Current Protocols in Toxicology

We have recently described a new model to study gamete interaction in mammalian species. The model recreates the spherical surface of the oocyte by using magnetic Sepharose beads coated with a layer of a recombinant protein involved in gamete interaction (such as ZP2, or the IZUMO1 receptor JUNO) and an external layer of cumulus oophorus cells, thus mimicking, to some extent, a native cumulus-oocyte complex. Once generated, this 3D model can be used in a sperm-binding assay to obtain valuable information about the molecular basis of gamete interaction, since different recombinant proteins can be used to coat the bead surface, thus generating a variety of models to be used for several species...

Several cell lines of different origin are routinely used in research and drug development as important models to study human health and disease. Studying cells in culture represents an easy and convenient tool to approach complex biological questions, but the disadvantage is that they may not necessarily reflect what is effectively occurring in vivo. Human primary cells can help address this limitation, as they are isolated directly from human biological samples and can preserve the morphological and functional features of their tissue of origin...

Open-globe injuries can result in permanent vision loss, partly due to extended delays between injury and medical intervention. Even with early intervention, the management of open-globe injuries remains a challenge for ophthalmologists, mostly due to inadequate or suboptimal current therapies. To aid in the development of novel therapeutics and track toxicological and pathophysiological changes, this article details an open-globe injury platform capable of inducing injuries in enucleated porcine eyes. The injury platform relies on a high-speed solenoid device to mimic explosive injury scenarios, allowing for large, complex injury shapes and sizes that are often observed in casualties and are more difficult to treat...

Mitochondrial dysfunction is a central component in the pathophysiology of multiple neuropsychiatric and degenerative disorders. Evaluating mitochondrial function in human-derived neural cells can help characterize dysregulation in oxidative metabolism associated with the onset of brain disorders, and may also help define targeted therapies. Astrocytes play a number of different key roles in the brain, being implicated in neurogenesis, synaptogenesis, blood-brain-barrier permeability, and homeostasis, and, consequently, the malfunctioning of astrocytes is related to many neuropathologies...

The nuclear factor-erythroid 2 p45-related factor 2 (Nrf2)-mediated stress response is a major cellular defense mechanism against endogenous and exogenous oxidants, electrophiles, and pro-inflammatory agents. A number of Nrf2 inducers are being developed to therapeutically stimulate this pathway. Inducers are typically sensed by Kelch-like ECH-associated protein 1 (Keap1), a negative regulator and a binding partner of Nrf2. Modifications of Keap1 by oxidants or electrophiles, or its targeting by compounds that disrupt its interaction with Nrf2, alter the conformation of the Keap1-Nrf2 protein complex, which initiates the accumulation of Nrf2 required for mounting a stress response...

Culture of human peripheral blood mononuclear cells (PBMCs) still remains a convenient and sensitive method for measurement of a person's immune system health. Basic elements of the process, namely PBMC purification and culture medium formulation, were first reported in the late 1960s, and the utility of the method for clinical application was reported in the 1970s. Clinically, the approach can provide information about the ability of an individual's immune system to fight off attacks by various pathogens. Over the years, the method has undergone many improvements, which have been aided by advancements made in flow cytometry technology and the development of fluorescent reagents...

Bone marrow transplantation is used to treat particular types of cancers such as lymphoma, leukemia, and multiple myeloma. Appropriate dosing of busulfan during the preparative phase is critical for a successful allograft; if blood concentrations get too high significant liver toxicity can occur, if blood concentrations are too low, then graft-versus-host disease (GVHD) can develop. Busulfan monitoring in blood allows hospitals with the opportunity to provide individualized medicine to patients and improve overall patient outcome...

Caenorhabditis elegans is a free-living animal that is used as a powerful experimental model in biological sciences. The natural habitat of the animal are areas rich in material from rotting plants or fruits being decomposed by a growing number of microorganisms. The ecology of the natural habitat of C. elegans is a complex interactive network involving many species, including numerous types of bacteria, viruses, fungi, slugs, snails, and isopods, among which bacteria play multifaceted roles in the natural history of C...

Immunosuppressant medications help suppress the immune system response through inhibition of various checkpoints in the regulatory biochemical pathway. This is useful in prevention of organ rejection in transplantation or in the treatment of autoimmune diseases such as lupus or rheumatoid arthritis. Quantification of immunosuppressive drugs in blood is needed clinically for optimization of treatment and to avoid toxicity or unwanted side effects. Here, we describe a quantitative method to determine the concentration of cyclosprine A, tacrolimus, sirolimus, and everolimus in whole blood...

B lymphocytes, or B cells, are important players in immunity that produce antigen-specific immunoglobulins. As a result, they are involved in various immune-linked pathologies. To better understand, prevent, or treat B cell-associated disease and immunotoxicity, we developed an in vitro assay to model early murine B cell differentiation within the bone marrow. This model uses sorted B cell precursors cultured on a supporting stromal cell layer, which over time acquire markers of further differentiated B cells, such as surface antigens and rearranged immunoglobulin light chain...

