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DOE OSTI2013

Detection of genotoxic and non-genotoxic carcinogens in Xpc{sup −/−}p53{sup +/−} mice

An accurate assessment of the carcinogenic potential of chemicals and pharmaceutical drugs is essential to protect humans and the environment. Therefore, substances are extensively tested before they are marketed to the public. Currently, the rodent two-year bioassay is still routinely used to assess the carcinogenic potential of substances. However, over time it has become clear that this assay yields false positive results and also has several economic and ethical drawbacks including the use of large numbers of animals, the long duration, and the high cost. The need for a suitable alternative assay is therefore high. Previously, we have proposed the Xpa*p53 mouse model as a very suitable alternative to the two-year bioassay. We now show that the Xpc*p53 mouse model preserves all the beneficial traits of the Xpa*p53 model for sub-chronic carcinogen identification and can identify both genotoxic and non-genotoxic carcinogens. Moreover, Xpc*p53 mice appear to be more responsive than Xpa*p53 mice towards several genotoxic and non-genotoxic carcinogens. Furthermore, Xpc*p53 mice are far less sensitive than Xpa*p53 mice for the toxic activity of DNA damaging agents and as such clearly respond in a similar way as wild type mice do. These advantageous traits of the Xpc*p53 model make it a better alternative for in vivo carcinogen testing than Xpa*p53. This pilot study suggests that Xpc*p53 mice are suited for routine sub-chronic testing of both genotoxic and non-genotoxic carcinogens and as such represent a suitable alternative to possibly replace the murine life time cancer bioassay. Highlights: ► The Xpc*p53 mouse model is able to identify genotoxic and non-genotoxic carcinogens. ► Time, animals and cost can be significantly reduced compared to the 2-year bioassay. ► Xpc*p53 mice are more advantageous for carcinogen identification than Xpa*p53 mice. ► Xpc*p53 mice exhibit a wild type response upon exposure to genotoxicants.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2013

Acetaminophen-induced acute liver injury in HCV transgenic mice

The exact etiology of clinical cases of acute liver failure is difficult to ascertain and it is likely that various co-morbidity factors play a role. For example, epidemiological evidence suggests that coexistent hepatitis C virus (HCV) infection increased the risk of acetaminophen-induced acute liver injury, and was associated with an increased risk of progression to acute liver failure. However, little is known about possible mechanisms of enhanced acetaminophen hepatotoxicity in HCV-infected subjects. In this study, we tested a hypothesis that HCV-Tg mice may be more susceptible to acetaminophen hepatotoxicity, and also evaluated the mechanisms of acetaminophen-induced liver damage in wild type and HCV-Tg mice expressing core, E1 and E2 proteins. Male mice were treated with a single dose of acetaminophen (300 or 500 mg/kg in fed animals; or 200 mg/kg in fasted animals; i.g.) and liver and serum endpoints were evaluated at 4 and 24 h after dosing. Our results suggest that in fed mice, liver toxicity in HCV-Tg mice is not markedly exaggerated as compared to the wild-type mice. In fasted mice, greater liver injury was observed in HCV-Tg mice. In fed mice dosed with 300 mg/kg acetaminophen, we observed that liver mitochondria in HCV-Tg mice exhibited signs of dysfunction showing the potential mechanism for increased susceptibility. -- Highlights: ► Acetaminophen-induced liver injury is a significant clinical challenge. ► HCV-infected subjects may be at higher risk for acetaminophen-induced liver injury. ► We used HCV transgenics to test if liver injury due to acetaminophen is exacerbated.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2013

A long-term three dimensional liver co-culture system for improved prediction of clinically relevant drug-induced hepatotoxicity

