Direct and Indirect Neurotoxic Potential of Metal/Metalloids in Plants and Fungi Used for Food, Dietary Supplements, and Herbal Medicine

Plants and mushrooms bioconcentrate metals/metalloids from soil and water such that high levels of potentially neurotoxic elements can occur in cultivated and wild species used for food. While the health effects of excessive exposure to metals/metalloids with neurotoxic potential are well established, overt neurological disease from prolonged ingestion of contaminated botanicals has not been recognized. However, the presence of metal elements may affect levels of botanical neurotoxins in certain plants and mushrooms that are established causes of acute and chronic neurological disease.


Introduction
Plants and mushrooms that are/are not used for food can sequester metals/metalloids drawn from soil and water that have the potential for human neurotoxic effects. Although this property of botanicals is exploited for the bioremediation of metal-contaminated soils, the possibility of toxic/neurotoxic effects in the consumer of metal-concentrating plants and mushrooms has been rarely addressed. This requires a synthesis of mycology, botany, and toxicology, not only of the human and mammal but also of the plant and mushroom.
Three scenarios are considered here: certain metals contained in plants and mushrooms used for food pose: (a) a direct threat to human neurological function and/or (b) an indirect threat because they can modulate the concentration of botanical compounds (hydrazine, dencichine, cyanogens) that by themselves pose a neurotoxic hazard to the consumer. Examples of the latter include certain plants (grasspea, cassava) and mushrooms (false morel), food use of which can precipitate neurotoxic disease.

Botanical Uptake of Metal Elements Required for Biological Function
The major elements in soil (>100 mg/kg), and minerals derived therefrom, include aluminum, calcium, carbon, iron, magnesium, nitrogen, oxygen, potassium, silicon, sodium, sulfur, and titanium. Other soil elements include barium, chlorine, manganese, phosphorus, strontium, and zirconium [1,2]. Common trace elements in soil (<100 mg/kg) comprise arsenic, chromium, cobalt, copper, lead, lithium, nickel, selenium, and zinc [1]. Whereas Ca, Fe, K, Mn, and P are required for normal human physiological function, prolonged excessive exposure to some of the listed elements (Al, As, Cu, Fe, Li, Pb), among others (Cd, Hg, Tl, Zn), can result in neurologic dysfunction or overt disease.
Soil goes through various stages of aging that result in changes in its chemistry and that of associated water. The resulting chemical environment is reflected in the organisms that feed on soil nutrients [1,2]. Fungi play a vital role in the soil environment by acting as a bridge between soil microbes and plants, thereby facilitating nutrient cycling and plant health and disease control [3]. Fungi are particularly effective in changing the solubility

Heavy Metals in Mushrooms with Potential Neurotoxicity
The True Morel Morchella esculenta, a facultative mycorrhizal mushroom widely prized by gourmets, illustrates the potential human health threat of consuming mushrooms that bioaccumulate metals from contaminated soil. In the USA, lead arsenate (PbHAsO4) was widely used as a moth pesticide from the late 1800s, replaced by DDT in the 1950s, and banned from use in fruit orchards in 1988. However, the potentially neurotoxic elements (Pb, As) persist in the topsoils on which morels grow. A study of 29 abandoned apple Toxics 2021, 9, 57 4 of 13 orchards in the northeastern USA revealed a range of lead and arsenic in soil (19.20-2450 and 3.08-244.00 mg/kg, respectively) and in the fruit bodies of M. esculenta (0.5-13.00 and 0.15-2.85 mg/kg, respectively) growing on polluted soils. The respective concentrations were statistically associated (r = 0.81) for lead and arsenic in soil, and for soil and morel lead content (r = 0.94, r = 0.57, respectively). Almost all (94%) of the arsenic stored in morel tissues was in the inorganic form, and the levels of the two elements in morel fruitbodies were considered to pose a human health risk [49]. A mycologist who consumed M. esculenta collected from apple orchards had elevated levels of urinary arsenic and lead; he complained of symptoms consistent with sensory (arsenic) neuropathy that resolved following chelation therapy [50].
