Modulation of extracellular homocysteine concentration in human cell lines

Clinica Chimica Acta 330 (2003) 151 – 159 www.elsevier.com/locate/clinchim

Modulation of extracellular homocysteine concentration in human cell lines Bjo¨rn Hultberg * Department of Clinical Chemistry, Institute of Laboratory Medicine, University Hospital, S-22185 Lund, Sweden Received 10 June 2002; received in revised form 10 December 2002; accepted 23 December 2002

Abstract Background: Despite the growing evidence that plasma homocysteine is a cardiovascular risk factor, the mechanism behind the vascular injuries is still unknown. Information about the metabolism of homocysteine is, therefore, essential for an understanding of its role in atherogenesis, thereby enabling a modulation of that risk. Methods: In the present study, we have examined the modulation of extracellular homocysteine in HeLa and hepatoma cell cultures in relation to a changed extracellular thiol redox status and in the presence of specific inhibitors of amino acid transporters. Results: The findings in the present study show that a changed thiol redox status by copper ions, copper chelator or the monothiol, N-acetylcysteine (NAC), affects extracellular homocysteine in the same way in hepatoma cell cultures, but not to the same extent as observed in HeLa cell cultures. However, the dithiols, dithiothreitol (DTT) and a-lipoic acid (LA), which lowered extracellular homocysteine concentration in HeLa cell cultures, increased the extracellular total homocysteine concentration in hepatoma cell cultures, probably mainly as a result of increased release of homocysteine extracellularly. Studies with specific inhibitors of amino acid transporters in HeLa cell cultures showed that homocysteine uptake occurred mainly by system A and glutamate transporters. Hepatoma cells seemed to have a much smaller uptake capacity of homocysteine compared to HeLa cells. Conclusion: The lack of uptake capacity of homocysteine in hepatoma cells indicates that hepatocytes only play a small role in the elimination of homocysteine from circulation. Intracellular metabolism, cellular export and the complex pattern of homocysteine uptake in different cells are important to examine further in order to possibly be able to lower plasma homocysteine levels. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Amino acid transporters; Copper; Dithiothreitol; Human cell lines; a-lipoic acid; N acetylcysteine

1. Introduction Homocysteine, a sulfhydryl amino acid, is the demethylated derivative of methionine [1]. Greatly elevated plasma levels of homocysteine are found in subjects with homocystinuria [1]. These patients exhibit early arteriosclerosis, arterial and venous thrombosis. * Tel.: +46-46173447; fax: +46-46189114. E-mail address: [email protected] (B. Hultberg).

Approximately 100 clinical and epidemiological studies published during the last 10 years show that even mild hyperhomocysteinemia is associated with cardiovascular disease [2,3]. Despite the growing evidence that plasma homocysteine is a cardiovascular risk factor, the mechanism behind the vascular injuries is still unknown. Information about the metabolism of homocysteine is, therefore, essential for an understanding of its role in atherogenesis, thereby enabling a modulation of that risk.

0009-8981/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0009-8981(03)00052-4

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Adenosyl methionine is a precursor of homocysteine and is the principal methyl donor in mammals [1]. After a methyl transfer reaction (transmethylation), adenosyl homocysteine is hydrolyzed by S-adenosylhomocysteine hydrolase to homocysteine and adenosine. Homocysteine may either be catabolized in the transsulfuration pathway via cystathionine to cysteine or remethylated back to methionine, mainly by the folate- and cobalamin-dependent enzyme methionine synthase [1]. The intracellular concentration of homocysteine seems to be regulated within strict limits by export mechanisms [4– 7] because a high homocysteine concentration will increase levels of adenosylhomocysteine leading to inhibition of transmethylation [7,8]. Under normal conditions, cells export considerable amounts of the intracellularly produced homocysteine. The export occurs at a rate reflecting the balance between intracellular production and further cellular metabolism. This mechanism is likely the biological basis for accumulation of homocysteine in extracellular fluids [4– 7], such as plasma and urine in patients with inborn errors of homocysteine metabolism [1], and with folate or cobalamin deficiency [9]. Knowledge of the metabolism and transport of homocysteine is important in order to understand physiological and pathological mechanisms of homocysteine homeostasis. Intracellular metabolism of homocysteine, thus, depends on pertinent tissue enzymes and vitamin levels, whereas extracellular homocysteine levels reflect not only metabolic changes within cells and cellular export but also cellular uptake of homocysteine. We have recently, in HeLa cell cultures [6,10,11], shown that even low concentrations of copper ions (0.1 – 1 Amol/l) had a profound effect on extracellular levels of homocysteine. Copper ions increased the concentration of extracellular homocysteine, possibly by decreasing the proportion of reduced thiol fractions, which leads to a lowered cellular uptake of extracellular homocysteine [6]. Consequently, the addition of antioxidant agents, such as thiols, was accompanied by a decreased concentration of extracellular homocysteine [12]. The cellular uptake systems for homocysteine have been little studied. It has been reported that uptake of extracellular homocysteine in human umbilical vein endothelial cells and in an endothelial cell line occurred via the sodium independent system L and the sodium dependent system ASC [13,14]. Homocysteine uptake

