Comparative investigation of multiple organs of mice and rats in the comet assay

Mutation Research 517 (2002) 53–74

Comparative investigation of multiple organs of mice and rats in the comet assay Kaoru Sekihashi a , Ayumu Yamamoto a , Yukie Matsumura a , Shunji Ueno b , Mie Watanabe-Akanuma c , Fekadu Kassie d , Siegfried Knasmüller d , Shuji Tsuda e , Yu F. Sasaki a,∗ a

Laboratory of Genotoxicity, Faculty of Chemical and Biological Engineering, Hachinohe National College of Technology, Tamonoki Uwanotai 16-1, Hachinohe, Aomori 039-1192, Japan b Veterinary Public Health, School of Veterinary Medicine and Animal Sciences, Kitasato University, Higashi 23-35-1, Towada, Aomori 034, Japan c Institute of Environmental Toxicology, Suzuki-cho 2-772, Kodaira, Tokyo 187, Japan d Institute of Cancer Research, Borschkegasse 8A, Vienna, Austria e Laboratory of Veterinary Public Health, Department of Veterinary Medicine, Faculty of Agriculture, Iwate University, Ueda 3-18-8, Morioka, Iwate 020-8550, Japan Received 11 April 2001; received in revised form 15 February 2002; accepted 15 February 2002

Abstract Mice and/or rats are usually used to detect chemical carcinogenicity and it has been known that there are species differences in carcinogenicity. To know whether there are species difference in genotoxicity, we conducted comparative investigation of multiple organs of mice and rats in the comet assay. Since the sensitivity to xenobiotics is different for different species, we queried species difference in the genotoxic sensitivity at one equitoxic level but not at one equidose. Therefore, groups of four mice or rats were treated once intraperitoneally or orally with a chemical at highest dose without death and distinct toxic manifestation. When the death was not observed at 2000 mg/kg of a chemical, 2000 mg/kg was used for the comet study. The stomach, colon, liver, kidney, bladder, lung, brain, and bone marrow were sampled 3, 8, and 24 h after treatment. Among chemicals tested, benzyl acetate, chlorodibromomethane and p-chloro-o-toluidine are carcinogenic to mice but not rats, and aniline, azobenzene, o-phenylphenol Na, and d-limonene are carcinogenic to rats but not mice. Although the two species differed in genotoxicity target organs and migration values, the judgement of a positive or negative response was the same for all chemicals studied except for 2,4-dimethoxyaniline, 2,5-diaminotoluene, and p,p -DDT when chemicals with positive responses in at least one organ are judged to be comet assay-positive. 2,4-Dimethoxyaniline and 2,5-diaminotoluene that are Ames test-positive non-carcinogens in both species were positive in one organ (urinary bladder for 2,4-dimethoxyaniline and stomach for 2,5-diaminotoluene) in rats, but negative in all mouse organs. p,p -DDT, which is an Ames test-negative but in vitro cytogenetic test-positive hepatic carcinogen in mice and rats, was positive in multiple rat organs, but not in any mouse organ. These results suggest that species differences in genotoxicity at one equitoxic level are not consistent with species difference in carcinogenicity and that the use of both species is appropriate to indicate a carcinogenic potential in the

∗ Corresponding author. Tel.: +81-178-27-7296. E-mail address: [email protected] (Y.F. Sasaki).

1383-5718/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 ( 0 2 ) 0 0 0 3 4 - 7

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comet assay with multiple organs, when chemicals being positive in at least one organ are judged to be comet assay-positive. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Species difference; Mouse; Rat; Genotoxicity; Multiple organs; Comet assay; Alkaline single cell gel electrophoresis (SCG) assay

