Journal of the Neurological Sciences 262 (2007) 15 26 www.elsevier.

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Autism: Transient in utero hypothyroxinemia related to maternal flavonoid ingestion during pregnancy and to other environmental antithyroid agents
Gustavo C. Romn
University of Texas Health Sciences Center at San Antonio, San Antonio, Texas, USA Veterans Administration Hospital, San Antonio, Texas, USA Available online 24 July 2007

Abstract The incidence and prevalence of autism have increased during the past two decades. Despite comprehensive genetic studies the cause of autism remains unknown. This review emphasizes the potential importance of environmental factors in its causation. Alterations of cortical neuronal migration and cerebellar Purkinje cells have been observed in autism. Neuronal migration, via reelin regulation, requires triiodothyronine (T3) produced by deiodination of thyroxine (T4) by fetal brain deiodinases. Experimental animal models have shown that transient intrauterine deficits of thyroid hormones (as brief as 3 days) result in permanent alterations of cerebral cortical architecture reminiscent of those observed in brains of patients with autism. I postulate that early maternal hypothyroxinemia resulting in low T3 in the fetal brain during the period of neuronal cell migration (weeks 812 of pregnancy) may produce morphological brain changes leading to autism. Insufficient dietary iodine intake and a number of environmental antithyroid and goitrogenic agents can affect maternal thyroid function during pregnancy. The most common causes could include inhibition of deiodinases D2 or D3 from maternal ingestion of dietary flavonoids or from antithyroid environmental contaminants. Some plant isoflavonoids have profound effects on thyroid hormones and on the hypothalamuspituitary axis. Genistein and daidzein from soy (Glycine max) inhibit thyroperoxidase that catalyzes iodination and thyroid hormone biosynthesis. Other plants with hypothyroid effects include pearl millet (Pennisetum glaucum) and fonio millet (Digitaria exilis); thiocyanate is found in Brassicae plants including cabbage, cauliflower, kale, rutabaga, and kohlrabi, as well as in tropical plants such as cassava, lima beans, linseed, bamboo shoots, and sweet potatoes. Tobacco smoke is also a source of thiocyanate. Environmental contaminants interfere with thyroid function including 60% of all herbicides, in particular 2,4-dichlorophenoxyacetic acid (2,4-D), acetochlor, aminotriazole, amitrole, bromoxynil, pendamethalin, mancozeb, and thioureas. Other antithyroid agents include polychlorinated biphenyls (PCBs), perchlorates, mercury, and coal derivatives such as resorcinol, phthalates, and anthracenes. A leading ecological study in Texas has correlated higher rates of autism in school districts affected by large environmental releases of mercury from industrial sources. Mercury is a well known antithyroid substance causing inhibition of deiodinases and thyroid peroxidase. The current surge of autism could be related to transient maternal hypothyroxinemia resulting from dietary and/or environmental exposure to antithyroid agents. Additional multidisciplinary epidemiological studies will be required to confirm this environmental hypothesis of autism. 2007 Elsevier B.V. All rights reserved.
Keywords: Autism; Hypothyroxinemia; Pregnancy; Antithyroid agents; Iodine; Endemic cretinism; Herbicides; Neuronal migration; Soy; Mercury; Polyphenols

1. Introduction The Centers for Disease Control and Prevention (CDC) estimate at half million the number of individuals with autism and autism spectrum disorders in the United States [1]. From a prevalence of 0.4 to 1/1000 children aged 8 years in the 1980s,

University of Texas Health Sciences Center at San Antonio, San Antonio, Texas, USA. Tel.: +1 210 617 5161; fax: +1 210 567 4659. E-mail address: romang@uthscsa.edu. 0022-510X/$ - see front matter 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2007.06.023

