Michael R. Franklin

Most rodent models of porphyria cutanea tarda (PCT) share in common the administration of iron and agents that induce transcription of cytochrome P450s. Dissection of changes related to porphyrin accumulation required generation of a genetic model free from exogenous precipitants. Mice heterozygous for a null Urod mutation and homozygous for null Hfe alleles spontaneously develop major increases in hepatic and urinary porphyrins several months after weaning but the high % uroporphyrin signature of PCT is established earlier, before total hepatic and urinary porphyrins rise. Total porphyrin levels eventually plateau at higher levels in females than in males. Porphyrinogens were the dominant tetrapyrroles accumulating in hepatocytes. Hepatic Urod activity is markedly reduced but total hepatic heme content does not diminish. Microsomal heme, however, is reduced and in vitro metabolism of prototype substrates showed that some but not all cytochrome P450 activities are reduced. High hepatic levels of uroporphyrinogen are also associated with increased glutathione S-transferase activity and elevated mRNA of 2 transporters, Abcc1 and Abcc4. This murine model of familial PCT affords the opportunity to study changes in porphyrinogen and porphyrin accumulation and transport in the absence of exogenous factors that alter P450 activity and transmembrane transporters.

Inhibition of cytochrome P450(CYP)-selective reactions in a single human and a single mouse hepatic microsome preparation by fourteen 1-substituted imidazoles provides a simultaneous ranking of reaction susceptibility to a specific imidazole and the relative inhibitory potency of the imidazoles for a given reaction. CYP3A4/5 activity was inhibited (IC₅₀ 50 values for CYP3A4/5 50 value 50 50 values than in human microsomes.

In epidemiology and human supplementation studies, as well as many animal models, selenium has shown antitumorigenic activity. The mechanism of action, however, has not been satisfactorily resolved. Selenium supplementation affects many enzymes in addition to those where selenocysteine is an essential component. Such enzymes include cytoprotective detoxifying enzymes, and the regulation of these enzymes by a set of 2‐substituted selenazolidine‐4(R)‐carboxylic acids (SCAs) has been investigated. Following seven consecutive daily doses of these prodrugs of L‐selenocysteine, changes in hepatic enzyme activities and/or mRNA levels of glutathione transferase (GST), microsomal epoxide hydrolase (mEH), NAD(P)H‐quinone oxidoreductase (NQO), UDP‐glucuronosyltransferase (UGT), glutathione peroxidase (GPx), and thioredoxin reductase (TR) have been observed. Among the enzymes examined, UGTs and GPx were found to be the least affected. Among the compounds, 2‐oxoSCA produced the most changes and 2‐phenylSCA produced the least, none. For no two compounds was the pattern of changes identical, and for a single compound, few changes were reproduced in common by the two routes of administration investigated. In general, more changes were elicited following intraperitoneal (i.p.) administration than with the intragastric (i.g.) route. This dominance was typified by 2‐butylSCA and 2‐cyclohexylSCA where enzyme activity elevations (TR and mEH with both, NQO with 2‐butylSCA) were seen only with the i.p. route. With 2‐oxoSCA, however, GST, TR, and NQO activities were found to be elevated independent of route. Only with GST (both routes) and TR (i.p. route), elevations in mRNAs accompanied the 2‐oxoSCA elicited elevations of activities at the time of sacrifice. For some enzymes, most notably mEH with compounds administered i.p., elevations in mRNAs were not manifest as increased enzyme activity. Thus, although constituting a closely related series of compounds, each 2‐substituted SCA produced its own unique pattern of changes, and for most members, changes were predominant following i.p. administration. © 2006 Wiley Periodicals, Inc. J Biochem Mol Toxicol 20:292–301, 2006; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20148

Several methods have been used to develop rodent models with the hepatic manifestations of porphyria cutanea tarda (PCT). Acute iron administration or mutations of the hemochromatosis gene (Hfe) have been used to generate hepatic siderosis, a nearly uniform finding in PCT. Heterozygosity for a null mutation at the uroporphyrinogen decarboxylase (Uro-D+/−) locus has been developed to mimic familial PCT in humans. This study examines the interplay of these 2 genetic risk factors and their influence, alone and combined with polychlorinated-biphenyl exposure. Neither an Hfe-null mutation nor iron-dextran administration alone or in combination with polychlorinated biphenyl exposure was porphyrinogenic in a 3-week model using mice wild-type at the Uro-D locus. Homozygosity for an Hfe-null mutation significantly elevated hepatic iron but not to the extent seen with parenteral iron-dextran administration. Homozygosity for an Hfe-null mutation but not iron-dextran administration was porphyrinogenic in animals heterozygous for the Uro-D mutation. Polychlorinated biphenyls were also porphyrinogenic in these animals. Uroporphyria in Uro-D+/− animals was exacerbated by combinations of the homozygous Hfe-null mutation and polychlorinated biphenyls and iron-dextran and polychlorinated biphenyls. In all cases in which uroporphyria developed, a greater degree of experimental uroporphyria was seen in female animals. All elevated hepatic uroporphyrin concentrations were accompanied by depressed uroporphyrinogen decarboxylase activity and the presence of a factor in cytosol that inhibits recombinant human uroporphyrinogen decarboxylase. In conclusion, the expression of the uroporphyric phenotype, dependent on the susceptibility imparted by a genetic mutation, provides a uniquely facile model for dissecting the molecular pathogenesis of the disease.

Selective induction of Phase II over Phase I drug‐metabolizing enzymes has been proposed as a mechanism for reduction of chemical carcinogenesis. Enzymes likely to play a role in this amelioration include the glutathione S‐transferases (GSTs) and among compounds that selectively induce key GSTs are tert‐butylhydroquinone (tBHQ) and oltipraz [4‐methyl‐5‐(2‐pyrazinyl)‐3H‐1,2‐dithiole‐3‐thione]. In vivo, and in hepatoma cells (H4IIE), these two agents induce rat GSTA2 mRNA to a similar extent. However, with a luciferase reporter construct containing 1651 bp of the proximal 5′ flanking region of the rGSTA2 gene in the same cell line and under similar conditions, luciferase activity was induced to a much greater extent by tBHQ than by oltipraz. A similar large intercompound differential was seen with reporter constructs containing either the rGSTA2 ARE enhancer and HNF1 site (−872 to −582) or XRE enhancer and HNF1 site (−1110 to −812). In H4IIE cells, the rGSTA2 mRNA response to each agent was completely inhibited by 1 μM actinomycin‐D cotreatment. With 1 μM cycloheximide cotreatment however, some induction by tBHQ remained, while induction by oltipraz was completely abolished. The induction response to tBHQ but not oltipraz was augmented by pretreatment with PD98059, a MEK1/2 specific inhibitor. Notwithstanding induction characteristics in common, oltipraz, and tBHQ have sufficient dissimilarities to indicate that rGSTA2 upregulation by the two agents is not identical. © 2002 Wiley Periodicals, Inc. J Biochem Mol Toxicol 16:154–161, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.10033