Effects of lipopolysaccharide (LPS) and peptidoglycan (PGN) on estradiol production in bovine granulosa cells from small and large follicles.

In cows, postpartum uterine infection due to bacteria that produce lipopolysaccharide (LPS) or peptidoglycan (PGN) leads to ovarian dysfunction. The aim of this study was to determine the effect of LPS and/or PGN on estradiol production from granulosa cells from small and large follicles in the bovine ovary. Granulosa cells from small and large ovarian follicles were exposed to LPS and/or PGN in vitro. LPS inhibited the expression of TLR4, CD14, MD2 and NOD1 genes in FSH-treated granulosa cells from small follicles. LPS suppressed estradiol (E2) production in granulosa cells from small and large follicles, while PGN inhibited E2 production in granulosa cells from large follicles. LPS or PGN did not affect granulosa cell survival. Although LPS alone suppressed E2 production in granulosa cells from small and large follicles, E2 production was not further suppressed when PGN was added to culture medium with LPS alone. Our data demonstrated that susceptibility to LPS or PGN in granulosa cells depends on the follicle developmental stage. The results of the present study suggest that ovarian dysfunction in cows with postpartum uterine infection may be caused by inhibitory effects of LPS and PGN on E2 production in granulosa cells.

Oestradiol enhances plasma growth hormone and insulin-like growth factor-I concentrations and increased the expression of their receptors mRNAs in the liver of ovariectomized cows.

Many metabolic hormones, growth hormone (GH), insulin-like growth factor-I (IGF-I) and insulin affect ovarian functions. However, whether ovarian steroid hormones affect metabolic hormones in cattle remains unknown. This study aimed to determine the effect of sex steroids on the plasma profiles of GH, IGF-I and insulin and their receptors in the liver and adipose tissues of dairy cows. Ovariectomized cows (n = 14) were randomly divided into four groups: control group (n = 3) was treated with saline on Day 0; oestradiol (E2) group (n = 3), with saline and 1 mg oestradiol benzoate (EB) on Day 0 and 5, respectively; progesterone (P4) group (n = 4) with two CIDRs (Pfizer Inc., Tokyo, Japan) from Day 0; and E2 + P4 group (n = 4) with two CIDRs on Day 0 that were removed on Day 6 and were immediately injected with 1 mg EB. The animals were euthanized after the experiment, and liver and adipose tissues samples were quantitatively analysed using real-time PCR for the expression of mRNA for the GH (GHR), IGF-I (IGFR-I) and insulin (IR) receptor mRNAs. Oestradiol benzoate significantly increased the number of peaks (p < 0.05), pulse amplitude (p < 0.05) and area under the curve (AUC; p < 0.01) for plasma GH; moreover, it increased plasma IGF-I concentration (p < 0.05), but it had no effect on the plasma insulin profile. P4 significantly decreased the AUC (p < 0.01), compared with the control group, whereas it did not affect the number of peaks and the amplitude of GH pulses. P4 + E2 did not affect the GH pulse profile. E2 increased the mRNA expression of GHR, IGFR-I and IR in the liver (p < 0.05), whereas both P4 and E2 + P4 did not change their expressions. Our results provide evidence that the metabolic and reproductive endocrine axes may regulate each other to ensure optimal reproductive and metabolic function.

Phase I/II study of twenty-four-hour infusion of irinotecan in combination with oral UFT plus leucovorin for metastatic colorectal cancer.

