Morphometric study of human hepatic cell modifications induced by fenofibrate

Metabolism Clinical and Eqmimental VOL XXXVI, NO 3

MARCH 1987

Morphometric

Study of Human Hepatic Cell Modifications by Fenofibrate

P. Gariot. E. Barrat, P. Drouin, P. Genton,

J.P. Pointel,

B. Foliguet,

Induced

M. Kolopp, and G. Debry

We have studied liver biopsies obtained in 12 hyperlipoproteinemic (HLP) patients (type II, 6: type IV, 6) treated with diet and fenofibrate, and in 15 patients (type II, 11; type IV, 4) receiving diet only. Electron microscopy of liver biopsies and the morphometric analysis according to the method of Weibel and Rohr showed mitrochondrial changes in patients treated with fenofibrate, these changes depending on the type of hyperlipoproteinemia. In type II HLP, we found a decreased volume of normal mitochondria (fenofibrate, 125.72 + 17.04 x 10m3 cm3/cmS; diet only, 185.84 + 8.96 lo-‘, P < .05). In type IV HLP we found a decreased number of giant mitochondria (fenofibrate. 0.08 + 0.03 x 10” cm-3; diet only, 0.32 f 0.08 x IO” cm?, P < .05) and a decreased volume of altered mitochondria (fenofibrate, 6.00 + 1.44 x 10-j cm3/cm3; diet only, 13.61 + 1 .I 7 x 1 Om3,P c: .05). In contrast with the rodent studies, the present study shows no change in the number of volume of peroxisomes. o 1987 by Grune & Strattom Inc.

HE HEPATOCYTE plays an important part in the metabolism of lipids, being both the site of the synthesis of endogenous lipoproteins and the site of catabolism of fatty acids through beta-oxidation in mitochondria and peroxisomes.’ Few authors have studied the morphology of human hepatocytes in primary hyperlipoproteinemia (HLP),2A and the effect of lipid-lowering drugs on this target cell of the lipid metabolism. In rodents, lipid-lowering drugs such as clofibrate (ethyld-p-chlorophenoxyisobutyrate) and fenofibrate (Lipanthyl, Fournier Laboratories, Dijon, France) (isopropyl-(4-p-(chlorobenzoyl)-2 phenoxy-2 methyl) propionate) have been shown to produce hepatomegaly and alterations of hepatocytes. The latter include both hypertrophy and hyperplasia of hepatocytes, increased smooth endoplasmic reticulum (SER), decreased rough endoplasmic reticulum (RER), polymorphism of mitochondria, and proliferation of peroxisomes.’ Such proliferation is noteworthy because peroxisomes are a site of beta oxidation of fatty acids.6 In man at therapeutic levels of these drugs, comparable changes in mitochondria and both SER and RER have been reported, as well as liver steatosis. But neither proliferation of peroxisomes nor changes in the size of hepatocytes have been found.’ These studies’,’ report subjective evaluations of liver biopsies and have not used methods of cellular quantification in order to assess discrete changes in hepatocytes and organelles. Fenofibrate is a commonly prescribed lipid-lowering drug in Europe; we thus decided to study and quantify the effects of therapeutic levels of the drug on the hepatocyte, and to

T

Metabolism, Vol 36, No 3 (March),

1987:

pp 203-210

compare two groups of patients with HLP treated with a diet only and with diet + fenofibrate, respectively. MATERIALS

AND METHODS

Patients Twenty-seven patients (21 men, 6 women), aged 19 to 70 years (46 + 12, mean + SD) (Tables 1 and 2) were selected among patients with primary HLP in the Department of Metabolic Diseases of the University Hospital, Nancy, France. Secondary HLP due to hypothyroidism, diabetes, nephrotic syndrome, bile obstruction, pancreatitis, or dysglobulinemia have been excluded. Blood lipids were assessed in the following manner: preparative ultracentrifugation of lipoproteins was carried out according to the technique of HaveI;’ total blood and selective lipoprotein lipid levels were assessed according to the technique of Klose et aI9 for cholesterol and to the procedure reported by Wahlefeld,” on the Technicon Auto-analyzer for triacylglycerols. HLP was classified according to WHO criteria” as modified by Carlson and Ericsonn.12 The patients were divided into four groups according to the type of HLP and the treatment that was given: Group 1: Type II HLP treated with diet only, exclusive of drug

