Repeated dose liver and gastrointestinal tract micronucleus assays using N-methyl-N′-nitro-N-nitrosoguanidine in young adult rats

Mutation Research 780-781 (2015) 100–106

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Repeated dose liver and gastrointestinal tract micronucleus assays using N-methyl-N -nitro-N-nitrosoguanidine in young adult rats Tomomi Takayanagi a , Yumi Wako b , Kazufumi Kawasako b , Hisako Hori a , Wataru Fujii a , Wakako Ohyama c,∗ a

Suntory Business Expert Limited, 1-1-1 Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618-8503, Japan LSI Medience Corporation, 14-1 Sunayama, Kamisu-shi, Ibaraki 314-0255, Japan c Yakult Honsha Co., Ltd., 1796 Yaho, Kunitachi-shi, Tokyo 186-8650, Japan b

a r t i c l e

i n f o

Article history: Received 25 December 2014 Accepted 28 December 2014 Available online 4 January 2015 Keywords: Micronucleus assay Repeated dose study Liver Glandular stomach Colon N-methyl-N -nitro-N-nitrosoguanidine (MNNG)

a b s t r a c t N-Methyl-N -nitro-N-nitrosoguanidine (MNNG) is a direct-acting mutagen that induces tumors in the glandular stomach, but not in the liver or colon, of rats after oral administration. To evaluate the performance of repeated dose liver and gastrointestinal tract micronucleus (MN) assays in young adult rats, MNNG was administered by oral gavage to male CD (SD) rats aged 6 weeks at doses of 0 (vehicle; 2.5% DMSO aqueous solution), 3.125, 6.25, 12.5, and 25 mg/kg/day once daily for 14 and 28 days, and the MN frequencies were examined in the hepatocytes, glandular stomach cells, and colonic cells. The MN induction in immature erythrocytes in the bone marrow of these animals was also simultaneously evaluated. The frequencies of micronucleated (MNed) glandular stomach cells were significantly increased in all MNNG treatment groups in a dose-dependent manner in both repeated dose studies. In contrast, the frequencies of MNed hepatocytes and colonic cells were not significantly increased compared to the vehicle control. In the bone marrow, a small but significant increase in the frequency of MNed immature erythrocytes was observed only at the highest dose in the 28-day study. Since a clear positive result in the glandular stomach agrees with the tissue specificity of tumor induction by this chemical, the MN assay with the glandular stomach, which is a direct contact site with high concentrations of test substances administered by oral gavage, may be useful for detecting genotoxic compounds that are short-lived in vivo, such as MNNG. © 2015 Elsevier B.V. All rights reserved.

1. Introduction A repeated dose liver micronucleus (MN) assay with young adult rats was recently developed, and this assay can detect several hepatocarcinogens that yield negative results in the bone marrow MN assay [1,2]. This test method can be integrated into repeated dose general toxicological studies because no specific treatment is necessary for the MN analysis, which helps to reduce the number of experimental animals under the principle of the 3Rs (replacement, reduction and refinement of animal studies). MN assays with rat glandular stomach and colon have also been developed, and these assays can detect typical stomach- and colon-targeted carcinogens following single or quadruple dosing [3,4]. The liver is a primary tissue for metabolism, and the gastrointestinal (GI) tract is a direct contact site with compounds administered by oral gavage.

∗ Corresponding author. Tel.: +81 42 577 8960; fax: +81 42 577 3020. E-mail address: [email protected] (W. Ohyama). http://dx.doi.org/10.1016/j.mrgentox.2014.12.009 1383-5718/© 2015 Elsevier B.V. All rights reserved.

Additionally, these tissues are common sites of cancer development, which is associated with a high incidence and/or mortality in humans [5]. Therefore, these are important target tissues for risk assessments of chemicals. Verifying the effectiveness of both the liver and GI tract MN assays following a long-term repeated dosing allows the simultaneous assessment of the genotoxic effects of a chemical in multiple tissues, including the bone marrow of the same animal. Moreover, the MN assays with these tissues (some or all of them as required) can be integrated into a repeated dose general toxicological study, which is useful to obtain information on both the genotoxicity and general toxicity and will help to more accurately assess the risk of the chemical. A collaborative study was conducted by the Mammalian Mutagenicity Study (MMS) Group, which is a subgroup of the Japanese Environmental Mutagen Society (JEMS), to evaluate the performance of the repeated dose liver and GI tract MN assay in young adult rats using several chemicals, including hepatocarcinogens and non-liver-targeted genotoxic carcinogens. As a part of the collaborative study, we performed 14and 28-day repeated-oral-dose MN assays with the liver, glandular

