Rat Glandular Stomach Research Articles

Inhibitory effect of calcium chloride on gastric carcinogenesis in rats after treatment with N-methyl-N'-nitro-N-nitrosoguanidine and sodium chloride.

The effects of calcium chloride on glandular stomach carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and sodium chloride were investigated in male outbred Wistar rats. Animals were given MNNG solution (100 p.p.m.) as drinking water and simultaneously fed a diet supplemented with 5% sodium chloride for 8 weeks. Matched negative controls received neither MNNG nor sodium chloride. Rats were then fed basal diet and given calcium chloride solution (1 or 0.2%) or tap water for the following 52 weeks. The incidences and multiplicities of preneoplastic hyperplasias in the glandular stomachs of rats given MNNG/sodium chloride followed by 1 and 0.2% calcium chloride were significantly lower than those in rats given MNNG/sodium chloride alone. The inhibitory effects of calcium were exerted in a dose-dependent manner. Calcium treatment also showed a tendency to inhibit the development of gastric adenocarcinomas although this was not statistically significant. Rats without carcinogen treatment had neither carcinomas nor preneoplastic hyperplasias in the glandular stomach. Calcium intake also significantly reduced the levels of malondialdehyde, a measure of lipid peroxidation, in the gastric mucosa and urine, the former in a dose-dependent manner. Thus, calcium chloride exerted inhibitory effects when given during the post-initiation phase of two-stage glandular stomach carcinogenesis in rats.

An alternative theory of tissue specificity by tumor promotion of okadaic acid in glandular stomach of SD rats.

To challenge the theory of tissue specificity of tumor promoters, the biochemical and tumor promoting effects of okadaic acid (OA), a potent tumor promoter on mouse skin, were studied in the mucosa of rat glandular stomach. OA strongly inhibited protein phosphatases 1 and 2A, and increased 4-fold the phosphorylation of elongation factor 2 in vitro in the mucosa. Intubation of 10 micrograms (12.4 nmol) OA induced ornithine decarboxylase in the mucosa. Tumor promotion of OA was studied in the glandular stomach initiated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in a two-stage carcinogenesis experiment. OA in drinking water, 10 micrograms (12.4 nmol) per rat per day from weeks 9-55 of the experiment, and 20 micrograms (24.8 nmol) from weeks 56-72, significantly enhanced development of the neoplastic changes in the glandular stomach (P < 0.05). The neoplastic changes included adenomatous hyperplasias and adenocarcinomas, both of which correspond to papillomas and carcinomas in a two-stage mouse skin carcinogenesis experiment. The percentages of neoplastic change-bearing rats of the groups treated with MNNG plus OA, MNNG alone or OA alone were 75.0, 46.4 and 0% respectively. OA enhanced tumorigenesis in the MNNG-initiated glandular stomach of rats through the same mechanisms of action as in mouse skin. The OA pathway mediated through inhibition of protein phosphatases 1 and 2A is applicable to various organs as a general mechanism of tumor promotion.

Molecular heterogeneity of platelet‐activating factor (PAF) in rat glandular stomach determined by gas chromatography/mass spectrometry. PAF molecular species changes upon water‐immersion stress

The molecular heterogeneity of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (alkylacetyl-GPC) and 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (acylacetyl-GPC) in normal rat glandular stomach was studied by gas chromatography/mass spectrometry (GC/MS) and tandem mass spectrometry. The percentage compositions of the molecular species of 1-alkyl-2-acetyl-GPC and 1-acyl-2-acetyl-GPC in the antrum were, respectively, 1-alkyl [16:0 (34%) and 18:0 (66%)]-2-acetyl-GPC and 1-acyl [16:0 (60%), 18:0 (14%) and 18:1 (26%)]-2-acetyl-GPC. The alkyl chain composition of 1-alkyl-2-acetyl-GPC was quite different from that of 1-alkyl-2-acyl-GPC in both the antrum and corpus, demonstrating a high degree of selectivity of alkyl chain utilization in PAF biosynthesis. The amount of 1-acyl-2-acetyl-GPC was much greater than that of 1-alkyl-2-acetyl-GPC. The molecular heterogeneity of 1-alkyl-2-acetyl-GPC and 1-acyl-2-acetyl-GPC in the corpus was similar to that in the antrum. Water-immersion stress affected not only the amount of 1-alkyl-2-acetyl-GPC and 1-acyl-2-acetyl-GPC, but also their molecular heterogeneity in the antrum and corpus. Whereas the amounts of 1-hexadecyl-2-acetyl-GPC and 1-acyl [16:0, 18:0 and 18:1]-2-acetyl-GPC decreased markedly (to less than one-fifth) in the antrum after such stress for 1 hr, the amount of 1-octadecyl-2-acetyl-GPC increased markedly (up to 4-fold) in the corpus and severe lesions were observed after stress for 7 hr.(ABSTRACT TRUNCATED AT 250 WORDS)

