Toxicological Sciences - Advance Access

Cadmium Induces Intracellular Ca2+- and H2O2-dependent Apoptosis through JNK- and p53-mediated Pathways in Skin Epidermal Cell line

Son, Y.-O., Lee, J.-C., Hitron, J. A., Pan, J., Zhang, Z., Shi, X. — Tue, 03 Nov 2009 07:18:39 PST

Cadmium is a toxic heavy metal and has been widely used in industry. The skin is an important target for this metal. The mechanisms by which cadmium leads to damage to the skin are unclear at present. The aims of this study were to examine whether cadmium induces apoptosis in mouse skin epidermal cell line, JB6 Cl41 cells, and to investigate the cellular mechanisms by which cadmium causes cytotoxicity in the cells. The present study showed that cadmium induced cell death by apoptosis in a dose-dependent manner, as proven by the appearance of cell shrinkage, the increase of Annexin V-positively stained cells, and the formation of nuclear DNA ladders. Cadmium-induced apoptosis involved a mitochondria-mediated mechanism but not caspase-dependent pathway, in that the critical apoptotic events induced by cadmium, such as the decrease of Bcl-2/Bcl-xL, the increase of GADD45, and the nuclear translocation of AIF, were not affected by the inhibition of executive caspases. In contrast, blockage of p53 and JNK by pharmacological inhibitors or si-RNA transfection suppressed the cadmium-induced apoptosis with the concomitant inhibition of anti-apoptotic Bcl-2 family proteins and GADD45, respectively. Further, the activation of p53 and JNK and their downstream proteins in cadmium-exposed cells were inhibited by individual treatment with catalase and Bapta-AM. These results suggest that cadmium induces apoptosis via the activation of JNK- and p53-mediated signaling, where calcium ion and hydrogen peroxide act as the pivotal mediators of the apoptotic signaling.

Cytochrome P4501A induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin and two chlorinated dibenzofurans in primary hepatocyte cultures of three avian species

Sun, 01 Nov 2009 23:06:30 PST

Relative potencies of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) and 2,3,7,8-tetrachlorodibenzofuran (TCDF) were determined in vitro in primary hepatocyte cultures of chicken (Gallus gallus), ring-necked pheasant (Phasianus colchicus) and Japanese quail (Corturnix japonica) embryos. Concentration-dependent effects on ethoxyresorufin O-deethylase (EROD) activity, and expression of cytochrome P4501A4 and 1A5 (CYP1A4 and CYP1A5) mRNA were determined in hepatocytes exposed to serial dilutions of TCDD, PeCDF or TCDF for 24 h. In chicken hepatocytes, the three compounds were equipotent inducers of EROD activity and CYP1A4/5 mRNA expression. However, in ring-necked pheasant and Japanese quail hepatocytes, PeCDF was more potent than TCDD (3- to 5-fold in ring-necked pheasant; 13- to 30-fold in Japanese quail). Among species, the rank order of sensitivity (most to least) to EROD and CYP1A4/5 mRNA induction for TCDD and TCDF was: chicken > ring-necked pheasant > Japanese quail. In contrast, the three species were approximately equisensitive to EROD and CYP1A4/5 mRNA induction by PeCDF. It has generally been assumed that TCDD is the most potent ‘dioxin-like compound’ (DLC) and that the chicken is the most sensitive avian species to CYP1A induction by all DLCs. This study indicates that PeCDF is more potent than TCDD in ring-necked pheasant and Japanese quail hepatocytes, and that ring-necked pheasant, Japanese quail and chicken hepatocytes are equally sensitive to CYP1A induction by PeCDF.

Creation of a Hyper-permeable Yeast Strain to Genotoxic Agents through Combined Inactivation of PDR and CWP Genes

Zhang, M., Hanna, M., Li, J., Butcher, S., Dai, H., Xiao, W. — Sun, 01 Nov 2009 23:06:30 PST

We previously established a genotoxicity detection system based on the transcriptional response of the yeast RNR3 gene to DNA damage. In order to further improve its sensitivity to genotoxicants, we have attempted to increase cell permeability by removing cell wall mannoproteins (CWPs). Here, we report that selected deletion of pleiotropic drug resistance (PDR) genes encoding membrane efflux transporters also enhanced cellular sensitivity to treatment by various genotoxic agents. Furthermore, we have validated our hypothesis that PDR and CWP protect cells through different mechanisms by demonstrating that simultaneous inactivation of the above two pathways resulted in a synergistic enhancement of assay sensitivity as measured by RNR3-lacZ expression, and that this effect is at the cell permeability level. The quadruple mutation results in RNR3-lacZ assay sensitivity to tested chemicals that apparently surpasses the industry standard Ames test. We argue that this hyper-permeable yeast mutant strain would be suitable for other chemical-based genotoxic assays.

Airway Trefoil Factor Expression during Naphthalene Injury and Repair

Greeley, M. A., Van Winkle, L. S., Edwards, P. C., Plopper, C. G. — Fri, 30 Oct 2009 08:08:00 PDT

While the role of trefoil factors (TFF) in the maintenance of epithelial integrity in the gastrointestinal tract is well known, their involvement in wound healing in the conducting airway is less well understood. We defined the pattern of expression of TFF1, TFF2 and TFF3 in the airways of mice during repair of both severe (300mg/kg) and moderate (200mg/kg) naphthalene induced Clara cell injury. Quantitative real-time PCR for TFF mRNA expression and immunohistochemistry for protein expression were applied to airway samples obtained by microdissection of airway trees or to fixed lung tissue from mice at 6 and 24 hours and 4 and 7 days after exposure to either naphthalene or an oil (vehicle) control. All three frefoil factors were expressed in normal whole lung and airways. TFF2 was the most abundant and was enriched in airways. Injury of the airway epithelium by 300 mg/kg naphthalene caused a significant induction of TFF1 gene expression at 24hrs, 4 and 7 days. In contrast TFF2 was decreased in the high dose group at 24 hrs and 4 days but returned to baseline levels by 7 days. TFF3 gene expression was not significantly changed at any timepoint. Protein localization via immunohistochemistry did not directly correlate with the gene expression measurements. TFF 1 and 3 expression was most intense in the degenerating Clara cells in the injury target zone at 6 and 24 hrs. Following the acute injury phase TFF1 and 3 were localized to the luminal apices of repairing epithelial cells and to the adjacent mesenchyme in focal regions that correlated with bifurcations and the bronchoalveolar duct junction. The temporal pattern of increases in TFF1, TFF2 and TFF3 indicate a role in cell death as well as proliferation, migration and differentiation phases of airway epithelial repair.

Quantitative Risk Analysis for N-Methyl Pyrrolidone using Physiologically Based Pharmacokinetic and Benchmark Dose Modeling

Thu, 29 Oct 2009 12:06:43 PDT

Establishing an occupational exposure limit (OEL) for N-methyl pyrrolidone (NMP) is important due to its widespread use as a solvent. Based on studies in rodents, the most sensitive toxic endpoint is a decrease in fetal/pup bodyweights observed after oral, dermal, and inhalation exposures of dams to NMP. Evidence indicates the parent compound is the causative agent. To reduce the uncertainty in rat to human extrapolations, physiologically based pharmacokinetic (PBPK) models were developed to describe the pharmacokinetics of NMP in both species. Since in utero exposures are of concern, the models considered major physiological changes occurring in the dam or mother over the course of gestation. The rat PBPK model was used to determine the relationship between NMP concentrations in maternal blood and decrements in fetal/pup body weights following exposures to NMP vapor. Body weight decrements seen after vapor exposures occurred at lower NMP blood levels than those observed after oral and dermal exposures. Benchmark dose (BMD) modeling was used to better define a point of departure (POD) for fetal/pup body weight changes based on dose-response information from two inhalation studies in rats. The POD and human PBPK model were then used to estimate the human equivalent concentrations (HEC) that could be used to derive an OEL value for NMP. The geometric mean of the PODs derived from the rat studies was estimated to be 350 mg*hr/L (expressed in terms of internal dose), a value which corresponds to a HEC of 480 ppm (occupational exposure of 8 hours/day, 5 days/week). The HEC is much higher than recently-developed internationally-recognized OELs for NMP of 10 ppm to 20 ppm, suggesting that these OELs adequately protect workers exposed to NMP vapor.

Aflatoxin B1 transfer and metabolism in human placenta

Thu, 29 Oct 2009 12:06:42 PDT

Aflatoxin B1 (AFB1), a common dietary contaminant, is a major risk factor of hepatocellular carcinoma (HCC). Early onset of HCC in some countries in Africa and South-East Asia indicates the importance of early life exposure. Placenta is the primary route for various compounds, both nutrients and toxins, from the mother to the fetal circulation. Furthermore, placenta contains enzymes for xenobiotic metabolism. AFB1, AFB1-metabolites and AFB1-albumin adducts have been detected in cord blood of babies after maternal exposure during pregnancy. However, the role that the placenta plays in the transfer and metabolism of AFB1 is not clear. In this study, placental transfer and metabolism of AFB1 were investigated in human placental perfusions and in in vitro studies. Eight human placentas were perfused with 0.5 or 5 µM AFB1 for 2-4 hours. In vitro incubations with placental microsomal and cytosolic proteins from eight additional placentas were also conducted. Our results from placental perfusions provide the first direct evidence of the actual transfer of AFB1 and its metabolism to aflatoxicol (AFL) by human placenta. In vitro incubations with placental cytosolic fraction confirmed the capacity of human placenta to form AFL. AFL was the only metabolite detected in both perfusions and in vitro incubations. Since AFL is less mutagenic, but putatively as carcinogenic as AFB1, the formation of AFL may not protect the fetus from the toxicity of AFB1.

Airway exposure to silica coated TiO2 nanoparticles induces pulmonary neutrophilia in mice

Thu, 29 Oct 2009 12:06:42 PDT

The importance of nanotechnologies and engineered nanoparticles has grown rapidly. It is therefore crucial to acquire up-to-date knowledge of the possible harmful health effects of these materials. Since a multitude of different types of nanosized TiO₂ particles are used in industry we explored their inflammatory potential using mouse and cell models. BALB/c mice were exposed by inhalation for either 2 hours, 2 hours on four consecutive days or 2 hours on four consecutive days for four weeks to several commercial TiO₂ nanoparticles, SiO₂ nanoparticles and to nanosized TiO₂ generated in a gas-to-particle conversion process at 10 mg/m³. In addition, effects of in vitro exposure of human macrophages and fibroblasts (MRC-9) to the different particles were assessed. SiO₂-coated rutile TiO₂ nanoparticles (cnTiO₂) was the only sample tested that elicited clear-cut pulmonary neutrophilia. Uncoated rutile and anatase as well as nanosize SiO₂ did not induce significant inflammation. Pulmonary neutrophilia was accompanied by increased expression of TNF- and neutrophil attracting chemokine CXCL1 in the lung tissue. TiO₂ particles accumulated almost exclusively in the alveolar macrophages. In vitro exposure of murine and human macrophages to cnTiO₂ elicited significant induction of TNF- and neutrophil attracting chemokines. Stimulation of human fibroblasts with cnTiO₂-activated macrophage supernatant induced high expression of neutrophil attracting chemokines, CXCL1 and CXCL8. Interestingly, the level of lung inflammation could not be explained by the surface area of the particles, their primary or agglomerate particle size or radical formation capacity, but is rather explained by the surface coating. Our findings emphasize that it is vitally important to take into account in the risk assessment that alterations of nanoparticles, e.g. by surface coating, may drastically change their toxicological potential.

In Utero and Lactational Exposure to Bisphenol A, in contrast to Ethinyl Estradiol, Does not Alter Sexually Dimorphic Behavior, Puberty, Fertility and Anatomy of Female LE Rats

Ryan, B. C., Hotchkiss, A. K., Crofton, K. M., Gray, L. E. — Wed, 28 Oct 2009 07:41:33 PDT

Many chemicals released into the environment display estrogenic activity including the oral contraceptive ethinyl estradiol (EE2) and the plastic monomer bisphenol A (BPA). EE2 is present in some aquatic systems at concentrations sufficient to alter reproductive function of fishes. Many concerns have been raised about the potential effects of BPA. The National Toxicology Program rated the potential effects of low doses of BPA on behavior and CNS as an area of "some concern", whereas most effects were rated as of "negligible" or "minimal" concern. However, the number of robust studies in this area was limited.

