Toxicological Sciences - recent issues

Nicotine and Type 2 Diabetes

Borowitz, J. L., Isom, G. E. — 2008-05-07

miRNAs: Effectors of Environmental Influences on Gene Expression and Disease

Hudder, A., Novak, R. F. — 2008-05-07

Discovered less than a decade ago, micro-RNAs (miRNAs) have emerged as important regulators of gene expression in mammals. They consist of short nucleic acids, on average ~22 nucleotides in length. The miRNAs exert their effect by binding directly to target messenger RNAs (mRNAs) and inhibiting mRNA stability and translation. Each miRNA can bind to multiple targets and many miRNAs can bind to the same target mRNA, allowing for a complex pattern of regulation of gene expression. Once bound to their targets, miRNAs can suppress translation of the mRNA by either sequestration or degradation of the message. Thus, miRNAs function as powerful and sensitive posttranscriptional regulators of gene expression. This review will summarize what is known about miRNA biogenesis, expression, regulation, function, mode of action, and role in disease processes with an emphasis on miRNAs in mammals. We discuss some of the methodology employed in miRNA research and the potential of miRNAs as therapeutic targets. The role of miRNAs in signal transduction and cellular stress is reviewed. Lastly, we identify new exciting avenues of research on the role of miRNAs in toxicogenomics and the possibility of epigenetic effects on gene expression.

Tissue Exposures to Free and Glucuronidated Monobutylyphthalate in the Pregnant and Fetal Rat following Exposure to Di-n-butylphthalate: Evaluation with a PBPK Model

2008-05-07

Human exposure to phthalic acid diesters occurs through a variety of pathways as a result of their widespread use in plastics. Repeated doses of di-n-butylphthalate (DBP) from gestation day (GD) 12 to 19 disrupt testosterone synthesis and male sexual development in the fetal rat. To gain a better understanding of the relationship of the target tissue (testes) dose to observed developmental effects, the pharmacokinetics of monobutyl phthalate (MBP) and its glucuronide (MBP-G) were examined in pregnant and fetal rats following single and repeated administration of DBP from GD 12–19. These data, together with results from previously published studies, were used to develop a physiologically based pharmacokinetic model for DBP and its metabolites in the male, pregnant and fetal rat. The model structure accounts for the major metabolic (hydrolysis, glucuronidation, oxidative metabolism) and transport processes (enterohepatic recirculation, urinary and fecal excretion, placental transfer). Extrapolation of the validated adult male rat model to gestation successfully predicts MBP and MBP-G levels in maternal plasma, placenta and urine, as well as the fetal plasma and testes. Sensitivity analysis indicates that plasma MBP kinetics are particularly sensitive to glucuronidation and enterohepatic recirculation: a decrease in the uridine 5'-diphospho-glucuronosyltransferase (UDPGT) capacity during gestation results in an increased MBP residence time, and saturation of UDPGT at the highest doses (> 100 mg/kg/day) causes a flattening out of the plasma time course data. Oxidative metabolism plays a significant role in elimination only at low doses (< 50 mg/kg DBP). Insights gained from modeling of the rat data will be used to support development of a human PBPK model for DBP.

Tumor Promotion in Liver of Mice with a Conditional Cx26 Knockout

2008-05-07

Connexin (Cx) 26 and 32 are the major gap junction proteins in liver. We recently demonstrated that Cx32 is essential for phenobarbital (PB)–mediated tumor promotion in mouse liver. To investigate whether Cx26 plays a similar role, an initiation-promotion experiment was conducted using mice with a liver-specific knockout of Cx26. Control and Cx26-deficient mice were injected a single dose of N-nitrosodiethylamine (DEN, 90 µg/g b.wt.) at 6 weeks of age and groups of mice were subsequently kept on a PB (0.05%) containing or control diet for 35 weeks. At the end of the experiment, the carcinogenic response in the liver was monitored. Mice from PB treatment groups showed strongly increased liver weights compared with mice treated with DEN alone, which was mostly due to a much higher tumor burden. The tumor response in PB-treated mice of both strains was quite similar, but the number of smaller tumors and of enzyme-altered neoplastic lesions was somewhat larger in PB-treated Cx26 knockout (Cx26 KO) compared with wild-type mice, whereas the volume fraction of enzyme-altered lesions was slightly reduced in PB-treated Cx26-deficient mice. There was no significant difference in tumor prevalence between Cx26 KO and wild-type mice. Altogether our present data show that elimination of Cx26 has only minor effects on chemically induced mouse hepatocarcinogenesis, in striking contrast to the effects seen in Cx32 KO mice.

Phthalate Induction of CYP3A4 is Dependent on Glucocorticoid Regulation of PXR Expression

Cooper, B. W., Cho, T. M., Thompson, P. M., Wallace, A. D. — 2008-05-07

Cytochrome P450 3A4 (CYP3A4) is responsible for oxidative metabolism of more than 60% of all pharmaceuticals. CYP3A4 is inducible by xenobiotics that activate pregnane X receptor (PXR), and enhanced CYP3A4 activity has been implicated in adverse drug interactions. Recent evidence suggest that the widely used plasticizer, di-2-ethylhexyl phthalate (DEHP), and its primary metabolite mono-2-ethylhexyl phthalate (MEHP) may act as agonists for PXR. Hospital patients are uniquely exposed to high levels of DEHP as well as being administered glucocorticoids. Glucocorticoids positively regulate PXR expression in a glucocorticoid receptor (GR)–mediated mechanism. We suggest that the magnitude of CYP3A4 induction by phthalates is dependent on the expression of PXR and may be significantly higher in the presence of glucocorticoids. DEHP and MEHP induced PXR-mediated transcription of the CYP3A4 promoter in a dose-dependent fashion. Coexposure to phthalates and dexamethasone (Dex) resulted in enhanced CYP3A4 promoter activity; furthermore, this induction was abrogated by both the GR antagonist RU486 and GR small interfering ribonucleic acid. Dex induced PXR protein expression in human hepatocytes and a liver-derived rat cell line. CYP3A4 protein was highly induced by Dex and DEHP coadministration in human hepatocyte cultures. Finally, enhanced 6β-hydroxytestosterone formation in Dex and phthalate cotreated human hepatocytes confirmed CYP3A4 enzyme induction. Concomitant exposure to glucocorticoids and phthalates resulting in enhanced metabolic activity of CYP3A4 may play a role in altered efficacy of pharmaceutical agents. Understanding the role of glucocorticoid regulation of PXR as a key determinant in the magnitude of CYP3A4 induction by xenobiotics may provide insight into adverse drug effects in a sensitive population.

Clinical Manifestations and Arsenic Methylation after a Rare Subacute Arsenic Poisoning Accident

Xu, Y., Wang, Y., Zheng, Q., Li, B., Li, X., Jin, Y., Lv, X., Qu, G., Sun, G. — 2008-05-07

One hundred and four workers ingested excessive levels of arsenic in an accident caused by leakage of pipeline in a copper-smelting factory. Clinical examinations were performed by physicians in a local hospital. Excreted urinary arsenic species were determined by cold trap hydride generation atomic absorption spectrometry. In the initial toxic phase, gastrointestinal symptoms were predominant (83 people, 79.8%). Most patients showed leucopenia (72 people, 69.2%), and increased serum alanine aminotransferase (84 people, 80.8%) and aspartate aminotransferase (58 people, 55.8%). Thirty-five patients (33.6%) had elevated red blood cells in urine. After 17 days of admission, many subjects (45 people, 43.3%) developed peripheral neuropathy and 25 of these 45 patients (24.0%) showed a decrease in motor and sensory nerve conduction velocity. In the comparison of urinary arsenic metabolites among subacute arsenic-poisoned, chronic high arsenic–exposed and control subjects, we found that subacute arsenic-poisoned patients had significantly elevated proportions of urinary inorganic arsenic (iAs) and methylarsonic acid (MMA) but reduced proportion of urinary dimethylarsinic acid (DMA) compared with chronic high arsenic–exposed and control subjects. Chronic exposed subjects excreted higher proportions of iAs and MMA but lower proportions of DMA in urine compared with control subjects. These results suggest that gastrointestinal symptoms, leucopenia, and hepatic and urinary injury are predominant in the initial phase of subacute arsenic poisoning. Peripheral neuropathy is the most frequent manifestation after the initial phase. The biomethylation of arsenic decreases in a dose rate–dependent manner.

