Dose-response curves for liver DNA fragmentation induced in rats by sixteen N-nitroso compounds as measured by viscometric and alkaline elution analyses.

A new viscometric technique, capable of detecting DNA strand breaks and alkali-labile sites by monitoring time-dependent changes of DNA-reduced viscosity, has been used to analyze dose-response curves for the induction of DNA damage in liver of rats treated with single p.o. doses of sixteen N-nitroso compounds. Statistically significant changes of DNA viscometric parameters, which are considered indicative of DNA fragmentation, were produced by N-nitrosodimethylamine (0.022 mg/kg), N-nitrosomethylethylamine (0.025 mg/kg), N-nitrosodiethylamine (0.067 mg/kg), N-nitrosodiethanolamine (1.03 mg/kg), N-nitrosodi-n-propylamine (0.31 mg/kg), N-nitrosodi-n-butylamine (0.083 mg/kg), N-nitroso-N-methylurea (0.56 mg/kg), N-nitroso-N-ethylurea (0.37 mg/kg), N-nitroso-N-butylurea (0.16 mg/kg), streptozotocin (20 mg/kg), N-nitrosomorpholine (0.4 mg/kg), N-nitrosopiperidine (2.22 mg/kg), N-nitrosopyrrolidine (5.0 mg/kg), 1-nitroso-2-imidazolidinone (0.31 mg/kg), and N-methyl-N'-nitro-N-nitrosoguanidine (5.57 mg/kg). The contemporary measurement of liver DNA fragmentation by the alkaline elution technique revealed that in our experimental conditions higher doses are needed to produce a statistically significant increase of DNA elution rate. This suggests that the viscometric method is capable of detecting smaller levels of N-nitroso compound-induced DNA fragmentation, but it does not exclude that the sensitivity of alkaline elution can be improved by appropriate modifications of the experimental procedure. With both techniques DNA damage was undetectable in liver of rats treated with 540 mg/kg of the non-hepatocarcinogen N-nitrosodiphenylamine. With the exception of N-nitrosodiethanolamine, that exhibited a plateau effect, all the other N-nitroso compounds examined displayed a linear dose-response curve over the entire wide range of doses tested. Consequently, a nonlinearity of the relationship between dose and tumor response cannot be attributed to a nonlinearity of the pharmacokinetic processes involved in the formation of DNA damage.

Chronic administration of levosulpiride and glycemic control in IDDM patients with gastroparesis.

OBJECTIVE: We evaluated the effect of chronic administration of levosulpiride, a prokinetic drug that is a selective antagonist for D2 dopamine receptors, on the glycemic control of IDDM subjects. RESEARCH DESIGN AND METHODS: The study was performed on 40 long-standing IDDM subjects with clinical signs of autonomic neuropathy and delayed gastric emptying. Gastric emptying time and glycemic parameters (diurnal glycemic profile and HbA1c) were checked under double-blind conditions before and after the administration of levosulpiride at the dosage of 25 mg t.i.d. orally for 6 months, or placebo. RESULTS: No significant differences were noted in the glycemic and HbA1c values before and after 6 months of placebo administration. In contrast, after 6 months of levosulpiride, glycemic control had improved (HbA1c 6.7 +/- 0.4 and 5.7 +/- 0.3%, P < 0.01; mean daily glycemia 10.9 +/- 0.8 and 8.8 +/- 0.4 mmol/l, P < 0.05, at the start and at the end of the study), while the dosage of injected insulin (0.65 +/- 0.02 IU.kg-1.day-1) and the number of severe hypoglycemic episodes remained unchanged. After 6 months of levosulpiride therapy, the time of gastric emptying was significantly reduced from 321 +/- 14 to 261 +/- 9 min (P < 0.001) and dyspeptic symptoms had improved. CONCLUSIONS: Our results show the importance of gastric emptying in the maintenance of glycemic control and the usefulness of chronic administration of levosulpiride in diabetic subjects with gastroparesis.

In vitro effects on monoamine uptake and release by the reversible monoamine oxidase-B inhibitors lazabemide and N-(2-aminoethyl)-p-chlorobenzamide: a comparison with L-deprenyl.

