L-Methionine (Met) has been implicated in parenteral nutritionassociated cholestasis in infants and, at high levels, it causes liver toxicity by mechanisms that are not clear. In this study, Met toxicity was characterized in freshly isolated male and female mouse hepatocytes incubated with 5 to 30 mM Met for 0 to 5 h. In male hepatocytes, 20 mM Met was cytotoxic at 4 h as indicated by trypan blue exclusion and lactate dehydrogenase leakage assays. Cytotoxicity was preceded by reduced glutathione (GSH) depletion at 3 h without glutathione disulfide formation. Exposure to 30 mM Met resulted in increased cytotoxicity and GSH depletion. It is interesting to note that female hepatocytes were resistant to Met-induced cytotoxicity at these concentrations and showed increased cellular GSH levels compared with hepatocytes exposed to medium alone. The effects of amino-oxyacetic acid (AOAA), an inhibitor of Met transamination, and 3-deazaadenosine (3-DA), an inhibitor of the Met transmethylation pathway enzyme S-adenosylhomocysteine hydrolase, on Met toxicity in male hepatocytes were then examined. Addition of 0.2 mM AOAA partially blocked Met-induced GSH depletion and cytotoxicity, whereas 0.1 mM 3-DA potentiated Met-induced toxicity. Exposure of male hepatocytes to 0.3 mM 3-methylthiopropionic acid (3-MTP), a known Met transamination metabolite, resulted in cytotoxicity and cellular GSH depletion similar to that observed with 30 mM Met, whereas incubations with D-methionine resulted in no toxicity. Female hepatocytes were less sensitive to 3-MTP toxicity than males, which may partially explain their resistance to Met toxicity. Taken together, these results suggest that Met transamination and not transmethylation plays a major role in Met toxicity in male mouse hepatocytes.

ABSTRACT:L-Methionine-dl-sulfoxide (MetO) is an L-methionine (Met) metabolite, but its role in Met metabolism and toxicity is not clear. In this study, MetO uptake, metabolism to Met, cytotoxicity, and glutathione (GSH) and glutathione disulfide (GSSG) status were characterized in freshly isolated mouse hepatocytes incubated at 37°C with 0 to 30 mM MetO for 0 to 5 h. In male hepatocytes, dose-dependent cytotoxicity concomitant with GSH depletion without GSSG formation occurred after exposure to 20 or 30 mM MetO but not after exposure to 10 mM MetO. Interestingly, female hepatocytes exposed to 30 mM MetO showed no cytotoxicity and exhibited increased intracellular GSH levels compared with control hepatocytes.

Flavin-containing monooxygenases (FMOs) are microsomal enzymes that catalyze the NADPH-and O 2 -dependent oxidation of heteroatoms (nitrogen, sulfur, phosphorus) present in the chemical structure of a variety of drugs and xenobiotics. Five functional forms (FMO1-5) have been characterized to date (Ziegler, 2002). In humans, FMO1 is the primary isoform expressed in neonate liver, but a switch occurs shortly after birth to FMO3, the primary isoform expressed in adult human liver (Koukouritaki et al., 2002). In mice, females, but not males, also express FMO3 in the liver (Falls et al., 1995;Ripp et al., 1999b). This sex-specific expression of FMO3 in mice makes them an attractive model for studying the role of FMO3 in human drug metabolism and disease.Our laboratory first identified Met as a substrate for cDNA-expressed rabbit FMO1-3 with K m values of 48.0, 29.9, and 6.5 mM, respectively (Duescher et al., 1994). The V/K values (0.9, 1.7, and 6.1 for FMO1-3, respectively) also suggested that FMO3 was the most efficient Met S-oxidizer of these three FMO isoforms. FMO3 Soxidation of Met was highly stereoselective, forming 8.4 times more methionine-d-sulfoxide (Met-d-O) than methionine-l-sulfoxide (Metl-O), whereas the d:l diastereomeric ratios for FMO1 and FMO2 were 1.5:1 and 0.7:1, respectively; FMO5 S-oxidation of Met was not detected. Recombinant human FMO3 exhibited a K m value of 3.7 mM with a V/K value of 4.6 and resulted in stereoselective formation of (Ripp et al., 1999b). Recombinant human FMO4 S-oxidation of Met exhibited a K m value greater than 10 mM with only 30% of the total sulfoxide formed being Met-d-O (Ripp et al., 1999a). The V/K value for FMO4 was not determined. These data indicated that FMO3 S-oxidation of Met proceeds with the highest affinity and greatest diastereomeric selectivity among FMO1-5.Stereoselective formation of Met-d-O was also detected in rabbit and rat liver microsomes incubated with Met, and exhibited K m and V/K values similar to those of cDNA-expressed FMO3 (Duescher et al., 1994;Krause et al., 1996). The latter results provided evidence for FMO3 being the primary isoform involved in Met S-oxidation in rabbit and rat liver. Additional experiments also provided evidence that FMOs, but not cytochrome P450s, peroxidases, or reactive oxygen species, mediated the Met S-oxidase activity (Krause et al., 1996).

