In vitro binding of potent mutagenic pyrolysates to intestinal bacteria.

The ability of 22 strains of intestinal bacteria to bind the mutagenic pyrolyzates--3-amino-1,4-dimethyl-5H-pyrido-[4,3-b]indole [(Trp-P-1) CAS: 62450-06-0], 3-amino-1-methyl-5H-pyrido [4,3-b]indole [(Trp-P-2) CAS: 62450-07-1], 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole [(Glu-P-1) CAS: 67730-11-4], 2-aminodipyrido[1,2-a:3',2'-d]imidazole [(Glu-P-2) CAS: 67730-10-3], 2-amino-3-methylimidazo[4,5-f]quinoline [(IQ) CAS: 76180-96-6], 2-amino-3,4-dimethylimidazo[4,5-f]quinoline [(MeIQ) CAS: 77094-11-2], and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline [(MeIQx) CAS: 77500-04-0]--was investigated and compared to their ability to bind to some dietary fibers (corn bran, apple pulp, soy bean fiber, cellulose, chitin, and chitosan). The pyrolyzates are potent mutagenic and carcinogenic heterocyclic amines formed during cooking. Solution of these amines was mixed with aqueous suspension of bacterial cells or dietary fibers, and removal of these amines from the reaction mixture by centrifugation was defined as the binding. Trp-P-1 and Trp-P-2 were effectively bound to all gram-positive and some gram-negative bacterial cells, corn bran, apple pulp, and soy bean fiber. Binding of Trp-P-1 and Trp-P-2 to Escherichia coli, Klebsiella pneumoniae, and cellulose was moderate, and to chitin and chitosan it was little. None but corn bran bound Glu-P-1 and Glu-P-2 effectively. Corn bran effectively bound all mutagens tested. The quantity of the binding of IQ, MeIQ, and MeIQx was dependent on the strain of bacteria and the kind of fiber. The mechanism of binding of Trp-P-2 to freeze-dried feces, Lactobacillus casei YIT 9018 (LC9018), and corn bran was investigated. The binding was pH dependent, occurred instantaneously, and was inhibited by the addition of metal salts. These results indicate that the binding was mostly due to a cation-exchange mechanism, but some irreversible binding of Trp-P-2 was observed, most notably to freeze-dried feces. The mutagenicity of Trp-P-2 for Salmonella typhimurium TA98 in the presence of S9 mix was inhibited by the addition of LC9018 or corn bran to the reaction mixture. The results indicate that bound Trp-P-2 did not cause mutation under the assay conditions.

Rapid regression of squamous cell hyperplasia and slow regression of basal cell hyperplasia in the forestomach of F344 rats treated with N-methyl-N'-nitro-N-nitrosoguanidine and/or butylated hydroxyanisole.

Cell kinetics of reversible and persistent forestomach lesions induced by the genotoxic agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and/or the nongenotoxic antioxidant butylated hydroxyanisole (BHA) was investigated. A total of 184 male F344 rats were divided into four groups: Group 1 given an initial single gastric intubation of MNNG received 2% BHA diet from the third wk to the 26th wk and then basal diet; Group 2 receiving 2% BHA without MNNG initiation; Group 3 given MNNG alone; and Group 4 serving as a nontreated control. Rats were sequentially sacrificed at 6, 16, 26, 30, and 46 wk. Bromodeoxyuridine was administered either as a single i.p. injection (100 mg/kg of body weight) 1 h before killing or continuously via an osmotic minipump (120 micrograms/h) for 1, 3, or 7 days prior to sacrifice, in each case labeled cells being detected by immunohistochemistry. Squamous cell hyperplasia (SCH) and basal cell hyperplasia (BCH), each characterized by different phenotypic keratin expression, were induced in Groups 1 to 3. After withdrawal of BHA, rapid regression of SCH and extremely slow regression of BCH were observed. Papillomas and squamous cell carcinomas developed irreversibly in Group 1 and 3, BHA significantly (P less than 0.01) enhancing the incidence of SCC in Group 1. Flash and continuous bromodeoxyuridine labeling revealed SCH to consist of cells of high mitotic activity and short life span, whereas BCH consisted of cells with low mitotic activity and long life span. In addition, highly labeled areas were observed in SCH after cessation of BHA feeding in Group 1 without regression, and similar lesions were also evident in Group 3. The results suggest that rapid regression of SCH and slow regression of BCH reflect different cell kinetic patterns and that highly labeled areas after release from stimulating agents might be preneoplastic changes related to cancer development.

