A weight of evidence review on the mode of action, adversity, and the human relevance of xylene's observed thyroid effects in rats.

Xylene substances have wide industrial and consumer uses and are currently undergoing dossier and substance evaluation under Registration, Evaluation, Authorization and Restriction of Chemicals (REACH) for further toxicological testing including consideration of an additional neurotoxicological testing cohort to an extended one-generation reproduction toxicity (EOGRT) study. New repeated dose study data on xylenes identify the thyroid as a potential target tissue, and therefore a weight of evidence review is provided to investigate whether or not xylene-mediated changes on the hypothalamus-pituitary-thyroid (HPT) axis are secondary to liver enzymatic induction and are of a magnitude that is relevant for neurological human health concerns. Multiple published studies confirm xylene-mediated increases in liver weight, hepatocellular hypertrophy, and liver enzymatic induction the oral or inhalation routes, including an increase in uridine 5'-diphospho-glucuronosyltransferase (UDP-GT) activity, the key step in thyroid hormone metabolism in rodents.

Comparative evaluation of rat and human in vitro assays for evaluation of thyroid toxicity.

The effects of ten test chemicals towards thyroid sodium-iodide symporter (NIS), thyroid peroxidase (TPO), and deiodinases (DIOs) type I, II, and III were evaluated in in vitro rat and human systems and compared. Test chemicals known to directly affect TH levels in vivo were confirmed to effectively inhibit at least one of the tested in vitro endpoints, without significant disparities between species, and the tested compounds known to not affect thyroid function, were found ineffective. Interestingly, Iodide Transport Blocker 5, a potent non-competitive iodine uptake inhibitor, exhibited effects beyond direct NIS inhibition, by impacting NIS function through ATP depletion, and also inhibited TPO and DIO1/2 enzymes, although to a lesser extent.

Xylene: weight of evidence approach case study to determine the need for an extended one generation reproductive study with a developmental neurotoxicity animal cohort.

Xylene is a high production volume chemical that is widely used as a solvent and polymer precursor, and is currently undergoing substance evaluation under Registration, Evaluation, Authorization and Restriction of Chemicals (REACH). Xylenes recently received testing decisions on one-generation reproductive toxicity (EOGRT) studies with additional developmental neurotoxicity (DNT) cohorts for each of the three isomers. Xylene presents a unique opportunity to investigate the need for additional animal DNT toxicology testing because it is a legacy industrial chemical for which a significant amount of animal and human data already exists on its toxicity profile, including central nervous system effects.

The short-term toxicity and metabolome of Benzene.

A 14-day rat study with plasma metabolomics was conducted to evaluate the toxicity of Benzene. Wistar rats were orally administered Benzene daily at doses of 0, 300 and 1000 mg/kg bw. The study identified liver and kidneys as target organs of Benzene toxicity and found reductions in total white blood cells, absolute lymphocyte and eosinophil cell counts, and increased relative monocyte counts suggesting bone marrow as a target organ.

The short-term toxicity and metabolome of dicyclopentadiene.

Dicyclopentadiene (DCPD) was investigated in a 14-day oral rat toxicity study based on the OECD 407 guideline in combination with plasma metabolomics. Wistar rats received the compound daily via gavage at dose levels of 0, 50 and 150 mg/kg bw. The high dose induced transient clinical signs of toxicity and in males only reduced body weight gain.

Differential lymphatic versus portal vein uptake of the synthetic pyrethroids deltamethrin and cis-permethrin in rats.

The objective of this study was to obtain data on pathways of absorption of the synthetic pyrethroids deltamethrin (DLM) and cis-permethrin (CPM) following oral administration to rats. Adult male Sprague-Dawley rats with cannulated mesenteric lymph ducts and hepatic portal veins were given single doses of either 5 mg/kg DLM or 60 mg/kg CPM via the duodenum and lymph and portal blood samples collected for up to 300 min. The pyrethroid dosing vehicles (5 mL/kg body weight) were either corn oil or glycerol formal.

Kinetics of metabolism of deltamethrin and - and -permethrin . Studies using rat and human liver microsomes, isolated rat hepatocytes and rat liver cytosol.

