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Mutagenesis Advance Access published online on September 2, 2008
Mutagenesis, doi:10.1093/mutage/gen047
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© The Author 2008. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org.

Evaluation of a liver micronucleus assay with 12 chemicals using young rats (II): a study by the Collaborative Study Group for the Micronucleus Test/Japanese Environmental Mutagen Society–Mammalian Mutagenicity Study Group

Hiroshi Suzuki*, Hironao Takasawa1, Kazuo Kobayashi2, Yukari Terashima2, Yasushi Shimada3, Izumi Ogawa4, Jin Tanaka5, Tadashi Imamura, Atsushi Miyazaki and Makoto Hayashi5

Ina Research Inc., 2148-188 Nishiminowa, Ina-shi, Nagano 399-4501, Japan 1Mitsubishi Chemical Safety Institute Ltd, 14 Sunayama, Kamisu-shi, Ibaraki 314-0255, Japan 2Toxicology Research Laboratory, R&D Kissei Pharmaceutical Co., Ltd, 2320-1 Maki, Hotaka, Azumino, Nagano 399-8305, Japan 3Hokko Chemical Industry Co., Ltd, 2165 Toda, Atsugi-shi, Kanagawa 243-0023, Japan 4Biological Research Laboratories, Nissan Chemical Industries, Ltd, 1470, Shiraoka, Minamisaitama-gun, Saitama 349-0294, Japan 5Biosafety Research Center, Foods, Drugs and Pesticides, 582-2 Shioshinden, Iwata, Shizuoka 437-1213, Japan

The partial hepatectomy method, co-treatment method with mitogens and an in vivo/in vitro assay method have been reported as in vivo liver micronucleus (MN) assays. These methods have disadvantages with respect to widespread use as an in vivo assay, i.e. they are time consuming, labour intensive and there is the possibility of interaction with the mitogens used. Therefore, we have attempted to develop a new method to overcome these disadvantages. The assay as described herein utilises the autonomous proliferation of hepatocytes of young rats. Nine chemicals have been evaluated using this method thus far. We have also assessed the sensitivity and detectability according to the following methods. A liver MN assay was performed in two strains of young rats using one or two doses of 12 chemicals to investigate the inducibility of micronucleated hepatocytes. For some of the chemicals, a peripheral blood MN assay was performed concurrently in the same animals. The following chemicals were used: diethylnitrosamine (DEN), 2-acetylaminofluorene (2AAF), 2,4-diaminotoluene (2,4-DAT), quinoline, p-dimethylaminoazobenzene (DAB), dimethylnitrosamine (DMN), ethylmethanesulphonate, 5-fluorouracil, mitomycin C (MMC), 1,2-dimethylhydrazine·2HCl, cyclophosphamide and 2,4-dinitrotoluene (2,4-DNT). The rodent hepatocarcinogens, quinoline, DAB and DMN showed positive responses in previous assays. The results of the present assay revealed new positive responses for single doses of 2AAF, 2,4-DAT, MMC, 1,2-dimethylhydrazine·2HCl and 2,4-DNT. These chemicals are known rodent hepatocarcinogens, whose clastogenicity is believed to be related to the formation of reactive metabolites generated through enzymatic activation, or the chemicals act directly. Two doses of 2AAF and DMN appeared to be more effective than a single dose in terms of MN induction. Although there were quantitative differences in the incidences of MNs, both strains of rat (F344 and SD) responded positively after treatment with DEN, DMN, 2,4-DAT, DAB, quinoline and 2AAF, suggesting that both strains are appropriate for the assay. Based on these results, it is concluded that this technique could be effective for detecting chemical clastogenicity in hepatocytes in vivo.


