Inhibitory effects of combined administration of antibiotics and anti-inflammatory drugs on lung tumor development initiated by N-nitrosobis(2-hydroxypropyl)amine in rats.

The effects of antibiotics and anti-inflammatory drugs on the promotion stage of lung carcinogenesis initiated with N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats were investigated in two experiments with a similar protocol. In experiment 1, rats received tap water containing 2000 p.p.m. BHP for 12 weeks followed by basal diet or basal diet containing 0.02% erythromycin (EM), 0. 04% ampicillin (ABPC), 1.5% sho-saiko-to, 0.02% EM plus 1.5% sho-saiko-to or 0.04% ABPC plus 1.5% sho-saiko-to for 8 weeks after BHP administration. The development of adenocarcinomas (AC), squamous cell carcinomas (SqC) and adenosquamous carcinomas (ASqC) was completely inhibited in rats given ABPC plus sho-saiko-to and the numbers of lung lesions including alveolar hyperplasias, adenomas and carcinomas were decreased in rats given EM plus sho-saiko-to or ABPC plus sho-saiko-to. Neutrophil and macrophage infiltration into alveolar spaces of the lung were also markedly suppressed. In experiment 2, rats received BHP in the same manner as in experiment 1 and basal diet or basal diet containing 0.04% ABPC, 0.006% piroxicam, 0.04% ABPC plus 0.006% piroxicam and 0.04% ABPC plus 0.75% ougon for 8 weeks. The incidence and number of carcinomas, including ACs, SqCs and ASqCs were decreased in rats given ABPC plus piroxicam or ABPC plus ougon. Bacteria, mainly Escherichia coli, were detected in broncho-alveolar lavage of rats receiving BHP. The results suggest that chronic inflammation might be involved in the progression of lung carcinogenesis by BHP in rats and its suppression may therefore be useful as a chemopreventive strategy in lung cancer clinics.

Treatment of obstructive sleep apnea syndrome with a Kampo-formula, San'o-shashin-to: a case report.

The following describes a 76-year-old male with obstructive sleep apnea syndrome successfully treated with a Kampo-formula, San'o-shashin-to (Formula medicamentorum tres ad dispellendi cordis). Polysomnography, performed before and after administration of San'o-shashin-to, revealed that the apnea index decreased from 11.1 events/hour to 4.1 events/hour, and that the apnea plus hypopnea index decreased from 18.4 events/hour to 10.7 events/hour. The patient was normo-weight (body mass index: 20.4 kg/m2), and events of sleep apnea and hypopnea were mostly noted during a non-rapid eye movement sleep. It is possible that San'o-shashin-to has some alleviating effects on the upper airway resistance during sleep.

Delay in administration of CDDP until completion of AGM-1470 treatment enhances antimetastatic and antitumor effects.

The efficacy of cis-diammine dichloroplatinum (CDDP) therapy in combination with continuous administration of angiogenesis inhibitor o-(chloroacetyl-carbamoyl) fumagillol (AGM-1470) was evaluated experimentally using a transplantable rat osteosarcoma line previously established in our laboratory. AGM-1470 (2.5 mg/kg body weight/week) was administered by Alzet osmotic pumps for 2 weeks starting from 7 days after tumor inplantation and CDDP (1.25 mg/kg) was given on days 21 and 24. The number of lung metastatic nodules was counted and the wet weights of the primary tumors were measured 5 weeks after tumor inplantation. Values with administration of CDDP 3 days after discontinuation of AGM-1470 were significantly lower than when the two agents were coadministered (P < 0.05). This animal model should facilitate optimization of the timing of combination therapy.

Telomerase activity correlates with growth of transplantable osteosarcomas in rats treated with cis-diammine dichloroplatinum or the angiogenesis inhibitor AGM-1470.

