N-acetyl-cysteine reduces neointimal thickening and procoagulant activity after balloon-induced injury in abdominal aortae of New Zealand white rabbits.

BACKGROUND: Procoagulant activity and oxidative stress generated by balloon injury to normal vessels promote the migration of medial smooth muscle cells and their proliferation in the intima. We hypothesised that administering levo N-acetyl-cysteine (NAC) i.v. at the time of injury, and s.c. before and after injury would reduce neointimal formation 4 weeks later and would regulate procoagulant activity in vessels with neointima undergoing ballooning a second time. METHODS AND RESULTS: at the time of injury rabbits received: NAC, unfractionated heparin (HEP) or both (NAC + HEP). Neointimal thickening at 28 days, calculated as the ratio between the intimal and medial area, was attenuated after NAC, HEP and NAC+HEP by 39%, 30% and 47% respectively when compared to untreated injured animals (CONTROLS) (p <0.05). At 28 days, bound thrombin activity and platelet adhesion 1 h after a repeated balloon injury decreased in animals receiving NAC, HEP and NAC+HEP bv 54%, 63% and 64% for thrombin activity (p <0.05 vs CONTROLS), and by 56%, 66% and 75% respectively for 111Indium-platelet deposition (p <0.05 vs CONTROLS). CONCLUSIONS: NAC in-vivo was effective in reducing neointimal thickening and procoagulant response after balloon injury.

Assay of stanozolol for tumor initiating and promoting activity in two rat liver foci bioassays.

In this study stanozolol, one of the most abused anabolic steroids, was investigated for tumor initiating and promoting activity in two rat liver foci bioassays, using gamma-glutamyltranspeptidase (GGT) as marker for detection of putative preneoplastic foci. Stanozolol, orally administered for 2 weeks, at a dose level approximately 400-times larger than the human therapeutic dose, in rats initiated with N-nitroso-diethylamine according to the Solt-Farber system assay, did not produce any increase in the number and volume of GGT-positive liver foci. A 6-week oral treatment with stanozolol (430 ppm in the diet) followed by 2 weeks of 2-acetylaminofluorene (AAF) diet (200 ppm), carried out according to the Tatematsu assay system to evaluate the initiating activity, did not provoke any significant modification of the number and volume of GGT-positive foci as compared to the controls. In the rats receiving AAF (200 ppm in the diet for 2 weeks) followed by 6 weeks of stanozolol, to evaluate the promoting activity, an increase in number and volume of the GGT-positive foci was observed at the highest oral dose, but the differences from the corresponding control values which resulted were not statistically significant. Taken as a whole the results of this study do not provide any substantial evidence of carcinogenic activity of stanozolol in rat liver, even when orally administered at high doses.

Increased frequency of N-methyl-N'-nitro-N-nitrosoguanidine-induced nuclear anomalies in the forestomach of rats pretreated with sodium chloride.

The frequency of nuclear anomalies (micronuclei, pyknosis, and karyorrhexis) in the forestomach mucosa was examined in Sprague-Dawley male rats given a single oral dose of 50 or 150 mg/kg of the gastric carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) 17 h after the administration of 2 ml of a 3 M NaCl solution. Rats pretreated with NaCl displayed an incidence of nuclear anomalies approximately 3-fold greater than the one observed in rats given MNNG alone, and micronucleated cells accounted to a significant extent for this increase. These findings confirm that NaCl presumably acts as co-carcinogen in the initial phase of gastric carcinogenesis, and suggest that its administration before the carcinogen might increase the sensitivity of short-term tests for the preliminary screening of potential gastric carcinogens.

Induction and promotion of gamma-glutamyltranspeptidase-positive foci in the liver of female rats treated with ethinyl estradiol, clomiphene, tamoxifen and their associations.

The objective of the present study was to determine whether a short exposure (6 weeks) to high doses of ethinyl estradiol (EE) could not only promote but also initiate hepatocarcinogenesis, and whether two antiestrogens, clomiphene (C) and tamoxifen (T), could influence EE activity. 2-Acetylaminofluorene (AAF), which has been shown to produce rat liver hyperplastic lesions characterized by the presence of estrogen receptors, was used either as a promoter to test for initiating activity, or as an initiator to test for promoting activity. Putative preneoplastic lesions were identified by means of a positive gamma-glutamyltranspeptidase (GGT) reaction. The results revealed that when administered alone in female Sprague-Dawley rats, not only E, but also C and T were clearly active in both initiating and promoting the development of GGT-positive foci. Moreover, in rats of the same strain treated with EE + C or EE + T a significant increase in the incidence of GGT foci demonstrated the occurrence of an additive effect in terms of both initiating and promoting activity. Fischer 344 rats were more susceptible than Sprague-Dawley rats to promotion by EE, C and T, but any substantial evidence of an additive effect was absent when the two anti-estrogens were administered in association with the estrogen.