Drug-induced liver injury (DILI) is the major cause for liver failure and post-marketing drug withdrawals. Due to species-specific differences in hepatocellular function, animal experiments to assess potential liabilities of drug candidates can predict hepatotoxicity in humans only to a certain extent. In addition to animal experimentation, primary hepatocytes from rat or human are widely used for pre-clinical safety assessment. However, as many toxic responses in vivo are mediated by a complex interplay among different cell types and often require chronic drug exposures, the predictive performance of hepatocytes is very limited. Here, we established and characterized human and rat in vitro three-dimensional (3D) liver co-culture systems containing primary parenchymal and non-parenchymal hepatic cells. Our data demonstrate that cells cultured on a 3D scaffold have a preserved composition of hepatocytes, stellate, Kupffer and endothelial cells and maintain liver function for up to 3 months, as measured by the production of albumin, fibrinogen, transferrin and urea. Additionally, 3D liver co-cultures maintain cytochrome P450 inducibility, form bile canaliculi-like structures and respond to inflammatory stimuli. Upon incubation with selected hepatotoxicants including drugs which have been shown to induce idiosyncratic toxicity, we demonstrated that this model better detected in vivo drug-induced toxicity, including species-specific drug effects, when compared to monolayer hepatocyte cultures. In conclusion, our results underline the importance of more complex and long lasting in vitro cell culture models that contain all liver cell types and allow repeated drug-treatments for detection of in vivo-relevant adverse drug effects. - Highlights: ► 3D liver co-cultures maintain liver specific functions for up to three months. ► Activities of Cytochrome P450s remain drug- inducible accross three months. ► 3D liver co-cultures recapitulate drug-induced liver toxicity observed in vivo. ► 3D liver co-cultures can detect species-specific drug toxicity observed in vivo. ► This in vitro model may improve assessment of human relevance of preclinical findings.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2013

In vivo application of a small molecular weight antifungal protein of Penicillium chrysogenum (PAF)

The antifungal protein of Penicillium chrysogenum (PAF) inhibits the growth of important pathogenic filamentous fungi, including members of the Aspergillus family and some dermatophytes. Furthermore, PAF was proven to have no toxic effects on mammalian cells in vitro. To prove that PAF could be safely used in therapy, experiments were carried out to investigate its in vivo effects. Adult mice were inoculated with PAF intranasally in different concentrations, up to 2700 μg·kg{sup −1} daily, for 2 weeks. Even at the highest concentration – a concentration highly toxic in vitro for all affected molds – used, animals neither died due to the treatment nor were any side effects observed. Histological examinations did not find pathological reactions in the liver, in the kidney, and in the lungs. Mass spectrometry confirmed that a measurable amount of PAF was accumulated in the lungs after the treatment. Lung tissue extracts from PAF treated mice exerted significant antifungal activity. Small-animal positron emission tomography revealed that neither the application of physiological saline nor that of PAF induced any inflammation while the positive control lipopolysaccharide did. The effect of the drug on the skin was examined in an irritative dermatitis model where the change in the thickness of the ears following PAF application was found to be the same as in control and significantly less than when treated with phorbol-12-myristate-13-acetate used as positive control. Since no toxic effects of PAF were found in intranasal application, our result is the first step for introducing PAF as potential antifungal drug in therapy. - Highlights: • PAF, the antifungal protein of Penicillium chrysogenum, was not toxic in mice. • Its intranasal application didn't induce pathological reactions in the lung. • PAF retained its antifungal activity in lung extracts. • Its application on the skin did not cause inflammation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2013

An integrated approach for prospectively investigating a mode-of-action for rodent liver effects

Registration of new plant protection products (e.g., herbicide, insecticide, or fungicide) requires comprehensive mammalian toxicity evaluation including carcinogenicity studies in two species. The outcome of the carcinogenicity testing has a significant bearing on the overall human health risk assessment of the substance and, consequently, approved uses for different crops across geographies. In order to understand the relevance of a specific tumor finding to human health, a systematic, transparent, and hypothesis-driven mode of action (MoA) investigation is, appropriately, an expectation by the regulatory agencies. Here, we describe a novel approach of prospectively generating the MoA data by implementing additional end points to the standard guideline toxicity studies with sulfoxaflor, a molecule in development. This proactive MoA approach results in a more robust integration of molecular with apical end points while minimizing animal use. Sulfoxaflor, a molecule targeting sap-feeding insects, induced liver effects (increased liver weight due to hepatocellular hypertrophy) in an initial palatability probe study for selecting doses for subsequent repeat-dose dietary studies. This finding triggered the inclusion of dose-response investigations of the potential key events for rodent liver carcinogenesis, concurrent with the hazard assessment studies. As predicted, sulfoxaflor induced liver tumors in rats and mice in the bioassays. The MoA data available by the time of the carcinogenicity finding supported the conclusion that the carcinogenic potential of sulfoxaflor was due to CAR/PXR nuclear receptor activation with subsequent hepatocellular proliferation. This MoA was not considered to be relevant to humans as sulfoxaflor is unlikely to induce hepatocellular proliferation in humans and therefore would not be a human liver carcinogen. - Highlights: • We prospectively generated MoA data into standard guideline toxicity studies. • A proactive MoA approach integrates all end points while minimizing animal use. • MoA data predicted the rodent carcinogenicity of sulfoxaflor via CAR/PXR. • Liver MoA was considered not relevant to humans and hence not a human carcinogen.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2013

