Evaluation of teratogenicity and genotoxicity induced by kramecyne (KACY).

Kramecyne (KACY), a polymer isolated from Krameria cytisoides Cav, has anti-inflammatory, anti-nociceptive, anti-arthritic and anti-ulcerogenic properties. As a part of standard preclinical safety tests, the present study sought to determine potential developmental toxicity (in female rats) and genotoxicity (in male mice) of KACY. Pregnant female rats were divided into six groups: the negative control (vehicle), the positive control (250 mg/kg of acetylsalicylic acid (ASA)), and four experimental groups (50, 250, 500 and 1000 mg/kg of KACY). To evaluate genotoxicity by in vivo micronuclei (MN) and sister chromatid exchange (SCE) tests, male mice were divided into five groups: the negative control (vehicle), the positive control (1.5 and 2.5 mg/kg of doxorubicin for MN and SCE, respectively), and three experimental groups (50, 500 and 1000 mg/kg of KACY). All treatments were administered by oral gavage. A slight maternal toxicity was evidenced by lower weight gain for rats receiving 500 and 1000 mg/kg of KACY, but no fetal malformations were found. However, there were less live fetuses/litter and greater post-implantation loss/litter at these two doses. Manifestations of developmental toxicity were limited to a higher rate of skeletal alterations. The MN tests did not evidence genotoxicity or cytotoxicity. KACY caused a slightly but significantly increased frequency of SCE. Although KACY-treated rats had skeletal alterations, these apparently were not caused by a mechanism of genotoxicity. Furthermore, the same administration in adult male mice did not produce genotoxicity. Hence, KACY herein proved to be safe for rats during the period of organogenesis.

Genotoxic and cytotoxic evaluation of venlafaxine in an acute and a subchronic assay in mouse.

The present research was made to determine the micronuclei and cytotoxic capacity of the antidepressant venlafaxine in an in vivo acute and subchronic assays in mouse. In the first study, we administered once 5, 50, and 250 mg/kg of the drug, and included a negative and a daunorubicin treated group. Observations were daily made during four days. The subchronic assay lasted 5 weeks with daily administration of venlafaxine (1, 5, and 10 mg/kg) plus a negative and an imipramine administered groups. Observations were made each week. In the first assay results showed no micronucleated polychromatic erythrocytes (MNPE) increase, except with the high dose at 72 h. The strongest cytotoxic effect was found with 250 mg/kg at 72 h (a 51% cytotoxic effect in comparison with the mean control level). In the subchronic assay no MNPE increase was found; however, with the highest dose a significant increase of micronucleated normochromatic erythrocytes was observed in the last three weeks (a mean of 51% respect to the mean control value). A cytotoxic effect with the two high doses in the last two weeks was observed (a polychromatic erythrocyte mean decrease of 52% respect to the mean control value). Results suggest caution with venlafaxine.

Chemoprotective effect of Spirulina (Arthrospira) against cyclophosphamide-induced mutagenicity in mice.

The aim of this study was to investigate the antimutagenic effects of Spirulina (SP) on male and female mice by the dominant lethal test using cyclophosphamide (CP) as a mutagen. Animals of both sex were given SP orally at 0, 200, 400 or 800 mg/kg body weight (b.w.) for 2 weeks prior to starting the CP treatment. CP was i.p. injected daily for 5 days at 40 mg/kg b.w. For the male-dominant lethal test, each male was caged with untreated females per week for 3 weeks. For the female-dominant lethal test the above doses and schedule treatments were used and treated females were caged for one week with untreated males (1-2). On days 13-15 after breeding was |started all the females were evaluated for incidence of pregnancy, total corpora lutea, total implants and pre- and post-implant losses. In the male-dominant lethal test, the CP induced pre- and post-implant losses in untreated females were inhibited at all SP doses. In the female-dominant lethal test only post-implantation losses were prevented at the same doses. Semen examination of a separate group of mice showed that SP improved its quality. Our results illustrate protective effects of SP in relation to CP-induced genetic damage to germ cells.