The current study was designed to determine if maternal exposure to relatively low oral doses of EE2 or BPA in utero and during lactation would alter the expression of well characterized sexually dimorphic behaviors or alter the age of puberty or reproductive function in the female LE rat offspring.

Pregnant rats were gavaged with vehicle, EE2 (0.05 to 50 µg/kg/day) or BPA (2, 20 and 200 µg/kg/day) from day 7 of gestation to postnatal day 18 and the female offspring were studied. EE2 (50 µg/kg/day) increased anogenital distance and reduced pup body weight at PND2, accelerated the age at vaginal opening, reduced F1 fertility and F2 litter sizes and induced malformations of the external genitalia (5 µg/kg). F1 females exposed to EE2 also displayed a reduced (male-like) saccharin preference (5 µg/kg) and absence of lordosis behavior (15 µg/kg), indications of defeminization of the CNS. BPA had no effect on any of the aforementioned measures. These results demonstrate that developmental exposure to pharmacologically-relevant dosage levels of EE2 can permanently disrupt the reproductive morphology and function of the female rat.

Developmental Neurotoxicity of Pyrethroid Insecticides in Zebrafish Embryos

DeMicco, A., Cooper, K. R., Richardson, J. R., White, L. A. — Tue, 27 Oct 2009 12:28:43 PDT

Pyrethroid insecticides are one of the most commonly used residential and agricultural insecticides. Based of the increased use of pyrethroids and recent studies showing that pregnant women and children are exposed to pyrethroids, there are concerns over the potential for developmental neurotoxicity. However, there have been relatively few studies on the developmental neurotoxicity of pyrethroids. In this study, we sought to investigate the developmental toxicity of six common pyrethroids, 3 type I compounds (permethrin, resmethrin, and bifenthrin) and 3 type II compounds (deltamethrin, cypermethrin, and -cyhalothrin), and to determine whether zebrafish embryos may be an appropriate model for studying the developmental neurotoxicity of pyrethroids. Exposure of zebrafish embryos to pyrethroids caused a dose dependent increase in mortality and pericardial edema, with type II compounds being the most potent. At doses approaching the LC₅₀, permethrin and deltamethrin caused craniofacial abnormalities. These findings are consistent with mammalian studies demonstrating that pyrethroids are mildly teratogenic at very high doses. However, at lower doses, body axis curvature and spasms were observed, which were reminiscent of the classic syndromes observed with pyrethroid toxicity. Treatment with Diazepam ameliorated the spasms, while treatment with the sodium channel antagonist MS-222 ameliorated both spasms and body curvature, suggesting that pyrethroid-induced neurotoxicity is similar in zebrafish and mammals. Taken in concert, these data suggest that zebrafish may be an appropriate alternative model to study the mechanism(s) responsible for the developmental neurotoxicity of pyrethroid insecticides and aid in identification of compounds that should be further tested in mammalian systems.

Effects of Pioglitazone, a Peroxisome Proliferator-Activated Receptor Gamma Agonist, on the Urine and Urothelium of the Rat

Sun, 25 Oct 2009 19:27:49 PDT

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors which belong to the nuclear receptor superfamily. Some PPAR agonists, such as pioglitazone, and dual PPAR/PPAR agonists, such as muraglitazar, induced urothelial bladder tumors in rats but not in mice. In this study, we investigated the early effects in the urine and bladder of rats treated with pioglitazone to evaluate the possible relation between urinary solids formation and urothelial cytotoxicity and regenerative proliferation. In a 4-week experiment, treatment of rats with 16 mg/kg pioglitazone induced cytotoxicity and necrosis of the urothelial superficial layer, with increased cell proliferation measured by bromodeoxyurindine labeling index and hyperplasia by histology. It also produced alterations in urinary solid formation, especially calcium-containing crystals and calculi. PPAR agonists (pioglitazone and troglitazone) in vitro reduced rat urothelial cell proliferation and induced uroplakin synthesis, a specific differentiation marker in urothelial cells. Our data support the hypothesis that the bladder tumors produced in rats by pioglitazone are related to the formation of urinary solids. This strongly supports the previous conclusion in studies with muraglitazar that this is a rat specific phenomenon and does not pose a urinary bladder cancer risk to humans treated with these agents.

Effects of cytochrome P450 inhibitors on the biotransformation of fluorogenic substrates by adult male rat liver microsomes and cDNA-expressed rat cytochrome P450 isoforms

Makaji, E., Trambitas, C. S., Shen, P., Holloway, A. C., Crankshaw, D. J. — Sun, 25 Oct 2009 19:27:48 PDT

We have evaluated the use of a panel of six fluorogenic cytochrome P450 substrates as a potential tool for rapid screening for global changes in CYP activity in rats under different physiological conditions. The biotransformation of 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin, 7-benzyloxy-4-(trifluoromethyl)-coumarin ,7-benzyloxyquinoline, 3-cyano-7-ethoxycoumarin, 7-methoxy-4-(trifuoromethyl)-coumarin and 7-ethoxy-4-trifluoromethyl-coumarin by microsomes from adult male rat liver were characterized, their sensitivities to fifteen putative inhibitors were determined and compared to similar experiments using nine different cDNA-expressed rat CYPs. Inhibitory profiles of the substrates in microsomes were different from each other, with some overlap, suggesting that each substrate is to some extent biotransformed by a different CYP isoform. Ketoconazole and clotrimazole were non-selective inhibitors while ticlopidine selectively inhibited biotransformation of 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin. CYP 2A1 did not biotransform any of the substrates and CYP 2E1 was insensitive to all the inhibitors tested. Some inhibitors did not affect the biotransformation of the fluorogenic substrates by cDNA-expressed isoforms as predicted by their effects on conventional substrates, for example chlorzoxazone and diethyldithiocarbamate were inactive against CYP2E1, and CYP2C6 was not inhibited by sulfaphenazole. When results in microsomes and cDNA-expressed CYPs were compared, only the majority of the biotransformation of 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin by microsomes could be assigned with full confidence to a specific CYP isoform, namely CYP2D2. Nevertheless different inhibitory profiles of the substrates indicate that the panel will be useful for rapid functional quantification of global CYP activity in rats under different experimental conditions. Our results also demonstrate the inappropriateness of extrapolating inhibitory data between conventional and fluorogenic CYP substrates.

Characterization of Peroxisome Proliferator-Activated Receptor {alpha} (PPAR{alpha}) - Independent Effects of PPAR{alpha} Activators in the Rodent Liver: Di-(2-ethylhexyl) phthalate Also Activates the Constitutive Activated Receptor

Thu, 22 Oct 2009 05:14:06 PDT

Peroxisome proliferator chemicals (PPC) are thought to mediate their effects in rodents on hepatocyte growth and liver cancer through the nuclear receptor peroxisome proliferator-activated receptor alpha (PPAR). Recent studies indicate that the plasticizer di-2-ethylhexyl phthalate (DEHP) increased the incidence of liver tumors in PPAR-null mice. We hypothesized that some PPC, including DEHP, induce transcriptional changes independent of PPAR but dependent on other nuclear receptors, including the constitutive activated receptor (CAR) that mediates phenobarbital (PB) effects on hepatocyte growth and liver tumor induction. To determine the potential role of CAR in mediating effects of PPC, a meta-analysis was performed on transcript profiles from published studies in which rats and mice were exposed to PPC and compared the profiles to those produced by exposure to PB. Valproic acid, clofibrate and DEHP in rat liver and DEHP in mouse liver induced genes including Cyp2b family members that are known to be regulated by CAR. Examination of transcript changes by Affymetrix ST 1.0 arrays and RT-PCR in the livers of DEHP-treated wild-type, PPAR-null and CAR-null mice demonstrated that 1) most (~94%) of the transcriptional changes induced by DEHP were PPAR-dependent, 2) many PPAR-independent genes overlapped with those regulated by PB, 3) induction of genes Cyp2b10, Cyp3a11 and metallothionine-1 by DEHP was CAR-dependent but PPAR-independent and 4) induction of a number of genes (Cyp8b1, Gstm4, Gstm7) was independent of both CAR and PPAR. Our results indicate that exposure to PPAR activators including DEHP activate multiple nuclear receptors in the rodent liver.

Mechanisms Mediating the Vesicant Actions of Sulfur Mustard after Cutaneous Exposure

Thu, 15 Oct 2009 06:22:51 PDT

Sulfur mustard, a chemical weapon first employed during World War I, targets the skin, eyes, and lung. It remains a significant military and civilian threat. The characteristic response of human skin to sulfur mustard involves erythema of delayed onset, followed by edema with inflammatory cell infiltration, the appearance of large blisters in the affected area, and a prolonged healing period. Several in vivo and in vitro models have been established to understand the pathology and investigate the mechanism of action of this vesicating agent in the skin. Sulfur mustard is a bifunctional alkylating agent which reacts with many targets including lipids, proteins and DNA, forming both intra- and intermolecular cross-links. Despite the relatively non-selective chemical reactivity of this agent, basal keratinocytes are more sensitive, and blistering involves detachment of these cells from their basement membrane adherence zones. The sequence and manner in which these cells die and detach is still unresolved. Much has been discovered over the past two decades with respect to the mechanisms of sulfur mustard-induced cytotoxicity, and the intracellular and extracellular targets of this vesicant. In this review, the effects of sulfur mustard exposure on the skin are described, as well as potential mechanisms mediating its actions. Successful therapy for sulfur mustard poisoning will depend on following new mechanistic leads to develop drugs that target one or more of its sites of action.

Oxidative Stress Caused by Ozone Exposure Induces Loss of Brain Repair in The Hippocampus of Adult Rats

Thu, 15 Oct 2009 06:22:49 PDT

Oxidative stress is involved in many neurodegenerative diseases. Chronic ozone exposure causes a secondary increase of reactive oxygen species, which cause an oxidative-stress state in the organism. Ozone is one of the main components of photochemical pollution. Our purpose was to test that oxidative stress caused by chronic low doses of ozone, by itself, alters adult neurogenesis and causes progressive neurodegeneration in the hippocampus, which actions lead to the loss of brain plasticity in the mature central nervous system of rats. Animals were exposed to an ozone-free air stream and for 15, 30, 60, and 90 days to low doses of ozone to cause oxidative stress. Each group was then tested by 1) a spectrophotometer test to quantify lipid peroxidation levels, 2) immunohistochemistry testing against doublecortine, Neu-N, p53, microglia, and glial fibrillary-acidic protein (GFAP), 3) western blot tests for doublecortine and Neu-N, and 4) a one-trial passive avoidance test. Our results indicated that ozone causes an increase of lipid peroxidation levels, morphological changes in the nucleus and the cytoplasm, and cell swelling in neurons. The western blot shows a decrease for Neu-N and doublecortine. Activated and later phagocytic microglia and an increased number of astrocytes were found. There was a memory deficiency positively related to the amount of ozone exposure. These alterations suggest that oxidative stress caused by low doses of ozone causes dysregulation of inflammatory processes, progressive neurodegeneration, chronic loss of brain repair in the hippocampus, and brain plasticity changes in the rat analogous to those seen in Alzheimer's disease.

Arsenic (+3 Oxidation State) Methyltransferase and the Methylation of Arsenicals in the Invertebrate Chordate Ciona intestinalis

Thu, 15 Oct 2009 06:22:47 PDT

Biotransformation of inorganic arsenic (iAs) involves methylation catalyzed by arsenic (+3 oxidation state) methyltransferase (As3mt), yielding mono- , di- , and trimethylated arsenicals. To investigate the evolution of molecular mechanisms that mediate arsenic biotransformation, a comparative genomic approach focusing on the invertebrate chordate Ciona intestinalis, was used. Bioinformatic analyses identified an As3mt gene in the C. intestinalis genome. Constitutive As3mt RNA expression was observed in heart, branchial sac and gastrointestinal tract. Adult animals were exposed to zero or 1 ppm of iAs for 1 or 5 days. Steady-state As3mt RNA expression in the gastrointestinal tract was not modulated significantly by 5 days of exposure to iAs. Tissue levels of iAs and its methylated metabolites were determined by hydride generation-cryotrapping-gas chromatography-atomic absorption spectrometry. At either time point, exposure to iAs significantly increased concentrations of iAs and its methylated metabolites in tissues. After 5 days of exposure, total speciated arsenic concentrations were highest in branchial sac (3705 ng/g) followed by heart (1019 ng/g) and gastrointestinal tract (835 ng/g). At this time point, the sum of the speciated arsenical concentrations in gastrointestinal tract and heart equaled or exceeded that of iAs; in branchial sac, iAs was the predominant species present. Ciona intestinalis metabolizes iAs to its methylated metabolites, which are retained in tissues. This metabolic pattern is consistent with the presence of an As3mt ortholog in its genome and constitutive expression of the gene in prominent organs, making this basal chordate a useful model to examine the evolution of arsenic detoxification.

