Environmental chemicals impact dog semen quality in vitro and may be associated with a temporal decline in sperm motility and increased cryptorchidism.

Adverse temporal trends in human semen quality and cryptorchidism in infants have been associated with exposure to environmental chemicals (ECs) during development. Here we report that a population of breeding dogs exhibit a 26 year (1988-2014) decline in sperm quality and a concurrent increased incidence of cryptorchidism in male offspring (1995-2014). A decline in the number of males born relative to the number of females was also observed. ECs, including diethylhexyl phthalate (DEHP) and polychlorinated biphenyl 153 (PCB153), were detected in adult dog testes and commercial dog foods at concentrations reported to perturb reproductive function in other species. Testicular concentrations of DEHP and PCB153 perturbed sperm viability, motility and DNA integrity in vitro but did not affect LH stimulated testosterone secretion from adult testis explants. The direct effects of chemicals on sperm may therefore contribute to the decline in canine semen quality that parallels that reported in the human.

Evaluation of spot and passive sampling for monitoring, flux estimation and risk assessment of pesticides within the constraints of a typical regulatory monitoring scheme.

In many agricultural catchments of Europe and North America, pesticides occur at generally low concentrations with significant temporal variation. This poses several challenges for both monitoring and understanding ecological risks/impacts of these chemicals. This study aimed to compare the performance of passive and spot sampling strategies given the constraints of typical regulatory monitoring. Nine pesticides were investigated in a river currently undergoing regulatory monitoring (River Ugie, Scotland). Within this regulatory framework, spot and passive sampling were undertaken to understand spatiotemporal occurrence, mass loads and ecological risks. All the target pesticides were detected in water by both sampling strategies. Chlorotoluron was observed to be the dominant pesticide by both spot (maximum: 111.8ng/l, mean: 9.35ng/l) and passive sampling (maximum: 39.24ng/l, mean: 4.76ng/l). The annual pesticide loads were estimated to be 2735g and 1837g based on the spot and passive sampling data, respectively. The spatiotemporal trend suggested that agricultural activities were the primary source of the compounds with variability in loads explained in large by timing of pesticide applications and rainfall. The risk assessment showed chlorotoluron and chlorpyrifos posed the highest ecological risks with 23% of the chlorotoluron spot samples and 36% of the chlorpyrifos passive samples resulting in a Risk Quotient greater than 0.1. This suggests that mitigation measures might need to be taken to reduce the input of pesticides into the river. The overall comparison of the two sampling strategies supported the hypothesis that passive sampling tends to integrate the contaminants over a period of exposure and allows quantification of contamination at low concentration. The results suggested that within a regulatory monitoring context passive sampling was more suitable for flux estimation and risk assessment of trace contaminants which cannot be diagnosed by spot sampling and for determining if long-term average concentrations comply with specified standards.

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.

A study on temporal trends and estimates of fate of Bisphenol A in agricultural soils after sewage sludge amendment.

Temporal concentration trends of BPA in soils were investigated following sewage sludge application to pasture (study 1: short term sludge application; study 2: long term multiple applications over 13 years). The background levels of BPA in control soils were similar, ranging between 0.67-10.57 ng g(-1) (mean: 3.02 ng g(-1)) and 0.51-6.58 ng g(-1) (mean: 3.22 ng g(-1)) for studies 1 and 2, respectively. Concentrations in both treated and control plots increased over the earlier sampling times of the study to a maximum and then decreased over later sampling times, suggesting other sources of BPA to both the treated and control soils over the study period. In study 1 there was a significant treatment effect of sludge application in the autumn (p=0.002) although no significant difference was observed between treatment and control soils in the spring. In study 2 treated soils contained considerably higher BPA concentrations than controls ranging between 12.89-167.9 ng g(-1) (mean: 63.15 ng g(-1)). This and earlier studies indicate the long-term accumulation of multiple contaminants by multiple sewage sludge applications over a prolonged period although the effects of the presence of such contaminant mixtures have not yet been elucidated. Fugacity modelling was undertaken to estimate partitioning of Bisphenol A (soil plus sewage: pore water: soil air partitioning) and potential uptake into a range of food crops. While Bisphenol A sorbs strongly to the sewage-amended soil, 4% by mass was predicted to enter soil pore water resulting in significant uptake by crops particularly leafy vegetables (3.12-75.5 ng g(-1)), but also for root crops (1.28-31.0 ng g(-1)) with much lower uptake into cereal grains (0.62-15.0 ng g(-1)). This work forms part of a larger programme of research aimed at assessing the risks associated with the long-term application of sewage sludge to agricultural soils.

