The application of the Comet assay in fish cell lines

Mutat Res Gen Tox En 842 (2019) 72–84

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The application of the Comet assay in fish cell lines ⁎

Bojana Žegura , Metka Filipič

T

Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Ljubljana, Slovenia

A R T I C LE I N FO

A B S T R A C T

Keywords: Fish cell lines Comet assay Rainbow trout cells Zebrafish cells

The use of fish models has been proven to be an effective and sensitive tool for the evaluation of genotoxicity of pure compounds and complex mixtures of chemicals in the context of environmental screening of pollutants and hazard assessment in aquatic toxicology. In particular, fish cell lines have been successfully introduced for detection of genotoxic effects and can serve as an alternative to animal testing in preliminary eco-/genotoxicological studies. For this purpose comet assay has been extensively used in fish cell lines for the evaluation of genotoxic potential of chemicals and complex environmental matrices. The most often used fish cell lines in the comet assay are RTG-2, RTgill-W1 and RTL-W1 derived from rainbow trout (Onchorhynchus mykiss) gonads, gills and liver, respectively, and ZFL and ZF4 cells established from zebrafish (Danio rerio) liver and embryos, respectively. The present review gives an overview of the most often-used permanent fish cell lines in genotoxicology and discusses their application in the comet assay.

1. Introduction Chemical pollution of aquatic ecosystems represents a threat to aquatic organisms as well as humans particularly when chemicals are present in complex mixtures due to the combined (synergistic, antagonistic, potentiating) effects that can occur between the compounds. As the adverse effects cannot be predicted based only on chemical analyses, bioassays represent important tools to accurately evaluate the effects of pollutants on aquatic organisms [1]. In environmental toxicology fish models in vivo are used for this purpose since they are the most diverse group of vertebrates found in the aquatic ecosystem [2]. In addition, fish can serve as laboratory models for studying toxicants of concern to human health. The results obtained on whole organisms can be effectively extrapolated to the natural environment as well as humans for the prediction of the adverse outcomes. However, due to a demand to reduce the use of animals for the experimental purposes (the “3Rs”initiative, standing for Refinement, Reduction and Replacement) to protect animal welfare [3], fish cell lines are of growing importance in eco/genotoxicology. They represent standardized experimental systems that are carried out in a controlled environment, giving fast, affordable and ethically eligible results. Cell cultures allow for the comparison between species for their relative sensitivity to environmental contaminants at the cellular level and to understand the mechanisms of action of contaminants [4]. They are considered as alternatives to animal use in aquatic toxicity testing and are proposed as in vitro-in vivo toxicity extrapolation tools. Therefore, fish cell cultures have gained

⁎

high application in toxicology for evaluating the effects of pure chemicals, including pesticides, metals, drugs, nanoparticles, complex environmental samples such as extracts from sediments, surface waters, wastewaters and many more. The first permanent fish cell line, RTG-2, was established from gonads of juvenile rainbow trout (Oncorhynchus mykiss) in 1962 and was used for studying fish viruses [5]. Since then development of fish cell lines mainly from freshwater fish is progressing. Lakra et al. [6] reported 283 cell lines derived from different tissues and various species from fish and the number continues to increase. Fish cell lines grow attached to the surface, and only a few have been adapted to grow in suspension [3]. They are cultivated over a wide temperature range, but due to the poikilothermic nature at lower temperatures than mammalian cells, usually below 30 °C. The cultivation temperatures are similar to the habitat temperature of the donor species. Even at optimal growth temperatures, fish cell lines grow more slowly than mammalian cells, which has to be taken into consideration in eco/genotoxicological studies. The fish cells are cultivated similarly to mammalian cells using the same growth media containing basal culture media supplemented with 5–15% mammalian sera. Detection of genotoxic effects induced by chemicals is dependent on the exposure and bioavailability of compounds, the uptake and metabolism, the intracellular concentration, the mode of toxic action and the balance between toxicity and protective cellular responses. In addition, the rates of DNA repair in fish cells appear to be lower relative to mammalian cells [7,8]. Therefore, when studying the effects of particular contaminants and/or biochemical

Corresponding author at: National Institute of Biology, Department of Genetic Toxicology and Cancer Biology, Večna pot 111, 1000 Ljubljana, Slovenia. E-mail addresses: [email protected] (B. Žegura), metka.fi[email protected] (M. Filipič).

https://doi.org/10.1016/j.mrgentox.2019.01.011 Received 14 August 2018; Received in revised form 18 January 2019; Accepted 21 January 2019 Available online 21 January 2019 1383-5718/ © 2019 Elsevier B.V. All rights reserved.

Mutat Res Gen Tox En 842 (2019) 72–84

B. Žegura, M. Filipič

DNA damage. These observations are in line with the results in mammalian cells [21], which makes RTG-2 cells as an useful model for studying oxidative stress-induced DNA damage. Modified comet assay with endonuclease III, which detects oxidised pyrimidines [16], has been applied in fish cells in a study on non-dioxin-like and low-affinity dioxin-like congeners of polychlorinated biphenyls (PCBs) as it is known that PCBs are linked with reactive oxygen species (ROS) production and they form metabolites through a CYP450-dependent mechanisms [24]. The results showed significant increase in oxidative damage induced by PCBs, which again confirms the suitability of RTG-2 cells for detection of indirect acting mutagens and compounds that increase the formation of ROS. Recently, RTG-2 cell line has been used for the assessment of genotoxicity of textile dyes that can be found in wastewaters from textile industry and consequently in aquatic environments. Alkaline and hOGG1-modified comet assay confirmed that studied textile dyes induced direct (DNA strand breaks) and indirect (oxidized bases) DNA damage [23]. Altogether, these studies indicate that the comet assay employing RTG-2 cells can be used as a cost-effective and reliable tool for genotoxicity assessments of direct and indirect acting genotoxic compounds found in aquatic environments. Another very often used fish cell line for genotoxicity assessment is RTgill-W1, an epithelial cell line isolated from rainbow trout gills [26]. RTgill-W1 cells have been validated as a model cell line for the evaluation of DNA damage by a comet assay induced by well known direct acting mutagens such as hydrogen peroxide (H2O2) [4,10,28,29], methyl methane sulfonate (MMS) [10,29] and indirect acting benzo(a) pyrene (BaP) [10] as well as UVC radiation [8]. The cells have been used to evaluate the genotoxic potential of heterocyclic synthetic compounds, benzotriazoles (BTRs) and their mixture tolytriazole (TT) [4,28], which can enter the environment through sewage and industrial wastewaters and can therefore affect aquatic organisms. The results revealed that in gill cells the studied compounds increased the formation of DNA damage but to a lower extend than in liver RTL-W1 cells (see below) [4,28]. In another study alkaline and formamidopyrimidine DNA-glycosylase-modified comet assay have been applied in RTgill-W1 cells for eco/genotoxicological study of complex environmental sediment samples containing polycyclic aromatic hydrocarbons (PAHs) and other contaminants such as polychlorinated biphenyls, organochlorine and organophosphate pesticides. The complex sediment extracts increased DNA damage as well as oxidative stress in RTgill-W1 cells [29], which confirms that this cell line represents a very important model for studying water-born toxicants, especially as the gill epithelia is the primary uptake site for contaminants into fish. Modified comet assay with Fpg on RTgill-W1 cells has been validated with model genotoxicants MMS, H2O2 [10,28] and BaP as well as environmental contaminants such as cadmium (Cd), the herbicide diuron and one of its metabolite, 3,4-dichloroaniline (3,4-DCA) [10]. When using the Fpgdigestion step, genotoxicity has been detected at lower concentrations compared to alkaline comet assay showing higher sensitivity of the modified assay and demonstrating measurable base-excision repair (BER) activity [10,29]. The RTgill-W1 cells showed weak biotransformation capacity (CYP450 1 A) when exposed to BaP that was however much lower compared to other cell lines (RTL-W1 and PLHC1) [10]. Further on, Kienzler et al. [8], have characterised RTgill-W1 cells for the presence of two repair processes for UV-induced photolesions, nucleotide excision repair (NER) and photoreactivation repair (PER), by means of an endonuclease T4-modified comet assay, which specifically allows the assessment of cyclobutane pyrimidine dimers (CPDs) repair. By using the UVC exposure as a model DNA damaging agent a clear UVC dose/DNA damage response and slow repair kinetics of cyclobutyl-pyrimidine dimers have been described in this fish cell line supporting the hypothesis that fish cells use both NER and PER to repair photo-damage, and that PER repairs damage more quickly than NER. By this the study has shown that RTgill-W1 cells have similar UV lesion repair profile as adult fish in vivo [8] and can be used as models to study DNA repair in fish cells and in genotoxicity assessment of

