Effect of Combination of Anticancer Agents and Nitroimidazoles on the Survival of Human Hepatocellular Carcinoma Cells under Hypoxic Conditions

PURPOSE: In a previous study, we have shown that anticancer agents inhibiting topoisomerases improve survival of tumor cells under hypoxic condition. In the present study, we evaluated whether and how cell survival effect of the anticancer agents under hypoxic conditions could be eliminated by the addition of nitroimidazoles, a class of bioreductive agents. METHODS: Human hepatocellular carcinoma cells (HepG2) were incubated with different combinations of pimonidazole (1~1,000 microg/ml) and doxorubicin (0.1 or 1 microg/ml) concentrations under different O2 concentrations [1, 3, 5, 10 and 21 O2]. Then cell numbers, glucose concentrations and lactic acid concentrations in the medium were measured, and DNA fragmentation assay was performed. Finally, different combinations of nitroimidazoles, such as pimonidazole, misonidazole, etanidazole, tinidazole, metronidazole, ornidazole or dimetridazole, and anticancer agents, such as doxorubicin, campothecin, epirubicin, dactinomycin, etoposide or mitomycin C was added to the cell culture medium under hypoxic conditions (1% O2). RESULTS: Pimonidazole at a concentration of 100 microg/ml eliminated cell survival effect of doxorubicin at the concentrations of 0.1 and 1 microg/ml under hypoxic condition (1% O2) by promoting apoptosis. Almost all the cells died even after 24 hours of incubation for all the oxygen concentrations at a combination of 100 microg/ml pimonidazole and 1 microg/ml doxorubicin. Finally, pimonidazole at a concentration of 100 microg/ml, and misonidazole or etanidazole at a concentration of 1,000 microg/ml eliminated cell survival effect of all the anticancer agents tested under hypoxic condition. CONCLUSION: Combination therapy of doxorubicin (adriamycin) with pimonidazole can maximize dororubicin efficacy by eliminating cell survival effect of doxorubicin under hypoxic conditions in treating solid tumors, such as breast cancer.

Arylamine N-methyltransferase and thiol methyltransferase activities in cholestatic rat liver induced by common bile duct ligation

Methylation catalyzed by methyltransferases is a major metabolic pathway for an inactivation of some catecholamines, niacinamide as well as aliphatic sulfhydryl drugs and toxic hydrogen sulfides. To investigate the effects of obstructive jaundice in an animal model, common bile duct ligation (CBDL) was performed in the rat and enzyme activities of S-adenosyl-L-methionine-dependent arylamine N-methyltransferase and thiol methyltransferase were examined in liver cell fractions and serum for a period of 42 d after CBDL. Both mitochondrial and microsomal arylamine N-methyltransferase showed significant increases in their activities between the 1st through the 7th day (P < or = 0.05 to 0.001), and between the 1st through the 28th day (P X or = 0.01 to 0.001) post-ligation, although the cytosolic arylamine N-methyltransferase activity did not show a significant change compared to the activities from the sham-operated control. The mitochondrial as well as microsomal thiol methyltransferase showed significant increases in their activities between the 1st through the 28th day (P < or = 0.05 to 0.01 and P < or = 0.01 to 0.001, respectively) post-ligation, although the cytosolic thiol methyltransferase activity did not show a significant change compared to the activities from the sham-operated control. Arylamine N-methyltransferase and thiol methyltransferase in the serum from cholestatic rats also showed significant increases in their activities between the 1st through 28th day (P < or = 0.01 to 0.001), and between the 0.5th through the 42nd day (P < or = 0.05 to 0.001) post-ligation compared to the sham-operated control, respectively. Enzyme kinetic parameters (Km and Vmax) of hepatic membrane-bound arylamine N-methyltransferase and thiol methyltransferase were analyzed with the preparation from the 7th day post-ligation, using tryptamine or 4-chlorothiophenol as substrates and S-Adenosyl-L-[methyl-3H]methionine as co-substrate. The results indicate that although the Km values were about the same as the sham-operated control, the Vmax values of both enzymes increased significantly (P < or = 0.01 and 0.001, respectively). These results suggest that the biosynthesis of arylamine N-methyltransferase and thiol methyltransferase have been induced in response to obstructive jaundice.