Evaluation of the potential carcinogenic activity of Senna and Cascara glycosides for the rat colon.

Anthraquinone glycosides of Senna and Cascara were investigated for their ability to induce aberrant crypt foci (ACF) in the rat colon mucosa, which are considered putative preneoplastic lesions. Dietary exposure to high doses of these glycosides for 56 successive days did not cause the appearance of ACF or increase in incidence of ACF induced by 1,2-dimethyl-hydrazine (DMH). However, in rats treated with both DMH and the highest dose of glycosides, the average number of aberrant crypts per focus, considered a consistent predictor of tumor outcome, was higher than in rats given DMH alone. These findings suggest that Senna and Cascara glycoside might behave as weak promoters in rat colon carcinogenesis.

A possible medium-term assay for detecting the effects of liver and colon carcinogens in rats.

The aim of the present study was to verify whether the tumorigenic effect of a rat liver carcinogen, 2-acetylaminofluorene (AAF), and of a promoter of rat colon carcinogenesis, chenodeoxycholic acid (CDCA), could be detected with a single medium-term assay using as markers gamma-glutamyltranspeptidase (GGT)-positive foci in the liver and aberrant crypt foci (ACF) in colon mucosa. In rats given in the first 2 weeks of treatment both N-nitrosodiethylamine (NDEA), as initiator of liver carcinogenesis, and azoxymethane (AOM), as initiator of colon carcinogenesis, the subsequent 6-week feeding on a diet containing AAF (0.01%) produced a significant marked increase of the number and area of GGT-positive foci which is consistent with the results of long term assays. When rats initiated with both NDEA and AOM were fed for 6 weeks on a diet containing CDCA (0.1%) a significant increase of large ACF as well as of crypt multiplicity was observed, consistently with the promoting effect of CDCA in colon carcinogenesis. The results obtained in this preliminary study suggest that this medium-term assay might be able to screen both liver and colon carcinogens in rats.

Effect of aspirin on incidence and growth of aberrant crypt foci induced in the rat colon by 1,2-dimethylhydrazine.

The aim of this work was to investigate if the possible chemopreventive effect of aspirin (ASA) on rat colon carcinogenesis could be detected with a medium-term assay. The end-point chosen was the inhibition of incidence and growth of putative preneoplastic lesions, the aberrant crypt foci (ACF) induced in the rat colon by two administrations of 1,2-dimethylhydrazine (DMH, 25 mg/kg p.o.). At both 4 and 8 weeks after the starting of the carcinogenic treatment the incidence of total ACF was reduced of 60% in rats receiving ASA (10 mg/kg/day p.o.) for 12 consecutive days during the initiation treatment with DMH. Also the number of the larger foci (with 3 or more crypts) was significantly lower in ASA-treated rats at both time-points (about 70% reduction). Moreover, concomitant ASA treatment determined a significant decrease of the mean number of crypts per focus at week 8. These results indicate that the chemopreventive effect of ASA on chemically-induced rat colon carcinogenesis observed in long-term studies may be detected in this relatively short assay.

Oral administration of chlordiazepoxide plus sodium nitrite investigated for tumor initiating activity in the rat liver.

Chlordiazepoxide (CDE) reacts with sodium nitrite at acid pH yielding the genotoxic derivative N-nitrosochlordiazepoxide (NO-CDE). In the present study oral administration of CDE plus NaNO2, previously found to produce DNA fragmentation in the rat liver, was examined for its ability to initiate hepatocarcinogenesis. The oral treatment for 6 successive weeks with CDE + NaNO2, added to the diet at the levels of 290 + 270 and 870 + 800 ppm, did not significantly increase the number or volume of gamma-glutamyltranspeptidase-positive foci (putative preneoplastic lesions). These findings are in agreement with the negative results previously obtained in rodent carcinogenesis assays and indicate that NO-CDE belongs to the progressively expanding list of genotoxic non-carcinogens.

Effects of ethinyl estradiol and epidermal growth-factor on DNA-synthesis in human cultured-hepatocytes.

