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Toxicological Sciences

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Recent studies in both humans and animals suggest that air pollution is an important risk factor for type 2 diabetes mellitus (T2DM). However, the mechanism by which air pollution mediates propensity to diabetes is not fully understood. While a number of epidemiologic studies have shown a positive association between ambient air pollution exposure and risk for T2DM, some studies have not found such a relationship. Experimental studies in susceptible disease models do support this association and suggest the involvement of tissues involved in the pathogenesis of T2DM such as the immune system, adipose, liver, and central nervous system. This review summarizes the epidemiologic and experimental evidence between ambient outdoor air pollution and T2DM.

Acrolein, a highly reactive unsaturated aldehyde, is a ubiquitous environmental pollutant and its potential as a serious environmental health threat is beginning to be recognized. Humans are exposed to acrolein per oral (food and water), respiratory (cigarette smoke, automobile exhaust, and biocide use) and dermal routes, in addition to endogenous generation (metabolism and lipid peroxidation). Acrolein has been suggested to play a role in several disease states including spinal cord injury, multiple sclerosis, Alzheimer’s disease, cardiovascular disease, diabetes mellitus, and neuro-, hepato-, and nephro-toxicity. On the cellular level, acrolein exposure has diverse toxic effects, including DNA and protein adduction, oxidative stress, mitochondrial disruption, membrane damage, endoplasmic reticulum stress, and immune dysfunction. This review addresses our current understanding of each pathogenic mechanism of acrolein toxicity, with emphasis on the known and anticipated contribution to clinical disease, and potential therapies.

FutureTox II, a Society of Toxicology Contemporary Concepts in Toxicology workshop, was held in January, 2014. The meeting goals were to review and discuss the state of the science in toxicology in the context of implementing the NRC 21st century vision of predicting in vivo responses from in vitro and in silico data, and to define the goals for the future. Presentations and discussions were held on priority concerns such as predicting and modeling of metabolism, cell growth and differentiation, effects on sensitive subpopulations, and integrating data into risk assessment. Emerging trends in technologies such as stem cell-derived human cells, 3D organotypic culture models, mathematical modeling of cellular processes and morphogenesis, adverse outcome pathway development, and high-content imaging of in vivo systems were discussed. Although advances in moving towards an in vitro/in silico based risk assessment paradigm were apparent, knowledge gaps in these areas and limitations of technologies were identified. Specific recommendations were made for future directions and research needs in the areas of hepatotoxicity, cancer prediction, developmental toxicity, and regulatory toxicology.

Drug-induced liver injury (DILI) is a leading cause of acute liver failure and the major reason for withdrawal of drugs from the market. Preclinical evaluation of drug candidates has failed to detect about 40% of potentially hepatotoxic compounds in humans. At the onset of liver injury in humans, currently used biomarkers have difficulty differentiating severe DILI from mild, and/or predict the outcome of injury for individual subjects. Therefore, new biomarker approaches for predicting and diagnosing DILI in humans are urgently needed. Recently, circulating microRNAs (miRNAs) such as miR-122 and miR-192 have emerged as promising biomarkers of liver injury in preclinical species and in DILI patients. In this study, we focused on examining global circulating miRNA profiles in serum samples from subjects with liver injury caused by accidental acetaminophen (APAP) overdose. Upon applying next generation high-throughput sequencing of small RNA libraries, we identified 36 miRNAs, including 3 novel miRNA-like small nuclear RNAs, which were enriched in the serum of APAP overdosed subjects. The set comprised miRNAs that are functionally associated with liver-specific biological processes and relevant to APAP toxic mechanisms. Although more patients need to be investigated, our study suggests that profiles of circulating miRNAs in human serum might provide additional biomarker candidates and possibly mechanistic information relevant to liver injury.

