins/pyrethroids determined in urine as biomarkers are summarized in Table 23.2. Barr et al. (2010) reported that 3-phenoxybenzoic acid (3-PBA), a metabolite common with many pyrethroid insecticides, was detected in . 70% of urine samples tested in the US. Non-Hispanic blacks had significantly higher 3-PBA concentrations than non-Hispanic whites and Mexican Americans, and children had significantly higher concentrations of 3-PBA than adolescents and adults. Cis- and trans-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (cis- and trans-Cl2CA) were highly correlated with each other and with 3-PBA, suggesting that 3-PBA was primarily derived from exposure to permethrin, cypermethrin, or their degradates. In a recent investigation, Wei et al. (2012) found that the flight attendants working on pyrethroid-disinsected commercial aircraft had significantly higher concentrations of 3PBA and cis- and trans-Cl2CA in the post-flight urine samples than those working on non-disinsected aircrafts and the general US population. Increase of pyrethroid metabolites in the preflight urine samples suggested an elevated body burden from a long-term exposure for those flight attendants routinely working on pyrethroid treated aircraft. Interestingly, flight attendants working on international flights connected to Australia had higher urinary levels of 3-PBA and cis- and trans-Cl2CA than those on either domestic and other international flights

TABLE 23.2 Metabolites of pyrethroids in urine used as biomarkers for an exposure to pyrethroid insecticides Parent Compound

Specific Metabolite(s)

Allethrin Cyfluthrin

trans-Chrysanthemumdicarboxylic acid 4-Fluoro-3-phenoxybenzoic acid 3-Phenoxybenzoic acid cis- and trans-3-(2,2-Dichlorovinyl)-2,2dimethylcyclopropane carboxylic acid trans-Chrysanthemumdicarboxylic acid cis- and trans-3-(2,2-Dichlorovinyl)-2,2dimethylcyclopropane carboxylic acid; 3-Phenoxybenzoic acid cis- and trans-3-(2,2-Dichlorovinyl)-2,2dimethylcyclopropane carboxylic acid; cis- 3-(2,2- Dibromovinyl)-2,2dimethylcyclopropane carboxylic acid; 3-Phenoxybenzoic acid cis- and trans-3-(2,2-Dichlorovinyl)-2,2dimethylcyclopropane carboxylic acid; 3-Phenoxybenzoic acid trans-Chrysanthemumdicarboxylic acid trans-Chrysanthemumdicarboxylic acid trans-Chrysanthemumdicarboxylic acid trans-Chrysanthemumdicarboxylic acid

Cypermethrin

Deltamethrin

Permethrin

Phenothrin Pyrethrum Resmethrin Tetramethrin

Note: Adapted from Malik et al. (2011).

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flying between Asia, Europe and North America. At veterinary diagnostic labs, residues of pyrethrins/pyrethroids have been detected in the brains of cats that died from overexposure to these insecticides. Recently, Tornero-Velez et al. (2012) proposed a pharmacokinetic model for cis- and trans-permethrin disposition in rats and humans with aggregate exposure application. In this investigation, the description of pharmacokinetics in humans was based on the properties of permethrin, PBPK models of deltamethrin in rats, and permethrin in vitro clearance data. The model was adapted with a biomarker submodel to evaluate exposure estimation in probabilistic risk assessment applications. Starr et al. (2012) investigated cumulative risk assessment of pyrethroid pesticides (permethrin, cypermethrin, β-cyfluthrin, deltamethrin, and esfenvalerate) taking into account pharmacokinetics, pharmacodynamics, and neurobehavioral assays. Findings supported the additive model of pyrethroid effect on motor activity and suggested that variation in the neurotoxicity of individual pyrethroids is related to toxicodynamic rather than toxicokinetic differences. In addition to residue detection of pyrethrins and pyrethroids and their metabolites, hematological, biochemical, and histopathological alterations have been reported in target and nontarget tissues, and these changes can be used as biomarkers of pyrethrins/pyrethroids toxicity (Sayim et al., 2005; Yavasoglu et al., 2006).

