Hyperlipemia and early pancreatic injury induced by ethanol intake in rats.

The pathogenesis of alcoholic pancreatitis is unknown, and even though hyperlipemia has been hypothesized to be a risk factor for alcoholic pancreatitis, no studies directly investigating whether there is a relationship between the two have ever been reported. Therefore, to determine if a relationship exists between hyperlipemia and alcoholic pancreatitis, especially the early stage of alcoholic pancreatic injury, we administered a regular liquid Lieber-DeCarli diet, with and without ethanol as 35% of total calories, to rats for 2 wk. Thereafter we measured their plasma lipid concentrations, pancreatic zymogen granule fragility, and plasma lipase activity and subsequently investigated the correlations between these parameters. Significant increases in plasma triglyceride, total cholesterol, phospholipid, nonesterified fatty acid, pancreatic zymogen granule fragility, and plasma lipase activity were observed in the ethanol liquid diet group, compared with the values of the control liquid diet group, and pancreatic zymogen granule fragility was correlated with plasma triglyceride (r=0.62), total cholesterol (r=0.77), phospholipid (r=0.76), nonesterified fatty acid concentrations (r=0.62), and lipase activity (r=0.63). These results show a possible relationship between hyperlipemia and the early stage of alcoholic pancreatic injury, and they may support the hypothesis that hyperlipemia contributes to the etiology of alcoholic pancreatitis.

Oral administration of sepimostat mesilate prevents acute alcohol pancreatic injury in rats.

The preventive effect of a novel synthetic serine protease inhibitor, sepimostat mesilate (sepimostat), on acute alcohol pancreatic injury, induced by exocrine hyperstimulation and ethanol administration, was assessed and compared with that of a similar protease inhibitor, camostat mesilate (camostat). Conscious rats were infused with 1 microg mL(-1) h(-1) caerulein intravenously for 6 h and with 0.1 g mL(-1) h(-1) ethanol for 9 h, with the latter infusion beginning 3 h after the start of the caerulein infusion. Sepimostat or camostat was administered orally 1 h before the caerulein infusion. Rats infused with caerulein plus ethanol showed increased plasma amylase and lipase activities, and aggravated pancreatic interstitial oedema when compared with rats given caerulein alone. Sepimostat at 10 and 30 mg kg(-1) prevented the increase in plasma amylase and lipase activities caused by caerulein plus ethanol infusion. Sepimostat at 30 mg kg(-1) suppressed the histological change. Camostat did not show any preventive effects at the equivalent dose. When conscious rats were infused with 1 microg mL(-1) h(-1) caerulein alone intravenously for 6 h, plasma amylase and lipase activities were increased compared with rats given saline. Neither drug prevented the increase in these activities at 30mg kg(-1). Our results suggest that sepimostat has superior preventive effects on alcohol-induced acute pancreatic injury compared with camostat. Sepimostat may thus be a useful drug in the therapy of alcohol-induced pancreatitis.

Ethanol administration delays recovery from acute pancreatitis induced by exocrine hyperstimulation.

Alcohol consumption causes acute alcohol pancreatitis and worsens the prognosis; however, there is no useful model for elucidation of the mechanism underlying this worsening. The aim of our study was to establish a new prognostic model of acute alcohol pancreatitis in rats. To ascertain the effect of continuous infusion of ethanol on each phase, i.e., progression and recovery, in caerulein-induced pancreatic injury in rats, we infused a physiological or supramaximal dose of caerulein intravenously to conscious Wistar rats for up to 6 h (time: 0-6 h) with or without ethanol infusion for 9 h (time: 3-12 h). Ethanol did not induce the pancreatic injury alone or when combined with a physiological dose of caerulein. In the progression phase, ethanol infusion for 3 h (time: 6 h) did not aggravate the pancreatic injury induced by a supramaximal dose of caerulein in terms of plasma amylase and lipase activities but did increase the pancreatic calcium level. In the recovery phase, however, ethanol infusion for 9 h (time: 12 h) significantly restrained the recovery from pancreatic injury as monitored in terms of these activities. Further, ethanol infusion for 9 h significantly increased the cumulative urinary excretion of amylase from 12 to 27 h but did not do the same from 0 to 12 h. In the histological evaluation at 27 h, the induction of acinar cell vacuolization and dilation of the glandular lumina and ducts were significant in the caerulein plus ethanol-treated group. Our findings suggest that ethanol administration delays the recovery rather than worsens the progression in acute pancreatic injury induced by exocrine hyperstimulation, and we consider our experimental model to be a useful tool for studying the pathogenesis of worsening prognosis in acute alcohol pancreatitis.