To determine the role of telomerase activity in the growth of tumors in rats undergoing chemotherapy, a comparison of the volumes of telomerase-positive transplantable osteosarcomas was made in rats treated with the antineoplastic agent cis-diammine dichloroplatinum (CDDP) or the angiogenesis inhibitor O-(chloroacetylcarbamoyl)fumagillol (AGM-1470). Male F344 rats, 8 weeks old, received transplants of macroscopic lung metastatic nodules into the subcutaneous back space and treatment was started on day 14 thereafter. CDDP was injected i.v. at doses of 0, 0.625, 1.25 and 2.5 mg/kg body weight (b.w.) and AGM-1470 was administered at total doses of 0, 2.5, 5 and 10 mg/kg b.w. over 2 weeks by osmotic pumps, also implanted into the subcutaneous back space, but remote from the transplanted tumors. On day 28, all animals were killed for measurement of transplanted tumor size and determination of telomerase activities by telomeric repeat amplification protocol (TRAP) assay. The results showed telomerase activity to be highly correlated with the treated/non-treated (T/C) tumor size ratio (r = 0.96, P < 0.0001). In a second experiment, CDDP at 2.5 mg/kg b.w. and AGM-1470 at 10 mg/kg b.w., these being the most effective doses, were given as in the first experiment, and animals were serially killed on days 14, 21, 28, 35 and 42. Tumors in rats treated with CDDP and AGM-1470 showed 18.2% and 20.5% of the control telomerase activity on days 35 and 21, respectively, when tumor growth was inhibited. However, on day 42, the activities increased to 46.5% and 92.5%, this correlating with re-growth (r = 0.73, P < 0.0001). These results suggest that decline of telomerase activity may be involved in tumor growth retardation induced by chemotherapeutic agents. This possibility clearly warrants further mechanistic studies.

Inhibitory effects of beta-carotene, palm carotene, and green tea polyphenols on pancreatic carcinogenesis initiated by N-nitorsobis(2-oxopropyl)amine in Syrian golden hamsters.

The effects of alpha-carotene, beta-carotene, palm carotene, and green tea polyphenols (GTP) on the progression stage of pancreatic carcinogenesis after rapid production of ductal lesions were studied in Syrian hamsters. Dose threshold inhibitory effects were noted for beta-carotene, 25 ppm, and palm carotene, 40 ppm, which includes 24 ppm beta-carotene reducing the numbers of putative preneoplastic lesions of duct epithelial hyperplasia and atypical hyperplasia, as well as carcinoma in situ and invasive carcinomas. GTP at doses of 500 and 5000 ppm, but not 100 ppm, also significantly decreased the numbers of hyperplasia and total duct lesions. Combined administration of 40 ppm palm carotene, and 50 ppm GTP similarly inhibited the lesion development. Alpha-carotene, however, did not affect pancreatic carcinogenesis. The results suggest that chemopreventive effects are exerted by beta-carotene and GTP above critical doses and that combined administration of palm carotene and GTP might be a candidate chemoprevention strategy for pancreatic cancer in humans.

Inhibitory effect of green tea extract on the process of pancreatic carcinogenesis induced by N-nitrosobis-(2-oxypropyl)amine (BOP) and on tumor promotion after transplantation of N-nitrosobis-(2-hydroxypropyl)amine (BHP)-induced pancreatic cancer in Syrian hamsters.

