Phthalate induced toxicity in prostate cancer cell lines and effects of alpha lipoic acid.

OBJECTIVE: The effects of dimethyl phthalate, diethyl phthalate, diisobutyl phthalate, di-n-butyl phthalate, benzylbutyl phthalate, di-2-ethylhexyl phthalate were investigated on human prostate cancer cell lines DU145 and PC3 in vitro. MATERIALS AND METHODS: Standards of dimethyl phthalate, diethyl phthalate, di-isobutyl phthalate, dibutyl phthalate, benzyl butyl phthalate, and di-ethyl hexyl phthalate were used. Alpha lipoic acid was used as antioxidant compound. DU145 and PC3 human prostate carcinoma cells were used. MTT assay were used for cytotoxicity assay. RESULTS: A low dose proliferative effect of phthalates in vitro was observed. With the hypothesis of the inhibition of aerobic glycolysis activity in cancer treatment, α-lipoic acid was applied to cells; where as a contrary to previous studies, no change in the cell proliferation was observed. In combination with ALA, at IC50 and lower doses, an increase of the cytotoxic effect was found for DIBP, DBP and BBP; while for DMP, DEP and DEHP, a decrease was observed for DU145 cells. In PC3 cells, a decrease was observed for DMP, DEP and DBPs; while no significant difference were observed for DEHP, DIBP and BBP. CONCLUSSION: The present study demonstrates preliminary information regarding the low dose proliferative effects of phthalates in prostate cancer in vitro (Tab. 2, Fig. 2, Ref. 65).

Hormonal treatment of male infertility: FSH.

FSH plays a crucial role in spermatogenesis. In the fetal and neonatal development stages, FSH activates the proliferation of the Sertoli cells and successively, during the pubertal phase, it influences the mitotic activity of the spermatogonia and encourages cellular differentiation, until arrival at the round spermatid stage. Because of its physiological role in spermatogenesis, various attempts have been made to treat idiopathic oligozoospermic men with FSH. However, the results obtained so far are still controversial. In this research, attention was focused on the possible criteria able to predict a seminal response to this specific hormonal treatment. Furthermore, the effectiveness of FSH therapy was evaluated in terms of sperm count and pregnancy rate. Thus far, based on more recent knowledge about the FSH receptor gene, the authors have correlated different polymorphisms of this gene with the outcome of FSH treatment. In this paper, the literature is reviewed and the authors' experience on using FSH in male infertility is discussed.

Effects of hazelnut-enriched diet on plasma cholesterol and lipoprotein profiles in hypercholesterolemic adult men.

OBJECTIVE: Frequent consumption of nuts is associated with favorable plasma lipid profiles and reduced risk of coronary heart disease (CHD). This study was conducted to investigate the effects of hazelnut-enriched diet on plasma cholesterol and lipoprotein profiles in hypercholesterolemic adult men compared with baseline and control diet, and also to measure the anthropometric parameters, habitual physical activities, nutrient intake and endothelial function. SUBJECTS AND DESIGN: Fifteen hypercholesterolemic men aged 48+/-8 years were recruited voluntarily. A well-controlled, 2-period (P1 and P2) study design with a total of 8-week was implemented. In the P1, subjects consumed a control diet (low-fat, low-cholesterol and high-carbohydrate). During the P2, the control diet was supplemented with MUFA-rich hazelnut (40 g/day), which provided 11.6% of total energy content. Anthropometric parameters and habitual physical activities were recorded. Plasma total and HDL cholesterol, TAG, ApoA-1, Apo B, total homocysteine and glucose concentrations were measured. All parameters and measurements were obtained at baseline and end of each 4-week diet period. RESULTS: Body weights of subjects remained stable throughout the study. Compared with baseline, the hazelnut-enriched diet decreased (P<0.05) the concentrations of VLDL cholesterol, triacylglycerol, apolipoprotein B by 29.5, 31.8, and 9.2%, respectively, while increasing HDL cholesterol concentrations by 12.6%. Total/HDL cholesterol and LDL/HDL cholesterol ratios favorably decreased (P<0.05). Although insignificant there was a decreasing trend for the rest of parameters, particularly in total (5.2%) and LDL cholesterol (3.3%) in subjects consuming a hazelnut-enriched diet compared to that of the baseline. No changes were found in fasting levels of glucose, Apo A-1 and homocysteine between the control and hazelnut-enriched diets. CONCLUSIONS: This study demonstrated that a high-fat and high-MUFA-rich hazelnut diet was superior to a low-fat control diet because of favorable changes in plasma lipid profiles of hypercholesterolemic adult men and, thereby positively affecting the CHD risk profile. SPONSORSHIP: Funding provided by a grant from the Hazelnut Promotion Group, Giresun, Turkey.

