Gut microbiota, which is known to be remarkably modulated in autistic patients, can be easily related to the elevated level of Hg. Gut microbes can modulate Hg via either methylation of less toxic inorganic Hg, Hg+2, or demethylation (i.e., detoxification) of methylmercury (MeHg) [15–18]. In studying bacterial diversity in relation to the MeHg level, Rothenberg et al. found that, among the studied bacterial species *Clostridiales*, *Subdoligranulum*, and *Akkermansia* spp., positive correlations for stool MeHg, hair total mercury (THg), and stool inorganic Hg were evident, while negative correlations for *Streptococcus* were determined using Spearman's and/or Pearson's [19]. These relative effects were related to approximately tenfold higher *Clostridium difficile* levels in the stool from autistic subjects, which may help to support the use of probiotics to ameliorate MeHg elevations which may lead to the development of autistic

In relation to the antioxidant and protective effect of selenium (Se), a recent review was written by Bjorklund and Causey on the molecular interactions between Hg and Se which result in neurotoxicity. Selected studies revealed associations between autism and Hg and Se concentration changes in hair and/or nails of autistic patients [21]. These studies reported significant increases in the levels of Hg and concurrent decreases in the levels of Se in hair and nails and

**Figure 1.** Role of fish in the bioaccumulation of mercury (Hg) and role of methylating and demethylating bacteria in the

control of methyl mercury (MeHg) concentration as environmental neurotoxin related to autism.

possible mechanism to cause Hg-induced brain damage [14].

features [20]. This is summarized in **Figure 1**.

In relation to the modern lifestyles—cell phones, microwaves, mercury and lead exposures, plastic, fast food—place children at higher risk of neurotoxicity that might lead to brain damage during early development. Moreover, environmental toxins such as misoprostol, valproic acid (VPA), and thalidomide have been reported as neurotoxins and linked to ASD. Pathogenic bacteria or their metabolites, cytomegalovirus, rubella, toxoplasmosis, and herpes simplex have also been characterized as neurotoxins which greatly contribute to autism.

This chapter summarizes the most important routes of exposure to environmental neurotoxins and explains how these toxins are related to the remarkable increase in the prevalence of autism. Moreover, the role of the stages of development and timing of exposures (prenatal, perinatal, and postnatal) will be discussed.

Based on the recorded biomarkers of autism, the role of selected environmental toxins in the induction of oxidative stress, neuroinflammation, impaired neurochemistry, and glutamate excitotoxicity as etiological mechanisms related to autism will be highlighted and illustrated.

#### **1.1. Heavy metals as neurotoxins**

A relationship between rises in environmental levels of Hg and the increase in both rates of autism and special education students has been reported [1]. In an attempt to find the relationship between elevated mercury levels and oxidative stress as etiological mechanisms in autistic individuals, Sajdel-Sulkowska et al. [2] found that mercury concentrations in the cerebellar areas of the brain were positively correlated with neurotrophin-3 (NT-3), as an oxidative stress marker. Khan et al. reported on NT-3 associated with much higher levels in autistic patients but without an association with Hg levels in blood, which was nonsignificantly different between autistic and control subjects [3]. This suggests that the same concentration of Hg may promote oxidative stress only in autistic patients but not in control subjects. In relation to Hg levels in hair as an indicator of neurotoxicity in autistic patients, the "poor excretor theory" asserts that autistic children are more prone to accumulate Hg because they are unable to readily excrete it when compared to age- and gender-matched controls [4]. However, other studies recorded that the higher the Hg levels in hair, the worse the autism symptoms [5]. A direct relationship between elevated blood levels of Hg and the degree of autism severity according to scales childhood autism rating scales (CARS), social responsiveness scales (SRS), and short sensory profile (SSP) was ascertained [6, 7].

In an attempt to better understand the role of heavy metal neurotoxicity, investigations of air pollution, Hg, lead (Pb), and arsenic (As) have been shown to stimulate oxidative stress and inflammation in humans, which may contribute to the pathogenesis of autism [8, 9]. Based on multiple studies, Pb, Hg, and As were recorded as neurotoxins related to autism [10–12]. Associations between autism prevalence and proximity to industrial facilities were ascertained by Dickerson et al. [13]. Most recently, a disruption of complex neuro-immune signaling as a mechanism necessary for neuronal migration and brain growth was accepted to be a possible mechanism to cause Hg-induced brain damage [14].

repetitive behavior. Environmental factors as significant contributors to children's health through a wide range of routes are greatly involved in the remarkable increase of autism spectrum disorder (ASD). It is well known and accepted that young children are more vulnerable to environmental toxins compared to adults because they breathe more air and consume more food relative to their body size in order to meet requirements of growth and development. Common hand-to-mouth behavior among infants and even young children of course

In relation to the modern lifestyles—cell phones, microwaves, mercury and lead exposures, plastic, fast food—place children at higher risk of neurotoxicity that might lead to brain damage during early development. Moreover, environmental toxins such as misoprostol, valproic acid (VPA), and thalidomide have been reported as neurotoxins and linked to ASD. Pathogenic bacteria or their metabolites, cytomegalovirus, rubella, toxoplasmosis, and herpes simplex have also been characterized as neurotoxins which greatly contribute to autism.

This chapter summarizes the most important routes of exposure to environmental neurotoxins and explains how these toxins are related to the remarkable increase in the prevalence of autism. Moreover, the role of the stages of development and timing of exposures (prenatal,

Based on the recorded biomarkers of autism, the role of selected environmental toxins in the induction of oxidative stress, neuroinflammation, impaired neurochemistry, and glutamate excitotoxicity as etiological mechanisms related to autism will be highlighted and illustrated.

A relationship between rises in environmental levels of Hg and the increase in both rates of autism and special education students has been reported [1]. In an attempt to find the relationship between elevated mercury levels and oxidative stress as etiological mechanisms in autistic individuals, Sajdel-Sulkowska et al. [2] found that mercury concentrations in the cerebellar areas of the brain were positively correlated with neurotrophin-3 (NT-3), as an oxidative stress marker. Khan et al. reported on NT-3 associated with much higher levels in autistic patients but without an association with Hg levels in blood, which was nonsignificantly different between autistic and control subjects [3]. This suggests that the same concentration of Hg may promote oxidative stress only in autistic patients but not in control subjects. In relation to Hg levels in hair as an indicator of neurotoxicity in autistic patients, the "poor excretor theory" asserts that autistic children are more prone to accumulate Hg because they are unable to readily excrete it when compared to age- and gender-matched controls [4]. However, other studies recorded that the higher the Hg levels in hair, the worse the autism symptoms [5]. A direct relationship between elevated blood levels of Hg and the degree of autism severity according to scales childhood autism rating scales (CARS), social responsive-

In an attempt to better understand the role of heavy metal neurotoxicity, investigations of air pollution, Hg, lead (Pb), and arsenic (As) have been shown to stimulate oxidative stress and

ness scales (SRS), and short sensory profile (SSP) was ascertained [6, 7].

increases their risk of exposures to environmental toxins.

perinatal, and postnatal) will be discussed.

**1.1. Heavy metals as neurotoxins**

56 Neurotoxins

Gut microbiota, which is known to be remarkably modulated in autistic patients, can be easily related to the elevated level of Hg. Gut microbes can modulate Hg via either methylation of less toxic inorganic Hg, Hg+2, or demethylation (i.e., detoxification) of methylmercury (MeHg) [15–18]. In studying bacterial diversity in relation to the MeHg level, Rothenberg et al. found that, among the studied bacterial species *Clostridiales*, *Subdoligranulum*, and *Akkermansia* spp., positive correlations for stool MeHg, hair total mercury (THg), and stool inorganic Hg were evident, while negative correlations for *Streptococcus* were determined using Spearman's and/or Pearson's [19]. These relative effects were related to approximately tenfold higher *Clostridium difficile* levels in the stool from autistic subjects, which may help to support the use of probiotics to ameliorate MeHg elevations which may lead to the development of autistic features [20]. This is summarized in **Figure 1**.

In relation to the antioxidant and protective effect of selenium (Se), a recent review was written by Bjorklund and Causey on the molecular interactions between Hg and Se which result in neurotoxicity. Selected studies revealed associations between autism and Hg and Se concentration changes in hair and/or nails of autistic patients [21]. These studies reported significant increases in the levels of Hg and concurrent decreases in the levels of Se in hair and nails and

**Figure 1.** Role of fish in the bioaccumulation of mercury (Hg) and role of methylating and demethylating bacteria in the control of methyl mercury (MeHg) concentration as environmental neurotoxin related to autism.

the association of these changes with the severity of an autistic phenotype [5, 22]. Other studies showed significant elevations in Hg in hair [23] and urine [22] of autistic patients without significant decrease of Se concentration or Se/Hg ratio. Moreover, some studies reported a lower Zn/Cu ratio in blood from autistic subjects as compared to healthy control subjects [24, 25] yet strong causal relationships for Hg neurotoxicity.

mitochondrial protectant prevented anesthesia-induced neuronal apoptosis and downstream

The use of antiepileptic drugs (AEDs) by pregnant mothers was found to be involved in major congenital abnormalities seen in neurodevelopmental disorders among which is autism [36]. Animal studies demonstrated that exposure to AEDs may result in neurotoxicity; for example, VPA, phenytoin, and phenobarbital, cause impaired neurodevelopment after prenatal exposure [37]. Despite the therapeutic effects of VPA, it is also associated with neurotoxicity [38] as evidenced in in vitro models. VPA neurotoxicity is usually related to the increased of ROS production, as a critical contributor to brain damage and dysfunction [38–40]. Mitochondrial dysfunction has been proposed as one of the most common deleterious effects of VPA neurotoxicity [41]. In relation to autism, VPA is most commonly related to the etiopathology and development of most of phenotypic features of autism. Many cases, population database studies, prospective studies, and retrospective studies, ascertained increased incidences of ASD and cognitive deficits in VPA-exposed children with a reported risk of 6–8% [42–45].

Chaudhary and Parvez observed a significant decrease in acetylcholinesterase (AChE) activity [46]. The inhibition of AChE activity by VPA in the cerebellum and cerebral cortex results in the accumulation of ACh at cholinergic synapses, leading to ACh receptor stimulation, decreased cellular metabolism, induction of cell membrane alterations, and disturbances in

/K+

transport of CNS sodium and potassium ions in a dose-dependent manner [48]. The marked

membrane depolarization and excessive Ca2+ entry inside neurons which may in turn induce glutamate excitotoxicity. This can be ascertained through the recent work of Kim et al. in which agmatine was used to treat VPA-induced animal models of neurotoxicity, whereto, the amelioration of glutamate excitability improves sociability and decreases the repetitive behavior appeared as two important autistic features [49]. In a recent study by Videman et al., carbamazepine, oxcarbazepine, and VPA were associated with impaired early language abilities at the age of 7 months. In contrast, face perception or social attention may be less affected by the neurotoxic effects of the studied AEDs [50]. Previously, the association between prenatal exposure to AEDs and the increased risk of cognitive impairment and ASD was detected

It is well accepted that healthy gut microbiota provides an effective barrier against colonization by opportunistic bacteria [51, 52]. This protective microbiota is severely disrupted with abuse of broad-spectrum antibiotics frequently administered during early childhood [53]. *C. difficile* produces two exotoxins: toxin A and toxin B. Acting together, these toxins damage intestinal mucosa and cells and result in watery diarrhea, which is the primary clinical symptom of *C. difficile* infection [54]. In addition to *C. difficile*, *Clostridium tetani* is another opportunistic pathogen that can lay dormant in spore form for long periods of time. Both species

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cognitive impairment in developing rat brains [33, 35].

neuronal activities [47]. VPA-inhibited Na+

/K+

**1.4. Clostridium neurotoxins and autism**

inhibition of Na+

at ages of 2 to 6 years.