1. Introduction One important objective of genotoxicity tests is to distinguish genotoxins from non-genotoxins as prescreening in the safety evaluation of chemicals and to assess the mutagenic and carcinogenic hazards of chemicals to humans [1]. Although general toxicity studies are usually conducted using rats, in vivo genotoxicity has been commonly assessed as induction of micronuclei in mice. This is partly because the micronucleus test in hematopoietic cells is a very convenient in vivo testing system. Unlike in mice, the spleen of the rats eliminates circulating micronucleated erythrocytes and thus makes rat peripheral blood micronucleus test difficult [2]. Therefore, up to now, reliable database for in vivo genotoxicity as revealed by micronucleus induction is established in mice but not rats [3]. Reliable carcinogenicity database, in which both species were used by the US NTP, demonstrated clearly species difference in carcinogenicity; out of 162 rodent carcinogens, 80 are carcinogens to both mice and rats, and 82 are carcinogens to a single species (39 are carcinogens to mice and 43 to rats) [4]. These findings show that the use of a single species of test animals for the indication of a carcinogenic potential of chemicals might not be reliable. Recent developments in the comet (alkaline single cell gel electrophoresis, SCG) assay procedure make it possible to use this assay in genetic toxicology [5,6]. Since one of the most important advantages of this technique is that DNA lesions can be measured in the absence of mitotic activity, genotoxicity in any mammalian organ can be detected. At the International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington, DC (25–26 March 1999), an expert panel met to develop guidelines for the use of the comet assay in genetic toxicology [6]. The expert panel reached a consensus that the optimal version of the comet assay for identifying agents with genotoxic activity was the alkaline (pH > 13) version of the assay developed by Singh et al. [7]. The goal of the expert panel was to identify minimal standards for

obtaining reproducible and reliable comet data deemed suitable for regulatory uses [6]. Guideline topics considered include initial considerations, principles and description of the test method, procedure, results, data analysis and reporting [6]. To date, we have been evaluating the in vivo genotoxicity in mouse, eight organs of 208 chemicals evaluated by the International Agency for Research on Cancer (IARC) and/or the US NTP [8–14]. Although our data support many of the important consensus’s for in vivo assay guideline developed by the expert panel, any reliable information as to animal species selection was not submitted in the IWGPT. If species difference in carcinogenicity is consistent with that in genotoxicity, species selection in the comet assay is one important issue to develop guidelines for regulatory submission. In this study, we query whether both species carcinogens are genotoxic in both species. The studied chemicals were mainly selected from the NTP database [4] and systematic overall assessment was made based on their own data.

2. Materials and methods 2.1. Chemicals and mice The chemicals and CAS numbers, abbreviations, sources, and carcinogenicity classification by NTP [4] and IARC (see Table 4 for references), and the vehicles for their administration, are shown in Table 1. Regular (GP-42) and low melting point (LGT) agarose were obtained from Nacalai Tesque Inc. (Kyoto, Japan), and diluted to 1 and 2%, respectively, with physiological saline. Male ddY mice and Wistar rats were obtained from Japan SLC Co. (Shizuoka, Japan) at 7 weeks of age and used after 1 week of acclimatization. They were fed commercial pellets MF (Oriental Yeast Industries Co., Tokyo, Japan) and tap water ad libitum throughout the acclimatization period and the experiment. The animal room was maintained at 20–24 ◦ C and 55–65% humidity with a 12 h light–dark cycle.

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Table 1 List of chemicals tested Chemicals (Abbreviation)

CAS number

IARCc Both species carcinogens Alkylating agents Methyl methanesulfonate (MMS) N-Nitrosodiethylamine (DEN)

Purity (%)

Sourcea

Vehicleb

99 >99.0

A W

S S

A

>98.0

A A A A A

97

W S W W T W W To

O O O T S T O O

W T T

T O O

S T W W

S O O S

Classification NTPd

66-27-3 55-18-5

2B 2A

Aromatic amines o-Anisidine Benzidine 4-Chloro-o-phenylenediamine p-Cresidine 2,4-Diaminoanisole 2HCl 2,4-Diaminotoluene o-Toluidine PhIP

