Glomerular hyperfiltration with hyperglycemia in the spontaneously diabetic Torii (SDT) fatty rat, an obese type 2 diabetic model.

Glomerular hyperfiltration is observed in an early stage of kidney diseases including diabetic nephropathy. A better understanding of pathophysiological changes in glomerular hyperfiltration is essential for development of new therapies to prevent kidney disease progression. In this study, we investigated glomerular changes including glomerular filtration rate (GFR) and glomerular size in the Spontaneously Diabetic Torii (SDT) fatty rat, an obese type 2 diabetic model, and we also evaluated pharmacological effects of the sodium glucose cotransporter 2 inhibitor dapagliflozin on the renal lesions. Dapagliflozin was administered to SDT fatty rats from 5 to 17 weeks of age. Blood and urinary biochemical parameters were periodically measured. GFR was determined by transdermal GFR monitor at 16 weeks of age and histopathological analysis was performed at 17 weeks of age. SDT fatty rat developed severe hyperglycemia and exhibited pathophysiological abnormalities in the kidney, such as an increased GFR, glomerular hypertrophy and tissue lesions. Dapagliflozin achieved good glycemic control during the experimental period, inhibited the increase in GFR, and improved histopathological abnormalities in tubules. These results suggest that the SDT fatty rat is a useful model for analyzing the pathogenesis of diabetic nephropathy during its early stage and dapagliflozin improves not only hyperglycemia but also glomerular hyperfiltration and tubule lesions in SDT fatty rat.

Toxicological evaluation of L-proline in a 90-day feeding study with Fischer 344 rats.

L-proline (L-Pro) is a non-essential amino acid, and has become widely used as supplements and health foods, recently. A subchronic oral toxicity study of L-Pro was conducted with groups of 10 male and 10 female Fischer 344 rats fed a powder diet containing 0%, 0.625%, 1.25%, 2.5% and 5.0% of L-Pro for 90 days. No treatment-related clinical signs and mortality were noted. We observed no clear treatment-related effects with regard to body weight, food intake or urinalysis data. The average daily water intakes of the treated female groups were significantly increased compared to the controls. The hematology (red blood cell parameter) and serum biochemistry (glucose, blood urea nitrogen, creatinine or uric acid) of the treated male and/or female groups were lower than those of the control groups. However, these changes were lacked dose-dependence, and no abnormalities were found in corresponding pathological findings. In conclusion, the no-observed-adverse-effect-level (NOAEL) for L-Pro was determined to be a dietary dose of 5.0% (2772.9 mg/kg body weight/day for males and 3009.3mg/kg body weight/day for females) under the present experimental conditions.

Toxic effects of l-aspartic acid at high dose levels on kidneys and salivary glands in Fischer 344 rats detected in a 90-day feeding study.

A subchronic oral toxicity study of l-aspartic acid (l-Asp) was conducted with groups of 10 male and 10 female Fischer 344 rats fed a powder diet containing 0%, 0.05%, 1.25%, 2.5% and 5.0% concentrations for 90 days. Serum biochemistry showed treatment-related decreases of blood urea nitrogen, creatinine and uric acid levels in both sexes. In addition, incidences of urinary ketone and protein were significantly increased in treated both sexes, while relative kidney weight was significantly increased in the 5.0% male rat, and regenerative renal tubules with tubular dilation were histopathologically observed in male rats of the 2.5% or greater groups. The observed renal injury was confirmed not to be due to accumulation of alpha2u-globulin. Acinar cell hypertrophy of salivary glands was histopathologically evident in male and female rats of the 2.5% or greater groups. The present results indicate that l-Asp causes toxic effects on kidneys and possibly salivary glands at high dose levels in male and female Fischer 344 rats. Such toxic effects were observed only in animals given 2.5% and/or higher doses of l-Asp. In conclusion, the no-observed-adverse-effect-level (NOAEL) for l-Asp is 1.25% (696.6 mg/kg body weight/day for males and 715.2 mg/kg body weight/day for females) under the present experimental conditions.

Existence of a threshold for induction of aberrant crypt foci in the rat colon with low doses of 2-amino-1-methyl-6-phenolimidazo[4,5-b]pyridine.

