Analysis of Bordetella pertussis populations in European countries with different vaccination policies.

Despite the widespread use of pertussis vaccines during the last decades, pertussis has remained an endemic disease with frequent epidemic outbreaks. Currently two types of vaccines are used: whole-cell vaccines (WCVs) and recently developed acellular vaccines (ACVs). The long-term aim of our studies is to assess the effect of different vaccination policies on the population structure of Bordetella pertussis and ultimately on the disease burden in Europe. In the present study, a total of 102 B. pertussis isolates from the period 1998 to 2001 from five European countries (Finland, Sweden, Germany, The Netherlands, and France) were characterized. The isolates were analyzed by typing based on variable number of tandem repeats (VNTR); by sequencing of polymorphic genes encoding the surface proteins pertussis toxin S1 and S3 subunits (ptxA and ptxC), pertactin (prn), and tracheal colonization factor (tcfA); and by fimbrial serotyping. The results reveal a relationship between geographic location and VNTR types, the frequency of the ptxC alleles, and serotypes. We have not observed a relationship between the strain characteristics we studied and vaccination programs. Our results provide a baseline which can be used to reveal changes in the B. pertussis population in Europe in the coming years.

Biomarkers of genetic damage and inflammation in blood and bronchoalveolar lavage fluid among former German uranium miners: a pilot study.

Former East German uranium miners who are known to have been exposed to radon are estimated to be at high risk for lung carcinogenesis. Among these miners over 200 occupationally caused lung cancer cases are expected to occur each year, resulting in a total of 7,000-24,000 excess lung cancer cases in the coming years. It is still unknown whether there is a correlation between biomarkers and the exposure of the uranium miners to ionizing radiation that might enable us to trace those miners with high lung cancer risk. The primary aim of this pilot study was to test the possibility of performing a biomarker study in this unique cohort of former uranium miners in spite of several limitations that had to be taken into consideration when comparing them with healthy controls, such as old age, age-dependent diseases and potential confounding artefacts from dissimilar smoking patterns. The second aim was to test a range of biomarkers for DNA damage and inflammation in leukocytes and bronchoalveolar fluid for their ability to detect biological effects. In this cohort of miners we found an increased frequency of chromosomal aberrations in blood lymphocytes and an increased prevalence of both fibronectin and tumour necrosis factor alpha in the bronchoalveolar fluid.

p53 mutations and codon 213 polymorphism of p53 in lung cancers of former uranium miners.

PURPOSE: There is a high prevalence of G-->T transversions of p53 in lung cancers of smokers. One study has reported a special "hotspot" mutation at codon 249 of p53 in lung cancers of former uranium miners. The aim of our study was to look for mutational spectra of p53 in former German uranium miners with lung cancers. METHODS: We investigated 16 patients with lung cancer who had worked as uranium miners in Germany and 13 lung cancer patients without a mining history of the same region. By means of the polymerase chain reaction and sequencing we looked for mutations in exons 5 7 of the p53 gene. RESULTS: We could not find any suggestion of hotspot mutations. The only G-->T mutation in former uranium miners was detected in the only nonsmoker. In 3 patients (19% of the total) we found a codon 213/3 polymorphism. CONCLUSIONS: The results indicate that G-->T transversions do not seem to be very common mutations in p53 in lung cancers probably caused by radiation. Therefore, p53 may be mutated early in lung cancer development if radiation exposure is a critical factor in carcinogenesis. In accordance with studies of thyroid cancer patients in the Chernobyl region, our results may indicate an overrepresentation of codon 213/3 polymorphism in p53 in radiation-caused cancers.

Elevated DNA single-strand breakage frequencies in lymphocytes of welders exposed to chromium and nickel.