To investigate whether the reversible monoamine oxidase-B (MAO-B) inhibitors lazabemide and Ro 16-6491 have any additional effect on monoamine uptake and release, in vitro experiments were performed on rat forebrain synaptosomes and blood platelets. The effects of the two drugs were compared with those of L-deprenyl, the well-known irreversible MAO-B inhibitor which is reported to affect amine uptake. Both lazabemide and Ro 16-6491 behaved as weak inhibitors of [3H]monoamine uptake by synaptosomes, with a similar rank order of potency for amine uptake inhibition (noradrenaline (NA) > or = 5-hydroxytryptamine (5 HT) > dopamine (DA)). The IC50 values for lazabemide and Ro 16-6491, respectively, were: 86 microM and 90 microM for NA uptake; 123 microM and 90 microM for 5HT uptake; > 500 microM and > 1000 microM for DA uptake. L-Deprenyl (rank order of inhibitory potency: NA > DA > 5 HT) was four to 10 times more potent than either compound in inhibiting [3H]catecholamine uptake (IC50 = NA 23 microM, DA 109 microM), and two to three times less potent in inhibiting 5 HT uptake (IC50 233 microM). Lazabemide and Ro 16-6491 also differed from L-deprenyl in their ability to induce release of endogenous monoamines from synaptosomes. Thus, Ro 16-6491 (500 microM) induced a greater 5 HT release than did L-deprenyl, but was less effective than L-deprenyl in releasing DA. On the contrary, lazabemide was almost completely inactive on either 5 HT and DA release. The differential effect of the three MAO-B inhibitors on synaptosome 5 HT uptake and release was confirmed by [14C]5HT uptake and liberation experiments with isolated rat platelets. The data indicate that the reversible MAO-B inhibitors lazabemide and Ro 16-6491 at relatively high concentrations possess amine uptake-inhibiting properties. With regard to the effects examined, lazabemide markedly differs from L-deprenyl since it does not interfere with DA uptake nor induce amine release from synaptosomes.

Effect of D2-dopamine receptor antagonist levosulpiride on diabetic cholecystoparesis: a double-blind crossover study.

BACKGROUND: Abnormal gall-bladder motility has been reported in diabetics. The objective was to evaluate the effect of chronic D2-dopamine receptor inhibition on gall-bladder emptying in diabetic patients. METHODS: Under double-blind placebo-controlled conditions and according to a crossover design, patients were randomly assigned to receive either 4 weeks treatment with levosulpiride 25 mg t.d.s. or 4 weeks treatment with placebo, with an interval of 15 days. Twenty-three consecutive long-standing, insulin-treated diabetics with autonomic neuropathy were studied. MEASUREMENTS: At the beginning of the study and after levosulpiride or placebo treatment, gall-bladder emptying was measured ultrasonically by evaluating the gall-bladder volume in basal conditions and every 15 min for 90 min after the ingestion of a standard meal. Statistical analysis of the results was performed by means of analysis of variance. RESULTS: Levosulpiride treatment reduced the basal mean gall-bladder volume from 21.6 +/- 2.3 to 18.6 +/- 2.3 mL (P < 0.05). Furthermore, the residual gall-bladder volume (9.3 +/- 1.4 mL) was significantly reduced compared to the corresponding pre-treatment volume (14.6 +/- 1.5 mL (P < 0.05). In placebo-treated patients, no significant differences were observed in gall-bladder volumes before and after treatment. CONCLUSION: These results show that chronic oral administration of the D2-dopamine antagonist levosulpiride has a significant effect on gall-bladder motility in diabetic patients.

DNA damage and repair induced by diazoacetyl derivatives of amino acids with different mechanism of cytotoxicity. Correlations with mutagenicity and carcinogenicity.

