Safety of Bifidobacterium breve, Bif195, employing a human exercise-induced intestinal permeability model: a randomised, double-blinded, placebo-controlled, parallel group trial.

We have previously shown that the probiotic Bifidobacterium breve strain Bif195 alleviates mucosal injury including ulcer formation in the upper intestine induced by non-steroid anti-inflammatory drugs (NSAIDs). Here, we report additional safety use of Bif195 in 126 healthy humans undergoing an exercise-induced intestinal permeability challenge in a double-blinded, placebo-controlled randomised 6-week intervention trial. Intestinal permeability was assessed by urinary lactulose/rhamnose (L/R) ratio. L/R ratio, plasma intestinal fatty acid binding protein (I-FABP) and gastrointestinal symptom rating scale (GSRS) questionnaire were measured resting and after a 1 h treadmill challenge, prior to and at the end of the intervention. To be able to compare the equivalence of resting state at baseline, of this cohort of well-trained subjects, to non-trained subjects, a cohort of 63 healthy and non-trained subjects (<2 h/week of endurance sports) was included. Study subjects (well-trained) were 35.7% women with a mean age and body mass index (in kg/m2) of 35.0 years and 24.8, respectively. There were no differences between the Bif195 and placebo groups in effects on L/R ratio, I-FABP and GSRS questionnaire score. In addition, there were no differences between Bif195 and placebo in number of adverse events and change in cytokines, liver or kidney biomarkers. The exercise model successfully induced intestinal permeability by statistically significantly increasing L/R ratio by ~100% (P<0.0001) and cytokines after the exercise challenge. No significant difference was found between well-trained and non-trained subjects in baseline resting L/R ratio. In conclusion, the reported cytoprotective effects of Bif195 are unlikely to be primarily related to small bowel permeability, and the safety of Bif195 in individuals with increased permeability is supported by the present data. ClinicalTrials.gov: NCT03027583.

Functional in vitro screening of probiotic strains for inoculation of piglets as a prophylactic measure towards Enterotoxigenic Escherichia coli infection.

Enterotoxigenic Escherichia coli (ETEC), being the major cause of post-weaning diarrhoea (PWD) in newly weaned piglets, induces poor performance and economic losses in pig production. This functional in vitro screening study investigated probiotic strains for use in suckling piglets as a prophylactic strategy towards PWD. Nine strains were evaluated based on their ability to: enhance intestinal epithelial barrier function, reduce adherence of ETEC F18 to intestinal cells, inhibit growth of ETEC F18, and grow on porcine milk oligosaccharides. Strains included in the screening were of the species Lactobacillus, Enterococcus, Bifidobacterium and Bacillus. Our in vitro screening demonstrated genus-, species and strain-specific differences in the mode of action of the tested probiotic strains. Some of the tested bifidobacteria were able to grow on the two porcine milk oligosaccharides, 3'-sialyllactose sodium salt (3'SL) and Lacto-N-neotetraose (LNnT), whereas most lactic acid bacteria strains and both Bacillus subtilis strains failed to do so. All probiotic strains inhibited growth of ETEC F18 on agar plates. All but the bifidobacteria reduced binding of ETEC F18 to Caco-2 cell monolayers, with the Enterococcus faecium strain having the most profound effect. All three lactic acid bacteria and Bifidobacterium animalis subsp. lactis counteracted the ETEC F18-induced permeability across Caco-2 cell monolayers with the E. faecium strain exhibiting the most pronounced protective effect. The findings from this in vitro screening study indicate that, when selecting probiotic strains for suckling piglets as a prophylactic strategy towards PWD, it would be advantageous to choose a multi-species product including strains with different modes of action in order to increase the likelihood of achieving beneficial effects in vivo.

Four weeks supplementation with Lactobacillus paracasei subsp. paracasei L. casei W8® shows modest effect on triacylglycerol in young healthy adults.

