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The induction of DNA-strand breaks and unscheduled DNA synthesis in F-344 rat hepatocytes following in vivo administration of caprolactam or benzoin
Mutation Research, 224 (1989) 361-364
361
Elsevier MUTGEN 02661
The induction of DNA-strand breaks and unscheduled D N A synthesis in F-344 rat hepatocytes following in vivo administration of caprolactam or benzoin Edilberto Bermudez, Tracy Smith-Oliverand Loretta Lyon Delehanty Department of Genetic Toxicology, Chemical Industry Institute of Toxicology, Research Triangle Park, NC (U.S.A.)
Keywords: Benzoin; Caprolactam; DNA-strand breaks, induction; DNA synthesis, unscheduled, induction; Hepatocytes
Summary Benzoin (ZOIN) and caprolactam (CAP) were administered by gavage to Fischer 344 rats at a dose of 750 m g / k g and the hepatocytes isolated 12, 24 or 48 h after treatment. The isolated hepatocytes were subsequently examined for the induction of DNA-strand breaks (SB) and unscheduled D N A synthesis (UDS). Neither ZOIN nor CAP induced SB or UDS in hepatocytes, however ZOIN did induce an increase in the fraction of cells in S-phase 24 h after treatment. These results correlate well with the observed lack of carcinogenicity of these compounds.
The presence of strand breaks in DNA may be assessed using the alkaline elution assay (Kohn and Ewig, 1973) and this assay has been utilized to determine the DNA-damaging effects of chemical treatment in vitro and in vivo (Bermudez et al., 1982; Brambilla et al., 1978; Erickson et al., 1980; Parodi et al., 1978; Petzold et al., 1978; Sina et al., 1983; Swenberg et al., 1976). Various lesions leading to SB are detectable using the alkaline elution assay including: frank breaks, excision sites, and alkali-labile sites. The induction of UDS has been utilized as an endpoint for the DNA-damaging activity of chemicals in various systems including primary rat hepatocyte cultures (Martin et al., 1977; Mirsalis et al., 1982; Williams, 1977, 1985). Two methods for assessing UDS rely upon the insertion of ra-
diolabeled bases into D N A sites that have been repaired by excision-type repair mechanisms followed by detection of the sites by autoradiography or scintillation counting. ZOIN and CAP have both been tested for carcinogenicity in rats and mice and have been found to be negative (DHHS, 1980, 1981). These two chemicals have also been tested in a variety of prokaryotic and eukaryotic short-term in vitro assays for genotoxicity and found to be generally, but not unanimously, inactive (Ashby et al., 1985). The present study was undertaken with the objective of determining if ZOIN or CAP can induce SB or UDS in F-344 rat hepatocytes when these compounds are administered to the animals by gavage.
Methods Correspondence: Dr. Edilberto Bermudez, Department of Genetic Toxicology, Chemical Industry Institute of Toxicology, Research Triangle Park, NC (U.S.A.).
