The conducted study revealed that hypothyreosis had an important effect on the cardiac oxidative stress in rats exposed to doxorubicin. The direction of these changes was dependent on the concentration of iodothyronine hormones and the cytostatic dose. In case of hypothyreosis, the intensity of the doxorubicin-dependent oxidative stress was not related to the P450R, iNOS, and OX level.
The oxidative stress in cardiomyocytes triggered by doxorubicin is a widely accepted hypothesis in cardiotoxicity leading to the fatal congestive failure of the heart. As it was pointed above (), the one-electron doxorubicin reduction, a key process in oxidative stress development, is catalyzed by microsomal [12
], cytoplasmic [12
], and mitochondrial enzymes [17
]. Among microsomal and cytoplasmic enzymes, the P450R [12
], iNOS [23
], and XO [15
] play important role. As it was mentioned in the introduction, T3 and T4 via genome mechanism may change the activity of various enzymes engaged in doxorubicin redox activation [18
] and in antioxidative defence [19
To confirm the proper dose of methimazole, the plasma FT3 and FT4 was determined and thyroid histology (data not shown) was examined. The significant dose-dependent reduction of both FT3 and FT4 level and decreased amounts of colloid in thyroid follicles confirmed the properly chosen regiment of methimazole.
Clinical sensitivity of the analyzed oxidative stress markers varies in the order oxDNA > CG > MDA + 4HNE > GSHT
since an increase in oxidative DNA damages was observed in all tested periods in the group of 15DOX and after treatment with the middle dose of DOX (5
mg) in 4 and 48
h. A significant doxorubicin-dependent increase in CG concentration was also observed at 4, 48, and 96
h but only in groups administered with the highest dose of DOX. An important increase in the lipid peroxidation was revealed exclusively at 96
h from the administration of the highest doxorubicin dose. Moreover, a lack of the GSHT
cardiac level changes was found.
Similar data was previously found in other studies. Palmeira et al. [28
] observed that a single bolus of doxorubicin (15
mg/kg) caused a significant increase of the cardiac level of 8-hydroxydeoxyguanosine (8OHdG), another marker of DNA oxidative changes. The abundance of adducts was highest at the earliest time-point examined (24
h) and decreased to control values within 2 weeks. On the other hand, a significant increase in the level of the cardiac carbonyl group 10 days from single administration of doxorubicin in the dose of 20
mg was also pointed out [29
]. Moreover, the single doxorubicin dose in the range of 10–20
mg/kg caused an increase of lipid peroxidation products, which was revealed after several days from the drug injection [29
], whereas a lower single dose of doxorubicin (2.5
mg/kg) caused an increase of MDA level during the first four hours and normalised within 24
]. It seems that the dose of 2
mg, which was used in the current study, is too low to elicit a marked increase of the lipid peroxidation after single injection.
The question arises about the cause of different sensitivity determined markers after doxorubicin treatment. Based on the current knowledge, it is not possible to explain incompatible changes of various biochemical markers in case of the doxorubicin exposition. However, a biological half-life and sensitiveness to different kinds of reactive oxygen species should be considered. The carbonyl group appears relatively early and is stable during hours, and even days [34
], while the lifespan of the main lipid peroxidation products is estimated for minutes [36
]. Lipid peroxidation products (e.g., MDA) form DNA adducts, which may explain the observed disproportion between the level of MDA and oxDNA in this study [37
]. Moreover, oxidative destruction of lipids, protein, and DNA depends on the type of reactive oxygen species, for example, HOCl, a product of the reaction catalysed by myeloperoxidase, easily elevates the carbonyl group concentration but has a very little or no activity to create oxidative changes in lipids and DNA in the intracellular environment [41
]. It should be also stressed that in a study conducted by Fadillioglu et al. [29
] a single dose of DOX (20
mg/kg) caused a significant increase of the cardiac myeloperoxidase activity.
It was previously assumed that hypothyreosis may change redox status in the heart of rats receiving doxorubicin, since triiodothyronine regulates P450R [18
] and is suspected to regulate iNOS expression [21
], which plays an important role in bioreductive activation of DOX. Furthermore, the hormone upregulates the genes responsible for synthesis of G6PDH, malic enzyme, and 6-phosphogluconate dehydrogenase [27
]. These enzymes are a crucial cellular source of NADPH which via P450 and iNOS trigger ROS synthesis and, at the same time seemingly paradoxically, are indispensable in cell's antioxidative defence by reduced glutathione regeneration.
The current study revealed that hypothyroid conditions may increase the cardiac oxidative stress caused by doxorubicin. The most affected oxidative stress marker by hypothyreosis in rats treated with doxorubicin was CG. Similar result was also found for GSHT
that was influenced by the lowest and middle doses of DOX at 48
h and in the lowest dose at 96
h after drug administration.
The significant influence of hypothyreosis on cardiac lipid peroxidation in rats administered with doxorubicin appears only one time, and no such changes were observed in the case of oxDNA damages. For that reason, the comments will be focused on changes in carbonyl groups.
After treatment with the lowest and middle doses of doxorubicin only, there were no significant changes in CG in any period of time comparing to the control. However, when both mentioned doses of the drug were given to rats with hypothyreosis, a significant increase of oxidative protein damages was revealed. That rise was even 2-3 times higher than in proper DOX group. In cases when rats were treated with doxorubicin only, oxidative protein damages were higher comparing to control in all periods of time. The protein oxidative damages in rats administered with the highest dose of the drug changed depending on hypothyreosis state. When doxorubicin was administered to the rats exposed to a lower methimazole concentration, the level of carbonyl groups dramatically dropped, but in animals with more intense hypothyreosis the carbonyl groups levels were significantly elevated.
The obtained finding referring to the P450R protein level is somewhat surprising because referring to the assumption based on Ram and Waxman's [20
] results, who observed that T3 upregulates gene expression of this enzyme, and on the basis of their study, it might be expected that inhibition of T3 synthesis caused by methimazole should diminish the concentration of the enzyme.
Taking into consideration that a very high increase in protein oxidative damages in all groups receiving the higher dose of methimazole with DOX was not accompanied by any changes in P450R protein level comparing to the DOX only proper group, it may be concluded that the oxidative stress is not related to the expression of the enzyme.
Similarly, iNOS was not responsible for the increased oxidative protein level because these elevations in group DOX + METH were not accompanied by an increase of the enzyme concentration. Moreover, for the lowest and middle doses of doxorubicin, the immunoexpression of iNOS was lower in group DOX + METH comparing to euthyroid rats (proper group of DOX only).
Generally, there were no effects of iodothyronine hormone status on the activity of xanthine oxidase in rats receiving doxorubicin. Furthermore, this enzyme cannot be responsible for the observed oxidative protein elevation in all groups of DOX + METH.
Collectively, hypothyroid-like state may intensify the oxidative stress caused by doxorubicin, but P450R, iNOS, and XO may be excluded as being responsible for these phenomena. Further studies are needed to explain the role of other doxorubicin biactivating enzymes, especially of the mitochondrial fraction.