Effects of phenolic antioxidants in low dose combination on forestomach carcinogenesis in rats pretreated with N-methyl-N'-nitro-N-nitrosoguanidine.

The combined effects of low doses of promoters or carcinogens on two-stage forestomach carcinogenesis were examined in rats pretreated with N-methyl-N'-nitro-N-nitrosoguanidine. Groups of 15 rats were given a single 150 mg/kg body weight intragastric dose of N-methyl-N'-nitro-N-nitrosoguanidine. Starting 1 week later they were fed a diet containing low doses of known forestomach promoters/carcinogens (0.5% caffeic acid, 0.2% catechol, 0.5% butylated hydroxyanisole, or 0.25% 2-tert-butyl-4-methylphenol), alone or in combination, or basal diet without antioxidant supplement for 35 weeks. Histopathological examination revealed the incidences of forestomach squamous cell carcinomas in animals treated with N-methyl-N'-nitro-N-nitrosoguanidine followed by caffeic acid, catechol, butylated hydroxyanisole, 2-tert-butyl-4-methylphenol, and basal diet to be 27, 20, 13, 13, and 7%, respectively, whereas the incidence increased to 80% by the combined treatment with these four chemicals. The present results thus show that although the low doses of individual promoters/carcinogens did not have significant promoting activity, their combination exerted a strong enhancing influence on rat forestomach carcinogenesis. The findings indicate the importance of summation and synergism at a low dose for agents present in the human environment.

Antitumor activity of 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxy-camptothec in, a novel water-soluble derivative of camptothecin, against murine tumors.

The search for new water-soluble analogues of camptothecin (CPT) with higher activity and less toxicity has led to the development of a novel compound, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxy-camptothecin (CPT-11), which showed significant antitumor activity against a broad spectrum of experimental tumor models by i.p., i.v., or oral administration. When its activity against L1210 was compared with that of CPT and known derivatives, CPT-11 was most effective, giving the highest maximum increase in life span (ILS) and showing good activity over a wide dose range. The antitumor activity of CPT-11 was shown against tumors not only in the ascites form but also in the solid form. Included among the more susceptible murine tumors are S180, Meth A fibrosarcoma, Lewis lung carcinoma, Ehrlich carcinoma, MH134 hepatoma, mammary carcinoma of C3H/HeN mice, L1210, and P388 leukemia. Probable cures of these tumors were induced frequently by CPT-11. The antitumor activity of CPT-11 against i.p.-implanted L1210 was superior to that of Adriamycin in maximum ILS, the number of cured mice, and the therapeutic ratio. CPT-11 at a dose of 100 mg/kg produced an ILS in excess of 300% with five of six mice surviving tumor free, and effected 100% tumor regression at 200 mg/kg, whereas the optimum dose of Adriamycin, 12.5-25 mg/kg, brought about 114-129% ILS with one of six mice surviving. The acute toxicity of CPT-11 was extremely low, particularly in the case of oral administration. CPT-11 is expected to be clinically useful.

Selective changes in gangliosides of human milk during lactation: a molecular indicator for the period of lactation.

Gangliosides of human milk from women at various periods of lactation were analyzed. GD3 in colostrum, particularly in the early period of lactation, was the major ganglioside, and the molar ratio of GM3 to GD3 was 0.2-0.3 in the milk at 2-6 days postpartum. In contrast, milk from women at 60-390 days postpartum contained GM3 as the major ganglioside and the molar ratio of GM3 to GD3 was more than 3. Milk at 8-40 days postpartum represented an intermediate stage in terms of the ratio of GM3 to GD3. The selective change in the molar ratio of gangliosides was observed as a phenomenon common to all human milk from different individuals at different periods of lactation, indicating that the periods of lactation can be defined on the basis of the ratio. Since glycolipids in human milk are preferentially localized in the milk fat globule membrane, which is derived from the plasma membrane of epithelial cells in the mammary gland, the changes in the ganglioside composition reported in this communication may reflect a qualitative change of the cells in the mammary gland.

