The effect of chronic dosing and p53 status on the genotoxicity of pro-oxidant chemicals in vitro

Abstract

In this study, we have studied the cytotoxicity and genotoxic potency of 3 pro-oxidants; H₂O₂, menadione and KBrO₃ in different dosing scenarios, namely acute (1-day dosing) and chronic (5-days). For this purpose, relative population doubling (RPD%) and mononucleated micronucleus (MN) test were used. TK6 cells and NH32 were employed in in vitro experiments. In the study, the total acute dose was divided into 5 days for each prooxidant chemicals by dose fractionation (1/5th per day) method. Acute dosing was compared to chronic dosing. The oxidative stress caused by the exposure of cells with pro-oxidant chemicals to the cells was determined by an optimized 2′,7′-dichlorofluorescein diacetate (DCFHDA) test method. The antioxidant levels of the cell lines were altered with buthionine sulfoxide (BSO) and N-acetyl cysteine (NAC), and the effect of antioxidant capacity on the MN formation in the cells was observed with this method. In the case of H₂O₂ and menadione, fractional dosing has been observed to result in lower toxicity and lower genotoxicity. But in the case of KBrO₃, unlike the other 2 pro-oxidants, higher MN induction was observed with fractionated doses. DCFHDA test clearly demonstrated ROS induction with H₂O₂ and menadione but not with KBrO₃. Unexpectedly, DCFHDA test demonstrated that KBrO₃ did not cause an increase ROS levels in both acute and chronic dosing, suggesting an alternative ROS induction mechanism. It was also observed that, treatment with BSO and NAC, caused increasing and decreasing of MN fold change respectively, allowing further ROS specific mechanisms to be explored. Hence, dose fractionation expectedly caused less MN, cytotoxicity and ROS formation with H₂O₂ and menadione exposure, but not with KBrO₃. This implies a unique mechanism of action for KBrO₃ induced genotoxicity. Chronic dosing in vitro may be a valuable approach allowing better understanding of how chemicals damage DNA and pose human hazards.

Pro-oxidants, micronucleus (MN), reactive oxygen species, DNA damage, antioxidants, H₂O₂, menadione, KBrO₃, glutathione, genotoxicity

Introduction

Genetic toxicology, refers to the ability of harmful substances to damage the genetic information in cells. It plays an important role in hazard identification and risk assessment of chemicals which are used in the production of drugs, pesticides, flavours etc. (1,2). The assessment of DNA damage plays a major role in safety assessment. DNA damage constantly occurs in our cells as a result of various intrinsic factors such as intracellular metabolism, DNA replication and exposure to genotoxic agents such as prooxidant chemicals (3). Throughout early stages of drug development, the ability of a substance to damage DNA through genotoxic mechanisms is fully explored to ensure accurate and timely hazard and risk assessment (2).

Through the induction of oxidative DNA damage, pro-oxidants are responsible for many disease aetiologies such as ageing, neurodegeneration and cancer. This is due to an imbalance caused by oxidative stress-induced through excessive ROS production and/or inhibition of antioxidant systems (4). Mammalian cells are constantly exposed to potentially damaging ROS arising from multiple sources (5,6). DNA damage induced by ROS results in a spectrum of DNA lesions including DNA adducts, single-strand breaks (SSBs) and double-strand breaks (DSBs) (7).

Cellular antioxidant defences act to protect cells against ROS through processes including; direct ROS sequestration or indirectly regulating antioxidant enzyme defences or via blocking ROS production in the first place. Although most DNA damage is not fatal to the cell the location and the nature of DNA damage, in addition to the total number of damaging events, is critical in determining cell survival (8). Cellular defences exist to combat longstanding attack from ROS, including the antioxidant enzyme system, glutathione (9). However, when the number of ROS produced exceeds the antioxidant capacity of the cell, oxidative stress ensues and cellular macromolecules, including DNA can be damaged. Numerous types of DNA lesion can potentially occur when exposed to ROS, including at guanine and thymine bases. These are susceptible to oxidative modifications such as 8-OHdG and thymine glycol (TG) (10).

Potassium bromate (KBrO₃) is used as a food additive (in baking as a flour improver and in the production of fish-pastes). It is also used in hair straightening products (11,12). Also it is formed as a by-product of reactions such as the disinfection of water by ozonation but it is not known to form naturally. Although acute high-dose exposure is mainly via voluntary ingestion of hair products or accidental contamination of bread preparations, chronic low dose exposure is most often due to trace levels in food and water. In vitro genotoxicity tests have shown positive results for KBrO₃, including the bacterial reverse mutation assay (Ames test) (13), the chromosomal aberration (CA) test conducted in Chinese hamster cells (14) and the mouse lymphoma assay (15). It has also been shown to be chromosome damaging in vivo with the micronucleus test (MN) (16,17). In vivo and in vitro results indicate that KBrO₃ treatment increases 8OHdG incidence (18,19). The genotoxicity of KBrO₃ is reported to result in DSBs that cause large deletions instead of 8-hydroxydeoxyguanosine (8OHdG) leading to GC>TA transversions (11). These deletions are believed to be responsible for mutagenesis, carcinogenesis and aging (20,21). The mechanism of oxidative stress induced by KBrO₃ is not fully understood. Its mechanism of action (MOA) differs from the other general types of oxidative stress (11,22). Several studies stated that KBrO₃ causes renal cell adenomas and carcinomas by administration in drinking water after long-term oral administration, especially in rats and mice (13,23). Due to inadequate information on human exposure, International Agency of Research on Cancer Institute (IARC) classified its as a possible carcinogen to human category, Group 2B (24).

