A human relevant mixture of persistent organic pollutants induces reactive oxygen species formation in isolated human leucocytes: Involvement of the ß2-adrenergic receptor.

Exposure to chlorinated (Cl), brominated (Br) and perfluoroalkyl acid (PFAA) persistent organic pollutants (POPs) is associated with immunotoxicity and other adverse effects in humans and animals. Previous studies on POPs have mainly focused on single chemicals, while studies on complex mixtures are limited. Using DCF and luminol assays we examined effects on ROS generation in isolated human neutrophils, monocytes and lymphocytes, after in vitro exposure to a total mixture and sub-mixtures of 29 persistent compounds (Cl, Br, and PFAA). The mixtures were based on compounds prominent in blood, breast milk, and/or food. All mixture combinations induced ROS production in one or several of the cell models, and in some cases even at concentrations corresponding to human blood levels (compound range 1 pM - 16 nM). Whilst some interactions were detected (assessed using a mixed linear model), halogenated subgroups mainly acted additively. Mechanistic studies in neutrophils at 500× human levels (0.5 nM - 8 µM) indicated similar mechanisms of action for the Cl, PFAA, the combined PFAA + Cl and total (PFAA + Br + Cl) mixtures, and ROS responses appeared to involve ß2-adrenergic receptor (ß2AR) and Ca2+ signalling, as well as activation of NADPH oxidases. In line with this, the total mixture also increased cyclic AMP at levels comparable with the non-selective ßAR agonist, isoproterenol. Although the detailed mechanisms involved in these responses remain to be elucidated, our data show that POP mixtures at concentrations found in human blood, may trigger stress responses in circulating immune cells. Mixtures of POPs, further seemed to interfere with adrenergic pathways, indicating a novel role of ßARs in POP-induced effects.

Maternal exposure to a human relevant mixture of persistent organic pollutants reduces colorectal carcinogenesis in A/J Min/+ mice.

An increased risk of developing colorectal cancer has been associated with exposure to persistent organic pollutants (POPs) and alteration in the gut bacterial community. However, there is limited understanding about the impact of maternal exposure to POPs on colorectal cancer and gut microbiota. This study characterized the influence of exposure to a human relevant mixture of POPs during gestation and lactation on colorectal cancer, intestinal metabolite composition and microbiota in the A/J Min/+ mouse model. Surprisingly, the maternal POP exposure decreased colonic tumor burden, as shown by light microscopy and histopathological evaluation, indicating a restriction of colorectal carcinogenesis. 1H nuclear magnetic resonance spectroscopy-based metabolomic analysis identified alterations in the metabolism of amino acids, lipids, glycerophospholipids and energy in intestinal tissue. In addition, 16S rRNA sequencing of gut microbiota indicated that maternal exposure modified fecal bacterial composition. In conclusion, the results showed that early-life exposure to a mixture of POPs reduced colorectal cancer initiation and promotion, possibly through modulation of the microbial and biochemical environment. Further studies should focus on the development of colorectal cancer after combined maternal and dietary exposures to environmentally relevant low-dose POP mixtures.

Stress Resilience of Spermatozoa and Blood Mononuclear Cells without Prion Protein.

The cellular prion protein PrPC is highly expressed in neurons, but also present in non-neuronal tissues, including the testicles and spermatozoa. Most immune cells and their bone marrow precursors also express PrPC. Clearly, this protein operates in highly diverse cellular contexts. Investigations into putative stress-protective roles for PrPC have resulted in an array of functions, such as inhibition of apoptosis, stimulation of anti-oxidant enzymes, scavenging roles, and a role in nuclear DNA repair. We have studied stress resilience of spermatozoa and peripheral blood mononuclear cells (PBMCs) derived from non-transgenic goats that lack PrPC (PRNPTer/Ter) compared with cells from normal (PRNP+/+) goats. Spermatozoa were analyzed for freeze tolerance, DNA integrity, viability, motility, ATP levels, and acrosome intactness at rest and after acute stress, induced by Cu2+ ions, as well as levels of reactive oxygen species (ROS) after exposure to FeSO4 and H2O2. Surprisingly, PrPC-negative spermatozoa reacted similarly to normal spermatozoa in all read-outs. Moreover, in vitro exposure of PBMCs to Doxorubicin, H2O2 and methyl methanesulfonate (MMS), revealed no effect of PrPC on cellular survival or global accumulation of DNA damage. Similar results were obtained with human neuroblastoma (SH-SY5Y) cell lines stably expressing varying levels of PrPC. RNA sequencing of PBMCs (n = 8 of PRNP+/+ and PRNPTer/Ter) showed that basal level expression of genes encoding DNA repair enzymes, ROS scavenging, and antioxidant enzymes were unaffected by the absence of PrPC. Data presented here questions the in vitro cytoprotective roles previously attributed to PrPC, although not excluding such functions in other cell types or tissues during inflammatory stress.