Role of colony-stimulating activity in antitumor activity of Lactobacillus casei in mice.

Wide-ranging differences were observed between the antitumor activities of 23 lactobacilli (13 species; 23 strains) and their capacities to elevate the level of serum colony-stimulating activity (CSA) by intraperitoneal administration in mice, and a good correlation existed between the two activities. The mechanism of enhanced production of CSA by administration of Lactobacillus casei YIT 9018 (LC 9018), one of the bacteria that had the strongest activities, and the role of CSA in antitumor activity of LC 9018 were studied. Colony-stimulating activity in the washing fluid from the peritoneal cavity of mice that had been administered LC 9018 intraperitoneally was elevated at 3 to 24 h after the injection, and CSA was also detected at elevated levels in the serum of the mice 6 to 12 h after injection. The cells responsible for the production of CSA after stimulation with LC 9018 seem to be the resident macrophages at the site of administration, because the resident macrophages of mice lavaged 1 h after an intraperitoneal administration of LC 9018 released CSA when they were cultured in vitro. Moreover, resident peritoneal macrophages of normal mice cultured with LC 9018 in vitro also produced CSA. Similar results were obtained with athymic nude mice, and the CSA-inducing activity of LC 9018 was diminished in the mice pretreated with carrageenan, which is selectively toxic to mature macrophages. Bone marrow cells matured to macrophages and polymorphonuclear cells by culture with the CSA induced by LC 9018 for 7 days. These matured macrophages showed strong antitumor activity both in vivo and in vitro. These results suggest that CSA plays important roles in the antitumor activity of LC 9018: it enhances not only the multiplication of committed precursor cells for macrophages and polymorphonuclear cells, but also the functional maturation of the precursor cells for macrophages which serve as potent effectors for tumor cells.

Increased production of cytotoxic macrophage progenitors by Lactobacillus casei in mice.

Heat-killed Lactobacillus casei YIT9018 (LC9018), when administered intravenously to normal mice, induced increase in Mac-1+ cells and Mac-2+ cells but not in Mac-3+ cells in spleen. The number of both populations changed in the same time course and was maximal 14 d after the administration. To know the effect of LC9018 on hematopoietic progenitor level, we examined the number of macrophage colony-forming cells (M-CFC), granulocyte-macrophage CFC (GM-CFC), and colony-forming units in spleen (CFU-S) in bone marrow 3 d after the administration. LC9018 stimulated the proliferation of M-CFC but not that of GM-CFC and CFU-S. LC9018-induced M-CFC were similar to normal M-CFC in dependence on macrophage colony-stimulating factor (M-CSF) and buoyant density. M-CFC-derived macrophages cultured in the presence of M-CSF expressed Mac-1 and Mac-2 but not Mac-3. They showed cytotoxic activity against syngenic tumor cells, Meth A, via direct contact, when assayed by using an in vitro colony inhibition assay or an in vivo Winn test. These results indicate that LC9018 stimulates the proliferation of cytotoxic macrophage progenitors in bone marrow and induces their differentiation in spleen. These effects may be one of the ways in which LC9018 suppresses tumor growth.

Modulatory interaction between initial clofibrate treatment and subsequent administration of 2-acetylaminofluorene or sodium phenobarbital on glutathione S-transferase positive lesion development.

The effects of 2-acetylaminofluorene (2-AFF) or sodium phenobarbital (PB) treatment subsequent to clofibrate (CF) administration in terms of preneoplastic lesion development and induction of hepatocellular carcinomas (HCC) were studied using Fischer 344 rats. Animals received CF (0.3% in diet) for the initial 30 weeks, and then either 2-AAF (0.01% in diet), PB (0.05% in diet) or basal diet until week 78. Further groups were initially given basal diet, and then treated with 2-AAF or PB week 30. Two-thirds partial hepatectomy was carried out on all animals at week 3, sacrifice of representative groups being performed at weeks 30, 48 and 78. No glutathione S-transferase placental form positive (GST-P+) or negative focal or nodular lesions were apparent at the cessation of CF administration. The induction of GST-P+ focal lesions by 2-AAF was markedly decreased at week 48 in the group previously given CF (P less than 0.05) and furthermore, the respective incidences of HCC at week 78 were 4/17 (23.5%) in the CF----2-AAF group and 7/17 (41.2%) in the 2-AAF alone case. No significant differences between CF----PB and PB alone groups were evident with regard to either GST-P+ lesions and HCC at weeks 48 and 78. No CF-specific GST-P negative neoplastic nodules or HCC were observed in any of the experimental groups. These results suggest that pretreatment with CF may inhibit the induction of GST-P+ focal lesions and HCC by subsequently administrated 2-AAF and that CF demonstrates no initiating activity for liver carcinogenesis under the present condition.

