Endocrine disruptors exposure and risk of male congenital malformations N.Olea Hospital Clínico University of Granada Granada, Spain June 2005 Kos, Greece Environment and Childhood: The INMA Spanish Study

Study cohorts

Exposures and other determinants

Background An previous ecological study* searching for geographical variations in male reproductive health and its relationships with the exposure to pesticides suggested that: The incidence of cryptorchidism (orchidopexy rates) in south-eastern Spain was higher in areas with greater use of pesticides. García-Rodriguez et al., EHP 104:1090-5, 1996

GR Along the Mediterranean coast, extensive areas are devoted to intensive farming in plastic greenhouses.

Study findings 270 criptorchidic boys <16 y.o. The frequency of orchidopexy increases in parallel with the degree of pesticide use OR = 2.32, 95%IC: (level 0 to level 3) Analysis limited to rural areas, excluding municipal areas OR = 2.35, 95%IC: *García-Rodriguez et al., EHP 104:1090-5, 1996

Study limitations Ecologic fallacy the failure of ecologic effect estimates to reflect the biologic effect al individual level among other limitations *Vidaeff & Sever, Reproductive Toxicology, 20:5-20, 2005

Ecologic studies are good generating hypothesis Background: Male sexual differentiation and reproductive functioning are critically dependent on a balance androgen/estrogen ratio. The exposure of the developing foetus to environmental pollutants (xenohormones) may be responsible for anomalies of sexual maturation and reproductive function in adult life.

Some difficulties The xenohormone hypothesis should be extended to encompass the multitude of weakly “estrogenic” agents: but how to assess exposure? what kind of epidemiology design?

How these points have been addressed? Chemical uses Pesticide sales Machinery as pesticide sprayers Questionnaires Parent occupation Mother exposure during pregnancy Biologic samples Determination of selected chemicals Biomarkers of exposure/effect Hormone/Xenohormone activity

1. Restrepo et al., Scan J Work Environ Health 16:239-46, 1990 Colombia: Offspring 8867 floriculture workers Nested case-control: cryptorchidism (16/222 congenital malformations) Controls: 443 boys Pesticide exposure: Questionnaire OR: %CI (Cryptorchidism) OR: %CI (Hypospadias)

2. García-Rodriguez et al., EHP 104:1090-5, 1996

3. Kristensen et al., Epidemiology 8:537-44,1997 Norway: register of newborn 192,417 births Retrospective cohort: cryptorchidism (251) and hypospadias (270) Pesticide purchase Tractor pesticide spraying equipment OR: %CI (Pesticide purchase) OR: %CI (Vegetable farming)

4. Weidner et al., Environ Health Perspect 106:793-6, 1998 Denmark: Register of newborn malformations Register based case-control: cryptorchidism (6177) and hypospadias (1345) Controls: 23,273 boys Parent occupation in farming and gardening (year of conception: ) OR: %CI (Mother in farming/gardening) OR: %CI (Mother in gardening)

5. Longnecker, et al. Am J Epidemiol 155:313-22, 2002 USA: Cohort of newborn Nested case-control: cryptorchidism (219) and hypospadias (199) Controls: 599 boys Pesticide measurements: DDE in mother blood, third trimester of pregnancy OR: %CI (Cryptorchidism) OR: %CI (Hypospadias)

Some problems detected in these publications regarding misclassification of exposure* The ecologic fallacy The use of registries and census The use of proxies instead of direct determinations The choice of a single reference exposure (DDE) *Vidaeff & Sever, Reproductive Toxicology, 20:5-20, 2005

5 papers! that’s all?

Working hypothesis Recent animal and human data suggest that exposure to pesticides during pregnancy may play a role in the development of some male sexual disorders, such as cryptorchidism and hypospadias. Pesticides as endocrine disruptors (EDs), include some organochlorine compounds with oestrogen or anti-androgen properties.

- Some (few) methods have been proposed by Soto and coworkers (1995) and Kortenkamp and coworkers (1999) to overcome the unpredictability of xenoestrogen interactions. Exposure scenario - Low level exposure to large numbers of chemicals. - Synergetic, additive, and/or antagonistic interactions between chemicals and hormones should be considered.

