Evaluation of contaminant removal of reverse osmosis and advanced oxidation in full-scale operation by combining passive sampling with chemical analysis and bioanalytical tools.

Advanced water treatment of secondary treated effluent requires stringent quality control to achieve a water quality suitable for augmenting drinking water supplies. The removal of micropollutants such as pesticides, industrial chemicals, endocrine disrupting chemicals (EDC), pharmaceuticals, and personal care products (PPCP) is paramount. As the concentrations of individual contaminants are typically low, frequent analytical screening is both laborious and costly. We propose and validate an approach for continuous monitoring by applying passive sampling with Empore disks in vessels that were designed to slow down the water flow, and thus uptake kinetics, and ensure that the uptake is only marginally dependent on the chemicals' physicochemical properties over a relatively narrow molecular size range. This design not only assured integrative sampling over 27 days for a broad range of chemicals but also permitted the use of a suite of bioanalytical tools as sum parameters, representative of mixtures of chemicals with a common mode of toxic action. Bioassays proved to be more sensitive than chemical analysis to assess the removal of organic micropollutants by reverse osmosis, followed by UV/H2O2 treatment, as many individual compounds fell below the quantification limit of chemical analysis, yet still contributed to the observed mixture toxicity. Nonetheless in several cases, the responses in the bioassays were also below their quantification limits and therefore only three bioassays were evaluated here, representing nonspecific toxicity and two specific end points for estrogenicity and photosynthesis inhibition. Chemical analytical techniques were able to quantify 32 pesticides, 62 PCPPs, and 12 EDCs in reverse osmosis concentrate. However, these chemicals could explain only 1% of the nonspecific toxicity in the Microtox assay in the reverse osmosis concentrate and 0.0025% in the treated water. Likewise only 1% of the estrogenic effect in the E-SCREEN could be explained by the quantified EDCs after reverse osmosis. In comparison, >50% of the estrogenic effect can typically be explained in sewage. Herbicidal activity could be fully explained by chemical analysis as the sampling period coincided with an illegal discharge and two herbicides dominated the mixture effect. The mass balance of the reverse osmosis process matched theoretical expectations for both chemical analysis and bioanalytical tools. Overall the investigated treatment train removed >97% estrogenicity, >99% herbicidal activity, and >96% baseline toxicity, confirming the suitability of the treatment train for polishing water for indirect potable reuse. The product water was indistinguishable from local tap water in all three bioassays. This study demonstrates the suitability and robustness of passive sampling linked with bioanalytical tools for semicontinuous monitoring of advanced water treatment with respect to micropollutant removal.

Recovery of a freshwater wetland from chemical contamination after an oil spill.

In March 2009, a cargo ship spilled 250 tons of heavy fuel oil off the Queensland coast of Australia. The pristine National Park Moreton Island, seven nautical miles to the east of the spill site, was most affected by the oil slick. Contamination of the island's shoreline was widespread, with freshwater wetlands particularly slow to recover as clean-up needed to be carefully managed to avoid damage to this sensitive ecosystem. During the clean-up process on Moreton Island a monitoring program was initiated using traditional chemical analysis in combination with bioanalytical techniques to assess the extent and variability in contamination at sites on the shoreline and freshwater wetlands. Water accommodated fractions (WAF) of oil residues from samples taken directly after the spill on the shoreline showed the same level of toxic potency as samples from the wetland while baseline-toxicity equivalent concentrations (baseline-TEQ) and 2,3,7,8-tetrachlorodibenzodioxin equivalent concentrations (TCDDEQ) were much lower in oil collected from the sandy beach. The umuC assay for genotoxicity and the E-SCREEN assay for estrogenic effects indicated the extracts were not genotoxic or estrogenic. PAH concentrations and toxicity in grab water samples were below detectable levels, however, extracts from time integrated silicone passive samplers deployed for several weeks at the contaminated sites gave measurable responses in the bioassays with TCDDEQ levels increased relative to the control site. The low levels of baseline-TEQ and TCDDEQ present after 8 months had further decreased 6 months later indicating satisfactory recovery of this pristine ecosystem after an oil spill.