AMITRAZ Amitraz is a triazapentadiene compound of the formamidine pesticide family, which has a chemical formula of C19H23N3 and a molecular weight of 293.41. Amitraz is a broad spectrum insecticide and acaricide used in agriculture, horticulture, and veterinary medicine throughout the world since 1974. In agriculture and horticulture, amitraz is used to control red spider mites, leaf miners, scale insects, aphids, and other infestations. In veterinary medicine, it is currently approved in the United States for various applications, including use on both food-producing and companion animals. Amitraz is used to control ticks, mites, lice, and many other pests on dogs, sheep, cattle, and pigs. It persists on hair and wool long enough to control all stages of the parasite. Amitraz poisoning is frequently encountered in dogs and cats. With dogs, poisoning is most often associated with accidental ingestion of the flea tick collar, resulting in severe toxicity and sometimes fatal poisoning. In humans, poisoning occurs due to oral ingestion of amitraz (Shitole et al., 2010). The major signs of acute poisoning are nausea, vomiting, coma, somnolence, miosis

or mydriasis, bradycardia, hypo- or hyperthermia, polyuria, and respiratory failure. Amitraz should also not be used in diabetic animals as it adversely affects the levels of glucose and insulin (Hsu and Schaffer, 1988).

Mechanism of toxicity Amitraz kills parasites by paralyzing their nervous system and sharp barbed mouth parts, thereby causing them to detach from animals. Unlike in insects, amitraz has been found to cause toxicity in animals by stimulating α2- adrenergic receptors (α2-AR), resulting in impairment of consciousness, respiratory depression, convulsions, bradycardia, hypotension, and hyperglycemia. Hypothermia occurs due to the inhibitory effect of amitraz on prostaglandin E2 synthesis. Amitraz acts centrally to influence blood pressure and heart rate by α2-AR agonism, which causes a reduction in peripheral sympathetic tone (Cullen and Reynoldson, 1990). In a recent experimental study, Marafon et al. (2010) observed significant declines in the heart rate and respiration rate of cats intoxicated with amitraz. Electrocardiography on an amitraz-poisoned English bulldog revealed prolonged QT intervals (Malmasi and Ghaffari, 2010). In the peripheral vasculature, both α1 and α2-ARs are involved in the vasopressor action of amitraz, which results in hypotension. It is suggested that the central α2-AR agonist activity of amitraz is responsible for CNS depression (Cullen and Reynoldson, 1990). Amitraz is also a potent inhibitor of the enzyme monoamine oxidase (MAO), which is responsible for degrading neurotransmitters (norepinephrine and serotonin), resulting in neurotoxicity and behavioral toxicity (Moser, 1991). Poisoning is most often encountered in dogs and cats and is generally acute in nature. Onset of clinical signs is noted within 30 min to 2 h after ingestion. Common clinical signs include GI disturbance (i.e. prolonged gastric transit time), CNS and respiratory depression, bradycardia, hypotension, and hypothermia. Death usually occurs due to respiratory failure. Biochemical changes include hyperglycemia, glucosuria, suppressed insulin release, and elevation of liver transaminases activities. Histopathological changes may include hepatic and renal cortical necrosis, hemorrhage, and renal failure. Clinical symptoms reported in humans were giddiness, vomiting, drowsiness, and gastric dilatation (Shitole et al., 2010). Unconscious patients’ CT brain scans revealed brain edema.

Biomarkers Amitraz is a highly lipid soluble compound that is rapidly absorbed following oral ingestion or dermal

NEONICOTINOIDS

application, thus making exposure potentially dangerous for animals and humans. In the stomach, amitraz can be metabolized to as many as six different metabolites and some of them are potentially toxic. The two major metabolites of amitraz are 2,4-dimethylformamide and N-(2,4-dimthylphenyl)-N’-methylformamide. The former metabolite is a relatively weak methemoglobinformer in dogs and humans. These metabolites are further catabolized to 2,4-dimethylaniline and ultimately to 4-amino-3-methylbenzoic acid, which is the principal metabolite found in the liver and urine. A pharmacokinetic study in dogs revealed that amitraz is significantly absorbed and has a long elimination half-life, which is responsible for most of the observed clinical signs (Hugnet et al., 1996). Marafon et al. (2010) reported similar findings in cats. In ponies and sheep, amitraz has a brief half-life after IV administration because it is hydrolyzed in the blood by formaminidases (Pass and Mogg, 1995). The proposed biomarkers of amitraz exposure and its toxicosis include detection of the residue of amitraz and its major metabolites in the plasma of poisoned animals or humans using GC with a nitrogen phosphorus detector or thermionic specific detector (Marafon et al., 2010) or HPLC with a UV detector (Hugnet et al., 1996). Clinical signs of amitraz toxicity associated with α2-AR agonism, and biochemical and histopathological alterations, can be used as biomarkers of amitraz toxicity. In a chronic study in rats, amitraz was reported to be embryotoxic and a teratogen at a maternally toxic dose of . 10 mg/kg/day (Kim et al., 2007), and the noobserved-adverse-effect level (NOAEL) of amitraz for both dams and embryo-fetal development was estimated to be 3 mg/kg/day. For further details on amitraz toxicity, see Gupta (2012a).

