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To study the ways in which compounds can induce adverse effects, toxicologists have been constructing Adverse Outcome Pathways (AOPs). An AOP can be considered as a pragmatic tool to capture and visualize mechanisms underlying different types of toxicity inflicted by any kind of stressor, and describes the interactions between key entities that lead to the adverse outcome on multiple biological levels of organization. The construction or optimization of an AOP is a labor intensive process, which currently depends on the manual search, collection, reviewing and synthesis of available scientific literature. This process could however be largely facilitated using Natural Language Processing (NLP) to extract information contained in scientific literature in a systematic, objective, and rapid manner that would lead to greater accuracy and reproducibility. This would support researchers to invest their expertise in the substantive assessment of the AOPs by replacing the time spent on evidence gathering by a critical review of the data extracted by NLP. As case examples, we selected two frequent adversities observed in the liver: namely, cholestasis and steatosis denoting accumulation of bile and lipid, respectively. We used deep learning language models to recognize entities of interest in text and establish causal relationships between them. We demonstrate how an NLP pipeline combining Named Entity Recognition and a simple rules-based relationship extraction model helps screen compounds related to liver adversities in the literature, but also extract mechanistic information for how such adversities develop, from the molecular to the organismal level. Finally, we provide some perspectives opened by the recent progress in Large Language Models and how these could be used in the future. We propose this work brings two main contributions: 1) a proof-of-concept that NLP can support the extraction of information from text for modern toxicology and 2) a template open-source model for recognition of toxicological entities and extraction of their relationships. All resources are openly accessible via GitHub (https://github.com/ontox-project/en-tox)., Competing Interests: Author TL was employed by ToxTrack. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Corradi, Luechtefeld, de Haan, Pieters, Freedman, Vanhaecke, Vinken and Teunis.)

Wildfire events are becoming increasingly common across many areas of the United States, including North Carolina (NC). Wildfires can cause immediate damage to properties, and wildfire smoke conditions can harm the overall health of exposed communities. It is critical to identify communities at increased risk of wildfire events, particularly in areas with that have sociodemographic disparities and low socioeconomic status (SES) that may exacerbate incurred impacts of wildfire events. This study set out to: (1) characterize the distribution of wildfire risk across NC; (2) implement integrative cluster analyses to identify regions that contain communities with increased vulnerability to the impacts of wildfire events due to sociodemographic characteristics; (3) provide summary-level statistics of populations with highest wildfire risk, highlighting SES and housing cost factors; and (4) disseminate wildfire risk information via our online web application, ENVIROSCAN. Wildfire hazard potential (WHP) indices were organized at the census tract-level, and distributions were analyzed for spatial autocorrelation via global and local Moran's tests. Sociodemographic characteristics were analyzed via k -means analysis to identify clusters with distinct SES patterns to characterize regions of similar sociodemographic/socioeconomic disparities. These SES groupings were overlayed with housing and wildfire risk profiles to establish patterns of risk across NC. Resulting geospatial analyses identified areas largely in Southeastern NC with high risk of wildfires that were significantly correlated with neighboring regions with high WHP, highlighting adjacent regions of high risk for future wildfire events. Cluster-based analysis of SES factors resulted in three groups of regions categorized through distinct SES profiling; two of these clusters (Clusters 2 and 3) contained indicators of high SES vulnerability. Cluster 2 contained a higher percentage of younger (<5 years), non-white, Hispanic and/or Latino residents; while Cluster 3 had the highest mean WHP and was characterized by a higher percentage of non-white residents, poverty, and less than a high school education. Counties of particular SES and WHP-combined vulnerability include those with majority non-white residents, tribal communities, and below poverty level households largely located in Southeastern NC. WHP values per census tract were dispersed to the public via the ENVIROSCAN application, alongside other environmentally-relevant data., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Winker, Payton, Brown, McDermott, Freedman, Lenhardt, Eaves, Fry and Rager.)

Background and Purpose: Growth of subependymal giant cell tumor and subependymal nodules has not been well-characterized. The purpose of this study was to determine whether growth curves can differentiate subependymal giant cell tumors from subependymal nodules., Materials and Methods: Brain MR imaging of patients with tuberous sclerosis complex were retrospectively reviewed from 2002 to 2018. All lesions in the region of the foramen of Monro were measured. Lesions were categorized on the basis of maximal diameter at the most recent scan: small lesions (<1 cm), indeterminate lesions (>1 cm), and resected lesions (>1 cm and surgically resected). Growth velocity and acceleration on serial imaging were analyzed, and growth rates were calculated between 0 and 20 years of age and compared among the 3 categories., Results: Forty-one patients were analyzed. The average age at the earliest scan was 5.9 (SD = 5.7) years. One hundred twenty-six small, 27 indeterminate, and 10 resected lesions were measured. Subependymal giant cell tumors grew faster than indeterminate lesions between 6 and 15 years of age. Indeterminate lesions grew faster than small lesions at 0-10 years of age. Resected lesions showed increased velocity and acceleration of growth compared with indeterminate lesions and small lesions on serial imaging., Conclusions: Growth differentiates subependymal nodules and subependymal giant cell tumors within the first 20 years of life, and the use of velocity and acceleration of growth may refine the diagnostic criteria of subependymal giant cell tumors. Additionally, 6-15 years of age may be an important period to monitor subependymal giant cell tumors at the foramen of Monro because increased growth may help to identify subependymal giant cell tumors that will continue to grow and result in obstructive hydrocephalus., (© 2021 by American Journal of Neuroradiology.)

The effects of exposure to the environmental toxicant cadmium, in combination with obesity, on the metal content in mouse testis were evaluated. Starting in utero and continuing through to 10 or 24 weeks post-weaning, male mice were exposed to cadmium (0, 0.5 or 5 ppm), and fed either a low (LFD) or high fat diet (HFD) post-weaning. Testicular levels of cadmium and essential metals were determined 10 and 24 weeks post-weaning by ICP-MS. Similar to what has been previously observed in the liver, kidney, heart and brain, significant levels of cadmium accumulated in the testis under all exposure conditions. Additionally, HFD-fed animals accumulated more cadmium than did their LFD-treated counterparts. Both treatments affected essential metal homeostasis in the testis. These findings suggest that cadmium and obesity may compromise the reproductive potential in the male mouse by disrupting essential metal levels., Competing Interests: The authors report no declarations of interest., (© 2021 The Author(s).)

Nematode Caenorhabditis elegans (C. elegans) exhibited a vigorous swimming behavior in liquid medium. Addition of dopamine inhibited the swimming behavior, causing paralysis in 65% of wild-type nematodes. Interestingly, phytocannabinoids cannabidiol (CBD) or cannabidivarin (CBDV), caused paralysis in 40% of the animals. Knockout of DOP-3, the dopamine D2-like receptor critical for locomotor behavior, eliminated the paralysis induced by dopamine, CBD, and CBDV. In contrast, both CBD and CBDV caused paralysis in animals lacking CAT-2, an enzyme necessary for dopamine synthesis. Co-administration of dopamine with either CBD or CBDV caused paralysis similar to that of either phytocannabinoid treatment alone. These data support the notion that CBD and CBDV act as functional partial agonists on dopamine D2-like receptors in vivo. The discovery that dopamine receptor is involved in the actions of phytocannabinoids moves a significant step toward our understanding of the mechanisms for medical uses of cannabis in the treatment of neurological and psychiatric disorders., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Analyses of human cohort data support the roles of cadmium and obesity in the development of several neurocognitive disorders. To explore the effects of cadmium exposure in the brain, mice were subjected to whole life oral cadmium exposure. There were significant increases in cadmium levels with female animals accumulating more metal than males (p < 0.001). Both genders fed a high fat diet showed significant increases in cadmium levels compared to low fat diet fed mice (p < 0.001). Cadmium and high fat diet significantly affected the levels of several essential metals, including magnesium, potassium, chromium, iron, cobalt, copper, zinc and selenium. Additionally, these treatments resulted in increased superoxide levels within the cortex, amygdala and hippocampus. These findings support a model where cadmium and high fat diet affect the levels of redox-active, essential metal homeostasis. This phenomenon may contribute to the underlying mechanism(s) responsible for the development of neurocognitive disorders., Competing Interests: The authors report no declarations of interest., (© 2020 The Author(s).)

