Inhibition of N-nitrosodiethylamine-induced hepatocarcinogenesis by Picroliv

Picroliv, an iridoid glycoside mixture prepared from the roots and rhizomes of Picrorhiza kurroa was found to be an effective inhibitor of hepatocarcinogenesis induced by N-Nitrosodiethylamine (NDEA) in rats. Animals administered with NDEA had large hepatic nodules and the liver weight was increased to 6.17 +/- 1.0 g/100 g.b.wt. as compared to the normal liver weight 2.84 +/- 0.08 g/100 g.b.wt. Picroliv administration (200 mg/Kg.b.wt) reduced the liver weight to 3.30 +/- 0.23 g/100 g.b.wt. Oral administration of Picroliv reduced NDEA-induced elevation of gamma-glutamyltranspeptidase (gamma-GT) in serum and liver to that of normal rats. Moreover, elevated levels of bilirubin, alkaline phosphatase (ALP), glutamatepyruvate transaminase (GPT) and serum peroxides were also found to be significantly reduced by Picroliv administration. Similar observations were noticed in glutathione (GSH) and glutathione S-transferase (GST) levels. Histopathological analysis of the Picroliv treated rat liver resembles that of a normal liver except for a few alterations such as hepatocytomegalia and karyomegalia in some focii. The results are indicative of the chemopreventive potential of Picroliv against chemically-induced liver tumours.     

Chemopreventive and Hepatoprotective Effects of Embelin on N-Nitrosodiethylamine and Carbon Tetrachloride Induced Preneoplasia and Toxicity in Rat Liver

Embelin, an active constituent isolated from the fruits of Embelia tsjeriam–cottam was investigated for its chemopreventive and hepatoprotective effects against N-nitrosodiethylamine (NDEA) induced liver preneoplasia or carbon tetrachloride (CCl4) induced liver damage. Rats received NDEA, 1ppm/g b.w. in drinking water for 6 weeks or CCl4, 0.7ml/kg i.p. once a week for 4 weeks and embelin 50mg, 100mg/kg b.w. orally prior, during and after exposure to NDEA/CCl4 for 20 or 5 weeks, respectively. Embelin treatment significantly prevented NDEA or CCl4 induced increase in biochemical marker enzymes: glutamate pyruvate transaminase, glutamate oxaloacetate transaminase, alkaline phosphatase, γ-glutamyl transpeptidase, glutathione-S-transferase, lipid peroxidase as well as hypoproteinemia, hypoalbuminuria and glutathione depletion. This was further substantiated by marked decrease in incidence of preneoplastic foci, and inflammatory cells on histopathological and transmission electron microscopic analysis. The present study suggests embelin is a promising chemopreventive and hepatoprotective agent.     

Effect of Emblica officinalis, Phyllanthus amarus and Picrorrhiza kurroa on N-nitrosodiethylamine induced hepatocarcinogenesis

Extracts of Emblica officinalis (EO), Phyllanthus amarus (P. amarus) and Picrorrhiza kurroa (P. kurroa) significantly inhibited hepatocarcinogenesis induced by N-nitrosodiethylamine (NDEA) in a dose dependent manner. The anticarcinogenic activity of these extracts were evaluated by their effect on tumour incidence, levels of carcinogen metabolizing enzymes, levels of liver cancer markers and liver injury markers. Animals treated with NDEA alone showed 100% tumour incidence and significantly elevated tissue levels of drug metabolizing enzymes such as glutathione S-transferase (GST) and aniline hydroxylase (AH). Treatment of extracts significantly reduced these levels. Levels of gamma-glutamyl transpeptidase (GGT) were also found to be elevated both in serum and tissues of tumour bearing animals, while they were significantly reduced in the treated group. Similar reduction was seen in tissue levels of reduced glutathione. Serum levels of lipid peroxide (LPO), alkaline phosphatase (ALP) and glutamate pyruvate transaminase (OPT), which are markers of liver injury, were also elevated. Morphology of liver tissue and levels of marker enzymes indicated that these extracts offered protection against chemical carcinogenesis.     

