A mouse model of severe halothane hepatitis based on human risk factors

Abstract

Halothane (2-bromo-2-chloro-1,1,1-trifluoro-ethane) is an inhaled anesthetic that induces severe, idiosyncratic liver injury, i.e., "halothane hepatitis," in approximately 1 in 20,000 human patients. We used known human risk factors (female sex, adult age, and genetics) as well as probable risk factors (fasting and inflammatory stress) to develop a murine model with characteristics of human halothane hepatitis. Female and male BALB/cJ mice treated with halothane developed dose-dependent liver injury within 24 h; however, the liver injury was severe only in females. Livers had extensive centrilobular necrosis, inflammatory cell infiltrate, and steatosis. Fasting rendered mice more sensitive to halothane hepatotoxicity, and 8-week-old female mice were more sensitive than males of the same age or than younger (4-week-old) females. C57BL/6 mice were insensitive to halothane, suggesting a strong genetic predisposition. In halothane-treated females, plasma concentration of tumor necrosis factor-alpha was greater than in males, and neutrophils were recruited to liver more rapidly and to a greater extent. Anti-CD18 serum attenuated halothane-induced liver injury in female mice, suggesting that neutrophil migration, activation, or both are required for injury. Coexposure of halothane-treated male mice to lipopolysaccharide to induce modest inflammatory stress converted their mild hepatotoxic response to a pronounced, female-like response. This is the first animal model of an idiosyncratic adverse drug reaction that is based on human risk factors and produces reproducible, severe hepatitis from halothane exposure with lesions characteristic of human halothane hepatitis. Moreover, these results suggest that a more robust innate immune response underlies the predisposition of female mice to halothane hepatitis.

Fig. 1.

Halothane-induced hepatotoxicity in male and female mice. A, plasma ALT activity evaluated 24 h after halothane treatment (intraperitoneal) of mice fed ad libitum (n = 3–6/group). *, significantly different from males given the same dose. #, significantly different from 7.5 mmol/kg-treated sex-matched animals. B and C, hematoxylin and eosin (H&E)-stained liver sections from 30 mmol/kg halothane-treated male and female mice, respectively. Liver section from the male mouse shows minimal necrosis, whereas the lesion is more severe in the female mouse. Labeled in picture are central vein (CV) and portal triad (PT).

Fig. 2.

Fasting enhances sensitivity to halothane-induced liver injury. Female mice were either fasted overnight or not and then given halothane at the doses indicated (n = 3–5/group). Blood was collected 24 h later for the determination serum ALT activity. *, significantly different from fed mice given the same dose. #, significantly different from 3.75-mmol/kg group.

Fig. 3.

Genetic background is a sensitivity factor for severe halothane hepatotoxicity in mice. Fasted female C57BL/6 and BALB/cJ mice were given halothane at the doses indicated (n = 3–5/group), and blood was collected 24 h later. A, ALT activity in plasma. B, immunoblot detection of TFA-protein adducts in liver homogenates from mice treated with 15 mmol/kg halothane. *, significantly different from BALB/cJ mice.

Fig. 4.

Lack of isoflurane-induced hepatotoxicity in female mice. Fasted female BALB/cJ mice were given halothane or isoflurane at the doses indicated (n = 3–5/group), and blood was collected at 12 h for plasma ALT activity. All isoflurane-treated mice had plasma ALT activities <50 U/l. *, significantly different from the respective halothane-treated group.

Fig. 5.

Age is a sensitivity factor for severe halothane hepatotoxicity in BALB/cJ mice. Fasted 4- and 8-week-old male and female BALBc/J mice (n = 5/group) were given 15 mmol/kg halothane i.p., and blood was collected 24 h later. *, significantly different from age-matched males. #, significantly different from 4-week-old females.

Fig. 6.