To characterize the clinical phenotype of Sunflower syndrome. Sunflower syndrome is a rare photosensitive epilepsy syndrome characterized by highly stereotyped seizures, photosensitivity, and heliotropism. We retrospectively reviewed the medical records of patients seen in the Massachusetts General Hospital for Children (MGHfC) pediatric epilepsy program with a history of Sunflower syndrome. Twenty-four patients were identified; 18 were female. At the time of initial MGHfC evaluation, patients' ages ranged from 6.4 to 25 years, with a median age of 11.5 years. All patients presented with hand-waving episodes (HWEs), although one patient no longer demonstrates this, but now has eye blinking episodes on exposure to light. Four have associated eye fluttering as a component of their most prevalent light-induced seizures. The average age at onset of HWEs was six years. Seventeen developed other symptoms prior to the onset of HWEs. The most prevalent symptom was an attraction to light and possible absence seizures. Light-induced seizures were generally refractory to broad-spectrum antiepileptic drugs (AEDs). Only three patients had a reduction of HWEs with the use of AEDs. Several non-pharmacological strategies reduced seizure frequency, however, efficacy varied. These non-pharmacological strategies included avoiding stimulus, focusing on other tasks, and occupying or restraining the hand that was involved in hand-waving. The use of tinted glasses reduced seizure frequency in 17 patients, however, no patient achieved seizure freedom. Twenty-two patients had available EEGs, 20 of which showed interictal epileptiform discharges. Additionally, many of the patients experienced a negative impact on their self-concept due to anxiety, depression, or negative interactions with peers. Sunflower syndrome is a generalized, pharmacoresistant epilepsy with childhood onset and remains poorly understood. To improve clinical care and scientific understanding, long-term prospective research exploring the natural history, etiology, and effective treatments for Sunflower syndrome should be conducted. [Published with video sequence].

Exposure to the environmental toxicant cadmium (Cd) contributes to the development of obesity-associated diseases. Obesity is a risk factor for a spectrum of unhealthy conditions including systemic metabolic dyshomeostasis. In the present study, the effects of whole-life exposure to environmentally-relevant concentrations of Cd on systemic essential metal distribution in adult mice fed a high-fat diet (HFD) were examined. For these studies, male and female mice were exposed to Cd-containing drinking water for >2 weeks before breeding. Pregnant mice and dams with offspring were exposed to Cd-containing drinking water. After weaning, offspring were continuously exposed to the same Cd concentration as their parents, and divided into HFD and normal (low) fat diet (LFD) groups. At 10 and 24 weeks, mice were sacrificed and blood, liver, kidney and heart harvested for metal analyses. There were significant concentration dependent increases in Cd levels in offspring with kidney > liver > heart. Sex significantly affected Cd levels in kidney and liver, with female animals accumulating more metal than males. Mice fed the HFD showed > 2-fold increase in Cd levels in the three organs compared to similarly treated LFD mice. Cadmium significantly affected essential metals levels in blood, kidney and liver. Additionally, HFD affected essential metal levels in these three organs. These findings suggest that Cd interacts with HFD to affect essential metal homeostasis, a phenomenon that may contribute to the underlying mechanism responsible for the development of obesity-associated pathologies.

Both obesity and arsenic exposure are global public health problems that are associated with increased risk of renal disease. The effect of whole-life exposure to environmentally relevant levels of arsenic within dietary high fat diet on renal pathogenesis were examined. In this study, C57BL/6 J mice were parentally exposed to 100 ppb arsenic before conception. After weaning, both male and female offspring were maintained on 100 ppb arsenic and fed either a normal (LFD) or high fat diet (HFD). At 10 and 24 weeks of age, the offspring were sacrificed and kidneys collected. Exposure to arsenic led to an increase body-weight in LFD diet-fed female but not male mice. This response was not observed in HFD-fed female mice; however male mice showed significant increases in body weight in both As- and non-treated animals. Histological analysis shows that arsenic exposure significantly increases HFD-induced glomerular area expansion, mesangial matrix accumulation and fibrosis compared to LFD control animals. HFD alone increases renal inflammation and fibrosis; reflected by increases in IL-1β, ICAM-1 and fibronectin levels. Arsenic exposure significantly increases HFD-induced inflammatory and oxidative stress responses. In general, male mice have more severe responses than female mice to HFD or arsenic treatment. These results demonstrate that arsenic exposure causes sex-dependent alterations in HFD-induced kidney damage., (Copyright © 2019. Published by Elsevier B.V.)

Childhood obesity, which is prevalent in developed countries, is a metabolic risk factor for cardiovascular disease. Cadmium (Cd), a ubiquitous environmental toxic metal, also has deleterious effects on the cardiovascular system. However, the combined effects of a high-fat diet (HFD) and lifelong, low-dose Cd exposure on cardiac remodeling remain unclear. This study aims to determine the effects of combined HFD and Cd exposure on cardiac remodeling, as well as gender-specific differences in the response. C57BL/6J mice were exposed to Cd at a low dose (L-Cd, 0.5 ppm) or high dose (H-Cd, 5 ppm) via drinking water from conception to sacrifice. After being weaned, the offspring mice were fed with a HFD (42% kcal from fat) for an additional 10 weeks. H-Cd exposure significantly increased Cd accumulation in the hearts of both parents and their offspring; a HFD showed no added effects on cardiac Cd content. H-Cd exposure increased cardiac metallothionein protein levels only in female mice, regardless of dietary intake. Histological analysis revealed that H-Cd exposure combined with a HFD induced cardiac hypertrophy and fibrosis only in female mice. This was further supported by elevated expression of ANP and COL1A1 protein levels along with COL1A1, COL1A2, and COL3A1 mRNA levels. Profibrotic markers PAI-1, CTGF, and FN were also elevated in HFD/H-Cd-exposed female mice. Levels of the oxidative stress marker 3-NT significantly increased in the hearts of HFD-fed female mice, whereas Cd exposure showed no additional effects. Of all the antioxidant markers examined, levels of CAT significantly increased in mice fed a HFD, regardless of gender and Cd exposure. In summary, a HFD combined with lifelong, low-dose Cd exposure induces cardiac hypertrophy and fibrosis in female but not male mice, a response that is independent of oxidative stress.