Epidemiologic studies have shown a lower risk of gastrointestinal cancer in green tea drinkers. In the present study, the inhibitory effect of green tea extract (GTE) on the process of pancreatic carcinogenesis induced by N-nitrosobis-(2-oxypropyl)amine (BOP) and on tumor promotion after transplantation of N-nitrosobis-(2-hydroxypropyl)amine (BHP)-induced pancreatic cancer were investigated in hamsters. In the first experiment, shortly after the initiation of pancreatic carcinogenesis by BOP, the animals in the GTE group were given GTE (0.5 mg/L) in their drinking water and the control group was given tap water. All animals were sacrificed 24 weeks later. There were no significant differences in body weight, water intake, or food consumption between the two groups during the experiments. GTE consumption was approximately 1.25 mg/day/100 g body weight during this experiment. Seven of the 13 hamsters (54%) in the control group were found to have pancreatic tumors, versus six of the 18 hamsters (33%) in the GTE group. The average number of tumors in the control group was 1.0/hamster, compared with 0.5/hamster in the GTE group. The overall incidence of macroscopic pancreatic tumors in the GTE group was about half that in the control group. The incidence of pancreatic cancer was 54% (12/13) in the control group and 44% (8/18) in the GTE group. The number of pancreatic cancers, including invasive carcinoma and carcinoma in situ, in the GTE group was 0.88/hamster, significantly lower than in the control group (1.68/hamster) (p < 0.05). The incidence of atypical ductal hyperplasia, which is thought to be an early pancreatic cancer, was also significantly lower in the GTE group than in the control group (1.50/hamster vs. 4.65/hamster) (p < 0.05). In the second experiment, 1-mm3 pieces of BHP-induced pancreatic cancer were transplanted into the back of hamsters. The control group (N = 16) was maintained on the basal diet and tap water throughout the experiment, and the GTE group (N = 16) was also maintained on the basal diet and tap water for the first 3 weeks after transplantation, when successful transplantation was confirmed and, thereafter, given tap water containing GTE (0.5 mg/L) for an additional 12 weeks. Tumor growth was similar in both groups until 11 weeks after transplantation, but inhibition of tumor growth became apparent after 11 weeks in the GTE group. At 13 weeks, the average tumor volume in the GTE group was 1.01 +/- 0.11 x 104 mm3, significantly smaller than that in the control group (1.98 +/- 0.37 x 104 mm3) (p < 0.05). The results demonstrated that GTE has an inhibitory effect on the process of pancreatic carcinogenesis and on tumor promotion of transplanted pancreatic cancer. These results suggest that GTE may come to serve as a chemopreventive and chemotherapeutic agent for pancreatic cancer.

Efficacy of CDDP and AGM-1470 chemotherapy against lung metastasis in rat osteosarcoma depends on the timing of combined administration.

The efficacy of combination therapy with cis-diammine-dichloroplatinum (II) (CDDP) and o-(chloroacetyl-carbamoyl) fumagillol (AGM-1470) was evaluated experimentally using a transplantable rat osteosarcoma line, previously established in our laboratory, with a high potential for metastasis. Tumor-bearing male Fischer 344 rats were administered CDDP (2.5 mg/kg) together with, or after discontinuation of, AGM-1470 treatment (10 mg/kg/body weight/week). When CDDP was administered three days after discontinuation of AGM-1470 the most pronounced antimetastatic effects were observed, although the antitumor effect was approximately the same.

Increased telomerase activity in hyperplastic nodules and hepatocellular carcinomas induced by a choline-deficient L-amino acid-defined diet in rats.

Activation of telomerase has been reported in several human cancers, including hepatocellular carcinomas (HCCs). We investigated telomerase activity during hepatocarcinogenesis induced by a choline-deficient L-amino acid-defined (CDAA) diet in rats. Male F344 rats were given a CDAA diet or a choline-supplemented L-amino acid-defined (CSAA) diet from 6 weeks of age for 75 weeks, and subgroups were killed 10 weeks, 50 weeks and 75 weeks after the beginning of the experiment. Hyperplastic nodules and HCCs were noted in rats fed a CDAA diet for 50 weeks and 75 weeks, respectively. Normal control liver specimens were obtained from 6-week-old rats. Telomerase activity was assessed by using a telomeric repeat amplification protocol (TRAP). Normal liver and background parenchyma of rats fed either of the diets for 10 weeks or 50 weeks showed weak telomerase activity. In contrast, markedly increased levels were demonstrated in hyperplastic nodules and HCCs. These results suggest that increased telomerase activity may be a biological feature of preneoplastic lesions that evolve to HCCs in rat liver.

