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Year : 2021  |  Volume : 39  |  Issue : 1  |  Page : 61-66

Metallic insignia in primary teeth: A biomarker for Autism Spectrum Disorders

1 Department of Paediatric and Preventive Dentistry, Baba Jaswant Singh Dental College and Hospital and Research Institution, Ludhiana, Punjab, India
2 Associate Professor, Prosthodontics, King Khalid University, Saudi Arabia

Date of Submission09-Nov-2020
Date of Decision28-Feb-2021
Date of Acceptance03-Mar-2021
Date of Web Publication22-Apr-2021

Correspondence Address:
Dr. Kanwalpreet Kaur
92/2, Sant Nagar, Near College Road, Civil Lines, Ludhiana, Punjab
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jisppd.jisppd_485_20

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Background: Lead accumulations have been found in teeth and related to behavior deficits in children, but there is a dearth of studies in exploring the role of zinc and manganese dysregulations in autism spectrum disorders (ASD) using the primary tooth as biomarker. Aims: The objectives of the study were to evaluate and compare the concentrations of zinc and manganese in the primary teeth serving as biomarker, in typically developing children and children with ASD. Settings and Design: Twelve primary incisors indicated for extraction were collected from children between the age group 6 and 9 years, for the study. Six primary incisors were obtained from children who had been diagnosed with ASD (study group). The other six teeth were obtained from typically developing children, in the similar age group. Methods: The primary incisors obtained were analyzed for metal concentrations using the technique Inductively Coupled Plasma Optical Emission Spectrometry. Statistical Analysis: This study was statistically analyzed by student's t-test. Results: It was observed that there are significant differences in metal concentrations found between tooth samples of ASD children and typically developing children. Zinc concentrations were double and manganese concentrations were three times, in teeth of ASD children group as compared to the children in the control group. Conclusions: Results of the current study indicate that there are considerable differences in concentrations of zinc and manganese between the two groups and support the contention that there might be an association between metal exposures of a pregnant mother and child during early years of childhood and incidence of ASD.

Keywords: Autism spectrum disorder, biomarker, manganese, metals, primary teeth, zinc

How to cite this article:
Kaur K, Suneja B, Jodhka S, Kaur J, Singh A, Singh SR. Metallic insignia in primary teeth: A biomarker for Autism Spectrum Disorders. J Indian Soc Pedod Prev Dent 2021;39:61-6

How to cite this URL:
Kaur K, Suneja B, Jodhka S, Kaur J, Singh A, Singh SR. Metallic insignia in primary teeth: A biomarker for Autism Spectrum Disorders. J Indian Soc Pedod Prev Dent [serial online] 2021 [cited 2022 Nov 30];39:61-6. Available from: http://www.jisppd.com/text.asp?2021/39/1/61/314364

   Introduction Top

Autism is not a single entity, but a spectrum of closely related disorders with a shared core of symptoms. These are a set of neurological disorders and are now considered to be one of the new pediatric morbidities.[1] These disorders currently are a leading cause of disability in children and impose a substantial emotional, economic, and physical burden on families whose children are affected.[2]

It has been estimated worldwide, that one in 160 children have ASD.[3] There is a dramatic rise in the prevalence of autistic spectrum disorders in India in the past few decades, affecting almost 0.15- 1.00% of the children (one in 400-500).[4] The diagnosis of autistic spectrum disorders is multifactorial and depends on its etiology, which itself is poorly understood.[5] Previously, it had been the school of thought, that all such disorders are the result of genetic inheritance. And one of the promises of human genetics is, that it revolutionizes the knowledge of the hidden etiology of diseases and helps in inventing their preventions and cures. Unfortunately, genetics have been found to account for only 10% of the autism spectrum disorders (ASDs) and the remaining causes appear to come from the environment. Therefore, it is now being increasingly recognized that such disorders can be caused by environmental factors and prenatal environmental exposures have been implicated in the occurrence of developmental disabilities in children.[6] Extensive research in the recent years, taking into account the presentation of many cases which have no family history of autistic spectrum disorders, the attention has been shifted toward potential roles of environmental intoxicants, specially exposure to metals and causation of behavioral deficits.[7]

