Rat Two-stage Carcinogenesis Model Research Articles

Combined Ascorbic Acid and Sodium Nitrite Treatment Induces Oxidative DNA Damage-Associated Mutagenicity In Vitro, but Lacks Initiation Activity in Rat Forestomach Epithelium

Combination treatment with sodium nitrite (NaNO(2)) and ascorbic acid (AsA) is well known to promote forestomach carcinogenesis in rats and weakly enhance esophageal carcinogenesis under acid reflux conditions. Nitric oxide generation and oxidative DNA damage are considered to be related to the enhancement of carcinogenesis. The purpose of the present study was to investigate whether oxidative DNA damage-associated genotoxicity and tumor initiating potential are involved in the carcinogenesis. In the bacterial reverse mutation assay using Escherichia coli deficient in the mutM gene encoding 8-hydroxydeoxyguanosine (8-OHdG) DNA glycosylase, the combination with NaNO(2) and AsA increased the mutation frequency dramatically, slight increase being evident in the parental strain. In vivo, a significant increase in 8-OHdG levels in the rat forestomach epithelium occurred at 24 h after combined treatment. Six-week-old F344 male rats were given drinking water containing 0.2% NaNO(2) and a diet supplemented with 1% AsA in combination, or the chemicals individually or basal diet alone for 12 weeks. After an interval of 2 weeks, they received 1% butylated hydroxyanisole in the diet for promotion until the end of weeks 52 and 78. Although one squamous cell carcinoma was observed in the combined group, there was no significant variation in tumor development among the groups. The study indicated that the combination of NaNO(2) with AsA induces genotoxicity due to oxidative DNA damage in vitro, and elevates 8-OHdG levels in the forestomach epithelium, but lacks initiating activity in the rat two-stage carcinogenesis model.

Combined treatment with green tea catechins and sodium nitrite selectively promotes rat forestomach carcinogenesis after initiation with N‐methyl‐N′‐ nitro‐N‐nitrosoguanidine

Combined treatment with several phenolic antioxidants and sodium nitrite (NaNO(2)) has already shown to enhance rat forestomach carcinogenesis. In the present experiment, effects of green tea catechins (GTC) alone or in combination with NaNO(2) on gastric carcinogenesis were investigated in a rat two-stage carcinogenesis model. Groups of eight, 6-week-old F344 male rats were given 0.01%N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in their drinking water and 5% NaCl in the diet for 10 weeks for glandular stomach initiation and a single intragastric administration of 100 mg/kg/bodyweight of MNNG at week 9 for forestomach initiation. From week 11, they received either drinking water containing 0.2% NaNO(2) and a diet supplemented with 1% GTC in combination, each individual chemical alone or a basal diet until the end of week 42. In the forestomach, incidences and multiplicities of neoplastic lesions were clearly increased by the combined treatment, in spite of GTC alone suppressing the occurrence of papillomas. In a short-term experiment with similar protocol without MNNG pretreatment, a significant increase of 8-hydroxydeoxyguanosine (8-OHdG) levels in forestomach DNA occurred 24 h after the combined treatment, concomitant with erosion and inflammatory cell infiltration. In an in vitro study, electron spin resonance demonstrated hydroxyl radical formation after incubation of epigallocatechin gallate or epicatechin gallate with the NO generator, NOC-7. Thus, GTC alone showed a weak chemopreventive effect on forestomach carcinogenesis, but in the presence of NaNO(2) it exerted a promotive effect which might involve hydroxyl-radical-associated oxidative DNA damage. However, no influence was exerted in the glandular stomach.

Enhancing effects of simultaneous treatment with sodium nitrite on 2-amino-3-methylimidazo[4,5-f]quinoline-induced rat liver, colon and Zymbal's gland carcinogenesis after initiation with diethylnitrosamine and 1,2-dimethylhydrazine

