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Event Title

UROD, Inhibition

Key Event Overview

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AOPs Including This Key Event

AOP Name Event Type Essentiality
AhR activation leading to uroporphyria KE Strong

Taxonomic Applicability

Name Scientific Name Evidence Links

Level of Biological Organization

Biological Organization

How this Key Event works

Figure 1: Disruption of the normal heme biosynthesis pathway by uroporphyrinogen decarboxylase (UROD) inhibition. Formation of the inhibitor (suggested as being uroporphomethene) is thought to require the action of the phase I metabolizing enzyme, CYP1A2. Synergistic induction of ALA synthase 1 and increases in oxidative stress (reactive oxygen species (ROS)), caused by alcohol, estrogens and xenobiotics, potentiate the accumulation of porphyrins and therefore the porphyric phenotype. (Modified from Caballes (2012) Liver Int. 32 (6), 880-893.)

Uroporphyrinogen decarboxylase (UROD) is the fifth enzyme in the heme biosynthesis pathway and catalyzes the step-wise conversion of uroporphyrinogen to coproporphyrinogen. Each of the four acetic acid substituents is decarboxylated in sequence with the consequent formation of hepta-, hexa-, and pentacarboxylic porphyrinogens as intermediates[1]. Impairment of this enzyme, either due to heterozygous mutations in the UROD gene or chemical inhibition of the UROD protein, leads to accumulation of uroporphyrins (and other highly carboxylated porphyrins) and ultimately a disorder known as uroporphyria[2]. Sufficient overproduction of porphyrins to cause symptoms does not usually occur until hepatic UROD activity is reduced by at least 70%[3].

Several lines of evidence support the concept that a UROD inhibitor is formed within the liver by the oxidation of uroporphyrinogen. Phillips et al.[4] identified this inhibitor as being uroporphomethene using a murine model for porphyria; however, their interpretation of the mass spectroscopy results has been criticized as inaccurate[5], leaving the exact characterization of the UROD inhibitor unresolved. None the less, it has been demonstrated that inhibitor formation is increased by (a) the induction/activation of the phase I metabolizing enzyme cytochrome P4501A2 (orthologous to avian CYP1A5), (b) iron loading, (c) alcohol excess, and (d) estrogen therapy (Figure 1)[6]. A negative-feedback loop exists in which the end-product (heme) represses the enzyme ALA synthase 1 and prevents excess formation of heme. When UROD activity is low, the regulatory heme pool is potentially depleted, causing a repression of the negative feedback loop, thereby increasing levels of precursors and furthering the accumulation of porphyrins.

How it is Measured or Detected

Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above. All other methods, including those well established in the published literature, should be described here. Consider the following criteria when describing each method: 1. Is the assay fit for purpose? 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? 3. Is the assay repeatable? 4. Is the assay reproducible?

Due to the high instability of porphyrinogens, they must be synthesized as an integral part of the enzyme assay for use as a substrate. Uroporphyrinogen can either be generated by enzymatic synthesis or chemical reduction[7]. The former makes use of bacterial porphobilinogen deaminase to prepare the porphyrinogen substrate and the latter often utilizes sodium amalgam or sodium borohydride under an inert gas. Chemical reduction however often involves large quantities of mercury or extremely alkaline conditions and requires significant purification before the enzyme assay can be performed. Bergonia and colleagues[8] suggest palladium on carbon (Pd/C) to be the most efficient and environmentally friendly chemical preparation of porphyrinogens as Pd/C is more stable than sodium amalgam and can easily be removed by filtration, eliminating the need for laborious purification.

Once uroporphyrinogen is synthesized it is co-incubated with UROD under standardized conditions. The reaction is then stopped, reaction products and un-metabolized substrate are esterified, and the porphyrin esters are separated and quantified using high performance liquid chromatography[7]. This enzyme assay classically utilizes milliliter quantities but has been modified to a microassay, minimizing cost and enhancing sensitivity[9].

Evidence Supporting Taxonomic Applicability


  1. Elder, G. H., and Roberts, A. G. (1995). Uroporphyrinogen decarboxylase. J Bioenerg. Biomembr. 27 (2), 207-214.
  2. Frank, J., and Poblete-Gutierrez, P. (2010). Porphyria cutanea tarda--when skin meets liver. Best. Pract. Res. Clin Gastroenterol. 24 (5), 735-745.
  3. Smith, A. G., and Elder, G. H. (2010). Complex gene-chemical interactions: hepatic uroporphyria as a paradigm. Chem. Res. Toxicol. 23 (4), 712-723.
  4. Phillips, J. D., Bergonia, H. A., Reilly, C. A., Franklin, M. R., and Kushner, J. P. (2007). A porphomethene inhibitor of uroporphyrinogen decarboxylase causes porphyria cutanea tarda. Proc. Natl. Acad. Sci. U. S. A 104 (12), 5079-5084.
  5. Danton, M., and Lim, C. K. (2007). Porphomethene inhibitor of uroporphyrinogen decarboxylase: analysis by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry. Biomed. Chromatogr. 21 (7), 661-663
  6. Caballes F.R., Sendi, H., and Bonkovsky, H. L. (2012). Hepatitis C, porphyria cutanea tarda and liver iron: an update. Liver Int. 32 (6), 880-893.
  7. 7.0 7.1 Phillips, J. D., and Kushner, J. P. (2001). Measurement of uroporphyrinogen decarboxylase activity. Curr. Protoc. Toxicol. Chapter 8, Unit.
  8. Bergonia, H. A., Phillips, J. D., and Kushner, J. P. (2009). Reduction of porphyrins to porphyrinogens with palladium on carbon. Anal. Biochem. 384 (1), 74-78.
  9. Jones, M. A., Thientanavanich, P., Anderson, M. D., and Lash, T. D. (2003). Comparison of two assay methods for activities of uroporphyrinogen decarboxylase and coproporphyrinogen oxidase. J Biochem. Biophys. Methods 55 (3), 241-249.