Sustained-hepatic arterial infusion of oxaliplatin: pharmacokinetic advantages over hepatic arterial infusion using a preclinical animal tumour model.

Hepatic arterial infusion (HAI) of oxaliplatin allows greater liver tumour drug exposure compared to systemic infusion. However, the therapeutic index of HAI oxaliplatin remains poor. Using Pickering emulsion technology, we developed a platform able to provide sustained releases of oxaliplatin. The goal of this study was to evaluate the pharmacokinetic advantages of sustained-HAI oxaliplatin over HAI using a preclinical animal tumour model. Injections of 0.6 mg oxaliplatin in 20 min were selectively done in left hepatic arteries of 20 rabbits bearing a VX2 liver tumour in the middle left-lobe, using HAI (n = 10) or sustained-HAI (n = 10). In each group, half of the rabbits were sacrificed at 24 h and half at 72 h. Mass spectrometry was used to quantify drug pharmacokinetics in blood and oxaliplatin concentrations in tumour tissues, right- and middle left-liver lobes, spleen and lung. Compared to HAI, sustained-HAI of oxaliplatin resulted in lower plasmatic peak (Cmax: 275 ± 41 vs. 416 ± 133 ng/mL, p = 0.02) and higher concentration in the tumour at 24 h (2118 ± 2107 vs. 210 ± 93 ng/g, p = 0.008). After HAI, oxaliplatin concentration in tumours was significantly higher than in lung at 24 h (p = 0.03) but no other difference was found between oxaliplatin concentrations in tumours and in liver lobes, spleen or lung, neither at 24 h nor at 72 h. On the opposite, sustained-HAI resulted in higher concentrations of oxaliplatin in tumour compared to oxaliplatin concentrations in the middle left lobe (163 ± 86 ng/g at 24 h, p = 0.01, and 90 ± 15 ng/g at 72 h, p = 0.04), right lobe (174 ± 112 ng/g at 24 h, p = 0.01, and 112 ± 35 ng/g, p = 0.04 at 72 h), spleen (142 ± 21 ng/g at 24 h, p = 0.01, and 98 ± 12 ng/g at 72 h, p = 0.04), and lung (85 ± 11 ng/g at 24 h, p = 0.01, and 52 ± 4 ng/g at 72 h, p = 0.03). Sustained-HAI improves the therapeutic index of HAI oxaliplatin and offers a great potential for patients suffering from unresectable colorectal liver metastases or hepatocellular carcinoma.

Evaluation of adhesion barrier types in a rat hepatectomy-induced adhesion model.

BACKGROUND: Adhesion formation after hepatectomy creates problems for repeat hepatectomy. This study aimed to compare the effectiveness of a spray (AdSpray) and sheet adhesion barrier (Seprafilm) in a rat hepatectomy-induced adhesion model. METHODS: Thirty male Sprague-Dawley rats underwent partial resection of the left lateral liver lobe. They were randomly assigned to control (n = 10), AdSpray (n = 10), and Seprafilm groups. Seven days after surgery, the animals were sacrificed, and adhesions at the hepatic resection surface were blindly evaluated. RESULTS: In the control group, adhesions were formed in all 10 animals (100%), with a 69% adhesion extent (mean). In the AdSpray group, the incidence of adhesions (40%) and the adhesion extent (mean, 10%) were significantly lower than in the control group (incidence; p = 0.0147, adhesion extent; p = 0.0007). In the Seprafilm group, the incidence of adhesions was 70%. The adhesion extent of Seprafilm (mean, 30%) was significantly lower than in the control group (p = 0.0492). No significant differences were observed between the AdSpray and Seprafilm groups. As for histopathological examination, animals in the AdSpray group showed a similar healing profile to that of the control group without delayed healing and regeneration of mesothelial cells. In contrast, the Seprafilm group showed ongoing foreign body reaction to Seprafilm, and regeneration of mesothelial cells was immature at 7 days. CONCLUSIONS: Both the spray-type gel and sheet adhesion barriers significantly reduced adhesion formation after hepatectomy. The spray-type adhesion barrier caused no adverse events and induced favorable healing. These adhesion barriers may be effective in hepatectomy. Further animal studies and clinical trials are required to determine their benefits in clinical use.