OBJECTIVE: Irinotecan has, in general, been administered as a 90-min infusion. However, several studies have demonstrated that continuous infusion seems to be a promising method of delivering irinotecan. This phase I/II trial was performed to evaluate the efficacy and safety of continuous infusion of irinotecan combined with UFT plus leucovorin (LV) for metastatic colorectal cancer. METHODS: Escalating doses of irinotecan (90-110 mg/m(2)) were administered by 24-hour infusion on day 1. UFT 300 mg/m(2)/day and LV 75 mg/day were administered orally, in 3 divided daily doses, on days 3-7 and 10-14. The treatment cycles were repeated every 2 weeks. RESULTS: In the phase I study, the maximum tolerated dose of irinotecan was 110 mg/m(2) and the recommended dose for the phase II study was determined to be 100 mg/m(2). Thirty-six patients, including 3 patients at the recommended dose in the phase I study, were evaluated in the phase II study. The common grade 3/4 toxicities were leucopenia, neutropenia, diarrhea and anorexia. The response rate was 63.9%, and the median progression-free and overall survival times were 8.3 and 24.6 months, respectively. CONCLUSION: A 24-hour infusion of irinotecan combined with UFT/LV is feasible and active for metastatic colorectal cancer.

Preoperative radio/chemo-radiotherapy in combination with intraoperative radiotherapy for T3-4Nx rectal cancer.

AIMS: To analyse the results of a single institution experience of combined preoperative radio/chemo-radiotherapy and intraoperative electron-radiation therapy (IORT) for locally advanced rectal cancer and to compare the results with surgery alone retrospectively. METHODS: The study cohort comprised 99 patients with clinical T3-4NxM0 adenocarcinoma of the rectum who had received preoperative radio/chemo-radiotherapy, radical surgery, and IORT [Group I]. Until 1998, 67 patients were treated with radiation only [Group Ia], and after 1999, 32 patients were concurrently given tegafur and uracil (UFT) [Group Ib]. 68 patients with clinical T3-4NxM0 rectal cancer were treated with surgery alone [Group II]. RESULTS: The median follow-up was 67 months in Group I and 83 months in Group II. Local recurrence rate was 2% in Group I, which was significantly lower than 16% in Group II (p=0.002) Both disease-free survival and overall survival in Group I were significantly better than those in Group II (p=0.04, p=0.02, respectively). Sphincter preservation was possible in 78% in Group Ib, which was significantly more than 42% in Group Ia (p=0.002). CONCLUSIONS: The combined preoperative radio/chemo-radiotherapy and IORT for clinical T3-4Nx rectal cancer significantly reduces local recurrence and improves prognosis. Combination of preoperative radiotherapy and oral UFT improves the feasibility of sphincter-preservation.

Intraoperative radiation therapy for curatively resected rectal cancer.

PURPOSE: Intraoperative radiotherapy has been used for local control of locally advanced rectal cancer. The aim of this study was to investigate the efficacy of intraoperative radiotherapy for curatively resected rectal cancer. METHODS: Between 1982 and 1998, intraoperative radiotherapy was administered in combination with curative resection in 78 patients with adenocarcinoma of the middle or lower third of the rectum (intraoperative radiotherapy group). Sixty-two of the patients had received preoperative radiotherapy with 20 Gy. Intraoperative radiotherapy was performed by a new strategy in which an electron beam was administered as uniformly as possible to the entire dissected surface of the pelvis. Retrospective comparisons were made with 248 patients treated by surgery alone during the same period (non-intraoperative radiotherapy group). RESULTS: The differences in tumor stage or surgical procedures between the two groups were not statistically significant. Survival, disease-free survival, and local recurrence-free survival in the intraoperative radiotherapy group were significantly more favorable than in the non-intraoperative radiotherapy group (P = 0.01, P = 0.04, and P = 0.02). Differences in survival were observed in Stage II patients but not in Stage I or Stage III patients. The local failure rate was 2.6 percent in the intraoperative radiotherapy group and 11.3 percent in the non-intraoperative radiotherapy group, and the difference was significant (P = 0.02). The distant metastasis rate was 18.0 percent in the intraoperative radiotherapy group and 19.5 percent in the non-intraoperative radiotherapy group, and the difference was not significant. There was a significantly higher rate of wound infection in the intraoperative radiotherapy group, but no infections were serious. CONCLUSIONS: In patients with adenocarcinoma of the middle or lower third of the rectum, intraoperative radiotherapy to the entire dissected surface of the pelvis reduced local recurrence in Stage II and Stage III patients and improved survival in Stage II patients.