Assessing indoor air exposures using passive sampling with bioanalytical methods for estrogenicity and aryl hydrocarbon receptor activity.

Passive air sampling was undertaken using polyurethane foam passive air samplers at three types of locations, including indoors (six offices) at buildings in the central business district (CBD) and at a private suburban home (indoor and outdoor) located 9 km from the CBD in Brisbane, Queensland, Australia. Estrogenic (E-SCREEN--MCF7-BOS) and aryl hydrocarbon receptor (AhR) (CAFLUX--H4G1.1c2) activity were assessed for samples collected from each of these locations. The samples were tested either as crude extracts ("untreated") or were subjected to H2SO4 silica gel ("treated") for each location in order to determine whether chemicals, which are not resistant to this treatment like polycyclic aromatic hydrocarbons, potentially account for the observed activity. In most cases, H2SO4 treatment resulted in a statistically significant reduction of potency for both endpoints, suggesting that chemicals less resistant to treatment may be responsible for much of the detected biological activity in these locations. Estrogenic potency measurements (<0.22-185 pg m(-3)) were highest in the indoor offices, followed by the indoor suburban home and finally the outdoor suburban home (which was not estrogenic). Total AhR activity for crude extracts (1.3-10 pg m(-3)) however was highest for the outdoor suburban home site. Levels of polycyclic aromatic hydrocarbons were monitored indoors and outdoors at the suburban home. At that location, polycyclic aromatic hydrocarbon air concentrations were on average approximately two times higher outdoor than indoor, while AhR potency was five times higher outdoor than indoor. No significant correlation was found between the estrogenic and AhR activity (P = 0.88) for the sites in this study.

Estrogen reduces aldosterone, upregulates adrenal angiotensin II AT2 receptors and normalizes adrenomedullary Fra-2 in ovariectomized rats.

We studied the effect of ovariectomy and estrogen replacement on expression of adrenal angiotensin II AT1 and AT2 receptors, aldosterone content, catecholamine synthesis, and the transcription factor Fos-related antigen 2 (Fra-2). Ovariectomy increased AT1 receptor expression in the adrenal zona glomerulosa and medulla, and decreased adrenomedullary catecholamine content and Fra-2 expression when compared to intact female rats. In the zona glomerulosa, estrogen replacement normalized AT1 receptor expression, decreased AT1B receptor mRNA, and increased AT2 receptor expression and mRNA. Estrogen treatment decreased adrenal aldosterone content. In the adrenal medulla, the effects of estrogen replacement were: normalized AT1 receptor expression, increased AT2 receptor expression, AT2 receptor mRNA, and tyrosine hydroxylase mRNA, and normalized Fra-2 expression and catecholamine content. We demonstrate that the constitutive adrenal expression of AT1 receptors, catecholamine synthesis and Fra-2 expression are partially under the control of reproductive hormones. Our results suggest that estrogen treatment decreases aldosterone production through AT1 receptor downregulation and AT2 receptor upregulation. AT2 receptor upregulation and modulation of Fra-2 expression may participate in the estrogen-dependent normalization of adrenomedullary catecholamine synthesis in ovariectomized rats. The AT2 receptor upregulation and the decrease in AT1 receptor function and in the production of the fluid-retentive, pro-inflammatory hormone aldosterone partially explain the protective effects of estrogen therapy.

Effect-based monitoring of the Danube River using mobile passive sampling.

