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TOXICOLOGY

L-Methionine Toxicity in Freshly Isolated Mouse Hepatocytes Is Gender-Dependent and Mediated in Part by Transamination

Department of Comparative Biosciences and Molecular and Environmental Toxicology Center, University of Wisconsin-Madison, Madison, Wisconsin

Received for publication May 9, 2008
Accepted June 12, 2008.

	Abstract

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L-Methionine (Met) has been implicated in parenteral nutrition-associated 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.

Many studies have provided insight into the potential mechanisms of Met-induced liver toxicity. Several metabolites from the Met transmethylation (TM) pathway have been studied for their toxicological contributions (Fig. 1). Met TM begins with the ATP-dependent conversion of Met to S-adenosylmethionine (SAM), an important biological methyl donor molecule (Finkelstein, 1990). ATP depletion from excessive SAM formation as well as the accumulation of SAM itself has been linked to Met-induced hepatotoxicity; however, the evidence is largely correlative (Hardwick et al., 1970; Regina et al., 1993). Methyl group donation from SAM results in the formation of S-adenosylhomocysteine (SAH), which is subsequently hydrolyzed to homocysteine (Hcy) (Finkelstein, 1990). Elevated levels of Hcy, which may be converted to the lysine-reactive metabolite Hcy thiolactone, have been clearly linked to arteriosclerosis, and they are an independent risk factor for cardiovascular disease in humans (Chwatko and Jakubowski, 2005). However, the toxicological role of Hcy and/or Hcy thiolactone in Met-induced liver toxicity has not been investigated.

View larger version (20K): [in this window] [in a new window]   Fig. 1. Schematic of the Met TM and TA pathways and the points of inhibition of AOAA and 3-DA. Bolded metabolites have been of particular toxicological interest.

Alternatively to Met TM, the Met transamination (TA) pathway has also been investigated for its involvement in Met toxicity (Fig. 1). Met TA is catalyzed by multiple transaminases including glutamine transaminase K and L and results in the formation of 2-keto-4-methylthiobutyric acid (Cooper, 1989; Scislowski and Pickard, 1993). 2-Keto-4-methylthiobutyric acid is then oxidatively decarboxylated by branched chain 2-oxo acid dehydrogenase complex to form 3-methylthiopropionic acid (3-MTP) (Steele and Benevenga, 1979; Jones and Yeaman, 1986). 3-MTP is further metabolized to the highly toxic and volatile molecules methanethiol, hydrogen sulfide, and dimethyl sulfide (Steele and Benevenga, 1979; Blom et al., 1988a; Gahl et al., 1988; Tangerman et al., 2000). Methanethiol has been shown to inhibit enzymes involved in protection from peroxidative damage in a similar manner to Met (Finkelstein and Benevenga, 1986). Furthermore, rats fed 3-MTP-spiked chow exhibited markedly similar toxicological symptoms as rats fed chow spiked with excess Met, which included growth retardation and hemolytic anemia (Steele et al., 1979). Taken together, these studies suggest that Met TA may also be involved in Met-induced liver toxicity.

In summary, the Met TM and TA pathways have both been implicated as toxicologically relevant pathways, but their relative roles in the liver toxicity associated with hypermethionemia remain unclear. The primary purpose of this study was to assess and compare the relative toxicological roles of Met TM and TA in freshly isolated male or female mouse hepatocytes exposed to high levels of Met through the use of inhibitors of these pathways. Met TA was inhibited using 0.2 mM amino-oxyacetic acid (AOAA), which was previously shown to inhibit Met TA in vitro (Mitchell and Benevenga, 1978). The formation of the Met TM metabolites Hcy and Hcy thiolactone was inhibited using 0.1 mM 3-deazaadenosine (3-DA), a known inhibitor of SAH hydrolase (García-Trevijano et al., 2000). Trypan blue exclusion, LDH leakage, reduced glutathione (GSH) and glutathione disulfide (GSSG) levels, and caspase-3/7 activation were measured to characterize and quantitate the Met-induced cytotoxic and biochemical effects. The toxicity of D-methionine (D-Met) and 3-MTP was also characterized using these endpoints to gain further insight into the mechanism of Met-induced toxicity.

