Daily supplementation with the Lab4P probiotic consortium induces significant weight loss in overweight adults.

This 9-month randomised, parallel, double-blind, single-centre, placebo-controlled study (PROBE, ISRCTN18030882) assessed the impact of probiotic supplementation on bodyweight. Seventy overweight Bulgarian participants aged 45-65 years with BMI 25-29.9 kg/m2 received a daily dose of the Lab4P probiotic comprising lactobacilli and bifidobacteria (50 billion cfu/day). Participants maintained their normal diet and lifestyle over the duration of the study. The primary outcome was change from baseline in body weight and secondary outcomes included changes in waist circumference, hip circumference and blood pressure. A significant between group decrease in body weight (3.16 kg, 95% CI 3.94, 2.38, p < 0.0001) was detected favouring the probiotic group. Supplementation also resulted in significant between group decreases in waist circumference (2.58 cm, 95% CI 3.23, 1.94, p < 0.0001) and hip circumference (2.66 cm, 95% CI 3.28, 2.05, p < 0.0001) but no changes in blood pressure were observed. These findings support the outcomes of a previous shorter-term Lab4P intervention study in overweight and obese participants (PROMAGEN, ISRCTN12562026). We conclude that Lab4P has consistent weight modulation capability in free-living overweight adults.

A randomised controlled study shows supplementation of overweight and obese adults with lactobacilli and bifidobacteria reduces bodyweight and improves well-being.

In an exploratory, block-randomised, parallel, double-blind, single-centre, placebo-controlled superiority study (ISRCTN12562026, funded by Cultech Ltd), 220 Bulgarian participants (30 to 65 years old) with BMI 25-34.9 kg/m2 received Lab4P probiotic (50 billion/day) or a matched placebo for 6 months. Participants maintained their normal diet and lifestyle. Primary outcomes were changes in body weight, BMI, waist circumference (WC), waist-to-height ratio (WtHR), blood pressure and plasma lipids. Secondary outcomes were changes in plasma C-reactive protein (CRP), the diversity of the faecal microbiota, quality of life (QoL) assessments and the incidence of upper respiratory tract infection (URTI). Significant between group decreases in body weight (1.3 kg, p < 0.0001), BMI (0.045 kg/m2, p < 0.0001), WC (0.94 cm, p < 0.0001) and WtHR (0.006, p < 0.0001) were in favour of the probiotic. Stratification identified greater body weight reductions in overweight subjects (1.88%, p < 0.0001) and in females (1.62%, p = 0.0005). Greatest weight losses were among probiotic hypercholesterolaemic participants (-2.5%, p < 0.0001) alongside a significant between group reduction in small dense LDL-cholesterol (0.2 mmol/L, p = 0.0241). Improvements in QoL and the incidence rate ratio of URTI (0.60, p < 0.0001) were recorded for the probiotic group. No adverse events were recorded. Six months supplementation with Lab4P probiotic resulted in significant weight reduction and improved small dense low-density lipoprotein-cholesterol (sdLDL-C) profiles, QoL and URTI incidence outcomes in overweight/obese individuals.

In vitro neuroprotective activities of two distinct probiotic consortia.

Neurodegeneration has been linked to changes in the gut microbiota and this study compares the neuroprotective capability of two bacterial consortia, known as Lab4 and Lab4b, using the established SH-SY5Y neuronal cell model. Firstly, varying total antioxidant capacities (TAC) were identified in the intact cells from each consortia and their secreted metabolites, referred to as conditioned media (CM). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Crystal Violet (CV) assays of cell viability revealed that Lab4 CM and Lab4b CM could induce similar levels of proliferation in SH-SY5Y cells and, despite divergent TAC, possessed a comparable ability to protect undifferentiated and retinoic acid-differentiated cells from the cytotoxic actions of rotenone and undifferentiated cells from the cytotoxic actions of 1-methyl-4-phenylpyridinium iodide (MPP+). Lab4 CM and Lab4b CM also had the ability to attenuate rotenone-induced apoptosis and necrosis with Lab4b inducing the greater effect. Both consortia showed an analogous ability to attenuate intracellular reactive oxygen species accumulation in SH-SY5Y cells although the differential upregulation of genes encoding glutathione reductase and superoxide dismutase by Lab4 CM and Lab4b CM, respectively, implicates the involvement of consortia-specific antioxidative mechanisms of action. This study implicates Lab4 and Lab4b as potential neuroprotective agents and justifies their inclusion in further in vivo studies.