T. Takayanagi et al. / Mutation Research 780-781 (2015) 100–106

stomach, colon and bone marrow of rats using N-methyl-N -nitroN-nitrosoguanidine (MNNG). MNNG is a direct-acting mutagen that induces tumors at the direct contact sites of rodents [6,7]. When MNNG is orally administered to rats, it causes tumors primarily in the glandular stomach. Tumors are also often observed in the upper parts of the GI tract, e.g., the esophagus, forestomach and small intestine, but rarely in the liver or other tissues [8–10]. Regarding the in vivo genotoxicity, MNNG yields a positive result in the unscheduled DNA synthesis (UDS) assay using the glandular stomach of rats after oral exposure [11]. MNNG also elicits positive responses in the glandular stomach, but not in the liver or bone marrow in the gene mutation assay with MutaTM mice [12]. This chemical also generates positive results in the MN assays in the glandular stomach after short-term oral treatment, but not in the bone marrow of mice/rats [3,4,13]. In the present study, we administered MNNG to rats via oral gavage for 14 and 28 days and examined the MN frequencies in the hepatocytes (HEPs), glandular stomach epithelial cells (GSCs), colonic epithelial cells (COCs), and immature erythrocytes (IMEs) of the bone marrow. 2. Materials and methods 2.1. Chemicals MNNG (CAS No. 70-25-7) was purchased from Sigma Chemical Co., Ltd. (St. Louis, MO) and dissolved in a 2.5% aqueous solution of dimethyl sulfoxide (DMSO) at a concentration of 2.5 mg/mL. The solution was diluted to 1.25, 0.625, and 0.313 mg/mL with a 2.5% DMSO solution and administered to rats immediately after preparation. 2.2. Animals Male Crl:CD (SD) rats were purchased from Charles River Japan Inc. (Shiga, Japan) and acclimated for 5–8 days. The animals were 6 weeks of age and weighed approximately 180–230 g at the beginning of the experiments. Two or three animals were housed in each plastic cage with wood-chip bedding in an air-conditioned room under constant temperature (21–25 ◦ C) and humidity (45–65%) with a 12-h light/dark cycle. They were allowed free access to food and drinking water. All protocols were approved by the Animal Care and Use Committee of Suntory Holdings, Ltd. and the experiments followed the guidelines of the above-mentioned company. 2.3. Dose levels, treatment and clinical observation A preliminary study was conducted to determine the dose levels of MNNG for the MN assays. Based on the literature of the short-term treatment study [4], MNNG was administered to rats by oral gavage at doses of 25, 50 and 100 mg/kg/day for 4 days. During the dosing period, the 100 mg/kg/day group experienced body weight loss, while the body weight did not increase in the 50 mg/kg/day group. A decrease in body weight gain was observed in the animals treated with 25 mg/kg/day compared to the nontreated controls after the second dose. The mean body weights on day 5 (the day after the last dosing) in the 100, 50, and 25 mg/kg/day groups were approximately 23%, 12%, and 5% lower than that in the control group, respectively. Therefore, 25 mg/kg/day was selected as the maximum tolerated dose level for these longer duration MN assays. The lower doses were 12.5, 6.25 and 3.125 mg/kg/day. All animals were administered the test chemical solution or vehicle (2.5% DMSO) by oral gavage in a volume of 10 mL/kg once daily for 14 or 28 days. Five animals were randomly assigned to each control or treatment group, and they were weighed daily prior to dosing and on

