Acetaminophen inhibits hemoprotein-catalyzed lipid peroxidation and attenuates rhabdomyolysis-induced renal failure

Abstract

Hemoproteins, hemoglobin and myoglobin, once released from cells can cause severe oxidative damage as a consequence of heme redox cycling between ferric and ferryl states that generates radical species that induce lipid peroxidation. We demonstrate in vitro that acetaminophen inhibits hemoprotein-induced lipid peroxidation by reducing ferryl heme to its ferric state and quenching globin radicals. Severe muscle injury (rhabdomyolysis) is accompanied by the release of myoglobin that becomes deposited in the kidney, causing renal injury. We previously showed in a rat model of rhabdomyolysis that redox cycling between ferric and ferryl myoglobin yields radical species that cause severe oxidative damage to the kidney. In this model, acetaminophen at therapeutic plasma concentrations significantly decreased oxidant injury in the kidney, improved renal function, and reduced renal damage. These findings also provide a hypothesis for potential therapeutic applications for acetaminophen in diseases involving hemoprotein-mediated oxidative injury.

Conflict of interest statement

Conflict of interest statement: J.A.O., O.B., and L.J.R. have filed a patent for the use of acetaminophen in rhabdomyolysis-induced renal failure. J.A.O. is a consultant for McNeil Pharmaceuticals.

Figures

Fig. 1.

Inhibition by ApAP of Mb- and Hb-induced oxidation of AA. (A) Ferric Mb (10 μM) was incubated with 10 μM AA. The reaction was initiated by adding H2O2 and proceeded for 3 h at 37 °C. The residual AA and the products of oxidation were extracted and analyzed as described in Methods. The oxidation is represented as nanomoles of AA oxidized by Mb in 3 h. Each data point represents the average of six different values. (B) Ferric Mb (10 μM) was incubated with 10 μM AA and ApAP. The reaction was initiated by adding 5 μM of H2O2 and proceeded for 3 h at 37 °C. The oxidation is represented as the percentage of AA oxidized by Mb in 3 h compared to the control in which no ApAP is present. Each data point represents the average of eight different values. (C) Ferric Hb (45 μM) was incubated with 10 μM AA. The reaction was initiated by adding H2O2 and proceeded for 3 h at 37 °C. The oxidation was analyzed as described above. Each data point represents the average of six different values. (D) Ferric Hb (45 μM) was incubated with 10 μM AA and ApAP. The reaction was initiated by adding 30 μM of H2O2 and proceeded for 3 h at 37 °C. Each data point represents the average of six different values.

Fig. 2.

Effect of ApAP on the state of oxidation of Mb. (A) Transition from ferric to ferryl Mb and its reduction by ApAP were monitored by recording visible spectra (350–650 nm). Ferryl Mb was generated by incubating ferric Mb (10 μM) with 17.5 μM H2O2 until there was no more change at 425 nm (B). At this time, ApAP (174 μM) was added and spectra were recorded every 2 min. The arrow indicates the time of addition of ApAP. (C and D) Effect of ApAP on the rate constant of ferryl decay. Ferryl Mb (10 μM) was reacted with ApAP in sodium acetate (pH 5.0) (C) or sodium phosphate (pH 7.4) (D). The pseudofirst order rate constants for reduction of ferryl to ferric Mb were measured from the time course (425–408 nm) and plotted as a function of ApAP concentration. With no ApAP, the rate constant for ferryl reduction is not zero due to autoreduction, which is more apparent at pH 5 than at pH 7.4. The data are fitted to a double rectangular hyperbola function (n = 3).

Fig. 3.

ApAP prevents the H2O2-dependent formation of DMPO adducts on Mb residues. Ferric Mb (100 μM) was incubated with, without, or with different combinations of H2O2 (250 μM), DMPO (100 mM), and ApAP (1 mM). After 2 h at 37 °C, the protein was analyzed by ESI mass spectrometry. The resulting deconvoluted mass spectra are shown for Mb (A), Mb plus DMPO and H2O2 (B), and Mb plus DMPO, H2O2, and ApAP (C). Mb + xNa+: peaks corresponding to salt adducts resulting from addition of 1 or 2 sodium ions per molecule of Mb.

Fig. 4.

Visual examination of a representative kidney from a normal rat (Left), a rat with rhabdomyolysis (Center), and a rat with rhabdomyolysis treated with ApAP (Right).

Fig. 5.

Effect of treatment with ApAP on oxidative injury (A and B) and on kidney function (C and D) in normal rats (controls), in normal rats treated with ApAP (control + ApAP), in rats with rhabdomyolysis (Rhabdo), and in rats with rhabdomyolysis treated with ApAP (Rhabdo + ApAP). (A) Urinary excretion of F2-IsoPs. **, P < 0.0001 for Rhabdo vs. controls; *, P < 0.005 for Rhabdo + ApAP vs. controls. The bracket indicates that ApAP significantly reduced urinary F2-IsoPs compared with Rhabdo alone (P = 0.015) (n ≥ 6 in each group). (B) Plasma levels of F2-IsoPs. **, P = 0.0002 for Rhabdo vs. controls. The bracket denotes a significant difference (P = 0.01) between Rhabdo + ApAP and Rhabdo, and NS denotes no significant difference from controls (n ≥ 6 in each group). (C) Creatinine clearance. **, P < 0.0001 for Rhabdo vs. controls; *, P = 0.012 for Rhabdo + ApAP vs. controls; NS denotes no significant difference from controls (n ≥ 6 in each group). (D) Plasma levels of creatinine. **, P < 0.0001 for Rhabdo vs. controls; *, P = 0.0002 for Rhabdo + ApAP vs. controls (n ≥ 6 in each group).

Fig. 6.

ApAP inhibits the formation of the hemoprotein cross-links in urine from rats with rhabdomyolysis. The amount of cross-linked hemoprotein in urine from rats with rhabdomyolysis (Rhabdo) and with rhabdomyolysis treated with ApAP (Rhabdo + ApAP) was determined by HPLC analysis as described in Methods. Chromatograms were measured at 400 nm as shown in Fig. S3. The peak corresponding to the cross-linked Mb at 19 min was integrated and the concentration was determined. The values represent the means ± SEM (n ≥ 5 in each group). An unpaired double-tailed Student’s t test was performed after having verified the normal distribution using the D’Agostino and Pearson omnibus normality test.