Until recently it has been generally considered that genotoxic carcinogens have no threshold in exerting their potential for cancer induction. However, the nonthreshold theory can be challenged with regard to assessment of cancer risk to humans. In the present study we show that a food derived, genotoxic hepatocarcinogen, 2-amino-1-methyl-6-phenolimidazo[4,5-b]pyridine (PhIP), does not induce aberrant crypt foci (ACF) as preneoplastic lesions at low dose (below 50 ppm) or 8-hydroxy-2'-deoxyguanosine (below 400 ppm) in the rat colon. Moreover PhIP-DNA adducts were not formed at the lowest dose (below 0.01 ppm). Thus, the dose required to initiate ACF is approximately 5000 times higher than that needed for adduct formation. The results imply a no-observed effect level (existence of a threshold) for colon carcinogenesis by a genotoxic carcinogen.

The copper-chelating agent, trientine, suppresses tumor development and angiogenesis in the murine hepatocellular carcinoma cells.

Angiogenesis is now recognized as a crucial process in tumor development, including hepatocellular carcinoma (HCC). Since HCC is known as a hypervascular tumor, anti-angiogenesis is a promising approach to inhibit the HCC development. Trientine dihydrochloride (trientine) is used in clinical practice as an alternative copper (Cu)-chelating agent for patients with Wilson's disease of penicillamine intolerance. In our study, we examined the effect of Cu-chelating agents on tumor development and angiogenesis in the murine HCC xenograft model. Although both trientine and penicillamine in the drinking water suppressed the tumor development, trientine exerted a more potent inhibitory effect than penicillamine. In combination with a Cu-deficient diet, both trientine and penicillamine almost abolished the HCC development. Trientine treatment resulted in a marked suppression of neovascularization and increase of apoptosis in the tumor, whereas tumor cell proliferation itself was not altered. In vitro studies also exhibited that trientine is not cytotoxic for the tumor cells. On the other hand, it significantly suppressed the endothelial cell proliferation. These results suggested that Cu plays a pivotal role in tumor development and angiogenesis in the murine HCC cells, and Cu-chelators, especially trientine, could inhibit angiogenesis and enhance apoptosis in the tumor with consequent suppression of the tumor growth in vivo. Since trientine is already used in clinical practice without any serious side effects as compared to penicillamine, it may be an effective new strategy for future HCC therapy.

Decreased expression of Bcl-x protein during hepatocarcinogenesis induced exogenously and endogenously in rats.

Dysregulations of apoptosis have been widely recognized as important events in multi-stage carcinogenesis. Bcl-x, a member of the Bcl-2 family, is known to act as a regulator of apoptosis. The present study was conducted to assess the role of altered Bcl-x protein expression in exogenous and endogenous hepatocarcinogenesis in rats. In the short-term exogenous models, male Fischer 344 rats, 6 weeks old, were given a single intraperitoneal injection of diethylnitrosamine (DEN) at a dose of 200 mg / kg body weight, partially hepatectomized at the end of week 3, administered phenobarbital at a concentration of 0.05% from the end of week 2 for 6 weeks, and sacrificed. In the livers, glutathione S-transferase (GST-P)-positive, putative preneoplastic lesions were induced, and Bcl-x protein expression was decreased in 24.7% of such lesions. The incidence of GST-P-positive lesions with decreased Bcl-x increased depending on the size of the lesions; 18.9%, 32.4% and 86.5% in the lesions smaller than 0.03, between 0.03 and 0.3, and larger than 0.3 mm(2), respectively. In GST-P-positive lesions larger than 0.3 mm(2), both apoptosis induction and cell proliferation activity were enhanced when Bcl-x protein expression was decreased. In the long-term exogenous models, rats were given 10 mg / kg of DEN, partially hepatectomized 4 h after treatment, administered 0.5 mg / kg of colchicine at the end of days 1 and 3, subjected to a selection procedure, and sacrificed at the end of week 45. Hepatocellular carcinomas were induced with the decreased Bcl-x protein expression. In the endogenous model, rats were fed a choline-deficient, L-amino acid-defined diet for 16 or 80 weeks and sacrificed. Bcl-x protein expression was decreased both in GST-P-positive lesions and hepatocellular carcinoma. These results suggest that this decrease of Bcl-x protein might serve as an indicator of the advanced form of preneoplastic lesions, and that this decrease could also be associated with a potential to progress into carcinoma in both exogenous and endogenous hepatocarcinogenesis of rats.