Decreased expression of the voltage-dependent anion channel in differentiated PC-12 and SH-SY5Y cells following Low-level Pb exposure

Prins, J. M., Park, S., Lurie, D. I. — Mon, 12 Oct 2009 04:29:50 PDT

Lead (Pb) has been shown to disrupt cellular energy metabolism which may underlie the learning deficits and cognitive dysfunctions associated with environmental Pb exposure. The voltage dependent anion channel (VDAC) plays a central role in regulating energy metabolism in neurons by maintaining cellular ATP levels and regulating calcium buffering, and studies have shown that VDAC expression is associated with learning in mice. In this study, we examined the effect of 5µM and 10µM Pb on VDAC expression in-vitro in order to determine whether Pb alters VDAC expression levels in neuronal cell lines. PC-12 and SH-SY5Y cells were used since they differentiate to resemble primary neuronal cells. VDAC expression levels were significantly decreased 48 h after exposure to Pb in both cell lines. In contrast, exposure to 24 h of hypoxia failed to produce a decrease in VDAC, suggesting that decreased VDAC expression is not a general cellular stress response, but is a result of Pb exposure. This decreased VDAC expression was also correlated with a corresponding decrease in cellular ATP levels. Real-time RT-PCR demonstrated a significant decrease in mRNA levels for the VDAC 1 isoform, indicating that Pb reduces transcription of VDAC1. These results demonstrate that exposure to 5µM and 10µM Pb reducesVDAC transcription and expression and is associated with reduced cellular ATP levels.

Proteomic analysis of hepatic tissue of zebrafish (Danio rerio) experimentally exposed to chronic microcystin-LR

Wang, M., Chan, L. L., Si, M., Hong, H., Wang, D. — Mon, 12 Oct 2009 04:29:47 PDT

Microcystin-LR (MCLR) is the most toxic and most frequently encountered hepatotoxin in the aquatic environment. This study investigated the protein profiles of zebrafish (Danio rerio) livers chronically exposed to MCLR concentrations (2 or 20 µg/l) using the proteomic approach as well as cell ultrastructure, protein phosphatase (PP) activity, protein phosphatase 2A (PP2A) abundance and toxin content analysis of the hepatic tissue. The results showed that, after 30 d exposure, the presence of MCLR strikingly enhanced toxin accumulation and the PP activity in zebrafish livers. However, the PP2A amounts were independent of toxin treatments. MCLR caused a noticeable damage to liver ultrastructure, a widespread swelling in the rough endoplasmatic reticulum and mitochondria was observed in the MCLR-exposed hepatocytes, and a honeycomb-like structure was formed in the treated nucleoli. Comparison of two-dimensional electrophoresis (2-DE) protein profiles of MCLR-exposed and non-exposed zebrafish livers revealed that the abundance of 22 proteins, measured by 2-DE, was remarkably altered in response to toxin exposure. These proteins were involved in cytoskeleton assembly, macromolecule metabolism, oxidative stress and signal transduction, indicating that MCLR toxicity in fish liver is complex and diverse. Thus, proteomics provides a new insight into MCLR toxicity; that chronic toxicity of MCLR is different from acute toxicity; and we speculate that the reactive oxygen species pathway might be the main toxic pathway instead of the PP one. Moreover, even a low concentration of MCLR in water could significantly interrupt cellular processes, and more care should be taken in determining the criterion for MCLR content in drinking water.

Subchronic 13-week Inhalation Exposure of Rats to Multiwalled Carbon Nanotubes: Toxic Effects are determined by Density of Agglomerate Structures, not fibrillar Structures

Pauluhn, J. — Mon, 12 Oct 2009 04:29:43 PDT

Wistar rats were nose-only exposed to Multi-walled Carbon Nanotubes (MWCNT, Baytubes^®) in a subchronic 13-week inhalation study. The focus of study was on respiratory tract and systemic toxicity, including analysis of MWCNT biokinetics in the lungs and lung-associated lymph nodes (LALNs). The time-course and concentration-dependence of pulmonary effects were examined by bronchoalveolar lavage (BAL), and histopathology up to 6 months postexposure. Particular emphasis was directed to the comparative characterization of MWCNT structures prior to and after micronization and dry powder dispersion into inhalation chambers. These determinations were complemented by additional analyses in digested BAL-cells. Animals were exposed on 6 hrs/day, five days per week for 13 consecutive weeks to 0, 0.1, 0.4, 1.5, and 6 mg/m³. The subchronic exposure to respirable solid aerosols of MWCNT was tolerated without effects suggestive of systemic toxicity. Kinetic analyses demonstrated a markedly delayed clearance of MWCNT from lungs at overload conditions. Translocation into LALNs occurred at 1.5 and 6 mg/m³ and required at least 13 weeks of study to become detectable. At these exposure levels the lung and LALN weights were significantly increased. Sustained elevations in BAL PMNs and soluble collagen occurred at these concentrations with borderline effects at 0.4 mg/m³. Histopathology revealed principal exposure-related lesions at 0.4 mg/m³ and above in the upper respiratory tract (goblet cell hyper- and/or metaplasia, eosinophilic globules, focal turbinate remodeling) and the lower respiratory tract (inflammatory changes in the bronchiolo-alveolar region, increased interstitial collagen staining). Granulomatous changes and a time-dependent increase of a bronchiolio-alveolar hyperplasia occurred at 6 mg/m³. All endpoints examined were unremarkable at 0.1 mg/m³ (NOAEL). In summary, this study demonstrates that the induced pathological changes are consistent with overload-related phenomena. Hence, the etiopathological sequence of inflammatory events caused by this type of MWCNT appears to be related to the high displacement volume of the low-density MWCNT-assemblage structure rather than to any yet ill-defined intrinsic toxic property. Thus, the hypothesis of study is verified, namely common denominators between carbon black and MWCNT do exist.

Cell volume decrease as a link between azaspiracid-induced cytotoxicity and c-jun-N-terminal kinase activation in cultured neurons

Vale, C., Nicolaou, K. C., Frederick, M. O., Vieytes, M. R., Botana, L. M. — Thu, 08 Oct 2009 10:58:29 PDT

Azaspiracids are a group of marine toxins recently described that currently includes 20 analogues. Not much is known about their mechanism of action, although the predominant analogue in nature, azaspiracid-1 (AZA-1) targets several organs in vivo, including the central nervous system and exhibits high neurotoxicity in vitro. Azaspiracid distribution is increasing globally with mussels being most widely implicated in azaspiracid-related food poisoning events and human poisoning by azaspiracids has emerged as an increasing worldwide problem in recent years. We used pharmacological tools to inhibit the cytotoxic effect of the toxin in primary cultured neurons. Several targets for azaspiracid-induced neurotoxicity were evaluated. AZA-1 elicited a concentration-dependent hyperpolarization in cerebellar granule cells of 2-3 days in vitro (div), however it did not modify membrane potential in mature neurons. Furthermore, in immature cells AZA-1 decreased the membrane depolarization evoked by exposure of the neurons to 50 mM K⁺. Preincubation of the neurons with 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), 4-Acetamido-4'-isothiocyanato-2,2'-stilbenedisulfonic acid (SITS), 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), amiloride or ouabain before addition of AZA-1 decreased the AZA-1 induced neurotoxicity and the increase in phosphorylated JNK caused by the toxin, indicating that disruption in ion fluxes was involved in the neurotoxic effect of AZA-1. Furthermore, short exposures of cultured neurons to AZA-1 caused a significant decrease in neuronal volume that was reverted by preincubation of the neurons with DIDS or amiloride before addition of the toxin. The results presented here indicate that the JNK activation induced by AZA-1 is secondary to the decrease in cellular volume elicited by the toxin.

Expression of Tyrosine Hydroxylase Increases the Resistance of Human Neuroblastoma Cells to Oxidative Insults

Thu, 08 Oct 2009 10:58:29 PDT

In this study we demonstrate that human neuroblastoma SH-SY5Y cells transfected with human tyrosine hydroxylase isoform 1 (SH+TH cells) were substantially more resistant to cell death induced by hydrogen peroxide and 6-hydroxydopamine, when compared to wild type SH-SY5Y cells (SH cells). SH+TH cells exhibit increased levels of dopamine compared to SH cells. Incubation with hydrogen peroxide or 6-hydroxydopamine (10 – 100 µM) for 24 hours caused a significant reduction in cell viability and increased apoptosis in both cell types. However, these effects were significantly reduced in the SH+TH cells when compared to the SH cells. The SH+TH cells showed an improved ability to detoxify peroxide, which correlated with an increase in glutathione peroxidase and glutathione reductase activity, while catalase activity was unchanged. Our data suggests that a preconditioning-like mechanism linked to higher dopamine levels increased the resistance of SH+TH cells against oxidative insults, which is at least in part related to an augmentation in the activity of glutathione-related antioxidant enzymes.

Reduction of glutamatergic neurotransmission by prolonged exposure to dieldrin involves NMDA receptor internalization and metabotropic glutamate receptor 5 down-regulation

Briz, V., Galofre, M., Sunol, C. — Wed, 07 Oct 2009 11:02:06 PDT

Dieldrin was previously used as a pesticide. Although its use has been discontinued, humans are still exposed to it due to its high environmental persistence and because it accumulates in the adipose tissue of animals. Acute exposure to dieldrin provokes convulsions, due to its antagonism on the GABA_A receptor. However little is known about the effects of low chronic exposure to this pollutant. In the present work we use primary cultures of cortical neurons to study the mechanisms involved in the toxic action of dieldrin. We found that 2 and 6 days in vitro (DIV) exposure to a subcytotoxic concentration (60nM) of dieldrin reduced the increase in intracellular calcium concentration ([Ca²⁺]_i) and the excitotoxicity caused by glutamate. Exposure to dieldrin for 6 DIV induced NMDA receptor (NMDAR) internalization and reduced metabotropic glutamate receptor 5 (mGLUR5) levels. Double immunostaining for NMDAR and mGLUR5 showed that these receptors lose colocalization on the cell membrane in neurons treated with dieldrin. No changes were observed in receptor functionalities or receptor levels after 2 DIV of exposure to dieldrin. However, the increase in [Ca²⁺]_i induced by coactivation of NMDAR and mGLUR5 was significantly reduced. Thus, a functional interaction between the two receptors seems to play an important role in glutamate-induced excitotoxicity. We confirm that permanent blockade of the GABA_A receptor by this persistent pesticide triggers adaptive neuronal changes consisting of a reduction of glutamatergic neurotransmission. This might explain the cognitive and learning deficits observed in animals after chronic treatment with dieldrin.

In Utero Exposure to Benzene Disrupts Fetal Hematopoietic Progenitor Cell Growth Via Reactive Oxygen Species

Badham, H. J., Winn, L. M. — Wed, 07 Oct 2009 11:02:06 PDT

It is hypothesized that the increasing incidence of childhood leukemia may be due to in utero exposure to environmental pollutants such as benzene, but the mechanisms involved remain unknown. We hypothesize that reactive oxygen species (ROS) contribute to the deregulation of fetal hematopoiesis caused by in utero benzene exposure. To evaluate this hypothesis, pregnant C57Bl/6N mice were exposed to benzene or PEG-catalase (antioxidative enzyme) and benzene. Colony formation assays on fetal liver cells were performed to measure erythroid and myeloid progenitor cell growth potential. The presence of ROS in CD117⁺ fetal liver cells was measured by flow cytometric analysis. Oxidative cellular damage was assessed by Western blot analysis of 4-hydroxynonenol and nitrotyrosine products, as well as reduced to oxidized glutathione ratios. Alterations in the redox sensitive signaling pathway NF-B were measured by Western blot analysis of IB- protein levels in fetal liver tissue. Results: In utero exposure to benzene caused a significant increase in ROS production and significantly altered fetal liver erythroid and myeloid colony numbers, but did not increase the levels of 4-HNE or nitrotyrosine products or alter reduced to oxidized glutathione ratios. However, in utero exposure to benzene did cause a significant decrease in fetal liver IB- protein levels suggesting activation of the NF-B pathway. Benzene-induced ROS formation, abnormal colony growth, and decreased IB- levels were all abrogated by pre-treatment with PEG-catalase. These results suggest that ROS play a key role in the development of in utero-initiated benzene toxicity potentially through disruption of hematopoietic cell signaling pathways.

