Antonina Cebulska-Wasilewska

Chromosomal aberration frequency in lymphocytes predicts the risk of cancer: results from a pooled cohort study of 22 358 subjects in 11 countries.

Mechanistic evidence linking chromosomal aberration (CA) to early stages of cancer has been recently supported by the results of epidemiological studies that associated CA frequency in peripheral lymphocytes of healthy individuals to future cancer incidence. To overcome the limitations of single studies and to evaluate the strength of this association, a pooled analysis was carried out. The pooled database included 11 national cohorts and a total of 22 358 cancer-free individuals who underwent genetic screening with CA for biomonitoring purposes during 1965–2002 and were followed up for cancer incidence and/or mortality for an average of 10.1 years; 368 cancer deaths and 675 incident cancer cases were observed. Subjects were classified within each laboratory according to tertiles of CA frequency. The relative risk (RR) of cancer was increased for subjects in the medium [RR = 1.31, 95% confidence interval (CI) = 1.07–1.60] and in the high (RR = 1.41; 95% CI = 1.16–1.72) tertiles when compared with the low tertile. This increase was mostly driven by chromosome-type aberrations. The presence of ring chromosomes increased the RR to 2.22 (95% CI = 1.34–3.68). The strongest association was found for stomach cancer [RRmedium = 1.17 (95% CI = 0.37–3.70), RRhigh = 3.13 (95% CI = 1.17–8.39)]. Exposure to carcinogens did not modify the effect of CA levels on overall cancer risk. These results reinforce the evidence of a link between CA frequency and cancer risk and provide novel information on the role of aberration subclass and cancer type.

Correlations between DNA and cytogenetic damage induced after chemical treatment and radiation

The induction of damage in human lymphocytes has been compared after treatment in vitro with two different agents, the chemical o-phenylenediamine (o-PDA) and gamma irradiation, in the alkaline single cell gel electrophoresis (Comet) assay, and after cytogenetic analysis. The chemical treatment caused dose-related increases in DNA damage in the Comet assay and cytogenetic damage in the first and second metaphases. The results revealed a very strong association between the two types of damage. Correlation coefficients were from 0.95 to 0.97. From previous studies, high correlation coefficients of 0.99 and 0.97 in the same assays were also evaluated for X-rays and fast neutrons, respectively. On the basis of such results, we suggest that the Comet assay responses provide a good prediction of cytogenetic damage. Thus, because of its simplicity and rapidity, the Comet assay would appear to be a very useful tool for determining the genotoxicity of environmental agents.

Tradescantia stamen-hair mutation bioassay on the mutagenicity of radioisotope-contaminated air following the Chernobyl nuclear accident and one year later.

This paper presents results of the research on the mutagenic effect of ambient air in the Cracow area. Initial studies were conducted in May 1986, following the Chernobyl accident. Other studies were performed at various sites within the Cracow area in the Spring of 1987. Counts were made of stunted hairs and pink cells in the stamen hairs of Tradescantia clone 4430. Mutations scored from the 11th day after the beginning of exposure were used as a measure of the mutagenic effect. The mean mutation frequencies measured in 1986 and 1987 were 0.43 and 0.21 per 100 hairs respectively. The time-dependent development of mutation frequencies observed after the Chernobyl accident showed a correlation with the time-dependent development of total radioactivities measured in the air at that time. The results obtained in 1987 showed on average a significant decrease of ambient air mutagenicity. Still, the variation of mutation rates observed during the investigated period at different sites in the Cracow area was rather high (0.09-0.38 mut/100 hairs). Only the highest frequencies observed in the Spring of 1987 were comparable to the level detected after the Chernobyl accident.

ras oncoproteins in human plasma from lung cancer patients and healthy controls

In order to explore the significance of ras oncoproteins in plasma in the carcinogenic process, we have examined samples from 40 Polish human lung cancer patients prior to treatment. They were compared with 35 healthy donors and have been screened using a direct analysis of the plasma. Proteins were separated by gel electrophoresis, transferred to a nitrocellulose membrane by Western blotting and detected by chemiluminescence, using monoclonal ras antibody as the primary antibody. Elevated increases in ras oncoproteins were determined where an increase was considered to be greater than 2 standard deviations above the mean negative control values. The results showed that in 45% of cancer patients ras oncoprotein levels were statistically significantly increased (P < 0.001, pooled two-sample t-test untransformed, and non-parametric Mann-Whitney test) in the plasma by comparison with 6% in the controls. This would suggest that an increase in ras oncoproteins in plasma could be a possible prognostic marker or biomarker for lung cancer.

Ras p21 protein levels in human plasma from patients with chronic obstructive pulmonary disease (COPD) compared with lung cancer patients and healthy controls.

