Cancer risk in humans predicted by increased levels of chromosomal aberrations in lymphocytes: Nordic study group on the health risk of chromosome damage.

Cytogenetic assays in peripheral blood lymphocytes (PBL) have been used extensively to survey the exposure of humans to genotoxic agents. The conceptual basis for this has been the hypothesis that the extent of genetic damage in PBL reflects critical events for carcinogenic processes in target tissues. Until now, no follow-up studies have been performed to assess the predictive value of these methods for subsequent cancer risk. In an ongoing Nordic cohort study of cancer incidence, 3182 subjects were examined between 1970 and 1988 for chromosomal aberrations (CA), sister chromatid exchange or micronuclei in PBL. In order to standardize for the interlaboratory variation, the results were trichotomized for each laboratory into three strata: low (1-33 percentile), medium (34-66 percentile), or high (67-100 percentile). In this second follow-up, a total of 85 cancers were diagnosed during the observation period (1970-1991). There was no significant trend in the standardized incidence ratio with the frequencies of sister chromatid exchange or micronuclei, but the data for these parameters are still too limited to allow firm conclusions. There was a statistically significant linear trend (P = 0.0009) in CA strata with regard to subsequent cancer risk. The point estimates of the standardized incidence ratio in the three CA strata were 0.9, 0.7, and 2.1, respectively. Thus, an increased level of chromosome breakage appears to be a relevant biomarker of future cancer risk.

Micronuclei in erythropoietic bone marrow cells: relation to cytogenetic pattern and prognosis in acute nonlymphocytic leukemia.

The bone marrow karyotype and the frequency of micronuclei in erythropoietic bone marrow cells (Howell-Jolly bodies) were determined in 25 adults with acute nonlymphocytic leukemia (ANLL). Ten patients had exclusively normal diploid bone marrow cells; 11 had a mixture of normal and abnormal cells, and 4 had abnormal bone marrow metaphases only. The frequency of micronuclei ranged frm 3 to 28/2000 erythropoietic bone marrow cells (median 10). The number of micronuclei was significantly higher in patients with abnormal metaphases than in those with normal metaphases; in patients with a mixture of normal and abnormal bone marrow metaphases there was an association between the frequency of abnormal metaphases and the number of micronuclei. A striking difference in median survival time was found between patients with low and high numbers of micronuclei, irrespective of the cytogenetic bone marrow patterns. Patients with fewer than 10 micronuclei per 2000 erythropoietic bone marrow cells had a median survival of 148 days; those with more than 10/2000 had a median survival of only 34 days (0.002 less than p less than 0.02).

An inter-Nordic prospective study on cytogenetic endpoints and cancer risk. Nordic Study Group on the Health Risk of Chromosome Damage.

To investigate whether high rates of chromosomal aberrations (CAs), sister chromatid exchange (SCE), or micronuclei(MN) in peripheral lymphocytes indicate an increased risk for subsequent cancer, a prospective cohort study of 2,969 subjects cytogenetically examined between 1970 and 1988 in four Swedish, two Finnish, and two Norwegian laboratories was initiated. To standardize for the interlaboratory variation, the results of the three cytogenetic endpoints were trichotomized for each laboratory into "low" (1st to 33rd percentile), "medium" (34th to 66th percentile), and "high" (67th to 100th percentile]. Thirty-four cancers had been diagnosed in the cohort during the observation period (1970 to 1985). The point-estimates of the standardized morbidity ratio (SMR) in the three CA strata were 90, 92, and 180, respectively. This trend for a positive association was not statistically significant (p = 0.06). There was no significant trend between SMR and the trichotomized rates of SCE. In the subcohort examined for MN only two cases of cancer had been diagnosed until now. If subjects with "high" frequencies of CA or SCE have a two-fold (or greater) risk of developing cancer as compared with individuals who have "medium" or "low" frequencies, we estimate that there is a likelihood of 80% and 70%, respectively, that this will be detectable as significant (p less than or equal to 0.05) differences after a further follow-up period of 5 years. Weaker associations between cancer risk and the cytogenetic endpoints would not be possible to evaluate until even later follow-ups.

