Neuronal calcium sensor proteins are unable to modulate NFAT activation in mammalian cells

Calcium activated gene transcription through Nuclear Factor of Activated T-cells, (NFAT) proteins, is emerging as a ubiquitous mechanism for the control of important physiological processes. Of the five mammalian NFAT isoforms, transcriptional activities of NFATs 1-4 are stimulated by a calcium driven association between the ubiquitous phosphatase calcineurin and the calcium-sensing protein calmodulin. Published in vitro evidence has suggested that other members of the calmodulin super-family, in particular the neuronal calcium sensor (NCS) proteins, can similarly modulate calcineurin activity. In this study we have assessed the ability of NCS proteins to interact directly with calcineurin in vitro and report a specific if weak association between various NCS proteins and the phosphatase. In an extension to these analyses we have also examined the effects of over-expression of NCS-1 or NCS-1 mutants on calcineurin signalling in HeLa cells in experiments examining the dephosphorylation of an NFAT-GFP reporter construct as a readout of calcineurin activity. Results from these experiments indicate that NCS-1 was not able to detectably modulate calcineurin/NFAT signalling in a live mammalian cell system, findings that are consistent with the idea that calmodulin and not NCS-1 or other NCS family proteins is the physiologically relevant modulator of calcineurin activity.
1
Introduction
Calcineurin (CN) [1] is a serine/threonine specific protein phosphatase present in all eukaryotes that is responsible for the modulation of numerous cell signalling pathways. Calcineurin interacts with the ubiquitous EF-hand containing calcium (Ca2+) sensor calmodulin (CaM) [2] and this obligate association drives calcium dependent phosphatase activity, one of the primary cellular targets of which are the Nuclear Factor of Activated T-cell (NFAT) family of transcription factors. This family of gene regulatory proteins is composed of five members (NFAT1-5) [3,4], that were first identified as fundamental components of the recombinatorial immune system of vertebrates [5], but which have now been implicated in a far broader range of diverse and important physiological processes. Expression of NFAT proteins is not restricted to cells of the immune system, demonstrated by their presence and effect on key functions in a selection of other cell and tissue types including brain and muscle. In a neuronal setting, NFATs have been implicated in axonal remodelling, synaptic plasticity and memory function [6-9]. Transcriptional events mediated by activated NFATs have been linked to aspects of cardiovascular development [10,11] and they have also been suggested to drive proliferative and apoptotic pathways in epithelial cells, fibroblasts, pre-adipocytes, pancreatic β-cells and osteoblasts [12-16]. The discovery that NFAT proteins can influence cell cycle regulation has led to the proposal that they may also be involved in certain aspects of tumorigenesis [17].
NFAT proteins in unstimulated cells are heavily phosphorylated on multiple serine residues by of a number of constitutively active kinases [18]. This modification acts to mask a nuclear localisation signal and under such circumstances NFATs remain predominantly cytosolic and transcriptionally inactive. Stimulation of cells with agonists coupled to the phospholipase-C/IP3 generating pathway, depletion of endoplasmic reticulum Ca2+ stores and subsequent sustained elevation of intracellular Ca2+ concentration ([Ca2+]i) due to opening of plasma membrane Ca2+ release activated Ca2+ channels elicits the rapid dephosphorylation of NFAT proteins, their nuclear import and upregulation of target gene expression [3]. Under such circumstances, the Ca2+ signal is integrated upstream of NFAT by CN in conjunction with CaM and it is this protein complex that represents the critical regulatory mechanism for NFAT dephosphorylation and hence activation of NFAT dependent gene expression. The relationship between CN/CaM and NFATs is so deeply intertwined that nuclear translocation of NFAT proteins has been employed as a robust assay of CN activity in intact mammalian cells [19-21] and has even found application in large scale screens for proteins involved in calcium induced calcium release [22].
CaM is the primordial member of a superfamily of small EF-hand containing Ca2+ binding proteins that function to transduce spatial and temporal cellular Ca2+ signals. Other members of the CaM family include the neuronal calcium sensor (NCS) subgroup of neuronal/neuroendocrine specific proteins [23,24]. Although only distantly related (21% sequence identity between CaM and NCS-1) published in vitro studies have identified potential overlap in target protein interactions between CaM and NCS-1 including an apparent positive interaction of both proteins with CN [25]. Since CaM exhibits high and ubiquitous expression these observations led to the speculation that NCS proteins may simply represent redundant or alternative modulators of classical CaM targets. Evidence from our laboratory and others has gone some way to disproving this idea with the identification of NCS specific binding proteins that, at present, have no documented interaction with CaM [26]. Further evidence for distinct functionality of the NCS protein family is apparent from their higher Ca2+ affinity compared to CaM, their differential cellular/tissue distributions and their non-redundancy revealed in genetic studies [23,27]. Together these data indicate that NCS proteins possess the requisite biochemical properties to allow them respond to unique Ca2+ signals and to bind to distinct effector proteins. The possibility exists however that overlap between CaM and NCS target specificities may be of physiological significance and in the case of CN, identification of a functionally relevant interaction with NCS proteins would be of great interest in view of recent findings implicating CN activity in aspects of neuronal function.
In order to resolve some of the outstanding questions concerning potentially redundant functions of NCS proteins we have examined whether or not they are able to elicit CN activation in the physiologically relevant setting of intact mammalian cells using NFAT activation as a coupled reporter. We present in vitro biochemical data indicating that various recombinantly expressed NCS family members are able to directly bind CN although to lower levels compared with CaM. Consistent with these data we have also determined that expression of NCS-1, mutants of this protein that have previously been demonstrated to disrupt NCS-1 function in vivo, along with other NCS family members have no effect on NFAT dephosphorylation in live HeLa cells in response to elevation of [Ca2+]i. These data are supported by studies indicating that the nuclear translocation of NFAT on elevation of [Ca2+]i to cell nuclei is unaffected in cells expressing NCS-1 or NCS-1 mutants. We therefore suggest that NCS proteins, although exhibiting a detectable in vitro affinity for CN, may not be likely to act as physiologically relevant modulators of CN activity or NFAT mediated gene transcription in intact cells.
2
Materials and methods
2.1
Recombinant proteins
All recombinant GST fusion proteins used in this study were expressed and purified as previously described [26]. Purified recombinant calcineurin was obtained from Sigma (Poole, UK) or Biomol (Exeter, UK). Unless otherwise stated all chemicals were of analytical grade and obtained from Sigma.
2.2
Small Scale GST protein binding assays
In all binding assays, recombinant GST-fusions proteins (1 μM) were immobilised by incubation with 40 μl of glutathione cellulose (GST cellulose) resin (50% bead slurry, Bioline, London, UK) that had been pre-washed in binding buffer (KCl 50 mM, HEPES 20 mM (pH 7.4), EGTA 5 mM, NTA 5 mM, CaCl2 4.6 mM (giving a free [Ca2+] of 1 μM)) (+Ca2+ conditions) or binding buffer with no added CaCl2 (- Ca2+ conditions) by incubation with constant agitation for 30 min/4 °C. Recombinant calcineurin (1 μM) or bovine brain cytosol (∼ 1 mg, dialysed against the appropriate ± Ca2+ binding buffer) were then added to immobilised GST proteins in a 100 μl total binding reaction volume and samples incubated with constant agitation for 2 hrs at 4 °C. GST cellulose beads were pelleted by centrifugation (5,000 rpm/1 min/4 °C) and washed with 1 ml of the appropriate binding buffer. This wash step was repeated three times and final bead pellets extracted into 50 μl SDS dissociation buffer (125 mM HEPES pH (6.8), 10% (v/v) sucrose, 10% (v/v) glycerol, 4% (w/v) SDS, 1% β-mercaptoethanol, 2 mM EDTA) and boiling for 5 min. Samples were resolved on SDS-PAGE (12.5% gel) and transferred to nitrocellulose filters for western blotting by transverse electrophoresis. Bound calcineurin was detected using a monoclonal antibody (1:1000, Sigma) followed by incubation with anti-mouse-HRP (1:400, Sigma) and visualisation with ECL reagents. All western blots were quantitated using ImageJ (National Institutes of Health) densitometry software.
