Intestinal microbiota contributes to individual susceptibility to alcoholic liver disease.

OBJECTIVE: There is substantial inter-individual diversity in the susceptibility of alcoholics to liver injury. Alterations of intestinal microbiota (IM) have been reported in alcoholic liver disease (ALD), but the extent to which they are merely a consequence or a cause is unknown. We aimed to demonstrate that a specific dysbiosis contributes to the development of alcoholic hepatitis (AH). DESIGN: We humanised germ-free and conventional mice using human IM transplant from alcoholic patients with or without AH. The consequences on alcohol-fed recipient mice were studied. RESULTS: A specific dysbiosis was associated with ALD severity in patients. Mice harbouring the IM from a patient with severe AH (sAH) developed more severe liver inflammation with an increased number of liver T lymphocyte subsets and Natural Killer T (NKT) lymphocytes, higher liver necrosis, greater intestinal permeability and higher translocation of bacteria than mice harbouring the IM from an alcoholic patient without AH (noAH). Similarly, CD45+ lymphocyte subsets were increased in visceral adipose tissue, and CD4(+)T and NKT lymphocytes in mesenteric lymph nodes. The IM associated with sAH and noAH could be distinguished by differences in bacterial abundance and composition. Key deleterious species were associated with sAH while the Faecalibacterium genus was associated with noAH. Ursodeoxycholic acid was more abundant in faeces from noAH mice. Additionally, in conventional mice humanised with the IM from an sAH patient, a second subsequent transfer of IM from an noAH patient improved alcohol-induced liver lesions. CONCLUSIONS: Individual susceptibility to ALD is substantially driven by IM. It may, therefore, be possible to prevent and manage ALD by IM manipulation.

Short-chain fatty acids and polyamines in the pathogenesis of necrotizing enterocolitis: Kinetics aspects in gnotobiotic quails.

Despite years of investigation, pathogenesis of necrotizing enterocolitis (NEC) remains elusive. Bacterial metabolites were implicated by several authors but their roles remain controversial. The aim of our study was to investigate the role of SCFAs and polyamines through a kinetic study of histological and macroscopical digestive lesions in monobiotic quails. Germ-free quails, inoculated with a Clostridium butyricum strain involved in a NEC case, were fed or not with a diet including lactose (7%). Quails were sacrificed at various times between D7 and D24 after bacterial inoculation. NEC-like lesions, i.e. thickening, pneumatosis, and hemorrhages, occurred only in lactose-fed quails and increased with time. The main histological characteristics were infiltrates of mononuclear cells, then heterophilic cells, then gas cyst and necrosis. The first event observed, before histological and macroscopical lesions, is a high production of butyric acid, which precedes an increase of iNOS gene expression. No difference in polyamines contents depending on the diet was observed. These results show the major role of butyric acid produced by commensal bacteria in the onset of the digestive lesions.

Allergic sensitization to bovine beta-lactoglobulin: comparison between germ-free and conventional BALB/c mice.

BACKGROUND: The 'hygiene hypothesis' suggests that high hygienic standards met in western countries lead to a lack of microbial exposure, thus promoting the development of atopy by preventing the proper maturation of the immune system. Germ-free animals are deprived of the immune stimulation that occurs during postnatal gut colonization by commensal bacteria. Germ-free mice could thereby provide an attractive model for studying the impact of gut microbiota on the development of Th2-mediated disorders such as allergy. METHODS: Germ-free and conventional BALB/c mice were sensitized to beta-lactoglobulin (BLG), a major cow's milk allergen, by means of intraperitoneal injections in the presence of incomplete Freund's adjuvant. Time courses of serum and fecal BLG-specific antibody responses were monitored and cytokine production was assayed in BLG-reactivated splenocytes. RESULTS: Serum BLG-specific IgG1 and IgE concentrations were significantly higher in germ-free mice during the primary immune response and IgE production persisted longer in germ-free mice. Furthermore, secretion of BLG-specific IgA was evidenced only in feces from germ-free mice while, in contrast, fecal IgG1 concentrations were at least 3-fold higher in conventional mice than in germ-free mice. Production of IL-5, IL-10 and IFN-gamma was 3-fold enhanced in BLG-reactivated splenocytes from germ-free mice. CONCLUSION: The absence of gut microbiota significantly affects the BLG-specific immune response in BALB/c mice, thus suggesting that this model might be of interest for further studies exploring the influence of gut colonization by different bacterial strains on the development of an allergic-type sensitization.