   Introduction
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 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
In vivo rodent bone marrow micronucleus (MN) assays are widely performed to test the genotoxicity of chemicals. Positive responses correlate well with carcinogenicity in multiple organs (1Go). Chemicals that show genotoxic potential following metabolic activation, however, such as rodent hepatocarcinogens, tend to be negative in bone marrow MN assays (1Go). Some chemicals that are positive in in vitro assays, but not in in vivo bone marrow MN assays, may induce MN in other tissues (2Go,3Go). Currently, the unscheduled DNA synthesis (UDS) assay and the in vivo bone marrow MN assay are the most internationally accepted in vivo genotoxicity assays and have the largest historical databases (4Go). The UDS assay, however, along with the in vivo single-cell gel electrophoresis (comet) assay, is the test method used for detecting DNA damage. Although the established and widely used in vivo MN assay targets bone marrow, in vivo MN assays that target other tissues, such as the skin, colon or liver, have also been developed (5Go–10Go). Using the liver instead of the bone marrow is important for the following reasons: (i) many chemicals are metabolised in the liver but not in the bone marrow, (ii) short-lived metabolites generated in the liver (or other tissue) may not allow efficient exposure in bone marrow, (iii) bone marrow is not a target organ for some classes of carcinogens. Zhurkov et al. recommend using both bone marrow and liver for evaluating systemic genetic toxicity of chemicals (11Go).

Based on these backgrounds, we have developed a liver MN assay using 4-week-old F344 rats (10Go). This method utilises the developmental proliferation of hepatocytes in young rats up to approximately 5 weeks of age. The metabolic capacity of young rats of this age is comparable to that of adult rats (12Go), although some cytochrome P450s may be absent (10Go). Some rodent hepatocarcinogens show positive responses in this assay (13Go), but many others have not been tested, nor have the effect of multiple dosing or usability of other strain of rats been evaluated. Here, we carried out a young rat liver MN assay to further evaluate the sensitivity of this assay using 12 direct- or indirect-acting genotoxins.


   Materials and methods
Top
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Animals
Male Fischer F344 and Crl:CD(SD) rats, 3 weeks of age, were purchased from Charles River Laboratories Japan, Inc. (Yakohama). The animals were observed for general health conditions once daily for at least 5 days (quarantine period) and used in the assay at 4 weeks of age. The animals were housed under a 12-h light–dark cycle and allowed free access to food and water.

Chemicals and dosages
2-Acetylaminofluorene (2AAF; CAS No. 53-96-3, >98% purity), cyclophosphamide (CP; CAS No. 50-18-0, 99.0% purity), 2,4-diaminotoluene (2,4-DAT; CAS No. 95-80-7, 95% purity), N,N'-dimethylhydrazine dihydrochloride (1,2-DMH·2HCl; CAS No. 306-37-6, 99.4% purity), 2,4-dinitrotoluene (2,4-DNT; CAS No. 606-20-2, 98% purity), ethylmethanesulphonate (EMS; CAS No. 62-50-0, >98% purity), 5-fluorouracil (5FU; CAS No. 51-21-8, >98.5% purity) and mitomycin C (MMC; CAS No. 50-07-7, >850 µg/ml potency) have not been tested previously in the liver MN assay. 2AAF was purchased from Tokyo Chemical Industry Co. Ltd (Tokyo, Japan), and 5FU was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). 1,2-DMH·2HCl and EMS were purchased from Sigma Aldrich Co. (St. Louis, MO, USA). All other chemicals were purchased from Wako Pure Chemical Industries Ltd (Osaka, Japan). For use, 2AAF, 2,4-DAT and 2,4-DNT were suspended in olive oil or corn oil, and the other chemicals were dissolved in physiological saline or distilled water. We based dose levels on those shown to be positive in the rat bone marrow MN assay (14Go), with the exception that the doses for 2,4-DAT and 2,4-DNT were calculated according to the method of Lorke (15Go) because the 50% lethal dose values were unclear. The dose levels were set up about 1/2 and 1/4 of the 50% lethal dose. CP and 5FU were also tested in the peripheral blood (PB) MN assay using samples collected from the same animals used for the liver MN assay (16Go). Dosing was conducted once intraperitoneally for MMC and 5FU, twice intraperitoneally or orally for EMS and orally for the other chemicals. Positive control animals received diethylnitrosamine (DEN; CAS No. 55-18-5, >99.0% purity, Tokyo Chemical Industry Co. Ltd) at 40 or 50 mg/kg (liver MN assays) via the oral route. 2AAF, dimethylnitrosamine (DMN; CAS No. 62-75-9, 99.9% purity), 2,4-DAT, quinoline (CAS No. 91-22-5, >95% purity), EMS and p-dimethylaminoazobenzene (DAB; CAS No. 60-11-7, indicator grade) were used to investigate the influence of a two-dose protocol and 2AAF, DMN, 2,4-DAT, quinoline, DEN and DAB were used to investigate the existence of any differences in sensitivity between rat strains after oral dosing. We purchased quinoline from Wako Pure Chemical Industries Ltd, DMN from Wako Pure Chemical Industries Ltd or Tokyo Chemical Industry Co. Ltd and DAB from Sigma Aldrich Co. Dose levels were selected based on the results of previous, single-dose assays (13Go). Each group consisted of four or five animals.

