The toxicity of organic fractions from aged oil sands process-affected water to aquatic species.

The process of surface mining and extracting bitumen from oil sand produces large quantities of tailings and oil sands process-affected water (OSPW). The industry is currently storing OSPW on-site while investigating strategies for their detoxification. One such strategy relies on the biodegradation of organic compounds by indigenous microbes, resulting in aged tailings waters with reduced toxicity. This study assessed the toxicity of OSPW aged statically for approximately 18 years. Dissolved organics in aged OSPW were fractionated using a preparative solid-phase extraction method that generated three organic fractions (F1-F3) of increasing polarity. Eight aquatic species from different trophic levels were exposed to whole OSPW (WW) and the derived OSPW organic fractions to assess toxicity: Pimephales promelas, Oryzias latipes, Vibrio fischeri, Daphnia magna, Lampsilis cardium, Hyalella azteca, Ceriodaphnia dubia, and Hexagenia spp. Broad comparisons revealed that P. promelas and H. azteca were most sensitive to dissolved organics within aged OSPW, while WW was most toxic to L. cardium and H. azteca. Three cases of possible contaminant interactions occurred within whole OSPW treatments, as toxicity was higher than organic fractions for H. azteca and L. cardium, and lower for P. promelas. As such, the drivers of toxicity appeared to be dependent on the species exposed. Of the organic fractions assessed, F3 (most polar) was the most toxic overall while F2 (intermediate polarity) displayed little toxicity to all species evaluated. This presents strong evidence that classical mono-carboxylic naphthenic acids, mostly present in F1 (least polar), are not primarily responsible for the toxicity in aged tailings. The current study indicates that although the aged tailings source (≥18 years) did not display acute toxicity to the majority of organisms assessed, inorganic components and polyoxygenated organics may pose a persistent concern to some aquatic organisms.

Stable nitrogen isotopes of nestling tree swallows indicate exposure to different types of oil sands reclamation.

Tree swallows (Tachycineta bicolor) inhabiting reclaimed wetlands on the oil sands in northern Alberta are potentially exposed to elevated levels of oil sands constituents such as polycyclic aromatic compounds (PAC) through diet. While increased detoxification enzyme activity as measured using 7-ethoxyresorufin O-deethylase in nestlings is a generally accepted indicator of exposure to oil sands constituents, there is no apparent method to detect dietary exposure specific to oil sands processed material (OSPM). In this study, stable C and N isotopes were analyzed from muscle and feathers of nestling tree swallows (15 d old) to distinguish dietary exposure of birds near reference and OSPM wetlands. High δ¹5N and low δ¹³C values in the nestling tissues differentiated those from the OSPM wetlands and reference sites. Lower δ¹5N values of nestlings compared to the δ¹5N values of larval chironomids from an earlier study suggested that the majority of the diet of the nestlings was derived from non-OSPM sources, despite residence near and on the OSPM wetlands. Our finding of limited utilization of OSPM resources by tree swallows indicates either low abundance or diversity of dietary items emerging from OSPM wetlands, or sensory avoidance of prey from those wetlands. Minimal consumption of OSPM-derived dietary sources may be attributed to published findings of limited adverse effects on tree swallow reproduction, or growth and development for these same nestlings. This study demonstrated that stable isotope analysis, particularly for N isotopes, may serve as a useful tool to trace dietary exposure to OSPM constituents as part of avian ecotoxicology assessments of reclaimed wetlands on the oil sands.

Food web structure in oil sands reclaimed wetlands.