by endothelial cells was also inhibited by both cysteine and cystine [13]. It is probable that liver cells, with their great blood contact area and high activity of enzymes in both the remethylation and transsulfuration pathways [15], play an important role for the fate of homocysteine in the circulation. The human hepatoma (HepG2) cell line exhibits most of the cellular features of normal human hepatocytes [16]. In the present study, we have, therefore, in order to elucidate cellular homocysteine transport, examined the modulation of extracellular homocysteine in both hepatoma cell and HeLa cultures in relation to a changed extracellular thiol redox status and in the presence of specific inhibitors of amino acid transporters.

2. Materials and methods 2.1. Cell culture The established HepG2 and HeLa cell lines were obtained from American Type Culture Collection, Rockville, MD, USA. The cells were cultured at 37 jC in 75-cm2 flasks, in 12 ml of RPMI 1640 (Gibco Laboratories, Santa Clara, CA, USA) containing 10% fetal calf serum (Gibco Laboratories). The cells were grown in humidified air with 5% CO2. The cells were routinely screened for and shown to be free from mycoplasma using a kit with a specific DNA probe from Gen-Probe, San Diego, CA, USA. New medium with or without test substances was added at the start of the experiments. In some experiments, the medium was changed daily for 3 days, and in other experiments, a 24-h incubation was used. The substances [bathocuproinedisulfonic acid (BCS), 1, 10 and 100 Amol/l; h-2-aminobicyclo(2,2,1-heptane)-2-carboxylic acid (BCH), 20 mmol/l; dithiothreitol (DTT), 100 and 500 Amol/l; glutamic acid (Glut), 10 mmol/ l; 2-methylaminoisobutyric acid (MEA), 20 mmol/l; N-acetylcysteine (NAC), 800 and 4000 Amol/l; alipoic acid (LA), 10, 50, 100, 200 and 500 Amol/l; CuSO4, 1, 10 and 100 Amol/l; all concentrations expressed as the final concentration in the medium] were prepared and added to the cell culture experiment immediately before the start of the experiment. All chemicals were bought from Sigma, St Louis, MO, USA.

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2.2. Methods After the experiments, the cells were washed with physiological saline before being detached by trypsin. The assay of total thiol concentration (including all thiol moieties, whether disulfide-bound or not) in the cells and medium was performed after an initial DTT reducing step, whereas in the assay of the reduced thiol concentration, the initial DTT reducing step was omitted as previously described [6,17]. The thiol analyses were performed with a high-performance liquid chromatographic method, which utilized isocratic reversedphase ion-pair liquid chromatography at pH 2.4 and postcolumn derivatization with 4,4V-dithiopyridine and colorimetric detection at 324 nm. The cellular content of homocysteine is expressed as the amount (nmol) per milligram cell protein. In the medium, homocysteine is expressed as the amount (nmol) present per milliliter of medium. Protein content was analyzed according to Lowry et al. [18] and expressed as milligram protein per dish. 2.3. Statistics Results are expressed as means and S.D. Statistical significance between the different groups was assessed by the Wilcoxon rank test. p < 0.05 was considered significant.

