TOXICOLOGYANDAPPLIEDPHARMACOLOGY 64,482-485 (1982)

ln Vitro Cytotoxicity and Genotoxicity of Dibutyltin Dichloride and Dibutylgermanium Dichloride

A. P. LI, A. R. DAHL, AND J. 0. HILL’

Lovelace Inhalation Toxicology Research Institute, P.O. Box 5890, Albuquerque, New Mexico 87185

Received December 28, 1981; accepted March 19, 1982

In Vitro Cytotoxicity and Genotoxicity of Dibutyltin Dichloride and Dibutylgermanium Dichloride. LI, A. P., DAHL, A. R., AND HILL, J. 0. (1982). Toxicol. Appl. Pharrnacol. 64, 482-485. The cytotoxicity and genotoxicity of dibutyltin dichloride (DBTC) and dibutylger- manium dichloride (DBGC) were studied with in vitro tests. DBTC was approximately lOOO- fold higher in cytotoxicity and genotoxicity than DBGC. Both compounds decreased the antibody production by lymphocytes in vitro at noncytotoxic doses, which may explain the immunosuppressive effect of the compounds in vivo. Both compounds induced mutation in Chinese hamster ovary cells, therefore suggesting that they are potentially carcinogenic.

Organotin compounds are widely used in plastics as stabilizers, catalysts, and curing agents and in agriculture as fungicides (Ross, 1965). The annual production of organotin compounds exceeds 17,000 tons, (Van der Kerk, 1975). Human exposure to organotin compounds can occur through the produc- tion and use of tin-containing plastics or through occupational or accidental exposure to biocidal compounds. Because of the wide- spread distribution and consequent potential for human exposure, it is important to es- tablish the biological effects of the organotin compounds.

The acute and chronic toxicity of organ- otin compounds has been studied, (Magee et al., 1957; Barnes and Stoner, 1958). The triorganotin compounds assert their toxicity in part by interference with mitochondrial ATP production. The generally less toxic diorganotins are potent immunosuppressants in vivo (Seinen et al., 1977; Seinen and Pen- ninks, 1979; Hill et al., 1980). The genotox- icity of organotin compounds has not been

’ Present address: Trudeau Institute, P.O. Box 59, Saranac Lake, N.Y. 12983.

extensively studied. Triphenyltin hydroxide was found to be mutagenic in the mouse dominant-lethal assay (Epstein et al., 1972). Tributyltin oxide, dibutyltin bis trifluoroac- etate, and monobutyltin trilaurate were found to induce chromosomal aberrations in mam- mals (Belyaeva et al., 1976).

In this paper we report our findings on the cytotoxicity and genotoxicity of dibutyltin dichloride (DBTC) and the germanium an- alog, dibutylgermanium dichloride (DBGC). DBGC was included because, since it differs from DBTC only in the substitution of a tin periodic congener at a single position, effects seen only for DBTC could reasonably be attributed to the presence of the tin atom. Two in vitro assays were used. To assay for potential immunosuppressive properties, we used rat lymphocytes to test both for cyto- toxicity and impairment of lymphocyte func- tion (impairment of antibody formation). To assay for mutagenicity, we used the well- established Chinese hamster ovary cell (CHO)/hypoxanthine guanine phosphori- bosyl transferase (HGPRT) mutation assay (Hsie et al., 1979; Li, 1981a).

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CYTOTOXICITY AND GENOTOXICITY OF ORGANOMETALS 4x3

DOSE ( pg/ml)

FIG. 1. Toxicity of DBTC (0, 0) and DBGC (A, A) toward rat lymphocytes, using dye-exclusion as a

measurement of cytotoxicity (open symbols) and inhi-

bition of antibody formation as a measurement of im- pairment of lymphocyte function (closed symbols).

METHODS

Cell culture. Chinese hamster ovary (CHO) cells clone K,-BH, was originally obtained from Dr. A. W. Hsie of Oak Ridge National Laboratory. The CHO cells

are now routinely maintained in our laboratory. The

cells were maintained in Ham’s F12 medium (Flow Labs) supplemented with 10% heat-inactivated newborn

calf serum (K.C. Biological Inc.), penicillin (50 units/ ml), and streptomycin (50 pg/ml) (K.C. Biological

Inc.). The stock cultures were maintained in the loga- rithmic phase of growth by subculturing twice weekly.