**1.3. Antiepileptic drugs (AEDs) as neurotoxins in autism**

#### **1.2. Anesthetic drugs as neurotoxins in autism**

An early study of the role of anesthesia in relation to neurotoxicity was described by Ikonomidou et al. [26]. They observed the effects of N-methyl-D-aspartate (NMDA) antagonist injections in rat pups, which led to acute postnatal neuronal apoptosis [26]. Also, they hypothesized that anesthetics such as ketamine blocked endogenous glutamate stimulation via NMDA receptors, leading to neuronal apoptosis. Apoptosis of neurons as an invasive marker of neurotoxicity was repeatedly demonstrated especially in animals that received multiple, high doses of ketamine during periods of developmental vulnerability [27, 28]. Upregulation of NMDA receptor expression levels in response to ketamine administration may modulate intracellular calcium homeostasis, possibly leading to apoptosis [29, 30]. Additionally, activation of gamma-aminobutyric acid (GABA) receptors through the inhalation of isoflurane-induced neurotoxicity in hippocampal culture cells was also associated with excessive neuronal influx of calcium [31]. Based on observation, it was suggested that anesthesia-induced neuronal toxicity may appear secondary to loss of calcium homeostasis within mitochondria as evident by mitochondrial dysfunction, accumulation of reactive oxygen species (ROS), and overexpression of caspases as pro-apoptotic markers (**Figure 2**) [32–34]. Although the actual mechanism for ROS accumulation was not identified, administration(s) of either an antioxidant or a

**Figure 2.** Neurotoxic effect of ketamine through loss of calcium homeostasis leading to neuronal death.

mitochondrial protectant prevented anesthesia-induced neuronal apoptosis and downstream cognitive impairment in developing rat brains [33, 35].

#### **1.3. Antiepileptic drugs (AEDs) as neurotoxins in autism**

the association of these changes with the severity of an autistic phenotype [5, 22]. Other studies showed significant elevations in Hg in hair [23] and urine [22] of autistic patients without significant decrease of Se concentration or Se/Hg ratio. Moreover, some studies reported a lower Zn/Cu ratio in blood from autistic subjects as compared to healthy control subjects [24, 25] yet

An early study of the role of anesthesia in relation to neurotoxicity was described by Ikonomidou et al. [26]. They observed the effects of N-methyl-D-aspartate (NMDA) antagonist injections in rat pups, which led to acute postnatal neuronal apoptosis [26]. Also, they hypothesized that anesthetics such as ketamine blocked endogenous glutamate stimulation via NMDA receptors, leading to neuronal apoptosis. Apoptosis of neurons as an invasive marker of neurotoxicity was repeatedly demonstrated especially in animals that received multiple, high doses of ketamine during periods of developmental vulnerability [27, 28]. Upregulation of NMDA receptor expression levels in response to ketamine administration may modulate intracellular calcium homeostasis, possibly leading to apoptosis [29, 30]. Additionally, activation of gamma-aminobutyric acid (GABA) receptors through the inhalation of isoflurane-induced neurotoxicity in hippocampal culture cells was also associated with excessive neuronal influx of calcium [31]. Based on observation, it was suggested that anesthesia-induced neuronal toxicity may appear secondary to loss of calcium homeostasis within mitochondria as evident by mitochondrial dysfunction, accumulation of reactive oxygen species (ROS), and overexpression of caspases as pro-apoptotic markers (**Figure 2**) [32–34]. Although the actual mechanism for ROS accumulation was not identified, administration(s) of either an antioxidant or a

**Figure 2.** Neurotoxic effect of ketamine through loss of calcium homeostasis leading to neuronal death.

strong causal relationships for Hg neurotoxicity.

58 Neurotoxins

**1.2. Anesthetic drugs as neurotoxins in autism**

The use of antiepileptic drugs (AEDs) by pregnant mothers was found to be involved in major congenital abnormalities seen in neurodevelopmental disorders among which is autism [36]. Animal studies demonstrated that exposure to AEDs may result in neurotoxicity; for example, VPA, phenytoin, and phenobarbital, cause impaired neurodevelopment after prenatal exposure [37]. Despite the therapeutic effects of VPA, it is also associated with neurotoxicity [38] as evidenced in in vitro models. VPA neurotoxicity is usually related to the increased of ROS production, as a critical contributor to brain damage and dysfunction [38–40]. Mitochondrial dysfunction has been proposed as one of the most common deleterious effects of VPA neurotoxicity [41]. In relation to autism, VPA is most commonly related to the etiopathology and development of most of phenotypic features of autism. Many cases, population database studies, prospective studies, and retrospective studies, ascertained increased incidences of ASD and cognitive deficits in VPA-exposed children with a reported risk of 6–8% [42–45].

Chaudhary and Parvez observed a significant decrease in acetylcholinesterase (AChE) activity [46]. The inhibition of AChE activity by VPA in the cerebellum and cerebral cortex results in the accumulation of ACh at cholinergic synapses, leading to ACh receptor stimulation, decreased cellular metabolism, induction of cell membrane alterations, and disturbances in neuronal activities [47]. VPA-inhibited Na+ /K+ -ATPase is an enzyme controlling the active transport of CNS sodium and potassium ions in a dose-dependent manner [48]. The marked inhibition of Na+ /K+ -ATPase activity may compromise neurotransmission, leading to partial membrane depolarization and excessive Ca2+ entry inside neurons which may in turn induce glutamate excitotoxicity. This can be ascertained through the recent work of Kim et al. in which agmatine was used to treat VPA-induced animal models of neurotoxicity, whereto, the amelioration of glutamate excitability improves sociability and decreases the repetitive behavior appeared as two important autistic features [49]. In a recent study by Videman et al., carbamazepine, oxcarbazepine, and VPA were associated with impaired early language abilities at the age of 7 months. In contrast, face perception or social attention may be less affected by the neurotoxic effects of the studied AEDs [50]. Previously, the association between prenatal exposure to AEDs and the increased risk of cognitive impairment and ASD was detected at ages of 2 to 6 years.

#### **1.4. Clostridium neurotoxins and autism**

It is well accepted that healthy gut microbiota provides an effective barrier against colonization by opportunistic bacteria [51, 52]. This protective microbiota is severely disrupted with abuse of broad-spectrum antibiotics frequently administered during early childhood [53]. *C. difficile* produces two exotoxins: toxin A and toxin B. Acting together, these toxins damage intestinal mucosa and cells and result in watery diarrhea, which is the primary clinical symptom of *C. difficile* infection [54]. In addition to *C. difficile*, *Clostridium tetani* is another opportunistic pathogen that can lay dormant in spore form for long periods of time. Both species produce cytotoxins known to cause cellular damage. Additionally, both *C. tetani* and *C. difficile* produce phenolic metabolites [55]. Some toxigenic strains of *C. tetani* produce an extremely potent neurotoxin. Clostridia toxins can enter the circulation and accumulate at nerve terminals through binding to host-independent receptors [56, 57]. Following endocytosis of the neurotoxin-receptor complex, acidification of the presynaptic vesicle triggers a conformational change in the N-terminal translocation domain. Acidification is mediated by the vesicular ATPase proton pump, whose function is to ensure the reuptake of neurotransmitters into the synaptic vesicle. This might provide a plausible mechanism for the abnormal reuptake of glutamate in autistic patients. Several studies have assessed the fecal flora of autistic and control individuals, reporting an overgrowth of pathogenic bacterial species in autistic patients when compared to controls. Among these species is *Clostridium* species, including *C. difficile* [58–60]. In addition, Parracho et al. found a higher incidence of the *Clostridium histolyticum* group in the fecal flora of 58 ASD children compared to 10 healthy children [61].

with impaired communicative and social behavior representative as autistic features [67]. Moreover, MIA offspring displayed altered gut microbiota and leaky gut. Treatment of mice with *Bacteroides fragilis* was effective in restoring normal gut permeability and reduces anxiety-like behavioral deficits in this autism model [67]. Most recently, Yang and Chiu reported that through the soluble NSF attachment protein receptor (SNARE) complex, clostridial neurotoxins block neurotransmission to or from neurons. In addition, the gut microbiota produces molecules that act on enteric neurons to reduce gastrointestinal motility and metabolites that stimulate the "gut-brain axis" to alter neural circuits and brain function and behavior [68]. Aljarallah reported that water extract of myrrh plant demonstrates high antimicrobial effect against *C. difficile* strains, which could support its use as a natural prod-

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By using retrospective epidemiological studies, environmental factors such as pesticides have been linked to autism. Experimental research in mouse cortical neuron-enriched cultures exposed to hundreds of chemicals commonly found in the environment and on food showed how such chemicals greatly affect brain development. Pearson et al. have been found that rotenone, a pesticide associated with Parkinson's disease risk, and certain fungicides, including pyraclostrobin, trifloxystrobin, famoxadone, and fenamidone, produce transcriptional changes in vitro that are similar to those seen in brain samples from humans with autism [70]. These chemicals stimulate ROS production and disrupt microtubules in neurons, effects that can be reduced by pretreating with a microtubule stabilizer and an antioxidant such as sulforaphane. In this study, 283 autistic children showed neuronal tube defects with potential relationships between maternal residential proximity and agricultural use of neurotoxic

Organophosphates (OPs) are the most generally utilized pesticides in agriculture, as well as bug sprays in residential, commercial, and industrial settings. Fetus may be exposed to OPs via the placenta or infants through breast milk, food, and inhalation. These small children appear particularly vulnerable to OPs and oxidative stress compared to adults, because of their lower activity levels of the enzyme paraoxonase, involved in OP inactivation and lipid peroxide degradation [72]. This enzyme was found to be significantly lower in autistic patients compared to healthy controls [73]. Prenatal exposure to OPs has been connected to neurodevelopmental disorder, which appears to be maintained during childhood, including deficits in cognitive abilities, working memory, and perceptual reasoning [74–76]. Attention deficits, receptive language, social cognition problems, reward, and behavioral dysfunction have been correlated with lower intelligence quotient (IQ) scores in humans prenatally exposed to chlorpyrifos [76]. Prenatal OP exposure has also been linked to increase autism risk [77]. Acetylcholinesterase (AChE) has been shown to be inhibited with OPs, determining excessive cholinergic transmission; however, OP's main neurotoxic actions are seemingly

There is a growing body of evidence that links the exposure to organochlorines (OCs) and autism. Despite their neurotoxic liabilities, OCs are frequently used in agriculture. The

uct for the amelioration of *C. difficile* in autistic patients [69].