Tissue factor pathway inhibitor prevents airway obstruction, respiratory failure and death due to sulfur mustard analog inhalation

Sulfur mustard (SM) inhalation causes airway injury, with enhanced vascular permeability, coagulation, and airway obstruction. The objective of this study was to determine whether recombinant tissue factor pathway inhibitor (TFPI) could inhibit this pathogenic sequence. Methods: Rats were exposed to the SM analog 2-chloroethyl ethyl sulfide (CEES) via nose-only aerosol inhalation. One hour later, TFPI (1.5 mg/kg) in vehicle, or vehicle alone, was instilled into the trachea. Arterial O{sub 2} saturation was monitored using pulse oximetry. Twelve hours after exposure, animals were euthanized and bronchoalveolar lavage fluid (BALF) and plasma were analyzed for prothrombin, thrombin–antithrombin complex (TAT), active plasminogen activator inhibitor-1 (PAI-1) levels, and fluid fibrinolytic capacity. Lung steady-state PAI-1 mRNA was measured by RT-PCR analysis. Airway-capillary leak was estimated by BALF protein and IgM, and by pleural fluid measurement. In additional animals, airway cast formation was assessed by microdissection and immunohistochemical detection of airway fibrin. Results: Airway obstruction in the form of fibrin-containing casts was evident in central conducting airways of rats receiving CEES. TFPI decreased cast formation, and limited severe hypoxemia. Findings of reduced prothrombin consumption, and lower TAT complexes in BALF, demonstrated that TFPI acted to limit thrombin activation in airways. TFPI, however, did not appreciably affect CEES-induced airway protein leak, PAI-1 mRNA induction, or inhibition of the fibrinolytic activity present in airway surface liquid. Conclusions: Intratracheal administration of TFPI limits airway obstruction, improves gas exchange, and prevents mortality in rats with sulfur mustard-analog-induced acute lung injury. - Highlights: • TFPI administration to rats after mustard inhalation reduces airway cast formation. • Inhibition of thrombin activation is the likely mechanism for limiting casts. • Rats given TFPI had improved tissue oxygenation, and mortality was prevented.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2013

Phycocyanobilin promotes PC12 cell survival and modulates immune and inflammatory genes and oxidative stress markers in acute cerebral hypoperfusion in rats

Since the inflammatory response and oxidative stress are involved in the stroke cascade, we evaluated here the effects of Phycocyanobilin (PCB, the C-Phycocyanin linked tetrapyrrole) on PC12 cell survival, the gene expression and the oxidative status of hypoperfused rat brain. After the permanent bilateral common carotid arteries occlusion (BCCAo), the animals were treated with saline or PCB, taking samples 24 h post-surgery. Global gene expression was analyzed with GeneChip Rat Gene ST 1.1 from Affymetrix; the expression of particular genes was assessed by the Fast SYBR Green RT-PCR Master Mix and Bioplex methods; and redox markers (MDA, PP, CAT, SOD) were evaluated spectrophotometrically. The PCB treatment prevented the H{sub 2}O{sub 2} and glutamate induced PC12 cell injury assessed by the MTT assay, and modulated 190 genes (93 up- and 97 down-regulated) associated to several immunological and inflammatory processes in BCCAo rats. Furthermore, PCB positively modulated 19 genes mostly related to a detrimental pro-inflammatory environment and counteracted the oxidative imbalance in the treated BCCAo animals. Our results support the view of an effective influence of PCB on major inflammatory mediators in acute cerebral hypoperfusion. These results suggest that PCB has a potential to be a treatment for ischemic stroke for which further studies are needed. - Highlights: • Phycocyanobilin (PCB) prevents H{sub 2}O{sub 2} and glutamate induced PC12 cell viability loss. • Anterior cortex and striatum are highly vulnerable to cerebral hypoperfusion (CH). • PCB modulates 190 genes associated to inflammation in acute CH. • PCB regulates 19 genes mostly related to a detrimental pro-inflammatory environment. • PCB restores redox and immune balances showing promise as potential stroke therapy.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2013

