Hepatocyte injury in tyrosinemia type 1 is induced by fumarylacetoacetate and is inhibited by caspase inhibitors

Abstract

Tyrosinemia type 1, caused by mutations in the fumarylacetoacetate hydrolase gene (Fah), is characterized by severe liver injury. We earlier developed a tyrosinemic mouse model with two genetic defects, Fah and 4-hydroxyphenylpyruvate dioxygenase (Hpd) deficiencies. Apoptosis of hepatocytes was induced and an acute onset of liver failure occurred after administration of homogentisic acid (HGA), the intermediate metabolite between the enzymes HPD and FAH. Cytochrome c was released from mitochondria prior to liver failure in the Fah-/- Hpd-/- double-mutant mice after the administration of HGA. In a cell-free system, the addition of fumarylacetoacetate induced the release of cytochrome c from the mitochondria. We also found that caspase inhibitors were highly effective in preventing the liver failure induced by HGA in the double-mutant mice. Therefore, fumarylacetoacetate apparently induces the release of cytochrome c, which in turn triggers activation of the caspase cascade in hepatocytes of subjects with hereditary tyrosinemia type 1.

Figure 1

Characterization of Fah^−/− Hpd^−/− double-mutant mice. (a) Scheme of tyrosine catabolism and the model mouse for tyrosinemia. The Fah^−/−Hpd^−/− mice were generated by intercrossing III and albino lethal mice. (b–d) Genotype analysis for Fah^−/−Hpd^−/− mice by PCR. Deletion in the c^14cos mice disrupted the Fah gene, resulting in the absence of exon 1 and 2 sequences (7, 11, 14). The Fah^−/− mice were identified by the absence of exon 2 sequences (111 bp) and the presence of exon 8 sequences (132 bp) of the Fah gene after PCR amplification of regions containing each exon, respectively (11). The results of PCR amplification of regions containing exon 2 (b) and exon 8 (c) of the Fah gene are shown. (d) Results of PCR amplification and restriction enzyme digestion of a region containing exon 7 of the Hpd gene. The left lane contains an undigested fragment with normal sequences (386 bp) after treatment with the restriction enzyme HindIII. Lanes 2–4 show products digested by HindIII because of the mutated sequences of the Hpd⁻ allele (272 and 114 bp). DNA fragments were analyzed by electrophoresis on 4% NuSieve agarose gel (FMC BioProducts) and stained with ethidium bromide. (e and f) Immunoblot analysis of FAH (e) and HPD (f) protein in the liver. FAH and HPD proteins were absent from the liver from the Fah^−/−Hpd^−/− mouse.

Figure 2

Effect of HGA or exogenous expression of human HPD on cell viability in primary cultured hepatocytes derived from III and Fah^−/− Hpd^−/− mice. (a) The primary cultured hepatocytes were incubated in medium containing various concentrations of neutralized HGA. The viability of cells was calculated 24 h after exposure to HGA. ○, Means of survival for hepatocytes from III mice (n = 4); ■, those from the Fah^−/− Hpd^−/− mice (n = 6). The bars represents SD. Fah^−/− Hpd^−/− hepatocytes are significantly vulnerable to HGA, in a dose-dependent manner, compared with control III hepatocytes. (b) The primary cultured hepatocytes were transfected with recombinant adenovirus bearing human Hpd cDNA (AdHPD) to express HPD exogenously. The primary cultured hepatocytes were treated with various amounts of AdHPD, and the viable cells were calculated 24 h after infection by the recombinant virus. ○, means of survival for hepatocytes from III mice (n = 4); ■, those from the Fah^−/− Hpd^−/− mice (n = 6). The Fah^−/− Hpd^−/− hepatocytes are significantly and dose-dependently vulnerable to the infection by AdHPD, compared with control III hepatocytes. (c) Time-dependent progress of apoptotic death of primary cultured hepatocytes from Fah^−/− Hpd^−/− mice in the presence of 1 mM HGA. DNA samples were prepared from the cells after appropriate intervals and analyzed as described in the text. Lane M, DNA size markers. Fragmentation of genomic DNA from the hepatocytes of Fah^−/− Hpd^−/− mice was evident 6 h after the administration of HGA.

Figure 3

Prevention of cell death by exogenous expression of FAH. (a) Exogenous expression of FAH by recombinant adenovirus bearing human FAH cDNA (AdFAH) in primary cultured hepatocytes. Lane 1, III cell; lane 2, Fah^+/− Hpd^−/− cell; Lane 3, Fah^−/− Hpd^−/− cell; and lane 4, Fah^−/− Hpd^−/− cells infected with AdFAH at moi 10. Twenty-four hours after infection, cells were harvested, sonicated, and subjected to SDS/PAGE. (b) Effect of AdFAH on HGA-induced apoptosis of primary cultured hepatocytes derived from Fah^−/− Hpd^−/− mice. The primary cultured hepatocytes were preinfected with AdFAH (■, n = 4) or AdOTC (○, n = 4), in various amounts. The viabilities of cultured hepatocytes, from either III or Fah^−/− Hpd^−/− mice, after treatment with various moi of AdFAH or AdOTC were similar (data not shown). Twenty-four hours after the infection of recombinant adenovirus, cell death was induced by 1 mM HGA. The viability of the cell was evaluated 24 h after the induction of cell death (adenovirus-infected but not HGA-treated control = 100%). The pretreatment of AdFAH protects Fah^−/− Hpd^−/− hepatocytes from HGA-induced apoptosis, in a dose-dependent manner. (c) Effect of AdFAH on AdHPD-induced apoptosis of primary cultured hepatocytes derived from Fah^−/− Hpd^−/− mice. The primary cultured hepatocytes were preinfected with AdFAH (■, n = 4) or AdOTC (○, n = 4), in various amounts. Twenty-four hours after treatment with the recombinant adenovirus, cell death was induced by infection with AdHPD at moi 10. Viability of cells was evaluated 24 h after the induction of cell death (AdFAH or AdOTC-infected but not HGA-treated control = 100%). Pretreatment with AdFAH protected Fah^−/− Hpd^−/− hepatocytes from AdHPD-induced apoptosis, in a dose-dependent manner.