90-04-0 92-87-5 95-83-0 120-71-8 615-05-4 95-80-7 95-53-4 105650-23-5

2B 1 2B 2B 2B 2B 2B 2B

Halides 1,2-Dibromo-3-chloropropane (DBCP) p,p -DDT 1,2,3,4,5,6-Hexachlorobenzene

96-12-8 50-29-3 118-74-1

2B 2B 2B

Others Aflatoxin B1 o-Aminoazotoluene Benzene 1,2-Dimethylhydrazine HCl

1162-65-8 97-56-3 71-43-2 306-37-6

1 2B 1 2B

Mouse carcinogens Benzyl acetate Chlorodibromomethane p-Chloro-o-toluidine

140-11-4 124-48-1 95-69-2

3 3 2A

B C C

>99.0 >98.0 >95

T W W

O O O

Rat Carcinogens Aniline Azobenzene o-Phenylphenol Na (OPP Na) d-Limonene

62-53-3 103-33-3 132-27-4 5989-27-5

3 3 2B

B B

>99 >95.0 >98

W T W T

O O O O

>99

T T

O O

>98 >98 >97 98.0

T T T T

S O O O

Carcinogens, rat MN positive and mouse MN negative Acrylonitrile 107-13-1 Sudan I (CI Solvent Yellow 14) (CI 12055) 842-07-9 Rodent non-carcinogens 2,5-Diaminotoluene sulfate 2,6-Diaminotoluene 2,4-Dimethoxyaniline HCl 1,4-Phenylenediamine 2HCl

6369-59-1 823-40-5 54150-69-5 624-18-0

A A

>99 >95 >99.0

>97.0 >98 >99.0

>95 A

D 2A 3 3

F F F F

a A: Aldrich Chemical Co. Inc., Milwaukee, WI (USA); K: Kanto Chemical Co. Inc., Tokyo (Japan); N: Nacalai Tesque Inc., Kyoto (Japan); S: Sigma Chemical Co., St. Louis, MO (USA); T: Tokyo Kasei Kogyo, Tokyo (Japan); To: Toronto Research Chemicals Inc., North York (Canada), W: Wako Pure Chemical Industries, Ltd., Osaka (Japan). b O: olive oil; S: saline; T: 2% Tween 80. c 1: Carcinogenic to humans; 2A: probably carcinogenic to humans; 2B: possibly carcinogenic to humans; 3: unclassifiable as carcinogenic to humans. d A: carcinogens to both rat and mouse; B: carcinogens affecting a single species at multiple sites; C: carcinogens affecting a single species at a single site; D: carcinogens affecting a single species/sex/site; E: chemicals with equivocal evidence of carcinogenicity; F: rat and mouse non-carcinogens.

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2.2. Animal treatment All chemicals except for DEN, MMS, acrylonitrile, and DBCP were administered orally. DEN, MMS, acrylonitrile, and DBCP were given intraperitoneally. Since the sensitivity to xenobiotics is different for different species, we queried species difference in the genotoxic sensitivity at equitoxic levels but not at equidoses. DNA damage is usually detected shortly after administration of a relatively high dose. Therefore, to know species difference at high level to cause systemic genotoxic effects, the used dose of each chemical was set at highest dose without death and distinct toxic manifestation, with a limit of 2000 mg/kg. In order to set appropriate dose of each chemical for the comet assay, approximate LD50s were determined by simple acute toxicity experiments using four–five animals. In the comet assay, in order to avoid false-positive results of the comet assay due to cytotoxicity, animals are recommended to be treated with a chemical at the doses at which gross necrosis is not observed [6]. With test animals receiving a single dose and then even died within 24 h, pathology findings are limited to grossly apparent sings with microscopic signs being either absent or such as to only confirm what was seen grossly [15]. Except for MMS and DEN, since no distinctive clinical signs were observed within 24 h after the treatment at 0.5 × LD50 in simple acute toxicity experiments, the used doses for them were set at 0.5 × LD50 for each species, with a limit of 2000 mg/kg. For DEN, although an accentuated lobular pattern was found in the liver at 0.5 × LD50, no microscopic signs for necrosis were observed. Therefore, the used dose for DEN was set at 0.5 × LD50 for each species. For MMS, since gross intraperitoneal hemorrhage was observed at 0.5 × LD50, the used dose for MMS was set at 0.25 × LD50 at which no intraperitoneal hemorrhage was observed. In the comet assay, groups of four mice or rats were treated once intraperitoneally or orally with a chemical. After 3, 8, and/or 24 h, slides for the comet assay were prepared at each set time as described below. 2.3. Vehicle control experiments Separate vehicle control experiments were performed to confirm if there are any significant differences among control animals sampled at different