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current estimates range from 3.3 to 10.6 (mean, 6.6) or 1 case per 152 children [13]. Increased prevalence has been demonstrated in large birth cohorts of US school-aged children [4,5], as well as in other parts of the world [6,7]. The rise of autism incidence during the last 20 years (19761997) is illustrated by population-based data from Olmstead County, Minnesota [8]. From 1976 to 1979 there were no cases of autism; thereafter, the incidence rate (per 100,000 children) increased as follows: 5.5 (19801983), 7.9 (19841987), 11.8 (19881991), 29.4 (19921994), and 44.9 (1995 to 1997). The 8-fold increase in incidence affected mainly children born after 1987. In contrast, the corresponding incidence of all other childhood developmental, neurologic or psychiatric disorders increased 1.8-fold [8]. Even conceding that some increment in cases could be due to changes in diagnostic criteria and to heightened awareness, it is clear that autism has become an important Public Health problem [1,3]. The descriptive epidemiology of autism provides few clues to its etiology [9]. Boys are more affected than girls (4.3:1) but this ratio changes with the degree of cognitive impairment [5] without consistent socioeconomic or racial distribution; however, autism is less common in children of immigrant mothers from Mexico [5]. There is increased risk in siblings but monozygotic twin concordance is b 100% [5]. The growing incidence indicates that causal environmental factors are more likely to cause autism than a pure genetic etiology. 2. Neuropathology of Autism Morphological changes in the brain of patients with autism indicate a prenatal time of onset of this pathology. In 1998, Bailey et al. [10] reported 4 megalencephalic cases of autism with increased brain size and weight; in most cases the cerebral cortex had an abnormal pattern of convolutions, with large hyperconvoluted temporal lobes and upward rotation of the hippocampus bilaterally. Microscopic examination revealed dysgenesis of the cerebral cortex with increased cortical thickness, abnormal laminar patterns, high density of hippocampal neurons, presence of neurons in the molecular layer, neuronal disorganization, poor differentiation of the greywhite matter boundary, and neuronal heterotopias. The white matter showed areas of focal increase in the number of single neurons and presence of ectopic grey matter. In 1993, Kemper and Bauman [11] confirmed the presence of abnormally small, densely packed neurons in all areas of the hippocampus, subiculum, mamillary body, septal nuclei and medial nuclei of the amygdala. Other constant changes [1015] include reduction in Purkinje cell and cerebellar granule cell density, and developmental abnormalities of the inferior olives. These findings are consistent with abnormal neuronal migration and alterations in the number, survival and orientation of neurons affecting the brain up to the time of olivary cell migration [14,15]; the latter occurs before the end of the 3rd month. Migration of neocortical neurons from periventricular regions occurs in humans mostly between weeks 12

and 24 of gestation [16]. Brain imaging data [1720] also date the possible onset of the morphological alterations of autism to early pregnancy. 3. Thyroid effects on brain development Deficiency of thyroid hormones during critical periods of brain development, both in utero and in the early postpartum period, is a well-recognized cause of brain damage leading to mental retardation, decreased intellectual capacity, psychomotor delay, and deafness [21]. Iodine is essential for the production of thyroid hormones. Lack of iodine in the diet is the most important worldwide cause of hypothyroidism, goiter and other iodine deficiency disorders (IDD) [22]. In 2005, nearly two billion individuals or almost one-third of the population of the world suffered from insufficient iodine intake and were at risk for IDD [23]. Women of reproductive age are at highest risk for IDD due to the effects of the thyroid on ovulation, fertility, and pregnancy outcome [22]. The main thyroid hormone active in the brain is 3,5,3triiodothyronine (T3) derived in large part from 5 deiodination of maternal thyroxine (T4) by local brain deiodinases D2 and D3. Maternal T4 and T3 are transported to the fetal brain from maternal blood across the bloodbrain barrier [24,25]. Prior to the formation of the fetal thyroid around midgestation the fetus is unable to produce thyroxine and is therefore completely dependent on maternal thyroxine [25]. Endemic cretinism is the most severe degree of in utero brain damage from maternal hypothyroxinemia ensuing from dietary iodine deficiency [26,27]. Clinical features include profound mental retardation, deafmutism, squint of eyes, signs of bulbar damage, spastic diplegia, pyramidal and extrapyramidal signs, and typical gait with laxity and deformities of the joints [27]. Endemic cretinism is different from congenital hypothyroidism, which occurs in about 1 in 3500 newborns as a result of morphological or functional deficiencies of thyroid function in the fetus and the newborn unrelated to dietary iodine deficiency [28]. In endemic cretinism, Cao et al. [29] demonstrated that correction of iodine deficiency was effective within a fairly narrow period up to the beginning of the third trimester, indicating a critical in utero window of time [30] for thyroxine effects on normal brain growth and development [3134]. Maternal serum levels of thyrotropin (thyroid stimulating hormone, TSH) increase progressively during normal pregnancy causing a corresponding rise of serum thyroglobulin [31]. Transfer of maternal T4 to the fetus occurs up to birth [32,35,36]. Therefore, the availability of free T4 to embryonic and fetal tissues depends on circulating maternal T4. Decreases in free T4 occur in hypothyroxinemic women even if clinically euthyroid [32]. With iodine deficiency, a steady decrease of free thyroxine occurs during gestation further increasing thyrotropin (TSH) and leading to goiter. Serum TSH and thyroglobulin values are even higher in neonates born to iodine deficient mothers given that the newborn is extremely sensitive to iodine deficiency. These