Boreal wetlands play an important role in global carbon balance. However, their ecosystem function is threatened by direct anthropogenic disturbance and climate change. Oil sands surface mining in the boreal regions of Western Canada denudes tracts of land of organic materials, leaves large areas in need of reclamation, and generates considerable quantities of extraction process-affected materials. Knowledge and validation of reclamation techniques that lead to self-sustaining wetlands has lagged behind development of protocols for reclaiming terrestrial systems. It is important to know whether wetlands reclaimed with oil sands process materials can be restored to levels equivalent to their original ecosystem function. We approached this question by assessing carbon flows and food web structure in naturally formed and oil sands-affected wetlands constructed in 1970-2004 in the postmining landscape. We evaluated whether a prescribed reclamation strategy, involving organic matter amendment, accelerated reclaimed wetland development, leading to wetlands that were more similar to their natural marsh counterparts than wetlands that were not supplemented with organic matter. We measured compartment standing stocks for bacterioplankton, microbial biofilm, macrophytes, detritus, and zoobenthos; concentrations of dissolved organic carbon and residual naphthenic acids; and microbial production, gas fluxes, and aquatic-terrestrial exports (i.e., aquatic insect emergence). The total biomass of several biotic compartments differed significantly between oil sands and reference wetlands. Submerged macrophyte biomass, macroinvertebrate trophic diversity, and predator biomass and richness were lower in oil sands-affected wetlands than in reference wetlands. There was insufficient evidence to conclude that wetland age and wetland amendment with peat-mineral mix mitigate effects of oil sands waste materials on the fully aquatic biota. Although high variability was observed within most compartments, our data show that 20-year-old wetlands containing oil sands material have not yet reached the same level of function as their reference counterparts.

Chemical fingerprinting of naphthenic acids and oil sands process waters-A review of analytical methods for environmental samples.

This article provides a review of the routine methods currently utilized for total naphthenic acid analyses. There is a growing need to develop chemical methods that can selectively distinguish compounds found within industrially derived oil sands process affected waters (OSPW) from those derived from the natural weathering of oil sands deposits. Attention is thus given to the characterization of other OSPW components such as oil sands polar organic compounds, PAHs, and heavy metals along with characterization of chemical additives such as polyacrylamide polymers and trace levels of boron species. Environmental samples discussed cover the following matrices: OSPW containments, on-lease interceptor well systems, on- and off-lease groundwater, and river and lake surface waters. There are diverse ranges of methods available for analyses of total naphthenic acids. However, there is a need for inter-laboratory studies to compare their accuracy and precision for routine analyses. Recent advances in high- and medium-resolution mass spectrometry, concomitant with comprehensive mass spectrometry techniques following multi-dimensional chromatography or ion-mobility separations, have allowed for the speciation of monocarboxylic naphthenic acids along with a wide range of other species including humics. The distributions of oil sands polar organic compounds, particularly the sulphur containing species (i.e., OxS and OxS2) may allow for distinguishing sources of OSPW. The ratios of oxygen- (i.e., Ox) and nitrogen-containing species (i.e., NOx, and N2Ox) are useful for differentiating organic components derived from OSPW from natural components found within receiving waters. Synchronous fluorescence spectroscopy also provides a powerful screening technique capable of quickly detecting the presence of aromatic organic acids contained within oil sands naphthenic acid mixtures. Synchronous fluorescence spectroscopy provides diagnostic profiles for OSPW and potentially impacted groundwater that can be compared against reference groundwater and surface water samples. Novel applications of X-ray absorption near edge spectroscopy (XANES) are emerging for speciation of sulphur-containing species (both organic and inorganic components) as well as industrially derived boron-containing species. There is strong potential for an environmental forensics application of XANES for chemical fingerprinting of weathered sulphur-containing species and industrial additives in OSPW.

The use of stable isotopes ((13)C/(12)C and (15)N/(14)N) to trace exposure to oil sands processed material in the Alberta oil sands region.

Various oil sands reclamation strategies incorporate oil sands processed material (OSPM) such as mature fine tailings (MFT), engineered tailings (consolidated tailings, CT), and tailings pond water (TPW) into reclamation components that need to develop into viable aquatic ecosystems. The OSPM will contain elevated salinity and organics such as naphthenic acids (NA) and polycyclic aromatic compounds (PAC) that can be chronically toxic to aquatic organisms depending upon levels and age. Due to the complexity of the chemical mixtures, analysis of these compounds in exposed organisms can be challenging. In this study, the stable carbon and nitrogen isotope signatures of selected invertebrates from various types of oil sands reclamation sites were analyzed to determine whether stable isotopes can be used to trace the exposure of aquatic organisms to organic constituents of OSPM. In a series of experimental reclamation ponds of similar age and size, there were trends of (13)C depletion and (15)N enrichment for benthic invertebrates along a gradient of increased levels of MFT and/or TPW. A survey of 16 sites revealed high delta(15)N values for invertebrates in aquatic systems containing MFT and CT (gypsum-treated mixes of MFT and tailings sand), which was attributed to the presence of NH(4)(+), a process by-product in OSPM. Findings of this study indicate a potential for the use of stable nitrogen isotopes to define exposure of biota to OSPM during environmental effects monitoring programs both in surface waters and in cases where groundwater seepage containing oil sands processed water enters surface receiving environments in the region.

