Novel in vivo TDP-43 stress reporter models to accelerate drug development in ALS.

The development of therapies to combat neurodegenerative diseases is widely recognized as a research priority. Despite recent advances in understanding their molecular basis, there is a lack of suitable early biomarkers to test selected compounds and accelerate their translation to clinical trials. We have investigated the utility of in vivo reporters of cytoprotective pathways (e.g. NRF2, p53) as surrogate early biomarkers of the ALS degenerative disease progression. We hypothesized that cellular stress observed in a model of ALS may precede overt cellular damage and could activate our cytoprotective pathway reporters. To test this hypothesis, we generated novel ALS-reporter mice by crossing the hTDP-43tg model into our oxidative stress/inflammation (Hmox1; NRF2 pathway) and DNA damage (p21; p53 pathway) stress reporter models. Histological analysis of reporter expression in a homozygous hTDP-43tg background demonstrated a time-dependent and tissue-specific activation of the reporters in tissues directly associated with ALS, before moderate clinical signs are observed. Further work is warranted to determine the specific mechanisms by which TDP-43 accumulation leads to reporter activation and whether therapeutic intervention modulates reporters' expression. We anticipate the reporter strategy could be of great value in developing treatments for a range of degenerative disorders.

Simulating BRAFV600E-MEK-ERK signalling dynamics in response to vertical inhibition treatment strategies.

In vertical inhibition treatment strategies, multiple components of an intracellular pathway are simultaneously inhibited. Vertical inhibition of the BRAFV600E-MEK-ERK signalling pathway is a standard of care for treating BRAFV600E-mutated melanoma where two targeted cancer drugs, a BRAFV600E-inhibitor, and a MEK inhibitor, are administered in combination. Targeted therapies have been linked to early onsets of drug resistance, and thus treatment strategies of higher complexities and lower doses have been proposed as alternatives to current clinical strategies. However, finding optimal complex, low-dose treatment strategies is a challenge, as it is possible to design more treatment strategies than are feasibly testable in experimental settings. To quantitatively address this challenge, we develop a mathematical model of BRAFV600E-MEK-ERK signalling dynamics in response to combinations of the BRAFV600E-inhibitor dabrafenib (DBF), the MEK inhibitor trametinib (TMT), and the ERK-inhibitor SCH772984 (SCH). From a model of the BRAFV600E-MEK-ERK pathway, and a set of molecular-level drug-protein interactions, we extract a system of chemical reactions that is parameterised by in vitro data and converted to a system of ordinary differential equations (ODEs) using the law of mass action. The ODEs are solved numerically to produce simulations of how pathway-component concentrations change over time in response to different treatment strategies, i.e., inhibitor combinations and doses. The model can thus be used to limit the search space for effective treatment strategies that target the BRAFV600E-MEK-ERK pathway and warrant further experimental investigation. The results demonstrate that DBF and DBF-TMT-SCH therapies show marked sensitivity to BRAFV600E concentrations in silico, whilst TMT and SCH monotherapies do not.

Acceleration of infectious disease drug discovery and development using a humanized model of drug metabolism.

A key step in drug discovery, common to many disease areas, is preclinical demonstration of efficacy in a mouse model of disease. However, this demonstration and its translation to the clinic can be impeded by mouse-specific pathways of drug metabolism. Here, we show that a mouse line extensively humanized for the cytochrome P450 gene superfamily ("8HUM") can circumvent these problems. The pharmacokinetics, metabolite profiles, and magnitude of drug-drug interactions of a test set of approved medicines were in much closer alignment with clinical observations than in wild-type mice. Infection with Mycobacterium tuberculosis, Leishmania donovani, and Trypanosoma cruzi was well tolerated in 8HUM, permitting efficacy assessment. During such assessments, mouse-specific metabolic liabilities were bypassed while the impact of clinically relevant active metabolites and DDI on efficacy were well captured. Removal of species differences in metabolism by replacement of wild-type mice with 8HUM therefore reduces compound attrition while improving clinical translation, accelerating drug discovery.

