Immunohistochemical evaluation of P21ras and P53 proteins expression in human non-small-cell lung cancers.

Human non-small-cell lung cancers (NSCLCs) of 48 patients were analyzed immunohistochemically to detect P21 ras and P53 proteins expression. The relationship between P21 ras and P53 proteins expression and clinicopathologic findings was also assessed. DAKO EnVision TM detection system was employed in the study. The P21 ras and P53 proteins expression was shown in 75% (36/48) and 33.3% (16/48) studied NSCLCs, respectively. In both cases the difference was significant when compared with adequate negative control. Simultaneous expression of both studied proteins was observed in all cases in which P53 expression was noticed. No significant association of P21 ras and P53 expression was found with age, histologic type, histologic grade, tumor size or lymph node metastasis of the studied NSCLCs. Therefore, our study suggests that P21 ras and P53 protein play a role in the pathogenesis of NSCLCs but they have no value as a prognostic markers in the case of lung cancers.

H-RAS, K-RAS, and N-RAS gene activation in human bladder cancers.

Bladder cancer is one of the leading causes of cancer death in most developed countries. In this work, 19 bladder cancer specimens, along with their infiltrations of the urinary bladder wall from the same patients, were examined for the presence of H-RAS, K-RAS, and N-RAS activation using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. The H-RAS activation was found in 15 (about 84%) of the 19 bladder cancers studied. The same results were obtained in the infiltrating urinary bladder wall samples. N-RAS gene mutations were observed in all cases (except 1) in which H-RAS gene mutations were detected. The results suggest a strong relationship between H-RAS and N-RAS gene activation in bladder cancer. Changes in the K-RAS gene in bladder cancers seem to be a rare event; this is in agreement with findings of other authors. We found activation of the gene in one specimen of bladder cancer and its infiltration of the urinary bladder wall in the same patient.

Assessment of aniline derivatives-induced DNA damage in the liver cells of B6C3F1 mice using the alkaline single cell gel electrophoresis ("comet") assay.

The alkaline single cell gel electrophoresis (SCGE) or "comet" assay under alkaline conditions was used to measure DNA damage in the liver cells of B6C3F1 male mice exposed to 2,4-dimethylaniline and 2, 4,6-trimethylaniline. Cells embedded in agarose were lysed, subjected briefly to an electric field, stained with a fluorescent DNA-binding stain, and viewed using a fluorescence microscope. The effect of 2,4-dimethylaniline and 2,4,6-trimethylaniline was studied after a single intraperitoneal injections at doses equal to 100, 200 mg/kg and 150, 300 mg/kg b.w., respectively. It was found that 2, 4-dimethylaniline and 2,4,6-trimethylaniline were able to damage DNA in the liver cells of the mice. As has been published elsewhere, the DNA damaging effect of the studied compounds were also observed in bone marrow cells of the mice. In conclusion, taking into account the results mentioned above and the results obtained by other researchers who reported mutagenic activity of 2,4-dimethylaniline and 2,4,6-trimethylaniline in Salmonella typhimurium assay and in a DNA repair test using Chinese hamster hepatocytes, it can be stated that both aromatic amines are genotoxic. Teratogenesis Carcinog. Mutagen. 19:323-327, 1999.

Assessment of aniline derivatives-induced DNA damage in the liver cells of B6C3F1 mice using the alkaline single cell gel electrophoresis ('comet') assay.

The alkaline single cell gel electrophoresis (SCGE) or 'comet' assay under alkaline conditions was used to measure DNA damage in the liver cells of B6C3F1 male mice exposed to 2,4-dimethylaniline and 2,4,6-trimethylaniline. Cells embedded in agarose were lysed, subjected briefly to an electric field, stained with a fluorescent DNA-binding stain, and viewed using a fluorescence microscope. The effect of 2,4-dimethylaniline and 2,4,6-trimethylaniline was studied after single intraperitoneal injections at doses equal to 100, 200 mg/kg and 150, 300 mg/kg body wt., respectively. It was found that 2,4-dimethylaniline and 2,4,6-trimethylaniline were able to damage DNA in the liver cells of the mice. As has been published elsewhere, the DNA damaging effect of the studied compounds was also observed in bone marrow cells of the mice. In conclusion, taking into account the results mentioned above and the results obtained by the other researchers who reported mutagenic activity of 2,4-dimethylaniline and 2,4,6-trimethylaniline in a Salmonella typhimurium assay and DNA repair test using Chinese hamster hepatocytes, it can be stated that both aromatic amines are genotoxic.