All cell cultures were kept at 37’C in a 100% hu- midified atmosphere of 5% CO, and 95% air.

Lymphocyte toxicity assays. The cells for the lym- phocyte studies were obtained from pooled thoracic

lymph nodes from Fischer 344 rats (laboratory-reared, specific-pathogen free). The animals were immunized

intratracheally with sheep red blood cells 7 days before the lymphocytes were harvested, in the time when peak

response in lung-associated lymph nodes was observed as previously described (Bite et al., 1979). The lym-

phocytes were plated in RPM1 1640 Medium (Grand Island Biological Co.) with 10% heat-inactivated fetal

calf serum (Howell Labs). Triplicate cultures containing 2 X IO6 cells per tube were used per treatment. Lym-

phocytes were treated with final concentrations of 9 to 75 pg/ml DBTC and 8 to 64 pg/ml DBGC dissolved in DMSO. Treatment was performed for 24 hr at 37°C. Trypan blue exclusion tests for viability and the in vitro Cunningham plaque assay for direct (IgM) antibody- forming cells (AFC) were performed (Cunningham and Szenberg, 1968). Data were expressed as percentage of

control value, which was approximately 540 Al?<‘/ 10’

lymphocytes. Cytotoxicity and mutagenicity assay with CHO cells.

The cytotoxicity and mutagenesis procedures have been previously described (Li, 1981a; Li, 1981 b). Briefly, CHO cells were plated in growth medium one day before treatment. On the day of treatment, medium was

changed to growth medium without serum. Graded con- centrations of DBTC and DBGC dissolved in DMSO

were added. The final concentrations of DBTC and DBGC were 0.05 to 0.3 pg/ml and 100 to 600 pg/ml,

respectively. The final concentration of DMSO was kept at 1%. The cells were then incubated at 37°C for 3 hr after which they were trypsinized. Two hundred ceils

were plated in triplicate from each sample in 35-mm-

diameter tissue culture plates for the determination of cytotoxicity. These plates were incubated for 7 days

after which the colonies that developed were fixed with 70% methanol, stained with 10% giemsa, and counted.

Cytotoxicity data were expressed as relative survival which was the ratio of the cloning efficiency (C.E.) of

the treated cells to that of the control:

C.E. = No. colonies/No. cells plated:

relative survival = E:i: :~~~t~o~~

To determine mutagenicity, the cells were subcul-

tured for an 8-day expression period (Li. 198111). The cells were then plated in selective medium consisting of

10 WM 6-thioguanine (Sigma) in hypoxanthine-free Ham’s F12 medium (KC Biological Incorporation) sup-

plemented with 5% dialyzed newborn calf serum. A

number of lo6 ceils per sample was selected for mutant frequency by plating 2 X lo5 cells in each of the five

selection plates. A total of 200 cells were plated in trip- licate in selective medium without 6-thioguanine in 35- mm diameter tissue culture plates for the determination

of cloning efficiency (C.E.). The plates were incubated for 9 days, after which the colonies that developed were

fixed, stained, and counted. The mutant frequency (M F) was calculated as

MF= No. of mutant colonies x -1.

No. of cells plated C.E.

MF was expressed as mutants per lo6 survivors.

Orgunometnls. DBTC and DBGC were obtained from Organometallics Inc., East Hamstead, New

Hampshire, and were used as supplied.

RESULTS

The LCsO value for lymphocytes as deter- mined by dye-exclusion was approximately 50 pg/ml (0.16 mM) for DBTC (Fig. 1). At

484 LI, DAHL, AND HILL

the same concentration of DBTC, the num- ber of antibody-forming cells (AFC) was reduced to approximately 10% of the con- trol. DBGC was not cytotoxic up to 64 pg/ ml (0.25 mM) at that concentration; how- ever, the number of AFC was reduced to approximately 50% of the control.

