0145-6008/95/1905-1327$03.00/0 ALCOHOLISM: CLINICAL AND EXPERIMENTAL RESEARCH

Vol. 19, No. 5 October 1995

Effect of Ethanol Consumption on Adult Rat Liver Mitochondria1 Populations Analyzed by Flow Cytometry

Antonio Mateos, Alberto Orfao, Angeles Almeida, Maria Isabel Martin, Casilda Lopez-Mediavilla, Jose Maria Medina, and Julio Fermoso

In the present study, the effects of administering ethanol to adult male rats on the distribution of the low fluorescence population (LFP) and high fluorescence population (HFP), and the hodamine-123 flu- orescence intensity of these groups of mitochondria are analyzed by flow cytometry. Our results show that ethanol administration to adult male rats induces a redistribution of the HFP and LFP mitochondria1 populations leading to an increase of the less functional HFP mito- chondria. In addition, ethanol induced an increase in the mean inten- sity of green fluorescence of the HFP that is probably related to an increased number of rhodamine-123 binding sites per mitochondria resulting from mitochondria enlargement.

Key Words: Alcohol, Rat Liver, Mitochondria, Flow Cytometry, Rhodamine-123.

T HAS been established for some time that chronic I alcohol intake, with increased levels of ethanol in the plasma, exerts a toxic effect on liver cells and their or- ganelles, including mit~chondria.'-~ The effects of ethanol on mitochondria have been shown to be influenced by several factors, such as the amount of ethanol consumed, continuance of ethanol consumption, the state of nutrition, and age and genetic predispositi~n.~,~ Enlargement and hyalin degeneration of mitochondria are prominent mor- phological features of the toxic effects of Func- tional studies of mitochondria have shown the existence of different abn~rmalities~.'~-~' after ethanol intake, such as an impaired functionality of the electron transport chain with a significant reduction of the liver mitochondrial res- piration associated with a decrease in functional cyto- chrome oxidase content and impaired oxidative phosphor- ylation.

Recently, we have shown" the existence of two rat liver mitochondria populations identified by their rhodamine- 123 uptake, as analyzed by flow cytometry: a low fluores- cence population (LFP) and a high fluorescence population (HFP). The proportion between both mitochondrial popu- lations changes during development, and the percentage of

each population is affected by the energization state of mitochondria.20 In addition, these HFP and LFP mitochon- dria show different density, enzyme activities, and respira- tory beha~i0rs. l~ Thus, it has been suggested that the HFP mitochondria may represent a less functional compartment of mitochondria, because they decrease both during devel- opment and during mitochondrial energizatioq2O whereas HFP mitochondria increase in the presence of protein synthesis inhibitors.21

The aim of the present study was to analyze the effects of administering ethanol in a liquid diet to adult rat males during 6 weeks on the distribution of both the HFP and the LFP mitochondrial populations and their rhodamine-123 uptake as analyzed by flow cytometry.

MATERIALS AND METHODS

Animals

Thirty adult male rats from the Wistar strain were purchased from Pan-lab (Barcelona, Spain) and housed in a room with controlled illumi- nation (12-hr light/l2-hr darkness) and temperature (23 2 l T ) , with constant access to food and water. One week later, half of the animals (alcoholic group) were fed a liquid diet containing ethanol (36% of the calories present in the diet) as previously reported:' in accordance with the indications of Lieber and DeCarkZ3 This liquid diet containing etha- nol was administered continuously for 6 weeks; the remaining 15 animals (control group) were fed an isocaloric liquid diet, whereas ethanol was replaced by a mixture of dextrin-maltose. During the 6 weeks, three animals of each group were killed by decapitation at 1, 3, and 6 weeks (days +7, +21, and +42) after the initiation of the administration of ethanol.

After 6 weeks, the remaining 12 adult Wistar male rats (6 from the alcoholic group and 6 from the control group) were fed an isocaloric liquid diet with dextrin-maltose and without ethanol. From these animals, half were killed by decapitation 1 week later (day +49) and the remaining animals at day +70. From the 6 animals killed at each of these time periods, 3 belonged to the alcoholic group and 3 belonged to the control group.

