Environmental and Molecular Mutagenesis 20:127-133 (1992)

Effects of Captan on DNA and DNA Metabolic-Processes in Human Diploid Fibroblasts Ronald D. Snyder Marion Merrell Dow Research Institute, Cincinnati, Ohio 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 for cell killing, approximately 0.8 pM, none of the above genotoxic events could be detected by the methods employed. Q 1992 WiIey-Liss, Inc.

Key words: Captan, DNA repair, DNA adducts, mutagenesis

INTRODUCTION The fungicide Captan has been shown to be mutagenic and to induce other genetic effects in procaryotes [Bridges et al., 1973; Bridges, 1975; Shirasu et al., 1976; Siebertet al., 19761, lower eucaryotes [Waters et al., 19801, and mammalian cells [Arlett et al., 1975; Fry and Fiscor, 1978; O’Neill et al., 1981; Garrett et al., 1986; Jingyie and Baoyeng, 19871. In addition, chronic feeding studies of Captan in mice have demonstrated an induction of duodenal hyperplasia and tumor formation [Stauffer Chemical Company, 1985; Chevron Chemical Company, 19801. While the mechanism underlying these genotoxic effects is not known, several reports have suggested that Captan may interact with DNA [Anderson and Rosenkranz, 1974; Stauffer Chemical Company, 19811 and induce DNA repair [Swenberg et al., 1976; Ahmed et al., 1977; Simmon et al., 19771, but a systematic examination of this question has been lacking. This study was designed to examine the nature of the genotoxic events associated with Captan exposure to human cells.

MATERIALS AND METHODS

serum at 5% CO, and 37°C in a humidified incubator. Where appropriate, cellular DNA was radiolabeled by the addition to the growth medium of 0.7 p,Ci/ml [3H]-thymidine (6.7 Ci/mmol, Amersham) or 0.17 p,Ci/ml [14C]-thymidine (543 mCi/mmol, Amersham) for 24 hr. Captan Technical (Stauffer Chemical Company, 98% purity) treatment was conducted in pH-adjusted Hanks’ balanced salt solution (HBSS).

Colony Forming Ability Approximately 5 X lo5 HSBP cells were dosed with Captan for 30 min at 37°C in HBSS. The drug was then washed from the dishes with three changes of HBSS and the cells were incubated for an additional 30 min in complete growth medium. Cells were harvested by trypsinization and replated at 500/dish for colony formation. After 10 days, the dishes were stained with Giemsa and colonies counted.

Alkaline Sucrose Sedimentation Studies For DNA strand break determination, ‘H-labeled cultures received Captan and 14C-labeled cultures served as untreated controls. For repair studies, both 3H- and I4C-labeled

Cell Culturing and Labeling Human foreskin fibroblasts (HSBP, Dr. J.D. Regan, Oak Ridge, TN) were seeded into 60 mm plastic tissue culture dishes or T25 flasks at a density of 5 X 104/5ml. They were grown in modified Eagle’s medium with 10% fetal bovine 0 1992 Wiley-Liss, Inc.

Received October 17, 1991; revised and accepted April 29, 1992 Address reprint requests to Ronald D. Snyder, Marion Merrell Dow Research Institute, Cincinnati, OH 45215.

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cultures received Captan but the 'H-labeled cultures also received the repair inhibitors 1-P-D-arabinofuranosyl cytosine (ara-C; final concentration 20 pM, Sigma) and hydroxyurea (HU; final concentration 2 mM, Sigma). Cells for both studies were harvested and cosedimented in linear 5-20% alkaline sucrose gradients and analyzed for DNA molecular weights as described previously [Francis et al., 1981, and references therein]. Nucleoside Uptake, Replicative DNA Synthesis, and Unscheduled DNA Synthesis

