Safety assessment of the dietary supplement OxyELITE™ Pro (New Formula) in inbred and outbred mouse strains.

Herbal dietary supplements have gained wide acceptance as alternatives to conventional therapeutic agents despite concerns regarding their efficacy and safety. In 2013, a spate of severe liver injuries across the United States was linked to the dietary supplement OxyELITE Pro-New Formula (OEP-NF), a multi-ingredient product marketed for weight loss and exercise performance enhancement. The principal goal of this study was to assess the hepatotoxic potential of OEP-NF in outbred and inbred mouse models. In an acute toxicity study, significant mortality was observed after administering 10X and 3X mouse-equivalent doses (MED) of OEP-NF, respectively. Increases in liver/body weight ratio, ALT and AST were observed in female B6C3F1 mice after gavaging 2X and 1.5X MED of OEP-NF. Similar findings were observed in a 90-day feeding study. These alterations were paralleled by altered expression of gene- and microRNA-signatures of hepatotoxicity, including Cd36, Nqo1, Aldoa, Txnrd1, Scd1 and Ccng1, as well as miR-192, miR-193a and miR-125b and were most pronounced in female B6C3F1 mice. Body weight loss, observed at week 1, was followed by weight gain throughout the feeding studies. These findings bolster safety and efficacy concerns for OEP-NF, and argue strongly for implementation of pre-market toxicity studies within the dietary supplement industry.

EPR spectroscopy solutions for assessment of decellularization of intrathoracic organs and tissues.

Using EPR spectroscopy it was established that the determination of the concentration of paramagnetic centers in lyophilized tissues allows indirect evaluation of the quality of decellularization of intrathoracic organs (diaphragm, heart, and lungs), since the content of paramagnetic particles in them can serve as a criterion of cell viability and points to the necessity to repeat decellularization. Experiments in rats showed that the EPR spectra of the native thoracic organs contained paramagnetic centers with g-factor values ranging from 2.007 to 2.011 at a concentration of 10(-8) to 6.62 × 10(-7) mol/g of lyophilized tissue, whereas in all decellularized tissues of the same organs paramagnetic particles were not detected.

Behavioral differences of male Wistar rats from different vendors in vulnerability and resilience to chronic mild stress are reflected in epigenetic regulation and expression of p11.

Outbred rat lines such as Wistar rats are commonly used for models of depressive disorders. Such rats arise from random mating schedules. Hence, genetic drift occurs in outbred populations which could lead to genotypic and phenotypic heterogeneity between rats from different vendors. Additionally, vendor specific rearing conditions could contribute to intrastrain variability. In the present study differences in behavioral responses to the chronic mild stress (CMS) model of depression within Wistar rat strains from different vendors are described. DNA methylation studies and mRNA expression analysis of p11 revealed that the behavioral differences between the substrains are reflected at the epigenetic and genetic level. The results suggest that there are breeder-dependent differences in vulnerability to stress in the CMS model of depression, which might bear on the validity of the model and contribute to contradictory findings and difficulties of replication between laboratories. P11 mRNA expression seems to be differently regulated depending on the quality of the stress response evoked by CMS exposure.

Isolation of the sapogenin from defatted seeds of Camellia oleifera and its neuroprotective effects on dopaminergic neurons.

Sasanqua saponin is a major active compound in the defatted seeds of Camellia oleifera but is always discarded without effective utilization. The sapogenin from hydrolysis of sasanqua saponin was purified, and its amination derivative was investigated on its neuroprotective effects, which were evaluated by animal models of Parkinson disease in mice induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The results showed that the sapogenin and its derivative increased dopamine content in striatum and tyrosine hydroxylase (TH) positive cells in substantia nigra and relieved inflammation and behavioral disorder, but the effect on movement was reversed by dopamine receptor antagonist haloperidol and was not intervened by adenosine receptor antagonist CGS 15943. Molecular simulation showed the interaction between dopamine receptor and the sapogenin or its derivative. It is proven that the sapogenin can protect dopamine neurons through antineuroinflammation and activation of dopamine receptor rather than adenosine receptor, and its amination improves the effects. This research provides the prospective prodrugs for Parkinson disease and a new medicinal application of sasanqua saponin.

Effects of olive polyphenols administration on nerve growth factor and brain-derived neurotrophic factor in the mouse brain.

