PA YEAST SC-1, Polyamine-Rich Saccharomyces cerevisiae, Induces Muscle Hypertrophy in C2C12 Myotubes.

Maintenance of appropriate muscle mass is necessary for good quality of life as skeletal muscles play critical roles in locomotion, metabolic homeostasis, and thermogenesis. Polyamines are essential metabolites that regulate several important cellular functions. In C57BL6 mice who underwent sciatic nerve transection of the hind limb, compensatory muscle hypertrophy is enhanced by the administration of polyamines. However, the action mechanisms of polyamines in muscle hypertrophy remain unclear. Here, we isolated PA YEAST SC-1, a polyamine-rich Saccharomyces cerevisiae, from Baker's yeast. We examined whether PA YEAST SC-1 induces muscle hypertrophy and elucidated the underlying action mechanisms of polyamines and the active ingredients in PA YEAST SC-1 using C2C12 myotubes. PA YEAST SC-1 at 1 mg/mL increased myosin heavy chain expression in C2C12 myotubes. Mechanistically, PA YEAST SC-1 induced the activation of Akt/mechanistic target of rapamycin kinase/p70S6K signaling. Furthermore, PA YEAST SC-1 decreased the expression levels of the ubiquitin ligases, atrogin-1 and muscle RING finger-1, via forkhead box O1 phosphorylation. These findings suggest PA YEAST SC-1 as an effective food ingredient for the treatment of muscle hypertrophy.

Allyl Isothiocyanate Maintains DHA-Containing Glycerophospholipids and Ameliorates the Cognitive Function Decline in OVX Mice.

Low-temperature-induced fatty acid desaturation is highly conserved in animals, plants, and bacteria. Allyl isothiocyanate (AITC) is an agonist of the transient receptor potential ankyrin 1 (TRPA1), which is activated by various chemophysiological stimuli, including low temperature. However, whether AITC induces fatty acid desaturation remains unknown. We showed here that AITC increased levels of glycerophospholipids (GP) esterified with unsaturated fatty acids, especially docosahexaenoic acid (DHA) in TRPA1-expressing HEK cells. Additionally, GP-DHA including phosphatidylcholine (18:0/22:6) and phosphatidylethanolamine (18:0/22:6) was increased in the brain and liver of AITC-administered mice. Moreover, intragastrical injection of AITC in ovariectomized (OVX) female C57BL/6J mice dose-dependently shortened the Δlatency time determined by the Morris water maze test, indicating AITC ameliorated the cognitive function decline in these mice. Thus, the oral administration of AITC maintains GP-DHA in the liver and brain, proving to be a potential strategy for preventing cognitive decline.

Diosgenin prevents dexamethasone-induced myotube atrophy in C2C12 cells.

Several pathophysiological abnormalities, including a sedentary lifestyle, chronic diseases, and oxidative stress, can contribute to muscle atrophy triggered by an imbalance in muscle protein synthesis and degradation. Resolving muscle atrophy is a critical issue as it can reduce the quality of life. Here, one of the promising functional food factors, diosgenin (a steroidal sapogenin) showed strong preventive activities against dexamethasone (Dex)-induced muscle atrophy, as determined by the expression levels and morphology of the myosin heavy chain in C2C12 myotubes. Diosgenin inhibited protein expressions of Dex-induced skeletal muscle-specific ubiquitin ligase, including muscle RING finger 1 (MuRF1) and casitas B-lineage lymphoma protooncogene b (Cbl-b) but not atrogin-1. Diosgenin ameliorated Dex-induced declines of Akt phosphorylation at Ser473 and FoxO3a phosphorylation at Ser253, which probably at least partially contributed to the suppression of MuRF1, Cbl-b, and atrogin-1 gene expression. Additionally, diosgenin inhibited Dex-induced nuclear translocation of the glucocorticoid receptor (GR), diosgenin therefore may competitively inhibit the interaction between Dex and GR. These findings suggest that diosgenin is an effective functional food for preventing glucocorticoid-induced skeletal muscle atrophy.

Mucosal damage and γ-H2AX formation in the rat urinary bladder induced by aromatic amines with structures similar to o-toluidine and o-anisidine.

