Hazard characterization of Alternaria toxins to identify data gaps and improve risk assessment for human health.

Fungi of the genus Alternaria are ubiquitous plant pathogens and saprophytes which are able to grow under varying temperature and moisture conditions as well as on a large range of substrates. A spectrum of structurally diverse secondary metabolites with toxic potential has been identified, but occurrence and relative proportion of the different metabolites in complex mixtures depend on strain, substrate, and growth conditions. This review compiles the available knowledge on hazard identification and characterization of Alternaria toxins. Alternariol (AOH), its monomethylether AME and the perylene quinones altertoxin I (ATX-I), ATX-II, ATX-III, alterperylenol (ALP), and stemphyltoxin III (STTX-III) showed in vitro genotoxic and mutagenic properties. Of all identified Alternaria toxins, the epoxide-bearing analogs ATX-II, ATX-III, and STTX-III show the highest cytotoxic, genotoxic, and mutagenic potential in vitro. Under hormone-sensitive conditions, AOH and AME act as moderate xenoestrogens, but in silico modeling predicts further Alternaria toxins as potential estrogenic factors. Recent studies indicate also an immunosuppressive role of AOH and ATX-II; however, no data are available for the majority of Alternaria toxins. Overall, hazard characterization of Alternaria toxins focused, so far, primarily on the commercially available dibenzo-α-pyrones AOH and AME and tenuazonic acid (TeA). Limited data sets are available for altersetin (ALS), altenuene (ALT), and tentoxin (TEN). The occurrence and toxicological relevance of perylene quinone-based Alternaria toxins still remain to be fully elucidated. We identified data gaps on hazard identification and characterization crucial to improve risk assessment of Alternaria mycotoxins for consumers and occupationally exposed workers.

Adverse Toxic Effects of Tyrosine Kinase Inhibitors on Non-Target Zebrafish Liver (ZFL) Cells.

Over the past 20 years, numerous tyrosine kinase inhibitors (TKIs) have been introduced for targeted therapy of various types of malignancies. Due to frequent and increasing use, leading to eventual excretion with body fluids, their residues have been found in hospital and household wastewaters as well as surface water. However, the effects of TKI residues in the environment on aquatic organisms are poorly described. In the present study, we investigated the cytotoxic and genotoxic effects of five selected TKIs, namely erlotinib (ERL), dasatinib (DAS), nilotinib (NIL), regorafenib (REG), and sorafenib (SOR), using the in vitro zebrafish liver cell (ZFL) model. Cytotoxicity was determined using the MTS assay and propidium iodide (PI) live/dead staining by flow cytometry. DAS, SOR, and REG decreased ZFL cell viability dose- and time-dependently, with DAS being the most cytotoxic TKI studied. ERL and NIL did not affect viability at concentrations up to their maximum solubility; however, NIL was the only TKI that significantly decreased the proportion of PI negative cells as determined by the flow cytometry. Cell cycle progression analyses showed that DAS, ERL, REG, and SOR caused the cell cycle arrest of ZFL cells in the G0/G1 phase, with a concomitant decrease of cells in the S-phase fraction. No data could be obtained for NIL due to severe DNA fragmentation. The genotoxic activity of the investigated TKIs was evaluated using comet and cytokinesis block micronucleus (CBMN) assays. The dose-dependent induction of DNA single strand breaks was induced by NIL (≥2 µM), DAS (≥0.006 µM), and REG (≥0.8 µM), with DAS being the most potent. None of the TKIs studied induced micronuclei formation. These results suggest that normal non-target fish liver cells are sensitive to the TKIs studied in a concentration range similar to those previously reported for human cancer cell lines. Although the TKI concentrations that induced adverse effects in exposed ZFL cells are several orders of magnitude higher than those currently expected in the aquatic environment, the observed DNA damage and cell cycle effects suggest that residues of TKIs in the environment may pose a hazard to non-intentionally exposed organisms living in environments contaminated with TKIs.

