N-Nitrosamino phosphates are unlikely transport forms for activated nitrosamines

Summary Some of the target organs for nitrosamine carcinogenicity have a low activating capacity but many carcinogenic nitrosamines can be activated in the liver. Conjugates, such as phosphates, are chemically accessible reaction products of 1-OH-nitrosamines, and are either potential detoxication products or potential transport forms for activated nitrosamines.¹⁴C-labeled 1-(N-ethyl-N-nitrosamino)ethyl phosphate was tested for its ability to enter primary rat hepatocytes but no uptake was detectable. No uptake was observable into fibroblasts and human leukocytes.N-Nitrosomethylbenzylamine is efficiently 1-C-hydroxylated by hepatocytes but the corresponding 1-C-phosphate was detectable neither in the cells nor in the surrounding medium.N-Nitrosamino-1-phosphates, unlike 1-glucuronides, therefore, do not seem to be important for nitrosamine toxicokinetics.Abbreviations MNEP 1-(N-methyl-N-nitrosamino)ethyl phosphate - ENEP 1-(N-ethyl-N-nitrosamino)ethyl phosphate - [¹⁴C]ENEP 1-(N ¹⁴C]ethyl-N-nitrosamino)-ethyl phosphate) - N[¹⁴]MBzA N-nitroso-[¹⁴C]methylbenzylamine Dedicated to Professor Dr. D. Schmähl on the occasion of his 65th birthday     

Pleiotrophin Expression in Human Pancreatic Cancer and Its Correlation with Clinicopathological Features, Perineural Invasion, and Prognosis

Pleiotrophin (PTN), a heparin-binding growth factor also known as neurite growth-promoting factor, exhibits several properties related with tumor development. PTN and its receptor, N-syndecan, may play a very important role in tumor growth and neural invasion of pancreatic cancer. We investigated PTN and N-syndecan protein levels in 38 patients with pancreatic cancer by immunohistochemistry, and analyzed for its correlation with clinicopathological features, perineural invasion, and prognosis. The results showed that PTN and N-syndecan proteins were found in 24 (63.2%) and 22 (57.9%) specimens, respectively. PTN and N-syndecan expressions were associated with perineural invasion (P = 0.016 and P = 0.029, respectively). High PTN expression was closely related to an advanced TNM stage (P = 0.007), lymph node metastasis (P = 0.040), and decreased postoperative survival at 3 years (50.0% versus 20.8%, respectively; P = 0.001). We conclude that high expression of PTN combined with N-syndecan may contribute to the increased perineural invasion and poor prognosis of pancreatic cancer.     

Role of prostaglandins in the early phases of experimental gastric carcinogenesis in the rat

Summary The study was initiated to evaluate the effect of N-methyl-N-nitro-N-nitrosoguanidine (NG) on gastric intraluminal prostaglandin release during a 30-day treatment period and to investigate the effect of a stable prostaglandin E¹ analogue (misoprostol) on NG-induced gastric mucosal damage during the same time period. Samples of gastric juice (1 h) were obtained from 40 male Sprague-Dawley rats with chronic gastric fistulas, in basal conditions and after 5, 15 and 30 days of continuous oral administration of NG (120 mg/l) or tap water. Aliquots of gastric juice were titrated with 0.1 M NaOH. Other aliquots were extracted with ethyl acetate and subjected to specific radioimmunoassay for prostaglandin E₂. The severity of gastric mucosal lesions was evaluated in 60 rats after 5 days and 30 days of continuous oral administration of NG (120 mg/l) or NG plus misoprostol (200 g/kg^-1/day^-1) or tap water, and a histological study was carried out. Administration of NG induced a significant decrease of gastric intraluminal prostaglandin E₂ concentration at 15 and 30 days. Oral administration of misoprostol, at non-antisecretory doses, protected the rats against NG-induced gastric mucosal damage. Prostaglandins may be involved in the early phases of experimental gastric carcinogenesis.This work was supported by grants from the Ministry of Education (MPI)     

