faculty of life sciences & medicine analytical & environmental … · 2017-07-29 · oral...

Postgraduate Research Symposium - 14th May, 2015

Faculty of Life Sciences & Medicine

Analytical & Environmental Sciences Division

Welcome

Cover illustration by James Gooch [OP2]

Introduction

Welcome to the 2015 Analytical & Environmental Sciences Division Postgraduate Research Symposium. The format for today’s Research Symposium includes poster presentations from our 1st and 2nd year graduate students and oral presentations from our final (3rd or 4th) year students. Postgraduate student training is a key objective for the Analytical & Environmental Sciences Division and staff will be present to provide helpful feedback to students. The poster format allows discussion between students and staff resulting in direct feedback. The aim is to help build our students confidence and presentational skills. For students in their final year the symposium provides an opportunity to improve presentational skills in the more formal setting of the lecture room. Furthermore, it provides them with experience in dealing with questions from a friendly and interested audience and this will help identify possible gaps in understanding in preparation for the oral examination of their thesis. Feedback will also be provided after the event to help students address any perceived weaknesses in their communication skills.

All presentations will be assessed by a panel of judges drawn from our academic and research staff. The panels’ goal is to not only identify the best oral and poster presentations but importantly to provide feedback to all participants so that they might learn from the experience. Prizes will be awarded for the best poster and the best oral presentation at the reception at the end of the Symposium. This year we also are pleased to include the Metrohm Student Award which has been won by Elizabeth Gilchrist.

We are always looking for ways to improve the usefulness of the annual symposium for our students and this year we include a lecture by Professor Debbie Jarvis, Professor of Public Health at the National Health and Lung Institute.

I would like to take this opportunity to thank all the participating students for their hard work. My thanks is also extended to all staff involved in the event including the Chairs of the oral presentations and the judges. Special thanks go to Prof Stephen Stürzenbaum and Dr Leon Barron the Divisions’ Postgraduate co-coordinators who, with the assistance of Angela Lewis and Valentina Lotti organized today’s event. I’m looking forward to the day immensely and I hope everyone enjoys the event.

Professor Frank Kelly Director, Analytical & Environmental Sciences Division

Analytical and Environmental Sciences Division Postgraduate Research Symposium

14th May, 2015

13:00 – 13:10 Introduction: Prof Frank Kelly (room: FWB 1.11)

13:10 - 14:50 Oral Presentation Session 1 (room: FWB 1.11) Chair: Dr. Leon Barron

13:10 – 13:30 [O1] Max Priestman

13:30 – 13:50 [O2] Wael Aljohani

13:50 – 14:10 [O3] Kelly Munro

14:15– 14:35 Short Oral Presentation Session 2 (room FWB 1.11)

Chair: Prof Stephen Stürzenbaum

14:15 – 14:20 [OP1] Yona Essig

14:20 – 14:25 [OP2] James Gooch

14:30 – 14:35 [OP3] Bronagh Murphy

14.40 – 15.00 Refreshments (room: FWB 1.11) 15:00-15:50 Key Note Speaker (room FWB 1.11) 15:00 – 15:40 Prof Deborah Jarvis:

Asthma and COPD - the research behind the headlines

15:40 – 15:50 Discussion

16:00-17:30 Poster Session and Reception (room FWB 1.13)

[OP1]- [OP3] and [P1]- [P7] Poster Presentations

17:30 Concluding Remarks and Prize Giving (room FWB 1.13)

Best Poster, Best Talk and Metrohm Student Award


14th May, 2015

Abstracts

Oral Presentations

[O1] Max Priestman Measurements of traffic-related CO2 emissions in London [O2] Wael Aljohani Fabrication and evaluation of an organic monolithic column based upon the polymerisation of hexyl methacrylate with 1,6-hexanediol ethoxylate diacrylate for the separation of small molecules by capillary liquid chromatography [O3] Kelly Munro ‘Semi-targeted’ analysis of the river Thames: combining targeted and non-targeted approaches for pharmaceutical and illicit drug residue detection

Oral Presentation [1]

Measurements of traffic-related CO2 emissions in London

Max Priestman1, Benjamin Barratt1, Sue Grimmond2

1Analytical and Environmental Sciences Division, Faculty of Life Sciences & Medicine, King’s College London, 2Department of Meteorology, University of Reading

