All animal studies were carried out under the Regulations for Animal Experiments at Gifu University. Male F344 rats (Shizuoka Laboratory Animal Center, Shizuoka, Japan) aged 4 weeks were housed in wire cages (three or four rats per cage). The rats had free access to drinking water and diets, under controlled conditions of humidity (50±10%), lighting (12-h light/dark cycle) and temperature (23±2°C). The rats were quarantined for 7 days and then randomized into experimental and control groups. Powdered CE-2 (Clea Japan, Tokyo, Japan) diet was used as the basal diet throughout the study. 6-MSITC (purity >99%) was kindly donated by Kinjirushi Co., Ltd. (Nagoya, Japan) and incorporated into the experimental diet at concentrations of 200 and 400 ppm. The diets were stored in a cold room (4°C) until use. 1,2-Dimethylhydrazine (DMH) was obtained from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan) and administered by subcutaneous (s.c.) injection (40 mg/kg) between 10:00 and 11:00 am to induce colorectal preneoplastic lesions.

A total of 66 male F344 rats were divided into seven groups. Groups 1–5 were initiated with DMH by four weekly s.c. injections (40 mg/kg body weight). Rats in groups 2 and 3 were fed the diet containing 200 and 400 ppm of 6-MSITC, respectively, for 5 weeks, starting 1 week before the first dosing of DMH until the end of week 5. The rats were then switched to and maintained on the basal diet until the termination. Starting 1 week after the cessation of DMH treatment, rats in groups 4 and 5 were fed the diet mixed with 200 and 400 ppm 6-MSITC, respectively, and this was continued until the termination. Rats in group 6 were fed the diet containing 400 ppm 6-MSITC throughout the experiment and group 7 served as the untreated control. At termination, all animals were sacrificed to assess the incidences of ACF and BCAC in the large bowel. Tissues were embedded in paraffin blocks and processed for routine histological observation with hematoxylin and eosin (H&E) staining.

After fixation of resected colons in 10% buffered formalin for at least 24 h at room temperature, the colons were stained using 0.5% methylene blue (in saline) for 1–3 min. After staining, ACF were counted and recorded according to the procedure described by Bird (20). The size of the lesions was scored as crypt multiplicity (ACF/lesion). In this study, we divided the colon into three portions after excision of the caecum (proximal, middle and distal) and used all of the segments for the analysis (21).

After ACF counting, colon tissues were embedded in paraffin and processed for immunohistochemistry of BCAC. Briefly, serial sections (4-μm thick) were prepared to include the middle portion between the surface and the bottom of the crypt. Some of these sections were used for immunohistochemistry of β-catenin and routine H&E staining. Immunohistochemistry was performed after exposure to 3% H₂O₂ for 20 min to block the endogenous peroxidase activity using a primary antibody to the β-catenin protein (1:1000 dilution; Transduction Laboratories, Lexington, KY, USA) at room temperature for 60 min. Then, the sections were stained with an LSAB kit (Dako, Glostrup, Denmark).

To assess the proliferative activity of cells in ACF and BCAC, immunohistochemical staining for PCNA was performed by using anti-PCNA (1:200 dilution) antibody and an LSAB kit (both from Dako). The nuclei that densely immunoreacted with PCNA were regarded as PCNA-positive. PCNA labeling indices were determined by counting the number of positive cells among at least 200 epithelial cells in ACF and BCAC, and were indicated as percentages.

Nine male F344 rats were divided into three groups. At 6 weeks of age, rats in group 1 were given corn oil through an intragastric tube and served as a control. Animals in groups 2 and 3 were given 6-MSITC at a dose of 20 and 40 mg/kg body weight in corn oil using a gastric tube, respectively (equivalent to daily consumption of 6-MSITC at 200 or 400 ppm). The animals were decapitated 24 h after the last dose of the vehicle or 6-MSITC. Livers were perfused in situ with ice-cold sterile 1.15% KCl, and 25% homogenates in 1.15% KCl were prepared. Microsomal fractions from these tissues were prepared using established procedures (22).

