Mátyás G. Jakab

Follow-up genotoxicological monitoring of nurses handling antineoplastic drugs.

Most of the antineoplastic drugs used in the treatment of tumors are carcinogenic to humans. Hospital nurses are often subject to possible occupational carcinogen exposure. Exposure may occur during handling and administration of infusion solutions containing cytostatics. A genotoxicological monitoring system to detect genotoxic changes was developed in our laboratory, helping to improve working conditions and subserving primary prevention. Multiple-endpoint follow-up genotoxicological monitoring was performed in peripheral blood lymphocytes (PBLs) among 4 groups of 95 nurses (152 investigations) occupationally exposed to cytostatics. The results were compared to those of historical and industrial controls. The genotoxicological endpoints were the determination of the frequency of sister chromatid exchanges (SCEs) and the cells with high-frequency SCEs (HFC), the frequency of structural and numerical chromosome aberrations, and the measurement of ultraviolet-light-induced unscheduled DNA-repair synthesis (UDS). In Hospital 1, where nurses worked without a safety cabinet, the percentage of cells with chromosome aberrations (AC) was significantly higher than that of the controls. In Hospital 2, where nurses used inadequate safety cabinets (with horizontal airflow), significantly elevated levels of AC, SCE, HFC, and UDS were detected. During follow-up, in Hospital 2 at the time of the second investigation AC was still significantly higher, although safety conditions had been improved. The results indicate the presence of genotoxic damage in hospital nurses working with no or inadequate safety equipment. In Hospitals 3 and 4 where nurses using biological safety cabinets, the results were lower than those in the previous two groups. In Hospital 3 in the first year of the study AC was as at the level of industrial controls. During follow-up in the course of the repeated investigations a fluctuation in AC above the control level and an increase in HFC in yr 4 and 6 of the study were observed. In this group, the fluctuation in AC and HFC during the study points to the possibility of genotoxic exposure with cytostatics despite of the use of suitable safety cabinets, drawing attention to other possible routes of exposure. In Hospital 4, both AC and HFC were elevated. These data corroborate the need to maintain safety measures to avoid exposure, and the necessity of intervention in the case of exposure when using and handling hazardous carcinogenic agents. The results also indicate a certain expression time for genotoxic changes, which can lead to late somatic mutations as well as to a possible higher risk of cancer.

The frequency of induced premature centromere division in human populations occupationally exposed to genotoxic chemicals

Premature (early) centromere division (PCD, i.e., the separation of centromeres during the prometaphase/metaphase of the mitotic cycle) seems to be a possible manifestation of chromosome instability in human chromosome-breakage syndromes. Chromosome instability also frequently occurs in the peripheral blood lymphocytes (PBL) of humans occupationally exposed to clastogenic agents, and is considered an etiologic factor of neoplastic diseases. In order to investigate the importance of PCD in cancer risk assessment, we studied the frequency of PCDs in PBL of 400 Hungarian subjects. The various groups comprised 188 control donors and 212 subjects occupationally exposed to different genotoxic chemicals, such as acrylonitrile (ACN) and/or dimethylformamide (DMF), benzene, cytostatic drugs, ethylene oxide (ETO), mixed exposure in the rubber industry, mixed organic solvents including CCl4, hot oil-mist, bitumen, and polychlorinated biphenyls (PCB). Data were compared with chromosomal aberration frequencies determined in the same samples. PCD yields are significantly higher in populations exposed to mixed chemicals, crude oil and cytostatic drugs, compared with controls. PCDs involving more than three chromosomes are also more frequent in ETO- and oil mist-exposed groups than in the others. The results indicate that the induction of PCDs is neither incidental nor artificial. As a consequence, we suggest that PCD can be developed into a new, exposure-related cytogenetic biomarker for a more adequate occupational cancer risk assessment. A further, follow-up epidemiological and cytogenetic investigation of PCD is in progress.

