Quantum dots (QDs), an important class of emerging nanomaterial, are widely anticipated to find application in many consumer and clinical products in the near future. Premarket regulatory scrutiny is, thus, an issue gaining considerable attention. Previous review papers have focused primarily on the toxicity of QDs. From the point of view of product regulation, however, parameters that determine exposure (e.g., 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 QDs 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.

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 h 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 cytochrome P450 (CYP) 1A1 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 versus direct effects on enzyme activity for DIM, we evaluated the effects of pretreatment alone (transcriptional activation) versus cotreatment 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 downregulation 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.

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 3 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 fivefold 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 that a small subset of chemicals contributed disproportionately to the misclassification rate. For these chemicals, the misclassification was more closely associated with genotoxicity status than with 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 overpredicted the tumor response, but the variability in predictions was significantly higher. The study provides both a set of gene expression biomarkers for predicting chemically induced mouse lung tumors and a broad assessment of important experimental and analysis criteria for developing microarray-based predictors of safety-related end points.

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 incubating BV2 cells with SAH (1–50nM) 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 concentrations 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 messenger RNA expression of MMP-2 in BV2 cells induced by SAH. In BV2 cells treated with SAH, the binding abilities of nuclear factor-kappa B (NF-B) and stimulatory protein-1 (Sp1) to the MMP-2 promoter were increased, whereas the level of NF-B inhibitor was decreased. In addition, SAH significantly increased the phosphorylation of extracellular signal–regulated kinase (ERK) and phosphatidylinositol-3-kinase/serine/threonine protein kinase (or protein kinase B) (PI3K/Akt) proteins but did not affect that of c-Jun NH2-terminal kinase or p38. Pretreatment 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 promoter.

The objectives of this study were to find a minimal dose of 17-ethinyl estradiol (EE) that is detrimental to the developing penis and fertility and to compare estrogenic effects between EE and diethylstilbestrol (DES). Neonatal rats received EE at 10 ng (1 µg/kg), 100 ng, 1 µg, or 10 µg per pup on alternate days from postnatal days 1 to 11 (dose-response study) or received EE or DES at 100 ng per pup daily from postnatal days 1 to 6 (comparative study). Effects of EE were dose dependent, with ≥ 100-ng dose inducing significant (p < 0.05) reductions in penile length, weight, and diameter. Additionally, the penis was malformed, characterized by underdeveloped os penis and accumulation of fat cells. Fertility was 0% in the ≥ 1-µg groups, in contrast to 60% in the 100-ng group and 100% in the 10-ng and control groups. Animals treated with ≥ 10 ng 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 10 ng (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.

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 (CYP) 19 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/l prochloraz, 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 upregulation 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 upregulated 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.

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 (IL-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.

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, 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.

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 nongenotoxic (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 data sets normalized by Robust Multi-array Average, 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.

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 the following: (1) data from quantitative high–throughput screening cytotoxicity and caspase activation assays conducted at the National Institutes of Health Chemical Genomics Center, (2) data from Salmonella typhimurium reverse mutagenicity assays conducted by the U.S. National Toxicology Program, and (3) hepatotoxicity data published in the Registry of Toxic Effects of Chemical Substances. 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 tested also at the National Institutes of Health Chemical Genomics Center. We compared the performance of our WFS approach with classical classification methods such as Naive 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.

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 preexposed 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 preexposed 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 preexposed as juveniles. Levels of Mn, Fe, and Cu were determined by inductively coupled plasma-mass spectrometry, with the greatest accumulation of Mn detected in juvenile mice in the striatum, substantia nigra (SN), and cortex. Only modest changes in Fe and Cu were detected in Mn-treated mice, primarily in the SN. 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 relative to adult animals without juvenile exposure.

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/6J 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 with controls. More robust activation of microglia was observed in juveniles, whereas astrogliosis was more prominent in adult mice preexposed 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-nitrotyrosine protein adducts were detected in dopamine- and cAMP-regulated phosphoprotein-32–positive neurons of the St of Mn-treated adult mice preexposed as juveniles. These data indicate that subchronic 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.

