Accepted Manuscript Title: Autism: A form of Lead and Mercury Toxicity Author: heba atia yassa PII: DOI: Reference:
S1382-6689(14)00241-5 http://dx.doi.org/doi:10.1016/j.etap.2014.10.005 ENVTOX 2100
To appear in:
Environmental Toxicology and Pharmacology
Received date: Revised date: Accepted date:
16-7-2014 7-10-2014 10-10-2014
Please cite this article as: yassa„Autism: A Mercury Toxicity, Environmental Toxicology and http://dx.doi.org/10.1016/j.etap.2014.10.005
form of Lead and Pharmacology (2014),
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Highlights - Autism is a developmental disability characterized by severe, pervasive deficits in
social interaction and communications.
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- Lead and mercury two of the most common heavy metals in the environment. - Lead and Mercury can lead to autistic disorders.
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- Many risk factors contribute to the high level of heavy metals in autistic children.
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- Defect in the metabolism of the heavy metals in autistic children also contribute to the high level of these heavy metals in their body.
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- Chelating agents can be used in the treatment of the autistic disorders.
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Title
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Autism: A Form of Lead and Mercury Toxicity Abstract:
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Autism is a developmental disability characterized by severe deficits in social interaction and communication. The definite cause of autism is still unknown. The aim of this study is to find out the relation between exposure to Lead and/or mercury as heavy metals and autistic symptoms, dealing with the heavy metals with chelating agents can improve the autististic symptoms. Method: blood and hair samples were obtained from 45 children from Upper Egypt with autism between the ages of 2 and 10 years and 45 children served as controls in the same age range, after taken an informed consent and fill a questionnaire to assess the risk factors. The samples were analyzed blindly for lead and mercury by using atomic absorption and ICP -MS. Data from the two groups were compared, then follow up of the autistic children after treatment with chelating agents were done. Results: The results obtained showed significant difference among the two groups, there was high level of mercury and lead among those kids with autism. Significant decline in the blood level of lead and mercury with the use of DMSA as a chelating agent. In addition, there was decline in the autistic symptoms with the decrease in the lead and mercury level in blood.Conclusion: lead and mercury considered as one of the main causes of autism. Environmental exposure as well as defect in heavy metal metabolism is responsible for the high level of heavy metals. Detoxification by chelating agents had great role in improvement of those kids.
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Keywords: Autism; heavy metals; autistic symptoms; chelating agents.
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1.1 Introduction: Autistic disorder (ASD) is a neurodevelopmental syndrome with onset before the age of 36 months. Diagnostic criteria consist of
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impairments in sociality and communication plus repetitive and stereotypic behaviors (Bernard et al., 2001; and Blaurock-Busch et al.,
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2011).
The reported prevalence of ASD has increased in recent decades.
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Information from the Centers for Disease Control and Prevention (CDC) and National Health Interview Survey (NHIS) revealed a nearly fourfold
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increase in parent-reported ASD between the 1997–1999 and 2006–2008 surveillance periods. CDC’s Autism and Developmental Disabilities
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Monitoring (ADDM) Network revealed a 78% increase in ASD prevalence between 2002 and 2008 in USA (Boyle et al., 2011and CDC, 2012). Approximately one in 54 boys and one in 252 girls living in the
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ADDM Network communities were identified as having ASDs. Also, as a
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Comparison of 2008 findings with those for earlier surveillance years
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revealed an increase in estimated ASD prevalence of 23% when the 2008 data were compared with the data for 2006. These data confirm that the estimated prevalence of ASDs identified in the ADDM network surveillance populations continues to increase. The extent to which these increases reflect better case ascertainment as a result of increases in awareness and access to services or true increases in prevalence of ASD symptoms. ASDs continue to be an important public health concern in the United States. Under scoring of these symptoms indicate the need for continued resources to identify potential risk factors and to provide essential supports for persons with ASDs and their families (CDC, 2012; Blumberg et al., 2013).
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According to the US census Bureau, international database (2004) in the USA the extrapolated prevalence is 587,310 autistic persons in 293,655,405-estimated population, while in Egypt 152,234 extrapolated
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prevalence in estimated population 76,117,421 that is considered by the US census bureau as a warning estimation. Although autism occurs in all
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cultures and countries, most of the published research came from Western
countries, little known about its clinical correlates and co-morbidity in
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Middle Eastern and Arab countries (Al-Salehi and Ghaziuddin, 2009 and Hussein et al., 2011) and although autism is not classified as degenerative
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disorders, life expectancy in these groups is somewhat reduced compared to the general population (Tyler et al., 2007; and Levy and Perry, 2011).
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The cause of autism remains vague, it is considered a mutifactorial disorder that influenced by genetic, environmental, and immunological factors as well as increased vulnerability to oxidative stress. No single
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gene has been found to be associated with autism, and involvement of
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multiple genes has been postulated (Keller and Persico, 2003 and Sung et al., 2005). Environmental agents, such as mercury, lead, measles, rubella
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virus, retinoic acid, maternal thalidomide, valproic acid and alcohol use during gestation have been evoked to be implied in the etiology of autism (London, 2000; and Mutter et al., 2005). Some possible sources of heavy metal poisoning include chemical
products, fertilizers, industrial paint, building materials, fish that is high in mercury, silver dental fillings, mercury-containing preservatives (thiomersal) in vaccines, nasal sprays, and many more. Lead may be found in the dirt near roads, leaded gasoline and can still be found in the paint from older houses. Children eating paint chips or those with pica may develop toxic lead levels (Blaurock-Busch et al., 2011). Most children get lead poisoning from living or staying in older homes that 4 Page 4 of 29
have lead paint. Many homes built before 1978 have lead paint on the inside and outside of the building. When old paint cracks and peels, it makes lead dust. Children get lead poisoning from swallowing dust on
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their hands and toys, also can get lead poisoning from the plumbing (CDC, 2013).
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In autistic children, the problem usually appears to, not due to high
exposure, but rather decreased excretion. The half-life of lead, mercury,
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and other toxic metals in the blood is weeks to months, so those metals rapidly leave the blood and accumulate in tissue and/or bone (Adams et Heavy metals considered as reproductive and developmental
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al., 2013).
toxins, they could cause birth defect and fetal developmental damage,
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neurological defects, developmental delay, learning disabilities and behavioral abnormalities (Blaurock-Busch et al., 2011). This paper hypothesized that exposure to variable environmental
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risk factors may affect tissue concentration of lead and / or mercury, thus
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contributing to the genesis of autistic spectrum disorder. This vulnerability may be either prenatal, postnatal, or in cumulative pattern.
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the study proposed to test some potential environmental risk factors and sources of exposure to lead and/or quicksilver, as they are the most usual types of heavy metals that can cause neural defects in children with autism spectrum disorder versus controls. Level of lead and/or mercury were measured in blood and hair samples in both autistic and control groups, in the promise of building connections between environmental exposures to heavy metals and appearence of autistic spectrum disorder. In summation, This paper
hypothesized that decrease exposure and
treatment of these heavy metals toxicity with chelating agents may improve the symptoms of the autistic children and hence improve their quality of life and learning ability. 5 Page 5 of 29
1.2 Subjects and Methods: Forty-five Autistic spectrum disorder (ASD) children (32 boys and 13 girls) between the age of 3 and 10 years (all were diagnosed as autistic
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spectrum by pediatricians and psychologist, they diagnosed by using the DSM - 5 criteria according to Carpenter, (2013). This criteria includes; A.
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Persistent deficits in social communication and social interaction across contexts, not accounted for by general development delays, and manifest
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by 3 of 3 symptoms. B. Restricted, repetitive patterns of behavior, interests, or activities as manifested by at least 2 of 4 symptoms. C.
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Symptoms must be present in early childhood (but may not become fully manifest until social demands exceed limited capacities). D. Symptoms
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together limit and impair everyday functioning.
All children attended special care in center for these cases in Assiut city, Egypt. This work was performed in the period from January 2012 to
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January 2014. This research follows all ethical considerations of human
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research according to (Kapp, 2006), All ethical rules for the Assiut Univesrity, Faculty of Medicine were followed, informed consent from
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parents, consent from the special care center, confidentiality of the results were saved.
Blood and hair samples were collected after taking informed
consent from their parents and filled a structured questionnaire which is used to collect the following information about (age - gender - parents education - parents occupation - age of the mother during pregnancytype of delivery and if there was any complications during pregnancy or not - presence of other cases in the family - environmental exposure to any toxin during pregnancy - type of feeding of the mother during pregnancy - environmental exposure to any toxin for the father - cigarette
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smoking or shisha smoking - child symptoms- symptoms related to vaccination- and pica) (Boseila et al., 2004). A control group was selected, which included 45 age-matched and
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sex-matched children without any psychiatric, medical disorders or developmental delay. These children were from the pediatric department
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diagnosed as normal children without any autistic disorders.