Long-term rodent carcinogenicity studies for evaluation of chemicals and pharmaceuticals concerning their carcinogenic potential to humans are currently receiving critical revision. Additional data from mechanistic studies can support cancer risk assessment by clarifying the underlying mode of action. In the course of the IMI MARCAR project, a European consortium of EFPIA partners and academics, which aims to identify biomarkers for nongenotoxic carcinogenesis, a toxicogenomic mouse liver database was generated. CD-1 mice were orally treated for 3 and 14 days with 3 known genotoxic hepatocarcinogens: C.I. Direct Black 38, Dimethylnitrosamine and 4,4'-Methylenedianiline; 3 nongenotoxic hepatocarcinogens: 1,4-Dichlorobenzene, Phenobarbital sodium and Piperonyl butoxide; 4 nonhepatocarcinogens: Cefuroxime sodium, Nifedipine, Prazosin hydrochloride and Propranolol hydrochloride; and 3 compounds that show ambiguous results in genotoxicity testing: Cyproterone acetate, Thioacetamide and Wy-14643. By liver mRNA expression analysis using individual animal data, we identified 64 specific biomarker candidates for genotoxic carcinogens and 69 for nongenotoxic carcinogens for male mice at day 15. The majority of genotoxic carcinogen biomarker candidates possess functions in DNA damage response (eg, apoptosis, cell cycle progression, DNA repair). Most of the identified nongenotoxic carcinogen biomarker candidates are involved in regulation of cell cycle progression and apoptosis. The derived biomarker lists were characterized with respect to their dependency on study duration and gender and were successfully used to characterize carcinogens with ambiguous genotoxicity test results, such as Wy-14643. The identified biomarker candidates improve the mechanistic understanding of drug-induced effects on the mouse liver that result in hepatocellular adenomas and/or carcinomas in 2-year mouse carcinogenicity studies.

The role of bile acids (BAs) as biomarkers for liver injury has been proposed for decades. However, the large inter- and intra-individual variability of the BA profile has prevented its clinical application. To this end, we investigated the effect of covariates such as food, gender, age, BMI, and moderate alcohol consumption on the BA profile in healthy human subjects. The BA profile was characterized by the calculation of indices that describe the composition, sulfation, and amidation of total and individual BAs. Both inter- and intra-individual variabilities of BA indices were low in serum and even lower in urine compared with those of absolute concentrations of BAs. Serum BA concentrations increased with consumption of food, whereas urinary BA concentrations were mildly affected by food. Gender differences in the urinary and serum BA profile were minimal. The serum and urinary BA profiles were also not affected by age. BMI showed minimal effect on the urine and serum BA profile. Moderate alcohol consumption did not have a significant effect on the BA profile in both urine and serum. When the effect of the type of alcohol was studied, the results indicate that moderate drinking of beer does not affect BA concentrations and has minimal effect on BA indices, whereas moderate wine consumption slightly increases BA concentrations without affecting the BA indices. In summary, urinary BA indices showed lower variability and higher stability than absolute BA concentrations in serum and showed minimal changes to covariate effects suggesting their utility as biomarkers in clinic.

Hepatobiliary diseases result in the accumulation of bile acids (BAs) in the liver, systemic blood, and other tissues leading to an unfavorable prognosis. The BA profile was characterized by the calculation of indices that describe the composition, sulfation, and amidation of total and individual BAs. Comparison of the urinary BA profiles between healthy subjects and patients with hepatobiliary diseases demonstrated significantly higher absolute concentrations of individual and total BAs in patients. The percentage sulfation of some individual BAs were different between the two groups. The percentage amidation of overall and most individual BAs was higher in patients than controls. The percentage of primary BAs (CDCA and CA) was higher in patients, whereas the percentage of secondary BAs (DCA and LCA) was lower in patients. BA indices belonging to percentage amidation and percentage composition were better associated with the severity of the liver disease as determined by the model for end-stage liver disease (MELD) score and disease compensation status compared with the absolute concentrations of individual and total BAs. In addition, BA indices corresponding to percentage amidation and percentage composition of certain BAs demonstrated the highest area under the receiver operating characteristic (ROC) curve suggesting their utility as diagnostic biomarkers in clinic. Furthermore, significant increase in the risk of having liver diseases was associated with changes in BA indices.