Male Fischer-344 rats [CDF(G-344)/CrlBr, Charles River Breeding Laboratories, Kingston, NY] weighing 200-280 g were housed in hanging
0165-1218/89/$03.50 © Elsevier Science Publishers B.V. (Biomedical Division)
362 stainless steel-mesh cages (Hazleton Systems, Vienna, VA) in a mass air displacement room (Bioclean, Hazleton Systems, Vienna, VA) with controlled t e m p e r a t u r e (22 _+ 1 °C), relative humidity (50 _+ 10%), and light cycles (12 h). The animals had free access to food (NIH-07 open formula) and water. Randomly selected animals were tested and found free of known pathogens (Standard Screening Necropsy, Microbiological Associates, Bethesda, MD). Z O I N and CAP were administered by gavage (1.0 m l / 1 0 0 g body weight) using corn oil or water, respectively, as the vehicle. Control animals received only vehicle. 12, 24 or 48 h after treatment with 750 m g / k g of chemical the animals were anesthetized and the livers perfused with collagenase to obtain single cell suspensions of hepatocytes as previously described (Bermudez et al., 1979; Williams et al., 1977). The cell suspensions were filtered through gauze, centrifuged at 50 × g for 4 rain and resuspended in cold Hanks' balanced salt solution (Flow Laboratories). At this time the cell density and viability, as determined by trypan blue exclusion, were assessed. The alkaline elution assay (Kohn, 1979) was performed as described by Bermudez et al. (1982). Control and treated cells (5 × 105) suspended in 5 m M phosphate-buffered saline (PBS, p H 7.2) were deposited onto PVC filters (2 txm, BSWP25, Millipore), lysed (0.02 M N a 3 E D T A , 2 M NaC1, 0.3% sarkosyl, 0.5 m g / m l proteinase K, p H 10.00) for 30 min and eluted for 10 h with 0.02 M H 4 E D T A (brought to p H 12.20 with 10% tetrapropylammonium hydroxide) at a flow rate of 0.05 m l / m i n . Two sets of triplicate filters were prepared for each sample, one set to be eluted and one to serve as a reference for the quantity of D N A initially deposited on the filters. The relative amounts of D N A present on the eluted and reference filters were determined by quantitation of the fluorescent product obtained after reacting D N A with 3,5-diaminobenzoic acid hydrochloride (0.3 g / m l , Aldrich, Milwaukee, WI) at 6 0 ° C for 90 min. Fluorescence was measured at an excitation wavelength of 414 nm and an emission wavelength of 508 nm. The data is expressed as the percent of the D N A retained (percent retention) and is determined by dividing the adjusted mean relative fluorescence of the eluted filters by the mean
relative fluorescence of the reference filters. Significance at the p < 0.05 level was determined by comparing the percent retention of the treated samples to the percent retention of the pooled control samples (78.3 _+ 4.4 [SEM]), using the t-test. Unscheduled D N A synthesis induction was assessed as described by Mirsalis et al. (1982). Cells (4 × 105) were seeded into dishes containing plastic coverslips and Williams medium E supplemented with 10% fetal calf serum. The cells were allowed to attach to the coverslips for 1.5 h, washed, and incubated for an additional 4 h in Williams medium E containing 10 /~Ci/ml [3H]thymidine. U n i n c o r p o r a t e d radioactivity was chased for 14-16 h with a 0.25 m M unlabeled thymidine solution. Control cells treated with 1 m M dimethylnitrosamine ( D M N , Aldrich, Milwaukee, WI) in vitro for 18 h, in the presence of [3H]thymidine, were used as the positive control. Fixation of the cells with 3 : 1 ethanol : acetic acid was preceded by nuclear swelling with 1% sodium citrate and followed by water washes. The coverslips were air-dried, mounted on glass slides and dipped in NTB2 nuclear track emulsion. Exposure of the emulsion for 12-14 days at - 2 0 ° C was followed by development and staining. The number of silver grains over the nucleus and cytoplasm were determined, using an automatic grain counting system, for at least 25 cells on each of 3 slides for each time and dose examined. The data is expressed as the net grains/nucleus and is determined by subtracting the number of grains in a nuclear sized area of the cytoplasm from the number of grains in the nucleus. A positive response is assumed where the net grains/nucleus is equal to or greater than 5. The percent of cells in S-phase was also determined by counting 3000 cells from each animal. Results and discussion
The status of CAP and Z O I N as noncarcinogens in animals has been established by National Toxicology Program bioassays (DHHS, 1980, 1981). Both of these chemicals were utilized as 'negative controls' in the evaluation of various short-term tests for carcinogens and CAP was found to be inactive in the majority, with the exception of assays using clastogenesis and somatic
363 TABLE 1 I N D U C T I O N OF STRAND BREAKS BY CAP A N D ZOIN IN RAT HEPATOCYTES F R O M TREATED ANIMALS Chemical
Time (h)
Dose (mg/kg)
n (animals)
% retention a ±S.D.