FHIT alterations in lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine in rats.

Alteration of the FHIT gene was investigated in lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl) amine (BHP) in male Wistar rats. Animals at 6 weeks of age were given 2000 p.p.m. of BHP in drinking water for 12 weeks, then maintained without further treatment until killed at the end of week 25. A total of 25 lung adenocarcinomas were obtained and total RNAs were extracted from each for assessment of aberrant transcription of the FHIT gene by reverse transcription (RT)-polymerase chain reaction (PCR) analysis. Aberrant transcripts were detected in 15 adenocarcinomas (60%) as absence in the regions of nucleotides (nt) -9 to 279, -98 to 279, -98 to 348 or -98 to 447. Genomic DNAs were also extracted from all 25 adenocarcinomas and exons 5-9 were examined for mutations, using PCR-single strand conformation polymorphism (SSCP) analysis and sequencing. A mutation was detected in only one adenocarcinoma (4%), an ACC to ATC (Thr to IIe) transition at codon 76. Southern blot analysis of eight tumors did not show any evidence of gross rearrangement or deletion of the FHIT gene. Western blot analysis revealed reduced expression of Fhit protein in six out of 10 adenocarcinomas (60%). These results suggest that alteration of the FHIT gene may be involved in the development of lung adenocarcinomas induced by BHP in rats.

Interleukin-15 expression is associated with malignant potential in colon cancer cells.

Interleukin 15 (IL-15 mRNA expression was detected in human colorectal cancer cells (Colo320, WiDr, TCO and DLD1) by the reverse transcriptase-polymerase chain reaction (RT-PCR). Only Colo320 and WiDr cells secreted IL-15 culture medium. With IL-15 treatment, all cell lines grew at a rate of 120-180% of that of nontreated cells. A binding assay with (125)I-labeled IL-15 showed binding activity to IL-15 in Colo320 (K(d): 0.098 nM) cells. IL-15 also reversed the growth inhibition caused by serum starvation in Colo320 cells. IL-15-induced cell growth in regular and serum-free media was abrogated by anti-IL-15 antibody treatment in Colo320 cells. Moreover, IL-15 treatment reduced doxorubicin-induced cytostasis and cytolysis in Colo320 cells by 50%. The invasion capacity of IL-15-treated Colo320 cells was 5.3 times that of untreated cells. Immunoblotting showed that IL-15-treated Colo320 cells exhibited downregulation of p21Waf1 and Bax, and upregulation of Bcl-2, phospho-AKT, MMP9/MMP2, and VEGF. Finally, immunostaining of human colon cancer revealed that 33 (70%) of 47 Dukes' C cases showed IL-15 expression in cancer cells, whereas only 16% of Dukes' B cases did (p < 0.0001). IL-15 may play important roles in cell proliferation, invasion, and metastasis of human colorectal cancer.

Elevated expression of transforming growth factor betas and the tumor necrosis factor family in lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine in rats.

Expression of transforming growth factor betas (TGF betas), tumor necrosis factor (TNF) family members, interferons (IFNs), macrophage migration inhibitory factor (MIF) and granulocyte macrophage colony stimulating factor (GM-CSF) in lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats was investigated using a multiprobe RNase protection assay (RPA) followed by densitometric quantification. Male Wistar rats, 6 weeks of age, were given 2,000 ppm BHP in their drinking water for 12 weeks and maintained without further treatment until killed at week 25. Total RNAs were extracted from 15 adenocarcinomas. Four samples of normal lung tissue from untreated rats served as controls. The expression of TGFbeta1, TGFbeta2, TGFbeta3, TNFalpha, TNFbeta and lymphotoxin beta (Ltbeta) was significantly higher in adenocarcinomas than in normal lung tissues. In contrast, MIF was expressed at the same level in neoplasms and normal tissue and no expression of IFNbeta, IFNgamma and GM-CSF was apparent in either adenocarcinomas or normal lung tissues. These results suggest that elevated expression of TGFbetas and TNF family members may contribute to the development and progression of lung adenocarcinomas induced by BHP in rats.

