Structure-Mutagenicity and Structure-Cytotoxicity Studies on Bromine

conjugates, are mutagenic in the Ames test, although both types of S-conjugates are cytotoxic and nephrotoxic. Recent studies showed that bromine- con...
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Chem. Res. Toxicol. 1994, 7, 157-163

157

Structure-Mutagenicity and Structure-Cytotoxicity Studies on Bromine-Containing Cysteine S-Conjugatesand Related Compounds Martin B. Finkelstein,t Spyridon Vamvakas,t Detlef Bittner,S and M. W. Anders*tt Department of Pharmacology, University of Rochester, 601 Elmwood Avenue, Rochester, New York 14642, and Institut fur Toxikologie, Universitiit Wiirzburg, Versbacher Strasse 9, 0-97078 Wiirzburg, Federal Republic of Germany Received September 27, 1 9 9 3

Glutathione and cysteine S-conjugates of several haloalkenes are nephrotoxic and cytotoxic. Chloroalkene-derived S-(1-chloroalkeny1)-L-cysteineconjugates, but not fluoroalkene-derived S-(2,2-dihalo-l,l-difluoroethyl)-~-cysteine conjugates, are mutagenic in the Ames test, although both types of S-conjugates are cytotoxic and nephrotoxic. Recent studies showed that brominecontaining S-(2,2-dihalo-l,l-difluoroethyl)-~-cysteine conjugates are mutagenic in the Ames test,thus challenging the generalization that S-(2,2-dihalo-l,l-difluoroethyl)-~-cysteine conjugates are not mutagenic. Hence a series of bromine-containing and bromine-lacking S-(2,2-dihalo1,l-difluoroethy1)-L-cysteineconjugates was prepared, and their mutagenicity was assessed in the Ames test with Salmonella t y p h i m u r i u m TA2638 as the test strain. In addition, several indices of cytotoxicity, including cytotoxicity in LLC-PK1 cells, induction of Ca2+release from pig kidney mitochondria, and DNA double-strand breaks in LLC-PK1 cells, were measured. The bromine-containing S-conjugates S-(2-bromo-2-chloro-l,l-difluoroethyl)-~-cysteine (BCD(BTFC),and S-(2,2-dibromo-l,l-difluoroethyl)FC), S-(2-bromo-1,1,2-trifluoroethyl)-~-cysteine L-cysteine (DBDFC) were mutagenic in the Ames test, whereas S-(2-chloro-1,1,2-trifluoroethyl)L-cysteine (CTFC), S-(2,2-dichloro-l,l-difluoroethyl)-~-cysteine (DCDFC), and S-(1,1,2,2tetrafluoroethy1)-L-cysteine(TFC), which lack bromine, were not. BCDFC, BTFC, CTFC, DBDFC, and TFC were cytotoxic in LLC-PK1 cells, and their cytotoxicity was blocked by the cysteine conjugate @-lyaseinhibitor (aminooxy)acetic acid. DCDFC showed little cytotoxicity in LLC-PK1 cells. BCDFC, BTFC, CTFC, DBDFC, DCDFC, and TFC induced Ca2+release from pig kidney mitochondria and DNA double-strand breaks in LLC-PK1 cells. The data show a clear difference in the mutagenicity of bromine-containing and bromine-lacking cysteine S-conjugates in the Ames test, but no remarkable differences among the S-conjugates in other indices of toxicity. Because mutagenicity is associated with the presence of bromine, it is possible that its presence affords a route t o novel metabolites that react with both DNA and protein. mitochondrial dysfunction and attendant perturbations in Ca2+homeostasisin renal tubular cells (21-24) and also Several haloalkenes are nephrotoxic and cytotoxic in induce expression of the protooncogenes c-fos and c-myc cultured and freshly isolated kidney cells. The target(25). organ selective toxicity of haloalkenes is associated with The reaction of glutathione with haloalkenes is catalyzed their glutathione-dependentbioactivation, which involves by the hepatic microsomal glutathione S-transferase (for hepatic glutathione S-conjugate formation, conversion of review, see ref 6). With 1,l-dichloroalkenes as substrates, glutathione S-conjugates to cysteine S-conjugates and S-(1-chloroalkeny1)glutathionesare formed, whereas 1,ltranslocation to the kidney, and bioactivation by renal difluoroalkenes usually afford S-(1,l-difluoroalky1)glucysteine conjugate @-lyase(@-lyase)l(for reviews, see refs tathione conjugates as products. Bacterial mutagenicity 1-8). Moreover, some haloalkene-derived glutathione and studies lead to the generalizationthat S-(1-chloroalkeny1)cysteine S-conjugates express 0-lyase-dependentmutageL-cysteine conjugates are mutagenic, but that S-(1,lnicity in bacterial test systems (9-14) and genotoxicity in difluoroalky1)-L-cysteine conjugates are not (11, 12). mammalian cell test systems (15-18). The 0-lyase pathway 2-Bromo-2-chloro-l,l-difluoroethene is a halothane-derived 1,l-difluoroalkene that is apparently metabolized has also been implicated in haloalkene-induced nephroto the mercapturate S-(2-bromo-2-chloro-1,l-difluorocarcinogenicity (19,201. Cysteine S-conjugates induce ethyl)-N-acetyl-L-cysteine (26,27). The 2-bromo-2-chloro1,l-difluoroethene-derived conjugates S-(2-bromo-2-chloroUniversity of Rochester. 1,l-difluoroethy1)glutathione and S-(2-bromo-2-chloro-1,lt Universitiit Wiirzburg. Abstract published in Advance ACS Abstracts, February 1, 1994. difluoroethy1)-L-cysteine (BCDFC) undergo @-lyase1 Abbreviations: ,%lyase, cysteine conjugate j3-lyase; BCDFC, S-(2dependent bioactivation and are nephrotoxic in rats and bromo-2-chloro-l,l-difluoroethyl)-~-cysteine; BTFC, S-(S-bromo-l,1,2trifluproethylbL-cysteine; CTFC, S-(2-chloro-1,1,2-trifluoroethyl)-~- cytotoxic in LLC-PK1 cells (28). Preliminary studies also cysteme;DBDFC, S-(2,2-dibromo-l,l-difluoroethyl)-~-cysteine; DCDFC, showed that BCDFC and S-(2,2-dibromo-1,l-difluoroS-(2,2-dichloro-l,l-difluoroethyl)-~-c~teine; DCVC, S-(l,2-dichloroviethyl)-L-cysteine (DBDFC) are mutagenic in the Ames FAB-MS, ny1)-i-cysteine; TFC, S-(1,1,2,2-tetrafluoroethyl)-~-cysteine; test (29). fast atom bombardment mass spectrometry.