Exclusion criteria include: refusal to participate, physically
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handicapped children and children with progressive neurological disorders and unstable epilepsy. We excluded children who were taking
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regular medications including stimulants, anticonvulsants, atypical antipsychotic drugs, chelating agents, and any history of allergy to
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DMSA (meso 2,3 dimercaptosuccinic acid).
Lead and mercury levels were determined in the blood and hair samples of all children by using atomic absorption and ICP-MS, the
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samples were checked twice for accuracy.
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Autistic children with high levels of lead and/or mercury; after confirmation of the result twice; and fill an informed consent by their
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parents; took treatment in the form of chelating agent, DMSA. Follow up of their lead and/ or mercury levels every two months was done, in addition to follow up of the improvement of their autistic symptoms. Liver and kidney function also with the complete blood picture were done before treatment and every 2 months. 1.2.1 Regimen of the DMSA dose (meso 2,3 dimercaptosuccinic acid): DMSA were given in oral doses of no more than 10 mg/kg/dose, and no more than 30 mg/kg/day with a maximum dose of 500 mg/dose (1500 mg/day maximum). Typical treatment periods are 3 days, followed by 11 days off. This cycle continued for several months until urinary 7 Page 7 of 29
excretion is decreased to near the reference range (Autism research institute, 2005).
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1.2.2 Safety: DMSA slightly increases the excretion of zinc and copper, so those
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elements were monitored, and zinc should be supplemented during therapy .
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Appearance of side effects which is rarely indicated stoppage of treatment or decrease the dose till the body tolerate it which is in the form of skin Serious side effects of
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rash, cough, drowsiness, ear /eye irritation.
DMSA are extremely rare and include allergic reaction; toxic epidermal
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necrolysis (TEN) and erythema multiforme indicate just reducing the dose (Autism research institute, 2005). Supplementaion by multiple vitamins containing zinc were given with the DMSA.
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Kidney and liver fuctions were measured before beginig of the
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treatment, also complete blood picture were done to exclude anaemia.
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120 Enrolled Kids
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40 not qualified diagnosed as neurological and CNS defects 15 refuse to share in the study 55 diagnosed as autistic child
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55 autistic child
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10 child excluded by exclusion criteria
10 Positive for mercury
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17 Positive for lead
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Negative for Lead and / or mercury
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45 autistic child at the beginning of the study
12 Positive for both lead and mercury
25 Underwent DMSA therapy 2 refused
Diagram (1): Study design
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1.2.3 Blood sample collection: Blood samples collected from all participant autistic and control as a whole blood sample in a vacutainer tube contain heparin (Heparinized,
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lead-free, "blue-top" blood collection tubes); blood samples can be stable for 3 days in 4°C (NIOSH Manual of Analytical Methods, 1994).
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Digestion of the sample:
Digestion was done by using acids one ml blood in 3:1:1 (v / v / v)
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HNO3: HClO4: H2SO4. Mix three volumes cons. HNO3 with one volume conc. HClO4 and one volume conc. H2SO4. Hot plate temperature was
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increased to 250 °C and continued until volume is 1 ml for blood remained (2 to 3 h) (NIOSH Manual of Analytical Method, 1994).
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1.2.4 Equipment and quality control:
Atomic absorption and the sample repeated for assurance by ICP MS (Inductively Coupled Plasma Mass Spectrometry (ICP-MS). The
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blank used was an acid blank and 10 μg/ml multielement solutions are
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used (NIOSH Manual of Analytical Method, 1994). 1.2.5 Hair specimen collection: Hair samples collected from all participants diseased and control
by single cutting from the occipital region. The samples cut to lengths of about 1.5–2 cm using clean stainless steel scissors. A minimum of 5–10 mg of hair was required for the hair analysis assay. The samples placed in a sealed plastic bag (El-baz et al., 2010). Washing procedure: Accurate weights of each sample prepared were obtained using microbalance. Each hair sample was washed sequentially with acetone, deionized water, and acetone using three successive portions, these can
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adequately wash any external contamination. The samples were then dried in an oven at 105°C (Razagui and Haswell, 1997). Digestion of hair samples:
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Digestion of hair was performed according to Razagui and Haswell, (1997
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Equipment:
The ICP-MS instrument used was a Fisons Instruments Plasma
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Quad 2 Plus. Optimization of the instrument's operating parameters was effected by means of a tuning solution containing the element beryllium,
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yttrium, indium, lanthanum, bismuth, and uranium at 10 ~g/L in a matrix of 20% nitric acid (v/v). The blank used was an acid blank and 10 μg/ml
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1.2.6 Statistical Analysis:
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multielement solutions are used.
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The paired T test was used to compare the autistic children and the normal ones. Statistical analysis was done by using SPSS version 17.
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Some of the data for toxic levels of lead and mercury in blood and hair were not normally distributed. A non-parametric Wilcoxon test was used instead of t test in these cases. 1.3 Results:
Table (1) shows the age and sex distribution of the autistic children
and the control group, the autistic group was 45 children (32 males, 71.1% and 13 females, 28.9%), while the control group was 45 children (31 males, 68.9% and 14 females, 31.1%). Table (2) shows a developmental comparison between the milestones/ months in both autistic and control groups, no significance difference between both groups except in talking which appeared to be significantly delayed in 11 Page 11 of 29
autistic children (P value = 0.001), ability to speak (one or two words) in autistic children delayed up to 25.5± 4.1 months while in control group ability to speak one or two words began 11.3± 1.02 months. The control studied autistic children can not perform sentences.
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children could perform sentences after 12.4 ± 2.04 months while all
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Risk factors in autistic and control groups presented in table (3)
and table (4). Table (3) shows that the mother's age had no significance
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in both groups. Hazards during pregnancy, type of delivery, complications during delivery, exposure to smoking either active or
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passive, exposure to pesticide, number of teeth filled with amalgam and seafood intake during pregnancy were not related to appearance of the
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autistic child in the family. Whereas exposure to paint during pregnancy, living in an old house before 1978, using henna during pregnancy, and type of water used during pregnancy had significant value if compared
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with the control group (P value was as follows 0.001, 0.05, 0.005, 0.001
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respectively). Table (4) shows the risk factors related to the children themselves. Many factors were studied and had non-significant difference
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as the number of the child in the family, the presence of another child with autism in the family, and presence of brothers or sisters with the same symptoms. Otherwise, behavioral changes after vaccination had significant value if compared with the control group (P value was less than 0.001). Changes of behavior after vaccination was mostly after the vaccine of MMR (measels - mumps and rubella) at the age of 18 months and the form of behavioral changes were delayed in acquiring new words, irritable movements in the form of rapid hand movements and decrease attention. Lead and mercury were measured in total blood and hair, and their values before treatment were given in table (5) and after treatment with 12 Page 12 of 29
DMSA in table (7). There was significant difference between the autistic and control group in both levels of lead and mercury in total blood and hair. Table (7) and figure (1) show the difference in lead and mercury
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level in total blood before treatment, after 2 months, after 4 months, 6 months and 8 months, there was a significant difference in the level of
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both lead and mercury.
Follow up of these autistic children during treatment, especially in
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the milestones (ability to speak and perform sentences) reveal that improvement of the level of speech with the decrease of the lead and
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mercury level, in the form of increase number of words, and perform understandable sentences.
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Table (6) shows the percentage of autistic children with high levels of lead and mercury (37.8% and 22.2% respectively). In addition to the
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percentage of those took treatment of heavy metals in the form of DMSA.
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1.4 Discussion: This study was designed to determine the risk factors that may
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directly or indirectly lead to autism, and to study the effects of heavy metals (lead and/or mercury) on autism and its severity in children. In
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addition, examination of the effects of chelating agents (DMSA) as a treatment for symptoms in such children was done. The study revealed
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that some factors related to lead an mercury exposure, had significant effects on autism in children; these included exposure to paint during
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pregnancy, living in an old house, use of hair dyes or henna during pregnancy, use of tape water as the main source of drinking water during
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pregnancy, and behavioral changes after vaccination. Other risk factors studied, including the age of the mother, paternal age, type of delivery, risks during pregnancy, complications during delivery, seafood intake
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during pregnancy, passive or active smoking by the mother during
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pregnancy, exposure to pesticides, and tooth amalgam had no significant
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effects on whether an autistic child was present in the family. Many studies have estimated the risk factors that may lead to autism in children, but in this study, we measured the most significant heavy metals hypothesized to be a cause of autism, lead and/ or mercury, and found that there is a significant relationship between these two heavy metals and the appearance of autism. In their study of the autism spectrum, Windham et al. (2006) suggested that there is a potential association between autism and metal concentration. Lyall et al. (2014), Rossignol et al. (2014), Blaurock-Busch et al. (2011), Blaurock- Busch et al., (2012), all showed that heavy metals, are common environmental factors, that lead to autism. In addition, Halsey and Hyman (2001) found that the vaccine of MMR (measles, mumps and rubella) contribute to the appearance of autistic 14 Page 14 of 29
child in the family. Demicheli et al., (2012) in their study about the relation between vaccination and autism, they conclude that adverse after MMR cannot be excluded. Some studies did not support the relation
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between the vaccination and autism as Taylor et al., (1999); and(Destefano and Chen, (2001). This study found a positive correlation
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between three factors, presence of positive history about exposure to lead and/ or mercury during the prenatal period or early postnatal period,
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elevation of the level of lead and/ or mercury in hair and blood in those children, and the improvement of the symptoms after treatment with
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chelating agents.