Liver MN assay
Specimens were prepared 3, 4 or 5 days after one or two dosings of the test chemical. Specimens from animals receiving 2AAF at 750 mg/kg were prepared 4, 5 or 6 days after dosing. Hepatocytes were isolated from anaesthetised rats by the collagenase perfusion method (collagenase, 45°C; room temperature, ~25°C), rinsed with 10% neutral formalin three times and centrifuged at 42 x g for 1 min. The hepatocyte pellets were suspended in 10% neutral buffered formalin and stored under refrigeration. Immediately prior to evaluation, 10–20 µl of hepatocyte suspension was mixed with an equal volume of acridine orange (AO)–DAPI (4',6-diamidino-2-phenylindole dihydrochloride) stain solution (AO, 0.5 mg/ml; DAPI, 10 µg/ml) for fluorescent staining. Approximately 10–20 µl of the mixture was dropped onto a glass slide and covered with a cover glass (24 x 40 mm).

Specimens of well-isolated hepatocytes were evaluated with the aid of a fluorescence microscope (x400 or greater with UV excitation) counting the number of micronucleated hepatocytes (MNHEPs) in 2000 hepatocytes (two fields) for each animal. MNHEPs were defined as hepatocytes with round or distinct MNs that stained like the nucleus, with a diameter 1/4 or less than that of the nucleus (17Go,18Go), and confirmed by focusing up and down, taking into account hepatocyte thickness. The incidence of M-phase cells in 2000 hepatocytes was also determined. M-phase cells were defined as cells in prophase to telophase having a poorly defined nuclear envelope, identifiable chromosomes and two unevenly shaped nuclei that strongly fluoresce.

PB MN assay
Blood samples were collected 2 days after dosing. Each animal was held in a restricted position while the tail was pricked with an injection needle and approximately 5–10 µl blood was collected into a micropipette. The sample was dropped onto an AO-stained glass slide and covered with a cover glass, or it was mixed with approximately 15–30 µl of 10% neutral buffered formalin, with care being taken to avoid coagulation, and stored in a refrigerator. Immediately prior to evaluation, the blood sample was mixed with an equal volume of AO stain solution (0.5 mg/ml) for fluorescent staining, and 5–10 µl was dropped onto a glass slide and covered with a cover glass. Specimens were evaluated with the aid of a fluorescence microscope (x600 or greater) with B excitation. The number of micronucleated reticulocytes (MNRETs) in 1000 or 2000 reticulocytes was counted for each animal.

Statistical analysis
Differences in the incidence of MNHEPs or MNRETs in test versus vehicle controls were analysed using the Kastenbaum and Bowman method (19Go).


   Results
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 Materials and methods
 Results
Discussion
 Funding
 References
 
Table I shows the results of the liver MN assay in F344 rats. The mean incidence (±standard deviation) of MNHEPs was 0.08 ± 0.07% in the solvent control group of 37 rats following a single dosing and 0.18 ± 0.18% in 28 rats following a double dosing. 5FU did not induce a statistically significant increase in % MNHEPs in a single dosing, but it did induce a marked increase in micronucleated cells in the PB MN assay (Table II). Both MMC and 1,2-DMH·2HCl significantly increased the incidence of MNHEPs. CP, an indirect mutagen was only weakly positive, although it induced a marked increase in micronucleated cells in the PB MN assay (Table II). 2,4-DNT, another indirect mutagen, also induced a statistically significant increase in % MNHEPs. EMS was negative by both the intraperitoneal and oral routes. 2AAF was slightly positive at 500 and 750 mg/kg in the single-dose assay, but induced a higher incidence of MNHEPs at 500 mg/kg in the two-dose assay. 2,4-DAT induced a statistically significant increase in MNHEPs in the single- and two-dose assays, and the magnitude of the quantitative response was not markedly dependent upon the number of doses administered. Quinoline induced a statistically significant increase in the incidence of MNHEPs in the two-dose assay. DAB at 150 mg/kg induced a statistically significant increase in the incidence of MNHEPs in both the single- (Figure 1D) and two-dose assays (Table I). DMN was positive in the two-dose assay. Marked decreases in the mitotic index (MI, %) were seen with EMS, 2AAF and 2,4-DAT (Table I). MNHEP results were qualitatively similar for both the F344 and SD rats (Figure 1).