Carcinogenicity and organ specificity of N-trimethylsilylmethyl-N-nitrosourea (TMS-MNU), N-neopentyl-N-nitrosourea (neoPNU), and N-methyl-N-nitrosourea (MNU) in rats

Summary The carcinogenicity and organ specificity of TMS-MNU and neoPNU, a carbon-analogue of TMS-MNU, in rats were investigated and compared with those of MNU. Compounds were dissolved in olive oil and rats in the experimental groups received 20 weekly intragastric intubations of 10 mg/kg of MNU or equimolar amounts of TMS-MNU or neoPNU in the same manner. The experiment was terminated when the survivors were sacrificed at the 52nd week after the final adminstration. In the TMS-MNU and MNU groups, tumors of the forestomach were induced and the incidence was 100% in the groups of both sexes. In addition, tumors of the glandular stomach, nervous system, kidney, and lung were also observed in these groups. Neurogenic tumors were found more frequently in the MNU group than in the TMS-MNU group. The incidence of lung tumors, however, was higher in the TMS-MNU group than in the MNU group. On the other hand, in the control and neoPNU groups, no tumor was found in these organs except the lung, and all tumors observed in these two groups were histologically similar to spontaneous ones in this strain of rats. These results indicate that the carcinogenicity of N-alkyl-N-nitrosoureas is dependent on the chemical structure of their alkyl chain. The result of the present study coincides with the previous result that the species of TMS-MNU in the alkylating step is the same as that of MNU, but different from neoPNU. The difference in the organ specificity between TMS-MNU and MNU demonstrates that the organ specificity is dominantly dependent on the distribution of the chemicals, since TMS-MNU may possibly be distributed differently from MNU because of its different partition property.Abbreviations TMS-MNU N-trimethylsilylmethyl-N-nitrosourea - neoPNU N-neopentyl-N-nitrosourea - MNU N-methyl-N-nitrosourea Part of this work was presented at the 46th Annual Meeting of the Japanese Cancer Association in Tokyo September, 1987     

Heparan sulfate Ndst1 regulates vascular smooth muscle cell proliferation, vessel size and vascular remodeling

Heparan sulfate proteoglycans are abundant molecules in the extracellular matrix and at the cell surface. Heparan sulfate chains are composed of groups of disaccharides whose side chains are modified through a series of enzymatic reactions. Deletion of these enzymes alters heparan sulfate fine structure and leads to changes in cell proliferation and tissue development. The role of heparan sulfate modification has not been explored in the vessel wall. The goal of this study was to test the hypothesis that altering heparan sulfate fine structure would impact vascular smooth muscle cell (VSMC) proliferation, vessel structure, and remodeling in response to injury. A heparan sulfate modifying enzyme, N-deacetylase N-sulfotransferase1 (Ndst1) was deleted in smooth muscle resulting in decreased N- and 2-O sulfation of the heparan sulfate chains. Smooth muscle specific deletion of Ndst1 led to a decrease in proliferating VSMCs and the circumference of the femoral artery in neonatal and adult mice. In response to vascular injury, mice lacking Ndst1 exhibited a significant reduction in lesion formation. Taken together, these data provide new evidence that modification of heparan sulfate fine structure through deletion of Ndst1 is sufficient to decrease VSMC proliferation and alter vascular remodeling.     