The study did not find any association between mother's age and
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the appearance of autistic symptoms. Parner et al., (2012) in their study found that the prevalence of autism in families with advanced aging parents were the same as families with young age parents. Larsson et al.,
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(2005) in their study about the risk factors of autism found that there was
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no statistically significant association between risk of autism and parity, parental age or socioeconomic status, these factors if present seem to act
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independently. In addition, Tsuchiya et al., (2008) found that maternal age effect may be demonstrable in some cases, but not all, and may be related to societal and cultural factors. This study excludes maternal age, the order of the child in the family, and the presence of other siblings or relatives with autistic manifestation as risk factors for autism. This study revealed that lead and/ or mercury exposure in either
prenatal or postnatal period had a positive correlation with the presence of an autistic child in the family. In addition, treatment of those kids with one of the chelating agents had a positive effect on decreasing the level of heavy metal and decreased the symptoms of autism in those kids. Against this, is the presence of other children in the same environment and same 15 Page 15 of 29
family with no autistic symptoms, this may be explained by the presence of some defects in methylation of these toxins so they retain these toxins in their bodies for long times, which can lead to the toxic effects and
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appearance of the autistic symptoms. James et al., (2004), Priya and Geetha, (2011) and Hartzell and Seneff (2012) they found that there was
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decrease in the capacity of methylation contributes to the development of the autism manifestations. Walsh et al., (2001) also, found that in their
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study there was metallothionein disorder in those children with autism and suggested that it is an inborn error of metallothionein function; this
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can explain the high levels of heavy metals in those children. In addition, Obrenovich et al., (2011) reported that these children with autistic
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manifestation revealed evidence of abnormal thiol metabolism, as well as significant alteration in deposition of several heavy metals. They suspected that these kids accumulate heavy metals in their bodies due to
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trouble in excreting thiol toxic heavy metals. DMSA were used in this study as a chelating agent, and found to be
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effective in decreasing the level of lead and mercury. Follow up of these kids showed improvment in word and sentences formation and also improvment in the unintentional movement. Adams, et al. (2009A) and Adams, et al. (2009B) agree with this result as they gave the same dose of DMSA. they found that DMSA therapy seems to be reasonably safe, effective in removing several toxic metals (especially lead), dramatically effective in normalizing RBC glutathione, and effective in normalizing platelet counts. Only 1 round (3 days) was sufficient to improve glutathione and platelets. Additional rounds increased excretion of toxic metals, and also improve the behavioral symptoms of autism. 1.5 Conclusion and recommendations: 16 Page 16 of 29
This study presented the hypothesis that there is a strong link between heavy metal toxicity, especially lead and mercury with autism spectrum disorder. Accumulation of these heavy metals in those children
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was the responsible for the appearance of the autistic symptoms. The current results revealed that accumulation of heavy metals may be due to
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many risk factors either during the neonatal period or postnatal period,
also accumulation of these heavy metals is due to defects in metabolism
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of those children, and so use of chelating agents can improve the autistic symptoms. The outcomes of the study support the conjecture that heavy
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metals have a cracking part in development of autistic symptoms, also confirm the use of chelating agents in improving the symptoms of those
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youngsters. Further studies to cover more numbers of autistic children are required for more verification of this theory. We strongly recommend
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accumulation in the body.
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avoidance of many risk factors that increase the heavy metal
1.6 Acknowledgments
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Many grateful to center of "Kaian" in Assiut and Sohag, which
helped us in performing this study with all endeavors by their pediatrician, psychologist and the audio and conversational specialist. Medical students in the final year of the faculty, especially Mai Sallah, made the day of the autistic children better with toys and sweets. 1.7 References:
1. Adams, J.; Bara, M.; Geis, E.; Mitchell, J.; Ingram, J.; Hensley, A.; Zappia, I.; Newmark, S.; Gehn, E.; Rubin, R.; Mitchell, K.; Bradstreet, J.; and El-Dahr, J. (2009): "Safety and efficacy of oral DMSA therapy for children with autism spectrum disorders: Part A - Medical results". BMC Clinical Pharmacology, 9:16 doi:10.1186/1472-6904-9-16. 17 Page 17 of 29
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2. Adams, J.; Bara, M.; Geis, E.; Mitchell, J.; Ingram, J.; Hensley, A.; Zappia, I.; Newmark, S.; Gehn, E.; Rubin, R.; Mitchell, K.; Bradstreet, J.; and El-Dahr, J. (2009): "Safety and efficacy of oral DMSA therapy for children with autism spectrum disorders: Part B - Behavioral results". BMC Clinical Pharmacology, BMC Clinical Pharmacology, 9:17 doi:10.1186/1472-6904-9-17
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3. Adams, J.; Audhya, T.; McDonough-Means, S.; Rubin, R.; Quig, D.; Geis, E.; Gehn, E.; Loresto, M.; Mitchell, J.; Atwood, S.; Barnhouse, S.; and Lee, W. (2013): "Toxicological Status of Children with Autism vs. Neurotypical Children and the Association with Autism Severity". Biol Trace Elem Res;151(2):171-180
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16.DeStefano, F ; and Chen, R.T. (2001): "Autism and measlesmumps-rubella vaccination: controversy laid to rest?". CNS Drugs.; 15(11):831-837. 17.El-baz, F.; Elhossiny, R.; Elsayed, A.; and Gaber, G. (2010): "Hair mercury measurement in Egyptian autistic children". The Egyptian Journal of Medical Human Genetics;11:135–141. 19 Page 19 of 29
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21.James, S.; Cutler, P.; Melny, k. S.; Jernigan, S.; Janak, L.; Gaylor, D.; and Neubrander, J. (2004): "Metabolic biomarkers of increased oxidative stress and impaired methylation capacity in children with autism". Am J Clin Nutr; 80:1611–1617.
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22. Kapp, M. (2006): "Ethical and legal issues in research involving human subjects: do you want a piece of me?". J Clin Pathol.; 59(4): 335–339. 23.Keller, F.; and Persico, A. M. (2003): "The neurobiological context of autism". Mol Neurobiol.; 28(1):1-22. 24.Larsson, H.; Eaton, W.; Madsen, K.; Vestergaard, M.; Olesen, A.; Agerbo, E.; Schendel, D.; Thorsen, P.; and Mortensen, P. (2005): "Risk Factors for Autism: Perinatal Factors, Parental Psychiatric History, and Socioeconomic Status". American Journal of Epidemiology, 161(10): 916-925. 25.Levy, A.; and Perry, A. (2011): "Outcomes in adolescents and adults with autism: A review of the literature". Research in Autism Spectrum Disorders; 5:1271–1282
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26.London, E. (2000): "The environment as an etiologic factor in autism: a new direction for research". Environ. Health Perspect.;108: 401–404.
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27.Lyall, K.; Schmidt, R.J.; and Hertz-Picciotto, I. (2014): "Maternal life style and environmental risk factors for autism spectrum disorders". Int J Epidemiol; 43(2):443-464.
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28.Mutter, J.; Naumann, J.; Schneider, R.; Walach, H.; and Haley, B. (2005): "Mercury and autism: accelerating evidence?". Neuro Endocrinol Lett.; 26(5): 439-446.
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29.NIOSH Manual of Analytical Methods (1994): Elements in blood or tissue. Form 8005.
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30.Obrenovich, M.; Shamberger, R.; and Lonsdale, D. (2011): "Altered Heavy Metals and Transketolase Found in Autistic Spectrum Disorder". Biol Trace Elem Res 144 ( 1-3): 475-486.
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31.Parner, E.; Cohen, S.; Lauritsen, M.; Jorgensen, M.; Schieve, L.; Allsopp, M.; and Obel, C. (2012): "Parental Age and Autism Spectrum Disorders". Annals of Epidemiology; 22(3): 143-150. 32.- Priya, L.; and Geetha, A. (2011): "Level of trace elements (copper, zinc, magnesium and selenium) and toxic elements (lead and mercury) in the hair and nail of children with autism". Biol Trace Elem Res.; 142(2):148-158. 33.Razagui, I.; and Haswell, S. (1997): "The Determination of Mercury and Selenium in Maternal and Neonatal Scalp Hair by Inductively Coupled Plasma-Mass Spectrometry". Journal of Analytical Toxicology, 21: 149-153. 34.Rossignol, D.A.; Genuis, S.J.; and Frye, R.E. (2014): "Environmental toxicants and autism spectrum disorders: a systematic review". Transl Psychiatry; 11:4-e360.
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.