	Materials and Methods

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Chemicals. Trypsin inhibitor (type II-O), collagenase (type IV), Met, D-Met, AOAA, 3-DA, GSH, GSSG, GSH reductase, 5,5'-dithio-bis(2-nitrobenzoic acid), pyruvate, NADH, NADPH, 2-vinylpyridine, 5-sulfosalicylic acid (SSA), EDTA, and Triton X-100 were obtained from Sigma-Aldrich (St. Louis, MO). 3-Methylthiopropionic acid (3-MTP) was purchased from Lancaster (Pelham, NH). Hanks' balanced salt solution was obtained from Invitrogen (Carlsbad, CA). Dulbecco's modified Eagle's medium (DMEM) (1x) with 4500 mg/l glucose, and sodium pyruvate but without L-glutamine, methionine, and cystine was purchased from HyClone Laboratories (Logan, UT). All other chemicals and reagents were of the highest quality commercially available.

Animals. Male and female B6C3F1 mice (7–11 weeks old) were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were maintained on a 12-h light/dark cycle and were allowed feed and water ad libitum. Hepatocytes were isolated at the same time of day to minimize the effects of circadian variation on GSH levels and other enzymatic processes of interest. Hepatocytes were isolated using the two-step EDTA/collagenase perfusion method as described by Kemper et al. (2001), except that livers were perfused with 0.6 mg/ml collagenase solution. The collagenase solution was also centrifuged at 39,000g for 30 min to remove nonsoluble components. Initial cell yield and viability were determined by trypan blue exclusion using a hemacytometer. Only hepatocytes with an initial overall viability of greater than 85% after isolation were used in experiments. Cells were then diluted to a concentration of 1 x 10⁶ cells/ml in DMEM and maintained on ice until use.

Cell Incubations. Incubations were carried out in 24-ml vials with screw caps fitted with Teflon-faced septa. Samples of suspended hepatocytes (2.5 ml of 1 x 10⁶ cells/ml) were transferred to the vials. Vial samples were purged with 95% O₂, 5% CO₂ (carbogen) before incubation at 37°C with gentle shaking (140 rpm). After a 4-min preincubation, 131.5 µl of substrate dissolved in DMEM was added to each 2.5-ml cell sample, resulting in a final concentration of 5 to 30 mM Met. For D-Met, the final concentration was 30 mM. For 3-MTP, the final concentration was 0.3 mM. For inhibitor studies 118.4 µl of substrate solution was added followed by 13.1 µl of inhibitor dissolved in DMEM resulting in final concentrations of 0.1 mM for 3-DA and 0.2 mM for AOAA. Samples were then repurged with carbogen and incubated for 0 to 5 h. Cell incubations were terminated by being placed on ice. After gentle mixing, aliquots were collected for biochemical and toxicological analysis.

Determination of Cell Viability. Trypan blue exclusion was measured by adding 12 µl of cell sample to 12 µl of 0.4% trypan blue. Viability was then determined using a hemacytometer. LDH leakage was analyzed by a method adapted from Cummings et al. (2000). The percentage viability was defined as (LDH in solubilized cell sample)/(LDH in solubilized cell sample + LDH in cell medium sample).

Measurement of Apoptosis. Apoptosis was measured using the Caspase-Glo 3/7 luminescence assay as described by Promega (Madison, WI). In brief, 50 µl of caspase-3/7 reagent was added to 50 µl of cell sample. The reaction was allowed to proceed at room temperature for 30 min before luminescence was measured. Background luminescence from the medium in each sample was also measured and subtracted from the value obtained with cells in medium to obtain the final corrected value. Dimethyl sulfoxide (5% solution) was used as a positive control.