The microbiota has been shown to have the potential to affect appetite and blood lipids positively in animal studies. We investigated if four weeks supplementation with Lactobacillus paracasei subsp. paracasei L. casei W8® (L. casei W8) had an effect on subjective appetite sensation, ad libitum energy intake, glucagon-like peptide 1 (GLP-1), glucose and insulin response in humans. Secondarily, we explored potential effects on blood lipids, fatty acids and stearoyl-CoA desaturase-1 (SCD1) activity in humans as well as SCD1 expression in piglets given L. casei W8 for two weeks. 64 healthy participants completed the double-blinded, randomised, controlled, parallel four weeks study with supplementation of L. casei W8 (1010 cfu) or placebo capsules. A meal test was conducted before and after the intervention, where subjective appetite, ad libitum energy intake, GLP-1, glucose and insulin response were measured. Additionally fasting blood lipids and fatty acids concentrations were measured. Sixteen piglets were randomised into two groups: L. casei W8 (1010 cfu/day) as top dressing on morning fed or no treatment. After two weeks piglets were sacrificed and tissue from ileum, jejunum and skeletal muscle were sampled for mRNA analyses of SCD1 expression. Compared to placebo, L. casei W8 did not affect appetite, ad libitum energy intake, GLP-1, glucose and insulin response and total, high-density or low-density lipoprotein cholesterol levels after four weeks intervention. Triacylglycerol decreased in the L. casei W8 group compared to placebo at week 4 (P=0.03). The C16:1n-7/C16:0 ratio, reflecting SCD1 activity, tended to decrease when having L. casei W8 (P=0.06) compared to placebo. Muscle SCD1 expression decreased in piglets supplemented with L. casei W8 compared to control. In conclusion, supplementation with L. casei W8 did not affect appetite parameters, glucose or insulin responses; but appear to be able to lower triacylglycerol levels, possibly by reducing its production.

2-(Cyclohexylamino)-1-(4-cyclopentylpiperazin-1-yl)-2-methylpropan-1-one, a novel compound with neuroprotective and neurotrophic effects in vitro.

Focusing on development of novel drug candidates for the treatment of neurodegenerative diseases, we developed and synthesized a new compound, 2-(cyclohexylamino)-1-(4-cyclopentylpiperazin-1-yl)-2-methylpropan-1-one (amido-piperizine 1). The compound demonstrated robust neuroprotective properties after both glutamate excitotoxicity and peroxide induced oxidative stress in primary cortical cultures. Furthermore, amido-piperizine 1 was found to significantly induce neurite outgrowth in vitro which could suggest central reparative and regenerative potential of the compound. With these potential beneficial effects in CNS, the ability of the amido-piperizine 1 to penetrate the blood-brain barrier was tested using MDR1-MDCK cells. Amido-piperizine 1 was found not to be a P-gp substrate and to have a high blood-brain barrier penetration potential, indicating excellent availability to the CNS. Moreover, amido-piperizine 1 had a fast metabolic clearance rate in vitro, suggesting that parenteral in vivo administration seems preferable. As an attempt to elucidate a possible mechanism of action, we found that amido-piperizine 1 bound in nano-molar range to the sigma-1 receptor, which could explain the observed neuroprotective and neurotrophic properties, and with a 100-fold lower affinity to the sigma-2 receptor. These results propose that amido-piperizine 1 may hold promise as a drug candidate for the treatment of stroke/traumatic brain injury or other neurodegenerative diseases.

Oxidative DNA damage in male Wistar rats exposed to di-n-butyl phthalate.

Dialkyl phthalate esters are used in the plastic industry and widely distributed in the environment. Previously, it has been shown that di-n-butyl phthalate (DBP) produces testicular atrophy and liver enlargement in rodents, and the mechanisms behind this could involve reactive oxygen species (ROS). In this study, oxidative DNA damage was measured in terms of the premutagenic modified nucleoside 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) in nuclear DNA from liver, kidneys, and testes from rats exposed to DBP in the perinatal or preadult period. In one experiment, pregnant rats were administered 0 or 0.5 g DBP/kg/d by gavage from d 7 after conception to d 17 after delivery and organs from male offspring were analyzed. In a second experiment, 25-d-old rats were administered 0, 0.5, or 2 g DBP/kg/d by gavage for 10 d. After perinatal exposure, body and organ weights were unchanged. The 8-oxodG/10(6) dG ratio in liver DNA increased significantly in the exposed group. In contrast, the 8-oxodG/10(6) dG ratio was significantly decreased in kidney DNA, whereas it remained unchanged in the testis. After preadult exposure (postnatal d 25 to 34) the testes weight of the exposed animals were significantly decreased and severe atrophy of the seminiferous tubules was observed. The body weight of the animals in the high-dose group was significantly decreased compared to the control. The 8-oxodG levels in liver, kidney, and testis DNA remained unchanged. Although ROS has been suspected of being involved in the formation of testicular atrophy in phthalate-exposed rats, no apparent sign of oxidative DNA damage was found after phthalate exposure perinatally or during the preadult stage. With respect to phthalate-induced oxidative DNA damage in the liver, it appears that the developmental stage during exposure is important.