Augmentation of the chemotherapeutic effectiveness of UFT, a combination of tegafur [1-(2-tetrahydrofuryl)-5-fluorouracil] with uracil, by oral l-leucovorin.

UFT, combination of tegafur [1-(2-tetrahydrofuryl)-5-fluorouracil] with uracil, is widely-used as an anti-neoplastic agent in Japan. We evaluated the anti-tumor efficacy of the combined modality of UFT with oral l-leucovorin. The augmentation of anti-tumor activity of UFT by co-administration of l-leucovorin was observed over a dose of 1.85 mg/kg (5.55 mg/m2) and was significant at a dose of 5.56 mg/kg (16.7 mg/m2). Using ten human tumor xenografts, l-leucovorin significantly enhanced the growth-suppressive ability of UFT against colon carcinoma (KM20C, Col-1) and mammary carcinoma (H-31, MX-1). Among various 5-fluorouracil (FUra) derivatives, such as UFT, 5'-deoxy-5-fluorouridine (5'-DFUR) and FUra, l-leucovorin gave the maximum augmentation to the anti-tumor activity of UFT, due to the prolonged half-life of FUra in plasma. Enhancement of the cytotoxic activity of FUra by l-leucovorin against KM20C colon carcinoma cell line was observed in a time-dependent manner at a concentration of 0.01 microM l-leucovorin. Based on these results, we conclude that the combination of UFT with oral l-leucovorin has significant antitumor activity and represents an interesting regimen to be evaluated in the clinical setting.

Activity of menogaril against various malignant lymphoma cell lines and a human lymphoma xenograft in mice.

Menogaril is an antitumor agent different from other anthracyclines in that it is active after oral administration; therefore, extravasation is not a side effect. In this basic study, we examined the antitumor activity of menogaril against malignant lymphoma. We compared its activity towards experimental malignant lymphoma with that of Adriamycin, epirubicin, pirarubicin, vincristine, and etoposide, treating mice with each drug at the dose schedule usually used for patients. Menogaril rapidly penetrated lymphoma cells and remained there at least 3 hours after the drug was washed out. Menogaril cleaved more double-stranded DNA in lymphoma cells than Adriamycin, epirubicin, pirarubicin, or etoposide. Menogaril had stronger antitumor activity against experimental malignant lymphoma in mice than Adriamycin, epirubicin, vincristine, and etoposide. Menogaril significantly lengthened the life span of mice bearing one of three lymphoma cell lines resistant to cisplatin, vincristine, or cyclophosphamide. Menogaril had stronger antitumor activity against the human malignant lymphoma xenograft LM-3 than Adriamycin. The strength of the cytotoxic activity of Menogaril might arise from its ready penetration into cells and its cleavage of double-stranded DNA. Therefore, Menogaril might become a useful drug for the treatment of patients with malignant lymphoma by oral administration; 7 days of administration was effective in the in vivo experiments.

[Augmentation of chemotherapeutic efficaciousness of UFT by oral l-leucovorin--l-leucovorin factors enhancing cytotoxic activity of 5-fluorouracil].

