The Correlation between the Level of Doxorubicin-Induced Cardiac Damage and Serum Soluble Fas in an Experimental Rat Model

Keywords

Cardiac - doxorubicin - fas - rat

Introduction

Chronic cardiomyopathy, which develops based on dose, is the most significant adverse effect of doxorubicin, which is one of the most essential medications used in the treatment of childhood cancers.[1] Many studies have been conducted to determine doxorubicin-related myocardial injuries at an earlier phase; however, no noninvasive method with high sensitivity and specificity has been found yet.

The aim of this study was to research the possible relation between the doxorubicin-induced cardiac injury level and soluble Fas (sFas) level. In this vein, by determining the existing cardiac injury level and predicting possible long-term cardiac complications, it has been aimed to perform early-phase dose modification or continue the treatment with alternative medications.

Material and Methods

Ethical declaration

The ethical approval of this study was obtained from the ethical board of experimental animals at the center for experimental research and application at our university medical school (approval number: 2013-083), and all the stages of the research were inspected by the same board.

Animal selection

In this study, 42 3-month-old Wistar Albino rats, randomly selected in terms of gender, with 300 g average weight were used.

Study groups and procedure

Two groups of rats, control group and study group which were administrated medicine, each consisting of 21 rats, were included in the study. The control group was given 2, 5 ml/kg/week serum physiologic, while the study group was intraperitoneally given 2.5 mg/kg/week dose of doxorubicin for 10 weeks. Two hours after this application, blood tail samples were taken from each animal, and 2 mg/ml of acetaminophen was given through tap water as analgesics. After the animals were anesthetized with xylazine hydrochloride in groups of three at weeks 2, 4, 6, 7, 8, 9, and 10, intracardiac blood samples were taken and they were sacrificed, and then the heart muscles were removed. By sacrificing animals at different weeks, it was aimed to determine at what phase the heart muscle injury started, the possible additional tissue changes in animals that were given higher doses of medicine, and the changes in cardiac tissue and sFas level in the peripheric blood due to dose and the correlation between them.

The cardiac tissue samples were divided and placed into two different boxes for pathologic and flow cytometric evaluations.

The tissues, following 10% formaldehyde and alcohol solution procedures, were changed into paraffin-embedded blocks for the pathologic evaluation. Some of the sections with 4 μm thickness taken from these blocks were evaluated under the light microscope by staining with hematoxylin and eosin to determine the injuries and morphological changes (interfibrillar hemorrhage, vascular dilatation, inflammatory cell infiltration, myocardial necrosis, and separation of muscle fibers) in cardiac muscle tissue. The changes determined after the evaluation were scored as: 0, absent; 1, mild; 2, moderate; and 3, severe. For the rest of the sections, Fas (CD95, APO-1) antibody was administrated using Ventana, Benchmark XT model (Ventana, Tucson, AZ, USA), immunohistochemical staining device, and Ultraview Universal DAB Detection Rat Kit (Roche, Ventana, NY, USA), and Fas expression was evaluated by the light microscope. All sections were taken in cross-section to evaluate all cardiac muscle tissue layers. All the evaluations were performed by the same pathologist who did not know which rats these preparats belong to.

The Fas expression on the surface of the cell was evaluated with 3-laser flow cytometer device (BD FACSARIA III, BD Biosciences, USA) for the flow cytometric assessment after the cardiac tissues were changed into suspension. Complete blood was analyzed with lysis method using a florescent monoclonal antibody marked with PE-CF594 hamster antimouse CD95 (BD Biosciences). Lysing was performed with FACS lysing solution (FACS lyse, Becton Dickinson, San Jose, CA, USA). Cells stained with PE-CF594 hamster IgG2 λ1 isotype control were used for negative control. After the data were analyzed with “dot plots” and “histogram plots” on “BD FACS DivaR” package program (Becton Dickinson, CA, USA), the findings were defined as relative rate and median expression.

For biochemical analysis, the blood serums were centrifuged within 2 h and kept at 80°C until the study date. The serums, melted at room temperature on the day of the study, were used to assess sFas levels using Rayto, Elisa reader device, and Elisa Kit for Rat Factor Related Apoptosis (FAS) (Uscn Life Science Inc. Wuhan, China).