The kinetics of metabolism of deltamethrin (DLM) and - and -permethrin (CPM and TPM) was studied in male Sprague-Dawley rat and human liver microsomes. DLM metabolism kinetics was also studied in isolated rat hepatocytes, liver microsomes and cytosol. Apparent intrinsic clearance (CL) values for the metabolism of DLM, CPM and TPM by cytochrome P450 (CYP) and carboxylesterase (CES) enzymes in rat and human liver microsomes decreased with increasing microsomal protein concentration.

Combining In Vivo and Organotypic In Vitro Approaches to Assess the Human Relevance of Basimglurant (RG7090), a Potential CAR Activator.

Basimglurant (RG7090), a small molecule under development to treat certain forms of depression, demonstrated foci of altered hepatocytes in a long-term rodent-toxicity study. Additional evidence pointed toward the activation of the constitutive androstane receptor (CAR), an established promoter of nongenotoxic and rodent-specific hepatic tumors. This mode of action and the potential human relevance was explored in vivo using rodent and cynomolgus monkey models and in vitro using murine and human liver spheroids.

An assay for screening xenobiotics for inhibition of rat thyroid gland peroxidase activity.

1. A number of chemicals have been shown to produce disruption of the thyroid gland, resulting in reduced thyroid hormone synthesis, by a mechanism involving inhibition of thyroid peroxidase (TPO) activity (EC 1.11.

Comparison of the effects of sodium phenobarbital in wild type and humanized constitutive androstane receptor (CAR)/pregnane X receptor (PXR) mice and in cultured mouse, rat and human hepatocytes.

Phenobarbital (PB), a constitutive androstane receptor (CAR) activator, produces liver tumours in rodents by a mitogenic mode of action involving CAR activation. In this study, the hepatic effects of sodium phenobarbital (NaPB) were compared in male C57BL/6J wild type (WT) mice and in humanized mice, where both the mouse CAR and pregnane X receptor (PXR) have been replaced by their human counterparts (hCAR/hPXR mice). Investigations were also performed in cultured male C57BL/6J and CD-1 mouse, male Sprague-Dawley rat and male and female human hepatocytes.

Mode of action and human relevance of THF-induced mouse liver tumors.

In a National Toxicology Program (NTP) bioassay, inhalation of tetrahydrofuran (THF) induced liver tumors in female B6C3F mice but not in male mice or rats of either sex. Since THF is not genotoxic, the NTP concluded this carcinogenic activity was likely mediated via non-genotoxic modes of action (MOA). Based on evidence that THF and phenobarbital share a similar MOA, female Car/Pxr knock-out mice were orally exposed to THF to evaluate the potential role of CAR activation in the MOA for THF-induced liver tumors.

PPARα Is Required for PPARδ Action in Regulation of Body Weight and Hepatic Steatosis in Mice.

Peroxisome proliferator activated receptors alpha (PPARα) and delta (PPARδ) belong to the nuclear receptor superfamily. PPARα is a target of well established lipid-lowering drugs. PPARδ (also known as PPARβ/δ) has been investigated as a promising antidiabetic drug target; however, the evidence in the literature on PPARδ effect on hepatic lipid metabolism is inconsistent.

Analysis of the role of Nrf2 in the expression of liver proteins in mice using two-dimensional gel-based proteomics.

Background: The transcription factor Nrf2 regulates expression of multiple cellular defence proteins through the antioxidant response element (ARE). Nrf2-deficient mice (Nrf2(-/-)) are highly susceptible to xenobiotic-mediated toxicity, but it is not known whether this reflects low basal expression or reduced inducibility of Nrf2-regulated genes in response to chemical insults.

Methods: Wild type and Nrf2(-/-) mice were fed diet supplemented with the established Nrf2 inducer butylated hydroxyanisole (BHA) [0.

Conditional Expression of Human PPARδ and a Dominant Negative Variant of hPPARδ In Vivo.