The anti-cholesterolaemic effect of a consortium of probiotics: An acute study in C57BL/6J mice.

Hypercholesterolaemia is a major risk factor for cardiovascular disease and it has been found that some probiotic bacteria possess cholesterol-lowering capabilities. In this study, the ability of the Lab4 probiotic consortium to hydrolyse bile salts, assimilate cholesterol and regulate cholesterol transport by polarised Caco-2 enterocytes was demonstrated. Furthermore, in wild-type C57BL/6J mice fed a high fat diet, 2-weeks supplementation with Lab4 probiotic consortium plus Lactobacillus plantarum CUL66 resulted in significant reductions in plasma total cholesterol levels and suppression of diet-induced weight gain. No changes in plasma levels of very low-density lipoprotein/low-density lipoprotein, high-density lipoprotein, triglycerides, cytokines or bile acids were observed. Increased amounts of total and unconjugated bile acids in the faeces of the probiotic-fed mice, together with modulation of hepatic small heterodimer partner and cholesterol-7α-hydroxylase mRNA expression, implicates bile salt hydrolase activity as a potential mechanism of action. In summary, this study demonstrates the cholesterol-lowering efficacy of short-term feeding of the Lab4 probiotic consortium plus L. plantarum CUL66 in wild-type mice and supports further assessment in human trials.

The vascular Ca2+-sensing receptor regulates blood vessel tone and blood pressure.

The extracellular calcium-sensing receptor CaSR is expressed in blood vessels where its role is not completely understood. In this study, we tested the hypothesis that the CaSR expressed in vascular smooth muscle cells (VSMC) is directly involved in regulation of blood pressure and blood vessel tone. Mice with targeted CaSR gene ablation from vascular smooth muscle cells (VSMC) were generated by breeding exon 7 LoxP-CaSR mice with animals in which Cre recombinase is driven by a SM22α promoter (SM22α-Cre). Wire myography performed on Cre-negative [wild-type (WT)] and Cre-positive (SM22α)CaSR(Δflox/Δflox) [knockout (KO)] mice showed an endothelium-independent reduction in aorta and mesenteric artery contractility of KO compared with WT mice in response to KCl and to phenylephrine. Increasing extracellular calcium ion (Ca(2+)) concentrations (1-5 mM) evoked contraction in WT but only relaxation in KO aortas. Accordingly, diastolic and mean arterial blood pressures of KO animals were significantly reduced compared with WT, as measured by both tail cuff and radiotelemetry. This hypotension was mostly pronounced during the animals' active phase and was not rescued by either nitric oxide-synthase inhibition with nitro-l-arginine methyl ester or by a high-salt-supplemented diet. KO animals also exhibited cardiac remodeling, bradycardia, and reduced spontaneous activity in isolated hearts and cardiomyocyte-like cells. Our findings demonstrate a role for CaSR in the cardiovascular system and suggest that physiologically relevant changes in extracellular Ca(2+) concentrations could contribute to setting blood vessel tone levels and heart rate by directly acting on the cardiovascular CaSR.

Young women with polycystic ovary syndrome have raised levels of circulating annexin V-positive platelet microparticles.