Infrequent Ki-ras and an absence of p53 mutations in hepatocellular carcinomas induced by a choline deficient L-amino acid defined diet in rats.

Mutations of Ki-ras and p53 genes in hepatocellular carcinomas (HCCs) induced by the choline deficient L-amino acid defined (CDAA) diet in rats were investigated by polymerase chain reaction (PCR), single strand conformation polymorphism (SSCP) analysis followed by direct sequencing. Male Fischer 344 rats, 6 weeks old, were continuously given a CDAA diet for 70 weeks and then sacrificed. Macroscopically detectable nodules which were histologically confirmed to be well-differentiated HCCs were dissected free from the surrounding tissue and subjected to gene mutation analysis along with samples of non-tumor areas. Conformational change in the Ki-ras gene was detected in 1 out of 7 HCCs, involving a GGC to GTC transversion at codon 13. No p53 mutations were detected in 7 HCCs and also neither Ki-ras nor p53 mutations were found in non-tumor areas. The results suggest that neither Ki-ras nor p53 genes play an important role in hepatocarcinogenesis caused by long term expose to a CDAA diet in rats.

Inhibitory effects of N,N'-diphenyl-p-phenylenediamine on the early stage of the enhanced hepatocarcinogenesis caused by coadministration of ethionine and a choline-deficient L-amino acid-defined diet in rats.

Effects of N,N'-diphenyl-p-phenylenediamine (DPPD), an antioxidant, on liver carcinogenesis caused by a choline-deficient L-amino acid-defined (CDAA) diet containing ethionine were studied in Fischer 344 rats. Male animals, 6 weeks old, were fed a CDAA diet, a choline-supplemented L-amino acid-defined (CSAA) diet or a CDAA diet containing 0.05% ethionine with or without 0.2% DPPD. Histological changes and lesions positive for gamma-glutamyltransferase (GGT) were analyzed 12 weeks after the beginning of the experiment. The levels of 8-hydroxyguanine (8-OHGua) in DNA and 2-thiobarbituric acid-reacting substances (TBARS) were measured as the parameters for cellular oxidative damage after 4 and 11 days of treatment. Expression of c-myc and c-Ha-ras was also investigated in relation to cell proliferation after 2, 4, 8 and 11 days. Histologically, development of diffuse fatty liver observed in rats fed a CDAA diet was inhibited, while massive oval cell proliferation and cholangiofibrosis resulted from the addition of ethionine with/without DPPD. The sizes but not numbers of GGT-positive lesions seen in the liver of rats fed a CDAA diet were increased and the levels of 8-OHGua formation and TBARS generation were also increased by the ethionine supplement. Both numbers and sizes of GGT-positive lesions were decreased and the level of TBARS, but not 8-OHGua, was decreased by adding DPPD. The increased expression of c-myc and c-Ha-ras detected in the liver of rats fed a CDAA diet was further increased by addition of ethionine and again reduced by DPPD. These results indicate that an antioxidant DPPD can inhibit the early stage of enhanced hepatocarcinogenesis caused by coadministration of ethionine and a CDAA diet, by blocking cellular oxidative damage as well as c-myc and c-Ha-ras expression.

Prevention by methionine of enhancement of hepatocarcinogenesis by coadministration of a choline-deficient L-amino acid-defined diet and ethionine in rats.