**1.5. Pesticides as neurotoxins in autism**

exerted by their axon metabolites [78].

pesticides [71].

Clayton hypothesized that impaired gut microbiota can be linked to autism through the abnormal gut bacterial metabolism of phenylalanine and tyrosine resulting in the production of P-cresol, with *C. difficile* as one of the most notable p-cresol producers [62]. Overgrowth of *C. difficile* can be linked to the etiology of autism through the inhibitory effect of p-cresol on dopamine β-hydroxylase as a rate-limiting enzyme of dopamine metabolism [63]. Moreover, *C. difficile-induced production of p-cresol can inhibit sulfonation as a detoxification mechanism of special importance when considering neonatal inactive* glucuronidation as an alternative detoxification reaction for xenobiotic excretion [64, 65]. Based on this information, p-cresol may be linked to autism through impaired gut microbiota and the overgrowth of *C. difficile* [66]. **Figure 3** summarizes the role of *C. difficile* overgrowth in the etiology of autism. Hsiao et al. reported that bacterial toxin-induced metabolic changes can trigger autistic behavior. Maternal immune-activated (MIA) females produce offspring

**Figure 3.** Role of *C.difficile* overgrowth in the etiology of autism through the inhibition of dopamine-beta hydroxylase by p-cresol as bacterial metabolite of tyrosine and phenylalanine.

with impaired communicative and social behavior representative as autistic features [67]. Moreover, MIA offspring displayed altered gut microbiota and leaky gut. Treatment of mice with *Bacteroides fragilis* was effective in restoring normal gut permeability and reduces anxiety-like behavioral deficits in this autism model [67]. Most recently, Yang and Chiu reported that through the soluble NSF attachment protein receptor (SNARE) complex, clostridial neurotoxins block neurotransmission to or from neurons. In addition, the gut microbiota produces molecules that act on enteric neurons to reduce gastrointestinal motility and metabolites that stimulate the "gut-brain axis" to alter neural circuits and brain function and behavior [68]. Aljarallah reported that water extract of myrrh plant demonstrates high antimicrobial effect against *C. difficile* strains, which could support its use as a natural product for the amelioration of *C. difficile* in autistic patients [69].

#### **1.5. Pesticides as neurotoxins in autism**

produce cytotoxins known to cause cellular damage. Additionally, both *C. tetani* and *C. difficile* produce phenolic metabolites [55]. Some toxigenic strains of *C. tetani* produce an extremely potent neurotoxin. Clostridia toxins can enter the circulation and accumulate at nerve terminals through binding to host-independent receptors [56, 57]. Following endocytosis of the neurotoxin-receptor complex, acidification of the presynaptic vesicle triggers a conformational change in the N-terminal translocation domain. Acidification is mediated by the vesicular ATPase proton pump, whose function is to ensure the reuptake of neurotransmitters into the synaptic vesicle. This might provide a plausible mechanism for the abnormal reuptake of glutamate in autistic patients. Several studies have assessed the fecal flora of autistic and control individuals, reporting an overgrowth of pathogenic bacterial species in autistic patients when compared to controls. Among these species is *Clostridium* species, including *C. difficile* [58–60]. In addition, Parracho et al. found a higher incidence of the *Clostridium histolyticum* group in the

Clayton hypothesized that impaired gut microbiota can be linked to autism through the abnormal gut bacterial metabolism of phenylalanine and tyrosine resulting in the production of P-cresol, with *C. difficile* as one of the most notable p-cresol producers [62]. Overgrowth of *C. difficile* can be linked to the etiology of autism through the inhibitory effect of p-cresol on dopamine β-hydroxylase as a rate-limiting enzyme of dopamine metabolism [63]. Moreover, *C. difficile-induced production of p-cresol can inhibit sulfonation as a detoxification mechanism of special importance when considering neonatal inactive* glucuronidation as an alternative detoxification reaction for xenobiotic excretion [64, 65]. Based on this information, p-cresol may be linked to autism through impaired gut microbiota and the overgrowth of *C. difficile* [66]. **Figure 3** summarizes the role of *C. difficile* overgrowth in the etiology of autism. Hsiao et al. reported that bacterial toxin-induced metabolic changes can trigger autistic behavior. Maternal immune-activated (MIA) females produce offspring

**Figure 3.** Role of *C.difficile* overgrowth in the etiology of autism through the inhibition of dopamine-beta hydroxylase by

p-cresol as bacterial metabolite of tyrosine and phenylalanine.

fecal flora of 58 ASD children compared to 10 healthy children [61].

60 Neurotoxins

By using retrospective epidemiological studies, environmental factors such as pesticides have been linked to autism. Experimental research in mouse cortical neuron-enriched cultures exposed to hundreds of chemicals commonly found in the environment and on food showed how such chemicals greatly affect brain development. Pearson et al. have been found that rotenone, a pesticide associated with Parkinson's disease risk, and certain fungicides, including pyraclostrobin, trifloxystrobin, famoxadone, and fenamidone, produce transcriptional changes in vitro that are similar to those seen in brain samples from humans with autism [70]. These chemicals stimulate ROS production and disrupt microtubules in neurons, effects that can be reduced by pretreating with a microtubule stabilizer and an antioxidant such as sulforaphane. In this study, 283 autistic children showed neuronal tube defects with potential relationships between maternal residential proximity and agricultural use of neurotoxic pesticides [71].

Organophosphates (OPs) are the most generally utilized pesticides in agriculture, as well as bug sprays in residential, commercial, and industrial settings. Fetus may be exposed to OPs via the placenta or infants through breast milk, food, and inhalation. These small children appear particularly vulnerable to OPs and oxidative stress compared to adults, because of their lower activity levels of the enzyme paraoxonase, involved in OP inactivation and lipid peroxide degradation [72]. This enzyme was found to be significantly lower in autistic patients compared to healthy controls [73]. Prenatal exposure to OPs has been connected to neurodevelopmental disorder, which appears to be maintained during childhood, including deficits in cognitive abilities, working memory, and perceptual reasoning [74–76]. Attention deficits, receptive language, social cognition problems, reward, and behavioral dysfunction have been correlated with lower intelligence quotient (IQ) scores in humans prenatally exposed to chlorpyrifos [76]. Prenatal OP exposure has also been linked to increase autism risk [77]. Acetylcholinesterase (AChE) has been shown to be inhibited with OPs, determining excessive cholinergic transmission; however, OP's main neurotoxic actions are seemingly exerted by their axon metabolites [78].

There is a growing body of evidence that links the exposure to organochlorines (OCs) and autism. Despite their neurotoxic liabilities, OCs are frequently used in agriculture. The association between autism and pesticide exposure during the third trimester has been observed from mothers living near agricultural areas where pesticides were used [79]. Shelton et al. found that increased exposure to insect repellent can lead to autism [80]. Eskenazi et al. and Rauh et al. reported that autistic children had higher OP metabolites during early to midpregnancy [77, 81]. Case-control studies reported that exposure to imidacloprid, insecticide, through the consistent use of flea/tick pet treatment throughout pregnancy period was associated with ASD [79, 82]. Rauh et al. reported that children exposed to OP insecticides showed psychomotor and mental development delays, attention, hyperactivity disorders, and pervasive developmental issues by 3 years of age [81].

Robert and English (2012) described ASD and applications of OCs in proximity to maternal residence before, during, and after pregnancy. Bayesian model as a flexible step function was formulated to measure the time that is needed by pesticide to affect fetus or children for mother who lived near agricultural area [83]. The association between autism and OCs was high, and the time of this association was extending from approximately 4 months prior to fertilization to 8 months into pregnancy. Roberts et al. have suggested that the risk of ASD is increased by 6.1-fold in children with maternal exposure to OCPs during the first trimester of pregnancy, a key period of gestation and neurodevelopmental processes in neonates [84]. Here, 465 children were enrolled in retrospective study to assess pesticide type, exposure time, and residential distance from pesticide application. This study reported an association between prenatal exposure to dicofol and endosulfan pesticide during the 8 weeks immediately following the time of cranial neural tube, and increased risk of autism in children of mothers who lived within fields that had the highest quartile of estimated pesticide exposure compared with children whose mothers lived more than far from exposure, and therefore had the lowest exposure levels [84].

Chlorinated biphenyl (CB) is used as dispersant in pesticide [85]. Because is a CB are pollutant with potentially persistent immunological and neurological effects [86, 87]. It has also been reported that CB can increase the production of ROS and cytotoxicity [88]. The neurological and immunological abnormalities as well as oxidative stress due to CB exposure have also been observed in autistic children [89–91]. **Figure 4** presents structures for selected insecticides linked to neurotoxicity and autism.

#### **1.6. Endocrine-disrupting chemicals**

Endocrine-disrupting chemicals (EDCs) such as polychlorinated biphenyl (PCB), polybrominated diphenyl ethers, bisphenol A (BPA), dioxins, and phthalate have strong associations with neurological disorders [92]. These EDCs are able to interfere with hormone functions because they may alter hormone-dependent processes and/or disrupt endocrine gland function. Certain EDCs are able to alter synaptic function and neural networks [93]. The EDCs are also termed neural-disrupting chemicals since they may increase the prevalence of neurodevelopmental disorders including autism. Prospective epidemiological studies are warranted to better understand EDC-related effects in humans [94]. Braun et al. found that midpregnancy BPA concentrations were associated with an increase in impaired neurodevelopment in early childhood [95]. In a study with 137 children, mothers with high phthalate metabolites in urine during the third trimester gave birth to infants that were more susceptible to

**Figure 4.** Structures of selected insecticides recorded as neurotoxin related to autism.

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**Figure 4.** Structures of selected insecticides recorded as neurotoxin related to autism.

association between autism and pesticide exposure during the third trimester has been observed from mothers living near agricultural areas where pesticides were used [79]. Shelton et al. found that increased exposure to insect repellent can lead to autism [80]. Eskenazi et al. and Rauh et al. reported that autistic children had higher OP metabolites during early to midpregnancy [77, 81]. Case-control studies reported that exposure to imidacloprid, insecticide, through the consistent use of flea/tick pet treatment throughout pregnancy period was associated with ASD [79, 82]. Rauh et al. reported that children exposed to OP insecticides showed psychomotor and mental development delays, attention, hyperactivity disorders, and perva-

Robert and English (2012) described ASD and applications of OCs in proximity to maternal residence before, during, and after pregnancy. Bayesian model as a flexible step function was formulated to measure the time that is needed by pesticide to affect fetus or children for mother who lived near agricultural area [83]. The association between autism and OCs was high, and the time of this association was extending from approximately 4 months prior to fertilization to 8 months into pregnancy. Roberts et al. have suggested that the risk of ASD is increased by 6.1-fold in children with maternal exposure to OCPs during the first trimester of pregnancy, a key period of gestation and neurodevelopmental processes in neonates [84]. Here, 465 children were enrolled in retrospective study to assess pesticide type, exposure time, and residential distance from pesticide application. This study reported an association between prenatal exposure to dicofol and endosulfan pesticide during the 8 weeks immediately following the time of cranial neural tube, and increased risk of autism in children of mothers who lived within fields that had the highest quartile of estimated pesticide exposure compared with children whose mothers lived more than far from exposure, and therefore had

Chlorinated biphenyl (CB) is used as dispersant in pesticide [85]. Because is a CB are pollutant with potentially persistent immunological and neurological effects [86, 87]. It has also been reported that CB can increase the production of ROS and cytotoxicity [88]. The neurological and immunological abnormalities as well as oxidative stress due to CB exposure have also been observed in autistic children [89–91]. **Figure 4** presents structures for selected insecti-

Endocrine-disrupting chemicals (EDCs) such as polychlorinated biphenyl (PCB), polybrominated diphenyl ethers, bisphenol A (BPA), dioxins, and phthalate have strong associations with neurological disorders [92]. These EDCs are able to interfere with hormone functions because they may alter hormone-dependent processes and/or disrupt endocrine gland function. Certain EDCs are able to alter synaptic function and neural networks [93]. The EDCs are also termed neural-disrupting chemicals since they may increase the prevalence of neurodevelopmental disorders including autism. Prospective epidemiological studies are warranted to better understand EDC-related effects in humans [94]. Braun et al. found that midpregnancy BPA concentrations were associated with an increase in impaired neurodevelopment in early childhood [95]. In a study with 137 children, mothers with high phthalate metabolites in urine during the third trimester gave birth to infants that were more susceptible to

sive developmental issues by 3 years of age [81].

