Co-oxidation by cyclooxygenases.
Authors: Szewczuk LM, Penning TM
Abstract
Cyclooxygenases (COXs; prostaglandin H(2) synthases) catalyze the bis-dioxygenation of arachidonic acid (AA) to generate prostaglandin (PG) G(2) followed by the peroxidative cleavage of PGG(2) to yield PGH(2), the precursor to all of the vasoactive PGs. These enzymes utilize a Fe-protoporhyrin IX (heme) co-factor to catalyze peroxide bond cleavage, which puts the Fe at a higher oxidation state (Fe(3+) → Fe(5+)). The heme Fe requires two electrons (e(-)) to return to its resting state (Fe(3+)) for the next round of catalysis. Peroxide bond cleavage thus occurs via...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Measurement of Cysteine Dioxygenase Activity and Protein Abundance.
Authors: Stipanuk MH, Dominy JE, Ueki I, Hirschberger LL
Abstract
Cysteine dioxygenase is an iron (Fe(2+))-dependent thiol dioxygenase that uses molecular oxygen to oxidize the sulfhydryl group of cysteine to generate 3-sulfinoalanine (commonly called cysteinesulfinic acid). Cysteine dioxygenase activity is routinely assayed by measuring cysteinesulfinate formation from substrate L-cysteine at pH 6.1 in the presence of ferrous ions to saturate the enzyme with metal cofactor, a copper chelator to diminish substrate oxidation, and hydroxylamine to inhibit pyridoxal 5'-phosphate-dependent degradation of produ...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Analysis of arsenical metabolites in biological samples.
Authors: Hernandez-Zavala A, Drobna Z, Styblo M, Thomas DJ
Abstract
Quantitation of iAs and its methylated metabolites in biological samples provides dosimetric information needed to understand dose-response relations. Here, methods are described for separation of inorganic and mono-, di-, and trimethylated arsenicals by thin layer chromatography. This method has been extensively used to track the metabolism of the radionuclide [(73)As] in a variety of in vitro assay systems. In addition, a hydride generation-cryotrapping-gas chromatography-atomic absorption spectrometric method is described for the quanti...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Morphometric analysis in neurodegenerative disorders.
Authors: Milatovic D, Montine TJ, Zaja-Milatovic S, Madison JL, Bowman AB, Aschner M
Abstract
The study of dendritic length and spine density has become a standard in the analysis of neuronal abnormalities since a considerable number of neurological diseases have their foundation in alterations in these structures. One of the best ways to study possible alterations in neuronal morphometry is the use of Golgi impregnation. Introduced more than a century ago, it is still the standard and state-of-the-art technique for visualization of neuronal architecture. We successfully applied the Golgi method to mouse, ...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Assessment of mitochondrial dysfunction arising from treatment with hepatotoxicants.
Authors: King AL, Bailey SM
Abstract
Mitochondrial dysfunction from toxicants is recognized as a causative factor in the development of numerous liver diseases including steatohepatitis, cirrhosis, and cancer. Toxicant-mediated damage to mitochondria result in depressed ATP production, inability to maintain proper cellular calcium homeostasis, and increased reactive oxygen species production. These disruptions contribute to hepatocellular death and lead to liver pathology. Herein, we describe a series of basic and advanced methodologies that can be incorporated into research projects aimed to understand th...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
In vitro assays of inorganic arsenic methylation.
Authors: Drobna Z, Styblo M, Thomas DJ
Abstract
Inorganic arsenic is extensively metabolized to produce mono-, di-, and trimethylated products. The formation of these metabolites produces a variety of intermediates that differ from inorganic arsenic in terms of patterns of distribution and retention and in toxic effects. In order to elucidate the pathway for arsenic methylation, it was necessary to develop a reliable in vitro assay system in which the formation of methylated metabolites could be monitored. Here, in vitro assay system that uses the postmicrosomal supernate from rat liver is used as the sour...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Transcription factor Nrf2: examination of nuclear protein levels by immunoblotting and promoter response element binding by chromatin immunoprecipitation (ChIP).
Authors: Shay KP, Smith EJ, Hagen TM
Abstract
Nuclear factor erythroid 2 (NF-E2) related factor 2 (Nrf2) is a transcription factor that governs the expression of over a hundred so-called phase II detoxification and antioxidant genes that are regulated through the antioxidant response element (ARE). Loss of Nrf2 activity has been implicated in cardiovascular disease, inflammation, aging, and cancer. Nrf2 is induced to accumulate in the nucleus when the cell encounters an oxidative stress, a fact that has been exploited experimentally to test the conditions under which ARE-containing genes are expressed. The...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Purification of arsenic (+3 oxidation state) methyltransferase from rat liver cytosol.
