Induction of kinetochore positive and negative micronuclei in mouse bone marrow cells by salicylazosulfapyridine and sulfapyridine.

Salicylazosulfapyridine (SASP) and its major metabolite sulfapyridine (SP) have been shown to induce chromosomal damage in vivo. Both chemicals were tested in the micronucleus (MN)/kinetochore (KC) staining test to gain insight into the question of whether chromosomal breakage, aneuploidy-inducing events, or both were important to the observed production of MN in bone marrow cells of mice. In this test, both SASP and SP were shown to be strong inducers of kinetochore positive (KC+) MN. Although small increases in kinetochore negative (KC-) MN were also observed in SP treated mice, as well as in mice receiving the highest dose of SASP tested, the results suggest that both chemicals induce predominantly aneuploidogenic type damage.     

The oxygen-centered radicals scavenging activity of sulfasalazine and its metabolites. A direct protection of the bowel.

Oxygen-centered radicals, such as superoxide (O2-) and hydroxyl radicals (.OH) generated by phagocytes have been suggested to be involved in the pathogenesis of chronic inflammations of the bowel, such as Crohn's disease and colitis ulcerosa. Recently, sulfasalazine (SASP) and its metabolites have been reported to exert their effects as a direct scavenger of oxygen-centered radicals in the bowel. To scavenge oxygen-centered radicals in vivo, however, SASP and its metabolites have to react with O2- and/or .OH in vitro very rapidly, furthermore they have to reach an appropriate (possible millimolar) concentration range at the site of inflammation. To test this possibility, we investigated the direct O2- and .OH scavenging activity of SASP and its metabolites using the specific electron paramagnetic resonance/spin trapping method, and we compared the 50% inhibition rates of SASP and its metabolites with their known concentrations in the bowel and in the human plasma. It was found that SASP and its metabolites, such as 5-amino-salicylic acid (5-ASA), and acetyl-5-amino-salicylic acid (AC-5-ASA), but not sulfapyridine (SP) and acetyl-sulfapyridine (Ac-SP) have a direct O2- and .OH scavenging activity in vitro systems. Among the compounds, SASP and 5-ASA can reach a concentration which is appropriate to scavenge oxygen-centered radicals in the bowel but not in the human plasma. It was concluded that the in vivo antiinflammatory effects of SASP and its metabolites are, at least partly, due to the direct oxygen-centered scavenging activity of these drugs.     

Influence of sulfasalazine, 5-aminosalicylic acid and sulfapyridine on prostanoid synthesis and metabolism in rabbit colonic mucosa

The effects of sulfasalazine (SASP) and its cleavage products 5-aminosalicylic acid (5-ASA) and sulfapyridine (SP) on prostanoid (PG) synthesis and degradation were determined in rabbit colonic mucosa fractions in vitro. When the microsomal fraction was incubated with (14C)arachidonic acid, 10(-3) M SASP and SP did not markedly change the formation of labeled PGE2, PGF2 alpha, TxB2 and 6-keto-PGF1 alpha X 10(-4) M 5-ASA increased synthesis about 2.7-fold; the pattern of PG identified was unaltered. In the presence of the 10-fold higher concentration of 5-ASA, PG synthesis remained elevated at a similar level. When the cytosolic fraction was incubated with (3H)PGE2, 10(-3) M 5-ASA was without influence and 10(-3) M SP decreased slightly PGE2 breakdown. However, SASP showed a pronounced inhibitory effect at 10(-5) M and inhibition of PGE2 degradation was complete at 10(-3) M SASP. The results are compatible with the assumption that stimulation of PG synthesis by 5-ASA is related to therapeutic benefit in the treatment of ulcerative colitis.     