False Morel mushrooms, such as Gyromitra esculenta, a poisonous species that is nevertheless eaten by some, contain gyromitrin (acetaldehyde N-methyl-N-formyl-hydrazone) (Figure 1, center). While the function of fungal gyromitrin is unknown, hydrazones can form complexes with metals, such as Ni 2+ , Cu 2+ , Zn 2+ , and Cd 2+ [51]. The principal metabolite of gyromitrin, monomethylhydrazine (MMH) (Figure 1, left), is an acutely neurotoxic compound that interferes with pyridoxine utilization by both glutamic acid decarboxylase and γ-aminobutyric acid (GABA) transaminase, leading to decreased concentrations of the inhibitory neurotransmitter GABA in the brain and consequent induction of seizures [52]. Levels of MMH in G. esculenta vary and can be reduced by prolonged desiccation [53]. Consumption of the False Morel G. gigas collected from soils near a closed lead mine in the French Alps has recently been linked to a focus of amyotrophic lateral sclerosis; while the authors did not measure lead levels in local False Morels, they attributed induction of neurodegenerative disease to the genotoxic properties of MMH [54].
The True Morel Morchella esculenta, a facultative mycorrhizal mushroom widely prized by gourmets, illustrates the potential human health threat of consuming mushrooms that bioaccumulate metals from contaminated soil. In the USA, lead arsenate (PbHAsO4) was widely used as a moth pesticide from the late 1800s, replaced by DDT in the 1950s, and banned from use in fruit orchards in 1988. However, the potentially neurotoxic elements (Pb, As) persist in the topsoils on which morels grow. A study of 29 abandoned apple orchards in the northeastern USA revealed a range of lead and arsenic in soil (19.20-2450 and 3.08-244.00 mg/kg, respectively) and in the fruit bodies of M. esculenta (0.5-13.00 and 0.15-2.85 mg/kg, respectively) growing on polluted soils. The respective concentrations were statistically associated (r = 0.81) for lead and arsenic in soil, and for soil and morel lead content (r = 0.94, r = 0.57, respectively). Almost all (94%) of the arsenic stored in morel tissues was in the inorganic form, and the levels of the two elements in morel fruitbodies were considered to pose a human health risk [49]. A mycologist who consumed M. esculenta collected from apple orchards had elevated levels of urinary arsenic and lead; he complained of symptoms consistent with sensory (arsenic) neuropathy that resolved following chelation therapy [50].
False Morel mushrooms, such as Gyromitra esculenta, a poisonous species that is nevertheless eaten by some, contain gyromitrin (acetaldehyde N-methyl-N-formyl-hydrazone) (Figure 1, center). While the function of fungal gyromitrin is unknown, hydrazones can form complexes with metals, such as Ni 2+ , Cu 2+ , Zn 2+ , and Cd 2+ [51]. The principal metabolite of gyromitrin, monomethylhydrazine (MMH) (Figure 1, left), is an acutely neurotoxic compound that interferes with pyridoxine utilization by both glutamic acid decarboxylase and γ-aminobutyric acid (GABA) transaminase, leading to decreased concentrations of the inhibitory neurotransmitter GABA in the brain and consequent induction of seizures [52]. Levels of MMH in G. esculenta vary and can be reduced by prolonged desiccation [53]. Consumption of the False Morel G. gigas collected from soils near a closed lead mine in the French Alps has recently been linked to a focus of amyotrophic lateral sclerosis; while the authors did not measure lead levels in local False Morels, they attributed induction of neurodegenerative disease to the genotoxic properties of MMH [54]. Hydrazine is a reducing agent that is used industrially to reduce metal salts and oxides to pure metals. Phenylhydrazines, notably agaritine (N2-(γ-L-glutamyl)-4-hydroxymethylphenylhydrazine) (Figure 1, right), are found in Agaricus mushrooms. Concentrations range from 200 to 500 mg agaritine/kg fresh weight in cultivated species of the universally eaten A. biporus (Button Mushroom). Wild samples of A. elvensis have been reported to contain up to 10,000 mg agaritine/kg fresh weight [55,56]. Heavy metals (mg/kg) in the fruiting bodies of A. bisporus growing wild in Poland included: Cd, 0.68- Hydrazine is a reducing agent that is used industrially to reduce metal salts and oxides to pure metals. Phenylhydrazines, notably agaritine (N2-(γ-L-glutamyl)-4-hydroxymethylph enylhydrazine) (Figure 1, right), are found in Agaricus mushrooms. Concentrations range from 200 to 500 mg agaritine/kg fresh weight in cultivated species of the universally eaten A. biporus (Button Mushroom). Wild samples of A. elvensis have been reported to contain up to 10,000 mg agaritine/kg fresh weight [55,56]. Heavy metals (mg/kg) in the fruiting bodies of A. bisporus growing wild in Poland included: Cd, 0.68-6.14; Cr, 0. 38 [57]. Samples collected near Kermanshah City, Iran, contained levels of arsenic (mean 65.23 ± 13.57 mg/kg) and zinc (mean 66.23 ± 2.80 mg/kg) that exceeded the maximum permissible limit [58].