Effects of carbon tetrachloride administration on initiation of liver cell foci by the non-hepatocarcinogens N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and benzo(a)pyrene (B(a)P).

In a development trial for an initiation bioassay system, cell proliferation kinetics after partial hepatectomy (PH) or CCl4 administration (1 ml/kg b.w., i.g.) and the effect of administration time after PH or CCl4 treatment on liver cell foci induction by the direct and indirect non-hepatocarcinogens, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and benzo(a)pyrene (B(a)P) were investigated. Male F344 rats were killed 12, 18, 24, 36, 48, 72 or 96 h after PH or CCl4 treatment and liver cell proliferation was examined with the bromodeoxiuridine (BrdU) labeling method. Appreciable increase in the BrdU labeling index was observed 18-36 h after PH with a peak at 24 h, and 18-72 h following treatment with CCl4 with a peak at 48 h. MNNG (80 mg/kg i.g.) or B(a)P (100 mg/kg i.g.) were administered to 7-week-old male F344 rats at various times after PH or CCl4 treatment and lesion induction was assessed using the resistant hepatocyte model. MNNG caused significant numbers of glutathione S-transferase placental form (GST-P)-positive liver cell foci in rats when given 12-36 h after PH, with a peak at 24 h. In contrast, the numbers of foci induced by B(a)P were maximal with exposure at 12 h after PH. In the CCl4 study, both MNNG and B(a)P induced significant increase in GST-P-positive liver cell foci when given 12-72 h after CCl4 treatment, with a peak at 48 h, the results being directly in line with the changes in BrdU labeling. From these findings, it is concluded that initiation assay protocols with a CCl4 proliferative stimulus to hepatocytes may prolong the appropriate administration period for effective detection of the initiation potential of both direct and indirect carcinogens targeting sites other than the liver.

Sequential observation of micrometastasis formation by bacterial lacZ gene-tagged Lewis lung carcinoma cells.

Sequential events in micrometastasis formation including entry into the blood circulation and arrest, extravasation and initial growth in the lung was investigated using bacterial lacZ gene-tagged Lewis lung carcinoma cells (4A1-1). Micrometastases in the lung could thereby be specifically detected at the single cell level by X-Gal staining. After intravenous injection, X-Gal positive tumor cells appeared to extravasate within hours, but most cells then degenerated or died in the alveolar space by 2-3 days postinjection. A decreased BrdU labeling index to a negligible level at 2 days postinjection and reduction of X-Gal positive foci to a basal level (less than 0.1% of injected cells) by 4 days are in line with rapid clearance of tumor cells from the lung. The size and BrdU labeling indices of the persisting X-Gal positive foci, however, started to increase from 4 days postinjection. Type IV collagen immunostaining demonstrated loss of pre-existing basement membranes with growth of micrometastases: When 4A1-1 cells were inoculated subcutaneously, lung micrometastases from resulting tumors were detected as single or small numbers of X-Gal positive cells at 2 weeks postinjection. Progressive development of micrometastasis to macroscopic metastasis was noted by 4-5 weeks postinjection. The results indicate that micrometastasis formation by Lewis lung carcinoma cells involves a sequence of events starting with rapid extravasation after arrest in the lung within 1 day, followed by death of most cells at 2-3 days and subsequent new growth and expansion of persisting tumor cells from 4 days postinjection.

Appearance of fucolipid after conventionalization of germ-free mice.