Effect of N-acetylcysteine on the Murine Model of Colitis Induced by Dextran Sodium Sulfate Through Up-Regulating PON1 Activity

Reactive oxygen species (ROS) are increased in inflammatory bowel disease (IBD) and have been implicated as mediators of intestinal inflammation. We investigated the hypothesis that N-acetylcysteine (NAC) as a glutathione (GSH) precursor attenuates disease progression in a murine dextran sodium sulfate (DSS)-induced colitis model. A colitis model was induced by adding 5% DSS into the drinking water for 7 days. BALB/c mice were injiciatur enema with saline, 5-ASA, N-acetylcysteine, respectively, and free drinking water as control group. DSS-treated mice developed severe colitis as shown by bloody diarrhea, weight loss, and pathologic involvement. Colon lengths were significantly decreased in DSS-treated mice with decreased GSH activity too (P < 0.01). ROS in the colon, the level of interleukin 1β (IL-1β) in colonic mucosa, serum tumor necrosis factor a (TNF-α), MPO, and MDA were significantly increased in DSS-treated animals (P < 0.01), with decreased PON1 activity (P < 0.01). However, NAC significantly decreased colonic MPO activity, ROS, TNF-α and IL-1β levels and increased PON1 activity and GSH concentration. Moreover, NAC attenuated the macroscopic colonic damage and the histopathologic changes-induced by DSS while similar to 5-ASA group. These results suggest that NAC may be effective in the treatment of colitis through its up-regulating PON1 and scavenging oxygen-derived free radicals.     

Blood flow and mucoid cap protect against penetration of carcinogens into superficially injured gastric mucosa of rats

In this study we tested the influence of blood flow and the mucoid cap on the penetration of carcinogens to the proliferative cells in the injured rat gastric mucosa. Ten minutes after mucosal exposure to 4.5 mol/liter NaCl,N-[³H]methyl-N-nitro-N-nitrosoguanidine was instilled intragastrically. Hypertonic saline caused superficial mucosal damage, formation of a mucoid cap, high gastric mucosal blood flow, and a large flux of fluid into the gastric lumen. The mean percentage of S-phase cells labeled with carcinogen (the cell population at risk forN-methyl-N-nitro-N-nitrosoguanidine-induced carcinogenesis) in the antrum and corpus was 0.2 and 0.2, respectively, in the injury control group, 10.1 and 2.0 after removal of the mucoid cap, 1.5 and 9.8 after celiac artery ligation, and 28.2 and 21.9 after removal of the mucoid cap and celiac artery ligation. These results show that both the mucoid cap and gastric mucosal blood flow protect against penetration of carcinogens into the superficially injured gastric mucosa.This work was supported by grants from the Norwegian Cancer Society and Helga Sembs fond. Dr Sørbye is a Research Fellow of the Norwegian Cancer Society.     

New records of mononchid nematodes from forests in the Slovak Republic

Summary  Three mononchid nematode species new for the Slovak Republic were recorded in forest ecosystems. In regularly-flooded forests with Fraxineto-Salicetum vegetation, the species Mylonchulus andrassyi and Tigronchoides ginglymodontus were recorded; in soil with the Querceto-Carpinetum forest type, the species Miconchus hopperi was observed. Comments on the morphometrical characteristics and ecology of the recorded species are presented here.     

DNA methylation in the digestive tract of F344 rats during chronic exposure toN-methyl-N-nitrosourea

