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Differential cytotoxicity of ifosfamide and its metabolites in renal epithelial cell cultures.

Broadhead, C.L., Walker, D., Skinner, R. and Simmons, N.L.

Toxicology in Vitro, 12(3), 209-217 (1998).

Ifosfamide (IF) chemotherapy is dependent on bioactivation by cytochrome P-450 and may be limited by concurrent nephrotoxicity. An in vitro approach utilizing the renal cell lines G401, MDCK Strains I and II, LLCPK1 and OK has been adopted to study nephrotoxic actions of IF and its known metabolites, 4-hydroperoxy-ifosfamide (4-OOH-IF, 4-OH-IF in solution), chloroacetaldehyde (CAA), isofosphoramide mustard (IF-mus) and acrolein using an MTT assay. IF is virtually non-toxic to the panel of renal cell lines used; prolonged incubation with ifosfamide led to a progressive reduction in cell MTT activity only in MDCKI (days 3, 4). 4-hydroxylation of IF was directly measured in MDCK microsomes; MDCKI microsomes gave an activity of 0.045 nmol/min/mg, while in the MDCKII microsome preparation only 0.004 nmol/min/mg was detected. The limited toxicity to IF observed in MDCKI may be explained in part by the ability of cell cytosol to activate IF by 4-hydroxylation. In contrast to IF, its metabolites applied extracellularly were toxic to the panel of renal cell lines used. This cytotoxicity varied within and between cell lines. In LLCPK1 cells after 48 hr of exposure to IF metabolites, the toxicity as measured as a percentage of control MTT activity was: CAA (30 μImage) 11±0.5%>4-OH-IF, (75 μImage), 22.9±1.2%,>acrolein (75 μImage), 42.0±2.9%much greater-thanIF (75 μImage) 69.8±2.8%. The toxicity to IF-mus (exposure at 150 μImage for 48 hr) varied between cell lines, MTT activity measured as a percentage of controls gave: MDCKII 38.1±3.2>G401 41.6±1.7%>MDCKI 60.1±1.7%not, vert, similarLLCPK1 62.4±2.5>OK cells 92.0±5.4%. Whereas G401 and OK cells show marked sensitivity to exposure to acrolein (75 μImage, 48 hr, 1.7±0.2%, 14.2±0.6% of control MTT, respectively), MDCKII cells are relatively insensitive to acrolein toxicity (86.5±4.2% of control MTT). In contrast to the toxicity of acrolein, G401 cells are relatively insensitive to CAA (30 μImage, 48 hr exposure, 48.4±1.7% of control MTT), while MDCKII and OK cells are markedly sensitive (MTT 8.7±0.5, 3.3±0.4% of controls, respectively). Finally, the toxicity of 4-OH-IF (75 μImage, 48 hr exposure) varied considerably between cell lines, with MTT values as a percentage of control values of: G401 3.1±0.3,>LLCPK1, 22.9±1.2%not, vert, similarOK, 23.6±1.4,>MDCKI cells 43.6±0.8%.