Three in vitro methods for the prediction of the skin sensitization hazard have been validated. However, predicting sensitizer potency is a key requirement for risk assessment. Here, we report a database of 312 chemicals tested in the KeratinoSens™ assay and for kinetic peptide binding. These data were used in multiple regression analysis against potency in the local lymph node assay (LLNA). The dataset covers the majority of chemicals from the validation of the LLNA to predict human potency and this subset was analyzed for prediction of human sensitization potency by in vitro data. Global analysis yields a regression of in vitro data to LLNA pEC3 with an R2 of 60% predicting LLNA EC3 with a mean error of 3.5-fold. The highest weight in the regression has the reaction rate with peptides, followed by Nrf2-induction and cytotoxicity in KeratinoSens™. The correlation of chemicals tested positive in vitro with human data has an R2 of 49%, which is similar to the correlation between LLNA and human data. Chemicals were then grouped into mechanistic domains based on experimentally observed peptide-adduct formation and predictions from the TIMES SS software. Predictions within these domains with a leave-one-out approach were more accurate, and for several mechanistic domains LLNA EC3 can be predicted with an error of 2- to 3-fold. However, prediction accuracy differs between domains and domain assignment cannot be made for all chemicals. Thus, this comprehensive analysis indicates that combining global and domain models to assess sensitizer potency may be a practical way forward.

One endocrine disruption mechanism is through binding to nuclear receptors such as the androgen receptor (AR) and estrogen receptor (ER) in target cells. The concentration of a chemical in serum is important for its entry into the target cells to bind the receptors, which is regulated by the serum proteins. Human sex hormone-binding globulin (SHBG) is the major transport protein in serum that can bind androgens and estrogens and thus change a chemical’s availability to enter the target cells. Sequestration of an androgen or estrogen in the serum can alter the chemical elicited AR- and ER-mediated responses. To better understand the chemical-induced endocrine activity, we developed a competitive binding assay using human pregnancy plasma and measured the binding to the human SHBG for 125 structurally diverse chemicals, most of which were known to bind AR and ER. Eighty seven chemicals were able to bind the human SHBG in the assay, whereas 38 chemicals were nonbinders. Binding data for human SHBG are compared with that for rat α-fetoprotein, ER and AR. Knowing the binding profiles between serum and nuclear receptors will improve assessment of a chemical’s potential for endocrine disruption. The SHBG binding data reported here represent the largest data set of structurally diverse chemicals tested for human SHBG binding. Utilization of the SHBG binding data with AR and ER binding data could enable better evaluation of endocrine disrupting potential of chemicals through AR- and ER-mediated responses since sequestration in serum could be considered.

Particulate matter (PM) exposure contributes to respiratory diseases and cardiopulmonary mortality. PM toxicity is related to sources and composition, such as abundance of polycyclic aromatic hydrocarbons (PAHs). We exposed adult male BALB/c mice, via oropharyngeal aspiration, to a range of doses of PM2.5 collected during the winter in downtown Sacramento near a major freeway interchange (SacPM). Two preparation methods (spin-down and multi-solvent extraction) were tested to remove particles from collection filters. Three doses were analyzed 24 h after treatment for (1) leukocytes and total protein in bronchoalveolar lavage fluid (BALF), (2) airway-specific and whole lobe expression of PAH-sensitive genes (CYP1B1 and CYP1A1) and IL-1 b, (3) lung histology, and (4) platelet function. Both extraction methods stimulated biological responses, but the spin-down method was more robust at producing IL-1 b and CYP1B1 gene responses and the multi-solvent extraction induced whole lung CYP1A1. Neutrophils in the BALF were increased 5- to 10-fold at the mid and high dose for both preparations. Histopathology scores indicated dose-dependent responses and increased pathology associated with spin-down-derived PM exposure. In microdissected airways, spin-down PM increased CYP1B1 gene expression significantly, but multi-solvent extracted PM did not. Platelet responses to the physiological agonist thrombin were approximately twice as potent in the spin-down preparation as in the multi-solvent extract. We conclude (1) the method of filter extraction can influence the degree of biological response, (2) for SacPM the minimal effective dose is 27.5–50 µg based on neutrophil recruitment, and (3) P450s are upregulated differently in airways and lung parenchyma in response to PAH-containing PM.

