Uroporphyrin accumulation associated with cytochrome P4501A induction in fish hepatoma cells exposed to aryl hydrocarbon receptor agonists, including 2,3,7,8-tetrachlorodibenzo-p-dioxin and planar chlorobiphenyls

Mark E. Hahn, Kartik Chandran

Research output: Contribution to journalArticle

54 Citations (Scopus)

Abstract

Hepatic uroporphyria is a well-known effect of halogenated aromatic hydrocarbons in mammalian and. avian systems, including primary cell cultures, but attempts to produce uroporphyria in vertebrate (mammalian) hepatoma lines have been unsuccessful. In this study, the ability of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), and selected chlorobiphenyl congeners to cause uroporphyria was examined in a fish hepatoma cell line (PLHC-1) that expresses aryl hydrocarbon (Ah) receptors and an inducible cytochrome P4501A (CYP1A). Dose-dependent accumulation of porphyrins was observed in cells treated for 48 h with TCDD or 3,3′,4,4′-tetrachlorobiphenyl (3,3′,4,4′-TCB; IUPAC 77) when the heme precursor δ-aminolevulinic acid (ALA) was present during the last 5 h of treatment. HPLC analysis identified the porphyrins as uroporphyrin (∼80%) and heptacarboxylporphyrin (∼20%). Uroporphyria did not occur in cells treated with TCDD or 3,3′,4,4′-TCB in the absence of added ALA. ALA-dependent porphyrin accumulation was also seen following treatment of PLHC-1 cells with TCDF or with the non-ortho-substituted chlorobiphenyls 3,4,4′,5-tetrachlorobiphenyl (IUPAC 81) and 3,3′,4,4′,5-pentachlorobiphenyl (IUPAC 126). Neither of the mono-ortho-substituted chlorobiphenyls 2,3,3′,4,4′-pentachlorobiphenyl (IUPAC 105) or 2,3′,4,4′,5-pentachlorobiphenyl (IUPAC 118) increased the porphyrin content of PLHC-1 cells. The ability of the PCB congeners to cause porphyria correlated with their ability to induce the CYP1A catalytic activity ethoxyresorufin O-deethylase (EROD) and immunodetectable CYP1A protein in these cells, suggesting direct or indirect regulation of porphyrin accumulation via the Ah receptor and/or the induced CYP1A. Induction of EROD activity by TCDD, TCDF, and the planar polychlorinated biphenyls was biphasic, with increases at lower concentrations of inducer followed by decreased induction at higher concentrations, as seen previously. EC50 values for porphyrin accumulation were similar to, or slightly higher than, the concentrations at which peak EROD activities were obtained, suggesting a relationship between the decline in EROD activity and enhanced porphyrin accumulation. α-Naphthoflavone inhibited TCDD-induced EROD activity and porphyrin accumulation, providing further evidence for the involvement of a fish CYP1A in the mechanism of this porphyria. Addition of 3,3′,4,4′-TCB to TCDD-treated cells also inhibited EROD activity, but enhanced porphyrin accumulation, suggesting that an interaction between the halogenated inducer and the induced CYP1A is necessary for the porphyrogenic response. PLHC-1 cells grown in medium supplemented with ALA may be a useful model system for studying mechanisms of chemical uroporphyria induced by Ah receptor agonists.

Original languageEnglish (US)
Pages (from-to)163-174
Number of pages12
JournalArchives of Biochemistry and Biophysics
Volume329
Issue number2
DOIs
StatePublished - May 15 1996
Externally publishedYes

Fingerprint

Uroporphyrins
Aryl Hydrocarbon Receptors
Porphyrins
Cytochromes
Fish
Hepatocellular Carcinoma
Fishes
Cytochrome P-450 CYP1A1
Aminolevulinic Acid
Porphyrias
Polychlorinated Biphenyls
Cells
Halogenated Hydrocarbons
Polychlorinated Dibenzodioxins
1,4-dioxin
Aromatic Hydrocarbons
Primary Cell Culture
Heme
Cell culture
Vertebrates

Keywords

  • Ah receptor
  • CYP1A
  • Cytochrome P450
  • Dioxin
  • Fish hepatoma cells
  • Heme
  • Polychlorinated biphenyl
  • Porphyria
  • Porphyrin