Sex-specific differences in hyperoxic lung injury in mice: Implications for acute and chronic lung disease in humans

Sex-specific differences in pulmonary morbidity in humans are well documented. Hyperoxia contributes to lung injury in experimental animals and humans. The mechanisms responsible for sex differences in the susceptibility towards hyperoxic lung injury remain largely unknown. In this investigation, we tested the hypothesis that mice will display sex-specific differences in hyperoxic lung injury. Eight week-old male and female mice (C57BL/6J) were exposed to 72 h of hyperoxia (FiO{sub 2} > 0.95). After exposure to hyperoxia, lung injury, levels of 8-iso-prostaglandin F{sub 2} alpha (8-iso-PGF 2α) (LC–MS/MS), apoptosis (TUNEL) and inflammatory markers (suspension bead array) were determined. Cytochrome P450 (CYP)1A expression in the lung was assessed using immunohistochemistry and western blotting. After exposure to hyperoxia, males showed greater lung injury, neutrophil infiltration and apoptosis, compared to air-breathing controls than females. Pulmonary 8-iso-PGF 2α levels were higher in males than females after hyperoxia exposure. Sexually dimorphic increases in levels of IL-6 (F > M) and VEGF (M > F) in the lungs were also observed. CYP1A1 expression in the lung was higher in female mice compared to males under hyperoxic conditions. Overall, our results support the hypothesis that male mice are more susceptible than females to hyperoxic lung injury and that differences in inflammatory and oxidative stress markers contribute to these sex-specific dimorphic effects. In conclusion, this paper describes the establishment of an animal model that shows sex differences in hyperoxic lung injury in a temporal manner and thus has important implications for lung diseases mediated by hyperoxia in humans. - Highlights: • Male mice were more susceptible to hyperoxic lung injury than females. • Sex differences in inflammatory markers were observed. • CYP1A expression was higher in females after hyperoxia exposure.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2013

DNA damage and apoptosis of endometrial cells cause loss of the early embryo in mice exposed to carbon disulfide

Carbon disulfide (CS{sub 2}) may lead to spontaneous abortion and very early pregnancy loss in women exposed in the workplace, but the mechanism remains unclear. We designed an animal model in which gestating Kunming strain mice were exposed to CS{sub 2} via i.p. on gestational day 4 (GD4). We found that the number of implanted blastocysts on GD8 was significantly reduced by each dose of 0.1 LD{sub 50} (157.85 mg/kg), 0.2 LD{sub 50} (315.7 mg/kg) and 0.4 LD{sub 50} (631.4 mg/kg). In addition, both the level of DNA damage and apoptosis rates of endometrial cells on GD4.5 were increased, showed definite dose–response relationships, and inversely related to the number of implanted blastocysts. The expressions of mRNA and protein for the Bax and caspase-3 genes in the uterine tissues on GD4.5 were up-regulated, while the expressions of mRNA and protein for the Bcl-2 gene were dose-dependently down-regulated. Our results indicated that DNA damage and apoptosis of endometrial cells were important reasons for the loss of implanted blastocysts induced by CS{sub 2}. - Highlights: • We built an animal model of CS2 exposure during blastocyst implantation. • Endometrial cells were used in the comet assay to detect DNA damage. • CS2 exposure caused DNA damage and endometrial cell apoptosis. • DNA damage and endometrial cell apoptosis were responsible for embryo loss.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Accumulation of lipids and oxidatively damaged DNA in hepatocytes exposed to particles

