ARCHIVES OF BIOCHEMISTRYAND BIOPHYSICS Vol. 196, No. 2, September, pp. 557-565, 1979

Studies

on the Transport Mechanism of 5-Methyltetrahydrofolic in Freshly Isolated Hepatocytes: Effect of Ethanol1

DONALD W. HORNE, WILLIAM

Acid

T. BRIGGS, AND CONRAD WAGNER

Biochemistry Research Laboratory, VA Medical Center, Nashville, Department

of Biochemistry Vanderbilt University Nashville, Tennessee $7292

Tennessee 37208, and School of Medicine,

Received January 16, 19’79;revised May 9, 1979 The effect of ethanol on the transport of 5-methyltetrahydrofolate in freshly isolated hepatocytes in vitro resulted in about a 30% increase in accumulation of substrate. It was shown that this was not due to differences in metabolism, nor to an inhibition of efflux. Preincubation with 40 mM ethanol for 45 min resulted in a significantly increased rate of entry of 5-methyltetrahydrofolate into the cells. The stimulatory effect was specific to 5-methyltetrahydrofolate since ethanol inhibited uptake of folate and methotrexate. The increased uptake was due to metabolism of ethanol aa shown by studies with pyrazole. Also, the n-alkanols, propanol through pentanol, and sorbitol but not methanol were stimulatory. Anaerobiosis and sodium azide stimulated uptake of 5-methyl-tetrahydrofolate but were inhibitory to methotrexate uptake. These data, taken together, suggest that the ethanol effect is due to increased entry of 5CH,-H,PteGlu into the cells possibly as the result of an increased cellular NADWNAD ratio.

Megaloblastic anemia in alcoholics is most frequently associated with decreased dietary intake of folates. The well-nourished alcoholic only rarely manifests this condition. (See Ref. (1) for discussion and references.) However, when folate intake is marginal alcohol can precipitate megaloblastic erythropoiesis as first shown by Sullivan and Herbert (2) and later confirmed by Eichner et al. (3, 4). The manner in which alcohol interferes with folate metabolism is uncertain. The ethanol effect has been attributed to malabsorption of folates (5-8) and to decreased folate polyglutamate synthesis (9). Malabsorption of folates in alcoholics may be caused, not by ethanol, but by intestinal lesions resulting from folate deficiency (2, 5, 8, 10, 11). Acute administration of ethanol has been shown to result in a fall in the serum folate level (12, 13). Subsequently, Lane et al. (14) concluded that ethanol blocks the release of folates ’ This study was supported by the Veterans Administration and by Grants 469 from the Nutrition Foundation and AM-15289 from the United States Public Health Service.

from tissues to plasma and Hillman et al. (15) reported evidence that ethanol interferes with the folate enterohepatic cycle. Recently we have demonstrated that ethanol stimulates the accumulation of 5-methyltetrahydrofolate (5-CH,-H,PteGlL# in isolated hepatocytes (16). The results reported here further characterize this ethanol effect and extend the findings to include the effects of alcohol on transport of methotrexate and folic acid (PteGlu). MATERIALS

AND METHODS

Animals. Male Sprague-Dawley rats (Harlan Industries) weighing 150-250 g were used for the isolation of hepatocytes. The rats were maintained on Wayne LabBlox and tap water fed ad lib&m. Preparation of hepatocytes. Hepatocytes were isolated by the collagenase-perfusion technique described by Berry and Friend (17)as mod&d by Horne et al. (18). Determination of uptake of fohte derivatives. The uptake of various folate derivatives was determined as

* Abbreviations used: 5-CH,-H,PteGlu, 5-methyltetrahydrofolate; PteGlu, pteroylglutamate, folate; Hepes, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. 557 0003-9861/79/100557-09$02.00/O Copyright 6 1979by AcademicPress, Inc. All rights of reproductionin any form reserved.

558

HORNE ET AL.