From the Service de Mkdecine G. Hbpiial Jeanne dilrc, Toul Cedex, France and Laboratoire d’Histologie, Embryologie et CytogMiique de I’lJniversitt de Nancy I, Vandoeuvre les Nancy. France. Address reprint requests to P. Gariot, MD. Service de MSdecine G. Hbpital Jeanne d’Arc, CHR Nancy, BP 303, 54201, Toul Cedex France. o 1987 by Crune & Swatton. Inc. 0026-0495/87/3603-0001$03.00/0

203

204

GARiOT ET AL

Table 1. Clinical and Biometrical

Patient NO.

Age (Vr)

sex

Type of HLP

Durationof Treatment

Characteristics

of HLP Patients Treated

Fenofibrate DOW (mgld)

BodyMass Index (w/h?)

With Fenofibrate

DailyAlcohol AbsorptionWhen PracticingBiopsy (kJ)

Heightof LiverMass (cm) Before Treatment

After Treatment

49

F

IIA

18mo

300

27.8

334

11

10

39

M

IIA

10 mo

300

21.8

0

10

10

61

M

IIA

86 mo

400

25.2

502

10

10

63

F

II8

19mo

300

24.2

418

11

11

58

F

IIB

18 mo

300

24.1

0

15

11

44

F

IIB

12mo

300

32.6

0

11

11

62

M

IV

4mo

400

23.9

0

10

10

8

46

M

IV

4 mo

300

20.6

9

49

M

IV

15 mo

300

26.3

10

30

M

IV

4mo

600

21.0

11

38

M

IV

4mo

600

22.5

12

47

M

IV

5 mo

400

20.8

1 kJ = 0.239

1,254

16

13

0

11

11

0

15

12

14

11

15

11

1,463 0

kcal.

medication (DA HLP, four patients; IIB HLP, seven patients; mean duration of the diet, eight months; range 4 to 14 months). Group 2: Type II HLP treated with diet and fenofibrate. (IIA HLP, three patients; IIB HLP, three patients). The mean daily dose of fenofibrate was 317 mg (range 300 to 400 mg). The mean duration of treatment was 27 months (range 10 to 86 months). Group 3: Type IV HLP treated by diet alone, exclusive of drug medication (four patients: mean duration of the diet, seven months, range 4 to 16 months). Group 4: Type IV HLP treated by diet and fenofibrate (six patients). The daily dose of fenofibrate was 433 mg (range 300 to 600 mg) and the mean duration of treatment was six months (range 4 to 15 months). Dietary prescriptions were adapted to patients’ weight and to the type of HLP”,” (type II, hypocholesterolemic diet ~200 mg/d; increase of polyunsaturated fats and decrease of saturated fats (P/S) >l.O) (type IV, alcohol and sucrose restriction; caloric restriction in overweight patients). The observance was evaluated by dietary recall at the time of the biopsy. Groups were statistically similar according the Mann and Whitney rank sum test in body mass index, age, daily alcohol intake, the duration of diet, and lipid levels on the day of biopsy.

Methods The study was approved by the local Ethical Committee. The patients gave their informed consent for percutaneous liver biopsy, which was carried out under local anaesthesia in aseptic conditions. The height (cm) of the liver mass was measured along the medial clavicular line before fenofibrate treatment and on the day of biopsy, in order to screen for clinical hepatomegaly.15 Preparation of rhe sections. The biopsy material was fixed during one hour in 2.5% giutaraldehyde solution buffered in 0.1 mol/L sodium cacodylate (pH 7.2). After wash-out in the buffer solution and postfixation by osmium tetroxide, the samples were dehydrated and embedded in epoxy resin. This is the recommended method to avoid artifact of fixation.16Semithin sections were stained in azure blue for light microscopy in order to select homogeneous hepatocytic areas of centrolobular location for ultrathin sectioning: this was done using a Reichert microtome, and the ultrathin sections were stained with uranyl acetate and lead citrate before examination on a Siemens Elmiskop 101 electron microscope (Siemens, Saint Denis, France). Morphomefric analysis of the sections. Morphometric analysis of the hepatocytes (on semithin sections by light microscopy) and of

