Gene expression profiling analysis reveals arsenic-induced cell cycle arrest and apoptosis in p53-proficient and p53-deficient cells through differential gene pathways

Arsenic (As) is a well-known environmental toxicant and carcinogen as well as an effective chemotherapeutic agent. The underlying mechanism of this dual capability, however, is not fully understood. Tumor suppressor gene p53, a pivotal cell cycle checkpoint signaling protein, has been hypothesized to play a possible role in mediating As-induced toxicity and therapeutic efficiency. In this study, we found that arsenite (As³⁺) induced apoptosis and cell cycle arrest in a dose-dependent manner in both p53^+/+ and p53^−/− mouse embryonic fibroblasts (MEFs). There was, however, a distinction between genotypes in the apoptotic response, with a more prominent induction of caspase-3 in the p53^−/− cells than in the p53^+/+ cells. To examine this difference further, a systems-based genomic analysis was conducted comparing the critical molecular mechanisms between the p53 genotypes in response to As³⁺. A significant alteration in the Nrf2-mediated oxidative stress response pathway was found in both genotypes. In p53^+/+ MEFs, As³⁺ induced p53-dependent gene expression alterations in DNA damage and cell cycle regulation genes. However, in the p53^−/− MEFs, As³⁺ induced a significant up-regulation of pro-apoptotic genes (Noxa) and down-regulation of genes in immune modulation. Our findings demonstrate that As-induced cell death occurs through a p53-independent pathway in p53 deficient cells while apoptosis induction occurs through p53-dependent pathway in normal tissue. This difference in the mechanism of apoptotic responses between the genotypes provides important information regarding the apparent dichotomy of arsenic's dual mechanisms, and potentially leads to further advancement of its utility as a chemotherapeutic agent.     

Cadmium modulates adipocyte functions in metallothionein-null mice

Our previous study has demonstrated that exposure to cadmium (Cd), a toxic heavy metal, causes a reduction of adipocyte size and the modulation of adipokine expression. To further investigate the significance of the Cd action, we studied the effect of Cd on the white adipose tissue (WAT) of metallothionein null (MT^−/−) mice, which cannot form atoxic Cd–MT complexes and are used for evaluating Cd as free ions, and wild type (MT^+/+) mice. Cd administration more significantly reduced the adipocyte size of MT^−/− mice than that of MT^+/+ mice. Cd exposure also induced macrophage recruitment to WAT with an increase in the expression level of Ccl2 (MCP-1) in the MT^−/− mice. The in vitro exposure of Cd to adipocytes induce triglyceride release into culture medium, decrease in the expression levels of genes involved in fatty acid synthesis and lipid hydrolysis at 24 h, and at 48 h increase in phosphorylation of the lipid-droplet-associated protein perilipin, which facilitates the degradation of stored lipids in adipocytes. Therefore, the reduction in adipocyte size by Cd may arise from an imbalance between lipid synthesis and lipolysis. In addition, the expression levels of leptin, adiponectin and resistin decreased in adipocytes. Taken together, exposure to Cd may induce unusually small adipocytes and modulate the expression of adipokines differently from the case of physiologically small adipocytes, and may accelerate the risk of developing insulin resistance and type 2 diabetes.     

Comparison of citrus coumarins on carcinogen-detoxifying enzymes in Nrf2 knockout mice

