SNAPSHOT

Created at: 2017-02-09 12:59

AOP ID and Title:

AOP 131: AhR activation leading to uroporphyria
Short Title: AHR activation-uroporphyria

Authors

Amani Farhat and Gillian Manning

Major contributor

Sean W. Kennedy


Status

Author status OECD status OECD project SAAOP status
Open for comment. Do not cite EAGMST Under Review 1.7 Included in OECD work plan

Abstract

Hepatic uroporphyria is a disorder where the disturbance of heme biosynthesis results in accumulation and excretion of uroporphyrin, heptacarboxylic acid and hexacarboxylic acid: collectively referred to as highly carboxylated porphyrins (HCPs)[1][2][3]. The disorder can be genetically acquired, due to a dysfunction in any of the 7 enzymes involved in the heme biosynthesis pathway [4], or may be chemically induced, which involves the inhibition of uroporphyrinogen decarboxylase (UROD). This adverse outcome pathway (AOP) describes the linkages leading to chemically induced porphyria through the activation of the aryl hydrocarbon receptor (AHR), a transcription factor that plays important endogenous roles in reproduction, liver and heart development, cardiovascular function, immune function and cell cycle regulation [5][6][7][8][9][10][11][12][13][14]. This AOP was developed in accordance with OECD guidelines and demonstrates a high degree of confidence as a qualitative AOP. The quantitative understanding of this AOP however is not yet complete, preventing the accurate prediction of uroporphyria from lower level key events.



Summary of the AOP

Molecular Initiating Event

Title Short name
Activation, AHR Activation, AHR

18: Activation, AHR

Short Name: Activation, AHR

AOPs Including This Key Event

AOP ID and Name Event Type
21: AhR activation leading to embryo toxicity in fish MolecularInitiatingEvent
57: AhR activation leading to hepatic steatosis MolecularInitiatingEvent
131: AhR activation leading to uroporphyria MolecularInitiatingEvent
150: Aryl hydrocarbon receptor activation leading to embryolethality via cardiotoxicty MolecularInitiatingEvent

Stressors

Name
Benzidine
Dibenzo-p-dioxin
Polychlorinated biphenyl
Polychlorinated dibenzofurans
Hexachlorobenzene

Evidence for Perturbation of this Molecular Initiating Event by Stressor

The AHR can be activated by several structurally diverse chemicals, but binds preferentially to planar halogenated aromatic hydrocarbons and polycyclic aromatic hydrocarbons. Dioxin-like compounds (DLCs), which include polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and certain polychlorinated biphenyls (PCBs), are among the most potent AHR ligands[38]. Only a subset of PCDD, PCDF and PCB congeners has been shown to bind to the AHR and cause toxic effects to those elicited by TCDD. Until recently, TCDD was considered to be the most potent DLC in birds[39]; however, recent reports indicate that 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) is more potent than TCDD in some species of birds.[40][13][41][21][42][43] When screened for their ability to induce aryl hydrocarbon hydroxylase (AHH) activity, dioxins with chlorine atoms at a minimum of three out of the four lateral ring positions, and with at least one non-chlorinated ring position are the most active[44]. Of the dioxin-like PCBs, non-ortho congeners are the most toxicologically active, while mono-ortho PCBs are generally less potent[45][9]. Chlorine substitution at ortho positions increases the energetic costs of assuming the coplanar conformation required for binding to the AHR [45]. Thus, a smaller proportion of mono-ortho PCB molecules are able to bind to the AHR and elicit toxic effects, resulting in reduced potency of these congeners. Other PCB congeners, such as di-ortho substituted PCBs, are very weak AHR agonists and do not likely contribute to dioxin-like effects [9].

The role of the AHR in mediating the toxic effects of planar hydrophobic contaminants has been well studied, however the endogenous role of the AHR is less clear [1]. Some endogenous and natural substances, including prostaglandin PGG2 and the tryptophan derivatives indole-3-carbinol, 6-formylindolo[3,2-b]carbazole (FICZ) and kynurenic acid can bind to and activate the AHR. [6][46][47][48][49] The AHR is thought to have important endogenous roles in reproduction, liver and heart development, cardiovascular function, immune function and cell cycle regulation [50][38][51][52][53][54][46][55][56][57] and activation of the AHR by DLCs may therefore adversely affect these processes.



Biological Organization

Level of Biological Organization
Molecular

The AHR structure has been shown to contribute to differences in species sensitivity to DLCs in several animal models. In 1976, a 10-fold difference was reported between two strains of mice (non-responsive DBA/2 mouse, and responsive C57BL/6 14 mouse) in CYP1A induction, lethality and teratogenicity following TCDD exposure[3]. This difference in dioxin sensitivity was later attributed to a single nucleotide polymorphism at position 375 (the equivalent position of amino acid residue 380 in chicken) in the AHR LBD[30][19][31]. Several other studies reported the importance of this amino acid in birds and mammals[32][30][22][33][34][35][31][36]. It has also been shown that the amino acid at position 319 (equivalent to 324 in chicken) plays an important role in ligand-binding affinity to the AHR and transactivation ability of the AHR, due to its involvement in LBD cavity volume and its steric effect[35]. Mutation at position 319 in the mouse eliminated AHR DNA binding[35].

The first study that attempted to elucidate the role of avian AHR1 domains and key amino acids within avian AHR1 in avian differential sensitivity was performed by Karchner et al.[22]. Using chimeric AHR1 constructs combining three AHR1 domains (DBD, LBD and TAD) from the chicken (highly sensitive to DLC toxicity) and common tern (resistant to DLC toxicity), Karchner and colleagues[22], showed that amino acid differences within the LBD were responsible for differences in TCDD sensitivity between the chicken and common tern. More specifically, the amino acid residues found at positions 324 and 380 in the AHR1 LBD were associated with differences in TCDD binding affinity and transactivation between the chicken (Ile324_Ser380) and common tern (Val324_Ala380) receptors[22]. Since the Karchner et al. (2006) study was conducted, the predicted AHR1 LBD amino acid sequences were been obtained for over 85 species of birds and 6 amino acid residues differed among species[14][37] . However, only the amino acids at positions 324 and 380 in the AHR1 LBD were associated with differences in DLC toxicity in ovo and AHR1-mediated gene expression in vitro[14][37][16]. These results indicate that avian species can be divided into one of three AHR1 types based on the amino acids found at positions 324 and 380 of the AHR1 LBD: type 1 (Ile324_Ser380), type 2 (Ile324_Ala380) and type 3 (Val324_Ala380)[14][37][16].


How this Key Event Works

The AHR Receptor

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that belongs to the basic helix-loop-helix Per-ARNT-Sim (bHLH-PAS) superfamily and consists of three domains: the DNA-binding domain (DBD), ligand binding domain (LBD) and transactivation domain (TAD)[1]. Other members of this superfamily include the AHR nuclear translocator (ARNT), which acts as a dimerization partner of the AHR [2][3]; Per, a circadian transcription factor; and Sim, the “single-minded” protein involved in neuronal development [4][5]. This group of proteins shares a highly conserved PAS domain and is involved in the detection of and adaptation to environmental change[4].

The molecular Initiating Event

The molecular mechanism of activation of gene expression by AHR1.

The molecular mechanism for AHR-mediated activation of gene expression is presented in the figure to the right. In its unliganded form, the AHR is part of a cytosolic complex containing heat shock protein 90 (HSP90), the HSP90 co-chaperone p23 and AHR-interacting protein (AIP)[6]. Upon ligand binding, the AHR migrates to the nucleus where it dissociates from the cytosolic complex and forms a heterodimer with ARNT[7]. The AHR-ARNT complex then binds to a xenobiotic response element (XRE) found in the promoter of an AHR-regulated gene and recruits co-regulators such as CREB binding protein/p300, steroid receptor co-activator (SRC) 1, SRC-2, SRC-3 and nuclear receptor interacting protein 1, leading to induction or repression of gene expression[6]. Expression levels of several genes, including phase I (e.g. cytochrome P450 (CYP) 1A, CYP1B, CYP2A) and phase II enzymes (e.g. uridine diphosphate glucuronosyl transferase (UDP-GT), glutathione S-transferases (GSTs)), as well as genes involved in cell proliferation (transforming growth factor-beta, interleukin-1 beta), cell cycle regulation (p27, jun-B) and apoptosis (Bax), are regulated through this mechanism [6][8][7][9].

AHR Isoforms

Two AHR isoforms (AHR1 and AHR2) have been identified in the black-footed albatross (Phoebastria nigripes), great cormorant (Phalacrocorax carbo) and domestic chicken (Gallus gallus domesticus)[10]. AHR1 mRNA levels were similar in the kidney, heart, lung, spleen, brain, gonad and intestine from the great cormorant but were lower in muscle and pancreas. AHR2 expression was mainly observed in the liver, but was also detected in gonad, brain and intestine. AHR1 levels represented a greater proportion (80%) of total AHR levels than AHR2 in the cormorant liver[10], and while both AHR isoforms bound to TCDD, AHR2 was less effective at inducing TCDD-dependent transactivation compared to AHR1 in black-footed albatross, great cormorant and domestic chicken[11][10].


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?

Transactivation Reporter Gene Assays (recommended approach)

Transient transfection transactivation

Transient transfection transactivation is the most common method for evaluating nuclear receptor activation[12]. Full-length AHR cDNAs are cloned into an expression vector along with a luminescent reporter gene construct (chimeric luciferase, P-lactamase or CAT reporter vectors containing the appropriate response elements for the gene of interest). There are a number of commercially available cell lines that can serve as recipients for these vectors (CV-1, HuH7, FLC-7, LS174T, LS180 MCF-7, HEC1, LLC-PK1, HEK293, HepG2, and Caco-2 cells)[12]. The greatest advantage of using transfected cells, rather than primary cell cultures, is the assurance that the nuclear receptor of interest is responsible for the observed induction. This would not be possible in a primary cell culture due to the co-regulation of different receptors for the same target genes. This model makes it easy to compare the responsiveness of the AHR across multiple species under the same conditions simply by switching out the AHR clone. One disadvantage to the transient transfection assay is the inherent variability associated with transfection efficiency, leading to a movement towards the use of stable cell lines containing the nuclear receptor and reporter gene linked to the appropriate response elements[12].