Plant Uptake of Metals/Metalloids with Neurotoxic Potential
The presence of heavy metals in plants used for food and their associated human health threats have been comprehensively reviewed by Rai and colleagues [59]. The root causes of this problem are attributed to the rapid pace of urbanization, changes in land use, and industrialization, especially in countries with high populations, such as India and China. Whereas in parts of Asia and Africa, inadequately treated wastewater, effluent, and sludge used for irrigation are the main sources of plant contamination, sources of heavy metals in food plants grown in America, Europe, and Oceania arise predominantly from particulate matter (from industrial and transport sectors) and agricultural practices. Sludge from distilleries and the chemical, electroplating, textile, and leather industries is often found to contain significantly high concentrations of heavy metals, such as Cr, Cu, Fe, Mn, Ni and Pb. Gold mining is a leading source of heavy metal contamination (especially Cd, Hg, Pb) in soil and food crops. Plants used in Asian herbal medicines acquire metal contaminants (e.g., As, Cd, Cr, Cu, Fe, and Pb) when grown near smelting or other industrial areas [60][61][62].
Certain plant species can immobilize metals in soil and groundwater through absorption and accumulation by roots, adsorption onto roots, or precipitation within the root zone, a process known as phytostabilization. Other species absorb and hyperaccumulate metals/metalloid contaminants through a phytoextraction process. Several plants, such as Brassica spp., can accumulate lead in concentrations >50 mg/g dw. The roots of B. juncea (Oriental Mustard) concentrated mercury 100-270X (dw) above initial solution concentrations, but only 0.7-2% was translocated to the shoots [63]. Numerous metals have been measured in plants used for cereals, fruits, and vegetables; for example, samples of rice (Oryza sativa) contained chromium (15-465 mg/kg), manganese (61-356 mg/kg), and lead (16-16,500 mg/kg), and high levels of arsenic have been found in rice and lettuce (Lactuca sativa) [59].

Grasspea
The legume Lathyrus sativus (grasspea) (Figure 2), prolonged heavy consumption of which triggers the irreversible central motorsystem disease lathyrism [64,65], is a strong metal accumulator of lead and cadmium in all parts of the plants [66][67][68]. Root tissues of lead-exposed grasspea showed a six-fold, two-fold, and three-and-a-half-fold reduction in calcium, zinc, and copper contents, respectively, which indicated the plant tolerates a deficiency in essential nutrients while storing large amounts of lead [69]. The uptake of cadmium and copper in grasspea shoots was exponential over the range of concentrations tested [70]. The distribution of lead in L. sativus has a selective character: leaves > roots > stems > seeds. While lead is also found in the seed, soil pollution with heavy metals does not affect seed quality, such that the high nutrient (K, O, Cu, Fe, Mn, Zn) and protein content (23.18-29.54%) is preserved [71]. Grasspea can also bioaccumulate arsenic in roots > shoots [72]. These properties are important because grasspea is considered an ideal crop for resource-poor farmers, is widely eaten on the Indian subcontinent, and serves as both a regular and famine food in the northern Ethiopian highlands [73,74].