NEONICOTINOIDS Neonicotinoids are a new class of insecticides, which includes imidacloprid, acetamiprid, thiacloprid, dinotefuran, nitenpyram, thiamethoxam, and clothianidin. They are commonly used in agriculture and veterinary medicine. Neonicotinoids have a high target specificity to insects, a relatively low risk for nontarget mammalian species and the environment, and a versatility in application methods (Ensley, 2012c). Among all neonicotinoids, imidacloprid is the most studied compound used as an insecticide for dermal application on animals, for grub control and as an insecticide for crop protection. The neonicotinoids class accounts for about 20% of the current global insecticide market.

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Mechanism of toxicity The neonicotinoids act on postsynaptic nicotinic acetylcholine receptors (nAChRs). In insects, these receptors are located entirely in the CNS. Mammalian tissue also contains multiple nAChRs subtypes, which are formed from different combinations of nine α, four β, and γ, δ, and ε subunits. In insects, there are binding sites in addition to nAChRs for neonicotinoids that are suggested to be used as mechanism-based biomarkers of exposure and effects (Badiou-Be´ne´teau et al., 2012). The selective toxicity of neonicotinoids in insects and mammals is attributed largely to the differential sensitivity of the insect and vertebrate nAChR subtypes. In insects, neonicotinoids act on at least three different subtypes of nAChRs, and cause a biphasic response, i.e., an initial increase in the frequency of spontaneous discharge followed by a complete block to nerve propagation. In mammals, nAChRs are located in the brain, autonomic ganglia, skeletal muscle, and spinal cord. Neonicotinoids have a much lower activity in vertebrates as compared to insects due to the different binding properties of the various receptor subtypes. Tomizawa and Casida (2011) suggested that the highaffinity for neonicotinoids at the insect nAChR is related to a single dominant binding orientation, whereas relative insensitivity at vertebrate nAChRs is caused by multiple binding confirmations in the agonist-binding pocket. Acute toxicity of the neonicotinoids in mammals is related to the potency at the α7 nicotinic receptor subtype with the activity at the α4, β2, α3 and α1 receptors having a decreased effect on toxicity. Toxicity in mammals involves complex interactions at multiple receptor sites with some of the receptor types even having a combination of agonist and antagonist effects on the synapse (Ensley, 2012c). Furthermore, neonicotinoids have relatively poor penetration of the blood–brain barrier (BBB) in mammals (Rose, 2012). Following an acute exposure to imidacloprid, clinical signs and symptoms can be observed as early as 15 min, and with recovery within 8 to 24 h. At low doses, neonicotinoids cause decreased activity and tremors, mydriasis or miosis, and incoordination; and at higher doses, they cause hypothermia, staggering gait, salivation, trembling, and spasms. At lethal doses, death is observed within 4 h. Humans, after deliberately ingesting imidacloprid, show symptoms of aspiration pneumonia, CNS effects (agitation, confusion, and coma) and respiratory failure. Cardiovascular effects include tachycardia, palpitation, and ventricular fibrillation. It needs to be emphasized that nicotinoid metabolites derived from some neonicotinoids (e.g. imidacloprid and thiacloprid) are more potent vertebrate nAChR agonists than their parent molecules and are likely to be more toxic (Rose, 2012). From chronic studies conducted in

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lab animals, NOAEL of imidacloprid was reported to be 9.3 mg/kg body wt/day. For further details of neonicotinoids toxicity, readers should refer to Ensley (2012c) and Rose (2012). Recently, Swenson and Casida (2013) demonstrated an additional mechanism of action for thiamethoxam causing hepatotoxicity and hepatocarcinogenicity. The proposed mechanism of action involves formation of reactive intermediates, such as formaldehyde and Nmethylol, and appears to be species-specific, i.e. in mice but not in rats.