Quantitation of GSH and GSSG. Samples were obtained to measure intracellular and medium levels of GSH and GSSG. In brief, 500 µl of cell sample was centrifuged at 50g for 2 min. An aliquot of the supernatant (200 µl) was then added to 800 µl of 5% SSA to be used for analysis of GSH and GSSG levels in the medium. The cell pellet was then washed with 1 ml of phosphate-buffered saline, pH 7.4. After centrifugation at 50g for 2 min, the supernatant was removed from the pellet and 1.25 ml of 5% SSA was added. The resulting solution was transferred to a clean microcentrifuge tube and stored at -80°C until analysis. GSH and GSSG levels were measured using the enzymatic recycling method of Tietze (1969). All samples were first centrifuged at 10,000g for 5 min. Total GSH (GSH + GSSG) levels and GSSG levels alone were then determined as described by Gunnarsdottir and Elfarra (2003). Standard curves were run in all experiments to allow for quantitation of the results. The concentration of reduced GSH in a sample was determined by subtracting the molar amount of GSH equivalents coming from GSSG from the molar amount of total GSH calculated from the standard curve.

View larger version (28K): [in this window] [in a new window]   Fig. 2. Time course of the cell viability of male (n = 4) or female (n = 3) hepatocytes exposed to medium alone or medium spiked with Met as determined by TB exclusion (A and B) and LDH leakage (C and D). The symbol (*) indicates values that were significantly lower than cells incubated with medium alone (*, p < 0.05; **, p < 0.01).

	Results

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To characterize the toxicity of Met in freshly isolated male and female mouse hepatocytes, cell viability along with cellular and medium GSH/GSSG levels were monitored in hepatocytes exposed to medium alone or medium containing increasing concentrations of Met for 0 to 5 h at 37°C.

Male hepatocytes exposed to 30 mM Met had decreased cell viability as determined by trypan blue exclusion (Fig. 2A) and LDH leakage (Fig. 2C) starting at 3 h compared with hepatocytes exposed to medium alone. In addition, Met-exposed male hepatocytes had depleted intracellular GSH levels starting at 2 h compared with hepatocytes incubated in medium alone (Fig. 3A). Incubations with 30 mM D-Met resulted in no cytotoxicity or GSH depletion (Fig. 4). Exposure of male hepatocytes to 20 mM Met led to GSH depletion by 3 h (Fig. 3A) and cytotoxicity at 4 h (Fig. 2, A and C), indicating a dose-dependent response. Incubations with 5 and 10 mM Met resulted in no detectable cytotoxicity. No caspase-3/7 activation was detected at any time point or Met concentration. It is interesting to note that female hepatocytes exposed to 30 mM Met showed no cytotoxic effects (Fig. 2, B and D) and significantly higher GSH levels at 2 and 3 h (Fig. 3B) compared with hepatocytes exposed to medium alone. Medium GSH levels in Met-exposed male and female hepatocytes were lower than hepatocytes exposed to medium alone starting at 2 and 3 h, respectively (Fig. 3, C and D). Cellular and medium GSSG levels in Met-exposed hepatocytes of both genders were similar to or lower than levels in hepatocytes exposed to medium alone (data not shown).

View larger version (25K): [in this window] [in a new window]   Fig. 3. Time course of intracellular (A and B) and medium (C and D) GSH levels of male (n = 7 for 20 mM Met; n = 4 for 30 mM Met) or female (n = 3) hepatocytes exposed to medium alone or medium spiked with Met. The symbol (*) indicates values that were significantly different from cells incubated with medium alone (*, p < 0.05; **, p < 0.01).