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35.-Sung, Y.J.; Dawson, G.; Munson, J.; Estes, A.; Schellenberg, G.D.; and Wijsman, E.M. (2005): "Genetic investigation of quantitative traits related to autism: use of multivariate polygenic models with ascertainment adjustment". Am J Hum Genet.; 76 (1): 68-81.
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36.Taylor, B ; Miller, E ; Farrington, C.P.; Petropoulos, M.C.; Favot-Mayaud, I.; Li, J.; and Waight, P. A. (1999): "Autism and measles, mumps, and rubella vaccine: no epidemiological evidence for a causal association". Lancet;12: 353(9169):20262069.
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37.Tsuchiya, K.; Matsumoto, M.; Miyachi, T.; Tsujii, M.; Nakamura, K.; Takagai, S.; Kawai, M.; Yagi, A.; Iwaki, K.; Suda, S.; Sugihara, G.; Iwata, Y.; Matsuzaki, H.; Sekine, Y.; Suzuki, K.; Sugiyama, T.; Mori, N.; and Takei, N. (2008): "Paternal age at birth and high-functioning autistic-spectrum disorder in offspring". The British Journal of Psychiatry; 193: 316– 321.
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38.Tyler, F.; Smith, L. K.; McGrother, C. W.; and Taub, N. A. (2007): "The impact of physical, intellectual and social impairments on survival in adults with intellectual disability: A population-based register study". Journal of Applied Research in Intellectual Disabilities, 20: 360–367. 39.-US census Bureau, international database RightDiagnosis.com.htm accessed in March 2014.
(2004):
40.Walsh, W.; Usman, A.; and Tarpey, J. (2001): "Disordered Metal Metabolism in a Large Autism Population". American Psychiatric Association Annual Meeting. New Orleans. http://www.safeminds.org/research/library/20010501.pdf
.
.
.
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41.Windham, G.C ; Zhang, L ; Gunier, R ; Croen, L.A , and Grether, J.K. (2006): "Autism spectrum disorders in relation to
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distribution of hazardous air pollutants in the san francisco bay area". Environ Health Perspect; 114(9):1438-1444.
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Table (1): Age distribution of both autistic and control children:
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2˂4 4˂6 6˂8 8 ˂ 10 Total
Autistic Children Control Total Male Female Total Male Female Total Male Female Total 16 4 20 14 5 19 30 9 39 8 2 10 8 4 12 16 6 22 3 1 4 5 1 6 8 2 10 5 6 11 4 4 8 9 10 19 32 13 45 31 14 45 63 27 90
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Age/ Sex
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Table (2): Comparison of the developmental milestones/months in autistic and control children (T test): Developmental Milestones / month Sitting
6.2 ± 0.51
6.1 ± 0.22
Crawling
9.8 ± 0.18
9.6 ± 0.41
Teething
7.4 ± 0.32
6.9 ± 0.12
Walking
14.7 ± 0.59
13.1 ± 0.87
Ability to speak (one or two words)
25.5 ± 4.1
11.3 ± 1.02
0.001**
Can not form phrase
12.4 ± 2.04
0.001**
Control
P value
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0.43 0.51
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0.41
0.38
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Ability to speak (sentences)
Autistic Children
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Table (3): Risk factors in autistic and control children (T test and compare means): factors not related to child
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Hazards during pregnancy
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Type of delivery
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Percent of sea food taken/month during pregnance
Painting inside house during pregnancy Passive smoking pregnancy Active smoking pregnancy Exposure to pesticide
during during
Living in old house before 1978 Number of filling teeth with amalgam Using hair pregnancy
dye
during
Using henna during pregnancy
10
20 10 10 31 14 34 11 0 45 27 18 19 26 19 23 3 20 22 3 20
20 10 5 9 36 32 13 0 45 23 22 10 35 15 21 9 27 15 3 17
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Mother level of education
Complications during delivery
5
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Paternal age
18 - ˂ 23 23 - ˂ 28 28 - ˂ 33 33 - ˂ 38 38- ˂ 43 25 - ˂ 30 30 - ˂ 35 35 - ˂ 40 40 - ˂ 45 45 - ˂ 50 None Secondary school High education Master or doctoral degree None Renal troubles Pre eclampsia Drug treatment Normal labor Cesarean None Obstruction Oxygen deficiency for fetus None
Control
10% 20% 30% or more Yes No Yes No Yes No Yes No Yes No None 1-2 More than 2 None 1-3 More than 3 None
7 17 12 7 2 2 21 15 3 4 0 2 37 8 31 2 5 7 15 30 33 7 5
P value
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Mother age in pregnancy
Autistic Children 5 20 15 4 1 2 20 17 4 2 0 1 34 10 30 5 4 6 25 20 35 6 4
0.47
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Mother risk factors
0.54
0.58
0.55
0.34
0.51
0.24
0.001** 0.24 0.78 0.22 0.05* 0.51
0.057
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28 0 15 28
2 0
1 1
0.001**
0.001**
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Type of drinking water during pregnancy
20 5 24 19
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1-3 More than 3 Tap water Filtered water (free of metals) Bottled water Boiled water
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Table (4): Risk factors autistic and control children (T test) (factors related to the child):
Vccination (MMR) In the form of changes of behavior
Yes
35
0.81
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Presence of autistic child in relatives
10 13 11 8 3 10 35 7 38
No
P value
0.74
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Presence of other autistic child in same family
1st 2nd 3rd 4th 5th Yes No Yes No
Control
0.76
0
10
45
0.001**
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Order of the autistic child in the family
Autistic Children 22 6 7 9 1 12 33 9 36
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Mother risk factors
Table (5): Comparison of blood and hair levels of lead and mercury in autistic and control children before treatment (T test):
Autistic Control (Mean± child (Mean± SD) SD) 55.24±10.02 9.75±0.58
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Blood level in (mcg/dl)
P value
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Lead
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Heavy metal
Mercury
4.02±0.54
0.00±0.02
Hair level in (mcg/gr)
Autistic child (Mean± SD)
Control (Mean± SD)
P value
0.001**
60.045±9.75
12.03±0.45 0.001**
0.001**
5.21±0.08
0.11±0.05
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0.001**
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Table (6): Percentage of autististic children with high level of lead and/or mercury, and percentage of children took DMSA: Percentage %
No. of autistic children agree to be treated with DMSA
Positive Lead
17
37.8
16
Positive Mercury
10
22.2
10
Positive for both metals
12
26.7
11
Negative for metals
18
40
Total
45
100
Percentage %
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No. of autistic child
35.5
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22.2
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0
26
24.4 0 57.7
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Heavy Metal
Before treatment (Mean± SD)
Lead
55.24±10.02 4.02±0.54
2 months after treatment (Mean± SD)
4 months after treatment (Mean± SD)
6 months after treatment (Mean± SD)
43.15± 7.02
30.24±7.14
18.04± 5.07
10.04±1.01
0.001**
3.01±0.11
1.41± 0.9
0.9±0.12
0.00±0.04
0.001**
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Heavy metal
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Table (7): Blood Lead and mercury level in Autistic children before and after treatment with DMSA (One Way Anova):
Mercury
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8 months after treatment (Mean± SD)
P value
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S1382-6689(14)00241-5 http://dx.doi.org/doi:10.1016/j.etap.2014.10.005 ENVTOX 2100
To appear in:
Environmental Toxicology and Pharmacology
Received date: Revised date: Accepted date:
16-7-2014 7-10-2014 10-10-2014
Please cite this article as: yassa„Autism: A Mercury Toxicity, Environmental Toxicology and http://dx.doi.org/10.1016/j.etap.2014.10.005
form of Lead and Pharmacology (2014),
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights - Autism is a developmental disability characterized by severe, pervasive deficits in
social interaction and communications.
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- Lead and mercury two of the most common heavy metals in the environment. - Lead and Mercury can lead to autistic disorders.
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- Many risk factors contribute to the high level of heavy metals in autistic children.
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- Defect in the metabolism of the heavy metals in autistic children also contribute to the high level of these heavy metals in their body.
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- Chelating agents can be used in the treatment of the autistic disorders.
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Title
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Autism: A Form of Lead and Mercury Toxicity Abstract:
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Autism is a developmental disability characterized by severe deficits in social interaction and communication. The definite cause of autism is still unknown. The aim of this study is to find out the relation between exposure to Lead and/or mercury as heavy metals and autistic symptoms, dealing with the heavy metals with chelating agents can improve the autististic symptoms. Method: blood and hair samples were obtained from 45 children from Upper Egypt with autism between the ages of 2 and 10 years and 45 children served as controls in the same age range, after taken an informed consent and fill a questionnaire to assess the risk factors. The samples were analyzed blindly for lead and mercury by using atomic absorption and ICP -MS. Data from the two groups were compared, then follow up of the autistic children after treatment with chelating agents were done. Results: The results obtained showed significant difference among the two groups, there was high level of mercury and lead among those kids with autism. Significant decline in the blood level of lead and mercury with the use of DMSA as a chelating agent. In addition, there was decline in the autistic symptoms with the decrease in the lead and mercury level in blood.Conclusion: lead and mercury considered as one of the main causes of autism. Environmental exposure as well as defect in heavy metal metabolism is responsible for the high level of heavy metals. Detoxification by chelating agents had great role in improvement of those kids.