Naturally occurring coumarins possess anti-carcinogenic activities in part by inducing carcinogen-detoxifying enzymes glutathione S-transferase (GST) and/or NAD(P)H quinone oxidoreductase (NQO1). Our goal was to determine whether citrus coumarins induce hepatic GST and/or NQO1 via activation of Nrf2 and the antioxidant response element (ARE). First, HepG2 cells stably transfected with the ARE and a green fluorescent protein (GFP) reporter were treated with increasing concentrations of coumarins and compared to positive controls. tert-Butylhydroquinone (TBHQ) and oltipraz increased GFP fluorescence, as did coumarin, limettin, auraptene, imperatorin, and 7,8-benzoflavone, suggesting that they activate the ARE, whereas isopimpinellin did not increase GFP fluorescence. Next, the effects of orally administered coumarins and oltipraz on hepatic GST and NQO1 activities were compared in Nrf2 knockout mice or Nrf2 heterozygous mice exhibiting the wild-type phenotype. Oltipraz, auraptene, imperatorin, isopimpinellin, and auraptene all significantly increased liver cytosolic GST activities in Nrf2 heterozygous mice. This effect was abrogated in Nrf2(−/−) mice dosed with oltipraz, attenuated in mice Nrf2(−/−) mice treated with auraptene and imperatorin, and still significant in Nrf2(−/−) mice treated with isopimpinellin. Of these compounds, only isopimpinellin significantly increased liver cytosolic NQO1 activities, and this effect was not attenuated in Nrf2(−/−) mice. These results strongly suggest that imperatorin and auraptene induce murine liver cytosolic GST activities via the Nrf2/ARE mechanism. Although structurally similar, isopimpinellin did not appear to activate HepG2–ARE–GFP and the Nrf2 knockout mouse study suggests that isopimpinellin may induce GST and NQO1 via additional mechanisms.     

PYDDT,a novel phase 2 enzymes inducer,activates Keap1–Nrf2 pathway via depleting the cellular level of glutathione

Keap1–Nrf2 pathway has emerged as a regulator for the endogenous antioxidant response, which is critical in defending cells against carcinogenesis. Herein, we demonstrated that depleting the cellular level of glutathione (GSH) by a novel electrophilic agent 2-(pro-1-ynyl)-5-(5,6-dihydroxypenta-1,3-diynyl) thiophene (PYDDT) could activate Keap1–Nrf2 pathway. In above process, it was found that Keap1 was modified by S-glutathionylation, an important post-translational modification of protein cysteines with critical roles in oxidative stress and signal transduction. We concluded from our findings that conjugation with intracellular GSH by PYDDT might lead to Keap1 S-glutathionylation and was a key event involved in its Nrf2 inducing activity.     

In utero exposure to benzo[a]pyrene increases adiposity and causes hepatic steatosis in female mice,and glutathione deficiency is protective

Polycyclic aromatic hydrocarbons (PAHs), including benzo[a]pyrene (BaP), are ubiquitous environmental pollutants found in tobacco smoke, air pollution, and grilled foods. Reactive metabolites and reactive oxygen species generated during PAH metabolism are detoxified by reactions involving glutathione (GSH). Early life exposures to tobacco smoke and air pollution have been linked to increased risk of obesity and metabolic syndrome. We investigated the independent and interactive effects of prenatal exposure to BaP and GSH deficiency due to deletion of the modifier subunit of glutamate cysteine ligase (Gclm), the rate-limiting enzyme in GSH synthesis, on adiposity and hepatic steatosis in adult female F1 offspring. We mated Gclm^+/− dams with Gclm^+/− males and treated the pregnant dams with 0, 2, or 10 mg/kg/day BaP in sesame oil by oral gavage daily from gestational day 7 through 16. We analyzed metabolic endpoints in female Gclm^−/− and Gclm^+/+ littermate F1 offspring. Prenatal BaP exposure significantly increased visceral adipose tissue weight, weight gain between 3 weeks and 7.5 months of age, hepatic lipid content measured by oil red O staining, and hepatic fatty acid beta-oxidation gene expression in Gclm^+/+, but not in Gclm^−/−, female offspring. Hepatic expression of lipid biosynthesis and antioxidant genes were decreased and increased, respectively, in Gclm^−/− mice. Our results suggest that reported effects of pre- and peri-natal air pollution and tobacco smoke exposure on obesity may be mediated in part by PAHs. GSH deficiency is protective against the metabolic effects of prenatal BaP exposure.     

Participation of nuclear factor (erythroid 2-related), factor 2 in ameliorating lithocholic acid-induced cholestatic liver injury in mice

BACKGROUND AND PURPOSE

Lithocholic acid (LCA), the most toxic bile acid, induces cholestatic liver injury in rodents. We previously showed that LCA activates the oxidative stress-responsive nuclear factor (erythroid-2 like), factor 2 (Nrf2) in cultured liver cells, triggering adaptive responses that reduce cell injury. In this study, we determined whether Nrf2 protects the liver against LCA-induced toxicity in vivo.