Comparative Temporal Toxicogenomic Analysis of TCDD- and TCDF-Mediated Hepatic Effects in Immature Female C57BL/6 Mice

2008-05-07

Temporal analyses were performed on hepatic tissue from immature female C57BL/6 mice in order to compare the gene expression profiles for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 2,3,7,8-tetrachlorodibzofuran (TCDF). Time course studies conducted with a single oral dose of 300 µg/kg TCDF or 30 µg/kg TCDD were used to compare differential gene expression on complementary DNA microarrays containing 13,361 features, representing 8194 genes at 2, 4, 8, 12, 24, 72, 120, and 168 h. One hundred and ninety-five genes were identified as differentially regulated by TCDF, of which 116 genes were in common with TCDD, with 109 exhibiting comparable expression profiles (correlation coefficients > 0.3). In general, TCDF was less effective in eliciting hepatic vacuolization, and differential gene expression compared with TCDD when given at an equipotent dose based on a toxic equivalence factor (TEF) of 0.1 for TCDF, especially 72-h postadministration. For example, the induction of Cyp1a1 messenger RNA by TCDF was less when compared TCDD. Moreover, TCDF induced less severe hepatocyte cytoplasmic vacuolization consistent with lower lipid accumulations which significantly subsided by 120 and 168 h when compared with TCDD. TCDF-elicited responses correlated with their hepatic tissue levels which gradually decreased between 18 and 168 h. Although both compounds elicited comparable gene expression profiles, especially at early time points, the TCDF responses were generally weaker. Collectively, these results suggest that the weaker TCDF responses could be attributed to differences in pharmacokinetics. However, more comprehensive dose–response studies are required at optimal times for each end point of interest in order to investigate the effect of pharmacokinetic differences on relative potencies that are important in establishing TEFs.

Liver Genomic Responses to Ciguatoxin: Evidence for Activation of Phase I and Phase II Detoxification Pathways following an Acute Hypothermic Response in Mice

2008-05-07

Ciguatoxins (CTX) are polyether neurotoxins that target voltage-gated sodium channels and are responsible for ciguatera, the most common fish-borne food poisoning in humans. This study characterizes the global transcriptional response of mouse liver to a symptomatic dose (0.26 ng/g) of the highly potent Pacific ciguatoxin-1 (P-CTX-1). At 1 h post-exposure 2.4% of features on a 44K whole genome array were differentially expressed (p ≤ 0.0001), increasing to 5.2% at 4 h and decreasing to 1.4% by 24 h post-CTX exposure. Data were filtered (|fold change| ≥ 1.5 and p ≤ 0.0001 in at least one time point) and a trend set of 1550 genes were used for further analysis. Early gene expression was likely influenced prominently by an acute 4°C decline in core body temperature by 1 h, which resolved by 8 h following exposure. An initial downregulation of 32 different solute carriers, many involved in sodium transport, was observed. Differential gene expression in pathways involving eicosanoid biosynthesis and cholesterol homeostasis was also noted. Cytochrome P450s (Cyps) were of particular interest due to their role in xenobiotic metabolism. Twenty-seven genes, mostly members of Cyp2 and Cyp4 families, showed significant changes in expression. Many Cyps underwent an initial downregulation at 1 h but were quickly and strongly upregulated at 4 and 24 h post-exposure. In addition to Cyps, increases in several glutathione S-transferases were observed, an indication that both phase I and phase II metabolic reactions are involved in the hepatic response to CTX in mice.

Khat (Catha edulis) Induces Reactive Oxygen Species and Apoptosis in Normal Human Oral Keratinocytes and Fibroblasts

2008-05-07

Khat chewing is widely practiced in Eastern Africa and the Middle East. Khat is genotoxic to cells within the oral mucosa, and several studies have suggested an association between khat use and oral lesions like hyperkeratosis and oral cancer. This study investigated the mechanism of khat-induced cytotoxicity using primary normal human oral keratinocytes (NOK) and fibroblasts (NOF). Khat induced rounding up of cells, plasma membrane blebbing, and condensation of nuclear chromatin within 3–6 h of exposure. The cells also showed externalization of phosphatidylserine and fragmentation of DNA. Morphological and biochemical features were compatible with cell death by apoptosis. Khat also induced an increase in cytosolic reactive oxygen species (ROS) and a depletion of intracellular glutathione (GSH) within 1 h of exposure. Antioxidants reduced ROS generation, GSH depletion and delayed the onset of cytotoxicity in both cell types. Generally, NOF cells were more sensitive to khat-induced cytotoxicity than NOK cells. These effects were elicited at concentrations of khat expected to occur in the oral cavity during khat chewing. In summary, khat induced apoptotic cell death in primary normal oral keratinocytes and fibroblasts by an early effect on mechanisms that regulate oxidative stress.

Heme-oxygenase 1 Gene Expression is a Marker for Hexavalent Chromium-Induced Stress and Toxicity in Human Dermal Fibroblasts

Joseph, P., He, Q., Umbright, C. — 2008-05-07

Several adverse health effects, including irritant and allergic contact dermatitis, have been reported among workers who are occupationally exposed to chromium-containing compounds. Human dermal fibroblasts were used as an in vitro experimental model to study the potential mechanisms underlying hexavalent chromium [Cr(VI)]–induced dermal toxicity. Exposure of the fibroblasts to 5µM Cr(VI) (LC50 for a 24-h exposure period) followed by microarray analysis of the gene expression profile revealed overexpression of several genes including those involved in cell stress response. The cellular level of glutathione, the major antioxidant molecule present in the cells, was significantly lower in the Cr(VI)-treated cells compared to the corresponding control cells. The Cr(VI)-induced overexpression of heme-oxygenase 1 messenger RNA (HO-1) in the fibroblasts was significantly blocked by actinomycin D and by inhibitors of MAP kinase pathways. The Cr(VI)-induced cytotoxicity and the overexpression of the HO-1 gene were dependent on the glutathione level of the fibroblasts. Buthionine sulfoximine-mediated GSH depletion resulted in enhanced Cr(VI) cytotoxicity and further overexpression of the HO-1 gene. On the other hand, elevated cellular levels of glutathione resulting from pretreating the cells with GSH significantly protected the cells against the Cr(VI)-induced cytotoxicity and blocked the HO-1 gene's overexpression. Pretreating the fibroblasts with N-acetyl cysteine also significantly reduced the Cr(VI)-induced cytotoxicity and overexpression of the HO-1 gene. In conclusion, depletion of GSH leading to cellular stress is a major mechanism responsible for Cr(VI)-induced cytotoxicity. Furthermore, the expression level of HO-1 gene is a marker for Cr(VI)-induced cell stress leading to cytotoxicity.

In Vitro Assessment of Mitochondrial Dysfunction and Cytotoxicity of Nefazodone, Trazodone, and Buspirone

2008-05-07

Mitochondrial toxicity is increasingly implicated in a host of drug-induced organ toxicities, including hepatotoxicity. Nefazodone was withdrawn from the U.S. market in 2004 due to hepatotoxicity. Accordingly, we evaluated nefazodone, another triazolopyridine trazodone, plus the azaspirodecanedione buspirone, for cytotoxicity and effects on mitochondrial function. In accord with its clinical disposition, nefazodone was the most toxic compound of the three, trazodone had relatively modest effects, whereas buspirone showed the least toxicity. Nefazodone profoundly inhibited mitochondrial respiration in isolated rat liver mitochondria and in intact HepG2 cells where this was accompanied by simultaneous acceleration of glycolysis. Using immunocaptured oxidative phosphorylation (OXPHOS) complexes, we identified Complex 1, and to a lesser amount Complex IV, as the targets of nefazodone toxicity. No inhibition was found for trazodone, and buspirone showed 3.4-fold less inhibition of OXPHOS Complex 1 than nefazodone. In human hepatocytes that express cytochrome P450, isoform 3A4, after 24 h exposure, nefazodone and trazodone collapsed mitochondrial membrane potential, and imposed oxidative stress, as detected via glutathione depletion, leading to cell death. Our results suggest that the mitochondrial impairment imposed by nefazodone is profound and likely contributes to its hepatotoxicity, especially in patients cotreated with other drugs with mitochondrial liabilities.

Na+/H+ Exchanger-1 Inhibitors Reduce Neuronal Excitability and Alter Na+ Channel Inactivation Properties in Rat Primary Sensory Neurons

Liu, C.-N., Somps, C. J. — 2008-05-07

Inhibitors of the Na⁺/H⁺ exchanger isoform 1 (NHE-1) have been associated with peripheral neuropathy in rats and dogs. Recent studies suggest that NHE-1 plays an important role in mediating neuronal excitability. To investigate potential NHE-1-mediated mechanisms contributing to neuronal toxicity, we studied the effects of NHE-1 inhibitors on nerve and dorsal root ganglion (DRG) neurons isolated from the adult rat. Compound action potentials (CAPs) were recorded from electrically stimulated sections of isolated sciatic nerve/DRG/root preparations. Whole-cell patch-clamp technique was used to record fast and slow voltage-dependent Na⁺ currents from dissociated DRG neurons (29–41 µm). Exposures to 1 and 10µM of a selective NHE-1 inhibitor reduced the amplitude of the CAP recorded from the dorsal root by 33% and 58%, respectively (p < 0.05). The compound had no effect on CAPs recorded from the ventral root. Perfusion of dissociated DRG neurons with NHE-1 inhibitors at 10 and 100µM shifted voltage-dependent inactivation curves of fast Na⁺ current by as much as 11 mV (p < 0.001) in the hyperpolarizing direction. No shift was observed in slow Na⁺ currents. No statistically significant drug effects were observed on voltage-dependent activation or recovery from inactivation of either fast or slow Na⁺ currents. These results suggest that NHE-1 inhibitors may reduce peripheral neuronal excitability by shifting fast Na⁺ channels into the inactivated state under physiological conditions. Such effects may underlie peripheral neuropathies reported in rats and dogs with NHE-1 inhibitors.