Vascular complications are common pathologies associated with type 1 diabetes. In recent years, histone deacetylation enzyme (HDAC) inhibitors have been shown to be successful in preventing atherosclerosis. To investigate the mechanism for HDAC3 inhibition in preventing diabetic aortic pathologies, male OVE26 type 1 diabetic mice and age-matched wild-type (FVB) mice were given the HDAC3-specific inhibitor RGFP-966 or vehicle for 3 mo. These mice were then euthanized immediately or maintained for an additional 3 mo without treatment. Levels of aortic inflammation and fibrosis and plasma and fibroblast growth factor 21 (FGF21) levels were determined. Because the liver is the major organ for FGF21 synthesis in diabetic animals, the effects of HDAC3 inhibition on hepatic FGF21 synthesis were examined. Additionally, hepatic miR-200a and kelch-like ECH-associated protein 1 (Keap1) expression and nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation were measured. HDAC3 inhibition significantly reduced aortic fibrosis and inflammation in OVE26 mice at both 3 and 6 mo. Plasma FGF21 levels were significantly higher in RGFP-966-treated OVE26 mice compared with vehicle-treated mice at both time points. It also significantly reduced hepatic pathologies associated with diabetes, accompanied by increased FGF21 mRNA and protein expression. HDAC3 inhibition also increased miR-200a expression, reduced Keap1 protein levels, and increased Nrf2 nuclear translocation with an upregulation of antioxidant gene and FGF21 transcription. Our results support a model where HDAC3 inhibition may promote Nrf2 activity by increasing miR-200a expression with a concomitant decrease in Keap1 to preserve hepatic FGF21 synthesis. The preservation of hepatic FGF21 synthesis ultimately leads to a reduction in diabetes-induced aorta pathologies.

Background: A central challenge in toxicity testing is the large number of chemicals in commerce that lack toxicological assessment. In response, the Tox21 program is re-focusing toxicity testing from animal studies to less expensive and higher throughput in vitro methods using target/pathway-specific, mechanism-driven assays., Objectives: Our objective was to use an in-depth mechanistic study approach to prioritize and characterize the chemicals affecting mitochondrial function., Methods: We used a tiered testing approach to prioritize for more extensive testing 622 compounds identified from a primary, quantitative high-throughput screen of 8,300 unique small molecules, including drugs and industrial chemicals, as potential mitochondrial toxicants by their ability to significantly decrease the mitochondrial membrane potential (MMP). Based on results from secondary MMP assays in HepG2 cells and rat hepatocytes, 34 compounds were selected for testing in tertiary assays that included formation of reactive oxygen species (ROS), upregulation of p53 and nuclear erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE), mitochondrial oxygen consumption, cellular Parkin translocation, and larval development and ATP status in the nematode Caenorhabditis elegans ., Results: A group of known mitochondrial complex inhibitors (e.g., rotenone) and uncouplers (e.g., chlorfenapyr), as well as potential novel complex inhibitors and uncouplers, were detected. From this study, we identified four not well-characterized potential mitochondrial toxicants (lasalocid, picoxystrobin, pinacyanol, and triclocarban) that merit additional in vivo characterization., Conclusions: The tier-based approach for identifying and mechanistically characterizing mitochondrial toxicants can potentially reduce animal use in toxicological testing. https://doi.org/10.1289/EHP2589.

Cardiovascular diseases (CVD) are the leading cause of morbidity and mortality in the world. Although considerable progress has been made in the diagnosis, treatment and prognosis of CVD, there is still a critical need for novel diagnostic biomarkers and new therapeutic interventions to decrease the incidence of this disease. Recently, there is increasing evidence that circulating miRNAs (miRNAs), i.e. endogenous, stable, single-stranded, short, non-coding RNAs, can be used as diagnostic biomarkers for CVD. Furthermore, miRNAs represent potential novel therapeutic targets for several cardiovascular disorders. In this review we provides an overview of the effects of several CVD; including heart failure, acute myocardial infarction, arrhythmias and pulmonary hypertension; on levels of circulating miRNAs. In addition, the use of miRNA as therapeutic targets is also discussed, as well as challenges and recommendations in their use in the diagnosis of CVD.

Autism spectrum disorders (ASD) are the most prevalent set of pediatric neurobiological disorders. The etiology of ASD has both genetic and environmental components including possible dysfunction of the immune system. The relationship of the immune system to aberrant neural circuitry output in the form of altered behaviors and communication characterized by ASD is unknown. Dysregulation of neurotrophins such as BDNF and their signaling pathways have been implicated in ASD. While abnormal cortical formation and autistic behaviors in mouse models of immune activation have been described, no one theory has been described to link activation of the immune system to specific brain signaling pathways aberrant in ASD. In this paper we explore the relationship between neurotrophin signaling, the immune system and ASD. To this effect we hypothesize that an interplay of dysregulated immune system, synaptogenic growth factors and their signaling pathways contribute to the development of ASD phenotypes.

Chronic exposure to cadmium is known to be a risk factor for human prostate cancer. Despite over-whelming evidence of cadmium causing carcinogenicity in humans, the specific underlying molecular mechanisms that govern metal-induced cellular transformation remain unclear. Acute exposure (up to 72 h) to cadmium induces apoptosis in normal prostate epithelial cells (RWPE-1), while chronic exposure (>1 year) transforms these cells to a malignant phenotype (cadmium-transformed prostate epithelial cells; CTPE). Increased expression of autophagy-regulated genes; Plac8, LC3B and Lamp-1; in CTPE cells was associated with cadmium-induced transformation. Increased expression of Plac8, a regulator of autophagosome/autolysosome fusion, facilitates the pro-survival function of autophagy and upregulation of pAKT (ser473) and NF-κβ, to allow CTPE to proliferate. Likewise, inhibition of Plac8 suppresses CTPE cell growth. Additionally, overexpression of Plac8 in RWPE-1 cells induces resistance to cadmium toxicity. Pharmacological inhibitors and an inducer of autophagy failed to affect Plac8 expression and CTPE cell viability, suggesting a unique role for Plac8 in cadmium-induced prostate epithelial cell transformation. These results support a role for Plac8 as an essential component in the cadmium-induced transformation of normal prostate epithelial cells to a cancerous state., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Cadmium, an established carcinogen, is a risk factor for prostate cancer. Induction of autophagy is a prerequisite for cadmium-induced transformation and metastasis. The ability of Psoralidin (Pso), a non-toxic, orally bioavailable compound to inhibit cadmium-induced autophagy to prevent prostate cancer was investigated., Methods: Psoralidin was studied using cadmium-transformed prostate epithelial cells (CTPE), which exhibit high proliferative, invasive and colony forming abilities. Gene and protein expression were evaluated by qPCR, western blot, immunohistochemistry and immunofluorescence. Xenograft models were used to study the chemopreventive effects in vivo., Results: Cadmium-transformed prostate epithelial cells were treated with Pso resulting in growth inhibition, without causing toxicity to normal prostate epithelial cells (RWPE-1). Psoralidin-treatment of CTPE cells inhibited the expression of Placenta Specific 8, a lysosomal protein essential for autophagosome and autolysosome fusion, which resulted in growth inhibition. Additionally, Pso treatment caused decreased expression of pro-survival signalling proteins, NFκB and Bcl2, and increased expression of apoptotic genes. In vivo, Pso effectively suppressed CTPE xenografts growth, without any observable toxicity. Tumours from Pso-treated animals showed decreased autophagic morphology, mesenchymal markers expression and increased epithelial protein expression., Conclusions: These results confirm that inhibition of autophagy by Pso plays an important role in the chemoprevention of cadmium-induced prostate carcinogenesis.