times after the vehicle treatments. These animals in the control experiments were treated in the same manner as those of the chemical treated animals. Twelve male ddY mice and Wistar rats were orally given by olive oil at 10 ml/kg and sacrificed 3, 8, and/or 24 h after the administration. As controls, untreated 12 male ddY mice and Wistar rats were used. 2.4. The comet assay This experiment was principally designed according to our methods for the comet assay with mouse multiple organs [8–14]. From shortly after treatment until just before they were killed, the animals were carefully observed for pharmacotoxic signs. The animals were sacrificed 3, 8, and 24 h after treatment, and eight organs—stomach, colon, liver, kidney, urinary bladder, lung, brain, and bone marrow—were removed. In our previous studies with the comet assay [10,14] and vehicle control experiments in this study (data are shown in Table 2), we observed no significant differences in mean migration between DNA from vehicle control groups and the corresponding untreated groups at any sampling time for any organ. Therefore, we used untreated animals as controls rather than concurrent vehicle controls. Necropsies were performed, and the organs were examined for changes in size, color, and texture. A small portion of each organ was fixed in 10% formaldehyde, dehydrated, and embedded in paraffin. Sections were cut and stained with hematoxylin and eosin. Histopathological examination was conducted when positive results were obtained in the comet assay. Slides prepared from nuclei isolated by homogenization [8] were placed in a chilled lysing solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Trizma, 1% sarkosyl, 10% DMSO, and 1% Triton X-100, pH 10) [7] and kept at 0 ◦ C in the dark for >60 min, then in chilled alkaline solution (300 mM NaOH and 1 mM Na2EDTA, pH 13) for 10 min in the dark at 0 ◦ C [8]. Electrophoresis was conducted at 0 ◦ C in the dark for 15 min at 25 V (0.96 V/cm) and approximately 250 mA. The slides were neutralized and thereafter stained with 50 ␮l of 20 ␮g/ml ethidium bromide (Wako Pure Chemical Industries Ltd.) [8]. We examined and photographed 50 nuclei per slide at 200× magnification with the aid of a fluorescence microscope. The length of the whole comet (“length”)

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Table 2 Migration of nuclear DNA from five organs of mice and rats treated with vehicles Species

Mouse

Treatment

Sampling time (h)

Stomach

Colon

Liver

Kidney

Bladder

Lung

Brain

Bone marrow

–

–

Mean S.E.M.

6.19 0.44

5.15 0.44

2.55 0.24

2.32 0.34

4.66 0.42

2.70 0.24

1.48 0.20

0.94 1.01

Olive oil

3

Mean S.E.M. Mean S.E.M. Mean S.E.M.

6.46 0.57 7.22 0.49 6.23 0.40

4.56 0.45 5.09 0.40 4.70 0.35

2.06 0.29 2.86 0.43 2.49 0.20

1.90 0.32 2.64 0.43 1.80 0.24

4.33 0.33 5.63 0.49 4.20 0.39

2.33 0.37 2.49 0.60 2.27 0.27

1.56 0.38 1.83 0.41 1.70 0.41

0.90 0.30 1.08 0.41 1.23 0.27

8 24 Rat

Migration (␮m, mean of 12 animals)

–

–

Mean S.E.M.

12.5 0.84

11.4 0.78

2.09 0.43

2.29 0.41

8.54 0.95

3.43 0.33

1.72 0.39

0.94 0.30

Olive oil

3

Mean S.E.M. Mean S.E.M. Mean S.E.M.