Although aromatic amines are widely used as raw materials for dyes, some, such as o-toluidine and o-anisidine, have shown concerning results regarding carcinogenicity in the urinary bladder. We have recently developed a short-term detection method for bladder carcinogens using immunohistochemistry for γ-H2AX, a DNA damage marker. Here, using this method, we evaluated aromatic amines with structures similar to o-toluidine and o-anisidine for bladder mucosal damage and potential carcinogenicity. In total, 17 aromatic amines were orally administered to male F344 rats for 28 days, and histopathological examination and γ-H2AX immunostaining of the urinary bladder were performed. Histopathological analysis revealed that seven aromatic amines, including 4-chloro-o-toluidine (4-CT), o-aminoazotoluene, 2-aminobenzyl alcohol (ABA), o-acetotoluidine (o-AT), 3,3'-dimethoxybenzidine, 4-aminoazobenzene (AAB), and 4,4'-methylenedianiline (MDA), induced various bladder lesions, such as hemorrhage, necrosis, and urothelial hyperplasia. The morphological characteristics of mucosal damage induced by these substances were divided into two major types: those resembling o-toluidine and those resembling o-anisidine. Six of these aromatic amines, excluding MDA, also caused significant increases in γ-H2AX formation in the bladder urothelium. Interestingly, 4-CT did not cause mucosal damage or γ-H2AX formation at the lower dose applied in previous carcinogenicity studies. These results showed for the first time that o-AT and ABA, metabolites of o-toluidine, as well as AAB caused damage to the bladder mucosa and suggested that they may be bladder carcinogens. In addition, 4-CT, which was thought to be a noncarcinogen, was found to exhibit bladder toxicity upon exposure to high doses, indicating that this compound may contribute to bladder carcinogenesis.

Theophylline Prevents Dexamethasone-Induced Atrophy in C2C12 Myotubes.

Skeletal muscle mass is maintained by a balance between the synthesis and degradation of muscle proteins, the collapse of which causes muscle wasting. The prevention of muscle wasting improves the quality of life and extends a healthy life. The methyl xanthine theophylline showed strong preventive activity against dexamethasone-induced muscle atrophy, as determined using the expression level of myosin heavy chain in C2C12 myotubes. Mechanistically, theophylline inhibited the expression of ubiquitin ligases MuRF1 and Cbl-b, but not that of atrogin-1. Furthermore, theophylline inhibits glucocorticoid receptor translocation to the nucleus. A pull-down assay using a theophylline probe revealed that theophylline and dexamethasone competitively interacted with the glucocorticoid receptor, suggesting an antagonistic activity of theophylline on glucocorticoid receptors. Additionally, theophylline inhibited the dexamethasone-induced phosphorylation of p38 and FoxO3a in C2C12 myotubes. These findings suggest that theophylline is an effective food ingredient in the prevention of glucocorticoid-induced skeletal muscle atrophy.

Anti-Inflammatory Effects of Dietary Polyphenols through Inhibitory Activity against Metalloproteinases.

Matrix metalloproteinases (MMPs) are zinc-dependent metalloproteinases that play important roles in a variety of diseases, including cancer, cardiovascular disease, diabetes, obesity, and brain diseases. Dietary polyphenols are thought to have a variety of beneficial effects on these diseases characterized by inflammation. Clinical studies have demonstrated that MMPs are in most cases upregulated in various inflammatory diseases, including osteoarthritis, rheumatoid arthritis, inflammatory bowel disease, and Alzheimer's disease. Studies using patient-derived human samples, animal studies, and cellular experiments have suggested that polyphenols may be beneficial against inflammatory diseases by suppressing MMP gene expression and enzyme activity. One important mechanism by which polyphenols exert their activity is the downregulation of reactive oxygen species that promote MMP expression. Another important mechanism is the direct binding of polyphenols to MMPs and their inhibition of enzyme activity. Molecular docking analyses have provided a structural basis for the interaction between polyphenols and MMPs and will help to explore new polyphenol-based drugs with anti-inflammatory properties.

Enokitake Mushroom and Its Active Component, Adenosine, Which Restores Testosterone Production in Impaired and Fatigued Mouse Models.