Chemopreventive activity of a macrofungus Phellinus rimosus against N-nitrosodiethylamine induced hepatocellular carcinoma in rat

Chemoprevention is an important alternative approach to control cancer. Chemical substances with multiple inhibitory properties would be a welcome addition to the class of chemopreventive drugs. In this study, we investigated the antioxidant, anti-inflammatory, antimutagenic and cancer preventive activities of aqueous extract of a macrofungus Phellinus rimosus (Berk) Pilat. The extract exhibited superoxide anion (O2-), hydroxyl radical (*OH), nitric oxide (NO*) scavenging and lipid peroxidation inhibiting activities. The inhibitory concentrations required by the extract to scavenge 50% (IC50) of the superoxide anion, hydroxyl radical and nitric oxide generated were 126 +/- 5.1, 71 +/- 4.7 and 31 +/- 4.5 microg/ml respectively. The concentration required to inhibit 50% of Fe2+ induced lipid peroxidation in rat liver homogenate was 318 +/- 2.4 microg/ml. The extract showed significant (P<0.05) anti-inflammatory activity in a dose dependent manner. Extract (100 mg/kg body wt, p.o) inhibited 44.5, 45.4 and 47% carrageenen, dextran and formalin induced inflammations respectively. The antimutagenic activity was determined by the Ames' Salmonella mutagenecity assay using histidine mutant Salmonella typhimurium strains. The extract at concentration of 5 mg/plate showed antimutagenecity against benzo[a]pyrene (B[a]P) and 4-nitro-o-pheneylenediamine (NPDA) induced mutations of TA98 and TA100 respectively. Anticarcinogenic activity was evaluated using N-nitrosodiethylamine (NDEA) induced hepatocellular carcinoma (HCC) in rats. Serum gamma glutamyl transpeptidase (GGT), glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT) and alkaline phosphatase (ALP) activities and lipid peroxidation level (MDA) were elevated significantly (P<0.05) in the NDEA alone treated group of animals. Treatment of the extract (25 and 50 mg/kg body wt, p.o.) prior to the NDEA administration decreased the serum GGT, GOT, GPT and ALP activities and MDA level in a dose dependent manner. The NDEA alone treated animals showed altered serum albumin/globulin ratio (A:G ratio), hyperfibrinogenaemia, increased hepatic glutathione S-transferase (GST) activity, glutathione-peroxidsae (GPx) activity and reduced glutathione (GSH) level compared to the extract plus NDEA treated group. The extract also inhibited in vitro aniline hydroxylase (AH) activity of rat liver induced by phenobarbitone in a dose dependent manner. The results, thus suggest the significant chemopreventive properties of the aqueous extract of the Phellinus rimosus against NDEA induced hepatocellular carcinoma by its antioxidant, anti-inflammatory and antimutagenic activities.     

Anti-Mutagenic and Anti-Carcinogenic Potential of the Carotenoid Meso-Zeaxanthin

Meso-zeaxanthin was investigated for antimutagenic and anticarcinogenic activity, using the Ames test(Salmonella typhimurium strains TA 98, TA 100, TA 102 and TA 1535) with direct acting mutagens like sodiumazide (NaN3) (5 μg/ plate), nitro-o-phenylendiamin (NPD) (20 μg/ plate), N-methyl- N’-nitro-N-nitrosoguanidine(MNNG) (1μg/ plate) and tobacco extract 50 mg/ plate) and with a mutagen needing microsomal activation,acetamidofluorene (AAF) ( 20 μg/ plate). The carotenoid was found to inhibit the mutagenicity induced byNaN3, NPD and MNNG in a concentration dependent manner, as well as that with AAF and the tobacco extract.Concentrations needed for 50 % inhibiton was found to be 50 μg/ plate for the chemical mutagens and 100 μg/plate for tobacco extract. Using specific resorufin derivatives as substrates in vitro, the concentration of mesozeaxanthinneeded for 50 % inhibition of CYP1A2 (7-methoxyresorufin-O-demethylase) was 5 μg/ml, for CYP2B1/2 (7- pentoxyresorufin-O-depentylase) was 8 μg/ml and for CYP1A1 (7-ethoxyresorufin-O-deethylase) was 12μg/ml, while that of CYP 2E1 (aniline hydroxylase) was 7μg/ml and for CYP 1A, 2A, 2B, 2D and 3A (aminopyrene-N-demethylase) was 10.5 μg/ml. Evaluated using nitroso diethyl amine (NDEA) induced hepatocellular carcinomain rats, treatment with meso-zeaxanthin reduced the tumor incidence when compared to the control group. Theactivity of glutamate oxaloacetate transaminase, glutamate pyruvate transaminase and alkaline phosphatase wasdrastically elevated in both serum and liver tissue of NDEA alone treated control animals and meso-Zeaxanthinpretreated animals showed significant decrease to normal levels, in line with histopathological findings.     

