Elsevier

Chemosphere

Volume 165, December 2016, Pages 529-538
Chemosphere

Combined cyto/genotoxic activity of a selected antineoplastic drug mixture in human circulating blood cells

https://doi.org/10.1016/j.chemosphere.2016.09.058Get rights and content

Highlights

  • An antineoplastic drug mixture was tested in human blood cells.

  • The mixture induced a time- and dose-dependent cyto/genotoxic effect.

  • Antineoplastic drug combinations can pose risks to human health.

  • Further toxicological screening of antineoplastic drug mixtures are warranted.

Abstract

Antineoplastic drugs are highly cytotoxic chemotherapeutic agents that can often interfere directly or indirectly with the cell's genome. In an environmental or medical setting simultaneous exposure may occur. Such multiple exposures may pose a higher risk than it could be assumed from the studies evaluating the effect of a single substance. Therefore, in the present study we tested the combined cyto/genotoxicity of a mixture of selected antineoplastic drugs with different mechanisms of action (5-fluorouracil, etoposide, and imatinib mesylate) towards human lymphocytes in vitro. The results suggest that the selected antineoplastic drug mixture is potentially cyto/genotoxic and that it can induce cell and genome damage even at low concentrations. Moreover, the changes in the measured oxidative stress parameters suggest the participation of reactive oxygen species in the cyto/genotoxicity of the selected mixture. The obtained results indicate not only that such mixtures may pose a risk to cell and genome integrity, but also that single compound toxicity data are not sufficient for the predicting toxicity in a complex environment. Altogether, the results emphasise the need for further toxicological screening of antineoplastic drug mixtures, especially at low environmentally relevant concentrations, as to avoid any possible adverse effects on the environment and human health.

Introduction

Antineoplastic drugs have been widely used for chemotherapy. However, many of them have been categorised as persistent carcinogenic, mutagenic and teratogenic compounds, triggering widespread concerns about their occupational exposure and ecotoxicological risks (Besse et al., 2012, Toolaram et al., 2014, Villarini et al., 2016, Zhang et al., 2013). The occurrence of antineoplastic drugs residues in the environment is, compared to many other pharmaceuticals, much lower. Their mechanisms of action (MoA) mainly target the prevention of growth and division of tumour cells via interference with the genetic material of the cells. However, antineoplastic drugs may not be selective enough to target only tumour cells but can also act on other types of dividing cells and tissues of exposed organisms (Besse et al., 2012, Deblonde and Hartemann, 2013, Kosjek and Heath, 2011, Toolaram et al., 2014, Zhang et al., 2013, Zounkova et al., 2010).

Antineoplastic drugs, their metabolites and transformation products (TPs) are detected worldwide in the aquatic environment and they usually occur at ng/L to μg/L levels or even below (Kosjek and Heath, 2011, Toolaram et al., 2014, Zhang et al., 2013). In fact, they have been unrestrictedly discharged into the environment not only through industry and sewage treatment plants but also from hospital effluents. The knowledge regarding their effects on humans and wild life once they have entered the environment is rather limited (Besse et al., 2012, Deblonde and Hartemann, 2013, Kosjek and Heath, 2011, Toolaram et al., 2014, Zhang et al., 2013, Zounkova et al., 2010). Antineoplastic drugs are designed to have specific biological interferences on targeted tissues in human bodies; therefore, many of them have been determined or suspected for their harmful effects even at low environmental levels (Zhang et al., 2013). Hence, the continuous life-long exposure to antineoplastic drugs, their metabolites and TPs including their possible additive and/or synergistic effects may put the ecosystem and humans to further threat if such exposure lingers.

There is a large number of antineoplastic drugs currently on the market with different MoA and their adverse effects have been well established in various test systems (Brezovšek et al., 2014, Gajski et al., 2016, Gerić et al., 2014, Novak et al., 2016, Parrella et al., 2015, Toolaram et al., 2014, Zounkova et al., 2010). However, the effects of low environmentally relevant concentrations of these drugs are still a matter of debate. The genotoxic effects of antineoplastic drugs in non-tumour cells are of special significance due to the possibility that they may induce genetic alterations in normal cells and/or secondary tumours in cancer patients. Antineoplastic drugs can interact with DNA directly or indirectly causing DNA damage and/or inhibiting DNA synthesis, as well as affecting mitosis and inhibiting cell proliferation (Toolaram et al., 2014). These actions can involve the unspecific inhibition of normal cells thus presenting a danger not only to humans but also to different environmental organisms. Besides, with increasing life expectancy and standard of living on a world scale, it has to be expected that the contribution of antineoplastic drugs to the environment will increase further.

Therefore, the aim of the present study was to investigate a possible combined cyto/genotoxic and oxidative potential of three antineoplastic drugs in a mixture; 5-fluorouracil (5-FU), etoposide (ET) and imatinibe mesylate (IM) (Table 1) in an experimental model with human peripheral blood lymphocytes (HPBLs) in vitro. Specific antineoplastic drugs were selected by the fact that they are among the most consumed anticancer drugs and based on their different MoA′.

