Heart failure in patients is associated with downregulation of mitochondrial quality control genes.

BACKGROUND: Mitochondrial dysfunction is one of key factors causing heart failure. We performed a comprehensive analysis of expression of mitochondrial quality control (MQC) genes in heart failure. METHODS: Myocardial samples were obtained from patients with ischemic and dilated cardiomyopathy in a terminal stage of heart failure and donors without heart disease. Using quantitative real-time PCR, we analysed a total of 45 MQC genes belonging to mitochondrial biogenesis, fusion-fission balance, mitochondrial unfolded protein response (UPRmt), translocase of the inner membrane (TIM) and mitophagy. Protein expression was analysed by ELISA and immunohistochemistry. RESULTS: The following genes were downregulated in ischemic and dilated cardiomyopathy: COX1, NRF1, TFAM, SIRT1, MTOR, MFF, DNM1L, DDIT3, UBL5, HSPA9, HSPE1, YME1L, LONP1, SPG7, HTRA2, OMA1, TIMM23, TIMM17A, TIMM17B, TIMM44, PAM16, TIMM22, TIMM9, TIMM10, PINK1, PARK2, ROTH1, PARL, FUNDC1, BNIP3, BNIP3L, TPCN2, LAMP2, MAP1LC3A and BECN1. Moreover, MT-ATP8, MFN2, EIF2AK4 and ULK1 were downregulated in heart failure from dilated, but not ischemic cardiomyopathy. VDAC1 and JUN were only genes that exhibited significantly different expression between ischemic and dilated cardiomyopathy. Expression of PPARGC1, OPA1, JUN, CEBPB, EIF2A, HSPD1, TIMM50 and TPCN1 was not significantly different between control and any form of heart failure. TOMM20 and COX proteins were downregulated in ICM and DCM. CONCLUSIONS: Heart failure in patients with ischemic and dilated cardiomyopathy is associated with downregulation of large number of UPRmt, mitophagy, TIM and fusion-fission balance genes. This indicates multiple defects in MQC and represents one of potential mechanisms underlying mitochondrial dysfunction in patients with heart failure.

The genetic diversity and structure of 18 sheep breeds exposed to isolation and selection.

The phylogenetic layout of the genotyped (30 microsatellite) 18 sheep breeds in this study demands and provides the opportunity to evaluate both neutral and adaptive components of genetic diversity in a naturally and artificially selected and subdivided sheep population. Seven Pramenka strains from Bosnia and Herzegovina and Croatia characterized by a very low intensity of artificial selection, preserved the highest neutral genetic variability. Eight central and north-western European breeds under considerable artificial isolation and selection preserved the lowest genetic variability. Only combinations of various phylogenetic parameters offer a reasonable explanation for underlying evolutionary forces working in the investigated island and mainland sheep breeds under variable natural and artificial selection. More than 60% of total genetic, diversity was allocated to virtually unselected Pramenka strains, and an additional 25% to native moderately selected Graue Gehoernte Heidschnucke and intensively selected Ostfriesische Milchschafe. Some economically very important breeds and strains did not contribute to a pool with maximal genetic diversity, while they play an important role in the cultural heritage of respective countries.

Metastases development following local tumour treatment.

Transplantable mouse methylcholanthrene- induced fibrosarcoma (CMC4 tumour growing in CBA/HZgr mice), characterized by lung metastases developing shortly after local tumour cell transplantation, was used as an experimental model to investigate the problem of tumour metastases after local tumour treatment. Surgery and/or irradiation were performed on locally growing tumour of particular size. Further, heavily irradiated, viable but not dividing tumour cells, imitating the situation in treated tumour-bearing organism, were injected intraperitoneally in a parallel group of treated tumour-bearing mice. The animals were killed 35 days after tumour transplantation and the number and volume of lung metastases were determined. Depending on the treatment performed, when the tumour mass was reduced or even eliminated, the number of lung metastases and their volume were significantly lower than in control mice, but the addition of tumour mass (injection of heavily irradiated tumour cells) resulted in a significant increase in lung metastases parameters, pointing to a possible role of the host's immune reaction against the tumour. Further, the release of a simple molecule, such as nitric oxide, from tumour mass seems to be detrimental for the survival of tumour cells and subsequently their metastases through the induction of angiogenesis and possible suppression of immune reaction. Thus, complex mechanisms could be involved when a locally growing tumour is exposed to a particular therapeutic approach.

