As summarized in , the study participants examined were between 1 to 6 years-old with a mean age of 3.5 ± 1.1 years-old. There were more male than female participants examined (male/female ratio = 5:1). Overall, the mean year of birth was 2001 ± 1.1 (range = 1999 to 2003). Among study participants, Caucasians (72%) were more preponderant than minorities (28%). The overall mean CARS score was 41.4 ± 4.1 (range = 37 to 51) for study participants. Further, more of the study participants had experienced a regressive event in development some time following birth (55.6%) than had not (44.4%).
summarizes the correlation between hair toxic metal concentrations and ASD severity as established by the Spearman’s rank correlation test statistic. Increasing hair Hg concentrations were found to significantly correlate with increasing ASD severity (Rho = 0.58, p = 0.013). Further, removing the anomalous highest Hg data point (CARS score = 43, hair Hg level = 4.4 µg of Hg per g of hair) from the sample set being evaluated still revealed a significant correlation (Rho = 0.57, p = 0.018). In contrast, no significant correlations were observed between any of the other hair toxic metal concentrations and ASD severity.
A summary of the correlation1 between hair toxic metals concentrations and ASD severity2.
summarizes the scatter plot distribution of hair Hg concentrations in comparison to CARS scores. The results of the present study suggest a significant correlation between increasing ASD severity and increasing hair Hg concentrations among study participants diagnosed with a moderate to severe ASD. In contrast, the other toxic metals showed no significant association with ASD severity. As a result, the present study helps to provide additional mechanistic support for Hg in the etiology of the clinical severity of an ASD diagnosis. The results observed in the present study are supported by an increasing number of recent critical reviews that provide biological plausibility for Hg exposure playing a significant etiological role in the pathogenesis of ASDs.
A summary of the scatter plot distribution of hair Hg concentrations in comparison to CARS scores. The scatter plot distribution excludes the highest anomalous hair Hg observation.
For example, Garrecht and Austin reported that Hg is recognized as a ubiquitous environmental neurotoxin and that there is mounting evidence linking it to ASDs from methods focusing on biomarkers of Hg damage, measurements of Hg exposure, epidemiological data, and animal studies [28
]. Among the key areas shown to associate Hg exposure with an ASD diagnosis examined by these investigators are: (1) the route and cellular mechanisms of Hg exposure in ASDs; (2) examples of possible genetic variables that are linked to both Hg sensitivity and ASDs; (3) the role Hg may play as an environmental toxin fueling the oxidative stress found in ASDs; (4) the role of mitochondrial dysfunction; and (5) the role of Hg in abnormal neuroexcitory and excitotoxity that may play a role in the immune dysregulation found in those with an ASD diagnosis.
Similarly, investigators described that Hg is widespread and persistent in the environment [29
]. Hg is a ubiquitous source of danger in fish, drugs, fungicides/herbicides, dental fillings, thermometers, vaccines, and many other products/food stuffs. Elevated Hg concentrations may remain in the brain from several years to decades following exposure. This is important because investigators have long recognized that Hg is a bio-accumulative neurodevelopmental poison; it can cause problems in neuronal cell migration and division, and can ultimately cause cell degeneration and death. Case-reports of subjects have described developmental regressions with ASD symptoms following fetal and/or early childhood Hg exposure, and epidemiological studies have linked exposure to Hg with an elevated risk of a subject being diagnosed with an ASD. Immune, sensory, neurological, motor, and behavioral dysfunctions similar to traits defining or associated with ASDs have been reported following Hg intoxication with similarities extending to neuroanatomy, neurotransmitters, and biochemistry.
Finally, Kern et al
. examined the parallels between the effects Hg intoxication on the brain and the brain pathology found in those diagnosed with an ASD [32
]. These investigators found evidence of many parallels between the two, including: (1) microtubule degeneration, specifically large, long-range axon degeneration with subsequent abortive axonal sprouting (short, thin axons); (2) dentritic overgrowth; (3) neuroinflammation; (4) microglial/astrocytic activation; (5) brain immune response activation; (6) elevated glial fibrillary acidic protein; (7) oxidative stress and lipid peroxidation; (8) decreased reduced glutathione levels and elevated oxidized glutathione; (9) mitochondrial dysfunction; (10) disruption in calcium homeostasis and signaling; (11) inhibition of glutamic acid decarboxylase (GAD) activity; (12) disruption of GABAergic and glutamatergic homeostasis; (13) inhibition of IGF-1 and methionine synthase activity; (14) impairment in methylation; (15) vascular endothelial cell dysfunction and pathological changes of the blood vessels; (16) decreased cerebral/cerebellar blood flow; (17) increased amyloid precursor protein; (18) loss of granule and Purkinje neurons in the cerebellum; (19) increased pro-inflammatory cytokine levels in the brain (TNF-α, IFN-γ, IL-1β, IL-8); and (20) aberrant nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB). These investigators also examined the ability of Hg to potentiate and work synergistically with other toxins and pathogens in a way that may contribute to the brain pathology in ASD. These investigators concluded that the evidence suggests that Hg may be either causal or contributory in the brain pathology in ASD, possibly working synergistically with other toxic compounds or pathogens, to produce the brain pathology observed in those diagnosed with an ASD.