Lymphocyte phenotype analysis and chromosome aberration frequency of workers occupationally exposed to styrene, benzene, polycyclic aromatic hydrocarbons or mixed solvents

The aim of our study was to investigate the immunotoxicity of benzene, styrene and polycyclic aromatic hydrocarbon exposure, to establish the correlation between immunological and genotoxicological parameters, and to assess the possible effect of confounding factors such as age and smoking. The immune status of the donors was characterized by measuring the surface antigens of peripheral lymphocytes. The studied antigens were the following: CD3, CD4, CD8, CD14, CD19, CD25, CD38, CD45, CD45RO, CD54, CD56, CD62L, CD71 and HLA-DR. In our studies, we compared the immunological and genotoxicological parameters (chromosome aberration, sister chromatid exchange frequency, unscheduled DNA synthesis) of the different groups with healthy controls. Analysis revealed changes in the expression of surface antigens on peripheral lymphocytes in correlation with exposure. Confounding factors, such as smoking, increased the proportion of CD4 positive T lymphocytes and influenced the surface expression of several antigens. In our investigation the occurrence of chromosome aberrations negatively correlated with CD25 (IL-2R) expression in both CD4 and CD8 positive T cells. The presented data suggest that solvents such as benzene, styrene and PAHs activate peripheral lymphocytes, and cause changes in the incidence of CD25+/CD4+ T lymphocytes that may represent a distinct subset of immune-regulatory T cells.

Formaldehyde-induced chromosomal aberrations and apoptosis in peripheral blood lymphocytes of personnel working in pathology departments

Peripheral blood lymphocytes (PBL) of 37 formaldehyde-exposed women from four pathology departments in Hungary were investigated to collect data on the effects of occupational exposures to formaldehyde and to find a possible relationship between in vivo formaldehyde-induced apoptosis and genotoxic effects. The subjects were divided into two groups: 16 donors exposed to formaldehyde together with various organic solvents, and 21 subjects exposed mainly to formaldehyde. The results were compared with 37 controls (all women) without known occupational exposure. Ambient air concentrations of formaldehyde were measured in three work places, and ranged from 0.23 to 1.21mg/m(3) (mean 0.9mg/m(3)). Measures of genotoxicity included the determination of the frequencies of chromosomal aberrations (CA), sister-chromatid exchange (SCE), HPRT mutations (variant frequency, VF) and the measurement of UV-induced unscheduled DNA-repair synthesis (UDS). The percentages of premature centromere division (PCD) and of cells with a high frequency of SCE (HF/SCE) were also scored. Apoptosis and cell proliferation were determined by flow cytometry. In both formaldehyde-exposed groups, the apoptotic activity and the CA levels in PBLs were significantly higher than in controls. The CA were mostly breaks of the chromatid type. In the second group, which was mainly exposed to formaldehyde, CA were slightly lower in comparison with the group exposed to formaldehyde and solvents, which may be attributed to a different rate of elimination of damaged lymphocytes as a consequence of formaldehyde-induced apoptotic activity. In the second group, a significant decrease of VF and a non-significant increase in HF/SCE were found compared with the control and the other group. In conclusion, the results demonstrate that exposure to formaldehyde induces apoptosis and CA, indicating an excess cancer risk among subjects occupationally exposed to formaldehyde. The results also emphasize the importance of the measurement of occupational air pollutants, such as formaldehyde, in order to avoid genotoxic effects in the workers.

Genotoxicological investigation of hospital nurses occupationally exposed to ethylene-oxide: I. Chromosome aberrations, sister-chromatid exchanges, cell cycle kinetics, and UV-induced DNA synthesis in peripheral blood lymphocytes

