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A Quantitative Morphological Analysis of Ethanol Effect Upon Rat Liver
Vol. 62. No.5
GASTROENTEROLOGY
Printed in U.S.A.
Copyright © 1972 by The Williams & Wilkins Co.
A QUANTITATIVE MORPHOLOGICAL ANALYSIS OF ETHANOL EFFECT UPON RAT LIVER WILLIAM
O.
AND HAROLD
DOBBINS.
J.
III,
M.D., EMORY
L.
ROLLINS, SUSAN
G.
BROOKS,
FALLON, M.D.
Departments of Medicine, George Washington University Medical Center, Washington, D. C., and University of North Carolina School of Medicine, Chapel Hill, North Carolina and from Gastrointestinal Research Laboratory, Veterans Administration Hospital, Washington, D. C.
Ethanol ingestion causes morphological changes in the livers of man and animals. This report describes a quantitative morphometric analysis of ethanol effects upon rat liver and a comparison with some biochemical determinations. Twenty-four rats were divided into three pair-fed groups: chow and 25% ethanol in drinking water; chow and an amount of sucrose isocaloric to the ethanol; and 25% ethanol in water and chow supplemented with 2% choline. After 35 days, a portion of liver was prepared for electron microscopy by standard techniques. Liver was analyzed for total phospholipid, triglyceride, lecithin, nitroreductase activity, and aniline hydroxylase activity. Twenty-five electron micrographs were obtained randomly from each specimen. Micrographs were analyzed by the lineal analysis method for cell and nuclear dimensions, and fractional volume of cytoplasmic components including mitochondria, peroxisomes, lysosomes, lipid bodies, and glycogen. Surface area of the mitochondrial envelope and the endoplasmic reticulum membrane and the approximate volume of mitochondria and peroxisomes were determined. Subjective analysis of electron micrographs failed to distinguish clearly the controls from the ethanol-treated rats. However, lineal analysis with statistical evaluation showed increased hepatic cell size (P < 0.001) and total volume of mitochondria, peroxisomes, lysosomes, and lipid bodies in the rats fed 25% ethanol. All of these changes were partially reversed by choline supplementation. The surface area of both smooth and rough endoplasmic reticulum membrane was reduced 50% in the ethanol-treated animals. This change was partially reversed by choline. Ethanol-induced increases in total liver phospholipid and lecithin were largely reversed by choline. Ethanol increased triglyceride content 2-fold and this was partially reversed by choline. Aniline hydroxylase increased 2-fold in the ethanol-treated rats when compared with control rats and 3-fold in the rats fed ethanol plus choline. Received June 14, 1971. Accepted January 12, 1972. Presented in part to the American Society for the Study of Liver Diseases, Chicago, Illinois, November 3, 1970. Address reprint requests to: Dr. William O. Dobbins, III, 2150 Pennsylvania Avenue, N.W., Washington, D.C. 20037.
This work was supported by United States Public Health Service Grants AM-13725 and AM-09000, and by Part I funds from the Veterans Administration. Standard deviations and errors were calculated in part by Mr. Emanuel Lerner of the Eastern Research Support Center, Veterans Administration Hospital, West Haven, Connecticut.
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LIVER PHYSIOLOGY AND DISEASE
Ethanol, either by a direct toxic effect on the liver cell or in combination with an associated nutritional imbalance, may result in fatty infiltration of the liver l - 14 and an increase in phospholipid content_ IS, 16 Choline has been reported to partially reverse or prevent many of the biochemical changes induced by ethano!.I 4 , IS, 17 Ethanol also has been reported to produce marked mitochondrial changes in either an Inhepatocytes 2 - 10 , 18, 19; crease 4 , 6, 9, II , 18, 19 or a decrease 12, 13 in the amount of endoplasmic reticulum; focal cytoplasmic degradation with an increase in the number of lysosomes 2 - 14 ; an increased number of peroxisomes 11; and increased glycogen content of liver cells. 4 Most of the morphological changes described are not specific for ethanol and depend on subjective analysis. However, subjective review of morphology in this study failed to distinguish with certainty all livers of control animals from livers of experimental animals given ethanol with or without choline supplementation. Therefore, data were obtained by a quantitative morphometric analysis of the effects of ethanol upon rat liver. Hepatic lipid content and drug-metabolizing enzyme systems were measured concurrently to facilitate comparisons with previous studies of ethanol-induced hepatic changes and to provide preliminary data on the correlation of structural and biochemical alterations.
Material and Methods Male Sprague-Dawley rats weighing 150 to 200 g were caged individually. Animals were fed Purina laboratory chow and water or 25% ethanol in water (v/v) on the following schedule: animals 1 through 9 were given chow and 25% ethanol; animals 11 through 18 received chow and 25% ethanol in the same amounts as the first group by pair feeding, but the chow was supplemented with 2% choline by weight; animals 21 through 30 were pair-fed an identical amount of chow, plus an amount of sucrose isocaloric to the 25% ethanol taken by the first group. The last group had unlimited access to water. One rat in the first group and 2 in the second died during the 1st week of the experiment. Thereafter, rat 30 was pair-fed with rat 9. The total caloric intake was con-
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stant for control and experimental animals, Mean weight gain was 3.1 g per day and mean caloric intake was 74.8 per day. The mean proportions of total calories were: protein, 19%; fat, 7.5%; mixed starches, 43.5%; and ethanol or sucrose, 30%. The rats were given constant access to food and were fed for 35 days. Rats were killed in groups of three pair-fed animals between 8 and 10 AM. Food, ethanol, and water were removed at 7 AM the same day. The animals were anesthetized with ether, the abdominal cavity was opened, and the rat exsanguinated from the aorta. A small fragment of the free edge of the right lobe of the liver was frozen immediately in liquid N 2 for lipid determinations and the remaining liver was then removed for preparation of cell fractions. 15 Each liver fragment was placed in ice-cold 1 % osmium tetroxide in 0.067 cacodylate buffer at pH 7.4. After 5-min fixation, the fragments were minced into 0.5-mm cubes and returned to the fixative for a total of 1 to 2 hr. The tissues were dehydrated in alcohol and embedded in epoxy resin. Sections (1.0- Jl thick) were obtained of each liver specimen and stained with toluidine blue. Portal and central vein areas were identified and the liver specimen was trimmed to appropriate size for thin sectioning. Thin sections were prepared from centrolobular fragments and an additional thick section was made after completion of thin sectioning. In order to obtain random electron microscopic photographs of each specimen, the thick section was examined by light microscopy and diagrammed on paper. Hepatocytes at a depth of 3 cells from portal and central areas (20% of hepatic area) were avoided. Five areas of midzonal cells (80% of hepatic area) were randomly marked. These areas were identified at the electron microscope and five photographs of each area were obtained at magnifications of 5,000 to 20,000 diameters. No condition of cytoplasmic or nuclear appearance was imposed in selection for photography. Only those cells showing technical artifacts such as knife scratches or heavy stain contamination were avoided. Repeated calibration of the AEI-EM6B electron microscope during this study showed that the microscope magnification was stable within a range of 2%, a range compatible with accurate quantitative measurements. Twenty-five electron micrographs were obtained from each of the 24 specimens for a total of 600 micrographs. Specimens were satisfactory for morphological analysis from 8 ani-
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LIVER PHYSIOLOGY AND DISEASE
Measurements of cellular components were mals fed chow plus ethanol, from 6 animals fed chow and 2% choline plus ethanol, and performed with the use of 8- by lO-inch enfrom 10 animals fed chow plus an isocaloric larged prints of the micrographs. In order to avoid bias, the micrographs for lineal analyamount of sucrose. The specimens were coded and analyzed sis of proper magnification were selected ranwithout reference to grouping. Light micro- domly from the micrographs of each specimen. scopic sections were graded randomly for the A square grid of white lines was superimposed presence of fat droplets using a scale of 1 + to on each print by lengths of 7-mil wire placed 3 +. Portions of all thick sections were photo- on a frame at I-inch intervals. All enlargements graphed at an original magnification of x 270 were done over a few days' time during which and enlarged photographically to x 540. Cell no change in enlargement magnification could and nuclear dimensions are shown in table 1. be demonstrated. A lineal analysis of hepatoHepatocytes tended to be oblong in shape and cyte cytoplasm was performed using prints hence were measured in two diameters. Nu- with a magnification of x 12,500. This allowed clear diameters were determined for each of the statistically accurate calculation of the perthree groups by direct measurement of the centage of cytoplasmic volume for various organelle fractions and the total area of cytodiameter of all easily identified nuclei. The electron photomicrographs of each plasm in square microns (table 2). Prints with a magnification of x 25,000 were specimen were evaluated on the basis of overall subjective morphological impression with- used to calculate membrane areas per square out reference to treatment group. This random micron of cytoplasm. Intersections of the and entirely subjective evaluation failed to grid lines with mitochondrial envelope, smooth separate clearly livers of control animals from endoplasmic reticulum, and rough endoplasmic the livers of experimental animals given ethanol. reticulum were counted (table 3). These data Therefore, a partial quantitative analysis of were not analyzed statistically. However, the the electron photomicrographs was performed large number of intersects counted gives vausing modifications of the methods of Weibel lidity to the results. Golgi membranes were and Loud. 20 • 2 2 counted as smooth endoplasmic reticulum. 1. Cell and nuclear dimensions"
TABLE
Sucrose control
No. of cells measured .... .. .. . Average cell length (Il) .. , , ..... ... . . Average cell width (Il) Average nuclear diameter (Il) ...
Et hanol
227 24.2 ± 4 . 1 16.3 ± 3 . 1 7.6±1.1
268 26 .6 ± 4.6 17 .6 ± 3 .0 8 .0 ± 1.3
P
+ choline
Ethanol
p
< 0.001 < 0 .001 < 0.001
167 26.5 ± 3 .9 16.7 ± 3.0 7.6 ± 1.1
<0.001 NS NS
"The values represent the arithmetic mean ± SEM. P values were calculated usmg Student's t-test and refer to comparison of the two ethanol groups to the controls. Length and width of cells of ethanol-treated animals were not significantly different (NS) from those of the ethanol plus choline-treated animals with P values> 0.1
Volume fraction of cy toplasmic components·
TABLE 2.