It has been proposed that aging results from the lifelong accumulation of intracellular damage via reactions with reactive oxygen species (ROS). Metallothioneins are conserved cysteine-rich proteins that function as efficient ROS scavengers and may affect longevity. To better understand mechanisms controlling metallothionein expression, the regulatory factors and pathways that controlled cadmium-inducible transcription of the C. elegans metallothionein gene, mtl-1, were identified. The transcription factor ATF-7 was identified in both ethylmethanesulfonate mutagenesis and candidate gene screens. PMK-1 and members of the insulin signaling pathway, PDK-1 and AKT-1/2, were also identified as mtl-1 regulators. Genetic and previous results support a model for the regulation of cadmium-inducible mtl-1 transcription based on the derepression of the constitutively active transcription factor ELT-2. In addition, knockdown of the mammalian homologs of PDK1 and ATF7 in HEK293 cells resulted in changes in metallothionein expression, suggesting that this pathway was evolutionarily conserved. The insulin signaling pathway is known to influence the aging process; however, various factors responsible for affecting the aging phenotype are unknown. Identification of portions of the insulin signaling pathway as regulators of metallothionein expression supports the hypothesis that longevity is affected by the expression of this efficient ROS scavenger.

Obesity often leads to obesity-related cardiac hypertrophy (ORCH), which is suppressed by zinc-induced inactivation of p38 mitogen-activated protein kinase (p38 MAPK). In this study, we investigated the mechanisms by which zinc inactivates p38 MAPK to prevent ORCH. Mice (4-week old) were fed either high fat diet (HFD, 60% kcal fat) or normal diet (ND, 10% kcal fat) containing variable amounts of zinc (deficiency, normal and supplement) for 3 and 6 months. P38 MAPK siRNA and the p38 MAPK inhibitor SB203580 were used to suppress p38 MAPK activity in vitro and in vivo, respectively. HFD activated p38 MAPK and increased expression of B-cell lymphoma/CLL 10 (BCL10) and caspase recruitment domain family member 9 (CARD9). These responses were enhanced by zinc deficiency and attenuated by zinc supplement. Administration of SB203580 to HFD mice or specific siRNA in palmitate-treated cardiomyocytes eliminated the HFD and zinc deficiency activation of p38 MAPK, but did not significantly impact the expression of BCL10 and CARD9. In cultured cardiomyocytes, inhibition of BCL10 expression by siRNA prevented palmitate-induced increased p38 MAPK activation and atrial natriuretic peptide (ANP) expression. In contrast, inhibition of p38 MAPK prevented ANP expression, but did not affect BCL10 expression. Deletion of metallothionein abolished the protective effect of zinc on palmitate-induced up-regulation of BCL10 and phospho-p38 MAPK. HFD and zinc deficiency synergistically induce ORCH by increasing oxidative stress-mediated activation of BCL10/CARD9/p38 MAPK signalling. Zinc supplement ameliorates ORCH through activation of metallothionein to repress oxidative stress-activated BCL10 expression and p38 MAPK activation., (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

With the phasing-out of the polybrominated diphenyl ether (PBDE) flame retardants due to concerns regarding their potential developmental toxicity, the use of replacement compounds such as organophosphate flame retardants (OPFRs) has increased. Limited toxicity data are currently available to estimate the potential adverse health effects of the OPFRs. The toxicological effects of 4 brominated flame retardants, including 3 PBDEs and 3,3',5,5'-tetrabromobisphenol A, were compared with 6 aromatic OPFRs and 2 aliphatic OPFRs. The effects of these chemicals were determined using 3 biological endpoints in the nematode Caenorhabditis elegans (feeding, larval development, and reproduction). Because C. elegans development was previously reported to be sensitive to mitochondrial function, results were compared with those from an in vitro mitochondrial membrane permeabilization (MMP) assay. Overall 11 of the 12 flame retardants were active in 1 or more C. elegans biological endpoints, with only tris(2-chloroethyl) phosphate inactive across all endpoints including the in vitro MMP assay. For 2 of the C. elegans endpoints, at least 1 OPFR had similar toxicity to the PBDEs: triphenyl phosphate (TPHP) inhibited larval development at levels comparable to the 3 PBDEs; whereas TPHP and isopropylated phenol phosphate (IPP) affected C. elegans reproduction at levels similar to the PBDE commercial mixture, DE-71. The PBDEs reduced C. elegans feeding at lower concentrations than any OPFR. In addition, 9 of the 11 chemicals that inhibited C. elegans larval development also caused significant mitochondrial toxicity. These results suggest that some of the replacement aromatic OPFRs may have levels of toxicity comparable to PBDEs., (Published by Oxford University Press on behalf of the Society of Toxicology 2016. This work is written by US Government employees and is in the public domain in the US.)

Background: Metallothioneins (MT) are small proteins, which are crucial for the distribution of heavy and transition metals. Previously, we found in mice that knockout of MT 1 and 2 genes (MTKO) impaired spatial learning and potentiated the learning impairment caused by developmental mercury exposure. The current study examined the neurocognitive and neurochemical effects of MTKO with the developmental copper (Cu) supplementation., Methods: Wildtype (WT) and MTKO mice were given supplemental Cu (0, 10 or 50 mg/l) in their drinking water during gestation and until weaning. When the mice were young adults they were trained on the win-shift 8-arm radial maze test of spatial learning and memory. After cognitive testing, their brains were analyzed for norepinepherine, dopamine and serotonin levels., Results: In the spatial learning test, wildtype mice showed the normal sex difference with males performing more accurately than the females. This effect was eliminated by MTKO and restored by moderate Cu supplementation during development. In neurochemical studies, MTKO caused a significant overall increase in serotonin in all of the regions studied: the frontal cortex, posterior cortex, hippocampus, striatum, midbrain, and brainstem. MTKO also caused a significant increase in norepinepherine in the brainstem and hippocampus. In wildtype mice, Cu supplementation during development caused a significant decline in dopamine and norepinepherine in the midbrain and dopamine in the frontal cortex. These effects were blocked by MTKO., Conclusions: The normal sex difference in spatial working memory accuracy, which was eliminated by MTKO, was restored by moderate copper supplementation. MTKO increased serotonin across all brain areas studied and increased norepinepherine only in the hippocampus and brainstem. MTKO blocked copper-induced decreases in dopamine and norepinepherine in the midbrain and dopamine in the frontal cortex.

Background: Modern toxicology is shifting from an observational to a mechanistic science. As part of this shift, high-throughput toxicity assays are being developed using alternative, nonmammalian species to prioritize chemicals and develop prediction models of human toxicity., Methods: The nematode Caenorhabditis elegans (C. elegans) was used to screen the U.S. Environmental Protection Agency's (EPA's) ToxCast™ Phase I and Phase II libraries, which contain 292 and 676 chemicals, respectively, for chemicals leading to decreased larval development and growth. Chemical toxicity was evaluated using three parameters: a biologically defined effect size threshold, half-maximal activity concentration (AC50), and lowest effective concentration (LEC)., Results: Across both the Phase I and Phase II libraries, 62% of the chemicals were classified as active ≤ 200 μM in the C. elegans assay. Chemical activities and potencies in C. elegans were compared with those from two zebrafish embryonic development toxicity studies and developmental toxicity data for rats and rabbits. Concordance of chemical activity was higher between C. elegans and one zebrafish assay across Phase I chemicals (79%) than with a second zebrafish assay (59%). Using C. elegans or zebrafish to predict rat or rabbit developmental toxicity resulted in balanced accuracies (the average value of the sensitivity and specificity for an assay) ranging from 45% to 53%, slightly lower than the concordance between rat and rabbit (58%)., Conclusions: Here, we present an assay that quantitatively and reliably describes the effects of chemical toxicants on C. elegans growth and development. We found significant overlap in the activity of chemicals in the ToxCast™ libraries between C. elegans and zebrafish developmental screens. Incorporating C. elegans toxicological assays as part of a battery of in vitro and in vivo assays provides additional information for the development of models to predict a chemical's potential toxicity to humans., Citation: Boyd WA, Smith MV, Co CA, Pirone JR, Rice JR, Shockley KR, Freedman JH. 2016. Developmental effects of the ToxCast™ Phase I and II chemicals in Caenorhabditis elegans and corresponding responses in zebrafish, rats, and rabbits. Environ Health Perspect 124:586-593; http://dx.doi.org/10.1289/ehp.1409645.

