Dill Shows Potential for Herb-Drug Interactions via Up-Regulation of CYP1A2, CYP2C19, SULT1A1, NAT2 and ABCB1 in Caco-2 Cells.

<b>Background and Objective:</b> Dill<i> </i>(<i>Anethum graveolens</i> L.) has the potential to develop as a new alternative medicine due to its pharmacological activities. However, studies into its safety regarding herb-drug interactions have been neglected. This study investigated the risk of dill-induced herb-drug interactions (HDI) by examining its effect on the expression of phase I and II drug-metabolizing enzyme and transporter genes in Caco-2 cells. <b>Materials and Methods:</b> Caco-2 cells (5×10⁵ cells/well) were treated with 10 µM ketoconazole, 20 µM rifampicin or dill extract (60-240 µg mL¹) for 72 hrs. Cell viability was assessed using the resazurin assay and reactive oxygen species (ROS) content was determined with 2 ,7 -dichlorofluorescein diacetate. Aspartate (AST) and alanine aminotransferase (ALT) levels were measured using L-aspartate and L-alanine with α-ketoglutarate as substrate. Expression of phase I (<i>CYP1A2</i>, <i>CYP2C19</i>, <i>CYP2D6</i>, <i>CYP2E1 </i>and <i>CYP3A4</i>) and II (<i>UGT1A6</i>,<i> SULT1A1</i>,<i> NAT1</i>,<i> NAT2 </i>and<i> GSTA1/2</i>) metabolizing genes and transporters (<i>ABCB1</i>,<i> ABCC2</i>,<i> ABCG2 </i>and <i>SLCO1B1</i>) were determined by RT/qPCR. <b>Results:</b> All tested concentrations of dill did not affect cell viability or AST and ALT levels. The highest concentration of dill extract (240 µg mL¹) significantly lowered the ROS level. Expression of <i>CYP1A2</i>, <i>CYP2C19</i>, <i>SULT1A1</i>, <i>NAT2 </i>and <i>ABCB1 </i>mRNA was significantly up-regulated by dill extract. <b>Conclusion:</b> Dill extract did not directly damage Caco-2 cells but prolonged use of dill may increase the risk of HDI via the up-regulation of the drug-metabolizing genes <i>CYP1A2</i>, <i>CYP2C19</i>, <i>SULT1A1</i>, <i>NAT2 </i>and the transporter <i>ABCB1</i>.

Differential alteration of drug-metabolizing enzyme activities after cyclophosphamide/adriamycin administration in breast cancer patients.

Cyclophosphamide (CPA) and adriamycin (ADR) are widely used drugs for cancer chemotherapy. It has been reported that CPA and ADR singly or in combination could alter activities of a variety of drug-metabolizing enzymes in animals via multiple mechanisms. However, the effects of CPA/ADR on drug metabolism are largely unknown in human beings. Losartan metabolism has been suggested as a marker for determination of CYP2C9 activity. Caffeine is a commonly used probe to assess the metabolic activities of CYP1A2, CYP2A6, N-acetyltransferase 2 (NAT2) and xanthine oxidase (XO). The present study was designed to analyze the effects of CPA/ADR on these drug-metabolizing enzymes by using losartan and caffeine as probe drugs. A single oral dose of 25 mg losartan and a cup of instant coffee was given to 15 breast cancer patients on three occasions (before, and 2-4 h and 3 weeks after the adjuvant CPA/ADR chemotherapy [600 mg CPA/m2/day, 60 mg ADR/m2/day]). Losartan, caffeine and their metabolites were analyzed by using high-pressure liquid chromatography. When compared with baseline, CYP1A2 activity was increased by 20% and CYP2C9 activity was decreased by 315% 3 weeks after the administration of CPA/ADR chemotherapy (p = 0.05). The chemotherapy did not change the activities of CYP2A6, NAT2 or XO. CPA/ADR treatment caused a differential effect on drug-metabolizing enzyme activities, and this may contribute to predicting the efficacy and toxicity of chemotherapeutics, as well as understanding the drug-drug interactions.

The effect of Shoseiryuto, a traditional Japanese medicine, on cytochrome P450s, N-acetyltransferase 2 and xanthine oxidase, in extensive or intermediate metabolizers of CYP2D6.