Characterisation and toxicological activity of three different Pseudo-nitzschia species from the northern Adriatic Sea (Croatia).

Diatoms of the genus Pseudo-nitzschia are cosmopolitans spread in seas and oceans worldwide, with more than 50 described species, dozens of which have been confirmed to produce domoic acid (DA). Here, we characterized and investigated the toxicological activity of secondary metabolites excreted into the growth media of different Pseudo-nitzschia species sampled at various locations in the northern Adriatic Sea (Croatia) using human blood cells under in vitro conditions. The results revealed that three investigated species of the genus Pseudo-nitzschia were capable of producing DA indicating their toxic potential. Moreover, toxicological data suggested all three Pseudo-nitzschia species can excrete toxic secondary metabolites into the surrounding media in addition to the intracellular pools of DA, raising concerns regarding their toxicity and environmental impact. In addition, all three Pseudo-nitzchia species triggered oxidative stress, one of the mechanisms of action likely responsible for the DNA damage observed in human blood cells. In line with the above stated, our results are of great interest to environmental toxicologists, the public and policy makers, especially in light of today's climate change, which favours harmful algal blooms and the growth of DA producers with a presumed negative impact on the public health of coastal residents.

Evaluation of potential toxicity of Steriplant©N aerosolization toward human alveolar cells A459 in vitro.

Protection of patients against hospital-acquired infections is of major importance. Disinfection of magnetic resonance imaging suites is, due to their unique properties and environment particularly, difficult to implement. We developed an OPTI-JET CS MD 2ZE aerosolizator for disinfection of a magnetic resonance imaging suite using the electrolyzed oxidizing water biocide Steriplant©N. The disinfection of the magnetic resonance imaging suite with this system reduced from the number of colony formed unit/m3 air by 87% and 96% in 6 and 15 min of disinfection, respectively. It is well known that exposure of personnel or patients to aerosols may represent risk to the respiratory system; therefore, the aim of this study was to assess potential cytotoxicity and genotoxicity of Steriplant©N aerosolization toward human alveolar cells A459 in vitro. The A459 cells were exposed to aerosol containing different concentrations (50% and 100% v/v) of Steripalnt©N for 6 min in a chamber that had been constructed to simulate the conditions in the magnetic resonance imaging suite. The cytotoxicity was evaluated by measuring iodide uptake, and the genotoxicity was determined by measuring formation of phosphorylated H2AX histones, a marker for deoxyribonucleic acid double-strand breaks, immediately after the aerosolization and after 1, 4, and 24 h postincubation. The results demonstrated that aerosolization with Steriplant©N at conditions reflecting aerosolization in a magnetic resonance imaging suite is not cytotoxic and does not exhibit genotoxic potential in vitro.

Chemoprotective Effects of Xanthohumol against the Carcinogenic Mycotoxin Aflatoxin B1.

The present study addresses the chemoprotective effects of xanthohumol (XN), a prenylated flavonoid found in the female inflorescences (hops) of the plant Humulus lupulus L., against the carcinogenic food contaminant aflatoxin B1 (AFB1). The chemical reactions of XN and its derivatives (isoxanthohumol (IXN), 8-prenylnaringenin (8-PN), and 6-prenylnaringenin (6-PN)) with the AFB1 metabolite, aflatoxin B1 exo-8,9-epoxide (AFBO), were investigated in silico, by calculating activation free energies (ΔG‡) at the Hartree-Fock level of theory in combination with the 6-311++G(d,p) basis set and two implicit solvation models. The chemoprotective effects of XN were investigated in vitro in the metabolically competent HepG2 cell line, analyzing its influence on AFB1-induced cytotoxicity using the MTS assay, genotoxicity using the comet and γH2AX assays, and cell cycle modulation using flow cytometry. Our results show that the ΔG‡ required for the reactions of XN and its derivatives with AFBO are comparable to the ΔG‡ required for the reaction of AFBO with guanine, indicating that XN, IXN, 8-PN, and 6-PN could act as scavengers of AFBO, preventing DNA adduct formation and DNA damage induction. This was also reflected in the results from the in vitro experiments, where a reduction in AFB1-induced cytotoxicity and DNA single-strand and double-strand breaks was observed in cells exposed to combinations of AFB1 and XN, highlighting the chemoprotective effects of this phytochemical.