Conclusion b
ZOIN ZOIN ZOIN
12 24 48
750 750 750
3 3 3
69_+ 9 86 _+4 86 _+3
NEG NEG NEG
CAP CAP CAP
12 24 48
750 750 750
3 3 3
81-4- 6 71 _+4 76 +_3
NEG NEG NEG
a % Retention
Mean of eluted filters × 100 Mean of reference filters
b NEG, where no significant ( P < 0.05) difference from controis was observed. The mean retention for all controls (n = 4) was 78_+4 (S.E.M.). Comparisons were made using the t-test.
cell mutation as endpoints (Ashby et al., 1985). Similarly, ZOIN was found to be inactive in various short-term tests, however it was positive in
TABLE 2 U N S C H E D U L E D D N A SYNTHESIS I N D U C T I O N IN HEPATOCYTES FROM RATS TREATED WITH ZOIN A N D CAP Chemical
Time (h)
Control
Dose (mg/kg)
n (animals)
NG a + S.D.
% of cells with > 5 NG
% of cells b in Sphase
-
4
-4+
1
0
0.37
ZOIN ZOIN ZOIN
12 24 48
750 750 750
3 3 3
-3 + 1 -7+ 1 -6___ 1
0 0 0
0.02 3.53 0.77
CAP CAP CAP
12 24 48
750 750 750
3 3 3
-3 + 1 -4+ 1 -4+ 1
0 0 0
0.03 0.10 0.10
DMN
c
1 mM
3
46+28
98
-
a NG, net grains/nucleus. The number or grains over the nucleus is determined by subtracting the number of grains in an area of the cytoplasm, equal to that of the nucleus, from the nuclear counts. Each data point represents the mean of the net grains per nucleus of 75-150 cells. Net grains/nucleus of greater than 5 are considered a positive response. b 3000 cells were counted per animal. c Control cells treated in vitro for 18 h.
some of the assays for SB, UDS and mammalian gene mutations (Ashby et al., 1985). Increases in SB were not detected 12, 24 or 48 h following the administration of 750 m g / k g ZOIN or CAP (Table 1). The reported in vitro induction of SB by ZOIN as measured by alkaline elution is attributable to a cytotoxic effect (Bradley, 1985). ZOIN and CAP have been reported to induce SB in the hepatocytes isolated from treated SpragueDawley rats (Parodi et al., 1978; Russo et al., 1989). The differences in route of administration, strain, and methodology prevent direct comparisons between the results of that laboratory and those of the present study. UDS was not induced in hepatocytes from the livers of rats treated with ZOIN or CAP when the cells were examined at 12, 24 or 48 h following treatment (Table 2). This finding is consistent with the absence of induced strand breaks in the same population of cells. Dimethylnitrosamine treatment of hepatocytes in vitro resulted in the induction of UDS (Table 2) indicating that the system was operational. Examination of the cell populations for the fraction in S-phase revealed that ZOIN induced an 8-9-fold increase over control (Tables 2) 24 h after administration. An effect on S-phase in the liver by ZOIN indicates that a lack of absorption is not a likely explanation for the lack of induction of D N A damage. This induction of replicative synthesis may also explain the positive UDS results