Due to their toxicity and persistence in the environment, brominated flame retardants (BFRs) are being phased out of commercial use, leading to the increased use of alternative chemicals such as the organophosphorus flame retardants (OPFRs). There is, however, limited information on the potential health effects of OPFRs. Due to the structural similarity of the OPFRs to organophosphorus insecticides, there is concern regarding developmental toxicity and neurotoxicity. In response, we evaluated a set of OPFRs (triphenyl phosphate [TPHP]), isopropylated phenyl phosphate [IPP], 2-ethylhexyl diphenyl phosphate [EHDP], tert-butylated phenyl diphenyl phosphate [BPDP], trimethyl phenyl phosphate [TMPP], isodecyl diphenyl phosphate [IDDP], (tris(1,3-dichloroisopropyl) phosphate [TDCIPP], and tris(2-chloroethyl)phosphate [TCEP]) in a battery of cell-based in vitro assays and alternative model organisms and compared the results to those obtained for two classical BFRs (3,3',5,5'-tetrabromobisphenol A [TBBPA] and 2,2'4,4'-brominated diphenyl ether [BDE-47]). The assays used evaluated the effects of chemicals on the differentiation of mouse embryonic stem cells, the proliferation and growth of human neural stem cells, rat neuronal growth and network activity, and development of nematode (Caenorhabditis elegans) and zebrafish (Danio rerio). All assays were performed in a concentration-response format, allowing for the determination of the point of departure (POD: the lowest concentration where a chemically-induced response exceeds background noise). The majority of OPFRs (8/9) were active in multiple assays in the range of 1-10 μM, most of which had comparable activity to the BFRs TBBPA and BDE-47. TCEP was negative in all assays. The results indicate that the replacement OPFRs, with the exception of TCEP, showed comparable activity to the two BFRs in the assays tested. Based on these results, more comprehensive studies are warranted to further characterize the potential hazard of some of these OPFR compounds., (Published by Elsevier Inc.)

Cadmium (Cd) is a toxic and carcinogenic metal naturally occurring in the Earth's crust. A common route of human exposure is via diet and cadmium accumulates in the liver. The effects of Cd exposure on gene expression in human hepatocellular carcinoma (HepG2) cells were examined in this study. HepG2 cells were acutely-treated with 0.1, 0.5, or 1.0 μM Cd for 24h; or chronically-treated with 0.01, 0.05, or 0.1 μM Cd for three weeks and gene expression analysis was performed using Affymetrix GeneChip® Human Gene 1.0 ST Arrays. Acute and chronic exposures significantly altered the expression of 333 and 181 genes, respectively. The genes most upregulated by acute exposure included several metallothioneins. Downregulated genes included the monooxygenase CYP3A7, involved in drug and lipid metabolism. In contrast, CYP3A7 was upregulated by chronic Cd exposure, as was DNAJB9, an anti-apoptotic J protein. Genes downregulated following chronic exposure included the transcriptional regulator early growth response protein 1. Ingenuity Pathway Analysis revealed that the top networks altered by acute exposure were lipid metabolism, small molecule biosynthesis, cell morphology, organization, and development; while top networks altered by chronic exposure were organ morphology, cell cycle, cell signaling, and renal and urological diseases/cancer. Many of the dysregulated genes play important roles in cellular growth, proliferation, and apoptosis, and may be involved in carcinogenesis. In addition to gene expression changes, HepG2 cells treated with cadmium for 24h indicated a reduction in global levels of histone methylation and acetylation that persisted 72 h post-treatment., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The extent of the innate immune response is regulated by many positively and negatively acting signaling proteins. This allows for proper activation of innate immunity to fight infection while ensuring that the response is limited to prevent unwanted complications. Thus mutations in innate immune regulators can lead to immune dysfunction or to inflammatory diseases such as arthritis or atherosclerosis. To identify novel innate immune regulators that could affect infectious or inflammatory disease, we have taken a comparative genomics RNAi screening approach in which we inhibit orthologous genes in the nematode Caenorhabditis elegans and murine macrophages, expecting that genes with evolutionarily conserved function also will regulate innate immunity in humans. Here we report the results of an RNAi screen of approximately half of the C. elegans genome, which led to the identification of many candidate genes that regulate innate immunity in C. elegans and mouse macrophages. One of these novel conserved regulators of innate immunity is the mRNA splicing regulator Eftud2, which we show controls the alternate splicing of the MyD88 innate immunity signaling adaptor to modulate the extent of the innate immune response., (Copyright © 2014 by the Genetics Society of America.)

Recent evidences indicated Nrf2 is more potent than Nrf1 in the activation of antioxidant genes. However, the roles of Nrf proteins in the regulation of copper-responsive transcription have not been well addressed. We took the toxicogenomic approach and the present network and Gene Ontology analyses results showed that Nrf1 and Nrf2 are distinctively involved in copper-responsive transcriptional regulation in HepG2 transcriptome. Cells deficient in either Nrf1 or Nrf2 were more susceptible to copper exposure than wild type cells. Nrf1 and Nrf2 null cells were transfected with the luciferase reporters containing either ARE(s) or a combination of ARE(s) and MREs, and then treated with copper. In Nrf2-null (Nrf2(-/-)) cells, copper did not activate transcription of reporter genes, whereas Nrf1 deficiency did not affect copper-inducible activation. Ectopic expression of Nrf2 restored copper-inducible transcription in Nrf2(-/-) cells. However, the changes in the intrinsic mRNA levels of MT-1 in Nrf null cells following copper treatment showed that Nrf1 and Nrf2 equally contributed to MT-1 activation after 4h, while Nrf1involved more than Nrf2 following 24h exposure. These results suggest that while Nrf2 is crucial for MRE/ARE-mediated transcription in response to copper, Nrf1 may activate MT-1 expression by a mechanism different from that Nrf2 employs., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Fluorides are commonly added to drinking water in the United States to decrease the incidence of dental caries. Silicofluorides, such as sodium hexafluorosilicate (Na2 SiF6 ) and fluorosilicic acid (H2 SiF6 ), are mainly used for fluoridation, although fluoride salts such as sodium fluoride (NaF) are also used. Interestingly, only the toxicity of NaF has been examined and not that of the more often used silicofluorides. In the present study, the toxicities of NaF, Na2 SiF6 , and H2 SiF6 were compared. The toxicity of these fluorides on the growth, feeding, and reproduction in the alternative toxicological testing organism Caenorhabditis elegans was examined. Exposure to these compounds produced classic concentration-response toxicity profiles. Although the effects of the fluoride compounds varied among the 3 biological endpoints, no differences were found between the 3 compounds, relative to the fluoride ion concentration, in any of the assays. This suggests that silicofluorides have similar toxicity to NaF., (© 2013 SETAC.)