12.2 1.09 11.9 0.73 11.6 0.96

10.9 0.69 11.0 0.75 11.4 0.95

2.00 0.34 1.75 0.23 1.87 0.41

2.58 0.45 2.34 0.44 2.31 0.40

9.45 0.48 9.14 0.84 9.37 0.70

2.74 0.34 2.89 0.46 2.67 0.38

2.06 0.36 1.74 0.38 2.16 0.31

0.94 0.22 0.86 0.24 1.02 0.28

8 24

and the diameter of the head (“diameter”) were measured for 50 nuclei per organ per animal. We calculated migration as the difference between length and diameter for each of the 50 nuclei. Mean migration of 50 nuclei from each organ was calculated for each individual animal. The differences between the averages of four treated animals and the untreated control animals were compared with the Dunnett test after one-way ANOVA. A P-value less than 0.05 was considered statistically significant.

the results are summarized in Table 4 with carcinogenicity study results. No death, morbidity, or distinctive clinical and/or microscopic signs were observed following any treatment with the exception of DEN for which an accentuated lobular pattern was found in the liver (rough surface) during necropsy 24 h after exposure (see Table 3) but without microscopic signs. Therefore, for the other test compounds, any DNA damage observed was not likely to be secondary to cytotoxicity. 3.1. Both species carcinogens

3. Results Table 2 shows DNA migration of nuclei from eight organs of untreated animals and animals treated orally with olive oil at 10 ml/kg. There were no significant differences in the mean migration between vehicle treated groups and corresponding untreated groups at any sampling points for any organs. The results enabled us to use untreated control for examining the effect of chemicals without sacrificing the larger number of vehicle concurrent control animals. The migration of DNA from the organs examined for each treatment group are shown in Table 3, and

3.1.1. Alkylating agents MMS was tested in mice and rats by i.p. administration; DNA damage was induced in all organs tested in both species. DEN yielded a statistically significant increase in DNA damage in all mouse and rat organs with the exception of bone marrow in mouse and brain and bone marrow in rat. 3.1.2. Aromatic amines o-Anisidene was positive in mouse colon and urinary bladder, and in all rat organs studied except for the liver and bone marrow; p-cresidine was positive

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in mouse urinary bladder, and rat stomach, colon, kidney, and urinary bladder. 2,4-Diaminoanisole induced DNA damage in mouse liver and brain and in all rat organs studied except for the brain; o-toluidine was positive in mouse stomach, liver, urinary bladder, lung, and brain, and in rat stomach, colon, kidney, and urinary bladder. 2,4-Diaminotoluene was positive in mouse stomach, liver, and kidney, and in rat stomach, colon, kidney, and lung. 4-Chloro-o-phenylenediamine was positive only in the liver in mice, and in the stomach, colon, kidney, and urinary bladder in rats. While benzidine induced DNA damage in all mouse organs studied except for the colon, it was positive in rat stomach, colon, liver, kidney, urinary bladder, and lung. PhIP induced DNA damage in the stomach and colon in both species, in the liver and kidney in mice, and in the urinary bladder in rats. 3.1.3. Halides DBCP was tested in the comet assay by i.p. injection in mice and rats, inducing a statistically significant increase in DNA damage in mouse stomach, colon, liver, kidney, lung, and bone marrow and in all organs tested in the rat except for the brain. DDT and hexachlorobenzene were tested by oral route. p,p -DDT induced DNA damage in all rat organs tested except for brain and bone marrow, while it did not yield a statistically significant increase in DNA damage in any of the mouse organs studied. Hexachlorobenzene did not induce DNA damage in any of mouse and rat organs studied. 3.1.4. Others Aflatoxin B1 was tested by the comet assay in mice and rats by i.p. treatment, inducing DNA damage in mouse stomach, colon, urinary bladder, lung, and brain, and in all rat organs studied. o-Aminoazotoluene induced DNA damage in mouse colon, liver, and lung by oral treatment. In rats, o-aminoazotoluene given orally was positive in the stomach, colon, urinary bladder, lung, and brain. Benzene given orally induced DNA damage in mouse stomach, liver, lung, and brain, and in all rat organs studied except for the bone marrow. 1,2-Dimethylhydrazine given orally induced DNA damage in mouse stomach, colon, liver, and kidney, and in all rat organs studied.

