Genotoxic effects of formaldehyde in the human lung cell line A549 and in primary human nasal epithelial cells

The alkaline comet assay was used to further characterize the induction of DNA-protein crosslinks (DPX) by formaldehyde (FA) and their removal in the human lung cell line A549 and in primary human nasal epithelial cells (HNEC). DPX were indirectly measured as the reduction of gamma ray-induced DNA migration. FA induced DPX in A549 cells in a concentration-related manner in the range of 100-300 microM. This result is in agreement with previous studies using different mammalian cell lines. The main new findings of the present study are: (i) Determination of cytotoxicity in relation to genotoxicity strongly depend on the method used. Cytotoxicity measured as the reduction in cell counts 48 hr after addition of FA to the cultures occurred parallel to the induction of DPX while colony forming ability was already reduced at 10 times lower FA concentrations; (ii) DPX induced by a 1-hr FA treatment were completely removed within 8 hr cultivation in fresh medium while in the presence of FA in the medium DPX levels remained unchanged for 24 hr; (iii) Induction and removal of DPX did not fundamentally differ between exponentially growing and confluent A549 cultures; (iv) Slowly proliferating HNEC showed the same sensitivity towards FA-induced DPX as A549 cells (i.e. the same FA concentrations induced DPX under the same experimental conditions) and removed DPX with a similar efficiency. In summary, these results contribute to a better understanding of the genotoxic activity of FA in vitro and indicate that the tested cultured primary and permanent human cells do not differ fundamentally with regard to the processing of FA-induced primary genotoxic effects.     

Genotoxic effects induced by formaldehyde in human blood and implications for the interpretation of biomonitoring studies

Formaldehyde (FA) was tested for its genotoxicity in human blood cultures. We treated blood samples at the start of the culture to follow FA-induced DNA damage (DNA-protein crosslinks, DPX), its repair and its genetic consequences in form of sister chromatid exchanges (SCE) and micronuclei (MN). Our results clearly indicate that DPX (determined by the comet assay) are induced at FA concentrations of > or =25 microM. DPX induced by FA concentrations up to 100 microM are completely removed before lymphocytes start to replicate. SCE are induced at concentrations >100 microM parallel to the induction of cytotoxicity (measured as reduction of the replication index). MN were not induced by FA concentrations up to 250 microM (the highest analyzable concentration) added at the start of the blood cultures in the cytokinesis-block micronucleus (CBMN) test. FA-induced cytotoxicity (measured as reduction of the nuclear division index) possibly prevented division of damaged cells. MN were only significantly induced in human blood when proliferating cells were exposed to FA during the last cell cycle before preparation. Several human biomonitoring studies reported increased frequencies of SCE and MN in lymphocytes of subjects exposed to FA. Our results characterize the genotoxic potential of FA in cultured lymphocytes and lead to the conclusion that cytogenetic effects of FA are very unlikely to occur in blood cultures of FA-exposed subjects.     

The replication of viral and cellular DNA in human herpesvirus 6-infected cells

Human herpesvirus 6 (HHV-6) is a newly identified lymphotropic herpesvirus. We have analyzed viral and host DNA replication in peripheral blood lymphocytes infected in the absence of drugs or infected in the presence of phosphonoacetic acid (PAA) or acyclovir (ACV). The results revealed the following: (i) Infection with HHV-6 resulted in the shutoff of host DNA replication. (ii) PAA at concentrations of 100 and 300 micrograms/ml significantly reduced virus replication. The drug inhibited viral DNA replication, whereas host cell DNA replication was not affected. This strongly suggests that HHV-6 encodes a PAA sensitive viral DNA polymerase. (iii) ACV at 20 microM did not interfere with virus production and virus spread. ACV at 100 microM only partly interfered with virus replication, whereas at 400 microM the block was more complete. Viral DNA replication was not affected by ACV at 20 microM. However, approximately 60 and 85% inhibition in viral DNA replication was observed in the presence of 100 and 400 microM of ACV. (iv) Assays for viral thymidine kinase (TK) revealed no significant increase in TK activity, whereas increased TK activity was noted following infection of the same peripheral blood lymphocytes with herpes simplex virus. Thus, either HHV-6 does not encode a tk enzyme which can phosphorylate ACV or the inefficient block may reflect lower sensitivity of the HHV-6 DNA polymerase to the drug.     

