Modulation of steroidogenesis by coastal waters and sewage effluents of Hong Kong, China, using the H295R assay

BACKGROUND, AIM, AND SCOPE: The presence of a variety of pollutants in the aquatic environment that can potentially interfere with the production of sex steroid hormones in wildlife and humans has been of increasing concern. The aim of the present study was to investigate the effects of extracts from Hong Kong marine waters, and influents and effluents from wastewater treatment plants on steroidogenesis using the H295R cell bioassay. After exposing H295R cells to extracts of water, the expression of four steroidogenic genes and the production of three steroid hormones were measured. MATERIALS AND METHODS: Water samples were collected during the summer of 2005 from 24 coastal marine areas and from the influents and effluents of two major waste water treatment plants (WWTPs) in Hong Kong, China. Samples were extracted by solid phase extraction (SPE). H295R cells were exposed for 48 h to dilutions of these extracts. Modulations of the expression of the steroidogenic genes CYP19, CYP17, 3betaHSD2, and CYP11beta2 were determined by measuring mRNA concentrations by real-time polymerase chain reaction (Q-RT-PCR). Production of the hormones progesterone (P), estradiol (E2), and testosterone (T) was quantified using enzyme linked immunosorbent assays (ELISA). RESULTS: Extracts from samples collected in two fish culture areas inhibited growth and proliferation of H295R cells at concentrations greater or equal to 10(5) L equivalents. The cells were exposed to the equivalent concentration of active substances in 10,000 L of water. Thus, to observe the same level of effect as observed in vitro on aquatic organisms would require a bioaccumulation factor of this same magnitude. None of the other 22 marine samples affected growth of the cells at any dilution tested. Twelve of the marine water samples completely inhibited the expression of CYP19 without affecting E2 production; inhibition of CYP17 expression was observed only in one of the samples while expression of CYP11beta2 was induced as much as five- and ninefold after exposure of cells to extracts from two locations. The expression of the progesterone gene 3betaHSD2 was not affected by any of the samples; only one sample induced approximately fourfold the production of E2. Although more than twofold inductions were observed for P and T production, none of these values were statistically significant to conclude effects on the production of these two hormones. While influents from WWTPs did not affect gene expression, an approximately 30% inhibition in the production of E2 and a 40% increase in P occurred for the exposure with influents from the Sha Tin and Stonecutters WWTPs, respectively. Effluents from WWTPs did not affect the production of any of the studied hormones, but a decrement in the expression of the aldosterone gene CYP11beta2 was observed for the Sha Tin WWTP exposure. No direct correlation could be established between gene expression and hormone production. DISCUSSION: Observed cytotoxicity in the two samples from fish culture areas suggest the presence of toxic compounds; chemical analysis is required for their full identification. Although effluents from WWTPs did not affect hormone production, other types of endocrine activity such as receptor-mediated effects cannot be ruled out. Interactions due to the complexity of the samples and alternative steroidogenic pathways might explain the lack of correlation between gene expression and hormone production results. CONCLUSIONS: Changes observed in gene expression and hormone production suggest the presence in Hong Kong coastal waters of pollutants with endocrine disruption potential and others of significant toxic effects. The aromatase and aldosterone genes seem to be the most affected by the exposures, while E2 and P are the hormones with more significant changes observed. Results also suggest effectiveness in the removing of compounds with endocrine activity by the WWTPs studied, as effluent samples did not significantly affect hormone production. The H295R cell showed to be a valuable toll in the battery required for the analysis of endocrine disrupting activities of complex environmental samples. RECOMMENDATIONS AND PERSPECTIVES: Due to the intrinsic complexity of environmental samples, a combination of analytical tools is required to realistically assess environmental conditions, especially in aquatic systems. In the evaluation of endocrine disrupting activities, the H295R cell bioassay should be used in combination with other genomic, biological, chemical, and hydrological tests to establish viable modes for endocrine disruption and identify compounds responsible for the observed effects.     