Menadione (2-methyl-1,4-naphthoquinone) also known as vitamin K₃ is among the quinone derivatives presenting vitamin K activity, which promotes the hepatic biosynthesis of blood clotting factors. Menadione is a dietary synthetic component, and it can be converted into vitamin K2 in the intestinal canal of the body (25). Menadione is used in the treatment of hypoprothrombinemia and cancer (4). The general reactivity of quinone compounds, resides in their ability to undergo redox-cycling and or to react directly with cellular nucleophiles such as protein and DNA. It has been reported that menadione caused the induction of CYP450 activity (26) and combination therapy with vitamin C exhibits synergistic antitumor activity (27). In the presence of flavo enzymes, it may undergo reduction to a semiquinone, which is an extremely unstable compound. It was shown that treatment with menadione caused SSBs using in vitro approaches and this indicates that menadione induced the formation of ROS including superoxide anion radical (O₂^-) (25,28,29). The genotoxic potency of menadione is dose-dependent and was clearly observed with in vitro studies (4,25,28) accomplished in mammalian cell lines using chromosomal approaches (e.g. MN, Sister Chromatographic Exchange (SCE) and CA.

Hydrogen peroxide (H₂O₂) is a natural physiological constituent of living cells, continuously produced by a variety of cellular pathways such as the inflammatory respiratory burst and during oxidative phosphorylation. It is also used in a wide range of external applications, for instance, in bleaching and in treatment of water and sewage (30–32). It is a natural source of oxidative damage in cells, causing a spectrum of DNA lesions, including single and double strand breaks (33). H₂O₂ is a non-radical ROS, but it can react by radical-mediated means; for example, in the presence of ferrous ions, H₂O₂ reacts through Fenton’s reaction to form a highly reactive hydroxyl radical (HO •) (34). Intracellular steady-state concentration of H₂O₂ above 1 µM is considered to cause oxidative stress inducing growth arrest and cell death (35,36). Dose dependent genotoxic effects of H₂O₂ were demonstrated in a study conducted in AHH-1 lymphoblastoid cell line between 0 – 30 µM (4). In another study which employed HepG2 cells to show DNA damaging effects of H₂O₂, it was demonstrated that genotoxic effects occurred between 25 µM and 50 µM (33).

Although the majority of human exposures involve long-term, chronic exposure (37) to low levels of compounds, high dosing and acute dosing regimes currently dominate in vitro testing (38). The US National Academy of Sciences report, ‘Toxicity Testing in the 21st Century [TT21C]: a Vision and a Strategy’ envisions that most of this safety testing will eventually be carried out in vitro using human cells. Current tests which identify cellular toxicity, specifically genotoxicity, include the Ames assay, SCE test and the in vitro micronucleus assay. These use a single, acute chemical dose, which requires administration with a suitable solvent, and exposure is over a short time period (3–6 hours, or 24 hours) (39). This short exposure time is not representative of everyday human exposure, which is often repeated and over a long period of time. A seminal publication (40) from our laboratory suggested that chronic dosing provides a reduced genotoxic response and is more in tune with the human exposure scenarios. In this recently published study, the effect of fractionated (chronic) doses of DNA alkylating agents methylmethane sulphonate (MMS) and N-methyl-N-nitrosourea (MNU) on TK6 human lymphoblast cells, over five and ten days were investigated (40). It was demonstrated in this study that acute dosing can overwhelm cellular defences and provide misleading positive results, chronic (fractionated dosing) produced reduced (MMS) and absent (MNU) genotoxicity.

The tumour suppressor gene p53 is mutated, deleted, or rearranged in more than half of all human tumours, confirming that the p53 protein is important in the aetiology of cancer (41). The p53 protein mediates key cellular responses to DNA damage and is involved in cell cycle regulation (42,43). For example, p53 function is required for the activation of a G1 cell cycle checkpoint after DNA damage and facilitates efficient DNA repair before replication (44). It is also well known that p53 mediates apoptosis caused by DNA damage (45). Cells deficient in the p53 protein can not arrest in G1 phase nor induce cell elimination with apoptosis after DNA damage and consequently may attempt replication of a damaged genome. Predictions based on this situation are that p53-deficient cells will have a mutator phenotype because cells with unrepaired DNA damage after genotoxic insult will continue to divide and thus accumulate further mutations. The increase in the mutations that occur will increase the cell’s neoplastic transformation potential. Therefore, p53 appears to be a molecular ‘genome protector’ by maintaining genetic stability in mammalian cells (44).

NH32 cells which are p53 deficient derivatives of TK6, were utilized in this study as they have been widely used in genetic toxicology studies and represent a versatile and reproducible system for examining p53’s role in genotoxic responses. TK6 (Thymine kinase heterozygote cell line) is one of standard cell lines used in vitro for genotoxicity testing and has been isolated from the lymphoblastoid cell line HH4 (46). NH32 is a p53 gene abrogated cell line derived from TK6. In order to examine whether p53 abrogation affects the susceptibility to pro-oxidant compounds, parallel studies were conducted in TK6 and NH32 (40,47).

In the study, we have compared acute v chronic dosing for 3 pro-oxidant chemicals, using TK6 and NH32 cells. We have deliberately used low dose ranges to prevent excessive toxicity in accordance with OECD guideline 487. For this purpose, before starting the main study dose range finding studies and the literature (4,11,28,33) were used to identify the lowest dose causing cellular damage.