Development of halothane-induced liver injury. Fasted female and male BALB/cJ mice were given vehicle or 15 mmol/kg halothane i.p. (n = 4–6/group). Blood was collected at various times for the determination of plasma ALT activity. There was no time- or sex-related difference in ALT activity in the vehicle-treated mice, so the results were combined and represented as zero time. *, significantly different from male mice at the same time. #, significantly different from vehicle-treated animals (zero time).

Fig. 7.

Histopathology of livers from halothane-treated mice. female BALB/cJ mice were fasted overnight and then given vehicle or halothane (15 mmol/kg i.p.). Representative liver sections from mice treated 12 h earlier were examined after hematoxylin and eosin (H&E) staining or after immunohistochemical staining for PMNs, in which PMNs appear as pink cells. Liver sections taken from mice 6 h after halothane administration were stained with Oil Red O, in which lipid appears as red dots. Labeled in the picture are central vein (CV) and portal triad (PT). Photomicrographs were taken at 200× magnification.

Fig. 8.

Halothane metabolism is similar in male and female mice. Fasted male and female BALB/cJ mice were treated with vehicle or 15 mmol/kg halothane, and liver samples were collected 12 h later. A, representative liver section from a halothane-treated, male mouse stained immunohistochemically for TFA-adducts and visualized with green color. 4,6-Diamidino-2-phenylindole nuclear stain appears blue. B, ratio of positive pixels in the centrilobular and periportal regions [central vein (CV)/portal triad (PT)] (n = 3/group). C, immunoblot detection of TFA-protein adducts in liver homogenates.

Fig. 9.

Sex-specific difference in plasma TNF-α concentration after halothane exposure. Female and male BALB/cJ mice were given vehicle or 15 mmol/kg halothane (n = 4–6/group), and blood was collected at various times. Plasma TNF-α concentration was determined using an OptEIA mouse enzyme-linked immunosorbent assay kit (BD Biosciences). The average plasma concentration of TNF-α in vehicle-treated animals was less than 40 pg/ml. *, significantly different from males at the same time. #, significantly different from vehicle controls (zero time).

Fig. 10.

Hepatic PMN recruitment is sex- and strain-dependent. Female and male BALB/cJ mice were given vehicle or 15 mmol/kg halothane (n = 4–5/group), and female BALB/cJ and C57BL/6 mice were given 30 mmol/kg halothane (n = 4). The number of PMNs in liver sections was determined by counting PMNs in immunostained tissue. A, hepatic PMNs in male and female BALB/cJ mice. The number of PMNs was less than eight for vehicle-treated mice and did not change with time, so those values were combined and represented as zero time. *, significantly different from halothane-treated males. #, significantly different from vehicle controls (zero time). B, hepatic PMNs in female, BALB/cJ and C57BL/6 mice. *, significantly different from halothane-treated BALB/cJ mice.

Fig. 11.

CD18 neutralization attenuates severe halothane hepatotoxicity. Fasted female BALB/cJ mice were treated with 15 mmol/kg halothane i.p. and either saline, control rabbit serum (NRS), or anti-CD18 rabbit serum (CD18 RS intravenous) as described under Materials and Methods. Plasma was collected 12 h after halothane administration and evaluated for ALT activity (n = 6/group). *, significantly different from all other groups.

Fig. 12.

Inflammation enhances sensitivity to halothane hepatotoxicity in male BALB/cJ mice. Mice were treated with vehicle or halothane and then either 5 × 10⁶ EU/kg LPS or saline vehicle 6 h later (n = 3–8/group). Blood and liver samples were collected 24 h after halothane administration. A, plasma ALT activity. *, significantly different from halothane-treated and from LPS-treated animals. B, hematoxylin and eosin (H&E) section of liver from a representative halothane/LPS-cotreated mouse. C, immunohistochemical staining for PMNs in a cotreated mouse; PMNs stain bright pink and nuclei stain blue. Both photomicrographs were taken at 200× magnification. Labeled in the pictures are central vein (CV) and portal triad (PT).