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The uptake and accumulation of metals occurs in the kidney, which is a key site for interaction between metal nephrotoxicants. The uptake/accumulation and interaction of CdCl2, HgCl2, K2Cr2O7, and NaAsO2 was examined in precision-cut rabbit renal cortical slices. Slices were incubated with 10(-6) to 10(-3) M of a single metal toxicant or combinations of metal toxicants for 12 hr in DME-F12 media. Slices were blotted and sandwiched between two mylar films stretched across XRF sample cups. Quantitation of the metal in the slices was performed by proton-induced X-ray emission analysis (PIXE). The uptake of the metals was rapid, often reaching a maximum between 3 to 6 hr; the accumulation of Hg was highest, followed in order by Cd, Cr, and As. When two metals were present together, substantial alterations were observed in the uptake of the metals in the slices. HgCl2 hindered the uptake of K2Cr2O7, NaAsO2, CdCl2 (in this order), whereas these metals facilitated the uptake of HgCl2. However, a decreased uptake of both metals was often noted after exposure to other combinations of metals. PIXE analysis of metal content in slices is attractive since all elements (atomic number > 20) can be determined simultaneously. This information will be particularly useful in studying potential toxic interactions.

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Methods for analyzing elements in biological samples are important for studying the effects of intoxication. Since its introduction in 1970 (1), proton-induced X-ray emission spectroscopy (PIXE) has been shown to be a powerful, multielemental analysis technique with high sensitivity (2). The PIXE technique involves bombarding a sample with protons which eject innershell electrons of the elements in the sample; the subsequent decay of the induced electronic states results in the emission of X-rays whose energies are characteristic of the elements present in the sample and whose intensities are proportional to the concentrations of these elements. A liquidnitrogen-cooled solid-state Si(Li) detector and a multichannel analyzer are used to determine the energies as well as the intensities of the emitted X-rays, and a computer program identifies the elements present and calculates their concentrations. Although the application of PIXE to biological samples is not new (3), and recent reviews have emphasized the usefulness of this technique (4-7), the optimization of PIXE for use in typical animal toxicity studies has not been described in the toxicology literature. Here we present the optimization of the instrumentation, sample preparation, and the application of PIXE to a disposition study of metals in renal tissue.The implementation of the PIXE technique has been limited mostly to physics and nuclear research laboratories because no commercial instrumentation is available. The proton beams for PIXE are generated by Van de Graaff accelerators that are not in high demand for applications in physics because much higher-energy ion beams are preferred in most nuclear physics research. The availability of high-energy accelerators has therefore made PIXE an attractive alternative to other conventional, multielemental analytical methods. The simultaneous quantification of a large number of elements in a sample makes PIXE an important analytical tool for use in diverse toxicological studies.Overview of PIXE Theory and Technique X-rays are produced as electrons from outer shells rapidly decay to replace innershell electrons ejected by high-energy protons. The energies of the X-ray emissions are characteristic of the elements, and the intensities of the emissions are proportional to the concentrations of the elements in the sample. The useful range of emitted X-ray energies for PIXE is 1-30 keV; the Si(Li) detector is capable of resolving X-ray emissions as closely spaced as 150 eV, and, as a consequence, 20-30 elements can be readily identified and determined simultaneously.An X-ray spectrum of a thin copper film,deposited on a carbon substrate is shown in Figure 1. The dominant features of the spectrum are the copper K. and K X-ray emission lines resulting from the ejection of the K (innermost) shell electrons of copper. The shape of the X-ray emission peaks in the spectrum is primarily Gaussian; the width of the peak is defined by the statistical distribution of the electrical pulses generated by X-ray photons in the Si(L...