ABSTRACT
Large-scale gene expression analysis of legacy* and emerging** brominated flame retardants were conducted in the male Harlan Sprague Dawley rat [1]. Each animal was dosed for 5 days with the chemical at concentrations of 0.1 - 1000 µmol/kg body weight per day. Following the last dose, a specimen of the left liver was removed for RNA extraction. The amplified RNA (aRNA) was fragmented and then hybridized to Affymetrix Rat Genome 230 2.0 Arrays. Each GeneChip® array was scanned using an Affymetrix GeneChip® Scanner 3000 7â¯G to generate raw expression level data (.CEL files). Statistical contrasts were used to find pairwise gene expression differences between the control group and each dose group using the R/maanova package [2]. The transcriptomic data can be used to provide insights into the degree of toxicity, toxic mechanisms, disease pathways activated by exposure, and for benchmark dose analysis. The gene expression data for each of the nine flame retardants discussed here accompanies the research article entitled, "Comparative Toxicity and Liver Transcriptomics of Legacy and Emerging Brominated Flame Retardants following 5-Day Exposure in the Rat" [1]. * polybrominated diphenyl ether 47 (PBDE 47), decabromodiphenyl ether (decaBDE), hexabromocyclododecane (HBCD); ** 2-ethylhexyl-2,3,4,5-tetrabromobenzoate (TBB); bis(2-ethylhexyl) tetrabromophthalate (TBPH); tetrabromobisphenol A-bis(2,3-dibromopropyl ether (TBBPA-DBPE); 1,2-bis(tribromophenoxy)ethane (BTBPE); decabromodiphenylethane (DBDPE); hexachlorocyclopentadienyl-dibromocyclooctane (HCDBCO).
ABSTRACT
AV7909 is a next-generation anthrax vaccine candidate indicated for post-exposure prophylaxis of exposure to Bacillus anthracis. AV7909 consists of the Anthrax Vaccine Adsorbed (AVA) bulk drug substance and the immunostimulatory Toll-like receptor 9 agonist oligodeoxynucleotide adjuvant, CPG 7909. Safety testing for pediatric population is warranted to support the potential emergency use of AV7909 in children. This study was conducted to investigate the local tolerance and potential systemic toxicity and their reversibility in juvenile rats by repeat intramuscular injections of the AV7909 vaccine candidate. Animals were dosed on postnatal day (PND) 21 (at weaning), PND 28, and PND 35, with the test article (AV7909), the adjuvant alone (Alhydrogel + CPG 7909), or sterile water for injection. Core group animals were necropsied on PND 37 and recovery group on PND 49. Study end points included survival, clinical observations, injection site observations, body weights, clinical pathology (hematology, coagulation, and clinical chemistry), pro-inflammatory biomarker analysis (alpha-2 macroglobulin [A2M] and alpha-1 acid glycoprotein [AGP]), and anatomic pathology. Immune response to vaccination was measured using the high-throughput anthrax lethal toxin neutralization assay (htpTNA). The AV7909 vaccine candidate produced no apparent systemic or local toxicity. The AGP and A2M levels were elevated in both the adjuvant-alone and AV7909 groups at the end of treatment but were comparable to control levels by the end of the recovery period. All animals in the AV7909 group demonstrated a robust neutralizing antibody response. The results indicate that AV7909 has a favorable safety profile in juvenile rats.
ABSTRACT
The relative toxicity of three legacy and six emerging brominated flame retardants* was studied in the male Harlan Sprague Dawley rat. The hepatocellular and thyroid toxicity of each flame retardant was evaluated following five-day exposure to each of the nine flame retardants (oral gavage in corn oil) at 0.1-1000⯵mol/kg body weight per day. Histopathology and transcriptomic analysis were performed on the left liver lobe. Centrilobular hypertrophy of hepatocytes and increases in liver weight were seen following exposure to two legacy (PBDE-47, HBCD) and to one emerging flame retardant (HCDBCO). Total thyroxine (TT4) concentrations were reduced to the greatest extent after PBDE-47 exposure. The PBDE-47, decaBDE, and HBCD liver transcriptomes were characterized by upregulation of liver disease-related and/or metabolic transcripts. Fewer liver disease or metabolic transcript changes were detected for the other flame retardants studied (TBB, TBPH, TBBPA-DBPE, BTBPE, DBDPE, or HCDBCO). PBDE-47 exhibited the most disruption of hepatocellular toxic endpoints, with the Nrf2 antioxidant pathway transcripts upregulated to the greatest extent, although some activation of this pathway also occurred after decaBDE, HBCD, TBB, and HCBCO exposure. These studies provide information that can be used for prioritizing the need for more in-depth brominated flame retardant toxicity studies.