Does Early Environmental Complexity Influence Tyrosine Hydroxylase in the Chicken Hippocampus and "Prefrontal" Caudolateral Nidopallium?

In adult chickens, the housing system influences hippocampal morphology and neurochemistry. However, no work has been done investigating the effects of the early life environment on chicken brain development. In the present study, we reared 67 commercial laying hens (Gallus gallus domesticus) in two environments that differed in the degree of complexity (aviary or cage system). These two groups were further divided into two age groups. At 20 weeks of age, 18 aviary-reared birds and 15 cage-reared birds were humanely euthanized and their brains dissected. At 24 weeks of age, a further 16 brains from aviary-reared birds and 18 brains from cage-reared birds were collected. These brains were prepared for immunohistochemical detection of tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of dopamine, in the hippocampus and the caudolateral nidopallium (NCL). There were no differences between the treatment groups in TH staining intensity in the hippocampus or the NCL. In the medial hippocampus, the right hemisphere had higher TH staining intensity compared to the left hemisphere. The opposite was true for the NCL, with the left hemisphere being more strongly stained compared to the right hemisphere. The present study supports the notion that the hippocampus is functionally lateralized, and our findings add to the body of knowledge on adult neural plasticity of the avian brain.

Fetal adrenal development: comparing effects of combined exposures to PCB 118 and PCB 153 in a sheep model.

This study investigated the effects of exposure to the ubiquitous contaminants polychlorinated biphenyls (PCBs) on the fetal adrenal cortex and on plasma cortisol using the domestic sheep (Ovis aries) as a model. Pregnant ewes were intendedly subjected to oral treatment with PCB 153 (98 µg/kg bw/day), PCB 118 (49 µg/kg bw/day) or the vehicle corn oil from mating until euthanasia on gestation day 134 (±0.25 SE). However, because of accidental cross-contamination occurring twice causing a mixed exposure scenario in all three groups, the focus of this paper is to compare three distinct groups of fetuses with different adipose tissue PCB levels (PCB 153high, PCB 118high and low, combined groups) rather than comparing animals exposed to single PCB congeners to those of a control group. When comparing endocrine and anatomical parameters from fetuses in the PCB 153high (n = 13) or PCB 118high (n = 14) groups with the low, combined group (n = 14), there was a significant decrease in fetal body weight (P < 0.05), plasma cortisol concentration (P < 0.001) and adrenal cortex thickness (P < 0.001). Furthermore, adrenal weight was decreased and plasma ACTH was increased only in the PCB 118high group. Expression of several genes encoding enzymes and receptors related to steroid hormone synthesis was also affected and mostly down-regulated in fetuses with high PCB tissue levels. In conclusion, we suggest that mono-and di-ortho PCBs were able to interfere with growth, adrenal development and cortisol production in the fetal sheep model. © 2011 Wiley Periodicals, Inc. Environ Toxicol, 2013.

In utero and lactational exposure to PCB 118 and PCB 153 alter ovarian follicular dynamics and GnRH-induced luteinizing hormone secretion in female lambs.

The effects of in utero and lactational exposure to two structurally different polychlorinated biphenyl (PCB) congeners on follicular dynamics and the pituitary-gonadal axis in female lambs were investigated. Pregnant ewes received corn oil, PCB 118, or PCB 153, and offspring was maintained until 60 days postpartum. Ovarian follicles were quantified using stereology. Plasma luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured using radioimmunoassay before and after administration of a gonadotropin releasing hormone (GnRH) analog. PCB 118 exposure increased numbers of transitional, secondary, and the sum of secondary, early antral, and antral (Σsecondary-antral) follicles, PCB 153 exposure only increased the number of primary follicles. GnRH-induced LH levels were significantly elevated in the PCB 153 exposure group. We conclude that PCB 153 and PCB 118 alter follicular dynamics in lambs and modulate the responsiveness of the pituitary gland to GnRH.