Evaluation of a Spray-type, Novel Dextrin Hydrogel Adhesion Barrier Under Laparoscopic Conditions in a Porcine Uterine Horn Adhesion Model.

STUDY OBJECTIVE: To establish a porcine uterine horn adhesion model that mimicked laparoscopic procedures and use it to investigate the effect of a spray-type, novel dextrin hydrogel adhesion barrier (AdSpray; Terumo Corporation, Tokyo, Japan) on postsurgical adhesions. DESIGN: A single-blind randomized controlled trial (Canadian Task Force Classification I). SETTING: A Certified animal research facility. SUBJECTS: Sixteen female pigs. INTERVENTIONS: All animals underwent laparoscopically assisted adhesion-inducing surgery. The uterine horns and the peritoneum of the pelvic sidewall were injured. In the experimental group, AdSpray was applied to the injured site, and the handling of the sprayer was assessed. At 28 ± 1 days after surgery, animals were sacrificed, and adhesions at the injured site were evaluated. Uterine horn suture sites were examined under a light microscope to assess healing of the incised wound, the inflammatory reaction, abscess, and the foreign body reaction to the surgical suture. MEASUREMENTS AND MAIN RESULTS: The control group showed severe adhesions over the entire surface interface at the uterine horn suture sites and peritoneal resection site. Compared with the control treatment, AdSpray exhibited a higher percentage of adhesion-free sites (p < .001) and reduced the total adhesion score (p < .001). In the AdSpray group, no inflammation or abscess formation was observed on histopathological examination, and ideal healing of the suture sites was confirmed in all cases. CONCLUSION: Based on the results of the present study, the novel dextrin hydrogel shows excellent adhesion prevention and can be easily applied during laparoscopy using a dedicated sprayer.

Histopathological background data of the systemic organs of CLAWN miniature swine with coronary artery stent implantation.

The aim of this study was to identify potential changes that could occur during histological evaluations of CLAWN miniature swine, with potential consequences for subsequent experiments. The systemic organs from male and female CLAWN miniature swine (16.3-42.3 months old) that had been used in long-term studies of coronary stent implantation were examined histologically. Commonly observed histopathological findings were testicular/epididymal atrophy, cyst-like follicles in the ovaries, hemosiderin deposition in the spleen, lipofuscin deposition in the proximal tubular epithelia and presence of eosinophilic globules in the Bowman's space and the lumen of the proximal tubules in the kidneys, and cellular infiltration in several organs, including the eyelids, respiratory organs, and digestive tract. However, none of these changes were serious enough to indicate a significant impact on research. In conclusion, this study identified CLAWN miniature swine as a suitable animal model for various experiments.

Lack of nrf2 results in progression of proliferative lesions to neoplasms induced by long-term exposure to non-genotoxic hepatocarcinogens involving oxidative stress.

To explore the role of oxidative stress in chemical carcinogenesis driven by non-genotoxic mechanisms, nrf2-deficient (nrf2(-/-)) and nrf2-wild-type (nrf2(+/+)) mice were exposed to pentachlorophenol (PCP) at concentrations of 600 or 1200 ppm for 60 weeks, or piperonyl butoxide (PBO) at concentrations of 3000 or 6000 ppm in the diet for 52 weeks, respectively. Additional studies were performed to examine 8-hydroxydeoxyguanosine (8-OHdG) levels in liver DNA and hepatotoxicological parameters in serum following 8 weeks of exposure of each group to PBO at the same doses as in the long-term study. Exposure to 600 ppm PCP caused cholangiofibrosis (CF) only in nrf2(-/-) mice, while 1200 ppm PCP induced CF in both genotypes. Moreover, cholangiocarcinomas were found with significant incidence only in nrf2(-/-) mice treated with 1200 ppm PCP. Short-term exposure to 6000 ppm PBO caused significant elevation of 8-OHdG levels in both genotypes, while exposure to 3000 ppm caused a significant increase in 8-OHdG only in nrf2(-/-) mice. There were no inter-genotype changes in the incidences of regenerative hepatocellular hyperplasia (RHH) following long-term exposure to PBO. However, the incidence and multiplicity of hepatocellular adenomas, especially those observed in RHH, were much higher in nrf2-/- mice treated with 6000 ppm PBO than in nrf2+/+ mice treated with 6000 ppm PBO. Therefore, oxidative stress generated through PCP or PBO metabolism may promote the proliferation and progression of preneoplastic lesions to neoplasms.