STUDY QUESTION: Are circulating microparticles (MPs) altered in young women with polycystic ovary syndrome (PCOS)? SUMMARY ANSWER: Women with PCOS have elevated concentrations of circulating platelet-derived MPs, which exhibit increased annexin V binding and altered microRNA (miR) profiles compared with healthy volunteers. WHAT IS KNOWN ALREADY: Some studies have shown that cardiovascular risk is increased in young women with PCOS but the mechanisms by which this occurs are uncertain. Circulating MPs are elevated in patients with cardiovascular disease but the characteristics of MPs in patients with PCOS are unclear. STUDY DESIGN, SIZE, DURATION: Case-control study comprising 17 women with PCOS (mean ± SD; age 31 ± 7 years, BMI 29 ± 6 kg/m(2)) and 18 healthy volunteers (age 31 ± 6 years, BMI 30 ± 6 kg/m(2)). PARTICIPANTS/MATERIALS, SETTING, METHODS: The study was conducted in a University hospital. Nanoparticle tracking analysis (NTA) and flow cytometry (CD41 platelet, CD11b monocyte, CD144 endothelial) were used to determine MP size, concentration, cellular origin and annexin V positivity (reflecting phosphatidylserine exposure). Fatty acid analysis was performed by gas chromatography and MP miR expression profiles were compared by microarray. MAIN RESULTS AND THE ROLE OF CHANCE: PCOS subjects showed increased MP concentrations compared with healthy volunteers (mean ± SD; 11.5 ± 5 × 10(12)/ml versus 10.0 ± 4 × 10(12)/ml, respectively; P = 0.03), which correlated with the homeostasis model of insulin resistance (r = 0.53, P = 0.03). This difference was predominantly seen in MPs whose size was in the small exosomal range (<150 nm in diameter, P< 0.05). PCOS patients showed a greater percentage of annexin V(+) MPs compared with healthy volunteers (84 ± 18 versus 74 ± 24%, respectively, P = 0.05) but the cellular origin of MPs, which were predominantly platelet-derived (PCOS: 99 ± 0.9%; controls: 99 ± 2.5%), did not differ. MP fatty acid concentration and composition was similar between groups but 16 miRs were differentially expressed (P < 0.05). LIMITATIONS, REASON FOR CAUTION: Patients with PCOS were classified by the Rotterdam criteria, which describes a less severe metabolic phenotype than other definitions of the syndrome. Our findings may thus not be generalizable to all patients with PCOS. MicroRNA expression analysis was only undertaken in an exploratory subset of the overall study population hence, validation of our findings in a larger cohort is mandatory. Furthermore, miR levels were unaltered for the highly expressed miRs and it is unclear whether differences in the lowly expressed miRs carries pathological relevance. WIDER IMPLICATIONS OF THE FINDINGS: This study suggests that women with PCOS have an altered MP profile but further studies are needed to confirm this, to explore the mechanisms by which these alterations develop and to establish whether therapies that improve insulin sensitivity are able to reduce circulating MP concentrations. STUDY FUNDING/COMPETING INTERESTS: The study was funded by grants from the Wales Heart Research Institute and Mrs John Nixon Scholarship. The authors have no conflicts of interest to declare.

Rodent carcinogenicity tests need be no longer than 18 months: an analysis based on 210 chemicals in the IARC monographs.

The IARC Monographs (Vols 1-70) were studied to determine the time of onset of treatment-related tumorigenicity in long-term rodent studies for chemicals classified by IARC as having sufficient evidence of carcinogenicity in animals. The analysis excluded studies on metals and their salts, studies on particulates, studies by parenteral routes of administration that resulted in tumours only at the site of exposure, and studies that did not approximate to the current standard long-term rodent carcinogenicity bioassay, for instance transplacental or multigeneration studies, initiator-promoter studies, lung tumour assays in Strain A mice and studies in newborn animals. Data from a total of 210 chemicals revealed that, overall, evidence of treatment-related tumorigenicity was first apparent within 12 months for 66% of the chemicals and for only 7% were studies of longer than 18 months necessary. All IARC Group 1 chemicals were detected in animals within 18 months, and most within 12 months. Most of the tumour types that required more than 18 months for detection were of dubious relevance to human risk assessment. Termination of rodent carcinogenicity studies at 18 months or earlier would greatly reduce the complications that arise in interpreting the findings in aged animals which often have defective hepatic or renal function and would also markedly reduce the time required for histopathological examination of dozens of tissues taken from the approximately 500 animals routinely employed in these studies.

Preclinical toxicological evaluation of sertraline hydrochloride.

The toxicity profile of the antidepressant drug sertraline was determined in a series of preclinical studies in mice, rats, rabbits and dogs. Acute, subchronic, reproductive, chronic and carcinogenicity studies were conducted by the oral route. The highest doses tested in these studies were the maximum tolerated doses based on clinical signs, decreased food consumption, body weight effects, organ weight changes or clinical/anatomical pathology findings. Genetic toxicity studies were also performed. The liver was identified as a target organ in the mouse, rat and dog. The observed liver findings were consistent with hepatic xenobiotic-metabolizing enzyme induction and included hepatomegaly, hepatocellular hypertrophy, slightly increased serum transaminase activity and proliferation of smooth endoplasmic reticulum. Hepatocellular fatty change, a minimal toxic effect, was seen in mice and rats. There was no teratogenicity in studies conducted at maternally toxic doses in rats and rabbits. Decreased neonatal survival and growth observed in these studies have been previously reported in reproduction studies with other serotonin reuptake inhibitors. Sertraline was not genotoxic in an extensive battery of tests. Carcinogenicity tests were negative in rats, while benign liver tumors were slightly increased in drugtreated male mice. Liver tumors were considered secondary to the enzyme inducing potential of sertraline and not indicative of human risk.

Preclinical toxicological evaluation of sertraline hydrochloride.

