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The goal of this policy note is twofold: first, to identify and propose how to address some of the key regulatory and implementation hurdles that Croatia and potentially other EU Member States are facing in greening their HD infrastructure while improving HD outcomes ; and second, to compile best practices and examples in green design, construction, and renovation of public HD buildings. The Note will also provide guidance and encourage dialogue among relevant policy makers at national, regional, and local levels, and with targeted clients. Furthermore, the recommendations would address the importance of green skills development and other related topics relevant to the implementation of EUGD. Overall, the analysis results and the recommendations on these issues could also be useful for World Bank experts and other external stakeholders focused on the green economy and human development.

Fumonisin B(1) (FB(1)) is a worldwide corn contaminant and has been epidemiologically linked to the high incidence of human esophageal cancer in South Africa and China. FB(1) is hepatocarcinogenic in rats by an unknown mechanism. Inhibition of ceramide synthase and disruption of membrane phospholipids have been shown to be mechanisms of toxicity. Here we show overexpression of cyclin D1 protein in both preneoplastic and neoplastic liver specimens obtained from a long-term feeding study of FB(1) in rats. In rats fed FB(1) short-term, cyclin D1 protein levels in liver were increased up to five-fold in a dose-responsive manner. Northern blot analysis demonstrated no increase in mRNA levels of cyclin D1. 2D electrophoresis of cyclin D1 protein in FB(1)-treated samples showed a distinct pattern of migration (presence of less negatively charged form of the protein) that differed from controls. Recently, it has been shown that phosphorylation of cyclin D1 by glycogen synthase kinase 3beta (GSK-3beta) on a single threonine residue (Thr-286) positively regulates proteosomal degradation of cyclin D1. In FB(1)-treated samples we detected GSK-3beta phosphorylated on serine 9; activated protein kinase B (Akt) appears to be responsible for this activity-inhibiting phosphorylation. These findings suggest that overexpression of cyclin D1 results from stabilization due to a lack of phosphorylation mediated by GSK-3beta. We also observed an increase in cyclin dependent kinase 4 (Cdk4) complexes with cyclin D1 in FB(1)-treated samples; additionally, elevated Cdk4 activity was shown by increased phosphorylation of the retinoblastoma protein. In summary, the activation of Akt leads to increased survival, inhibition of GSK-3beta activity and post-translational stabilization of cyclin D1, all events responsible for disruption of the cell cycle G(1)/S restriction point in hepatocytes. This is the first report suggesting the mechanism by which FB(1) acts as a carcinogen.

Growth factors bind to membrane receptor tyrosine kinases, resulting in autophosphorylation and subsequent binding to proteins with SH2 domains, including growth factor receptor-bound protein 2 (Grb2). Grb2 bridges receptors to tyrosine kinase substrates such as SHC and SOS, which in turn facilitate the activation of downstream signaling pathways, including Ras and mitogen-activated protein kinase (MAPK). Overexpression of Grb2 has been demonstrated in several types of neoplasia but has not been investigated in liver tumorigenesis. Here we investigated Grb2 expression in liver lesions in N-nitrosodimethylamine (NDMA)-treated Helicobacter hepaticus-infected and -noninfected A/J mice at 1 year of age. Previously, we reported (6) that infection promotes the development of these NDMA-initiated tumors. In controls, Grb2 immunostaining was absent from normal hepatic tissues, whereas the inflammatory lesions in infected livers were positive for cytoplasmic Grb2 in both hepatocytes and infiltrating leukocytes. All preneoplastic foci (7 of 7), 15 of 27 adenomas, and 3 of 7 carcinomas were positive for Grb2 by immunostaining in both infected and noninfected NDMA-initiated livers. Involvement of Grb2 was confirmed by immunoblotting of similarly infected mice at 9 to 18 months of age, showing a 2.5- to 3.3-fold increase in Grb2 protein in infected livers (p < 0.05 compared with uninfected controls) as well as in preneoplastic foci, adenomas, and carcinomas. These livers also showed a 2.5- to 2.8-fold increase in total Ras protein. The results suggest that upregulation of Grb2 is an early event in liver carcinogenesis, whether caused by the bacterial infection or by NDMA. Concomitant upregulation of Ras p21 would ensure transmission of amplified signal from growth factors via Grb2.