In utero exposure to cigarette chemicals induces sex-specific disruption of one-carbon metabolism and DNA methylation in the human fetal liver.

BACKGROUND: Maternal smoking is one of the most important modifiable risk factors for low birthweight, which is strongly associated with increased cardiometabolic disease risk in adulthood. Maternal smoking reduces the levels of the methyl donor vitamin B12 and is associated with altered DNA methylation at birth. Altered DNA methylation may be an important mechanism underlying increased disease susceptibility; however, the extent to which this can be induced in the developing fetus is unknown. METHODS: In this retrospective study, we measured concentrations of cobalt, vitamin B12, and mRNA transcripts encoding key enzymes in the 1-carbon cycle in 55 fetal human livers obtained from 11 to 21 weeks of gestation elective terminations and matched for gestation and maternal smoking. DNA methylation was measured at critical regions known to be susceptible to the in utero environment. Homocysteine concentrations were analyzed in plasma from 60 fetuses. RESULTS: In addition to identifying baseline sex differences, we found that maternal smoking was associated with sex-specific alterations of fetal liver vitamin B12, plasma homocysteine and expression of enzymes in the 1-carbon cycle in fetal liver. In the majority of the measured parameters which showed a sex difference, maternal smoking reduced the magnitude of that difference. Maternal smoking also altered DNA methylation at the imprinted gene IGF2 and the glucocorticoid receptor (GR/NR3C1). CONCLUSIONS: Our unique data strengthen studies linking in utero exposures to altered DNA methylation by showing, for the first time, that such changes are present in fetal life and in a key metabolic target tissue, human fetal liver. Furthermore, these data propose a novel mechanism by which such changes are induced, namely through alterations in methyl donor availability and changes in 1-carbon metabolism.

In utero exposure to cigarette smoke dysregulates human fetal ovarian developmental signalling.