The neurotoxic property of grasspea is attributable to its content of the excitotoxic nonprotein amino acid β-N-oxalylamino-L-alanine (L-BOAA, syn. dencichine) (Figure 3), also known as β-oxalyldiaminopropionic acid (β-ODAP) and, perhaps, also to its low content of methionine and cysteine [68]. L-BOAA/β-ODAP is found in all parts of the plant, with concentrations of 0.5-2.5% in traditional varieties of grasspea [75]. Zinc deficiency and oversupply of iron to the roots of L. sativus induce increases in the L-BOAA/β-ODAP content in ripe seed [76]. The biosynthesis of L-BOAA/β-ODAP and its genetic and environmental regulation are under intensive study because grasspea is tolerant of drought and waterlogging, flourishes without inputs, and the seed has significant protein content, such that grasspea varieties with low-L-BOAA/β-ODAP content may potentially serve as a safe and valuable foodstuff [68,77,78]. However, environmental factors such as drought, zinc deficiency, iron oversupply, and the presence of heavy metals in the soil reportedly can considerably increase the level of L-BOAA/ß-ODAP in the seed of grasspea [79]. The neurotoxic property of grasspea is attributable to its content of the excitotoxi nonprotein amino acid β-N-oxalylamino-L-alanine (L-BOAA, syn. dencichine) (Figure 3 also known as β-oxalyldiaminopropionic acid (β-ODAP) and, perhaps, also to its low con tent of methionine and cysteine [68]. L-BOAA/β-ODAP is found in all parts of the plan with concentrations of 0.5-2.5% in traditional varieties of grasspea [75]. Zinc deficienc and oversupply of iron to the roots of L. sativus induce increases in the L-BOAA/β-ODAP content in ripe seed [76]. The biosynthesis of L-BOAA/β-ODAP and its genetic and envi ronmental regulation are under intensive study because grasspea is tolerant of drough and waterlogging, flourishes without inputs, and the seed has significant protein conten such that grasspea varieties with low-L-BOAA/β-ODAP content may potentially serve a a safe and valuable foodstuff [68,77,78]. However, environmental factors such as drough zinc deficiency, iron oversupply, and the presence of heavy metals in the soil reportedl can considerably increase the level of L-BOAA/ß-ODAP in the seed of grasspea [79]. The addition of arbuscular mycorrhizal fungi (AMF) to grasspea seedlings promoted plant growth of grasspea under sulfate stress [80]. AMF are associated with most terres trial plants: they penetrate the roots and form symbiotic relationships that protect th plant from diverse stressors, including resistance to temperature extremes, drought, wa terlogging, salinity, and heavy metals [81]. L. sativus is highly tolerant of such environ  The neurotoxic property of grasspea is attributable to its content of the excit nonprotein amino acid β-N-oxalylamino-L-alanine (L-BOAA, syn. dencichine) (Figu also known as β-oxalyldiaminopropionic acid (β-ODAP) and, perhaps, also to its low tent of methionine and cysteine [68]. L-BOAA/β-ODAP is found in all parts of the with concentrations of 0.5-2.5% in traditional varieties of grasspea [75]. Zinc defic and oversupply of iron to the roots of L. sativus induce increases in the L-BOAA/β-O content in ripe seed [76]. The biosynthesis of L-BOAA/β-ODAP and its genetic and ronmental regulation are under intensive study because grasspea is tolerant of dr and waterlogging, flourishes without inputs, and the seed has significant protein co such that grasspea varieties with low-L-BOAA/β-ODAP content may potentially se a safe and valuable foodstuff [68,77,78]. However, environmental factors such as dro zinc deficiency, iron oversupply, and the presence of heavy metals in the soil repo can considerably increase the level of L-BOAA/ß-ODAP in the seed of grasspea [79 The addition of arbuscular mycorrhizal fungi (AMF) to grasspea seedlings prom plant growth of grasspea under sulfate stress [80]. AMF are associated with most t trial plants: they penetrate the roots and form symbiotic relationships that prote plant from diverse stressors, including resistance to temperature extremes, drough terlogging, salinity, and heavy metals [81]. L. sativus is highly tolerant of such en mental stressors, which suggests AMF are normally associated with the plant and The addition of arbuscular mycorrhizal fungi (AMF) to grasspea seedlings promoted plant growth of grasspea under sulfate stress [80]. AMF are associated with most terrestrial plants: they penetrate the roots and form symbiotic relationships that protect the plant from diverse stressors, including resistance to temperature extremes, drought, waterlogging, salinity, and heavy metals [81]. L. sativus is highly tolerant of such environmental stressors, which suggests AMF are normally associated with the plant and thus able to provide some protection against heavy metals. The subject is of importance to the health of people with dietary reliance on grasspea in countries such as Bangladesh, Ethiopia, India, and Nepal [77].