Sucrose control
Number of micrographs . . . . ... . .. .... . Total area of cytoplasm (Il 2) . . . . . .. . · · .0· · ' . .. Cytoplasmic volume (% ± SEM) Mitochondria .. . .. . . . .. ..... . Peroxisomes . . .. . . . . . ... . . Lysosomes .... . .. . ... . . . ....... . .... . . .. Lipid bodies . . ... . . . . .. ... .... .. .... .. . . .. Glycogen . . . . . . .. . . . . . . ... Other .. . . ...... ..... . . . . ...... . . . . ... 0
••
•
•••
28 5994
Ethanol
27 7056
22 .6 ± 1.1 26 .0 1.6 ± 0.44 1.8 0.3 ± 0.45 0.7 1.0± 1.5 1.4 20 . 3 ± 1.4 18 . 7 54 .2 ± 1.9 51.4 " The method of measurmg, calculatmg, and expressmg volume fractIOns Methods." bNot statistically significant. '
••
•
•
0
•
••
0
0
••
•
••
••
•
••
•
•
••
•
•
••
•
Ethanol + choline
17 3663 ± 1.4 ± 0.44 ± 0.35 ± 1.7 ± 1.9 ± 2. 2 IS
23 . 6 1.5 0.4 0 .3 18 .7 55 . 5
± 1.2 ± 0.41 ± 0.46 ± 5.9 b ± 1.5 ± 2.0
detailed m "Material and
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LIVER PHYSIOLOGY AND DISEASE
These same prints at magnifications of 25,000 were used to calculate dimensions and approximate volumes of mitochondria and peroxisomes (table 4). The number of mitochondria, peroxisomes, and lysosomes per unit area was determined (table 5). Lysosomal dimensions were not measured because of their paucity. Calculations were performed as follows: u = volume of individual organelle V = total volume of hepatocyte cytoplasm Vum = relative volumes of mitochondria Vup = relative volumes of peroxisomes Vul = relative volumes of lysosomes Vuu = relative volumes of lipid droplets Vug relative volumes of glycogen Vuo = relative volumes of all other cytoplasmic components Grid lines over m, p, I, Ii, g, and 0 were measured using a Keuffel and Esser scale calibrated in 0.5-mm intervals. Grid lines over nuclei and extracellular space were not measured. TABLE
Total number of points (length in millimeters) over cytoplasm is expressed as Pt. Pt
=
Pm + Pp + Pi + Pli + Pg + Po
Volume fractions of each component are thus calculated by the formula Vum
=
Pm/Pt and is expressed as
J.l.2
Measurements of surface area are expressed as surface density, Sui. The area of membrane included in a unit test volume is termed the surface density and is given by the formula Sui
2Nlior [2(c)]/[d]
=
where Nli is the number of membrane intersections (c) per unit length of sampling line (d). Thus Sui = [2(c) lid and is expressed as J.I. 2/J.I. 3.
For mitochondria, the expression relates number of intersections of mitochondrial envelope to the corresponding length of line
3. Surface areas of mitochondrial envelope and endoplasmic reticulum membrane Cytoplasm analyzed
Mitochondrial envelope/ IJ. 3 mitochondria
IJ. '1
1J'1
~
Sucrose control ······. 0 · · . Ethanol + choline .. .. .. . .. Ethanol . . .. . . . . . . . . . . .
5395 2894 3794
6 .08 (3703)b
5.16 (1764) 5.60 (2752)
Endoplasmic reticulumo /
11 3
cytoplasm
SER
RER
2 .34 (6286) 1.48 (2145) l.38 (2633)
1.54 (4154) l.00 (1456) 0.76 (1459)
" SER, smooth endoplasmic reticulum; RER, rough endoplasmic reticulum. b The number of intersections counted is in parentheses. The method of calculating surface areas of these membranes is outlined in the text. TABLE 4.
Organelle measurements Mitochondria
Length
Width
~
~
l.21 l.26 l.34
0 . 75 0.77 0 . 74
Sucrose control .... .. .. , . , .. Ethanol + choline . . .... Ethanol . . . . . . . . ... . . .
Peroxisomes Volume ~'
0.53 (1470)" 0.59 (972) 0.58 (1545)
Diameter ~
0 .59 (300) 0.66 (164) 0 .53 (382)
" Number of organelles measured is listed in parentheses. TABLE
5. Organelle counts per square micron of cytoplasm Cytoplasmic length
Mitochondria
Peroxisomes
Lysosomes
0.21 (1l25)" 0 . 17 (502) 0.20 (749)
0.036 (193) 0.031 (89) 0.038 (144)
0.010 (52) 0.021 (62) 0 .012 (46)
~
Sucrose control . .. . . . . . Ethanol + choline . . . . . ... Ethanol " . . .. .. .. ... . . ..
5395 2894 3794
" Number of each organelle counted is listed in parentheses.
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LIVER PHYSIOLOGY AND DISEASE
traversing the mitochondria (d) alone. For endoplasmic reticulum (ER), the expression relates the measure of ER per unit volume of cytoplasm with (d), the length of sampling line traversing the cytoplasm. Mitochondrial volume was roughly estimated assuming that mitochondria are closed right circular cylinders of diameter D and length L:
V
=
(7r D3)/(4
d where
f
=
D/L
The number of particles (mitochondria, peroxisomes, lysosomes) was determined by counting these particles and expressing this count as a ratio to the cytoplasmic length traversed in the same micrographs. Sampling. In order to obtain statistical validity in the description of tissues, representative samples of these tissues must be obtained. In this study, tissue samples were obtained from a similar portion of the liver of each animal. The method of obtaining random micrographs of the tissues and consistent instrumental magnification and photogmphic magnification have been described. Observations were confined to midzonal liver cells because these cells are structurally homogeneous in normal rats, while significant differences are found in cells immediately surrounding central veins. 21 Cells immediately adjacent to portal areas show only minor variations from mid zonal cells. 21 The validity of using linear measurements to compute relative volumes has been widely applied by geologists who have used thin sections of rock in computing relative volumes of components of rock.20-22 The validity of this method has been demonstrated for biological applications. 20, 21 The accuracy of the quantitative description of a tissue is improved by increasing the number of micrographs analyzed. The standard deviation of a random sample counted, such as the length of mitochondrial profiles traversed by a grid line, is given by the square root of TABLE
mg/g liver
Sucrose control .. . .... . , Ethanol ...... .... . . Ethanol + 2% choline ' . ,
'"
'"
Results Light microscopy. Control rat livers were found by light microscopic grading to have o to 1+ fat with a single exception which was 2+. Rats fed ethanol plus 2% choline in chow had 0 to 1+ liver fat. Five of the livers from ethanol-treated rats had greater than 1 + fat. Lineal analysis of the electron micrographs (table 2) supported these light microscopic findings. Quantitative triglyceride determinations for these groups are shown in table 6 and conform roughly to the morphological findings.
6. Quantitative triglyceride determinations·
Triglyceride
Diet
the total count collected, Ci. For optimum efficiency the sample size, Ci, should be adjusted so that Ci is approximately equal to the standard deviation among the values of Ci obtained from a number of different micrographs from the sample analyzed. A larger sample is necessary when the volume fraction of a component is small, when the component varies in size, or when the component is irregularly distributed. This condition was generally utilized in this study. Mean values for tissue characteristics have been calculated from the combined raw data of all micrographs in each of the three groups. Standard errors reported are equal to the standard deviation among the corresponding values calculated independently for each micrograph. Other methods. Cell fractions were prepared and hepatic phospholipid and lecithin content measured as previously described. 15 Hepatic triglyceride was determined by a modification of the method of Kessler and Lederer 23 for serum triglyceride. Aniline hydroxylase activity was measured in liver homogenates" and nitroreductase activity in microsomal preparations. 25 Protein was estimated by the method of Lowry et a1. 26
2.93 5.65 4.11
± ± ±
0.33 0 . 74 0.42
Phospholipid ~moles/g
20.41 28.18 23.30
± ± ±
liver
1.18 0.89 0.74
Lecithin ~moles/g
11.03 16.17 12.93
± ± ±
liver
0.59 0.56 1.19
Microsomal protein
mg/g liver
25.68 23.48 24.88
± ± ±
0.72 0.96 0.58
Values are means ± SEM for each group of rats. Wet weight of liver was used for reference. Differences in triglyceride, phospholipid, and lecithin content between the ethanol and the control groups are significant (P < 0.05). The differences in triglyceride, phospholipid, and lecithin content between control and ethanol plus 2% choline groups are not significant. There is no significant difference in the recovery of microsomal protein from the three groups. a
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LIVER PHYSIOLOGY AND DISEASE
Average cell length and width varied very little among the three groups (table 1). Hepatocytes from both ethanol-treated groups were slightly larger than hepatocytes of the control rats. The nuclei of hepatocytes of the ethanol-treated rats were slightly larger than nuclei of hepatocytes of the control rats and of the ethanol plus choline-treated rats. Electron microscopy. Subjective evaluation of electron photomicrographs was done without knowledge of treatment group. The electron photomicrographs obtained from the liver of 1 animal in each of the ethanol groups were considered to be normal in appearance. Electron micrographs from control rats were found to vary from normal in appearance to abnormal with many of the mitochondrial changes characteristic of ethanol-treated livers. The prominence of mitochondrial changes in both ethanol groups was readily apparent and resembled those described previously.2.14. 18. 19 No regular discernible difference in the smooth and rough endoplasmic reticulum structure could be detected in the three groups. Lineal analysis of the electron micrographs was undertaken (table 2, figs. 1 to 4) to permit statistical evaluation of results. Our findings in normal rat hepatocytes are in general agreement with those published by Loud.21 The lineal analysis confirmed (table 2) that the mitochondrial volume did increase in the livers of ethanoltreated rats and that the addition of 2% choline to the diet resulted in mitochondrial volume intermediate between that of the ethanol group and the pair-fed controls. Lysosomal volume doubled in the livers of the ethanol-treated group but increased only slightly in the livers of rats given ethanol plus choline. Peroxisome volume increased slightly in the ethanol group and diminished very slightly in the ethanol plus choline group. The volume of lipid droplets increased approximately 50% in the ethanol group. There was an insufficient number of lipid droplets in the sample analyzed from the ethanol-plus choline-treated group to permit a statistically valid comparison with the other
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groups. Glycogen volume decreased slightly in both treated groups. As often occurs when cacodylate buffer is utilized, glycogen varied in staining qualities. This presented no problem, however, in its identification and quantitation. Surface areas of the mitochondrial envelope and endoplasmic reticulum membrane showed major differences among the three groups (table 3). Mitochondrial envelope surface area decreased in both ethanol groups reflecting the increased mitochondrial volume (table 4). Mitochondria in the livers of rats treated with ethanol plus choline had a slightly greater increase in individual volume than did mitochondria of the livers of rats treated with ethanol alone. However, the total volume of mitochondria of the ethanol plus choline group was less than that of the ethanol-treated group as shown by the lower count of mitochondria per square micron of cytoplasm in the ethanol plus choline group (table 5) . Hence, there were slightly fewer mitochondria in both ethanoltreated groups but these mitochondria were larger and had a greater individual and total volume than did mitochondria from control livers. Peroxisomes increased in diameter in the group given ethanol plus choline but decreased in diameter in the ethanol group (table 4). In contrast, there were fewer peroxisomes per square micron of cytoplasm in the ethanol plus choline group and increased numbers in the ethanol-treated group (table 5). Lysosomes per square micron of cytoplasm increased in number and volume in both ethanol-treated groups (tables 2 and 5). There was a moderate decrease in surface area of both smooth endoplasmic reticulum (SER) and rough endoplasmic reticulum (RER) in both ethanol-treated groups, with the ethanol plus choline group showing a smaller decrease in surface area than the ethanol-treated group (table 3). The Golgi apparatus did not appear to vary significantly in the three groups, but this observation was not quantitated. Lipids and other measurements. A nearly 2-fold increase in hepatic triglyc-
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Vol. 62, No . 5
FIG. 1. Electron micrography of a portion of an hepatocyte of sucrose control rat liver. Micrographs at this magnification were utilized for the lineal analysis. It illustrates the average appearance of normal hepatocytes showing slightly elongated mitochondria (M), modest deposits of glycogen (Gly) , occasionallysosomes (Lys), more frequent peroxisomes (1'), rough endoplasmic reticulum (RER) , smooth endoplasmic reticulum (SER) , lipid droplets (Lip), and a bile ductule (B) [double stained (uranyl acetate and lead citrat e) x 12,500j.