3.1. Study Strengths
Key among the strengths of the present study is the correspondence of its results to those reported by previous investigators. This is especially true with respect to a previous study by Elsheshtawy et al
]. These investigators examined a cohort of study subjects diagnosed with an ASD of similar age and sex distribution as those examined in the present study. These investigators observed a significant correlation between increasing ASD severity measured by CARS and increasing Hg in the hair of study subjects, comparable to the results observed in the present study. Just as in the present study, these investigators failed to find a significant relationship between Pb hair concentrations and ASD severity measured by CARS. They even observed, consistent with the present study, a non-significant trend toward a correlation between increasing Pb hair concentrations and decreasing ASD severity measured by CARS.
Another significant strength of the present study is that only study participants with an ASD diagnosis of moderate to severe severity were included. This strength of the present study occurred because previous studies suggested first that there might be differences in toxic metal hair excretion patterns among subjects diagnosed with an ASD in comparison to neurotypical controls [3
]. Secondly, previous studies also suggested that even among subjects diagnosed with an ASD, it had been observed previously observed that study subjects diagnosed moderate to severe ASD severity had significantly different concentrations of toxic metals than study subjects diagnosed with an ASD of mild severity [12
]. As a result, the aforementioned concerns did not need to be considered in the present study because all of the study participants examined were diagnosed with a moderate to severe ASD. In addition, the present study utilized an a priori
definition of moderate to severe ASD severity for CARS scores (37 to 60) derived from previously published investigations, so that no post data collection biases could be introduced by the present investigators in regard to methods of defining ASD severity.
3.2. Study Limitations
In considering the limitations of the present study, the sample of study participants examined was small. As a result, it is possible that with additional numbers of study participants, the study design employed would have had additional statistical power to detect potential associations between hair toxic metal concentrations and ASD severity measured by CARS. Despite this potential limitation, the present study did have adequate statistical power to detect a significant association between Hg hair concentrations and ASD severity. Another potential limitation of the present study was that some of the results observed may have occurred because of a statistical chance or because of some unknown bias in the data examined. This potential limitation should have had a small impact on the observations made because a limited number of statistical tests were conducted on the present data sample, and the size and specificity of the correlations observed argues against the results of this study being consequence of statistical chance or because of some unknown bias. Also, the statistical robustness of the present findings indicates a significant correlation between Hg hair concentrations and ASD severity, even after eliminating the anomalous highest hair Hg value from the results reported. This argues against attributing the results observed to statistical chance or some unknown bias. Further, the consistency of the results in the present study with previous studies additionally argues against statistical chance or unknown bias in the data. Another potential limitation of the present study was that CARS testing was utilized to help confirm an ASD diagnosis and measure overall ASD severity. It is possible that other tests such as Autism Diagnostic Observation Schedule (ADOS) or Autism Diagnostic Interview, Revised (ADI-R) may have yielded different results than those observed in the present study. It would be of use in future studies to employ other ASD tests to determine the comparability of results obtained with those found in the present study. It is also not possible with the prospective cross-sectional ASD cohort design of the present study to assign a causal relationship between Hg exposure and ASD severity or to draw conclusions between Hg concentrations in subjects diagnosed with an ASD in comparison to neurotypical controls. Despite these limitations, the significant relationship between increasing hair Hg concentrations and ASD severity argues that the Hg concentrations did have a significant impact on ASD severity. Therefore, future studies should evaluate the potential etiological basis of Hg exposure with ASD severity in different populations. Finally, the present study also did not allow us to determine the sources of Hg significantly contributing to hair Hg concentrations observed. Interestingly, investigators in a previous small study published on hair Hg concentrations in children diagnosed with an ASD, with similar demographics to the participants examined in the present study, observed that Hg exposure from vaccines containing Thimerosal significantly contributed to hair Hg concentrations, where as a mild non-significant increases of mean hair Hg concentrations were observed in subjects diagnosed with an ASD whose mothers had increasing numbers of dental amalgams during pregnancy or had increasing fish consumption during pregnancy [33
]. As a result, future studies should evaluate the relationship between different sources of Hg exposure and hair Hg concentrations in subjects diagnosed with an ASD.