their organelles (on microphotographs by electron microscopy) was done according to the method of Weibel as modified by Rohr.” For each patient, three different blocks were randomly taken from the same biopsy fragment and cut at 40 nm. We then microphotographed, at the original magnification of 2,400, ten randomly chosen areas from sections in each preparation. The microphotographs were then enlarged by 2 (x4800) and by 4 (x9600). The semithin sections (50 test areas were examined by light microscopy in each biopsy) and the microphotographs (60 pictures from the electron microscope were examined in each biopsy) were morphometrically assessed, using a 9874 A digitalizer table (Hewlett Packard, Orsay, France). We measured the number of surface area of hepatocytes, of nuclei and of intracellular organelles (mitochondria, peroxisomes, fat vesicles, reticulum, lysosomes). From these data, using the mathematical model developed by Weibel,‘s we computed the primary morphometric parameters: volume density (Vvi, total volume of cells, nuclei, or organelles per cm3 of liver tissue) and numerical density (Nvi, total number of same per cm3 of liver tissue). Given the heterogeneity of mitochondria, we computed the primary parameters of normal mitochondria, of altered mitochondria (twisted, dilated of showing disorganization of cristae), of mitochondria showing inclusions, and of giant mitochondria. Following Slabodsky-Brousse et a1,19we define giant mitochondria as a population of biggest organelles corresponding to the second part of the bimodal curve of frequency distribution of total mitochondria. The high number of mitochondria we studied (with a minimum of 700 measurements per biopsy) allowed to establish such frequency distributions in each patient. According to Shnitka?’ peroxisomes appear as rounded or oval organelles surrounded by single tripartite members containing finely granular matrices of moderate electron density without nucleoid in human. These single-membrane limited bodies can be distinguished on morphologic grounds from lysosomes by their uniformity of size, homogeneous matrice, and frequent close association with loops of endoplasmic reticulum. For surface measurement of studied cells and organelles, the interassay and intra-assay coefficient of variation remained below 2%.

Statistical Methods For statistical comparisons between the groups, we used the Mann and Whitney rank sum test. For correlations we performed the Spearman test2’

FENOFIBRATE

AND

HUMAN

LIVER

205

Table 2. Clinical and Biometrical

Characteristics

of HLP Patients Treated Daily Alcohol Consumption

Patient NO.

Type of

Age (vr)

HLP

Body Mass Index (wt/ht’j

When

With Diet Alone Height of Liver Mass (cm)

Practicing Biopsy

S&x?

After

(kJ)

Treatment

Treatment

13

19

M

IIA

19.3

0

11

14

52

M

IIA

23.4

1,609

12

12

15

32

F

IIA

18.6

0

10

10 11

10

16

32

M

IIA

29.7

0

11

17

42

F

IIB

21.0

0

10

11

18

60

M

IIB

24.7

0

11

11 11

19

70

M

IIB

26.5

0

11

20

50

M

IIB

27.9

1,097

10

11

21

39

M

IIB

21.9

0

10

10

22

30

M

IIB

22.5

0

10

10

23

37

M

II8

27.9

731

11

10

24

43

M

IV

25.0

1,254

11

11

25

39

M

IV

23.2

0

10

10

26

48

M

IV

27.7

836

11

10

27

54

M

IV

29.0

836

15

11

1 kJ = 0.239kcal.