previously reported for this compound in vitro (Glauert et al., 1985). In conclusion, no induction of SB or UDS was observed in hepatocytes following the administration to rats of ZOIN and CAP. These results are supportive of the reported noncarcinogenicity of these compounds and indicate that they may serve as true negative controls. References Ashby, J. (1985) Overview and conclusions of the IPCS collaborative study on in vitro assay systems, in: J. Ashby, F.J. de Serres, M. Draper, M. Ishidate Jr., B.H. Margolin, B.E. Matter and M.D. Shelby (Eds.), Evaluation of Short-Term Tests for Carcinogens, Progress in Mutation Research, Vol. 5, Elsevier, Amsterdam pp. 117-174. Bermudez, E., D. Tillery and B.E. Butterworth (1979) The effect of 2,4-diaminotoluene and isomers of dinitrotoluene
364 on unscheduled DNA synthesis in primary rat hepatocytes, Environ. Mutagen., 1, 391-398. Bermudez, E., J.C. Mirsalis and H.C. Eales (1982) Detection of DNA damage in primary cultures of rat hepatocytes following in vivo and in vitro exposure to genotoxic agents, Environ. Mutagen., 4, 667-679. Bradley, M.O. (1985) Measurement of DNA single-strand breaks by alkaline elution in rat hepatocytes, in: J. Ashby, F.J. de Serres, M. Draper, M. Ishidate Jr., B.H. Margolin, B.E. Matter and M.D. Shelby (Eds.), Evaluation of ShortTerm Tests for Carcinogens, Progress in Mutation Research, Vot. 5, Elsevier, Amsterdam, pp. 353-357. Brambilla, G., M. Cavanna, S. Parodi, L. Sciaba, A. Pino and L. Robiano (1978) DNA damage in liver, colon, stomach, lung and kidney of BALB/c mice treated with 1,2-dimethylhydrazine, Int. J. Cancer, 22, 174-180. DHHS (1980) Bioassay of benzoin for possible carcinogenicity, DHHS Publ. No. (NIH) 80-1770, U.S. Dept. Health and Human Services. DHHS (1981) Bioassay of caprolactam for possible carcinogenicity, DHHS Publ. No. (NIH) 80-1760, U.S. Dept. Health and Human Services. Erickson, L.C., R. Osieka, N.A. Sharkey and K.W. Kohn (1980) Measurement of DNA damage in unlabeled mammalian cells analyzed by alkaline elution and a fluorometric DNA assay, Anal. Biochem., 106, 169-174. Glauert, H.P., W.S. Kennan, G.L. Sattler and H.C. Pitot (1985) Assays to measure the induction of unscheduled DNA synthesis in cultured hepatocytes, in: J. Ashby, F.J. de Serres, M. Draper, M. Ishidate Jr., B.H. Margolin, B.E. Matter and M.D. Shelby (Eds.), Evaluation of Short-Term Tests for Carcinogens, Progress in Mutation Research, Vol. 5, Elsevier, Amsterdam, pp. 371-373. Kohn, K.W. (1979) DNA as a target in cancer chemotherapy: A new approach to the study of macromolecular damage produced in mammalian cells by anticancer agents and carcinogens, Methods of Cancer Research, Drug Develop., Part A, 291-345. Kohn, K.W., and R.A.G. Ewig (1973) Alkaline elution analysis, a new approach to the study of DNA single-strand interruptions in cells, Cancer Res., 33, 1849-1853.
