Barry Elliott

The genetic toxicity of time : Importance of DNA-unwinding time to the outcome of single-cell gel electrophoresis assays

Single-cell gel electrophoresis assays (comet assays) are described in which DNA damage is assessed in mouse skin keratinocytes treated with N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and beta-propiolactone (BPL) either in vitro or in vivo. The positive results observed under both conditions of test encourage the further development of the mouse skin comet assay as a screen for direct-acting in vivo genotoxins. From the outset of the present experiments we were struck by the compacted nature of the DNA in mouse skin keratinocytes. Under similar conditions of assay, rodent hepatocytes presented a uniform unwound distribution of DNA over the whole nuclear region. In order to study this effect we varied what seemed to be the most obviously related assay parameter: the DNA-unwinding time. A series of experiments was conducted in which control and MNNG-treated cells were exposed to a range of alkaline DNA-unwinding times (0.3-18 h) followed by measurement of the three comet tail parameters (length, DNA content, and their product, tail moment). Each of these parameters increased with increasing time of unwinding such that the tails observed for MNNG-treated cells with 0.3 h of DNA unwinding were similar in length to the tails of control cells exposed to an 8 h DNA-unwinding time. It is concluded that DNA-unwinding time is a critical parameter of the comet assay and that it may require optimisation for each tissue/cell type studied. Further, the data alert to the prospect that agents that uniquely affect chromosomal protein superstructure may increase comet tail length/DNA content in the absence of chemically induced DNA damage. Thus, there may be two discrete classes of chemical interaction with chromosomal DNA that yield identical comet assay results, but which have different implications for the genetic toxicity of the test agent. Similar effects were observed for rat hepatocytes or mouse lymphoma cells exposed to an 18 h DNA-unwinding time, but no comet tails were produced by exposure of cells to the lysis conditions (pH 10.0) for 18 h.

Assessment of developmental effects, cytotoxicity and genotoxicity in the marine polychaete (Platynereis dumerilii) exposed to disinfected municipal sewage effluent.

While sodium hypochlorite is widely used as a disinfectant for municipal sewage effluents and power station cooling waters discharged into coastal environments, there is limited information on the potential in vivo genotoxicity of such disinfection procedures to marine organisms. Using a recently developed test system based on the marine polychaete Platynereis dumerilii, we have evaluated impacts based on embryo-larval development, cytotoxicity and genotoxicity following exposure to disinfected settled (primary) effluent from a municipal sewage treatment works (STW). Sewage samples were collected from Newton Abbot STW, Devon, UK and then disinfected with sodium hypochlorite based on standard operational procedures. Exposure of polychaetes to dilutions of disinfected sewage in seawater (20 +/- 1 degree C) led to a marked reduction in normal embryo-larval development (7 h EC50 from 0.57-1.88% (v/v), n = 4), with a simultaneous increase in cytotoxicity. Following the calculation of the Maximum Tolerated Dose (MTD), based on developmental and cytotoxic effects, the organisms were also analysed for the induction of chromosomal aberrations. This investigation demonstrated the absence of genotoxicity in polychaetes exposed in vivo to sewage disinfected with sodium hypochlorite. These observations extend our previously published studies in which polychaetes exposed to non-disinfected sewage, while showing developmental toxicity and cytotoxicity, did not exhibit any evidence of cytogenetic damage.

Review of the genotoxicity of 2-butoxyethanol

The available data on the genotoxicity of 2-butoxyethanol have been reviewed. 2-Butoxyethanol has been examined for genotoxic activity in a range of in vitro and in vivo assays, including the mouse bone marrow micronucleus assay. The in vitro assays used range from well validated and generally accepted assays such as the Salmonella/microsome and in vitro cytogenetic assay, through to less well validated assays such as assessment of the inhibition of metabolic cooperation in V79 cells. The levels of experimental details and data reporting vary across the studies with some papers presenting only limited information. Taking the above factors into consideration, the available data indicate that 2-butoxyethanol has no significant genotoxic activity. This conclusion is also consistent with the chemical structure of 2-butoxyethanol, which is not alerting for likely genotoxic activity. These collected considerations indicate that 2-butoxyethanol is unlikely to be a genotoxic carcinogen to rodents, a prediction that supplements seventeen published predictions of the outcome of the ongoing NTP rodent carcinogenicity bioassays.

Evaluation of the genotoxicity of municipal sewage effluent using the marine worm Platynereis dumerilii (Polychaeta: Nereidae).

Samples of settled (primary) effluent were collected from a municipal sewage treatment works at Newton Abbot, Devon, UK, a site which discharges primary effluent via long sea pipeline into the English Channel (minimum of 200-fold initial dilution). Sewage samples were collected during the period February-April 1995 and were analysed for standard physico-chemical parameters (ammonia, chemical oxygen demand, conductivity, non-purgeable organic carbon and settled solids). Samples were also tested for cytotoxicity, genotoxicity, and for developmental effects in the embryo-larval stages of the marine worm, Platynereis dumerilii. Exposure to sewage concentrations of > or = 10% (v/v) in seawater at 20 +/- 1 degrees C led to a marked reduction in normal embryo-larval development (7 h EC50 values from 10% to 18% v/v, n = 5). There was also evidence of a simultaneous delay in the cell cycle progression (as determined by sister chromatid differential staining) following embryo-larval exposures to sewage concentrations of > or = 10% (v/v). Following the calculation of the Maximum Tolerated Dose (MTD), based on cytotoxic and developmental effects, cells from the same embryo-larvae were analysed for chromosomal aberrations (CAs). Results were consistent for all samples tested, demonstrating the absence of cytogenetic damage following the in vivo exposure of polychaete embryo-larvae to settled sewage.

An assessment of the genetic toxicology of antimony trioxide

Antimony trioxide (Sb2O3, CAS 1309-64-4) has been examined in a range of in vitro and in vivo genotoxicity assays. Negative results were obtained with the Salmonella/microsome assay and the L5178Y mutation assay, but a positive response was observed in the in vitro cytogenetic assay using isolated human peripheral lymphocytes. However, in vivo, antimony trioxide was non-clastogenic in the mouse bone marrow micronucleus assay, following oral gavage administration for 1, 7, 14 or 21 days at dose levels of up to 5000 mg/kg (single dose) or 1000 mg/kg (repeat dose). A negative result was also obtained in the in vivo rat liver DNA repair (unscheduled DNA synthesis) assay following a single oral gavage administration of doses up to 5000 mg/kg. These data show no genotoxicity for antimony trioxide in vivo and do not confirm a previous report of clastogenicity in the mouse on repeated dosing. It is concluded that antimony trioxide is not genotoxic in vivo and does not present a genotoxic hazard to humans.