responses it is very important to select the most adequate cell line in terms of metabolic activity, type of cells related to the rout of exposure, resistance to selected compounds and similar. Several fish cell lines such as RTG-2, RTL-W1, RTH-149, PLHC-1, ZFL, BF2, FHM etc. have retained cytochrome P450-dependent monooxygenase activities and are able to metabolise indirect acting genotoxic compounds [9,10]. Alternatively, exogenous metabolic activation system (hepatic microsomal fraction S9) can be employed [11,12]. The application of fish cell lines in environmental toxicology has been reviewed mainly in relation to the cytotoxicity of chemicals [3]; however, genotoxicity is of great importance due to its relevance in chronic exposure situation and its possible delayed consequences at the population level. The cultured fish cells were used for the first time for genotoxicity assessment in 1979 when transformed cells derived from a primary culture of embryonic tissue of butterfly splitfin (Ameca splendens) were used for the evaluation of the induction of sister chromatid exchanges (SCEs) by carcinogens/ mutagens [13]. Since then a variety of different fish cell lines have been successfully introduced for detection of a large variety of genotoxic endpoints using different genotoxicity assays including the comet assay. The most often used cell lines in the comet assay are RTG-2, RTgill-W1 and RTL-W1 established from rainbow trout (Onchorhynchus mykiss), and ZFL and ZF4 from zebrafish (Danio rerio). Apart to these comet assay has been applied on many other fish cell lines (summarized in Tables 1 and 2). The comet assay is a sensitive, reliable, rapid and low-cost assay for detection of DNA single and double strand breaks, alkali-labile sites, and incomplete excision repair events at the level of individual cells. In principle, any cell type can be used for genotoxicity testing with the comet assay [14]. The sensitivity of the comet assay can be significantly increased by introducing an additional digestion step with specific restriction endonucleases that recognize one or several DNA lesions and convert unrepaired lesions to additional DNA strand breaks. For this purpose various enzymes such as endonuclease III (endoIII), formamido pyrimidine DNA glycosylase (fpg), hOGG1, T4 endonuclease V and Alk A have been successfully used [15,16]. The comet assay has the application in different fields ranging from genetic, environmental and occupational toxicology to biomonitoring and human epidemiology [17–19] using not only human but also animal models [19]. 2. Rainbow trout (Onchorhynchus mykiss) cell lines One of the most frequently used fish cell lines in aquatic toxicity is a fibroblast-like cell line, RTG-2 derived from rainbow trout gonads [5]. The cells contain cytochrome P450-dependent monooxygenase activity and aryl hydrocarbon receptors [20]. RTG-2 cells have been validated as a model cell line for the evaluation of DNA damage induced by nanoparticles [22,23], dyes [23], indirect acting genotoxic compounds such as polycyclic aromatic hydrocarbons [11,12,21,25], polychlorinated biphenyls [24], nitrofurantoin [11], 2-acetylaminofluorene [11], dimethylnitrosamine [11] and direct acting genotoxic compounds including hydrogen peroxide [22,24,26], 4-nitroquinoline-1-oxide [11,21], ethyl methane sulfonate [12], methyl methane sulfonate [23,24], N-methyl-N9-nitro-N-nitrosoguanidine [11], metals [25], physical stressor such as ultra violet radiation [21] as well as complex environmental samples [11,21]. The cells have also been proved to be a suitable model for genotoxicity screening of native un-concentrated surface water samples [11]. Moreover, RTG-2 cells displayed higher sensitivity than standard rodent Chinese hamster ovary-K1 (CHO-K1) cells towards direct acting genotoxicant, hydrogen peroxide, and two environmentally relevant forms of inorganic arsenicals, namely trivalent sodium arsenite (As3+) and pentavalent sodium arsenate (As5+). This might be due the differences in metabolic activity, lower activities in DNA repair and lower antioxidant enzymes, or the glutathione levels compared to rodent cells [25]. Vevers et al. [21], showed that DNA strand breaks induced by titanium dioxide nanoparticles alone or in combination with UVA irradiation were mediated through oxidative 73

Oncorhynchus mykiss (rainbow trout) Oncorhynchus mykiss (rainbow trout) Oncorhynchus mykiss (rainbow trout)

Oncorhynchus mykiss (rainbow trout)

Danio rerio (zebrafish)

Danio rerio (zebrafish)

Poeciliopsis lucida (topminnow) Paralichthys olivaceus (olive flounder)

Etroplus suratensis (green chromide)

Catla catla (Indian major carp)

Catla catla (Indian major carp)

Labeo rohita (roho labeo)

Channa striata (striped snakehead, murrel) Pimephales promelas (fathead minnow)

RTG-2

RTgill-W1

RTH-149

ZFL

ZF4

PLHC-1 FG

IEG

SICH

ICG

LRG

CSG

74 skin

gill

gill

gill

heart

gill

liver gill

embryo

liver

liver

liver

gill

gonad

Tissue

ATCC (CCL-2872) or ECACC (90102529)

A.S. Sahul Hameed, Tamilnadu, India

A.S. Sahul Hameed, Tamilnadu, India

A.S. Sahul Hameed, Tamilnadu, India

A.S. Sahul Hameed, Tamilnadu, India

A.S. Sahul Hameed, Tamilnadu, India

ATCC (CRL-2406) Hong-Zhi Miao, Qingdao, China

ATCC (CRL-2050)

ATCC (CRL-2634)

N. Bols and L. Lee, Waterloo, University, Canada ATCC (CRL-1710)

ATCC (CCL-2523)

ATCC (CCL-55) or ECACC (90102529)

Source/ Provider

FBS: fetal bovine serum; BCS: bovine calf serum; FCS: fetal calf serum. * At least two studies have been reported in the literature.

EPC

RTL-W1

Fish species

Cell line*

Table 1 The most often used fish cell lines for genotoxicity assessment with the comet assay.

Earle’s minimal essential medium supplemented with 10% FBS and 1% non-essential amino acids, in a humidified incubator with 5% CO2 at 19-22 °C. Leibovitz's L15 medium supplemented with 10% FBS, in a humidified incubator with atmospheric air at 20 °C. Leibovitz's L15 medium supplemented with 10% FBS, in a humidified incubator with atmospheric air at 20 °C. Eagle's minimum essential medium supplemented with 10% FBS, in a humidified incubator with 5% CO2 at 21 °C Leibovitz L-15 (50%), DMEM (35%), and Ham F-12 (15%), supplemented with 5% FBS, in a humidified incubator with atmospheric air at 28 °C. Dulbecco’s modified Eagle’s minimum-Ham’s F12 medium, supplemented with 20% FBS, in a humidified incubator with atmospheric air at 28 °C. Leibovitz's L15 medium supplemented with 10% FBS, in a humidified incubator with 5% CO2 at 30 °C. Earle’s minimal essential medium supplemented with 10% BCS, in a humidified incubator with 5% CO2 at 19-22 °C. Leibovitz's L15 medium supplemented with 10% FBS, in a humidified incubator with atmospheric air at 28 °C. Leibovitz's L15 medium supplemented with 10-15% FBS, in a humidified incubator with atmospheric air at 28 °C. Leibovitz's L15 medium supplemented with 10% FBS, in a humidified incubator with atmospheric air at 28 °C. Leibovitz's L15 medium supplemented with 10% FBS, in a humidified incubator with atmospheric air at 28 °C. Leibovitz's L15 medium supplemented with 10% FBS, in a humidified incubator with atmospheric air at 28 °C. Earle’s minimal essential medium supplemented with 10% FCS, in a humidified incubator with 5% CO2 at 25 °C.