Neurotoxicological Evaluation of Long-Term Lanthanum Chloride Exposure in Rats

He, X., Zhang, Z., Zhang, H., Zhao, Y., Chai, Z. — 2008-05-07

With their widespread application in industry, agriculture, medicine, and daily life, rare earth elements (REEs) are widely used in various fields and eventually accumulated in human body. Therefore, understanding the effects of REEs on health has become more and more important. In this work, the neurotoxicity of lanthanum (La) was evaluated. Wistar rats were exposed to lanthanum chloride through oral administration at 0, 0.1, 2, and 40 mg/kg doses from gestation day 0 through 6 months of age. Experiments were carried out to reveal the effects of La exposure on brain functions from four aspects including behavioral performance, [Ca²⁺]_i level and the activity of Ca²⁺-ATPase (adenosine triphosphatase) in hippocampal cells, oxidative stress, and Nissl staining. Adverse effects were observed in 2 and 40 mg/kg dose groups and increased with dose. Morris water maze test showed that La exposure at 2 and 40 mg/kg could significantly impair the behavioral performance. (The preference for the target quadrant decreased by 16.6% and 19.4% versus control, respectively.) The neurotoxicological consequences demonstrated that the alteration in homeostasis of [Ca²⁺]_i/Ca²⁺-ATPase (the ratio of [Ca²⁺]_i vs. Ca²⁺-ATPase activity increased by 44% in rats of 40 mg/kg group), the inhibition to activities of antioxidant enzymes, and the subsequent cell damage (18% and 23% cell loss in CA3 subregion of rats in 2 and 40 mg/kg group, respectively) might be involved in the neurological adverse effects of REEs exposure.

Increased Pancreatic Beta-Cell Apoptosis following Fetal and Neonatal Exposure to Nicotine Is Mediated via the Mitochondria

Bruin, J. E., Gerstein, H. C., Morrison, K. M., Holloway, A. C. — 2008-05-07

In Canada, nicotine replacement therapy is recommended as a safe smoking cessation aid for pregnant women. However, we have shown in an animal model that fetal and neonatal nicotine exposure causes increased beta-cell apoptosis and loss of beta-cell mass, which leads to the development of postnatal dysglycemia and obesity. The goal of this study was to determine whether the observed beta-cell apoptosis is mediated via the mitochondrial and/or death receptor pathway. Female Wistar rats were given saline (control) or nicotine bitartrate (1 mg/kg/day) via sc injection for 2 weeks prior to mating until weaning (postnatal day 21). At weaning, pancreas tissue was collected for Western blotting, electron microscopy (EM), and immunohistochemistry. Key markers of each apoptotic pathway were examined in whole pancreas homogenates and mitochondrial/cytosolic pancreas fractions. In the death receptor pathway, Fas and soluble Fas ligand (FasL) protein were significantly increased in the nicotine-exposed offspring compared to control animals; there was no difference in the ratio of inactive/active caspase-8 or membrane-bound FasL expression. In the mitochondrial pathway, there was a significant increase in the ratio of Bcl2/Bax, Bax translocation to the mitochondria, cytochrome c release to the cytosol, and the ratio of active/inactive caspase-3 in nicotine-exposed offspring relative to control animals. Furthermore, increased mitochondrial swelling was observed by EM in the pancreatic beta cells of nicotine-exposed offspring. Taken together, these data suggest that beta-cell apoptosis following developmental nicotine exposure is mediated via the mitochondria.

Evaluation of Putative Biomarkers of Nephrotoxicity after Exposure to Ochratoxin A In Vivo and In Vitro

Rached, E., Hoffmann, D., Blumbach, K., Weber, K., Dekant, W., Mally, A. — 2008-05-07

The kidney is one of the main targets of xenobiotic-induced toxicity, but early detection of renal damage is difficult. Recently, several novel biomarkers of nephrotoxicity have been identified by transcription profiling, including kidney injury molecule-1 (Kim-1), lipocalin-2, tissue inhibitor of metalloproteinases-1 (Timp-1), clusterin, osteopontin (OPN), and vimentin, and suggested as sensitive endpoints for acute kidney injury in vivo. However, it is not known if these cellular marker molecules may also be useful to predict chronic nephrotoxicity or to detect nephrotoxic effects in vitro. In this study, a panel of new biomarkers of renal toxicity was assessed via quantitative real-time PCR, immunohistochemistry, and immunoblotting in rats treated with the nephrotoxin ochratoxin A (OTA) for up to 90 days and in rat proximal tubule cells (NRK-52E) treated with OTA in vitro. Repeated administration of OTA to male F344/N rats for 14, 28, or 90 days resulted in a dose- and time-dependent increase in the expression of Kim-1, Timp-1, lipocalin-2, OPN, clusterin, and vimentin. Changes in gene expression were found to correlate with the progressive histopathological alterations and preceded effects on traditional clinical parameters indicative of impaired kidney function. Induction of Kim-1 messenger RNA expression was the earliest and most prominent response observed, supporting the use of this marker as sensitive indicator of chronic kidney injury. In contrast, no significant increase in the expression of putative marker genes and proteins were evident in NRK-52E cells after exposure to OTA for up to 48 h, suggesting that they may not be suitable endpoints for sensitive detection of nephrotoxic effects in vitro.

Fabp3 as a Biomarker of Skeletal Muscle Toxicity in the Rat: Comparison with Conventional Biomarkers

2008-05-07

Fatty acid binding protein 3 (Fabp3) has been used as a serological biomarker of cardiac injury, but its utility as a preclinical biomarker of injury to skeletal muscle is not well described. Fabp3 concentrations were determined for tissues from Sprague–Dawley rats and found to occur at highest concentrations in cardiac muscle and in skeletal muscles containing an abundance of type I fibers, such as the soleus muscle. Soleus is also a primary site of skeletal muscle (SKM) injury caused by lipid-lowering peroxisome proliferator–activated receptor alpha (PPAR-) agonists. In rats administered repeat doses of a PPAR- agonist, the kinetics and amplitude of plasma concentrations of Fabp3 were consistent with plasma compound concentrations and histopathology findings of swollen, hyalinized, and fragmented muscle fibers with macrophage infiltration. Immunohistochemical detection of Fabp3 revealed focal depletion of Fabp3 protein from injured SKM fibers which is consistent with increased serum Fabp3 concentrations in treated rats. We then assessed the predictivity of serological Fabp3 for SKM necrosis in short duration toxicology studies. Rats were treated with various doses of 27 different compounds, and the predictivity of serological biomarkers was assessed relative to histology in individual rats and in treatment groups. Under these study conditions, Fabp3 was the most useful individual biomarker based on concordance, sensitivity, positive and negative predictive values, and false negative rate. In addition, the combination of Fabp3 and aspartate aminotransferase (AST) had greater diagnostic value than the conventional combination of creatine kinase-MM isoenzyme (CK) and AST.

Renal Anemia Induced by Chronic Ingestion of Depleted Uranium in Rats

2008-05-07

Kidney disease is a frequent consequence of heavy metal exposure and renal anemia occurs secondarily to the progression of kidney deterioration into chronic disease. In contrast, little is known about effects on kidney of chronic exposure to low levels of depleted uranium (DU). Study was performed with rats exposed to DU at 40 mg/l by chronic ingestion during 9 months. In the present work, a ~20% reduction in red blood cell (RBC) count was observed after DU exposure. Hence, three hypotheses were tested to determinate origin of RBC loss: (1) reduced erythropoiesis, (2) increased RBC degradation, and/or (3) kidney dysfunction. Erythropoiesis was not reduced after exposure to DU as revealed by erythroid progenitors, blood Flt3 ligand and erythropoietin (EPO) blood and kidney levels. Concerning messenger RNA (mRNA) and protein levels of spleen iron recycling markers from RBC degradation (DMT1 [divalent metal transporter 1], iron regulated protein 1, HO1, HO2 [heme oxygenase 1 and 2], cluster of differentiation 36), increase in HO2 and DMT1 mRNA level was induced after chronic exposure to DU. Kidneys of DU-contaminated rats had more frequently high grade tubulo-interstitial and glomerular lesions, accumulated iron more frequently and presented more apoptotic cells. In addition, chronic exposure to DU induced increased gene expression of ceruloplasmin (x12), of DMT1 (x2.5), and decreased mRNA levels of erythropoietin receptor (x0.2). Increased mRNA level of DMT1 was associated to decreased protein level (x0.25). To conclude, a chronic ingestion of DU leads mainly to kidney deterioration that is probably responsible for RBC count decrease in rats. Spleen erythropoiesis and molecules involved in erythrocyte degradation were also modified by chronic DU exposure.

