In Vitro Cytotoxicity Study of Cyclophosphamide, Etoposide and Paclitaxel on Monocyte Macrophage Cell Line Raw 264.7

Abstract

The presence of antineoplastic compounds in aquatic ecosystem is an emerging challenge for the society. Antineoplastic compounds released into the aquatic environment exhibit a potential threat to normal aquatic life. Particularly, antineoplastic compounds are responsible for direct or indirect interference with the cellular DNA of an organism and cause toxicity to cells. The present study focused on the assessment of in vitro toxic effect of cyclophosphamide, etoposide and paclitaxel on Raw 264.7 cell line (mouse monocyte macrophage cells). The inhibitory concentration of cyclophosphamide, etoposide, and paclitaxel was determined. The IC₅₀ values of these compounds were 145.44, 5.40, and 69.76 µg ml⁻¹ respectively. This is the first report on toxicity analysis of cyclophosphamide, paclitaxel and etoposide on Raw 264.7 cell line by reducing cell viability and indicating the cell cytotoxicity i.e., 69.58% for cyclophosphamide, 92.01% for etoposide and 88.85% for paclitaxel on concentration 250 µg ml⁻¹. The results of their cytotoxicity assessment highlight the need of improvement in sewage treatment technology for the efficient removal of these compounds from aquatic environment.

Introduction

Worldwide, cancer is the second highest non-communicable disease after cardiovascular disease. The incidence of new cancer cases in year 2012 was 14.1 million and it becomes increases to 18.07 million in the year 2018 [1]. Consequently, this increment in cancer incidence leads to the demand, production and consumption of antineoplastic drugs [2–4]. Unexpectedly, through the oncology wards of hospitals, discharge of hospitalized patients, outpatients and due to lack of treatment facility in STPs (sewage treatment plant), antineoplastic compounds are persistently coming into water bodies. Several studies investigated the presence of these compounds in aquatic the environment and the occurrence is due to persistence or recalcitrant nature of antineoplastic drugs after going through treatment plants and remain dynamic after pass through wastewater treatment plant [5–11]. Antineoplastic drugs are non-specific in nature and have a property to kill or inhibit cell growth by blocking the cell cycle. So, due to their lack of specificity and negative interaction with cellular DNA, they are cytostatic and mutagenic for normal cells even present at very low concentrations in water bodies [12].

Researchers reported the toxicity (cytotoxicity, mutagenicity, and ecotoxicity) of antineoplastic compounds on different models in terms of EC₅₀ (effective concentration), LC₅₀ (median lethal dose), IC₅₀ (inhibitory concentration), LOEC (Lowest observed effect concentration), and NOEC (No observed effect concentration) [13–17]. But the effect of cyclophosphamide, etoposide and paclitaxel on the immune system of any organism is not elucidated yet. Every organism has a defence mechanism against pathogens and other toxic substances [18, 19]. The immune system has different specialized cells which protect the body from harmful substance. Among these cells, the macrophage is one of the most important immune cells which protect the body from invasion of harmful substance inside the body by playing a role in innate immunity [20, 21]. Cyclophosphamide, paclitaxel, and etoposide are the most widely used antineoplastic drugs by oncologists for the treatment of cancer patients [22, 23]. So, heavy use of these compounds, they are reaching into the aquatic ecosystem and their presence in an aquatic environment having a hazardous effect on human health.

This study aims to predict the cytotoxicity of three antineoplastic compounds i.e., cyclophosphamide, paclitaxel and etoposide on immune cells line Raw 264.7 (mouse macrophage). This will help to determine the toxic level of antineoplastic compounds and their impact on aquatic life if present in an open environment.

Materials and Methods

Cell line (Raw 264.7) was procured from NCCS Pune for cytotoxicity testing. Analytical grade chemicals were used in the growth and maintenance of cell line. Chemical like DMEM medium (AT183), Sodium bicarbonate (TC230M), Phenol red (I010), Foetal Bovine Serum (FBS) (RM10432) from, Trypan Blue (TC193), Penicillin–Streptomycin (A004), Antineoplastic drugs [Cyclophosphamide (RM8152), Paclitaxel (RM9750), Etoposide (E0675)], DMSO (TC185), MTT [3-4, 5-dimethylthiazol-2, 5 diphenyl tetrazolium bromide] (M2128) were purchased from Hi-media (Mumbai-India).

