Lindy L. Thomsen

Inhibition of growth of colon 38 adenocarcinoma by vinblastine and colchicine: Evidence for a vascular mechanism

Vinblastine or colchicine, administered intraperitoneally to B6D2F1 mice with advanced subcutaneous colon 38 tumours, induced substantial tumour growth delays with progressive development of haemorrhagic necrosis beginning within 8 hours of treatment. Two multidrug-resistant P388 leukaemia sublines, refractory to vinblastine and vincristine when grown as intraperitoneal ascites, were sensitive to necrosis induction when grown as subcutaneous tumours. Vascular labelling with two fluorescent markers indicated that vincristine substantially reduced tumour blood flow within 4 hours after treatment. The effects of vinblastine, vincristine and colchicine were similar to those of tumour necrosis factor alpha in that: (a) similar tumour necrosis and blood flow changes were induced, (b) coadministration of the serotonin antagonist cyproheptidine prevented tumour necrosis and (c) plasma nitrate levels were elevated, indicative of the stimulation of oxidation of L-arginine to nitric oxide. The results suggest that vinca alkaloids and colchicine act on solid tumours by host cell-mediated vascular effects as well as by direct tubulin-mediated cytotoxicity.

Serotonin involvement in the antitumour and host effects of flavone-8-acetic acid and 5,6-dimethylxanthenone-4-acetic acid

The relationship of serotonin (5-HT) receptors to the action of the experimental antitumour drugs flavone-8-acetic acid (FAA) and 5,6-dimethylxanthenone-4-acetic acid (5,6-MeXAA) was studied. Both FAA and 5,6-MeXAA are known to induce the synthesis of tumour necrosis factor-α (TNF) and to stimulate nitric oxide synthesis in vivo, as measured by elevation of plasma nitrate. Serotonin potentiated the effect of a subtherapeutic dose of 5,6-MeXAA (20 mg/kg) as measured both by plasma nitrate increase and by growth delay of s.c. implanted colon 38 tumours. On the other hand, administration of the serotonin 5-hydroxytryptamine-2 (T-HT2) antagonist cyproheptadine (20 mg/kg) inhibited both the plasma nitrate response and, to a lesser extent, the induction of tumour haemorrhagic necrosis by 5,6-MeXAA, FAA and TNF. Reduction of circulating plasma serotonin by pre-treatment withp-chlorophenylalanine and reserpine reduced the plasma nitrate response, but not the tumour necrosis response, to 5,6-MeXAA (30 mg/kg). It is suggested that serotonin is necessary for the induction of nitric oxide synthases and acts, either directly or indirectly, in concert with TNF. Serotonin agonists may have utility in increasing nitric oxide synthesis in response to TNF or to agents that induced TNF as part of their antitumour action.

Plasma nitrate clearance in mice : modeling of the systemic production of nitrate following the induction of nitric oxide synthesis

Nitric oxide (NO) is produced in mammals by the enzyme NO synthase (NOS) in response to a number of agents, including the experimental antitumour agent flavone acetic acid (FAA) and the cytokine tumour necrosis factor-α (TNF). NO is converted rapidly in the presence of oxygen, water and haemoglobin to oxidation products, largely nitrate. To quantitate the production of nitric oxide it is necessary to know the clearance of nitrate. The concentration of nitrite and nitrate ion in the plasma of C3H and BDF1 (C57BL6×DBA2) mice was assessed before and after injection of sodium nitrate and sodium nitrite. Nitrite was converted rapidly to nitrate and the kinetics of elimination of nitrate were determined. There was no significant difference between results obtained with different mouse strains, between levels of nitrite and nitrite, or between i.p. and i.v. administration, and the observations were therefore combined. The volume of distribution of nitrate was 0.71±0.04 l/kg and the clearance was 0.32±0.02 l/h−1/kg−1 (plasma half-life, 1.54 h). Using previously published data, we developed a pharmacokinetic-pharmacodynamic model that relates the production of TNF in response to administration of FAA, the enhancement of NOS activity in response to TNF, and the elevation of plasma nitrate in response to NO production. This information permits the prediction from observed plasma nitrate values of the amount of NOS induced in vivo.

