Gender influence on antioxidant capacity and oxidative damage in saliva of children with autism spectrum disorder: a preliminary study

Introduction: Autism is a complex neurological disorder with multifactorial causes, which compromises physical, psychological, emotional and social skills. This disorder also impairs dental care and patient oral health. Saliva can be easily obtained using a non-invasive technique and has been used in the diagnosis and screening of many pathologies. Total antioxidant capacity of saliva is reduced in the presence of caries in children and has been studied as a possible marker for autism. However, data on salivary antioxidants in children with autism spectrum disorder (ASD) is scarce. Objective: This preliminary study evaluated the existence of gender influence on non-enzymatic antioxidant capacity and protein oxidative damage in the saliva of children with this disorder. Our hypothesis is that there are gender differences in these biochemical parameters in children with austim.


INTRODUCTION
Autism Spectrum Disorder (ASD) is a neurodevelopmental condition that affects 10 out of every 1,000 children worldwide.Impairment of communication and social interaction, shown by withdrawal and lack of interest in other people, repetitive or restricted behaviors and/or interests are common in these circumstances, as well as intellectual deficit and epilepsy 1,2 .All these behaviors and alterations can complicate dental care in children and adults with ASD 3 .
Reduced release of neurotransmitters in the cerebellum, prefrontal and temporal cortices are associated with several behavioral changes presented by patients with ASD 4,5 .Besides, an increased oxidative stress due to reduced antioxidant defense 6 and consequently greater damage to proteins are identified as causes of neurodegeneration in these individuals 7 .
Studies from our research group observed alterations in oxidative stress markers in the saliva of patients with other neurological diseases, such as cerebral palsy 8 and microcephaly 9 .Other authors observed a reduction in the total antioxidant capacity (TAC) in the saliva of individuals with ASD in comparison to the neurotypical group, associated with poor oral hygiene 10 .In neurotypical children, changes in the redox state, with a prevalence of antioxidants, were also described in the presence of caries 11 and the magnitude of these variations seems to be associated with the severity of the injury 12 .
There are several conditions that cause oral problems in individuals with ASD, such as eating habits, chewing difficulties, continuous use of medication and poor oral hygiene [13][14][15] .The study of possible markers of oxidative stress associated with caries in ASD patients depends primarily on comprehensive knowledge of the characteristics of the saliva of these individuals, but information about the influence of gender on these parameters in the saliva of these individuals is scarce.Therefore, the aim of this preliminary study was to evaluate the existence of gender influence on non-enzymatic antioxidant capacity and protein oxidative damage in the saliva of patients with ASD, aged between 5 and 15 years.Our hypothesis is that there are gender differences in these biochemical parameters in children with ASD.

MATERIAL AND METHOD
This cross-sectional observational study is part of a larger project entitled "Evaluation of the oral condition of patients with Autism Spectrum Disorder", approved by the Research Ethics Committee of the Araçatuba School of Dentistry FOA-UNESP (CAAE: 24597019.5.0000.5420).

Sample Calculation
Values of average and standard deviation of salivary total antioxidant capacity 8 , were used for the sample calculation, with the aid of the Open Epi (Atlanta, Georgia, USA).Considering 95% for the confidence interval and 80% for the test power, the sample number (n) was 12 (twelve)/group.

Selection of Patients
Among the patients diagnosed with ASD treated in an outpatient clinic by the Dental Assistance Center for People with Disabilities (CAOE) of the São Paulo University (UNESP), School of Dentistry, Araçatuba, 15 females and 15 males aged between 5 and 15 years were invited to participate in the study.Inclusion's criteria were previous diagnosis for ASD; patients undergoing treatment at the CAOE and in good health and oral hygiene, certified by a dentist from the Center's team.Exclusion's criteria were non-collaborating patients, need for sedation, unstable health, carriers of blood disorders, edentulous patients, patients supplemented with vitamins two months prior to collection, and those whose legal guardians did not sign the free and informed consent form.

Collection of Saliva Samples
Whole saliva samples were obtained between 8:00 and 11:00 am, on a pre-scheduled date for the patients follow-up appointment at the CAOE.Two hours prior to collection, food intake, toothpaste or rinsing solutions were not allowed.Mouth cleaning was done 2 hours before the procedure using water and a brush.
During sample collection, patients remained seated in a room with pleasant temperature and light while the fluid was aspirated from the mouth floor into a sterile, sealed bottle in an ice bath, with the aid of a suction device for a maximum of 10 minutes 8 .Saliva was centrifuged at 10,000 g (Centrifuge 5810 R, Eppendorf, Hamburg, Germany) at 4°C for 10 min to remove cells and reduce turbidity 16 .The supernatant was fractionated and stored at -80°C until the time of analysis.

Protein Concentration
Protein concentration was evaluated by Lowry's method, as described by Hartree 17 using bovine serum albumin (Sigma Aldrich, Saint Louis, Mussouri, U.S.A.) as standard curve, ranging from 0.1 to 1.0 mg/mL.Absorbance was determined at 660 nm wavelength in a microplate reader (Power Wave 340-Biotek, Winooski, Vermont, USA).

Total Non-Enzymatic Antioxidant Capacity
The FRAP assay 18 was used with a concentration of 0.2 to 3.0 nmol/L ferrous sulfate (Synth, Diadema, São Paulo, Brazil) as a standard curve and absorbance was estimated at 595 nm in a microplate reader.

Uric Acid Concentration
A commercial colorimetric enzymatic system with an end-point reaction (Labtest S.A., Lagoa Santa, MG, Brazil) was used, according to the manufacturer's recommendations and with the aid of a semi-automatic analyzer (BIO 2000, Bioplus, Barueri, SP, Brazil).