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the necropsy day for both the 14- and 28-day studies. Clinical signs were observed once daily. Approximately 24 h after the final dosing, the animals were euthanized by exsanguination from the abdominal aorta under anesthesia with pentobarbital sodium (Kyoritsu Seiyaku Corporation, Tokyo, Japan) following overnight fasting, and the liver, glandular stomach, colon, and femur were excised. The liver was weighed. 2.4. MN assays MN assays using the liver, glandular stomach, colon, and bone marrow were performed according to the protocol described in the summary report of this collaborative study [14]. Briefly, approximately 1 g of the left lateral lobe was sliced and treated with a digestion solution containing 100 units/mL collagenase (Collagenase Yakult-S, Yakult Pharmaceutical Industry, Co., Ltd., Tokyo, Japan) to isolate the HEPs. The glandular stomach and colon were each everted on a glass rod and then incubated in a tube with ethylenediaminetetraacetic acid disodium salt (EDTA) solution to isolate the epithelial cells. The solution contained 1 mM EDTA and 2 mM dithiothreitol in Mg2+ /Ca2+ -free Hanks’ balanced salt solution (HBSS) for the stomach and 1 mM EDTA in Mg2+ /Ca2+ -free HBSS for the colon. The cells collected from the liver, glandular stomach, and colon were fixed with 10% neutral-buffered formalin and refrigerated until analysis. Both ends of the femur were cut, and the bone marrow cells were then collected by washing the femur cavity with fetal bovine serum (FBS) (PAA laboratories Inc. ON, Canada) [15]. After centrifugation, the collected cells were re-suspended in FBS and smeared on a glass slide. The smears were dried, fixed with methanol and stored until analysis. Immediately prior to scoring, the cells were stained with a staining solution containing 500 ␮g/mL acridine orange (AO) and 10 ␮g/mL 4 ,6-diamidino-2-phenylindole dihydrochloride (DAPI) for the liver, 50 ␮g/mL AO and 2.5 ␮g/mL DAPI for the glandular stomach, 125 ␮g/mL AO and 2.5 ␮g/mL DAPI for the colon, and 40 ␮g/mL AO for the bone marrow. The glandular stomach staining solution was also used for colon samples that contained few cells. The slides were coded, and 2000 intact HEPs, GSCs, COCs, and IMEs were scored per tissue per animal to determine the frequency of micronucleated (MNed) cells using a fluorescence microscope (magnification: 400×; liver, 600×; glandular stomach, colon, and bone marrow) with UV excitation (365 nm) for the liver, glandular stomach, and colon and blue excitation (450–490 nm) for the bone marrow. In addition, the number of mitotic phase cells out of 2000HEPs, and the number of IMEs out of 500 erythrocytes were counted for each animal to determine the mitotic index (MI) and percentage of IMEs to total erythrocytes (%IMEs), respectively. 2.5. Pathological examination Internal organs were examined macroscopically. The histopathological examination of the liver, which was required in this collaborative study, was performed. Additionally, we histopathologically examined the glandular stomach which was the target tissue of MNNG. The liver (left lateral lobe) and stomach were fixed with 10% neutral buffered formalin, embedded in paraffin, and then sectioned. The sections were deparaffinized and stained with hematoxylin and eosin according to standard methods. Histopathological examination of the liver and glandular stomach was performed under a light microscope by LSI Medience Corporation and Suntory Business Expert Limited, respectively. For examination of the glandular stomach in the 14-day study, only the animals receiving the highest dose of MNNG and vehicle were examined.

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B. 28-day

450

450

400

400

350

350

300

300

250 *

** **

200 150

** **

** **

Control (2.5% DMSO) MNNG 3.125mg/kg MNNG 6.25mg/kg MNNG 12.5mg/kg MNNG 25mg/kg

100 50

Body weight (g)

Body weight (g)

A. 14-day

**

250 * 200

** ** ** ** ** ** ** ** ** ** ** * ** ** ** ** ** ** ** **

150 Control (2.5% DMSO) MNNG 3.125mg/kg MNNG 6.25mg/kg MNNG 12.5mg/kg MNNG 25mg/kg

100 50

0

0

1

2

3

4

5

6

7

8

9

10

11 12

13 14

1

2

3

4

5

6

7

8

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

Day

Day

Fig. 1. Changes in the body weight of rats treated with MNNG for 14 (A) or 28 (B) days. The data represent the mean ± SD. *P < 0.05, **P < 0.01; significant difference from the control (Dunnett’s test).

2.6. Statistical analysis

statistical analysis was performed with either SAS (Ver. 9.3) or JMP (Ver. 5).