Luciferase reporter gene (LRG) assay

For demonstrative purposes, a luciferase reporter gene assay used to measure AHR1-mediated transactivation for avian species is described here. A monkey kidney cell line (Cos-7) that has low endogenous AHR1 expression was transfected with the appropriate avian AHR1 clone, cormorant ARNT1, a CYP1A5 firefly luciferase reporter construct and a Renilla luciferase vector to control for transfection efficiency. After seeding, the cells were exposed to DLC and luciferase activity was measured using a luminometer. Luminescence, which is proportional to the extent of AHR activation, is expressed as the ratio of firefly luciferase units to Renilla luciferase units [13]. This particular assay was modified from its original version to increase throughput efficiency; (a) cells were seeded in 96-well plates rather than Petri dishes or 48- well plates, (b) DLCs were added directly to the wells without changing the cell culture medium, and (c) the same 96-well plates were used to measure luminescence without lysing the cells and transferring to another plate. Similar reporter gene assays have been used to measure AHR1 activation in domestic and wild species of birds, including the chicken, ring-necked pheasant (Phasianus colchicus), Japanese quail (Coturnix japonica), great cormorant, black-footed albatross and peregrine falcon (Falco peregrinus).[14][13][15][11][16][17]

Transactivation in stable cell lines

Stable cell lines have been developed and purified to the extent that each cell contains both the nuclear receptor and appropriate reporter vector, eliminating the variability associated with transfection [12]. A stable human cell line containing a luciferase reporter driven by multiple dioxin response elements has been developed that is useful in identifying AhR agonists and antagonists[18]. An added benefit of this model is the potential to multiplex 3 assays in a single well: receptor activation, cell viability and enzyme activity[12]. Such assays are used extensively in drug discovery due to their high throughput efficiency, and may serve just as useful for risk assessment purposes.

Ligand-Binding Assays

Ligand binding assays measure the ability of a test compound to compete with a labeled, high-affinity reference ligand for the LBD of a nuclear receptor. It is important to note that ligand binding does not necessitate receptor activation and therefore cannot distinguish between agonists and antagonists; however, binding affinities of AHR ligands are highly correlated with chemical potencies[19] and can explain differences in species sensitivities to DLCs[20][21][22]; they are therefore worth mentioning. Binding affinity and efficacy have been used to develop structure-activity relationships for AHR disruption[20][23] that are potentially useful in risk-assessment. There has been tremendous progress in the development of ligand-binding assays for nuclear receptors that use homogenous assay formats (no wash steps) allowing for the detection of low-affinity ligands, many of which do not require a radiolabel and are amenable to high throughput screening[24][12]. This author however was unable to find specific examples of such assays in the context of AHR binding and therefore some classic radioligand assays are described instead.

Hydroxyapatite (HAP) binding assay

The HAP binding assay makes use of an in vitro transcription/translation method to synthesize the AHR protein, which is then incubated with radiolabeled TDCPP and a HAP pellet. The occupied protein adsorbs to the HAP and the radioactivity is measured to determine saturation binding. An additional ligand can also be included in the mixture in order to determine its binding affinity relative to TCDD (competitive binding)[25][22]. This assay is simple, repeatable and reproducible; however, it is insensitive to weak ligand-receptor interactions[22][21][26].

Whole cell filtration binding assay

Dold and Greenlee[27] developed a method to detect specific binding of TCDD to whole mammalian cells in culture and was later modified by Farmahin et al.[21] for avian species. The cultured cells are incubated with radiolabeled TCDD with or without the presence of a competing ligand and filtered. The occupied protein adsorbs onto the filter and the radioactivity is measured to determine saturation binging and/or competitive binding. This assay is able to detect weak ligand-receptor interactions that are below the detection limit of the HAP assay[21].

Protein-DNA Interaction Assays

The active AHR complexed with ARNT can be measured using protein-DNA interaction assays. Two methods are described in detail by Perez-Romero and Imperiale[28]. Chromatin immunoprecipitation measures the interaction of proteins with specific genomic regions in vivo. It involves the treatment of cells with formaldehyde to crosslink neighboring protein-protein and protein-DNA molecules. Nuclear fractions are isolated, the genomic DNA is sheared, and nuclear lysates are used in immunoprecipitations with an antibody against the protein of interest. After reversal of the crosslinking, the associated DNA fragments are sequenced. Enrichment of specific DNA sequences represents regions on the genome that the protein of interest is associated with in vivo. Electrophoretic mobility shift assay (EMSA) provides a rapid method to study DNA-binding protein interactions in vitro. This relies on the fact that complexes of protein and DNA migrate through a nondenaturing polyacrylamide gel more slowly than free DNA fragments. The protein-DNA complex components are then identified with appropriate antibodies. The EMSA assay was found to be consistent with the LRG assay in chicken hepatoma cells dosed with dioxin-like compounds[29].