Background: Currently, transport accounts for one-quarter of global energy use and energy-related CO2 emissions. It is the only sector of the UK economy for which current green house gas emissions, primarily as CO2, are greater than in 1990, estimated at 121 Mt CO2 or 26% of total CO2 emissions(1). These estimates are typically based on emission factors calculated from the consumption of fossil fuels or from specific drive cycle testing under laboratory conditions, rather than direct ‘real-world’ measurements of the emissions themselves. Our research has the aim of providing measured assessments of the impact of local and national CO2 reduction schemes within the transport sector, thereby allowing cross-validation of standard emission inventory-based estimates. Methods: For the first phase of research, we implemented a fixed, long-term continuous CO2 monitoring network at ground level across Greater London with the aim of quantifying vehicle exhaust CO2 emissions from ambient CO2 and co-located NOX measurements. Measurements are taken at three roadside monitoring stations across London and at two urban background sites. The second phase focused on coupling emissions factors calculated from the CO2 monitoring network to new emissions factors derived by an on-road remote sensing device (RSD), developed at the University of Denver(2) (figure 1). The RSD measures emissions ratios of NOX to CO2 directly from the exhaust plumes of passing vehicles. Results: Between 2010 and 2014, ambient CO2 concentrations were elevated by between 9 and 35 ppm at roadside monitoring stations in comparison to urban background locations during a typical day. During daylight hours alone this enhancement was as high as 55 ppm at the kerbside in a street canyon. However, no significant trend in traffic-related CO2 was detected from four years of monitoring. When coupled with co-located NOX measurements, NOX/CO2 emissions ratios of 3.6 – 4.1 ppb ppm-1 from the static network were somewhat lower than the 5.5 ppb ppm-1 from the RSD. Conclusions: There is considerable ambient CO2 enhancement at roadside locations across London. The various ratios of NOX to CO2 imply that CO2 does not have a unique imprint in the traffic plume, but rather leaves various signatures. The CO2 signal at each location is influenced by a combination of meteorological conditions, mixing layer height and atmospheric stability in combination with traffic source characteristics such as fleet composition and vehicle speed. References: (1) Department of Energy & Climate Change (DECC). 2011 Greenhouse Gas emissions by Fuel Type and End User. Statistical Release 29th March 2012 (2) Bishop, G. A. & Stedman, D., H. (1996). “Measuring the Emissions of Passing Cars.” Accounts of Chemical Research 29: 489-495.

Figure 1: Set up of remote sensing equipment for monitoring of vehicle exhaust emissions


Fabrication and evaluation of an organic monolithic column based upon the polymerisation of hexyl methacrylate with 1,6-hexanediol ethoxylate diacrylate for the separation of small molecules by capillary liquid chromatography

Wael Aljohani, Cristina Legido Quigley, Norman Smith Analytical and Environmental Sciences Division, Faculty of Life Sciences & Medicine,

King’s College London

Background: Monolithic columns are an appealing substitute to conventional particulate stationary phases for capillary liquid chromatography, being continuous interconnected networks with large through-pores channels. Often separation efficiencies using organic monoliths for small molecules are not high compared with silica monoliths, since the number of mesopores (less than 50 nm) is not as high as in silica monoliths. However, recently approaches have been adopted including using a single cross linker, hyper-cross linking, using shorter polymerisation times, and using longer crosslinkers. In this study, the monomer HMA was employed with two different cross linkers including EDMA and 1, 6 HEDA in order to investigate the effect of cross-linker length on efficiencies for small molecules (1).

Method:

Figure 1: A scheme of preparation of polymeric monolithic columns

Results: It was found that the efficiency of the separation improved tenfold when using the longer cross linker, 1,6HEDA compared with the shorter crosslinker, EDMA. This improvement was associated with the increase in number of methylene groups resulting in an increased number of mesopores, less than 50 nm in dimension. The 1, 6 HEDA monolith showed a porosity of 90 % and no evidence of swelling or shrinking with the use of organic solvents. Moreover, the 1,6 HEDA monolith demonstrated high reproducibility for the separation of the retained compounds anisole and naphthalene. These showed retention time RSDs of 1.79% and 2.74% respectively. A van Deemter plot was also constructed for the most retained molecule, naphthalene, within the range 0.82 mm/s to 2.01mm/s and showed that the 1,6 HEDA monolith was not noticeably affected by higher linear velocities demonstrating the wide range of usable linear velocities with low peak dispersion. The fabricated monolith also demonstrated slight tailing with basic molecules, which is often a challenge with silica based monoliths due to secondary interactions between basic moieties and the hydroxyl groups of the silica. Additionally, the 1,6 HEDA monolith was utilised successfully to quantify the drug amitriptyline in pharmaceutical tablets. Conclusion: longer cross linkers have proved to be an effective approach to enable organic monolithic columns to be employed with small molecules. The 1, 6 HEDA monoliths showed good performance with basic molecules.

References: 1) Jandera, P., et al., Cross-linker effects on the separation efficiency on (poly)methacrylate capillary monolithic columns. Part I. Reversed-phase liquid chromatography. Journal of Chromatography A, 2013. 1274: p. 97-106; 2) Jiang, Z.J., et al., Preparation and characterization of long alkyl chain methacrylate-based monolithic column for capillary chromatography. Journal of Biochemical and Biophysical Methods, 2007. 70(1): p. 39-45.