Biomarkers Pharmacokinetic studies of imidacloprid revealed its rapid and complete absorption and distribution following oral administration in rats. Peak plasma levels were achieved within 2 h and the initial and terminal halflives in plasma were about 3 h and 26 118 h, respectively. The highest tissue residues after 48 h were found in the liver, kidney, lung, and skin. Low concentrations of imidacloprid were found in the brain due to poor penetration of the BBB, and a low concentration was found in fat due to low lipophilicity. Elimination of 90% of the dose occurred within 24 h (75 80% in the urine and the remainder in the feces originating from biliary excretion). Ensley (2012c) has described two pathways for imidacloprid metabolism in mammals. The first pathway involves oxidative cleavage of imidacloprid to imidazolidine and 6-chloronicotinic acid. The imidazolidine moiety is excreted in the urine. The 6-chloronicotinic acid is further degraded by glutathione conjugation to a derivative of mercapturic acid and then to methyl mercaptonicotinic acid. The mercaptonicotinic acid is then conjugated with glycine to form a hippuric acid conjugate, which is excreted. The second pathway involves hydroxylation of the imidazolidine ring, with two metabolites (5-hydroxyimidacloprid and 4-hydroxyimidacloprid) that are detected in the urine. Residue detection of neonicotinoids and their metabolites in the body tissue/fluids serves as a biomarker of exposure. HPLC coupled with a UV or MS detector is commonly used to detect the residue of neonicotinoids and their metabolites. Signs and symptoms of toxicity and histopathological changes in the liver can be used as biomarkers of neonicotinoid toxicity.

FIPRONIL Fipronil is an insecticide of the phenylpyrazoles class and an active ingredient of one of the popular

ectoparasiticide veterinary products, Frontline. Frontline is commonly used on dogs and cats to kill fleas, and all stages of ticks (brown dog ticks, American dog ticks, lone star ticks) which may carry Lyme disease, and mites. Fipronil is also formulated as insect bait for roaches, ants, and termites; as a spray for pets; and as a granular form on turf and golf courses. The USEPA has determined fipronil to be safe for use on dogs and cats, with no harm to humans who handle these pets. Most of the time poisoning cases of fipronil occur in dogs and cats due to accidental ingestion of the product Frontline. In humans, poisoning is mainly due to accidental ingestions or suicidal attempt. In agriculture, fipronil is widely used for soil treatment, seed coating, and crop protection.

Mechanism of toxicity The mechanism of action of fipronil is better understood in insects than it is in mammals. In insects, fipronil or its major metabolite (fipronil sulfone) noncompetitively binds to GABAA-gated chloride channels, thereby blocking the inhibitory action of GABAA in the CNS. This leads to hyperexcitation at low doses, and paralysis and death at higher doses. Fipronil exhibits a . 500-fold selective toxicity to insects over mammals, primarily because of affinity differences in receptor binding between insect and mammalian receptors. In other words, fipronil binds more tightly to GABAA receptors in insects than in mammals. Fipronil is more selective at this receptor through the β3 subunit in insects than in mammals. This selectivity is less pronounced with fipronil metabolites (sulfone and desulfinyl). It needs to be emphasized that fipronil sulfone is rapidly formed in humans and experimental animals, and fipronil sulfone can persist much longer than fipronil; therefore the toxicological effects are also likely due to the sulfone metabolite. The toxicity of another metabolite, fipronil desulfinyl, is qualitatively similar to that of fipronil, but the dose-effect curve for neurotoxic effects appears to be steeper for fipronil desulfinyl. Also, fipronil desulfinyl appears to have a much greater affinity to bind to sites in the chloride ion channel of the rat brain GABA receptor. This finding appears to be consistent with the greater toxicity of fipronil desulfinyl in the CNS of mammals. Comparatively recently, Narahashi et al. (2010) has explained the mechanism of action of fipronil in detail in insects and mammals. In laboratory animals, fipronil administration by the oral route can produce the signs of neurotoxicity, including convulsions, tremors, abnormal gait, and hunched posture. Similar signs can be produced following inhalation exposure. Poisoned dogs and cats usually show signs of tremors, convulsions, seizures, and death.