The nuclear receptor, NR1C2 or peroxisome proliferator-activated receptor (PPAR)-δ, is ubiquitously expressed and important for placental development, fatty acid metabolism, wound healing, inflammation, and tumour development. PPARδ has been hypothesized to function as both a ligand activated transcription factor and a repressor of transcription in the absence of agonist. In this paper, treatment of mice conditionally expressing human PPARδ with GW501516 resulted in a marked loss in body weight that was not evident in nontransgenic animals or animals expressing a dominant negative derivative of PPARδ.

Mechanisms of induction of cytosolic and microsomal glutathione transferase (GST) genes by xenobiotics and pro-inflammatory agents.

Glutathione transferase (GST) isoezymes are encoded by three separate families of genes (designated cytosolic, microsomal and mitochondrial transferases), with distinct evolutionary origins, that provide mammalian species with protection against electrophiles and oxidative stressors in the environment. Members of the cytosolic class Alpha, Mu, Pi and Theta GST, and also certain microsomal transferases (MGST2 and MGST3), are up-regulated by a diverse spectrum of foreign compounds typified by phenobarbital, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, pregnenolone-16α-carbonitrile, 3-methylcholanthrene, 2,3,7,8-tetrachloro-dibenzo-p-dioxin, β-naphthoflavone, butylated hydroxyanisole, ethoxyquin, oltipraz, fumaric acid, sulforaphane, coumarin, 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole, 12-O-tetradecanoylphorbol-13-acetate, dexamethasone and thiazolidinediones. Collectively, these compounds induce gene expression through the constitutive androstane receptor (CAR), the pregnane X receptor (PXR), the aryl hydrocarbon receptor (AhR), NF-E2-related factor 2 (Nrf2), peroxisome proliferator-activated receptor-γ (PPARγ) and CAATT/enhancer binding protein (C/EBP) β.

The cap'n'collar transcription factor Nrf2 mediates both intrinsic resistance to environmental stressors and an adaptive response elicited by chemopreventive agents that determines susceptibility to electrophilic xenobiotics.

Transcription factor Nrf2 regulates genes encoding drug-metabolising enzymes and drug transporters, as well as enzymes involved in the glutathione, thioredoxin and peroxiredoxin antioxidant pathways. Using mouse embryonic fibroblast (MEF) cells from Nrf2(+/+) and Nrf2(-/-) mice, in conjunction with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay, we have shown that loss of Nrf2 diminishes the intrinsic resistance of mutant fibroblasts towards isothiocyanates (i.e.

Proteomic analysis of Nrf2 deficient transgenic mice reveals cellular defence and lipid metabolism as primary Nrf2-dependent pathways in the liver.

The transcription factor Nrf2 regulates expression of multiple cellular defence proteins through the antioxidant response element (ARE). Nrf2-deficient mice (Nrf2(-/-)) are highly susceptible to xenobiotic-mediated toxicity, but the precise molecular basis of enhanced toxicity is unknown. Oligonucleotide array studies suggest that a wide range of gene products is altered constitutively, however no equivalent proteomics analyses have been conducted.

Activation of the NRF2 signaling pathway by copper-mediated redox cycling of para- and ortho-hydroquinones.

Transcription factor NF-E2 p45-related factor 2 (Nrf2) mediates adaptation to oxidants and electrophiles through up-regulating genes that contain antioxidant response elements (AREs) in their promoters. Using the stably transfected human AREc32 reporter cell line, we found that copper and other transition metals enhanced induction of ARE-driven luciferase by 2-tert-butyl-1,4-hydroquinone (tBHQ) as a result of increased oxidation to 2-tert-butyl-1,4-benzoquinone (tBQ). Following exposure to tBHQ for 30 min, ARE-luciferase activity measured after 24 hr was dependent on the presence of Cu(2+).

Expression and localization of rat aldo-keto reductases and induction of the 1B13 and 1D2 isoforms by phenolic antioxidants.

