In
vitro bioassays for toxicity testing of
wastewater - an evaluation of different sample
treatment techniques
Ellinor Berkelind
Degree project inbiology, Master ofscience (2years), 2020 Examensarbete ibiologi 45 hp tillmasterexamen, 2020
Biology Education Centre, Uppsala University, and SLU, Institutionen för biomedicin och veterinär folkhälsovetenskap
Supervisor: Johan Lundqvist
External opponent: Agneta Oskarsson
Abstract
In vitro bioassays can be used to detect bioactive compounds in environmental water samples.
There is an ongoing issue with the release of unknown chemicals into the environment, where combined chemical mixtures can cause adverse effects. Solid phase extraction is a common method used to extract compounds from water samples, however, there is a risk of losing compounds during the sample preparation. To overcome this issue, an additional sample treatment was used where concentrated cell culture media was added to untreated water samples. The activity from extracted water samples was compared with the activity from concentrated cell culture media, to evaluate if bioactive/toxic compounds are lost during solid phase extraction procedure. Water samples had previously been collected and the samples were analyzed for aryl hydrocarbon receptor, oxidative stress, androgen receptor, and estrogen receptor by using reporter gene assays. Extracted water samples induced higher activity in the reporter gene assays compared to samples with concentrated cell culture media, except for in oxidative stress. The calculated bioanalytical equivalents were similar for the different sample treatment techniques for aryl hydrocarbon receptor, oxidative stress and estrogen receptor, whereas the bioanalytical equivalent for extracted water samples were much higher than samples for concentrated cell culture media for androgen receptor. The present result does not support the idea of loss of important bioactive/toxic compound during sample extraction, at least for the studied sample. The observed effects could also be due to antagonistic compounds present in the unconcentrated samples, which are lost during sample extraction. However, the additional sample treatment technique with adding concentrated cell culture media to untreated water sample shows to promise as a method to examine the water quality for environmental water samples with minimal sample preparation.
Table of contents
Abbreviations ... i
1. Introduction ... 1
1.1 Background ... 1
1.2 Risk assessment ... 1
1.3 In vitro bioassays ... 2
1.3.1 Aryl hydrocarbon receptor ... 2
1.3.2 Nuclear factor erythroid 2-related factor 2 ... 2
1.3.3 Estrogen receptor and androgen receptor ... 3
1.4 Solid phase extraction ... 3
1.5 Project ... 4
2. Material and methods ... 5
2.1 Cell culturing ... 5
2.2 Water sample collection ... 6
2.3 Preparation of SPE: Extracted wastewater samples ... 6
2.4 Preparation of concentrated cell culture media: untreated wastewater samples ... 6
2.5 MTS assay ... 7
2.6 Reporter gene assays ... 7
2.7 Data evaluation ... 8
3. Results ... 8
3.1 Cell viability ... 8
3.2 AhR activity ... 10
3.3 Nrf2 activity ... 11
3.4 ER activity ... 12
3.5 AR activity ... 13
3.6 BEQ values ... 14
4. Discussion ... 15
5. Conclusion ... 17
6. Acknowledgement ... 17
7. References ... 17
Abbreviations
AhR - Aryl hydrocarbon receptor BEQ – Bioanalytical equivalent
DCC- Dextran-charcoal treated fetal bovine serum DOC – Dissolved organic carbon
DHT – Dihydrotestosterone
DMEM - Dulbecco’s Modified Eagle Medium DMSO – Dimethyl sulfoxide
ER – Estrogen receptor E2 - Estradiol
FBS – Fetal bovine serum HR – Hormone receptor Hsp – Heat shock protein LLE – Liquid-liquid extraction
MTS - 5-[3-(carboxymethoxy)phenyl]-3-(4,5-dimethyl-2-thiazolyl)-2-(4-sulfophenyl)-2H- tetrazolium inner salt
NOM – Natural organic matter
Nrf2 - Nuclear factor erythroid 2-related factor 2 PBS - Phosphate buffer saline
PLB - Passive lysis buffer
REF – Relative enrichment factor SPE – Solid phase extraction STP – Sewage treatment plant
TCDD - 2,3,7,8-tetrachlorodibenzo-p-dioxin tBHQ- Tert-Butylhydroquinone
WWTP – Wastewater treatment plant XRE - Xenobiotic response enhancer
1
1. Introduction 1.1 Background
There is an ongoing issue with the release of unknown compounds into the environment.
Chemicals from pharmaceuticals, health care products and detergents are some examples of what can be found in effluent water from sewage treatment plants (STP) (Fent et al. 2006).
Furthermore, municipal, industries and agriculture also release chemicals through wastewater into the environment (Heeb et al. 2012). As of today, it is not possible to monitor all the chemicals being released. It is estimated that more than 100 000 chemicals are being used daily in commercial use, where 4000 are pharmaceuticals. Common chemical analyses (target screening) can detect up to a few hundred different chemicals in a sample, however
environmental water samples can contain up to thousands of different chemicals (Escher &
Leusch 2011). Therefore, it is not possible to monitor all chemicals being release with chemical analyses.
Releasement of unknown chemicals is of concern for both the human health and for the aquatic environment, since the combined effect of these chemicals are not known (Escher &
Leusch 2011). Chemicals used in pharmaceuticals are biologically active and are designed to have a specific target or to interact with specific pathways. It has been reported that some of these can stay in the environment for a long time (Boxall et al. 2012). It is not only the active substance in pharmaceuticals that may cause an issue, the interaction between chemicals need to be considered as it can cause adverse effects (Ankley et al. 2016). Interaction between chemicals can cause agonist, antagonistic or synergistic effects. As it is not known which compound that are being released, their combined effects cannot be estimated with methods that rely on toxicity testing of single compounds. Furthermore, also naturally occurring compounds can be toxic. Many of the chemicals in environmental water samples occurs in low concentrations, many far below any concentrations that would cause an adverse effect on its own. Due to low concentration of chemicals in water samples, it is common to preserve and concentrate the compounds through extraction to examine the water quality (Abbas et al.
2019).