From the Department of Medicine (AO), Unit of Cytometry, and Depart- ment of Biochemistry and Molecular Biology (AA., C.L.-M., J.M.M.), Uni- versity of Salamanca, Salamanca, Spain.

Received for publication October 7, 1994; accepted April 28, 1995 This study was supported in part by the D. G.I. C. Y. T. and by the Consejeria

de Cultura de la Junta de Castilla-Leh A A . was the recipient of a fellowship from the Ministerio de Educacibn y Ciencia.

Reprint requests: Alberto Orfao, M.D., Ph.D., Servicio General de Cito- metria, Laboratorio de Hematologia, Hospital Universitario de Salamanca, 37007 Salamanca, Spain.

Copyright 0 1995 by The Research Society on Alcoholism.

Alcohol Clin Exp Res, Val 19, No 5, 1995: pp 1327-1330

Isolation of Rat Liver Mitochondria

The whole mitochondrial fraction (WMF) was isolated from the liver of adult male Wistar rats. Liver was homogenized with 5 ml of isolation medium (250 mM sucrose, 1 mM EDTA, and 10 mM Hepes; pH 7.4)/1 g of tissue. Afterward, 2 ml of this homogenate was layered onto a discon- tinuous Percoll gradient, based on the method reported by Reinhart et al.,24 with some modification^.'^ The density of the WMF measured by colored density-marker beads (Pharmacia LKB, Uppsala, Sweden) was 1.082 g/ml. In agreement with Pertoft and Laurent,z isolation with Percoll did not interfere with mitochondria viability, as assessed by measuring their respiratory rate using succinate as substrate as previously de-

1327

1328 MATEOS E l AL.

scribed"; mitochondria from control rats showed a suitable respiratory control index.

Mitochondria Rhodamine-I23 Staining

Isolated adult rat liver mitochondria were stained by incubation with rhodamine-123 (10 pgiml) (Sigma Chemical Co., St. Louis, MO) at 37°C for 15 min in the dark. The unbound dye was removed by centrifugation, and the pellet was washed twice with isolation medium.

Flow Cytometry Analysis of Mitochondria

All measurements of mitochondria fluorescence and side (90" angle) light scatter (SSC) were made for at least 10,000 eventshest using a FACStar flow cytometer (BectonDickinson, San Jose, CA) and the CON- SORT 30 software program (BectonDickinson). Data on mitochondria1 fluorescence and light scatter were obtained using a 5-W argon ion laser tuned at 488 nm and 250 mW, rhodamine-123 green fluorescence was measured through a 530 t 15 nm bandpass filter placed in front of the green photomultiplier tube using a four decade log amplifier. The mean intensity of the green fluorescence (IGF), caused by rhodamine-123 up- take by isolated mitochondria, was determined for each of the LFP and HFP mitochondrial populations using the LYSYS I1 software program (Becton/Dickinson) and expressed as fluorescence channels (scaled from 0 to 10,000 arbitrary units). Graphics were plotted using the LYSYS I1 software program. Each day, all measurements were performed within a 30-min period of time using the same instrument set-up and conditions to obtain comparable results for the different measurements.

Statistical Analyses

Statistical analyses of the proportion between the HFP and LFP mito- chondrial populations were conducted using Student's t test. For the comparison between the mean IGF of the HFP and LFP at different days, the Mann-Whitney U test was used.

RESULTS

As previously reported,13 two distinct mitochondrial pop- ulations were obtained upon analyzing the rat liver WMF by flow cytometry: an LFP and an HFP. Both populations were present in all animals analyzed both from the alco- holic and the control groups (Fig. 1).

