Oxidative stress/reactive metabolite gene expression signature in rat liver detects idiosyncratic hepatotoxicants

Previously we reported a gene expression signature in rat liver for detecting a specific type of oxidative stress (OS) related to reactive metabolites (RM). High doses of the drugs disulfiram, ethinyl estradiol and nimesulide were used with another dozen paradigm OS/RM compounds, and three other drugs flutamide, phenacetin and sulindac were identified by this signature. In a second study, antiepileptic drugs were compared for covalent binding and their effects on OS/RM; felbamate, carbamazepine, and phenobarbital produced robust OS/RM gene expression. In the present study, liver RNA samples from drug-treated rats from more recent experiments were examined for statistical fit to the OS/RM signature. Of all 97 drugs examined, in addition to the nine drugs noted above, 19 more were identified as OS/RM-producing compounds—chlorpromazine, clozapine, cyproterone acetate, dantrolene, dipyridamole, glibenclamide, isoniazid, ketoconazole, methapyrilene, naltrexone, nifedipine, sulfamethoxazole, tamoxifen, coumarin, ritonavir, amitriptyline, valproic acid, enalapril, and chloramphenicol. Importantly, all of the OS/RM drugs listed above have been linked to idiosyncratic hepatotoxicity, excepting chloramphenicol, which does not have a package label for hepatotoxicity, but does have a black box warning for idiosyncratic bone marrow suppression. Most of these drugs are not acutely toxic in the rat. The OS/RM signature should be useful to avoid idiosyncratic hepatotoxicity of drug candidates.     

Type 2 diabetic rats are sensitive to thioacetamide hepatotoxicity

Previously, we reported high hepatotoxic sensitivity of type 2 diabetic (DB) rats to three dissimilar hepatotoxicants. Additional work revealed that a normally nonlethal dose of CCl4 was lethal in DB rats due to inhibited compensatory tissue repair. The present study was conducted to investigate the importance of compensatory tissue repair in determining the final outcome of hepatotoxicity in diabetes, using another structurally and mechanistically dissimilar hepatotoxicant, thioacetamide (TA), to initiate liver injury. A normally nonlethal dose of TA (300 mg/kg, ip), caused 100% mortality in DB rats. Time course studies (0 to 96 h) showed that in the non-DB rats, liver injury initiated by TA as assessed by plasma alanine or aspartate aminotransferase and hepatic necrosis progressed up to 48 h and regressed to normal at 96 h resulting in 100% survival. In the DB rats, liver injury rapidly progressed resulting in progressively deteriorating liver due to rapidly expanding injury, hepatic failure, and 100% mortality between 24 and 48 h post-TA treatment. Covalent binding of 14C-TA-derived radiolabel to liver tissue did not differ from that observed in the non-DB rats, indicating similar bioactivation-based initiation of hepatotoxicity. S-phase DNA synthesis measured by [3H]-thymidine incorporation, and advancement of cells through the cell division cycle measured by PCNA immunohistochemistry, were substantially inhibited in the DB rats compared to the non-DB rats challenged with TA. Thus, inhibited cell division and compromised tissue repair in the DB rats resulted in progressive expansion of liver injury culminating in mortality. In conclusion, it appears that similar to type 1 diabetes, type 2 diabetes also increases sensitivity to dissimilar hepatotoxicants due to inhibited compensatory tissue repair, suggesting that sensitivity to hepatotoxicity in diabetes occurs in the absence as well as presence of insulin.     

Neurofilament a biomarker of neurodegeneration in autoimmune encephalomyelitis

Monitoring neuroaxonal loss in multiple sclerosis is an important objective to study the pathogenesis and response to treatment of the disease. The release of neurofilaments is a potential surrogate biomarker of neurodegeneration. One route to explore this aspect further is through the use of animal models that have well-defined, and predictable, disease courses. The release of neurofilaments into plasma across the course of relapsing-remitting and secondary progressive experimental autoimmune encephalomyelitis in Biozzi ABH was assessed, as well as measuring anti-neurofilament antibodies using ELISA. It was found that there was an immediate release in neurofilaments into the blood following the initial paralytic attack. Neurofilament release was continuous in relapse and remission but returned towards baseline in chronic disease, as animals entered the post-relapsing progressive phase of the disease. This was mirrored by a loss of neurofilament-specific antibodies. In contrast neurofilament levels increased dramatically as neurodegeneration and clinical disease occurred in the G93A SOD1 transgenic C57BL/6 x SJL mice, model of amyotrophic lateral sclerosis. These data further support neurofilament levels as a good surrogate measure of neurodegeneration and their potential use as a surrogate endpoint in neuroprotective studies.     

Rhinitis in Older Adults

Rhinitis symptoms of rhinorrhea, congestion, sneezing, nasal/ocular pruritis, and postnasal drainage can significantly affect the quality of life for older adults. As the US population ages, it will be increasingly important for health-care providers to effectively diagnose and manage rhinitis. Rhinitis is categorized broadly into allergic rhinitis and non-allergic rhinitis. Environmental changes and avoidance measures are a primary means of intervention. In addition, there are several topical therapies (nasal sprays) that can be effective for symptom control.     