Materials and methods

Chemicals

H₂O₂, KBrO₃, methyl methanesulfonate (MMS), N-Acetyl-L-cysteine (NAC), L-Buthionine- sulfoximine (BSO) and DCFHDA were all purchased from Sigma (Dorset, U.K.). Menadione was obtained from Merck Millipore. Phosphate buffer saline (PBS), dimethyl sulfoxide (DMSO), RPMI 1640 (without L glutamine) and glutamine were obtained from Gibco (Thermo Fisher Scientific, U.K.). All chemical dilutions used for treatment of cell line cultures were freshly prepared from stock solutions with ultra-pure water.

Cell culture

The human lymphoblastoid cell lines TK6 and NH32 (p53-competent and p53-deficient, respectively) were cultured in RPMI 1640 supplemented with 10% horse serum and 1% L-glutamine in an 80-cm² flask at 37°C, 5% CO₂ with 100% humidity. The cells were maintained at a concentration of 1–2 x 10⁵/ml.

Cell culture was initiated by thawing a frozen cell stock in 50 mL medium in TS-75 culture flasks. Cells were then cultured for experimental use. Cell density was determined by an automatic cell counter, Coulter Counter Z2, (Beckman Coulter, Inc., CA, US), and the cells were appropriately diluted to prevent overgrowth (<1 x 10⁶cells/min). Cell suspensions were prepared at 1 x 10⁵ cells/mL and treated with serial dilution with one of the chemicals described above for 24 h and 5 days (acute and chronic, respectively).

In the 5-day chronic exposure study, when the 3^rd dosing day was reached, the cells in the flasks were counted to keep the number of cells within the desired range. Then, the volume was adjusted to keep cells at 1 x 10⁵/mL. The cell volume used at this stage was recorded and used as a dilution factor for calculating RPD%. MMS was used to make positive control groups in all study, these concentrations were 0.585 μg/mL for chronic study and 2.925 μg/mL for acute study.

The mononucleate Micronucleus assay

MN frequency was utilized to assess the level of DNA damage. To allow chronic exposures and MN assessment, the mononucleate version of the MN assay was employed. TK6 and NH32 suspensions with cells at 1 x 10⁵/mL were seeded for 5 days at 37°C and 5% CO₂. Each flask (n=3, independently produced on different days) was dosed with appropriately diluted test chemical for chronic (5 day x 24 h) treatment, after which the cells were centrifuged, washed once in PBS, resuspended in 10-mL fresh media. In this study, two dosing methods were used. The first method is acute dosing, in which cells are exposed to the chemical agent for 24 hours. In the second method used, the total dose was divided into 5 days by dose fractionation (5 x 24 hours) and the cells were exposed to the 1/5 of the chemical every day. For example, 20 µM acute dose value for H₂O₂, by dose fractionation, cells were exposed to a concentration of 4 µM for each of 5 days. A total concentration of 10 µM Menadione was applied to the cells for 5 days at 2 µM. Likewise, the acute dose of KBrO3 of 0.8 mM was administered to the cells for 5 days x 1.6 mM in chronic dosing. Medium containing chemical was removed by centrifugation and replaced with fresh medium. Treated cells were then harvested after a further 24h recovery period, during which no further test chemical was added. Treated cells were resuspended in 10 mL of hypotonic solution (0.56% KCl) and centrifuged immediately. Cells were resuspended in fixative-1 (methanol:acetic acid:0.9% NaCl [5:1:6 parts]) and centrifuged after a 10-min incubation period. Cells were transferred to fixative-2 (methanol:acetic acid [5:1 parts] centrifuged, washed 3 times and maintained in the final fixative-2 wash at 4°C for 24 h. Fixed cells were centrifuged, and 100 µL was spread onto polished, fixed and hydrated slides, stained with DAPI (4’.5-d,amino-2-phenylindole) and viewed under a Carl Zeiss Axio Imager fluorescence microscope. Slides were scored utilizing the Metafer 4 software version 3.5 (MetaSystems, Altlussheim, Germany). The criteria for identifying MN were as previously described (48). A minimum of 2000 mononucleated cells were scored per replicate, and each dose was performed in triplicate (an average of 6000 mononucleated cell per dose).

To allow acute exposure and MN assessment, the mononucleate version of the MN assay was employed. TK6 and NH32 cell suspensions at 1 x 10⁵/mL were seeded for 24 h at 37°C and 5% CO₂ exactly as in the chronic exposure. Then, each flask (n=3, independently produced on different days) was dosed with appropriately diluted test chemical for acute (24 h) treatment. Unlike chronic dosing, chemical agents were applied to the cells with a single cumulative dose not exceeding 100 µL. Medium containing chemical was removed by centrifugation and replaced with fresh media. Treated cells were harvested after a 24-hour recovery period when no further test chemicals were added. After this stage, the cells are treated properly with hypotonic solution, fixative 1 and fixative 2 solutions, as applied in chronic dosing, and then fixed to the slides.

