Use of human-liver microsomes from kidney-transplant donors for the induction of chromatid aberrations and sister-chromatid exchanges by means of pre-carcinogens in Chinese hamster cells in vitro.

Samples of two human livers taken during operation of kidney donor patients were processed for microsome fractions and used for metabolization of cyclophosphamide (CP) and dimethylnitrosamine (DMN) in combination with the NADPH-generating system. Rat-liver microsomes were checked for comparison. Induction of chromatid aberrations and sister-chromatid exchanges in a newly isolated clone of Chinese hamster fibroblasts served as indicators of activity. Human S-9 fractions standardized on protein content showed strong variations of CP and DMN activation. Whereas liver microsomes of one patient (who also suffered from Gaucher's disease) were highly active for both pre-carcinogens and metabolized DMN at the same level as the uninduced rat-liver microsomes, the S-9 fraction from the second patient failed to activate CP, but was distinctly positive for DMN. It is suggested that samples of liver and other organs of renal transplant donors might be a practicable source of freshly prepared human microsome fractions usable in biochemical, genetic and carcinogenetic studies. Problems concerning the extrapolation of results are discussed.     

Apoptosis and cell proliferation in proximal tubular cells exposed to apoptotic bodies. Novel pathophysiological implications in cisplatin-induced renal injury

The therapeutic efficacy of the antineoplastic drug cisplatin is limited by its nephrotoxicity, which affects particularly to proximal tubular cells (PTC). Cisplatin-induced cytotoxicity appears to be multifactorial and involves inflammation, oxidative stress as well as apoptosis. We have recently shown that the cyclo-oxygenase-2 (COX-2)/intracellular prostaglandin E₂ (iPGE₂)/EP receptor pathway mediates the apoptotic effect of cisplatin on human proximal tubular HK-2 cells. Here, we studied the effects on HK-2 cells of apoptotic bodies (ABs) generated after treatment of HK-2 cells with cisplatin. We found that ABs inhibited cell growth, induced apoptosis and increased COX-2 expression and iPGE₂ in ABs-recipient HK-2 cells. Inhibition of the COX-2/iPGE₂/EP receptor pathway in these cells prevented the effects of ABs without interfering with their internalization. Interestingly, 2nd generation ABs (i.e. ABs released by cells undergoing apoptosis upon treatment with ABs) did not trigger apoptosis in naïve HK-2 cells, and stimulated cell proliferation through the COX-2/iPGE₂/EP receptor pathway. These results suggest that ABs, through iPGE₂-dependent mechanisms, might have a relevant role in the natural history of cisplatin-induced acute kidney failure because they contribute first to the propagation of the noxious effects of cisplatin to non-injured PTC and then to the promotion of the proliferative tubular response required for proximal tubule repair. Since iPGE₂ also mediates both cisplatin-induced HK-2 cell apoptosis, intervention in the COX-2/iPGE₂/EP receptor pathway might provide us with new therapeutic avenues in patients with cisplatin-induced acute kidney injury.     

The combination of glutamate receptor antagonist MK-801 with tamoxifen and its active metabolites potentiates their antiproliferative activity in mouse melanoma K1735-M2 cells

Recent reports suggest that N-methyl-d-aspartate receptor (NMDAR) blockade by MK-801 decreases tumor growth. Thus, we investigated whether other ionotropic glutamate receptor (iGluR) antagonists were also able to modulate the proliferation of melanoma cells. On the other hand, the antiestrogen tamoxifen (TAM) decreases the proliferation of melanoma cells, and is included in combined therapies for melanoma. As the efficacy of TAM is limited by its metabolism, we investigated the effects of the NMDAR antagonist MK-801 in combination with TAM and its active metabolites, 4-hydroxytamoxifen (OHTAM) and endoxifen (EDX). The NMDAR blockers MK-801 and memantine decreased mouse melanoma K1735-M2 cell proliferation. In contrast, the NMDAR competitive antagonist APV and the AMPA and kainate receptor antagonist NBQX did not affect cell proliferation, suggesting that among the iGluR antagonists only the NMDAR channel blockers inhibit melanoma cell proliferation. The combination of antiestrogens with MK-801 potentiated their individual effects on cell biomass due to diminished cell proliferation, since it decreased the cell number and DNA synthesis without increasing cell death. Importantly, TAM metabolites combined with MK-801 promoted cell cycle arrest in G1. Therefore, the data obtained suggest that the activity of MK-801 and antiestrogens in K1735-M2 cells is greatly enhanced when used in combination.