The LC5,, values of DBTC and DBGC for CHO cells, as determined by cloning effi- ciency, were approximately 0.35 @g/ml ( 1.12 PM) and 600 Kg/ml (2.33 mrvr), respectively (Fig. 2). Both compounds induced mutations at the HGPRT gene locus in CHO cells. The mutant frequency increased with dose up to 0.2 pg/ml (0.66 PM) for DBTC and 400 pg/ml (1.55 mM) for DBGC. A decrease in mutant frequency was observed at higher concentrations. DBTC appeared to have a three-order-of-magnitude higher cytotoxic- ity and mutagenicity than DBGC.

DISCUSSION

Both DBTC and DBGC impaired anti- body formation by lymphocytes at similar concentrations. This impairment of lympho- cyte function may be a specific biological effect since it was observed at doses of DBTC and DBGC with low cytotoxicity. This ob- servation is similar to that observed by Kutz et al. (1980) and is consistent with the high immunosuppressant effects of dialkyltin compounds in vivo (Seinen et al., 1977). The lymphocyte system described here is there- fore a possible in vitro model for the studying of the immunosuppressant effects of organ- otin compounds.

The high in vitro cytotoxicity of DBTC is not surprising since it had been found to have high acute toxicity in vivo. The positive genotoxicity of DBTC at the low concentra- tions (< 1 ppm) tested in the CHO/HGPRT mutation assay, however, is a matter of con- cern. Our in vitro data are consistent with the previously reported in vivo genotoxicity data obtained from the mouse dominant le- thal assay (Epstein et al., 1972) and the bone

DOSE ( pglml)

FIG. 2. Cytotoxicity (open symbols) and mutagenicity (closed symbols) of DBTC (0, 0) and DBGC (A, A) in CHO cells.

marrow chromosomal aberration assay (Be- lyaeva et al., 1976) indicating that dialkyltin compounds are genotoxic and are therefore potentially carcinogenic. It is interesting to note that DBTC was not mutagenic in the Ames test for mutagenicity (Clark, personal communication). It appears that mamma- lian genotoxicity assays are required for the testing of DBTC and similar compounds.

The genotoxicity observed for both dibu- tyltin dichloride and the germanium analog may be due to a similar mechanism. How- ever, the concentrations of DBGC required to achieve toxic effects were 1000 times those for DBTC. Simple physical differences such as solubility effects probably are not respon- sible for the observed quantitative toxicity differences since both compounds are quite insoluble in water but freely soluble in or- ganic solvents. One possible mechanism is that both compounds directly bind as elec- trophiles to the DNA bases to form penta- or hexa-coordinate compounds. Such com- pounds are highly stable and readily formed (Neumann, 1967; Aldridge et al., 198 1; Pal- lerito et al., 1976). This reasoning would also explain the quantitative differences in DBGC and DBTC since the tendency for penta- or hexa-coordination is greater for tin than for germanium (Neumann, 1967; Glockling, 1969). We are currently investigating DNA binding of DBTC to test our hypothesis.

The observed genotoxicity effects of DBTC and DBGC may extend to other organotin

CYTOTOXICITY AND GENOTOXICITY OF ORGANOMETALS 4x5

and organogermanium compounds, although the potency would probably depend on a sub- tle admixture of chemicophysical properties. Moreover, since electrophilic binding to ni- trogen bases is common to most metals, there is a potential for genotoxicity for other lipid soluble metal compounds which are able to penetrate the cell membrane and to reach the genetic material. The genotoxicity data for DBTC presented here suggest possible carcinogenicity for the compound.

ACKNOWLEDGMENTS

The authors thank Millie L. Deland and M. S. Giere for their excellent technical assistance, E. Gaff for the graphics, and Drs. A. L. Brooks, J. S. Dutcher, W. M. Hadley, D. L. Lundgren, R. F. Henderson, and R. 0. McClellan for their critical review of this manu- script. Research supported in part by the Environmental Protection Agency via Interagency Agreement No. EPA-81-D-X0533 under U.S. Department of Energy Contract DE-AC04-76EV01013.

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