eride content was observed in the rats fed ethanol (table 6) . A lesser increase was noted in the rats given 2% choline. The rise in hepatic phospholipid and lecithin
content in this experiment was similar to that previously reported. 1 5 There was no difference in total hepatic protein content in the three groups. However, a small de-
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LIVER PHYSIOLOGY AND DISEASE
crease in the recovery of microsomal protein per gram of liver was noted in the rats given ethanol. Aniline hydroxylase activity was increased 2-fold in rats fed ethanol and
1027
nearly 3-fold when choline was given in addition to ethanol (table 7). No change was observed in microsomal nitroreductase activity in rats given ethanol. At the end of the experiment the mean
FIG. 2. Electron micrograph portions of two hepatocytes of an ethanol-treated rat. The mitochondria (M) are large and often encircle organelles and portions of cytoplasm (arrows). Mitochondrial cristae are often absent and atypical in shape. Glycogen (Gly) is present as a large conglomerate as well as being scattered diffusely throughout the cytoplasm. The rough endoplasmic reticulum (RER) might be described in one area as "degranulated." The peroxisomes (Pi possess prominent nucleoids in this particular micrograph. Lysosomes (Lys), Golgi apparatus (G), and a bile ductule are normal in appearance (double stained, x 12,5(0).
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FIG. 3. Electron micrograph of portion of an hepatocyte from an ethanol-treated rat. Micrographs at this magnification were utilized for counting mitochondrial and endoplasmic reticulum membrane intersections. Findings illustrated in this micrograph are similar to those described in figure 2. The smooth endoplasmic reticulum (SER) appears slightly dilated while the rough endoplasmic reticulum (RER) is normal in appearance. Mitochondria (M). Peroxisomes (P). Golgi apparatus (G). Portion of nucleus (N) (double stained, x 25,000).
serum level of ethanol was 153 mg per 100 ml and varied from a low of 33 to a high of 270 mg per 100 ml.
Discussion Observations were made of "midzonal" (centrolobular) liver cells, excluding only
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LIVER PHYSIOLOGY AND DISEASE
those cells within a depth of 3 cells from portal and central areas. This midzonal area clearly reflects 80% of hepatocytes. 21 Further, Iseri et al. 4 showed that, in normal rats, the RER of hepatocytes im-
1029
mediately adjacent to the central vein is often vesiculated and may be confused with alcohol-induced changes in midzonal hepatocytes. 4 Thus we chose to quantitate alcohol-induced changes m midzonal
FIG. 4. Electron micrograph of portion of two hepatocytes from an ethanol-plus choline-treated rat. The mitochondria (M) are intermediate in appearance between those of sucrose control and ethanol-treated rats. Glycogen (Gly) is prominent while the smooth endoplasmic reticulum (SER) and rough endoplasmic reticulum (RER) are normal in appearance. Peroxisomes (P), Golgi apparatus (G), lysosomes (Lys), nucleus (N), and a bile ductule (B) appear unaltered (double stained, x 12,5(0).
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LIVER PHYSIOLOGY AND DISEASE TABLE 7 .
Enzyme determinations'
Diet
Sucrose control .. Ethanol . . . Ethanol + 2% choline
Aniline hydroxylase
reductase
nmoles/mg protein
nmoles/mg protein
24 .76 ± 1.84 53 .12 ± 12.46
56.07 ± 8 .37 51.67 ± 5.82
71 .65 ± 19.50
50. 30 ± 8.57
Nit ro·
• Values are means ± SEM for 10 rats in each group. Aniline hydroxylase is measured in micromoles of paraaminophenol formed per milligram of homogenate protein per 30 min. The nitroreductase is measured as micro moles of paraaminobenzoic acid formed per milligram of microsomal protein per 30 min.
hepatocytes, excluding 20% of liver area. This may account for some discrepancies in our study and other studies cited in this report. The effect of long term ethanol administration on the ultrastructure and the lipid content of liver cells of a variety of animal species has been described. 2, 4·9, 15 Acute administration of ethanol has been reported to produce similar biochemical and morphological effects, 3, 18,19, 27 Prominent mitochondrial changes in hepatocytes of ethanol-treated animals were emphasized by Hartroft's group 2, 11-14 and have subsequently been described by other investigators, 3-10, 18, 19 French 28 studied membrane structure and function of mitochondrial fractions isolated from rat liver after chronic ingestion of ethanol and observed that the outer membrane of mitochondria in livers of ethanol-treated rats also has been noted in other electron microscopic studies,' We have confirmed these morphological changes in mitochondria of livers of rats after chronic ethanol feeding under the dietary conditions described, Porta and Gomez-Dumm" used a planimetric method to show that individual liver mitochondria of ethanol-treated rats had a mean area 2-fold greater than that of control rats while the total number of mitochondria per unit area of cytoplasm was reduced in the ethanol-treated animals, This study shows similar changes in ethanol-fed rats with mitochondrial volume increasing 15%, a change partially
Vol . 62, No . 5
prevented by supplementation of the diet with 2% choline, In human alcoholic hepatitis, the organelle change which reportedly correlates best with the clinical condition is mitochondrial alteration, 18 Therefore, structural and functional changes in the mitochondria may have clinical consequences, 27, 29, 30 Isolated hepatic mitochondria of rat livers obtained after prolonged ethanol ingestion have decreased oxidative properties for pyruvate and fatty acids, 31 an effect which may also be attributed to ethanol-induced mitochondrial inJUry.