Effects of background substraction on differential kidney function measured by static scintigraphy with DMSA and dynamic scintigraphy with MAG 3.

UNLABELLED: The aim of this study was to assess the influence of background subtraction (BS) on estimation of differential kidney function (DF) on the static scintigraphy with (99m)Tc dimercaptosuccinic acid (DMSA) and dynamic scintigraphy with (99m)Tc mercaptoacetyltriglicine (MAG3) and to establish possible differences between DF values estimated with these methods. PATIENTS, METHODS: Patients (n=106) were selected among those scheduled to static and dynamic scintigraphies within 3 months, with no interim clinical and laboratory changes, regardless the kidney pathology. DF was estimated according to the uptake ratio method. Four background regions of interest (ROIs) were applied, identical for both studies, and DF values were recalculated after BS. The corrected values were compared to the values before correction, separately for DMSA and MAG3, and between the studies. The results showed that ROIs used introduce variable results for the same patients, predominantly when noncorrected DF values were <45%. There were no significant differences between DF values (corrected and noncorrected) obtained from static and dynamic scintigraphy in all groups of patients. Since numerous reasons can bring to the errors in DF estimation when BS is used, the conclusions are that it would probably be more accurate to avoid BS, particularly when DF values are compared in a patient follow-up, and when kidney function is normal. BS should be used, but always in the same way, only when there is a significant difference in kidney size, or when DF is <25%, since background activity is then considerable. MAG3 and DMSA can be equally used for DF estimation and their results compared in patient follow-up.

Initial presentation of scintigraphic changes during the first episode of acute pyelonephritis in children: simultaneous evaluation with MAG3 and DMSA.

UNLABELLED: 99mTc-DMSA scintigraphy is generally accepted as the method of choice for detecting renal parenchymal damage in pyelonephritis. 99mTc-MAG3 dynamic scintigraphy is not routinely used for this purpose. The AIM of this study was to evaluate the MAG3 scintigraphic presentation in the acute phase of pyelonephritis in children and re-evaluate them at least 6 months later, as well as to establish whether a MAG3 in the parenchymal phase is as reliable and sensitive in the detection of a renal parenchymal damage as the DMSA. PATIENTS, METHODS: The MAG3 scintigraphic pattern was evaluated during the first episode of acute pyelonephritis in 31 children (median age: 2.5 years) and compared to the DMSA scan. The scintigraphy was performed on the same day with both radiopharmaceuticals. After at least 6 months the whole procedure was repeated on 28 patients. A scoring system was designed to evaluate the parenchymal lesions, and categorize them as positive or equivocal. The findings on the initial scans were compared to those obtained in the follow up studies. RESULTS: When all lesions (equivocal + positive) were analysed, MAG3 sensitivity was 98%, and specificity 78%, while for positive lesions only, the values were 83 and 100%, respectively. The average acute severity score was significantly lower for both MAG3 and DMSA then the follow up score (p < 0.0001). These results corresponded to a clinical convalescence, which was observed in 26/28 children in the follow up. CONCLUSION: With the MAG3 scintigraphy a reliable semi quantitative and qualitative detection of the renal inflammatory lesions can be obtained in acute pyelonephritis, as well as their recovery, thus obviating the need for a DMSA scan. Moreover, the duration of the MAG3 procedure is shorter, enabling the visualization of the entire collecting system as well, while the radiation exposure is approximately a half of that delivered by the DMSA scan.

Antitumor efficiency of novel fluoro-substituted 6-amino-2-phenylbenzothiazole hydrochloride salts in vitro and in vivo.

The purpose of this study was to investigate antitumor activity of novel fluoro-substituted 6-amino-2-phenylbenzothiazole hydrochloride salts in vitro and in vivo. A novel series of hydrochloride or dihydrochloride salts of the novel 2-(fluoro-substituted phenyl)-6-aminobenzothiazoles (5-7) have been prepared in multistep synthesis starting from 3- or 4-fluorobenzaldehydes and 2-amino-5-nitrothiophenol and evaluated for their antiproliferative activity against human cervical (HeLa), breast (MCF-7), colon (CaCO-2), and laryngeal (Hep-2) carcinomas and against fibroblast cell lines (WI-38). Also, antitumor activity of these compounds was evaluated in vitro and in vivo against murine melanoma (B16-F10), fibrosarcoma (FsaR), and squamous cell carcinoma (SCCVII). The tested compounds were found to exert good cytotoxic activity in vitro. The cytotoxic effect was selective, cell specific, and dose dependent, between 33 microM for MCF-7 and 110 microM for WI-38. Benzothiazoles reduced de novo protein and DNA synthesis up to 75%. All examined benzothiazoles had significant antitumor activity in vivo against melanoma B16-F10, fibrosarcoma, and squamous cell carcinoma. The best therapeutic results were achieved when therapy started 7 days after tumor cell implantation and when benzothiazoles were given repeatedly five times every 2 days, i.e., on day 7, 9, 11, 13, and 15 after transplantation of tumor cells.

