Farnesol is glucuronidated in human liver, kidney and intestine in vitro, and is a novel substrate for UGT2B7 and UGT1A1

Abstract

Farnesol is an isoprenoid found in many aromatic plants and is also produced in humans, where it acts on numerous nuclear receptors and has received considerable attention due to its apparent anticancer properties. Although farnesol has been studied for over 30 years, its metabolism has not been well characterized. Recently, farnesol was shown to be metabolized by cytochromes P450 in rabbit; however, neither farnesol hydroxylation nor glucuronidation in humans have been reported to date. In the present paper, we show for the first time that farnesol is metabolized to farnesyl glucuronide, hydroxyfarnesol and hydroxyfarnesyl glucuronide by human tissue microsomes, and we identify the specific human UGTs (uridine diphosphoglucuronosyltransferases) involved. Farnesol metabolism was examined by a sensitive LC (liquid chromatography)-MS/MS method. Results indicate that farnesol is a good substrate for glucuronidation in human liver, kidney and intestine microsomes (values in nmol/min per mg). Initial analysis using expressed human UGTs indicated that UGTs 1A1 and 2B7 were primarily responsible for glucuronidation in vitro, with significantly lower activity for all the other UGTs tested (UGTs 1A3, 1A4, 1A6, 1A9 and 2B4). Kinetic analysis and inhibition experiments indicate that, in liver microsomes, UGT1A1 is primarily responsible for farnesol glucuronidation; however, in intestine microsomes, UGT2B7 is probably the major isoform involved, with a very-low-micromolar K(m). We also show the first direct evidence that farnesol can be metabolized to hydroxyfarnesol by human liver microsomes and that hydroxyfarnesol is metabolized further to hydroxyfarnesyl glucuronide. Thus glucuronidation may modulate the physiological and/or pharmacological properties of this potent signalling molecule.

Scheme 1. The mevalonate synthesis pathway showing the role of farnesol

Farnesol originates from three sources: (i) synthesis of FPP via the mevalonate pathway, followed by action by FPP phosphatase, (ii) degradation of prenylated proteins (although this has not been proved experimentally), or (iii) external sources, such as dietary or pharmaceutical. The broken arrow indicates the feedback role that farnesol has in controlling HMG-CoA reductase levels. Farnesol can be metabolized to farnesyl phosphate and then to FPP, to hydroxyfarnesol, farnesal or to the farnesyl glucuronide. F, farnesol; FP, farnesyl phosphate; P-450, unknown CYP; ADH, alcohol dehydrogenase.

Figure 1. LC–MS/MS trace of farnesol glucuronidation in human liver microsomes using MRM detection of farnesyl glucuronide

For details, see the Experimental section.

Figure 2. Structures of farnesol and the farnesyl glucuronide product

Figure 3. Mass spectra of farnesyl β-D-glucuronide and hydroxyfarnesyl β-D-glucuronide

(A) Mass spectrum of farnesyl β-D-glucuronide was run from 100 to 400 m/z in negative-ion mode; the spectrum was derived from a daughter ion scan at the farnesyl glucuronide mass (m/z 397), from an incubation of farnesol with human liver microsomes and UDPGA. The inset shows the proposed fragmentation of farnesyl β-D-glucuronide with main fragment masses. Masses at 175, 157 and 113 m/z are common fragments of glucuronide. No peak was observed for a farnesol aglycone fragment (221 m/z). (B) Mass spectrum of hydroxyfarnesyl β-D-glucuronide. Main peaks observed at 193, 175 and 113 m/z are indicative of the glucuronide, and the peak at 237 is the hydroxyfarnesol aglycone. As in the case of the farnesyl β-D-glucuronide fragmentation, no peak was observed for the farnesol moiety.

Figure 4. Kinetic analysis of farnesol glucuronidation by microsomes or cell lines expressing human UGTs

(A) Human liver microsomes, (B) human kidney microsomes, (C) human intestine microsomes, (D) human UGT2B4, (E) human UGT2B7. For (C) and (E), data from the low concentration values (0–10 μM) are shown in the insets. The data points are based on the mean of a duplicated experiment.

Figure 5. Eadie–Hofstee plots of farnesol glucuronidation

Farnesol glucuronidation was carried out by (A) human liver microsomes, (B) human kidney microsomes, (C) human intestine microsomes or (D) human UGT 2B7. The K_m values for the different lines are indicated.