The effects of methionine on hepatocarcinogenesis induced by coadministration of a choline-deficient L-amino acid-defined (CDAA) diet and ethionine were examined. F344 male rats were divided into 4 experimental groups. Groups 1 and 2 received the CDAA diet and a choline-supplemented L-amino acid-defined (CSAA)++ diet, respectively. Group 3 received the CDAA diet containing 0.05% ethionine, and group 4 the CDAA diet containing 0.05% ethionine and 0.47% methionine. Animals were killed after 12 weeks of treatment. Histologically, the CDAA diet induced intracellular fat accumulation and foci. In contrast, ethionine caused not only foci, but also hyperplastic nodules, cholangiofibrosis and the proliferation of oval cells without such fat accumulation. Methionine abolished the development of all of the liver lesions induced by coadministration of the CDAA diet and ethionine. To investigate the effects of methionine on induction of c-myc and c-Ha-ras expression, as well as generation of 8-hydroxyguanine (8-OHGua) and 2-thiobarbituric acid-reacting substances (TBARS), by coadministration of the CDAA diet and ethionine, subgroups of 3 to 5 animals were killed at 2, 4, 8, or 11 days after the beginning of the experiment. Coadministration of the CDAA diet and ethionine markedly enhanced the level of expression of c-myc and c-Ha-ras, 8-OHGua formation and TBARS generation as compared with the CDAA or CSAA diet within 11 days, and methionine blocks these actions. These results indicate that addition of methionine prevents the induction of c-myc and c-Ha-ras expression, 8-OHGua formation and TBARS generation, as well as hepatocellular lesions, by coadministration of the CDAA diet and ethionine in rats, and suggest a possible involvement of oxidative stress and gene expression in hepatocarcinogenesis by these agents.

Efficacy of the angiogenesis inhibitor O-(chloroacetyl-carbamoyl)fumagillol (AGM-1470) on osteosarcoma growth and lung metastasis in rats.

The efficacy of the anti-angiogenic agent, O-(chloroacetyl-carbamoyl)fumagillol (AGM-1470), against primary tumor growth and spontaneous lung metastasis was evaluated experimentally using a transplantable osteosarcoma line in rats previously established in our laboratory. Male Fischer 344 rats bearing the tumor with a high potential for metastasis received intermittent or continuous subcutaneous administrations of AGM-1470. Both treatment regimens resulted in significant inhibitions of spontaneous lung metastasis and primary tumor growth in a dose-dependent manner, with continuous administration of AGM-1470 exerting the most pronounced inhibitory effects on both parameters.

Inhibitory effects of sulfation inhibitors on initiation of pancreatic ductal carcinogenesis by N-nitrosobis(2-oxopropyl)amine in hamsters.

The effects of dehydroepiandrosterone sulfate (DHAS), a typical hydroxysteroid sulfotransferase (HSTase) inhibitor, and of 3'-phosphoadenosine 5'-phosphate (PAP), a nonspecific sulfation inhibitor on N-nitrosobis(2-oxopropyl)-amine (BOP)-induced initiation were examined in a rapid production model for pancreatic carcinomas in hamsters in order to elucidate the involvement of sulfotransferase in the metabolic activation of beta-oxypropylnitrosamines. While neither low nor high doses of DHAS and PAP exerted any significant influence on the incidence of ductal lesions including carcinomas, the high dose of DHAS (350 mg/kg body wt) and a both low (90 mg/kg) and high (180 mg/kg) doses of PAP reduced the mean numbers of pancreatic ductal adenocarcinomas. The high dose of PAP also reduced the number of all ductal lesions combined. The results thus suggest that metabolic activation with STase is involved in BOP-induced pancreatic ductal carcinogenesis in hamsters, and support the hypothesis that BOP is metabolized to beta-hydroxyalkylnitrosamines followed by activation to proximate sulfuric acid esters by HSTase.

Age-related accumulation of phosphatidylcholine hydroperoxide in cultured human diploid cells and its prevention by alpha-tocopherol.

The levels of phosphatidylcholine hydroperoxide in serially cultured human fetal diploid fibroblasts at various population doubling levels were determined by high-performance liquid chromatography combined with chemiluminescence detection. This methodology utilizes a mixture of cytochrome c and luminol as post-column hydroperoxide group specific luminescent reagents. The cellular hydroperoxide content increased with age from 0.34 to 27.72 pmol/10(6) cells. At the end of the cells' in vitro lifespan (51st population doubling level), the hydroperoxide content per 10(6) cells reached about 80 times the level found in cells of the 20th population doubling level. Supplementation of exogenous alpha-tocopherol to the culture medium prevented hydroperoxide accumulation, but did not extend the lifespan in vitro. The results indicate that substantial intracellular phospholipid hydroperoxide accumulation occurred in the course of aging of human fetal diploid fibroblasts.