Safety and efficacy of arbutamine stress echocardiography / 대한내과학회지

BACKGROUND: Exercise and pharmacologic stress echocardiography are widely used for detecting coronary artery disease. Arbutamine is a new synthetic mild alpha1-receptor and - receptor agonist developed specifically for stress echocardiography. Arbutamine is superior to dobutamine owing to its enforced chronotropic action than that of dobutamine. We intended to know safety and efficacy of arbutamine stress echocardiography in inducing myocardial ischemia and detecting coronary artery disease. METHODS: We underwent arbutamine stress echocardiography on 52 patients, dobutamine stress echocardiography in 35 patients. Alteration of blood pressure, heart rate, regional wall motion on echocardiography were evaluated. Sensitivity and specificity were determined by coronary angiography for 61 patients(Arbutamine: 31, Dobutamine : 30) RESULTS: 1) Hemodynamic alterations respect to stress agents Baseline Maximal Baseline Maximal Interval for Blood pressure Blood pressure Heartrate Heart rate maximal heartrate Arbutamine 122/70mmHg 138/72mmHg 69BPM 137BPM 8.2 min* Dobutamine 126/73mmHg 136/77mmHg 74BPM 102BPM 11.4 min* (* p < 0.05) 2) Comparison of Arbutamine and Dobutamine in sensitivity Sensitivity(Specificity) Side effects Atropine Arbutamine 80.1% (90%) 33(63.5%) 8(15.4%) Dobutamine 78.2% (71.4%) 21(60%) 7(20%) 3) Side effects of stress agents Hypotension Palpitation, tremor Arrhythmia Chest pain Arbutamine 15(28.8%)* 4(7.7%)* 21(40.4%) 8(9.2%) Dobutamine 3(8.6%)* 9(25.7%)* 12(34.3%) 5(5.7%) (* p < 0.05) 4) Premature ventricular contraction was most common arrhythmia in both group. There was no fatal or significant complication, and most complications were subsided after discontinuation of stress agents. CONCLUSION: Arbutamine is an effective and safe pharmacologic stress agent in detecting myocardial ischemia and superior to dobutamine in increasing heart rate. Sensitivity and specificity of arbutamine were higher than that of dobutamine.

Long-term Follow-Up Results of the Patients with Clinically Inapparent Pericardial Effusion

BACKGROUND: Pericardiocentesis is not routinely recommended in most patients with pericardial effusion (PE), except for patients with cardiac tamponade. However, the long-term follow-up results in patients with clinically not significant PE are few. METHODS: Sixty-five consecutive patients (mean age:57 yrs, 26 males) out of 87 patients with PE, who were clinically not serious, were studied prospectively once in every two month for mean 6 months (2-12 months) without any specific treatment. The amount of PE was measured at the enddiastole period of parasternal long axis view and apical four chamber view. RESULTS: The incidence of insignificant PE in our echocardiographic laboratory is 3.4% (n=87 from 2461). The maximal accumulation site of PE was posterior (n=51, 79%). The next is anterior (n=11, 17%) and right ventricular side (3, 5%). The amount of PE is less (0.37+/-0.17cm vs 0.64+/-0.54cm, p=0.018) in localized PE (n=24, 37%) than that of diffuse form (n=41, 63%), which spreads to more than 2 chambers. The presumptive etiologies of PE were unknown (n=41), heart failure (n=5), myocardial infarction (n=6), viral (n=3), and others (n=10). The amount of PE was decreased from 0.54+/-0.46 cm to 0.30+/-0.26 cm, 0.23+/-0.24 cm, and 0.21+/-0.23 cm 2, 4, and 6 months after intial evaluation, respectively, without any complication. CONCLUSION: The patients with PE, not combining >KERN=