Culture conditions

[72,73]

[67–71]

[67,69]

[66,69,70]

[66–69]

[65–67]

[10,56,57] [62–64]

[53–55]

[45–48,59–61]

[40,43]

[4,20,30,34–38,58]

[4,10,27–29,34]

[11,12,20–25]

Reference

B. Žegura, M. Filipič

Mutat Res Gen Tox En 842 (2019) 72–84

RTgill-W1; Oncorhynchus mykiss (rainbow trout) Gill cell line

RTG-2; Oncorhynchus mykiss (rainbow trout) Fibroblast-like from gonadal tissue derived cell line Radiosensitive cell line

Cell line/ Species

75

Polychlorinated biphenyls: 2 and 24 h -PCB153 (2,20,4,40,5,50-hexachlorobiphenyl; 30 μM) -PCB 138 (2,20,3,4,40,50-hexachlorobiphenyl; 70 μM) -PCB118 (2,30,4,40,5-pentachlorobiphenyl; 30 μM) -PCB 101 (2,20,40,5,50-pentachlorobiphenyl; 50 μM) Benzo-a-pyrene (B(a)P, 20 μM) Hydrogen peroxide (H2O2, 125 μM) Cadmium sulphide (CdS) quantum dots coated with methyl Polyethylene glycol (M-PEG) (0.01, 0.1, 1 μg mL−1; 24 h) Silver sulphide (Ag2S) nanoparticles coated with methyl polyethylene glycol (M-PEG) (0.01, 0.1 and 1 μg mL−1; 24 and 48 h) Methyl methane sulfonate (MMS; 0.5 mM) textile dye Direct Black 38 (DB38; azo dye) and textile dye Reactive Blue 15 (RB15; copper phthalocyanine dye; 32.25, 62.5, 125, 250, 500, 750, 1000 μg mL−1; 3 h) methyl methanesulfonate (MMS; 0.5 mM, 10 min) hydrogen peroxide (H2O2; 50 μM, 10 min μM; for hOGG1-modified alkaline) Organic solvent sediment extracts (containing polycyclic aromatic hydrocarbons, polychlorinated biphenyls, organochlorine and organophosphate pesticides) from Lagos lagoon, Nigeria; 7 mg and 35 mg eQsed/mL Benzothiazoles: Benzotriazol (BTR); 1H-benzotriazole (1H-BTR), 5chlorobenzotriazole (5CBTR), 1 hydroxybenzotriazole (1OHBTR), 5,6-dimethyl-1H-benzotriazole monohydrate (DM)); Tolytriazole

Hydrogen peroxide (H2O2; 1-100 μM) Trivalent sodium arsenite (As3+; 1-10 μM) Pentavalent sodium arsenate (As5+; 1-10 μM)

4-nitroquinoline-1-oxide (NQO; 6.5 nM - 210 nM) Benzo(a)pyrene (BaP; 0.94 – 50 μM) Leachate water samples from a gaswork remediation site containing xylene (17.7-20 mg L−1), benzene (0.5-1.1 mg L−1), ethylbenzene (0.88-1.4 mg L−1), 16 PAHs (6.4-12.1 μg L−1) 4-nitroquinoline-N-oxide (NQO; 13-210 nM) N-methyl-N9-nitro-N-nitrosoguanidine (MNNG; 0.034–1.08 μM) Benzo(a)pyrene (BaP; 0.93-50 μM; +S9 at 0.244 mg/mL) Nitrofurantoin (NF; 131.2 μM –1.049 mM + S9) 2-acetylaminofluorene (AAF; 0.0027–26.8697 μM; +S9) Dimethylnitrosamine (DMN; 0.1930 μM –134.989 mM; +S9) Non-concentrated environmental river surface water samples Benzo(a)pyrene (BaP; 0.1–10 μM; +/- S9 mixture) Ethyl methanesulfonate (EMS, 0.1–1 mM)

Titanium dioxide (TiO2) nanoparticles (5 and 50 μg mL−1; 24 h) + UVA (1.53 kJ m-2 (20 min) and 3 kJ m-2 (40 min)); cells exposed in PBS Hydrogen peroxide (H2O2; 5, 25 μM)

Agent/stressor (type, concentrations used, exposure time)

Table 2 Studies using the comet assay for the evaluation of DNA damage in permanent fish cell lines.

% tail DNA

Comet score (VS)

20 °C, air 24 h: 125 mg L−1 for 1H-BTR, 4MBTR (↑), 5MBTR (↑), TT (no ↑)); at 60 mg L−1 for 1OHBTR (no ↑), at 30 mg L−1 for 5CBTR (↑); at

% tail DNA

22 °C, air DB38 (3 h): ↑ DNA sb and hOOG1 sensitive sites at 125 μg mL−1 RB15 (3 h): : no ↑ DNA sb and hOOG1 sensitive sites; MMS (0.5 mM): ↑ DNA sb; H2O2 (50 μM): ↑ hOOG1 sensitive sites 18 °C, air 24 h; ↑ at 7 mg eQsed/mL

% tail DNA

22 °C, 5% CO2 CdS: ↑ DNA sb at ≥ 0.1 μg mL−1 (24 h), no ↑ DNA sb (48 h) Ag2S: no ↑ DNA sb (24 and 48 h)

[4]

[29]

[23]

[22]

[24]

[25]

[12]

[11]

[20]

[21]

Ref.

(continued on next page)

Alkaline comet assay

Alkaline and fpgmodified comet assay

Alkaline and hOGG1modified comet assay

Alkaline comet assay

Alkaline and endoIIImodified comet assay

TM

Alkaline comet assay

% tail DNA

22 °C, air BaP (3-day): ↑ DNA sb; BaP (3-day exposure, followed by 3- day recovery): no ↑ DNA sb EMS (3-day): ↑ DNA sb at 1 mM; EMS: (3-day exposure, followed by 3- day recovery) ↑ DNA sb at 1 mM 21 ± 1 °C; 5% CO2 H2O2: at ≥ 1 μM ↑ DNA sb As3+: 3.2 1 μM ↑ DNA sb As5+: 1 μM ↑ DNA sb 2 h: PCBs: 101, 118, 153↑ DNA sb 24 h: PCBs: 118, 138, 153↑ DNA sb and 101, 118, 153↑ DNA sb (endo III) B(a)P: ↑ DNA sb at 20 μM H2O2: ↑ DNA sb at 125 μM

Alkaline comet assay

Alkaline comet assay

% tail DNA, TL, TM

19 °C, air LOEC (2 h): 13 nM for NQO, 3.7 μM for BaP, 131.2 μM NF, 0.136 μM MNNG, no ↑ DNA sb for AAF and DMN Water samples: ↑ DNA sb

% tail DNA

Alkaline comet assay

% tail DNA, TL, TM

Remarks

Alkaline and fpgmodified comet assay

Parameter measured % tail DNA

20 ± °C, 5% CO2 TiO2 NP (24 h): no ↑ DNA sb TiO2 NP (50 μg mL−1) + UVA (3 kJ m-2; 30 min): ↑ DNA sb (without and with fpg) H2O2 (5, 25 μM; 10 min): ↑ 19 °C, air LOEC (2 h): 13 nM for NQO, 3.74 μM for BaP Leachate water samples: no ↑ DNA sb

Culture conditions/ Response

B. Žegura, M. Filipič

Mutat Res Gen Tox En 842 (2019) 72–84

RTgutGC; Oncorhynchus mykiss (rainbow trout) Gut cell line RTL-W1; Oncorhynchus mykiss (rainbow trout); Fibroblast-like liver derived cell line (normal liver)

Cell line/ Species

Table 2 (continued)

76

Cadmium chloride (CdCl2; 0.02, 0.18, 0.92, 1.83 mg L−1) Methyl methane sulfonate (MMS; 5.5, 25.5, 55 mg L−1) benzo(a)pyrene (B(a)P; 0.13, 0.25, 1.26 mg L−1) polychlorinated biphenyl 126 (PCB126; 3 × 10 − 4, 3.3 × 10 − 3, 0.33 mg L−1) Arabian Light Crude Oil (BAL 110; 87 mg/mL) Erika Heavy Oil (HFO n°2; 50 mg/mL) UVC: 1.75–2.5 J m−2 −0, 1, 3 and 9 h after UVC exposure to follow photoreactivation repair (PER) process; −0, 1, 3, 16, 24 h and 48 h after UVC exposure to follow nucleotide excision repair (NER) process Benzo[a]pyrene (BaP; 0, 0.001, 0.01, 0.1 and 1 μM; 24 h) Hydrogen peroxide (H2O2; 1, 5, 10, 50 μM; 10 min, 4 °C)

Acetone extracts of sediments from Danube River (5, 10, 20, 40 mg SEQ/mL) Acetonic sediment extracts from Tieteˆ River (1.5, 3, 6 and 12 SEQ/ mL; 48 h)

