title: Slow O-demethylation of methyl triclosan to triclosan, which is rapidly glucuronidated and sulfonated in channel catfish liver and intestine.
abstract: The antibacterial personal care product triclosan is discharged in municipal waste, and converted in part by bacteria in sewage sludge and soil to its more lipid-soluble methyl ether, methyl triclosan. Triclosan and methyl triclosan have been detected in water, sediment, fish and invertebrates near sewage treatment facilities. Understanding the biotransformation of methyl triclosan and triclosan in a model food fish, the channel catfish, will be of value in assessing the likelihood that these compounds will bioaccumulate in exposed fish, and therefore potentially pass up the food chain. We hypothesize that cytochrome P450 will catalyze the O-demethylation of methyl triclosan to yield triclosan, which is likely to undergo glucuronidation or sulfonation of the phenolic hydroxyl group. Conversion of methyl triclosan to triclosan was measured by LC/MS/MS following aerobic incubation of varying concentrations of methyl triclosan with NADPH and hepatic and intestinal microsomes from untreated, 3-methylcholanthrene-treated (10 mg/kg, i.p.) or PCB-126-treated (0.1 mg/kg, i.p.) channel catfish (n=4 per treatment group). The K(m) values for methyl triclosan were similar for untreated, 3-methylcholanthrene-treated and PCB-126-treated catfish liver microsomes, ranging from 80 to 250 μM. V(max) values for O-demethylation ranged from 30 to 150 pmol/min/mg protein, with no significant differences between controls, PCB-126-treated or 3-methylcholanthrene-treated fish, suggesting that methyl triclosan O-demethylation was not a CYP1-catalyzed reaction. Methyl triclosan O-demethylation activities in intestinal microsomes were similar to or lower than those found with liver microsomes. The calculated rate of O-demethylation of methyl triclosan in catfish liver at 1 μM, a concentration reported in exposed fish, and 21°C, an early summer water temperature, is 0.10 pmol/min/mg protein. This slow rate of metabolism suggests that upon continued exposure, methyl triclosan may bioaccumulate in the channel catfish. Triclosan itself, however, was readily glucuronidated by hepatic and intestinal microsomes and sulfonated by hepatic and intestinal cytosol. Triclosan glucuronidation followed Michaelis-Menten kinetics when rates were measured across a concentration range of 5-1000 μM, whereas triclosan sulfonation exhibited substrate inhibition at concentrations above 10-20 μM in both intestinal and hepatic cytosol. Based on the enzyme kinetic constants measured in hepatic and intestinal fractions at 21°C, triclosan at 1 μM could be glucuronidated at rates of 23 and 3.2 pmol/min/mg protein respectively in liver and intestine, and sulfonated at rates of 277 (liver) and 938 (intestine) pmol/min/mg protein. These rates are much higher than the rates of demethylation of methyl triclosan, and suggest that triclosan would be rapidly cleared and unlikely to bioaccumulate in catfish tissues.
authors.affiliation: Department of Medicinal Chemistry, University of Florida, Gainesville, FL 32610-0485, United States. mojames@ufl.edu
authors.forename: Margaret O
authors.initials: MO
authors.lastname: James
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Marth
authors.forename: Laura
authors.initials: L
authors.lastname: Rowland-Faux
mesh_terms: D000818:Animals; D001711:Biotransformation; D003600:Cytosol / Q000378:metabolism; D004032:Diet; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016166:Free Radical Scavengers / Q000494:pharmacology; D020719:Glucuronides / Q000378:metabolism; D007059:Ictaluridae / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D008745:Methylation / Q000187:drug effects; D008861:Microsomes / Q000201:enzymology / Q000378:metabolism; D013696:Temperature; D014260:Triclosan / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D014874:Water Pollutants, Chemical / Q000737:chemistry / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D016166:Free Radical Scavengers; D020719:Glucuronides; D014874:Water Pollutants, Chemical; D014260:Triclosan; C502486:methyl triclosan; D004121:Dimethyl Sulfoxide