Protein Oxidative Damage
The oxidative damage to proteins was evaluated based on carbonylation, through an alkaline reaction with 2,4-dinitrophenylhydrazine (Sigma Aldrich, Saint Louis, Mussouri, U.S.A.) as described by Mesquita et al. 19 .

Statistical Analysis
Values presented as mean ± standard deviation were tested for adherence to normality using the Shapiro-Wilk test.To compare groups, the Mann Whitney test was adopted, considering a 5% significance level (GraphPad Prim 8.02).

RESULT
Of the 30 patients invited to participate in the study, 24 remained, being 12 girls and 12 boys.The others were excluded for showing discomfort during saliva collection, which was interrupted and not resumed.The average ages were 9.08 ± 2.84 for girls and 9.17 ± 2.98 for boys.The minimum and maximum ages for girls were 6 and 15 years old, respectively.For boys, these values were 5 and 14 years old, respectively.
Regarding the use of medication, 50% of the girls used antipsychotics, 33% used anticonvulsants, 25% used antidepressants and 25% used anxiolytics.In this group, 50% use an association of two or three classes of drugs.In the group of boys, 100% were medicated with antipsychotics, 33% anticonvulsants, 25% antidepressant and 8% anxiolytic.Drug association was present in 58% of boys.
There was no significant difference between groups in values for total protein (girls: 1.075 ± 0.370 mg/mL; boys: 1.430 ± 0.7480; p = 0.3259), or for antioxidant defense (TAC and UA) and oxidative damage to proteins (protein carbonyl, PC) (Table 1).

DISCUSSION
ASD is a complex neurological disorder with multifactorial causes which compromises physical, psychological, emotional and social skills of patients 20 .The preference for foods with sweeter taste and pasty consistency, poor oral hygiene due to lack of dexterity, supervision or acceptance, as well as continuous use of medications that reduce salivary flow can compromise the oral health of these individuals, increasing susceptibility to caries and gingivitis 3,[13][14][15] .In addition, behavioral changes in these children can make dental care difficult, resulting in further deterioration of oral health.Antipsychotic, antidepressant, anxiolytic and anticonvulsant medications are commonly used to minimize behavioral, social and neurological changes in these patients, providing better quality of life for them and their caregivers.The most used drug in the studied groups was antipsychotics, followed by anticonvulsants.All boys in the studied group used antipsychotics, while only half of the girls were medicated with this class of drug.The use of anticonvulsants and antidepressants was similar between girls and boys.These drug classes, which in most patients used in association, interfere with salivary flow 14 , therefore we chose not to determine this parameter in this preliminary study.Still, in order to normalize the oxidative stress markers results, we used total protein concentrations.For that reason, all results are expressed as a function of the amount of protein in the sample, expressed in milligrams (mg) 8,9,11,12 .
Saliva can be easily obtained using a non-invasive technique and has been used in the diagnosis and screening of many pathologies.This multifunctional fluid is essential for the maintenance of oral health and homeostasis and is composed of water, electrolytes, proteins, enzymes, volatile compounds, hormones of endogenous origin and antioxidant compounds, such as uric acid (UA), glutathione, sialic acid and albumin.The individual measurement of antioxidants is very expensive and time-consuming.Thus, an alternative approach is to measure the TAC using FRAP assay to estimate UA which contributes approximately 70% to TAC of saliva and other non-enzymatic antioxidants, such as vitamins C and E and bilirubin 21 .Therefore, saliva also plays an important role in the control of oxidative stress, reflecting the imbalance between the generation of reactive oxygen species and the ability to scavenge such harmful substances or repair them, resulting damage through enzymatic and non-enzymatic antioxidant defense systems, in favor of the oxidants.These reactive species are produced by physiological metabolism, medication use, local and systemic pathologies and conditions 22 .
Recent studies have shown that in neurotypical children, caries severity has a direct effect on TAC and UA, which gradually reduces salivary oxidative damage 12 .In children with ASD, salivary TAC was significantly reduced in relation to their healthy counterparts 10 .However, studies with saliva of children with ASD are scarce, particularly those investigating the possible variations associated with gender, which was the purpose of the present study.
The absence of gender-associated differences in salivary TAC values was not observed in a study conducted on the saliva of neurotypical individuals with mean ages of 23.4 ± 3.0 years for the female group and 25.4 ± 3.1 years, for the male group 21 .In that case, the authors observed significantly higher TAC values in women's saliva, but they did not carry out the uric acid assay.In the present study, no gender difference was observed in UA.This TAC discrepancy may be related to changes in the composition of salivary non-enzymatic antioxidants, due to age, and needs to be further investigated using specific assays for each antioxidant.The oxidative damage to proteins was greater in the saliva of adult men than in women 21 , different of our results in saliva of children with ASD, but the methodology used to analyze this parameter was different.The absence of statistical differences could be associated with the sample size, suggesting an insufficient sample to determine the statistical difference, because this is a preliminary study.Therefore, other studies are being conducted by our group in order to know the redox profile in the saliva of patients with ASD, before using them as indicative of caries disease.

CONCLUSION
Within the limits of this study, it was concluded that in saliva of children with autism spectrum disorder gender does not influence the oxidative stress biomarkers evaluated in this study.

Table 1 .
Values of total non-enzymatic antioxidant capacity (TAC), uric acid (UA) and protein carbonyl (PC) in saliva of girls and boys with autism spectrum disorder (ASD) Mean values ± standard deviation.Mann Whitney test, significance level 5%.