It has been demonstrated that the liver tumor promoter ethinyl estradiol (EE) potentiates the DNA synthetic response of cultured rat hepatocytes to epidermal growth factor (EGF), probably acting as a comitogen with a growth factor of the EGF/TGF family. The present study investigated the effects of EE and EGF on DNA synthesis in cultures of rat and human hepatocytes obtained from 5 female donors. The cultures were given the following treatments: a. 15 muM EE for 30 h; b. 25 ng/ml of EGF for 12 h; c. combination of treatments a and b. The results obtained in rat hepatocytes confirm that EE potentiates the.DNA synthetic response to EGF, as evaluated by the labelling index. As to human hepatocytes, in only one case the response to EGF produced a significant increase of DNA synthesis and the EE pretreatment did not potentiate this effect. The results of this study indicate that, in contrast to the response observed in rat hepatocytes, EE does not seem to affect the DNA synthesis in primary cultures of human hepatocytes, and the association EE+EGF does not produce any synergic effect.

Evaluation of omeprazole genotoxicity in a battery of in vitro and in vivo assays.

Omeprazole, a proton pump inhibitor of wide use in the treatment of gastric acid-related disorders, was evaluated for its genotoxic effects in both rat and human cultured cells and in the intact rat. DNA repair synthesis, as revealed by autoradiography, was detected in primary cultures of metabolically competent rat hepatocytes exposed to concentrations ranging from 10 to 100 mg/l, but the responses cannot be considered as clearly positive. Under the same experimental conditions any significant evidence of DNA repair was absent in primary hepatocytes from two human donors. At the same concentrations a modest but dose-related increase of micronucleated cells, that reached the level of statistical significance at 33 mg/l, was present in primary rat hepatocytes and in one of two human donors. In human lymphocytes exposed to subtoxic concentrations ranging from 0.78 to 12.5 mg/l a reproducible concentration dependent clastogenic effect was absent. In partially hepatectomized female rats treated with a single p.o. dose of 1000 mg/kg, the frequency of micronucleated cells was 5.2-fold higher than in controls in the liver, but only 2.0-fold higher in polychromatic erythrocytes of the bone marrow. In rats of the same sex given azoxymethane as initiator of colon carcinogenesis the oral administration for 8 successive weeks of 10 mg/kg omeprazole on alternate days increased the response to azoxymethane, as indicated by the occurrence in colon mucosa of a modest but statistically significant increase in both the average number and size of aberrant crypt foci. Taken as a whole, our results suggest that omeprazole behaves as a weak genotoxic agent for the rat liver. Reliable information about the potential genotoxic risk to humans requires further studies on primary cells from a wide number of donors.

Effect of bisacodyl and cascara on growth of aberrant crypt foci and malignant tumors in the rat colon.

Laxatives abuse has been associated with an increased risk for colon cancer. However, little is known about laxatives long-term carcinogenic potential in experimental studies. The present study was designed to investigate the effects of bisacodyl (4.3 and 43 mg/kg) and cascara (140 and 420 mg/kg) on azoxymethane (AOM)-induced aberrant crypt foci (ACF) and tumors. Animals, divided in 10 groups were treated with AOM and laxatives (alone or in combination) for 13 weeks. At the end of treatment animals were killed and the colon removed and analysed for the determination of ACF and tumors. Bisacodyl (4.3 and 43 mg/kg), given alone, did not induce the development of colonic ACF and tumors. Bisacodyl (4.3 mg/kg) coupled with AOM increased the number of crypt per focus, but not the number of tumors. Bisacodyl (43 mg/kg) significantly increased the number of crypt per focus and tumors. Cascara (140 and 420 mg/kg) did not induce the development of colonic ACF and tumors and did not modify the number of AOM-induced ACF and tumors. The results of the present study indicate a possible promoting effect of bisacodyl on rat colon carcinogenesis (especially at higher doses) and absence of any promoting or initiating activity of a laxative and diarrhoeal dose of cascara.

Effects of reduced glutathione and n-acetylcysteine on lidocaine metabolism in cimetidine treated rats.

The aim of this work was to evaluate the effects of exogenous glutathione (GSH) and N-acetylcysteine (NAC) on the formation of monoethylglycinexylidide (MEGX) from lidocaine in rats with and without the administration of cimetidine. GSH and NAC were administered intraperitoneally (i.p.) (1 mmol/kg) 1 hour before treatment with cimetidine (0.5 mmol/kg) or saline, and 1 hr later all rats were injected i.p. with lidocaine (1 mg/kg). Blood samples were drawn 30 min after the lidocaine injection. MEGX and lidocaine serum concentrations were determined by means of fluorescence polarization immuno-assay using the TDX system. Cimetidine produced a decrease in MEGX levels (from 210 +/- 18 to 164 +/- 13 ng/mL) and a parallel increase in lidocaine levels (from 73 +/- 22 to 172 +/- 47 ng/mL), consistent with cytochrome P-450 3A inhibition. Both GSH and NAC produce a significant decrease in MEGX levels (151 +/- 16 and 139 +/- 14 ng/mL, respectively), but no significant increase in lidocaine levels were found. As compared to the cimetidine group, pre-treatment using either GSH or NAC with cimetidine produced a marked decrease in lidocaine levels (37 +/- 27 and 63 +/- 28 ng/mL, respectively) and no modification of MEGX levels (155 +/- 12 and 165 +/- 22 ng/mL, respectively). These results suggest that GSH and NAC might accelerate the lidocaine metabolism while counteracting the inhibitory effect of cimetidine.