Bare Capillary

Flushing un‐reacted material out using mechanical Pump

Testing monolithic column with test mixture using HPLC

Treatment capillary’s wall with γ‐MAPS prior to polymerisation process

Filling the capillary with Polymerisation mixture

Initiation the polymerisation

process thermally


‘Semi-Targeted’ analysis of the river Thames: combining targeted and non-targeted approaches for pharmaceutical and illicit drug residue detection

Kelly Munro,1 Anthony Edge,2 Claudia Martins,3 David Cowan,4 Leon Barron1

1Analytical and Environmental Sciences Division, Faculty of Life Sciences &

Medicine, King’s College London, 2Thermo Fisher Scientific, Tudor Road, Runcorn, 3Thermo Fisher Scientific, 355 River Oaks Parkway, San Jose, CA 95134, USA,

4Drug Control Centre, King’s College London Background: The potential of suspect/non-targeted analytical approaches using high resolution analytical techniques for pharmaceuticals and related contaminant identification in the environment has been widely acknowledged in recent years. By developing better post-data acquisition mining tools, a larger number of unknown contaminants can be potentially identified in parallel with a traditional targeted analysis. Presented herein is the application of a novel ‘semi-targeted’ screening method using solid phase extraction (SPE) and liquid chromatography-high resolution mass spectrometry (LC-HRMS) for a temporal and spatial study of drug occurrence in the River Thames.

Figure 1: Temporal and spatial variation of pharmaceutical occurrence in the River Thames

Methods: A broad SPE extraction method was developed using 45 structurally diverse compounds. Detection was performed using HRMS, permitting simultaneous quantification of target analytes and qualitative analysis for additional pharmaceutical content. Data-mining software was used to develop and apply an optimised screening method, using matrix-matched reference standards to confirm the presence of unknown contaminants within the river. Results: In general, no overall temporal or spatial trends were observed, with any variation remaining on a compound specific basis. The optimised data-mining screening method was used to shortlist > 400 potential contaminants within the river water samples. The presence of 4 shortlisted unknown suspects was confirmed using a matrix-matched standard. Conclusions: Overall, by combining a targeted quantification method with a qualitative non-target approach, the applied semi-targeted method resulted in a more comprehensive analysis of the river, underlying its potential in the analysis of pharmaceuticals in the environment.


14th May, 2015

Abstracts

Short Oral Presentations and Poster

[OP1] Yona Essig C. elegans metallothioneins: dissection of trace metal detoxification and homeostasis [OP2] James Gooch Fluorogenic substrates for the detection of biological fluids and fingerprints [OP3] Bronagh Murphy Semi-targeted screening for 51 organic explosive-related compounds using liquid chromatography-high resolution mass spectrometry and in silico data-mining

Short Oral Presentation and Poster [1]

Fig 1: C.elegans exposed to 500µM zinc for 48h. The left panel shows the accumulation of zinc in the nematodes body. The right panel shows an average XAS (X-ray Absorption Spectroscopy) spectrum of zinc hotspots in nematodes.

C. elegans metallothioneins: dissection of trace metal detoxification and homeostasis

Yona Essig*, Samuel M. WebbϮ, Stephen Sturzenbaum* *Analytical and Environmental Sciences Division, Faculty of Life Sciences and Medicine, King’s

College London, ϮStanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, CA 94025, USA

Background: Metallothioneins (MTs) are metal binding proteins of low molecular weight, they lack aromatic amino acids and contain a high proportion of cysteines (1). The cysteine residues are involved in the binding of divalent heavy metals. Some heavy metals are essential to organisms however at a certain concentration any heavy metal is toxic. Therefore metallothioneins play a substantial role in heavy metal detoxification (2). The C. elegans genome contains two different isoforms encoding for MT-1 and MT-2. Intriguingly, MT-1 has 15 additional amino acids in the C-terminal region, of which 3 are metal-binding residues. Both isoforms are induced by heavy metals in the gut, in addition MT-1 (but not MT-2) is constitutively expressed in the lower bulb of the pharynx. Metallothioneins are overall important in protecting the organism against Cadmium toxicity but are also thought to be essential in Cu(I) and Zn(II) homeostasis and responses to oxidative stress. Methods: By means of a high energy electron beam accelerator (SLAC-Institute Stanford Linear Accelerator Centre/ National Accelerator Laboratory, Menlo Park, USA) it is possible to quantify metal concentration at 5 micron resolution, therefore heavy metal accumulation can be visualized in the body of the nematode C. elegans. Several knock out worm strains were examined (namely the MT-1 knock out mtl-1 (tm1770), the MT-2 knock out mtl-2 (gk125) and the MT-1 and MT-2 double knock out mtl-1;mtl-2 (zs1)) following the exposure to different metal concentration (500µM zinc, 150µM zinc, 100µM cadmium, 30µM cadmium) for different time periods (4h,48h). Furthermore exposures to metal mixtures were examined (150µM zinc and 30µM cadmium, 150µM zinc and 500µM cadmium, 500µM zinc and 500µM cadmium). Results: Exposure of nematodes to zinc and cadmium significantly increased the metal accumulation in comparison to the respective controls. This increase was dose dependent. Exposing the nematodes to a mixture of zinc and cadmium resulted in a significantly reduced accumulation of metals (compared to the single metal exposure). Altogether the WT zinc exposed worm displays the lowest zinc accumulation compared to the mutants. Similar levels of cadmium is accumulated in WT and mutant worms. Conclusion: This study demonstrates that MT-1 and MT-2 are important for detoxification and homeostasis of zinc. The interaction between these two genes with other genes is currently being studied in more detail. Furthermore, the XAS spectra will be analysed to study potential changes in the binding energy within the different mutant backgrounds. References: 1. Hamer, D. H. (1986). Metallothionein. Annual Rev Biochem 55, 913-951. 2. Beyersmann, D., Hechtenberg, S. (1997). Cadmium, gene regulation, and cellular signalling in mammalian cells. Toxicol Appl Pharmacol 144, 247-261.