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  		Table I. Results of the liver MN assay in rats

 


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  		Table II. Results of the PB MN assay in rats

 


Figure 1
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  		Fig. 1. Incidence of MNHEPs (%) and MI (%) 3–6 days after a single administration of DEN (A), DMN (B), 2,4-DAT (C), DAB (D), quinoline (E) and 2AAF (F, G) to F344 and SD rats. *P < 0.05, **P < 0.01, Kastenbaum and Bowman test.

 

   Discussion
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 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
The low incidence of MNHEPs (%) in solvent control rats suggests the robustness of this assay, as previously reported (13Go). 2AAF, 2,4-DAT, MMC, 1,2-DMH·2HCl and 2,4-DNT were judged positive for MN-inducing potential in the present study. Although CP significantly increased MNHEP incidence, the increase was small. CP was negative in an earlier published liver MN assay using young rats and in vivo/in vitro UDS assays (9Go,20Go). The increase in MN frequency in the liver after CP treatment was weak to equivocal and requires further independent evaluation. 2AAF, an indirect mutagen, is positive in the bacterial reverse mutation assay (Ames assay) through metabolic activation (21Go). It is weakly positive in the absence of metabolic activation and more strongly positive in the presence of metabolic activation in the in vitro chromosomal aberration (CA) assay (22Go) and positive in the rat bone marrow MN assay and the in vivo/in vitro UDS assay (14Go,23Go). 2AAF is also carcinogenic in rats, producing liver and urinary bladder tumours (21Go). It is converted by N-hydroxylation by CYP1A2, and the metabolites, which are further metabolised by sulphotransferase and other enzymes, are thought to react with DNA (21Go). Since young rats show CYP1A2 activity (24Go), it is likely that 2AAF metabolites damage hepatocyte DNA, resulting in MN induction. 2,4-DAT is positive in the Ames assay with metabolic activation and in the in vitro CA assay without activation (25Go,26Go). It is negative in the mouse PB MN assay (1Go) and questionably positive in the rat bone marrow MN assay (25Go). It is weakly positive in the in vivo/in vitro UDS assay (23Go,25Go) and carcinogenic to rat liver, where it binds covalently to DNA (27Go). The positive response in the present study may indicate that bone marrow and liver assays are particularly useful for the detection of the in vivo genotoxic effects of 2,4-DAT, by providing the necessary metabolic activation pathways. In fact, clear positive responses for DAB in the rat were detected in the liver MN assay but not in the bone marrow MN assay (28Go). MMC induced MNHEPs at incidences of ≥1% and was judged to be positive for MN-inducing potential. MMC, which was positive in this study is strongly positive in the in vitro CA assay at low dose levels (22Go), mostly due to cross-linking (29Go), and is positive in the liver MN assay using young rats. 1,2-DMH is directly positive in the Ames assay and in the in vitro CA assay (22Go,30Go) as well as in the mouse and rat bone marrow MN assay and the in vivo/in vitro UDS assay (23Go,31Go,32Go). It binds covalently to protein, DNA and RNA in many tissues (33Go). The positivity of 1,2-DMH in the present study is consistent with these data. 1,2-DMH is also carcinogenic to mice and rats in many tissues including liver (33Go). 2,4-DNT is positive in the Ames assay (34Go), but negative in the in vitro CA assay (22Go), and it is negative in the mouse bone marrow MN assay (35Go) but positive in the in vivo/in vitro UDS assay (23Go). 2,4-DNT is carcinogenic in the liver and hepatotoxic as shown by serum liver enzyme elevation (36Go). Nitro-reduction by gut flora through the enterohepatic circulation is an important step for genotoxicity of this compound. Therefore, it is likely that a compound with that kind of metabolic requirements would be less active when tested in vitro using standard methods of metabolic activation, i.e. with S9-mix. In the present study, active metabolites generated through nitro-reduction by gut flora probably damaged hepatocyte DNA, resulting in a positive response. The young rat liver MN assay may be a useful in vivo assay for compounds like 2,4-DNT that target the liver, as with the in vivo/in vitro UDS assay. EMS was negative in the present study, as has been reported for other types of liver MN assays (37Go), probably due to the rapid disappearance of DNA damage (38Go). 5FU was also negative in the present assay. 5FU is a potent inhibitor of thymidylate synthetase and inhibits DNA synthesis (39Go). Since the MI of bone marrow is several times higher than that of the liver, one of the reasons 5FU did not induce MNHEPs may be the comparatively low level of mitotic activity in the liver (9Go). 5FU is also negative in the in vivo comet assay using mice (40Go). There were some marked decreases, some slight increases and some large increases in MI observed for specific chemicals. This result indicates that the assay was performed on animals with dividing hepatocytes. Quinoline, DAB and DMN, which had been judged positive following single injection in a previous study (13Go), also showed positive results following two dosings. 2AAF and DMN showed a clear tendency to increase the incidence of % MNHEPs, as the dosings increased (Table I and Figure 1B). These results suggest that a more significantly positive result may be obtained using multiple dosing, if a single-dose protocol generates only weak to equivocal results. Decreases of % MNHEPs with time (single dosings of 2,4-DNT at 200 mg/kg and 2,4-DAT at 250 mg/kg ; double dosings of 2,4-DAT at 62.5 and 125 mg/kg and DAB at 150 mg/kg) were probably due to inhibition or delayed cell proliferation or cell death due to chemical cytotoxicity (37Go,41Go). Our observation of a time-dependent increase in MI% exceeding the control value following a single dose of 2,4-DAT at 250 mg/kg was consistent with the compound's effect on cell proliferation, which may be related to its mutagenicity or carcinogenicity (42Go,43Go). In addition, we observed no correlation between % MNHEPs and MI% values in this assay.