Dimethylarsinic acid induces 8-hydroxy-2'-deoxyguanosine formation in the kidney of NCI-Black-Reiter rats.

Dirnethylarsenic peroxyl radical [(CH(3))(2)AsOO] has been postulated to be responsible for DNA damage induced by dimethylarsinic acid (DMA). In an effort to elucidate the possible mechanism of tumor-inducing potential of DMA, an experiment was designed to investigate the formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a specific marker of oxidative base damage in the kidney tissues of NCI-Black Reiter (NBR) rats. Animals were divided into four groups and administered the vehicle - saline, 5, 10 and 20 mg/kg body weight respectively of DMA by gavage, once a day, 5 days a week, for a period of 4 weeks. DMA induced increase of 8-OHdG levels in the kidney of the rats treated, with the highest level at the dose of 10 mg/kg body weight. Analysis of the kidney for cell proliferation employing PCNA-positive index showed greater proliferation in the tissues of treated rats. However, DMA did not have any influence on apoptosis in this regimen. Histopathological examination of the kidney selections revealed the presence of vacuolated degeneration and dilation of the proximal tubule cells in two groups (10 and 20 mg/kg body weight). This study provides evidence to substantiate the role of DMA in inducing oxidative DNA damage in the kidney.

Increased oxidative DNA damage in mammary tumor cells by continuous epidermal growth factor stimulation.

BACKGROUND: Growth factors can enhance the malignant potential of tumor cells. To examine the relationship between growth factors and tumor progression, we previously established a weakly malignant cell line, ER-1. We found that a 24-hour exposure of ER-1 cells to epidermal growth factor (EGF) induced malignant properties (tumor progression) that were reversible but that, after a 1-month exposure, these changes were irreversible. In this study, we investigated the irreversible changes induced in ER-1 cells by a 1-month exposure to EGF and the possible involvement of oxidative stress. METHODS: ER-1 cells were treated with EGF (100 ng/mL) for 1 month in the presence or absence of an antioxidant, N-acetylcysteine or selenium, and compared with untreated control ER-1 cells. We assessed tumor progression by measuring intracellular peroxide levels, 8-hydroxydeoxyguanosine (a marker for oxidative DNA damage) levels, in vitro invasiveness, and in vivo tumorigenicity and metastatic ability. All statistical tests are two-sided. RESULTS: After ER-1 cells were treated for 1 month with EGF, levels of intracellular peroxide and 8-hydroxyguanosine in the DNA of treated cells were higher than those in the DNA of control cells, and treated ER-1 cells were more tumorigenic and metastatic in vivo and more invasive in vitro than untreated control cells (all P<.001). Levels of 8-hydroxyguanosine in DNA increased as the length of the EGF treatment increased (P<.001). However, when N-acetylcysteine or selenium was added with EGF for 1 month, levels of intracellular peroxide and 8-hydroxyguanosine in DNA were comparable to those in control cells (r =.795). Both tumorigenicity (P =.008) and metastatic ability (P<.001) decreased after addition of N-acetylcysteine or selenium. CONCLUSION: The irreversible changes caused by continuous EGF stimulation of ER-1 cells result from increased oxidative damage in the DNA, which generates tumor cells with more malignant characteristics.

Enhancement of hepatocarcinogenesis initiated with diethylnitrosamine or N-nitrosobis(2-hydroxypropyl)amine by a choline-deficient, L-amino acid-defined diet administered prior to the carcinogen exposure in rats.