The orphan nuclear receptor NR4A2 (Nurr1) constitutively regulates inflammatory gene expression in glial cells by suppressing DNA binding activity of NF-B. We recently reported that novel 1,1-bis(3'-indolyl)-1-(p-substitutedphenyl)methane (C-DIM) compounds that activate NR4A family nuclear receptors in cancer lines also suppress inflammatory gene expression in primary astrocytes and prevent loss of dopaminergic neurons in mice exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and probenecid (MPTPp). In this study, we postulated that the basis for this neuroprotection involves blockade of glial activation and subsequent expression of NF-B-regulated inflammatory genes. To examine this mechanism, we treated transgenic NF-B/EGFP reporter mice with MPTPp for 7 days (MPTPp7d) followed by daily oral gavage with either vehicle (corn oil; MPTPp14d) or C-DIMs containing p-methoxyphenyl (C-DIM5), p-hydroxyphenyl (C-DIM8), or p-chlorophenyl (C-DIM12) groups. Each compound conferred significant protection against progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc), even when given after 7 days of dosing with MPTPp. C-DIM12 had the greatest neuroprotective activity in MPTPp-treated mice, and was also the most potent compound in suppressing activation of microglia and astrocytes, expression of cytokines and chemokines in quantitative polymerase chain reaction (qPCR) array studies, and in reducing expression of NF-B/EGFP in the SN. C-DIM12 prevented nuclear export of Nurr1 in dopaminergic neurons and enhanced expression of the Nurr1-regulated proteins tyrosine hydroxylase and the dopamine transporter. These data indicate that NR4A-active C-DIM compounds protect against loss of dopamine neurons in the MPTPp model of PD by preventing glial-mediated neuronal injury and by supporting a dopaminergic phenotype in TH-positive neurons in the SNpc.

Sorafenib is associated with adverse cardiac effects, including left ventricular dysfunction. However, the precise mechanism remains unclear. Here, we aimed to establish the genes responsible for this cardiotoxicity using zebrafish and human cardiomyocytes. Fluorescent cardiac imaging using pigmentless zebrafish with green fluorescent protein hearts revealed that the ventricular dimensions of the longitudinal axis with sorafenib were significantly shorter than those of the control group. Transcriptome analysis of their hearts revealed that stanniocalcin 1 (stc1) was downregulated by sorafenib. stc1 knockdown in zebrafish revealed that reduction of stc1 decreased the longitudinal dimensions of zebrafish ventricles, similar to that which occurs during sorafenib treatment. STC1 downregulation and cytotoxicity were also seen in human cardiomyocytes exposed to sorafenib. To clarify the molecular function of stc1 in sorafenib-induced cardiotoxicity, we focused on oxidative stress in cardiomyocytes treated with sorafenib. Reactive oxygen species (ROS) production significantly increased in both species of human cardiomyocytes and zebrafish exposed to sorafenib and STC1 knockdown compared with the controls. Finally, we found that forced expression of stc1 normalized impairment, decreasing the longitudinal dimensions in zebrafish treated with sorafenib. Our study demonstrated that STC1 plays a protective role against ventricular dysfunction and ROS overproduction, which are induced by sorafenib treatment. We discovered for the first time that STC1 downregulation is responsible for sorafenib-induced cardiotoxicity through activated ROS generation.

