1. Outdoor air pollution exposures and micronuclei frequencies in lymphocytes from pregnant women and newborns in Crete, Greece (Rhea cohort) Cristina O’Callaghan-Gordo ISEE Conference, São Paulo, 31/08/2015

2. The authors declare they have no actual or potential competing financial interests. Disclosure of competing financial interests

3. Air pollution and cancer Outdoor air pollution carcinogenic to humans (group 1) Increased risk of lung cancer at increasing levels of exposure to PM Association with other cancers is less clear  childhood cancer ?? Air Pollution and Cancer, 2013. IARC Scientific Publication No. 161 Editors: K Straif, A Cohen, and J Samet

4. Increase in childhood cancer in Europe in the last decades (E. Steliarova-Foucher, Lancet 2004)

5. Causes of childhood cancer are not well understood No clear associations with environmental factors Research of childhood cancer presents several limitations: –Exposures may start in pregnancy –Rare outcome Need of: –Study exposures during pregnancy – Large numbers and prospective studies Use of biomarkers of cancer risk as a research tool (NewGeneris EU project)

6. MN in maternal and cord blood (image: M.Pedersen, 2010) OBJECTIVE Outdoor air pollution exposure during pregnancy Smoking during pregnancy Vitamin C

7. Small extra-nucleus formed as a result of chromosome breaks or loss during cell division (Krisch-Volders, Mutation Research, 2014) Micronuclei (MN) - a biomarker of DNA damage (I) Bi-nucleated cell with MN (image: Pedersen, 2010)

8. MN in T-lymphocytes associated with increased cancer risk in adults (Bonassi et al., Carcinogenesis, 2007): Micronuclei (MN) - a biomarker of DNA damage (II) Figure 1. Probability curves of cancer free survival by tertile of MN frequency (pooled data from the HUMN cohort). Cancer free survival refers to time from MN test to the first cancer diagnosis (Bonassi et al., Carcinogenesis, 2007) Association between MN in newborns and increased risk of childhood cancer not clear Some studies have evaluated association between carcinogenic exposure during pregnancy and MN frequency in the newborn  Small studies

9. METHODS (I) Subjects: –Mothers & newborns from Rhea cohort (Heraklion, Crete, Greece) –Pregnancies between February 2007-2008 Exposure: Air pollution levels at maternal home address (estimated annual mean concentration-ESCAPE project): –PM 2.5 –PM 10 –PM 2.5-10 –PM 2.5 absorbance –NO 2 –NO x Outcome: MN frequency in T-lymphocytes in maternal blood and MN in cord blood. Semi-automated readings using cytokinesis block micronucleus assay (CBMN)

10. METHODS (II) Potential effect modifiers: –Smoking: self-reported (consistency checked by cotinine levels in urinary samples) –Vitamin C: frequency food questionnaires (FFQ) Potential confounders : –Maternal age / Gestational age –Residence area (urban/rural) –Education (low/medium/high) –Maternal origin (Greek/others) –Season of delivery Statistical methods: –negative binomial regression –Imputation of missing values (covariates) –Adjusted models Face to face interviews, self- administrated questionnaires and medical records

11. Study population Mothers (n=181) 86% Greek origin Mean age 29.7 ±5.0 years 31% active smoking during pregnancy 55% medium educational level 71% lived in urban areas Median MN: 2.63 (IQR:2.53) Differences in median MN frequency by: maternal age, educational level, origin, season of delivery Newborns (n=183) 53% newborns were boys Mean gestational age was 38.4 ± 1.3 weeks Mean birth weight was 3,225 ± 412 g. 6% preterm birth Median MN: 1.48 (IQR:1.83) Differences in median MN frequency by season of birth

12. Air pollution levels – Heraklion (Greece) Median concentrations (IQR): PM 2.5 : 14.4 (1.3) µg/m 3 PM 2.5-10 : 22.5 (3.0) µg/m 3 PM 10 :37.0 (3.0) µg/m 3 PM 2.5 absorbance: 1.1 (0.3) 10 -5 per m Distribution of average concentrations in different study areas - ESCAPE project- (a, b, c, d from Eeftens et al, Atmospheric Environment, 2012; e, f, from Cyrys et al Atmospheric Environment, 2012) NO 2 : 12.2 (4.1) µg/m3 NO x : 19.5 (9.6) µg/m3 e. NO 2 NO x a. PM 2.5 b. PM 2.5 abs d. PM 2.5-10 c. PM 10

13. Air pollution-MN Air pollutants Maternal blood*Cord blood** IRR (95%CI) PM 2.5 (5μg/m 3 )1.53 (1.02, 2.29)0.97 (0.63, 1.50) PM 2.5-10 (5μg/m 3 )1.14 (0.94, 1.38)0.96 (0.79, 1.17) PM 10 (10μg/m 3 )1.16 (0.82, 1.64)1.14 (0.79, 1.65) PM 2.5 absorbance (10 -5 per m)0.85 (0.58, 1.25)0.85 (0.55, 1.34) NO 2 (10μg/m 3 )1.08 (0.84, 1.40)1.07 (0.79, 1.45) NO x (20μg/m 3 )1.09 (0.88, 1.34)1.11 (0.83, 1.48) Association between air pollution exposure during pregnancy and incidence rate ratio [IRR (95%CI)] of MN frequency in maternal and cord blood samples *Maternal models adjusted for maternal age at delivery, maternal education, maternal residence, maternal origin and season of delivery; ** Cord models adjusted for gestational age, maternal education, maternal residence and season of birth 53% increase in maternal MN frequency for each 5µg/m 3 increment of PM 2.5 levels.

