Non-invasive differentiation of hepatic steatosis and steatohepatitis in a mouse model using nitroxyl radical as an MRI-contrast agent

Abstract

Drug-induced steatohepatitis is considered more serious than drug-induced hepatic steatosis, so that differentiating between the two is crucial in drug development. In addition, early detection of drug-induced steatohepatitis is considered important since recovery is possible with drug withdrawal. However, no method has been established to differentiate between the two. In the development of drug-induced steatohepatitis, reactive oxygen species (ROS) is excessively generated in the liver. It has been reported that ROS can be monitored with electron spin resonance (ESR) and dynamic nuclear polarization-magnetic resonance imaging (DNP-MRI) by using nitroxyl radicals, which are known to participate in various in vivo redox reactions. The decay/reduction rate, which is an index for monitoring nitroxyl radicals, has been reported to be increased in tissues with excessive ROS levels other than liver, but decreased in methionine choline deficient (MCD) diet-induced steatohepatitis with excess ROS. Therefore, looking to differentiate between drug-induced hepatic steatosis and steatohepatitis, we examined whether the reduction rate decreases in steatohepatitis other than the MCD-diet induced disease and whether the decrease could be detected by MRI. We used STAM™ mice in which hepatic steatosis and steatohepatitis developed sequentially under diabetic conditions. 3-carbamoyl-PROXYL (CmP), one of the nitroxyl radicals, was injected intravenously during the MRI procedure and the reduction rate was calculated. The reduction rate was significantly higher in early steatohepatitis than in hepatic steatosis and the control. Excess ROS in early steatohepatitis was detected by an immunohistochemical marker for ROS. Therefore, it was indicated that the increase or decrease in the reduction rate in steatohepatitis differs depending on the model, and early steatohepatitis could be noninvasively differentiated from hepatic steatosis using CmP in MRI. Since the change in direction of the reduction rate in steatohepatitis in clinical studies could be predicted by confirming the reduction rate in preclinical studies, the present method, which can be used consistently in clinical and preclinical studies, warrants consideration as a candidate monitoring method for differentiating between early drug-induced steatohepatitis and hepatic steatosis in drug development.

Keywords: Magnetic resonance imaging; Nitroxyl radicals; Reactive oxygen species; Steatohepatitis.

Graphical abstract

Fig. 1

Redox transformation of the nitroxyl radical. The nitroxyl radical is converted to hydroxylamine by reduction or by oxidation to an oxoammonium cation and further reduction. Those conversions are reversible and the equilibrium between the nitroxyl radical and hydroxylamine occurs after in vivo administration. The nitroxyl radical provides T1 contrast because it has an unpaired electron. The hydroxylamine doesn’t provide T1 contrast because it doesn’t have an unpaired electron.

Fig. 2

Phantom images. (A) T1-weighted phantom image with 3-Carbamoyl-PROXYL (0–50 mM). (B) The mean contrast to noise ratios (CNRs) increased with increasing dose. The doses plotted are the same as in A. The mean CNRs at ≥ 1 mM exceeded 5 and meets the definition that is apparently distinguishable from background.

Fig. 3

Histopathological images in the liver. (A, B, C, and D) Typical histopathological images in the hepatic steatosis group (A), steatohepatitis (NAS of 2) group (B), steatohepatitis (NAS of 3) group (C), and control group (D). Vacuolation in hepatocytes is observed in the hepatic steatosis and steatohepatitis groups. Ballooning cells and/or inflammatory cells are indicated by black arrowheads. Bar, 100 µm.

Fig. 4

4-hydroxynoneal (4-HNE) immunolabeling and analysis in the liver. (A, B, C, and D) Typical immunohistochemical images of 4-HNE in the hepatic steatosis group (A), steatohepatitis (NAS of 2) group (B), steatohepatitis (NAS of 3) group (C), and control group (D). Immunolabeling of 4-HNE in the cytoplasm is indicated by white arrowheads in hepatocytes and black arrowheads in mononuclear cells. Bar, 30 µm. (E) Comparison of the 4-HNE positive area ratio in the steatohepatitis (NAS of 3) group to that in other groups. Significantly different from the control group: **p < 0.01, from the hepatic steatosis group: †† p < 0.01, and from the steatohepatitis group (NAS of 2): ‡‡ p < 0.01 (Dunnett’s test). Bars indicate the mean values.

Fig. 5

T1-weighted sequential images with 3-Carbamoyl-PROXYL (CmP) and its analysis in the liver. (A) Typical T1-weighted sequential images with CmP. From the left, images are of the control liver, steatotic liver, and steatohepatitic liver (NAS of 3). Circle indicates region of interest. Hyperintensity of the liver in T1-weighted MRI was lost earlier in the steatohepatitic liver (NAS of 3) than in the control liver and steatotic liver. (B) Images show the reduction rate per pixel for three mice in A. White and black, the extremes of the color spectrum, represent the highest and lowest reduction rate, respectively. The color of the whole liver is mostly yellowish green in the steatohepatitic liver (NAS of 3), while it is mostly light blue in the control liver and steatotic liver. It showed that the reduction rate in the steatohepatitic liver (NAS of 3) was higher than in the other two. (C) The graph shows the sequential signal intensity change rate in the liver. Semilogarithmic plots of signal intensity change rate in the control liver (squares), steatotic liver (crosses), and steatohepatitic liver (NAS of 3; circles). The reduction rate was obtained from the slope of the linear regression line, and the range of data used for analysis is indicated by blue, green, and yellow dot plots. The graph shows that the slope was higher in steatohepatitic liver (NAS of 3) than in the control liver and steatotic liver. (D) The vertical dot plot shows a significantly higher reduction rate in the liver in the steatohepatitis (NAS of 3) group than in other groups. Significantly different from the control group: **p < 0.01, from the hepatic steatosis group: †† p < 0.01, and from the steatohepatitis group (NAS of 2): ‡‡ p < 0.01 (Dunnett’s test). Bars indicate the mean values.

Fig. 6

Reduction rate in the stomach. The vertical dot plot shows that no statistically significant difference was observed in the reduction rate in the stomach between the control group and the other groups or between the hepatic steatosis group and steatohepatitis (NAS of 3) group. Bars indicate the mean values.