Revised relative potency values for PCDDs, PCDFs, and non-ortho-substituted PCBs for the optimized H4IIE-luc in vitro bioassay

While the World Health Organization 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalency factors are useful estimates of relative potencies of mixtures when conducting risk assessments, they are not useful when comparing the results of bioassays such as the H4IIE-luc to concentrations of TCDD equivalents calculated from instrumental analyses. Since there are thousands of dioxin-like compounds (DLCs), one use of screening assays is to determine if all of the aryl hydrocarbon receptor (AhR) active DLCs in a mixture have been accounted for in instrumental analyses. For this purpose, bioassay-specific relative potency (ReP) values are needed. RePs of 21 polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans, and dioxin-like polychlorinated biphenyls that exhibit effects mediated through the AhR were determined by use of the H4IIE-luc assay. Different values of RePs are derived, depending on the statistical, curve-fitting methods used to derive them from the dose–response relationships. Here, we discuss the various methods for deriving RePs from in vitro data and their assumptions and effects on values of RePs. Full dose–response curves of 2,3,7,8-TCDD and other representative DLCs were used to estimate effective concentrations at multiple points (e.g., EC20-50-80), which were then used to estimate ReP of each DLC to 2,3,7,8-TCDD.     

In vitro assessment of AhR-mediated activities of TCDD in mixture with humic substances

Humic substances (HS) are ubiquitous natural products of decomposition of dead organic matter. HS is present in most freshwaters at concentrations ranging from 0.5 to 50 mg L⁻¹. Organic carbon can represent 20% dry weight of sediments. Recently, the interaction of dissolved HS with the aryl hydrocarbon receptor (AhR) has been demonstrated. The AhR is a cytosolic receptor to which persistent organic pollutants (POPs) can bind and many of their toxic effects are mediated through interactions with this receptor. We describe in vitro effects (using H4IIE-luc cells) of binary mixtures of various HS with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), since in contaminated environments these compounds occur simultaneously. Six out of 12 HS samples activated AhR even at environmentally relevant concentrations (17 mg L⁻¹), but did not reach the full AhR-activation even at excessive concentration. In simultaneous exposure of H4IIE-luc to HS (17 mg L⁻¹) and TCDD (1.2 pM) without any preincubation prior to exposure, either significant additive or facilitative effects were observed. No negative interactions, due to possible sorption of TCDD to HS was observed. Nevertheless, if the HS–TCDD binary mixture was preincubated for 6 days prior to the exposure on H4IIE-luc cells, the additive and facilitative effects were less due to possible sorption of TCDD onto HS. Similar results were obtained from analogous experiments with greater concentrations of both TCDD and HS.     

Stabilization of potassium permanganate particles with manganese dioxide

A new potassium permanganate reagent with slow-release properties was designed and tested for possible application in in situ chemical oxidation. For this purpose, MnO₂-coated KMnO₄ particles (MCP) were prepared by partial reduction of solid KMnO₄ using the acid-catalyzed reaction with n-propanol or the comproportionation of Mn(VII) and Mn(II) in n-propanol as reaction medium. Column tests showed that, for MCP with a residual KMnO₄ fraction of 70 wt%, the duration of permanganate release under flow-through conditions was prolonged by a factor of 10 compared to untreated KMnO₄. While KMnO₄ is too soluble to be used in reactive barriers, MCP could be introduced into the aquifer by filling of trenches or boreholes; this would allow a prolonged passive dosing of permanganate into the flowing groundwater. In addition, experiments were conducted in order to determine the oxidation capability of native KMnO₄ particles and MCP in CH₂Cl₂, a representative non-polar non-aqueous phase liquid (NAPL). It may be possible to utilize the significantly higher reactivity of MCP under these conditions for the design of slow-release permanganate particles for NAPL source treatment.     

Longer-term and short-term variability in pollution of fluvial sediments by dioxin-like and endocrine disruptive compounds