Possible tumor-initiating and -promoting activities of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5 H)-furanone (MX) in rat glandular stomach

Possible tumor-initiating and -promoting activities of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5 H)-furanone (MX) in rat glandular stomach

Possible tumour-initiating and -promoting activities of hickory smoke condensate in rat glandular stomach

Possible tumour-initiating and -promoting activities of hickory smoke condensate in rat glandular stomach

Induction of preneoplastic lesions by sequential treatment of rats with three carcinogens: N-butyl-N-(4-hydroxybutyl) nitrosamine, 7,12-dimethylbenz(a)-anthracene, and N-methyl-N-nitro-N-nitrosoguanidine.

N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), 7, 12-dimethylbenz(a)anthracene (DMBA), and N-methyl-N-nitro-N-nitrosoguanidine (MNNG) were given by gastric intubation, in sequence, to 6-week-old female SD rats. After receiving BBN (600mg/kg body wt.) three times, every other day, in the 1st week, the animals were administered a single dose of DMBA (50mg/kg body wt.) at the beginning of week 2 and MNNG (100mg/kg body wt.) three times every other day in the 3rd week. Groups were sacrificed at the end of weeks 12, 16, 20, and 24 of the experiment. The putative preneoplas-tic lesion, papillary or nodular hyperplasia as well as papillomas were found in the urinary bladder after week 16 but the incidences were very low. Adenocarcinoma in the mammary gland appeared after week 12 at a high incidence. Papilloma and carcinoma in the forestomach were induced after week 16. Pepsinogen isoenzyme 1 altered pyloric glands could be detected immunohistochemically after week 12. These results indicate that treatment with the three carcinogens given in sequence is effective for induction of preneoplastic and neoplastic lesions in the urinary bladder, mammary gland, forestomach, and glandular stomach of rats in a relative short-period.

Genotoxicity and cell proliferative activity of omeprazole in rat stomach mucosa

Induction of unscheduled DNA synthesis (UDS) as a marker of genotoxicity and induction of ornithine decarboxylase (ODC) activity as a marker of cell proliferative activity by omeprazole were determined in the glandular stomach mucosa of male F344 rats after oral administration. Commercial enteric-coated omeprazole (Losec) at doses of 30 and 100 mg/kg body weight induced a dose-dependent increase in UDS but not replicative DNA synthesis in the pyloric mucosa of rat stomach 4 h after its administration. Dosedependent significant induction of ODC activity was observed in fundic and pyloric mucosa with a maximum 8 h after administration of omeprazole at doses of 37.5–100 mg/kg body weight. These results show that omeprazole has genotoxicity and cell proliferative activity in the rat glandular stomach mucosa