Iseri et al. 4 noted that, in rats fed ethanol for 6 to 27 days, centrolobular hepatocytes were characterized by vesiculation and prominence of both smooth SER and, to a lesser degree, of RER with some detachment of ribosomes from the RER. Vesiculation and apparent increase in the amount of SER with degranulation of RER and decreased RER was described later by this same group under a variety of conditions. 6 , 9, 18, 19 Hartroft's group"-14 has noted similar changes in the RER and SER although they described increased SER in only one study. 11 There is general agreement that the RER is reduced in amount,2- 14, 18, 19 a finding quantitatively confirmed in this report. Under these experimental conditions, a decrease in amount of both the SER and RER was observed in livers of ethanoltreated rats. Addition of choline to the diets resulted in partial prevention of this change, especially in the RER. Degranulation of the RER was not documented in the ethanol group by double blind subjective assay of the photomicrograph. However, no attempt was made to quantitate the possible degranulation of the RER. The discrepancy between our SER findings and those of other reports require explanation. Several considerations may be important in understanding these differences. Vesiculation of SER has been subjectively interpreted as an increase or proliferation of SER in studies of cortisone effect on liver. However, quantitative estimates of SER have not supported this subjective impression and have shown
May 1972
LIVER PHYSIOLOGY AND DISEASE
decreased amounts of SER. 32 Therefore, the quantitative analysis of SER content as performed here may be a better reflection than subjective impressions of SER surface area under these experimental conditions. Possibly the most convincing published electron photomicrograph of increased SER in livers of ethanol-fed rats is that of Rubin et al. 9 They report this change as most prominent in rats fed ethanol and a diet deficient in choline and reduced in casein, methionine, and cystine. 9 The SER was only "moderately hypertrophied" in the nondeficient, ethanolfed rats and the surface area was not quantified morphologically.9 It is possible that quantitative analysis under these conditions will not support the initial impression of these authors. However, Rubin et al. 33 and Lieber and DeCarli 34 have observed that ethanol administration in combination with a liquid, relatively high fat diet results in increased microsomal protein in rats. This may represent the biochemical counterpart of proliferation of hepatic SER. 33. 34 However, no increase in microsomal protein content was observed in this study. The differences in both electron microscopic and biochemical findings between the data of Rubin et al. 9 and those reported here might be related to significant differences in experimental design. Although total ethanol levels werecomparabIe, the other dietary conditions were markedly different. The relatively low fat intake and use of sucrose rather than Dextri-Maltose as carbohydrate source in this study may account for quantitative differences in the two experiments and the discrepancy in SER results. If this is the case, it strongly implies that the long term effects of ethanol ingestion on the liver are significantly influenced by other dietary factors as suggested by previous studies. 12. 15 . 17 The rise observed in aniline hydroxylase specific activity was not accompanied by an increased amount of SER on morphological analysis, demonstrating that these may be independent phenomena. Changes in endoplasmic reticulum proteins may be highly selective as shown by the study of
1031
Arias et al. 35 in the case of phenobarbital. It is likely that ethanol also causes increases in a limited number of specific proteins of the endoplasmic reticulum but without changes in the total amount of endoplasmic reticulum. Disorganization of the RER is a common response of hepatocytes to injury and is not a specific reaction to chemical agents alone. 36 For example, reduced amounts of RER have been found in rapidly proliferating cells, in association with an increased number of free ribosomes. 3 6 This may occur in ethanol-treated hepatocytes also, ·although quantitative documentation of increased free ribosomes is not available. Subjectively, we did not detect an increase in free ribosomes. The reduction in total microsomal protein content in rats fed ethanol was too small and did not conform to the decrease in RER and SER determined by electron microscopy. Several explanations for this discrepancy may be offered. The amount endoplasmic reticulum recovered by the centrifugational techniques used represents only a portion of the total endoplasmic reticulum as estimated by P-450 determinations. 37 Therefore, relatively small changes in the portion of endoplasmic reticulum recovered by centrifugation (approximately 30 to 50%) would produce significant variations in the microsomal protein levels obtained. Alternatively, differences in endoplasmic reticulum width, protein concentration, or contamination of microsomal pellets with protein derived from other particulate fractions could explain the discrepancy. Moreover, the content of the vesicular structures seen in electron microscopic study of these pellets is unknown and variations in size and content of these vesicles could markedly affect protein estimates of the pellets recovered. Further studies of this problem are in progress. The increase in hepatic phospholipid content of ethanol-treated rats cannot be explained by the changes in the endoplasmic reticulum but may relate to the increase in mitochondrial volume. Focal cytoplasmic degradation in the form of increased number of autophagic
1032
LIVER PHYSIOLOGY AND DISEASE
vacuoles (lysosomes) has been commonly noted in livers of ethanol-fed animals 2 - 14 , 19 and was confirmed here. Lysosomal volume increased from 0.3% in normals to 0.7% in ethanol-fed rats but only to 0.4% in the rats fed ethanol plus choline (table 2) . Lysosomes are increased in many forms of hepatic injury,18 probably indicating increased turnover of damaged cellular components. Increased number of peroxisomes have been noted in livers of ethanoltreated animals. 11 Peroxisome volume of ethanol-treated rats was 1.8% compared to 1.6% of control rats (table 2). Peroxisome volume fraction decreased to 1.5% in ethanol plus choline-treated rats. The significance of the changes in peroxisome size and volume is not known but it is tempting to speculate that they relate to the known catalase content of peroxisomes. 38 Catalase may potentiate ethanol metabolism in vitro. 39 Recently it has been suggested that ethanol may be metabolized, in part, by a microsomal pathway. 33, 34 Many drugs also are metabolized by the microsomal mixed function oxidase system. In man and in rats, the blood clearance of drugs metabolized in hepatic microsomes is slowed by concomitant ethanol administration despite increased activity of some drug detoxification enzymes. 33 , 34 This effect may reflect either competitive inhibition of ethanol and certain drugs for microsomal enzyme(s) or a toxic effect of ethanol upon these systems. The fact that 3 days of phenobarbital administration in rats inhibits ethanol-induced fatty liver may correlate with the increased amount of SER induced by phenobarbital. 40, 41 Choline also increases the amount of RER in the rat given ethanol and partially prevents some of the associated alterations in lipid content, suggesting the possibility of a mechanism similar to that of phenobarbital. This study supports previous evidence concerning the effect of large amounts of dietary choline upon ethanol-induced fatty liver, phospholipid metabolism, and structural changes. 17 Lieber and DeCarli 17 showed that choline protects against the fatty liver produced by prolonged ethanol
Vol. 62, No.5
intake. Porta et al. 2 , 14 and Takada et al. 7 also have demonstrated protection against fatty liver by choline supplementation. REFERENCES 1. Scheig R: Effects of ethanol on the liver. Am J Clin Nutr 23:467-473, 1970 2. Porta EA, Hartroft WS, de la Iglesia FA: Hepatic changes associated with chronic alcoholism in rats. Lab Invest 14:1437-1455, 1965 3. Ashworth CT, Wrightsman F, Cooper B, et al: Cellular aspects of ethanol-induced fatty liver: a correlated ultrastructural and chemical study. J Lipid Res 6:258-268, 1965 4. Iseri OA, Lieber CS, Gottlieb LS: The ultrastructure of fatty liver induced by prolonged ethanol ingestion. Am J Pathol 48:535-555, 1966 5. Thorpe MEC, Shorey CO: Long-term alcohol administration. Its effects on the ultrastructure and lipid content of the rat liver cell. Am J Pathol 48:557-577, 1966 6. Lane BP, Lieber CS: Ultrastructural alterations in human hepatocytes following ingestion of ethanol with adequate diets. Am J Pathol 49: 593-603, 1966 7. Takada A, Porta EA, Hartroft WS: Correlation of structural and functional recovery from cirrhosis in rats treated with lipotropic diets. Am J Pathol 49:841- 869, 1966 8. Lieber CS, Spritz N: Effects of prolonged ethanol intake in man: role of dietary, adipose, and endogenously synthesized fatty acids in the pathogenesis of the alcoholic fatty liver. J Clin Invest 45:1400-1410, 1966 9. Rubin E, Hutterer F, Lieber CS: Ethanol increases hepatic smooth endoplasmic reticulum and drug-metabolizing enzymes. Science 159: 1469-1470, 1968 10. Lieber CS, Rubin E : Alcoholic fatty liver in man on a high protein and low fat diet. Am J Med 44: 200-206, 1968 11. Porta EA, Gomez-Dumm CLA: A new experimental approach in the study of chronic alcoholism. I. Effects of high alcohol intake in rats fed a commercial laboratory diet. Lab Invest 18:352364,1968 12. Gomez-Dumm CLA, Porta EA, Hartroft WS, et al : A new experimental approach in the study of chronic alcoholism. II. Effects of high alcohol intake in rats fed diets of various adequacies. Lab Invest 18:365-378, 1968 13. Koch OR, Porta EA, Hartroft WS: A new experimental approach in the study of chronic alcoholism. nI. Role of alcohol versus sucrose or fat-derived calories in hepatic damage. Lab Invest 18:379-386, 1968 14. Porta EA, Koch OR, Hartroft WS: A new experimental approach in the study of chronic
May 1972
15.
16. 17.
18.
19. 20.
21. 22.
23.
24. 25.
26.
27.
28.
LIVER PHYSIOLOGY AND DISEASE
alcoholism. IV. Reproduction of alcoholic cirrhosis in rats and the role of lipotropes versus vitamins. Lab Invest 20:562-572, 1969 Fallon HJ, Gertman PM, Kemp EL: The effects of ethanol ingestion and choline deficiency on hepatic lecithin biosynthesis in the rat. Biochim Biophys Acta 187:94-103, 1969 French SW: Effect of chronic ethanol feeding on rat liver phospholipid. J Nutr 91:292-298, 1967 Lieber CS, DeCarli LM : Study of agents for the prevention of the fatty liver produced by prolonged alcohol intake. Gastroenterology 50: 316-322, 1966 Rubin E, Lieber CS: Early fine structural changes in the human liver induced by alcohol. Gastroenterology 52:1-13, 1967 Rubin E, Lieber CS: Alcohol-induced hepatic injury in nonalcoholic volunteers. N Engl J Med 278:869-876, 1968 Weibel ER, Kistler GS, Scherle WF: Practical sterological methods for morphometric cytology. J Cell Bioi 30:23-38, 1966 Loud AV: A quantitative sterological description of the ultrastructure of normal rat liver parenchymal cells. J Cell Bioi 37:27-46, 1968 Henning A, Meyer-Arendt JR: Microscopic volume determination and probability. Lab Invest 12:460-464, 1963 Kessler G, Lederer H: Fluorometric measurements of triglycerides, Automation in Analytical Chemistry. Edited by LT Skeggs Jr. New York, Mediad Inc, 1965, p 341 Imai Y, Ito A, Sato R: Evidence for biochemically different types of vesicles in the hepatic microsomal fraction. J Biochem 60:417-428, 1966 Fouts JR, Brodie BB: The enzymatic reduction of chloramphenicol, p-nitrobenzoic acid, and other aromatic nitro compounds in mammals. J Pharmacol Exp Ther 119:197-207, 1957 Lowry OH, Rosebrough NJ, Farr AL, et al: Protein measurements with folin phenol reagent. J Bioi Chern 193:265-269, 1951 Takeuchi J, Takada A, Ebata K, et al: Effect of alcohol on the livers of rats. I. Effect of a single intoxicating dose of alcohol on the livers of rats fed a choline-deficient diet or a commercial ration. Lab Invest 19:211-217, 1968 French SW: Fragility of liver mitochondria in ethanol-fed rats. Gastroenterology 54:1106-1114, 1968
1033
29. Lee SH, Torack RM: Electron microscope studies of glutamic oxalacetic transaminase in rat liver cell. J Cell Bioi 39:716-724, 1968 30. Lee SH, Torack RM: A biochemical and histochemical study of glutamic oxalacetic transaminase activity of rat hepatic mitochondria fixed in situ and in vitro. J Cell Bioi 39:725-732, 1968 31. Kiessling KH, Tilander K: The effect of prolonged alcohol treatment on the respiration of the liver and brain mitochondria from male and female rats. Exp Cell Res 30:476-480, 1963 32. Wiener J, Loud AV, Kimberg DV, et al: A quantitative description of cortisone-induced alterations in the ultrastructure of rat liver parenchymal cells. J Cell Bioi 37:47-61, 1968 33. Rubin E, Bacchin P, Gang H, et al: Induction and inhibition of hepatic microsomal and mitochondrial enzymes by ethanol. Lab Invest 22: 569-580, 1970 34. Lieber CS, DeCarli LM: Hepatic microsomal ethanol-oxidizing system: In vitro characteristics and adaptive properties in vivo. J Bioi Chern 245:2505-2512, 1970 35. Arias 1M, Doyle D, Schimke RT: Studies on the synthesis and degradation of proteins of the endoplasmic reticulum of rat liver. J Bioi Chern 244:3303-3315, 1969 36. Stenger RJ: Organelle pathology of the liver. The endoplasmic reticulum. Gastroenterology 58: 554-574, 1970 37. Greim H: Synthesisteigerung und abbauhemmung bei der vermehrung der mikrosomalen sytochrome p-450 und b-5 durch phenobarbital. Naunyn Schmiedebergs Arch Pharmakol 266: 260-275, 1970 38. Poole B, Leighton F, De Duve C: The synthesis and turnover of rat liver peroxisomes. II. Turnover of peroxisome proteins. J Cell Bioi 41: 536- 546, 1969 39. Isselbacher KJ, Carter EA: Ethanol oxidation by liver microsomes: Evidence against a separate and distinct enzyme system. Biochem Biophys Res Commun 39:530-537, 1970 40. Staubli W, Hess R, Weibel ER: Correlated morphometric and biochemical studies on the liver cell. II. Effects of phenobarbital on rat hepatocytes. J Cell Bioi 42:92-112, 1969 41. Koff RS, Carter EA, Lui S, et al: Prevention of the ethanol-induced fatty liver in the rat by phenobarbital. Gastroenterology 59:50-61, 1970
GASTROENTEROLOGY
Printed in U.S.A.