DNA damage (alkaline filter elution) and sister chromatid exchange (SCE) frequencies were measured in lymphocytes of 39 welders and 39 controls. The welders showed a significantly higher rate of DNA single-strand breakages and significantly elevated SCE values. These results are not in accordance with those of a former study in which only DNA-protein cross-links were measured. The different results may be explained on the basis of different exposure levels for chromium(VI) and nickel. Both methods are not specific but sensitive enough to measure genotoxic damage after occupational exposure to chromium(VI) and nickel in the range of threshold values for the workplace on a collective basis. Additionally, the results indicate that DNA single-strand breakage and DNA-protein cross-links show different increases depending on the exposure levels for chromium and nickel.

DNA single strand breakage, DNA adducts, and sister chromatid exchange in lymphocytes and phenanthrene and pyrene metabolites in urine of coke oven workers.

OBJECTIVES: To investigate the specificity of biological monitoring variables (excretion of phenanthrene and pyrene metabolites in urine) and the usefulness of some biomarkers of effect (alkaline filter elution, 32P postlabelling assay, measurement of sister chromatid exchange) in workers exposed to polycyclic aromatic hydrocarbons (PAHs). METHODS: 29 coke oven workers and a standardised control group were investigated for frequencies of DNA single strand breakage, DNA protein cross links (alkaline filter elution assay), sister chromatid exchange, and DNA adducts (32P postlabelling assay) in lymphocytes. Phenanthrene and pyrene metabolites were measured in 24 hour urine samples. 19 different PAHs (including benzo(a)pyrene, pyrene, and phenanthrene) were measured at the workplace by personal air monitoring. The GSTT1 activity in erythrocytes and lymphocyte subpopulations in blood was also measured. RESULTS: Concentrations of phenanthrene, pyrene, and benzo(a)pyrene in air correlated well with the concentration of total PAHs in air; they could be used for comparisons of different workplaces if the emission compositions were known. The measurement of phenanthrene metabolites in urine proved to be a better biological monitoring variable than the measurement of 1-hydroxypyrene. Significantly more DNA strand breaks in lymphocytes of coke oven workers were found (alkaline filter elution assay); the DNA adduct rate was not significantly increased in workers, but correlated with exposure to PAHs in a semiquantitative manner. The number of sister chromatid exchanges was lower in coke oven workers but this was not significant; thus counting sister chromatid exchanges was not a good variable for biomonitoring of coke oven workers. Also, indications for immunotoxic influences (changes in lymphocyte subpopulations) were found. CONCLUSIONS: The measurement of phenanthrene metabolites in urine seems to be a better biological monitoring variable for exposure to PAHs than measurement of hydroxypyrene. The alkaline filter elution assay proved to be the most sensitive biomarker for genotoxic damage, whereas the postlabelling assay was the only one with some specificity for DNA alterations caused by known compounds.

32P-postlabeling analysis of DNA adducts in different populations.

Blood samples were obtained from different populations exposed occupationally or by lifestyle habits to polycyclic aromatic hydrocarbons (PAH). DNA adducts were determined by 32P-postlabeling assay either in white blood cells (WBC) or lymphocytes. The level of DNA adducts ranged from 1.5 per 10(9) nucleotides in one of the control groups up to 7.1 per 10(9) nucleotides in one group of PAH-exposed workers. Comparison of the adduct thin layer chromatography (TLC) profiles revealed individual variation in both pattern and level of DNA adducts. Significant differences of adduct levels were detected between one group of PAH-exposed coke-oven workers and the corresponding control group. Only a weak influence of the smoking habits on the amount of adducts was detectable in occupationally exposed or unexposed individuals.

Sister chromatid exchange frequencies in lymphocytes of oral cancer patients seem to be influenced by drinking habits.