In so doing, it was planned to evaluate the relationship of sFas values, obtained from peripheric blood, both with histopathologically determined cardiac injury and Fas expression and flow cytometrically determined positive Fas cell numbers in cardiac tissue.

Statistical analysis

The statistical analyses were performed through SPSS software package program (Version 21; Chicago, IL, USA). Since the numerical data used in this study were not appropriate to the normal distribution approach, the data were analyzed with nonparametric Mann–Whitney U-test and Wilcoxon signed-rank test. In this study, categoric variables were given as “n” and descriptive statistic was defined as percentage values; for numeric variables, minimum–maximum value and median–standard deviation were used. In all analysis techniques, the level of significance was accepted as 0.05, and thus, decisions were defined as 95% confidence interval.

Results

When the sFas levels of the study group were compared within the group, the average of weeks 2nd–9th was higher than that of the 1st week before the administration of the medicine, and the higher rate found in the averages of weeks 2nd–7th was statistically meaningful [Table 1].

Table 1

Comparison of mean values of 1^st and the other weeks in the study group

Week	n	1st week (n=21)	P
2nd	21	−48.9±125.2	0.021
3rd	18	−107.8±138.9	0.007
4th	18	−174.2±138.8	0.001
5th	15	−200.0±124.1	0.001
6th	15	−139.6±172.3	0.017
7th	12	−104.7±137.8	0.031
8th	9	−45.8±187.1	0.441
9th	6	−4.4±164.1	0.917
10th	3	18.7±156.5	-

Similarly, the averages found in the 3rd–8th weeks were also higher than that of the 2nd week, and the higher rate in weeks 3rd–6th was statistically meaningful [Table 2].

Table 2

The comparison of mean values of 2^nd week and the other weeks in the study group

Week	n	2nd week (n=21)	P
3rd	18	−76.6±142.5	0.025
4th	18	−143.0±113.8	0.001
5th	15	−175.2±115.3	0.001
6th	15	−114.8±127.5	0.005
7th	12	−79.8±161.0	0.131
8th	9	−31.5±127.5	0.594
9th	6	34.2±138.4	0.345
10th	3	63.2±31.5	0.109

Averages of the 4th–6th weeks were also higher than that of the 3rd week, and the higher rate in 4th week was statistically meaningful [Table 3].

Table 3

The comparison of mean values of 3^rd week and the other weeks in the study group

Week	n	3rd week (n=18)	P
4th	18	−66.3±145.8	0.040
5th	15	−68.8±177.4	0.069
6th	15	−8.4±175.2	0.649
7th	12	27.1±147.8	0.346
8th	9	42.6±152.5	0.441
9th	6	127.9±145.6	0.074
10th	3	145.4±19.9	0.109

The sFas values of the study group regularly increased each week between 1st and 5th weeks when compared to the previous week, and the increase within the first 4 weeks was statistically meaningful. However, following the 5th week, sFas values decreased regularly each week, although they were not statistically meaningful. Of these rates, only the one in week 10 was higher than that of week 9 [Table 4].

Table 4

Comparison of progression of soluble Fas values in time and sequential values per week in medication group

Week	1st (n=21)	2nd (n=21)	3rd (n=18)	4th (n=18)	5th (n=15)	6th (n=15)	7th (n=12)	8th (n=9)	9th (n=6)
2nd	−48.9±125.2
P	0.021
3rd		−76.6±142.5
P		0.025
4th			−66.3±145.8
P			0.040
5th				−16.1±159.3
P				0.733
6th					60.3±181.1
P					0.320
7th						27.0±214.5
P						0.504
8th							29.6±168.6
P							0.767
9th								71.3±156.8
P								0.345
10th									−16.9±24.5
P									0.285

When the sFas values of the control group were compared within the group, only the mean rate of week 4 was seen to be significantly higher than that of week 6 (P = 0.006).

When the average sFas levels of the groups were compared to each other weekly, it was seen that the rates of 1st–8th weeks were higher in the study group, and the higher rates in 2nd–8th weeks were statistically meaningful [Table 5].