Background: Mercury is a ubiquitous environmental toxicant that exists in multiple chemical forms. A paucity of information exists regarding the differences or similarities by which different mercurials act at the molecular level., Results: Transcriptomes of mixed-stage C. elegans following equitoxic sub-, low- and high-toxicity exposures to inorganic mercuric chloride (HgCl2) and organic methylmercury chloride (MeHgCl) were analyzed. In C. elegans, the mercurials had highly different effects on transcription, with MeHgCl affecting the expression of significantly more genes than HgCl2. Bioinformatics analysis indicated that inorganic and organic mercurials affected different biological processes. RNAi identified 18 genes that were important in C. elegans response to mercurial exposure, although only two of these genes responded to both mercurials. To determine if the responses observed in C. elegans were evolutionarily conserved, the two mercurials were investigated in human neuroblastoma (SK-N-SH), hepatocellular carcinoma (HepG2) and embryonic kidney (HEK293) cells. The human homologs of the affected C. elegans genes were then used to test the effects on gene expression and cell viability after using siRNA during HgCl2 and MeHgCl exposure. As was observed with C. elegans, exposure to the HgCl2 and MeHgCl had different effects on gene expression, and different genes were important in the cellular response to the two mercurials., Conclusions: These results suggest that, contrary to previous reports, inorganic and organic mercurials have different mechanisms of toxicity. The two mercurials induced disparate effects on gene expression, and different genes were important in protecting the organism from mercurial toxicity.

The innate immune response plays a critical role in fighting infection; however, innate immunity also can affect the pathogenesis of a variety of diseases, including sepsis, asthma, cancer, and atherosclerosis. To identify novel regulators of innate immunity, we performed comparative genomics RNA interference screens in the nematode Caenorhabditis elegans and mouse macrophages. These screens have uncovered many candidate regulators of the response to lipopolysaccharide (LPS), several of which interact physically in multiple species to form an innate immunity protein interaction network. This protein interaction network contains several proteins in the canonical LPS-responsive TLR4 pathway as well as many novel interacting proteins. Using RNAi and overexpression studies, we show that almost every gene in this network can modulate the innate immune response in mouse cell lines. We validate the importance of this network in innate immunity regulation in vivo using available mutants in C. elegans and mice.

The presence of toxic amounts of transition metals in the environment may originate from a range of human activities and natural processes. One method for the removal of toxic levels of metals is through chelation by small molecules. However, chelation is not synonymous with detoxification and may not affect the bioavailability of the metal. To test the bioavailability of chelated metals in vivo, the effects of several metal/chelator combinations were tested in the environmentally relevant organism Caenorhabditis elegans. The effect of metal exposure on nematode growth was used to determine the toxicity of cadmium, copper, nickel, and zinc. The restoration of growth to levels observed in nonexposed nematodes was used to determine the protective effects of the polydentate chelators: acetohydroxamic acid (AHA), cyclam, cysteine, calcium EDTA, desferrioxamine B, 1,2-dimethyl,3-hydroxy,4-pyridinone, and histidine. Cadmium toxicity was removed only by EDTA; copper toxicity was removed by all of the chelators except AHA; nickel toxicity was removed by cyclam, EDTA, and histidine; and zinc toxicity was removed by only EDTA. These results demonstrate the utility of polydentate chelators in the remediation of metal-contaminated systems. They also demonstrate that although the application of a chelator to metal contaminants may be effective, binding alone cannot be used to predict the level of remediation. Remediation depends on a number of factors, including metal complex speciation in the environment.

The spread of chemicals, including insecticides, into the environment often raises public health concerns, as exemplified by a recent epidemiologic study associating in utero piperonyl butoxide (PBO) exposure with delayed mental development. The insecticide synergist PBO is listed among the top 10 chemicals detected in indoor dust; a systematic assessment of risks from PBO exposure, as for many toxicants unfortunately, may be underdeveloped when important biological targets that can cause toxicity are unknown. Hedgehog/Smoothened signaling is critical in neurological development. This study was designed to use novel high-throughput in vitro drug screening technology to identify modulators of Hedgehog signaling in environmental chemicals to assist the assessment of their potential risks. A directed library of 1408 environmental toxicants was screened for Hedgehog/Smoothened antagonist activity using a high-content assay that evaluated the interaction between Smoothened and βarrestin2 green fluorescent protein. PBO was identified as a Hedgehog/Smoothened antagonist capable of inhibiting Hedgehog signaling. We found that PBO bound Smoothened and blocked Smoothened overexpression-induced Gli-luciferase reporter activity but had no effect on Gli-1 downstream transcriptional factor-induced Gli activity. PBO inhibited Sonic Hedgehog ligand-induced Gli signaling and mouse cerebellar granular precursor cell proliferation. Moreover, PBO disrupted zebrafish development. Our findings demonstrate the value of high-throughput target-based screening strategies that can successfully evaluate large numbers of environmental toxicants and identify key targets and unknown biological activity that is helpful in properly assessing potential risks.

Cadmium is an environmental toxicant whose exposure is associated with multiple human pathologies. To prevent cadmium-induced toxicity, organisms produce a variety of detoxification molecules. In response to cadmium, the nematode Caenorhabditis elegans increases the steady-state levels of several hundred genes, including two metallothioneins, mtl-1 and mtl-2, and the cadmium-specific response gene, cdr-1. Despite the presumed importance in metal detoxification of mtl-1 and mtl-2, knockdown of their expression does not increase cadmium hypersensitivity, which suggests that these genes are not required for resistance to metal toxicity in C. elegans. To determine whether cdr-1 is critical in metal detoxification and compensates for the loss of mtl-1 and/or mtl-2, C. elegans strains were generated in which one, two, and all three genes were deleted, and the effects of cadmium on brood size, embryonic lethality, the Bag phenotype, and growth were determined. Growth at low cadmium concentrations was the only endpoint in which the triple mutant displayed more sensitivity than the single and double mutants. A lack of hypersensitivity in these strains suggests that other factors may be involved in the response to cadmium. Caenorhabditis elegans produces phytochelatins (PCs) that are critical in the defense against cadmium toxicity. PC levels in wild type, cdr-1 single, mtl-1, mtl-2 double, and triple mutants were measured. PC levels were constitutively higher in the mtl-1, mtl-2 double, and triple mutants compared with wild type. Following cadmium exposure, PC levels increased. The lack of cadmium hypersensitivity when these genes are deleted may be attributed to the compensatory effects of increases in PCs.

We previously identified Tbc1d23 as a candidate novel regulator of innate immunity using comparative genomics RNA interference screens in Caenorhabditis elegans and mouse macrophages. Using Tbc1d23 knockout mice and macrophages engineered to overexpress Tbc1d23, we now show that Tbc1d23 is a general inhibitor of innate immunity signaling, strongly inhibiting multiple TLR and dectin-signaling pathways. Tbc1d23 likely acts downstream of the TLR-signaling adaptors MyD88 and Trif and upstream of the transcription factor XBP1. Importantly, like XBP1, Tbc1d23 affects the maintenance, but not the initiation, of inflammatory cytokine production induced by LPS. Tbc1d23 acts as a RAB-GAP to regulate innate immunity signaling. Thus, Tbc1d23 exerts its inhibitory effect on innate immunity signaling in a spatiotemporal fashion. The identification of a novel spatiotemporal regulator of innate immunity signaling validates the comparative genomics approach for innate immunity gene discovery.