Incorporation of [6-3H]fucose and [1-14C]glucosamine into the lipid fraction of microvillus membrane (MVM) was studied in germ-free and conventionalized mice after intraperitoneal injection of the radioactive precursors. Incorporation of [6-3H]fucose was clearly detected in conventionalized mice but not detectable in germ-free mice. There was no difference in the incorporation of [1-14C]glucosamine between the two groups. The lipid fraction of MVM labeled with [6-3H]fucose showed a spot of slower mobility than asialo GM1 on TLC with autoradiography and it was confirmed to be a fucolipid on abolishing the radiolabeled original spot by alpha-L-fucosidase treatment. These results suggest that the synthesis of the fucolipid is induced by conventionalization of germ-free mice.

Biosynthesis of microvillus membrane-associated glycoproteins of small intestinal epithelial cells in germ-free and conventionalized mice.

We studied the effect of intestinal microorganisms on the synthesis of membrane-associated glycoproteins in the upper small intestine by intraperitoneally administering L-[3H]fucose, D-[14C]glucosamine, or L-[3H]leucine to germ-free mice and mice exposed to microorganisms for 4 weeks (conventionalized). The incorporation of the labeled compounds into sucrase-isomaltase complex and maltase was determined by immunoprecipitating Triton X-100-solubilized microvillus membranes with their antibodies. Purified microvillus membranes from germ-free and conventionalized mice differed in the activities of some marker enzymes but not in the number and mobility of the components on SDS-polyacrylamide gel electrophoresis. Maximal incorporation of [3H]fucose and [14C]glucosamine into the microvillus membrane and two enzymes was reached 2-3 h post-injection in both groups, however, the amounts incorporated were larger in conventionalized mice. There was little difference in [3H]leucine incorporation into the total glycoproteins of microvillus membranes between the two groups. Our results suggest that the introduction of microorganisms stimulates the synthesis of sugar chains of microvillus membrane-associated glycoproteins. The enhanced in vitro fucosyltransferase activity in conventionalized mice partly supports this suggestion.

Structure of 6-deoxytalose-containing polysaccharide from the cell wall of Bifidobacterium adolescentis.

The isolation and analysis of the cell wall and the polysaccharide-glycopeptide complexes of Bifidobacterium adolescentis YIT4011 are presented. Polysaccharide-glycopeptide complexes, PS-GP1 and PS-GP2, were solubilized from the cell wall by treatment with N-acetylmuramidase. PS-GP1 and PS-GP2 were found to be composed of glucose, 6-deoxytalose and a small amount of glycopeptide. The products of Smith degradation of the PS-GPs had no glucose-containing fraction, but were composed of 1,2/1,3-linked 6-deoxytalose. Furthermore, a second Smith degradation of this fraction yielded trisaccharide-glyceraldehyde. These results and methylation analysis led to the conclusion that PS-GP1 or 2 has a repeating unit of----3)6dTal(beta 1----3)6dTal(beta 1----3)6dTal(beta 1----2)-6dTal(alpha 1----2)6dTal(alpha 1----2)6dTal(alpha 1-, and that glucose residues are linked to position C-3 of the 2-O-substituted 6-deoxytalose residues.

Presence of asialo GM1 and glucosylceramide in the intestinal mucosa of mice and induction of fucosyl asialo GM1 by conventionalization of germ-free mice.

Two major neutral glycolipids of the intestinal mucosa were purified by a series of column chromatographies and the structures were determined to be glucosylceramide and asialo GM1 by gas liquid chromatography. The carbohydrate structure of asialo GM1 was also confirmed from the reactivity of the glycolipid to rabbit anti-asialo GM1 antibody by an enzyme linked-immunosorbent antibody. The ceramide portion of both glycolipids had an extremely hydrophilic nature and more than 90% of the ceramide was composed of phytosphingosine and alpha-hydroxy fatty acids. In the previous paper we reported that the induction of a fucolipid in the microvillus membrane of mouse intestinal mucosa by conventionalization was observed on monitoring the incorporation of radiolabeled fucose in vivo. A fucoglycolipid having the same mobility on an autoradiogram as the fucolipid labeled in vivo was produced by in vitro incubation of intestinal asialo GM1 and GDP-[14C]fucose with the mucosal homogenates. Moreover, asialo GM1 prepared from brain gangliosides exhibited a similar ability to accept fucose and it was converted to fucosyl asialo GM1 which moved faster than the product from intestinal asialo GM1. The difference is considered to be due to the ceramide composition. These results suggest that conventionalization can induce the fucosyl asialo GM1 in the microvillus membrane probably through the induction of fucosyltransferase. Understanding the molecular mechanism of interaction between the physiological flora and host is the matter of further study.

