Mutational spectra of aflatoxin B1 in vivo establish biomarkers of exposure for human hepatocellular carcinoma.

Aflatoxin B1 (AFB1) and/or hepatitis B and C viruses are risk factors for human hepatocellular carcinoma (HCC). Available evidence supports the interpretation that formation of AFB1-DNA adducts in hepatocytes seeds a population of mutations, mainly G:C→T:A, and viral processes synergize to accelerate tumorigenesis, perhaps via inflammation. Responding to a need for early-onset evidence predicting disease development, highly accurate duplex sequencing was used to monitor acquisition of high-resolution mutational spectra (HRMS) during the process of hepatocarcinogenesis. Four-day-old male mice were treated with AFB1 using a regimen that induced HCC within 72 wk. For analysis, livers were separated into tumor and adjacent cellular fractions. HRMS of cells surrounding the tumors revealed predominantly G:C→T:A mutations characteristic of AFB1 exposure. Importantly, 25% of all mutations were G→T in one trinucleotide context (CGC; the underlined G is the position of the mutation), which is also a hotspot mutation in human liver tumors whose incidence correlates with AFB1 exposure. The technology proved sufficiently sensitive that the same distinctive spectrum was detected as early as 10 wk after dosing, well before evidence of neoplasia. Additionally, analysis of tumor tissue revealed a more complex pattern than observed in surrounding hepatocytes; tumor HRMS were a composite of the 10-wk spectrum and a more heterogeneous set of mutations that emerged during tumor outgrowth. We propose that the 10-wk HRMS reflects a short-term mutational response to AFB1, and, as such, is an early detection metric for AFB1-induced liver cancer in this mouse model that will be a useful tool to reconstruct the molecular etiology of human hepatocarcinogenesis.

Keap1/Nrf2 pathway activation leads to a repressed hepatic gluconeogenic and lipogenic program in mice on a high-fat diet.

The Keap1/Nrf2 pathway, known to regulate the expression of a series of cytoprotective and antioxidant genes, has been studied in the context of obesity and type 2 diabetes; diseases that are characterized by chronic oxidative stress. There is increasing evidence, however, that the transcription factor Nrf2 can crosstalk with pathways not directly related to cytoprotection. Our present work focuses on the effect of Nrf2 on hepatic gluconeogenesis and lipogenesis, two metabolic processes which are dysregulated in the obese/diabetic state. To this end, a genetic mouse model of Nrf2 pathway activation was used (Keap1-hypo; both Keap1 alleles are hypomorphic) and was exposed to a 3-month high-fat diet along with the relevant control wild-type mice. The Keap1-hypo mice were partially protected from obesity, had lower fasting glucose and insulin levels and developed less liver steatosis compared to the wild-type. Key gluconeogenic and lipogenic enzymes were repressed in the Keap1-hypo livers with concomitant activated Ampk signaling. Primary Keap1-hypo hepatocyte cultures also show increased Ampk signaling and repressed glucose production. In conclusion, increased Keap1/Nrf2 signaling in the liver is accompanied by repressed gluconeogenesis and lipogenesis that can, at least partially, explain the ameliorated diabetic phenotype in the Keap1-hypo mice.

Nrf2: control of sensitivity to carcinogens.

Induction of enzymes that enhance the detoxication of chemical carcinogens has been a broadly effective strategy for chemoprevention of experimental carcinogenesis in rodent models. Several inducing agents are now in clinical trials to evaluate utility for prevention of cancers associated with unavoidable high exposures to environmental carcinogens. The successes of these pre-clinical and clinical interventions lead to studies to define the molecular basis for protection by these agents, which now include phenolic antioxidants, dithiolethiones, isothiocyanates, and triterpenoids. In the mid-1990s, the NF-E2-related factor 2 (Nrf2) transcription factor was identified as a key regulator of the inducible expression of enzymes such as glutathione S-transferases and NAD(P)H: quinone oxidoreductase in catalyzing the detoxication of reactive electrophiles and oxidants that contribute to the formation of mutations and ultimately cancers. Nrf2 is now recognized to regulate a broad cytoprotective, transcriptional response leading to prevention of damage to DNA, proteins and lipids; recognition, repair and removal of macromolecular damage; and tissue renewal following toxic assaults. Highlighting the importance of this pathway as a determinant of susceptibility to carcinogenesis, multiple studies now demonstrate enhanced incidence, multiplicity, and/or tumor burden in Nrf2-disrupted mice compared to wild-type in models of inflammation and colon cancer, bladder cancer, lung disease and cancer, stomach cancer, mammary cancer, skin cancer, and hepatocarcinogenesis.