Combined effects of sodium nitrite (NaNO2) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) on liver, colon and Zymbal's gland carcinogenesis were assessed using a rat two-stage carcinogenesis model, with a focus on involvement of oxidative stress. Male 6-week-old F344 rats were given a single intraperitoneal injection of 200 mg/kg of diethylnitrosamine and 4 subcutaneous injections of 40 mg/kg of 1,2-dimethylhydrazine for initiation. Then, they were administered 0 or 300 ppm IQ in the diet or 0, 0.1 or 0.2% NaNO2 in their drinking water for 27 weeks. The treatment with NaNO2+IQ significantly enhanced colon and Zymbal's gland carcinogenesis and tended to enhance hepatocarcinogenesis. The incidence of lung tumors in the IQ-treated groups was significantly increased as compared with the initiation alone group. In a second experiment, male rats were given IQ or NaNO2 under the same conditions as before for 1 week, and at sacrifice, their liver and colon tissue or mucosa were collected for analysis of 8-hydroxydeoxyguanosine (8-OHdG), thiobarbituric acid reactive substances (TBARS), acrolein-modified protein and the bromodeoxyuridine-labeling index (BrdU-LI) (in the colon). In the colon, 8-OHdG, acrolein-modified protein levels and BrdU-LI were significantly increased by the combined treatment. These results indicate that the treatment with NaNO2 enhances IQ-induced colon and Zymbal's gland carcinogenesis in rats and that oxidative DNA damage and lipid peroxidation may partly be involved, especially in the colon. In addition, this experiment showed that IQ can act as a potent lung carcinogen in rats.

Lack of modifying effects of atrazine and/or tamoxifen on thyroid carcinogenesis in rats pretreated with N-bis(2-hydroxypropyl)nitrosamine (DHPN)

The modifying effects of atrazine, and/or tamoxifen, on thyroid carcinogenesis were investigated in a rat two-stage carcinogenesis model following N-bis(2-hydroxypropyl)nitrosamine (DHPN) initiation. Five-week-old male F344 rats were given a single subcutaneous injection of DHPN (2800 mg/kg, body weight) or vehicle alone. Starting 1 week later, the animals were fed a diet supplemented with 0, 5, 50 or 500 ppm of atrazine, 500 ppm atrazine plus 5 ppm tamoxifen, or 5 ppm tamoxifen in the DHPN-treated groups, and 0 or 500 ppm of atrazine in the DHPN-untreated groups for 24 weeks. At autopsy major organs, including the thyroid, pituitary, liver, kidney, testis, epididymis, and brain, were collected and histopathologically examined. Body weights were significantly ( P<0.05) decreased by the high doses of atrazine or tamoxifen, the effect being enhanced in combination. Relative thyroid weights were significantly increased ( P<0.05) only in the tamoxifen-treated group and pituitary weights were elevated with 500 ppm atrazine plus tamoxifen ( P<0.05). Relative liver weights were increased by the high dose of atrazine. However, the atrazine and/or tamoxifen treatments did not induce significant histopathological changes in the major organs, including the thyroid, nor cause significant changes in serum TSH levels. These results suggest that neither atrazine nor tamoxifen may promote thyroid carcinogenesis, alone as well as in combination.

Site-dependent modulating effects of conjugated fatty acids from safflower oil in a rat two-stage carcinogenesis model in female Sprague–Dawley rats

Modifying effects of dietary administration of conjugated fatty acids from safflower oil (CFA-S), rich in conjugated linoleic acid, on major organs were examined in the post-initiation stage of a two-stage carcinogenesis model in female rats. Groups of 21 or 22 F344 female rats were treated sequentially with 2,2′-dihydroxy-di- n-propylnitosamine (intragastrically, i.g.), 7,12-dimethylbenz[ a]anthracene (i.g.), 1,2-dimethylhydrazine (subcutaneously) and N-butyl- N-(4-hydroxybutyl)nitrosamine (in drinking water) during the first 3 weeks for initiation, and then administered diet containing 1 or 0.1% CFA-S for 33 weeks. Further groups of animals were treated with carcinogens or 1% CFA-S alone, or maintained as non-treated controls. All surviving animals were killed at week 36, and major organs were examined histopathologically for development of pre-neoplastic and neoplastic lesions. The 1 and 0.1% CFA-S treatment significantly decreased the incidence and multiplicity of mammary carcinomas, though a clear dose response was not observed. In the urinary bladder, the incidence of papillary or nodular hyperplasia but not tumors was significantly increased in the 1% CFA-S-treated group. The results indicate that low dose CFA-S may find application as a potent chemopreventor of mammary carcinogenesis.