Glycine decreases developmental damage induced by hyperglycaemia in mouse embryos.

Hyperglycaemia induces neural tube defects and growth retardation in cultured mouse and rat embryos. In this study the possibility that glycine could prevent hyperglycaemia-induced embryopathy was researched. Early somite mouse embryos were cultured in normal medium, hyperglycaemic medium (50 mmol L(-1) glucose), or with glycine (1 mmol L(-1)) supplementation of normal and hyperglycaemic rat serum for 48 h. The embryo growth and differentiation were determined to estimate developmental and congenital malformations as well as lipid peroxidation levels. Adding glycine to the control culture medium did not affect embryonic development. Whereas the amino acid protected against telencephalon dysmorphogenesis, the decreased DNA content and number of somites, and the morphological score affectation induced by the hyperglycaemic medium, it had no preventive effect on the retarded differentiation of the otic system. Moreover, it prevented the high hyperglycaemia-induced lipoperoxidation levels of embryonic tissues. Embryos were partially protected from the hyperglycaemia-induced teratogenesis due to the antioxidative effect of glycine. As no other mechanisms related to the antiglycation or other protective effects of glycine were examined, the mechanism whereby it acted as an antiteratogenic agent needs further study.

Genotoxic and oxidative damage induced by Helicobacter pylori in Meriones unguiculatus.

Infection with Helicobacter pylori has been shown to be at the origin of various gastric pathologies. However, it has not yet been established whether the etiology of such diseases, particularly of gastric cancer, is related to the production of free radicals or to mutagenesis. The aim of this study was to determine whether a six-month infection with Helicobacter pylori increased the amount of lipid peroxidation, nitric oxide, and DNA damage in Mongolian gerbils (Meriones unguiculatus). H. pylori was characterized genotypically and administered orally to the animals. Four tests were applied to identify the presence of bacteria at one, two, four, and six months after the inoculation, namely, isolation and identification in culture, the urease test, the ELISA assay, and immunohistochemical staining of gastric biopsies. The infection was considered to be successful when three of the above-mentioned tests were positive. The infection occurred in 30% of the animals in the first month after the H. pylori inoculation and in 60-70% of the animals in the later stages. Levels of malondialdehyde, nitric oxide, and DNA damage (using the "comet" assay) were determined in the gastric tissue of the animals at one, two, four, and six months. We found statistically significant increases in malondialdehyde and nitric oxide levels from the second month on. The comet assay in animals infected with H. pylori showed a significant increase in the mean tail length throughout the observation period. We conclude that our results support the assumption that oxidative damage and DNA breakage produced by the infection with H. pylori are some of the initial alterations occurring in the development of gastric diseases.

Antimutagenicity of Stevia pilosa and Stevia eupatoria evaluated with the Ames test.

Stevia pilosa and Stevia eupatoria are plants used for various purposes in traditional medicine. In this report we studied the antimutagenic effect of methanolic extracts obtained from leaves, root, and flowers of the two species using the Ames test with and without metabolic activation. We tested the effect of the extracts on the damage induced by three mutagens with the following results: 1 - we found an inhibitory effect of both species on the mutagenicity induced by 2-aminoanthracene in the strain TA98. The best antimutagenic effect was obtained with leaves of both species and the flowers of S. eupatoria (99%), 2 - the mutations induced with N-ethyl-N'-nitro-N-nitrosoguanidine in the strain TA100 was also reduced. The flowers of S. pilosa and the root of S. eupatoria showed about 93% of inhibition, 3 - finally, the mutations induced by mitomycin-C on the strain TA102 had a reduction of 87% with the leaves of S. eupatoria. Besides, we determined the radical scavenging potential of the extracts with the DPPH method, and found a potent effect produced by all extracts, with an efficacy of more than 90%. The present study showed both antimutagenic and antioxidant potential of the tested extracts, and suggest the pertinence to confirm these effects in other models, and to accurately determine their mechanism of action.