References

  1. ↑ 1.0 1.1 Okey, A. B. (2007). An aryl hydrocarbon receptor odyssey to the shores of toxicology: the Deichmann Lecture, International Congress of Toxicology-XI. Toxicol.Sci. 98, 5-38.
  2. ↑ Hoffman, E. C., Reyes, H., Chu, F. F., Sander, F., Conley, L. H., Brooks, B. A., and Hankinson, O. (1991). Cloning of a factor required for activity of the Ah (dioxin) receptor. Science 252, 954-958.
  3. ↑ 3.0 3.1 Poland, A., Glover, E., and Kende, A. S. (1976). Stereospecific, high affinity binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin by hepatic cytosol. Evidence that the binding species is receptor for induction of aryl hydrocarbon hydroxylase. J.Biol.Chem. 251, 4936-4946.
  4. ↑ 4.0 4.1 Gu, Y. Z., Hogenesch, J. B., and Bradfield, C. A. (2000). The PAS superfamily: sensors of environmental and developmental signals. Annu.Rev.Pharmacol.Toxicol. 40, 519-561.
  5. ↑ Kewley, R. J., Whitelaw, M. L., and Chapman-Smith, A. (2004). The mammalian basic helix-loop-helix/PAS family of transcriptional regulators. Int.J.Biochem.Cell Biol. 36, 189-204.
  6. ↑ 6.0 6.1 6.2 6.3 Fujii-Kuriyama, Y., and Kawajiri, K. (2010). Molecular mechanisms of the physiological functions of the aryl hydrocarbon (dioxin) receptor, a multifunctional regulator that senses and responds to environmental stimuli. Proc.Jpn.Acad.Ser.B Phys.Biol.Sci. 86, 40-53.
  7. ↑ 7.0 7.1 Mimura, J., and Fujii-Kuriyama, Y. (2003). Functional role of AhR in the expression of toxic effects by TCDD. Biochimica et Biophysica Acta - General Subjects 1619, 263-268.
  8. ↑ Giesy, J. P., Kannan, K., Blankenship, A. L., Jones, P. D., and Newsted, J. L. (2006). Toxicology of PCBs and related compounds. In Endocrine Disruption Biological Bases for Health Effects in Wildlife and Humans (D. O. Norris, and J. A. Carr, Eds.), pp. 245-331. Oxford University Press, New York.
  9. ↑ 9.0 9.1 9.2 Safe, S. (1994). Polychlorinated biphenyls (PCBs): Environmental impact, biochemical and toxic responses, and implications for risk assessment. Critical Reviews in Toxicology 24, 87-149.
  10. ↑ 10.0 10.1 10.2 Yasui, T., Kim, E. Y., Iwata, H., Franks, D. G., Karchner, S. I., Hahn, M. E., and Tanabe, S. (2007). Functional characterization and evolutionary history of two aryl hydrocarbon receptor isoforms (AhR1 and AhR2) from avian species. Toxicol.Sci. 99, 101-117.
  11. ↑ 11.0 11.1 Lee, J. S., Kim, E. Y., and Iwata, H. (2009). Dioxin activation of CYP1A5 promoter/enhancer regions from two avian species, common cormorant (Phalacrocorax carbo) and chicken (Gallus gallus): association with aryl hydrocarbon receptor 1 and 2 isoforms. Toxicol.Appl.Pharmacol. 234, 1-13.
  12. ↑ 12.0 12.1 12.2 12.3 12.4 12.5 Raucy, J. L., and Lasker, J. M. (2010). Current in vitro high throughput screening approaches to assess nuclear receptor activation. Curr. Drug Metab 11 (9), 806-814.
  13. ↑ 13.0 13.1 13.2 Farmahin, R., Wu, D., Crump, D., Hervé, J. C., Jones, S. P., Hahn, M. E., Karchner, S. I., Giesy, J. P., Bursian, S. J., Zwiernik, M. J., and Kennedy, S. W. (2012). Sequence and in vitro function of chicken, ring-necked pheasant, and Japanese quail AHR1 predict in vivo sensitivity to dioxins. Environ.Sci.Technol. 46, 2967-2975.
  14. ↑ 14.0 14.1 14.2 14.3 Farmahin, R., Manning, G. E., Crump, D., Wu, D., Mundy, L. J., Jones, S. P., Hahn, M. E., Karchner, S. I., Giesy, J. P., Bursian, S. J., Zwiernik, M. J., Fredricks, T. B., and Kennedy, S. W. (2013b). Amino acid sequence of the ligand binding domain of the aryl hydrocarbon receptor 1 (AHR1) predicts sensitivity of wild birds to effects of dioxin-like compounds. Toxicol.Sci. 131, 139-152.
  15. ↑ Fujisawa, N., Ikenaka, Y., Kim, E. Y., Lee, J. S., Iwata, H., and Ishizuka, M. (2012). Molecular evidence predicts aryl hydrocarbon receptor ligand insensitivity in the peregrine falcon (Falco peregrines). European Journal of Wildlife Research 58, 167-175.
  16. ↑ 16.0 16.1 16.2 Manning, G. E., Farmahin, R., Crump, D., Jones, S. P., Klein, J., Konstantinov, A., Potter, D., and Kennedy, S. W. (2012). A luciferase reporter gene assay and aryl hydrocarbon receptor 1 genotype predict the embryolethality of polychlorinated biphenyls in avian species. Toxicol.Appl.Pharmacol. 263, 390-399.
  17. ↑ Mol, T. L., Kim, E. Y., Ishibashi, H., and Iwata, H. (2012). In vitro transactivation potencies of black-footed albatross (Phoebastria nigripes) AHR1 and AHR2 by dioxins to predict CYP1A expression in the wild population. Environ.Sci.Technol. 46, 525-533.
  18. ↑ Yueh, M. F., Kawahara, M., and Raucy, J. (2005). Cell-based high-throughput bioassays to assess induction and inhibition of CYP1A enzymes. Toxicol. In Vitro 19 (2), 275-287.
  19. ↑ 19.0 19.1 Poland, A., and Knutson, J. C. (1982). 2,3,7,8-tetrachlorodibenzo-p-dioxin and related halogenated aromatic hydrocarbons: examination of the mechanism of toxicity. Annu. Rev. Pharmacol. Toxicol. 22, 517-554.
  20. ↑ 20.0 20.1 Hestermann, E. V., Stegeman, J. J., and Hahn, M. E. (2000). Relative contributions of affinity and intrinsic efficacy to aryl hydrocarbon receptor ligand potency. Toxicol. Appl. Pharmacol 168 (2), 160-172.
  21. ↑ 21.0 21.1 21.2 21.3 21.4 Farmahin, R., Jones, S. P., Crump, D., Hahn, M. E., Giesy, J. P., Zwiernik, M. J., Bursian, S. J., and Kennedy, S. W. (2014). Species-specific relative AHR1 binding affinities of 2,3,4,7,8-pentachlorodibenzofuran explain avian species differences in its relative potency. Comp Biochem. Physiol C. Toxicol. Pharmacol. 161C, 21-25.
  22. ↑ 22.0 22.1 22.2 22.3 22.4 22.5 22.6 Karchner, S. I., Franks, D. G., Kennedy, S. W., and Hahn, M. E. (2006). The molecular basis for differential dioxin sensitivity in birds: Role of the aryl hydrocarbon receptor. Proc. Natl. Acad. Sci. U. S. A 103 (16), 6252-6257.
  23. ↑ Lee, S., Shin, W. H., Hong, S., Kang, H., Jung, D., Yim, U. H., Shim, W. J., Khim, J. S., Seok, C., Giesy, J. P., and Choi, K. (2015). Measured and predicted affinities of binding and relative potencies to activate the AhR of PAHs and their alkylated analogues. Chemosphere 139, 23-29.
  24. ↑ Jones, S. A., Parks, D. J., and Kliewer, S. A. (2003). Cell-free ligand binding assays for nuclear receptors. Methods Enzymol. 364, 53-71.
  25. ↑ Gasiewicz, T. A., and Neal, R. A. (1982). The examination and quantitation of tissue cytosolic receptors for 2,3,7,8-tetrachlorodibenzo-p-dioxin using hydroxylapatite. Anal. Biochem. 124 (1), 1-11.
  26. ↑ Nakai, J. S., and Bunce, N. J. (1995). Characterization of the Ah receptor from human placental tissue. J Biochem. Toxicol. 10 (3), 151-159.
  27. ↑ Dold, K. M., and Greenlee, W. F. (1990). Filtration assay for quantitation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) specific binding to whole cells in culture. Anal. Biochem. 184 (1), 67-73.
  28. ↑ Perez-Romero, P., and Imperiale, M. J. (2007). Assaying protein-DNA interactions in vivo and in vitro using chromatin immunoprecipitation and electrophoretic mobility shift assays. Methods Mol. Med. 131, 123-139.
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  30. ↑ 30.0 30.1 Ema, M., Ohe, N., Suzuki, M., Mimura, J., Sogawa, K., Ikawa, S., and Fujii-Kuriyama, Y. (1994). Dioxin binding activities of polymorphic forms of mouse and human arylhydrocarbon receptors. J.Biol.Chem. 269, 27337-27343.
  31. ↑ 31.0 31.1 Poland, A., Palen, D., and Glover, E. (1994). Analysis of the four alleles of the murine aryl hydrocarbon receptor. Mol.Pharmacol. 46, 915-921.
  32. ↑ Backlund, M., and Ingelman-Sundberg, M. (2004). Different structural requirements of the ligand binding domain of the aryl hydrocarbon receptor for high- and low-affinity ligand binding and receptor activation. Mol.Pharmacol. 65, 416-425.
  33. ↑ Murray, I. A., Reen, R. K., Leathery, N., Ramadoss, P., Bonati, L., Gonzalez, F. J., Peters, J. M., and Perdew, G. H. (2005). Evidence that ligand binding is a key determinant of Ah receptor-mediated transcriptional activity. Arch.Biochem.Biophys. 442, 59-71.
  34. ↑ Pandini, A., Denison, M. S., Song, Y., Soshilov, A. A., and Bonati, L. (2007). Structural and functional characterization of the aryl hydrocarbon receptor ligand binding domain by homology modeling and mutational analysis. Biochemistry 46, 696-708.
  35. ↑ 35.0 35.1 35.2 Pandini, A., Soshilov, A. A., Song, Y., Zhao, J., Bonati, L., and Denison, M. S. (2009). Detection of the TCDD binding-fingerprint within the Ah receptor ligand binding domain by structurally driven mutagenesis and functional analysis. Biochemistry 48, 5972-5983.
  36. ↑ Ramadoss, P., and Perdew, G. H. (2004). Use of 2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin as a probe to determine the relative ligand affinity of human versus mouse aryl hydrocarbon receptor in cultured cells. Mol.Pharmacol. 66, 129-136.
  37. ↑ 37.0 37.1 37.2 Head, J. A., Hahn, M. E., and Kennedy, S. W. (2008). Key amino acids in the aryl hydrocarbon receptor predict dioxin sensitivity in avian species. Environ.Sci.Technol. 42, 7535-7541.
  38. ↑ 38.0 38.1 Denison, M. S., Soshilov, A. A., He, G., DeGroot, D. E., and Zhao, B. (2011). Exactly the same but different: promiscuity and diversity in the molecular mechanisms of action of the aryl hydrocarbon (dioxin) receptor. Toxicol.Sci. 124, 1-22.
  39. ↑ van den Berg, M., Birnbaum, L. S., Bosveld, A. T., Brunström, B., Cook, P., Feeley, M., Giesy, J. P., Hanberg, A., Hasegawa, R., Kennedy, S. W., Kubiak, T. J., Larsen, J. C., Van Leeuwen, F. X. R., Liem, A. K. D., Nolt, C., Peterson, R. E., Poellinger, L., Safe, S., Schrenk, D., Tillitt, D. E., Tysklind, M., Younes, M., Wærn, F., and Zacharewski, T. R. (1998). Toxic equivalency factors (TEFs) for PCBs, PCDDs, PCDFs for humans and wildlife. Environ.Health Perspect. 106, 775-792.
  40. ↑ Cohen-Barnhouse, A. M., Zwiernik, M. J., Link, J. E., Fitzgerald, S. D., Kennedy, S. W., Hervé, J. C., Giesy, J. P., Wiseman, S. B., Yang, Y., Jones, P. D., Wan, Y., Collins, B., Newsted, J. L., Kay, D. P., and Bursian, S. J. (2011b). Sensitivity of Japanese quail (Coturnix japonica), Common pheasant (Phasianus colchicus), and White Leghorn chicken (Gallus gallus domesticus) embryos to in ovo exposure to TCDD, PeCDF, and TCDF. Toxicol.Sci. 119, 93-103.
  41. ↑ Farmahin, R., Crump, D., Jones, S. P., Mundy, L. J., and Kennedy, S. W. (2013a). Cytochrome P4501A induction in primary cultures of embryonic European starling hepatocytes exposed to TCDD, PeCDF and TCDF. Ecotoxicology 22(4), 731-739.
  42. ↑ Hervé, J. C., Crump, D., Jones, S. P., Mundy, L. J., Giesy, J. P., Zwiernik, M. J., Bursian, S. J., Jones, P. D., Wiseman, S. B., Wan, Y., and Kennedy, S. W. (2010a). Cytochrome P4501A induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin and two chlorinated dibenzofurans in primary hepatocyte cultures of three avian species. Toxicol. Sci. 113(2), 380-391.
  43. ↑ Hervé, J. C., Crump, D. L., McLaren, K. K., Giesy, J. P., Zwiernik, M. J., Bursian, S. J., and Kennedy, S. W. (2010b). 2,3,4,7,8-pentachlorodibenzofuran is a more potent cytochrome P4501A inducer than 2,3,7,8-tetrachlorodibenzo-p-dioxin in herring gull hepatocyte cultures. Environ. Toxicol. Chem. 29(9), 2088-2095.
  44. ↑ Poland, A., and Glover, E. (1973). Studies on the mechanism of toxicity of the chlorinated dibenzo-p-dioxins. Environ.Health Perspect. 5, 245-251.
  45. ↑ 45.0 45.1 McFarland, V. A., and Clarke, J. U. (1989). Environmental occurrence, abundance, and potential toxicity of polychlorinated biphenyl congeners: Considerations for a congener-specific analysis. Environ.Health Perspect. 81, 225-239.
  46. ↑ 46.0 46.1 Omiecinski, C. J., Vanden Heuvel, J. P., Perdew, G. H., and Peters, J. M. (2011). Xenobiotic metabolism, disposition, and regulation by receptors: from biochemical phenomenon to predictors of major toxicities. Toxicol.Sci. 120 Suppl 1, S49-S75.
  47. ↑ Swedenborg, E., and Pongratz, I. (2010). AhR and ARNT modulate ER signaling. Toxicology 268, 132-138.
  48. ↑ Diani-Moore, S., Ma, Y., Labitzke, E., Tao, H., David, W. J., Anderson, J., Chen, Q., Gross, S. S., and Rifkind, A. B. (2011). Discovery and biological characterization of 1-(1H-indol-3-yl)-9H-pyrido[3,4-b]indole as an aryl hydrocarbon receptor activator generated by photoactivation of tryptophan by sunlight. Chem. Biol. Interact. 193(2), 119-128.
  49. ↑ Wincent, E., Bengtsson, J., Mohammadi, B. A., Alsberg, T., Luecke, S., Rannug, U., and Rannug, A. (2012). Inhibition of cytochrome P4501-dependent clearance of the endogenous agonist FICZ as a mechanism for activation of the aryl hydrocarbon receptor. Proc. Natl. Acad. Sci. U. S. A 109(12), 4479-4484.
  50. ↑ Baba, T., Mimura, J., Nakamura, N., Harada, N., Yamamoto, M., Morohashi, K., and Fujii-Kuriyama, Y. (2005). Intrinsic function of the aryl hydrocarbon (dioxin) receptor as a key factor in female reproduction. Mol.Cell Biol. 25, 10040-10051.
  51. ↑ Fernandez-Salguero, P. M., Pineau, T., Hilbert, D. M., McPhail, T., Lee, S. S., Kimura, S., Nebert, D. W., Rudikoff, S., Ward, J. M., and Gonzalez, F. J. (1995). Immune system impairment and hepatic fibrosis in mice lacking the dioxin-binding Ah receptor. Science 268, 722-726.
  52. ↑ Ichihara, S., Yamada, Y., Ichihara, G., Nakajima, T., Li, P., Kondo, T., Gonzalez, F. J., and Murohara, T. (2007). A role for the aryl hydrocarbon receptor in regulation of ischemia-induced angiogenesis. Arterioscler.Thromb.Vasc.Biol. 27, 1297-1304.
  53. ↑ Lahvis, G. P., Lindell, S. L., Thomas, R. S., McCuskey, R. S., Murphy, C., Glover, E., Bentz, M., Southard, J., and Bradfield, C. A. (2000). Portosystemic shunting and persistent fetal vascular structures in aryl hydrocarbon receptor-deficient mice. Proc.Natl.Acad.Sci U.S.A 97, 10442-10447.
  54. ↑ Mimura, J., Yamashita, K., Nakamura, K., Morita, M., Takagi, T. N., Nakao, K., Ema, M., Sogawa, K., Yasuda, M., Katsuki, M., and Fujii-Kuriyama, Y. (1997). Loss of teratogenic response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice lacking the Ah (dioxin) receptor. Genes Cells 2, 645-654.
  55. ↑ Schmidt, J. V., Su, G. H., Reddy, J. K., Simon, M. C., and Bradfield, C. A. (1996). Characterization of a murine Ahr null allele: involvement of the Ah receptor in hepatic growth and development. Proc.Natl.Acad.Sci U.S.A 93, 6731-6736.
  56. ↑ Thackaberry, E. A., Gabaldon, D. M., Walker, M. K., and Smith, S. M. (2002). Aryl hydrocarbon receptor null mice develop cardiac hypertrophy and increased hypoxia-inducible factor-1alpha in the absence of cardiac hypoxia. Cardiovasc.Toxicol. 2, 263-274.
  57. ↑ Zhang, N., Agbor, L. N., Scott, J. A., Zalobowski, T., Elased, K. M., Trujillo, A., Duke, M. S., Wolf, V., Walsh, M. T., Born, J. L., Felton, L. A., Wang, J., Wang, W., Kanagy, N. L., and Walker, M. K. (2010). An activated renin-angiotensin system maintains normal blood pressure in aryl hydrocarbon receptor heterozygous mice but not in null mice. Biochem.Pharmacol. 80, 197-204.

