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.

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.

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.

Evaluation of flutamide genotoxicity in rats and in primary human hepatocytes.

Flutamide, an effective competitive inhibitor of the androgen receptor used orally for palliative treatment of prostatic carcinoma and regulation of prostatic hyperplasia was evaluated for its genotoxic effects in the intact rat and in primary cultures of human hepatocytes. Negative responses were obtained in all the in vivo assays as well as in the in vitro assay. In rats given a single oral dose of 500 mg/kg flutamide, fragmentation and repair of liver DNA were absent, and no increase was observed in the frequency of micronucleated hepatocytes. In the liver of rats given flutamide as initiating agent at the dose of 500 mg/kg/week for 6 successive weeks, gamma-glutamyltraspeptidase-positive foci were detected only in 3 of 10 rats. There was no evidence of a promoting effect on the development of aberrant crypt foci in rats given 100 mg/kg flutamide on alternate days for 8 successive weeks. In primary cultures of human hepatocytes from one male and one female donor DNA fragmentation as measured by the Comet assays, and DNA repair synthesis as revealed by quantitative autoradiography, were absent after a 20 hr exposure to flutamide concentrations ranging from 18 to 56 microM. Taken as a whole, our results seem to indicate that flutamide is a non-genotoxic drug.

Does senna extract promote growth of aberrant crypt foci and malignant tumors in rat colon?

Current evidence suggests that aberrant crypt foci (ACF) can be used to evaluate agents for their potential colon carcinogenic activity. The aim of the present study was to determine whether senna pod extract (SE) itself induces ACF and tumors in the rat colon or increases the development of ACF and tumors induced by azoxymethane (AOM). A daily administration of SE 10 mg/kg by mouth for 13-28 weeks produced a weak laxative effect but did not itself cause the appearance of ACF or tumors. The numbers of ACF and tumors induced by AOM were, however, increased by a dose of SE (100 mg/kg) able to induce chronic diarrhea over three months. These results suggest that SE does not cause the appearance of ACF or tumors in the rat colon nor does it have a promoting effect when given to rats at a dose that produces laxation (10 mg/kg), whereas a diarrhogenic dose (100 mg/kg) increases the appearance of tumors induced by AOM.

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.

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.

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.

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.

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.

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.

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.

Comparative study of DNA repair induced by cyproterone acetate, chlormadinone acetate and megestrol acetate in primary cultures of human and rat hepatocytes.

Cyproterone acetate (CPA), a synthetic progestin recently found to induce genotoxic effects in hepatocytes from female rats and from humans of both genders, and two structural analogues, chlormadinone acetate (CMA) and megestrol acetate (MGA), have been compared for their capacity to induce DNA repair synthesis as measured by quantitative autoradiography. Exposure of primary human hepatocytes for 20 h to concentrations of CPA, CMA and MGA ranging from 2 to 50 microM induced positive responses in cultures from donors of both genders and the amounts of DNA repair elicited by the three progestins were similar. Under the same experimental conditions substantial differences were observed in the amounts of DNA repair elicited by the three progestins in primary hepatocytes from female rats, their potency decreasing in the following order CPA > CMA > MGA, and the three compounds failed to induce DNA repair in hepatocytes from male rats. These results, which agree with previous findings, suggest that for these sex steroids extrapolation to humans of results obtained in rats might be questionable.

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.

Induction of micronuclei and initiation of enzyme-altered foci in the liver of female rats treated with cyproterone acetate, chlormadinone acetate, or megestrol acetate.

The synthetic anti-androgen and progestin cyproterone acetate (CPA), recently found to be genotoxic for the liver, and two structurally similar progestins, chlormadinone acetate (CMA) and megestrol acetate (MGA), have been compared for clastogenic and tumor-initiating activities in female rats. In the micronucleus assay, carried out in rats given a single p.o. dose of 100 mg/kg, CPA induced the maximum increase in the frequency of micronucleated hepatocytes (6.6-fold as compared to controls) when treatment was performed 3 days before partial hepatectomy and cell sampling 2 days later. Under the same experimental conditions the clastogenic potencies of CMA and MGA were 69% and 36% of that of CPA respectively. In the liver foci assay, p.o. dosing with 100 mg/kg CPA once a week for 6 successive weeks induced, as compared to controls, a significant increase in the number and area of gamma-glutamyltranspeptidase-positive foci. At the same dosage schedule the tumor-initiating activity of CMA and MGA was 7- to 10-fold lower than that of CPA. These findings suggest that the 1,2 alpha-methylene group, present in CPA but absent in both CMA and MGA, favours the activation to a reactive species and/or hinders the biotransformation to non-toxic metabolites.

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.

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.

In vitro and in vivo testing of hydralazine genotoxicity.

Hydralazine (HDZ), a p.o. effective antihypertensive drug, was evaluated for its genotoxic effects in both rodent and human cultured cells and in the intact rat. Dose-dependent amounts of DNA fragmentation, as measured by the alkaline elution technique, and of DNA repair synthesis, as revealed by autoradiography, were produced in primary cultures of metabolically competent rat hepatocytes by subtoxic HDZ concentrations ranging from 0.32 to 1.0 mM. A similar potency in inducing DNA repair synthesis was displayed by HDZ in primary cultures of hepatocytes from four human donors. A modest reduction of both DNA fragmentation (-13%) and DNA repair (approximately -50%) was observed in hepatocytes obtained from rats pretreated with indomethacin in order to reduce prostaglandin synthetase activity. In contrast, neither in rat nor in human hepatocytes, differences in N-acetyltransferase activity resulted in meaningful changes of the same end points. V79 cells, which are essentially deficient of monooxygenases catalyzing the biotransformation of xenobiotics, were as sensitive as hepatocytes to the DNA-damaging activity of HDZ. Moreover, after exposure to 0.1 to 0.3 mM HDZ, a modest (2.1- to 2.8-fold), but significant, increase in the frequency of mutation to 6-thioguanine resistance was observed in V79 cells in the absence of a metabolic activation system. In rats, a single p.o. dose of 80 mg/kg produced a clastogenic effect in the liver, but not in the bone marrow, and the p.o. administration for 14 successive days of approximately 46 mg/kg/day increased the average diameter of liver basophilic foci initiated by diethylnitrosamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of DNA-damaging, clastogenic, and promoting activities of metoclopramide and procainamide in rats.

The DNA-damaging and clastogenic activities of metoclopramide (MCA) and procainamide (PCA), two substituted benzamides not systematically tested for genotoxicity before clinical use, were investigated in rats given a single high oral dose (500 mg/kg) of these drugs. Neither MCA nor PCA induced DNA fragmentation in liver, kidney, gastric mucosa, spleen, and bone marrow, as detected by the alkaline elution technique. Moreover, neither drug increased the frequency of micronucleated hepatocytes and the frequency of micronucleated polychromatic erythrocytes in the bone marrow of partially hepatectomized rats. However, in rats initiated with N-nitrosodiethylamine and given water containing 0.125% MCA for 14 successive days a clear-cut and statistically significant increase in the number and size of liver gamma-glutamyltranspeptidase-positive foci and basophilic foci, which are consistent with potential promoting activity, was observed. Under the same experimental conditions the effect of PCA was markedly lower, only limited to a modest increase of the number and area of gamma-glutamyltranspeptidase-positive foci.

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.