Many aquatic pollutants can be present at low concentrations, but their mixtures can still affect health or behavior of exposed organisms. In this study, toxicological and chemical analyses were combined for spatial contamination profiling using an innovative passive sampling approach. A novel Dynamic Passive Sampler (DPS) was employed as a mobile sampler from a ship cruising along 2130km of the Danube river during the Joint Danube Survey 3 (JDS3). The sampling was performed in eight subsequent river stretches with two types of complementary passive samplers: silicone rubber sheets (SR) used for non-polar chemicals and SDB-RPS Empore™ disks (ED) for more hydrophilic compounds. Besides extensive chemical analyses, the bioactivity of samples was characterized by a battery of reporter gene bioassays. Cross-calibration of the employed passive samplers enabled robust estimation of water concentrations applicable for compounds with a wide range of physicochemical properties. DPS was suitable for sampling of water contaminants even at pgL-1 levels, with 209 of 267 analyzed compounds detected in the samples. Biological effects were detected in both ED and SR extracts across all river stretches by bioassays focused on xenobiotic metabolism mediated by the aryl hydrocarbon and pregnane X receptors, endocrine disruptive potential mediated by estrogen and androgen receptors and the oxidative stress response. The bioassay responses expressed as bioanalytical equivalent concentrations (BEQbio) were comparable with data obtained from large volume active sampling. The extracts of the ED samplers were more biologically active than extracts of SR samplers. Except of estrogenicity, where the analyzed chemicals explained on average 62% of the effects in ED samples, the detected chemicals explained <8% of BEQbio values. The study shows the utility of the combination of the innovative passive sampling approach with effect-based tools for efficient and fast monitoring even in water bodies with relatively low levels of contamination.

AT1 receptor blockade regulates the local angiotensin II system in cerebral microvessels from spontaneously hypertensive rats.

BACKGROUND AND PURPOSE: Blockade of angiotensin II AT1 receptors in cerebral microvessels protects against brain ischemia and inflammation. In this study, we tried to clarify the presence and regulation of the local renin-angiotensin system (RAS) in brain microvessels in hypertension. METHODS: Spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) controls were treated with an AT1 receptor antagonist (candesartan, 0.3 mg/kg per day) via subcutaneous osmotic minipumps for 4 weeks. The expression and localization of RAS components and the effect of AT1 receptor blockade were assessed by Affymetrix microarray, qRT-PCR, Western blots, immunohistochemistry and immunofluorescence. RESULTS: We found transcripts of most of RAS components in our microarray database, and confirmed their expression by qRT-PCR. Angiotensinogen (Aogen), angiotensin-converting enzyme (ACE) and AT1 receptors were localized to the endothelium. There was no evidence of AT2 receptor localization in the microvascular endothelium. In SHR, (pro)renin receptor mRNA and AT1 receptor mRNA and protein expression were higher, whereas Aogen, ACE mRNA and AT2 receptor mRNA and protein expression were lower than in WKY rats. Candesartan treatment increased Aogen, ACE and AT2 receptor in SHR, and increased ACE and decreased Aogen in WKY rats, without affecting the (pro)renin and AT1 receptors. CONCLUSIONS: Increased (pro)renin and AT1 receptor expression in SHR substantiates the importance of the local RAS overdrive in the cerebrovascular pathophysiology in hypertension. AT1 receptor blockade and increased AT2 receptor stimulation after administration of candesartan may contribute to the protection against brain ischemia and inflammation.

A centrally acting, anxiolytic angiotensin II AT1 receptor antagonist prevents the isolation stress-induced decrease in cortical CRF1 receptor and benzodiazepine binding.

Long-term pretreatment with an angiotensin II AT1 antagonist blocks angiotensin II effects in brain and peripheral organs and abolishes the sympathoadrenal and hypothalamic-pituitary-adrenal responses to isolation stress. We determined whether AT1 receptors were also important for the stress response of higher regulatory centers. We studied angiotensin II and corticotropin-releasing factor (CRF) receptors and benzodiazepine binding sites in brains of Wistar Hannover rats. Animals were pretreated for 13 days with vehicle or a central and peripheral AT1 antagonist (candesartan, 0.5 mg/kg/day) via osmotic minipumps followed by 24 h of isolation in metabolic cages, or kept grouped throughout the study (grouped controls). In another study, we determined the influence of a similar treatment with candesartan on performance in an elevated plus-maze. AT1 receptor blockade prevented the isolation-induced increase in brain AT1 receptors and decrease in AT2 binding in the locus coeruleus. AT1 receptor antagonism also prevented the increase in tyrosine hydroxylase mRNA in the locus coeruleus. Pretreatment with the AT1 receptor antagonist completely prevented the decrease in cortical CRF1 receptor and benzodiazepine binding produced by isolation stress. In addition, pretreatment with candesartan increased the time spent in and the number of entries to open arms of the elevated plus-maze, measure of decreased anxiety. Our results implicate a modulation of upstream neurotransmission processes regulating cortical CRF1 receptors and the GABA(A) complex as molecular mechanisms responsible for the anti-anxiety effect of centrally acting AT1 receptor antagonists. We propose that AT1 receptor antagonists can be considered as compounds with possible therapeutic anti-stress and anti-anxiety properties.