Publisher Summary This chapter examines the signaling pathways in the normal and neoplastic breast. The epidermal growth factor (EGF) family members that bind to EGFR are EGF, transforming growth factor α (TGFα), amphiregulin (AR, a heparin-binding factor), heparin-binding EGF (HbEGF), epiregulin, and β-cellulin. Cripto (CR-1) is an EGF family member that plays an important role in embryogenesis and mammary gland development. CR-1 is overexpressed in several human tumors. However, CR-1 binds to a type I serine/threonine kinase receptor for activin (ALK4), which is expressed on the cell surface of mammary epithelial cells, rather than binding to EGFR or one of the other ErbB receptors. ErbB receptors undergo homo- or heterodimerization following ligand binding. After ligand binding, EGFR/ErbB1/HER-1 undergoes either endocytosis and degradation by both proteasomal and lysosomal pathways, or the receptor is recycled to plasma membrane. ErbB2/HER-2 is considered to be endocytosis-impaired, and is the most stable protein among ErbBs in the plasma membrane. Ductal growth in the mammary epithelium is defective when ErbB2 is disrupted in the mammary gland, implying a prominent role for ErbB2 in mammary ductal growth. ErbB2 plays a significant role in ductal morphogenesis. While kinase-dead, ErbB3/HER-3 frequently forms a high-affinity co-receptor for heregulin by heterodimerization with ErbB2/HER-2. ErbB3/HER-3 possesses several docking sites for PI3K, and can initiate PI3K/Akt signaling when transactivated by ErbB2/HER-2. EGF can act as an oncogene-like molecule when transfected and overexpressed in immortalized rodent fibroblasts. Forced overexpression of EGFR in the mammary gland, under the control of the MMTV or β-lactoglobulin (BLG) promoters, results in abnormal mammary gland development and the production of epithelial hyperplasias.

Inositol hexaphosphate (IP6) is a naturally occurring polyphosphorylated carbohydrate with demonstrated anti-proliferative and anti-cancer activity in mammary cells. We hypothesized that IP6 modulates cell cycle proteins by action on cytoplasmic signaling molecules. The effects of both pharmacological (2 mM) and physiological (100 microM) doses of IP6 on major PKC isoforms (PKCalpha, delta, epsilon, beta and zeta), PI3-K/Akt and ras/Erk1/2 were evaluated. Treatment of MCF-7 human breast cancer cells with 2 mM IP6 for 24 h caused a 3.1-fold increase in the expression of anti-proliferative PKCdelta. Similar results were observed with 100 microM IP6 at only 30-60 min post-treatment. IP6 also caused an increase in PKCdelta activity, shown by its translocation from cytosol to membrane. No changes in expression of PKC alpha, delta, epsilon, beta and zeta were detected. Additionally, IP6 caused a decrease of Erk1/2 and Akt activity. Among cell cycle control proteins, IP6 resulted in increased p27Kip1 protein levels and marked reduction of pRb phosphorylation. Specificity of the IP6 effects on p27Kip1 and pRb in MCF-7 cells (hormone-dependent) were additionally confirmed in highly invasive hormone-independent MDA-MB 231 breast cancer cells. Use of specific pharmaclogical inhibitors of PKC delta, MEK/Erk, and PI3K/Akt pathways indicated that the IP6-mediated effects on PKC delta were responsible for up-regulation of p27Kip, and pRb hypo-phosphorylation. In addition, IP6-induced apoptosis detected in MCF-7 cells appeared also to be PKC delta-dependent. Our data suggest potential usefulness of IP6 as a novel therapeutic modulator of PKC delta and p27Kip1, an important prognostic factor in human breast cancers.

A naturally occurring, cocoa-derived pentameric procyanidin (pentamer) was previously shown to cause G0/G1 cell cycle arrest in human breast cancer cells by an unknown molecular mechanism. Here, we show that pentamer selectively inhibits the proliferation of human breast cancer cells (MDA MB-231, MDA MB-436, MDA MB-468, SKBR-3, and MCF-7) and benzo(a)pyrene-immortalized 184A1N4 and 184B5 cells. In contrast, normal human mammary epithelial cells in primary culture and spontaneously immortalized MCF-10A cells were significantly resistant. We evaluated whether this differential response to pentamer may involve depolarization of the mitochondrial membrane. Pentamer caused significant depolarization of mitochondrial membrane in MDA MB231 cells but not the more normal MCF-10A cells, whereas other normal and tumor cell lines tested gave variable results. Further investigations, using a proteomics approach with pentamer-treated MDA MB-231, revealed a specific dephosphorylation, without changes in protein expression, of several G1-modulatory proteins: Cdc2 (at Tyr15), forkhead transcription factor (at Ser256, the Akt phosphorylation site) and p53 (Ser392). Dephosphorylation of p53 (at Ser392) by pentamer was confirmed in MDA MB-468 cells. However, both expression and phosphorylation of retinoblastoma protein were decreased after pentamer treatment. Our results show that breast cancer cells are selectively susceptible to the cytotoxic effects of pentameric procyanidin, and suggest that inhibition of cellular proliferation by this compound is associated with the site-specific dephosphorylation or down-regulation of several cell cycle regulatory proteins.