2.3
Large scale GST protein binding assays
This binding assay is essentially as described in [26]. Briefly, each recombinant GST-fusion protein (5–10 mg), including free GST as control, was immobilised onto 3 ml glutathione-Sepharose 4B resin that had been pre-washed extensively with binding buffer by incubation for 2 h at 4 °C with constant agitation. Clarified bovine brain cytosolic extract was applied to the GST affinity columns and binding allowed to proceed for 16 h at 4 °C with constant agitation. Each column was washed with a minimum 50 volumes of binding buffer and specific Ca2+-dependent binding proteins eluted using binding buffer containing no added Ca2+(calcium-free or CF buffer). An additional high-salt elution step with binding buffer supplemented with 1 M NaCl and no added Ca2+ (High Salt or HS buffer) was used to isolate potential Ca2+-independent binding interactions. Eluted protein fractions were concentrated by methanol precipitation and pellets extracted into SDS dissociation buffer by boiling for 5 min. All western blots were quantitated using ImageJ (National Institutes of Health) densitometry software.
2.4
Sulfo-SBED cross-linking experiments
The manufacturers protocol provided by Pierce (Rockford, IL, USA) was followed with minor modifications. All steps, except those indicated, were carried out in the dark. Bait recombinant protein of interest (∼5 mg) was dialysed against binding buffer overnight at 4 °C. Immediately before use, the contents of one tube of No-Weigh Sulfo-SBED (Pierce) was dissolved in 22 μl of DMSO, this was then added to 1 ml of dialysed protein. Sulfo-SBED/protein mixes were incubated at room temperature for 30 minutes, centrifuged briefly to remove any precipitated, hydrolysed, Sulfo-SBED, and dialysed overnight against binding buffer. Bovine brain cytosol (500 μl, ∼4 mg protein) dialysed against binding buffer was added to 500μl of the Sulfo-SBED/protein mix. This mixture was incubated on a rotator at room temperature for 1 hour. After this step, samples were exposed to long-wave UV illumination (365 nm) at a distance of ∼5 cm from source for 15 minutes. During this step an appropriate volume of Neutravidin beads (Pierce) were washed extensively in binding buffer, 100 μl added to the bait/cytosol mixture and samples incubated on a rotator for 1 hour at room temperature. Bait/cytosol mixes were then centrifuged at 13,000 rpm for 1 minute, supernatants removed and beads washed with 1 ml of binding buffer. This step was repeated 6 times. Disulfide bond reduction was then achieved by incubating with 1 ml of 50 mM DTT for 1 hour at room temperature. Beads were subsequently centrifuged and washed extensively with binding buffer. After the final wash, 100 μl of SDS dissociation buffer was added to the beads and samples boiled for 10 minutes. Samples were separated on SDS PAGE (12.5% gel) and western blotted with a monoclonal anti-calcineurin antibody (1:1000, Sigma) followed by detection with anti-mouse-HRP (1:400, Sigma) and ECL reagents.
2.5
HeLa cell cultures and transfections
HeLa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Paisley, UK) containing 5% foetal bovine serum (Invitrogen), 1% non-essential amino acids (Invitrogen), and 1% penicillin/streptomycin (Invitrogen) at 37 °C in an atmosphere of 5% CO2 and maintained at ∼ 1,000,000 cells per 75 cm2 flask. 24 hrs prior to transfection, cells were seeded onto glass cover slips on a 24-well plate at ∼ 50,000 cells per well. Transfection mixtures comprised 3 μl Fugene™ (Roche, UK) per 1 μg plasmid DNA in a 100 μl total volume of DMEM and were incubated at room temperature for 30 minutes prior to being added drop-wise to cells. Cells were assayed 24-48 hrs post-transfection. The NFAT-GFP reporter construct was a kind gift from Professor Anjana Rao (Harvard Medical School, Boston, MA, USA).
2.6
Ionomycin induced NFAT translocation
Transfected HeLa cells were washed twice in Krebs buffer (NaCl 145 mM; HEPES 20 mM (pH 7.4); D-(+)-Glucose 10 mM; KCl 5 mM; CaCl2 3 mM; MgCl2 1.3 mM; NaH2PO4 1.2 mM). Cells were subsequently incubated in 500 μl Krebs containing 3 μM ionomycin or 100 μM histamine for required times before either solubilisation in SDS dissociation buffer, to investigate the phosphorylation state of NFAT via Western blotting, or fixing in 4% formaldehyde to observe the resulting sub-cellular localisation of NFAT post-treatment via confocal microscopy. For western blotting all samples were resolved on SDS-PAGE (12.5% gel) and proteins transferred to nitrocellulose filters by transverse electrophoresis. Filters were probed with monoclonal anti-GFP (1:1000, JL-8 clone, Invitrogen) followed by incubation with anti-mouse-HRP (1:400, Sigma) and protein visualised with ECL reagents.
2.7
Confocal microscopy
For confocal laser scanning microscopy, transfected cells were examined with either a Leica TCS-SP-MP microscope or a Leica TCS-SP2-AOBS microscope (Leica Microsystems, Heidelberg, Germany) using a 22 μm pinhole and a 63× water immersion objective with a 1.2 numerical aperture. EGFP was imaged using excitation at 488 nm and light collection at 500-550 nm.
3
Results
3.1
Direct binding of calcineurin to NCS proteins
A previous in vitro study demonstrated that NCS-1 is capable of stimulating the phosphatase activity of CN but did not examine whether the two proteins directly associate. To confirm a direct interaction we first performed a series of in vitro binding assays utilising various recombinantly expressed and purified NCS proteins to act as baits and where either purified recombinant CN or bovine brain cytosol were employed as a source of prey protein. Initially, purified CN (1 μM) was incubated in the presence of various GST-tagged NCS proteins (also at 1 μM) in binding buffer containing 1 μM free Ca2+ (Fig. 1a, upper panel). CaM was included in this assay as a positive control for CN binding (Fig. 1a, upper panel, Calmodulin Sepharose). CN was observed to bind directly to GST-NCS-1, GST-Hippocalcin, GST-Neurocalcin-δ and GST-CaBP1 but not GST-KChIP1, despite equal loading of the proteins (Fig. 1a, lower panel), indicating some degree of interaction specificity. It was also apparent from this analysis that, under identical experimental conditions, seven-fold less CN was bound by GST-tagged NCS proteins than by CaM. We next examined if GST-tagged NCS proteins could efficiently interact with CN present as a component of a complex protein mixture. GST control protein, GST-NCS-1 and GST-CaBP1 (Calcium Binding Protein-1, a calcium sensor more closely related to CaM than the NCS family proteins [28]) were incubated with bovine brain cytosolic protein extract in a large scale pull-down experiment [26] in the presence of 1 μM free Ca2+ and Ca2+ dependent binding partners eluted by application of Ca2+ free buffer containing EGTA and NTA (Fig. 1b, Calcium Free (CF) lanes). An additional elution step using 1 M NaCl supplemented buffer was also applied to the samples to elute potential Ca2+ independent binding proteins (Fig. 1b, High Salt (HS) lanes). Western blotting for the presence of CN in eluates confirmed a strictly Ca2+ dependent interaction with both GST-NCS-1 and GST-CaBP1. No binding was detectable to GST control protein. The signal for CN in the bovine brain cytosolic extract was compared in the same experiment (Fig. 1b, Bovine brain cytosol) and it was clear that CN was not enriched over the brain extract in the Ca2+ free eluates.
