Influence of hOGG1, XRCC1 and XRCC3 genotypes on biomarkers of genotoxicity in workers exposed to cobalt or hard metal dusts.

Identification of genetic polymorphisms responsible for reduced DNA repair capacity may allow better cancer prevention. We examined whether variations in genes involved in base-excision (hOGG1, XRCC1) and double strand break (XRCC3) DNA repair contribute to inter-individual differences in genotoxic effects induced in the lymphocytes of 21 cobalt (Co) exposed, 26 hard metal (WC-Co) exposed and 26 matched control male workers. Genotyping was performed by PCR-RFLP. DNA single strand breaks and alkali-labile sites were measured by the alkaline Comet assay. Chromosomal rearrangements resulting from chromosome loss or acentric fragments were assessed as micronucleated mononucleates (MNMC) and binucleates (MNCB) with the cytokinesis-block micronucleus test. Urinary 8-hydroxydeoxyguanosine (8-OHdG) levels were used as an indicator of systemic oxidative DNA damage. A significantly higher frequency of MNMC was observed in WC-Co exposed workers with variant hOGG1(326) genotype. Multivariate analysis performed with genotypes, age, exposure status, type of plant, smoking and their interaction terms as independent variables indicated that MNMC and Comet tail DNA (TD) were influenced by genetic polymorphisms. In the exposed and total populations, workers variant for both XRCC3 and hOGG1 had elevated MNMC frequencies. Further studies will demonstrate whether genotyping for hOGG1 and XRCC3 polymorphisms is useful for a better individual monitoring of workers.

Update on the genotoxicity and carcinogenicity of cobalt compounds.

OBJECTIVE: To integrate recent understandings of the mechanisms of genotoxicity and carcinogenicity of the different cobalt compounds. METHOD: A narrative review of the studies published since the last IARC assessment in 1991 (genotoxicity, experimental carcinogenesis, and epidemiology). RESULTS: Two different mechanisms of genotoxicity, DNA breakage induced by cobalt metal and especially hard metal particles, and inhibition of DNA repair by cobalt (II) ions contribute to the carcinogenic potential of cobalt compounds. There is evidence that soluble cobalt (II) cations exert a genotoxic and carcinogenic activity in vitro and in vivo in experimental systems but evidence in humans is lacking. Experimental data indicate some evidence of a genotoxic potential for cobalt metal in vitro in human lymphocytes but there is no evidence available of a carcinogenic potential. There is evidence that hard metal particles exert a genotoxic and carcinogenic activity in vitro and in human studies, respectively. There is insufficient information for cobalt oxides and other compounds. CONCLUSION: Although many areas of uncertainty remain, an assessment of the carcinogenicity of cobalt and its compounds requires a clear distinction between the different compounds of the element and needs to take into account the different mechanisms involved.

Absence of significant genotoxicity in lymphocytes and urine from workers exposed to moderate levels of cobalt-containing dust: a cross-sectional study.

Mortality studies have shown that, in the past, lung cancer occurred after exposure to mixtures of cobalt metal and metallic carbide particles, the main constituents of hard metals, but apparently not when exposure was to cobalt alone. The major objective of this biomonitoring study was to assess genotoxic effects as a measure for carcinogenic risk in workers from cobalt refineries and hard metal plants currently exposed to the threshold limit value/time-weighted average (TLV-TWA) for cobalt-containing dust. The study comprised three groups of workers: 35 workers exposed to cobalt dust from three refineries, 29 workers exposed to hard metal dust from two producing plants, and 35 matched control subjects recruited from the respective plants. The study design integrated complementary methodologies to assess biomarkers of effects that represent both initial DNA damage (8-hydroxydeoxyguanosine [8-OHdG] in urine and comet assay on lymphocytes) and definitive chromosome breakage/loss (micronuclei in lymphocytes). Cobalt and cotinine were determined in urine as a measure for cobalt exposure and recent smoking, respectively. No significant increase of genotoxic effects was detected in workers exposed to cobalt-containing dust as compared to controls. No difference in any genotoxicity biomarker was found between workers exposed to cobalt and hard metal dusts. Multiple regression analysis indicated that workers who smoked and were exposed to hard metal dusts had elevated 8-OHdG and micronuclei values. Because this observation is in line with a previous epidemiological study of an increased risk of dying from lung cancer in workers from the hard metal industry who smoked, it is concluded that this specific occupational group needs closer medical surveillance.