TABLE I previously described (16, 18). The procedure consists of incubating the cells at 37°C with shaking for variEFFECTOF SODIUMAND ETHANOLON ous times with the radiolabeled folate derivatives, cenUPTAKE OF 5-CH,-HJ’teGlu” trifuging rapidly to pellet the cells, and washing the pellet twice with ice-cold medium (Krebs-Ringer soluPercentage of control tion buffered at pH 7.4 with 25 mM Hepes). The radioMajor cation activity in the pellet was counted in 1.0 ml of Scinin medium Control 40 mM Ethanol P tisol (Isolab, Inc.) or ACS (Fisher) in a Searle Isocap liquid scintillation counter. 100.0 ? 2.85 Na+ 124.0 + 6.80 co.02 Statistical treatment ofdata. All values are reported 100.0 + 2.75 115.0 ” 3.05 0.5), respectively, for control and ethanol-treated cells indicating that rate of exit of 5-CH,-H,PteGlu is identical for both groups of cells. Metabolism of Accumulated 5-CH,-H,PteGlu To assess the magnitude of the effect of ethanol on the metabolism of 5-CH3H,PteGlu, hepatocytes were incubated with 0.25 PM 5-CH,-H,[G-3H]PteGlu for 60 min in the absence and presence of 40 mM ethanol. Cell extracts were made and column chromatography was performed as stated under Materials and Methods. Figure 3 illustrates these results. Comparing Fig. 3a

0

102030405060706090 TIME

102030405060

bin)

FIG. 2. Effect of ethanol on efflux of SCH,-H,PteGlu. Freshly isolated hepatocytes were incubated for 30 min with 0.25 PM 5CH,-H,PteGlu in the presence or absence of 40 mM ethanol. The cells were washed once with fresh 3’7°C buffer (with or without 40 mM ethanol, as appropriate) and resuspended in fresh buffer with 40 mM ethanol (0) or without ethanol (0) and the efflux of 5-CH,H,PteGlu was estimated over the next SO min (A). In (B) the amount of 5-CH,-H,PteGlu which is free to efflux from the cells has been plotted on a log scale as a function of time. The results are reported as the average of three separate experiments.

560

HORNE ET AL.

1.0 A

260

0.5

0

b IO-

6‘: 0 x E 3

6-

FRACTION

NUMBER

FIG. 3. Cellex-T chromatography of extracts of hepatocytes incubated for 60 min at 37°C with 0.25 pM labeled 5-CH,-H,PteGlu. (-) Radioactivity; (- - -) absorbance of marker compounds (in order of elution, 5CH,H,PteGlu and H,PteGlu). Details of the experiment are in the text and under Materials and Methods. (a) Control; (b) cells incubated in the presence of 40 mM ethanol.

(control) with 3b (40 IrIM ethanol) it can be seen that there is no qualitative difference in the elution profiles under the two conditions. Also, in control cells, 71.2% of the radioactivity (recovery, 97.8%) eluted along with marker 5-CH,-H,PteGlu and in ethanol-treated cells 75.3% of the radioactivity

(recovery, 103%) eluted with authentic 5CH,-H,PteGlu. Effect of Ethanol on Uptake of Other Folate Derivatives Table II reports the uptakes of methotrexate, PteGlu, and 5-CH,-H,PteGlu as

TABLE II EFFECT OF ETHANOL ON UPTAKE OF VARIOUS FOLATE DERIVATIVES’

Substrate

Concentration (PM)

30-min 46 Control

P

60-min % Control

Methotrexate PteGlu 5-CH,-H,PteGlu

0.25 0.28 0.34

92.2 2 5.9 100.2 + 2.9 124.9 * 3.9

0.25 0.9 0.05

75.6 k 4.6 93.9 -c 1.8 138.6 k 4.2

P CO.01

co.05 -Co.001

a Hepatocytes were incubated at 37°C with 13H]methotrexate, [SHIPteGlu, and 5CH,-HJ3H]PteGlu either without (control) or with 40 mM ethanol. At 30 and 60 min duplicate samples were taken to estimate uptake. The results are expressed as percentage of control uptake 2 SEM (n = 4).