Induction of 8-hydroxydeoxyguanosine but not initiation of carcinogenesis by redox enzyme modulations with or without menadione in rat liver.

Inducibility of oxidative stress in rat liver in vivo by menadione-associated redox cycling activation under redox enzyme modulating conditions was examined by monitoring hepatocyte injury and 8-hydroxydeoxyguanosine (8-OHdG) levels of liver DNA. In addition, the treatment-associated liver tumor initiating activity was assessed in terms of development of gamma-glutamyl-transpeptidase (GGT)- and glutathione S-transferase placental form (GST-P)-positive foci and hyperplastic nodules. With or without following menadione treatment (50 mg/kg, i.g.), redox enzyme modulations of increased cytochrome P450 reductase activity induced by phenobarbital (PB)-Na (100 mg/kg, i.p. for 5 days), inhibition of DT-diaphorase by dicumarol (25 mg/kg, i.p.) and depletion of glutathione by phorone (200 mg/kg, i.p.), with or without further supplement of iron EDTA-Na-Fe(III) (70 mg/kg, i.p.), caused both substantial hepatocyte necrosis and 8-OHdG production in Fischer 344 male rats. Subsequent feeding with a 0.05% PB diet for 64 weeks resulted in slightly increased development of GGT-positive foci but not GST-P positive lesions or hyperplastic nodules, suggesting a lack of tumor-initiating activity of the oxidative DNA damage associated with redox enzyme modulations with or without menadione.

Therapeutic trial of hematological disorders with intermittent administration of high-dose 1-alpha-hydroxyvitamin D3.

Aplastic anemia and myelodysplastic syndrome were successfully treated with an intermittent administration of high-dose 1 alpha-hydroxy-vitamin D3, an active analogue of 1,25-dihydroxyvitamin D3. This effect was considered to be through the differentiation-inducing and immunomodulatory actions of 1,25-dihydroxyvitamin D3. The only adverse effect was hypercalciuria which was controllable by decreasing the dose.

Effect of 1,25-dihydroxyvitamin D3 on phospholipid composition of rat renal brush border membrane.

To analyze the effects of vitamin D on renal tubular cell membrane phospholipid metabolism, the effects of vitamin D depletion and repletion on renal brush border membrane (BBM) phosphatidylethanolamine (PE), phosphatidylcholine (PC), and phosphatidylinositol (PI) were studied. The PC content of BBM from kidneys of rats deprived of vitamin D for 5-6 wk was 33.5 +/- 2.2 nmol Pi/mg protein. This was significantly lower than the PC content of BBM from kidneys of rats supplemented with vitamin D2 for 2 wk, 41.0 +/- 0.4 nmol Pi/mg protein. Vitamin D depletion also decreased the content of BBM PE. The fatty acid composition of BBM PC was altered by vitamin D depletion. Vitamin D depletion increased palmitic acid and decreased stearic, linoleic, and arachidonic acid. Vitamin D repletion with a single physiological dose of 1,25(OH)2D3 (30 pmol), 16 h prior to study tended to increase membrane content of PC and significantly increased the linoleic acid content of the PC fraction. Single-dose vitamin D repletion with a pharmacological dose of 1,25(OH)2D3 (2.4 nmol) produced a significant increase in BBM content of PC and also significantly increased the linoleic acid content of PC. These results demonstrate that vitamin D deficiency affects PC and PE content of rat renal BBM and their fatty acid composition, and that vitamin D repletion with 1,25(OH)2D3 for 16 h partially normalizes the changes in PC.