Cancer predictive value of cytogenetic markers used in occupational health surveillance programs.

It has not previously been clear whether cytogenetic biomarkers in healthy subjects will predict cancer. Earlier analyses of a Nordic and an Italian cohort indicated predictivity for chromosomal aberrations (CAS) but not for sister chromatid exchanges (SCES). A pooled analysis of the updated cohorts, forming a joint study base of 5271 subjects, will now be performed, allowing a more solid evaluation. The importance of potential effect modifiers, such as gender, age at testing, and time since testing, will be evaluated using Poisson regression models. Two other potential effect modifiers, occupational exposures and smoking, will be assessed in a case-referent study within the study base.

Increased frequency of chromosome aberrations in long-term cultured cerebrospinal fluid lymphocytes of patients with multiple sclerosis.

A method employing long-term lymphocyte culturing was developed to study chromosome aberrations in samples with very few cells. It was used to examine lymphocytes from the cerebrospinal fluid (CSF) and peripheral blood (PB) in 23 patients with clinically definite multiple sclerosis (MS), nine patients with other neurological diseases (OND), and eight healthy individuals. MS patients had significantly more aberrations in CSF lymphocytes than in PB lymphocytes (6.4 vs 4.1; P = 0.003). In contrast, no such difference was noted among patients with OND (3.8 vs. 3.7; P = 0.89) or healthy controls (3.6 vs 3.5; P = 0.90). CSF lymphocytes from MS patients had more aberrations than CSF lymphocytes from healthy controls (P = 0.012), but there was no difference between PB lymphocytes from MS patients and controls (P = 0.58). The patients with OND were similar to healthy controls both in CSF (3.8 vs 3.6; P = 0.91) and PB lymphocytes (3.7 vs 3.5; P = 0.90).

Mechanism of benzene toxicity. Effects of benzene and benzene metabolites on bone marrow cellularity, number of granulopoietic stem cells and frequency of micronuclei in mice.

The effects of benzene and benzene metabolites *hydroquinone and catechol) on bone marrow cellularity, number of granulopoietic stem cells and on the frequency of micronuclei in polychromatic erythrocytes were investigated in mice. The dose-effect curve for benzene revealed that there was a threshold dose (approx. 100 mg benzene/kg body wt./day injected subcutaneously on 6 consecutive days) above which severe toxicity occurred in all three parameters. Also hydroquinone gave rise to adverse effects in the parameters studied, but the sequence of occurrence was different from that observed with benzene. These data are interpreted to indicate that hydroquinone is a hemotoxic metabolite of benzene in mice in vivo, but that other metabolites, or benzene itself, also probably contribute to the toxicity. Catechol gave no effects. However, due to acute effects like tremor and convulsions only rather low doses could be tested. Simultaneous administration of toluene dramatically reduced the toxicity of benzene, but gave only a small reduction of the hydroquinone-induced effects.

Micronuclei in lymphocytes with preserved cytoplasm. A method for assessment of cytogenetic damage in man.

The micronucleus method for studying cytogenetic effects in lymphocytes in man was modified. The cells were analyzed with preserved cytoplasm, which allowed a more precise identification of micronuclei. The method was tested on 58 individuals 38 of whom were exposed to styrene. Higher frequencies of micronuclei were obtained in 96-h cultures than in 72-h ones. In cells X-rayed in vitro a culture time of 80-88 h gave a maximal frequency of micronuclei. There was a very close correlation between the results of cultures from whole blood and from 'buffy coat' (r = 0.99). There was also a good correlation between two observers (r = 0.95), but a systematic difference of about 30% existed between the two sets of observations. The method error (variation coefficient) was 11 and 6% in preparations with mean micronuclei frequencies of 4 and 38%, respectively. The styrene-exposed group displayed weak but statistically significant correlations between frequencies of micronuclei and numerical chromosome aberrations. There were statistically significant effects of age, smoking and low levels of styrene exposure but these factors explained only 12-24% of the total variance.