OBJECTIVE: Polyphenols are chemicals derived from plants known to possess antioxidant and anti-inflammatory properties. High intake of fruit and vegetables is believed to be beneficial to human health. Various studies have suggested that dietary polyphenols may protect against cancer and cardiometabolic and neurodegenerative diseases. Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are neurotrophins that play key roles in brain cell development, growth, and survival. The aim of this study was to investigate whether or not administration of olive (Olea europaea L.) polyphenols could have an effect on NGF and BDNF content and the expression of their receptors, TrkA and TrkB, respectively, in the mouse brain. METHODS: NGF and BDNF were measured by enzyme-linked immunosorbent assay. TrkA and TrkB were measured by Western blotting. RESULTS: We found NGF and BDNF elevation in the hippocampus and olfactory bulbs and a decrease in the frontal cortex and striatum. These data were associated with potentiated expression of TrkA and TrkB in the hippocampus and olfactory bulbs but no differences between groups in the striatum and frontal cortex. Polyphenols did not affect some behavioral mouse parameters associated with stressing situations. CONCLUSIONS: Altogether, this study shows that olive polyphenols in the mouse may increase the levels of NGF and BDNF in crucial areas of the limbic system and olfactory bulbs, which play a key role in learning and memory processes and in the proliferation and migration of endogenous progenitor cells present in the rodent brain.

Assessment of the genotoxic, antigenotoxic, and cytotoxic activities of the ethanolic fruit extract of Solanum lycocarpum A. St. Hill. (Solanaceae) by micronucleus test in mice.

Solanum lycocarpum A. St. Hill. (Family Solanaceae), popularly known in Brazil as lobeira, is a common weed in the Brazilian Cerrado vegetation. The fruits of this species have been used in Brazil for culinary purposes and in folk medicine as a sedative, diuretic, antiepileptic, antispasmodic, hypoglycemic, and hypocholesterolemic agent as well as in the control of obesity. Due to the spreading use of this plant as a therapeutic resource and food, the present study aimed to evaluate the genotoxic, antigenotoxic, and cytotoxic effects of S. lycocarpum ethanolic fruit extract using the mouse bone marrow micronucleus test. Both genotoxicity and antigenotoxicity of this ethanolic fruit extract were evaluated by using the frequency of micronucleated polychromatic erythrocytes, whereas cytotoxicity was assessed by the polychromatic and normochromatic erythrocytes ratio. Our results indicated that although S. lycocarpum ethanolic fruit extract did not exhibit genotoxic effect in mice bone marrow, both cytotoxic and antigenotoxic actions were evidenced at all tested doses.

Solanum paniculatum L. leaf and fruit extracts: assessment of modulation of cytotoxicity and genotoxicity by micronucleus test in mice.

Solanum paniculatum L. is a plant species widespread throughout tropical America, especially in the Brazilian Savanna region. It is used in Brazil for culinary purposes and in folk medicine to treat liver and gastric dysfunctions, as well as hangovers. Previous studies with S. paniculatum ethanolic leaf extract (ELE) or ethanolic fruit extract (EFE) demonstrated that they have no genotoxic activity meant either in the micronucleus test in mice or in the phage induction SOS Inductest in bacterial strains; however, cytotoxicity was demonstrated in both tests. Because of the spread use of this plant as a therapeutic resource and food, the present study aimed at evaluating the modulator effects of S. paniculatum ELE or EFE against mitomycin C (MMC) using the mouse bone marrow micronucleus test. This short-term test was used to detect the acute effects of responsive erythropoiesis after 24- and 48-hour exposure periods. Swiss-Webster mice were orally treated with three different concentrations (100, 200, or 300 mg/kg) of ELE or EFE simultaneously with a single dose of MMC (4 mg/kg i.p.). Antigenotoxicity was evaluated using the frequency of micronucleated polychromatic erythrocytes (MNPCEs), whereas anticytotoxicity was assessed by the polychromatic/normochromatic erythrocyte ratio. Our results demonstrated that neither the ELE nor EFE of S. paniculatum protected cells against the cytotoxic action of MMC. Nevertheless, the present study showed the antimutagenic effect of ELE after a 24-hour treatment (reduction in the frequencies of MNPCEs after a 48-hour treatment with ELE can be due to toxicity) and no antimutagenic action of the EFE treatment against the aneugenic and/or clastogenic activities of MMC.