Reproductive and stress hormone levels in goldfish (Carassius auratus) exposed to oil sands process-affected water.

Athabasca oil sands mining in northern Alberta produces process-affected waters that are characterized by the presence of naphthenic acids, polycyclic aromatic hydrocarbons, and high salinity. The purpose of this study was to examine the impact of these process-affected waters on reproductive and stress related endpoints in mature goldfish, Carassius auratus. In two separate studies, testosterone and 17beta-estradiol levels in the plasma were significantly reduced in both male and female goldfish caged for 19 days in process-affected waters relative to controls. This effect was most pronounced in goldfish caged at a site containing mature fine tailing and tailings pond water (P5). Ovarian and testicular tissues from fish in the caging studies were incubated in vitro to evaluate potential differences in basal steroid production levels and responsiveness to human chorionic gonadotropin (hCG). Basal levels of testosterone were reduced significantly in males and females from P5 compared with the control pond (P1) demonstrating that the gonads from exposed fish had a diminished steroidogenic capacity. Gonadal tissues of fish from all ponds responded similarly to hCG suggesting that the steroid biosynthetic pathway remained functionally intact. Plasma cortisol levels were significantly higher in male goldfish caged in a pond containing mature fine tailings and capped with uncontaminated water (P3) and in P5 compared with P1. Collectively, these studies suggest that waste products of oil sands mining have the potential to disrupt the normal endocrine functioning in exposed fish through alterations to both reproductive and glucocorticoid hormone biosynthesis. In additional laboratory studies, exposure of goldfish to a naphthenic acid extract for 7 days failed to replicate the effects of processes-affected waters on plasma steroid levels and the causative agent(s) responsible for the effects on steroid biosynthesis remains to be identified.

The effects of salinity on naphthenic acid toxicity to yellow perch: gill and liver histopathology.

Naphthenic acids (NAs) are naturally occurring saturated linear and cyclic carboxylic acids found in petroleum, including the bitumen contained in the Athabasca Oil Sands deposit in Alberta, Canada. The processing of these oil sands leads to elevated concentrations of NAs, as well as increased salinity from produced waters as a result of ions leaching from the ores, the process aids, and the water associated with the deeper aquifers. These changes can result in waters that challenge reclamation of impacted waters associated with oil sands development. Laboratory tests examined the effects of salinity on NA toxicity using local young-of-the-year yellow perch exposed to a commercially available mixture of NAs (CNA) and an NA mixture that was extracted from oil sands process-affected water (ENA), with and without the addition of sodium sulfate (Na(2)SO(4)). Gill and liver histopathological changes were evaluated in the surviving fish after 3 weeks of exposure. At 6.8 mg/L ENA and 3.6 mg/L CNA, 100% mortality was observed, both with and without the addition of salt. Exposure of yellow perch to 25% of the NA required to give an LC100 (0.9 mg/L CNA; 1. 7 mg/L ENA) resulted in high levels of gill proliferative (epithelial, mucous, and chloride cell) changes, a response that was increased with the addition of 1g/L salt (Na2SO4) for the ENA. The significance of these changes was a reduced gill surface area, which likely caused a reduction in both the transport of NAs within the fish and the exchange of vital respiratory gases. While the gills were affected, no liver alterations were identified following NA or NA+salt exposures. Differences in the chemical composition of the NAs tested may explain the differences in the lethality and histopathology of yellow perch.

Gill and liver histopathological changes in yellow perch (Perca flavescens) and goldfish (Carassius auratus) exposed to oil sands process-affected water.