Angiotensin II AT1 receptor blockade abolishes brain microvascular inflammation and heat shock protein responses in hypertensive rats.

Endothelial dysfunction and inflammation enhance vulnerability to hypertensive brain damage. To explore the participation of Angiotensin II (Ang II) in the mechanism of vulnerability to cerebral ischemia during hypertension, we examined the expression of inflammatory factors and the heat shock protein (HSP) response in cerebral microvessels from spontaneously hypertensive rats and their normotensive controls, Wistar Kyoto rats. We treated animals with vehicle or the Ang II AT(1) receptor antagonist candesartan, 0.3 mg/kg/day, via subcutaneously implanted osmotic minipumps for 4 weeks. Spontaneously hypertensive rats expressed higher Angiotensin II AT(1) receptor protein and mRNA than normotensive controls. Candesartan decreased the macrophage infiltration and reversed the enhanced tumor necrosis factor-alpha and interleukin-1beta mRNA and nuclear factor-kappaB in microvessels in hypertensive rats. The transcription of many HSP family genes, including HSP60, HSP70 and HSP90, and heat shock factor-1 was higher in hypertensive rats and was downregulated by AT(1) receptor blockade. Our results suggest a proinflammatory action of Ang II through AT(1) receptor stimulation in cerebral microvessels during hypertension, and very potent antiinflammatory effects of the Ang II AT(1) receptor antagonist. These compounds might be considered as potential therapeutic agents against ischemic and inflammatory diseases of the brain.

Anti-stress and anti-anxiety effects of centrally acting angiotensin II AT1 receptor antagonists.

The brain and the peripheral (hormonal) angiotensin II systems are stimulated during stress. Activation of brain angiotensin II AT(1) receptors is required for the stress-induced hormone secretion, including CRH, ACTH, corticoids and vasopressin, and for stimulation of the central sympathetic activity. Long-term peripheral administration of the angiotensin II AT(1) antagonist candesartan blocks not only peripheral but also brain AT(1) receptors, prevents the hormonal and sympathoadrenal response to isolation stress and prevents the formation of stress-induced gastric ulcers. The mechanisms responsible for the prevention of stress-induced ulcers by the AT(1) receptor antagonist include protection from the stress-induced ischemia and inflammation (neutrophil infiltration and increase in ICAM-1 and TNF-alpha) in the gastric mucosa and a partial blockade of the stress-induced sympathoadrenal stimulation, while the protective effect of the glucocorticoid release during stress is maintained. AT(1) receptor antagonism prevents the stress-induced decrease in cortical CRH(1) and benzodiazepine binding and is anxiolytic. Blockade of brain angiotensin II AT(1) receptors offers a novel therapeutic opportunity for the treatment of anxiety and other stress-related disorders.

Estrogen upregulates renal angiotensin II AT1 and AT2 receptors in the rat.