Changes in pollutant loads in relatively dynamic river sediments, which contain very complex mixtures of compounds, can play a crucial role in the fate and effects of pollutants in fluvial ecosystems. The contamination of sediments by bioactive substances can be sensitively assessed by in vitro bioassays. This is the first study that characterizes detailed short- and long-term changes in concentrations of contaminants with several modes of action in river sediments. One-year long monthly study described seasonal and spatial variability of contamination of sediments in a representative industrialized area by dioxin-like and endocrine disruptive chemicals. There were significant seasonal changes in both antiandrogenic and androgenic as well as dioxin-like potential of river sediments, while there were no general seasonal trends in estrogenicity. Aryl hydrocarbon receptor-dependent potency (dioxin-like potency) expressed as biological TCDD-equivalents (BIOTEQ) was in the range of 0.5–17.7 ng/g, dry mass (dm). The greatest BIOTEQ levels in sediments were observed during winter, particularly at locations downstream of the industrial area. Estrogenicity expressed as estradiol equivalents (EEQ) was in the range of 0.02–3.8 ng/g, dm. Antiandrogenicity was detected in all samples, while androgenic potency in the range of 0.7–16.8 ng/g, dm dihydrotestosterone equivalents (DHT-EQ) was found in only 30 % of samples, most often during autumn, when antiandrogenicity was the least. PAHs were predominant contaminants among analyzed pollutants, responsible, on average, for 13–21 % of BIOTEQ. Longer-term changes in concentrations of BIOTEQ corresponded to seasonal fluctuations, whereas for EEQ, the inter-annual changes at some locations were greater than seasonal variability during 1 year. The inter- as well as intra-annual variability in concentrations of both BIOTEQ and EEQ at individual sites was greater in spring than in autumn which was related to hydrological conditions in the river. This study stresses the importance of river hydrology and its seasonal variations in the design of effective sampling campaigns, as well as in the interpretation of any monitoring results.     

Levels and risks of particulate-bound PAHs in indoor air influenced by tobacco smoke: a field measurement

Considering tobacco smoke as one of the most health-relevant indoor sources, the aim of this work was to further understand its negative impacts on human health. The specific objectives of this work were to evaluate the levels of particulate-bound PAHs in smoking and non-smoking homes and to assess the risks associated with inhalation exposure to these compounds. The developed work concerned the application of the toxicity equivalency factors approach (including the estimation of the lifetime lung cancer risks, WHO) and the methodology established by USEPA (considering three different age categories) to 18 PAHs detected in inhalable (PM₁₀) and fine (PM_2.5) particles at two homes. The total concentrations of 18 PAHs (Σ_PAHs) was 17.1 and 16.6 ng m^?3 in PM₁₀ and PM_2.5 at smoking home and 7.60 and 7.16 ng m^?3 in PM₁₀ and PM_2.5 at non-smoking one. Compounds with five and six rings composed the majority of the particulate PAHs content (i.e., 73 and 78 % of Σ_PAHs at the smoking and non-smoking home, respectively). Target carcinogenic risks exceeded USEPA health-based guideline at smoking home for 2 different age categories. Estimated values of lifetime lung cancer risks largely exceeded (68–200 times) the health-based guideline levels at both homes thus demonstrating that long-term exposure to PAHs at the respective levels would eventually cause risk of developing cancer. The high determined values of cancer risks in the absence of smoking were probably caused by contribution of PAHs from outdoor sources.     

The potential of advanced technologies to reduce carbon capture costs in future IGCC power plants

Over the next two decades, our nation will need to add a substantial amount of new power generation capacity. The possibility of more stringent environmental regulations for greenhouse gas emissions in the utility sector has provided a window of opportunity for integrated gasification combined cycles (IGCCs) equipped with carbon capture and sequestration (CCS) to participate significantly in this expansion. This paper analyzes several advanced technologies under development in the Department of Energy (DOE) research and development (R&D) portfolio that have the potential to improve process efficiency, reduce capital and operating expense, and increase plant availability resulting in a significant reduction in the cost of electricity for plants that capture carbon.     

The United States Department of Energy's Regional Carbon Sequestration Partnerships program: a collaborative approach to carbon management