Defining the in vivo mechanism of air pollutant toxicity using murine stress response biomarkers.

Air pollution can cause a wide range of serious human diseases. For the informed instigation of interventions which prevent these outcomes there is an urgent need to develop robust in vivo biomarkers which provide insights into mechanisms of toxicity and relate pollutants to specific adverse outcomes. We exemplify for a first time the application of in vivo stress response reporters in establishing mechanisms of air pollution toxicity and the application of this knowledge in epidemiological studies. We first demonstrated the utility of reporter mice to understand toxicity mechanisms of air pollutants using diesel exhaust particles compounds. We observed that nitro-PAHs induced Hmox1 and CYP1a1 reporters in a time- and dose-dependent, cell- and tissue-specific manner. Using in vivo genetic and pharmacological approaches we confirmed that the NRF2 pathway mediated this Hmox1-reporter induction stress reporter activity. We then correlated the activation of stress-reporter models (oxidative stress/inflammation, DNA damage and Ah receptor -AhR- activity) with responses in primary human nasal cells exposed to chemicals present in particulate matter (PM; PM2.5-SRM2975, PM10-SRM1648b) or fresh roadside PM10. To exemplify their use in clinical studies, Pneumococcal adhesion was assessed in exposed primary human nasal epithelial cells (HPNEpC). The combined use of HPNEpC and in vivo reporters demonstrated that London roadside PM10 particles induced pneumococcal infection in HPNEpC mediated by oxidative stress responses. The combined use of in vivo reporter models with human data thus provides a robust approach to define the relationship between air pollutant exposure and health risks. Moreover, these models can be used in epidemiological studies to hazard ranking environmental pollutants by considering the complexity of mechanisms of toxicity. These data will facilitate the relationship between toxic potential and the level of pollutant exposure in populations to be established and potentially extremely valuable tools for intervention studies for disease prevention.

Through a glass, darkly? HepaRG and HepG2 cells as models of human phase I drug metabolism.

The pharmacokinetic and safety assessment of drug candidates is becoming increasingly dependent upon in vitro models of hepatic metabolism and toxicity. Predominant among these is the HepG2 cell line, although HepaRG is becoming increasingly popular because of its perceived closer resemblance to human hepatocytes. We review the functionality of these cell lines in terms of Phase I protein expression, basal cytochrome P450-dependent activity, and utility in P450 induction studies. Our analysis indicates that HepG2 cells are severely compromised: proteomic studies show that they express few key proteins in common with hepatocytes and they lack drug-metabolizing capacity. Differentiated HepaRGs are more hepatocyte-like than HepG2s, but they also have limitations, and it is difficult to assess their utility because of the enormous variability in data reported, possibly arising from the complex differentiation protocols required to obtain hepatocyte-like cells. This is exacerbated by the use of DMSO in the induction protocol, together with proprietary supplements whose composition is a commercial secret. We conclude that, while currently available data on the utility of HepaRG generates a confusing picture, this line does have potential utility in drug metabolism studies. However, to allow studies to be compared directly a standardized, reproducible differentiation protocol is essential and the cell line's functionality in terms of known mechanisms of P450 regulation must be demonstrated. We, therefore, support the development of regulatory guidelines for the use of HepaRGs in induction studies as a first step in generating a database of consistent, reliable data.

Potential of in vivo stress reporter models to reduce animal use and provide mechanistic insights in toxicity studies.