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changes in neonatal TSH frequently occur with levels of iodine deficiency that would not affect the thyroid function in non-pregnant adults [32]. The effect of thyroid hormones is through binding of T3 to nuclear thyroid hormone receptors regulating gene expression in different brain regions by interacting with regulatory sites in target genes [34]. Thyroid hormone receptors (TRs) are present in the cerebral cortex of the human fetus by 89 weeks of gestation [37,38] and increase about 10-fold between 10 and 18 weeks gestation [34]. During the second trimester, Kester et al. [39] found that brain T3 levels greatly increased in the cerebral cortex along with increasing iodothyronine deiodinase (D2) activity. Therefore, moderately low T4 maternal levels may be damaging to the fetus [32,40,41]. Haddow et al. [42] found that the strongest predictor of mental development of the infant was the mothers' free T4 levels at 12 weeks of gestation. Furthermore, low levels of free T4 at both 12 and 32 weeks of gestation resulted in worse cognitive outcome of the child. The importance of maternal T4 for fetal brain development within a very restricted time window has received experimental support in rat models of maternal hypothyroxinemia. 4. Animal models of congenital hypothyroidism The Spanish School of Endocrinology (Gabriella Morreale de Escobar, Juan Bernal Carrasco, Francisco Escobar del Rey, Pere Berbel, and collaborators) contributed substantially to our understanding of the molecular basis of the action of thyroid hormones on the developing brain. In a number of experimental and clinical studies they have shown the crucial effects of thyroid hormones on neuronal differentiation, on oligodendrocytes, astrocytes and microglia, as well as on neuronal migration, formation of cortical layers, neural networking and synaptogenesis [summarized in [3234]]. Experimental models of endemic cretinism in the rat have demonstrated that absence of thyroid hormone during pregnancy (induced by methods such as surgical thyroidectomy or use of radioactive iodine, propylthiouracil or methimazole), followed by elimination of thyroid hormones in the pups, results in numerous brain alterations [43] including faulty differentiation of neurons, particularly Purkinje cells, glial cells, myelination, and abnormal migration of cortical layers and callosal connections in visual and auditory areas [44]. The intrauterine timing of thyroid hormone effects in the brain was recently clarified by Lavado-Autric et al. [45] and Aus et al. [46] from the Spanish School of Endocrinology. In an elegant series of experiments they produced in the rat a model of maternal hypothyroxinemia-low T4 but normal T3demonstrating that transient and mild thyroid function deficits in the mother during early gestation produced permanent abnormalities in cortical development. Lavado-Autric et al. [45] induced hypothyroxinemia by maternal low iodine diet and demonstrated faulty neuronal migration in hippocampus and somatosensory cortex of the progeny. Neurons were found in abnormal locations, cortical barrels were absent and cortical layers were blurred. The

timing of thyroid effect on the fetal cortex has been defined even more closely by Aus et al. [46] who produced mild and transient hypothyroxinemia using methimazole for 3 days only, from embryonic day 12 (E12) to E15, and induced permanent alterations of the cytoarchitecture and radial distribution of neurons in the somatosensory cortex and hippocampus of the progeny. Cuevas et al. [47] demonstrated similar irreversible alterations of radial neuronal migration after transient maternal hypothyroxinemia at onset of corticogenesis (E10E13) in mice [47]. Therefore, transient and limited thyroid hormone deficiency in utero may cause the morphological brain lesions of autism. Migration of cortical neurons along the scaffolding provided by radial glia is regulated by the reelin-dab signaling system [16,48]. Reelin, an extracellular glycoprotein secreted by Cajal-Retzius neurons of layer I and by GABAergic neurons, binds to membrane receptors on migrating neurons phosphorylating the Disabled homolog-1 (Dab1) to guide cells to their destination [49]. Reelin receptors include the apolipoprotein E receptor 2 (ApoER2), the very-low-density lipoprotein receptor (VLDL-R) and the 31 integrin protein [16,49]. Hypothyroidism reduces reelin expression and enhances Dab1 expression [50] explaining the migratory alterations observed in experimental models of congenital hypothyroidism and maternal hypothyroxinemia. In fact, reelin signaling abnormalities have been reported in autism [51,52]. Using postmortem material from superior frontal, parietal, and cerebellar cortex from autistic brains and matched controls, Fatemi et al. [53] demonstrated significant reductions in reelin protein, reelin mRNA, and Dab 1 mRNA along with elevations in Reln receptor, and VLDL-R mRNA in frontal and cerebellar cortex, indicative of impairments in the reelindab signaling system in autism. Some studies [5457] have found genetic susceptibility polymorphisms of the reelin (RELN) gene in autism; however, other comprehensive studies have been negative [5861] suggesting that environmental factors probably play an important role in the disruption of this signaling system. 5. Potential causes of maternal hypothyroxinemia that could be associated with autism The most common causes of maternal hypothyroxinemia are dietary iodine deficiency [62] and exposure to environmental antithyroid agents. Table 1 modified from the pioneering work of Gaitan [63] summarizes some of the numerous and relatively unknown environmental antithyroid and goitrogenic agents. 5.1. Iodine deficiency According to recent WHO estimates [64], some 2 billion people, including 285 million school-age children, have iodine deficiency, defined as urinary iodine excretion of less than 100 g per liter [62]. Iodine deficiency is present throughout the world, including industrialized nations [64]. Utiger [65] emphasized that most T3 is produced by