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3.2. Mouse carcinogens Benzyl acetate given orally was positive in the comet assay in mouse stomach, colon, kidney, and brain, and in all rat organs tested except for the brain and bone marrow. Chlorodibromomethane was positive in mouse liver and brain, and in all rat organs studied except for the brain and bone marrow. p-Chloroo-toluidine was positive in mouse liver, urinary bladder, lung, and brain, and in rat liver and kidney. 3.3. Rat carcinogens Aniline given orally yielded a statistically significant increase in DNA damage in mouse colon, liver, urinary bladder, lung, brain, and bone marrow, and in rat stomach, colon, liver, kidney, urinary bladder, and lung. Azobenzene given orally was positive in mouse stomach, colon, urinary bladder, and lung, and in all rat organs studied except for the brain. o-Phenylphenol Na given orally was positive in all mouse organs tested except for the brain and bone marrow, and in all rat organs tested except for the bone marrow. On the other hand, d-limonene did not induce DNA damage in any of mouse and rat organs studied. 3.4. Carcinogens, rat MN positive and mouse MN negative Acrylonitrile was tested by the comet assay in mice and rats by i.p. treatment, inducing DNA damage in mouse stomach, colon, urinary bladder, lung, and brain, and in rat stomach, colon, kidney, urinary bladder, and lung. Sudan I given orally induced DNA damage in mouse colon, and in rat stomach, colon, kidney, and lung. (There are no reliable carcinogenicity data for acrylonitrile and Sudan I in mice and rats, respectively.) 3.5. Rodent non-carcinogens In mice and rats, 2,6-diaminotoluene and 1,4-phenylenediamine did not yield a statistically significant increase in DNA damage in any of the organs studied. While 2,5-diaminotoluene and 2,4-dimethlxyaniline were negative in all mouse organs studied, the former was positive in rat stomach and the latter in rat urinary bladder.

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4. Discussion

were lower in rats than mice for the liver (0.83-fold) and brain (0.66-fold). Those results suggest that genotoxicity sensitive organs are different for mice and rats.

4.1. Species difference in genotoxicity Up to date, much information about in vivo genotoxicity has been obtained by the micronucleus assay using rodent hematopoietic system and some chemicals are known to differ in micronucleus induction between rats and mice [2,3]. Although mice are more susceptible than rats to o-aminoazotoluene and benzidine and rats are more susceptible than mice to azobenzene and Sudan I in micronucleus induction [2], they were positive in the comet assay in at least one organ of both species. Among 30 chemicals studied, only three chemicals (2,4-dimethoxyaniline, 2,5-diaminotoluene, and p,p -DDT) showed species difference in their responses in the comet assay. Of 30 studied chemicals, 23 yielded DNA damage in at least one organ of mice and 26 in at least one organ of rats (Table 5). When chemicals which induce comet assay-positive effect in at least one organ are judged to be comet assay-positive, an almost identical proportion of positive genotoxic responses is produced in mice and rats. In mice, organs in which positive ratios were over 50% were stomach (16/23), colon (14/23), liver (16/23), urinary bladder (14/23), lung (13/23), and brain (12/23). In rats, high positive response ratios were observed in the stomach (24/26), colon (23/26), liver (15/26), kidney (22/26), urinary bladder (22/26), and lung (18/26). At one equitoxic level, kidney cells were more sensitive to genotoxicity in rats than in mice; positive ratio was about two-fold higher in rats than mice (1.95-fold). Also, for the stomach (1.33-fold), colon (1.45-fold), urinary bladder (1.39-fold), lung (1.22-fold), and bone marrow (1.11-fold), positive ratios were higher in rats than in mice. On the other hand, positive ratios