RESULTS

Clinical Data In drug-treated groups, five patients had clinical hepatomegaly (height 212 cm)” before fenofibrate (type II HLP, n = I ; type IV HLP, n = 4). After fenofibrate, no new case of hepatomegaly was noted while the previous cases either decreased (n = 2) or disappeared (n = 3) (Tables 1 and 2). Blood Lipids The results of the effect of treatments on lipids profiles have been reported in Table 3. Improvement of HLP was noted under diet treatment (group 1, total cholesterol, P c .05; group 3, triacylglycerol, P < .05) and under diet + fenofibrate treatment (group 2, total cholesterol, P -C .Ol; group 4, triacylglycerol, P -C.01). However the improvement was more pronounced in patients treated by diet and fenofibrate than in patients treated by diet alone, both for cholesterol (P < .05) and for triacylglycerol (P < .Ol). Study of Hepatocyte in the Type II HLP Group Patient treated by diet only (group I). In morphological study by electron microscopy, giant and altered mitochondria were found, some containing paracristalline inclusions. SER as well as RER were sometimes found to be dilated (Figs 1 and 2). Peroxisomes were morphologically normal with dense, homogeneously displayed matrices without inclusions. Steatosis was moderate. Other liver cells (Kuppfer cells, endothelial cells, Ito cells) had a normal aspect. The results of the morphometric study are shown on Table 4. Patient treated by diet + fenojibrate (group 2). By electron microscopy, no morphologic difference was noticed between groups 1 and 2 (Figs 3 and 4). After morphometric study of the hepatocytes, the statistical analysis showed significant differences between groups 1 and 2. In group 2, there was a decreased volume of normal

mitochondria when compared to group 1 (Vvi, P < .05). There was no further statistically significant difference between groups 1 and 2 in the morphometric results of hepatocytes and other organelles (Table 4). Study of Hepatocytes in the HLP T)jpe IV Groups By electron microscopy, no difference was found in the morphologic study between groups 3 (diet only) and 4 (diet + fenofibrate), nor between patients with type IV HLP (groups 3 and 4) and type II HLP (groups 1 and 2). Morphometric study of hepatocytes yielded statistically significant differences between groups 3 and 4. We found in patients treated with fenofibrate a smaller number of giant mitochondria (Nvi, P < .05) and a smaller volume of altered mitochondria (Vvi, P < .05). There was no further statistically significant difference between groups 3 and 4 in the morphometric analysis of hepatocytes and other organelles (Table 5). Study of Peroxisomes By electron microscopy, peroxisomes were found to be normal in all groups. The morphometric study did not show statistically significant differences between patients treated by diet alone and patients treated by diet + fenofibrate for volume density and numerical density. There was no correlation between the total amount of fenofibrate ingested and volume density (r = .22, NS) or numerical density (r = .33, NS) of peroxisomes. DISCUSSION

The effect of treatment (diet and diet + fenofibrate) on the lipid profile of patients with various HLP is in accordance with the results of the literature.22-25 In this study, we intended to assess the short- and medium-term effect of fenofibrate on the hepatocyte. The use of morphometric methods allowed us to find moderate

206

GARIOT ET AL

Table 3.

Lipid Profiles Before and After Treatment Cholesterol(mmol/L)

BeforeTreatment

Type of HLP

Triacylglycerol (mmol/L)

After Treatment

BeforeTreatment

After Treatment

Diet 9.17

+ 0.40

8.40

+ 0.33’

2.88

t 1.50

2.46

+ 1.54

IIA (n = 4)

9.20

f 0.38

a.43

+ 0.31

1.06 + 0.02

0.95

f 0.06

IIB (n - 7)

9.16

k 0.23

8.38

? 0.30

3.67

+ 1.11

3.09

+ 1.22

IV (group 3, n = 4)

6.30

i 0.73

6.47

f 0.82

3.12

+ 0.90

1.51 + 0.70*

II (group 2, n = 61

10.80

+ 2.71

7.07

+ 1.oot

2.38

+ 1.51

1.62 k 1.06

IIA (n = 3)

10.50

+ 1.99

6.94

+ 1.06

1.29 f 0.12

1.28 + 0.20

IIB (n = 3)

11.10

f 2.46

7.20

k 0.93

3.47

f 0.97

1.96 + 0.89

6.02

k 0.48

5.33

+ 0.62

19.36

f 6.20

2.54

II(groupl,n=

11)