STUDY QUESTION: How does maternal cigarette smoking disturb development of the human fetal ovary? SUMMARY ANSWER: Maternal smoking increases fetal estrogen titres and dysregulates several developmental processes in the fetal ovary. WHAT IS KNOWN ALREADY: Exposure to maternal cigarette smoking during gestation reduces human fetal ovarian cell numbers, germ cell proliferation and subsequent adult fecundity. STUDY DESIGN, SIZE, DURATION: The effects of maternal cigarette smoking on the second trimester human fetal ovary, fetal endocrine signalling and fetal chemical burden were studied. A total of 105 fetuses were studied, 56 from mothers who smoked during pregnancy and 49 from those who did not. PARTICIPANTS/MATERIALS, SETTING METHODS: Ovary, liver and plasma samples were collected from electively terminated, normally progressing, second trimester human fetuses. Circulating fetal hormones, levels of 73 fetal ovarian transcripts, protein localization, density of oocytes/primordial follicles and levels of 16 polycyclic aromatic hydrocarbons (PAHs) in the fetal liver were determined. MAIN RESULTS AND THE ROLE OF CHANCE: Circulating fetal estrogen levels were very high and were increased by maternal smoking (ANOVA, P = 0.055-0.004 versus control). Smoke exposure also dysregulated (two-way ANOVA, smoking versus gestation weeks interaction, P = 0.046-0.023) four fetal ovarian genes (cytochrome P450 scc [CYP11A1], NOBOX oogenesis homeobox [NOBOX], activator of apoptosis harakiri [HRK], nuclear receptor subfamily 2, group E, member 1 [NR2E1]), shifted the ovarian Inhibin ßA/inhibin α ratio (NHBA/INHA) transcript ratio in favour of activin (ANOVA, P = 0.049 versus control) and reduced the proportion of dominant-negative estrogen receptor 2 (ERß: ESR2) isoforms in half the exposed fetuses. PAHs, ligands for the aryl hydrocarbon receptor (AHR), were increased nearly 6-fold by maternal smoking (ANOVA, P = 0.011 versus control). A fifth transcript, COUP transcription factor 1 (nuclear receptor subfamily 2, group F, member 1: NR2F1, which contains multiple AHR-binding sites), was both significantly increased (ANOVA, P = 0.026 versus control) and dysregulated by (two-way ANOVA, smoking versus gestation weeks interaction, P = 0.021) maternal smoking. NR2F1 is associated with repression of FSHR expression and smoke-exposed ovaries failed to show the normal increase in FSHR expression during the second trimester. There was a significantly higher number of DEAD (Asp-Glu-Ala-Asp) box polypeptide 4 (DDX4) VASA-positive (ANOVA, P = 0.016 versus control), but not POU domain, class 1, transcription factor 1 (POU5F1) OCT3/4-positive, oocytes in smoke-exposed fetuses and this matched with a significantly higher number of primordial follicles (ANOVA, P = 0.024 versus control). LIMITATIONS, REASONS FOR CAUTION: The effects of maternal smoking on establishment of the maximum fetal primordial follicle pool cannot be reliably studied in our population since the process is not completed until 28 weeks of gestation and normal fetuses older than 21 weeks of gestation are not available for study. Our data suggest that some fetal ovaries are affected by smoke exposure while others are not, indicating that additional studies, with larger numbers, may show more significant effects. WIDER IMPLICATIONS OF THE FINDINGS: Fetal exposure to chemicals in cigarette smoke is known to lead to reduced fecundity in women. Our study suggests, for the first time, that this occurs via mechanisms involving activation of AHR, disruption of inhibin/activin and estrogen signalling, increased exposure to estrogen and dysregulation of multiple molecular pathways in the exposed human fetal ovary. Our data also suggest that alterations in the ESR2 positive and dominant negative isoforms may be associated with reduced sensitivity of some fetuses to increased estrogens and maternal smoking. STUDY FUNDING/COMPETING INTEREST(S): The study was supported by grants from the Chief Scientist Office (Scottish Executive, CZG/1/109, and CZG/4/742), NHS Grampian Endowments (08/02), the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 212885, a Society for Reproduction & Fertility summer studentship, Medical Research Scotland (research grant 354 FRG) and the Medical Research Council (WBS: U.1276.00.002.00001 and G1100357). The authors declare they have no competing interests, be it financial, personal or professional.

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.

Maternal undernutrition does not alter Sertoli cell numbers or the expression of key developmental markers in the mid-gestation ovine fetal testis.

BACKGROUND: The aim of this study was to determine the effects of maternal undernutrition on ovine fetal testis morphology and expression of relevant histological indicators. Maternal undernutrition, in sheep, has been reported, previously, to alter fetal ovary development, as indicated by delayed folliculogenesis and the altered expression of ovarian apoptosis-regulating gene products, at day 110 of gestation. It is not known whether or not maternal undernutrition alters the same gene products in the day 110 fetal testis. DESIGN AND METHODS: Mature Scottish Blackface ewes were fed either 100% (Control; C) or 50% (low; L) of estimated metabolisable energy requirements of a pregnant ewe, from mating to day 110 of gestation. All pregnant ewes were euthanized at day 110 and a sub-set of male fetuses was randomly selected (6 C and 9 L) for histology studies designed to address the effect of nutritional state on several indices of testis development. Sertoli cell numbers were measured using a stereological method and Ki67 (cell proliferation index), Bax (pro-apoptosis), Mcl-1 (anti-apoptosis), SCF and c-kit ligand (development and apoptosis) gene expression was measured in Bouins-fixed fetal testis using immunohistochemistry. RESULTS: No significant differences were observed in numbers of Sertoli cells or testicular Ki67 positive cells. The latter were localised to the testicular cords and interstitium. Bax and Mcl-1 were localised specifically to the germ cells whereas c-kit was localised to both the cords and interstitium. SCF staining was very sparse. No treatment effects were observed for any of the markers examined. CONCLUSIONS: These data suggest that, unlike in the fetal ovary, maternal undernutrition for the first 110 days of gestation affects neither the morphology of the fetal testis nor the expression of gene products which regulate apoptosis. It is postulated that the effects of fetal undernutrition on testis function may be expressed through hypothalamic-pituitary changes.

