Evaluation of estrogenic activity of organic biocides using ER-binding and YES assay.

Biocides have been used not only in everyday items such as clothes, kitchenware, daily necessities, and infant utensils, but also in cosmetics and wrapping papers for foodstuffs. Since there is a high possibility of exposure to biocides from such materials, their safety must be assessed adequately using a range of methods. We investigated the estrogenic activity of 20 organic biocides using two in vitro screen assays: estrogen receptor (ER) binding assay and yeast estrogen screen (YES) assay. Twelve of the biocides were positive in the ER-binding assay. Regardless of the presence or absence of rat S9Mix for metabolic activation, 4-chloro-3-methylphenol was positive in both ER-binding and YES assay. 4-Chloro-3,5-dimethylphenol was positive in the ER-binding assay and showed a pseudopositive manner in the YES assay that was observed the dose-independent estrogenic activity at only one dose point. Hiba oil was positive in the ER-binding assay but was positive in the YES assay only in the presence of rat S9Mix. These results suggest that ER-binding and YES assay could be adapted for evaluation of the endocrine-disrupting activity of biocides. The biocides found to be positive in vitro now require assessment by in vivo screening methods.

Prevalence and contamination levels of Listeria monocytogenes in retail foods in Japan.

Retail foods in Japan were surveyed for the presence and contamination levels of L. monocytogenes. It was isolated from 12.2, 20.6, 37.0 and 25.0% of 41 minced beef, 34 minced pork, 46 minced chicken and 16 minced pork-beef mixture samples, respectively. MPN values were higher than 100/g in five (10.9%) minced chicken samples, but lower than 100/g in all minced beef, pork and pork-beef mixture samples. The organism was also isolated from 5.4% of the 92 smoked salmon samples at MPN values lower than 10/g, and from 3.3% of 213 ready-to-eat raw seafood samples at MPN values from lower than 0.3 to higher than 100/g. None of the 285 vegetable samples were contaminated with L. monocytogenes. These findings indicate that ready-to-eat raw seafoods are relatively high risk among the foods surveyed in this study.

Modulation by extracellular matrices of monooxygenase and CYP1A1 induction in Hep G2 cells in serum-free culture.

The in vitro cellular functions of differentiated cells are influenced by culture conditions. Effects of several extracellular matrices (ECMs) on cytochrome P450-dependent monooxygenases (MFOs) induction and cytochrome P4501A1 (CYP1A1) gene expression were estimated in Hep G2 cells cultured in a serum-free medium. The cells were cultured on collagen type I- and II-, fibronectin-, and matrigel-coated dishes and MFO activities were induced by the addition of 3-methylcholanthrene (MC). The induction of ethoxycoumarin O-deethylase (ECOD) and alkoxyresorufin O-dealkylase activities as well as the expression of CYP1A1 mRNA were also determined. ECOD and methoxy- and ethoxyresorufin O-dealkylase activities in Hep G2 cells were enhanced by culturing the cells using a serum-free medium on fibronectin- or matrigel-coated dishes. ECOD activity on fibronectin-coated dishes was about 3-fold higher than that using a serum-supplemented medium on untreated dishes. Furthermore, both immobilized and soluble fibronectin enhanced the induction of MFOs. The expression of CYP1A1 mRNA using fibronectin-coated dishes was about 2-fold higher than that using a serum-supplemented medium on untreated dishes. These findings suggest that the gene expression in cultured cells is greatly influenced by ECMs. By using fibronectin-coated dishes to cell culture in a serum-free medium, reproducible and highly sensitive results can be obtained in experiments using cultured cells.

Disturbed pyloric motility in Ws/Ws mutant rats due to deficiency of c-kit-expressing interstitial cells of Cajal.