Diet + fenofibrate

IV (group 4, n = 6)

k0.56t

lf < .05, before Y after treatment. tf

< .O 1, before Y after treatment.

alterations that might have been neglected by the usual morphologic examinations. Hepatomegaly and Alterations of Hepatocytes No hepatomegaly was found in patients after fenofibrate, while hepatomegaly existing prior to the drug treatment either decreased or disappeared. Furthermore, the morphometric study showed that the number and volume of hepatocytes were identical in all the groups. In contrast to animal experimentation, fenofibrate does not seem to induce hepatomegaly or abnormalities in the number and volume of hepatocytes in man. Study of Liver Peroxisomes

According to Reddy et aLz6drugs that induce proliferation of peroxisomes may be considered as a new class of carcino-

Fig 1. HLP type II patients treated intramitochondrial inclusions (MI.

by diet

(magnification

genetic agent. However, this study is not devoted to the study of carcinogenicity, as this aim would require a different methodology and a much longer follow-up. We nevertheless focused our attention on peroxisomes; these organelles are the site of fatty acid beta oxidation and show early changes in untreated HLP.” The morphometric study of peroxisomes did not yield results statistically different between patients treated by diet alone and patients treated by diet + fenofibrate. There was no correlation between the total amount of fenofibrate ingested and the results of the morphometric study. These findings suggest that in our sample population fenofibrate does not alter the number of volume of liver peroxisomes. Our results give quantitative confirmation of the lack of proliferation of liver peroxisomes noted by Blumke et al’ by microscopic observation, but in our sample and under the

x4.OW1.

Note

a dilated

reticulum

(R), altered

mitochondria,

and

FENOFIBRATE AND HUMAN

Fig 2.

207

LIVER

HLP type II patients

treated

by diet (magnification

x20,DDO).

Dilated reticulum

(R) and intra mitochondrial inclusions (Ml are

noted in the hepatocyte.

experimental

conditions of our study, we can also eliminate

in the number and volume of peroxisomes as a consequence of fenofibrate therapy. These results are in complete contradiction with the animal experiments: in rodents, fenofibrate induces a rapid and large increase of the number and size of liver peroxisomes.’ These discrepancies can be explained by (1) a difference in dosage; minimun dose levels of 50 mg/kg/d were used in rodents, while the average daily dosage was 6 mg/kg in man;

discrete changes

Table 4. Morphometric Diet(n=

(2) a different metabolism of lipoproteins**; and (3) a difference in peroxisome enzymes equipment according to the difference in species. Comparisons between our results and those obtained with other lipid-lowering drugs of the fibrate class allow us to think that the hepatic cell effect of each drug is different. Hanefeld et a129have reported an increase in the number of peroxisomes in patients treated by clofibrate, while De la lglesia et al” have seen rarefaction and densification of the peroxisomal matrix after treatment by gemfibrozil.

Study in Type II HLP Groups

11)

Vvi (x 10-T

Diet + FenofibrateIn - 6) Nvi

Vvi (x 10-Y

Hepatocyte

888 + 18

194 * 13 x los

Nucleus

101 + 7

196 I+ 13 x 10’

88*

Steatosis

106 + 45

857 + 208 x 10’

69 i 35

621

Peroxisomes 185.84

f 8.96

10.04

f 0.84

x 10”

Altered mitochondria

11.03

i 2.19

0.60

+ 0.13

x 1o’O

Giant mitochondna

11.40

5 3.09

0.20

f 0.05

5.89

2 3.73

0.29

+ 0.17

Rough endoplasmic reticulum

54+

Smooth endoplasmic reticulum

61 * 21

Values are given as mean + SEM. lP < .05.