Introduction

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0 1994 American Chemical Society

158 Chem. Res. Toxicol., Vol. 7, No. 2, 1994 T h e observed mutagenicity of BCDFC and DBDFC challenged t h e generalization that 1,l-difluoroalkenederived S-conjugates are not mutagenic. Hence experiments were designed t o test t h e hypothesis that t h e mutagenicity of BCDFC and DBDFC was associated with t h e presence of bromine. Accordingly, a range of S-(2,2dihalo-1,l-difluoroethy1)-L-cysteine conjugates was prepared, a n d their mutagenicity a n d cytotoxicity were studied. T h e results show that t h e presence of bromine confers mutagenicity, whereas other indices of toxicity differ little between bromine-containing and brominelacking S-(2,2-dihalo-l,l-difluoroethyl)-~-cysteine conjugates.

Experimental Procedures Materials. Reagents for tissue culture were obtained from Sigma Chemical Co. (St. Louis, MO; Deisenhofen, FRG) or GibcoBRL (Grand Island, NY; Eggenstein, FRG). Oxoid Nutrient Broth No. 2 was purchased from Oxoid GmbH (Wesel, FRG). Polycarbonate filters were obtained from Millipore (Tubingen,FRG). Proteinase K was purchased from Boehringer (Mannheim, FRG). 1,l-Difluoro-l,2,2,2-tetrachloroethane, 1,ldibromo-2,2-difluoroethene, and bromotrifluoroethene were obtained from PCR Inc. (Gainesville, FL). Other reagents were purchased from Sigma Chemical Co. or Aldrich Chemical Co. (Milwaukee, WI). Instrumental Analyses. lH NMR spectra were recorded with a Bruker WP270 spectrometer operating at 270.13 MHz and are reported in ppm downfield from tetramethylsilane. l9F NMR spectra were acquired with a Bruker WP270 spectrometer equipped witha dedicated 5-mm 1QFprobe and operating at 254.18 MHz for fluorine and are referenced to external trifluoroacetamide in D20 (6 0 ppm). Fast atom bombardment mass spectra (FAJ3-MS)were recorded with a VG TS-250double-focusingmass spectrometer with a glycerol matrix. Syntheses. (A)S-(2-Bromo-2-chloro-l,l-difluoroethyl)L-cysteine. This compound was prepared as described by Finkelstein et al. (28). (B)S-(2-Bromo-2-chloro1,l-difluoroethyl)-DL-a-met hylcysteine. This analog was prepared as described previously (28). (C)S-(2-Bromo-1,1,2-trifluoroethyl)-~-cysteine (BTFC). Sodium hydroxide (2.0 g, 50 mmol) was added to an ice-cold mixture of water (20 mL) and methanol (20 mL). L-Cysteine (2.0 g, 16.5 mmol) was then added, and the double-necked flask was sealed with a balloon and a septum. The flask was purged with nitrogen, and bromotrifluoroethene was added until the balloon was full. The balloon was refilled as needed for 3 h. Caution: Bromotrifluoroethene is pyrophoric and should be used in an efficient fume hood. After 3 h, the system was purged with nitrogen, and concentrated HC1 was added to bring the pH to 4.0. The crystals that formed were harvested by vacuum filtration; 2.01 g (7.10 mmol) of the product was obtained (43% yield). TLC (silica gel: 1-butanol/acetic acid/water, 4 l : l ) with ninhydrin detection showed a single spot (Rf = 0.41). The lH and lgF NMR data for the new compound S-(P-bromo-l,1,2trifluoroethy1)-L-cysteineare shown in Table 1. (D)S-(2-Chloro1,1,2-trifluoroethyl)-~-cysteine (CTFC). This S-conjugate was synthesized according to Dohn et al. (30). (E)S-(2,2-Dibromo-l,l-difluoroethyl)-~-cysteine (DBDFC). L-Cysteine (1.0 g, 8.3 mmol) was added to an ice-cold mixture of water (10 mL) and methanol (10 mL) containing sodium hydroxide (0.7 g, 17.5 mmol) in a flask fitted with a balloon and septum. The balloon was filled with l,l-dibromo-2,2-difluoroethene, and the mixture was stirred on ice for 3 h. After adding concentrated HCI to pH 2.0, crystalsformed. The flask was placed in the refrigerator overnight, and the crystals were collected by vacuum filtration; 1.35 g (3.94 mmol) of product was obtained (48% yield). TLC (silicagel: 1-butanol/aceticacid/water, 4:l:l) with ninhydrin detection showed a single spot (Rf = 0.58). lH