This paper reviews the Regional Carbon Sequestration Partnerships (RCSP) concept, which is a first attempt to bring the U.S. Department of Energy's (DOE) carbon sequestration program activities into the "real world" by using a geographically-disposed-system type approach for the U.S. Each regional partnership is unique and covers a unique section of the U.S. and is tasked with determining how the research and development activities of DOE's carbon sequestration program can best be implemented in their region of the country. Although there is no universal agreement on the cause, it is generally understood that global warming is occurring, and many climate scientists believe that this is due, in part, to the buildup of carbon dioxide (CO(2)) in the atmosphere. This is evident from the finding presented in the National Academy of Science Report to the President on Climate Change which stated "Greenhouse gases are accumulating in Earth's atmosphere as a result of human activities, causing surface air temperatures and subsurface ocean temperatures to rise. Temperatures are, in fact, rising. The changes observed over the last several decades are likely mostly due to human activities, ...". In the United States, emissions of CO(2) originate mainly from the combustion of fossil fuels for energy production, transportation, and other industrial processes. Roughly one third of U.S. anthropogenic CO(2) emissions come from power plants. Reduction of CO(2) emissions through sequestration of carbon either in geologic formations or in terrestrial ecosystems can be part of the solution to the problem of global warming. However, a number of steps must be accomplished before sequestration can become a reality. Cost effective capture and separation technology must be developed, tested, and demonstrated; a database of potential sequestration sites must be established; and techniques must be developed to measure, monitor, and verify the sequestered CO(2). Geographical differences in fossil fuel use, the industries present, and potential sequestration sinks across the United States dictate the use of a regional approach to address the sequestration of CO(2). To accommodate these differences, the DOE has created a nationwide network of seven Regional Carbon Sequestration Partnerships (RCSP) to help determine and implement the carbon sequestration technologies, infrastructure, and regulations most appropriate to promote CO(2) sequestration in different regions of the nation. These partnerships currently represent 40 states, three Indian Nations, four Canadian Provinces, and over 200 organizations, including academic institutions, research institutions, coal companies, utilities, equipment manufacturers, forestry and agricultural representatives, state and local governments, non-governmental organizations, and national laboratories. These partnerships are dedicated to developing the necessary infrastructure and validating the carbon sequestration technologies that have emerged from DOE's core R&D and other programs to mitigate emissions of CO(2), a potent greenhouse gas. The partnerships provide a critical link to DOE's plans for FutureGen, a highly efficient and technologically sophisticated coal-fired power plant that will produce both hydrogen and electricity with near-zero emissions. Though limited to the situation in the U.S., the paper describes for the international scientific community the approach being taken by the U.S. to prepare for carbon sequestration, should that become necessary.     

In vitro studies of copper release from powder particles in synthetic biological media

The aim of this paper is to provide quantitative data on copper release from powder particles of different copper materials, including artificial copper patina, Cu(2)O and metallic Cu, when exposed to different synthetic biological media to simulate an inhalation scenario and/or skin contact. Generated data may contribute in risk assessment of potential health effects following exposure to and handling of various copper materials. All tests were performed in vitro to determine total copper concentrations, release rates of total copper, and to elucidate its time-dependence. The copper release process was interpreted in terms of specific surface area, surface morphology-, and composition. All powder materials show a time-dependent release process with total copper release rates less than 3 microg/cm(2) per hour at steady state conditions, for all media investigated. The importance of using relevant test media when simulating different interstitial lung conditions and difficulties encountered when comparing powder particles of essentially different properties are thoroughly discussed.     

The importance of the will to improve: how ‘sustainability’ sidelined local livelihoods in a carbon-forestry investment in Uganda

This paper describes a ‘win–win’ discourse on local sustainable development and global climate change mitigation regarding Kachung, a Swedish–Norwegian climate forestry investment in Uganda certified under the Clean Development Mechanism (CDM). In many ways, this investment is a typical example of how private interests and capital accumulation are prioritised over local concerns in natural resource management under neoliberalism. This study, however, indicated that investors had genuine intentions of creating mutual benefits for the global environment and local people. Drawing on Li (2007), we show that this ‘will to improve’ was nevertheless constructed in ways that resulted in prioritisation of global climate change mitigation over local context-specific concerns.

We identify three core factors making the win–win discourse around Kachung plantation especially resilient: (i) the perceived urgency of climate change mitigation, (ii) the apolitical framing of ‘sustainability’ as an environmental issue that can be fixed through external technical interventions and (iii) the devaluation of local and context-specific knowledge. We end by suggesting that research on the neoliberalisation of nature focus more on analysing the rationales behind specific interventions. This would leave us better equipped to suggest how such interventions should be modified to produce true wins for local contexts.