Table 5

Comparison of soluble Fas values in both groups with each other per week

Week	n	Control group	Drug group	P
1st	21	72.7±75.4	99.0±90.9	0.199
2nd	21	59.9±43.4	157.5±79.8	<0.001
3rf	18	96.0±75.5	210.6±126.6	0.001
4th	18	87.9±36.9	276.9±100.5	<0.001
5th	15	100.6±36.9	298.1±106.8	0.001
6th	15	57.8±46.6	237.7±111.5	<0.001
7th	12	98.1±69.0	204.1±144.0	0.031
8th	9	86.3±134.5	162.4±106.6	0.041
9th	6	113.0±62.5	89.3±116.4	0.336
10th	3	72.0±36.0	60.3±42.3	0.513

When the groups are compared with each other and within themselves in terms of flow cytometrically determined Fas-positive cell numbers, no statistically meaningful difference was detected at any of the weeks.

In the immunohistochemical investigation performed with Fas antibodies, no positive staining was detected neither in control nor in the study group. In control group, while interfibrillar hemorrhage, vascular dilatation, and inflammatory cell infiltration were seen as 62%, 90.5%, and 43% but only slightly, respectively, no myocardial necrosis and separation of muscle fibers were observed. However, in the study group, these pathological findings increased with time, and interfibrillar hemorrhage at 10th week, vascular dilatation between 7th and 10th weeks, inflammatory cell infiltration at 9th and 10th weeks, separation of muscle fibers at 10th week, and myocardial necrosis also at 10th week became severe [Table 6] and [Figure 1].

Table 6

Pathological evaluation of cardiac tissue of rats in the control and drug groups

Rat number	Week	Staining with Fas antibody		Interflbrillar hemorrhage		Vascular dilatation		Inflammatory cell infiltration		Separation of muscle fibers		Myocardial necrosis
		Control	Drug	Control	Drug	Control	Drug	Control	Drug	Control	Drug	Control	Drug
0 – Absent; 1 – Mild; 2 – Moderate; 3 – Severe
1	2	0	0	1	1	1	1	0	1	0	0	0	0
2		0	0	0	1	0	1	0	1	0	1	0	0
3		0	0	1	1	1	1	1	1	0	1	0	0
4	4	0	0	1	2	1	2	0	1	0	1	0	0
5		0	0	1	2	1	2	0	1	0	1	0	0
6		0	0		1	1	1	1	1	0	1	0	1
7	6	0	0	1	2	1	2	1	2	0	1	0	2
8		0	0	1	1	1	1	0	2	0	1	0	2
9		0	0	1	2	1	2	0	2	0	1	0	2
10	7	0	0	1	3	1	3	1	2	0	1	0	2
11		0	0	1	3	1	3	1	2	0	2	0	2
12		0	0	0	2		3	0	2	0	2	0	2
13	8	0	0	1	2	1	3	0	2	0	2	0	3
14		0	0	0	3	1	3	1	2	0	2	0	3
15		0	0	0	3	1	3	0	2	0	2	0	2
16	9	0	0	0	2	1	3	1	3	0	2	0	3
17		0	0	1	3	1	3	1	3	0	2	0	2
18		0	0	0	3	1	3	0	3	0	2	0	3
19	10	0	0	1	3	1	3	0	3	0	3	0	3
20		0	0	1	3	1	3	1	3	0	3	0	3
21		0	0	0	3	1	3	0	3	0	3	0	3

Figure 1: (a) In the drug group, significant interfibrillar hemorrhage and vascular dilatation (H and E, ×200) (b) in the drug group, significant lymphocyte infiltration (H and E, ×200) (c) in the drug group, extensive myocardial necrosis (H and E, ×200), (d) immunohistochemical CD95 negativity (CD95, ×400)

Discussion

Various studies had been performed previously to detect doxorubicin-related myocardial injuries, including the investigation of serum natriuretic peptide levels,[2] radionuclide screening, monitoring of systolic and diastolic parameters through echocardiography, stress echocardiography performed with exercise and dobutamine, and performing cardiac biopsy.[3] Nevertheless, these studies were regarded insufficient both due to technical challenges and negative physical conditions of oncology patients and the results of such studies.