Sister chromatid exchange (SCE) values were determined in the lymphocytes of 24 oral cancer patients before therapy and in the lymphocytes of 24 control persons standardized with respect to sex, age and smoking habits. Oral cancer patients showed significantly elevated SCE values (mean 7.82 versus 6.42). In both groups the highest SCE values were found in the subgroups with the highest alcohol consumption. A significant correlation between SCE and gamma-glutamyltranspeptidase (GGT) values by Spearman correlation analysis was detected in the combined group (cancer patients and control persons) (n = 32, r = 0.40, P = 0.023). The SCE values in the oral cancer patients were weakly correlated (Pearson) to DNA adduct levels (n = 22, r = 0.39, P = 0.068) and DNA single-strand breakage frequencies (n = 12, r = 0.56, P = 0.054) in lymphocytes. The correlation (Pearson) between SCE values and DNA strand breakage values in lymphocytes was significant (n = 10, r = 0.67, P = 0.036) in smoking cancer patients. The increase of SCE values with respect to alcohol drinking habits underlines epidemiologic findings that alcohol is an important co-carcinogen in many cancers, especially in oral cancers. Because of the influences on SCE and adduct levels in lymphocytes, alcohol drinking habits should be controlled as broadly as possible in biomarker studies.

DNA-protein cross-links and sister chromatid exchange frequencies in lymphocytes and hydroxyethyl mercapturic acid in urine of ethylene oxide-exposed hospital workers.

The lymphocytes of 25 hospital workers exposed to ethylene oxide and of a standardized control group were investigated for DNA damage (measured by alkaline filter elution) and sister chromatid exchange (SCE) frequencies. Additionally, the excretion of hydroxyethyl mercapturic acid (HEMA) in the 24-h urine of ten workers and ten control persons was determined. The peak levels of ethylene oxide in air during the first 8 min after opening of the sterilization unit were measured. Peak levels of ethylene oxide in the air of up to 417 ppm after opening of the sterilization unit were detected. In the alkaline filter elution assay we found significantly reduced elution rates in the exposed workers, indicating DNA-protein cross-links. The reduction of the elution rates through HVLP filters correlated significantly with the exposure classification (low, medium, high) (r = -0.45, P < 0.05) and the ethylene oxide peak level after opening of the sterilization unit (r = -0.42, P < 0.05). The SCE frequencies in the standardized control group were significantly elevated. With respect to (n = 78) historic control SCE values of our institute, the SCE values of the disinfectors were not significantly elevated (6.54 vs 6.27). The ethylene oxide-exposed workers did not have a greater percentage of high-frequency SCE cells. The mean HEMA concentration in the urine of the exposed workers was significantly elevated, but there were wide variations in HEMA concentrations and no correlation to ethylene oxide exposure. We conclude that the alkaline filter elution assay may be a sensitive parameter for ethylene oxide-exposed workers.(ABSTRACT TRUNCATED AT 250 WORDS)

Genotoxic and cytotoxic effects of 4-aroyl-1-nitrosohydrazine-carboxamides on O6-alkylguanine-DNA alkyltransferase-positive and -negative human cell lines.

Five different representatives (I-V) of a new class of bifunctional alkylating agents, the 4-aroyl-1-nitrosohydrazinecarboxamides ("nitrososemicarbazides"), were evaluated for their potential interaction with DNA and for their cytotoxic activity in vitro to O6-alkylguanine-DNA alkyltransferase-positive (Mer+) and -negative (Mer-) human cell lines. The HeLa MR cell line (Mer-) showed up to 20-fold higher sensitivity at IC50 (dose that inhibits colony formation by 50%) to agents I-V than did the HeLa S3 cell line (Mer+) in a colony-formation assay. These data were compared to those obtained by treatment of the two cell lines with carmustine, a currently used antitumor drug. In Mer+ cells comparable results to those with carmustine were obtained with compounds III, IV and V; in Mer- cells compounds I and II showed nearly the same effects as carmustine. Whether compounds I-V produce DNA strand breaks and/or DNA-protein cross-links was investigated using an alkaline filter elution technique. In this assay all compounds produced DNA single-strand breaks; no correlation could be detected between the strand breakage frequency and cytostatic, mutagenic and antitumor activity.

DNA strand breakage and DNA adducts in lymphocytes of oral cancer patients.