Short Oral Presentation and Poster [2]

Fluorogenic Substrates for the Detection of Biological Fluids and Fingerprints

James Gooch, Barbara Daniel, Vincenzo Abbate, Elizabeth Gilchrist, Nunzianda Frascione

Analytical and Environmental Sciences Division, Faculty of Life Sciences & Medicine,

King’s College London Background: The discovery of biological evidence at the scene of a crime is often useful in clarifying the circumstances surrounding an event and, more importantly, identifying those involved. However at present, there are no techniques available that possess the ability to simultaneously locate and identify both fingerprints and body fluids within a single step. The recognition of fluid-specific biomarkers by fluorogenic peptide substrates may provide a novel resolution to this issue [1].

Figure 1: Rhodamine-110 – A highly fluorescent leaving group produced upon

fluorogenic substrate cleavage by fluid-specific protease activity. Methods: This study utilises a number of fluorogenic peptide substrates specific to intra-fluidic protease enzymes in an effort to detect human semen, sweat and fingerprints deposited on a number of surfaces typical to criminal investigation. Results: For the first time the ability of fluorescent substrates to identify biological fluid and fingerprint depositions in situ has been successfully demonstrated. Both human semen and sweat was detected across a range of surfaces typical to forensic investigation with additional visualisation via a direct spraying application. These particular substrates exhibited ideal increases in fluorescence intensity upon target interaction, even at sensitive 1:1000 fluid dilutions, giving opportunity for their use in contaminated deposits or those washed in removal attempts. Importantly, substrates were found to have no effect on DNA profiling processes after application to biological fluids and thereby negate the potential forfeit of fluid identification in order to maximise genetic material recovery. Conclusions: Displaying both an immediate and specific response to analyte presence, fluorogenic substrates have the potential to prevent month-long visual evidence searches by localising fluid and fingerprint depositions within a matter of seconds. Serious thought should therefore be given to the use of fluorogenic substrates as replacements to current presumptive testing techniques. References. 1. Gooch, J., Daniel, B., Frascione, N., 2014. Talanta, (125), 210-214.

Semi-targeted screening for 51 organic explosive-related compounds using liquid chromatography-high resolution mass spectrometry and in silico data-

mining Bronagh Murphy, Mark Parkin, Leon Barron


King’s College London Background: The development of flexible analytical methods which can be applied to the confirmatory identification of conventional organic explosives as well as their precursors, transformation products and new or emerging compounds is highly advantageous. Normally, in order to confirm the identity of a suspected explosives-related compound a reference standard is required to match both chromatographic retention time and high resolution mass spectrometry (HRMS) data. However, for new or unknown compounds, such reference standards may not yet exist. Therefore, we present for the first time the application of predictive modelling tools to generate chromatograhic retention times of explosive-type compounds.

Experimental LC‐HRMS data

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Figure 1: Positive correlation between experimental retention times and those predicted using an artificial neural network (y = 0.9221x + 0.3829, R² = 0.929).

Methods: A liquid chromatography-high resolution mass spectrometry (LC-HRMS) screening method for >50 explosives-related compounds was developed. Separations were optimised on a C18-aromatic type stationary phase using 0.2 mM ammonium chloride as an additive to enhance atmospheric pressure chemical ionisation of target analytes and a 22 minute water:methanol mobile phase gradient. A database of chromatographic retention time (tR) as well as HRMS data was generated for all compounds. A range of artificial neural networks (ANN) types were then investigated for prediction of tR based on a number of targeted chemical descriptors. Results: A generalised regression-type neural network was found to generate the best correlation between predicted and measured tR (Fig. 1). This model was trained using 27 analytes as case examples and was sufficient to predict the tR of the remaining 24 compounds in the reference mixture with an average deviation from the true value of within 1.3 minutes (<6 % of runtime). HRMS data for these compounds was also easily predicted using the instrument software (TraceFinder 3.0), offering a potential in silico approach for the preliminary direction of new compounds and for better direction of reference standard synthesis efforts in the future for confirmatory identification. Conclusions: Overall, this approach provides the potential for semi-targeted analysis with a realistic capability for potential preliminary identification of new compounds without the need for reference materials.