Subject(s)
Chemical and Drug Induced Liver Injury/pathology , Flame Retardants/toxicity , Hydrocarbons, Brominated/toxicity , Liver/metabolism , Transcriptome/drug effects , Animals , Cell Size/drug effects , Environmental Monitoring , Hepatocytes/drug effects , Liver/drug effects , Liver/pathology , Male , Organ Size/drug effects , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship , Thyroid Diseases/chemically induced , Thyroid Diseases/pathology , Thyroxine/metabolism , ToxicogeneticsABSTRACT
JD5037 is a novel peripherally restricted CB1 receptor (CB1R) inverse agonist being developed for the treatment of visceral obesity and its metabolic complications, including nonalcoholic fatty liver disease and dyslipidemia. JD5037 was administered by oral gavage at 10, 40, and 150â¯mg/kg/day dose levels for up to 34 days to Sprague Dawley rats, and at 5, 20, and 75â¯mg/kg/day dose levels for 28 consecutive days to Beagle dogs. In rats, higher incidences of stereotypic behaviors were observed in 10â¯mg/kg females and 40â¯mg/kg males, and slower responses for reflex and sensory tests were observed only in males at 10 and 40â¯mg/kg during neurobehavioral testing. Sporadic minimal incidences of decreased activity (males) and seizures (both sexes) were observed in rats during daily clinical observations, without any clear dose-relationship. Male dogs at 75â¯mg/kg during treatment period, but not recovery period, had an increased incidence of gut associated lymphoid tissue hyperplasia and inflammation in the intestine. In both species, highest dose resulted in lower AUCs indicative of non-linear kinetics. Free access to food increased the plasma AUC∞ by ~4.5-fold at 20â¯mg/kg in dogs, suggesting presence of food may help in systemic absorption of JD5037 in dogs. Based on the study results, 150â¯mg/kg/day in rats, and 20 and 75â¯mg/kg/day doses in male and female dogs, respectively, were determined to be the no-observed-adverse-effect-levels (NOAELs).
Subject(s)
Drugs, Investigational/toxicity , Pyrazoles/toxicity , Receptor, Cannabinoid, CB1/agonists , Seizures/chemically induced , Stereotyped Behavior/drug effects , Sulfonamides/toxicity , Animals , Area Under Curve , Behavior, Animal/drug effects , Disease Models, Animal , Dogs , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Drugs, Investigational/therapeutic use , Female , Humans , Investigational New Drug Application , Male , No-Observed-Adverse-Effect Level , Non-alcoholic Fatty Liver Disease/drug therapy , Pyrazoles/pharmacokinetics , Pyrazoles/therapeutic use , Rats , Rats, Sprague-Dawley , Sex Factors , Sulfonamides/pharmacokinetics , Sulfonamides/therapeutic useABSTRACT
There is a need for fast, efficient, and cost-effective hazard identification and characterization of chemical hazards. This need is generating increased interest in the use of zebrafish embryos as both a screening tool and an alternative to mammalian test methods. A Collaborative Workshop on Aquatic Models and 21st Century Toxicology identified the lack of appropriate and consistent testing protocols as a challenge to the broader application of the zebrafish embryo model. The National Toxicology Program established the Systematic Evaluation of the Application of Zebrafish in Toxicology (SEAZIT) initiative to address the lack of consistent testing guidelines and identify sources of variability for zebrafish-based assays. This report summarizes initial SEAZIT information-gathering efforts. Investigators in academic, government, and industry laboratories that routinely use zebrafish embryos for chemical toxicity testing were asked about their husbandry practices and standard protocols. Information was collected about protocol components including zebrafish strains, feed, system water, disease surveillance, embryo exposure conditions, and endpoints. Literature was reviewed to assess issues raised by the investigators. Interviews revealed substantial variability across design parameters, data collected, and analysis procedures. The presence of the chorion and renewal of exposure media (static versus static-renewal) were identified as design parameters that could potentially influence study outcomes and should be investigated further with studies to determine chemical uptake from treatment solution into embryos. The information gathered in this effort provides a basis for future SEAZIT activities to promote more consistent practices among researchers using zebrafish embryos for toxicity evaluation.
