Long-term exposure to chemicals in sewage sludge fertilizer alters liver lipid content in females and cancer marker expression in males.

BACKGROUND: The increased incidence of diseases, including metabolic syndrome and infertility, may be related to exposure to the mixture of chemicals, which are ubiquitous in the modern environment (environmental chemicals, ECs). Xeno-detoxification occurs within the liver which is also the source of many plasma proteins and growth factors and plays an important role in the regulation of homeostasis. OBJECTIVES: The objective of this study was to investigate the effects of ECs on aspects of liver function, in a well characterized ovine model of exposure to a real-life EC mixture. METHODS: Four groups of sheep (n = 10-12/sex/treatment) were maintained long-term on control or sewage sludge-fertilized pastures: from conception to culling at 19 months of age in females and from conception to 7 months of age and thereafter in control plots until culling at 19 months of age in males. Environmental chemicals were measured in sheep livers and RNA and protein extracts were assessed for exposure markers. Liver proteins were resolved using 2D differential in-gel electrophoresis and differentially expressed protein spots were identified by liquid chromatography/tandem mass spectroscopy. RESULTS: Higher levels of polycyclic aromatic hydrocarbons (PAHs) and lower levels of polychlorinated biphenyls (PCBs) in the livers of control males compared to control females indicated sexually dimorphic EC body burdens. Increased levels of the PAHs Benzo[a]anthracene and chrysene and reduced levels of PCB 153 and PCB 180 were observed in the livers of continuously exposed females. EC exposure affected xenobiotic and detoxification responses and the liver proteome in both sexes and included major plasma-secreted and blood proteins, and metabolic enzymes whose pathway analysis predicted dysregulation of cancer-related pathways and altered lipid dynamics. The latter were confirmed by a reduction in total lipids in female livers and up-regulation of cancer-related transcript markers in male livers respectively by sewage sludge exposure. CONCLUSIONS: Our results demonstrate that chronic exposure to ECs causes major physiological changes in the liver, likely to affect multiple systems in the body and which may predispose individuals to increased disease risks.

The human placental proteome is affected by maternal smoking.

Detrimental effects of maternal smoking on the term placental proteome and steroid-metabolizing activities, and maternal hormone levels, were studied by using seven non-smoker and seven smoker placentae. Smoking significantly affected 18% of protein spots. The functional networks affected were i) cell morphology, cellular assembly and organization, cellular compromise (15 hits) and ii) DNA replication, recombination, and repair, energy production, nucleic acid metabolism (6 hits). Smoking significantly up-regulated such proteins as, SERPINA1, EFHD1 and KRT8; and down-regulated SERPINB2, FGA and HBB. Although maternal plasma steroids were not significantly altered, the catalytic activity of CYP1A1 was increased whereas CYP19A1 activity was reduced by smoking. Furthermore, transcript expression of CYP1A1 and CYP4B1 were induced while HSD17B2, NFKB and TGFB1 were repressed by smoking. The observed smoking induced wide-spread changes on placental proteome and transcript levels may contribute to the lowered birth weights of the new-born child and placenta.

The fetal ovary exhibits temporal sensitivity to a 'real-life' mixture of environmental chemicals.

The development of fetal ovarian follicles is a critical determinant of adult female reproductive competence. Prolonged exposure to environmental chemicals (ECs) can perturb this process with detrimental consequences for offspring. Here we report on the exposure of pregnant ewes to an environmental mixture of ECs derived from pastures fertilized with sewage sludge (biosolids): a common global agricultural practice. Exposure of pregnant ewes to ECs over 80 day periods during early, mid or late gestation reduced the proportion of healthy early stage fetal follicles comprising the ovarian reserve. Mid and late gestation EC exposures had the most marked effects, disturbing maternal and fetal liver chemical profiles, masculinising fetal anogenital distance and greatly increasing the number of altered fetal ovarian genes and proteins. In conclusion, differential temporal sensitivity of the fetus and its ovaries to EC mixtures has implications for adult ovarian function following adverse exposures during pregnancy.

Exposure to chemical cocktails before or after conception--- the effect of timing on ovarian development.