Summary The formation ofO ⁶-methyldeoxyguanosine (O ⁶-MedGuo) was determined by an immuno-slot-blot assay in DNA of various tissues of F344 rats exposed toN-methyl-N-nitrosourea (MNU) in the drinking water at 400 ppm for 2 weeks. Although the pyloric region of the glandular stomach is a target organ under these experimental conditions, the extent of DNA methylation was highest in the forestomach (185 molO ⁶-MedGuo/mol guanine). Fundus (91 mol/mol guanine) and pylorus (105 mol/mol guanine) of the glandular stomach, oesophagus (124 mol/mol guanine) and duodenum (109 mol/mol guanine) showed lower levels ofO ⁶-MedGuo but differed little between each other. Thus, no correlation was observed between target organ specificity and the extent of DNA methylation. This is in contrast to the gastric carcinogen,N-methyl-N-nitro-N-nitrosoguanidine (MNNG), which preferentially alkylates DNA of the pylorus, the main site of induction of gastric carcinomas by this chemical. In contrast to MNU, the non-enzymic decomposition of MNNG is accelerated by thiol compounds (reduced glutathione,l-cysteine), which are present at much higher concentrations in the glandular stomach than in the forestomach and oesophagus. During chronic exposure to MNNG (80 ppm), mucosal cells immunoreactive toO ⁶-MedGuo are limited to the luminal surface [Kobori et al. (1988) Carcinogenesis 9:2271–2274]. Although MNU (400 ppm) produced similar levels ofO ⁶-MedGuo in the pylorus, no cells containing methylpurines were detectable by immunohistochemistry, suggesting a more uniform methylation of mucosal cells by MNU than by MNNG. After a single oral dose of MNU (90 mg/kg) cells containing methylpurines were unequivocally identified using antibodies toO ⁶-MedGuo and the imidazole-ring-opened product of 7-methyldeoxyguanosine. In the gastric fundus, their distribution was similar to those methylated by exposure to MNNG, whereas the pyloric region contained immunoreactive cells also in the deeper mucosal layers. After a 2-week MNU treatment, the rate of cell proliferation, as determined by bromodeoxyuridine immunoreactivity, was only slightly enhanced in the oesophagus and in the fundus, but markedly in the forestomach and the pyloric region of the glandular stomach. It is concluded that the overall extent of DNA methylation, the distribution of alkylated cells within the mucosa and the proliferative response all contribute to the organ-specific carcinogenicity of MNU.Abbreviations MNU N-methyl-N-nitrosourea - MNNG N-methyl-N-nitro-N-nitrosoguanidine - O ⁶-MedGuo O ⁶-methyldeoxyguanosine - 7-MedGuo 7-methyldeoxyguanosine (imidazole ring open) - BrdUrd bromodeoxyuridine     

Hyperglycaemia is associated with changes in the regional concentrations of glucose and myo-inositol within the brain

Aims/hypothesis  The aim of the study was to assess the effect of hyperglycaemia on regional concentrations of glucose and other substrates within the brain in non-diabetic individuals and in patients with type 1 diabetes. Methods  The brain metabolites of 17 men with type 1 diabetes and 12 age-matched non-diabetic men (22–43 years old) were studied after an overnight fast (plasma glucose 9.2 ± 3.0 vs 4.8 ± 0.5 mmol/l, respectively). N-Acetylaspartate (NAA), creatine, choline, myo-inositol (mI) and glucose in the frontal cortex, frontal white matter and thalamus were quantified with proton magnetic resonance spectroscopy. Results  In the non-diabetic participants, the glucose level was 47% higher (p < 0.01) in the frontal cortex than in the frontal white matter. In contrast, this regional variation was not observed in the diabetic participants, in whom the glucose level in the frontal white matter was 64% higher (p < 0.001) and in the frontal cortex 25% higher (p = 0.033) than that of the non-diabetic participants. In the diabetic participants, the glucose level in each of the three regions studied correlated with fasting plasma glucose (r = 0.88–0.67, p < 0.01). In addition, in the diabetic participants, mI was 20% higher (p < 0.001) and NAA 6% lower (p = 0.037) in the frontal white matter, and mI was 8% higher (p = 0.042) in the frontal cortex, than in the non-diabetic participants. Conclusions/interpretation  In type 1 diabetes, hyperglycaemia is associated with accumulation of glucose and mI in the cortex and in the white matter. The results of this study were presented in abstract form at the 17th Annual Meeting of the Diabetic Neuropathy Study Group (NEURODIAB) of the EASD in Utrecht, the Netherlands, 14–16 September 2007.     