For uptake analysis, approximately 5 X lo5 cells were incubated for 1 hr in the presence of either 1.0 pCi/ml [3H]-thymidine (6.7 Ci/mmol, ICN) or 2.5 pCi/ml ['HIdeoxycytidine (28 Ci/mmol, ICN) and then extensively washed free of excess label with five changes of PBS. Dishes were extracted with 1 ml ice-cold 5% TCA for 2 hr at 4°C. Extractable radioactivity represented acid-soluble label (unincorporated) and remaining cellular label represented acid-insoluble (incorporated) counts. Replicative DNA synthesis was measured in exponentially growing cells by incubation for 2 hr in medium containing label as used in the uptake studies. The cells were harvested, counted, and acid insoluble radioactivity determined by TCA precipitation on Whatman 3MM discs. Unscheduled DNA synthesis following ultraviolet irradiation was measured in cells which had been held in confluency for 20 days (25 days post-seeding) with frequent media changes. The same labeling conditions were used as for replicative DNA synthesis measurement. Under these conditions, background incorporation was extremely low and 10 joules/M2 ultraviolet irradiation induced approximately four to seven times the nonirradiated background levels of incorporation when measured with labeled thymidine or deoxycytidine, respectively.

Nick Translation Assay The nick translation assay was performed essentially according to the technique of Nose and Okamoto (1983) with modifications [Snyder and Matheson, 19851. Briefly, 1 x lo6 Captan-treated or untreated fibroblasts were harvested by trypsinization from confluent cultures and suspended in 0.25 M sucrose, 0. I M Tris-HC1 pH 7.4, 10 mM MgCl,, and 0.5 mM dithiothreitol. Lysolecithin (Sigma) was added to a final concentration of 100 pg/ml. The cell suspension was kept on ice for 2 4 min and pelleted at 6,500g. This pellet was then resuspended in nick translation assay mix containing 50 mM Tris-HC1 pH 7.4, 5 mM MgCl,, 10 mM 2-mercaptoethanol, 50 pg/ml bovine serum albumin, 0.05 mM each dATP, dGTP, and dTTP, and 5 pCi/ml [3H]dCTP(28 Ci/mmol). E. coli DNA polymerase I was added to 40 units/ml. The reaction was allowed to proceed for 30 min at room temperature, then the entire assay mix was spotted onto a Whatman 3MM filter disc

prewetted with 2% pyrophosphate and the discs processed for acid insoluble radioactivity. DNA- Protein Cross- Linking

Analysis of DNA-protein cross-linking was carried out according to the procedure of Smith [ 19621. Briefly, appropriately treated cells prelabeled with tritiated thymidine were harvested, suspended in 2% SDS, 1 M sodium citrate pH 7, and gently stirred at room temperature for 1 hr. The solution was precipitated with two volumes of cold ethanol and the pellet collected by centrifugation was resuspended in SDS-citrate. This was treated with an equal volume of 1 M KCI to precipitate the protein and detergent, leaving the bulk of the DNA in the supernatant. Radioactivity associated with this precipitate was measured after successive cycles of dissolving and resuspension until no further radioactivity could be washed from the precipitate. Remaining label was taken as a measure of DNA-protein cross-linking.

DNA Isolation, Hydrolysis, and HPLC Analysis Herring sperm DNA

Three milligrams [ ''C]-trichloromethyl Captan (56 mCi/ mmol, Amersham) were dissolved in 750 pL DMSO and reacted with 4 or 4.6 mg herring sperm DNA (Sigma) for I hr at 37°C in 7.5 ml PBS (pH 6.0 or 7.5). DNA was purified by pheno1:chloroform extraction and ethanol precipitation followed by repeated cycles of precipitation and dissolution to remove all unincorporated label. The DNA was then hydrolysed for HPLC analysis as described below. Human fibroblast DNA

Tritiated thymidine-prelabeled confluent cultures were harvested by trypsinization and resuspended in 10 ml HBSS containing 4.5 mg [I4C] Captan for 1 hr at 37°C. DNA was then isolated by the hydroxyapatite method of Muller and Rajewsky [ 19801. Tritium and carbon-14 radioactivity associated with the DNA was monitored throughout exhaustive dialysis against distilled water until no further reduction in the I4U3Hratio was observed. Thermal acid hydrolysis to release purine bases from DNA was conducted by heating DNA to 100°C for 30 min in 10 mM cacodylate, cooling on ice, and addition of 0.1 volume 1 N HCI. The DNA was then heated at 70°C for 30 min and the supernatant following 6,500g centrifugation was used for HPLC analysis. HPLC analysis was conducted with two Waters p Bondapak C18 columns in series utilizing a linear gradient from 100% 0.02 M KH,P04 to 60% methanol. One-milliliter fractions were collected for scintillation counting. Fidelity Assay

Synthetic double stranded template-primers Poly (dA-dT) and Poly d(G).Oligo d(C)12-18(P-L Biochemicals) were