Key Events

Title Short name
Oxidation, Uroporphyrinogen Oxidation, Uroporphyrinogen
Inhibition, UROD Inhibition, UROD
Accumulation, Highly carboxylated porphyrins Accumulation, Highly carboxylated porphyrins
Induction, CYP1A2/CYP1A5 Induction, CYP1A2/CYP1A5

844: Oxidation, Uroporphyrinogen

Short Name: Oxidation, Uroporphyrinogen

AOPs Including This Key Event

AOP ID and Name Event Type
131: AhR activation leading to uroporphyria KeyEvent

Biological Organization

Level of Biological Organization
Cellular

How this Key Event Works

Figure 1:Oxidation of the heme precursor uroporphyrinogen III to uroporphyrin III leading to symptoms of the disorder porphyria. UROD: uroporphyrinogen decarboxylase. (Modified from Smith and Elder (2010) Chem. Res. Toxicol. 23 (4), 712-723.

Uroporphyrinogen III is the first cyclic metabolic intermediate in the biosynthesis of heme. Under normal conditions, it is converted into coproporphyrinogen III by the enzyme uroporphyrinogen decarboxylase (UROD), and subsequently processed to heme following three further steps[1]. In the event that UROD activity is reduced (due to genetic disorders or chemical inhibition) uroporphyrinogen III, and other porphyrinogen substrates of UROD, are oxidized to highly stable porphyrins, which accumulation and lead to a heme disorder known as porphyria (Figure 1)[2].


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?

Porphyrins fluoresce red when exposed to UV light; therefore, uroporphyrinogen oxidation (UROX) can be directly measured as uropororphyrin fluorescence in a spectrophotofluorimeter. UROX has been measured spectrofluorimetrically in avian[3] mammalian[4] and aquatic[5] species.


References

  1. ↑ [1]"Wikipedia:Uroporphyrinogen III"
  2. ↑ Smith, A. G., and Elder, G. H. (2010). Complex gene-chemical interactions: hepatic uroporphyria as a paradigm. Chem. Res. Toxicol. 23 (4), 712-723.
  3. ↑ Sinclair, P. R., Gorman, N., Walton, H. S., Sinclair, J. F., Lee, C. A., and Rifkind, A. B. (1997). Identification of CYP1A5 as the CYP1A enzyme mainly responsible for uroporphyrinogen oxidation induced by AH receptor ligands in chicken liver and kidney. Drug Metab. Dispos. 25 (7), 779-783.
  4. ↑ Jacobs, J. M., Sinclair, P. R., Bement, W. J., Lambrecht, R. W., Sinclair, J. F., and Goldstein, J. A. (1989). Oxidation of uroporphyrinogen by methylcholanthrene-induced cytochrome P-450. Essential role of cytochrome P-450d. Biochem. J 258 (1), 247-253.
  5. ↑ Wainwright, J. S., Hopkins, K. M., Bums Jr., T.A., and Di Giulio, R. T. Investigation of potential biomarkers of exposure to bleached kraft mill effluent in North Carolina rivers. 27708-0328. 1995. Durham, NC. Ref Type: Report

845: Inhibition, UROD

Short Name: Inhibition, UROD

AOPs Including This Key Event

AOP ID and Name Event Type
131: AhR activation leading to uroporphyria KeyEvent

Biological Organization

Level of Biological Organization
Molecular

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].


References

  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.

846: Accumulation, Highly carboxylated porphyrins

Short Name: Accumulation, Highly carboxylated porphyrins

AOPs Including This Key Event

AOP ID and Name Event Type
131: AhR activation leading to uroporphyria KeyEvent

Biological Organization

Level of Biological Organization
Organ

How this Key Event Works

Under normal conditions, the heme biosynthesis pathway is tightly regulated and porphyrins (other than protoporphyrin) are only present in trace amounts[1]. However, when the regulatory process is disturbed, a variety of porphyrin precursors of heme accumulate in various organs including the liver and urinary and fecal excretion is elevated[2]). The pattern of porphyrin accumulation in chicken and rodents is similar following exposure to a variety of chemicals, and can be used to identify which enzyme in the heme pathway is predominately affected. In the case of uroporphyrinogen decarboxylase inhibition, uroporphyrin, hepta-, and hexacarboxylic acid porphyrin (highly carboxylated porphyrins) are elevated[1].


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?

The hepatic and urinary/fecal porphyrin patters can be determined using a high-performance liquid chromatograph equipped with a fluorescence detector. Kennedy et al.[3] describe the method for tissue extraction and porphyrin quantification in detail, which is rapid and highly sensitive.


References

  1. ↑ 1.0 1.1 Marks, G. S., Powles, J., Lyon, M., McCluskey, S., Sutherland, E., and Zelt, D. (1987). Patterns of porphyrin accumulation in response to xenobiotics. Parallels between results in chick embryo and rodents. Ann. N. Y. Acad. Sci. 514, 113-127.
  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. ↑ Kennedy, S. W., Wigfield, D. C., and Fox, G. A. (1986). Tissue porphyrin pattern determination by high-speed high-performance liquid chromatography. Anal. Biochem. 157 (1), 1-7.

850: Induction, CYP1A2/CYP1A5

Short Name: Induction, CYP1A2/CYP1A5

AOPs Including This Key Event

AOP ID and Name Event Type
131: AhR activation leading to uroporphyria KeyEvent

Biological Organization

Level of Biological Organization
Molecular

How this Key Event Works

The aryl hydrocarbon receptor (AHR) is involved in the regulation of several genes, including phase I (e.g. CYP1A, CYP1B, CYP2A) and phase II enzymes (e.g. UDP-GT, GSTs)[1][2][3]. Upon ligand binding, the AHR migrates to the nucleus where it dissociates from the cytosolic complex and forms a heterodimer with ARNT[3]. The AHR-ARNT complex then binds to a xenobiotic response element (XRE) found in the promoter of an AHR-regulated gene and recruits co-regulators such as CREB binding protein/p300, steroid receptor co-activator (SRC) 1, SRC-2, SRC-3 and nuclear receptor interacting protein 1, leading to induction or repression of gene expression[1].

The Cyp1A2/Cyp1A5 gene encodes a member of the cytochrome P450 superfamily of enzymes. The cytochrome P450 proteins are monooxygenases which catalyze many reactions involved in drug metabolism and synthesis of cholesterol, steroids and other lipids. The protein encoded by this gene localizes to the endoplasmic reticulum and its expression is induced by some polycyclic aromatic hydrocarbons (PAHs), some of which are found in cigarette smoke. The enzyme's endogenous substrate is unknown; however, it is able to metabolize some PAHs to carcinogenic intermediates. Other xenobiotic substrates for this enzyme include caffeine, aflatoxin B1, and acetaminophen. [4]

Of all AHR-regulated genes, the CYP1A subfamily of enzymes is the most studied and is often used as a biomarker of Dioxin-like compound (DLC) exposure and toxicity[5][6][7][8]. CYP1A5 is the avian isoform and is orthologous to the mammalian CYP1A2[9]. CYP1A5 is expressed in avian heart, liver and kidney tissues[10][11], and DLC-mediated induction of CYP1A5 mRNA has been measured in avian hepatocyte and cardiomyocyte cultures[12][13][10][14]. Positive correlations between hepatic CYP1A expression and DLC concentrations have also been reported in wild bird species, including bald eagles (Haliaeetus leucocephalus)[15], great blue herons (Ardea herodias)[16], double-crested cormorants (Phalacrocorax auritus)[17], black-crowned night herons (Nycticorax nycticorax)[18] and ospreys (Pandion haliaetus)[19]. Mouse CYP1A2 is only constitutively expressed in the liver, but is inducible in the liver, lung, and duodenum[20].