Exposure to particles has been suggested to generate hepatosteatosis by oxidative stress mechanisms. We investigated lipid accumulation in cultured human hepatocytes (HepG2) and rat liver after exposure to four different carbon-based particles. HepG2 cells were exposed to particles for 3 h and subsequently incubated for another 18 h to manifest lipid accumulation. In an animal model of metabolic syndrome we investigated the association between intake of carbon black (CB, 14 nm) particles and hepatic lipid accumulation, inflammation and gene expression of Srebp-1, Fasn and Scd-1 involved in lipid synthesis. There was a concentration-dependent increase in intracellular lipid content after exposure to CB in HepG2 cells, which was only observed after co-exposure to oleic/palmitic acid. Similar results were observed in HepG2 cells after exposure to diesel exhaust particles, fullerenes C{sub 60} or pristine single-walled carbon nanotubes. All four types of particles also generated oxidatively damaged DNA, assessed as formamidopyrimidine DNA glycosylase (FPG) sensitive sites, in HepG2 cells after 3 h exposure. The animal model of metabolic syndrome showed increased lipid load in the liver after one oral exposure to 6.4 mg/kg of CB in lean Zucker rats. This was not associated with increased iNOS staining in the liver, indicating that the oral CB exposure was associated with hepatic steatosis rather than steatohepatitis. The lipid accumulation did not seem to be related to increased lipogenesis because there were unaltered gene expression levels in both the HepG2 cells and rat livers. Collectively, exposure to particles is associated with oxidative stress and steatosis in hepatocytes. - Highlights: • Oral exposure to nanosized carbon black was associated with hepatosteatosis in rats. • In vitro studies included carbon black, C{sub 60}, diesel exhaust particles and SWCNTs. • Exposure to particles and free fatty acids increased lipid load in HepG2 cells. • Unaltered expression of lipogenesis genes despite oxidative stress in hepatocytes • Particles evoke hepatosteatosis by increased uptake rather than synthesis of lipids.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Developmental defects in zebrafish for classification of EGF pathway inhibitors

One of the major challenges when testing drug candidates targeted at a specific pathway in whole animals is the discrimination between specific effects and unwanted, off-target effects. Here we used the zebrafish to define several developmental defects caused by impairment of Egf signaling, a major pathway of interest in tumor biology. We inactivated Egf signaling by genetically blocking Egf expression or using specific inhibitors of the Egf receptor function. We show that the combined occurrence of defects in cartilage formation, disturbance of blood flow in the trunk and a decrease of myelin basic protein expression represent good indicators for impairment of Egf signaling. Finally, we present a classification of known tyrosine kinase inhibitors according to their specificity for the Egf pathway. In conclusion, we show that developmental indicators can help to discriminate between specific effects on the target pathway from off-target effects in molecularly targeted drug screening experiments in whole animal systems. - Highlights: • We analyze the functions of Egf signaling on zebrafish development. • Genetic blocking of Egf expression causes cartilage, myelin and circulatory defects. • Chemical inhibition of Egf receptor function causes similar defects. • Developmental defects can reveal the specificity of Egf pathway inhibitors.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Model-based analysis of thromboxane B{sub 2} and prostaglandin E{sub 2} as biomarkers in the safety evaluation of naproxen