EXPERIMENTAL APPROACH

Nrf2 disrupted (Nrf2^−/−) and wild-type mice were treated with LCA (125 mg·kg⁻¹ body weight) to induce liver injury. Levels of mRNA, protein and function of important Nrf2 target genes coupled with liver histology and injury biomarkers of mice were examined.

KEY RESULTS

In 4 day LCA treatments, we observed a significantly higher hepatic induction of Nrf2 target, cytoprotective genes including thioredoxin reductase 1, glutamate cysteine ligase subunits, glutathione S-transferases, haeme oxygenase-1 and multidrug resistance-associated proteins 3 and 4 in the wild type as compared with the Nrf2^−/− mice. Moreover, basal and LCA-induced hepatic glutathione and activities of glutathione S-transferases and thioredoxin reductases were higher in wild-type than in Nrf2^−/− mice. This reduced production of cytoprotective genes against LCA toxicity rendered Nrf2^−/− mice more susceptible to severe liver damage with the presence of multifocal liver necrosis, inflamed bile ducts and elevation of lipid peroxidation and liver injury biomarkers, such as alanine aminotransferase and alkaline phosphatase.

CONCLUSIONS AND IMPLICATIONS

Nrf2 plays a crucial cytoprotective role against LCA-induced liver injury by orchestrating adaptive responses. The pharmacological potential of targeting liver Nrf2 in the management of cholestatic liver diseases is proposed.     

Molecular mechanism of trichloroethylene-induced hepatotoxicity mediated by CYP2E1

Cytochrome P450 (CYP) 2E1 was suggested to be the major enzyme involved in trichloroethylene (TRI) metabolism and TRI-induced hepatotoxicity, although the latter molecular mechanism is not fully understood. The involvement of CYP2E1 in TRI-induced hepatotoxicity and its underlying molecular mechanism were studied by comparing hepatotoxicity in cyp2e1^+/+ and cyp2e1^−/− mice. The mice were exposed by inhalation to 0 (control), 1000, or 2000 ppm of TRI for 8 h a day, for 7 days, and TRI-hepatotoxicity was assessed by measuring plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities and histopathology. Urinary metabolites of trichloroethanol and trichloroacetic acid (TCA) were considerably greater in cyp2e1^+/+ compared to cyp2e1^−/− mice, suggesting that CYP2E1 is the major P450 involved in the formation of these metabolites. Consistent with elevated plasma ALT and AST activities, cyp2e1^+/+ mice in the 2000 ppm group showed histopathological inflammation. TRI significantly upregulated PPARα, which might function to inhibit NFκB p50 and p65 signalling. In addition, TRI-induced NFκB p52 mRNA, and significantly positive correlation between NFκB p52 mRNA expression and plasma ALT activity levels were observed, suggesting the involvement of p52 in liver inflammation. Taken together, the current study directly demonstrates that CYP2E1 was the major P450 involved in the first step of the TRI metabolism, and the metabolites produced may have two opposing roles: one inducing hepatotoxicity and the other protecting against the toxicity. Intermediate metabolite(s) from TRI to chloral hydrate produced by CYP2E1-mediated oxidation may be involved in the former, and TCA in the latter.     

Thermodynamics of A2B adenosine receptor binding discriminates agonistic from antagonistic behaviour

Thermodynamic parameters ΔG°, ΔH° and ΔS° of the binding equilibrium of 12 ligands (six agonists and six antagonists) to the A_2B adenosine receptor subtype have been determined by affinity measurements carried out on HEK 293 cells stably transfected with human A_2B adenosine receptors at six different temperatures (4, 10, 15, 20, 25, 30 °C) and van’t Hoff plot analysis have been performed. Affinity constants were obtained from saturation experiments of [³H]MRE 2029-F20 or by its displacement in inhibition assays for the other compounds. van’t Hoff plots were essentially linear in the temperature range investigated, showing that the ΔCp° of the binding equilibrium is nearly zero. Thermodynamic parameters are in the range 7 ≤ ΔH° ≤ 23 kJ mol⁻¹and 123 ≤ ΔS° ≤ 219 J K⁻¹ mol⁻¹ for agonists and −40 ≤ ΔH° ≤ −20 kJ mol⁻¹ and 10 ≤ ΔS° ≤ 91 J K⁻¹ mol⁻¹ for antagonists indicating that agonistic binding is always totally entropy-driven while antagonistic binding is enthalpy and entropy-driven. In the −TΔS° versus ΔH° plot the thermodynamic data are clearly arranged in separate clusters for agonists and antagonists, which, therefore, turn out to be thermodynamically discriminated.     