3D Pharmacophore-Based Discovery of Novel KV10.1 Inhibitors with Antiproliferative Activity.

(1) Background: The voltage-gated potassium channel KV10.1 (Eag1) is considered a near- universal tumour marker and represents a promising new target for the discovery of novel anticancer drugs. (2) Methods: We utilized the ligand-based drug discovery methodology using 3D pharmacophore modelling and medicinal chemistry approaches to prepare a novel structural class of KV10.1 inhibitors. Whole-cell patch clamp experiments were used to investigate potency, selectivity, kinetics and mode of inhibition. Anticancer activity was determined using 2D and 3D cell-based models. (3) Results: The virtual screening hit compound ZVS-08 discovered by 3D pharmacophore modelling exhibited an IC50 value of 3.70 µM against KV10.1 and inhibited the channel in a voltage-dependent manner consistent with the action of a gating modifier. Structural optimization resulted in the most potent KV10.1 inhibitor of the series with an IC50 value of 740 nM, which was potent on the MCF-7 cell line expressing high KV10.1 levels and low hERG levels, induced significant apoptosis in tumour spheroids of Colo-357 cells and was not mutagenic. (4) Conclusions: Computational ligand-based drug design methods can be successful in the discovery of new potent KV10.1 inhibitors. The main problem in the field of KV10.1 inhibitors remains selectivity against the hERG channel, which needs to be addressed in the future also with target-based drug design methods.

Lethal and Sub-Lethal Effects and Modulation of Gene Expression Induced by T Kinase Inhibitors in Zebrafish (Danio Rerio) Embryos.

Tyrosine kinase inhibitors (TKIs) are designed for targeted cancer therapy. The consumption of these drugs during the last 20 years has been constantly rising. In the zebrafish (Danio rerio) embryo toxicity test, we assessed the toxicity of six TKIs: imatinib mesylate, erlotinib, nilotinib, dasatinib, sorafenib and regorafenib. Imatinib mesylate and dasatinib induced lethal effects, while regorafenib, sorfenib and dasatinib caused a significant increase of sub-lethal effects, predominantly oedema, no blood circulation and formation of blood aggregates. The analyses of the changes in the expression of selected genes associated with the hormone system after the exposure to imatinib mesylate, dasatinib and regorafenib demonstrated that all three tested TKIs deregulated the expression of oestrogen receptor esr1, cytochrome P450 aromatase (cypa19b) and hydroxysteroid-dehydrogenase (hsd3b), regorafenib, and also thyroglobulin (tg). The expression of genes involved in the DNA damage response (gadd45 and mcm6) and apoptosis (bcl2) was deregulated only by exposure to regorafenib. The data indicate that common mechanisms, namely antiangiogenic activity and interference with steroidogenesis are involved in the TKI induced sub-lethal effects and potential hormone disrupting activity, respectively. The residues of TKIs may represent an environmental hazard; therefore, further ecotoxicological studies focusing also on the effects of their mixtures are warranted.

Deregulation of whole-transcriptome gene expression in zebrafish (Danio rerio) after chronic exposure to low doses of imatinib mesylate in a complete life cycle study.

