Cancer Letters

Cancer Letters

Volume 220, Issue 2, 8 April 2005, Pages 145-154
Cancer Letters

Expression of cytochrome P450 1A1 and its contribution to oxidation of a potential human carcinogen 1-phenylazo-2-naphthol (Sudan I) in human livers

https://doi.org/10.1016/j.canlet.2004.07.036Get rights and content

Abstract

Cytochrome P450 1A1 (CYP1A1) is one of the most important enzymes implicated in the metabolic activation of carcinogens. To date, there is still conflicting evidence for the expression of enzymatically functional CYP1A1 in human liver. In the present work, we clearly demonstrate that CYP1A1 capable of metabolizing a carcinogen 1-phenylazo-2-naphthol (Sudan I) is expressed in livers of eight American Caucasian donors. Using two independent methods (immunoblotting and N-terminal sequencing), CYP1A1 protein was detected and quantified in all human hepatic microsomes tested in the study. Its levels, ranging from 0.97 to 3.0 pmol/mg protein, correlated with activities catalyzed by this enzyme [7-ethoxyresorufin O-deethylation (EROD) and oxidation of Sudan I], indicating the presence of enzymatically active CYP1A1. Even though levels of CYP1A1 expression are low, <0.7% of total hepatic CYP, the CYP1A1 contribution to oxidation of carcinogenic Sudan I in the test set of human liver microsomes ranges from 12 to 30%.

Introduction

The cytochrome P450 (CYP) monooxygenase system is a superfamily of enzymes, which play a critical role in the metabolism of numerous carcinogens. The activity of this system can occasionally result in their activation into a reactive species generating DNA adducts, which is believed to be one of the primary steps in the initiation of chemical carcinogenesis [1]. Five of the CYPs have been implicated for most of the metabolic activation of xenobiotics: CYP1A1, 1A2, 1B1, 2E1 and 3A4 [1], [2]. Specifically, CYP1A1 is of particular interest because it is the most active of the CYPs in metabolizing polycyclic aromatic hydrocarbon into active species forming DNA adducts [3]. The relationship between CYP1A1 expression and increased susceptibility to chemical-induced carcinogenesis remains an area of continued research and debate, due to the occurrence of a multitude of conflicting studies (see for a review Refs. [4], [5]). Therefore, it is important to determine the expression of CYP1A1 in humans, especially in the liver, which is the major site of first-pass metabolism. The finding that the CYP1A1 polymorphism is linked to hepatocellular carcinoma [6] is of interest since CYP1A1 was though to be only expressed in extrahepatic organs.

Numerous studies have examined hepatic CYP1A1 expression by western immunoblotting using polyclonal and monoclonal anti-CYP1A1 antibodies or antibodies raised against a synthetic peptide of a CYP1A1 protein molecule [7], [8], [9], [10], [11], [12]. The highest selectivity of these exhibited the anti-CYP1A1 anti-peptide antibody prepared by Murray et al. [7]. This antibody clearly recognized only recombinant human CYP1A1, while it was found not to be bound to any human hepatic microsomal protein. The crucial issue for detection of CYP1A1 expression in human liver by immunoblotting is the sensitivity of antibodies, besides their specificity [4]. In light of this, the sensitivity of the anti-CYP1A1 anti-peptide antibody prepared by Murray et al. [7] might be under the detection limit to recognize such low levels of CYP1A1 expressed in human livers [4], [5], [13], [14]. In contrast, Drahushuk et al. [4], using a monoclonal antibody directed against a marine scup P450E that was highly sensitive to human CYP1A1, detected CYP1A1 protein in all 20 human liver samples they studied. Likewise, recently we reported the presence of CYP1A1 in human hepatic microsomes, employing a partially purified chicken polyclonal anti-rat CYP1A1 antibody exhibiting an extremely high sensitivity to recognize human CYP1A1 [15]. Additionally, N-terminal sequencing confirmed that the protein band detected by immunoblotting was CYP1A1 [15]. However, two fundamental questions remain to be answered: (i) whether the enzymatically active CYP1A1 protein is expressed in human liver, and (ii) whether such low amounts of CYP1A1 in human hepatic microsomes [4], [15] might participate (and to which extent) in oxidation of carcinogens known to be effectively oxidized by human recombinant CYP1A1. To answer these questions, correlation between the immunoreactive content of CYP1A1 in a set of microsomal samples from livers of eight Caucasian donors and EROD activity known to be associated with CYP1A enzymes [16] or oxidation of a potential human carcinogen, 1-phenylazo-2-naphthol (Sudan I) [15], [17], [18], was utilized.

Section snippets

Chemicals

Chemicals were obtained from the following sources: NADP+, NADPH and glucose 6-phosphate from Sigma Chemical Co. (St Louis, MO, USA); 7-ethoxyresorufin from Fluka Chemie AG (Buchs, Switzerland), glucose 6-phosphate dehydrogenase from Serva (Heidelberg, Germany) and Sudan I (1-phenylazo-2-naphthol) from British Drug Houses (UK). All these and other chemicals were reagent grade or better. The derivatives 1-(4-hydroxyphenylazo)-2-naphthol (4′-OH-Sudan I), 1-(phenylazo)-naphthalene-2,6-diol

Detection of CYP1A1 in human hepatic microsomes

Using the combination of a chicken polyclonal antibody raised against rat recombinant CYP1A1 and western immunoblotting with enzymatic (alkaline phosphatase) detection, we developed a highly sensitive system to detect human CYP1A1 [15]. However, because this antibody cross-reacted with human recombinant CYP1A2 [15], here, we tested its cross-reactivity also with other human recombinant CYPs (CYP1B1, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4). Fig. 1A demonstrates the specificity of used

Discussion

The CYP1A1 enzyme has been shown to be responsible for activation of many polycyclic aromatic hydrocarbons and related chemicals to species binding to DNA and has, therefore, been considered to be involved in initiation steps of carcinogenesis. In animals and humans, CYP1A1 seems not to be constitutively expressed, but is readily inducible via the aryl hydrocarbon receptor. To date, the results from various laboratories studying expression of CYP1A1 in human liver have differed considerably,

Acknowledgements

Supported in part by Grant Agency of the Czech Republic (grant 203/03/0283) and the Ministry of Education of the Czech Republic (grant MSM 113100001).

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