RPD% measurement

Initially, cells were seed in the T25 flasks as 1 x 10⁵ cell/mL. After 24 hours, they were counted by a Coulter counter and then were dosed with the appropriate concentration of chemical. For 24 hours after the cells were treated with these chemicals, they were transferred to flasks containing fresh media and allowed to recover for a day. At end of the recovery time cell were counted by the same way and it was calculated according to the RPD% equation. On third day out of 5 days chronic treatment, cells were counted and diluted again to ensure cell number was 1 x 10⁵ cell/mL.

$${\displaystyle \begin{array}{l}{\displaystyle \mathrm{R}\mathrm{P}\mathrm{D}\mathrm{\%}=\frac{Numberofpopulationdoublingsintreatedcultures}{Numberofpopulationdoublingsincontrolcultures}\mathrm{x}100}\end{array}}$$

$${\displaystyle \begin{array}{ll}& \mathrm{P}\mathrm{o}\mathrm{p}\mathrm{u}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{d}\mathrm{o}\mathrm{u}\mathrm{b}\mathrm{l}\mathrm{i}\mathrm{n}\mathrm{g}=\\ & \left[\mathrm{l}\mathrm{o}\mathrm{g}(\mathrm{P}\mathrm{o}\mathrm{s}\mathrm{t}-\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r}÷\mathrm{I}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r})\right]÷\mathrm{l}\mathrm{o}\mathrm{g}2\end{array}}$$

DCFHDA analysis

After treatment performing with chemicals, 100 µL cell suspensions were transferred into a black-clear bottom 96 well plate (n=3) which has 100 µL DCFHDA chemical (20 μM) on each well. Then plate was incubated and it was measured at 30 min, 1, 2, 3, 4,5, 6, 24 h a plate reader to control. Excitation wavelength was 485 nm and emission was 520 nm.

Alteration of cellular GSH levels

In order to modulate GSH levels prior to measuring the genotoxicity of the 3 pro-oxidants, GSH levels were both supplemented and depleted in independent experiments. Cells were dosed with 100 µM NAC to promote GSH levels prior 2 hours to pro-oxidant exposure and to increase cellular antioxidant status. Cells were also treated with 100 µM BSO, an inhibitor of GSH synthesis, for 2 hours prior to pro-oxidant treatment to assess the impact of GSH depletion.

Statistical analysis

Genotoxicity data were analysed using a one-way analysis of variance, followed by a Dunnett’s posthoc test, to determine if any of the treatment doses were significantly different from the untreated control. BSO or NAC data were evaluated compared to matched pro-oxidant treatment with a Student t test. Similarly, comparison of data obtained from acute and chronic dosing was evaluated with a Student t test.

Results

Chronic (fractionated) dosing reduces micronucleus frequency compared to acute dosing for H₂O₂ and menadione, but not KBrO₃ in TK6 cells

Dose-responses for chronic exposures (5 day x 24 h treatment + 24 h recovery) and acute exposures (24 h treatment + 24 h recovery) to the pro-oxidants H₂O₂, menadione and KBrO₃ were generated in both TK6 and NH32 cells (Fig. 1, 2 and 3). Cell viability was measured using the RPD% test, which is a sensitive early measure of cytotoxicity. The in vitro micronucleus assay was applied using a semi-automated image analysis system (Metafer, Zeiss) scoring 6000 cells per dose. In Fig. 1–3, clear dose dependent increases in MN are seen following the acute (24 h + 24 h) study in TK6 cells with H₂O₂, menadione and KBrO₃. The first significant doses showing increased MN frequencies in TK6 cells after acute dosing are 5 µM, 15 µM and 0.2 mM for H₂O₂, menadione and KBrO₃, respectively (Fig. 1–3). The H₂O₂ dose response in TK6 cells (Fig. 1) was accompanied by low levels of toxicity (reduced RPD%), however with menadione (Fig. 2) and KBrO₃ (Fig. 3), there were dramatic reductions in RPD% values in TK6 cells. The recommendations of the OECD 487 testing guideline for the in vitro MN assay, suggest RPD% values that are reduced by >55±5% (60% reduction) should be considered in the ‘too toxic’ category. Hence for menadione and for KBrO₃, the higher doses should be interpreted with caution.

Figure 1.

Cytotoxic effects and chromosomal damages in response to pro-oxidants. Comparative representation of MN frequency and RPD% results after acute and chronic (fractionated) dosing of TK6 and NH32 cell lines with 0 – 20 μM H₂O₂. Dose-response and relationships of hydrogen peroxide in the TK6 and NH32 cell lines with respect to micronucleus frequency and RPD% which as an indicator of cytotoxicity. On the left side, the genotoxic and cytotoxic responses of TK6 and NH32 cells to acute exposure with the concentrations indicated in the figure of H₂O₂, respectively, are observed. On the right of the figure, genotoxic and cytotoxic responses of their cells to chronic exposure to H₂O₂ are observed. Statistical evaluation was obtained by comparing the genotoxicity results of cells exposed to H₂O₂ with the result from the negative control located in the far left bar in the figures. P-value less than 0.05, 0.01 and 0.001 were summarized with one (*), two (**) and three (***) asterisks, respectively.

Figure 2.

Cytotoxic effects and chromosomal damages in response to pro-oxidants. Comparative representation of MN frequency and RPD% results after acute and chronic (fractionated) dosing of TK6 and NH32 cell lines with 0 – 15 μM menadione. Dose-response and relationships of menadione in the TK6 and NH32 cell lines with respect to micronucleus frequency and RPD% which as an indicator of cytotoxicity. On the left, the genotoxic and cytotoxic responses of TK6 and NH32 cells to acute exposure with the concentrations indicated in the figure of menadione, respectively, are observed. On the right of the figure, genotoxic and cytotoxic responses of their cells to chronic exposure to menadione are observed. Statistical evaluation was obtained by comparing the genotoxicity results of cells exposed to menadione with the result from the negative control located in the far left bar in the figures.