Genotoxicity of Captan

129

type of DNA damage recognized by the excision repair system (Table I). Two additional conclusions may be drawn from the data in Table I: 1 ) The production of DNA strand breaks is dependent on the treatment pH with much more DNA damage occurring at pH 6.6 than at pH 7.6. This could reflect the fact that Captan degrades rapidly in aqueous solutions at pH 7.5 and above [Environmental Protection Agency, 19761. 2) The strand breaking activity of Captan may reside in a volatile component. This is suggested by the nearly complete absence of DNA strand breaks in cells treated in 0 01 02 03 04 05 loosely covered tissue culture dishes (open system) as opDose (pg/ml) posed to tightly capped flasks (closed system). This same Fig. 1. Cloning ability of human diploid fibroblasts treated with Captan. phenomenon has been reported for Captan in the Ames Salmonella mutagenesis assay (J. McGregor, personal comProcedure as outlined in Materials and Methods. 0.1 pg/ml is 0.33 pM. munication) and in CHO mutagenesis studies [Stauffer Chemical Company, 19841. The most likely active constitueither untreated or incubated with Captan or methyl ni- ent of Captan is the trichloromethylsulfenyl group. This trosourea (MNU) for 2 hr at 37°C in 67 mM phosphate moiety easily splits off [Couch et al., 19771 and could exert buffer pH 6.2. Precipitated Captan was removed by centrif- DNA damaging effects directly or by its hypothesized conugation and the remaining supernatant was dialyzed over- version to the highly reactive thiophosgene [DeBaun et al., night against 50 mM Tris-HC1 pH 7.4. Resultant dialyzed 1974; Environmental Protection Agency; 19761. In studies templates were used within 2 days of preparation. Error-rate not presented here, perchloromethyl mercaptan (the trichloof DNA synthesis on these templates was determined from romethylsulfenyl moiety of Captan) was also shown to inthe ratio of noncomplementary to complementary nucleotide duce DNA strand breaks and to induce ara-C inhibitable incorporated. The reaction mixture contained 100-p,M repair. Recent studies have demonstrated that Captan is an inhib(DNA phosphate) or template-primer, 50 pM complemenitor of DNA synthesis [Decloitre and Martin, 19801 and that tary dNTP, 5 p M noncomplementary dNTP, 5 mM MgCl,, this inhibition derives from an inhibitory interaction of the 50 p,g/ml bovine serum albumin, 2.5 units E. coli DNA trichloromethylsulfenyl side chain with DNA polymerase polymerase I, and 100 mM Tris-HC1 pH 7.4 in a final [Dillwith and Lewis, 1980, 19821. Captan is known to block volume of 200 pl. [3H] complementary or noncomplementhe uptake of uridine into acid soluble pools (Roger Lewis, tary dNTPs were used to quantitate incorporation and misinpersonal communication) and Table I1 demonstrates a simicorporation, respectively. The reaction was incubated at lar inhibition of thymidine uptake, the mechanism of which 37°C for 30 min and then spotted on Whatman 3MM discs has not been investigated. All DNA synthesis studies were and processed for acid insoluble radioactivity measurement. therefore conducted utilizing radiolabeled deoxycytidine, the uptake of which was not affected by Captan treatment. RESULTS AND DISCUSSION Table I1 demonstrates that both replicative synthesis (SCS) Figure 1 shows the effects of Captan treatment on the and ultraviolet radiation-induced repair synthesis (UDS) cloning ability of HSBP fibroblasts. Exposure for 30 min to were reduced in a dose-dependent fashion by Captan. In our 1.65 pM Captan reduced cloning ability to about 20%. hands, Captan by itself did not induce UDS at doses between However, much higher doses (up to 300 pM) could be 0.01 and 3 pM (not shown). Captan has previously been employed in short-term studies with no detectable overt reported to induce positive responses in the UDS assay [Ahmed et al., 19771. However, it is difficult to assess this toxicity or impairment of cellular DNA repair functions. The results in Table I demonstrate that DNA strand breaks observation in light of the strong inhibition of thymidine and repairing sites were observed in alkaline sucrose sedi- uptake and DNA synthesis reported above. The DNA damaging and repair-inducing character of mentation studies of Captan-treated fibroblasts. These dosedependent strand breaks were only detected after 2-3 hr Captan was verified using the nick translation assay [Nose incubation (not shown). Sites undergoing “long-patch” exci- and Okamoto, 1983; Snyder and Matheson, 19851. This sion repair are susceptible to inhibition by agents such as assay measures incorporation by exogenously added E. coli ara-C and hydroxyurea, resulting in stable single strand DNA polymerase of radiolabeled dNTPs into cellular DNA breaks [Collins et al., 1977; Snyder et al., 1981; Francis et sites that have been nicked by test chemical treatment. It is al., 1981 ; Collins et al., 1984, and references therein]. Such shown in Table 111 that DNA damage appears immediately inhibitor-dependent breaks were observed following expo- following removal of high doses of Captan. It is also shown sure to Captan (Table I), indicating that Captan induced a that ara-C/HU treatment leads to a further small but repro-