Determination of sulphasalazine and its main metabolite sulphapyridine and 5-aminosalicylic acid in human plasma by liquid chromatography/tandem mass spectrometry and its application to a pharmacokinetic study

A simple and sensitive liquid chromatography/positive-ion electrospray ionization mass spectrometry (LC-ESI-MS/MS) method has been developed for the simultaneous determination of sulphasalazine (SASP) and its main metabolite sulphapyridine (SP) and 5-aminosalicylic acid (5-ASA) with 100 μL of human plasma using dimenhydrinate as the internal standard (I.S.). The API-3000 LC-MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. Protein precipitation process was used to extract SASP, SP, 5-ASA and I.S. from human plasma. The total run time was 9.0 min and the elution of SASP, SP and 5-ASA was at 4.8 min, 2.5 min and 2.0 min, respectively. The separation was achieved with a mobile phase consisting of 0.2% formic acid, 2 mM ammonium acetate in water (mobile phase A) and 0.2% formic acid, 2 mM ammonium acetate in methanol (mobile phase B) by using gradient elution on a XBP Phenyl column (100 mm × 2.1 mm, 5 μm). The developed method was validated in human plasma with a lower limit of quantitation of 10 ng/mL for SASP, SP and 5-ASA, respectively. A linear response function was established for the range of concentrations 10-10,000 ng/mL (r>0.99) for SASP and 10-1000 ng/mL (r>0.99) for SP and 5-ASA. The intra and inter-day precision values for SASP, SP and 5-ASA met the acceptance as per FDA guidelines. SASP, SP and 5-ASA were stable during stability studies, i.e., long term, auto-sampler and freeze/thaw cycles. The method was successfully applied for the evaluation of pharmacokinetics of SASP, SP and 5-ASA after single oral doses of 250 mg SASP to 10 healthy volunteers.     

Comparison of the level of sister chromatid exchanges and chromosome aberrations induced by chemical mutagens in vitro and in vivo

The paper presents results of a study of a dose dependence of induction of SCE and chromosomal aberrations at the exposure of human lymphocytes in vitro and bone marrow cells of mice in vivo to 5 alkylating chemicals. The efficiency of SCE induction in vitro is found to be 300-30 times as high as that of arising of chromosomal aberrations. The same regularity is observed in experiments in vivo, but the ratio is reduced to 60-20 times.     

The in vitro micronucleus assay for detection of cytogenetic effects induced by mutagen-carcinogens: comparison with the in vitro sister-chromatid exchange assay

The sensitivity of a cytogenetic assay, as expressed by the in vitro induction of micronuclei (MN), was compared to the in vitro induction of sister-chromatid exchanges (SCEs). Chinese hamster lung (V79) cells were exposed to 3 known alkylating agents: methyl methanesulphonate (MMS), ethyl methanesulphonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and to 5 newly synthesized naphthofurans: 2-nitro-7-methoxynaphtho[2,1-b]furan (A), 2-nitro-8-methoxynaphtho[2,1-b]furan (B), 2-nitronaphtho[2,1-b]furan (C), 2-nitro-7-bromonaphtho[2,1-b]furan (D) and 7-methoxynaphtho[2,1-b]furan (E). The induction of MN only was also analysed after exposure of the cells to 4 alcohols: ethanol, methanol, butanol and propanol. The lowest dose at which a significant effect could be observed was determined. In both assays, MNNG, MMS and EMS were equally active with the following order of potency: MNNG greater than MMS greater than EMS, the latter being a very weak inducer of MN and SCE. Compounds A and B were also very effective in both assays. Compound C was a more active inducer of SCE than MN. Compounds D and E were not active in either assay. None of the 4 alcohols induced MN. Our results are compared with the previously published data on in vitro and in vivo induction of SCE and MN. We conclude that the MN in vitro assay which detects clastogens as well as agents affecting the spindle apparatus, is a good indicator of genotoxicity, though slightly less sensitive than the in vitro SCE test. It could provide a rapid, simple and inexpensive complementary short-term test for the evaluation of potentially mutagenic chemicals.     

Evaluation of the mutagenic potential of the hair dye N-methyl-amino-2-nitro-4-N',N'-bis-(2-hydroxyethyl)-aminobenzene in a battery consisting of Drosophila and mammalian cytogenetic assays

The compound N-methyl-amino-2-nitro-4-N', N'-bis(2-hydroxyethyl)-aminobenzene was tested for mutagenic activity in the sex-linked recessive lethal test with Drosophila melanogaster, the induction of chromosomal aberrations and sister-chromatid exchanges (SCEs) with Chinese hamster ovary (CHO) cells in vitro, and the micronucleus test with mouse bone-marrow cells in vivo. Consistently negative results were obtained with the 3 tests. The SCE tests gave positive results with prolonged treatments. It is concluded that reliable decisions about mutagenic activity cannot be based on the induction, in vitro, of SCEs alone.     