Emerging role of endoglin (CD105) as a marker of angiogenesis with clinical potential in human malignancies.

Angiogenesis is crucial for tumor development and progression, and antiangiogenetic therapy represents a promising approach for cancer treatment. Thus, the in-depth understanding of the molecular mechanism(s) regulating angiogenesis, together with the characterization of molecules expressed by endothelial cells and involved in distinct steps of the angiogenetic process, will greatly improve the design of new and more effective therapeutic strategies in human malignancies. Endoglin (CD105), a cell membrane glycoprotein predominantly expressed on cellular lineages within the vascular system, and over-expressed on proliferating endothelial cells, is involved in blood vessels development and represents a powerful marker of neovascularization. CD105 binds several factors of the Transforming Growth Factor (TGF)-beta superfamily, a pleiotropic cytokine that regulates different cellular functions including proliferation, differentiation and migration. In human malignancies of different histotype, CD105 is highly expressed on endothelial cells of both peri- and intra-tumoral blood vessels, while it is weakly expressed or absent on neoplastic cells. This unique tissue distribution strongly suggests for a prognostic, diagnostic and therapeutic potential of CD105 in neoplastic diseases. In this review we will summarize the structural and functional features of CD105, as well as its tissue distribution in normal and neoplastic tissues. Furthermore, the practical implications of CD105 in human malignancies will also be discussed.

Endoglin (CD105): a target for anti-angiogenetic cancer therapy.

Targeting of tumor vasculature is a promising strategy for cancer treatment. Among endothelial cell markers, Endoglin, a cell membrane glycoprotein, is emerging as an attractive therapeutic target on angiogenetic blood vessels, and it currently represents a powerful marker to quantify tumor angiogenesis. In normal human tissues, Endoglin is weakly expressed on erytroid precursors, stromal cells and activated monocytes, whereas it is strongly expressed on proliferating endothelial cells. In human neoplasias of different histotype, Endoglin is mainly present on endothelial cells of both peri- and intra-tumoral blood vessels, while it is weakly expressed or absent on neoplastic cells. Endoglin is an accessory component of the receptor complex of Transforming Growth Factor (TGF)-beta, a pleiotropic cytokine that modulates angiogenesis by the regulation of different cellular functions including proliferation, differentiation and migration. Interestingly, the over-expression of Endoglin antagonizes several cellular responses to TGF-beta1, while its down-regulation potentiates cellular responses to TGF-beta1. In animal models, administration of radiolabeled anti-Endoglin monoclonal antibodies (mAb) efficiently images primary tumors, and naked or conjugated anti-Endoglin mAb suppress angiogenesis and tumor growth. In this review we will summarize the complex of experimental evidences pointing to Endoglin as a vascular target to design innovative bioimmunotherapeutic strategies in human neoplasias.

Plasma lipids in Turkish children: impact of puberty, socioeconomic status, and nutrition on plasma cholesterol and HDL.