The size of micronuclei in human lymphocytes varies according to inducing agent used.

Micronuclei were induced in vitro in human lymphocytes by mitomycin C, X-rays, vincristine, and colcemid and analyzed in cells with preserved cytoplasm. The micronucleus/cell nucleus ratio was measured. It was found that micronuclei induced by mitomycin C and X-rays were significantly smaller than those formed by vincristine and colcemid. Thus, in spite of the wide size span of human chromosomes, it could be shown that it is possible to differentiate between micronuclei formed by spindle-damaging agents (vincristine and colcemid) and those induced by agents directly damaging the chromosomes (mitomycin C and X-rays). Mitomycin C-induced micronuclei were smaller than those induced by X-rays, probably because the former agent preferentially produces chromatid fragments and the latter chromosome fragments.

Micronuclei and mitotic index in B-, T4- and T8-cells treated with mitomycin C and gamma-irradiation.

Lymphocytes were treated in vitro with mitomycin C and gamma-radiation at different doses (0-250 nmol/l and 0-2 Gy, respectively). After incubation in RPMI 1640 medium and stimulation with phytohemagglutinin for 72 h, the lymphocyte subgroups T4 (CD4), T8 (CD8) and B (CD19) were separated by an immunomagnetic method and analyzed for the presence of micronuclei. With mitomycin C the highest levels were found in T4- and B-cells. When micronuclei were induced by irradiation the T4-cells showed the highest frequencies and the B-cells the lowest. The outcome of B-cells with gamma-irradiation was probably due to a pronounced cytotoxic reaction in this cell type, which could be measured as a decrease in mitotic index.

Cytogenetic effects in rotogravure printers exposed to toluene (and benzene).

Comparing 21 rotogravure printers exposed to toluene (medians: time-weighted air level 150 mg/m3, blood toluene 1.6 mumole/l) and 21 unexposed controls (median blood toluene less than or equal to 0.01 mumole/l) there was a significant increase in the frequency of micronuclei (MN) in pokeweed mitogen (PWM)-stimulated peripheral blood lymphocytes in the printers, as compared to the controls (2.8% vs. 1.5%; p = 0.03; all p adjusted for age and smoking). The frequency of small MN (size ratio MN/main nucleus less than or equal to 0.03) in PWM-stimulated lymphocytes was associated with the exposure (1% vs. 0.3%; p = 0.05). Furthermore, among the exposed subjects there was an association between blood toluene and small MN (0.17% per mumole/l; p = 0.0005). Small MN in phytohemagglutinin (PHA) cultures displayed no association with any exposure parameter. However, in the printers, an estimated cumulative exposure index was weakly correlated with the frequency of total MN in PHA-stimulated cells (0.00003% per mg/m3 x year; p = 0.07). Among the printers, chromosomal breaks in PHA-stimulated cells were associated with the duration of earlier benzene exposure (0.03% per year; p = 0.01). The results of this study strongly indicate that toluene causes a clastogenic effect on the B-cells even at low exposure levels. Further, earlier benzene exposure seems to have caused chromosomal breaks in T-cells.

Increased frequencies of micronuclei in T8 lymphocytes of smokers.

Micronucleus frequencies and mitotic indices were analyzed in B, T4, and T8 lymphocytes from 40 smokers and 42 non-smoking referents. The highest level of micronuclei was found in T4 cells followed by T8 and B cells. These differences were statistically significant. There were statistically significant linear correlations between the micronucleus frequencies of all three subsets. There was a statistically significant effect of smoking only in the T8 cells. Smoking also increased the number of neutrophilic granulocytes and lymphocytes in the peripheral blood. There was a statistically significant effect of age on the micronucleus frequencies in T4 and T8 lymphocytes. The mitotic indices did not have any effect on the micronucleus frequencies and they were not influenced by smoking, age or sex.