Cytotoxicity and Regenerative Proliferation as the Mode of Action for Diuron-Induced Urothelial Carcinogenesis in the Rat

Wed, 07 Oct 2009 11:02:05 PDT

Diuron, a substituted urea herbicide, is carcinogenic to the urinary bladder of rats at high dietary levels. Its proposed carcinogenic mode of action (MOA) includes urothelial cytotoxicity and necrosis followed by regenerative cell proliferation and sustained urothelial hyperplasia. Cytotoxicity could be induced either by urinary solids or by chemical toxicity by diuron and/or metabolites excreted in the urine. Diuron was not genotoxic in a previous single cell gel (comet) assay, but possible cross-linking activity remained to be evaluated. The present study explored the MOA of diuron and the effect of urinary acidification on the development of urothelial lesions. Male Wistar rats were fed diuron (2,500 ppm, about 130 mg/kg of body weight) either with or without NH₄Cl 10,000 ppm to acidify the urine. Reversibility of urothelial changes was also examined. The animals were euthanized after 15, 25 or 30 weeks. Diuron-fed rats had urinary amorphous precipitate and magnesium ammonium phosphate crystals similar to control animals. Groups treated with diuron+NH₄Cl showed decreased urinary pH and reduced amounts of urinary crystals and precipitate. Urothelial necrosis and simple hyperplasia were observed by light microscopy and scanning electron microscopy both in diuron and in diuron+NH₄Cl treated groups. Cytotoxicity and proliferative changes were mostly reversible. A modified comet assay developed in vitro with CHO cells showed that diuron did not induce DNA cross-links. These data suggest that cytotoxicity with consequent regenerative cell proliferation is the predominant MOA for diuron rat urothelial carcinogenesis, the cytotoxicity being chemically induced and not due to urinary solids.

Developmental exposure to manganese (Mn) increases adult susceptibility to inflammatory activation of glia and neuronal protein nitration

Moreno, J. A., Streifel, K. M., Sullivan, K. A., Legare, M. E., Tjalkens, R. B. — Wed, 07 Oct 2009 11:02:05 PDT

Chronic exposure to manganese (Mn) produces a neurodegenerative disorder affecting the basal ganglia characterized by reactive gliosis and expression of neuroinflammatory genes including inducible nitric oxide synthase (NOS2). Induction of NOS2 in glial cells causes overproduction of nitric oxide (NO) and injury to neurons that is associated with parkinsonian-like motor deficits. Inflammatory activation of glia is believed to be an early event in Mn neurotoxicity but specific responses of microglia and astrocytes to Mn during development remain poorly understood. In this study we investigated the effect of juvenile exposure to Mn on the activation of glia and production of NO in C57Bl/6 mice, postulating that developmental Mn exposure would lead to heightened sensitivity to gliosis and increased expression of NOS2 in adult mice exposed again later in life. Immunohistochemical analysis indicated that Mn exposure caused increased activation of both microglia and astrocytes in the striatum (St), globus pallidus (Gp), and substantia nigra pars reticulata (SNpr) of treated mice compared to controls. More robust activation of microglia was observed in juveniles, whereas astrogliosis was more prominent in adult mice pre-exposed during development. Co-immunofluorescence studies demonstrated increased expression of NOS2 in glia located in the Gp and SNpr. Additionally, greater increases in the level of 3-nitro-tyrosine protein adducts were detected in DARPP32-positive neurons of the striatum of Mn-treated adult mice pre-exposed as juveniles. These data indicate that sub-chronic exposure to Mn during development leads to temporally distinct patterns of glial activation that result in elevated nitrosative stress in distinct populations of basal ganglia neurons.

Age-dependent susceptibility to manganese-induced neurological dysfunction

Wed, 07 Oct 2009 11:02:04 PDT

Chronic exposure to manganese (Mn) produces a spectrum of cognitive and behavioral deficits associated with a neurodegenerative disorder resembling Parkinson's disease. The effects of high-dose exposure to Mn in occupational cohorts and in adult rodent models of the disease are well described but much less is known about the behavioral and neurochemical effects of Mn in the developing brain. We therefore exposed C57Bl/6 mice to Mn by intragastric gavage as juveniles, adults, or both, postulating that mice exposed as juveniles and then again as adults would exhibit greater neurological and neurochemical dysfunction than mice not pre-exposed as juveniles. Age- and sex-dependent vulnerability to changes in locomotor function was detected, with juvenile male mice displaying the greatest sensitivity, characterized by a selective increase in novelty seeking and hyperactive behaviors. Adult male mice pre-exposed as juveniles had a decrease in total movement and novelty seeking behavior and no behavioral changes were detected in female mice. Striatal dopamine levels were increased in juvenile mice but were decreased in adult pre-exposed as juveniles. Levels of Mn, Fe, and Cu were determined by ICP-MS, with the greatest accumulation of Mn detected in juvenile mice in the striatum, substantia nigra and cortex. Only modest changes in Fe and Cu were detected in Mn-treated mice, primarily in the substantia nigra. These results reveal that developing mice are more sensitive to Mn than adult animals and that Mn exposure during development enhances behavioral and neurochemical dysfunction dysfunction relative to adult animals without juvenile exposure.

The Role of Hypoxia in 2-Butoxyethanol-induced Hemangiosarcoma

Wed, 07 Oct 2009 11:02:04 PDT

To understand the molecular mechanisms underlying compound-induced hemangiosarcomas in mice, and therefore their human relevance, a systems biology approach was undertaken using transcriptomics and Causal Network^TM Modeling from mice treated with 2-butoxyethanol (2-BE). 2-BE is a hemolytic agent that induces hemangiosarcomas in mice. We hypothesized that the hemolysis induced by 2-BE would result in local tissue hypoxia, a well-documented trigger for endothelial cell proliferation leading to hemangiosarcoma. Gene expression data from bone marrow, liver, and spleen of mice exposed to a single dose (4h) or 7 daily doses of 2-BE were used to develop a mechanistic model of hemangiosarcoma. The resulting mechanistic model confirms previous work proposing that 2-BE induces macrophage activation and inflammation in the liver. In addition, the model supports local tissue hypoxia in the liver and spleen, coupled with increased Epo signaling and erythropoiesis in the spleen and bone marrow, and suppression of mechanisms that contribute to genomic stability, events that could be contributing factors to hemangiosarcoma formation. Finally, an immunohistochemistry method (Hypoxyprobe^TM) demonstrated that tissue hypoxia was present in the spleen and bone marrow. Together, the results of this study identify molecular mechanisms that initiate hemangiosarcoma, a key step in understanding safety concerns that can impact drug decision processes, and identified hypoxia as a possible contributing factor for 2-BE-induced hemangiosarcoma in mice.

P19 neuronal differentiation and retinoic acid metabolism as criteria to investigate atrazine, nitrite and nitrate developmental toxicity

Solari, M., Paquin, J., Ducharme, P., Boily, M. — Tue, 06 Oct 2009 07:17:10 PDT

Atrazine and nitrogenous fertilizers are agrochemical contaminants frequently detected in water systems in North America. Several studies reported their ability to affect amphibian and mammalian development. Retinoids, supplied in the diet or synthesized by cells, are essential to embryogenesis. Disturbance of their homeostasis may lead to teratogenic effects. Retinoic acid (RA) is a major retinoid regulator of cell proliferation and differentiation. Previous studies reported alterations of retinoid stores in bullfrogs of Yamaska River subwatersheds (Québec, Canada), a region of intensive agricultural activities associated with atrazine, nitrate and nitrite contaminants. These contaminants could affect RA metabolism and RA-mediated processes. Mouse P19 embryonic stem cells, which can differentiate to neurons in response to RA, were used to test this hypothesis. Cells were cultured in the absence or presence of contaminants during neuroinduction with RA, and assayed by flow cytometry for expression of SSEA1 (embryonic marker) and βIII-tubulin (neuronal marker). Cell cultures were also analysed for RA metabolism by HPLC. Downregulation of SSEA1 paralleled βIII-tubulin upregulation in a RA concentration-dependent manner. Atrazine, nitrate and nitrite did not affect differentiation at environmentally-encountered micromolar concentrations. However, low molar nitrite prevented RA-induced SSEA1 downregulation, and decreased βIII-tubulin appearance. Decreased cell viability/proliferation accompanied these differentiation effects. P19 cells metabolized RA to polar retinoids. RA metabolism was not affected at any concentration of atrazine, nitrate or nitrite. Environmentally-relevant levels of these contaminants thus had no gross effect on neurodifferentiation and RA catabolism of embryonic stem cells. P19 cell-based bioassays may provide valuable tools in monitoring developmental toxicity.

Use of gene expression of neural markers in cultured neural cells to identify developmental neurotoxicants

Sunol, C. — Tue, 06 Oct 2009 07:17:09 PDT

Rapamycin inhibits yeast nucleotide excision repair independently of Tor kinases

Limson, M. V., Sweder, K. S. — Mon, 05 Oct 2009 09:01:43 PDT

The yeast target of rapamycin kinases, Tor1 and Tor2, belong to the phosphatidylinositol 3-kinase related family of proteins, which are involved in the cellular response to DNA damage and changes in nutrient conditions. In contrast to yeast, many eukaryotes possess a single Tor kinase. Regardless of the number of Tor kinases in an organism, two distinct complexes involving Tor proteins exist in eukaryotes, TORC1 and TORC2. The yeast TORC1, containing Tor1 or Tor2, is sensitive to the antibiotic rapamycin. The yeast TORC2 is insensitive to rapamycin. We examined the influence of rapamycin treatment upon yeast transcription-coupled nucleotide excision repair in a gene transcribed by RNA polymerase II. We also examined tor mutants for their ability to perform transcription-coupled repair in the absence or presence of rapamycin. Ostensibly lacking TORC1 and TORC2 function, a tor1tor2^ts mutant grown at the nonpermissive temperature exhibited similar rates of repair as the wild-type strain. However, repair of both strands in genes decreases in the wild-type strain and the tor1tor2^ts mutant exposed to rapamycin. Rapamycin may be inhibiting DNA repair independently of the Tor kinases. In yeast, FPR1 encodes the rapamycin-binding protein Fpr1 that inhibits the TORC1 kinase in the presence of rapamycin. Fap1 competes with rapamycin for Fpr1 binding. Deletion of the FPR1 or FAP1 gene abolishes the inhibitory effect of rapamycin on repair. Thus, the decreased repair observed following rapamycin treatment is independent of TORC1/2 function and likely due to a function of Fap1. We suggest that Fap1 and peptidyl prolyl isomerases, particularly Fpr1, function in the cellular response to genotoxic stress. Our findings have clinical implications for genetic toxicities associated with genotoxic agents when co-administered with rapamycin.

Differential impact of diesel particle composition on pro-allergic dendritic cell function

Sun, 04 Oct 2009 22:28:24 PDT

Diesel exhaust particles (DEP) were described as potent adjuvant in the induction and maintenance of allergic diseases suggesting that they might play a role in the increase of allergic diseases in the industrialized countries. However, the cellular basis by which these particles enhance allergic immune responses is still a matter of debate.

Thus, we exposed immature murine bone marrow-derived dendritic cells (BMDC) to different particles or particle-associated organic compounds in the absence or presence of the maturation stimuli lipopolysachharide and analyzed the cellular maturation, viability, and cytokine production. Furthermore, we monitored the functionality of particle-exposed BMDC to suppress B cell isotype switching to IgE.

Only highly polluted DEP (standard reference material SRM1650a) but not particle-associated organic compounds or less polluted DEP from modern diesel engines were able to modulate the dendritic cell phenotype. SRM1650a particles significantly suppressed LPS-induced IL-12p70 production in murine BMDC, whereas cell surface marker expression was not altered. Furthermore, SRM1650a-exposed immature BMDC lost the ability to suppress IgE isotype switch in B cells.

This study revealed that highly polluted DEP not only interfere with dendritic cell maturation but additionally with dendritic cell function, thus suggesting a role in Th2 immune deviation.