An evaluation of the DNA damaging effect of selected aniline derivatives using the alkaline single cell gel electrophoresis ("comet") assay.

The alkaline (pH > 13) single cell gel electrophoresis (SCGE) assay or "comet" assay was used to evaluate for DNA damage induced in bone marrow cells of B6C3F1 mice by four aniline derivatives 2,4-dimethylaniline (2,4-xylidine). 2,4,6-trimethylaniline (mesidine), 2-chloro-4-methylaniline and 4-chloro-N-methylaniline. The study revealed that two of the four compounds studied, i.e. 2,4-dimethylaniline and 2,4,6-trimethylaniline, increased the extent of DNA migration in bone marrow cells of mice. Two others. 2-chloro-4-methylaniline and 4-chloro-N-methylaniline, had no effect on the DNA of the cells in test conditions. The results of this study, in combination with those of other researchers. leads to the conclusion that 2,4-dimethylaniline and 2,4,6-trimethylaniline are genotoxic and potentially carcinogenic.

An evaluation of the genotoxic properties of some chosen dyes using the micronucleus test in vivo.

The frequency of micronucleated polychromatic erythrocytes and the polychromatic to normochromatic erythrocyte ratio was studied in BalbC mice treated with four azo dyes: Direct Blue 74, Direct Blue 296, Direct Blue 297 and Direct Green 98 at two (40% and 80% LD50/kg body weight) concentrations. None of the studied compounds revealed a genotoxic activity in the micronucleus test. However, it was found that two dyes, direct Blue 297 at doses 40% and 80% LD50 and Direct Green 98 at dose 80% LD50, cause a significant decrease in the ration of polychromatic to normochromatic erythrocytes in bone marrow of mice, which means that at the doses specified above they can affect the proliferation of the blood cells.

Nucleotide sequence of c-H-ras-1 gene from B6C3F1 mice.

The c-H-ras-1 gene of an B6C3F1 mouse was isolated and nucleotide sequence determined. Our study has revealed that this c-H-ras-1 gene consists of four exons, separated by three introns ranging in size from 150 to 649 bp. The coding parts of the sequence of mouse c-H-ras-1 gene show no important differences as compared with those of the rat, hamster and human gene. More numerous changes were found in introns. The identity of mouse c-H-ras-1 gene with rat, hamster and human ones at the nucleotide level is 86.40%, 80.04% and 67.87%, respectively. Comparison of amino acids in protein sequence of c-H-ras gene of mouse, rat, hamster and human points to high degree of conservation of the gene.

Potential carcinogenicity assessment of alpha-aescin and phenbendasol.

Potential carcinogenic activity of alpha-aescin and phenbendasole made by "Polfa" (Poland) as well as phenbendasole produced by "Hoechst" (Germany) was studied using Salmonella/microsome test, DNA repair test and micronucleus assay. None of tested preparations were mutagenic or genotoxic what suggest that none of them possess potential carcinogenic activity. Besides, it was established that alpha-aescin exhibits strong and phenbendasol weak acute systemic toxicity for mice. Alpha-aescin and phenbendasole produced in Poland have been found to be toxic for bone marrow cells of mice but only when administered at a high dose of 80% LD50.

Cyanuric chloride has no genotoxic and mutagenic properties in bacteria and bone marrow cells.

Three short-term tests were used; Salmonella/microsome assay, in vivo sister chromatid exchange assay (SCE) and micronucleus assay to evaluate mutagenic and genotoxic properties of 2,4,6-trichlorotriazine; cyanuric chloride. Mutagenicity assays were carried out using the standard top agar overplay plate assay described by Maron and Ames (9). Tester strains TA97a, TA98, TA100 and TA102 were used. Compound was dissolved in 0.1 ml of DMSO and doses of 1, 10, 100 and 500 micrograms/plate were tested in the absence and in the presence of the S9-mix. From the results obtained it appeared that incubation of the test substance with the bacteria did not increase the number of His+ revertants with any of the strains of S. typhimurium, either in the absence or in the presence of the S9-mix. At the high dose level used i.e. 100 and 500 micrograms/plate, the test substance appeared to be slightly toxic for strain TA 97a (in the absence of the S9-mix), as was seen from a diminished number of revertant colonies. The SCE test was performed according to the GENE-TOX programme. No significant increase was noted in the incidence of SCE in the groups treated with all tested doses of cyanuric chloride. Thus, in this test cyanuric chloride did not induce chromosomal damage resulting in SCE formation in bone marrow cells of mice. The micronucleus assay in vivo was performed on mice bone marrow cells. The incidence of micronucleated polychromatic erythrocytes after administration of all doses of cyanuric chloride used were not statistically different (p > 0.05) as compared to negative controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Genotoxicity assessment of Rokanol B2 and Rokamid R1.