Imatinib mesylate (IM) is an anticancer drug that belongs to tyrosine kinase inhibitors. We report the results of the first investigation of the chronic exposure of zebrafish (Danio rerio) to IM. The exposure to IM (0.01, 1 and 100 µg/L) was initiated in adult fish and continued through hatching and the offspring generation for seven months. In addition to standard toxicological endpoints, induction of genotoxic effects and whole-genome transcriptome of liver samples of offspring generation of zebrafish were analysed. Exposure to IM did not affect the survival and growth of zebrafish, did not cause any histopathological changes, but it induced a marginal increase in the chromosomal damage in blood cells. The whole-genome transcriptome analyses demonstrated dose-dependent increase in the number of differentially expressed genes with a significantly higher number of deregulated genes in female fish compared to male. Differentially expressed genes included genes involved in response to DNA damage, cell cycle control and regulation of circadian rhythm. Based on the low genotoxic activity and the pattern of the changes in DNA damage responsive genes we consider that at current environmental exposure levels, IM represents low risk for genotoxic effects in aquatic organisms. Exposure to IM also induced deregulation of the expression of genes associated with steroidogenesis and hormone metabolism and function, which indicates hormone-disrupting activity of IM that has not been studied so far. The study provide new information on the potential consequences of chronic exposure to the residues of tyrosine kinase inhibitors, which remain to be further explored.

Minimum Information for Reporting on the Comet Assay (MIRCA): recommendations for describing comet assay procedures and results.

The comet assay is a widely used test for the detection of DNA damage and repair activity. However, there are interlaboratory differences in reported levels of baseline and induced damage in the same experimental systems. These differences may be attributed to protocol differences, although it is difficult to identify the relevant conditions because detailed comet assay procedures are not always published. Here, we present a Consensus Statement for the Minimum Information for Reporting Comet Assay (MIRCA) providing recommendations for describing comet assay conditions and results. These recommendations differentiate between 'desirable' and 'essential' information: 'essential' information refers to the precise details that are necessary to assess the quality of the experimental work, whereas 'desirable' information relates to technical issues that might be encountered when repeating the experiments. Adherence to MIRCA recommendations should ensure that comet assay results can be easily interpreted and independently verified by other researchers.

The comet assay in animal models: From bugs to whales - (Part 2 Vertebrates).

The comet assay has become one of the methods of choice for the evaluation and measurement of DNA damage. It is sensitive, quick to perform and relatively affordable for the evaluation of DNA damage and repair at the level of individual cells. The comet assay can be applied to virtually any cell type derived from different organs and tissues. Even though the comet assay is predominantly used on human cells, the application of the assay for the evaluation of DNA damage in yeast, plant and animal cells is also quite high, especially in terms of biomonitoring. The present extensive overview on the usage of the comet assay in animal models will cover both terrestrial and water environments. The first part of the review was focused on studies describing the comet assay applied in invertebrates. The second part of the review, (Part 2) will discuss the application of the comet assay in vertebrates covering cyclostomata, fishes, amphibians, reptiles, birds and mammals, in addition to chordates that are regarded as a transitional form towards vertebrates. Besides numerous vertebrate species, the assay is also performed on a range of cells, which includes blood, liver, kidney, brain, gill, bone marrow and sperm cells. These cells are readily used for the evaluation of a wide spectrum of genotoxic agents both in vitro and in vivo. Moreover, the use of vertebrate models and their role in environmental biomonitoring will also be discussed as well as the comparison of the use of the comet assay in vertebrate and human models in line with ethical principles. Although the comet assay in vertebrates is most commonly used in laboratory animals such as mice, rats and lately zebrafish, this paper will only briefly review its use regarding laboratory animal models and rather give special emphasis to the increasing usage of the assay in domestic and wildlife animals as well as in various ecotoxicological studies.

The comet assay in animal models: From bugs to whales - (Part 1 Invertebrates).

