Histochem Cell Biol (2008) 129:735–749 743 Fig. 3 MDBs formed in DDC- fed animals resemble inclusion bodies observed in human dis- eases. Liver sections were dou- ble labeled with antibodies to K8/K18 (green) and p62 (red). Samples from mouse fed DDC for 12 weeks (d) and from a pa- tient with alcoholic steatohepati- tis (b) exhibit multiple irregularly shaped inclusion bodies, which appear yellow due to presence of both epitopes. In contrast, control human (a) and mouse liver (c) display an un- aVected keratin network with no apparent p62 staining. Note that DDC feeding leads to deposition of protoporphyrin, which can be seen as occasional blue pigment in (d) Zatloukal et al., unpublished data). During DDC detoxiWca- tion, N-methylprotoporphyrin is formed through transfer of a methyl group to heme moieties (Tephly et al. 1981). N-methylprotoporphyrin subsequently acts as a potent inhib- itor of ferrochelatase, thereby causing porphyria (Tephly et al. 1980). Accordingly, MDB formation and DDC eVects can be eVectively attenuated by feeding S-adenosylmethio- nine, that is a compound involved in methyl group transfer (Li et al. 2008). This is reminiscent of the situation in ASH, which is also associated with disrupted methyl group metab- olism (Schalinske and Nieman 2005). DDC targets mitochondria, where it reacts with cyto- chrome P450 (Marks et al. 1985). An unbiased microarray analysis identiWed cytochrome P450 (Cyp) 2a5 as a major gene induced both after DDC exposure and particularly after DDC re-challenge. Moreover, Cyp2e5 overexpression spacially coincides with MDB formation (Zatloukal et al., unpublished data). Cyp2a5 is a “leaky” cytochrome which produces reactive oxygen species (Lewis et al. 1989). In that sense, it resembles human Cyp2E1, which was impli- cated as a source of oxidative stress in the pathogenesis of ASH and NASH (Villeneuve and Pichette 2004). To address the role of mitochondrial stress in MDB for- mation, we re-fed DDC-primed mice with DDC alone or in combination with the mitochondria-targeted antioxidant mito Q (Smith et al. 2003). Mito Q co-administration atten- uated both MDB formation and DDC-induced liver dam- age. Therefore, mitochondrial oxidative stress seems to be involved in MDB formation in mice which is in good con- cordance to the mitochondrial dysfunction seen both in ASH and NASH patients (Pessayre 2007; Mantena et al. 2008; Zatloukal et al., unpublished data). Ad 2. Keratins are major constituents of MDBs and both altered K8/K18 expression and keratin modiWcation seems to aVect MDB formation (Zatloukal et al. 2007; Ku et al. 2007). Griseofulvin/DDC feeding leads to rapid induction of K8/K18 expression with disproportional K8 > K18 levels (Denk et al. 2000). The elevated K8/K18 ratio is crucial for MDB formation as shown in K18-knockout and K8 over- expressing animals, who are predisposed to MDB formation already upon short exposure to DDC and even form MDBs spontaneously during aging (Magin et al. 1998; Nakamichi et al. 2005). Accordingly, K8-null or K18 overexpressing mice are resistant to MDB formation and the protective function of K18 is not aVected by its phosphorylation status or mutation (Zatloukal et al. 2000; Harada et al. 2007). However, the exclusive MDB inducing property of K8 in vivo cannot be reproduced in vitro, where aggregates resem- 123