Impacts of liver macrophages, gut microbiota, and bile acid metabolism on the differences in iHFC diet-induced MASH progression between TSNO and TSOD mice.

BACKGROUND: Tsumura-Suzuki non-obese (TSNO) mice exhibit a severe form of metabolic dysfunction-associated steatohepatitis (MASH) with advanced liver fibrosis upon feeding a high-fat/cholesterol/cholate-based (iHFC) diet. Another ddY strain, Tsumura-Suzuki diabetes obese (TSOD) mice, are impaired in the progression of iHFC diet-induced MASH. AIM: To elucidate the underlying mechanisms contributing to the differences in MASH progression between TSNO and TSOD mice. METHODS: We analyzed differences in the immune system, gut microbiota, and bile acid metabolism in TSNO and TSOD mice fed with a normal diet (ND) or an iHFC diet. RESULTS: TSOD mice had more anti-inflammatory macrophages in the liver than TSNO mice under ND feeding, and were impaired in the iHFC diet-induced accumulation of fibrosis-associated macrophages and formation of histological hepatic crown-like structures in the liver. The gut microbiota of TSOD mice also exhibited a distinct community composition with lower diversity and higher abundance of Akkermansia muciniphila compared with that in TSNO mice. Finally, TSOD mice had lower levels of bile acids linked to intestinal barrier disruption under iHFC feeding. CONCLUSIONS: The dynamics of liver macrophage subsets, and the compositions of the gut microbiota and bile acids at steady state and post-onset of MASH, had major impacts on MASH development.

Beneficial Effects of Dietary Fiber in Young Barley Leaf on Gut Microbiota and Immunity in Mice.

The health benefits of young barley leaves, rich in dietary fiber, have been studied for several decades; however, their beneficial effects on the intestinal microenvironment remain to be elucidated. To investigate the effects of young barley leaf-derived dietary fiber (YB) on the gut microbiota and immunity, mice were fed an AIN-93G diet containing cellulose or YB and subjected to subsequent analysis. The population of MHC-II-positive conventional dendritic cells (cDCs) and CD86 expression in the cDCs of Peyer's patches were elevated in the YB-fed mice. MHC-II and CD86 expression was also elevated in the bone marrow-derived DCs treated with YB. 16S-based metagenomic analysis revealed that the gut microbiota composition was markedly altered by YB feeding. Among the gut microbiota, Lachnospiraceae, mainly comprising butyrate-producing NK4A136 spp., were overrepresented in the YB-fed mice. In fact, fecal butyrate concentration was also augmented in the YB-fed mice, which coincided with increased retinaldehyde dehydrogenase (RALDH) activity in the CD103+ cDCs of the mesenteric lymph nodes. Consistent with elevated RALDH activity, the population of colonic IgA+ plasma cells was higher in the YB-fed mice than in the parental control mice. In conclusion, YB has beneficial effects on the gut microbiota and intestinal immune system.

Dynamics of Liver Macrophage Subsets in a Novel Mouse Model of Non-Alcoholic Steatohepatitis Using C57BL/6 Mice.

Macrophages are critical for the development of non-alcoholic steatohepatitis (NASH). Our previous findings in TSNO mouse livers showed that an iHFC (high-fat/cholesterol/cholate) diet induced liver fibrosis similar to human NASH and led to the accumulation of distinct subsets of macrophage: CD11c+/Ly6C- and CD11c-/Ly6C+ cells. CD11c+/Ly6C- cells were associated with the promotion of advanced liver fibrosis in NASH. On the other hand, CD11c-/Ly6C+ cells exhibited an anti-inflammatory effect and were involved in tissue remodeling processes. This study aimed to elucidate whether an iHFC diet with reduced cholic acid (iHFC#2 diet) induces NASH in C57BL/6 mice and examine the macrophage subsets accumulating in the liver. Histological and quantitative real-time PCR analyses revealed that the iHFC#2 diet promoted inflammation and fibrosis indicative of NASH in the livers of C57BL/6 mice. Cell numbers of Kupffer cells decreased and recruited macrophages were accumulated in the livers of iHFC#2 diet-fed C57BL/6 mice. Notably, the iHFC#2 diet resulted in the accumulation of three macrophage subsets in the livers of C57BL/6 mice: CD11c+/Ly6C-, CD11c-/Ly6C+, and CD11c+/Ly6C+ cells. However, CD11c+/Ly6C+ cells were not distinct populations in the iHFC-fed TSNO mice. Thus, differences in cholic acid content and mouse strain affect the macrophage subsets that accumulate in the liver.