The chemicals that induced MNHEPs in F344 rats also induced them in SD rats, indicating that SD rats are also suitable for the liver MN assay, though the number of chemicals tested in SD rats is small. The incidence of MNHEPs tended to be higher in F344 rats treated with DAB and in SD rats treated with quinoline. These results may be related to strain differences in drug-metabolising enzyme activity (44Go–46Go).

The chemicals judged positive in the present assay were rodent hepatocarcinogens and the results are considered to be related either to the generation of reactive metabolites as a consequence of enzymatic activation or that the compounds are direct-acting mutagens. In young rats, the expression of CYP450 species, namely CYP1A2, CYP2A1, 2B1, 2B2, 2E1, 3A1 and 3A2, was confirmed, but that of 2C7, 2C11, 2C12 and 2C22 was not confirmed (24Go). An assessment of a test result should be made in consideration of the expression pattern of CYP species. In addition to expressing enzymes that catalyse phase I reactions, young rats express enzymes that catalyse phase II reactions. Uridinediphosphate glucuronosyltransferase activity, e.g. increases rapidly between 18 and 25 days after birth, and then remains unchanged until 60 days (47Go). Selenium-dependent glutathione peroxidase and non-protein sulfhydryl activity are also expressed between 18 and 60 days after birth (47Go).

From these results, we conclude that this technique would be appropriate for detecting MN induction in vivo in the liver, taking into account previous liver MN assay results (13Go) and the characteristics of each chemical that yielded positive and/or negative responses. If the liver MN assay is carried out in combination with the PB MN assay, more useful information could be obtained by comparing MN incidences in two different tissues, as demonstrated by the results obtained with CP and 5FU in this paper.


   Funding
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 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
Japanese Environmental Mutagen Society (JEMS)-Mammalian Mutagenicity Study Group (MMS)


   Acknowledgments
 
This article was communicated by the Mammalian Mutagenicity Study Group of the Environmental Mutagen Society of Japan.


   Notes
 
* To whom correspondence should be addressed. Tel: +81 265 73 8611; Fax: +81 265 73 8612; Email: h-suzuki{at}ina-research.co.jp


   References
Top
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received on May 16, 2008; revised on July 28, 2008; accepted on July 29, 2008.


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