Chemical risk assessment ensures protection from the toxic effects of drugs and manmade chemicals. To comply with regulatory guidance, studies in complex organisms are required, as well as mechanistic studies to establish the relevance of any toxicities observed to man. Although in vitro toxicity models are improving, in vivo studies remain central to this process. Such studies are invariably time-consuming and often involve large numbers of animals. New regulatory frameworks recommend the implementation of "smart" in vivo approaches to toxicity testing that can effectively assess safety for humans and comply with societal expectations for reduction in animal use. A major obstacle in reducing the animals required is the time-consuming and complexity of the pathological endpoints used as markers of toxicity. Such endpoints are prone to inter-animal variability, subjectivity and require harmonisation between testing sites. As a consequence, large numbers of animals per experimental group are required. To address this issue, we propose the implementation of sophisticated stress response reporter mice that we have developed. These reporter models provide early biomarkers of toxic potential in a highly reproducible manner at single-cell resolution, which can also be measured non-invasively and have been extensively validated in academic research as early biomarkers of stress responses for a wide range of chemicals at human-relevant exposures. In this report, we describe a new and previously generated models in our lab, provide the methodology required for their use and discuss how they have been used to inform on toxic risk (likelihood of chemical causing an adverse health effect). We propose our in vivo approach is more informative (refinement) and reduces the animal use (reduction) compared to traditional toxicity testing. These models could be incorporated into tiered toxicity testing and used in combination with in vitro assays to generate quantitative adverse outcome pathways and inform on toxic potential.

Improving the predictive power of xenograft and syngeneic anti-tumour studies using mice humanised for pathways of drug metabolism.

Drug development is an expensive and time-consuming process, with only a small fraction of drugs gaining regulatory approval from the often many thousands of candidates identified during target validation. Once a lead compound has been identified and optimised, they are subject to intensive pre-clinical research to determine their pharmacodynamic, pharmacokinetic and toxicological properties, procedures which inevitably involve significant numbers of animals - mainly mice and rats, but also dogs and monkeys in much smaller numbers and for specific types of drug candidates. Many compounds that emerge from this process, having been shown to be safe and efficacious in pre-clinical studies, subsequently fail to replicate this outcome in clinical trials, therefore wasting time, money and, most importantly, animals. The poor predictive power of animal models in pre-clinical studies is predominantly due to lack of efficacy or safety reasons, which in turn can be attributed mainly to the significant species differences in drug metabolism between humans and animals. To circumvent this, we have developed a complex transgenic mouse model - 8HUM - which faithfully replicates human Phase I drug metabolism (and its regulation), and which will generate more human-relevant data [ REFINEMENT] from fewer animals [ REDUCTION] in a pre-clinical setting and reduce attrition in the clinic. One key area for the pre-clinical application of animals in an oncology setting - almost exclusively mice - is their use in anti-tumour studies. We now further demonstrate the utility of the 8HUM mouse using a murine melanoma cell line as a syngeneic tumour and also present an immunodeficient version 8HUM_Rag2 -/- - for use in xenograft studies. These models will be of significant benefit not only to Pharma for pre-clinical drug development work, but also throughout the drug efficacy, toxicology, pharmacology, and drug metabolism communities, where fewer animals will be needed to generate more human-relevant data.

Glutathione-S-transferase P promotes glycolysis in asthma in association with oxidation of pyruvate kinase M2.

BACKGROUND: Interleukin-1-dependent increases in glycolysis promote allergic airways disease in mice and disruption of pyruvate kinase M2 (PKM2) activity is critical herein. Glutathione-S-transferase P (GSTP) has been implicated in asthma pathogenesis and regulates the oxidation state of proteins via S-glutathionylation. We addressed whether GSTP-dependent S-glutathionylation promotes allergic airways disease by promoting glycolytic reprogramming and whether it involves the disruption of PKM2. METHODS: We used house dust mite (HDM) or interleukin-1ß in C57BL6/NJ WT or mice that lack GSTP. Airway basal cells were stimulated with interleukin-1ß and the selective GSTP inhibitor, TLK199. GSTP and PKM2 were evaluated in sputum samples of asthmatics and healthy controls and incorporated analysis of the U-BIOPRED severe asthma cohort database. RESULTS: Ablation of Gstp decreased total S-glutathionylation and attenuated HDM-induced allergic airways disease and interleukin-1ß-mediated inflammation. Gstp deletion or inhibition by TLK199 decreased the interleukin-1ß-stimulated secretion of pro-inflammatory mediators and lactate by epithelial cells. 13C-glucose metabolomics showed decreased glycolysis flux at the pyruvate kinase step in response to TLK199. GSTP and PKM2 levels were increased in BAL of HDM-exposed mice as well as in sputum of asthmatics compared to controls. Sputum proteomics and transcriptomics revealed strong correlations between GSTP, PKM2, and the glycolysis pathway in asthma. CONCLUSIONS: GSTP contributes to the pathogenesis of allergic airways disease in association with enhanced glycolysis and oxidative disruption of PKM2. Our findings also suggest a PKM2-GSTP-glycolysis signature in asthma that is associated with severe disease.

