The natural product betulin is under investigation for several therapeutic indications, however l... more The natural product betulin is under investigation for several therapeutic indications, however little is known about its metabolism. In the present study, the glucuronidation and sulfation of betulin in human and rat liver microsomes and cytosol were tested. We further identified the main UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) involved in these two metabolism pathways. Results showed that one betulin glucuronide metabolite was observed after incubation with human and rat liver microsomes. The glucuronidation of betulin in human liver microsomes had a Km value of 21.1 ± 5.93 μM and a Vmax value of 6.39 ± 0.66 pmol/min/mg protein. The glucuronidation activity in rats was too low to get enzyme kinetic parameters. Among the 11 recombinant UGT enzymes investigated, UGT1A3 and UGT1A4 were identified as the major enzymes catalyzing the glucuronidation of betulin [Km values of 10.12 ± 8.09 and 8.04 ± 3.96 μM, Vmax values of 6.71 ± 1.51 and 5.98 ± 0.76 nmol/min/(mg protein)]. Two betulin sulfate metabolites were found in human and rat liver cytosols. Human and rat liver had similar affinity for the formation of these two metabolites, the apparent Vmax for betulin sulfate I was higher than that for betulin sulfate II in both species. Among the SULT isoforms studied, SULT2A1 was the major isoenzyme involved in the betulin sulfation metabolism in human liver cytosol. The results suggest that glucuronidation and sulfation are important metabolism pathways for betulin, and UGT1A3, UGT1A4 and SULT2A1 play the major roles in betulin glucuronidation and sulfation.
Methoxylated bromodiphenyl ethers (MeO-BDEs), marine natural products, can be demethylated by cyt... more Methoxylated bromodiphenyl ethers (MeO-BDEs), marine natural products, can be demethylated by cytochrome P450 to produce hydroxylated bromodiphenyl ethers (OH-BDEs), potentially toxic metabolites that are also formed by hydroxylation of BDE flame retardants. The OH-BDEs may be detoxified by glucuronidation and sulfonation. This study examined the demethylation of 6-MeO-BDE47, 2'-MeO-BDE68 and 4'-MeO-BDE68, in hepatic microsomes from the red snapper, Lutjanus campechanus, a marine fish likely to be exposed naturally to MeO-BDEs, and the channel catfish, Ictalurus punctatus, a freshwater fish in which pathways of xenobiotic biotransformation have been studied. We further studied the glucuronidation and sulfonation of the resulting OH-BDEs as well as of 6-OH-2'-MeO-BDE68 in hepatic microsomes and cytosol fractions of these fish. The three studied biotransformation pathways were active in both species, with high individual variability. The range of activities overlapped in the two species. Demethylation of MeO-BDEs, studied in the concentration range 10-500 μM, followed Michaelis-Menten kinetics in both fish species, however enzyme efficiencies were low, ranging from 0.024 to 0.334 μL min.mg protein. Conjugation of the studied OH-BDEs followed Michaelis-Menten kinetics in the concentration ranges 1-50 μM (glucuronidation) or 2.5-100 μM (sulfonation). These OH-BDEs were readily glucuronidated and sulfonated in the fish livers of both species, with enzyme efficiencies one to three orders of magnitude higher than for demethylation of the precursor MeO-BDEs. The relatively low efficiencies of demethylation of the MeO-BDEs, compared with higher efficiencies for OH-BDE conjugation, suggests that MeO-BDEs are more likely than OH-BDEs to bioaccumulate in tissues of exposed fish.
Escalating levels of antibiotic resistance in mycoplasmas, particularly macrolide resistance in M... more Escalating levels of antibiotic resistance in mycoplasmas, particularly macrolide resistance in Mycoplasma pneumoniae and M. genitalium , have narrowed our antibiotic arsenal. Further, mycoplasmas lack a cell wall and do not synthesize folic acid, rendering common antibiotics, such as beta-lactams, vancomycin, sulfonamides, and trimethoprim, of no value.