Effects of diets containing soybean meal on trypsin mRNA expression and activity in Atlantic salmon (Salmo salar L).

Atlantic salmon develop subacute enteritis in the distal intestine (DI) when fed diets containing soybean meal (SBM) at high levels, a condition accompanied by increased trypsin activity in the DI intestinal content compared to fish fed conventional fishmeal (FM) based diets. To further investigate the responses of Atlantic salmon to dietary SBM, we measured trypsin activity in intestinal contents, quantified pancreatic trypsin mRNA expression, surveyed trypsin mRNA expression in selected tissues and characterized active forms of trypsin in the intestinal wall and brain. Enzyme measurements showed that trypsin activity in the intestinal content of SBM fed fish was lower in the proximal segments of the intestine, but higher in the DI compared to FM fed fish. The difference in enzyme activity was not reflected in a differential expression of pancreatic trypsin mRNA between fish fed the different diets (FM or SBM). Trypsin mRNA was expressed in 18 different tissues (esophagus, stomach, pancreas, pyloric tissue, midintestine, distal intestine, liver, head kidney, kidney, heart, spleen, thymus, brain, eye, gills, gonads, muscle and skin) but was most prominently expressed in tissues of the gastrointestinal (GI) tract and brain. We report for the first time an upregulation of trypsin-like activity in the DI wall using an in-gel trypsin activity assay, as well as modulated activity in the brain of fish fed SBM. The increased activity in the DI wall may contribute to disease severity and higher trypsin activity in the intestinal content.

In vivo oocyte hydration in Atlantic halibut (Hippoglossus hippoglossus); proteolytic liberation of free amino acids, and ion transport, are driving forces for osmotic water influx.

The in vivo swelling and hydration of maturing oocytes of Atlantic halibut Hippoglossus hippoglossus were studied in order to characterise the osmotic mechanism underlying oocyte hydration in oviparous marine teleosts that spawn pelagic eggs. Sequential biopsies from two females, spanning four hydration cycles, were examined by osmometry, solute analysis and electrophoresis of dissected hydrating oocytes and ovulated eggs. The hydration cycle of the biopsied halibuts lasted 33-54 h. The majority of ovarian oocytes existed in a pre-hydrated condition (individual wet mass approx. 3.7 mg, diameter approx. 1.87 mm, 63 % H(2)O) with easily visible, non-coalesced, yolk platelets. Group-synchronous batches of the pre-hydrated oocytes increased in individual wet mass, diameter and water content to reach the ovulated egg stage of approximately 15 mg, 3.0 mm and 90 % H(2)O, respectively. The yolk osmolality of the hydrating oocytes was transiently hyperosmotic to the ovarian fluid (range 305-350 mOsmol l(-1)) with a peak osmolality of about 450 mOsmol l(-1) in oocytes of 6-8 mg individual wet mass. The transient hyperosmolality was well accounted for by the increase in oocyte content of free amino acids (FAAs; approx. 2300 nmol oocyte(-1)), K(+) (approx. 750 nmol oocyte(-1)), Cl(-) (approx. 900 nmol oocyte(-1)), total ammonium (approx. 300 nmol oocyte(-1)) and inorganic phosphate (P(i); approx. 200 nmol oocyte(-1)) when relating to the increase in cellular water. The oocyte content of Na(+) did not increase during the hydration phase. Extensive proteolysis of yolk proteins, in particular a 110 kDa protein, correlated with the increase in the FAA pool, although the latter increased by approx. 20 % more than could be accounted for by the decrease in the oocyte protein content. Both indispensable and dispensable amino acids increased in the FAA pool, and particularly serine, alanine, leucine, lysine, glutamine and glutamate. Taurine content remained stable at approx. 70 nmol oocyte(-1) during oocyte hydration. The results show that final hydration of Atlantic halibut oocytes is caused by an osmotic water uptake in which FAAs, derived mainly from the hydrolysis of a 110 kDa yolk protein, contribute approximately 50 % of the yolk osmolality and ions (Cl(-), K(+), P(i), NH(4)(+)) make up the balance.