Rifampin successfully induced CYP2C9 and CYP3A4 activity and mRNA expression in the Fa2N-4 cells, which confirms the responsiveness of this immortalized cell line to CYP induction ( and ). As shown in , CYP2C9 activity in Fa2N-4 cells was induced by rifampin to 0.95–2.48 fold compared to media control exposure. Under the same conditions, exposure of Fa2N-4 cells to either 100 or 200 μg/mL garlic significantly reduced CYP2C9 activity in a concentration- and time-dependent manner. By days three and four, both 100 and 200 μg/mL concentrations of garlic lowered CYP2C9 activity to nearly undetectable levels (). We next replicated the experiment to determine whether at lower garlic concentrations, CYP2C9 activity could be induced (). We found that at 50 μg/mL, garlic could still suppress hydroxylation of diclofenac by >90% at day four. With respect to CYP3A4 activity, exposure of Fa2N-4 to varying concentrations of garlic extract did not alter CYP3A4 activity ().
Effects of Garlic on CYP2C9 activity
Effects of Garlic on CYP2C9 and CYP3A4 activity and mRNA expression by day four of treatmenta
We also determined the effects of garlic on RNA transcripts of CYP2C9 and CYP3A4 in Fa2N-4 cells. The RNA levels were determined on day four using a validated, quantitative RT-PCR assay for each of the target gene transcripts. As shown in , exposure of these cells to rifampin induces both CYP2C9 and CYP3A4 mRNA transcripts by about four-fold compared to control medium. When the same cells were exposed to garlic, CYP2C9 showed a garlic concentration-dependent reduction in transcript levels. In contrast, exposure to even up to 100 μg/mL of garlic extract did not appear to significantly alter the CYP3A4 transcript concentration in hepatocytes. These results suggest that garlic extract had a selective suppressive effect on the mRNA expression of CYP2C9 in Fa2N-4 hepatocytes. Taken together, the enzymatic activity of CYP2C9 and CYP3A4 in these hepatocytes appeared to have tracked the effects of garlic on RNA transcript levels.
Although it has been suggested that garlic supplements may induce CYP activity in vivo, there is little direct evidence that garlic extract up-regulates human CYP2C9 or CYP3A4 expression. Using a novel immortalized hepatocyte Fa2N-4 cell line that exhibits similar characteristics as those of primary human hepatocytes, we have systematically studied the modulating effects of garlic extract on CYP 2C9 and CYP3A4 mRNA expression and catalytic activity. Our results show that garlic suppresses CYP2C9 expression and activity in a remarkable, concentration-dependent manner, but that garlic has no discernable effect on CYP3A4 within the same cells. The changes in CYP2C9 activity paralleled those of RNA transcript levels. The decrease in transcript concentration is not due to any detrimental effects of garlic extract on cell viability, since expression and activity of CYP3A4 were maintained in the same cells. Also, the degree of induction observed with rifampin in our Fa2N-4 cultures () is comparable to that reported by others [11
]. In regards to mechanism, the decrease in CYP2C9 transcript could reflect either reduction in transcription or increased RNA degradation, or both. As CYP3A4 transcription is not affected by garlic exposure, it appears that the putative effects of garlic on intracellular transcription or message destabilization are a target-gene specific process. The exact mechanism awaits further investigation. It should be noted that garlic extract could also directly inhibit CYP2C9, but not CYP3A4 activity. Using cDNA expressed recombinant CYP3A4 enzyme, in microsome (cell-free) preparations, Foster et al. have shown that various preparations of garlic extract at a fixed 25 mg/mL concentration inhibited CYP3A4 and CYP2C9 activity [9
]. It is not known whether garlic extract at the much lower concentrations employed in our experiments (between 5–200 μg/mL) can directly inhibit CYP2C9 activity ( and ). Nonetheless, it is clear that the activity of CYP3A4 in Fa2N-4 was not affected by the chosen medium concentrations of garlic extract ().
It has been proposed that garlic supplements’ effects on CYP3A4 in humans is likely to be biphasic in nature; that is, inhibition during the early phase of treatment [8
], and simultaneous inhibition and induction following chronic treatment. This hypothesis is supported by the minimal effects of short term exposure (four to five days) to garlic on modifying the area under the concentration-time curve (AUC) of a single dose of ritonavir, a substrate for CYP3A4 [12
]. Chronic (i.e., 21-days) administration of dehydrated garlic (Natrol's GarliPure™ caplets BID) significantly reduced the AUC and Cmax of the HIV protease inhibitor saquinavir (a CYP3A4 and P glycoprotein substrate) in nine healthy volunteers [6
]. It was proposed that garlic constituents lowered the saquinavir plasma concentrations by inducing the drug-metabolizing enzyme CYP3A4 in the intestine and the liver, and/or the membrane efflux transporter P-glycoprotein at the intestinal mucosa. CYP3A4 activity was not affected by 14-day administration of a different dehydrated garlic product (Kwai garlic supplement, three tablets BID) [13
]. Only one study to date has examined the effects of steam-distilled garlic oil on CYP3A4; midazolam hydroxylation, a marker of CYP3A4, was not affected by steam-distilled garlic oil (500 mg TID for 28 days) [14
]. It appears that garlic has an equivocal effect on hepatic CYP3A4 that may be substrate-dependent.
Given the remarkable ability for garlic extract to suppress the expression and activity of CYP2C9 in Fa2N-4 cells and previous reports of its direct competitive inhibition of CYP2C9, garlic supplements may interact with drugs that are CYP2C9 substrates with narrow therapeutic indices or notable adverse reactions. For example, celecoxib is extensively metabolized by CYP2C9 [15
] for which dose-dependent cardiotoxicity has been a concern. In addition to celecoxib, garlic supplements may interact with other drugs metabolized by CYP2C9, such as the antiplatelet drug cilostazol, and the antidiabetic agent glyburide [18