OBJECTIVE: Shoseiryuto (TJ-19) contains eight herbal components, including Ephedra sinica, and has been used for treating asthma and allergic rhinitis in Asian countries for several centuries. In this study, we investigated the potential herb-drug interaction of TJ-19 in healthy volunteers and attempted to ascertain whether or not the interaction might be affected by the cytochrome P450 (CYP) 2D6 genotype. METHODS: We assessed the effect of TJ-19 on the activities of CYP1A2, CYP2D6, CYP3A, xanthine oxidase (XO), and N-acetyltransferase 2 (NAT2) in 37 healthy subjects. The subject pool consisted of 19 extensive metabolizers (EMs) with CYP2D6*Wild/*Wild, and 18 intermediate metabolizers (IMs) with CYP2D6*10/*10. The baseline activities of five enzymes were ascertained by their respective urinary metabolic ratios from an 8-h urine sample, after an oral 150-mg and 30-mg dose of caffeine and dextromethorphan were administrated, respectively. Thereafter, the subjects received 4.5 g of TJ-19 twice daily for 7 days, and underwent the same phenotyping test on postdose day 7. RESULTS: The activities of all enzymes examined did not differ before or after the 7-day administration of TJ-19. Consequently, the influence of the CYP2D6 genotype on the herb-drug interaction remained unsolved. CONCLUSION: Our results indicate that TJ-19 at the generally recommended dosage is unlikely to cause pharmacokinetic interaction with co-administered medications primarily dependent on the CYP1A2, CYP2D6, CYP3A, XO, and NAT2 pathways for elimination.

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

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.

Inhibition of N-acetyltransferase activity and gene expression in human colon cancer cell lines by diallyl sulfide.

Diallyl sulfide (DAS) is one of the major components of garlic (Allium sativum) and is widely used in the world for food. In this study, DAS was selected for testing the inhibition of arylamine N-acetyltransferase (NAT) activity (N-acetylation of 2-aminofluorene) and gene expression (mRNA NAT) in human colon cancer cell lines (colo 205, colo 320 DM and colo 320 HSR). The NAT activity was examined by high performance liquid chromatography and indicated that a 24 h DAS treatment decreases N-acetylation of 2-aminofluorene in three colon (colo 205, 320 DM and colo 320 HSR) cancer cell lines. The NAT enzymes (protein) were analyzed by western blotting and flow cytometry and it indicated that DAS decreased the levels of NAT in three colon (colo 205, 320 DM and colo 320 HSR) cancer cell lines. The gene expression of NAT (mRNAT NAT) was determined by polymerase chain reaction (PCR), it was shown that DAS affect mRNA NAT expression in examined human colon cancer cell lines. This report is the first to demonstrate that DAS does inhibit human colon cancer cell NAT activity and gene expression.

Effects of garlic components diallyl sulfide and diallyl disulfide on arylamine N-acetyltransferase activity and 2-aminofluorene-DNA adducts in human promyelocytic leukemia cells.

Two components of garlic, diallyl sulfide (DAS) and diallyl disulfide (DADS), inhibited arylamine N-acetyltransferase (NAT) activity and 2-aminofluorene-DNA adduct in human promyelocytic leukemia cells (HL-60). The NAT activity was measured by high performance liquid chromatography assaying for amounts of N-acetyl-2-aminofluorene (2-AAF) and remaining 2-aminofluorene (2-AF). Cellular cytosols and intact cell suspensions were assayed. The inhibition of NAT activity and 2-AF-DNA adduct formation in human leukemia cells by DAS and DADS were dose-dependent and were directly proportional. The data also indicated that DAS and DADS decrease the apparent values of Km and Vmax from human leukemia cells in both assays. This is the first report of garlic components affecting human leukemia cell NAT activity and 2-AF-DNA adduct formation.

Effects of berberine on arylamine N-acetyltransferase activity and 2-aminofluorene-DNA adduct formation in human leukemia cells.

Berberine is an alkaloid occurring in the plant genera Berberis and Coptis. Although berberine had been demonstrated to have antineoplastic function by inhibiting DNA-synthesis in activated lymphocytes, there is no available information to address berberine affects on human leukemia cell N-acetyltransferase (NAT) activity and 2-aminofluorene (AF)-DNA adduct formation. Thus, berberine was tested for inhibition of arylamine NAT activity and AF-DNA adduct formation in human leukemia cells. The NAT activity was measured by a high performance liquid chromatography assaying for the amounts of N-acetyl-2-aminofluorene (AAF) and N-acetyl-p-aminobenzoic acid (N-Ac-PABA) and the remaining AF and p-aminobenzoic acid (PABA). The NAT activity and AF-DNA adduct formation in human leukemia cells were inhibited by berberine in a dose-dependent manner, i.e. the higher the concentration of berberine, the higher the inhibition of NAT activity and AF-DNA adduct. The data also indicate that berberine decreased the apparent values of Km and Vmax from human leukemia cells in both cytosol and intact cells.

Effects of luteolin on arylamine N-acetyltransferase activity in rat blood and liver.

In this study we investigated inhibition of Arylamine N-acetyltransferase (NAT) activity in rat blood and liver tissue cytosols by luteolin. Using high-performance liquid chromatography, NAT activity for acetylation of 2-aminofluorene and remaining unacetylated 2-aminofluorene were examined. The NAT activity in rat blood and liver tissue was inhibited by luteolin in a dose-dependent manner: higher concentrations of luteolin in the reaction resulted in greater inhibition of NAT activities in both examined tissues. The data also indicated that luteolin decreased apparent Km and Vmax of NAT enzymes from rat blood and liver tissue cytosols. This report is the first demonstration that luteolin can affect rat blood and liver tissue NAT activity.