DNA-damaging effect of cyclophosphamide on human blood cells in vivo and in vitro studied with the single-cell gel test (comet assay).

Abstract

The single-cell gel (SCG) test was used to study the induction and persistence of DNA damage by cyclophosphamide (CP) in human blood cells after treatment in vitro and in vivo. S9-mix-activated CP (from 0.1 mM upward) induced DNA effects in a concentration-dependent manner in the in-vitro SCG test. Blood cells from various donors showed considerable intra- and interindividual variability. Incubation of CP-treated blood samples at 37 degrees C caused a rapid decrease in DNA effects, but DNA migration was still significantly increased 1 hr after the end of the CP treatment. Comparative studies with the in vitro sister chromatid exchange (SCE) test were performed that demonstrated that much lower CP concentrations (about 100 times) were required for a significant induction of SCEs. A group of 11 patients who suffered from vasculitis/collagen disease and were treated with low CP doses (50-200 mg/day) exhibited an elevated level of DNA damage in the SCG test with peripheral blood cells, compared with a group of 11 control persons or 5 patients without chemotherapy. Increases in DNA damage were variable and not clearly related to the CP dose. SCE tests could successfully be performed with 5 out of the 11 CP-treated patients; all showed significantly increased SCE frequencies. For six patients no result could be obtained with the SCE test due to a failure of lymphocyte proliferation. Three multiple sclerosis patients who received high doses of CP were investigated with the SCG test before, during, and after the treatment. The results indicate that CP-induced DNA effects that are detectable with the SCG test persist in vivo for a period of several days, but for less than 2 weeks. The results of our study provide information with regard to the use of the SCG test in human monitoring. The advantages and limitations of the test are discussed.