Authors: Drobna Z, Styblo M, Thomas DJ
Abstract
Demonstrating the enzymatic basis of arsenic methylation is critical to further studies of the pathway for the conversion of inorganic arsenic into a variety of methylated metabolites. This protocol describes a procedure for the purification of an arsenic methyltransferase from rat liver cytosol. Purification of this enzyme and subsequent cloning of its gene has permitted studies of enzyme structure and function, and has lead to the identification of orthologous genes in genomes of organisms ranging in complexity from sea urchins to humans. These proteins are...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Manipulation of expression of arsenic (+3 oxidation state) methyltransferase in cultured cells.
Authors: Drobna Z, Styblo M, Thomas DJ
Abstract
Methylation of inorganic arsenic to produce mono-, di-, or trimethylated products is the central process in the cellular metabolism of arsenic. Identification of arsenic (+3 oxidation state) methyltransferase (As3mt) as the enzyme that could catalyze all the steps in the pathway for arsenic methylation suggests that expression of this enzyme could be a useful target for manipulation. Here, methods are described for heterologous expression of the rat As3mt gene in a human urothelial cell line that normally does not express this enzyme and for silencing of the ...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Measurement of cysteine S-conjugate β-lyase activity.
Authors: Cooper AJ, Krasnikov BF, Pinto JT, Bruschi SA
Abstract
Cysteine S-conjugate β-lyases are pyridoxal 5'-phosphate (PLP)-containing enzymes that catalyze the conversion of cysteine S-conjugates [RSCH(2)CH(NH(3) (+))CO(2) (-)] and selenium Se-conjugates [RSeCH(2)CH(NH(3) (+))CO(2) (-)] that contain a leaving group in the β position to pyruvate, ammonium and a sulfur-containing fragment (RSH) or selenium-containing fragment (RSeH), respectively. In mammals, at least ten PLP enzymes catalyze β-elimination reactions with such cysteine S-conjugates. All are enzymes involved in amino acid metabolism tha...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Application of single-cell microfluorimetry to neurotoxicology assays.
Authors: Limke TL, Atchison WD
Abstract
Intracellular signaling events play fundamental roles in regulating physiological function. In neurons, these include inducing growth and differentiation, secretion, gene expression, and controlling processes associated with learning and memory. All of these processes have in common the vital dependence on changes in intracellular Ca²(+) [Ca²(+)](i). Numerous toxicants, including metals, polychlorinated biphenyls, and biological neurotoxins, can disrupt [Ca²(+)](i). Understanding how toxicants disrupt Ca²(+)-dependent neuronal signaling, and thus induce neuronal ...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Neonatal rat primary microglia: isolation, culturing, and selected applications.
Authors: Ni M, Aschner M
Abstract
Microglial cells elaborate trophic factors and cytokines and remove toxins and debris from the extracellular space in the central nervous system, acting analogously to peripheral macrophages. Over the past two decades, increased attention has been directed at the role of microglia, not only in normal physiology, but also in mediating neurotoxicity. Activation of microglia is inherent to multiple neurodegenerative disorders and exposure to toxic compounds. In large measure, these revelations have come about as a result of technologies that enable researchers to obtain high ...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Overview of gastrointestinal toxicology.
Authors: Coruzzi G
Abstract
The gastrointestinal (GI) tract has the unique feature of having a huge area for exposure to potentially harmful agents, including concentrated acid, food, chemicals, and pathogens. Research over the past decades has identified some of the key events that are involved in mucosal damage and defense. The enteric nervous system, immune system, and a variety of endocrine and paracrine mediators act in concert to endure mucosal barrier integrity and gut homeostasis. Disruptors of mucosal defense mechanisms include drugs, food allergens and contaminants, metals, chemicals, radiation, ...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Methods to measure gastric mucosal lesions in the rat.
Authors: Morini G, Grandi D
Abstract
The maintenance of gastric mucosal integrity is ensured by a dynamic balance between protective and noxious factors. The gastric mucosa has multiple protective mechanisms that allow the mucosa to withstand frequent exposure to potentially damaging agents such as acid and peptic secretions, bacterial products, ingested food, alcoholic beverages, and certain drugs. The imbalance between defensive and aggressive factors is at the basis of the formation of erosions/lesions or ulcerations of the gastric mucosa. The difference between an erosion/lesion and ulceration is that ...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Assessment of enteral bacteria.
Authors: Menozzi A, Ossiprandi MC
Abstract
The disruption of intestinal barrier leads to the penetration of noxious luminal compounds into the gut wall, causing further damage. This unit describes the assessment of enteric bacteria translocation into the intestinal wall of rats, an established method for the evaluation of bowel damage to the mucosal epithelial barrier. The Basic Protocol provided in the present unit describes collection and preparation of small intestine sample, performing of sample serial dilutions for bacterial culture, performing of the culture of aerobic and anaerobic bacteria on petri...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