Use of the Pup as the Statistical Unit in Developmental Neurotoxicity Studies: Overlooked Model or Poor Research Design?

Hardy, M., Stedeford, T. — 2008-05-07

Response to: Use of the Pup as the Statistical Unit in Developmental Neurotoxicity Studies: Overlooked Model or Poor Research Design?

Eriksson, P. — 2008-05-07

Modeling Neurodevelopment Outcomes and Ethylmercury Exposure from Thimerosal-Containing Vaccines

Dorea, J. G., Marques, R. C. — 2008-05-07

Modeling Neonatal Thimerosal Exposure in Mice

Berman, R. F., Pessah, I. N., Mouton, P. R., Mav, D., Harry, G. J. — 2008-05-07

ERRATUM

2008-05-07

Why Does ARNT2 Behave Differently from ARNT?

Hankinson, O. — 2008-04-07

Pathophysiological Role of Osteopontin in Hepatic Inflammation, Toxicity, and Cancer

Ramaiah, S. K., Rittling, S. — 2008-04-07

Osteopontin (OPN) is a highly modified integrin-binding extracellular matrix glycophosphoprotein produced by cells of the immune system, epithelial tissue, smooth muscle cells, osteoblasts, and tumor cells. Extensive research has elucidated the pivotal role of OPN in cell signaling that controls inflammation, tumor progression, and metastasis. OPN interaction with the integrin receptors expressed on inflammatory cells through its arginine–glycine–aspartate (RGD) and non-RGD motifs promote migration and adhesion of cells. In the liver, it has been reported that hepatic Kupffer cells secrete OPN facilitating macrophage infiltration into necrotic areas following carbon tetrachloride liver toxicity. Recent work has highlighted the role of OPN in inflammatory liver diseases such as alcoholic and nonalcoholic liver disease and T-cell–mediated hepatitis. The role of OPN in hepatocellular carcinoma (HCC) has also generated significant interest, especially with regards to its role as a prognostic factor. OPN therefore appears to play an important role during liver inflammation and cancer. In this review we will present data to demonstrate the key role played by OPN in mediating hepatic inflammation (neutrophils, monocytes/macrophages, and lymphocytes) and its role in HCC. Greater understanding of the pathophysiologic role of OPN in hepatic inflammation and cancer may enable development of novel inflammation and cancer treatment strategies.

Computational Toxicology--A State of the Science Mini Review

2008-04-07

Advances in computer sciences and hardware combined with equally significant developments in molecular biology and chemistry are providing toxicology with a powerful new tool box. This tool box of computational models promises to increase the efficiency and the effectiveness by which the hazards and risks of environmental chemicals are determined. Computational toxicology focuses on applying these tools across many scales, including vastly increasing the numbers of chemicals and the types of biological interactions that can be evaluated. In addition, knowledge of toxicity pathways gathered within the tool box will be directly applicable to the study of the biological responses across a range of dose levels, including those more likely to be representative of exposures to the human population. Progress in this field will facilitate the transformative shift called for in the recent report on toxicology in the 21st century by the National Research Council. This review surveys the state of the art in many areas of computational toxicology and points to several hurdles that will be important to overcome as the field moves forward. Proof-of-concept studies need to clearly demonstrate the additional predictive power gained from these tools. More researchers need to become comfortable working with both the data generating tools and the computational modeling capabilities, and regulatory authorities must show a willingness to the embrace new approaches as they gain scientific acceptance. The next few years should witness the early fruits of these efforts, but as the National Research Council indicates, the paradigm shift will take a long term investment and commitment to reach full potential.

Interlaboratory Evaluation of Genomic Signatures for Predicting Carcinogenicity in the Rat

2008-04-07

The Critical Path Institute recently established the Predictive Safety Testing Consortium, a collaboration between several companies and the U.S. Food and Drug Administration, aimed at evaluating and qualifying biomarkers for a variety of toxicological endpoints. The Carcinogenicity Working Group of the Predictive Safety Testing Consortium has concentrated on sharing data to test the predictivity of two published hepatic gene expression signatures, including the signature by Fielden et al. (2007, Toxicol. Sci. 99, 90–100) for predicting nongenotoxic hepatocarcinogens, and the signature by Nie et al. (2006, Mol. Carcinog. 45, 914–933) for predicting nongenotoxic carcinogens. Although not a rigorous prospective validation exercise, the consortium approach created an opportunity to perform a meta-analysis to evaluate microarray data from short-term rat studies on over 150 compounds. Despite significant differences in study designs and microarray platforms between laboratories, the signatures proved to be relatively robust and more accurate than expected by chance. The accuracy of the Fielden et al. signature was between 63 and 69%, whereas the accuracy of the Nie et al. signature was between 55 and 64%. As expected, the predictivity was reduced relative to internal validation estimates reported under identical test conditions. Although the signatures were not deemed suitable for use in regulatory decision making, they were deemed worthwhile in the early assessment of drugs to aid decision making in drug development. These results have prompted additional efforts to rederive and evaluate a QPCR-based signature using these samples. When combined with a standardized test procedure and prospective interlaboratory validation, the accuracy and potential utility in preclinical applications can be ascertained.

Characterization of Organic Anion Transporting Polypeptide 1b2-null Mice: Essential Role in Hepatic Uptake/Toxicity of Phalloidin and Microcystin-LR

2008-04-07

The liver-specific importer organic anion transporting polypeptide 1b2 (Oatp1b2, Slco1b2, also known as Oatp4 and Lst-1) and its human orthologs OATP1B1/1B3 transport a large variety of chemicals. Oatp1b2-null mice were engineered by homologous recombination and their phenotype was characterized. Oatp1b2 protein was absent in livers of Oatp1b2-null mice. Oatp1b2-null mice develop normally and breed well. However, adult Oatp1b2-null mice had moderate conjugated hyperbilirubinemia. Compared with wild-types, Oatp1b2-null mice had similar hepatic messenger RNA expression of most transporters examined except a higher Oatp1a4 but lower organic anion transporter 2. Intra-arterial injection of the mushroom toxin phalloidin (an Oatp1b2-specific substrate identified in vitro) caused cholestasis in wild-type mice but not in Oatp1b2-null mice. Hepatic uptake of fluorescence-labeled phalloidin was absent in Oatp1b2-null mice. Three hours after administration of microcystin-LR (a blue-green algae toxin), the binding of microcystin-LR to hepatic protein phosphatase 1/2a was much lower in Oatp1b2-null mice compared with wild-type mice. In contrast, Oatp1b2-null mice were transiently protected from decrease in bile flow induced by estradiol-17β-D-glucuronide, a common substrate for Oatps. Oatp1b2-null mice were completely resistant to the hepatotoxicity induced by phalloidin and microcystin-LR, but were similarly sensitive to -amanitin–induced hepatotoxicity compared with wild-type mice. In conclusion, Oatp1b2-null mice display altered basic physiology and markedly decreased hepatic uptake/toxicity of phalloidin and microcystin-LR. Oatp1b2-null mice are useful in elucidating the role of Oatp1b2 and its human orthologs OATP1B1/1B3 in hepatic uptake and systemic disposition of toxic chemicals and therapeutic drugs.

Toxicogenomic Dissection of the Perfluorooctanoic Acid Transcript Profile in Mouse Liver: Evidence for the Involvement of Nuclear Receptors PPAR{alpha} and CAR

2008-04-07

A number of perfluorinated alkyl acids including perfluorooctanoic acid (PFOA) elicit effects similar to peroxisome proliferator chemicals (PPC) in mouse and rat liver. There is strong evidence that PPC cause many of their effects linked to liver cancer through the nuclear receptor peroxisome proliferator–activated receptor alpha (PPAR). To determine the role of PPAR in mediating PFOA transcriptional events, we compared the transcript profiles of the livers of wild-type or PPAR-null mice exposed to PFOA or the PPAR agonist WY-14,643 (WY). After 7 days of exposure, 85% or 99.7% of the genes altered by PFOA or WY exposure, respectively were dependent on PPAR. The PPAR–independent genes regulated by PFOA included those involved in lipid homeostasis and xenobiotic metabolism. Many of the lipid homeostasis genes including acyl-CoA oxidase (Acox1) were also regulated by WY in a PPAR–dependent manner. The increased expression of these genes in PPAR-null mice may be partly due to increases in PPAR expression upon PFOA exposure. Many of the identified xenobiotic metabolism genes are known to be under control of the nuclear receptor CAR (constitutive activated/androstane receptor) and the transcription factor Nrf2 (nuclear factor erythroid 2–related factor 2). There was excellent correlation between the transcript profile of PPAR–independent PFOA genes and those of activators of CAR including phenobarbital and 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) but not those regulated by the Nrf2 activator, dithiol-3-thione. These results indicate that PFOA alters most genes in wild-type mouse liver through PPAR, but that a subset of genes are regulated by CAR and possibly PPAR in the PPAR-null mouse.