Finkelstein et al. Table 1. 1H and

19F

N M R Data for

S-(2-Bromo-l,1,2-trifluoroethyl)-~-cysteine

chemical shift (ppm) 4.24

multiplicity dofd doft

Fa

3.26-3.49 6.62-6.68 6.80-6.85 -10.0

Fb

-14.0

dofd

Fc

-77.0

m

nucleus Ha Hb, Hc Hd

m

d of d

coupling constants (Hz) H,-Hb = 4.6 Ha-Hc = 7.1 Hb-Hc 15.6 Fa-Fb = 210 Fa-Fc = 22 Fa-& = 4.7 Fb-F, = 20 Fb-Hd = 8.2 Fc-Hd = 48

and lgF NMR spectra were consistent with those reported by Commandeur et al. (31). (F)S-(2,2-Dichloro-l,l-difluoroethyl)-~-cysteine (DCDFC). This compoundwas prepared as described for BTFC except that l,l-dichloro-2,2-difluoroethene, prepared accordingto Sauer (32), was used instead of bromotrifluoroethene. The yield was 34% (1.43g, 5.61 mmol) of product. TLC (silicagel: 1-butanol/ acetic acid/water, 4l:l) with ninhydrin detection showed a single spot (Rf= 0.60). lH and 19FNMR spectra were consistent with those reported by Commandeur et al. (31). FAB-MS, m/z (re1 intensity) 234 (M + H - HF, 56), 254 (M + H, loo), 256 (M + 2, 64), 258 (M + 4, 6). (G)5-(1,2-Dichlorovinyl)-~-cysteine (DCVC).DCVC was prepared by the method of McKinney et al. (33). (H)S-(1,1,2,2-Tetrafluoroethyl)-~-cysteine (TFC). TFC was obtained by synthesis (34). Mutagenicity Assay in Salmonella typhimurium. The mutagenicity of the S-conjugates was investigated with S. typhimurium TA2638 as the test strain. S-Conjugatesthat failed to induce a mutagenic effect in this strain were also tested in S. typhimurium strains TA100, TA102, and TA98. The bacterial strains were originally provided by Prof. B. Ames, and their properties (UV and crystal violet sensitivity, ampicillin or tetracycline resistance, and mutability by UV irradiation) were regularlytested (35). Preincubations (2 h, 37 "C) were performed in 500 pL of 0.1 M phosphate buffer (pH 7.4) with 100 pL of bacterial culture (grown for 10 h in Oxoid Nutrient Broth No. 2) and 20 pL of the test compound dissolved in methanol. To some preincubation mixtures was added (aminooxy)aceticacid, dissolved in 20 pL methanol, to a final concentration of 500 pM. At the end of the preincubation time, 2 mL of top agar containing histidine (0.05 mM) and biotin (0.05 mM) was added, and the mixture was plated on Vogel Bonner agar medium. After incubation for 2 days at 37 "C, coloniesof revertants were counted with an automated colony counter. All determinations were done in duplicate, and all experiments were performed four times. Differences in colony counts of analogous plates usually did not exceed 10% . Cytotoxicity Studies. LLC-PK1 cells (American Type Culture Collection, Rockville, MD) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 5.6 mM glucose, and 20 mM N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (pH 7.4). Cells were seeded in 12-wellplates at 2.5 X lo5cells/wellfor cytotoxicity experiments. After 24 h of cell growth to subconfluent levels, the cells were rinsed twice with Dulbecco's phosphate-buffered saline solution. The S-conjugates, dissolved in 1 mL of Earle's balanced salt solution, were added to the cells and incubated for 24 h a t 37 "C. In some experiments, (aminooxy)aceticacid (100 pM) was added along with the conjugates. After 24 h, the medium was removed

