A cross-platform approach to characterize and screen potential neurovascular unit toxicants

Development of the neurovascular unit (NVU) is a complex, multistage process that requires orchestrated cell signaling mechanisms across several cell types and ultimately results in formation of the blood-brain barrier. Typical high-throughput screening (HTS) assays investigate single biochemical or single cell responses following chemical insult. As the NVU comprises multiple cell types interacting at various stages of development, a methodology combining high-throughput results across pertinent cell-based assays is needed to investigate potential chemical-induced disruption to the development of this complex cell system. To this end, we implemented a novel method for screening putative NVU disruptors across diverse assay platforms to predict chemical perturbation of the developing NVU. HTS assay results measuring chemical-induced perturbations to cellular key events across angiogenic and neurogenic outcomes in vitro were combined to create a cell-based prioritization of NVU hazard. Chemicals were grouped according to similar modes of action to train a logistic regression literature model on a training set of 38 chemicals. This model utilizes the chemical-specific pairwise mutual information score for PubMed MeSH annotations to represent a quantitative measure of previously published results. Taken together, this study presents a methodology to investigate NVU developmental hazard using cell-based HTS assays and literature evidence to prioritize screening of putative NVU disruptors towards a knowledge-driven characterization of neurovascular developmental toxicity. The results from these screening efforts demonstrate that chemicals representing a range of putative vascular disrupting compound (pVDC) scores can also produce effects on neurogenic outcomes and characterizes possible modes of action for disrupting the developing NVU.     

ReProComet: a new in vitro method to assess DNA damage in mammalian sperm.

The increasing request of chemical safety assessment demands for the validation of alternative methods to reduce the resort to animal experimentation. Methods that evaluate reproductive toxicity are among those requiring the largest use of animals. Presently, no validated in vitro alternative exists for the assessment of reproductive toxicity. Mammalian sperm are sensitive targets of DNA-reactive chemicals, which form premutagenic adducts. Here, we propose a new method based on comet assay to detect DNA damage induced by potential germ cell mutagens in bull sperm available from assisted reproduction practices. In somatic cells, chemical-induced adducts can be revealed by comet assay that detects DNA breaks produced during adduct repair. Mature sperm, however, are devoid of repair enzymes, and adducts are processed only after fertilization. For this reason, comet assay is not sensitive to detect DNA lesions induced in sperm by most chemicals. To overcome such limitation, we developed a modified comet assay based on the addition of a protein extract from HeLa cells to agarose-embedded sperm on microscopic slides. To test the method, sperm were treated in vitro with methyl methanesulfonate (MMS) or melphalan (MLP) and comet assay was conducted both with and without protein supplementation. No effect of MMS or MLP was detected without protein supplementation; on the contrary, a clear-cut dose-dependent effect was measured after addition of the cell extract. These results represent a proof of concept of a novel in vitro mutagenicity test on sperm that could offer a promising approach to complement previously validated in vivo germ cell genotoxicity assays.     

Semiconductor manufacturing: an introduction to processes and hazards

Recent studies suggest that semiconductor workers have an increased incidence of work-related illness. Semiconductor manufacturing is a chemically intensive industry involving many potentially hazardous operations. As this industry moves into new geographic areas, health care professionals will be asked to evaluate medical or workplace conditions associated with unfamiliar and complex production processes. This paper provides an overview of semiconductor manufacturing processes for these health practitioners. Each step of device fabrication is detailed with its attendant chemical and physical hazards. Broader concepts of industrial control technology, clean room ventilation, and ergonomics are explained. The hazards are tabulated to allow rapid assessment of the risks inherent to each processing step. References have been chosen to guide the reader to more indepth information.     

A developmental role for ataxia-telangiectasia mutated in protecting the embryo from spontaneous and phenytoin-enhanced embryopathies in culture.

Ataxia-telangiectasia (A-T) is characterized by impaired recognition and repair of DNA damage and increased sensitivity to ionizing radiation (IR), cancer, and neurodegeneration. We previously showed pregnant knockout mice lacking the A-T gene product ataxia-telangiectasia mutated (Atm) are highly susceptible to the embryopathic effects of IR, which damages DNA, possibly via generation of reactive oxygen species (ROS). Here we show that Atm more broadly protects against both spontaneous and phenytoin-enhanced embryopathies. In the absence of drug exposure, cultured embryos from pregnant Atm knockout mice showed more embryopathies than wild-type littermates, with a gene dose-dependent decrease in susceptibility from -/- to +/- to +/+ embryos (p < 0.05). A similar but significantly enhanced gene dose-dependent pattern of embryopathic susceptibility was evident in Atm knockout embryos exposed to the ROS-initiating teratogen phenytoin (p < 0.05). These results provide the first evidence that Atm has a broad developmental importance beyond IR embryopathies, possibly by protecting the embryo from constitutive and xenobiotic-enhanced oxidative stress, with even heterozygotes showing increased risk. This developmental role of Atm further implicates DNA damage in ROS-mediated teratogenesis and DNA damage response and repair as risk factors for individual susceptibility.     

Endocrine toxicology: A Review of the application of endocrinology in experimental toxicology

In the past endocrine toxicology has not been a common subject in routine toxicity studies. However, since the endocrine system is an important integrating system of the body, controlling the major physiological functions, it is important to investigate the mechanism of action of exogenous compounds in endocrine target organs or hormonal target cells. The following procedure is suggested to detect effects on the endocrine system in routine toxicity experiments: (1) determination of the weight of endocrine organs and histology as screening parameters; (2) determination of circulating hormones in combination with morphological or immunocytochemical methods: (3) specific function tests and in vitro methods to determine dysfunction of specific endocrine organs or cells. That the use of such an approach has provided insight into the mechanism of action of chemical compounds will be demonstrated by results of endocrine toxicity studies with the antibiotic compound sulphadimidine, interfering with thyroid hormone synthesis as a secondary mechanism leading to thyroid tumour formation, the androgenic compound trenbolone acetate, used for growth promotion, for which the disturbance of the gonadal function formed the basis for the establishment of the no-observed-hormonal-effect level, the antibacterial compound furazolidone, suspected of having an oestrogenic activity which was hypothesized as the underlying mechanism for the observed mammary tumour formation, and the antimicrobial agent carbadox, used as feed additive for pigs, for which the interference with adrenal function, resulted in a severe disturbance of the water and salt balance in target animals. Originally presented at ECCP 93.