Mutagenic activation of biliary metabolites of benzo(a)pyrene by beta-glucuronidase-positive bacteria in human faeces.

Human faeces hydrolysed synthetic beta-D-glucuronides of both p-nitrophenol and phenolphthalein. The origin of this activity in faeces was localised in the bacterial pellet fraction after centrifugation. Ninety-seven bacterial strains with beta-glucuronidase activity isolated from fresh human faeces were identified as species of Bacteroides, Peptostreptococcus, Fusobacterium, Propionibacterium, Clostridium, Eubacterium and Bifidobacterium. They were classified into two groups according to their activity against two synthetic beta-D-glucuronides. One group hydrolysed p-nitrophenyl glucuronide and phenolphthalein glucuronide to the same extent and the other hydrolysed p-nitrophenyl glucuronide much more strongly than phenolphthalein glucuronide. The bile of rats given benzo(a)pyrene by mouth was tested for mutagenicity in the presence and absence of cell-free extracts of human faeces and bacteria. Extracts of beta-glucuronidase-positive bacteria increased the mutagenicity of metabolites of benzo(a)pyrene, as did faecal extracts, but extracts of beta-glucuronidase-negative bacteria did not. D-Saccharic acid-1,4-lactone inhibited the increase in mutagenicity produced by the faecal extracts and extracts of beta-glucuronidase-positive bacteria except for Peptostreptococcus strains 204 and 952. These results indicate that some intestinal bacteria have beta-glucuronidases heterogenous in substrate specificity and that they may be involved in mutagenicity of benzo(a)pyrene in the intestinal tract.

Inhibition of spontaneous and experimental metastasis by a new derivative of camptothecin, CPT-11, in mice.

The antimetastatic effect of a new water-soluble derivative of camptothecin, 7-ethyl-10-(4-(1-piperidino)-1-piperidino) carbonyloxy-camptothecin (CPT-11), were examined in several metastatic murine tumor systems. Intravenous (i.v.) injection of CPT-11 into BALB/c mice inhibited lung metastasis by i.v. inoculated, metastatic colonic adenocarcinoma 26 (C26) cells, C26NL-17, in BALB/c mice. This treatment was also effective in C57BL/6 mice against lung metastasis by i.v. inoculated B16-F10 and B16-BL6 cells, highly metastatic variants of the B16 melanoma. Furthermore, intraperitoneal (i.p.) injection of CPT-11 significantly inhibited the growth of C26NL-22 cells, a highly metastatic variant of C26, inoculated subcutaneously (s.c.) into the left front footpads of BALB/c mice. Also, i.p. or i.v. injection of CPT-11 effectively inhibited the growth of 3LL tumors inoculated s.c. into the hind footpads of C57BL/6 mice. Moreover, following s.c. inoculation of either C26NL-22 or 3LL cells, combined surgical excision of the primary tumor and either i.p. or i.v. CPT-11 injections given before or after surgery markedly inhibited the formation of pulmonary metastases. These results show that a new derivative of camptothecin, CPT-11, has a potent inhibitory effect against both spontaneous and experimental lung metastasis.

Somatotopic organization of motoneurons innervating the pronators, carpal and digital flexors and forepaw muscles in the dog: a retrograde horseradish peroxidase study.

Central distribution of motor neurons innervating the individual forearm and forepaw muscle through the median and ulnar nerves was studied in the dog using the retrograde horseradish peroxidase (HRP) method. All HRP-labeled cells were seen ipsilaterally in restricted parts within the dorsolateral (DL) and retrodorsolateral nucleus (rDL) of the ventral horn at levels from the cranial tip of C7 to the cranial third of T2. The results showed that motoneurons situated more cranially supply the more proximal muscles and those situated more caudally supply the more distal ones, and that the pronator motoneurons occupy longitudinally the central part of the DL; the carpal flexor motoneurons, the medial part of the DL; the digital flexor motoneurons, the dorsal central part of the DL and the lateral fourth of the rDL; and the forepaw muscle motoneurons, the medial three-fourths of the rDL. These somatotopic arrangements of each motor pool could be correlated with the location and action of the muscles.

