The ER activation assay was not successful due to a high background activation of the assay. 28 Table 4-1: The % viability and standard deviations (mean viability ± standard . deviation) of the three highest concentrations of PVC leachates after 24, 48 and 96 hours of leaching at 4°C and 30°C using the MDA approx. 2 cell lines. 31 Table 4-2 The % viability and standard deviations (mean viability ± standard .deviation) of the three highest concentrations of PVC leachates after 24, 48 and 96 hours of leaching at 4°C and 30°C.
INTRODUCTION
Background
These compounds can include polyaromatic hydrocarbons (PAHs), metals and persistent organic pollutants (POPs) such as polychlorinated biphenyls (PCBs), and organochlorine pesticides such as dichlorodiphenyltrichloroethane (DDT) (Bouwmeester et al., 2015). Most of the research conducted on these contaminants considers their effects on the endocrine system as an individual compound (Fent et al., 2006). Essentially, activation or inhibition can be compared to known trigger values that indicate risks of these compounds to the environment or human health (Tang et al., 2013).
Aims and objectives
The luciferase gene is encoded downstream of the promoter, which is where the hormonal receptor will attach after binding to its ligand, enabling expression of the gene upon activation. Inhibition of the receptors can also be quantified when no activation of the receptor occurs due to a lack of luciferase being produced (Wilson, 2002). Use a colorimetric MTT assay to determine the cytotoxic effects of the leachate on a mammalian colon cancer cell line, HuTu-80.
LITERATURE REVIEW
- Plastic
- Sources of plastic
- Types of plastic
- Polyvinyl chloride
- Microplastics
- Sources of environmental microplastics
- Leaching and leachates
- PVC-specific additives
- Desorption
- Effects of plastic on the environment and organisms
- Physical effects
- Chemical effects
- Endocrine disruption
- Health risks associated with endocrine disruption
- Detection of EDCs
- Reporter gene assays
- Trigger values
- Cytotoxicity
- Conclusion
All plastics can be found in the environment in MP form (De Haan et al., 2019). Andrady (2011) summarizes the classifications of degradation according to its cause: i) biodegradation is caused by living organisms; ii) photodegradation by light; iii) hydrolysis by reaction with water; iv) thermo-oxidative degradation (a slow oxidative degradation at moderate temperatures); v) thermal degradation), even if it is not an environmental degradation mechanism, is caused by high temperatures) (Blettler et al., 2019). These include polyaromatic hydrocarbons (PAHs) and persistent organic pollutants (POPs), such as polychlorinated biphenyls (PCBs), and organochlorine pesticides such as dichlorodiphenyltrichloroethane (DDT) (Bouwmeester et al., 2015).
METHODS AND MATERIALS
- Leaching of virgin and recycled PVC
- Maintenance of cells
- MDA-kb2 cell line
- T47D-KBluc cell line
- HuTu-80 cell line
- General maintenance
- Preparation of exposure concentrations
- Leachates
- Reference compounds
- Reporter gene assays
- AR activation assay using MDA-kb2 cell line
- GR activation assay using MDA-kb2 cell line
- AR inhibition assay using MDA-kb2 cell line
- ER activation assay using T47D-KBluc cell line
- ER inhibition assay using T47D-KBluc cell line
- Reading the plates
- Viability assay (MTT)
- Data processing and statistical analysis
Similarly, heat sterilization of leachate could alter the chemical integrity of the leachate. After the initial incubation, 2.5 µL of extracts and dexamethasone as a positive control were added to the wells with cells. In seeding the AR inhibition assay, cells were treated with 0.283 ng/mL testosterone to activate a small fraction of receptors before exposure to leachate.
The plates were incubated for 48 hours, after which the inner wells were dosed with 2.5 µl of the leachates and flutamide as a positive control. The cell-containing wells were dosed with 2.5 µl of the leachates and E2 as a positive control before being incubated for 24 hours. After the final incubation of each of the previous assays, the plates were washed with DPBS containing Mg 2+ and Ca 2+ .
The optical density within the well is directly proportional to the viability of the cells. The measured absorbance is directly proportional to cell viability (Ulukaya et al., 2008). The results of the viability assay were exported to Microsoft Excel to determine cell viability after exposure to the compounds.
The %viability was determined for each well based on the average of the SC, which is considered 100% viable.
RESULTS
AR inhibition
- Cytotoxicity
- Reporter gene assay
The leachates at 96 hours appear to be the least cytotoxic over the time variant with the least number of viabilities below 80%. An example of this is PVC B leached for 48 hours at both 4°C and 30°C, where the light produced by the highest concentrations is much lower than that of the other concentrations (Figure 4-3) because the light that is produced by the corresponding concentrations the highest concentrations decreased due to the decrease in cell number. The MDA-kb2 cell line, with endogenous ARs, was used to determine the inhibition potential of the leachates.
A dose-response curve was produced in which the highest concentrations of flutamide produced the least amount of light. The inhibition caused by the lye fluids was compared to the maximum amount of light produced by the flutamide (%Flu max) from the standard curve (Figure 4-1). The lack of inhibition is evident from the lack of dose-response curves of the compounds, even if some of the values are below 100%.
However, there is no dose response, indicating that the observed responses are not due to AR inhibition. The most obvious is PVC A (Figure 4-2), which was leached for 96 hours at 30 °C, as its highest concentration produced light that was measured at 158% of the maximum flutamide. However, this is inconsistent with the activation data, so activation cannot be assumed for any leachate.