Validation and implementation of an internal standard in comet assay analysis.

The comet assay is widely used to detect DNA damage in single cells. However, only moderate attention has been paid to the experimental variability of this assay, especially during electrophoresis. To take into account this variation and to be able to compare measurements from different electrophoretic runs, as would be necessary when large numbers of samples need to be analysed, it is important to integrate an internal standard into the assay. This study presents a first step in the validation and implementation of an internal standard in the alkaline comet assay. Untreated and ethyl methanesulfonate treated cells (K562 human erythroleukemia cell line) were used as negative and positive internal standards, respectively, in each electrophoresis run. Three steps were followed: (1) assessment of the different levels of variability which may influence the damage levels of the internal standards, (2) evaluation of the variability across separate electrophoresis runs on the quantification of DNA damage in the internal standards by three experimenters involved in different studies and (3) proposal of an adequate calculation system to integrate the internal standards into test sample data. The application of the two proposed models to samples from a human biomonitoring study is presented. The model which calibrates the measurements against the negative internal standard is the most useful since this negative standard was the most stable across experiments and among the three experimenters. The percentage of DNA in the tail is the most appropriate parameter to analyse induced DNA damage, because its interelectrophoresis and interexperimenter variation is less pronounced than that of tail length.

Evaluation of the in vitro direct and indirect genotoxic effects of cobalt compounds using the alkaline comet assay. Influence of interdonor and interexperimental variability.

The mechanisms of cobalt-induced pulmonary interstitial fibrosis and cancer are incompletely understood. DNA damage, either induced by genotoxic (direct or via oxygen radicals) or co-genotoxic (e.g. inhibition of DNA repair) processes may play an important role in the initiation of cancer. The alkaline comet assay provides a sensitive tool to investigate these two processes. Cobalt metal, a mixture of cobalt with tungsten carbide and cobalt chloride, were compared for their DNA-damaging capacity. Concentrations from 0 to 6.0 microg Co-equivalent/ml were tested. All three compounds were able to induce DNA damage in isolated human lymphocytes from three donors, in a dose- and time-dependent way. A relatively large interexperimental and interdonor variability in response was observed. This was ascribed to technical parameters and unidentified individual factors. This confirms the importance of repeating experiments using the same and different donors. The DNA-damaging potential of the cobalt-tungsten carbide mixture was higher than that of cobalt metal and cobalt chloride, which had comparable responses. No significant increase of DNA migration was observed when the DNA of cells treated with cobalt metal, cobalt-tungsten carbide or tungsten carbide were incubated with the oxidative lesion-specific enzyme formamidopyrimidine DNA glycosylase. This suggests that during the short treatment period no substantial oxidative damage to DNA was produced. Cobalt metal was able to inhibit the repair of methylmethanesulphonate-induced DNA damage. This was concluded from simultaneous exposure to cobalt and methyl methanesulphonate, post-incubation and post-treatment with 1.2 microg/ml cobalt of methyl methanesulphonate-treated cells.

Nereis virens (Annelida: Polychaeta) is not an adequate sentinel species to assess the genotoxic risk (comet assay) of PAH exposure to the environment.