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Keywords: Autism; heavy metals; autistic symptoms; chelating agents.
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1.1 Introduction: Autistic disorder (ASD) is a neurodevelopmental syndrome with onset before the age of 36 months. Diagnostic criteria consist of
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impairments in sociality and communication plus repetitive and stereotypic behaviors (Bernard et al., 2001; and Blaurock-Busch et al.,
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2011).
The reported prevalence of ASD has increased in recent decades.
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Information from the Centers for Disease Control and Prevention (CDC) and National Health Interview Survey (NHIS) revealed a nearly fourfold
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increase in parent-reported ASD between the 1997–1999 and 2006–2008 surveillance periods. CDC’s Autism and Developmental Disabilities
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Monitoring (ADDM) Network revealed a 78% increase in ASD prevalence between 2002 and 2008 in USA (Boyle et al., 2011and CDC, 2012). Approximately one in 54 boys and one in 252 girls living in the
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ADDM Network communities were identified as having ASDs. Also, as a
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Comparison of 2008 findings with those for earlier surveillance years
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revealed an increase in estimated ASD prevalence of 23% when the 2008 data were compared with the data for 2006. These data confirm that the estimated prevalence of ASDs identified in the ADDM network surveillance populations continues to increase. The extent to which these increases reflect better case ascertainment as a result of increases in awareness and access to services or true increases in prevalence of ASD symptoms. ASDs continue to be an important public health concern in the United States. Under scoring of these symptoms indicate the need for continued resources to identify potential risk factors and to provide essential supports for persons with ASDs and their families (CDC, 2012; Blumberg et al., 2013).
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According to the US census Bureau, international database (2004) in the USA the extrapolated prevalence is 587,310 autistic persons in 293,655,405-estimated population, while in Egypt 152,234 extrapolated
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prevalence in estimated population 76,117,421 that is considered by the US census bureau as a warning estimation. Although autism occurs in all
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cultures and countries, most of the published research came from Western
countries, little known about its clinical correlates and co-morbidity in
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Middle Eastern and Arab countries (Al-Salehi and Ghaziuddin, 2009 and Hussein et al., 2011) and although autism is not classified as degenerative
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disorders, life expectancy in these groups is somewhat reduced compared to the general population (Tyler et al., 2007; and Levy and Perry, 2011).
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The cause of autism remains vague, it is considered a mutifactorial disorder that influenced by genetic, environmental, and immunological factors as well as increased vulnerability to oxidative stress. No single
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gene has been found to be associated with autism, and involvement of
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multiple genes has been postulated (Keller and Persico, 2003 and Sung et al., 2005). Environmental agents, such as mercury, lead, measles, rubella
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virus, retinoic acid, maternal thalidomide, valproic acid and alcohol use during gestation have been evoked to be implied in the etiology of autism (London, 2000; and Mutter et al., 2005). Some possible sources of heavy metal poisoning include chemical
products, fertilizers, industrial paint, building materials, fish that is high in mercury, silver dental fillings, mercury-containing preservatives (thiomersal) in vaccines, nasal sprays, and many more. Lead may be found in the dirt near roads, leaded gasoline and can still be found in the paint from older houses. Children eating paint chips or those with pica may develop toxic lead levels (Blaurock-Busch et al., 2011). Most children get lead poisoning from living or staying in older homes that 4 Page 4 of 29
have lead paint. Many homes built before 1978 have lead paint on the inside and outside of the building. When old paint cracks and peels, it makes lead dust. Children get lead poisoning from swallowing dust on
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their hands and toys, also can get lead poisoning from the plumbing (CDC, 2013).
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In autistic children, the problem usually appears to, not due to high
exposure, but rather decreased excretion. The half-life of lead, mercury,
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and other toxic metals in the blood is weeks to months, so those metals rapidly leave the blood and accumulate in tissue and/or bone (Adams et Heavy metals considered as reproductive and developmental
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al., 2013).
toxins, they could cause birth defect and fetal developmental damage,
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neurological defects, developmental delay, learning disabilities and behavioral abnormalities (Blaurock-Busch et al., 2011). This paper hypothesized that exposure to variable environmental
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risk factors may affect tissue concentration of lead and / or mercury, thus
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contributing to the genesis of autistic spectrum disorder. This vulnerability may be either prenatal, postnatal, or in cumulative pattern.
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the study proposed to test some potential environmental risk factors and sources of exposure to lead and/or quicksilver, as they are the most usual types of heavy metals that can cause neural defects in children with autism spectrum disorder versus controls. Level of lead and/or mercury were measured in blood and hair samples in both autistic and control groups, in the promise of building connections between environmental exposures to heavy metals and appearence of autistic spectrum disorder. In summation, This paper
hypothesized that decrease exposure and
treatment of these heavy metals toxicity with chelating agents may improve the symptoms of the autistic children and hence improve their quality of life and learning ability. 5 Page 5 of 29
1.2 Subjects and Methods: Forty-five Autistic spectrum disorder (ASD) children (32 boys and 13 girls) between the age of 3 and 10 years (all were diagnosed as autistic
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spectrum by pediatricians and psychologist, they diagnosed by using the DSM - 5 criteria according to Carpenter, (2013). This criteria includes; A.
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Persistent deficits in social communication and social interaction across contexts, not accounted for by general development delays, and manifest
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by 3 of 3 symptoms. B. Restricted, repetitive patterns of behavior, interests, or activities as manifested by at least 2 of 4 symptoms. C.
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Symptoms must be present in early childhood (but may not become fully manifest until social demands exceed limited capacities). D. Symptoms
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together limit and impair everyday functioning.
All children attended special care in center for these cases in Assiut city, Egypt. This work was performed in the period from January 2012 to
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January 2014. This research follows all ethical considerations of human
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research according to (Kapp, 2006), All ethical rules for the Assiut Univesrity, Faculty of Medicine were followed, informed consent from
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parents, consent from the special care center, confidentiality of the results were saved.
Blood and hair samples were collected after taking informed
consent from their parents and filled a structured questionnaire which is used to collect the following information about (age - gender - parents education - parents occupation - age of the mother during pregnancytype of delivery and if there was any complications during pregnancy or not - presence of other cases in the family - environmental exposure to any toxin during pregnancy - type of feeding of the mother during pregnancy - environmental exposure to any toxin for the father - cigarette
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smoking or shisha smoking - child symptoms- symptoms related to vaccination- and pica) (Boseila et al., 2004). A control group was selected, which included 45 age-matched and
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sex-matched children without any psychiatric, medical disorders or developmental delay. These children were from the pediatric department
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diagnosed as normal children without any autistic disorders.
Exclusion criteria include: refusal to participate, physically
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handicapped children and children with progressive neurological disorders and unstable epilepsy. We excluded children who were taking
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regular medications including stimulants, anticonvulsants, atypical antipsychotic drugs, chelating agents, and any history of allergy to
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DMSA (meso 2,3 dimercaptosuccinic acid).
Lead and mercury levels were determined in the blood and hair samples of all children by using atomic absorption and ICP-MS, the
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samples were checked twice for accuracy.
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Autistic children with high levels of lead and/or mercury; after confirmation of the result twice; and fill an informed consent by their
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parents; took treatment in the form of chelating agent, DMSA. Follow up of their lead and/ or mercury levels every two months was done, in addition to follow up of the improvement of their autistic symptoms. Liver and kidney function also with the complete blood picture were done before treatment and every 2 months. 1.2.1 Regimen of the DMSA dose (meso 2,3 dimercaptosuccinic acid): DMSA were given in oral doses of no more than 10 mg/kg/dose, and no more than 30 mg/kg/day with a maximum dose of 500 mg/dose (1500 mg/day maximum). Typical treatment periods are 3 days, followed by 11 days off. This cycle continued for several months until urinary 7 Page 7 of 29
excretion is decreased to near the reference range (Autism research institute, 2005).
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1.2.2 Safety: DMSA slightly increases the excretion of zinc and copper, so those
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elements were monitored, and zinc should be supplemented during therapy .
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Appearance of side effects which is rarely indicated stoppage of treatment or decrease the dose till the body tolerate it which is in the form of skin Serious side effects of
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rash, cough, drowsiness, ear /eye irritation.
DMSA are extremely rare and include allergic reaction; toxic epidermal
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necrolysis (TEN) and erythema multiforme indicate just reducing the dose (Autism research institute, 2005). Supplementaion by multiple vitamins containing zinc were given with the DMSA.
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Kidney and liver fuctions were measured before beginig of the
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treatment, also complete blood picture were done to exclude anaemia.
