AN ULTRASTRUCTURAL ANALYSIS OF SMOOTH ENDOPLASMIC RETICULUM IN HEPATOCYTES OF ALCOHOL-FED RATTUS  NORVEGICUS

 

MYRNA BEJAR

ANALYTICAL CYTOLOGY CORE FACILITY

DR. JOANNE T. ELLZEY AND LAURA DADER

UNIVERSITY OF TEXAS AT EL PASO

JULY 29, 1998

 

 

 

ABSTRACT

Through the “Bridges to the Future” program, the research project was conducted.  I began the program with several goals in mind that I wished to accomplish by working with Dr. Ellzey and Laura Dader and that I feel I reached.  I obtained a better understand the effects of ethanol on the liver cells (hepatocytes) and reviewed the latest literature.  I also had the opportunity to learn the techniques of transmission electron microscopy.  During my research, I was introduced to and assimilated biomedical research.  This program also offered me the time to establish meaningful relationships with UTEP students, staff, and faculty.  I am excited about returning to UTEP and continuing my education and eventually receiving a Bachelors degree.

            During the eight weeks of the program, I compared the smooth endoplasmic reticulum (SER) of alcohol-fed rats (Rattus norvegicus) to that of control rats.  The hypothesis was in the presence of intoxicating levels of ethanol, induction of enzymes that metabolize ethanol is anticipated.  The hypertrophy of smooth endoplasmic reticulum is correlated with the induction of cytochrome P450 2e1.  Therefore, an increase in the volume density of SER is expected in the presence of intoxicating levels of ethanol.    Micrographs were analyzed using a point count method to obtain the data that would be used with the Student t-test.  The Student t-test gave a statistical analysis of the data.  The mean SER volume density for the control group is 5.22% and for the experimental group is 4.79%. In comparing the two groups, no significant difference was found.  Using the Student t-test and other programs for statistical analysis gave me the opportunity to learn about statistics.  This gave me an understanding of how important statistics are in the field of science.

 

 

 TABLE OF CONTENTS

Introduction

Methods and Materials

Results

Discussion

Conclusion

Acknowledgements

Literature Cited

Appendix

 

 

INTRODUCTION

 

            The study of alcohol and its effects is of great interest.  The scientific community is continuously updating information by conducting research.  This area is of great importance and concern due to cirrhosis of the liver being the seventh leading cause of death in the United States (Lieber, 1994). The metabolism of ethanol has been linked to alcohol-induced tissue damage (Lieber, 1997).  Ethanol metabolism occurs through different pathways.  The main pathway is through the activity of the enzyme alcohol dehydrogenase (ADH).  It is found in the hepatocytes (liver cells) and serves as a catalyst for the oxidation of ethanol.  As ethanol oxidizes, it changes to acetaldehyde and it is this toxic form that is detrimental to the liver. The microsomal ethanol oxidizing system (MEOS) involves the ethanol-inducible cytochrome P450 2e1 (CYP2e1) which has more severe toxic manifestations.  CYP2e1 yields an increase in acetaldehyde generation, enzyme inactivation, decreased DNA repair, impaired utilization of oxygen, lipid peroxidation, and increased collagen synthesis (Lieber, 1997).  This collagen along with continued alcohol consumption may lead to the development of liver fibrosis and eventually cirrhosis. 

Liver enzymes and the effects of alcohol have also been studied in the developing fetus.  It has been found that ADH is expressed in very low concentrations in the human embryonic tissues and CYP2e1 is present.  A recent study has given results that indicate CYP2e1 is present in the human fetal liver and the enzyme is functionally similar to CYP2e1 in adults (Carpenter , et al, 1996).  The detrimental effects on the livers of adults who consume alcohol is possible to develop in the human fetal livers that are exposed to alcohol.  With this finding, it is with great urgency that the science and medical community come to better understand the effects of alcohol. 

Alcoholism is being researched as far as the genetic influence that may be a factor in alcoholic liver disease.  The isozymes encoded by ADH2*2 and ALDH2*2 have been found to influence alcohol drinking behavior or alcoholism substantially (Crabb, 1995).  According to Holden (1998), “Alcoholism genes are multiple, they interact in unknown ways, and they have incomplete penetrance, which means you can have the genes but not be an alcoholic.”  A better understanding of alcohol and how it affects the body is necessary to begin to possibly control what may eventually lead to alcoholism.

In this study, in the presence of intoxicating levels of ethanol, induction of rat liver enzymes that metabolize ethanol is anticipated. One of those enzymes is the cytochrome P450 2e1 located in the smooth endoplasmic reticulum (SER).  The hypertrophy of SER is correlated with the induction of cytochrome P450 2e1.

