Induction of CYP1A by Benzo[k]fluoranthene in Human Hepatocytes: CYP1A1 or CYP1A2?

doi:10.1006/abbi.2001.2323

Archives of Biochemistry and Biophysics

Volume 389, Issue 1, 1 May 2001, Pages 130-134

https://doi.org/10.1006/abbi.2001.2323 Get rights and content

Abstract

Whilefresh human hepatocyte cultures are widely used to model hepatic cytochrome P450 (CYP) regulation and activity, their CYP1A subfamily composition induced by, e.g., polycyclic aromatic hydrocarbons is ambiguous. CYP1A1, CYP1A2, or both have been reported to be expressed, and their varied roles in chemical carcinogenesis makes resolution of which CYPs are expressed essential. We have used an immunoblot system with Bis-Tris–HCl-buffered polyacrylamide gel, which clearly resolves human CYP1A1 and CYP1A2, and polyclonal goat anti-human CYP1A1/CYP1A2 and rabbit anti-human CYP1A2 antibodies to probe the expressed CYP1A1 and CYP1A2 composition of seven individual human hepatocyte cultures induced with 5 μM benzo[k]fluoranthene (BKF) for 24 h. In six of the cultures only CYP1A1 was detected, and in the seventh both CYPs were detected. In most vehicle-treated hepatocyte cultures, neither CYP1A1 nor CYP1A2 was detected. In three additional hepatocyte cultures treated individually with BKF and 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD), the resultant induced CYP1A1/1A2 profiles were essentially not influenced by the nature of the inducing agents. To develop an activity-based assay to differentiate between CYP1A1 and CYP1A2 expression in human hepatocytes, our previously published R warfarin assay (Drug Metab. Disp. (1995) 23, 1339–1345) was applied to TCDD (10 nM)-treated hepatocyte culture. The low concentration of TCDD did not produce inhibition of the warfarin metabolism—such inhibition could confound the results. Based on the ratios of 6- to 8-hydroxywarfarin formed in two cultures, the ratios of CYP1A1/CYP1A2 expressed in these cultures were determined and they agreed with the ratios determined by immunoblot analysis. Thus each individual human hepatocyte culture must be characterized for induced CYP1A1 and CYP1A2 expression in studies of CYP1A activity. The warfarin assay provides a means of characterizing the cultures.