Cellular Differentiation and Histogenesis of Rat Glandular Stomach Cancers

The gastric and intestinal phenotypic expressions of tumor cells in 18 adenomatous hyperplasias, 33 well‐differentiated adenocarcinomas, and 16 undifferentiated adenocarcinomas (4 poorly differentiated adenocarcinomas, 10 signet‐ring cell carcinomas and 2 mucinous adenocarcinomas) induced by N‐methyl‐N’‐nitro‐N‐nitrosoguanidine or 4‐nitroquinoline‐I‐oxide in the rat glandular stomach were studied by histochemical stainings for mucin and immunohistochemical staining for pepsinogen isozyme 1 (Pg 1). By histochemical staining for mucin [by the paradoxical concanavalin A method, the modified method with labeled peanut lectin, the galactose oxidase‐Schiff (GOS) reaction, and the sialidase‐GOS reaction] and immunohistochemical staining of Pg 1, gastric cancer cells of each histological group could be clearly classified into a gastric type, including mucous neck cell pyloric gland cell, and surface mucous cell subtypes, and an intestinal type, including goblet‐cell, and intestinal absorptive cell subtypes. All tumors examined in this work consisted mainly of gastric‐type cells but intestinal‐type tumor cells were occasionally found among the gastric‐type tumor cells. The incidences of intestinal‐type cells in adenomatous hyperplasias (11.1%) and small well‐differentiated adenocarcinomas (28.6%) were significantly less (P<0.05) than that in large well‐differentiated adenocarcinomas (68.4%). The incidence of intestinal‐type cells in small undifferentiated adenocarcinomas (25.0%) was also less than that in large ones (58.3%). The present results suggest the occurrence of change of phenotypic expression of tumor cells from the gastric type to the intestinal type during growth of tumors.

Effects of restraint stress on catecholamine concentrations in the glandular stomach of rats

Restraint stress is known to induce gastric ulcers in rats. Peripheral sympathetic activity and catecholamines are involved in the pathogenesis of these gastric ulcers. The aim of the present study was to evaluate the effects of restraint on mucosal and muscle catecholamine concentrations in the glandular stomach of rats. In unrestrained rats, noradrenaline concentration was higher in the muscle than in the mucosa of the glandular stomach (629 +/- 106 vs 18 +/- 3 pg/mg and 217 +/- 37 vs 18 +/- 8 pg/mg, respectively in the corpus and the antrum, p less than 0.01). This can be explained by the existence of an abundant noradrenergic innervation in the muscle layer. After 20 hours of restraint, adrenaline and noradrenaline concentrations were significantly decreased in adrenals, in comparison with unrestrained animals (255 +/- 53 vs 638 +/- 160 ng/mg and 113 +/- 17 vs 198 +/- 37 ng/mg, respectively for adrenaline and noradrenaline, p less than 0.05). In the glandular stomach, noradrenaline and adrenaline concentrations in restrained rats were not significantly different from those in unrestrained rats. However, adrenaline concentrations in the muscle of restrained rats were higher than in the mucosa. Moreover, restraint induced a significant decrease in dopamine concentration in the antral mucosa (from 100 +/- 12 pg/mg in unrestrained rats to 15 +/- 5 pg/mg in restrained rats), suggesting that a depletion in dopamine in the antral mucosa could be one of the pathogenetic factors involved in antral gastric stress-induced ulcers in rats.

Effect of cold-restraint stress on glutathione and lipid peroxide levels in the liver and glandular stomach of rats

The effect of starvation and cold-restraint stress on glutathione and lipid peroxide levels in the liver, stomach and plasma of rats was investigated. Hepatic and gastric glutathione levels were significantly decreased in starvation and cold-restraint groups when compared with values obtained from the control group. In both tissues, lipid peroxide levels were significantly decreased after starvation but were not significantly different from control values after cold-restraint treatment. However, when compared with the values obtained from the starvation group there was a significant increase in both hepatic and gastric lipid peroxide levels after cold-restraint. Plasma lipid peroxide levels were slightly decreased in the starvation group and significantly increased in the cold-restraint group. Our results suggest that pathological consequences of stress on different tissues could be due to stimulation of lipid peroxidation.

Glycosphingolipid Composition of the Gastric Mucosa

The glycosphingolipid (GSL) compositions of the human, rabbit, and rat gastric mucosa were studied and compared. In neutral GSLs, although GalCer was the most abundant component in every tissue examined, species-specific differences were clearly observed in their composition. Acidic GSLs were composed of sulfatides and gangliosides. Every gastric mucosa contained sulfatides (GalCer-sulfate) in a high concentration. The concentrations of sulfatides per gram of dry weight were 416.0, 933.8, 394.0, 365.9, and 135.9 nmol for the human fundus, human antrum, rabbit fundus, rabbit antrum, and rat glandular stomach, respectively. On the other hand, gangliosides were less in these tissues. The molar ratios of sulfatides to GM3, a major ganglioside, were 4.3, 6.7, 11.1, 2.1, and 1.2 for each gastric mucosa described above, respectively. A high sulfatide concentration in animal gastric mucosa, with its structural similarity to sucralfate, implies that sulfatides may play an important role for gastrointestinal mucosal protection.