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?

Enzyme activity

There are a number of substrates that are preferentially metabolized by Cyp1A2 and CYP1A5 allowing for CYP1A activity to be measured as a function metabolite formation. Methoxyresorufin O-demethylation (MROD) is a classic marker of Cyp1A2/5 activity[21] and is often used due to the ease of fluorometric techniques; however, Burke et al.[21] suggest that a ratio of MROD to ethoxyresorufin O-demethylation (EROD) is a better measure of CYP1A2 activity due to the contribution of CYP1A1 to MROD. CYP1A2/5 activity can also be measured as the metabolic rate of arachidonic acid[11], oroporphyrinogen[22], acetanilide 4-hydroxylase and caffeine[23]. Caffeine metabolism has been used in clinical studies as a biomarker for CYP1A2 activity in humans[24].

Quantitative polymerase chain reaction (QPCR)

Levels of CYP1A2/5 messenger RNA can be measured using QPCR. This technique monitors the amplification of a targeted gene during PCR as accumulative fluorescence [25]. For example, Head and Kennedy[26] developed a multiplex QPCR assay utilizing dual-labeled fluorescent probes to measure CYP1A4 and CYP1A5 mRNA levels simultaneously from samples already analyzed for EROD activity. QPCR has high throughput capability and a low detection limit relative to other methods.

Luciferase reporter gene (LRG) assay

An LRG assay can be used to measure AHR1-mediated transactivation of a target gene. This assay is particularly useful as it can measures CYP1A4/5 induction exclusively caused by activation of the AHR, through which many DLCs exert their toxic effects. This assay is easily modified to measure AHR1-mediated transactivation in various species, simply by transfecting the desired AHR cDNA clone and reporter gene construct (containing the appropriate reporter gene) into the chosen cell line. This has been demonstrated to be an efficient high throughput method in various avian and mammalian studies.[27][28][29]


References

  1. ↑ 1.0 1.1 Fujii-Kuriyama, Y., and Kawajiri, K. (2010). Molecular mechanisms of the physiological functions of the aryl hydrocarbon (dioxin) receptor, a multifunctional regulator that senses and responds to environmental stimuli. Proc.Jpn.Acad.Ser.B Phys.Biol.Sci. 86, 40-53.
  2. ↑ Giesy, J. P., Kannan, K., Blankenship, A. L., Jones, P. D., and Newsted, J. L. (2006). Toxicology of PCBs and related compounds. In Endocrine Disruption Biological Bases for Health Effects in Wildlife and Humans (D. O. Norris, and J. A. Carr, Eds.), pp. 245-331. Oxford University Press, New York.
  3. ↑ 3.0 3.1 Mimura, J., and Fujii-Kuriyama, Y. (2003). Functional role of AhR in the expression of toxic effects by TCDD. Biochimica et Biophysica Acta - General Subjects 1619, 263-268.
  4. ↑ [1]"Entrez Gene: cytochrome P450; Gene ID: 1544."
  5. ↑ Harris, M. L., and Elliott, J. E. (2011). Effects of Polychlorinated Biphenyls, Dibenzo-p-Dioxins and Dibenzofurans, and Polybrominated Diphenyl Ethers in Wild Birds. In Environmental Contaminants in Biota (J. P. Meador, Ed.), pp. 477-528. CRC Press.
  6. ↑ Head, J. A., Farmahin, R., Kehoe, A. S., O'Brien, J. M., Shutt, J. L., and Kennedy, S. W. (2010). Characterization of the avian aryl hydrocarbon receptor 1 from blood using non-lethal sampling methods. Ecotoxicology 19, 1560-1566.
  7. ↑ Rifkind, A. B. (2006). CYP1A in TCDD toxicity and in physiology - With particular reference to CYP dependent arachidonic acid metabolism and other endogenous substrates. Drug Metabolism Reviews 38, 291-335.
  8. ↑ Safe, S. (1987). Determination of 2,3,7,8-TCDD toxic equivalent factors (TEFs): Support for the use of the in vitro AHH induction assay. Chemosphere 16, 791-802.
  9. ↑ Goldstone, H. M. H., and Stegeman, J. J. (2006). A revised evolutionary history of the CYP1A subfamily: Gene duplication, gene conversion, and positive selection. Journal of Molecular Evolution 62, 708-717.
  10. ↑ 10.0 10.1 Jones, S. P., and Kennedy, S. W. (2009). Chicken embryo cardiomyocyte cultures--a new approach for studying effects of halogenated aromatic hydrocarbons in the avian heart. Toxicol.Sci 109, 66-74.
  11. ↑ 11.0 11.1 Rifkind, A. B., Kanetoshi, A., Orlinick, J., Capdevila, J. H., and Lee, C. A. (1994). Purification and biochemical characterization of two major cytochrome P-450 isoforms induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in chick embryo liver. J.Biol.Chem. 269, 3387-3396.
  12. ↑ Farmahin, R., Crump, D., Jones, S. P., Mundy, L. J., and Kennedy, S. W. (2013a). Cytochrome P4501A induction in primary cultures of embryonic European starling hepatocytes exposed to TCDD, PeCDF and TCDF. Ecotoxicology.
  13. ↑ Hervé, J. C., Crump, D., Jones, S. P., Mundy, L. J., Giesy, J. P., Zwiernik, M. J., Bursian, S. J., Jones, P. D., Wiseman, S. B., Wan, Y., and Kennedy, S. W. (2010a). Cytochrome P4501A induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin and two chlorinated dibenzofurans in primary hepatocyte cultures of three avian species. Toxicol.Sci. 113, 380-391.
  14. ↑ Manning, G. E., Mundy, L. J., Crump, D., Jones, S. P., Chiu, S., Klein, J., Konstantinov, A., Potter, D., and Kennedy, S. W. (2013). Cytochrome P4501A induction in avian hepatocyte cultures exposed to polychlorinated biphenyls: comparisons with AHR1-mediated reporter gene activity and in ovo toxicity. Toxicol.Appl.Pharmacol. 266, 38-47.
  15. ↑ Elliott, J. E., Norstrom, R. J., Lorenzen, A., Hart, L. E., Philibert, H., Kennedy, S. W., Stegeman, J. J., Bellward, G. D., and Cheng, K. M. (1996). Biological effects of polychlorinated dibenzo-p-dioxins, dibenzofurans, and biphenyls in bald eagle (Haliaeetus leucocephalus) chicks. Environ.Toxicol.Chem. 15, 782-793.
  16. ↑ Bellward, G. D., Norstrom, R. J., Whitehead, P. E., Elliott, J. E., Bandiera, S. M., Dworschak, C., Chang, T., Forbes, S., Cadario, B., Hart, L. E., and . (1990). Comparison of polychlorinated dibenzodioxin levels with hepatic mixed-function oxidase induction in great blue herons. J.Toxicol.Environ.Health 30, 33-52.
  17. ↑ Sanderson, J. T., Norstrom, R. J., Elliott, J. E., Hart, L. E., Cheng, K. M., and Bellward, G. D. (1994). Biological effects of polychlorinated dibenzo-p-dioxins, dibenzofurans, and biphenyls in double-crested cormorant chicks (Phalacrocorax auritus). J.Toxicol.Environ.Health 41, 247-265.
  18. ↑ Rattner, B. A., Hatfield, J. S., Melancon, M. J., Custer, T. W., and Tillitt, D. E. (1994). Relation among cytochrome-P450, Ah-active PCB congeners and dioxin equivalents in pipping black-crowned night-heron embryos. Environ.Toxicol.Chem. 13, 1805-1812.
  19. ↑ Elliott, J. E., Wilson, L. K., Henny, C. J., Trudeau, S. F., Leighton, F. A., Kennedy, S. W., and Cheng, K. M. (2001). Assessment of biological effects of chlorinated hydrocarbons in osprey chicks. Environ.Toxicol.Chem. 20, 866-879.
  20. ↑ Dey, A., Jones, J. E., and Nebert, D. W. (1999). Tissue- and cell type-specific expression of cytochrome P450 1A1 and cytochrome P450 1A2 mRNA in the mouse localized in situ hybridization. Biochem. Pharmacol. 58 (3), 525-537.
  21. ↑ 21.0 21.1 Burke, M. D., Thompson, S., Weaver, R. J., Wolf, C. R., and Mayer, R. T. (1994). Cytochrome P450 specificities of alkoxyresorufin O-dealkylation in human and rat liver. Biochem. Pharmacol. 48 (5), 923-936.
  22. ↑ Sinclair, P. R., Gorman, N., Walton, H. S., Sinclair, J. F., Lee, C. A., and Rifkind, A. B. (1997). Identification of CYP1A5 as the CYP1A enzyme mainly responsible for uroporphyrinogen oxidation induced by AH receptor ligands in chicken liver and kidney. Drug Metab. Dispos. 25 (7), 779-783.
  23. ↑ Staskal, D. F., Diliberto, J. J., DeVito, M. J., and Birnbaum, L. S. (2005). Inhibition of human and rat CYP1A2 by TCDD and dioxin-like chemicals. Toxicol. Sci. 84 (2), 225-231.
  24. ↑ Kalow, W., and Tang, B. K. (1991). Use of caffeine metabolite ratios to explore CYP1A2 and xanthine oxidase activities. Clin Pharmacol. Ther. 50 (5 Pt 1), 508-519.
  25. ↑ [2]"Real-time polymerase chain reaction"
  26. ↑ Head, J. A., and Kennedy, S. W. (2007). Same-sample analysis of ethoxyresorufin-O-deethylase activity and cytochrome P4501A mRNA abundance in chicken embryo hepatocytes. Anal. Biochem. 360 (2), 294-302.
  27. ↑ Farmahin, R., Manning, G. E., Crump, D., Wu, D., Mundy, L. J., Jones, S. P., Hahn, M. E., Karchner, S. I., Giesy, J. P., Bursian, S. J., Zwiernik, M. J., Fredricks, T. B., and Kennedy, S. W. (2013). Amino acid sequence of the ligand-binding domain of the aryl hydrocarbon receptor 1 predicts sensitivity of wild birds to effects of dioxin-like compounds. Toxicol. Sci. 131 (1), 139-152.
  28. ↑ Farmahin, R., Wu, D., Crump, D., Hervé, J. C., Jones, S. P., Hahn, M. E., Karchner, S. I., Giesy, J. P., Bursian, S. J., Zwiernik, M. J., and Kennedy, S. W. (2012). Sequence and in vitro function of chicken, ring-necked pheasant, and Japanese quail AHR1 predict in vivo sensitivity to dioxins. Environ. Sci. Technol. 46 (5), 2967-2975.
  29. ↑ Garrison, P. M., Tullis, K., Aarts, J. M., Brouwer, A., Giesy, J. P., and Denison, M. S. (1996). Species-specific recombinant cell lines as bioassay systems for the detection of 2,3,7,8-tetrachlorodibenzo-p-dioxin-like chemicals. Fundam. Appl. Toxicol. 30 (2), 194-203.