<Emphasis Type="Italic">N</Emphasis>-Acetyl-<Emphasis Type="SmallCaps">l</Emphasis>-Glutamine,A Liquid-Stable Source of Glutamine,Partially Prevents Changes in Body Weight and on Intestinal Immunity Induced by Protein Energy Malnutrition in Pigs

The goal of this study was to evaluate the preventive effect of free glutamine versus N-acetyl-l-glutamine, a liquid-stable source of glutamine, on gut damage induced by protein energy malnutrition in pigs. Healthy pigs (n=6) were fed a liquid formula for 30 days. Three subgroups of malnourished pigs (n=6) received daily 20% of the food intake recorded in control group, supplemented with calcium caseinate, glutamine, or N-acetyl-l-glutamine. Body weight was recorded, and small intestinal samples were evaluated for biochemical and immunologic parameters. Suppression in body weight gain was significantly lower in pigs fed with N-acetyl-l-glutamine than in the rest of malnourished pigs. Total number of lymphocytes, CD21⁺ B cells and CD4⁺ T cells in ileal Peyer patches were not significantly different in malnourished pigs fed with N-acetyl-l-glutamine and in healthy pigs. In conclusion, N-acetyl-l-glutamine has a moderate protective effect, partially preventing changes induced by protein energy malnutrition.     

Morphology and modes of cell proliferation in earliest signet-ring-cell carcinomas induced in canine stomachs byN-ethyl-N′-nitro-N- nitrosoguanidine

Summary Signet-ring-cell carcinomas were induced in the stomach of 12 beagle dogs by p.o. administration ofN-ethyl-N-nitro-N-nitrosoguanidine (ENNG), and the morphology and modes of cell proliferation in an incipient stage of cancer growth were studied with bromodeoxyuridine (BrdUrd) incorporation. From 5 to 27 months after the completion of 8 months' carcinogen treatment, minute carcinomas were found in the stomachs of 9 dogs. Before sacrifice, the dogs were given a single or repeated i.v. injections of BrdUrd for 1–3 days. Minute signet-ring-cell carcinomas were found to form a layered structure, in which the cancer cells proliferated in the lamina propria at the gland-neck level and differentiated to postmitotic signet-ring cells at the upper and lower levels of the mucosa. From repeated injections of BrdUrd, the time required for all the proliferative cells to be labelled with BrdUrd (reflecting the maximum cellcycle time) was estimated to be 1.7 days for the normal glands, and 2.7 days for minute signet-ring-cell carcinomas. From the labelling index with BrdUrd as well as from the morphology, earliest carcinomas were identified in the single gland. There remained atrophic normal epithelium commonly in the single-gland lesions. Proliferative atypical cells appeared to be shed into the stroma passively through the atrophy and subsequent collapse of the gland rather than through active invasion. This may be a reason why cancer cells in minute signet-ring cell carcinomas preserved the normal pattern of cell renewal movement to form the layered structure.Abbreviations used ENNG N-ethyl-N-nitro-N-nitrosoguanidine - BrdUrd bromodeoxyuridine - LI labelling index - t _c cell cycle time - t _s duration of DNA synthetic phase This work was supported by Grants-in Aid for Cancer Research from the Ministry of Education. Science and Culture, Japan.     

Detection of oncogene mutation from neoplastic colonic cells exfoliated in feces

Purpose: Best chances of a cure from colorectal cancer are obtained before metastatic spread. Lack of specific tests allowing early diagnosis of the tumor accounts for investigation of gene alterations involved in carcinogenesis by a noninvasive method. In the present study, K-ras codons 12 and 13 mutations were studied in neoplastic cells shed from the bowel into the stool and those contained in the tumor and normal mucosa. Moreover, healthy patients and a few others with precancerous conditions were examined. METHODS: Stool, tumor, and mucosa samples were taken from 25 patients with colorectal adenocarcinoma. Stool and mucosa samples were obtained from 11 healthy patients, and stool, pathologic bowel tissue, and normal mucosa samples were obtained from 3 patients with adenoma (1) or ulcerative colitis (2). Polymerase chain reaction amplification and restriction enzyme analysis were performed. RESULTS: K-ras codon 12 mutations were detected in both tumor and stool samples of 10 cancer patients, and no gene alterations were observed in 14 patients. In one patient with a tumor, a mutation was shown in only the tumor tissue. The agreement rate in tumor and stool analysis was 96 percent. A normal pattern of K-ras codons 12 and 13 was observed in the bowel mucosa. All stool and mucosa samples from healthy patients were not altered in K-ras.Agreement was registered between samples taken from patients with preneoplastic lesions. CONCLUSIONS: These preliminary findings show a high rate of accuracy in the investigation of K-ras alterations in the colorectal cells shed into the feces, suggesting that such an approach could be used to study other gene alterations and, prospectively, to identify early colorectal cancers.Read at the meeting of The American Society of Colon and Rectal Surgeons, Montreal, Quebec, Canada, May 7 to 12, 1995.     