IVERMECTIN AND SELAMECTIN

Following dermal exposure, fipronil toxicity is more pronounced in rabbits than in rats and mice. Humans exposed to fipronil by ingestion may show symptoms of headache, tonic-clonic convulsions, seizures, paresthesia, pneumonia, and death. Neurotoxic symptoms of fipronil poisoning in humans are typically associated with the antagonism of central GABA receptors. It has been suggested that fipronil is a developmental neurotoxicant. In an in vitro study using differentiating N2a neuroblastoma cells, Sidiropoulou et al. (2011) demonstrated that fipronil caused severe disruption of the developmentally important ERK {1/2}-MAP kinase signal transduction pathway, as evidenced by significant reductions in the activation state of MAP kinase (MEK {1/2}), and particularly ERK {1/2}. These findings supported the contention that fipronil is a developmental neurotoxicant, and unrelated to GABA receptor inhibition. Recently, Roques et al. (2012) demonstrated that fipronil and fipronil sulfone induced thyroid disruption in rats. But, fipronil sulfone has greater potential than fipronil for thyroid disruption because it persists much longer in the organism than fipronil itself.

Biomarkers After topical application of Frontlinet, fipronil spreads and sequesters in the lipids of the skin and hair follicles, and continues to be released onto the skin and coat, resulting in long-lasting residual activity against fleas and ticks. Jennings et al. (2002) reported the maximum concentration of fipronil on the canine hair coat 24 h after a single application of Frontlinet top spot. With a descending concentration trend, fipronil residue was detected on dog’s hair coat for a period of 30 days. Fipronil is metabolized to fipronil sulfone in the liver by cytochrome P450 (Roques et al., 2012). Fipronil and its metabolites (primarily sulfone) can persist in the tissues, particularly in fat and fatty tissues, for one week after treatment. The long half-life (150 245 h) of fipronil in blood may reflect a slow release of metabolites from fat. Pharmacokinetic studies in rats suggest that fipronil excretes mainly in the feces (45 75%) and little in the urine (5 25%). Detection and confirmation of residue of fipronil and its metabolites is usually performed using GS-MS. Detection of residue of fipronil or its metabolites in the body tissue, urine, feces, or on skin or hair can be used as biomarkers of fipronil exposure. Clinical signs and symptoms and pathological changes in liver are not specific, and are of little value in terms of toxicological biomarkers. For further details on toxicity of fipronil, readers are referred to Anadon and Gupta (2012).

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IVERMECTIN AND SELAMECTIN Ivermectin is a semisynthetic macrocyclic lactone (ML) which was first isolated from Streptomyces avermitilis. Ivermectin is a mixture of two homologs (80% B1a and 20% B1b), and it was introduced to the market as abamectin in 1981 as a potent antiparasitic animal health drug. The drug is approved at a very low dosage for the control of parasites in many animal species (cattle, sheep, swine, horses, dogs, and cats), but not approved in lactating cows, sheep, and goats. Although ivermectin poisoning can occur in any animal species, dogs of certain breeds (collies, Old English sheepdog, German shepherd, and others), Murray Grey cattle, and young animals (with less developed BBB) are particularly ¯ more sensitive. Recently in Japan, Crump and Omura (2011) described ivermectin as a “wonder drug” in humans against onchocerciasis and lymphatic filariasis. In agriculture, ivermectin is used for its insecticidal, miticidal, and acaricidal activities. Selamectin is a novel semi-synthetic avermectin, which is marketed as Revolutiont for topical application on dogs and cats six weeks of age and older. Selamectin is used to kill fleas, ticks, and ear mites in dogs and cats. With ivermectin, selamectin, and other MLs (doramectin, eprinomectin, milbemycin, and moxidectin), poisoning in animals may occur due to overdosage or accidental exposure.

Mechanism of toxicity Ivermectin Ivermectin is effective against nematodes and arthropods, but not against cestodes and trematodes. This is because ivermectin acts as a GABA receptor agonist, and cestodes and trematodes lack a GABA system. In mammals, a GABA system is present only in the CNS, and ivermectin exerts toxicity by blocking the postsynaptic transmission of nerve impulses by potentiating the release and binding of GABA, thus blocking GABAmediated transmission. In general, ivermectin does not cross the BBB in most animal species. The defective pglycoprotein transporter (ABCB1) in the BBB has been found in at least 11 breeds of dogs (including Collies), and in many other animals. Therefore, when the pglycoprotein transporter is defective or overwhelmed by ivermectin overdosage, the integrity of the BBB is compromised, leading to ivermectin toxicosis in the animal. Some adverse effects of ivermectin in dogs, horses, and cattle also appear to be due to GABA-mediated cholinergic effects. Both homologs of ivermectin are neurotoxicants and equally potent. The clinical signs and symptoms of