To provide an independent method to examine the interaction between NCS proteins and CN, we exploited a recently developed bi-functional cross-linking reagent [29] which permits the transfer of a biotin tag from a bait protein of interest to any specifically interacting prey proteins captured following a binding reaction. Samples from a GST-Neurocalcin-δ binding assay prepared via this method were probed with an anti-CN antibody and the presence of the phosphatase confirmed only in samples treated with the cross-linker (Fig. 1c, (+) Sulfo) and no CN was detectable in samples devoid of the reagent (Fig. 1c, (-) Sulfo). Despite use of cross-linking to maintain any low affinity interactions, CN was not enriched in the eluate over brain cytosol input into each condition (Fig. 1c (+) Sulfo vs. Bovine brain cytosol).
The data presented in Fig. 1a suggests that the affinity of CN for CaM may be substantially greater than for the NCS proteins tested. In order to independently verify these observations we further examined the binding of CN from bovine brain cytosol to GST control protein and GST-NCS-1 in direct comparison to CaM (Fig. 1d). We observed Ca2+ dependent binding of CN to CaM (Fig. 1d, Calmodulin Sepharose (+)) that was significantly enriched in the eluate compared to bovine brain cytosolic extract, in contrast, at this exposure of the western blot, binding to GST-NCS-1 was barely detectable. On longer exposure to film, NCS-1 bound CN was detectable although it should be noted that results obtained from this small scale binding assay are not directly comparable to data presented in Fig. 1b from a large scale binding assay which employed a greater amount of bovine brain cytosolic prey protein. These data are consistent with a high affinity Ca2+ dependent interaction between CaM and CN in contrast to a comparatively weak interaction between NCS-1 and CN.
3.2
Overexpression of NCS proteins has no effect on the dephosphorylation of an NFAT-GFP reporter construct in response to cytosolic Ca2+ elevation in HeLa cells
Although our in vitro studies pointed to CaM as most likely the predominant interacting/activating partner for CN, we speculated that the situation in the complex physiological setting of mammalian cells might not reflect the results obtained from such a comparatively simple analysis. We therefore attempted to resolve this issue through an examination of a possible interaction between CN and NCS proteins in intact HeLa cells. For these experiments we obtained a GFP tagged NFAT reporter construct [30], encoding the phosphorylated regulatory domain of the phosphatase, that undergoes a detectable dephosphorylation and nuclear translocation in response to sustained increases in [Ca2+]i. Our rationale was that should there be a functionally relevant NCS protein/CN interaction in cells then there might be discernable alterations in dephosphorylation kinetics/nuclear translocation of the NFAT reporter in cells [30,31] over-expressing NCS proteins as observed for other NCS-1 specific functions [32-35].
In initial studies, we saw no effect on NFAT dephosphorylation or nuclear translocation of over-expression of wild-type NCS-1, hippocalcin or neurocalcin-δ. Since NCS-1 had been identified in a previous report [25] as an in vitro activator of CN enzymatic activity we decided, however, to focus on this NCS family member for this part of the study. A second reason for examining NCS-1 in further detail also related to the fact that we had at our disposal several mutant NCS-1 constructs one of which has been previously characterised as a dominant inhibitor of NCS-1 function [32]. Such mutants, we reasoned, might assist in unmasking potential functional interactions between NCS-1, CN and NFAT. The first of these mutants, NCS-1E120Q, has been employed in a number of studies due to its ability to interact normally with NCS-1 effectors however, as a consequence of impaired Ca2+ binding and conformational change, it can then exert a dominant inhibitory phenotype [32-36]. The second NCS-1 mutant we employed in this study, NCS-1G2A, is myristoylation deficient and, unlike wild type NCS-1 which is constitutively membrane associated through this lipid modification, is entirely cytosolic [37,38]. This protein was included in these studies due to the fact that the majority of cellular CN is similarly cytosolic and hence we hypothesised that the G2A mutation may exaggerate any effects of a potential NCS-1/CN interaction.
In an initial analysis, HeLa cells were co-transfected with NFAT-GFP along with control empty vector, NCS-1 or NCS-1 mutant constructs (Fig. 2a). Cells were treated for 7 minutes with ionomycin/Ca2+ to elevate [Ca2+]i, lysed and NFAT-GFP detected by western blotting. A band corresponding to the fully phosphorylated form of NFAT was apparent in 0 time point samples from all transfections (Fig. 2a, P, upper arrow). Dephosphorylation of NFAT to two more rapidly migrating species after sustained [Ca2+]i for 7 minutes was observable in all transfected samples and there was no detectable difference in the dephosphorylation pattern obtained between control transfected cells and NCS-1 or NCS-1 mutant transfected conditions (Fig. 2a, DeP, lower arrows). These data are fully consistent with results obtained from parallel confocal imaging analyses of NFAT-GFP nuclear translocation in HeLa cells transfected and treated in an identical manner but which were then fixed for microscopy (Fig. 2b). For all transfection conditions at time 0 of Ca2+/ionomycin treatment NFAT-GFP was excluded from cell nuclei and was diffusely cytosolic (Fig. 2b, control, left hand panels), compatible with the presence of the phosphorylated form of the protein (Fig. 2a). After 7 minutes of Ca2+/ionomycin treatment NFAT-GFP had redistributed to cell nuclei for all transfection conditions (Fig. 2b, ionomycin, right hand panels) again consistent with the rapid dephosphorylation of the protein (Fig. 2a).
We additionally verified that all NCS-1 expression constructs used in these studies over-expressed in HeLa cells to similar extents when co-expressed with NFAT-GFP (Fig. 2c). Western blotting with an anti-NCS-1 antiserum [39] of total cell lystates from HeLa cells transfected identically to those used in functional assays (Figs. 2a and b) highlighted essentially equivalent levels of over-expression of NCS-1, NCS-1G2A and NCS-1E120Q proteins. There was no detectable NCS-1 protein expression in control, pcDNA transfected, cells (Fig. 2c, pcDNA).
3.3
Overexpression of NCS-1, NCS-1E120Q and NCS-1G2A has no effect on the dephosphorylation of an NFAT-GFP reporter construct in response to cytosolic Ca2+ elevation in HeLa cells over an acute time course
Our analyses suggested that, over a 7 minute time course, none of the NCS-1 constructs under examination were capable of affecting the dephosphorylation of NFAT-GFP in response to [Ca2+]i elevation and, by inference, nor were they able to influence CN activity in intact mammalian cells. These whole cell data were consistent with our in vitro binding studies however we decided to complete our investigation with two further analyses, firstly examining whether or not NCS-1 might exert a far more subtle effect on the CN/NFAT pathway over an acute time course of [Ca2+]i elevation (Fig. 3a). In these experiments, HeLa cells were again transfected with NFAT-GFP along with control or NCS-1 expression constructs however dephosphorylation was monitored over a short 5 minute time course. For all constructs tested NFAT dephosphorylation was clearly apparent by the 5 minute time point (a 25 minute time point was also included to demonstrate complete dephosphorylation of NFAT-GFP) as determined by gel shift of the NFAT-GFP. Analysis of these earlier time points indicated there was no discernable difference in the pattern of dephosphorylation between any of the NCS-1 constructs tested and control transfected cells.
In these experiments the calcium induced elevation in cytosolic free Ca2+ was artificially generated by high concentrations of ionomycin and external Ca2+. Since NCS-1 has a high affinity for Ca2+ (300 nM) one possibility is that it could activate CN under conditions of limited Ca2+ elevation thus increasing the overall cellular sensitivity to a Ca2+ signal. We completed, therefore, our analyses with an examination as to whether over-expression of NCS-1 or its mutants could influence NFAT dephosphorylation in response to a physiological stimulus. Histamine is a well characterised HeLa cell agonist that generates oscillatory intracellular Ca2+ signals [40] and activation of protein kinase C with phorbol esters has been shown to potentiate NFAT driven gene expression elicited by such Ca2+ oscillations [41-43]. We stimulated HeLa cells transfected identically to those in Figs. 2 and 3 with 100 μM histamine in both the presence and absence of 50 nM PMA over a three hour time course and assessed the phosphorylation state of NFAT-GFP by western blotting. No dephosphorylation of NFAT-GFP was detectable in these experiments in control vector expressing cells indicating that the stimulus was below the threshold for activation of CN/NFAT-GFP dephosphorylation. Significantly, cells expressing NCS-1, NCS-1E120Q or NCS-1G2A also failed to exhibit dephosphorylation of NFAT-GFP indicating that over-expression of NCS-1 did not enhance the sensitivity of the CN/NFAT signalling pathway to Ca2+ signals under these conditions (data not shown).