The toxicity profile of the antidepressant drug sertraline was determined in a series of preclinical studies in mice, rats, rabbits and dogs. Acute, subchronic, reproductive, chronic and carcinogenicity studies were conducted by the oral route. The highest doses tested in these studies were the maximum tolerated doses based on clinical signs, decreased food consumption, body weight effects, organ weight changes or clinical/anatomical pathology findings. Genetic toxicity studies were also performed. The liver was identified as a target organ in the mouse, rat and dog. The observed liver findings were consistent with hepatic xenobiotic-metabolizing enzyme induction and included hepatomegaly, hepatocellular hypertrophy, slightly increased serum transaminase activity and proliferation of smooth endoplasmic reticulum. Hepatocellular fatty change, a minimal toxic effect, was seen in mice and rats. There was no teratogenicity in studies conducted at maternally toxic doses in rats and rabbits. Decreased neonatal survival and growth observed in these studies have been previously reported in reproduction studies with serotonin reuptake inhibitors. Sertraline was not genotoxic in an extensive battery of tests. Carcinogenicity tests were negative in rats, while benign liver tumors were slightly increased in drug-treated male mice. Liver tumors were considered secondary to the enzyme inducing potential of sertraline and not indicative of human risk.

The case for an upper dose limit of 1000 mg/kg in rodent carcinogenicity tests.

We examined two rodent carcinogenicity data bases comprising 301 chemicals from the US National Toxicology Program (NTP) and 241 pharmaceuticals from the US Physicians' Desk Reference (PDR) to determine the nature of the tumors produced at dose levels > 1000 mg/kg (or equivalent dietary concentrations). Ten chemicals increased tumors only at dose levels greater than 1000 mg/kg. For six of these, the lowest dose tested was > 1000 mg/kg, so they may be active at dose levels below 1000 mg/kg. One chemical was active in rats and mice, 2 in rats only and 7 in mice only. Four of the chemicals were mutagenic to Salmonella. The tumor types produced by the other six putatively non-genotoxic chemicals suggest that a high dose limit of 1000 mg/kg is appropriate for rodent bioassays. Overtly genotoxic chemicals are no longer routinely subjected to bioassays.

The rodent carcinogenicity bioassay produces a similar frequency of tumor increases and decreases: implications for risk assessment.

We examined the overall results of 124 consecutive rodent carcinogenesis assays carried out at the maximum tolerated dose on 37 chemicals reported recently by the Toxicology Program of the United States. In 31 experiments each in male and female F-344 rats and in male and female B6C3F1 mice, tumor increases and decreases occurred in 41 and 46% of the experiments, respectively. In 22 experiments both increases and decreases in tumor incidence were reported. Of the experiments with decreases in tumor incidence, about 70% were associated with lower body weights of the treated animals. However, of the 30 chemicals producing some tumor decreases, 12 showed decreases in some experiments without any association with bodyweight. Ten chemicals that were Salmonella positive produced increases and decreases in tumor incidences and three produced only decreases in tumor incidence. If it is considered that the bioassay provides information relevant to the carcinogenic potential of a chemical, then logically it must also be considered that information about the cancer-preventive potential of a chemical is provided. When a chemical causes increases and decreases in tumors, several questions follow. First, which are more relevant to the health of an exposed individual: tumor increases or tumor decreases? Second, should such a chemical be stigmatized as a "carcinogen," in view of all the legal and economic implications that ensue from such a label? Third, how should one define a carcinogen?

High dose levels are not necessary in rodent studies to detect human carcinogens.

Guidelines for the conduct of rodent carcinogenicity studies stipulate that when the test substance is administered via the diet, its concentration need not exceed 5% of the diet. Since it is now apparent that human carcinogens are amongst the most potent of rodent carcinogens, it should be possible to detect accurately potential human carcinogens by using only relatively low dose levels in rodent studies. Our analysis of the potency of human carcinogens in rodent studies leads to the conclusion that, even after applying a safety factor of 10, there is no purpose in using dose levels higher than 500 mg/kg body weight or 1% in the diet.

Dose-response studies with nitrosoheptamethyleneimine and its alpha-deuterium-labeled derivative in F344 rats.