Effects of pre-administration of a choline-deficient, L-amino acid-defined (CDAA) diet on hepatocarcinogenesis initiated with diethylnitrosamine (DEN) or N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats were investigated. A pre-administrating period was set as 1 week, because CDAA diet induces liver injuries by this time-point. In a time-course study, male Fischer 344 rats, 6 weeks old, received a 1-week pre-administration of choline-supplemented, L-amino acid-defined (CSAA) or CDAA diet, DEN at a dose of 100 mg/kg body weight by a single intraperitoneal injection, then CSAA or CDAA diet for up to 8 weeks, and were sacrificed 4, 6 and 8 weeks after DEN. CDAA diet administered only after DEN significantly increased the numbers of glutathione S-transferase placental form (GST-P)-positive lesions 4, 6 and 8 weeks after DEN and their sizes 6 and 8 weeks after DEN. CDAA diet administered both before and after DEN similarly increased the numbers and sizes of GST-P-positive lesions, but with a significantly greater degree than obtained by the diet administered only after DEN. In a dose response study, rats received vechicle or DEN, at a dose of 0.001, 0.01, 0.1, 1, 10, 20, 50, 100 or 200 mg/kg body weight, 1 week after the commencement of CSAA or CDAA diet, and sacrificed 8 weeks after vehicle or DEN. The significant increases of the numbers of GST-P-positive lesions were obtained after 50-200 mg/kg body weight of DEN under the CSAA diet administration, whereas those were detected after 10-200 mg/kg under CDAA diet administration. Sizes became significantly larger with only 200 mg/kg body weight of DEN in the CSAA case but with 50-200 mg/kg in the CDAA case. Male Wistar rats received a 1-week pre-administration of CSAA or CDAA diet, vehicle or BHP, at a dose of 600 or 1200 mg/kg body weight, by a single intraperitoneal injection, then CSAA or CDAA diet for 8 weeks, and were then sacrificed. The numbers of GST-P-positive lesions demonstrated significant increment with 1200 mg/kg body weight of BHP by CDAA diet administered only after BHP and, to a significantly greater degree, by the diet administered both before and after BHP. While CDAA diet administered only after BHP did not alter the sizes of GST-P-positive lesions, the diet administered both before and after 600 and 1200 mg/kg body weight of BHP significantly increased the sizes of the lesions. These results indicate that the pre- plus post-administration of CDAA diet enhances hepatocarcinogenesis initiated with DEN or BHP, more than the post-administration only, thus providing a sensitive model to detect weak liver carcinogenic potency of environmental chemicals.

Dietary choline restriction causes complex I dysfunction and increased H(2)O(2) generation in liver mitochondria.

Removal of choline from the diet results in accumulation of triglycerides in the liver, and chronic dietary deficiency produces a non-genotoxic model of hepatocellular carcinoma. An early event in choline deficiency is the appearance of oxidized lipid, DNA and protein, suggesting that increased oxidative stress may facilitate neoplasia in the choline deficient liver. In this study, we find that mitochondria isolated from rats fed a choline-deficient, L-amino acid defined diet (CDAA) demonstrate impaired respiratory function, particularly in regard to complex I-linked (NADH-dependent) respiration. This impairment in mitochondrial electron transport occurs coincidentally with alterations in phosphatidylcholine metabolism as indicated by an increased ratio of long-chain to short-chain mitochondrial phosphatidylcholine. Moreover, hydrogen peroxide (H(2)O(2)) generation is significantly increased in mitochondria isolated from CDAA rats compared with mitochondrial from normal rats, and the NADH-specific yield of H(2)O(2) is increased by at least 2.5-fold. These findings suggest an explanation for the rapid onset of oxidative stress and energy compromise in the choline deficiency model of hepatocellular carcinoma and indicate that dietary choline withdrawal may be a useful paradigm for the study of mitochondrial pathophysiology in carcinogenesis.

Age and organ dependent spontaneous generation of nuclear 8-hydroxydeoxyguanosine in male Fischer 344 rats.