5-FU is a pyrimidine analogue that belongs to the group of antimetabolites and is among the most consumed anticancer drugs (Besse et al., 2012, Longley et al., 2003, Zhang et al., 2008). It interferes with nucleoside metabolism and can be incorporated into RNA and DNA, leading to cytotoxicity and cell death (Noordhuis et al., 2004, Thomas and Zalcberg, 1998). Besides, treatment of cells with 5-FU leads to an accumulation of cells in S-phase and has been shown to induce p53 dependent apoptosis (Longley et al., 2003, Zhang et al., 2008).

ET is a topoisomerase inhibitor that causes an increase in DNA and chromosomal damage and cell death. It is used to treat a wide spectrum of human cancers and is often used in combination with other antineoplastic drugs (Hande, 1998, Meresse et al., 2004, Valkov and Sullivan, 2003). The primary cytotoxic target for ET is topoisomerase II, which regulates DNA under- and over-winding, and removes knots and tangles from the genome by generating transient double-stranded breaks (Baldwin and Osheroff, 2005).

IM is a protein kinase inhibitor developed for targeted chemotherapy. IM selectively inhibits the Bcr-Abl tyrosine kinase, and is used as the first therapeutic choice against chronic myelogenous leukaemia (Al-Hadiya et al., 2014, Hartmann et al., 2009, Moen et al., 2007). It also inhibits some other tyrosine kinase activities, which indicates its potential use for the treatment of other cancers (Palmberg et al., 2009). The consumption of protein kinase inhibitors, including IM, is increasing rapidly and is at present one of the most consumed anticancer drugs (Besse et al., 2012).

In order to evaluate the induction of DNA strand breaks and genomic instability, the comet assay (Azqueta and Collins, 2013) and its formamidopyrimidine-DNA glycosylase (Fpg)-modification for the detection of oxidative DNA damage (Collins, 2014), cytokinesis-block micronucleus (CBMN) assay (Fenech et al., 2011), and sister chromatid exchange (SCE) assay (Wilson and Thompson, 2007) were used. Moreover, the participation of oxidative stress in the cyto/genotoxicity of the selected mixture was assayed by measuring malondialdehyde (MDA) level as a measure of lipid peroxidation (LPO) (Del Rio et al., 2005) and protein carbonyls’ (PC) level as a measure of oxidised proteins (OXP) (Augustyniak et al., 2015). Overall, the study provides new knowledge about the impact of antineoplastic drug mixtures on non-target, human blood cells, which is necessary for future human and environmental risk assessment.

Section snippets

Chemicals

Chromosome kit P was from Euroclone (Milano, Italy); 5-bromodeoxyuridine (BrdU), 5-fluorouracil (5-FU; MW 130.08 g/mol; CAS 51-21-8), acridine-orange (AO), bleomycin, colchicine, cytohalasin-B, ethidium-bromide (EtBr), histopaque, 1,1,3,3-tetramethoxy propane (TMP), thiobarbituric acid (TBA), low melting point agarose (LMP), mitomycin C and normal melting point (NMP) agarose were from Sigma (St Louis, USA); etoposide (ET; MW 588.57 g/mol; CAS 33419-42-0) and imatinib mesylate (IM; MW

Cytotoxicity and proliferation kinetics

The viability of HPBLs exposed to the antineoplastic drug mixture was significantly (P < 0.05) affected as determined by vital staining using AO/EtBr. A significant decrease was noticed at ≥0.01 and ≥ 0.00001 μg/mL after 4 and 24 h, respectively. At the applied concentration range, the reduction of NDI, which reflects the cytostatic effect of drugs, was significant at ≥ 1 μg/mL after both exposure times. Moreover, the reduction of PRI was also observed and was significant at ≥ 0.01 μg/mL after

Discussion

The occurrence of antineoplastic drugs, their metabolites and TPs in the environment is of great concern, especially as these drugs may induce adverse effects on the environment and human health. They are released into the aquatic environment mainly via sewage treatment plants but can also be excreted by patients after drug administration. Furthermore, if unused they can often be improperly disposed in drains (Besse et al., 2012, Deblonde and Hartemann, 2013, Kosjek and Heath, 2011, Toolaram

Conclusions

Based on our results, the tested antineoplastic drugs proved to be cyto/genotoxic in vitro towards the selected cells even at low environmentally relevant concentrations. This opens a question of the negative impact of these pharmaceuticals on wild life if discharged excessively in the aquatic environment since the target of anticancer agents is nuclear DNA and the effects are not restricted to cancerous cells only. Given the nature of antineoplastic drugs, that is, to kill or inhibit cell

Conflict of interests

Authors declare no conflict of interest.

Acknowledgments

This work was supported by the Seventh Framework Programme FP7/2007-2013 under grant agreement No. 265264, the Ministry of Science, Education and Sports of the Republic of Croatia under the grant agreement No. 022-0222148-2125, and the Program of bilateral collaboration between Croatia and Slovenia (Grant Nos. 533-19-14-0003 (Croatia) and BI-HR/14-15-004 (Slovenia)).

