A comprehensive study of the harmful effects of ZnO nanoparticles using Drosophila melanogaster as an in vivo model.

This study planned to determine the range of biological effects associated with ZnO-NP exposure using Drosophila melanogaster as an in vivo model. In addition, ZnCl2 was used to determine the potential role of Zn ions alone. Toxicity, internalization through the intestinal barrier, gene expression changes, ROS production, and genotoxicity were the end-points evaluated. No toxicity or oxidative stress induction was observed in D. melanogaster larvae, whether using ZnO-NPs or ZnCl2. Internalization of ZnO-NPs through the intestinal barrier was observed. No significant changes in the frequency of mutant clones (wing-spot test) or percentage of DNA in tail (comet assay) were observed although significant changes in Hsp70 and p53 gene expression were detected. Our study shows that ZnO-NPs do not induce toxicity or genotoxicity in D. melanogaster, although uptake occurs and altered gene expression is observed.

Genotoxic and cell-transforming effects of titanium dioxide nanoparticles.

The in vitro genotoxic and the soft-agar anchorage independent cell transformation ability of titanium dioxide nanoparticles (nano-TiO2) and its microparticulated form has been evaluated in human embryonic kidney (HEK293) and in mouse embryonic fibroblast (NIH/3T3) cells. Nano-TiO2 of two different sizes (21 and 50 nm) were used in this study. The comet assay, with and without the use of FPG enzyme, the micronucleus assay and the soft-agar colony assay were used. For both the comet assay and the frequency of micronuclei a statistically significant induction of DNA damage, was observed at the highest dose tested (1000 µg/mL). No oxidative DNA damage induction was observed when the comet assay was complemented with the use of FPG enzyme. Furthermore, long-term exposure to nano-TiO2 has also proved to induce cell-transformation promoting cell-anchorage independent growth in soft-agar. Results were similar for the two nano-TiO2 sizes. Negative results were obtained when the microparticulated form of TiO2 was tested, indicating the existence of important differences between the microparticulated and nanoparticulated forms. As a conclusion it should be indicated that the observed genotoxic/tranforming effects were only detected at the higher dose tested (1000 µg/mL) what play down the real risk of environmental exposures to this nanomaterial.

Antioxidant and antigenotoxic properties of CeO2 NPs and cerium sulphate: Studies with Drosophila melanogaster as a promising in vivo model.

Although in vitro approaches are the most used for testing the potential harmful effects of nanomaterials, in vivo studies produce relevant information complementing in vitro data. In this context, we promote the use of Drosophila melanogaster as a suitable in vivo model to characterise the potential risks associated to nanomaterials exposure. The main aim of this study was to evaluate different biological effects associated to cerium oxide nanoparticles (Ce-NPs) and cerium (IV) sulphate exposure. The end-points evaluated were egg-to-adult viability, particles uptake through the intestinal barrier, gene expression and intracellular reactive oxygen species (ROS) production by haemocytes, genotoxicity and antigenotoxicity. Transmission electron microscopy images showed internalisation of Ce-NPs by the intestinal barrier and haemocytes, and significant expression of Hsp genes was detected. In spite of these findings, neither toxicity nor genotoxicity related to both forms of cerium were observed. Interestingly, Ce-NPs significantly reduced the genotoxic effect of potassium dichromate and the intracellular ROS production. No morphological malformations were detected after larvae treatment. This study highlights the importance of D. melanogaster as animal model in the study of the different biological effects caused by nanoparticulated materials, at the time that shows its usefulness to study the role of the intestinal barrier in the transposition of nanomaterials entering via ingestion.

Genotoxicity and DNA repair processes of zinc oxide nanoparticles.

Two different sizes of zinc oxide nanoparticles (ZnO NP, ≤ 35 nm and 50-80 nm) were tested in the human lymphoblastoid cell line TK6 to increase our knowledge on their genotoxic potential. The comet assay was the system used, and the results obtained showed that the highest concentration tested (100 µg/ml) for the two selected compounds was genotoxic. The percent DNA in tail obtained after treatment with ZnO NP (≤ 35 nm) was significantly higher than that of ZnO NP (50-80 nm) at all concentrations tested. To investigate the nature of the induced genotoxic damage, specific enzymes recognizing oxidized DNA bases were used. Treatments with endonuclease III (Endo III) and formamidopyrimidine DNA glycosylase (FPG) demonstrated that only ZnO NP (50-80 nm) were able to induce significant levels of net oxidative DNA damage. Further DNA repair kinetics studies revealed that DNA damage initially induced was removed in approximately 5 h. DNA damage induced by ZnO NP was repaired more slowly than damage following microparticulated ZnO exposure. No marked differences in repair kinetics of both forms of ZnO NP were observed. Evidence indicates that a high proportion of DNA damage induced by ZnO NP (50-80 nm) correlated with induction of oxidative damage, and that both forms of ZnO NP interfere with mechanisms involved in DNA damage repair.