The comet assay, also called single cell gel electrophoresis, is a sensitive, rapid and low-cost technique for quantifying and analysing DNA damage and repair at the level of individual cells. The assay itself can be applied on virtually any cell type derived from different organs and tissues of eukaryotic organisms. Although it is mainly used on human cells, the assay has applications also in the evaluation of DNA damage in yeast, plant and animal cells. Therefore, the purpose of this review is to give an extensive overview on the usage of the comet assay in animal models from invertebrates to vertebrates, covering both terrestrial and water biota. The comet assay is used in a variety of invertebrate species since they are regarded as interesting subjects in ecotoxicological research due to their significance in ecosystems. Hence, the first part of the review (Part 1) will discuss the application of the comet assay in invertebrates covering protozoans, platyhelminthes, planarians, cnidarians, molluscs, annelids, arthropods and echinoderms. Besides a large number of animal species, the assay is also performed on a variety of cells, which includes haemolymph, gills, digestive gland, sperm and embryo cells. The mentioned cells have been used for the evaluation of a broad spectrum of genotoxic agents both in vitro and in vivo. Moreover, the use of invertebrate models and their role from an ecotoxicological point of view will also be discussed as well as the comparison of the use of the comet assay in invertebrate and human models. Since the comet assay is still developing, its increasing potential in assessing DNA damage in animal models is crucial especially in the field of ecotoxicology and biomonitoring at the level of different species, not only humans.

Dose-Modifying Factor of Radiation Therapy with Concurrent Cisplatin Treatment in HPV-Positive Squamous Cell Carcinoma: A Preclinical Study.

Human papillomavirus (HPV) is an important etiological factor in oropharyngeal squamous cell carcinoma (SCC). Compared to HPV-negative tumors, HPV-positive oropharyngeal SCC has shown a better response to nonsurgical treatments. In this study, we determined the dose-modifying factors for HPV-positive tumors with single-dose irradiation, with or without low radiosensitizing doses of cisplatin. In vitro, we determined an increased radiosensitivity of HPV-positive SCC, which might be a consequence of HPV-induced changes in the cell cycle regulation and DNA damage response, leading to increased cell death. Additionally, compared to HPV-negative tumors, 30% higher radiosensitivity of HPV-positive tumors was determined by tumor growth delay monitoring in immunodeficient mice in vivo. Concurrent cisplatin treatment had an additive effect in both HPV-negative and HPV-positive tumors, resulting in 20% better response in HPV-positive tumors than in HPV-negative tumors.

Cytotoxicity and genotoxicity of anticancer drug residues and their mixtures in experimental model with zebrafish liver cells.

Anticancer drugs enter aquatic environment predominantly via hospital and municipal wastewater effluents where they may, due to their genotoxic potential, cause adverse environmental effects even at very low doses. In this study we evaluated cytotoxic and genotoxic potential of two widely used anticancer drugs, cyclophosphamide (CP) and ifosfamide (IF) as individual compounds and in a complex mixture together with 5-fluorouracil (5-FU) and cisplatin (CDDP) because these four drugs have been frequently detected in an oncological ward effluents. As an experimental model we used zebrafish liver cell (ZFL) line. The cytotoxicity was determined with the MTS assay and genotoxicity with the comet assay and cytokinesis block micronucleus (CBMN) assay that measure the formation of DNA strand breaks and genomic instability, respectively. CP and IF exerted low cytotoxicity towards ZFL cells. Both compounds induced DNA strand breaks and genomic instability, however at relatively high concentrations that are not relevant for the contamination of aquatic environment. The mixture of CP, IF, 5-FU and CDDP was tested at maximal detected concentrations of each drug as determined in the effluents from the oncological ward. The mixture was not cytotoxic and did not induce genomic instability, but it induced significant increase in the formation of DNA strand breaks at concentrations of individual compounds that were several orders of magnitude lower from those that were effective when tested as individual compounds. The results indicate that such mixtures of anticancer drugs may pose a threat to aquatic organisms at environmentally relevant concentrations and contribute to the accumulating evidence that it is not always possible to predict adverse effects of complex mixtures based on the toxicological data for individual compounds.

Assessment of the genotoxicity of the tyrosine kinase inhibitor imatinib mesylate in cultured fish and human cells.