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120 Enrolled Kids
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40 not qualified diagnosed as neurological and CNS defects 15 refuse to share in the study 55 diagnosed as autistic child
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55 autistic child
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10 child excluded by exclusion criteria
10 Positive for mercury
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17 Positive for lead
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Negative for Lead and / or mercury
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45 autistic child at the beginning of the study
12 Positive for both lead and mercury
25 Underwent DMSA therapy 2 refused
Diagram (1): Study design
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1.2.3 Blood sample collection: Blood samples collected from all participant autistic and control as a whole blood sample in a vacutainer tube contain heparin (Heparinized,
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lead-free, "blue-top" blood collection tubes); blood samples can be stable for 3 days in 4°C (NIOSH Manual of Analytical Methods, 1994).
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Digestion of the sample:
Digestion was done by using acids one ml blood in 3:1:1 (v / v / v)
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HNO3: HClO4: H2SO4. Mix three volumes cons. HNO3 with one volume conc. HClO4 and one volume conc. H2SO4. Hot plate temperature was
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increased to 250 °C and continued until volume is 1 ml for blood remained (2 to 3 h) (NIOSH Manual of Analytical Method, 1994).
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1.2.4 Equipment and quality control:
Atomic absorption and the sample repeated for assurance by ICP MS (Inductively Coupled Plasma Mass Spectrometry (ICP-MS). The
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blank used was an acid blank and 10 μg/ml multielement solutions are
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used (NIOSH Manual of Analytical Method, 1994). 1.2.5 Hair specimen collection: Hair samples collected from all participants diseased and control
by single cutting from the occipital region. The samples cut to lengths of about 1.5–2 cm using clean stainless steel scissors. A minimum of 5–10 mg of hair was required for the hair analysis assay. The samples placed in a sealed plastic bag (El-baz et al., 2010). Washing procedure: Accurate weights of each sample prepared were obtained using microbalance. Each hair sample was washed sequentially with acetone, deionized water, and acetone using three successive portions, these can
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adequately wash any external contamination. The samples were then dried in an oven at 105°C (Razagui and Haswell, 1997). Digestion of hair samples:
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Digestion of hair was performed according to Razagui and Haswell, (1997
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Equipment:
The ICP-MS instrument used was a Fisons Instruments Plasma
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Quad 2 Plus. Optimization of the instrument's operating parameters was effected by means of a tuning solution containing the element beryllium,
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yttrium, indium, lanthanum, bismuth, and uranium at 10 ~g/L in a matrix of 20% nitric acid (v/v). The blank used was an acid blank and 10 μg/ml
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1.2.6 Statistical Analysis:
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multielement solutions are used.
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The paired T test was used to compare the autistic children and the normal ones. Statistical analysis was done by using SPSS version 17.
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Some of the data for toxic levels of lead and mercury in blood and hair were not normally distributed. A non-parametric Wilcoxon test was used instead of t test in these cases. 1.3 Results:
Table (1) shows the age and sex distribution of the autistic children
and the control group, the autistic group was 45 children (32 males, 71.1% and 13 females, 28.9%), while the control group was 45 children (31 males, 68.9% and 14 females, 31.1%). Table (2) shows a developmental comparison between the milestones/ months in both autistic and control groups, no significance difference between both groups except in talking which appeared to be significantly delayed in 11 Page 11 of 29
autistic children (P value = 0.001), ability to speak (one or two words) in autistic children delayed up to 25.5± 4.1 months while in control group ability to speak one or two words began 11.3± 1.02 months. The control studied autistic children can not perform sentences.
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children could perform sentences after 12.4 ± 2.04 months while all
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Risk factors in autistic and control groups presented in table (3)
and table (4). Table (3) shows that the mother's age had no significance
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in both groups. Hazards during pregnancy, type of delivery, complications during delivery, exposure to smoking either active or
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passive, exposure to pesticide, number of teeth filled with amalgam and seafood intake during pregnancy were not related to appearance of the
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autistic child in the family. Whereas exposure to paint during pregnancy, living in an old house before 1978, using henna during pregnancy, and type of water used during pregnancy had significant value if compared
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with the control group (P value was as follows 0.001, 0.05, 0.005, 0.001
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respectively). Table (4) shows the risk factors related to the children themselves. Many factors were studied and had non-significant difference
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as the number of the child in the family, the presence of another child with autism in the family, and presence of brothers or sisters with the same symptoms. Otherwise, behavioral changes after vaccination had significant value if compared with the control group (P value was less than 0.001). Changes of behavior after vaccination was mostly after the vaccine of MMR (measels - mumps and rubella) at the age of 18 months and the form of behavioral changes were delayed in acquiring new words, irritable movements in the form of rapid hand movements and decrease attention. Lead and mercury were measured in total blood and hair, and their values before treatment were given in table (5) and after treatment with 12 Page 12 of 29
DMSA in table (7). There was significant difference between the autistic and control group in both levels of lead and mercury in total blood and hair. Table (7) and figure (1) show the difference in lead and mercury
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level in total blood before treatment, after 2 months, after 4 months, 6 months and 8 months, there was a significant difference in the level of
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both lead and mercury.
Follow up of these autistic children during treatment, especially in
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the milestones (ability to speak and perform sentences) reveal that improvement of the level of speech with the decrease of the lead and
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mercury level, in the form of increase number of words, and perform understandable sentences.
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Table (6) shows the percentage of autistic children with high levels of lead and mercury (37.8% and 22.2% respectively). In addition to the
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percentage of those took treatment of heavy metals in the form of DMSA.
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1.4 Discussion: This study was designed to determine the risk factors that may
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directly or indirectly lead to autism, and to study the effects of heavy metals (lead and/or mercury) on autism and its severity in children. In
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addition, examination of the effects of chelating agents (DMSA) as a treatment for symptoms in such children was done. The study revealed
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that some factors related to lead an mercury exposure, had significant effects on autism in children; these included exposure to paint during
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pregnancy, living in an old house, use of hair dyes or henna during pregnancy, use of tape water as the main source of drinking water during
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pregnancy, and behavioral changes after vaccination. Other risk factors studied, including the age of the mother, paternal age, type of delivery, risks during pregnancy, complications during delivery, seafood intake
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during pregnancy, passive or active smoking by the mother during
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pregnancy, exposure to pesticides, and tooth amalgam had no significant
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effects on whether an autistic child was present in the family. Many studies have estimated the risk factors that may lead to autism in children, but in this study, we measured the most significant heavy metals hypothesized to be a cause of autism, lead and/ or mercury, and found that there is a significant relationship between these two heavy metals and the appearance of autism. In their study of the autism spectrum, Windham et al. (2006) suggested that there is a potential association between autism and metal concentration. Lyall et al. (2014), Rossignol et al. (2014), Blaurock-Busch et al. (2011), Blaurock- Busch et al., (2012), all showed that heavy metals, are common environmental factors, that lead to autism. In addition, Halsey and Hyman (2001) found that the vaccine of MMR (measles, mumps and rubella) contribute to the appearance of autistic 14 Page 14 of 29
child in the family. Demicheli et al., (2012) in their study about the relation between vaccination and autism, they conclude that adverse after MMR cannot be excluded. Some studies did not support the relation
ip t
between the vaccination and autism as Taylor et al., (1999); and(Destefano and Chen, (2001). This study found a positive correlation
cr
between three factors, presence of positive history about exposure to lead and/ or mercury during the prenatal period or early postnatal period,
us
elevation of the level of lead and/ or mercury in hair and blood in those children, and the improvement of the symptoms after treatment with
an
chelating agents.
