Demonstrating the reliability of in vivo metabolomics based chemical grouping: towards best practice.

While grouping/read-across is widely used to fill data gaps, chemical registration dossiers are often rejected due to weak category justifications based on structural similarity only. Metabolomics provides a route to robust chemical categories via evidence of shared molecular effects across source and target substances. To gain international acceptance, this approach must demonstrate high reliability, and best-practice guidance is required. The MetAbolomics ring Trial for CHemical groupING (MATCHING), comprising six industrial, government and academic ring-trial partners, evaluated inter-laboratory reproducibility and worked towards best-practice. An independent team selected eight substances (WY-14643, 4-chloro-3-nitroaniline, 17α-methyl-testosterone, trenbolone, aniline, dichlorprop-p, 2-chloroaniline, fenofibrate); ring-trial partners were blinded to their identities and modes-of-action. Plasma samples were derived from 28-day rat tests (two doses per substance), aliquoted, and distributed to partners. Each partner applied their preferred liquid chromatography-mass spectrometry (LC-MS) metabolomics workflows to acquire, process, quality assess, statistically analyze and report their grouping results to the European Chemicals Agency, to ensure the blinding conditions of the ring trial. Five of six partners, whose metabolomics datasets passed quality control, correctly identified the grouping of eight test substances into three categories, for both male and female rats. Strikingly, this was achieved even though a range of metabolomics approaches were used. Through assessing intrastudy quality-control samples, the sixth partner observed high technical variation and was unable to group the substances. By comparing workflows, we conclude that some heterogeneity in metabolomics methods is not detrimental to consistent grouping, and that assessing data quality prior to grouping is essential. We recommend development of international guidance for quality-control acceptance criteria. This study demonstrates the reliability of metabolomics for chemical grouping and works towards best-practice.

A 3-mirror surface force balance for the investigation of fluids confined to nanoscale films between two ultra-smooth polarizable electrodes.

We present a new technique, based on the Surface Force Balance (SFB), for the direct measurement of surface forces between two ultra-smooth and polarizable gold electrode surfaces across thin fluid films. Combining the direct interferometric measurement of surface separation and contact geometry with smooth electrode surfaces has proved challenging in the past, and for this reason, previous measurements with the SFB typically involved two insulating mica surfaces, or one mica surface and one electrode surface, or an alternative less direct measure of the surface separation. Here, we demonstrate that a 3-mirror interferometer can overcome these difficulties: the setup involves two ultra-smooth electrode/mirror surfaces between which the fluid is confined and a third mirror to allow for interferometric detection of the liquid thickness with nanometer resolution and at thicknesses much smaller than the diffraction limit of the light. We conclude with a proof-of-concept measurement across dry nitrogen gas. The technique should prove useful for studying the properties of fluids confined at the nanoscale inside a slit-pore of controlled electrical potential or subject to applied electric fields.

Lithium-drug interactions altering the intracellular lithium level: an in vitro study.

An in vitro incubation system utilizing a bicarbonate-CO2 buffer system accurately predicts the in vivo red cell:plasma lithium ratio (LR). Piperazine phenothiazines produced the most marked elevations of intracellular lithium, doubling the LR when added to the system at 3 X 10(-5) M. The aminoalkyl phenothiazines and thioxanthenes were somewhat less active, while the non-phenothiazine antipsychotics, such as loxapine, haloperidol and molindone, produced only minor increases in the LR. Tricyclic antidepressants produced a 20-30% increase, while other types of antidepressants, the major neurotransmitters and their metabolites, hormones, benzodiazepines and diuretics did not show any activity. Of the antihypertensives studied, only hydralazine had an effect on the LR. The effect of fluphenazine on the intracellular lithium level was paralleled by an increase in intracellular sodium and was blocked by dipyridamole. Several important lithium-drug interactions may contribute to an increased LR and suggest caution in the interpretation of studies of the relationship between the LR and clinical aspects of the affective disorders.

Evidence for the existence of a sexually dimorphic nucleus in the preoptic area of the rat.

The volume of an intensely staining component of the preoptic area of the male rat is markedly larger than that of the female. Moreover, its volume in both sexes is altered by perinatal hormone exposure consistent with the view that this brain region undergoes hormone dependent sexual differentiation. The present study was carried out to determine if this sexually dimorphic area of the brain has a greater cell density than that of the surround, and if a unique population or distribution of cells, either within one sex or between males and females, characterized this region. A single coronal paraffin section (10 micrometer) through the approximate center of this sexually dimorphic area in four adult gonadectomized rats of each sex was evaluated systematically. Each cell was labelled as being inside or outside of the sexually dimorphic area. In addition to cell density per unit area the following parameters were evaluated through a closed-circuit video system: cell size, staining intensity, shape, and the presence of processes and of a nucleolus. The presence of a nucleolus was further used to identify neurons within the total population of almost 5000 cells that was evaluated. In both sexes, the sexually dimorphic area was characterized by a significantly increased cell density per unit area compared to that of the surround. On this basis, the term, the Sexually Dimorphic Nucleus of the Preoptic Area (SDN-POA) is proposed, for this region. Moreover, the SDN-POA of the male was characterized by increased neuronal density per unit area. The SDN-POA in the male was also found to contain larger cells and neurons, as determined by direct measurement of their greatest diameter, as well as a greater percentage of cells and neurons rated large on a three-point scale (small, medium, and large). No consistent differences in frequency distribution by stain intensity, shape, or the presence of cell processes were found to characterize the SDN-POA or contribute to the sexual dimorphism. It is concluded that the marked sex difference in the volume of the SDN-POA is due principally to an increase in the male of the total area of higher cell and neuronal density. However, the present results do not eliminate the possibility that more subtle differences in neuronal characteristics may exist in the SDN-POA.

Evidence for a morphological sex difference within the medial preoptic area of the rat brain.

The present report demonstrates the existence of a marked sexual difference in the volume of an intensely staining cellular component of the medial preoptic nucleus (MPON) of the rat. Moreover, this sexual dimorphism is shown to be independent of several specific hormonal conditions in the adult, but significantly influenced, perhaps determined, by the perinatal hormone environment. Adult rats were gonadectomized and sacrificed 2 or 5-6 weeks later, or sacrificed after gonadectomy and priming with estradiol benzoate (2 microgram/day x 3) and 500 microgram progesterone, or testosterone propionate (TP, 500 microgram/day x 14), or the ingestion of propylthiouracil (0.15% of the diet) for one month, or following water deprivation for 24 h. These treatments did not affect the sexual dimorphism in the MPON and, in all groups, nuclear volume in the male animals was significantly greater than that of females whether nuclear volume was expressed in absolute terms or relative to brain weight. On the other hand, the volume of the MPON of the adult male castrated neonatally was significantly reduced when compared to that of the male castrated at the time of weaning, i.e. after the period of sexual differentiation of the brain. Consistent with the view that this nuclear region undergoes sexual differentiation is the fact that the volume of the MPON was significantly greater in female rats injected with 1 mg TP on day 4 of life than in oil-treated females. More subtle sex differences in the volume of the suprachiasmatic nucleus were also detected, as were several treatment effects. Although these differences may fall within the error of the analytical procedure, it is possible that hormone- or sex-dependent morphological differences exist elsewhere in the brain. Nevertheless, the gross sexual dimorphism in the MPON clearly demonstrates a possible morphological basis for the sexual differentiation of brain function.