The natural product betulin is under investigation for several therapeutic indications, however l... more The natural product betulin is under investigation for several therapeutic indications, however little is known about its metabolism. In the present study, the glucuronidation and sulfation of betulin in human and rat liver microsomes and cytosol were tested. We further identified the main UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) involved in these two metabolism pathways. Results showed that one betulin glucuronide metabolite was observed after incubation with human and rat liver microsomes. The glucuronidation of betulin in human liver microsomes had a Km value of 21.1 ± 5.93 μM and a Vmax value of 6.39 ± 0.66 pmol/min/mg protein. The glucuronidation activity in rats was too low to get enzyme kinetic parameters. Among the 11 recombinant UGT enzymes investigated, UGT1A3 and UGT1A4 were identified as the major enzymes catalyzing the glucuronidation of betulin [Km values of 10.12 ± 8.09 and 8.04 ± 3.96 μM, Vmax values of 6.71 ± 1.51 and 5.98 ± 0.76 nmol/min/(mg protein)]. Two betulin sulfate metabolites were found in human and rat liver cytosols. Human and rat liver had similar affinity for the formation of these two metabolites, the apparent Vmax for betulin sulfate I was higher than that for betulin sulfate II in both species. Among the SULT isoforms studied, SULT2A1 was the major isoenzyme involved in the betulin sulfation metabolism in human liver cytosol. The results suggest that glucuronidation and sulfation are important metabolism pathways for betulin, and UGT1A3, UGT1A4 and SULT2A1 play the major roles in betulin glucuronidation and sulfation.
Methoxylated bromodiphenyl ethers (MeO-BDEs), marine natural products, can be demethylated by cyt... more Methoxylated bromodiphenyl ethers (MeO-BDEs), marine natural products, can be demethylated by cytochrome P450 to produce hydroxylated bromodiphenyl ethers (OH-BDEs), potentially toxic metabolites that are also formed by hydroxylation of BDE flame retardants. The OH-BDEs may be detoxified by glucuronidation and sulfonation. This study examined the demethylation of 6-MeO-BDE47, 2'-MeO-BDE68 and 4'-MeO-BDE68, in hepatic microsomes from the red snapper, Lutjanus campechanus, a marine fish likely to be exposed naturally to MeO-BDEs, and the channel catfish, Ictalurus punctatus, a freshwater fish in which pathways of xenobiotic biotransformation have been studied. We further studied the glucuronidation and sulfonation of the resulting OH-BDEs as well as of 6-OH-2'-MeO-BDE68 in hepatic microsomes and cytosol fractions of these fish. The three studied biotransformation pathways were active in both species, with high individual variability. The range of activities overlapped in the two species. Demethylation of MeO-BDEs, studied in the concentration range 10-500 μM, followed Michaelis-Menten kinetics in both fish species, however enzyme efficiencies were low, ranging from 0.024 to 0.334 μL min.mg protein. Conjugation of the studied OH-BDEs followed Michaelis-Menten kinetics in the concentration ranges 1-50 μM (glucuronidation) or 2.5-100 μM (sulfonation). These OH-BDEs were readily glucuronidated and sulfonated in the fish livers of both species, with enzyme efficiencies one to three orders of magnitude higher than for demethylation of the precursor MeO-BDEs. The relatively low efficiencies of demethylation of the MeO-BDEs, compared with higher efficiencies for OH-BDE conjugation, suggests that MeO-BDEs are more likely than OH-BDEs to bioaccumulate in tissues of exposed fish.
Escalating levels of antibiotic resistance in mycoplasmas, particularly macrolide resistance in M... more Escalating levels of antibiotic resistance in mycoplasmas, particularly macrolide resistance in Mycoplasma pneumoniae and M. genitalium , have narrowed our antibiotic arsenal. Further, mycoplasmas lack a cell wall and do not synthesize folic acid, rendering common antibiotics, such as beta-lactams, vancomycin, sulfonamides, and trimethoprim, of no value.
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