The selective tyrosine kinase inhibitor imatinib mesylate (IM) is a widely used anticancer drug. Recent studies showing that IM can induce DNA and chromosomal damage in crustaceans and higher plants prompted us to re-examine its potential genotoxicity. IM was not mutagenic in the Ames assay (Salmonella typhimurium). Cytotoxicity and genotoxicity were evaluated in vitro in zebrafish (Danio rerio) liver (ZFL), human hepatoma (HepG2), and human peripheral blood lymphocyte (HPBL) cells. Genotoxicity was determined with the comet assay and with the cytokinesis-block micronucleus cytome assay. ZFL and HPBL cells showed comparable sensitivity to IM cytotoxicity, while HepG2 cells were less sensitive. At non-cytotoxic concentrations, IM induced DNA strand breaks in ZFL and HepG2 cells. An increase in the number of micronuclei was observed in ZFL and HPBL cells. In HPBLs, IM also induced an increase in the number of nucleoplasmic bridges and nuclear buds. Based on the data of the consumption of IM in European countries the predicted environmental concentrations (PEC) were calculated to be in the range between 3.3 and 5.0ng/L, which are several orders of magnitude lower from those that caused adverse effects in fish and human derived cells. However, based on the in vitro studies it is not possible to quantitatively predict the hazard for wildlife and humans, therefore further studies are warranted to explore the underlying molecular mechanisms of induced IM genotoxic effects as well as the studies of the occurrence of IM in the aquatic and occupational environment to establish the relevance of these observations for aquatic organisms and occupationally exposed personnel.

Chemical and toxicological characterisation of anticancer drugs in hospital and municipal wastewaters from Slovenia and Spain.

Anticancer drugs are continuously released into hospital and urban wastewaters, where they, most commonly, undergo conventional treatment in wastewater treatment plants (WWTPs). Wastewaters contain complex mixtures of substances including parent compounds, their metabolites and transformation products (TPs). In this study, samples of hospital effluents and WWTP influents and effluents from Slovenia and Spain were analyzed for twenty-two selected anticancer drugs, their metabolites and transformation products. Acute and chronic toxicity tests were performed on the crustacean Ceriodaphnia dubia, genotoxicity was determined with Tradescantia and Allium cepa micronucleus (MN) assays and in vitro comet assay in zebrafish (Danio rerio) liver cell line (ZFL cells). Sixty of the two hundred-twenty determinations revealed detectable levels of anticancer drug residues. Among the targeted compounds, platinum based were most frequently detected (90%). Furthermore, erlotinib was detected in 80%, cyclophosphamide and tamoxifen in 70% and methotrexate in 60% of the samples. Seven of ten samples were toxic to C. dubia after acute exposure, whereas after chronic exposure all samples reduced reproduction of C. dubia at high sample dilutions. Allium cepa proved insensitive to the potential genotoxicity of the tested samples, while in Tradescantia increased MN frequencies were induced by a hospital effluent and WWTP influents. In ZFL comet assay all but one sample induced a significant increase of DNA strand breaks. Correlations of chemotherapeutics or their TPs were detected for all bioassays except for Allium cepa genotoxicity test, however for each test the highest correlations were found for different substances indicating differential sensitivities of the test organisms.

Assessment of the mutagenic and genotoxic activity of cyanobacterial toxin beta-N-methyl-amino-L-alanine in Salmonella typhimurium.

A neurotoxin ß-N-methylamino-L-alanine (L-BMAA) is a non-protein amino acid produced by most cyanobacteria ubiquitously present in aquatic and terrestrial environments. Due to its global presence in surface waters, a widespread human exposure is possible and therefore this toxin represents a health risk for humans and animals. L-BMAA has been linked to the development of a variety of neurodegenerative diseases. Its neurotoxic activity has been extensively studied, while nothing is known on its genotoxic properties. In the present study we evaluated for the first time L-BMAA mutagenic potential using Ames assay on several Salmonella typhimurium strains (TA97a, TA98, TA100, TA102 and TA1535). The results showed that the toxin (up to 0.9 mg/plate) did not induce mutations without or with S9 metabolic activation. Its genotoxic activity was further studied with the SOS/umuC assay on S. typhimurium TA1535/pSK1002 and the results showed that it was not cytotoxic nor genotoxic for bacteria. The present study represents the first evidence that L-BMAA is not mutagenic nor genotoxic for bacteria even at concentrations much higher than those typically found in the environment. However, as most of the cyanobacterial toxins are not bacterial mutagens it is very important to further elucidate its genotoxic activity in eukaryotic cells.