The extraction of bitumen from the Athabasca oil sands (Alberta, Canada) produces significant volumes of process-affected water containing elevated levels of naphthenic acids (NAs), ions, and polycyclic aromatic hydrocarbons (PAHs). The sublethal response of aquatic organisms exposed to oil sands constituents in experimental aquatic environments that represent possible reclamation options has been studied. In this study, the effects of process-affected waters on gill and liver tissues in yellow perch (Perca flavescens) and caged goldfish (Carassius auratus) held in several reclamation ponds at Syncrude's Mildred Lake site have been assessed. Following a 3-week exposure, significant gill (epithelial cell necrosis, mucous cell proliferation) and liver (hepatocellular degeneration, inflammatory cell infiltration) histopathological changes were noted in fish held in waters containing high levels of oil sands process-affected water. In addition, measurements of gill dimensions (gill morphometrical indices) proved sensitive and provided evidence of a physiological disturbance (gas exchange) with exposure to oil sands materials. Due to the complexity of oil sands process-affected water, the cause of the alterations could not be attributed to specific oil sands constituents. However, the histopathological parameters were strong indicators of exposure to oil sands process-affected water and morphometrical data were sensitive indicators of pathological response, which can be used to identify the interactive effects of ionic content, NAs, and PAHs in future laboratory studies.

Real-time visualization of processive myosin 5a-mediated vesicle movement in living astrocytes.

Recycling endosomes in astrocytes show hormone-regulated, actin fiber-dependent delivery to the endosomal sorting pool. Recycling vesicle trafficking was followed in real time using a fusion protein composed of green fluorescent protein coupled to the 29-kDa subunit of the short-lived, membrane-bound enzyme type 2 deiodinase. Primary endosomes budded from the plasma membrane and oscillated near the cell periphery for 1-4 min. The addition of thyroid hormone triggered the processive, centripetal movement of the recycling vesicle in linear bursts at velocities of up to 200 nm/s. Vesicle migration was hormone-specific and blocked by inhibitors of actin polymerization and myosin ATPase. Domain mapping confirmed that the hormone-dependent vesicle-binding domain was located at the C terminus of the motor. In addition, the interruption of normal dimerization of native myosin 5a monomers inactivated vesicle transport, indicating that single-headed myosin 5a motors do not transport cargo in situ. This is the first demonstration of processive hormone-dependent myosin 5a movement in living cells.

Myosin V plays an essential role in the thyroid hormone-dependent endocytosis of type II iodothyronine 5'-deiodinase.

In astrocytes, thyroxine modulates type II iodothyronine 5'-deiodinase levels by initiating the binding of the endosomes containing the enzyme to microfilaments, followed by actin-based endocytosis. Myosin V is a molecular motor thought to participate in vesicle trafficking in the brain. In this report, we developed an in vitro actin-binding assay to characterize the thyroid hormone-dependent binding of endocytotic vesicles to microfilaments. Thyroxine and reverse triiodothyronine (EC(50) levels approximately 1 nm) were >100-fold more potent than 3,5,3'-triiodothyronine in initiating vesicle binding to actin fibers in vitro. Thyroxine-dependent vesicle binding was calcium-, magnesium-, and ATP-dependent, suggesting the participation of one or more myosin motors, presumably myosin V. Addition of the myosin V globular tail, lacking the actin-binding head, specifically blocked thyroid hormone-dependent vesicle binding, and direct binding of the myosin V tail to enzyme-containing endosomes was thyroxine-dependent. Progressive NH(2)-terminal deletion of the myosin V tail and domain-specific antibody inhibition studies revealed that the thyroxine-dependent vesicle-tethering domain was localized to the last 21 amino acids of the COOH terminus. These data show that myosin V is responsible for thyroid hormone-dependent binding of primary endosomes to the microfilaments and suggest that this motor mediates the actin-based endocytosis of the type II iodothyronine deiodinase.

Cloning, expression, and functional characterization of the substrate binding subunit of rat type II iodothyronine 5'-deiodinase.

