Primary DNA damage but not mutagenicity correlates with ciprofloxacin concentrations in German hospital wastewaters.

Recently, we showed for the wastewater of a large Swiss university hospital that primary DNA damage, assessed by a bacterial SOS repair assay (umuC test), could be largely assigned to a specific class of antibiotics, the fluoroquinolones (FQs) (Hartmann et al. [1998] Environ Toxicol Chem 17:377-382). In an attempt to confirm the significance of FQs for the bacterial DNA damaging effects in native hospital wastewaters, 25 samples from five German clinics were screened in this study by the umuC test. The results were compared to HPLC-derived concentrations of ciprofloxacin, an important member of the FQs. Ten samples (40%) were umuC-positive and ciprofloxacin concentrations ranged from 0.7 to 124.5 microg/L (n = 24). Primary DNA damage, as indicated by the umuC test, correlated strongly with ciprofloxacin concentrations in a logistic, dose-dependent manner (r2 = 0.896), almost irrespective of the use of S9 metabolic activation. The lowest observed effect concentration (LOEC) for ciprofloxacin was 5.2 microg/L (+S9) and 5.9 microg/L (-S9). Similar to our previous findings, these results indicate that positive umuC results in hospital wastewater are strongly dependent on the presence of fluoroquinolone antibiotics. In a second part of the study, previously generated Ames and V79 chromosomal aberration data of the same samples (Gartiser and Brinker [1995] in Umweltbundesamt Texte 74/95) were compared with the newly generated results. Neither the mutagenic effects detected by the Ames assay (8%, n = 25) nor the positive V79 results (46% n = 13) seemed to be caused by ciprofloxacin. Therefore, the Ames and V79 results suggest the presence of additional mutagens that are yet to be identified.

Correlation between on-line PAH detection in airborne particle samples and their bacterial genotoxicity.

The photoelectric aerosol sensor (PAS) is a technique suitable for on-line monitoring of particle-bound polycyclic aromatic hydrocarbons (PPAHs). Although this is a very fast and inexpensive technique, it does not measure individual PAH species but gives a measure of the total amount of PPAHs. Because of the suitability of this sensor for air-pollution screening, it is desirable to know whether a correlation exists between the PPAHs detected with this method and the biological relevance of the respective particle samples. To test the DNA damaging potential of the organic fraction of collected particles, the umuC test with Salmonella typhimurium TA1535/pSK1002 was used. The primary source for particle sampling was a stationary diesel engine, but samples from a parking garage and two locations in the city of Zürich have also been included. The total mass of PPAHs as determined by the PAS was plotted against the induced genotoxicity. This resulted in a linear correlation (r2 = 0.82), indicating that the PAS detects biologically relevant PPAHs.

A plasmid rescue to investigate mutagenesis in transgenic D. melanogaster.

We present a plasmid rescue from transgenic Drosophila to study spontaneous and mutagen-induced mutations in vivo. Transgenic Drosophila lines were established by transformation with a shuttle vector containing the bacterial lacZ gene as a target for mutagenesis. The target gene can be recovered into bacteria by restriction endonuclease treatment of total genomic DNA, followed by ligation of the recircularized shuttle vectors. The resulting circular plasmids are then transformed back into E. coli lacZ- mutants, where the activity of the lacZ genes is scored on the induction substrate X-Gal. The number of inactivated versus intact lacZ genes directly indicates the mutation frequency. By the described target gene rescue procedure up to 5000 lacZ gene copies can be rescued from one fly routinely. Spontaneous background mutation rates using this system are 2.6 +/- 0.6 x 10(-4). Treatment of larvae with ethylnitrosourea (ENU) resulted in a dose-dependent increase of the mutation frequency to 4.8 +/- 0.6 x 10(-4) for 0.5 mM and 6.9 +/- 1.2 x 10(-4) for 1 mM ENU, respectively.

Detection of genotoxic activity in native hospital waste water by the umuC test.

The genotoxic potential of the waste water of a hospital was evaluated by the umuC test. Within 2 years over 800 native waste water samples were analysed. Genotoxic activity was found in 13% of the samples. The highest genotoxic activity occurred in the morning hours, but genotoxic samples were detected also during the day and at night. 96% of the genotoxic waste water samples revealed a genotoxic potential without growth inhibition of test bacteria monitored as OD600, in the same way as antineoplastic drugs like mitomycin C or cisplatin. 4% of the genotoxic waste water samples showed combined cytotoxic and genotoxic activities as seen in control experiments using glutaraldehyde containing disinfectants and certain antibiotics.

Two different chromatin structures coexist in ribosomal RNA genes throughout the cell cycle.

The structure of ribosomal chromatin in exponentially growing Friend cells, in stationary cells, and in metaphase chromosomes was studied by psoralen photocrosslinking. It is shown that in intact cells, two distinct types of ribosomal chromatin coexist in Friend cells, one that contains nucleosomes and represents the inactive copies and one that lacks a repeating structure and corresponds to the transcribed genes. A single gene copy is either in one or the other chromatin state. The relative amounts of the two types of structures are similar in interphase and metaphase, however, their run-on activities differ significantly. This suggests that the two states of chromatin are maintained independently of the transcriptional process and that they are stably propagated through the cell cycle.

Analysis of the psoralen-crosslinking pattern in chromatin DNA by exonuclease digestion.

When chromatin is photoreacted with psoralen, crosslinks occur preferentially in the linker DNA between nucleosomes. The pattern of these crosslinks can be analysed by exonuclease digestion of random DNA fragments, since the exonucleases tested stop at sites of psoralen-crosslinks. Further digestion of these fragments with S1-nuclease leads to DNA fragments of nucleosomal and polynucleosomal size, which presumably carry psoralen-crosslinks at both ends. This method of analysis of chromatin structure complements the classical micrococcal nuclease digestion analysis, since it can be performed in vitro as well as in vivo, and since it is independent of pH and ionic conditions.