S-Conjugate Mutagenicity and Cytotoxicity from the plates and centrifuged. Lactate dehydrogenaseactivity in the medium was assayed according to Stevens et al. (36). Cytotoxicityis expressedas the percent of lactate dehydrogenase released in the presence of test compounds compared with that of Triton X-100-lysed cells. Isolation of Pig Kidney Mitochondria and Determination of Cas+ Release. Fresh pig kidney cortices were homogenized in ice-cold 20 mM triethanolamine hydrochloridebuffer (pH 7.4) containing 250 mM sucrose, 1 mM EGTA, 10 mM KHzPOr, 5 mM MgC12, 20 mM KCl, and 0.1% bovine serum albumin. Mitochondrial fractions were isolated from renal cortical homogenates as described by Johnson and Lardy (37). Mitochondria (1.5 mg of protein/mL) were suspended in 20 mM Tris-HC1 buffer (pH 7.6) containing 250 mM sucrose. Calcium release into the incubation medium was measured with Arsenazo I11 by dual-wavelength spectroscopy at 665 and 685 nm with an Ultraspect I1 spectrophotometer (LK13 Biochrom, Cambridge, U.K.). For the determination of Ca2+release, the mitochondria were incubated for 3 min with Arsenazo I11 (30 pM), succinate (2.5 mM), rotenone (0.2 mg/mL), and CaClz (50 mM). Calcium release was determined before and after addition of 1mM of the S-conjugates, as described by Richter and Kass (38). Determination of DNA Double-Strand Breaks with the Neutral Filter DNA Elution Assay. LLC-PK1cella (American Type Culture Collection, passage 196-205) were grown in 150cm2 plastic tissue-culture flasks in Dulbecco’s modified Eagle’s medium supplemented with 20 mM N-(2-hydroxyethyl)piperazine-N’-2-ethanesulfonicacid, 10% fetal calf serum, 100 units of penicillin/mL, 1mg of streptomycin/mL, 1.7 g of NaHCOdL, and 3 g of glucose/L. Cells (1P)were plated in 35-mm wells (6 wells per plate). One day later, monolayers in exponential growth were washed twice with phosphate-buffered saline and exposed for 4 h to the S-conjugates at a concentration of 400 pM. After exposure to the S-conjugates,the cells were incubated for 48 h in S-conjugatefree Dulbecco’s modified Eagle’s medium. Control and exposed LLC-PK1 cells were harvested with a rubber policeman and washedwithice-coldphosphate-bufferedsaline. Finally, the cells were centrifuged at 500g for 5 min, and the pellet was stored at -20 OC. DNA double-strand breaks were quantified with the neutral filter elution method (39). The cell pellets were thawed at room temperature and transferred with 2 mL of ice-cold phosphate-buffered saline to 25-mm, 2-pm pore size polycarbonate filters. The cells were lysed for 1h in the dark at room temperature with 1.5mL of 0.05 M Tris buffer (pH 9.6) containing 0.05 M glycine, 0.025 M NaaEDTA, 2% (w/v) sodium dodecyl sulfate, and 0.5 mg/mL freshly dissolved proteinase K. After lysis, 40 mL of the elution buffer [0.05 M Tris (pH 9.6) containing 0.05 M glycine, 0.025 M NaZEDTA, and 2 % (w/v) sodium dodecyl sulfate] was added to the reservoir above each filter, and the DNA was eluted at 2 mL/h for 15 h with an Ismatec IPN 8 pump (Ismatec,Zbich, Switzerland). Samplesof the eluates from each fiiter were collected B t 90-min intervals. The DNAconcentrations in eluate samples were determined fluorimetrically with the fluorescent dye Hoechst 33258 (excitation wavelength 360 nm, emission wavelength 450 nm) (40). The DNA remaining on the filter was extracted by incubating the filters for 3 h in the elution buffer and was quantified with Hoechst 33258.