1.2 Risk assessment
Many toxicological studies and studies of health hazards are often performed with one chemical to examine the toxic effects on humans or animals. These studies are important to increase knowledge about the effects caused by a chemical. By only testing one chemical at the time, several parameters can be excluded, and the mechanisms of toxicity caused by the chemical can easier be established. The problem with this approach is that it is not reflecting the current situation, where humans are exposed to several chemical mixture at the same time.
Often in wastewater samples, the toxicity is caused by mixtures of unknown chemicals rather than known chemicals. Therefore it is important to study the combined effect of chemical mixtures (Yang 2016, Neale et al. 2017). A study made by Silva et al. 2002 showed that different estrogenic agents act together and cause significant effects, even at concentrations below the their no observed effect concentrations. Studies similar to Silva et al, 2002 are important as it consider the effects from combine agents. Risk assessment would be more applicable if it was based on combined effects from a mixture of chemicals rather than from a single agent. The current risk assessments for human health does not consider the potential interactions between different chemicals (Monosson 2005).
2 1.3 In vitro bioassays
The use of in vitro bioassays is becoming more frequent. Bioassays can be used to analyze different mode of actions and water quality. In this report, in vitro bioassays will be used to observe toxicity in wastewater by examine different mode of actions, such as activation of different receptors. An advantage with using in vitro bioassays is that different mammalian or human cell lines are used, which is more ethical than performing animal studies. Many toxicity studies are performed on animals with endpoints as mortality, growth, behavioral effects and reproduction rate. Whereas cell-based assays exclude these endpoints and are additionally more sensitive, cheaper and more time-efficient. By using cell bases assays, the result is more applicable to the human health than animal testing would be (Escher & Leusch 2011).
Bioanalytical tools as cell bases assays can detected mixtures of known and unknown compounds, where it is otherwise is common to test already known chemicals (Escher &
Leusch 2011). The following reporter gene assays, aryl hydrocarbon receptor (AhR), nuclear factor erythroid 2-related factor 2 (Nrf2), estrogen receptor (ER) and androgen receptor (AR) will be used for this project.
1.3.1 Aryl hydrocarbon receptor
The aryl hydrocarbon receptor is found in the cytoplasm and it is found in many different cell types such as epithelia cells. While in the cytoplasm, proteins as heat shock protein 90
(hsp90) binds to the AhR, acting as chaperones and preventing AhR to bind to DNA and enabling ligand binding to the receptor. When a ligand binds to AhR, the chaperones are removed and the AhR complex is transported into the nucleus where it forms a heterodimer with AhR nuclear translocator. The heterodimer binds to xenobiotic response enhancer (XRE) which is located at the promotor region of specific genes, which are activated when the heterodimer binds to the XRE. The most known activated genes are CYP1A1, CYP2A1, CYP1B1 (Boelsterli 2007). The dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD) is the most potent AhR inducer known. The half-life of TCDD in humans is more than 7 years and it is classified as a human carcinogen. TCDD causes many different adverse effects by binding to the AhR, such as inducing tumors, shrinking of thymus, craniofacial abnormalities, alterations in immunological and reproductive systems (Baccarelli et al. 2004).
1.3.2 Nuclear factor erythroid 2-related factor 2
Nuclear factor erythroid 2-related factor 2 (Nrf2)is a transcript factor activated to prevent oxidative stress. It is a cellular defense mechanism which activates transcription of
antioxidant and detoxifying enzymes (Escher et al. 2012, Wu et al. 2014). Nrf2 is activated upon oxidative stress to degrade reactive oxygen species and environmental carcinogens which are harmful to the cell. Nrf2 activates genes which contain an antioxidant response element which encodes detoxifying enzymes such as glutathione-S-transferase and
nicotinamide adenine dinucleotide phosphate (NADPH) (Escher et al. 2012). The importance of Nrf2 to induce cytoprotective enzymes has been well studied. Where depletion of Nrf2 in micecan give rise to many different adverse effects, such as inducing hepatotoxicity,
neurotoxicity, carcinogenicity and inflammations. Compounds as benzo(a)pyrene which is a polycyclic aromatic hydrocarbon and the neurotoxin 1-metyl-4-fenyl-1,2,3,6-
tetrahydropyridine (MPTP) are some examples of compounds which activates Nrf2 (Osburn
& Kensler 2008).
3 1.3.3 Estrogen receptor and androgen receptor
Both estrogen and androgen belongs to steroid hormones, steroid hormones are lipophilic which allows them to diffuse over the cell membrane. A steroid hormone can either bind to the receptor in the cytoplasm or in the nucleus. Androgens and estrogens belong to different subgroups of steroid hormones as they bind to different receptors. Hormones as testosterone and its more potent metabolite 5-dihydrotestosterone are examples of androgens that binds to the androgen receptor (Li & Al-Azzawi 2009), whereas estrogens such as 17B-estradiol (E2) bind to estrogen receptor (Klinge 2001).
Binding to a hormone receptor (HR) can either cause an agonist or antagonistic effect. Where agonistic effect increases the transcription factor and antagonistic effect inhibits gene
activation. An agonist effect occurs when a steroid hormone binds to its receptor, it induces a conformational change in the receptor and dislocate the chaperones as hsp90 and hsp70.
Thereafter, dimerization occurs, and the receptor is translocated to the hormone response element (HRE), androgen response element for androgens and estrogen response element for estrogenic compounds (Klinge 2001, Boonyaratanakornkit & Edwards 2004). Binding to the HRE induce gene activity and transcription is initiated. An antagonistic effect occurs when a xenobiotic ligand binds to a hormone receptor and prohibits natural steroids to bind.
Tamoxifen act as an antagonist as it binds to ER which prevents E2 to bind to the receptor, this inhibits gene activation. Alterations of ER can lead to different types of cancer, where it can give rise to breast cancer in women. Alterations of ER can also cause different adverse effects as cardiovascular disease and insulin resistance (Deroo & Korach 2006). Alterations of AR caused by xenobiotics can cause many different adverse effects, such as prostate and testicular cancer (Hess-Wilson & Knudsen 2006, Kharlyngdoh et al. 2018). Different organophosphate flame retardants, which can be found in building materials, plastic, textile fabrics, can cause both agonistic and antagonistic effects by binding to ER and AR (Kojima et al. 2013).