Chemotherapy of the transplantable acute leukemia L5222 in rats.

This study presents results of single-drug and combination chemotherapy of the transplantable acute leukemia L5222 in BD IX rats. In leukemia L5222 there is a direct relationship between the number of transplanted cells and mean life expectancy. After single-drug therapy with L-asparaginase, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), cyclophosphamide, cytosine arabinoside, daunomycin, 6-mercaptopurine, methylglyoxal bis(guanylhydrazone) dihydrochloride, prednisolone, or vincristine, the best therapeutic effect was observed with BCNU and cyclophosphamide. A massive-dose therapy with BCNU repeated twice or a conbination of vincristine with cyclophosphamide or BCNU with cyclophosphamide yielded a high percentage of cures. Morever, leukemia L5222 seems to be suitable for studying the influence of drugs on the proliferation kinetics of leukemia cells.

Incidence of radiation lymphomas in C57BL/6 mice is not promoted after intraperitoneal treatment with the phorbol ester tetradecanoylphorbol acetate.

Female C57BL/6 mice, given 4 X-ray irradiations each with 1.7 Gy, according to H.S. Kaplan and M.S. Brown (J. Natl. Cancer Inst., 13 (1952) 185-192) developed lethal lymphomas in more than 90%, 270 days after irradiation. Intraperitoneal application of tetradecanoylphorbol acetate (TPA), 30 ng/g, twice weekly for 240 days had no influence on survival of the animals and incidence of the malignant lymphomas.

Carcinogenic effect of cisplatin (cis-diammine-dichloroplatinum (II), CDDP) in BD IX rats.

The potent antitumor agent cis-diammine-dichloroplatinum(II) (CDDP) also has carcinogenic properties. CDDP was administered i.p. to 50 BD IX rats for 3 weeks, 3 X 1 mg/kg body weight per week. All animals were pretreated by hydration before each CDDP application, but only half the animals additionally received mannitol together with the CDDP solution, as a nephroprotective measure. To date, 455 days after the first application, 33 animals have died, 13 of them of malignancies: 12 leukemias and 1 renal fibrosarcoma. So far, no significant differences in the frequency and type of malignancies have been observed between animals which additionally received mannitol and the others which did not. In the control group of 25 animals, which received NaCl solution 0.9% i.p. (3 X 1 ml/kg per week, for 3 weeks) malignancies have not yet occurred. The high tumor incidence determined in this study reveals that the risk of secondary tumor development in patients treated with CDDP should not be disregarded.

Inhibition of gastric tumorigenesis by alpha-difluoromethylornithine in rats treated with N-methyl-N'-nitro-N-nitrosoguanidine.

Male Wistar rats were treated concurrently with a combination of the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG; CAS 70-25-7) and the polyamine-synthesis inhibitor alpha-difluoromethylornithine (DFMO) at two different doses of 0.5% and 1.0% (w/v). Experimental groups were treated with (I) MNNG alone (n = 25), (II) MNNG plus 0.5% (w/v) DFMO (n = 25), (III) MNNG plus 1.0% (w/v) DFMO (n = 25), (IV) 1.0% (w/v) DFMO alone (n = 25). Group V represented untreated controls (n = 20). Both the carcinogen and DFMO were administered in drinking water. The treatment time with the carcinogen and DFMO was 35 weeks. After treatment was completed animals were followed for an additional 50 weeks to cover a total observation time of 85 weeks. Significantly fewer animals developed gastric adenocarcinoma in the two groups of animals that received a combined treatment of MNNG plus DFMO compared to animals treated with the carcinogen alone (P < 0.05 and 0.005). No benign or malignant neoplastic lesions were observed in the stomach or duodenum of animals treated with DFMO alone or in untreated controls. It is concluded that concurrent treatment with DFMO prevents the development of malignant gastric epithelial tumors induced by MNNG in rats.

DNA methylation in maternal, fetal and neonatal rat tissues following perinatal administration of procarbazine.