Detection of tumor cells in the portal and peripheral blood of patients with colorectal carcinoma using competitive reverse transcriptase-polymerase chain reaction.

BACKGROUND: In spite of many reports, it remains unclear whether the presence of tumor cells in circulating blood flow predicts a poor prognosis. METHODS: Competitive seminested reverse transcriptase-polymerase chain reaction (RT-PCR), a technique for the quantitative detection of tumor cells, was applied to detect the presence of tumor cells in portal and peripheral blood samples from 121 patients with colorectal carcinoma and to clarify their clinical significance. This technique can detect one carcinoembryonic antigen (CEA) mRNA-expressing tumor cell in 1 x 10(5) normal lymphocytes. RESULTS: Six of 33 healthy volunteers (18%) demonstrated a positive reaction to this technique. CEA mRNA expression was detected in the portal blood in 51% of patients and in the peripheral blood in 42% of patients. The results from the two blood samples were consistent in 91% of patients. The positive expression rates for portal blood in patients with T1 tumors and those with TNM Stage I disease were 38% and 45%, respectively. The positive rate was significantly higher in patients with colon carcinoma and those with Stage III or IV disease. CEA mRNA expression, quantitatively measured (x 10(-8)/beta-actin), was 22.9 +/- 35.1 in the portal blood and 19.9 +/- 40.0 in the peripheral blood, with no statistically significant difference. A significant positive correlation was noted between portal and peripheral CEA mRNA expression levels according to Spearman correlation analysis (correlation coefficient = 0.78; P < 0.01). Multivariate analysis revealed that the positive rate and level of CEA mRNA expression in the portal and peripheral blood did not appear to be influenced by the established prognostic factors. CONCLUSIONS: The presence of circulating tumor cells might be of less value as a prognostic factor because they also can be detected in patients with early-stage colorectal carcinoma and appeared to be independent of the conventional prognostic factors.

Promotion of radiation-induced formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine by nitro 5-deazaflavin derivatives.

6-Nitro- and 8-nitro-5-deazaflavin derivatives have been found to enhance prominently the radiation-induced formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) at the expense of formation of 2,6-diamino-4-hydroxy-5-formamidopyrimidine nucleosides (FapydGuo) both in deaerated and in N(2)O saturated aqueous 2'-deoxyguanosine solutions. The radiosensitizing capacity of a 9-nitro-5-deazflavin derivative was observed only in the N(2)O saturated aqueous solutions.

Radiosensitization effect by combination with paclitaxel in vivo, including the effect on intratumor quiescent cells.

PURPOSE: To evaluate the radiosensitization effect on solid tumors upon combination treatment with paclitaxel (TXL), including the effect on intratumor quiescent (Q) cells. METHODS AND MATERIALS: Mice bearing SCC VII or EL4 solid tumors received 5-bromo-2'-deoxyuridine (BrdU) continuously for 5 days to label all proliferating (P) cells. The mice then received gamma-irradiation with or without tirapazamine (TPZ) at various time points after TXL administration. Another group of mice received a series of test doses of gamma-rays while alive or after tumor clamping to obtain hypoxic fractions (HFs) in the tumors at various time points after TXL administration. Immediately after irradiation, the tumor cells were isolated and incubated with a cytokinesis blocker. The micronucleus (MN) frequency in cells without BrdU labeling (Q cells) was determined using immunofluorescence staining for BrdU. Meanwhile, 6 h after irradiation, the tumor cells were isolated from the solid tumors in another group of mice, and the apoptosis frequency in Q cells was also determined with immunofluorescence staining for BrdU. The MN and apoptosis frequency in total (P + Q) tumor cells were determined from the tumors that were not pretreated with BrdU. For the measurement of the HFs, the MN or apoptosis frequency of Q cells was then used to calculate the surviving fraction of Q cells from the regression line for the relationship between the MN or apoptosis frequency and the surviving fraction of total tumor cells. RESULTS: In both SCC VII and EL4 tumors, maximum values of mitotic index (MI) and apoptosis frequency were observed 9 and 24 h after TXL administration, respectively. However, on the whole, the apoptosis frequency for SCC VII was very low. gamma-Irradiation 9 h after TXL administration induced significant radiosensitization effects on the total cells of both tumors. Irradiation at 60 h had a more significant effect on total cells of EL4 tumor, but no significant effect on total cells of SCC VII tumor. Combined treatment with TXL induced no radiosensitization effect on Q cells in either tumor. The effect on Q cells was observed only after TPZ was administered. The HF of total cells in EL4 tumors decreased significantly 60 h after TXL administration. CONCLUSION: No radiosensitization effect upon combination treatment with TXL is induced in Q tumor cells. However, the effect on P cells is produced by irradiation at the time when the maximum values of MI are induced following TXL administration. In addition, for tumors that are susceptible to apoptosis after TXL administration alone, irradiation at the time of sufficient reoxygenation in tumors after TXL administration produces a greater radioenhancement effect on P cells.

