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Reproductive effects of environmental chemicals in females

Final Report Summary - REEF (Reproductive effects of environmental chemicals in females)

Executive summary:

Many of the chemicals that are present in the environment (Environmental chemicals (ECs)) enter the mother by routes such as eating, drinking and skin contact. Some of these chemicals are broken down by the mother's liver and by the placenta, but significant quantities pass to the fetus in the womb. Once in the fetus, some of these ECs may cause damage. Such chemicals include Endocrine-disrupting compounds (EDCs), which either mimic or block the effects of hormones in the fetus. Resulting reproductive damage has been seen in wildlife, domestic animals and those used to test toxic chemicals and in humans themselves. Although the reproductive system in females is sensitive to the damaging effects of ECs, most focus has been on the male or has relied on a toxicological approach, which uses short-lived rodent species exposed to high doses of single ECs for short periods of time. While these studies have generated important findings, they are not necessarily a good way of understanding what is occurring in humans and domestic animals which are inadvertently exposed to ECs for long periods of time to low doses of complex cocktails of chemicals in the environment.

The REEF project has used three models to investigate the effects of ECs on the female reproductive system. Firstly, sheep exposed to cocktails of chemicals present in processed sewage sludge used to fertilise pasture according to agricultural guidelines. This is a real-life model. Secondly, mice and sheep exposed to selected chemicals, which we have shown are increased in the livers of sheep on sewage-sludge fertilised pastures. This model is designed to investigate mechanisms and pathways that lead from exposure to damage. Thirdly, normal human fetuses obtained by elective terminations from women in the second trimester of pregnancy, with a second real-life exposure of the fetus to chemicals imposed when the women smoked cigarettes.

The objectives of the REEF project were to:

- examine the effects of sewage sludge exposure during specific periods of fetal ovarian development in the sheep;
- examine the effects of environmental concentrations of specific chemicals on female reproductive development and health in the sheep and mouse. Both cell culture and whole animal approaches were used;
- investigate the effects of these chemicals on to subsequent generations;
- examine the effects of cigarette smoke on the human fetus;
- integrate human and animal models in order to better understand the processes leading to reduced fertility and other problems such as increased obesity.

Main conclusions of the REEF project:

- exposure to sewage sludge chemicals has different effects on the female fetus than the male, with the fetal ovary most sensitive to ECs late in pregnancy;
- different individual chemicals have different effects from each other and from sewage sludge and have more damaging effects on the mouse ovary than the sheep;
- in the mouse, the damaging effects can pass through to the great-grandchildren of the animal exposed to ECs while pregnant;
- in sheep, grand-daughters of exposed animals did not have abnormal ovaries;
- Diethylhexyl phthalate (DEHP) and Polychlorinated biphenyls (PCBs) act as obesogens? making mice fatter;
- a women who smokes while pregnant increases the quantity of ECs reaching her fetus and causes fetal ovary changes likely to reduce fertility in adulthood.

In conclusion, the REEF project has shown complex effects of ECs on the female reproductive system, with differences in the nature and degree of damage varying depending on the stage of pregnancy during exposure, the sex of the fetus and the species being investigated. Since some findings were also not predicted, REEF suggests that caution must be used to extrapolate EC exposure findings to humans.

Project context and objectives:

During the last decade, concerns have emerged about the increasing incidence of abnormalities in reproductive function and other aspects of health and wellbeing in humans, farm animals and some wildlife species and the possible correlation with exposure to ECs, including EDCs, heavy metals and other Potentially toxic elements (PTEs) and other pollutants (Toppari et al, 1996 Environ Health Perspect 104 (Suppl 4) 741-803). Moreover, it is increasingly recognised that animals are most vulnerable to such adverse effects during early stages of development (Rhind, 2005. Reprod Dom Anim 40 282-290) and there is evidence that effects of exposure in one generation can be expressed in the unexposed offspring of later generations.

In humans, problems in gametogenesis, oocyte transport, hormonal preparation of the uterine lining, implantation and viability of the conceptus all produce difficulty in achieving a recognised pregnancy, but an estimated 30 % of infertility cases are unexplained (Evers, 2002. Lancet 360 151-159). Premature ovarian failure (POF) is a primary ovarian defect characterised by absent menarche (primary amenorrhea) or premature depletion of ovarian follicles before the age of 40 years (secondary amenorrhea). Amongst the possible causes, environmental pollutants, as well as genetic defects, drugs and autoimmunity have been implicated. Populations of numerous wildlife species are in decline and in many cases exposure to ECs and EDCs, in particular, have been implicated in this process. The proposed effects have been detected in various components of reproductive systems (Rhind, 2009. Phil Trans Royal Soc 364 3391-3401).