The cytostatic drug procarbazine has previously been shown to be a potent transplacental neurotropic carcinogen in rats. Following a single IP administration of (14C-methyl)procarbazine (110 mg/kg) on day 22 of gestation, methylation products with cellular DNA were determined in fetal and maternal rat organs. The concentration of the major adduct N7-methylguanine was highest in the maternal liver (224 mumol/mol guanine). Fetal and nonhepatic maternal tissues exhibited significantly lower levels, but differed little from each other. In brain, lung, intestines, and placenta the O6-methylguanine/N7-methylguanine ratio was close to 0.11, indicating that procarbazine, like other methylating carcinogens, initiates malignant transformation via methyldiazonium hydroxide as the ultimate reactant. Following a single dose of (14C-methyl)procarbazine to newborn animals, methylpurine values were 30-60 times lower than after prenatal administration. This suggests that DNA alkylation in nonhepatic tissues occurs by systemic distribution of a proximate carcinogen formed in the adult rat liver.

[Selective induction of tumors in Wistar rats with loop colostomy after administration of methyl-(acetoxymethyl)-nitrosamine (MAOMN) (author's transl)]. / Selektive Erzeugung von Tumoren an Wistar-Ratten an einem doppelläufigen Anus praeter nach Gabe von Methyl-(acetoxy-methyl)-Nitrosamin (MAOMN).

A single dose of 7 mg/kg methyl-(acetoxymethyl)-nitrosamine in Wistar rats with double loop colostomy induced adenomas, papillomas, and adenocarcinomas at the site of colostomy in 83% of the treated animals. The mean induction time was 320 days. The pronounced local effect as well as the high tumor yield are discussed in regard to the evident high epithelial sensitivity at the colostomy site.

Investigation of the tumorigenic response to benzo(a)pyrene in aqueous caffeine solution applied orally to Sprague-Dawley rats.

In a lifetime experiment benzo(a)pyrene (B(a)P) was administered to Sprague-Dawley rats either as an admixture to the diet or by gavage in an aqueous 1.5% caffeine solution. Dissolved benzo(a)pyrene induced more tumors of the forestomach than undissolved benzo(a)pyrene. The 1.5% caffeine solution (annual dose 27 g/kg) did not exert any carcinogenic activity under the conditions of this bioassay.

Regenerative effects of tetrachlorodecaoxide in BD IX rats after total-body gamma irradiation.

Tetrachlorodecaoxide (TCDO) was tested for its effects in BD IX rats when combined with a single dose nearing LD50 of total-body irradiation (gamma rays, 60Co). In pilot tests we found that TCDO administrations prior to or immediately after irradiation led to a very high mortality rate (up to 90%), whereas the initiation of TCDO treatment on Day 2, 3, or 4 after irradiation lowered the death rate noticeably, with optimum results when TCDO application was started on Day 4. In our major experiment on 100 BD IX rats, it was demonstrated that the following treatment schedule considerably decreased the death rate (from 44 to 4%): 15.5 mumol TCDO/kg body wt/day on Days 4-6 after irradiation and 7.75 mumol/kg body wt/day on Days 7-11. The animals treated with TCDO showed only mild anemia in the peripheral blood, accompanied by reticulocytosis and low-grade leukocytopenia. Examination of the bone marrow on Day 12 after irradiation revealed X-ray-induced agranulocytosis in the animals that had received only physiological saline solution, whereas in the bone marrow of the animals treated with TCDO there was erythropoiesis as well as myelopoiesis. In addition, the degree of hair loss and depigmentation occurring about 1 month after irradiation was considerably reduced by TCDO. From these results it can be postulated that TCDO has two different effects: as an oxygen donator it causes radiosensitization in the tissue when given before or immediately after irradiation; as an agent stimulating phagocytes and tissue regeneration, it promotes regeneration very effectively when damage is already evident in the tissue.

Anticarcinogenic effect of tetrachlorodecaoxide after total-body gamma irradiation in rats.