Disacchandase Levels of the Rat Jejunum Are Altered by Dietary Fibre

Rat jejunal disaccharidases were measured after feeding high fat fibre-free, high fat pectin, high fat cellulose, high fat galactomannan and high fat tannin diets for 27 weeks. Compared with the high fat fibre-free diet, pectin lowered both lactase and sucrase levels (p less than 0.025). Galactomannan and tannin lowered only the lactase level (p less than 0.05), while cellulose had no significant effect on disaccharidase levels. These data suggest that dietary fibre components have individual and specific effects on jejunal disaccharidase levels, in the presence of a high fat diet.

Nitric oxide: its production in host-cell-infiltrated EMT6 spheroids and its role in tumour cell killing by flavone-8-acetic acid and 5,6-dimethylxanthenone-4-acetic acid

SummaryFlavone-8-acetic acid (FAA) and its more dose-potent analogue 5,6-dimethylxanthenone-4-acetic acid (5,6-MeXAA), appear to exert their antitumour effects through vascular and other host-mediated mechanisms and are known to induce the synthesis of nitric oxide by murine macrophages. We investigated the role of nitric oxide in the cytotoxic effects of these drugs in host-cell-infiltrated spheroids. EMT6 murine mammary adenocarcinoma cells were grown in culture to produce multicellular spheroids in vitro spheroids), which were then inoculated i. p. into mice. After 6 days the spheroids were removed ex vivo spheroids). Exposure to FAA (890 μm) and 5,6-MeXAA (80 μm) in vitro for 20 h increased nitrite concentrations to 6.7 and 9.7 nmol/spheroid, respectively, as compared with 0.7 nmol/spheroid in the absence of drug. FAA and 5,6-MeXAA did not increase nitrite production in in vitro spheroids in cells obtained by peritoneal lavage. However, mixed cultures of in vitro spheroids and peritoneal cells treated with 5,6-MeXAA produced nitrite (2.5 nmol/spheroid), indicating that interactions between host cells and tumour cells were important for induction. The effets of these drugs on ex vivo spheroids were prevented by co-incubation withNG-monomethyl-l-arginine, indicating that nitrite originated from the oxidation ofl-arginine to nitric oxide. Cell sorting of disaggregated spheroids into EMT6 cells andMac-1-positive macrophage populations indicated that both of these cell populations could be induced to synthesise nitric oxide by subsequent incubation with 5,6-MeXAA. Incubation of ex vivo spheroids with FAA and 5,6-MeXAA decreased the clonogenicity of EMT6 cells, and this effect was wholly (FAA) or partially (5,6-MeXAA) reversed by the presence ofNG-monomethylarginine (250 μm). FAA and 5,6-MeXAA may therefore exert some of their cytotoxic effects on tumour cells through the production of nitric oxide.

Intra-Caecal Short Chain Fatty Acids Are Altered by Dietary Pectin in the Rat

When Dark Agouti rats were changed from a conventional pellet diet containing 3.3% crude fibre and 4% fat to the experimental diet containing 5% pectin and 17% fat, the levels of n-butyrate in the caecum rapidly decreased more than fivefold. Such a change could be important as n-butyrate is known to be a major energy source for colonocytes, and to affect colon tumour cell development in vitro. Striking decreases in the caecal concentrations of the short chain fatty acids isobutyrate, n-valerate and isovalerate occurred when Wistar rats were fed an experimental diet containing 5% pectin as the only dietary fibre, compared to rats fed the experimental diet containing no dietary fibre. These large changes in the short chain fatty acid profile, caused by pectin supplementation, have important implications for the metabolism and health of the colon.