In lymphocytes of 12 oral cancer patients (and two control groups) the frequencies of DNA single-strand breaks and DNA-protein cross-linking were determined by alkaline filter elution. We found elevated DNA elution rates, which must be interpreted as an increased strand breakage frequency. There were significant correlations between the DNA strand breakage frequency and smoking habits. Using the 32P-postlabelling assay we determined the DNA adduct level in lymphocytes of 23 oral cancer patients (and two control groups). No significant influence of smoking habit on the DNA adduct level could be detected. There was a significant correlation between the DNA adduct level and the gamma-glutamyltranspeptidase (GGT) value, suggesting systemic influences of alcohol drinking habits on the adduct level.

Mitochondrial DNA of Chlamydomonas reinhardtii: the structure of the ends of the linear 15.8-kb genome suggests mechanisms for DNA replication.

The mitochondrial genome of Chlamydomonas reinhardtii is a linear double-stranded DNA of 15.8 kb. With the exception of the termini its DNA sequence has been published. Here we describe the unique structure of the two termini determined from cloned fragments or, for the very terminal sequences, by the Maxam and Gilbert method after 5' labeling of uncloned terminal fragments. The 15.8-kb DNA is characterized by terminal inverted repeats of 531 or 532 bp in length including long 3' extensions. The 3' single-stranded extensions of the left and right ends are non-complementary, identical in sequence, and comprise 39 to 41 nucleotides. Remarkably, the linear genome possesses in addition an internal 86-bp repeat of the two outermost sequences. The unusual structure of the 15.8-kb DNA termini is compared with those of other linear mitochondrial DNAs. Possible mechanisms of 15.8-kb DNA replication are discussed.

[Polychlorinated biphenyls (PCBs) and reproduction disturbances]. / Polychlorierte Biphenyle (PCBs) und Reproduktionsstörungen.

A review of the impact of chlorinated biphenyls (PCBs) on reproduction is presented. PCBs are able to pass the placenta; accumulation in offspring is mainly achieved by breast feeding. Disturbances of fertility and embryotoxicity were detected in animals; teratogenicity without maternal toxicity was only observed in mice. In animal experiments disturbances of fetal and postnatal development were detected as well as in human studies. Neurotoxicity was observed even at low-level exposures in monkeys, which are most comparable to humans because of the development of the same clinical symptoms. Technical PCBs which are dominating in occupational and environmental exposure until now must be judged as toxic for reproduction: dermal exposure (especially in accidents) should be considered as an important route of exposure beside inhalation. Work to list possible PCB exposure risks and to develop ways to reduce these risks are necessary now.

Investigations of the frequency of DNA strand breakage and cross-linking and of sister chromatid exchange frequency in the lymphocytes of patients with multiple myeloma undergoing cytostatic therapy with melphalan and prednisone.

An investigation was made of 17 patients with multiple myeloma using the method of alkaline filter elution for the detection of DNA damage and the determination of sister chromatid exchange (SCE) frequency in peripheral lymphocytes during a course of chemotherapy with melphalan and prednisone. We were able to detect elevated SCE frequencies in pretreated patients that approximately doubled during the therapeutic cycle. An appreciable level of DNA cross-linking was detected by alkaline filter elution; DNA cross-linking scarcely increased during a course of chemotherapy. The increase in the SCE frequency during the first therapy cycle was even greater in the case of patients with newly diagnosed multiple myelomas. The results obtained by alkaline filter elution and measuring SCE frequencies suggest that these techniques are suitable as methods in molecular epidemiology, especially if applied to persons who are chronically exposed to cytostatic drugs. Whether or not the methods could be valuable in evaluating therapy response needs further investigation.

[Experiences with alkaline filter elution for the detection of DNA damage by genotoxic compounds]. / Erfahrungen mit der Alkalischen Filterelution beim Nachweis von DNA-Schäden durch gentoxische Verbindungen.