Although tungsten carbide-cobalt (WC-Co) nanoparticles (NPs) have been widely used because of their robustness, their risk to human health remains poorly studied, despite the International Agency for Research on Cancer (IARC) classifying them as "probably carcinogenic" for humans (Group 2A) in 2006. Our current study aimed at defining the cytotoxicity and genotoxicity of one set of commercially available 60-nm diameter WC-Co NPs on three human cell lines representative of potential target organs: A549 (lung), Hep3B (liver), and Caki-1 (kidney). The cytotoxicity of WC-Co NPs was determined by evaluating cell impedance (xCELLigence), cell survival/death, and cell cycle checkpoints. Flow cytometry was used to not only evaluate cell cycle checkpoints, but to also estimate reactive oxygen species (ROS) generation. In addition, -H2Ax foci detection (confocal microscopy), considered to be the most sensitive technique for studying DNA double-strand breaks, was utilized to evaluate genotoxicity. As a final part of this study, we assessed the cellular incorporation of WC-Co NPs, first byflow cytometry (side scatter), and then by confocal microscopy (light reflection) to ensure that the NPs had entered cells. Overall, our current findings demonstrate that WC-Co NPs induce cell mortality, DNA double-strand breaks, and cell cycle arrest in human renal (Caki-1) and liver (Hep3B) cell lines, but do not induce significant cytotoxic effects in A549 lung cells. Interestingly, although WC-Co NPs effectively entered the cells in all 3 lines tested, ROS were detected in Caki-1 and Hep3B, but not in A549. This may explain the great differences in the cytotoxic and genotoxic effects we observed between these lines.

Ligand-activated receptors regulate numerous genes, and mediate effects of a broad set of endogenous and exogenous chemicals in vertebrates. Understanding the roles of these transcription factors in zebrafish (Danio rerio) is important to the use of this non-mammalian model in toxicological, pharmacological, and carcinogenesis research. Response to a potential agonist for the pregnane X receptor (Pxr) [pregnenolone (PN)] was examined in developing zebrafish, to assess involvement of Pxr in regulation of selected genes, including genes in cytochrome P450 subfamilies CYP2 and CYP3. We also examined interaction of Pxr and the aryl hydrocarbon receptor (Ahr) signaling pathways. Pregnenolone caused a dose-dependent increase in mRNA levels of pxr, ahr2, CYP1A, CYP2AA1, CYP2AA12, CYP3A65, and CYP3C1, most of which peaked at 3 µM PN. The well-known Ahr agonist 3,3’,4,4’,5-pentachlorobiphenyl (PCB126) also upregulated expression of pxr, ahr2, CYP1A, CYP2AA12, CYP3A65, and CYP3C1 in a dose-dependent manner. Inhibition of pxr translation by morpholino antisense oligonucleotides (MO) suppressed PN-induced expression of pxr, ahr2, CYP3A65, and CYP3C1 genes. Levels of CYP2AA1 and CYP2AA12 mRNA were increased in the control-MO group exposed to PN; this was prevented by knocking down Pxr. Similarly, Ahr2-MO treatment blocked PCB126-induced mRNA expression of pxr, CYP1A, CYP2AA12, CYP3A65, and CYP3C1. The present study shows self-regulation of pxr by PN in developing zebrafish. Selected zebrafish CYP1, CYP2 (including several CYP2AAs) and CYP3 genes appear to be under the regulation of both Pxr and Ahr2.

The International Agency for Research on Cancer has recently reclassified diesel engine exhaust (DEE) as a Group 1 carcinogen. Micronucleus (MN), nucleoplasmic bridge (NPB), and nuclear bud (NBUD) frequencies in peripheral blood lymphocytes (PBLs) are associated with cancer risk. However, the impact of DEE exposure on MN frequency has not been thoroughly elucidated due to mixed exposure and its impact on NPB and NBUD frequencies has never been explored in humans. We recruited 117 diesel engine testing workers with exclusive exposure to DEE and 112 non-DEE-exposed workers, and then we measured urinary levels of 4 mono-hydroxylated polycyclic aromatic hydrocarbons (OH-PAHs) using high-performance liquid chromatography-mass spectrometry as well as MN, NPB, and NBUD frequencies in PBLs using cytokinesis-block MN assay. The DEE-exposed workers exhibited significantly higher MN, NPB, and NBUD frequencies than the non-DEE-exposed workers (P < 0.05). Among all study subjects, increasing levels of all 4 urinary OH-PAHs, on both quartile and continuous scales, were associated with increased MN, NPB, and NBUD frequencies (all P < 0.05). When the associations were analyzed separately in DEE-exposed and non-DEE-exposed workers, we found that the association between increasing quartiles of urinary 9-hydroxyphenanthrene (9-OHPh) and MN frequencies persisted in DEE-exposed workers (P = 0.001). The percent of MN frequencies increased, on average, by 23.99% (95% confidential interval, 9.64–39.93) per 1-unit increase in ln-transformed 9-OHPh. Our results clearly show that exposure to DEE can induce increases in MN, NPB, and NBUD frequencies in PBLs and suggest that DEE exposure level is associated with MN frequencies.