View larger version (15K): [in this window] [in a new window]   Fig. 4. Time course of TB exclusion viability (A), LDH leakage viability (B), and intracellular GSH levels (C) of male hepatocytes (n = 3) exposed to medium alone, medium spiked with 30 mM Met, or medium spiked with 30 mM D-Met. The symbol (*) indicates values that were significantly lower than cells incubated with medium alone (*, p < 0.05; **, p < 0.01; ***, p < 0.001). The symbol () indicates values that were significantly higher than cells incubated with Met (, p < 0.05; , p < 0.01; , p < 0.001).

View larger version (11K): [in this window] [in a new window]   Fig. 5. Effect of 0.2 mM AOAA (n = 4) and 0.1 mM 3-DA (n = 3) on intracellular GSH levels in male hepatocytes exposed to 30 mM Met for 2 h. The symbol (*) indicates values that were significantly lower than cells incubated with medium alone (*, p < 0.05; **, p < 0.01; ***, p < 0.001). The symbol () indicates values that were significantly higher than cells incubated with Met alone (p < 0.05).

View larger version (32K): [in this window] [in a new window]   Fig. 6. Time course of the effect of 0.2 mM AOAA (n = 4) and 0.1 mM 3-DA (n = 3) on the cell viability of male hepatocytes exposed to medium alone or medium spiked with 30 mM Met as determined by TB exclusion (A and B) and LDH leakage (C and D). The symbol (*) indicates values that were significantly lower than cells incubated with medium alone (*, p < 0.05; **, p < 0.01; ***, p < 0.001). The symbol () indicates values that were significantly different from cells incubated with Met alone (, p < 0.05; , p < 0.01; , p < 0.001).

To assess the role of Met TM and homocysteine formation in Met toxicity in male mouse hepatocytes, the effect of the inhibitor 3-DA was examined. In contrast to AOAA, addition of 0.1 mM 3-DA to Met-exposed cells seemed to increase the magnitude of GSH depletion compared with hepatocytes exposed to Met alone at 2 h (Fig. 5B), although the results were not statistically significant (p = 0.12). 3-DA also potentiated Met-induced cytotoxicity, resulting in a significant decrease in cell viability starting at 2 h (Fig. 6, B and D). At 3 and 4 h, the viability of cells exposed to 30 mM Met and 0.1 mM 3-DA was nearly half that of cells exposed to Met alone.

Because the above-mentioned studies indicated that Met TA was an important pathway for Met toxicity, the toxicity of 3-MTP, a known human Met TA metabolite, was investigated. Exposure of male hepatocytes to 0.3 mM 3-MTP resulted in toxicity that was similar in profile to that of 30 mM Met, including trypan blue and LDH cytotoxicity starting at 3 h (Fig. 7, A and C, respectively) preceded by GSH depletion starting at 2 h (Fig. 7E) without concomitant GSSG formation (data not shown). The toxicity of 3-MTP was also assessed in female hepatocytes to further investigate potential mechanisms responsible for the gender-dependent toxicity of Met. Exposure of female hepatocytes to 0.3 mM 3-MTP resulted in GSH depletion at 3 h (Fig. 7F), but little to no cytotoxicity (Fig. 7, B and D).

View larger version (23K): [in this window] [in a new window]   Fig. 7. Time course of TB exclusion viability (A and B), LDH leakage viability (C and D), and intracellular GSH levels (E and F) of male (n = 4) and female (n = 4) hepatocytes exposed to medium alone or medium spiked with 0.3 mM 3-MTP. The symbol (*) indicates values that were significantly lower than cells incubated with medium alone (*, p < 0.05).

	Discussion

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In male hepatocytes, exposure to as low as 20 mM Met resulted in dose-dependent GSH depletion without increased formation of GSSG that preceded necrotic cell death. Addition of AOAA decreased both the Met-induced GSH depletion and cytotoxicity, whereas addition of 3-DA potentiated these effects. The Met TA metabolite 3-MTP caused nearly identical toxicity as Met at 100-fold lower concentrations. Taken together, these results suggest a major role for Met TA in Met hepatotoxicity. Additional support for this hypothesis is provided by the lack of hepatotoxicity of D-Met, which is a poor substrate for L-amino acid-specific transaminases. Transamination of D-Met can be accomplished by D-amino acid oxidase, but this enzyme is not present in mouse liver (Konno et al., 1997).