Differences in the frequencies of micronucleated hepatocytes (MNHEPs), micronucleated glandular stomach cells (MNGSCs), micronucleated colonic cells (MNCOCs), and micronucleated immature erythrocytes (MNIMEs) between the MNNG and vehicle control groups were statistically analyzed using Kastenbaum–Bowman tables [16]. The dose–response relationships in the frequencies of MNGSCs were analyzed with the Cochran–Armitage trend test. Body weight and liver weight (absolute and relative) data were statistically analyzed using Bartlett’s test for the homogeneity of variance, followed by a one-way analysis of variance for homogeneous data or Kruskal–Wallis test for heterogeneous data. When a significant difference was observed between groups, Dunnett’s test or the Dunnett-type multiple comparison test was conducted. The

3. Results 3.1. Clinical signs and body weight Decreases in locomotor activity and salivation were observed in the MNNG-treated animals at doses of 12.5 mg/kg/day or more approximately 1 week after the initiation of dosing in both the 14and 28-day studies. The animals in the highest dose group in the 28day study appeared pale starting on day 24 and were emaciated at the end of the dosing period. The mean body weight of the highest dose group was significantly lower than the vehicle control group approximately 1 week after the initiation of dosing in both the 14and 28-day studies (Fig. 1).

Table 1 Frequency of MNed cells and cell proliferation in the liver and bone marrow after MNNG administration for 14 and 28 days. Dosing period

14 days

28 days

Dose (mg/kg/day)

No. of animals

0

5

3.125

5

6.25

5

12.5

5

25

5

0

5

3.125

5

6.25

5

12.5

5

25

5

Liver

Bone marrow

MNHEPs (%) Mean ± SD (Individual data)

MI (%) Mean ± SD (Individual data)

MNIMEs (%) Mean ± SD (Individual data)

%IMEs Mean ± SD (Individual data)

0.07 ± 0.04 (0.15, 0.05, 0.05, 0.05, 0.05) 0.07 ± 0.04 (0.00, 0.05, 0.10, 0.10, 0.10) 0.05 ± 0.05 (0.05, 0.10, 0.00, 0.00, 0.10) 0.05 ± 0.04 (0.05, 0.10, 0.05, 0.00, 0.05) 0.14 ± 0.07 (0.20, 0.15, 0.20, 0.10, 0.05)

0.06 ± 0.02 (0.05, 0.05, 0.10, 0.05, 0.05) 0.05 ± 0.05 (0.10, 0.00, 0.10, 0.00, 0.05) 0.05 ± 0.04 (0.05, 0.10, 0.05, 0.05, 0.00) 0.05 ± 0.05 (0.00, 0.05, 0.10, 0.00, 0.10) 0.07 ± 0.08 (0.15, 0.05, 0.00, 0.15, 0.00)

0.21 ± 0.15 (0.35, 0.15, 0.35, 0.20, 0.00) 0.20 ± 0.18 (0.05, 0.20, 0.15, 0.50, 0.10) 0.21 ± 0.08 (0.10, 0.30, 0.15, 0.25, 0.25) 0.20 ± 0.13 (0.15, 0.15, 0.05, 0.40, 0.25) 0.14 ± 0.09 (0.05, 0.15, 0.05, 0.25, 0.20)

59.7 ± 8.1 (49.4, 56.8, 71.8, 59.8, 60.6) 57.5 ± 1.8 (55.4, 58.4, 56.4, 57.4, 60.0) 59.2 ± 2.7 (61.0, 60.6, 61.2, 58.0, 55.0) 56.5 ± 3.6 (53.6, 58.2, 57.0, 52.4, 61.2) 51.7 ± 6.1 (55.8, 47.6, 43.2, 57.8, 54.2)

0.03 ± 0.07 (0.00, 0.15, 0.00, 0.00, 0.00) 0.06 ± 0.04 (0.00, 0.05, 0.05, 0.10, 0.10) 0.07 ± 0.03 (0.05, 0.05, 0.10, 0.05, 0.10) 0.04 ± 0.04 (0.00, 0.05, 0.10, 0.05, 0.00) 0.03 ± 0.04 (0.00, 0.00, 0.10, 0.05, 0.00)

0.06 ± 0.05 (0.05, 0.00, 0.05, 0.15, 0.05) 0.08 ± 0.12 (0.15, 0.25, 0.00, 0.00, 0.00) 0.02 ± 0.03 (0.00, 0.05, 0.05, 0.00, 0.00) 0.05 ± 0.04 (0.05, 0.00, 0.10, 0.05, 0.05) 0.03 ± 0.04 (0.00, 0.00, 0.00, 0.10, 0.05)

0.18 ± 0.09 (0.20, 0.20, 0.05, 0.30, 0.15) 0.25 ± 0.07 (0.30, 0.15, 0.30, 0.20, 0.30) 0.19 ± 0.10 (0.15, 0.10, 0.15, 0.35, 0.20) 0.29 ± 0.10 (0.45, 0.20, 0.25, 0.30, 0.25) 0.37 ± 0.08* (0.30, 0.45, 0.45, 0.35, 0.30)

