DNA damage and repair induced in vitro by nitrilotriacetic acid (NTA) in human lymphocytes

:!.lutation Research, 209 11988) 149-154

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Iclse~ier MTRI_. 0154

D N A damage and repair induced in vitro by nitrilotriacetic acid (NTA) in human lymphocytes L. Celotti, D. Furlan, P. Ferraro and A.G. Levis Department q[ Biology. Via Loredan 10. 35131 Padua r ltalv j

(Accepted 29 July 1988) Kevwordw l tuman lymphocytes:DNA repair: DNA replication: NTA

Summar) In cultured human lymphocytes we determined the ability of nitrilotriacetic acid (NTA) to inhibit DNA replication and to stimulate DNA repair synthesis (UDS), as well as to influence the UDS induced by UV irradiation. In phytohemagglutinin-stimulated lymphocytes a strong inhibition of DNA replication was induced by NTA concentrations above 10-3 M, which was accompanied by a marked cell lethality, whereas at lower doses the incorporation of tritiated thymidine (SH-TdR) into DNA or treated cells was slightly increased in comparison to untreated cells. When, after NTA pretreatment, UDS was determined by scintillation spectrometry or autoradiography in unstimulated Go lymphocytes, UV-irradiated or unirradiated, an increased incorporation ot" SH-TdR was observed, positively correlated with the NTA doses. This effect was only partially due to the expansion of the intracellular TdR pool as a consequence of the stimulation of ~HTdR uptake by NTA. Even after normalization of the scintillometric data by the radioactivities of the soluble nucleotide fraction, significant increase of DNA repair synthesis was detected after treatment with 7.5 x 10- t - 1 0 - 2 M NTA.

Nitrilotriacetic acid (NTA), a chelating agent used as a substitute for polyphosphates in household detergents (Perry et al., 1984), induces tumors of the urinary tract in rodents chronically treated with high dose levels (Anderson et al., 1985). NTA is generally inactive in short-term mutagenicity tests (reviewed by Venier et al., 1987 and Costa et al., 1988a), thus its carcinogenicity was attributed to epigenetic mechanisms (Hodges, 1982), involving chronic toxicity (Anderson et al., 1983) and tumor promotion (Malcolm et al., 1983; Hiasa et al., 1985). As a matter o f fact NTA, although not inducing Correspondence: Dr. L. Celoni, Department of Biology, Via Loredan 10, 35131 Padua (Italy).

gene mutation in a variety of cell systems (Loprieno et al., 1985; Venier et al., 1985; Celotti et al., 1987; Costa et al., 1988b), significantly enhanced the resistance to dyphtheria toxin in human cells cultured in vitro (Grilli and Capucci, 1985). NTA did not produce chromosomal effects in mammalian cells in vivo and in vitro (Epstein et al., 1972; Jorgenson et al., 1975; Montaldi et al., 1987, 1988), but chromosomal aberrations a n d / o r micronuclei were sometimes observed in cultured plant and animal cells (Kihlman and Sturelid, 1970; De Marco et al., 1986) and human lymphocytes (Bora, 1975) treated with high doses of NTA. Moreover, NTA induced aneuploidy in Drosophila and mouse germ-line cells (Costa et al., 1988a). As regards the induction of DNA damage and

0165-7992/88/$ 03.50 '-; 1988 Elsevier Science Publishers B.V. (Biomedical Division)

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repair, NTA bound to mammalian cell DNA in vitro (Colacci, 1985) and caused differential lethality in repair-deficient strains of Escherichia coli (Venicr et al., 1987), whereas it did not induce mitotic gene conversion in Saccharomyces cerevisiae (Loprieno et al., 1985), nor DNA damage and repair synthesis (Williams et al., 1982) or sister-chromatid exchanges (Montaldi et al., 1985, 1987) in cultured mammalian cells. Also the in vitro mammalian cell transformation assays gave contrasting results with NTA. No transformation was obtained in BHK 21/c13 hamster ceils (Lanfranchi el al., 1988), whereas positive results were described with BAI.B/c 3"F3 mouse cells (Sivak, 1983) and rat embryo cells infected with leukemia virus (Traul et al., 1981). Therefore it cannot be excluded that in some cell systems and at particular dose levels NTA exerts a weak genotoxic action. To test this hyphothesis we examined in cultured human lymphocytes the ability of NTA to inhibit DNA replication and to stimulate DNA repair synthesis (UDS), as well as to influence the UDS induced by UV irradiation. Materials and methods

Cells [.ymphocytes were isolated from samples of peripheral blood of 2 healthy donors (Boym, 1977). The cells were cultured in Eagle's Minimum Essential Medium (E-MEM) supplemented with 20O7o fetal calf serum (FCS; Seromed) in a humidified incubator with 5o70 CO2. Viable cells were determined by trypan blue exclusion. U V irradiation The lymphocytes were exposed for 6 sec in open petri dishes to UV irradiation (254 nm) using a UVS-II mineral light lamp, which gave a fluence rate of 4 J/mZ/sec. UV fluence was measured by a VLX radiometer UV (Vilber Lourmat, Marne La Vallet Cedex 2, France). Drug treatment The sodium salt of nitrilotriacetic acid (NTA) was obtained from Ega-Chemie (West Germany).