Polychaetes, because of their bioturbation capacity, play an important role in the distribution of anthropogenic contaminants (including polycyclic aromatic hydrocarbons [PAHs]) throughout the sediments. In this work the use of Nereis virens (Annelida: Polychaeta) as a bioindicator to assess the genotoxic risk of PAH exposure for the environment was evaluated. For this purpose the alkaline single cell gel electrophoresis [comet] assay was applied on the coelomocytes of in vivo exposed Nereis virens. Benzo[a]pyrene (B[a]P) was chosen because it is classified by the IARC (International Agency for Research on Cancer) as "probably carcinogenic to humans" and because its mechanisms of action are well-known. Nereis virens was exposed to B[a]P in concentrations of 0.3, 0.6, 10, 20, 35 and 45 mg/ml by an intracoelomic injection of B[a]P (20 microliters) dissolved in dimethyl sulphoxide (DMSO). A solvent control with DMSO, a positive control with ethyl methane sulphonate (EMS) (12.1 mg/ml) and a negative control were included in each experiment. For each treatment four animals were analysed. After 1 hr treatment coelomocytes were harvested by puncturing the coelomic cavity with a sharpened Pasteur pipette, mixed with 0.5% low melting point agarose and sandwiched between two other gel layers. Ethidium bromide stained nuclei were analysed for tail length and tail moment. 12.1 mg/ml EMS, pure DMSO (98.9%) and B[a]P in all tested concentrations induced a statistically significant increase of DNA single strand breaks in the comet assay. The effect of B[a]P, however, was only at the highest concentration (45 mg/ml) significantly stronger than the effect of DMSO alone. Although a relatively large heterogeneity in the results could be observed, these experiments clearly showed that Nereis virens is not suited as a sentinel species for the assessment of the genotoxic risk of PAH exposure because this species seems to be very resistant to benzo[a]pyrene.

Is "Campylobacter upsaliensis" an unrecognised cause of human diarrhoea?

For 3 years a filtration system for the isolation of "new" campylobacter was included in the culture protocol of 15,185 stool specimens. "C upsaliensis" was isolated in 99 patients, C jejuni subsp doylei in 4, and C hyointestinalis in 2. "C upsaliensis" was the only organism isolated in 83 patients. Clinical information was available for 77 out of these 83 patients. 92% of the patients had diarrhoea; vomiting and fever were rare (14% and 7%, respectively); the onset was mostly sudden; and the symptoms usually lasted for less than a week. Gross or occult blood was present in a quarter of cases and neutrophils were detected in faecal smears in about a fifth. "C upsaliensis" may be an unrecognised and frequent cause of diarrhoea in man, and selective isolation media should be combined with non-selective isolation systems.

Modified selective medium for isolation of Campylobacter spp. from feces: comparison with Preston medium, a blood-free medium, and a filtration system.

Our previously described (H. Goossens, M. De Boeck, and J. P. Butzler, Eur. J. Clin. Microbiol. 2:389-393, 1983) selective medium, consisting of cefoperazone (15 mg/liter), rifampin (10 mg/liter), colistin (10,000 IU/liter), and amphotericin B (2 mg/liter) (medium M1), for the isolation of Campylobacter jejuni and Campylobacter coli from stool specimens was modified as follows: cefoperazone (30 mg/liter), rifampin (10 mg/liter), and amphotericin B (2 mg/liter) (medium M2). A comparative study of the isolation of Campylobacter spp. from stool specimens was carried out with medium M1; medium M2; a selective blood-free medium consisting (per liter) of charcoal (4 g), ferrous sulfate (0.25 g), sodium pyruvate (0.25 g), casein hydrolysate (3 g), sodium deoxycholate (1 g), nutrient broth no. 2 (25 g), agar (12 g), and cefoperazone (32 mg) (medium M3); and Preston medium containing (per liter) trimethoprim (10 mg), rifampin (10 mg), polymyxin B (5,000 IU), and cycloheximide (100 mg) (medium M4). We also included a filtration system in which membrane filters were applied directly to the surface of the nonselective blood-free medium distributed in small petri dishes. A total of 5,276 stool specimens were tested: 2,788 stool specimens were tested on M1 and M3 in study 1; 2,488 stool specimens were inoculated on the four selective media in study 2, and the last 986 specimens of the 2,488 were tested in parallel with the filtration system. In study 2, 128 Campylobacter strains were isolated from 126 different patients; 85.0, 88.3, 82.5, and 66.6% of these strains were isolated on M1, M2, M3, and M4, respectively. No contaminating fecal flora was found on 65.4, 70.7, 62.4, and 40.3% of the M1, M2, M3, and M4 plates, respectively. Furthermore, C. coli was found to be more susceptible to antibiotics present in the selective media, particularly colistin and polymyxin B, than was C. jejuni. We therefore recommend M2 for the isolation of Campylobacter spp. Finally, the filtration method was found to be easy and cheap; although the sensitivity was low, this method allowed the isolation of new Campylobacter spp. which seem to be associated with diarrhea.