Structural transition in inactive Balbiani ring chromatin of Chironomus during micrococcus nuclease digestion.

We have analysed by micrococcus nuclease digestion the chromatin structure of genes in the Balbiani ring (BR) regions of a Chironomus cell line. Gel electrophoresis of the DNA fragments reveals a repeating structure which consists of two repeat sizes, a long repeat seen in the large fragments and a small repeat seen in the small fragments. The two repeats hardly overlap, except in a narrow transition zone which is at a different fragment size in the BR 2.2 and the BR 2.1 gene. The sizes of the large repeats fit the repeat of the underlying DNA sequence. The short repeats are between 170 and 180 bp, and after H1 depletion the short repeat in the BR 2.2 gene is 160 bp. Our most favoured interpretation of these data is that in intact chromatin the nucleosomes in the BR genes are phased with respect to the repeating DNA sequence, whereas micrococcus nuclease digestion leads to loss of a nucleosome-positioning constraint and hence to rearrangement of the nucleosomes. Our results imply a possible artefact of nuclease digestion of chromatin, which has to be taken into account in mapping nucleosome positions.

Two genes in Balbiani ring 2 with metabolically different 75S transcripts.

Balbiani ring 2 (BR2) in salivary glands of Chironomus pallidivittatus and C. tentans (two sibling species of the subgenus Camptochironomus) is a favoured model system for studies of gene organization and transcript formation. Here we show that BR2 is more complex than hitherto believed, containing two 75S RNA-producing genes, BR2a and BR2b, present in different 35-40 kb blocks of DNA. The transcripts hybridizing to two different repeat units originating in BR2 differ in size. Further support for the presence of two genes comes from RNA studies during experimentally induced BR2 regression. The amounts of BR2a RNA per cell remain more or less constant throughout the course of the experiment, whereas the BR2b RNA decreases considerably. Under normal conditions there is 5-6 times more BR2a RNA than BR2b RNA. This ratio increases 3-fold under experimental conditions. BR2a and BR2b, although partially homologous, contain repeat units with characteristic differences. BR2a contains a repeat unit that is much more similar to a BR1 repeat than it is to the BR2b repeat. The possibility is discussed that a Balbiani ring in general represents an integrated set of active genes rather than a singe gene.

Psoralen-crosslinking of DNA as a probe for the structure of active nucleolar chromatin.

Trimethylpsoralen was used to crosslink the extrachromosomal ribosomal DNA in nucleoli or nuclei of growing Dictyostelium discoideum cells. The DNA was extracted and was examined by spreading under denaturing conditions for electron microscopy. Intact 95,000 base ribosomal DNA molecules were seen, showing regularly spaced, single-stranded bubbles of about 200 to 400 bases in size, interrupted twice by 11,000 base heavily crosslinked stretches, which correspond to the known positions of the coding regions. The bubbles on the nontranscribed regions indicate the presence of nucleosomes during crosslinking. The DNA was digested with restriction enzymes and analysed by gel electrophoresis in parallel with DNA not treated with psoralen. Fragments from the non-coding region had the same mobility as untreated DNA, while those from the coding region had a markedly lower mobility, though not as low as that of crosslinked pure DNA. This shifting of the bands, specific to the coding region, was also seen when whole cells were treated with psoralen. Treatment of nucleoli with 2 m-NaCl (which is known to dissociate histones) before addition of psoralen led to strong crosslinking all along the ribosomal DNA, resulting in a decreased electrophoretic mobility of bands from the non-coding region, but no further retardation of those from the coding region. In differentiating Dictyostelium cells, slugs, where ribosomal RNA synthesis is very much reduced, the extent of psoralen-crosslinking in the coding region was reduced, but not completely to the level of that of the non-transcribed spacer. In order to test whether psoralen itself alters chromatin structure, crosslinked and non-crosslinked nucleoli from growing cells were lysed with heparin and spread for electron microscopy. There was no difference in the appearance or the frequency of the transcription units seen. Digestion of crosslinked nuclei with micrococcal nuclease indicated an undisturbed structure for bulk chromatin, as well as for the chromatin in the non-transcribed spacer of the ribosomal DNA. Thus psoralen-crosslinking does not lead to extensive disruption or distortion of the structure of either inactive or active chromatin. We conclude, taking the results presented in the Appendix into account, that the extent of psoralen-crosslinking in chromatin DNA is diagnostic for the structure of undistorted chromatin.(ABSTRACT TRUNCATED AT 400 WORDS)

Chromatin structure of a hyperactive secretory protein gene (in Balbiani ring 2) of Chironomus.

We examined the chromatin structure of a Balbiani ring (secretory protein gene) in the salivary glands of Chironomus larvae in its hyperactive state after stimulation with pilocarpine. For the inactive state of the gene an established tissue culture cell line, not expressing the gene, was used. Electron microscopy showed an RNA polymerase density of approximately 38/microns. Micrococcal nuclease digestion of purified nuclei followed by DNA transfer and hybridization revealed a smear with no recognizable discrete DNA fragments. Without pilocarpine stimulation a faint nucleosomal repeat was superimposed upon the smear, and in tissue culture cells a clear nucleosomal repeat was revealed. The restriction enzyme XbaI, which has a 6-bp recognition sequence, cut the gene in the hyperactive chromatin state, but not in its inactive conformation. The combined results are best explained by the absence of most of the nucleosomes in this hyperactive RNA polymerase II transcribed gene.