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DOE OSTI2024

The impact of sampling scale: A comparison of methods for estimating external contaminant exposure in free-ranging wildlife

The impacts of contaminants on wildlife are dose dependent, and thus being able to track or predict exposure following contamination events is important for monitoring ecosystem health. However, the ability to track exposure in free-ranging wildlife is often severely limited. Consequently, researchers have predominantly relied on simple methods for estimating contaminant exposures in wildlife with little regard for spatial contaminant heterogeneity or an animal’s use of diverse habitats. We evaluated the influence sampling scale (i.e., how finely contaminant distribution and organism’s spatial use of the landscape is mapped) has on (1) realism and (2) conservativeness of exposure estimates. To do this, we monitored the actual exposure of wild boar (Sus scrofa) in Fukushima, Japan to radioactive contamination using GPS27 coupled contaminant monitors placed on individual animals. We compared empirical exposures to estimates generated by combining varying amounts of information about an individual boar’s location and/or movement, with the distribution of contamination on the landscape. Here we found that the most realistic exposure estimates were produced when finer-scale contaminant distribution surveys (e.g., airborne surveys) were combined with more accurate estimates of an individual’s space use (e.g., home ranges or core areas). Importantly, estimates of exposure based on single point surveys at a trap site (a simple method commonly used in the literature), did not correlate with actual exposure rates, suggesting dose-effects studies using this method may result in spurious conclusions. These results suggest that researchers seeking realistic estimates of exposure, such as in dose-effect studies, should ensure they have adequately accounted for fine-scale contaminant distribution patterns and areas of higher use by study organisms. However, conservative estimates of exposure i.e., intentionally over-predicting exposure as is done in initial tiers of ecological risk analyses) were not as scale sensitive and could be achieved with a single known location and coarse contaminant distribution maps.

63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTAN↗

DOE OSTI2012

Co-exposure to nickel and cobalt chloride enhances cytotoxicity and oxidative stress in human lung epithelial cells

Nickel and cobalt are heavy metals found in land, water, and air that can enter the body primarily through the respiratory tract and accumulate to toxic levels. Nickel compounds are known to be carcinogenic to humans and animals, while cobalt compounds produce tumors in animals and are probably carcinogenic to humans. People working in industrial and manufacturing settings have an increased risk of exposure to these metals. The cytotoxicity of nickel and cobalt has individually been demonstrated; however, the underlying mechanisms of co-exposure to these heavy metals have not been explored. In this study, we investigated the effect of exposure of H460 human lung epithelial cells to nickel and cobalt, both alone and in combination, on cell survival, apoptotic mechanisms, and the generation of reactive oxygen species and double strand breaks. For simultaneous exposure, cells were exposed to a constant dose of 150 μM cobalt or nickel, which was found to be relatively nontoxic in single exposure experiments. We demonstrated that cells exposed simultaneously to cobalt and nickel exhibit a dose-dependent decrease in survival compared to the cells exposed to a single metal. The decrease in survival was the result of enhanced caspase 3 and 7 activation and cleavage of poly (ADP-ribose) polymerase. Co-exposure increased the production of ROS and the formation of double strand breaks. Pretreatment with N-acetyl cysteine alleviated the toxic responses. Collectively, this study demonstrates that co-exposure to cobalt and nickel is significantly more toxic than single exposure and that toxicity is related to the formation of ROS and DSB. -- Highlights: ► Decreased survival following simultaneous exposure to NiCl{sub 2} and CoCl{sub 2}. ► Enhanced caspase and PARP cleavage following co-exposure. ► Increased formation of ROS in dual exposed cells. ► N-acetyl cysteine pretreatment decreases Co and Ni toxicity. ► Co-exposure to Ni and Co enhances the formation of double strand breaks.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2012

Biotransformation of 2,3,3,3-tetrafluoropropene (HFO-1234yf) in male, pregnant and non-pregnant female rabbits after single high dose inhalation exposure