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Table 1 Environmental agents producing antithyroid effects or goiter (modified from Gaitan, E. Environmental Goitrogenesis. Boca Raton, Florida: CRC Press [63]) Compounds Inorganic Iodine deficiency Iodine excess Lithium Mercury Sulfurated organics Thiocyanate (SCN) Isothiocyanates L-5-vinyl-2-thiooxazolidone (goitrin) Disulfides (R-S-S-R) Flavonoids (polyphenols) Glycosides Aglycones C-ring fission metabolites (i.e., phloroglucinol, phenolic acids) Polyhydroxyphenols and phenol derivatives Phenol Catechol (1,2-dihydroxybenzene) Resorcinol (1,3-dihydroxybenzene) Hydroquinone (1,4-dihydroxybenzene) m-dihydroxyacetophenones 2-methylresorcinol 5-methylresorcinol (orcinol) 4-methylcatechol Pyrogallol (1,2,3-trihydroxybenzene) Phloroglucinol (1,3,5-trihydroxybenzene) 4-chlororesorcinol 3-chloro-4-hydroxybenzoic acid 2,4,-dinitrophenol Pyridines 3-hydroxypyridine Dihydroxypyridines Phthalate esters and metabolites Diisobutyl phthalate Dioctyl phthalate o-phthalic acid m-phthalic acid 3,4-dihydroxybenzoic acid (DHBA) 3,5-dihydroxybenzoic acid Polychlorinated (PCB) and polybrominated (PBB) biphenyls PCBs (Aroclor) PBBs and PBB oxides Other organochlorines (2,4-dichlorophenoxyacetic acid) 2,4-D Dichlorodiphenyltrichloroethane (p,p-DDT) Dichlorodiphenyldichloroethane (p,p-DDE) Dieldrin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) Polycyclic aromatic hydrocarbons 3,4-benzopyrene 3-methylcolanthrene 7,12-dimethylbenzanthracene

extrathyroidal deiodination of T4 and the iodine released by deiodination is rapidly excreted in the urine. Therefore, when thyroxine needs are higher, such as in pregnant and lactating women, infants, and children, more dietary iodine is needed. According to Morreale de Escobar et al. [32] early maternal hypothyroxinemia (weeks 812) may be 150200 times more common than congenital hypothyroidism, potentially affecting 1 in 20 newborns. As demonstrated in experimental models, maternal hypothyroxinemia can produce permanent brain lesions in the fetus. Therefore, it is recommended that pregnant and lactating women receive daily iodine supplements (i.e., 200 g of iodine) from the onset of pregnancy or earlier [66]. In the United States, a seven-fold increase in the frequency of moderate iodine deficiency among pregnant women has occurred since the 1970s, along with a four-fold increase in the frequency of moderate iodine deficiency in the total population, coincident with a greater than 50% decline in urinary iodine excretion [67], along with subclinical signs of thyroid deficiency [68]. Presence of urinary perchlorate (Cl04 ) in adolescents and adult men and women could partly explain this trend [69]. Ammonium perchlorate, a major component of solid rocket fuels, is readily soluble in water and has long-term stability in aqueous solution; perchlorate has been detected in water from areas associated with the aerospace industry [70]. Perchlorate interferes with iodide uptake at the sodium/iodide symporter reducing the amount of iodide available for production of thyroid hormones. Nonetheless, a study in Nevada failed to correlate perchlorate levels in drinking water with pediatric neurobehavioral disorders, including autism [71]. As mentioned below (Table 2), a long list of environmental agents could explain also the progressive decline in urinary iodine excretion in the USA. 5.2. Foodstuffs with antithyroid effects 5.2.1. Thiocyanate (SCN) The antithyroid and goitrogenic effects of Brassicae plants (Family Cruciferae) have long been known [72], including cabbage (Brassica oleracea), broccoli, cauliflower, kale, kohlrabi, Brussels sprouts, and rutabaga (swede or yellow turnip, Brassica napobrassica), rapeseed and mustard. SCN interferes with active uptake and concentration of inorganic iodide by the thyroid and inhibits the enzyme thyroperoxidase thereby preventing the incorporation of iodine into thyroglobulin. Rutabaga and turnips contain a thiourea-like product (progoitrin), a precursor of goitrin that also interferes with thyroperoxidase [72]. A number of staple foods in the tropics contain large amounts of cyanogenic glycosides that are detoxified as SCN [21]. These plants include cassava (Manihot esculenta Crantz), millet, yam, sweet potato, corn, bamboo shoots, and lima beans (Phaseolus vulgaris). Tobacco smoke (Nicotiana tabacum) also contains considerable amounts of cyanide (150300 g per cigarette) in addition to other goitrogenic products such as resorcinol derivatives, flavonoids, and hydroxypyridines [63]. Thiocyanate toxicity from

