Mesenchyme-derived factors enhance preneoplastic growth by non-genotoxic carcinogens in rat liver.

Many frequently prescribed drugs are non-genotoxic carcinogens (NGC) in rodent liver. Their mode of action and health risks for humans remain to be elucidated. Here, we investigated the impact of two model NGC, the anti-epileptic drug phenobarbital (PB) and the contraceptive cyproterone acetate (CPA), on intrahepatic epithelial-mesenchymal crosstalk and on growth of first stages of hepatocarcinogenesis. Unaltered hepatocytes (HC) and preneoplastic HC (HCPREN) were isolated from rat liver for primary culture. DNA replication of HC and HCPREN was increased by in vitro treatment with 10 µM CPA, but not 1 mM PB. Next, mesenchymal cells (MC) obtained from liver of rats treated with either PB (50 mg/kg bw/day) or CPA (100 mg/kg bw/day), were cultured. Supernatants from both types of MC raised DNA synthesis of HC and HCPREN. This indicates that PB induces replication of HC and HCPREN only indirectly, via growth factors secreted by MC. CPA, however, acts on HC and HCPREN directly as well as indirectly via mesenchymal factors. Transcriptomics and bio-informatics revealed that PB and CPA induce extensive changes in the expression profile of MC affecting many growth factors and pathways. MC from PB-treated rats produced and secreted enhanced levels of HBEGF and GDF15, factors found to suppress apoptosis and/or induce DNA synthesis in cultured HC and HCPREN. MC from CPA-treated animals showed enhanced expression and secretion of HGF, which strongly raised DNA replication of HC and HCPREN. In conclusion, our findings reveal profound effects of two prototypical NGC on the hepatic mesenchyme. The resulting release of factors, which suppress apoptosis and/or enhance cell replication preferentially in cancer prestages, appears to be crucial for tumor promotion by NGC in the liver.

Proinflammatory mesenchymal effects of the non-genotoxic hepatocarcinogen phenobarbital: a novel mechanism of antiapoptosis and tumor promotion.

Many environmental pollutants and drugs, including steroid hormones, hypolipidemics and antiepileptics, are non-genotoxic carcinogens (NGC) in rodent liver. The mechanism of action and the risk for human health are still insufficiently known. Here, we study the effects of phenobarbital (PB), a widely used model NGC, on hepatic epithelial-mesenchymal crosstalk and the impact on hepatic apoptosis. Mesenchymal cells (MC) and hepatocytes (HC) were isolated from control and PB-treated rat livers. PB induced extensive changes in gene expression in MC and much less in HC as shown by transcriptomics with oligoarrays. In MC only, transcript levels of numerous proinflammatory cytokines were elevated. Correspondingly, ELISA on the supernatant of MC from PB-treated rats revealed enhanced release of various cytokines. In cultured HC, this supernatant caused (i) nuclear translocation and activation of nuclear factor-κB (shown by immunoblots of nuclear extracts and reporter gene assays), (ii) elevated expression of proinflammatory genes and (iii) protection from the proapoptotic action of transforming growth factor beta 1 (TGFß1). PB treatment in vivo or in vitro elevated the production and release of tumor necrosis factor alpha from MC, which was identified as mainly responsible for the inhibition of apoptosis in HC. In conclusion, our findings reveal profound proinflammatory effects of PB on hepatic mesenchyme and mesenchymal-epithelial interactions. The resulting release of cytokines acts antiapoptotic in HC, an effect crucial for tumor promotion and carcinogenesis by NGC.

Superoxide deficiency attenuates promotion of hepatocarcinogenesis by cytotoxicity in NADPH oxidase knockout mice.