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Table 2 The ratio expressed as percentage between the total amount of the intracellular homocysteine and the total amount of homocysteine in hepatoma cell cultures incubated for 24 h Cell protein (mg/dish)

Ratio (%)

<2 2–4 4–6

3.2 F 0.9 4.5 F 1.8* 5.4 F 1.6*

Means and S.D. are presented. Eight dishes are used in each group. * p < 0.05 compared to cell dishes with cell protein below 2 mg.

homocysteine in hepatoma cell cultures are given in Table 1. The relation between intra- and extracellular total homocysteine concentrations and cell density, measured as total cell protein, in hepatoma cell cultures incubated for 24 h was studied in 20 different experiments, with cell protein varying between 1.5 and 6.3 mg/dish. Extracellular homocysteine concentration increased with increasing cell protein (q = 0.71, p < 0.01), whereas there was no significant relation between intracellular homocysteine concentration and cell density. The ratio between intracellular and total amount of homocysteine (the sum of the intracellular and extracellular amount of homocysteine) in the hepatoma cell cultures after 24 h of incubation varied between 1.6% and 8.7%, depending on the cell density (Table 2). 3.2. Homocysteine concentrations and copper ions

3. Results 3.1. Homocysteine concentrations and relation to cell density in hepatoma cell cultures The total concentrations and the reduced proportions (expressed in percent) of intra- and extracellular Table 1 Concentrations and percentages of the reduced proportions of intraand extracellular homocysteine in hepatoma cell cultures incubated for 24 h Cell-Hcy (nmol/mg)

Cell-Hcy (%)

Medium-Hcy (nmol/ml)

Medium-Hcy (%)

1.1 F 0.3

5F3

7.1 F 1.8

4F3

Means and S.D. are presented. Eight dishes are used. Cell protein was 4.2 F 0.8 mg.

Hepatoma cell cultures incubated in the same medium for 3 days showed a higher concentration of extracellular total homocysteine in the presence of copper ions compared to control cell cultures on day 1, whereas the cell cultures without any addition exhibited a decreased concentration of extracellular homocysteine after 3 days compared to day 1 (Table 3). Similar but more pronounced changes were observed in HeLa cell cultures. Likewise, the effect of copper ions and a copper(I) chelator (bathocuproinedisulfonic acid, BCS) on the extracellular total homocysteine concentrations was much more pronounced in HeLa cell cultures than in hepatoma cell cultures (Fig. 1). Hepatoma cell cultures showed an increased concentration of homocysteine at 100 Amol/l of copper ions ( p < 0.05) and a decreased concentration at 100

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Table 3 The extracellular concentration (nmol/ml) of homocysteine in hepatoma and HeLa cell cultures incubated for 1 or 3 days in the same medium with and without 10 Amol/l of copper ions

Day Day Day Day

1, 3, 1, 3,

control control Cu 10 AM Cu 10 AM

Table 4 Concentrations and percentages of the reduced proportions of extracellular homocysteine after incubation for 3 days (daily change of medium) with different thiols in hepatoma and HeLa cell cultures

Hepatoma cells

HeLa cells

Hepatoma cells

HeLa cells

7.0 F 1.9 4.9 F 1.2* 7.6 F 1.7 9.4 F 2.1a,*

6.8 F 1.2 0.6 F 0.4* 8.5 F 1.9* 8.0 F 1.5a,*

MediumHcy (nmol/ml)

MediumHcy (%)

MediumHcy (nmol/ml)

MediumHcy (%)

4F3

6.8 F 1.3

12 F 4

29 F 5*

7.0 F 1.9 3.0 F 0.7*

53 F 17*

Means and S.D. are presented. Six dishes are used in each experiment. Cell protein at the start of the experiment varied between 3.6 and 4.0 mg/dish. a p < 0.05 compared to control day 3. * p < 0.05 compared to control day 1.

