1 Urine sediment as DNA source in the study of susceptibility biomarkersINAIL- Department of Occupational and Environmental Medicine, Epidemiology and Hygiene. Monte Porzio Catone - Rome - Italy Urine sediment as DNA source in the study of susceptibility biomarkers Pieranna Chiarella, PhD
2 The Biomonitoring of Occupational ExposurePAHs Benzene Styrene is the determination of exposure biomarkers to chemicals agents (carcinogens)
3 Human Biomonitoring & BiomarkersRisk assessment is based mainly on the measure of dose biomarker, supported by effect and susceptibility biomarkers. 1. dose biomarkers (metabolites) 2. effect biomarkers (genotoxicity) 3. susceptibility biomarkers (gene polymorphisms)
4 Benzene is a known as toxic chemical and carcinogenic agentdose biomarkers (metabolites) 3. susceptibility biomarkers (gene polymorphisms) 1 3 1 3 1 1 3 Carbonari et al Biomark. Med. 10 (2)
5 Glutathione-S-Transferase (GST) enzymes conjugate glutathione to toxic molecules (benzene) forming metabolites easier to be excreted
6 Phase II enzymes synthesized from polymorphic genesInvolved in the metabolism of several toxic, carcinogenic agents (benzopyrene, benzene, styrene, PAH, heavy metals, aflatoxin B1, tobacco smoke, chemotherapeutics). GSTT1 and GSTM1 Phase II enzymes synthesized from polymorphic genes Homozygous deletions of GSTT1 and GSTM1 gene result in the absence of enzyme activity, increasing the susceptibility of individuals to the effect of toxic and carcinogenic agent exposure.
7 GSTT1 and GSTM1 allele frequencyGST-T1 null GST-M1 null Asia 0.301 0.449 Europe 0.182 0.517 Africa 0.371 0.316 America and Canada 0.197 0.529 South America 0.267 0.397 Adapted from Kasthurinaidu SP et al. GST M1-T1 null Allele Frequency Patterns in Geographically Assorted Human Populations: A Phylogenetic Approach. PLoS ONE 10(4): e (2015). Frequencies are expressed as mean.
8 Procedure for dose and susceptibility biomarker assessmentPhase 1: SPMA, TTMA and creatinine on urine samples Phase 2: Blood DNA analysis for GSTs coding genes Questionnaires: personal information, working and life habits (smoking).
9 Occupational Exposure to Benzene StudiesStudy Population Work Environment Benzene exposure SPMA in GSTT1 null vs positive SPMA in GSTM1 null Kim et al. 2007 250 shoe-workers 136 controls Shoe-factories clothes- factories Tianjin (China) 0.512 ppm Lower SPMA Mansi et al. 2012 315 Petrochemical workers Petrochemical Industry (Italy) 0.01 ppm Lower SPMA in non-smokers and smokers Lower SPMA only in smokers Sorensen et al. 2004 50 oil shale mineworkers 50 controls Mine (Estonia) 0.035 ppm (Surface workers) 0.058 ppm (Underground workers) No significant difference Qu Q et al. 2005 181 Factory workers Unspecified Factory (China) 0.06 to 122 ppm (median 3.2 ppm) Not analyzed Manini et al. 2006 37 Taxi drivers Urban area 0.018 ppm Manini et al. 2010 100 Traffic Policemen 102 Gasoline pump attendants Urban Area Not reported Carrieri M et al. 2012 28 Petrochemical plant workers Petrochemical Plant outdoor (Italy) ppm Lower SPMA Lin et al. 2008 70 workers in a chemical factory Chemical synthesis factory (Taiwan) 7.2 ppm (entire population) High exp. (>1ppm) 15 ppm Low exp. (<1 ppm) 0.2 ppm
10 SPMA is lower in GSTT1/M1 null than in positive genotypesGSTT1/M1 null genotype may influence this benzene metabolite Importance of genetic profile in the susceptibility to certain occupational exposures
11 315 Urine specimens (exposure assessment)630 biological specimens 0.78 0.22 0.47 0.53 315 Blood specimens (genotyping) GSTT1 GSTM1 Positive (245) Null (70) Positive (148) Null (167) 315 Urine specimens (exposure assessment) Mansi A et al Toxicol. Letters 213(1):57-62.
12 Can we use a unique matrix for simultaneous biomonitoring and genotyping?The most widely used biological matrices for gene polymorphism analysis are: Blood (first choice as DNA source) Buccal cells (second choice) URINE ?