The relationship between augmentation of cytotoxic activity of 5-fluorouracil (FUra) by l-leucovorin (l-LV) and combination schedule was investigated using human colon adenocarcinoma KM 20 C cell line. The enhancement of cytotoxic activity of FUra by l-LV or 5-CH3FH4, a main metabolite after oral administration of l-LV, was observed after 96-hr exposure to either l-LV or 5-CH3FH4 at concentrations of greater 0.001 microM or 0.3 microM, respectively. We found that 1.5 microM FUra, which has no effect on cell-growth without l-LV, could suppress strongly the proliferation if cells were co-treated with l-LV for more than 8 hr. Therefore, enhancement of cytotoxic activity of FUra by l-LV depended on the time cell exposure to l-LV. Moreover, this effect was observed even at a concentration of 0.01 microM l-LV after 48-hr exposure. Likewise, it is known that the antitumor activity of FUra correlates with the time-schedule of treatment with FUra. These results suggest that in addition to FUra derivatives which continuously release FUra, it is beneficial for the combination therapy of FUra with l-LV to consider continuous administration of l-LV as well.

Long-term maintenance of functional rat hepatocytes in primary culture by additions of pyruvate and various hormones.

Mature adult rat hepatocytes were cultured as monolayers in serum-free Williams medium E containing 10(-7) M each of insulin (Ins), dexamethasone (Dex) and triiodothyronine (T3) and 30 mM pyruvate. The hepatocytes remained morphologically intact for at least 14 days, during which period they maintained normal liver functions such as the expressions of cytochrome P-450 mRNA and glucokinase and secretion of albumin. They also retained the ability to resume proliferation. Cells cultured with pyruvate had a much higher ATP level than those without pyruvate, suggesting that pyruvate can sustain functional hepatocytes for a long period in culture in the presence of Ins, Dex and T3, probably by producing enough energy for their maintenance.

Importance of cell aggregation for expression of liver functions and regeneration demonstrated with primary cultured hepatocytes.

Adult rat hepatocytes aggregated to form floating multicellular spheroids when cultured in Primaria dishes, which have a positively charged surface, in serum-free Williams' medium E (WE) supplemented with insulin and epidermal growth factor (EGF). These hormones were essential for maintenance of the spheroids, whereas the size of the spheroids depended on the inoculum cell density. The spheroids retained in vivo levels of expressions of albumin and glucokinase and synthesized scarcely any DNA even in the presence of insulin and EGF. On transfer to type I collagen-coated dishes, the spheroids gradually disaggregated and the cells formed monolayers, in which the expressions of albumin and glucokinase were suppressed and DNA synthesis and hexokinase activity were increased. DNA synthesis of hepatocytes in monolayer culture was maximal 24 hr after transfer of the spheroids, approximately 80% of the hepatocyte nuclei were labelled with bromodeoxyuridine during culture for 48 hr, and the mitotic index was approximately 70% after 60 hr. These results suggest that, in spheroids, hepatocytes remained in the G0 phase, but that when they formed monolayers, they progressed to the G1 phase and proceeded through the cell cycle in the presence of insulin and EGF. This work shows that the cell cycle of hepatocytes in culture can be manipulated by providing conditions for quiescence as spheroids or growth as monolayers and that the shape of hepatocytes is important for regulating their growth and liver-specific functions.

Toxicity of D, L-mandelonitrile-beta-D-glucoside, "prulaurasin" in rat.

Toxicity of cyanogenetic glucoside was studied with rats using synthesized D, L-mandelonitrile-beta-D-glucoside. Considering from blood cyanide level, the cause of death was presumed to be cyanide intoxication. Hydrolysis of cyanogenetic glucoside occurred mainly in small and large intestines. Cyanide level in the intestine seemed to be independent of the amount of glucoside present. LD50 value was calculated to 560 mg/kg but good dose dependent result was not obtained. Relatively rapid absorption of the glucoside from the gastrointestinal tract was observed with the highest absorption rate of 53.4% within 165 min. About 30-45% of the administered glucoside was excreted within 24 hr without alteration and dose dependence appeared in an excreted ratio. The ratio of D-mandelonitrile-beta-D-glucoside to L-form excreted in the urine or remained in the gastrointestinal tract was almost in the same as at administration, and hence biological behaviors, such as absorption, excretion and hydrolysis, of both D- and L-forms of glucoside were almost the same.