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

@article{81b258c082b9476293310116446b638f,
title = "Uroporphyrin accumulation associated with cytochrome P4501A induction in fish hepatoma cells exposed to aryl hydrocarbon receptor agonists, including 2,3,7,8-tetrachlorodibenzo-p-dioxin and planar chlorobiphenyls",
abstract = "Hepatic uroporphyria is a well-known effect of halogenated aromatic hydrocarbons in mammalian and. avian systems, including primary cell cultures, but attempts to produce uroporphyria in vertebrate (mammalian) hepatoma lines have been unsuccessful. In this study, the ability of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), and selected chlorobiphenyl congeners to cause uroporphyria was examined in a fish hepatoma cell line (PLHC-1) that expresses aryl hydrocarbon (Ah) receptors and an inducible cytochrome P4501A (CYP1A). Dose-dependent accumulation of porphyrins was observed in cells treated for 48 h with TCDD or 3,3′,4,4′-tetrachlorobiphenyl (3,3′,4,4′-TCB; IUPAC 77) when the heme precursor δ-aminolevulinic acid (ALA) was present during the last 5 h of treatment. HPLC analysis identified the porphyrins as uroporphyrin (∼80{\%}) and heptacarboxylporphyrin (∼20{\%}). Uroporphyria did not occur in cells treated with TCDD or 3,3′,4,4′-TCB in the absence of added ALA. ALA-dependent porphyrin accumulation was also seen following treatment of PLHC-1 cells with TCDF or with the non-ortho-substituted chlorobiphenyls 3,4,4′,5-tetrachlorobiphenyl (IUPAC 81) and 3,3′,4,4′,5-pentachlorobiphenyl (IUPAC 126). Neither of the mono-ortho-substituted chlorobiphenyls 2,3,3′,4,4′-pentachlorobiphenyl (IUPAC 105) or 2,3′,4,4′,5-pentachlorobiphenyl (IUPAC 118) increased the porphyrin content of PLHC-1 cells. The ability of the PCB congeners to cause porphyria correlated with their ability to induce the CYP1A catalytic activity ethoxyresorufin O-deethylase (EROD) and immunodetectable CYP1A protein in these cells, suggesting direct or indirect regulation of porphyrin accumulation via the Ah receptor and/or the induced CYP1A. Induction of EROD activity by TCDD, TCDF, and the planar polychlorinated biphenyls was biphasic, with increases at lower concentrations of inducer followed by decreased induction at higher concentrations, as seen previously. EC50 values for porphyrin accumulation were similar to, or slightly higher than, the concentrations at which peak EROD activities were obtained, suggesting a relationship between the decline in EROD activity and enhanced porphyrin accumulation. α-Naphthoflavone inhibited TCDD-induced EROD activity and porphyrin accumulation, providing further evidence for the involvement of a fish CYP1A in the mechanism of this porphyria. Addition of 3,3′,4,4′-TCB to TCDD-treated cells also inhibited EROD activity, but enhanced porphyrin accumulation, suggesting that an interaction between the halogenated inducer and the induced CYP1A is necessary for the porphyrogenic response. PLHC-1 cells grown in medium supplemented with ALA may be a useful model system for studying mechanisms of chemical uroporphyria induced by Ah receptor agonists.",
keywords = "Ah receptor, CYP1A, Cytochrome P450, Dioxin, Fish hepatoma cells, Heme, Polychlorinated biphenyl, Porphyria, Porphyrin",
author = "Hahn, {Mark E.} and Kartik Chandran",
year = "1996",
month = "5",
day = "15",
doi = "10.1006/abbi.1996.0205",
language = "English (US)",
volume = "329",
pages = "163--174",
journal = "Archives of Biochemistry and Biophysics",
issn = "0003-9861",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - Uroporphyrin accumulation associated with cytochrome P4501A induction in fish hepatoma cells exposed to aryl hydrocarbon receptor agonists, including 2,3,7,8-tetrachlorodibenzo-p-dioxin and planar chlorobiphenyls

AU - Hahn, Mark E.

AU - Chandran, Kartik

PY - 1996/5/15

Y1 - 1996/5/15

N2 - Hepatic uroporphyria is a well-known effect of halogenated aromatic hydrocarbons in mammalian and. avian systems, including primary cell cultures, but attempts to produce uroporphyria in vertebrate (mammalian) hepatoma lines have been unsuccessful. In this study, the ability of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), and selected chlorobiphenyl congeners to cause uroporphyria was examined in a fish hepatoma cell line (PLHC-1) that expresses aryl hydrocarbon (Ah) receptors and an inducible cytochrome P4501A (CYP1A). Dose-dependent accumulation of porphyrins was observed in cells treated for 48 h with TCDD or 3,3′,4,4′-tetrachlorobiphenyl (3,3′,4,4′-TCB; IUPAC 77) when the heme precursor δ-aminolevulinic acid (ALA) was present during the last 5 h of treatment. HPLC analysis identified the porphyrins as uroporphyrin (∼80%) and heptacarboxylporphyrin (∼20%). Uroporphyria did not occur in cells treated with TCDD or 3,3′,4,4′-TCB in the absence of added ALA. ALA-dependent porphyrin accumulation was also seen following treatment of PLHC-1 cells with TCDF or with the non-ortho-substituted chlorobiphenyls 3,4,4′,5-tetrachlorobiphenyl (IUPAC 81) and 3,3′,4,4′,5-pentachlorobiphenyl (IUPAC 126). Neither of the mono-ortho-substituted chlorobiphenyls 2,3,3′,4,4′-pentachlorobiphenyl (IUPAC 105) or 2,3′,4,4′,5-pentachlorobiphenyl (IUPAC 118) increased the porphyrin content of PLHC-1 cells. The ability of the PCB congeners to cause porphyria correlated with their ability to induce the CYP1A catalytic activity ethoxyresorufin O-deethylase (EROD) and immunodetectable CYP1A protein in these cells, suggesting direct or indirect regulation of porphyrin accumulation via the Ah receptor and/or the induced CYP1A. Induction of EROD activity by TCDD, TCDF, and the planar polychlorinated biphenyls was biphasic, with increases at lower concentrations of inducer followed by decreased induction at higher concentrations, as seen previously. EC50 values for porphyrin accumulation were similar to, or slightly higher than, the concentrations at which peak EROD activities were obtained, suggesting a relationship between the decline in EROD activity and enhanced porphyrin accumulation. α-Naphthoflavone inhibited TCDD-induced EROD activity and porphyrin accumulation, providing further evidence for the involvement of a fish CYP1A in the mechanism of this porphyria. Addition of 3,3′,4,4′-TCB to TCDD-treated cells also inhibited EROD activity, but enhanced porphyrin accumulation, suggesting that an interaction between the halogenated inducer and the induced CYP1A is necessary for the porphyrogenic response. PLHC-1 cells grown in medium supplemented with ALA may be a useful model system for studying mechanisms of chemical uroporphyria induced by Ah receptor agonists.