Nrf2/ARE signaling protects against oxaliplatin-induced hepatotoxicity in mice

Nuclear factor erythroid 2-related factor 2 (Nrf2) controls the expression of a wide array of antioxidant response element (ARE)-driven genes, which are involved in stress response and metabolism regulation. The role of Nrf2/ARE signaling in resistances of cancer cells to radiotherapy and chemotherapy has been widely accepted. However, much less is known about the relevance of Nrf2 to chemotherapy-associated toxicities, such as hepatotoxicity. In the present study, nine chemotherapeutic agents were firstly tested in embryonic fibroblasts (MEFs) and hepatocytes isolated from Nrf2 deficient or wild-type mice. The results indicate that the cytotoxicity of oxaliplatin in hepatocytes was significantly higher than that in MEFs and enhanced by Nrf2 deficiency. Furthermore, oxaliplatin treatment caused more pronounced steatosis and severer liver injury in Nrf2^–/–mice compared with wild-type counterparts, as evidenced by dramatically elevated serum transaminase and bilirubin, increased accumulation of fat, inflammatory infiltration and blood congestion. The increased hepatotoxicity in Nrf2 deficient mice was possibly caused by decreased expression of antioxidant genes and glutathione depletion. Our results demonstrated that oxaliplatin-inducedhepatotoxicity was significantly impacted by Nrf2 status, therefore Nrf2 could potentially serve as a biomarker to predict or a target to prevent hepatotoxicity of oxaliplatin.     

Binding thermodynamics at the human cannabinoid CB1 and CB2 receptors

The thermodynamic parameters ΔG°, ΔH° and ΔS° of the binding equilibrium of agonists and antagonists at cannabinoid CB₁ and CB₂ receptors were determined by means of affinity measurements at different temperatures and van’t Hoff plots were constructed. Affinity constants were measured on CHO cells transfected with the human CB₁ and CB₂ receptors by inhibition assays of the binding of the cannabinoid receptor agonist [³H]-CP-55,940. van’t Hoff plots were linear for agonists and antagonists in the temperature range 0-30 °C. The thermodynamic parameters for CB₁ receptors fall in the ranges 17 ≤ ΔH° ≤ 59 kJ/mol and 213 ≤ ΔS° ≤ 361 kJ/mol for agonists and −52 ≤ ΔH° ≤ −26 kJ/mol and −12 ≤ ΔS° ≤ 38 kJ/mol for antagonists. The thermodynamic parameters for CB₂ receptors fall in the ranges 27 ≤ ΔH° ≤ 48 kJ/mol and 234 ≤ ΔS° ≤ 300 kJ/mol for agonists and −19 ≤ ΔH° ≤ −17 kJ/mol and 43 ≤ ΔS° ≤ 74 kJ/mol for antagonists. Collectively, these data show that agonist binding is always totally entropy-driven while antagonist binding is enthalpy and entropy-driven, indicating that CB₁ and CB₂ receptors are thermodynamically discriminated. These data could give new details on the nature of the forces driving the CB₁ and CB₂ binding at a molecular level. Enthalpy, entropy, free energy and binding affinity for each ligand to its receptor can all be assessed and therefore the optimal binding profile discovered. Carrying out these binding investigations as early as possible in the discovery process increases the probability that a lead compound will become a successful pharmaceutical compound.     

Metabolism and Tissue Distribution of Sulforaphane in Nrf2 Knockout and Wild-Type Mice

Purpose  

To determine the metabolism and tissue distribution of the dietary chemoprotective agent sulforaphane following oral administration to wild-type and Nrf2 knockout (Nrf2^−/−) mice.