Cytotoxicity evaluation of ketoprofen found in pharmaceutical wastewater on HEK 293 cell growth and metabolism

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Highlights

  • Ketoprofen is a nonsteroidal anti-inflammatory drug commonly used in medicine.

  • Inefficient ketoprofen removal could risk human health and environments.

  • HEK293 cells treated with ketoprofen < 30 μg/L showed insignificant cytotoxic effects.

  • However, the treated cells did manifest the behaviour of cancer cells.

  • Upregulation of COX-1 expression among the treated cells might indicate inflammation.

Abstract

Inefficient ketoprofen removal from pharmaceutical wastewater may negatively impact the ecosystem and cause detrimental risks to human health. This study was conducted to determine the cytotoxicity effects of ketoprofen on HEK 293 cell growth and metabolism, including cyclooxygenase-1 (COX-1) expression, at environmentally relevant concentrations. The cytotoxic effects were evaluated through the trypan blue test, DNS assay, MTT assay, and the expression ratio of the COX-1 gene. The results of this study show insignificant (p > 0.05) cytotoxic effects of ketoprofen on cell viability and cell metabolism. However, high glucose consumption rates among the treated cells cause an imitation of the Warburg effect, which is likely linked to the development of cancer cells. Apart from that, the upregulation of COX-1 expression among the treated cells indicates remote possibility of inflammation. Although no significant cytotoxic effects of ketoprofen were detected throughout this study, the effects of prolonged exposure of residual ketoprofen need to be evaluated in the future.

Graphical abstract

Ketoprofen treatments on HEK 293 cells at environmentally relevant concentrations caused cytotoxicity effects as listed on the right side of the figure.

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Introduction

The increasing release of pharmaceutical wastewater into the environment has caused concern among communities worldwide since those discharging activities have been found to have negative impacts on the ecosystem and will become toxic above a certain concentration (Ekpeghere et al., 2016). Pharmaceutical wastewater mainly originates from pharmaceutical processing plants and through residual pharmaceuticals produced by consumers (Gadipelly et al., 2014). A study conducted by Chen et al. (2016) showed the dominant presence of ibuprofen, caffeine, paracetamol, diclofenac, metoprolol, furosemide, ketoprofen, triclosan, and tramadol in the wastewater treatment area, with 100 % detection.

To address this problem, researchers are studying approaches to treat wastewater containing pharmaceutical residues through constructed wetlands (Marsidi et al., 2016), advance ozonation processes (Ewadh et al., 2019, 2018a), coagulation, electrocoagulation (Haan et al., 2018), activated carbon adsorption, membrane separation, floatation, and sedimentation (Guo et al., 2017; Kaur et al., 2019). However, incomplete removal of pharmaceutical and personal care product wastes by wastewater treatment plants (WWTPs) and/or from unintended sources leads to the diffusion of these compounds into surface water (Bebianno and Gonzalez-Rey, 2015; Ewadh et al., 2017).

In a previous study, the presence of ketoprofen in effluents generated from WWTPs was recorded to be approximately 1 μg/L as a result of the inefficient removal of ketoprofen (5–70 %) (Cuklev et al., 2012). In surface water and sewage sludge, a higher concentration of ketoprofen (28–53 μg/L) was found (Narvaez and Jimenez, 2012). The occurrence of ketoprofen was also detected in tap water and drinking water at a concentration of 0.273 μg/L and 0.110 μg/L, respectively (Kermia et al., 2016).

Nonsteroidal anti-inflammatory drugs (NSAIDs) are common drugs used widely for medical purposes, with 90 % of consumers being adults that are more than 65 years of age (Badri et al., 2016). Ketoprofen, which belongs to the NSAID group because of its analgesic, anti-inflammatory, anti-rheumatic, and anti-pyretic properties, has been found to potentially cause cytotoxicity and genotoxicity at high concentrations of exposure (Hamdani et al., 2014). In the medical field, ketoprofen has been used to treat rheumatic diseases (Kennedy et al., 1994) and non-rheumatic diseases, such as dysmenorrhea (Cailleteau, 1988). Ketoprofen can reduce inflammation and fever and can heal severe pain through the inhibition of prostaglandin production by cyclooxygenase-2 (COX-2) (Sostres et al., 2010).