Long-term exposure to carcinogens combined with chronic hepatitis contributes greatly to the worldwide high incidence of hepatocellular carcinoma (HCC). It is still unclear to which extent the release of pro-inflammatory reactive oxygen or nitrogen species contributes to the development of this malignancy. Here, we aim to elucidate the role of superoxide in a model of chemical hepatocarcinogenesis. p47(phox) knockout mice (KO), lacking superoxide formation by phagocytic NADPH oxidase (phox), and wild-type animals (WT) were subjected to two different initiation-promotion protocols: (1) single dose of diethylnitrosamine (DEN) at 6 weeks of age followed by phenobarbital (PB) via diet, or ethanol (EtOH) in drinking water; (2) DEN at neonatal age followed by three cytotoxic doses of DEN at intervals of 6-7 weeks. The appearance of tumors and prestages was quantified. There was no obvious difference in the capacity of DEN to initiate hepatocarcinogenesis in KO and WT mice. PB promoted tumor development in both genotypes without significant difference. EtOH induced steatosis significantly less in KO than in WT liver, but had no effect on tumor formation in either genotype. However, hepatocarcinogenesis by three cytotoxic DEN doses after neonatal initiation was attenuated significantly in KO. Macrophages/monocytes identified by the specific antigen F4/80 were more abundant in KO than in WT liver, possibly reflecting a compensatory response. We conclude that phox-derived superoxide is not essential but is supportive for the promotion of hepatocarcinogenesis by cytotoxic doses of DEN. The production of superoxide may therefore contribute to the promotion of hepatocarcinogenesis by cytotoxic/pro-inflammatory stimuli.

FAPI Uptake in Gallbladder Is It Normal Biodistribution?

ABSTRACT: Radiolabeled fibroblast activation protein inhibitors (FAPIs) have been extensively used in different types of cancers, although not yet FDA approved. Normal patterns of FAPI biodistribution have been investigated, and it is known that FAPI is expressed in nonmalignant pathophysiological lesions, characterized by tissue remodeling such as atherosclerosis, arthritis, and scar/fibrotic tissues. In this interesting image, we are presenting the accumulation of 68 Ga-FAPI in the gallbladder. This finding could be related to a normal distribution of the radiotracer as a physiologic finding. This is a potentially important finding as FAPI may be used as theragnostic agent in the future.

Food-derived peroxidized fatty acids may trigger hepatic inflammation: a novel hypothesis to explain steatohepatitis.

BACKGROUND & AIMS: Obesity and hepatic steatosis are frequently associated with the development of a non-alcoholic steatohepatitis (NASH). The mechanisms driving progression of a non-inflamed steatosis to NASH are largely unknown. Here, we investigated whether ingestion of peroxidized lipids, as being present in Western style diet, triggers the development of hepatic inflammation. METHODS: Corn oil containing peroxidized fatty acids was administered to rats by gavage for 6 days. In a separate approach, hepatocytes (HC), endothelial (EC) and Kupffer cells (KC) were isolated from untreated livers, cultured, and incubated with peroxidized linoleic acid (LOOH; linoleic acid (LH) being the main fatty acid in corn oil). Samples obtained from in vivo and in vitro studies were mainly investigated by qRT-PCR and biochemical determinations of lipid peroxidation products. RESULTS: Rat treatment with peroxidized corn oil resulted in increased hepatic lipid peroxidation, upregulation of nitric oxide synthetase-2 (NOS-2), cyclooxygenase-2 (COX-2), interleukin-1ß (IL-1ß), and tumor necrosis factor-α (TNFα), elevation of total nitric oxides, and increase in cd68-, cd163-, TNFα-, and/or COX-2 positive immune cells in the liver. When investigating liver cell types, LOOH elevated the secretion of TNFα, p38MAPK phosphorylation, and mRNA levels of NOS-2, COX-2, and TNFα, mainly in KC. The elevation of gene expression could be abrogated by inhibiting p38MAPK, which indicates that p38MAPK activation is involved in the pro-inflammatory effects of LOOH. CONCLUSIONS: These data show for the first time that ingestion of peroxidized fatty acids carries a considerable pro-inflammatory stimulus into the body which reaches the liver and may trigger the development of hepatic inflammation.

Case Report of RANBP2 Mutation and Familial Acute Necrotizing Encephalopathy.

INTRODUCTION: Acute necrotizing encephalopathy (ANE), a rare entity with unique clinical presentation, can be associated significant morbidity and mortality. The majority of ANE reported cases are sporadic. However, reports of extremely rare familial cases are scarce. Case Presentation. We described three cases, two siblings and their cousin, affected by ANE, all of them exhibiting RAN-binding protein 2 (RANBP2) gene mutation. They all presented with seizure and decreased level of consciousness. Unlike the siblings, the cousin eventually expired mainly due to the delay in diagnosis, resulting from late presentation of typical brain involvements of ANE in magnetic resonance imaging (MRI). CONCLUSION: The presented cases are the first reports of familial ANE in Iran. Attempt was made to raise awareness on this disease, because high clinical suspicion plays an important role in the early diagnosis and proper management of these patients.