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, isozyme 1 (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 peroxide or tert-butyl peroxide 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 with 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 (1) GPx-1 is an important molecular target involved in MeHg-induced neurotoxicity and (2) 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.

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 (≤ 300µM) 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.

Metallothionein (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 the 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 nuclear factor-B (NF-B) in neuronal cells in a dose-dependent manner. The MT-III–induced antiapoptotic effects and increase in NF-B activity were blocked by specific inhibitors of TrkA, phosphatidylinositol-3 kinase (PI3K), Akt, or NF-B, indicating that MT-III provides neuronal protection by activating NF-B through the TrkA/PI3K/Akt signaling pathway.

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 Lethal Dose₅₀ [LD₅₀]), via gavaging from gestation day (GD) 6 to postnatal day (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 luteinizing hormone (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 (~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 GD length and stillbirths 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.

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 analogs 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 fumonisin B₁ (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) body weight (bw) HFB₁ on embryonic day (E)7–E8. 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 per 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% of the fetuses were affected. The findings indicate that HFB₁ does not cause NTD in the sensitive LM/Bc mouse model and only weakly disrupts sphingolipid metabolism at doses up to sevenfold higher (micromole per kilogram body weight basis) than the previously reported lowest observed adverse effect level for FB₁.

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 multiwall CNT (MWCNT) material (Nanocyl NC 7000) according to Organisation for Economic Co-operation and Development test guideline 413. Wistar rats were head-nose exposed for 6 h/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.

Sulfur mustard (SM) is a strong alkylating agent. Inhalation of SM 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 explant-outgrowth 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 500µM). 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 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 300µM 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 interleukin (IL)-6 and IL-8 were also observed. Apoptotic markers such as cytochrome c, p53, Fas-associated protein with death domain, and procaspase-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 coculture model to understand further the pathogenesis of SM-induced barrier injury and to search for novel approaches in SM therapy.

Linear and nonlinear toxicity criteria were derived for 2,3,7,8-tetrachlorodibenzo(p)dioxin (TCDD) using the recent National Toxicology Program rat cancer bioassay. Dose-response relationships were assessed for combined liver tumors based on lifetime average liver concentrations (LALCs) 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 1 x 10^–4 per pg/kg/day was calculated as the ratio between the benchmark response rate and the HED at the lower confidence limit of the benchmark dose (BMDL)₀₁. A nonlinear reference dose (RfD) with a value of 100 pg/kg/day 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/day 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 U.S. Environmental Protection Agency's Cancer Guidelines.

Butterbur extracts (Petasites hybridus) are recommended for the prevention of migraine, but pharmacovigilance reports may be suggestive of rare hepatobiliary toxicity. 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 ~200-fold of therapeutic doses, induced liver transaminases and bilirubin elevations were observed. In a subsequent 6-month 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 (lactate dehydrogenase, MTT, and ATP), transaminase activities (alanine aminotransferase and aspartate aminotransferase), albumin synthesis, urea and testosterone metabolism to assay for cytochrome P450 monooxygenase activity. Only with extracts rich in petasin (37% petasin) and at high and 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 and lipid and protein metabolism. Importantly, neither chronic rat in vivo nor rat in vitro studies predicted reliably hepatotoxicity, therefore reemphasizing 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.

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 liquid chromatography-tandem mass spectrometry 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. Hypoacetylated 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 activity. We have further identified a hyperacetylated 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 N-benzyloxycarbonyl-Val-Ala-Asp(OMe) fluoromethyl ketone 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 further assist in vitro, in vivo, and clinical bridging in toxicological research.

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 (s-ROFA) particles (450 µg/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 (3 ppm), 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 hour postexposure, urethane-anesthetized rats were monitored for heart rate (HR), electrocardiogram, 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 postexposure. 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.