Eight synthetic N-diazoacetyl amino acids, prepared by inserting a diazoacetyl group onto the alpha-nitrogen of a natural amino acid, and two natural diazoazetyl amino acids, azaserine (9-diazoacetyl-L-serine) and DON (6-diazo-5-oxo-L-norleucine), have been studied by autoradiography for their capacity to induce DNA repair synthesis in mouse cells cultivated "in vitro". Dose-dependent unscheduled DNA synthesis was present in cells treated with the eight N-diazoacetyl derivatives, and was absent in cells exposed to approximately equitoxic concentrations of azaserine and DON. Azaserine and DON, unlike N-diazoacetyl derivatives, did not alkylate gamma-(4-nitrobenzyl) pyridine at an appreciable extent. When DNA damage (single stranded breaks or weak points in alkali) was measured by the sensitive technique of alkaline elution, DGA was found about 4 times as potent as azaserine and about 12 times as DON on a molar basis, but about 800 and 17,000 times as potent as azaserine and DON respectively by extrapolating to equitoxic concentrations. Carcinogenicity and mutagenicity seem to follow mainly the capability of inducing DNA damage.

Cytotoxicity, DNA fragmentation and sister-chromatid exchange in Chinese hamster ovary cells exposed to the lipid peroxidation product 4-hydroxynonenal and homologous aldehydes.

The cytotoxic and genotoxic activities of 4-hydroxypentenal (HPE), 4-hydroxyhexenal (HHE), 4-hydroxyoctenal (HOE), 4-hydroxynonenal (HNE) and 4-hydroxyundecenal (HUE) were investigated in Chinese hamster ovary (CHO) cells. All five 4-hydroxyalkenals reduced plating efficiency in a concentration (ranging from 7 to 170 microM) lower than that producing a parallel reduction of trypan blue-excluding cells, but with both methods the increase in molarity needed to obtain a lethal effect was constantly rather small. With all five 4-hydroxyalkenals a significant amount of DNA fragmentation, as revealed either by the alkaline elution assay or by alkaline denaturation followed by chromatographic partition of single- and double-stranded DNA, was detected only after cell exposure to a cytotoxic concentration. HPE, HHE and HOE induced a clear-cut increase of sister-chromatid exchange (SCE) frequency, while that displayed by cells treated with HNE and HUE was minimal, even if dose-dependent and statistically significant. Since 4-hydroxyalkenals have been shown to originate from biomembrane lipids peroxidation, these findings should be taken into consideration in the assessment of the genotoxic role of lipoperoxidation in humans.

A practical procedure for testing DNA damage in vivo, proposed for a pre-screening of chemical carcinogens.

The alkaline elution method was adapted to the evaluation of DNA damage induced in vivo through a practical and reliable microfluorometric procedure, without any need for tissue pre-labeling. The DNA damage induced in vivo by treatment with a single dose of N-nitrosodimethylamine (DMNA), N-methyl-N-nitroso-urea (MNU), 1,2-dimethylhydrazine (DMH) or cycasin has been detected in different organs of mice or rats. The results obtained are rather consistent with the organotropism of these carcinogens, and show a satisfactory dose dependent of DNA damage. DMH and cycasin, both negative in the Ames' Salmonella/microsome mutagenicity test, are clearly positive with in vivo DNA damage/alkaline elution assay. This latter method, complemented with other short-term tests, may play a useful role in the pre-screening of chemical carcinogens.

A procedure for the assay of DNA damage in mammalian cells by alkaline elution and microfluorometric DNA determination.

The microfluorimetric assay of Kissane and Robins (J. Biol. Chem. 233, 184-188, 1958) has been modified to monitor DNA content in alkaline elution fractions. The elution profiles of DNA from treated and control cultured cells demonstrated a good correlation whether DNA in the elution fractions was determined by the fluorometric method or by measuring its radioactivity. The DNA recovery was of about 80% and the measured material was DNase digestible.

DNA-damaging activity of biotic and xenobiotic aldehydes in Chinese hamster ovary cells.

Alkaline elution was employed to study DNA damage in CHO-Kl cells treated with a series of biotic and xenobiotic aldehydes. DNA cross-linking was measured in terms of the reduction in the effect of methyl methanesulphonate on the kinetics of DNA elution and was observed in cells treated with formaldehyde, acetaldehyde, methylglyoxal and malonaldehyde. Propionaldehyde, valeraldehyde, hexanal and 4-hydroxynonenal produced DNA single-strand breaks, or lesions which were converted to breaks in alkali. Both types of DNA damage occurred in cells exposed to malealdehyde. These findings support the hypothesis of a carcinogenic effect of the aldehydic products (malonaldehyde, methylglyoxal, propionaldehyde, hexanal, 4-hydroxynonenal) released in biomembranes during lipid peroxidation.