Ecotoxicity and genotoxicity of cyclophosphamide, ifosfamide, their metabolites/transformation products and their mixtures.

Cyclophosphamide (CP) and ifosfamide (IF) are commonly used cytostatic drugs that repress cell division by interaction with DNA. The present study investigates the ecotoxicity and genotoxicity of CP, IF, their human metabolites/transformation products (TPs) carboxy-cyclophosphamide (CPCOOH), keto-cyclophosphamide (ketoCP) and N-dechloroethyl-cyclophosphamide (NdCP) as individual compounds and as mixture. The two parent compounds (CP and IF), at concentrations up to 320 mg L(-1), were non-toxic towards the alga Pseudokirchneriella subcapitata and cyanobacterium Synecococcus leopoliensis. Further ecotoxicity studies of metabolites/TPs and a mixture of parent compounds and metabolites/TPs performed in cyanobacteria S. leopoliensis, showed that only CPCOOH (EC50 = 17.1 mg L(-1)) was toxic. The measured toxicity (EC50 = 11.5 mg L(-1)) of the mixture was lower from the toxicity predicted by concentration addition model (EC50 = 21.1 mg L(-1)) indicating potentiating effects of the CPCOOH toxicity. The SOS/umuC assay with Salmonella typhimurium revealed genotoxic activity of CP, CPCOOH and the mixture in the presence of S9 metabolic activation. Only CPCOOH was genotoxic also in the absence of metabolic activation indicating that this compound is a direct acting genotoxin. This finding is of particular importance as in the environment such compounds can directly affect DNA of non-target organisms and also explains toxicity of CPCOOH against cyanobacteria S. leopoliensis. The degradation study with UV irradiation of samples containing CP and IF showed efficient degradation of both compounds and remained non-toxic towards S. leopoliensis, suggesting that no stable TPs with adverse effects were formed. To our knowledge, this is the first study describing the ecotoxicity and genotoxicity of the commonly used cytostatics CP and IF, their known metabolites/TPs and their mixture. The results indicate the importance of toxicological evaluation and monitoring of drug metabolites as they may be for certain aquatic species more hazardous than parent compounds.

Genotoxic potential of selected cytostatic drugs in human and zebrafish cells.

Due to their increasing use, the residues of anti-neoplastic drugs have become emerging pollutants in aquatic environments. Most of them directly or indirectly interfere with the cell's genome, which classifies them into a group of particularly dangerous compounds. The aim of the present study was to conduct a comparative in vitro toxicological characterisation of three commonly used cytostatics with different mechanisms of action (5-fluorouracil [5-FU], cisplatin [CDDP] and etoposide [ET]) towards zebrafish liver (ZFL) cell line, human hepatoma (HepG2) cells and human peripheral blood lymphocytes (HPBLs). Cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and acridine orange/ethidium bromide staining. All three drugs induced time- and dose-dependent decreases in cell viability. The sensitivity of ZFL and HepG2 cells towards the cytotoxicity of 5-FU was comparable (half maximal inhibitory concentration (IC50) 5.3 to 10.4 µg/mL). ZFL cells were more sensitive towards ET- (IC50 0.4 µg/mL) and HepG2 towards CDDP- (IC50 1.4 µg/mL) induced cytotoxicity. Genotoxicity was determined by comet assay and cytokinesis block micronucleus (CBMN) assay. ZFL cells were the most sensitive, and HPBLs were the least sensitive. In ZFL cells, induction of DNA strand breaks was a more sensitive genotoxicity endpoint than micronuclei (MNi) induction; the lowest effective concentration (LOEC) for DNA strand break induction was 0.001 µg/mL for ET, 0.01 µg/mL for 5-FU and 0.1 µg/mL for CDDP. In HepG2 cells, MNi induction was a more sensitive genotoxicity endpoint. The LOEC values were 0.01 µg/mL for ET, 0.1 µg/mL for 5-FU and 1 µg/mL for CDDP. The higher sensitivity of ZFL cells to cytostatic drugs raises the question of the impact of such compounds in aquatic ecosystem. Since little is known on the effect of such drugs on aquatic organisms, our results demonstrate that ZFL cells provide a relevant and sensitive tool to screen genotoxic potential of environmental pollutant in the frame of hazard assessment.