Subject(s)
Embryo, Nonmammalian , Toxicity Tests/methods , Zebrafish/embryology , Animals , Chorion/metabolism , Drug Evaluation, Preclinical/methods , Embryonic Development/drug effects , High-Throughput Screening AssaysABSTRACT
The hallmark of the extended one-generation reproductive toxicity study (EOGRTS) is that, based on certain criteria or triggers, selected offspring are assigned at weaning to different cohorts for further investigation of sexual maturation, reproductive organ integrity and function, neuropathological and behavioral endpoints, and/or immune function. The triggers allow for a more customizable design based directly on the data, while minimizing animal usage. Compared to the two-generation reproductive toxicity study, the EOGRTS design increases the number, extent, and duration of F1-offspring assessments resulting in more thorough and efficient utilization of the first generation while excluding the second generation of offspring unless triggered. Therefore, the EOGRTS has the potential to reduce the number of rats required by nearly 1200 animals per study. When performing the EOGRTS, the complexity of this study should not be underestimated and experienced flexible testing laboratories with sufficient resources and historical control data for all parameters are essential. The aim of this review is to discuss the important aspects of this challenging study design and to share our knowledge on the implementation of this study in our laboratories. In addition, we elaborate on the type of criteria for expansion of the study and logistical considerations. Altogether, this review can be used as guidance by other labs, study monitors, and registration officers.
Subject(s)
Reproduction/drug effects , Toxicity Tests/methods , Animals , Cohort Studies , European Union , Government Regulation , Guidelines as Topic , Rats , Toxicity Tests/standardsABSTRACT
To assess the efficacy of reproduction/developmental screening studies (OECD 421 and 422), a retrospective evaluation of 134 studies was performed. The major findings were: (1) for up to half of the studies with developmental and reproductive toxicity, these effects would have been missed in other types of studies, which underscores that reproduction/developmental screening studies should not be waived by default based on negative 28-day and/or prenatal developmental data, (2) the required number of animals as stated in the guidelines, is appropriate for detecting developmental and reproductive toxicity, and (3) adding measurements like anogenital distance, internal sex determination and nipple retention, plus extending the postnatal period would add predictive value. Overall, the current reproduction/developmental screening studies are effective in providing unique data, especially considering the limited number of animals used. Some simple additions would enrich its value in risk assessment even further.
Subject(s)
Abnormalities, Drug-Induced , Hazardous Substances/toxicity , Reproduction/drug effects , Toxicity Tests/methods , Animals , Female , Guidelines as Topic , Male , Pregnancy , Prenatal Exposure Delayed Effects , Rats , Toxicity Tests/standardsABSTRACT
Developmental neurotoxicity (DNT) testing assesses potentially adverse effects on the developing nervous system. The present DNT study was conducted to generate historical data with the Wistar Han (WH) and Sprague Dawley (SD) rat strains, commonly used in Europe and the US, respectively. Potential differences between these strains in DNT endpoints have not been extensively investigated. Motor activity, startle response, learning and memory testing, and neurological (quantitative and qualitative) examinations were conducted using three groups of control, prenatally exposed (to Methylazoxymethanol [MAM] on gestation Day 15) and acutely treated (with IDPN, MK-801 or Chlorpromazine) animals for each strain. The positive controls produced clear effects in most endpoints investigated, with limited functional differences in baseline behavior and positive control sensitivity. However, SD rats were considerably more susceptible to MAM-induced learning and memory impairments and neurological damage. These data highlight differential sensitivity between the strains, which may require risk assessment consideration for developmental neurotoxicants.