Nrf2 activation does not affect adenoma development in a mouse model of colorectal cancer.

Transcription factor nuclear factor erythroid 2 p45-related factor 2 (Nrf2) and its main negative regulator, Kelch-like ECH associated protein 1 (Keap1), are at the interface between redox and intermediary metabolism. Nrf2 activation is protective in models of human disease and has benefits in clinical trials. Consequently, the Keap1/Nrf2 protein complex is a drug target. However, in cancer Nrf2 plays a dual role, raising concerns that Nrf2 activators may promote growth of early neoplasms. To address this concern, we examined the role of Nrf2 in development of colorectal adenomas by employing genetic, pharmacological, and metabolomic approaches. We found that colorectal adenomas that form in Gstp-/-: ApcMin/+ mice are characterized by altered one-carbon metabolism and that genetic activation, but not disruption of Nrf2, enhances these metabolic alterations. However, this enhancement is modest compared to the magnitude of metabolic differences between tumor and peri-tumoral tissues, suggesting that the metabolic changes conferred by Nrf2 activation may have little contribution to the early stages of carcinogenesis. Indeed, neither genetic (by Keap1 knockdown) nor pharmacological Nrf2 activation, nor its disruption, affected colorectal adenoma formation in this model. We conclude that pharmacological Nrf2 activation is unlikely to impact the early stages of development of colorectal cancer.

Application of the in vivo oxidative stress reporter Hmox1 as mechanistic biomarker of arsenic toxicity.

Inorganic arsenic (iAs) is a naturally occurring metalloid present in drinking water and polluted air exposing millions of people globally. Epidemiological studies have linked iAs exposure to the development of numerous diseases including cognitive impairment, cardiovascular failure and cancer. Despite intense research, an effective therapy for chronic arsenicosis has yet to be developed. Laboratory studies have been of great benefit in establishing the pathways involved in iAs toxicity and providing insights into its mechanism of action. However, the in vivo analysis of arsenic toxicity mechanisms has been difficult by the lack of reliable in vivo biomarkers of iAs's effects. To address this issue we have applied the use of our recently developed stress reporter models to study iAs toxicity. The reporter mice Hmox1 (oxidative stress/inflammation; HOTT) and p21 (DNA damage) were exposed to iAs at acute and chronic, environmentally relevant, doses. We observed induction of the oxidative stress reporters in several cell types and tissues, which was largely dependent on the activation of transcription factor NRF2. We propose that our HOTT reporter model can be used as a surrogate biomarker of iAs-induced oxidative stress, and it constitutes a first-in-class platform to develop treatments aimed to counteract the role of oxidative stress in arsenicosis. Indeed, in a proof of concept experiment, the HOTT reporter mice were able to predict the therapeutic utility of the antioxidant N-acetyl cysteine in the prevention of iAs associated toxicity.

Drug-induced chromatin accessibility changes associate with sensitivity to liver tumor promotion.