Assessment of serum (1->3)-beta-D-glucan concentration as a measure of disease burden in a murine model of invasive pulmonary aspergillosis.

Serum (1-->3)-beta-D-glucan concentrations were serially measured in the presence and absence of antifungal therapy in a murine model of invasive pulmonary aspergillosis. Serum (1-->3)-beta-D-glucan was detected early during the course of infection, and reductions in this biomarker were associated with improved survival in animals treated with antifungal agents.

Immunolocalization assessment of metastasis-associated protein 1 in human and mouse mature testes and its association with spermatogenesis.

AIM: To investigate the stage-specific localization of metastasis-associated protein 1 (MTA1) during spermatogenesis in adult human and mouse testis. METHODS: The immunolocalization of MTA1 was studied by immunohistochemistry and Western blot analysis. The distribution pattern of MTA1 in mouse testis was confirmed by using quantitative analysis of purified spermatogenic cells. RESULTS: The specificity of polyclonal antibody was confirmed by Western blot analysis. MTA1 was found expressed in the nucleus of germ cells, except elongate spermatids, and in the cytoplasm of Sertoli cells; Leydig cells did not show any specific reactivity. MTA1 possessed different distribution patterns in the two species: in humans, the most intensive staining was found in the nucleus of round spermatids and of primary spermatocytes while in mice, the most intense MTA1 staining was in the nucleus of leptotene, zygotene and pachytene spermatocytes. In both species the staining exhibited a cyclic pattern. CONCLUSION: The present communication initially provides new evidence for the potential role of MTA1 in mature testis. In addition, its distinctive expression in germ cells suggests a regulatory role of the peptide during spermatogenesis.

2D and 3D assessment of neuropathology in rat brain after prenatal exposure to methylazoxymethanol, a model for developmental neurotoxicty.

To evaluate the ability of a tiered quantitative morphological approach to reveal developmental neurotoxicity, morphometric parameters were measured in the offspring of rats treated with methylazoxymethanol (MAM) during days 13-15 of pregnancy. Treatment was aimed at inhibiting the proliferation phase of hippocampal neurons while leaving cerebellar neurons unaffected. 2D and 3D assessment of brain morphology combined with straightforward measurement of brain size, weight and volume, and the usefulness of estimation of total neuron numbers were studied. Each tier indicated major effects of MAM, from macroscopic effects in the cerebrum (first tier) to a considerable loss of neurons in the hippocampal CA1 pyramidal layer (third tier). The cerebellum and the number of cerebellar granular neurons were not changed. Along with each step of the proposed tiered approach (brain size-->linear morphometry-->stereology), the discriminative strength of the endpoints, and thus the probability to pinpoint the extent and location of developmental brain lesions increased.

Bayesian mapping of QTL in outbred F2 families allowing inference about whether F0 grandparents are homozygous or heterozygous at QTL.

In this paper, we propose a new Bayesian method for QTL analysis in outbred F2 families based on Markov chain Monte Carlo (MCMC) estimation allowing inference about whether each of F0 founders (grandparents) is homozygous or heterozygous at QTL. This, in turn, allows us to select a model accurately explaining observations of phenotypes for F2 individuals. The proposed method performs the fitting a statistical model of the two possible QTL states in each F0 grandparent, that is, homozygous and heterozygous at QTL, and gives a posterior distribution for the QTL states in each F0 grandparent. We confine ourselves to the discrimination of two QTL states, homozygous or heterozygous, for each of the F0 grandparents without taking into consideration whether common alleles are shared by F0 grandparents. The statistical model includes allelic effects and dominance effects for each QTL. The number of parameters representing allelic effects and dominance effects is therefore changed depending on the QTL states. A Reversible Jump MCMC technique is used for transition between the models of different dimensions. The effectiveness of the proposed method was investigated using simulation experiments. It was practicable to estimate the QTL states of F0 grandparents as well as the number, the locations and the effects of QTL segregating in an outbred F2 family.

Rodent vendor apparent source of mouse parvovirus in sentinel mice.