Occurrence in human urine of new sulphur-containing N-nitrosamino acids N-nitrosothiazolidine 4-carboxylic acid and its 2-methyl derivative,and their formation

Summary To quantitate endogenous nitrosation reactions in man, the quantity of N-nitrosoproline (NPRO) excreted in the urine after ingestion of proline and/or nitrate was estimated. When this monitoring method (NPRO test) was applied in clinical and field studies, several hitherto unidentified N-nitroso compounds were frequently detected. These were recently identified as sulphur-containing N-nitrosamino acids, N-nitrosothiazolidine 4-carboxylic acid (NTCA), and trans- and cis-isomers of N-nitroso-2-methylthiazolidine 4-carboxylic acid (NMTCA).NTCA and NMTCA were readily formed in vitro following nitrosation at acidic pH of the respective precursor, thiazolidine 4-carboxylic acid (TCA) or of 2-methylthiazolidine 4-carboxylic acid (MTCA). As the latter compounds can be formed by reaction of l-cysteine with formaldehyde or acetaldehyde, respectively, NTCA and NMTCA were also formed by reacting l-cysteine with the respective aldehyde and with nitrite at optimal pH (2.5 for NTCA and 4.5 for NMTCA).Up to 95% of NTCA and NMTCA given orally to fasted rats was recovered as such in urine and faeces within 2 days. Administration of TCA or MTCA, together with nitrite increased the urinary excretion of NTCA and NMTCA, as did co-administration of l-cysteine, nitrite, and the respective aldehyde.NTCA and NMTCA were also detected in the 24-h urine of human volunteers, and smokers tended to excrete higher levels than nonsmokers. Daily excretion levels varied, however, and a diet supplemented with ascorbic acid significantly descreased the total amount of nitrosamino acids. NTCA and NMTCA may occur in human urine as a result of (i) intake of preformed N-nitroso compounds; (ii) intake of thiazolidine 4-carboxylic acid or its 2-methyl derivative and subsequent nitrisation in vivo; (iii) endogenous two-step synthesis by the reaction of l-cysteine with the respective aldehyde and a nitrosating agent.Thus, measurement of NTCA and NMTCA together with NPRO in urine may provide an index for the exposure of human subjects to nitrosamines or their precursors, i. e., nitrosating agents, certain aldehydes, or aldehyde-generating compounds. Our data demonstrate unequivocally that N-nitroso compounds are formed in the human body, as suggested previously by Druckrey. Their relevance to human cancer at specific sites should now be investigated.Abbreviations NPRO N-nitrosoproline - NSAR N-nitrososarcosine - NTCA N-nitrosothiazolidine 4-carboxylic acid - NMTCA N-nitroso-2-methylthiazolidine 4-carboxylic acid - TCA thiazolidine 4-carboxylic acid - MTCA 2-methylthiazolidine 4-carboxylic acid - NPIC N-nitrosopipecolic acid - GC gas chromatography Dedicated to Professor Hermann Druckrey on the occasion of his 80th birthday     