Chemopreventive effects of 4-methylthio-3-butenyl Isothiocyanate (Raphasatin) but not curcumin against pancreatic carcinogenesis in hamsters.

The modifying effects of 4-methylthio-3-butenyl isothiocyanate (MTBITC) and curcumin were investigated in N-nitrosobis(2-oxopropyl)amine (BOP)-initiated hamsters. Male 6-week-old Syrian hamsters were subcutaneously injected with 10 mg/kg body weight (b.w.) of BOP four times a week, and fed a diet supplemented with 80 mg/kg diet of MTBITC, equivalent to 4.6 mg/kg b.w./day for the initiation stage or 3.8 mg/kg b.w./day for the postinitiation stage administration, respectively, or 2000 mg/kg diet of curcumin, equivalent to 118.8 mg/kg b.w./day for the initiation stage or 100.8 mg/kg b.w./day for the postinitiation stage administration, respectively. The incidence of combined pancreatic lesions, including atypical hyperplasias and adenocarcinomas, was significantly decreased to 55% (P < 0.05) by the 80 mg/kg diet MTBITC given during the initiation stage as compared to the BOP alone group (85%) but not by the curcumin administration at 16 weeks after the BOP-treatment. In the second study, the multiplicity of combined pancreatic lesions was also significantly decreased to 0.50 ± 0.51 (P < 0.05) by 700 mg/kg diet MTBITC given in the initiation stage (equivalent to 59.0 mg/kg b.w./day) as compared to the BOP alone group (1.10 ± 1.02). Our results indicate that the naturally occurring isothiocyanate MTBITC may exert preventive effects against BOP-initiation of hamster pancreatic carcinogenesis.

Oxidative DNA damage and in vivo mutagenicity caused by reactive oxygen species generated in the livers of p53-proficient or -deficient gpt delta mice treated with non-genotoxic hepatocarcinogens.

Oxidative stress is thought to participate in chemical carcinogenesis and may trigger gene mutations. To accurately assess the carcinogenesis risk posed to humans by chemical exposure, it is important to understand the pathways by which reactive oxygen species (ROS) are generated and the effects of the resulting oxidative stress. In the present study, p53-proficient and -deficient gpt delta mice were given pentachlorophenol (PCP), phenobarbital (PhB) or piperonyl butoxide (PBO), which are classified as non-genotoxic hepatocarcinogens in rodents, at the respective carcinogenic doses for 13 weeks. Exposure to PCP or PBO, but not PhB, invoked significant increases in liver DNA 8-hydroxydeoxyguanosine (8-OHdG) levels. Treatment with PCP significantly increased mRNA levels of the gene encoding NAD(P):quinone oxidoreductase 1 (NQO1) in the liver, suggesting that redox cycling of the PCP metabolite tetrachlorohydroquinone gave rise to ROS. Exposure to PhB or PBO significantly elevated CYP 2B10 mRNA levels while NQO1 levels were also significantly increased in PBO-treated mice. Therefore, in addition to involvement of the CYP catalytic pathway in the ROS-generated system of PBO, catechol derivatives produced from the opening of the PBO functional group methylenedioxy ring probably resulted in ROS generation. However, PCP, PBO and PhB failed to increase gpt and red/gam gene mutations in the liver independently of p53. Overall, the action of oxidative stress by ROS derived from the metabolism of these carcinogens might be limited to cancer-promoting activity, which supports the previous classification of these carcinogens as non-genotoxic.

Uterine Carcinosarcoma in a 2-year-old Female Wistar Hannover GALAS Rat.

Carcinosarcomas are rare tumors in humans as well as rats and most commonly occur in the uterus. Recently, we observed a case of incidental carcinosarcoma of the uterus in a female Wistar Hannover GALAS [BrlHan:WIST@ Jcl (GALAS)] rat at 2 years of age. Histopathologically, the tumor was characterized by an admixture of malignant epithelial and nonepithelial elements. The carcinomatous components represented a type of endometrial carcinoma, consisting of glandular and solid proliferation of large-sized tumor cells. Prominent mitoses and tumor cell invasion were observed. The sarcomatous components were characterized by multifocal proliferation of severe atypical cells with cartilage matrix and were diagnosed as chondrosarcoma. Transitions between carcinomatous and sarcomatous components were observed, and many tumor cells in the solid lesion showed immunohistochemical reactivity with both cytokeratin and vimentin. Based on these findings, this tumor was diagnosed as a uterine carcinosarcoma. This is the first report of uterine carcinosarcoma in Wistar Hannover GALAS [BrlHan:WIST@Jcl (GALAS)] rats.