"Toxicity Testing in the 21st Century: A View from the Chemical Industry

Bus, J. S., Becker, R. A. — Sun, 04 Oct 2009 22:28:24 PDT

Weighted Feature Significance (WFS): A Simple, Interpretable Model of Compound Toxicity Based on the Statistical Enrichment of Structural Features

Sun, 04 Oct 2009 22:28:23 PDT

In support of the U.S. Tox21 program, we have developed a simple and chemically intuitive model we call Weighted Feature Significance (WFS) to predict the toxicological activity of compounds, based on the statistical enrichment of structural features in toxic compounds. We trained and tested the model on: (1) data from quantitative high-throughput screening (qHTS) cytotoxicity and caspase activation assays conducted at the NIH Chemical Genomics Center (NCGC), (2) data from Salmonella typhimurium reverse mutagenicity assays conducted by the U.S. National Toxicology Program (NTP), and (3) hepatotoxicity data published in the Registry of Toxic Effects of Chemical Substances (RTECS). Enrichments of structural features in toxic compounds are evaluated for their statistical significance and compiled into a simple additive model of toxicity, and then used to score new compounds for potential toxicity. The predictive power of the model for cytotoxicity was validated using an independent set of compounds from the U.S. Environmental Protection Agency (EPA) tested also at the NCGC. We compared the performance of our WFS approach with classical classification methods such as Naïve Bayesian clustering and support vector machines. In most test cases, WFS showed similar or slightly better predictive power, especially in the prediction of hepatotoxic compounds, where WFS appeared to have the best performance among the three methods. The new algorithm has the important advantages of simplicity, power, interpretability, and ease of implementation.

2,3,7,8-Tetrachlorodibenzo-p-dioxin Inhibits Fibroblast Growth Factor 10-Induced Prostatic Bud Formation in Mouse Urogenital Sinus

Vezina, C. M., Hardin, H. A., Moore, R. W., Allgeier, S. H., Peterson, R. E. — Sun, 04 Oct 2009 22:28:23 PDT

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) dorsalizes the pattern of prostatic buds developing from the urogenital sinus (UGS) of male fetal mice, causing some buds to form in inappropriate positions while blocking formation of others. This teratogenic TCDD action significantly reduces prostate main duct number and causes ventral prostate agenesis in exposed males. The purpose of this study was to determine whether inhibition of fibroblast growth factor 10 (FGF10) signaling is mechanistically linked to mouse prostatic budding impairment by TCDD. In utero TCDD exposure induced aryl hydrocarbon receptor (AHR)-responsive cytochrome P450 1b1 mRNA in ventral UGS regions where Fgf10 and FGF receptor 2 (Fgfr2) mRNA were expressed and where budding was most severely inhibited by TCDD. However, TCDD exposure did not reduce Fgf10 or Fgfr2 mRNA abundance in the UGS or alter their distribution. Addition of FGF10 protein to UGS organ culture media increased the abundance of UGS basal epithelial cells immuno-positive for phosphorylated extracellular signal-regulated kinase (ERK). FGF10 also increased the number of bromodeoxyuridine (BrdU)-labeled UGS epithelial cells and increased the number of prostatic buds formed per UGS. Addition of TCDD to UGS organ culture media did not alter FGF10-induced ERK activation in UGS basal epithelium but prevented FGF10-induced BrdU incorporation and blocked FGF10-induced prostatic bud formation. These results identify basal UGE cells as the key site of FGF10 action during fetal prostate development and suggest that TCDD likely acts downstream of FGFR2 and ERK to restrict UGS epithelial cell proliferation and prevent prostatic bud formation.

Suppression of Insulin-Like Growth Factor Acid-Labile Subunit (IGFALS) Expression - A Novel Mechanism for Deoxynivalenol-Induced Growth Retardation

Amuzie, C. J., Pestka, J. J. — Sun, 04 Oct 2009 22:28:22 PDT

Consumption of deoxynivalenol (DON), a trichothecene mycotoxin commonly detected in cereal-based foods, causes impaired growth in many animal species. While growth retardation is used as a basis for regulating DON levels in human food, the underlying mechanisms remain poorly understood. Oral exposure of mice to DON rapidly induces multi-organ expression of proinflammatory cytokines and this is followed by upregulation of several suppressors of cytokine signaling (SOCS), some of which are capable of impairing growth hormone (GH) signaling. The purpose of this study was to test the hypothesis that impairment of the GH axis precedes DON-induced growth retardation in the mouse. Subchronic dietary exposure of young (4-wk old) mice to DON (20 ppm) over a period of 2 to 8 wk was found to: (1) impair weight gain, (2) result in a steady-state plasma DON concentration (40-60 ng/ml), (3) downregulate hepatic insulin-like growth factor acid labile subunit (IGFALS) mRNA expression and (4) reduce circulating insulin-like growth factor 1 (IGF1) and IGFALS levels. Acute oral exposure to DON at 0.5-12.5 mg/kg bw markedly suppressed hepatic IGFALS mRNA levels within 2 h in a dose-dependent fashion whereas 0.1 mg/kg bw was without effect. DON-induced IGFALS mRNA upregulation occurred both with and without exogenous GH treatment. These latter effects co-occurred with robust hepatic SOCS3 upregulation. Taken together, these data suggest that oral DON exposure perturbs GH axis by suppressing two clinically relevant growth-related proteins, IGFALS and IGF1. Both have potential to serve as biomarkers of effect in populations exposed to this common foodborne mycotoxin.

Diesel exhaust particles in the lung aggravate experimental acute renal failure

Nemmar, A., Al-Salam, S., Zia, S., Yasin, J., Al Husseni, I., Ali, B. H. — Thu, 01 Oct 2009 04:06:47 PDT

Inhaled particles are associated with pulmonary and extrapulmonary effects. Also, acute renal failure (ARF) is associated with increased mortality, related to pulmonary complications. Here, we tested the possible potentiating effect of diesel exhaust particles (DEP) in an animal model of ARF induced by a single intraperitoneal injection of cisplatin (CP, 6 mg/kg) in rats. Six days later, the rats were intratracheally (i.t.) instilled with either DEP (0.5 or 1 mg/kg) or saline (control), and renal, systemic and pulmonary variables were studied 24 h thereafter. CP increased the serum concentrations of urea, creatinine, and reduced glutathione (GSH) concentration and superoxide dismutase activity in renal cortex. CP caused renal tubular necrosis, and increased urine volume, protein concentrations and N-acetyl-β-D-glucosaminidase (NAG) activity, and decreased urine osmolality. The combination of DEP and CP aggravated the CP-induced effects on serum urea and creatinine, urine NAG activity, and renal GSH. The arterial O₂ saturation and PO₂ were significantly decreased in CP + DEP versus CP + saline, and CP+DEP versus DEP. The number of platelets was reduced in DEP compared to saline-treated rats, and CP + DEP versus DEP alone or CP + saline. Increases in macrophage and neutrophils numbers in bronchoalveolar lavage were found in DEP versus saline group, and CP + DEP versus CP. Histopathological changes in lungs of DEP –treated rats were aggravated by the combination of CP + DEP. These included marked interstitial cell infiltration and congestion. We conclude that the presence of DEP in the lung aggravated the renal, pulmonary and systemic effects of CP-induced ARF.

Long-term Inhalation Toxicity Studies with Multi-walled Carbon Nanotubes: Closing the Gaps or Initiating the Debate?

Warheit, D. B. — Sat, 26 Sep 2009 04:01:05 PDT

Capture Compound Mass Spectrometry (CCMS) Sheds Light on the Molecular Mechanisms of Liver Toxicity of two Parkinson Drugs

Sat, 26 Sep 2009 04:01:04 PDT

Capture Compound Mass Spectrometry (CCMS) is a novel technology that helps to understand the molecular mechanism of the mode of action of small molecules. The Capture Compounds are trifunctional probes: a selectivity function (the drug) interacts with the proteins in a biological sample, a reactivity function (phenylazide) irreversibly forms a covalent bond, and a sorting function (biotin) allows the captured protein(s) to be isolated for mass spectrometric analysis.

Tolcapone and entacapone are potent inhibitors of catechol-O-methyl transferase (COMT) for the treatment of Parkinson's disease. We aimed to understand the molecular basis for the difference of both drugs in respect to side effects. Using Capture Compounds with these drugs as selectivity functions we were able to unambiguously and reproducibly isolate and identify their known target COMT. Tolcapone Capture Compounds captured five times more proteins than entacapone Capture Compounds. Moreover, tolcapone Capture Compounds isolated mitochondrial and peroxisomal proteins. The major tolcapone – protein interactions occurred with components of the respiratory chain and of the fatty acid β-oxidation. Previously reported symptoms in tolcapone-treated rats suggested that tolcapone might act as decoupling reagent of the respiratory chain (Haasio et al).

Our results demonstrate that CCMS is an effective tool for the identification of a drug's potential off-targets. It fills a gap in currently used in-vitro screens for drug profiling that do not contain all of the toxicologically relevant proteins. Thereby CCMS has the potential to fill a technological need in drug safety assessment and helps to reengineer or to reject drugs at an early pre-clinical stage.

S-Adenosylhomocysteine Promotes the Invasion of C6 Glioma Cells via Increased Secretion of Matrix Metalloproteinase-2 in Murine Microglial BV2 Cells

Lin, H.-C., Song, T.-Y., Hu, M.-L. — Sat, 26 Sep 2009 07:21:38 PDT

S-Adenosylhomocysteine (SAH) is a risk factor for many diseases, including tumor progression and neurodegenerative disease. In this study, we examined the hypothesis that SAH may indirectly enhance the invasion of C6 glioma cells by induction of matrix metalloproteinase-2 (MMP-2) secreted from the murine microglia BV2 cells. We obtained conditioned medium (CM) by incubation of BV2 cells with SAH (1-50 nM) for 24 h. We found that the SAH-containing CM (SAH-BV2-CM) strongly enhanced the invasiveness of C6 glioma cells and that this effect increased with increasing concentration of SAH in the SAH-BV2-CM. The effect of CM could be attributed to its MMP-2 activity, as a result of increased protein and mRNA expression of MMP-2 in BV2 cells induced by SAH. In BV2 cells treated with SAH, the binding abilities of NF-B and Sp1 to the MMP-2 promoter were increased, whereas the level of IB was decreased. In addition, SAH significantly increased the phosphorylation of ERK and PI3K/Akt proteins but did not affect that of JUK or p38. Pre-treatment of BV2 cells with an inhibitor specific for ERK (U0126) markedly abated the expression of ERK and MMP-2. Furthermore, SAH significantly and dose-dependently decreased tissue inhibitor of metalloproteinase-2 (TIMP-2) in BV2 cells. Thus, SAH may induce the invasiveness of C6 glioma cells by decreased TIMP-2 expression and increased MMP-2 expression in BV2 cells. The latter effect is likely mediated through the ERK and PI3K/Akt pathways, with increased binding activities of NF-B and Sp1 to the MMP-2 gene promotor.

High Mobility Group Box-1 protein and Keratin-18, circulating serum proteins informative of acetaminophen-induced necrosis and apoptosis in vivo

Fri, 25 Sep 2009 07:58:36 PDT

Drug-induced hepatotoxicity represents a major clinical problem and an impediment to new medicine development. Serum biomarkers hold the potential to provide information about pathways leading to cellular responses within inaccessible tissues which can inform the medicinal chemist and the clinician with respect to safe drug design and use. Hepatocyte apoptosis, necrosis and innate immune activation have been defined as features of the toxicological response associated with the hepatotoxin acetaminophen (APAP). Within this investigation we have unambiguously identified and characterized by LC-MS/MS differing circulating molecular forms of High Mobility Group Box-1 Protein (HMGB1) and Keratin-18 (K18) which are linked to the mechanisms and pathological changes induced by APAP in the mouse. Hypo-acetylated HMGB1 (necrosis indicator), caspase-cleaved K18 (apoptosis indicator) and full-length K18 (necrosis indicator) present in serum showed strong correlations with the histological time course of cell death and was more sensitive than alanine aminotransferase (ALT) activity. We have further identified a hyper-acetylated form of HMGB1 (inflammatory indicator) in serum which indicated that hepatotoxicity was associated with an inflammatory response. The inhibition of APAP-induced apoptosis and K18 cleavage by the caspase inhibitor Z-VAD.fmk are associated with increased hepatic damage, by a shift to necrotic cell death only. These findings illustrate the initial verification of K18 and HMGB1 molecular forms as serum-based sensitive tools that provide insights into the cellular dynamics involved in APAP hepatotoxicity within an inaccessible tissue. Based on these findings, potential exists for the qualification and measurement of these proteins to assist in vitro, in vivo and clinical bridging in toxicological research.