Genomic instability in newborn with short telomeres.

UNLABELLED: Telomere length is considered to be a risk factor in adults due to its proved association with cancer incidence and mortality. Since newborn present a wide interindividual variation in mean telomere length, it is relevant to demonstrate if these differences in length can act also as an early risk indicator. To answer this question, we have measured the mean telomere length of 74 samples of cord blood from newborns and studied its association with the basal genetic damage, measured as the frequency of binucleated cells carrying micronuclei. In addition, we have challenged the cells of a subgroup of individuals (N = 35) against mitomycin-C (MMC) to establish their sensitivity to induced genomic instability. Results indicate that newborn with shorter telomeres present significantly higher levels of genetic damage when compared to those with longer telomeres. In addition, the cellular response to MMC was also significantly higher among those samples from subjects with shorter telomeres. Independently of the causal mechanisms involved, our results show for the first time that telomere length at delivery influence both the basal and induced genetic damage of the individual. IMPACT: Individuals born with shorter telomeres may be at increased risk, especially for those biological processes triggered by genomic instability as is the case of cancer and other age-related diseases.

Zinc oxide nanoparticles: genotoxicity, interactions with UV-light and cell-transforming potential.

The in vitro genotoxic and the soft agar anchorage independent cell transformation ability of zinc oxide nanoparticles (NPs) and its bulky forms have been evaluated in human embryonic kidney (HEK293) and in mouse embryonic fibroblast (NIH/3T3) cells, either alone or in combination with UVB-light. The comet assay, with and without the use of FPG and Endo III enzymes, the micronucleus assay and the soft-agar colony assay were used. For the comet assay a statistically significant induction of DNA damage, with and without the enzymes, were observed up of 100µg/mL. ZnO NPs were able to increase significantly the frequency of micronuclei, and similar results were observed in the cell transformation assay where such NPs were able to induce cell-anchorage independent growth. These effects were observed at doses up 100µg/mL. Although UVB-light was able to induce genotoxic damage and cell-anchorage growth, a significant antagonist interaction effect was observed in combination with ZnO NPs. These in vitro results, obtained with the selected cell lines, contribute to increase our genotoxicity database on the ZnO NPs effects as well as to open the discussion about their risk in photo-protection sun screens.

Micronucleus frequency in copper-mine workers exposed to arsenic is modulated by the AS3MT Met287Thr polymorphism.

Arsenic(III)methyltransferase (AS3MT) has been demonstrated to be the key enzyme in the metabolism of arsenic as it catalyses the methylation of arsenite and monomethylarsonic acid (MMA) to form methylated arsenic species, which have higher toxic and genotoxic potential than the parent compounds. The aim of this study is to evaluate if genetic variation in the AS3MT gene influences arsenic-induced cytogenetic damage, measured by the micronucleus (MN) assay. AS3MT Met287Thr allele frequencies and MN values were determined for 207 subjects working in the copper-mine industry, who were exposed to variable levels of arsenic. The urinary arsenic profile was used as individual biomarker of arsenic exposure. Results indicate that the MN frequencies found in peripheral blood lymphocytes of the exposed population poorly correlate with the levels of total arsenic content in urine. Nevertheless, when workers were classified according to their AS3MT Met287Thr genotypes, significantly higher MN values were observed for those carrying the variant allele [odds ratio (OR), 3.4 (1.6-5.2); P=0.0003)]. To our knowledge, these results are the first to show that genetic variation in AS3MT, especially the Met287Thr polymorphism, may play a role in modulating the levels of arsenic-induced cytogenetic damage among individuals chronically exposed to arsenic.

In vivo genotoxicity assessment of titanium, zirconium and aluminium nanoparticles, and their microparticulated forms, in Drosophila.

