Inflammation as a Possible Trigger for Mitoxantrone-Induced Cardiotoxicity: An In Vivo Study in Adult and Infant Mice

Figures

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  Figure 1. Schematic representation of the distribution of the animals by cumulative dose, as well as the timeline of administration of MTX and sacrifice.

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  Figure 2. Cardiac histopathology evaluation done by light microscopy from MTX-treated adult and infant animals and respective controls, as assessed by haematoxylin and eosin staining (experiment 1, mice were euthanized 14 days after the last administration) (A–D). Light micrograph from: (A) infant mice controls in experiment 1, showing normal morphology and structure; (B) infant mice given a cumulative dose of 7.0 mg/kg MTX; (C) adult mice in experiment 1 showed normal morphology and structure; (D) adult mice given a cumulative dose of 7.0 mg/kg MTX. Presence of vacuolization (orange arrow), inflammatory infiltration (yellow arrow), as well as large and uncondensed nucleus (cyan arrow). Cellular oedema (white arrow) and necrotic zones (blue arrow) are evident only in adult mice. Scale bar = 100 µm, n = 3. Images taken at 40× amplification.

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  Figure 3. Cardiac histopathology evaluation done by light microscopy from MTX-treated adult and infant animals and respective, as assessed by haematoxylin and eosin staining controls (experiment 2, adult and infant mice were euthanized 7 or 17 days after the last administration, respectively) (A–D). Light micrograph from: (A) infant mice controls in experiment 2, showing normal morphology and structure; (B) infant mice given a cumulative dose of 6.0 mg/kg MTX; (C) adult mice in experiment 2 showed normal morphology and structure; (D) adult mice given a cumulative dose of 6.0 mg/kg MTX. Presence of vacuolization (orange arrow), inflammatory infiltration (yellow arrow), vascular congestion (green arrow), necrotic zones (blue arrow), as well as large and uncondensed nucleus (cyan arrow) is evident. Scale bar = 100 µm, n = 3. Images taken at 40× amplification.

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  Figure 4. Sirius red cardiac staining assessed by light microscopy from MTX-treated adult and infant animals and respective controls (A–F). Light micrograph from (A) the control infant mice (experiment 1, mice were euthanized 14 days after the last administration); (B) infant mice injected with a cumulative dose of 7.0 mg/kg MTX; (C) control adult mice; (D) adult mice injected with a cumulative dose of 7.0 mg/kg MTX showing higher fibrosis. (E) Control adults (experiment 2, mice were euthanized 7 days after the last administration); (F) adult mice after receiving a cumulative dose of 6.0 mg/kg MTX. Scale bar = 100 µm, n = 3. Images taken at 40× amplification.

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  Figure 5. ATP synthase subunit beta (52 kDa), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (37 kDa) expression, and ratio GAPDH to ATP synthase subunit beta evaluated by Western blotting, from (A–C) infant MTX-treated (M), and control mice (C), and (D–F) adult mice exposed to a cumulative dose of 6.0 mg/kg MTX (M), and control (C) adult mice (experiment 2, infant and adult mice were euthanized 17 or 7 days after the last administration, respectively). Values are expressed as mean ± SD and were obtained from 6 (infant) and 6–7 (adult) animals from each treatment group. Statistical comparisons were made using the t-test: * p < 0.05, MTX 6.0 mg/kg vs. control. OD: optic density. Protein loading was confirmed by the Ponceau S staining (Figures S3 and S4).

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  Figure 6. Protein carbonylation cardiac content evaluated by slot blot in (A) infant mice and (B) adult mice exposed to a cumulative dose of 6.0 mg/kg MTX (experiment 2, infant and adult mice were euthanized 17 or 7 days after the last administration, respectively). Values are expressed as mean ± SD and were obtained from six animals from each treatment group. Statistical comparisons were made using the t-test: * p < 0.05, MTX 6.0 mg/kg vs. control. OD: optic density, M: mitoxantrone, C: control.

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  Figure 7. Superoxide dismutase 2/ manganese-dependent superoxide dismutase (SOD2/MnSOD) (26.6 kDa), catalase (60 kDa) and glutathione peroxidase (22 kDa) expression evaluated by Western blotting, from (A–C) infant MTX-treated (M) and control (C) mice, and (D–F) adult mice exposed to a cumulative dose of 6.0 mg/kg MTX (M) and their respective controls (C) (experiment 2, infant and adult mice were euthanized 17 or 7 days after the last administration, respectively). Values are expressed as mean ± SD and were obtained from 6 (infant) and 6–7 (adult) animals from each treatment group. Statistical comparisons were made using the t-test: * p < 0.05, MTX 6.0 mg/kg vs. control. OD: optic density. Protein loading was confirmed by the Ponceau S staining (Figures S3 and S4).

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  Figure 8. Representative photomicrographs of heart immunohistochemistry from 6.0 mg/kg MTX-treated adult and respective controls (experiment 2, mice euthanized 7 days after the last administration), by a marker (CD68+) for macrophages M1 (A,B) and by a marker for (CD206) macrophage M2 (C,D) detection, and indicated by yellow arrows. Light micrograph (A,C) from the control of M1 (A) and M2 staining (C); light micrograph (B,D) from adult mice given a cumulative dose of 6.0 mg/kg MTX and performed staining for M1 (B) or M2 (D). Scale bar = 100 µm, n = 3. Images taken at 40× amplification.

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  Figure 9. Representative photomicrographs of immunohistochemistry determination of NF-κB p65 in the cardiomyocytes-like cells from 6.0 mg/kg MTX-treated adult and respective controls (experiment 2, mice euthanized 7 days after the last administration) (A,B). Light micrograph (A) from the controls; and (B) from adult mice given a cumulative dose of 6.0 mg/kg MTX. Scale bar = 100 µm, n = 3. Images taken at 40× amplification.

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  Figure 10. Nuclear kappa B nuclear transcription factor (NF-κB) p52 (50 kDa) and NF-κB p65 (60 kDa) expression evaluated by Western blotting, from (A,B) infant MTX-treated (M) and control (C) mice, and (C,D) adult mice exposed to a cumulative dose of 6.0 mg/kg MTX (M) and respective controls (C) (experiment 2, infant and adult mice were euthanized 17 or 7 days after the last administration, respectively). Values are expressed as mean ± SD and were obtained from 5–6 (infant) and 6–7 (adult) animals from each treatment group. Statistical comparisons were made using the t-test: ** p < 0.01, MTX 6.0 mg/kg vs. control. OD: optic density. Protein loading was confirmed by the Ponceau S staining (Figures S3 and S4).

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  Figure 11. Tumour necrosis factor-α (TNF- α) (25 kDa), interleukin-6 (IL-6) (24 kDa), myeloperoxidase (48 kDa) expression evaluated by western blotting, from (A–C) infant MTX-treated (M) and control (C) mice, and (D–F) adult mice exposed to a cumulative dose of 6.0 mg/kg MTX (M) and respective controls (C) (experiment 2, infant and adult mice euthanized 17 or 7 days after the last administration, respectively). Values are expressed as mean ± SD and were obtained from 6 (infant) and 6–7 (adult) animals from each treatment group. Statistical comparisons were made using the t-test: * p < 0.05, MTX 6.0 mg/kg vs. control. OD: optic density. Protein loading was confirmed by the Ponceau S staining (Figures S3 and S4).