Abnormalities of tumor suppressor gene <Emphasis Type="Italic">p16</Emphasis>in pancreatic carcinoma: immunohistochemical and genetic findings compared with clinicopathological parameters

Background. Abnormalities of the tumor suppressor gene p16 have been reported in a variety of human tumors but are rare in pancreatic carcinoma except for cancer cell lines and xenografts. Their clinicopathological significance remains unknown. The purpose of this study was to examine immunohistochemical and genetic alterations of p16 in primary pancreatic carcinoma tissues and to investigate the relation between abnormalities of p16 and clinicopathological parameters to elucidate their clinicopathological significance. Methods. We investigated p16 expression in 60 pancreatic carcinoma cases by immunohistochemistry using a monoclonal antibody clone G175-405. In addition, we analyzed genetic alterations of the p16 gene using DNA extracted from microdissected tissue of pancreatic carcinoma, by polymerase chain reaction, nonradioisotopic single-strand conformation polymorphism (non-RI-SSCP), DNA sequencing, and hypermethylation analyses using restriction enzymes. We compared the abnormalities of p16 alterations with clinicopathological parameters to elucidate their significance. Results. On immunohistochemical study, staining for p16 protein was strongly positive in 22 (37%) of 60 pancreatic carcinoma cases, weakly positive in 24 (40%), and negative in 14 (23%). In contrast, p16 mutations were recognized in 9 (15%) of the 60 pancreatic carcinoma cases. The incidence of p16 mutations was 2 (9%) in 22 cases of pancreatic carcinoma with strongly positive staining, 4 (17%) in 24 with weakly positive staining, and 3 (21%) in 14 with negative staining. Hypermethylation of p16 was detected in the two pancreatic carcinoma cases with weakly positive staining, although homozygous deletions were not found in any case. There was no significant correlation between the expression of p16 protein and any of the clinicopathological parameters. However, there was a tendency for the tumor to be larger in patients with decreased expression of p16 protein than in those with normal expression levels. In contrast, the tumor was significantly larger and the survival period significantly shorter for patients with pancreatic carcinoma with p16 mutation or hypermethylation than for those with pancreatic carcinoma with an intact p16 gene (P 0.05). Conclusions. These findings suggest that p16 alterations may participate in the aggressiveness of pancreatic carcinoma.     

Different effects of sequential combinations ofN-methylformamide with 5-fluorouracil on human colon carcinoma cells growing in nude mice

Summary The effects of the combination ofN-methylformamide (NMF) with 5-fluorouracil (5-FU) on tumor growth and morphological features of human colon carcinoma cells (HT29) implanted in nude mice were assessed. Both agents were administered i.p. at tolerable doses: 5-FU at 19 mg/kg for 5 days and NMF at 200 mg/ kg for 12 days. Four main schedules were tested: 5-FU alone, NMF alone, NMF followed by 5-FU and 5-FU followed by NMF. The last sequence was the most effective, as compared with the other treatment regimens. In particular, the 5-FU NMF combination induced a tumor inhibition of about 75% at the end of the treatments (17th day) versus an inhibition of 23%–43% in the other schedules. Morphological observations, carried out by light and electron microscopy, indicated a possible relationship between the presence of structural changes and tumor growth inhibition. The results of this study renew interest in the use of NMF in sequential combination confirming sequence as a critical factor for the optimal combination of NMF and 5-FU.Abbreviations 5-FU 5-fluorouracil - NMF N-methylformamide - DMF N,N-dimethylformamide - cisplatin cis-diamminedichloroplatinum - m _T/m _c tumor weight inhibition - t _T-t _c tumor growth delay This work was supported by grants from the Italian National Research Council, contract 89.03979.CT04; Ministero della Sanità; Istituto Oncologico Romagnolo n 90152.1     

Effect of N-acetylcysteine on oxidative stress and ventricular function in patients with myocardial infarction