130

Snyder

TABLE 1. Alkaline Sucrose Sedimentation Analysis of DNA Strand Breaks and Sites Undergoing Repair Following Treatment of Human Fibroblasts With Captan Dose

Treatment time

System

16.5pM 49.5 pM

3 hr 3 hr

Closed Closed

99.0 pM

3 hr

Closed

198 p M

3 hr

Closed

49.5 pM

3 hr

Open

99.0 pM

3 hr

Open

198 pM

3 hr

Open

99 pM

0.5 hr 1 hr 2 hr

DNA strand breaks 10' daltons

Treatment PH

No inhibitors

6.6 6.6 1.6 6.6 7.6 6.6 7.6 6.6 7.6 6.6 1.6 6.6 7.6 6.6 6.6 6.6

0 0.06 0 1.3 0.3 2.4 1.2 0 0 0 0 0.12 0 0 0 0

Closed Closed Closed

+

Ara-C/HU" 0 0.14 0.16 1.24 0.94 2.4 2. I 0.04 0.06 0.52 0.38 0.36 0.38 0.62 0.64 0.94

Results presented are derived from one set of experiments and are representative of the results obtained in repeat trials. "DNA single strand breaks in excess of number observed in the absence of inhibitors. No strand breaks were observed in cells treated only with inhibitors. Ara-C/HU treatment at 20 p M and 2 mM final concentrations, respectively, for 3 hr.

TABLE 11. Effects of Captan on Ultraviolet Radiation-Induced Unscheduled DNA Synthesis (UDS), Semiconservative DNA Synthesis (SCS), and Nucleoside Uptake in Human Fibroblasts ~

CPM incorporated (% control) Dose 0 3.3 pM 9.8 pM 16.5 LLM

UDS

scs

2100*88 (100) 1827 2 168 (87) 609 2 133 (29) 210* 21 (10)

14,500 490 (100) 11,020 f 1542 (76) 3915 704 (27) 1015 40 (4)

*

* *

Uptake (% control) I'HldThd

13HldCvd

100

100

7.5

100

Assays performed as in Materials and Methods section. Confluent cultures received 10joule/M2 ultraviolet irradiation and were labeled for 2 hr for UDS determinations. SCS measurements were made with a similar 2-hr labeling of cells in exponential growth phase. CPM determinations were the average of three determinations SD. 3H-deoxycytidine was used for all incorporation studies.

*

ducible increase in radiolabel incorporation. We have previously documented the use of the nick translation assay to detect inhibition of excision repair by ara-C and HU [Snyder, 19861. Thus, the above results are consistent with the alkaline sucrose data and suggest that Captan-induced DNA damage may be at least partially repaired via a long-patch repair system. Thus, DNA damage and excision repair have been demonstrated by two different methods following Captan treatment. It should be pointed out, however, that excessive doses of Captan must be used in order to detect these events. Two micromolar Captan reduces cloning efficiency to zero, 3.3 p M Captan depresses cellular DNA synthesis, but doses of at least 50 p M must be employed in order to see DNA damage and repair. Thus, either cytotoxicity associated with Captan exposure is mediated by cellular interactions other

than direct DNA binding, or very small numbers of lesions (below the limits of detection with the present methods) are needed for cytotoxicity. In addition to DNA strand breaks, Captan may also induce DNA-protein cross-linking. Figure 2 shows that Captan-treated cells bind threefold to fourfold more DNA to protein than untreated controls. Similar results have been obtained with this technique using known cross-linking agents such as formaldehyde [Snyder and Van Houten, 19861, novobiocin [Snyder et al., 19821, and ultraviolet radiation [Smith, 19621. The nature of this cross-linking is unknown but Captan has been reported to bind to histones and thus alter their ability to stabilize DNA structure [Couch and Siegel, 19771. Herring sperm DNA when reacted with radiolabeled Captan acquired 1,293 and 868 adducts/108 daltons DNA de-