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.

Risk assessment of the use of PAS100 green composts in sheep and cattle production in Scotland.

A generalized quantitative risk assessment for the use of source-segregated green waste (SSGW) compost use in livestock production is presented. This assessment focussed on potential risks associated with a specific product, PAS100 compost that meets the UK publicly available specification 100 and represents the majority of compost available for use in extensive Scottish livestock systems. A hazard screening approach was used to identify all potentially hazardous agents present in compost. A total of 497 potentially hazardous agents were screened, with 147 finally put forward for quantitative risk assessment. Scenarios modelled in the assessment included surface application of compost to grazing land and also incorporation into soil and subsequent uptake by fodder crops. Risk estimates were compared to those associated with six comparator materials, including various sludges, slurries and farm yard manures. Overall, five potentially hazardous agents (PCB28, PCB138, PCB153, 1,2,3,4,6,7,8-HpCDD, Clopyralid) returned a hazard quotient >1 but within margins of uncertainty, indicating that further investigation may be required. Within the limitations of available information, SSGW compost was found to pose less risk to grazing livestock, or the environment, than other commonly-used soil amendments. While this assessment relates to a specific product/standard used in the UK, the methodology could easily be applied to other composts/products/situations. Therefore these results have wider applicability.

Protein-energy malnutrition during early gestation in sheep blunts fetal renal vascular and nephron development and compromises adult renal function.

A nutritionally poor maternal diet can reduce nephron endowment and pre-empt premature expression of markers for chronic renal disease in the offspring. A mechanistic pathway from variation in maternal diet through altered fetal renal development to compromised adult kidney structure and function with adult-onset obesity has not been described. We show that maternal protein-energy malnutrition in sheep blunts nephrogenic potential in the 0.44 gestation (65 days gestation, term ∼147 days) fetus by increasing apoptosis and decreasing angiogenesis in the nephrogenic zone, effects that were more marked in male fetuses. As adults, the low-protein-exposed sheep had reduced glomerular number and microvascular rarefaction in their kidneys compensated for, respectively, by glomerular hypertrophy and increased angiogenic support. In this study, the long-term mild anatomical deficits in the kidney would have remained asymptomatic in the lean state, but when superimposed on the broad metabolic challenge that obesity represents then microalbuminuria and blunted bilateral renal function revealed a long-term physiological compromise, that is only predicted to worsen with age. In conclusion, maternal protein-energy malnutrition specifically impacts fetal kidney vascular development and prevents full functionality of the adult kidney being achieved; these residual deficits are predicted to significantly increase the expected incidence of chronic kidney disease in prenatally undernourished individuals especially when coupled with a Western obesogenic environment.

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.

Effects of polychlorinated biphenyls in CD-1 mice: reproductive toxicity and intergenerational transmission.