Iron-induced oxidative DNA damage in rat sperm cells in vivo and in vitro.

We investigated whether acute iron intoxication causes oxidative DNA damage, measured in terms of 7-hydro-8-oxo-2'-deoxyguanosine, 8-oxodG, in nuclear DNA in testes and epididymal sperm cells in vivo and in vitro in rats. In addition, we investigated levels of the modified nucleoside in liver and kidney and measured its urinary excretion. Sperm cells were isolated from the epididymides and the testes cells were isolated after homogenisation. In vitro, the sperm and testes cells were incubated with increasing concentrations of FeCl2 ranging from 0 to 600 microM. The median (range) levels of 8-oxodG/10(5) dG in the epididymal sperm cells increased from 0.48 (0.42-0.90) to 15.1 (11.4-17.6) (p < 0.05), whereas the level rose from 0.63 (0.22-0.81) to 8.8 (4.5-11.6) (p < 0.05) at 0 and 600 microM, respectively, in the testicular cells. In vivo groups of 7-8 rats received 0, 200 or 400 mg iron/kg as dextran i.p. After 24 h, epididymal sperm cells, testes, kidneys and liver were collected for analysis. Kidney and sperm DNA showed a significant increase in 8-oxodG in the iron-treated animals. The median (range) values of the 8-oxodG/10(5) dG in the epididymal sperm cells rose from 0.66 (0.38-1.09) to 1.12 (0.84-5.88) (p < 0.05) at 0 and 400 mg iron/kg, respectively, whereas the values in the testes and liver showed no significant change. In the kidneys the 8-oxodG/10(5) dG median (range) values were 0.98 (0.73-1.24), 1.21 (1.13-1.69) and 1.34 (1.12-1.66) after 0, 200 and 400 mg iron/kg, respectively (p < 0.05). The 8-oxodG-excretion rate was measured in 24h urine before and after iron treatment. The rate of urinary 8-oxodG excretion increased from 129 (104-179) pmol/24 h before treatment to 147 (110-239) pmol/24 h after treatment in the group receiving 400 mg iron/kg (p < 0.05). The results indicate that acute iron intoxication may increase oxidative damage to sperm and kidney DNA.

Experimental study of oxidative DNA damage.

Animal experiments allow the study of oxidative DNA damage in target organs and the elucidation of dose-response relationships of carcinogenic and other harmful chemicals and conditions as well as the study of interactions of several factors. So far the effects of more than 50 different chemical compounds have been studied in animal experiments mainly in rats and mice, and generally with measurement of 8-oxodG with HPLC-EC. A large number of well-known carcinogens induce 8-oxodG formation in liver and/or kidneys. Moreover several animal studies have shown a close relationship between induction of dative DNA damage and tumour formation. In principle the level of oxidative DNA damage in an organ or cell may be studied by measurement of modified bases in extracted DNA by immunohistochemical visualisation, and from assays of strand breakage before and after treatment with repair enzymes. However, this level is a balance between the rates of damage and repair. Until the repair rates and capacity can be adequately assessed the rate of damage can only be estimated from the urinary excretion of repair products albeit only as an average of the entire body. A number of model compounds have been used to induce oxidative DNA damage in experimental animals. The hepatocarcinogen 2-nitropropane induces up to 10-fold increases in 8-oxodG levels in rat liver DNA. The level of 8-oxodG is also increased in kidneys and bone marrow but not in the testis. By means of 2-nitropropane we have shown correspondence between the increases in 8-oxodG in target organs and the urinary excretion of 8-oxodG and between 8-oxodG formation and the comet assay in bone marrow as well potent preventive effects of extracts of Brussels sprouts. Others have shown similar effects of green tea extracts and its components. Drawbacks of the use of 2-nitropropane as a model for oxidative DNA damage relate particularly to formation of 8-aminoguanine derivatives that may interfere with HPLC-EC assays and have unknown consequences. Other model compounds for induction of oxidative DNA damage, such as ferric nitriloacetate, iron dextran, potassium bromate and paraquat, are less potent and/or more organ specific. Inflammation and activation of an inflammatory response by phorbol esters or E. coli lipopolysaccharide (LPS) induce oxidative DNA damage in many target cells and enhance benzene-induced DNA damage in mouse bone marrow. Experimental studies provide powerful tools to investigate agents inducing and preventing oxidative damage to DNA and its role in carcinogenesis. So far, most animal experiments have concerned 8-oxodG and determination of additional damaged bases should be employed. An ideal animal model for prevention of oxidative DNA damage has yet to he developed.