Effects of Captan on DNA and DNA metabolic processes in human diploid fibroblasts.

The fungicide Captan has been examined for its effects on DNA and DNA processing in order to better understand the genotoxicity associated with this agent. Captan treatment resulted in production of DNA single strand breaks and DNA-protein cross-links and elicited an excision repair response in human diploid fibroblasts. Captan was also shown to inhibit cellular DNA synthesis and to form stable adducts in herring sperm and human cellular DNA. Misincorporation of nucleotides into Captan-treated synthetic DNA templates was significantly elevated in an in vitro assay using E. coli DNA polymerase I, suggesting that DNA adduct formation by Captan could have mutagenic consequences. In sum, these studies demonstrate that Captan is capable of interacting with DNA at a number of levels and that these interactions could provide the basis for Captan's genotoxicity. The extreme cytotoxicity of this fungicide, however, could be due to other cellular effects since at the IC50 for cell killing, approximately 0.8 microM, none of the above genotoxic events could be detected by the methods employed.     

Detection of DNA strand breaks, DNA-protein crosslinks, and telomerase activity in nickel-transformed BALB/c-3T3 cells.

Although nickel compounds are known carcinogens, the underlying carcinogenic mechanisms are not fully understood. The objective of this research was to determine if the genotoxic lesions of DNA strand breaks and DNA-protein crosslinks are present in nickel-transformed BALB/c-3T3 cells, and to further elucidate the potential carcinogenesis of insoluble and soluble nickel compounds through telomerase activity in nickel-transformed BALB/c-3T3 cell lines. DNA strand breaks, DNA-protein crosslinks and telomerase activity were investigated by single cell gel electrophoresis (comet assay), (125)I-postlabelling techniques, and the TRAP-silver staining assay, respectively. Results showed that both DNA strand breaks and DNA-protein crosslinks were present in nickel-transformed BALB/c-3T3 cells. However, the highest levels of DNA strand breaks and DNA-protein crosslinks were found in insoluble crystalline NiS-transformed cells and high levels of DNA strand breaks and DNA-protein crosslinks were also found in the transformed cells induced by two water-soluble NiCl(2) and NiSO(4) at moderate concentrations of cytotoxicity. These data suggest that these two genetic endpoints are useful biomarkers and are associated with cell transformation and carcinogensis of insoluble and soluble nickel compounds. Also, we found that the crystalline NiS- and NiCl(2)-transformed cells possessed a high telomerase activity. A weak telomerase was found in NiSO(4)-transformed cells. The results seem to indicate that in addition to crystalline NiS, some water-soluble nickel compounds such as NiCl(2) are also highly carcinogenic. These results may partly explain the cell transformation and relative carcinogenic potency of insoluble crystalline NiS, soluble NiCl(2), and NiSO(4).     

3Rs-friendly approach to exogenous metabolic activation that supports high-throughput genetic toxicology testing