Several studies indicate that in utero and perinatal exposure to polychlorinated biphenyls (PCBs) induces adverse reproductive effects, but it remains unclear whether such effects may be transmitted to subsequent generations. We therefore investigated the association between maternal exposure to PCBs and reproductive health in male and female offspring over three generations. Mouse dams were fed 0, 1, 10, and 100 µg/kg/day of a PCB mixture (101 + 118) during pregnancy and lactation. PCB levels were measured in the tissues of both dams and offspring. PCB concentrations at all doses investigated were greater in the offspring than in the dams (p ≤ 0.0001) confirming that the progeny were exposed as a result of maternal exposure. In F1 offspring, exposure to PCBs resulted in reductions in (1) testis weight (p ≤ 0.05) and seminiferous tubule diameter (p ≤ 0.05), (2) sperm viability (p ≤ 0.0001) and developmental capacity (p ≤ 0.05), (3) ovary weight (p ≤ 0.05), (4) oocyte developmental capacity (p ≤ 0.05), and (5) increased follicular atresia (p ≤ 0.0001). In females, adverse effects were observed only in the F1 animals. In contrast, male offspring exhibited reduced sperm viability and altered seminiferous tubule distribution up to the third generation, showing intergenerational transmission. In summary, our data indicate that exposure to PCBs at the time of gonadal sex determination perturbed, significantly, the reproductive physiology of male and female offspring in adulthood. Furthermore, male reproductive deficiencies may be observed in at least two further generations. These findings have significant implications for reproductive health and fertility of animals and humans.

Optimized determination of polybrominated diphenyl ethers and polychlorinated biphenyls in sheep serum by solid-phase extraction-gas chromatography-mass spectrometry.

We describe a solid-phase extraction (SPE) method, followed by gas chromatography-mass spectrometry (GC-MS), for the simultaneous determination of polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs) in sheep serum samples. The denaturation of serum proteins by formic acid, water-1-propanol and water-2-propanol were compared and optimized. Seven different solid-phase sorbents were tested and it was found that Strata-X cartridge (200mg, 6 mL) gave the best recoveries (92-106%, SD<6%, n=3) for all the target analytes. The different extraction solvents (iso-hexane and dichloromethane), either alone or in combination, were used to extract these persistent organic compounds from spiked serum samples by SPE. Removal of co-extracted biogenic materials was achieved using adsorption chromatography with acid modified silica and activated silica. Iso-hexane was found to be the most appropriate solvent for clean-up providing good recoveries and clear chromatographic separation; its use is preferable to that of DCM because it is less environmentally toxic. The limits of detection (LOD) of the proposed method were 47-105 pg g(-1) and 16-24 pg g(-1) for the different PBDEs and PCBs studied, respectively. The developed method was linear over the range from 0.05 to 30 ng g(-1), for all PBDEs except PBDE 183 (0.10-30 ng g(-1)), and from 0.02 to 30 ng g(-1) for all tested PCB congeners. The established method was successfully applied to sheep serum samples from Scotland, UK, for the determination of the target PBDEs and PCBs.

Simultaneous extraction and clean-up of polybrominated diphenyl ethers and polychlorinated biphenyls from sheep liver tissue by selective pressurized liquid extraction and analysis by gas chromatography-mass spectrometry.

We describe a selective pressurized liquid extraction (SPLE) method, followed by gas chromatography-mass spectrometry (GC-MS), for the simultaneous extraction and clean-up of polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs) in sheep liver tissue samples. The on-line clean-up of liver tissue by SPLE was tested using differing amount of acid-modified silica (sulphuric acid:silica gel, 1:2, w/w), the most effective amount being 20 g. Different extraction solvents (iso-hexane and dichloromethane), either alone or in various combinations, were used to extract these target compounds from spiked liver samples. Variables affecting the SPLE extraction efficiency, including temperature, pressure, number of extraction cycles and static extraction time were studied; the optimum parameters were 80 °C, 10.3 MPa, 2 cycles and 5 min, respectively. The SPLE based method was compared with more traditional Soxhlet, off-line PLE, ultrasonic and heating extraction methods. Overall the mean percentage recoveries for all target chemicals using SPLE were 86-103% (n=3, SD < 9%), and compared favourably with the Soxhlet (63-109%, n=3, SD < 8%), off-line PLE (82-104%, n=3, SD < 18%), ultrasonic (86-99%, n=3, SD < 11%) and heating (72-102%, n=3, SD < 21%) extraction methods. The limits of detection of the proposed method were 5-96 pg g⁻¹ and 2-29 pg g⁻¹ for the different PBDE and PCB chemicals studied, respectively. The outputs of the proposed method were linear over the range from 0.02 to 30 ng g⁻¹, for all PCB and PBDE congeners except for PBDE 100 and 153 (0.05-30 ng g⁻¹) and PBDE 183 (0.1-30 ng g⁻¹). The method was successfully applied to sheep liver samples for the determination of the target PBDE and PCB compounds.