Genetic Testing in Various Neurodevelopmental Disorders Which Manifest as Cerebral Palsy: A Case Study From Iran.

Purpose: Cerebral palsy (CP) is a heterogeneous permanent disorder impacting movement and posture. Investigations aimed at diagnosing this disorder are expensive and time-consuming and can eventually inconclusive. This study aimed to determine the diagnostic yield of next generation sequencing in patients with atypical CP (ACP). Methods: Patient eligibility criteria included impaired motor function with onset at birth or within the first year of life, and one or more of the following conditions: severe intellectual disability, positive family history, brain imaging findings not typical for cerebral palsy, abnormal neurometabolic profile, intractable seizure, normal neuroimaging despite severe psychomotor disability, after pediatric neurologist assessment including neuroimaging and biochemical-metabolic study offered for genetic study. Results: Exome sequencing was done for 66 patients which revealed pathogenic, likely pathogenic, and variants of unknown significance in 36.2, 9, and 43.9%, respectively. We also found 10 new mutations and were able to suggest specific and personalized treatments for nine patients. We also found three different mutations with different phenotypical spectrum in one gene that have not been reported for cerebral palsy. Conclusion: An accurate history and physical examination and determination of patients with atypical cerebral palsy for doing exome sequencing result in improved genetic counseling and personalized management.

Analysis of hematological parameters as prognostic markers for toxicity and survival of 223Radium treatment.

223Radium (223Ra) has emerged as treatment prolonging survival in patients with metastatic castration-resistant prostate cancer (CRPC). As 223Ra can cause hematotoxicity (HT), pre-existing hematopoiesis might influence the efficacy of 223Ra and the rate of hematotoxicity, but as to our knowledge such data has not been published yet, we retrospectively conducted an analysis on patients receiving 223Ra. 54 patients treated with 223Ra had a median survival of 67 weeks, which was significantly reduced in patients with pre-existing Hb toxicity (Tox) grade 2 (48 weeks P = 0.008) as compared to grade 1 (67 weeks) and normal levels of Hb (not reached); survival in patients with Plt Tox grade 1 was significantly reduced (44 weeks) as compared to normal Plt counts (71 weeks, P = 0.033). Patients with impaired hematopoiesis regarding Hb and Plts developed significantly more grade 3 and 4 HT (Hb < 10 g/dl: 42.9% [3/7] vs 10.6% [5/47], P < 0.001; Plt < 150 G/L: 28.6% [2/7] vs 6.4% [3/47], P = 0.002) and received significantly fewer treatment cycles (Hb <10 g/dl: 5.1 vs 5.8, P = 0.04; Plt < 150 G/L: 3.4 vs 5.6, P < 0.001). These results imply that pre-existing impaired hematopoiesis, in particular thrombocytopenia and anemia, before 223Ra therapy, is an important risk factor for worse outcome of treatment with 223Ra.

[11C]acetate PET as a tool for diagnosis of liver steatosis.

PURPOSE: To investigate [11C]acetate PET-surrogate parameter of fatty acid synthase activity-as suitable tool for diagnosis and monitoring of liver steatosis. METHODS: In this retrospective study, data were obtained from 83 prostatic carcinoma patients from 1/2008 to 1/2014. Mean HU was calculated from unenhanced CT of all patients from liver with liver HU less than 40 as threshold for liver steatosis. SUVmax of the liver and of the blood pool in thoracic aorta (as background for calculation of a liver/background ratio [SUVl/b]) was measured. t test was used with a P < 0.05 considered as statistically significant difference and ROC analysis was used for calculating specificity and sensitivity. RESULTS: 19/83 patients (20%) had diagnosis of hepatic steatosis according to CT. Uptake of [11C]acetate was significantly higher in patients with hepatic steatosis as compared to control group (SUVmax 7.96 ± 2.0 vs. 5.48 ± 2.3 [P < 0.001]). There was also a significant correlation between both SUVmax (r = - 0.52, P < 0.001) and SUVl/b (r = - 0.59, P < 0.001) with the density (HU) of the liver. In ROC analysis for detection of liver steatosis SUVmax (threshold: 5.86) had a sensitivity of 94% and specificity of 69% with an AUC of 0.81. Increasing body mass index is correlated with the severity of steatosis. CONCLUSION: We showed for the first time that hepatic steatosis associates with increased [11C]acetate uptake. Also, severity of steatosis correlates with [11C]acetate uptake. [11C]acetate uptake PET seems promising for the assessment of liver steatosis.