Prolonged uses of NSAIDs, especially ketoprofen, may cause gastrointestinal diseases due to non-specific inhibition of cyclooxygenase (COX), either COX-1 or COX-2 (Buttgereit et al., 2001). A naturally existing enzyme, COX-1 plays important roles throughout the body, mainly gastrointestinal protection and platelet production control, while COX-2 is known as an inducible enzyme that causes inflammation (Hawkey, 2001). Other than gastrointestinal diseases, the consumption of ketoprofen has also been reported to cause other side effects, such as central nervous system diseases, cardiovascular diseases, respiratory diseases, urogenital diseases, dermatologic diseases, and metabolic diseases (Schattenkirchner, 1991). The previous uses of ketoprofen in veterinary medicine also led to the extinction of Gyps scavengers and the death of eider ducks (Cuklev et al., 2012).

The cytotoxic effects of ketoprofen found in surface water originating from WWTPs have not been clearly determined. Currently, only a few studies of the cytotoxic effects of ketoprofen found in the environment have been performed (Cuklev et al., 2012; Ewadh et al., 2018b; Hamdani et al., 2014). A cytotoxic evaluation of ketoprofen at very low concentrations (1–100 μg/L) was conducted by Cuklev and co-workers (2012) using rainbow trout fish, but an evaluation on mammalian cells was only evaluated at concentrations of 25–75 μg/L (Ewadh et al., 2018b). Therefore, this study was conducted to evaluate the effect of ketoprofen on HEK 293 cell growth and metabolism, including COX-1 expression, at concentrations lower than 30 μg/L, which represent the relevant ketoprofen concentrations in the environment.

Section snippets

Cell culture maintenance

Human embryonic kidney cell lines (HEK 293) were purchased from American Type Culture Collection (ATCC). The cells were grown in Roswell Park Memorial Institute Medium (RPMI 1640) (Gibco, UK) supplemented with 5–10 % foetal bovine serum (Sigma Aldrich, USA), 100 U/mL penicillin (Gibco, UK), 100 μg/mL streptomycin (Gibco, UK), and 2.5 μg/mL amphotericin B (Gibco, UK). The cells were nurtured at 37 °C with the presence of 5% carbon dioxide (CO2). The medium growth was renewed twice a week. When

Results and discussion

According to Cuklev and co-workers (2012), ketoprofen treatment at low concentrations in a controlled system poses a lower risk to wild fish compared to other NSAIDs based on an approximately negligible bioconcentration factor (0.6 %). In terms of ketoprofen treatment on mammalian cells, Hamdani and co-workers (2014) reported a significant cytotoxic effect of ketoprofen on the Vero cell line at concentration above 1.5 g/L and insignificant cytotoxic effects in the concentration range of

Conclusion

The cytotoxicity of ketoprofen on HEK 293 cells at very low concentrations (<30 μg/L) is negligible; however, ketoprofen has not yet been declared safe. Distinguishable effects can still be observed between treated and untreated cells, which contributes to an ambiguous conclusion. The growth analysis showed insignificant (p > 0.05) reduction of cell proliferation and unpredictable cell viability throughout the 5-day ketoprofen treatment. The unexpected Warburg effect discovered in the metabolic

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CRediT authorship contribution statement

Nurul Nadiah Zulkarnain: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data curation, Writing - original draft, Writing - review & editing, Visualization. Nurina Anuar: Conceptualization, Methodology, Resources, Writing - review & editing, Supervision. Nor Azfa Johari: Methodology, Writing - review & editing, Supervision. Siti Rozaimah Sheikh Abdullah: Resources, Writing - review & editing, Supervision. Ahmad Razi Othman: Methodology, Validation, Writing - review &

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

Funding for this study was provided by Universiti Kebangsaan Malaysia (UKM) using university grant DCP-2017-010/1. Staff assistance from the Department of Chemical and Process Engineering, UKM, and Malaysia Genome Institute is appreciated and gratefully acknowledged.

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