In Turkish adults, HDL cholesterol (HDL-C) levels are 10-15 mg/dl lower than those of adults in western Europe and the United States. In this study, we determined whether HDL-C levels in Turks are low from birth to adulthood and assessed the effect of socioeconomic status (SES) on plasma lipids and lipoproteins. Analyses of cord blood from 105 Turkish newborns showed low levels of plasma cholesterol ( approximately 60 mg/dl) and HDL-C (approximately 30 mg/dl), consistent with results from other Western ethnic groups. Prepubescent 8- to 10-year-old Turkish boys and girls of upper (n = 82) and lower (n = 143) SES had high HDL-C levels (50-60 mg/dl) similar to those of western European children. However, the cholesterol (154-158 mg/dl) and HDL-C (55-58 mg/dl) levels of upper SES children were approximately 25 and approximately 12 mg/dl higher, respectively, than those of lower SES children. Height, weight, skinfold thickness, and estimated body fat were greater in the upper SES children and appeared to reflect dietary differences. Upper SES children consumed more total fat (approximately 35% vs. 25% of total calories), including more saturated fat of animal origin, and less carbohydrate (approximately 50% vs. 62% of total calories), consistent with their elevated plasma cholesterol levels. Carbohydrate intake correlated inversely with the HDL-C level. The HDL-C levels in the prepubescent children, especially those of higher SES, who consumed diets more like western Europeans, decreased markedly to adult levels, with males exhibiting a approximately 20 mg/dl decrease (from 58 to 37 mg/dl) and females a approximately 13 mg/dl decrease (from 55 to 42 mg/dl). SES did not affect HDL-C levels in adults. The profound decrease may reflect alterations in androgen/estrogen balance in Turks at puberty and a modulation of hepatic lipase affecting HDL-C levels.

Inhibition of fasL sustains phagocytic cells and delays myogenesis in regenerating muscle fibers.

Macrophage-muscle cell interactions are complex, and the majority is unknown. The persistence of inflammatory cells in skeletal muscle could be critical for myofiber viability. In the present paper, we show that FasL plays a role in the resolution of muscle inflammation. We analyzed inflamed muscles of normal mice treated from day 3 to day 8 with a FasL inhibitor (Fas-Ig) or with control Ig. Treated muscles were collected at 3, 5, and 10 days. The treatment with recombinant Fas-Ig protein induced a severe persistence of inflammatory cells at 5 days (115,000+/-27,838 vs. 41,661+/-6848, p<0.01) and 10 days from injury (145,500+/-40,850 vs. 5000+/-1000, p<0.001). Myofiber regeneration was highly impaired (37+/-14 vs. 252+/-28, p<0.01). Apoptosis of phagocytic cells was absent during Fas-Ig treatment (0.9+/-0.6 vs. 1300+/-150, p<0.0001), but apoptotic, mononucleated cells appeared at day 10, 2 days after the suspension of Fas-Ig administration. The time course of FasL expression during muscle inflammation, at mRNA and protein level, reveals a peak during myoblast proliferation. The peak of FasL expression coincides with the peak of apoptosis of phagocytic cells. In situ hybridization shows the co-expression of FasL and MyoD mRNA in mononucleated cells, i.e., myoblasts. Experiments on the myoblast cell culture confirmed the expression of FasL in myoblasts. The findings shown here indicate one of the pathways to control myoblast-macrophage interaction and might be relevant for the control of inflammatory cells in muscle tissue. Perhaps altering FasL expression with recombinant proteins could ameliorate inflammation in degenerative myopathies and up-regulate muscle regeneration.

The p53 gene in patients under the age of 40 with gastric cancer: mutation rates are low but are associated with a cardiac location.

BACKGROUND: Determining both the frequency and the spectrum of p53 gene mutation in young patients with gastric cancer might provide clues to the host related genetic mechanism(s) in gastric carcinogenesis. PATIENTS AND METHODS: p53 mutations were assessed (by means of polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), followed by DNA sequencing) in a cohort of 105 consecutive Italian patients in whom gastric cancer was ascertained before the age of 41. RESULTS: A low prevalence of p53 mutations (eight of 105) was observed, with no significant difference between intestinal (three of 31; 10%) and diffuse (five of 74; 7%) phenotypes. A significantly higher prevalence of p53 mutations was associated with the cardiac location (odds ratio, 7.09; confidence interval, 1.56 to 32.11). In all but one case, p53 mutations were associated with a stage higher than I. All eight mutations were located at CpG sites, where G : C to A : T transitions have been associated with frequent methylation at the C5 position of cytosine. CONCLUSIONS: These findings show that, unlike what has been consistently demonstrated in the general population, p53 mutations are uncommon in gastric cancer occurring in young patients, and in such patients, p53 alterations are significantly associated with the cardiac location.