Comparison of induction of DNA single-strand breaks and initiation of rat hepatocarcinogenesis by different nitrosamines

The capacities of nitrosamines to induce DNA single-strand breaks (SSB) and to initiate carcinogenesis in rat liver were compared. N-Nitrosodiethanolamine (NDELA), N-nitrosoethylhydroxyethylamine (NEHEA) and N-nitrosodiethylamine (NDEA) were equipotent in inducing DNA SSB when administered by gavage at doses of 0.35 mmol/kg, 0.015 mmol/kg and 0.37 mmol/kg, respectively. Male Wistar rats were injected with these nitrosamines and were then submitted to a selection procedure. Ten rats per group were sacrificed one week after the end of the selection to see the effects of the nitrosamines on the development of preneoplastic lesions. The numbers of gamma-glutamyl transferase (GGT)-positive lesions per cm2 were 0.8, 2.1, 5.2 and 40.1 in rats treated with saline, NDELA, NEHEA and NDEA, respectively. N-Nitrosobis(2,2,2-trifluorethyl)amine (6F-NDEA), a nongenotoxic and noncarcinogenic nitrosamine, induced 0.7 GGT-positive lesions per cm2. Ten rats per group also received 0.05% phenobarbital in their drinking-water and were killed six months after initiation in order to see the effect of the different nitrosamines on the incidence and yield of tumours. Two extrahepatic cancers were found after administration of NEHEA, whereas two hepatocellular carcinomas were detected after injection of NDEA. No cancer developed in the other groups. Although other factors may influence the process, these results indicate that no simple correlation can be established between induction of SSB in DNA and initiation of tumours by nitrosamines in rat liver.     

Inhibition of N-nitrosodiethylamine- and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced tumorigenesis in A/J mice by green tea and black tea.

The effect of p.o. administration of tea on nitrosamine-induced carcinogenesis was investigated. Female A/J mice were given N-nitrosodiethylamine (NDEA) (10 mg/kg) p.o. once a week for 8 weeks and were killed 16 weeks after the last dose. More than 90% of the mice had forestomach and lung tumors. The animals had an average of 8.3 forestomach and 2.5 lung tumors/mouse. With 0.63 or 1.25% green tea infusion (12.5 g green tea leaves brewed with 1 liter of boiling water) as the sole source of drinking water for the entire experimental period, the pulmonary tumor incidence was decreased by 18 or 44%, and the tumor multiplicity was reduced by 36 or 60%, respectively. The treatments also decreased the forestomach tumor incidence by 18 or 26% and tumor multiplicity by 59 or 63%, respectively. Administration of 0.63 or 1.25% green tea infusion, either during the NDEA treatment period only or starting 1 week after the completion of NDEA treatment, also decreased the pulmonary tumor incidence and multiplicity and the forestomach tumor multiplicity. The inhibitory effects of green tea infusion were also observed in a similar experiment using a higher dosage of NDEA (20 mg/kg). Treatment of female A/J mice with a single dose (103 mg/kg) of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) resulted in the formation of pulmonary adenomas in almost all of the animals with an average of 9.3 tumors/mouse after 16 weeks. When 0.6% decaffeinated green tea or black tea extract was given during the NNK-treatment period, tumor multiplicity was reduced by 67 or 65%, respectively. When the tea extract was given after the NNK-treatment period until the end of the experiment, 0.6% green tea extract decreased the tumor incidence and multiplicity by 30 and 85%, respectively. In this protocol, 0.6% black tea extract reduced tumor multiplicity by about 63% but did not significantly affect the tumor incidence. The results clearly demonstrated an inhibitory action of green tea and black tea on nitrosamine-induced tumorigenesis.     

Mechanisms of fat-related modulation of N-nitrosodiethylamine-induced tumors in rats: organ distribution, blood lipids, enzymes and pro-oxidant state.