Martin, C.N., A.C. McDermid and R.C. Garner (1977) Measurement of unscheduled DNA synthesis in HeLa cells by liquid scintillation counting after carcinogen treatment, Cancer Lett., 2, 355 360. Mirsalis, J.. C.K. Tyson and B.E. Butterworth (1982) Detection of genotoxic carcinogens in the in vivo/in vitro hepatocyte DNA repair assay, Environ. Mutagen., 4. 553-562. Parodi, S., M. Taningher, L. Santi, M. Cavanna, k. Sciaba, A. Maura and G. Brambilla (1978) A practical procedure for testing DNA damage in vivo proposed for a prescreening of chemical carcinogens, Mutation Res., 53, 39 46. Petzold, G.L., and J.A. Swenberg (1978) Detection of DNA damage induced in vivo following exposure of rats to carcinogens, Cancer Res.. 38, 1589 1594. San, R.H.C., and H.F. Stich (1975) DNA repair synthesis of cultured human cells as a rapid bioassay for chemical carcinogens, Int. J. Cancer, 16, 284-291. Sina, J.F., C.L Bean, G.R. Dysart, V.I. Taylor and M.O. Bradley (1983) Evaluation of the alkaline elution/rat hepatocyte assay as a predictor of carcinogenic/mutagenic potential, Mutation Res., 113, 357-391. Swenberg, J.A., G.L. Pedzold and P.R. Harbach (1976) In vitro DNA damage/alkaline elution assay for predicting carcinogenic potential, Biochem. Biophys. Res. Commun., 72, 732 738. Williams, G.M. (1977) Detection of chemical carcinogens by unscheduled DNA synthesis in rat liver primary cell cultures, Cancer Res., 37, 1845-1851. Williams, G.M. (1985) Summary report on the performance of the assays for DNA damage, in: J. Ashby, F.J. de Serres, M. Draper, M. Ishidate Jr., B.H. Margolin, B.E. Matter and M.D. Shelby (Eds.), Evaluation of Short-Term Tests for Carcinogens, Progress In Mutation Research, Vol. 5, Elsevier, Amsterdam, pp. 59 67. Williams, G.M., E. Bermudez and D. Scaramuzzino (1977) Rat hepatocyte primary cultures, lII. Improved dissociation and attachment techniques and the enhancement of survival by culture medium, In Vitro, 13, 809-817.
361
Elsevier MUTGEN 02661
The induction of DNA-strand breaks and unscheduled D N A synthesis in F-344 rat hepatocytes following in vivo administration of caprolactam or benzoin Edilberto Bermudez, Tracy Smith-Oliverand Loretta Lyon Delehanty Department of Genetic Toxicology, Chemical Industry Institute of Toxicology, Research Triangle Park, NC (U.S.A.)
Keywords: Benzoin; Caprolactam; DNA-strand breaks, induction; DNA synthesis, unscheduled, induction; Hepatocytes
Summary Benzoin (ZOIN) and caprolactam (CAP) were administered by gavage to Fischer 344 rats at a dose of 750 m g / k g and the hepatocytes isolated 12, 24 or 48 h after treatment. The isolated hepatocytes were subsequently examined for the induction of DNA-strand breaks (SB) and unscheduled D N A synthesis (UDS). Neither ZOIN nor CAP induced SB or UDS in hepatocytes, however ZOIN did induce an increase in the fraction of cells in S-phase 24 h after treatment. These results correlate well with the observed lack of carcinogenicity of these compounds.
The presence of strand breaks in DNA may be assessed using the alkaline elution assay (Kohn and Ewig, 1973) and this assay has been utilized to determine the DNA-damaging effects of chemical treatment in vitro and in vivo (Bermudez et al., 1982; Brambilla et al., 1978; Erickson et al., 1980; Parodi et al., 1978; Petzold et al., 1978; Sina et al., 1983; Swenberg et al., 1976). Various lesions leading to SB are detectable using the alkaline elution assay including: frank breaks, excision sites, and alkali-labile sites. The induction of UDS has been utilized as an endpoint for the DNA-damaging activity of chemicals in various systems including primary rat hepatocyte cultures (Martin et al., 1977; Mirsalis et al., 1982; Williams, 1977, 1985). Two methods for assessing UDS rely upon the insertion of ra-
diolabeled bases into D N A sites that have been repaired by excision-type repair mechanisms followed by detection of the sites by autoradiography or scintillation counting. ZOIN and CAP have both been tested for carcinogenicity in rats and mice and have been found to be negative (DHHS, 1980, 1981). These two chemicals have also been tested in a variety of prokaryotic and eukaryotic short-term in vitro assays for genotoxicity and found to be generally, but not unanimously, inactive (Ashby et al., 1985). The present study was undertaken with the objective of determining if ZOIN or CAP can induce SB or UDS in F-344 rat hepatocytes when these compounds are administered to the animals by gavage.