Micronuclei in lymphocyte subsets in relation to immune proteins and allergic disease.

This is an investigation of 54 boys and 23 girls with a median age of 19 years (range 18-22 years). The study group contained 12 boys and five girls with asthma and 23 boys and seven girls with allergic rhinitis. Sensitivity to pollen and furred animals were reported by 22 boys and eight girls and by 17 boys and six girls, respectively. The levels of serum immune proteins (IgA, IgE and IgG with subclasses, and IgM) were determined by immunological techniques. As a biomarker of chromosomal damages, the lymphocyte micronuclei was used. We analyzed the frequencies of micronuclei in 3000 B-lymphocytes and in equal numbers of T4- and T8-lymphocytes. The lymphocytes were separated by magnetic attraction in T4 (CD4), T8 (CD8) and B (CD19) fractions using Dynabeads(R). The most interesting finding of this investigation was that the three markers of atopic disease, asthma, hypersensitivity to pollen and IgE levels, associated significantly with increased frequencies of micronuclei in B-lymphocytes. There was also a relation between IgA and the frequency of micronuclei in B-cells. In an epidemiological study of 7000 individuals with allergic diseases, we have found an over-risk for lymphomas in the group with positive skin prick test. Hypothetically, we think that there may be a link between our present finding of an increased mutagenic activity and the lymphoma over-risk among individuals with allergic disease since most lymphomas stem from B-lymphocytes.

In vivo and in vitro ethylene oxide exposure of human lymphocytes assessed by chemical stimulation of unscheduled DNA synthesis.

Factory workers exposed to ethylene oxide (EO), 0.5-1.0 ppm in factory air, together with matched controls from the same factory, were examined for evidence of toxic exposure by measurement of unscheduled DNA synthesis (UDS) induced by N-acetoxy-2-acetylaminofluorene (NA-AAF) and of chromosome aberrations in peripheral lymphocytes. The total chromatid gaps plus breaks were significantly elevated and NA-AAF-induced UDS was significantly reduced in the EO-exposed group as compared with the unexposed control group. The NA-AAF-induced UDS values negatively correlated to the duration (yr) of EO exposure (r = -0.45, p less than 0.02) and the number of chromosome breaks (r = -0.61, p less than 0.05), indicating an inhibition in vivo of DNA-repair capacity by EO. These data were verified in vitro by biochemical and autoradiographic studies of EO-induced UDS in human blood cells. Above 2 mM EO, UDS was inhibited in lymphocytes whether they were cultured for 24 or 122 h after alkylation with EO. Even at the subtoxic EO dose of 0.1 mM, lymphocytes were sensitized to additional exposures of NA-AAF, so that cytotoxicity was increased to 40% compared with 5% for the controls even though UDS was unaffected. It is concluded that EO was toxic to lymphocytes, even when they were sensitized at non-toxic EO doses to the cytotoxic action of other mutagens (e.g. NA-AAF), and the cells that did survive above 2 mM EO were inhibited in their DNA-repair capacity as judged by reduced UDS.

B- and T-lymphocyte micronuclei in chimney sweeps with respect to genetic polymorphism for CYP1A1 and GST1 (class Mu).