Amol/l of BCS ( p < 0.05) compared to control cell cultures without any addition, whereas all HeLa cell cultures with any addition except that with 1 Amol/l of BCS exhibited significant changes ( p < 0.05) compared to control cell cultures without any addition. 3.3. Homocysteine concentrations and thiols Hepatoma cell cultures without any addition had a lower ( p < 0.05) reduced proportion of extracellular homocysteine compared to HeLa cell cultures (Table 4). As expected, the addition of thiols (LA/DTT/ NAC) increased the reduced proportions of extracel-

Control LA, 10 AM LA, 50 AM LA, 100 AM LA, 200 AM LA, 500 AM DTT, 10 AM DTT, 100 AM DTT, 500 AM NAC, 200 AM NAC, 800 AM NAC, 4000 AM

7.2 F 1.5 16.1 F 2.9* 27.3 F 4.7* 27.0 F 5.3* 20.8 F 4.5* 8.9 F 3.1* 7.7 F 1.9 10.2 F 1.9* 10.0 F 2.3* 7.4 F 1.7 6.2 F 2.1 4.8 F 1.7*

87 F 16* 7F4 18 F 4* 55 F 12*

1.5 F 0.7* 3.4 F 0.5* 1.4 F 0.5*

33 F 11* 56 F 13*

25 F 5* 29 F 8*

4.6 F 1.1* 2.0 F 0.8*

15 F 7 35 F 9*

Means and S.D. are presented. Eight dishes are used for each experiment. Cell protein varied between 3.8 and 4.2 mg/dish except in hepatoma cells incubated in the presence of 500 Amol/l of LA, where a significantly lowered cell protein was obtained (2.9 F 0.5, p < 0.05). LA, a-lipoic acid. DTT, dithiothreitol. NAC, N-acetylcysteine. * p < 0.05 compared to controls.

lular homocysteine in both hepatoma and HeLa cell cultures. Extracellular total homocysteine concentrations were lowered in HeLa cell cultures in the

Fig. 1. Mean concentrations (nmol/ml) of extracellular homocysteine after the addition of copper(II) ions (Cu), bathocuproinedisulfonic acid (BCS) or a combination of both after 3 days of incubation with daily change of medium. Six dishes were used for each experiment.

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presence of thiols. In hepatoma cell cultures, the presence of NAC also lowered extracellular homocysteine concentration, whereas the presence of DTT and particularly LA increased extracellular total homocysteine. Intracellular total homocysteine concentration was not significantly changed, except in HeLa cell cultures at 500 Amol/l of DTT, where a slight decrease was observed (not shown). In hepatoma cell cultures, there was a significantly lowered cell protein at 500 Amol/l of LA. 3.4. Homocysteine uptake in HeLa and hepatoma cells The characteristics of the homocysteine uptake by HeLa and hepatoma cells were examined with the use of specific inhibitors of different amino acid uptake systems (Table 5). The findings indicate that homocysteine uptake in HeLa cells occurred by system A (inhibited by MEA) and glutamate transporters (inhibited by glutamic acid), whereas in hepatoma cells, no inhibition was observed with any of the tested systems. The addition of thiols to these inhibition experiments showed that in HeLa cell cultures with inhibition of glutamate transporters (by glutamic acid), all thiols added decreased extracellular homocysteine compared to cultures with addition of glutamic acid alone (Table 6). The addition of thiols in HeLa cell cultures with inhibition of system A

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Table 6 The extracellular concentration of homocysteine in HeLa and hepatoma cell cultures after incubation for 24 h with specific inhibitors of amino acid transporters and different thiols Homocysteine (nmol/ml)

MEA, 20 mM MEA and DTT, 100 AM MEA and LA, 100 AM MEA and NAC, 2000 AM Glut, 10 mM Glut and DTT, 100 AM Glut and LA, 100 AM Glut and NAC, 2000 AM

HeLa cell culture

Hepatoma cell culture

6.0 F 1.3 4.8 F 1.0* 5.3 F 0.9* 3.7 F 0.7* 6.2 F 1.1 4.8 F 1.0* 3.8 F 0.8* 2.2 F 0.5*

8.4 F 1.7 12 F 2.1* 23 F 3.9* 7.7 F 1.8 8.2 F 1.5 11 F 1.9* 21 F 4.1* 3.8 F 1.1*

Means and S.D. are given. Cell protein at the start of the experiments varied between 3.6 and 4.0 mg/dish. Eight dishes were used for each experiment. * p < 0.05 compared to cell cultures with addition of the specific inhibitor.