Enhancing effects of carbon tetrachloride on in vivo mutagenicity in the liver of mice fed 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx).

Chronic stimulus subsequent to cell injury plays an important role in cancer development, but the precise mechanisms remain unknown partly because appropriate animal models are lacking. In the present study, the effects of hepatotoxicant carbon tetrachloride (CCl(4)) on in vivo mutagenicity were investigated using gpt delta mice with or without p53. Female B6C3F(1) p53-proficient or -deficient gpt delta mice were given a diet containing 300 ppm of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) for 13 weeks, concurrently with intraperitoneal injection of 1 ml/kg CCl(4) solution once a week. Mutant frequencies of gpt and red/gam in p53-proficient mice fed MeIQx were both significantly elevated by CCl(4)co-treatment. Enhancing effects of CCl(4) treatment were also noted in p53-deficient mice. In the mutation spectra analysis of gpt mutant colonies, G:C to T:A transversions were predominantly observed regardless of CCl(4) injection, and clonal expansion of gpt colonies were increased in the co-treated group as compared with MeIQx alone group. The present data showing no significant changes in mRNA expression levels of CYP1A2 and GSTa4 between MeIQx-treated groups with and without CCl(4). In the Western blotting analysis, CYP1A2 protein levels were significantly decreased in the co-treated group as compared to MeIQx alone group, and GSTα protein levels were not changed among any groups. It is suggested that the mutant frequency by co-treatment with CCl(4) might result from some factors other than p53 or MeIQx metabolism/excretion. Thus, our data clearly demonstrate that this model could be a powerful tool for identifying the mechanisms underlying combinatorial effects on carcinogenesis.

Effects of co-treatment of dextran sulfate sodium and MeIQx on genotoxicity and possible carcinogenicity in the colon of p53-deficient mice.

To investigate the effects of dextran sulfate sodium (DSS) and/or 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) on in vivo genotoxicity in the colon, male C57BL/6 p53 (+/+), p53 (+/-) or p53 (-/-) gpt delta mice were twice given 1-week treatment with DSS, 2 weeks apart, and then sacrificed after 2 and 14 weeks. Although colon length was significantly shortened after DSS treatment in all genotypes at each time point, no significant difference in gpt mutant frequency (MF) and tumorigenicity was found between DSS and control groups regardless of genotype. Then, male B6C3F(1) p53 (+/+) or p53 (+/-) gpt delta mice were given DSS as described above and/or fed 300 ppm MeIQx for 7 weeks. Colon length was significantly shortened with DSS in either genotype at weeks 7 and 26, but no effects of co-treatment with MeIQx or p53 deficiency were evident. MeIQx showed a tendency to increase gpt MF in the colon of mice with either genotype, but co-treatment with DSS did not affect these increments. Appreciable incidences of colonic aberrant crypt foci (ACFs) were reported in DSS as well as co-treatment groups of each genotype. Colonic adenomas were observed in co-treatment groups of both genotypes as well as the DSS-only group of p53 (+/+). No effects of the combination of DSS and MeIQx on colon pre- and neoplastic lesions were reported. Our results indicate that MeIQx may take more than 7 weeks to induce genotoxicity in the colon and that the co-treatment of mice did not enhance colon tumorigenicity even in p53-deficient mice.

Oxidative DNA damage and reporter gene mutation in the livers of gpt delta rats given non-genotoxic hepatocarcinogens with cytochrome P450-inducible potency.