Figure 4

Cytochrome c release from mitochondria in vivo and in vitro. (a) Immunoblot analysis of cytochrome c in the liver of Fah^−/− Hpd^−/− mice. Livers from variously treated mice were resected, and immediately mitochondrial and S-100 cytosolic fractions were prepared and subjected to SDS/PAGE and immunoblotting. Lane 1, untreated III mouse treated with HGA; lane 2, III mouse treated with HGA; lane 3, untreated Fah^−/− Hpd^−/− mouse; lane 4, Fah^−/− Hpd^−/− mouse 1 h after treatment with HGA; lane 5, Fah^−/− Hpd^−/− mouse 6 h after treatment with HGA; lane 6, DEVD-pretreated HGA-treated Fah^−/− Hpd^−/− mouse; lane 7, YVAD-pretreated HGA-treated Fah^−/− Hpd^−/− mouse. While a small amount of cytochrome c was detected in the cytosolic fraction (lanes 1–3), significant amounts of cytochrome c were detected in each lane in the case of cytosolic fractions of livers from Fah^−/− Hpd^−/− mice treated with HGA (lanes 4–7). The amounts of cytosolic cytochrome c remained unchanged by pretreatment with caspase inhibitors DEVD or YVAD (lanes 6 and 7). (b) Cytochrome c is released from control mitochondria in a cell-free system by the addition of cytosolic fraction from the HGA-treated Fah^−/− Hpd^−/− mouse. Mitochondria (1 mg) from control mice were incubated with S-100 fractions prefiltered to remove cytochrome c and other proteins, as described in the text. After incubation, the mitochondria and the supernatants were separated by centrifugation and analyzed for cytochrome c, using immunoblotting. Lane 1, III mouse treated with HGA; lane 2, untreated Fah^−/− Hpd^−/− mouse; lane 3, Fah^−/− Hpd^−/− mouse 1 h after treatment with HGA; lane 4, Fah^−/− Hpd^−/− mouse 6 h after treatment with HGA; lane 5, DEVD-pretreated HGA-treated Fah^−/− Hpd^−/− mouse; lane 6, YVAD-pretreated HGA-treated Fah^−/− Hpd^−/− mouse. A significant release of cytochrome c from mitochondria into the medium was evident in the fractions from HGA-treated Fah^−/− Hpd^−/− mice, whether pretreated with caspase inhibitors or not (lanes 3, 4, 5, and 6). (c) Cytochrome c is released from mitochondria by purified FAA. Mitochondria (1 mg) from the control mouse were incubated in the presence of purified FAA. After the incubation, the supernatant was analyzed for cytochrome c. Lane 1, buffer A only; lane 2, 1 μM FAA; lane 3, 10 μM FAA; lane 4, 100 μM FAA; lane 5, 10 μM HGA; lane 6, 100 μM HGA. Incubation of the purified FAA with control mitochondria resulted in release of cytochrome c into the medium.

Figure 5

Prevention of cell death by treatment with caspase inhibitors. (a) Effect of caspase inhibitors on HGA-induced apoptosis of primary cultured hepatocytes derived from Fah^−/− Hpd^−/− mice. Two hours after the treatment with the inhibitors, cell death was induced by 1 mM HGA. Pretreatment with caspase inhibitors, YVAD (•, n = 4) or DEVD (■, n = 4), protected Fah^−/− Hpd^−/− hepatocytes from HGA-induced apoptosis in a dose-dependent manner. Open symbols indicate means of survival rate for hepatocytes treated with YVAD (○, n = 3) or DEVD (□, n = 3) without HGA. (b) Effect of caspase inhibitors on AdHPD-induced apoptosis of primary cultured hepatocytes derived from Fah^−/− Hpd^−/− mice. Two hours after treatment with the inhibitors, cell death was induced by infection with AdHPD at moi 10. Pretreatment with caspase inhibitors YVAD (•, n = 4) or DEVD (■, n = 4), protects Fah^−/− Hpd^−/− hepatocytes from AdHPD-induced apoptosis in a dose-dependent manner. Open symbols indicate means of survival for hepatocytes that were treated by YVAD (○, n = 3) or DEVD (□, n = 3) without AdHPD.

Figure 6

HGA-induced apoptosis of hepatocytes in the Fah^−/− Hpd^−/− mice and in vivo protective effect of caspase inhibitors. The liver sections were obtained from variously treated mice and investigated as described in the text. The controls were untreated III (a), III treated with HGA (b), and untreated Fah^−/− Hpd^−/− (c). The positive signals of in situ detection of DNA fragmentation were seen in 30–80% of hepatocytes in the sections from Fah^−/− Hpd^−/− mice treated with HGA (800 mg/kg body weight) (d), 2–4% of YVAD-pretreated Fah^−/− Hpd^−/− treated with HGA (e), and below 1% in DEVD-pretreated Fah^−/− Hpd^−/− treated with HGA (f), compared with below 1% of the controls (a, III without HGA; b, III with HGA; c, Fah^−/− Hpd^−/− without HGA.). (Original magnification, ×400.)