14. Air pollution-MN. Vitamin C Maternal blood: Interaction for PM 2.5 (p-value=0.030) PM 2.5 associated with MN only among women with vitamin C intake below recommended valued during pregnancy (<85 ng/day) Air pollutantsDaily levels of vitamin C (ng/day) < 85 (n= 20)≥85 - <170 (n=41)≥170 (n=69) IRR* (95%CI) PM 2.5 (5μg/m 3 )9.35 (2.77, 31.61)1.86 (0.85, 4.09)1.17 (0.65, 2.08) *Models adjusted for maternal age at delivery, maternal education, maternal residence, maternal origin and season of delivery

15. Air pollution-MN. Vitamin C Cord blood: Interaction for PM 10 (p-value=0.021) Decreased MN at higher exposure among newborns of mothers with vitamin C intake below recommended valued during pregnancy (<85 ng/day) Air pollutantsDaily levels of vitamin C (ng/day) < 85 (n=22)≥85 - <170 (n=43)≥170 (n=69) IRR* (95%CI) PM 10 (10μg/m 3 )0.33 (0.13, 0.86)1.72 (0.69, 4.27)1.46 (0.79, 2.71) *Cord models adjusted for gestational age, maternal education, maternal residence and season of birth

16. Air pollution-MN. Smoking Maternal blood: Interaction for PM 2.5-10, PM 10, NO 2 and NO x. These air pollutant were strongly associated with increased MN frequency among smoker women, but not among non-smokers. Air pollutantsMaternal blood (n=172)* non-smokers (n=116)smokers (n=56)p-value # IRR* (95%CI) PM 2.5-10 (5μg/m 3 ) 1.05 (0.86, 1.29)1.39 (0.94, 2.05)0.036 PM 10 (10μg/m 3 ) 0.93 (0.66, 1.29)2.33 (1.16, 4.67)0.017 NO 2 (10μg/m 3 ) 0.87 (0.69, 1.10)1.62 (1.06, 2.47)0.001 NO x (20μg/m 3 ) 0.86 (0.69, 1.08)1.46 (1.08, 1.96)0.001 *Models adjusted for maternal age at delivery, maternal education, maternal residence, maternal origin and season of delivery; # p-value for interaction (Wald test) Cord blood: No interaction observed

17. SUMMARY OF RESULTS-DISCUSSION (I) 1. PM 2.5 associated with MN frequency in maternal blood, particularly among those that did not fulfil the recommended vitamin C allowances during pregnancy: PM 2.5 is the PM fraction most strongly associated with adverse health effects (WHO, 2013) DNA damage associated to PM occurs via oxidative damage, i.e. production of reactive oxidative species - ROS- (Møller et al, Mutational Research, 2014) Oxidative damage in one of the mechanisms known to induce MN formation (Kirsch-Volders et al, Mutation Research, 2014)  Our results support the hypothesis that vitamin C neutralizes ROS produced after exposure to PM 2.5

18. SUMMARY OF RESULTS-DISCUSSION (II) 2. Among women smoking during pregnancy, PM 10, NO 2, NO x associated with increase MN frequency with in maternal blood: Results suggest higher vulnerability of smokers to the effect of air pollution Higher vitamin C daily allowances are recommended for smokers (Institute of Medicine, 2001)  Differences observed between smokers and non- smokers could be driven by insufficient vitamin C intake among smokers

19. SUMMARY OF RESULTS-DISCUSSION (III) 3.No association was detected between air pollution exposure during pregnancy and MN frequency in cord blood: Few studies on maternal exposure to air pollution during pregnancy and MN in the newborn (Pedersen et at, Environmental Research, 2009; Rossnerova et al, Mutational Research, 2011) These studies found association between air pollution and MN in newborns but not in mothers (contrary to our results) Previous studies estimated exposure at area level, whereas we used individual level estimates.  Our results suggest that air pollution exposure during pregnancy does not increase MN frequency in the newborn

20. STRENGTHS & LIMITATIONS Limitations Annual mean concentrations might have lead to some exposure misclassification as air pollution levels can vary within a year  We adjusted all our models for season of delivery/birth to help control for some of the variability Potential misclassification of mothers who moved during pregnancy  Sensitivity analysis excluding women who moved during pregnancy (7% of women) yield similar results. Strengths MN quantification using semi-automated image base scoring system that reduced potential variation and subjectivity of manual scoring (Decordier et al., 2009) Air pollution exposures were measured using the ESCAPE project models for air pollution that have been previously established and used in large European studies.

21. Acknowledgments CREAL Marie Pedersen Ana Espinosa Mark Nieuwenhuijsen Manolis Kogevinas University of Crete Eleni Fthenou Leda Chatzi Georgia Chalkiadaki Theano Roumeliotaki Marina Vafeiadi Euripides Stephanou Maastricht University Jos Kleinjans Contact: cocallaghan@creal.cat Institute for Risk Assessment Science, University of Utrecht Rob Beelen Gerard Hoek Laboratory of Cell Genetics, Vrije University Ilse Decordier Kim Vande Loock Micheline Kirsch-Volders Istituto Nazionale per la Ricerca sul Cancro Domenico Franco Merlo,