Figure 3.

Cytotoxic effects and chromosomal damages in response to pro-oxidants. Comparative representation of MN frequency and RPD% results after acute and chronic (fractionated) dosing of TK6 and NH32 cell lines with 0 – 0.8 mM KBrO₃. Dose-response and relationships of potassium bromate in the TK6 and NH32 cell lines with respect to micronucleus frequency and RPD% which as an indicator of cytotoxicity. On the left, the genotoxic and cytotoxic responses of TK6 and NH32 cells to acute exposure with the concentrations indicated in the figure of KBrO₃, respectively, are observed. On the right of the figure, genotoxic and cytotoxic responses of their cells to chronic exposure to KBrO3 are observed. Statistical evaluation was obtained by comparing the genotoxicity results of cells exposed to KBrO₃ with the result from the negative control located in the far left bar in the figures.

In all 3 datasets, chronic (fractionated) application of the pro-oxidants across 5 days led to reduced toxicity (increased RPD% level relative to control), in contrast to the highly toxic acute treatment for menadione and KBrO₃ (Fig. 2–3). In addition, in the case of H₂O₂ and menadione, the chronic exposure regime also reduced overall MN induction in TK6 cells (Fig. 1 and 2). However, this was not the case with KBrO₃, where chronic treatment appeared to actually increase overall levels of MN induction compared to acute dosing (Fig. 3). Whilst the dose giving the first statistically significant increase in micronucleus frequency above control levels was the same in both acute and chronic treatment for H₂O₂ and menadione, the scale of the induction was clearly greater under acute treatment. For example, in TK6 cells at 15 µM and 20 µM H₂O₂, the acute MN frequencies were 3 fold and 3.6 fold higher than control values respectively, but for chronic exposure these was reduced to 2.3 and 2.5 fold the control values respectively. Similarly, for menadione at 5 µM and 10 µM (15 µM too toxic in acute data), in TK6 cells the acute MN levels were 1.4 fold and 1.6 fold that of control respectively, but these were reduced to 1.2 and 1.6 fold after chronic dosing.

In the case of KBrO₃, it is interesting to note that chronic (fractionated) dosing led to greater increases in MN induction. After dosing with 0.2 and 0.4 µM (0.8 µM too toxic with acute data), the MN induction after acute dosing was 3.7 fold and 6.5 fold the control TK6 values respectively, after chronic treatment, this was increased to 4.0 fold and 7.1 fold the control values.

p53 deficiency increases the sensitivity of the cells to pro-oxidant chemicals

Untreated NH32 cells displayed a significant (for acute dosing p<0.001; chronic dosing p<0.01) increase in background MN level compared to untreated TK6 cells (Fig. 1–3) after both acute and chronic dosing. When examining the pro-oxidant dose responses obtained in parallel in TK6 and NH32 cells, it was clear that the NH32 cells were much more sensitive to the pro-oxidants in general (Fig. 1–3). NH32 responses show less induced cytotoxicity after exposure to the pro-oxidants with less of a reduction in RPD% levels, suggesting a lower level of ROS induced necrosis and/or apoptosis in these p53 null cells. In terms of MN induction, the NH32 cells display more obvious increased levels of MN induction compared to TK6 cells. This is true particularly for menadione and KBrO₃, where the MN induction doubled compared to TK6 cells at the higher doses. In the acute dosing experiments, TK6 MN levels reached a maximum of 5% (6.5 fold that of control) excluding the highly toxic dose points. In NH32 cells after acute dosing, the equivalent MN frequencies reached up to 17% (12.2 fold the control level). Given the lower level of induced cytotoxicity in NH32 cells, it’s important to note that this increased MN induction occurred in surviving cells at non-toxic doses. This is particularly evident for KBrO₃, where for acute dosing the RPD% level hits around 30% at 0.4 mM for TK6 cells but 30% RPD% is achieved at 0.8 mM for NH32.

In terms of the chronic v acute responses in NH32 cells, a similar overall effect as was seen in TK6 cells is noted for NH32 cells, for H₂O₂ and menadione. That is, less MN induction was noted in chronic (fractionated) treatment compared to acute treatment (Fig. 1, 2). For example, for H₂O₂ at 15 µM and 20 µM, the MN level was 2.2 fold and 3.2 fold that of control respectively, whereas for the chronic treatment this was 1.5 fold and 2.7 fold the control levels respectively. These changes between H₂O₂ and acute and chronic dosing were non-significant (p>0.05). For menadione, at the 5 µM and 10 µM dose (15 µM too toxic in the acute study), the MN fold difference rose to 2.1 fold and 2.9 fold that of control, but for chronic treatment it was 1.3 and 1.9 fold control values respectively. These changes between menadione and acute and chronic treatment with 10 µM was significant (p<0.05) thought dosing with 5 µM was non-significant. Whereas for KBrO₃, the same (or slightly greater) enhanced responses was seen for chronic treatment compared to acute treatment, as was noted above in TK6 cells (Fig. 3). For example, at the doses of 0.6 and 0.8 mM KBrO₃, the MN level was 8.7 fold and 12.2 fold the control values for acute treatment respectively, whilst for chronic treatment these equivalent values were 9.2 fold and 11.1 fold the control values respectively. However, these changes between KBrO₃ and acute and chronic dosing were not significant (p>0.05).

The impact of antioxidant status on pro-oxidant induced DNA damage.