Mouse parvovirus (MPV) has been increasingly prevalent in laboratory animal facilities, and the source of infection often can be difficult to determine. After 4 years of sporadic MPV detected in our sentinel mice and continual failure to identify index cases in colony mice, we developed a regimen to house newly arrived vendor mice in large sterile cages with a high stocking density. Some of these mice were retained in isolation after the remaining mice were deployed as sentinels. After detecting MPV seropositive sentinel mice 4 weeks after introduction to the mouse colonies in one facility, the remaining naïve mice that had been previously housed with those sentinels also tested positive for MPV, despite never having been exposed to colony mice. These results suggest that commercially bred mice intended for use as sentinels may, in fact, arrive at animal facilities already infected with MPV. Depending upon numerous factors, including the health surveillance methods used, it is possible that a low prevalence of MPV may exist undetected at rodent vendors.

Detection of multiple QTL with epistatic effects under a mixed inheritance model in an outbred population.

A quantitative trait depends on multiple quantitative trait loci (QTL) and on the interaction between two or more QTL, named epistasis. Several methods to detect multiple QTL in various types of design have been proposed, but most of these are based on the assumption that each QTL works independently and epistasis has not been explored sufficiently. The objective of the study was to propose an integrated method to detect multiple QTL with epistases using Bayesian inference via a Markov chain Monte Carlo (MCMC) algorithm. Since the mixed inheritance model is assumed and the deterministic algorithm to calculate the probabilities of QTL genotypes is incorporated in the method, this can be applied to an outbred population such as livestock. Additionally, we treated a pair of QTL as one variable in the Reversible jump Markov chain Monte Carlo (RJMCMC) algorithm so that two QTL were able to be simultaneously added into or deleted from a model. As a result, both of the QTL can be detected, not only in cases where either of the two QTL has main effects and they have epistatic effects between each other, but also in cases where neither of the two QTL has main effects but they have epistatic effects. The method will help ascertain the complicated structure of quantitative traits.

Assessment of retinal degeneration in outbred albino mice.

Evaluation of a pharmaceutical's safety includes assessment of the potential for ophthalmologic toxicity. These nonclinical studies commonly use various outbred stocks of mice. Pretest indirect ophthalmoscopic examinations in the commonly used outbred stock Hsd:ICR(CD-1) indicated that retinal degeneration was a problem in this particular outbred stock of mice. This prompted the authors to examine other stocks of outbred mice routinely used in the performance of nonclinical safety studies. Groups of mice were observed over a 13-week period to determine the progression and changing incidence of retinal degeneration. Light intensity in the room and caging was measured during the study, and it was determined that light did not play a direct role in the progression of the retinal degeneration observed during the study. Histomorphologic examination of the mouse eyes was performed at the end of the study to confirm the presence of retinal degeneration observed after ophthalmoscopic examination. The incidence of retinal atrophy in the various outbred stocks of mice was: Crl:CFW(SW)BR (98.3%), Tac(SW)fBR (80%), Tac:Icr:Ha(ICR)fBR (75%), Hsd:ICR(CD-1) (43.3%), and Crl:CF-1BR (3.0%). Retinal atrophy was not observed in the following outbred mice stocks: Crl:CD-1(ICR)BR, HsdWin:CFW1, and Hsd:NSA(CF-1). On the basis of these findings, it is highly recommended that pretest ophthalmologic screening be performed on mice to obviate pre-existing conditions from confounding or invalidating nonclinical study results.

Pharmacological assessment of the role of nitric oxide in mice infected with lethal and nonlethal species of malaria.

This pharmacological investigation sought to determine whether nitric oxide (NO) had an antiparasitic effect and/or mediated pathology in mice infected with nonlethal P. chabaudi or lethal P. berghei. Nitric oxide synthase (NOS) inhibitors were evaluated for their ability to inhibit the rise in reactive nitrogen intermediates (RNI) induced by bacterial lipopolysaccharide (LPS) in mice. The more effective compound, aminoguanidine (AG) inhibited the rise in RNI induced by P. chabaudi and increased mortality, but had no effect on parasitaemia. Inducers and donors of NO were screened for their ability to increase RNI and the most effective agents evaluated for their ability to modify P. berghei infection. S-Nitrosoglutathione had little effect, but LPS decreased parasitaemia and mortality. An inconsistent relationship is evident between the abilities of these agents to modify NO activity and their effects on malaria in mice. Increased mortality in mice with P. chabaudi treated with AG indicates a reduction in resistance. The absence of an effect on parasitaemia by a NOS inhibitor or NO donor indicates either RNI have insignificant antimalarial action in vivo or the efficacy of the compounds is inadequade. Resistance to P. berghei in LPS-treated mice demonstrates an antiparasitic effect, but this may be attributable to factors other than NO.

