The toxicity of APAP has been extensively characterized in mouse models and in humans. ALT measurements are commonly used indicators of toxicity in experimental and clinical studies of APAP injury. However, measurement of these serum markers does not per se
quantify liver function [6
]. The current study was designed to examine ICG as a functional marker of APAP toxicity in the mouse model and to examine the effect of treatment with NAC on ICG clearance. Our data demonstrate that the pharmacokinetic profiles of ICG varied significantly between the APAP only and saline treated mice. The most striking findings were the changes in MRT and ClT
observed between the APAP only treated mice and the saline treated controls ( and ). Moreover, the pharmacokinetics of ICG in APAP toxicity in the mouse were significantly altered by the administration of NAC, the common clinical antidote used in the management of APAP overdose. Remarkably, mice treated with NAC 1 h after APAP had a pharmacokinetic profile for ICG (eg., CLT
and MRT) that was similar to that of the saline treated mice. However, mice treated with NAC 4 h after APAP had a pharmacokinetic profile for ICG (eg., CLT
and MRT) that was similar to the APAP only treated mice.
ICG is used in the clinical setting to evaluate hepatosplanchnic circulation and as a marker for graft viability in liver transplantation [25
]. The clearance of ICG has been previously examined as a functional marker in experimental models of liver injury or toxicity [12
]. Inage et al.
] examined the pharmacokinetics of ICG in rats with partial hepatectomy and in rats with fibrosis secondary to carbon tetrachloride toxicity and found that the clearance of ICG correlated with the functional reserve and liver mass [6
The clearance of ICG is regarded to be a strong correlate of hepatic blood flow [30
]. Hepatic blood flow has been shown to be altered in APAP toxicity [31
] and very early changes occur in the microcirculation of the liver in APAP toxicity in the mouse. Electron microscopy studies have demonstrated a number of ultrastructural changes in the hepatic sinusoids [33
]. For example, swelling of sinusoidal endothelial cells occurs before
there is evidence of biochemical injury as indicated by ALT elevation at 2 h. In addition, others have observed hepatic sinusoidal endothelium swelling at 30 min with subsequent reduced blood flow as early as 2 h after the oral administration of APAP [33
]. Our laboratory recently reported the initial elevation of plasma hyaluronic acid, a marker of endothelial cell dysfunction, at 2 h in mice treated with APAP [21
]. Alterations in blood flow in APAP toxicity are also thought to occur in the later phases of toxicity. For example, elevation of hemoglobin in mouse liver has been demonstrated at 24 h, indicating venous congestion [34
] and the onset of hypoxia in mouse liver in APAP toxicity is a relatively late event (Chaudhuri, in submission). In the present study, we chose to examine the pharmacokinetics of ICG at 24 h because of our interest in late events in the toxicity and in particular the recovery stages of APAP toxicity. In previous work, we have reported that the proliferation of hepatocytes (ie., hepatocyte regeneration) following APAP injury occurs at 24 h after the administration of APAP [21
Of interest, ICG administered to mice immediately prior to APAP was previously shown to reduce the extent of tissue necrosis at 24 h, but not at earlier time points, in APAP toxicity in mice [36
]. It was postulated that competition between APAP-GSH and ICG for excretion via the biliary route may underlie the hepatoprotection of ICG. Thus, it is possible that the higher accumulation of oxidized GSH in the ICG treated mice led to increased regeneration of reduced GSH, potentially limiting the effects of the toxic metabolite NAPQI. In the present study, ICG was administered late in the toxicity (24 h) at a point in time when ICG would not be expected to interfere with the biliary elimination of APAP and its conjugates [37
A primary finding of the present study was the preservation of hepatic function in APAP treated mice that received NAC treatment 1 h after APAP. The clearance of ICG was normal in these mice (). Interestingly, hepatic APAP protein adducts were elevated in the mice. Previous data from our laboratory utilizing immunohistochemical approaches for detection of APAP protein adducts (22) suggests that the timely administration of NAC prevented the rupture of hepatocytes and the release of both adducts and ALT into the blood (–). However, a 4 h delay in NAC treatment offered no protection from toxicity and no functional benefits. The time course for the development of toxicity in mice [24
] is highly compressed compared to the time course for the development of toxicity observed in humans in which evidence of hepatic injury may not be apparent until 24 h after the APAP ingestion [38
]. Thus, identifying time points in the progression of hepatic injury in the mouse model that reflect toxicity versus those that reflect liver function may be difficult due to the compressed time course of APAP toxicity in the mouse. Examination of ICG clearance at alternative NAC treatment times may be able to segregate the effect of NAC on parameters that represent injury versus function. Nevertheless, the data presented in this study may have relevance for future investigations testing novel therapies for APAP toxicity. Despite the diminishing efficacy of NAC at later stages of APAP toxicity in humans [17
], NAC is being utilized more widely in the clinical setting for non-APAP related causes of hepatotoxicity [1
]. Some data support the hypothesis that NAC has beneficial effects that extend beyond replacement of GSH. For example, Devlin et al
] found that NAC treatment substantially increased oxygen consumption and delivery, as well as the clearance of ICG, in human subjects with hepatic failure of diverse etiologies [37
]. In the present study, we noted that mice treated with NAC 1 h after APAP had a clearance for ICG that was comparable to that of the saline treated mice. Thus, the findings of the present study would be consistent with the hypothesis offered by Devlin et al
] that NAC may affect oxygen consumption and delivery.
We conclude that the clearance of ICG is a sensitive indicator of hepatic dysfunction resulting from APAP toxicity. Furthermore, the clearance of ICG is affected by the timing of NAC administration. Future studies of novel therapies for APAP toxicity could utilize ICG clearance to test the effect of these therapies on the restoration or maintenance of hepatic function.