Interstitial cells of Cajal (ICC) are believed to initiate the basic contractile activity of the gastrointestinal tract. Interstitial cells of Cajal express c-kit receptor tyrosine kinase and are deficient in Ws/Ws mutant rats with a small deletion of the c-kit gene. As Ws/Ws rats show remarkable bile reflux to the stomach, the contraction pressure of the pylorus was compared between Ws/Ws and control +/+ rats. The contraction pressure of the pylorus was measured using a microtransducer, which was inserted through a pin-hole in the anterior wall of the stomach under anesthesia. The magnitude of bile reflux was estimated by measuring the content of bile acids in the stomach. The c-kit messenger RNA-expressing cells were detected by in situ hybridization. Frequency and the maximum pressure of the contraction were comparable between Ws/Ws and +/+ rats, but the duration of the contraction was significantly shorter in Ws/Ws rats than in +/+ rats. The number of c-kit messenger RNA-expressing ICC in the pylorus of Ws/Ws rats was 1.7% that of +/+ rats. The bile reflux observed in Ws/Ws rats was attributed to the decrease in the duration of the pyloric contraction, which appeared to result from the deficiency of c-kit messenger RNA-expressing ICC.

Increase of mast cells in the liver and lung may be associated with but not a cause of fibrosis: demonstration using mast cell-deficient Ws/Ws rats.

Tissue fibrosis is frequently associated with an increase of mast cells, and mast cells are regarded as playing a role in the induction of tissue fibrosis. We attempted to examine whether mast cells influenced the induction of fibrosis using Ws/Ws mast cell-deficient rats. The mast cell deficiency of Ws/Ws rats is due to a 12-base pair deletion of the c-kit gene. The activity of c-kit receptor tyrosine kinase is remarkably reduced in Ws/Ws rats. Liver fibrosis was induced by the repeated injections of pig serum, and lung fibrosis was induced by the instillation of bleomycin. Marked fibrosis in the liver and lung did occur in the Ws/Ws rats, and the magnitude of fibrosis was more severe in Ws/Ws rats than in control normal (+/+) rats. The mast cell increase was observed in the liver of +/+ and Ws/Ws rats and in the lung of +/+ rats. However, the number of mast cells in the liver of treated Ws/Ws rats with marked fibrosis was comparable to that observed in the liver of nontreated +/+ rats without fibrosis. Histamine content increased in the liver and lung of +/+ rats after the treatment, but it remained in low levels even after the treatment in Ws/Ws rats. Mast cells and histamine did not appear to play important roles in the induction of fibrosis. Thus, an increase in mast cell number and histamine content may be associated with but not a cause of fibrosis.

A comparison of Listeria monocytogenes serovar 4b isolates of clinical and food origin in Japan by pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis (PFGE) patterns of 102 L. monocytogenes serovar 4b isolates from patients and foods examined in Japan were compared with 16 isolates from foodborne listeriosis episodes which occurred in North America or Europe. Using a combination of PFGE patterns with the restriction enzymes SmaI, ApaI, AscI and Sse8387I, 82 clinical isolates from Japan were categorized into 45 PFGE types: the largest group of 17 isolates (20.7%) were of the same PFGE type as cultures from the large foodborne outbreaks which occurred in California (1985) and Switzerland (1983-1987). Twenty cultures from foods on retail sale in Japan were classified into 12 PFGE types: four isolates were of three PFGE types also recognized among isolates of clinical origin from Japan, including the predominant clinical type.

Molecular typing of Listeria monocytogenes isolated in Japan by pulsed-field gel electrophoresis.

A total of 40 strains of Listeria monocytogenes which have been demonstrated to be serovar 1/2a, 1/2b, and 4b were genotyped by pulsed-field gel electrophoresis (PFGE) after separate digestion with Apa I, Asc I, Sma I, and Sse 8387 I. Twenty-seven unrelated strains including four representative strains showed distinctly different genotypes according to their PFGE profiles. Then nine strains isolated from shredded cheese of different lots and four strains isolated from the cheese-processing environment were shown to display the same genotype. Therefore, it is suggested that the Listeria was spread in cheese by cross-contamination from the cheese-processing environment. Thus, PFGE analysis has a good typeability and excellent discriminatory power, and has provided a useful tool for investigation of the source of Listeria contamination.

Flow cytometric analysis of cell proliferation kinetics during duodenal ulcer healing.