For NCS-1E120Q to be able to exert a dominant negative effect it would have to bind to CN. In order to validate our use of the NCS-1E120Q mutant as a dominant negative construct in this study we therefore generated a recombinantly purified GST-tagged fusion construct encoding this protein which was used to confirm its ability to bind to purified CN (Fig. 3b). GST-NCS-1E120Q retained almost wild-type CN binding activity in this assay although with less strict Ca2+-dependency validating its use in functional assays of NFAT dephosphorylation and nuclear translocation. For GST-NCS-1 we observed a 13.7-fold increase in binding of CN in the presence of Ca2+ compared to conditions where Ca2+ was absent. In contrast, there was only a 2.2-fold increase in CN binding to GST-NCS-1E120Q in the presence of Ca2+.
4
Discussion
Diverse Ca2+ signals are transduced and decoded within cells by a variety of Ca2+-sensing proteins. Of these Ca2+ responsive proteins the most intensively studied and best understood is calmodulin (CaM) [2]. CaM is the prototypical member of a super-family of evolutionarily diverse EF-hand containing Ca2+ sensors to which the NCS sub-family belongs [23]. Whilst the NCS proteins are only distantly related to CaM, comparative studies have identified significant in vitro overlap in their effector specificities [25]. It was proposed that NCS proteins might represent a set of redundant or alternative modulators of bone fide CaM target interactions [25] but the ubiquitous and high level expression of CaM would make this doubtful.
Calcineurin (CN) is a cellular serine/threonine phosphatase critical to the activation of NFAT transcription factors and hence in the control of a variety of fundamental physiological processes [3]. CaM has been identified as the essential co-activator for CN function, however CN has also been identified as a potential target for NCS-1 [25] and as such represents a candidate dual CaM/NCS effector.
A series of possibilities exist as a direct consequence of these observations. Firstly, NCS proteins could indeed simply represent redundant modulators for CaM targets and as such might be expected to elicit responses and to exhibit biochemical properties closely approximating those observed with CaM. Secondly, NCS proteins could feasibly interact with CaM targets in biochemically distinct ways to modulate their activities in specific, CaM independent, manners leading to the overall generation of unique physiological endpoints. The latter has been suggested for dual regulation of voltage-gated Ca2+-channels by both CaBP1 and CaM [44]. With specific regard to possible CN/NFAT activation by NCS-1, this possibility is intriguing in light of data identifying NFAT dependent gene expression as important to aspects of neuronal cell function. Thirdly, the possibility also exists that interactions observed between NCS proteins and CaM targets are physiologically irrelevant in cells. In this study we have attempted to resolve these issues through an investigation of firstly the biochemical characteristics of NCS protein interactions with CN, and secondly by examining the potential consequences of NCS protein function on activation of the classical CaM-CN-NFAT pathway in mammalian cells.
In a series of in vitro biochemical assays we were able to show that there exists a selective and Ca2+ dependent interaction between various NCS proteins and both purified recombinant CN, and CN derived from bovine brain cytosolic protein extract. We noted during these analyses however that significantly more CN bound to immobilized CaM. These data are consistent with the findings of Schaad et al. indicating that NCS-1 is able to stimulate the phosphatase activity of CN in vitro but to a lesser extent than observed for CaM activation. In experiments utilising bovine brain cytosol as a source of prey protein the difference in binding of the NCS proteins for CN in comparison to CaM was more greatly magnified, and whilst considerable quantities of CN could be affinity purified by a calmodulin sepharose column, the amount of CN associated with NCS family members under identical experimental conditions was considerably lower. Previous work using a biotinylated protein overlay assay also found much less binding of CN to NCS-1 compared to CaM [45]. These data led us to conclude that the CaM/CN interaction was likely to be of a higher affinity than for interactions between CN and NCS proteins. Indeed, published biochemical parameters suggest that the in vitro Kd for CaM/CN interaction may be ≤ 0.1 nM [46] with that reported for NCS-1/CN being 350 nM [25]. The in vitro affinity of CaM for CN is therefore three orders of magnitude greater than that determined for NCS-1 which would agree with the binding data presented in this study. Importantly, CaM is expressed at 5-200 fold higher concentrations in cells than NCS-1 [25] and so CN would be predicted to be bound to CaM rather than NCS-1 at elevated [Ca2+]i levels.
To assess whether NCS proteins were capable of modulating CN activity in a cellular environment we employed the dephosphorylation and nuclear translocation of a characterised NFAT-GFP reporter construct as a probe of CN phosphatase activity. Co-expression of NCS-1 did not alter the basal phosphorylation state of NFAT-GFP compared with control transfected cells nor did it exert a detectable effect on NFAT-GFP dephosphorylation/nuclear translocation after sustained elevation of [Ca2+]i during single endpoint and acute time course analyses. Co-expression of the NCS proteins Hippocalcin, Neurocalcin-δ [26], and the more closely related CaBP1 (which shares 56% similarity with CaM [47]) were similarly without effect in analyses of NFAT-GFP dephosphorylation (data not shown). From these analyses we cannot formally exclude the possibility that NCS family proteins may activate a pool of cellular CN that does not signal via NFAT dephosphorylation. This seems unlikely however taking into account our biochemical data and the fact that in numerous other studies assessing diverse triggers of CN activity, concomitant activation of the NFAT pathway is always detected.
In an extension to these studies we also examined the effects of two NCS-1 mutant proteins, NCS-1E120Q and NCS-1G2A, on NFAT-GFP dephosphorylation in HeLa cells. We were able to demonstrate that the E120Q mutant of NCS-1 [32] retained approximately wild type binding properties with respect to interaction with purified CN. However, this protein, which has been routinely observed to exert a dominant negative phenotype in various other studies of NCS-1 function [32-35], was unable to generate detectable alterations in NFAT-GFP dephosphorylation. In an effort to try and understand if NCS-1 was unable to mediate an in vivo modulation of CN activity due to its restricted membrane localisation we also examined NFAT-GFP phosphorylation in the presence of co-expressed NCS-1G2A, a protein that fails to become myristoylated and as a result remains cytosolic. Since the bulk of cellular CN is also cytosolic we reasoned that expression of the G2A mutant might bias any interaction with CN to generate an observable alteration in dephosphorylation of NFAT-GFP. Our analysis of this mutant indicated that it elicited no significant change in the kinetics of NFAT-GFP dephosphorylation compared to control transfected cells. In related experiments we tested the ability of NCS-1 and NCS-1 dominant inhibitory mutants to influence NFAT dephosphorylation in response to physiological stimuli. We observed no detectable changes in NFAT dephosphorylation in the presence of over-expressed NCS-1 proteins suggesting that in addition to sustained elevation of cytosolic free Ca2+, NCS-1 proteins are similarly unable to stimulate NFAT dephosphorylation in response to agonist derived Ca2+ signals that were below the threshold for significant activation of CN and NFAT dephosphorylation.
Collectively our findings would argue that, although NCS proteins are capable of interacting with CN in vitro, they may be unable to act as functionally relevant activators of the enzyme in a cellular context. The possibility remains that there may be some level of interplay between NCS proteins and CaM within protein complexes, exemplified by observations that both CaM and CaBP1 interact with IP3-receptors [40,48,49], that both NCS-1 and CaM are able to interact with Ca2+ channels [50] and G-protein coupled receptor kinases [34,51,52] and that CaM, NCS-1 and CaBP1 have all been shown to modulate the activity of the transient receptor potential channel, TRPC5 [35]. It is impossible to formally rule out direct modulation of CN by NCS proteins under all physiological conditions based on the data presented in this study however we have provided data to challenge the idea that CN is a cellular target of the NCS protein family and instead that CaM appears the more likely regulator of this key signalling phosphatase.