Dystrophin deficient myotubes undergo apoptosis in mouse primary muscle cell culture after DNA damage.

Apoptosis has been demonstrated to occur in differentiated myocardial muscle, neonatal skeletal muscle and skeletal myoblasts in response to injury. In this report, we studied differentiated normal and dystrophin deficient murine skeletal muscle cell cultures that have been injured by a pulse of cis-platinum (2 h). Forty-eight hours after DNA damage, dystrophin positive myotubes appeared almost normal though some myoblasts showed DNA fragmentation. On the other hand, dystrophin deficient myotubes presented progressive degeneration via apoptosis detected either by TUNEL or by nuclear morphology. Degeneration of mdx muscle fibers was confirmed by counting both the number of myotubes observed by contrast phase microscopy and myonuclei viewed by immunoreaction for MyoD. A 6-fold decrease in the number of muscle cells was observed in the dystrophin-deficient cell culture compared to the parental culture (P < 0.001). Direct evidence of degenerating myotubes displaying MyoD- and TUNEL-positive nuclei was obtained. Like myoblasts, differentiated dystrophin deficient myotubes were able to degenerate via apoptosis, showing that mature dystrophin deficient cells are fragile and undergo apoptosis when subjected to a mild injury which would normally be repaired in parental cells.

ED2+ macrophages increase selectively myoblast proliferation in muscle cultures.

We have previously shown by coculturing myoblasts and macrophages that myotube formation is strongly increased in vitro by the presence of an acid stable, heat-labile, soluble growth factor(s) secreted by macrophages. In this paper we obtained macrophages from peritoneal washing which also contained limited amounts of other cells such as lymphocytes and mesothelial cells. We here demonstrate that an ED2-positive (ED2+) macrophage subpopulation is responsible for myoblast enhanced proliferation. ED2+ macrophages were separated by a magnetic-activated cell sorter (MACS) using a monoclonal antibody against ED2, a membrane antigen peculiar to macrophages. Both ED2+ macrophages and their conditioned medium increased myotube formation when added to primary muscle cultures. Furthermore we demonstrate that muscle growth induced by macrophages is mainly the consequence of an increased myoblast proliferation by showing the presence of an increased number of MyoD-positive (MyoD+) myonuclei.

Exercise induces myonuclear ubiquitination and apoptosis in dystrophin-deficient muscle of mice.

Apoptosis plays a major role in several diseases, including viral infections, autoimmune diseases, cancer, cardiac infarct, and neurological disorders. To investigate the role of apoptosis in muscular dystrophy, dystrophin-deficient (mdx) mice were subjected to spontaneous exercise and skeletal muscles were analyzed for apoptosis and ubiquitin. The increase of apoptotic myonuclei after exercise was detected by TUNEL, by electron microscopy, and by DNA analyses for high molecular weight and for ladder fragments. Expression of ubiquitin correlated with exercise and with positive myonuclei for apoptosis. Biochemical analysis confirmed a high level of ubiquitination both in sarcoplasmic and myofibrillar proteins. Muscles from sedentary congenit control mice (C57B ) were negative for apoptosis, while after exercise some nuclei were positive. We also revealed that normal myoblasts committed to apoptosis in vitro showed an increased expression of ubiquitin. Western blot for bcl-2, FasL, and BAG1 showed a significant decrease of bcl-2 product only in mdx mice after exercise. Thus, spontaneous exercise results in the increase of ubiquitin expression and in the reduction of bcl-2 tightly related to programmed cell death in mdx mice. These findings confirm that DNA fragmentation, absent in muscles of sedentary normal mice but present in mdx mice at rest, dramatically increases after exercise, shedding new light on exercise-induced muscle damage and on its progression in dystrophinopathies.

Apoptosis, DNA damage and ubiquitin expression in normal and mdx muscle fibers after exercise.

