Intensification of doxorubicin-related oxidative stress in the heart by hypothyroidism is not related to the expression of cytochrome P450 NADPH-reductase and inducible nitric oxide synthase, as well as activity of xanthine oxidase

Abstract

Cytochrome P450 NADPH-reductase (P450R), inducible synthase (iNOS) and xanthine oxidase play an important role in the antracycline-related cardiotoxicity. The expression of P450R and iNOS is regulated by triiodothyronine. The aim of this study was to evaluate the effect of methimazole-induced hypothyreosis on oxidative stress secondary to doxorubicin administration. 48 hours after methimazole giving cessation, rats were exposed to doxorubicin (2.0, 5.0 and 15 mg/kg). Blood and heart were collected 4, 48 and 96 h after the drug administration. Animals exposed exclusively to doxorubicin or untreated ones were also assessed. The hypothyreosis (0.025% of methimazole) significantly increased the doxorubicin effect on the cardiac carbonyl group and they may increase the glutathione level. An insignificant effect of methimazole was noticed in case of the cardiac lipid peroxidation product, the amount of DNA oxidative damages, iNOS and xanthine oxidase-enzymes responsible for red-ox activation of doxorubicin. However, the concentration of P450R was affected by a lower dose of methimazole in rats administered with doxorubicin. Since in rats receiving doxorubicin changes in oxidative stress caused by methimazole were not accompanied by elevation of bioreductive enzymes, it may be concluded that these changes in the oxidative stress were not related to the tested enzymes.

Figure 1

Mechanism expressed doxorubicin-dependent free radical generation and reportedly proposed role of triiodothyronine in redox equilibrium. P450R and iNOS transfer one electron to doxorubicin (DOX) from NADPH₂ leading to synthesis of doxorubicin radical (DOX*). Subsequently, electron is taken by O₂ and to produce superoxide anion radical (O₂ ^−∗). That is staring point to oxidative stress. Seemingly paradoxically NADPH₂ is indispensable to antioxidative activity, and its synthesis is transcriptionally regulated by triiodothyronine (T3).

Figure 2

Representative Western Blot analysis for P450R protein in cardiac muscle homogenates. Beta-actin is shown as a loading control. Densitometric analysis (mean ± SD) of total P450R content, expressed as percent changes with respect to the control group, which has been set at 100%. *P < 0.05 versus control and proper DOX group.

Figure 3

Strong positive cytoplasmic immunostaining for inducible nitric oxide synthase (iNOS) in cardiomyocytes. (a) 5DOX/96 h. (b) 5DOX + MET_L/96 h; (DakoEnvision+/HRP; objective magnification (a) and (b): 20x).

Figure 4

Representative mRNA iNOS content in cardiac muscle. HPRT was used as endogenous control. The results express percent changes (mean ±SD) with respect to the control group, which has been set at 100%. *P < 0.05 versus control.