14th May, 2015

Abstracts

Poster Presentations

[P1] Danielle Moncrieffe Towards quantifying PIIINP in human serum by LC-MS – identifying suitable surrogate peptides

[P2] Sana Alsaleh Exposure to perchlorate anion affects key life history parameters of the nematode Caenorhabditis elegans

[P3] Nuria Camiña Establishing the oxidative potential of roadside particulate matter in London from personal monitors

[P4] Eleonora Nicolosi Characterising organic carbon sources in London’s air

[P5] Lindsay Reed Metabolic activation of the environmental carcinogen benzo[a]pyrene in Hepatic Cytochrome b5/P450 Reductase Null (HBRN) mice

[P6] Diana Silva Can personalised air pollution exposure information influence individuals’ behaviour to protect public health?

[P7] Marjaana Viljanto Investigating the use of steroid ratios for the detection of the use of pseudo-endogenous anabolic-androgenic steroids in the horse

Poster Presentation [1]

Towards quantifying PIIINP in human serum by LC-MS – identifying suitable surrogate peptides

Danielle Moncrieffe, Mark C. Parkin, Alessandro Musenga, David A. Cowan


King’s College London

Background: Procollagen III amino-terminal propeptide (PIIINP), a 42 kDa protein with a mean basal blood concentration in man of 5 ng/mL (120 pM), is a biomarker for growth hormone in clinical and anti-doping samples. In the absence of an international human PIIINP standard, current measurements of PIIINP rely on immunoassays that employ bovine PIIINP (sharing 97% homology) as reference. Here we identify peptides for quantifying human PIIINP in limited serum sample volumes (<500 µL), by assessing a trypsin digest of bovine PIIINP in human serum albumin (HSA) by liquid chromatography tandem mass spectrometry (LC-MS/MS).

PQQEAVEGGCSHLGQSYADR DVWK PEPCQICVCDSGSVLCDDIICDDQELDCPNPEIPFGECCAVCPQPPTAPTR

PPNGQGPQGPK GDHPGPHPGIHPGR NGDHPGIHPGQHPGSHPGSHPGPHPGICESCPTGGQNYSP

Figure 1: Illustration of human PIIINP with post-translational modifications, showing a map of tryptic (Tryp) cleavage and specificity (%) according to Keil rules[1] (image modified from O. Niemela[2])

Methods: Bovine PIIINP in HSA was digested with trypsin in silico and ex silico. The latter was analysed by LC-high resolution MS (LC-HRMS), where peptides were selected for targeted LC-MS/HRMS analysis based on their uniqueness, stability, reproducibility and MS suitability (size and sensitivity). Human equivalents of T1 and T5 were synthesized and their LOD (without matrix) was determined by LC-MS/MS.

Results: T1 and T5 were identified with 100% sequence coverage from the bovine PIIINP digest. These peptides contained pyroglutamate and hydroxyproline residues that deviate from their in silico peptides. A T6 peptide with a spontaneously deamidated asparagine was also observed, with 85% sequence coverage. T3+T4 peptide (7 kDa) is not ideal for MS, and is therefore not targeted in our analyses. The LOD of synthesized human equivalents of T1 and T5 is 50 pM.

Conclusions: T1 and T5 were identified as the most suitable surrogate PIIINP peptides. With LODs of 50 pM, these peptides show promise for quantifying PIIINP at basal blood concentration (120 pM) by LC-MS methods.

References: [1] B. Keil, Specificity of proteolysis, Springer-Verlag GmbH 1992. [2] O. Niemela, Clin. Chem., 31 (1985) 1301-1304.

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Tryp 94% Tryp 100%

T1 T3

T4 T5 T6


Exposure to perchlorate anion affects key life history parameters of the

nematode Caenorhabditis elegans

Sana Alsaleh, Leon Barron, Stephen Sturzenbaum

Analytical and Environmental Sciences Division, School of Life Sciences & Medicine, King’s College London

Background: The perchlorate anion is a powerful oxidizer used in the construction of low order explosives, rockets, missiles, some ammunition, air bags and fireworks1. Perchlorate is periodically replaced and disposed of because of its limited shelf life. However, perchlorate salts are very stable and highly soluble and once released into aquatic systems, will dissociate into the corresponding cations and the perchlorate anion. It has been reported that ground and surface waters are contaminated by perchlorate and such contamination can persist for decades2. Perchlorate is known to inhibit thyroid hormone synthesis in vertebrates. However, less is known about its effects independent from the thyroid3.