62 Neurotoxins

the lowest exposure levels [84].

cides linked to neurotoxicity and autism.

**1.6. Endocrine-disrupting chemicals**

autism [96]. Larsson et al. have found a correlation between EDC and autism [97]. High serum phthalate concentrations have been recorded in autistic children [98]. Experimental animals show changes in development, synaptic organization, neurotransmitter synthesis and release, and brain structural organization due to exposure to EDC [99]. Cock et al. described links between brominated flame retardants, perfluorinated compounds, and ASD, yet additional weights of evidence are needed [100].

aminotransferase and glutamate dehydrogenase. These authors pointed out that oxidative stress was consistent with some changes in proteomic markers. Alteration in blood and brain glutathione status and deficiencies of reduced glutathione are increasingly associated with autism. Fortunately, certain studies demonstrating that supplementation with antioxidants such as vitamins C and E reduced oxidative impacts on rat endometrium from due to vitamins E and C reduced adverse impacts on rat endometrium due to exposure to 900 MHz EMR [118]. Ilhan et al. proved that *Ginkgo biloba* has also prevented mobile phone-induced increases in lipid peroxides and nitric oxide levels in brain tissue as well as decreases in brain superoxide dismutase and glutathione peroxidase activities and increases in brain xanthine oxidase and adenosine deaminase activities, together with the relief of the histopathological cell injury [119]. **Figure 5** demonstrates the role of cell phone radiation in the etiology of

Neurotoxins and Autism

65

http://dx.doi.org/10.5772/intechopen.69431

**Figure 5.** Role of *cell phone radiation* in the etiology of autism through oxidative stress as a major etiological mechanism.

autism through oxidative stress as a major etiological mechanism.

Several EDCs are known to disturb sex steroid and affect thyroid hormone (TH) levels which in turn are known to affect synaptogenesis, neuronal differentiation, migration, and myelination [101]. They play critical roles in brain development and potentially also for development of the connectome. TH receptor mutation in mice showed reduced density of GABAergic in the hippocampus, which was accompanied by more depressive and anxious behavior [102]. It is demonstrated that BPA inhibits the GABAAR-mediated response and that BPA affects development of GABAergic and dopaminergic systems [103]. Some studies indicate EDCs as a cause for neurodevelopmental disorders through GABAergic system changes [104, 105].

#### **1.7. Radio frequency energy (RFE) of cell phone as neurotoxin in autism**

During pregnancy, the possibility of fetal damage is increased as mothers are exposed to RFE [106]. Notably, the fetus may not be fully protected by amniotic fluid. It is well known that the pelvic structure permits deep penetration of the RFE to be absorbed within the developing fetus. Based on this, many investigations proposed that the dramatic increase in the incidence of autism since 1980 can be related to the neurotoxicity of cell phone radiation [107–109].

Based on our understanding on the etiological mechanisms in autism, such as oxidative stress, neuroinflammation, and glutamate excitotoxicity, the remarkable increase in the prevalence of autism about tenfolds since 1980 can be related to this dramatic increase which reach 1:45 on 2015 [110]. It is well documented that exposure to radio frequency radiation (RFR) can be accompanied by oxidative stress in human and animal models of autism [111, 112]. Unfortunately, these effects of cellular phone use can occur even at low and legal intensity which are now common environmental risk factors for infants, young children, adults, pregnant women, and fetuses. Cell phone radiations enhance free radical formation through the Fenton reaction as catalytic process through which iron converts hydrogen peroxides, a product of oxidative respiration in the mitochondria, into hydroxyl free radical, which is very potent and can induce damage of macromolecules, such as membrane phospholipids, DNA, and protein. Radio frequency radiation at very low intensities can also impair mitochondrial metabolism and modulate glutathione, glutamate, and GABA, which are substances related to the pathophysiology of autism [90, 113–116].

Fragopoulou et al. reported that through proteomic analysis of brain regulatory proteins from mice following prolonged exposure to electromotive force (EMF) led to either downregulation or overexpression of 143 proteins [117]. These altered proteins include neural functionrelated proteins, alpha-synuclein, glia maturation factor beta, cytoskeletal proteins, heat shock proteins, apolipoprotein E, as well as proteins of brain metabolism such as aspartate aminotransferase and glutamate dehydrogenase. These authors pointed out that oxidative stress was consistent with some changes in proteomic markers. Alteration in blood and brain glutathione status and deficiencies of reduced glutathione are increasingly associated with autism. Fortunately, certain studies demonstrating that supplementation with antioxidants such as vitamins C and E reduced oxidative impacts on rat endometrium from due to vitamins E and C reduced adverse impacts on rat endometrium due to exposure to 900 MHz EMR [118]. Ilhan et al. proved that *Ginkgo biloba* has also prevented mobile phone-induced increases in lipid peroxides and nitric oxide levels in brain tissue as well as decreases in brain superoxide dismutase and glutathione peroxidase activities and increases in brain xanthine oxidase and adenosine deaminase activities, together with the relief of the histopathological cell injury [119]. **Figure 5** demonstrates the role of cell phone radiation in the etiology of autism through oxidative stress as a major etiological mechanism.

autism [96]. Larsson et al. have found a correlation between EDC and autism [97]. High serum phthalate concentrations have been recorded in autistic children [98]. Experimental animals show changes in development, synaptic organization, neurotransmitter synthesis and release, and brain structural organization due to exposure to EDC [99]. Cock et al. described links between brominated flame retardants, perfluorinated compounds, and ASD, yet additional

Several EDCs are known to disturb sex steroid and affect thyroid hormone (TH) levels which in turn are known to affect synaptogenesis, neuronal differentiation, migration, and myelination [101]. They play critical roles in brain development and potentially also for development of the connectome. TH receptor mutation in mice showed reduced density of GABAergic in the hippocampus, which was accompanied by more depressive and anxious behavior [102]. It is demonstrated that BPA inhibits the GABAAR-mediated response and that BPA affects development of GABAergic and dopaminergic systems [103]. Some studies indicate EDCs as a cause for neurodevelopmental disorders through GABAergic system

During pregnancy, the possibility of fetal damage is increased as mothers are exposed to RFE [106]. Notably, the fetus may not be fully protected by amniotic fluid. It is well known that the pelvic structure permits deep penetration of the RFE to be absorbed within the developing fetus. Based on this, many investigations proposed that the dramatic increase in the incidence of autism since 1980 can be related to the neurotoxicity of cell phone radiation [107–109].

Based on our understanding on the etiological mechanisms in autism, such as oxidative stress, neuroinflammation, and glutamate excitotoxicity, the remarkable increase in the prevalence of autism about tenfolds since 1980 can be related to this dramatic increase which reach 1:45 on 2015 [110]. It is well documented that exposure to radio frequency radiation (RFR) can be accompanied by oxidative stress in human and animal models of autism [111, 112]. Unfortunately, these effects of cellular phone use can occur even at low and legal intensity which are now common environmental risk factors for infants, young children, adults, pregnant women, and fetuses. Cell phone radiations enhance free radical formation through the Fenton reaction as catalytic process through which iron converts hydrogen peroxides, a product of oxidative respiration in the mitochondria, into hydroxyl free radical, which is very potent and can induce damage of macromolecules, such as membrane phospholipids, DNA, and protein. Radio frequency radiation at very low intensities can also impair mitochondrial metabolism and modulate glutathione, glutamate, and GABA, which are substances related

Fragopoulou et al. reported that through proteomic analysis of brain regulatory proteins from mice following prolonged exposure to electromotive force (EMF) led to either downregulation or overexpression of 143 proteins [117]. These altered proteins include neural functionrelated proteins, alpha-synuclein, glia maturation factor beta, cytoskeletal proteins, heat shock proteins, apolipoprotein E, as well as proteins of brain metabolism such as aspartate

**1.7. Radio frequency energy (RFE) of cell phone as neurotoxin in autism**

weights of evidence are needed [100].

to the pathophysiology of autism [90, 113–116].

changes [104, 105].

64 Neurotoxins

**Figure 5.** Role of *cell phone radiation* in the etiology of autism through oxidative stress as a major etiological mechanism.

#### **Author details**

Afaf El-Ansary1,3\*, Abeer Al-Dbass2 and Hanan Qasem3

\*Address all correspondence to<EMAIL_ADDRESS>
1 Central Laboratory, Female Center for Medical Studies and Scientific Section, King Saud University, Riyadh, Saudi Arabia

[11] Sanders T, et al. Neurotoxic effects and biomarkers of lead exposure: A review. Reviews

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2 Biochemistry Department, College of Science, King Saud University, Riyadh, Saudi Arabia

3 Autism Research and Treatment Centre, King Saud University, Riyadh, Saudi Arabia

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**Author details**

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**Chapter 5**

**Provisional chapter**

**Botulinum Neurotoxin: A Multifunctional Protein for**

Botulinum neurotoxin (BoNT) is a major therapeutic agent licensed in neurological indications such as dystonia and spasticity. In recent years, its use has steadily increased in other neurological areas and new therapeutic areas and also in the aesthetic setting. Paradoxically, BoNT is also the causative agent of the disease botulism and a potential bioterrorism toxin. The BoNT family of toxins comprised more than 40 individual members, classified into 7 serotypes and are produced by Gram-positive obligate anaerobic bacteria. BoNTs are enzymatic multi-modular proteins with a complex multistep mechanism of action. Their target site is at peripheral neurons, particularly the neuromuscular junction, at which they inhibit acetylcholine neurotransmission. Despite intense activity in the BoNT field, today there are still gaps in knowledge both in clinical practice and in basic research. The discovery of the structure-function of BoNT and its domains has allowed rational design of new features using molecular engineering. The diversity of BoNT molecules, both natural and engineered, is an invaluable pool from which to design future new therapeutics with unique pharmacological properties for current and

**Keywords:** botulinum neurotoxin, therapeutic agent, botulism, recombinant protein,

**Botulinum Neurotoxin: A Multifunctional Protein for**

DOI: 10.5772/intechopen.69433

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

There are currently four botulinum neurotoxin (BoNT) clinical products available in the Western hemisphere: abobotulinumtoxinA (Dysport®, Ipsen, Paris, France), incobotulinumtoxinA (Xeomin®, Merz Pharmaceuticals GmbH, Frankfurt, Germany), onabotulinumtoxinA (Botox®, Allergan, Irvine, CA, USA) and rimabotulinumtoxinB (Myobloc®, Solstice

**the Development of New Therapeutics**

**the Development of New Therapeutics**

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.69433

Elena Fonfria

**Abstract**

novel indications.

