Utility of in vivo metabolomics to support read-across for UVCB substances under REACH.

Structure-based grouping of chemicals for targeted testing and read-across is an efficient way to reduce resources and animal usage. For substances of unknown or variable composition, complex reaction products, or biological materials (UVCBs), structure-based grouping is virtually impossible. Biology-based approaches such as metabolomics could provide a solution. Here, 15 steam-cracked distillates, registered in the EU through the Lower Olefins Aromatics Reach Consortium (LOA), as well as six of the major substance constituents, were tested in a 14-day rat oral gavage study, in line with the fundamental elements of the OECD 407 guideline, in combination with plasma metabolomics. Beyond signs of clinical toxicity, reduced body weight (gain), and food consumption, pathological investigations demonstrated the liver, thyroid, kidneys (males only), and hematological system to be the target organs. These targets were confirmed by metabolome pattern recognition, with no additional targets being identified. While classical toxicological parameters did not allow for a clear distinction between the substances, univariate and multivariate statistical analysis of the respective metabolomes allowed for the identification of several subclusters of biologically most similar substances. These groups were partly associated with the dominant (> 50%) constituents of these UVCBs, i.e., indene and dicyclopentadiene. Despite minor differences in clustering results based on the two statistical analyses, a proposal can be made for the grouping of these UVCBs. Both analyses correctly clustered the chemically most similar compounds, increasing the confidence that this biological approach may provide a solution for the grouping of UVCBs.

Studies on the environmental fate, ecotoxicology and toxicology of 2-methyl 1,3-propanediol.

2-methyl 1,3-propandiol (MPD) is a low molecular weight, colorless glycol used in polymer and coating applications. The log Kow of -0.6 suggests partitioning to aqueous phases with a low concern for possible bioaccumulation. MPD was found to be inherently biodegradable. Ecotoxicological results in several aquatic and terrestrial species found no significant hazard potential. MPD is rapidly absorbed via the oral and dermal routes, metabolized to 3-hydroxybutyrate, and excreted in urine with a half-life of 3.6 h. Acute toxicity testing found low toxicity via all routes. Barely perceptible skin irritation was observed in human volunteers, whereas there was no evidence of irritation in rabbits. Skin sensitization in Guinea pigs was negative. Human skin patch results indicated minimal response in about 1% of individuals. There was no evidence of mutagenicity using bacterial and mammalian test systems. A 90-day oral study in rats found no adverse effects at any dose. Three developmental toxicity studies in rats and rabbits, found no treatment-related maternal toxicity, fetal toxicity or malformations. A two-generation reproduction study in rats found no consistent treatment-related adverse effects on reproduction in either generation. No carcinogenicity studies with MPD were identified. MPD presents a low degree of toxicological and ecotoxicological or environmental hazard.

Drosophila D-titin is required for myoblast fusion and skeletal muscle striation.

An ethylmethane sulfonate (EMS) mutagenesis of Drosophila melanogaster aimed at discovering novel genes essential for neuromuscular development identified six embryonic lethal alleles of one genetic locus on the third chromosome at 62C. Two additional lethal P element insertion lines, l(3)S02001 and l(3)j1D7, failed to complement each other and each of the six EMS alleles. Analysis of genomic sequence bracketing the two insertion sites predicted a protein of 16,215 amino acid residues, encoded by a 70 kb genomic region. This sequence includes the recently characterized kettin, and includes all known partial D-Titin sequences. We call the genetic locus, which encodes both D-Titin and kettin, D-Titin. D-Titin has 53 repeats of the immunoglobulin C2 domain, 6 repeats of the fibronectin type III domain and two large PEVK domains. Kettin appears to be the NH2-terminal one third of D-Titin, presumably expressed via alternative splicing. Phenotype assays on the allelic series of D-Titin mutants demonstrated that D-Titin plays an essential role in muscle development. First, D-Titin has an unsuspected function in myoblast fusion during myogenesis and, second, D-Titin later serves to organize myofilaments into the highly ordered arrays underlying skeletal muscle striation. We propose that D-Titin is instrumental in the development of the two defining features of striated muscle: the formation of multi-nucleate syncitia and the organization of actin-myosin filaments into striated arrays.

