DNA damage by haloalkanes in human lymphocytes cultured in vitro

Cancer Letters, 13 (1981) 213-218 o Elsevier/North-Holland Scientific

213 Publishers Ltd.

DNA DAMAGE BY HALOALKANES CULTURED IN VITRO*

IN HUMAN LYMPHOCYTES

PAOLO PEROCCO and GIORGIO PROD1

Zstituto di Cancerologia, Univereitd di Bologna, Via S. Giacomo 14, l-40100 (Ztaly) (Received (Accepted

Bologna

7 April 1981) 5 May 1981)

SUMMARY

The DNA damaging activity of 7 haloalkanes was studied in a short-term in vitro system which utilized human lymphocytes. The parameters studied were the inhibition of scheduled (duplicative) and unscheduled (reparative) DNA syntheses seen as tritiated thymidine uptake. The results obtained suggested that chloromethyl methyl ether (CMME), 1,2dibromoethane (DBE), trichloroethylene (TCE) and 1,2dichloroethane (DCE) gave positive results such as DNA damaging agents, while carbon tetrachloride (CTC), chloroform (TCM) and dichloromethane (DCM) gave low or negative results.

INTRODUCTION

Halogenated chemicals such as saturated and unsaturated hydrocarbons and ethers are of wide industrial application as fumigants, solvents or intermediates in the production of other chemicals [9]. It has been shown that many of these substances are toxic and that in many cases they possess mutagenic, teratogenic and/or carcinogenic properties. Studies on these noxious actions were carried out in studies, in vivo or in vitro, which utilized rodents or bacterial cells and lower eukaryotes respectively [2,4,7-9,121. However, the wide utilization of these chemicals would render it necessary to obtain data on their action on human cells directly. In this connection, we describe in this work the action of 7 of these substances as toxic or DNA damaging agents of human lymphocytes cultured in vitro. The parameters taken into account were the scheduled (replicative) and unscheduled (reparative) DNA syntheses seen as tritiated thymidine uptake *This work was supported by a grant from Minister0 della Sanitd: “Piano di ricerca nel camp0 delle malattie sociali”. No. 500.4/RSC/135/L/1208.

214

in a well-known short-term cell culture system with or without activation of the tested compounds [6,11,13,14].

metabolic

MATERIALS AND METHODS

Chemicals The following halogenated chemicals were tested: 12dibromoethane (C,H,Br,) (DBE), 1,2dichloroethane (CzH4C12) (DCE), trichloroethylene (C2HC13) (TCE), chloroform (CHCIJ) (TCM), carbon tetrachloride (Ccl,) (CTC), dichloromethane (CH&) )DCM) and chloromethyl methyl ether (ClCH20CH3) (CMME). They were provided from Carlo Erba, Milan, Italy or from Merck-Schuchardt, Darmstadt, F.R.G. and were 97-99s pure substances. Taking into account the low water-solubility or water-insolubility of most of these chemicals, they were dissolved in culture medium containing 1% dimethylsulfoxide (DMSO; Sigma, St. Louis, MO, U.S.A.) before their addition to the cell cultures at final concentrations of 10,5 and 2.5 pi/ml and 0.5% DMSO. Cell cultures The in vitro test system utilized in this study was based on that previously described [14] with modifications. Briefly, 50-60 ml of blood from healthy adult donors were collected and lymphocytes were separated according to BGyum [ 31. Lymphocytes, 5 X lO’/well of microtest plate 3040 (Falcon Plastics, Los Angeles, CA, U.S.A.) were cultured in a volume of 0.2 ml of medium RPM1 1640 supplemented with 2 mM glutamine, containing 0.5% DMSO (final concentration) and 0.25 PCi of tritiated thymidine ([ 3H]TdR; 20 Ci/mmol, The Radiochemical Centre, Amersham, England). Cells were cultured for a 4-h period at 37°C in humidified atmosphere containing 5% CO, in presence (treated) or in absence (controls) of each dose of the chemicals in sextuplicate wells. The toxic effects of the substances were measured by [ 3H]TdR uptake for the scheduled DNA synthesis (SDS). To study the effects of the halogenated compounds as DNA damaging agents, the [3H]TdR uptake for unscheduled DNA synthesis (UDS) was determined by adding to the cultures 10 mM hydroxyurea (HU; Sigma) to block the [ 3H]TdR uptake due to SDS [ 6,111. The metabolic activation of the chemicals was obtained following the method proposed by Ames et al. [l] : 1 ml of S-9 mix contained 0.2 ml of rat liver phenobarbital-induced S-9; 8 I.tmol MgClz; 33 pmol KCl; 5 pmol glucose 6-phosphate; 4 pm01 NADP. At the end of the culture period, [ 3H]TdR uptake by lymphocytes for SDS or for UDS in sextuplicate wells was determined by a liquid scintillation spectrometer as previously described [14] and it was exposed in dpm f standard error (S.E.).