Cell proliferation in the gastroduodenal mucosa of patients with duodenal ulcers was evaluated using flow cytometry. Forty patients with duodenal ulcers and 12 normal subjects were investigated. Biopsy samples were obtained during endoscopic examination and subjected to DNA analysis by flow cytometry. Thirty patients with duodenal ulcers were healed within 3 months with H2 blockers (tractable or responsive ulcers), whereas 10 patients did not respond to treatment (intractable ulcers). The percentage of cells at the DNA-synthetic phase, an index of cell proliferation, was constant in the adjacent duodenal mucosa 2 cm from ulcer margin and antral mucosa during duodenal ulcer healing. The index at the margin of tractable ulcers was elevated during the active stage (12.9 +/- 1.3), peaked during the healing stage (15.4 +/- 2.8) and returned to the same level at the scarring stage (10.9 +/- 2.0) as normal controls (10.3 +/- 1.7). However, the index was not elevated in intractable ulcers (10.3 +/- 1.7 in the healing stage) and was smaller than in tractable ulcers. These data indicate that augmented mucosal cell proliferation at the ulcer margin plays an important role in duodenal ulcer healing and intractable ulcers are characterized by an abnormal failure to accelerate DNA synthesis to achieve ulcer repair.

Detection of Listeria monocytogenes in humans, animals and foods.

The Listeria monocytogenes-carrying rates were 100% for listeriosis patients and 1.3% for healthy humans. The L. monocytogenes contamination rates for retail sliced beef (34.2%) and pork (36.4%) were significantly higher (p < 0.05) than those for cattle (2.0%) and pigs (0.8%) and for cattle (4.9%) and swine (7.4%) carcasses. The percentages of serotypes 1/2a, 1/2b and 4b which are most dominant in human patients were high in isolates from fresh (90.0%) and processed (100%) fish and shellfish and imported natural cheese (96.7%).

The ratio of plasma levels of IL-10/TNF-alpha and its relationship to disease severity and survival in patients with leptospirosis.

Twelve patients with leptospirosis were enrolled in a study to evaluate whether a correlation exists between disease severity and plasma levels of TNF-alpha and IL-10 as determined by enzyme-linked immunoassay. Six patients had less severe disease by analysis of liver, kidney and lung involvement, and 6 patients had severe disease, 2 of whom died. The results of the study confirm an association between high levels of TNF-alpha and poor outcome in leptospirosis. Levels of IL-10 were in a similar range in the 10 surviving and the 2 non-surviving patients. The ratio of IL-10/TNF-a correlated with disease severity in that a high ratio was associated with less severe disease and survival. The possibility is raised that treatment approaches to change this cytokine profile may be more effective in this type of inflammatory condition than it has been in bacterial sepsis.

Expression of the cyclooxygenase-2 gene in gastric epithelium.

This study examined the mRNA level of cyclooxygenase-2 (cox-2) in a rat gastric mucosal cell line after growth stimulation in vitro and in rat gastric mucosa before and after acid-induced injury in vivo. RGMI cells were stimulated with fetal calf serum in the in vitro study. Thereafter, the cox-2 mRNA level was examined by Northern analysis. Effects of NS-398, a specific inhibitor of cox-2, and indomethacin on prostaglandin production and cell proliferation were examined in RGM1 cells. In the in vivo study, rats were given 1 ml of 0.6 N hydrochloric acid into the stomach. The level of cox-2 mRNA was examined in rat gastric mucosa after acid administration. Cox-2 mRNA increased 20-60 min after growth stimulation in RGM1 cells. Both NS-398 and indomethacin suppressed prostaglandin production and cell proliferation after growth stimulation. Expression of cox-2 mRNA was observed 40 and 60 min after administration of 0.6 N HCl. Gastric lesions developed within 60 min after HCl administration and healed significantly within 48 h. The present study shows the expression of cox-2 in gastric epithelial cells in vitro and in gastric mucosa after injury in vivo. The results also suggest that cox-2 is involved in de novo synthesis of prostaglandins and cell proliferation in gastric epithelium.

[Molecular approach for analyses of gut function: an overview].