Prenatal and environmental exposures have been implicated in the occurrence of developmental disabilities in children, but the research relating to the roles of metals or chemicals in the causation of these disorders has been slow.[8] It has been hypothesized that exposure to environmental factors such as metals like zinc and manganese can be a causative factor for autistic spectrum disorders. Pregnant mothers living close to industrial centers have increased risk of their offspring being affected by ASD.[9] The maximum susceptibility is prominent during the prenatal period when the placenta is permeable and the fetal blood–brain barrier and the neuronal growth is immature.[10] Children, in their early childhood also are particularly vulnerable to the neurotoxic effects of exposures to heavy metals because their brains are still in the developing stage.[11]

The contributions of multiple metal exposures and the occurrence of ASDs are unclear, although dysregulation of the metals zinc and manganese has been implicated because of their potential roles in the central nervous system. These elements play an important role in the synaptic transmission of impulses across the central nervous system. Their toxicity or deficiency can lead to neurological or psychological disorders. Studies in the last decades clearly prove that zinc has a crucial role in neonatal development. Zinc is one of the essential trace elements in humans and participates in many metabolic and signaling pathways in the human body. It plays an important role in the immune and the central nervous system, being one of the most prevalent metal ions in the brain and is involved in the regulation of neurogenesis, neuronal migration, and differentiation, consequently maintaining healthy brain function and shaping cognitive development.[12] Zinc is involved in the regulation of the shank family of proteins which are present in the synapses in the brain. These proteins have zinc-binding sites and excess or deficit of zinc causes mutations in this protein. Shank proteins are involved in the maturation of gabaminergic receptors which are involved in the transmission of impulses.[13]

Manganese (Mn) is also a naturally occurring trace element which is essential for health and development in humans. Manganese is an essential cofactor for metalloenzyme-superoxide dismutase, which protects cells against antioxidant processes.[14] Dysregulation of manganese and the consequences of neurotoxicity are well documented.[15] Increased level of manganese causes mitochondrial inactivation as a primary route of action. This results in the enhanced formation of oxygen reactive species in the areas of the brain which can lead to oxidative stress and cause impaired propagation of impulses.[16] Exposure to manganese in the prenatal period has been related to higher than normal levels of disruptive behavior in children at age of three.[17]

To assess the effects of metals on the neurological development of children, there is a need of a biomarker or a parameter. The choice of the biomarker is critical to the measurement of exposures to metals. The commonly used biological matrices to assess the exposure of the child, such as blood and urine, provide access to single point serum concentrations only, the values of which show diurnal variations. Hair and nails are other biomarkers which can be used to provide information about exposures in this period, but these can be contaminated by shampoos, nail paints, etc., and do not provide access to the prenatal period.[18] These markers cannot retrospectively assess the prenatal and early postnatal exposure. Therefore, using these biomarkers to aid in the diagnosis of ASD is not feasible. Consequently, the children suspected to have ASD are subjected to psychological interventions and multiple behavior screening tests which are also not very definitive of diagnosing autism and can be very exhausting for the child and the parent.

A primary tooth can suffice as a reliable biomarker to assess the environmental exposure of both the mother and the child in early childhood years. The mineralization of these teeth proceeds in an incremental pattern, akin to growth rings of trees, spanning the entire prenatal and early postnatal phases. Enamel formation in primary teeth begins in utero and completes by approximately 3 months to 1-year postbirth as according to the particular tooth period of mineralization.[19] The metals are accessible through the placental barrier which go into the fetal bloodstream and deposit these metals in the teeth while they are mineralizing. Enamel provides an archival record of these fetal periods as when the enamel formation is complete, blood flow ceases in this tissue.[20] The neonatal line delineates prenatal and postnatal developmental regions. Data on the fine incremental microstructure of teeth can be used to uncover the cumulative chemical exposures which the mother might have had during her gestation period.