MultiFlow® DNA Damage—p53, γH2AX, Phospho-Histone H3 is a miniaturized, flow cytometry-based assay that provides genotoxic mode of action information by distinguishing clastogens, aneugens, and nongenotoxicants. Work to date has focused on the p53-competent human cell line TK6. While mammalian cell genotoxicity assays typically supply exogenous metabolic activation in the form of concentrated rat liver S9, this is a less-than-ideal approach for several reasons, including 3Rs considerations. Here, we describe our experiences with low concentration S9 and saturating co-factors which were allowed to remain in contact with cells and test chemicals for 24 continuous hours. We exposed TK6 cells in 96-well plates to each of 15 reference chemicals over a range of concentrations, both in the presence and absence of 0.25% v/v phenobarbital/β-naphthoflavone-induced rat liver S9. After 4 and 24 hr of treatment cell aliquots were added to wells of a microtiter plate containing the working detergent/stain/antibody cocktail. After a brief incubation robotic sampling was employed for walk-away flow cytometric data acquisition. PROAST benchmark dose (BMD) modeling was used to characterize the resulting dose–response curves. For each of the 8 reference pro-genotoxicants studied, relative nuclei count, γH2AX, and/or p53 biomarker BMD values were order(s) of magnitude lower for 0.25% S9 conditions compared to 0% S9. Conversely, several of the direct-acting reference chemicals exhibited appreciably lower cytotoxicity and/or genotoxicity BMD values in the presence of S9 (eg, resorcinol). These results prove the efficacy of the low concentration S9 system, and indicate that an efficient and highly scalable multiplexed assay can effectively identify chemicals that require bioactivation to exert their genotoxic effects.     

L-asparaginase bound to collagen membranes: effect of glutaraldehyde crosslinking on stabilization of catalytic activity.

Reconstituted bovine collagen has been used extensively in our laboratory as a carrier for immobilized E. coli L-asparaginase. The activity and catalytic stability of these collagen-asparaginase membranes can be altered substantially by conditions used in membrane crosslinking with glutaraldehyde. As the concentration of glutaraldehyde used in tanning is increased, the initial specific activity of collagen-asparaginase membranes decreased asymptotically to a limiting value. Similar results occurred when membranes were subjected to increasing time periods of tanning at a constant glutaraldehyde concentration. These observations point to a time-concentration relationship for glutaraldehyde tanning and its effect on the specific activity of collagen-asparaginase membranes. Specific activities of membranes tanned a glutaraldehyde concentrations of 5% or higher appear to be very stable over long periods of alternate storage and assay. This result, however, is not observed with membranes tanned at glutaraldehyde concentrations lower than 5% for short periods of time (approximately 30 sec to 1 min). It is not clear whether the instability of membranes tanned at lower concentrations of glutaraldehyde or shorter intervals of tanning is due to enzyme elution from the membrane or denaturation of the bound enzyme.     

Influence of culture conditions on the DNA-damaging effect of benzene and its metabolites in human peripheral blood mononuclear cells

The DNA-damaging ability of benzene and its metabolites on peripheral blood mononuclear cells (PBMC) has been investigated by using the alkaline comet assay. The PBMC were incubated with different compounds in two different media for 2 and 24 hr at concentrations that did not affect cell viability and the DNA damage was quantified by a computerized image analysis system. Benzene and phenol (5 mM) did not show any genotoxic activity after 2 hr of incubation in the two media tested, phosphate-buffered saline (PBS) and RPMI containing 5% of heat-inactivated fetal calf serum (RPMI + 5% FCS), whereas phenol was genotoxic and cytotoxic at 10 mM after 24 hr of incubation in RPMI + 5% FCS. All other benzene metabolites were genotoxic at micromolar concentrations when incubated in PBS with the following decreasing order of potency: benzenetriol, catechol, hydroquinone, and benzoquinone. When the PBMC were incubated in RPMI + 5% FCS, the effect of catechol (200-600 microM) and benzenetriol (10 microM) was reduced, whereas the genotoxicity of benzenetriol at high concentrations (50-100 microM) and hydroquinone (150-2500 microM) was not affected. In contrast, the effect of benzoquinone at 5 and 10 microM was greatly enhanced when the cells were incubated in RPMI + 5% FCS. This effect resulted mainly from the presence of serum in the medium and it was almost completely inhibited by boiling the serum (100 degrees C, 5 min) and was partially reduced by extensive dialysis. Benzoquinone was the most damaging compound when tested under more physiological conditions, thereby supporting the general observation that it is the most myelotoxic benzene metabolite.     