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ivermectin toxicosis, primarily involving the CNS, have been described in ivermectin-sensitive dogs. Further support for CNS involvement was the finding that the ivermectin concentrations in the brain were higher in dogs with p-glycoprotein defect displaying symptoms of ivermectin toxicosis than in non-sensitive dogs (Pullium et al., 1985). Clinical signs include dehydration, bradycardia, respiratory depression, cyanosis, mydriasis, and a diminished gag reflex. Toxic signs may also include vomiting, ataxia, tremors, hypersalivation, coma, and death. Ivermectin should not be given to kittens, as it can cross the BBB. At higher doses, other animals show similar signs of ivermectin toxicosis. In a recent experimental study in rats, abamectin has been found to cause testicular damage, thereby impairing male fertility (Celic-Ozenci et al., 2011).

Selamectin In insects and parasites, selamectin binds to glutamate gated chloride channels in the nervous system, causing them to remain open. This causes chloride ions to continuously flow into the nerve cell, changing the charge of the cell membrane. The continuous flow of chloride ions blocks neurotransmission, and transmission of stimuli to the muscle is prevented. Selamectin binding is irreversible, and thereby it causes prolonged channel opening and permanent hyperpolarization. Selamectin has no such effect in the mammalian nervous system and, therefore, it is considered to be a safe insecticide. However, in acute cases, signs of toxicoses may include hair loss at the site of application, vomiting, diarrhea with or without blood, lethargy, salivation, tachypnea, pruritus, urticaria, erythema, ataxia, and fever. In rare instances, seizures followed by death occur with overt acute overdose. In a recent study, dogs treated with a single topical application of Revolutiont (6 mg/kg body wt) showed no signs of any poisoning, though detectable residue of selamectin persisted on skin and hair coat for a month (Gupta et al., 2005). For more details on ivermectin and selamectin toxicity, readers should refer to Gwaltney-Brant et al. (2012).

Biomarkers In general, these insecticides are well absorbed, distributed widely throughout the body (higher concentrations in fat and fatty tissues), and tend to have long tissue residence times. They undergo some metabolism and are excreted unchanged in the feces via the bile. Because of the enterohepatic recycling, the half-lives of these compounds are in the range of days to weeks. Interestingly, residues of these compounds are excreted in the milk. Residue analysis of ivermectin, selamectin,

or any other MLs, along with clinical signs, can serve as biomarkers of exposure and effects. These compounds are analyzed using HPLC with UV, fluorescence, or photo diode array detector (Anastaseo et al., 2002; Gupta et al., 2005).

ROTENONE Rotenone is one of the naturally occurring insecticides present in a number of plants of Derris, Lonchocarpus, Tephrosia, and Mundulea species. It has a molecular formula of C23H22O6 and a molecular weight of 394.42. Rotenone is used worldwide because it has broad spectrum insecticidal, acaricidal, and other pesticidal properties. Its formulations include crystalline preparations (about 95%), emulsifiable solutions (about 50%), and dust (0.75%). In veterinary medicine, rotenone is used in powder form to control parasitic mites on chicken and other fowl, and lice and ticks on dogs, cats, and horses. Rotenone dust is also used to control beetles and aphids on vegetables, fruits, berries, and flowers. Rotenone emulsions are used for eliminating unwanted fish in the management of bodies of water. It is also formulated along with other pesticides, such as carbaryl, pyrethrins, piperonyl butoxide, and lindane, in products to control insects, mites, ticks, lice, spiders, and undesirable fish. According to a survey conducted by the USEPA in 1990, rotenone was found to be one of the pesticides most commonly used in and around the home. Rotenone is a very safe compound when properly used, but in higher doses it is toxic to humans, animals, and fish. WHO classifies rotenone as a moderately hazardous Class II pesticide. It has been involved in suicidal attempts, in which acute congestive heart failure was the characteristic feature at autopsy. During the past decade, rotenone has received enormous attention because of its link to Parkinson’s disease (Pan-Montojo, et al., 2010; Xiong et al., 2012). Currently, rotenone is extensively used by researchers as an experimental drug to produce mitochondrial dysfunction and reproduce Parkinson’s disease in animal models (Drolet et al., 2009; Xiong et al., 2012).

