Steve Wiseman

Biological impact of phthalates

Esters of phthalic acid are chemical agents used to improve the plasticity of industrial polymers. Their ubiquitous use in multiple commercial products results in extensive exposure to humans and the environment. This study investigated cytotoxicity, endocrine disruption, effects mediated via AhR, lipid peroxidation and effects on expression of enzymes of xenobiotic metabolism caused by di-(2-ethy hexyl) phthalate (DEHP), diethyl phthalate (DEP), dibutyl phthalate (DBP) and benzyl butyl phthalate (BBP) in developing fish embryos. Oxidative stress was identified as the critical mechanism of toxicity (CMTA) in the case of DEHP and DEP, while the efficient removal of DBP and BBP by phase 1 enzymes resulted in lesser toxicity. DEHP and DEP did not mimic estradiol (E(2)) in transactivation studies, but at concentrations of 10mg/L synthesis of sex steroid hormones was affected. Exposure to 10mg BBP/L resulted in weak transactivation of the estrogen receptor (ER). All phthalates exhibited weak potency as agonists of the aryl hydrocarbon receptor (AhR). The order of potency of the 4 phthalates studied was; DEHP>DEP>BBP>>DBP. The study highlights the need for simultaneous assessment of: (1) multiple cellular targets affected by phthalates and (2) phthalate mixtures to account for additive effects when multiple phthalates modulate the same pathway. Such cumulative assessment of multiple biological parameters is more realistic, and offers the possibility of more accurately identifying the CMTA.

Hydroxylated Polybrominated Diphenyl Ethers and Bisphenol A in Pregnant Women and Their Matching Fetuses: Placental Transfer and Potential Risks

Hydroxylated polybrominated diphenyl ethers (OH-PBDEs) are suspected endocrine disruptors, which can pass through the mammalian placenta and accumulate in the human maternal-fetal-placental unit. However, little is known about mechanisms of placental transfer and the associated risk(s). Ten OH-PBDE congeners, bisphenol A (BPA), total 17beta-estradiol (E2), and total thyroxine (T4) were quantified in blood serum from 26 pregnant women and 28 matching fetuses, including three pairs of twins from South Korea. Only 6-OH-BDE-47, a naturally occurring OH-PBDE, was detected at relatively great concentrations (maternal serum: 17.5 +/- 26.3 pg/g ww, fetal cord blood serum: 30.2 +/- 27.1 pg/g ww), which suggests that exposure was related to diets among Korean women. Concentrations of 6-OH-BDE-47 in maternal and cord serum were positively correlated, with concentrations being significantly greater in cord blood serum. The placental transfer ratio between fetal and maternal blood serum for 6-OH-BDE-47 (F/M ratio: 1.4 +/- 1.1) was different than the observed placental transfer ratio of BPA and previously reported values for hydroxylated polychlorinated biphenyls (OH-PCBs). This result is possibly due to large affinities to T4 transport proteins. Lesser concentrations of E2 and T4 were detected in cord blood serum (E2: 4.7 +/- 2.2 ng/mL, T4: 8.5 +/- 1.7 microg/dL) compared to maternal blood serum (E2: 8.0 +/- 3.0 ng/mL, T4: 9.7 +/- 1.8 microg/dL). A major effect of OH-PBDE exposure might be a decrease in serum T4 concentrations. Potential risks associated with disruption of T4 transport to the developing fetus such as negative consequences for fetal neurological development should be considered in further studies.