Results Syntheses. Reaction of L-cysteine in the presence of base with haloethenes gave the expected cysteine S-conjugates in satisfactory (34-48% ) yields. With DBDFC, side reactions predominated unless stoichiometric amounts of base and L-cysteine were used. All compounds were pure by TLC, ‘HNMR, and l9F NMR. The structures of the cysteine S-conjugates studied are shown in Figure 1. Mutagenicity in S. typhimurium TA2638. DCVC is a potent mutagen in the Ames test, and its mutagenicity

Chem. Res. Toxicol., Vol. 7, No. 2, 1994 159

BTFC: Xl Br, X2 = F BCDFC: Xi = Br, X2 = CI CTFC: X i = CI, X2 = F DBDFC: X1 =X2 = Br DCDFC: X i =& = CI TFC: Xi = X2 = F

HJ+sJfH 0 DCVC Figure 1. Structures of cysteine S-conjugates. CI

12001

8ooil 1 600

200 0

600 800 Amount (nmol/plate)

200

400

1000

Figure 2. Mutagenicity of S-(1,2-dichlorovinyl)-~-cysteine (O), S-(2-bromo-2-chloro-l,l-difluoroethyl)-~-cysteine (A), S-(2,2dibromo-1,l-difluoroethy1)-L-cysteine(n),S-(2-bromo-l,1,?-trifluoroethy1)-L-cysteine (+), S-(2-chloro-l,1,2-trifluoroethyl)-~cysteine (O), S-(2,2-dichloro-l,l-difluoroethyl)-ccyste(A),and S-(1,1,2,2-tetrduoroethyl)-~-cysteine (m) in the Ames mutagenicity assay with S. typhimurium TA2638. Mutagenicity was determined as described in Experimental Procedures. Data are shown from one experimenttypical of four; values among different experiments varied less than 105%.

is due t o the high activities of @-lyase present in S. typhimurium (10). The mutagenicity of DCVC was confirmed in the present study: in the presence of 20 nmol of DCVC per incubation plate, the number of revertants increased up to 20-fold compared with unexposed controls (Figure 2). At higher concentrations, the mutagenicity of DCVC was masked by ita bactericidal effects. Brominecontaining S-(2,2-dihalo-l,l-difluoroethyl)-~-cysteine Sconjugates (BCDFC, BTFC, and DBDFC) were also mutagenic t o S. typhimurium TA2638 (Figure 2), but the analog S-(2-bromo-2-chloro-l,l-difluoroethyl)-~~-amethylcysteine, which cannot undergo a j3-lyase-catalyzed @-eliminationreaction (71, was not mutagenic (data not shown). Both the absolute number of induced revertants and t h e relative mutagenic potency (number of revertants/ nmol of S-conjugate) of BCDFC, BTFC, and DBDFC were lower than with DCVC. The @-lyaseinhibitor (aminooxy)acetic acid (500pM) inhibited the mutagenicity of all S-conjugates by approximately 50% (data not shown); (aminooxy)acetic acid is not mutagenic at the concentrations used (12). Bromine-lacking S-(2,2-

160 Chem. Res. Toxicol., Vol. 7, No. 2, 1994

Finkelstein et al.

100 . A

. c

. B

80 -

I

0

-2

d

I

2

60-

1

40-

x

20

-

40t

A

20

"

0

25

7 50 100 (UM)

Concentration

0

12.5

25

50

100

0

3200 6400 (uM)

1600

Concentration

Concentration (pM)

Figure 3. Cytotoxicityof S-(2-bromo-2-chloro-l,l-difluoroethyl)-~-cysteine (A), S-(2-bromo-1,1,2-trifluoroethyl)-~-cysteine (B),S-(2,2dibromo-1,l-difluoroethy1)-L-cysteine (C),S-(2-chloro-1,1,2-trifluoroethyl)-~-cysteine (D),S-(l,1,2,2-tetrafluoroethyl)-~-cysteine (E), and S-(2,2-dichloro-l,l-difluoroethyl)-~-cysteine (F)in LLC-PK1 cells. Cells were incubated with the indicated concentrations of cysteine S-conjugatesin the absence (open bars) or presence (solid bars) of (aminooxy)acetic acid for 24 h, and cytotoxicity waa measured by quantifying release of lactate dehydrogenase activity into the medium. Data are shown as means i SD, n = 3.