(by MEA) also decreased extracellular homocysteine. The thiols were also added to hepatoma cell cultures with inhibition of system A and glutamate transporters (Table 6). The addition of NAC decreased extracellular homocysteine in the presence of glutamic acid but not in the presence of MEA. The addition of dithiols in the presence of MEA or glutamic acid showed similar results to control cell cultures without any addition of an inhibitor of amino acid transporter.

Table 5 The extracellular concentration of cysteine and homocysteine in HeLa and hepatoma cell cultures after incubation for 24 h without (controls) and with specific inhibitors of amino acid transporters HeLa cell culture

Medium without cell contact Control cell BCH, 20 mM MEA, 20 mM Glut, 10 mM

Hepatoma cell culture

Cysteine (nmol/ml)

Homocysteine (nmol/ml)

Cysteine (nmol/ml)

Homocysteine (nmol/ml)

351 F 33

0

351 F 33

0

250 F 26 278 F 23* 275 F 25* 310 F 30*

4.5 F 1.0 4.1 F 0.9 6.8 F 1.2* 7.0 F 1.4*

264 F 31 292 F 26* 304 F 23* 322 F 29*

6.5 F 1.1 4.8 F 1.1* 6.8 F 1.3 6.6 F 0.9

Means and S.D. are given. Cell protein at the start of the experiments varied between 3.6 and 4.0 mg/dish. Eight dishes were used for each experiment. BCH, h-2-aminobicyclo(2,2,1-heptane)-2-carboxylic acid. MEA, 2-methylaminoisobutyric acid. * p < 0.05 compared to control cell cultures.

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4. Discussion 4.1. Homocysteine concentrations and relation to cell density in hepatoma cell cultures Extracellular total homocysteine in hepatoma cell cultures is released to about the same extent and also related to cell density, as previously observed in HeLa cell cultures [6]. 4.2. Homocysteine concentrations and relation to copper ions and thiols We have previously shown in HeLa cell cultures that the extracellular concentration of homocysteine is increased in the presence of copper ions [6,10,11]. The presence of copper ions increases oxidative activity in cell cultures, thereby decreasing the proportion of reduced thiols extracellularly [10], and since the reduced fraction of homocysteine seems to be more available for cellular uptake than oxidized homocysteine in HeLa cell cultures [6,12], the extracellular concentration of homocysteine was lowered. BCS is a specific copper(I) ion chelator, which can strongly bind copper ions and form stable complexes [19]. We have previously reported [12] that an inhibition of the oxidation of the extracellular thiols by the addition of BCS occurs in HeLa cell culture, and the reduced fraction of extracellular homocysteine is increased, which then probably leads to an increased cellular uptake and, as a consequence, a lowered concentration of total homocysteine extracellularly. The addition of thiols also increased the reduced fraction of extracellular homocysteine and was accompanied by a decrease of extracellular homocysteine concentration [12]. Thus, the redox status of homocysteine is important for its cellular uptake in HeLa cell cultures. We suggested that increased oxidation catalyzed by copper ions leads to increased disulfide formation between homocysteine and protein cysteinyl groups or low molecular weight thiols, probably resulting in a lowered cellular uptake of homocysteine [6], whereas the presence of antioxidative agents such as BCS and thiols leads to a higher proportion of the reduced form of homocysteine and consequently a higher cellular uptake [12]. In hepatoma cell cultures, there are similarities to the findings of copper ions and copper chelator in