Tetrachlorodecaoxygen (TCDO) therapy of acute radiation syndrome was tested for a possible influence on the development of X-ray-induced malignancies. BD IX rats were exposed to total-body irradiation (TBI, gamma rays, 9 or 11 Gy) and received daily intravenous injections of either TCDO or physiological saline solution from days 4 through 11 after TBI. The short-term TCDO therapy reduced the acute death rate markedly, but survival rates after 4 months were similar with and without TCDO. The first malignancy after TBI occurred on day 103, and over the lifetime of the animals the tumor incidence in the group given TBI (11 Gy) without TCDO treatment was 73% vs 20% in animals with short-term TCDO therapy after TBI. In particular, there was a highly significant prevention of radiation-induced leukemia [P (one-sided) < 0.001] by TCDO, and a significantly reduced incidence of malignant epithelial tumors [P (one-sided) < 0.05]. The development of sarcomas was not affected by TCDO. Long-term survival was not enhanced by TCDO due to the occurrence of bronchopneumonial infections about 1 year after TBI. In conclusion, TCDO is not only a potent therapeutic agent in acute radiation syndrome, but it also significantly reduced the carcinogenic risk in rats after exposure to ionizing radiation.

Promotion of gastric tumorigenesis by duodenal contents in rats induced with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG).

Reflux of duodenal contents into the stomach occurs in patients with pyloric incompetence and after gastric resection when bile-diverting procedures are omitted. In such settings duodenal contents have been considered to favor the development of gastric cancer. We have studied the effect of chronic duodenogastric reflux on gastric tumor promotion in rats treated with N-methyl-N'-nitrosoguanidine (MNNG) in an experimental design that avoids physical trauma to the glandular stomach. Thus the effect of trauma-induced tissue repair on carcinogenesis is eliminated, and duodenogastric reflux is isolated as an experimental parameter. To achieve such reflux the first jejunal loop was anastomosed to the forestomach in rats. Animals were exposed to MNNG in drinking water (83 mg/L) for 12 weeks before induction of reflux. Experimental groups were as follow: I, reflux plus MNNG (n = 32); II, MNNG alone (n = 27); III, reflux alone (n = 28); IV, control (n = 25). The experiment was terminated after 56 weeks. Only animals that had survived for 90 days were included in the effective number of animals, which allowed for equal chances of tumor development. In no animal that died earlier had tumors developed. Animals with reflux plus MNNG treatment had significantly more glandular neoplasms (12/32) than did animals with MNNG treatment alone (4/27; p less than 0.05). Similarly, more animals with squamous cell neoplasms were recorded in group I (9/32) than in group II (2/27; p less than 0.05). In consideration of all tumors of epithelial and mesenchymal origin, more gastric malignant tumors were observed in group I (9/32) than in group II (2/27; p less than 0.05). It is concluded that chronic exposure to duodenal contents promotes the development of gastric neoplasia.

Experimental studies on the effect of duodenal contents on the epithelium of the esophagus.

10 male BD IX rats and 15 male and female Wistar rats weighing between 250 and 300 g had an esophago-jejunostomy according to the method described by Levrat et al. (1962). Four weeks after surgery all 40 animals were sacrificed and examined macroscopically as well as histologically. All animals had deep ulcerative lesions in the lower half of the esophagus associated with hyperplasia, hyperkeratosis and akanthosis.

Comparative study on the effects of chlorite oxygen reaction product TCDO (tetrachlorodecaoxygen) and sodium chlorite solution (NaClO2) with equimolar chlorite content on bone marrow and peripheral blood of BDIX rats.

The effects of the chlorite-oxygen reaction product TCDO (tetrachlorodecaoxygen, active ingredient of the systemic application form of WF 10) were investigated on bone marrow and peripheral blood of BDIX rats in comparison to a sodium chlorite solution with a chlorite content identical to that of WF 10. Despite difficulties in determining the chemical differences between TCDO and a sodium chlorite solution, their differing effects on cells, tissue and organism were striking. The following characteristics have been observed: Stimulation of the bone marrow, evidenced by the pronounced increase in mature granulocytes, pronormo- and normoblasts, or increased cell proliferation rate, determined by means of the BrdUrd method, was achieved only with WF 10 (TCDO). Stimulation of the bone marrow led in turn to increased numbers of leucocytes and monocytes in the peripheral blood. In addition, WF 10 induced the production of large granular lymphocytes (LGLs), referred to as natural killer cells (NK-cells). In contrast, NaClO2 solution suppressed bone marrow function, exhibiting a toxic effect when given on a long-term basis. At the same time the number of mature granulocytes as well as pronormo- and normoblasts decreased, while the presence of LGLs was not observed. The results showed that TCDO is a potent stimulator of the bone marrow function and an effective modulator of the entire immune system. The toxic effect of chlorite, derived from the TCDO matrix, is not noticeable, being completely compensated by the favourable effects of TCDO.