This paper reviewed recent progress in analyses of gut function by the aid of molecular biology. Particularly noted were 1) involvement of immediate early genes in mucosal epithelial proliferation in vitro and in ulcer healing in rat stomach in vivo; and 2) possible involvement of c-kit/stem cell factor, endothelin-receptor B, endothelin-3 and ret in regulation of gut motility and Hirschsprung's disease. These cases show that molecular biology, together with novel approaches through medical engineering, significantly expands our knowledge on regulation of gut functions.

Induction of cytochrome P450-dependent monooxygenase in serum-free cultured Hep G2 cells.

We examined the induction of cytochrome P450-dependent mixed-function monooxygenase (MFO) in the human hepatoma cell line Hep G2 by means of several factors. The MFO activities induced in the cells cultured in medium containing five commercial sera varied significantly, and the activity in the cells cultured in the absence of serum was about twice as high as that in cells supplemented with serum. The activity of ethoxycoumarin O-deethylase was highest 12 hr after adding 3-methylcholanthrene, and it was induced by several polycyclic aromatic hydrocarbons such as benzo(a)anthracene and benzo(a)pyrene, which are usually found in urban air as environmental contaminants. Furthermore, an extract from the total suspended particles collected using a high volume air sampler, which was mutagenic in the Ames assay using Salmonella typhimurium TA98, induced the same enzyme activities in Hep G2 cells. These findings suggest that serum-free culture allows the stable and highly sensitive measurement of induced MFO activity, and that studies of MFO induction by environmental samples using human hepatoma Hep G2 cells should provide helpful information regarding the risk associated with environmental contaminants.

Disturbed intestinal movement, bile reflux to the stomach, and deficiency of c-kit-expressing cells in Ws/Ws mutant rats.

BACKGROUND & AIMS: Interstitial cells of Cajal (ICCs) are believed to initiate the basic contractile activity of the gastrointestinal tract. Because ICCs in the intestine of mice express c-kit receptor tyrosine kinase and because rats are more commonly used than mice for pathophysiological investigations of the gastrointestinal tract, the number of the c-kit messenger RNA-expressing cells was compared with gastrointestinal movement in rats. METHODS: The c-kit messenger RNA-expressing cells were detected by in situ hybridization. The autonomous contraction of excised segments of the ileum was recorded. The function of the pyloric sphincter was evaluated by measuring the content of bile acids in the stomach. RESULTS: The c-kit messenger RNA-expressing cells were not detectable in the stomach of Ws/Ws mutant rats with a small deletion at the tyrosine kinase domain of c-kit, and the number of c-kit messenger RNA-expressing cells decreased to 7% that of normal control rats in the ileum of Ws/Ws rats. The contractile activity of the ileum was apparently impaired, and the content of bile acids in the stomach was significantly increased in Ws/Ws rats. CONCLUSIONS: The abnormalities in the ileal movement and pyloric sphincter function in Ws/Ws rats were attributable to the deficiency of c-kit messenger RNA-expressing cells.

Infection with Nippostrongylus brasiliensis induces invasion of mast cell precursors from peripheral blood to small intestine.

Precursors of mast cells were defined as cells that formed mast-cell colonies in methylcellulose culture (CFU-mast). Mononuclear cells (MNC) were obtained from the bone marrow, peripheral blood, and small intestine of Ws/Ws rats with a small deletion at the tyrosine kinase domain of c-kit and of control normal (+/+) rats. In the culture containing concanavalin A-stimulated spleen cell conditioned medium (ConA-SCM) alone, the numbers of mast-cell colonies produced by Ws/Ws MNC were comparable with those of +/+ MNC. In the culture containing both ConA-SCM and stem cell factor (a ligand of c-kit), however, the numbers of mast-cell colonies produced by +/+ blood MNC were 107 times as great as that of Ws/Ws blood MNC. Using this culture condition, we investigated changes in concentration of CFU-mast in the marrow, blood, and intestine of +/+ rats after infection with Nippostrongylus brasiliensis (NB), which induced marked mast-cell accumulation in the small intestine. The concentration of CFU-mast in blood dropped to 21% of preinfection levels 1 week after the NB infection. In contrast, a sevenfold increase of CFU-mast occurred in the small intestine. The proportion of CFU-mast in S phase of the cell cycle remained at low levels in the marrow and blood after NB infection, but it increased significantly in the small intestine. The present result suggests that NB infection induces the invasion of CFU-mast into the intestine from blood and their subsequent proliferation in the tissue site.