Endosulfan is an organochlorine insecticide and has been implicated in neurotoxicity, hepatotoxicity, immunosuppression and teratogenicity. However, the molecular mechanism of endosulfan toxicity is not yet clear. Recent studies demonstrated that oxidative stress induced by endosulfan is involved in its toxicity and accumulating evidence suggests that endosulfan can modulate the activities of stress-responsive signal transduction pathways including extracellular signal regulated kinases (ERK) 1/2. However, none of the previous studies investigated the ability of endosulfan to modulate activating protein-1 (AP-1) binding and antioxidant response element (ARE)-mediated transcription as an underlying mechanism of endosulfan toxicity. In this report, we show that treatment of HepG2 cells with endosulfan significantly increased oxidative stress-responsive transcription via AP-1 activation. In addition, endosulfan-induced transcription was enhanced in cells depleted of glutathione by buthionine sulfoximine (BSO) treatment. Exposure to endosulfan resulted in a significant increase in the activities of MAPKs, ERK1/2 and p38. Endosulfan-induced increases in enzymatic activities of these MAPKs were consistent with MAPK phosphorylation. Endosulfan exposure also caused an increase in c-Jun phosphorylation. These results suggest a model for endosulfan toxicity in which endosulfan increases ERK1/2 and p38 activities and these activated MAPKs then increase c-Jun phosphorylation. Phosphorylated c-Jun, in turn, increases AP-1 activity, which results in activation of ARE-mediated transcription., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

A number of practical advantages have made the nematode Caenorhabditis elegans a useful model for genetic and developmental biological research. These same advantages, along with conservation of disease and stress response pathways, availability of mutant and transgenic strains, and wealth of biological information, have led to the increased use of C. elegans in toxicological studies. Although the potential to study the mechanisms of developmental toxicology in C. elegans is promising, embryonic and larval growth tests to identify compounds that affect the nematode have remained the primary use of C. elegans in developmental toxicology. Here, we describe a C. elegans larval growth and development assay for medium- and high-throughput screening using the COPAS Biosort flow cytometer and provide descriptions of the data and subsequent analysis.

The base excision DNA repair (BER) pathway known to occur in Caenorhabditis elegans has not been well characterized. Even less is known about the DNA polymerase (pol) requirement for the gap-filling step during BER. We now report on characterization of in vitro uracil-DNA initiated BER in C. elegans. The results revealed single-nucleotide (SN) gap-filling DNA polymerase activity and complete BER. The gap-filling polymerase activity was not due to a DNA polymerase β (pol β) homolog, or to another X-family polymerase, since computer-based sequence analyses of the C. elegans genome failed to show a match for a pol β-like gene or other X-family polymerases. Activity gel analysis confirmed the absence of pol β in the C. elegans extract. BER gap-filling polymerase activity was partially inhibited by both dideoxynucleotide and aphidicolin. The results are consistent with a combination of both replicative polymerase(s) and lesion bypass/BER polymerase pol θ contributing to the BER gap-filling synthesis. Involvement of pol θ was confirmed in experiments with extract from pol θ null animals. The presence of the SN BER in C. elegans is supported by these results, despite the absence of a pol β-like enzyme or other X-family polymerase.

Mercury (Hg) is a toxic metal that can exist in multiple chemical species. Humans are commonly exposed to methylmercury and Hg vapor, which are converted to mercuric species in the body. Despite years of research, little information exists on the similarities and differences in the mechanisms of Hg toxicity. The relative toxicity of mercuric chloride (HgCl(2)) and methylmercury chloride (MeHgCl) in Caenorhabditis elegans was determined in assays that measured growth, feeding, reproduction, and locomotion. The effect of HgCl(2) and MeHgCl on the expression of several archetypal stress-response genes was also determined. There was no significant difference between the EC50s of the two mercurials in terms of C. elegans growth. However, MeHgCl was more toxic to C. elegans than HgCl(2) when assessing feeding, movement, and reproduction, all of which require proper neuromuscular activity. Methylmercury chloride exposure resulted in increased steady-state levels of the stress response genes at lower concentrations than HgCl(2). In general, MeHgCl was more toxic to C. elegans than HgCl(2), particularly when assaying behaviors that require neuromuscular function., (Copyright © 2011 SETAC.)

Previous global transcriptome and interactome analyses of copper-treated HepG2 cells identified hepatocyte nuclear factor 4α (HNF4α) as a potential master regulator of copper-responsive transcription. Copper exposure caused a decrease in the expression of HNF4α at both mRNA and protein levels, which was accompanied by a decrease in the level of HNF4α binding to its consensus DNA binding sequence. qRT-PCR and RNAi studies demonstrated that changes in HNF4α expression ultimately affected the expressions of its down-stream target genes. Analysis of upstream regulators of HNF4α expression, including p53 and ATF3, showed that copper caused an increase in the steady-state levels of these proteins. These results support a model for copper-responsive transcription in which the metal affects ATF3 expression and stabilizes p53 resulting in the down-regulation of HNF4α expression. In addition, copper may directly affect p53 protein levels. The suppression of HNF4α activity may contribute to the molecular mechanisms underlying the physiological and toxicological consequences of copper toxicity in hepatic-derived cells., (Published by Elsevier B.V.)

Background: Exposure to cadmium is associated with human pathologies and altered gene expression. The molecular mechanisms by which cadmium affects transcription remain unclear. It has been proposed that cadmium activates transcription by altering intracellular calcium concentration ([Ca(2+)](i)) and disrupting calcium-mediated intracellular signaling processes. This hypothesis is based on several studies that may be technically problematic; including the use of BAPTA chelators, BAPTA-based fluorescent sensors, and cytotoxic concentrations of metal., Methodology/principal Finding: In the present report, the effects of cadmium on [Ca(2+)](i) under non-cytotoxic and cytotoxic conditions was monitored using the protein-based calcium sensor yellow cameleon (YC3.60), which was stably expressed in HEK293 cells. In HEK293 constitutively expressing YC3.60, this calcium sensor was found to be insensitive to cadmium. Exposing HEK293::YC3.60 cells to non-cytotoxic cadmium concentrations was sufficient to induce transcription of cadmium-responsive genes but did not affect [Ca(2+)](i) mobilization or increase steady-state mRNA levels of calcium-responsive genes. In contrast, exposure to cytotoxic concentrations of cadmium significantly reduced intracellular calcium stores and altered calcium-responsive gene expression., Conclusions/significance: These data indicate that at low levels, cadmium induces transcription independently of intracellular calcium mobilization. The results also support a model whereby cytotoxic levels of cadmium activate calcium-responsive transcription as a general response to metal-induced intracellular damage and not via a specific mechanism. Thus, the modulation of intracellular calcium may not be a primary mechanism by which cadmium regulates transcription.