2,3,3,3-Tetrafluoropropene (HFO-1234yf) is a novel refrigerant intended for use in mobile air conditioning. It showed a low potential for toxicity in rodents studies with most NOAELs well above 10,000 ppm in guideline compliant toxicity studies. However, a developmental toxicity study in rabbits showed mortality at exposure levels of 5,500 ppm and above. No lethality was observed at exposure levels of 2,500 and 4,000 ppm. Nevertheless, increased subacute inflammatory heart lesions were observed in rabbits at all exposure levels. Since the lethality in pregnant animals may be due to altered biotransformation of HFO-1234yf and to evaluate the potential risk to pregnant women facing a car crash, this study compared the acute toxicity and biotransformation of HFO-1234yf in male, female and pregnant female rabbits. Animals were exposed to 50,000 ppm and 100,000 ppm for 1 h. For metabolite identification by {sup 19}F NMR and LC/MS-MS, urine was collected for 48 h after inhalation exposure. In all samples, the predominant metabolites were S-(3,3,3-trifluoro-2-hydroxypropanyl)-mercaptolactic acid and N-acetyl-S-(3,3,3-trifluoro-2-hydroxypropanyl)-L-cysteine. Since no major differences in urinary metabolite pattern were observed between the groups, only N-acetyl-S-(3,3,3-trifluoro-2-hydroxypropanyl)-L-cysteine excretion was quantified. No significant differences in recovery between non-pregnant (43.10 ± 22.35 μmol) and pregnant female (50.47 ± 19.72 μmol) rabbits were observed, male rabbits exposed to 100,000 ppm for one hour excreted 86.40 ± 38.87 μmol. Lethality and clinical signs of toxicity were not observed in any group. The results suggest that the lethality of HFO-1234yf in pregnant rabbits unlikely is due to changes in biotransformation patterns or capacity in pregnant rabbits. -- Highlights: ► No lethality and clinical signs were observed. ► No differences in metabolic pattern between pregnant and non-pregnant rabbits. ► Rapid and similar metabolite excretion in all groups. ► Very low amount of biotransformation in all groups (< 0.1%).

60 APPLIED LIFE SCIENCES↗

DOE OSTI2012

The role of the anaesthetised guinea-pig in the preclinical cardiac safety evaluation of drug candidate compounds

Despite rigorous preclinical and clinical safety evaluation, adverse cardiac effects remain a leading cause of drug attrition and post-approval drug withdrawal. A number of cardiovascular screens exist within preclinical development. These screens do not, however, provide a thorough cardiac liability profile and, in many cases, are not preventing the progression of high risk compounds. We evaluated the suitability of the anaesthetised guinea-pig for the assessment of drug-induced changes in cardiovascular parameters. Sodium pentobarbitone anaesthetised male guinea-pigs received three 15 minute intravenous infusions of ascending doses of amoxicillin, atenolol, clonidine, dobutamine, dofetilide, flecainide, isoprenaline, levosimendan, milrinone, moxifloxacin, nifedipine, paracetamol, verapamil or vehicle, followed by a 30 minute washout. Dose levels were targeted to cover clinical exposure and above, with plasma samples obtained to evaluate effect/exposure relationships. Arterial blood pressure, heart rate, contractility function (left ventricular dP/dt{sub max} and QA interval) and lead II electrocardiogram were recorded throughout. In general, the expected reference compound induced effects on haemodynamic, contractility and electrocardiographic parameters were detected confirming that all three endpoints can be measured accurately and simultaneously in one small animal. Plasma exposures obtained were within, or close to the expected clinical range of therapeutic plasma levels. Concentration–effect curves were produced which allowed a more complete understanding of the margins for effects at different plasma exposures. This single in vivo screen provides a significant amount of information pertaining to the cardiovascular risk of drug candidates, ultimately strengthening strategies addressing cardiovascular-mediated compound attrition and drug withdrawal. -- Highlights: ► Evaluation of the anaesthetised guinea-pig to determine cardiac liability. ► Haemodynamic, contractility, ECG parameters and plasma exposure all measurable. ► Single small animal model offering extensive evaluation of a drug's CV risk. ► Potential to strengthen drug discovery cardiovascular strategy. ► Potential to halt progression of drugs with CV liability, reducing drug attrition.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2012