Reproductive and Sphingolipid Metabolic Effects of Fumonisin B1 and its Alkaline Hydrolysis Product in LM/Bc Mice: Hydrolyzed Fumonisin B1 Did Not Cause Neural Tube Defects

Voss, K. A., Riley, R. T., Snook, M. E., Waes, J. G.-v. — Fri, 25 Sep 2009 07:58:36 PDT

Fumonisins are mycotoxins produced by Fusarium verticillioides. They are toxic to animals and exert their effects through mechanisms involving disruption of sphingolipid metabolism. Fumonisins are converted to their hydrolyzed analogues by alkaline cooking (nixtamalization). Both fumonisins and hydrolyzed fumonisins are found in nixtamalized foods such as tortillas and consumption of tortillas has been implicated as a risk factor for neural tube defects (NTD). Fumonisin B₁ (FB₁) induced NTD when given (ip) to pregnant LM/Bc mice, however, neither the NTD-induction potential of hydrolyzed FB₁ (HFB₁) nor its affect on sphingolipid metabolism in pregnant mice have been reported. The teratogenic potential of FB₁ and HFB₁ was therefore compared using the LM/Bc mouse model. Dams were dosed (ip) with 2.5, 5.0, 10 or 20 mg/kg (< 49 µmol/kg) bw HFB₁ on E7-8. Negative and positive control groups were given vehicle or 10 mg/kg (14 µmol/kg) bw FB₁, respectively. The high dose of HFB₁ disrupted sphingolipid metabolism, albeit slightly, but did not cause maternal liver lesions or NTD (n = 8-10 litters/group). In contrast, 10 mg/kg bw FB₁ markedly disrupted maternal sphingolipid metabolism, caused hepatic apoptosis in the dams, increased fetal death rates and decreased fetal weights. Furthermore, NTD were found in all FB₁-exposed litters (n = 10) and 66 + 24 percent of the fetuses were affected. The findings indicate that HFB₁ does not cause NTD in the sensitive LM/Bc mouse model and only weakly disrupted sphingolipid metabolism at doses up to 7-fold higher (µmol/kg body weight basis) than the previously reported LOAEL for FB₁.

Use of Short-term Transcriptional Profiles to Assess the Long-term Cancer-Related Safety of Environmental and Industrial Chemicals

Wed, 23 Sep 2009 08:21:17 PDT

The process for evaluating chemical safety is inefficient, costly, and animal intensive. There is growing consensus that the current process of safety testing needs to be significantly altered to improve efficiency and reduce the number of untested chemicals. In this study, the use of short-term gene expression profiles was evaluated for predicting the increased incidence of mouse lung tumors. Animals were exposed to a total of 26 diverse chemicals with matched vehicle controls over a period of three years. Upon completion, significant batch-related effects were observed. Adjustment for batch effects significantly improved the ability to predict increased lung tumor incidence. For the best statistical model, the estimated predictive accuracy under honest five-fold cross-validation was 79.3% with a sensitivity and specificity of 71.4 and 86.3%, respectively. A learning curve analysis demonstrated that gains in model performance reached a plateau at 25 chemicals indicating that the size of current data set was sufficient to provide a robust classifier. The classification results showed a small subset of chemicals contributed disproportionately to the misclassification rate. For these chemicals, the misclassification was more closely associated with genotoxicity status than efficacy in the original bioassay. Statistical models were also used to predict dose-response increases in tumor incidence for methylene chloride and naphthalene. The average posterior probabilities for the top models matched the results from the bioassay for methylene chloride. For naphthalene, the average posterior probabilities for the top models over-predicted the tumor response, but the variability in predictions were significantly higher. The study provides both a set of gene expression biomarkers for predicting chemically-induced mouse lung tumors as well as a broad assessment of important experimental and analysis criteria for developing microarray-based predictors of safety-related endpoints.

Estimates of Cancer Potency of 2,3,7,8-Tetrachlorodibenzo(p)dioxin using Linear and Non-linear Dose-Response Modeling and Toxicokinetics

Simon, T., Aylward, L. L., Kirman, C. R., Rowlands, J. C., Budinsky, R. A. — Wed, 23 Sep 2009 08:21:16 PDT

Linear and non-linear toxicity criteria were derived for 2,3,7,8-tetrachlorodibenzo(p)dioxin (TCDD) using the recent National Toxicology Program (NTP) rat cancer bioassay. Dose-response relationships were assessed for combined liver tumors based on lifetime average liver concentrations (LALC) estimated with a toxicokinetic model. Rat LALC estimates at the 1% point of departure (POD) were obtained with benchmark dose (BMD) modeling to yield the BMD₀₁ in terms of LALC. The same toxicokinetic model was used to back-extrapolate the human-equivalent external dose (HED). A linear cancer slope factor (CSF) with a value of 1E-04 per pg/kg-d was calculated as the ratio between the benchmark response rate (BMR) and the HED at the BMDL₀₁. A non-linear reference dose (RfD) with a value of 100 pg/kg-d was developed from the BMD₀₁ value by applying uncertainty factors to rat internal and human external doses. The RfD was 100 times higher than the 10^-4 risk specific dose (RSD) based on the linear CSF. For comparison, BMD₀₁ and BMDL₀₁ values were developed for key events in the tumor promotion mode of action (MOA) of TCDD. This MOA involves dysregulation of the normal function of the aryl hydrocarbon receptor and its associated biological processes, and results in pathologies that drive tumor promotion and progression. The BMD₀₁ values for key events were consistent with the timing of the key events within the MOA and provided support for the choices of the 1% tumor rate as a POD and dichotomous Hill model for representing receptor-mediated carcinogenicity. Because a threshold toxicity criterion most accurately reflects the MOA, the RfD for TCDD with a value of 100 pg/kg-d is considered appropriate for regulatory purposes, consistent with a 2006 NRC panel's recommendation to develop a threshold-based cancer potency factor for TCDD and with the methodology in USEPA's Cancer Guidelines.

Discrimination for genotoxic and non-genotoxic carcinogens by gene expression profiling in primary mouse hepatocytes improves with exposure time

Mon, 21 Sep 2009 13:15:38 PDT

Assessing the potential carcinogenicity of chemicals for humans represents an ongoing challenge. Chronic rodent bioassays predict human cancer risk at only limited reliability and are simultaneously expensive and long-lasting. In order to seek for alternatives, the ability of a transcriptomics-based primary mouse hepatocyte model to classify carcinogens by their modes of action was evaluated. As it is obvious that exposure will induce a cascade of gene expression modifications, in particular, the influence of exposure time in vitro on discriminating genotoxic (GTX) carcinogens from non-genotoxic (NGTX) carcinogens class discrimination was investigated. Primary mouse hepatocytes from male C57Bl6 mice were treated for 12, 24, 36 and 48 h with two GTX and two NGTX carcinogens. For validation, two additional GTX compounds were studied at 24 and 48 h. Immunostaining of H2AX foci was applied in order to phenotypically verify DNA damage. It confirmed significant induction of DNA damage after treatment with GTX compounds but not with NGTX compounds. Whole genome gene expression modifications were analyzed by means of Affymetrix microarrays. When using differentially expressed genes from datasets normalized by RMA, the two classes and various compounds were better separated from each other by hierarchical clustering when increasing the treatment period. Discrimination of GTX and NGTX carcinogens by Prediction Analysis of Microarray improved with time, and resulted in correct classification of the validation compounds. The present study shows that gene expression profiling in primary mouse hepatocytes is promising for discriminating GTX from NGTX compounds and that this discrimination improves with increasing treatment period.

Probucol Increases Glutathione Peroxidase-1 Activity and Displays Long-Lasting Protection Against Methylmercury Toxicity in Cerebellar Granule Cells

Mon, 21 Sep 2009 13:15:38 PDT

Methylmercury (MeHg) is an environmental neurotoxicant whose molecular mechanisms underlying toxicity remain elusive. Here we investigated molecular events involved in MeHg-induced neurotoxicity in cultured cerebellar granule cells (CGCs) as well as potential protective strategies for such toxicity. Glutathione peroxidase (GPx-1) activity was significantly (p = 0.0017) decreased at 24 h before MeHg-induced neuronal death (day in vitro 4). This event was related to enhanced susceptibilities to hydrogen- or tert-butyl-peroxides and increased lipid peroxidation. However, intracellular calcium levels, glutamate uptake and glutathione levels, as well as glutathione reductase and catalase activities, were not changed by MeHg exposure at this time-point. Probucol (PB), a lipid-lowering drug, displayed a long-lasting protective effect against MeHg-induced neurotoxicity. The beneficial effects of PB were correlated to increased GPx-1 activity and decreased lipid peroxidation. The protection afforded by PB was significantly higher when compared to the antioxidants ascorbic acid and trolox. In vitro studies with the purified GPx-1 proved that MeHg inhibits and PB activates the enzyme activity. Overexpression of GPx-1 prevented MeHg-induced neuronal death. These data indicate that (i) GPx-1 is an important molecular target involved in MeHg-induced neurotoxicity and (ii) PB, which increases GPx-1 activity in CGCs, induces enduring protection against such toxicity. The results bring out new insights on the potential therapeutic strategies for poisonings to MeHg and other pathological conditions related to increased production and/or decreased detoxification of peroxides.

Approaches for Assessing Risks to Sensitive Populations: Lessons Learned from Evaluating Risks in the Pediatric Population

Mon, 21 Sep 2009 13:15:39 PDT

Assessing the risk profiles of potentially sensitive populations requires a ‘tool chest’ of methodological approaches to adequately characterize and evaluate these populations. At present, there is an extensive body of literature on methodologies that apply to the evaluation of the pediatric population. The HESI Subcommittee on Risk Assessment of Sensitive Populations evaluated key references in the area of pediatric risk to identify a spectrum of methodological approaches. These approaches are considered in this manuscript for their potential to be extrapolated for the identification and assessment of other sensitive populations. Recommendations as to future research needs and/or alternate methodological considerations also are made.

Toxicogenomics applied to cultures of human hepatocytes enabled an identification of novel petasites hybridus extracts for the treatment of migraine with improved hepatobiliary safety

Anderson, N., Meier, T., Borlak, J. — Mon, 21 Sep 2009 13:15:36 PDT

Butterbur extracts (petasites hybridus) are recommended for the prevention of migraine, but pharmacovigilance reports may be suggestive for rare hepatobiliary toxicity. In order to evaluate its hepatotoxic potential a series of in vivo and in vitro studies were carried out. Essentially, there were no signs of hepatocellular toxicity at estimated therapeutic C_max levels of 60 ng/mL. Nonetheless, in a 28-day toxicity study at approximately 200-fold of therapeutic doses induced liver transaminases and bilirubin elevations were observed. In a subsequent 6-months chronic toxicity study the initial hepatobiliary effects were reproduced, but at the end of the study liver function recovered and returned to normal as evidenced by clinical chemistry measurements. To identify possible mechanisms of hepatotoxicity, we investigated liver function in vitro at >170-fold of therapeutic C_max levels, including cytotoxicity (LDH, MTT, ATP), transaminase activities (ALT, AST), albumin synthesis, urea and testosterone metabolism to assay for CYP monooxygenase activity. Only with extracts rich in petasin (37% petasin) and at high well above therapeutic doses liver toxicity was observed. A toxicogenomic approach applied to hepatocyte cultures enabled hypothesis generation and was highly suggestive for extracts high in petasin content to impair bile acid transport, lipid and protein metabolism. Importantly, neither chronic rat in vivo nor rat in vitro studies predicted reliably hepatotoxicity, therefore re-emphasising the utility of human-based in vitro investigations for the development of safe medicinal products. Finally, toxicogenomics enabled the characterization of a novel butterbur extract with no signals for hepatotoxicity.

Modulation of aflatoxin B1-mediated genotoxicity in primary cultures of human hepatocytes by diindolylmethane, curcumin and xanthohumols

Mon, 21 Sep 2009 13:15:35 PDT

This study employed cultured human primary hepatocytes to investigate the ability of the putative chemopreventive phytochemicals- curcumin (CUR), 3,3’-diindolylmethane (DIM), isoxanthohumol (IXN), or 8-prenylnaringenin (8PN) to reduce DNA-adduct formation of the hepatocarcinogen aflatoxin B₁ (AFB). Following 48 hr of pretreatment DIM and 8PN significantly increased AFB-DNA adduct levels, whereas CUR and IXN had no effect. DIM greatly enhanced the transcriptional expression of CYP1A1 and CYP1A2 mRNA. Glutathione S-transferase mRNAs were not increased by any of the treatments. In vitro enzyme activity assays demonstrated that 8PN and DIM, but not CUR or IXN, inhibited human CYP1A1, CYP1A2, and CYP3A4 activities. To distinguish between treatment effects on transcription vs. direct effects on enzyme activity for DIM, we evaluated the effects of pre-treatment alone (transcriptional activation), vs. co-treatment alone (enzyme inhibition). The results demonstrated that effects on gene expression, but not catalytic activity, are responsible for the observed effects of DIM on AFB-DNA adduct formation. The increase in AFB-DNA damage following DIM treatment may be explained through its substantial induction of CYP1A2 and/or its down-regulation of GSTM1, both of which were significant. The increase in DNA damage by DIM raises potential safety risks for dietary supplements of DIM and its precursor indole-3-carbinol.