References (76)

  • M. Fenech et al.

    Measurement of micronuclei in lymphocytes

    Mutat. Res.

    (1985)
  • Z. Gačić et al.

    The impact of in vivo and in vitro exposure to base analogue 5-FU on the level of DNA damage in haemocytes of freshwater mussels Unio pictorum and Unio tumidus

    Environ. Pollut.

    (2014)
  • G. Gajski et al.

    In vitro effect of the antimalarial drug proguanil hydrochloride on viability and DNA damage in human peripheral blood lymphocytes

    Environ. Toxicol. Pharmacol.

    (2010)
  • G. Gajski et al.

    Cytogenetic status and oxidative DNA-damage induced by atorvastatin in human peripheral blood lymphocytes: standard and Fpg-modified comet assay

    Toxicol. Appl. Pharmacol.

    (2008)
  • M. Gerić et al.

    γ-H2AX as a biomarker for DNA double-strand breaks in ecotoxicology

    Ecotoxicol. Environ. Saf.

    (2014)
  • K.R. Hande

    Etoposide: four decades of development of a topoisomerase II inhibitor

    Eur. J. Cancer

    (1998)
  • B.D. Kevadiya et al.

    Layered inorganic nanocomposites: a promising carrier for 5-fluorouracil (5-FU)

    Eur. J. Pharm. Biopharm.

    (2012)
  • M. Kirsch-Volders et al.

    Report from the in vitro micronucleus assay working group

    Mutat. Res.

    (2003)
  • T. Kosjek et al.

    Occurrence, fate and determination of cytostatic pharmaceuticals in the environment

    Trac. - Trends Anal. Chem.

    (2011)
  • R. Kovács et al.

    Assessment of toxicity and genotoxicity of low doses of 5-fluorouracil in zebrafish (Danio rerio) two-generation study

    Water Res.

    (2015)
  • L. Lamberti et al.

    Cell kinetics and sister-chromatid-exchange frequency in human lymphocytes

    Mutat. Res.

    (1983)
  • P. Lebailly et al.

    Assessment of DNA damage induced in vitro by etoposide and two fungicides (carbendazim and chlorothalonil) in human lymphocytes with the comet assay

    Mutat. Res.

    (1997)
  • P. Noordhuis et al.

    5-Fluorouracil incorporation into RNA and DNA in relation to thymidylate synthase inhibition of human colorectal cancers

    Ann. Oncol.

    (2004)
  • A. Padjas et al.

    Cytogenetic damage in lymphocytes of patients undergoing therapy for small cell lung cancer and ovarian carcinoma

    Toxicol. Appl. Pharmacol.

    (2005)
  • A. Parrella et al.

    Eco-genotoxicity of six anticancer drugs using comet assay in daphnids

    J. Hazard. Mater

    (2015)
  • G. Ribas et al.

    Sister-chromatid exchanges (SCE) induction by inhibitors of DNA topoisomerases in cultured human lymphocytes

    Mutat. Res.

    (1996)
  • P. Sánchez-Suárez et al.

    DNA damage in peripheral blood lymphocytes in patients during combined chemotherapy for breast cancer

    Mutat. Res.

    (2008)
  • N.P. Singh et al.

    A simple technique for quantitation of low levels of DNA damage in individual cells. Exp

    Cell Res.

    (1988)
  • A.P. Toolaram et al.

    Environmental risk assessment of anti-cancer drugs and their transformation products: a focus on their genotoxicity characterization-state of knowledge and short comings

    Mutat. Res. - Rev. Mutat. Res.

    (2014)
  • J.D. Tucker et al.

    Sister-chromatid exchange: second report of the Gene-Tox Program

    Mutat. Res.

    (1993)
  • N.I. Valkov et al.

    Tumor p53 status and response to topoisomerase II inhibitors

    Drug resist. Updat

    (2003)
  • D.M. Wilson et al.

    Molecular mechanisms of sister-chromatid exchange

    Mutat. Res.

    (2007)
  • J. Zhang et al.

    Removal of cytostatic drugs from aquatic environment: a review

    Sci. Total Environ.

    (2013)
  • R. Zounkova et al.

    Ecotoxicity and genotoxicity assessment of cytotoxic antineoplastic drugs and their metabolites

    Chemosphere

    (2010)
  • G.R. Angstreich et al.

    Treatment options for chronic myeloid leukemia: imatinib versus interferon versus allogeneic transplant

    Curr. Opin. Oncol.

    (2004)
  • A. Azqueta et al.

    The essential comet assay: a comprehensive guide to measuring DNA damage and repair

    Arch. Toxicol.

    (2013)
  • S.D. Baker

    Pharmacology of fluorinated pyrimidines: eniluracil

    Invest. New Drugs

    (2000)
  • E.L. Baldwin et al.

    Etoposide, topoisomerase II and cancer

    Curr. Med. Chem. Anticancer. Agents

    (2005)
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