Gel electrophoresis and blot analysis were carried out as described in detail previously, according to the established methods of Laemmli (23) and Towbin et al (24), respectively. Separated proteins were transferred by semi-dry electroblotting from sodium dodecylsulfate-polyacrylamide gels to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA, USA) in 25 mM Tris buffer (pH 8.3) containing 0.19 M glycine and 20% (v/v) methanol. The membranes were blocked by incubation with phosphate-buffered saline containing 5% skim milk for 1 h before incubation with goat anti-rat polyclonal antibodies for CYP1A1/2, CYP2B1/2, CYP2E1 and CYP3A2 (Daiichi Pure Chemicals, Tokyo, Japan), and then stained using biotinylated anti-goat immunoglobulin G (Vector Laboratories, Burlingame, CA, USA) using a Wako ABC-POD kit (Wako Pure Chemicals, Osaka, Japan).

UDPGT activity towards bilirubin and 4-nitrophenol in liver microsomes was assayed according to the methods of Heirwegh et al (25) and Isselbacher et al (26), respectively, and towards testosterone with UDP-[¹⁴C(U)]glucuronic acid, as described by Matern et al (27). Each test was carried out with liver microsomes pooled from three rats. Experiments were performed six to eight times, and then the average value was calculated.

Data are expressed as the mean ± SD. The statistical significance of the difference in mean values was analyzed using one-way analysis of variance (ANOVA) and the unpaired t-test. Significance was defined as P<0.05.

The rats tolerated s.c. injection of DMH and/or 6-MSITC feeding well. During the study, no clinical signs of toxicity were present in any group. The mean body, liver and kidney weights did not significantly differ among the groups (Table I). Histologically, there were no pathological alterations indicative of toxicity of 6-MSITC in the major organs, such as the liver or kidney.

The rats in groups 1–5 developed ACF and BCAC in the colonic mucosa. No such lesions were noted in any of the rats in groups 6 and 7. In group 1, the frequency of ACF and BCAC were 323.8±69.7/colon and 3.80±1.05/cm² colonic mucosa, respectively. The number of ACF/colon was significantly lower in group 3 DMH-treated rats fed a 400 ppm 6-MSITC diet during the initiation phase than that in rats treated with DMH alone (group 1) (153.9±62.4, 52% reduction, P<0.0001; Table II). The reduction was also significant for the larger ACF with ≥4 crypts: from 155.1 to 65.4 (58% reduction, P<0.001; Table II, group 3 vs. group 1). In group 3, 400 ppm 6-MSITC also caused a significant decrease in the mean number of BCAC/cm² colon when compared with the control rats in group 1 (0.91±0.58, 76% reduction, P<0.00001; Table III). Thus, both ACF and BCAC were reduced to approximately the same extent. Regarding crypt multiplicity, a significant decrease in the number of crypts/lesion in group 3 was found in both ACF and BCAC when compared with that in group 1 (P<0.05 and P<0.001, respectively). The number of crypts/BCAC in groups 2 (DMH + 200 ppm 6-MSITC) and 5 (DMH→400 ppm 6-MSITC) were smaller than those in group 1, with a statistical significance of P<0.05 and P<0.01, respectively.

In group 3, treatment of rats with 400 ppm levels of 6-MSITC resulted in a significant decrease in the labeling index of PCNA in BCAC when compared to group 1 treated with DMH alone (P<0.01; Table IV). The mean PCNA labeling indices of groups 4 and 5 in ACF were lower than group 1, but the differences did not reach statistical significance.

Fig. 1 shows immunoblots and levels of liver microsomal CYP proteins in male F344 rats 24 h after 6-MSITC treatment. Neither hepatic CYP2B1 nor CYP1A1 were constitutively expressed. Treatment with 40 mg/kg b.w. 6-MSITC significantly decreased CYP1A2, -2B2, -2E1 and -3A2 proteins by 20, 35, 44 and 20%, respectively, relative to the vehicle group. Exposure to 20 mg/kg b.w. 6-MSITC also significantly decreased hepatic CYP1A2, -2B2 and -2E1 protein levels when compared to group 1.

Table V summarizes the effects of 6-MSITC treatment on hepatic UDPGT activity towards 4-nitrophenol, bilirubin and testosterone in liver microsomes. No significant differences were noted among the three groups.