Constitutive delivery of bovine beta-lactoglobulin to the digestive tracts of gnotobiotic mice by engineered Lactobacillus casei.

The gut microbiota is critical for maturation of the immune system. Recent evidence suggests that early establishment of lactobacilli in the intestinal microbiota, during neonatal colonization or by probiotic supplementation, could prevent the development of allergic disorders. Postnatal maturation of the gut immune system with allergen-producing lactobacilli colonizing the digestive tract could then affect the development of further allergic sensitization. In this paper, we describe construction of a recombinant Lactobacillus casei strain that can constitutively deliver bovine beta-lactoglobulin (BLG), a major cow's milk allergen, to the guts of gnotobiotic mice. The blg gene was inserted into the L. casei chromosome downstream of an endogenous promoter. BLG production was improved by fusing the propeptide LEISSTCDA (LEISS) to the BLG mature moiety. This led to a 10-fold increase in LEISS-BLG production compared to the production obtained without the propeptide and also led to enhanced secretion corresponding to 5% of the total production. After inoculation into germfree C3H/HeN mice, the genetic stability of the recombinant strain and in vivo BLG production were confirmed for at least 10 weeks. BLG stimulation of spleen cells from mice monoassociated with the BLG-producing lactobacilli induced secretion of the Th1 cytokine gamma interferon and, to a lesser extent, the Th2 cytokine interleukin-5. No BLG-specific immunoglobulin G1 (IgG1), IgG2a, or IgA was detected in sera or in fecal samples. These results suggest that gut colonization with allergen-producing lactobacilli could provide a useful model for studying the modulation of allergic disorders.

The activities of several detoxication enzymes are differentially induced by juices of garden cress, water cress and mustard in human HepG2 cells.

It has been previously demonstrated in a human-derived hepatoma cell line (HepG2) that juices from cruciferous vegetables protect against the genotoxicity caused by dietary carcinogens. HepG2 cells possess different enzymes involved in the biotransformation of xenobiotics. Therefore, we investigated the effect of cruciferous juices on the activities of CYP 1A and several phase II enzymes in this cell model. For each experiment, 1 x 10(6) cells were seeded on Petri dishes. After 2 days, the juices (0.5-8 microl/ml of culture medium) were added for 48 h prior to cell harvesting. The addition of juice from water cress (Nasturtium officinalis R. Br) significantly increased the activities of ethoxyresorufin-O-deethylase at high doses only and NAD(P)H-quinone reductase in a dose-dependent manner (1.8- and 5-fold, respectively). The addition of juice from garden cress (Lepidum sativum L.) significantly increased the activities of NAD(P)H-quinone reductase and UDP-glucuronosyl-transferase with a maximal effect around the dose of 2 microl/ml juice (1.4- and 1.2-fold, respectively) while the other enzymes were not altered. Mustard (Sinapis alba L.) juice increased the activities of NAD(P)H-quinone reductase (2.6-fold at the dose of 8 microl/ml), and N-acetyl-transferase (1.4-fold at the dose of 8 microl/ml) in a dose-dependent manner while a maximal induction of UDP-glucuronosyl-transferase was obtained with a dose of 2 microl/ml (1.8-fold). These observations show that the three juices have different induction profiles: only water cress acted as a bifunctional inducer by enhancing both phase I and phase II enzymes. As a consequence, each juice may preferentially inhibit the genotoxicity of specific compounds.

Identification of new derivatives of sinigrin and glucotropaeolin produced by the human digestive microflora using 1H NMR spectroscopy analysis of in vitro incubations.