The assessment of safety in traditional toxicology protocols relies on evidence arising from observed adverse events (AEs) in animals and on establishing their correlation with different measures of drug exposure (e.g., C{sub max} and AUC). Such correlations, however, ignore the role of biomarkers, which can provide further insight into the underlying pharmacological mechanisms. Here we use naproxen as a paradigm drug to explore the feasibility of a biomarker-guided approach for the prediction of AEs in humans. A standard toxicology protocol was set up for the evaluation of effects of naproxen in rat, in which four doses were tested (7.5, 15, 40 and 80 mg/kg). In addition to sparse blood sampling for the assessment of exposure, thromboxane B{sub 2} and prostaglandin E{sub 2} were also collected in satellite groups. Nonlinear mixed effects modelling was used to evaluate the predictive performance of the approach. A one-compartmental model with first order absorption was found to best describe the pharmacokinetics of naproxen. A nonlinear relationship between dose and bioavailability was observed which leads to a less than proportional increase in naproxen concentrations with increasing doses. The pharmacodynamics of TXB{sub 2} and PGE{sub 2} was described by direct inhibition models with maximum pharmacological effects achieved at doses > 7.5 mg/kg. The predicted PKPD relationship in humans was within 10-fold of the values previously published. Moreover, our results indicate that biomarkers can be used to assess interspecies differences in PKPD and extrapolated data from animals to humans. Biomarker sampling should be used systematically in general toxicity studies. - Highlights: • Prediction of a drug's safety profile from preclinical protocols remains challenging. • Pharmacokinetic measures of safe exposure (e.g., AUC) ignore the role of biomarkers. • PKPD relationships enable the evaluation of adverse events in a mechanistic manner. • Major differences exist between rats and humans in the effects of naproxen on TXB{sub 2}. • A biomarker-guided approach may facilitate the prediction of adverse events in humans.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Downregulation of immediate-early genes linking to suppression of neuronal plasticity in rats after 28-day exposure to glycidol

We previously found that the 28-day oral toxicity study of glycidol at 200 mg/kg/day in rats resulted in axonopathy in both the central and peripheral nervous systems and aberrations in the late-stage of hippocampal neurogenesis targeting the process of neurite extension. To capture the neuronal parameters in response to glycidol toxicity, these animals were subjected to region-specific global gene expression profiling in four regions of cerebral and cerebellar architectures, followed by immunohistochemical analysis of selected gene products. Expression changes of genes related to axonogenesis and synaptic transmission were observed in the hippocampal dentate gyrus, cingulate cortex and cerebellar vermis at 200 mg/kg showing downregulation in most genes. In the corpus callosum, genes related to growth, survival and functions of glial cells fluctuated their expression. Immunohistochemically, neurons expressing gene products of immediate-early genes, i.e., Arc, Fos and Jun, decreased in their number in the dentate granule cell layer, cingulate cortex and cerebellar vermis. We also applied immunohistochemical analysis in rat offspring after developmental exposure to glycidol through maternal drinking water. The results revealed increases of Arc{sup +} neurons at 1000 ppm and Fos{sup +} neurons at ≥ 300 ppm in the dentate granule cell layer of offspring only at the adult stage. These results suggest that glycidol suppressed neuronal plasticity in the brain after 28-day exposure to young adult animals, in contrast to the operation of restoration mechanism to increase neuronal plasticity at the adult stage in response to aberrations in neurogenesis after developmental exposure. - Highlights: • Neuronal toxicity parameters after 28-day glycidol treatment were examined in rats. • Region-specific global gene expression profiling was conducted in brain regions. • Cortical tissues downregulated genes on axonogenesis and synaptic transmission. • Cortical tissues decreased immunoreactive neurons for Arc, Fos or Jun. • The results suggest that 28-day glycidol treatment suppressed neuronal plasticity.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

In vivo doses of butadiene epoxides as estimated from in vitro enzyme kinetics by using cob(I)alamin and measured hemoglobin adducts: An inter-species extrapolation approach

1,3-Butadiene (BD) is a rodent and human carcinogen. In the cancer tests, mice have been much more susceptible than rats with regard to BD-induced carcinogenicity. The species-differences are dependent on metabolic formation/disappearance of the genotoxic BD epoxy-metabolites that lead to variations in the respective in vivo doses, i.e. “area under the concentration-time curve” (AUC). Differences in AUC of the most gentoxic BD epoxy-metabolite, diepoxybutane (DEB), are considered important with regard to cancer susceptibility. The present work describes: the application of cob(I)alamin for accurate measurements of in vitro enzyme kinetic parameters associated with BD epoxy-metabolites in human, mouse and rat; the use of published data on hemoglobin (Hb) adduct levels of BD epoxides from BD exposure studies on the three species to calculate the corresponding AUCs in blood; and a parallelogram approach for extrapolation of AUC of DEB based on the in vitro metabolism studies and adduct data from in vivo measurements. The predicted value of AUC of DEB for humans from the parallelogram approach was 0.078 nM · h for 1 ppm · h of BD exposure compared to 0.023 nM · h/ppm · h as calculated from Hb adduct levels observed in occupational exposure. The corresponding values in nM · h/ppm · h were for mice 41 vs. 38 and for rats 1.26 vs. 1.37 from the parallelogram approach vs. experimental exposures, respectively, showing a good agreement. This quantitative inter-species extrapolation approach will be further explored for the clarification of metabolic rates/pharmacokinetics and the AUC of other genotoxic electrophilic compounds/metabolites, and has a potential to reduce and refine animal experiments. - Highlights: • In vitro metabolism to in vivo dose extrapolation of butadiene metabolites was proposed. • A parallelogram approach was introduced to estimate dose (AUC) in humans and rodents. • AUC of diepoxybutane predicted in humans was 0.078 nM h/ppm h butadiene exposure. • The presented approach has a potential to reduce, refine and replace animal experiments.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