Selective pressurized liquid extraction of estrogenic compounds in soil and analysis by gas chromatography-mass spectrometry.

A selective pressurized liquid extraction (SPLE) method, followed by gas chromatography-mass spectrometry (GC-MS), for the simultaneous extraction and clean-up of estrone (E1), 17ß-estradiol (E2), 17α-ethynylestradiol (EE2), estriol (E3) and bisphenol A (BPA) from soil samples is described. The on-line clean-up of soil by SPLE was achieved using different organic matter retainers, including silica, alumina and Florisil, the most effective being silica. Thus, different amounts of silica, in conjunction with different extraction solvents (acetone, ethyl acetate, isohexane and dichloromethane), either alone or in combination, were used to extract the target chemicals from spiked soil samples. It was shown that 3g silica resulted in satisfactory rates of recovery of target compounds and acetone:dichloromethane (1:3, v/v) was efficient in extracting and eluting estrogenic compounds for SPLE. Variables affecting the SPLE efficiency, including temperature and pressure were studied; the optimum parameters were 60°C and 1500 psi, respectively. The limits of detection (LODs) of the proposed method were 0.02-0.37 ng g(-1) for the different estrogenic chemicals studied. The outputs using the proposed method were linear over the range from 0.1 to 120 ng g(-1) for E1, E2, EE2, 0.2-120 ng g(-1) for E3, and 0.5-120 ng g(-1) for BPA. The optimized method was further verified by performing spiking experiments in natural soil matrices; good rates of recovery and reproducibility were achieved for all selected compounds and the method was successfully applied to soil samples from Northeast Scotland, for the determination of the target compounds.

Pregnant ewes exposed to multiple endocrine disrupting pollutants through sewage sludge-fertilized pasture show an anti-estrogenic effect in their trabecular bone.

Pregnant ewes were maintained on pastures fertilized, twice yearly, with either sewage sludge (2.25tonnes dry matter/ha; Treated; T) or inorganic fertilizer containing equivalent amounts of nitrogen (Control; C), to determine effects on maternal and fetal bone structures, density and mechanical properties of exposure to environmental concentrations of multiple endocrine disrupting compounds (EDCs) and heavy metal pollutants. The ewes were maintained on the respective pastures from the age of about 8months until they were 4-6years of age and they were slaughtered at 110d gestation. Metaphyseal parts of adult ewe femurs exhibited a significantly reduced mean, total cross sectional area (CSA, -4%; p<0.05), lower trabecular bone mineral content (BMC, mg/mm; -18%; p<0.05), trabecular bone mineral density (BMD, mg/cm(3), -8.0%; p<0.05) and trabecular CSA, mm(2), -11.1%; p<0.05) in T compared with C animals. Femurs of T ewes were stronger than those of C ewes but this may reflect greater body weights. At the mid-diaphyseal part of the fetal bones, there was a reduction in endosteal circumference (-6.7%, p<0.05) and marrow cavity area (-13.8%, p<0.05) in the female T fetuses compared with female C fetuses. In the male fetuses the mid-diaphyseal part total bone mineral content was higher (+3.0%, p<0.05) in T than in C animals. No treatment difference in biomechanical bending was detected in the fetuses. It is concluded that ewes grazing pasture fertilized with sewage sludge exhibited an anti-estrogenic effect on their trabecular bone in the form of reduced mineral content and density, despite increased body weight. It is suggested that human exposure to low levels of multiple EDCs may have implications for bone structure and human health.

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.