Methods Correspondence: Dr. Edilberto Bermudez, Department of Genetic Toxicology, Chemical Industry Institute of Toxicology, Research Triangle Park, NC (U.S.A.).
Male Fischer-344 rats [CDF(G-344)/CrlBr, Charles River Breeding Laboratories, Kingston, NY] weighing 200-280 g were housed in hanging
0165-1218/89/$03.50 © Elsevier Science Publishers B.V. (Biomedical Division)
362 stainless steel-mesh cages (Hazleton Systems, Vienna, VA) in a mass air displacement room (Bioclean, Hazleton Systems, Vienna, VA) with controlled t e m p e r a t u r e (22 _+ 1 °C), relative humidity (50 _+ 10%), and light cycles (12 h). The animals had free access to food (NIH-07 open formula) and water. Randomly selected animals were tested and found free of known pathogens (Standard Screening Necropsy, Microbiological Associates, Bethesda, MD). Z O I N and CAP were administered by gavage (1.0 m l / 1 0 0 g body weight) using corn oil or water, respectively, as the vehicle. Control animals received only vehicle. 12, 24 or 48 h after treatment with 750 m g / k g of chemical the animals were anesthetized and the livers perfused with collagenase to obtain single cell suspensions of hepatocytes as previously described (Bermudez et al., 1979; Williams et al., 1977). The cell suspensions were filtered through gauze, centrifuged at 50 × g for 4 rain and resuspended in cold Hanks' balanced salt solution (Flow Laboratories). At this time the cell density and viability, as determined by trypan blue exclusion, were assessed. The alkaline elution assay (Kohn, 1979) was performed as described by Bermudez et al. (1982). Control and treated cells (5 × 105) suspended in 5 m M phosphate-buffered saline (PBS, p H 7.2) were deposited onto PVC filters (2 txm, BSWP25, Millipore), lysed (0.02 M N a 3 E D T A , 2 M NaC1, 0.3% sarkosyl, 0.5 m g / m l proteinase K, p H 10.00) for 30 min and eluted for 10 h with 0.02 M H 4 E D T A (brought to p H 12.20 with 10% tetrapropylammonium hydroxide) at a flow rate of 0.05 m l / m i n . Two sets of triplicate filters were prepared for each sample, one set to be eluted and one to serve as a reference for the quantity of D N A initially deposited on the filters. The relative amounts of D N A present on the eluted and reference filters were determined by quantitation of the fluorescent product obtained after reacting D N A with 3,5-diaminobenzoic acid hydrochloride (0.3 g / m l , Aldrich, Milwaukee, WI) at 6 0 ° C for 90 min. Fluorescence was measured at an excitation wavelength of 414 nm and an emission wavelength of 508 nm. The data is expressed as the percent of the D N A retained (percent retention) and is determined by dividing the adjusted mean relative fluorescence of the eluted filters by the mean
relative fluorescence of the reference filters. Significance at the p < 0.05 level was determined by comparing the percent retention of the treated samples to the percent retention of the pooled control samples (78.3 _+ 4.4 [SEM]), using the t-test. Unscheduled D N A synthesis induction was assessed as described by Mirsalis et al. (1982). Cells (4 × 105) were seeded into dishes containing plastic coverslips and Williams medium E supplemented with 10% fetal calf serum. The cells were allowed to attach to the coverslips for 1.5 