Antigenotoxic effect of Saccharomyces cerevisiae on the damage produced in mice fed with aflatoxin B(1) contaminated corn.

The potential of Saccharomyces cerevisiae (Sc) was evaluated for reducing the micronucleated normochromatic erythrocytes (MNNE) rate in mice fed AFB(1) contaminated corn. The study included two groups fed AFB(1) contaminated corn (0.4 and 0.8 mg/kg), a control fed uncontaminated corn, another group fed uncontaminated corn and 0.3% of Sc (1 x 10(8) live cells/g), and two groups fed AFB(1) contaminated corn (0.4 and 0.8 mg/kg) plus 0.3% Sc. Weight and MNNE were determined weekly for six weeks. Subsequently, the same determinations were made for another three-week period, but in mice receiving only a normal diet, without AFB(1) and Sc. Results in the first period revealed the following: control and Sc fed mice had similar constant weight increase, and low MNNE rate; mice fed only AFB(1) showed weight decrease and significant MNNE increase; finally, Sc improved weight gain and reduced MNNE produced by AFB(1). In the second period, results exhibited a tendency similar to that of the previous phase in the control and Sc fed mice; the weight and MNNE values improved in the other groups. We also determined the capacity of Sc for adsorbing and modifying the mycotoxin structure. The mixture was filtered to obtain two phases, and AFB(1) content was measured. Sc revealed a potent adsorbent capacity; however, chromatographic determination suggested no structural modification.

In vitro genotoxic evaluation of three alpha-asarone analogues.

alpha-Asarone has shown a significant capacity to reduce the level of lipids, including cholesterol. However, several toxic and genotoxic studies have determined that its use may pose a risk to human health. Therefore, a series of compounds structurally analogous to alpha-asarone were prepared in order to maintain the same pharmacological properties but with low toxicity. In this study we evaluated the potential of three alpha-asarone analogues to induce mutagenicity using the Ames test (strains TA98 and TA100 in the presence of metabolic activation), as well as the induction of sister chromatid exchanges (SCE) in cultured human lymphocytes. The tested compounds were: 1-(2,4,5-trimethoxyphenyl)propan-1-one (D1), 1-(2-chloro-4,5-dimethoxyphenyl)propan-1-one (D2), and 1-(4,5-dimethoxy-2-nitrophenyl)propan-1-ol (D3). The results in the first assay showed no mutagenic effect for the three tested analogues; in the TA100 strain, certain cytotoxicity did appear in the case of D2 and D3 only at high concentrations. In regard to the SCE assay, compounds D1 and D2 presented no statistical differences in comparison with the control culture values; however, the high dose of D3 (300 microg/ml) produced a significant increment in SCE (68% above the control value). With respect to the mitotic index and the cellular proliferation kinetics, we observed a reduction when compounds D2 and D3 were used at the higher concentrations. Our results encourage further preclinical studies of these compounds in both in vitro and in vivo models (particularly for analogues D1 and D2), to determine their toxicological profile and establish the possibility of using them in humans.

SCE frequencies induced by ethanol, tequila and brandy in mouse bone marrow cells in vivo.

The genotoxicity of ethanol, tequila and brandy was evaluated by scoring the frequency of sister chromatid exchanges (SCE) and determining the values of the average generation time (AGT). We studied four dosages of each substance i.p. inoculated into mice. The cytogenetic analysis was performed in bone marrow cells. The results showed that all three substances were weak genotoxicants. Tequila showed the strongest response followed by brandy and ethanol. None of them modified the cell proliferation kinetics as demonstrated by the AGT results.

Genotoxic evaluation of letimide with the mouse bone marrow micronucleus test.