AB - Hepatic uroporphyria is a well-known effect of halogenated aromatic hydrocarbons in mammalian and. avian systems, including primary cell cultures, but attempts to produce uroporphyria in vertebrate (mammalian) hepatoma lines have been unsuccessful. In this study, the ability of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), and selected chlorobiphenyl congeners to cause uroporphyria was examined in a fish hepatoma cell line (PLHC-1) that expresses aryl hydrocarbon (Ah) receptors and an inducible cytochrome P4501A (CYP1A). Dose-dependent accumulation of porphyrins was observed in cells treated for 48 h with TCDD or 3,3′,4,4′-tetrachlorobiphenyl (3,3′,4,4′-TCB; IUPAC 77) when the heme precursor δ-aminolevulinic acid (ALA) was present during the last 5 h of treatment. HPLC analysis identified the porphyrins as uroporphyrin (∼80%) and heptacarboxylporphyrin (∼20%). Uroporphyria did not occur in cells treated with TCDD or 3,3′,4,4′-TCB in the absence of added ALA. ALA-dependent porphyrin accumulation was also seen following treatment of PLHC-1 cells with TCDF or with the non-ortho-substituted chlorobiphenyls 3,4,4′,5-tetrachlorobiphenyl (IUPAC 81) and 3,3′,4,4′,5-pentachlorobiphenyl (IUPAC 126). Neither of the mono-ortho-substituted chlorobiphenyls 2,3,3′,4,4′-pentachlorobiphenyl (IUPAC 105) or 2,3′,4,4′,5-pentachlorobiphenyl (IUPAC 118) increased the porphyrin content of PLHC-1 cells. The ability of the PCB congeners to cause porphyria correlated with their ability to induce the CYP1A catalytic activity ethoxyresorufin O-deethylase (EROD) and immunodetectable CYP1A protein in these cells, suggesting direct or indirect regulation of porphyrin accumulation via the Ah receptor and/or the induced CYP1A. Induction of EROD activity by TCDD, TCDF, and the planar polychlorinated biphenyls was biphasic, with increases at lower concentrations of inducer followed by decreased induction at higher concentrations, as seen previously. EC50 values for porphyrin accumulation were similar to, or slightly higher than, the concentrations at which peak EROD activities were obtained, suggesting a relationship between the decline in EROD activity and enhanced porphyrin accumulation. α-Naphthoflavone inhibited TCDD-induced EROD activity and porphyrin accumulation, providing further evidence for the involvement of a fish CYP1A in the mechanism of this porphyria. Addition of 3,3′,4,4′-TCB to TCDD-treated cells also inhibited EROD activity, but enhanced porphyrin accumulation, suggesting that an interaction between the halogenated inducer and the induced CYP1A is necessary for the porphyrogenic response. PLHC-1 cells grown in medium supplemented with ALA may be a useful model system for studying mechanisms of chemical uroporphyria induced by Ah receptor agonists.

KW - Ah receptor

KW - CYP1A

KW - Cytochrome P450

KW - Dioxin

KW - Fish hepatoma cells

KW - Heme

KW - Polychlorinated biphenyl

KW - Porphyria

KW - Porphyrin

UR - http://www.scopus.com/inward/record.url?scp=0029876239&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029876239&partnerID=8YFLogxK

U2 - 10.1006/abbi.1996.0205

DO - 10.1006/abbi.1996.0205

M3 - Article

VL - 329

SP - 163

EP - 174

JO - Archives of Biochemistry and Biophysics

JF - Archives of Biochemistry and Biophysics

SN - 0003-9861

IS - 2

ER -