Effects Of Secondary Bile Acids Research Articles

Abstract 6682: Bile acid profiles and racial disparities in colorectal cancer: Exploring the protective role of secondary bile acid derivatives

Abstract Background: Colorectal cancer (CRC) is the third leading cause of cancer death in the United States, with diet and gut microbiota-derived bile acids implicated as long-term risk factors. The interplay between bile acids and immune cell differentiation, particularly of macrophages and T-cells, is a crucial aspect of the CRC microenvironment. Given the high-animal-fat diets' influence on the microbiome’s production of hydrophobic secondary bile acids such as deoxycholic acid (DCA) and lithocholic acid (LCA), this study investigates the potential immunomodulatory effects of their lower concentrations and their isomers in possibly fostering M2-like macrophage expansion. Methods: Clinical samples were obtained during colonoscopies with no tumor findings at Rush University Medical Center, Chicago. RNA was extracted from colonoscopy biopsies for NanoString RNA expression analysis, while bile acids were isolated from both lyophilized stool and frozen serum samples. To explore the immunological effects of secondary bile acids, the macrophage RAW 264.7 cell line, alongside peritoneal macrophages and bone marrow-derived macrophages (BMDMs) from C57BL/6J mice, were treated with LCA and LCA isomers. Following the bile acid treatment, LPS (100 ng/mL) or IL-4 (40 ng/ml) was added to elicit pro-inflammatory or anti-inflammatory states, respectively. After 48 hours, conditioned media were collected for enzyme-linked immunosorbent assay (ELISA), while cells were analyzed by flow cytometry and rt-qPCR. Results: Contrary to initial hypotheses, the levels of DCA and LCA levels were not significantly different between African American and non-Hispanic White subjects. Notably, allo-isomers and oxo-derivatives of these bile acids, which may promote Treg and M2-like macrophage expansion, were substantially lower in African American subjects. Ex vivo treatments with isoalloLCA and IsoLCA led to notable changes in the M2 macrophage differentiation and shifts in PGE2 production upon exposure to inflammatory or anti-inflammatory stimuli, suggesting a nuanced alteration in the macrophage-mediated inflammatory response. Conclusion: This study highlights a significant ethnic variation in bile acid profiles, with a potential impact on immune modulation and CRC risk. The reduced presence of certain bile acid isomers in African American subjects may influence the risk of CRC through altered macrophage differentiation and function. These findings emphasize the need for personalized dietary and therapeutic strategies to modulate the bile acid metabolome, potentially reducing CRC disparities. Citation Format: Hanchu Dai, Patricia G. Wolf, Lisa Tussing-Humphreys, Ece Mutlu, Jason M. Ridlon, Rex Gaskins. Bile acid profiles and racial disparities in colorectal cancer: Exploring the protective role of secondary bile acid derivatives [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6682.

Chronic jetlag reprograms gene expression in the colonic smooth muscle layer inducing diurnal rhythmicity in the effect of bile acids on colonic contractility.

Secondary bile acids entrain peripheral circadian clocks and inhibit colonic motility via the bile acid receptor GPBAR1. We aimed to investigate whether chronodisruption affected the rhythm in serum bile acid levels and whether this was associated with alterations in clock gene and Gpbar1 mRNA expression in the colonic smooth muscle layer. We hypothesized that this in turn may affect the rhythm in the inhibitory effect of secondary bile acids on colonic contractility. Mice were exposed to 4 weeks of chronic jetlag induction. The expression of Gpbar1 and clock genes was measured in colonic smooth muscle tissue using RT-qPCR over 24 h (4h time interval). The effect of secondary bile acids on electrical field-induced neural contractions was measured isometrically in colonic smooth muscle strips. Chronic jetlag abolished the rhythmicity in serum bile acid levels. This was associated with a phase-shift in diurnal clock gene mRNA fluctuations in smooth muscle tissue. Chronic jetlag induced a rhythm in Gpbar1 expression in the colonic smooth muscle layer. In parallel, a rhythm was induced in the inhibitory effect of taurodeoxycholic acid (TDCA), but not deoxycholic acid, on neural colonic contractions that peaked together with Gpbar1 expression. Chronodisruption abolished the rhythm in bile acid levels which might contribute to a shift in smooth muscle clock gene expression. Our findings suggest that chronodisruption caused a transcriptional reprogramming in the colonic smooth layer thereby inducing a rhythm in the expression of Gpbar1 and in the inhibitory effect of TDCA on colonic contractility.