G.C. Romn / Journal of the Neurological Sciences 262 (2007) 1526 Table 2 Synthetic chemicals that interfere with the production, transport, and metabolism of thyroid hormone (according to Howdeshell [103]) Thyroid mechanism and interfering chemical Uptake of iodide by thyroid gland 2,4-D (2,4-dichlorophenoxyacetic acid) 3-Amino-1,2,4-triazole Aldrin Amitrole Aroclor 1254 1,2-Dihydroxybenzene (catechol) 4-Chlororesorcinol Clofentezine o-Cresol p-Cresol Cythion 1,3-Dihydroxynaphthalene 1,5-Dihydroxynaphthalene 2,3-Dihydroxynaphthalene 2,7-Dihydroxynaphthalene 2,4-Dihydroxybenzaldehyde 2,4-Dihydroxybenzoic acid Ethiozin Ethylene thiourea Fipronil Hexachlorobenzene Hexadrin 4-Hexylresorcinol 1,3,4-Trihydroxybenzene (hydroxyquinol) Hydroxyquinol triacetate Lead Mancozeb Mercuric chloride 3-Methylcholanthrene Methylmercuric chloride Methylparathion 2-Methylresorcinol Mull-Soy Nabam 5-Methylresorcinol (orcinol) Pendimethalin Pentachloronitrobenzene Phenobarbital Phenol 1,3,5-Trihydroxybenzene (phloroglucinol) Polybrominated biphenyls Pregnenolone-16-carbonitrile Propylthiouracil 1,2,3-Trihydroxybenzene (pyrogallol) Pyrimenthanil 1,3-Dihyroxybenzene (resorcinol) o-Hydroxybenzyl alcohol (saligenin) Selenium Thiocyanate Sodium/iodide symporter Perchlorate Perrhenate Serum protein-bound iodide level 2,4-D 2,4-Dinitrophenol 3-Methylcholanthrene Amitrole Aroclor Cythion (continued on next page) Table 2 (continued) Thyroid mechanism and interfering chemical Serum protein-bound iodide level Malathion Mancozeb Mercuric chloride o,p-DDD Hexadrin Thyroid peroxidase action general information Amitrole Ammonia Ethylene thiourea Fipronil Mancozeb 4,4-Methylenedianiline Thiocyanate Thyroid peroxidase action oxidation of iodide Aminotriazole Ammonia Cadmium chloride Endosulfan Ethylene thiourea 1,2,3,4,5,6-Hexachlorocyclohexane (lindane) Malathion Mancozeb Mercury chloride Methamizole Polybrominated biphenyls Thiourea Thyroid peroxidase action iodination of tyrosine Polybrominated biphenyls Binding to thyroglobulin o,p-DDD Pentachlorophenol Binding to transthyretin Bromoxynil (3,5-bibromo-4-hydroxybenzonitril) 4-(Chloro-o-tolyloxy) acetic acid 4-(4-Chloro-2-methylphenoxy) butyric acid Chlorophenol Chloroxuron 2,4-D (2,4-dichlorophenoxyacetic acid) 2,4-Dichlorophenoxybutric acid Dioxtylphthalate o,p-DDD p,p-DDD ((1,1-dichloro-2,2-bis(p-chlorophenyl) ethane) 2,3-Dichlorophenol 2,4-Dichlorophenol 2,6-Dichlorophenol 2-(2,4-Dichlorophenoxy) propionic acid [dichloroprop] 1,1,1-Trichloro-2,2-bis (chlorophenol) ethanol [difocol] 2,4-Dinitrophenol 2,4-Dinitro-6-methylphenol Ethyl-bromophos Ethyl-parathion 2-(2,4,5-Trichlorophenoxy) propionic acid [fenoprop] Hexachlorobenzene Hexachlorophene 2-Hydroxybiphenyl 4-Hydroxybiphenyl Binding to transthyretin

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G.C. Romn / Journal of the Neurological Sciences 262 (2007) 1526 Table 2 (continued) Thyroid mechanism and interfering chemical Catabolism and biliary elimination of T4/T3 in the liver Aroclor 1254 3,4-Benzopyrene DDT Hexachlorobenzene 3-Methylcholanthrene Phenobarbital Polybrominated biphenyls