Neurotoxicity of gadolinium contrast agents for magnetic resonance imaging in rats with osmotically disrupted blood-brain barrier.

The neurotoxicity of intravenously injected Gadolinium (Gd) complexes to rats with disrupted blood-brain barrier (BBB) was evaluated. After disruption of the BBB by infusion of mannitol solution, one of several contrast agents tested was injected intravenously at a dose of 1 or 3 mmol Gd/kg, and neurological symptoms were graded. The concentrations of Gd in brain and plasma were also measured. Injection of Gd-DTPA at a dose of 3 mmol Gd/kg did not change behavior. On the other hand, Gd-DTPA-BMA, Gd-DO3A-butrol, and Gd-DO3A-HP each induced behavioral impairments, and some animals died within 1 h after injection. Gd-DO3A-HP showed lethal effect even at a dose of 1 mmol/kg. The concentration of Gd in the brain of the animals injected with Gd-DO3A-HP at 3 mmol Gd/kg was essentially the same as that of animals injected with Gd-DTPA at the same dose. The neurotoxicity of the contrast agents tested was graded as follows: Gd-DTPA < or = Gd-DTPA-BMA = Gd-DO3A-butrol < Gd-DO3A-HP.

Intracarotid blood pressure changes during contrast medium injection.

PURPOSE: To investigate changes in blood pressure during intracarotid injection of contrast material. METHODS: Two catheters were inserted into the ipsilateral common carotid artery of dogs. The proximal catheter was used for injection of contrast material and the other was positioned distally to monitor blood pressure. RESULTS: Distal intracarotid pressure rose significantly during injection of contrast material at all rates (5, 7, and 10 mL/s) and at all doses (0.2, 0.5, 0.7, and 1.0 mL/kg). In addition, these blood pressure elevations were shown to be correlated with injection rates and doses. Even when the pressure-monitoring coaxial catheter was advanced into smaller arteries, no decline in the impact of injection was observed. CONCLUSION: Intracarotid injection of contrast material causes a temporal elevation of cerebral blood pressure in dogs.

Comparison of iopamidol and ioversol in vitro and in animal studies.

In the present series of studies we investigated differences in vitro and in animal experiments between iopamidol (Iopamiron, CAS 60166-93-0) and ioversol (CAS 87771-40-2). The studies included the in vitro investigations partition coefficient, lysozyme inhibition, coagulation time and erythrocyte morphology as well as the in vivo paradigms acute toxicity, neural toxicity, general behavior/locomotor activity and angiography. Iopamidol was superior to ioversol in most of the tests. In spite of its higher hydrophilicity, ioversol did not show improved tolerance in comparison to iopamidol.

A convenient screening test for hypoxic cell radiosensitizers/cytotoxins.

