5 research outputs found
Impact of pregnancy related hormones on drug metabolizing enzyme and transport protein concentrations in human hepatocytes
Pregnancy alters the disposition and exposure to multiple drugs indicated for pregnancy-related complications. Previous in vitro studies have shown that pregnancy-related hormones (PRHs) alter the expression and function of certain cytochrome P450s (CYPs) in human hepatocytes. However, the impact of PRHs on hepatic concentrations of non-CYP drug-metabolizing enzymes (DMEs) and transport proteins remain largely unknown. In this study, sandwich-cultured human hepatocytes (SCHH) from five female donors were exposed to vehicle or PRHs (estrone, estradiol, estriol, progesterone, cortisol, and placental growth hormone), administered individually or in combination, across a range of physiologically relevant PRH concentrations for 72Â h. Absolute concentrations of 33 hepatic non-CYP DMEs and transport proteins were quantified in SCHH membrane fractions using a quantitative targeted absolute proteomics (QTAP) isotope dilution nanoLC-MS/MS method. The data revealed that PRHs altered the absolute protein concentration of various DMEs and transporters in a concentration-, isoform-, and hepatocyte donor-dependent manner. Overall, eight of 33 (24%) proteins exhibited a significant PRH-evoked net change in absolute protein concentration relative to vehicle control (ANOVA p < 0.05) across hepatocyte donors: 1/11 UGTs (9%; UGT1A4), 4/6 other DMEs (67%; CES1, CES2, FMO5, POR), and 3/16 transport proteins (19%; OAT2, OCT3, P-GP). An additional 8 (24%) proteins (UGT1A1, UGT2B4, UGT2B10, FMO3, OCT1, MRP2, MRP3, ENT1) exhibited significant PRH alterations in absolute protein concentration within at least two individual hepatocyte donors. In contrast, 17 (52%) proteins exhibited no discernable impact by PRHs either within or across hepatocyte donors. Collectively, these results provide the first comprehensive quantitative proteomic evaluation of PRH effects on non-CYP DMEs and transport proteins in SCHH and offer mechanistic insight into the altered disposition of drug substrates cleared by these pathways during pregnancy
Species-Specific Differences in the <i>in Vitro</i> Metabolism of Lasiocarpine
There are species-related differences
in the toxicity of pyrrolizidine
alkaloids (PAs) partly attributable to the hepatic metabolism of these
alkaloids. In this study, the metabolism of lasiocarpine, a potent
hepatotoxic and carcinogenic food contaminant, was examined <i>in vitro</i> with human, pig, rat, mouse, rabbit, and sheep
liver microsomes. A total of 12 metabolites (M1–M12) were detected
with the human liver microsomes, of which M1, M2, M4, and M6 were
unstable in the presence of reduced glutathione (GSH). With the exception
of M3 and M8, the formation of all metabolites of lasiocarpine was
catalyzed by CYP3A4 in humans. Tandem mass spectra (MS/MS) detected
several new metabolites, termed M4–M7; their toxicological
significance is unknown. M9 (<i>m</i>/<i>z</i> 398), identified as a demethylation product, was the main metabolite
in all species, although the relative dominance of this metabolite
was lower in humans. The level of the reactive metabolites, as measured
by M1 ((3<i>H</i>-pyrrolizin-7-yl)Âmethanol) and the GSH
conjugate, was higher with the liver microsomes of susceptible species
(human, pig, rat, and mouse) than with the species (rabbit and sheep)
resistant to PA intoxication. In general, in addition to the new metabolites
(M4–M7) that could make humans more susceptible to lasiocarpine-induced
toxicity, the overall metabolite fingerprint detected with the human
liver microsomes differed from that of all other species, yielding
high levels of GSH-reactive metabolites
<i>In Silico</i> Prediction of the Site of Oxidation by Cytochrome P450 3A4 That Leads to the Formation of the Toxic Metabolites of Pyrrolizidine Alkaloids
In
humans, the metabolic bioactivation of pyrrolizidine alkaloids
(PAs) is mediated mainly by cytochrome P450 3A4 (CYP3A4) via the hydroxylation
of their necine bases at C3 or C8 of heliotridine- and retronecine-type
PAs or at the N atom of the methyl substituent of otonecine-type PAs.
However, no attempts have been made to identify which C atom is the
most favorable site for hydroxylation <i>in silico</i>.