To better understand the role of cellular antioxidant enzymes in the DNA damage responses seen in Fig. 1–3, TK6 cells were exposed to fractionated doses of the 3 pro-oxidants as above, but co-treated with a glutathione precursor (NAC) or inhibitor (BSO) to modulate glutathione protection. To supplement glutathione, TK6 cells were co-treated with 100µM N acetyl cysteine and glutathione was inhibited in parallel using the known glutathione inhibitor BSO (100 µM). The results of these experiments are shown in Fig. 4 and Supplementary Table-I. NAC had a non-significant effect on MN levels in untreated TK6 cells (a 1.3 fold reduction). NAC, however significantly abrogated the MN induction by both H₂O₂ (1.6 fold reduction) and menadione (1.6 fold reduction), but had no significant effect on KBrO₃ treated cells (1.2 fold reduction). BSO had the opposite effect, as expected, significantly increasing MN induction in untreated cells (1.7 fold increase) and in TK6 cells treated with KBrO₃ (1.3 fold increase). BSO non-significantly increased MN levels in H₂O₂ (1.3 fold increase) and menadione treated TK6 cells (1.4 fold increase). This data confirms the role of glutathione (and the cellular antioxidant machinery) in abrogating pro-oxidant induced DNA damage.

Figure 4.

The effect of changing the antioxidant levels of the cells on the genotoxic activities caused by prooxidants.. Cells were exposed to NAC and BSO for two hours before being exposed to the prooxidant chemicals indicated in the x-axis of the figure. Statistical evaluation was obtained by comparing the genotoxicity results of cells exposed to chemicals with the result from the negative control located in the far left bar in the figures.

In the study, although the cells were exposed to only 2 hours of incubation with BSO and NAC, the observed changes in MN values as seen in Fig. 4 and Supplementary Table I showed that this time was sufficient for the efficacy of BSO to be evident.

Table I.

The frequency of MN, MN Fold Change and RPD% Changes After Acute and Chronic Exposure of TK6 and NH32 Cells to Prooxidant Chemicals The data shown individual MN levels as % MN frequency plus fold increase over control value (in brackets)

Acute									Chronic
	TK6				NH32					TK6				NH32
H2O2 (µM)	MN	Fold change	p value	RPD%	MN	Fold change	p value	RPD%	H2O2 (µM)	MN	Fold change	p value	RPD%	MN	Fold change	p value	RPD%
Cont.	0.78	-	-	100.00	1.41	-	-	100.00	Cont.	0.81	-	---	100.00	1.51	-	---	100,00
5	1.38	1.8	**	99.92	1.87	1.3	-	95.97	5	1.44	1.8	*	89.78	1.63	1.1	-	98.48
10	1.88	2.4	***	99.38	2.80	2.0	**	95.02	10	1.60	2.0	*	91.63	1.94	1.3	-	99.53
15	2.31	3.0	***	99.13	3.05	2.2	**	95.43	15	1.86	2.3	**	89.02	2.30	1.5	-	103.85
20	2.77	3.6	***	67.09	4.49	3.2	***	88.28	20	2.06	2.5	***	87.69	4.02	2.7	**	92.75
	TK6				NH32					TK6					NH32
Mena (µM)	MN	Fold change	p value	RPD%	MN	Fold change	p value	RPD%	Mena (µM)	MN	Fold change	p value	RPD%	MN	Fold change	p value	RPD%
Cont.	0.78	-	---	100.00	1.41		---	100.00	Cont.	0.81	-	---	100.00	1.51	-	---	100.00
2	0.81	1.0	-	99.54	1.93	1.4	-	101.89	2	0.86	1.1	-	101.34	1.70	1.1	-	99.91
5	1.09	1.4	-	72.92	2.90	2.1	***	89.11	5	1.00	1.2	-	101.56	1.96	1.3	-	98.38
10	1.26	1.6	-	32.12	4.10	2.9	***	40.07	10	1.31	1.6	-	100.94	2.81	1.9	***	95.18
15	1.91	2.5	***	-68.72	5.32	3.8	***	-3.45	15	1.34	1.7	**	96.03	3.31	2.2	***	90.45
	TK6				NH32					TK6						NH32
KBrO3 (mM)	MN	Fold change	p value	RPD%	MN	Fold change	p value	RPD%	KBrO3 (mM)	MN	Fold change	p value	RPD%	MN	Fold change	p value	RPD%
Cont.	0.78	-	---	100.00	1.41	-	---	100.00	Cont.	0.81	-		100.00	1.51	-	---	100.00
0.2	2.90	3.7	***	81.27	5.25	3.7	**	88.97	0.2	3.27	4.0	*	90.11	7.62	5.1	***	96.75
0.4	5.05	6.5	***	27.47	9.68	6.9	***	62.91	0.4	5.76	7.1	***	76.09	10.90	7.2	***	87.68
0.6	6.82	8.7	***	-13.15	12.19	8.7	***	54.37	0.6	7.88	9.7	***	55.00	13.81	9.2	***	85.77
0.8	8.20	10.5	***	-17.78	17.19	12.2	***	30.41	0.8	10.11	12.5	***	38.26	16.78	11.1	***	74.72

H₂O₂ and menadione induce ROS detectable by the fluorescent reporter DCFHDA

To further explore ROS induction within TK6 cells and NH32 cells, we employed the fluorescent reporter DCFHDA to measure the time course of ROS induction in the cells after treatment with H₂O₂, menadione and KBrO₃. For H₂O₂ and menadione, significant ROS induction was observed from as early as 1 hour post exposure in both TK6 and NH32 cells (Fig. 5). However, very little ROS induction was detectable with KBrO₃, with some higher doses and later time point (24 hour) data points only showing an effect. There was no detectable difference in response between the TK6 cells and NH32 cells in terms of DCFHDA fluorescence.