A rat model of nerve agent exposure applicable to the pediatric population: The anticonvulsant efficacies of atropine and GluK1 antagonists

Inhibition of acetylcholinesterase (AChE) after nerve agent exposure induces status epilepticus (SE), which causes brain damage or death. The development of countermeasures appropriate for the pediatric population requires testing of anticonvulsant treatments in immature animals. In the present study, exposure of 21-day-old (P21) rats to different doses of soman, followed by probit analysis, produced an LD{sub 50} of 62 μg/kg. The onset of behaviorally-observed SE was accompanied by a dramatic decrease in brain AChE activity; rats who did not develop SE had significantly less reduction of AChE activity in the basolateral amygdala than rats who developed SE. Atropine sulfate (ATS) at 2 mg/kg, administered 20 min after soman exposure (1.2 × LD{sub 50}), terminated seizures. ATS at 0.5 mg/kg, given along with an oxime within 1 min after exposure, allowed testing of anticonvulsants at delayed time-points. The AMPA/GluK1 receptor antagonist LY293558, or the specific GluK1 antagonist UBP302, administered 1 h post-exposure, terminated SE. There were no degenerating neurons in soman-exposed P21 rats, but both the amygdala and the hippocampus were smaller than in control rats at 30 and 90 days post-exposure; this pathology was not present in rats treated with LY293558. Behavioral deficits present at 30 days post-exposure, were also prevented by LY293558 treatment. Thus, in immature animals, a single injection of atropine is sufficient to halt nerve agent-induced seizures, if administered timely. Testing anticonvulsants at delayed time-points requires early administration of ATS at a low dose, sufficient to counteract only peripheral toxicity. LY293558 administered 1 h post-exposure, prevents brain pathology and behavioral deficits. - Highlights: • The LD{sub 50} of soman was determined in postnatal-day-21 rats. • Rats with no seizures after 1.2XLD{sub 50} soman had less reduction of AChE in the amygdala. • Atropine sulfate (ATS) at 2 mg/kg, given at 20 min after soman, blocked seizures. • With ATS at 0.5 mg/kg, LY293558 or UBP302 at 1 h after exposure terminated seizures. • LY293558 prevented brain pathology and behavioral deficits.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Neuronal changes and oxidative stress in adolescent rats after repeated exposure to mephedrone

Mephedrone is a new designer drug of abuse. We have investigated the neurochemical/enzymatic changes after mephedrone administration to adolescent rats (3 × 25 mg/kg, s.c. in a day, with a 2 h interval between doses, for two days) at high ambient temperature (26 ± 2 °C), a schedule that intends to model human recreational abuse. In addition, we have studied the effect of mephedrone in spatial learning and memory. The drug caused a transient decrease in weight gain. After the first dose, animals showed hypothermia but, after the subsequent doses, temperature raised over the values of saline-treated group. We observed the development of tolerance to these thermoregulatory effects of mephedrone. Mephedrone induced a reduction of the densities of dopamine (30% in the frontal cortex) and serotonin (40% in the frontal cortex and the hippocampus and 48% in the striatum) transporters without microgliosis. These deficits were also accompanied by a parallel decrease in the expression of tyrosine hydroxylase and tryptophan hydroxylase 2. These changes matched with a down-regulation of D{sub 2} dopamine receptors in the striatum. Mephedrone also induced an oxidative stress evidenced by an increase of lipid peroxidation in the frontal cortex, and accompanied by a rise in glutathione peroxidase levels in all studied brain areas. Drug-treated animals displayed an impairment of the reference memory in the Morris water maze one week beyond the cessation of drug exposure, while the spatial learning process seems to be preserved. These findings raise concerns about the neuronal long-term effects of mephedrone. - Highlights: • We studied the dopaminergic and serotonergic neurotoxicity of mephedrone in rats. • Mephedrone induced a transient hypothermia following sustained hyperthermia. • In a weekend consumption pattern, mephedrone induced selective neurotoxicity. • Mephedrone generated oxidative stress. • Mephedrone induced an impairment in memory function.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Comparing probabilistic and descriptive analyses of time–dose–toxicity relationship for determining no-observed-adverse-effect level in drug development