In earlier studies, we and others have established that activation of EGFR can promote survival in association with upregulation of Bcl-x(L). However, the mechanism responsible for upregulation of Bcl-x(L) is unknown. For the current studies we have chosen pro-apoptotic, c-Myc-overexpressing murine mammary epithelial cells (MMECs) derived from MMTV-c-Myc transgenic mouse tumors. We now demonstrate that EGFR activation promotes survival through Akt and Erk1/2. Blockade of EGFR kinase activity and the PI3-K/Akt and MEK/Erk pathways with pharmacological inhibitors resulted in a significant induction of cellular apoptosis, paralleled by a downregulation of both Akt and Erk1/2 proteins. Consistent with a survival-promoting role of Akt, we observed that constitutively activated Akt (Myr-Akt) inhibited apoptosis of pro-apoptotic, c-Myc-overexpressing cells following the inhibition of EGFR tyrosine kinase activity. In addressing possible downstream effectors of EGFR through activated Akt, we detected significant upregulation of Bcl-x(L) protein, suggesting this pro-survival protein is a target of Akt in MMECs. By using pharmacological inhibitors of PI3-K/Akt and MEK/Erk together with dominant-negative Akt and Erk1 we observed the decrease in Bcl-x(L) protein. Our findings may be of importance for understanding the emerging role of Bcl-x(L) as a potential marker of poor prognosis in breast cancer.

This chapter highlights the importance of some of the molecules in breast signaling. Clearly, the importance of signaling by the c-ErbB family, TGF-β/Smads, several other growth factors, PI3K/Akt, MEK/Erk, and Stats in mammary gland development and mammary tumorigenesis has been well documented. Despite the fact that the recent insights revealed a significant amount of evidence for their role, mostly by research using in vitro human cellular models and mouse models of human breast cancer, it appears that many more questions remain unanswered. Indeed, studies to date have only started to answer some of the fundamental questions about how the signaling molecules mentioned here contribute to physiological and pathological activities in the breast tissue. New and more sophisticated insights will undoubtedly provide crucial information that will help our understanding of the role of these molecules in breast cancer and will help in providing valuable information for future therapies.

A new murine Helicobacter species, Helicobacter hepaticus, infects the livers of mice, causing a progressive chronic active hepatitis culminating in hepatocellular tumors. To examine the role of chronic H. hepaticus infection in carcinogenesis, H. hepaticus-infected male infant mice of A/JCr strain were given a single ip dose of N-nitrosodimethylamine (NDMA). Noninfected A/J mice similarly treated with NDMA served as controls. The effect of hepatitis induced by H. hepaticus was studied for 64 wk. At 31-36 wk, the incidence of hepatocellular adenomas in infected mice was significantly higher than in noninfected mice (82 vs 52%; p = 0.05). The multiplicity of hepatocellular tumors was also significantly higher in infected mice compared to noninfected mice (3.2 ± 0.09 vs 0.09 ± 0.2; p = 0.03). At 51-64 wk, many (10/18) infected mice developed hepatocellular carcinomas while only 2 of 19 control mice developed such tumors ( p = 0.005). Overexpression of cyclin D was observed in hepatocytes as well as adenomas induced by NDMA in H. hepaticus-infected mice, suggesting its role in inflammation, abnormal cell growth, and early neoplasia. High molecular weight keratins were highly expressed in hyperplastic oval cells in hepatitis and in liver tumors in mice with hepatitis, establishing a reliable marker for oval cells in formalin-fixed, paraffin-embedded tissue. Thus, chronic H. hepaticus infection significantly stimulated cyclin D expression, accelerated the development of liver tumors, increased the multiplicity of such lesions, and enhanced the progression of benign to malignant tumors.