Cassava
The tubers and leaves of Manihot esculenta (cassava) provide a source of food for huge numbers of people in Asia, Africa, and South America (Figure 4). For example, cassava tubers supply~70% of the daily calorie input for >50 million people in Nigeria, the world's largest cassava producer (49 million metric tons/year). Fresh cassava root contains >30% carbohydrate, >2% protein, 0.1% fat, and >75% moisture content [82,83]. However, M. esculenta also synthesizes cyanogenic glucosides (linamarin and its methylated relative lotaustralin; 97:3), the concentration of which must be substantially reduced before the tuber is used for human consumption. The method of processing cassava roots, the duration of tuber storage, and the type of meal preparation determine the amount of consumer exposure to cassava cyanogens and heavy metals [84]. Failure to remove the cyanogenic glucosides and their products before human ingestion can precipitate acute cyanide (HCN) poisoning or, with continued daily consumption, the subacute onset of an irreversible motorsystem disease (cassavism), which is known as konzo in the Democratic Republic of Congo and mantakassa in Mozambique [85,86]. Whether the daily consumption of cassava-derived gari in Nigeria is also responsible for endemic peripheral neuropathy with optic atrophy and sensorineural deafness has been questioned. numbers of people in Asia, Africa, and South America (Figure 4). For example, cassava tubers supply ~70% of the daily calorie input for >50 million people in Nigeria, the world's largest cassava producer (49 million metric tons/year). Fresh cassava root contains >30% carbohydrate, >2% protein, 0.1% fat, and >75% moisture content [82,83]. However, M. esculenta also synthesizes cyanogenic glucosides (linamarin and its methylated relative lotaustralin; 97:3), the concentration of which must be substantially reduced before the tuber is used for human consumption. The method of processing cassava roots, the duration of tuber storage, and the type of meal preparation determine the amount of consumer exposure to cassava cyanogens and heavy metals [84]. Failure to remove the cyanogenic glucosides and their products before human ingestion can precipitate acute cyanide (HCN) poisoning or, with continued daily consumption, the subacute onset of an irreversible motorsystem disease (cassavism), which is known as konzo in the Democratic Republic of Congo and mantakassa in Mozambique [85,86]. Whether the daily consumption of cassavaderived gari in Nigeria is also responsible for endemic peripheral neuropathy with optic atrophy and sensorineural deafness has been questioned. Acute toxicity from cassava processing and/or ingestion results from cyanide ion (CN -) binding to iron and copper sites in cytochrome c oxidase, which is required for electron transport and energy generation. Cassavism is associated with elevated levels of urinary thiocyanate (SCN -) produced in the consumer by the addition of endogenous methionine/cystine sulfur to cyanide by thiosulfate sulfurtransferase (rhodanese) [87], the enzyme activity of which is inhibited by certain metal ions (Au, Ni, Pt, and Zn) [88]. Laboratory animals given a sulfur-free diet also eliminate CN as cyanate (OCN -) [89], prolonged exposure to which (in the form of NaOCN) can precipitate peripheral neuropathy [90]. All three anions (CN -, OCN -, and SCN -) form metal complexes, at least in the chemistry laboratory [91][92][93]. Cyanide also forms stable complexes with cobalt, gold, iron, and mercury, a property used commercially to leach the metal from gold-bearing ore [94]. Linamarin may be involved in the uptake of gold and mercury into cassava roots [95].