Modifying Effects of Propolis on MeIQx Promotion of Rat Hepatocarcinogenesis and in a Female Rat Two-Stage Carcinogenesis Model After Multiple Carcinogen Initiation

The modifying effects of the dietary administration of water- and ethanol-extracted propolis produced in Brazil (WB and EB, respectively) on 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) promotion of rat hepatocarcinogenesis were investigated in a medium-term liver bioassay system with use of male Fischer 344 rats. The number and area of glutathione S-transferase placental form (GST-P)-positive foci in rats given 0.5% WB were significantly increased compared with the group given MeIQx alone. Furthermore, the numbers of GST-P-positive foci were higher in rats given 0.1% WB or EB than in those given the basal diet alone. The modifying effects of propolis on other organs were also examined in female Fischer 344 rats given multiple carcinogens for initiation. Rats received water- and ethanol-extracted propolis produced in Brazil and Uruguay (WB, EB, WU, and EU, respectively) in the diet after exposure to three different carcinogens. The incidence of total mammary tumors was significantly lower in rats given EU than in the control group. These results indicate that a water extract of propolis exerts a cocarcinogenic effect on MeIQx hepatocarcinogenesis while promoting the effect at low dose in a two-stage hepatocarcinogenesis model. Moreover, they suggest that ethanol-extracted propolis may be an inhibitor of mammary gland carcinogenesis.

Post-initiation effects of a super critical extract of propolis in a rat two-stage carcinogenesis model in female F344 rats

Post-initiation modifying effects of dietary administration of a super critical extract of propolis on major organs were examined using a two-stage carcinogenesis model. Groups of 21 or 22 F344 female rats were treated sequentially with 2,2′-dihydroxy-di- n-propylnitrosamine (DHPN, i.g.), 7,12-dimethylbenz[ a]anthracene (DMBA, i.g.), 1,2-dimethylhydrazine (DMH, s.c.) and N-butyl- N-(4-hydroxybutyl)nitrosamine (BBN, in drinking water) during the first 3 weeks for initiation, and then administered diet containing 0.1 or 0.01% propolis for 33 weeks. Further groups were treated with the carcinogens alone, 0.1% propolis alone or basal diet alone. All surviving animals were killed at week 36, and major organs were examined histopathologically for development of preneoplastic and neoplastic lesions. The incidence and multiplicity of mammary carcinomas were significantly decreased by the 0.1 and 0.01% propolis treatments. In the urinary bladder, the incidence of PN hyperplasia but not tumors was, in contrast, significantly increased by 0.1% propolis. Similarly, the number and area of glutathione S-transferase placental form (GST-P)-positive liver foci were significantly elevated with this high dose. The results indicate that a low dose of a super critical extract of propolis may find application as a potent chemopreventor of mammary carcinogenesis.

Promoting Effect of Nalidixic Acid on Preputial/Clitoral Gland Tumor Development in a Rat Two-stage Carcinogenesis Model.

The promoting effect of nalidixic acid (NA), an old quinolone antibacterial agent, was investigated in a two-stage preputial/clitoral gland carcinogenesis model in F344 rats initiated with 3, 2'-dimethyl-4-aminobiphenyl (DMAB). 3, 3'-Dimethoxybenzidine dihydrochloride (DMOB) was used as a genotoxic carcino-gen for the preputial/clitoral gland. After that, ofloxacin (OFLX), a new quinolone antibacterial agent, was examined. Male and female rats were given weekly subcutaneous injections of DMAB at a dose of 50mg/kg body weight, for a total of 8 times. Ten weeks after the commencement of DMAB treatment, rats were given 4, 000ppm NA in the diet, 330ppm DMOB in the drinking water or 4, 000ppm OFLX in thediet for the next 26 or 42 weeks. Upon histopathological investigation at experimental weeks 36 and 52, preputial/clitoral gland adenomas and carcinomas, respectively, were found in rats given DMAB alone. NA or DMOB administration after DMAB treatment enhanced the incidences of adenomas or carcinomas of the preputial/clitoral gland whereas OFLX did not. Meanwhile, NA is known to be a non-genotoxicagent. The present results indicate that NA is a tumor promoter in the preputial/clitoral gland of rats.