Several studies have reported the effects of the consumption of various mushroom species on the testes in animal experimental models. Mushrooms, including enokitake mushrooms (Flammulina velutipes), and vegetables contain adenosine may affect testosterone production. Here, we aimed to elucidate the effects of enokitake and its active component, adenosine, on testosterone production in primary cultures of testicular cells in vivo using mice models and in vitro. The administration of enokitake ethanolic extract increased testosterone production in the cisplatin-impaired mouse model. The direct effect of mushroom extracts on testicular cells was examined and liquid chromatography-mass spectrometry analysis confirmed that the mushroom- and vegetable-induced increase in testosterone production mainly involved adenosine. Additionally, the administration of enokitake extract or adenosine to wet floor fatigue model mice promoted testicular testosterone production and enhanced Leydig cell function through insulin-like peptide three level upregulation. Structurally related compounds, including cordycepin, showed lower bioactivity than adenosine. This study showed that the ingestion of adenosine-containing mushrooms and vegetables may effectively increase testicular testosterone production. We conclude that mushrooms with a relatively high adenosine content, such as enokitake, may be useful against aging and fatigue.

Non-target GC-MS analyses of fecal VOCs in NASH-hepatocellular carcinoma model STAM mice.

The increased incidence of obesity in the global population has increased the risk of several chronic inflammation-related diseases, including non-alcoholic steatohepatitis (NASH)-hepatocellular carcinoma (HCC). The progression from NASH to HCC involves a virus-independent liver carcinogenic mechanism; however, we currently lack effective treatment and prevention strategies. Several reports have suggested that fecal volatile organic compounds (VOCs) are strongly associated with NASH-HCC; therefore, we explored the biomarkers involved in its pathogenesis and progression. Fecal samples collected from control and NASH-HCC model STAM mice were subjected to headspace autosampler gas chromatography-electron ionization-mass spectrometry. Non-target profiling analysis identified diacetyl (2,3-butandione) as a fecal VOC that characterizes STAM mice. Although fecal diacetyl levels were correlated with the HCC in STAM mice, diacetyl is known as a cytotoxic/tissue-damaging compound rather than genotoxic or mutagenic; therefore, we examined the effect of bioactivity associated with NASH progression. We observed that diacetyl induced several pro-inflammatory molecules, including tumor necrosis factor-α, cyclooxygenase-2, monocyte chemoattractant protein-1, and transforming growth factor-ß, in mouse macrophage RAW264.7 and Kupffer KPU5 cells. Additionally, we observed that diacetyl induced α-smooth muscle actin, one of the hallmarks of fibrosis, in an ex vivo cultured hepatic section, but not in in vitro hepatic stellate TWNT-1 cells. These results suggest that diacetyl would be a potential biomarker of fecal VOC in STAM mice, and its ability to trigger the macrophage-derived inflammation and fibrosis may partly contribute to NASH-HCC carcinogenesis.

LPGAT1/LPLAT7 regulates acyl chain profiles at the sn-1 position of phospholipids in murine skeletal muscles.

Skeletal muscle consists of both fast- and slow-twitch fibers. Phospholipids are important structural components of cellular membranes, and the diversity of their fatty acid composition affects membrane characteristics. Although some studies have shown that acyl chain species in phospholipids differ among various muscle fiber types, the mechanisms underlying these differences are unclear. To investigate this, we analyzed phosphatidylcholine (PC) and phosphatidylethanolamine (PE) molecules in the murine extensor digitorum longus (EDL; fast-twitch) and soleus (slow-twitch) muscles. In the EDL muscle, the vast majority (93.6%) of PC molecules was palmitate-containing PC (16:0-PC), whereas in the soleus muscle, in addition to 16:0-PC, 27.9% of PC molecules was stearate-containing PC (18:0-PC). Most palmitate and stearate were bound at the sn-1 position of 16:0- and 18:0-PC, respectively, and 18:0-PC was found in type I and IIa fibers. The amount of 18:0-PE was higher in the soleus than in the EDL muscle. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) increased the amount of 18:0-PC in the EDL. Lysophosphatidylglycerol acyltransferase 1 (LPGAT1) was highly expressed in the soleus compared with that in the EDL muscle and was upregulated by PGC-1α. LPGAT1 knockout decreased the incorporation of stearate into PC and PE in vitro and ex vivo and the amount of 18:0-PC and 18:0-PE in murine skeletal muscle with an increase in the level of 16:0-PC and 16:0-PE. Moreover, knocking out LPGAT1 decreased the amount of stearate-containing phosphatidylserine (18:0-PS), suggesting that LPGAT1 regulated the acyl chain profiles of phospholipids, namely, PC, PE, and PS, in the skeletal muscle.