Hydrogen peroxide (H2O2; 10 μM; 10 min, 4 °C; recovery followed for 4 h) Methyl methane sulfonate (MMS; 0.4 μM; 15 min, RT; recovery followed for 24 h) Benzo(a)pyrene (BaP; 0.02, 0.2, 2, 10, 50 μM; 24 and 48 h)

Benzothiazoles: 2,20-Dithiobis (benzothiazole) (DBTH; 5 mg L−1), N,N-Dicyclohexyl-2-benzothiazolsulfene amide (NNA; 250 mg L−1), MBTHS (250 mg L−1), C.I. Vat yellow 2 (VY; 15 mg L−1), 2Aminobenzothiazole (2ABTH; 30 mg L−1), 2-Hydroxybenzothiazole (OHBTH; 15 mg L−1), Sodium 2-mercaptobenzothiazole (NaMBTH; 12.5 mg L−1), Zinc 2-mercaptobenzothiazole (ZnMBTH; 5 mg L−1), mercaptobenzothiazole (2MBTH; 12.5 mg L−1), C.I. Sulphur orange 1 (SO; 3 mg L−1), 3,30-diethylthiadicar bocyanine iodide (DTDC; 0.025 mg L−1), Benzothiazole (BTH; 30 mg L−1); Hydrogen peroxide (H2O2; 100 μM) UVC (254 nm): 1.75–2.5 J m−2 −0, 1, 3 and 9 h after UVC exposure to follow photoreactivation repair (PER) process; −0, 1, 3, 16, 24 h and 48 h after UVC exposure to follow nucleotide excision repair (NER) process. Benzo[a]pyrene (BaP; 0, 0.001, 0.01, 0.1 and 1 μM; 24 h) Hydrogen peroxide (H2O2; 1, 5, 10, 50 μM; 10 min, 4 °C) Methyl methane sulfonate (MMS; 0.1, 0.5, 1, 5 μM; 15 min, RT) CdCl2 (0.1, 1, 10 μM; 24 h) Diuron (0.1, 1, 10, 100 μg L−1; 24 h) 3,4-dichloroaniline (3–4 DCA; 0.1, 1, 10, 100 μg L−1 24 h)

(TT; 4-methyl-1H-benzotriazole 4MBTR) and5-methyl-1Hbenzotriazole (5MBTR)); Hydrogen peroxide (H2O2; 100 μM)

Agent/stressor (type, concentrations used, exposure time)

% tail DNA

% tail DNA

20 °C, air; H2O2: ↑ DNA sb ≥ 5 μM (↑ ≥ 1 μM; Fpg)

[10]

[34]

[31]

[38]

[58]

[39]

[28]

[10]

[34]

[27]

Ref.

(continued on next page)

Alkaline and fpgmodified comet assay

T4-modified comet assay

Alkaline and fpgmodified comet assay

Alkaline comet assay

TM

% tail DNA

Alkaline comet assay

Alkaline comet assay

Alkaline and fpgmodified comet assay

TM

% tail DNA

20 °C, air; no DNA repair (standard alkaline comet assay) ↑ DNA repair (T4-modified comet assay)

20 °C, air ↑ DNA sb at 5-40 mg SEQ/mL 21 °C, air; 48 h: ↑ DNA sb at ≥ 1.5 mg SEQ/ml depending on the sampling location 20 °C, air 24 h (BaP, PCB126, BAL, HFO n°2), 15 min (MMS): ↑ DNA sb at 0.25 mg L−1 B(a)P; ↑ DNA sb at 0.18 mg L−1 CdCl2; no ↑ DNA sb in PCB126; ↑ DNA sb at BAL and HFO n°2 only in the presence of Fpg. EC50 for BAL: 345.00 ± 87.00 mg L−1 EC50 for HFO n°2: 150.00 ± 15.00 mg L−1

21 °C, air 24 and 48 h: ↑ DNA sb ≥10 μM

% tail DNA

% tail DNA

20 °C, air; H2O2: ↑ DNA sb ≥ 5 μM (↑ ≥ 1 μM; Fpg) MMS: ↑ DNA sb ≥ 0.5 μM (↑ ≥ 0.1 μM; Fpg) Cd: ↑ DNA sb ≥ 0.1 μM (↑ ≥ 0.1 μM; Fpg) BaP: no ↑ DNA sb (↑ ≥ 0.1 μM; Fpg) Diuron: ↑ DNA sb ≥ 100 μg L−1 (↑ ≥ 0.1 μg L−1; Fpg) 3,4-DCA: ↑ DNA sb ≥ 1 μg L−1 (↑ ≥ 0.1 μg L−1; Fpg) 20 °C, air; H2O2: ↑ DNA sb and Fpg sites at 10 μM MMS: ↑ DNA sb and Fpg sites at 0.4 μM

Alkaline and fpgmodified comet assay

T4-modified comet assay

% tail DNA

20 °C, air; no DNA repair (standard alkaline comet assay) ↑ DNA repair (T4-modified comet assay)

Remarks

Alkaline comet assay

Parameter measured

Comet score (VS)

10 mg L−1 for DM (no ↑); 12 days (no ↑: at 60 mg L−1 for 1H-BTR, at 30 mg L−1 for 4MBTR, 5MBTR, TT, 1OHBTR, at 7.5 mg L−1 for 5CBTR, at 10 mg L−1 for DM). H2O2 (100 μM; 5 min): ↑ 20 °C, air 24 h: ↑ BTHs, ↑ 2ABT, ↑ OHBTH; 12 days: No ↑ DNA sb H2O2 (100 μM; 5 min): ↑ DNA sb

Culture conditions/ Response

B. Žegura, M. Filipič

Mutat Res Gen Tox En 842 (2019) 72–84

77

ZFL; Danio rerio (zebrafish) Normal liver cell line

RTH-149; Oncorhynchus mykiss (rainbow trout); Hepatoma cell line

Cell line/ Species

Table 2 (continued)

Soluble fraction of biodiesel extracted from sunflower oil produced by the methanol transesterification and the ethanol transesterification 5-fluorouracil (5-FU; 0.01, 0.1, 1, 10 μg mL−1; 4, 24 and 72 h) Cisplatin (CDDP; 0.01, 0.1, 1, 10 μg mL−1; 4, 24 and 72 h) etoposide (ET; 0.01, 0.1, 1 μg mL−1; 4 and 24 h) Benzo[a]pyrene (BaP; 50 μM; 72 h) Imatinib-mesylate (IM; 0.001, 0.01, 0.1, 1 μg mL−1; 4, 24 and 72 h) Benzo[a]pyrene (BaP; 50 μM; 72 h) tert-butyl hydroperoxide (TBHP; 1 mM; 20 min)

Hydrogen peroxide (H2O2; 53, 130 and 2650 μM) Surface water samples from Kishon river outflow (2 h; 50 v/v %)

Complex river water samples (50 v/v %)

Hydrogen peroxide (H2O2; 1, 5, and 10 μM)

2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD; 0, 20, 40, 60, 80, and 100 mg L−1; 48 h) UV light radiation (5 min) Surface sediment extracts of Lake Skadar (0.75, 1.5, 3 mg/mL; 12 h) UV light (240-280 nm, 5 min)

18 °C, 5% CO2 2 h: ↑ DNA sb ≥ 130 μM H2O2; ↑ DNA sb at 50 v/v% surface water samples 28 °C, air; 5, 10, 20 v/v % 1, 3, 6 and 12 h; ↑ 28 °C, air; LOEC (72 h): 0.001 μg/mL for ET, 0.01 μg/mL for 5-FU and 0.1 μg/ mL for CDDP; ↑ BaP 28 °C, air; LOEC (72 h): 0.01 μg/mL for IM ↑ BaP, ↑ TBHP

n.d. Sediment extracts: ↑ DNA sb at ≥ 1.5 mg/ml depending on the location 20 °C, 5% CO2 H2O2: 2 h; ↑ ≥1 μM 20 °C, 5% CO2 ↑ 50 v/v %; 2 h

Alkaline comet assay

% tail DNA

[49]

[47]

[46]

[59]

(continued on next page)

Alkaline comet assay

% tail DNA

% tail DNA

Alkaline and fpg- and endoIII-modified comet assay Alkaline comet assay

DNA damage score (VS)

[41]

[43]

Alkaline comet assay

Alkaline comet assay

[40]

[33]

[35]

[32]

[28]

[37]

[20]

[36]

Ref.