Menadione biphasically controls JNK-linked cell death in leukemia Jurkat T cells

Signals for cell-death induction by menadione were studied in Jurkat T cells. Low concentrations of menadione (10-20 microM) and H(2)O(2) (10-50 microM) induced cell death accompanying low (menadione: <5%) or moderate (H(2)O(2): 10-15%) levels of DNA fragmentation in Jurkat cells. These concentrations of menadione (10 microM) and H(2)O(2) also caused membrane (necrotic) cell death at unproportionally high (80%) and proportional (10-30%) levels, respectively. Higher concentrations (100-5,000 microM) of H(2)O(2) exclusively induced membrane cell death. Unexpectedly, 30-300 microM menadione induced ever-decreasing levels of necrotic cell death in a concentration-dependent manner. An in vitro kinase assay showed that 20-50 microM, but not >100 microM, menadione induced activation of c-Jun NH(2)-terminal kinase (JNK), whereas a striking activation of JNK was induced by 500-5,000 microM H(2)O(2). Induction of cell death by a low concentration of menadione was partially inhibited in dominant negative JNK gene-transfected Jurkat/VPF cells. A high concentration (300 microM) of menadione was found to inhibit cell-death induction by high concentrations (200-5,000 microM) of H(2)O(2). The JNK inhibitory activity of menadione was also demonstrated in a cell-free system. However, menadione did not activate JNK in vitro. These results suggest that JNK is required for induction of not only apoptotic cell death, but also necrotic cell death in Jurkat T cells and that menadione biphasically controls this JNK-linked signal for inducing cell death.     

Relationship between DNA adduct formation and unscheduled DNA synthesis (UDS) in cultured mouse epidermal keratinocytes

Primary cultures of mouse epidermal keratinocytes from SENCAR mice were treated with 7,12-dimethylbenz(a)anthracene (DMBA), benzo(a)pyrene [B(a)P], (+/-)7 beta-8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene [(+/-)anti-BPDE], and (+/-)7 beta, 8 alpha-dihydroxy-9 beta, 10 beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene [(+/-)syn-BPDE] to examine the relationship between DNA adduct formation and the induction of unscheduled DNA synthesis (UDS). DNA adducts were measured as pmol hydrocarbon bound per mg of DNA, and UDS was quantitated autoradiographically as net grains per nucleus. A good correlation was observed between the levels of UDS detected and the amount of DNA adducts present in the cell population when comparing similar compounds within the linear dose-response range of 0.005 micrograms/ml-0.25 micrograms/ml. A higher rate of UDS for a given level of DNA adducts was interpreted as an increased efficiency of DNA repair. In some cases, an increase in the efficiency of DNA excision repair correlated with lower tumor-initiating activity. For this family of PAH, the concentration below which UDS could no longer be detected was approximately 0.01 microgram/ml. However, DNA adducts were measurable at concentrations of 0.01 and 0.005 micrograms/ml. The limits of detection of the current UDS assay in the SENCAR MEK culture system occurred at hydrocarbon adduct levels of approximately 10 pmol/mg DNA, or approximately 1 adduct per 3 x 10(5) bases. Additionally, the UDS assay was unable to detect DNA repair induced by the weakly carcinogenic PAHs, dibenz(aj)anthracene and 7-methyl-dibenz(aj)anthracene. The UDS assay did detect DNA repair by the more strongly carcinogenic PAH, 6-methylcholanthrene. These results suggest that the present UDS assay with MEKs is a useful assay for the rapid screening of potential genotoxic agents. However, the limits of sensitivity are such that the current assay may be unable to detect a low level of DNA damage induced by some weakly genotoxic (carcinogenic) agents. In addition, while the limits of sensitivity determined in these experiments apply to the polycyclic aromatic hydrocarbon class, other classes of genotoxic compounds such as alkylating agents or crosslinking agents may exhibit different thresholds of detection.     