dihalo-1,l-difluoroethyb-L-cysteine S-conjugates (DCDFC, CTFC, and TFC) failed to increase the number of revertants per plate above control values (Figure 2). The lack of mutagenicity of DCDFC, CTFC, and TFC was confirmed with S. typhimurium strains TA100, TA102, and TA98 (data not shown). Cytotoxicity. BCDFC, BTFC, DBDFC, CTFC, and TFC produced concentration-dependent cytotoxicity in LLC-PK1 cells, as shown by release of lactate dehydrogenase into the medium (Figure 3). The cytotoxicity of the S-conjugates was blocked by (aminooxy)acetic acid, an inhibitor of the pyridoxal phosphate-dependent &lyase (41). The differences in the cytotoxicity of BCDFC,BTFC, DBDFC, CTFC, and TFC were not remarkable. DCDFC showed little cytotoxicity in LLC-PK1 cells. Ca2+ Efflux from Isolated Pig Kidney Mitochondria. Both control and S-conjugate-exposedmitochondria retained Ca2+for about the first 15 min of the incubation, after which a time-dependent release of Ca2+was observed (Figure 4). DCVC (1mM), which induces mitochondrial Ca2+ release (24), was used as a positive control in these experiments. By 30 min, all cysteine S-conjugates studied stimulated Ca2+efflux, resulting in an increase in the Ca2+ concentration in the medium to 25-35 nmol/mL (Figure 4). Differences were observed in the time course of Ca2+ efflux among the S-conjugates tested in the presence of 1mM CTFC, Ca2+was retained by the mitochondria for 25 min, whereas incubation with BTFC induced significant Ca2+efflux by 17.5 min. DNA Double-Strand Breaks in LLC-PK1 Monolayers. The induction of Ca2+-dependent, DNA doublestrand breaks by the mitochondrial Ca2+ depletor DCVC (24) and the results obtained with isolated pig kidney

50,

0

10

20 Time (min)

30

0

10

20

30

Time (min)

Figure 4. Inductionof Ca2+efflux from pig kidney mitochondria by cysteine S-conjugates. Isolated pig kidney mitochondria (1.5 mg of protein/mL) were incubated with 1mM S-(2-bromo-l,1,2trifluoroethy1)-L-cysteine(+,panel A), S-(2-bromo-2-chloro-l,1difluoroethy1)-L-cysteine (A, panel A), S-(2,2-dibromo-l,ldifluoroethy1)-L-cysteine (0,panel A), S-(2,2-dichloro-l,ldifluoroethy1)-L-cysteine (A,panel B), S-(1,2-dichlorovinyl)-~cysteine (0,panel B), or S-(2-chloro-1,1,2-trifluoroethyl)-~-

cysteine (0,panel B),and Ca2+efflux was quantifiedas described in Experimental Procedures. Data are shown as means f SD, n = 4.

mitochondria indicated that DNA fragmentation caused by the activation of Ca2+-and Mg2+-dependentendonucleases may be involved in the cytotoxicity of S-conjugates. A 4-h-pulse treatment of LLC-PK1 monolayers with 400 pM BCDFC, BTFC, CTFC, DBDFC, DCDFC, or DCVC, which did not induce cytotoxicity,increased the percentage of fragmented DNA compared with unexposed controls (Figure 5).

Discussion Previous studies showed that S-(1-chloroalkeny1)-Lcysteine conjugates are mutagenic in S. typhimurium and

Chem. Res. Toricol., Vol. 7, No. 2, 1994 161

S-Conjugate Mutagenicity and Cytotoxicity

i

a

501 0 ~ ' ' ' " ' ' ' ' 0 3 6 9 1 2 1 5 0 Time (h)

3

6

9

1 2 1 5

Time (h)

Figure 5. Induction of DNA double-strand breaks in cultured LLC-PK1 cells incubated with cysteine S-conjugates. Subconfluent monolayers of LLC-PK1 cells were incubated in the absence (0) or presence of 400 p M S-(2-bromo-l,l,2-trifluoroethyl)-~cysteine (+, panel A), S-(2-bromo-2-chloro-l,l-difluoroethyl)L-cysteine (A, panel A), S-(2,2-dibromo-l,l-difluoroethyl)-~cysteine (0, panel A), S-(2,2-dichloro-l,l-difluoroethyl)-~-cysteine (A, panel B), S-(1,2-dichlorovinyl)-~-cysteine (0, panel B), or S-(2-chloro-l,l,2-trifluoroethyl)-~-cysteine ( 0 ,panel B) for 4 h. The percentage of fragmented DNA was quantified after 48 h of incubation in S-conjugate-free medium. Data are shown from one experiment typical of four; values among different experiments varied less than 10%.