Two surface-active compounds, Rokanol B2 and Rokamid R1, were examined with three types of screening tests: 1. standard Ames test in vitro using S. typhimurium TA97a, TA98, TA100 and TA102 strains; 2. micronucleus test in vivo; 3. sister chromatid exchange (SCE) on bone marrow cells of Balb C mice. Negative results of the testing, in terms of the effects of activity of both the technical preparations on S. typhimurium cells and bone marrow of Balb C mice, were found for both tested chemicals.

[Cellular protooncogenes--role in cancer development and activation under the influence of chemical substances]. / Protoonkogeny komórkowe--rola w procesie rozwoju nowotworów i aktywacja pod wplywem substancji chemicznych.

This review describes the role of cellular protooncogenes in cancer development. The chemical activation of cellular protooncogenes is also presented.

[Mechanism and kinetics of micronuclei formation in mouse bone marrow]. / Mechanizm i kinetyka powstawania mikrojader w szpiku kostnym myszy.

The paper presents a review of results concerning mechanism and kinetics of micronuclei formation. Special attention has been paid to differences in the induction of micronuclei by clastogenic compounds and spindle poisons as well as modifying factors which play important role in micronuclei formation.

[Identification of potential carcinogenic dyes and intermediates on the basis of their genotoxicity]. / Identyfikacja potencjalnie rakotwórczych barwników i pólproduktów na podstawie oceny ich genotoksycznosci.

41 dyes, pigments and surface-active compounds were tested for mutagenic and genotoxic properties. It was found that 43% of the studied dyes of Polish make which belonged to the azo, triarylmethane and anthraquinone compounds were mutagenic or genotoxic. All the studied pigments and surface active compounds did not reveal potential carcinogenic activity except for NNO Dyspergator whose possible genotoxic activity needs confirmation in further study.

Genotoxicity assessment of sulfosuccinate IO-5, Rokamid MRZ 17, Rokacet RZG7P2 and Roksol TL-7 using bacteria and bone marrow rodent cells.

Three short-term tests were used for evaluation of the genotoxic activity of four surface active agents. These were: Ames, Salmonella reversion assay using 4 tester strains (TA97a, TA98, TA100 and TA102), the micronucleus test int the bone marrow of Balb C mice and in vivo sister chromatid exchange (SCE) analysis in SFIS or Balb C mice. The frequency of micronucleated PCEs did not exceed the control values in mice of both sexes after intraperitoneal administration of all four compounds. Three preparations--Sulfosuccinate IO-5, Rokamid MRZ 17 and Rokacet RZG7P2 produced a negative response in Salmonella strains gene mutation assay and SCE induction test in mouse bone marrow cells. The Roksol TL-7 induced frameshift and base-pair substitution mutations in Salmonella tester strains both with and without S9 (prepared from the liver of rats which had been pretreated with Aroclor 1254). Evidently positive results (more than a twofold increase in the number of revertants per plate) were observed in tester strain S. typhimurium TA97a (with and without S9 metabolic activation) and S. typhimurium TA100 (with S9 metabolic activation) at a dose of 0.2 microliter Roksol TL-7 per plate. Roksol TL-7 caused slight increase in the SCE level in mouse bone marrow cells. A significant increase in SCE frequency was observed at doses of 50, 75 and 100 mg/kg.

Statistical analysis of the genotoxicity study results.

One of the methods used in the genotoxicity study of chemical compounds is micronucleus test in vivo. There is no clear consensus as to which statistical method is most suitable to study the frequency of occurrence of micronucleated polychromatic erythrocytes. This paper proposes two statistical tests: approximate normal test and conditional binomial test (Kastenbaum & Bowman).

Examination of the potential mutagenicity and genotoxicity of some synthetic dyes.