Genotoxicity testing of fluconazole in vivo and in vitro

The genotoxic effects of the antifungal drug fluconazole (trade name triflucan) were assessed in the chromosome aberration (CA) test in mouse bone-marrow cells in vivo and in the chromosome aberration, sister chromatid exchange (SCE) and micronucleus (MN) tests in human lymphocytes. Fluconazole was used at concentrations of 12.5, 25.0 and 50.0 mg/kg for the in vivo assay and 12.5, 25.0 and 50.0 microg/ml were used for the in vitro assay. In both test systems, a negative and a positive control (MMC) were also included. Six types of structural aberration were observed: chromatid and chromosome breaks, sister chromatid union, chromatid exchange, fragments and dicentric chromosomes. Polyploidy was observed in both the in vivo and in vitro systems. In the in vivo test, fluconazole did not significantly increase the frequency of CA. In the in vitro assays, CA, SCE and MN frequencies were significantly increased in a dose-dependent manner compared with the negative control. The mitotic, replication and cytokinesis-block proliferation indices (CBPI) were not affected by treatments with fluconazole. According to these results, fluconazole is clastogenic and aneugenic in human lymphocytes, but these effects could not be observed in mice. Further studies should be conducted in other test systems to evaluate the full genotoxic potential of fluconazole.     

Genotoxicity evaluation of sesamin and episesamin

Sesamin is a major lignan that is present in sesame seeds and oil. Sesamin is partially converted to its stereoisomer, episesamin, during the refining process of non-roasted sesame seed oil. We evaluated the genotoxicity of these substances through the following tests: a bacterial reverse mutation assay (Ames test), a chromosomal aberration test in cultured Chinese hamster lung cells (CHL/IU), a bone marrow micronucleus (MN) test in Crlj:CD1 (ICR) mice, and a comet assay using the liver of Sprague-Dawley (SD) rats. Episesamin showed negative results in the Ames test with and without S9 mix, in the in vitro chromosomal aberration test with and without S9 mix, and in the in vivo comet assay. Sesamin showed negative results in the Ames test with and without S9 mix. In the in vitro chromosomal aberration test, sesamin did not induce chromosomal aberrations in the absence of S9 mix, but induced structural abnormalities at cytotoxic concentrations in the presence of S9 mix. Oral administration of sesamin at doses up to 2.0g/kg did not cause a significant increase in either the percentage of micronucleated polychromatic erythrocytes in the in vivo bone marrow MN test or in the % DNA in the comet tails in the in vivo comet assay of liver cells. These findings indicate that sesamin does not damage DNA in vivo and that sesamin and episesamin have no genotoxic activity.     

Chromosomal damage induced by maleic hydrazide in mammalian cells in vitro and in vivo

The induction of sister-chromatid exchanges (SCE) and chromosomal aberrations (Ch.Ab.) by the herbicide maleic hydrazide (MH) has been investigated in Chinese hamster ovary (CHO) cells grown in vitro and in bone marrow cells of mice treated in vivo. MH induces SCE and Ch.Ab. in CHO cells without metabolic activation; however, no induction of SCE was found in the in vivo experiments.     

Genotoxic effects of triphenyltin acetate and triphenyltin hydroxide on mammalian cells in vitro and in vivo.