HeLa cell cultures, but the effects of these agents seem to be much weaker in hepatoma cell cultures. However, the effect of thiol addition differs between the two cell types, even if the addition of thiols increased the reduced proportion of extracellular homocysteine in both hepatoma and HeLa cell cultures. In HeLa cell cultures, we observed, in agreement with previous findings [12], a decreased concentration of extracellular homocysteine. In hepatoma cell cultures, only the addition of the monothiol NAC, and only at the highest concentration used, lowered the extracellular concentration of homocysteine, whereas the dithiols DTT and LA increased the extracellular total homocysteine concentration, despite increasing the reduced proportions of extracellular homocysteine. The reason for this discrepancy is not obvious. The findings in the present study, thus, show that a changed redox status by copper ions, BCS or NAC in hepatoma cell cultures affects extracellular homocysteine in the same way, but not to the same extent as observed in HeLa cell cultures. The fact that hepatoma cells do not respond to redox changes as much as HeLa cells might have some connection to the finding of a very low proportion of reduced extracellular homocysteine in hepatoma cell cultures compared to HeLa cell cultures (the same is valid for extracellular cysteine and glutathione in hepatoma cell culture— unpublished findings), which indicates that hepatoma cells normally only export a smaller part of their cellular reducing equivalents extracellularly. Particularly, the presence of LA (even at low concentration—10 Amol/l) strongly increased the extracellular total homocysteine concentration in hepatoma cell cultures, in contrast to a lowered extracellular concentration of homocysteine in HeLa cell cultures. In this context, it is interesting that the properties of LA distinguish it from other thiols. When LA is added to cells, it is rapidly reduced intracellularly to the dithiol dihydrolipoate, which is released outside the cell [20]. LA continuously regenerates its reductive dithiol form by using cellular reducing equivalents. Since extracellular homocysteine (like cysteine and glutathione) has a lower reduced proportion in hepatoma cell cultures than in HeLa cell cultures, it is likely that these cells use a larger part of their reducing equivalents for different intracellular metabolic pathways compared with HeLa

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cells. Therefore, in the presence of LA, some of these metabolic processes, e.g. the reducing steps in the folate pathway, might be more severely influenced in hepatoma cells than in HeLa cells. This might lead to hampered remethylation and result in increasing amounts of intracellular homocysteine being formed and released extracellularly. Another possibility is that oxidative stress may occur in hepatoma cells because the cellular amount of reducing equivalents is decreased in the presence of LA. It is known that the activity of methionine synthase is reduced under oxidizing conditions, whereas the activity of cystationine synthase is enhanced under the same conditions [15,21]. In response to oxidative stress, the metabolic response would, therefore, favor transsulfuration that would facilitate the synthesis of glutathione. This may explain the findings of an initial increase of extracellular homocysteine (assuming a preferential inhibition of methionine synthase at lower concentrations of LA), which thereafter decreases at higher concentration of LA. At these concentrations of LA, there is a high level of oxidative stress, and a larger amount of homocysteine is, therefore, used for glutathione synthesis. 4.3. Homocysteine uptake in HeLa and hepatoma cells The transport of amino acids across the mammalian cell membrane is performed by specific systems. It is known [13] that transport of cysteine is mediated by at least three distinct systems (system L, system A and system ASC). Previously, it has been shown that homocysteine was transported by systems L and ASC in human umbilical vein endothelial cells [13]. It was also shown that not only cysteine but also cystine inhibited the uptake of homocysteine [13]. It is known that cysteine added to growth medium is rapidly autoxidized to cystine [22,23]. The uptake of cystine is mediated by a family of glutamate transporters [24,25]. Cysteine is also known to be transported by the glutamate transporters [24,26]. On the basis of these facts, we examined the concentration of extracellular cysteine and homocysteine in HeLa/hepatoma cell cultures after addition of different inhibitors of the above-mentioned systems. BCH is a wellknown analogue of system L and MEA of system A, whereas system ASC cannot be inhibited specifically