Cyclic vomiting syndrome in children: experience with 181 cases from southern Iran.

AIM: To evaluate the clinical presentation, response to prophylactic therapy and outcome of children with cyclic vomiting syndrome (CVS) in Shiraz, Iran. METHODS: During a period of 11 years (March 1994 to March 2005), 181 consecutive children with a final diagnosis of CVS were evaluated, treated and followed in our center. Patients were randomized to receive either amitriptyline or propranolol as prophylactic treatments. RESULTS: There were 88 boys and 93 girls with mean age of onset of symptoms of 4.9 +/- 3.3 years (range, neonatal period to 14 years), the mean age at final diagnosis was 6.9 years (range, 1.5 to 14), and the mean duration between the onset of the first attack and the final diagnosis of CVS was 2 +/- 1.81 years (range, 1/6 to 8). The mean duration of each attack was 4.26 days (range, from few hours to 10 d) and the mean interval between the attacks was 1.8 mo (range, 1 wk to 12 mo). The time of onset of the attacks was midnight to early morning in about 70% of cases. Amitriptyline was effective in 46 out of 81 (56%) patients (P < 0.001). Propranolol appeared to have a superior action and was effective in 74 out of 83 (92%) patients (P < 0.0001). CONCLUSION: There is a significant lag time between the onset of clinical symptoms and the final diagnosis of CVS in our area. In patients with typical clinical presentations of CVS, who are examined by an experienced physician, invasive workup is not necessary. Propranolol appears more effective than amitriptyline for prophylactic use in children with CVS.

Irritable bowel syndrome in adults over 35 years in Shiraz, southern Iran: prevalence and associated factors.

BACKGROUND: The symptoms of irritable bowel syndrome (IBS) are common in the general population. The aim of this population-based study was to determine the prevalence of IBS and describe the associated factors including demographic, life style and health-seeking behaviors in Shiraz city, southern Iran. METHODS: From April to September 2004, 1978 subjects aged > 35 years old completed a validated and reliable questionnaire on IBS. RESULTS: The prevalence rate of IBS was 10.9%, higher in females, in 35-44 years old age group and among subjects eating fast food (14.1%) but was lower in those taking more fruits and vegetables (10.5%). The occurrence of anxiety, nightmare and restlessness was also significantly higher in subjects with IBS. It had an association with psychological distress and recurrent headaches but not with drinking tea/coffee, smoking or physical activity. CONCLUSIONS: In our area, IBS was correlated with gender, age, psychological distress, recurrent headaches and consumption of fast foods that necessitate health planning programs by health policy makers.

Prevalence of celiac disease in Shiraz, southern Iran.

BACKGROUND/AIM: This study was performed to evaluate the prevalence of celiac disease (CD) in Shiraz, southern Iran. MATERIALS AND METHODS: Serum samples were collected from 1440 persons (age range = 20-83 years, mean age = 45.4 years) in 2004 and screened for endomysial and tissue transglutaminase antibodies. A questionnaire was completed for all subjects in relation to gastrointestinal (GI) symptoms and cases with positive serology were requested to undergo small-bowel biopsy. RESULTS: Seven cases (0.5%) were positive for IgA anti-tissue transglutaminase (anti-tTG), and only two (0.14%) were positive for IgA anti-endomysial antibody (anti-EMA), both of whom had highly positive anti-tTg levels (40.4 and 48.0 IU/l). The major clinical symptoms of CD, such as recurrent abdominal pain and change in bowel habits were present in all patients with positive anti-tTG assays. Only five subjects with positive serology agreed to undergo upper GI endoscopy and duodenal biopsy. Three of these cases were reported with Marsh I histologic findings, while in the two cases with positive serologic anti-EMA, more advanced forms of CD were present. CONCLUSION: The prevalence of CD in apparently healthy adults was lower than the reported series from northern parts of the country; therefore, we suggest a more long-term follow-up study in high-risk groups, especially in the apparently healthy subjects in our region.