Table 1 shows the distribution of the LFP and the HFP mitochondria from adult male rat liver at the different time periods analyzed in the present study. As seen herein, during the first 6 weeks (days +7, +21, and +42), there was a progressive increase in the percentage of the HFP with a decrease of the LFP mitochondria in the alcoholic rats, whereas no major changes were detected in the control group. The maximum difference was observed 6 weeks after ethanol administration was initiated ( p = 0.05).

Upon removing ethanol from the alcoholic rats diet, the proportion between the two mitochondria populations pro- gressively returned to the initial values, which were achieved at day +70, 4 weeks after the alcohol insult was removed (Table 1).

Regarding the mean intensity of the green fluorescence (IGF) caused by rhodamine-123 uptake by the LFP mito- chondria, no major changes were detected during alcohol administration nor after its elimination from the diet, with the exception of a slight decrease in the LFP mean IGF

" --.,

Fig. I. Flow cytometry of rhodamine-123-stained mitochondria from both the control (A) and the alcoholic (6) rats at different time periods analyzed during ethanol administration [day + 7 (l), day +21 (2), and day +42 (3)] and after it was removed [day +49 (4) and day +70 (5)]. X-axis, rhodamine-123 green fluores- cence; Z-axis, number of events; Y-axis, 90" angle light scatter (side scatter). HFP and LFP mitochondria populations are also identified.

detected at day +7 (26 ? 3 vs. 16 5 2 fluorescence chan- nels; p = 0.07) (Table 2). By contrast, an important in- crease on the mean IGF resulting from rhodamine-123 uptake by the HFP was observed at day +42 (228 5 14 vs. 327 5 15 fluorescence channels; p = 0.005) after ethanol consumption was initiated (Table 2), this being coincident with the time period wherein the maximum effect of etha- nol on the redistribution of the LFP and HFP mitochon- drial populations was observed. After removal of ethanol from the diet of the alcoholic rats, the increased mean IGF was still observed 1 week later (day +49: 199 2 23 vs. 288

ETHANOL CONSUMPTION IN ADULT RAT LIVER 1329

Table 1. Relative Numbers of the LFP and the HFP Mitochondria at Different Time Periods During Ethanol Administration (Days +7, +21, and +42) and

After It Was Removed (Days +49 and +70)

Control group Alcoholic group

% LFP % HFP % LFP % HFP pvalues

Day+7 9 7 2 1 3 2 1 9 7 2 1 3 2 1 0.77

Day+42 91 2 1 9 2 1 8 3 2 5 1 7 2 2 0.05 Day +21 9 4 2 1 6 2 1 91 2 2 9 2 2 0.10

Day +49 9 4 2 1 6 t 1 8 6 2 4 1 4 . ~ 4 0.08 Day+70 91 t 1 9 - t 1 9 3 2 2 7 2 2 0.40

Values are mean 2 SD. Each experiment includes three different animals.

2 18 fluorescence channels; p = 0.01) although it returned to normal levels at day +70 (Table 2).

DISCUSSION

Flow cytometry is a relatively recent and innovative tech- nology that allows a quite rapid, objective, and sensitive multiparametric analysis of cells and their organelles as they flow aligned one by one in front of a light source, usually a In recent years, there has been increas- ing interest in analyzing cell organelles by flow cytometry, including chromosomes2’ and m i t ~ c h o n d r i a . ~ ~ - ~ ~ Accord- ingly, two mitochondrial populations have been identified in rat liver cells on the basis of their rhodamine-123 uptake and side light scatter properties: the LFP and the HFP.19 The proportion between these two mitochondrial popula- tions changes under different physiological conditions.20 In addition, both mitochondrial populations display distinct characteristics. The HFP represents a less functional com- partment of mitochondria displaying both a lower respira- tory rate and decreased NADH UQ-oxidase, succinate-DH and cytochrome c-oxidase enzyme activities, compared with LFP.’9,21,33 In the present study, these two mitochondrial populations were identified in the WMF from all the ani- mals analyzed, with the LFP being the predominant popu- lation in accordance with previous reports?’ Our data show that ethanol consumption induces a progressive increase in the HFP:LFP ratio, with this effect being maximal 6 weeks after ethanol administration was initiated. These results would support the notion that the existence of an impaired mitochondrial function after ethanol intake may be caused by decreased oxygen c~nsumption~’~~~,~~,’~~~’ associated

with an impaired phosphorylation and a de- creased cytochrome oxidase that could be at least partially related to the increase of the less functional HFP mitochondria.