Endogenous nitric oxide modulates ethanol-induced gastric mucosal injury in rats.

BACKGROUND/AIMS: Endothelium-derived relaxing factor regulates vascular tone via vasodilation. The relative contribution of endogenous nitric oxide to the pathophysiology of ethanol-induced gastric mucosal microcirculatory disturbances was investigated in anesthetized rats. METHODS: Macroscopic and microscopic gastric mucosal damage and gastric mucosal hemodynamics including blood flow and hemoglobin oxygen saturation (ISO2) were assessed by pretreatment with a specific NO synthase inhibitor, N omega-nitro-L-arginine (L-NNA), before and after intragastric administration of ethanol. RESULTS: Pretreatment with L-NNA significantly increased macroscopic (7.7-fold) and microscopic damage caused by 30% ethanol. Concurrent administration of L-arginine, but not D-arginine, significantly reduced the increase in mucosal damage. Similar results were obtained with 60% ethanol. Pretreatment with L-NNA decreased both mucosal blood flow and ISO2 in the basal period and enhanced decreases in both mucosal blood flow (2.7-fold) and ISO2 (4.3-fold) induced by 30% ethanol compared with controls. Concurrent administration of L-arginine, but not D-arginine, significantly inhibited the effect of L-NNA on blood flow and ISO2 in the basal period as well as after intragastric administration of 30% ethanol. CONCLUSIONS: Endogenous NO modulates ethanol-induced gastric mucosal injury through the regulation of gastric mucosal microcirculation.

Gastric mucosal cell proliferation and TGF alpha in the healing of peptic ulcers in human subjects.

We investigated the role of gastric mucosal proliferation and mucosal transforming growth factor-alpha (TGF alpha) in the healing of peptic ulcers in human subjects. During endoscopic observation, mucosal biopsy specimens were taken from the ulcer margin for flow cytometric analysis of the mucosal cell cycle and ELISA for mucosal TGF alpha content. Immunohistochemistry confirmed that biopsy specimens contained proliferating gastric epithelial cells, TGF alpha, and the TGF alpha receptor. When the stage of ulcer healing was categorized according to Sakita and Miwa, mucosal cell proliferation was less at active stages and gradually increased during the healing process. The mucosal TGF alpha level at the ulcer margin was not detectable at the A1 stage but increased to within the normal range during ulcer healing. After re-establishment of the epithelial layer at the site of the ulcer, cell proliferation returned to normal and TGF alpha remained within the normal range. Immunohistochemistry showed coexistence of TGF alpha and its receptor within PCNA-positive proliferating cells. These results suggest that TGF alpha and its receptor play an important role in gastric epithelial cell replication. The increase in TGF alpha at the ulcer margin might indicate a role in stimulation of gastric cell replication during ulcer healing.

Serum-free culture of human hepatoma Hep G2 cells with egg yolk low density lipoprotein.

We prepared serum-free medium supplemented with growth factors, hormones, nutrients, and egg yolk low density lipoprotein (LDL), and examined the growth-promoting activities of growth factors and culture conditions on human hepatoma Hep G2 cells. Collagen type I was appropriate for precoating tissue culture vessels in which Hep G2 cells were cultured in serum-free medium. William's medium E and a mixture of Dulbecco and Vogt's modification of Eagle's medium and Ham's F-12 (1:1) were used as basal medium. LDL was the most effective growth promoter of Hep G2 cells among the supplemented factors. High density lipoprotein (HDL) prepared from human serum showed 10 times more growth promoting activity than LDL, but HDL is more expensive. Therefore, the serum-free medium supplemented with growth factors, hormones, nutrients, and egg yolk LDL was suitable for Hep G2 cell culture.