55.3 ± 3.1 (50.4, 55.6, 55.6, 59.1, 56.0) 55.9 ± 5.4 (50.0, 50.4, 61.8, 58.7, 58.6) 52.0 ± 6.7 (42.8, 53.8, 58.8, 57.0, 47.4) 55.6 ± 3.1 (59.4, 56.2, 57.2, 51.4, 53.8) 64.4 ± 1 2.0 (60.3, 58.0, 85.8, 58.8, 59.2)

MNHEPs: micronucleated hepatocytes; MI: mitotic index; MNIMEs: micronucleated immature erythrocytes; IMEs: immature erythrocytes. * P < 0.01; significant difference from the control (Kastenbaum and Bowman test).

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Table 2 Frequency of MNed cells in the glandular stomach and colon after MNNG administration for 14 and 28 days. Dosing period

14 days

28 days

Dose (mg/kg/day)

No. of animals

0

5

3.125

5

6.25

5

12.5

5

25

5

0

5

3.125

5

6.25

5

12.5

5

25

5

Glandular stomach

Colon

MNGSCs (%) Mean ± SD (Individual data)

MNCOCs (%) Mean ± SD (Individual data)

0.11 ± 0.05 (0.20, 0.10, 0.10, 0.10, 0.05) 0.29 ± 0.14** (0.35, 0.20, 0.45, 0.10, 0.35) 0.41 ± 0.20** (0.35, 0.20, 0.65, 0.25, 0.60) 0.61 ± 0.31** (0.55, 0.40, 0.25, 0.90, 0.95) 0.85 ± 0.62** (1.75, 0.20, 0.45, 1.20, 0.65)

0.14 ± 0.08 (0.10, 0.10, 0.05, 0.20, 0.25) 0.23 ± 0.10 (0.20, 0.40, 0.20, 0.20, 0.15) 0.24 ± 0.04 (0.30, 0.25, 0.20, 0.20, 0.25) 0.23 ± 0.04 (0.30, 0.20, 0.20, 0.20, 0.25) 0.20 ± 0.15 (0.00, 0.40, 0.25, 0.15, 0.20)

0.06 ± 0.07 (0.00, 0.10, 0.05, 0.15, 0.00) 0.15 ± 0.09* (0.10, 0.10, 0.10, 0.15, 0.30) 0.23 ± 0.19** (0.10, 0.10, 0.20, 0.20, 0.55) 0.32 ± 0.19** (0.55, 0.10, 0.45, 0.35, 0.15) 0.44 ± 0.19** (0.50, 0.25, 0.50, 0.25, 0.70)

0.31 ± 0.14 (0.20, 0.40, 0.50, 0.15, 0.30) 0.26 ± 0.19 (0.15, 0.10, 0.35, 0.55, 0.15) 0.34 ± 0.15 (0.40, 0.35, 0.20, 0.20, 0.55) 0.26 ± 0.16 (0.30, 0.10, 0.25, 0.50, 0.15) 0.27 ± 0.14 (0.45, 0.35, 0.10, 0.25, 0.20)

MNGSCs: micronucleated glandular stomach cells; MNCOCs: micronucleated colonic cells. P < 0.05. ** P < 0.01; significant difference from the control (Kastenbaum and Bowman test).

*

3.2. Liver MN assay

The frequencies of MNGSCs were significantly increased in all MNNG treatment groups compared to the vehicle control in both the 14- and 28-day studies (Table 2), and the increases were dosedependent (P < 0.01, Cochran–Armitage trend test). The mean value of each treatment group in the 28-day study was nearly half the value of the corresponding dose group in the 14-day study.

observed in the MNNG-treated animals at doses of 12.5 mg/kg/day or more, and the small seminal vesicle was seen at the highest dose. A significant increase in the relative liver weight was observed in the animals that received the highest dose of MNNG compared to the controls, even though the absolute liver weight did not significantly change in either study (Table 3). In the histopathological examination, minimal hepatocyte hypertrophy was seen in 3 of 5 animals at the highest dose, and minimal to mild inflammatory cell infiltration in the perilobular region of the liver was observed in the majority of animals at doses of 12.5 mg/kg/day or more, with a dose dependent increase in incidence and severity in the 14-day study (Table 4). In the 28-day study, mild centrilobular hepatic single cell necrosis was noted in an animal in the highest dose group, and minimal inflammatory cell infiltration in the perilobular region of the liver was observed in a few animals that received doses of 12.5 mg/kg/day or more (Table 4). In the glandular stomach, minimal to severe erosion/ulcers, and minimal to moderate inflammatory cell infiltration and fibrosis were noted in the majority of animals at the highest dose in the 14-day study (Table 5). In the 28-day study, minimal to mild erosion/ulcers and mild to severe fibrosis were observed in all animals at doses of 12.5 mg/kg or more. Minimal to mild erosion and fibrosis were also present in a few animals that received 6.25 mg/kg/day MNNG (Table 5).