Effect of Oral Administration of Polyethylene Glycol 400 on Gut Microbiota Composition and Diet-Induced Obesity in Mice.

Polyethylene glycol (PEG) is a commonly used dispersant for oral administration of hydrophobic agents. PEG is partly absorbed in the small intestine, and the unabsorbed fraction reaches the large intestine; thus, oral administration of PEG may impact the gut microbial community. However, to the best of our knowledge, no study evaluated the effects of PEG on gut commensal bacteria. Herein, we aimed to determine whether oral administration of PEG modifies the gut microbiota. Administration of PEG400 and PEG4000 altered gut microbial diversity in a concentration-dependent manner. Taxonomic analysis revealed that Akkermansia muciniphila and particularly Parabacteroides goldsteinii were overrepresented in mice administered with 40% PEG. PEG400 administration ameliorated the high-fat diet (HFD)-induced obesity and adipose tissue inflammation. Fecal microbiome transplantation from PEG400-administered donors counteracted the HFD-induced body and epididymal adipose tissue weight gain, indicating that PEG400-associated bacteria are responsible for the anti-obesity effect. Conversely, carboxymethyl cellulose, also used as a dispersant, did not affect the abundance of these two bacterial species or HFD-induced obesity. In conclusion, we demonstrated that oral administration of a high concentration of PEG400 (40%) alters the gut microbiota composition and ameliorates HFD-induced obesity.

Data on plasma cortisol levels in nibbler fish Girella punctata reared under high-density conditions in either surface seawater or deep ocean water.

Deep ocean water (DOW) is the water obtained from depth of >200 m below the surface of Earth's oceans and is characterized by rich nutrients and cleanliness [1,2]. We have recently reported that DOW suppresses the high-density-induced increase of plasma cortisol levels (i.e., a stress marker) in Japanese flounder (Paralichthys olivaceus) [1]. The current study aimed to examine whether the cortisol-reducing effect of DOW was observed in other marine organisms as well by comparing the plasma cortisol levels of nibbler fish Girella punctata reared under high-density conditions between surface seawater (SSW) and DOW. The nibbler fish were caught from Tsukumo Bay of Noto Peninsula (Ishikawa Prefecture, Japan). The DOW was obtained from seawater 320 m below the Noto Bay surface at a facility (Aquas Noto, Ishikawa Prefecture, Japan), whereas SSW was obtained from Tsukumo Bay (Noto Peninsula, Ishikawa Prefecture). The dissolved oxygen was maintained at approximately 7 mg/L in DOW as well as in SSW. Before they were transferred to the high-density condition, nibbler fish were acclimated in SSW at 20°C for 1 week at a mean density of 100 g/62.5 L. To expose them to the high-density stress, each of fish was kept at a density of 10 kg/m3 in a single aquarium (60 × 25 × 30 cm) containing either SSW or DOW (n = 8). Subsequently, the fish were reared with SSW or DOW for 10 days at 20°C ± 1°C under a 12:12-h light-dark cycle. A heparin containing syringe was used to obtain the blood samples from the caudal vessels of the fish anesthetized with a 0.04% 2-phenoxyethanol (FUJIFILM Wako Pure Chemical Corporation). The blood sampling was performed on days 0, 5, and 10 after rearing in the small aquaria. The plasma samples were prepared from the collected blood by centrifuging it at 5200 × g for 5 min and the cortisol concentrations were determined using an enzyme-linked immunosorbent assay (ELISA) kit (Cosmo Bio Co. Ltd., Tokyo, Japan) from those samples. The plasma cortisol concentration of nibbler fish reared in SSW on day 10 was significantly higher than that on day 0, whereas those reared in DOW did not show significant difference on the respective days. The current data contributes to the generalization of the cortisol-reducing effect of DOW on fish, which has been proposed in Japanese flounder [1]. These data could be used for developing and designing experiments to analyze the mechanisms underlying the cortisol-reducing effects by using small fish such as zebrafish, a well-established animal model.

The innate immune receptor RP105 promotes metabolic syndrome by altering gut microbiota composition and intestinal barrier function.