**1. Introduction**

targeted secretion inhibitor

Elena Fonfria

**Provisional chapter**

#### **Botulinum Neurotoxin: A Multifunctional Protein for the Development of New Therapeutics the Development of New Therapeutics**

**Botulinum Neurotoxin: A Multifunctional Protein for**

DOI: 10.5772/intechopen.69433

#### Elena Fonfria Elena Fonfria Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.69433

#### **Abstract**

Botulinum neurotoxin (BoNT) is a major therapeutic agent licensed in neurological indications such as dystonia and spasticity. In recent years, its use has steadily increased in other neurological areas and new therapeutic areas and also in the aesthetic setting. Paradoxically, BoNT is also the causative agent of the disease botulism and a potential bioterrorism toxin. The BoNT family of toxins comprised more than 40 individual members, classified into 7 serotypes and are produced by Gram-positive obligate anaerobic bacteria. BoNTs are enzymatic multi-modular proteins with a complex multistep mechanism of action. Their target site is at peripheral neurons, particularly the neuromuscular junction, at which they inhibit acetylcholine neurotransmission. Despite intense activity in the BoNT field, today there are still gaps in knowledge both in clinical practice and in basic research. The discovery of the structure-function of BoNT and its domains has allowed rational design of new features using molecular engineering. The diversity of BoNT molecules, both natural and engineered, is an invaluable pool from which to design future new therapeutics with unique pharmacological properties for current and novel indications.

**Keywords:** botulinum neurotoxin, therapeutic agent, botulism, recombinant protein, targeted secretion inhibitor

#### **1. Introduction**

There are currently four botulinum neurotoxin (BoNT) clinical products available in the Western hemisphere: abobotulinumtoxinA (Dysport®, Ipsen, Paris, France), incobotulinumtoxinA (Xeomin®, Merz Pharmaceuticals GmbH, Frankfurt, Germany), onabotulinumtoxinA (Botox®, Allergan, Irvine, CA, USA) and rimabotulinumtoxinB (Myobloc®, Solstice

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

Neurosciences, Louisville, KY, USA) [1]. Several other products are available for use in other countries, in particular in the Asian markets, and new formulations and products are underdevelopment [2]. By 2022, it is expected that the market size for botulinum products will reach \$6.6 billion, driven by the expansion of their therapeutic uses and also the appetite for noninvasive aesthetic applications [3].

Around 200 years ago (between 1817 and 1822), a German medical officer, Justinus Kerner, published a series of papers to provide the first accurate and complete description of the symptoms of food-borne botulism, which led to the discovery of BoNT as the causative agent and the prediction by Kerner of its potential clinical utility [4]. This fascinating class of proteins present a modular molecular architecture with distinct binding, translocation and enzymatic domains. The different structural and functional domains can be regarded as 'building blocks' and have facilitated a number of engineering approaches aimed, amongst other purposes, at extending the therapeutic applications of BoNTs to other cell types beyond their natural target of the neuromuscular junction [5].

The aim of this chapter is to (1) provide an overview of the current clinical uses and a historical perspective of botulinum neurotoxin discovery, the disease it causes and the threats and opportunities that it poses and (2) present the current understanding of the structure-function of the toxin and its application in the development of new therapeutics.

#### **2. Clinical uses**

BoNT products are neuromuscular blocking agents which exert their effect through inhibition of acetylcholine release. BoNTs are amongst the most tissue-selective drugs known in clinical pharmacology and are characterised by high potency, high specificity and long duration of action of around 3–6 months following a single injection [6]. These characteristics have made BoNTs highly successful and effective therapeutic agents for the management of several chronic and debilitating diseases of neuronal hyperactivity. Although initially thought to inhibit acetylcholine release only at the neuromuscular junction, BoNTs are recognised to also inhibit release of neurotransmitters from autonomic nerve terminals, for example, in glands (e.g. in hyperhidrosis), and nociceptive neurons in pain states [6, 7].

Currently, there are four formulations of BoNTs approved by the US Food and Drug Administration (FDA) for several clinical applications (see **Table 1**). Cervical dystonia, also known as spasmodic torticollis (disorder characterised by involuntary contractions of neck and upper shoulder muscles resulting in abnormal postures and/or movement of the neck, shoulder and head and that may be associated with neck pain), is the only condition for which all four formulations are approved. Other neurological conditions include spasticity (disorder characterised by tight or stiff muscles and an inability to control those muscles), with approved formulations both for adult and paediatric populations, migraine and blepharospasm (dystonia that can cause disabling eye closure). Other non-neurological therapeutic FDA-approved uses are strabismus (eye misalignment), overactive bladder, urinary incontinence and hyperhidrosis (excessive sweating).

**FDA-approved**

**Treatment population**

**(Dysport®)**

Approved Approved

**AbobotulinumtoxinA**

**IncobotulinumtoxinA**

**OnabotulinumtoxinA**

**RimabotulinumtoxinB**

**(Myobloc®)**

Approved

**(Botox®)**

Approved Approved Approved

na

na

na

na

**(Xeomin®)**

Approved Approved

na na

**indication**

Cervical dystonia

Upper limb spasticity

Lower limb spasticity

Lower limb spasticity

Migraine Blepharospasm

Strabismus Glabellar lines

Overactive bladder

Urinary incontinence

Hyperhidrosis

na = indication not FDA-approved.

**Table 1.**

Food and Drug Administration (FDA)-approved indications for the use of marketed botulinum neurotoxins products [1].

 Several other products are available for use in other countries, in particular in the Asian markets, and new formulations and products are underdevelopment [2]. By 2022, it is expected that the market size for botulinum products will reach \$6.6 billion, driven by the expansion of their therapeutic uses and also the appetite for non-

Around 200 years ago (between 1817 and 1822), a German medical officer, Justinus Kerner, published a series of papers to provide the first accurate and complete description of the symptoms of food-borne botulism, which led to the discovery of BoNT as the causative agent and the prediction by Kerner of its potential clinical utility [4]. This fascinating class of proteins present a modular molecular architecture with distinct binding, translocation and enzymatic domains. The different structural and functional domains can be regarded as 'building blocks' and have facilitated a number of engineering approaches aimed, amongst other purposes, at extending the therapeutic applications of BoNTs to other cell types beyond their

The aim of this chapter is to (1) provide an overview of the current clinical uses and a historical perspective of botulinum neurotoxin discovery, the disease it causes and the threats and opportunities that it poses and (2) present the current understanding of the structure-function

BoNT products are neuromuscular blocking agents which exert their effect through inhibition of acetylcholine release. BoNTs are amongst the most tissue-selective drugs known in clinical pharmacology and are characterised by high potency, high specificity and long duration of action of around 3–6 months following a single injection [6]. These characteristics have made BoNTs highly successful and effective therapeutic agents for the management of several chronic and debilitating diseases of neuronal hyperactivity. Although initially thought to inhibit acetylcholine release only at the neuromuscular junction, BoNTs are recognised to also inhibit release of neurotransmitters from autonomic nerve terminals, for example, in glands (e.g. in hyperhidrosis), and nociceptive neurons in pain states [6, 7]. Currently, there are four formulations of BoNTs approved by the US Food and Drug Administration (FDA) for several clinical applications (see **Table 1**). Cervical dystonia, also known as spasmodic torticollis (disorder characterised by involuntary contractions of neck and upper shoulder muscles resulting in abnormal postures and/or movement of the neck, shoulder and head and that may be associated with neck pain), is the only condition for which all four formulations are approved. Other neurological conditions include spasticity (disorder characterised by tight or stiff muscles and an inability to control those muscles), with approved formulations both for adult and paediatric populations, migraine and blepharospasm (dystonia that can cause disabling eye closure). Other non-neurological therapeutic FDA-approved uses are strabismus (eye misalignment), overactive bladder, urinary inconti-

of the toxin and its application in the development of new therapeutics.

invasive aesthetic applications [3].

76 Neurotoxins

**2. Clinical uses**

natural target of the neuromuscular junction [5].

nence and hyperhidrosis (excessive sweating).

**Table 1.** Food and Drug Administration (FDA)-approved indications for the use of marketed botulinum neurotoxins products [1]. Historically, BoNT products have been considered as a single pharmacological class [8]. However, the existing BoNT products vary in the identity and amount of toxin present, their formulations, the manufacturing processes and the potency methods used to determine the strength of the products [9, 10]. As a result, the different products are not considered to be interchangeable, and their respective clinical efficacy and safety are unique to each specific product [11].

the eyebrows): abobotulinumtoxinA (Dysport®, Ipsen as the marketing authorisation holder with Galderma as distributor in the aesthetic indication), incobotulinumtoxinA (Xeomin®/ Bocouture®, Merz) and onabotulinumtoxinA (Botox®/Vistabel®, Allergan) (see **Table 1**). The facial aesthetic uses of BoNTs are extensive, mainly not approved and under investigation, and patient satisfaction with treatment is very high, with significant improvement in patientreported outcomes. Rhytides (skin wrinkles) regions for treatment include forehead, brow, region between the eyebrows, around the eyes (crow's feet) and nose (bunny lines), smile

Botulinum Neurotoxin: A Multifunctional Protein for the Development of New Therapeutics

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79

Despite intense use of BoNTs in clinical practice, approval and labelling guidance does not exist to address key questions such as where BoNTs fit amongst various treatment options for a given condition, recommendations of one product over another for a given indication or

Botulism is a rare but potentially fatal disease caused by BoNT intoxication. Botulism is characterised by a descending flaccid paralysis with symptoms of cranial nerve dysfunction such as diplopia (double vision), dysphagia (difficulty in swallowing), pupillary dilation and ptosis (drooping eyelids), progressing to respiratory failure and, in rare occasions if not provided with suitable intensive care and life support, ultimately death. Fever and altered mental status are absent. The diagnosis of botulism is largely clinical and is confirmed by laboratory tests, sometimes including the detection of BoNTs in contaminated materials, food or bodily waste [19]. Botulism in humans is classified according to the route of entry of the toxin: food-borne botulism occurs after the ingestion of BoNT-contaminated food that contains the preformed toxin; infant botulism is the result of bacteria colonising the immature gastrointestinal tract of infants which then produce and release the toxin in situ; wound botulism results from spore contamination into the tissue and is mostly associated with injection drug abuse; and iatrogenic botulism can occur as a result of excessive BoNT use either for therapeutic or cosmetic use [20]. Inhalation botulism is also a possibility, if the toxin were to enter through the respira-

tory tract. However, inhalation botulism is rare and does not occur naturally [21].