that their mutagenicity is dependent on bioactivation by P-lyase (10). In contrast, S-(1,l-difluoroalky1)-L-cysteine conjugates are not mutagenic in the Ames test (11, 12). Hence the observed mutagenicity of BCDFC and DBDFC in the Ames test was unexpected (29). I t was, therefore, of interest to ask whether the mutagenicity of BCDFC and DBDFC was a unique event or whether mutagenicity was associated with the presence of bromine. Accordingly, a series of bromine-containing and bromine-lacking cysteine S-conjugates was prepared, and their mutagenicity in the Ames test, their cytotoxicity in LLC-PK1 cells, their ability to stimulate Ca2+ release from pig kidney mitochondria, and their ability to induce DNA double-strand breaks in LLC-PK1 cells were investigated. All of the cysteine S-conjugates studied are substrates for rat renal 0-lyase (42, 43). The bromine-containing cysteine S-conjugates studied (BCDFC, BTFC, and DBDFC) were mutagenic in the Ames test, whereas the analogs that lacked bromine (CTFC, DCDFC, and TFC) were not. DCVC is a known mutagen in the Ames test (11, 12), and its mutagenicity w a confirmed in the present experiments. The mutagenic potency of the S-(haloalkeny1)-L-cysteineconjugate DCVC was much greater than that of the bromine-containing S-(haloalky1)-L-cysteineconjugates tested, This may indicate a qualitative or quantitative difference in the production of mutagenic metabolites from these compounds. (Aminooxy)aceticacid-inhibitable @-lyaseactivity is present in S. typhimurium (lo), and the mutagenicity of BCDFC, BTFC, and DBDFC was inhibited by (aminooxy)acetic acid, indicating a role for 8-lyase-dependent bioactivation of the cysteine S-conjugates. Moreover, the analog S-(2-bromo-2-chloro- 1,l-difluoroethyl)-~~-crmethylcysteine, which cannot undergo a 8-lyase-catalyzed @-eliminationreaction (7), was not mutagenic. BCDFC, BTFC, DBDFC, CTFC, and TFC were cytotoxic in LLC-PK1 cells. The differences in cytotoxicity among these S-conjugates were not remarkable, although TFC was somewhat more potent than the other S-conjugates studied. (Aminooxy)acetic acid, an inhibitor of 8-lyase (41),blocked the cytotoxicity of the S-conjugates, confirming a role for the P-lyase in the bioactivation of the compounds. Previous studies demonstrated that DBDFC,

CTFC, and TFC are cytotoxic in a range of in vitro test systems, including freshly isolated rat renal proximal tubular cells,primary cultures of rat renal proximal tubular cells, and rat renal tissue slices (31,44-46). BCDFC has previously been shown to be cytotoxic in LLC-PK1 cells (28). In the present study, the new compound BTFC was also cytotoxic in LLC-PK1 cells. DCDFC showed little cytotoxicity in LLC-PK1 cells.2 The failure of DCDFC to exert cytotoxicity in LLC-PK1 cells comparable to other S-conjugates tested is not understood. DCDFC is nephrotoxic and mildly hepatotoxic in rats (47); the nephrotoxicity of DCDFC has been confirmed in this l a b ~ r a t o r y .DCDFC ~ and the corresponding mercapturic acid [S-(2,2-dichloro-l,l-difluoroethyl)-N-acetyl-~-cysteinelhave been reported to be cytotoxic in rat renal proximal tubular cells and in rat renal tissue slices (31, 44, 46). All of the cysteine S-conjugates studied stimulated Ca2+ release from isolated pig kidney mitochondria. There was little difference in the stimulation of ea2+release between bromine-containingand bromine-lackingS-conjugates,but the onset of BTFC-stimulated ea2+ release occurred somewhat earlier (and that of BCDFC and CTFC somewhat later) than that observed with other compounds. Previous studies have demonstrated that DCVC also stimulates Ca2+release from pig kidney mitochondria (24) and that perturbations in cellular Ca2+homeostasis are an early event in DCVC-induced cytotoxicity in LLC-PK1 cells (23). Moreover, CTFC and DCVC induce mitochondrial dysfunction, including reduced DNA, RNA, and protein synthesis (21). Both bromine-containing and bromine-lacking cysteine S-conjugates induced DNA double-strand breaks in LLCP K I cells. In the neutral elution assay used, DBDFC and DCDFC induced the largest and smallest increase, respectively,in DNA double-strand breaks. Previous studies showed that DCVC also induces DNA double-strand breaks in LLC-PK1 cells (24) and DNA single-strand breaks in rabbit kidneys (15). DCVC-induced DNA double-strand breaks are associated with activation of ea2+-and Mg2+-dependentendonucleases (24). Although the calcium dependence of the observed DNA doublestrand breaks was not specifically investigated in the present studies, mitochondrial Ca2+release was stimulated by all of the cysteine S-conjugates tested. The results presented herein demonstrate a clear difference in the mutagenicity of bromine-containing and bromine-lacking S-(2,2-dihalo-l,l-difluoroethyl)-~-cysteine conjugates, whereas several indices of cytotoxicity did not differ remarkably among the compounds studied. This may indicate that the selective mutagenicity of bromine-containing cysteine S-conjugates is associated with the formation of metabolites that react with DNA in addition to reacting with proteins. The cytotoxicity of cysteine S-conjugates is associated with the &lyasedependent formation of reactive metabolites that bind covalently to cellular macromolecules (36,48,49). Thioacylating metabolites are formed by @-lyase-catalyzed @-elimination reactions of DCVC, CTFC, TFC, and S-(1,2,2-trichlorovinyl)-~-cysteine (50-58). 2 The basis for the low cytotoxicityof DCDFC in LLC-PK1 cells is not understood. Cytotoxicitywaa asseased independently at the University of Rochester and at the Universitiit Wiuzburg, and similar results were obtained. 3 M. B. Finkelstein and M. W. Anders, unpublished observations.