We studied renal AT1 and AT2 receptors in male, female, ovariectomized and ovariectomized-estrogen-treated Wistar-Hanover and Wistar-Kyoto rats. AT1 receptors and AT1A receptor mRNA predominated, with no significant differences between males and females. AT2 receptor expression was restricted in female rats to the capsule, the transition zone between outer and inner medulla, the endothelium lining the papilla, and arcuate arteries and veins. There were no AT2 receptors in male rats, while male mice express substantial numbers of estrogen-dependent AT2 receptors. Arcuate arteries and veins expressed AT1B mRNA in males and females, and AT2 mRNA in females only. AT1 receptor and AT2 receptor expression were estrogen-dependent, with increases in AT1 and AT2 receptor expression after estrogen treatment in ovariectomized rats. Estrogen treatment increased prostaglandin E2 (PGE2) and cGMP concentrations in the renal medulla, and eNOS expression in cortical arteries. In rodents, expression of renal Angiotensin II receptor types is estrogen-dependent, with significant species, strain and area differences. Our results support an important role for AT2 receptors in the regulation of renal function and in the protective effects of estrogen in the kidney.

Angiotensin II AT1 receptor blockade reverses pathological hypertrophy and inflammation in brain microvessels of spontaneously hypertensive rats.

BACKGROUND AND PURPOSE: The spontaneously hypertensive rat (SHR) is vulnerable to brain ischemia and stress and exhibits a chronically stimulated brain angiotensin II system, cerebrovascular hypertrophy, and inflammation. Pretreatment with angiotensin II type 1 (AT1) receptor antagonists protects from brain ischemia and from stress and prevents the development of stress-induced gastric ulcers in part by reducing inflammation in the gastric mucosa. We studied whether AT1 receptor antagonists could exert antiinflammatory effects in the brain vasculature as a mechanism for their protective effects against ischemia. METHODS: Ten-week-old SHR and normotensive Wistar-Kyoto male rats received the AT1 receptor antagonist candesartan (0.3 mg/kg per day) or vehicle for 28 days via osmotic minipumps. We studied AT1 receptors, intercellular adhesion molecule-1 (ICAM-1), endothelial nitric oxide synthase (eNOS), and number of macrophages by immunohistochemistry and Western blots. RESULTS: We found increased endothelial AT1 receptor expression of brain microvessels and middle cerebral artery of SHR. Brain AT1 receptor inhibition reversed the pathological vascular hypertrophy, increased and normalized eNOS expression, and decreased ICAM-1 expression and the number of adherent and infiltrating macrophages in cerebral vessels of SHR. CONCLUSIONS: The antiinflammatory effects of AT1 receptor antagonists may be an important mechanism in protecting against ischemia.

Bioanalytical tools for the evaluation of organic micropollutants during sewage treatment, water recycling and drinking water generation.

A bioanalytical test battery was used for monitoring organic micropollutants across an indirect potable reuse scheme testing sites across the complete water cycle from sewage to drinking water to assess the efficacy of different treatment barriers. The indirect potable reuse scheme consists of seven treatment barriers: (1) source control, (2) wastewater treatment plant, (3) microfiltration, (4) reverse osmosis, (5) advanced oxidation, (6) natural environment in a reservoir and (7) drinking water treatment plant. Bioanalytical results provide complementary information to chemical analysis on the sum of micropollutants acting together in mixtures. Six endpoints targeting the groups of chemicals with modes of toxic action of particular relevance for human and environmental health were included in the evaluation: genotoxicity, estrogenicity (endocrine disruption), neurotoxicity, phytotoxicity, dioxin-like activity and non-specific cell toxicity. The toxicity of water samples was expressed as toxic equivalent concentrations (TEQ), a measure that translates the effect of the mixtures of unknown and potentially unidentified chemicals in a water sample to the effect that a known reference compound would cause. For each bioassay a different representative reference compound was selected. In this study, the TEQ concept was applied for the first time to the umuC test indicative of genotoxicity using 4-nitroquinoline as the reference compound for direct genotoxicity and benzo[a]pyrene for genotoxicity after metabolic activation. The TEQ were observed to decrease across the seven treatment barriers in all six selected bioassays. Each bioassay showed a differentiated picture representative for a different group of chemicals and their mixture effect. The TEQ of the samples across the seven barriers were in the same order of magnitude as seen during previous individual studies in wastewater and advanced water treatment plants and reservoirs. For the first time a benchmarking was performed that allows direct comparison of different treatment technologies and covers several orders of magnitude of TEQ from highly contaminated sewage to drinking water with TEQ close or below the limit of detection. Detection limits of the bioassays were decreased in comparison to earlier studies by optimizing sample preparation and test protocols, and were comparable to or lower than the quantification limits of the routine chemical analysis, which allowed monitoring of the presence and removal of micropollutants post Barrier 2 and in drinking water. The results obtained by bioanalytical tools were reproducible, robust and consistent with previous studies assessing the effectiveness of the wastewater and advanced water treatment plants. The results of this study indicate that bioanalytical results expressed as TEQ are useful to assess removal efficiency of micropollutants throughout all treatment steps of water recycling.