A stable number of cases of botulism have been reported in Europe (i.e. European Economic Area, comprised of 31 countries) in recent years. During the period 2007–2014, an average of 115 cases per year of confirmed botulism occurred, and 5% of those were fatal [22]. A very similar numbers in the US were reported by the Centres for Disease Control and Prevention (CDC) for the same period (2007–2014), with an average of 143 confirmed cases per year, with 2% of those being fatal. According to the CDC, the most numerous cases were of infant botulism, but wound and food-borne botulisms were also presented yearly, plus a minor percent-

There is currently no approved pharmacological treatment for BoNT intoxication in humans, and recent efforts have focussed on the development of (1) vaccines from partially purified toxins, (2) use of specific antitoxin antibodies and (3) small molecule inhibitors [20, 24]. Once an

(gummy smile), upper lip, corners of the mouth, jaw, chin and neck area [16, 17].

clinical differences in potency and duration of action (see Refs. [8, 18]).

**3. Disease and bioterrorism threat**

age of cases of unknown aetiology [23].

In 2016, the American Academy of Neurology (AAN) published updated guidelines for the clinical use of BoNT [12]. The 2016 AAN recommendations for BoNT use, based on evidence from clinical trials, do not fully match the FDA-approved indications or AAN's previous guidelines from 2008, which is a reflection of the expanding uses of BoNTs [8]. Multiple clinical trials are being conducted to investigate the efficacy and safety of BoNTs for various clinical conditions and, in addition, pilot studies are being conducted to test the efficacy of BoNTs for new indications [9, 13, 14]. A summary of not approved new indications for which botulinum toxins are under investigation is presented in **Table 2**.

The use of BoNTs has been extended to aesthetic applications for the reduction of facial lines. According to recent statistics, BoNT injections are now the most popular of all cosmetic procedures worldwide, both surgical and non-surgical [15], and, in the US, more than 6.6 million injections were performed in 2014 alone for aesthetic reasons [16]. There are currently three BoNT products approved by the FDA for use in glabellar lines (wrinkles that appear between

**Table 2.** Not approved new indications for which botulinum toxins are under investigation [9, 13, 14].

the eyebrows): abobotulinumtoxinA (Dysport®, Ipsen as the marketing authorisation holder with Galderma as distributor in the aesthetic indication), incobotulinumtoxinA (Xeomin®/ Bocouture®, Merz) and onabotulinumtoxinA (Botox®/Vistabel®, Allergan) (see **Table 1**). The facial aesthetic uses of BoNTs are extensive, mainly not approved and under investigation, and patient satisfaction with treatment is very high, with significant improvement in patientreported outcomes. Rhytides (skin wrinkles) regions for treatment include forehead, brow, region between the eyebrows, around the eyes (crow's feet) and nose (bunny lines), smile (gummy smile), upper lip, corners of the mouth, jaw, chin and neck area [16, 17].

Despite intense use of BoNTs in clinical practice, approval and labelling guidance does not exist to address key questions such as where BoNTs fit amongst various treatment options for a given condition, recommendations of one product over another for a given indication or clinical differences in potency and duration of action (see Refs. [8, 18]).

#### **3. Disease and bioterrorism threat**

Historically, BoNT products have been considered as a single pharmacological class [8]. However, the existing BoNT products vary in the identity and amount of toxin present, their formulations, the manufacturing processes and the potency methods used to determine the strength of the products [9, 10]. As a result, the different products are not considered to be interchangeable, and their respective clinical efficacy and safety are unique to each specific

In 2016, the American Academy of Neurology (AAN) published updated guidelines for the clinical use of BoNT [12]. The 2016 AAN recommendations for BoNT use, based on evidence from clinical trials, do not fully match the FDA-approved indications or AAN's previous guidelines from 2008, which is a reflection of the expanding uses of BoNTs [8]. Multiple clinical trials are being conducted to investigate the efficacy and safety of BoNTs for various clinical conditions and, in addition, pilot studies are being conducted to test the efficacy of BoNTs for new indications [9, 13, 14]. A summary of not approved new indications for which botuli-

The use of BoNTs has been extended to aesthetic applications for the reduction of facial lines. According to recent statistics, BoNT injections are now the most popular of all cosmetic procedures worldwide, both surgical and non-surgical [15], and, in the US, more than 6.6 million injections were performed in 2014 alone for aesthetic reasons [16].  Not approved new indications for which botulinum toxins are under investigation [9, 13, 14].

num toxins are under investigation is presented in **Table 2**.

Anismus Exotropia, esotropia,

entropion

product [11].

78 Neurotoxins

Benign prostatic hyperplasia

Botulism is a rare but potentially fatal disease caused by BoNT intoxication. Botulism is characterised by a descending flaccid paralysis with symptoms of cranial nerve dysfunction such as diplopia (double vision), dysphagia (difficulty in swallowing), pupillary dilation and ptosis (drooping eyelids), progressing to respiratory failure and, in rare occasions if not provided with suitable intensive care and life support, ultimately death. Fever and altered mental status are absent. The diagnosis of botulism is largely clinical and is confirmed by laboratory tests, sometimes including the detection of BoNTs in contaminated materials, food or bodily waste [19]. Botulism in humans is classified according to the route of entry of the toxin: food-borne botulism occurs after the ingestion of BoNT-contaminated food that contains the preformed toxin; infant botulism is the result of bacteria colonising the immature gastrointestinal tract of infants which then produce and release the toxin in situ; wound botulism results from spore contamination into the tissue and is mostly associated with injection drug abuse; and iatrogenic botulism can occur as a result of excessive BoNT use either for therapeutic or cosmetic use [20]. Inhalation botulism is also a possibility, if the toxin were to enter through the respiratory tract. However, inhalation botulism is rare and does not occur naturally [21].

A stable number of cases of botulism have been reported in Europe (i.e. European Economic Area, comprised of 31 countries) in recent years. During the period 2007–2014, an average of 115 cases per year of confirmed botulism occurred, and 5% of those were fatal [22]. A very similar numbers in the US were reported by the Centres for Disease Control and Prevention (CDC) for the same period (2007–2014), with an average of 143 confirmed cases per year, with 2% of those being fatal. According to the CDC, the most numerous cases were of infant botulism, but wound and food-borne botulisms were also presented yearly, plus a minor percentage of cases of unknown aetiology [23].

There is currently no approved pharmacological treatment for BoNT intoxication in humans, and recent efforts have focussed on the development of (1) vaccines from partially purified toxins, (2) use of specific antitoxin antibodies and (3) small molecule inhibitors [20, 24]. Once an outbreak occurs, medical treatment includes treatment with the botulin heptavalent antitoxin and consideration of admission to an intensive care unit with mechanical ventilation until recovery. Botulism is not contagious, and standard precautions are sufficient for infection control [19].

produce seven different serotypes of botulinum toxin, termed BoNT/A to BoNT/G. BoNT/A, BoNT/B and BoNT/F were discovered following incidences of food-borne botulism, reminiscent of the original 'sausage poisoning', whereas BoNT/C, BoNT/D and BoNT/E were discovered following incidences of botulism in animals [27] (see **Figure 1**). BoNT/G, discovered in 1970, was reported in a sample extracted from soil, and to date there has not been reported cases of botulism caused by BoNT/G in the wild affecting either humans or animals [30]. A possible eighth type, initially termed BoNT/H was reported in 2013, but later reclassified as a

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81

Despite Kerner suggesting the potential of BoNT as a therapeutic agent in conditions of muscular hypercontraction and glandular hypersecretion, it was not until around 150 years later

**Figure 1.** Timeline of the discovery of the seven botulinum toxin types. For context, also depicted are the dates of the first accurate description of botulism and the first use of botulinum toxins as therapeutic agent. A proposed eighth type was

initially reported in 2013, now classified as an F/A hybrid toxin. For details see Refs. [2, 27, 28].

BoNT/FA hybrid [31].

Botulism also occurs in animals and begins with the growth of the BoNT-producing bacteria in decaying carcasses followed by the release of the toxin into the environment. Both toxin and bacteria can spread via transmission of BoNT-insensitive animals such as maggots and other invertebrates that are consumed by healthy BoNT-sensitive animals, which eventually die and allow the growth of the bacteria and the subsequent production of the toxin to self-amplify the cycle [20].

Partly due to the fact that no effective treatment is available for BoNT intoxication in humans and the perceived ease in which it could be used in a bioterror attack, BoNT is classified as a potential bioterrorism weapon by the US CDC. BoNT belongs to the category A, the highest level of concern regarding public health and need of preparedness. Only five other agents are classified as category A agents, those being anthrax (*Bacillus anthracis*), bubonic plague (*Yersinia pestis*), smallpox (*Variola major*), tularemia (*Francisella tularensis*) and arenaviruses causing viral hemorrhagic fevers [19]. A contentious paper from 2005 regarding ease of BoNT intoxication through cow's milk destined for human consumption calculated if would take only 4 g of BoNT, e.g. roughly equivalent to 1 teaspoon of granulated sugar, to poison over 400,000 people [25]. The publication of that research opened a public safety debate within the scientific community regarding BoNT dual-use research [26], which was reopened when the allegedly new BoNT/H type was originally reported [27]. However, it should be noted that BoNTs are much more toxic (in the range of 100–1000 times) when injected than when administered orally; and delivery by aerosols is considered inefficient [20].

#### **4. Historical overview of BoNT discovery**

BoNT/A is the most potent toxin known to man, with a reported estimated human lethal dose of 1.3–2.1 ng/kg intravenously or intramuscularly and 10–13 ng/kg when inhaled [4]. Not surprisingly, its effects have been known throughout history long before the molecular identity of the toxin was elucidated. Botulism-like symptoms were known by ancient Greeks and Egyptians, and the Byzantine emperor Leo IV (886–911 AD) banned 'blood sausage' as it caused a fatal illness. It was not until around a thousand years later that following a number of sausage poisoning outbreaks in Germany the first accurate and complete description of the symptoms of food-borne botulism was described between 1817 and 1822 by J. Kerner. The extracted causative agent was named 'sausage poison' and was believed to be a 'fatty acid'. Later, a German physician named Muller referred to the sausage poisoning as botulism from the Latin name for sausage, 'botulus' [4, 28].