Effect of the dithiocarbamate pesticide zineb and its commercial formulation azzurro. I. Genotoxic evaluation on cultured human lymphocytes exposed in vitro.

The in vitro cytogenetic effects exerted by the dithiocarbamate fungicide zineb and one of its commercial formulations currently used in Argentina, azzurro, were studied in whole blood human lymphocyte cultures. The genotoxicity of the fungicides was measured by analysis of the frequency of chromosomal aberrations and sister chromatid exchanges (SCEs) and cell cycle progression assays. Both zineb and azzurro activities were tested within the range 0.1-100.0 microg/ml immediately after in vitro lymphocyte stimulation. Only concentrations of 50.0 and 100.0 microg/ml zineb and azzurro induced a significant increase in SCE frequency over control values. Furthermore, this genotoxicity appears to be correlated with its cytotoxicity, measured as cell cycle kinetics, since both a significant delay in cell cycle progression and a significant reduction in proliferative rate index were only observed in those cultures treated with these fungicide concentrations. For both chemicals, a progressive dose-related inhibition of the mitotic activity of cultures was observed when increasing the fungicide concentration. Moreover, only the mitotic activity statistically differed from control values when doses of zineb or azzurro <10 microg/ml were employed. For both fungicides the mitotic index reached the minimal value at doses of 100 microg/ml. Both products induced a significant dose-dependent increase in the number of abnormal cells, chromatid-type and chromosome-type aberrations as well as in the total number of aberrations in the 0.1-100.0 microg/ml dose range. Based on these results, the evaluation of zineb as a controversial genotoxic/non-genotoxic compound for human health should be reconsidered. Instead, we demonstrate that the fungicide induces large DNA alterations and should be considered as a clastogenic mutagen.     

Activation of 3-nitrobenzanthrone and its metabolites to DNA-damaging species in human B lymphoblastoid MCL-5 cells

3-Nitrobenzanthrone (3-NBA) is one of the most potent mutagens in the Ames Salmonella typhimurium assay and a suspected human carcinogen recently identified in diesel exhaust and in airborne particulate matter. 3-Aminobenzanthrone (3-ABA), 3-acetylaminobenzanthrone (3-Ac-ABA) and N-acetyl-N-hydroxy-3-aminobenzanthrone (N-Ac-N-OH-ABA) have been identified as 3-NBA metabolites. In the present study we investigated the genotoxic effects of 3-NBA and its metabolites in the human B lymphoblastoid cell line MCL-5. DNA strand breaks were measured using the Comet assay, chromosomal damage was assessed using the micronucleus assay and DNA adduct formation was determined by 32P-post-labelling analysis. DNA strand-breaking activity was observed with each compound in a concentration-dependent manner (1-50 microM, 2 h incubation time). At 50 microM median comet tail lengths (CTLs) were 25.0 microm for 3-NBA, 48.0 microm for 3-ABA, 54.5 microm for 3-Ac-ABA and 65.0 microm for N-Ac-N-OH-ABA. Median CTLs in control incubations were in the range 7.7-13.1 micro m. Moreover, the strand-breaking activity of 3-NBA was more pronounced in the presence of a DNA repair inhibitor, hydroxyurea. Depending on the concentration used (1-20 microM, 24 h incubation time), 3-NBA and its metabolites also showed clastogenic activity in the micronucleus assay. 3-NBA and N-Ac-N-OH-ABA were the most active at low concentrations; at 1 microM the total number of micronuclei per 500 binucleate cells was 4.7 +/- 0.6 in both cases, compared with 1.7-3.0 for controls (P < 0.05). Furthermore, multiple DNA adducts were detected with each compound (1 microM, 24 h incubation time), essentially similar to those found recently in vivo in rats treated with 3-NBA or its metabolites. DNA adduct levels ranged from 1.3 to 42.8 and from 2.0 to 39.8 adducts/10(8) nt using the nuclease P1 and butanol enrichment procedures, respectively. DNA binding was highest for N-Ac-N-OH-ABA, followed by 3-NBA, and much lower for 3-ABA and 3-Ac-ABA. All major 3-NBA-derived DNA adducts produced in MCL-5 cells were found to be formed from reductive metabolites bound to purine bases and lacked an N-acetyl group. These results demonstrate that 3-NBA and its metabolites are effectively activated to DNA-damaging species in human MCL-5 cells, which may reflect the genotoxic potential of 3-NBA in humans. Environmental exposure to 3-NBA may be a health hazard for large sections of the population and the risks associated with such exposure require further assessment.     

