| Abstract |
Persistent organic pollutants (POPs) are considered to be neurotoxic, influencing the synthesis and activity of neurotransmitters and the organization of the developing brain through alterations in basic cellular signaling processes and endocrine function. Human studies on a longitudinal and/or cross-sectional level have associated exposure to POPs in utero and in early childhood with adverse neurodevelopmental outcomes, such as reduced intelligence, ADHD, decreased performance in memory, autism spectrum disorders and other behavioral problems.
Inflammation is a complex natural defense mechanism by body tissues in response to injurious stimuli. This response may stop being protective for the organism and become harmful if it occurs chronically. Inflammatory markers, such as cytokines, are proteins involved in normal aspects of neurodevelopment and there is growing evidence associating them in complex, higher order neurological functions, such as cognition and memory. Imbalanced cytokine production, signaling and regulation may have various neurological consequences. A body of research has evolved around the role of prenatal cytokines as markers of risk for cognitive dysfunction in special and vulnerable populations and extensive research findings report that cytokine levels in plasma and/ or sera are altered in neurodevelopmental disorders, e.g. Autism Spectrum Disorders.
The specific objectives of this thesis are the following:
To explore the role of high concentration levels of prenatal exposure to HCB, DDE and PCBs in offspring cognitive development at 4, 6 and 11 years of age.
To explore the role of high concentration levels of prenatal exposure to HCB, DDE and PCBs in offspring behavioral and emotional difficulties at 4, 6 and 11 years of age.
To explore the role of high concentration levels of several inflammatory markers (IFN-γ, IL-1β, IL-6, IL-8, IL-10, IL-17α, MΙP-1α, TNF-α) measured in child serum at 4 years of age in child cognitive development at 4, 6 and 11 years of age.
To explore the role of high concentration levels of several inflammatory markers (IFN-γ, IL-1β, IL-6, IL-8, IL-10, IL-17α, MΙP-1α, TNF-α) measured in child serum at 4 years of age in child behavioral and emotional difficulties at 4, 6 and 11 years of age.
Methods
This study uses data from the Rhea Study, a prospective mother-child cohort established in 2007 in Crete, Greece. Pregnant women were recruited in the study at the time of the first ultrasound examination, around the 12th gestational week, from two public and two private prenatal clinics in Heraklion, during a twelve-month period, from 02/2007 until 02/2008. Trained midwives described in detail the study to pregnant women, obtained written informed consent, measured height, weight, and blood pressure, collected urine and blood samples, and completed a thorough questionnaire concerning participants’ diet, sociodemographic and lifestyle characteristics, and exposure to various environmental agents. Mother-child pairs were invited to participate in child follow-up assessments when the children were 18 months, 4, 6 and 11 years of age.
POPs: Maternal serum samples were collected at the first prenatal visit around the 3rd and 4th month of pregnancy, in 10 ml Silicone gel separator vacutainer tubes (Becton Dickinson, UK). Tubes were centrifuged within 2 hours from blood collection at 2500rpm for 10min and were then stored in aliquots at -80°C until assayed. The POP analyses were performed in the National Institute for Health and Welfare, Chemicals and Health Unit, Kuopio, Finland with an Agilent 7000B gas chromatograph triple quadrupole mass spectrometer (GC-MS/MS). Serum concentrations of six individual PCB congeners (IUPAC numbers: 118, 138, 153, 156, 170 and 180), HCB, and DDE were determined. All the results were reported on whole weight and expressed in pg/ml serum, while samples below the limit of quantification (LOQ) were assigned the value 0.5×LOQ. LOQ was 6 pg/ml for PCB118 and PCB156; 10 pg/ml for HCB, DDE, PCB138, PCB153, PCB170, PCB180. We chose to use wet-weight levels for the POPs but adjusted for maternal serum triglycerides and cholesterol as continuous variables in all multivariable models to minimize potential biases associated with automatic lipid adjustment. POPs were treated as categorical variables. We calculated total PCB concentrations by summing the concentrations of the 6 individual PCB congeners and studied the associations of interest for the sum of PCBs. Inflammatory markers: At the 4-year-follow-up assessment, blood samples were collected by venipuncture for each child (10ml) in SST gel separator vacutainer (BD vacutainers, UK), after written parental consent. For the reduction of pain and discomfort of the children, anesthetic cream 5% EMLA with composition 2.5% lidocaine and 2.5% prilocaine (AstraZeneca, UK) was used. Analyses were performed in the Laboratory of Clinical Nutrition and Epidemiology