Early proliferative responses of forestomach and glandular stomach of rats treated with five different phenolic antioxidants

The effects of 8 weeks of oral administration of five different phenolic antioxidants, e.g. catechol (CC), resorcinol, hydroquinone (HQ), 2-tert-butyl-4-methylphenol (TBMP) and propylparabene (PP), on forestomach and glandular stomach epithelium of male F344 rats were evaluated using a combined immunohistochemical and histopathological approach. Treatment with CC and TBMP induced a significant elevation of DNA synthesis in the forestomach epithelium, associated with hyperplasia. CC administration also brought about an increase of DNA synthesis in the pyloric gland mucosa, cell proliferation in this case being reflected by an increment in the crypt height. In addition to causing an increase in the pepsinogen-isozyme-1-altered pyloric glands (PAPG), which are considered to be putative preneoplastic precursor lesions in the rat glandular stomach, CC treatment was associated with submucosal growth of pyloric mucosal cells tending to decreased pepsinogen isoenzyme 1 binding. However, DNA synthesis values in these latter areas were lower than in pyloric glands of the control group. In contrast, other phenolic compounds, resorcinol, HQ and PP, did not induce any changes in the stomach mucosa. The present results demonstrated strong proliferative responses in the stomach epithelium for CC and TBMP, indicative of promoting potential in both cases, and suggest that CC and TBMP may exert detrimental effects leading to promotion of stomach carcinogenesis or cancer development via an early proliferative response.

Epithelial cell proliferation in rat forestomach and glandular stomach mucosa induced by catechol and analogous dihydroxybenzenes

An investigation of antioxidant-induced lesions in stomach epithelia was performed using F344 rats of both sexes. Histopathological changes, levels of DNA synthesis and pepsinogen isozyme 1 altered pyloric gland (PAPG) induction were assessed following 4 weeks oral administration of catechol (CC) or analogs such as hydroxyhydroquinone (HHQ), protocatechuic acid (PCA), protocatechualdehyde (PCAH), dopamine and DL-dopa. While epithelial hyperplasia of the forestomach was only observed in the groups given CC, DNA synthesis in this epithelium was increased in groups of both sexes treated with CC, HHQ or dopamine. In the glandular stomach, CC induced submucosal growth of pyloric mucosal cells and an increase in crypt height associated with an elevation of DNA synthesis and numbers of PAPG. In contrast, dopamine brought about significant reduction in DNA synthesis in the pyloric mucosa of both sexes. The other CC analogs did not exert any obvious influence on glandular stomach mucosa. Since cell proliferation is well correlated to tumor promotion, the results suggested that HHQ and dopamine may have promoting potential for rat two-stage forestomach carcinogenesis in common with CC, while dopamine might be expected to inhibit glandular stomach carcinogenesis.

Alternation of Gastric Mucosal Glycoprotein (Lectin-Binding Pattern) in Gastric Mucosa in Stress

Gastric mucosal cells of the rat glandular stomach were studied by light and electron microscopic procedures by use of lectins in the development of acute gastric mucosal lesions. Effects of the H2-receptor antagonist sofalcone (2'carboxymethoxy-4,4'-bis 3-methyl-2) and truncal vagotomy with pyloroplasty on lectin binding sites and distribution were also investigated. Biotinylated lectins in combination with ABC (avidin-biotinyl peroxidase complex) method were used for light and horseradish peroxidase (HRP)-labeled lectins for electron microscopic studies. Gastric mucosal cells showed the specific binding pattern for each lectin by light microscopy. Especially, binding sites and distribution of peanut agglutinin (PNA) were characteristic after induction of stress, truncal vagotomy, and administration of each drug. Staining and distribution increased in the gastric mucosa upward and downward after that. In electron microscopic studies, PNA strongly stained the membranes of the intracellular secretory canaliculi of a parietal cell. These results suggested that alternation of binding sites and distribution was regulated by change of gastric mucosal blood flow and of acidity in the parietal cells. Therefore, increase of glycoconjugate distribution is supposed to be a possibility of cytoprotective effect for a change of environment in the parietal cells.