Influence of culture conditions on monoamine oxidase A and B activity in rat astrocytes.

Astroglial cells dispersed from newborn rat hemispheres were established in medium supplemented with 20 per cent fetal calf serum (FBS) and then grown to a confluent monolayer in the presence of 10 per cent FBS or charcoal-stripped FBS (CS). Type 1 astrocytes were subcultured and either maintained under the same conditions of the primary cultures or converted to serum-free chemically defined medium (CDM). No differences were found in either MAO A or MAO B activity of astrocytes grown in the presence of FBS or CS after 15 and 21 days in vitro (day 1 and 6 of subculture). In contrast, on day 21 both MAO A and MAO B activities were markedly higher in astrocytes subcultured in CDM compared with cells maintained in serum-supplemented medium. This difference appeared to be due to increased number of enzyme molecules, since kinetic analysis showed an increase in Vmax of both MAO isoenzymes in serum-free medium, but no change in Km. Consistently, the recovery of MAO A and MAO B activity after irreversible enzyme inhibition by clorgyline and deprenyl was faster in CDM than in FBS-supplemented medium, indicating enhanced enzyme synthesis under serum-free condition. Estimates of half-lives for the recovery of MAO A and MAO B activity indicated that, under both culture conditions, type A activity had a higher turnover rate than type B. The effect of CDM on astrocyte MAO does not appear to be due to selection of a subpopulation of cells, but rather linked to a morphological change (differentiation) with increased synthesis of both MAO isoenzymes.

DNA damage in liver, colon, stomach, lung and kidney of BALB/c mice treated with 1,2-dimethylhydrazine.

DNA single-strand breaks induced in various organs of BALB/c mice by treatment with a single dose of 1,2-dimethylhydrazine (DMH) were studied by means of the alkaline elution method modified in order to allow the evaluation of DNA damage in vivo with no need of radioactive prelabelling. DNA damage was detected in liver, lung, kidney, stomach and colon mucosa, with the liver showing the greatest amount of damage. Its degree was dependent on the dose and route of administration. A differential effect was evident in colon mucosa from Swiss and C57BL/6 mice which are respectively susceptible and resistant to the induction of bowel tumors by DMH. The higher degree of DNA damage found in liver in comparison with colon mucosa is consistent with the previously reported higher degree of DNA methylation, but does not correlate with the specificity of this carcinogen in inducing tumors of the large intestine in mice given repeated subcutaneous injections.

Different effects of intraperitoneal or subcutaneous administration of H2-antagonists on pancreatic response to chronic administration of caerulein in rats.

This study deals with the effect of H2-receptor antagonists on pancreatic response to chronic administration of caerulein. Caerulein was administered alone or combined with cimetidine, ranitidine or famotidine twice a day in various regimes. At the end of treatment, pure pancreatic juice was collected after hormonal stimulation. Then, the rats were killed, and growth and composition of the pancreatic tissue were determined. Caerulein increased pancreatic weight and enzyme content as well as volume and enzyme activity of pancreatic juice. When given alone the three H2-receptor antagonists were totally ineffective. Both ranitidine and famotidine, but not cimetidine, significantly reduced pancreatic response to chronic administration of caerulein only when given intraperitoneally together with caerulein. On the contrary, separate applications of caerulein and ranitidine (or famotidine) did not influence caerulein-stimulated pancreatic growth or enzyme secretion. Moreover, in rats treated both intraperitoneally and subcutaneously with caerulein, the H2-antagonists reduced the pancreatic response only partially and in proportion to the intraperitoneal dose of caerulein. The responsiveness of pancreatic tissue to subcutaneous caerulein was not modified. The results suggest that H2-receptor antagonists induce (1) impaired uptake of caerulein when given together with peptide, but (2) have no specific inhibitory effect on pancreatic response to caerulein.

Alkaline elution assay as a potentially useful method for assessing DNA damage induced in vivo by diazoalkanes.