Methods: Wild type C. elegans worms were exposed to sodium perchlorate added to both NGM agar and the food source, OP50 bacteria, at concentrations ranging from 1ng/ml to 5mg/ml. Phenotypic changes including growth, development, reproductive performances and lifespan indices were investigated. Results: Perchlorate was found to stimulate growth at low concentrations (1ug/ml) but to induce an arrested development at high concentrations (5mg/ml). Reproductive performance was also decreased in a dose responsive manner in which the total brood size decreased by almost 50% at 1mg/ml. However, C. elegans life span, pharyngeal pumping and defecation were not affected by sodium perchlorate. Conclusions: These observations suggest that the exposure to perchlorate might trigger a hormetic effect at low concentrations but at higher concentrations toxic effects are pronounced. Future experiments will explore the toxicogenomic response of C. elegans challenged with perchlorate. References: 1) M.A. Greer, G. Goodman, R.C. Pleus, S.E. Greer. (2002). Environ. Health Perspect., 110, pp. 927–937. 2) J.J. Clark. (2000). Kluwer Academic/Plenum Publishers, New York, USA (2000), pp. 15–29. 3) J. Wolff. (1998). Pharmacol. Rev., 50, pp. 89–105.

Figure: C. elegans exposure to perchlorate induces a toxicogenomic response that results in a change in key life history parameters such as growth. C. elegans growth/size is increased at low concentrations (1µg/ml) and is reduced at higher concentrations (1mg/ml).


Establishing the oxidative potential of roadside particulate matter in London from personal monitors

Nuria Camiña, Benjamin Barratt, Christina Dunster, Frank J. Kelly, Ian S. Mudway

Analytical and Environmental Sciences Division, Faculty of Life Sciences & Medicine, King’s College London

Background: It has been hypothesised that exposure to primary traffic emissions from diesel traffic worsens symptoms of patients with chronic obstructive lung disease (COPD). To test this, healthy non-smoking volunteers (n=40) and patients with Stage 2 COPD (n=40) were invited to perform alternate 2 hour walks along Oxford Street (kerb side exposures to exclusively diesel traffic) and Hyde Park (as an urban background location), with the independent walks separated by at least 3–8 weeks. In addition to assessing the patients physiological responses, real time measurements of pollutants at these two locations were made to estimate the patient’s exposure to gaseous and particulate pollutants. PM2.5 filters were also collected to assess the particulate oxidative and compositional characteristics at each site.

Methods: PM2.5 filters (n=75) were collected from Oxford Street and Hyde Park between Oct 2012 to Jan 2014, with filter loadings ranging from 4.0–73.0 μg. The particulate oxidative potential (OP) was measured by incubating 8mm discs cut from each filter in a synthetic respiratory tract lining fluid and quantifying the decrease in glutathione (GSH) and ascorbate (AA) concentrations over 4 hours (pH7.0, 37oC). Antioxidant quantification was achieved using reverse phase HPLC with electrochemical detection (for ascorbate) and the GSSG-reductase-5, 5′-dithio-bis (2-nitrobenzoic acid) recycling assay (for glutathione). The % loss of AA and GSH over the incubation was determined and adjusted for the concentration of particles on the cut discs to derive 3 measures of OP: glutathione dependent OP (OPGSH/μg), ascorbate dependent OP (OPAA/μg) and the aggregate sum of the two (OPTOT/μg).

Results: The personal monitoring demonstrated increased PM2.5 concentrations during the Oxford Street walks, compared to Hyde Park: 15.6 (12.6-24.3) vs. 5.6 (4.0 – 9.9) μg/m3, P<0.001 (data presented as medians with 25th and 75th percentiles). Despite this, no difference in the PM2.5 oxidative potential was observed; this was the case when assessed using either the ascorbate or glutathione dependent OP metrics, or the sum of the two, expressed per μg of sampled PM, or per m3 of ambient air (see table below). Due to the low PM2.5 filter loadings OP was not measurable in 31% and 40% of the filters from Oxford Street and Hyde Park respectively.

Metric Hyde Park (n=40) Oxford Street (n=35) Mann-Whitney U Test OPGSH/μg 3.4 (0.0-17.6) 3.8 (0.0-10.0) NS OPAA/μg 4.8 (0.0-26.0) 3.7 (0.6-9.5) NS OPTOT/μg 12.9 (2.0-35.0) 10.0 (1.3-21.7) NS OPGSH/m3 52.1 (0.0-123.4) 62.4 (0.0-189.0) NS OPAA/m3 74.9 (0.0-140.7) 83.0 (12.8-172.7) NS OPTOT/m3 142.2 (24.4-275.8) 195.8 (27.5-406.8) NS

The extent to which the measured OP was influenced by season was also examined; grouping the exposure days into warm (April-September) and cold (October-March) periods. This sub-analysis revealed that OPAA expressed both per μg and m3 was significantly elevated during the cold period (P<0.05 and <0.01 respectively). A similar seasonal pattern was not seen with the GSH dependent metrics.