Alkaline comet assay

Alkaline comet assay

TM

TEM, MATL, TA, CTL (VS) CS, TL, CTL (VS); measured: head diameter and total TL TL DNA damage score (VS)

Alkaline comet assay

OTM

Alkaline comet assay

Alkaline and fpgmodified comet assay

% tail DNA

20 °C, air; H2O2: ↑ DNA sb and Fpg sites at 10 μM MMS: ↑ DNA sb and Fpg sites at 0.4 μM

TM

Alkaline and fpgmodified comet assay

% tail DNA

20 °C, air; 24 h: ↑ DNA sb ≥ 50 % equivalent effluent concentration of the sample (without and with Fpg)

20 °C, air; 24 h: Sediment extracts from Northwest Bay sites: ↑ DNA sb Sediment extracts from South Bay site: no DNA sb H2O2: ↑ DNA sb at 1 μM 20 °C, air TMDD (48 h): ↑ DNA sb at 100 mg L−1

Alkaline comet assay

TL, TM, % tail DNA

19 °C, air LOEC (2 h): 13 nM for NQO, 1.2 μM for BaP Leachate water samples: no ↑ DNA sb

Remarks

Alkaline and fpg -modified comet assay

MMS: ↑ DNA sb ≥ 0.5 μM (↑ ≥ 0.1 μM; Fpg) Cd: no ↑ DNA sb (↑ ≥ 1 μM; Fpg) BaP: no ↑ DNA sb (↑ ≥ 0.5 μM; Fpg) Diuron: no ↑ DNA sb (↑ ≥ 1 μg L−1; Fpg) 3,4-DCA: no ↑ DNA sb (↑ ≥ 0.1 μg L−1; Fpg) 20 °C, air; CTB: no ↑ DNA sb CTB (≥ 1%) + UVA: ↑ DNA sb AS-blend (10%) + UVA: ↑ DNA sb

Methyl methane sulfonate (MMS; 0.1, 0.5, 1, 5 μM; 15 min, RT) Cd (0.1, 1, 10 μM; 24 h) Herbicide Diuron (0.1, 1, 10, 100 μg L−1; 24 h) Metabolite 3,4-dichloroaniline (3–4 DCA; 0.1, 1, 10, 100 μg L−1 24 h) Complex runoff samples were prepared under controlled conditions and samples after various time points (4 h- 36 day): -runoff from asphalt pavement with coal-tar-based (CTB) sealcoat containing PAHs -runoff from asphalt pavement with an asphalt-based sealcoat hypothesized to contain about 7% CTB sealcoat (AS-blend). 24 h 1 and 10 % dilution + 2 h UVA (333 μWcm−2) 4-nitroquinoline-1-oxide (NQO; 6.5 nM - 210 nM) benzo(a)pyrene (BaP; 0.94 – 50 μM) Leachate water samples from a gaswork remediation site containing xylene (17.7-20 mg L−1), benzene (0.5-1.1 mg L−1), ethylbenzene (0.88-1.4 mg L−1), 16 PAHs (6.4-12.1 μg L−1) Organic extracts of effluents from textile industry (containing dyes and aromatic amines); 5, 10, 50, 75, 100 % equivalent effluent concentration Effluents released to Piracicaba River, São Paulo State, Brazil Hydrogen peroxide (H2O2; 10 μM; 10 min, 4 °C; recovery followed for 4 h) Methyl methane sulfonate (MMS; 0.4 μM; 15 min, RT; recovery followed for 24 h) Organic extracts of sediments from lake Laguna, Philippines (3.730 mg SEQ/ml water) Hydrogen peroxide (H2O2; 1 μM; 1 h)

Parameter measured

% tail DNA

Culture conditions/ Response

Agent/stressor (type, concentrations used, exposure time)

B. Žegura, M. Filipič

Mutat Res Gen Tox En 842 (2019) 72–84

78

PLHC-1; Poeciliopsis lucida (topminnow) Hepatocellular carcinoma

Chromium (K2Cr2O7; 10, 20, 30, 40 and 50 mg L−1; 24 h) Hydrogen peroxide (H2O2; 5 μM)

Benzo[a]pyrene (BaP; 0, 0.001, 0.01, 0.1 and 1 μM; 24 h) Hydrogen peroxide (H2O2; 1, 5, 10, 50 μM; 10 min, 4 °C) Methyl methane sulfonate (MMS; 0.1, 0.5, 1, 5 μM; 15 min, RT) CdCl2 (0.1, 1, 10 μM; 24 h) Diuron (0.1, 1, 10, 100 μg L−1; 24 h) 3,4-dichloroaniline (3–4 DCA; 0.1, 1, 10, 100 μg L−1 24 h)

Benzo[a]pyrene (B[a]P; 0.1–10 μM) Ethyl methanesulfonate (EMS; 10–1000 μM) Extracts of sediment samples (0.08–20 mg/mL; containing PAHs, PCBs and metals) Arsenic trioxide (As2O3; 0–100 μM) for 10, 20 and 40 h

Cadmium chloride (CdCl2; 0.02, 0.18, 0.92, 1.83 mg L−1) Methyl methane sulfonate (MMS; 5.5, 25.5, 55 mg L−1) Benzo(a)pyrene (B(a)P; 0.13, 0.25, 1.26 mg L−1) Polychlorinated biphenyl 126 (PCB126; 3 × 10 − 4, 3.3 × 10 − 3, 0.33 mg L−1) Arabian Light Crude Oil (BAL 110; 87 mg/mL) Erika Heavy Oil (HFO n°2; 50 mg/mL)

Benzo[a]pyrene (BaP; 0.1, 1 and 10 μM; 2 h and 6 days); Ethyl methanesulfonate (EMS; 0.1, 0.5 and 1 mM; 2 h and 6 days)

PAC2; Danio rerio (zebrafish)

OLCAB-e3; Oryzias latipes (Japanese medaka) Embryos cell line

Gamma irradiation using 137Cs source (24 h; 1, 10, 100, and 750 mGy/d) Uranium [uranyl nitrate UO2(NO3)2•6H2O]: (0, 1, 2, 5, 10, 30, 50, 75, 100 and 250 μM; 24 h) Aluminium (AlCl3; 0, 20, 50, 100 μM; 24 h) Cadmium (CdCl2; 0, 30, 50 and 100 μM; 24 h

Hydrogen peroxide (H2O2; 20 μM; 10 min; recovery up to 180 min)

Cyclophosphamide (CP; 10, 37.5, 75, 150, 300 μg mL−1; 12, 120 μg L−1; 72 h) Ifosfamide (IF; 10, 37.5, 75, 150, 300 μg mL−1; 10, 100 μg L−1; 72 h) 5-fluorouracil (5-FU; 0.09, 0.9 μg L−1; 72 h) Cisplatin (CDDP; 0.6, 6 μg L−1; 72 h) Complex mixtures of low doses of CP, IF, 5-FU, CDDP; 72 h Hospital effluents and wastewater treatment plants influents and effluents (10, 20 and 30 v/v %; 72 h) Benzo[a]pyrene (BaP; 50 μM; 72 h); Etoposide (ET; 100 ng/mL; 72 h) Gasoline water-soluble fraction (5, 10, 25 and 50 %; 1, 3 and 6 h)

Agent/stressor (type, concentrations used, exposure time)

ZF4; Danio rerio (zebrafish) Embryonic zebrafish fibroblasts

Cell line/ Species

Table 2 (continued)

30 °C, 5% CO2 10h: no ↑ DNA sb 20h: at 100 μM ↑ DNA sb 40h: 2-100 μM ↑ DNA sb 30 °C, air; 10 min: H2O2: ↑ ≥ 1 μM (↑ ≥ 1 μM; Fpg) 15 min: MMS: ↑ ≥ 0.5 μM (↑ ≥ 0.1 μM; Fpg) 24 h: CdCl2: ↑ 1 μM (↑ ≥ 0.1 μM; Fpg) 24 h: BaP: ↑ ≥ 1 μM (↑ ≥ 1 μM; Fpg) 24 h: Diuron: 10 μg L−1 (↑ ≥ 0.1 μg L−1; Fpg) 24 h: 3,4-DCA: 10 μg L−1 (↑ ≥ 1 μg L−1; Fpg) 28 °C, air; Cr (24 h): ↑ DNA sb ≥ 10 μM;