The hypothesis is that an increase in the volume density of smooth endoplasmic reticulum is expected in the presence of intoxicating levels of ethanol.  The null hypothesis is the point count (volume density) of SER in the experimental group will be the same as the point count in the control group.  The alternate hypothesis to this is the point count (volume density) of SER in the experimental group will be greater than the point count in the control group.  Either the null or the alternate hypothesis will be rejected using the statistical analysis results.

METHODS AND MATERIALS

 

The control and experimental adult, male rats (Rattus norvegicus) were match-paired according to weight.  There were five rats in each group.  The control rats were fed liquid diets equivalent to that consumed by the match-paired experimental rats for two weeks.  The experimental group was fed ethanol in their diet.  The animals were fed, sacrificed, and their livers fixed and embedded by Delfina Dominguez.  The specimens were provided in the form of plastic BEEM capsules.  These capsules were used to collect data for my research.  A trapezoid was made on the capsules that would later be sectioned for viewing. To cut the trapezoids, glass knives were made with the LKB Glass Knife Maker.  These knives were used on the Sorval MT-1 Ultramicrotome to cut thick and ultrathin sections from the BEEM capsules.  The thick sections were viewed with the Zeiss Axioskop light microscope.  The ultrathin sections (90 nm thick) were viewed with the Zeiss EM-10 a transmission electron microscope (TEM).  These sections were poststained with uranyl acetate and lead citrate.  This allows for contrast on the specimen sections.  Photographs using the Zeiss EM-10 TEM were taken of the thin sections with Kodak SO-163 film.  Negatives were developed and photographs were printed on Kodabromide F5 paper.  These prints were then used to interpret and qualitatively determine the smooth endoplasmic reticulum volume density in the control and experimental rats.

            A total of 100 prints were evaluated (50 per group).  These prints were also provided by Delfina Dominguez.  The method used to determine the volume density of the smooth endoplasmic reticulum was the point count method.  A grid made up of 450 squares, each square measuring 1cm, was placed over the print.  Any squares that did not fall on the photograph of the hepatocyte were subtracted from the total number of squares on the grid.  I took count of all the points that fell on the smooth endoplasmic reticulum. 

A count of the points that fell on the nucleus and sinusoids was subtracted from the total number of points that were on the print.  This was to determine the ratio of the smooth endoplasmic reticulum to the cytoplasm (PP).  This ratio determines the volume density of smooth endoplasmic reticulum (Vv) = (PP) i.e. the portion of the hepatocyte cytoplasm that is occupied by SER.

            The computer program Excel was used to record the data in the form of tables and bar graphs.  The Student F-test was used to determine the means of each group due to a wide variance within the control group.  The Student t-test was also utilized in analyzing the statistics of the data.

 

 

RESULTS

 

Figure 7.  Mean SER (%) Control/Alcohol-fed rat comparison.

Control Rats

Alcohol-fed Rats

 

 

 

 

Mean

5.224

Mean

4.788

Standard Error

0.381

Standard Error

0.611

Median

5.08

Median

4.82

Standard Deviation

0.853

Standard Deviation

1.367

Sample Variance

0.728

Sample Variance

1.870

Range

2.26

Range

3.33

Minimum

4.07

Minimum

3.57

Maximum

6.33

Maximum

6.9

Sum

26.12

Sum

23.94

Count

5

Count

5

 

 

DISCUSSION

 

The mean SER volume density (%) from each mouse was calculated using Microsoft EXCEL ’97 (Table 1).  Using the mean instead of the raw data of 50 measurements from each group was done so that my results would reflect the correct degrees of freedom (df), that is 4 instead of 49.  This is important because you get greater statistical power with the greater df.  The df correlated to the number of animals used and not the number of observations recorded. 

            The means obtained were then entered into the F-test Two Sample for Variances (Table 2).  This was to determine whether I should use the t-Test assuming equal or unequal variances.  I selected the t-Test Assuming Unequal Variances, because the variance from the F-test was great, 1.871 within the control group and 0.370 within the alcohol-fed group (Table 3).  The

t-test showed a P-value of 0.196 (19%).  This value would have had to have been less than 0.05 (5%) to reject the null hypothesis.

            In the control rats, the mean value for the volume density of SER was 5.20% with a standard error of +/- 0.38.  In the experimental group, the mean value for the volume density of SER was 4.79% with a standard error of +/- 0.61.  Results from the Student t-test comparing the volume densities show that there was no statistically significant difference between the control and the experimental groups.

 

 

CONCLUSIONS

 

It was believed that by increasing the amount of CYP2e1 in the hepatocyte, the volume of the smooth endoplasmic reticulum would also increase.  The lack of a statistical difference may have been due to the amount of ethanol that was fed to the rats or the amount of time.  A daily measure of the blood alcohol levels may have indicated whether the alcohol-fed rats were reaching intoxicating levels of ethanol consumption.  In future experiments, an increase in ethanol consumption may show a more significant difference in the volume density of the smooth endoplasmic reticulum of the experimental vs. the control group.