Several studies have shown heavy metals in cassava and the processed flour derived from its root. Cassava in communities in Nigeria's River State contained metal elements in both the leaves (Fe > Cu > Cr > Ni > Pb > Cd) and tubers (Fe > Cu > Cr > Ni > Pb > Cd) Acute toxicity from cassava processing and/or ingestion results from cyanide ion (CN -) binding to iron and copper sites in cytochrome c oxidase, which is required for electron transport and energy generation. Cassavism is associated with elevated levels of urinary thiocyanate (SCN -) produced in the consumer by the addition of endogenous methionine/cystine sulfur to cyanide by thiosulfate sulfurtransferase (rhodanese) [87], the enzyme activity of which is inhibited by certain metal ions (Au, Ni, Pt, and Zn) [88]. Laboratory animals given a sulfur-free diet also eliminate CN as cyanate (OCN -) [89], prolonged exposure to which (in the form of NaOCN) can precipitate peripheral neuropathy [90]. All three anions (CN -, OCN -, and SCN -) form metal complexes, at least in the chemistry laboratory [91][92][93]. Cyanide also forms stable complexes with cobalt, gold, iron, and mercury, a property used commercially to leach the metal from gold-bearing ore [94]. Linamarin may be involved in the uptake of gold and mercury into cassava roots [95].
Several studies have shown heavy metals in cassava and the processed flour derived from its root. Cassava in communities in Nigeria's River State contained metal elements in both the leaves (Fe > Cu > Cr > Ni > Pb > Cd) and tubers (Fe > Cu > Cr > Ni > Pb > Cd) [96]. Metal concentrations in the tubers (Cd, Ni, and Pb) and leaves (Cd, Cr, and Pb) of cassava plants grown on soils around a lead sulfide (galena) mining area in Nigeria's Ebonyi State exceeded WHO standards [97]. Exceedances for Al, As, Ca, Cr, Cu, Fe, K, Mn, Pb, and Zn were found in cassava tubers growing around two cement factories in Ogun State, Nigeria [98]. Cassava grown on soil contaminated with crude oil in the Niger Delta Region contained heavy metals (Fe > Zn > Ni > Pb > Cd > Cr) in their leaves and tubers [99].
Automobile emissions added to the metal content of soil samples and cassava plants collected from farmlands along a major expressway in Nigeria's Delta State [100]. Mean levels (mg/kg) of heavy metals (Fe> Zn> Ni> Cr > Pb) in soil samples were 142. 93  farmlands along highways in Owerri, Nigeria, contained leaf concentrations of Zn > Cu >Pb> Ni > Cd [101].
While several studies have revealed the potential for heavy metal contamination of cassava roots and leaves used for food in sub-Saharan Africa, whether and which metals can influence the content, speciation, or neurotoxic potential of the plant's cyanogens and their metabolites are unknown. Metal neurotoxicity has not been reported or looked for in those who use cassava as a food staple.

Key Conclusions
Botanicals used for food, nutritional supplements, and medicinal purposes can bioaccumulate metals/metalloids with neurotoxic potential in concentrations that, in some circumstances, exceed permissible limits for human consumption. While certain heavy metals/metalloids, such as arsenic, lead, manganese, and mercury [105][106][107][108], have established human neurotoxic potential, especially during brain development, their effects from ingestion of contaminated botanicals have rarely if ever resulted in diagnosed neurological illness. Associations have been reported between heavy metals and certain neurodegenerative diseases, including amyotrophic lateral sclerosis [109,110], Parkinson's disease [111,112], and Alzheimer's disease [113,114], but evidence of cause-effect relationships is generally lacking. Chemical principles in some mushrooms (hydrazone/hydrazines) and plants (dencichine, cyanogens) have established human neurotoxicity [115,116], their concentration varying in response to the uptake of metal elements from soil and water. Such botanical species normally pose serious health hazards to humans because of their potential for acute, chronic, and possibly long-latency neurological disease, the risk for such effects varying with the amount and duration of consumption. Witness the extraordinarily high prevalence (5.5% in 2019) of spastic paraparesis (lathyrism) in northeast Ethiopia, with somewhat higher rates (6.2%) among those who use traditional iron-containing clay pots to cook grasspea [117]. The metallic content of botanicals used for food, thus has the potential for direct and indirect adverse effects on the human nervous system. On the other hand, the ability of certain botanicals to bioaccumulate certain metals/metalloids is exploited to protect human health by reducing soil and water pollution via phytoremediation and mycoremediation [118,119].  Data Availability Statement: All data were drawn from published works as cited.