The first isolation of the bacteria responsible for producing the toxic agent causing botulism was performed by the Belgian professor Emile Pierre van Ermengem and was termed *Bacillus botulinus*. Its name was changed to *Clostridium botulinum* when the aerobic *Bacillus* genus was separated from the anaerobic *Clostridium* genus [29]. To date, six different BoNT-producing bacterial groups are known; all have been taxonomically classified as clostridia. These clostridia produce seven different serotypes of botulinum toxin, termed BoNT/A to BoNT/G. BoNT/A, BoNT/B and BoNT/F were discovered following incidences of food-borne botulism, reminiscent of the original 'sausage poisoning', whereas BoNT/C, BoNT/D and BoNT/E were discovered following incidences of botulism in animals [27] (see **Figure 1**). BoNT/G, discovered in 1970, was reported in a sample extracted from soil, and to date there has not been reported cases of botulism caused by BoNT/G in the wild affecting either humans or animals [30]. A possible eighth type, initially termed BoNT/H was reported in 2013, but later reclassified as a BoNT/FA hybrid [31].

outbreak occurs, medical treatment includes treatment with the botulin heptavalent antitoxin and consideration of admission to an intensive care unit with mechanical ventilation until recovery. Botulism is not contagious, and standard precautions are sufficient for infection control [19]. Botulism also occurs in animals and begins with the growth of the BoNT-producing bacteria in decaying carcasses followed by the release of the toxin into the environment. Both toxin and bacteria can spread via transmission of BoNT-insensitive animals such as maggots and other invertebrates that are consumed by healthy BoNT-sensitive animals, which eventually die and allow the growth of the bacteria and the subsequent production of the toxin to self-amplify the cycle [20]. Partly due to the fact that no effective treatment is available for BoNT intoxication in humans and the perceived ease in which it could be used in a bioterror attack, BoNT is classified as a potential bioterrorism weapon by the US CDC. BoNT belongs to the category A, the highest level of concern regarding public health and need of preparedness. Only five other agents are classified as category A agents, those being anthrax (*Bacillus anthracis*), bubonic plague (*Yersinia pestis*), smallpox (*Variola major*), tularemia (*Francisella tularensis*) and arenaviruses causing viral hemorrhagic fevers [19]. A contentious paper from 2005 regarding ease of BoNT intoxication through cow's milk destined for human consumption calculated if would take only 4 g of BoNT, e.g. roughly equivalent to 1 teaspoon of granulated sugar, to poison over 400,000 people [25]. The publication of that research opened a public safety debate within the scientific community regarding BoNT dual-use research [26], which was reopened when the allegedly new BoNT/H type was originally reported [27]. However, it should be noted that BoNTs are much more toxic (in the range of 100–1000 times) when injected than when admin-

istered orally; and delivery by aerosols is considered inefficient [20].

BoNT/A is the most potent toxin known to man, with a reported estimated human lethal dose of 1.3–2.1 ng/kg intravenously or intramuscularly and 10–13 ng/kg when inhaled [4]. Not surprisingly, its effects have been known throughout history long before the molecular identity of the toxin was elucidated. Botulism-like symptoms were known by ancient Greeks and Egyptians, and the Byzantine emperor Leo IV (886–911 AD) banned 'blood sausage' as it caused a fatal illness. It was not until around a thousand years later that following a number of sausage poisoning outbreaks in Germany the first accurate and complete description of the symptoms of food-borne botulism was described between 1817 and 1822 by J. Kerner. The extracted causative agent was named 'sausage poison' and was believed to be a 'fatty acid'. Later, a German physician named Muller referred to the sausage poisoning as botulism from

The first isolation of the bacteria responsible for producing the toxic agent causing botulism was performed by the Belgian professor Emile Pierre van Ermengem and was termed *Bacillus botulinus*. Its name was changed to *Clostridium botulinum* when the aerobic *Bacillus* genus was separated from the anaerobic *Clostridium* genus [29]. To date, six different BoNT-producing bacterial groups are known; all have been taxonomically classified as clostridia. These clostridia

**4. Historical overview of BoNT discovery**

80 Neurotoxins

the Latin name for sausage, 'botulus' [4, 28].

Despite Kerner suggesting the potential of BoNT as a therapeutic agent in conditions of muscular hypercontraction and glandular hypersecretion, it was not until around 150 years later

**Figure 1.** Timeline of the discovery of the seven botulinum toxin types. For context, also depicted are the dates of the first accurate description of botulism and the first use of botulinum toxins as therapeutic agent. A proposed eighth type was initially reported in 2013, now classified as an F/A hybrid toxin. For details see Refs. [2, 27, 28].

that the first clinical application was made. In 1981, Dr. Alan B. Scott at what was formerly known as the Smith-Kettlewell Institute of Visual Science, San Francisco, California, USA, used BoNT/A for the treatment of strabismus as an alternative to surgical intervention. The original name of the drug was Oculinum®, and its rights were later acquired by Allergan Inc., which changed the name of the drug to Botox®.

#### **5. The producing bacteria: types of toxin and nontoxin proteins**

Upon the first description of the botulism symptoms (see above), the initial hypothesis was that botulism was caused by a toxin produced by a single bacterial organism, as is the case for the closely related toxin tetanus toxin and its producing bacteria *Clostridium tetani* [32]. However, it soon became apparent that different types of toxin and different producing bacteria existed for BoNT [29].

In 1910–1919, serological methods were introduced for categorisation of the toxin-producing bacteria and for the toxins themselves, that are still in use today [33]. Biochemical and molecular techniques have complemented those initial classifications and have confirmed the presence of multiple species of BoNT-producing clostridia and multiple species of BoNT proteins. BoNT-producing bacteria are Gram-positive, anaerobic, spore-forming and rod-shaped organisms and are commonly found in any soil or water environment. The seven distinct serotypes differ by 37–70% in amino acid sequence [34]. Early observations pointed to a level of intratypic serological diversity that led to variants within serotypes to be called sub(sero)types and a proposal that new subtypes would differ by 2.6% at the protein sequence level. However, this rule is not consistently applied today throughout all the subtypes [35]. It is considered that 41 individual toxins exist and the various toxin subtypes are given a letter designation for the toxin serotype followed by a sequential number in order of discovery, e.g. BoNT/A1 and BoNT/E11. Only 4 serotypes currently present subserotypes, namely BoNT/A (8 subtypes), BoNT/B (8 subtypes), BoNT/E (12 subtypes) and BoNT/F (7 subtypes) (see **Figure 2**). Interestingly, BoNT/C and BoNT/D occur naturally as well as hybrid toxins, termed BoNT/CD and BoNT/DC. A third naturally occurring hybrid, BoNT/FA, was initially proposed as the new serotype BoNT/H following its discovery in 2013 but later reclassified as a hybrid toxin [36].

Current classification of BoNT-producing clostridia is according to group designation based on metabolic biochemical criteria (see **Table 3**). The metabolic groups represent distinct species of *Clostridium botulinum* (Groups I to III) and *Clostridium argentinense* (Group IV), and these species include non-toxigenic as well as neurotoxigenic members. In addition, *Clostridium baratii* and *Clostridium butyricum* are also known to produce BoNTs (Groups V and VI). To add to the confusion, some *Clostridium botulinum* strains do not produce BoNT, in particular if subcultured repeatedly in the laboratory; and some additional toxins are produced by the neurotoxigenic *Clostridium botulinum*, such as C2 toxin, C3 exoenzyme and botulinolysin. However, no alternative nomenclature for this group of organisms has been accepted [32].

When more than one toxin is produced by a single strain, such as Ba or Bf, the capital letter designates the toxin produced in greater amounts. If a gene is present but not expressed, it is denoted between brackets, for example, A(B); and if a gene is present but truncated, it will have an apostrophe to indicate this fact, such as A(B′). This diversity in BoNT-producing bacterial strains is the result of toxin gene associations with transposases such as insertion sequence elements, recombinases, the acquisition of plasmids or infection by phage [37], within and between the

**Figure 2.** Phylogenetic tree depicting the relationship of the 41 known botulinum neurotoxins. Individual FASTA files were accessed through the NCBI portal (NIH, USA), and the protein alignment and phylogram were constructed using

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the online software Clustal Omega (EMBL-EBI, Germany).

Clostridial strains in different groups can produce the same toxin (e.g. Groups I, II and V produce BoNT/F), and bivalent toxin combinations within the same strain have been identified. Botulinum Neurotoxin: A Multifunctional Protein for the Development of New Therapeutics http://dx.doi.org/10.5772/intechopen.69433 83

that the first clinical application was made. In 1981, Dr. Alan B. Scott at what was formerly known as the Smith-Kettlewell Institute of Visual Science, San Francisco, California, USA, used BoNT/A for the treatment of strabismus as an alternative to surgical intervention. The original name of the drug was Oculinum®, and its rights were later acquired by Allergan Inc.,

Upon the first description of the botulism symptoms (see above), the initial hypothesis was that botulism was caused by a toxin produced by a single bacterial organism, as is the case for the closely related toxin tetanus toxin and its producing bacteria *Clostridium tetani* [32]. However, it soon became apparent that different types of toxin and different producing bac-

In 1910–1919, serological methods were introduced for categorisation of the toxin-producing bacteria and for the toxins themselves, that are still in use today [33]. Biochemical and molecular techniques have complemented those initial classifications and have confirmed the presence of multiple species of BoNT-producing clostridia and multiple species of BoNT proteins. BoNT-producing bacteria are Gram-positive, anaerobic, spore-forming and rod-shaped organisms and are commonly found in any soil or water environment. The seven distinct serotypes differ by 37–70% in amino acid sequence [34]. Early observations pointed to a level of intratypic serological diversity that led to variants within serotypes to be called sub(sero)types and a proposal that new subtypes would differ by 2.6% at the protein sequence level. However, this rule is not consistently applied today throughout all the subtypes [35]. It is considered that 41 individual toxins exist and the various toxin subtypes are given a letter designation for the toxin serotype followed by a sequential number in order of discovery, e.g. BoNT/A1 and BoNT/E11. Only 4 serotypes currently present subserotypes, namely BoNT/A (8 subtypes), BoNT/B (8 subtypes), BoNT/E (12 subtypes) and BoNT/F (7 subtypes) (see **Figure 2**). Interestingly, BoNT/C and BoNT/D occur naturally as well as hybrid toxins, termed BoNT/CD and BoNT/DC. A third naturally occurring hybrid, BoNT/FA, was initially proposed as the new serotype BoNT/H fol-

Current classification of BoNT-producing clostridia is according to group designation based on metabolic biochemical criteria (see **Table 3**). The metabolic groups represent distinct species of *Clostridium botulinum* (Groups I to III) and *Clostridium argentinense* (Group IV), and these species include non-toxigenic as well as neurotoxigenic members. In addition, *Clostridium baratii* and *Clostridium butyricum* are also known to produce BoNTs (Groups V and VI). To add to the confusion, some *Clostridium botulinum* strains do not produce BoNT, in particular if subcultured repeatedly in the laboratory; and some additional toxins are produced by the neurotoxigenic *Clostridium botulinum*, such as C2 toxin, C3 exoenzyme and botulinolysin. However, no alternative nomenclature for this group of organisms has been accepted [32].

Clostridial strains in different groups can produce the same toxin (e.g. Groups I, II and V produce BoNT/F), and bivalent toxin combinations within the same strain have been identified.

**5. The producing bacteria: types of toxin and nontoxin proteins**

lowing its discovery in 2013 but later reclassified as a hybrid toxin [36].

which changed the name of the drug to Botox®.

teria existed for BoNT [29].

