Preferential inducibility of CYP1A1 and CYP1A2 by TCDD: Differential regulation in primary human hepatocytes versus transformed human cells

https://doi.org/10.1016/j.bbrc.2005.12.203Get rights and content

Abstract

Cytochrome P4501A1 (CYP1A1) induction, a marker of aryl hydrocarbon (Ah) receptor activation, has been associated with carcinogenicity of the environmental agent 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Consistently, we show that TCDD treatment led to induction of CYP1A1 in responsive human cancer cell lines including HepG2, LS174T, and MCF-7, as determined by Western blotting and CYP1A form-selective R-warfarin 6- and 8-hydroxylation. TCDD, however, preferably induced CYP1A2, not CYP1A1, in primary human hepatocytes. Such CYP1A form-preferred induction at the protein level was apparently uncorrelated with non-preferred mRNA induction in any cells studied. Moreover, while both genes were up-regulated by TCDD in primary hepatocytes and HepG2 cells, the induction of CYP1A1 and CYP1A2 at the mRNA level was distinguishable, indicated by the marked differences in activation kinetics and the response to the protein synthesis inhibitors, anisomycin and cycloheximide. Furthermore, formation of total benzo(a)pyrene (BaP)–DNA adducts was not altered following BaP exposure in TCDD-treated primary hepatocytes, whereas significantly elevated, in a CYP1A1-dependent manner, in the treated HepG2 cells. Taken together, our findings, demonstrating the complexities of TCDD-associated human Ah receptor function and differential regulations of CYP 1A enzymes, suggest clearly the need for caution when extrapolating data obtained in cell-based models.

Section snippets

Materials and methods

Cell culture and treatment. HepG2, LS174T, and MCF-7 cells (American Type Culture Collection, Manassas, VA) were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and transferred to hepatocyte culture medium as used for primary hepatocytes (In Vitro Technologies, Baltimore, MD). Two days later when the cells grew to cover approximately 75% of the well surface, the treatment was initiated. Primary cultures of hepatocytes (In Vitro Technologies, Baltimore, MD,

Cell type-preferred TCDD inducibility in enzyme activity

The activities of CYP1A1 and CYP1A2 were unambiguously determined in cultured cells applying the quantitative LC/MS/MS method for warfarin metabolic profiling [21]. Although both convert R-warfarin to 6-hydroxywarfarin and 8-hydroxywarfarin, CYP1A1 has a slight preference for forming 8-hydroxywarfarin while CYP1A2 dominantly forms 6-hydroxywarfarin [14]. In initial experiments, we repeatedly observed distinct R-warfarin metabolic profiles in primary human hepatocytes and HepG2 cells following

Acknowledgment

We thank B. King for her technical assistance.

References (30)

  • D.B. McGregor et al.

    An IARC evaluation of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans as risk factors in human carcinogenesis

    Environ. Health Perspect.

    (1998)
  • M.H. Sweeney et al.

    Human health effects after exposure to 2,3,7,8-TCDD

    Food Addit. Contam.

    (2000)
  • J.Y. Park et al.

    Induction of cytochrome P4501A1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin or indolo(3,2-b)carbazole is associated with oxidative DNA damage

    Proc. Natl. Acad. Sci. USA

    (1996)
  • D.W. Nebert et al.

    Human drug-metabolizing enzyme polymorphisms: effects on risk of toxicity and cancer

    DNA Cell Biol.

    (1996)
  • Y. Shimizu et al.

    Benzo[a]pyrene carcinogenicity is lost in mice lacking the aryl hydrocarbon receptor

    Proc. Natl. Acad. Sci. USA

    (2000)
  • Cited by (43)

    • Assessment of the carcinogenic effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin using mouse embryonic stem cells to form teratoma in vivo

      2019, Toxicology Letters
      Citation Excerpt :

      The potential evil changes in TCDD-exposed mESCs were thus evaluated by analyzing transcriptional levels of CYP1A1 in different treatments, and the results confirmed significant up-regulation of CYP1A1 induced by TCDD stimulation in mESCs (Fig. 2A). As a marker of AhR receptor activation, CYP1A1 induction has been associated with carcinogenicity of TCDD (Zhang et al., 2006), the cellular tumor antigen, p53 mRNA levels remained unchanged in mESCs after chemical treatment though (Fig. 2B). The antagonist experiments based on both CH223191 and ANF revealed the significant decrease in CYP1A1 mRNA expressions of mESCs when compared with that in TCDD exposure group (Fig. S1), further confirming the crucial role of AhR signaling pathway in TCDD-induced effects.

    • Induction of cytochrome P450 mRNA in porcine primary hepatocytes cultured under serum free conditions: Comparison of freshly isolated cells and cryopreserved

      2017, Experimental Cell Research
      Citation Excerpt :

      Thus, the hepatocytes were treated with known activators of the transcription factors regulating the major CYP1-3 families. In accordance with previous studies, TCDD induced a large increase in CYP1A2 mRNA expression [5,27,28]. The increase was not affected by the presence of serum in the culturing media.

    • Cyp1a reporter zebrafish reveals target tissues for dioxin

      2013, Aquatic Toxicology
      Citation Excerpt :

      Bioassays are essential to assess the effect of TCDD on living organisms, and cultured animal cells and animals have been used for the bioassays. Because bioassays using cultured animal cells are convenient to perform, they have been widely used (Carvan et al., 2000; Postlind et al., 1993; Wiebel et al., 1996; Yueh et al., 2005; Zhang et al., 2006). However, this approach has several limitations.

    • Multidrug resistance protein (MRP) 4 attenuates benzo[a]pyrene-mediated DNA-adduct formation in human bronchoalveolar H358 cells

      2012, Toxicology Letters
      Citation Excerpt :

      We have also shown in H358, human lung bronchoalveolar cells that CYP1A1/1B1 are responsible for the metabolism of benzo[a]pyrene (B[a]P), to the ultimate carcinogen (+)-B[a]PDE (Jiang et al., 2007). Detoxification of B[a]PDE occurs through glutathione (GSH) S-transferase (GST)-mediated adduct formation or further hydrolysis by epoxide hydrolase to the corresponding tetraols (Uno et al., 2004; Hukkanen et al., 2002; Zhang et al., 2006). B[a]PDE that escapes detoxification is able to enter to the nucleus and react with DNA to form adducts, particularly at the exocyclic amines of 2′-deoxyguanosine (dGuo) and 2′-deoxyadenosine (dAdo) (Fig. 1) (Ruan et al., 2007).

    View all citing articles on Scopus

    Abbreviations: TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; Ah, aryl hydrocarbon; CYP, cytochrome P450; BaP, benzo(a)pyrene; ANM, anisomycin; CHX, cycloheximide; PBS, phosphate-buffered saline; LC/MS/MS, liquid chromatography–tandem mass spectrometry; α-NF, α-naphthoflavone; RT-PCR, reverse transcription-polymerase chain reaction; AhR, aryl hydrocarbon receptor; AhRR, aryl hydrocarbon receptor repressor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CT, calf thymus.

    View full text