Figure 5.

Comparative representation of oxidative stress measured by DCFHDA method caused by prooxidant chemicals (H₂O₂, menadione and KBrO₃) in TK6 and NH32 cell lines. Statistical evaluation was obtained by comparing the ROS results of cells exposed to chemicals with the result from the negative control located in the left bar in each part of the figure.

Discussion

Here we compared the effects of acute and chronic dosing with 3 pro-oxidant chemicals in p53 proficient and p53 deficient lymphoblastoid cells. Overall, as we expected, we saw for H₂O₂ and menadione a reduction in toxicity and genotoxicity when the exposure is fractionated (but the same cumulative dose). However, surprisingly, with KBrO₃ we see equivalent, if not enhanced genotoxicity with the fractionated dosing compared to acute dosing. The effect was seen in both TK6 (p53 competent) and NH32 (p53 deficient) cells. This suggests that the DNA damage response controlled by p53 is not responsible for these noted effects.

Acute v chronic dosing

We were particularly interested in the acute v chronic responses to pro-oxidants, as chronic exposures are more physiologically relevant in terms of human exposure to these types of agents. It is possible that much of the existing genotoxicity data generated in vitro using acute exposure regimes over-estimates the genotoxic threat of chemicals as acute dosing can overwhelm cellular defences. Our acute data for pro-oxidants here shows similar responses to other previously published data (Table II). It has previously been noted that lowest observed adverse effect level (LOAEL) for H₂O₂ range from 18 – 40 μM compared to our 5–10 μM here. For KBrO₃, published LOAEL range from 0.6 – 5 mM compared to our 0.2 mM here. Finally, for menadione, previously published LOELs range from 2.9–5.8 μM compared to our 5–15 μM here.

Table II.

LOELs reported in other studies for H₂O₂, menadione and KBrO₃

	Cell line	Pro-oxidant chemical	Treatment/Exposure time	Doses	Statistically significant dose (LOEL)	Study
1	TK6	H2O2	4 h	6 – 30 µM	18 µM	Wilde et al. 2018 (49)
2	TK6	H2O2	4 h	20 – 320 µM	-40 µM for MN, -80 µM for Alkaline COM assay	Kimura et al. (50)
	AHH-1	H2O2	4 h	2 – 30 µM	-25 µM for automated method, -25 µM for visual method	Seager et al. 2014 (51)
3	TK6	KBrO3	4 h	0.5– 5 mM	-5 mM for MN, -2.5 mM for Alkaline COM -1 mM for Neutral COM assay	Luan et al. (2007) (11)
4	TK6	KBrO3	24 h	6.25 – 100 µM	N.S.	Platel et al. 2009 (52)
5	AHH-1	KBrO3	4 h	0.1 – 0.8 mM	-0.6 mM for automated method, -0.5 mM visual method	Seager et al. 2014 (51)
6	AHH-1	Menadione	4 h	0.8 – 8.6 µM	-2.9 µM for automated method, -5.8 µM for visual method	Seager et al. 2014 (51)

N.S.: Not significant; COM: Comet Assay

In the case of H₂O₂ and menadione, our original hypothesis was supported, that is, that fractionated dosing would lead to lower toxicity and lower genotoxicity compared to equivalent acute dosing. This is evident in (Fig. 1,2) where we saw a 23% reduction in micronucleus formation at 15 µM H₂O₂ and a 32% reduction in MN with menadione, when dosed chronically. In particular, we noted that fractionated dosing led to less toxicity as measured by RPD%. This is important as acute dosing with menadione induced clear toxicity leading to the MN induction at the higher doses having to be interpreted with caution due to the OECD guidelines for the MN assay (39) which state that toxicity should be <60% maximum. The fractionation of these doses reduced this toxicity allowing these same doses to be suitable for MN analysis after chronic exposure (see Fig. 2).

The reduced genotoxicity observed in the fractionated dosing with H₂O₂ and menadione is presumably due to several mechanisms. This could include antioxidant neutralisation of the low levels of ROS generated by these 2 chemicals. This protective antioxidant response would include both cell culture antioxidant reagents (e.g. sequestration by serum proteins) and cellular antioxidant enzymes (SOD, glutathione, catalase etc). In addition, the reduced genotoxicity observed with the fractionation of doses could be due to DNA repair processes which readily remove oxidised DNA bases in the chronic dosing scenario, but become overwhelmed with the acute dosing. Hence, the low dose ranges used here are even lower when they are fractionated (fifths per day) and allow cellular tolerance to the genotoxic effects.

KBrO₃

In contrast to the other 2 pro-oxidants, we observed that KBrO₃ showed higher MN induction in all fractionated doses. This was unexpected and is contrary to the situation with H₂O₂ and Menadione. If we accept that antioxidant capacity and DNA repair lead to reduced genotoxicity for fractionation of H₂O₂ and menadione, then why is KBrO₃ different? We saw that fractionated KBrO₃ dosing caused increasing MN induction, for example at 0.6 mM, KBrO₃ induced 11.5% and 19.1% MN in acute and chronic dosing, respectively (see Table III)

Table III.