13 ADVANTAGE OF USING URINE IN THE OCCUPATIONAL BIOMONITORINGSINGLE BIOLOGICAL SAMPLE Cell fraction (Renal tubular, transitional urothelial, squamous cells white blood cells) Acellular fraction Benzene: SPMA, tt-MA Styrene: MA, PGA Biomarkers of exposure Biomarkers of susceptibility
14 Multiple harvesting in the same day Rare Feasible Biological sample Blood Buccal cells Urine Harvesting technique Invasive Non-invasive Volume Limited Partially limited Unlimited Multiple harvesting in the same day Rare Feasible Trained staff and special materials required Yes No Special conditions for transport and shipment Participant response Low Medium High Infection Risk (HIV, HBV, pathogens) Presence of interference proteins and PCR inhibitors Utility in metabolite detection Possible Need for special laboratory equipment BSLII hood, swinging rotor centrifuge Time for cell isolation procedure Long (hours) Short (10 min) Total DNA yield 20-30 mg/mL (Qiagen DNA extraction kit and Lahiri et al. 1995) 2-4 mg/single mouthwash (Mulot C et al. 2005) 0.4 mg/mL (Van der Hel 2002) Gender-dependent DNA yield
15 Urine DNA isolation Kit for DNA extractionTraditional in-house protocols -phenol-chloroform -proteinase K incubation -salting out and alcohol DNA precipitation Our choice Salting out the cellular proteins and alcohol precipitation (In-house protocol)(Van der Hel et al 2002, modified protocol). 20 donors (11 male and 9 female) of our Institute enrolled to test the protocol feasibility
16 METHOD DNA extraction, quantification and electrophoretic gel analysisCell centrifugation /PBS washes/ Cell lysis at 55oC Protein precipitation with 5M NaCl Ethanol-DNA precipitation DNA resuspension in TE pH 8.0 Multiplex-PCR (single run for both GSTT1 and GSTM1 polymorphisms) MW #11 #8 #9 #10 #2 #18 #20 #16 #19 #12 C+ C- GSTT1 (480 bp) GSTM1 (215 bp) primer pairs
17 DNA recovery (ng/mL urine)ID Gender Age Total DNA recovery (ng) DNA recovery (ng/mL urine) DNA purity (260/280) GSTT1 polymorphism GSTM1 1 Male 41 1320 16.5 1.76 Positive Null 2 39 390 5.6 1.43 Positive 3 40 12 0.17 ? - 4 43 960 1.41 5 41 9 0.13 1.5 - - 6 7 8 10 11 13 14 15 16 17 18 19 20 Female 48 40 59 42 50 44 38 32 45 52 56 225 270 186 75 7000 5200 3400 5440 300 1072 212 0.042 0.085 3.2 3.85 2.65 1.07 87.5 65 48.5 0.68 77.7 0.04 4.3 13.4 3.5 1.42 1.2 0.76 1.8 1.78 1.83 1.62 1.7 1.68 1.74 1.6 Positive Null Null
18 MALE Gender difference FEMALE Total Urine Volume: 60-80 mLDNA yield: ng/mL urine DNA yield Mean: 6 ng/mL urine Total DNA recovery: mg Ratio A 260/A280 nm: 1.35 DNA yield: ng/mL urine DNA yield Mean: 43 ng/mL urine Total DNA recovery: 0 - 7mg Ratio A 260/A280 nm: 1.73 Ratio>2: RNA contamination Ratio<1.8 residual proteins, reagents MALE Gender difference FEMALE
19 URINE DNA RECOVERY Protocol/ kit Manufacturer DNA recovery/ml of urineProtocol/ kit Manufacturer DNA recovery/ml of urine Starting Urine amount Urine Total DNA recovery Preparation Time Disadvantage Reference Zymo- Research Quick-DNA™ Urine Kit 0-20 ng/mL El Bali et al. 2014 40 mL 5 μg 60 min Low DNA yield Requires high sample volume Zymo- Research Handbook Norgen Urine DNA Isolation Kit 0-40 ng/mL 3-25 mL Unknown (lower yield) 30 min Low yield and very expensive for large-scale use Norgen Abcam Urine kit DNA Isolation Kit 0-230 ng/mL 5 mL mg 20 min - Abcam QIAamp Viral RNA Mini Kit* 0-900 ng/mL 1 mL Not specified 60 min Higher yield Lower fragmentation vs other kits Qiagen Handbook QiAmp blood DNA micro Kit from body fluids (Qiagen) 0-900 ng/mL 4-12 μg 20-30 min Low fragmentation vs other kits Salting out & Ethanol precipitation protocol 0-400 ng/mL Van der Hel 0-75 ng/mL Chiarella et al. 50-80 mL mg 4 hours Van der Hel et al. 2002 KIT * inclusion of AVL buffer recommended to inactivate PCR inhibitors found in urine
20 KIT protocol In-house protocol•Fast and standardized •Relatively Fast •Low amount of urine (1 mL) •Variable amount of urine •Variable quality of DNA •Variable quality of DNA (Mulot C 2005) •Expensive (2.70 € up to € /prep) •Cheap •Expiry date (12 months) •No expiry date •Beta-Mercaptoethanol •No toxic reagents •Kit-dependent DNA yield •Subject-dependent DNA yield • Limited urine volume (up to 40 mL) •Unlimited urine volume •Not validated for urine •Not validated for urine
21 CONCLUSION √ Urine sample can be used to assess both exposure and susceptibility biomarkers, giving opportunity for large-scale-studies. √ In-house urine DNA extraction protocols can be used as alternative to expensive kit. √ Association of gene polymorphisms with altered metabolite production following exposure to toxic chemicals may help to identify susceptible genotypes and to protect categories of workers with higher exposure risk. In-house protocol Weakness √ Individual and gender variability of DNA √ Low DNA yield/quality in male samples Implementation √ Increase DNA yield -multiple sampling: harvest urine at the start and end of work-shift -pool cell fractions of the same subject √ Possibility of extending the analysis to a wider panel of enzymes and large cohort of workers
22 ACKNOWLEDGEMENTS Carbonari D, Capone P, Cavallo D, Iavicoli S , Mansi A, Sisto R, Tranfo G. INAIL DIMEILA Research Staff Members