Previous reports have proposed that reactive oxygen species resulting from induction of cytochrome P450 (CYP) isozymes might be involved in the modes of action of hepatocarcinogens with CYP-inducible potency. In the present study, we investigated 8-hydroxydeoxyguanosine (8-OHdG) levels, in vivo mutagenicity and glutathione S-transferase placental form (GST-P)-positive foci in the livers of gpt delta rats treated with piperonyl butoxide (PBO) or phenobarbital (PhB) for 4 and 13 weeks. Significant elevations in Cyp 1A1 and Cyp 1A2 mRNA levels after PBO treatment, and in Cyp 2B1 mRNA levels after PBO or PhB treatment, appeared together with remarkable hepatomegaly through the experimental period. Time-dependent and statistically significant increases in 8-OHdG levels were observed in the PBO treatment group along with significant increases in proliferating cell nuclear antigen (PCNA)-positive hepatocytes at 4 weeks, while no increase in 8-OHdG levels was found in PhB-treated rats. No changes in mutant frequencies of gpt and red/gam (Spi(-)) genes in liver DNA from PBO- or PhB-treated rats were observed at 4 or 13 weeks. A 13-week exposure to either PBO or PhB did not affect the number and area of GST-P-positive hepatocytes. CYP 1A1 and 1A2 induction may be responsible for elevated levels of 8-OHdG in PBO-treated rats. However, neither GC:TA transversions nor deletion mutations, typically regarded as 8-OHdG-related mutations, were observed in any of the treated rats. We conclude that reactive oxygen species, possibly produced through CYP catalytic pathways, likely induced genomic DNA damage but did not give rise to permanent gene mutation.

Simultaneous induction of non-neoplastic and neoplastic lesions with highly proliferative hepatocytes following dietary exposure of rats to tocotrienol for 2 years.

It was recently shown that 1-year chronic exposure of rats to tocotrienol (TT) induced highly proliferative liver lesions, nodular hepatocellular hyperplasia (NHH), and independently increased the number of glutathione S-transferase placental form (GST-P)-positive hepatocytes. Focusing attention on the pathological intrinsic property of NHH, a 104-week carcinogenicity study was performed in male and female Wistar Hannover rats given TT at concentrations of 0, 0.4 or 2% in the diet. The high-dose level was adjusted to 1% in both sexes from week 51 because the survival rate of the high-dose males dropped to 42% by week 50. At necropsy, multiple cyst-like nodules were observed, as in the chronic study, but were further enlarged in size, which consequently formed a protuberant surface with a partly pedunculated shape in the liver at the high dose in both sexes. Unlike the chronic study, NHH was not always accompanied by spongiosis, and instead angiectasis was prominent in some nodules. However, several findings in the affected hepatocytes such as minimal atypia, no GST-P immunoreactivity and heterogeneous proliferation, implied that NHH did not harbor neoplastic characteristics from increased exposure despite sustained high cell proliferation. On the other hand, in the high-dose females, the incidence of hepatocellular adenomas was significantly higher than in the control. There was no TT treatment-related tumor induction in any other organs besides the liver. Thus, the overall data clearly suggested that NHH is successively enlarged by further long-term exposure to TT, but does not become neoplastic. In contrast, TT induces low levels of hepatocellular adenomas in female rats.

Dietary catechol causes increased oxidative DNA damage in the livers of mice treated with acetaminophen.

We have shown that direct reaction of catechol with nitric oxide (NO) results in generation of reactive oxygen and nitrogen species (RNS) through semiquinone radical formation, leading to oxidative DNA damage in rat forestomach. In the present study, we investigated whether dietary catechol systemically exerts the same effects under NO-rich circumstances, when given before and during induction of inflammatory lesions. Male ICR mice were treated with or without 0.8% catechol in the diet for 2 weeks followed by acetaminophen (APAP) administration at a dose of 300mg/kg by single i.p. injection. Along with several indicators of APAP-induced hepatitis, 8-hydroxydeoxyguanosine (8-OHdG) levels and immunohistochemistry for 3-nitrotyrosine (NO(2)Tyr) in the livers were examined at 1.5, 4 and 24h after APAP injection. 8-OHdG was significantly increased at 24h in the co-treatment group, but not with either catechol or APAP alone. Elevation of serum ALT and AST activities, decrease of reduced glutathione levels and histopathological liver changes were observed to the same extents in both APAP-treated groups. In view of the finding of positive hepatocytes for NO(2)Tyr prior to generation of 8-OHdG, the process of oxidative DNA damage might involve RNS formation. Precise quantitative analysis of NO(2)Tyr by means of liquid chromatography with tandem mass spectrometry (LC-MS/MS) in an additional study with the same experimental protocol confirmed increase of RNS due to the reaction of catechol with NO produced after APAP-induced hepatitis. The overall data imply that antioxidants with a catechol structure can cause oxidative DNA damage under inflammatory conditions.