Letimide, 3[2-(diethylamino)ethyl]-2H-1,3-benzoxazine-2,4(3H)-dione, a new analgesic, is a cyclic derivative of a salicylamide with a higher pharmacological potency than acetylsalicylic acid. In this study we evaluated its clastogenic activity using the micronucleus test in vivo. Our results did not show any clastogenic effect produced by letimide as compared with control mice and animals treated with cyclophosphamide.

Cytogenetic and teratogenic evaluation of letimide.

Letimide is a new efficient analgesic salicylate derivative. In this study we evaluated its cytogenetic and teratogenic potential. For chromosomal aberrations and sister chromatid exchange analysis we tested 250, 375, 500 and 625 micrograms/ml of letimide in human lymphocyte cultures in vitro, and for the in vivo cytogenetic study analysing the same parameters we studied the effects of 30, 50 and 100 mg/kg in mouse bone marrow cells. The teratogenic study was performed at dosages of 50, 100 and 200 mg/kg/d of letimide in mice. The results agree with the systems studied previously, showing no significant effect in the rate of aberrations, sister chromatid exchanges or congenital malformations induced by this new analgesic.

Inhibitory effect of chamomile essential oil on the sister chromatid exchanges induced by daunorubicin and methyl methanesulfonate in mouse bone marrow.

Different preparations of chamomile (Matricaria chamomilla) are used to treat various diseases, including inflammation and cancer; however, no studies on the plant's antigenotoxic capacity have been made. The aim of the present work was to determine the inhibitory effect of the chamomile essential oil (CO), on the sister chromatid exchanges (SCEs) produced by daunorubicin and methyl methanesulfonate (MMS) in mouse bone marrow cells. CO was analyzed and was found to contain 13 compounds, mainly bisabolol and its oxides, chamazulene, farnesene, germacrene and other sesquiterpenes. Initially, a toxic and a genotoxic analysis of CO were made; both showed negative results. To determine whether CO can inhibit the mutagenic effects induced by daunorubicin, one group of mice was administered corn oil, another group was treated with the mutagen (10 mg/kg), a third group was treated with 500 mg/kg of CO; three other groups were treated first with CO (5, 50 and 500 mg/kg) and then with 10 mg/kg of daunorubicin. In the case of MMS, the experimental groups consisted of the following: the negative control group which was administered corn oil, a group treated with 25 mg/kg of MMS, a group treated with 1000 mg/kg of CO, and three groups treated first with CO (250, 500 and 1000 mg/kg) and then with MMS (25 mg/kg). The results indicated a dose-dependent inhibitory effect on the SCEs formed by both mutagens. In the case of daunorubicin, a statistically significant result was observed in the three tested doses: from the lowest to the highest dose, the inhibitory values corresponded to 25.7, 63.1 and 75.5%. No alterations were found with respect to the cellular proliferation kinetics, but a reduction in the mitotic index was detected. As regards MMS, the inhibitory values were 24.8, 45.8 and 60.6%; no alterations were found in either the cellular proliferation kinetics or in the mitotic indices. Our results suggest that CO may be an effective antimutagen that could be considered for further study.

Antigenotoxic and antioxidant effect of grapefruit juice in mice treated with daunorubicin.

Grapefruit juice (GJ) is widely consumed in many countries. Several of its constituents possess nutritive value, as well as antigenotoxic and antioxidant effects. Therefore, the aim of this investigation was to evaluate the capacity of GJ to inhibit the micronucleated polychromatic erythrocytes (MNPE) produced by daunorubicin (Dau) in an acute assay in mice, as well as to determine its antioxidant potential in mouse hepatic microsomes, and its capacity to trap free radicals in vitro. In regard to the first point, GJ produced no toxic or genotoxic damage; on the contrary, it generated a significant reduction of the MNPE formed by Dau. The effect was found throughout the examined schedule (from 24 to 96 h). The two high doses produced inhibition of about 60% at 48 h, 86% at 72 h and 100% at 96 h after the treatment. With respect to the GJ antioxidant potential, a 50% decrease in liver microsomal lipid peroxidation produced by Dau was found by quantifying malondialdehyde formation. Finally, a strong GJ scavenging activity evaluated with the 1,1-diphenyl-2-picryl-hydrazyl (DPPH) was observed, giving rise to a concentration-dependent curve with a correlation coefficient of 0.98. Overall, our results established an efficient anticlastogenic potential of GJ, probably related to its antioxidant capacity, or to alterations of Dau metabolism, suggesting the pertinence of extending research on the matter using other mutagens and biological models.

