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The boy of 15, named by German newspapers as Marvin K, was found in the same clothes that he went missing in. Neighbours in Recklinghausen, about 60 miles north of Cologne, said they had not seen the teenager but had occasionally heard the screams of a man coming from the flat.
Police were last night holding a year-old unemployed handyman called Lars H on suspicion of sharing images of child abuse. They said there were no signs that Marvin had been held by force.
At the time he went missing he was in local authority care. His mother Manuela, 53, said: 'He held on to me really tightly and he began to tremble.
He said 'Mummy, take me home. I was locked away for two and a half years and I couldn't get any fresh air'. We both cried. He took my hand and wouldn't let go.
Marvin was 13 when he went missing after being taken into a nearby care home after the death of his father. He said goodbye to his carers before leaving to meet friends on June 11, in the town centre.
He sent a final WhatsApp message at Police had been prepared to give up. His discovery seems to have been by accident. At 7am on Friday police visited the home of Lars H with a van and a dog trained to sniff out hidden computer hard drives - suspected of containing child abuse images.
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All other arsenicals tested, including the trivalent compounds sodium arsenite and monomethylarsonous acid, were found to be stable over a minimum two month period.
All arsenicals used in the cytotoxicity, genotoxicity, uptake, and electroporation experiments are listed in Table 1. Trypan blue, Cytochalasin B, May-Grünwald and Giemsa solutions were purchased from Sigma, and the hypoosmotic buffer for electroporation from Eppendorf Germany.
CHO-9 and Hep G2 cells were treated with the arsenicals at different concentrations for 24 h, and all experiments were performed in duplicate. Cell viability was evaluated immediately after exposure.
Treated and untreated cells were harvested by trypsin treatment Sigma. Cell counting was performed following trypan blue staining.
The cell suspension was mixed with an equivalent volume of 0. Cell viability is expressed as percentage of surviving cells compared to the total number of cells.
Significance was tested by using the Student's t -test. Then the arsenic compounds were applied for 24 h at different concentrations 0.
The frequency of MN formation was expressed as percentage of binucleated bn cells with MN. Using the cytokinesis-blocked MN assay, the extent and progression of nuclear division could be assessed by analysing the NDI.
A number of effects, including a decreased optimal pulse voltage, ensure that the plasma membrane can be permeated more easily. The tolerance of CHO-9 cells to hypoosmolar conditions was tested in preliminary experiments.
A suspension of cells 0. The cells were transferred to electroporation cuvettes with a 4 mm gap. Following an incubation period of 24 h recovery time , the CBMN-assay was carried out.
The experiments were set up as follows: a unexposed control, b unexposed control and electroporation, c exposed with arsenicals, and d exposed with arsenicals and electroporation.
All experiments were performed in triplicate. To assess the membrane permeability of the arsenic compounds under normal and forced conditions, CHO-9 cells were incubated with the arsenic compounds at different concentrations 0.
Since the results obtained from experiments with exposure periods of 1 h and 24 h did not differ significantly, all uptake experiments were performed over a period of 1 h.
For comparison of arsenic uptake in different cell lines, 8. Following incubation, cells were washed with PBS and subsequently resuspended in 10 ml fresh culture medium.
The absence of intact cells was confirmed by microscopic examination. Whole-cell and cell-free extract samples were prepared as described above.
Solutions up to 1 in dilutions were delivered at 0. The signals 75 As ms , 77 Cl ms , and In ms were monitored. The chi-square-test was used for the statistical analysis of the results of the micronucleus assay and the two-tailed Student's t -test for evaluating the data from the cytotoxicity test and the NDI.
A concentration-dependency was observed for these species with a maximum uptake at the highest external concentration applied.
Cells were exposed to arsenicals for 1 h. The uptake of the organoarsenic compounds by Hep G2 cells was tested using concentrations from 0.
The difference between the arsenic concentration in whole-cell extract and the cell-free extract was not significant in all cases. The intracellular concentration of arsenic was in most cases lower for Hep G2 cells than in CHO-9 cells.
The cellular uptake of the arsenic species by CHO-9 cells increased after treatment of cells using electroporation.
Uptake of arsenic compounds by CHO-9 cells under forced uptake electroporation condition. Cytotoxicity of pentavalent and trivalent arsenic compounds in CHO-9 cells.
The percentage of decreased cell viability is shown in relation to the untreated control. The cytotoxicity was determined by trypan blue-staining.
Cytotoxicity of pentavalent and trivalent arsenic compounds in Hep G2 cells. All experiments were performed in duplicate. The number of micronucleated cells was not significantly increased when cells were exposed to MMA V for 24 h Fig.
The formation of MN rose significantly at a tested concentration of 7. Micronucleus induction after exposure of CHO-9 cells to different concentrations of arsenite and arsenate for 24 h.
Only incubation of cells with 0. Using longer recovery periods 28 h and 35 h , this delay was not statistically significant anymore data not shown.
These compounds were chosen for electroporation experiments because they were negative in the MN assay under normal exposure conditions Figs.
No significant differences in MN induction were observed between the unexposed control and the unexposed control after electroporation.
MN induction after electroporation of untreated and treated CHO cells. After exposure and electroporation the cells were treated with cytochalasin B for additional 24 h recovery time.
The experiments were repeated four times. We have shown that most of the arsenic species except DMA III and As i III in CHO-9 cells are not bound to cellular membranes, but are able to enter the cytosol no significant difference in arsenic concentration in whole-cell extract and membrane-removed cell extract.
The differences between DMA III as well as As i III concentrations in whole-cell extract and in membrane-removed cell extract may be explained by the presence of a specific exporter for these species.
The apparent lack of As i III export in Hep G2 cells may be explained by considering the high arsenic biotransformation capacity of hepatocytes. Efficient biotransformation of inorganic arsenic to methylarsenic species would remove the need for the extrusion of As i III as a significant resistance mechanism.
Kessel et al. Arsenic can either enhance or reduce nitric oxide NO production, depending on the type of cell, the arsenic species, and the concentration of the tested arsenical.
Suppression of NO production has been shown to reduce arsenite-induced oxidative DNA damage and micronucleus formation Gurr et al. An elevated resistance to As-induced genotoxicity in Hep G2 cells was reported by Gebel et al.
The authors did not find elevated numbers of induced micronuclei in Hep G2 cells after arsenic exposure. A cell-cycle delay in arsenite-treated human leukaemia cells was reported by McCabe et al.
Kligerman et al. In our study, a recovery period was instituted, i. Hence, in contrast to the results of McCabe et al. Taken together, these data indicate that arsenic compounds are able to temporarily delay the cell cycle, but that this delay is reversible.
These results are in agreement with data of previous studies published by Dopp et al. After an extended exposure time of 24 h and a recovery period of 14 h in the present study, the DMA III -induced cell-cycle delay was still detectable.
In conclusion, the presented results reveal that the uptake capabilities of arsenic compounds are dependent upon both the cell type and the arsenic species: We noted a resistance to intracellular accumulation of arsenic, either due to increased resistance at the uptake level or an enhanced efflux mechanism by Hep G2 cells in comparison to CHO-9 cells.
The induced genotoxic effects were directly correlated to the increased intracellular arsenic concentrations. What is sustainability?
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