As in vivo system, we propose Drosophila melanogaster as a useful model for study the genotoxic risks associated with nanoparticle exposure. In this study we have carried out a genotoxic evaluation of titanium dioxide (TiO2), zirconium oxide (ZrO2) and aluminium oxide (Al2O3) nanoparticles and their microparticulated forms in D. melanogaster by using the wing somatic mutation and recombination assay. This assay is based on the principle that loss of heterozygosis and the corresponding expression of the suitable recessive markers, multiple wing hairs and flare-3, can lead to the formation of mutant clones in treated larvae, which are expressed as mutant spots on the wings of adult flies. Third instar larvae were feed with TiO2, ZrO2 and Al2O3 nanoparticles, and their microparticulated forms, at concentrations ranging from 0.1 to 10mM. Although a certain level of aggregation/agglomeration was observed in solution, it must be noted than the constant digging activity of larvae ensures that treated medium pass constantly through the digestive tract ensuring exposure. The results showed that no significant increases in the frequency of all spots (e.g. small single, large single, twin, total mwh and total spots) were observed, indicating that these nanoparticles were not able to induce genotoxic activity in the wing spot assay of D. melanogaster. Negative data were also obtained with the microparticulated forms. This indicates that the nanoparticulated form of the selected nanomaterials does not modify the potential genotoxicity of their microparticulated versions. These in vivo results contribute to increase the genotoxicity database on the TiO2, ZrO2 and Al2O3 nanoparticles.

Genotoxicity of cobalt nanoparticles and ions in Drosophila.

Nanogenotoxicology is an emergent area of research, relevant for estimating the potential carcinogenic risk of nanomaterials. Since most of the approaches use in vitro studies, and neglecting the whole organism limits the accuracy of the obtained results, we have used Drosophila melanogaster to study the possible genotoxic potential of cobalt nanoparticles (Co NPs). The wing somatic mutation and recombination test has been the test of choice. This test is based on the principle that the loss of heterozygosis and the corresponding expression of the suitable recessive markers, multiple wing hairs and flare-3 can lead to the formation of mutant clone cells in growing up larvae, which are expressed as mutant spots on the wings of adult flies. Co NPs, as well as the ionic form cobalt chloride, were given to third instar larvae through the food, at concentrations ranging from 0.1 to 10 mM. The results obtained indicate that both cobalt forms are able to induce significant increases in the frequency of mutant clones. Although at low concentrations only Co NPs were genotoxic, the level of genetic damage obtained at the highest dose tested of cobalt chloride (10 mM) showed a significant higher increase in the frequency of total spots than those observed after the treatment with cobalt nanoparticles. As conclusion, our results indicate that Co NPs were able to induce genotoxic activity in the wing-spot assay of D. melanogaster, mainly via the induction of somatic recombination. The differences observed in the behaviour of the two selected cobalt forms may result from differences in the uptake.

Mutagenic/recombinogenic effects of four lipid peroxidation products in Drosophila.

The human diet is an important factor in the development of different diseases. Lipid peroxidation during frying in edible vegetable liquid oils of food components is a mechanism leading to the formation of free radicals. Such radicals induce tissue damage and are implicated in diverse pathological conditions, including aging, atherosclerosis, brain disorders, cancer, lung disorders and various liver disorders. In the present study, we decided to investigate the genotoxic effects of four lipid peroxidation products in the in vivo Drosophila wing somatic mutation and recombination test. In this test, point mutation, chromosome breakage and mitotic recombination produce single spots; while twin spots are produced only by mitotic recombination. Drosophila is a suitable eukaryotic organism for mutagenicity studies and also its metabolism is quite similar to that of mammalians. Since conflicting data exist on the possible risk of several lipid peroxidation products for humans, we have selected four of them, namely acrolein, crotonaldehyde, 4-hydroxy-hexenal (4-HHE) and 4-oxo-2-nonenal (4-ONE). Especially at the highest concentrations tested all exert both mutagenic and recombinogenic effects in the Drosophila SMART assay, showing a direct dose-effect relationship. This is the first study reporting genotoxicity data in Drosophila for these compounds.

Genotoxic analysis of four lipid-peroxidation products in the mouse lymphoma assay.