A dose-response study of the carcinogenicity of nitrosoheptamethyleneimine (N-HEP) in inbred F344 male and female rats was performed by administration of the nitrosamine at several concentrations in drinking water to groups of 20 rats. The concentrations differed by a factor of nearly 2.5 and ranged from 1.0 to 100 mg/liter. The duration of treatment was 13, 25, 50, or 100 weeks, after which the animals were allowed to die naturally of tumors induced. In most treated groups the incidence of tumors of the upper gastrointestinal tract approached 100%. However, at the higher doses there was an inverse relationship between total dose and survival time of the rats. Matched treatment of groups of rat with N-HEP labeled with deuterium in the alpha positions resulted in longer survival. The slower action of the deuterium-labeled compound, as measured by a lower rate of death from tumors, suggests that cleavage of a carbon-hydrogen bond at an alpha position is a rate-limiting step in carcinogenesis by N-HEP in rats.

Dose-response studies in carcinogenesis by nitroso-N-methyl-N-(2-phenyl)ethylamine in rats and the effects of deuterium substitution.

A dose-response study of the carcinogenicity of nitroso-N-methyl-N-(2-phenyl)ethylamine was carried out in male Fischer 344 rats. The compound was given in drinking water at concentrations of 115, 28, 9.5, 3.2, 1.1 and 0.4 mg/litre. The highest concentration proved toxic leading to the early death of several animals; the remainder of this group were treated for 21 wk. All of the other concentrations were given for 33 wk, except the 28 mg/litre treatment which ceased at 30 wk. An additional group of rats was given 0.4 mg/litre for 104 wk. In all groups of animals, except those in the high-dose group that died early and those given 0.4 mg/litre for 33 wk, 50% or more of the animals had tumours of the oesophagus or forestomach or both when they died. In several groups the number of rats with these tumours approached 100%. The total dose of carcinogen received by the rats in the lowest dose group was 1.3 mg and 45% of them had tumours of the upper gastro-intestinal tract. The effect on carcinogenicity of labelling the nitrosamine with deuterium in either the methyl group or the alpha-methylene of the phenylethyl group was determined by treating groups of rats with equimolar concentrations of the deuterium-labelled and unlabelled nitrosamine. A very significant increase in carcinogenic effectiveness was observed with the compound containing deuterium in the alpha-methylene of the phenylethyl group, suggesting that methylation might not be the important event in carcinogenesis by this compound in rats.

Pathology of subacute methylmercurialism in cats.

Clinical signs of toxicosis, neurologic lesions, and increased tissue residues of methylmercury (MM)were produced in 9 cats by oral administration of 1.29 and 0.86 mg of Hg/kg of body weight/day as methylmercuric hydroxide. Clinical signs, which began after 15 days of exposure, included anorexia, ataxia, hypermetria, proprioceptive impairment, blindness, vertical nystagmus, and grand mal convulsions. Significant lesions occurred in cerebrum, brainstem, and cerebellum and correlated well with clinical signs. Microscopic central nervous system lesions consisted of neuronal degeneration, necrosis and loss of neurons, swollen axons, demyelination, loss of nerve cell processes, vacuolation of neuropil, gliosis, neuronophagia, perivascular cuffs, endothelial hypertrophy and hyperplasia, leptomeningitis, and infrequent vascular necrosis. Overall distribution of central nervous system lesions was unrelated to daily dose, but more advanced lesions were produced by the smaller daily dose. Mean tissue residues of MM were generally directly related to daily dose, and the average distribution among tissues was constant, with highest concentrations in liver, followed by kidney, spleen, muscle, and brain. In utero exposure of kittens to MM, revealed transplacental accumulation.

The pathology of subacute methylmerculialism in swine.

Clinical signs of toxicosis, neurologic lesions, and elevated tissue residues of methylmercury (MM) were produced in 12 pigs by oral administration of 1.29, 0.86, 0.64, and 0.43 mg mercury/kg of body weight daily as methylmercuric hydroxide (MMH). Clinical signs which began on day 17 were ataxia, dysmetria, blindness, convulsions, paresis, and death. Time of onset of signs was inversely related to size of daily dose. Microscopic lesions were found in the cerebrum brain stem, and spinal cord, and correlated well with clinical signs. The cerebrum in which severity of lesions was directly related to length of exposure was the most severely affected region of the central nervous system (CNS). Lesions were neuronal necrosis, neuronophagia, cortical vacuolation, axon swelling, gliosis, leptomeningitis, and vascular fibrinoid necrosis. Neuronal necrosis was most extensive within mid and deep cerebrocortical laminae. Brain residues of MM were directly proportional to the size of daily dose, and statistically significant. Distribution of MM among different tissues was rather uniform with highest concentrations found in liver, followed by kidney, muscle, spleen, and brain.