3.5. Colon MN assay

4. Discussion

There was no significant difference in the frequency of MNCOCs between the vehicle control and MNNG treatment groups in both the 14- and 28-day studies (Table 2).

In the present study, we administered a stomach carcinogen, MNNG, to rats via oral gavage for 14 and 28 days and examined the frequencies of MNHEPs, MNGSCs, MNCOCs and MNIMEs. In both treatment periods, significant and clear dose-dependent increases in the frequencies of MNed cells were observed only in the glandular stomach of the rats. MNNG is a direct-acting mutagen that produces positive results in the bacterial reverse mutation test and in vitro chromosomal aberration test without metabolic activation [17]. After short-term oral treatment, MNNG induced DNA damage and micronuclei in rat

There was no significant difference in the frequency of MNHEPs and the MI value between the vehicle control and MNNG treatment groups in both the 14- and 28-day studies (Table 1). 3.3. Bone marrow MN assay The frequencies of MNIMEs and %IMEs in the MNNG-treated rats were similar to the controls in the 14-day study (Table 1). In the 28-day study, a small but significant increase in the frequency of MNIMEs was observed only at the highest dose of MNNG compared to the vehicle control. The %IMEs did not significantly differ between the vehicle control and MNNG treatment groups, although the %IMEs tended to increase in the highest dose group. 3.4. Glandular stomach MN assay

3.6. Pathological examination At necropsy, distention of the stomach and colon, and small seminal vesicle were observed in the animals receiving the highest dose of MNNG in the 14-day study. In the 28-day study, a watery content in the stomach and colon as well as swelling of the liver were

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Table 3 Liver weight after MNNG administration for 14 and 28 days.

*

Dosing period

Dose (mg/kg/day)

Body weight (g)a

Liver weighta Absolute weight (g)

Relative weight (%)

14 days

0 3.125 6.25 12.5 25

281.6 ± 11.9 288.9 ± 15.5 286.9 ± 9.9 261.8 ± 16.1 197.4 ± 13.5**

9.3 ± 0.5 9.4 ± 0.8 9.6 ± 0.3 8.8 ± 0.9 9.4 ± 0.8

3.29 ± 0.08 3.24 ± 0.18 3.26 ± 0.06 3.35 ± 0.15 4.78 ± 0.52*

28 days

0 3.125 6.25 12.5 25

351.4 ± 33.8 354.8 ± 14.1 361.8 ± 46.2 327.6 ± 23.7 217.1 ± 22.8**

10.6 ± 1.5 11.1 ± 1.1 11.5 ± 2.0 10.4 ± 1.5 9.3 ± 0.9

3.02 ± 0.20 3.13 ± 0.24 3.18 ± 0.15 3.17 ± 0.24 4.29 ± 0.24**

P < 0.05. ** P < 0.01; significant difference from the control (Dunnett’s test). a The data represent the mean ± SD.

Table 4 Histopathological findings in the liver of rats after MNNG administration for 14 and 28 days. Findings

Dosing period

14 days

Dose (mg/kg/day) No. of animals

0 5

28 days

3.125 5

6.25 5

12.5 5

25 5

0 5

3.125 5

6.25 5

12.5 5

25 5

Hypertrophy of hepatocytes, perilobular

Grade +

0

0

0

0

3

0

0

0

0

0

Inflammatory cell infiltration, perilobular

+ ++

0 0

0 0

0 0

3 1

0 5

0 0

0 0

0 0

1 0

2 0

Single cell necrosis of hepatocytes, centrilobular

+ ++

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 1

Grade: +, minimal; ++, mild.

glandular stomach cells in the UDS assay and the MN assay, respectively [3,4,11]. Our results in the glandular stomach are consistent with the positive results of these in vivo genotoxicity studies. We used 2.5% DMSO as a vehicle in the present study and showed that the vehicle did not induce MNed cells in the tissues examined. Namely, the mean vehicle control values of MNHEPs,