Type II iodothyronine 5'-deiodinase catalyzes the bioactivation of thyroid hormone in the brain. In astrocytes, this approximately 200-kDa, membrane-bound enzyme is composed of at least one p29 subunit, an approximately 60-kDa, cAMP-induced activation protein, and one or more unidentified catalytic subunit(s). Recently, an artificial type II-like selenodeiodinase was engineered by fusing two independent cDNAs together; however, no native type II selenodeiodinase polypeptide is translated in the brain or brown adipose tissue of rats. These data suggest that the native type II 5'-deiodinase in rat brain is unrelated to this artificial selenoprotein. In this report, we describe the cloning of the 29-kDa subunit (p29) of type II 5'-deiodinase from a lambdazapII cDNA library prepared from cAMP-induced astrocytes. The 3.3-kilobase (kb) cDNA encodes an approximately 30-kDa, 277-amino acid long, hydrophobic protein lacking selenocysteine. Northern blot analysis showed that a 3.5-kb p29 mRNA was present in tissues showing type II 5'-deiodinase activity such as brain and cAMP-stimulated astrocytes. Domain-specific, anti-p29 antibodies specifically immunoprecipitated enzyme activity. Overexpression of exogenous p29 or a green fluorescence protein (GFP)-tagged p29 fusion protein led to a >100-fold increase in deiodinating activity in cAMP-stimulated astrocytes, and the increased activity was specifically immunoprecipitated by anti-GFP antibodies. Steady-state reaction kinetics of the enzyme in GFP-tagged p29-expressing astrocytes are identical to those of the native enzyme in brain. Direct injection of replication-deficient Ad5-p29(GFP) virus particles into the cerebral cortex of neonatal rats leads to a approximately 2-fold increase in brain type II 5'-deiodinating activity. These data show 1) that the 3.3-kb p29 cDNA encodes an essential subunit of rat type II iodothyronine 5'-deiodinase and 2) identify the first non-selenocysteine containing subunit of the deiodinase family of enzymes.

Thyroid hormone regulates the extracellular organization of laminin on astrocytes.

Astrocytes produce laminin, a key extracellular matrix guidance molecule in the developing brain. Laminin is bound to transmembrane receptors on the surface of astrocytes known as integrins, which are, in turn, bound to the microfilament meshwork inside the astrocyte. Previous studies have shown that T4 regulates the pattern of integrin distribution in astrocytes by modulating the organization of the microfilaments. In this study, the effect of thyroid hormone on the secretion and topology of laminin in astrocytes was examined. Linear arrays of secreted laminin were observed on the surface of the T4-treated astrocytes within 10 h after seeding the cells onto poly-D-lysine-coated coverslips and became an organized meshwork by 24 h. In contrast, little if any laminin was identified on the surface of either hormone-deficient or T3-treated cells until 36 h after seeding and then was restricted to punctate deposits. Secretion of laminin into the medium by hormone-deficient and T3-treated cells was significantly greater than that by T4-treated cells. Conversely, deposition of laminin into the extracellular matrix was significantly greater in T4-treated cells than in hormone-deficient and T3-treated cells. Thyroid hormone had no effect on the production of laminin by astrocytes. These data show that T4 regulates the extracellular deposition and organization of laminin on the surface of astrocytes and provide a mechanism by which this morphogenic hormone can influence neuronal migration and axonal projection in the developing brain.

Thyroid hormone regulates the expression of laminin in the developing rat cerebellum.

In the rat cerebellum, migration of neurons from the external granular layer to the internal granular layer occurs postnatally and is dependent upon the presence of thyroid hormone. In hypothyroidism, many neurons fail to complete their migration and die. Key guidance signals to these migrating neurons are provided by laminin, an extracellular matrix protein that is fixed to the surface of astrocytes. Expression of laminin in the brain is developmentally timed to coincide with neuronal growth spurts. In this study, we examined the role of thyroid hormone on the expression and distribution of laminin in the rat cerebellum. We show that laminin content steadily increased 2- to 3-fold from birth to maximal levels on postnatal day 8-10 then steadily decreased to a plateau by postnatal day 12 in the euthyroid cerebellum. Immunoreactive laminin appeared in the molecular layer of the euthyroid cerebellum by postnatal day 4, reached maximal intensity by postnatal day 8-10, and was gone by postnatal day 14. In contrast, laminin content in the hypothyroid cerebellum remained unchanged from birth until postnatal day 10 and then increased to maximal levels over the next two days; maximal levels were approximately 35% less than those levels in the euthyroid cerebellum. Laminin staining did not appear in the molecular layer of the hypothyroid rat cerebellum until postnatal day 10, reached maximal intensity by postnatal day 15 and disappeared by postnatal day 18, despite the continued presence granular neurons in the external granular layer. These data indicate that the disruption of the timing of the appearance and regional distribution of laminin in the absence of thyroid hormone may play a major role in the profound derangement of neuronal migration observed in the cretinous brain.