Characterization of the ATPase activity of the Escherichia coli RecG protein reveals that the preferred cofactor is negatively supercoiled DNA.

RecG is a member of the superfamily 2 helicase family. Its possible role in vivo is ATP hydrolysis driven regression of stalled replication forks. To gain mechanistic insight into how this is achieved, a coupled spectrophotometric assay was utilized to characterize the ATPase activity of RecG in vitro. The results demonstrate an overwhelming preference for negatively supercoiled DNA ((-)scDNA) as a cofactor for the hydrolysis of ATP. In the presence of (-)scDNA the catalytic efficiency of RecG and the processivity (as revealed through heparin trapping), were higher than on any other cofactor examined. The activity of RecG on (-)scDNA was not due to the presence of single-stranded regions functioning as loading sites for the enzyme as relaxed circular DNA treated with DNA gyrase, resulted in the highest levels of ATPase activity. Relaxation of (-)scDNA by a topoisomerase resulted in a 12-fold decrease in ATPase activity, comparable to that observed on both linear double-stranded (ds)DNA and (+)scDNA. In addition to the elevated activity in the presence of (-)scDNA, RecG also has high activity on model 4Y-substrates (i.e. chicken foot structures). This is due largely to the high apparent affinity of the enzyme for this DNA substrate, which is 46-fold higher than a 2Y-substrate (i.e. a three-way with two single-stranded (ss)DNA arms). Finally, the enzyme exhibited significant, but lower activity on ssDNA. This activity was enhanced by the Escherichia coli stranded DNA-binding protein (SSB) protein, which occurs through stabilizing of the binding of RecG to ssDNA. Stabilization is not afforded by the bacteriophage gene 32 protein, indicating a species specific, protein-protein interaction is involved. These results combine to provide significant insight into the manner and timing of the interaction of RecG with DNA at stalled replication forks.

Editor's Highlight: Pregnancy Alters Aflatoxin B1 Metabolism and Increases DNA Damage in Mouse Liver.

Pregnancy is a complex physiological state, in which the metabolism of endogenous as well as exogenous agents is ostensibly altered. One exogenous agent of concern is the hepatocarcinogen aflatoxin B1 (AFB1), a foodborne fungal toxin, that requires phase I metabolic oxidation for conversion to its toxic and carcinogenic form, the AFB1-8,9-exo-epoxide. The epoxide interacts with cellular targets causing toxicity and cell death; these targets include the covalent modification of DNA leading to mutations that can initiate malignant transformation. The main detoxification pathway of the AFB1-epoxide involves phase II metabolic enzymes including the glutathione-S-transferase (GST) family. Pregnancy can modulate both phase I and II metabolism and alter the biological potency of AFB1. The present work investigated the impact of pregnancy on AFB1 exposure in mice. A single IP dose of 6 mg/kg AFB1 was administered to pregnant C57BL/6 J mice at gestation day 14 and matched non-pregnant controls. Pregnant mice accumulated 2-fold higher AFB1-N7-guanine DNA adducts in the liver when compared with nonpregnant controls 6 h post-exposure. Enhanced DNA adduct formation in pregnant animals paralleled elevated hepatic protein expression of mouse CYP1A2 and mouse homologs of human CYP3A4, phase I enzymes capable of bioactivating AFB1. Although phase II enzymes GSTA1/2 showed decreased protein expression, GSTA3, the primary enzymatic protection against the AFB1-epoxide, was unaffected at the protein level. Taken together, our results reveal that pregnancy may constitute a critical window of susceptibility for maternal health, and provide insight into the biochemical factors that could explain the underlying risks.

Genetic or pharmacologic activation of Nrf2 signaling fails to protect against aflatoxin genotoxicity in hypersensitive GSTA3 knockout mice.