Presynaptic glutamic acid decarboxylase is required for induction of the postsynaptic receptor field at a glutamatergic synapse.

We have systematically screened EMS-mutagenized Drosophila for embryonic lethal strains with defects in glutamatergic synaptic transmission. Surprisingly, this screen led to the identification of several alleles with missense mutations in highly conserved regions of Dgad1. Analysis of these gad mutants reveals that they are paralyzed owing to defects in glutamatergic transmission at the neuromuscular junction. Further electrophysiological and immunohistochemical examination reveals that these mutants have greatly reduced numbers of postsynaptic glutamate receptors in an otherwise morphologically normal synapse. By overexpressing wild-type Dgad1 in selected neurons, we show that GAD is specifically required in the presynaptic neuron to induce a postsynaptic glutamate receptor field, and that the level of postsynaptic receptors is closely dependent on presynaptic GAD function. These data demonstrate that GAD plays an unexpected role in glutamatergic synaptogenesis.

Leonardo, a Drosophila 14-3-3 protein involved in learning, regulates presynaptic function.

The leonardo gene encodes a conserved member of the 14-3-3 protein family, which plays a role in Drosophila learning. Immunological localization of the protein shows that it is expressed at synaptic connections and enriched in presynaptic boutons of the neuromuscular junction (NMJ). Null leonardo mutants die as mature embryos. Electrophysiological assays of the mutant NMJ demonstrate that basal synaptic transmission is reduced by 30% and that transmission amplitude, fidelity, and fatigue resistance properties are reduced at elevated stimulation frequencies and in low external [Ca2+]. Moreover, transmission augmentation and post-tetanic potentiation (PTP) are disrupted in the mutant. These results suggest that Leonardo plays a role in the regulation of synaptic vesicle dynamics, a function which may underlie synaptic modulation properties enabling learning.

Presynaptic development at the Drosophila neuromuscular junction: assembly and localization of presynaptic active zones.

We describe the extent to which presynaptic structures at the embryonic neuromuscular junction of Drosophila can form in mutants where development of postsynaptic somatic muscles is affected. Although twist mutant embryos lack mesoderm, motor axons still grow out of the CNS and form morphologically normal presynaptic active zones, independent of their target cells. In myoblast city mutant embryos, myoblasts do not fuse but form fully differentiated mononucleate muscles, which make functional neuromuscular synapses with correctly localized presynaptic active zones. Myoblasts also fail to fuse but still attract appropriate innervation in mef2 mutant embryos. However, these myoblasts fail to differentiate into muscles and presynaptic active zones fail to localize at neuromuscular contacts. Thus, the process of synapse formation can be genetically separated from the process of target recognition, revealing that localization of presynaptic active zones requires mef2-dependent muscle differentiation.

Mutations in a novel gene, myoblast city, provide evidence in support of the founder cell hypothesis for Drosophila muscle development.

We have used mutations in the newly identified gene myoblast city to investigate the founder cell hypothesis of muscle development in Drosophila melanogaster. In embryos mutant for myoblast city the fusion of myoblasts into multinucleate muscles is virtually abolished. Nevertheless, a subset of the myoblasts develop specific muscle-like characteristics, including gene expression appropriate to particular muscles, migration to the appropriate part of the segment, correct position and orientation, and contact by motor neurons. We suggest that this subset of myoblasts represents the proposed muscle founder cells and we draw an analogy between these founder cells and the muscle pioneers described for grasshopper muscle development.

dpp induces mesodermal gene expression in Drosophila.