Thyroid mechanism and interfering chemical Lindane Linuron Malathion Pentachlorophenol Phenol Pyrogallol 2,4,5-Trichlorophenoxyacetic acid 1,4-Tetrachlorophenol PCB-77 Trichloroacetic acid Trichlorobenzene 2,3,4-Trichlorophenol 2,4,5-Trichlorophenol 2,4,6-Trichlorophenol 2,4,5-Trichlorophenoxyacetic acid methyl ester Binding to albumin Pentachlorophenol Catabolism of T4 or T3: type I or II 5-deiodinase 3,3,4,4,5,5-Hexachlorobiphenyl 3-Methylcholanthrene Aminotriazole Amiodarone Aroclor 1254 Cadmium chloride Diphenylthiohydantoin Dimethoate Fenvalerate Hexachlorobenzene Lead Phenobarbital Propylthiouracil PCB 77 TCDD Glucuronidation of T4/T3 Acetochlor Aroclor 1254 3,4-Benzpyrene Clofentenzine Clofibrate DDT Fenbuconazole 3,3,4,4,5,5-Hexabromobiphenyl Hexachlorobenzene 2,3,3,4,4,5-Hexachlorobiphenyl 3,3,4,4,5,5-Hexachlorobiphenyl 3-Methylcholanthrene Pendimethalin PCB 126 Phenobarbital Polybrominated biphenyls PCBs Pregnenolone-16-carbonitrile Promadiamine Pyrimethanil PCB 77 TCDD Thiazopyr

cassava consumption, plus the combined effect of iodine and selenium deficiency, causes endemic goiter and play a major role in the myxedematous form of endemic cretinism observed in Africa [72]. 5.2.2. Polyphenols and flavonoids Polyphenols are common dietary constituents of plant origin characterized by the presence of cyclic benzene compounds [73,74]. Polyphenols are strong antioxidants that protect plants from ultraviolet radiation damage, as well as from bacterial, fungal, and viral pathogens, insects and other pests. Flavonoids also act as metal chelators, and as visible color signals to attract pollinators and disperse fruits and seeds. Polyphenols in the diet include flavonoids, phenolic acids, stilbenes (resveratrol), and other complex molecules such as lignans found in oil seeds (flax, soy, and canola or rapeseed) [75,76]. Resveratrol from red wine has been shown to increase life expectancy and to protect against cancer, heart disease, ischemic lesions, diabetes, inflammation, and viral infection [77]. Natural flavonoids are usually conjugated to sugars or carbohydrates (glycosides) and polymerized to other flavonoids and nonflavonoids (acyl derivatives); non-conjugated forms are called aglycones. Food flavonoids and polyphenols can polymerize into large molecules called tannins. Polyphenols exhibit multiple mechanisms of action, including quenching of oxygen and free radicals, metal chelation, hormonal estrogen activity, regulation of gene expression, effects on cell signaling pathways, inhibition of cancer cell growth, anti-inflammatory, anti-atherosclerotic and antithrombotic effects on platelets, endothelial cells, nitric oxide, vascular smooth muscle cells, monocytes, macrophages, and LDL-cholesterol [7683]. Unfortunately, the emphasis on the potential health benefits of polyphenols has overshadowed the profound effects of some plant isoflavonoids on thyroid hormones and other hormones. Many isoflavonoids structurally resemble T3 and T4 and could act as these hormones influencing the hypothalamuspituitary feedback system [84,85]. Genistein and daidzein from soy (Glycine max) are among the most important plant isoflavonoids with antithyroid effects; both inhibit thyroperoxidase, the enzyme that catalyzes iodination and thyroid hormone biosynthesis [85]. This effect is magnified under conditions of iodine deficiency. More worrisome for the potential impact of soy on the fetal brain

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during pregnancy is the inhibition of 5-deiodinases (D2 and D3) in neural tissues by genistein [86] and other flavonoids as a result of conformational changes in the enzyme structure [84]. Moreover, in North America as many as 25% of infant formulas are based on soy protein [87]; children receive up to 11 mg/kg of soy per day compared with 1 mg/kg in adults increasing the risk of hypothyroidism in newborns [8789]. Flavonoid aglycones resulting from intestinal digestion are rapidly and readily absorbed [90] increasing the likelihood of development of antithyroid effects and goiter in infants receiving soy-based formulas as milk substitute [9197]. It has been postulated that the fermentation of soybeans commonly used in China and the Orient could decrease some of its antithyroid effects [98]. However, kojic acid, a fungal metabolite produced by Aspergillus flavus, used traditionally in the production of miso (soybean paste), shoyu (soy sauce) and sake, also has antithyroid effects including iodine uptake inhibition, decrease in T3 and T4, increase in TSH and induction of goiter [99]. Deiodinase I is also affected by other plant flavonoids [100] including catechin from tea (Camellia sinensis), quercetin (found in apples, onions, red grapes, citrus fruits, broccoli, cherries, berries, and prickly-pear cactus), kaempferol (from Delphinium, Witch-hazel, and grapefruit), rutin (found in buckwheat), and baicalein, isolated from the roots of baikal or Chinese skullcap (Scutellaria baicalensis Georgi). In the presence of iodine deficiency, some anthocyanins, catechins and tannins from nuts exhibit goitrogenic effects [84]; these include peanuts (Arachis hypogea), cashew nuts (Anacardium occidentale), almonds (Prunus amygdalus), and the areca nut (Areca catechin). Other plants with hypothyroid effects include millets such as pearl millet (Pennisetum glaucum) and fonio millet (Digitaria exilis) [101]; bugleweed (Lycopus virginicus), gypsywort (Lycopus europaeus), water horehound (Lycopus lucidus or Lycopus americanus), gromwell (Lithospermum ruderale), European gromwell (Lithospermum officinale), lemonbalm (Melissa officinalis), and perhaps rosemary (Rosmarinus officinalis) and sage (Salvia officinalis) [102]. 5.3. Environmental chemicals with antithyroid effects Howdeshell [103] and Colborn [104,105] have recently reviewed the potential effects of environmental chemical agents on endocrine functioncausing in particularly thyroid disruption and abnormal brain development. Low doses of toxins could become deleterious in cases of low maternal iodine [104]. Table 2 [103] provides a comprehensive list of chemical agents with effects at several levels of thyroid metabolism. More than 60% of herbicides are endocrine disruptors [104] in particular the widely used 2,4-dichlorophenoxyacetic acid (2,4-D), mancozeb, acetochlor, aminotriazole, amitrole, bromoxynil, pendamethalin, and the thioureas. Contamination of drinking water from areas rich in coal and shale by naturally occurring powerful antithyroid products such as resorcinol (1,3-dihydroxyben-