Toxicity of untreated and ozone-treated oil sands process-affected water (OSPW) to early life stages of the fathead minnow (Pimephales promelas)

Due to a policy of no release, oil sands process-affected water (OSPW), produced by the surface-mining oil sands industry in North Eastern Alberta, Canada, is stored on-site in tailings ponds. Currently, ozonation is considered one possible method for remediation of OSPW by reducing the concentrations of dissolved organic compounds, including naphthenic acids (NAs), which are considered the primary toxic constituents. However, further work was needed to evaluate the effectiveness of ozonation in reducing the toxicity of OSPW and to ensure that ozonation does not increase the toxicity of OSPW. This study examined effects of untreated, ozone-treated, and activated charcoal-treated OSPW (OSPW, O3-OSPW, and AC-OSPW) on the early life stage (ELS) of fathead minnow (Pimephales promelas). Success of hatching of eggs, spontaneous movement, and incidences of hemorrhage, pericardial edema, and malformation of the spine of embryos were examined. To elucidate the mechanism of toxicity, concentrations of reactive oxygen species (ROS) were measured, and the abundances of transcripts of genes involved in biotransformation of xenobiotics, response to oxidative stress, and apoptosis were quantified by real-time PCR. Compared to the control group, which had an embryo survival rate of 97.9 ± 2.08%, survival was significantly less when exposed to OSPW (43.8 ± 7.12%). Eggs exposed to untreated OSPW exhibited a significantly greater rate of premature hatching, and embryos exhibited greater spontaneous movement. Incidences of hemorrhage (50.0 ± 3.40%), pericardial edema (56.3 ± 7.12%), and malformation of the spine (37.5 ± 5.38%) were significantly greater in embryos exposed to OSPW compared to controls. These effects are typical of exposure to dioxin-like compounds, however, abundance of transcripts of cyp1a was not significantly greater in embryos exposed to OSPW. Significantly greater concentrations of ROS, and greater abundances of transcripts cyp3a, gst, sod, casp9, and apopen compared to controls, indicated that exposure to OSPW caused oxidative stress, which can result in damage to mitochondria and promote activation of caspase enzymes and apoptotic cell death. Removal of dissolved organic constituents by ozone treatment, or by activated charcoal, significantly attenuated all of the adverse effects associated with untreated OSPW. The results suggest that the organic fraction of OSPW can negatively impact the development of fathead minnow embryos through oxidative stress and apoptosis, and that ozonation attenuates this developmental toxicity.

Alberta oil sands development

Polycyclic aromatic hydrocarbons (PAHs) are a ubiquitous class of hundreds of organic compounds composed of two or more aromatic rings that are released naturally and from human activities. Natural sources of PAHs include forest fires, oil seeps, and volcanic eruptions; anthropogenic sources include burning of fossil fuels and wood, production of coke and charcoal, petroleum refining, and petroleum spills (1). The US Agency for Toxic Substances and Disease Registry has ranked PAHs eighth on their list of hazardous substances (2).

Interconversion of hydroxylated and methoxylated polybrominated diphenyl ethers in Japanese medaka.

Polybrominated diphenyl ethers (PBDEs), hydroxylated (OH) and methoxylated (MeO), have been widely detected in aquatic environments. However, relationships among these structurally related compounds in exposed organisms are unclear. To elucidate biotransformation relationships among BDE-47, 6-OH-BDE-47, and 6-MeO-BDE-47, dietary accumulation, maternal transfer, and tissue distribution of these compounds and their transformation products were investigated in sexually mature Japanese medaka (Oryzias latipes). In addition, transformation of each compound was determined in vitro using liver microsomes of medaka. OH-PBDEs and MeO-PBDEs were not detected in fish exposed to BDE-47. However, significant concentrations of 6-OH-BDE-47 were detected in medaka or microsomes exposed to 6-MeO-BDE-47. Significant concentrations of 6-MeO-BDE-47 were also measured in fish exposed to 6-OH-BDE-47, but 6-MeO-BDE-47 was not detected in microsomes exposed to 6-OH-BDE-47. Similar patterns of transformation products were observed in medaka eggs from adult fish during exposure. This study presents direct in vivo evidence of biotransformation of 6-MeO-BDE-47 to 6-OH-BDE-47. In addition, this is the first study to demonstrate biotransformation of 6-OH-BDE-47 to 6-MeO-BDE-47. Demethylation of 6-MeO-BDE-47 was the primary transformation pathway leading to formation of 6-OH-BDE-47 in medaka, while the previously hypothesized formation of OH-PBDEs from synthetic BDE-47 did not occur. Biotransformation products formed in adult female medaka were transferred to eggs.