Squalene epoxidase drives cancer cell proliferation and promotes gut dysbiosis to accelerate colorectal carcinogenesis

ObjectiveAberrant lipid metabolism is a hallmark of colorectal cancer (CRC). Squalene epoxidase (SQLE), a rate-limiting enzyme in cholesterol biosynthesis, is upregulated in CRC. Here, we aim to determine oncogenic function...

Secondary Bile Acids and Short Chain Fatty Acids in the Colon: A Focus on Colonic Microbiome, Cell Proliferation, Inflammation, and Cancer.

Secondary bile acids (BAs) and short chain fatty acids (SCFAs), two major types of bacterial metabolites in the colon, cause opposing effects on colonic inflammation at chronically high physiological levels. Primary BAs play critical roles in cholesterol metabolism, lipid digestion, and host–microbe interaction. Although BAs are reabsorbed via enterohepatic circulation, primary BAs serve as substrates for bacterial biotransformation to secondary BAs in the colon. High-fat diets increase secondary BAs, such as deoxycholic acid (DCA) and lithocholic acid (LCA), which are risk factors for colonic inflammation and cancer. In contrast, increased dietary fiber intake is associated with anti-inflammatory and anticancer effects. These effects may be due to the increased production of the SCFAs acetate, propionate, and butyrate during dietary fiber fermentation in the colon. Elucidation of the molecular events by which secondary BAs and SCFAs regulate colonic cell proliferation and inflammation will lead to a better understanding of the anticancer potential of dietary fiber in the context of high-fat diet-related colon cancer. This article reviews the current knowledge concerning the effects of secondary BAs and SCFAs on the proliferation of colon epithelial cells, inflammation, cancer, and the associated microbiome.

Secondary bile acids inhibit Candida albicans growth and morphogenesis.

Candida albicans is one of the most common causes of fungal infections in humans with a significant mortality rate. However, the factors involved in C. albicans gastrointestinal (GI) colonization remain unclear. We hypothesize that secondary bile acids have direct antifungal activity against C. albicans and may play a critical role in maintaining GI colonization resistance against C. albicans. In this study, we investigated the effect of secondary bile acids including lithocholic acid (LCA) and deoxycholic acid (DCA) on C. albicans growth and morphogenesis. Results indicate that LCA and DCA at in vivo cecal micelle concentrations inhibit C. albicans growth in vitro. Interestingly, LCA and DCA also significantly inhibited the germ tube, hyphae and biofilm formation in C. albicans. In addition, pre-treatment of C. albicans with LCA and DCA significantly reduced the percentage of C. albicans cells attached to a colon cancer cell line. Collectively, our results demonstrate that secondary bile acids play an important role in controlling the growth and morphological switching of C. albicans. Results from this study demonstrate that secondary bile acid possess direct antifungal activity against C. albicans, explaining a potential mechanism for gastrointestinal colonization resistance against C. albicans.

Ursodeoxycholic acid and chemoprevention of colorectal cancer

Colorectal cancer is respectively the third and second most common cancer among men and women in France. Interest in chemoprevention for colorectal cancer has increased over the last two decades. Beside non-steroidal anti-inflammatory drugs, ursodeoxycholic acid (UDCA) may have chemopreventive action in colorectal cancer with a likely better tolerance. In high-risk populations such as patients with inflammatory bowel disease or prior colorectal adenoma or carcinoma, retrospective and prospective studies have suggested a beneficial effect of UDCA. In azoxymethane model, UDCA inhibits tumor development by countering the tumor-promoting effects of secondary bile acids, such as deoxycholic acid (DCA). The opposing effects of UDCA and DCA on lipid raft composition may be central to their effects on colonic tumorigenesis. Differential effects of DCA and UDCA on growth factor and inflammatory signals involved in colorectal carcinogenesis, such as epidermal growth factor receptors (EGFR) signaling and Cox-2 expression, likely mediate their opposing effects on colonic tumor promotion and tumor inhibition, respectively.

A Nonenzymatic Modification of the Amino-terminal Domain of Histone H3 by Bile Acid Acyl Adenylate

Although it has been proposed that the secondary bile acids, deoxycholic acid and lithocholic acid, increase the number of aberrant crypt foci in the colon and may act as colon tumor promoters, there is little evidence detailing their mechanism of action. Histones play an important role in controlling gene expression, and the posttranslational modification of histones plays a role in regulation of intracellular signal transduction. In particular, the amino-terminal tail domain of histone H3 is sensitive to several posttranslational modifications, and acetylation of this domain changes its electrostatic environment and results in the loss of native folding. Therefore, we studied the modification of epsilon-amino groups on human histone H3 by deoxycholyl adenylate, which is an active intermediate in deoxycholyl thioester biosynthesis. After incubation of recombinant human histone H3 with a smaller amount of acyl adenylate, followed by enzymatic digestion, the peptide fragment mixtures were analyzed by matrix-assisted laser desorption ionization mass spectrometry. These data showed the formation of only one adduct fragment, which corresponded to amino acids 3-8 with a deoxycholate adduct, suggesting that the epsilon-amino group of Lys(4) had the highest reactivity. This novel modification, formation of a bile acid adduct on the histone H3 amino-terminal tail domain through an active acyl adenylate, may relate to the carcinogenesis-promoting effects of secondary bile acids.