15

12

Nvi 200&8x

10’

202*8x

lo6

571 * 175 X lo6 6 z 1 x 10”

7+1

5 * 1 x 1o’O

Normal mitochondria

Mitochondria with inclusion

793 f 33

+ 17.04’

8.30

+ 0.75

7.34

+ 1.83

0.44

t 0.09

x 1o’O

x 1O’O

6.38

+ 1.79

0.13

i 0.04

x 1o’O

x 1o’O

4.32

” 3.23

0.32

2 0.23

x 10”

125.72

27 -r 5 602

16

x 10”

208

GARIOT ET Al

Fig 3. HLP type II patients treated by diet + fenogbrate (magnification x4DCMN. The same alterations are noted in patients treated by diet and in patients treated by diet and fenofibrate concerning reticulum IF?)and mitochondria (M).

Fig 4. HLP type II patient treated by diet + fenofibrate (magnification x28,ooO). There is abundant smooth endoplasmic reticulum (RI interspersed among glycogen particles IG). The peroxisomes appear regularly shaped with homogeneous matrix IPI.

FENOFIBRATE AND HUMAN LIVER

209

Table 5. Morphometric Study in Type IV HLP Groups Diet (n = 4)

Diet 4 Fenofibrate In = 61

Vvi (x 10m3)

Hepatocyte

Nvi

882 + 32

Nucleus

79*

Steatosis

90 + 45

Peroxisomes

Normal mnochondria

10

Vvi (x 103

NW

154 t- 13 x loa

862 f 17

200+30x

157 ? 13 x 10s

61 +8

203k31

40 + 22

696 r 398 x lo6

1,481

+- 707 x 10’

7?2

6 + 2 x 10”

10 + 3

215.78 + 12.82

10.60 t- 1.60 x 10”

197.64 & 14.23

lo6 x lo6

9 i 4 x 1o’O 10.91

_+ 1.24 x 10”

Altered mitochondria

13.61

f 1.17

0.69

c 0.13

x 10”

6.00

+ 1.44.

0.34

+ 0.09

x 1o’O

Giant mitochondria

23.99

+ 8.22

0.32

+ 0.08

x 10”

7.99

k 3.36

0.08

+ 0.03

x 10”’

5.21

+ 3.35

0.18

+ 0.11

x 10”

4.92

i- 2.17

0.30

t 0.16

x IO”

Mitochondria with inclusion Rough endoplasmic reticulum

74 + 23

-

47 + 10

Smooth endoplasmic reticulum

95 + 33

-

97 f 34

Values are given as mean f SEM. fP

<

.05.

Heterogeneity

of Mitochondria

Changes in mitochondria seen in patients treated by fenotibrate depended on the type of HLP. The decrease in number of giant mitochondria and in volume of altered mitochondria seems to reflect positive effect of the drug. On the other hand, the decrease in volume of normal mitochondria seen in type II HLP group is difficult to explain; it may suggest a modification of fatty acid catabolism at the level of mitochondrial beta-oxidation. Our results do not give any clue as to the positive or negative significance of these changes. The morphometric study showed that number and volume of mitochondria with paracristalline inclusions were identical in all groups, and we cannot confirm the results of Blumke et al’ who have reported increased mitochondria with paracristalline inclusion in patients treated by fenofi-

brate.

fenofibrate. The discrepancy probably stems from methodological differences, as these authors have not quantified the hepatic changes that were seen. Epidemiologic studies have shown the efficiency of lipid reduction in the primary and secondary prevention of cardiovascular diseases. 3’.32In our sample population, we have not found significant cellular changes with treatment periods ranging from 4 to 86 months, except modifications of mitochondria, which are difficult to explain. These findings clearly need to be confirmed on larger samples, but they solidify the impression that fenofibrate, an efficient, welltolerated,” lipid-lowering drug, does not produce substantial pathologic changes. The discrepancies between the results of human and rodent studies are such that another animal model may be necessary to evaluate properly the pharmacology and toxicity of lipid-lowering drugs of the fibrate class.

Study of Other Organelles

ACKNOWLEDGMENT

Likewise, we cannot confirm the results of Blumke et al’ who have described increased steatosis and dilation of SER and

a reduced

amount

of RER

in patients

treated

by

The authors would like to thank the editorial board and reviewers for the constructive discussions of this manuscript. We are indebted to Chantal Veuvas for excellent secretarial assistance.

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