Biosynthetic Gene Expression and Tissue Distribution of Diosgenin in Dioscorea japonica.

Diosgenin is an aglycone of dioscin, a major bioactive steroidal saponin found in plants, including Himalayan Paris (Paris polyphylla), fenugreek (Trigonella foenum-graecum), and yam (Dioscorea spp.). We have previously demonstrated that a species of natural yam, Dioscorea japonica, contains a promising bioactive compound diosgenin, which induces anti-carcinogenic and anti-hypertriacylglycerolemic activities. Here, we found for the first time that Japanese yam (D. japonica) bulbils are richer in diosgenin than the edible tubers (rhizomes) and leaves. LC-MS and imaging-MS analyses revealed that diosgenin accumulated in the peripheral region of D. japonica bulbils. Additionally, we performed RNA-seq analysis of D. japonica, and multiple sequence alignment identified D. japonica CYP90 (DjCYP90), the orthologous gene of CYP90G4 in P. polyphylla, CYP90B50 in T. foenum-graecum, CYP90G6 in Dioscorea zingiberensis, and CYP90G in Dioscorea villosa, which encodes a diosgenin biosynthetic rate-limiting enzyme. The expression levels of DjCYP90 were significantly upregulated in D. japonica bulbils than in its rhizomes and leaves. Since diosgenin is one of the most promising functional food factors executing several favorable bioactivities, D. japonica bulbils rich in diosgenin would be a beneficial natural resource.

Screening method toward ClbP-specific inhibitors.

BACKGROUND: Colibactin is a genotoxin produced by Escherichia coli and other Enterobacteriaceae that is believed to increase the risk of colorectal cancer (CRC) of their symbiosis hosts, including human. A peptidase ClbP is the key enzyme for activation of colibactin. Inhibition of ClbP is considered to impede maturation of precolibactin into genotoxic colibactin. Therefore, ClbP-specific inhibitors could potentially prevent the onset of CRC, one of the leading causes of cancer-related deaths in the world. This study intends to establish an efficient screening system for identifying inhibitors that are specific to ClbP. METHODS: Two types of assays were applied in the screening procedure: a probe assay and an LC-MS assay. For the probe assay, we employed the synthesized probe which we described in our previous report. This probe can be hydrolyzed efficiently by ClbP to release a fluorophore. Hence it was applied here for detection of inhibition of ClbP. For the LC-MS assay, formation of the byproduct of precolibactin maturation process, N-myristoyl-D-asparagine, was quantified using a liquid chromatography-mass spectrometry (LC-MS) technique. The probe assay can be performed much faster, while the LC-MS assay is more accurate. Therefore, our method employed the two assays in sequence to screen a large number of compounds for inhibition of ClbP. RESULTS: A library of 67,965 standard compounds was evaluated by the screening method established in the current study, and one compound was found to show a moderate inhibitory activity against ClbP. CONCLUSION: A simple screening method for ClbP-specific inhibitors was established. It was proven to be reliable and is believed to be useful in developing potential prophylactic agents for CRC.

Involvement of microRNA modifications in anticancer effects of major polyphenols from green tea, coffee, wine, and curry.

Epidemiological studies have shown that consumption of green tea, coffee, wine, and curry may contribute to a reduced risk of various cancers. However, there are some cancer site-specific differences in their effects; for example, the consumption of tea or wine may reduce bladder cancer risk, whereas coffee consumption may increase the risk. Animal and cell-based experiments have been used to elucidate the anticancer mechanisms of these compounds, with reactive oxygen species (ROS)-based mechanisms emerging as likely candidates. Chlorogenic acid (CGA), curcumin (CUR), epigallocatechin gallate (EGCG), and resveratrol (RSV) can act as antioxidants that activate AMP-activated protein kinase (AMPK) to downregulate ROS, and as prooxidants to generate ROS, leading to the downregulation of NF-κB. Polyphenols can modulate miRNA (miR) expression, with these dietary polyphenols shown to downregulate tumor-promoting miR-21. CUR, EGCG, and RSV can upregulate tumor-suppressing miR-16, 34a, 145, and 200c, but downregulate tumor-promoting miR-25a. CGA, EGCG, and RSV downregulate tumor-suppressing miR-20a, 93, and 106b. The effects of miRs may combine with ROS-mediated pathways, enhancing the anticancer effects of these polyphenols. More precise analysis is needed to determine how the different modulations of miRs by polyphenols relate to the cancer site-specific differences found in epidemiological studies related to the consumption of foods containing these polyphenols.