nfat, nuclear factor of activated t-cells

kchip1, potassium channel interacting protein-1

page, polyacrylamide gel electrophoresis

egta, ethylene glycol-bis(2-aminoethylether)-n,n,n′n′-tetra-acetic acid

P P P P R R R M R R M M M M M M M M M M M


Analysis of pharmaceuticals in wastewater and removal using a membrane bioreactor

Much attention has recently been devoted to the life and behaviour of pharmaceuticals in the water cycle. In this study the behaviour of several pharmaceutical products in different therapeutic categories (analgesics and anti-inflammatory drugs, lipid regulators, antibiotics, etc.) was monitored during treatment of wastewater in a laboratory-scale membrane bioreactor (MBR). The results were compared with removal in a conventional activated-sludge (CAS) process in a wastewater-treatment facility. The performance of an MBR was monitored for approximately two months to investigate the long-term operational stability of the system and possible effects of solids retention time on the efficiency of removal of target compounds. Pharmaceuticals were, in general, removed to a greater extent by the MBR integrated system than during the CAS process. For most of the compounds investigated the performance of MBR treatment was better (removal rates >80%) and effluent concentrations of, e.g., diclofenac, ketoprofen, ranitidine, gemfibrozil, bezafibrate, pravastatin, and ofloxacin were steadier than for the conventional system. Occasionally removal efficiency was very similar, and high, for both treatments (e.g. for ibuprofen, naproxen, acetaminophen, paroxetine, and hydrochlorothiazide). The antiepileptic drug carbamazepine was the most persistent pharmaceutical and it passed through both the MBR and CAS systems untransformed. Because there was no washout of biomass from the reactor, high-quality effluent in terms of chemical oxygen demand (COD), ammonium content (N-NH4), total suspended solids (TSS), and total organic carbon (TOC) was obtained.
Introduction
Most pharmaceutical substances are, by nature, biologically active and hydrophilic, in order that the human body can take them up easily, and persistent, to avoid degradation before they have a curing effect. Depending on the pharmacology of a medical substance it will be excreted as a mixture of metabolites, as unchanged substance, or conjugated with an inactivating compound attached to the molecule [1]. When they enter a wastewater-treatment plant, xenobiotics are not usually completely mineralized. They are either partially retained in the sludge, or metabolized to a more hydrophilic but still persistent form and, therefore, pass through the wastewater-treatment plant (WWTP) and end up in the receiving waters. Their removal in WWTPs is variable and depends on the properties of the substance and process conditions (e.g. sludge retention time (SRT), hydraulic retention time (HRT), temperature) [2, 3]. Levels of many pharmaceutically active compounds (PhACs) are barely reduced and they are, therefore, detected in WWTP effluents. The presence of PhACs in surface, drinking, and wastewaters is well documented in the literature [1, 4–12]. Although present at low concentrations in the environment, drugs can have adverse effects on aquatic organisms. These effects are chronic rather than acutely toxic, and depend on exposure (bioavailability), susceptibility to the compound in question, and the degradability of the compound [13].
To ensure compliance with future discharge requirements, upgrading of existing wastewater-treatment facilities and implementation of new technologies is envisaged as the next step in improvement of wastewater treatment. In the last ten to fifteen years the use of membranes in wastewater reclamation has attracted much interest. Membrane technology has become a technically and economically feasible alternative for water and wastewater treatment, especially because of high SRTs achieved within compact reactor volumes. In the MBR the concentration of microorganisms can be increased to up to 20 mg L−1. Because of this high biomass concentration the rate of degradation is higher and specialists are grown for problematic compounds. Another advantage of membrane treatment is separation of suspended solids by membranes, so they are not limited by the settling characteristics of the sludge. The performance, in terms of effluent quality, is believed to be better, but there is a striking lack of knowledge about the behaviour of trace-pollutants. Literature data on this subject is still very limited and contradictory [2, 11, 14–16]. Ultrafiltration membranes do not enable greater retention of the drugs investigated in this study as a result of filtration effects—the molecular sizes of the compounds selected are at least a factor of 100 smaller than the pore size of the membranes. Additional removal of hydrophobic compounds by membranes can, nevertheless, be expected, because they are adsorbed by particles deposited as a layer on the membrane surface; this effect for hydrophilic compounds is not yet very well defined, however [11, 17].
The objectives of this work were detection of target pharmaceuticals in wastewater influents and effluents, observation of their elimination in the CAS process, and comparison with results obtained for a laboratory-scale MBR provided with a plate-and-frame submerged membrane module. The pharmaceutical products investigated were analgesics and anti-inflammatory drugs (ibuprofen, ketoprofen, naproxen, diclofenac, indomethacin, acetaminophen, mefenamic acid, and propyphenazone), lipid regulators and cholesterol-lowering statin drugs (clofibric acid, gemfibrozil, bezafibrate, pravastatin, and mevastatin), antibiotics (erythromycin, azithromycin, sulfamethoxazole, trimethoprim, and ofloxacin), psychiatric drugs (fluoxetine and paroxetine), an antiepileptic drug (carbamazepine), β-blockers (atenolol, sotalol, metoprolol, and propranolol), anti-histaminics (famotidine and loratidine), anti-ulcer agents (lansoprazole and ranitidine), an anti-diabetic (glibenclamide), and a diuretic (hydrochlorothiazide). These compounds had different physicochemical properties (i.e. neutral, ionic, hydrophilic, hydrophobic). Their chemical structures and CAS numbers are listed in the Appendix. If their behaviour during wastewater treatment could be more reliably related to process design and operating conditions, process performance could possibly be improved by alteration of these conditions in accordance with the types of molecule that are difficult to eliminate.
Experimental
Materials and standards
Chemical standards of carbamazepine, lansoprazole, loratidine, famotidine, trimethoprim, ofloxacin, atenolol, metoprolol, azithromycin dihydrate, erythromycin hydrate, fluoxetine hydrochloride, ranitidine hydrochloride, sulfamethoxazole, propranolol hydrochloride, indomethacin, acetaminophen, mefenamic acid, clofibric acid, bezafibrate, mevastatin, and sotalol hydrochloride were purchased from Sigma–Aldrich (Steinheim, Germany), propyphenazone, pravastatin, and paroxetine hydrochloride from LGC Promochem (London, UK), ketoprofen, diclofenac, gemfibrozil, ibuprofen, and naproxen from Jescuder (Rubí, Spain), glibenclamide from SIFA Chemicals (Liestal, Switzerland), and hydrochlorothiazide from Pliva (Zagreb, Croatia). All pharmaceutical standards were of high-purity grade (>90%).
Isotopically labelled compounds used as internal standards were 13C-Phenacetin, from Sigma–Aldrich, mecoprop-d3, from Dr Ehrenstorfer (Augsburg, Germany), and ibuprofen-d3, atenolol-d7, and carbamazepine-d10 from CDN Isotopes (Quebec, Canada).
All solvents (methanol, acetonitrile, and water) were HPLC-grade and were purchased from Merck (Darmstadt, Germany), as also was hydrochloric acid (HCl, 37%), ammonium acetate (NH4Ac), and acetic acid (HAc). Nitrogen for drying, purity 99.995%, was from Air Liquide (Spain).
Stock solutions of individual standards (1 g L−1) and internal standards were prepared in methanol and stored at −20 °C. Stock solutions of ofloxacin, pravastatin, and sulfamethoxazole were renewed monthly because of their limited stability. A standard mixture in which the compounds were at a concentration of approx. 20 mg L−1 was prepared from the stock solutions. Further dilutions of this mixture were prepared in 25:75 (v/v) methanol–water and were used as working standard solutions. A mixture of internal standards prepared by dilution of individual stock solutions in methanol was used for internal standard calibration.