131

Genotoxicity of Captan

TABLE Ill. Nick Translation of DNA Strand Breaks and Sites Undergoing Repair Following Treatment of Human

60

Fibroblasts With Captan Dose“

0 26.4 pM 132 pM 264 pM

Incubationb

1420 18 1480 f 42 1434 f 21 1478 f 19 2672 & 57 2598 f 107 3404 2 71 3408 f 58 3038 f 38 4070 f 23

10

20

30

40

50

h

*

2 hr 2 hr plus inhibitors 2 hr 2 hr plus inhibitors None 2hr 2 hr plus inhibitors None 2hr 2 hr ulus inhibitors

G

:

i

40

30

-

2 c

x

20 10

Results are the average f SD of five separate determinations as measured as described in Materials and Methods. ”1-hr treatment at 3TC, pH 6.6, closed system. bNo Captan present during incubation; inhibitors were ara-C (20 p M , final concentration) and hydroxyurea (2 mM, final concentration).

OO 0

A

CPM incorporated (% Control)

?

50

Wash Number

Fig. 2. DNA-protein cross-linking in Captan-treated human fibroblasts. Procedures as in Materials and Methods. O-O untreated cells; 0-0 cells treated with 200 pM Captan, 30 min at 37°C.

pending on whether the initial treatment was conducted at pH 6.0 or 7.5, respectively. These values translate to one adduct every 129 or 189 base pairs, respectively. Treatment of human fibroblasts with equal amounts of labeled Captan resulted in approximately 240 adduck/ 10’ daltons DNA. HPLC analysis of thermal acid hydrolysed Captan-treated Herring sperm DNA (Fig. 3) demonstrates that discrete peaks are observed which elute with similar times to the purine bases. Unreacted Captan elutes at 7 min. These data are suggestive of covalent binding consistent with previous observations [Anderson and Rosenkranz, 1974; Stauffer Chemical Company, 19811. Alkylation at the O6 position of guanine results in increased base pairing with thymine instead of cytosine [Abbott and Saffhill, 19791due to alteration in normal hydrogen bonding and 0-alkylations of thymine result in increased mispairing with guanosine [Abbott and Saffhill, 19771. These base modifications, then, have the potential of “fixing” mutations in the DNA sequence if not repaired prior to

+

Inject

5

10

15

20

25

9 -

30

Elution h e (rnin)

Fig. 3. HPLC analysis of thermal acid hydrolyzed herring sperm DNA treated with Captan. The positions of guanine ( G ) and adenine ( A ) are marked. See Materials and Methods section.

replication. In order to establish whether the addition of Captan to DNA has mutagenic potential, Captan was reacted with synthetic DNA templates and the fidelity of replication catalyzed by E. coli DNA polymerase I was measured. Table IV demonstrates that poly (dA-dT) and poly d(G).oligo d(C)12-18treated with Captan directed increased misincorporationof dGTP and dlTP, respectively, over that seen on untreated templates. The degree of misincorporation was similar to that seen on templates treated with MNU. dCTP incorporation was not significantly increased by MNU or Captan. Decreased fidelity of replication is consistent with bacterial mutagenesis studies which have demonstrated Captan to induce primarily base pair substitutions [Bridges et al., 1973; Bridges, 1975; Shirasu eta]., 19761. A strong mutagenic response was also observed with Chinese hamster cells [O’Neill et al., 19811 but mouse cells appear resistant to rnutagenesis by Captan [Litton Bionetics, Inc., 1980; Miller, 19801. We have also observed that Captan induces only a very weak mutagenic response in human fibroblasts [Stauffer Chemical Company, 19841. The reason for this species specificity in mutagenic response is not known. In conclusion, these studies have demonstrated that Captan 1) inhibits cellular DNA synthetic processes, 2) induces both DNA-protein cross-links and DNA base modifications, a fraction of which are apparently removed from cellular DNA by excision repair processes, and 3) has mutagenic potential via decreased fidelity of polymerization on Captan-modified templates. Doses of Captan far in excess of the ICso are required in order to demonstrate DNA interactive effects with the methodology employed here. Therefore, the biological relevance of these findings might well be called into question. However, it must be assumed that even very low environmental exposures to any DNA-reactive agent will result in the same spectrum of damages seen in the