There is relatively little information regarding the critical xenobiotic-metabolizing cytochrome P450 (CYP) enzymes in Caenorhabditis elegans, despite this organism's increasing use as a model in toxicology and pharmacology. We carried out experiments to elucidate the capacity of C. elegans to metabolically activate important promutagens via CYPs. Phylogenetic comparisons confirmed an earlier report indicating a lack of CYP1 family enzymes in C. elegans. Exposure to aflatoxin B(1) (AFB(1)), which is metabolized in mammals by CYP1, CYP2, and CYP3 family enzymes, resulted in significant DNA damage in C. elegans. However, exposure to benzo[a]pyrene (BaP), which is metabolized in mammals by CYP1 family enzymes only, produced no detectable damage. To further test whether BaP exposure caused DNA damage, the toxicities of AFB(1) and BaP were compared in nucleotide excision repair (NER)-deficient (xpa-1) and NER-proficient (N2) strains of C. elegans. Exposure to AFB(1) inhibited growth more in xpa-1 than N2 nematodes, but the growth-inhibitory effects of BaP were indistinguishable in the two strains. Finally, a CYP-nicotinamide adenine dinucleotide phosphate reductase-deficient strain (emb-8) of C. elegans was found to be more resistant to the growth-inhibitory effect of AFB(1) exposure than N2, confirming that the AFB(1)-mediated growth inhibition resulted from CYP-mediated metabolism. Together, these results indicate that C. elegans lacks biologically significant CYP1 family-mediated enzymatic metabolism of xenobiotics. Interestingly, we also found that xpa-1 nematodes were slightly more sensitive to chlorpyrifos than were wild type. Our results highlight the importance of considering differences between xenobiotic metabolism in C. elegans and mammals when using this alternative model in pharmaceutical and toxicological research.

Metallothioneins (MTs) protect cells from oxidative damage by scavenging reactive oxygen species (ROS). Concurrent with protecting cells from ROS-mediated damage, MT transcription is induced by ROS. ROS activate transcription by affecting several signal transduction pathways, many of which have been implicated in regulating MT transcription. ROS-activated intracellular signaling is mediated by the stable lipid peroxide 4-hydroxynonenal (HNE). After determining the level of sensitivity of Hepa 1-6 cells to HNE, MT-1 mRNA expression was shown to be induced in a concentration and time-dependent manner after HNE exposure. Finally, using MT-based reporters, HNE was found to induce MT transcription via both antioxidant response and metal response elements. Thus, ROS may activate MT transcription by generating HNE that in turn affects signaling pathways that regulate MT transcription via the transcription factors AP-1 and MTF-1., (Copyright 2010 Wiley Periodicals, Inc.)

Base excision repair (BER) is a DNA repair pathway designed to correct small base lesions in genomic DNA. While DNA polymerase beta (pol beta) is known to be the main polymerase in the BER pathway, various studies have implicated other DNA polymerases in back-up roles. One such polymerase, DNA polymerase lambda (pol lambda), was shown to be important in BER of oxidative DNA damage. To further explore roles of the X-family DNA polymerases lambda and beta in BER, we prepared a mouse embryonic fibroblast cell line with deletions in the genes for both pol beta and pol lambda. Neutral red viability assays demonstrated that pol lambda and pol beta double null cells were hypersensitive to alkylating and oxidizing DNA damaging agents. In vitro BER assays revealed a modest contribution of pol lambda to single-nucleotide BER of base lesions. Additionally, using co-immunoprecipitation experiments with purified enzymes and whole cell extracts, we found that both pol lambda and pol beta interact with the upstream DNA glycosylases for repair of alkylated and oxidized DNA bases. Such interactions could be important in coordinating roles of these polymerases during BER.

To define the mechanisms involved in the molecular response to the carcinogenic metal cadmium, two novel metal-inducible genes from C. elegans were characterized: numr-1 and numr-2 (nuclear localized metal responsive). numr-1 and numr-2 sequences and cellular patterns of expression are identical, indicating that these are functionally equivalent genes. Constitutive transcription of numr-1 and numr-2 is developmentally regulated and occurs in the intestine, in head and tail neurons, and vulva muscles. Exposure to metals induces numr-1 and numr-2 transcription in pharyngeal and intestinal cells. Other environmental stressors do not affect transcription, indicating that these are metal-specific, stress-responsive genes. NUMR-1 and NUMR-2 target to nuclei and colocalize with HSF-1, suggesting that they may be components of nuclear stress granules. Nematodes overexpressing NUMR-1 and NUMR-2 are resistant to stress and live longer than control animals; likewise reducing expression increases sensitivity to metals and decreases neuromuscular functions. Upstream regulatory regions of both genes contain potential binding sites for DAF-16 and SKN-1, which are components of the insulin-IGF-like signaling pathway. This pathway regulates longevity and stress responses in C. elegans. NUMR-1 and NUMR-2 may function to promote resistance to environmental stressors and longevity, which is mediated by the insulin-IGF-like signaling pathway.

The National Research Council has outlined the need for non-mammalian toxicological models to test the potential health effects of a large number of chemicals while also reducing the use of traditional animal models. The nematode Caenorhabditis elegans is an attractive alternative model because of its well-characterized and evolutionarily conserved biology, low cost, and ability to be used in high-throughput screening. A high-throughput method is described for quantifying the reproductive capacity of C. elegans exposed to chemicals for 48 h from the last larval stage (L4) to adulthood using a COPAS Biosort. Initially, the effects of exposure conditions that could influence reproduction were defined. Concentrations of DMSO vehicle

The relationship between the mechanisms that control an organism's lifespan and its ability to respond to environmental challenges are poorly understood. In Caenorhabditis elegans, an insulin-like signaling pathway modulates lifespan and the innate immune response to bacterial pathogens via a common mechanism involving transcriptional regulation by the DAF-16/FOXO transcription factor. The C. elegans germ line also modulates lifespan in a daf-16-dependent manner. Here, we show that the germ line controls the innate immune response of C. elegans somatic cells to two different Gram-negative bacteria. In contrast to the insulin-like signaling pathway, the germ line acts via distinct signaling pathways to control lifespan and innate immunity. Under standard nematode culture conditions, the germ line regulates innate immunity in parallel to a known p38 MAPK signaling pathway, via a daf-16-independent pathway. Our findings indicate that a complex regulatory network integrates inputs from insulin-like signaling, p38 MAPK signaling, and germ line stem cells to control innate immunity in C. elegans. We also confirm that innate immunity and lifespan in C. elegans are distinct processes, as nonoverlapping regulatory networks control survival in the presence of pathogenic and nonpathogenic bacteria. Finally, we demonstrate that the p38 MAPK pathway in C. elegans is activated to a similar extent by both pathogenic and nonpathogenic bacteria, suggesting that both can induce the nematode innate immune response.

We performed experiments to characterize the inducibility of nucleotide excision repair (NER) in Caenorhabditis elegans, and to examine global gene expression in NER-deficient and -proficient strains as well as germline vs. somatic tissues, with and without genotoxic stress. We also carried out experiments to elucidate the importance of NER in the adult life of C. elegans under genotoxin-stressed and control conditions. Adult lifespan was not detectably different between wild-type and NER-deficient xpa-1 nematodes under control conditions. However, exposure to 6J/m(2)/day of ultraviolet C radiation (UVC) decreased lifespan in xpa-1 nematodes more than a dose of 100 J/m(2)/day in wild-type. Similar differential sensitivities were observed for adult size and feeding. Remarkably, global gene expression was nearly identical in young adult wild-type and xpa-1 nematodes, both in control conditions and 3h after exposure to 50 J/m(2) UVC. Neither NER genes nor repair activity were detectably inducible in young adults that lacked germ cells and developing embryos (glp-1 strain). However, expression levels of dozens of NER and other DNA damage response genes were much (5-30-fold) lower in adults lacking germ cells and developing embryos, suggesting that somatic and post-mitotic cells have a much lower DNA repair ability. Finally, we describe a refinement of our DNA damage assay that allows damage measurement in single nematodes.