Conclusions: These data demonstrate the feasibility of performing OP analysis on very low particle concentrations from filters collected from personal monitoring campaign work. They do not support the contention that PM from high diesel sites have elevated oxidative properties, but do suggest that season may strongly influence this metric. These data will ultimately be associated with the symptomatic responses from the patients performing the walks on these days, to examine the extent that particulate OP influences symptoms.


Characterising organic carbon sources in London’s air

Eleonora Nicolosi1, Gary W. Fuller1 and Paul Quincey2

1MRC-HPA Centre for Environment and Health, King’s College London

2 National Physical Laboratory, Teddington

Introduction: There is poor quantification of the sources and processes that determines organic carbon (OC) particulate concentrations in ambient air. Daily filter samples were collected from rural, urban background and roadside locations in the London region to explore their different characteristics using their volatility and chemical properties. Methods: A Sunset Laboratory Thermal-Optical Carbon Aerosol Analyzer was used for the OC analysis following the EUSAAR2 protocol (Cavalli et al, 2010). This determined total OC for each sample and separated OC fractions using four temperature steps in a He atmosphere and by pyrolysis during the heating process. To account for seasonality, PM10 samples were collected during winter (6 Jan - 11 Feb 2012) and summer (21 July - 23 Aug 2012). Exhaust samples from the main air pollutant sources (diesel and petrol vehicles) (Watson, 1994) were also analysed to characterize them by their relative OC peak abundances. Results: Fig. 1 compares the mean OC desorbed during each temperature step (OC1 to OC4), and the pyrolysed OC (POC), from the three sites in winter and summer. A clear spatial gradient could be seen with concentrations of OC1 to 4 and POC increasing from rural to roadside indicating primary sources. Greater concentrations of OC4 were measured during the summer, indicative of a more oxidised and less volatile organic carbon from photochemical processing.

Figure 1: Mean OC evolved at each temperature step by site and season.

References: Cavalli, F et al 2010. Toward a standardised thermal-optical protocol for measuring atmospheric organic and elemental carbon: the EUSAAR protocol. AMT 3, 79-89 201. Watson, J. G., et al 1994. Differences in the carbon composition of source profiles for diesel-powered and gasoline-powered vehicles. Atmos Env 28, 2493-2505.


Metabolic activation of the environmental carcinogen benzo[a]pyrene in Hepatic Cytochrome b5/P450 Reductase Null (HBRN) mice

Lindsay Reed1, Iveta Mrizova2, Frantisek Barta2, Radek Indra2, Michaela Moserova2, Colin J. Henderson3, David H. Phillips1, Marie Stiborova2, and Volker M. Arlt1

1Analytical and Environmental Sciences Division, MRC-PHE Centre for Environment and Health, King’s College London; 2Department of Biochemistry, Charles University,

Prague; 3Division of Cancer Research, University of Dundee Background: Benzo[a]pyrene (BaP) is an environmental pollutant that exerts its carcinogenic effects after metabolic activation by cytochrome P450s1. In the present study Hepatic Reductase Null (HRN) and Hepatic Cytochrome b5/P450 Reductase Null (HBRN) mice have been used to study the role of P450-mediated metabolism and disposition of BaP. In HRN mice cytochrome P450 oxidoreductase (Por), the electron donor to P450, is deleted specifically in hepatocytes. In HBRN mice the microsomal haemoprotein cytochrome b5, which can also act as an electron donor from cytochrome b5 reductase to P450 enzymes, is also deleted in the liver2.

Methods: Wild type (WT), HRN and HBRN mice were treated once with 125 mg/kg body weight of BaP for 24 hours. Hepatic microsomal fractions were also incubated with BaP and DNA in the presence of NADPH as the enzymatic cofactor in vitro. BaP-DNA adduct levels were measured using 32P-Postlabelling analysis. Metabolites formed during in vitro incubations were analysed using HPLC. Cyp1a enzyme activity in the microsomal fractions was measured using EROD. Results: BaP-DNA adduct levels were significantly higher in liver of HRN mice than WT mice while no significant difference in adduct formation was observed in liver between HBRN and WT mice. BaP-DNA adduct formation in the livers of these mouse lines correlated with the formation of BaP-7,8-dihydrodiol in vitro in hepatic microsomes from BaP-treated mice in the presence of NADPH. Two BaP-DNA adducts were formed in vitro, one of them being dG-N2-BPDE. BaP-DNA adduct formation was significantly higher with microsomal fractions from WT mice than with those from HRN or HBRN mice, which correlated with EROD activity. Conclusions: In this study, our results demonstrate that POR and cytochrome b5

modulate the P450-mediated bioactivation on BaP in vitro and in vivo. References: 1. Baird, W.M., Hooven, L.A., Mahadevan, B., 2005. Carcinogenic polycyclic aromatic hydrocarbon-DNA adducts and mechanism of action. Environ. Mol. Mutagen. 45: 106-114. 2. Henderson, C.J., McLaughlin, L.A., Wolf, C.R., 2013. Evidence that cytochrome b5 and cytochrome b5 reductase can act as sole electron donors to the hepatic cytochrome P450 system. Mol. Pharmacol. 83: 1209-1217

Figure 1: Autoradiogram images showing DNA

adducts in vivo (liver) and in vitro (NADPH + BaP).