of Diseases of Medical School, University of Crete. Blood samples were centrifuged (Kubota 4000, Japan) at 2500rpm 10min within 2 hours after collection and stored at -80o C until assayed. The Milliplex Map human high sensitivity T cell magnetic bead panel (Cat. # HSTCMAG-28SK) from Millipore (Billerica, MA) was used for the simultaneous quantification of IFN-γ IL-1β, IL-6, IL-8, IL-10, IL-17α, MIP-1α and TNF-α in the supernatants. The principle of the assay is based on the quantification of multiple bio-molecules concurrently employing fluorescent-coded magnetic beads (MagPlex-C microspheres). The microspheres were incubated with the samples and then were allowed to pass rapidly through laser systems that distinguish the different sets of microspheres and the fluorescent dyes on the reporter bio-molecules. The sensitivity of the assay for every bio-molecule was: 0.3 pg/ml IFN-γ, 0.1 pg/ml IL-1β, 0.1 pg/ml IL-6, 0.1 pg/ml IL-8, 0.6 pg/ml IL-10, 0.3 pg/ml IL-17α, 0.9 pg/ml MIP-1α and 0.2 pg/ml TNF-α. We used a limit of 4 SD based on the statistical convention that observations 4 or more SD from the expected mean can be considered to be “extreme outliers” and thus, excluded from the statistical analyses. The intra-assay precision (%CV) for all biomolecules was <5%. The inter-assay precision (%CV) for IFNγ, IL-6, IL-10 and IL-17α was <20%, for IL-1β, IL-8, MIP-1α and TNF-α was <15%. The above analyses were performed on an automated analyzer Luminex 100 connected with the Luminex xPONENT software.
Cognitive development assessment at 4 years was conducted using the McCarthy Scales of Children’s Abilities (MSCA), which evaluate child development across five domains: verbal, perceptual, quantitative, memory, and motor and offers a composite index of general cognitive development. Cognitive development assessment at 6 years of age was carried out using the Raven’s Colored Progressive Matrices (RCPM), which assess non-verbal general intelligence, the Finger Tapping Test (FTT), which assess motor speed, and the Trail Making Test part A & part B (TMT-Part A & TMT-Part B), which assess visual search, speed of processing, mental flexibility, and executive functions. Cognitive ability at 11 years was carried out using the Wechsler Intelligence Scale for Children, fifth edition (WISC-V), assessing intellectual functioning in children across five Primary Index Scales [including Verbal Comprehension Index (VCI); Visual Spatial Index (VSI); Fluid Reasoning Index (FRI); Working Memory Index (WMI); and Processing Speed Index (PSI)] and the four Ancillary Index Scales [including Quantitative Reasoning Index (QRI); Nonverbal Index (NI); General Ability Index (GAI) and Cognitive Proficiency Index (CPI)]. Behavioral and emotional development were overall assessed through the parent-report questionnaires. At 4 years of age the Attention Deficit Hyperactivity Disorder Test (ADHDT) and the Strengths and Difficulties Questionnaire (SDQ) were completed by participants’ parents. The ADHDT is based on ADHD criteria of DSM-IV and provides 4 indexes, corresponding to 3 subscales (hyperactivity, inattention, impulsivity) and a total ADHD difficulties index. The SDQ is a brief screening questionnaire designed to assess behavioral strengths and difficulties in children and evaluates emotional symptoms, conduct problems, hyperactivity and inattention, peer-relationship problems, and prosocial behaviour. SDQ provides two additional composite indexes of internalizing and externalizing problems. Behavioral and emotional problems at 6and 11 years of age were assessed through Child Behaviour Checklist – Parent Report Form (CBCL) and the Conner’s Parent Rating Scale, Revised, Short Form (CPRS-R: S). The CBCL includes 6 scales that correspond to different diagnostic categories of the DSM-IV: affective problems, anxiety problems, somatic problems, attention deficit/hyperactivity problems, oppositional defiant problems, and conduct problems, and two composite indexes of internalizing and externalizing problems. The CPRS-R: S assess oppositional problems, cognitive problems/inattention, and hyperactivity, and provides an index of total ADHD symptoms.
Descriptive analyses on the characteristics of the population, and the distribution of the exposures, and the outcomes were conducted. Generalized additive models were used to assess the linearity of the associations of interest. Multiple linear and logistic regression models were used to explore the associations of interest, accordingly.
Results
POPs
High prenatal HCB exposure was associated with decreased performance in i) cognitive, perceptual, executive and working memory functions at the age of 4, ii) non-verbal general intelligence, processing speed and mental flexibility and motor speed at 6 years of age and iii) visual spatial, fluid reasoning, working memory, quantitative reasoning, nonverbal, general ability and cognitive proficiency functions at 11 years of age.