1.4 Solid phase extraction
Solid phase extraction (SPE) is a technique used for sample preparation. It is used for
isolation, concentration clean up and medium exchange, normally from gas, fluid or liquid. In SPE, analytes are extracted in the solid phase and thereafter isolated from the sample. The analytes are then recovered by elution by using a liquid or a fluid. SPE have become widely used and is used in areas as chemistry, environmental pharmaceutical, clinical food and industrial chemistry. SPE comes with a low cost, it has simple procedures and short processing times (Poole 2003).
SPE targets organic micropollutants, an advantage of using SPE for environmental water samples, is that extraction prevents microbial degradation. During SPE, compounds as natural organic matter can be decreased and compounds as ions, nutrients and salts can be removed.
This facilitates detection of toxicological compounds which are extracted from the environmental water samples (Abbas et al. 2019, Neale et al. 2018).
An issue with sample preparation is that there is a risk of losing compounds from the sample (Abbas et al. 2019). This becomes an even bigger issues when working with water samples with unknown compounds and since contaminants in environmental water samples normally occurs in low concentrations (Escher & Leusch 2011). A study made by Niss et al. 2018 developed an alternative method for sample preparation, by using concentrated the cell culture media diluted in untreated water samples to reduce the risk of losing compounds from the sample. This showed to be effective.
4 1.5 Project
Due to the ongoing release of unknown chemicals into the environment, in vitro bioassays are a good method to use to examine if samples, in this case, wastewater samples may cause toxicity to human cells. In vitro bioassays will be used to detect if the samples contain any chemicals which will activate important toxicity pathways, regardless if the chemicals are known or unknown.
As for this project, water samples had previously been collected from different STP in Sweden. From each STP, extracted water samples was made through SPE. As of the known issue with losing chemicals through SPE, an additional method was used to reduce the risk of losing compounds from the sample, where cells were exposed to concentrated cell culture media diluted in untreated water samples. By doing this, there will not be loss of compounds during the preparation steps. The extracted samples and samples with concentrated cell culture media were tested in different bioassays. For this project wastewater samples were analyzed for AhR, oxidative stress, ER and AR. Cell lines with stably transfected AhR, Nrf2,
ER and AR sensitive plasmids were used to evaluate if chemicals in the wastewater will activate these receptors.
A study made by Niss et al. 2018 have already tested bioassays with concentrated cell culture media, where activity in AhR and ER was shown. Their study showed that using concentrated cell culture media can be used to asses water quality. The aim of this thesis was to evaluate the two different sample preparation techniques in detection of toxicity in wastewater (Figure 1). The activity from extracted water samples was compared with the activity from samples with concentrated cell culture media, to evaluate if bioactive/toxic compounds are lost during the SPE procedure.
Figure 1. Sample extraction was made through SPE, the recovered extraction (blue drop) was diluted in cell medium, which was used during the reporter gene assays. The additional sample preparation technique is concentrated cell culture media (red drop) diluted in untreated water samples collected from different WWTP, which was used during the reporter gene assays.
Cell medium à
Concentrated cell media
Water samples from WWTP à
5
2. Material and methods 2.1 Cell culturing
Cells were subcultured two to three times a week and medium was changed every second to third day. While subculturing, previous culture medium was removed, and cells were washed with phosphate buffer saline (PBS) (Gibco) to remove remaining medium. 0.05% Trypsin- EDTA (1X) (Gibco) was added to the flask (3 ml to a 75cm2 flask or 4 ml to a 175cm2 flask) and cells were incubated at 37 °C with 5 % CO2 for 3 to 5 minutes to allow cells to detach from the surface. Cell medium was added to the flask and mixed thoroughly to stop the action of trypsin and to separate the cells. Cells were transferred to a falcon tube and centrifuged for 3 minutes at 1000 rmp. The supernatant was removed, and the pellet was resuspended with culture medium. Cell cultures were incubated in a humidified atmosphere at 37 °C with 5 % CO2 until further analyses.
The DR-EcoScreen cells are stably transfected with an AhR sensitive plasmid. The cell line is developed from mouse cancer cell line and is used for the aryl hydrocarbon receptor (AhR) transcriptional activation reporter gene assay (Takeuchi et al. 2008). The cells were cultured in MEM Alpha (1X) (Gibco), 5% fetal bovine serum (FBS) (Gibco), 1%
penicillin/streptomycin (100 U mL-1 penicillin and 100 μg mL-1 streptomycin) (Gibco) and 150 µg/ml selective antibiotic Hygromycin B Gold (100 mg/ml) (Invivogen). The
experimental medium for DR-EcoScreen cells contained the same components as the culture medium but without Hygromycin B Gold.
The cell line MCF7AREc32 was used for the Nrf2 reporter gene assay (Wang et al. 2006). A stably transfected human mammary MCF7 cell line is developed by transfecting ARE driven antioxidant gene which regulates Nrf2. The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (1X) GlutaMAXTM (Gibco), 10% FBS, 1% penicillin/streptomycin and 0.8 mg/ml geneticin (G418) (100 mg/ml) (InvivoGen). The experimental medium contained the same components as the culture medium but without geneticin.
The VM7Luc4E2 cell line is human breast cancer (MCF7) cells, stably transfected with an estrogen receptor sensitive luciferase plasmid controlled by estrogen response elements (Brennan et al. 2016) . Cells were cultured in RPMI 1640 with L-glutamine (Gibco), 8% FBS, 0.9% penicillin/streptomycin and 0.55 mg/mL gentamicin (Gentamicin Sulfate, 50mg/ml) (Lonza). Two to three days prior to experimental set-up, VM7Luc4E2 cells were transferred to experimental estrogen free medium consisting of DMEM (with 4.5 g/L glucose) (Lonza), 4.5% dextran-charcoal treated FBS (DCC-FBS) (Gibco), 2% L-glutamine, 0.9%
penicillin/streptomycin and 0.38 mg/mL gentamicin.