Exposure of female fetuses to environmental chemicals (ECs) during pregnancy results in a disturbed ovarian adult phenotype. We investigated the influence of pre- and/or post-conception exposure to low-level mixtures of ECs on the structure and function of the fetal ovine ovary. We examined ovarian morphology, expression of oocyte and granulosa cell-specific genes and proteome. Female fetuses were collected at day 110 of gestation, from dams exposed continuously until, and after mating, by grazing in pastures treated with sewage sludge as a fertiliser (TT) or in control fields treated with inorganic fertiliser (CC). In addition, in a cross-over design, fetal ovaries were collected from dams maintained on sludge pastures up to the time of mating but then transferred to control pastures (TC) and, reciprocally, those transferred from control to treated pastures at mating (CT). On examination, the proportion of type 1a follicles (activating primordial follicles) was significantly lower in animals from the CT groups compared with CC and TT groups (P<0.05). Of the 23 ovarian gene transcripts studied, 14 were altered in the ovaries of exposed fetuses (CT, TC, and TT) relative to controls, with the largest number of changes observed in cross-exposure pattern groups (CT or TC). Continuous EC exposure (TT) produced fewer transcript alterations and only two genes (INHBA and GSN) presented differential profiles between CC and TT. Fetal ovarian proteome analysis (2-DE gels) showed, across all exposure groups, 86 differentially expressed protein spots compared to controls. Animals in the CT group exhibited the highest number (53) while TC and TT presented the same number of affected protein spots (42). Fetal ovarian proteins with altered expression included MVP (major vault protein) and several members of the heat-shock family (HSPA4L, HSP90AA1 and HSF1). The present findings indicate that continuous maternal EC exposure before and during gestation, are less deleterious for fetal ovarian development than a change in maternal EC exposure between pre and post-conception. The pathways by which the ovary responds to this chemical stress were common in TT, CT, TC exposed foetuses. In addition to the period of pregnancy, the pre-conception period appears also as crucial for conditioning long-term effects of EC exposure on ovarian development and primordial follicle reserve and hence future fertility.

Peri-conceptional changes in maternal exposure to sewage sludge chemicals disturbs fetal thyroid gland development in sheep.

Ewes were exposed to sewage sludge-fertilized pastures in a study designed investigate pre-conceptual and/or gestational exposure to environmental chemicals. The in utero impact on fetal thyroid morphology and function at day 110 (of 145) of pregnancy was then determined. Pre-conceptual exposure increased the relative thyroid organ weights in male fetuses. The number of thyroid follicles in thyroids of fetuses after pre-conceptual or gestational exposure was reduced. This correlated with an increase in Ki67 positive cells. Pre-conceptual exposure to sewage sludge reduced small blood vessels in fetal thyroids. Thyroid tissues of exposed fetuses contained regions where mature angio-follicular units were reduced exhibiting decreased immunostaining for sodium-iodide symporter (NIS). Fetal plasma levels of fT3 and fT4 in exposed animals, however, were not different from controls suggesting compensatory changes in the thyroid gland to maintain homeostasis in exposed fetuses. The regional aberrations in thyroid morphology may impact on the post-natal life of the exposed offspring.

Impact of sublethal levels of environmental pollutants found in sewage sludge on a novel Caenorhabditis elegans model biosensor.

A transgenic strain of the model nematode Caenorhabditis elegans in which bioluminescence reports on relative, whole-organism ATP levels was used to test an environmentally-relevant mixture of pollutants extracted from processed sewage sludge. Changes in bioluminescence, following exposure to sewage sludge extract, were used to assess relative ATP levels and overall metabolic health. Reproductive function and longevity were also monitored. A short (up to 8 h) sublethal exposure of L4 larval stage worms to sewage sludge extract had a concentration-dependent, detrimental effect on energy status, with bioluminescence decreasing to 50-60% of the solvent control (1% DMSO). Following longer exposure (22-24 h), the energy status of the nematodes showed recovery as assessed by bioluminescence. Continuous exposure to sewage sludge extract from the L4 stage resulted in a shorter median lifespan relative to that of solvent or medium control animals, but only in the presence of 400-600 µM 5-fluoro-2'-deoxyuridine (FUdR), which was incorporated to inhibit reproduction. This indicated that FUdR increased lifespan, and that the effect was counteracted by SSE. Exposure to sewage sludge extract from the L1 stage led to slower growth and a delayed onset of egg laying. When L1 exposed nematodes reached the reproductive stage, no effect on egg laying rate or egg number in the uterus was observed. DMSO itself (1%) had a significant inhibitory effect on growth and development of C. elegans exposed from the L1 stage and on reproduction when exposed from the L4 stage. Results demonstrate subtle adverse effects on C. elegans of a complex mixture of environmental pollutants that are present, individually, in very low concentrations and indicate that our biosensor of energy status is a novel, sensitive, rapid, quantitative, whole-organism test system which is suitable for high throughput risk assessment of complex pollutant mixtures.