Here, in order to determine the site of hydroxylation that eventually
leads to the formation of the toxic metabolites produced from lasiocarpine,
retrorsine, and senkirkin, we utilized the ligand-based electrophilic
Fukui function <i>f</i><sup>–</sup>(<b>r</b>) and hydrogen-bond dissociation energies (BDEs) as well as structure-based
molecular docking. The ligand-based computations revealed that the
C3 and C8 atoms of lasiocarpine and retrorsine and the C26 atom of
senkirkin were chemically the most susceptible locations for electrophilic
oxidizing reactions. Similarly, according to the predicted binding
orientation in the active site of the crystal structure of human CYP3A4
(PDB code: 4I4G), the alkaloids were positioned in such a way that the C3 atom of
lasiocarpine and retrorsine and the C26 of senkirkin were closest
to the catalytic heme Fe. Thus, it is concluded that the C3 atom of
lasiocarpine and retrorsine and C26 of senkirkin are the most favored
sites of hydroxylation that lead to the production of their toxic
metabolites
DataSheet1_Impact of pregnancy related hormones on drug metabolizing enzyme and transport protein concentrations in human hepatocytes.PDF
Pregnancy alters the disposition and exposure to multiple drugs indicated for pregnancy-related complications. Previous in vitro studies have shown that pregnancy-related hormones (PRHs) alter the expression and function of certain cytochrome P450s (CYPs) in human hepatocytes. However, the impact of PRHs on hepatic concentrations of non-CYP drug-metabolizing enzymes (DMEs) and transport proteins remain largely unknown. In this study, sandwich-cultured human hepatocytes (SCHH) from five female donors were exposed to vehicle or PRHs (estrone, estradiol, estriol, progesterone, cortisol, and placental growth hormone), administered individually or in combination, across a range of physiologically relevant PRH concentrations for 72Â h. Absolute concentrations of 33 hepatic non-CYP DMEs and transport proteins were quantified in SCHH membrane fractions using a quantitative targeted absolute proteomics (QTAP) isotope dilution nanoLC-MS/MS method. The data revealed that PRHs altered the absolute protein concentration of various DMEs and transporters in a concentration-, isoform-, and hepatocyte donor-dependent manner. Overall, eight of 33 (24%) proteins exhibited a significant PRH-evoked net change in absolute protein concentration relative to vehicle control (ANOVA p < 0.05) across hepatocyte donors: 1/11 UGTs (9%; UGT1A4), 4/6 other DMEs (67%; CES1, CES2, FMO5, POR), and 3/16 transport proteins (19%; OAT2, OCT3, P-GP). An additional 8 (24%) proteins (UGT1A1, UGT2B4, UGT2B10, FMO3, OCT1, MRP2, MRP3, ENT1) exhibited significant PRH alterations in absolute protein concentration within at least two individual hepatocyte donors. In contrast, 17 (52%) proteins exhibited no discernable impact by PRHs either within or across hepatocyte donors. Collectively, these results provide the first comprehensive quantitative proteomic evaluation of PRH effects on non-CYP DMEs and transport proteins in SCHH and offer mechanistic insight into the altered disposition of drug substrates cleared by these pathways during pregnancy.</p
Identification of a New Reactive Metabolite of Pyrrolizidine Alkaloid Retrorsine: (3<i>H</i>‑Pyrrolizin-7-yl)methanol
Pyrrolizidine alkaloids (PAs) such
as retrorsine are common food
contaminants that are known to be bioactivated by cytochrome P450
enzymes to putative hepatotoxic, genotoxic, and carcinogenic metabolites
known as dehydropyrrolizidine alkaloids (DHPs). We compared how both
electrochemical (EC) and human liver microsomal (HLM) oxidation of
retrorsine could produce short-lived intermediate metabolites; we
also characterized a toxicologically important metabolite, (3<i>H</i>-pyrrolizin-7-yl)Âmethanol. The EC cell was coupled online
or offline to a liquid chromatograph/mass spectrometer (LC/MS), whereas
the HLM oxidation was performed in 100 mM potassium phosphate (pH
7.4) in the presence of NADPH at 37 °C. The EC cell oxidation
of retrorsine produced 12 metabolites, including dehydroretrorsine
(<i>m</i>/<i>z</i> 350, [M + H<sup>+</sup>]),
which was degraded to a new reactive metabolite at <i>m</i>/<i>z</i> 136 ([M + H<sup>+</sup>]). The molecular structure
of this small metabolite was determined using high-resolution mass
spectrometry and NMR spectroscopy followed by chemical synthesis.
In addition, we also identified another minor but reactive metabolite
at <i>m</i>/<i>z</i> 136, an isomer of (3<i>H</i>-pyrrolizin-7-yl)Âmethanol. Both (3<i>H</i>-pyrrolizin-7-yl)Âmethanol
and its minor isomer were also observed after HLM oxidation of retrorsine
and other hepatotoxic PAs such as lasiocarpine and senkirkin. In the
presence of reduced glutathione (GSH), each isomer formed identical
GSH conjugates at <i>m</i>/<i>z</i> 441 and <i>m</i>/<i>z</i> 730 in the negative ESI-MS. Because
(3<i>H</i>-pyrrolizine-7-yl)Âmethanol) and its minor isomer
subsequently reacted with GSH, it is concluded that (3<i>H</i>-pyrrolizin-7-yl)Âmethanol may be a common toxic metabolite arising
from PAs