Targeting the mTOR/4E-BP pathway in endometrial cancer

Endometrial cancer is the most common gynecologic malignancy. Although it is highly treatable in the early stages of disease, therapies for advanced and recurrent disease are rarely curative. A molecular and genetic understanding of endometrial cancer involves the mTOR signaling pathway, an emerging target for treatment of type I disease (the most common presentation). Endometrial cancers show a significant reliance on the mTOR pathway for survival, and studies to date have revealed a clinical advantage in targeting this pathway. Less well developed in the study of endometrial cancer is an understanding of mTOR signaling to its major downstream effector, translational control. Given the poor rate of success for treatment of late-stage endometrial cancer, increasing attention is being directed to the development of new therapeutic approaches, including targeting the mTOR pathway. Here, we discuss the potential benefit of targeting mTOR combined with existing chemotherapies by monitoring its impact on translational regulatory pathways and key translation targets in endometrial cancer. We also highlight laboratory and clinical research findings that will provide new avenues for future research and clinical development.     

Changes in immune function in asthma in the elderly

Asthma is an inflammatory disorder of the airway. The airway inflammation of asthma is typically an allergic inflammation characterized by cells and mediators described as a "Th2" inflammatory response. There is a growing body of evidence describing changes in the function of immune cells upon aging, a phenomenon referred to as "immunosenescence". Several studies utilizing animal models and human subjects with asthma have begun to explore age-related effects on the airway inflammation in asthma. This review explores the existing data on the presence and effects of immunosenescence or age-related changes in immune function in asthma.     

Hepatic expression of heme oxygenase-1 and antioxidant response element-mediated genes following administration of ethinyl estradiol to rats

Heme oxygenase-1 (HO-1) is one of several enzymes induced by hepatotoxicants, and is thought to have an important protective role against cellular stress during liver inflammation and injury. The objective of the present study was to evaluate the role of HO-1 in estradiol-induced liver injury. A single dose of ethinyl estradiol (500 mg/kg, po) resulted in mild liver injury. Repeated administration of ethinyl estradiol (500 mg/kg/day for 4 days, po) resulted in no detectable liver injury or dysfunction. Using RT-PCR analysis, we demonstrate that HO-1 gene expression in whole liver tissue is elevated (>20-fold) after the single dose of ethinyl estradiol. The number and intensity of HO-1 immunoreactive macrophages were increased after the single dose of ethinyl estradiol. HO-1 expression was undetectable in hepatic parenchymal cells from rats receiving Methocel control or a single dose of ethinyl estradiol, however cytosolic HO-1 immunoreactivity in these cells after repeated dosing of ethinyl estradiol was pronounced. The increases in HO-1 mRNA and HO-1 immunoreactivity following administration of a single dose of ethinyl estradiol suggested that this enzyme might be responsible for the observed protection of the liver during repeated dosing. To investigate the effect of HO-1 expression on ethinyl estradiol-induced hepatotoxicity, rats were pretreated with hemin (50 micromol/kg, ip, a substrate and inducer of HO-1), with tin protoporphyrin IX (60 micromol/kg, ip, an HO-1 inhibitor), or with gadolinium chloride (10 mg/kg, iv, an inhibitor/toxin of Kupffer cells) 24 h before ethinyl estradiol treatment. Pretreatment with modulators of HO-1 expression and activity had generally minimal effects on ethinyl estradiol-induced liver injury. These data suggest that HO-1 plays a limited role in antioxidant defense against ethinyl estradiol-induced oxidative stress and hepatotoxicity, and suggests that other coordinately induced enzymes are responsible for protection observed with repeated administration of high doses of this compound.     

Dual mTORC1/2 inhibition in a preclinical xenograft tumor model of endometrial cancer

Objectives

Up to 70% of endometrioid endometrial cancers carry PTEN gene deletions that can upregulate mTOR activity. Investigational mTOR kinase inhibitors may provide a novel therapeutic approach for these tumors. Using a xenograft tumor model of endometrial cancer, we assessed the activity of mTOR and downstream effector proteins in the mTOR translational control pathway after treatment with a dual mTOR complex 1 and 2 (mTORC1/2) catalytic inhibitor (PP242) compared to that of an allosteric mTOR complex 1 (mTORC1) inhibitor (everolimus, RAD001).

Methods

Grade 3 endometrioid endometrial cancer cells (AN3CA) were xenografted into nude mice. Animals were treated with PP242, PP242 and carboplatin, carboplatin, RAD001, and RAD001 and carboplatin. Mean tumor volume was compared across groups by ANOVA. Immunoblot analysis was performed to assess mTORC1/2 activity using P-Akt, P-S6 and P-4E-BP1.

Results

The mean tumor volume of PP242 + carboplatin was significantly lower than in all other treatment groups, P < 0.001 (89% smaller). The RAD001 + carboplatin group was also smaller, but this did not reach statistical significance (P = 0.097). Immunoblot analysis of tumor lysates treated with PP242 demonstrated inhibition of activated P-Akt.

Conclusions

Catalytic mTORC1/2 inhibition demonstrates clear efficacy in tumor growth control that is enhanced by the addition of a DNA damage agent, carboplatin. Targeting mTORC1/2 leads to inhibition of Akt activation and strong downregulation of effectors of mTORC1, resulting in downregulation of protein synthesis. Based on this study, mTORC1/2 kinase inhibitors warrant further investigation as a potential treatment for endometrial cancer.