MNGSCs, and MNCOCs in both the 14- and 28-day studies were within the mean ± 3SD in the negative controls of 8-week-old rats administered water by oral gavage (MNHEPs, 0.06 ± 0.21%; MNGSCs, 0.09 ± 0.24%; MNCOCs, 0.11 ± 0.27%; unpublished data); these latter data were obtained in the collaborative trial to confirm the inter-laboratory reproducibility [14], and the MNIME data

Table 5 Histopathological findings in the glandular stomach of rats after MNNG administration for 14 and 28 days. Dosing period

14 daysa

Dose (mg/kg/day) No. of animals

0 5

25 5

0 5

3.125 5

6.25 5

12.5 5

25 5

+ ++

0 0

1 0

0 0

0 0

1 0

1 2

0 0

Ulcer

+ ++ +++ ++++

0 0 0 0

1 1 0 1

0 0 0 0

0 0 0 0

0 0 0 0

1 1 0 0

2 2 1 0

Fibrosis, mucous neck

+ ++ +++ ++++

0 0 0 0

1 3 1 0

0 0 0 0

0 0 0 0

1 1 0 0

0 4 1 0

0 3 1 1

Inflammatory cell infiltration, neutrophilic, eosinophilic

+ ++

0 0

1 3

0 0

0 0

0 0

4 1

5 0

+ ++ +++

0 0 0

2 2 1

0 0 0

0 0 0

0 0 0

1 0 0

4 0 0

+ ++ +++

0 0 0

0 0 0

0 0 0

1 0 0

1 1 1

0 5 0

3 2 0

Findings

28 days

Grade Mucosal tissue Erosion

Submucosal tissue Inflammatory cell infiltration, neutrophilic, eosinophilic

Inflammatory cell infiltration, lymphocytic

Grade: +, minimal; ++, mild; +++, moderate; ++++, severe. a Animals in the MNNG treatment groups at the doses of 3.125–12.5 mg/kg/day were not analyzed in the 14-day study.

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were within the range of the negative controls in the testing facility. No histopathological changes were observed in the glandular stomach and liver of the vehicle controls, which demonstrates that a repeated dose of 2.5% DMSO did not exert a toxic effect on these tissues. These results are consistent with a study that showed no increases in the frequencies of the MNGSCs, MNCOCs and MNIMEs in rats treated with 5% DMSO by oral gavage for 4 days [4] and with the report that showed no adverse effects of DMSO when rats were administered 50% DMSO via a stomach tube at a dose level equivalent to 2000 mg undiluted DMSO/kg/day for 45 days [18]. In the glandular stomach of the animals that received doses of 6.25 mg/kg/day or more, several histopathological changes were observed, such as minimal to severe erosion/ulcers, inflammatory cell infiltration and fibrosis. However, the individual values of the MNGSCs frequency in those groups did not correlate with the severity of the histopathological changes. In addition, histopathological changes were not observed in the animals treated with the lowest dose (3.125 mg/kg/day), while the frequencies of MNGSCs were significantly increased. These findings suggest that the increases in the frequencies of MNGSCs were due to the direct genotoxic activity of this chemical and not due to tissue toxicity. In contrast, the frequencies of MNed cells in the colon of the MNNG-treated rats were not increased compared to the vehicle controls in both the 14- and 28-day studies. These findings are consistent with the negative result obtained from the 4-day treatment study [4]. In the liver, there was no significant difference in the frequency of MNHEPs between the vehicle control and MNNG treatment groups in both the 14- and 28-day studies. However, some histopathological changes, such as inflammation, were observed in the liver of rats in the 2 higher dose groups. Additionally, the blood chemistry data, which were obtained from 4 animals per group in the 28-day study, showed a significant increase in ALT and tendency for ALP and ␥-GTP to increase (Supplementary data; Appendix 1) in the animals that received the highest dose as compared to the vehicle controls. Both of these results indicated that liver injury was induced in the animals. Besides the stomach, the liver of rats is reportedly the most prominent site for histopathological lesions as a result of a single dose of MNNG (ca. 150 mg/kg; a sesame oil suspension) by oral gavage [19]. In addition, adverse effects were not observed in the vehicle controls in the present study. Therefore, the liver lesions observed in our study were attributed to the toxicity of MNNG. Interestingly, the histopathological changes in the liver were milder and the incidence was lower in the 28-day study compared to the 14-day study. The reason for this discrepancy is unclear but may be the adaptation to the toxic effects of MNNG in the liver. On the other hand, the absolute and relative liver weights as well as the MI values did not decrease at any doses, suggesting that cell proliferation in the liver was not depressed. These results indicate that MNNG exerts toxic effects, but not genotoxic effects, on rat livers after oral exposure. The present negative results are consistent with reports indicating that MNNG yielded a negative result in a gene mutation assay with MutaTM mice [11] and rarely induced liver tumors in rat carcinogenicity studies [7,9]. MNNG generates the ultimate carcinogen, methyldiazonium hydroxide, during its non-enzymatic decomposition, which is accelerated by high concentrations of cellular thiols in the glandular stomach [20,21]. Weistler et al. [20] measured the alkylation products of DNA, N7 -methylguanine and O6 -methylguanine in the GI tract (from esophagus to ileum) and liver in rats after a single oral gavage of [14 C-methyl]- MNNG. The level of N7 -methylguanine was highest in the glandular stomach, followed by the duodenum and forestomach. The levels were much lower in the jejunum and liver than in the glandular stomach. No amount of N7 -methylguanine was detected in the ileum. O6 -Methylguanine, which is related