Membrane bioreactor (MBR)
A submerged MBR of approximately 21 L active volume equipped with two flat sheet membranes (A4 size, area 0.106 m2, pore size 0.4 μm), purchased from Kubota (Osaka, Japan), was installed in a municipal WWTP in Rubí (Barcelona, Spain). Although the nominal porosity of the membranes was 0.4 μm (microfiltration) a fouling layer of proteins and microorganisms formed on the surface of the membranes reduced the effective porosity to 0.01 μm, which brought the type of filtration into the ultrafiltration range [17].
The MBR was operated in parallel with the CAS process (aeration tank and secondary settling tank). The biocenosis of the MBR was grown from inoculated sludge from the municipal WWTP (aeration basin) and cultivated over a period of approximately 1 month to reach steady-state conditions. The hydraulic retention time was set to 14 h by regulating the effluent flow and the SRT was infinite, because no sludge was discharged from the reactor.
The laboratory-scale MBR was operated dynamically in intermittent permeation mode—cycles of permeation for 8 min interrupted with 2 min of halt. Influent and permeate flows were controlled by use of flow meters and computer-controlled pumps. Continuous aeration was provided by means of a sparger pipe situated at the bottom of the reaction vessel; the oxygen concentration was kept between 1 and 2 mg L−1. The temperature inside the reactor was 20 ± 2 °C throughout sampling.
Wastewater-treatment plant (WWTP)
Rubí WWTP was designed for 125,550 inhabitant equivalents. During the sampling programme the WWTP was operating with an average daily flow of 22,000 m3 day−1. A mixture of municipal, hospital, and industrial wastewater is treated. Treatment consists of pretreatment, preliminary treatment, primary sedimentation, and secondary (biological) treatment. Pretreated wastewater goes through a physical process of settling in a primary clarifier. Secondary treatment occurs in pre-denitrification (anaerobic) and nitrification (aerobic) tanks, and two secondary clarifiers. Secondary sludge is recirculated to a primary clarifier which improves the settling characteristics of the primary sludge and increases sludge age. A mixture of primary and secondary (activated) sludge is processed (thickening, dewatering) and anaerobically digested, and biogas produced is used to heat a digester. The hydraulic retention time of CAS treatment in WWTP Rubí, calculated for the average daily flow, is approximately 12 h. During the sampling programme the plant was operating with an SRT of approximately 3 days. WWTP effluent is discharged into the river Riera de Rubí, which flows into the Mediterranean sea.
Sampling and sample preparation
Sampling was conducted during May and June, 2005. Twenty-eight samples were analyzed. All wastewater samples were taken as time-proportional grab-samples, bearing in mind the HRT of the MBR and CAS processes. The sampling points were:
primary sedimentation tank effluent, as the inflow to the conventional treatment plant and membrane bioreactor,CAS effluent, andMBR effluent.
Wastewater samples were collected, in amber glass bottles pre-rinsed with ultra-pure water, as 24-h composite samples; the volume depended on the type of sample (100 mL for influent wastewater and 200 mL for effluent). Immediately on arrival at the laboratory they were filtered through 1-μm glass fibre filters and then through 0.45 μm Nylon membrane filters from Whatman (UK). The target compounds were extracted in one step, by a method described elsewhere [18], using a Baker vacuum system (J.T. Baker, The Netherlands) and Waters (Milford, MA, USA) Oasis HLB cartridges (60 mg, 3 mL) previously conditioned at neutral pH with 5 mL methanol then 5 mL deionised water (HPLC grade). Elution was performed twice with 4 mL methanol at a flow of 1 mL min−1. The extracts were then evaporated under a nitrogen stream and reconstituted with 1 mL 25:75 (v/v) methanol–water.
Chemical analysis
LC analysis was performed with a Waters (Milford, MA, USA) 2690 HPLC system coupled to a Micromass Quattro (Manchester, UK) triple quadrupole mass spectrometer equipped with a Z-spray electrospray interface. Chromatographic separation was achieved on a Purospher Star RP-18 endcapped column (125 mm × 2.0 mm, particle size 5 μm) and a C18 guard column, both from Merck.
A specific multi-residue analytical method was set up for measurement of the concentrations of the pharmaceutical compounds in wastewaters [18]. Analysis was performed in multiple-reaction-monitoring (MRM) mode, in both positive and negative electrospray-ionization mode. This method was refined for analysis of hydrochlorothiazide and glibenclamide. MRM transitions selected for each compound are summarized in Table 1. In accordance with the performance characteristics defined in EU Commission Decision 2002/657/EC for confirmation and identification of pharmaceuticals when using LC–tandem MS as the instrumental technique, a minimum of three identification points are required. When using LC–MS–MS (QqQ) analysis two MRM transitions are sufficient to confirm the identity of a compound. The MRM ratio, calculated as the relationship between the abundances of both transitions and the LC retention time are also criteria used to confirm the presence of an analyte in the samples. In this study, therefore, transitions between a precursor ion and the two most abundant fragment ions were chosen for each analyte when working in MRM mode, resulting in four identification points, enough to conform with the aforementioned EU directive. When poor fragmentation was observed for the compounds, only one transition could be monitored. Confirmation of the identities of these was achieved by matching their LC retention times with those of standards. Shifts in retention times were less than 3%, so the confirmation was regarded as sufficiently reliable. For internal standards only one transition was selected, because they were isotopically labelled compounds unlikely to be found in environmental samples.
Table 1MRM transitions of the compounds analyzedCompoundMRM 1MRM 2MRM 3Ibuprofen205→161Ketoprofen253→209253→197Naproxen229→170229→185Diclofenac294→250294→214Indomethacin356→297356→255Acetaminophen152→110152→93Mefenamic acid240→196240→180Propyphenazone231→201231→189Clofibric acid213→127213→85Gemfibrozil249→121Bezafibrate360→274360→154Pravastatin447→327Mevastatin391→185391→159Carbamazepine237→194237→192Fluoxetine310→44310→148Paroxetine330→192330→123Lansoprazole370→252370→205Famotidine338→189338→259Ranitidine315→176315→130Loratidine383→337383→267383→259Erythromycin734.5→158734.5→576.4Azithromycin749.5→591.4749.5→158Sulfamethoxazole254→92254→156Trimethoprim291→230291→261Ofloxacin362→316Atenolol267→190267→145Sotalol273→255273→213Metoprolol268→133268→159Propranolol260→166260→183Hydrochlorothiazide296→269296→205Glibenclamide494→369
To compensate for matrix effects from sample matrices internal standard calibration and adequate dilution of sample extracts were used, on the basis of the previously published method [18].
Recoveries of the method were determined by spiking. Influent samples and CAS and MBR effluents were spiked in triplicate with a standard mixture of selected compounds to a final concentration of 1 μg L−1. Spiked samples and a blank sample were analysed by the above mentioned method. Recoveries of the target pharmaceuticals are listed in Table 2, with method detection limits (MDL) for influent and effluent samples. MDLs and method quantification limits (MQL) were calculated on the basis of signal-to-noise ratios (S/N) of 3 and 10, respectively.