Radioprotective 105 (RP105) plays a key role in the development of high-fat diet (HFD)-induced metabolic disorders; however, the underlying mechanisms remain to be understood. Here, we aimed to uncover whether RP105 affects metabolic syndrome through the modification of gut microbiota. We confirmed that body weight gain and fat accumulation by HFD feeding were suppressed in Rp105-/- mice. Fecal microbiome transplantation from HFD-fed donor Rp105-/- mice into HFD-fed recipient wild-type mice significantly improved various abnormalities associated with metabolic syndrome, including body weight gain, insulin resistance, hepatic steatosis, macrophage infiltration and inflammation in the adipose tissue. In addition, HFD-induced intestinal barrier dysfunction was attenuated by fecal microbiome transplantation from HFD-fed donor Rp105-/- mice. A 16S rRNA sequence analysis indicated that RP105 modified gut microbiota composition and was involved in the maintenance of its diversity. Thus, RP105 promotes metabolic syndrome by altering gut microbiota composition and intestinal barrier function.

Kynurenine promotes Calcitonin secretion and reduces cortisol in the Japanese flounder Paralichthys olivaceus.

Deep ocean water (DOW) exerts positive effects on the growth of marine organisms, suggesting the presence of unknown component(s) that facilitate their aquaculture. We observed that DOW suppressed plasma cortisol (i.e., a stress marker) concentration in Japanese flounder (Paralichthys olivaceus) reared under high-density condition. RNA-sequencing analysis of flounder brains showed that when compared to surface seawater (SSW)-reared fish, DOW-reared fish had lower expression of hypothalamic (i.e., corticotropin-releasing hormone) and pituitary (i.e., proopiomelanocortin, including adrenocorticotropic hormone) hormone-encoding genes. Moreover, DOW-mediated regulation of gene expression was linked to decreased blood cortisol concentration in DOW-reared fish. Our results indicate that DOW activated osteoblasts in fish scales and facilitated the production of Calcitonin, a hypocalcemic hormone that acts as an analgesic. We then provide evidence that the Calcitonin produced is involved in the regulatory network of genes controlling cortisol secretion. In addition, the indole component kynurenine was identified as the component responsible for osteoblast activation in DOW. Furthermore, kynurenine increased plasma Calcitonin concentrations in flounders reared under high-density condition, while it decreased plasma cortisol concentration. Taken together, we propose that kynurenine in DOW exerts a cortisol-reducing effect in flounders by facilitating Calcitonin production by osteoblasts in the scales.

Effect of Wheat-Derived Arabinoxylan on the Gut Microbiota Composition and Colonic Regulatory T Cells.

The health benefits of wheat-derived arabinoxylan, a commonly consumed dietary fiber, have been studied for decades. However, its effect on the gut microenvironment and inflammatory bowel disease remains unclear. The objective of this study was to understand the effect of wheat-derived arabinoxylan on gut microbiota, colonic regulatory T cells (Tregs), and experimental colitis. In this study, healthy and chronic colitis model mice were fed chow containing cellulose or wheat-derived arabinoxylan for 2-6 weeks and subjected to subsequent analysis. A 16S-based metagenomic analysis of the fecal DNA revealed that Lachnospiraceae, comprising butyrate-producing and Treg-inducing bacteria, were overrepresented in arabinoxylan-fed mice. In line with the changes in the gut microbiota, both the fecal butyrate concentration and the colonic Treg population were elevated in the arabinoxylan-fed mice. In a T cell transfer model of chronic colitis, wheat-derived arabinoxylan ameliorated body weight loss and colonic tissue inflammation, which may, in part, be mediated by Treg induction. Moreover, wheat-derived arabinoxylan suppressed TNFα production from type 1 helper T cells in this colitis model. In conclusion, wheat-derived arabinoxylans, by altering the gut microenvironment, may be a promising prebiotic for the prevention of colitis.

Adaptation to the shallow sea floor environment of a species of marine worms, Oligobrachia mashikoi, generally inhabiting deep-sea water.