Synergism between sodium chloride and sodium taurocholate and development of pepsinogen-altered pyloric glands: relevance to a medium-term bioassay system for gastric carcinogens and promoters in rats.

In an approach to early detection of gastric carcinogens and promoters in an in vivo test system, promotion by sodium chloride (NaCl) and the synergistic effects of NaCl and sodium taurocholate (Na‐TC) on development of pepsinogen‐altered pyloric glands (PAPG) in rat glandular stomach after initiation with N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG) were investigated. A total of 205 male WKY/NCrj rats were divided into 8 groups. Group 1 was given a single dose of MNNG of 160 mg/ kg body weight by gastric intubation, and starting 2 weeks later basal diet containing Na‐TC for 18 weeks. In addition, 1 ml doses of saturated NaCl solution were given by gastric intubation at weeks 4, 6, 8 and 10. Similarly, group 2 was treated with MNNG and Na‐TC, while group 3 animals received MNNG and NaCl. Group 4 was given MNNG alone. Groups 5–8 served as equivalent controls without MNNG initiation. The results revealed significantly enhanced induction of immunohisto‐chemically defined PAPG in the Na‐TC + NaCl (P< 0.001), Na‐TC (P<0.01) and NaCl (P<0.01) treated animals initiated with MNNG. Sodium chloride demonstrated a clear synergistic effect with Na‐TC in promoting the development of PAPG, suggesting possible advantage for its use in medium‐term in vivo assays for detection of gastric carcinogens and promoters.

Effects of Chronic Ethanol Consumption on Carcinogen Activating and Detoxifying Systems in Rat Upper Alimentary Tract Tissue

The effect of chronic ethanol consumption on enzyme systems directly involved in carcinogen activation and detoxification were studied in rat upper alimentary tract tissue. Microsomal cytochrome P-450 (P-450) levels and glutathione levels as well as glutathione transferase and UDP-glucuronic acid transferase (UDPGT) activities were measured in mucosa scraped from esophagus, forestomach and glandular stomach of rats which had been pair-fed ethanol or dextrimaltose-containing diets. Esophageal and forestomach P-450 levels were increased in the ethanol-fed rats. The ethanol diet also produced a small but significant increase in esophageal glutathione transferase levels. Glutathione levels and UDPGT activity were unaffected. Since P-450 is directly involved in the activation of many chemical carcinogens, these results are consistent with the hypothesis that the increase in upper alimentary tract cancer risk associated with alcohol abuse is due, at least in part, to ethanol's altering the balance between carcinogen activation and detoxification.

Toxicity studies of amphetamine sulfate, ampicillin trihydrate, codeine, 8-methoxypsoralen, α-methyldopa, penicillin VK and propantheline bromide in rats and mice

Thirteen-week toxicity studies in F344/N rats and B6C3F 1 mice were conducted to determine general toxicity and target organ toxicity with amphetamine sulfate, ampicillin trihydrate, codeine, 8-methooxypsoralen, α-methyldopa sesquihydrate, penicillin VK, and propantheline bromide. This paper discusses the toxicity observed; use of the toxicity data to set dose levels for subsequent 2-year studies; and comparison of dose levels administered to rodents with doses used in the treatment of human disease. Drugs were administered orally in the feed or by gavage. The lowest doses in the 13-week studies were comparable to therapeutic doses in man on a mg/m 2 (body surface area) basis or 5–10 times doses used in man on a mg/kg body weight basis. Little toxicity was seen at the low dose level with ampicillin, penicillin VK, 8-methoxypsoralen or propantheline bromide. At higher doses, target organ toxicity seen included the urinary bladder in male rats after propantheline bromide; the glandular stomach in rats and mice after penicillin VK; the liver and adrenals in rats after 8-methoxypsoralen; and the kidney in mice and rats after α-methyldopa. After amphetamine, codeine, or ampicillin administration, no target organ toxicity was seen in rats or mice, even at doses which caused body weight gain depression. The high doses chosen for subsequent 2-year studies were within 10 times human dose levels when compared on a mg/m 2 body surface area.