ETHANOL

AND 5-METHYLTETRAHYDROFOLATE

TRANSPORT

561

only 5-CH3-H,PteGlu was significantly affected, showing an uptake some 125% of the control without ethanol. At 60 min, however, there was significant inhibition of uptake of methotrexate and of PteGlu by ethanol, whereas 5-CH,-H,PteGlu uptake was still greater than the control. Effect of Axide on Methotrexate Uptake It has previously been shown that sodium azide has a stimulatory effect on 5-CH,-H,80 100 20 40 60 0 PteGlu uptake in hepatocytes (20). In conMINUTES trast, as shown in Fig. 4, azide is inhibiFIG. 4. Effect of sodium azide on methotrexate up- tory toward methotrexate uptake. After 90 take. Hepatocytes were incubated at 37°C with 4.0 min 1.0 mM sodium azide led to about 45% PM [3H]methotrexate (0) alone and in addition to 1.0 inhibition and 10.0 ITIM azide to about 87% mM sodium azide (x), or 10 mM sodium azide (0). inhibition of methotrexate uptake. Uptake was performed as described under Materials and Methods.

Effect of Anaerobiosis on 5-CH,-HPteGlu Uptake

they are affected by 40 mM ethanol. Uptake Figure 5 illustrates the effect of various of these folates was performed at comparable concentrations and sampling times OJN, mixtures on the uptake of 5-CH3-H,were 30 and 60 min. At 30 min of uptake PteGlu. As the percentage 0, is decreased

.E E

% f 0 \ Z E *

200 -

100 -

I 0

I 5

I IO

1 IS

20

% o2

FIG. 5. Effect of anaerobiosis and ethanol on the uptake of 5-CH,-H,PteGlu. After isolation the hepatocytes were placed in 20 ml plastic scintillation vials fitted with a septum. Air was replaced with N2. N1 was removed and Oe added with a syringe to give the initial percentages of 0, shown. 5CH,-H,PteGlu (0.25 pM) was injected through the septum and uptake was measured at 45 min (0) no addition: (0) 10 mM ethanol.

562

HORNE ET AL. TABLE III EFFECTOFPYRAZOLE ONTHE ETHANOL EFFECTS Concentration (mM)

Addition

40.0 1.0 1.0 40.0 5.0 5.0 40.0

None Ethanol Pyrazole Pyrazole and ethanol Pyrazole Pyrazole and ethanol

Percentage control

P

5.5(6) 7.3(6) 5.6(6) 14.5(6)

co.05 >0.50 >0.07

100.7 t 5.7(4) 101.5 k 3.6(4)

>0.90 BO.80

100.0 c 127.0 + 105.0 ? 131.1 r

a Hepatocytes were incubated at 37°C with 0.25 pM 5-CH,-H,PteGlu and the additions shown. At 60 min uptake was estimated. Results are expressed as percentage control uptake ? SEM. Values in parentheses are number of determinations.

there is a progressive increase in the uptake of 5-CH,-H,PteGlu such that in 0% O2 the uptake is about 40% greater than in 20% OZ. Also, the presence of 10 mM ethanol resulted in increased uptake in all atmospheres other than 100% N,. Effect of Pyraxole

on the Ethanol

Effect

It is well known that pyrazole inhibits metabolism of ethanol by inhibiting alcohol dehydrogenase (see Ref. (21) for discussion and further references). Thus if the ethanol effect on 5-CH,-H,PteGlu is due to metabolism of ethanol pyrazole should reverse the effect. This is indeed the case as shown in Table III. Incubation of the cells with ethanol again stimulated 5-CH,-H,PteGlu uptake. Pyrazole at 1.0 mM was without effect on uptake in control cells and did not block the ethanol effect. However, at 5.0 mM pyrazole effectively abolished the ethanol-induced stimulation of uptake. Thus a metabolite, such as acetaldehyde, or another consequence of ethanol metabolism, such as an increased NADH/NAD ratio, and not ethanol, per se, is responsible for the ethanol effect.