Beard worms from the family Siboglinidae, are peculiar animals and are known for their symbiotic relationships with sulfur bacteria. Most Siboglinids inhabit the deep-sea floor, thus making difficult to make any observations in situ. One species, Oligobrachia mashikoi, occurs in the shallow depths (24.5 m) of the Sea of Japan. Taking advantage of its shallow-water habitat, the first ecological survey of O. mashikoi was performed over a course of 7 years, which revealed that its tentacle-expanding behavior was dependent on the temperature and illuminance of the sea water. Furthermore, there were significantly more O. mashikoi with expanding tentacles during the nighttime than during the daytime, and the prevention of light eliminated these differences in the number of expending tentacles. These results confirmed that the tentacle-expanding behavior is controlled by environmental light signals. Consistent with this, we identified a gene encoding a photoreceptor molecule, neuropsin, in O. mashikoi, and the expression thereof is dependent on the time of day. We assume that the described behavioral response of O. mashikoi to light signals represent an adaptation to a shallow-water environment within the predominantly deep-sea taxon.

Effect of soluble oat fiber on intestinal microenvironment and TNBS-induced colitis.

Soluble oat fibers, including ß-glucan, have been shown to alter the gut microbiome composition and ameliorate DSS-induced colitis; however, the beneficial effect of soluble oat fiber on colonic inflammation is not yet fully understood. In this study, we demonstrated that soluble oat fibers ameliorate T cell-dependent colitis through the induction of peripherally induced regulatory T cells (pTregs). Soluble oat fibers elevated colonic butyrate production dose-dependently, which coincided with the overrepresentation of Faecalibaculum rodentium (an analog of butyrate-producing Holdemanella biformis) in the gut microbiome. Soluble oat fibers promoted the growth of F. rodentium and H. biformis even in vitro, and increased the concentration of butyrate in the culture supernatant. These results indicate that soluble oat fibers are an energy source for butyrate-producing bacteria and are a fermentation substrate. Soluble oat fibers increased the percentage of colonic pTregs and ameliorated the weight loss and inflammation in acute 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis; this may in part be mediated by the increase in IL-10-producing T cells. In conclusion, our results suggest that the administration of soluble oat fibers is a promising prebiotic treatment for the prevention of colitis mediated via altered gut microbiota composition and elevated butyrate production.

Impact of Vancomycin Treatment and Gut Microbiota on Bile Acid Metabolism and the Development of Non-Alcoholic Steatohepatitis in Mice.

The potential roles of the gut microbiota in the pathogenesis of non-alcoholic fatty liver disease, including non-alcoholic steatohepatitis (NASH), have attracted increased interest. We have investigated the links between gut microbiota and NASH development in Tsumura-Suzuki non-obese mice fed a high-fat/cholesterol/cholate-based (iHFC) diet that exhibit advanced liver fibrosis using antibiotic treatments. The administration of vancomycin, which targets Gram-positive organisms, exacerbated the progression of liver damage, steatohepatitis, and fibrosis in iHFC-fed mice, but not in mice fed a normal diet. F4/80+-recruited macrophages were more abundant in the liver of vancomycin-treated iHFC-fed mice. The infiltration of CD11c+-recruited macrophages into the liver, forming hepatic crown-like structures, was enhanced by vancomycin treatment. The co-localization of this macrophage subset with collagen was greatly augmented in the liver of vancomycin-treated iHFC-fed mice. These changes were rarely seen with the administration of metronidazole, which targets anaerobic organisms, in iHFC-fed mice. Finally, the vancomycin treatment dramatically modulated the level and composition of bile acid in iHFC-fed mice. Thus, our data demonstrate that changes in inflammation and fibrosis in the liver by the iHFC diet can be modified by antibiotic-induced changes in gut microbiota and shed light on their roles in the pathogenesis of advanced liver fibrosis.

Physiological consequences of space flight, including abnormal bone metabolism, space radiation injury, and circadian clock dysregulation: Implications of melatonin use and regulation as a countermeasure.

Exposure to the space environment induces a number of pathophysiological outcomes in astronauts, including bone demineralization, sleep disorders, circadian clock dysregulation, cardiovascular and metabolic dysfunction, and reduced immune system function. A recent report describing experiments aboard the Space Shuttle mission, STS-132, showed that the level of melatonin, a hormone that provides the biochemical signal of darkness, was decreased during microgravity in an in vitro culture model. Additionally, abnormal lighting conditions in outer space, such as low light intensity in orbital spacecraft and the altered 24-h light-dark cycles, may result in the dysregulation of melatonin rhythms and the misalignment of the circadian clock from sleep and work schedules in astronauts. Studies on Earth have demonstrated that melatonin regulates various physiological functions including bone metabolism. These data suggest that the abnormal regulation of melatonin in outer space may contribute to pathophysiological conditions of astronauts. In addition, experiments with high-linear energy transfer radiation, a ground-based model of space radiation, showed that melatonin may serve as a protectant against space radiation. Gene expression profiling using an in vitro culture model exposed to space flight during the STS-132 mission, showed that space radiation alters the expression of DNA repair and oxidative stress response genes, indicating that melatonin counteracts the expression of these genes responsive to space radiation to promote cell survival. These findings implicate the use of exogenous melatonin and the regulation of endogenous melatonin as countermeasures for the physiological consequences of space flight.