Adverse Outcomes

Title Short name
N/A, Uroporphyria N/A, Uroporphyria

369: N/A, Uroporphyria

Short Name: N/A, Uroporphyria

AOPs Including This Key Event

AOP ID and Name Event Type
131: AhR activation leading to uroporphyria AdverseOutcome

Biological Organization

Level of Biological Organization
Individual

Chemical-induced uroporphyria has only been detected in birds[7][1][8] and mammals[5] , including an accidental outbreak in humans due to hexachlorobenzen-contaminated grain in the 1950s[9]. Fish are less susceptible to chemical-induced uroporphyria, but elevated levels of HCP have been documented in highly contaminated environments[10].

Differences in avian species sensitivity to chemical-induced porphyrin accumulation have been observed. Common tern embryo hepatocyte cultures did not accumulate porphyrins in response to dioxin-like compound (DLC) exposure and were 50 to over 1600 times less sensitive than chicken embryo hepatocyte cultures to DLC-mediated CYP1A induction[11]. Porphyrin accumulation in ring-necked pheasant embryo hepatocyte cultures following PCDD, PCDF and PCB exposure was also significantly less than that observed in chicken embryo hepatocytes[12]. These results are consistent with the predicted species sensitivity based on AHR1 sequence, as ring-necked pheasants are type 2 (Ile324_Ala380; moderately sensitive), and common terns are type 3 (Val324_Ala380; least sensitive) species[13][14][15].



How this Key Event Works

Figure 1: The heme biosynthetic pathway. Deficiency in a particular gene along the pathway results in the indicated form of porphyria: 8 separate disorders that are characterized by hepatic accumulation and increased excretion of porphyrins. Source: Frank, J., and Poblete-Gutierrez, P. (2010) Porphyria cutanea tarda--when skin meets liver. Best. Pract. Res. Clin Gastroenterol. 24 (5), 735-745.

Porphyria is a disorder in which the disturbance of heme biosynthesis results in accumulation and excretion of porphyrins[1]. A variety of porphyrias exist depending on which enzyme in the pathway is deficient (Figure 1). In the case of chemically induced porphyria, uroporphyrinogen decarboxylase (UROD), which converts uroporphyrinogen to coproporphyrinogen, is inhibited. The phase I metabolizing enzyme CYP1A2/CYP1A5 is believed to catalyzes the oxidation of uroporphyrinogen to uroporphyrin, leading to uroporphyrin accumulation and liver damage[2][3]. In humans, this disorder is known as porphyria cutanea tarda (PCT) and may be caused by chemical exposure or a hereditary deficiency in UROD[4]. The accumulation of porphyrins in the liver causes cirrhosis, mild fatty infiltration, patchy focal necrosis, and inflammation of portal tracts. When the activity of UROD is reduced to less than 30% of normal, the disorder manifests as an overt skin disease; the accumulation of porphyrins in the skin causes photosensitization that is characterized by fragile skin, superficial erosions, sub-epidermal bullae, hypertrichosis, patchy pigmentation and scarring[5].


How it is Measured or Detected

Porphyria is easily confirmed through a urinary or fecal analysis to measure the levels and pattern of excreted porphyrins. Samples are quantified using a high-performance liquid chromatograph equipped with a fluorescence detector[6]. Frank and Poblete-Gutiérrez[4] illustrate how the types of porphyria can be differentiated by the relative abundance of different porphyrins (Figure 2). Uroporphyria is the animal model equivalent to human porphyria cutanea tard [5]

Figure 2: Biochemical characteristics of the porphyrias in urine, stool, and blood (plasma and erythrocytes). Source: http://www.mayomedicallaboratories.com/articles/communique/2015/03-porphyria-testing/; Accessed December 9, 2015

References

  1. ↑ 1.0 1.1 Kennedy, S. W., and Fox, G. A. (1990). Highly carboxylated porphyrins as a biomarker of polyhalogenated aromatic hydrocarbon exposure in wildlife: Confirmation of their presence in Great Lakes herring gull chicks in the early 1970s and important methodological details. Chemosphere 21, 407-415.
  2. ↑ Rifkind, A. B. (2006). CYP1A in TCDD toxicity and in physiology - With particular reference to CYP dependent arachidonic acid metabolism and other endogenous substrates. Drug Metabolism Reviews 38, 291-335.
  3. ↑ Smith, A. G., Clothier, B., Carthew, P., Childs, N. L., Sinclair, P. R., Nebert, D. W., and Dalton, T. P. (2001). Protection of the Cyp1a2(-/-) null mouse against uroporphyria and hepatic injury following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol.Appl.Pharmacol. 173, 89-98.
  4. ↑ 4.0 4.1 Frank, J., and Poblete-Gutierrez, P. (2010) Porphyria cutanea tarda--when skin meets liver. Best. Pract. Res. Clin Gastroenterol. 24(5), 735-745.
  5. ↑ 5.0 5.1 5.2 Smith, A. G., and Elder, G. H. (2010) Complex gene-chemical interactions: hepatic uroporphyria as a paradigm. Chem. Res. Toxicol. 23 (4), 712-723.
  6. ↑ Kennedy, S. W., Wigfield, D. C., and Fox, G. A. (1986). Tissue porphyrin pattern determination by high-speed high-performance liquid chromatography. Anal. Biochem. 157 (1), 1-7.
  7. ↑ Fox, G. A., Norstrom, R. J., Wigfield, D. C., and Kennedy, S. W. (1988) Porphyria in herring gulls: A biochemical response to chemical contamination of great lakes food chains. ‘’Environmental Toxicology and Chemistry’’ ‘’’7’’’ (10), 831-839
  8. ↑ Kennedy, S. W., Fox, G. A., Trudeau, S. F., Bastien, L. J., and Jones, S. P. (1998) Highly carboxylated porphyrin concentration: A biochemical marker of PCB exposure in herring gulls. Marine Environmental Research 46 (1-5), 65-69.
  9. ↑ Cripps, D. J., Peters, H. A., Gocmen, A., and Dogramici, I. (1984) Porphyria turcica due to hexachlorobenzene: a 20 to 30 year follow-up study on 204 patients. Br. J Dermatol. 111 (4), 413-422.
  10. ↑ Wainwright, J. S., Hopkins, K. M., Bums Jr., T.A., and Di Giulio, R. T. (1995) Investigation of potential biomarkers of exposure to bleached kraft mill effluent in North Carolina rivers. Durham, NC.
  11. ↑ Lorenzen, A., Shutt, J. L., and Kennedy, S. W. (1997b). Sensitivity of common tern (Sterna hirundo) embryo hepatocyte cultures to CYP1A induction and porphyrin accumulation by halogenated aromatic hydrocarbons and common tern egg extracts. Archives of Environmental Contamination and Toxicology 32, 126-134.
  12. ↑ Lorenzen, A., and Kennedy, S. W. (1995). Sensitivities of Chicken and Pheasant Embryos and Cultured Embryonic Hepatocytes to Cytochrome P4501A Induction and Porphyrin Accumulation by TCDD, TCDF and PCBs. Organohalogen Compounds 25, 65-68.
  13. ↑ Farmahin, R., Manning, G. E., Crump, D., Wu, D., Mundy, L. J., Jones, S. P., Hahn, M. E., Karchner, S. I., Giesy, J. P., Bursian, S. J., Zwiernik, M. J., Fredricks, T. B., and Kennedy, S. W. (2013b). Amino acid sequence of the ligand binding domain of the aryl hydrocarbon receptor 1 (AHR1) predicts sensitivity of wild birds to effects of dioxin-like compounds. Toxicol.Sci. 131, 139-152.
  14. ↑ Head, J. A., Hahn, M. E., and Kennedy, S. W. (2008). Key amino acids in the aryl hydrocarbon receptor predict dioxin sensitivity in avian species. Environ.Sci.Technol. 42, 7535-7541.
  15. ↑ Manning, G. E., Farmahin, R., Crump, D., Jones, S. P., Klein, J., Konstantinov, A., Potter, D., and Kennedy, S. W. (2012). A luciferase reporter gene assay and aryl hydrocarbon receptor 1 genotype predict the embryolethality of polychlorinated biphenyls in avian species. Toxicol.Appl.Pharmacol. 263, 390-399.

Scientific evidence supporting the linkages in the AOP

Upstream Event Relationship Type Downstream Event Evidence Quantitative Understanding
Accumulation, Highly carboxylated porphyrins directly leads to N/A, Uroporphyria Strong Strong
Induction, CYP1A2/CYP1A5 directly leads to Oxidation, Uroporphyrinogen Moderate Weak
Activation, AHR directly leads to Induction, CYP1A2/CYP1A5 Strong Strong
Oxidation, Uroporphyrinogen directly leads to Inhibition, UROD Moderate Weak
Inhibition, UROD directly leads to Accumulation, Highly carboxylated porphyrins Moderate Moderate

Graphical Representation

Overall Assessment of the AOP

Domain of Applicability

Life Stage Applicability
Life Stage Evidence
Adult Strong
Juvenile Strong
Taxonomic Applicability
Term Scientific Term Evidence Links
mouse Mus musculus Strong NCBI
rat Rattus norvegicus Strong NCBI
human Homo sapiens Strong NCBI
chicken Gallus gallus Strong NCBI
herring gull Larus argentatus Strong NCBI
Japanese quail Coturnix japonica Strong NCBI
Common Starling Common Starling Moderate NCBI

Life Stage Applicability, Taxonomic Applicability, Sex Applicability
Elaborate on the domains of applicability listed in the summary section above. Specifically, provide the literature supporting, or excluding, certain domains.