DNA damage score (VS)

28 °C, air; 1, 3 and 6 h; ↑ ≥ 5% 28 °C, air; H2O2 (10 min): at 20 μM ↑ DNA sb; no ↑ DNA sb after 180 min of recovery 28 °C, 5% CO2 ↑ DNA sb at 750 mGy/d 28 °C, 5% CO2 ↑ DNA sb ≥ 10 μM 28 °C, 5% CO2 AlCl3 (24 h): ↑ DNA sb at 50 μM CdCl2 (24 h): no ↑ DNA sb EMS (2 h): ↑ DNA sb at 1 mM; EMS (6-days): ↑ DNA sb ≥ 0.5 mM; (+ 6-days recovery): ↑ DNA sb at 1 mM; BaP (2 h): ↑ DNA sb only at 0.1 μM (-S9); ↑ DNA sb ≥ 0.1 μM (+S9); BaP (6 days): no ↑ DNA sb; (+ 6-days recovery): no ↑ DNA sb 28 °C, air 24 h (BaP, PCB126, BAL, HFO n°2), 15 min (MMS); ↑ at 1.26 mg L−1 B(a)P ↑ at 0.02 mg L−1 CdCl2 ↑ at 0.0033 mg L−1 PCB126 ↑ at BAL and HFO n°2 only with Fpg EC50 for BAL: 295.00 ± 52.20 mg L−1 HFO n°2: no ↑ DNA sb 30 °C, air 24 h: ↑ DNA sb at ≥ 0.1 μM BaP; at ≥ 10 μM EMS Sediment extracts: ↑ DNA sb

Alkaline comet assay

MTL

TL

[66]

[10]

[56]

[57]

[31]

[75]

[55]

[54]

[53]

[45]

[61]

[74]

Ref.

(continued on next page)

Alkaline comet assay

Alkaline and fpgmodified comet assay

Alkaline comet assay

TL, TM, % tail DNA

% tail DNA

Alkaline comet assay

Alkaline and fpgmodified comet assay

% tail DNA

% tail DNA

Alkaline comet assay

Alkaline comet assay

MTL

% tail DNA

Alkaline comet assay

Alkaline comet assay

Alkaline comet assay

Alkaline comet assay

Remarks

MTL

TL

% tail DNA

Parameter measured

28 °C, air; ↑ at 10-30 v/v %; ↑ BaP (72 h); ↑ ET

28 °C, air; LOEC (72 h): 37.5 μg/mL for CP and IF; ↑ complex mixture (consisiting of 12 μg/L CP + 10 μg/L IF + 0.09 μg/L 5-FU + 0.6 μg/L CDDP)

Culture conditions/ Response

B. Žegura, M. Filipič

Mutat Res Gen Tox En 842 (2019) 72–84

CB; Catla catla (major carp) Brain cell line LRG Labeo rohita (roho labeo) Gill cell line EPC; Pimephales promelas (fathead minnow) *previously reported to be isolated from Cyprinus carpio L. (common carp) epithelioma papillosum cyprinid cell line derived from skin tumor of carp [76]

ICG; Catla catla (major carp) Gill cell line

SICH; Catla catla (major carp) Heart cell line

SISS; Lates calcarifer (Asian sea bass) Spleen cell line SISSK; Lates calcarifer (Asian sea bass) Kidney cell line IEE; Etroplus suratensis (green chromide) Eye cell line IEK; Etroplus suratensis (green chromide) Kidney cell line IEG; Etroplus suratensis (green chromide) Gill cell line

Cell line/ Species

Table 2 (continued)

28 °C, air; Cr (24 h): ↑ DNA sb ≥ 10 μM;

Chromium (K2Cr2O7; 10, 20, 30, 40 and 50 mg L−1; 24 h) Hydrogen peroxide (H2O2; 5 μM)

79

Silver nanoparticles (Ag-NPs; 2, 4, 8, 16, 32 and 64 μg/mL; 24 h) Hydrogen peroxide (H2O2; 5 μM) Herbicides: Dezormon (0.1–1000 mg L−1) and Optica trio (0.1–10,000 mg L−1) active ingredients: −4-chloro-o-tolyloxyacetic acid (MCPA; 0.01–100 mg L−1) −2,4-dichlorophenoxyacetic acid (2,4-D; 0.01–100 mg L−1) −2-(4-chloro-2-methylphenoxy)propionic acid (mecoprop; 0.01–100 mg L−1) −2-(2,4-dichlorophenoxy)propionic acid) (dichlorprop; 0.01–100 mg L−1) - ternary mixture of MCPA, dichlorpop, and mecoprop in the ratio of 0.56:0.36:0.11, respectively (0.1–1000 mg L−1) Hydrogen peroxide (H2O2; 1 mM; 15 min) Organic sediment extracts from the North Sea (24 h; 7 to 307 mg sediment dry weight/mL; supplemented with fish enzyme suspension for metabolic activation) Benzo[a]pyrene (BaP; 25 ng/mL; 2 or 24 h) Organic extracts of marine sediments from the North Sea and the Baltic Sea (24 h; dry weight mg/mL; supplemented with fish enzyme suspension for metabolic activation) Benzo[a]pyrene (BaP; 2.5 or 25 ng/mL; 24 h) Hydrogen peroxide (H2O2; 5 min)

Insecticide cypermethrin (CYP; 1.25, 2.5, 5, 10 and 20 ng/mL; 24 h)

Silver nanoparticles (Ag-NPs; 2, 4, 8, 16, 32 and 64 μg/mL; 24 h) Hydrogen peroxide (H2O2; 5 μM) Insecticide cypermethrin (CYP; 1.25, 2.5, 5, 10 and 20 ng/mL; 24 h)

Chromium (K2Cr2O7; 10, 20, 30, 40 and 50 mg L−1; 24 h) Hydrogen peroxide (H2O2; 5 μM) Silver nanoparticles (Ag-NPs; 2, 4, 8, 16, 32 and 64 μg/mL; 24 h) Hydrogen peroxide (H2O2; 5 μM) Chromium (K2Cr2O7; 10, 20, 30, 40 and 50 mg L−1; 24 h) Hydrogen peroxide (H2O2; 5 μM) Insecticide cypermethrin (CYP; 1.25, 2.5, 5, 10 and 20 ng/mL; 24 h)

25 °C, 5% CO2 (culture conditions) 8 °C, 5% CO2 (exposure conditions) 24 h: ↑ DNA sb at ≥ 200 mg/mL (depending of the type of the extract) 25 °C, 5% CO2 (culture conditions) 8 °C, 5% CO2 (exposure conditions) 24 h: ↑ DNA sb at ≥ 1 mg/mL (depending of the type of the extract)

28 °C, air CYP (24 h): ↑ DNA sb ≥ 5 ng/mL 28 °C, air; Ag-NPs (24 h): ↑ DNA sb ≥ 8 ng/mL 15 °C, air 24 h: ↑ DNA sb at 1000 mg L−1 dezormon; at 10,000 mg L−1 Optica trio; at 10 and 100 mg L−1 2,4-D; at 100 mg L−1 MCPA; no ↑ DNA sb in mecoprop, dichloroprop and ternary mixture.

28 °C, air; Cr (24 h): ↑ DNA sb ≥ 10 μM; 28 °C, air CYP (24 h): ↑ DNA sb ≥ 5 ng/mL 28 °C, air; Cr (24 h): ↑ DNA sb ≥ 10 μM; 28 °C, air; Ag-NPs (24 h): ↑ DNA sb ≥ 8 ng/mL 28 °C, air; Cr (24 h): ↑ DNA sb ≥ 10 μM; 28 °C, air CYP (24 h): ↑ DNA sb ≥ 5 ng/mL 28 °C, air; Ag-NPs (24 h): ↑ DNA sb ≥ 8 ng/mL 28 °C, air CYP (24 h): ↑ DNA sb ≥ 5 ng/mL

28 °C, air; Cr (24 h): ↑ DNA sb ≥ 10 μM;

Chromium (K2Cr2O7; 10, 20, 30, 40 and 50 mg L−1; 24 h) Hydrogen peroxide (H2O2; 5 μM)

Chromium (K2Cr2O7; 10, 20, 30, 40 and 50 mg L−1; 24 h) Hydrogen peroxide (H2O2; 5 μM) Insecticide cypermethrin (CYP; 1.25, 2.5, 5, 10 and 20 ng/mL; 24 h)

28 °C, air; Cr (24 h): ↑ DNA sb ≥ 10 μM;

Culture conditions/ Response

Chromium (K2Cr2O7; 10, 20, 30, 40 and 50 mg L−1; 24 h) Hydrogen peroxide (H2O2; 5 μM)

Agent/stressor (type, concentrations used, exposure time)

[78]

Alkaline comet assay

(continued on next page)

[77]

Alkaline comet assay

DNA damage score (VS)

[73]

[72]

Alkaline comet assay

Alkaline comet assay

% tail DNA

[69]

[67]

[67]

[69]

[67]

[66]

[69]

[66]

[67]

[66]

[66]

[66]

[66]

Ref.