Semisolid selective-motility enrichment medium for isolation of salmonellae from fecal specimens.

A semisolid selective-motility enrichment medium for the isolation of salmonellae from fecal specimens was developed which was based on Rappaport enrichment broth. During a 7-year period more than 30,000 stool samples were tested. The medium showed a high specificity (95.1%) and sensitivity (80.3%) when compared with MacConkey agar, SS agar, and brilliant green agar (after Selenite-F Enrichment [BBL Microbiology Systems]). Furthermore, our isolation rate of Salmonella species from fecal samples showed an increase of 22.3% when this semisolid medium was added to the routine culture media. Growth could easily be interpreted. The medium has a bias toward the isolation of Salmonella paratyphi B, but it is unsatisfactory for detecting the nonmotile strains Salmonella typhi and S. paratyphi A.

Increased prostate-type acid phosphatase activity in serum and typical bone lesions simulating the presence of prostatic carcinoma.

In a man with myelomonocytic leukemia, the association of increased prostatic acid phosphatase activity in serum and the presence of typical bone lesions on roentgenography suggested the existence of disseminated prostatic carcinoma. During the clinical observation period, however, prostatic involvement could not be proved. Moreover, bone pain and prostatic-type acid phosphatase activity in serum closely paralleled monocyte counts and the degree of hepatosplenomegaly and leukemic skin lesions. Finally, meticulous postmortem examination of the prostate showed no prostatic carcinoma. This clinical picture appears to be entirely explicable in terms of leukemic organ infiltration and the proliferation of monocytes, which are known to contain acid phosphatase isoenzymes like those in the prostate.

The accessory costotransverse foramen: a radioanatomical study.

One hundred fifty-six anatomical specimens of cervical vertebrae and 55 C1 and 53 C2 vertebrae were examined for the presence of an accessory costotransverse foramen. We also reviewed 60 cervical spine computed tomographic (CT) scans. The variations of the costotransverse and accessory foramen are discussed. The frequency of the latter is 19% in the anatomical specimens and 45% in CT scans. The local anatomy and the excellent visualization on axial transverse CT are stressed.

An echo-free silicone elastomer block for ultrasonography.

The authors use a silicone elastomer block, rather than less-convenient water baths, to reduce echo reverberations during ultrasound examinations of superficial anatomic structures.

Monocytic leukaemia associated with myeloid metaplasia, resembling metastatic bone disease.

A case of lethal, subacute monocytic leukaemia is described in which the development of multiple sclerotic bone lesions, resembling metastases, was due to secondary myeloid metaplasia. The spectrum of leukaemic involvement of the skeleton is discussed with emphasis on sclerotic bone lesions. The differential diagnosis of other focal areas of bone sclerosis is considered.

A new selective medium for the isolation of Campylobacter jejuni from human faeces.

A new selective medium, Butzler's medium Virion, for the isolation of Campylobacter jejuni from human faeces is described. This medium contains the following antibiotics per liter: cefoperazone 15 mg, rifampicin 10 mg, colistin 10,000 IU, and amphotericin B 2 mg. At 42 degrees C there was no difference in the isolation rate on Butzler's medium Virion and Butzler's medium Oxoid, but the competing faecal flora was best suppressed by the new medium which allows easier reading of plates and better recognition of campylobacter colonies. Also, the isolation rate and growth of faecal flora were comparable when this new selective medium was incubated at both 37 degrees C and 42 degrees C in a candle jar or a special incubator. These results suggest that Butzler's medium Virion is a very reliable medium for the isolation of campylobacter. In comparison with other selective media it has important advantages for investigators who have little experience in isolating this enteropathogen, or for developing countries where special 42 degrees C incubators are often not available.