            This experiment failed to reject the null hypothesis that the smooth endoplasmic reticulum in both groups would be the same. This is based on the statistics of the data collected.

 

 

ACKNOWLEDGEMENTS

 

Dr. Joanne T. Ellzey, Laura Dader, and Delfina Dominguez provided invaluable assistance in this project.

Dr. Melchor Ortiz and Dr. Joan Staniswalis were of great assistance in the statistical analysis of the data.

 

 

LITERATURE CITED

 

Dominguez, D.  1988.  Morphometric Analysis of Peroxisomes in

     Hepatocytes of Alcohol-fed Rattus norvegicus.  Masters thesis. The University

     of Texas at El Paso.

 

Lieber, C.S.  1997.  Ethanol metabolism, cirrhosis and alcoholism.  Clinica

     Chimica Acta. 257 (1):59-84, Jan.

 

Carpenter, SP.,  Lasker, J.M.,  Raucy, JL. 1996.  Expression, induction, and

     catalytic activity of the ethanol- inducible cytochrome P450 (CYP2e1) in

     human fetal liver and hepatocytes.  Molecular Pharmacology. 49(2):260-268.

 

Crabb, D.W.  1995.  Ethanol oxidizing enzymes: roles in alcohol metabolism and

     alcoholic liver disease.  Progress in Liver Diseases.  13:151-72.

 

 Holden.  Science 280: 1349, 1998.

 

 

APPENDIX

 

Mean SER Volume Density (%)

Control

Alcohol-fed

5.07

4.92

3.58

4.97

3.57

5.72

6.9

6.33

4.82

5.08

Table 1.  Mean SER volume density (%) for control and alcohol-fed rats.

F-Test Two-Sample for Variances

 

Control

Alcohol

Mean

4.788

5.407

Variance

1.871

0.370

Observations

5

5

df

4

4

F

5.062

 

P(F<=f) one-tail

0.073

 

F Critical one-tail

6.388

 

Table 2.  F-Test, for variances between volume density of control and alcohol-fed rats.

t-Test: Two-Sample Assuming Unequal Variances

 

Control

Alcohol

Mean

4.788

5.407

Variance

1.871

0.370

Observations

5

5

Hypothesized Mean Difference

0

 

df

4

 

t Stat

-0.924

 

P(T<=t) one-tail

0.196

 

t Critical one-tail

1.943

 

Table 3.  T-Test, assuming unequal variances between volume density of control and alcohol-fed rats.

 

 

 

magnification

Negative #

# of SER points

475-(B.C.+Nucleus)

Cont.Rat #

SER %

1

19,050

1286

29

415

1

6.99

2

19,050

1287

29

368

1

7.88

3

19,050

1288

22

423

1

5.20

4

19,050

1289

24

380

1

6.32

5

19,050

1290

29

442

1

6.56

6

19,050

1291

16

423

1

3.78

7

19,050

1293

21

350

1

6.00

8

19,050

1295

17

451

1

3.77

9

19,050

1296

11

432

1

2.55

10

19,050

1297

7

419

1

1.67

11

19,050

1298

14

384

2

3.65

12

19,050

1299

13

415

2

3.13

13

19,050

1300

12

336

2

3.57

14

19,050

1301

25

454

2

5.51

15

19,050

1302

9

449

2

2.00

16

19,050

1308

17

417

2

4.08

17

19,050

1309

16

409

2

3.91

18

19,050

1310

16

396

2

4.04

19

19,050

1311

14

450

2

3.11

20

19,050

1313

12

423

2

2.84

21

19,050

1314

8

403

3

1.99

22

19,050

1315

7

321

3

2.18

23

19,050

1316

15

371

3

4.04

24

19,050

1317

5

421

3

1.19

25

19,050

1318

5

428

3

1.17

26

19,050

1319

9

375

3

2.40

27

19,050

1321

5

370

3

1.35

28

19,050

1322

17

363

3

4.68

29

19,050

1325

13

412

3

3.16

30

19,050

1329

40

463

3

8.64

31

19,050

1331

35

411

3

8.52

32

19,050

1332

19

434

4

4.38

33

19,050

1334

42

403

4

10.42

34

19,050

1335

32

395

4

8.10

35

19,050

1336

15

397

4

3.78

36

19,050

1337

51

436

4

11.70

37

19,050

1338

41

423

4

9.69

38

19,050

1339

28

324

4

8.64

39

19,050

1341

19

382

4

4.97

40

19,050

1342

26

365

4

7.12

41

19,050

1343

34

368

4

9.24

42

19,050

1344

26

429

5

6.06

43

19,050

1345

37

409

5

9.05

44

19,050

1346

33

475

5

6.95

45

19,050

1348

20

372

5

5.38

46

19,050

1350

10

404

5

2.48

47

19,050

1351

10

383

5

2.61

48

19,050

1353

8

475

5

1.68

49

19,050

1354

12

475

5

2.53

50

19,050

1358

29

435

5

6.67

 

Table 4. Data for control Rattus norvegicus.