Pre-clinical studies of toxin-specific Nanobodies: Evidence of in vivo efficacy to prevent fatal disturbances provoked by scorpion envenoming

Scorpions represent a significant threat to humans and animals in various countries throughout the world. Recently, we introduced Nanobodies (Nbs) to combat more efficiently scorpion envenoming and demonstrated the performance of NbAahIF12 and NbAahII10 to neutralize scorpion toxins of Androctonus australis hector venom. A bispecific Nb construct (NbF12-10) comprising these two Nbs is far more protective than the classic Fab′{sub 2} based therapy and is the most efficient antivenom therapy against scorpion sting in preclinical studies. Now we investigate the biodistribution and pharmacokinetics of {sup 99m}Tc labeled Nbs by in vivo imaging in rodents and compared these data with those of the Fab′{sub 2} product (PAS). The pharmacodynamics of the Nbs was investigated in rats by in vivo echocardiography and it is shown that NbF12-10 prevents effectively the hemodynamic disturbances induced by a lethal dose of venom. Moreover, even a late injection of NbF12-10 restores the heart rate and brings the blood pressure to baseline values. Histology confirms that NbF12-10 prevents lung and heart lesions of treated mice after envenoming. In conjunction, in this preclinical study, we provide proof of concept that NbF12-10 prevents effectively the fatal disturbances induced by Androctonus venom, and that the Nanobody based therapeutic has a potential to substitute the classic Fab′{sub 2} based product as immunotherapeutic in scorpion envenoming. Further clinical study using larger cohorts of animals should be considered to confirm the full protecting potential of our NbF12-10. -- Highlights: ► Nanobody therapy prevents the hemodynamic disturbances induced by a lethal dose. ► Late injection of Nanobody restores hemodynamic parameters to baseline values. ► Nanobody therapy prevents lung and heart lesions of treated mice after envenoming. ► Labeled Nanobody and Fab’2 pharmacokinetics curves reach plateau in favour of Nanobody.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2012

Improved preclinical cardiovascular therapeutic indices with long-term inhibition of norepinephrine reuptake using reboxetine

Norepinephrine reuptake inhibitors (NRIs) acutely increase norepinephrine (NE) levels, but therapeutic antidepressant activity is only observed after weeks of treatment because central NE levels progressively increase during continued drug exposure. Similarly, while NRIs acutely increase blood pressure (BP) and heart rate (HR) due to enhanced sympathetic neurotransmission, chronic treatment changes the responsiveness of the central noradrenergic system and suppresses these effects via autonomic regulation. To better understand the relationship between NE increases and cardiovascular safety, we investigated acute and chronic effects of the NRI reboxetine on central NE release and on BP and HR and electrical alternans, a measure of arrhythmia liability, in guinea pigs. NE release was assessed by microdialysis in medial prefrontal cortex (mPFC) and hypothalamic paraventricular nucleus (PVN); BP and HR were measured by telemetry. Animals were treated for 28 days with 15 mg/kg/day of reboxetine or vehicle via an osmotic minipump and then challenged with acute intravenous doses of reboxetine. Animals chronically treated with reboxetine had 2-fold higher extracellular basal NE levels in mPFC and PVN compared to basal levels after chronic vehicle treatment. BP was significantly increased after the first day of treatment, and gradually returned to vehicle levels by day 21. These data indicate that chronic NRI treatment may lead to an increase in central NE levels and a concomitant reduction in BP based on exposure–response curves compared to vehicle treatment, suggesting a larger separation between preclinical estimates of efficacy vs. safety compared to acute NRI treatment. -- Highlights: ► Acute RBX produces blood pressure increases acutely that decrease with chronic RBX ► Chronic RBX increases brain NE levels, a preclinical surrogate of improved efficacy ► Short-term screening of NRI often underestimates the chronic therapeutic index ► Chronic cardiovascular safety and efficacy more adequately address therapeutic index ► Similar paradigms may exist with other centrally and peripherally acting drugs.

60 APPLIED LIFE SCIENCES↗

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↗