One- and two-dimensional (1)H NMR spectroscopy were used to study the biotransformation of two dietary glucosinolates, sinigrin (SIN), and glucotropaeolin (GTL) by the human digestive microflora in vitro. The molecular structures of the new metabolites issued from the aglycone moiety of the glucosinolate were identified, and the modulation of carbon metabolism was studied by quantifying bacterial metabolites issued from the xenobiotic incubation in the presence or absence of a source of free glucose. Unambiguously and for the first time, it was shown that SIN and GTL were transformed quantitatively into allylamine and benzylamine, respectively. The comparison of the kinetics of transformation of SIN and GTL with and without glucose clearly showed that the presence of glucose did not modify either the nature of the metabolites or the rate of transformation of the glucosinolates (complete degradation within 30 h). The main end products of the glucose moiety of glucosinolates were characteristic of anaerobic carbon metabolism in the digestive tract (acetate, lactate, ethanol, propionate, formate, and butyrate) and similar to those released from free glucose. This work represents the first application of (1)H NMR spectroscopy to the study of xenobiotic metabolism by the human digestive microflora, demonstrating allyl- and benzylamine production from glucosinolates. Whether these amines are produced in vivo from dietary glucosinolates remains to be established. This would reduce the availability of other glucosinolate metabolites, notably cancer-protective isothiocyanates.

Intestinal microflora plays a crucial role in the genotoxicity of the cooked food mutagen 2-amino-3-methylimidazo [4,5-f]quinoline.

We investigated the impact of the intestinal microflora on the genotoxicity of 2-amino-3-methylimidazo[4,5-f] quinoline (IQ), a mutagenic/carcinogenic heterocyclic amine commonly found in fried meats and fish. In parallel, we also examined the effect of the microflora on the protective effect of glucotropaeolin (GT), a glucosinolate contained in cruciferous vegetables, towards IQ-induced genotoxic effect. Conventional (NF), human flora associated (HFA) and germ free (GF) rats were treated either with 90 mg/kg IQ alone, 150 mg/kg GT alone or a combination of the two by gavage and DNA damage was determined in liver and colon cells using the alkaline single cell gel electrophoresis (SCGE) or comet assay. IQ caused a significant effect in both organs of all groups. However, DNA damage was most pronounced in NF animals. In colon cells, DNA migration was 6-fold more in IQ-exposed rats as compared with untreated controls. The effect measured with liver cells was similar. In comparison to NF rats, in HFA rats, tail length of the comets was 22 and 53% lower in liver and colon cells, respectively. Significantly weaker effects were seen in GF animals (66 and 75% lower damage in hepatocytes and colonocytes, respectively, than in NF animals). Pretreatment with GT led to a complete reduction of IQ-induced DNA damage regardless of the microbial status of the animals. In addition, a moderate decrease in spontaneous DNA damage was seen in animals that received GT alone. Our results show that the microflora has a strong impact on the genotoxic effects of IQ. We conclude that the alkaline SCGE assay with rats harbouring different flora opens new possibilities to investigate the role of intestinal bacteria on health risks caused by dietary carcinogens.

Impact of bacteria in dairy products and of the intestinal microflora on the genotoxic and carcinogenic effects of heterocyclic aromatic amines.

This article gives a short overview on the present state of knowledge of the effects of the intestinal microflora on the health hazards of heterocyclic aromatic amines (HAs). Results of single cell gel electrophoresis assays with conventional, germ free and human flora associated rats indicate that the presence of intestinal microorganisms strongly enhances the induction of DNA-damage in colon and liver cells by IQ. Furthermore, it was found that supplementation of the feed with Lactobacilli attenuates the induction of colon cancer by this same amine. These recent findings suggest that the intestinal microflora and lactic acid bacilli in dairy products strongly affect the health risks of HAs. Nevertheless, most previous experiments with HAs focused on the involvement of mammalian enzymes in the biotransformation of these compounds and only a few articles are available which concern interactions of bacteria with HAs. Some of these studies suggested that the formation of directly mutagenic hydroxy-metabolites of the amines by fecal bacteria might be an important activation pathway but it turned out that the hydroxy-derivative of IQ is not genotoxic in mammalian cells and does not cause colon cancer in laboratory rodents. There is some evidence that hydrolysis of HA-metabolites by bacterial ss-glucuronidase might play a role in the activation of HAs but experimental data are scarce and no firm conclusions can be drawn at present. The most important detoxification mechanism appears to be the direct binding of the HAs to the cell walls of certain bacterial strains contained in fermented foods. It was shown that these effects do also take place under physiologically relevant conditions. Overall, it seems that intestinal bacteria play a key role in the activation and detoxification of HAs which has been an area of research long ignored. The elucidation of these mechanisms may enable the development of biomarkers for colon cancer risk and nutritional strategies of protection.

