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Key Event Title
|Level of Biological Organization|
Key Event Components
|catalytic activity||uroporphyrinogen decarboxylase||decreased|
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP||Point of Contact||Author Status||OECD Status|
|AHR activation-uroporphyria||KeyEvent||Amani Farhat (send email)||Open for citation & comment||WPHA/WNT Endorsed|
|All life stages||Not Specified|
Key Event Description
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. 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), which are normally only present in trace amounts.
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. 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 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. This enzyme assay classically utilizes milliliter quantities but has been modified to a microassay, minimizing cost and enhancing sensitivity.
Another method based on reverse-phase HPLC was developed. This assay system uses either uroporphyrinogen III or pentacarboxyporphyrinogen I as substrate and liver homogenate in sucrose treated with a suspension of cellulose phosphate as enzyme source.
Domain of Applicability
UROD inhibition has been measured in mouse rat and human liver, Japanese quail kidney and chicken erythrocytes and hepatocytes.
- ↑ Elder, G. H., and Roberts, A. G. (1995). Uroporphyrinogen decarboxylase. J Bioenerg. Biomembr. 27 (2), 207-214.
- ↑ Frank, J., and Poblete-Gutierrez, P. (2010). Porphyria cutanea tarda--when skin meets liver. Best. Pract. Res. Clin Gastroenterol. 24 (5), 735-745.
- ↑ Smith, A. G., and Elder, G. H. (2010). Complex gene-chemical interactions: hepatic uroporphyria as a paradigm. Chem. Res. Toxicol. 23 (4), 712-723.
- ↑ 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.
- ↑ 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
- ↑ 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.
- ↑ 7.0 7.1 Phillips, J. D., and Kushner, J. P. (2001). Measurement of uroporphyrinogen decarboxylase activity. Curr. Protoc. Toxicol. Chapter 8, Unit.
- ↑ 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.
- ↑ 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.
- Smith, A. G., Francis, J. E., Kay, S. J., and Greig, J. B. (1981) Hepatic toxicity and uroporphyrinogen decarboxylase activity following a single dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin to mice. Biochem. Pharmacol. 30 (20), 2825-2830.
- Rios de Molina, M.D., Wainstok de Calmanovici, R., San Martin de Viale, L.C. (1980) Investigations of the presence of porphyrinogen carboxy-lase inhibitor in the liver of rats intoxicated with hexachlorobenzene. Int. J. Biochem. 12, 1027-32.
- Miranda, C.L., Henderson, M.C., Wang, J.-L, Nakaue, H.S., and Buhler, D.R. (1992) Comparative effects of the polychlorinated biphenyl mixture, Aroclor 1242, on porphyrin and xenobiotic metabolism in kidney of Japanese quial and rat. Comp. Biochem. Physiol. 103C(1), 149-52.
- Kawanishi, S., Seki, Y., and Sano, S. (1983) Uroporphyrinogen decarboxilase: Purification, properties, and inhibition by polychlorinated biphenyl isomers. J. Biol. Chem. 258(7), 4285-92.
- Lambrecht, R. W., Sinclair, P. R., Bement, W. J., Sinclair, J. F., Carpenter, H. M., Buhler, D. R., Urquhart, A. J., and Elder, G. H. (1988). Hepatic uroporphyrin accumulation and uroporphyrinogen decarboxylase activity in cultured chick-embryo hepatocytes and in Japanese quail (Coturnix coturnix japonica) and mice treated with polyhalogenated aromatic compounds. Biochem. J. 253(1), 131-138.
- Francis, J. E., & Smith, A. G. (1984). Assay of mouse liver uroporphyrinogen decarboxylase by reverse-phase high-performance liquid chromatography. Analytical biochemistry, 138(2), 404-410.