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to the carcinogenic effects of alkylating agents, was present in detectable amounts only in the glandular stomach, duodenum and forestomach. These findings explain that the glandular stomach is the primary target tissue for tumor induction by oral administration of MNNG. Moreover, the metabolism of MNNG after oral administration may also be related to the absence of tumor development in the colon; when [14 C-methyl]- or [14 C-guanidino]-MNNG was administered to rats via oral gavage, almost 90% of the radioactivity was excreted in the urine within 10 h, and less than 1% was egested in the feces for 24 h after administration [22]. The major metabolite in the urine is N-methyl-N -nitroguanidine, which is not a mutagenic compound [23]. These findings mean that MNNG or its metabolites scarcely reach the colon following oral administration. The present positive result in the glandular stomach and negative results in the colon and liver agree with the tissue specificity of MNNG carcinogenicity. These results suggest that the repeated dose MN assay using the glandular stomach, colon and liver can be utilized to evaluate the tissue specificity of a test substance. Regarding the effects of MNNG on the bone marrow, positive results are reported in most of the rodent bone marrow MN tests via the intraperitoneal route, while weakly positive or negative results are shown in the tests via the oral route [3,4,12,24–29]. There is also a report showing that MNNG is rapidly decomposed in the presence of whole blood or purified hemoglobin obtained from rats [30]. In the present study, the %IMEs did not significantly decrease in any treatment groups for either treatment period. Additionally, micronuclei were not induced in HEPs by MNNG, as stated above. Therefore, the negative result in the bone marrow in the 14-day study may be due to the failure of sufficient amounts of active molecules to reach the bone marrow and/or the instability of MNNG. In the animals that received the highest dose in the 28-day study, a small but significant increase in the frequency of MNIMEs was observed compared to the vehicle controls. The hematology data, which were obtained from 4 animals per group in the 28-day study, showed significant decreases in the erythrocyte counts, hemoglobin concentration, and hematocrit value (Supplementary data; Appendix 2) in the animals that received the highest dose, indicating that these animals were anemic. The %IMEs also tended to increase in these animals. These data suggest that erythropoiesis increased in the bone marrow of these animals to compensate for the loss of erythrocytes in the peripheral blood. Because an increase in erythropoiesis may contribute to the increase in the MNed erythrocyte frequency [31], the increased frequency of MNIMEs at the highest dose may be due to a secondary effect of anemia. The frequency of MNGSCs in each treatment group in the 28day study was approximately half that in the corresponding dose group in the 14-day study. Analyzing the cell proliferation with an appropriate index may help to elucidate this reason. However, the repeated dose glandular stomach MN assay was sufficiently sensitive to detect the genotoxicity of MNNG for both treatment periods. In conclusion, the present results indicate that the tissuespecific genotoxicity of MNNG can be detected by conducting repeated dose MN assays with the liver, glandular stomach, colon and bone marrow. A repeated dose MN assay with the glandular stomach, which is a direct contact site with high concentrations of test substances administered by oral gavage, may also be useful for detecting genotoxic compounds that are short-lived in vivo. Further studies with various chemicals will identify the characteristics (sensitivity and specificity) of the repeated dose liver and GI tract MN assays. Conflict of interest We declare that there are no conflicts of interest.

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