Effects of cruciferous vegetables and their constituents on drug metabolizing enzymes involved in the bioactivation of DNA-reactive dietary carcinogens.

Epidemiological studies give evidence that cruciferous vegetables (CF) protect humans against cancer, and also results from animal experiments show that they reduce chemically induced tumor formation. These properties have been attributed to alterations in the metabolism of carcinogens by breakdown products of glucosinolates, which are constituents of CF. The present article gives an overview on the present state of knowledge on the impact of CF and their constituents on enzymes that are involved in the metabolism of DNA-reactive carcinogens. The development of in vitro models with metabolically competent cell lines led to the detection of potent enzyme inducers contained in CF such as sulforaphane. Recently, we showed that Brassica juices induce glutathione-S-transferases (GST) and cytochrome P-450 1A2 in human hepatoma cells (HepG2) and protect against the genotoxic effects of B(a)P and other carcinogens. Earlier in vivo experiments with rodents indicated that indoles and isothiocyanates, two major groups of glucosinolate breakdown products, attenuate the effects of polycyclic aromatic hydrocarbons (PAHs) and nitrosamines via induction of GST and inhibition of cytochrome-P450 isoenzymes, respectively. Our own investigations showed that CF are also protective towards heterocyclic amines (HAs): Brussels sprouts- and garden cress juices attenuated IQ-induced DNA-damage and preneoplastic lesions in colon and liver of rats. These effects were paralleled by induction of uridine-di-phospho-glucuronosyl transferase (UDPGT) which is very probably the mechanism of protection against HAs by cruciferous vegetables. There is also evidence that consumption of CF might protect humans against cancer. In matched control intervention studies with these vegetables, it was shown that they induce GST-activities in humans but overall, results were inconclusive. Recently, we carried out crossover intervention studies and found pronounced GST-induction upon consumption of Brussels sprouts and red cabbage, whereas no effects were seen with white cabbage and broccoli. Furthermore, we found that the isoenzyme induced was GST-pi which plays an important role in protection against breast, bladder, colon and testicular cancer. No induction of the GST-alpha isoform could be detected. Urinary mutagenicity experiments gave further evidence that CF affect drug metabolism in humans. Consumption of red cabbage led to changes in the pattern of meat-derived urinary mutagenicity. Overall, CF are among the most promising chemopreventive dietary constituents and further elucidation of their protective mechanisms and the identification of active constituents may contribute to the development of highly protective Brassica varieties.

Formation of allyl isothiocyanate from sinigrin in the digestive tract of rats monoassociated with a human colonic strain of Bacteroides thetaiotaomicron.

A human digestive strain of Bacteroides thetaiotaomicron was tested for its ability to metabolise sinigrin, a glucosinolate commonly found in Brassica vegetables. Gnotobiotic rats harbouring the bacterial strain were orally dosed with 50 micromol sinigrin. HPLC analysis of the digestive contents showed that sinigrin was degraded in the large bowel, where B. thetaiotaomicron was established at a high level. Concurrently, a hydrolysis product of sinigrin, allyl isothiocyanate, was identified by GC-MS analysis, following headspace solid-phase microextraction of the digestive contents; its production peaked at ca. 200 nmol, 12 h after dosing. This is the first study to demonstrate in vivo the involvement of a human colonic predominant bacterium in the bioconversion of a dietary glucosinolate to a potentially anticarcinogenic isothiocyanate.

Polymeric proanthocyanidins are catabolized by human colonic microflora into low-molecular-weight phenolic acids.

Polymeric proanthocyanidins are common constituents of many foods and beverages. Their fate in the human body remains largely unknown. Their metabolism by human colonic microflora incubated in vitro in anoxic conditions has been investigated using nonlabeled and (14)C-labeled purified proanthocyanidin polymers. Polymers were almost totally degraded after 48 h of incubation. Phenylacetic, phenylpropionic and phenylvaleric acids, monohydroxylated mainly in the meta or para position, were identified as metabolites by gas chromatography coupled to mass spectrometry (GC-MS). Yields were similar to those previously reported for flavonoid monomers. These results provide the first evidence of degradation of dietary phenolic polymers into low-molecular-weight aromatic compounds. To understand the nutritional properties of proanthocyanidins, it is therefore essential to consider the biological properties of these metabolites.