Copyright © 1972 by The Williams & Wilkins Co.
A QUANTITATIVE MORPHOLOGICAL ANALYSIS OF ETHANOL EFFECT UPON RAT LIVER WILLIAM
O.
AND HAROLD
DOBBINS.
J.
III,
M.D., EMORY
L.
ROLLINS, SUSAN
G.
BROOKS,
FALLON, M.D.
Departments of Medicine, George Washington University Medical Center, Washington, D. C., and University of North Carolina School of Medicine, Chapel Hill, North Carolina and from Gastrointestinal Research Laboratory, Veterans Administration Hospital, Washington, D. C.
Ethanol ingestion causes morphological changes in the livers of man and animals. This report describes a quantitative morphometric analysis of ethanol effects upon rat liver and a comparison with some biochemical determinations. Twenty-four rats were divided into three pair-fed groups: chow and 25% ethanol in drinking water; chow and an amount of sucrose isocaloric to the ethanol; and 25% ethanol in water and chow supplemented with 2% choline. After 35 days, a portion of liver was prepared for electron microscopy by standard techniques. Liver was analyzed for total phospholipid, triglyceride, lecithin, nitroreductase activity, and aniline hydroxylase activity. Twenty-five electron micrographs were obtained randomly from each specimen. Micrographs were analyzed by the lineal analysis method for cell and nuclear dimensions, and fractional volume of cytoplasmic components including mitochondria, peroxisomes, lysosomes, lipid bodies, and glycogen. Surface area of the mitochondrial envelope and the endoplasmic reticulum membrane and the approximate volume of mitochondria and peroxisomes were determined. Subjective analysis of electron micrographs failed to distinguish clearly the controls from the ethanol-treated rats. However, lineal analysis with statistical evaluation showed increased hepatic cell size (P < 0.001) and total volume of mitochondria, peroxisomes, lysosomes, and lipid bodies in the rats fed 25% ethanol. All of these changes were partially reversed by choline supplementation. The surface area of both smooth and rough endoplasmic reticulum membrane was reduced 50% in the ethanol-treated animals. This change was partially reversed by choline. Ethanol-induced increases in total liver phospholipid and lecithin were largely reversed by choline. Ethanol increased triglyceride content 2-fold and this was partially reversed by choline. Aniline hydroxylase increased 2-fold in the ethanol-treated rats when compared with control rats and 3-fold in the rats fed ethanol plus choline. Received June 14, 1971. Accepted January 12, 1972. Presented in part to the American Society for the Study of Liver Diseases, Chicago, Illinois, November 3, 1970. Address reprint requests to: Dr. William O. Dobbins, III, 2150 Pennsylvania Avenue, N.W., Washington, D.C. 20037.
This work was supported by United States Public Health Service Grants AM-13725 and AM-09000, and by Part I funds from the Veterans Administration. Standard deviations and errors were calculated in part by Mr. Emanuel Lerner of the Eastern Research Support Center, Veterans Administration Hospital, West Haven, Connecticut.
1020
May 1972
LIVER PHYSIOLOGY AND DISEASE
Ethanol, either by a direct toxic effect on the liver cell or in combination with an associated nutritional imbalance, may result in fatty infiltration of the liver l - 14 and an increase in phospholipid content_ IS, 16 Choline has been reported to partially reverse or prevent many of the biochemical changes induced by ethano!.I 4 , IS, 17 Ethanol also has been reported to produce marked mitochondrial changes in either an Inhepatocytes 2 - 10 , 18, 19; crease 4 , 6, 9, II , 18, 19 or a decrease 12, 13 in the amount of endoplasmic reticulum; focal cytoplasmic degradation with an increase in the number of lysosomes 2 - 14 ; an increased number of peroxisomes 11; and increased glycogen content of liver cells. 4 Most of the morphological changes described are not specific for ethanol and depend on subjective analysis. However, subjective review of morphology in this study failed to distinguish with certainty all livers of control animals from livers of experimental animals given ethanol with or without choline supplementation. Therefore, data were obtained by a quantitative morphometric analysis of the effects of ethanol upon rat liver. Hepatic lipid content and drug-metabolizing enzyme systems were measured concurrently to facilitate comparisons with previous studies of ethanol-induced hepatic changes and to provide preliminary data on the correlation of structural and biochemical alterations.
Material and Methods Male Sprague-Dawley rats weighing 150 to 200 g were caged individually. Animals were fed Purina laboratory chow and water or 25% ethanol in water (v/v) on the following schedule: animals 1 through 9 were given chow and 25% ethanol; animals 11 through 18 received chow and 25% ethanol in the same amounts as the first group by pair feeding, but the chow was supplemented with 2% choline by weight; animals 21 through 30 were pair-fed an identical amount of chow, plus an amount of sucrose isocaloric to the 25% ethanol taken by the first group. The last group had unlimited access to water. One rat in the first group and 2 in the second died during the 1st week of the experiment. Thereafter, rat 30 was pair-fed with rat 9. The total caloric intake was con-
1021
stant for control and experimental animals, Mean weight gain was 3.1 g per day and mean caloric intake was 74.8 per day. The mean proportions of total calories were: protein, 19%; fat, 7.5%; mixed starches, 43.5%; and ethanol or sucrose, 30%. The rats were given constant access to food and were fed for 35 days. Rats were killed in groups of three pair-fed animals between 8 and 10 AM. Food, ethanol, and water were removed at 7 AM the same day. The animals were anesthetized with ether, the abdominal cavity was opened, and the rat exsanguinated from the aorta. A small fragment of the free edge of the right lobe of the liver was frozen immediately in liquid N 2 for lipid determinations and the remaining liver was then removed for preparation of cell fractions. 15 Each liver fragment was placed in ice-cold 1 % osmium tetroxide in 0.067 cacodylate buffer at pH 7.4. After 5-min fixation, the fragments were minced into 0.5-mm cubes and returned to the fixative for a total of 1 to 2 hr. The tissues were dehydrated in alcohol and embedded in epoxy resin. Sections (1.0- Jl thick) were obtained of each liver specimen and stained with toluidine blue. Portal and central vein areas were identified and the liver specimen was trimmed to appropriate size for thin sectioning. Thin sections were prepared from centrolobular fragments and an additional thick section was made after completion of thin sectioning. In order to obtain random electron microscopic photographs of each specimen, the thick section was examined by light microscopy and diagrammed on paper. Hepatocytes at a depth of 3 cells from portal and central areas (20% of hepatic area) were avoided. Five areas of midzonal cells (80% of hepatic area) were randomly marked. These areas were identified at the electron microscope and five photographs of each area were obtained at magnifications of 5,000 to 20,000 diameters. No condition of cytoplasmic or nuclear appearance was imposed in selection for photography. Only those cells showing technical artifacts such as knife scratches or heavy stain contamination were avoided. Repeated calibration of the AEI-EM6B electron microscope during this study showed that the microscope magnification was stable within a range of 2%, a range compatible with accurate quantitative measurements. Twenty-five electron micrographs were obtained from each of the 24 specimens for a total of 600 micrographs. Specimens were satisfactory for morphological analysis from 8 ani-
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Vol. 62, No . 5
LIVER PHYSIOLOGY AND DISEASE
Measurements of cellular components were mals fed chow plus ethanol, from 6 animals fed chow and 2% choline plus ethanol, and performed with the use of 8- by lO-inch enfrom 10 animals fed chow plus an isocaloric larged prints of the micrographs. In order to avoid bias, the micrographs for lineal analyamount of sucrose. The specimens were coded and analyzed sis of proper magnification were selected ranwithout reference to grouping. Light micro- domly from the micrographs of each specimen. scopic sections were graded randomly for the A square grid of white lines was superimposed presence of fat droplets using a scale of 1 + to on each print by lengths of 7-mil wire placed 3 +. Portions of all thick sections were photo- on a frame at I-inch intervals. All enlargements graphed at an original magnification of x 270 were done over a few days' time during which and enlarged photographically to x 540. Cell no change in enlargement magnification could and nuclear dimensions are shown in table 1. be demonstrated. A lineal analysis of hepatoHepatocytes tended to be oblong in shape and cyte cytoplasm was performed using prints hence were measured in two diameters. Nu- with a magnification of x 12,500. This allowed clear diameters were determined for each of the statistically accurate calculation of the perthree groups by direct measurement of the centage of cytoplasmic volume for various organelle fractions and the total area of cytodiameter of all easily identified nuclei. The electron photomicrographs of each plasm in square microns (table 2). Prints with a magnification of x 25,000 were specimen were evaluated on the basis of overall subjective morphological impression with- used to calculate membrane areas per square out reference to treatment group. This random micron of cytoplasm. Intersections of the and entirely subjective evaluation failed to grid lines with mitochondrial envelope, smooth separate clearly livers of control animals from endoplasmic reticulum, and rough endoplasmic the livers of experimental animals given ethanol. reticulum were counted (table 3). These data Therefore, a partial quantitative analysis of were not analyzed statistically. However, the the electron photomicrographs was performed large number of intersects counted gives vausing modifications of the methods of Weibel lidity to the results. Golgi membranes were and Loud. 20 • 2 2 counted as smooth endoplasmic reticulum. 1. Cell and nuclear dimensions"
TABLE
Sucrose control
No. of cells measured .... .. .. . Average cell length (Il) .. , , ..... ... . . Average cell width (Il) Average nuclear diameter (Il) ...