Two organotin pesticides, triphenyltin acetate (TPTA) and triphenyltin hydroxide (TPTH), were evaluated for their ability to induce micronuclei (MN) and sister chromatid exchange (SCE) in vitro using cultured Chinese hamster ovary (CHO) cells and in vivo BALB/c mouse erythrocytes. Both pesticides induced a dose-dependent increase but only TPTH induced a significant increase in MN at the highest dose (150 ng/ml) tested in CHO cells. With adding S9 microsomal fractions, both pesticides induced a meaningful MN induction at 150 ng/ml and a dose-dependent significant increase in SCE. In vivo MN induction in erythrocytes was conducted by treating BALB/c mice orally or intraperitoneally with these pesticides either in a single or triple treatments. Oral gavage (p.o.) of TPTA resulted in a dose-related significant increase of MN induction in peripheral blood and of TPTH induced a significant increase in micronucleated reticulocyte (MNRETs) only in a single treatment. Intraperitoneal administration of TPTA or TPTH, however, resulted in meaningless random increases in MN though these increases might be attributable to toxic effects. The MNRETs levels in the treatment with both pesticides were independent to the sampling time. This study demonstrated that TPTA and TPTH was potential chromosome mutagens.     

Correlation of the sister chromatid exchange level with chromosome aberrations induced by chemical mutagens in vivo

The data on the dose dependencies of the induction of sister chromatid exchanges (SCE) and chromosomal aberrations during exposure of mouse bone marrow cells in vivo to 5 alkylating substances are provided. The efficacy of SCE induction was found to be higher than that of chromosomal aberrations. It was established that SCE induced by chemical mutagens in vivo and in vitro are more sensitive and stable tests than chromosomal aberrations.     

Induction of chromosome aberrations in vitro by phenolphthalein: mechanistic studies

Phenolphthalein induces tumors in rodents but because it is negative in assays for mutation in Salmonella and in mammalian cells, for DNA adducts and for DNA strand breaks, its primary mechanism does not seem to be DNA damage. Chromosome aberration (Ab) induction by phenolphthalein in vitro is associated with marked cytotoxicity. At very high doses, phenolphthalein induces weak increases in micronuclei (MN) in mouse bone marrow; a larger response is seen with chronic treatment. All this suggests genotoxicity is a secondary effect that may not occur at lower doses. In heterozygous TSG-p53((R)) mice, phenolphthalein induces lymphomas and also MN, many with kinetochores (K), implying chromosome loss. Induction of aneuploidy would be compatible with the loss of the normal p53 gene seen in the lymphomas.Here we address some of the postulated mechanisms of genotoxicity in vitro, including metabolic activation, inhibition of thymidylate synthetase, cytotoxicity, oxidative stress, DNA damage and aneuploidy. We show clearly that phenolphthalein does not require metabolic activation by S9 to induce Abs. Inhibition of thymidylate synthetase is an unlikely mechanism, since thymidine did not prevent Ab induction by phenolphthalein. Phenolphthalein dramatically inhibited DNA synthesis, in common with many non-DNA reactive chemicals that induce Abs at cytotoxic doses. Phenolphthalein strongly enhances levels of intracellular oxygen radicals (ROS). The radical scavenger DMSO suppresses phenolphthalein-induced toxicity and Abs whereas H(2)O(2) potentiates them, suggesting a role for peroxidative activation. Phenolphthalein did not produce DNA strand breaks in rat hepatocytes or DNA adducts in Chinese hamster ovary (CHO) cells. All the evidence points to an indirect mechanism for Abs that is unlikely to operate at low doses of phenolphthalein. We also found that phenolphthalein induces mitotic abnormalities and MN with kinetochores in vitro. These are also enhanced by H(2)O(2) and suppressed by DMSO. Our findings suggest that induction of Abs in vitro is a high-dose effect in oxidatively stressed cells and may thus have a threshold. There may be more than one mechanism operating in vitro and in vivo, possibly indirect genotoxicity at high doses and also chromosome loss, both of which would likely have a threshold.     

Resveratrol, a naturally occurring polyphenol, induces sister chromatid exchanges in a Chinese hamster lung (CHL) cell line.

