A novel, integrated in vitro carcinogenicity test to identify genotoxic and non-genotoxic carcinogens using human lymphoblastoid cells

Human exposure to carcinogens occurs via a plethora of environmental sources, with 70–90% of cancers caused by extrinsic factors. Aberrant phenotypes induced by such carcinogenic agents may provide universal biomarkers for cancer causation. Both current in vitro genotoxicity tests and the animal-testing paradigm in human cancer risk assessment fail to accurately represent and predict whether a chemical causes human carcinogenesis. The study aimed to establish whether the integrated analysis of multiple cellular endpoints related to the Hallmarks of Cancer could advance in vitro carcinogenicity assessment. Human lymphoblastoid cells (TK6, MCL-5) were treated for either 4 or 23 h with 8 known in vivo carcinogens, with doses up to 50% Relative Population Doubling (maximum 66.6 mM). The adverse effects of carcinogens on wide-ranging aspects of cellular health were quantified using several approaches; these included chromosome damage, cell signalling, cell morphology, cell-cycle dynamics and bioenergetic perturbations. Cell morphology and gene expression alterations proved particularly sensitive for environmental carcinogen identification. Composite scores for the carcinogens’ adverse effects revealed that this approach could identify both DNA-reactive and non-DNA reactive carcinogens in vitro. The richer datasets generated proved that the holistic evaluation of integrated phenotypic alterations is valuable for effective in vitro risk assessment, while also supporting animal test replacement. Crucially, the study offers valuable insights into the mechanisms of human carcinogenesis resulting from exposure to chemicals that humans are likely to encounter in their environment. Such an understanding of cancer induction via environmental agents is essential for cancer prevention

Bromate minimization during ozonation: Mechanistic considerations

Bromate formation during ozonation of bromide-containing natural waters is somewhat inversely connected to the ozone characteristics: an initial fast increase followed by a slower formation rate. During the initial phase mostly OH radical reactions contribute to bromate formation, whereas in the secondary phase both ozone and OH radicals are important. To minimize bromate formation several control options are presented: ammonia addition, pH depression, OH radical scavenging, and scavenging or reduction of hypobromous acid (HOBr) by organic compounds. Only the two first options are applicable in drinking water treatment. By both methods a similar effect of a bromate reduction of approximately 50% can be achieved. However, bromate formation during the initial phase of the ozonation cannot be influenced by either method. Ammonia (NH3) efficiently scavenges HOBr to NH2Br. However, this reaction is reversible which leads to higher required NH3 concentrations than expected. The rate constant k(NH2Br) for the hydrolysis of NH2Br by OH- to NH3 and OBr- was found to be 7.5.10(6) M-1 s(-1). pH depression shifts the HOBr/OBr- equilibrium to HOBr and also affects the ozone chemistry. The effect on ozone chemistry was found to be more important for bromate formation. For a given ozone exposure, the OH radical exposure decreases with decreasing pH. Therefore, for pH depression the overall oxidation capacity for a certain ozone exposure decreases which in turn leads to a smaller bromate formation

Ozonation of bromide-containing drinking waters: a delicate balance between disinfection and bromate formation

The occurrence of Cryptosporidium in raw waters and bromate formation during ozonation of bromide-containing waters leads to a difficult optimisation of ozonation processes. On the one hand inactivation of Cryptosporidium requires high ozone exposures, on the other hand under these conditions bromate formation is favored. In order to overcome this problem we need information about (i) the oxidant concentrations (ozone and OH radicals) during an ozonation process, (ii) kinetics of the inactivation of Cryptosporidium, (iii) kinetics and mechanism of bromate formation, and (iv) the reactor hydraulics. The strong temperature dependence of the inactivation of Cryptosporidium which results in a higher ozone exposure (time-integrated action of ozone) at low temperatures makes it more difficult to fulfil disinfection and bromate standards at low temperatures. Cinder these conditions control options for bromate formation can be applied. Depression of pH and addition of ammonia have been selected to be the best options. For a given ozone exposure both measures lead to a reduction of bromate formation in the order of 50%

Methods for the photometric determination of reactive bromine and chlorine species with ABTS

New methods for the determination of reactive bromine and chlorine species are presented. Hypobromous acid (HOBr) and all three bromamines species (NH2Br, NHBr2, NBr3) are analyzed as a sum parameter and hypochlorous acid (HOCl), monochloramine (NH2Cl) and chlorine dioxide (ClO2) can be determined selectively. However, no distinction is possible between HOCl and the active bromine species. The bromine and chlorine species react with ABTS (2,2-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid-diammonium salt) to a green colored product that is measured at 405 or 728 nm. Free chlorine and NH2Cl can be measured in the presence of ozone. The method is therefore suitable if combinations of disinfectants are used, such as chlorine/chlorine dioxide or chlorine/ozone. In natural waters, the method provides a detection limit for all chlorine/bromine species of less than 0.1 muM. The colored reaction product is very stable and allows a fixation of the chlorine/bromine species in the field and subsequent determination of the absorption in the laboratory. (C) 2000 Elsevier Science Ltd. All rights reserved

Inactivation of Bacillus subtilis spores and formation of bromate during ozonation

Inactivation of B. subtilis spores with ozone was investigated to assess the effect of pH and temperature, to compare the kinetics to those for the inactivation of C. parvum oocysts, to investigate bromate formation under 2-log inactivation conditions, and to assess the need for bromate control strategies. The rate of B. subtilis inactivation with ozone was independent of pn. decreased with temperature (activation energy of 42,100 J mol(-1)), and was consistent with the CT concept. B, subtilis was found to be a good indicator for C. poruum at 20 30 degreesC, but at lower temperatures R. subtilis was inactivated more readily than C. parvum. Bromate formation increased as both pH and temperature increased. For water with an initial bromide concentration of 33 mu gl(-1), achieving 2-logs of inactivation, without exceeding the 10 mu gl(-1) bromate standard, was most difficult at 30 degreesC for B. subtilis and at midrange temperatures(10-20 degreesC) for C. parvum pH depression and ammonia addition were found to reduce bromate formation without affecting B. subtilis inactivation, and may be necessary for waters containing more than 50 mu gl(-1) bromide. (C) 2001 Elsevier Science Ltd. All rights reserved