The aldo-keto reductase (AKR) phase I drug metabolism enzyme superfamily is implicated in detoxification or bioactivation of a wide variety of carbonyl-bearing compounds. In this study, we have used antibodies raised against purified recombinant rat AKR isoforms 1A3, 1B4, 1C9, 1D2, and 7A1 to characterize the expression profile of these superfamily members in the rat and define their localization by immunohistochemistry. Western blotting showed that AKR1A3, AKR1B4, and AKR1C9 are ubiquitously expressed, whereas AKR1D2 and AKR7A1 are present in liver, adrenal gland, and kidney, with the latter also present in testis, spleen, and stomach.

Characterization of the cancer chemopreventive NRF2-dependent gene battery in human keratinocytes: demonstration that the KEAP1-NRF2 pathway, and not the BACH1-NRF2 pathway, controls cytoprotection against electrophiles as well as redox-cycling compounds.

To better understand the role of transcription factor NF-E2-related factor (NRF) 2 in the human and its contribution to cancer chemoprevention, we have knocked down its negative regulators, Kelch-like ECH-associated protein 1 (KEAP1) and broad-complex, tramtrack and bric à brac and cap'n'collar homology 1 (BACH1), in HaCaT keratinocytes. Whole-genome microarray revealed that knockdown of KEAP1 resulted in 23 messenger RNAs (mRNAs) being up-regulated > or = 2.0-fold.

Transcription factor Nrf2 mediates an adaptive response to sulforaphane that protects fibroblasts in vitro against the cytotoxic effects of electrophiles, peroxides and redox-cycling agents.

Sulforaphane can stimulate cellular adaptation to redox stressors through transcription factor Nrf2. Using mouse embryonic fibroblasts (MEFs) as a model, we show herein that the normal homeostatic level of glutathione in Nrf2(-/-) MEFs was only 20% of that in their wild-type counterparts. Furthermore, the rate of glutathione synthesis following its acute depletion upon treatment with 3 micromol/l sulforaphane was very substantially lower in Nrf2(-/-) MEFs than in wild-type cells, and the rebound leading to a approximately 1.

Induction of sulfiredoxin expression and reduction of peroxiredoxin hyperoxidation by the neuroprotective Nrf2 activator 3H-1,2-dithiole-3-thione.

Peroxiredoxins are an important family of cysteine-based antioxidant enzymes that exert a neuroprotective effect in several models of neurodegeneration. However, under oxidative stress they are vulnerable to inactivation through hyperoxidation of their active site cysteine residues. We show that in cortical neurons, the chemopreventive inducer 3H-1,2-dithiole-3-thione (D3T), that activates the transcription factor Nuclear factor erythroid 2-related factor (Nrf2), inhibits the formation of inactivated, hyperoxidized peroxiredoxins following oxidative trauma, and protects neurons against oxidative stress.

Induction of cancer chemopreventive enzymes by coffee is mediated by transcription factor Nrf2. Evidence that the coffee-specific diterpenes cafestol and kahweol confer protection against acrolein.

Mice fed diets containing 3% or 6% coffee for 5 days had increased levels of mRNA for NAD(P)H:quinone oxidoreductase 1 (NQO1) and glutathione S-transferase class Alpha 1 (GSTA1) of between 4- and 20-fold in the liver and small intestine. Mice fed 6% coffee also had increased amounts of mRNA for UDP-glucuronosyl transferase 1A6 (UGT1A6) and the glutamate cysteine ligase catalytic (GCLC) subunit of between 3- and 10-fold in the small intestine. Up-regulation of these mRNAs was significantly greater in mice possessing Nrf2 (NF-E2 p45 subunit-related factor 2) than those lacking the transcription factor.

Transcription factor Nrf2 is essential for induction of NAD(P)H:quinone oxidoreductase 1, glutathione S-transferases, and glutamate cysteine ligase by broccoli seeds and isothiocyanates.

Cruciferous vegetables contain glucosinolates that, after conversion to isothiocyanates (ITC), are capable of inducing cytoprotective genes. We examined whether broccoli seeds can elicit a chemoprotective response in mouse organs and rodent cell lines and investigated whether this response requires nuclear factor-erythroid 2 p45-related factor 2 (Nrf2). The seeds studied contained glucosinolate at 40 mmol/kg, of which 59% comprised glucoiberin, 19% sinigrin, 8% glucoraphanin, and 7% progoitrin.