The current view indicates that after eccentric exercise myofibers are mechanically damaged and therefore an inflammatory and necrotic process occurs. In the present paper we examine the possibility that apoptosis plays a role in normal and dystrophin-deficient muscles after running. We analysed for apoptosis normal and dystrophin-deficient mouse muscles after a night of spontaneous wheel-running followed by two days of rest. Terminal deoxynucleotidyl transferase-mediated end-labeling of DNA in nuclei in tissue sections and gel electrophoresis of extracted DNA showed the presence of fragmented DNA. Furthermore, ubiquitin, a protein whose appearance is related to apoptosis, increased in muscles of both dystrophic and normal runner mice. The present findings which confirm that DNA damage is absent in muscles of sedentary mice but present in muscles of runner mice offer a new hypothesis on early events of muscle damage.

Analysis of muscle cell culture medium by size-exclusion chromatography.

The application of both low-pressure (preparative) and high-performance (analytical) size-exclusion chromatography to the fingerprinting of muscle cell culture supernatant is reported. The chromatograms showed significant differences between fresh media and muscle cell culture media. In addition, only one fraction derived from muscle culture medium contained factor(s) of proteic nature able to interfere with the cell cycle, of a continuous proliferating cell line.

Pharmacokinetics and tumor concentration of intraarterial and intravenous cisplatin in patients with head and neck squamous cancer.

Tumor-tissue platinum levels and major pharmacokinetic parameters were determined in 11 patients with head and neck squamous cancer (HNSC) who were given cisplatin (50 mg/m2 daily x 2 days) and 5-fluorouracil (5-FU; 1000 mg/m2, continuous infusion x 5 days) either i.a. or i.v. The plasma peak platinum concentrations (cmax) and the areas under the curve for total platinum concentration versus time (AUC) during i.a. infusions were lower than the i.v. cmax (mean, 1.92 +/- 0.28 and 4.08 +/- 2.80 mg/l, for i.a. and i.v. infusions, respectively) and AUC values (mean, 22.55 +/- 4.96 and 40.66 +/- 10.71 mg h-1 l-1 for i.a. and i.v. treatment, respectively), suggesting a first-passage extraction of the drug by the tumor mass during i.a. infusion. However, no statistically significant difference was found in platinum tumor concentrations after i.a. administration versus i.v. infusion. The lack of a difference in tumor platinum concentrations between the i.a. and the i.v. administration routes might be explained either by a relatively high blood supply to the tumor area, enabling efflux of the surplus free platinum from the tissue, or by the delay between drug infusion and biopsy. After three cycles of i.a. treatment good tumor remission was obtained with minimal local toxicity. Larger clinical studies testing the advantages of the i.a. administration route over i.v. infusion appear to be necessary.

[Leukotriene B4 in gastritis and peptic ulcer]. / Il leucotriene B4 nella gastrite e nell'ulcera peptica.

To contribute to a better understanding of the role of leukotrienes in inflammatory conditions of the upper gastrointestinal tract, leukotriene B4 (LTB4) levels were measured by specific radioimmunoassay in extracts of mucosal biopsies from the stomach and duodenum of control subjects and of patients with gastritis, benign gastric ulcer and duodenal ulcer. The control mucosa contained measurable levels of LTB4. Levels were 20% higher in gastritis than in controls and the lowest levels were found in the patients with no bile reflux. LTB4 levels were more than 3 times higher in duodenal ulcer (p less than 0.025) than in controls, whereas gastric ulcer margins had very low levels. No clear relationship was found between LTB4 levels and the drugs used in treatment. While these data do not explain the role of LTB4, they suggest that LTB4 may be an important mediator of the inflammation accompanying or leading to development of duodenal ulcer.

Effect of L-carnitine and L-aminocarnitine on calcium transport, motility, and enzyme release from ejaculated bovine spermatozoa.

Experiments were performed to further elucidate the role of gamma-amino-beta-hydroxybutyric acid trimethylbetaine (carnitine) on the metabolism and functions of spermatozoa. Addition of 20 mM L-carnitine to suspensions of ejaculated bovine spermatozoa resulted in an increase of cellular calcium transport, whereas 20 mM L-aminocarnitine (an inhibitor of carnitine palmitoyltransferase) caused an inhibition of this process. Both L-carnitine and L-aminocarnitine inhibited the progressive motility of spermatozoa, and the oxygen consumption as well as the release of the enzymes hyaluronidase and glutamate-oxaloacetate transaminase from spermatozoa. Labeled carnitine was rapidly taken up by spermatozoa by a process strongly dependent on temperature and extracellular concentration of carnitine. It is concluded that the effects produced by high concentrations of carnitine or aminocarnitine are mainly due to interactions of these compounds with the cellular membranes of spermatozoa.