Life Stage Applicability: Uroporphyria occurs following chemical exposure in juvenile or adult individuals. Fetal exposure to dioxin-like compounds causes developmental abnormalities and embryolethality rather than HCP accumulation[15][16][17][18][19]. Turkish children under the age of two that were exposed to HCB through breastmilk passed away from a condition called "pink sore”[20].

Taxonomic Applicability: Although the AHR is highly conserved in evolution[21], chemical-induced uroporphyria has only been detected in birds[1][2][3] and mammals[22] , including an accidental outbreak in humans due to hexachlorobenzen-contaminated grain in the 1950s[20]. Fish are less susceptible to chemical-induced uroporphyria, but elevated levels of HCP have been documented in highly contaminated environments[23].

Sex Applicability: Although this AOP applies broadly to both males and females, sexual dimorphism for uroporphyria has been observed in rats exposed to hexachlorobenzene (HCB). Hepatic uroporphyrin III was markedly increased in female rats exposed to HCB whereas exposed males showed levels of hepatic porphyrins similar to controls[24].

Essentiality of the Key Events

Molecular Initiating Event Summary, Key Event Summary
Provide an overall assessment of the essentiality for the key events in the AOP. Support calls for individual key events can be included in the molecular initiating event, key event, and adverse outcome tables above.

Every Key event in this AOP is absolutely essential for downstream events to occur. A summary of evidence for essrntiality of each key event is given below.

Molecular Initiating Event: AHR activation (Essentiality=strong)

  • Mice with a high-affinity Ahr allele (C57BL/6J ) are much more sensitive to uroporphyria than mice with low-affinity Ahr allele (DBA/2)[25][26][27][28][29];
  • The Ah locus influences the susceptibility of C57BL/6J mice to HCB-induced porphyria[30];
  • Ahr knockout mice (C57BL/6) are resistant to development of porphyria, even in the presence of iron loading[25];
  • Primary hepatocytes of avian species indicate that species that are highly sensitive to AHR activation are more sensitive to uroporphyrin accumulation than species with lower sensitivity to AHR activation[31].

Key Event 1: CYP1A2/Cyp1A5 induction (Essentiality=strong)

  • CYP1A2 knockout in mice prevents chemical-induced uroporphyria[32][33][34];
  • CYP1A2 knockout prevents porphyria in genetically predisposed mice (Hfe-/-, Urod-/+) that normally develop porphyria in absence of external stimuli[35];
  • CYP1A2 levels are correlated with the extent of urophorphyrin accumulation in mice[36];
  • 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and non-ortho substituted PCBs that are potent inducers of CYP1A4/5 cause accumulation of only HCPs in chicken embryonic hepatocytes cultures, whereas PCBs that do not induce CYP1A4/5 cause a porphyrin pattern that is not consistent with inhibition of UROD[37];
  • Common tern (Sterna hirundo) embryonic hepatocyte cultures, which are ~50 to > 1600 times less sensitive than chicken embryonic hepatocyte cultures to CYP1A5 induction by TCDD and PCBs, do not accumulate HCPs upon chemical exposure[31].

It should be noted that a recent study by Davies et al.[25] found that both C57BL/6J mice (susceptible to chemical-induced porphyria) and DBA/2 mice (resistant to porphyria due to polymorphism in AHR gene) showed increased expression of CYP1A2 when exposed to TCDD, even though the DBA/2 strain did not develop porphyria. Furthermore AHR-/- mice showed a mild uroporphyric response in the presence of iron loading and 5-aminolevulinic acid (a heme precursor). These findings suggest that the induction of CYP1A2 is not crucial for chemical-induced porphyria, but a basal level of expression is absolutely essential.

Key Event 2: Uroporphyrinogen oxidation (UROX) (Essentiality=strong)

  • Uroporphyria is characterized biochemically by increased formation of HCPs derived by oxidation of the porphyrinogen substrates of uroporphyrinogen decarboxylase (UROD); secondary to decreased activity of this enzyme in the liver[22];
  • Uroporphomethane, derived from oxidizing a single carbon bridge in uroporphyrinogen, has been identified as the UROD inhibitor that leads to chemically- and genetically-induced uroporphyria in mice[38];
  • UROX activity is positively correlated with uroporphyrin levels in mice[36].

Key Event 3: Uroporphyrinogen decarboxylase (UROD) inhibition (Essentiality=strong)

  • Mutations in the UROD gene that reduce or eliminate UROD activity lead to porphyria in mammals; a decrease in hepatic UROD activity of at least 70% is necessary to observe symptoms from overproduction of porphyrins[22];
  • A marked progressive decrease in UROD enzyme activity is a common feature in animal models of chemical-induced porphyria[22][34][39][40][41];
  • Liver cytosol UROD activity in female rats exposed to HCB was decreased more than 70% and correlated with elevated hepatic uroporphyrin levels, whereas male rats, which did not develop porphyria, showed UROD activity similar to controls[24];
  • UROD activity is inversely proportional to uroporphyrin levels in mice[36];
  • In chicken hepatocytes, the strongest inducers of porphyrin accumulation were also the strongest inhibitors of UROD activity[41];
  • Reduced UROD enzyme activity, not protein levels, is characteristic of uroporphyria in humans and rats[24][42][43].

Key Event 4: Highly carboxylated porphyrin (HCP) accumulation (Essentiality=strong)

  • Under normal heme biosynthesis, porphyrins are only present in trace amounts in the liver; however, in the absence of UROD activity, the oxidation of Uroporphorynogen to uroporphyrins dominates, leading to an accumulation of HCPs;
  • Porphyrins are strongly fluorescent compounds resulting in a characteristic red fluorescence of hepatic tissue under UV light that is proportional to the level of porphyrins[44][45]. Increased urinary excretion of porphyrins is also indicative of their accumulation and can lead to dark red/brown urine[22]. HCPs also accumulate in the skin causing solar hypersensitivity and increased skin fragility[46];
  • HCP accumulation was observed in avian embryo hepatocyte cultures following exposure potent AHR agonists (dioxin-like compounds)[37][47][48][49] and in the livers of Japanese quails and chickens exposed to PCBs[50][51][52];
  • HCP accumulation was evident in mice treated with polyhalogenated aromatic compounds[36] and TCDD-treated rats[25].

Weight of Evidence Summary

Summary Table
Provide an overall summary of the weight of evidence based on the evaluations of the individual linkages from the Key Event Relationship pages.

Dose concordance

Table 1 demonstrates that upstream KEs (monooxygenase activity/quantity) are significantly affected at lower doses than downstream KEs (porphyrin levels). After a 6 month recovery period, CYP450 and hepatic porphyrin levels were dramatically reduced, however, they did not return to normal. Furthermore, urinary porphyrin excretion remained maximally elevated[53]

Uroporphyria Table 1 TCDD recovery.png

Temporal Concordance

Table 2 demonstrates that upstream KEs (CYP1A2 expression and UROD inhibition) are significantly affected at earlier time-points than downstream KEs (porphyrin levels). These studies also show that upstream KEs are more sensitive to change than downstream KEs; ddY mice showed a 44% reduction in UROD activity but did not develop uroporphyria[25][54].

Uroporphyria Table 2 sensitive vs resistant.png

Key Events Relationships

Table 3 shows a sampling of the literature that demonstrates changes in KEs at multiple levels of organization leading to uroporphyria. The use of animal models resistant to porphyria (low AHR affinity or AHR/CYP1A2 knockout) illustrates the essentiality of these KEs in for downstream effects.

Uroporphyria Table 3 KER Summary.png

Quantitative Consideration

Summary Table
Provide an overall discussion of the quantitative information available for this AOP. Support calls for the individual relationships can be included in the Key Event Relationship table above.

The overall quantitative understanding of this AOP is poor. Quantitative models have been developed that predict the AHR transactivation potential of various compounds [55][56][57], but the extent of AHR activation necessary to produce porphyria is not known. It has been established that a reduction in UROD activity of at least 70% is required to lead to overt uroporphyrin in mammals[58][24][54]. Additionally, numerous in vitro systems have been developed to study porphyrin accumulation and UROD inhibition simultaneously; therefore, this KER provides the most feasible target for a predictive, quantitative model. However, care must be taken when reading across to other species; UROD inhibition is not always observed in avian models of porphyria, and when it is, it is less pronounced[59][60][47].