Liver cancer susceptibility varies amongst humans and between experimental animal models because of multiple genetic and epigenetic factors. The molecular characterization of such susceptibilities has the potential to enhance cancer risk assessment of xenobiotic exposures and disease prevention strategies. Here, using DNase I hypersensitivity mapping coupled with transcriptomic profiling, we investigate perturbations in cis-acting gene regulatory elements associated with the early stages of phenobarbital (PB)-mediated liver tumor promotion in susceptible versus resistant mouse strains (B6C3F1 versus C57BL/6J). Integrated computational analyses of strain-selective changes in liver chromatin accessibility underlying PB response reveal differential epigenetic regulation of molecular pathways associated with PB-mediated tumor promotion, including Wnt/ß-catenin signaling. Complementary transcription factor motif analyses reveal mouse strain-selective gene regulatory networks and a novel role for Stat, Smad, and Fox transcription factors in the early stages of PB-mediated tumor promotion. Mapping perturbations in cis-acting gene regulatory elements provides novel insights into the molecular basis for susceptibility to xenobiotic-induced rodent liver tumor promotion and has the potential to enhance mechanism-based cancer risk assessments of xenobiotic exposures.

Application of hepatic cytochrome b5/P450 reductase null (HBRN) mice to study the role of cytochrome b5 in the cytochrome P450-mediated bioactivation of the anticancer drug ellipticine.

The anticancer drug ellipticine exerts its genotoxic effects after metabolic activation by cytochrome P450 (CYP) enzymes. The present study has examined the role of cytochrome P450 oxidoreductase (POR) and cytochrome b5 (Cyb5), electron donors to P450 enzymes, in the CYP-mediated metabolism and disposition of ellipticine in vivo. We used Hepatic Reductase Null (HRN) and Hepatic Cytochrome b5/P450 Reductase Null (HBRN) mice. HRN mice have POR deleted specifically in hepatocytes; HBRN mice also have Cyb5 deleted in the liver. Mice were treated once with 10 mg/kg body weight ellipticine (n = 4/group) for 24 h. Ellipticine-DNA adduct levels measured by 32P-postlabelling were significantly lower in HRN and HBRN livers than in wild-type (WT) livers; however no significant difference was observed between HRN and HBRN livers. Ellipticine-DNA adduct formation in WT, HRN and HBRN livers correlated with Cyp1a and Cyp3a enzyme activities measured in hepatic microsomes in the presence of NADPH confirming the importance of P450 enzymes in the bioactivation of ellipticine in vivo. Hepatic microsomal fractions were also utilised in incubations with ellipticine and DNA in the presence of NADPH, cofactor for POR, and NADH, cofactor for Cyb5 reductase (Cyb5R), to examine ellipticine-DNA adduct formation. With NADPH adduct formation decreased as electron donors were lost which correlated with the formation of the reactive metabolites 12- and 13-hydroxy-ellipticine in hepatic microsomes. No difference in adduct formation was observed in the presence of NADH. Our study demonstrates that Cyb5 contributes to the P450-mediated bioactivation of ellipticine in vitro, but not in vivo.

Constitutive androstane receptor 1 is constitutively bound to chromatin and 'primed' for transactivation in hepatocytes.

The constitutive androstane receptor (CAR) is a xenobiotic sensor expressed in hepatocytes that activates genes involved in drug metabolism, lipid homeostasis, and cell proliferation. Much progress has been made in understanding the mechanism of activation of human CAR by drugs and xenobiotics. However, many aspects of the activation pathway remain to be elucidated. In this report, we have used viral constructs to express human CAR, its splice variants, and mutant CAR forms in hepatocytes from Car-/- mice in vitro and in vivo. We demonstrate CAR expression rescued the ability of Car-/- hepatocytes to respond to a wide range of CAR activators including phenobarbital. Additionally, two major splice isoforms of human CAR, CAR2 and CAR3, were inactive with almost all the agents tested. In contrast to the current model of CAR activation, ectopic CAR1 is constitutively localized in the nucleus and is loaded onto Cyp2b10 gene in the absence of an inducing agent. In studies to elucidate the role of threonine T38 in CAR regulation, we found that the T38D mutant was inactive even in the presence of CAR activators. However, the T38A mutant was activated by CAR inducers, showing that T38 is not essential for CAR activation. Also, using the inhibitor erlotinib, we could not confirm a role for the epidermal growth factor receptor in CAR regulation. Our data suggest that CAR is constitutively bound to gene regulatory regions and is regulated by exogenous agents through a mechanism which involves protein phosphorylation in the nucleus.