The study was designed to investigate mutagenic and genotoxic properties of six synthetic dyes (Acid Green M, Acid Brown ZMSz, Acid Brown MSzCz, Anionic Black M, Anionic Brown ZM, Anionic Deep Red SM) obtained from All-Union Cancer Research Centre (AUCRC) in Moscow. To study mutagenicity and genotoxicity two short-term tests--Salmonella/microsome test using four strains of Salmonella-typhimurium TA97a, TA98, TA 100, TA102 and micronuclear test on mice were performed. The bacterial test was carried out with and without S9 mix fraction. The micronuclear test was conducted on mice Balb C with doses equal to 40 and 80% LD50 for male and 80% for female mice. Two of the studied dyes (Acid Green M. Anionic Brown ZM) proved to be direct and one (Anionic Deep Dark SM) indirect mutagens in the Salmonella/microsome assay. None of the studied compounds revealed a gentoxic activity in the micronucleus test. However, it was found that four dyes (Acid Brown ZMSz, Acid Brown MSzCz, Anionic Brown ZM and Anionic Deep Red SM) cause a significant decrease in the ratio of polychromatic to normochromatic erythrocytes in bone marrow of mice, which means that at doses used in the experiment they are toxic to erythrocyte series cells.

Mutagenic and genotoxic activity of chosen dyes and surface active compounds used in the textile industry.

This study was designed to investigate the mutagenic and genotoxic properties of ten dyes and four surface active compounds using Salmonella/microsome assay and the micronucleus test. Five of the investigated dyes (Acid Blue 7, Acid Green 16, Direct Black 19:1, Basic Red 22, Basic Orange 28) possessed mutagenic activity with regard to test strains of Salmonella. In addition, all of them increased the frequency of micronucleated polychromatic erythrocytes in the bone marrow of mice. Three other compounds (Acid Blue 62, Direct Yellow 12, Direct Red 81), which were not mutagenic in the Salmonella/microsome assay, were genotoxic in the micronucleus test. The other two dyes (Reactive Blue 13, Acid Red 213), as well as tested surface active compounds, did not exert mutagenic and genotoxic effects, and therefore, it is most probable that they do not have carcinogenic properties. Besides, it was noted that Acid Blue 62, Direct Black 19:1, Direct Red 81 and Basic Orange 28 cause a significant decrease in the ratio polychromatic to normochromatic erythrocytes in the bone marrow of mice, which means that, at the doses used in the experiment, they are toxic to the erythrocyte series cells of bone marrow. The other compounds under consideration have no such effect.

Mutagenic activity of some textile dyes in different test systems.

The Salmonella/microsome mammalian test, the micronucleus and the dominant lethal tests on mice were used to study mutagenic effects of three dyes widely used in the textile industry. Direct Black 19:1, Direct Red 81 and Acid Blue 62 increased the frequency of micronucleated polychromatic erythrocytes in bone marrow of mice. Out of all dyes tested, only Direct Black 19:1 appeared to be an indirect mutagen inducing reverse mutations in two strains of Salmonella typhimurium TA 1538 and TA 98. None of them produced dominant lethal mutations in germ cells of male mice.

[Methods used for evaluating mutagenic and genotoxic properties of chemical compounds. I. The micronucleus test in vivo (an abridged version)]. / Metodyki stosowane do oceny wlasciwosci mutagennych i genotoksycznych zwiazków chemicznyoh. Czesc I. Test mikrojadrowy in vivo (wersja skrócona).

Methods applied within the micronuclear test in vivo for evaluation of genotoxic effects of chemical substances upon the bone marrow cells of mammals are presented. The test consists of administering the test compound to animals and then, after 30, 48 and 72 hours, making preparations from the bone marrow and calculating the percentage of polychromatic erythrocytes with micronuclei (index of genotoxic effects of the compound) and the ratio of polychromatic to normochromatic erythrocytes (index of toxic effects of the substance on bone marrow cells). As a criterion of genotoxic effects of the compound, adopted is a significant and reproducible difference of results at least once or a significant increase in the percentage of polychromatic erythrocytes with micronuclei and dose-response relationship.

Genotoxic effects of dioxolane and trioxane in mice evaluated by the micronucleus test.

The possible genotoxic effects of dioxolane and trioxane were studied using the micronucleus test. Mice were given dioxolane and trioxane i.p. in two doses at 24-h intervals. Dioxolane at doses from 1500 mg/kg to 6000 mg/kg caused a significant increase of micronucleated polychromatic erythrocytes in bone marrow of mice as compared to the negative control value. Positive results in the micronucleus test provide evidence of genotoxic activity of dioxolane. I.p. administration of trioxane produced no chromosomal damage resulting in erythrocyte micronucleus formation, even at highly toxic doses.