Epidemiological studies have shown an increased incidence of lung cancer, bladder cancer, and esophageal cancer in chimney sweeps, probably due to their exposure to PAH in soot. The work environment for sweeps has, however, improved during the last decades. It was thus important to assess whether the present exposure still may cause genotoxic effects. A further objective was to assess whether genetic polymorphisms in metabolic enzyme activities could explain some of the variation in the parameters of genotoxicity. Venous blood samples were drawn from 71 chimney sweeps and 59 control subjects. Micronuclei were analyzed in activated peripheral B- and T-lymphocytes with preserved cytoplasm. Polymorphisms for CYP1A1 and GST1 in the sweeps were analyzed by a PCR technique. The sweeps did not have higher frequencies of micronuclei in B- or T-lymphocytes than the control subjects, when allowance was made for age and smoking in a multiple regression analysis. Further, there was no association between years of active work as a sweep and any of the two micronucleus parameters. None of the sweeps had the rare CYP1A1 genotype val/val and only one individual had the m2/m2 genotype. The presence of at least one GST1 allele (GST1+) was observed in 36 subjects (51.4%). Thirteen individuals (18.6%) were of the m1/m2 or m2/m2 genotype. And among those only seven had the combined GST1- and m1/m2 genotype. No difference was observed in B- or T-lymphocyte micronucleus frequencies between sweeps with the rare CYP1A1 genotypes m1/m2, m2/m2 or ile/val compared to individuals with the m1/m1 and ile/ile genotypes. Moreover, the GST1 deficient sweeps (GST1-) did not show any altered micronucleus frequency compared to the GST1 positive sweeps. A possible reason for the lack of genotoxic effect in sweeps is the improved hygienic conditions and change in fuels, which has decreased the exposure levels for PAH. Host polymorphisms for metabolizing enzymes did not influence the micronucleus frequencies. As the sweeps did not differ from the control subjects, with respect to micronucleus frequencies, no conclusion on the importance of host polymorphisms for genotoxic risk can be drawn.

Gasoline pump mechanics had increased frequencies and sizes of micronuclei in lymphocytes stimulated by pokeweed mitogen.

Fifteen gasoline pump mechanics and 15 controls were investigated with the lymphocyte micronucleus assay and also with differential count of leukocytes in the peripheral blood. The lymphocytes were stimulated with phytohemagglutinin and pokeweed mitogen in parallel cultures. The pump mechanics had increased frequencies and sizes of micronuclei in the pokeweed mitogen-stimulated cultures but not in those stimulated with phytohemagglutinin. The difference in effect between the 2 mitogens is probably due to an increased sensitivity of the B-lymphocytes to mutagens. The leukocyte count was significantly higher in the gasoline-exposed group. The effect of the occupational exposure was probably caused by the benzene content of the gasoline. The time-weighted average for 8 h was around 1 mg/m3 of benzene, but there were peak levels of 20 mg/m3.

Cancer predictive value of cytogenetic markers used in occupational health surveillance programs: a report from an ongoing study by the European Study Group on Cytogenetic Biomarkers and Health.

The cytogenetic endpoints in peripheral blood lymphocytes: chromosomal aberrations (CA), sister chromatid exchange (SCE) and micronuclei (MN) are established biomarkers of exposure for mutagens or carcinogens in the work environment. However, it is not clear whether these biomarkers also may serve as biomarkers for genotoxic effects which will result in an enhanced cancer risk. In order to assess this problem, Nordic and Italian cohorts were established, and preliminary results from these two studies indicated a predictive value of CA frequency for cancer risk, whereas no such associations were observed for SCE or MN. A collaborative study between the Nordic and Italian research groups, will enable a more thorough evaluation of the cancer predictivity of the cytogenetic endpoints. We here report on the establishment of a joint data base comprising 5271 subjects, examined 1965-1988 for at least one cytogenetic biomarker. Totally, 3540 subjects had been examined for CA, 2702 for SCE and 1496 for MN. These cohorts have been followed-up with respect to subsequent cancer mortality or cancer incidence, and the expected values have been calculated from rates derived from the general populations in each country. Stratified cohort analyses will be performed with respect to the levels of the cytogenetic biomarkers. The importance of potential effect modifiers such as gender, age at test, and time since test, will be evaluated using Poisson regression models. The remaining two potential effect modifiers, occupational exposures and smoking, will be assessed in a case-referent study within the study base.

Allergic disease and the infestation of Enterobius vermicularis in Swedish children 4-10 years of age.