Preparation of Media and Stocks

Media Preparation

The medium was prepared using DMEM (1.0 g), sodium bicarbonate (0.37 g) and phenol red (1.5 mg) dissolve into 100 ml double distilled water. After mixing the medium component pH was adjusted to 7.2–7.4. 11 ml FBS was added to make 10% FBS containing medium, 1 ml penicillin–streptomycin solution into prepared media and filter the medium through filter assembly in sterile environment. After filtration, takes 1 ml medium and add it into a 5 ml LB (Luria broth) tube. The medium was kept at 4 °C and LB tube at 37 °C overnight for sterility check.

Preparation of Stock Solution

The stock solution of cyclophosphamide, etoposide and paclitaxel were prepared of 1000 µg l⁻¹ in absolute DMSO. After the preparation of stock, dilution of stock was made into 250, 200, 150, 100, 50, 50, 25, and 10 µg ml⁻¹ for each drug.

Cell Culture

After the sterility check of prepared medium, Raw 264.7 cells were grown in the prepared medium using a T-75 flask at 37 °C and 5% CO₂ in an incubator [24]. After the sufficient growth of adherent cells, takes out cells from a flask with the help of PBS in a sterile centrifuge tube and centrifuge at 2500 rpm for 5 min for pellet formation. Then, remove the supernatant and mix the pellet gently by adding 5 ml of fresh media. After mixing of cells, take out 100 µl cell suspension and 400 µl trypan blue. The cell suspension with trypan blue was used for cell count in the haemocytometer.

Cytotoxicity Assay

Primarily, 1 × 10⁴ of RAW 264.7 cells were seeded (200 µl/well) into 96 well plates. Keep 96 well plate in the incubator at 37 °C and 5% CO₂ for 24 h. After 24 h, take out the plate from the incubator and add 50 µl of drugs sample (concentration 250, 200, 150, 100, 50, 25, and 10 µg ml⁻¹) into wells of the plate. DMSO was used as solvent control, 5000 µg ml⁻¹ concentration of drugs was used as positive control and fresh media was used as a negative control. Each well in the plate was containing the final volume 250 µl. All control and test sample were added in triplicate into wells. After adding the sample, keep the plate at 37 °C with 5% CO₂ for 48 h for treatment.

After 48 h, remove the medium from each well, then added 200 µl fresh medium without FBS and 25 µl MTT (5 mg ml⁻¹) into each well. Then, put the plate at 37 °C with 5% CO₂ for four h. After four h of incubation, remove MTT containing media from each well and added 200 µl DMSO into each well and cover the plate with foil to avoid direct light and shake in the orbital shaker for 15 min at 37 °C. Finally, check the absorbance of the plate at 590 nm in the microtitre plate reader.

The following equation determined the cytotoxicity of antineoplastic compounds:

$$\%\text{of Cytotoxicity}=100-\left[\frac{\text{Absorbance}\left(\text{Sample}\right)}{\text{Absorbance}\left(\text{Control}\right)}\right]\times 100.$$

Statistical Analysis and IC₅₀ Determination

All experiments were repeated three times to minimize errors during experiments. The statistical analysis during this experiment was performed through estimating the relative potency of above antineoplastic compounds. The IC₅₀ (Inhibitory concentration) were analysed by the equation y = mx + c using MS-Excel software.