132

Snyder

TABLE IV. Misincorporation on Synthetic DNA Templates Modified by Captan or MNU* Correct incorporation Template

-

Poly d (AT)

Agent Untreated

Incorrect incorporation (pmoles)

(pmoles)

dGTP

5465 (100)a

0.39

dCTP

dlTP

11.2

0.28 MNU 1 6 m M

5275(97)

4.2

MNU 160 m M

3185 (58)

6.8

Captan 1.3 mM

Poly d(G) . oligo d(C)

5765 (105)

Fold increase error rate over controls

0.4

1.5

0.21

29.9 1.3 13.9

5.7 0.31

I .o

0.2

31.1 1.3

6.5

26.0 mM

2930 (54)

Untreated MNU 15.4 mM Captan 5.8 mM 12.0 mM

710 (100)

0.71

-

330 (46)

0.97

2.9

644 (91)

0.97 2.2

3.9

565 (80)

1.5

*Values are derived from a single experiment. aPercent control incorporation.

high-dose studies required to detect these lesions and that very few lesionskell may be required for genotoxicity.

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Genotoxicity of Captan by nick translation in permeable cells. Biochem Biophys Res Commun I 1 I :38>389. O’Neill JP, Forbes NL, Hsie AW (1981): Cytotoxicity and mutagenicity of the fungicides captan and folpet in cultured mammalian cells (CHO/ HGPRT system). Environ Mutagen 3:233-237. Shirasu Y,Moriya M,Kato K, Furuhashi A, Kada T (1976): Mutagenicity screening of pesticides in the microbial system. Mutat Res 40:1930. Siebert D, Zimmerman FK, Lemperle E (1976): Genetic effects of fungicides. Mutat Res 10:533-540. Simmon VF, Mitchell AD, Jorgenson TA (1977): Evaluation of selected pesticides as chemical mutagens: in vitro and in vivo studies. EPA Report 600/1-77-028. Smith KC (1 962): Dose dependent decrease in extractibility of DNA from bacteria following irradiation with UV light or with visible light plus dye. Biochem Biophys Res Commun 8: 157-163. Snyder RD (1986): Evaluation of putative inhibitors of DNA excision repair in cultured human cells by the rapid nick translation assay. Mutat Res 173:279-286. Snyder RD, Matheson DW (1985): Nick translation, a sensitive assay for monitoring DNA damage and repair in cultured human fibroblasts. Environ Mutagen 7:267-279. Snyder RD, Van Houten B (1986): Genotoxicity of formaldehyde and an zevaluation of its effects on the DNA repair process in human diploid fibroblasts. Mutat Res 165:21-30.

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Snyder RD, Carrier WL, Regan JD (1981): Application of arabinofuranosyl cytosine in the kinetic analysis and quantitation of DNA repair in human cells after ultraviolet irradiation. Biophys J 35:339-350. Snyder RD, Van Houten B, Regan JD (1982): Studies on the inhibition of repair of ultraviolet and methyl methanesulfonate-induced DNA damage in the DNA of human fibroblasts by novobiocin. Nucleic Acids Res 10:6207-6219. Stauffer Chemical Company (1981): The association of captan with mouse and rat deoxyribonucleic acid. Report No. T-10435. Stauffer Chemical Company (1984): Mutagenicity of captan in mammalian cells. Report No. T-10436. Stauffer Chemical Company ( 1985): Identification of a preneoplastic alteration following dietary administration of captan technical to CD-I mice. Report No. T-11007. Swenberg JA, Petzold GL, Harbach PR (1976): In vitro DNA damage/ alkaline elution assay for predicting carcinogenic potential. Biochem Biophys Res Commun 72:732-738. Waters MD, Simmons VF, Mitchell AD, Jorgenson TA, Valencia R (1980): An overview of short-term tests for the mutagenic and carcinogenic potential of pesticides. J Environ Sci Health [B] 15:867-906.

Accepted byJ. J. McCormick