Sister chromatid exchanges produced by imipramine and desipramine in mouse bone marrow cells treated in vivo.

Imipramine and desipramine are two widely used tricyclic antidepressants which have shown conflicting results in regard to their in vitro genotoxic evaluation. The aim of this investigation was to determine the capacity of these compounds to induce in vivo sister-chromatid exchanges (SCEs) in mouse bone marrow cells. For each compound, the animals were organized in five groups constituted by five individuals. They were intraperitoneally (ip) administered with the test substances as follows: a negative control group treated with 0.4 ml of distilled water, a positive control group administered with cyclophosphamide (70 mg/kg), three groups treated with imipramine (7, 20 and 60 mg/kg), and three other groups treated with desipramine (2, 20 and 60 mg/kg). The general procedure included the subcutaneous implantation to each mouse of a 5-bromodesoxyuridine tablet (45 mg), and 1 h later, the administration of the chemicals involved. Twenty-one hours after the tablet implantation, the mice received colchicine, and 3 h later their femoral bone marrow was obtained in KCL, fixed, and stained with the Hoechst-Giemsa method. The results showed that both compounds were SCE inducers, starting from the second tested dose. The response of these compounds was dose-dependent, and showed that the highest tested dose increased about four times the SCE control level. The cellular proliferation kinetics was not affected by the chemicals, and the mitotic indexes were slightly diminished with the highest dose. These results indicate an in vivo genotoxic potential for both chemicals, and suggest that it is pertinent to follow their evaluation in other models.

In vivo and in vitro genotoxic evaluation of indorenate.

5-Methoxytryptamine, beta-methylcarboxylate hydrochloride (indorenate) is a new antihypertensive serotonin derivative. We evaluated its genotoxic activity using the mouse bone marrow and cytogenetic test and the human lymphocyte culture cytogenetic assay. As endpoints we measured chromosomal aberrations, sister-chromatid exchanges and cellular proliferation kinetics. Our results agree in both systems showing that indorenate is a non-genotoxic agent in these assays.

Sister-chromatid exchange analysis in vivo using different 5-bromo-2'-deoxyuridine-labeling systems.

The quality of sister-chromatid differentiation, the basal rate of sister-chromatid exchanges (SCE) and the rate of cellular proliferation were studied in untreated and mitomycin C(MMC)-treated mice, using 4 different systems for administering 5-bromodeoxyuridine (BrdU): BrdU adsorbed to charcoal, tablets of BrdU mixed with cholesterol, tablets of BrdU coated with agar and tablets of BrdU partially coated with paraffin. The quality of sister-chromatid differentiation with the studied methods showed a useful stain contrast in an average of 75.4% second-division mitosas, with the lowest average occurring in mice implanted with agar-coated tablets. The frequency of SCE and the replicative index were similar in mice administered BrdU by all 4 systems both in control and in MMC-treated mice. From a practical point of view, the charcoal method could be done the fastest.

The effect of tequila in the synaptonemal complex structure of mouse spermatocytes.

The effect of tequila in the synaptonemal complex (SC) of mouse spermatocytes was determined. We tested 3 dosages (2.1, 4.2 and 8.4 g/kg) administered in a single intraperitoneal inoculation. The frequency of SC alterations was established in pachytenic nuclei 5 days after the administration using a silver impregnation technique. Three types of alterations were observed (desynapses, breaks and multiaxials) and the rate of each alteration was compared with that obtained with appropriate controls, including cyclophosphamide (CP) (150 mg/kg). The results showed a significant increase induced by tequila only in the frequency of desynapses. This damage began at the second highest dose (4.2 g/kg). The other SC alterations were in the control range. CP, however, induced a significant increase in all 3 types of SC alterations.