Roles of Macrophages in Advanced Liver Fibrosis, Identified Using a Newly Established Mouse Model of Diet-Induced Non-Alcoholic Steatohepatitis.

Macrophages play critical roles in the pathogenesis of non-alcoholic steatohepatitis (NASH). However, it is unclear which macrophage subsets are critically involved in the development of inflammation and fibrosis in NASH. In TSNO mice fed a high-fat/cholesterol/cholate-based diet, which exhibit advanced liver fibrosis that mimics human NASH, we found that Kupffer cells (KCs) were less abundant and recruited macrophages were more abundant, forming hepatic crown-like structures (hCLS) in the liver. The recruited macrophages comprised two subsets: CD11c+/Ly6C- and CD11c-/Ly6C+ cells. CD11c+ cells were present in a mesh-like pattern around the lipid droplets, constituting the hCLS. In addition, CD11c+ cells colocalized with collagen fibers, suggesting that this subset of recruited macrophages might promote advanced liver fibrosis. In contrast, Ly6C+ cells were present in doughnut-like inflammatory lesions, with a lipid droplet in the center. Finally, RNA sequence analysis indicates that CD11c+/Ly6C- cells promote liver fibrosis and hepatic stellate cell (HSC) activation, whereas CD11c-/Ly6C+ cells are a macrophage subset that play an anti-inflammatory role and promote tissue repair in NASH. Taken together, our data revealed changes in liver macrophage subsets during the development of NASH and shed light on the roles of the recruited macrophages in the pathogenesis of advanced fibrosis in NASH.

The Impact of Low-Viscosity Soluble Dietary Fibers on Intestinal Microenvironment and Experimental Colitis: A Possible Preventive Application of Alpha-Cyclodextrin in Intestinal Inflammation.

SCOPE: The purpose of this study is to compare the impact of four low-viscosity soluble dietary fibers (DFs) on the intestinal microenvironment, in terms of microbiota composition, short-chain fatty acid (SCFA) production, proportion of colonic peripherally induced regulatory T cells (pTregs), and experimental colitis in mice. METHODS AND RESULTS: Mice are administered 5% w/v low-viscosity soluble DFs in drinking water for 2 weeks. The gut microbiota composition is determined using 16S rRNA sequencing. Luminal SCFAs are quantified by gas chromatography, and colonic pTregs are analyzed using flow cytometry. All low-viscosity soluble DFs promote the growth of beneficial bacteria such as Akkermansia muciniphila and Bacteroides acidifaciens, while eliminating pathogenic bacteria such as Clostridium perfringens. Moreover, two low-viscosity soluble DFs significantly increase the abundance of commensal bacteria and promote the accumulation of propionate and butyrate, leading to marked induction of colonic pTregs. Consistently, these two fibers, in particular α-cyclodextrin, show remarkable anti-inflammatory properties in a colitis mouse model. CONCLUSION: Mice administered any low-viscosity soluble DF show comparable gut microbiota compositions, but differ in terms of bacterial abundance, SCFA concentration, pTreg population, and colitis development. This exploratory study suggests that administration of α-cyclodextrin may be a possible strategy for the prevention of colitis.

Osteoclastic and Osteoblastic Responses to Hypergravity and Microgravity: Analysis Using Goldfish Scales as a Bone Model.