In vivo localization of lymphocytes labelled with low concentrations of Hoechst 33342

Hoechst 33342 (HO 33342) is a fluorescent dye which binds specifically to DNA and can be used to label lymphocytes for in vivo migration studies. Lymphocytes were treated with varying concentrations of HO 33342 and assayed in vitro for effects on viability, mitogen-stimulated proliferation, and motility. In vivo traffic studies were performed to determine a dye concentration with minimal toxicity for lymphocytes, but sufficient fluorescence for detection of cells in frozen sections. The concentration reported to yield quantitative staining of nuclear DNA (10.7 microM, or 6 micrograms/ml) reduced motility and proliferative response, and resulted in an altered lymphocyte migration pattern compared to untreated lymphocytes. A concentration of 0.25 microM, however, produced no toxicity in the in vitro assays, and an in vivo migration pattern similar to that of untreated cells; lymphocytes stained with 0.25 microM HO 33342 for 30 min were readily observable in histological sections. This study indicates that the concentration of HO 33342 optimal for DNA staining may exert deleterious effects on in vivo lymphocyte traffic studies, and that far lower dye concentrations are more suitable for such studies.     

Suppression of tumor necrosis factor secretion from white blood cells by synthetic antisense phosphorothioate oligodeoxynucleotides

In this ex vivo, rather than in vitro, experiment, a synthetic antisense oligodeoxynucleotide was tested to suppress tumor necrosis factor - alpha(TNF) secretion from lipopolysaccharide-stimulated white blood cells. Antisense oligomer showed significant and specific suppressive effect to the secretion of TNF at concentrations of 1.0 and 10 microM. At the concentration of 1 microM, there were 68.4 and 63.9% suppression of TNF secretion at 2 and 24 h after resuspension of blood cells. At the concentration of 10 microM, the suppressions were slightly higher than those at 1 microM, which were 71.8 and 76.2%, respectively. A 50%-matched scrambler showed suppressive effect only at 10 microM concentration, and the suppression only occurred at 2 and 24 h after incubation. Sense oligomer showed no suppressive effects at any of the concentrations. The specificity of this oligomer was documented by dose-effect phenomenon, sequence-dependent suppression and absence of effect on the synthesis of another cytokine (interleukin-6). A series of parallel studies was performed and showed that all three oligomers at any concentration tested had no effect on the interleukin-6 secretion after LPS stimulation.In conclusion, properly designed antisense oligodeoxynucleotide can significantly and specifically suppress the secretion of TNF by blood cells in an ex vivo system and it may be a good "information" drug to treat diseases that are caused by over production of TNF.     