Protein-protein interactions are important in human disease. Developing and refining tools to understand physical contacts between signaling proteins is crucial. This article describes a split luciferase complementation (SLC) method designed to discover inhibitors of protein-protein interaction. Different fusion proteins with split luciferase are constructed, expressed, and purified, and then assessed to determine the best pair that generates the strongest luminescence. SLC specificity and affinity are further confirmed...

We describe a detailed protocol to establish a newborn rat whole ovary culture, which enables the study of direct effects (independent of hypothalamic-pituitary-gonadal axis) of endocrine disrupting chemicals (EDCs), such as benzophenone-3 (BP-3). This method is useful to understand changes in follicle formation, primordial to primary transition, and expression of regulatory molecules linked to these processes and also provides an alternative to animal models. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Rat ovarian surgery Basic Protocol 2: Whole organ/ovarian culture Basic Protocol 3: RNA isolation and quantitative real-time PCR Basic Protocol 4: Histological processing and staining...

In order to circumvent ethical, technical, and economic drawbacks regarding the use of animal serum in cell culturing, it is possible to adapt mammalian cells to serum-free media. Nowadays, there are several serum-free formulations available, including fully animal derived-free and chemically defined media, and different adaptation techniques. This article focuses on the gradual adaptation of a mammalian suspension cell culture to a chemically defined medium. The first step is to transfer the cells cultured in medium supplemented with fetal bovine serum (FBS) to a chemically defined medium of your choice, containing the same amount of FBS...

Tissue-specific knockout mice are widely used throughout scientific research. A principle method for generating tissue-specific knockout mice is the Cre-loxP system. Here, we give a detailed description of the steps required to generate and validate tissue-specific knockout mice using the Cre-loxP system. The first protocol describes how to use gene targeting in mouse embryonic stem cells to generate mice with conditional alleles. Subsequent protocols describe how to recover Cre transgenic mice from cryopreserved sperm using in vitro fertilization and present a breeding strategy for obtaining tissue-specific knockouts...

Herein, we describe a protocol for the preparation and analysis of primary isolated rat hepatocytes in a 3D cell culture format described as spheroids. The hepatocyte cells spontaneously self-aggregate into spheroids without the need for synthetic extracellular matrices or hydrogels. Primary rat hepatocytes (PRHs) are a readily available source of primary differentiated liver cells and therefore conserve many of the required liver-specific functional markers, and elicit the natural in vivo phenotype when compared with common hepatic cells lines...

The cornea provides a functional barrier separating the outside environment from the intraocular environment, thereby protecting posterior segments of the eye from infection and damage. Pathological changes that compromise the structure or integrity of the cornea may occur as a result of injury or disease and can lead to debilitating effects on visual acuity. Over 10 million people worldwide are visually impaired or blind due to corneal opacity. Thus, physiologically relevant in vitro approaches to predict corneal toxicity of chemicals or effective treatments for disease prior to ocular exposure, as well as to study the corneal effects of systemic, chronic conditions, such as diabetes, are needed to reduce use of animal testing and accelerate therapeutic development...

The correct functioning of Sertoli cells (SCs) is pivotal for successful spermatogenesis. They are major targets for hormones, endocrine disruptors, and other substances that men are subjected to every day. One of the main SC functions that quickly responds to a deleterious stimulus is proliferation. This is directly related to the in vivo capacity of these cells to sustain a good number of developing germ cells. The protocols in this article can be tested on SCs of different origin. For the case of human SCs from small human testicular biopsies, a short and simple protocol to isolate and culture these cells is provided...

Using galactose instead of glucose in the culture medium of hepatoma cell lines, such as HepG2 cells, has been utilized for a decade to unmask the mitochondrial liability of chemical compounds. A modified glucose-galactose assay on HepG2 cells, reducing the experimental period for screening of mitochondrial toxicity to 2 to 4 hr, has been previously reported. HepaRG cells are one of the few cell lines that retain some of the important characteristics of human hepatocytes, offering advantages of working with a cell line, therefore, are considered an alternative for HepG2 cells in drug toxicity screening...

Light microscopy has played a central role in science for the past couple of hundred years and will continue to do so. Multiple super-resolution microscopy techniques have been in the headlines for smashing what for more than 100+ years was believed to be the limits of optical microscopy. This resolution improvement enables the visualization of molecular structures and processes on the nano scale. While certain scientific questions in toxicology can benefit from modalities within the super-resolution suite, due diligence is required for efficiency and to achieve optimal results...

Macrophages are innate immune cells that play important roles in various physiological and pathological processes. Evaluation of pro-inflammatory effects of drugs on macrophages has become commonplace in preclinical drug development prior to human clinical trials. Despite their body-wide distribution, tissue macrophages are often difficult to collect from large animals and humans in a noninvasive manner. Therefore, in vitro-differentiated macrophages are important tools to facilitate cross-species analysis of macrophage function...