Effect based monitoring of seasonal ambient air exposures in Australia sampled by PUF passive air samplers.

There has been relatively little bioanalytical effect based monitoring conducted using samples derived from polyurethane foam (PUF) passive air samplers. Combining these techniques may provide a more convenient and cost effective means of monitoring the potential for biological effects resulting from exposure to complex mixtures in a range of scenarios. Seasonal polycyclic aromatic hydrocarbon (PAH) levels were monitored at sites around Australia using direct chemical analysis. In addition, both indirect acting genotoxicity (umuC assay) and aryl hydrocarbon receptor (AhR) activity (chemically activated fluorescent gene expression [CAFLUX assay]), which are effects potentially relevant to subsequent carcinogenesis for these compounds, were measured. The levels of PAHs as well as genotoxicity and AhR activity were all higher in winter compared to summer and for sites in urban capital cities compared to other locations. Statistically significant relationships were found between the levels of PAHs and both genotoxicity and AhR activity. The dominant contributors to the total AhR activity, were found to be for compounds which are not resistant to H(2)SO(4)/silica gel treatment and were relatively rapidly metabolised that is consistent with a PAH type response. Relative potency estimates for individual PAHs determined for the first time on the CAFLUX assay were used to estimate the proportion of total AhR activity (≤ 3.0%) accounted by PAHs monitored. Observed responses are thus largely due to non-quantified AhR active compounds.

A peripherally administered, centrally acting angiotensin II AT2 antagonist selectively increases brain AT1 receptors and decreases brain tyrosine hydroxylase transcription, pituitary vasopressin and ACTH.

The physiological actions of brain Angiotensin II AT(2) receptors and their relationship to Angiotensin II AT(1) receptors remain controversial. To further clarify their role, we determined to what extent systemic administration of an AT(2) receptor antagonist affected AT(2) receptor binding within the brain and the expression of AT(1) receptors. For this purpose, we subcutaneously administered the AT(2) receptor antagonist PD123319 (1 mg/kg/day) to adult male rats for two weeks via osmotic minipumps. We also studied the content of pituitary adrenocorticotropic hormone and vasopressin, representative of hypothalamic-pituitary-adrenal axis activation, and the tyrosine hydroxylase gene expression in the locus coeruleus as a measure of central norepinephrine function. We found significant decreases in AT(2) receptor binding in brain areas inside the blood brain barrier, the inferior olive and the locus coeruleus. AT(2) receptor blockade increased AT(1) receptor binding and mRNA expression not only in the subfornical organ and the median eminence, situated outside the blood brain barrier, but also in the hypothalamic paraventricular nucleus, located inside the blood brain barrier. These changes paralleled decreased expression of tyrosine hydroxylase mRNA in the locus coeruleus and decreased pituitary adrenocorticotropic and vasopressin content. Our results demonstrate that sustained peripheral administration of an AT(2) antagonist decreases binding to brain AT(2) receptors, indicating that this drug is a useful tool for the study of their central role. AT(2) receptor activity inhibition up-regulates AT(1) receptor expression in specific brain areas. Blockade of brain AT(2) receptors is compatible with enhanced hypothalamic-pituitary-adrenal axis and decreased central sympathetic system activity.