Et hanol
227 24.2 ± 4 . 1 16.3 ± 3 . 1 7.6±1.1
268 26 .6 ± 4.6 17 .6 ± 3 .0 8 .0 ± 1.3
P
+ choline
Ethanol
p
< 0.001 < 0 .001 < 0.001
167 26.5 ± 3 .9 16.7 ± 3.0 7.6 ± 1.1
<0.001 NS NS
"The values represent the arithmetic mean ± SEM. P values were calculated usmg Student's t-test and refer to comparison of the two ethanol groups to the controls. Length and width of cells of ethanol-treated animals were not significantly different (NS) from those of the ethanol plus choline-treated animals with P values> 0.1
Volume fraction of cy toplasmic components·
TABLE 2.
Sucrose control
Number of micrographs . . . . ... . .. .... . Total area of cytoplasm (Il 2) . . . . . .. . · · .0· · ' . .. Cytoplasmic volume (% ± SEM) Mitochondria .. . .. . . . .. ..... . Peroxisomes . . .. . . . . . ... . . Lysosomes .... . .. . ... . . . ....... . .... . . .. Lipid bodies . . ... . . . . .. ... .... .. .... .. . . .. Glycogen . . . . . . .. . . . . . . ... Other .. . . ...... ..... . . . . ...... . . . . ... 0
••
•
•••
28 5994
Ethanol
27 7056
22 .6 ± 1.1 26 .0 1.6 ± 0.44 1.8 0.3 ± 0.45 0.7 1.0± 1.5 1.4 20 . 3 ± 1.4 18 . 7 54 .2 ± 1.9 51.4 " The method of measurmg, calculatmg, and expressmg volume fractIOns Methods." bNot statistically significant. '
••
•
•
0
•
••
0
0
••
•
••
••
•
••
•
•
••
•
•
••
•
Ethanol + choline
17 3663 ± 1.4 ± 0.44 ± 0.35 ± 1.7 ± 1.9 ± 2. 2 IS
23 . 6 1.5 0.4 0 .3 18 .7 55 . 5
± 1.2 ± 0.41 ± 0.46 ± 5.9 b ± 1.5 ± 2.0
detailed m "Material and
May 1972
1023
LIVER PHYSIOLOGY AND DISEASE
These same prints at magnifications of 25,000 were used to calculate dimensions and approximate volumes of mitochondria and peroxisomes (table 4). The number of mitochondria, peroxisomes, and lysosomes per unit area was determined (table 5). Lysosomal dimensions were not measured because of their paucity. Calculations were performed as follows: u = volume of individual organelle V = total volume of hepatocyte cytoplasm Vum = relative volumes of mitochondria Vup = relative volumes of peroxisomes Vul = relative volumes of lysosomes Vuu = relative volumes of lipid droplets Vug relative volumes of glycogen Vuo = relative volumes of all other cytoplasmic components Grid lines over m, p, I, Ii, g, and 0 were measured using a Keuffel and Esser scale calibrated in 0.5-mm intervals. Grid lines over nuclei and extracellular space were not measured. TABLE
Total number of points (length in millimeters) over cytoplasm is expressed as Pt. Pt
=
Pm + Pp + Pi + Pli + Pg + Po
Volume fractions of each component are thus calculated by the formula Vum
=
Pm/Pt and is expressed as
J.l.2
Measurements of surface area are expressed as surface density, Sui. The area of membrane included in a unit test volume is termed the surface density and is given by the formula Sui
2Nlior [2(c)]/[d]
=
where Nli is the number of membrane intersections (c) per unit length of sampling line (d). Thus Sui = [2(c) lid and is expressed as J.I. 2/J.I. 3.
For mitochondria, the expression relates number of intersections of mitochondrial envelope to the corresponding length of line
3. Surface areas of mitochondrial envelope and endoplasmic reticulum membrane Cytoplasm analyzed
Mitochondrial envelope/ IJ. 3 mitochondria
IJ. '1
1J'1
~
Sucrose control ······. 0 · · . Ethanol + choline .. .. .. . .. Ethanol . . .. . . . . . . . . . . .
5395 2894 3794
6 .08 (3703)b
5.16 (1764) 5.60 (2752)
Endoplasmic reticulumo /
11 3
cytoplasm
SER
RER
2 .34 (6286) 1.48 (2145) l.38 (2633)
1.54 (4154) l.00 (1456) 0.76 (1459)
" SER, smooth endoplasmic reticulum; RER, rough endoplasmic reticulum. b The number of intersections counted is in parentheses. The method of calculating surface areas of these membranes is outlined in the text. TABLE 4.
Organelle measurements Mitochondria
Length
Width
~
~
l.21 l.26 l.34
0 . 75 0.77 0 . 74
Sucrose control .... .. .. , . , .. Ethanol + choline . . .... Ethanol . . . . . . . . ... . . .
Peroxisomes Volume ~'
0.53 (1470)" 0.59 (972) 0.58 (1545)
Diameter ~
0 .59 (300) 0.66 (164) 0 .53 (382)
" Number of organelles measured is listed in parentheses. TABLE
5. Organelle counts per square micron of cytoplasm Cytoplasmic length
Mitochondria
Peroxisomes
Lysosomes
0.21 (1l25)" 0 . 17 (502) 0.20 (749)
0.036 (193) 0.031 (89) 0.038 (144)
0.010 (52) 0.021 (62) 0 .012 (46)
~
Sucrose control . .. . . . . . Ethanol + choline . . . . . ... Ethanol " . . .. .. .. ... . . ..
5395 2894 3794
" Number of each organelle counted is listed in parentheses.
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Vol. 62, No.5
LIVER PHYSIOLOGY AND DISEASE
traversing the mitochondria (d) alone. For endoplasmic reticulum (ER), the expression relates the measure of ER per unit volume of cytoplasm with (d), the length of sampling line traversing the cytoplasm. Mitochondrial volume was roughly estimated assuming that mitochondria are closed right circular cylinders of diameter D and length L:
V
=
(7r D3)/(4
d where
f
=
D/L
The number of particles (mitochondria, peroxisomes, lysosomes) was determined by counting these particles and expressing this count as a ratio to the cytoplasmic length traversed in the same micrographs. Sampling. In order to obtain statistical validity in the description of tissues, representative samples of these tissues must be obtained. In this study, tissue samples were obtained from a similar portion of the liver of each animal. The method of obtaining random micrographs of the tissues and consistent instrumental magnification and photogmphic magnification have been described. Observations were confined to midzonal liver cells because these cells are structurally homogeneous in normal rats, while significant differences are found in cells immediately surrounding central veins. 21 Cells immediately adjacent to portal areas show only minor variations from mid zonal cells. 21 The validity of using linear measurements to compute relative volumes has been widely applied by geologists who have used thin sections of rock in computing relative volumes of components of rock.20-22 The validity of this method has been demonstrated for biological applications. 20, 21 The accuracy of the quantitative description of a tissue is improved by increasing the number of micrographs analyzed. The standard deviation of a random sample counted, such as the length of mitochondrial profiles traversed by a grid line, is given by the square root of TABLE
mg/g liver
Sucrose control .. . .... . , Ethanol ...... .... . . Ethanol + 2% choline ' . ,
'"
'"
Results Light microscopy. Control rat livers were found by light microscopic grading to have o to 1+ fat with a single exception which was 2+. Rats fed ethanol plus 2% choline in chow had 0 to 1+ liver fat. Five of the livers from ethanol-treated rats had greater than 1 + fat. Lineal analysis of the electron micrographs (table 2) supported these light microscopic findings. Quantitative triglyceride determinations for these groups are shown in table 6 and conform roughly to the morphological findings.