8-Hydroxydeoxyguanosine (8-OHdG) is a major oxidative DNA adduct playing roles in senescence, carcinogenesis and various disease processes. High-performance liquid chromatography with an electrochemical detection (HPLC-ECD) method has been widely used to assess organ levels of 8-OHdG, and a recently introduced immunohistochemical approach has made it possible to clarify intra-organ localization. In the present study, these methods were employed to reveal age-dependent changes in nuclear 8-OHdG within various tissues of male Fischer 344 rats between 18 fetal days and 104 weeks of age. 8-OHdG was detected in the nuclei of cerebellar small granule and small cortical cells, cerebral nerve cells, and choroid plexus epithelia of the brain and ependymal cells of the spinal cord; parenchymal cells in the anterior lobe of the pituitary and adrenal glands (mainly cortex); bronchial epithelium of the lung; intra-hepatic bile duct, pancreatic duct, glandular gastric and intestinal epithelial cells; renal tubular epithelial cells (mainly medulla); and spermatogonia and spermatocytes of the testis and seminal vesicle epithelia. The nuclear 8-OHdG levels were high (more than two lesions per 10(6) deoxyguanosines) from 7 days to 104 weeks of age in the brain, 3 to 6 weeks in the adrenal gland, 6 to 104 weeks in the lung, and 3 to 52 weeks in the testis. In the other organs, the nuclear 8-OHdG levels remained low throughout. These findings provide a basis for research dealing with oxidative stress by indicating organ-specific and age- but not aging-dependent changes in the localization of spontaneously generated nuclear 8-OHdG in intact rats. The immunohistochemical approach has advantages for assessing variation of 8-OHdG formation at the cellular level not accessible to the HPLC-ECD method.

Hepatotoxicity and consequently increased cell proliferation are associated with flumequine hepatocarcinogenesis in mice.

It has been reported that flumequine (FLU) induces hepatic tumors in mice when given orally for 18 months. We investigated possible underlying mechanisms using a two-stage mouse hepatocarcinogenesis model. After initiation with a single intraperitoneal injection of 100 mg/kg body weight diethylnitrosamine (DEN) or saline, male CD-1 mice were given 4000 ppm FLU in the diet or 500 ppm phenobarbital (PB) in drinking water for 9, 19, 24 or 30 weeks. Toxicity, evidenced by centrilobular swollen and polar hepatocytes with fatty droplets, infiltration of inflammatory cells and increased numbers of mitosis in hepatocytes, was apparent in the livers of mice treated with FLU at all time points, but its severity declined towards the termination. FLU did not induce cytochrome P-450 enzymes such as 1A1, 2B1 and 3A2 as assessed immunohistochemically, while positive expression of 8-hydroxy-2'-deoxyguanosine (8-OHdG) was increased in hepatocytes of both DEN + FLU and FLU groups compared with the relevant controls. In animals given PB, eosinophilic swelling of hepatocytes was prominent, and the hepatocytes showed strongly positive reactions for CYP 1A1 and 3A2. Altered cell foci were induced in the livers of FLU-treated animals both with and without DEN initiation, especially the former, and their development paralleled the degree of hepatic toxicity. These results suggest that FLU hepatocarcinogenicity in mice is dependent on hepatotoxic damage and consequently increased cell proliferation. Oxidative damage to DNA may also be a crucial factor.

Single treatment with cisplatin or UFT, but not their combination treatment enhances the metastatic capacity of mouse fibrosarcoma cells.

To elucidate the roles of chemotherapeutic agents in tumor progression, we examined whether cis-diamminedichloroplatinum (II) (cisplatin) and UFT would promote malignant progression of a weakly tumorigenic and poorly metastatic fibrosarcoma cell line OR-32SK in vivo. C57BL/6 mice, transplanted with QR32SK, were treated with either cisplatin or UFT alone and in combination. After treatment with or without cisplatin and/or UFT, we established in vitro culture cell lines from the tumors arising in the mice on day 21 and then i.v. injected the established cell lines into syngeneic mice. As a result, the cell lines established from cisplatin-treated mice and UFT-treated mice had significantly higher metastatic capacity in lung compared to the ones from control untreated mice (64 and 65%, respectively, versus 26.7%). The cell lines established from the mice with the combination therapy showed lower lung metastasis (11%) than the ones from control untreated mice. Furthermore, we found the incidences of these experimental metastases were closely related with in vitro chemotactic activities and the production of MMP-9 of the cultured cell lines. These results indicate that cisplatin and UFT as a single agent promote the chemotactic activities and the production of MMP-9 in non-metastatic fibrosarcoma cells, resulting in the conversion to highly metastatic ones, and that cisplatin and UFT in combination failed to promote the chemotactic activities and the conversion. These results suggest that the combination therapy with cisplatin and UFT is useful in preventing the emergence of more malignant tumor cells after chemotherapy.