BACKGROUND: The possible association between certain childhood infections and the propensity to develop allergic disease may include intestinal helminth infections. The experiences of such associations derive mainly from studies in tropical areas, and the results are not clear-cut. OBJECTIVE: To explore the association between Enterobius vermicularis and allergic disease in Swedish children 4 to 10 years of age. METHOD: The occurrence of E. vermicularis was examined by perianal tape tests in 70 allergic children recovered from a pediatric register of positive skin-prick tests. A nonallergic control group (n = 102) was gathered from a cohort of children previously examined for the prevalence of E. vermicularis and allergic symptoms. RESULTS: In the allergic group 26 of the 70 cases (37%) had a positive tape-test for E. vermicularis, compared to 23 of the 102 cases (23%) in the nonallergic control group (p = .037). CONCLUSION: The results indicate that E. vermicularis could be more frequent in children with allergic disease as defined by allergic symptoms and a positive skin-prick test compared to nonallergic children, i. e., those without a history of allergic disease. These data, however, do not allow any conclusion on the nature of the possible association between E. vermicularis and allergic disease.

Cytogenetic and hematological effects in plastics workers exposed to styrene.

For 20 glass-reinforced plastics workers exposed to styrene and 22 unexposed referents, the frequency and size distribution of micronuclei were determined for lymphocytes stimulated with phytohemagglutinin or pokeweed mitogen, and white blood cell counts were made. Furthermore, chromosome aberrations were scored for 11 of the exposed subjects and 15 of the referents. The mean level of styrene in the breathing zone of the workers was 56 mg/m3. Workers exposed to styrene did not show an increase in any of the cytogenetic end points studied when the effect of age and smoking was allowed for in a multiple regression analysis. A significant 30% increase in the number of peripheral monocytes was observed for the exposed workers. No correlations between the cytogenetic and hematological tests on one hand and the length of exposure time on the other could be detected.

Exposure to polycyclic aromatic hydrocarbons and genotoxic effects on nonsmoking Swedish road pavement workers.

OBJECTIVES: The objective of this study was to investigate exposure to polycyclic aromatic hydrocarbons (PAH) from asphalt fumes among Swedish road pavement workers and determine whether any effects could be detected with genotoxic tests. METHODS: The study included 28 nonsmoking road pavers and 30 nonsmoking referents. The concentration of PAH was determined in the breathing zone of the road pavers. 1-Hydroxypyrene was analyzed before and after shifts of asphalt work and during the afternoon for referents. Sister chromatid exchanges (SCE) and micronuclei (MN) were determined in peripheral lymphocytes. RESULTS: Several 3- or 4-ring PAH were found, and the analysis indicated that they occurred in bitumen fumes rather than in traffic fumes. The average total concentration of PAH was 2.3 (range 0.2-23.8) microg/m3. The concentration of 1-hydroxypyrene in urine was higher for the road pavers than for the referents, but there was no significant difference between the pre- and postshift values of the road pavers. The road pavers had no significant increase in SCE or MN. CONCLUSIONS: The study showed that Swedish road pavers have an increased exposure to PAH from bitumen fumes, but no genotoxic effects could be detected by SCE or MN tests.

Effects of occupational exposure to mercury vapor on lymphocyte micronuclei.

For 26 chloralkali workers exposed to inorganic mercury and 26 age-matched, occupationally unexposed referents, the frequency and size distribution of micronuclei were determined in peripheral lymphocytes stimulated with either phytohemagglutinin or pokeweed mitogen. For the exposed workers the mean concentrations of mercury in urine, plasma, and erythrocytes were 16 nmol/mmol of creatinine, 48 nmol/l, and 78 nmol/l, respectively, and their mean exposure time was 10 years. Neither the frequency nor the size of micronuclei was significantly different in the two groups; nor were there any correlations to current mercury levels. However, in the exposed group, and with phytohemagglutinin as the mitogen, a statistically significant correlation between previous exposure to mercury (cumulative exposure or number of blood mercury peaks) and the frequency of micronuclei was found. This association was also present when the effects of age and smoking were allowed for, and it may indicate an accumulation of cytogenetic effects in T-lymphocytes.