The mammalian homolog of the frog type II selenodeiodinase does not encode a functional enzyme in the rat.

Type II iodothyronine deiodinase is a short-lived, membrane-bound enzyme found in rat brain, brown adipose tissue, and cAMP-stimulated astrocytes. Recently, a full-length complementary DNA (cDNA) encoding a 30-kDa, type II-like selenodeiodinase was cloned from frog, and a homologous partial cDNA (rBAT 1.1), containing two in-frame selenocysteine codons (UGA), was isolated from rat brown adipose tissue. Importantly, the rBAT 1.1 cDNA was derived from a 7.5-kb messenger RNA (mRNA) and did not encode a functional selenoenzyne unless an enabling selenocysteine insertion sequence was appended to the presumed coding region and this cDNA. In this study we determined whether the native 7.5-kb SeD2 mRNA in rat tissues programmed the synthesis of the native type II deiodinase using specific antibodies that were raised against the C-terminus of full-length, 30-kDa SeD2 protein and against the catalytic core of SeD2. Direct analysis of the translation products programmed by the native SeD2 mRNA in cAMP-stimulated astrocytes was performed using antisense deoxynucleotides and hybrid selection strategies. (Bu)2cAMP-stimulated rat astrocytes expressed both type II deiodinase activity (approximately 2500 U/mg protein) and contained abundant levels of the 7.5-kb SeD2 mRNA. However, no immunoreactive 30-kDa SeD2 protein was identified by Western analysis, immunoprecipitation, or immunocytochemistry, and the specific C-terminus antiserum failed to immunoprecipitate deiodinase activity from (Bu)2cAMP-stimulated astrocytes, brown adipose tissue or brain. Instead, the native 7.5-kb SeD2 mRNA encoded a 15-kDa protein that terminated at the first UGA codon and contained the catalytically inactive, N-terminal 129 amino acids of SeD2. These data show that the native 7.5-kb SeD2 mRNA in stimulated astrocytes does not encode D2.

Thyroid hormone-regulated actin polymerization in brain.

Thyroid hormones play an important role in the growth and development of the brain. Central to the proper integration of neuronal circuitry is the ability of the growing neurite to interpret guidance cues during its migration. The action cytoskeleton is especially rich in the growth cone, and is a likely target for thyroid hormone regulation. This brief review summarizes work showing that thyroxine, but not T3, dynamically regulates the polymerization of the actin cytoskeleton in astrocytes. The ability of T4 to enhance actin polymerization, without directly affecting gene expression, has a profound effect on the ability of the cell to interact with laminin, the major extracellular matrix protein in the developing brain. T4 also regulates the formation of key cell contacts with extracellular matrix guidance cues. These processes are likely to participate in thyroid hormone's regulation of brain development.

Inflammatory thyroid disorders.

Inflammatory disorders of the thyroid, including autoimmune thyroiditis, are among the most common endocrine abnormalities encountered in clinical practice. The association of pain with these disorders, however, is relatively uncommon. Despite this observation, painful thyroid disorders comprise a significant component of the spectrum of thyroid disease. A rational approach to such patients, including history, physical examination, laboratory evaluation, radionuclide or ultrasonographic imaging, and fine needle aspiration biopsy, will allow the appropriate diagnosis to be made in the vast majority of cases.

Catalytic activity of type II iodothyronine 5'-deiodinase polypeptide is dependent upon a cyclic AMP activation factor.

Type II iodothyronine 5'-deiodinase is an approximately 200-kDa multimeric enzyme in the brain that catalyzes the deiodination of thyroxine (T4) to its active metabolite, 3,5,3'-triiodothyronine. In astrocytes, cAMP stimulation is required to express catalytically active type II iodothyronine 5'-deiodinase. The affinity ligand N-bromoacetyl-L-T4 specifically labels the 29-kDa substrate-binding subunit (p29) of this enzyme in cAMP-stimulated astrocytes. To determine the requirements for cAMP-induced activation of this enzyme, we optimized N-bromoacetyl-L-T4 labeling of p29 in astrocytes lacking type II iodothyronine 5'-deiodinase activity and examined the effects of cAMP on the hydrodynamic properties and subcellular location of the enzyme. We show that the p29 subunit is expressed in unstimulated astrocytes and requires 10-fold higher concentrations of N-bromoacetyl-L-T4 to achieve incorporation levels equal to those of p29 in cAMP-stimulated cells. Gel filtration showed that p29 was part of a multimeric membrane-associated complex in both cAMP-stimulated and unstimulated astrocytes and that cAMP stimulation led to an increase of approximately 60 kDa in the mass of the holoenzyme. In unstimulated astrocytes, p29 resides in the perinuclear space. Cyclic AMP stimulation leads to the translocation of p29 to the plasma membrane coincident with the appearance of deiodinating activity. These data show that cAMP-dependent activation of type II iodothyronine 5'-deiodinase activity results from the synthesis of additional activating factor(s) that associates with inactive enzyme and leads to the translocation of enzyme polypeptide(s) from the perinuclear space to the plasma membrane.