Subject(s)
Behavior, Animal/drug effects , Control Groups , Neurotoxins/toxicity , Animals , Brain/drug effects , Brain/pathology , Chlorpromazine/toxicity , Dizocilpine Maleate/toxicity , Female , Learning/drug effects , Male , Memory/drug effects , Methylazoxymethanol Acetate/analogs & derivatives , Methylazoxymethanol Acetate/toxicity , Motor Activity , Neurotoxicity Syndromes/etiology , Neurotoxicity Syndromes/pathology , Neurotoxicity Syndromes/physiopathology , Nitriles/toxicity , Pregnancy , Prenatal Exposure Delayed Effects , Rats , Rats, Sprague-Dawley , Rats, Wistar , Reflex, Startle/drug effects , Species SpecificityABSTRACT
An extended one-generation study is proposed to replace the standard two-generation study, and would eliminate breeding F1 animals unless triggered by effects on parental reproduction or pup development. Nine two-generation studies in rats were reviewed to determine whether reproductive or developmental toxicity was more pronounced in the F2 generation. Three studies lacked reproductive effects, one had reproductive toxicity only in parental animals, three had mostly equivalent effects between generations, and two showed greater toxicity in the second generation. In each study with relevant effects, criteria proposed by Cooper et al. [Cooper RL, Lamb IV JC, Barlow SM, Bently K, Brady AM, Doerrer NG, et al. A tiered approach to life stages testing for agricultural chemical safety assessment. Crit Rev Toxicol 2006;36:69-98.] were applied to determine whether additional breeding would be triggered in the extended one-generation design. Additional mating was triggered for all but one study with more pronounced F1 reproductive toxicity. Thus, while the extended one-generation design may be a useful substitution for the two-generation study, more stringent criteria must first be developed to determine whether additional mating is actually needed.
Subject(s)
Copulation/drug effects , Reproduction/drug effects , Research Design/statistics & numerical data , Toxicity Tests/methods , Xenobiotics/toxicity , Administration, Oral , Animal Feed , Animals , Female , Male , No-Observed-Adverse-Effect Level , Rats , Rats, Wistar , Retrospective StudiesABSTRACT
The basal forebrain cortical cholinergic system (BFCS) is critical for the regulation of attentional information processing. BFCS activity is regulated by several cortical and subcortical structures, including the nucleus accumbens (NAC) and prefrontal cortex (PFC). GABAergic projection neurons from NAC to basal forebrain are modulated by Glu receptors within NAC. We previously reported that intra-NAC perfusions of NMDA or its antagonist CPP stimulate ACh release in PFC. In this experiment we determined whether this trans-synaptic modulation of cortical ACh release is evident in multi-sensory associational areas like the posterior parietal cortex (PPC). Artificial cerebrospinal fluid (aCSF, control), NMDA (250 or 400 muM), or CPP (200 or 400 muM) were perfused into the NAC shell and ACh was measured in the ipsilateral PPC. Amphetamine (2.0 mg/kg, i.p), was systemically administered as a positive control in a fourth session, since it also stimulates cortical ACh release but via mechanisms known to not necessitate transmission within the NAC. Neither NMDA nor CPP increased ACh efflux in the PPC, yet both drugs increased ACh release in PFC, suggesting that NMDA receptor modulation in the NAC increases ACh in the cortex in a regionally-specific manner. Systemic amphetamine administration significantly increased (100-200%) ACh in the PPC, suggesting that levels of ACh in the PPC can be increased following certain pharmacological manipulations. The cortical region-specific modulation of ACh by NAC may underlie the linkage of motivational information with top-down controls of attention as well as guide appropriate motor output following exposure to salient and behaviorally relevant stimuli.