Ozonation attenuates the steroidogenic disruptive effects of sediment free oil sands process water in the H295R cell line.

There is concern regarding oil sands process water (OSPW) produced by the oil sands industry in Alberta, Canada. Little is known about the potential for OSPW, and naphthenic acids (NAs), which are the primary persistent and toxic constituents of OSPW, to affect endocrine systems. Although ozonation significantly reduces concentrations of NAs and OSPW toxicity, it was hypothesized that oxidation of OSPW might produce hydroxylated products with steroidogenic activity. Therefore, untreated and ozone treated OSPW were examined for effects on sex steroid production using the H295R Steroidogenesis Assay. Untreated OSPW significantly decreased testosterone (T) and increased 17beta-estradiol (E2) concentrations at OSPW dilutions greater or equal to 10-fold. This effect was mainly due to decreased E2 metabolism. Analysis of CYP19A (aromatase) mRNA abundance and enzyme activity suggested that induction of this enzyme activity may have also contributed to these effects. Reduction of parent NA concentrations by 24% or 85% decreased the effect of OSPW on E2 production. Although T production remained significantly reduced in cells exposed to ozone treated OSPW, the effect was diminished. Aromatase mRNA abundance and enzyme activity were significantly greater in cells exposed to ozone treated OSPW, however the magnitude was less than in cells exposed to untreated OSPW. No change of E2 metabolism was observed in cells exposed to ozone treated OSPW, which may account for recovery of E2 levels. The results indicate that OSPW exposure can decrease E2 and T production, but ozonation is an effective treatment to reduce NA concentrations in OSPW without increasing affects on steroidogenesis.

Simultaneous quantification of multiple classes of phenolic compounds in blood plasma by liquid chromatography-electrospray tandem mass spectrometry

A method using high performance liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS) in positive ion mode was developed for the simultaneous analysis of 30 phenolic compounds, including four estrogens, bisphenol A (BPA), 10 hydroxylated polybrominated dephenyl ethers (OH-PBDEs) and 15 bromophenols (BRPs), in blood plasma. In the present method, derivatization with dansyl chloride was employed, and all the derivertized target compounds were well resolved on a 100mm Xbridge C18 column with acetonitrile and 0.1% formic acid as the mobile phases. Purification procedures, such as liquid-liquid extraction and silica-gel chromatography, were applied to reduce matrix effects in the sample extract and remove excess derivatizing reagents, thus permitting selective and sensitive detection of the target phenolic compounds. The limit of quantification for all analytes, with a signal-to-noise ratio of approximately 10, was 2-30 pg/g (plasma weight) except for 6-OH-BDE-137 (30 pg/g) and 3-BRP (60 pg/g). The method was validated for recoveries (68-100%), accuracy (84-110%) and precision (3.7-11%) using charcoal-stripped bovine blood plasma spiked with all target compounds (500 and 5000 pg/mL). Finally, the method was applied to analyze six blood plasma samples from frogs and cormorants, where two natural estrogens, one BPA, one OH-PBDE and four BRPs were detected. The greatest total concentrations of estrogens coincided with the least total concentrations of other phenolic compounds for both species. The proposed method based on derivatization followed by LC-MS/MS provides a novel method to simultaneously monitor multiple groups of phenolic compounds in blood plasma.