h, washed, and incubated for an additional 4 h in Williams medium E containing 10 /~Ci/ml [3H]thymidine. U n i n c o r p o r a t e d radioactivity was chased for 14-16 h with a 0.25 m M unlabeled thymidine solution. Control cells treated with 1 m M dimethylnitrosamine ( D M N , Aldrich, Milwaukee, WI) in vitro for 18 h, in the presence of [3H]thymidine, were used as the positive control. Fixation of the cells with 3 : 1 ethanol : acetic acid was preceded by nuclear swelling with 1% sodium citrate and followed by water washes. The coverslips were air-dried, mounted on glass slides and dipped in NTB2 nuclear track emulsion. Exposure of the emulsion for 12-14 days at - 2 0 ° C was followed by development and staining. The number of silver grains over the nucleus and cytoplasm were determined, using an automatic grain counting system, for at least 25 cells on each of 3 slides for each time and dose examined. The data is expressed as the net grains/nucleus and is determined by subtracting the number of grains in a nuclear sized area of the cytoplasm from the number of grains in the nucleus. A positive response is assumed where the net grains/nucleus is equal to or greater than 5. The percent of cells in S-phase was also determined by counting 3000 cells from each animal. Results and discussion
The status of CAP and Z O I N as noncarcinogens in animals has been established by National Toxicology Program bioassays (DHHS, 1980, 1981). Both of these chemicals were utilized as 'negative controls' in the evaluation of various short-term tests for carcinogens and CAP was found to be inactive in the majority, with the exception of assays using clastogenesis and somatic
363 TABLE 1 I N D U C T I O N OF STRAND BREAKS BY CAP A N D ZOIN IN RAT HEPATOCYTES F R O M TREATED ANIMALS Chemical
Time (h)
Dose (mg/kg)
n (animals)
% retention a ±S.D.
Conclusion b
ZOIN ZOIN ZOIN
12 24 48
750 750 750
3 3 3
69_+ 9 86 _+4 86 _+3
NEG NEG NEG
CAP CAP CAP
12 24 48
750 750 750
3 3 3
81-4- 6 71 _+4 76 +_3
NEG NEG NEG
a % Retention
Mean of eluted filters × 100 Mean of reference filters
b NEG, where no significant ( P < 0.05) difference from controis was observed. The mean retention for all controls (n = 4) was 78_+4 (S.E.M.). Comparisons were made using the t-test.
cell mutation as endpoints (Ashby et al., 1985). Similarly, ZOIN was found to be inactive in various short-term tests, however it was positive in
TABLE 2 U N S C H E D U L E D D N A SYNTHESIS I N D U C T I O N IN HEPATOCYTES FROM RATS TREATED WITH ZOIN A N D CAP Chemical
Time (h)
Control
Dose (mg/kg)
n (animals)
NG a + S.D.
% of cells with > 5 NG
% of cells b in Sphase
-
4
-4+
1
0
0.37
ZOIN ZOIN ZOIN
12 24 48
750 750 750
3 3 3
-3 + 1 -7+ 1 -6___ 1
0 0 0
0.02 3.53 0.77
CAP CAP CAP
12 24 48
750 750 750
3 3 3
-3 + 1 -4+ 1 -4+ 1
0 0 0
0.03 0.10 0.10
DMN
c
1 mM
3
46+28
98
-
a NG, net grains/nucleus. The number or grains over the nucleus is determined by subtracting the number of grains in an area of the cytoplasm, equal to that of the nucleus, from the nuclear counts. Each data point represents the mean of the net grains per nucleus of 75-150 cells. Net grains/nucleus of greater than 5 are considered a positive response. b 3000 cells were counted per animal. c Control cells treated in vitro for 18 h.