Degradation and recycling of the substrate-binding subunit of type II iodothyronine 5'-deiodinase in astrocytes.

Thyroxine dynamically regulates levels of type II iodothyronine 5'-deiodinase (5'D-II) by modulating enzyme inactivation and targeting the enzyme to different pathways of internalization. 5'D-II is an approximately 200-kDa multimeric protein containing a 29-kDa substrate-binding subunit (p29) and an unknown number of other subunits. In the absence of thyroxine (T4), p29 is slowly endocytosed and transported to the lysosomes. T4 treatment rapidly activates an actin-mediated endocytotic pathway and targets the enzyme to the endosomes. In this study, we have characterized the influence of T4 on the intracellular trafficking of 5'D-II. We show that T4 accelerates the rate of 5'D-II inactivation by translocating the enzyme to the interior of the cell and by sequestering p29 in the endosomal pool without accelerating the rate of degradation of p29. This dichotomy between the rapid inactivation of catalytic activity and the much slower degradation of p29 is consistent with the reuse of p29 in the production of 5'D-II activity. Immunocytochemical analysis with a specific anti-p29 IgG shows that pulse affinity-labeled p29 reappears on the plasma membrane approximately 2 h after enzyme internalization in the presence of T4, indicating that p29 is recycled. Despite the ability of p29 to be recycled in the T4-treated cell, 5'D-II catalytic activity requires ongoing protein synthesis, presumably of another enzyme component(s) or an accessory enzyme-related protein. In the absence of T4, enzyme inactivation and p29 degradation are temporally linked, and pulse affinity-labeled p29 is internalized and sequestered in discrete intracellular pools. These data suggest that T4 regulates fundamental processes involved with the turnover of integral membrane proteins and participates in regulating the inter-relationships between the degradation, recycling, and synthetic pathways.

Selenium-regulated translation control of heterologous gene expression: normal function of selenocysteine-substituted gene products.

In eukaryotes, the synthesis of selenoproteins depends on an exogenous supply of selenium, required for synthesis of the novel amino acid, selenocysteine, and on the presence of a "selenium translation element" in the 3' untranslated region of mRNA. The selenium translation element is required to re-interpret the stop codon, UGA, as coding for selenocysteine incorporation and chain elongation. Messenger RNA lacking the selenium translation element and/or an inadequate selenium supply lead to chain termination at the UGA codon. We exploited these properties to provide direct translational control of protein(s) encoded by transfected cDNAs. Selenium-dependent translation of mRNA transcribed from target cDNA was conferred by mutation of an in-frame UGU, coding for cysteine, to UGA, coding for either selenocysteine or termination, then fusing the mutated coding region to a 3' untranslated region containing the selenium translation element of the human cellular glutathione peroxidase gene. In this study, the biological consequences of placing this novel amino acid in the polypeptide chain was examined with two proteins of known function: the rat growth hormone receptor and human thyroid hormone receptor beta 1. UGA (opal) mutant-STE fusion constructs of the cDNAs encoding these two polypeptides showed selenium-dependent expression and their selenoprotein products maintained normal ligand binding and signal transduction. Thus, integration of selenocysteine had little or no consequence on the functional activity of the opal mutants; however, opal mutants were expressed at lower levels than their wild-type counterparts in transient expression assays. The ability to integrate this novel amino acid at predetermined positions in a polypeptide chain provides selenium-dependent translational control to the expression of a wide variety of target genes, allows facile 75Se radioisotopic labeling of the heterologous proteins, and permits site-specific heavy atom substitution.