Gastric Lesions Induced by Kainic Acid Injection into the Dorsal Motor Nucleus of the Vagus Nerve in Rats

Kainic acid injection into the dorsal motor nucleus of the vagus nerve (DMN) induced lesions in the glandular stomach of rats. A decrease in mucus production seemed to play an important role in the pathogenesis of the kainic acid-induced gastric lesions. On the other hand, by means of horseradish peroxidase tracing method, the original cells projecting to the DMN were identified in the central nucleus of the amygdala, several hypothalamic nuclei and the insular cortex. Considering that kainic acid is a long-acting neuronal excitant, it was suggested that the continual stimulation of DMN neurons facilitated gastric ulcer formation.

Evidence of potential tumour-initiating and tumour-promoting activities of hickory smoke condensate when given alone or with nitrite to rats

Epidemiological studies have indicated that the consumption of smoked fish and meat products is associated with an increased risk of stomach cancer. A commercial hickory smoke condensate (HSC) was evaluated for its tumour-initiating and promoting activities in the glandular stomach using short-term methods in vivo. HSC (1 ml of a 10–100% v/v solution/rat) was given orally, either with or without nitrite (25–100 μmol/rat) to male F344 rats. The potential of HSC to act as a glandular stomach carcinogen was revealed by the induction in the pyloric mucosa of the stomach of ornithine decarboxylase (ODC), replicative DNA synthesis (RDS) and DNA single-strand breaks. The administration of HSC with nitrite also induced unscheduled DNA synthesis in the pyloric mucosa, but led to decreased induction of ODC, RDS and DNA single-strand breaks in comparison with treatment with HSC alone. These results suggest that HSC contains substance(s) that have potential tumour-initiating and/or tumour-promoting activities and that reaction with nitrite generates new substance(s) that could act as potential tumour-initiators in the rat glandular stomach.

Antioxidants-Carcinogenic and Chemopreventive Properties

Synthetic and naturally occurring antioxidants have a wide variety of biological actions in rodents in addition to their primary antioxidant activity. Some of the included biological effects are of direct interest in relation to studies of carcinogenicity and/or modulation of carcinogenesis. Since the synthetic antioxidant BHA was first found to exert carcinogenic potential in rat and hamster forestomach epithelium, many other synthetic and naturally occurring antioxidants have been examined for their ability to induce proliferative activity in the alimentary canal. These studies have revealed that caffeic acid and sesamol are also tumorigenic for rat forestomach epithelium, whereas catechol and p-methylcatechol induce neoplasia in rat glandular stomach epithelium. Although the proliferative response is very rapid, with inflammation and ulceration, it takes a very long time before carcinomas develop. The proliferative lesions in the forestomach induced by BHA or caffeic acid are largely reversible, in contrast to those induced by genotoxic carcinogens, which generally persist and develop into cancer. Therefore, chronic irritation is considered to be responsible for the induction of stomach cancer by antioxidants. Butylated hydroxyanisole can undergo oxidative metabolism in vitro, and some of the metabolites formed have the potential for binding to proteins. Neither BHA nor its metabolites binds to DNA in vivo, but protein binding in the forestomach was greater than 10 times higher than that in the glandular stomach. It is thus conceivable that BHA is oxidatively metabolized in the forestomach epithelium (possibly entering into redox cycling), and reactive metabolites including semiquinone radicals or active oxygen species are responsible for the carcinogenesis by a mechanism involving binding to macromolecules. Many antioxidants have been shown to modify carcinogenesis, and as a rule, they inhibit the initiation stage by reducing the interaction between carcinogen and DNA. However, both promotion and inhibition have been reported for second-stage carcinogenesis, depending on the organ site, species of animal, or initiating carcinogen. They can also block reaction of amine and nitrite to form nitrosamines or reduce TPA promotion of skin carcinogenesis. Generally high doses of antioxidants are required for carcinoma induction or modification of chemical carcinogenesis. The significance of the reported tumorigenicity and strong promoting activity of antioxidants for forestomach epithelium of animals to the development of human cancer appears limited mainly because humans do not have a forestomach. The carcinogenic and strong promoting activities of catechol and its structurally related compounds on rat glandular stomach epithelium are of greater concern because this tissue is directly analogous to human gastric epithelium.(ABSTRACT TRUNCATED AT 400 WORDS)