Elsevier

Phytomedicine

Volume 12, Issue 5, 16 May 2005, Pages 351-358
Phytomedicine

Berberine inhibits arylamine N-acetyltransferase activity and gene expression in mouse leukemia L 1210 cells

https://doi.org/10.1016/j.phymed.2003.11.008Get rights and content

Abstract

N-acetyltransferases (NATs) are recognized to play a key role in the primary step of arylamine compounds metabolism. Polymorphic NAT is coded for rapid or slow acetylators, which are being thought to involve cancer risk related to environmental exposure. Berberine has been shown to induce apoptosis and affect NAT activity in human leukemia cells. The purpose of this study is to examine whether or not berberine could affect arylamine NAT activity and gene expression (NAT mRNA) and the levels of NAT protein in mouse leukemia cells (L 1210). N-acetylated and non-N-acetylated AF were determined and quantited by using high performance liquid chromatography. NAT mRNA was determined and quantited by using RT-PCR. The levels of NAT protein were examined by western blotting and determined by using flow cytometry. Berberine displayed a dose-dependent inhibition to cytosolic NAT activity and intact mice leukemia cells. Time-course experiments indicated that N-acetylation of AF measured from intact mice leukemia cells were inhibited by berberine for up to 24 h. The NAT1 mRNA and NAT proteins in mouse leukemia cells were also inhibited by berberine. This report is the first demonstration, which showed berberine affect mice leukemia cells NAT activity, gene expression (NAT1 mRNA) and levels of NAT protein.

Introduction

The N-acetyltransferases (NATs) play an important role in the metabolism of arylamine chemicals and carcinogens that catalyze both detoxifying N-acetylation and O-acetylation that generate intermediate metabolites in electrophiles which then bind to DNA and form DNA adduct formation that finally generates cancer in specific target organs or tissues (Hein, 1988; Fretland et al., 2002). N-acetyltransferase-1 (NAT1) and N-acetyltransferase-2 (NAT2) are encoded by NAT1 and NAT2 in human chromosome 8 (Blum et al., 1990). The genetic determined the variants of both enzymes of individuals which then lead to the specific NAT activity for rapid or slow acetylation (Weber and Hein, 1985; Evans, 1989; Weber, 1987). Based on the NAT activity (N-acetylation of substrate) the individuals are classified into rapid and slow acetylators. From the epidemiological statistic analysis, it was demonstrated that slow acetylators have an increased bladder cancer risk after exposed to smoking-derived or occupational carcinogens (Risch et al., 1995; Brockmoller et al., 1996; Cartwright, 1982) but rapid acetylator with high intake of cooked meat containing carcinogenic heterocyclic amines could be a potential risk factor for colorectal cancer (Lang, 1986; Roberts-Thomson et al., 1996; Chen et al., 1998). Lenkemia usually occurs in animals and children. Usually, mutagen by oral administration was absorbed from the intestinal system, and then passed through the circulation system to the whole body. The in vivo studies of our research colleagues had been evaluated, and these mutagen and intermediates were deposited in the blood and had been accumulated in other tissues for a long time. Recently, comprehensive reviews on the molecular genetic analysis and epidemiology of the both NATs acetylation polymorphisms have been reported (Brockton et al., 2000; Cascorbi and Roots, 1999; Hein et al., 2000).

Berberine (5,6-Dihydro-9,10-dimethoxybenzo[g]-1,3-benzodioxolo[5,6-a]quinolizinium), a benzodioxoloquinolozine alkaloid present in the plant genera Berberis and Coptis, and also in many other plants, has been widely used to treat gastroenteritis and diarrhea patients in the Chinese population for a long time (Tang and Eisenbrand, 1992). Berberine acts as an antimicrobial (Amin et al., 1969), antidiarrhea (Tai et al., 1981; Yamamoto et al., 1993), and antineoplastic agent (Hoshi et al., 1976; Zhang et al., 1990) and it also has a high binding affinity for mast cells (Berlin and Enerback, 1983) and influenced mast cell-mediated chloride secretion in rat colons (Taylor and Baird, 1993). Recently, it was also demonstrated that berberine could be used as an anti-inflammatory agent which may arise in part from the inhibition of DNA-synthesis in human activated peripheral lymphocytes (Weber and Hein, 1985; Ckless et al. 1995). In our laboratory, we also found out that berberine affected NAT activity of human colon (Chung et al., 1999) and bladder (Lin et al., 1999a, Lin et al., 1999b) tumor cells. However, there is no available information to address berberine effects on NAT activity and gene expression in the mouse leukemia cells. Thus, the initial studies were focused on the effects of berberine on the NAT activity and gene expression of a mouse leukemia L 1210 cell line.

Section snippets

Chemicals and reagents

Berberine, ethylenediaminetetraacetic acid (EDTA), 2-aminofluorene (AF), N-acetyl-2-aminofluorene (AAF), dithiothreitol (DTT), Tris, acetylcarnitine, leupeptin, bovine serum albumin (BSA), phenylmethylsulfonylfluoride (PMSF), dimethyl sulfoxide (DMSO), acetyl-coenzyme A (Acetyl-CoA) and carnitine acetyltransferase were obtained from Sigma Chemical Co. (St. Louis, MO). All of the chemicals used were reagent grade.

Mouse leukemia cell line

Mouse lymphocytic leukemia cell line (L 1210) was obtained from the Food Industry

Effects of various concentrations of berberine on mouse leukemia cells in cytosol and intact cells

The possible effects of berberine on NAT activity in mouse leukemia cells cytosols were examined by HPLC assessing the percentage of acetylation of AF. For the cytosolic examinations, AF was added to the cytosol for N-acetylation of AF then the determination of AF and AAF by HPLC. The means±SD (standard deviation) of NAT activity co-treated with or without various concentrations of berberine with AF in cytosols is given in Table 1. The data indicated that there were decreased amounts of AAF

Discussion

Berberine had been demonstrated to inhibit DNA, RNA and protein synthesis in sarcoma S180 cells in vitro (Creasey, 1979). Berberine-treated HL-60 cells had an increased G2/M phase population (Kuo et al., 1995). Recently, it was also reported that berberine modulates expression of mdr1 gene product and the responses of digestive track cancer cells to paclitaxel (Lin et al., 1999a, Lin et al., 1999b). However, there is no available information to address berberine affect NAT gene expression on

Acknowledgments

This work was supported by Grant NSC90-2745-P-039-001 from the National Science Council of Taiwan, ROC.

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