Inflammatory cell-mediated tumour progression and minisatellite mutation correlate with the decrease of antioxidative enzymes in murine fibrosarcoma cells.

We isolated six clones of weakly tumorigenic fibrosarcoma (QR) from the tumorigenic clone BMT-11 cl-9. The QR clones were unable to grow in normal C57BL/6 mice when injected s.c. (1x10(5) cells). However, they formed aggressive tumours upon co-implantation with a 'foreign body', i.e. a gelatin sponge, and the rate of tumour take ranged from 8% to 58% among QR clones. The enhanced tumorigenicity was due to host cell-mediated reaction to the gelatin sponge (inflammation). Immunoblot analysis and enzyme activity assay revealed a significant inverse correlation between the frequencies of tumour formation by QR clones and the levels of manganese superoxide dismutase (Mn-SOD, P<0.005) and glutathione peroxidase (GPchi, P<0.01) in the respective tumour clones. Electron spin resonance (ESR) revealed that superoxide-scavenging ability of cell lysates of the QR clone with high level of Mn-SOD was significantly higher than that with low level of the antioxidative enzyme in the presence of potassium cyanide, an inhibitor for copper-zinc superoxide dismutase (CuZn-SOD) (P<0.001). Minisatellite mutation (MSM) induced by the inflammatory cells in tumour cells were investigated by DNA fingerprint analysis after QR clones had been co-cultured with gelatin-sponge-reactive cells. The MSM rate was significantly higher in the subclones with low levels of Mn-SOD and GPchi (P<0.05) than in the subclones with high levels of both enzymes. The MSM of the subclones with low levels of both enzymes was inhibited in the presence of mannitol, a hydroxyl radical scavenger. The content of 8-hydroxydeoxyguanosine (8-OHdG) by which the cellular DNA damage caused by active oxygen species can be assessed was significantly low in the tumours arising from the QR clone with high levels of Mn-SOD and GPchi even if the clone had been co-implanted with gelatin sponge, compared with the arising tumour from the QR clone with low levels of those antioxidative enzymes (P<0.001). In contrast, CuZn-SOD and catalase levels in the six QR clones did not have any correlation with tumour progression parameters. These results suggest that tumour progression is accelerated by inflammation-induced active oxygen species particularly accompanied with declined levels of intracellular antioxidative enzymes in tumour cells.

Endogenous liver carcinogenesis in the rat.

Carcinogenesis may be effected not only through exposure to exogenous stimuli but also by genetic and epigenetic influences derived from endogenous factors. In the latter case, the mechanisms are still largely obscure because of the limited availability of appropriate in vivo experimental models. However, continuous feeding of a diet deficient in choline and methionine is well known to cause hepatocellular carcinomas (HCC) in rats in the absence of any known exogenous carcinogens and can serve as a good research model. A semi-synthetic, choline-deficient, L-amino acid-defined (CDAA) diet, containing practically no choline and low methionine, induces HCC with a background of fatty liver and hepatocyte death, subsequent regeneration and fibrosis resulting in cirrhosis. Using the CDAA diet, we have revealed the participation of oxidative injury to DNA and other subcellular components and of alteration in intrahepatic signal transduction pathways in the mechanisms underlying this rat liver carcinogenesis model. In the present paper, the current understanding of endogenous rat liver carcinogenesis, due to dietary choline deficiency, is reviewed.

Increased telomerase activity in intrahepatic cholangiocellular carcinomas induced by N-nitrosobis(2-oxopropyl)amine in hamsters.

Telomerase activities in intrahepatic cholangiocarcinomas induced by N-nitrosobis(2-oxopropyl)amine (BOP) in female hamsters were determined using a telomeric repeat amplification protocol (TRAP) assay followed by densitometric quantification. Each determination was repeated to confirm the results and telomerase activity was also detected by gel electrophoresis. An increase was evident in all of 10 cholangiocarcinomas examined, with levels ranging from 2.48 to 4.40 times the normal liver value by densitometric quantification. This finding of a consistent increase suggests that telomerase activation is involved in the development of intrahepatic cholangiocarcinomas and immortalization of cancer cells.