Inhibition of the development of rat liver preneoplastic lesions by verapamil and dexverapamil.

The effect of verapamil and dexverapamil on the development of liver preneoplastic foci was investigated in male Sprague-Dawley rats initiated with N-nitrosodiethylamine (200 mg/kg i.p.), fed on a diet containing 0.01% 2-acetylaminofluorene and subjected to partial hepatectomy, according to the hepatocarcinogenesis model developed by Solt and Farber. Administration of drinking water containing 0.03% verapamil or dexverapamil resulted in a decrease in the incidence and size of gamma-glutamyl transpeptidase-positive foci. The chemopreventive effect of dexverapamil was more marked than that of verapamil. These findings support the hypothesis that these two calcium channel blockers act by reducing the resistance of initiated hepatocytes to the mitoinhibitory and cytotoxic effects of 2-acetylaminofluorene.

DNA damage induced by seven N-nitroso compounds in primary cultures of human and rat kidney cells.

Seven N-nitroso compounds (NOC), known to induce kidney tumors in rats, were assayed for DNA-damaging activity in primary cultures of human and rat kidney cells. DNA fragmentation was measured by the alkaline elution technique. Positive responses were obtained in cells of both species with N-nitrosodimethylamine (32 mM), N-nitrosodiethylamine (32 mM), N-nitrosodi-n-propylamine (10 mM), N-ethyl-N-hydroxyethylnitrosamine (18 mM), and streptozotocin (1 mM). N-nitrosodiethanolamine and N-nitrosomorpholine were inactive at the highest concentration tested (32 mM). The responses of human kidney cells were qualitatively similar to those of rat kidney cells, but statistically significant differences between the two species in the DNA-damaging potencies were observed with N-ethyl-N-hydroxyethylnitrosamine and streptozotocin, both more genotoxic in rat cells. Taken as a whole, the results suggest on the one hand that the five active NOC might be carcinogenic for the kidney in humans, and on the other hand that the rat kidney cell/DNA damage assay is a valid model for predicting the genotoxic potential of NOC in human kidney cells.

An in vivo micronucleus assay for detecting the clastogenic effect in rat kidney cells.

A micronucleus assay in vivo has been developed that is based on the use of freshly isolated kidney cells from mononephrectomized rats. In this validation study, a statistically significant increase in the frequency of micronucleated cells was detected in rats given i.p. a single dose of four kidney carcinogens, N-nitrosodimethylamine, N-nitrosodiethylamine, N-ethyl-N-hydroxyethylnitrosamine and N-nitroso-N-methylurea. The clastogenic effect was more marked when the same dose was injected for 3 successive days. As compared to controls, treated rats displayed a reduction in the frequency of binucleated cells, presumably due to a toxicity-induced inhibition of cellular proliferation. The proposed method should be suitable for the detection of the clastogenic effect of procarcinogens biotransformed into reactive species in the kidney.

Cinnamaldehyde-induced micronuclei in rodent liver.

Cinnamaldehyde, a widely used flavoring agent, has so far been subjected to a limited range of genotoxicity tests, mainly carried out in vitro, which produced contradictory results. Therefore we have examined cinnamaldehyde using additional in vivo genotoxicity end-points. In Sprague-Dawley rats, a single oral dose equal to 1/2 LD50 did not induce DNA fragmentation in liver and gastric mucosa as evaluated by the alkaline elution technique, increased the frequency of micronucleated hepatocytes but not of bone marrow micronucleated polychromatic erythrocytes, and gave rise to a significantly higher incidence of total nuclear anomalies but not of micronucleated cells in forestomach mucosa. In Swiss mice, the equitoxic dose of cinnamaldehyde caused the same clastogenic effect in the liver, whilst a negative response was observed in both bone marrow and forestomach mucosa. Finally, in rats initiated with N-nitrosodiethylamine the administration of 500 mg/kg/day cinnamaldehyde for 14 successive days produced a modest but statistically significant increase of the average diameter and area of gamma-glutamyltranspeptidase-positive foci that, together with changes observed in other parameters, might be considered indicative of a potential promoting activity. Taken as a whole, these findings confirm that high doses of cinnamaldehyde may induce genetic alterations at the chromosomal level, and suggest that the liver is the preferential target of its undesirable effects.