The study did not find any association between mother's age and
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the appearance of autistic symptoms. Parner et al., (2012) in their study found that the prevalence of autism in families with advanced aging parents were the same as families with young age parents. Larsson et al.,
d
(2005) in their study about the risk factors of autism found that there was
te
no statistically significant association between risk of autism and parity, parental age or socioeconomic status, these factors if present seem to act
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independently. In addition, Tsuchiya et al., (2008) found that maternal age effect may be demonstrable in some cases, but not all, and may be related to societal and cultural factors. This study excludes maternal age, the order of the child in the family, and the presence of other siblings or relatives with autistic manifestation as risk factors for autism. This study revealed that lead and/ or mercury exposure in either
prenatal or postnatal period had a positive correlation with the presence of an autistic child in the family. In addition, treatment of those kids with one of the chelating agents had a positive effect on decreasing the level of heavy metal and decreased the symptoms of autism in those kids. Against this, is the presence of other children in the same environment and same 15 Page 15 of 29
family with no autistic symptoms, this may be explained by the presence of some defects in methylation of these toxins so they retain these toxins in their bodies for long times, which can lead to the toxic effects and
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appearance of the autistic symptoms. James et al., (2004), Priya and Geetha, (2011) and Hartzell and Seneff (2012) they found that there was
cr
decrease in the capacity of methylation contributes to the development of the autism manifestations. Walsh et al., (2001) also, found that in their
us
study there was metallothionein disorder in those children with autism and suggested that it is an inborn error of metallothionein function; this
an
can explain the high levels of heavy metals in those children. In addition, Obrenovich et al., (2011) reported that these children with autistic
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manifestation revealed evidence of abnormal thiol metabolism, as well as significant alteration in deposition of several heavy metals. They suspected that these kids accumulate heavy metals in their bodies due to
te
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trouble in excreting thiol toxic heavy metals. DMSA were used in this study as a chelating agent, and found to be
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effective in decreasing the level of lead and mercury. Follow up of these kids showed improvment in word and sentences formation and also improvment in the unintentional movement. Adams, et al. (2009A) and Adams, et al. (2009B) agree with this result as they gave the same dose of DMSA. they found that DMSA therapy seems to be reasonably safe, effective in removing several toxic metals (especially lead), dramatically effective in normalizing RBC glutathione, and effective in normalizing platelet counts. Only 1 round (3 days) was sufficient to improve glutathione and platelets. Additional rounds increased excretion of toxic metals, and also improve the behavioral symptoms of autism. 1.5 Conclusion and recommendations: 16 Page 16 of 29
This study presented the hypothesis that there is a strong link between heavy metal toxicity, especially lead and mercury with autism spectrum disorder. Accumulation of these heavy metals in those children
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was the responsible for the appearance of the autistic symptoms. The current results revealed that accumulation of heavy metals may be due to
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many risk factors either during the neonatal period or postnatal period,
also accumulation of these heavy metals is due to defects in metabolism
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of those children, and so use of chelating agents can improve the autistic symptoms. The outcomes of the study support the conjecture that heavy
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metals have a cracking part in development of autistic symptoms, also confirm the use of chelating agents in improving the symptoms of those
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youngsters. Further studies to cover more numbers of autistic children are required for more verification of this theory. We strongly recommend
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accumulation in the body.
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avoidance of many risk factors that increase the heavy metal
1.6 Acknowledgments
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Many grateful to center of "Kaian" in Assiut and Sohag, which
helped us in performing this study with all endeavors by their pediatrician, psychologist and the audio and conversational specialist. Medical students in the final year of the faculty, especially Mai Sallah, made the day of the autistic children better with toys and sweets. 1.7 References:
1. Adams, J.; Bara, M.; Geis, E.; Mitchell, J.; Ingram, J.; Hensley, A.; Zappia, I.; Newmark, S.; Gehn, E.; Rubin, R.; Mitchell, K.; Bradstreet, J.; and El-Dahr, J. (2009): "Safety and efficacy of oral DMSA therapy for children with autism spectrum disorders: Part A - Medical results". BMC Clinical Pharmacology, 9:16 doi:10.1186/1472-6904-9-16. 17 Page 17 of 29
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2. Adams, J.; Bara, M.; Geis, E.; Mitchell, J.; Ingram, J.; Hensley, A.; Zappia, I.; Newmark, S.; Gehn, E.; Rubin, R.; Mitchell, K.; Bradstreet, J.; and El-Dahr, J. (2009): "Safety and efficacy of oral DMSA therapy for children with autism spectrum disorders: Part B - Behavioral results". BMC Clinical Pharmacology, BMC Clinical Pharmacology, 9:17 doi:10.1186/1472-6904-9-17
an
us
3. Adams, J.; Audhya, T.; McDonough-Means, S.; Rubin, R.; Quig, D.; Geis, E.; Gehn, E.; Loresto, M.; Mitchell, J.; Atwood, S.; Barnhouse, S.; and Lee, W. (2013): "Toxicological Status of Children with Autism vs. Neurotypical Children and the Association with Autism Severity". Biol Trace Elem Res;151(2):171-180
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4. Al-Salehi, S.M.; and Ghaziuddin, M. (2009): "G6PD deficiency in autism: a case-series from Saudi Arabia". Eur Child Adolesc Psychiatry, 18(4):227-230.
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.5 Autism Research Institute (2005): "Treatment Options for Mercury/Metal toxicity in autism and related developmental disabilities: consensus position paper". www.Autism Research Institute.com
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6. Bernard, S.; Enayati, A.; Redwood, L.; Roger, H.; and Binstock, T. (2001): "Autism: a novel form of mercury poisoning". Med Hypotheses; 56: 462-471.
7. Blaurock- Busch, E.; Amin, O.; Dessoki, H.; and Rabah, T. (2012): "Toxic Metals and Essential Elements in Hair and Severity of Symptoms among Children with Autism". A Journal of Clinical Medicine; 7 (1): 38-48 8. Blaurock-Busch, E.; Amin, O.; and Rabah, T. (2011): "Heavy Metals and Trace Elements in Hair and Urine of a Sample of Arab Children with Autistic Spectrum Disorder". A Journal of Clinical Medicine; 6(4): 247-257. 9. Blumberg, S.; Bramlett, M.; Kogan, M.; Schieve, L.; Jones, J.; and Lu, M. (2013): "Changes in Prevalence of Parent-reported 18 Page 18 of 29
Autism Spectrum Disorder in School-aged U.S. Children: 2007 to 2011–2012". National Health Statistics Reports, 65: 1-12.
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10.Boseila, S.; Gabr, A.; and Hakim, I. (2004): "Blood Lead Levels in Egyptian Children: Influence of Social and Environmental Factors". American Journal of Public Health; 94(1): 47-50.
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11.Boyle, C.A.; Boulet, S.; Schieve, L.A.; Cohen, R.A.; Blumberg, S.J.; Yeargin-Allsopp, M.; et al. (2011): "Trends in the prevalence of developmental disabilities in US children, 1997– 2008". Pediatrics 127(6):1034–1042.
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12.Carpenter, L. (2013): " DSM-5 autism spectrum disorder, guidelines and criteria". https:// depts.washington. edu/dbpeds/ Screening%20 Tools/ DSM-5(ASD.Guidelines) Feb2013.pdf
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13.CDC. Center for Disease Control (2012): "Prevalence of autism spectrum disorders—Autism and Developmental Disabilities Monitoring Network, 14 sites, United States, 2008". MMWR 61(SS03):1–19.
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14. CDC. Center for Disease Control (2013): Lead poisoning, words to know from A to Z. www.cdc.gov/nceh/lead
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15.Demicheli, V ; Rivetti, A.; Debalini, M.G ; and Di Pietrantonj, C. (2012): "Vaccines for measles, mumps and rubella in children". Cochrane Database Syst Rev.; 15(2): CD004407.
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16.DeStefano, F ; and Chen, R.T. (2001): "Autism and measlesmumps-rubella vaccination: controversy laid to rest?". CNS Drugs.; 15(11):831-837. 17.El-baz, F.; Elhossiny, R.; Elsayed, A.; and Gaber, G. (2010): "Hair mercury measurement in Egyptian autistic children". The Egyptian Journal of Medical Human Genetics;11:135–141. 19 Page 19 of 29
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18.Halsey, N.A.; and Hyman, S.L. (2001): "Measles-mumps-rubella vaccine and autistic spectrum disorder: report from the New Challenges in Childhood Immunizations Conference convened in Oak Brook, Illinois". Pediatrics; 107(5): E84.
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19.Hartzell, S.; and Seneff, S. (2012): "Impaired Sulfate Metabolism and Epigenetics: Is There a Link in Autism?". Entropy; 14: 19531977
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20.Hussein, H.; Taha, G.; and Almanasef, A. (2011): "Characteristics of autism spectrum disorders in a sample of Egyptian and Saudi patients: transcultural cross sectional study". Child and Adolescent Psychiatry and Mental Health; 5(34): 1-12.
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21.James, S.; Cutler, P.; Melny, k. S.; Jernigan, S.; Janak, L.; Gaylor, D.; and Neubrander, J. (2004): "Metabolic biomarkers of increased oxidative stress and impaired methylation capacity in children with autism". Am J Clin Nutr; 80:1611–1617.
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22. Kapp, M. (2006): "Ethical and legal issues in research involving human subjects: do you want a piece of me?". J Clin Pathol.; 59(4): 335–339. 23.Keller, F.; and Persico, A. M. (2003): "The neurobiological context of autism". Mol Neurobiol.; 28(1):1-22. 24.Larsson, H.; Eaton, W.; Madsen, K.; Vestergaard, M.; Olesen, A.; Agerbo, E.; Schendel, D.; Thorsen, P.; and Mortensen, P. (2005): "Risk Factors for Autism: Perinatal Factors, Parental Psychiatric History, and Socioeconomic Status". American Journal of Epidemiology, 161(10): 916-925. 25.Levy, A.; and Perry, A. (2011): "Outcomes in adolescents and adults with autism: A review of the literature". Research in Autism Spectrum Disorders; 5:1271–1282
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26.London, E. (2000): "The environment as an etiologic factor in autism: a new direction for research". Environ. Health Perspect.;108: 401–404.
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27.Lyall, K.; Schmidt, R.J.; and Hertz-Picciotto, I. (2014): "Maternal life style and environmental risk factors for autism spectrum disorders". Int J Epidemiol; 43(2):443-464.