Groups of rats, either dosed with N-nitrosodiethylamine (NDEA) for 10 weeks (from the age of 7 to 17 weeks) or untreated, were fed diets containing either 2% (low fat, LF) or 30% polyunsaturated fat (high fat, HF) on an equicaloric basis from 5 weeks until rats were 43 weeks old. Biochemical parameters were measured during and at the end of the experiment in various organs, blood, urine and exhaled air, for correlation with the presence or absence of tumors. The HF diet tended to increase the number of hepatic tumors induced by NDEA, while the number of extrahepatic tumors was higher in rats fed on the LF diet; also the overall tumor incidence was higher in the LF group. In the HF/NDEA group, only two benign extrahepatic tumors were found. Plasma total and free cholesterol and triglyceride concentrations were lower in the HF than the LF group without NDEA treatment. In animals bearing liver and/or extrahepatic tumors all plasma lipid concentrations were lower than in tumor-free animals. Only minor or no changes were detected in blood catalase activity, malondialdehyde level, reduced glutathione (GSH) level or GSH-related enzymes and excretion of thioethers in the urine due to dietary modulation or NDEA. Changes in the liver that were associated with the HF diet were: (i) increased amounts of some polyunsaturated fatty acids and of total phospholipids in liver microsomes; (ii) an enhanced level of lipid peroxidation in liver; (iii) a decrease in liver glutathione levels during NDEA treatment, with a simultaneous adaptive increase in superoxide dismutase levels, and a decrease in renal glutathione levels in both treated and untreated groups; (iv) enhanced microsomal induction of aminopyrine N-demethylase and epoxide hydrolase activities by NDEA, and (v) decreased hexose monophosphate shunt (HMS) activity. All mono-oxygenase activities were lower, and the activities of epoxide hydrolase, UDP-glucuronosyltransferase and HMS were higher, in liver tumors than in non-tumorous liver of similarly-treated rats. Neither diet nor NDEA had a major effect on drug-metabolizing enzyme activities in lung and kidney. HF diet significantly increased ethane exhalation (an indicator of the whole-body pro-oxidant state) over those on the LF diet: in rats on either diet, it was further increased when NDEA was given. Ethane exhalation was still elevated 30 weeks after the cessation of NDEA treatment. Our results suggest an association between the observed changes in biochemical parameters, notably oxidative stress, due to dietary modulation and the altered tumor incidence and organ distribution of tumors induced by NDEA.     

Clinical pharmacokinetics of epirubicin: the importance of liver biochemistry tests.

The influence of liver biochemistry tests on epirubicin pharmacokinetics has been investigated in 52 women with advanced breast cancer, 27 of whom had radiologically proven liver metastases. Patients received epirubicin 12.5-120 mg m-2 given as an i.v. bolus. Epirubicin levels were measured by HPLC following the first cycle of treatment. Epirubicin elimination, expressed as clearance (dose/AUC), in the 22 patients with normal AST and bilirubin was compared with that of 30 patients with a raised AST +/- raised bilirubin. Epirubicin clearance was significantly reduced in the patients with a raised AST, whether their serum bilirubin was normal (22 patients) or elevated (eight patients). In the 30 patients with a raised AST +/- raised bilirubin, epirubicin clearance correlated strongly with the level of AST (r = -0.72) but not with serum bilirubin, alkaline phosphatase, albumin or creatinine. Using a multiple regression analysis, AST was the only one of these biochemical variables predictive of epirubicin clearance (r2 = 0.47, P = 0.0006). We conclude that a raised serum AST is a more sensitive and reliable measure of abnormal epirubicin pharmacokinetics than increased bilirubin. These findings have implications for anthracycline treatment in patients with abnormal liver biochemistry.     

Biochemical and cellular effects of radiofrequency induced interstitial hyperthermia on normal canine liver

Interstitial hyperthermia (44.0 +/- 0.5 degrees C for 40 minutes) was delivered to the livers of 16 dogs to determine acute effects of treatment on blood chemistry, histology, and cellular appearance of normal liver. SGOT in treated animals peaked immediately at 300 +/- 21 U/L (within 2 hrs) and returned to control value within 7 days. LDH levels peaked at 1 day post-treatment and again at 2 weeks (300 +/- 16 U/L and 340 +/- 25 U/L respectively) and returned to pre-treatment values by week 4. SGPT remained elevated for 6 to 7 days following hyperthermia, but returned to control value at 2 weeks. There was also a rise in alkaline phosphatase (200 +/- 14 U/L 1 day post-treatment), which returned to a pre-treatment level by week 3. Changes in serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, lactate dehydrogenase, and alkaline phosphatase were attributed to both liver parenchymal damage induced by hyperthermia and to surgery. Other deviations in the blood chemistries and hematological parameters measured were ascribed to the stress response from surgical intervention, or to the resultant hemodilution from fluids during surgery. Microscopic examination upon necropsy, performed 4 weeks post-operatively, displayed limited fibrosis with some alteration of liver architecture, generalized sinusoidal dilation and red blood cells in the space of Disse. Cellular ultrastructure changes showed an increase of myelin figures, but mitochondria and other cellular organelles remained essentially normal. Localized tissue inflammation and some loss of function occurred in response to localized hyperthermia for this volume of tissue at therapeutic temperatures. This study showed that the technique was feasible and confirmed that the parenchymal damage caused by interstitial hyperthermia did not produce the severe loss of function that might have been expected.     