High maternal serum levels of PCBs were associated with offspring decreased performance in working memory tasks at preschool age, increased response time in TMT Part A and lower speed scores in FFT (non-dominant hand) at 6 years of age and lower scores in several WISC-V index scores (working memory, processing speed, quantitative reasoning, nonverbal, general ability and cognitive proficiency) at 11 years of age.
At 11 years of age we found statistically significant positive associations between high HCB levels and three behavioral scales scores in the basic models. However, in the adjusted models the estimates were weakened and it may be possible there is residual confounding implicated in the direction of the results,
No association was demonstrated between high prenatal POPs levels with behavioral outcomes at any age-timepoint, with the sole exception of the association of high prenatal HCB levels with child peer problems in SDQ at 4 years.
No associations were found between maternal DDE concentrations and neurodevelopmental and behavioral scores at 4 and 11 years of age. Regarding 6 years of assessment, we only found one associations of high prenatal DDE levels with increased response time in TMT Part B.
No indication for effect modification by child sex, maternal pre-pregnancy body mass index (BMI) and maternal TSH during pregnancy was found.
Inflammatory Markers
Preschoolers with elevated TNF-α concentrations in serum demonstrated decreased scores in memory, memory span and working memory and preschoolers with high IFN-γ serum levels showed lower scores in memory span scale. Children at 4 years with high levels of IL-8 showed lower prosocial behavior scores.
Children with elevated levels of IFN-γ at 4 years showed increased scores in oppositional and hyperactivity scales, as well as in internalized and externalized CBCL scores at 6 years of age; high IL-1β levels at 4 years were associated with more oppositional symptoms and externalized problems at 6 years of age. We also found that increased TNF-a/IL10 ratio was related with lower inattention and ADHD scores at 6 years.
Children with high levels of IL-8 at 4 years showed increased scores in processing speed; children with high levels of IL6/IL-10 ratio at 4 years showed increased scores in visual spatial performance at 11 years. High levels of the anti-inflammatory IL-10 at 4 years were associated with increased cognitive proficiency scores; high IL6/IL-10 ratio were related to increased general ability scores at 11 years. We also found that children with high levels of IFN-γ at 4 years demonstrated increased scores in hyperactivity and ADHD scales. High IL-17α levels at 4 years were associated with increased internalized problems scales at 11 years.
Our results showed greater risk for reduced verbal performance scores for boys with high IL-17α serum concentration levels, as well as lower motor scores at 4 years for boys with high IL-6 serum concentrations. We also found greater risk for lower scores in memory and memory span scales at 4 years for overweight/obese children with high TNF-α serum concentrations.
Conclusions
To conclude, the present thesis supports and extends previous knowledge that prenatal exposure to high levels of POPs can be associated with reduced offspring cognitive development. This is actually the first study highlighting the association between prenatal POPs concentration levels and child neurodevelopment across three different timepoints, at 4, 6 and 11 years of age, strengthening the evidence for this association. We conclude that those findings raise the possibility that exposure to HCB and PCBs may play a more crucial role in child cognition than previously considered and show new directions for research in birth cohort studies. A deeper understanding of environmental risk factors for impaired cognitive development could be of considerable public health importance because of their potential modifiability. While tens of thousands of industrial chemicals are still in use, evidence on their potential neurodevelopmental effects remains inadequate for the vast majority. Studies like the current one may signify a valuable initial step towards exploring environmental risk factors for cognitive disorders. To our best of knowledge, this the first study conducted in a general population sample of children which highlights the significant role of increased inflammatory levels during preschool years in child cognitive performance across multiple timepoints. In fact, whilst some studies have examined the possible link between child inflammatory biomarkers and neurodevelopment, most of them were carried out with samples of extremely premature infants or with clinical samples of children with autism spectrum disorders. Moreover, most of those studies have focused on the relationship between maternal inflammatory cytokines during pregnancy and their children's neurodevelopmental outcomes. As there are no studies to this date that explored how inflammatory biomarkers relate to measures of neurodevelopmental scores in a general population sample, these results may shed some light in new pathways of investigation. Our findings reinforce the existing evidence that elevated inflammatory activity may be involved in early pathophysiological processes, such as memory deficits at a cross-sectional level, and in behavioral difficulties at a longitudinal level. An increased understanding of the interactions between pro-and-anti-inflammatory cytokine patterns and levels, and cognitive functions could allow us to identify early at-risk children for targeted interventions and allow every child to meet their full developmental potential. Inflammatory biomarkers could also even serve as indicators and possibly lead to prognosis and therapy in order to prevent developmental delays and behavioral problems in at-risk children.
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