The herbicide atrazine (2-chloro-4-[ethylamino]-6-[isopropylamino]-s-triazine) is the most common water contaminant in the United States. Atrazine is a phosphodiesterase inhibitor and is classified as an estrogen disrupting compound because it elevates estrogen levels via induction of the enzyme aromatase. Previous studies have shown that atrazine exposure alters the function of innate immune cells such as NK cells, DC, mast cells, and macrophages. In this study we have examined the impact of in vitro atrazine exposure on the activation, proliferation, and effector cytokine production by primary murine CD4+ T lymphocytes. We found that atrazine exposure significantly inhibited CD4+ T cell proliferation and accumulation as well as the expression of the activation markers CD25 and CD69 in a dose-dependent manner. Interestingly, the effects were more pronounced in cells from male animals. These effects were partially mimicked by pharmacological reagents that elevate intracellular cAMP levels and addition of exogenous rmIL-2 further inhibited proliferation and CD25 expression. Consistent with these findings, atrazine exposure during T cell activation resulted in a 2- to 5-fold increase in the frequency of Foxp3+ CD4+ T cells.

Cyclooxygenase-1 (COX-1) is the constitutive form of the COX enzyme family, which produces bioactive lipids called prostanoids. Although the role of COX-2 in liver diseases has been studied, little is known about the function of COX-1 in liver injury. We aimed to investigate the role and mechanism of COX-1 in acute liver injury. Carbon tetrachloride (CCl4) was administered to induce acute liver injury in wild-type or COX-1-deficient mice. Both genetic (partially or completely) deletion of COX-1 expression and pharmacological inhibition of COX-1 activity in mice exacerbated acute liver injury induced by CCl4, revealing the (1) histopathological changes and increased serum levels of aminotransferases; (2) oxidative stress in the liver partly through the action of cytochrome P450 2E1-dependent pathway; (3) enhanced inflammatory and chemoattractive responses with increased number of activated macrophages; and (4) increased apoptosis through both intrinsic and extrinsic apoptotic pathways. These pathological changes were partly through the modulation of transcription factor-dependent pathways (eg, NF-B and C/EBP-α). Pre-treatment with prostaglandin E2 (PGE2) or 5-lipoxygenase (5-LO) inhibitor in homozygous COX-1 knockout mice significantly ameliorated CCl4-induced hepatic injury. In addition, level of hepato-protective molecules (eg, OSM and OSMR) and associated liver regeneration pathway were significantly inhibited by the deficiency of COX-1 but restored by the addition of PGE2 or the inhibition of 5-LO. Furthermore, the alternative arachidonic acid metabolism pathway of 5-LO, which induced additional inflammation in the liver, was activated in response to the deficiency of COX-1. In conclusion, basal expression of COX-1 is essential for the protection of liver against chemical-induced hepatotoxicity and required for hepatic homeostatic maintenance.

Glucocorticoids (GCs) are routinely administered systemically or injected into the eye when treating numerous ocular diseases; however, their toxicity on the retinal microvasculature has not been previously investigated. In this article, the effects of hydrocortisone (Hydro), dexamethasone, dexamethasone-phosphate and triamcinolone acetonide (TA) were evaluated in vitro on human skin microcirculation cells and, bovine endothelial retinal cells, ex-vivo, on flat mounted rat retinas. The degree of GCs induced endothelial cell death varied according to the endothelial cell type and GCs chemical properties. GCs toxicity was higher in skin microvascular endothelial cells and for hydrophobic GC formulations. The mechanism of cell death differed between GCs, Hydro and TA activated the leukocyte elastase inhibitor/L-DNase II pathways but did not activate caspases. The mechanisms of cell death observed in cell cultures were similar to those observed in rat retinal explants. Taken together these results indicate that particular attention should be paid to the potential vascular side effects when administrating GCs clinically and in particular when developing sustained-release intraocular devices.