Effect of specific antibody pretreatment on liver uptake of 111In-labeled anticarcinoembryonic antigen monoclonal antibody in nude mice bearing human colon cancer xenografts

Administration of a large dose (0.2 mg) of unlabeled specific anticarcinoembryonic antigen (anti-CEA) monoclonal antibody (MAB) to nude mice bearing LS174T human colon cancer xenografts significantly decreased normal liver uptake of 111In-labeled anti-CEA MAB (Indacea). Mice bearing tumors of approximately 1 g showed liver accumulation of indium-111 at 48 h following injection of 2 micrograms/10 microCi Indacea of 33.8 +/- 1.5% injected dose per gram (%ID/g) (N = 25). Treatment with 0.2 mg unlabeled anti-CEA MAB reduced this to 8.9 +/- 0.5% ID/g (N = 22; P less than 0.001). The dose of pretreatment was found to be critical. Increasing the amount of unlabeled MAB to 2.0 mg did not significantly improve the liver level of indium-111, but did compromise the tumor uptake of Indacea (15.9 +/- 1.3 versus 12.4 +/- 0.4% ID/g; P less than 0.05). Lowering the dose of pretreatment 10-fold resulted in increased (P less than 0.001) liver uptake of the label (26.5 +/- 2.8% ID/g). The unlabeled anti-CEA MAB treatment given as a single dose or fractionated over several days gave the same results. The decrease in liver uptake was the same for i.v. administration of the unlabeled MAB given 1 week prior to Indacea injection or mixed together with Indacea. With i.p. administration, simultaneous injection of the unlabeled MAB with Indacea was not as effective as pretreatment (20 min to 7 days) in decreasing the liver uptake of 111In (P less than 0.05). Epitope specificity and affinity were shown to be important considerations in the choice of MAB combinations used for pretreatment and imaging. Pretreatment with nonspecific MAB was ineffective in decreasing liver uptake of Indacea.     

Alleviation of cyclophosphamide-induced urotoxicity by naturally occurring sulphur compounds

Treatment of Swiss albino mice with naturally occurring sulphur compounds such as Diallyl sulphide (DAS) (500 microg/dose/animal, i.p) and Diallyl Disulphide (DADS) (250 microg/dose/animal, i.p) for five days along with cyclophosphamide (CTX) (1.5 mmol/kg.body.wt; i.p) reduced the CTX induced urotoxicity. Morphological analysis of the urinary bladders of the CTX treated group showed severe inflammation and dark colouration whereas the sulphur compounds treated group showed almost normal bladder morphology. Treatment with DAS and DADS was found to reduce the urine urea N2 level DAS (15.31+/-0.01 g/l), DADS (12.38+/-0.17 g/l) 4 hrs after CTX administration which was elevated in the CTX alone treated group (26.87+/-1.86 g/l). Urine protein level, which was enhanced drastically (8.66+/-0.47 g/l) after the CTX treatment, was significantly reduced (2.9+/-0.25 g/l) by the treatment with DADS. Similarly GSH content (which was drastically reduced by the CTX administration) in both bladder (0.87+/-0.1 nmol/mg protein) and liver (2.47+/-0.6 nmol/mg protein) was enhanced significantly (P<0.001) by the treatment with DAS and DADS both in bladder (DAS--1.56+/-0.17 nmol/mg protein; DADS--2.65+/-0.21 nmol/mg protein) and liver (DAS--5.30+/-0.07 nmol/mg protein; DADS--6.93+/-0.06 nmol/mg protein). Histopathological analysis of the bladder of the CTX alone treated group showed severe necrosis of the tissue whereas the sulphur compounds treated group showed normal bladder pathology.     