DNA damage score (VS)

Alkaline comet assay

Alkaline comet assay

Alkaline comet assay

% tail DNA

% tail DNA

% tail DNA

Alkaline comet assay

Alkaline comet assay

% tail DNA % tail DNA

Alkaline comet assay

Alkaline comet assay

% tail DNA TL

Alkaline comet assay

Alkaline comet assay

% tail DNA TL

Alkaline comet assay

Alkaline comet assay

Alkaline comet assay

Alkaline comet assay

Remarks

TL

TL

TL

TL

Parameter measured

B. Žegura, M. Filipič

Mutat Res Gen Tox En 842 (2019) 72–84

20 ± 1 °C, air H2O2: ↑ DNA sb at ≥ 5 μg mL−1 (alkaline, Endo III and Fpg) UVA: ↑ DNA sb at ≥ 5 kJ m−2 (alkaline, Endo III) and ↑ DNA sb at ≥ 2.5 kJ m−2 Fpg) TiO2/ no UVA (24 h): ↑ DNA sb at 100 μg mL−1; ↑ DNA sb (Fpg) at ≥ 1 μg mL−1; no DNA sb (Endo III) UVA (2 h, 2.5 kJ m−2): ↑ DNA sb and Fpg TiO2 (2 h, 1 μg mL−1): ↑ DNA sb and Fpg TiO2/+UVA (2 h, 1 μg mL−1 + 2.5 kJ m−2): ↑ DNA sb and Fpg 28 °C, air TiO2 (24 h): ↑ DNA sb at ≥ 12.5 μg mL−1 ZnO (24 h): ↑ DNA sb at ≥ 12.5 μg mL−1 30 °C, 5% CO2 WS: ↑ DNA sb at ≥ 30 v/v% depending on the sample SS: ↑ DNA sb depending on the sample H2O2: ↑ DNA sb

Hydrogen peroxide (H2O2; 1-20 μM; 5 min) UVA (2.5–10 kJm−2) Anatase TiO2 nanoparticles (size 5 nm; 0.1–100 μg mL−1; 2 and 24 h)

Surface river water samples (WS; 10-40 v/v%; 72 h) Organic extracts from sediments (SS; 0.5-20 mg/mL; 72 h) (sampled upstream of a gypsum mine in the Kosovčica Spring and downstream of the mine in the Kosovčica River) Hydrogen peroxide (H2O2; 50 μM; 10 min) Crude sediment extracts of coastal area (0.2, 0.5, 1, 5, 10 and 20 mg dry weight/mL; 24 h) Insecticide propoxur (1, 10, 25, 50, and 75 μg mL−1; 24 h)

80

Musk xylene (MX; 10.0 μg L ) Vitamin E (a-tocopherol; 1 mg L−1) Bilberry anthocyanins extract (Anthocyanin; 1 mg L−1) Benzene (1.0 μL/L) exposure: 5, 7, 14, 21 days

−1

TiO2 nanoparticles (0, 12.5, 25, 50 mg L−1, 24 h) ZnO nanoparticles (0, 12.5, 25, 50 mg L−1, 24 h)

Malachite green (C23 H26 N2O, MG; 0.001-10 μg/mL; 48 h)

20 °C, air; 24 h: Sediment extracts: ↑ DNA sb 20 °C, air; 24 h: ↑ DNA sb at 75 μg mL−1 28-28.5 °C, air MX (5, 7, 14, 21 days): ↑ DNA sb at all exposures MX + vitE: no ↑ DNA sb at 7, 14 and 21 days MX + Anthocyanin: no ↑ DNA sb at all exposures Benzene: ↑ DNA sb at all exposures

Alkaline comet assay

Alkaline comet assay

DNA damage score (VS) % tail DNA

Alkaline comet assay

Alkaline comet assay

Alkaline comet assay

Alkaline and endo III and fpg-modified comet assay

Alkaline comet assay

Alkaline comet assay

Alkaline comet assay

Alkaline comet assay

Remarks

DNA damage score (VS)

TL, TM, % tail DNA

% tail DNA

% tail DNA

% tail DNA

% tail DNA

VS

% tail DNA

Visual analyses (no scoring)

Parameter measured

[82]

[64]

[63]

[81]

[80]

[79]

[71]

[71]

[70]

[67]

Ref.

↑, significant increase; ↓, significant decrease; ≥, at and above; % tail DNA; AU, arbitrary units; CS, comet score; CTL, cumulative tail length; DNA sb – DNA strand breaks; fpg- formamidopyrimidine DNA-glycosylase; hhours; min- minutes; MATL, mean actual tail length, MTL, mean tail length; OTM, Olive tail moment; SEQ- sediment-equivalent per mL; TA, tail area; TEM, tail extent moment; TL, tail length; TM, tail moment; T4, T4PDG (pyrimidine dimer glycosylase) restriction enzyme allows detection of repair kinetics of cyclobutane pyrimidine dimmers; VS, visual scoring; 5-FU, 5-fluorouracil; AgNO3, silver nitrate; B[a]P, benzo(a)pyrene; CdCl2, cadmium chloride; CDDP, cisplatin; CP, cyclophosphamide; CuCl2, copper chloride; EMS, ethylmethanesulphonate; ET, etoposide; H2O2, hydrogen peroxide; H2S, MMC, mitomycin C; MMS, methylmethanesulfonate; NP, nanoparticles; NQO, 4-nitroquinoline-N-oxide; PAHs, polycyclic aromatic hydrocarbons; PC, positive control; PCB, polychlorinated biphenyl; PCP, pentachlorophenol; Pd(NO3)2, lead nitrate; QDs, quantum dots; TiO2, titanium dioxide; UV, ultra violet; ZnO, zinc oxide.

FG; Paralichthys olivaceus (olive flounder); marine species Gill cell line DLEC; Dicentrarchus labrax (european bass); marine species embryonic cell line

WAG; Wallago attu (wallago catfish) Gill cell line CCO; Ictalurus punctatus (channel catfish) Fibroblast-like cell line derived from ovaries

CSK; Channa striata (striped snakehead, murrel) Kidney cell line GFSk-S1; Carassius auratus (goldfish); Primary cell line developed from the skin with fibroblast-like morphology

Malachite green (C23 H26 N2O, MG; 0.001-10 μg/mL; 48 h)

Pesticide endosulfan (0.5 ng/mL; 24 h)

28 °C, air CYP (24 h): ↑ DNA sb ≥ 5 ng/mL 28 ± 2 °C, air 24 h, ↑ DNA sb at 0.5 ng/mL 28 °C, air 48 h: ↑ DNA sb at 0.1 μg mL−1 28 °C, air 48 h: ↑ DNA sb at 0.1 μg mL−1

Insecticide cypermethrin (CYP; 1.25, 2.5, 5, 10 and 20 ng/mL; 24 h)

28 °C, air H2O2(5 min): ↑ DNA sb

TTCF; Tor tor (tor mahseer); Caudal fin cell line CSG; Channa striata (striped snakehead, murrel) Gill cell line

Culture conditions/ Response

Agent/stressor (type, concentrations used, exposure time)

Cell line/ Species

Table 2 (continued)