The pathological role of α-synuclein (α-Syn) aggregation in neurodegeneration is well recognized, but the physiological function of normal α-Syn remains unknown. As α-Syn protein contains multiple divalent metal binding sites, herein we conducted a comprehensive characterization of the role of α-Syn in manganese-induced dopaminergic neurotoxicity. We established transgenic N27 dopaminergic neuronal cells by stably expressing human wild-type α-Syn at normal physiological levels. α-Syn-expressing dopaminergic cells significantly attenuated Mn-induced neurotoxicity for 24-h exposures relative to vector control cells. To further explore cellular mechanisms, we studied the mitochondria-dependent apoptotic pathway. Analysis of a key mitochondrial apoptotic initiator, cytochrome c, revealed that α-Syn significantly reduces the Mn-induced cytochrome c release into cytosol. The downstream caspase cascade, involving caspase-9 and caspase-3 activation, during Mn exposure was also largely attenuated in Mn-treated α-Syn cells in a time-dependent manner. α-Syn cells also showed a dramatic reduction in the Mn-induced proteolytic activation of the pro-apoptotic kinase PKC. The generation of Mn-induced reactive oxygen species (ROS) did not differ between α-Syn and vector control cells, indicating that α-Syn exerts its protective effect independent of altering ROS generation. Inductively coupled plasma-mass spectrometry (ICP-MS) revealed no significant differences in intracellular Mn levels between treated vector and α-Syn cells. Notably, the expression of wild-type α-Syn in primary mesencephalic cells also rescued cells from Mn-induced neurotoxicity. However, prolonged exposure to Mn promoted protein aggregation in α-Syn-expressing cells. Collectively, these results demonstrate that wild-type α-Syn exhibits neuroprotective effects against Mn-induced neurotoxicity during the early stages of exposure in a dopaminergic neuronal model of PD.

Polycyclic aromatic hydrocarbons (PAHs) induce developmental defects including cardiac deformities in fish. The aryl hydrocarbon receptor (AHR) mediates the toxicity of some PAHs. Exposure to a simple PAH mixture during embryo development consisting of an AHR agonist (benzo(a)pyrene-BaP) with fluoranthene (FL), an inhibitor of cytochrome p450 1(CYP1)—a gene induced by AHR activation—results in cardiac deformities. Exposure to BaP or FL alone at similar concentrations alters heart rates, but does not induce morphological deformities. Furthermore, AHR2 knockdown prevents the toxicity of BaP + FL mixture. Here, we used a zebrafish microarray analysis to identify heart-specific transcriptomic changes during early development that might underlie cardiotoxicity of BaP + FL. We used AHR2 morphant embryos to determine the role of this receptor in mediating toxicity. Control and knockdown embryos at 36 h post-fertilization were exposed to DMSO, 100 μg/l BaP, 500 μg/l FL, or 100 μg/l BaP + 500 μg/l FL, and heart tissues for RNA were extracted at 2, 6, 12, and 18 h-post-exposure (hpe), prior to the appearance of cardiac deformities. Data show AHR2-dependent BaP + FL effects on expression of genes involved in protein biosynthesis and neuronal development in addition to signaling molecules and their associated molecular pathways. Ca2+-cycling and muscle contraction genes were the most significantly differentially expressed category of transcripts when comparing BaP + FL-treated AHR2 morphant and control embryos. These differences were most prominent at 2 and 6 hpe. Therefore, we postulate that BaP + FL may affect cellular Ca2+ levels and subsequently cardiac muscle function, potentially underlying BaP + FL cardiotoxicity.