4.2. Species difference in the concordance between genotoxicity and carcinogenicity The mouse liver is the main target organ for the carcinogenic effect of the studied chemicals (13 out of 21 studied mouse carcinogens target the liver). Since one objective of genotoxicity tests is the assessment of the carcinogenic hazards of chemicals to humans [1], the liver, in which many pro-mutagens are activated, is generally selected as the target organ. We, therefore, compare mice and rats for the concordance of genotoxicity at equitoxic levels and carcinogenicity in the liver (Table 6). In mice, among 30 studied chemicals, 16 were comet assay-positive in the liver and 13 were hepatocarcinogen, from which there seem to be a good concordance between genotoxicity and carcinogenicity in the liver. However, out of 16 chemicals being genotoxic in the liver, only seven (DEN, 4-chloro-o-phenylenediamine, 2,4-diaminotoluene, o-toluidine, o-aminoazotoluene, chlorodibromomethane, and benzene) were mouse hepatocarcinogens and nine were not. In rats, although 15 chemicals were comet assay-positive in the liver, only four (azobenzene, aflatoxin B1, DEN, and p,p -DDT) were hepatocarcinogens and 11 were not. Furthermore, six out of 13 mouse hepatocarcinogens and five out of nine rat hepatocarcinogens were comet assay-negative in the liver of mice and rats, respectively. Therefore, for the chemicals investigated in this study, these results suggest that the concordance between hepatic genotoxicity and carcinogenicity is not high in both species.

Table 5 Comparison of comet assay-positive organs in mice and rats for 30 chemicals Species

Mouse Rat a

Na

23 (21) 26 (22)

Numbers of comet assay-positive chemicals in each organb S

C

L

K

Ub

Lu

Br

BM

Others

16 (5) 24 (3)

14 (0) 23 (4)

16 (13) 15 (9)

10 (1) 22 (4)

14 (4) 22 (5)

13 (8) 18 (2)

12 (0) 9 (2)

4 (4) 5 (0)

(10) (9)

Numbers of comet assay-positive chemicals. Numbers in parentheses represent the number of carcinogens in each species. Number of chemicals that were positive in each organ. Numbers in parentheses represent the number of carcinogens that targeted each organ. S, stomach; C, colon; L, liver; K, kidney; Ub, urinary bladder; Lu, lung; Br, brain; BM, bone marrow. b

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Table 6 Concordance of the results of comet assay and carcinogenicity study in the liver Comet assay

Mouse Concordant results

+ –

Carcinogenicity test

+ –

Subtotal Disconcordant results

Number of chemicals, (%)

– +

15

Total

30 + –

+ –

Subtotal Disconcordant results

– +

16

Total

30

b

53.8

52.9

44.4

52.4

50.0

50.0

46.7

11 (68.8) 5 (31.3)

Subtotal

a

Car−b

4 (28.6) 10 (71.4) 14

+ –

Car+a

9 (64.3) 6 (42.9)

Subtotal

Rat Concordant results

Comet + (%)

7 (50.0) 8 (57.1) 15

+ –

Total (%)

53.3

Proportion of chemicals that are comet assay-positive in the liver out of hepatic carcinogens. Proportion of chemicals that are comet assay-positive in the liver out of hepatic non-carcinogens.

Mice and rats differ in their responses to tumor induction; mice are more susceptible to benzyl acetate, chlorodibromomethane, and p-chloro-o-toluidine, and rats are to aniline, azobenzene, OPP Na, and d-limonene [4,18]. (For acrylonitrile, there are no reliable data of carcinogenicity in mice.) Although the two species differed in genotoxicity target organs and migration values, all the studied chemicals except for d-limonene were comet assay-positive in both species. (d-Limonene was negative in both species.) On the contrary, three chemicals that did not show species difference in carcinogenicity differed in the results of this assay. p,p -DDT, which is Ames test-negative but cytogenetics test-positive [19] hepatic carcinogen in mice and rats [16], was positive in rat multiple organs. 2,4-Dimethoxyaniline and 2,5-diaminotoluene are carcinognic to neither mice nor rats [4], but they were positive in one organ (urinary bladder for 2,4-dimethoxyaniline and stomach for 2,5-diaminotoluene) in rats. These results suggest that species difference in genotoxicity is not consistent with species difference in carcinogenicity.