#

magnification

Negative #

# of SER points

475-(B.C.+Nucleus)

Exp.Rat #

SER %

1

19,050

1455

25

473

1

5.29

2

19,050

1456

15

475

1

3.16

3

19,050

1458

10

471

1

2.12

4

19,050

1460

21

461

1

4.56

5

19,050

1461

34

411

1

8.27

6

19,050

1462

8

351

1

2.28

7

19,050

1464

13

393

1

3.31

8

19,050

1465

22

398

1

5.53

9

19,050

1472

30

456

1

6.58

10

19,050

1410

33

462

1

7.14

11

19,050

1409

26

427

2

6.09

12

19,050

1408

13

401

2

3.24

13

19,050

1404

18

469

2

3.84

14

19,050

1403

10

373

2

2.68

15

19,050

1402

9

372

2

2.42

16

19,050

1401

18

430

2

4.19

17

19,050

1400

24

369

2

6.50

18

19,050

1397

42

471

2

8.92

19

19,050

1393

23

452

2

5.09

20

19,050

1413

15

317

2

4.73

21

19,050

1416

18

475

3

3.79

22

19,050

1420

15

317

3

4.73

23

19,050

1423

30

445

3

6.74

24

19,050

1426

34

460

3

7.39

25

19,050

1424

22

468

3

4.70

26

19,050

1427

26

466

3

5.58

27

19,050

1428

10

395

3

2.53

28

19,050

1429

18

373

3

4.83

29

19,050

1430

19

456

3

4.17

30

19,050

1431

41

421

3

9.74

31

19,050

1435

13

453

4

2.87

32

19,050

1436

26

441

4

5.90

33

19,050

1437

14

418

4

3.35

34

19,050

1438

26

447

4

5.82

35

19,050

1440

12

437

4

2.75

36

19,050

1442

83

460

4

18.04

37

19,050

1443

37

381

4

9.71

38

19,050

1447

17

451

4

3.77

39

19,050

1448

21

475

4

4.42

40

19,050

1449

11

410

4

2.68

41

19,050

1451

12

472

5

2.54

42

19,050

1452

21

375

5

5.60

43

19,050

1453

21

475

5

4.42

44

19,050

1455

15

469

5

3.20

45

19,050

1493

19

468

5

4.06

46

19,050

1492

10

347

5

2.88

47

19,050

1490

32

462

5

6.93

48

19,050

1487

27

464

5

5.82

49

19,050

1481

27

449

5

6.01

50

19,050

1479

21

475

5

4.42

 

Table 5. Data for alcohol-fed Rattus norvegicus.

 

Control Rats

 

 

 

 

 

 

 

 

 

 

 

 

 

 

mean

Rat 1

Rat 2

Rat 3

Rat 4

Rat 5

 

6.99

3.65

1.99

4.38

6.06

7.88

3.13

2.18

1.42

9.05

5.2

3.57

4.04

8.1

6.95

6.32

5.51

1.19

3.78

5.38

6.56

2

1.17

11.7

2.48

3.78

4.08

2.4

9.69

2.61

6

3.91

1.35

8.64

1.68

3.77

4.04

4.68

4.97

2.53

2.55

3.11

3.16

7.12

6.67

1.67

2.84

8.64

9.24

 

 

 

8.52

 

 

 

 

 

 

 

5.07

3.58

3.57

6.90

4.82

Table 6. Mean volume density for control rats 1-5.

Alcohol-fed Rats

 

 

 

 

 

 

 

 

 

 

 

 

 

Rat 1

Rat 2

Rat 3

Rat 4

Rat 5

 

5.29

4.73

9.74

2.68

4.42

3.16

5.09

4.17

4.42

6.01

2.12

8.92

4.83

3.77

5.82

4.56

6.5

2.53

9.71

6.93

8.27

4.19

5.58

18.04

2.88

2.28

2.42

4.7

2.75

4.06

3.31

2.68

7.39

5.82

3.2

5.53

3.84

6.74

3.354

4.42

6.58

3.24

4.73

5.9

5.6

7.14

6.09

3.79

2.87

2.54

 

 

 

 

 

mean

4.92

4.97

5.72

6.33

5.08

 

Table 7. Mean volume density for alcohol-fed rats 1-5.

Figure 5.  Mean SER volume density for control rats (%).

 

Figure 6.  Mean SER volume density for alcohol-fed rats (%).

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