Protective effect of Andrographis paniculata and andrographolide on cyclophosphamide-induced urothelial toxicity

The protective effect of Andrograhis paniculata and andrographolide (ANDLE) against cyclophosphamide (CTX)-induced urothelial toxicity was investigated in this study. Pretreatment of Swiss albino mice with A paniculata extract (10 mg/dose/animal intraperitoneally [ip]) and ANDLE (500 microg/dose/animal ip) could significantly reduce CTX (1.5 nmol/kg body weight)-induced urothelial toxicity. Morphological and histopathological analysis of urinary bladder of CTX-treated mice showed severe inflammation and dark coloration, whereas A paniculata and ANDLE-treated mice showed almost normal bladder morphology. Elevation of urinary protein level (7.33 +/- 0.3 g/L) by CTX administration was reduced by A paniculata (3.78 +/- 0.4 g/L) and ANDLE treatment (4.19 +/- 0.1 g/L). Urinary urea N2 level, which was elevated after 48 hours of CTX administration (24.25 +/- 0.2 g/L) was found to be reduced by the treatment with A paniculata (14.19 +/- 0.5 g/L) and ANDLE (15.79 +/- 0.4 g/L). A decreased level of reduced glutahione (GSH) content in liver (2.81 +/- 0.1 nmol/mg protein) and bladder (1.20 +/- 0.2 nmol/mg protein) after CTX administration was also increased by the treatment with A paniculata (liver: 5.78 +/- 0.3 nmol/mg protein; bladder: 2.96 +/- 0.2 nmol/mg protein) and ANDLE (liver: 5.14 +/- 0.3 nmol/mg protein; bladder: 2.84 +/- 0.2 nmol/mg protein). Production of the proinflammatory cytokine, tumor necrosis factor-alpha, which was elevated during CTX administration, was found to be inhibited by A paniculata and ANDLE treatment. The lowered level of interleukin-2 and interferon-gamma during CTX treatment was elevated by the administration of A paniculata and ANDLE.     

Erythropoietin-induced reduction of hypoxia before and during fractionated irradiation contributes to improvement of radioresponse in human glioma xenografts

PURPOSE: Our study investigated the influence of recombinant human erythropoietin (rHuEPO) treatment, inducing raised hemoglobin levels in nonanemic mice, on intratumor oxygenation before and during fractionated irradiation. Furthermore, the consequences of rHuEPO administration on tumor response to fractionated radiotherapy (RT) were evaluated. METHODS AND MATERIALS: Experiments were performed on two human malignant glioma (GBM Nan1 and U87) xenografted in nude mice. RHuEPO was daily delivered (0.3 IU/g/day, 5 days/week). Tumor hypoxia was assessed before (T1) and during (T6) fractionated irradiation using (1) pO(2)-Histograph (Eppendorf, Hamburg, Germany) and (2) the EF5-binding assay. Vascular density was determined using type IV collagen immunostaining. To assess RT efficacy, the irradiation schedule was 20 fractions of 2 Gy, once daily, 5 days/week over 4 weeks. RESULTS: At T1, hemoglobin levels in rHuEPO-treated mice were significantly increased. Percentage of pO(2) values <2.5 mm Hg was reduced in rHuEPO-treated tumors as compared with control groups (37.1 +/- 19.1% vs. 58.5 +/- 27.0%; p = 0.009 for GBM Nan1; 81.6 +/- 13.4% vs. 91.5 +/- 8.3%; p = 0.035 for U87). The decrease of viable hypoxic tumor cells fraction after rHuEPO was confirmed by the EF5-binding assay. Vascular density was not altered after rHuEPO treatment. At T6, rHuEPO reduced the hypoxic fraction by about 20% (p = 0.036 and p = 0.171) in GBM Nan1 and U87 irradiated tumors. RHuEPO did not influence tumor growth by itself. RT alone or combined with rHuEPO induced a significant tumor growth delay. Finally, rHuEPO significantly enhanced RT efficacy (p = 0.012 in GBM Nan1 and p = 0.037 in U87), resulting in radiopotentiation ratios of 1.21 and 1.54 for respective models. CONCLUSIONS: Our results indicate that rHuEPO, by enhancing blood oxygen-carrying capacity, decreases intrinsic tumor hypoxia and maintains its effect during fractionated irradiation in malignant glioma xenografts. Therefore, rHuEPO contributes to radiosensitize these tumors.     

BW12C: effects on tumour hypoxia, tumour thermosensitivity and relative tumour and normal tissue perfusion in C3H mice