Interaction between polymorphisms in aspirin metabolic pathways, regular aspirin use and colorectal cancer risk: A case-control study in unselected white European populations.

Regular aspirin use is associated with reduced risk of colorectal cancer (CRC). Variation in aspirin's chemoprevention efficacy has been attributed to the presence of single nucleotide polymorphisms (SNPs). We conducted a meta-analysis using two large population-based case-control datasets, the UK-Leeds Colorectal Cancer Study Group and the NIH-Colon Cancer Family Registry, having a combined total of 3325 cases and 2262 controls. The aim was to assess 42 candidate SNPs in 15 genes whose association with colorectal cancer risk was putatively modified by aspirin use, in the literature. Log odds ratios (ORs) and standard errors were estimated for each dataset separately using logistic regression adjusting for age, sex and study site, and dataset-specific results were combined using random effects meta-analysis. Meta-analysis showed association between SNPs rs6983267, rs11694911 and rs2302615 with CRC risk reduction (All P<0.05). Association for SNP rs6983267 in the CCAT2 gene only was noteworthy after multiple test correction (P = 0.001). Site-specific analysis showed association between SNPs rs1799853 and rs2302615 with reduced colon cancer risk only (P = 0.01 and P = 0.004, respectively), however neither reached significance threshold following multiple test correction. Meta-analysis of SNPs rs2070959 and rs1105879 in UGT1A6 gene showed interaction between aspirin use and CRC risk (Pinteraction = 0.01 and 0.02, respectively); stratification by aspirin use showed an association for decreased CRC risk for aspirin users having a wild-type genotype (rs2070959 OR = 0.77, 95% CI = 0.68-0.86; rs1105879 OR = 0.77 95% CI = 0.69-0.86) compared to variant allele cariers. The direction of the interaction however is in contrast to that published in studies on colorectal adenomas. Both SNPs showed potential site-specific interaction with aspirin use and colon cancer risk only (Pinteraction = 0.006 and 0.008, respectively), with the direction of association similar to that observed for CRC. Additionally, they showed interaction between any non-steroidal anti-inflammatory drugs (including aspirin) use and CRC risk (Pinteraction = 0.01 for both). All gene x environment (GxE) interactions however were not significant after multiple test correction. Candidate gene investigation indicated no evidence of GxE interaction between genetic variants in genes involved in aspirin pathways, regular aspirin use and colorectal cancer risk.

Identification of Novel Pathways of Osimertinib Disposition and Potential Implications for the Outcome of Lung Cancer Therapy.

Purpose: Osimertinib is a third-generation inhibitor of the epidermal growth factor receptor used in treatment of non-small cell lung cancer. A full understanding of its disposition and capacity for interaction with other medications will facilitate its effective use as a single agent and in combination therapy.Experimental Design: Recombinant cytochrome P450s and liver microsomal preparations were used to identify novel pathways of osimertinib metabolism in vitro A panel of knockout and mouse lines humanized for pathways of drug metabolism were used to establish the relevance of these pathways in vivoResults: Although some osimertinib metabolites were similar in mouse and human liver samples there were several significant differences, in particular a marked species difference in the P450s involved. The murine Cyp2d gene cluster played a predominant role in mouse, whereas CYP3A4 was the major human enzyme responsible for osimertinib metabolism. Induction of this enzyme in CYP3A4 humanized mice substantially decreased circulating osimertinib exposure. Importantly, we discovered a further novel pathway of osimertinib disposition involving CPY1A1. Modulation of CYP1A1/CYP1A2 levels markedly reduced parent drug concentrations, significantly altering metabolite pharmacokinetics (PK) in humanized mice in vivoConclusions: We demonstrate that a P450 enzyme expressed in smokers' lungs and lung tumors has the capacity to metabolise osimertinib. This could be a significant factor in defining the outcome of osimertinib treatment. This work also illustrates how P450-humanized mice can be used to identify and mitigate species differences in drug metabolism and thereby model the in vivo effect of critical metabolic pathways on anti-tumor response. Clin Cancer Res; 24(9); 2138-47. ©2018 AACR.