Influence of selected anti-cancer drugs on the induction of DNA double-strand breaks and changes in gene expression in human hepatoma HepG2 cells.

In chemotherapy, various anti-cancer drugs with different mechanisms of action are used and may represent different risk of undesirable delayed side effects in treated patients as well as in occupationally exposed populations. The aim of the present study was to evaluate genotoxic potential of four widely used anti-cancer drugs with different mechanisms of action: 5-fluorouracil (5-FU), cisplatin (CDDP) and etoposide (ET) that cause cell death by targeting DNA function and imatinib mesylate (IM) that inhibits targeted protein kinases in cancer cells in an experimental model with human hepatoma HepG2 cells. After 24 h of exposure all four anti-cancer drugs at non-cytotoxic concentrations induced significant increase in formation of DNA double strand breaks (DSBs), with IM being the least effective. The analysis of the changes in the expression of genes involved in the response to DNA damage (CDKN1A, GADD45A, MDM2), apoptosis (BAX, BCL2) and oncogenesis (MYC, JUN) showed that 5-FU, CDDP and ET upregulated the genes involved in DNA damage response, while the anti-apoptotic gene BCL2 and oncogene MYC were downregulated. On the contrary, IM did not change the mRNA level of the studied genes, showing different mechanism of action that probably does not involve direct interaction with DNA processing. Genotoxic effects of the tested anti-cancer drugs were observed at their therapeutic concentrations that may consequently lead to increased risk for development of delayed adverse effects in patients. In addition, considering the genotoxic mechanism of action of 5-FU, CDDP and ET an increased risk can also not be excluded in occupationally exposed populations. The results also indicate that exposure to 5-FU, CDDP and ET represent a higher risk for delayed effects such as cancer, reproductive effects and heritable disease than exposure to IM.

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

Residues of anti-neoplastic drugs represent new and emerging pollutants in aquatic environments. Many of these drugs are genotoxic, and it has been postulated that they can cause adverse effects in aquatic ecosystems. 5-Fluorouracil (5-FU) is one of the most extensively used anti-neoplastic drugs in cancer therapy, and this article describes the results of the first investigation using a two-generation toxicity study design with zebrafish (Danio rerio). Exposure of zebrafish to 5-FU (0.01, 1.0 and 100 µg/L) was initiated with adult zebrafish (F0 generation) and continued through the hatchings and adults of the F1 generation, and the hatchings of the F2 generation, to day 33 post-fertilisation. The exposure did not affect survival, growth and reproduction of the zebrafish; however, histopathological changes were observed in the liver and kidney, along with genotoxic effects, at all 5-FU concentrations. Increases in DNA damage determined using the comet assay were significant in the liver and blood cells, but not in the gills and gonads. In erythrocytes, a significant, dose-dependent increase in frequency of micronuclei was observed at all 5-FU concentrations. Whole genome transcriptomic analysis of liver samples of F1 generation zebrafish exposed to 0.01 µg/L and 1 µg/L 5-FU revealed dose-dependent increases in the number of differentially expressed genes, including up-regulation of several DNA-damage-responsive genes and oncogenes (i.e., jun, myca). Although this chronic exposure to environmentally relevant concentrations of 5-FU did not affect the reproduction of the exposed zebrafish, it cannot be excluded that 5-FU can lead to degenerative changes, including cancers, which over long-term exposure of several generations might affect fish populations. The data from this study contribute to a better understanding of the potential consequences of chronic exposure of fish to low concentrations of anti-neoplastic drugs, and they demonstrate that further studies into multi-generation toxicity are needed.