Lipid-peroxidation products are formed by the thermal treatment of foodstuffs, as well as by endogenous processes. In addition, they are also common environmental pollutants originating from many different sources. Since conflicting data exist on their possible risk for humans, we have selected four lipid-peroxidation products namely acrolein, crotonaldehyde, 4-hydroxy-hexenal (4-HHE) and 4-oxo-2-nonenal (4-ONE) to determine their ability to induce mutagenicity in mammalian cells. There is an important lack of mutagenicity data on mammalian cells for such products, which presents an important gap for any risk-assessment estimation. We have used the mouse lymphoma assay (MLA) to determine the mutagenic potential of these four compounds. This assay detects a broad spectrum of mutational events, from point mutations to chromosome alterations. The results obtained indicate that the four selected compounds are mutagenic in the MLA assay, showing a direct dose-effect relationship. The relative mutagenic potencies according to the induced mutant frequency (IMF) are as follows: crotonaldehyde (IMF=758.5×10(-6)), 4-ONE (IMF=700.5×10(-6)), acrolein (IMF=660.5×10(-6)) and 4-HHE (IMF=572×10(-6)). Although the differences between the induced mutant frequencies for these compounds are not very large, the α,ß-unsaturated aldehyde 4-oxo-2-nonenal turned out to be the agent most mutagenic. This is because its induced mutant frequency was reached after treatment with 10µM, while 50µM of the other compounds was needed to reach the reported frequencies.

Genotoxicity testing of two lead-compounds in somatic cells of Drosophila melanogaster.

The in vivo genotoxic activity of two inorganic lead compounds was studied in Drosophila melanogaster by measurement of two different genetic endpoints. We used the wing-spot test and the comet assay. The comet assay was conducted with larval haemocytes. The results from the wing-spot test showed that neither lead chloride, PbCl(2), nor lead nitrate, Pb(NO(3))(2), were able to induce significant increases in the frequency of mutant spots. In addition, the combined treatments with gamma-radiation and PbCl(2) or Pb(NO(3))(2) did not show significant variations in the frequency of the three categories of mutant spots recorded, compared with the frequency induced by gamma-radiation alone. This seems to indicate that the lead compounds tested do not interact with the repair of the genetic damage induced by ionizing radiation. When the lead compounds were evaluated in the in vivo comet assay with haemocytes, Pb(NO(3))(2) was effective in inducing significant increases of DNA damage with a direct dose-response pattern. These results confirm the usefulness of the comet assay with haemocytes as an in vivo model and support the assumption that there is a genotoxic risk associated with lead exposure.

Chromium-induced genotoxicity and interference in human lymphoblastoid cell (TK6) repair processes.

Two model chromium (Cr) compounds, one hexavalent (sodium chromate) and one trivalent (chromium chloride), were investigated in a human lymphoblastoid cell line (TK6) to increase our knowledge regarding Cr-induced genotoxicity mechanisms. Both selected compounds were genotoxic using the comet assay, although the percentage of DNA in tail obtained after treatment with Cr(VI) was significantly higher than that obtained with Cr(III), at the higher concentrations tested. To determine the nature of the induced damage, enzymes recognizing oxidized bases were used. Treatments with formamidopyrimidine (FPG) and endonuclease III (EndoIII) displayed a greater degree of DNA damage, indicating that the induction of oxidized bases accounts for an important proportion of the damage induced by Cr compounds. In addition, the kinetic repair studies showed that generated DNA damage is removed in approximately 8 h, with the damage induced by Cr(III) being removed/repaired more rapidly than damage produced by Cr(VI). To detect Cr interferences with the repair process, a post-treatment was applied after exposure to 2 Gy gamma radiation. Post-treatment significantly delayed the repair kinetics of DNA damage induced by radiation. This interference effect induced by Cr(VI) was more pronounced. In conclusion, evidence indicates that a high proportion of the Cr-induced DNA damage is correlated with oxidative damage, and that both Cr compounds interfere with repair mechanisms involved in repair of DNA damage induced by gamma radiation.

Mutagenic analysis of six disinfection by-products in the Tk gene of mouse lymphoma cells.