Cytochrome b 5 impacts on cytochrome P450-mediated metabolism of benzo[a]pyrene and its DNA adduct formation: studies in hepatic cytochrome b 5 /P450 reductase null (HBRN) mice.

Benzo[a]pyrene (BaP) is an environmental pollutant that, based on evidence largely from in vitro studies, exerts its genotoxic effects after metabolic activation by cytochrome P450s. In the present study, Hepatic Reductase Null (HRN) and Hepatic Cytochrome b 5 /P450 Reductase Null (HBRN) mice have been used to study the role of P450s in the metabolic activation of BaP in vivo. In HRN mice, cytochrome P450 oxidoreductase (POR), the electron donor to P450, is deleted specifically in hepatocytes. In HBRN mice the microsomal haemoprotein cytochrome b 5 , which can also act as an electron donor from cytochrome b 5 reductase to P450s, is also deleted in the liver. Wild-type (WT), HRN and HBRN mice were treated by i.p. injection with 125 mg/kg body weight BaP for 24 h. Hepatic microsomal fractions were isolated from BaP-treated and untreated mice. In vitro incubations carried out with BaP-pretreated microsomal fractions, BaP and DNA resulted in significantly higher BaP-DNA adduct formation with WT microsomal fractions compared to those from HRN or HBRN mice. Adduct formation (i.e. 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-BaP [dG-N2-BPDE]) correlated with observed CYP1A activity and metabolite formation (i.e. BaP-7,8-dihydrodiol) when NADPH or NADH was used as enzymatic cofactors. BaP-DNA adduct levels (i.e. dG-N2-BPDE) in vivo were significantly higher (~ sevenfold) in liver of HRN mice than WT mice while no significant difference in adduct formation was observed in liver between HBRN and WT mice. Our results demonstrate that POR and cytochrome b 5 both modulate P450-mediated activation of BaP in vitro. However, hepatic P450 enzymes in vivo appear to be more important for BaP detoxification than its activation.

Measuring in vivo responses to endogenous and exogenous oxidative stress using a novel haem oxygenase 1 reporter mouse.

KEY POINTS: Haem oxygenase 1 (Hmox1) is a cytoprotective enzyme with anti-inflammatory and anti-oxidant properties that is induced in response to multiple noxious environmental stimuli and disease states. Tools to enable its expression to be monitored in vivo have been unavailable until now. In a new Hmox1 reporter model we provide high-fidelity, single-cell resolution blueprints for Hmox1 expression throughout the body of mice. We show for the first time that Hmox1 is constitutively expressed at barrier tissues at the interface between the internal and external environments, and that it is highly induced in muscle cells during systemic inflammation. These data suggest novel biological insights into the role of Hmox1 and pave the way for the use of the model to study the role of environmental stress in disease pathology. ABSTRACT: Hmox1 protein holds great promise as a biomarker of in vivo stress responses as it is highly induced in stressed or damaged cells. However, Hmox1 expression patterns have thus far only been available in simple model organisms with limited relevance to humans. We now report a new Hmox1 reporter line that makes it possible to obtain this information in mice, a premiere model system for studying human disease and toxicology. Using a state-of-the-art strategy, we expressed multiple complementary reporter molecules from the murine Hmox1 locus, including firefly luciferase, to allow long-term, non-invasive imaging of Hmox1 expression, and ß-galactosidase for high-resolution mapping of expression patterns post-mortem. We validated the model by confirming the fidelity of reporter expression, and its responsiveness to oxidative and inflammatory stimuli. In addition to providing blueprints for Hmox1 expression in mice that provide novel biological insights, this work paves the way for the broad application of this model to establish cellular stresses induced by endogenous processes and those resulting from exposure to drugs and environmental agents. It will also enable studies on the role of oxidative stress in the pathogenesis of disease and its prevention.