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28.Mutter, J.; Naumann, J.; Schneider, R.; Walach, H.; and Haley, B. (2005): "Mercury and autism: accelerating evidence?". Neuro Endocrinol Lett.; 26(5): 439-446.
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29.NIOSH Manual of Analytical Methods (1994): Elements in blood or tissue. Form 8005.
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30.Obrenovich, M.; Shamberger, R.; and Lonsdale, D. (2011): "Altered Heavy Metals and Transketolase Found in Autistic Spectrum Disorder". Biol Trace Elem Res 144 ( 1-3): 475-486.
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31.Parner, E.; Cohen, S.; Lauritsen, M.; Jorgensen, M.; Schieve, L.; Allsopp, M.; and Obel, C. (2012): "Parental Age and Autism Spectrum Disorders". Annals of Epidemiology; 22(3): 143-150. 32.- Priya, L.; and Geetha, A. (2011): "Level of trace elements (copper, zinc, magnesium and selenium) and toxic elements (lead and mercury) in the hair and nail of children with autism". Biol Trace Elem Res.; 142(2):148-158. 33.Razagui, I.; and Haswell, S. (1997): "The Determination of Mercury and Selenium in Maternal and Neonatal Scalp Hair by Inductively Coupled Plasma-Mass Spectrometry". Journal of Analytical Toxicology, 21: 149-153. 34.Rossignol, D.A.; Genuis, S.J.; and Frye, R.E. (2014): "Environmental toxicants and autism spectrum disorders: a systematic review". Transl Psychiatry; 11:4-e360.
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35.-Sung, Y.J.; Dawson, G.; Munson, J.; Estes, A.; Schellenberg, G.D.; and Wijsman, E.M. (2005): "Genetic investigation of quantitative traits related to autism: use of multivariate polygenic models with ascertainment adjustment". Am J Hum Genet.; 76 (1): 68-81.
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36.Taylor, B ; Miller, E ; Farrington, C.P.; Petropoulos, M.C.; Favot-Mayaud, I.; Li, J.; and Waight, P. A. (1999): "Autism and measles, mumps, and rubella vaccine: no epidemiological evidence for a causal association". Lancet;12: 353(9169):20262069.
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37.Tsuchiya, K.; Matsumoto, M.; Miyachi, T.; Tsujii, M.; Nakamura, K.; Takagai, S.; Kawai, M.; Yagi, A.; Iwaki, K.; Suda, S.; Sugihara, G.; Iwata, Y.; Matsuzaki, H.; Sekine, Y.; Suzuki, K.; Sugiyama, T.; Mori, N.; and Takei, N. (2008): "Paternal age at birth and high-functioning autistic-spectrum disorder in offspring". The British Journal of Psychiatry; 193: 316– 321.
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38.Tyler, F.; Smith, L. K.; McGrother, C. W.; and Taub, N. A. (2007): "The impact of physical, intellectual and social impairments on survival in adults with intellectual disability: A population-based register study". Journal of Applied Research in Intellectual Disabilities, 20: 360–367. 39.-US census Bureau, international database RightDiagnosis.com.htm accessed in March 2014.
(2004):
40.Walsh, W.; Usman, A.; and Tarpey, J. (2001): "Disordered Metal Metabolism in a Large Autism Population". American Psychiatric Association Annual Meeting. New Orleans. http://www.safeminds.org/research/library/20010501.pdf
.
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41.Windham, G.C ; Zhang, L ; Gunier, R ; Croen, L.A , and Grether, J.K. (2006): "Autism spectrum disorders in relation to
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distribution of hazardous air pollutants in the san francisco bay area". Environ Health Perspect; 114(9):1438-1444.
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Table (1): Age distribution of both autistic and control children:
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cr
2˂4 4˂6 6˂8 8 ˂ 10 Total
Autistic Children Control Total Male Female Total Male Female Total Male Female Total 16 4 20 14 5 19 30 9 39 8 2 10 8 4 12 16 6 22 3 1 4 5 1 6 8 2 10 5 6 11 4 4 8 9 10 19 32 13 45 31 14 45 63 27 90
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Age/ Sex
24 Page 24 of 29
Table (2): Comparison of the developmental milestones/months in autistic and control children (T test): Developmental Milestones / month Sitting
6.2 ± 0.51
6.1 ± 0.22
Crawling
9.8 ± 0.18
9.6 ± 0.41
Teething
7.4 ± 0.32
6.9 ± 0.12
Walking
14.7 ± 0.59
13.1 ± 0.87
Ability to speak (one or two words)
25.5 ± 4.1
11.3 ± 1.02
0.001**
Can not form phrase
12.4 ± 2.04
0.001**
Control
P value
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0.43 0.51
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0.41
0.38
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Ability to speak (sentences)
Autistic Children
25 Page 25 of 29
Table (3): Risk factors in autistic and control children (T test and compare means): factors not related to child
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Hazards during pregnancy
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Type of delivery
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Percent of sea food taken/month during pregnance
Painting inside house during pregnancy Passive smoking pregnancy Active smoking pregnancy Exposure to pesticide
during during
Living in old house before 1978 Number of filling teeth with amalgam Using hair pregnancy
dye
during
Using henna during pregnancy
10
20 10 10 31 14 34 11 0 45 27 18 19 26 19 23 3 20 22 3 20
20 10 5 9 36 32 13 0 45 23 22 10 35 15 21 9 27 15 3 17
an
Mother level of education
Complications during delivery
5
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Paternal age
18 - ˂ 23 23 - ˂ 28 28 - ˂ 33 33 - ˂ 38 38- ˂ 43 25 - ˂ 30 30 - ˂ 35 35 - ˂ 40 40 - ˂ 45 45 - ˂ 50 None Secondary school High education Master or doctoral degree None Renal troubles Pre eclampsia Drug treatment Normal labor Cesarean None Obstruction Oxygen deficiency for fetus None
Control
10% 20% 30% or more Yes No Yes No Yes No Yes No Yes No None 1-2 More than 2 None 1-3 More than 3 None
7 17 12 7 2 2 21 15 3 4 0 2 37 8 31 2 5 7 15 30 33 7 5
P value
ip t
Mother age in pregnancy
Autistic Children 5 20 15 4 1 2 20 17 4 2 0 1 34 10 30 5 4 6 25 20 35 6 4
0.47
cr
Mother risk factors
0.54
0.58
0.55
0.34
0.51
0.24
0.001** 0.24 0.78 0.22 0.05* 0.51
0.057
26 Page 26 of 29
28 0 15 28
2 0
1 1
0.001**
0.001**
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Type of drinking water during pregnancy
20 5 24 19
ip t
1-3 More than 3 Tap water Filtered water (free of metals) Bottled water Boiled water
27 Page 27 of 29
Table (4): Risk factors autistic and control children (T test) (factors related to the child):
Vccination (MMR) In the form of changes of behavior
Yes
35
0.81
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Presence of autistic child in relatives
10 13 11 8 3 10 35 7 38
No
P value
0.74
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Presence of other autistic child in same family
1st 2nd 3rd 4th 5th Yes No Yes No
Control
0.76
0
10
45
0.001**
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Order of the autistic child in the family
Autistic Children 22 6 7 9 1 12 33 9 36
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Mother risk factors
Table (5): Comparison of blood and hair levels of lead and mercury in autistic and control children before treatment (T test):
Autistic Control (Mean± child (Mean± SD) SD) 55.24±10.02 9.75±0.58
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Blood level in (mcg/dl)
P value
Ac ce p
Lead
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Heavy metal
Mercury
4.02±0.54
0.00±0.02
Hair level in (mcg/gr)
Autistic child (Mean± SD)
Control (Mean± SD)
P value
0.001**
60.045±9.75
12.03±0.45 0.001**
0.001**
5.21±0.08
0.11±0.05
28
0.001**
Page 28 of 29
Table (6): Percentage of autististic children with high level of lead and/or mercury, and percentage of children took DMSA: Percentage %
No. of autistic children agree to be treated with DMSA
Positive Lead
17
37.8
16
Positive Mercury
10
22.2
10
Positive for both metals
12
26.7
11
Negative for metals
18
40
Total
45
100
Percentage %
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No. of autistic child
35.5
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22.2
an
0
26
24.4 0 57.7
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Heavy Metal
Before treatment (Mean± SD)
Lead
55.24±10.02 4.02±0.54
2 months after treatment (Mean± SD)
4 months after treatment (Mean± SD)
6 months after treatment (Mean± SD)
43.15± 7.02
30.24±7.14
18.04± 5.07
10.04±1.01
0.001**
3.01±0.11
1.41± 0.9
0.9±0.12
0.00±0.04
0.001**
Ac ce p
Heavy metal
te
Table (7): Blood Lead and mercury level in Autistic children before and after treatment with DMSA (One Way Anova):
Mercury
29
8 months after treatment (Mean± SD)
P value
Page 29 of 29