Diverse effects of three furocoumarins on human lymphocyte proliferation.

The biological effects of three furocoumarins on the proliferation of human normal peripheral blood lymphocytes have been investigated. Mitogen-stimulated human lymphocytes were assayed "in vitro" by measuring 3H-thymidine (3H-TdR) incorporation in the presence and in the absence of 15-30 microM 3-carbethoxypsoralen (3-CPs), trimethylangelicin (TMA) and psoralen (PSR) with and without UV-A irradiation (365 nm). The three furocoumarins differ in their ability to form mono- and bi-functional adducts with DNA pyrimidine bases and in producing reactive species of oxygen. At low furocoumarin doses and short times of UV-A irradiation (15-30 sec) used in the present study, 3-CPs did not affect 3H-TdR incorporation in PHA-stimulated human lymphocytes, TMA strongly inhibited 3H-TdR incorporation, while, unexpectedly, PSR increased 3H-TdR incorporation in the absence of irradiation, likely acting, under these experimental conditions, as a co-mitogen.

Dark and photohemolysis of erythrocytes by furocoumarins.

It has been shown that various furocoumarins are able to cause dark hemolysis in red blood cells (RBC). However, this effect is evident only at relatively high furocoumarin concentrations (4.6 x 10(-4) M) - much higher than those used in photosensitization experiments or photochemotherapeutic treatments. Among the various furocoumarins examined in this study, only psoralen (Ps) and 3-carbethoxypsoralen (3-CPs) showed strong photohemolytic effects, while the other compounds revealed little or no activity. This fact indicates that Ps and 3-CPs are able to induce selective damage to the cell membrane of RBC. By pre-irradiating furocoumarin in ethanol or isotonic saline solutions and adding the irradiated solutions to a RBC suspension, hemolysis was observed in various compounds. The products of photolysis which form during pre-irradiation may be responsible, in terms of hemolysis, for toxic effects on RBC.

Uptake of chromate by rat thymocytes and role of glutathione in its cytoplasmic reduction.

1. Chromate is taken up by rat thymocytes over a wide range of extracellular chromate concentrations (0.074 +/- 5.0 mM). 2. Chromate uptake followed Michaelis-Menten kinetics and was inhibited by 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulphonic acid, suggesting that the anion carrier was responsible for the uptake by rat thymocytes. 3. The decrease in chromate uptake by the thiol(SH)-blocking agent diethyl maleate, the increased chromate uptake caused by the thiol-protecting agent dithiothreitol, and the reduction of glutathione/glutathione disulphide ratio indicated that the maintenance of the integrity of SH-groups greatly influenced the uptake and reduction of Cr(VI), and that glutathione may be responsible for the intracellular reduction of Cr(VI) to Cr(III).

Intracellular chromium reduction.

Two steps are involved in the uptake of Cr(VI): (1) the diffusion of the anion CrO4(2-) through a facilitated transport system, presumably the non-specific anion carrier and (2) the intracellular reduction of Cr(VI) to Cr(III). The intracellular reduction of Cr(VI), keeping the cytoplasmic concentration of Cr(VI) low, facilitates accumulation of chromate from extracellular medium into the cell. In the present paper, a direct demonstration of intracellular chromium reduction is provided by means of electron paramagnetic (spin) resonance (EPR) spectroscopy. Incubation of metabolically active rat thymocytes with chromate originates a signal which can be attributed to a paramagnetic species of chromium, Cr(V) or Cr(III). The EPR signal is originated by intracellular reduction of chromium since: (1) it is observed only when cells are incubated with chromate, (2) it is present even after extensive washings of the cells in a chromium-free medium; (3) it is abolished when cells are incubated with drugs able to reduce the glutathione pool, i.e., diethylmaleate or phorone; and (4) it is abolished when cells are incubated in the presence of a specific inhibitor of the anion carrier, 4-acetamido-4'-isothiocyanatostilbene-2-2'-disulfonic acid.