Upon removing ethanol from the diet, the HFP:LFP ratio returned to the level detected in the adult male rats from the control group. These results are in accordance with previous data that indicate that, under the same ex- perimental conditions when the ethanol insult is removed, liver mitochondria return to their normal functional and morphological state?734

Rhodamine-123 uptake by mitochondria measured as the mean IGF has been shown to be associated with the membrane potential and to reflect, at least to a certain extent, the functionality of m i t ~ c h o n d r i a . ~ ~ ~ ~ ~ , ~ ~ ” ’ Our re- sults show that regarding the mean IGF, the administration of ethanol induces an early slight decrease of the LFP together with a late significant increase of the HFP. The former finding would be in agreement with the induction of a decrease in the membrane potential of the LFP mito- chondria by ethanol. However, this would not explain the increase in the mean IGF of the HFP detected 6 weeks after ethanol administration was initiated. It has been shown’9220 that, with respect to their rhodamine-123 up- take, the HFP mitochondria display a low ability to react to energization and deenergization. Thus, the increased mean IGF/mitochondria, detected for the HFP mitochondria af- ter alcohol administration, would more likely be related to an increase in the number of rhodamine-123 binding sites/ mitochondria which, in turn, would be associated with the structural changes reported on liver mitochondria after ethanol con~umpt ion .~ ,~ ,~ Although rhodamine-123 has been extensively used for the study of mitochondria, and the mechanisms of rhodamine-123 uptake by mitochondria are well-established, the rhodamine-123 binding sites are still ~ n k n o w n . ~ ~ , ~ ~ , ~ ~ It has been shown that, after ethanol intake, mitochondria increase in size, become pleiornor- phic, show increased numbers of inclusion bodies, and display disorganization of cri~tae.~”” In this context, the presence of megamitochondria has been These megamitochondria have been shown to be formed by the fusion of mitochondria containing small crystalloids resulting from the dietary withdrawal of In- terestingly, in the present study, it is shown that, on remov-

Table 2. Mean Intensity of Green Fluorescence of the LFP and the HFP Mitochondria at Different Time Periods During Ethanol Administration (Days +7, +21, and +42) and After It Was Removed (Days +49 and +70)

Control group Alcoholic group p values.

LFP HFP LFP HFP LFP HFP

Day +7 26 2 4 153 -t 45 16 -C 2 176 t 18 0.07 0.54 Day +21 26 t 3 198 2 18 28 2 2 159 2 16 0.60 0.13 Day +42 31 2 4 228 t 14 29 t 2 327 2 15 0.48 0.005

Day +49 30 2 2 199 2 23 30 t 2 288 t 18 1 .oo 0.01 Day +70 38 2 5 190 2 20 34 -t 3 171 2 23 0.55 0.58

Values are mean .C SD of the mean lGF/mitochondria expressed as fluorescence channels (arbitrary units). Each experiment includes three different animals. * Control vs. alcoholic group.

1330 MATEOS ET AL.

ing the alcohol insult, the increased IGF of the HFP mito- chondria returns to the normal values in accordance with previous studies that have shown that, after the reintroduc- tion of riboflavin into the diet of alcoholic animals, cleavage or division sites are developed in megamitochondria that can be detected by electron micro~copy.~~

In conclusion, our results show that ethanol administra- tion to adult male rats induces a redistribution of the HFP and LFP mitochondria, with an increase of the less func- tional HFP. In addition, ethanol induced an increase in the mean IGF of the HFP. This is probably related to an increased number of rhodamine-123 binding sitedmito- chondria.

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