Genistein is an Efficient Estrogen in the Whole-Body throughout Mouse Development

2008-04-07

The widespread use of diets containing estrogenic compounds raises questions on how relevant the presence of phytoestrogens may be, in order to allow a correct development of the reproductive ability and sexual maturity in humans and animals. The isoflavone genistein is the most estrogenically active molecule present in soy. Here we show that genistein, through an estrogen receptor (ER)–mediated action, modulates gene expression in the whole body of male mice in a dose- and time-dependent manner, at all ages. By luciferase bioassays, we show that genistein-induced ER activation is present in reproductive and nonreproductive organs of the transgenic mice Estrogen Responsive Element (ERE)-tK-LUC, although to an extent that is lower than what observed with the administration of estradiol. Peak activity was registered at genistein doses of 500–5000 µg/kg, at 12 h from the administration by gavage. In the liver, ER- and ER-β messenger RNAs and two target genes, CYP17 and the progesterone receptor, were modulated by genistein. CYP17 and PR time-dependent induction was similar to that of luciferase. ER- protein level followed an opposite regulation by genistein and estradiol. Genistein passed from the lactating mother to the suckling offspring at levels sufficient to activate gene expression in reproductive and nonreproductive tissues of the pups, with maximal upregulation at 16–24 h. We also followed responsiveness to genistein in the testis, from early development to adult age. Testis are well responsive to genistein as well as to estradiol already at day 14.5 of fetal development, as determined by exposing organotypic cultures from mouse fetus testis. Ovaries were not responsive under the same conditions. Activation of luciferase correlates with an activation of cell proliferation in testis, but not in the ovaries. Prolonged exposure (15 days) to genistein also decreases prostate weight like estradiol. In conclusion, our results show that genistein affects reproductive and nonreproductive organs of male mice in a dose- and time-dependent manner, at all developmental ages.

Deletion of Yeast CWP Genes Enhances Cell Permeability to Genotoxic Agents

Zhang, M., Liang, Y., Zhang, X., Xu, Y., Dai, H., Xiao, W. — 2008-04-07

We have previously reported the development of a novel genotoxic testing system based on the transcriptional response of the yeast RNR3-lacZ reporter gene to DNA damage. This system appears to be more sensitive than other similar tests in microorganisms, and is comparable with the Ames test. In an effort to further enhance detection sensitivity, we examined the effects of altering major cell wall components on cell permeability and subsequent RNR3-lacZ sensitivity to genotoxic agents. Although inactivation of single CWP genes encoding cell wall mannoproteins had little effect, the simultaneous inactivation of both CWP1 and CWP2 had profound effects on the cell wall structure and permeability. Consequently, the RNR3-lacZ detection sensitivity is markedly enhanced, especially to high molecular weight compounds such as 4-nitroquinoline-N-oxide (> sevenfold) and phleomycin (> 13-fold). In contrast, deletion of genes encoding representative membrane components or membrane transporters had minor effects on cell permeability. We conclude that the yeast cell wall mannoproteins constitute the major barrier to environmental genotoxic agents and that their removal will significantly enhance the sensitivity of RNR-lacZ as well as other yeast-based genotoxic tests.

Gestational Exposure to Perfluorooctane Sulfonate Suppresses Immune Function in B6C3F1 Mice

Keil, D. E., Mehlmann, T., Butterworth, L., Peden-Adams, M. M. — 2008-04-07

Perfluorinated alkyl acids (PFAAs) are used in a multitude of applications and are categorized as high-production volume chemicals produced in quantities exceeding 10,000 lbs/year. As a result, widespread exposure has been documented in adults, children, and infants. It is generally accepted that children are more sensitive to the effects of xenobiotic exposures during fetal and postnatal periods of development; therefore, considerable efforts are required to investigate the potential impact of a model PFAA, perfluorooctane sulfonate (PFOS) on children's immunological health. Using the pairing of female C57BL/6N mice with male C3H/HeJ, developmental immunotoxicity was evaluated in B6C3F1 pups following oral maternal exposure to PFOS on gestations days 1–17. Exposure levels included 0.1, 1, and 5 mg/kg/day PFOS. Natural killer (NK) cell activity, SRBC IgM plaque assay, CD4/8 lymphocytic subpopulations, nitrite production in peritoneal macrophages, and body/organ weights were evaluated at 4 and 8 weeks of age in F1 pups. No significant dose-responsive changes in maternal or pup body weights, flow cytometry, or macrophage function were observed, yet hepatomegaly was indicated in F1 male pups at 4 weeks of age. Functional deficits were not evident until 8 weeks of age when NK cell function and IgM production were significantly decreased. When compared with females, male pups were more sensitive to the effects of PFOS thereby establishing a no observed adverse effect level and low observed adverse effect level of 0.1 and 1.0 mg/kg/day (males only) following maternal PFOS exposure level, respectively. This study establishes that the developing immune system is sensitive to the effects of PFOS and results in functional deficits in innate and humoral immunity detectable at adulthood.

Surface Marker-Defined Head Kidney Granulocytes and B Lymphocytes of Rainbow Trout Express Benzo[a]pyrene-Inducible Cytochrome P4501A Protein

Nakayama, A., Riesen, I., Kollner, B., Eppler, E., Segner, H. — 2008-04-07

Polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (BaP) are immunotoxic to fish. Metabolism of PAHs in immune cells has been implicated in PAH immunotoxicity in mammals, but for fish the presence of metabolic enzymes in immune cells is less clear. The objective of this study was to examine localization and induction of the BaP-metabolizing biotransformation enzyme, cytochrome P4501A (CYP1A), in head kidney immune cells of rainbow trout (Oncorhynchus mykiss). In the first step, we measured induction of CYP1A-dependent 7-ethoxyresorufin-O-deethylase (EROD) activity and CYP1A protein in head kidney of rainbow trout treated with a single intraperitoneal (ip) injection of 25 mg BaP/kg body weight. From days 3 to 10 postinjection, the BaP treatment led to a significant elevation of EROD and CYP1A protein in head kidney and liver, with CYP1A expression levels in the head kidney being much lower than in the liver. Next, we examined the cellular localization of CYP1A protein in the head kidney cell types: vascular endothelial, endocrine and lymphoid cells. CYP1A immunoreactivity was detectable only in BaP-treated trout, where it was localized in endothelial and lymphoid cells. Finally, we aimed to clarify which of the hematopoietic cell types possess CYP1A protein. Using double immunostaining for CYP1A and surface markers of rainbow trout immune cells, we identified B lymphocytes and granulocytes expressing inducible CYP1A protein and being the likely sites of BaP metabolism in the head kidney.

3,4-Dichloropropionanilide (DCPA) Inhibits T-Cell Activation by Altering the Intracellular Calcium Concentration following Store Depletion

Lewis, T. L., Brundage, K. M., Brundage, R. A., Barnett, J. B. — 2008-04-07

Stimulation of T cells through the T-cell receptor results in the activation of a series of signaling pathways that leads to the secretion of interleukin (IL)-2 and cell proliferation. Influx of calcium (Ca²⁺) from the extracellular environment, following internal Ca²⁺ store depletion, provides the elevated and sustained intracellular calcium concentration ([Ca²⁺]_i) critical for optimal T-cell activation. Our laboratory has documented that exposure to the herbicide 3,4-dichloropropionanilide (DCPA) inhibits intracellular signaling events that have one or more Ca²⁺ dependent steps. Herein we report that DCPA attenuates the normal elevated and sustained [Ca²⁺]_i that follows internal store depletion in the human leukemic Jurkat T cell line and primary mouse T cells. DCPA did not alter the depletion of internal Ca²⁺ stores when stimulated by anti-CD3 or thapsigargin demonstrating that early inositol 1,4,5-triphosphate–mediated signaling and depletion of Ca²⁺ stores were unaffected. 2-Aminoethyldiphenol borate (2-APB) is known to alter the store-operated Ca²⁺ (SOC) influx that follows Ca²⁺ store depletion. Exposure of Jurkat cells to either DCPA or 50µM 2-APB attenuated the increase in [Ca²⁺]_i following thapsigargin or anti-CD3 induced store depletion in a similar manner. At low concentrations, 2-APB enhances SOC influx but this enhancement is abrogated in the presence of DCPA. This alteration in [Ca²⁺]_i, when exposed to DCPA, significantly reduces nuclear levels of nuclear factor of activated T cells (NFAT) and IL-2 secretion. The plasma membrane polarization profile is not altered by DCPA exposure. Taken together, these data indicate that DCPA inhibits T-cell activation by altering Ca²⁺ homeostasis following store depletion.