Nitric oxide production in endotoxin-resistant C3H/HeJ mice stimulated with flavone-8-acetic acid and xanthenone-4-acetic acid analogues

The production of nitric oxide in endotoxin-resistant C3H/HeJ mice in response to flavone-8-acetic acid (FAA), derivatives of xanthenone-4-acetic (XAA), endotoxin and recombinant human tumour necrosis factor-alpha (TNF-alpha) was investigated and compared with the induction of haemorrhagic necrosis in subcutaneous M16/C tumours. FAA and XAA analogues stimulated nitric oxide production both in vitro (activated macrophages) and in vivo (plasma nitrate elevation) in both C3H/HeJ and C3H/HeN mice (5,6-dimethyl-XAA greater than 5-methyl-XAA greater than FAA greater than XAA greater than 8-methyl-XAA). Recombinant human TNF-alpha stimulated nitric oxide production equally from both murine strains while endotoxin stimulated nitric oxide production only by C3H/HeN mice. The extent of induction of haemorrhagic necrosis in tumour-bearing mice treated with FAA, 5,6-dimethyl XAA or endotoxin paralleled the effects on nitric oxide production, showing a differential between the two strains of mice only in the case of endotoxin.

Sucralfate interactions with gastric mucus

Sucralfate protects the stomach against a number of experimental damaging agents and is efficacious in the treatment of peptic ulcer disease. It binds with acidity to the base of an ulcer to form a protective barrier. Sucralfate also enhances prostaglandin synthesis and release in the mucosa. In this study, the rat stomach was examined to determine sucralfates interaction with gastric mucus. Mucus in the rat stomach forms a distinct and continuous blanket. In snap-frozen samples, pretreatment with phosphate-buffered saline as a control shows a layer of mucus of homogeneous structure thinner than the homogeneous layer after pretreatment with antibodies developed against rat gastric mucus. Pretreatment with the surface protective agent sucralfate shows some increase in the thickness of mucus with a thin dense sublayer adjacent to the epithelium and a less dense-appearing outer zone of variable thickness. Analysis of x-rays generated by the electron beam on windows of mucus and epithelium showed the expected gradients of sodium, potassium, chloride, and sulfur. The percentage of aluminum and sulfur in the mucus was higher in sucralfate-treated samples than in controls. Interaction between sucralfate and gastric mucus needs further investigation.

Toxin-Induced Cell Membrane Injury in Guinea Pigs given Lincomycin

Summary Guinea pigs treated with lincomycin developed colitis, acute cholecystitis and abnormalities in red blood cell morphology. The present study was designed to study the production of clostridial toxins after lincomycin treatment. Lincomycin produced abnormalities in conventional but not in germ‐free guinea pigs. Clostridium difficile was cultured from cecal contents of conventional guinea pigs treated with lincomycin. Cecal filtrate from sick guinea pigs was subjected to Sepharose 4B‐CL and Sephadex G‐200 column chromatography, yielding a partially purified toxin. Both cecal filtrate and partially purified toxin samples contained a heat labile substance which was cytotoxic to human lung fibroblast WI‐38 cells, and which was neutralized by Clostridium sordelli antitoxin. Toxin samples given orally or intraperitoneally killed normal guinea pigs. Finally, toxin samples induced red cell membrane changes in vitro as well as producing features of acute inflammation in healthy explants of guinea pig cecum and gallbladder in organ culture. Lincomycin treated guinea pigs produced Clostridium difficile toxin(s) which in turn caused diffuse cell membrane injury.

Cellulose and pectin alter intestinal β-glucuronidase (EC 3.2.1.31) in the rat

Groups of rats were given a fibre-free diet containing none or one of the three fibre components: pectin, cellulose or galactomannan. After feeding for 16 weeks, total protein level and beta-glucuronidase (EC 3.2.1.31) activity in the contents and mucosa of jejunum and ileum, and in the contents only of the caecum, were determined. The pectin supplement reduced protein concentration in jejunal contents while cellulose reduced protein concentration in the ileal and caecal contents. beta-Glucuronidase activity of caecal contents was significantly reduced in both the pectin- and cellulose-fed groups. Cellulose affected the beta-glucuronidase activity of both the ileal contents while pectin reduced the beta-glucuronidase of the ileal but not the jejunal contents. Dietary fibre components did not significantly affect jejunal or ileal mucosal beta-glucuronidase activity.