Structural, and numeric chromosome aberrations (CA), sister‐chromatid exchange (SCE), phytohemagglutinin stimulation (U), proliferative rate index (PRI), and UV light‐induced unscheduled DNA‐synthesis (UDS) were investigated in peripheral blood lymphocytes (PBL) of 48 historical controls (“Controls”); of 14 hospital controls in Budapest, Hungary (“Budapest controls”); of 9 nurses occupationally exposed to low‐dose ethylene‐oxide (ETO) in Budapest (“Budapest exposed”); of 10 hospital controls in Eger, Hungary (“Eger controls”); and of 27 high dose ETO‐exposed nurses in Eger (“Eger exposed”), where neoplasias, mainly breast cancers, were observed. ETO concentrations in the ambient air samples varied from 5 to 20 mg/m3 in Budapest; and from 5 to 100 mg/m3 in Eger. Both LI and PRI were depressed in Budapest exposed, indicating ETO‐induced cytotoxicity and, however, normal in Eger exposed. SCE was slightly elevated in Budapest exposed, but significantly increased in Eger exposed. The yields of cells with high frequency SCE (HFC) were only increased in Eger exposed. The expected low CA frequencies were found in Controls and in Budapest controls. ETO exposures significantly increased the CA frequencies in Budapest and Eger exposed. In Budapest exposed, as expected, we found deletions; in a lesser extent chromatid exchanges and dicentrics; but no rings were detected. These results are in a good accordance to the published data of other investigations carried out on ETO‐exposed human populations. However, in Eger exposed, beside the increased yields of deletions, the frequencies of dicentrics and rings showed a significant excess compared to the reviewed data. An unexpected, significant increase of dicentric and ring frequencies was also detected among the hospital controls in Eger controls without known clastogenic exposure. The role of confounding factors (age, smoking and drinking habit, total leukocyte count and hematocrit) was investigated by an analysis of variance on CA and SCE frequencies in Controls and in Eger exposed. Leukocyte count and mean age showed only significant effects on CA in Eger exposed and on SCE in Controls, respectively. A possible active confounding factor could be the temporary natural radioactivity of the local top water. UDS in Budapest exposed and in Eger control were significantly higher then in the Controls and in Budapest controls. In Eger exposed UDS was significantly decreased compared to the Budapest exposed and Eger control groups. The explanation of the present results is difficult on the basis of the reviewed data on ETO‐induced CA frequencies in exposed human populations, and it raises an issue of an independent genotoxic effect in Eger, which is common both in Eger controls and in Eger exposed, such as natural radioactivity.

Follow-up biological and genotoxicological monitoring of acrylonitrile- and dimethylformamide-exposed viscose rayon plant workers

In order to investigate the genotoxic effects of occupational acrylonitrile (ACN) and dimethylformamide (DMF) exposures, clinical serum and urine parameters and genotoxicological endpoints such as chromosome aberration (CA), sister chromatid exchange (SCE), high frequency SCE (HFC), cell cycle kinetics, and UV‐induced unscheduled DNA synthesis (UDS) were followed up three times during a 20‐month period in peripheral blood lymphocytes (PBL) of 26 workers (13 maintainers and 13 fiber producers) occupationally exposed to ANC and/or DMF in a viscose rayon plant, 26 matched control subjects, and six industrial controls (all males). Six of the 26 exposed subjects were hospitalized because of liver dysfunction that had developed due to inhalative DMF exposure. The rate of smoking was estimated on the basis of serum thiocyanate (SCN) levels. Average peak air ACN and DMF concentrations were over the maximum concentration limits at the time of both investigations. Urine ACN and monomethyl‐formamide (MMF) excretions of the exposed subjects were almost doubled after work shifts. An increase in lymphocyte count (in months 0 and 7), and severe alterations in the liver function were observed in the exposed subjects. In PBLs the proliferative rate index (PRI) was already increased in month 0 compared with the controls. In each study, significant increases in CA and SCE frequencies, as well as increases in UDS were found in PBLs of the exposed subjects. The frequencies of chromatid breaks and acentric fragments further increased in month 7 and remained constantly elevated in month 20. Increased yields of both chromatid and chromosome‐type exchange aberrations first appeared in month 20, when HFCs were 2.72 times more frequent in fiber producers than in maintainers. The role of some important biological confounding factors (age, white blood cell count, and hematocrit) and lifestyle confounding factors (smoking and drinking habits) were subjected to an analysis of variance during the second study. Increased CA, SCE, and UDS were found both in control and exposed smokers when current smoking was established on the basis of the serum SCN levels. The cytogenetic data suggest that occupational exposures to ACN and DMF induce considerable genotoxic consequences and may increase the cancer risk in the exposed human populations. Environ. Mol. Mutagen. 31:301–310, 1998