Metallothionein-III provides neuronal protection through activation of Nuclear Factor-{kappa}B via the TrkA/Phosphatidylinositol-3 kinase/Akt signaling pathway

Fri, 18 Sep 2009 23:12:03 PDT

Metallothionein-III (MT-III) is associated with resistance to neuronal injury. However, the underlying mechanism for its effects is unclear. The present study investigated the mechanisms of MT-III protection of neuronal cells from hypoxia or DNA damage-induced cell death. MT-III reduced the hydrogen peroxide- or DNA damage-induced effects on neuronal cells including cell death, the activation of caspase-3 and -9, and the release of mitochondrial cytochrome c to the cytoplasm in a dose-dependent manner. MT-III also increased the activation of Akt, the phosphorylation and degradation of IB, the nuclear translocation/accumulation and the transcriptional activity of NF-B in neuronal cells in a dose-dependent manner. The MT-III-induced anti-apoptotic effects and increase in NF-B activity were blocked by specific inhibitors of TrkA, PI3K, Akt, or NF-B, indicating that MT-III provides neuronal protection by activating NF-B through the TrkA/PI3K/Akt signaling pathway.

The Nrf2-Keap1-ARE toxicity pathway as a cellular sensor for skin sensitizers -functional relevance and a hypothesis on innate reactions to skin sensitizers

Natsch, A. — Fri, 18 Sep 2009 23:12:02 PDT

With the tight deadlines set both by the public and by regulatory authorities to replace animal tests for toxicological endpoints relevant to the development of cosmetic products, a large number of research projects have recently focused on cellular endpoints affected by skin sensitizing compounds. The general aim stated in these projects was to find ‘markers’ for skin sensitizers, be it at the level of the transcriptome or at the protein level. Rather then talking of ‘cellular markers’, the new paradigm ‘Toxicity testing in the 21th century’ formulated by the National Academy of Sciences in the United States focuses on ‘Toxicity pathways’. A specific ‘marker’ for any given toxicological endpoint can only exist, if specific toxicity pathways, comprising specific sensors, are linked to this endpoint. In the context of skin sensitization, one has to ask whether there is an innate cellular signaling pathway activated by skin sensitizers. Here a significant body of evidence, mainly accumulated in the last 20 months, is reviewed, indicating that indeed the Nrf2-Keap1-ARE regulatory pathway is such a toxicity pathway activated by cysteine-reactive skin sensitizers. Whereas first indications on the in vivo relevance are available, key questions remain open and can now specifically be addressed. A minority of sensitizers, more specifically reacting with lysine residues, appears not to activate the Nrf2-Keap1-ARE pathway, and might trigger yet another unknown toxicity pathway.

Dynamic Nature of Alterations in the Endocrine System of Fathead Minnows Exposed to the Fungicide Prochloraz

Fri, 18 Sep 2009 10:48:36 PDT

The vertebrate hypothalamic-pituitary-gonadal (HPG) axis is controlled through various feedback mechanisms that maintain a dynamic homeostasis in the face of changing environmental conditions, including exposure to chemicals. We assessed the effects of prochloraz on HPG axis function in adult fathead minnows (Pimephales promelas) at multiple sampling times during 8-day exposure and 8-day depuration/recovery phases. Consistent with one mechanism of action of prochloraz, inhibition of cytochrome P450 (CYP19) aromatase activity, the fungicide depressed ex vivo ovarian production and plasma concentrations of 17β-estradiol (E2) in female fish. At a prochloraz water concentration of 30 µg/L, inhibitory effects on E2 production were transitory, and did not persist during the 8-day exposure phase. At 300 µg prochloraz/L, inhibition of E2 production was evident throughout the 8-day exposure, but steroid titers recovered within 1 day of cessation of exposure. Compensation or recovery of steroid production in prochloraz-exposed females was accompanied by up-regulation of several ovarian genes associated with steroidogenesis, including cyp19a1a, cyp17 (hydroxylase/lyase), cyp11a (cholesterol side-chain cleavage) and follicle-stimulating hormone receptor. In male fathead minnows, the 8-day prochloraz exposure decreased testosterone (T) production, possibly through inhibition of CYP17. However, as for E2 in females, ex vivo testicular production and plasma concentrations of T recovered within 1 day of stopping exposure. Steroidogenic genes up-regulated in testis included cyp17 and cyp11a. These studies demonstrate the adaptability of the HPG axis to chemical stress, and highlight the need to consider the dynamic nature of the system when developing approaches to assess potential risks of endocrine-active chemicals.

Establishment of an Immunoglobulin M Antibody Forming Cell Response Model for Characterizing Immunotoxicity in Primary Human B Cells

Lu, H., Crawford, R. B., North, C. M., Kaplan, B. L.F., Kaminski, N. E. — Fri, 18 Sep 2009 10:48:33 PDT

Rodent models have been extensively utilized to identify putative human immunotoxicants; however, even when immunotoxicity is established, uncertainty remains whether the effects are predictive of human risk. Therefore, the objective of this study was to establish a polyclonal immunoglobulin M (IgM) antibody forming cell (AFC) response model to directly characterize immunotoxicity in primary mouse or human B cells. CD40 ligand (CD40L) was selected to activate B cells because it effectively drives both primary human and mouse B cells in vitro to AFC in a physiologically-relevant manner to mimic T cell-dependent antibody responses in vivo. In this model, the IgM AFC response is induced by cell surface-expressed CD40L and promoted by recombinant cytokines. Reported here are the conditions required to induce IgM AFC responses using mouse splenic B cells or human peripheral blood B cells, allowing for species comparisons. Moreover, less than one order of magnitude difference was observed in the CD40L-induced B cell AFC responses based on data from multiple donors. In addition to antibody production, proliferation and phenotypic changes characteristic of B cell activation as well as the plasma cell phenotype were also significantly induced. Finally, two well-characterized immunotoxicants, arsenic and benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), using the CD40L-induced IgM AFC response were compared in both mouse and human B cells. Collectively, an IgM AFC response model is described that can be applied to assess the sensitivity of antibody responses to modulation by xenobiotics using mouse as well as human primary B cells.

Differential contribution of the mitochondrial respiratory chain complexes to reactive oxygen species production by redox cycling agents implicated in parkinsonism

Drechsel, D. A., Patel, M. — Fri, 18 Sep 2009 10:48:37 PDT

Exposure to environmental pesticides can cause significant brain damage and has been linked with an increased risk of developing neurodegenerative disorders, including Parkinson's disease. Bipyridyl herbicides such as paraquat (PQ), diquat (DQ), and benzyl viologen (BV), are redox cycling agents known to exert cellular damage through the production of reactive oxygen species (ROS). We examined the involvement of the mitochondrial respiratory chain in ROS production by bipyridyl herbicides. In isolated rat brain mitochondria, H₂O₂ production occurred with the following order of potency: BV > DQ > PQ in accordance with their measured ability to redox cycle. H₂O₂ production was significantly attenuated in all cases by antimycin A, an inhibitor of complex III. Interestingly, at micromolar (≤ 300 µM) concentrations PQ-induced H₂O₂ production was unaffected by complex I inhibition via rotenone, whereas DQ-induced H₂O₂ production was equally attenuated by inhibition of complex I or III. Moreover, complex I inhibition decreased BV-induced H₂O₂ production to a greater extent than with PQ or DQ. These data suggest that multiple sites within the respiratory chain contribute to H₂O₂ production by redox cycling bipyridyl herbicides. In primary midbrain cultures, H₂O₂ differed slightly with the following order of potency: DQ > BV > PQ. In this model, inhibition of complex III resulted in roughly equivalent inhibition of H₂O₂ production with all three compounds. These data identify a novel role for complex III-dependence of mitochondrial ROS production by redox cycling herbicides while emphasizing the importance of identifying mitochondrial mechanisms by which environmental agents generate oxidative stress contributing to parkinsonism.

A single exposure to particulate or gaseous air pollution increases the risk of aconitine-induced cardiac arrhythmia in hypertensive rats

Hazari, M. S., Haykal-Coates, N., Winsett, D. W., Costa, D. L., Farraj, A. K. — Fri, 11 Sep 2009 05:26:14 PDT

Epidemiological studies demonstrate an association between arrhythmias and air pollution. Aconitine-induced cardiac arrhythmia is widely used experimentally to examine factors that alter the risk of arrhythmogenesis. In this study, Wistar-Kyoto (WKY) and Spontaneously Hypertensive (SH) rats acutely exposed to synthetic residual oil fly ash particles (s-ROFA, 450µg/m3) were "challenged" with aconitine to examine whether a single exposure could predispose to arrhythmogenesis. Separately, SH rats were exposed to varied particulate matter (PM) concentrations (0.45, 1.0, or 3.5 mg/m3 s-ROFA), or the irritant gas acrolein (3ppm), to better assess the generalization of this challenge response. Rather than directly cause arrhythmias, we hypothesized that inhaled air pollutants sensitize the heart to subsequent dysrhythmic stimuli. Twenty-four hours post-exposure, urethane-anesthetized rats were monitored for heart rate (HR), ECG, and blood pressure (BP). SH rats had higher baseline HR and BP, and significantly longer PR intervals, QRS duration, QTc, and JTc than WKY rats. PM Exposure caused a significant increase in the PR interval, QRS duration and QTc in WKY rats, but not in SH rats. Heart rate variability was significantly decreased in WKY rats after PM exposure, but increased in SH rats. Cumulative dose of aconitine that triggered arrhythmias in air-exposed SH rats was lower than WKY rats; and even lower for each strain post-exposure. SH rats exposed to varied concentrations of PM or acrolein developed arrhythmia at significantly lower doses of aconitine than controls; however, there was no PM concentration-dependent response. In conclusion, a single exposure to air pollution may increase the sensitivity of cardiac electrical conduction to disruption. Moreover, there seem to be host factors (e.g. cardiovascular disease) that increase vulnerability to triggered arrhythmias regardless of the pollutant or its concentration.

Ligand Activation of Peroxisome Proliferator-Activated Receptor-{beta}/{delta} (PPAR{beta}/{delta}) and Inhibition of Cyclooxygenase 2 (COX2) Enhances Inhibition of Skin Tumorigenesis

Bility, M. T., Zhu, B., Kang, B. H., Gonzalez, F. J., Peters, J. M. — Fri, 11 Sep 2009 05:26:14 PDT

Ligand activation of peroxisome proliferator-activated receptor-β/ (PPARβ/) and inhibition of cyclooxygenase-2 (COX2) activity by non-steroidal anti-inflammatory drugs (NSAID) can both attenuate skin tumorigenesis. There is also evidence that attenuation of skin tumorigenesis by inhibition of COX2 activity occurs through PPARβ/–independent mechanisms. The present study examined the hypothesis that combining ligand activation of PPARβ/ with inhibition of COX2 activity will cooperatively inhibit chemically-induced skin tumor progression using both in vivo and ex vivo models. A two-stage chemical carcinogenesis bioassay was performed in wild-type and Pparβ/-null mice. After twenty-two weeks, cohorts of both mouse lines were divided into four experimental groups: 1) control, 2) topical application of the PPARβ/ ligand GW0742, 3) dietary administration of the COX2 inhibitor nimesulide, or 4) both GW0742 and nimesulide. Ligand activation of PPARβ/ did not influence skin tumor progression while a modest decrease in skin tumor multiplicity was observed with dietary nimesulide. Interestingly, the combined treatment of GW0742 and nimesulide increased the efficacy of the decrease in papilloma multiplicity for six weeks in wild-type mice, but this effect was not found at later timepoints and was not found in similarly treated Pparβ/-null mice. Neoplastic keratinocyte lines cultured with GW0742 and nimesulide also exhibited enhanced inhibition of cell proliferation coincident with increased expression of Keratin mRNAs. Results from these studies support the hypothesis that combining ligand activation of PPARβ/ with inhibition of COX2 activity can inhibit chemically-induced skin tumor progression by modulating differentiation.