A convenient in vitro screening test using E. coli B/r for evaluating a variety of hypoxic cell radiosensitizers/hypoxic cell cytotoxins has been developed for the initial selection of candidates in medicinal/organic chemistry laboratories. E. coli cells were used for convenience since: (1) the bacterium is grown using commercially available broths, where it multiplies rapidly, and requires little specialized equipment for growth and handling. (2) More is known about the genetics and biochemistry of the radiation damage to these cells and their repair than any other organism.

In vivo radiosensitizing effect of nitroimidazole derivative KIN-804.

PURPOSE: In vivo characteristics of 2-nitroimidazole-1-methylacetohydroxamate (KIN-804), which is a newly developed hypoxic cell radiosensitizer, are presented. METHODS AND MATERIALS: The toxicity, pharmacokinetics, and radiosensitizing effect of KIN-804 were studied by in vivo experiments using C3H/He mice bearing the SCC-VII tumor. Results were compared with misonidazole (MISO). RESULTS: LD50(7) of KIN-804 and MISO were 3200 mg/kg and 2000 mg/kg, respectively. The peak of concentrations of KIN-804 in the tumor occurred 20 min after intraperitoneal injection and reached about 62% of the maximum concentration in the blood. The concentrations in brain and sciatic nerve were very low and clearance from sciatic nerve was rapid. Enhancement ratios of KIN-804 calculated using the growth delay method were 1.22, 1.50 and 1.71 at doses of 50, 100, and 200 mg/kg, respectively, compared with 1.36 for MISO at a dose of 100 mg/kg. In the TCD50 assay, enhancement ratios at a dose of 200 mg/kg were 1.69 for KIN-804 and 1.52 for MISO, respectively. CONCLUSION: KIN-804 is a promising radiosensitizer since it shows less toxicity and higher radiosensitizing activity than MISO.

Electrochemical prevention of marine biofouling with a carbon-chloroprene sheet.

A carbon-chloroprene sheet (CCS) electrode was used for the electrochemical disinfection of the marine gram-negative bacterium Vibrio alginolyticus. When the electrode was incubated in seawater containing 10 cells per ml for 90 min, the amount of adsorbed cells was 4.5 x 10 cells per cm. When a potential of 1.2 V versus a saturated calomel electrode was applied to the CCS for 20 min, 67% of adsorbed cells were killed. This disinfection was due to the direct electrochemical oxidation of cells and not to a change in pH or to the generation of toxic substances, such as chlorine. In a 1-year field experiment, marine biofouling of a CCS-coated cooling pipe caused by attachment of bacteria and invertebrates was considerably reduced by application of a potential of 1.2 V versus a saturated calomel electrode. Since this method requires low potential electrical energy, use of a CCS coating appears to be a suitable method for the clean prevention of marine biofouling.

Cerebral embolization in rats induced by red blood cells treated with hypertonic X-ray contrast medium.

RATIONALE AND OBJECTIVES: Hypertonic X-ray contrast media induce morphologic changes in red blood cells (RBCs) and reduce their deformability when measured in vitro. This study investigated whether RBCs treated with hypertonic contrast media could induce circulatory as well as metabolic disturbances in vivo. METHODS: Autologous blood was mixed with an equal volume of meglumine diatrizoate (306 mgI/mL); 50 microliters of this mixture was infused into the left internal carotid artery of rats 2 minutes after mixing. Five minutes after infusion, the cerebral blood flow was determined using iodo[14C]antipyrine. 31P-magnetic resonance spectroscopy (MRS), 1H-MRS and 1H-magnetic resonance imaging (MRI) of the brain were performed before and after infusion of the mixture to study changes in energy metabolites and the integrity of the blood-brain barrier (BBB), as well as the development of edema. RESULTS: The blood flow decreased by 70% to 80% in the left cerebral cortex and caudate-putamen. 31P-MRS and 1H-MRS demonstrated derangement of oxidative phosphorylation. 1H-MRI demonstrated instant destruction of the BBB and gradual progress of edema in the left hemisphere. CONCLUSIONS: These results indicate that intracarotid infusion of RBCs treated with hypertonic contrast medium can induce embolization and subsequent ischemic damage to the brain.