Is Breast Cancer Cluster Influenced by Environmental and Occupational Factors Among Hospital Nurses in Hungary

An unusual cluster of 8 breast cancer and 8 other malignant tumor cases (ovarian, uterus, lung, colon and brain tumors and malignant melanoma) developed in a period of 12 years among 98 nurses exposed to ethylene oxide (EtOx) for 5–15 years in a unit using gas sterilizer in a hospital of the archiepiscopal city of Eger, Hungary. EtOx concentration in air samples of the working area varied from 5 to 150 mg/m3. The question was, if there was any causal relationship between the elevated incidence of breast cancer and the EtOx exposure, the other possibility was, that this cluster appeared accidentally. EtOx is a human carcinogen, however, no increased breast cancer incidence in EtOx-exposed subjects was reported in the literature. We followed up for two consecutive years the 27 non cancer patients, EtOx-exposed nurses and 11 unexposed hospital controls with the aid of a multiple genotoxicology monitor including chromosomal aberration, sister-chromatide exchange, HPRT point mutation and DNA repair studies. The results were compared with data from 30 local historical controls, 48 historical controls from Budapest, 14 hospital controls and 9 EtOx exposed nurses from Budapest. Significantly high chromosome aberration yields (especially chromosome type exchanges) were alike detected in EtOx-exposed and the two other control groups in Eger. These results could not be interpreted as a consequence of EtOx exposure only, since in the EtOx-exposed group from Budapest, beside an increased total aberration frequency, the obtained exchange type aberration yields were as low as the historical controls. A plausible explanation can be the natural low dose radioactivity (222Rn) of the local tap-water due to a specific geological situation in Eger. The spontaneous breast cancer incidence in Hungary doubled in the last 10 years compared with the previous 20 years (1960–1980), especially in Eger. The appearence of the high breast cancer incidence in the hospital of Eger indicates the combined effect of EtOx and a more common local etiologic factor, such as the naturally radioactive tap-water. However, since the reported studies did not involve the investigation either of the genetic predisposition, or the effects of other possible environmental, occupational, and/or life style confounding factors, further studies (partly in progress) are necessary to clarify the importance of these factors.

Chemical Safety and Health Conditions among Hungarian Hospital Nurses

Abstract:  In the present study genotoxicological and immunotoxicological follow‐up investigations were made on 811 donors including 94 unexposed controls and 717 nurses with various working conditions from different hospitals (The Hungarian Nurse Study). The nurses were exposed to different chemicals: cytostatic drugs, anesthetic, and sterilizing gases, such as ethylene oxide (ETO) and formaldehyde. The measured biomarkers were: clinical laboratory routine tests, completed with genotoxicological (chromosome aberrations [CA], sister chromatid exchange [SCE]), and immune‐toxicological monitoring (ratio of lymphocyte subpopulations, lymphocyte activation markers, and leukocyte oxidative burst). The highest rate of genotoxicologically affected donors (25.4%) was found in the group of cytostatic drug‐exposed nurses. Comparing geno‐ and immunotoxicological effect markers, we found that among genotoxicologically affected donors the frequency of helper T cell (Th) lymphocytes, the ratio of activated T and B cells increased, whereas the oxidative burst of leukocytes decreased. In hospitals with lack of protective measures increased CA yields were observed compared to those with ISO 9001 quality control or equivalent measures. Anemia, serum glucose level, thyroid dysfunctions, benign, and malignant tumors were more frequent in the exposed groups than in controls. The hygienic standard of the working environment is the basic risk factor for the vulnerability of nurses. On the basis of these results, it is suggested, that the used cytogenetic and immunological biomarkers are appropriate to detect early susceptibility to diseases. The Hungarian Nurse Study proved that the use of safety measures could protect against occupational exposure at work sites handling cytostatic drugs, anesthetic, and sterilizing gases.

Genotoxicological monitoring of 175 subjects living in the green belts, inner town or near chemical industrial estates in Greater Budapest agglomeration, Hungary.