The current study was therefore undertaken - to compare the concentrations of zinc and manganese in the primary tooth, serving as a biomarker, in typically developing children versus children with ASD.

   Methods Top

The study setting for this research was within our research institute. Eligibility criteria included the children between 6 and 9 years of age having primary incisors with preshedding mobility/indicated for extraction. Children with systemic disease or medical conditions such as cerebral palsy or other genetic disorders related to developmental disabilities or having parents with known medical illness or substance abuse were excluded from the study.

Twelve primary incisors indicated for extraction were collected from children between the age group 6 and 9 years, for the study and divided into two groups. Six primary incisors were obtained from the children of a special school who had been diagnosed with autism spectrum disorders (study group). The other six teeth were obtained from typically developing children, in the similar age group, who had been screened for not having ASD (control group), by a qualified medical practitioner.

Teeth were collected and stored in acetone in small snap lock boxes, with numerical codings [Figure 1] and [Figure 2]. Tooth samples from both groups of children (test group, and control group) were sent to Shriram Institute for Industrial Research, New Delhi (A Unit Of Shriram Scientific And Industrial Research Foundation), where they were analyzed for concentrations of metals by a method called Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES). The investigator, for testing the metal levels, was blinded to the teeth samples of the two groups of children.
Figure 1: Tooth samples

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Figure 2: Tooth samples

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Sample preparation and inductively coupled plasma-optical emission spectrometry

The teeth were cleaned and rinsed in deionized and distilled water and dried. The samples were powdered with the help of pestle and mortar. The powdered sample was analyzed for manganese and zinc content using the technique of Inductively Coupled Plasma-Optical Emission Spectrometry in which the composition of elements in samples can be determined using argon plasma and a spectrometer [Figure 3].[21],[22]
Figure 3: Working of the Inductively Coupled Plasma Optical Emission Spectrometry from the sample introduction to detector[21]

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The limit of detection (LoD) of the instrument [Figure 4] for manganese and zinc was 0.03 ppm and limit of quantification (LoQ) of the instrument for manganese and zinc was 0.1 ppm. LoD and LoQ are terms used to describe the smallest concentrations of a measure that can be reliably measured by an analytical procedure.[23]
Figure 4: Inductively Coupled Plasma Optical Emission Spectrometry Instrument

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   Results Top

The data received from the Sriram Institute of Industrial Research for the concentrations of zinc and manganese in the two groups [Figure 5] and [Figure 6] were compiled and entered in a spreadsheet computer program (Microsoft Excel 2010). Descriptive statistics included computation means and standard deviations. Because of the small sample size, the analysis was performed with student's t-test, for drawing comparisons between the tooth samples of the two groups.
Figure 5: Result control group* (zinc - 502 ppm, manganese 7.2 ppm). *Result of metal concentrations in tooth sample of one subject in the control group is displayed, for the sake of convenience

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Figure 6: Result study group (zinc – 1329 ppm, manganese – 27.4 ppm). Result of metal concentrations in tooth sample of one subject in the study group is displayed, for the sake of convenience

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[Table 1] and [Table 2] depict the mean and standard deviations of concentrations of zinc and manganese in the sample teeth in the study group and the control group. The confidence interval and P value were set at 95% and ≤ 0.05, respectively, for both the groups. The calculated P value for differences in zinc concentration was 0.001 and for differences in manganese concentrations was 0.002 which is significant. The bar graphs [Figure 7] and [Figure 8] too present significant differences in concentrations of both the metals, between the tooth samples of the control and the study group.
Table 1: Comparison of mean values of zinc in the study and control groups (ppm)

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Table 2: Comparison of mean values of manganese in the study and control groups (ppm)

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Figure 7: Bar graph depicting the comparison of mean levels of zinc in the control group and the study group

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Figure 8: Bar graph depicting the comparison of mean levels of manganese in the control group and the study group

