Fig. 2. Preliminary experiment to define (A) the time course of Cu accumulation by freshly isolated intestinal cells, and (B) Cu retention by Cu-loaded cells placed in normal physiological saline. (A) The cells were incubated in external Cu (Cu_o) concentrations of no added Cu (control, filled circles), 10 (open circles), 50 (filled triangles), 100 (open triangles), 200 (filled squares), 400 (open squares), or 800 µmol l^–1 Cu (filled diamonds) for up to 30 min. Cells were then quickly washed in a 0.1 µmol l^–1 EDTA washing solution and pelleted prior to determination of cell Cu content (expressed as nmol Cu mg^–1 cell protein). Values are means ± S.E.M. (N=6 experiments). Rectangular hyperbola are fitted to the raw data using Sigma plot. All cells had reached a stable Cu content by 15 min of exposure time (all significantly higher than the control with no added Cu, Student's t-test, P<0.05, except for the 10 µmol l^–1 Cu_o treatment). (B) Cells were loaded with Cu by exposing them to 800 µmol l^–1 Cu for 15 min, and the subsequent loss of accumulated Cu was followed for 1 h by placing cells back into normal physiological saline (no added Cu). Values are means ± S.E.M. (N=9 experiments) and expressed as nmol Cu mg^–1 cell protein. Values in parentheses are the percent decrease in Cu content (means ± S.E.M.) of the cells relative to the start of the post-exposure period where the Cu content of the Cu-loaded cells is defined as 100%. ^*Statistically different from time 0 post-exposure by Kruskall–Wallis test (P=0.00005). Note, cells did not leak Cu for at least the first 15 min in normal saline (no statistical difference from time 0 post-exposure control).