To explore the value of an increase in ras p21 proteins in plasma as a biomarker for the carcinogenic process or for the general disease state, we have directly analysed for ras p21 proteins, plasma samples from Polish human patients with chronic obstructive pulmonary disease (COPD). They were compared with appropriate controls and also with the Polish lung cancer patients previously examined before treatment [D. Anderson, J.A. Hughes, A. Cebulska-Wasilewska, E. Nizankowska, B. Graca, Ras oncoproteins in human plasma from lung cancer patients and healthy controls, Mutat. Res. 349 (1996) 121-126]. An elevated level of ras p21 proteins was considered to be greater than 2 standard deviations (SD) above the mean negative control values. Nine out of 20 COPD patients (mean age = 65.9 years) had increased ras p21 protein levels when compared with 20 age-matched (mean age = 62.4 years) controls of the present study with a mean + 2 SD of 0.70. Eighteen out of 40 lung cancer patients (mean age = 60.1 years) had increased ras p21 protein levels compared with their concurrent controls (mean age = 40.2 years) with a mean + 2 SD of 2.53. However when compared with the age-matched controls of this present study, there were 35 out of 40 (87.5%) with increased levels. When the COPD patients and lung cancer patients were compared with 101 historical controls (age range 25-76 years, of those whose age was recorded) from unexposed healthy populations from Poland, Estonia and Spain with a mean + 2 SD of 1.83, then 4 out of 20 (20%) COPD patients and 30 out of 40 (75%) lung cancer patients had increased levels. Whether using concurrent controls, age-matched controls or historical controls, the data would suggest that an increase in ras p21 protein levels in plasma from lung cancer patients could be a possible prognostic marker or biomarker for lung cancer. COPD patients when compared with historical controls or age-matched controls had lower ras p21 protein values than cancer patients. Their ras p21 protein values might also be a biomarker for cancer. It is possible that some of these COPD patients were in the process of developing cancer or perhaps would die from COPD before cancer develops. It cannot be ruled out that the increases could be a biomarker of exposure since many of the lung cancer patients and most of the COPD patients were smokers.

Factors affecting various biomarkers inuntreated lung cancer patients and healthy donors

The purpose of the present communication was to determine in lung cancer patients and healthy donors if there was a possible association between cancer and biomarkers of cytogenetic damage and ras p21 oncoprotein levels, and if various exogenous confounding factors (such as smoking habit) and endogenous ones (age, sex, etc.) could affect these biomarkers. Peripheral blood and plasma were collected from 31 lung cancer patients prior to treatment and 35 healthy donors of a similar socioeconomic status and from the same region in Poland. Chromosomal aberrations (CA), sister chromatid exchanges (SCE), high frequency cells (HFC), and proliferative rate index (PRI) were examined from the blood and ras p21 oncoproteins from the plasma. These parameters were used as biomarkers of genotoxic anomalies. All the biomarkers were examined for their relationship to confounding factors of age, sex, smoking habit, and immediate family cancer history. Results were analyzed by a t‐test, analysis of variance (ANOVA), and stepwise multivariate regression analysis. All types of CA (including and excluding gaps), percent aberrant cells, SCE, and ras p21 oncoproteins were statistically significantly higher in cancer patients than in the healthy donors. Although there were smaller numbers of females in the cancer patients group who were older than the males, there was a difference due to sex (gender) with statistically significant increases in females for CA, SCE, and HFC, but there was no increase for ras p21 oncoproteins. Cytogenetic damage was not related to other cancers in the immediate families of the groups. All major CA parameters differed significantly between smokers and non‐smokers in the cancer patients group, and SCE and HFC differed in the healthy donors group. Such parameters also showed a significant variability with the number of cigarettes smoked and the years of smoking habit. Multivariate regression analyses showed a significant association between cytogenetic damage, ras p21 oncoproteins, and cancer. In conclusion, cytogenetic damage and ras p21 oncoproteins in this study appear to be biomarkers associated with cancer, but have not been proved causally, and confounding factors such as age, sex (gender), and smoking can have an impact on them. Environ. Mol. Mutagen. 30:205–216, 1997.

Cytogenetic damage and ras p21 oncoprotein levels from patients with chronic obstructive pulmonary disease (COPD), untreated lung cancer and healthy controls.