6. Quantitative triglyceride determinations·
Triglyceride
Diet
the total count collected, Ci. For optimum efficiency the sample size, Ci, should be adjusted so that Ci is approximately equal to the standard deviation among the values of Ci obtained from a number of different micrographs from the sample analyzed. A larger sample is necessary when the volume fraction of a component is small, when the component varies in size, or when the component is irregularly distributed. This condition was generally utilized in this study. Mean values for tissue characteristics have been calculated from the combined raw data of all micrographs in each of the three groups. Standard errors reported are equal to the standard deviation among the corresponding values calculated independently for each micrograph. Other methods. Cell fractions were prepared and hepatic phospholipid and lecithin content measured as previously described. 15 Hepatic triglyceride was determined by a modification of the method of Kessler and Lederer 23 for serum triglyceride. Aniline hydroxylase activity was measured in liver homogenates" and nitroreductase activity in microsomal preparations. 25 Protein was estimated by the method of Lowry et a1. 26
2.93 5.65 4.11
± ± ±
0.33 0 . 74 0.42
Phospholipid ~moles/g
20.41 28.18 23.30
± ± ±
liver
1.18 0.89 0.74
Lecithin ~moles/g
11.03 16.17 12.93
± ± ±
liver
0.59 0.56 1.19
Microsomal protein
mg/g liver
25.68 23.48 24.88
± ± ±
0.72 0.96 0.58
Values are means ± SEM for each group of rats. Wet weight of liver was used for reference. Differences in triglyceride, phospholipid, and lecithin content between the ethanol and the control groups are significant (P < 0.05). The differences in triglyceride, phospholipid, and lecithin content between control and ethanol plus 2% choline groups are not significant. There is no significant difference in the recovery of microsomal protein from the three groups. a
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LIVER PHYSIOLOGY AND DISEASE
Average cell length and width varied very little among the three groups (table 1). Hepatocytes from both ethanol-treated groups were slightly larger than hepatocytes of the control rats. The nuclei of hepatocytes of the ethanol-treated rats were slightly larger than nuclei of hepatocytes of the control rats and of the ethanol plus choline-treated rats. Electron microscopy. Subjective evaluation of electron photomicrographs was done without knowledge of treatment group. The electron photomicrographs obtained from the liver of 1 animal in each of the ethanol groups were considered to be normal in appearance. Electron micrographs from control rats were found to vary from normal in appearance to abnormal with many of the mitochondrial changes characteristic of ethanol-treated livers. The prominence of mitochondrial changes in both ethanol groups was readily apparent and resembled those described previously.2.14. 18. 19 No regular discernible difference in the smooth and rough endoplasmic reticulum structure could be detected in the three groups. Lineal analysis of the electron micrographs was undertaken (table 2, figs. 1 to 4) to permit statistical evaluation of results. Our findings in normal rat hepatocytes are in general agreement with those published by Loud.21 The lineal analysis confirmed (table 2) that the mitochondrial volume did increase in the livers of ethanoltreated rats and that the addition of 2% choline to the diet resulted in mitochondrial volume intermediate between that of the ethanol group and the pair-fed controls. Lysosomal volume doubled in the livers of the ethanol-treated group but increased only slightly in the livers of rats given ethanol plus choline. Peroxisome volume increased slightly in the ethanol group and diminished very slightly in the ethanol plus choline group. The volume of lipid droplets increased approximately 50% in the ethanol group. There was an insufficient number of lipid droplets in the sample analyzed from the ethanol-plus choline-treated group to permit a statistically valid comparison with the other
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groups. Glycogen volume decreased slightly in both treated groups. As often occurs when cacodylate buffer is utilized, glycogen varied in staining qualities. This presented no problem, however, in its identification and quantitation. Surface areas of the mitochondrial envelope and endoplasmic reticulum membrane showed major differences among the three groups (table 3). Mitochondrial envelope surface area decreased in both ethanol groups reflecting the increased mitochondrial volume (table 4). Mitochondria in the livers of rats treated with ethanol plus choline had a slightly greater increase in individual volume than did mitochondria of the livers of rats treated with ethanol alone. However, the total volume of mitochondria of the ethanol plus choline group was less than that of the ethanol-treated group as shown by the lower count of mitochondria per square micron of cytoplasm in the ethanol plus choline group (table 5) . Hence, there were slightly fewer mitochondria in both ethanoltreated groups but these mitochondria were larger and had a greater individual and total volume than did mitochondria from control livers. Peroxisomes increased in diameter in the group given ethanol plus choline but decreased in diameter in the ethanol group (table 4). In contrast, there were fewer peroxisomes per square micron of cytoplasm in the ethanol plus choline group and increased numbers in the ethanol-treated group (table 5). Lysosomes per square micron of cytoplasm increased in number and volume in both ethanol-treated groups (tables 2 and 5). There was a moderate decrease in surface area of both smooth endoplasmic reticulum (SER) and rough endoplasmic reticulum (RER) in both ethanol-treated groups, with the ethanol plus choline group showing a smaller decrease in surface area than the ethanol-treated group (table 3). The Golgi apparatus did not appear to vary significantly in the three groups, but this observation was not quantitated. Lipids and other measurements. A nearly 2-fold increase in hepatic triglyc-
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FIG. 1. Electron micrography of a portion of an hepatocyte of sucrose control rat liver. Micrographs at this magnification were utilized for the lineal analysis. It illustrates the average appearance of normal hepatocytes showing slightly elongated mitochondria (M), modest deposits of glycogen (Gly) , occasionallysosomes (Lys), more frequent peroxisomes (1'), rough endoplasmic reticulum (RER) , smooth endoplasmic reticulum (SER) , lipid droplets (Lip), and a bile ductule (B) [double stained (uranyl acetate and lead citrat e) x 12,500j.
eride content was observed in the rats fed ethanol (table 6) . A lesser increase was noted in the rats given 2% choline. The rise in hepatic phospholipid and lecithin
content in this experiment was similar to that previously reported. 1 5 There was no difference in total hepatic protein content in the three groups. However, a small de-
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crease in the recovery of microsomal protein per gram of liver was noted in the rats given ethanol. Aniline hydroxylase activity was increased 2-fold in rats fed ethanol and
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nearly 3-fold when choline was given in addition to ethanol (table 7). No change was observed in microsomal nitroreductase activity in rats given ethanol. At the end of the experiment the mean
FIG. 2. Electron micrograph portions of two hepatocytes of an ethanol-treated rat. The mitochondria (M) are large and often encircle organelles and portions of cytoplasm (arrows). Mitochondrial cristae are often absent and atypical in shape. Glycogen (Gly) is present as a large conglomerate as well as being scattered diffusely throughout the cytoplasm. The rough endoplasmic reticulum (RER) might be described in one area as "degranulated." The peroxisomes (Pi possess prominent nucleoids in this particular micrograph. Lysosomes (Lys), Golgi apparatus (G), and a bile ductule are normal in appearance (double stained, x 12,5(0).
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FIG. 3. Electron micrograph of portion of an hepatocyte from an ethanol-treated rat. Micrographs at this magnification were utilized for counting mitochondrial and endoplasmic reticulum membrane intersections. Findings illustrated in this micrograph are similar to those described in figure 2. The smooth endoplasmic reticulum (SER) appears slightly dilated while the rough endoplasmic reticulum (RER) is normal in appearance. Mitochondria (M). Peroxisomes (P). Golgi apparatus (G). Portion of nucleus (N) (double stained, x 25,000).
serum level of ethanol was 153 mg per 100 ml and varied from a low of 33 to a high of 270 mg per 100 ml.
Discussion Observations were made of "midzonal" (centrolobular) liver cells, excluding only
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those cells within a depth of 3 cells from portal and central areas. This midzonal area clearly reflects 80% of hepatocytes. 21 Further, Iseri et al. 4 showed that, in normal rats, the RER of hepatocytes im-
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mediately adjacent to the central vein is often vesiculated and may be confused with alcohol-induced changes in midzonal hepatocytes. 4 Thus we chose to quantitate alcohol-induced changes m midzonal
FIG. 4. Electron micrograph of portion of two hepatocytes from an ethanol-plus choline-treated rat. The mitochondria (M) are intermediate in appearance between those of sucrose control and ethanol-treated rats. Glycogen (Gly) is prominent while the smooth endoplasmic reticulum (SER) and rough endoplasmic reticulum (RER) are normal in appearance. Peroxisomes (P), Golgi apparatus (G), lysosomes (Lys), nucleus (N), and a bile ductule (B) appear unaltered (double stained, x 12,5(0).
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LIVER PHYSIOLOGY AND DISEASE TABLE 7 .
Enzyme determinations'
Diet
Sucrose control .. Ethanol . . . Ethanol + 2% choline
Aniline hydroxylase
reductase
nmoles/mg protein
nmoles/mg protein
24 .76 ± 1.84 53 .12 ± 12.46
56.07 ± 8 .37 51.67 ± 5.82
71 .65 ± 19.50
50. 30 ± 8.57
Nit ro·
• Values are means ± SEM for 10 rats in each group. Aniline hydroxylase is measured in micromoles of paraaminophenol formed per milligram of homogenate protein per 30 min. The nitroreductase is measured as micro moles of paraaminobenzoic acid formed per milligram of microsomal protein per 30 min.
hepatocytes, excluding 20% of liver area. This may account for some discrepancies in our study and other studies cited in this report. The effect of long term ethanol administration on the ultrastructure and the lipid content of liver cells of a variety of animal species has been described. 2, 4·9, 15 Acute administration of ethanol has been reported to produce similar biochemical and morphological effects, 3, 18,19, 27 Prominent mitochondrial changes in hepatocytes of ethanol-treated animals were emphasized by Hartroft's group 2, 11-14 and have subsequently been described by other investigators, 3-10, 18, 19 French 28 studied membrane structure and function of mitochondrial fractions isolated from rat liver after chronic ingestion of ethanol and observed that the outer membrane of mitochondria in livers of ethanol-treated rats also has been noted in other electron microscopic studies,' We have confirmed these morphological changes in mitochondria of livers of rats after chronic ethanol feeding under the dietary conditions described, Porta and Gomez-Dumm" used a planimetric method to show that individual liver mitochondria of ethanol-treated rats had a mean area 2-fold greater than that of control rats while the total number of mitochondria per unit area of cytoplasm was reduced in the ethanol-treated animals, This study shows similar changes in ethanol-fed rats with mitochondrial volume increasing 15%, a change partially
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prevented by supplementation of the diet with 2% choline, In human alcoholic hepatitis, the organelle change which reportedly correlates best with the clinical condition is mitochondrial alteration, 18 Therefore, structural and functional changes in the mitochondria may have clinical consequences, 27, 29, 30 Isolated hepatic mitochondria of rat livers obtained after prolonged ethanol ingestion have decreased oxidative properties for pyruvate and fatty acids, 31 an effect which may also be attributed to ethanol-induced mitochondrial inJUry.