The cell line AR-EcoScreen GR KO M1 was used for the androgen receptor (AR) reporter gene assay. AR-EcoScreen GR KO M1 is developed from the Chinese hamster ovary cell line and stably expresses human androgen reporter, which is linked to luciferase and renilla (Zwart et al. 2017). Furthermore, the cell line specifically knocked out the glucocorticoid receptor gene. AR-EcoScreen GR KO M1 cells were cultured in DMEM F-12 (Sigma), 10% DCC- FBS, 1% penicillin/streptomycin, 1% L-glutamine, 25 µg/ml Hygromycin B and 50 µg/ml Zeocin (100 mg/ml) (Invitrogen). The experimental medium contained DMEM F-12 (Sigma), 10% DDC-FBS (Gibco), 1% penicillin/streptomycin.
6 2.2 Water sample collection
Water samples were previously collected from six different STP in Sweden (Table 1). Water samples were sterile filtered with a 0.22 µm syringe filter (MillexTM, Millipore) when brought to the lab. Furthermore, concentrated water samples were extracted in the lab from each STP through SPE. All samples were kept at – 20 degrees until analysis were performed.
Table 1. The different sites were the water samples were collected from in Sweden.
Site STP Year Sampling point
Jönköping STP 1 2018 Influent
Jönköping STP 16 2018 Influent
Nolhaga, Alingsås STP 49 2018 Influent
Stadskvarn, Skövde STP 55 2018 Effluent
Jämtland STP 125 2018 Recipient
Gävleborg STP 127 2018 Effluent
2.3 Preparation of SPE: Extracted wastewater samples
Extractions from each STP were made from the water samples through SPE cartridges, Oasis HLB 20 cc, 1g (Waters). Where 50 ml of wastewater was filtered with 0.22 µm syringe filter (MillexTM, Millipore). SPE cartridges were conditioned with 30 ml methanol and 20 ml milli- Q water. Thereafter, water samples were added by a vacuum manifold through a small tube of at a speed of 1 drop per second. Finally, each sample was eluted twice with 10 ml methanol into glass tubes. The two eluates were pooled, and the volume was reduced to approximately 0.1 ml/dryness. The resulting pellet was resuspended in 0.5 ml of ethanol. Thereafter, the samples were transferred into a LC-MS glass vials and the concentrated water samples were stored at -20°C degrees until analysis.
The extraction of water samples lead to water sample enrichment by a factor 100. The water samples were diluted 100-fold in cell medium, leading to a final concentration of 1% ethanol.
The combination of the enrichment and dilution of the samples constitute the relative enrichment factor (REF) calculated as following equations:
𝑅𝐸𝐹 = 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟!"#$%%$&∗ 𝑒𝑛𝑟𝑖𝑐ℎ𝑚𝑒𝑛𝑡 𝑓𝑎𝑐𝑡𝑜𝑟'() where the dilution and enrichment factor is calculated by the following equations:
𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟!"#$%%$& = *#+,-. $00.0 1# !"#$%%$&
1#1$+ *#+,-. "2 !"#$%%$&
𝐸𝑛𝑟𝑖𝑐ℎ𝑚𝑒𝑛𝑡 𝑓𝑎𝑐𝑡𝑜𝑟'() = *#+,-. 3$1.4
*#+,-. .514$61
REF > 1 implies an enriched water sample and REF < 1 implies a diluted water sample.
In all experiments, vehicle controls consist of 1% ethanol, equivalent to water sample ethanol content.
2.4 Preparation of concentrated cell culture media: untreated wastewater samples
For the exposure experiments, a ten times concentrated cell culture media was prepared using Dulbecco’s Modified Eagle Medium (DMEM) powder media dissolved in deionized water, pH was adjusted to 7.0 and sterile filtered with a 0.22 µm syringe filter (MillexTM , Millipore).
The 10x cell culture media was then diluted to 1x using the STP wastewater samples. For AhR and Nrf2 reporter gene assays, the 10x DMEM (GIBCO) cell culture medium was supplemented with 3.7 g/L sodium bicarbonate (7,5%) (Sigma), 0.11 g/L sodium pyruvate 100 mM (Sigma), 5% FBS, 1% L-glutamine and 1% penicillin/streptomycin.
7
For AR and ER reporter gene assays, along with 10x DMEM (Sigma), 3,7 g/L sodium bicarbonate (7,5%), 0,11 g/L sodium pyruvate solution 100 mM, 4,5 g/L glucose solution (Gibco) were supplemented along with 5% DDC-FBS, 1% L-glutamine and 1%
penicillin/streptomycin. The final concentration factor for all reporter gene assays were 0.83, which indicate that the samples have been diluted as the concentration factor is below one.
2.5 MTS assay
All water samples were tested for cytotoxicity by measuring cell viability. Two different methods were used for the assessment of cell viability, MTS-based colorimetric assay (Cell Titer 96® Aqueous One Solution Cell Proliferation Assay) (Promega) and ATP based luminescent assay (CellTiter-Glo® Luminescent Cell Viability Assay) (Promega).
MTS measures the mitochondrial enzymatic activity of cells that are able to reduce MTS tetrazolium to a colored formazan dye. Only viable cells are able to reduce MTS tetrazolium to formazan. The quantity of formazan product measured at 490nm absorbance is directly proportional to the number of living cells in culture. This conversion is presumably done by NADPH or NADH produced by dehydrogenase enzymes in metabolically active cells. In ATP assay is luminescence based on the quantitation of ATP present which is directly
proportional to the number of metabolically active cells. Extracted water samples were tested at concentrations of REF 1, REF 0.5 and REF 0.1. Whereas the samples with concentrated cell culture media were tested at concentrations of REF 0.83, REF 0.4, REF 0.2 and REF 0.1.
