Application of Modelling Techniques to the Planning of In Vitro Arsenic Kinetic Studies

Elaina M. Kenyon, Michael Fea, Mirek Styblo and Marina V. Evans

A kinetic model describing the hepatic methylation of arsenite ([As[III]) was developed on the basis of limited data from in vitro mechanistic studies. The model structure is as follows: sequential enzymic methylation of arsenite to its monomethylated (MMA) and dimethylated (DMA) products by first-order and Michaelis-Menten kinetics, respectively; uncompetitive inhibition of the formation of DMA by As(III); and first-order reversible binding of As(III), MMA and DMA to cytosolic proteins. Numerical sensitivity analysis was used to evaluate systematically the impact of changes in input parameters on model responses. Sensitivity analysis was used to investigate the possibility of designing experiments for robust testing of the uncompetitive inhibition hypothesis, and for further refining the model. Based on the sensitivity analysis, the MMA concentration is the most important response on which to focus. The parameters Vmax and ki can be reliably estimated by using the same concentration time-course data at intermediate initial arsenite concentrations of 1–5μM at 30 ± 5 minutes. Km must be estimated independently of Vmax, since the two parameters are highly correlated at all times, and the optimal experimental conditions would include lower initial concentrations of arsenite (0.1–0.5μM) and earlier time-points (about 8–18 minutes). The use of initial arsenite concentrations much above 5μM would not yield additional useful information, because the sensitivity coefficients for MMA, protein-bound MMA, DMA and protein-bound DMA tend to become extremely small or exhibit erratic trends. Overall trends in the sensitivity analysis indicated the desirability of performing measurements at times shorter than 60 minutes. This work demonstrates that physiological modelling and sensitivity analysis can be efficient tools for experimental planning and hypothesis testing when applied in the earliest phases of kinetic model development, thus allowing more-efficient and more-directed experimentation, and minimising the use of laboratory animals.
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A Comparative Study of the Toxicity of Mercury Dichloride and Methylmercury, Assayed by the Frog Embryo Teratogenesis Assay–Xenopus (FETAX)

Mariangela Prati, Rosalba Gornati, Patrizia Boracchi, Elia Biganzoli, Salvador Fortaner, Romano Pietra, Enrico Sabbioni and Giovanni Bernardini

The Frog Embryo Teratogenesis Assay–Xenopus (FETAX) is a powerful and flexible bioassay that makes use of the embryos of the anuran amphibian Xenopus laevis. The FETAX can detect xenobiotics that affect embryonic development, when mortality, teratogenicity and growth inhibition are used as endpoints. The FETAX was used to compare the embryotoxic and teratogenic potentials of two chemical species of mercury: inorganic mercury(II) chloride (HgCl2) and organic methylmercury chloride (MeHgCl). MeHgCl, with an estimated median lethal concentration [LC50] of 0.313μM and a median teratogenic concentration [TC50] of 0.236μM, showed a higher toxicity than HgCl2, with estimated LC50 and TC50 values of 0.601μM and 0.513μM, respectively. On the basis of these results, HgCl2 and MeHgCl can be classified as “slightly teratogenic compounds”, as the ratio of LC50/TC50 is less than 1.5. There was a significant deviation from the commonly described monotonic behaviour of the concentration–response curves, suggesting a hormetic effect of both species of mercury. Uptake experiments, followed by neutron activation analysis, showed a higher incorporation of mercury in embryos exposed to MeHgCl compared with those exposed to HgCl2. Interestingly, Hg-exposed embryos showed a higher content of selenium and zinc than did control embryos.
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Arsenic Toxicity and HSP70 Expression in Xenopus laevis Embryos

Rosalba Gornati, Claudio Monetti, Davide Vigetti, Stefano Bosisio, Salvador Fortaner, Enrico Sabbioni, Giovanni Bernardini and Mariangela Prati

The evaluation of the effect of trace metals on health can be difficult, because of their presence in the environment in various chemical forms. Exposure to arsenic compounds is an example of this complexity, as it can be present in the environment in inorganic and organic forms. The effects of arsenic in vertebrates are complicated by several variables, such as speciation of the element, the exposure route, and the susceptibility of the particular animal species. The embryotoxicity and teratogenicity of three arsenic species - sodium arsenite (NaAsO2), disodium hydrogen arsenate (Na2HAsO4) and dimethylarsinic acid [(CH3)2AsOOH] - were evaluated by the modified frog embryo teratogenic assay on Xenopus (FETAX). We also show how the classical FETAX endpoints, based on morphological and morphometrical analysis, can conveniently be integrated with the study of molecular markers. Possible changes in the expression of the mRNA for the heat-shock protein HSP70, following exposure to NaAsO2, were examined by using the reverse transcriptase polymerase chain reaction. HSP70 mRNA is strongly induced by arsenic.
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