Mice are resistant to aflatoxin hepatotoxicity, primarily due to high expression of glutathione S-transferases (GSTs), and in particular the GSTA3 subunit. Nuclear factor erythroid 2 related factor 2 (Nrf2) signaling, which controls a broad-based cytoprotective response, was activated either genetically or pharmacologically in an attempt to rescue GSTA3 knockout mice from aflatoxin genotoxicity. Genetic activation of Nrf2 signaling was attained in a GSTA3: hepatocyte-specific Keap1 double knockout (DKO) mouse whereas pharmacologic activation of Nrf2 was achieved through pretreatment of mice with the triterpenoid 1-[2-cyano-3-,12-dioxoleana-1,9(11)-dien-28-oyl] imidazole (CDDO-Im) prior to aflatoxin B1 exposure. Following oral treatment with aflatoxin, urine was collected from mice for 24 h and hepatic and urinary aflatoxin metabolites then quantified using isotope dilution-mass spectrometry. Although Nrf2 was successfully activated genetically and pharmacologically, neither means affected the response of GSTA3 knockout mice to chemical insult with aflatoxin. Hepatic aflatoxin B1-N(7)-guanine levels were elevated 120-fold in GSTA3 knockout mice compared with wild-type and levels were not attenuated by the interventions. This lack of effect was mirrored in the urinary excretion of aflatoxin B1-N(7)-guanine. By contrast, urinary excretion of aflatoxin B1-N-acetylcysteine was >200-fold higher in wild-type mice compared with the single GSTA3 knockout or DKO mouse. The inability to rescue GSTA3 knockout mice from aflatoxin genotoxicity through the Nrf2 transcriptional program indicates that Gsta3 is unilaterally responsible for the detoxication of aflatoxin in mice.

Notch-Nrf2 axis: regulation of Nrf2 gene expression and cytoprotection by notch signaling.

The Notch signaling pathway enables regulation and control of development, differentiation, and homeostasis through cell-cell communication. Our investigation shows that Notch signaling directly activates the Nrf2 stress adaptive response pathway through recruitment of the Notch intracellular domain (NICD) transcriptosome to a conserved Rbpjκ site in the promoter of Nrf2. Stimulation of Notch signaling through Notch ligand expression in cells and by overexpression of the NICD in Rosa(NICD/-)::AlbCre mice in vivo induces expression of Nrf2 and its target genes. Continuous and transient NICD expression in the liver produces a Notch-dependent cytoprotective response through direct transcriptional activation of Nrf2 signaling to rescue mice from acute acetaminophen toxicity. This response can be reversed upon genetic disruption of Nrf2. Morphological studies showed that the characteristic phenotype of high-density intrahepatic bile ducts and enlarged liver in Rosa(NICD/-)::AlbCre mice could be at least partially reversed after Nrf2 disruption. Furthermore, the liver and bile duct phenotypes could be recapitulated with constitutive activation of Nrf2 signaling in Keap1(F/F)::AlbCre mice. It appears that Notch-to-Nrf2 signaling is another important determinant in liver development and function and promotes cell-cell cytoprotective signaling responses.

When NRF2 talks, who's listening?

Activation of the KEAP1-NRF2 signaling pathway is an adaptive response to environmental and endogenous stresses and serves to render animals resistant to chemical carcinogenesis and other forms of toxicity, whereas disruption of the pathway exacerbates these outcomes. This pathway, which can be activated by sulfhydryl-reactive, small-molecule pharmacologic agents, regulates the inducible expression of an extended battery of cytoprotective genes, often by direct binding of the transcription factor to antioxidant response elements in the promoter regions of target genes. However, it is becoming evident that some of the protective effects may be mediated indirectly through cross talk with additional pathways affecting cell survival and other aspects of cell fate. These interactions provide a multi-tiered, integrated response to chemical stresses. This review highlights recent observations on the molecular interactions and their functional consequences between NRF2 and the arylhydrocarbon receptor (AhR), NF-κB, p53, and Notch1 signaling pathways.

Regulation of notch1 signaling by nrf2: implications for tissue regeneration.

The Keap1-Nrf2-ARE signaling pathway elicits an adaptive response for cell survival after endogenous and exogenous stresses, such as inflammation and carcinogens, respectively. Keap1 inhibits the transcriptional activation activity of Nrf2 (p45 nuclear factor erythroid-derived 2-related factor 2) in unstressed cells by facilitating its degradation. Through transcriptional analyses in Keap1- or Nrf2-disrupted mice, we identified interactions between the Keap1-Nrf2-ARE and the Notch1 signaling pathways. We found that Nrf2 recognized a functional antioxidant response element (ARE) in the promoter of Notch1. Notch1 regulates processes such as proliferation and cell fate decisions. We report a functional role for this cross talk between the two pathways and show that disruption of Nrf2 impeded liver regeneration after partial hepatectomy and was rescued by reestablishment of Notch1 signaling.