MTS based assay was used for DR-EcoScreen cells and AR EcoScreen GR KO M1, both at plating density of 4000 cells/well and MCF7 AREc32 at 5200 cells/well in a transparent 384- plates (Costar ® Corning Incorporated). ATP based assay was used for VM7Luc4E2 cells with a plating density of 16000 cells/well in white 384-plates (Costar ® Corning Incorporated). Cells were left to incubate for 24 hours, after which cells were exposed to water samples for 24 hours.
For assays with concentrated medium, the medium from the plate was removed and replaced with 10x concentrated medium diluted in untreated water sample and incubated for 24 hours.
At experiment termination, medium was removed from the plate and 10 µl/well of CellTiter 96® AQueous One Solution Reagent (Promega) together with 40 µl/well of PBS, was added to the plate and cells were incubated for approximately 15 -30 minutes until the yellow color changes to brown/ violet color. For assay termination with VM7Luc4E2 cells, 25 µL well-1 of CellTiter-Glo® Luminescent reagent was added, the plate was put on a shaker for 2 minutes and successively incubated for 10 minutes.
Absorbance and luminescence for the MTS and ATP-based assays respectively, were measured on Infinite M1000 plate reader (Tecan). A decrease in viability of ≥ 20% was considered as cytotoxic. 1% ethanol is used as the vehicle/negative control and 10% DMSO as the positive control for all cell viability assays. The samples were normalized to vehicle/negative control.
All samples were tested in four replicates and 8 replicates for vehicle/negative control. The sample with highest concentration where no cytotoxicity occurred was used for further reporter gene assays.
2.6 Reporter gene assays
In all experiments, four replicates were tested for each sample. For extracted water samples, a vehicle control was included, and it consisted of 1% of ethanol with an exception of 0.1%
8
ethanol for E2 standards in concentrated medium for ER assay. The vehicle control used for concentrated cell culture media consisted of 1% of milli-q water. The reporter gene assay was performed at least two times. Positive controls were included for each reporter gene assay.
The positive controls were used for the standard curves.
All experiments in stably transfected cell lines were conducted over a three-day period with cell seeding on day 1, cell treatment with water samples and standards on day 2, and
luciferase measurement on day 3. The plating density is the same for each cell line as
described in cell viability assays and were plated in white -384 plates. On the second day for extracted water samples, the plate was incubated for 24 hours and then exposed to the SPE- concentrated water samples, vehicle control and positive controls whereas for water samples with concentrated cell culture media, the medium was removed and wastewater samples diluted in concentrated cell culture media, vehicle control and positive controls was added. At experiment termination on day 3, cells were lysed with passive lysis buffer (PLB) (Promega), 10µL per wellfor 15-30 minutes. Luciferase activity was measured using the Luciferase®
Reporter Assay System (Promega) according to the manufacturer’s instructions.
Luminescence was measured on Infinite M1000 plate reader (Tecan) with an automatic injection syringe. The injection volume for the Firefly luciferase reagent was 10 µl/well.
Luminescence measurement was conducted over a 5s period, 2s after reagent was automatically injected with Firefly luciferase reagent.
For the AhR bioassay in DR–ecoscreen cells, TCDD (Solveco) was used as the positive control with concentrations from 0.1 pM up to 10000 pM. Dihydrotestosterone (DHT) (Sigma) with concentrations from 0,001 pM to 1000 nM was used as the positive control for the AR bioassay in AR EcoScreen GR KO M1. The AR bioassay was tested for agonistic effect. Tert-Butylhydroquinone (tBHQ) (Sigma) was used as a positive control for Nrf2 activity in MCF7 AREc32 with concentrations from 0.78 µM to 25 µM. The ER bioassay was tested for agonistic effect. 17ß-estradiol (E2) (Sigma) was used as positive control with concentrations from 3.59 x 10-13 M to 3.67 x 10-10 M. The weak positive control for agonist mode is 9.06 x10-6 M of p,p’-Methoxychlor (Sigma-Aldrich).
2.7 Data evaluation
Bioactivities from water samples and positive controls were normalized to the plate vehicle controls, set to 1. The standard curves for positive controls were obtained by fitting data to a nonlinear regression curve fit, expect for Nrf2 where a linear regression was performed using GraphPad Prism 7.
3. Results 3.1 Cell viability
The cell viability assays showed that none of the extracted water samples caused any cytotoxicity, except for wastewater sample STP55-0.1 for oxidative stress (Figure 2). The wastewater sample STP55-0.1 (REF 0.1) has more than 20% decrease in cell viability compared to the vehicle control, which indicates cytotoxicity. It is unlikely that the lowest concentration indicates cytotoxicity and not the higher concentrations. The MTS assays was performed one more time for STP55 and no cytotoxicity was observed in the any
concentrations (Figure 3). For concentrated cell culture media, the only assay which showed cytotoxicity was for the AhR reporter gene assay. For wastewater samples STP1 and STP55 cytotoxicity was observed at the highest REF of 0.83, the wastewater sample STP125 showed cytotoxicity at REF 0.83 and REF 0.4 (Figure 2).