B. Žegura, M. Filipič

Mutat Res Gen Tox En 842 (2019) 72–84

Mutat Res Gen Tox En 842 (2019) 72–84

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been mainly used for detection of DNA strand breaks induced by model genotoxic compounds such as hydrogen peroxide [45] and benzo(a) pyrene [46–48], cytostatic drugs [46–49] and complex environmental mixtures [48–50]. ZFL cells were reported to be more sensitive and susceptible towards detection of primary DNA damage induced by cytostatic drugs 5-fluorouracil, cisplatin, etoposide [46] and imatinib mesylate [51] than human derived cells, HepG2 cells and human peripheral blood lymphocytes. This stresses the importance and the need to use the most appropriate cell model and exposure conditions when studying genotoxic effects of compounds with specific mechanism of action. Complex mixture of four cytostatic drugs 5-fluorouracil, cisplatin, cyclophosphamide and ifosfamide at concentrations detected in the effluents from the oncological ward that were several orders of magnitude lower from those being effective when tested as individual compounds induced increased level of DNA damage in ZFL cells determined with the comet assay [49]. Hospital effluents and wastewater treatment plant (WWTP) influents and effluents, which contained cytostatic drugs and their metabolic and transformation products, were reported to significantly increase the formation of DNA strand breaks in ZFL cells [48]. These studies confirmed the sensitivity of ZFL cells to identify the potential harmful effects of complex mixtures of cytostatic drugs for the aquatic organisms. In addition to studying cytostatic drugs, ZFL cells have been used to assess the genotoxic potential of gasoline water-soluble fraction consisting of monoaromatic hydrocarbons, mainly of ethylbenzene, xylene and benzene, and naphthalene, anthracene and phenanthrene among PAHs. Comet assay has revealed a concentration-dependent relationship between damage and dilution [50]. DNA base excision repair in ZFL cell line has been evaluated after exposure to hydrogen peroxide and the results showed that repair capacity of ZFL cells differs from zebrafish primary hepatocytes in terms of time needed to complete DNA repair [45]. Another zebrafish ZF4 cell line has been established from 1-day-old zebrafish embryos [52]. DNA damage and repair have been analysed in ZF4 cells after the irradiation to gamma rays and the results revealed an accelerated DNA repair at lower doses and accumulation of DNA double-strand breaks at higher doses suggesting a high radiosensitivity of embryonic ZF4 cells [53]. Further ZF4 cell line has been used for studying DNA damage repair and the molecular mechanisms underlying genotoxicity of uranium [54], aluminium and cadmium [55] and the results provided evidence of the cells sensitivity towards the studied compounds.

contaminants. When comparing NER kinetics observed in RTGill-W1 and RTL-W1 cell lines, RTGill-W1 cells have faster repair capacity then RTL-W1 cells [8]. RTL-W1 is a fibroblast-like non-transformed permanent cell line taken from the liver of adult male rainbow trout (Oncorhynchus mykiss) [30]. RTL-W1 cells have showed higher biotransformation capacities (including both constitutive and inductive cyp1 a1 activity) compared to other fish cell lines such as RTG-2 [20], RTgill-W1 [9,10], RTH-149 [9] and others. They contain an active aryl hydrocarbon receptor [30]. RTL-W1 cells have been widely used for the assessment of DNA damage with the comet assay. They have been applied for the measurement of the effects of the model toxicants such as 4-nitroquinoline-1-oxide (NQO) [20], MMS [10,29,32], H2O2 [10,29,32,33], CdCl2 [10,32], BaP [10,21,32], PCBs [31], heterocyclic synthetic compounds [4,28] and complex environmental samples [21,33–38] as well as UV radiation [33–35]. For example Pannetier et al. [31], showed moderate genotoxicity of oil samples containing a large number of PAHs and alkylated PAHs. In another study, the influence of complex mixtures of chemicals in coal tar (samples of runoffs from asphalt pavement) in co-exposure with and without UVA on DNA damage and DNA repair capacity has been investigated in RTL-W1 cells [36]. For samples of runoffs deleterious effects have been measured with standard and Fpg-modified comet assay, while co-exposure to UVA has shown impairment of BER capacity, which in addition to photosensitization induced also photomodification [36]. Complex organic extracts from textile industry effluents containing dyes and aromatic amines increased primary DNA damage and Fpg-sensitive sites [37]. Moreover several studies have confirmed RTL-W1 cells as a sensitive model for the assessment of genotoxic potential of sediment extracts from rivers and lakes [32,33,38]. In addition to the above mentioned cell lines from rainbow trout, comet assay has been applied also on the intestinal RTgutGC [39] and hepatoma RTH-149 [40–42] cells that are both metabolically competent cell lines [39] and in RTH-149 cells aryl hydrocarbon receptors have been identified [42]. RTgutGC cell line was validated with well known mutagen benzo(a)pyrene [39], while RTH-149 cells were validated using hydrogen peroxide [40,42] and complex river water samples [41,42]. 3. Zebrafish (Danio rerio) cell lines Apart of rainbow trout derived cell lines comet assay has been extensively applied also on cell lines (ZFL and ZF4) derived from small tropical fresh water teleost zebrafish (Danio rerio). ZFL cell line (Fig. 1) is derived from normal adult zebrafish liver and it expresses alanine aminotransferase, aspartate aminotransferase, glucose-6-phosphatase enzyme activities and inducible proteins related to cytochrome P450 1A1 [44] showing the metabolic competency of cells. The cell line has

4. Other fish cell lines In addition to cell lines derived from rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio) several other cell lines have been applied for genotoxicity testing with the comet assay; however, to a lesser extent. These cell lines have been derived from various freshwater species such as Oryzias latipes (OLCAB-e3 cells), Poeciliopsis lucida (PLHC-1 cells), Lates calcarifer (SISS and SISSK cells), Etroplus suratensis (IEE, IEK and IEG cells), Catla catla (SICH, ICB and CB cells), Labeo rohita (LRG cells), Channa striata (CSG and CSK cells), Pimephales promelas (EPC cells), Tor tor (TTCF cells), Wallago attu (WAG cells), Ictalurus punctatus (CCO cells) and marine species Paralichthys olivaceus (FG cells), Dicentrarchus labrax (DLEC cells) (for details see Table 2). Among these PLHC-1 cell line has been frequently used for genotoxicity assessment. It is a hepatocellular carcinoma cell line derived from the topminnow (Poeciliopsis lucida). It has the capacity to express the cytochrome P4501 A system and it contains the aryl hydrocarbon receptor (AhR) [3]. The cell line has been used to evaluate the formation of DNA strand breaks induced by arsenic trioxide [56], ethyl methane sulfonate [57], methyl methane sulfonate [10], hydrogen peroxide [10], benzo(a)pyrene [10], environmental pollutants [10], and complex extracts of marine sediment samples containing polycyclic aromatic hydrocarbons, polychlorinated biphenyls and metals [57]. However, as PLHC-1 cell line is of cancerous origin and due to its

Fig. 1. Photographic image of zebrafish (Danio rerio) liver (ZFL) cell line. 81

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inherent genetic instability and inadequate DNA repair capacities [10] it requires further studies to better assess its reliability in genotoxicity testing. In other cell lines listed in Table 2 application of the comet assay has been described in only one study therefore the suitability of these cell lines for genotoxicity assessment with the comet assay has to be further validated.

[11]

[12]

[13]

5. Conclusions [14]

When identifying the genotoxic potential of environmental pollutants to aquatic organisms it is necessary to select the most relevant bioassay and the comet assay represents a very sensitive and reliable assay for detection of primary DNA damage induced by pure compounds and complex environmental chemical mixtures. The assay allows efficient screening of a large number of physical and/or chemical agents. As fish cell lines are more relevant and representative for the aquatic environment they can be used as an experimental model for the identification of genotoxic hazard and risk of aquatic environmental pollutants. Several studies showed that fish cells are more susceptible to the genotoxic effect of specific environmental pollutants than mammalian cells. The difference in the sensitivities and the higher induction of DNA damage in fish cells compared to mammalian cells can be attributed the differences in cell growth and capacity of DNA repair systems between fish and mammals. Fish cell lines grow at lower temperatures than mammalian cells and have longer doubling time. They have lower rate and capacity of DNA repair than mammalian cells. Taken together it can be concluded that comet assay in fish cell lines represents a sensitive tool for the assessment of toxicological hazard of pure compounds and their complex mixtures to aquatic organisms.

[15]

[16]

[17]

[18]

[19]

[20]

Conflict of interest The authors declare that they have no conflict of interest.

[21]

Acknowledgements

[22]

The authors acknowledge the financial support from the Slovenian Research Agency (P1-0245) and bilateral collaborations between Republic of Slovenia and Republic of Serbia (BI-RS/18-19-029).

[23]

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