The purpose of the present communication was to determine in patients with chronic obstructive pulmonary disease (COPD), untreated lung cancer and healthy controls if there was a possible association between the disease state and biomarkers of cytogenetic damage and ras p21 oncoprotein levels, and if various exogenous confounding factors such as smoking habit and endogenous ones (sex, cancer in the immediate family) could affect these biomarkers. The individuals in all groups were as well-matched as possible for age to determine if this could be eliminated as a confounder. Peripheral blood and plasma were collected from 20 COPD patients, 31 cancer patients and 20 healthy controls. Chromosomal aberrations (CA), sister chromatid exchanges (SCE) and high frequency SCE cells (HFC) were examined from the blood and ras p21 oncoproteins from the plasma. These parameters were used as biomarkers of genotoxic anomalies. All the biomarkers were examined for their relationship to the confounding factors. Results were analysed by a t-test, analysis of variance (ANOVA) and stepwise multivariate regression analysis. There was an increase in CA, although not statistically so, in COPD and cancer patients by comparison with healthy controls, but there was a statistically significant increase in SCE, HFC and ras p21 oncoproteins. There was also a statistically significant difference between respiratory volume parameters in COPD patients and controls. Respiratory parameters were not measured in cancer patients. Ras p21 oncoproteins were also statistically significantly increased in the COPD and cancer patients, suggesting that the disease state alone might be sufficient to increase the oncoproteins, or that some of the COPD patients were in the process of developing cancer or perhaps some would die from COPD before cancer developed. Smoking was shown to have a marked effect on all parameters investigated. Ex-smokers showed less effects. Since age was very well controlled, there was little effect due to age. There was an effect due to sex, but cancer in the immediate family had little effect on any of the parameters.

Mutagenic analysis of 2,3-diaminophenazine and 2-amino-3-hydroxyphenazine in Salmonella strains expressing different levels of O-acetyltransferase with and without plant and mammalian activation

2,3-Diaminophenazine (DAP) and 2-amino-3-hydroxyphenazine (AHP) are products generated from oxidative-type phenylenediamine hair dyes and are also present in pesticide formulations as contaminants. Earlier studies demonstrated that DAP and AHP were mutagenic in Salmonella typhimurium strains after mammalian microsomal activation. Plant systems can activate structurally similar arylamines. S. typhimurium strains have been developed that express elevated levels of acetyl-CoA: N-hydroxyarylamine O-acetyltransferase (OAT). O-acetyltransferase expression is necessary for the generation of the ultimate arylamine promutagen after plant activation. A number of arylamines including 2-aminofluorene, benzidine and 4-aminobiphenyl were activated by plant cells into mutagens in the OAT over-expressing S. typhimurium strain, YG1024. The objectives of this research were to examine the mutagenicity of DAP and AHP with mammalian or plant activation in Salmonella strains with different acetyltransferase activities. The hypothesis tested was whether and to what degree a metabolite of DAP or AHP could serve as a substrate for bacterial O-acetyltransferase and induce mutation in Salmonella. DAP and AHP without activation induced both frameshift and base pair substitution mutations in S. typhimurium strains that exhibited elevated levels of O-acetyltransferase activity. The mutagenicity of DAP and AHP were greatly enhanced with mammalian hepatic microsomal activation resulting in a preferential induction of frameshift mutations. With the hisD3052 allele as the gene target, S9-activated DAP induced frameshift mutations in YG1024 and TA98 as well as the OAT deficient strain TA98/1,8-DNP6. S9-activated AHP induced mutation only in the OAT over-expressing strain, YG1024. With the hisG46 allele, O-acetyltransferase activity was necessary for the metabolism of DAP and AHP to products that induce base pair substitution mutations. An intriguing finding of this work was the antimutagenic capacity of TX1MX, a plant cell-free activation mixture. TX1MX repressed the mutagenic activity of DAP and AHP at frameshift and base pair substitution mutation targets.

Studies on the genotoxic effects of aminophenazines using two cellular models and three different methods.

This paper presents studies on the genotoxicity of two aminophenazines: 2,3-diaminophenazine (DAP) and 2-amino-3-hydroxyphenazine (AHP). The genotoxic activities of these compounds were evaluated with human lymphocytes using the alkaline single cell gel electrophoresis (SCGE) assay and two cytogenetic assays (chromosome aberrations (CA) and sister chromatid exchange (SCE) analysis). Results show that these chemicals elicited an increase in DNA and chromosomal damage under the studied ranges of concentration. Concentration-response curves were similar and there was a positive correlation between the damage observed at the DNA and chromosomal levels. DAP was more genotoxic than AHP and this agreed with the genotoxic potencies reported in bacterial systems.

Examination of ras oncoproteins in human plasma from healthy controls and workers exposed to petroleum emissions, including benzene-related compounds.

Ras oncoproteins in blood plasma from workers exposed to petroleum emissions and unexposed controls were examined from Polish and Estonian samples. Twenty-four workers and 35 unexposed controls were examined from Poland and 97 exposed and 40 unexposed controls from Estonia. Of the Estonian workers, 50 were exposed to benzene in a benzene production plant and 47 to polyaromatic hydrocarbons and benzene in a cokery. Blood plasma proteins were separated by gel electrophoresis, transferred to a nitrocellulose membrane by Western blotting and detected by chemiluminescence using a monoclonal antibody as the primary antibody. There were no statistically significant differences between the exposed and the control groups in either the Polish or the Estonian samples.