I h e treatment of lymphocytcs was performed in EMEM with 20% FCS for 72 h in the case of DNA replication and for 24 h in the case of UDS.

Determination o f D N A replication The cells were seeded at 10~/well in 96-well microtiter plates (Sterilin) with phytohemagglutinin (PHA P, Seromed) (10 #I/well). 48 h after seeding, 2 p.Ci/ml of tritiated thymidine (~H-TdR; specific activity 40 Ci/mmole; Amity PG) were added and maintaincd during the last 24 h of incubation. At the end of labelling, the cells were collected, rinsed with ice-cold Hanks' Balanced Salt Solution (HBSS) and transferred on glass fiber filters (GF/C, Whatmann). In order to extract soluble nucleotide pools, filters were washed with cold 5o70 trichloroacetic acid (TCA), 100% ethanol and dried. The radioactivity remaining on the filters was measured by liquid-scintillation spectroscopy in a Packard A 300C counter with an efficiency of 30%. UDS determination Immediately after isolation, lymphocytes were incubated for 24 h in E-MEM with 20% FCS, supplemented or not with NTA at different concentrations. After 21 h of incubation, the cells were irradiated with UV light, before adding 2.5 mM hydroxyurea (HU; Sigma) and 3H-TdR (10 #Ci/ml) for 3 h to determine the UV-induced UDS in lymphocytes treated or not with NTA. An additional set of cultures was treated with NTA but not irradiated and another one was the untreated control. All experiments were run in triplicate. Incorporated radioactivity was measured both by liquid-scintillation spectroscopy as described for DNA replication, and by autoradiography. Autoradiography was performed on samples labelled in the absence of HU. The slides were dipped into K2 emulsion (Ilford), developed after 14 days at 4°C and stained with Giemsa solution. UDS was measured by counting the number of grains on 100 non-S-phase nuclei. For each experimental condition we counted 4-6 slides. As NTA was shown to increase the uptake o f 3H-TdR into the intracellular pool, a correction of the original DNA

151 radioactivily scintillometric data was performed, following the procedure adopted by Bianchi et al. (1982). In summary, the radioactivities of TCAextracted nucleotide pool in the treated samples were determined and were expressed as percentages of control values. The DNA radioactivity was normalized by dividing its original value in cpm by the percem value of the corresponding intracellular pool. The normalized DNA radioactivity was assumed to better reflect the actual level of precursor incorporation into DNA (Bianchi et al., 1983). Results

3H-TdR incorporation, in the presence of hydroxyurea. The level of repair synthesis measured by scintillation counting was higher in NTA-pretreated lymphocytes in comparison to lymphocytes UV-irradiated only (Fig. 2b). A positive correlation between ~H-TdR incorporation and NTA doses was determined (P < 0.001). To check whether the treatment with NTA by itself causes 3H-TdR to be incorporated by Go quiescent lymphocytes, we repeated the experiment with unirradiated cells. In that case also the incorporation of radioactivity increased and was positively correlated with NTA dose (P < 0.001) (Fig. 2a).

The effect of NTA on DNA replicative synthesis was examined by measuring the uptake of 3H-TdR by PH A-stimulated human lymphocytes. The dose range of NTA tested was 10-4-10 -2 M. In Fig. 1 we report the values of radioactivity incorporated at different doses and the corresponding numbers of viable cells determined by trypan blue exclusion. DNA replication appeared strongly inhibited at NTA concentrations above 10- 3 M, while at lower doses Ihe incorporation of 3H-TdR in treated cells was slightly increased in comparison to untreated cells. We also investigated the influence of NTA on DNA repair induced by UV by measuring in Go lymphocytes treated with NTA the UV-stimulated

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These results were confirmed by autoradiography. The incorporation of radioactivity was monitored in experiments carried out with irradiated and unirradiated lymphocytes treated with high concentrations of NTA (7.5 x 10 - 3-10- 2 M) and in the absence of HU. Autoradiographic data are reported in Fig. 3. Grain numbers on nuclei of NTA-treated lymphocytes, both UV-irradiated and unirradiated, were higher in comparison with control cells (P < 0.01 and P < 0.001, respectively). Treatment with chemicals, alone or in association with HU, can modify the uptake ot" exogenous TdR, hence the availability of labelled DNA precursors in the intracellular pool (Bianchi et al., 1982, 1983). To evaluate the variations of the TdR pool induced by treatment with NTA, we measured the soluble radioactivities of the samples. The values in the treated cells were higher than the control, therefore we corrected the DNA radioactivities as detailed in the Materials and methods section. In Fig. 4 we report the radioactivity values of macromolecular DNA normalized by the

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Our experiments indicate that NTA affects ~HTdR incorporation into the DNA of human lymphocytes stimulated in vitro with PHA. This effect depends on treatment doses: at 10 4-10- 3 M NTA the incorporation of 3H-TdR was higher in treated than in control cells, while at doses above 10 - ~ M the values of 3H-TdR incorporation were much lower in treated cells. At the higher doses of treatment a marked cell lethality took place which the observed effects on 3H-TdR incorporation can bc referred to, rather than to a specific inhibition ot" DNA synthesis.