It is known that the bone matrix plays an important role in the response to physical stresses such as hypergravity and microgravity. In order to accurately analyze the response of bone to hypergravity and microgravity, a culture system under the conditions of coexistence of osteoclasts, osteoblasts, and bone matrix was earnestly desired. The teleost scale is a unique calcified organ in which osteoclasts, osteoblasts, and the two layers of bone matrix, i.e., a bony layer and a fibrillary layer, coexist. Therefore, we have developed in vitro organ culture systems of osteoclasts and osteoblasts with the intact bone matrix using goldfish scales. Using the scale culture system, we examined the effects of hypergravity with a centrifuge and simulated ground microgravity (g-µG) with a three-dimensional clinostat on osteoclasts and osteoblasts. Under 3-gravity (3G) loading for 1 day, osteoclastic marker mRNA expression levels decreased, while the mRNA expression of the osteoblastic marker increased. Upon 1 day of exposure, the simulated g-µG induced remarkable enhancement of osteoclastic marker mRNA expression, whereas the osteoblastic marker mRNA expression decreased. In response to these gravitational stimuli, osteoclasts underwent major morphological changes. By simulated g-µG treatments, morphological osteoclastic activation was induced, while osteoclastic deactivation was observed in the 3G-treated scales. In space experiments, the results that had been obtained with simulated g-µG were reproduced. RNA-sequencing analysis showed that osteoclastic activation was induced by the down-regulation of Wnt signaling under flight-microgravity. Thus, goldfish scales can be utilized as a bone model to analyze the responses of osteoclasts and osteoblasts to gravity.

Isoliquiritigenin Attenuates Adipose Tissue Inflammation and Metabolic Syndrome by Modifying Gut Bacteria Composition in Mice.

SCOPE: Isoliquiritigenin (ILG) has been reported to attenuate adipose tissue inflammation and metabolic disorder; however, the underlying mechanisms remain to be elucidated. The aim of this study is to elucidate whether ILG shows the anti-inflammatory and antimetabolic syndrome effects through gut microbiota modification. METHODS AND RESULTS: Mice are fed a high-fat diet (HFD) with or without ILG for up to 12 weeks. The effect of ILG on body weight, blood glucose level, adipose tissue inflammation, gut barrier function, and gut microbiota composition are investigated. ILG supplementation alleviates HFD-induced obesity, glucose tolerance, and insulin resistance and suppresses inflammatory gene expression in epididymal white adipose tissue (eWAT). Moreover, ILG supplementation modifies gut bacterial composition by increasing the abundance of antimetabolic disease-associated species (e.g., Parabacteroides goldsteinii and Akkemansia muciniphila) and up-regulated genes associated with gut barrier function. Fecal microbiome transplantation (FMT) from ILG-fed donors counteract HFD-induced body and eWAT weight changes, inflammation-related gene expression, glucose tolerance, and insulin resistance, thereby suggesting that ILG-responsive gut bacteria exerts anti-inflammatory and antimetabolic syndrome effects. CONCLUSION: Alterations in gut bacteria underly the beneficial effects of ILG against adipose tissue inflammation and metabolic disorders. ILG may be a promising prebiotic for the prevention and treatment of metabolic syndrome.

Ultrasound-Induced DNA Damage and Cellular Response: Historical Review, Mechanisms Analysis, and Therapeutic Implications.

The biological effects of ultrasound may be classified into thermal and nonthermal mechanisms. The nonthermal effects may be further classified into cavitational and noncavitational mechanisms. DNA damage induced by ultrasound is considered to be related to nonthermal cavitations. For this aspect, many in vitro studies on DNA have been conducted for evaluating the safety of diagnostic ultrasound, particularly in fetal imaging. Technological advancement in detecting DNA damage both in vitro and in vivo have elucidated the mechanism of DNA damage formation and their cellular response. Damage to DNA, and the residual damages after DNA repair are implicated in the biological effects. Here, we discuss the historical evidence of ultrasound on DNA damage and the mechanism of DNA damage formation both in vitro and in vivo, compared with those induced by ionizing radiation. We also offer a commentary on the safety of ultrasound over X-ray-based imaging. Also, understanding the various mechanisms involved in the bioeffects of ultrasound will lead us to alternative strategies for use of ultrasound for therapy.

Hydroxylated benzo[c]phenanthrene metabolites cause osteoblast apoptosis and skeletal abnormalities in fish.