Radiosensitization by a new potent nucleoside analog: 1-(1',3',4'-trihydroxy-2'-butoxy)methyl-2-nitroimidazole(RP-343).

PURPOSE: A new hypoxic cell sensitizer has been synthesized; this is a 2-nitroimidazole nucleoside analog having erythritol as a sugar moiety at the N-1 position of the imidazole ring (RP-343). Its possibility as a potent hypoxic cell sensitizer was compared with those of RP-170 and etanidazole. METHODS AND MATERIALS: Radiosensitization was tested in two murine tumors, EMT6 using in vitro and in vivo-in vitro assays and SCCVII using growth delay and TCD50 assays. Pharmacokinetic study was performed in Balb/c mice bearing EMT6 tumors and in Beagle dogs. LD50 of each sensitizer was obtained with ICR mice. RESULTS: As might be expected from the almost identical electron affinities of the three sensitizers, they were equally effective against hypoxic EMT6 cells in vitro. While having the lowest partition coefficient (0.035), RP-343 exhibited almost equally effective distribution to tumors and sensitizing radiation activity. An intravenous (i.v.) injection of 100 mg/kg of RP-343, RP-170 and etanidazole showed an almost equal sensitizer enhancement ratio (SER) of about 1.4 to solid EMT6 tumor under in vivo-in vitro assay and a virtually equal SER of 1.33-1.44 to solid SCCVII tumor under both tumor growth delay assay and TCD50 assay. A great advantage of RP-343 over RP-170 and etanidazole is its very much lower toxicity; their LD50 in mice were > 6.0, 4.3 and 4.8 g/kg, respectively, on i.v. injection. The lower toxicity of RP-343 was supported by its lower concentrations in the brain; the RP-343 AUC for brain was 0.43 times that of RP-170. Three indices were selected to compare the three nitroimidazoles. SER at 5% LD50 doses of RP-343, RP-170 and etanidazole was 1.66, 1.59 and 1.56. At the same toxicity levels, RP-343 was found to have better sensitization of solid tumors over both etanidazole and RP-170. The maximum tumor concentration/AUC for brain (Cmax,tumor/AUCbrain) ratios for RP-343 and RP-170 were 9.62 and 3.98. CONCLUSIONS: This extremely high ratio of RP-343 could explain its lower toxicity than RP-170 or etanidazole. The therapeutic risk index defined as D1.5/LD50 (D1.5 is the sensitizer dose to obtain the SER of 1.5 in vivo) for RP-343, RP-170 and etanidazole were 0.022, 0.033 and 0.036, respectively. Especially, the effectively lower therapeutic risk index for RP-343 presents the possibility of clinical advantage over etanidazole.

[Preclinical evaluation of iotrolan as a contrast medium for angiography and urography].

Efficacy and tolerability of iotrolan, a nonionic isotonic dimer, as a contrast medium for angiography and urography were investigated in animals. In the arteriography of rabbit femur, the efficacy of iotrolan 280 mgI/ml was as good as iopamidol 300 mgI/ml and better than meglumine diatrizoate 306 mgI/ml. In rat urography, the efficacy of iotrolan 280 mgI/ml was better than both iopamidol 370 mgI/ml and iohexol 350 mgI/ml. Vascular pain was less with iotrolan 280 mgI/ml than with iohexol 300 mgI/ml in rats. Effect of iotrolan on the pulmo-cardiovascular parameters, arterial pO2, hematocrit and plasma osmolality was less than iopamidol and diatrizoate in rabbits. Iotrolan induced no renal dysfunction and diuresis where iopamidol induced diuresis in rats. Effect of iotrolan on the blood coagulation was similar to nonionic monomers and less than diatrizoate in rabbits. Because of its isotonicity, iotrolan induced little water shift in the blood vessel and urinary tract, which would result in good efficacy and tolerability. These results suggest that iotrolan is superior to ionic and nonionic monomers for angiography and urography.