Acute Morphological and Toxicological Effects in a Human Bronchial Coculture Model after Sulphur Mustard Exposure

Fri, 11 Sep 2009 05:26:14 PDT

Sulphur mustard (SM) is a strong alkylating agent. Inhalation of sulphur mustard causes acute lung injury accompanied by severe disruption of the airway barrier. In our study we tested the acute effects after mustard exposure in an in vitro coculture bronchial model of the proximal barrier.

To achieve this we seeded normal human bronchial epithelial cells (HBEC) together with lung fibroblasts as a bilayer on filter plates and exposed the bronchial model after 31 days of differentiation to various concentrations of SM (30, 100, 300 and 500 µM).

The HBEC formed confluent layers, expressing functional tight junctions as measured by transepithelial electrical resistance (TER). Mucus production and cilia formation reappeared in the coculture model. TER was measured after 2 h and 24 h following treatment. Depending on the different concentrations TER decreased in the first 2 h up to 55% of the control at the highest concentration. After 24 h TER seemed to recover, because at concentrations up to 300 µM values were equal to the control. SM induced a widening of intercellular spaces and a loss in cell-matrix adhesion. Mucus production increased with the result that cilia ceased to beat. Changes in the proinflammatory cytokines IL-6 and IL-8 were also observed. Apoptotic markers such as cytochrome c, p53, FADD and pro-caspase 3 were significantly induced at concentrations of less than 100µM. In summary, SM induces morphological and biochemical changes that reflect pathological effects of SM-injury in vivo. It is hoped to use this co-culture model to understand further the pathogenesis of SM-induced barrier injury and to search for novel approaches in SM therapy.

Gene and Protein Responses of Human Monocytes to Extracellular Cysteine Redox Potential

Go, Y.-M., Craige, S. E., Orr, M., Gernert, K. M., Jones, D. P. — Fri, 11 Sep 2009 05:26:13 PDT

The redox potential of the major thiol/disulfide couple, cysteine (Cys) and its disulfide cystine (CySS) in plasma (E_hCys) is oxidized in association with oxidative stress, and oxidized E_hCys is associated with cardiovascular disease risk. In vitro exposure of monocytes to oxidized E_hCys increases expression of the proinflammatory cytokine, interleukin-1β, suggesting that E_hCys could be a mechanistic link between oxidative stress and chronic inflammation. Because cell membranes contain multiple Cys-rich proteins which could be sensitive to E_hCys, we sought to determine whether E_hCys specifically affects proinflammatory signaling or has other effects on monocytes. We used microarray analysis and mass spectrometry-based proteomics to evaluate global changes in protein redox state, gene expression and protein abundance in monocytes in response to E_hCys. Pathway analysis results revealed that in addition to IL-1β-related pathways, components of stress/detoxification and cell death pathways were increased by oxidized E_hCys while components of cell growth and proliferation pathways were increased by a reduced potential. Phenotypic studies confirmed that a cell stress response occurred with oxidized E_h and that cell proliferation was stimulated with reduced E_h. Therefore, plasma E_hCys provides a control over monocyte phenotype which could contribute to cardiovascular disease risk and provide a novel therapeutic target for disease prevention.

Mal-development of the Penis and Loss of Fertility in Male Rats Treated Neonatally with Female Contraceptive 17{alpha}-Ethinyl Estradiol (EE). A Dose-response Study and a Comparative Study with a Known Estrogenic Teratogen Diethylstilbestrol (DES)

Mathews, E, Braden, T., Williams, C., Williams, J., Bolden-Tiller, O, Goyal, H O — Thu, 03 Sep 2009 10:43:53 PDT

The objectives of this study were to find a minimal dose of EE that is detrimental to the developing penis and fertility, and to compare estrogenic effects between EE and DES. Neonatal rats received EE at 10ng (1µg/kg), 100ng, 1µg, or 10µg per pup on alternate days from postnatal days 1-11 (dose-response study); or received EE or DES at 100ng per pup daily from postnatal days 1-6 (comparative study). Effects of EE were dose-dependent, with ≥100ng dose inducing significant (P < 0.05) reductions in penile length, weight and diameter. Additionally, the penis was malformed, characterized by under-developed os penis and accumulation of fat cells. Fertility was 0% in the ≥1µg groups, in contrast to 60% in the 100ng group and 100% in the 10ng and control groups. Animals treated with ≥10ng had significant reductions in the weight of bulbospongious muscle, testis, seminal vesicle, epididymal fat pad, and in epididymal sperm numbers. A comparison of EE and DES effects showed similar reductions in penile weight and length and the weight of bulbospongiosus muscle, testis, seminal vesicle, epididymis, and epididymal fat pad in both adolescent and adult rats. While 5/6 control males sired, only 1/6 in the EE group and 0/6 in the DES group sired. Hence, neonatal exposure to EE at 10ng (environmentally relevant dose) adversely affects male reproductive organs. A dose ten-times higher than this leads to permanently mal-developed penis and infertility. Furthermore, EE and DES exposures show similar level of toxicity to male reproductive organs.

Early life exposure to Dimethoate induced Reproductive toxicity: Evaluation of effects on Pituitary-testicular axis of mice

Verma, R., Mohanty, B. — Wed, 02 Sep 2009 08:02:39 PDT

In utero and lactational exposure to organophosphate dimethoate exerted toxic impact on the reproductive system of male mice. Pregnant mice were exposed to 4, 8 and 16 mg/kg of the pesticide, the sublethal doses (2.5, 5 and 10% of LD₅₀), via gavaging from GD 6 to PND 21. The effects on the male reproductive system were evaluated at two stages; at prepubertal age (PND 22) and at the postpubertal age of PND 63. Gonadal inhibition was reflected in the significant reduction of weight and distinct histopathological alteration of testis and epididymis as well as in decreased sperm counts which could be linked to hormonal imbalance caused by dimethoate interference of reproductive axis. Disruption of pituitary-testicular axis was shown in the weak immunointensity of LH cells, reduction in their size and number and lowered plasma LH and testosterone levels as observed in the neonates exposed to two higher tested doses. In addition, the direct toxic impact of the pesticide on the testicular Leydig cells and inhibition of steroidogenesis could be suggested. Drastic reduction in the testosterone level (approximately by 70%) was suggestive of this effect. The adverse effects were persisted in the young adult mice. Developmental toxicity was evident in the highest dose-exposed (10% LD₅₀) group where gestation day length and still births were significantly increased along with a decrease of body weight and anogenital distance of the fetus. Maternal exposure of pesticide during gestation and lactation periods thus adversely affected the pituitary-testicular axis of mice neonates which further caused reproductive dysfunctioning of young adult mice.

State of Academic Knowledge on Toxicity and Biological Fate of Quantum Dots

Pelley, J. L., Daar, A. S., Saner, M. A. — Fri, 14 Aug 2009 12:33:19 PDT

Quantum dots, an important class of emerging nanomaterial, are widely anticipated to find application in many consumer and clinical products in the near future. Pre-market regulatory scrutiny is, thus, an issue gaining considerable attention. Previous review papers have focused primarily on the toxicity of quantum dots. From the point of view of product regulation, however, parameters that determine exposure (for example, dosage, transformation, transportation and persistence) are just as important as inherent toxicity. We have structured our review paper according to regulatory risk assessment practices, in order to improve the utility of existing knowledge in a regulatory context. Herein, we summarize the state of academic knowledge on quantum dots pertaining not only to toxicity, but also their physicochemical properties, and their biological and environmental fate. We conclude this review with recommendations on how to tailor future research efforts to address the specific needs of regulators.

mRNA expression is a relevant tool to identify developmental neurotoxicants using an in vitro approach

Mon, 03 Aug 2009 03:17:07 PDT

So far, only a few industrial chemicals have been identified as developmental neurotoxicants. Since the current developmental neurotoxicity (DNT) guidelines (OECD TG 426) are based entirely on in vivo studies that are both time consuming and costly, there is a need to develop alternative in vitro methods for initial screening to prioritise chemicals for further DNT testing. In this study, gene expression at the mRNA level was evaluated to determine whether this could be a suitable endpoint to detect potential developmental neurotoxicants. Primary cultures of rat cerebellar granule cells (CGCs) were exposed to well known (developmental) neurotoxicants (methyl mercury chloride, lead chloride, valproic acid and tri-methyl tin chloride) for different time periods. A significant down-regulation of the mRNA level for the neuronal markers (NF-68, NF-200, NMDA-R and GABAA-R) was observed after exposure to methyl mercury chloride, valproic acid and tri-methyl tin chloride. Moreover, a significant increase of the neural precursor marker nestin mRNA was also observed. The mRNA expression of the astrocytic markers (GFAP and S100β) was unchanged. In contrast, exposure to lead chloride significantly decreased the mRNA level of the astrocytic marker GFAP while the neuronal markers were less affected. These results suggest that gene expression could be used as a sensitive tool for the initial identification of DNT effects induced by different mechanisms of toxicity in both cell types (neuronal and glial) and at various stages of cell development and maturation.

INHALATION TOXICITY OF MULTI-WALL CARBON NANOTUBES IN RATS EXPOSED FOR 3 MONTHS

Tue, 07 Jul 2009 05:24:15 PDT

Carbon nanotubes (CNT) are of great commercial interest. Theoretically, during processing and handling of CNT and in abrasion processes on composites containing CNT, inhalable CNT particles might be set free. For hazard assessment, we performed a 90-day inhalation toxicity study with a multi-wall CNT (MWCNT) material (Nanocyl NC 7000) according to OECD test guideline 413. Wistar rats were head-nose exposed for 6 hours/day, 5 days/week, 13 weeks, total 65 exposures, to MWCNT concentrations of 0 (control), 0.1, 0.5 or 2.5 mg/m³. Highly respirable dust aerosols were produced with a proprietary brush generator which neither damaged the tube structure nor increased reactive oxygen species on the surface.

Inhalation exposure to MWCNT produced no systemic toxicity. However, increased lung weights, pronounced multifocal granulomatous inflammation, diffuse histiocytic and neutrophilic inflammation, and intra-alveolar lipoproteinosis were observed in lung and lung-associated lymph nodes at 0.5 and 2.5 mg/m³. These effects were accompanied by slight blood neutrophilia at 2.5 mg/m³. Incidence and severity of the effects were concentration-related. At 0.1 mg/m³, there was still minimal granulomatous inflammation in the lung and in lung-associated lymph nodes; a no observed effect concentration was therefore not established in this study.

The test substance has low dust-forming potential, as demonstrated by dustiness measurements, but nonetheless strict industrial hygiene measures must be taken during handling and processing. Toxicity and dustiness data such as these can be used to compare different MWCNT materials and to select the material with the lowest risk potential for a given application.

OGG1 and MYH are involved in the incision of trivalent arsenical-induced DNA adducts

Pu, Y.-S., Jan, K.-Y., Wang, T.-C., Wang, A. S. S., Gurr, J.-R. — Wed, 29 Nov 2006 06:47:56 PST

Since trivalent arsenicals are known to induce oxidative DNA damage in human cells, we asked if they induce other types of DNA damage and how these DNA damages are repaired. Treatment of human promyelocytic leukemia NB4 cells with 0.5 µM As₂O₃ for 30 min induced no DNA breaks, as analyzed by a standard comet assay. However, breaks were detected if these cells were then digested with endonuclease III (EnIII), formamidopyrimidine-DNA glycosylase (Fpg), or a nuclear extract (NE) of NB4 cells. Using either H₂O₂-Fe treated nuclei or As₂O₃-treated cells, digestion with either NE or EnIII+Fpg generated the same amount of breaks, and subsequent treatment with EnIII+Fpg resulted in no increase in breaks in NE-digested cells and vice versa. The human cell lines, defective in nucleotide excision protein, such as XPA, XPD, and XPG, excised UVC-induced adducts less rapidly than normal fibroblasts, but excised As₂O₃-adducts at the same rate as the normal cells. Immunodepletion of the NE with antibody against OGG1 or MYH decreased the incision of DNA adducts induced by As₂O₃, NaAsO₂, monomethylarsonic acid, and dimethylarsinic acid, while antibodies against XPA, XPB, XPD, XPF, or XPG, did not. These results suggest that these trivalent arsenicals induce the formation of only oxidative DNA adducts and that OGG1 and MYH are involved in these incision processes.