The no-observed-adverse-effect level (NOAEL) of a drug defined from animal studies is important for inferring a maximal safe dose in human. However, several issues are associated with its concept, determination and application. It is confined to the actual doses used in the study; becomes lower with increasing sample size or dose levels; and reflects the risk level seen in the experiment rather than what may be relevant for human. We explored a pharmacometric approach in an attempt to address these issues. We first used simulation to examine the behaviour of the NOAEL values as determined by current common practice; and then fitted the probability of toxicity as a function of treatment duration and dose to data collected from all applicable toxicology studies of a test compound. Our investigation was in the context of an irreversible toxicity that is detected at the end of the study. Simulations illustrated NOAEL's dependency on experimental factors such as dose and sample size, as well as the underlying uncertainty. Modelling the probability as a continuous function of treatment duration and dose simultaneously to data from multiple studies allowed the estimation of the dose, along with its confidence interval, for a maximal risk level that might be deemed as acceptable for human. The model-based data integration also reconciled between-study inconsistency and explicitly provided maximised estimation confidence. Such alternative NOAEL determination method should be explored for its more efficient data use, more quantifiable insight to toxic doses, and the potential for more relevant animal-to-human translation. - Highlights: • Simulations revealed issues with NOAEL concept, determination and application. • Probabilistic modelling was used to address these issues. • The model integrated time-dose-toxicity data from multiple studies. • The approach uses data efficiently and may allow more meaningful human translation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

The value of integrating pre-clinical data to predict nausea and vomiting risk in humans as illustrated by AZD3514, a novel androgen receptor modulator

Nausea and vomiting are components of a complex mechanism that signals food avoidance and protection of the body against the absorption of ingested toxins. This response can also be triggered by pharmaceuticals. Predicting clinical nausea and vomiting liability for pharmaceutical agents based on pre-clinical data can be problematic as no single animal model is a universal predictor. Moreover, efforts to improve models are hampered by the lack of translational animal and human data in the public domain. AZD3514 is a novel, orally-administered compound that inhibits androgen receptor signaling and down-regulates androgen receptor expression. Here we have explored the utility of integrating data from several pre-clinical models to predict nausea and vomiting in the clinic. Single and repeat doses of AZD3514 resulted in emesis, salivation and gastrointestinal disturbances in the dog, and inhibited gastric emptying in rats after a single dose. AZD3514, at clinically relevant exposures, induced dose-responsive “pica” behaviour in rats after single and multiple daily doses, and induced retching and vomiting behaviour in ferrets after a single dose. We compare these data with the clinical manifestation of nausea and vomiting encountered in patients with castration-resistant prostate cancer receiving AZD3514. Our data reveal a striking relationship between the pre-clinical observations described and the experience of nausea and vomiting in the clinic. In conclusion, the emetic nature of AZD3514 was predicted across a range of pre-clinical models, and the approach presented provides a valuable framework for predicition of clinical nausea and vomiting. - Highlights: • Integrated pre-clinical data can be used to predict clinical nausea and vomiting. • Data integrated from standard toxicology studies is sufficient to make a prediction. • The use of the nausea algorithm developed by Parkinson (2012) aids the prediction. • Additional pre-clinical studies can be used to confirm and quantify the risk.

60 APPLIED LIFE SCIENCES↗