fore, cells were incubated in the presence of acetaldehyde and uptake of 5-CH,H,PteGlu was measured. Table IV shows that after 5 min of uptake acetaldehyde at 0.04 InM was slightly inhibitory and at 10.0 mM was inhibitory to the extent of 40%, whereas 40.0 InM ethanol was without effect. After 30 min acetaldehyde at 10.0 lllM was still inhibitory while ethanol resulted in about 15% stimulation of uptake. At 60 min all treatment were stimulatory. Uptake in the Presence of Various Alcohols

In order to determine if the stimulation of uptake of 5-CH,-H,PteGlu were specific for ethanol, the n-alkanols from methanol through pentanol and sorbitol were tested (see Table V). At equimolar concentrations (40 mM) methanol was inhibitory. However, ethanol, propanol, butanol, and pentanol all stimulated uptake. Of particular interest is the fact that sorbitol was also stimulatory since it is oxidized to fructose via hexitol dehydrogenase (22) and does not give rise to a straight-chain aldehyde as do the other alcohols tested. DISCUSSION

Uptake of 5-CH,-HQteGlu of Acetaldehyde

in the Presence

It appeared that metabolism of ethanol is necessary for stimulation of 5-CH,H,PteGlu uptake (see Table III). There-

Numerous studies have documented the effects of ethanol on uptake of various nutrients such as amino acids (23-26), glucose (27-28), vitamins (8, 29-30), minerals (3133), and fats (34). In most cases, an excep-

ETHANOL

AND 5-METHYLTETRAHYDROFOLATE

TRANSPORT

563

TABLE IV EFFECT OF ACETALDEHYDE ON UPTAKE OF 5-CH,-H,PteGlu” Percentage control Additions None Acetaldehyde

Ethanol

Concentration ma

5 min

0.04 1.0 10.0 40.0

100.0 ? 1.6 93.7 +- 2.26 98.2 k 3.0 60.7 2 1.6b 103.2 f 2.9

30 min 99.8 k 94.4 + 104.7 k 82.3 k 115.5 f

2.4 3.2 7.8 4.4b 6.5*

60 min 100.0 + 2.1 109.8 ‘- 3.2* 115.4 +- 4.6b 115.6 ” 4.7* 147.1 2 4.6b

a Acetaldehyde or ethanol at the concentrations shown and 5-CH,-H,PteGlu at 0.3 PM were added to hepatocytes. At 5,30, and 60 min samples were taken to estimate uptake. The results are reported as percent of control uptake ?SEM (n = 6). * P 5 0.05 when compared to control.

tion being manganese absorption in the intestine (33), the effect was an inhibition of transport. In a previous communication (16) it was shown that incubation of hepatocytes in the presence of ethanol results in an increased accumulation of 5-CH,-H,PteGlu. The time course of the ethanol effect indicated that maximal stimulation occurred between 40 and 60 min of uptake. It was further determined that the effect was concentration dependent with saturation occurring at an extracellular ethanol concentration of approximately 30 mM. In the present study we have confirmed and extended these findings.

The increased accumulation of 5CH,HJ’teGlu induced by ethanol could be due to a quantitative difference in the metabolism of this folate coenzyme. However, as shown in Fig. 3 and under Results there was no significant difference in metabolism of accumulated 5CH,-H,PteGlu in ethanoltreated and control cells. Some insight into the mechanism of the ethanol effect on 5-CH,-H,PteGlu uptake is provided by studies with pyrazole, an inhibitor of ethanol metabolism. If ethanol, per se, enhanced accumulation pyrazole should either increase or not affect this accumulation of substrate. However, if the responsible agent were an ethanol metabolite, pyrazole would be expected LLIdecrease TABLE V the ethanol effect. Indeed, 5mM pyrazole EFFECT OF VARIOUS ALCOHOLSON abolished the ethanol effect as shown in TaUPTAKE OF 5-CH,-H,PteGlu” ble III. Thus, the stimulation of 5-CH3H,PteGlu uptake must be a consequence of Percentage ethanol metabolism and not due to an effect control P Addition of ethanol itself upon the uptake process. A likely candidate is acetaldehyde. AcetalControl 100.0 f 1.7(12) dehyde did lead to enhanced accumulation