Mapping viability loci using molecular markers.

In genetic mapping experiments, some molecular markers often show distorted segregation ratios. We hypothesize that these markers are linked to some viability loci that cause the observed segregation ratios to deviate from Mendelian expectations. Although statistical methods for mapping viability loci have been developed for line-crossing experiments, methods for viability mapping in outbred populations have not been developed yet. In this study, we develop a method for mapping viability loci in outbred populations using a full-sib family as an example. We develop a maximum likelihood (ML) method that uses the observed marker genotypes as data and the proportions of the genotypes of the viability locus as parameters. The ML solutions are obtained via the expectation-maximization algorithm. Application and efficiencies of the method are demonstrated and tested using a set of simulated data. We conclude that mapping viability loci can be accomplished using similar statistical techniques used in quantitative trait locus mapping for quantitative traits.

Comparison of methods used for recovering the line origin of alleles in a cross between outbred lines.

Here, we introduce the idea of probabilities of line origins for alleles in general pedigrees as found in crosses between outbred lines. We also present software for calculating these probabilities. The proposed algorithm is based on the linear regression method of Haley, Knott and Elsen (1994) combined with the Markov chain Monte Carlo (MCMC) method for estimating quantitative trait locus coefficients used as regressors. We compared the relative precision of our method and the original method as proposed by Haley et al. (1994). The scenarios studied varied in the allelic distribution of marker alleles in parental lines and in the frequency of missing marker genotypes. We found that the MCMC method achieves a higher accuracy in all scenarios considered. The benefits of using MCMC approximation are substantial if the frequency of missing marker data is high or the number of marker alleles is low and the allelic frequency distribution is similar in both parental lines.

Development and characterization of a novel, graded model of clip compressive spinal cord injury in the mouse: Part 2. Quantitative neuroanatomical assessment and analysis of the relationships between axonal tracts, residual tissue, and locomotor recovery.

A detailed examination of the histopathological features of the clip compression injury in mice was performed to understand the relationships between neurological function and existing pathology of the spinal cord. Adult, female CD1 mice underwent three grades of extradural clip compression injury (3-g, 8-g, and 24-g FEJOTA mouse clips), transection, and sham injury at T3-4. Quantitative behavioural assessments were performed for 4 weeks following SCI. After 4 weeks, Fluoro-Gold was introduced caudal to the SCI site, at T9, and was retrogradely transported for 5 days to the origin of spared axons through the injury site. Counts of retrogradely labeled neurons in the brain-stem, midbrain, and sensory-motor cortex indicated that the number of intact descending axons that traversed the lesion decreased with increasing injury severity (F > 28; df = 4; p < 0.0001; one-way ANOVA). Independent linear correlation analyses were performed between indices of neurological recovery (BBB and IP test), counts of retrogradely labeled neurons and morphometric assessments of normal residual tissue at the injury epicenter. The BBB test correlated strongly with the amount of residual tissue at the injury epicenter (R = 0.945, df = 28, p < 0.0001). Counts of neurons retrogradely labeled with Fluoro-Gold were also strongly correlated with the BBB scores. The extrapyramidal (raphespinal, reticulospinal, vestibulospinal, and rubrospinal) tracts had Pearson correlation coefficients (R) of 0.814, 0.812, 0.813, and 0.747, respectively (df = 28, p < 0.0001). The pyramidal (corticospinal) tract had a correlation of R = 0.747, df = 28, p < 0.0001 with the BBB scores. The IP scores also correlated strongly with the persistence of extrapyramidal (raphespinal, reticulospinal, vestibulospinal and rubrospinal) tracts with correlation coefficients of 0.801, 0.782, 0.790, and 0.836, respectively (df = 28, p < 0.0001). These data indicate that the counts of retrogradely labeled neurons at the origin of distinct descending motor pathways are predictors of the variance of the functional recovery measured by the BBB and IP tests following spinal cord injury. In addition, we provide a detailed neuroanatomical study of clip compression injury in mice that can be used to study the molecular mechanisms of SCI in knockout and transgenic mice.