162 Chem. Res. Toxicol., Vol. 7, No. 2, 1994

The observation that DCVC is a potent mutagen led to a search for novel metabolites. A mechanism for the formation of thioketenes from S-(1-chloroalkeny1)-Lcysteine conjugates, but not from the S-(2,2-dihalo-l,ldifluoroethy1)-L-cysteineconjugate CTFC, has been elaborated (52), but a role for thioketene metabolites in DNA modification has not been demonstrated. Indeed, little information is available about the covalent binding of metabolites of cysteine S-conjugates to DNA. Sulfurcontaining metabolites of [3SSlDCVCinteract with DNA (59,60),but no adducts thus formed have been chemically characterized. With S-(2,2-dihalo-l,l-difluoroethyl)-~-cysteine conjugates, there is an apparent correlation between terminal haloacetic acid formation and mutagenicity: chlorofluoroacetic acid and difluoroacetic acid are metabolites of the nonmutagenic S-conjugates CTFC (50) and TFC (311, respectively. Commandeur et al. (31) did not detect dihaloacetic acid metabolites of DBDFC, which is mutagenic, and of DCDFC, which is not mutagenic. Recent experiments have, however, demonstrated the formation of dichloroacetic acid as a metabolite of DCDFC, although dibromoacetic acid formation was not detected with DBDFC as the substrate (43). [It should be noted that Commandeur et al. (31) and Finkelstein et al. (43) used different analyticalmethods to determine dihaloacetic acid formation.] Moreover, preliminary studies indicate that, in addition to pyruvate, glyoxylateis formed as a metabolic of BCDFC, BTFC, and DBDFC, but no glyoxylate formation was observed with CTFC, DCDFC, and TFC as substrates (43). The formation of glyoxylate as a terminal metabolite of bromine-containing cysteine S-conjugates can be rationalized by the formation of an a-thiolactone or a-thionolactone intermediate, whose formation and reactivity may be associated with their mutagenicity. These findings point to a diversity of pathways of biotransformation of cysteine S-conjugates bearing different halogen substituents. Elaboration of these pathways and quantification of the metabolites formed may afford insight into the selective mutagenicity of bromine-containing S-(2,2-dihalo-l,l-difluoroethyl)-~-cysteine conjugates.

Acknowledgment. The authors thank Sandra E. Morgan for her assistance in preparing the manuscript. This research was supported by National Institute of Environmental Health SciencesGrants ES03127 (M.W.A.) and ES07026 (M.B.F.), by the Deutsche Forschungsgemeinschaft Sonderforschungsberich 172 (S.V.), and by NATO Grant 901032 (M.W.A.). References (1) Anders, M. W., Lash, L., Dekant, W., Elfarra, A. A., and Dohn, D. R. (1988)Biosynthesis and biotransformation of glutathione Sconjugates to toxic metabolites. CRC Crit. Rev. Toxicol. 18,311-

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