In order to evaluate directly in vivo the extent of correlation existing between mutagenic-carcinogenic activity of diazoalkanes and their DNA damaging activity, alkaline elution was used to study the induction of single-strand breaks in and repair of DNA from mice treated with N-diazoacetylglycine amide (DGA). A dose-dependent DNA fragmentation was present, 4 hr after ip injection of single doses of DGA, in liver, lung, kidney, spleen, thymus, and bone marrow. The differences among these organs in the amount of DNA damage were small, even if sometimes statistically significant. Elution profiles indicated that a progressive conversion of alkali-labile sites to single-strand breaks took place in the first 50 min of elution. Twenty-four hr after treatment with 1000 mg/kg, DNA damage was only slightly reduced in liver, lung and bone marrow. A detailed statistical analysis of the variability of experimental results is reported in order to give information about the reliability of the method, and to allow the possibility of calculating the number of separate experiments required to reach statistical significance for a given increase of DNA elution rate.

DNA fragmentation in some organs of rats and mice treated with cycasin.

Cycasin (methylazoxymethanol-beta-D-glucoside) is carcinogenic in several animal species. It produces a variety of malignant tumours, mainly in the liver of mice, and in the liver, kidney and large intestine in rats. It does not appear to be mutagenic in the Ames test, even in the presence of liver microsome fraction, and it is among those carcinogens (less than 10%) ranked as "false negatives" in this test. The ability of cycasin to damage in vivo liver, kidney, lung and colonic DNA of Wistar rats and C57BL/L mice was investigated by means of alkaline elution technique. Oral single-dose administration of cycasin, in the range of 50-400 mg/kg body weight, produced in the rat a clearly evident dose-dependent DNA fragmentation in the liver, and less marked damage to DNA from kidney and colon mucosa. In mice, the same treatment produced dose-dependent DNA damage only in the liver. DNA repair up to 18 h appeared to be incomplete both in mice and rats. Methylazoxymethanol acetate is considered to be an active form of cycasin. While in vivo methylazoxymethanol acetate caused DNA damage, in vitro it appeared inactive and required metabolic activation, possibly consisting in its hydrolysis by esterase activity, to be able to cause DNA fragmentation.

Methylglyoxal-induced DNA-protein cross-links and cytotoxicity in Chinese hamster ovary cells.

The technique of alkaline elution was applied to study the capacity of methylglyoxal to induce DNA damage and repair in Chinese hamster ovary cells. DNA cross-linking was observed after a 90-min exposure to a subtoxic dose (1.5 mM), and the cross-links were fully repaired by 24 h. The cross-linking appeared to be DNA-protein in nature, since proteinase treatment removed the effect. When the same cells were exposed to methylglyoxal in the presence of a rat liver metabolic system, both cytotoxicity and cross-linking frequency were significantly reduced.

Gastrokinetic effects of levosulpiride in dyspeptic patients with diabetic gastroparesis.

OBJECTIVE: Antidopaminergic drugs may be useful in diabetic gastroparesis because the inhibitory activity of hyperglycemia on gastric motility seems to be related to dopamine receptor stimulation. For this reason, we evaluated the effect of levosulpiride on gastric emptying, dyspeptic symptoms, and metabolic parameters of insulin-treated diabetic patients. METHODS: Under double-blind conditions, 40 longstanding, insulin-treated dyspeptic patients with autonomic neuropathy and delayed gastric emptying were randomly submitted, with an interval of 15 days, to 4 wk of administration of both levosulpiride 25 mg t.i.d. and placebo according to a cross-over design. At the beginning of the study and after levosulpiride or placebo treatment, the gastric emptying time of a standard meal was measured ultrasonically; gastrointestinal symptom scores and glycaemic control were also evaluated. RESULTS: Levosulpiride reduced significantly (p < 0.001) the gastric emptying time from 416 +/- 58 to 322 +/- 63 min, whereas placebo did not change it consistently (396 +/- 58 vs 372 +/- 72 min). Symptoms improved significantly (p < 0.001) with levosulpiride compared with placebo. However, there was no significant correlation between the acceleration of gastric emptying and the symptomatological improvement. The reduction of mean plasma glycosylated hemoglobin concentrations after levosulpiride (7.3 +/- 1.9 vs 5.8 +/- 1.3) was not significantly different (p = not significant) compared with placebo (6.8 +/- .7 vs 6.1 +/- 1.4). CONCLUSIONS: Our study first demonstrates that levosulpiride has an accelerating effect on the emptying of solids from the stomach of patients with diabetic gastroparesis. The drug is also effective in relieving upper gastrointestinal symptoms in patients whose gastric emptying times remain very slow. Our findings suggest, but do not prove, that better blood glucose control could be achieved with reduction of gastric emptying time; further trials are needed in this field.