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When, after NTA treatment, UDS was determined by scintillation spectrometry in Go lymphocytes, UV-irradiated or unirradiated, an increased incorporation of radioactive thymidine was observed, positively correlated with the NTA doses. The increase was not due to the experimental conditions required by the liquid scintillation technique adopted to detect UDS, e.g., by the use of HU. In fact, also when UDS was measured by autoradiography, that is in the absence of HU, the increased incorporation of tritiated precursor during DNA synthesis was confirmed. The increased incorporation of 3H-TdR into I)NA detected at high doses of NTA (above 10M) could be due to the incrcased uptake of 3H-TdR into the intracellular pool. However, also when DNA radioactivities were normalized by taking into account the increased radioactivities of the intracellular pool, the incorporation of 3H-TdR into DNA remained significantly higher in treated than in conlrol cells. The above data can be taken to indicate that in cuhurcd Go human lymphocytes prolonged treatments with high doses of NTA produce DNA damage and stimulate DNA excision repair. The possibility that NTA induces DNA damage and repair is scarcely supported by the literature, but cannot be completely ruled out. Negative results were obtained with systems usually employed to detect this kind of genetic effects, such as the gene conversion assay in the yeast S. cerevisiae (Loprieno et al., 1985), the autoradiographic determination of DNA repair synthesis in primary cultures of rat hepatocytes (Williams et al., 1982), and the sister-chromatid exchange assay in cultured mammalian cell lines and human lymphocyles (Ved Brat and Williams 1984; Montaldi et al., 1985, 1987). It must be noticed that the experiments concerning DNA repair in rat hepatocytes refer to NTA doses lower than 3 x 10 3 M (Williams et al., 1982) whereas in our present experiments the increased incorporation of 3H-TdR into DNA was observed by autoradiography and scintillation counting after treatment with high doses of NTA ( 7 . 5 x 1 0 - 3 - 1 0 2 M). Moreover, positive indications of NTA genotoxici-

ty were the binding of radioactive NTA to m a m malian cell DNA in vitro (Colacci, 1985) and differential lethality in strains of E. coil deficient for various systems of DNA repair (Venier et al., 1987). A different possibility is suggested by the wellknown ability of NTA to chelate cations inducing by this way cytotoxic and even carcinogenetic effects (Anderson et al., 1985). Chelation of intracellular cations by NTA could modify the organization of chromatin and alter the activity of the enzymatic systems involved in DNA replication and repair. The increased incorporation of "l'dR into DNA could bc due to the activation of new replication origins and the increase in size and rate of synthesis of the replicons or the patches related to DNA repair. In particular, DNA replication would be enhanced in stimulated lymphocytes treated with subtoxic doses of NTA, and treatment with high NTA doses would favor the incorporation of TdR during the repair of UV-induced or spontaneous DNA damages.

Acknowledgemenls This work was supported b'y' grants from the Italian National Research Council (C.N.P., 1-'rogetto Strategico 'Mutagenesi') and the Venetia Region.

References Anderson, R.L., W.E. Bishop and R.I.. Campbell (1985) A review of the en`'ironmental and m a m m a l i a n toxicology of nitrilotriacetic acid, CRC Crit. Re','. Toxicol., 15, 1-102. Bianchi, V., F. Nuzzo, A. Abbondandolo, S. Bonatti, E. Capelli, R. Fiorio, E. Giulotto, A. Mazzaccaro, M. Stefanini, L. Zaccaro, A. Zantedeschi and A . G . I . e v i s (1982) Scintillometric determination of I)NA repair in h u m a n cell lines: a critical appraisal, Mutation Re~,., 93, 447-463. Bianchi, V., A. Zantedeschi and A . G . t . e ` ' i s (1983)The scintillometric evaluation of I)NA repair ~,ynthesis can be distorted by changes of Ihymidine pool radioactivity, ( h e m . Biol. Interact., 43, 17-31. Bora, K.C. (1975) bff'ects of nitrilotriacetic acid (NTA) on c h r o m o s o m e replication and structure in h u m a n cells, Mutation Re,,., 31, 325 (abstract). Boym, A. (1977) Separation of lymphocytes, lymphocyte subgroups and monocytes: a review, [ y m p h o l o g y , 10, 71-76.

154 Celotti, 1., D. Furlan, L. Seccati and A.G.l.cvi~,