215

Data evaluation DNA repair values were calculated for each chemical in the experiments carried out in presence of 10 mM HU taking into account the values in dpm obtained in the cultures performed without S-9 mix versus those obtained in presence of the metabolizing mixture. Therefore, an expected proportional value (x) was calculated as follows: dpmt

(s-9

mix )

x

dpm,+ (s-9

mix)

(1)

x= ._

dpmF(s-9

mix)

where: *(S-9 mix) = cultures performed in presence or in absence of S-9 mix; t = values in dpm obtained in the cultures performed in presence of a chemical; c = values in dpm obtained in the cultures performed in absence of chemicals (controls). Effective DNA repair values (r) were: r

=

dpmt+ (s-9 mix) -

(2)

3~

RESULTS

Table 1 reports the data obtained in a representative experiment which utilized human lymphocytes cultured in vitro in presence of 7 haloalkanes expressed as [ 3H]TdR uptake after 4 h of culture. These values are taken as parameter of the toxic effect measured as inhibition of SDS. It appears that DBE, TCE, CTC and CMME exert the more relevant toxic effects. On the contrary, cells cultured in presence of DCE, TCM and DCM show [ 3H]TdR uptake values similar to those of the control. Table 2 reports the data obtained in 1 experiment carried out in presence of 3 doses of the chemicals and 10 mM HU with or without S-9 mix. The data of the cultures performed in absence of S-9 mix versus those carried out with the metabolizing mixture highly differ in the case of CMME and DBE and, at a lower extent, in the case of DCE and TCE. Owing to the fact that values obtained with the 3 doses employed for each chemical are very similar, the subsequent experiments were carried out with TABLE 1 [3H]TdR UPTAKE BY HUMAN PRESENCE OF 7 HALOALKANES 1,2-Dibromoethane 1,2-Dichloroethane Trichloroethylene Chloroform

1479 2287 1261 2511

f * f f

LYMPHOCYTES (5 r/ml) 70 60 36 61

Data, in dpm, are the mean of sextuplicate

CULTURED

IN VITRO

Carbon tetrachloride Dichloromethane Chloromethyl methyl ether Control samples * S.E.

IN

1120 2356 856 2661

* t * f

57 111 9 57

216 TABLE 2 [3H]TdR UPTAKE BY HUMAN LYMPHOCYTES CULTURED IN VITRO FOR A 4-h PERIOD IN PRESENCE OF 10 mM HU AND 3 DOSES OF 7 HALOALKANES IN PRESENCE OR IN ABSENCE OF S-9 MIX METABOLIZING SYSTEM Dosesa 10 pi/ml

5 pi/ml

2.5 pi/ml

Without S-9 mix

Control 1,2 -Dibromoethane 1,2 -Dichloroethane Trichloroethylene Chloroform Carbon tetrachloride Dichloromethane Chloromethyl methyl ether With S-9 mix Control 1,2-Dibromoethane 1,2 -Dichlorethane Trichloroethylene Chloroform Carbon tetrachloride Dichloromethane Chloromethyl methyl ether

24 33 37 22 39 23 31 26

715 520 586 532 659 481 491 278

612 f 26 882 f 28 532 f 21 439 f 23 578*22 488 f 21 537 f 39 1180 f 33

612 936 673 668 595 466 510 1320

715 604 483 392 661 391 532 313

f f f * f f f f

f 24 f 16 -f 11 f 26 f 38 f 41 f 27 * 13

f f f * + * f f

715124 582 f 43 571 f 32 473 * 27 795 f 30 690 f 26 593 * 24 346 * 20

26 63 45 34 26 18 40 57

612 911 630 533 568 478 573 1125

f f i * f f f f

26 29 34 23 13 31 35 59

Data, in dpm, are the mean of sextuplicate samples f S.E. a Chemicals were dissolved in medium containing 1% DMSO. Final concentration of the DMSO in the cultures was 0.5%. TABLE 3 UNSCHEDULED DNA REPARATIVE SYNTHESIS OF HUMAN LYMPHOCYTES CULTURED IN VITRO FOR A 4-h PERIOD IN PRESENCE OF 10 mM HU AND OF 10,5,2.5 &ml OF 7 HALOALKANES WITH OR WITHOUT S-9 MIX METABOLIZING SYSTEM

1,2-Dibromoethane 1,2-Dichloroethane Trichloroethylene Chloroform Carbon tetrachloride Dichloromethane Chloromethyl methyl ether

10 Ml/ml

5 rl/ml

2.5 r/ml

1st

1st

2nd

3rd

1st

365 118 103 12 153 81 912

490 171 212 30 54 89 1082

579 213 396 0 102 26 955

467 187 141 0 64 0 1009

412 141 128 0 0 65 828

The values, in dpm, were obtained from 3 experiments (lst, 2nd, 3rd) foUowing the equations reported in Materials and Methods. Values of the 1st experiment refer to the data reported in Table 2. Negative values are reported as 0.