Inductive interactions between germ layers are an essential feature of the development of many organisms. In several species these interactions are mediated by members of the transforming growth factor-beta (TGF beta) family. In amphibians, different concentrations of activin can induce different types of mesoderm in the animal cap assay. In Drosophila, a member of the TGF beta family, decapentaplegic (dpp), acts as an inductive signal. Midway through embryogenesis, dpp is expressed in the visceral mesoderm, and enhances the expression of the homeotic gene labial in the underlying midgut endoderm. Earlier in development, however, dpp expression is limited to the dorsal ectoderm. At this stage in development, thickveins, a dpp receptor, is expressed in the mesoderm, and this suggests that ectodermal dpp might not only be required for development of dorsal ectoderm, but could also act inductively to mediate pattern formation in the underlying mesoderm. Here we show, by expressing dpp ectopically in the ectoderm and mesoderm and by examining dpp null mutant embryos, that dpp regulates expression of mesodermal genes.

Myogenesis and muscle patterning in Drosophila.

Subsets of differentiating muscles in the Drosophila embryo express putative transcription factors, such as S59 and vestigial. These genes may control the development of specific muscle properties. Myogenesis in embryos mutant for wingless is grossly deranged. Mesodermal expression of S59 is lost, whereas some vestigial-expressing muscles develop. wingless dependence and independence of specific muscle subsets correlates with an early derangement of twist expression in wingless mutants. The possible role of the ectoderm in patterning Drosophila mesoderm is discussed.

Loss of function of the Drosophila zfh-1 gene results in abnormal development of mesodermally derived tissues.

The Drosophila zfh-1 gene encodes an unusual protein with nine Cys2His2 type zinc-finger motifs and one homeodomain that shows a complex pattern of expression in the embryonic mesoderm and nervous system. To study the function of zfh-1, we generated loss-of-function zfh-1 mutations. Phenotypic analysis of zfh-1 mutant embryos reveals that the gene is not required for the initial segregation of the mesoderm or for the differentiation of mesodermally derived tissues. Rather, loss of zfh-1 function results in various degrees of local errors in cell fate or positioning.

A dual requirement for neurogenic genes in Drosophila myogenesis.

In wild-type embryos of Drosophila melanogaster, the formation of differentiated larval muscles is preceded by the segregation of small numbers of progenitor or founder cells in the embryonic mesoderm. The founder cells, characterised by the expression of genes encoding putative transcription factors such as S59 or vestigial, fuse with neighbouring myoblasts to form syncytial precursors of individual muscles. Founder cell segregation is deranged in embryos mutant for any of the neurogenic genes: enlarged clusters of cells expressing S59 or vestigial are detected at the sites where small numbers of founder cells segregate in the wild type. In addition, muscle differentiation is deranged in such embryos in a way that appears to be closely linked to the extent of epidermal disruption caused by the neurogenic phenotype: myoblast fusion is limited to regions of the mesoderm beneath the residual epidermis left by the hyperplasia of the nervous system, and late expression of S59 and vestigial is lost from mesoderm not lying within the margins of the residual epidermis. Thus neurogenic gene functions appear to be required both for the normal segregation of founder cells and for muscle differentiation. It is not clear whether either of these requirements reflects an essential function for any or all of the neurogenic genes within the mesoderm itself.

Cells with persistent twist expression are the embryonic precursors of adult muscles in Drosophila.

twist expression in the embryonic mesoderm of Drosophila declines during germ band retraction to leave a residual population of twist-expressing cells in the late embryo. In the abdomen, the pattern of twist expression is a simple one: a single cell ventrally, pairs of cells laterally and three cells dorsally in each hemisegment. In the thorax, there are patches of cells associated with the imaginal discs and there are additional clusters in A8 and A9. During larval life, the twist-expressing cells proliferate and, in the abdomen, they form ventral, lateral and dorsal clusters, which are the precursors of the adult abdominal muscles, while in the thorax, they form populations of cells in the imaginal discs that correspond to the adepithelial cells described by previous authors. While most thoracic twist-expressing cells are associated with the discs, the abdominal cells are separate from the precursors of the adult abdominal epidermis, the abdominal histoblasts, and lie on branches of peripheral nerves. The distribution of these cells is tightly linked to the pattern of peripheral nerves, but they segregate normally in da/da embryos despite the absence of the peripheral nervous system.