The Usefulness of Pericardial Biopsy to Evaluate the Causes of Pericardial Disease

BACKGROUND AND OBJECTIVES: The identification of a specific etiology of effusive pericardial disease is difficult because of the limited yield of cytologic and microbiologic pericardial fluid analysis. We performed retrospective study to find out whether pericardial biopsy was superior to pericardial fluid analysis in search of the etiology of pericardial effusion. MATERIALS AND METHOD: We reviewed 76 cases of moderate to severe pericardial effusion on which we performed surgical pericardial biopsy from Sep. 1986 to Sep. 1996. The results of pericardial fluid analysis, clinical manifestation, pericardial biopsy were compared retrospectively. RESULTS: 1)Clinical diagnosis of pericardial effusion were as follow:neoplastic disease (7.9%), tuberculosis (72.4%), constrictive pericarditis (17.1%), and others (2.6%). 2)By the percutaneous pericardial biopsy, we confirmed 19 cases (25%). Etiology of 4 cases (5.3%) were malignancy and 15 cases (19.7%) tuberculosis. Fifteen out of 76 patients who were diagnosed by biopsy as tuberculous pericarditis and 28 patients who were suspected as tuberculous pericarditis clinically were treated with antituberculous medications. Ten patients (66.7%) of pathologically diagnosed patients and 18 patients (69.2%) of clinically diagnosed patients showed complete resolution of pericarditis. CONCLUSION: By pericardial biopsy, we only confirmed 19 cases (25.0%). It means that pericardial biopsy is not superior to pericardial fluid analysis in searching of etiology of pericardial effusion. Moreover, it is not sufficient for final diagnosis of pericardial effusion.

Effect of Hemodialysis on Levels of Malondialdehyde and Antioxidant Enzymes in Erythrocytes from Patients with End Stage Renal Disease / 대한신장학회잡지

To clarify the mechanism of the protective effect of hemodialysis on lipid peroxidation in RBC membrane structures, the level of malondialdehyde (MDA) which is the lipid peroxidation product, and the activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase, and glutathione peroxidase (GSH-Px) were determined before and after hemodialysis in the RBCs from 20 patients with end stage renal disease (ESRD), and from 14 healthy subjects. Before dialysis, MDA levels in the RBCs from the patients with ESRD were higher than those from healthy controls. SOD and catalase activities in the RBCs were lower. After hemodialysis, MDA, SOD, and catalase in the RBCs from the patients with ESRD were normalized. These results indicate that hemodialysis treatment is helpful to protect the peroxidative darnage through normalizing the activities of antioxidant enzymes.

Activities of Hepatic Antioxidant Enzymes in Rats during 14 Days of Head-down Suspension as a Model of Simulated Weightlessness

Cells and tissues of human and animal are protected against free radicals by several complex mechanisms including the action of antioxidant enzymes, such as superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase. There had been a few reports that simulated or actual weightlessness induced the decrease in activity of antioxidant enzymes and the increase in lipid peroxidation, The purpose of this study was to observe the time-course variation of antioxidant enzymes activities in rats during 14 days of head-down suspension(HDS) at -45 degrees as a model of simulated weightlessness. During HDS, the hepatic activity of SOD significantly decreased (p<0.05) at 3 day of HDS and then maintained a lower value compared to control horizontal position, GSH-Px activity also significantly decreased (p<0.05) at 3 and 7 day of HDS, thereafter showed a slight increasing trend to control horizontal value. The activity of hepatic catalase increased during HDS and the value at the end of HDS showed significant increase (p<0.05). From these results, there is a possibility that weightlessness induce the increase of oxygen free radicals according to the decrease of some antioxidant enzyme activities. The main scavenger to oxygen free radicals is operated via catalase system in rats during HDS. Therefore, we suggest that it is necessary to administrate the antioxidants for protection of the body against oxygen free radicals during first 1 week after exposure of weightlessness, at least.