Deoxycholic acid activates beta-catenin signaling pathway and increases colon cell cancer growth and invasiveness.

Colorectal cancer is often lethal when invasion and/or metastasis occur. Tumor progression to the metastatic phenotype is mainly dependent on tumor cell invasiveness. Secondary bile acids, particularly deoxycholic acid (DCA), are implicated in promoting colon cancer growth and progression. Whether DCA modulates beta-catenin and promotes colon cancer cell growth and invasiveness remains unknown. Because beta-catenin and its target genes urokinase-type plasminogen activator receptor (uPAR) and cyclin D1 are overexpressed in colon cancers, and are linked to cancer growth, invasion, and metastasis, we investigated whether DCA activates beta-catenin signaling and promotes colon cancer cell growth and invasiveness. Our results show that low concentrations of DCA (5 and 50 microM) significantly increase tyrosine phosphorylation of beta-catenin, induce urokinase-type plasminogen activator, uPAR, and cyclin D1 expression and enhance colon cancer cell proliferation and invasiveness. These events are associated with a substantial loss of E-cadherin binding to beta-catenin. Inhibition of beta-catenin with small interfering RNA significantly reduced DCA-induced uPAR and cyclin D1 expression. Blocking uPAR with a neutralizing antibody significantly suppressed DCA-induced colon cancer cell proliferation and invasiveness. These findings provide evidence for a novel mechanism underlying the oncogenic effects of secondary bile acids.

Deoxycholic acid stimulates migration in colon cancer cells.

Deoxycholic acid and other secondary bile acids have long been considered tumour promoters in the colon. However, their effect on cell migration, known to play an important role in colon carcinogenesis, has not been studied so far. To investigate the possible effects of deoxycholic acid on colon cancer-cell migration in culture. Human colon carcinoma cells (Caco-2) were seeded on basement membrane matrix. To evaluate replication-blocked cell migration, we wounded confluent monolayers of cells with a sterile scalpel, and inhibited cell replication with mitomycin C. Immediately after wounding, the cells were exposed to 0-100 micromol/l deoxycholic acid. Migration over 72 h was monitored using a phase contrast microscope. Replication-blocked migration was stimulated by deoxycholic acid in a dose-dependent manner, with the maximum effect at 20 micromol/l deoxycholic acid. Enhancement of migration rate was unaffected by immunoneutralization of transforming growth factor beta (a known migration-promoting peptide). However, specific inhibition of protein kinase C markedly inhibited deoxycholic acid-induced Caco-2 cell migration. In addition to its well-established role in the enhancement of proliferation, deoxycholic acid also stimulates colon cancer-cell migration along the basement membrane matrix. The mechanism of this stimulation is likely to involve protein kinase C. Deoxycholic acid-stimulated migration might additionally contribute to the tumour-promoting effects of secondary bile acids in the colon.

Deoxycholate induces DNA damage and apoptosis in human colon epithelial cells expressing either mutant or wild-type p53

Diets rich in fat result in higher concentrations of secondary bile acids or their salts in the colon, which may adversely affect cells of the colonic epithelium. Because secondary bile acids are thought to be genotoxic, exposing colon epithelial cells to secondary bile acids may induce DNA damage that might lead to apoptosis. The requirement for the p53 tumor suppressor gene in such events is unknown. In particular, the effects of secondary bile acids on colon epithelial cells having different p53 tumor suppressor gene status have not been examined. Therefore, HCT-116 and HCT-15 human colon adenocarcinoma cells, which express the wild-type and mutant p53 genes, respectively, were exposed to physiological concentrations of deoxycholate. The cells were then analyzed for evidence of DNA damage and apoptosis. After 2 h of incubation with 300 μM deoxycholate, both cell lines had greater levels of single-strand breaks in DNA as assessed by the comet assay. After 6 h of exposure to deoxycholate, HCT-116 and HCT-15 cells showed morphological signs of apoptosis, i.e., membrane blebbing and the presence of apoptotic bodies. Chromatin condensation and fragmentation were also seen after staining DNA with 4′,6-diamidino-2-phenylindole. Other apoptotic assays revealed greater binding of annexin V-fluorescein isothiocyanate, as well as greater post-enzymatic labeling with dUTP-fluorescein isothiocyanate, by both cell lines exposed to deoxycholate. These data suggest that deoxycholate caused DNA damage in colon epithelial cells that was sufficient to trigger apoptosis in a p53-independent manner.