Variations in digestive physiology of rats after short duration flights aboard the US space shuttle.

The purpose of this work was to assess the influence of microgravity on several endogenous and microbial parameters of digestive physiology. On the occasion of two Spacelab Life Sciences missions, SLS-1 (a 9-day space flight) and SLS-2 (a 14-day space flight), Sprague-Dawley rats flown aboard the US space shuttle were compared to age-matched ground-based controls. In both flights, exposure to microgravity modified cecal fermentation: concentration and profile of short-chain fatty acids were altered, whereas urea and ammonia remained unchanged. Only in SLS-1 was there an induction of intestinal glutathione-S-transferase. Additional analyses in SLS-2 showed a decrease of hepatic CYP450 and of colonic goblet cells containing neutral mucin. After a postflight recovery period equal to the mission length, only modifications of the hepatic and intestinal xenobiotic metabolizing enzymes still persisted. These findings should help to predict the alterations of digestive physiology and detoxification potential likely to occur in astronauts. Their possible influence on health is discussed.

Proanthocyanidins and human health: systemic effects and local effects in the gut.

Proanthocyanidins share common properties with other polyphenols, in particular their reducing capacity and ability to chelate metal ions. However, their polymeric nature clearly makes them different. They have a high affinity for proteins and their absorption through the gut barrier is likely limited to the molecules of low polymerization degree and to the metabolites formed by the colonic microflora, as suggested by in vitro experiments. The nutritional significance of proanthocyanidins is discussed in relation to their physico-chemical properties and bioavailability.

Development of test systems for the detection of compounds that prevent the genotoxic effects of heterocyclic aromatic amines: preliminary results with constituents of cruciferous vegetables and other dietary constituents.

Over the past decades, strong efforts have been made to identify dietary constituents that protect against the genotoxic effects of heterocyclic aromatic amines (HAAs). However, most of the methods that have been used, in particular in vitro assays that require the addition of exogenous enzyme homogenates, have only a limited predictive value because important protective mechanisms are not adequately represented and may give misleading results. Therefore, we attempted to develop improved test systems, namely assays, with human hepatoma cells and single-cell gel electrophoresis (SCGE) tests with rats. Genotoxicity tests with human derived Hep G2 cells reflect the genotoxic effects of HAAs better than other in vitro systems. They also enable the detection of protective effects since the human derived hepatoma cells possess phase I and phase II enzymes that are involved in the activation/ detoxification of the amines. The most appropriate endpoint for experiments with Hep G2 cells appears to be micronucleus induction, but protocols for other endpoints are available as well. The second promising model is the SCGE ("comet") assay with rats that was used successfully to measure protective effects of constituents of cruciferous vegetables against 2-amino-3-methylimidazo[4,5-flquinoline (IQ) in the liver and in the colon mucosa. The present study describes the experimental design of the new approaches, as well as results obtained with various dietary constituents.

Effects of the bacterial status of rats on the changes in some liver cytochrome P450 (EC 1.14.14.1) apoproteins consequent to a glucosinolate-rich diet.

The aim of the present work was to investigate the influence of the intestinal microflora on the changes in hepatic cytochrome P450 apoproteins induced by dietary glucosinolates. Ten rats harbouring a conventional digestive microflora were offered either a diet containing 390 g myrosinase-free rapeseed meal/kg (n 5) or a control diet devoid of glucosinolates (n 5). A similar trial was performed using germ-free rats. After 4 weeks of exposure to the dietary regimens, animals were slaughtered and their livers removed for preparation of microsomes and analysis of cytochrome P450 (EC 1.14.14.1). The glucosinolate-rich diet decreased the concentration of total cytochrome P450 in conventional rats only (-34%). The bacterial status did not modify the concentration of apoproteins CYP1A2 and CYP2B1/B2, but greatly decreased the concentration of the male constitutive isoform CYP2C11 (-53 and -45% respectively in conventional and germ-free rats). Germ-free rats fed on the glucosinolate-rich diet had a greater concentration of CYP3A (+139%) and a lower concentration of CYP2E1 (-32%) than their counterparts fed on the control diet. However, these differences were absent in conventional animals. On the whole, the influence of the intestinal microflora on the changes in hepatic cytochrome P450 due to the consumption of cruciferous vegetables is very complex and obviously involves different mechanisms according to the apoprotein.