Effect of cisplatin on cell death and DNA crosslinking in rat mammary. Adenocarcinoma in vitro

The pharmacodynamic effects of cisdiamminedichloroplatinum(II) (CDDP) in vitro have been reported, but the dosage and exposure time in vitro have not been based on clinical observations of the drug's actions in vivo. In this study, the authors attempted to evaluate the pharmacodynamic effects of CDDP in vitro in terms of cell survival and DNA crosslinking by simulating unbound CDDP administration at varying concentrations to a rat mammary adenocarcinoma line (known as line 66). CDDP exposure was conducted by both constant concentration procedures and a simulated in vivo procedure. Colony formation assay for the surviving fraction and alkaline elution assay for DNA crosslink measurement were performed in order to evaluate the pharmacodynamics of CDDP. Cell survival was a function of the area under the drug concentration time curve (AUC) of unbound CDDP (R2 = 0.77, P < 0.002) for all drug exposure procedures as analyzed by the analysis of covariance test. There was a strong correlation between the surviving fraction and the crosslink index of the total amount of DNA crosslinks (R2 = 0.85, P < 0.0005). Both the total amount of DNA-DNA crosslinks and the DNA-protein crosslinks, of which the latter were dominant, were affected not by the exposure procedures, but by the AUC value (P < 0.002). The thresholds of cytocidal effect were 1.59 mg.h/l for the AUC and 0.008 for the crosslink index. The pharmacodynamic effects in vitro by simulated in vivo exposure were identical to those of constant.     

Characterization of collagen gel solutions and collagen matrices for cell culture

The influence of glutaraldehyde as a crosslinking agent to increase the strength of collagen matrices for cell culture was examined in this study. Collagen solutions of 1% were treated with different concentrations (0-0.2%) of glutaraldehyde for 24 h. The viscoelasticity of the resulting collagen gel solution was measured using dynamic mechanical analysis (DMA), which demonstrated that all collagen gel solutions examined followed the same model pattern. The creep compliance model of Voigt-Kelvin satisfactorily described the change of viscoelasticity expressed by these collagen gel solutions. These crosslinked collagen gel solutions were freeze-dried to form a matrix with a thickness of about 0.2-0.3 mm. The break modulus of these collagen matrices measured by DMA revealed that the higher the degree of crosslinking. the higher the break modulus. The compatibility of fibroblasts isolated from nude mouse skin with these collagen matrices was found to be acceptable at a cell density of 3 x 10(5) cells/cm2 with no contraction, even when using a concentration of glutaraldehyde of up to 0.2%.     

Relative sensitivity of fish and mammalian cells to sodium arsenate and arsenite as determined by alkaline single-cell gel electrophoresis and cytokinesis-block micronucleus assay

To protect human and ecosystem health, it is necessary to develop sensitive assays and to identify responsive cells and species (and their life stages). In this study, the relative genotoxicity of two inorganic arsenicals: trivalent sodium arsenite (As(3+)) and pentavalent sodium arsenate (As(5+)), was evaluated in two cell lines of phylogenetically different origin, using alkaline single-cell gel electrophoresis (i.e., the Comet assay) and the cytokinesis-block micronucleus (MN) assay. The cell lines were the rainbow trout gonad-2 (RTG-2) and Chinese hamster ovary-K1 (CHO-K1) lines. Following optimization and validation of both assays using reference chemicals (i.e., 1-100 microM hydrogen peroxide for the Comet assay and 1-10 mM ethylmethane sulfonate for the MN assay), cells were exposed to 1-10 microM of both arsenicals to determine the relative extent of genetic damage. The unexposed controls showed similar (background) levels of damage in both cell lines and for both assays. Treatment with the arsenicals induced concentration-dependent increases in genetic damage in the two cell lines. Arsenite was more potent than arsenate in inducing DNA strand breaks in the Comet assay; at the highest concentration (10 microM) arsenite produced similar levels of DNA damage in CHO-K1 and RTG-2 cells, while 10 microM arsenate was significantly more genotoxic in RTG-2 cells. MN induction was consistently higher in RTG-2 cells than in CHO-K1 cells, with 10 microM arsenite inducing an approximate 10-fold increase in both cell lines. MN induction also was positively correlated with DNA strand breaks for both arsenicals. Overall, the study demonstrated that the fish cells are more sensitive than the mammalian cells at environmentally realistic concentrations of both arsenicals, with arsenite being more toxic.