Thiosulfate sulfurtransferase and UDP-glucuronosyltransferase activities in cholestatic rat liver induced by common bile duct ligation

We have investigated the effect of cholestasis on the hepatic thiosulfate sulfurtransferase (rhodanese) and UDP-glucuronosyltransferase (UDP-GT) activities in rats. Rhodanese activities in the liver cytosol, mitochondria and microsomal fractions as well as in the rat serum, and UDP-GT activity in the microsome have been investigated for a period of 42 days after common bile duct (CBD) ligation. The cytosolic rhodanese activity showed a significant decrease between the first through the 42nd day, and the mitochondrial activity showed a significant decrease between the 7th through the 42nd day after CBD ligation compared to the activities from the sham operated control, respectively. In the case of microsomal preparation, both rhodanese and UDP-GT also showed significant decrease in their activities after the ligation for the former enzyme between the 14th and the 42nd days, and for the latter enzyme between the third and 42nd days, respectively. On the other hand, the serum rhodanese activity increased markedly soon after the ligation, exhibiting the peak activity after 1 day of CBD ligation with about 4.6-fold increment. The activity subsequently decreased gradually reaching to the control level at the 42nd day post-ligation. Enzyme kinetic parameters of hepatic rhodanese and UDP-GT were analyzed using sodium thiosulfate and p-nitrophenol as substrates, respectively, with the preparations from the 28th day post-ligation. The results indicated that although the K-m values of these enzymes were about the same as the sham-operated control, the V-max values of the both enzymes decreased significantly. These results, therefore, suggest that the biosynthesis of rhodanese and UDP-GT have been reduced in response to cholestasis, and that the elevation of rhodanese activity in the serum is most likely due to leakage from the liver subsequent to CBD ligation.

Induction of Rat Liver gamma-Glutamyl Transpeptidase by Bile Acid Load / 대한간학회지

BACKGROUND/AIMS: In order to elucidate the possible mechanism of increase of y-glutamyl transpeptidase (y-GTP) activity in cholestatic liver and serum was studied. METHOD: Rats were divided into eight groups: Normal, sham operated control, bile duct obstruction (BDO) alone (BDO group), BDO plus taurocholic acid (TCA) injection (BDO plus TCA group), BDO plus tauroursodeoxycholic acid (TUDCA) injection (BDO plus TUDCA group), choledoco-caval shunt (CCS) operation (CCS groups), CCS plus TCA injection (CCS plus TCA group), and CCS plus TUDCA injection (CCS plus TUDCA group). Y-GTP activity was determined in the serum and liver cytosolic, mitochondrial and microsomal preparations isolated from above experimental rats. The values of Km and Vmax in this hepatic enzyme was measured. RESULT: the activities of liver cytosolic and microsomal y-GTP showed a significant increase in the CCS group. The activities of liver cytosolic, mitochondrial and microsomal y-GTP showed a significant increase in the BDO group. And the activity of serum y-GTP showed a marked increase in teth CCS and BDO poups. However, y-GTP activities in the serum and in liver microsomal prepatation rose more rapidly in the BDO group tban CCS. Y-GTP activity in liver cytosolic and microsomal preparatians, and its Vmax value incmmxl significantly in both CCS plus TCA group, and BDO plus TCA group than each control group, such as CCS and BDO group. On the other hand, the values of Km of the hepatic subcellular y-GTP did not change in the all experimental groups. Sennn y-GTP activity increased significantly in both CCS plus 7CA group, and BDO plus TCA group than each control group. However, these serum and hepatic enzyme activities did not change in both CCS plus TUDCA group and BDO plus TUDCA group. CONCLUSIONS: The above results suggest that 7CA stimulates biosynthesis of the y-GTP in the liver. And the elevations of the serum enzymes activity thought to be caused by increase of hepatocyte membrane permeability by a physical property (detergency) of TCA, which cause the enzyme to leak into the blood in large quantities.