Drinking water must be disinfected prior to its distribution for human consumption. This water treatment process generates disinfection by-products (DBPs), formed by the interaction of the disinfectant with organic matter, anthropogenic contaminants and inorganic (bromide/iodide) matter naturally present in source water. Due to the potential genotoxic/carcinogenic risk of these DBPs, we have investigated the mutagenic potential of six of such compounds on the thymidine kinase (Tk) gene in the well-validated mouse lymphoma assay (MLA). The MLA quantifies a wide range of genetic alterations affecting the expression of this gene in L5178Y/Tk(+/-)-3.7.2C cells. In this study we selected six emerging DBPs, corresponding to three different chemical classes: halonitromethanes (bromonitromethane and trichloronitromethane), halogenated acetaldehydes (tribromoacetaldehyde and chloral hydrate) and hydroxyfuranones (mucobromic and mucochloric acids), each class including one chlorinated and one brominated form. The results showed that after 4h of treatment, only mucobromic acid increased the frequency of mutant colonies, with a higher proportion of small colonies, which would indicate a clastogenic potential. This is the first study reporting mutagenicity data in mammalian cells for the six selected DBPs.

Proposal of an in vivo comet assay using haemocytes of Drosophila melanogaster.

This study presents the first application of an in vivo alkaline comet assay using haemocytes of Drosophila melanogaster larvae. These cells, which play a role similar to that of mammalian blood, can be easily obtained and represent an overall exposure of the treated larvae. To validate the assay, we evaluated the response of these cells to three well-known mutagenic agents: ethyl methanesulfonate (EMS), potassium dichromate (PD), and gamma radiation (γ-irradiation). Third-instar Drosophila larvae were exposed to different concentrations of EMS (1, 2, and 4 mM) and PD (0.5, 1, and 2.5 mM) and to different doses of γ-irradiation (2, 4, and 8 Gγ). Subsequently, haemolymph was extracted from the larvae, and haemocytes were isolated by centrifugation and used in the comet assay. Haemocytes exhibited a significant dose-related increase in DNA damage, indicating that these cells are clearly sensitive to the treatments. These results suggest that the proposed in vivo comet test, using larvae haemocytes of D. melanogaster, may be a useful in vivo assay for genotoxicity assessment.

Micronuclei and pesticide exposure.

Micronucleus (MN) is a biomarker widely used in biomonitoring studies carried out to determine the genetic risk associated to pesticide exposure. Many in vitro and in vivo studies, as well as epidemiological approaches, have demonstrated the ability of certain chemical pesticides to produce genetic effects including cancer and other chronic pathologies in humans; thus, biomonitoring studies have been carried out to characterise the genetic risk associated to pesticide exposure. It must be noted that 'pesticide exposure' is a broad term covering complex mixtures of chemicals and many variables that can reduce or potentiate their risk. In addition, there are large differences in pesticides used in the different parts of the world. Although pesticides constitute a wide group of environmental pollutants, the main focus on their risk has been addressed to people using pesticides in their working places, at the chemical industry or in the crop fields. Here, we present a brief review of biomonitoring studies carried out in people occupationally exposed to pesticides and that use MN in lymphocytes or buccal cells as a target to determine the induction of genotoxic damage. Thus, people working in the chemical industry producing pesticides, people spraying pesticides and people dedicated to floriculture or agricultural works in general are the subject of specific sections. MN is a valuable genotoxic end point when clear exposure conditions exist like in pesticide production workers; nevertheless, better study designs are needed to overcome the uncertainty in exposure, genetic susceptibility and statistical power in the studies of sprayers and floriculture or agricultural workers.

Genotoxic analysis of silver nanoparticles in Drosophila.

Health risk assessment of nanomaterials is an emergent field, genotoxicity being an important endpoint to be tested. Since in vivo studies offer many advantages, such as the study of the bioavailability of nanomaterials to sensitive target cells, we propose Drosophila as a useful model for the study of the toxic and genotoxic risks associated with nanoparticle exposure. In this work we have carried out a genotoxic evaluation of silver nanoparticles in Drosophila by using the wing somatic mutation and recombination test. This test is based on the principle that loss of heterozygosis and the corresponding expression of the suitable recessive markers, multiple wing hairs and flare-3, can lead to the formation of mutant clones in larval cells, which are expressed as mutant spots on the wings of adult flies. Silver nanoparticles were supplied to third instar larvae at concentrations ranging from 0.1-10 mM. The results showed that small but significant increases in the frequency of total spots were observed, thus indicating that silver nanoparticles were able to induce genotoxic activity in the wing spot assay of D. melanogaster, mainly via the induction of somatic recombination. These positive results obtained with silver nanoparticles contrast with the negative findings obtained when silver nitrate was tested.