The Met-induced depletion of GSH without GSSG formation in male hepatocytes suggested that GSH was conjugating with reactive metabolites. Thus, Met TA may be involved in the production of GSH-reactive electrophiles. Further metabolism of 3-MTP has been investigated by Blom et al. (1988a) who found that methanethiol, dimethyl sulfide, and methanethiol-mixed disulfides formed from 3-MTP metabolism in human and rat hepatocytes. Gahl et al. (1988) also detected increased levels of mixed disulfides (CH₃S-SX) in a human with hepatic methionine adenosyltransferase deficiency. Thus, it is possible that volatile sulfur molecules formed in the Met TA pathway react with free GSH to form mixed GSH disulfides. The excessive formation of both protein and nonprotein mixed disulfides in the cell may result in cellular damage and cytotoxicity.

The potentiation of Met toxicity by 3-DA indicated that formation of Hcy via the Met TM pathway did not play a major role in Met hepatotoxicity and suggested that Met TM acted as a detoxification pathway at these Met levels. Exposure to 3-DA is known to result in increased SAM and SAH levels and inhibition of methylation reactions in liver (Duerre, 1988; Prytz and Aarbakke, 1990). The buildup of SAM results in feedback inhibition of MAT activity (Finkelstein, 1990). Thus, more Met may be available for Met TA, which may explain the increased hepatotoxicity detected in this study. However, it cannot be ruled out that a toxic buildup of cellular SAM or SAH also played a role in the potentiation of Met hepatotoxicity by 3-DA.

The finding that female hepatocytes were completely resistant to Met hepatotoxicity and had higher cellular GSH levels after Met exposure was interesting and unexpected. Increases in cellular GSH levels in Met-exposed female hepatocytes may be due to increased GSH synthesis via cysteine formation from the Met TM pathway (Wang et al., 1997). Met has also been shown to inhibit cellular GSH efflux by 50 to 70% in Met-exposed rat hepatocytes, possibly by allosteric inhibition of the GSH transport system, resulting in higher cellular GSH levels compared with control cells (Aw et al., 1986). Consistent with this finding, we detected lower medium and higher cellular GSH levels at 3 h in Met-exposed female hepatocytes compared with hepatocytes exposed to medium alone. Although similar processes may occur in male hepatocytes, any cellular gains in GSH levels seem to be overwhelmed by GSH losses secondary to Met TA and formation of mixed disulfides.

Gender differences in 3-MTP toxicological sensitivity allowed for some insight to be gained regarding potential mechanisms of gender-dependent Met hepatotoxicity. Female hepatocytes were much less sensitive to 3-MTP-induced cytotoxicity compared with male hepatocytes, but they were only slightly less sensitive to 3-MTP-induced GSH depletion. These results suggest that cellular events independent of GSH depletion may play a role in gender differences in 3-MTP and Met cytotoxicity. Other mechanisms may also be involved in eliciting gender-dependent Met hepatotoxicity and GSH depletion. For example, male and female differences in cellular Met uptake and efflux could result in sensitivity differences to Met hepatotoxicity. Met is known to be transported by both the "A" and "L" amino acid transport systems. Hissin and Hilf (1979) reported that 17β-estradiol inhibited system A transport of proline in vitro in breast cancer cells, demonstrating that sex-hormones can modulate amino acid transport systems used by Met. Although the effect of 17β-estradiol on liver amino acid transport is not known, lower levels of Met uptake in female hepatocytes compared with males could contribute to their resistance to Met hepatotoxicity. Gender differences in Met metabolism such as increased Met TM and/or decreased Met TA in females could also contribute to the observed toxicological differences. In support of this hypothesis, Met-dosed female mice had higher levels of SAM present in the liver after 15 min compared with Met-dosed males (Dever and Elfarra, 2006). Further investigations are required to examine potential gender differences in 3-MTP metabolism and rates of Met TM and Met TA in mouse hepatocytes.

