No abstract

The purpose of this study was to continue our effort to determine how enzyme-transporter coupling affect disposition of flavonoids. The rat intestinal perfusion and Caco-2 cell models were used together with relevant microsomes. In perfusion model, isoflavone (i.e., formononetin and biochanin A) absorption and subsequent excretion of its metabolites were always site-dependent. Maximal amounts of intestinal and biliary conjugates excreted per 30 min were 31 and 51 nmol for formononetin, more than that for pure biochanin A (12 and 20 nmol). When a standardized red clover extract (biochanin A/formononetin ϭ 10:7) was used, the results indicated that more metabolites of biochanin A than formononetin were found in the perfusate (36.9 versus 22.8 nmol) and bile (78 versus 51 nmol). In metabolism studies, rat intestinal and liver microsomes always glucuronidated biochanin A faster (p Ͻ 0.05) than formononetin, whereas intestinal microsomes glucuronidated both isoflavones faster (p Ͻ 0.05) than liver microsomes. However, rapid metabolism in the microsomes did not translate into more efficient excretion in either the rat perfusion model as shown previously or in the Caco-2 model. In the Caco-2 model, both isoflavones were rapidly absorbed, efficiently conjugated, and the conjugates excreted apically and basolaterally. More formononetin conjugates were excreted than biochanin A when used alone, but much more biochanin A conjugates were found when using the isoflavone mixture. In conclusion, efficiency of enzyme-transporter coupling controls the amounts of metabolites excreted by the intestine and liver and determines the relative contribution of enteric and enterohepatic recycling to the in vivo disposition of isoflavones.Red clover (Trifolium pratense L.) extracts are sold as dietary supplements in supermarkets and health food stores in both industrialized and developing countries. Several defined extracts of red clover are sold in the United States to treat women who suffer from menopausal-related symptoms (Rijke et al., 2001;Oleszek and Stochmal, 2002). The extracts are available as tablets (e.g., Promensil), capsules, tea, liquid preparations, and several other forms. Limited clinical studies have demonstrated the effectiveness of Promensil in the management of hot flashes (van de Weijer and Barentsen, 2002), although the efficacy of such products is not always consistent (Baber et al., 1999;St. Germain et al., 2001).Red clover extract contains many of the same isoflavones as soy (Lin et al., 2000). However, a majority of isoflavones in red clover are present in aglycone forms, whereas they are present as glucosides in soy and soy extracts. Furthermore, the principal isoflavones are biochanin A and formononetin in red clover but are genistein and daidzein in soy. Nevertheless, these isoflavones all have similar biological activities, and biochanin A and formononetin can be converted to genistein and daidzein by cytochrome P450 (Tolleson et al., 2002;Hu et al., 2003b). Moreover, daidzein can be metabolized to h...

ABSTRACT:The purpose of this study is to determine the importance of coupling of efflux transporters and metabolic enzymes in the intestinal disposition of six isoflavones (genistein, daidzein, formononetin, glycitein, biochanin A, and prunetin), and to determine how isoflavone structural differences affect the intestinal disposition. A rat intestinal perfusion model was used, together with rat intestinal and liver microsomes. In the intestinal perfusion model, significant absorption and excretion differences were found between isoflavones and their respective glucuronides (p <0.05), with prunetin being the most rapidly absorbed and formononetin glucuronides being the most excreted in the small intestine. In contrast, glucuronides were excreted very little in the colon. In an attempt to account for the differences, we measured the glucuronidation rates of six isoflavones in microsomes prepared from rat intestine and liver. Using multiple regression analysis, intrinsic clearance (CL int ) and other enzyme kinetic parameters (V max and K m ) were determined using appropriate kinetic models based on Akaike's information criterion. The kinetic parameters were dependent on the isoflavone used and the types of microsomes. To determine how metabolite excretion rates are controlled, we plotted excretion rates versus calculated microsomal rates (at 10 M), CL int values, K m values, or V max values, and the results indicated that excretion rates were not controlled by any of the kinetic parameters. In conclusion, coupling of intestinal metabolic enzymes and efflux transporters affects the intestinal disposition of isoflavones, and structural differences of isoflavones, such as having methoxyl groups, significantly influenced their intestinal disposition.

Conjugating enzymes are traditionally recognized as one of the major biological barriers to the entry of xenobiotics/drugs into systemic circulation and represent one of the main pathways for their elimination. Similar to drugs that undergo extensive phase I metabolism, drugs that undergo extensive conjugation have poor bioavailability and are more prone to metabolism-based drug interactions. Previously, enterohepatic recycling is used to explain why certain xenobiotics have half-lives that are much longer than expected from intravenous injection studies. In addition, changes in expression levels of metabolic enzymes due to chemical induction or suppression are often recognized as the source of drug interaction or toxicity of pollutants and carcinogens. These traditional approaches, whereas yielding highly valuable information, fail to recognize the fact that many conjugates (especially hydrophilic ones) cannot permeate the cell membrane. In the present review, we will focus on the coupling process that involves both conjugating enzymes and efflux transporters. We will briefly review conjugating enzymes capable of producing highly hydrophilic metabolic products. The other focus of this review is on various transporters capable of moving negatively charged hydrophilic conjugates across the cellular membrane. Evidence will support the hypothesis that efficient coupling of the conjugating enzymes and efflux transporters enables enterohepatic recycling and enteric recycling processes. Termed as a "revolving door" theory, the hypothesis focuses on the role played by efflux transporter capable of modulating the cellular excretion of hydrophilic metabolites. Coupling process in intestine, liver and kidney will be discussed with an emphasis on the intestinal coupling process, since we have just begun to understand it. Biological consequence and new insights into how coupling process can impact bioavailability of xenobiotics, biological functions of drugs and carcinogens, and drug interactions will be discussed.

Alantolactone is a sesquiterpene lactone isolated from Inula helenium L. Although alantolactone possesses anti-inflammation and apoptosis-induction activities, the underlying mechanism of anti-cancer effect on human breast cancer cells remains largely unknown. In this study, we explored the possibility of alantolactone as an apoptosis-inducing cytotoxic agent using MDA-MB-231 cells as in vitro model. Alantolactone significantly induced its apoptosis, demonstrated by cell cycle analysis, annexin V-APC/7-AAD double staining and dUTP nick end labeling. Additionally, alantolactone triggered the mitochondrial-mediated caspase cascade apoptotic pathway, which was confirmed by increased Bax/Bcl-2 ratio, loss of MMP, release of cytc from mitochondria to cytoplasm, activation of caspase 9/3, and subsequent cleavage of PARP. Z-VAD-FMK partially prevented apoptosis induced by alantolactone. Alantolactone provoked the production of ROS, while NAC (a scavenger of ROS) reversed alantolactone-mediated depolarization of MMP and apoptosis. Alantolactone modulated the activities of MAPKs. As expected, cotreatment with SB203580, SP600125 or U0126 could reduced the apoptotic rate. Furthermore, alantolactone decreased the protein expressions of p-NF-kB p65 and p-STAT3, increased p-c-Jun level in a dose-dependent manner. These findings suggested that alantolactone possessed anticancer activity via ROS-mediated mitochondrial dysfunction involving MAPK pathway, and had an effect on the transcription factors of NF-kB, AP-1 and STAT3.