Health Effects of Soy Isoflavones and Green Tea Catechins on Cancer and Cardiovascular Diseases Based on Urinary Biomarker Levels.

Plant polyphenols have various health effects. Genistein, which is abundant in soybeans, and epigallocatechin-3-gallate, which is abundant in green tea, are major flavonoids, a subclass group of polyphenols. Several epidemiological studies have shown that these flavonoids have beneficial effects against cancer and cardiovascular diseases. However, other studies did not show such effects. Several confounding factors, including recall bias, are related to these inconsistent findings, and the determination of metabolites in the urine may be useful in reducing the number of confounding factors. Equipment, which can be used by research participants to collect samples from a portion of voided urine within 24 h without the help of medical workers, has been developed for epidemiological investigations. Previous studies, in which flavonoid metabolites in these urine samples were measured, revealed that soy intake was correlated with a reduced risk of certain types of cancer and cardiovascular diseases worldwide. Although soybeans and green tea consumption may have protective effects against cancer and cardiovascular diseases, further clinical studies that consider different confounding factors are required to provide evidence for the actual impact of dietary flavonoids on human diseases, including cancer and cardiovascular diseases. One possible mechanism involved is discussed in relation to the downregulation of reactive oxygen species and the upregulation of 5'-adenosine monophosphate-activated protein kinase elicited by these flavonoids.

Contribution of Non-Coding RNAs to Anticancer Effects of Dietary Polyphenols: Chlorogenic Acid, Curcumin, Epigallocatechin-3-Gallate, Genistein, Quercetin and Resveratrol.

Growing evidence has been accumulated to show the anticancer effects of daily consumption of polyphenols. These dietary polyphenols include chlorogenic acid, curcumin, epigallocatechin-3-O-gallate, genistein, quercetin, and resveratrol. These polyphenols have similar chemical and biological properties in that they can act as antioxidants and exert the anticancer effects via cell signaling pathways involving their reactive oxygen species (ROS)-scavenging activity. These polyphenols may also act as pro-oxidants under certain conditions, especially at high concentrations. Epigenetic modifications, including dysregulation of noncoding RNAs (ncRNAs) such as microRNAs, long noncoding RNAs, and circular RNAs are now known to be involved in the anticancer effects of polyphenols. These polyphenols can modulate the expression/activity of the component molecules in ROS-scavenger-triggered anticancer pathways (RSTAPs) by increasing the expression of tumor-suppressive ncRNAs and decreasing the expression of oncogenic ncRNAs in general. Multiple ncRNAs are similarly modulated by multiple polyphenols. Many of the targets of ncRNAs affected by these polyphenols are components of RSTAPs. Therefore, ncRNA modulation may enhance the anticancer effects of polyphenols via RSTAPs in an additive or synergistic manner, although other mechanisms may be operating as well.

Toxicological effects of two metabolites derived from o-toluidine and o-anisidine after 28-day oral administration to rats.

