Dithio-bis-mercaptoethanesulphonate (DIMESNA) does not prevent cellular damage by metabolites of ifosfamide and cyclophosphamide in LLC-PK1 cells

Abstract

Ifosfamide (IF) is an alkylating cytostatic with urotoxic (haemorrhagic cystitis) and nephrotoxic (Fanconi syndrome) side effects. Cyclophosphamide (CP), a structural isomer of IF, shows urotoxic but no nephrotoxic side effects. The development of haemorrhagic cystitis during therapy with IF or CP can be prevented by the uroprotective drug sodium-2-mercaptoethanesulphonate (MESNA). However, even in the presence of MESNA, Fanconi syndrome may still develop after therapy with IF. Using the renal tubular cell line LLC-PK1, we investigated whether there is a protective effect of either MESNA or of its major metabolite DIMESNA, in combination with metabolites of IF or CP, on thymidine incorporation, uridine incorporation or total protein. DIMESNA, the dimer of MESNA, is the dominant form of the molecule in the circulation; the proximal tubular cell must convert this back to MESNA at the expense of glutathione, before it can exert its uroprotective action. We did not find a protective effect of DIMESNA under any of the experimental conditions tested. LLC-PK1 cells exposed to 3 mmol/l DIMESNA did not convert DIMESNA to MESNA. The toxic effect of the CP metabolite 4-OOH-CP was more pronounced in the presence of DIMESNA than in its absence. MESNA completely prevented the toxic effects of acrolein and of 4-OOH-CP. The toxic effects of 4-OOH-IF and of chloracetaldehyde, two major metabolites of IF, were significantly reduced in the presence of MESNA. However, even at 30-fold molar excess of MESNA over a 4-OOH_IF, thymidine incorporation remained reduced by 40% compared with controls, indicating incomplete protection of tubular cells against metabolites of IF. Similarly, the effect of chloracetaldehyde was not completely reversed by MESNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Ifosfamide and Mesna: Effects on the Na/H Exchanger Activity in Renal Epithelial Cells in Culture (LLC-PK₁)

“…No comparable effect of Dimesna with any of the other metabolites of IF could be detected with respect to Na/Fl exchange. Flowever, we did show a similar combined effect for Dimesna and the CP metabolite 4-OH-CP [9], In those experiments thymidine incorporation into LLC-PK, cells was inhibited stronger by the combination ol'4-01 l-CP plus Dimesna than by 4-OH-CP alone.…”

“…The inability o f LLC-PK, cells to convert significant amounts of Dimesna to Mesna [9] explains the lack o f any protective effect o f Dimesna in all experiments presented here. The incomplete protection of LLC-PK| cells by the active compound Mesna against 4-OH-IF is an other very interesting finding.…”

“…In the presence of Di mesna the dose-response curve for acrolein is even displaced to the left, indicating a higher toxicity of the combination of both compounds. In a previous paper [9], we could show that in the supernatant o f LLC-PK, cells incubated with Dimesna no Mesna was detectable, show ing that there is no or insufficient conversion of Dimesna to Mesna. This finding might be ex plained by either an inability of LLC'-PK, cells to actively take up Dimesna and to secrete Mes na or by a lack of capacity to reduce Dimesna to Mesna.…”

“…The incomplete protection of LLC-PK| cells by the active compound Mesna against 4-OH-IF is an other very interesting finding. In our previous work [9], investigating the effect of4-OH-IF on thymidine incorporation, uridine incorpora tion, and total protein in LLC-PK| cells, we showed that Mesna, even at a concentration of 5 mmol/1, would not protect LLC-PK| cells completely from the toxic effects of 150 pmol/1 4-OF1-IF. In contrast to that, the toxic effects o f 4-OF1-CP were fully prevented even by 0.5 mmol/1 of Mesna [9], Thus, with respect to a tubuloprotective effect of Mesna, there is a sig nificant difference between the metabolite of CP which is not tubulotoxic in vivo and of IF which causes Fanconi syndrome.…”

“…Another important issue is the role of the uroprotectant Mesna which prevents hemorrhagic cystitis, while tubular damage may still develop. In order to investigate the pathomechanism of IF-induced tubular toxicity, we established an in vitro model of Fanconi syndrome in renal tubular cells in culture (LLC-PK,) [6][7][8][9]. We could show that metabo lites of IF have cytotoxic effects in LLC-PK.)…”