Results and Discussion

In this study, the MTT assay was performed by using Raw 264.7 cell line to evaluate the cytotoxicity of cyclophosphamide, etoposide and paclitaxel which are widely used for cancer treatment and showing presence in water bodies. The MTT assay was applied to detect [25] cell viability and the percentage of cytotoxicity on Raw 264.7 cells with different doses of cyclophosphamide, etoposide and paclitaxel i.e., 10, 25, 50, 100, 150, 200 and 250 µg.ml⁻¹(Fig. 1). After 48 h of antineoplastic compound treatment, the viability of cells was determined and the results of cytotoxicity for above mentioned antineoplastic compounds described in Table 1. The cytotoxicity percentage of cyclophosphamide on Raw 264.7 cell at concentration 10, 25, 50, 100, 150, 200 and 250 µg ml⁻¹ were 17.48%, 26.17%, 34.22%, 41.96%, 50.07%, 61.69% and 69.58% respectively. In case of etoposide the percentage of cytotoxicity on Raw 264.7 cell at an exposure concentration 10, 25, 50, 100, 150, 200 and 250 µg ml⁻¹ were 42.99%, 52.22%, 63.35%, 74.86%, 80.23%, 87.29% and 92.01% respectively. While, the cytotoxicity of paclitaxel on Raw 264.7 cell at concentration 10, 25, 50, 100, 150, 200 and 250 µg ml⁻¹ were 31.07%, 36.49%, 44.08%, 59.57%, 75.25%, 84.9% and 88.85% respectively. At 48 h of antineoplastic compounds treatment indicated the consistent decrease in cell viability of Raw 264.7 cells with an increase in the concentration of cyclophosphamide, etoposide and paclitaxel. These three antineoplastic compounds were significantly inhibiting proliferation in Raw 264.7 cell line by exhibiting cytotoxic effects. The IC₅₀ value for cyclophosphamide was 145.44 µg ml⁻¹, etoposide was 5.40 µg ml⁻¹ and paclitaxel was 69.76 µg ml⁻¹ on 10, 25, 50, 100, 150, 200 and 250 µg ml⁻¹ concentrations (Table 1). IC₅₀ of etoposide was the lowest as compared to cyclophosphamide and paclitaxel as indicated in Fig. 2 and Table 1. So, among these three antineoplastic compounds, etoposide indicating the highest cytotoxicity on Raw 264.7 cells because if the IC₅₀ value of the compound is lower, the compounds will be more cytotoxic and it was determined by designing a dose–response curve. The cytotoxic response of different concentrations of these three antineoplastic compounds on Raw 264.7 cell line is shown in Fig. 2a–c. The inhibitory concentration value showing half-maximal effective concentration obtained from the relative potency of selected antineoplastic compounds and the dose–response curve. In prior studies, acute and chronic toxicity of cyclophosphamide, etoposide and paclitaxel were analysed on Vibrio fischeri (V. fischeri), Brachionus calyciflorus (B. calyciflorus), Ceriodaphnia dubia (C. dubia), Danio rerio (D.rerio), Lemna minor (L. minor), Daphnia magna (D. magna) and Thamnocephalus platyurus (T. platyurus) (Table 2). The toxicity was determined in the form of EC₅₀/LC₅₀. The values of EC₅₀/LC₅₀ for cyclophosphamide on V. fischeri were > 100 µg.ml⁻¹ (acute) and 1396 µg ml⁻¹ (chronic), B. calyciflorus were 1924 µg ml⁻¹ (acute) and 89.84 µg ml⁻¹ (chronic), C. dubia were 986.6 µg ml⁻¹ (acute) and 58.03 µg ml⁻¹ (chronic), T. platyurus was 1396 µg ml⁻¹ (acute) and L. minor was> 100 µg ml⁻¹ (chronic) [13, 15, 17]. In etoposide, the value of toxicity on B. calyciflorus were > 120 µg ml⁻¹ (acute) and 3.7 µg ml⁻¹ (chronic), C. dubia was 0.204 µg ml⁻¹ (chronic), D. rerio was > 100 µg ml⁻¹ (acute), T. platyurus was 74.85 µg ml⁻¹ (acute) and D. magna was 0.239 µg ml⁻¹ (chronic) [26, 27]. In paclitaxel, toxicity was determined on D. magna was > 0.074 µg ml⁻¹ (acute) [28]. It shows that, the cytotoxic effect of cyclophosphamide and etoposide is lower as well as higher on Raw 264.7 cells as compared to previous studies but in the case of paclitaxel toxicity is lower as compared to prior studies. Researchers determined the acute and chronic toxicity of these antineoplastic compounds on different animal model and microorganism such as bacteria, algae etc. by estimating different parameters such as luminescence, growth inhibition mortality, immobilisation and reproduction inhibition is given in Table 2 but the cytotoxic effect of cyclophosphamide, etoposide and paclitaxel on Raw 264.7 cell is not yet analysed.

Fig. 1.

Schematic diagram of complete cytotoxicity assay of cyclophosphamide, etoposide and paclitaxel on Raw 264.7 cells

Table 1.

Determination of cytotoxicity and inhibitory concentration of cyclophosphamide, etoposide and paclitaxel

Antineoplastic compound	Concentration dose (µg ml−1)	% of cell cytotoxicity	IC50 (µg ml−1)
Cyclophosphamide	10	17.48	145.44
	25	26.17
	50	34.22
	100	41.96
	150	50.07
	200	62.69
	250	69.58
Paclitaxel	10	31.07	69.76
	25	36.49
	50	44.08
	100	59.57
	150	75.25
	200	84.9
	250	88.85
Etoposide	10	42.99	5.40
	25	52.22
	50	63.35
	100	74.86
	150	80.23
	200	87.29
	250	92.01

Fig. 2.

Dose-response curve of a cyclophosphamide, b etoposide and c paclitaxel

Table 2.

