Volume 39, Issue 6, Pages 901-909 (December 2003) Extensive changes in liver gene expression induced by Hereditary Tyrosinemia type I are not normalized by treatment with 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3- cyclohexanedione (NTBC)  Marjanka C Luijerink, Saskia M.M Jacobs, Ellen A.C.M van Beurden, Leander P Koornneef, Leo W.J Klomp, Ruud Berger, Inge E.T van den Berg  Journal of Hepatology  Volume 39, Issue 6, Pages 901-909 (December 2003) DOI: 10.1016/S0168-8278(03)00433-1

Fig. 1 Tyrosine degradation pathway and hereditary tyrosinemia type I. The principal metabolites involved in the pathway are tyrosine, 4-hydroxyphenylpyruvate, homogentisic acid, maleylacetoacetate (MAA), and fumarylacetoacetate. The enzymes involved are, from top to bottom, tyrosine aminotransferase (TAT), p-hydroxyphenylpyruvate dioxygenase (HPD), homogentisic acid dioxygenase (HGD), maleylacetoacetate dioxygenase (MAI) and fumaryl acetoacetate hydrolase (FAH). In hereditary tyrosinemia type I, the enzyme FAH is deficient. FAA and MAA accumulate and cause extensive liver damage. The drug NTBC blocks the enzyme HPD in the second step of the pathway and therefore prevents acute liver disease. Journal of Hepatology 2003 39, 901-909DOI: (10.1016/S0168-8278(03)00433-1)

Fig. 2 Morphology of livers of Fah−/− mice compared to Fah+/− mice. Haematoxylin staining was performed on 8 μm paraformaldehyde fixed liver sections of Fah−/− (A, B) and Fah+/− (C, D) mice either withdrawn from NTBC for 14 days (A, C) or receiving NTBC (B, D). The HT-I affected mouse (A) shows increased amounts of enlarged hepatocytes (triangles), infiltrating cells (thin arrows), and remnants of dead cells (thick arrows), indicating that the HT-1 phenotype has been established. Fah−/− mice treated with NTBC only show a slight irregularity in size of hepatocytes (B). Original magnifications ×400. Journal of Hepatology 2003 39, 901-909DOI: (10.1016/S0168-8278(03)00433-1)

Fig. 3 RNA blot analysis of a subset of identified transcripts. Northern blot analysis was performed on RNA from livers of male (M) and female (F) Fah−/− and Fah+/− mice, either on or off NTBC as indicated. HT-I-affected mice are marked (*). Probes were generated by random primed labelling of 45 ng of PCR-amplified inserts of subtracted clones and radioactively labeled. RNA blots were hybridized as described in Section 2. (A) Transcripts induced in livers of Fah−/− mice off NTBC. Glutathione reductase, retinoblastoma binding protein 7, and tubulin beta 2 are induced in Fah−/− mice off NTBC, normal in Fah−/− mice on NTBC and not induced by NTBC itself. Peroxiredoxin is induced in Fah−/− mice off NTBC and slightly induced in Fah−/− and Fah+/− mice on NTBC. (B) Transcripts suppressed in livers of Fah−/− mice off NTBC. Apolipoprotein AII, argininosuccinate synthetase 1, complement component C3, and serine protease inhibitor 2 are suppressed in Fah−/− mice off NTBC and normalized by NTBC. SPOT14 is induced by NTBC, but suppressed in Fah−/− mice off and on NTBC. Major urinary protein II is male-specific, and suppressed in livers of male Fah−/− mice off NTBC. Ethidium bromide staining of the 18S band is used as control for the applied amount of total RNA. Journal of Hepatology 2003 39, 901-909DOI: (10.1016/S0168-8278(03)00433-1)

Fig. 4 Diagrams of expression profiles of distinct induced and suppressed transcripts in livers of HT-I affected mice relative to livers of healthy mice. Different SSH experiments were performed as was described in Section 2; SSH-A (A) compares liver cDNA pools of Fah−/− mice off NTBC to liver cDNA pools from Fah+/− mice, SSH-B (B) compares liver cDNA pools of Fah−/− mice, on and off NTBC. Distinct transcripts were divided into nine main categories according to the cellular roles of their encoded proteins, and subdivided as indicated. The inner and outer circles represent the different categories and sub-categories, respectively. Journal of Hepatology 2003 39, 901-909DOI: (10.1016/S0168-8278(03)00433-1)