The brain subventricular zone (SVZ) is a source of neural precursor cells; these cells travel along the rostral migratory stream (RMS) to destination areas in the process of adult neurogenesis. Recent x-ray fluorescence (XRF) studies reveal an extensive accumulation of copper (Cu) in the SVZ. Earlier human and animal studies also suggest an altered Cu homeostasis after manganese (Mn) exposure. This study was designed to test the hypothesis that Mn exposure by acting on the divalent metal transporter-1 (DMT1) altered Cu levels in SVZ and RMS, thereby affecting adult neurogenesis. Adult rats received intraperitoneal (i.p.) injections of 6 mg Mn/kg as MnCl2 once daily for 4 weeks with concomitant injections of bromodeoxyuridine (BrdU) for 5 days in the last week. In control rats, Cu levels were significantly higher in the SVZ than other brain regions examined. Mn exposure significantly reduced Cu concentrations in the SVZ (P < 0.01). Immunohistochemical data showed that in vivo Mn exposure significantly increased numbers of BrdU(+) cells, which were accompanied with increased GFAP(+) astrocytic stem cells and DCX(+) neuroblasts in SVZ and RMS. Quantitative RT-PCR and Western blot confirmed the increased expression of DMT1 in SVZ following in vivo Mn exposure, which contributed to Mn accumulation in the neurogenesis pathway. Taken together, these results indicate a clear disruptive effect of Mn on adult neurogenesis; the effect appears due partly to Mn induction of DMT1 and its interference with cellular Cu regulation in SVZ and RMS. The future research directions based on these observations are also discussed.

Chronic exposure to single-walled carbon nanotubes (SWCNT) has been reported to induce apoptosis resistance of human lung epithelial cells. As resistance to apoptosis is a foundation of neoplastic transformation and cancer development, we evaluated the apoptosis resistance characteristic of the exposed lung cells to understand the pathogenesis mechanism. Passage control and SWCNT-transformed human lung epithelial cells were treated with known inducers of apoptosis via the intrinsic (antimycin A and CDDP) or extrinsic (FasL and TNF-α) pathway and analyzed for apoptosis by DNA fragmentation, annexin-V expression, and caspase activation assays. Whole-genome microarray was performed to aid the analysis of apoptotic gene signaling network. The SWCNT-transformed cells exhibited defective death receptor pathway in association with cellular FLICE-inhibitory protein (c-FLIP) overexpression. Knockdown or chemical inhibition of c-FLIP abrogated the apoptosis resistance of SWCNT-transformed cells. Whole-genome expression signature analysis confirmed these findings. This study is the first to demonstrate carbon nanotube-induced defective death receptor pathway and the role of c-FLIP in the process.

To provide useful alternatives to in vivo animal studies, in vitro assays for dose-response assessments of xenobiotic chemicals must use concentrations in media and target tissues that are within biologically-plausible limits. Determining these concentrations is a complex matter, which can be facilitated by applying physiologically-based pharmacokinetic (PBPK) models in an in vitro to in vivo extrapolation (IVIVE) paradigm. We used ethanol (EtOH), a ubiquitous chemical with defined metrics for in vivo and in vitro embryotoxicity, as a model chemical to evaluate this paradigm. A published series of life-stage PBPK models for rats was extended to mice, yielding simulations that adequately predicted in vivo blood EtOH concentrations (BECs) from oral, intraperitoneal, and intravenous routes in nonpregnant and pregnant adult mice. The models were then extrapolated to nonpregnant and pregnant humans, replicating BEC data within a factor of two. The rodent models were then used to conduct IVIVEs for rodent and whole-embryo culture embryotoxicity data (neural tube closure defects, morphological changes). A second IVIVE was conducted for exposure scenarios in pregnant women during critical windows of susceptibility for developmental toxicity, such as the first 6-to-8 weeks (prerecognition period) or mid-to-late pregnancy period, when EtOH consumption is associated with fetal alcohol spectrum disorders. Incorporation of data from human embryonic stem cell studies led to a model-supported linkage of in vitro concentrations with plausible exposure ranges for pregnant women. This effort demonstrates benefits and challenges associated with use of multispecies PBPK models to estimate in vivo tissue concentrations associated with in vitro embryotoxicity studies.