Bisphenol A Disrupts Steroidogenesis in Human H295R Cells

There is increasing concern over the risk of environmentally relevant doses of bisphenol A (BPA) on human endocrine systems. Effects of BPA on steroidogenesis and the related molecular mechanisms were investigated in H295R human adenocarcinoma cells. This immortal cell line is unique in expressing all the enzymes of the steroidogenic pathways. The effects of BPA on steroidogenesis, 17β-estradiol (E2) metabolism, and aromatase activity were examined in H295R cells exposed to BPA from 3.0 × 10(-1) to 3.0 × 10(3) ng/ml. Concentrations of BPA in basic cell culture materials were verified. Stable CYP17A-knockdown H295R cells were developed to verify the mechanism of inhibited steroidogenesis by BPA. Background concentrations of BPA in control cell culture media ranged from 0.03 to 0.38 ng/ml. Significantly lesser concentrations of androstenedione, testosterone, cortisol, and cortisone were caused by exposure to 30-3000 ng BPA/ml. In contrast, sconcentrations of estrone (E1) and E2 were significantly greater in BPA-exposed H295R cells. Lesser production of androstenedione and testosterone by H295R cells exposed to BPA was the most sensitive endpoint (no observable effect concentrations < 30 ng BPA/ml). CYP17A knockdown in H295R cells resulted in less production of both 17α hydroxyprogesterone and androstenedione. The results are consistent with the hypothesis that in H295R cells, BPA selectively inhibits 17,20-lyase but not 17α-hydroxylase. The primary mechanism causing increased E2 in the medium was inhibition of E2 metabolism rather than greater aromatase (CYP19) activity. These results suggest that BPA has the potential to interfere with cellular steroidogenesis in humans through multiple molecular mechanisms.

The Impact of Metallic Coagulants on the Removal of Organic Compounds from Oil Sands Process-Affected Water

Coagulation/flocculation (CF) by use of alum and cationic polymer polyDADMAC, was performed as a pretreatment for remediation of oil sands process-affected water (OSPW). Various factors were investigated and the process was optimized to improve efficiency of removal of organic carbon and turbidity. Destabilization of the particles occurred through charge neutralization by adsorption of hydroxide precipitates. Scanning electron microscope images revealed that the resultant flocs were compact. The CF process significantly reduced concentrations of naphthenic acids (NAs) and oxidized NAs by 37 and 86%, respectively, demonstrating the applicability of CF pretreatment to remove a persistent and toxic organic fraction from OSPW. Concentrations of vanadium and barium were decreased by 67-78% and 42-63%, respectively. Analysis of surface functional groups on flocs also confirmed the removal of the NAs compounds. Flocculation with cationic polymer compared to alum, caused toxicity toward the benthic invertebrate, Chironoums dilutus, thus application of the polymer should be limited.

Effect of ozonation on the estrogenicity and androgenicity of oil sands process-affected water.

There is increasing environmental concern about the volume of oil sands process-affected water (OSPW) produced by the oil sands industry in Alberta, Canada. There is limited knowledge of the toxic effects of OSPW and one of the primary organic constituents, naphthenic acids (NAs), which are thought to be one of the toxic constituents of OSPW. OSPW and NAs can have endocrine disrupting potential. The NAs in OSPW are persistent, but ozonation can significantly reduce concentrations of NA, while increasing their biodegradability, and consequently reduce OSPW toxicity. However, it is of concern that OSPW ozonation might generate hydroxylated cycloaliphatics with endocrine disrupting potential. In this study, the estrogen receptor- (ER) and androgen receptor- (AR) mediated effects of OSPW and ozone-treated OSPW were investigated in vitro by use of T47D-kbluc (estrogen responsive) and MDA-kb2 (androgen responsive) cells. Ozonation neither attenuated nor intensified the estrogenicity of OSPW. The estrogenic responses to untreated OSPW and ozone treated OSPW were 2.58(±0.22)-fold and 2.48(±0.13)-fold greater than those of controls, respectively. Exposure to untreated OSPW produced significant antiandrogenicity in the presence of 0.01, 0.05, or 0.1 nM testosterone (T), while ozone-treated OSPW produced significant antiandrogenicity in the presence of 0.01 or 0.05 nM T. Exposure to untreated and ozone-treated OSPW also caused potentiation of androgen receptor-mediated effects of T. OSPW could cause estrogenic and antiandrogenic effects through receptor mediated pathways, and ozonation can partially mitigate the OSPW antiandrogenicity as well as androgen potentiating effect, without increasing estrogen potency.