Trophic response and morphological changes in pancreas of caerulein treated rats: dose and time dependent effects.

The present study was performed to determine the effect of the duration of chronic caerulein administration given at different doses, on the rat pancreas. Four groups of rats, one treated with 0.9% NaCl (control) and the others with caerulein 2, 5 and 10 micrograms/Kg twice a day i.p. were used. After a treatment period of 15, 30 and 60 days, 6 rats from each group were anesthetized, the pancreas was removed, and growth and composition of pancreatic tissue were determined. Small samples were taken for histological examination. Caerulein induced pancreatic hyperplasia and hypertrophy. The dose of caerulein used and the length of the treatment did not significantly modify the trophic effect. Focal perivascular and periductular lymphomonocytic infiltrates associated with cellular abnormalities were seen at 30 and 60 days. The results suggest that 1) the trophic effect of caerulein is not dose-and-time dependent and 2) morphological abnormalities can appear during long term treatment with CCK analogous.

In vivo and in vitro genotoxicity of three antihypertensive hydrazine derivatives (hydralazine, dihydralazine, and endralazine).

Three antihypertensive hydrazine derivatives (hydralazine, dihydralazine, and endralazine) were found to be genotoxic in four in vivo or in vitro short-term test systems. a) In mice, a single ip administration of the LD50 of the three drugs caused a small but statistically significant increase over controls in DNA elution rate, ie, a modest amount of DNA fragmentation, in three of the four organs (liver, lung, kidney, and spleen) tested, DNA damage being absent in lung for hydralazine and endralazine and in liver for dihydralazine. Only for hydralazine DNA lesions were always repaired within 12 hr, in agreement with the constant lack of cumulative effects in mice given five successive daily doses. The rank of potencies was hydralazine greater than dihydralazine greater than endralazine. b) In mice bone marrow cells, all three hydrazine derivatives induced a modest but statistically significant increase over controls in the frequency of sister chromatid exchanges, the rank of potencies being in this case dihydralazine greater than endralazine greater than hydralazine. c) In the Ames reversion test all three drugs behaved as direct-acting mutagens of low potency, whose activity was not influenced by rat liver nor by mouse liver or lung S-9 fractions. Hydralazine and dihydralazine elicited mixed genetic mechanisms of mutations, while endralazine exclusively induced frameshift errors in Salmonella DNA. The recently developed strain TA97 was the most efficient in revealing frameshift errors with all three drugs. d) The selective lethality assays in a battery of two S typhimurium and five E coli strains confirmed the direct genotoxicity of hydralazine, dihydralazine, and endralazine, in order of potency. Potency was evaluated by means of a sensitive and reliable micromethod procedure. Among those investigated, the recA recombination repair and the lexA post-replication repair ("SOS functions") and, to a lesser extent, also the polymerase I mechanism, appeared to contribute to the specific DNA repair with all three drugs, while excision repair systems (uvrA and uvrB) did not appear to be involved.

A bioseparation apparatus with high-pressure fluid injection and fluid sampling.

A novel apparatus in which fluids may be injected and sampled at high pressure is described. Bioseparation applications of the apparatus were demonstrated in three model systems: (1) lambdaDNA was eluted under pressure from an anion exchange column into a low-salt (0.25 M) buffer, thereby eliminating conventional time-consuming desalting procedures required for downstream analysis of the DNA; (2) RNA was separated under pressure from a RNA/DNA mixture, thereby enabling rapid differential preparation of nucleic acids; and (3) an antibody was purified from a protein mixture by affinity capture at one pressure and dissociation from the antigen binding partner at a second pressure, thereby enabling the immunoreactivities of both antibody and antigen to be preserved during the separation process.