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Figure 2. The viability test shows the MTS assays for AhR, Nrf2 and AR, and the ATP assays for ER. The panels to the left show the viability for extracted water samples and the panels to the right shows the viability for
VC(MQ) 10% DMSO STP1-0.1 STP1-0.5 STP1-1.0 STP16-0.1 STP16-0.5 STP16-1.0 STP49-0.1 STP49-0.5 STP49-1.0 STP55-0.1 STP55-0.5 STP55-1.0 STP125-0.1 STP125-0.5 STP125-1.0 STP127-0.1 STP127-0.5 STP127-1.0 0.0
0.5 1.0 1.5
Cell viability (Fold change compared to vehicle control)
Extracted water samples DR-Ecoscreen
VC(MQ) 10% DMSO STP1-0.1 STP1-0.5 STP1-1.0 STP16-0.1 STP16-0.5 STP16-1.0 STP49-0.1 STP49-0.5 STP49-1.0 STP55-0.1 STP55-0.5 STP55-1.0 STP125-0.1 STP125-0.5 STP125-1.0 STP127-0.1 STP127-0.5 STP127-1.0 0.0
0.5 1.0 1.5
Cell viability (Fold change compared to vehicle control)
Extracted water samples MCF7AREc32
VC(MQ) 10% DMSO STP1-0.1 STP1-0.5 STP1-1.0 STP16-0.1 STP16-0.5 STP16-1.0 STP49-0.1 STP49-0.5 STP49-1.0 STP55-0.1 STP55-0.5 STP55-1.0 STP125-0.1 STP125-0.5 STP125-1.0 STP127-0.1 STP127-0.5 STP127-1.0 0.0
0.5 1.0 1.5
Cell viability (Fold change compared to vehicle control)
Extracted water samples VM7Luc4E2
VC(MQ) 10% DMSO STP1-0.1 STP1-0.5 STP1-1.0 STP16-0.1 STP16-0.5 STP16-1.0 STP49-0.1 STP49-0.5 STP49-1.0 STP55-0.1 STP55-0.5 STP55-1.0 STP125-0.1 STP125-0.5 STP125-1.0 STP127-0.1 STP127-0.5 STP127-1.0 0.0
0.5 1.0 1.5 2.0
Cell viability (Fold change compared to vehicle control)
Extracted water samples AR-Ecoscreen GR KO M1
VC(MQ) 10% DMSO STP1-0.1 STP1-0.2 STP1-0.4 STP1-0.83 STP16-0.83 STP49-0.83 STP55-0.1 STP55-0.2 STP55-0.4 STP55-0.83 STP125-0.1 STP125-0.2 STP125-0.4 STP125-0.83 STP127-0.83 0.0
0.5 1.0 1.5 2.0
Cell viability (Fold change compared to vehicle control)
Concentrated cell media DR-Ecoscreen
VC(MQ) 10% DMSO STP1-0.1 STP1-0.2 STP1-0.4 STP1-0.83 STP16-0.1 STP16-0.2 STP16-0.4 STP16-0.83 STP49-0.1 STP49-0.2 STP49-0.4 STP49-0.83 STP55-0.1 STP55-0.2 STP55-0.4 STP55-0.83 STP125-0.1 STP125-0.2 STP125-0.4 STP125-0.83 STP127-0.1 STP127-0.2 STP127-0.4 STP127-0.83 0.0
0.5 1.0 1.5 2.0
Cell viability (Fold change compared to vehicle control)
Concentrated cell media MCF7AREc32
VC(MQ) 10% DMSO STP1-0.1 STP1-0.4 STP1-0.83 STP16-0.1 STP16-0.4 STP16-0.83 STP49-0.1 STP49-0.4 STP49-0.83 STP55-0.1 STP55-0.4 STP55-0.83 STP125-0.1 STP125-0.4 STP125-0.83 STP127-0.1 STP127-0.4 STP127-0.83 0.0
0.5 1.0 1.5
Cell viability (Fold change compared to vehicle control)
Concentrated cell media VM7Luc4E2
VC(MQ) 10% DMSO STP1-0.1 STP1-0.2 STP1-0.4 STP1-0.83 STP16-0.1 STP16-0.2 STP16-0.4 STP16-0.83 STP49-0.1 STP49-0.2 STP49-0.4 STP49-0.83 STP55-0.1 STP55-0.2 STP55-0.4 STP55-0.83 STP125-0.1 STP125-0.2 STP125-0.4 STP125-0.83 STP127-0.1 STP127-0.2 STP127-0.4 STP127-0.83 0.0
0.5 1.0 1.5
Cell viability (Fold change compared to vehicle control)
Concentrated cell media AR-Ecoscreen GR KO M1
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concentrated cell culture media. Extracted water samples were tested with the following REF concentrations, 1, 0.5, 0.1. Whereas samples with concentrated cell culture media were tested with the following REF
concentrations, 0.83, 0.4, 0.2 and 0.1. Samples below the red line indicates that cytotoxicity occurs as the value represents a decrease in cell viability of more than 20% compared to the vehicle control. The red line is at 0.8- fold induction and the vehicle control for all samples are 1-fold induction.
Figure 3. The viability test for extracted water sample, STP55, for oxidative stress. No cytotoxicity is observed in the different concentrations.
3.2 AhR activity
All extracted water samples activated the AhR, where STP1, STP125 and STP127 induced the highest AhR activity with a six-fold induction compared to the vehicle control (Figure 4).
For concentrated cell culture media, STP1 and STP16 induced AhR activity above the threshold. STP1 with REF 0.4 induced a 1.9-fold induction and STP16 with REF 0.83
induced a 1,9-fold induction in AhR activity (Figure 4). The standard curve for TCDD shows an increasing AhR activity with increasing concentrations of TCDD (pM).
VC(EtOH) 10% DMSO STP55-0.1 STP55-0.5 STP55-1.0 0.0
0.5 1.0 1.5
Cell viability (Fold change compared to vehicle control)
Extracted water samples MCF7AREc32
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Figure 4. AhR activity from extracted water samples (upper left) and the standard curve from the positive control, TCDD (upper right). The AhR activity from concentrated cell culture media (lower left) and the standard curve for TCDD (lower right).
3.3 Nrf2 activity
For Nrf2 STP1 induced a 1.7-fold induction, STP16 induced a 1,5-fold induction, STP125 induced 1.4-fold induction and STP127 induced a 1.5-fold induction, all samples at REF 1.
For concentrated cell culture media, STP1 induced a 3.2-fold induction and STP16 a 2.3-fold- induction, both at REF 0.83.
VC(EtOH) STP1-1 STP16-1 STP49-1 STP55-1 STP125-1 STP127-1
0 2 4 6 8
AhR activity (Fold change vs vehicle control)
Extracted water samples
VC(MQ) STP1-0.4 STP16-0.83 STP49-0.83 STP55-0.4 STP125-0.2 STP127-0.83
0 2 4 6 8
AhR activity (Fold change vs vehicle control)
Concentrated cell media
0.01 0.1 1 10 100 1000 10000 100000
0 5 10 15 20
Concentration TCDD (pM) AhR activity (Fold change vs vehilce control)
Standard curve
0.01 0.1 1 10 100 1000 10000 100000
0 5 10 15
Concentration TCDD (pM) AhR activity (Fold change vs vehilce control)
Standard curve
12
Figure 5. Nrf2 activity from extracted water samples (upper left) and the standard curve from the positive control, tBHQ (upper right). The Nrf2 activity from concentrated cell culture media (lower left) and the standard curve for tBHQ (lower right).