Genotoxic effects of two nickel-compounds in somatic cells of Drosophila melanogaster.

In view of the scarcely available information on the in vivo mutagenic and co-mutagenic activity of nickel, the genotoxic potential of two nickel-compounds, nickel chloride (NiCl(2)) and nickel sulphate (NiSO(4)), was assessed in Drosophila melanogaster by measuring two different genetic endpoints. On the one hand, we used the wing-spot assay, which is based on the principle that the loss of heterozygosity of two suitable recessive markers, multiple wing hairs (mwh) and flare-3 (flr(3)), can lead to the formation of mutant clones in the imaginal disks of larval cells. On the other hand, the in vivo comet assay, which detects single- and double-strand DNA breaks, was also used with larval haemocytes. These cells offer several advantages: they are highly sensitive to genotoxic agents, the sampling and processing methodologies are quite simple and the level of basal DNA damage is relatively low. No significant increases in the frequencies of the three categories of mutant spots (i.e. small single spots, large single spots, and twin spots) were observed in the wing-spot assay; however, NiSO(4) induced significant dose-dependent increases in DNA damage in the comet assay. In addition, the combined treatments with gamma-radiation and NiCl(2) and NiSO(4) showed a slight but significant increase in the frequency of the three categories of mutant spots compared with the frequency induced by gamma-radiation alone, indicating that both nickel compounds have a synergistic interaction. These results support the assumption that both nickel compounds could act as co-mutagens interfering with DNA-repair processes and that the in vivo comet assay is a sensitive and effective method for detecting the DNA damage induced by NiSO(4) in haemocytes of D. melanogaster.

Genotoxicity testing of three monohaloacetic acids in TK6 cells using the cytokinesis-block micronucleus assay.

Chemical disinfection of water generates harmful chemical compounds, known as disinfection by-products (DBPs). One class of DBPs is constituted by haloacetic acids (HAAs), the second major group in prevalence (after trihalomethanes) detected in finished drinking water. In this article, we report the results obtained in the evaluation of the chromosome damage induced by three monohaloacetic acids, namely iodoacetic acid (IAA), bromoacetic acid (BAA) and chloroacetic acid (CAA). To evaluate the induction of chromosome damage, we used the cytokinesis-block micronucleus test that measures the ability of genotoxic agents to induce both clastogenic and/or aneugenic effects. No previous data exist on the effects of these compounds on human chromosomes. We tested five doses of each HAA, in addition to the negative and positive controls. The highest dose tested for each HAA was that immediately lower than the dose producing total cytotoxicity. Our results show that none of the three HAAs tested was able to increase significantly the frequency of micronucleus in binucleated TK6 cells, the rank order in decreasing cytotoxicity was IAA > BAA >> CAA.

DNA damage induction by two halogenated acetaldehydes, byproducts of water disinfection.

Drinking water contains disinfection byproducts, generated by the interaction of chlorine (or other disinfecting chemicals) with organic matter, anthropogenic contaminants, and bromide/iodide naturally present in most source waters. One class of these chemicals is the halogenated acetaldehydes (HAs), identified in high quantities when ozone is used as primary or secondary disinfectant. In this study, an analysis of the genotoxic potential of two HAs, namely tribromoacetaldehyde (TBA) and chloral hydrate (CH) has been conducted in human cells (TK6 cultured cells and peripheral blood lymphocytes). The comet assay was used to 1) measure the induction of single and double-strand DNA breaks, 2) evaluate the capacity of inducing oxidative DNA damage, and 3) determine the DNA repair kinetics of the induced primary genetic damage. In addition, chromosome damage, as a measure of fixed damage, was evaluated by means of the micronucleus test. The results of the comet assay show that both compounds are clearly genotoxic, inducing high levels of DNA breaks, TBA being more effective than CH. According to the comet results, both HAs produce high levels of oxidized bases, and the induced DNA damage is rapidly repaired over time. Contrarily, the results obtained in the micronucleus test, which measures the capacity of genotoxic agents to induce clastogenic and aneugenic effects, are negative for the two HAs tested, either using TK6 cells or human peripheral blood lymphocytes. This would indicate that the primary damage induced by the two HAs is not fixed as chromosome damage, possibly due to an efficient repair or the death of damaged cells, which is an important point in terms of risk assessment of DBPs exposure.