To study the toxicity of 3-hydroxybenzo[c]phenanthrene (3-OHBcP), a metabolite of benzo[c]phenanthrene (BcP), first we compared it with its parent compound, BcP, using an in ovo-nanoinjection method in Japanese medaka. Second, we examined the influence of 3-OHBcP on bone metabolism using goldfish. Third, the detailed mechanism of 3-OHBcP on bone metabolism was investigated using zebrafish and goldfish. The LC50s of BcP and 3-OHBcP in Japanese medaka were 5.7 nM and 0.003 nM, respectively, indicating that the metabolite was more than 1900 times as toxic as the parent compound. In addition, nanoinjected 3-OHBcP (0.001 nM) induced skeletal abnormalities. Therefore, fish scales with both osteoblasts and osteoclasts on the calcified bone matrix were examined to investigate the mechanisms of 3-OHBcP toxicity on bone metabolism. We found that scale regeneration in the BcP-injected goldfish was significantly inhibited as compared with that in control goldfish. Furthermore, 3-OHBcP was detected in the bile of BcP-injected goldfish, indicating that 3-OHBcP metabolized from BcP inhibited scale regeneration. Subsequently, the toxicity of BcP and 3-OHBcP to osteoblasts was examined using an in vitro assay with regenerating scales. The osteoblastic activity in the 3-OHBcP (10-10 to 10-7 M)-treated scales was significantly suppressed, while BcP (10-11 to 10-7 M)-treated scales did not affect osteoblastic activity. Osteoclastic activity was unchanged by either BcP or 3-OHBcP treatment at each concentration (10-11 to 10-7 M). The detailed toxicity of 3-OHBcP (10-9 M) in osteoblasts was then examined using gene expression analysis on a global scale with fish scales. Eight genes, including APAF1, CHEK2, and FOS, which are associated with apoptosis, were identified from the upregulated genes. This indicated that 3-OHBcP treatment induced apoptosis in fish scales. In situ detection of cell death by TUNEL methods was supported by gene expression analysis. This study is the first to demonstrate that 3-OHBcP, a metabolite of BcP, has greater toxicity than the parent compound, BcP.

Low-intensity ultrasound inhibits melanoma cell proliferation in vitro and tumor growth in vivo.

PURPOSE: To determine the effect of low-intensity ultrasound on cancer cell proliferation in vitro and tumor growth in vivo. METHODS: In vitro, several cancer cell lines were exposed to low-intensity ultrasound at 0.11 W/cm2 for 2 min. Of the cell lines screened, melanoma C32 is one of the cell lines that showed sensitivity to growth inhibition by ultrasound and was therefore used in succeeding experiments. In vivo, under the same ultrasound conditions used in vitro, C32 tumors in mice were exposed to ultrasound daily for 2 weeks, and the tumor volumes were monitored weekly using sonography. RESULTS: In vitro, C32 cell growth was inhibited, attaining 43.2% inhibition on the 3rd day. In vivo, tumor growth was significantly inhibited, with the treated tumors exhibiting 2.7-fold slowed tumor growth vs. untreated tumors at week 2. Such inhibition was not associated with increased cell death. Several genes related to the cell cycle and proliferation were among those significantly regulated. CONCLUSION: These findings highlight the potential of low-intensity ultrasound to inhibit tumor growth in a noninvasive, safe, and easy-to-administer way. In addition, this may suggest that the mechanical stress induced by ultrasound on C32 cells may have affected the intrinsic biomolecular mechanism related to the cell growth of this particular cell line. Further research is needed to identify which of the regulated genes played key roles in growth inhibition.

Symbiotic polyamine metabolism regulates epithelial proliferation and macrophage differentiation in the colon.

Intestinal microbiota-derived metabolites have biological importance for the host. Polyamines, such as putrescine and spermidine, are produced by the intestinal microbiota and regulate multiple biological processes. Increased colonic luminal polyamines promote longevity in mice. However, no direct evidence has shown that microbial polyamines are incorporated into host cells to regulate cellular responses. Here, we show that microbial polyamines reinforce colonic epithelial proliferation and regulate macrophage differentiation. Colonisation by wild-type, but not polyamine biosynthesis-deficient, Escherichia coli in germ-free mice raises intracellular polyamine levels in colonocytes, accelerating epithelial renewal. Commensal bacterium-derived putrescine increases the abundance of anti-inflammatory macrophages in the colon. The bacterial polyamines ameliorate symptoms of dextran sulfate sodium-induced colitis in mice. These effects mainly result from enhanced hypusination of eukaryotic initiation translation factor. We conclude that bacterial putrescine functions as a substrate for symbiotic metabolism and is further absorbed and metabolised by the host, thus helping maintain mucosal homoeostasis in the intestine.