Possible participation of oxidative stress in causation of cell proliferation and in vivo mutagenicity in kidneys of gpt delta rats treated with potassium bromate.

Clarifying the participation of oxidative stress among possible contributing factors in potassium bromate (KBrO(3))-induced carcinogenesis is of importance from the perspective of human health protection. In the present study, utilizing the antioxidative effects of alpha-tocopherol (alpha-TP) or sodium ascorbic acid (SAA) to attenuate oxidative stress, alterations in bromodeoxyuridine labeling indices (BrdU-LIs) and reporter gene mutations in kidneys of male and female gpt delta rats given KBrO(3) were examined. Five male and female gpt delta rats in each group were given KBrO(3) at a concentration of 500ppm in the drinking water for 9 weeks, with 1% of alpha-TP or SAA administered in the diet from 1 week prior to the KBrO(3) treatment until the end of the experiment. Increases in 8-hydroxydeoxyguanosine levels in kidney DNA of both sexes of rats given KBrO(3) were significantly inhibited by SAA, but not alpha-TP. While BrdU-LIs in the proximal tubules of female rats were also significantly reduced by SAA, those in the males and gpt mutant frequencies in kidney DNA of both sexes were not affected by SAA or alpha-TP. Immunohistochemical and Western blot analyses for alpha(2u)-globulin strongly suggested that induction of cell proliferation observed in the males might primarily result from accumulation of this protein, independent of oxidative stress. The overall data indicated that while oxidative stress well correlates with induction of cell proliferation in females, its role in males and in generation of in vivo mutagenicity by KBrO(3) in both sexes is limited.

Lack of promotion activity of diacylglycerol oil on 4-nitroquinoline 1-oxide induced carcinogenesis in the oral cavity of SD rats.

A recent study using c-Ha-ras proto-oncogene transgenic (rasTg) rats demonstrated possible enhancing effects of diacylglycerol (DAG) on 4-nitroquinoline 1-oxide (4NQO) induced carcinogenesis of the tongue. To assess effects in their Sprague-Dawley back strain, a two-stage carcinogenesis study using 4NQO as an initiator was performed. Groups of 30 male rats were initially treated with 4NQO at a dose of 10ppm in the drinking water for the first 10 weeks followed after a 1 week recovery interval by 11% DAG, 5.5% DAG+5.5% triacylglycerol (TAG), 2.75% DAG+8.25% TAG, 1.38 DAG+9.62% TAG, 11% TAG, 11% high linoleic acid TAG (HLTG), 5.5% DAG or 2.75% DAG in the diet for 35 weeks. Further groups of animals were treated with distilled water instead of 4NQO followed by 11% DAG, 11% TAG or 11% HLTG in the same manner. The final survival rates in 4NQO-treated groups were from 50 to 77%. However, incidences and multiplicities of squamous cell papillomas and carcinomas in the oral cavity induced by 4NQO were not affected by any of the dietary treatments. Thus, in contrast to the positive data using rasTg rats, DAG had no potential for enhancing 4NQO-induced tumorigenesis in their back strain.

Induction of characteristic hepatocyte proliferative lesion with dietary exposure of Wistar Hannover rats to tocotrienol for 1 year.

Tocotrienol is an antioxidant which has found commercial application as a food additive and health supplement all over the world. Since there have been no reports regarding toxicological effects of long-term exposure, we performed a 52-week chronic study using Wistar Hannover rats of both sexes given the compound at doses of 0, 0.08, 0.4 or 2% in powdered basal diet. Since 6 animals in the 2% male group died of hemorrhage of several organs by week 50, the maximum dose level was changed to 1% in both sexes for the last 2 weeks. Decrease of body weight gain was observed in the 2% males from week 5 and females from week 10, this persisting to the end of the study. With the high dose, prolongation of prothrombin time and increase of serum ALT in males, and increase of serum ALP in both sexes were observed with statistical significance. In male and female rats receiving 0.4% or less, there were no toxicological changes in any of the parameters examined. At necropsy, multiple cyst-like nodules on the liver surface were macroscopically pronounced in both sexes receiving 2%. On histopathological examination, hepatocellular nodules were evident with distortion of hepatic cords and compression of the surrounding tissue, almost all including areas of spongiosis hepatis. The constituent hepatocytes were immunohistochemically stained with proliferation cell nuclear antigen at high rates. Nevertheless, they did not exhibit overt atypia and the basic lobular architecture remained intact. Additionally, they were consistently negative for glutathione S-transferase placental form (GST-P). Accordingly, we propose the newly categorized but previously used name 'nodular hepatocellular hyperplasia', which may not necessarily have a neoplastic or regenerative nature. However, quantitative GST-P analysis of the liver sections overall showed numbers of GST-P foci in the high dose females to be significantly elevated as compared to the control value. Based on the present data demonstrating nodular liver lesions only at the high dose of both sexes, we conclude that the no-observed-adverse-effect level (NOAEL) is 0.4% (303 mg/kg/day for males, and 472 mg/kg/day for females).