Recent evidence suggests that postischemic myocardial dysfunction (“stunning”) may be mediated by oxygen free radicals. Various studies have reported the beneficial effects of antioxidants in ischemia–reperfusion injury. The aim of this study was to assess the effect of N-acetylcysteine (NAC) treatment on oxidative stress, infarct size, and left ventricular (LV) function, as adjunct therapy in myocardial infarction (MI). Patients with acute MI received either 15 g NAC infused over 24 h (n = 15) or no NAC (n = 15), combined with streptokinase. Peripheral venous blood was serially sampled to measure creatine kinase (CK)-MB levels. Plasma malondialdehyde (MDA) level was measured at admission and after 4 and 24 h. Echocardiography was performed within 3 days of MI and after 3 months. At admission, plasma MDA levels were not different between the groups. In the NAC-treated patients plasma MDA levels decreased, whereas in the nontreated NAC patients MDA levels increased at 4 and 24 h (P < 0.01 and P < 0.001, respectively). Left ventricular ejection fraction was higher (P < 0.05) and LV end-systolic and end-diastolic diameters were lower (P < 0.001 and P < 0.001) in patients receiving NAC on day 3. Left ventricular wall motion score index was significantly lower in patients treated with NAC on day 3 (P < 0.05). Left ventricular diastolic parameters were not different whether patients were treated with NAC or not. No difference in reduction of infarct size was detected between the groups according to CK-MB levels. It was thus demonstrated that administration of NAC in combination with streptokinase significantly diminished oxidative stress and improved LV function in patients with acute MI. These encouraging results would justify the performance of a larger controlled study.     

Measurement of melatonin and its metabolites: importance for the evaluation of their biological roles

Many physiologic changes related to lightdark cycles and antioxidant effects have been related to melatonin (N-acetyl-5methoxytryptamine) and its metabolites, N ¹-acetyl-N ²-formyl-5methoxykynuramine AMK) and N ¹-acetyl-5methoxykynuramine AMK). In this review, we discuss some methodologies, in particular, those employing high-performance liquid chromatography tandem mass spectrometry (HP (MS/MS)assays to quantitatively determine melatonin, AMK, and AMK. These approaches offer a highly specific and an accurate quantification of melatonin and its metabolites. These characteristics are essential to point out correctly the biological effects of these compounds in physiological and pathological conditions.     

Nephrotoxicity of acyclovir andcis-diamminedichloroplatinum(II)-effect of co-administration in rats

Summary The effect of co-administration of acyclovir andcis-diamminedichloroplatinum(II) (cisplatin) on nephrotoxicity in male Wistar rats was investigated. Animals received acyclovir (15 mg/kg body weight, s.c., three times per day for 5 days) or cisplatin (5 mg/kg body weight, i.p., one single injection) or a combination of both drugs. Acyclovir plasma levels were determined after one single acyclovir s.c. injection. Urines were monitored for volume, pH, osmolality and excretion ofN-acetyl--d-glucosaminidase (NAG), lysozyme and total protein. Concentrations of blood urea nitrogen and plasma creatinine were determined on day 6. Renal cortical slices were monitored to assess the accumulation of weak organic bases (tetraethylammonium) and acids (p-aminohippurate). Cisplatin induced a marked increase in the excretion of NAG, lysozyme and total protein and an increase in urine volume, plasma creatinine and blood urea nitrogen. Urine osmolality and accumulation ofp-aminohippurate were depressed by cisplatin. Acyclovir treatment alone caused no significant symptoms of nephrotoxicity. Co-administration did not impair renal function more than cisplatin treatment alone, excepting a slight rise in lysozyme excretion on day 6. Short-term antiviral therapy with acyclovir, concomitant to cisplatin treatment, may bring, if at all, a slightly increased nephrotoxic risk.Abbreviations Cisplatin cis-diamminedichloroplatinum(II) - NAG N-acetyl--d-glucosaminidase - BUN blood urea nitrogen - TEA tetraethylammonium - PAH p-aminohippurate