Alteration in sensitivity of transmural electrical response to propionate in rat colon after chronic luminal infusion of short-chain fatty acids.

The colon is always exposed to abundant short-chain fatty acids (SCFA) produced by gut fermentation. In order to know an effect of chronic load of SCFA on colonic functions, we studied that the acute and chronic effects of SCFA on transmural potential difference (p.d.) across the proximal colon of germ-free (GF), gnotobiotic (GB) and conventionalized (CV) rats in vivo. Intravenous administration of SCFA (acute effect), such as propionate, butyrate, valerate or caproate, caused a transient increase in the p.d. The acute effects of propionate were studied in detail. The dose-response curve of CV rats shifted markedly to the right compared to that of GF rats, suggesting that CV rats were less sensitive to the acute effects of propionate than GF rats. Decreased sensitivity also appeared in GB rats (monocontamination with Fusobacterium varium). By chronic luminal infusion of isotonic sodium propionate or butyrate (25.5 ml/day) into the proximal colon of GF rats for 7 days (chronic effect), the acute effects of propionate were reduced. Atropine reduced the p.d. increment produced by propionate and shifted the dose-response curve of propionate to the right. These results suggest that chronic luminal load of SCFA resulted in a type of chronic refractoriness.

Clofibrate-induced neoplastic development in the rat liver is associated with decreased connexin 32 expression but not with a co-ordinated shift in expression of marker enzymes.

Altered enzyme phenotype and expression of connexin 32 (Cx32), a gap junction protein were studied during the development of rat liver tumors induced by the non-genotoxic carcinogen, clofibrate. (1) In contrast to previous findings for nitrosamine-induced lesions, preneoplastic enzyme-altered foci (EAF) and neoplastic nodules (NN) lacked any clear association with degree of altered enzyme expression because of an almost complete negativity for GST-P and GGT. (2) Immunohistochemically demonstrated Cx32 spots on the hepatocyte membranes showed a clear decrease in clofibrate-induced lesions. (3) Naturally occurring EAF demonstrating GST-P and/or GGT positivity did not show a significant decrease of Cx32 counts suggesting a reversible nature. Therefore, the Cx32 decrease appears closely linked to progression of hepatocarcinogenesis irrespective of the enzyme phenotype of neoplastic focal lesions and the carcinogens used for their induction.

Mutagenic activation of biliary metabolites of 1-nitropyrene by intestinal microflora.

To investigate the modifying role of intestinal microflora in the metabolism of chemical carcinogens in vivo, we subjected bile from Fischer rats treated per os with chemical carcinogens and related compounds to a mutagenicity assay in the presence and absence of a cell-free extract from human feces. A mixture of the bile sample and potassium phosphate buffer was incubated in the presence or absence of human cell-free fecal extract and then further incubated with a bacterial suspension of Salmonella typhimurium tester strains TA98 or TA100. Bile from rats treated with 1-nitropyrene (1-NP) produced about 2700 and 400 revertants per plate in strain TA98 in the presence and absence of the fecal extract, respectively. There was a drug dose- and bile volume-related response. Treatment of 1-NP-bile with beta-glucuronidase, but not aryl sulfatase, enhanced its mutagenicity. Cell-free extracts of some strains of intestinal bacteria (Bacteroides fragilis ATCC 12044, B. vulgatus ATCC 8482, B. thetaiotaomicron ATCC 12290, Bacteroides sp. strain 524, Eubacterium eligens VPI C15-48, Peptostreptococcus sp. strain 204 and Escherichia coli A-5-18) also enhanced the mutagenicity of 1-NP-bile. These bacterial cell-free extracts hydrolyzed the synthetic beta-D-glucuronides of phenolphthalein and/or p-nitrophenol. These data indicate that the glucuronide(s) of 1-NP-metabolite(s) secreted into bile can be hydrolyzed in the intestine by bacterial beta-glucuronidases to potent mutagenic aglycone(s).