Table 2Mean recoveries of the selected compounds and method detection limits (MDL) in ng L−1CompoundRecovery (%)MDL (ng L−1)InfluentMBR effluentCAS effluentInfluentMBR and CAS effluentIbuprofen131 (18.1)a68.8 (11.0)90.4 (11.0)98.020.0Ketoprofen62.8 (2.94)71.3 (3.11)59.1 (0.897)19074.0Naproxen49.2 (20.0)59.4 (1.28)53.4 (2.31)79.020.0Diclofenac83.3 (1.17)94.9 (10.0)95.0 (12.6)16040.0Indomethacin113 (2.95)120 (5.63)110 (3.78)15031.0Acetaminophen123 (17.0)108 (10.5)56.0 (7.61)20.95.35Mefenamic acid93.3 (1.95)92.1 (1.02)91.5 (5.29)5.701.85Propyphenazone60.0 (8.00)71.0 (4.00)71.0 (4.00)4.801.45Clofibric acid86.0 (10.8)104 (6.87)74.5 (1.40)16.33.75Gemfibrozil91.0 (8.47)87.5 (1.36)108 (17.2)8.702.20Bezafibrate106 (3.43)94.4 (9.30)89.4 (4.62)18.54.35Pravastatin85.6 (2.56)78.0 (12.2)96.0 (19.5)12030.9Mevastatin103 (8.61)134 (15.6)123 (9.86)9.301.30Carbamazepine84.0 (7.84)89.5 (5.20)88.0 (9.24)2.200.600Fluoxetine46.7 (2.34)93.7 (17.6)59.0 (1.60)19.81.70Paroxetine62.2 (2.15)109 (5.73)71.4 (1.49)3.500.650Lansoprazole70.0 (10.0)87.0 (5.00)86.0 (4.00)10.94.20Famotidine58.2 (7.76)55.4 (6.30)66.6 (5.39)3.100.40Ranitidine41.5 (9.85)75.8 (14.8)125 (11.7)1.400.300Loratidine72.6 (1.81)78.0 (6.97)64.5 (4.98)8.002.75Erythromycin67.7 (3.15)50.0 (13.0)66.6 (12.0)12.42.00Azithromycin30.0 (7.00)73.0 (2.00)63.0 (3.00)1.000.300Sulfamethoxazole33.7 (2.76)95.5 (9.24)78.3 (1.08)16.13.10Trimethoprim58.8 (3.29)128 (6.58)60.8 (3.87)1.300.350Ofloxacin142 (19.0)135 (5.45)138 (4.47)29.37.85Atenolol83.5 (33.8)60.8 (10.8)131 (15.5)1.700.750Sotalol47.1 (2.91)31.9 (3.05)52.0 (3.63)4.800.700Metoprolol36.7 (1.44)120 (2.64)76.7 (1.43)6.301.60Propranolol60.2 (0.506)90.8 (4.02)70.5 (5.27)2.600.300Hydrochlorothiazide39.8 (7.43)58.9 (1.62)73.4 (22.9)4.500.900Glibenclamide100 (11.7)107 (10.3)98.5 (11.7)19.22.30aThe relative standard deviation (%) of the recoveries is given in parentheses (n = 3)
Results and discussion
It is well documented that WWTPs are major contributors of pharmaceuticals in the environment. WWTP Rubí mainly receives domestic, hospital, and industrial wastewater and effluent concentrations of several monitored compounds exceed μg L−1 levels. Ranges of output loads for WWTP Rubi for each pharmaceutical and mean values (g day−1) are reported in Table 3. The quantities of pharmaceuticals discharged into the environment are calculated by multiplying the detected effluent concentrations by an average daily flow rate of 22,000 m3 day−1. The total amount of pharmaceutical compounds discharged by WWTP Rubi into the environment exceeded 300 g day−1 (an average value). The most abundant compounds, with average individual loads of 21–56 g day−1, were the anti-inflammatory drugs ibuprofen, naproxen, and diclofenac, the lipid regulators gemfibrozil and bezafibrate, the diuretic hydrochlorothiazide, and the β-blocker atenolol.
Table 3Average daily output loads of the investigated pharmaceuticals for Rubí WWTPPharmaceuticalEffluent load (g day−1)MeanRangeAnalgesics and anti-inflammatory drugsNaproxen37.010.8–76.9Ketoprofen17.111.4–36.3Ibuprofen56.37.39–137.9Diclofenac27.317.3–43.8Indomethacin1.93nd–2.73Acetaminophen4.551.06–9.2Mefenamic acid0.440.27–0.85Propyphenazone0.680.43–0.96Anti-ulcer agentRanitidine2.770.55-5.30Psychiatric drugParoxetine0.08nda–0.16Antiepileptic drugCarbamazepine5.211.44-6.71AntibioticsOfloxacin6.932.40–11.2Sulfamethoxazole3.061.42–5.81Erythromycin2.290.95–4.51β-blockersAtenolol21.07.70–33.2Metoprolol3.321.14–5.43DiureticHydrochlorothiazide33.721.2–46.0Hypoglycaemic agentGlibenclamide0.74nd–0.98Lipid regulator and cholesterol lowering statin drugsGemfibrozil54.330.1–73.9Bezafibrate21.610.9–50.8Clofibric acid1.750.40–3.43PravastatinndndaNot detectable (below the LOQ)
To assess the efficiency of elimination by the MBR, substance-specific analysis must be performed and the bulk properties DOC and COD of wastewater influents and effluents must also be determined. The performance of the MBR system is summarized in Table 4. The data are presented for the sampling period. Removal efficiencies of 98.7% for TSS and 90.4% for total COD were achieved during the membrane process. Average effluent ammonia concentration was 1.01 μg L−1 in the MBR effluent, compared with 48.41 μg L−1 in the CAS effluent. It is known that membrane processes are quite efficient at removing COD and TOC from wastewater [19, 20]. Improved COD removal is attributed to the combination of complete retention of particulate material by the membrane, including suspended COD and high molecular weight organisms, and to avoidance of biomass washout problems common in activated sludge systems. Consequently, stable conditions are provided for growth of specialized microorganisms which are the able to remove poorly biodegradable components.
Table 4Summary of the performance of the MBR systemPropertyInfluentMBR effluentCAS effluentTSS (mg L−1)119.2 (17.37)a1.600 (1.770)26.72 (15.69)CODtotal (mg L−1)508.2 (124.3)48.58 (22.47)111.6 (53.35)TOC (mg L−1)67.67 (24.29)10.89 (3.470)27.33 (13.75)N-NH4 (mg L−1)49.13 (15.79)1.010 (0.4200)48.41 (12.87)pH7.52 (0.300)7.08 (0.270)7.63 (0.160)aValues are averages from n = 16 samples, with standard deviations in parentheses
Of 31 pharmaceutical products included in the analytical method, 22 were detected in the wastewater entering WWTP Rubí. Box plots of measured concentrations of each pharmaceutical are showed in Figs. 1, 2 and 3. Ten measured values are given for influent and MBR effluent concentrations and eight for CAS effluent (data are missing for two sampling programmes). For each variable the box has lines at the lower quartile (25%), median (50%), and upper quartile (75%) values. The whiskers are the lines extending from each end of the box to show the extent of the data up to 1.5 times the interquartile range (IQR). Outliers are marked with + symbols.