Toxicity data of cyclophosphamide, paclitaxel and etoposide on different organism [29]

Antineoplastic compounds	Toxicity type	Organism	Toxicity (EC50/LC50) µg ml−1	References
Cyclophosphamide	Acute	V. fischeri	> 100	[17]
		B. calyciflorus	1924 (1210–3036)	[13]
		C. dubia	986.6 (765.3–1272)	[13]
		D. magna	> 1000	[30]
			> 100	[17]
		T. platyurus	1396 (1304–1494)	[13]
	Chronic	V.fischeri	1396 (1304–1494	[15]
		S.leopoliensis	> 120	[14]
			> 320	[14]
			> 100	[17]
			> 200	[13]
		B.calyciflorus	89.84 (67.62–119.4)	[13]
		C.dubia	58.03 (37.43–89.98)	[13]
		D.magna	> 100	[31]
		L.minor	> 100	[17]
Paclitaxel	Acute	D.magna	> 0.074	[28]
Etoposide	Acute	B.calyciflorus	> 120	[32]
		C.dubia	16% at 120	[32]
		D.magna	25% at 120	[32]
			30 (16–40)	[30]
		T. platyurus	74.85 (56.36–99.40	[32]
		D.rerio	> 100	[27]
	Chronic	P.subcapitata	250 (120–460)	[30]
		B.calyciflorus	3.7 (2.7–5.3)	[32]
		C.dubia	0.204 (0.152–0.256)	[32]
		D.magna	0.239 (0.181–0.299)	[32]

In the above result, for cyclophosphamide the percentage of cell cytotoxicity was 17.48% minimum at an exposure concentration 10 µg ml⁻¹ and 69.58% maximum at 250 µg ml⁻¹, while the 50% of cell death occurred at 145.44 µg ml⁻¹. The linear regression value for cyclophosphamide was 0.9519 (Fig. 2 a). The etoposide was showing higher cell cytotoxicity that was 42.99% minimum at concentration 10 µg ml⁻¹ and 92.01% maximum at concentration 250 µg ml⁻¹, while the 50 percent cell death occurred at 5.40 µg ml⁻¹. The linear regression value for etoposide was 0.9185 (Fig. 2b). In present study, the cytotoxicity percentage of paclitaxel on Raw 264.7 cells was 31.07% minimum at concentration 10 µg ml⁻¹ and 88.85% maximum at concentration 250 µg ml⁻¹, while 50% of cell death after the treatment of paclitaxel was at concentration 69.76 µg ml⁻¹. The linear regression value for paclitaxel was 0.9717 (Fig. 2c). It reveals that etoposide is more toxic for Raw 264.7 cells as compare to cyclophosphamide and paclitaxel.

This is the first study in which MTT assay was used to detect cytotoxic effect of above antineoplastic compounds on the immune cell line. The test was based on the standard colorimetric assay by the estimation of cell proliferation or cell growth. The present study showed cyclophosphamide, etoposide and paclitaxel are cytotoxic for Raw 264.7 cells while their cytotoxicity is varying accordingly. The cytotoxic effect shown by antineoplastic compounds in MTT assay cause cell growth inhibition in Raw 264.7 cells which may be occur due to DNA strand breakage or topoisomerase activity blockage but the exact mechanism is unclear. So, the above finding indicates that these antineoplastic compounds considered as potential cytotoxic agent and cause damage of the immune system or body of an organism when come in contact with these compounds.

Conclusion

The study focused on toxic effect analysis of cyclophosphamide, etoposide, and paclitaxel on Raw 264.7 cell line (monocyte macrophage). MTT assay was performed to evaluate the in vitro cytotoxicity and the result indicates that these compounds put an adverse effect on mouse macrophage cell line. According to performed experiments all three drugs exhibiting cytotoxic effect on Raw 264.7 cells but etoposide will be classified as more toxic than paclitaxel and cyclophosphamide. It indicates towards a strong effort should be made to remove or degrade these compounds at source before reaching to the aquatic environment as they are harmful for the immune system of aquatic animal diversity. Further studies such as mutagenic, carcinogenic effects are required to help more understanding of the exposure and hazardous effect of these compounds on cell lines and organisms.

Authors’ contributions

All authors are equally contributed in this manuscript.

Funding

This work was financially supported by Department of Biotechnology (Ministry of Science and Technology), Govt. of India [Grant No: BT/IN/INNO-INDIGO/26/MKM/2015-16].

Compliance with ethical standards

Conflicts of 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.

Consent to participate

Not applicable.

Consent for publication

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