Although both o-toluidine and o-anisidine are known as aromatic amines with bladder carcinogenicity, the specific metabolites involved in carcinogenesis are still unclear. Here, we examined the toxicological effects of head-to-tail dimers of o-toluidine and o-anisidine, 2-methyl-N4-(2-methylphenyl) benzene-1,4-diamine (MMBD) and 2-methoxy-N4-(2-methoxyphenyl) benzene-1,4-diamine (MxMxBD), respectively, in rats. Six-week-old male F344 rats were orally administered MMBD, MxMxBD, o-toluidine, and o-anisidine at a dose of 100 mg/kg/day for 28 days. Rats administered 400 mg/kg o-toluidine and 600 mg/kg/day o-anisidine were set as high-dose groups for comparison. Histopathology and immunohistochemistry for γ-H2AX, a DNA damage biomarker, and bladder stem cell markers, including aldehyde dehydrogenase 1A1 (ALDH1A1), were performed. MMBD and MxMxBD caused different toxicities than their monomers, inducing hepatotoxicity such as vacuolar degeneration but not splenic lesions due to methemoglobinemia. Bladder lesions, including urothelial hyperplasia, were observed in the high-dose o-toluidine and o-anisidine groups, whereas no obvious changes were induced in the low-dose groups or their dimers. Although γ-H2AX formation was significantly increased by o-toluidine and o-anisidine treatment, γ-H2AX formation did not differ among the MMBD, MxMxBD, and control groups. Notably, immunohistochemistry revealed marked increases in ALDH1A1 expression in the bladder urothelium of the MMBD and MxMxBD groups and in the o-toluidine and o-anisidine groups, suggesting that the two dimers may contribute to the bladder carcinogenic effects of o-toluidine and o-anisidine to some extent. The degrees of bladder lesions and γ-H2AX formation did not correlate with the amount of unchanged o-toluidine and o-anisidine in urine, indicating the presence of other metabolites responsible for these findings.

Cytotoxic Homo- and Hetero-Dimers of o-toluidine, o-anisidine, and Aniline Formed by In Vitro Metabolism.

Several aromatic amine compounds are urinary bladder carcinogens. Activated metabolites and DNA adducts of polycyclic aromatic amines, such as 4-aminobiphenyl, have been identified, whereas those of monocyclic aromatic amines, such as o-toluidine (o-Tol), o-anisidine (o-Ans), and aniline (Ani), have not been completely determined. We have recently reported that o-Tol and o-Ans are metabolically converted in vitro and in vivo to cytotoxic and mutagenic p-semidine-type dimers, namely 2-methyl-N4-(2-methylphenyl) benzene-1,4-diamine (MMBD) and 2-methoxy-N4-(2-methoxyphenyl) benzene-1,4-diamine (MxMxBD), respectively, suggesting their roles in urinary bladder carcinogenesis. In this study, we found that when o-Tol and o-Ans were incubated with S9 mix, MMBD and MxMxBD as well as two isomeric heterodimers, MMxBD and MxMBD, were formed. Therefore, any two of o-Tol, o-Ans, and Ani (10 mM each) were incubated with the S9 mix for up to 24 h and then subjected to LC-MS to investigate their metabolic kinetics. Metabolic conversions to all nine kinds of p-semidine-type homo- and hetero-dimers were observed, peaking at 6 h of incubation with the S9 mix; MxMxBD reached the peak at 6.1 ± 1.4 µM. Homo- and hetero-dimers containing the o-Ans moiety in the diamine structure showed a faster dimerization ratio, whereas levels of these dimers, such as MxMxBD, markedly declined with further incubation. Dimers containing o-Tol and Ani were relatively stable, even after incubation for 24 h. The electron-donating group of the o-Ans moiety may be involved in rapid metabolic conversion. In the cytotoxic assay, dimers with an o-Ans moiety in the diamine structure and MMBD showed approximately two- to four-fold higher cytotoxicity than other dimers in human bladder cancer T24 cells. These chemical and biological properties of homo- and hetero-dimers of monocyclic aromatic amines may be important when considering the combined exposure risk for bladder carcinogenesis.

Anti-Cancer Effects of Dietary Polyphenols via ROS-Mediated Pathway with Their Modulation of MicroRNAs.

Consumption of coffee, tea, wine, curry, and soybeans has been linked to a lower risk of cancer in epidemiological studies. Several cell-based and animal studies have shown that dietary polyphenols like chlorogenic acid, curcumin, epigallocatechin-3-O-gallate, genistein, quercetin and resveratrol play a major role in these anticancer effects. Several mechanisms have been proposed to explain the anticancer effects of polyphenols. Depending on the cellular microenvironment, these polyphenols can exert double-faced actions as either an antioxidant or a prooxidant, and one of the representative anticancer mechanisms is a reactive oxygen species (ROS)-mediated mechanism. These polyphenols can also influence microRNA (miR) expression. In general, they can modulate the expression/activity of the constituent molecules in ROS-mediated anticancer pathways by increasing the expression of tumor-suppressive miRs and decreasing the expression of oncogenic miRs. Thus, miR modulation may enhance the anticancer effects of polyphenols through the ROS-mediated pathways in an additive or synergistic manner. More precise human clinical studies on the effects of dietary polyphenols on miR expression will provide convincing evidence of the preventive roles of dietary polyphenols in cancer and other diseases.

