Endocrine disruptors exposure and risk of male congenital malformations

N.OleaHospital Clínico University of GranadaGranada, Spain

June 2005Kos, Greece

Environment and Childhood: The INMA Spanish Study

Population Inclusion

year Target number of

infants

Ribera de l’Ebre 1997/99 100

Menorca 1997/98 500

Granada 2001/02 700

Valencia 2003/04 1300

Asturias 2003/04 500

Sabadell 2004 800

Study cohorts

Exposures and other determinants

Prenatal period Postnatal period

12 weeks 20 weeks 32 weeks Birth 1 year 4 years

Exposures

PAHS, PMs, Ozone, VOCs, and NO2

Outdoor

Outdoor Indoor (VOCs,

NO2) Questionnaire

Questionnaire

GIS Questionnaire

GIS

Hydroxypyrene Maternal urine Child urine

Trihalomethanes Outdoor Indoor

Questionnaire Questionnaire Questionnaire

Organochlorines, Policromates, Polybrominates,

Ftalates, Phenols and Polyphenols

Maternal serum Questionnaire Cord serum Mecomium1 Child serum

Other endocrine disrupters Questionnaire Placenta

Lead Cord blood Child blood

Arsenic Maternal nail Child nail

Mercury Newborn hair Child hair

Maternal occupation Questionnaire

Other determinants

Diet Questionnaire Questionnaire Questionnaire Questionnaire

Antioxidants Maternal serum Child serum

Folate Maternal serum

Fatty acids Maternal plasma Maternal milk2

Cord blood Child plasma

Genetic study Maternal blood Cord blood

Paternal psicoaffectivity Questionnaire

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 GRAlong 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: 1.26-4.29 (level 0 to level 3)

Analysis limited to rural areas, excluding municipal areas

OR = 2.35, 95%IC: 1.39-3.99

*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: 1.3 95%CI 0.7-2.4 (Cryptorchidism)OR: 1.2 95%CI 0.6-2.4 (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 purchaseTractor pesticide spraying equipment

OR: 1.70 95%CI 1.16-2.50 (Pesticide purchase)OR: 2.32 95%CI 1.34-4.01 (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: 1983-1992)

OR: 1.38 95%CI 1.10-1.73 (Mother in farming/gardening)OR: 1.67 95%CI 1.14-2.47 (Mother in gardening)

5. Longnecker, et al. Am J Epidemiol 155:313-22, 2002

USA: Cohort of newborn 1959-1966

Nested case-control: cryptorchidism (219) and hypospadias (199)

Controls: 599 boys

Pesticide measurements: DDE in mother blood, third trimester of pregnancy

OR: 1.3 95%CI 0.7-2.4 (Cryptorchidism)OR: 1.2 95%CI 0.6-2.4 (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 studyin 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, 17-43 yrs)

- 57% had low educational level (similar to that of fathers)

- 40% were multiparous

- 19.25% 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= 1.11-8.01, p for trend= 0.03)

- work of mothers in agriculture (OR= 3.47, 95%CI= 1.33-9.03)

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: 1.10-7.24; 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

JUANPEDRO

ROSA

RAMON