Although gender differences in Met hepatotoxicity have not been reported previously, interspecies differences were identified by Shinozuka et al. (1971), who found that guinea pigs were more sensitive than rats to Met-induced liver toxicity after i.p. injection of a single high dose of Met. However, it is interesting to note that guinea pigs of both sexes were used, whereas in rats, only females were dosed. Our finding that female mouse hepatocytes were resistant to Met hepatotoxicity complicates the conclusions of that study because male rats were not tested and it is unknown whether rats would also exhibit gender-dependent toxicity after exposure to excess Met.

In humans, the toxicological significance of the Met TA pathway remains unclear. Typical Met levels in hypermethionemic humans (0.5–2.5 mM) are significantly lower than the concentrations of Met required to elicit cytotoxicity in mouse hepatocytes in the present study (20 mM), suggesting that they may not be sufficient to cause liver injury (Gahl et al., 1988; Chou, 2000). However, Met toxicity has been implicated in TPN-cholestasis, a condition characterized by liver damage and high plasma Met levels (1–1.5 mM) in infants as well as decreased hepatic GSH levels in neonatal rats (Heyman et al., 1984; Moss et al., 1999). Furthermore, the Met TM pathway has been shown to be immature and not fully functional in infants (Balistreri et al., 1983). Taken together, these observations suggest that Met TA could play a toxicological role in TPN-cholestasis.

Gender differences in Met metabolism in humans have also been reported. Higher levels of Met transamination products were detected in premenopausal women given oral Met compared with men of similar age given oral Met (Blom et al., 1988b). It was suggested that increased Met TA in healthy humans may actually function as a protective pathway by preventing formation of Hcy and decreasing the long-term cardiovascular risk correlated with high levels of Hcy.

In conclusion, male freshly isolated mouse hepatocytes, but not female, exhibited GSH depletion followed by cytotoxicity when exposed to high levels of Met. The toxicity was at least partially mediated by Met TA, but not TM. Evidence has been provided that gender differences in metabolism and detoxification of 3-MTP may contribute to gender-specific Met cytotoxicity in male hepatocytes, but other mechanisms are probably involved. Further studies are required to elucidate the exact causes of gender differences in Met toxicity in mouse hepatocytes that may serve as a useful model for human Met metabolism and hepatotoxicity.

	Footnotes

 
This research was supported by National Institutes of Health Grants R01 DK44295 and T32-ES-007015.

A preliminary report of this work was previously presented at the following conference: Dever JT and Elfarra AA (2008) Sex-related differences in methionine toxicity in freshly isolated mouse hepatocytes and evidence for transamination, but not transmethylation playing a key mechanistic role. 2008 Society of Toxicology Annual Meeting; 2008 16–20 Mar; Seattle, WA. Society of Toxicology, Reston, VA.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.108.141044.

ABBREVIATIONS: Met, L-methionine; TPN, total parenteral nutrition; TM, transmethylation; SAM, S-adenosyl-L-methionine; SAH, S-adenosyl-L-homocysteine; Hcy, homocysteine; TA, transamination; 3-MTP, 3-methylthiopropionic acid; AOAA, amino-oxyacetic acid; 3-DA, 3-deazaadenosine; LDH, lactate dehydrogenase; GSH, glutathione; GSSG, glutathione disulfide; D-Met, D-methionine; SSA, 5-sulfosalicylic acid; DMEM, Dulbecco's modified Eagle's medium; TB, trypan blue.

Address correspondence to: Dr. Adnan A. Elfarra, School of Veterinary Medicine, University of Wisconsin-Madison, 2015 Linden Dr., Madison, WI 53706-1102. E-mail: elfarra{at}svm.vetmed.wisc.edu

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