some of the assays for SB, UDS and mammalian gene mutations (Ashby et al., 1985). Increases in SB were not detected 12, 24 or 48 h following the administration of 750 m g / k g ZOIN or CAP (Table 1). The reported in vitro induction of SB by ZOIN as measured by alkaline elution is attributable to a cytotoxic effect (Bradley, 1985). ZOIN and CAP have been reported to induce SB in the hepatocytes isolated from treated SpragueDawley rats (Parodi et al., 1978; Russo et al., 1989). The differences in route of administration, strain, and methodology prevent direct comparisons between the results of that laboratory and those of the present study. UDS was not induced in hepatocytes from the livers of rats treated with ZOIN or CAP when the cells were examined at 12, 24 or 48 h following treatment (Table 2). This finding is consistent with the absence of induced strand breaks in the same population of cells. Dimethylnitrosamine treatment of hepatocytes in vitro resulted in the induction of UDS (Table 2) indicating that the system was operational. Examination of the cell populations for the fraction in S-phase revealed that ZOIN induced an 8-9-fold increase over control (Tables 2) 24 h after administration. An effect on S-phase in the liver by ZOIN indicates that a lack of absorption is not a likely explanation for the lack of induction of D N A damage. This induction of replicative synthesis may also explain the positive UDS results previously reported for this compound in vitro (Glauert et al., 1985). In conclusion, no induction of SB or UDS was observed in hepatocytes following the administration to rats of ZOIN and CAP. These results are supportive of the reported noncarcinogenicity of these compounds and indicate that they may serve as true negative controls. References Ashby, J. (1985) Overview and conclusions of the IPCS collaborative study on in vitro assay systems, in: J. Ashby, F.J. de Serres, M. Draper, M. Ishidate Jr., B.H. Margolin, B.E. Matter and M.D. Shelby (Eds.), Evaluation of Short-Term Tests for Carcinogens, Progress in Mutation Research, Vol. 5, Elsevier, Amsterdam pp. 117-174. Bermudez, E., D. Tillery and B.E. Butterworth (1979) The effect of 2,4-diaminotoluene and isomers of dinitrotoluene
364 on unscheduled DNA synthesis in primary rat hepatocytes, Environ. Mutagen., 1, 391-398. Bermudez, E., J.C. Mirsalis and H.C. Eales (1982) Detection of DNA damage in primary cultures of rat hepatocytes following in vivo and in vitro exposure to genotoxic agents, Environ. Mutagen., 4, 667-679. Bradley, M.O. (1985) Measurement of DNA single-strand breaks by alkaline elution in rat hepatocytes, in: J. Ashby, F.J. de Serres, M. Draper, M. Ishidate Jr., B.H. Margolin, B.E. Matter and M.D. Shelby (Eds.), Evaluation of ShortTerm Tests for Carcinogens, Progress in Mutation Research, Vot. 5, Elsevier, Amsterdam, pp. 353-357. Brambilla, G., M. Cavanna, S. Parodi, L. Sciaba, A. Pino and L. Robiano (1978) DNA damage in liver, colon, stomach, lung and kidney of BALB/c mice treated with 1,2-dimethylhydrazine, Int. J. Cancer, 22, 174-180. DHHS (1980) Bioassay of benzoin for possible carcinogenicity, DHHS Publ. No. (NIH) 80-1770, U.S. Dept. Health and Human Services. DHHS (1981) Bioassay of caprolactam for possible carcinogenicity, DHHS Publ. No. (NIH) 80-1760, U.S. Dept. Health and Human Services. Erickson, L.C., R. Osieka, N.A. Sharkey and K.W. Kohn (1980) Measurement of DNA damage in unlabeled mammalian cells analyzed by alkaline elution and a fluorometric DNA assay, Anal. Biochem., 106, 169-174. Glauert, H.P., W.S. Kennan, G.L. Sattler and H.C. Pitot (1985) Assays to measure the induction of unscheduled DNA synthesis in cultured hepatocytes, in: J. Ashby, F.J. de Serres, M. Draper, M. Ishidate Jr., B.H. Margolin, B.E. Matter and M.D. Shelby (Eds.), Evaluation of Short-Term Tests for Carcinogens, Progress in Mutation Research, Vol. 5, Elsevier, Amsterdam, pp. 371-373. Kohn, K.W. (1979) DNA as a target in cancer chemotherapy: A new approach to the study of macromolecular damage produced in mammalian cells by anticancer agents and carcinogens, Methods of Cancer Research, Drug Develop., Part A, 291-345. Kohn, K.W., and R.A.G. Ewig (1973) Alkaline elution analysis, a new approach to the study of DNA single-strand interruptions in cells, Cancer Res., 33, 1849-1853.
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