Subject(s)
Acetylcholine/metabolism , Basal Nucleus of Meynert/metabolism , Cerebral Cortex/metabolism , Neural Pathways/metabolism , Nucleus Accumbens/metabolism , Receptors, Glutamate/metabolism , Amphetamine/pharmacology , Animals , Basal Nucleus of Meynert/drug effects , Cerebral Cortex/drug effects , Cholinergic Fibers/drug effects , Cholinergic Fibers/metabolism , Dopamine Uptake Inhibitors/pharmacology , Excitatory Amino Acid Agonists/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , Extracellular Fluid/metabolism , Glutamic Acid/metabolism , Male , Microdialysis , Neural Pathways/drug effects , Nucleus Accumbens/drug effects , Parietal Lobe/drug effects , Parietal Lobe/metabolism , Rats , Rats, Wistar , Receptors, Glutamate/drug effects , Synaptic Transmission/drug effects , Synaptic Transmission/physiologyABSTRACT
Microdialysis has been widely used to measure acetylcholine (ACh) release in vivo and has provided important insights into the regulation of cholinergic transmission. However, microdialysis can be constrained by limited spatial and temporal resolution. The present experiments utilize a microelectrode array (MEA) to rapidly measure ACh release and clearance in anaesthetized rats. The array electrochemically detects, on a second-by-second basis, changes in current selectively produced by the hydrolysis of ACh to choline (Ch) and the subsequent oxidation of choline and hydrogen peroxidase (H(2)O(2)) at the electrode surface. In vitro calibration of the microelectrode revealed linear responses to ACh (R(2) = 0.9998), limit of detection of 0.08 microm, and signal-to-noise ratio of 3.0. The electrode was unresponsive to ascorbic acid (AA), dopamine (DA), or norepinephrine (NE) interferents. In vivo experiments were conducted in prefrontal cortex (PFC) of anaesthetized rats. Pressure ejections of ACh (10 mm; 40 nL) through an adjoining micropipette produced a rapid rise in current, reaching maximum amplitude in approximately 1.0 s and cleared by 80% within 4-11 s. Endogenously released ACh, following local depolarization with KCl (70 mm; 40, 160 nL), was detected at values as low as 0.05 microm. These signals were volume-dependent and cleared within 4-12 s. Finally, nicotine (1.0 mm, 80 nL) stimulated ACh signals. Nicotine-induced signals reflected the hydrolysis of ACh by endogenous acetylcholinesterase (AChE) as inhibition of the enzyme following perfusion with neostigmine (10 microm) attenuated the signal (40-94%). Collectively, these data validate a novel method for rapidly measuring cholinergic transmission in vivo with a spatial and temporal resolution that far exceeds conventional microdialysis.
Subject(s)
Acetylcholine/metabolism , Prefrontal Cortex/metabolism , Analysis of Variance , Animals , Chromatography, High Pressure Liquid/methods , Electrochemistry/methods , Extracellular Space/metabolism , Male , Microdialysis/methods , Microelectrodes , Nicotine/pharmacology , Nicotinic Agonists/pharmacology , Prefrontal Cortex/drug effects , Rats , Rats, WistarABSTRACT
The nucleus accumbens (NAC) plays a key role in directing appropriate motor output following the presentation of behaviorally relevant stimuli. As such, we postulate that accumbens efferents also participate in the modulation of neuronal circuits regulating attentional processes directed toward the identification and selection of these stimuli. In this study, N-methyl-d-aspartate (NMDA) and D1 ligands were perfused into the shell region of the NAC of awake rats. Cortical cholinergic transmission, a mediator of attentional processes, was measured via microdialysis probes inserted into the prefrontal cortex (PFC). NMDA perfusions (150 or 250 microm) into NAC resulted in significant increases in acetylcholine (ACh) efflux in PFC (150-200% above baseline levels). Co-administration of the D1 antagonist SCH-23390 (150 microm) markedly attenuated (by approx. 70%) ACh efflux following perfusions of 150 microm NMDA but not following 250 microm NMDA, suggesting that D1 receptor activity contributes to the ability of the lower but not the higher concentration of NMDA to increase cortical ACh release. Collectively, these data reveal a positive modulation of NMDA receptors by D1 receptors in NAC that is expressed trans-synaptically at the level of cortical transmission. This modulation may underlie the coordinated linking of attentional processes and motor output following exposure to salient and behaviorally relevant stimuli.