References

  1. ↑ 1.0 1.1 Fox, G. A., Norstrom, R. J., Wigfield, D. C., and Kennedy, S. W. (1988) Porphyria in herring gulls: A biochemical response to chemical contamination of great lakes food chains. ‘’Environmental Toxicology and Chemistry’’ ‘’’7’’’ (10), 831-839
  2. ↑ 2.0 2.1 Kennedy, S. W., and Fox, G. A. (1990) Highly carboxylated porphyrins as a biomarker of polyhalogenated aromatic hydrocarbon exposure in wildlife: Confirmation of their presence in Great Lakes herring gull chicks in the early 1970s and important methodological details. Chemosphere 21 (3), 407-415.
  3. ↑ 3.0 3.1 Kennedy, S. W., Fox, G. A., Trudeau, S. F., Bastien, L. J., and Jones, S. P. (1998) Highly carboxylated porphyrin concentration: A biochemical marker of PCB exposure in herring gulls. Marine Environmental Research 46 (1-5), 65-69.
  4. ↑ Thunell, S. (2000) Porphyrins, porphyrin metabolism and porphyrias. I. Update. Scand. J. Clin. Lab Invest 60 (7), 509-540.
  5. ↑ Baba, T., Mimura, J., Nakamura, N., Harada, N., Yamamoto, M., Morohashi, K., and Fujii-Kuriyama, Y. (2005) Intrinsic function of the aryl hydrocarbon (dioxin) receptor as a key factor in female reproduction. Mol. Cell Biol. 25 (22), 10040-10051.
  6. ↑ Denison, M. S., Soshilov, A. A., He, G., DeGroot, D. E., and Zhao, B. (2011) Exactly the same but different: promiscuity and diversity in the molecular mechanisms of action of the aryl hydrocarbon (dioxin) receptor. Toxicol. Sci. 124 (1), 1-22.
  7. ↑ Fernandez-Salguero, P. M., Pineau, T., Hilbert, D. M., McPhail, T., Lee, S. S., Kimura, S., Nebert, D. W., Rudikoff, S., Ward, J. M., and Gonzalez, F. J. (1995) Immune system impairment and hepatic fibrosis in mice lacking the dioxin-binding Ah receptor. Science 268 (5211), 722-726.
  8. ↑ Ichihara, S., Yamada, Y., Ichihara, G., Nakajima, T., Li, P., Kondo, T., Gonzalez, F. J., and Murohara, T. (2007) A role for the aryl hydrocarbon receptor in regulation of ischemia-induced angiogenesis. Arterioscler. Thromb. Vasc. Biol. 27 (6), 1297-1304.
  9. ↑ Lahvis, G. P., Lindell, S. L., Thomas, R. S., McCuskey, R. S., Murphy, C., Glover, E., Bentz, M., Southard, J., and Bradfield, C. A. (2000) Portosystemic shunting and persistent fetal vascular structures in aryl hydrocarbon receptor-deficient mice. Proc. Natl. Acad. Sci U. S. A 97 (19), 10442-10447.
  10. ↑ Mimura, J., Yamashita, K., Nakamura, K., Morita, M., Takagi, T. N., Nakao, K., Ema, M., Sogawa, K., Yasuda, M., Katsuki, M., and Fujii-Kuriyama, Y. (1997) Loss of teratogenic response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice lacking the Ah (dioxin) receptor. Genes Cells 2 (10), 645-654.
  11. ↑ Omiecinski, C. J., Vanden Heuvel, J. P., Perdew, G. H., and Peters, J. M. (2011) Xenobiotic metabolism, disposition, and regulation by receptors: from biochemical phenomenon to predictors of major toxicities. Toxicol. Sci. 120 Suppl 1, S49-S75.
  12. ↑ Schmidt, J. V., Su, G. H., Reddy, J. K., Simon, M. C., and Bradfield, C. A. (1996) Characterization of a murine Ahr null allele: involvement of the Ah receptor in hepatic growth and development. Proc. Natl. Acad. Sci U. S. A 93 (13), 6731-6736.
  13. ↑ Thackaberry, E. A., Gabaldon, D. M., Walker, M. K., and Smith, S. M. (2002) Aryl hydrocarbon receptor null mice develop cardiac hypertrophy and increased hypoxia-inducible factor-1alpha in the absence of cardiac hypoxia. Cardiovasc. Toxicol. 2 (4), 263-274.
  14. ↑ Zhang, N., Agbor, L. N., Scott, J. A., Zalobowski, T., Elased, K. M., Trujillo, A., Duke, M. S., Wolf, V., Walsh, M. T., Born, J. L., Felton, L. A., Wang, J., Wang, W., Kanagy, N. L., and Walker, M. K. (2010) An activated renin-angiotensin system maintains normal blood pressure in aryl hydrocarbon receptor heterozygous mice but not in null mice. Biochem. Pharmacol. 80 (2), 197-204.
  15. ↑ Brunström, B. (1988) Sensitivity of embryos from duck, goose, herring gull, and various chicken breeds to 3,3',4,4'-tetrachlorobiphenyl. Poultry science 67 (1), 52-57.
  16. ↑ Carro, T., Taneyhill, L. A., and Ottinger, M. A. (2013) The effects of an environmentally relevant 58 congener polychlorinated biphenyl (PCB) mixture on cardiac development in the chick embryo. Environ. Toxicol. Chem. 23(6), 1325-31
  17. ↑ Gilbertson, M. (1983) Etiology of chick edema disease in herring gulls in the lower Great Lakes. Chemosphere 12 (3), 357-370.
  18. ↑ Lavoie, E. T., and Grasman, K. A. (2007) Effects of in ovo exposure to PCBs 126 and 77 on mortality, deformities and post-hatch immune function in chickens. J. Toxicol. Environ. Health A 70 (6), 547-558.
  19. ↑ Wells, P. G., Lee, C. J., McCallum, G. P., Perstin, J., and Harper, P. A. (2010) Receptor- and reactive intermediate-mediated mechanisms of teratogenesis. Handb. Exp. Pharmacol. (196), 131-162.
  20. ↑ 20.0 20.1 Cripps, D. J., Peters, H. A., Gocmen, A., and Dogramici, I. (1984) Porphyria turcica due to hexachlorobenzene: a 20 to 30 year follow-up study on 204 patients. Br. J Dermatol. 111 (4), 413-422.
  21. ↑ Kewley, R. J., Whitelaw, M. L., and Chapman-Smith, A. (2004) The mammalian basic helix-loop-helix/PAS family of transcriptional regulators. Int. J. Biochem. Cell Biol. 36 (2), 189-204.
  22. ↑ 22.0 22.1 22.2 22.3 22.4 Smith, A. G., and Elder, G. H. (2010) Complex gene-chemical interactions: hepatic uroporphyria as a paradigm. Chem. Res. Toxicol. 23 (4), 712-723.
  23. ↑ Wainwright, J. S., Hopkins, K. M., Bums Jr., T.A., and Di Giulio, R. T. (1995) Investigation of potential biomarkers of exposure to bleached kraft mill effluent in North Carolina rivers. Durham, NC.
  24. ↑ 24.0 24.1 24.2 24.3 Mylchreest, E., and Charbonneau, M. (1997) Studies on the mechanism of uroporphyrinogen decarboxylase inhibition in hexachlorobenzene-induced porphyria in the female rat. Toxicol. Appl. Pharmacol. 145 (1), 23-33.
  25. ↑ 25.0 25.1 25.2 25.3 25.4 Davies, R., Clothier, B., Robinson, S. W., Edwards, R. E., Greaves, P., Luo, J., Gant, T. W., Chernova, T., and Smith, A. G. (2008) Essential role of the AH receptor in the dysfunction of heme metabolism induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Chem. Res. Toxicol. 21 (2), 330-340.
  26. ↑ Jones, K. G., and Sweeney, G. D. (1977) Association between induction of aryl hydrocarbon hydroxylase and depression of uroporphyrinogen decarboxylase activity. Res. Commun. Chem. Pathol. Pharmacol. 17 (4), 631-637.
  27. ↑ Jones, K. G., and Sweeney, G. D. (1980) Dependence of the porphyrogenic effect of 2,3,7,8-tetrachlorodibenzo(p)dioxin upon inheritance of aryl hydrocarbon hydroxylase responsiveness. Toxicol. Appl. Pharmacol. 53 (1), 42-49.
  28. ↑ 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.
  29. ↑ Smith, A. G., Clothier, B., Robinson, S., Scullion, M. J., Carthew, P., Edwards, R., Luo, J., Lim, C. K., and Toledano, M. (1998) Interaction between iron metabolism and 2,3,7,8-tetrachlorodibenzo-p-dioxin in mice with variants of the Ahr gene: a hepatic oxidative mechanism. Mol. Pharmacol. 53 (1), 52-61.
  30. ↑ Hahn, M. E., Gasiewicz, T. A., Linko, P., and Goldstein, J. A. (1988) The role of the Ah locus in hexachlorobenzene-induced porphyria. Studies in congenic C57BL/6J mice. Biochem. J. 254 (1), 245-254.
  31. ↑ 31.0 31.1 Lorenzen, A., Shutt, J. L., and Kennedy, S. W. (1997) Sensitivity of common tern (Sterna hirundo) embryo hepatocyte cultures to CYP1A induction and porphyrin accumulation by halogenated aromatic hydrocarbons and common tern egg extracts. Archives of Environmental Contamination and Toxicology 32 (2), 126-134.
  32. ↑ Greaves, P., Clothier, B., Davies, R., Higginson, F. M., Edwards, R. E., Dalton, T. P., Nebert, D. W., and Smith, A. G. (2005) Uroporphyria and hepatic carcinogenesis induced by polychlorinated biphenyls-iron interaction: absence in the Cyp1a2(-/-) knockout mouse. Biochem. Biophys. Res. Commun. 331 (1), 147-152.
  33. ↑ Sinclair, P. R., Gorman, N., Dalton, T., Walton, H. S., Bement, W. J., Sinclair, J. F., Smith, A. G., and Nebert, D. W. (1998) Uroporphyria produced in mice by iron and 5-aminolaevulinic acid does not occur in Cyp1a2(-/-) null mutant mice. Biochem. J. 330 ( Pt 1'), 149-153.
  34. ↑ 34.0 34.1 Smith, A. G., Clothier, B., Carthew, P., Childs, N. L., Sinclair, P. R., Nebert, D. W., and Dalton, T. P. (2001) Protection of the Cyp1a2(-/-) null mouse against uroporphyria and hepatic injury following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol. Appl. Pharmacol. 173 (2), 89-98.
  35. ↑ Phillips, J. D., Kushner, J. P., Bergonia, H. A., and Franklin, M. R. (2011) Uroporphyria in the Cyp1a2-/- mouse. Blood Cells Mol. Dis. 47 (4), 249-254.
  36. ↑ 36.0 36.1 36.2 36.3 Gorman, N., Ross, K. L., Walton, H. S., Bement, W. J., Szakacs, J. G., Gerhard, G. S., Dalton, T. P., Nebert, D. W., Eisenstein, R. S., Sinclair, J. F., and Sinclair, P. R. (2002) Uroporphyria in mice: thresholds for hepatic CYP1A2 and iron. Hepatology 35 (4), 912-921.
  37. ↑ 37.0 37.1 Lorenzen, A., Kennedy, S. W., Bastien, L. J., and Hahn, M. E. (1997) Halogenated aromatic hydrocarbon-mediated porphyrin accumulation and induction of cytochrome P4501A in chicken embryo hepatocytes. Biochemical Pharmacology 53 (3), 373-384.
  38. ↑ 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.
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