Increased frequency of micronucleated kidney cells in rats exposed to halogenated anaesthetics.

Six halogenated anaesthetics were tested for their ability to induce micronuclei formation in the rat kidney. A statistically significant increase in the frequency of micronucleated cells was detected in rats given a single p.o. dose of 4 mmol/kg of halothane (3.48 x baseline), chloroform (3.32 x baseline), trichloroethylene (3.24 x baseline), sevoflurane (2.98 x baseline), and isoflurane (2.95 x baseline). In contrast, the response was substantially negative in rats given the same dose of enflurane. As compared to controls, rats treated with halothane and trichloroethylene displayed a reduction in the frequency of binucleated cells presumably due to a toxicity-induced inhibition of cellular proliferation. These findings suggest a potential genotoxic activity of halogenated anaesthetics for the rat kidney.

Induction of micronuclei and of enzyme-altered foci in the liver of female rats exposed to progesterone and three synthetic progestins.

Progesterone (PG) and three structurally similar synthetic progestins-norethisterone (NE), allylestrenol (AE), and dydrogesterone (DG)-have been compared for their ability to induce the formation of micronuclei and of enzyme-altered foci in the liver of female rats. In the micronucleus assay, carried out in rats given a single p.o. dose of 100 mg kg-1 3 days before partial hepatectomy and sacrificed for cell sampling 2 days later, the frequency of micronucleated hepatocytes was 3.5-fold higher than in controls with PG, 2.8-fold with DG, 2.2-fold with NE and 2.1-fold with AE, but the increase was statistically significant only for PG. In the liver foci assay, performed to evaluate the tumor initiating activity of p. o. dosing with 100 mg kg-1 once a week for 6 successive weeks, the values of the number and area of gamma-glutamyltranspeptidase-positive foci were, as compared to controls, 15.9- and 100-fold higher with NE, and 13.9- and 52-fold higher with AE, but only the increase of area produced by NE was statistically significant; PG and DG did not display in this test any activities. Considered together with previous findings, these results suggest that NE might be biotransformed in the liver into reactive species and thus behave as a weak genotoxic agent.

Evaluation of effective renal plasma flow with I-127 ortho-iodohippurate and I-123 ortho-iodohippurate in rabbits.

RATIONALE AND OBJECTIVES: Radiolabeled ortho-iodohippurate is commonly employed for evaluating effective renal plasma flow (ERPF) by means of either in vivo scintigraphy and/or plasma clearance curves. A new method has recently been developed for measuring levels of stable iodine (iodine-127) in biologic samples, based on the detection of x-ray fluorescence photons. In this study, the authors assessed the potential of the new system to evaluate ERPF by using an iodinated contrast medium with adequate glomerular filtration and tubular secretion properties. MATERIALS AND METHODS: A commercial system was used to evaluate ERPF after intravenous injection of stable I-127 ortho-iodohippurate. The results were compared with the clearance values of I-123 ortho-iodohippurate, considered the reference standard. Seven rabbits under general anesthesia were given intravenous injections of I-123 ortho-iodohippurate and I-127 ortho-iodohippurate. The corresponding plasma curves were evaluated from 4 to 60 minutes to calculate ERPF as the dose/integral of plasma curve. RESULTS: The initial distribution volumes of I-123 ortho-iodohippurate (149.4 mL/kg +/- 12.1) and I-127 ortho-iodohippurate (148.8 mL/kg +/- 11.8) were virtually superimposable, thus confirming the chemical identity of the two compounds. The plasma clearance values for I-127 ortho-iodohippurate (11.15 mL/min kg(-1) +/- 1.44) were slightly (not significantly) higher than those for I-123 ortho-iodohippurate (10.49 mL/min kg(-1) +/- 1.41), perhaps because of a relative "mass" load effect of the iodinated medium. CONCLUSION: The results obtained in this study demonstrate the feasibility of the new system for evaluating ERPF, provided that a compound with adequate glomerular filtration and tubular secretion properties is employed.

[Intestinal infarct: a critical review of the cases observed in the decade of 1981-1991]. / L'infarto intestinale: revisione critica dei casi osservati nel decennio 1981-1991.

The authors consider the causes of bowel infarction and report the up-to-date diagnostic tools for optional treatment. They verify management and outcome of 97 cases treated during the last decade.