zene) and the substituted resorcinol 2,4-dihydroxyacetophenone, methoxy-anthracene, phthalate esters and phthalic acid, can produce endemic goiter and hypothyroidism [106]. The developmental neurotoxicity of polychlorinated biphenyls (PCBs) and dioxins has been known since the 1990s [107,108]. Neurobehavioral alterations have been observed in newborn children exposed to PCBs including motor immaturity, hyporreflexia, and lower psychomotor scores between 6 months and 2 years of age; along with decrease in dopamine in basal ganglia and prefrontal cortex [107]. Similar dopamine alterations have been reported in children with autism [109]. These abnormalities could be mediated by disruption of thyroid hormones by competitive binding of PCBs (structurally similar to thyroid hormones) to serum transport proteins [110] and by alterations in thyroid hormone-responsive genes in the developing brain [103 105,110112]. Recently, Kimura-Kuroda et al. [113] demonstrated that several hydroxy-PCBs are capable of inhibiting the thyroid-hormone-dependent development of dendrites in cerebellar Purkinje cells and suggested that PCBs could be a cause of autism and other developmental disorders. 6. Existing evidence Experimental evidence has demonstrated the importance of appropriate maternal T4 and T3 during early stages of brain development. It is known that a transient thyroid hormone deficit for as little as 3 days [4547] produces permanent alterations of neuronal migration similar to those observed in autism [114]. Nonetheless, currently there is a dearth of information on the potential role of thyroid disorders in autism. In 1958, triiodothyronine was first used for the treatment of autism with some improvement [115], leading to the finding in Britain of low thyroid values in 45 of 62 autistic children [116]; however, controlled trials failed to confirm positive results of this therapy [117119]. Naturally, the developmental brain changes caused by in utero hypothyroidism are permanent and should not improve with postnatal thyroid hormone treatment. In 1995, marginal changes in circadian diurnal TSH levels were reported [120]; more recently, normal neonatal thyroxine (T4) levels were detected in a group of 6 autistic children found among 227 cases of childhood neurobehavioral disorders and 948 controls [121]. Nonetheless, to my knowledge, there is no information on autism and maternal thyroid function during the first or second trimesters of pregnancy. Of related interest is the finding that a family history of autoimmune thyroiditis doubled the risk of autism (OR = 2.09, 95% CI 1.283.41) [122]. Autoimmune thyroiditis may occur after exposure to polybrominated byphenils (PBB) [123] and other pollutants [124], and in rats exposed to carbon tetrachloride, methylcholantrene, and dimethyl-benzanthracene [63]. Although many authors have suggested that environmental pollutantssuch as those affecting thyroid hormones may play a role in autism [85,103105,112,113,125] only

