Highly protective alkalinization by ammonia vapor diffusion in viscosimetric DNA damage assessment

A method for the measurement of viscosities correlated to DNA alterations in alkaline homogenate suspensions is described. The alkaline pH shift to afford cell lysis, DNA unfolding, and denaturation is attained by gaseous ammonia diffusion, thus avoiding shear stress from mechanical mixing. At the same time a stabilizing density gradient is established. This solution is run through a plastic measuring tube that is wide enough to minimize the influence of uneven swelling of the lysing DNA-containing components. Flow times under a carefully controlled water head are registered, and their ratios to control solutions are evaluated. The relative viscosities show a strong and irreversible dependence on shear and on DNase treatment and therefore are considered as essentially DNA derived. The time dependence of the lysate viscosities with and without the DNA-damaging agent bleomycin is given and the dose:activity curves of this agent with sponge homogenates from two orders of Porifera are given with their 50% effective concentration values. The dose:activity curve of an extract from a polluted marine point source is demonstrated. The concentration changes in sponges exposed at differently polluted marine sites are shown. The idea of alkalinization through gaseous diffusion in conjunction with a simple measuring device has already proven a sensitive, reliable, and specific tool in the assessment of DNA damage produced under both laboratory and field conditions.