The comet assay which detects initial DNA lesions cannot indicate accurately which comet assay-positive organs would be tumor targets among the damaged organs, and, as described above, good concordance between organ specific genotoxicity and carcinogenicity was not observed for most of the chemicals we studied. When chemicals that are positive in at least one organ are judged to be comet assay-positive, among 30 studied chemicals, 23 that were carcinogenic to mice and/or rats are positive in the comet assay with mice. Among the remaining seven chemicals that were negative in the comet assay with mice, four were rodent non-carcinogens [4] and three (DDT, hexachlorobenzene, and d-limonene) were non-genotoxic (Ames test-negative) rodent carcinogens [16,17]. In rats, 26 out of 30 studied chemicals were positive in the comet assay, whereas two rodent non-carcinogens were positive in the rat in a single organ. Among the remaining four chemicals, two were rodent non-carcinogens and two (hexachlorobenzene and d-limonene) were non-genotoxic rodent carcinogens [4,17]. When chemicals that induce positive

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effect in at least one organ are judged to be comet assay-positive, our study suggests a good concordance between the positive response ratio of the comet assay in each species and carcinogenicity in rodents. 5. Conclusion The sensitivity of mouse and rat organs towards the genotoxicity of the test compounds was different and species difference in genotoxicity is not consistent with species difference in carcinogenicity. The concordance between hepatic genotoxicity and carcinogenicity is not high in both species. When chemicals that induce positive effect in at least one organ are judged to be comet assay-positive, however, our study suggests a good concordance between the positive response ratio of the comet assay in each species and carcinogenicity in rodents. Although genotoxicity sensitive organs are different for mice and rats, both mice and rats can be used in the comet assay as an in vivo genotoxicity testing system. References [1] ICH, Topic 2A Genotoxicity; Guidance on specific aspect of regulatory genotoxicity testing of pharmaceuticals, in: Proceedings of the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use, 1995, Step 4 Guidline (see Web site http://ifpma.org/ich1.html). [2] A. Wakata, Y. Miyamae, S.-i. Sato, T. Suzuki, T. Morita, N. Asano, T. Awogi, K. Kondo, M. Hayashi, Evaluation of the rat micronucleus test with bone marrow and peripheral blood: summary of the 9th collaborative study by CSGMT/ JEMS.MMS, Environ. Mol. Mutagen 21 (1998) 84–100. [3] T. Morita, N. Asano, T. Awogi, Y.F. Sasaki, S. Sato, H. Shimada, S. Sutou, T. Suzuki, A. Wakata, T. Sofuni, M. Hayashi, Evaluation of the rodent micronucleus assay to screen IARC carcinogens (group 1, 2A, and 2B), The summary report of the 6th collaborative study by CSGMT/JEMS, Mutat. Res. 389 (1997) 3–122. [4] J. Ashby, R.W. Tennant, Definitive relationships among chemical structure, carcinogenicity and mutagenicity for 301 chemicals tested by the US NTP, Mutat. Res. 257 (1991) 229–306. [5] D.W. Fairbairn, P.L. Olive, K.L. O’Neill, The comet assay: a comprehensive review, Mutat. Res. 339 (1995) 37–59. [6] R.R. Tice, D. Anderson, B. Burlinson, A. Hartmann, H. Kobayashi, Y. Miyamae, E. Rojas, J-C. Ryu, Y.F. Sasaki, The single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing, Environ. Mol. Mutagen 35 (2000) 206–221.

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