82 Neurotoxins

**Figure 2.** Phylogenetic tree depicting the relationship of the 41 known botulinum neurotoxins. Individual FASTA files were accessed through the NCBI portal (NIH, USA), and the protein alignment and phylogram were constructed using the online software Clustal Omega (EMBL-EBI, Germany).

When more than one toxin is produced by a single strain, such as Ba or Bf, the capital letter designates the toxin produced in greater amounts. If a gene is present but not expressed, it is denoted between brackets, for example, A(B); and if a gene is present but truncated, it will have an apostrophe to indicate this fact, such as A(B′). This diversity in BoNT-producing bacterial strains is the result of toxin gene associations with transposases such as insertion sequence elements, recombinases, the acquisition of plasmids or infection by phage [37], within and between the

Until recently, BoNTs were believed to be produced exclusively by clostridia organisms. In 2015, the first homologue of BoNTs was described within the genome of the rice fermentation bacteria *Weissella oryzae* SG25 [42]. Bioinformatic analysis of the genomic sequences of *W. oryzae* SG25 revealed one gene with a very similar structure to BoNTs, whereas a second gene showed partial similarity with the BoNT-associated NTNH proteins [42]. Recombinant expression of the BoNT-like protein revealed that it shares similarities with BoNT/B regarding its targeting profile and it is also expected to block neurotransmitter release. The new BoNTlike protein showed no serological cross-reactivity with the seven known BoNT serotypes,

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BoNTs are zinc metalloproteases consisting of three major domains. Produced as a single polypeptide of 150 KDa, BoNTs require activation by cleavage of the polypeptide post-translationally resulting in the so termed heavy chain, of ~ 100 KDa, and a light chain (LC), of ~ 50 KDa, held together by a disulphide bridge between the two chains [44]. Functionally, the light chain hosts the metalloprotease domain, and the heavy chain comprises both the binding domain (HC) and the translocation domain (HN). The producing bacteria in Groups I, III and IV (see **Table 3**) are proteolytic strains and will release the cleaved active product, whereas the products of the other producing bacteria are believed to be activated by proteases of the intoxicated organism [38].

The neuromuscular junction is the natural target of BoNTs, and intoxication follows an intricate multistep mechanism [20, 45], in which the toxin-associated proteins of the progenitor toxin complex play a crucial role. For an overview of the routes of entry and mechanism of

Unintentional BoNT entry into the organism occurs mainly through ingestion of contaminated foods leading to food-borne botulism (see above) or through wounds [20]. Alternatively, and in particular in cases of infant botulism, the producing bacteria can colonise the immature gastrointestinal tract and produce the exotoxin in situ. The progenitor complex allows BoNTs to effectively cross the intestinal trans-epithelial barrier and reach the lymphoid and general blood circulation. Under neutral and alkaline environments, such as in the bloodstream, the complex dissociates and the naked toxin is able to target neuromuscular junctions [46]. In clinical applications, the toxin is delivered locally to the site of action. BoNT entering the body undergoes a relatively short distribution phase which sees the toxin selectively targeting peripheral nerve endings, and an elimination phase that comprises both (1) an interneuronal metabolism following cellular entry and (2) systemic metabolism and elimination which are assumed to be

Upon reaching the neuromuscular junction, BoNTs are able to specifically target nerve terminals using their Hc-binding domain and internalise through endocytosis. Once in the acidic environment of the endosome, the BoNT HN domain translocates the LC domain into the cytosol, allowing the Zn+2 metalloprotease enzyme to cleave target soluble N-ethylmaleimide-sensitive

and it was dubbed BoNT/Wo by the authors [43].

**6. Structure-function of BoNT toxins**

action of the toxin, see **Figure 3**.

through the liver [47].

**Table 3.** BoNT-producing clostridial species, see Refs. [20, 29, 38].

groups and species. Groups IV–VI have the toxin genes located in the chromosome, considered less mobile, whereas Group III has the toxin genes in highly mobile elements such as plasmids and bacteriophages. Groups I and II have a mixture of chromosome and plasmid localisation [38]. A recent genetic study of *C. botulinum* strains causing human botulism in France showed that the genetic diversity of the BoNT-producing organism appeared as a result of multiple and independent genetic rearrangements and not from a single evolutionary lineage [39].

All seven BoNT serotype toxins are released from the producing bacteria as large protein complexes with a number of neurotoxin-associated proteins (NAPs) to become highly potent oral toxins, often ingested in contaminated foods [38, 40]. The NAPs are encoded together with the *bont* gene in one of two different gene clusters, the hemagglutinin (HA) cluster or the orfX cluster. Both clusters encode the nontoxic non-hemagglutinin (NTNHA) protein, which assembles with BoNT to form the smaller of the progenitor toxin complexes. BoNT/A, BoNT/B, BoNT/C and BoNT/D complexes contain HA, whereas BoNT/E and BoNT/F complexes do not contain HA. The components of the BoNT complex vary with neurotoxin serotypes and the *Clostridium* strain producing them. BoNTs are produced in three progenitor forms: M (medium), L (large) and LL (extralarge) complex. The M form consists of the neurotoxin (of 150 KDa) with NTNHA and has a total weight of ~ 300 KDa. The L and LL complexes consist of several HA proteins besides the BoNT and NTNHA, and its molecular weight is ~ 500 KDa for the L form and ~ 900 KDa for the LL form. The function of the proteins encoded in the orfX genes remains unknown [41]. NAPs are known to protect BoNTs against the proteases of the gastrointestinal tract and the acidic conditions of the stomach and to facilitate the intestinal trans-epithelial delivery to the toxin into the lymphoid and general circulation [38]. The role of NAPs in the producing bacteria is not known. Recently, it has been proposed that the primary role of NAPs and in particular that of NTNHA is to protect BoNTs from damage in the decaying biological material where the toxin is mostly produced in the wild [20].

Until recently, BoNTs were believed to be produced exclusively by clostridia organisms. In 2015, the first homologue of BoNTs was described within the genome of the rice fermentation bacteria *Weissella oryzae* SG25 [42]. Bioinformatic analysis of the genomic sequences of *W. oryzae* SG25 revealed one gene with a very similar structure to BoNTs, whereas a second gene showed partial similarity with the BoNT-associated NTNH proteins [42]. Recombinant expression of the BoNT-like protein revealed that it shares similarities with BoNT/B regarding its targeting profile and it is also expected to block neurotransmitter release. The new BoNTlike protein showed no serological cross-reactivity with the seven known BoNT serotypes, and it was dubbed BoNT/Wo by the authors [43].

#### **6. Structure-function of BoNT toxins**

groups and species. Groups IV–VI have the toxin genes located in the chromosome, considered less mobile, whereas Group III has the toxin genes in highly mobile elements such as plasmids and bacteriophages. Groups I and II have a mixture of chromosome and plasmid localisation [38]. A recent genetic study of *C. botulinum* strains causing human botulism in France showed that the genetic diversity of the BoNT-producing organism appeared as a result of multiple and

**Mixture of serotype(s) produced by a single**

IV G – *Clostridium argentinense*

– *Clostridium novyi*

**Nontoxinogenic bacteria belonging to the same group**

*Clostridium sporogenes*

*Clostridium subterminale Clostridium hastiforme*

**strain**

Bf, Bf/a

*Clostridium botulinum* II B, E, F – *Clostridium taeniosporum*

*Clostridium baratii* V F – *Clostridium baratii Clostridium butyricum* VI E – *Clostridium butyricum*

All seven BoNT serotype toxins are released from the producing bacteria as large protein complexes with a number of neurotoxin-associated proteins (NAPs) to become highly potent oral toxins, often ingested in contaminated foods [38, 40]. The NAPs are encoded together with the *bont* gene in one of two different gene clusters, the hemagglutinin (HA) cluster or the orfX cluster. Both clusters encode the nontoxic non-hemagglutinin (NTNHA) protein, which assembles with BoNT to form the smaller of the progenitor toxin complexes. BoNT/A, BoNT/B, BoNT/C and BoNT/D complexes contain HA, whereas BoNT/E and BoNT/F complexes do not contain HA. The components of the BoNT complex vary with neurotoxin serotypes and the *Clostridium* strain producing them. BoNTs are produced in three progenitor forms: M (medium), L (large) and LL (extralarge) complex. The M form consists of the neurotoxin (of 150 KDa) with NTNHA and has a total weight of ~ 300 KDa. The L and LL complexes consist of several HA proteins besides the BoNT and NTNHA, and its molecular weight is ~ 500 KDa for the L form and ~ 900 KDa for the LL form. The function of the proteins encoded in the orfX genes remains unknown [41]. NAPs are known to protect BoNTs against the proteases of the gastrointestinal tract and the acidic conditions of the stomach and to facilitate the intestinal trans-epithelial delivery to the toxin into the lymphoid and general circulation [38]. The role of NAPs in the producing bacteria is not known. Recently, it has been proposed that the primary role of NAPs and in particular that of NTNHA is to protect BoNTs from damage in the decaying biological material where the toxin is mostly produced in the wild [20].

independent genetic rearrangements and not from a single evolutionary lineage [39].

**Clostridial bacteria Group BoNT**

*Clostridium botulinum* III C, D,

*Clostridium argentinense*

84 Neurotoxins

**serotype(s) produced**

CD hybrids DC hybrids

*Clostridium botulinum* I A, B, F A(B), A(B′), Ab, Af, Ba,

**Table 3.** BoNT-producing clostridial species, see Refs. [20, 29, 38].

BoNTs are zinc metalloproteases consisting of three major domains. Produced as a single polypeptide of 150 KDa, BoNTs require activation by cleavage of the polypeptide post-translationally resulting in the so termed heavy chain, of ~ 100 KDa, and a light chain (LC), of ~ 50 KDa, held together by a disulphide bridge between the two chains [44]. Functionally, the light chain hosts the metalloprotease domain, and the heavy chain comprises both the binding domain (HC) and the translocation domain (HN). The producing bacteria in Groups I, III and IV (see **Table 3**) are proteolytic strains and will release the cleaved active product, whereas the products of the other producing bacteria are believed to be activated by proteases of the intoxicated organism [38].

The neuromuscular junction is the natural target of BoNTs, and intoxication follows an intricate multistep mechanism [20, 45], in which the toxin-associated proteins of the progenitor toxin complex play a crucial role. For an overview of the routes of entry and mechanism of action of the toxin, see **Figure 3**.

Unintentional BoNT entry into the organism occurs mainly through ingestion of contaminated foods leading to food-borne botulism (see above) or through wounds [20]. Alternatively, and in particular in cases of infant botulism, the producing bacteria can colonise the immature gastrointestinal tract and produce the exotoxin in situ. The progenitor complex allows BoNTs to effectively cross the intestinal trans-epithelial barrier and reach the lymphoid and general blood circulation. Under neutral and alkaline environments, such as in the bloodstream, the complex dissociates and the naked toxin is able to target neuromuscular junctions [46]. In clinical applications, the toxin is delivered locally to the site of action. BoNT entering the body undergoes a relatively short distribution phase which sees the toxin selectively targeting peripheral nerve endings, and an elimination phase that comprises both (1) an interneuronal metabolism following cellular entry and (2) systemic metabolism and elimination which are assumed to be through the liver [47].