We studied the effects of chronic low dose exposure to environmental pollutants on the peripheral blood lymphocytes (PBL) of subjects living in the green belts and the inner town, and/or working in the surrounding of industrial estates of Greater Budapest agglomeration, Hungary. The effects of some biological (gender, age, hematocrit and white blood cell counts), lifestyle (smoking, drinking habits and residential areas), and seasonal confounding factors were also considered. PBLs of 175 Hungarian donors of Budapest agglomeration, i.e. 45 subjects living in the green belts without significant genotoxic exposure (C1), 43 donors living in the inner town (C2), and 87 individuals living and/or working near chemical industrial estates (C3), were analysed for structural and numeric chromosome aberrations (CA), sister-chromatid exchanges (SCE), cell proliferation indices (lectine stimulation, LI; and proliferation rate index, PRI), and UV-light-induced unscheduled DNA synthesis (UDS). Each subject was interviewed personally and also investigated clinically. The three populations were matched for age, smoking and drinking habits. We excluded all donors with acute infectious and/or chronic non-infectious diseases, and/or with exposure to any known chemical hazard. For C1s, the base line CA and SCE frequencies were 0.25% and 6.41 per mitosis, respectively; in C2, these frequencies were 0.48% and 6.07 per mitosis, respectively, and in C3, these data were 1.60% and 5.71 per mitosis, respectively. A significant increase of CA due to chromosome type acentric fragments was demonstrated in the donors living in the inner town (C2), compared to those living in the green belts (C1). In C3s, significant (p < 0.05) elevations were found in the frequencies of gaps, aberrant cells, total aberrations (excluding gaps), chromatid and chromosome type aberrations, and in UDS, compared to C1s. Results indicate an increased genotoxicologic risk in donors living and/or working in industrial areas. Gender-related differences in SCE frequencies were demonstrated in C2 and C3; in the latter smoking-induced changes in UDS were also found. Light drinking did not appear to influence the results.

The somatostatin analogue peptide TT-232 induces apoptosis and chromosome breakage in cultured human lymphocytes

Somatostatin receptors are supposed to be important in the regulation of apoptosis. In this study, we measured apoptosis occurring spontaneously, or induced by the synthetic somatostatin analogue, the peptide TT-232. We examined isolated human peripheral blood lymphocytes (PBL) from 32 nurses exposed bedside to cytostatic drugs, 12 chronic lymphoid leukaemia (CLL) patients prior to treatment, and 19 unexposed, healthy donors without anamnestic occupational exposure to genotoxic agents. Cells were stimulated by phytohaemagglutinin-P (PHA) and cultured for 69 h with or without 15 microg/ml TT-232, respectively. Cell kinetic parameters and apoptosis were determined by flow cytometry after staining with FITC-labeled anti-BrdU and propidium iodide (PI) and the results on spontaneous and peptide-induced apoptosis were compared with the obtained chromosome aberration frequencies (CA). The peptide TT-232 unexpectedly induced chromosome breakage in addition to apoptosis. The mean spontaneous apoptotic fractions were 6.65+/-0.89%, 6.46+/-0. 53%, and 3.07+/-0.57%, and the mean CA yields in the samples without TT-232 were 1.74+/-0.46%, 2.44+/-0.40%, and 4.50+/-1.05%, for healthy subjects, nurses, and CLL patients, respectively. A total of 15 microg/ml TT-232 treatment in healthy subjects increased the mean CA frequency (10.38+/-1.57%), as well as the apoptotic cell fraction (2.63+/-0.45 times higher than the corresponding untreated sample). In TT-232-treated PBLs of nurses, CA remained unchanged and the mean apoptotic cell fraction showed only a slight increase (1.24+/-0.11 times higher than the untreated). Among CLL patients, TT-232 treatment significantly increased both CA (up to 17.83+/-4.04%) and the ratio of apoptotic cells (21.78+/-11.00 times higher than the untreated). These results demonstrated significant differences in apoptosis sensitivity in controls, nurses and CLL donors, after 15 microg/ml TT-232 treatment. Data also indicate that the induced CA yields in CLL donors with high CA are in correlation with TT-232-induced apoptosis.