Effects of chair-restraint on gastrointestinal transit time and colonic fermentation in male rhesus monkey (Macaca mulatta).

The incidence of an 18 day chair-restraint on the digestive physiology of male rhesus monkey was investigated for space research purposes, comparing four trained restraint subjects with two vivarium controls. Chair-restraint induced a 2.5-fold acceleration of the gastrointestinal transit time, which persisted throughout the 7 day postrestraint period, and an increase of the fecal dry matter content, which mean value rose from 40.7% to 69.6%. Fecal pH remained unaltered throughout the experiment. Modifications of fermentative metabolites produced by the colonic microflora and excreted through the breath (hydrogen and methane) or in the feces (short chain fatty acids and ammonia) could not be reliably related to chair-restraint and probably involved side-stress factors. On the whole, alterations due to chair-restraint are shown to be different from those reported in the literature, following a modification of the dietary composition. These data may help to predict the alterations of digestive physiology likely to occur in immobilized human patients.

Modulation of the biological effects of glucosinolates by inulin and oat fibre in gnotobiotic rats inoculated with a human whole faecal flora.

The influence of dietary fibre on the biological effects of glucosinolates was investigated in gnotobiotic rats harbouring a human whole faecal flora. Animals were fed for 6 wk with diets containing 12% rapeseed meal (RSM) supplemented or not supplemented with 10% inulin (INL) or oat fibre. Both fibre types enhanced the liver hypertrophy due to RSM to equal extents, but had different effects on the other glucosinolate-related toxic effects. INL partially restored a normal thyroid hormone status whereas kidney weight, goitre and growth deficit were increased on exposure to the diet containing oat fibre. Oat fibre and, to a lesser extent, INL modulated the alterations of digestive xenobiotic-metabolizing enzymes (XME) induced by RSM. They counter-balanced the induction of hepatic cytochrome P-450 and lessened the induction of uridine diphosphate-glucuronosyltransferase in the liver but did not modify depletion of its activity in the small intestine. On the other hand, they enhanced the induction of glutathione S-transferase in the liver and the large intestine but not in the small intestine. These findings give new evidence that the biological effects of naturally occurring non-nutrient compounds are closely dependent on the composition of the diet. Two mechanisms are proposed to explain the different influence of INL and oat fibre on RSM toxicity. Their different fermentative characteristics could lead to a modulation of the bacterial metabolism of glucosinolates in the caecum. Alternatively, their own action on the digestive XME could modify the subsequent metabolism of bacterial glucosinolate derivatives.

Rape-seed meal toxicity in gnotobiotic rats: influence of a whole human faecal flora or single human strains of Escherichia coli and Bacteroides vulgatus.

Gnotobiotic growing rats harbouring either a whole human faecal flora or single human strains of Escherichia coli (EM0) or Bacteroides vulgatus (BV8H1) were fed for 7 weeks on semi-synthetic diets in which the protein source was either soya-bean meal (SM) or rape-seed meal (RM). For each bacterial status the RM-diet group was compared with the control group fed on the SM diet. The association of human faecal flora with the RM diet was responsible for reduced feed intake and reduced weight gain, an enlargement of the liver and thyroid and a decrease in both thyroxine and triiodothyronine plasma levels. The association of the B. vulgatus BV8H1 strain with the RM diet reproduced all these effects, except that triiodothyronine plasma levels were not significantly modified. Rats inoculated with the E. coli EM0 strain and fed on the RM diet exhibited a goitre and lowered thyroxine and triiodothyronine plasma levels. These results show that the human intestinal microflora may be involved in glucosinolate metabolism when cruciferous vegetables are consumed by man. The specificity of the symptoms observed according to the rat bacterial status supports the hypothesis that bacteria yield specific toxic glucosinolate derivatives according to their enzymic potential.