Chloroacetaldehyde as a Sulfhydryl Reagent: The Role of Critical Thiol Groups in Ifosfamide Nephropathy

Abstract

Chloroacetaldehyde (CAA) is a metabolite of the alkylating agent ifosfamide (IFO) and putatively responsible for renal damage following anti-tumor therapy with IFO. Depletion of sulfhydryl (SH) groups has been reported from cell culture, animal and clinical studies. In this work the effect of CAA on human proximal tubule cells in primary culture (hRPTEC) was investigated. Toxicity of CAA was determined by protein content, cell number, LDH release, trypan blue exclusion assay and caspase-3 activity. Free thiols were measured by the method of Ellman. CAA reduced hRPTEC cell number and protein, induced a loss in free intracellular thiols and an increase in necrosis markers. CAA but not acrolein inhibited the cysteine proteases caspase-3, caspase-8 and cathepsin B. Caspase activation by cisplatin was inhibited by CAA. In cells stained with fluorescent dyes targeting lysosomes, CAA induced an increase in lysosomal size and lysosomal leakage. The effects of CAA on cysteine protease activities and thiols could be reproduced in cell lysate. Acidification, which slowed the reaction of CAA with thiol donors, could also attenuate effects of CAA on necrosis markers, thiol depletion and cysteine protease inhibition in living cells. Thus, CAA directly reacts with cellular protein and non-protein thiols, mediating its toxicity on hRPTEC. This effect can be reduced by acidification. Therefore, urinary acidification could be an option to prevent IFO nephropathy in patients.

The Nephrotoxic Ifosfamide-Metabolite Chloroacetaldehyde Interferes with Renal Extracellular Matrix Homeostasis

“…The measured increase in fluorescence/time was normalized for protein content. Possible direct effects of CAA on collagenase I were also tested, since CAA is capable of direct modification of proteins [21]. We found no alterations in collagenase I activity after 4 h incubation of the enzyme with CAA up to 150 µM.…”

“…In order to explain this discrepancy between the induction of total gelatinase activity and reduction of MMP-2 activity, we investigated the contribution of the lysosomal protease cathepsin B in CAA-treated RPTECs. Since CAA induces lysosomal protein overload and leakage of lysosomes [21], we further investigated the sensitivity of total gelatinase activity against the specific cathepsin B inhibitor CA-074. As shown in Figure 3A, CA-074 (10 µM) led to a significant reduction of total gelatinase activity in media of CAA-treated cells (15 µM CAA: 111.9 ± 14.6 vs. 77.5 ± 11.4 % of control; 150 µM CAA: 182.0 ± 19.8 vs. 101.4 ± 18.8 % of control), whereas no significant effect of CA-074 in media of control cells was observable (92.7 ± 6.4 % of control).…”

“…Since acute CAA toxicity can be partly reduced by extracellular acidification due to reduced sulfhydryl reactivity of the compound in acidic environment [21], the effects of lowering extracellular pH on collagen III secretion provoked by CAA was investigated. Figure 6 shows the amount of extracellular collagen III in acidified media (pH o 7.2 or 6.9) of RPTEC after 48 h CAA exposure.…”

“…The effects of CAA with regards to changes in lysosomal morphology and permeability could be enhanced by additional protein challenge [21]. Therefore, a possible contribution of an elevated protein load on collagen III secretion in response to CAA was investigated.…”

“…CAA acts as a sulfhydryl reagent and thereby impairs the proper function of proteins necessary for the balance of proliferation and apoptosis by inhibition of caspases. We could also show that CAA inhibits cathepsin B, a lysosomal cysteine protease of importance for digestion of endocytosed protein [21]. Depletion of non-protein-sulfhydryl groups such as reduced glutathione (GSH) impairs the capacity of the cells to scavenge free radicals, thereby increasing the susceptibility to oxidative stress.…”