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Measurement of human CYP1A2 induction by inhalation exposure to benzo(a)pyrene based on in vivo isotope breath method
2016, Environmental Pollution
Citation Excerpt :
This is not surprising given that enzymatic activity is highly specific and that different individual PAHs may undergo different metabolic pathways. The metabolic pathways of PAHs are very complex and involve many types of enzymes including many from the liver P450 family (e.g., CYP1A1, CYP1A2) (Vakharia et al., 2001; Liu et al., 2001). Our findings about a significant association between B(a)P ingestion and CYP1A2 induction were based on a relatively small sample size (n = 13) and may need to be further confirmed in a larger study, and extensive individual variability in CYP1A2 and a genetic polymorphism affecting the inducibility of CYP1A2 should be kept in mind (Larsen and Brosen, 2005; Zhou et al., 2009), as ingested BaP only explained 34% of variations in measured CYP1A2 induction.
Cytochrome P450 1A2 (CYP1A2) is an enzyme involved in the metabolic activation of certain carcinogens, and inducible by toxic substrates. To date, few studies have investigated in vivo CYP1A2 induction in humans and its relationship to polycylic aromatic hydrocarbons (PAHs) like benzo(a)pyrene (BaP). Non-smoking healthy male coke-oven workers (n = 30) were recruited as ‘exposure’ group, and non-smoking healthy office workers in the same city (n = 10) were selected as ‘control’ group, to test whether high inhalation exposure to PAHs can induce CYP1A2 activity in human livers. Significantly higher inhalation exposure of PAHs were found among the exposure group compared to the control. Inhalation BaP exposure concentration in the exposure group was more than 30 times higher than the control group (p < 0.001). However, the exposure group did not exhale significant higher levels of ¹³CO₂/¹²CO₂ in breath samples (p = 0.81), and no significant relationship was found between the inhaled BaP concentration and the ¹³CO₂/¹²CO₂ ratio (p = 0.91). A significant association was found between the ¹³CO₂/¹²CO₂ exhalation and dietary BaP intake level. Hepatic CYP1A2 activity/induction level was not effected by inhaled BaP but was altered by ingestion of BaP.
Differential expression of CYP1A1 and CYP1A2 genes in H4IIE rat hepatoma cells exposed to TCDD and PAHs
2015, Environmental Toxicology and Pharmacology
Citation Excerpt :
However, as most of these assays cannot distinguish between different categories of pollutants acting through the common signaling pathway, the information provided on the type of toxicants responsible for the observed responses often lacks specificity. In addition, studies that relied solely on activities catalyzed by CYP1A1 and -1A2 could not distinguish between the two, while mRNA-based studies readily differentiated between CYP1A1 and -1A2 mRNA expression (Liu et al., 2001). Several studies on different experimental models confirmed differential expression of CYP and GST gene isoforms after exposure to TCDD or PAHs (Garner and Di Giulio, 2012; Iwanari et al., 2002; Shimada et al., 2002; Švihálková-Šindlerová et al., 2007; Xu et al., 2000).
Rat hepatoma cells H4IIE were treated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and polycyclic aromatic hydrocarbons (PAHs) (dibenz(a,h)anthracene, benzo(a)pyrene, benz(a)anthracene, chrysene), low-concentration mixtures of PAHs and TCDD, and environmental mixtures contaminated by PAHs and their derivatives. Expression of the gene battery comprising cytochrome P450 Cyp1a1, Cyp1a2, Cyp1b1, and glutathione-s-transferase Gsta2 and Gstp was investigated using quantitative real time polymerase chain reaction (qRT-PCR) analysis. The results revealed that TCDD induce Cyp1a1 > Cyp1a2 > Cyp1b1, while PAHs and PAH-containing environmental mixtures induce Cyp1a2 > Cyp1a1 > Cyp1b1 gene expression pattern. While low-concentration mixtures elicited a more pronounced response in comparison to single treatments, the typical gene expression patterns were not observed. In all samples, Gsta2 was predominantly expressed relative to Gstp. These findings indicate that differential Cyp1a1 and Cyp1a2 expression in the H4IIE cells might be used for detection of PAHs in highly contaminated environmental mixtures, but not in low-concentration mixtures of these compounds.
Induction of CYP1A1 and CYP1B1 by benzo(k)fluoranthene and benzo(a)pyrene in T-47D human breast cancer cells: Roles of PAH interactions and PAH metabolites
2008, Toxicology and Applied Pharmacology
The interactions of polycyclic aromatic hydrocarbons (PAH) and cytochromes P450 (CYP) are complex; PAHs are enzyme inducers, substrates, and inhibitors. In T-47D breast cancer cells, exposure to 0.1 to 1 μM benzo(k)fluoranthene (BKF) induced CYP1A1/1B1-catalyzed 17β-estradiol (E₂) metabolism, whereas BKF levels greater than 1 μM inhibited E₂ metabolism. Time course studies showed that induction of CYP1-catalyzed E₂ metabolism persisted after the disappearance of BKF or co-exposed benzo(a)pyrene, suggesting that BKF metabolites retaining Ah receptor agonist activity were responsible for prolonged CYP1 induction. BKF metabolites were shown, through the use of ethoxyresorufin O-deethylase and CYP1A1-promoter-luciferase reporter assays to induce CYP1A1/1B1 in T-47D cells. Metabolites formed by oxidation at the C-2/C-3 region of BKF had potencies for CYP1 induction exceeding those of BKF, whereas C-8/C-9 oxidative metabolites were somewhat less potent than BKF. The activities of expressed human CYP1A1 and 1B1 with BKF as substrate were investigated by use of HPLC with fluorescence detection, and by GC/MS. The results showed that both enzymes efficiently catalyzed the formation of 3-, 8-, and 9-OHBKF from BKF. These studies indicate that the inductive effects of PAH metabolites as potent CYP1 inducers are likely to be additional important factors in PAH-CYP interactions that affect metabolism and bioactivation of other PAHs, ultimately modulating PAH toxicity and carcinogenicity.
Preferential inducibility of CYP1A1 and CYP1A2 by TCDD: Differential regulation in primary human hepatocytes versus transformed human cells
2006, Biochemical and Biophysical Research Communications
Citation Excerpt :
The reason for the deviation between the results from Liu et al. and the present study is unclear. However, CYP1A form-preferred induction by TCDD might not be sufficiently investigated in their study, in that neither the basal activities of R-warfarin 6- and 8-hydroxylation in untreated cells nor the comparison of the regioselectivities of R-warfarin hydroxylation, designated by the ratio of the 6- to 8-hydroxylation rates, in TCDD-treated and untreated cells was provided [13]. Gene activation at the mRNA level, possibly representing the likelihood of functional cellular events, has been used to gauge CYP1A1 induction, particularly in the studies performed in human population.
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.
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2019, Drug Metabolism and Disposition

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Regular ArticleInduction of CYP1A by Benzo[k]fluoranthene in Human Hepatocytes: CYP1A1 or CYP1A2?

Abstract

Biochem. Pharmacol.

J. Biol. Chem.

Biochem. Pharmacol.

Adv. Drug Delivery Rev.

Chem.-Biol. Interact.

Anal. Biochem.

Biol. Cell

Toxicology

Hepatology

J. Lab. Clin. Med.

Toxicol. In Vitro

Toxicol. Lett.

Biochem. Pharmacol.

Chem.-Biol. Interact.

Biochem. Pharmacol.

Chem.-Biol. Interact.

Methods Enzymol.

Biochem. Biophys. Res. Commun.

Gastroenterology

Biochem. Soc. Trans.

Drug Metab. Disp.

Mol. Pharmacol.

Regular Article
Induction of CYP1A by Benzo[k]fluoranthene in Human Hepatocytes: CYP1A1 or CYP1A2?