Role of Morphological Methods in the Analysis of Chemically Induced Colon Cancer in Rats

The role of selected histopathological methods was evaluated for the characterization of tumourous tissues resulting from chemically induced carcinogenesis in rats. Colon cancer was induced by treating rats with a direct carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), alone or with subsequent treatments with secondary bile acids. Morphological studies showed that the tumorigenic effect of MNNG resulted in changes in the shape of the crypts, disappearance of mucous (goblet) cells, and marked increases in the mitotic index, the size of nuclei and the number of aberrant nuclei. A significant number of lymphocytes infiltrated between the crypts and into their lumens. A sharp decrease in concentrations of neutral mucopolysaccharides and sulfomucins and an increase in the amount of vic-glycol groups and nonsulfated mucosubstances were demonstrated in tumourous epithelial cells. A moderate positive reaction to tissue polypeptide antibody was observed in these cells. The tumor-promoting effects of secondary bile acids were detected morphologically by an increase in the amount of connective tissue that infiltrated newly formed tumors. The increased number of micronuclei in otherwise histologically unaltered colon tissues adjacent to a tumor suggests that these regions represent a precancerous stage in the development of tumors. The important role of morphological methods in the evaluation of the effects of carcinogen and tumor promoters and in the detection of various phases in experimental carcinogenesis was discussed.

Effects of secondary bile acids on the intrauterine development in rats

The effects of secondary bile acids (lithocholic--LCA, and deoxycholic--DCA) on the in vivo development of rat embryos and fetuses were studied. Daily intraperitoneal injections of 2 ml of 1 mM LCA and of 5 mM DCA during days 6 till 15 of pregnancy resulted in an increase of resorptions among 20 day-old fetuses to 22.8% and 9.9%, respectively, vs. 6.2% in controls. Similar injections on days 12 to 19 resulted in an increase of resorptions to 10.3% after treatment with LCA and to 36% after treatment with DCA. Percent of retarded embryos was similar for both bile acids: 7.7 and 8.7% after injections on days 6-15 and 12.3-12.5% after injections on days 12-19 of gestation. This was accompanied by a significant increase in the wet weight of the placenta of living embryos. Intraamniotic injections of 2 microliters of 1 mM LCA into 10 day-old embryos resulted in 18.5% resorptions (vs. 7.5% in controls), 9.2% malformations, and 3.1% growth retardations observed on day 12 of pregnancy. The rate of resorptions following this treatment increased on day 20 of pregnancy to 71% vs. 16% in controls. No differences were found in the wet weight of 20 day-old living fetuses or their livers and placentas between experimental and control groups following i.p. or intraamniotic injections. In addition, single intrauterine instillation of 0.2 ml of 1 mM LCA 10-14 days before mating with normal isogeneic males resulted in 9% of malformations among 12 day-old embryos while malformations were absent in the saline-injected controls. The deleterious effects of secondary bile acids to the embryos were accompanied by damage to the visceral yolk sac. These findings may be significant in relation to the complications previously associated with cholestasis of pregnancy in humans.

Effects of Secondary Bile Acids on the in vitro Development of Early Somite Rat Embryos

Effects of secondary bile acids--lithocholic (LCA) and deoxycholic (DCA)--on the in vitro development of early somite (10.5 days old) rat embryos were studied. It was shown that an addition to the culture medium of 0.1 mM LCA (final concentration) resulted in 9% growth-retarded and 12% malformed embryos when the duration of exposure was 24 h. When treatment with LCA was prolonged to 48 h, the rate of growth retardation increased to 18% and that of malformations to 40% versus 0.5% for both parameters observed in controls. This could be interpreted as a reversible or time-dependent effect of LCA on the in vitro development of the mammalian embryo. Culture of embryos in medium with 0.5 mM DCA resulted in 22% of growth retardation and 50% of malformations. DCA in 0.1 mM final concentration had only slight and statistically nonsignificant effects. Retardation of growth development could be demonstrated by a decrease in crown-rump length and the number of somites. Among malformed embryos, abnormalities in the development of the neural tube and exencephaly were the most common types of malformations. Abnormalities as well as growth retardation were accompanied by significant pathological changes in structure and perhaps in function of the endodermal visceral yolk sac cells. It could be suggested that secondary bile acids when present in pathophysiological concentrations can affect the embryonic development by direct inhibitory effects and that these effects may be time and dose dependent.