In biological effect monitoring the alkaline filter elution is a suitable method to detect DNA strand breakage and cross-linking in peripheral blood lymphocytes. We applied this method to three groups of workers occupationally exposed to carcinogens: n = 39 welders with exposure to chromium and nickel, n = 20 female shoemakers with exposure to benzene and n = 25 disinfectors in hospitals with exposure to ethylene oxide. In comparison to standardised control groups our results must be interpreted as indicating an increased rate of DNA cross-linking in welders and disinfectors whereas the female shoemakers showed an increased rate of DNA strand breakage. The differences between the results of the exposed groups and the stratified control groups were significant in most cases. We could reexamine 6 shoemakers 4 months after cessation of exposure to benzene; at this moment the results were indicating a clear decrease of the DNA strand breakage. Therefore the method of alkaline filter elution seems to be a valuable tool in biological effect monitoring of groups occupationally or environmentally exposed to carcinogens, especially if the exposure is low, but persistent over a long period.

Incidence of bladder cancer in a cohort of workers exposed to 4-chloro-o-toluidine while synthesising chlordimeform.

Between 1982 and 1990 seven cases of bladder cancer were detected in a group of 49 workers who were synthesising chlordimeform from 4-chloro-o-toluidine. Latency periods ranged from 15 to 23 years. The incidence of bladder tumours in this group was significantly higher than that of the cancer registers of the former GDR, Saarland, and Denmark by factors of 89.7, 53.8, and 35.0 respectively. This provides further evidence that monocyclic aromatic amines such as 4-chloro-o-toluidine may be carcinogenic in humans.

Investigations of the frequency of DNA strand breakage and cross-linking and of sister chromatid exchange frequency in the lymphocytes of female workers exposed to benzene and toluene.

Peripheral lymphocyte DNA damage as measured by the method of alkaline filter elution and the frequency of sister chromatid exchange (SCE) in lymphocytes was investigated for a group of 20 female workers of a shoemaking plant who were exposed to benzene and toluene, primarily below the German threshold limit value of 5 and 100 p.p.m. respectively; the results were compared with those from a control group. In the female workers significantly raised (P less than 0.05) SCE values were found. The relative DNA elution rate through polycarbonate filters was significantly increased (P less than 0.001). The elution rate through polyvinylidene fluoride (HVLP) filters showed a tendency to increase (P = 0.052), which must be interpreted as indicating increased DNA strand breakage. The SCE rates of the female workers were significantly correlated (P less than 0.01) with the relative DNA elution rate through HVLP filters. There was no correlation with the actual benzene and toluene uptake measured by personal air monitoring. Four months after cessation of work, DNA strand breakage decreased significantly (P less than 0.05) in blood samples of six reinvestigated female workers.

Concentrations of tetrachloroethene in blood and trichloroacetic acid in urine in workers and neighbours of dry-cleaning shops.

Tetrachloroethene concentrations in blood and trichloroacetic acid concentrations in urine were determined--primarily over the course of a week--for 29 persons living in the vicinity of dry-cleaning shops. The mean levels of tetrachloroethene increased during the week. In some neighbours concentrations were exceeding the German biological threshold limit value for tetrachloroethene (1000 micrograms/l blood), persisting over the whole week in one case. The concentrations of tetrachloroethene in blood depended on the floor and the construction type of the building where these people were living, but not of the type of system used in the dry-cleaning shops. 5 of 12 dry-cleaners were found to have tetrachloroethene levels exceeding the German biological threshold limit value, some of them by a considerable amount.

Investigations of the frequency of DNA strand breakage and cross-linking and of sister chromatid exchange in the lymphocytes of electric welders exposed to chromium- and nickel-containing fumes.

A total of 39 electric welders exposed to chromium and nickel were compared with 18 controls standardized for age, smoking habits and sex with respect to the frequency of sister chromatid exchange (SCE) and of DNA strand breakage and cross-linking (measured by the method of alkaline filter elution) in their blood lymphocytes. A significant correlation was found between the frequency of SCE and of individual DNA strand breakage and the concentration of chromium in the urine. Less DNA from the welders than from the control group was eluted through the two filter types used (polycarbonate and polyvinylidene fluoride filters). This must be interpreted as resulting from the presence of DNA-protein cross-links, which has the secondary effect of leading to a relative reduction in the measurable frequency of strand breakage amongst the welders. The present results are in good agreement with in vitro and in vivo investigations that confirm the importance of DNA-protein cross-links for the carcinogenic effect of chromium.