Adduct 1 (dG-N2-BPDE) is present in both in vivo

and in vitro experiments whereas adduct 2 is only

present in vitro


Can personalised air pollution exposure information influence individuals’ behaviour to protect public health?

Diana Silva, Christopher McKevitt, Ben Barratt

Analytical and Environmental Sciences Division, Faculty of Life Sciences & Medicine, King’s College London

Background: Air pollution is a public health concern accountable for numerous health problems and thousands of premature deaths[1]. Despite the introduction of measures aiming to improve London’s air quality, levels remain above legal limits in many areas. While improvements to ambient air quality are essential to public health, recent studies have demonstrated that there are additional choices that individuals can make to reduce their exposure to air pollution, and hence the associated health impacts. Individuals can now measure the air pollution they are exposed to by using portable air pollution sensors. This study aims to assess the extent to which personalised air quality measurements can influence public behaviour, and evaluate how individuals disseminate information and outcomes. Methods: This project uses a translational research framework[2] and its methodology is rooted on a number of participatory models[3] drawing on observation, surveys and interviews. Preliminary results: The first of a series of linked projects is currently being carried out at a Primary school. All 400 children at the school learned about air pollution through tailored presentations. A subset of children then measured the air pollution they were exposed to as they travelled to and from school using portable exposure monitors and GPS watches (Figure 1).

Figure 1: A child from a South East London school measuring air pollution (Black carbon (BC)) on his way back home. Work is ongoing to assess the extent to which this personalised information influences the families’ behaviour in the short and medium term. This research is supported by a public website, BreatheLondon.org.uk. References: [1] Giller, B., Report on estimation of mortality impacts of particulate air pollution in London, in Consulting Report P951-001. 2010, Institute of Ocupational Medicine: London. [2] Ramirez-Andreotta, M.D., et al., Environmental Research Translation: Enhancing interactions with communities at contaminated sites. Science of The Total Environment, 2014. 497–498(0): p. 651-664. [3].Hughes, I., The Sage handbook of action research participative inquiry and practice Chapter 25: Action Research in Health Care. 2008, London.


Investigating the use of steroid ratios for the detection of the use of pseudo-

endogenous anabolic-androgenic steroids in the horse

Marjaana Viljanto1,2, James Scarth2, Mark Parkin1, Christopher J. Walker1, Andrew T. Kicman1, Lynn Hillyer3

1Analytical and Environmental Sciences Division, Faculty of Life Sciences &

Medicine, King’s College London; 2Sport and specialised Analytical Services, LGC; 3British Horseracing Authority

Background: The use of steroid ratios could provide an additional approach to aid in the differentiation between the administration of pseudo-endogenous androgenic-anabolic steroids from natural conditions that result in atypical analytical findings. This required the development and validation of a quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the determination of testosterone, epitestosterone and dehydroepiandrosterone (DHEA) in equine urine.

Methods: Urine samples were extracted and analysed to obtain a total concentration of free, sulphate and glucuronide conjugated fractions of testosterone, epitestosterone and DHEA to establish the steroid ratios. Sample treatment consisted of solid-phase extraction, methanolysis and derivatisation using methoxyamine hydrochloride (to enhance sensitivity). Sample analysis was performed using an LC-MS/MS system (Waters Xevo TQ-S). The developed method was quantitatively validated as fit-for-purpose using measures of linearity, precision and accuracy, selectivity and sensitivity. A matrix ‘standard addition’ approach was used to prepare calibration lines and quality control samples.

Figure 1: Total ion chromatogram of the methoxyamine derivatives of testosterone, epitestosterone and DHEA

Results: The method was successfully validated for testosterone, epitestosterone and DHEA covering the calibration range from endogenous concentrations to 500 ng/mL. The lower limit of quantification (LLOQ) was determined to be the endogenous concentration of the control urine, which was calculated as 1.5 ng/mL for testosterone and 1.4 ng/mL for epitestosterone and DHEA. The limit of detection was calculated to be 0.15 ng/mL for testosterone and epitestosterone and 0.9 ng/mL for DHEA. Intra- and inter-batch precision (CV%) and accuracy (RE%) were within ±15 % (±20 % at the LLOQ) from their theoretical concentrations and coefficient of determination was better than 0.995 for these three steroids. Conclusions: The method is ready to be applied to the analysis of urine from untreated horses in order to determine baseline concentrations and steroid ratios. This information could be used to develop statistical models to differentiate them from horses that may have been administered steroids.

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faculty of life sciences & medicine analytical & environmental … · 2017-07-29 · oral...

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