Cellular Response to Diesel Exhaust Particles Strongly Depends on the Exposure Method

Holder, A. L., Lucas, D., Goth-Goldstein, R., Koshland, C. P. — 2008-04-07

In vitro exposure to aerosols at the air–liquid interface (ALI) preserves the physical and chemical characteristics of aerosol particles. Although frequently described as being a more physiologic exposure method, ALI exposure has not been directly compared with conventional in vitro exposures where the particles are suspended in medium. We exposed immortalized human bronchial epithelial cells (16HBE14o) to aerosolized diesel exhaust particles at the ALI and to suspensions of collected particles. The response of the cells was determined from measurements of the cell viability and interleukin-8 (IL-8) secretion. The deposited size distribution at the cell surface was measured with transmission electron microscopy to obtain a dose for the ALI exposure. Although exposure by either method caused a slight decrease in cell viability and induced IL-8 secretion, the response to ALI exposure occurred at doses several orders of magnitude lower than exposure to particles in suspension. The most likely sources for the different dose responses are the artifacts introduced during the collection and resuspension of particles for conventional suspension exposures. The number concentration of particles deposited at the ALI is similar to the modeled deposition in the tracheal–bronchial region in a human lung, but the ALI size distribution is skewed toward particles larger than those deposited in the lung.

Measuring Brain Manganese and Iron Accumulation in Rats following 14 Weeks of Low-Dose Manganese Treatment Using Atomic Absorption Spectroscopy and Magnetic Resonance Imaging

2008-04-07

Chronic exposure to manganese (Mn) may lead to a movement disorder due to preferential Mn accumulation in the globus pallidus and other basal ganglia nuclei. Iron (Fe) deficiency also results in increased brain Mn levels, as well as dysregulation of other trace metals. The relationship between Mn and Fe transport has been attributed to the fact that both metals can be transported via the same molecular mechanisms. It is not known, however, whether brain Mn distribution patterns due to increased Mn exposure vs. Fe deficiency are the same, or whether Fe supplementation would reverse or inhibit Mn deposition. To address these questions, we utilized four distinct experimental populations. Three separate groups of male Sprague–Dawley rats on different diets (control diet [MnT], Fe deficient [FeD], or Fe supplemented [FeS]) were given weekly intravenous Mn injections (3 mg Mn/kg body mass) for 14 weeks, whereas control (CN) rats were fed the control diet and received sterile saline injections. At the conclusion of the study, both blood and brain Mn and Fe levels were determined by atomic absorption spectroscopy and magnetic resonance imaging. The data indicate that changes in dietary Fe levels (either increased or decreased) result in regionally specific increases in brain Mn levels compared with CN or MnT animals. Furthermore, there was no difference in either Fe or Mn accumulation between FeS or FeD animals. These data suggest that dietary Fe manipulation, whether increased or decreased, may contribute to brain Mn deposition in populations vulnerable to increased Mn exposure.

2,3,7,8-Tetracholorodibenzo-p-Dioxin Exposure Disrupts Granule Neuron Precursor Maturation in the Developing Mouse Cerebellum

2008-04-07

The widespread environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been linked to developmental neurotoxicity associated with abnormal cerebellar maturation in both humans and rodents. TCDD mediates toxicity via binding to the aryl hydrocarbon receptor (AhR), a transcription factor that regulates the expression of xenobiotic metabolizing enzymes and growth regulatory molecules. Our previous studies demonstrated that cerebellar granule neuron precursor cells (GNPs) express transcriptionally active AhR during critical developmental periods. TCDD exposure also impaired GNP proliferation and survival in vitro. Therefore, this study tested the hypothesis that TCDD exposure disrupts cerebellar development by interfering with GNP differentiation. In vivo experiments indicated that TCDD exposure on postnatal day (PND) 6 resulted in increased expression of a mitotic marker and increased thickness of the external granule layer (EGL) on PND10. Expression of the early differentiation marker TAG-1 was also more pronounced in postmitotic, premigratory granule neurons of the EGL, and increased apoptosis of GNPs was observed. On PND21, expression of the late GNP differentiation marker GABA_A6 receptor (GABAR_A6) and total estimated cell numbers were both reduced following exposure on PND6. Studies in unexposed adult AhR^–/– mice revealed lower GABAR_A6 levels and DNA content. In vitro studies showed elevated expression of the early differentiation marker p27/Kip1 and the GABAR_A6 in GNPs following TCDD exposure, and the expression patterns of proteins related to granule cell neurite outgrowth, βIII-tubulin and polysialic acid neural cell adhesion molecule, were consistent with enhanced neuroblast differentiation. Together, our data suggest that TCDD disrupts a normal physiological role of AhR, resulting in compromised GNP maturation and neuroblast survival, which impacts final cell number in the cerebellum.

Motorcycle Exhaust Induces Reproductive Toxicity and Testicular Interleukin-6 in Male Rats

2008-04-07

Motorcycle exhaust (ME) from two-stroke engines contains many toxicants and poses a potential health hazard. The major objectives of the present study were to investigate the male reproductive toxicity of ME and the underlying mechanisms of toxicity. Male Wistar rats were exposed to ME by inhalation 1 h each in the morning and afternoon, Monday through Friday. Exposures to 1:50 diluted ME for 4 weeks or to 1:10 diluted ME for 2 and 4 weeks showed concentration- and time-dependent decreases of testicular weight, spermatid number, and cauda epididymal sperm number. Subsequent studies were done using 4-week exposure to 1:10 diluted ME. ME caused histopathological changes including testicular spermatocytic necrosis and seminiferous tubule atrophy and cauda epididymal formation of clusters of pyknotic and necrotic sperm cells. ME-exposed male rats mated with untreated females showed decreases of male mating index and female fertility index and an increase of implantation site loss. ME decreased 7-ethoxycoumarin O-deethylase and superoxide dismutase activities but induced proinflammatory cytokine interleukin-6 (IL-6) messenger RNA (mRNA) in the testis. Male rats were exposed to ME with or without cotreatment with 50 mg/kg vitamin E orally for 4 weeks. ME decreased serum testosterone concentration. This effect was reversed by cotreatment with vitamin E. ME decreased testicular spermatid number and induced IL-6 mRNA and protein. These effects were also reversed by the vitamin E cotreatment. The present findings show that ME causes male reproductive effects and induces testicular IL-6 in rats by mechanisms involving induction of oxidative stress and inhibition of steroidogenesis.

Reactive Oxygen Species Contribute to Lipopolysaccharide-Induced Teratogenesis in Mice

2008-04-07

Lipopolysaccharide (LPS) has been associated with adverse developmental outcome, including embryonic resorption, fetal death and growth retardation, and preterm delivery. In the present study, we showed that an ip injection with LPS daily from gestational day (gd) 8 to gd 12 resulted in the incidence of external malformations. The highest incidence of malformed fetuses was observed in fetuses from dams exposed to 20 µg/kg LPS, in which 34.9% of fetuses per litter were externally malformed. In addition, 17.4% of fetuses per litter in 30 µg/kg group and 12.5% of fetuses per litter in 10 µg/kg group were externally malformed. Importantly, external malformations were also observed in fetuses from dams exposed to only two doses of LPS (20 µg/kg, ip) on gd 8, in which 76.5% (13/17) of litters and 39.1% of fetuses per litter were affected. LPS-induced teratogenicity seemed to be associated with oxidative stress in fetal environment, measured by lipid peroxidation, nitrotyrosine residues, and glutathione (GSH) depletion in maternal liver, embryo, and placenta. alpha-Phenyl-N-t-butylnitrone (PBN, 100 mg/kg, ip), a free radical spin-trapping agent, abolished LPS-induced lipid peroxidation, nitrotyrosine residues, and GSH depletion. Consistent with its antioxidant effects, PBN decreased the incidence of external malformations. Taken together, these results suggest that reactive oxygen species might be, at least partially, involved in LPS-induced teratogenesis.

Influence of TCDD on Zebrafish CYP1B1 Transcription during Development

2008-04-07

Cytochrome P450 1B1 (CYP1B1) is a heme-containing monooxygenase that metabolizes various polycyclic aromatic hydrocarbons and aryl amines, as well as retinoic acid and steroid hormones. Here we report the cloning of an ortholog of CYP1B1 from zebrafish and the demonstration that transcription of zebrafish CYP1B1 was modulated by two types of mechanisms during different developmental stage. First in late pharyngula stage before hatching, CYP1B1 was constitutively transcribed in retina, midbrain–hindbrain boundary and diencephalon regions through a close coordination between aryl hydrocarbon receptor 2 (AHR2)–dependent and AHR2-independent pathways. After hatching, the basal transcription was attenuated and it could not be elicited upon 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure. In contrast, TCDD exposure induced de novo CYP1B1 transcription in larval branchial arches and heart tissues via an AHR2-dependent pathway. Blocking AHR2 translation completely eliminated the TCDD-mediated CYP1B1 transcription. However, we did not detect any types of CYP1B1 transcription in liver and kidney tissues through the developmental stage. It suggests that the constitutive and TCDD-inducible types of CYP1B1 transcriptions are modulated by distinct pathways with different tissue specificities. Finally, we investigated the role of CYP1B1 in TCDD-mediated embryonic toxicity. Because knockdown of CYP1B1 did not prevent TCDD-induced pericardial edema and cranial defects, it suggests that CYP1B1 is not involved in the developmental toxicity of dioxin.