Iseri et al. 4 noted that, in rats fed ethanol for 6 to 27 days, centrolobular hepatocytes were characterized by vesiculation and prominence of both smooth SER and, to a lesser degree, of RER with some detachment of ribosomes from the RER. Vesiculation and apparent increase in the amount of SER with degranulation of RER and decreased RER was described later by this same group under a variety of conditions. 6 , 9, 18, 19 Hartroft's group"-14 has noted similar changes in the RER and SER although they described increased SER in only one study. 11 There is general agreement that the RER is reduced in amount,2- 14, 18, 19 a finding quantitatively confirmed in this report. Under these experimental conditions, a decrease in amount of both the SER and RER was observed in livers of ethanoltreated rats. Addition of choline to the diets resulted in partial prevention of this change, especially in the RER. Degranulation of the RER was not documented in the ethanol group by double blind subjective assay of the photomicrograph. However, no attempt was made to quantitate the possible degranulation of the RER. The discrepancy between our SER findings and those of other reports require explanation. Several considerations may be important in understanding these differences. Vesiculation of SER has been subjectively interpreted as an increase or proliferation of SER in studies of cortisone effect on liver. However, quantitative estimates of SER have not supported this subjective impression and have shown
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decreased amounts of SER. 32 Therefore, the quantitative analysis of SER content as performed here may be a better reflection than subjective impressions of SER surface area under these experimental conditions. Possibly the most convincing published electron photomicrograph of increased SER in livers of ethanol-fed rats is that of Rubin et al. 9 They report this change as most prominent in rats fed ethanol and a diet deficient in choline and reduced in casein, methionine, and cystine. 9 The SER was only "moderately hypertrophied" in the nondeficient, ethanolfed rats and the surface area was not quantified morphologically.9 It is possible that quantitative analysis under these conditions will not support the initial impression of these authors. However, Rubin et al. 33 and Lieber and DeCarli 34 have observed that ethanol administration in combination with a liquid, relatively high fat diet results in increased microsomal protein in rats. This may represent the biochemical counterpart of proliferation of hepatic SER. 33. 34 However, no increase in microsomal protein content was observed in this study. The differences in both electron microscopic and biochemical findings between the data of Rubin et al. 9 and those reported here might be related to significant differences in experimental design. Although total ethanol levels werecomparabIe, the other dietary conditions were markedly different. The relatively low fat intake and use of sucrose rather than Dextri-Maltose as carbohydrate source in this study may account for quantitative differences in the two experiments and the discrepancy in SER results. If this is the case, it strongly implies that the long term effects of ethanol ingestion on the liver are significantly influenced by other dietary factors as suggested by previous studies. 12. 15 . 17 The rise observed in aniline hydroxylase specific activity was not accompanied by an increased amount of SER on morphological analysis, demonstrating that these may be independent phenomena. Changes in endoplasmic reticulum proteins may be highly selective as shown by the study of
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Arias et al. 35 in the case of phenobarbital. It is likely that ethanol also causes increases in a limited number of specific proteins of the endoplasmic reticulum but without changes in the total amount of endoplasmic reticulum. Disorganization of the RER is a common response of hepatocytes to injury and is not a specific reaction to chemical agents alone. 36 For example, reduced amounts of RER have been found in rapidly proliferating cells, in association with an increased number of free ribosomes. 3 6 This may occur in ethanol-treated hepatocytes also, ·although quantitative documentation of increased free ribosomes is not available. Subjectively, we did not detect an increase in free ribosomes. The reduction in total microsomal protein content in rats fed ethanol was too small and did not conform to the decrease in RER and SER determined by electron microscopy. Several explanations for this discrepancy may be offered. The amount endoplasmic reticulum recovered by the centrifugational techniques used represents only a portion of the total endoplasmic reticulum as estimated by P-450 determinations. 37 Therefore, relatively small changes in the portion of endoplasmic reticulum recovered by centrifugation (approximately 30 to 50%) would produce significant variations in the microsomal protein levels obtained. Alternatively, differences in endoplasmic reticulum width, protein concentration, or contamination of microsomal pellets with protein derived from other particulate fractions could explain the discrepancy. Moreover, the content of the vesicular structures seen in electron microscopic study of these pellets is unknown and variations in size and content of these vesicles could markedly affect protein estimates of the pellets recovered. Further studies of this problem are in progress. The increase in hepatic phospholipid content of ethanol-treated rats cannot be explained by the changes in the endoplasmic reticulum but may relate to the increase in mitochondrial volume. Focal cytoplasmic degradation in the form of increased number of autophagic
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vacuoles (lysosomes) has been commonly noted in livers of ethanol-fed animals 2 - 14 , 19 and was confirmed here. Lysosomal volume increased from 0.3% in normals to 0.7% in ethanol-fed rats but only to 0.4% in the rats fed ethanol plus choline (table 2) . Lysosomes are increased in many forms of hepatic injury,18 probably indicating increased turnover of damaged cellular components. Increased number of peroxisomes have been noted in livers of ethanoltreated animals. 11 Peroxisome volume of ethanol-treated rats was 1.8% compared to 1.6% of control rats (table 2). Peroxisome volume fraction decreased to 1.5% in ethanol plus choline-treated rats. The significance of the changes in peroxisome size and volume is not known but it is tempting to speculate that they relate to the known catalase content of peroxisomes. 38 Catalase may potentiate ethanol metabolism in vitro. 39 Recently it has been suggested that ethanol may be metabolized, in part, by a microsomal pathway. 33, 34 Many drugs also are metabolized by the microsomal mixed function oxidase system. In man and in rats, the blood clearance of drugs metabolized in hepatic microsomes is slowed by concomitant ethanol administration despite increased activity of some drug detoxification enzymes. 33 , 34 This effect may reflect either competitive inhibition of ethanol and certain drugs for microsomal enzyme(s) or a toxic effect of ethanol upon these systems. The fact that 3 days of phenobarbital administration in rats inhibits ethanol-induced fatty liver may correlate with the increased amount of SER induced by phenobarbital. 40, 41 Choline also increases the amount of RER in the rat given ethanol and partially prevents some of the associated alterations in lipid content, suggesting the possibility of a mechanism similar to that of phenobarbital. This study supports previous evidence concerning the effect of large amounts of dietary choline upon ethanol-induced fatty liver, phospholipid metabolism, and structural changes. 17 Lieber and DeCarli 17 showed that choline protects against the fatty liver produced by prolonged ethanol
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intake. Porta et al. 2 , 14 and Takada et al. 7 also have demonstrated protection against fatty liver by choline supplementation. REFERENCES 1. Scheig R: Effects of ethanol on the liver. Am J Clin Nutr 23:467-473, 1970 2. Porta EA, Hartroft WS, de la Iglesia FA: Hepatic changes associated with chronic alcoholism in rats. Lab Invest 14:1437-1455, 1965 3. Ashworth CT, Wrightsman F, Cooper B, et al: Cellular aspects of ethanol-induced fatty liver: a correlated ultrastructural and chemical study. J Lipid Res 6:258-268, 1965 4. Iseri OA, Lieber CS, Gottlieb LS: The ultrastructure of fatty liver induced by prolonged ethanol ingestion. Am J Pathol 48:535-555, 1966 5. Thorpe MEC, Shorey CO: Long-term alcohol administration. Its effects on the ultrastructure and lipid content of the rat liver cell. Am J Pathol 48:557-577, 1966 6. Lane BP, Lieber CS: Ultrastructural alterations in human hepatocytes following ingestion of ethanol with adequate diets. Am J Pathol 49: 593-603, 1966 7. Takada A, Porta EA, Hartroft WS: Correlation of structural and functional recovery from cirrhosis in rats treated with lipotropic diets. Am J Pathol 49:841- 869, 1966 8. Lieber CS, Spritz N: Effects of prolonged ethanol intake in man: role of dietary, adipose, and endogenously synthesized fatty acids in the pathogenesis of the alcoholic fatty liver. J Clin Invest 45:1400-1410, 1966 9. Rubin E, Hutterer F, Lieber CS: Ethanol increases hepatic smooth endoplasmic reticulum and drug-metabolizing enzymes. Science 159: 1469-1470, 1968 10. Lieber CS, Rubin E : Alcoholic fatty liver in man on a high protein and low fat diet. Am J Med 44: 200-206, 1968 11. Porta EA, Gomez-Dumm CLA: A new experimental approach in the study of chronic alcoholism. I. Effects of high alcohol intake in rats fed a commercial laboratory diet. Lab Invest 18:352364,1968 12. Gomez-Dumm CLA, Porta EA, Hartroft WS, et al : A new experimental approach in the study of chronic alcoholism. II. Effects of high alcohol intake in rats fed diets of various adequacies. Lab Invest 18:365-378, 1968 13. Koch OR, Porta EA, Hartroft WS: A new experimental approach in the study of chronic alcoholism. nI. Role of alcohol versus sucrose or fat-derived calories in hepatic damage. Lab Invest 18:379-386, 1968 14. Porta EA, Koch OR, Hartroft WS: A new experimental approach in the study of chronic
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29. Lee SH, Torack RM: Electron microscope studies of glutamic oxalacetic transaminase in rat liver cell. J Cell Bioi 39:716-724, 1968 30. Lee SH, Torack RM: A biochemical and histochemical study of glutamic oxalacetic transaminase activity of rat hepatic mitochondria fixed in situ and in vitro. J Cell Bioi 39:725-732, 1968 31. Kiessling KH, Tilander K: The effect of prolonged alcohol treatment on the respiration of the liver and brain mitochondria from male and female rats. Exp Cell Res 30:476-480, 1963 32. Wiener J, Loud AV, Kimberg DV, et al: A quantitative description of cortisone-induced alterations in the ultrastructure of rat liver parenchymal cells. J Cell Bioi 37:47-61, 1968 33. Rubin E, Bacchin P, Gang H, et al: Induction and inhibition of hepatic microsomal and mitochondrial enzymes by ethanol. Lab Invest 22: 569-580, 1970 34. Lieber CS, DeCarli LM: Hepatic microsomal ethanol-oxidizing system: In vitro characteristics and adaptive properties in vivo. J Bioi Chern 245:2505-2512, 1970 35. Arias 1M, Doyle D, Schimke RT: Studies on the synthesis and degradation of proteins of the endoplasmic reticulum of rat liver. J Bioi Chern 244:3303-3315, 1969 36. Stenger RJ: Organelle pathology of the liver. The endoplasmic reticulum. Gastroenterology 58: 554-574, 1970 37. Greim H: Synthesisteigerung und abbauhemmung bei der vermehrung der mikrosomalen sytochrome p-450 und b-5 durch phenobarbital. Naunyn Schmiedebergs Arch Pharmakol 266: 260-275, 1970 38. Poole B, Leighton F, De Duve C: The synthesis and turnover of rat liver peroxisomes. II. Turnover of peroxisome proteins. J Cell Bioi 41: 536- 546, 1969 39. Isselbacher KJ, Carter EA: Ethanol oxidation by liver microsomes: Evidence against a separate and distinct enzyme system. Biochem Biophys Res Commun 39:530-537, 1970 40. Staubli W, Hess R, Weibel ER: Correlated morphometric and biochemical studies on the liver cell. II. Effects of phenobarbital on rat hepatocytes. J Cell Bioi 42:92-112, 1969 41. Koff RS, Carter EA, Lui S, et al: Prevention of the ethanol-induced fatty liver in the rat by phenobarbital. Gastroenterology 59:50-61, 1970