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recently Palmer and colleagues [126], from the University of Texas at San Antonio, reported an ecological association between autism and environmental mercury pollution. This study was based on quantitative data on mercury released in the Texas environment, provided by the Agency for Toxic Substances and Disease Registry (ATSDR) and the Environmental Protection Agency (EPA). The authors correlated environmentally released mercury (per 1000 lb) with rates of autism in Texas using county (N = 254) and school district (N = 1184) information by means of a multilevel Poisson regression model adjusted for racial composition, socioeconomic level and urbanicity. They concluded that the risk for autism increased 6.15% per each 100 lb of environmentally released mercury (RR = 1.614, 95% CI 1.4871.752); the highest increase in relative rates of autism in school districts was for urban relative to rural (RR = 4.726, 95% CI 3.85.9) and for suburban vs. rural districts (RR = 2.547, 95% CI 2.1 3.2). Autism accounted for the increase in special education rate whereby each 100 lb of mercury released increased 4.3% the rate of special education students (RR = 1.433, 95% CI 1.351.52) [126]. This study from our institution is one of the first to demonstrate an association between environmental pollution and autism. The bulk of the mercury pollution in Texas is from metallic mercury but increases in organic mercury (methylmercury) are expected to occur as metallic mercury enters the food chain. Both organic and inorganic mercury compounds have antithyroid effects, although metallic mercury is a stronger inhibitor of iodothyrosine deiodinase [127]. Moreover, inorganic mercuric chloride (HgCl2) is also a strong inhibitor of thyroid peroxidase, an effect not observed with methylmercury chloride [128,129]. Human exposure to low levels of metallic mercury vapors produces decrease in T3 [130,131] confirming the inhibition of 5-deiodinase type I in vivo; enzyme inhibition was higher in those with low urinary iodine excretion [131]. During pregnancy, Takser et al. [132] found a significant correlation between inorganic mercury levels in umbilical cord and lower maternal serum free thyroxin (T4) levels and concluded that even low levels of exposure to persistent environmental contaminants interfere with thyroid status during pregnancy. This report is important because it confirms the likelihood of fetal exposure to mercury and the possible inhibition of fetal brain deiodinases as postulated here. Moreover, mercury and PCB exposure in experimental animals produces alterations of gait and coordination, along with morphological changes in cerebellar Purkinje cells [133]. Other than industrial pollution, dental amalgam fillings are the most common source of metallic mercury in the general population. A recent autopsy study [134] demonstrated higher tissue levels of mercury in subjects with greater number of occlusal amalgam surfaces. Moreover, mercury levels were significantly higher in brain tissue compared with thyroid and kidney tissues in subjects with N 12 amalgam fillings (all P = 0.01) [134]. A related finding was the observed decrease in anti-thyroid antibodies in patients with autoimmune thyroiditis after removal of dental amalgam fillings [135].

The highly publicized hypothesis that linked autism with thimerosal, a mercury-containing vaccine preservative, has been amply refuted [136140]. The WHO advocates continued use of thimerosal-containing vaccines; mercury exposure in children is more likely to occur from breast feeding and the benefits of eliminating thimerosal from a vaccine are quite small (b 1% reduction over a lifetime) [140]. As emphasized in this review, the neuropathology of autism probably occurs early in the pregnancy. Parents usually link the first manifestations of autism with the routine childhood vaccinations, particularly with the MeaslesMumpsRubella (MMR) vaccine [136]. However, there is no scientific evidence that the MMR vaccine or the mercury preservative (thimerosal) used in some vaccines plays any causal or triggering role in autism. However, rare cases of mercury encephalopathy in children, characterized by excretion of significant amounts of mercury post chelation challenge, can be confused with regressive autism [141]. 7. Conclusions I postulate here that autism may result from the following chain of events: 1) Occurrence of transient maternal hypothyroxinemia (low T4) early in pregnancy (weeks 812). 2) Low T4 may result from iodine deficiency of dietary origin or from maternal exposure to antithyroid substance(s), or both. 3) Exposures may include: (a) dietary use of soybean products containing genistein and daidzein; or consumption of other plant products containing isoflavonoids or thiocyanate which inhibit thyroperoxidase and/or tissue deiodinases leading to low fetal T3; or, (b) contamination of water sources and foodstuffs with thyrotoxic environmental pollutants (man made or natural), including herbicides such as 2,4-D, PCBs, PBBs, perchlorate, mercury, or coal derivatives such as resorcinol, anthracenes, and phthalates. 4) Low maternal T4 plus inactivation of brain deiodinases by any one of the above mechanisms would lower tissue levels of triiodothyronine (T3) resulting in abnormal neuronal migration via reelin dysregulation. 5) reelin-dab signaling abnormalities occur in autistic brains [5153] including significant reductions in reelin protein, reelin mRNA, and Dab 1 mRNA, along with increase of the Reln receptor [5053]. In autistic children, some polymorphisms of the reelin (RELN) gene could confer susceptibility to environmental factors [5457] including those causing low tissue triiodothyronine, as described above. This hypothesis would explain the absence of complete concordance for autism among monozygotic twins (due to different degree of inhibition of deiodinases in each twin, since each one has separate prenatal environment with differences in the placenta, amniotic sac, and vascularization, leading to

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epigenic differences later in life [142]), and the limited results of genetic studies such as those mapping autism risk loci using genetic linkage and other techniques [143] that appear to implicate neuroligins and neurexins, adhesion molecules involved in synapse development and function [144]. Multidisciplinary epidemiological studies of dietary and environmental exposures to thyrotoxic products and quantification of the risk of autism from transient maternal hypothyroxinemia during the first and second trimesters of pregnancy will be required to confirm this hypothesis. Lastly, from the Public Health viewpoint, the major advantage of confirming the hypothesis proposed here is the possibility of early intervention to modify the multiple environmental factors that may lead to the development of autism. References
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