1. To identify the main risk factors for cryptorchidism and hypospadias in south-eastern Spain, and their possible association with environmental factors, with special emphasis on exposure to environmental chemicals with estrogenic activity (xenoestrogens). 2. To determine whether the combined effect of environmental estrogens measured as the total effective xenoestrogen burden (TEXB) is a risk factor for cryptorchidism and hypospadias. Cryptorchidism and hypospadias: A case-control study in South eastern Spain Objectives

Case Boys born at the University Granada hospital, who are diagnosed with cryptorchidism (one or both testes) or hypospadias at birth Control Boys born at the University Granada hospital, selected from the cohort, without cryptorchidism or hypospadias, matched with cases for gestational age (  7 days) day of birth (  15 days) parity (primiparous/multiparous)

Questionnaire: conducted by trained interviewers, at the hospital, and developed for the prospective multi-centre study Biomarker of exposure to mixtures of xenoestrogens: Total effective xenoestrogen burden (TEXB) Chemicals quantified in placentas: Bioaccumulative organochlorine compounds/xenohormones p-p´ DDT, aldrin, o-p´ DDT, dieldrin, mirex, p-p´ DDE, endrin, lindane, o-p´DDD, chlordane, methoxychlor, endosulfan  y , endosulfan diol, endosulfan sulfate, endosulfan ether, endosulfan lactone

Cohort (n=702) Cases Controls Eligible population 50 (100)118 (100) Decline to participate 2 (4) 4 (3) Final population 48 (96) 114 (97) Recruitment period ~2 years Subjects (%)

Results - Mean age of mothers was 29.5 yrs (range, yrs) - 57% had low educational level (similar to that of fathers) - 40% were multiparous % had preterm delivery - 21% of deliveries were by Caesarian

Risk of cryptorchidism and hypospadias increased with: -lower birth weight (p= 0.03) -the professional exposure of fathers to chemicals (OR= 2.98, 95%CI= , p for trend= 0.03) -work of mothers in agriculture (OR= 3.47, 95%CI= ) Results

Risk cryptorchidism and hypospadias decreased with: - mother age (p=0.09) - lower increase in maternal weight during pregnancy (p= 0.008) Results

TEXB was positive in: - 72% of the alpha fractions (mean 3.21 ± 9.26 pM estradiol/g of placenta) - 86% of the beta fractions (mean 8.61±2.88 pM estradiol/g of placenta). Higher TEXB alpha and beta levels were found in cases than in paired controls. Results

Conditional logistical regression analysis, adjusted for mother’s age at delivery and weight of newborn, showed that TEXB of the alpha fractions is a risk factor for cryptorchidism (OR = 2.82, 95% CI: ; p= 0.031) Results

DDT and metabolites, endosulfan and metabolites, lindane, aldrin/dieldrin/endrin, hexachlorobenzene, methoxychlor and mirex were found in placentas. The most frequent pesticides detected were p,p’DDE (84%), followed by lindane (61%). The number of residues found in placenta samples was significantly higher in cases (p= 0.002). Results

Conditional logistical regression analysis, adjusted for mother’s age at delivery and weight of newborn, showed a higher risk of cryptorchidism/hypospadias with the presence of: Lindane, DDTs, Endosulfan I, Mirex. This risk was also associated with higher concentrations of lindane (p = 0.007) and dieldrin (p = 0.052). Results

The estrogenicity of placenta tissue extracts due to bioaccumulated environmental estrogens, measured as the total effective xenoestrogen burden of the alpha fraction (TEXB-alpha) is a risk factor for genital tract malformations. TEXB: goes beyond the quantification of environmental estrogens. It measures the biological activity resulting from mixture of xenoestrogens Conclusions

The optimal information regarding harmful effects of xenoestrogens in human should come from epidemiologic studies It would be irresponsible to postpone the monitoring and regulatory measures until all the necessary evidence is available Recomendations Information Knowledge Concern Awareness Decision Making

The precautionary principle (Spanish version)

ANA MARGA PATRICIA JUAN PEDRO ROSA RAMON