Comparison of MN % inductions caused by the acute and chronic dosing of KBrO₃

KBrO3 (mM)	Acute dosing MN inductions (%)	Chronic dosing MN inductions (%)
Control	100	103.9
0.2	100	112.8
0.4	100	114.1
0.6	100	115.5
0.8	100	119.1

It is also interesting to note that although the toxicity (as measured by RPD%) was reduced with fractionated KBrO₃ compared to acute dosing with KBrO₃ (Fig. 3), it failed to be abrogated to the extent seen with menadione (Fig. 2). For example; in acute dosing of 5 and 10µM menadione, while RPD% values were 72.9% and 32.1%, respectively, it was observed that these values increased to 101.6% and 100.9% with chronic (fractionated) administration. Similarly, in acute dosing of 15 and 20 µM H₂O₂, RPD% values were 95.4% and 88.3%, respectively, it was observed that these values increased to 103.9% and 92.8% with chronic (fractionated) administration. However, with 0.4 and 0.6 mM acute KBrO₃ application, the RPD% values were 27.5% and (-13.15%), respectively. In the chronic dosing experiments of the same doses, RPD% values could only increase to 76.09 and 55.0%, respectively (Supplementary Fig. 1).

We are uncertain as to the exact mechanism behind the enhanced genotoxicity noted with chronically dosed KBrO₃ compared to acute dosing. However, it may be explained in some way by the fact that KBrO₃ behaves differently to other prooxidants. Here we were able to show strong ROS induction by H₂O₂ and menadione with the fluorescent reported DCFHDA, but little ROS induction was measured with KBrO₃ using DCFHDA (Fig. 5). In addition, previous reports have demonstrated that KBrO₃ operates in an unusual manner with respect to ROS generation and may be regarded more as a glutathione depletor (11,12,53–56). So, if antioxidant capacity is important for detoxification of the pro-oxidants, then the fact that KBrO₃ induces a different type of ROS might explain the differing response in chronic v acute dosing. It is also maybe possible that KBrO₃ induces DNA damage less amenable to DNA repair, which accumulates in the genome causing enhanced effects in the chronic dosing situation.

We also examined the dose responses of these 3 pro-oxidants in both p53 competent (TK6) and p53 null cell (NH32) models, which were isogenic. We showed that overall, the same effects were observed. That is in the case of H₂O₂ and menadione that fractionation reduced both toxicity and genotoxicity. Also, that with KBrO₃, this reduced effect with fractionated dosing was also not seen in NH32 cells. Hence, the same overall effects were observed suggesting that the unexpected effects seen with KBrO₃ are not p53 dependent. Hence, the DNA damage response and the downstream effects on cell cycle progression are perhaps not relevant to the KBrO₃ specific response noted here.

It was noted that the NH32 cell line had much higher background MN frequencies than that of TK6 cells. Other studies have noted similar enhanced sensitivities in NH32 cells (40,57–60). In the vehicle control group, the MN frequencies were 0.78 and 1.41 for TK6 and NH32 cells, respectively, for acute treatment. Similarly, in the chronic treatment study, vehicle control’s MN frequencies were 0.81 and 1.51, for TK6 and NH32 cell, respectively, showing the impact of p53 on genome stability. Also, the prooxidant induced toxicity (as measured by RPD%) was seen to be lower overall in NH32 cells compared to TK6 (see Fig. 3 in particular). This was particularly noted with the KBrO₃ exposures (acute and chronic). This may suggest a reduced apoptotic response in NH32 cells due to lack of p53 which would lead to less cell death and greater cell proliferation despite DNA damage.

In the future it may be possible to use 8-OHdG measurements to detect the specific genotoxic damage in prooxidant treated cells allowing the mechanism of DNA damage induction to be assessed. At the same time, it may be valuable to determine the 8-OHdG level in the cells in alongside the changes in MN fold change due to the application of NAC and BSO to the cells. However, the data shown here using MN, which were clearly dose-dependent in all Figures 1, 2, 3, 4, 5 and Supplementary Fig. 1) shows that the MN test performed is sensitive enough to detect oxidative damage to DNA. Similarly, FPG comet assay, is an important method in detecting oxidative DNA damage, and it would be interesting to compare data generated with FPG comet with the data obtained with MN measured with the Metafer system. The research method used in the detection of DNA damage due to acute and chronic proxidant exposure in this study has clearly shown that the chemical concentrations used in the study are reliable for detecting their genotoxic activities.

We show here the importance of chronic dosing to fully explore the hazards posed by exposures to relevant human carcinogens. Previously the sole focus on acute (and high dose) exposures has limited our understanding of how physiological levels of genotoxins damage DNA. We propose that chronic dosing is used more widely in vitro to explore chemically induced adverse outcomes. We have used a 5 day treatment period here, as we have used this exposure duration previously (40), however, other chronic dosing regimes should be explored. In particular, the fractionated dosing here shows us that for H₂O₂ and menadione, we observed less genotoxicity and less toxicity giving us a more holistic view of the hazards posed by these agents, without the confounding toxicity accompanied by acute dosing and high doses. Crucially, the chronic treatment has highlighted a mechanistic difference with KBrO₃ which would not have been evident with acute dosing alone. Whether this is a feature unique to KBrO₃, or is a more general effect with other genotoxins clearly requires further study.

To whom correspondence should be addressed at Faculty of Pharmacy, Sivas Cumhuriyet University, Sivas, 58140, Turkey, Fax: [phone]

Acknowledgements

This work was supported by The Scientific and Technological Research Council of Turkey (TUBITAK) under The Science Fellowships and Grand Programme [1059B191800378].

Conflict of interest statement: None declared.