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   Discussion Top

Trace elements play an important role in health, the deficiency, and excess of which can cause metabolic disturbances and can affect the cognitive abilities of children. Overexposure to certain metals in the prenatal phase, during the development of the central nervous system, can exert neurotoxic effects because the placenta is permeable and the blood brain of the child is still premature.[10],[11]

Previously, studies have been done on increased exposure to elements, such as lead, leading to children dying of encephalopathy. Their teeth removed later, upon analysis showed to have higher concentrations of lead than that found in controls.[24] In another study, tooth lead levels from shed incisor teeth of children living in the lead smelter area were analyzed. It provided evidence for the association between childhood lead exposure and neuropsychological impairment in terms of intellectual performance and motor integration.[25]

Apart from lead, few studies have also been conducted to explore whether exposure to other metals such as zinc and manganese can be potential causes for cognitive and behavioral deficits in children. Zinc is involved in all enzyme classes and the propagation of impulses in the brain. Studies have shown that baby teeth from children with autism contain more toxic lead and less of essential nutrients like zinc and manganese.[26],[27] Manganese is another metal, which has been shown to accumulate in dopaminergic receptors in the brain, and this fact prompted studies to determine whether manganese concentrations affect dopaminergic transmissions.[28] One study on research of manganese levels in ASD children showed that these children had lower levels of this metal in their postnatal years than typically developing children, using the tooth as a biomarker.[29] In another study, the levels of manganese in ASD children were found significantly declined, using hair as a biomarker.[30] Because this metal is implicated in causing neurodevelopmental disorders,[31],[32],[33] more research on manganese being a possible risk is needed.[34]

The studies conducted in past have shown lower levels of zinc and manganese being found in teeth of ASD children. However, our study is discrepant with correlational studies done before in that higher concentrations of zinc and manganese were found in ASD children' teeth.

This discrepancy may be explained by the evidence from the study in the Journal of Toxicology, 2007[35] which gives credence to the “poor excretor theory” and suggests that children with autism may be poor detoxifiers relative to normally developing children. Exposure to metals causes a lower total glutathione plasma levels. Glutathione is an enzyme which is important for the detoxification of heavy metals. Lowered glutathione levels make a person more susceptible to heavy metal toxicity. Metallothionein is another enzyme which is also important for the detoxification of heavy metals, and a metallothionein defect might increase a child's susceptibility to toxic metals. Besides detoxification of heavy metals, functions of metallothionein in the body include the development of brain neurons, maturation of the GI tract, antioxidation, boosting immune function, and delivery of zinc to cells. Metallothionein dysfunction may result in the inability to clear the body of heavy metals, a dysfunctional immune system, and ultimately to the neurological changes seen in ASD. It would also explain the male sex predominance (4:1) seen in autism because MT synthesis is enhanced by estrogen and progesterone.

The current study, however, had some limitations because of the small sample size. Although the participants were matched on age range and geographic location, the control for variates such as the age of the mother at childbirth, her access to health care during the gestation period, and the exact age of the child during removal of the tooth were not taken into consideration. Further, the method used, that is, ICP-OES, although demonstrates extreme sensitivity in the detection of trace elements,[36],[37] however does not preclude the possibility of measurement imprecisions which can occur in sample handling and processing. Furthermore, the information on the standard concentrations of trace elements in primary teeth is scarce and contradictory as the concentrations may show variance according to the geographic locations and other environmental factors.

   Conclusions Top

The results of the current study indicate that there are considerable differences in the concentrations of zinc and manganese between the two groups and support the fact that there are associations between metal exposures to a pregnant mother and a child during his early years of childhood. This study could be a surpassing contribution by pediatric dentistry to the world of medicine to aid in the diagnosis of ASD, where genomics can sometimes fail to unfold the jumble, specially for the diagnosis of a disorder in a country like India where parents are in the greatest denial for accepting their child's disability.

Future prospects

Based on the results of this study, a primary tooth may serve as a sound exposome for children health research for other medical conditions related to metallic concentrations and their dysregulation.


The authors would like to acknowledge Shriram Institute for Industrial Research, New Delhi.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

   References Top

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]

  [Table 1], [Table 2]


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