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.

beta-Naphthoflavone enhances oxidative stress responses and the induction of preneoplastic lesions in a diethylnitrosamine-initiated hepatocarcinogenesis model in partially hepatectomized rats.

The tumour-promoting effects of beta-naphthoflavone (BNF), a novel aryl hydrocarbon receptor (AhR) agonist, were investigated using a medium-term hepatocarcinogenesis model in rats. Six-week-old male F344 rats received an intraperitoneal injection of N-diethylnitrosamine (DEN) at a dose of 200mg/kg body weight and were fed a diet containing 0% (basal diet), 0.5% or 1% BNF for 6 weeks from 2 weeks after DEN treatment. All animals were subjected to two-thirds partial hepatectomy 1 week after the BNF treatment. The number and area of glutathione S-transferase placental form (GST-P) positive foci significantly increased in the livers of rats treated with BNF with concomitantly increased cell proliferation compared to those in the livers of the DEN alone group. Global gene expression analysis and subsequent quantitative real-time reverse transcription-polymerase chain reaction revealed that BNF induced not only the 'AhR gene battery'Cyp1a1, Cyp1a2, Cyp1b1, Nqo1, Aldh3a1 and Ugt1a6 but also the transcription factor NF-E2-related factor 2 (Nrf2)-regulated genes such as Gstm1, Gpx2, Akr7a3 and Yc2 (and also Nqo1), presumably due to the adaptive response against BNF-triggered oxidative stress responses. Reactive oxygen species production increased in microsomes isolated from the livers of BNF-treated rats, and this enhancement was suppressed by the P450 inhibitor SKF-525A. Furthermore, BNF enhanced oxidative DNA damage and lipid peroxidation, estimated by the levels of 8-hydroxydeoxyguanosine (8-OHdG) and thiobarbituric acid-reactive substances. These results suggest that the administration of BNF at a high dose and over a long-term enhance oxidative stress responses which may contribute to its hepatocarcinogenic potential in rats.

Enhancement of esophageal carcinogenesis in acid reflux model rats treated with ascorbic acid and sodium nitrite in combination with or without initiation.

Combined treatment with sodium nitrite (NaNO2) and ascorbic acid (AsA) has already been shown to promote rat forestomach carcinogenesis, possibly due to nitric oxide generation under acidic conditions. We hypothesized that a similar effect might occur in the esophagus when the luminal pH is decreased by acid reflux. To clarify this possibility, reflux esophagitis model rats (F344 male) were coadministered 0.2% NaNO2 in the drinking water and 1% AsA in the diet. After 32 weeks of the combined treatment, a significant increase in the incidence of epithelial hyperplasias of the lower-middle and lowest parts of the esophagus were observed compared with the basal-diet group, along with exacerbation of dysplasia and extension of the lesions. Additionally, one squamous cell papilloma was found only in the combined-treatment group. Subsequently, we confirmed the enhancing effects of NaNO2 and AsA cotreatment in the rat N-bis(2-hydroxypropyl)nitrosamine-initiated esophageal tumorigenesis model. The incidence of hyperplasia was enhanced in all segments, along with the incidence and multiplicity of squamous cell papillomas in the lowest segment of the esophagus. Thus, the data demonstrate that combined treatment with NaNO2 and AsA exerts promoting effects on rat esophageal carcinogenesis under acid reflux conditions, as in the forestomach. These findings suggest that the risk of excessive intake of a combination of nitrite and antioxidants for esophageal carcinogenesis is appreciable, particularly in patients with reflux esophagitis.