Department of occupational and environmental medicine A sensitive assay for proteases in work environmental samples based on zymography

Transcription

1 University hospital of North Norway Department of occupational and environmental medicine A sensitive assay for proteases in work environmental samples based on zymography Berit E. Bang, Nabin Malla, Sampada Bhagwat, Merethe Larsen and Jan Olof Winberg Good work is good health Presented by Berit E. Bang Senior Researcher Department of Occupational and Environmental Medicine University Hospital North Norway berit.bang@unn.no

2 Proteases are part of the bioaerosol in seafood industry work environment Microorganisms Allergens Endotoxins Enzymes proteases Water beams and mechanical processing liberates droplets and particles to the air Bioaerosols are airborne particles consisting of or originating from living organisms 2

3 Seafood industry workers have increased respiratory symptoms and lung functional impairment compared to controls Only few are sensitized to fish: 2.7 % IgE positive to cod in white fish industry 2.2 % IgE positive to salmon in salmon industry Exposure to total microorganisms and endotoxin is generally low in fish industry A dose response relationship between exposure to total protein in salmon industry and 1. Lung function (FEV 1 cross shift decline) 2. Symptoms (cough, chest tightness) 3

4 The rationale behind establishing a method for measuring proteases in exposure samples is the fact that fish industry workers have respiratory effects that cannot be explained by allergy, or exposure to endotoxins or microorganisms, but seem to be linked to the protein compartment of bioaerosols 4

5 Proteases in seafood tissues induce inflammation in cell models Use a human airway cell model (A549) Proteases from the fish digestion system and outer mucus coat stimulate the inflammatory mediator IL-8 in a dose-dependent manner The effect is inhibited by protease inhibitors and not endotoxin inhibitors, demonstrating that it is dependent on protease and not endotoxin activity IL-8 levels (pg/million cells) c. Skin mucus IL-8 levels (pg/million cells) Tissue alone + PIC + SPI Concentration of tissue homogenate (mg/ml) 0 Unstimulated Pyloric caeca Stomach Skin mucus 5

6 Sampling proteases in seafood industry work environments Liquid samples Liquid samples (fish juice/water) collected from work-benches, containing trays, etc 6

7 Seafood proteases are present in seafood industry work environments in levels above those eliciting inflammatory responses in cell studies Activity of trypsine-like enzymes in environmental samples (ES) compared to maximal effective activity in cell-studies 0,09 Trypsine-like activity (Units/ml) 0,08 0,07 0,06 0,05 0,04 0,03 0,02 0,01 0 maximal effectiv trypsin ES ES ES Environmental sample from factory A Environmental sample from factory B Maximal effective enzyme concentration of salmon trypsin in cell studies of inflammation (IL-8 stimulation)

8 Is exposure to seafood proteases linked to respiratory effects in worker populations? Need of method to quantify low protease levels in bioaerosol samples Department of occupational and environmental medicine 8

9 Need of sensitive assay for air samples Activity (da405nm/min) Chromogenic assay Chromogenic (or fluorescence based) assays are not sensitive enough for bioaerosol samples [Trypsin] nm 250 Zymographic assay [E]: 0 1 nm Maximal intensity [Trypsin] (nm) 9

10 Zymography, standardcurve A stock solution with known concentration of active enzyme is diluted and standards added to the zymographic gel The intensity (transparency) of the enzyme band is read by an image analyse software Concentrations in nm A standard curve is drawn using the maximal intensity given by the software Maximal intensity Maximal intensity of trypsin bands Maximal intensity Trypsin standard curve, linear region y = x R² = Enzyme concentration (nm) Enzyme concentration (nm) 10

11 Sampling of bioaerosols in seafood industry Filters carried in breathing zone Sampling pumps carried in rucksacks Three piece sampler Polytetrafluoroethylene filters (PTFE), 1 µm Flow: 3 L/min Sampling time (8 or 10 hours) Freeze (- 20 o C and 70 o C) Stationary sampling 11

12 Characterization of proteases in samples from salmon industry Zymographic gel containing samples from seafood industry and molecular weight standards Zymographic gel treated with 10 mm EDTA Zymographic gel treated with 1 mm Pefabloc Molecular weight studies based on migration distances (Rf values) show that bioaerosol samples from salmon industry contain proteases close to 133 kda, 137 kda, 110 kda and 54 kda and 24 kda Protease inhibition studies demonstrate that the dominating proteases are serine proteases (inhibited by Pefabloc but not EDTA) 12

13 Quantification of proteases in samples from salmon industry Trypsin standard [E] nm Unknown sample No Intensity (I-max) Unknown sample [E] nm Samples Standard curve Standards, nm , , , ,6 162 I-max y = x R² = [Enzyme], nm 0, ,3 87 S ,98 S ,34 S ,31 S ,61 S > 1 13

14 Examples of exposure levels in salmon industry Slaughtering 5,38 ng/m 3 Scraping 24,10 ng/m 3 Sorting 44,23 ng/m 3 14

15 Method validation based on 16 replicate measurements of filter extracts Linearity (r-squared) of standard curves Range Median Limit of Detection * (LOD) (nm) 0.27 Limit of quantification (LOQ**) (nm) 0.70 Precision (coefficient of variation, CV %) Sample 1 (0.4 nm) 45 % Sample 2 ( 0.5 nm) 33 % Sample 3 (0.7 nm) 27 % * LOD is defined as 3 X SD of 16 replicate measurements of a low concentration sample (0.03 nm) **LOQ is defined as 10 X SD of 16 replicate measurements of a low concentration sample (0.03 nm) CV % = (SD/mean)*100 of medium concentration samples 15

16 Proposed semiquantification scale for epidemiological studies Group the samples using percentiles based on increasing enzyme concentration Make a semi-quantitaive scale: Protease exposure level Part of data belonging to exposure level 1 < 25 percentile 2 25 percentile < 75 percentile 3 75 percentile 16

17 Conclusion Zymography is a sensitive assay, allowing the detection of picomolar concentrations of proteases in workplace bioaerosol samples The zymographic technique allow characterization of unknown proteases based on specific protease inhibition The zymograhic technique also allow the estimation of molecular weight based on migration distances compared to standards Zymography may be used for semi-quantification of airway exposure to proteases in epidemiological studies 17

18 Institutions: UiT The Arctic University of Norway University Hospital North Norway, Department of Occupational and Environmental Medicine Northern Norway Regional Health Authority (sponsor) PhD-students: Anett Kristin Larsen, Olga Shiryaeva, Marte Thomassen, Sampada Bhagwat, Cecilie Thon Heidelberg Co-supervisors: Ole Morten Seternes, Bjørn Straume, Jan-Olof Winberg, Lisbeth Aasmoe Reseacher: Nabin Malla Senior engineer: Merethe Larsen Occupational hygienists: Eva Kramvik, Marit Nøst Hegseth, Ann-Helen Olsen Foto by Olga Shiryaeva 18