217

a single dose (5 @l/ml) and the results were expressed taking into account the data from the cultures performedwithout S-9 mix versus those performed with S-9 mix following Eqns. 1 and 2 reported in Materials and Methods. Table 3 reports the data from 3 distinct experiments. The data of the first experiment refer to that reported in Table 2. Values obtained in the 3 experiments are very similar. Since they express the enhancement of [ ‘H]TdR uptake by cells cultured in presence of HU with or without S-9 mix, we can assume that these values represent the DNA reparative synthesis when the chemicals are metabolized by S-9 mix. CMME, DBE, DCE and TCE appear to elicit the higher UDS; on the contrary, TCM, CTC and DCM show very low or negative values.

DISCUSSION

The results of the present study indicate that CMME, DBE, TCE and DCE elicit DNA reparative synthesis in human lymphocytes (Table 3). This finding is taken as index of a DNA damaging action by these substances and, therefore, of their mutagenic power. On the contrary, the low or negative values of UDS observed for CTC, DCM and TCM exclude such an action by these chemicals in the test system utilized. The values on UDS and those on SDS inhibition are generally not dosedependent (Table 2 and 3), perhaps because the chemical solutions are saturated at all the doses employed. The data obtained are substantially in accord with those reported by other authors with experimental systems which utilized bacterial cells or lower eukaryotes [2,4,7-9,12). It must be pointed out that in our shortterm system positive results are observable only after metabolic activation of the chemicals, confirming previous results [2,8,123. On the contrary, other data reported in the literature, in very different experimental procedures, demonstrate mutagenic properties of some of these substances even in absence of a metabolizing system [9,10,12]. As far as the evaluation of the data is concerned, low UDS values could not be excluded in the experimental schedules which did not utilize the S-9 mix. In fact, it is not possible to distinguish this hypothetic low reparative synthesis from the inhibition of [ 3H]TdR uptake due to toxic effects exerted by the chemicals. In any case, the high values obtained in treated cells cultured with S-9 mix versus the controls are not observable in cultures performed without S-9 mix (Table 2). This finding makes us sure that the metabolic activation increases the DNA damaging properties of the tested chemicals. Finally, the high values of UDS observed for CMME (Table 3) could be ascribed to the action of bis(chloromethy1) ether (BCME) because it was demonstrated that in aqueous solution CMME is transformed into BCME, which was recognized as carcinogenic for man also [ 71.

218 REFERENCES 1 Ames, B.N., McCann, J. and Yamasaki, E. (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutat. Res., 31, 347-364. 2 Banarjee, S., Van Duuren, B.L. and Orvambo, F.I. (1980) Microsome-mediated covalent binding of 1,2dichloroethane to lung microsomal protein and salmon sperm DNA. Cancer Res., 40,2170-2173. 3 BSyum, A. (1968) Separation of leukocytes from blood and bone marrow. J. Clin. Iab. Invest., Suppl. 97, 21, 77-82. 4 Brem, H., Stein, A.B. and Rosenkranx, H.S. (1974) The mutagenic and DNA-modifying effect of haloalkanee. Cancer Res., 34, 2576-2579. 5 Cerna, M. and Kyp&novB, H. (1977) Mutagenic activity of chloroethylenes analyzed by screening system tests. Mutat. Res., 46, 214. 6 Evans, R.G. and Norman, A. (1968) Radiation stimulated incorporation of thymidine into the DNA of human lymphocytes. Nature, 217.455-456. 7 Fishbein, L. (1979) Potential halogenated industrial carcinogenic and mutagenic chemicals. III. Alkane halides, alkanols and ethers. Sci. Total Environ., 11, 223-257. 8 Greim, H., Bonse, G., Radwan, Z., Reichert, D. and Henschler, D. (1975) Mutagenicity in vitro and potential carcinogenicity of chlorinated ethylenes as a function of metabolic oxirane formation. Biochem. Pharmacol., 24, 2013-2017. 9 I.A.R.C. (1974-1979) Monographs on the evaluation of carcinogenic risk of chemicals to man. Vol. 4, 15, 20. I.A.R.C., Lyon, France. 10 Jongen, W.M.F., Alink, G.M. and Koeman, J.H. (1978) Mutagenic effect of dichloromethane on Salmonella typhimurium. Mutat. Res., 56, 245-248. 11 Lieberman, M.W., Baney, R.N., Lee, R.E., Sell, S. and Farber, E. (1971) Studies on DNA repair in human lymphocytes treated with proximate carcinogens and alkylating agents. Cancer Res., 31, 1297-1306. 12 McCann, J., Choi, E., Yamasaki, E. and Ames, B.N. (1975) Detection of carcinogens as mutagens in the SalmoneNa/microsome test: assay of 300 chemicals. Proc. Natl. Acad. Sci. U.S.A., 72, 5135-5139. 13 Perocco, P., Rocchi, P., Grilli, S., Franceschi, C. and Prodi, G. (1976) DNA repair after UV and gamma irradiation. II. Human lymphocytes. Arch. Sci. Biol., 60,79-85. 14 Rocchi, P., Perocco, P., Alberghini, W., Fini, A. and Prodi, G. (1980) Effect of pesticides on scheduled and unscheduled DNA synthesis of rat thymocytes and human lymphocytes. Arch. Toxicol., 45, 101-108.