Glyceryl trinitrate (GTN) was previously reported to induce hepatocellular carcinoma (HCC) in rats after prolonged feeding. The present experiments were undertaken to evaluate the histogenesis and molecular biology of these tumors and the possible role of nitric oxide (NO), a GTN metabolite, in their development. Male F344 rats received a single i.g. intubation of GTN (1.2 g/kg) at 6 weeks of age and/or a diet containing 1% GTN from 8 weeks of age until necropsy, i.e. for up to 78 weeks. Some animals were subjected to 2/3 partial hepatectomy (PH) at 9 weeks of age. Five sequential sacrifices (14, 32, 52, 78 and 84 weeks of age) were performed. No liver tumors developed in control rats or in rats that received GTN only by a single i.g. intubation, even when intubation was followed by PH. Preneoplastic foci, mainly of clear cell and mixed cell type (identified as positive for glutathione S-transferase placental form) were found from 14 weeks of age in rats receiving GTN in the diet. Focal eosinophilic areas (atypical foci) composed of atypical hepatocytes that often extended into the veins were observed beginning at 52 weeks of age. Some mixed hepatocholangiocellular adenomas and carcinomas arose in eosinophilic lesions. HCCs were seen beginning at 78 weeks of age, but only in rats receiving dietary GTN. Incidence of HCC in the latter animals was 50-75%. Most HCCs were well differentiated. The carcinogenic effect of GTN given in the diet was not affected by prior intubation of a large single dose followed by PH. No p53 mutations were found in 18 tumors but K-ras point mutations, all within codon 12, were found in 8/18 tumors, mostly those with cholangiocellular elements. These were first or second position G-->T transversions or second position G-->A transitions. While these mutation types have also been commonly seen in bacteria after NO-related DNA damage, the fact that tumors arose only on prolonged feeding of this potently bioactive agent at massive doses seems consistent with a more complex mechanism involving multiple (i.e. genetic and/or epigenetic) factors in carcinogenesis by GTN.

Fumonizini, nedavno otkriveni metaboliti plijesni Fusarium moniliforme Sheldon u inficiranom kukuruzu i drugim žitaricama, razlogom su zabrinutosti u mnogim državama. Trenutačno, čini se da su gospodarski i zdravstveni problemi uzrokovani drugim mikotsinima daleko manje značajni u usporedbi s FB, do sada najpotentnijim promotorom rasta tumora. Ovi mikotoksini imaju ulogu u pojavi leukoencefalomalacije konja (ELEM), pulmonarnog edema svinja (PPE), različitih zdravstvenih poremetnji u peradi, hepatokarcinoma štakora i moguće karcinoma jednjaka u ljudi. Također, fumonizini značajan su problem zbog proširenosti plijesni F. monilitorme, pojavljuju se u visokim količinama i jedinstvenog su načina djelovanja. Fumonizini interferiraju s biosintezom sfingolipida specifičnom inhibicijom enzima sfinganin-N-acil-transferaze što uzrokuje poremećaj u metabolizmu i prenošenju signala u stanici., Fumonisins, recently discovered metabolites of Fusarium moniliforme Sheldon, occuring naturally in infected corn and other cereals are an area of growing concern in many countries. Presently, it seems that the economic and health risk of other mycotoxins is far below the significans of potent tumor promoter fumonisin B₁. These compounds are involved in incidence of equine leukoencephalomalacia (ELEM), porcine pulmonary edema (PPE), disease problems in poultry, hepatocarcinoma in rats and possibly esophageal carcinoma (EG) in humans. Fumonisins are significant problem because F. moniliforme in corn is widespread, naturally they occur in ppm range, and their mode of action is unique. They interfere with sphingolipid biosynthesis by specifically inhibiting an enzyme sphinganine-N-acyl transferase. Because of this, the activity of secondar messanger system in the cells is affected.

We review the hepatocarcinogenic effects of fungal cultures of Fusarium verticillioides(= Fusarium moniliforme) strain MRC 826 in male BD IX rats. Subsequent chemical analyses of the fumonisin B (FB) mycotoxin content in the culture material used and long-term carcinogenesis studies with purified FB1 provide information about dose-response effects, relevance of hepatotoxicity during FB1-induced carcinogenesis, and the existence of a no-effect threshold. Fumonisin intake levels of between 0.08 and 0.16 mg FB/100 g body weight (bw)/day over approximately 2 years produce liver cancer in male BD IX rats. Exposure levels < 0.08 mg FB/100 g bw/day fail to induce cancer, although mild toxic and preneoplastic lesions are induced. The nutritional status of the diets used in the long-term experiments was marginally deficient in lipotropes and vitamins and could have played an important modulating role in fumonisin-induced hepatocarcinogenesis. Short-term studies in a cancer initiation/promotion model in rat liver provided important information about the possible mechanisms involved during the initial stages of cancer development by this apparently nongenotoxic mycotoxin. These studies supported the findings of long-term investigations indicating that a cytotoxic/proliferative response is required for cancer induction and that a no-effect threshold exists for cancer induction. The mechanisms proposed for cancer induction are highlighted and include the possible role of oxidative damage during initiation and the disruption of lipid metabolism, integrity of cellular membranes, and altered growth-regulatory responses as important events during promotion.