Fig. 1Removal, during MBR and CAS treatment, of the analgesics and anti-inflammatory drugs naproxen (a), ketoprofen (b), ibuprofen (c), mefenamic acid (d), diclofenac (e), indomethacin (f), acetaminophen (g), and propyphenazone (h)Fig. 2Removal during MBR and CAS treatment of the antibiotics ofloxacin (a), sulfamethoxazole (b), and erythromycin (c), the β-blockers atenolol (d) and metoprolol (e), the anti-ulcer agent ranitidine (f), the antiepileptic drug carbamazepine (g), and the psychiatric drug paroxetine (h)Fig. 3Removal during MBR and CAS treatment of the lipid regulator and cholesterol-lowering statin drugs gemfibrozil (a), bezafibrate (b), clofibric acid (c), and pravastatin (d), the diuretic hydrochlorothiazide (e), and the hypoglycaemic agent glibenclamide (f)
The highest influent concentrations (μg L−1) were measured for the analgesics and anti-inflammatory drugs naproxen, ibuprofen, ketoprofen, diclofenac, and acetaminophen, the antihyperlipoproteinaemic drugs gemfibrozil and bezafibrate, the β-blocker atenolol, and the diuretic hydrochlorothiazide. For other compounds input concentrations were usually in the range 10–100 ng L−1. Because the low concentrations measured were sometimes close to the limits of quantification, any hypothesis about the efficiency of their elimination is questionable. Mean removal was, nevertheless, calculated for each of the pharmaceutical compounds; the results are presented in Table 5. The most important removal pathways of organic compounds during wastewater treatment are:
biotransformation/biodegradation,adsorption by the sludge (excess sludge removal), andstripping by aeration (volatilization).Table 5Mean removal of selected pharmaceuticals by the MBR and CAS processesCompoundElimination (%) in:MBRaCASbAnalgesics and anti-inflammatory drugsNaproxen99.3 (1.52)85.1 (11.4)Ketoprofen91.9 (6.55)51.5 (22.9)Ibuprofen99.8 (0.386)82.5 (15.8)Diclofenac87.4 (14.1)50.1 (20.1)Indomethacin46.6 (23.2)23.4 (22.3)Acetaminophen99.6 (0.299)98.4 (1.72)Mefenamic acid74.8 (20.1)29.4 (32.3)Propyphenazone64.6 (13.3)42.7 (19.0)Anti-ulcer agentsRanitidine95.0 (3.74)42.2 (47.0)Psychiatric drugsParoxetine89.7 (6.69)90.6 (4.74)Antiepileptic drugsCarbamazepineNo eliminationcNo eliminationAntibioticsOfloxacin94.0 (6.51)23.8 (23.5)Sulfamethoxazole60.5 (33.9)55.6 (35.4)Erythromycin67.3 (16.1)23.8 (29.2)B-blockersAtenolol65.5 (36.2)No eliminationMetoprolol58.7 (72.8)No eliminationDiureticsHydrochlorothiazide66.3 (7.79)76.3 (6.85)Hypoglycaemic agentsGlibenclamide47.3 (20.1)44.5 (19.1)Lipid regulator and cholesterol lowering statin drugsGemfibrozil89.6 (23.3)38.8 (16.9)Bezafibrate95.8 (8.66)48.4 (33.8)Clofibric acid71.8 (30.9)27.7 (46.9)Pravastatin90.8 (13.2)61.8 (23.6)a,bValues are averages, with relative standard deviations (%) in parentheses, for n = 10a or n = 8b samplescCompounds were classified as “no elimination” if elimination was less than 10%
Because of the low values of the Henry coefficients (KH) of the compounds investigated [21], the fraction removed by volatilization can be neglected [16]. The two processes abiotic (adsorption) and biotic degradation (transformation by microorganisms) could not be distinguished, because no method was developed for analysis of the target compounds in sludge. The term “removal” is therefore used here for conversion of a micropollutant to compounds other than the parent compound.
Elimination efficiency of the laboratory-scale MBR and the full-scale CAS process was comparable for naproxen, ibuprofen, acetaminophen, hydrochlorothiazide, and paroxetine. All were removed to a large extent by both systems (removal was greater than 80% except for hydrochlorothiazide, for which it was between 56 and 85%). Hydrochlorothiazide and paroxetine were eliminated slightly better by conventional treatment. Similar results for the behaviour of these drugs during conventional treatment have been reported by several authors [2, 3, 9, 11].
For ketoprofen, diclofenac, bezafibrate, and gemfibrozil removal by the MBR system was very high and uniform (>90%), with the exception of two sampling programme. It is assumed this variation could have been a result of reduced microbial activity or altered sorption and flocculation conditions. No plausible explanation can be given for the drastically reduced efficiency of removal of clofibric and mefenamic acid by MBR in two sampling programmes; otherwise these were eliminated with efficiencies between 65 and 90%. High and steady removal (>80%) in the MBR was also observed for ranitidine and ofloxacin. In conventional treatment all these pharmaceuticals were eliminated with a wide range of efficiencies, always lower than those obtained by the MBR. Better removal of readily biodegradable micropollutants by the MBR could be because of the smaller flock size of the sludge, which enhances mass transfer by diffusion and therefore increases elimination. Taking into consideration the composition of sludge originating from a membrane bioreactor (specialized microorganisms, large amount of active biomass in suspended solids) improved removal is to be expected; this was confirmed by our experiments.
A possible explanation of substantially greater attenuation of diclofenac by the MBR (average removal efficiency 87% compared with 50% in CAS) could be the greater age of the MBR sludge. Improved removal is observed with increasing solids retention time [14]. Another explanation could be greater adsorption potential of the MBR sludge, because the organic matter content is greater than for CAS sludge. According to results from the EU project Poseidon [22], adsorption processes affect elimination of diclofenac. Literature data on this matter is still very contradictory. Clara et al. reported poor removal of diclofenac in laboratory-scale WWTPs whereas in full-scale plants removal varied from less than 20% to between 60 and 80% for some of the facilities investigated [2]. Heberer et al. [7] reported low removal efficiencies in a WWTP whereas Ternes et al. documented significant (69%) elimination of diclofenac [8].
Removal of carbamazepine was, in contrast, very poor (<20%), and effluent concentrations for both MBR and CAS were frequently greater than influent levels. Poor elimination of this neutral drug has been reported by many authors [9, 11, 23, 24]. Glucuronide conjugates of carbamazepine can, presumably, be cleaved in sewage, thus increasing environmental concentrations [8].
Rates of removal of the antibiotic sulfamethoxazole were very variable in both treatments investigated. According to Drillia et al. its microbial degradation will depend on the presence of readily biodegradable organic matter in wastewater; this varies during both MBR and CAS treatment [25]. Also, a substantial amount of sulfamethoxazole enters WWTPs as its human metabolite N4-acetylsulfamethoxazole, which can possibly be converted back to the original compound during treatment [26].
Efficiency of removal of atenolol, metoprolol, pravastatin, erythromycin, and indomethacin varied in both MBR and CAS treatment. This could not be explained. Fluctuation of elimination efficiency was also observed for propyphenazone (44.8–82.9% for MBR and 6.82–62.6% for CAS) and glibenclamide (14.8–73.7% for MBR and 11.9–79.7% for CAS).
Effluent concentrations greater than those recorded for the influent could be explained by the presence of input conjugate compounds that are transformed into the original compounds during treatment. Because these conjugates were not included in the analysis, no firm conclusion can be made about their biotransformation, especially because sampling inaccuracy can also lead to errors.
Conclusion
Several pharmaceutical products (e.g. ibuprofen, naproxen, acetaminophen, ketoprofen, diclofenac, bezafibrate, gemfibrozil, ranitidine, ofloxacin, hydrochlorothiazide, and paroxetine) with high rates of attenuation can be expected to be completely removed from wastewater by adsorption or degradation, or a combination of both, during membrane treatment. For most of the compounds investigated MBR effluent concentrations were significantly lower than in the effluent from conventional treatment. Elimination of hydrochlorothiazide and paroxetine was slightly better in CAS treatment. Some substances (e.g. carbamazepine) were not removed by either MBR or CAS treatment. No relationship was found between the structures of target compounds and their removal during wastewater treatment, however. The range of variation of the efficiency of removal by the MBR system was small for most of the compounds; in conventional treatment greater fluctuations were observed and removal efficiency was found to be much more sensitive to changes in operating conditions (temperature, flow rate, etc).
Although membrane technology seems a promising means of removal of pharmaceutical compounds, the MBR process investigated would not completely halt discharge of micropollutants. Membrane treatment processes should be optimized by modification of the membranes (variation of the materials and reduction of molecular mass cut-off limits) and/or by modification of the treatment process (inoculation of special microorganisms). The efficiencies of diverse microbial populations in elimination of selected pharmaceuticals, and optimization of design and operating conditions of a laboratory-scale MBR will be the main objectives of our future investigations. That would provide guidelines for scale-up of a biological pilot plant and its evaluation by integration into an industrial process water-recycling system. Because of the current lack of information on the behaviour of pharmaceuticals in surface and wastewaters, however, further studies are required on the occurrence, fate, and effects of these substances in the environment.

pharmaceuticals
membrane bioreactor
removal efficiency
wastewater treatment
conventional activated sludge treatment