Mechanism of toxicity In insects, rotenone is both a contact and a systemic insecticide. Rotenone inhibits the transfer of electrons from Fe-S centers in complex I to ubiquinone in the electron transport chain. This prevents NADH from being converted into usable cellular energy, i.e. ATP. In

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mammals and fish, rotenone inhibits the oxidation of NADH to NAD, thereby blocking the oxidation of NAD and the substrates such as glutamate, α-ketoglutarate, and pyruvate. Rotenone causes inhibition of mitochondrial respiratory chain complex I, which can cause oxidative stress and lead to selective degeneration of striatal-nigral dopamine neurons. Besides complex I inhibition, nitrosative stress, increased nitric oxide and malondialdehyde levels, aggregation of α-synuclein and polyubiquitin, activation of astrocytes and microglial cells, inflammatory reaction, glutamate excitotoxicity, and neuronal apoptosis are involved in the mechanisms of rotenone-evoked parkinsonism (reviewed in Xiong et al., 2012). In a recent in vitro study, Mundhall et al. (2012) demonstrated that 30 min superfusion of horizontal slices of rat midbrain with 100 nM rotenone caused significant injury to tyrosine hydroxylase-positive proximal dendrites in dorsal and ventral regions of the substantia nigra zona impacta and ventral tegmental area. Rotenone toxicity has been studied using various in vitro and in vivo models (reviewed in Xiong et al., 2012). Rotenone exerts selective toxicity, as it is highly toxic to fish because of its rapid absorption from the GI tract in comparison to mammalian species in which it is poorly absorbed. The selective toxicity of rotenone in insects and fish versus mammals can also be explained by its metabolism. Rotenone converts to highly toxic metabolites in large quantities in insects and fish, while it converts to nontoxic metabolites in mammals. Rotenone has been proven to be a neurotoxicant in all species tested. In mammals, rotenone acute exposure can produce vomiting, incoordination, muscle tremors, and clonic convulsions. Cardiovascular effects include tachycardia, hypotension, and impaired myocardial contractility. Death occurs due to cardio-respiratory failure. Chronic exposure to rotenone exerts Parkinson’s disease-like neuropathology in experimental lab animals (Drolet et al., 2009; Pan-Montojo et al., 2010). A NOAEL of 0.4 mg/kg/day has been determined in rats and dogs. For further details on rotenone toxicity, readers are referred to Gupta (2012b).

mice. Unabsorbed rotenone from the GI tract is excreted in the feces. Residue detection of rotenone and/or its metabolites in blood, urine, feces, or liver can serve as a biomarker of rotenone exposure. Rotenone residue can be determined using HPLC with a fluorescence detector or LC-MS-MS (Caboni et al., 2008). Characteristic toxicological symptoms and histopathological changes can be used as biomarkers of rotenone toxicity.

CONCLUDING REMARKS AND FUTURE DIRECTIONS The majority of insecticides are of chemical origin, and the rest are of biological origin. They are used worldwide in agriculture, horticulture, forestry, residential areas, gardens, homes and offices. Insecticides are of diverse chemical structures, and therefore their mechanism of action, pharmacokinetics, and toxicity significantly vary. Most insecticides are neurotoxicants in insects and nontarget mammalian (including humans) species, wildlife, and aquatic species. In nontarget species, the insecticides appear to be of lesser toxicity than that in insects, but their toxic effects involve other body organs and systems, in addition to the nervous system. In general, detection of the residue of insecticides and/ or their metabolites in body fluids (urine, blood serum, plasma, and milk) or tissue is often used as biomarker of exposure. Alterations in behavioral, biochemical, molecular, histopathological endpoints are used as biomarkers of effects. In insecticide toxicity, biomarkers of genetic susceptibility are very little understood and this needs to be explored in future studies. Future research will explore novel biomarkers with greater sensitivity, reliability, and reproducibility, preferably nondestructible and noninvasive for chemical toxicity and chemicalrelated long-term illnesses, such as neurodegenerative, metabolic, and carcinogenesis. The authors would like to thank Ms. Michelle A. Lasher and Ms. Robin B. Doss for their technical assistance in the preparation of this chapter.

Biomarkers In animals, rotenone has been found to be 100 times more toxic via the IV route than by the oral route, because of its poor absorption from the GI tract. Rotenone is metabolized in the liver by NADP-linked hepatic microsomal enzymes. Several metabolites of rotenone have been identified as rotenoids, such as rotenolone I and II, hydroxyl and dihydroxyrotenone, etc. Approximately 20% of a rotenone dose is excreted in the urine within 24 h of oral administration in rats and

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