Major stages of ovarian development (oogenesis, folliculogenesis and steroidogenesis) take place during fetal life and it is becoming increasingly clear that that female pathologies result from alterations in fetal ovary development. Indeed, oocyte production in adulthood depends on fetal ovary development since it is during this period that the germ cell stock is established, that meiosis initiates and follicles form and begin to differentiate. By breaking gestation down into discrete biologically relevant periods (sex determination and early ovary development, around primordial follicle formation, secondary and then antral follicle formation) and comparing sewage sludge exposed to non-exposed animals, we aimed to investigate the impact of real-life, environmentally relevant EC cocktails on critical events during ovary genesis. Studying maternal and fetal genes, proteins, hormones and ovarian morphology we will unravel key effects of such exposure and identify candidate genes involved in the mechanism of action of EC cocktails.

The involvement of ECs in the perturbation of female reproductive tissue development in the fetus and on subsequent adult reproductive function has been postulated (Hruska et al. Clin Obstet Gynecol 43 821-829, 2000). EDCs include a wide range of groups of chemicals, primarily anthropogenic in origin, such as dioxins, PCBs, organochlorine pesticides and plasticisers that have been/are used extensively in manufacturing and agriculture. Together with heavy metal pollutants, EDCs are ubiquitous in the environment, and domestic animals, as well as wildlife species, are potentially exposed to them (Colborn et al., 1993. Environ Health Perspect 101 378-384; Fries, 1995. J Anim Sci 73 1639 -1650). Through the consumption of meat and dairy products, and probably through many other routes, such as inhalation and absorption through skin, humans are also exposed. However, target tissue concentrations are poorly defined and their mechanisms of action are poorly understood.

Associations between tissue concentrations of EDCs and expression of variables relevant to reproductive dysfunction cannot, in isolation, demonstrate a causal relationship. This problem is compounded by the fact that mixtures of pollutants of different classes can operate in an additive (or even contrary) manner, possibly in conjunction with inorganic metal pollutants. A further difficulty arises when addressing effects on humans since deliberate contamination of human subjects to investigate possible causal relationships is ethically unacceptable. Thus, there is a need to study animal models with real-world rates of exposure, focussing on likely components of relevant reproductive circuits and mechanisms. To date, much of the work concerning effects of ECs, EDCs and other pollutants has focussed on rodent models, using single compounds administered for short periods, often at pharmacological doses. While such studies enhance understanding of the mechanisms of action of individual compounds, they do not elucidate risks to animal production, ecosystem wellbeing and human health. These depend on prolonged exposure to low real-world levels of a mixture of pollutants including low concentrations of multiple compounds. The work of this project was designed to take account of these limitations and to address effects of prolonged, low level exposure to a mixture of pollutants.

Chemicals of many classes have been shown to perturb reproductive function in animals and humans (IEH, 1999) and they are known to act additively (Rajapakse et al., 2002 Environ Health Perspect 110 917-921). Accordingly, some of our studies were based on sheep exposed to sewage sludge-treated pastures to simulate real-world exposure to a mixture of ECs. Clearly, it is impossible to measure every individual chemical, or even every class of chemical, partly because insufficient material is available in some cases. A range of chemicals representative of a number of key classes known to exert adverse biological effects was therefore selected for study. Soil and animal and human tissues were analysed in the Macaulay Institute's accredited laboratories.

The environmental burden of ECs and EDCs comprises something like 100 000 different chemicals and since it is clearly logistically impossible to measure them all, ubiquitous, environmentally-persistent chemicals with diverse chemical properties, all of which are known to exert endocrine disrupting effects, were selected for study:

(a) Diethylhexyl phthalate: Phthalates are high-production, synthetic chemicals that are ubiquitous environmental contaminants because of their use in plastics and other common consumer products. DEHP is the most abundant phthalate in the environment. Humans are exposed to these compounds through ingestion, inhalation and dermal exposure for their whole lifetime including intrauterine life. It has been suggested, previously, that tissue accumulation of phthalates was likely to be of little biological significance because they are readily degraded, either in the environment (Fries, 1996. Sci Total Environ 185 93 -108) or in the digestive tract (Heindel et al., 1989. Fundam Appl Toxicol 12 508-518) but they nevertheless they are present in sheep tissue (Rhind et al., 2005. Environ Health Perspect 113 447-453).
(b) PCBS: The ICES 7, a set of congeners (28, 52, 101, 118, 138, 153, 180) that is internationally recognised as an appropriate measure of PCB pollution, was used. These are also of special interest due to their known ubiquitous environmental persistence, tissue accumulation and reproductive toxicity. They are known to accumulate in the tissue and milk of many species of animals and humans (Bachour et al., 1998. Arch Environ Contam Toxicol 35 666-673). While concentrations of most PCB were found to be higher in maternal than fetal tissue, the reverse was found for PCBs 101 and 118 (Rhind et al., 2010; J Environ Monit 12 1582-1593). PCBs reach the embryo as early as shortly after conception by uterine fluid and while concentrations are low, it is known that early embryos are potentially sensitive to such low levels of PCB. Furthermore, in-utero exposure to PCB mix during pregnancy reduces the number of preantral and antral follicles of certain size classes in rats, indicating that the developing ovary is a sensitive target organ to PCB exposure, an important finding may account for reduced fecundity and fertility in adult offspring.
(c) Polybrominated diphenyl ethers (PBDE) congeners covering a range of bromination (28, 47, 99, 100, 153, 154, 183). While environmental levels of PCBs are generally declining, those of PBDEs have increased in recent years owing to their extensive use as fire retardants (Darnerud et al., 2001. Environ Health Perspect 109 (Suppl 1) 49-68). They are chemically related to PCBs and may exert similar effects.
(d) Polycyclic aromatic hydrocarbons (PAH): 16 compounds that have been defined by the US EPA as priority pollutants. Many of these are derived from the combustion of fossil fuels, as well as from forest fires and volcanoes and so they are highly ubiquitous in the environment.
(e) Metals (PTEs), as well as important elements in nutrition were measured Cd; Cr; Cu; Ni; Pb; Zn (toxic 6) and Hg; As, Mo, Mn, Na, K, Mg, Ba, Ti, V, W, Fe, Co, B, Al, Sn, P, Sb, Se, S. The toxicity of some metals at relatively high concentrations has long been recognised but they may also act at low concentrations, perhaps in conjunction with other chemicals.

We already know that DEHP and two PCBs preferentially accumulate in the sheep fetus following exposure of their mothers. We therefore investigated, in separate studies, effects of combinations of these chemicals on both the fetal sheep and mouse ovaries. EC-sensitive genes and proteins identified in our animal studies were used as signposts for studies of normal second trimester human fetal ovaries, to better understand the risks of ECs on human female reproductive development in the womb. The mouse was used to study the mechanisms by which ECs interfere with reproduction. We used both models to study whether or not the effects of chemicals on a developing fetus are passed on to her offspring in turn.

We hypothesised that altered female reproductive function and health could be due to the reprotoxic effects of environmental factors acting mainly during fetal life. Since our preliminary data in the sewage sludge ovine model indicated that DEHP and PCBs preferentially accumulate in the fetus, we aimed to focus on the impact of these chemicals, as well as a real-life cocktail of ECs in sewage sludge, on the developing female gonad. Through imprinting/methylation analyses, the importance of transgenerational effects of developmental insults on the fetus was investigated.

Our overall objective was to link measurements of tissue EC concentrations and associated physiological indices in animal models to parallel measurements of EC concentrations and physiological indices in human fetuses derived from mothers with different patterns of EC exposure (according to relevant indices: cigarettes/day, maternal age, weight, height, on-going illness and medication). By measuring human and animal tissue EDC concentrations and associated physiological changes in parallel, it was aimed to extrapolate animal responses to humans in a controlled manner and thereby improve understanding of relationships between exposure and effect in humans. Despite the best efforts of many research teams, this remains a critical blind-spot in our current understanding.

The objectives of the studies were:

1. To assess rates of accumulation of selected EDCs in animal tissues and, in particular, to determine:
a. relative concentrations in maternal and fetal tissues;
b. differences with chemical class in tissue burdens; and
c. effects of exposure on rates of accumulation stages of fetal development.

2. To assess impacts of real-life exposure at low concentration for a prolonged period of cocktail of chemicals on mammalian female reproduction (sheep).
3. To investigate molecular mechanisms of ovarian development affected by exposure to ECs (alone or in mixture) in-utero in sheep and mice.
4. To conduct a functional analysis of pollutant effects on female reproductive function in two animal models (sheep and mouse).
5.To investigate transgenerational transmission of reproductive defects in F2 and F3 animals.

Our ultimate objective was to determine how the developing human female fetus might be affected by exposure to ECs during development. One of our key objectives, therefore, was to integrate the mouse and sheep exposure studies with others on the human. Accordingly, we aimed to determine the expression and localisation of EC-sensitive genes in the human fetal ovary itself. This is based on the rationale that electively terminated, normal, fetuses from women who either did or did not, smoke cigarettes during pregnancy are an excellent model for the study the effects of complex mixtures of ECs on fetal human development (Fowler et al., 2012 Mol Cell Endocrinol in press). The expected outcomes of this study will allow the assessment of the nature and intensity of the reproductive responses to both selected compounds and complex mixtures.

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