Inhibitory effect of nordihydroguaiaretic acid on the frequency of micronuclei induced by methyl methanesulfonate in vivo.

Nordihydroguaiaretic acid (NDGA) is an antioxidant originally obtained from plants of the genus Larrea. This chemical has shown antigenotoxic activity measuring gene mutations and sister-chromatid exchanges. The aim of this investigation was to determine if NDGA is also an antigenotoxic agent and can inhibit the induction of micronucleus (MN) formation by methyl methanesulfonate (MMS) in mouse. The frequency of micronucleated polychromatic erythrocytes (MPE) was scored for 4 days, and a MN induction curve by a single injection of MMS (40 mg/kg) was obtained. The results of this experiment showed that the highest MN incidence was reached at the second day of exposure with a mean of 13.2%+/-1.0. This value is more than 4 times the control mean. Thus, the modulatory study by NDGA was established at a 2-day exposure time using three doses (6.0, 11.0, and 17.0 mg/kg) against the damage induced by 40 mg/kg of MMS. The results of this study showed a significant reduction of the clastogenic damage at the two highest doses, where the inhibitory values corresponded to 62.2% and 66.7%, respectively. With respect to the ratio polychromatic erythrocytes/normochromatic erythrocytes, a marked toxicity was detected with 2 days of MMS exposure; however, the combination of the two high doses of NDGA plus MMS significantly reduced the cytotoxic damage produced by MMS alone.

Genotoxic evaluation of ammonium inactivated aflatoxin B1 in mice fed with contaminated corn.

Aflatoxin B1 (AFB1) is a major contaminant in different agricultural products including maize. In an attempt to reduce this problem and the hazards to human health, an AFB1 inactivating system with ammonia has been developed. In this work we evaluated the efficiency of the system in mice using micronucleus (MN) and sister chromatid exchange (SCE) analysis. Four groups of animals were fed during 8 weeks with a special diet mainly composed of maize: (1) uncontaminated; (2) uncontaminated/inactivated; (3) contaminated/inactivated; and (4) contaminated. We evaluated MN at weekly intervals in peripheral blood, and in weeks 4 and 8 SCE frequencies were quantified in bone marrow cells. The results showed that animals fed with AFB1 contaminated/inactivated maize had a 45% lower level of induced cytogenetic damage than those animals fed with AFB1 contaminated, but not inactivated maize. A residual amount of AFB1 after the inactivating treatment and the reconversion back to AFB1 in the organism may account for the remaining increased levels of SCE and MN.

Inhibitory effect of chlorophyllin on the frequency of sister chromatid exchanges produced by benzo[a]pyrene in vivo.

The study was designed to determine the antigenotoxic potential of chlorophyllin (Chl), against the frequency of sister-chromatid exchanges (SCE) produced by benzo[a]pyrene (B[a]P) in vivo. We used the mouse bone marrow test system to measure the effect of a single injection of the compounds: 40 mg/kg of B[a]P, and 1 h later, 1.0, 2.0, 4.0 and 8.0 mg/kg of Chl. As controls we included both chemicals using the dosages mentioned above as well as mineral oil (0.25 mg/kg). The results indicated the following: (1) Chl per se was not genotoxic, showing SCE values in the range of the control level; (2) B[a]P increased the rate of SCEs three times in relation to the basal level; (3) the SCE level produced with B[a]P was diminished by all 4 doses of Chl, but better results were obtained with 2-4 mg/kg, a range which induced Inhibition Indices of 80.9% and 77.5% respectively; (4) the Average Generation Time Index was not modified by the compounds used in the experiment; and (5) the Mitotic Index also showed no significant modification induced by the chemicals, with respect to the control value.