3.4 ER activity
REF 1 for STP1 and STP127 induced a higher activity than the highest concentration of E2 in the standard curve (3,6 * 10-10M), indicating that the concentration of estrogenic compounds in these extracts were saturating the assay. Therefore, REF 0.5 was used for both samples to receive values within the standard curve. All extracted water samples except STP49 induced ER activity (Figure 6). STP1 with REF 0.5 induced a 5.5-fold induction, STP16 with REF 1 a 6-fold induction, STP55 with REF 1 a 2-fold induction, STP125 with REF 1 a 1.9-fold
induction and STP127 with REF 0.5 a 5,4-fold induction. For concentrated cell culture media, STP1 induced a 3-fold induction, STP16 a 3.7-fold induction, STP49 a 1.6-fold induction and STP127 a 3.1-fold induction in the ER activity. All samples for concentrated cell culture media were analyzed at REF 0.83 (Figure 6). The standard curve for ER shows an increasing ER activity with increasing concentrations of E2 (µM).
VC(EtOH) STP1-1 STP16-1 STP49-1 STP55-1 STP125-1 STP127-1
0 1 2 3 4
Nrf2 activity (fold change vs vehicle control)
Extracted water samples
VC(MQ) STP1-0.83 STP16-0.83 STP49-0.83 STP55-0.83 STP125-0.83 STP127-0.83 0
1 2 3 4
Nrf2 activity (fold change vs vehicle control)
Concentrated cell media
0 10 20 30
0 2 4 6 8
Concentration tBHQ (µM) Nrf2 activity (fold change vs vehicle control)
Standard curve
0 5 10 15
0 1 2 3 4
Concentration tBHQ (µM) Nrf2 activity (fold change vs vehicle control)
Standard curve
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Figure 6. ER activity from extracted water samples (upper left) and the standard curve from the positive control, estradiol (upper right). The ER activity from concentrated cell culture media (lower left) and the standard curve for estradiol (lower right).
3.5 AR activity
For extracted water samples, the REF values 1 and 0.5 for STP1 induced a higher activity than the highest concentration of DHT in the standard curve, indicating that the concentration of androgenic compounds in these extracts were saturating the assay. Therefore, REF 0.1 was used to receive values within the standard curve. STP1 at REF 0.1 induced a 2,8-fold
induction, STP16 at REF 1 a 3.9-fold induction and STP125 at REF 0.1 a 2.3-fold induction in the AR activity (Figure 7). REF 0.1 was used for STP125 as the higher REF values of STP125 induced potential cytotoxicity which was not shown during the MTS assay. This was noticed when the samples were analyzed in a dose-response study and exerted an inversely proportional dose-dependency, a decreasing activity with increasing REF values (data not shown). The activity for REF 1 and REF 0.5 showed one-fold induction, the same as the
VC(EtOH) STP1-0.5 STP16-1 STP49-1 STP55-1 STP125-1 STP127-0.5
0 2 4 6 8
ER activity (Fold change vs vehicle control)
Extracted water samples
VC(MQ) STP1-0.83 STP16-0.83 STP49-0.83 STP55-0.83 STP125-0.83 STP127-0.83
0 2 4 6 8
ER activity (Fold change vs vehicle control)
Concentated cell media
10-7 10-6 10-5 10-4 10-3
0 2 4 6 8
Estradiol (µM) ER activity (Fold change vs vehicle control)
Standard curve
10-7 10-6 10-5 10-4 10-3 0
1 2 3 4
Estradiol (µM) ER activity (Fold change vs vehicle control)
Standard curve
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vehicle control, whereas the AR activity for REF 0.1 showed a 2,3-fold induction. It indicated that the AR activity is suppressed for REF 0.5 and REF 1 possibly due to cytotoxicity. For concentrated cell culture media, the REF values 0.83 and 0.4 for STP1 and STP16 induced a higher activity than the highest concentration of DHT, hence was REF 0.2 used. STP1 and STP16 induced AR activity, with a 2,1-fold and 2-fold induction at REF 0.2 respectively.
(Figure 7).
Figure 7. AR activity from extracted water samples (upper left) and the standard curve from the positive control, DHT (upper right). The AR activity from concentrated cell culture media (lower left) and the standard curve for DHT (lower right).
3.6 BEQ values
To evaluate the two different sample treatment technologies, the bioanalytical equivalent concentration (BEQ) for each sample was calculated based on the two experimental set-ups (Table 2). The BEQ is derived from the standard curve of the different reporter gene assays to
VC(EtOH) STP1-0.1 STP16-1 STP49-1 STP55-1 STP125-0.1 STP127-1
0 1 2 3 4 5
AR activity (Fold change vs vehilce control)
Extracted water samples
VC(MQ) STP1-0.2 STP16-0.2 STP49-0.83 STP55-0.83 STP125-0.83 STP127-0.83
0 1 2 3 4 5
AR activity (Fold change vs vehicle control)
Concentrated cell media
10-10 10-5 100 105
0 2 4 6
Concentration DHT (nM) AR activity (Fold change vs vehilce control)
Standard curve DHT
0.000001 0.00001 0.0001 0.001 0.01 0.1 1 10 100 1000 10000 0
1 2 3 4
Concentration DHT (nM) AR activity (Fold change vs vehicle control)
Standard curve DHT
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translate the observed activity at REF 1 to the corresponding concentration of a known inducer that would cause the same activity. For extracted water samples which induced activity at REF1 and where no observed cytotoxicity occurred, BEQ was calculated based on the values from REF 1 using GraphPad. For samples with concentrated cell culture media which induced activity at REF 0.83 and where no observed cytotoxicity occurred, BEQ was calculated based on the values from REF 0.83. The BEQ value for 0.83 was then multiplied to REF 1. For samples with concentrated cell culture media where cytotoxicity was shown, the BEQ-value was calculated by doing a linear regression to estimate the value for REF 1 based on the results from multiple concentrations where no cytotoxicity occurred.