We tested the genotoxicity of 3,5,4'-trihydroxystilbene (resveratrol), a polyphenolic phytoalexin found in grapes, in a bacterial reverse mutation assay, in vitro chromosome aberration (CA) test, in vitro micronucleus (MN) test, and sister chromatid exchange (SCE) test. Resveratrol was negative in the strains we used in the bacterial reverse mutation assay (S. typhimurium TA98 and TA100 and E. coli WP2uvrA) in the absence and presence of a microsomal metabolizing system. It induced structural CAs at 2.5-20 microg/ml and showed weak aneuploidy induction in a Chinese hamster lung (CHL) cell line. It induced MN cells and polynuclear and karyorrhectic cells after 48h treatments in the in vitro MN test. In the SCE test, resveratrol caused a clear cell-cycle delay; at 10 microg/ml, the cell cycle took twice as long as it did in the control. Resveratrol induced SCEs dose-dependently at up to 10 microg/ml, at which it increased SCE six-fold, and the number was almost as large as mitomycin C, a strong SCE inducer. No second mitoses were observed at 20 microg/ml even after 54h. Cell cycle analysis by FACScan indicated that resveratrol caused S phase arrest, and 48h treatment induced apoptosis. Our results suggest that resveratrol may preferentially induce SCE but not CA, that is, it may cause S phase arrest only when SCEs are induced.     

Chromosomal changes induced in mouse bone marrow cells following six weeks inhalation of cyclohexanol

The effects of low doses of cyclohexanol exposure were studied in mouse bone marrow cells including chromosome aberrations (CA), micronucleus (MN) and sister chromatid exchanges (SCE) as biomarkers. Capillaries with a tested agent that was evaporated continuously were placed in an experimental chamber for six weeks. No clastogenic and/or aneugenic effect of CA and MN induction was observed. A significant elevation of induced damage was achieved in the SCE study (p < 0.001) that has confirmed the early exposure of cyclohexanol to mice.     

Scavenging of reactive oxygen and nitrogen species by the prodrug sulfasalazine and its metabolites 5-aminosalicylic acid and sulfapyridine

Sulfasalazine is a prodrug composed by a molecule of 5-aminosalicylic acid (5-ASA) and sulfapyridine (SP), linked by an azo bond, which has been shown to be effective in the therapy of inflammatory bowel diseases (IBD) such as ulcerative colitis and Crohn's disease, as well as of rheumatic diseases, such as rheumatoid arthritis and ankylosing spondylitis. The precise mechanism of action of sulfasalazine and/or its metabolites has not been completely elucidated, though its antioxidant effects are well established and are probably due to its scavenging effects against reactive oxygen and nitrogen species (ROS and RNS), as well as metal chelating properties, in association to its inhibitory effects over neutrophil oxidative burst. The present work was focused on screening and comparing the potential scavenging activity for an array of ROS (O(2)(?-), H(2)O(2), (1)O(2), ROO(?) and HOCl) and RNS ((?)NO and ONOO(-)), mediated by sulfasalazine and its metabolites 5-ASA and SP, using validated in vitro screening systems. The results showed that both 5-ASA and sulfasalazine were able to scavenge all the tested ROS while SP was practically ineffective in all the assays. For HOCl, (1)O(2), and ROO(?), 5-ASA showed the best scavenging effects. A new and important finding of the present study was the strong scavenging effect of 5-ASA against (1)O(2). 5-ASA was shown to be a strong scavenger of (?)NO and ONOO(-). Sulfasalazine was also able to scavenge these RNS, although with a much lower potency than 5-ASA. SP was unable to scavenge (?)NO in the tested concentrations but was shown to scavenge ONOO(-), with a higher strength when the assay was performed in the presence of 25 mM bicarbonate, suggesting further scavenging of oxidizing carbonate radical. In conclusion, the ROS- and RNS-scavenging effects of sulfasalazine and its metabolites shown in this study may contribute to the anti-inflammatory effects mediated by sulfasalazine through the prevention of the oxidative/nitrative/nitrosative damages caused by these species.     

Ah locus-associated differences in induction of sister-chromatid exchanges and in DNA adducts by benzo[a]pyrene in mice.