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[13]. Glutamic acid was used as inhibitor of the glutamate transporters [24,25]. The findings indicate that homocysteine uptake in HeLa cells occurred mainly by system A and by glutamate transporters. In hepatoma cells, hardly any inhibition of homocysteine uptake was observed with any of the tested systems. The lack of uptake capacity of homocysteine in hepatoma cells might indicate that hepatocytes only play a small role in the elimination of homocysteine from circulation. Our findings, and the observation that human umbilical cells and an endothelial cell line [13,14] used systems L and ASC for uptake of homocysteine, thus, indicate that the elimination of homocysteine from circulation is complex and that different cell types might have different roles. The addition of thiols to these inhibition experiments showed that in HeLa cell cultures with inhibition of glutamate transporters, all thiols added decreased extracellular homocysteine compared to cultures with addition of glutamic acid alone. Thus, the increase of the reduced proportion of homocysteine might lead to an augmented uptake of homocysteine possible by system A. The addition of thiols in HeLa cell cultures with inhibition of system A also decreased extracellular homocysteine. One possible explanation of the lowered extracellular homocysteine concentration could be an augmented uptake of reduced homocysteine via the glutamate transporters. The thiols were also added to hepatoma cell cultures with inhibition of system A or the glutamate transporters. The addition of NAC decreased extracellular homocysteine in the presence of glutamic acid, but not in the presence of MEA. A possible explanation for this finding is that the strongly increased reduced proportion of homocysteine by NAC leads to a measurable uptake of homocysteine by system A, even in hepatoma cells. The addition of dithiols in the presence of MEA or glutamic acid showed similar results to control cell cultures without any inhibition of amino acid transporters, which indicates that none of the tested transport systems have a role in increased extracellular homocysteine concentration observed after dithiol addition in hepatoma cells. Thus, these studies suggest that increased extracellular homocysteine in hepatoma cell cultures after the addition of dithiols is mainly attributed to increased release of homocysteine extracellularly.

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5. Conclusion The findings in the present study show that a changed redox status in hepatoma cell cultures affects extracellular homocysteine in the same way, but not to the same extent as observed in HeLa cell cultures. In HeLa cell cultures, in agreement with previous findings [12], we observed a decreased concentration of extracellular homocysteine after addition of thiols. In hepatoma cell cultures, only the addition of the monothiol NAC, and only at the highest concentration used, lowered the extracellular concentration of homocysteine, whereas dithiols DTT and LA increased the extracellular total homocysteine concentration, probably mainly attributed to increased homocysteine release extracellularly. Studies with specific inhibitors of amino acid transport in HeLa cells showed that homocysteine uptake occurred mainly by system A and the glutamate transporters. Hepatoma cells seemed to have a much smaller uptake capacity of homocysteine compared to HeLa cells. The lack of uptake capacity of homocysteine in hepatoma cells indicates that hepatocytes play only a small role in the elimination of homocysteine from circulation. The release of homocysteine extracellularly might be a reflection of a mechanism for the regulation of the remethylation and the transsulfuration pathways in the body. These two intersecting pathways compose the mechanisms for intracellular homocysteine metabolism in mammals [15]. The remethylation cycle occurs in all tissues and it conserves methionine. The transsulfuration pathway has a limited distribution and is found primarily in the liver, kidney, small intestine and pancreas [15]. Regulation of homocysteine metabolism might be achieved by changes in the quantity of homocysteine distributed between the two competing pathways. Homocysteine may, thus, be removed either by enzymatic transformation (remethylation/transsulfuration) or by transport from the cell of origin to other cells. It is likely that the normal plasma homocysteine concentration represents this transit from the site of synthesis to the site of catabolism. Intracellular metabolism, cellular export and the complex pattern of homocysteine uptake in different cells are, therefore, important to examine further in order to possibly be able to lower plasma homocysteine levels. Since elevated plasma homocysteine level is associated with vascular disease [2,3], a

lowered plasma level might reduce the risk for vascular disease. Furthermore, LA is widely used as a food supplement, and dihydrolipoate, its reduced form, is regarded as a potent and biologically safe reducing agent and antioxidant [20]. Pharmacokinetic studies of LA have shown that after a single oral dose of 10 mg/kg body weight, plasma concentrations can reach 60– 70 Amol/l [27]. Thus, with these doses, it is likely that homocysteine metabolism in hepatocytes is affected. Since increased plasma homocysteine concentration is associated with vascular disease [2,3], a reevaluation of LA as a biologically safe reductant might be made.

Acknowledgements This work was supported by grants from the Swedish Heart Lung Foundation and the Albert Pa˚hlsson Foundation.

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