α-Retinol (αR) is a structural isomer of retinol [vitamin A (VA)] that does not bind to serum retinol-binding protein (RBP). In this study, α-retinyl acetate (αRA) was synthesized and given orally (35 μmol) to VA-deficient lactating sows (n = 11) to assess its potential to trace RBP-independent retinol transport and tissue uptake. The αRA dose primarily appeared in sow serum as 4 α-retinyl esters (αRE) with peak serum total αR concentrations (the sum of the alcohol and ester forms) detected at 2 h (70 ± 23 nmol/L, mean ± SEM) postdose. From 0 to 40 h postdose, the percentage of serum total αR in the alcohol form did not increase. Rapid αR uptake into sow milk was observed with peak concentrations (371 ± 83 nmol/L) at 7.5 h postdose, consistent with the uptake of αRE from chylomicra. A high percentage of the αRA dose (62 ± 15%, mean ± SD) was present in the livers of sows (n = 6) killed 22-28 d postdose. Approximately 15-26% of the sow αRA dose was transferred to the livers of the nursing piglets (n = 17) after 3 d. In piglets and sows, a similar percentage of hepatic total αR was detected in the ester form as that of hepatic total retinol. Taken together, these data suggest that an oral dose of αRA effectively traces the uptake, esterification, chylomicron transport, and hepatic storage of retinol and may be useful for deciphering the role of RBP-independent delivery of retinol to other tissues.

Dietary α-carotene is found in orange and purple-orange carrots. Upon α-carotene’s central cleavage in the intestine, α-retinal and retinal are formed and reduced to α-retinol and retinol. Previous reports suggested that α-retinol has 2% biopotency of all-trans-retinyl acetate due in part to its inability to bind to retinol-binding protein. The current studies re-determined α-retinol's biopotency compared with retinol and 3, 4-didehydroretinol in a growth assay. Weanling rats (n 40) were fed vitamin A-deficient diet for 8 weeks, divided into 4 treatment groups (n 10/group), and orally dosed with 50 nmol/d retinyl acetate (14.3 µg retinol), α-retinyl acetate (14.3 µg α-retinol), 3, 4-didehydroretinyl acetate (14.2 µg 3, 4-didehydroretinol), or cottonseed oil (negative control). Supplementation continued until control rats showed deficiency signs 5 weeks after supplementation began. In comparison to retinol, body weights and area-under-the-response curves revealed that α-retinol and 3,4-didehydroretinol had 40–50 and 120–130% bioactivity, respectively, compared with retinol. In study 2, rats (n 40) received 70 nmol retinyl acetate and 0, 17.5, 35, or 70 nmol α-retinyl acetate daily for 3 weeks. Although liver retinol differed among groups, α-retinol did not appreciably interfere with retinol storage. In study 3, 3.5 µmol/d α-retinyl acetate was fed to rats (n 15) for 21 d and groups were killed at 1, 2, and 3 week intervals. No hepatic toxicity was observed. In conclusion, α-retinol and didehydroretinol are more biopotent than previously reported with sustained equimolar dosing at 50 nmol/d, which was an amount of retinol known to keep rats in vitamin A balance.