The effect of alleles of the Ah locus on the induction of sister-chromatid exchanges (SCE) was studied in C57Bl/6 and in DBA/2 mice treated twice intragastrically with benzo[a]pyrene (BP, 100 or 10 mg/kg b.w.). To measure the changes in the frequency of SCE, 2 protocols were used: in vivo in bone marrow cells after implantation of 5-bromodeoxyuridine (BrdU) tablets and in vivo/in vitro in spleen lymphocytes cultured with BrdU. On day 5 mice were killed and SCEs estimated in bone marrow cells. BP-DNA adducts in bone marrow and spleen were analyzed on day 5 after the same exposure to BP. In the spleen lymphocytes SCE frequencies were analyzed after an additional 48 h of culture. We found that at both doses of BP, the number of SCEs and BP-DNA adducts in bone marrow and in spleen cells was significantly higher in aryl hydrocarbon hydroxylase (AHH)-non-inducible (DBA/2) mice than in AHH-inducible (C57BL/6) mice. Only marginal induction of SCE was noted after the high dose of BP in C57BL/6 mice in bone marrow in vivo, whereas a highly significant increase in the frequency of SCEs was found in splenocytes in the in vivo/in vitro test. The spleen cells contained larger amounts of BP-DNA adducts and demonstrated higher absolute levels of SCEs than bone marrow cells. The sensitivity of both the in vivo/in vitro and the in vivo SCE test is high enough for assessment of Ah locus-linked differences in BP genotoxicity in mice at the prolonged time between treatment and cell preparation. The present data confirm the influence of inducibility of AHH in the intestine on the genotoxicity of BP to distal tissues after oral exposure to BP.     

Genotoxicity of cadmium chloride in human lymphocytes evaluated by the comet assay and cytogenetic tests

Peripheral blood lymphocytes were tested in vitro for genotoxic effects of cadmium chloride. Whole blood samples of four healthy, non-smoking subjects were preincubated with CdCl2 in concentrations of 10(-4), 10(-3), and 5 . 10(-3) mol/L for three hours before the cells were assessed for DNA-damage using the single cell alkaline gel electrophoresis assay (comet assay) or cultivated for chromosomal aberrations (CA), sister chromatid exchanges (SCE), and the micronucleus (MN) test. The comet assay showed notable interindividual differences. The results of the cytogenetic tests showed an increase in the frequency of CA, MN, and SCE with CdCl2 in the treated cultures, yet none was able to show a correlation between concentrations of cadmium chloride and the frequency of damages. The MN slides were stained with Giemsa and with DNA fluorochrome 4', 6'-diamidino-2-phenylindole (DAPI). The frequency of MN in slides stained with DAPI was significantly higher than in those stained with Giemsa, which might be due to an underestimation of small micronuclei in Giemsa-stained slides.     

In vitro micronucleus assay with Chinese hamster V79 cells - results of a collaborative study with in situ exposure to 26 chemical substances

A collaborative study with 10 participating laboratories was conducted to evaluate a test protocol for the performance of the in vitro micronucleus (MN) test using the V79 cell line with one treatment and one sampling time only. A total of 26 coded substances were tested in this study for MN-inducing properties. Three substances were tested by all 10 laboratories and 23 substances were tested by three or four laboratories in parallel. Six aneugenic, 7 clastogenic and 6 non-genotoxic chemicals were uniformly recognised as such by all laboratories. Three chemicals were tested uniformly negative by three laboratories although also clastogenic properties have been reported for these substances. Another set of three clastogenic substances showed inconsistent results and one non-clastogenic substance was found to be positive by one out of three laboratories. Within the study, the applicability of the determination of a proliferation index (PI) as an internal cytotoxicity parameter in comparison with the determination of the mitotic index (MI) was also evaluated. Both parameters were found to be useful for the interpretation of the MN test result with regard to the control of cell cycle kinetics and the mode of action for MN induction. The MN test in vitro was found to be easy to perform and its results were mainly in accordance with results from chromosomal aberration tests in vitro.     

Ratio of the frequency of chromosome aberrations and sister chromatid exchanges to the dose of the mutagenic exposure to cyclophosphamide in vivo and in vitro

Cyclophosphamide (CP) and its metabolites were used to compare the rate of chromosomal aberrations (CA) and sister chromatid exchange (SCE) in the rabbit lymphocytes in vivo and in vitro. The dose-dependent increase of cytogenetic effects rate appeared to be of linear and exponential dependence for SCE and CA, respectively, both in vivo and in vitro. The regression equation coefficients coincided in in vivo and in vitro experiments.