Respiratory Toxicity of Diacetyl in C57Bl/6 Mice

Morgan, D. L., Flake, G. P., Kirby, P. J., Palmer, S. M. — 2008-04-07

Diacetyl, a component of artificial butter flavoring, is a potential etiological agent of obliterative bronchiolitis (OB); however, the toxic dose and mechanisms of toxicity remain controversial. We evaluated the respiratory toxicity of diacetyl in a murine model using several exposure profiles relevant to workplace conditions at microwave popcorn packaging plants. Male C57Bl/6 mice were exposed to inhaled diacetyl across several concentrations and duration profiles, or by direct oropharyngeal aspiration. Effects of diacetyl on the respiratory tract were evaluated by histopathology and BALF analyses. Subacute exposure to 200 or 400 ppm diacetyl for 5 days caused deaths, necrotizing rhinitis, necrotizing laryngitis and bronchitis. Reducing the exposure to 1 h/day (100, 200, 400 ppm) for 4 weeks resulted in less nasal and laryngeal toxicity, but led to peribronchial and peribronchiolar lymphocytic inflammation. A similar pattern was observed with intermittent high-dose exposures at 1200 ppm (15 min, twice a day, 4 weeks). Subchronic exposures to 100 ppm (6 h/day, 12 weeks) caused moderate nasal injury, and peribronchial lymphocytic inflammation accompanied by epithelial atrophy, denudation, and regeneration. Treatment with 400 mg/kg by oropharyngeal aspiration to bypass the nose caused foci of fibrohistiocytic proliferation with little or no inflammation at the junction of the terminal bronchiole and alveolar duct. Depending on the route and duration of exposure, diacetyl causes significant epithelial injury, peribronchial lymphocytic inflammation, or fibrohistiocytic lesions in the terminal bronchioles. Collectively these results indicate that clinically relevant diacetyl exposures result in a pattern of injury that replicates features of human OB.

U-shaped Dose Response in Vasomotor Tone: A Mixed Result of Heterogenic Response of Multiple Cells to Xenobiotics

2008-04-07

U-shaped response has been frequently encountered in various biological areas including epidemiology, toxicology, and oncology. Despite its frequent observation, the theory of U-shaped response has been crippled by the lack of a robust mechanism underlying and incomplete in vitro and in vivo correlation. In the present study, a novel mechanism is provided for a U-shaped response, based on the findings of agonist-induced vasomotor tone change affected by menadione (MEN) (synthetic vitamin K₃), a reactive oxygen species generator, and arsenic, an environmental pollutant, which showed typical U-shaped responses in both in vitro aortic contractile response and in vivo blood pressure. U-shaped responses by MEN and arsenic were a combined result from heterogenic susceptibilities and responses of multiple target cells composing blood vessels, that is, endothelium and smooth muscle. Notably, endothelium, a regulator of vasomotor tone, was primarily affected by low-dose stimuli, whereas smooth muscle, an effector of vascular contraction, was affected later by high-dose. The dysfunction of smooth muscle was produced by high-dose MEN-induced hydrogen peroxide, resulting in the attenuation of vascular contractile reactivity, whereas low-dose MEN-induced superoxide led to the quenching of vasodilatory nitric oxide in endothelial cells, resulting in the enhancement of vasoconstriction. This mechanistic theory, the difference in susceptibilities and responses to a common stimulus between regulator and effector components of a system, could give a new insight into the explanation of various U-shaped responses and provide a new evidence for the need of the risk assessment of toxicants with a wider dose range.

Analysis of Ah Receptor-ARNT and Ah Receptor-ARNT2 Complexes In Vitro and in Cell Culture

Dougherty, E. J., Pollenz, R. S. — 2008-04-07

ARNT and ARNT2 proteins are expressed in mammalian and aquatic species and exhibit a high level of amino acid identity in the basic-helix loop-helix PER/ARNT/SIM domains involved in protein interactions and DNA binding. Since the analysis of ARNT2 function at the protein level has been limited, ARNT2 function in aryl hydrocarbon receptor (AHR)–mediated signaling was evaluated and compared to ARNT. In vitro, ARNT and ARNT2 dimerized equally with the AHR in the presence of 2,3,7,8-tetracholorodibenzo-p-dioxin (TCDD) and ARNT2 outcompeted ARNT for binding to the AHR when expressed in excess. In contrast, activation of the AHR with 3-methylcholanthrene or benzo[a]pyrene resulted in predominant formation of AHR•ARNT complexes. ARNT2 expressed in Hepa-1 cell culture lines with reduced ARNT protein resulted in minimal induction of endogenous CYP1A1 protein compared to cells expressing ARNT, and mutation of the putative proline residue at amino acid 352 to histidine failed to produce an ARNT2 that could function in AHR-mediated signaling. However, the expression of ARNT2 in wild-type Hepa-1 cells reduced TCDD-mediated induction of endogenous CYP1A1 protein by 30%, even though AHR•ARNT2 complexes could not be detected in nuclear extracts. Western blot analysis of numerous mouse tissues and various cell culture lines showed that both endogenous ARNT and ARNT2 could be detected in cells derived from kidney, central nervous system, and retinal epithelium. Thus, ARNT2 has the ability to dimerize with the liganded AHR in vitro and is influenced by the activating ligand yet appears to be limited in its ability to influence AHR-mediated signaling in cell culture.

Perinatal Lead Exposure Affects Nitric Oxide and Cyclooxygenase Pathways in Aorta of Weaned Rats

Grizzo, L. T., Cordellini, S. — 2008-04-07

Perinatal Pb exposure may modulate arterial tone through nitric oxide (NO) and cyclooxygenase products. To investigate this, Wistar dams received 1000 ppm of Pb or sodium acetate (control) in drinking water during pregnancy and lactation. Curves were constructed in phenylephrine-precontracted intact and/or denuded rings of thoracic aortas of weaned (23-day-old) male pups from their responses to N-nitro-L-arginine methyl ester (L-NAME, NO synthase inhibitor) and ACh in the absence or presence of indomethacin (10^–5M, cyclooxygenase inhibitor) or L-NAME (3 x 10^–7M and 3 x 10^–4M). Blood lead concentration and systolic blood pressure (SBP) were higher in intoxicated than control pups (blood lead µg/dl: control < 3.0, Pb 58.7 ± 6.5*; SBP mmHg: control 111.4 ± 2.3, Pb 135.5 ± 2.4*). In L-NAME–treated rings maximal responses increased in Pb-exposed rats, and were higher in intact than in denuded aortas (contraction [% of phenylephrine] intact: control 184.3 ± 23.7, Pb 289.1 ± 18.3*; denuded: control 125.1 ± 4.5, Pb 154.8 ± 13.3*). ACh-induced relaxation in intact aortas from Pb-exposed rats presented rightward shift in L-NAME presence (EC50 x 10^–7M: control 1.32 [0.33–5.18], Pb 4.88 [3.56–6.69]*) but moved left under indomethacin (EC50 x 10^–7M: control 8.95 [3.47–23.07], Pb 0.97 [0.38–2.43]*). *p < 0.05 significant relative to the respective control; N = 7–9. Endothelium removal abolished ACh-induced relaxation. Perinatal Pb exposure caused hypertension associated with alterations in the production and/or release of basal and stimulated endothelium-derived relaxing factors–NO and constricting cyclooxygenase products. These findings may help explain the contribution of NO and cyclooxygenase products to the etiology and/or maintenance of Pb-induced hypertension and could ultimately lead to therapeutic advantages in plumbism.

