Genotoxicity in Xenopus laevis exposed to sodium perchlorate and UV radiation



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Texas Tech University


The purpose ofthis work was to determine if simultaneous exposure of amphibian larvae to sodium perchlorate (SP) and UV-B radiation would affect mortality, final stage of development, single strand DNA breaks (SSB), pyrimidine dimer formation, and oxidative DNA damage. Two trials were performed where 450 Xenopus laevis larvae were exposed to combinations of UV-B and SP. Nominal SP exposure concentrations were 0.0 mg/L, 0.05 mg/L, or 100.0 mg/L. Perchlorate exposure groups were further subdivided into UV-B treatments where half the animals received 0.0mJ/cm 2/d and the remaining half received 100 mj/cm2 /d. Fifteen Nieuwkoop-Faber (N-F) stage 51 (trial 1) larvae and N-F stage 50 (trial 2) larvae were maintained in FETAX media in each of the 30 l.0L glass beakers. Exposures were carried out in a Precision Scientific model 815 microprocessor controlled low temperature incubator adapted to uniformly deliver UV-B radiation. A 12:12 light:dark cycle was maintained with digital timer-controlled cool white fluorescent bulbs. Ultraviolet light was similarly produced by digital timer controlled medical grade 20W UV-B fluorescent bulbs with an on:off cycle set appropriately to deliver daily dosage and to approximate a natural midday peak of UV. Completion of exposures was defined as when 80% of the larvae exposed to 0.0 mg/L SP plus 0.0 mJ/cm^/d UV-B reached N-F stage 60. Upon exposure completion, tadpoles were staged and tails were removed 0.5cm from the point of attachment and stored in liquid nitrogen until DNA extraction. Tail tissue from two animals from each replicate were combined for DNA extraction, purification, and analysis. Single strand break analyses were performed using agarose gel electrophoresis. Pyrimidine dimers were assessed using a combination of T4 endonuclease V digestion and agarose gel electrophoresis. Oxidative DNA damage was similarly assessed using simultaneous endonuclease III plus Formamidopyrimidine DNA-Glycosylase (fpg) digestion and agarose gel electrophoresis. Sodium perchlorate and UV-B interaction had a significant effect on mortality in the second trial. Perchlorate also had a significant effect on rates of metamorphosis in both trials in a dose-dependant manner. Average molecular length of DNA fragments (as a measure of SSB) was lower for all UV-B exposed groups, although not at significant levels. The incidence of cyclobutane pyrimidine dimers within the DNA was increased by UV-B treatment below significant levels. Perchlorate, UV-B, and their interaction had no apparent effects on oxidative damage within DNA samples. We believe that, while SP does pose a threat to normal development and growth to natural amphibian populations, SP exposure does not enhance the susceptibility of these populations to the DNA-damaging effects of UV-B. Likewise, UV-B exposure does not enhance the toxicity of SP to amphibian larvae.



Nucleotide sequence, Xenopus laevis, Polycarbonates, Ultraviolet radiation, Perchlorates, Genetic toxicology