Impact of endocrine-disrupting compounds (EDCs) on female reproductive health.

Evidence is accumulating that environmental chemicals (ECs) including endocrine-disrupting compounds (EDCs) can alter female reproductive development, fertility and onset of menopause. While not as clearly defined as in the male, this set of abnormalities may constitute an Ovarian Dysgenesis Syndrome with at least some origins of the syndrome arising during foetal development. ECs/EDCs have been shown to affect trophoblast and placental function, the female hypothalamo-pituitary-gonadal axis, onset of puberty and adult ovarian function. The effects of ECs/EDCs are complex, not least because it is emerging that low-level, 'real-life' mixtures of ECs/EDCs may carry significant biological potency. In addition, there is evidence that ECs/EDCs can alter the epigenome in a sexually dimorphic manner, which may lead to changes in the germ line and perhaps even to transgenerational effects. This review summarises the evidence for EC, including EDC, involvement in female reproductive dysfunction, it highlights potential mechanisms of EC action in the female and emphasises the need for further research into EC effects on female development and reproductive function.

Exposure to a complex cocktail of environmental endocrine-disrupting compounds disturbs the kisspeptin/GPR54 system in ovine hypothalamus and pituitary gland.

BACKGROUND: Ubiquitous environmental chemicals, including endocrine-disrupting chemicals (EDCs), are associated with declining human reproductive health, as well as an increasing incidence of cancers of the reproductive system. Verifying such links requires animal models exposed to "real-life," environmentally relevant concentrations/mixtures of EDC, particularly in utero, when sensitivity to EDC exposure is maximal. OBJECTIVES: We evaluated the effects of maternal exposure to a pollutant cocktail (sewage sludge) on the ovine fetal reproductive neuroendocrine axes, particularly the kisspeptin (KiSS-1)/GPR54 (G-protein-coupled receptor 54) system. METHODS: KiSS-1, GPR54, and ERalpha (estrogen receptor alpha) mRNA expression was quantified in control (C) and treated (T) maternal and fetal (110-day) hypothalami and pituitary glands using semiquantitative reverse transcription polymerase chain reaction, and colocalization of kisspeptin with LHbeta (luteinizing hormone beta) and ERalpha in C and T fetal pituitary glands quantified using dual-labeling immunohistochemistry. RESULTS: Fetuses exposed in utero to the EDC mixture showed reduced KiSS-1 mRNA expression across three hypothalamic regions examined (rostral, mid, and caudal) and had fewer kisspetin immunopositive cells colocalized with both LHbeta and ERalpha in the pituitary gland. In contrast, treatment had no effect on parameters measured in the adult ewe hypothalamus or pituitary. CONCLUSIONS: This study demonstrates that the developing fetus is sensitive to real-world mixtures of environmental chemicals, which cause significant neuroendocrine alterations. The important role of kisspeptin/GPR54 in regulating puberty and adult reproduction means that in utero disruption of this system is likely to have long-term consequences in adulthood and represents a novel, additional pathway through which environmental chemicals perturb human reproduction.

In utero exposure to low doses of environmental pollutants disrupts fetal ovarian development in sheep.

Epidemiological studies of the impact of environmental chemicals on reproductive health demonstrate consequences of exposure but establishing causative links requires animal models using 'real life' in utero exposures. We aimed to determine whether prolonged, low-dose, exposure of pregnant sheep to a mixture of environmental chemicals affects fetal ovarian development. Exposure of treated ewes (n = 7) to pollutants was maximized by surface application of processed sewage sludge to pasture. Control ewes (n = 10) were reared on pasture treated with inorganic fertilizer. Ovaries and blood were collected from fetuses (n = 15 control and n = 8 treated) on Day 110 of gestation for investigation of fetal endocrinology, ovarian follicle/oocyte numbers and ovarian proteome. Treated fetuses were 14% lighter than controls but fetal ovary weights were unchanged. Prolactin (48% lower) was the only measured hormone significantly affected by treatment. Treatment reduced numbers of growth differentiation factor (GDF9) and induced myeloid leukaemia cell differentiation protein (MCL1) positive oocytes by 25-26% and increased pro-apoptotic BAX by 65% and 42% of protein spots in the treated ovarian proteome were differently expressed compared with controls. Nineteen spots were identified and included proteins involved in gene expression/transcription, protein synthesis, phosphorylation and receptor activity. Fetal exposure to environmental chemicals, via the mother, significantly perturbs fetal ovarian development. If such effects are replicated in humans, premature menopause could be an outcome.