Table 2. The bioanalytical equivalent (BEQ) values for the different water samples at REF 1.
Extracted water samples Concentrated cell culture media
AhR
(pM) Nrf2
(µM) ER
(M) AR
(nM) AhR
(pM) Nrf2
(µM) ER
(M) AR
(nM)
STP1 4.9 4.8 1*10-10 115.5 2,7 16,6 0.4*10–10 15,4
STP16 2.9 3.9 1*10–10 190.4 1,6 11.3 2.9*10-10 7.1
STP49 2.5 N/A N/A N/A N/A N/A 3.3*10–12 N/A
STP55 2.0 N/A 0.9*10-12 N/A N/A N/A N/A N/A
STP125 4.4 3.5 0.8*10-12 7.2 N/A N/A N/A N/A
STP127 4.8 4.2 2,7*10-10 N/A N/A N/A 0.5*10–10 N/A
4. Discussion
It is common to extract environmental water samples to preserve and recover as much of the reactive compounds in the sample as possible. Extraction prevents microbial degradation, it decreases natural organic matter and removes ions, nutrients and salts. The issue with extraction preparations is the loss of compounds, where compounds with low affinity to the sorbent are washed away (Abbas et al. 2019). During the MTS and ATP based assays, cytotoxicity occurred in samples with concentrated cell culture media. Cytotoxicity occurred in three out of six samples for the AhR assay (Figure 2). A reason for why cytotoxicity occurred in samples with concentrated cell culture media and not in extracted water samples, could be that untreated water contains more complex mixtures of different compounds. As ions, organic matter and salt have not been removed from the sample as it does in extracted samples. The interaction of chemical mixtures in untreated samples then causes a higher risk of cytotoxicity in the sample.
For this project, chemicals in the collected STP samples induced AhR, Nrf2, ER and AR (Figure 4-7). Which chemicals in the mixtures that induced the activity will remain unknown.
The induction of different receptors is likely caused by combined effects from different chemicals in the mixtures. A study made of Neale et al. 2017, integrated chemical analyses and bioassays, to establish how much of the effect from chemicals present in surface water could be explained. Their result showed that the chemical analyses could explain 0-30% of the observed activity for induction of AhR, 0.1–0.3% of the observed activity for induction of ER and 0.04-0.4 % of the observed activity for induction of AR. Their study emphasizes that toxicity in environmental water samples is caused by unknown chemicals and unknown mixtures of chemicals. Using effect-based methods that can integrate effects from both known and unknown chemicals in a sample is therefore a necessity to fully evaluate the chemical hazards in an environmental sample.
As previously mention, there is a risk of losing compounds during SPE. Hence, the additional sample treatment technique with using concentrated cell culture media was used to overcome this issue. It has been hypothesized that the potential loss of compounds when samples are
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extracted by SPE could result in an underestimation of the presence of hazardous chemicals in the analyzed samples. The aim of this study was to compare two sample treatment techniques (concentrating the sample and dilute it in cell culture mediums versus concentrating the cell culture medium and diluting it in the water sample) to evaluate if the SPE procedure would result in an underestimation of the presence of hazardous chemicals. The results show that extracted water samples induces higher activity than samples with concentrated cell culture media, in three out of four endpoints (AhR, ER and AR). The present results do not lend support to the idea that extraction with SPE would result in a loss of chemicals during SPE and thereby an underestimation of the presence of hazardous chemicals, since extracted water samples induced the higher activity. For oxidative stress, the activity was higher in samples with concentrated cell culture media compared to extracted water samples (Figure 5). This could potentially support the idea that bioactive compounds are lost during SPE. However, further studies need to be done to establish the loss of chemicals during SPE. The content of the water samples has a big impact on the result, since every water samples contain different set up of chemicals depending on sampling time and collection point.
Furthermore, using concentrated cell culture media in untreated water can still be used as a technique to avoid losing compounds. In this study, it is shown that samples with
concentrated cell culture media induced activity in all reporter gene assays (Figure 4-7). A similar study made by Niss et al, 2018 used the same technique with concentrated cell culture media and their study showed induction of AhR and ER. This emphasize that the additional sample preparation technique with using concentrated cell culture media in undiluted water can be used to monitor water quality without risking loss of chemicals.
An issue with using concentrated cell culture media as a treatment technique in undiluted water is the risk of having cytotoxicity. As undiluted water contains more chemicals than extracted water samples. An issue with cytotoxicity is that it can mask the effects from samples (Abbas et al. 2019). This became noticeable while observing the MTS assay and AhR assay, no cytotoxicity occurred in the extracted water samples. Whereas cytotoxicity occurred in three out of six samples for samples with concentrated cell culture media (Figure 2). All of the extracted water samples at REF 1 induced AhR activity ranging from three-fold up to six-fold induction. For samples with concentrated cell culture media, due to
cytotoxicity, the samples had to be tested in lower concentrations. Where STP1 were tested with REF 0.4, STP55 with REF 0.4 and STP125 at REF 0.2 (Figure 4). With low
concentrations, STP55 and STP125 did not induce an activity above threshold. The issue with this approach is that by using lower concentrations, it correlates with lower activity and masking of the effects.
To overcome the issues with using different REF concentrations for different samples, the BEQ values was calculated to REF 1 for all samples which induced activity (Table 2). The BEQ values for AhR and ER are similar between extracted water samples and samples with concentrated cell culture media. This indicates that the method with concentrated cell culture media can be used as an alternative method to SPE, but it also indicates that we did not observe any significant loss of AhR and ER activating chemicals when samples were extracted with SPE.
Regarding the AR assay, neither here could we observe any loss of activating chemicals when samples were extracted with SPE. In fact, the BEQ for extracted water samples was
significantly higher than the BEQ for samples with concentrated cell culture media. It has been shown that natural organic matter (NOM) and dissolved organic carbon (DOC), which is