AR activation
The cytotoxicity evident from the highest concentrations of several of the samples, for example PVC E (Figure 4-13), where %Tmax decreased at the highest concentrations of leachates, is consistent with the cytotoxicity data for this cell line (Table 4-1) ) . The GR activation assay is performed only when it is necessary to clarify which receptors were activated.
ER inhibition
- Cytotoxicity
- Reporter gene assay
The T47D-KBluc cells were used, and the assay was performed under the ER inhibition conditions. Moderate cytotoxicity (40-80%) is indicated by orange and non-cytotoxic responses (>80%) are recorded in green. The most cytotoxic leachate was PVC A leached for 24 h at 4°C, which measured only 56.03% viability.
However, this is inconsistent with a dose response, as the lowest concentration has the lowest viability. In addition, there is no evidence of cytotoxicity in the reporter gene assay (Figure 4-18), which is usually shown by a downward curve at higher concentrations. The T47D-KBluc cell line was used to determine the ability of the leachates to inhibit ER.
The anti-estrogen ICI was used as a positive control, providing an effective dose-response curve (Figure 4-15) against which the leaching response was compared. However, there was no dose-responsive inhibition of the ER by either leachate (Figures 4-16-4-21). Due to the consistent amount of light produced across the concentrations, dose-related inhibition cannot be inferred.
ER activation
Cytotoxicity as an endpoint
An example of this is PVC B–F leached for 24 hours, where lower concentrations generally represent the highest persistence (Figure 4-23). During the preparation of the liquid dilutions, a visible “non-aqueous” layer was seen at the highest concentration (4 g/mL) of some of the liquids. An interesting phenomenon is that there are higher concentrations that appear to be less cytotoxic than lower ones.
The leachate from PVC A is generally the least cytotoxic, with no concentrations causing a viability <60% (Figure 4-23). Furthermore, the leachate from the PVC leached at 96 hours appears to be less cytotoxic compared to that leached at 24 and 48 hours (Figure 4-23). All PVCs leached at 30°C show similar dose-response effects to those leached at 4°C, with the most prominent dose responses evident in PVC B–F leached for 24 h (Figure 4- 24).
At a leaching temperature of 30°C, the leachates after 24 hours are the most cytotoxic, and those at 96 hours are the least cytotoxic.
Summary of results
DISCUSSION
Reporter gene assays
- Effects on the AR
- Anti-androgenic effects
- Androgenic effects
- Effects on the ER
- Anti-oestrogenic effects
- Oestrogenic effects
- Binding competition of mixtures
There was no measurable inhibition of the ER, which was evident by the lack of dose responses. However, unlike the responses of the AR, the leachate exposed to the ER produced less light than the positive control in a few cases. This study was unable to effectively determine the ER-activating effects of the leachate due to too high a background of estrogen activation existing in the cells.
There are a number of mechanisms of action that lead to target gene expression, particularly regarding the ER. However, there was still too much background activation to use the assay for definitive results. The inability to complete the ER activation assay is unfortunate, as many of the compounds thought to wash out are known ER agonists.
Chemical analysis of EDCs leached from marine microplastics revealed that ER agonists were present in the highest concentrations. Most EDC risk assessment methods focus on the effects of individual chemicals. However, the interaction of the ingredients within the mixture can have a much larger or smaller effect than expected.
Theoretically, a mixture of chemicals in leachate can interfere with any of the mechanisms mentioned above.
Cytotoxicity
These tests may involve the use of different cell lines with different sensitivities and receptors. However, further tests should also include whole-organism models, as there is currently a lack of sufficient knowledge to close the gap between in vitro and in vivo methods. The ability of phthalates to migrate to the surface of the plastic probably increases the leaching potential (Schyns . & Shaver, 2020).
However, there is still a need to establish a meaningful link between laboratory results and organismal health (Prinsloo et al., 2013). However, the HuTu-80 cell line is used as a good indication of the uptake of compounds in the digestive tract (Botha et al., 2019). It can be assumed that exposure to these leachates may affect the health of the digestive system.
After ingestion, MPs will travel through the organism's digestive tract, which has a significantly higher pH due to stomach acid.
Factors influencing leaching
- Plastic size
- Time
- Temperature
- Concentration
- pH
Smaller sized MPs had a detection frequency of 59% for EDCs, while medium and large plastics had 34% and 50%, respectively (Chen et al., 2019). The leaching times of this study were chosen based on the knowledge that leaching reaches equilibrium after 24 hours under isotropic conditions (Chen et al., 2019). Fluctuations in water temperature occur naturally due to seasonal changes and the speed of the water body's flow.
The water in the bottles incubated at the higher temperature appeared to have more EDCs (Aneck-Hahn et al., 2018). It is important to distinguish between the concentration of the MPs in the EtOH leached and the concentration of the compounds within the leachates. It was beyond the scope of this study to determine the concentrations of specific compounds within the leachate.
However, the reported concentrations are those of the amount of PVC leached into the EtOH. Trying to determine the exact concentration of microplastics within a body of water is not easy and also fell outside the scope of the study. Acidic and alkaline conditions are more harmful to MPs than neutral pHs (Mortula et al., 2021).
However, mixtures of compounds are known to react differently than individual compounds due to additive, synergistic and antagonistic effects on the receptor (Christen et al., 2012; Hansen et al., 2019).
CONCLUSION AND RECOMMENDATIONS
ADDENDUM