Induction of DNA Damage in Mouse Colorectum by Administration of Colibactin-producing Escherichia coli, Isolated from a Patient With Colorectal Cancer.

BACKGROUND/AIM: Among colorectal cancer-associated intestinal microbiota, colibactin-producing (clb+) bacteria are attracting attention. We aimed to clarify the interaction between clb+ Escherichia coli and normal colorectal epithelial cells in vivo and in vitro. MATERIALS AND METHODS: Five-week-old female Balb/c mice were divided in an untreated group, a group treated with clb+ E. coli isolated from a Japanese patient with colorectal cancer (E. coli-50), and a group treated with non colibactin-producing E. coli (E. coli-50/ΔclbP). Mice were sacrificed at 18 weeks of treatment. RESULTS: Treatment with clb+ E. coli increased positivity for H2A histone family member X phosphorylated at Ser-139 (γH2AX) in epithelial cells of the luminal surface of the mouse rectum but this did not occur in the E. coli-50/ΔclbP and untreated groups. In an in vitro setting, the ratio of apoptotic cells was increased and cell counts were reduced by treatment with clb+ E. coli more than in untreated cells and normal rat colorectal epithelial cells. CONCLUSION: E. coli-50 induced DNA damage in the mouse rectum, possibly by direct interaction between clb+ E. coli and normal colorectal epithelial cells. Our findings imply that regulation of clb+ E. coli infection may be a useful strategy for colorectal cancer control.

Toward Engineered Biosynthesis of Drugs in Human Cells.

Biosynthetic genes are not only responsible for the formation of bioactive substances but also suited for other applications including gene therapy. To test the feasibility of human cells producing antibiotics in situ when provided with a heterologous biosynthetic gene, we focused on cytochrome P450, the class of enzymes important in conferring bioactivity to natural product precursors. We selected Fma-P450 that plays a central role in the fumagillin antimicrobial biosynthesis in Aspergillus fumigatus to examine fungal metabolite production by HeLa cells that express fma-P450 heterologously. Here we show that HeLa cells harboring fma-P450 can biosynthesize 5-hydroxyl-ß-trans-bergamoten and cytotoxic 5-epi-demethoxyfumagillol when supplemented with the nontoxic precursor ß-trans-bergamotene. While the production level was insufficient to effect cell death, we demonstrate that programming human cells to autogenerate antibiotics by introducing a heterologous biosynthetic gene is feasible.

Establishment of Novel Genotoxicity Assay System Using Murine Normal Epithelial Tissue-Derived Organoids.

Short-/middle-term and simple prediction studies for carcinogenesis are needed for the safety assessment of chemical substances. To establish a novel genotoxicity assay with an in vivo mimicking system, we prepared murine colonic/pulmonary organoids from gpt delta mice according to the general procedure using collagenase/dispase and cultured them in a 3D environment. When the organoids were exposed to foodborne carcinogens-2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) and acrylamide (AA)-in the presence of metabolic activation systems, mutation frequencies (MFs) occurring in the gpt gene dose-dependently increased. Moreover, the mutation spectrum analysis indicated predominant G:C to T:A transversion with PhIP, and A:T to C:G and A:T to T:A transversion with AA. These data correspond to those of a previous study describing in vivo mutagenicity in gpt delta mice. However, organoids derived from the liver, a non-target tissue of PhIP-carcinogenesis, also demonstrated genotoxicity with a potency comparable to colonic organoids. Organoids and PhIP were directly incubated in the presence of metabolic activation systems; therefore, there was a lack of organ specificity, as observed in vivo. Additionally, PhIP-DNA adduct levels were comparable in hepatic and colonic organoids after PhIP exposure. Taken together, the organoids prepared in the present study may be helpful to predict chemical carcinogenesis.