Novel Pathways of Ponatinib Disposition Catalyzed By CYP1A1 Involving Generation of Potentially Toxic Metabolites.

Ponatinib, a pan-BCR-ABL tyrosine kinase inhibitor for the treatment of chronic myeloid leukemia (CML), causes severe side effects including vascular occlusions, pancreatitis, and liver toxicity, although the underlying mechanisms remain unclear. Modifications of critical proteins through reactive metabolites are thought to be responsible for a number of adverse drug reactions. In vitro metabolite screening of ponatinib with human liver microsomes and glutathione revealed unambiguous signals of ponatinib-glutathione (P-GSH) adducts. Further profiling of human cytochrome P450 (P450) indicated that CYP1A1 was the predominant P450 enzyme driving this reaction. P-GSH conjugate formation paralleled the disappearance of hydroxylated ponatinib metabolites, suggesting the initial reaction was epoxide generation. Mouse glutathione S-transferase p1 (mGstp1) further enhanced P-GSH adduct formation in vitro. Ponatinib pharmacokinetics were determined in vivo in wild-type (WT) mice and mice humanized for CYP1A1/2 and treated with the CYP1A1 inducers 2,3,7,8-tetrachlorodibenzodioxin or 3-methylcholanthrene. Ponatinib exposure was significantly decreased in treated mice compared with controls (7.7- and 2.2-fold for WT and humanized CYP1A1/2, respectively). Interestingly, the P-GSH conjugate was only found in the feces of CYP1A1-induced mice, but not in control animals. Protein adducts were also identified by liquid chromatography-tandem mass spectrometry analysis of mGstp1 tryptic digests. These results indicate that not only could CYP1A1 be involved in ponatinib disposition, which has not been previously reported, but also that electrophilic intermediates resulting from CYP1A1 metabolism in normal tissues may contribute to ponatinib toxicity. These data are consistent with a recent report that CML patients who smoke are at greater risk of disease progression and premature death.

Aldo-keto reductases are biomarkers of NRF2 activity and are co-ordinately overexpressed in non-small cell lung cancer.

This corrects the article DOI: 10.1038/bjc.2016.363.

Feedback control of AHR signalling regulates intestinal immunity.

The aryl hydrocarbon receptor (AHR) recognizes xenobiotics as well as natural compounds such as tryptophan metabolites, dietary components and microbiota-derived factors, and it is important for maintenance of homeostasis at mucosal surfaces. AHR activation induces cytochrome P4501 (CYP1) enzymes, which oxygenate AHR ligands, leading to their metabolic clearance and detoxification. Thus, CYP1 enzymes have an important feedback role that curtails the duration of AHR signalling, but it remains unclear whether they also regulate AHR ligand availability in vivo. Here we show that dysregulated expression of Cyp1a1 in mice depletes the reservoir of natural AHR ligands, generating a quasi AHR-deficient state. Constitutive expression of Cyp1a1 throughout the body or restricted specifically to intestinal epithelial cells resulted in loss of AHR-dependent type 3 innate lymphoid cells and T helper 17 cells and increased susceptibility to enteric infection. The deleterious effects of excessive AHR ligand degradation on intestinal immune functions could be counter-balanced by increasing the intake of AHR ligands in the diet. Thus, our data indicate that intestinal epithelial cells serve as gatekeepers for the supply of AHR ligands to the host and emphasize the importance of feedback control in modulating AHR pathway activation.