Reproductive / Developmental Effects of an Environmentally-Relevant Organochlorine Mixture

Principle Investigator
Pierre Ayotte
Unité de recherche en santé publique
Université Laval
2400 D'Estimauville
Beauport, QC G1E 7G9

payotte@cspq.qc.ca

Contributing Partner(s)
Janice Bailey, Laval University

Project Length
2 Years

Funding
1999-2000: $185,000
2000-2001: $168,000

Why Project Was Undertaken: Mixtures of chlorine-containing organic chemicals (such as PCBs and dioxins) in fish and marine animals are known to disrupt sex hormone systems in animals. Chemicals that can disrupt normal levels of these hormones are known as 'endocrine-disrupting' chemicals (EDCs). There is concern that exposure to organochlorine chemicals at levels found in the environment may affect sexual development and fertility in the offspring of exposed animals, including humans.

How Project Was Conducted: Female pigs were fed a mixture of endocrine disrupting chemicals at exposure levels similar to those found in traditional foods of the Inuit of Nunavik, Quebec. Chemical exposure continued through pregnancy and nursing. Male piglets were nursed by their exposed mother for a month and then fed an untreated diet. At sexual maturity, sperm of offspring were examined and testes were weighed to see if exposure to this chemical mixture affected male reproductive tissues. Eggs alone or egg and sperm cells taken from untreated pigs were directly exposed to the same chemical mixture. The development of the cells and embryo were evaluated.

What Was Found and Conclusions: The mother pigs were not affected by chemical mixtures, but the piglets from exposed mothers had shorter body lengths at birth. Mature offspring in the high exposure group had reduced sperm movement and lower testes weights. These findings indicate that exposure of the developing fetus and newborn to EDCs during pregnancy and nursing can affect male reproductive development at sexual maturity even if exposure does not continue past the first month of life. Levels of sex hormones in egg and sperm cells were altered by direct exposure to the chemical mixture. Egg cell development and embryo development were slower and sperm penetration into eggs was inhibited in the presence of the chemical mixtures. These findings may have significance for humans since the pig is similar to humans in terms of its digestive and reproductive systems. Further studies are planned to determine whether levels of organochlorine chemicals in Arctic fish and marine animals may adversely affect reproduction in humans.

Executive Summary

Several organochlorine compounds which are found as a complex mixture in northern aquatic food chains have been shown to interact with hormonal systems such as androgenic and oestrogenic systems. In particular, several DDT analogues display weak oestrogenic properties while p,p'-DDE is a potent anti-androgen. Dioxin-like compounds can also interfere with hormone levels and signalling pathways. Our main hypothesis is that in utero and lactational exposure to an environmentally relevant complex mixture of endocrine-disrupting organochlorines alters the development of the male reproductive tract, which in turn leads to adverse effects on male reproductive function during adulthood. Our secondary hypothesis is that this mixture of organochlorines modifies the maturation of gametes and early embryonic development. The pig was selected as the experimental model because of physiological similarities to humans especially with regard to digestive, reproductive and endocrine systems.

In the in vivo part of this study, sows were administered an organochlorine mixture similar to that found in traditional foods from the Arctic aquatic food chain, which are part of the diet of Inuit people living in Nunavik (northern Quebec, Canada). Animals were allocated to four treatment groups and received either corn oil alone or increasing doses of a mixture comprising 15 organochlorine compounds, either pure chemicals or commercial mixtures. Sows were inseminated with the semen of an untreated boar and treatment was continued throughout gestation and lactation. Sows in the low dose group displayed during gestation a polychlorinated biphenyl (PCB) body burden similar to that encountered in young women from the general southern Quebec population. The mid-dose group animals had a PCB body burden similar to the average value documented in Inuit women. Body burden in the high dose group is observed in highly-exposed Inuit women. Hence, PCB concentrations achieved in this developmental study are relevant for populations environmentally exposed to these compounds.

There was no maternal toxicity induced by the mixture. However, maternal exposure to the mixture caused piglets to be shorter at birth, an effect that may be related to disruption of thyroid hormone signalling by compounds present in the mixture or their metabolites, possibly hydroxylated PCBs. Male pigs were nursed during their first month of life and received a standard diet after weaning. Extensive sperm testing took place when animals reached sexual maturity. Boars from the high dose group had reduced sperm motility compared to controls. Organs were weighed during autopsy and a dose-response decrease in testicular weight was observed. Reduced testis weights in boars stands for an anti-androgenic effect resulting from maternal exposure to the mixture. p,p'-DDE could be responsible for this effect since it is a major constituent of the mixture and a well known blocker of the androgen receptor. PCBs, which are also present in the mixture, can also impair sex organ development through a mechanism involving activation of the aryl hydrocarbon receptor.

Using various cell systems expressing luciferase as the reporter gene, the endocrine disrupting potential of our mixture was investigated. We observed that our mixture displayed anti-androgenic activity which appeared to be due solely to its content in p,p'-DDE. No apparent interaction was seen between the various constituents of the mixture. The mixture also displayed weak estrogenic activity, which appeared to result mainly from the xenoestrogens p,p'-DDT, b-hexachlorocyclohexane and p,p'-DDE. In addition, the mixture exhibited a dioxin-like activity similar to that induced by mono-ortho PCB congeners, which possess a weak affinity for the aryl hydrocarbon receptor.

The last series of experiments involved exposure of gametes to the mixture during in vitro maturation (IVM) or in vitro fertilisation (IVF). In IVM experiments, inclusion of the mixture to the maturation milieu of porcine cumulus-oocytes complexes decreased the quality of cumulus cell expansion and increased the percentage of these cells undergoing programmed cell death (apoptosis). The latter effect was seen at low concentrations, similar to those encountered in plasma samples of some Inuit women of reproductive age. The mixture also decreased oocyte maturation and early embryonic development, following insemination with untreated boar sperm.

In the next series of experiments, oocytes were co-cultured with sperm in the IVF medium containing increasing concentrations of the mixture. The mixture decreased the rate of sperm penetration of the oocyte and early embryonic development. These effects were encountered only at high concentrations of the mixture. Incubation of pig sperm alone with the IVF medium containing the mixture reduced sperm motility and viability. These results suggest that exposing porcine oocytes and sperm to an environmentally pertinent organochlorine mixture in vitro disrupts oocyte and sperm fertility and further embryonic development.

The organochlorine mixture that was used in these studies, while similar to that found in the Arctic marine food chain, is not representative of the mixture found in humans and laboratory animals following chronic exposure. Further experiments will be conducted with plasma extracts of sows treated with the organochlorine mixture in order to assess the endocrine disrupting properties of both parent compounds and their metabolites. Furthermore, another in vivo reproductive/developmental study will be conducted using the rat as the animal model and the same organochlorine mixture to confirm in another animal species the results obtained with the porcine model.

Publications

Publications in scientific journals

Accepted/Published articles

Campagna, C., Sirard, M.A., Ayotte, P., Bailey, J.L. 2001. Impaired maturation, fertilization and embryonic development of porcine oocytes following exposure to an environmentally-relevant organochlorine mixture. In press.

Submitted articles

Campagna, C., Guillemette, C., Paradis, R., Sirard, M.-A., Ayotte, P., Bailey, J.L. 2001. An environmentally relevant organochlorine mixture damages sperm function and fertility in the porcine model.

Larochelle, C., Giroux, S., Pereg, D., Bailey, J. L., Ayotte P. 2002. Assessment of androgenic, estrogenic and dioxin-like potential of a complex environmentally-relevant organochlorine mixture using transactivation cell assays.