Letter regarding: "Paraquat: The Red Herring of Parkinson's Disease Research"

Cory-Slechta, D. A., Thiruchelvam, M., Di Monte, D. A. — 2008-04-07

Paraquat and Parkinson's Disease: Response by Dr. Miller

Miller, G. W. — 2008-04-07

Response to "Paraquat: The Red Herring of Parkinson's Disease Research"

LoPachin, R. M., Gavin, T. — 2008-04-07

Paraquat and Parkinson's Disease: Response by Dr. Miller, Part II

Miller, G. W. — 2008-04-07

The Vascular System as a Target of Metal Toxicity

2008-03-07

Vascular system function involves complex interactions among the vascular endothelium, smooth muscle, the immune system, and the nervous system. The toxic metals cadmium (Cd), arsenic (As), and lead (Pb) can target the vascular system in a variety of ways, ranging from hemorrhagic injury to subtle pathogenic remodeling and metabolic changes. Acute Cd exposure results in hemorrhagic injury to the testis, although some strains of animals are resistant to this effect. A comparison of Cd-sensitive with Cd-resistant mouse strains showed that expression of the Slc39a8 gene, encoding the ZIP8 transporter, in the testis vasculature endothelium is responsible for this difference. Endogenously, ZIP8 is a Mn²⁺/HCO₃^–symporter that may also contribute to Cd damage in the kidney. Chronic Cd exposure is associated with various cardiovascular disorders such as hypertension and cardiomyopathy and it is reported to have both carcinogenic and anticarcinogenic activities. At noncytotoxic concentrations of 10–100nM, Cd can inhibit chemotaxis and tube formation of vascular endothelial cells. These angiostatic effects may be mediated through disruption of vascular endothelial cadherin, a Ca²⁺-dependent cell adhesion molecule. With regard to As, ingestion of water containing disease-promoting concentrations of As promotes capillarization of the liver sinusoidal endothelium. Because capillarization is a hallmark precursor for liver fibrosis and contributes to an imbalance of lipid metabolism, this As effect on hepatic endothelial cells may be a pathogenic mechanism underlying As-related vascular diseases. With regard to Pb, perinatal exposure may cause sustained elevations in adult blood pressure, and genetically susceptible animals may show enhanced sensitivity to this effect. Taken together, these data indicate that the vascular system is a critical target of metal toxicity and that actions of metals on the vascular system may play important roles in mediating the pathophysiologic effects of metals in specific target organs.

Concentrations of the Propylene Metabolite Propylene Oxide in Blood of Propylene-Exposed Rats and Humans--a Basis for Risk Assessment

2008-03-07

Propylene (PE) was not carcinogenic in long-term studies in rodents. However, its biotransformation to propylene oxide (PO) raises questions about a carcinogenic risk. PO alkylates macromolecules, is a direct mutagen, and caused tumors in rodents at high concentrations. In order to acquire knowledge on the species-specific PO concentrations in blood resulting from PE exposure, we exposed male Fischer 344/N rats in closed exposure chambers to constant PE concentrations, between 20.1 and 3000 ppm (7 h at least), and four male volunteers to mean constant PE concentrations of 9.82 and 23.4 ppm (180 min) in inhaled air. In the animal experiments, PE and PO were measured in the chamber atmosphere, PE by gas chromatography with flame ionization detection (GC/FID), PO by GC/FID or GC with mass-selective detection (GC/MSD). In the human studies, PE was measured in inhaled and exhaled air by GC/FID. PO was quantified by GC/MSD from exhaled breath collected in gasbags. Blood concentrations of PO were calculated based on the measured PO concentrations in air using the blood-to-air partition coefficients of 60 (rat) and 66 (human). In rats, PO blood concentrations ranged from 53 nmol/l at 20.1 ppm PE to 1750 nmol/l at 3000 ppm PE. In humans, mean blood concentrations of PO were 0.44 and 0.92 nmol/l at mean PE concentrations of 9.82 and 23.4 ppm, respectively. These findings should be taken into consideration when estimating the carcinogenic risk of PE to humans based on carcinogenicity studies in PE- or PO-exposed rats.

Mdm2 as a Sensitive and Mechanistically Informative Marker for Genotoxicity Induced by Benzo[a]pyrene and Dibenzo[a,l]pyrene

Malmlof, M., Paajarvi, G., Hogberg, J., Stenius, U. — 2008-03-07

Mdm2 is an oncoprotein interacting with p53 and maintaining low p53 levels in unstressed cells. Here we investigated the effect of genotoxic compounds on Mdm2 phosphorylation levels. Employing the Mdm2 2A10 antibody and phosphatase treatment we found that Mdm2 accumulated in HepG2 cells when exposed to low concentrations of genotoxic compounds such as mitomycin C, etoposide, 5-fluorouracil, and benzo[a]pyrene (BP). The low-dose responses were not accompanied by p53 accumulation and the effect of low concentrations of BP on Mdm2 was not affected by small interfering RNA for p53. In human lymphoblasts 10nM BP induced an Mdm2 response. Low concentrations of BP also induced binding of Mdm2 to chromatin in HepG2 cells, but no p53 binding or H2AX phosphorylation. The more mutagenic dibenzo[a,l]pyrene as well as higher BP concentrations instead induced H2AX and p53 Ser15 association with chromatin. Acrolein potentiated the effect of BP on p53 stabilization and chromatin binding. Taken together, these data suggest that (1) Mdm2 is a sensitive biomarker for certain types of genotoxicity, and (2) that polycyclic aromatic hydrocarbons-induced Mdm2 binding to chromatin reflects repairable damage, whereas chromatin binding of p53 Ser15 and H2AX indicates more persistent DNA damage. The analysis of Mdm2 and related endpoints might be useful for evaluating mutagenic potentials of DNA damages. It is suggested that patterns documented here can be used for separating BP doses that induce readily repaired DNA adducts from doses that overwhelm this capacity.

A Biologically Based Dose-Response Model for Dietary Iodide and the Hypothalamic-Pituitary-Thyroid Axis in the Adult Rat: Evaluation of Iodide Deficiency

McLanahan, E. D., Andersen, M. E., Fisher, J. W. — 2008-03-07

A biologically based dose-response (BBDR) model was developed for dietary iodide and the hypothalamic-pituitary-thyroid (HPT) axis in adult rats. This BBDR-HPT axis model includes submodels for dietary iodide, thyroid-stimulating hormone (TSH), and the thyroid hormones, T₄ and T₃. The submodels are linked together via key biological processes, including (1) the influence of T₄ on TSH production (the HPT axis negative feedback loop), (2) stimulation of thyroidal T₄ and T₃ production by TSH, (3) TSH upregulation of the thyroid sodium (Na⁺)/iodide symporter, and (4) recycling of iodide from metabolism of thyroid hormones. The BBDR-HPT axis model was calibrated to predict steady-state concentrations of iodide, T₄, T₃, and TSH for the euthyroid rat whose dietary intake of iodide was 20 µg/day. Then the BBDR-HPT axis model was used to predict perturbations in the HPT axis caused by insufficient dietary iodide intake, and simulation results were compared to experimental findings. The BBDR-HPT axis model was successful in simulating perturbations in serum T₄, TSH, and thyroid iodide stores for low-iodide diets of 0.33–1.14 µg/day. Model predictions of serum T₃ concentrations were inconsistent with observations in some cases. BBDR-HPT axis model simulations show a steep dose-response relationship between dietary intake of iodide and serum T₄ and TSH when dietary iodide intake becomes insufficient (less than 2 µg/day) to sustain the HPT axis. This BBDR-HPT axis model can be linked with physiologically based pharmacokinetic models for thyroid-active chemicals to evaluate and predict dose-dependent HPT axis alterations based on hypothesized modes of action. To support continued development of this model, future studies should include time course data after perturbation of the HPT axis to capture changes in endogenous iodide, serum TSH, T₄, and T₃.

Synergistic Neurotoxic Effects of Arsenic and Dopamine in Human Dopaminergic Neuroblastoma SH-SY5Y Cells

Shavali, S., Sens, D. A. — 2008-03-07

Parkinson's disease is an environmentally influenced, neurodegenerative disease of unknown origin that is characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta of the brain. Arsenic is an environmental contaminant found naturally in ground water, industrial waste, and fertilizers. The initial goal of the present study was to determine if a mixture of arsenite (As⁺³) and dopamine (DA) could cause enhanced degeneration of dopaminergic neuronal cells. Additional goals were to determine the mechanism (apoptosis or necrosis) of As- and DA-induced cell death and if death could be attenuated by antioxidants. The cell culture model employed was the SH-SY5Y neuroblastoma cell line that has been shown to possess differentiated characteristics of dopaminergic neurons. The results demonstrated that a mixture of As⁺³ and DA was synergistic in producing the death of the SH-SY5Y cells when compared with exposure to either agent alone. A mixture of 10µM As⁺³ and 100µM DA produced almost a complete loss of cell viability over a 24-h period of exposure, whereas, each agent alone had minimal toxicity. It was shown that necrosis, and not apoptosis, was the mechanism of cell death produced by exposure of the SH-SY5Y cells to the mixture of As⁺³ and DA. It was also demonstrated that the antioxidants, N-acetylcysteine, and Sulforaphane, attenuated the toxicity of the mixture of As⁺³ and DA to the SH-SY5Y cells. This study provides initial evidence that As⁺³ and DA synergistically can cause enhanced toxicity in cultured neuronal cells possessing dopaminergic differentiation.