BW12C (5-[2-formyl-3-hydroxypenoxyl] pentanoic acid) is an agent which stabilises oxyhaemoglobin and thus reduces oxygen delivery to tissues. It is of interest as a possible potentiator of bioreductive agents and/or hyperthermia. The increases in radiobiological hypoxic fraction of RIF-1 and KHT tumours 30 min after 70 mg kg-1 BW12C i.v. were measured and shown to be similar; factors (+/- 2 s.e.) ranged from 3.87 (2.84-5.29) to 5.92 (1.92-18.2) despite the large variation in initial hypoxic fraction, from 0.30 (0.18-0.50) % for RIF-1 intramuscularly in the leg to 16.3 (14.7-18.1) % for subcutaneous KHT flank tumours. Thermosensitivity of intramuscular KHT leg tumours was not enhanced by 70 mg kg-1 BW12C 30 min before heating at 43 degrees C, 43.5 degrees C or 44 degrees C, assayed by regrowth delay. The effect of 70 mg kg-1 BW12C on relative tissue perfusion (RTP), assayed by 86Rb extraction, was measured from 0.5 h to 6 h after treatment. After 1 h RTP (+/- 2 s.e.) in RIF-1 tumours was reduced to 84 +/- 5.7% and 68 +/- 9.6% of control in leg and flank tumours respectively, and to 86 +/- 6.4% in leg muscle while flank skin RTP was unaltered at 109 +/- 8.6%. There were substantial increases in kidney (149 +/- 10.7%) spleen (173 +/- 22.1%) and lung (128 +/- 10.4%) at 1 h but in liver there was a decrease at 2 h to 85 +/- 8.4%. Dose response studies showed that the threshold dose for reduction of tumour RTP is between 55 and 70 mg kg-1, but perturbations in normal tissue RTP occur at lower doses, e.g. 40 mg kg-1 for spleen. BW12C had minimal effects on renal function measured by 51CrEDTA clearance. The data as a whole indicate that reduction in tumour perfusion is likely to be an important determinant in the increase in tumour hypoxia induced by BW12C.     

Once-weekly epoetin beta therapy in patients with solid tumours and chemotherapy-induced anaemia: a randomized, double-blind, dose-finding study

Anaemia is common in patients receiving chemotherapy, causing symptoms that have a major impact on quality of life (QoL). Epoetin beta rapidly increases haemoglobin (Hb) levels and improves QoL in anaemic patients with a variety of tumours. This was a randomized, double-blind, parallel-group, dose-finding study assessing the efficacy and safety of once-weekly epoetin beta in patients with solid tumours receiving chemotherapy. Adult patients with anaemia (Hb < 11 g/dL) were randomized to receive epoetin beta 30,000 IU or 20,000 IU once weekly for 12 weeks. All patients received oral iron supplementation. Haemoglobin levels, transfusion need and QoL [Functional Assessment of Cancer Therapy-fatigue (FACT-F) subscale score] were assessed at regular intervals. Fifty patients were randomized; 30 patients received epoetin beta 30,000 IU once weekly and 20 received 20,000 IU once weekly. Mean (+/- SD) increase in Hb from baseline to week 12 was 1.75 +/- 2.15 g/dL in the 30,000 IU group (P = 0.008 vs. baseline) and 1.04 +/- 1.75 g/dL in the 20,000 IU group (non-significant). Haemoglobin response (increase in Hb >or=2 g/dL from baseline) was observed in 78.3% of patients receiving epoetin beta 30,000 IU and 66.7% receiving epoetin beta 20,000 IU. Improvements in FACT-F subscale score were significantly (P < 0.001) correlated with increases in Hb level. Transfusion use was low during the study in both groups. Both epoetin beta regiments were well tolerated and there were no dose-dependent adverse events. Epoetin beta 30,000 IU once weekly is an effective and well-tolerated treatment of anaemia in patients with solid tumours.     

Rat oesophageal cytochrome P450 (CYP) monooxygenase system: comparison to the liver and relevance in N-nitrosodiethylamine carcinogenesis.

N-nitrosodiethylamine (NDEA) is able to induce tumours in the rat oesophagus. It has been suggested that this could be due to tissue specific expression of NDEA activating cytochrome P450 enzymes. We investigated this by characterizing the oesophageal monooxygenase complex of male Wistar rats and comparing it with that of the liver. Total amount of cytochrome P450, NADPH P450 reductase, cytochrome b5 and cytochrome b5 reductase of the oesophageal mucosa was approximately 7% of what was found in the liver. In addition, major differences were found in the cytochrome P450 isoenzyme composition between these organs: CYP 2B1/2B2 and CYP3A were found only in the liver, whereas CYP1A1 was constitutively expressed only in the oesophagus. Of the two well-known nitrosamine metabolizing enzymes, CYP2A3 was found only in the oesophagus whereas CYP2E1 was exclusively expressed in the liver. Catalytic studies, western blotting and RT-PCR analyses confirmed the expression of CYP2A3 in the oesophagus. CYP2A enzymes are known to be good catalysts of NDEA metabolism. Oesophageal microsomes had a K(m) for NDEA metabolism, which was about one-third of that of hepatic microsomes, but they showed similar activities when compared per nmol of total P450. NDEA activity in the oesophagus was significantly increased by coumarin (CO), which also induced oesophageal CYP2A3. Immunoinhibition of the microsomal NDEA activity showed that up to 70% of this reaction is catalysed by CYP2A3 in the oesophagus, whereas no inhibition of the hepatic NDEA activity could be achieved by the anti-CYP2A5 antibody. NDEA, but not N-nitrosodimethylamine (NDMA) inhibited the oesophageal metabolism of CO. The results of the present investigation show major differences in the enzyme composition of the oesophageal and hepatic monooxygenase complexes, and are in accordance with the hypothesis that the NDEA organotropism could, to a large extent, be due to the tissue specific expression of the activating enzymes.     