The doxorubicin-related myocardial injury is irreversible, and its severity is dependent on the dose.[4] The threshold of dose for irreversible myocardial injuries in rats is 27 mg/m2 or 3.8 mg/kg.[5] This threshold is the closest to that of human beings with 4.6 mg/kg.[6] While the heart failure risk in human is highly low in doses below 450 mg/m2 (0.1%), this rate reaches to 7% with 550 mg/m2 and to 50% with 1000 mg/m2.[7] In our study, however, severe interfibrillar hemorrhage and severe vascular dilatation were seen starting week 7 (cumulative dose of medication, 15 mg/kg), severe myocardial necrosis was seen starting week 8 (cumulative dose of medication, 17.5 mg/kg), severe inflammatory cell infiltration was seen starting week 9 (cumulative dose of medication, 20 mg/kg), and severe separation of muscle fibers was seen starting week 10 (cumulative dose of medication, 22.5 mg/kg).

In the pathogenesis of doxorubicin-related cardiomyopathy, free oxygen radicals, myocardial adrenergic function disorder, intracellular overload calcium, release of cardiotoxic cytokines, and apoptotic mechanisms were found responsible.[8] While apoptosis is controlled by several proteins such as death receptors, adaptor molecules, Bcl-2, and caspase sequence, caspase sequence is activated through two different ways, one mediated by mitochondria and the other mediated by Fas molecules.[9]

Fas is a membrane protein from the tumor necrosis factor family and coded by 10th chromosome. It is expressed in hepatocytes, various tumor cells, lungs, myocardial cells, lymphoid, and epithelial cells[10] and has two isoforms, one as membrane bounded and soluble other.[11] While membrane-bound form is composed of transmembranous and cytoplasmic parts, the soluble form has no transmembranous domain.[12] When agonist antibodies or natural ligands, also known as sFas ligand (sFasL), are bound,[11] death domain included by cytoplasmic part is bound to a protein called “Fas-Associated Death Domain,” and by becoming active caspase system subjects the cell to apoptosis.[13] In addition, Fas expression may also lead to doxorubicin-related cardiotoxicity by inducing inflammation, fibrosis, reactive oxygen species, and hypertrophy.[14]

Surface Fas expression on cardiac cells is increased by the effect of doxorubicin.[15] These apoptotic proteins expressed in the membrane may be released into the plasma after separated by proteolytic enzymes, and with this release, the level of soluble forms is increased.[16] In our study, sFas levels between 2nd and 7th weeks were significantly higher in the study group when compared to the levels found at 1st week before the medication. Besides, parallel to the increasing dose of medication, the sFas levels determined in the first four blood samples were also increased significantly when compared to the values of a previous week. However, at the end of the study, there was no meaningful increase in the Fas-positive cell number determined by flow cytometric evaluation. This situation may increase the injury of the tissues based on cumulative medicine dose increased until the end of the study, and therefore, it may also decrease the capacity of cells to express Fas. Since, in our study, sFas levels in the study group gradually decreased each week after the 5th week, and moderate/severe tissue injuries were generally determined at this phase.

Fas ligand is also a protein that is bound to the membrane. Both the form bound to the membrane and the one that can be changed into soluble form after separated by metalloproteinase may induce apoptosis. If regulated correctly, while sFasL–Fas apoptosis mechanisms clear off infected cells and cancerous cells, overworking of this mechanism cause tissue injury.[17] The sFas increased parallel to cell injury in acute phase may prevent inducing apoptosis by binding with natural sFasL in the mainstream. Since studies have shown that higher sFas levels are natural defense mechanisms against antineoplastic treatment.[16] High sFas levels determined during ischemic myocardial injury[18] and chemotherapy[19] are associated with a better prognosis. In addition, the decrease in the number of surface Fas antigens caused by release may decrease the possible injuries by narrowing the area where sFasL can bind to the cell surface and trigger apoptosis; it has been defined that the treatment carried with Fas ligand antibodies improved cardiac functions in doxorubicin-related cardiomyopathy.[14]

Conclusion

Both cell-surface Fas expression and plasma sFas level due to the release of these antigens may increase in the acute phase of doxorubicin-related cardiomyopathy, which increased parallel to the cumulative dose. However, parallel to the increase of the dose and tissue injury, there may be decrease on both eventually. The sFas level determined at acute phase may be beneficial in predicting existing tissue injury and late complications that may appear in the future.

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