Extracranial stereotactic radiotherapy: evaluation of PTV coverage and dose conformity

During the past few years the concept of cranial stereotactic radiotherapy has been successfully extended to extracranial tumoral targets. In our department, hypofractionated treatment of tumours in lung, liver, abdomen, and pelvis is performed in the Stereotactic Body Frame (ELEKTA Instrument AB) since 1997. We present the evaluation of 63 consecutively treated targets (22 lung, 21 liver, 20 abdomen/pelvis) in 58 patients with respect to dose coverage of the planning target volume (PTV) as well as conformity of the dose distribution. The mean PTV coverage was found to be 96.3% +/- 2.3% (lung), 95.0% +/- 4.5% (liver), and 92.1% +/- 5.2% (abdomen/pelvis). For the so-called conformation number we obtained values of 0.73 +/- 0.09 (lung), 0.77 +/- 0.10 (liver), and 0.70 +/- 0.08 (abdomen/pelvis). The results show that highly conformal treatment techniques can be applied also in extracranial stereotactic radiotherapy. This is primarily due to the relatively simple geometrical shape of most of the targets. Especially lung and liver targets turned out to be approximately spherically/cylindrically shaped, so that the dose distribution can be easily tailored by rotational fields.     

Hepatocarcinogenesis in mice with a conditional knockout of the hepatocyte growth factor receptor c‐Met

The receptor for the hepatocyte growth factor/scatter factor (HGF/SF), c‐Met, plays a role in tumour promotion, progression and metastasis. In this study, we analysed chemically induced hepatocarcinogenesis in mice lacking a functional HGF receptor in their liver. Control and c‐Met deficient mice were injected with a single dose of N‐nitrosodiethylamine (DEN, 90 μg/g b.wt.) at 6 weeks of age and mice were subsequently kept on a phenobarbital (PB) containing diet (0.05%) for 35 weeks or on control diet. At the end of the experiment, the carcinogenic response in liver of the animals was monitored. Conditional c‐met knockout (KO) mice showed a higher prevalence of macroscopically visible liver tumours and of glutamine synthetase positive and glucose‐6‐phosphatase deficient lesions in liver. Tumour promotion by PB led to significant increases in the number of preneoplastic and neoplastic lesions in liver of both wild‐type and c‐met knockout mice, with only minor differences in response. Our results indicate that a defect in c‐Met‐mediated signaling increases chemically induced tumour initiation in liver but does not significantly affect PB‐mediated tumour promotion. © 2008 Wiley‐Liss, Inc.     

Radioprotective activity of naturally occurring organosulfur compounds

The radioprotective effects of naturally occurring sulfur compounds and isothiocyanates such as diallyl sulfide (DAS), diallyl disulfide (DADS), allyl methyl sulfide (AMS), allyl isothiocyanate (AITC) and phenyl isothiocyanate (PITC) have been investigated in whole body irradiated Swiss albino mice. Administration of these sulfur compounds could reduce the serum content of alkaline phosphatase (ALP), which was elevated after irradiation (23.9 +/- 1.82 KA units). The elevated liver content of glutamate pyruvate transaminase (GPT) in control animals (76.2 +/- 2.2 U/mL) after irradiation was significantly reduced in DAS (58.93 +/- 4 U/mL) and AMS (55.7 +/- 2.2 U/mL) treated animals. Elevated levels of lipid peroxides in serum and liver of irradiated control animals were also significantly reduced by treatment with these sulfur compounds. The glutathione (GSH) content in liver and intestinal mucosa was drastically reduced after irradiation. All the sulfur compounds and isothiocyanates could effectively enhance the GSH content of intestinal mucosa and liver. Findings at histopathological analysis of the intestine proved to be correlated with the above results.